Quinazoline alkaloid enantiomers in Polygonum tinctorium, preparation method and application thereof

Abstract

The present invention belongs to the technical field of medicine, and relates to a pair of quinazoline alkaloid enantiomer compounds extracted and separated from the plant Polygonum tinctorium Ait. of the genus Polygonum in the Polygonaceae family, which has a quinazoline-fused pyrrolidine alkaloid 6 / 6 / 5 tricyclic mother nucleus. The present invention also relates to a preparation method of the compound, and its use in the preparation of a drug for treating neurodegenerative diseases. The preparation method of the present invention is simple and easy to operate, has good reproducibility, and has a relatively high purity. The obtained compound has a good neuroprotective effect.

Description

Technical Field:

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a method for preparing enantiomers of new quinazoline alkaloids from Polygonum tinctorium, and the application of such compounds in neuroprotection. Background Art:

[0002] Polygonum tinctorium: Polygonum tinctorium Ait. belongs to the genus Polygonum of the family Polygonaceae, and is an annual herbaceous plant, which is cultivated throughout the country or in a semi-wild state. The leaves of Polygonum tinctorium have a long history of medicinal use in China, and the dried leaves are used as Folium Isatidis in medicine. It is cold in nature and bitter in taste, and has the effects of clearing heat and detoxifying, cooling blood and removing spots.

[0003] With the process of global population aging, neurodegenerative diseases have received increasing attention. Among them, Alzheimer's disease and Parkinson's disease are becoming the main killers leading to the death of the middle-aged and elderly. In recent years, studies have shown that the SH-SY5Y cell line is very sensitive to the changes of oxygen free radicals and is often used to study the mechanism of neuronal cells affected by oxidative stress. Oxidative stress mediates cell damage in a variety of neurodegenerative diseases. Therefore, anti-oxidative stress is considered an effective treatment strategy for neurodegenerative diseases and has good prospects for the treatment of neurodegenerative diseases. Summary of the Invention:

[0004] The purpose of the present invention is to provide a method for preparing enantiomers of new quinazoline alkaloids from Polygonum tinctorium and their application in neuroprotection.

[0005] The structure of the enantiomers of the new quinazoline alkaloids isolated from the plant Polygonum tinctorium Ait. of the genus Polygonum of the family Polygonaceae according to the present invention is shown in the figure:

[0006]

[0007] The preparation technical solution of the present invention includes the following steps:

[0008] Take the dried leaves of Polygonum tinctorium, extract with ethanol, combine the extraction solutions and concentrate to obtain an extract. The extract is extracted with dichloromethane, and the obtained components are subjected to silica gel column chromatography, and isocratic gradient elution is carried out with a dichloromethane-methanol system of 100:0 - 0:100, and a total of 4 components (A - D) are collected.

[0009] Component A is subjected to polyamide column chromatography, and gradient elution is carried out with an ethanol-water system of 0:100 - 100:0 to obtain two components A1 and A2.

[0010] Component A1 is subjected to gradient elution with an ethanol-water system of 30:70 - 90:10 using HP20 column chromatography to obtain three components (A1-1 - A1-3).

[0011] The component A1-2 was gradient eluted with an ethanol-water system of 30:70 - 90:10 using an ODS column chromatography to obtain four components (a - b).

[0012] The obtained component a was subjected to silica gel column chromatography with a dichloromethane-methanol system of 50:1 - 10:1 on the basis of TLC analysis to obtain four sub-components (a1 - a4).

[0013] Compound 1 was obtained by separating the sub-component a3 using a mobile phase of methanol-water on a preparative reverse-phase high performance liquid chromatography.

[0014] Compound 1 was resolved on a Chiralpak AD-H chiral column using a mobile phase of n-hexane - isopropanol to obtain compound 1a and compound 1b.

[0015] In the above steps, the Polygonum tinctorium used was the dried leaves of Polygonum tinctorium Ait. of the genus Polygonum in the family Polygonaceae.

[0016] In the above steps, the dried leaves of Polygonum tinctorium were refluxed and extracted 2 - 3 times with 70 - 80% industrial ethanol for 2 - 3 h each time.

[0017] In the above steps, a3 was separated on a preparative reverse-phase high performance liquid chromatography with a mobile phase of methanol-water of 60:40.

[0018] In the above steps, compound 1 was separated on a Chiralpak AD-H chiral column with a mobile phase of n-hexane - isopropanol of 2:1.

