A quebrachine alkaloid compound and a preparation method and application thereof

Abstract

The application belongs to the technical field of medicine extraction, and provides a novel-skeleton quinazoline alkaloid compound, a preparation method and application thereof. The compound is a compound shown in the following formula (I). The preparation method is as follows: 95% EtOH is used to heat and reflux to extract dried whole periwinkle, and then low-temperature reduced-pressure concentration is used to obtain a crude extract; the crude extract is suspended in 3% tartaric acid water, and the pH is adjusted to be acidic. Then, ethyl acetate is used for extraction to obtain an ethyl acetate part and a water part, the water part solution is adjusted to be alkaline by using a Na2CO3 solution, and then dichloromethane is used for extraction to obtain a dichloromethane part; the dichloromethane part is subjected to silica gel column chromatography, and gradient elution is performed by using an eluent, target fractions containing the compound of formula (I) are collected, and the target fractions are sequentially subjected to ODS column chromatography, Sphadex LH-20 gel column chromatography and semi-preparative liquid phase separation to obtain a quinazoline alkaloid compound. The compound obtained in the application has acetylcholinesterase inhibition effect, has good potential medicinal value, and is expected to be used for the preparation of various drugs for treating Alzheimer's disease and other neurodegenerative diseases.

Description

Technical Field

[0001] This invention relates to the field of drug extraction and separation technology, specifically to a white pine alkaloid compound, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is one of the most common neurodegenerative diseases, characterized by memory loss, cognitive impairment, and behavioral abnormalities. Its pathological mechanism is closely related to decreased acetylcholine (ACh) levels. Acetylcholinesterase (AChE), a key enzyme in the breakdown of ACh, further exacerbates the decline in ACh levels due to excessive activity, leading to impaired neurotransmission. Therefore, AChE inhibitors are considered an important pharmacological strategy for treating Alzheimer's disease. Natural products, due to their unique structural diversity, novelty, and significant biological activity, have always been an important source of drug lead compounds and have attracted considerable attention from the scientific community. To date, drugs developed based on natural products occupy an important position in clinical applications, which further stimulates scientists to explore and research novel natural product drugs for treating neurodegenerative diseases such as Alzheimer's disease with higher selectivity, stronger efficacy, and better safety.

[0003] Periwinkle ( Catharanthus roseus (L.) G. Don), belonging to the Apocynaceae family and the genus Vinca ( Catharanthus The plant *Catharanthus roseus* (G. Don), commonly known in China as "Daily Grass," "Wild Goose Red," and "Four Seasons Flower," is a traditional Chinese herbal medicine. It is cold in nature and bitter in taste, possessing detoxifying, anti-cancer, diuretic, hemostatic, blood pressure-lowering, blood sugar-lowering, calming, and liver-clearing effects. Therefore, periwinkle is often used to treat various diseases such as diabetes, hypertension, burns, diarrhea, skin diseases, and tumors. It is suitable for treating various tumors, such as Hodgkin's disease, choriocarcinoma, breast cancer, lung cancer, lymphosarcoma, oral cancer, acute hemorrhage, ovarian cancer, and malignant melanoma. The vincristine and vinblastine contained in periwinkle have become indispensable drugs in modern medical anti-tumor treatment.

[0004] Studies have shown that periwinkle contains alkaloids and lignans, among other compounds. Pharmacological activity studies have revealed that its chemical components possess antitumor, antihypertensive, and hypoglycemic biological activities.

[0005] With the increasingly widespread application of periwinkle, natural product scientists both domestically and internationally have successively conducted basic research on this plant. Modern pharmacological studies have shown that periwinkle extract possesses biological activities such as anti-tumor, anti-angiogenic, antihypertensive, and hypoglycemic effects. To date, chemical components including alkaloids, aromatic compounds, ergosterols, and lignans have been isolated and identified from periwinkle. Among these, alkaloids, as the main chemical components, exhibit significant anti-tumor activity, inhibiting the proliferation and spread of tumor cells, inducing tumor cell apoptosis, and inhibiting tumor cell invasion and metastasis.

