Acegastrodin derivative compound as well as preparation method and application thereof

Abstract

The invention discloses an acegastrodin derivative compound with a chemical structural formula as shown in the specification, and the acegastrodin derivative compound has a protective effect on PC12 cell injury induced by H2O2 and can be used for preparing a neuroprotective preparation. The compound disclosed by the invention can be efficiently and sensitively combined with target protein so as to obtain potential target protein, a favorable tool can be provided for target research, potential targets of acegastrodin in treatment of neurological diseases can be explored, and the application of acegastrodin in treatment of neurological diseases can be promoted. The invention provides a new method for treating nerve diseases and ascertaining the mechanism of treating nerve diseases by acegastrodin. .

Description

Technical Field

[0001] This invention belongs to the field of molecular probe preparation, and mainly relates to the preparation and application of an acetylgastrodin functional probe. Background Technology

[0002] Acetyl gastrodin is an acetylated derivative of gastrodin. It is only slightly soluble in water, but highly lipid-soluble, making it easier to enter the central nervous system and exert its effects. It does not easily accumulate in the body and can restore the balance between excitation and inhibition processes in the cerebral cortex. Acetyl gastrodin tablets are widely used clinically to treat insomnia, neurasthenia, vascular headaches, and tension headaches, but its effect on neurodegenerative diseases has not been reported.

[0003] Activity-based proteomics analysis primarily utilizes active small molecule probes (ABPs) to identify the targets of active drug components. ABPs are probes modified from the structure of the active component; different tags on ABPs have different functions, primarily imaging localization and binding enrichment. The function of an ABP is mainly determined by its tag; for example, attaching an affinity tag enables enrichment, allowing for subsequent target enrichment and mechanistic studies. Attaching a biotin tag enhances its target localization function, enabling visualization and detection of target proteins. There are no reports on constructing active molecular probes based on activity-based proteomics analysis to study potential targets for the neuroprotective effects of acetylgastrodin. This invention provides a method for preparing a functional probe for acetylgastrodin and its application in target enrichment. Summary of the Invention

[0004] This invention provides an acetylgastrodin-derived compound, the chemical structural formula of which is as follows: .

[0005] The preparation method of the above-mentioned acetylgastrodin derivative compounds is as follows: 1. Acetyl gastrodin, succinic anhydride, 4-(dimethylamino)pyridine (DMAP) and tetrahydrofuran were mixed and esterified at 55-65℃. The reaction product was purified by silica gel column chromatography to obtain intermediate S1. ; 2. Biotin, o-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU), and N,N-dimethylformamide (DMF) were mixed, and then triethylamine (TEA) was added dropwise. The mixture was stirred until the solid was completely dissolved. N-Boc-1,6-hexanediamine was added, and an amidation reaction was carried out at room temperature. The reaction product was purified by silica gel column chromatography to obtain intermediate S2. ; 3. At room temperature, intermediate S2 was dissolved in a trifluoroacetic acid-dichloromethane mixed solution, and the reaction was stirred to remove the Boc protecting group. The reaction product was purified by silica gel column chromatography to obtain intermediate S3. ; The trifluoroacetic acid-dichloromethane mixed solution is prepared by mixing trifluoroacetic acid and dichloromethane in a volume ratio of 1:4-6.

[0006] 4. Under room temperature and in the presence of solvent, intermediate S1, intermediate S3, o-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU) and triethylamine (TEA) were mixed and stirred to react. The reaction product was purified by silica gel column chromatography to obtain acetylgastrodin derivative compound (P1). .

[0007] Another objective of this invention is to apply acetyl gastrodin derivatives in the preparation of neuroprotective agents.

[0008] The active ingredient of the formulation of the present invention is the above-mentioned acetyl gastrodin derivative compound, and one or more pharmaceutically acceptable excipients may be added to improve the stability, absorption effect or ease of use of the formulation. The drug can be prepared into a variety of suitable dosage forms, including but not limited to capsules, pills, powders, tablets, granules, oral liquids, etc.

[0009] Another objective of this invention is to apply acetylgastrodin derivatives in the preparation of reagents for identifying the target of acetylgastrodin.

