γ-aminobutyric acid analog, preparation method therefor, and use thereof

Abstract

The present invention relates to a γ-aminobutyric acid analog, a preparation method therefor, and use thereof. The γ-aminobutyric acid analog comprises a γ-aminobutyric acid moiety and a polyethylene glycol (PEG) moiety. The γ-aminobutyric acid analog of the present invention is an active GABA analog, which can promote the proliferation of pancreatic β cells and the secretion of insulin, has in‑vivo functions similar to those of GABA, exhibits improved in-vivo metabolic properties compared to GABA, and thus can be used as a drug substitute for the treatment of metabolic diseases and neurological diseases.

Description

γ-aminobutyric acid analogues, their preparation methods and applications Technical Field

[0001] This application relates to the field of medicinal chemistry, specifically to a γ-aminobutyric acid analogue, its preparation method, and its application. Background Technology

[0002] Gamma-aminobutyric acid (GABA) is an amino acid that plays a major neurotransmitter role in the central nervous system (CNS). GABA also has physiological functions in the periphery; for example, pancreatic β cells express and produce large amounts of GABA, and GABA can stimulate the secretion of insulin by pancreatic islets and pancreatic β cells. Therefore, GABA may play autocrine and paracrine roles in the pancreas.

[0003] GABA is found in high concentrations in pancreatic β cells, located in synaptic microvesicles and roughly dense core vesicles containing insulin, and plays an important role in regulating pancreatic endocrine function. There are three types of GABA receptors: type A GABA receptor (GABAAR), type B GABA receptor (GABABR), and type C GABA receptor (GABACR) (Solomon VR, Tallapragada VJ, Chebib M, et al. GABA allosteric modulators: an overview of recent developments in non-benzodiazepine modulators[J]. Eur J Med Chem, 2019, 171: 434-461.). The first two receptors, GABAAR and GABABR, are expressed on the surface of pancreatic α cells and β cells. Studies have shown that glutamate decarboxylase (GAD) is an autoantigen in type 1 diabetes (T1D), which can convert glutamate into GABA (Baekkeskov, S., et al., Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase. Nature, 1990, 347(6289): p.151-6.), thereby maintaining pancreatic islet homeostasis (Menegaz D, Hagan DW, J, et al. Mechanism and effects of pulsatile GABA secretion from cytosolic pools in the human beta cell[J].Nat Metab, 2019, 1(11): 1110-1126.). GABA plays a regulatory role in the pancreas by activating GABAAR in pancreatic α cells, thereby inhibiting glucagon secretion (Reetz, A., et al., GABA and pancreatic beta-cells: colocalization of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion.EMBO J, 1991, 10(5): p.1275-84.). In addition, exogenous GABA has the effect of lowering blood sugar and promoting the regeneration of pancreatic β cells (Soltani N, Qiu H, Aleksic M, et al. GABA exerts protective and regenerative effects on islet beta cells and reverses diabetes[J]. Proc Natl Acad Sci USA, 2011, 108(28): 11692-11697; Ben-Othman N, Vieira A, Courtney M, et al. Long-term GABA administration induces alpha cell-mediated beta-like cell neogenesis[J]. Cell, 2017, 168(1-2): 73-85.e11.), so GABA therapy can maintain the quality of pancreatic β cells and prevent diabetes.

[0004] However, GABA has a small molecular weight, a rapid metabolic rate in vivo, and very low oral bioavailability, which greatly limits its clinical application. Therefore, it is necessary to develop a new drug form of GABA with improved in vivo metabolic properties. Summary of the Invention

[0005] Technical Purpose

[0006] One objective of this invention is to provide a γ-aminobutyric acid (GABA) analogue, which is an agonist of GABAAR and GABABR, and can improve the in vivo metabolic properties of GABA to meet the needs of clinical applications. In insulin-secreting β cells of the pancreas (such as the INS-1 cell line), GABA or GABA receptor agonists can promote insulin secretion from β cells, and also enhance β cell proliferation and have anti-apoptotic effects.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned γ-aminobutyric acid analogue.

[0008] Another object of the present invention is to provide the pharmaceutical use of the above-mentioned γ-aminobutyric acid analogues.