[0006] A rare 6 / 5 / 8 / 5 tetracyclic carbon skeleton of eucalyptus-type alkaloids has been isolated from periwinkle for the first time. Although there are numerous research reports on the isolation of alkaloids from periwinkle, there have been no reports to date of isolating eucalyptus-type alkaloids with a 6 / 5 / 8 / 5 tetracyclic carbon skeleton from periwinkle. Summary of the Invention

[0007] The purpose of this invention is to address the aforementioned problems by providing a white pine-type alkaloid compound, its preparation method, and its application. The compound obtained by this invention has acetylcholinesterase inhibitory activity and has good potential medicinal value, and is expected to be used in the preparation of various drugs for treating neurodegenerative diseases such as Alzheimer's disease.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A white oak-type alkaloid compound, said compound being a compound of formula (I) or a pharmaceutically acceptable salt thereof: (I).

[0009] The present invention also provides a method for preparing the above-mentioned white oak-type alkaloid compound, comprising the following steps: (1) Using periwinkle as the extraction raw material, periwinkle extract was obtained; (2) The periwinkle extract was dissolved in tartaric acid solution (mass fraction of 3%), and the insoluble matter was removed by filtration to obtain the filtrate. The filtrate was then extracted with ethyl acetate to obtain the ethyl acetate extract and the acidic solution after the ethyl acetate extraction and separation. (3) Adjust the pH of the acidic solution after the ethyl acetate extraction and separation in (2) to alkaline, extract with dichloromethane, and finally concentrate under reduced pressure to obtain the dichloromethane fraction; (4) The dichloromethane fraction was subjected to silica gel column chromatography with gradient elution using eluents composed of different ratios of dichloromethane and methanol or eluents composed of petroleum ether and acetone. The fraction was analyzed by thin-layer chromatography and high-performance liquid chromatography. The target fraction containing the compound shown in formula (I) was collected. The target fraction containing the target compound was then separated sequentially using an ODS column, a Sphadex LH-20 gel column, and a semi-preparative liquid chromatography to obtain the target compound. In the above preparation method, the periwinkle mentioned in step (1) is a plant of the genus *Vinca* in the family Oleaceae. Catharanthus roseus (L.) G. Don, preferably the upper part is the extractable material.

[0010] The above preparation method for obtaining periwinkle extract is largely the same as the existing technology. Specifically, periwinkle can be used as raw material, and the solvent used for extraction can be chloroform, dichloromethane, acetone, ethanol with a volume concentration of 60-95% or methanol with a volume concentration of 60-100%. The specific extraction methods can be room temperature extraction, maceration, heating extraction, reflux extraction, continuous reflux extraction or ultrasonic extraction.

[0011] In the above preparation method, in step (2), after the extract is dissolved in 3% tartaric acid solution, the pH of the extract solution is adjusted to 2~3; after filtering out insoluble matter, the filtrate is extracted with ethyl acetate 3-4 times.

[0012] In the above preparation method, the pH adjustment to alkalinity in step (3) is to adjust the pH value to 9~12, preferably pH=9~11, more preferably pH=10~12, and the dichloromethane extraction is performed 3-4 times.

[0013] In the above preparation method, the temperatures involved in steps (1-4) are all below 60°C. The low-pressure range is -0.05 to -0.098 MPa.

[0014] In the above preparation method, in step (4), when the eluent used for silica gel column chromatography is composed of dichloromethane and methanol, gradient elution is performed according to the volume ratios of dichloromethane and methanol as 100:1, 80:1, 50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, 1:1, and 1:5; when the eluent is composed of petroleum ether and acetone, gradient elution is performed according to the volume ratios of dichloromethane and methanol as 100:1, 80:1, 50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, 1:1, 1:5, and 1:10; every 0.5 L of fraction is collected, and the collected fraction is analyzed and detected. The eluent volume for each gradient concentration is twice the column volume, preferably three times.