[0010] The acetyl gastrodin-derived compounds provided by this invention have a protective effect against H2O2-induced PC12 cell damage and can be used to prepare neuroprotective agents. The compounds of this invention can bind to target proteins efficiently and sensitively, thereby revealing potential target proteins. This invention provides a valuable tool for target research, exploring potential targets of acetyl gastrodin in the treatment of neurological diseases. Through peptide secondary mass spectrometry analysis, the potential target protein that specifically binds to the acetyl gastrodin functional probe was found to be PCCA. This invention provides a new method for the treatment of neurological diseases and for elucidating the mechanism of acetyl gastrodin in treating neurological diseases. Attached Figure Description

[0011] Figure 1 This is a diagram showing the silver staining results of the protein labeled with compound P1. Detailed Implementation

[0012] The method of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the scope of protection of the present invention is not limited to the content described. Unless otherwise specified, the reagents in the embodiments are all conventional reagents or reagents prepared by conventional methods. Example 1: Preparation of acetylgastrodin derivative compounds 1. Esterification reaction Acetyl gastrodin (90 mg, 0.2 mmol) was weighed and placed in a round-bottom flask. Succinic anhydride (36 mg, 0.36 mmol), DMAP (24 mg), and 5 mL of tetrahydrofuran were added. After mixing, the mixture was heated under reflux at 60 °C for 12 h. The reaction product was purified by silica gel column chromatography. The product was eluted with an ethyl acetate-petroleum ether mixture (ethyl acetate:petroleum ether volume ratio of 1:1). The eluent was collected and dried to obtain intermediate S1. 2. Amide reaction Biotin (100 mg, 0.41 mmol) and TBTU (145 mg, 0.45 mmol) were weighed and placed in a round-bottom flask. DMF (2 mL) was added, followed by TEA (34 μL). The mixture was stirred for 20 min until the solid was completely dissolved. Then, N-Boc-1,6-hexanediamine (98 mg, 0.45 mmol) was added. The mixture was stirred at room temperature for 4 h. The reaction was quenched with ethyl acetate. The mixture was washed with saturated brine. The ethyl acetate layer was collected, the solvent was removed, and the mixture was purified by silica gel column chromatography (methanol:ethyl acetate = 1:20). The eluent was collected and dried to obtain intermediate S2. 3. Deprotection Intermediate S2 was dissolved in a trifluoroacetic acid-dichloromethane mixed solution (1:5) and stirred at room temperature for 2 h to remove the Boc protecting group. After the reaction was completed, n-butanol was added to quench the reaction. The mixture was washed three times with saturated brine, and the n-butanol layers were collected and combined. After concentration, the mixture was purified by silica gel column chromatography (methanol:dichloromethane = 1:4). The eluent was collected and dried to obtain intermediate S3. 4. Amide-linked Weigh intermediate S1 (40 mg, 0.072 mmol) into a round-bottom flask, add intermediate S3 (30 mg, 0.088 mmol), TBTU (25 mg), 2 mL DMF, and 3 drops of TEA, and stir the reaction at room temperature for 6 h. The reaction product is purified by silica gel column chromatography (methanol: dichloromethane = 1:15), the eluent is collected and dried to obtain acetylgastrodin derivative compound P1; P1: 1 H NMR (400 MHz, CDCl3) δ 7.28 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.7 Hz,2H), 6.31 (m, 1H), 6.27 (m, 1H), 6.16 (s, 1H), 5.58 (s, 1H), 5.23-5.32 (m,2H), 5.16 (t, J= 9.6 Hz, 1H), 5.04-5.10 (m, 3H), 4.48-4.52 (m, 1H), 4.26-4.33(m, 2H), 4.16 (dd, J = 12.3, 2.4 Hz, 1H), 3.85-3.90 (m, 1H), 3.11-3.21 (m, 6H), 2.90 (dd, J = 12.6, 4.5 Hz, 1H), 2.66-2.74 (m, 3H), 2.48 (t, J = 6.8 Hz, 2H), 2.20 (t, J = 6.7 Hz, 2H), 2.07 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.03 (s,3H), 1.59-1.75 (m, 4H), 1.40-1.47 (m, 5H), 1.31 (m, 4H). 13 C NMR (100 MHz, CDCl3) δ 173.5, 173.2, 171.7, 170.7, 170.4, 169.6, 169.5, 164.1, 156.9, 131.0,130.0 (2C), 117.1 (2C), 99.1, 77.4, 72.8, 72.2, 71.3, 68.4, 66.1, 62.1, 60.4,55.7, 46.5, 40.7, 39.3, 39.2, 35.9, 31.0, 29.8, 29.4, 28.2, 28.1, 26.2, 26.1,25.7, 20.8 (2C), 20.7 (2C). HRMS (+ESI): m / z calcd for C 41 H 59 N4O 15 S [M+H] + :879.3698, found: 879.3699.

[0013] The structure of the final product P1 was analyzed by HRMS. 1 H-NMR and 13 C-NMR analysis confirmed the structure of the compound as follows: .