[0019] The obtained compound was subjected to systematic structure identification and the results were as follows:

[0020] The structure of compound 1 was identified using high-resolution mass spectrometry, one-dimensional NMR, and two-dimensional NMR. The relevant structure characterization results are shown in Table 1 and Figures 1 to 10 .

[0021] Compound 1, white powder (methanol). HRESIMS gave the quasi-molecular ion peak 298.1161 [M+Na] + (calcd. for C 14 H 17 N3O3Na, 298.1162), combined with 1 H and 13 C-NMR data to determine its molecular formula as C 14 H 17 N3O3, with an unsaturation degree of 8. 1 In the 1H-NMR (600 MHz, DMSO-d6) spectrum, δ H7.74 (1H, d, J = 8.0 Hz, H-5), 7.02 (1H, t, J = 8.0 Hz, H-6), 7.41 (1H, t, J = 8.0 Hz, H-7), 7.27 (1H, d, J = 8.0 Hz, H-8) suggest the presence of a 1,2-disubstituted benzene ring. δ H 8.01 (1H, t, J = 5.2 Hz, H-10) suggests the presence of an active hydrogen on nitrogen. δ H 3.35 (1H, m, H-11a), 3.12 (1H, m, H-11b), 3.54 (1H, m, H-12a), 3.49 (1H, overlapped, H-12b), 3.59 (1H, m, H-13a), 3.49 (1H, overlapped, H-13b), 1.98 (1H, m, H-14a), 1.83 (1H, overlapped, H-14b), 2.60 (1H, m, H-15a), 1.89 (1H, overlapped, H-15b) are signals of five methylene hydrogens. δ H 5.02 (1H, dd, J = 8.5, 5.0 Hz, H-2) is a methine hydrogen signal. δ H 4.75 (1H, t, J = 5.3 Hz, 12-OH) is a hydroxyl hydrogen signal. 13 The 13C-NMR (150 MHz, DMSO-d6) spectrum gives a total of 14 carbon signals. δ C 120.1, 127.2, 121.2, 132.7, 116.7, 144.4 suggest benzene ring carbon signals, δ C 160.5 (C-4), 155.3 (C-9) suggest two carbonyl carbon signals, and five methylene carbon signals δ C 42.8 (C-11), 59.4 (C-12), 44.2 (C-13), 21.0 (C-14), 32.7 (C-15), and one methine carbon signal δ C 71.4 (C-2). Combining the molecular formula and degree of unsaturation of the compound, in addition to the benzene ring and two carbonyl groups, there should be two rings in the structure.

[0022] In the HMBC spectrum, H-5 shows correlations with C-7, C-8a, H-6 shows a correlation with C-4a, H-7 shows a correlation with C-8a, H-8 shows a correlation with C-6, H-5 shows a correlation with C-4, which determines the presence of the 4-quinazolinone fragment. In addition, it can also be observed in the HMBC spectrum that H-10 shows a correlation with C-9, H-11 shows a correlation with C-9, H-12 shows a correlation with C-11, 12-OH shows correlations with C-11, C-12. Combining 1 H- 1Correlation signals between H-10 / H-11 / H-12 / 12-OH were observed in the H COSY spectrum, suggesting that the hydroxyethyl group is connected to the amide group. In the HMBC spectrum, H-13 has correlations with C-2 and C-15, H-14 has a correlation with C-2, and at the same time, 1 H- 1 HCOSY correlation signals were observed. Therefore, the presence of the pyrrolidine ring fragment was determined and fused to N-1 and C-2 of the 4-quinazolinone fragment. Thus, the planar structure of the compound was determined.

[0023] Compound 1 was resolved by a Chiralpak AD-H chiral column to obtain 1a and 1b, with a ratio of approximately 1:1. Their CD spectra are mirror images of each other. Their absolute configurations were determined by comparing the calculated and measured ECDs. The experimental CD curves of 1a and 1b are in agreement with the calculated ECD curves preset for the 2S and 2R configurations, respectively. Therefore, the absolute configurations of 1a and 1b were determined to be 2S and 2R, respectively.

[0024] Table 1 of Compound 1 1 H(600 MHz) and 13 C(150 MHz) data (DMSO-d6)

[0025]

[0026] The neuroprotective activities of the pair of new enantiomers of the invention were investigated. In the model of hydrogen peroxide (H2O2)-induced damage to human neuroblastoma SH-SY5Y cells, compounds 1a and 1b showed obvious in vitro neuroprotective effects, comparable to the positive control drug. Therefore, the new quinazoline alkaloid enantiomers of the present invention have the prospect of further developing drugs for preventing and treating neuroinflammation.