[0015] In the above preparation method, when the target fraction containing the target compound obtained in step (5) is separated sequentially using an ODS column, a Sphadex LH-20 gel chromatography column, and a semi-preparative liquid chromatography column, the eluent used is preferably: When separating using an ODS column, the eluent is methanol and water at different volume ratios (30%, 40%, 50%, 60%, 70%, 80%, 90%), and 0.2 L of fraction is collected per volume. The fractions are analyzed and the target fraction is collected. Alternatively, the eluent is acetonitrile and water at different volume ratios (20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%), and gradient elution is performed, with 0.2 L of fraction collected per volume. The fractions are analyzed and the target fraction is collected.

[0016] When using a Sphadex LH-20 gel chromatography column for separation, the eluent is methanol or 90% methanol, and fractions are collected in 30 ml volumes for analysis. When using a semi-preparative liquid chromatography system for separation, the flow rate was 7 mL / min, the detection wavelength was 210 nm, the mobile phase was 45:55:0.01 acetonitrile-water-trifluoroacetic acid (volume ratio), and the chromatographic peak with a retention time of 29.0 min was collected; or the mobile phase was 60:40:0.01 methanol-water-trifluoroacetic acid (volume ratio), and the chromatographic peak with a retention time of 25.5 min was collected.

[0017] In the above preparation method, when analyzing and detecting the fraction in step (4), the detection method can be TLC, preferably HPLC, and more preferably HPLC-MS.

[0018] This invention also protects the use of the above-mentioned compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating neurodegenerative diseases such as Alzheimer's disease.

[0019] This invention also protects drugs for treating neurodegenerative diseases such as Alzheimer's disease prepared using the above-mentioned compounds or their pharmaceutically acceptable salts as active ingredients.

[0020] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: This invention provides a novel skeletonized alkaloid compound of the *Acer buergerianum* type, along with its preparation method and applications. The preparation method is simple and easy to operate. Investigating the inhibitory effect of this compound on acetylcholinesterase shows that it possesses in vitro anti-acetylcholinesterase activity, indicating good potential medicinal value and promising application in the preparation of various drugs for treating neurodegenerative diseases such as Alzheimer's disease. Detailed Implementation

[0021] To more clearly illustrate the present invention, the following specific embodiments will be used to further explain the invention.

[0022] Example 1 A method for preparing a white oak-type alkaloid compound includes the following steps: (1) Take 25 kg of the dried whole plant of periwinkle after crushing, use 60% methanol as solvent, reflux extract 3 times, combine and concentrate under reduced pressure to obtain 2.1 kg of periwinkle extract. (2) Dissolve the periwinkle extract in a 3% tartaric acid solution and adjust the pH of the extract solution to 2; filter out the insoluble matter and extract the filtrate twice with ethyl acetate and concentrate under reduced pressure to obtain the ethyl acetate extract; (3) Adjust the pH of the acidic aqueous solution left after ethyl acetate extraction in step (2) to 8 with saturated sodium carbonate solution, extract it twice with dichloromethane, and concentrate it to obtain the dichloromethane fraction. (4) The dichloromethane fraction was separated by silica gel column chromatography using a gradient elution with dichloromethane and methanol as the eluent, at volume ratios of 100:1, 80:1, 50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, and 1:1. A gradient elution was performed at a 1:5 ratio, with each 0.5 L fraction collected. The samples were fragmented and combined by TLC to obtain eight fractions. HPLC-UV and HPLC-MS analysis revealed that the sixth fraction contained the target compound. The target fraction containing the compound shown in formula (I) was collected. This target fraction was separated using an ODS column with a gradient elution of methanol and water in different volume ratios (30%, 40%, 50%, 60%, 70%, 80%, 90%), with each 0.2 L fraction collected. The fractions were analyzed, and the target fraction was collected. Further separation was performed using a Sphadex LH-20 gel column with methanol as the eluent, with each 30 mL fraction collected. The fractions were analyzed, and the target fraction was collected. Finally, semi-preparative liquid chromatography was used for separation at a flow rate of 7 mL / min, a detection wavelength of 210 nm, and a mobile phase of 45:55:0.01. The target compound was obtained by collecting the chromatographic peak with a retention time of 29.0 min at a volume ratio of acetonitrile-water-trifluoroacetic acid. The obtained product was structurally identified by NMR, HRESIMS and ECD calculations, and was determined to be the target compound Velbanamine A.