[0009] Technical solution

[0010] On one hand, the present invention provides a γ-aminobutyric acid analog or a salt thereof, the γ-aminobutyric acid analog comprising a γ-aminobutyric acid moiety and a polyethylene glycol (PEG) moiety, preferably wherein the PEG moiety is covalently linked to the amino or carboxyl terminus of the γ-aminobutyric acid moiety.

[0011] In a specific embodiment, the γ-aminobutyric acid analogue is represented by the following formula I:

[0012] In equation I above, This indicates the PEG portion.

[0013] In a specific application method, the average molecular weight of the PEG portion ranges from 200 to 8000 Da.

[0014] In some embodiments, the average molecular weight of the PEG portion is about 200 Da, 300 Da, 400 Da, 600 Da, 800 Da, 1000 Da, 1500 Da, 2000 Da, 3000 Da, 4000 Da, 6000 Da, or 8000 Da.

[0015] In a specific embodiment, the average molecular weight of the PEG portion is approximately 200 Da.

[0016] In a specific embodiment, the average molecular weight of the PEG portion is approximately 400 Da.

[0017] In a specific embodiment, the average molecular weight of the PEG portion is approximately 800 Da.

[0018] In a specific embodiment, the average molecular weight of the PEG portion is approximately 1000 Da.

[0019] In some embodiments, the salt of the γ-aminobutyric acid analog is a salt formed by the γ-aminobutyric acid analog with an inorganic or organic acid.

[0020] In some embodiments, the inorganic acid includes hydrochloric acid, sulfuric acid, or nitric acid.

[0021] In some embodiments, the organic acid includes citric acid, fumaric acid, lactic acid, propionic acid, malic acid, formic acid, or acetic acid.

[0022] In a specific embodiment, the salt of the γ-aminobutyric acid analog is the hydrochloride salt of the γ-aminobutyric acid analog as shown in the following formula:

[0023] On the other hand, the present invention provides a method for preparing the above-mentioned γ-aminobutyric acid analogue or its salt, the method comprising the following steps S1-S3:

[0024] S1: Reaction of γ-aminobutyric acid (GABA) with di-tert-butyl dimethyl carbonate yields Boc-protected GABA (Boc-GABA);

[0025] S2: Boc-GABA undergoes an esterification reaction with PEG to obtain Boc-GABA-PEG;

[0026] S3: In the presence of inorganic or organic acids, remove the Boc protecting group from Boc-GABA-PEG to obtain the γ-aminobutyric acid analog or its salt.

[0027] In some embodiments, in step S2, the molecular weight of PEG is in the range of 200-8000 Da, for example, the PEG is PEG400 or PEG800.

[0028] In a specific embodiment, in step S2, dimethylformamide / dichloromethane is added as a solvent.

[0029] In a specific embodiment, in step S2, dicyclohexylcarbodiimide and 4-dimethylaminopyridine are added to carry out an esterification reaction.

[0030] In some embodiments, in step S3, the inorganic acid includes hydrochloric acid, sulfuric acid, or nitric acid.

[0031] In some embodiments, in step S3, the organic acid includes citric acid, fumaric acid, lactic acid, propionic acid, malic acid, formic acid, or acetic acid.

[0032] On the other hand, the present invention provides a pharmaceutical composition comprising the above-mentioned γ-aminobutyric acid analogue or its salt, and pharmaceutically acceptable excipients.

[0033] On the other hand, the present invention provides the use of the above-mentioned γ-aminobutyric acid analogue or its salt, or the above-mentioned pharmaceutical composition, in the preparation of a medicament for treating diabetes, cerebrovascular disorders, or related diseases.