[0014] Example 2: Neuroprotective effects of acetylgastrodin derivatives This study investigated the effect of acetylgastrodin derivative compound P1 on the survival rate of PC12 cells, and cell proliferation activity was detected using the CCK8 assay. Logarithmic growth phase PC12 cells were seeded in 96-well plates and cultured in an incubator (5% CO2, 37℃) until adherence. The model group and drug-treated groups were incubated with DMEM / F12 complete culture medium containing 200 μmol / L H2O2 for 24 h at 37℃. Then, the drug-treated groups were incubated with acetyl-gastrodin derivative compound P1 at final concentrations of 25 μmol / L, 50 μmol / L, 100 μmol / L, and 200 μmol / L for 24 h, respectively. The model group did not receive P1, and a blank group was set up without H2O2 or P1. After intervention, the cells were incubated with CCK8 solution for 2 h, and the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated as follows: Cell viability (%) = (A... 模型组 / 给药组 / A 空白组 ) × 100%.

[0015] The results are shown in Table 1. Compared with the blank group, the survival rate of PC12 cells induced by H2O2 was significantly reduced. Compared with the model group, the addition of acetyl gastrodin derivative compound P1 to the drug treatment group can increase the survival rate of PC12 cells in a concentration-dependent manner. The above results indicate that acetyl gastrodin derivative compound P1 has a protective effect against H2O2-induced PC12 cell damage. Table 1. Acetylgastrodin functional probes enhance H2O2-induced cell survival rate of PC12 neural cells.

[0016] Note: Compared with the blank group, # p <0.05; compared with the model group, * p <0.05.

[0017] Example 3: Acetylgastrodin-derived compound P1 as a probe-labeled specific target protein Brain tissue from C57BL / 6J mice was collected, RIPA lysis buffer was added, and the mixture was thoroughly ground. The mixture was centrifuged at 12000 rpm for 10 min in a refrigerated centrifuge at 4°C, and the supernatant was collected as the mouse brain tissue protein solution. Four groups were set up: a blank control group, an empty magnetic bead group, a probe P1 group, and a biotin group. In the blank control group, 75 μg of mouse brain tissue protein was quantitatively added using BCA; in the empty magnetic bead group, 200 μg of mouse brain tissue protein and 100 μg of magnetic beads were added; in the probe P1 group, 200 μg of mouse brain tissue protein, a final concentration of 50 μM probe P1, and 100 μg of magnetic beads were added; and in the biotin group, 200 μg of mouse brain tissue protein, 50 μM biotin, and 100 μg of magnetic beads were added. Add 200 μg of protein and 50 μM probe P1 to a centrifuge tube in probe P1 group. Incubate together at room temperature for 1 h on a rotary mixer. Then add 100 μg of magnetic beads and mix well. Incubate for another 1 h under the same conditions. After incubation, separate on a magnetic rack for 3 min. Discard the liquid and retain the magnetic bead-protein complex. Add 20 μL of Ripa lysis buffer, boil for 10 min, discard the magnetic beads, and collect the solution to obtain the enriched protein of probe P1. The operation is the same for other experimental groups.

[0018] Silver staining was used to observe the specific target protein labeled by the acetylgastrodin derivative compound P1. The results are shown in [Figure number missing]. Figure 1 The results showed that the acetylgastrodin-derived compound P1 group exhibited a specific band at approximately 75-100 kDa, which differed from the biotin group and the empty magnetic bead group. This indicates that compound P1 binds to certain specific proteins in the 75-100 kDa region and was enriched using streptavidin magnetic beads. The specific band of probe P1 group was excised and identified by protein mass spectrometry using LC-MS / MS. Analysis of the peptide secondary mass spectrometry revealed that the potential target protein specifically binding to acetylgastrodin-derived compound P1 is PCCA, with the amino acid sequence of its core peptide being HIEIQVLGDK. This demonstrates that acetylgastrodin functional probes can be applied to the enrichment and identification of natural product targets.

Claims

1. An acetyl gastrodin derivative compound having the following chemical structure: 。 2. The method of preparing the acetyl gttiko derivative compound according to claim 1, characterized by, comprising the following steps: (1) esterification of acetyl gastrodin with succinic anhydride in the presence of a solvent and a catalyst at 55-65°C to obtain an intermediate S1 having the following chemical structure: ； (2) amidation of biotin, N-Boc-1,6-hexanediamine and a coupling reagent in the presence of a solvent and a base at room temperature to obtain an intermediate S2 having the following chemical structure: ； (3) stirring and reacting the intermediate S2 in a trifluoroacetic acid-dichloromethane mixed solution at room temperature to remove the Boc protecting group and obtain an intermediate S3; ； (4) stirring and reacting the intermediate S1, the intermediate S3 and a coupling reagent in the presence of a solvent and a base at room temperature to obtain the acetyl gastrodin derivative compound; 。 3. Use of the acetyl gastrodin derivative compound of claim 1 in the preparation of a neuroprotective preparation.

4. Use of the acetyl gastrodin derivative compound of claim 1 in the preparation of an acetyl gastrodin target site identification reagent.

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