[0027] A pharmaceutical composition comprising the quinazoline alkaloid enantiomers prepared from Polygonum tinctorium or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0028] A Polygonum tinctorium extract comprising the above-mentioned quinazoline alkaloid enantiomers.

[0029] The present invention also provides the use of the quinazoline alkaloid enantiomers prepared from Polygonum tinctorium or a pharmaceutical composition comprising the above-mentioned enantiomers in the preparation of a drug having neuroprotective activity.

[0030] The present invention also provides the use of the quinazoline alkaloid enantiomers prepared from Polygonum tinctorium or a pharmaceutical composition comprising the above-mentioned enantiomers in the preparation of a drug for preventing and / or treating neurodegenerative diseases.

[0031] The advantages of the present invention are that the compounds are all new compounds with novel structures, and at the same time, they have strong anti-neuroinflammatory activity and have the value of further development. Description of the Drawings:

[0032] Figure 1 UV spectrum of Compound 1;

[0033] Figure 2 HR-ESIMS spectrum of Compound 1;

[0034] Figure 3 1H-NMR spectrum of Compound 1 (600 MHz, DMSO-d6);

[0035] Figure 4 13C-NMR spectrum of Compound 1 (150 MHz, DMSO-d6);

[0036] Figure 5 HSQC spectrum of Compound 1 (600 MHz, DMSO-d6);

[0037] Figure 6 HMBC spectrum of Compound 1 (600 MHz, DMSO-d6);

[0038] Figure 7 1H-1H COSY spectrum of Compound 1 (600 MHz, DMSO-d6);

[0039] Figure 8 Chiral resolution chromatogram of Compound 1;

[0040] Figure 9 CD spectrum of Compound 1a / 1b;

[0041] Figure 10 Calculated ECD spectrum of Compound 1a / 1b. Detailed Embodiments:

[0042] The following listed embodiments are helpful for those skilled in the art to better understand the present invention, but do not limit the present invention in any way.

[0043] Example 1:

[0044] Preparation of quinazoline alkaloid enantiomers, the specific operation is as follows:

[0045] Take the dry leaves of Polygonum tinctorium and reflux extract them 3 times with 70% industrial ethanol for 2 hours each time. Combine the extraction solutions and concentrate to obtain an extract. The extract is extracted with dichloromethane and the obtained components are subjected to silica gel column chromatography, and isocratic gradient elution is carried out with a dichloromethane-methanol system of 100:0 - 0:100. A total of 4 components (A - D) are collected.

[0046] Component A was subjected to polyamide column chromatography and eluted with an ethanol-water system in a gradient of 0:100 - 100:0 to obtain two components, A1 and A2.

[0047] Component A1 was subjected to gradient elution with an ethanol-water system of 30:70 - 90:10 using HP20 column chromatography to obtain three components (A1-1 - A1-3).

[0048] Component A1-2 was subjected to gradient elution with an ethanol-water system of 30:70 - 90:10 using ODS column chromatography to obtain four components (a - b).

[0049] The obtained component a was subjected to silica gel column chromatography with a dichloromethane-methanol system of 50:1 - 10:1 on the basis of TLC analysis to obtain four sub-components (a1 - a4).

[0050] Compound 1 was separated from sub-component a3 using a mobile phase of methanol-water (60:40, v / v) on preparative reverse-phase high-performance liquid chromatography.

[0051] Compound 1 was resolved on a Chiralpak AD-H chiral column using a mobile phase of n-hexane - isopropanol (2:1, v / v) to obtain compound 1a and compound 1b.

[0052] Example 2:

[0053] Investigation of the anti-neuroinflammatory activity of compound 1.

[0054] SH-SY5Y cultured cells, with 3 replicate wells in each group. H2O2 model group: After cell passage and routine culture, the cells were made into a cell suspension with serum-containing DMEM and inoculated into 96-well plates, and statically cultured in an incubator at 37°C and 5% CO2 for 12 h until the cells adhered. In the drug treatment groups, 12.5, 25, 50 μM of the drug was added for pre-treatment for 1 h, and then H2O2 with a final concentration of 200 μM was added and allowed to act for 4 h. Then, 200 μL of MTT was added to each well, and after incubation for 4 h, the absorbance value was measured at 490 nm using an enzyme-linked immunosorbent assay reader. Survival rate (%) = [A 490 (drug treatment group) - A 490 (blank control)] / [A 490 (negative control) - A 490 (blank control)] × 100%.