[0034] In a specific implementation, the diabetes includes type 1 diabetes or type 2 diabetes, and the cerebrovascular disorders include sequelae of stroke, cerebral arteriosclerosis, sequelae of traumatic brain injury, hemiplegia, or speech disorders. Beneficial effects

[0035] The γ-aminobutyric acid (GABA) analogue of the present invention can induce GABA-like currents and promote insulin secretion in pancreatic β cells, increase INS-1 cell proliferation, and prevent INS-1 cell apoptosis induced by high glucose or cytokines. In a mouse model, the GABA analogue improves glucose tolerance in mice. Therefore, the GABA analogue provided in this application is an active GABA analogue with similar functions to GABA in pancreatic β cells and can serve as a substitute for GABA. Attached Figure Description

[0036] Figure 1 shows the effects of GP-1 on GABA membrane currents (A) and insulin secretion (B) in INS-1 cells. (A) GP-1 induces GABA currents in INS-1 cells in a dihydrotestosterone-sensitive manner. (B) Results of RIA (radioimmunoassay) of insulin secretion using a rat insulin kit (Linco) in the presence of native GABA or 1 mM GP-1 at 2.8 mM glucose (28 mM glucose as a positive control). HG represents 28 mM high glucose, and LG represents 2.8 mM low glucose. **P < 0.01 compared to LG treatment.

[0037] Figure 2 shows the effect of GP-1 on glucose tolerance in mice. (A) Blood glucose levels measured at 0, 15, 30, 60, and 120 minutes after glucose injection, where GABA dose: 0.2 μmol / mouse. GP-1 dose: 0.2 μmol / mouse. (B) Area under the curve (AUC) of blood glucose levels obtained from the IPGTT in Figure A. *P < 0.05 compared to the PBS treatment group.

[0038] Figure 3 shows the results of the MTT assay for the effect of GP-1 on INS-1 cell proliferation. In the figure, FBS is fetal bovine serum; compared with 0% FBS treatment, **p<0.01, *p<0.05.

[0039] Figure 4 shows the protective effect of GP-1 on INS-1 cells induced by high glucose or cytotoxic factors. (A) Immunoblotting results of INS-1 cells induced by high glucose. (B) Immunoblotting results of INS-1 cells induced by cytotoxic cytokines. (C&D) Comparison of histograms of caspase 3 cleavage density and GAPDH band intensity in A&B. **p<0.01, NT is the negative control group without any treatment.

[0040] Figure 5 shows the FACS results of INS-1 cells treated with GP-1 or GABA followed by cytotoxic factor treatment. (A) Untreated cells; (B) Cells treated with the cytokine mixture alone for 24 hours; (C) Cells treated with 100 μM GP-1 for 24 hours, followed by the cytotoxic factor mixture for 24 hours; (D) Cells treated with 100 μM GABA for 24 hours, followed by the cytotoxic factor mixture for 24 hours. *p < 0.05 compared to the control group. Detailed Implementation

[0041] The technical content of the present invention will be described in detail below through specific embodiments, so that those skilled in the art can better understand the present invention.

[0042] In the above reaction formula, PEG represents The structures shown have an average molecular weight of 400 or 800 Da.

[0043] Reagents and conditions: i) Boc)₂O, Et₃N, methanol / H₂O 1:1, rt, yield 89%. ii) PEG₄00 or PEG₈00, DCC, DMAP, DMF / CH₂Cl₂; yields of 3 and 4 were 71% and 63%, respectively. iii) HCl(g), CH₂Cl₂; yields of 5 and 6 were 91% and 93%, respectively.

[0044] the term

[0045] In this application, "PEG" refers to polyethylene glycol. Depending on the average molecular weight, PEG can include PEG200, PEG300, PEG400, PEG600, PEG800, PEG1000, PEG1500, PEG2000, PEG3000, PEG4000, PEG6000, and PEG8000. For example, PEG200 represents an average molecular weight of approximately 200, PEG300 represents an average molecular weight of approximately 300, and so on.

[0046] Example 1: Synthesis of compound GABA-PEG400

[0047] Step 1: Compound 1 (GABA, 1.03 g, 10.0 mmol) was dissolved in H2O (10 mL), and MeOH (10 mL) was added dropwise. Then, triethylamine (Et3N) (2.1 mL, 15.0 mmol) and di-tert-butyl dicarbonate ((Boc)2O) (4.36 g, 20.0 mmol) were added dropwise. The mixture was stirred at room temperature for 8 h. After filtration through diatomaceous earth, the residue was washed with brine (10 mL × 2) and water (10 mL × 3), and dried to obtain a white solid compound 2 (Boc-GABA).