[0023] Example 2 A method for preparing a white oak-type alkaloid compound includes the following steps: (1) Take 25 kg of the dried whole plant of periwinkle after crushing, extract it three times at room temperature with 100% methanol as solvent, combine the extracts and concentrate them under reduced pressure to obtain periwinkle extract paste. (2) Dissolve the periwinkle extract in a 3% tartaric acid solution and adjust the pH of the extract solution to 2.5; after filtering out the insoluble matter, extract the filtrate three times with ethyl acetate and concentrate under reduced pressure to obtain the ethyl acetate extract; (3) The acidic aqueous solution left after ethyl acetate extraction in step (2) was adjusted to pH 9 with saturated sodium carbonate solution, extracted with dichloromethane 3 times, and concentrated under reduced pressure to obtain the dichloromethane fraction. (4) The dichloromethane fraction was separated by silica gel column chromatography using a gradient elution with dichloromethane and methanol as the eluent. The gradient elution was performed at volume ratios of dichloromethane and methanol of 100:1, 80:1, 50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, 1:1, and 1:5. Each 0.5 L fraction was collected and analyzed by HPLC-UV and HPLC-MS. The target fraction containing the compound shown in formula (I) was collected. The target fraction containing the target compound was separated by an ODS column using an eluent composed of acetonitrile and water at different volume ratios. The gradient elution was performed at acetonitrile contents of 30%, 40%, 50%, 60%, 70%, 80%, and 90%. Each 0.2 L fraction was collected and analyzed. The target fraction was collected. Sphadex was then used to separate the target fraction. Separation was performed using an LH-20 gel column with methanol as the eluent. Fractions were collected in 30 mL increments and analyzed to identify the target fraction. The fraction was then separated using a semi-preparative liquid chromatography (HPLC) system at a flow rate of 7 mL / min, a detection wavelength of 210 nm, and a mobile phase ratio of 60:40:0.01 (methanol-water-trifluoroacetic acid). The chromatographic peak with a retention time of 25.5 min was collected to obtain the target compound. The target compound was identified by NMR, HRESIMS and ECD calculations and was confirmed to be Velbanamine A. Example 3 A method for preparing enantiomeric alkaloid dimers includes the following steps: (1) Take 25 kg of dried whole plant parts of periwinkle, crush them, extract them with ultrasonically using chloroform as solvent, and recover the solvent under reduced pressure to obtain periwinkle extract. (2) Dissolve the periwinkle extract in a 3% tartaric acid solution and adjust the pH of the extract solution to 3; after filtering out the insoluble matter, extract the filtrate four times with ethyl acetate and concentrate under reduced pressure to obtain the ethyl acetate extract. (3) The acidic solution left after ethyl acetate extraction in step (2) was adjusted to pH 12 with saturated sodium carbonate solution, extracted with dichloromethane 4 times, and concentrated under reduced pressure to obtain the dichloromethane fraction. (4) The dichloromethane fraction was separated by silica gel column chromatography using a gradient elution with dichloromethane and methanol as the eluent. The gradient elution was performed with dichloromethane and methanol in volume ratios of 100:1, 80:1, 50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, 1:1, and 1:5. Each 0.5 L fraction was collected and analyzed by HPLC-UV and HPLC-MS. The target fraction containing the compound shown in formula (I) was collected. The target fraction containing the target compound was separated by an ODS column using methanol and water in different volume ratios as the eluent. The gradient elution was performed with methanol contents of 30%, 40%, 50%, 60%, 70%, 80%, and 90%. Each 0.2 L fraction was collected and analyzed. The target fraction was collected. Sphadex was then used to separate the target fraction. Separation was performed using an LH-20 gel column with methanol as the eluent. Fractions were collected in 30 mL increments and analyzed to identify the target fraction. The fraction was then separated using a semi-preparative liquid chromatography (HPLC) system at a flow rate of 7 mL / min, a detection wavelength of 210 nm, and a mobile phase ratio of 60:40:0.01 (methanol-water-trifluoroacetic acid). The chromatographic peak with a retention time of 25.5 min was collected to obtain the target compound. The target compound was identified by NMR, HRESIMS and ECD calculations and was confirmed to be Velbanamine A. Example 4 Repeat Example 1, except that the extraction solvent in step (1) is changed from 60% methanol to 90% methanol.

