A compound or a pharmaceutically acceptable salt thereof, and a preparation method and application thereof

Abstract

This application discloses a compound or a pharmaceutically acceptable salt thereof, its preparation method, and its application, belonging to the field of natural compound technology. The compound is a β-carboline alkaloid with the structural formula C2. 12 H 12 N2O possesses antitumor activity, laying the foundation for the preparation of antitumor products. Moreover, this compound is derived from Boletus thunbergii, which has been cultivated on a large scale, providing abundant raw materials. The separation and extraction steps are simple, rapid, highly operable, and yield stable results, enabling large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of natural compound technology, specifically to a β-carboline alkaloid or a pharmaceutically acceptable salt thereof derived from Boletus thunbergii, and its preparation method and application. Background Technology

[0002] Phleboporus portentosus, also known as the dark brown reticulated bolete or, more commonly, the black bolete, belongs to the order Boletales, family Boletinellaceae, and genus Phleboporus. Phleboporus portentosus has been successfully cultivated artificially and can now be mass-produced in industrial settings.

[0003] A tumor is a new growth formed by the proliferation of local tissue cells under the influence of various carcinogenic factors. Because these new growths often present as space-occupying, mass-like protrusions, they are also called neoplasms. A tumor is an abnormal disease formed when a cell in a local tissue loses normal regulation of its growth at the gene level under the influence of various carcinogenic factors, leading to its clonal abnormal proliferation. It is one of the major diseases threatening human health today and seriously endangers human life. Currently, there are more than 50 commonly used anti-tumor drugs in clinical practice, but most of them can only alleviate the symptoms and cannot achieve a complete cure. Therefore, the development of new anti-tumor drugs has always been a major aspect of drug research. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a β-carboline alkaloid compound with antitumor activity, laying the foundation for the development of related health products or pharmaceuticals.

[0005] To address the above problems, a first aspect of the present invention provides a β-carboline alkaloid compound, the compound having the molecular formula C2. 12 H 12 N2O, chemical structural formula as follows:

[0006]

[0007] The term "compound" as used in this invention is intended to include all stereoisomers (e.g., enantiomers and diastereomers), geometric isomers, tautomers, and isotopes of the structures shown. Unless otherwise specified, compounds identified by name or structure as a particular tautomer form in this invention are intended to include other tautomer forms.

[0008] In one specific embodiment of the present invention, the compound was isolated from Boletus glomeratus.

[0009] This compound is a novel β-carboline alkaloid derived from Boletus thunbergii, which exhibits antitumor activity and can inhibit the proliferation of tumor cells.

[0010] The specific name of this compound is N-formyl-1,2,3,4-tetrahydro-β-carboline.

[0011] Another aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable transporter. The salt may be a potassium salt, sodium salt, etc.

[0012] These pharmaceutical compositions can be prepared using methods known in the pharmaceutical field. In some embodiments, the active substance of the present invention can be mixed with a pharmaceutically acceptable carrier for the preparation of the pharmaceutical composition. The active substance includes the aforementioned β-carboline alkaloid or a pharmaceutically acceptable salt thereof.

[0013] As used in this invention, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for contact with human and animal tissues within a reasonable medical judgment without excessive toxicity, irritation, allergic reactions, or other problems or complications, and have a reasonable benefit / risk ratio. In some embodiments, pharmaceutically acceptable carriers, materials, compositions, and / or dosage forms refer to those approved by regulatory agencies (such as the U.S. Food and Drug Administration, the National Medical Products Administration, or the European Medicines Agency) or listed in generally accepted pharmacopoeias (such as the United States Pharmacopeia, the Chinese Pharmacopoeia, or the European Pharmacopoeia) for use in animals (more particularly for humans).

[0014] Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, pharmaceutically acceptable liquids, gels, or solid carriers, aqueous media (e.g., sodium chloride injection, Ringer's solution injection, isotonic glucose injection, sterile water injection, or glucose and lactated Ringer's solution injection), non-aqueous media (e.g., non-volatile plant-derived oils, cottonseed oil, corn oil, sesame oil, or peanut oil), antimicrobial substances, isotonic substances (e.g., sodium chloride or glucose), and buffer solutions (e.g., ... Phosphate or citrate buffers), antioxidants (e.g., sodium bisulfate), anesthetics (e.g., procaine hydrochloride), suspending / dispersing agents (e.g., sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid)), emulsifiers (e.g., polysorbate 80 (Tween-80)), diluents, adjuvants, or non-toxic excipients, other components known in the art, or combinations thereof. Suitable components may include, for example, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, or emulsifiers.

[0015] In some embodiments, the pharmaceutical composition may be used in combination with one or more other drugs. In some embodiments, the pharmaceutical composition comprises at least one other drug.

[0016] Another aspect of the present invention provides a method for preparing the above-mentioned compound, comprising the following steps:

[0017] S1: Extraction: The dark brown stalked Boletus is dissolved in an organic solvent and extracted to obtain an extract;

[0018] S2: Extraction: Extract the extract using an organic solvent to obtain the extract;

[0019] S3: Separation: The extract was separated by column chromatography to obtain the isolate;

[0020] S4: Purification: The isolated substance is purified to obtain the compound.