[0048] Step 2: Compound 2 was dissolved in dimethylformamide / dichloromethane (1:1, 30 mL), and dicyclohexyl carbodiimide (DCC) (2.06 g, 10.0 mmol) was added. The mixture was stirred at 0 °C for 30 min. 4-Dimethylaminopyridine (DMAP) (1.22 g, 10.0 mmol) and PEG400 (40.0 mmol) were added. After stirring overnight, the mixture was poured into water (100 mL), acidified to pH 3.0 with 3 M HCl, and then extracted with ethyl acetate (50 mL × 3). The organic layer was evaporated under reduced pressure to give the residue (3, Boc-GABA-PEG400), in 71% yield.

[0049] Step 3: Compound 3 was treated with anhydrous HCl in CH2Cl2 (100 mL) for 6 hours. The volatiles were then removed to obtain compound 5 (GABA-PEG400) in 91% yield. Compound 5: High-resolution ESI-MS m / z 412.2586, 456.2857, 500.3102, 544.3350, 588.3616. GABA-PEG400 [M+H] + The calculated values ​​are 412.2547 (n=7), 456.2809 (n=8), 500.3071 (n=9), 544.3333 (n=10), and 588.3595 (n=11).

[0050] Example 2: Synthesis of compound GABA-PEG800

[0051] Step 1: Compound 1 (GABA, 1.03 g, 10.0 mmol) was dissolved in H2O (10 mL), and MeOH (10 mL) was added dropwise. Then, Et3N (2.1 mL, 15.0 mmol) and di-tert-butyl dicarbonate (4.36 g, 20.0 mmol) were added dropwise. The mixture was stirred at room temperature for 8 h. After filtration through diatomaceous earth, the residue was washed with brine (10 mL × 2) and water (10 mL × 3), and dried to obtain a white solid compound 2 (Boc-GABA).

[0052] Step 2: Compound 2 was dissolved in dimethylformamide / dichloromethane (1:1, 30 mL), and DCC (2.06 g, 10.0 mmol) was added. The mixture was stirred at 0 °C for 30 min. DMAP (1.22 g, 10.0 mmol) and PEG800 (40.0 mmol) were added. After stirring overnight, the mixture was poured into water (100 mL), acidified to pH 3.0 with 3 M HCl, and then extracted with ethyl acetate (50 mL × 3). The organic layer was evaporated under reduced pressure to give the residue (4, Boc-GABA-PEG800) in 63% yield.

[0053] Step 3: Compound 4 was treated with anhydrous HCl in CH2Cl2 (100 mL) for 6 hours. The volatiles were then removed to obtain compound 6 (GABA-PEG800, hereinafter referred to as "GP-1"), in 93% yield.

[0054] Compound 6: High-resolution ESI-MS m / z 852.51, 88, 896.5451, 940.5699, 984.5972. GABA-PEG800 [M+H] + The calculated values ​​are 852.5168 (n=17), 896.5430 (n=18), 940.5692 (n=19), and 984.5954 (n=20).

[0055] Example 3: GP-1 induces GABA membrane currents and increases insulin secretion in INS-1 cells.

[0056] Transmembrane currents induced by GP-1 (100 μM) in INS-1 cells were generated using an EPC-9 amplifier and pulse software (HEKA, Germany) in whole-cell voltage-clamp mode. Rapid application of GP-1 induced an inward current when the cell voltage was clamped at -60 mV. The intracellular solution contained (in mM) 130 KCl, 10 NaCl, 0.5 MgCl2, 1 EGTA, 5 HEPES, and 0.15 mg / ml nystatin (pH 7.3). The standard bath solution (in mM) contained 138 NaCl, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, 5.6 glucose, and 5 HEPES (pH 7.4) (left panel of Figure 1, A). The current induction of GP-1 was blocked by the GABA receptor antagonist bicuculine (Bic) (right panel of Figure 1, A).

[0057] Furthermore, insulin secretion RIA (radioimmunoassay) was performed using a rat insulin kit (Linco) in the presence of natural GABA or 1 mM GP-1 at 2.8 mM glucose (28 mM glucose as a positive control) (Figure 1, B).