[0024] The target compound was identified by NMR, HRESIMS and ECD calculations and was confirmed to be Velbanamine A.

[0025] Example 5 Repeat Example 1, except that the extraction solvent in step (1) is changed from 60% methanol to 75% ethanol.

[0026] The target compound was identified by NMR, HRESIMS and ECD calculations and was confirmed to be Velbanamine A. Example 6 Repeat Example 1, except that the extraction solvent in step (1) is changed from 60% methanol to 95% ethanol, and the extraction method is heating extraction at 60°C for 3 hours.

[0027] The target compound was identified by NMR, HRESIMS and ECD calculations and was confirmed to be Velbanamine A. Example 7 Repeat Example 1, except that the extraction solvent in step (1) is changed from methanol with a volume concentration of 60% to acetone.

[0028] The target compound was identified by NMR, HRESIMS and ECD calculations and was confirmed to be Velbanamine A. II. Product Confirmation The products obtained in Examples 1-7 were structurally identified by NMR, HRESIMS and ECD calculations, and the spectral data are shown below; and the purity of the compounds was >95% by HPLC analysis.

[0029] Physicochemical data of compound 1 UV (MeOH) λ max 262 (4.12), 279 (4.01), 331 (3.83) nm; (+) HR-ESI-MS m / z 323.1755 [M + H] + , calcd for 323.1754.

[0030] Table 1 Compound 1 1 H (400 MHz) and 13 C (100 MHz) NMR (CD3OD) Data Sheet Therefore, the structural formula of the above compound 1 Velbanamine A can be determined as shown in the following formula (I): (I) II. Study on the determination of acetylcholinesterase inhibitory activity of the product The reaction mixture was incubated at 37°C for 20 min with phosphate-buffered saline (PBS) (0.1 mol / L; pH=8.0), test compounds (100, 40, 20, 10, and 1 μM), and acetylcholinesterase (0.1 U / mL). Donepezil was used as a positive control, and a blank control was also included. Acetylcholinesterase inhibitory activity was assessed by initiating the reaction with 40 μL of a solution containing DTNB (2.0 mmol / L) and ATCI (15.0 mmol / L). After a further 20 min incubation at 37°C, absorbance was measured at 412 nm every 2 min over 10 min using a microtiter plate spectrophotometer. The percentage of anti-acetylcholinesterase activity was calculated using the following formula: Antiacetylcholinesterase activity (%) = [( E - S ) / E ×100%, in E This represents the enzyme activity without the test compound. S The enzyme activity was represented when the test alkaloid was present. The cell proliferation inhibition rate of each well was calculated, and its IC50 was calculated. 50 The values ​​and results are shown in Table 2 below: Table 2: As shown in Table 1, the compounds of this invention have a good inhibitory effect on acetylcholinesterase, indicating that the compounds of this invention have good anti-acetylcholinesterase activity and are expected to be used in the preparation of various drugs for treating neurodegenerative diseases such as Alzheimer's disease.