[0055] Table 2 Neuroprotective activities of compound 1a and 1b

[0056]

[0057] a Lipid-soluble vitamin E was used as a positive control drug.

[0058] The above description is only a preferred embodiment of the present disclosure and an explanation of the applied technical principles. Those skilled in the art should understand that the scope of the invention involved in the present disclosure is not limited to the technical solutions formed by the specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. For example, the technical solutions formed by mutually replacing the above features with the technical features (but not limited to) disclosed in the present disclosure that have similar functions.

Claims

1. An enantiomer of quinazoline alkaloid in Polygonum tinctorium, characterized in that, The enantiomers have any one of the structures shown below: 。 2. The quinazoline alkaloid enantiomers in Polygonum tinctorium Lour. according to claim 1, characterized in that, The polygonum tinctorium refers to polygonum tinctorium, a plant of the genus polygonum in the family polygonaceae Polygonum tinctorium Ait.]。 3. A method for preparing the quinazoline alkaloid enantiomers in Polygonum tinctorium Lour. as described in claim 1 or claim 2, characterized in that, Comprising the following steps: Taking the dry leaves of Polygonum tinctorium, refluxing and extracting with ethanol, combining the extraction solutions and concentrating to obtain an extract, extracting the extract with dichloromethane, and subjecting the obtained components to silica gel column chromatography, performing isocratic gradient elution with a dichloromethane-methanol system of 100:1 - 0:100, and collecting a total of 4 components A - D; Component A is subjected to polyamide column chromatography, and gradient elution is performed with an ethanol-water system of 0:100 - 100:0 to obtain two components A1 and A2; Using HP20 column chromatography to perform gradient elution on component A1 with an ethanol-water system of 30:70 - 90:10 to obtain three components A1-1 - A1-3; Using ODS column chromatography to perform gradient elution on component A1-2 with an ethanol-water system of 30:70 - 90:10 to obtain four components a - b; The obtained component a is subjected to silica gel column chromatography with a dichloromethane-methanol system of 50:1 - 10:1 on the basis of TLC analysis to obtain four sub-components a1 - a4; Compound 1 was obtained by separating sub-component a3 using a mobile phase of methanol-water on a preparative reversed-phase high-performance liquid chromatography; Compound 1 was resolved using a mobile phase of n-hexane-isopropanol on a Chiralpak AD-H chiral chromatography column to obtain compound 1a and compound 1b.

4. The preparation method of the quinazoline alkaloid enantiomers in Polygonum tinctorium Lour. according to claim 3, characterized in that, The Polygonum tinctorium used refers to the plant Polygonum tinctorium in the genus Polygonum of the family Polygonaceae Polygonum tinctorium Ait.] 5. The method for preparing quinazoline alkaloid enantiomers from Polygonum tinctorium Lour. according to claim 4, characterized in that, Taking the dry leaves of dry Polygonum tinctorium, refluxing and extracting with 70 - 80% ethanol for 2 - 3 times, 2 - 3 h each time.

6. The preparation method of the quinazoline alkaloid enantiomers in Polygonum tinctorium Lour. according to claim 4, characterized in that, Sub-component a3 was separated with a mobile phase of methanol-water of 60:40, and compound 1 was separated with a mobile phase of n-hexane-isopropanol of 2:

1.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition is a composition prepared by mixing the quinazoline alkaloid enantiomers in Polygonum tinctorium or their pharmaceutically acceptable salts as claimed in claim 1 or claim 2 with a pharmaceutically acceptable excipient, and the excipient refers to a diluent, adjuvant or carrier that can be used in the pharmaceutical field.

8. Use of the quinazoline alkaloid enantiomers in Polygonum tinctorium or their pharmaceutically acceptable salts as claimed in claim 1 or claim 2 in the preparation of a drug having neuroprotective activity.

9. Use of the pharmaceutical composition as claimed in claim 7 in the preparation of a drug having neuroprotective activity.

10. Use of the quinazoline alkaloid enantiomers in Polygonum tinctorium or their pharmaceutically acceptable salts as claimed in claim 1 or claim 2 or the pharmaceutical composition as claimed in claim 7 in the preparation of a drug for preventing and / or treating neurodegenerative diseases.

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