[0058] As shown in Figure 1A, GP-1 induces GABA currents in INS-1 cells in a dihydrotestosterone-sensitive manner, indicating that GP-1 activates type A GABA receptors (GABAAR) in the β-cell line INS-1. As shown in Figure 1B, RIA of INS-1 cells demonstrates that GP-1 can stimulate insulin secretion.

[0059] Example 4: Effect of GP-1 on glucose tolerance in mice

[0060] All mice (C57BL / 6 mice, purchased from Shanghai Silex Laboratory Animal Co., Ltd., under standard feeding conditions) (n=5 / group) were fasted for 16 hours, and then injected intraperitoneally with GP-1, GABA, or PBS, respectively. Thirty minutes after injection, an intraperitoneal glucose tolerance test (IPGTT) was performed by intraperitoneal injection of 1.5 g / kg glucose, and blood glucose levels were measured using a glucometer. Blood was collected from the tip of the mouse tail for blood glucose measurement.

[0061] The results are shown in Figure 2. As can be seen from Figure 2, GP-1 significantly improved glucose tolerance in mice compared to GABA.

[0062] Example 5: MTT assay to detect the effect of GP-1 on INS-1 cell proliferation

[0063] INS-1 cells were incubated with serum-containing medium for 24 hours, and then incubated with different drugs (0% FBS, GP-1 1 μM, GP-1 10 μM, GP-1 100 μM, GABA 100 μM) for 24 hours before MTT assays were performed. Each treatment condition was applied in serum-free medium.

[0064] The results are shown in Figure 3. The absorbance rate represents the cell proliferation rate; the higher the absorbance, the more cells proliferate, which means higher cell viability. As can be seen from Figure 3, treatment of INS-1 cells with GP-1 (1, 10, 100 μM, 24 hours) significantly increased cell proliferation.

[0065] Example 6: Protective effect of GP-1 on INS-1 cells induced by high glucose or cytotoxic factors

[0066] Immunoblotting of high glucose-induced INS-1 cells: cells were treated with GP-1 at concentrations ranging from 1 to 100 μM for 24 hours, followed by treatment with 30 mM glucose for 24 hours, and detected with anti-caspase 3 antibody and anti-GAPDH antibody.

[0067] Immunoblotting of INS-1 cells induced by cytotoxic factors: cells were treated with GP-1 at concentrations ranging from 1 to 100 μM for 24 hours, followed by treatment with cytotoxic cytokines (10 ng / ml IL-1β, 50 ng / ml TNF-α, 50 ng / ml IFN-γ) for 24 hours, or with 100 μM GP-1 and bicucullin or nifedipine (20 μM). Detection was performed using anti-caspase 3 antibody and anti-GAPDH antibody.

[0068] The results are shown in Figure 4. Figure 4 shows that GP-1 pretreatment reduced apoptosis in INS-1 cells treated with cytokines or high concentrations of glucose (100 μM, 24 h). Western blotting indicated that GP-1 protected β cells from apoptosis induced by cytotoxic cytokines (10 ng / ml IL-1β, 50 ng / ml TNF-α, 50 ng / ml IFN-γ) or high concentrations of glucose (30 mM).

[0069] Example 7: Flow cytometry fluorescence sorting (FACS) of INS-1 cells treated with GP-1 or GABA followed by cytotoxic factors.

[0070] INS-1 cells were grouped and treated as follows: (A) untreated cells; (B) cells treated with a cytokine mixture (10 ng / ml IL-1β, 50 ng / ml TNF-α, 50 ng / ml IFN-γ) for 24 hours; (C) cells treated with 100 μM GP-1 for 24 hours, followed by treatment with a cytotoxic factor mixture (10 ng / ml IL-1β, 50 ng / ml TNF-α, 50 ng / ml IFN-γ) for 24 hours; and (D) cells treated with 100 μM GABA for 24 hours, followed by treatment with a cytotoxic factor mixture (10 ng / ml IL-1β, 50 ng / ml TNF-α, 50 ng / ml IFN-γ) for 24 hours, and then stained with propidium iodide (0.05 mg / ml). The samples were then analyzed by flow cytometry on a logarithmic scale.

[0071] The results are shown in Figure 5. As can be seen from Figure 5, flow cytometry analysis using PI staining showed that pretreatment of INS-1 cells with GP-1 (100 μM, 24 h) reduced cytokine-induced apoptosis compared to GABA.