Claims

1. A white oak-type alkaloid compound, characterized in that: The compound is a compound of formula (I) or a pharmaceutically acceptable salt thereof: (I)。 2. The method for preparing a white oak-type alkaloid compound according to claim 1, characterized in that: Includes the following steps: (1) Using periwinkle as the extraction raw material, periwinkle extract was obtained; (2) The periwinkle extract was dissolved in tartaric acid solution (mass fraction of 3%), and the insoluble matter was removed by filtration to obtain the filtrate. The filtrate was then extracted with ethyl acetate to obtain the ethyl acetate extract and the acidic aqueous solution left after the ethyl acetate extraction. (3) Adjust the pH of the acidic aqueous solution left after ethyl acetate extraction in (2) to alkaline, extract with dichloromethane, and finally concentrate under reduced pressure to obtain the dichloromethane fraction; (4) The dichloromethane fraction was subjected to silica gel column chromatography with gradient elution using eluents composed of different ratios of dichloromethane and methanol or eluents composed of petroleum ether and acetone. The fraction was analyzed by thin-layer chromatography and high-performance liquid chromatography. The target fraction containing the compound shown in formula (I) was collected. The target fraction containing the target compound was then separated sequentially using an ODS column, a Sphadex LH-20 gel chromatography column, and a semi-preparative liquid chromatography column to obtain the target compound.

3. The preparation method according to claim 2, characterized in that: The solvent used for extraction in step (1) is chloroform, dichloromethane, acetone, ethanol with a volume concentration of 60-95% or methanol with a volume concentration of 60-100%. The specific extraction methods are room temperature extraction, maceration, heating extraction, reflux extraction, continuous reflux extraction or ultrasonic extraction.

4. The preparation method according to claim 2, characterized in that: In step (2), after the extract is dissolved in a 3% tartaric acid solution, the pH value of the extract solution is 2-3; after filtering out the insoluble matter, the filtrate is extracted with ethyl acetate 3-4 times.

5. The preparation method according to claim 2, characterized in that: In steps (1-4), the temperatures involved are all below 60°C. The low-pressure range is -0.05 to -0.098 MPa.

6. The preparation method according to claim 2, characterized in that: In step (3), the pH is adjusted to alkaline by using a saturated sodium carbonate solution to adjust the pH to 9-10, and the dichloromethane extraction is performed 3-4 times.

7. The preparation method according to claim 2, characterized in that: In step (4), during silica gel column chromatography, when the eluent consists of dichloromethane and methanol, gradient elution is performed using dichloromethane and methanol at volume ratios of 100:1, 80:1, 50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, 1:1, and 1:5; when the eluent consists of petroleum ether and acetone, gradient elution is performed using dichloromethane and methanol at volume ratios of 100:1, 80:1, 50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, 1:1, 1:5, and 1:10; each 0.5 L fraction is collected and analyzed.

8. The preparation method according to claim 2, characterized in that: In step (4), during separation using an ODS column, the eluent is methanol and water in different volume ratios (30%, 40%, 50%, 60%, 70%, 80%, 90%) for gradient elution, and each 0.2L fraction is collected. The fractions are then analyzed, and the target fraction is collected. Alternatively, the eluent is acetonitrile and water in different volume ratios, and gradient elution is performed with acetonitrile content of 30%, 40%, 50%, 60%, 70%, 80%, 90%. Each 0.2L fraction is collected, and the fractions are then analyzed, and the target fraction is collected. When separating using a Sphadex LH-20 dextran gel column, the eluent is methanol or 90% methanol-water. Every 30 ml of fraction is collected, the fraction is analyzed, and the target fraction is collected. When using a semi-preparative liquid chromatography system for separation, the flow rate was 7 mL / min, the detection wavelength was 210 nm, the mobile phase was 45:55:0.01 acetonitrile-water-trifluoroacetic acid (volume ratio), and the chromatographic peak with a retention time of 29.0 min was collected; or the mobile phase was 60:40:0.01 methanol-water-trifluoroacetic acid (volume ratio), and the chromatographic peak with a retention time of 25.5 min was collected.

9. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating neurodegenerative diseases such as Alzheimer's disease.

10. A medicament for treating neurodegenerative diseases such as Alzheimer's disease, prepared using the compound of claim 1 or a pharmaceutically acceptable salt thereof as the active ingredient.

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