[0072] In summary, based on the results of the above embodiments, the GP-1 compound provided in this application exhibits the following superior biological activities: effectively inducing GABA-like currents and significantly enhancing insulin secretion function; significantly promoting INS-1 cell proliferation; and compared with traditional GABA compounds, GP-1 shows a more significant advantage in improving glucose tolerance in mice and preventing apoptosis of INS-1 cells induced by high glucose or cytokines. Therefore, GP-1 can be regarded as a biologically enhanced GABA analog, possessing similar and significantly improved biological characteristics to GABA in regulating pancreatic β-cell function. Furthermore, by repeating Examples 1 to 5 with compound 5 (GABA-PEG400), the same experimental results as GP-1 were obtained, further confirming the functional effectiveness and mechanism of action of the compounds of this invention.

Claims

1. A γ-aminobutyric acid analog or a salt thereof, comprising a γ-aminobutyric acid moiety and a polyethylene glycol (PEG) moiety, preferably wherein the PEG moiety is covalently attached to the amino or carboxyl terminus of the γ-aminobutyric acid moiety.

2. The γ-aminobutyric acid analogue or its salt according to claim 1, wherein it is represented by the following formula I: In equation I above, This indicates the PEG portion.

3. The γ-aminobutyric acid analogue or its salt according to claim 1 or 2, wherein, The average molecular weight of the PEG portion is 200Da, 300Da, 400Da, 600Da, 800Da, 1000Da, 1500Da, 2000Da, 3000Da, 4000Da, 6000Da, or 8000Da.

4. The γ-aminobutyric acid analogue or its salt according to claim 1 or 2, wherein, The average molecular weight of the PEG portion is 200 Da, 400 Da, 800 Da, or 1000 Da.

5. The γ-aminobutyric acid analogue or its salt according to claim 1 or 2, wherein, The salt of the γ-aminobutyric acid analog is a salt formed by the γ-aminobutyric acid analog and an inorganic or organic acid. Preferably, The inorganic acid includes hydrochloric acid, sulfuric acid, or nitric acid, and / or The organic acids include citric acid, fumaric acid, lactic acid, propionic acid, malic acid, formic acid, or acetic acid.

6. The γ-aminobutyric acid analogue or its salt according to claim 1 or 2, wherein, The salt of the γ-aminobutyric acid analog is the hydrochloride salt of the γ-aminobutyric acid analog as shown in the following formula:

7. A method for preparing a γ-aminobutyric acid analog or a salt thereof as described in any one of claims 1-6, the method comprising the following steps: S1: Reaction of γ-aminobutyric acid (GABA) with di-tert-butyl dimethyl carbonate yields Boc-protected GABA (Boc-GABA); S2: Boc-GABA undergoes an esterification reaction with PEG to obtain Boc-GABA-PEG; S3: In the presence of inorganic or organic acids, remove the Boc protecting group from Boc-GABA-PEG to obtain the γ-aminobutyric acid analog or its salt.

8. The method according to claim 7, wherein, In step S2, The molecular weight range of PEG is 200-8000 Da, for example, the PEG is PEG400 or PEG800; and / or Add dimethylformamide / dichloromethane as a solvent; and / or Esterification reaction is carried out by adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and / or In step S3, the inorganic acid includes hydrochloric acid, sulfuric acid, or nitric acid; the organic acid includes citric acid, fumaric acid, lactic acid, propionic acid, malic acid, formic acid, or acetic acid.

9. A pharmaceutical composition comprising a γ-aminobutyric acid analog or a salt thereof as described in any one of claims 1-6, and pharmaceutically acceptable excipients.

10. The use of a γ-aminobutyric acid analogue or a salt thereof as described in any one of claims 1-6, or the use of a pharmaceutical composition as described in claim 9 in the preparation of a medicament for treating diabetes, cerebrovascular disorders, for example, the diabetes including type 1 diabetes or type 2 diabetes, and the cerebrovascular disorders including sequelae of stroke, cerebral arteriosclerosis, sequelae of traumatic brain injury, hemiplegia, or speech disorders.

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