[0021] The compound is derived from Boletus pumilus, which has been successfully cultivated artificially, providing a large quantity of raw materials. Moreover, the preparation method described above is simple, rapid, and highly operable, enabling large-scale industrial production.

[0022] In one specific embodiment of this application, step S1 specifically includes:

[0023] The dark brown stalked Boletus was mixed with methanol solution and extracted. The extract was then concentrated under reduced pressure and evaporated to dryness to obtain a crude extract.

[0024] In one specific embodiment of this application, step S1 further includes:

[0025] The fruiting bodies or mycelia of Boletus glomeratus are added to a 70-85% methanol solution, mixed thoroughly, and then extracted.

[0026] In one specific embodiment of the present invention, the extraction time is 20-28 hours. For example, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, or 28 hours.

[0027] In one specific embodiment of the invention, the extraction is performed 3-6 times. For example, 3 times, 4 times, 5 times, or 6 times.

[0028] In one specific embodiment of the present invention, step S2 specifically includes:

[0029] The crude extract was suspended in water, the pH was adjusted to 9-10, chloroform was added for extraction, and the mixture was concentrated under reduced pressure and evaporated to dryness to obtain the extract.

[0030] In one specific embodiment of the present invention, step S3 specifically includes:

[0031] The extract was separated by MCI column chromatography, yielding a total of 12 components, numbered Fr.1 to Fr.12.

[0032] In one specific embodiment of the present invention, the MCI column chromatography separation process is as follows: the extract is eluted by a 20%–100% methanol-water gradient through an MCI column.

[0033] In one specific embodiment of the present invention, step S4 specifically includes:

[0034] The compound can be obtained by purifying Fr.7 by HPLC.

[0035] In one specific embodiment of the present invention, the specific steps of the HPLC purification are as follows: an X-Bridge C18 column is selected, and the mobile phase is acetonitrile-water with a volume ratio of 35:65.

[0036] In one specific embodiment of the invention, diethylamine is added to the aqueous phase of the mobile phase at a volume ratio of 0.05%. The addition of diethylamine alters the peak shape and resolution of the compound, making it easier to separate.

[0037] In one specific embodiment of the present invention, the method for preparing the compound is as follows:

[0038] S1: Take dark brown stamen Boletus fruiting bodies or mycelial samples, add 80% methanol solution at room temperature, mix well, extract for 24 hours, extract 5 times, combine the extracts, concentrate under reduced pressure and evaporate to dryness to obtain crude extract.

[0039] S2: After suspending the above crude extract in water, adjust the pH to 9-10 with Na2CO3, then extract with an equal volume of chloroform 5 times, and concentrate under reduced pressure to dryness to obtain the extract.

[0040] S3: The above extracts were separated by MCI column chromatography. After elution with MeOH-H2O (20%-100%, v / v), a total of 12 fractions were obtained, which were named Fr.1 to Fr.12 in sequence.

[0041] S4: Fr.7 was purified by HPLC using an X-Bridge C18 column and an acetonitrile-water mobile phase of 35:65, v / v. 0.05% (v / v) diethylamine (DEA) was added to the aqueous phase, and the solution was collected. R =15.5~18.2min.

[0042] According to another aspect of the present invention, the use of the above-described compounds in the preparation of antitumor articles for prevention and / or treatment is provided. The above-described compounds possess antitumor activity, laying the foundation for the preparation of antitumor articles.

[0043] In the first specific embodiment of the present invention, the tumor is caused by cervical cancer cells or gastric cancer cells.

[0044] The above-mentioned technical solution of the present invention has at least one of the following beneficial technical effects: The present invention provides a new compound, which is a β-carboline alkaloid compound with antitumor activity, laying the foundation for the preparation of antitumor products. Moreover, this compound is derived from Boletus thunbergii, which has been artificially cultivated on a large scale, with abundant raw materials, simple and rapid separation and extraction steps, strong operability, and stable results, enabling large-scale industrial production. Attached Figure Description

[0045] Figure 1 The present invention provides N-formyl-1,2,3,4-tetrahydro-β-carbazoline. 1 H-NMR spectrum;

[0046] Figure 2 The present invention provides N-formyl-1,2,3,4-tetrahydro-β-carbazoline. 13 C-NMR spectrum;

[0047] Figure 3 This is the HMBC spectrum of N-formyl-1,2,3,4-tetrahydro-β-carbamoline provided by the present invention. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and the accompanying drawings. It should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0049] Unless otherwise specified, all products or components mentioned in this application are commercially available products.

[0050] Example 1: Preparation of β-carboline alkaloids

[0051] 1. Chop 14.6 kg of fresh dark brown Boletus thunbergii, place it in a bucket, add 10.00 L of 80% methanol solution at room temperature, mix well, soak for 24 h, soak 5 times, combine the extracts, concentrate under reduced pressure and evaporate to dryness to obtain 831.5 g of brown crude extract.

[0052] 2. After suspending the above crude extract in water, adjust the pH to 9-10 with Na2CO3, then extract with an equal volume of chloroform 5 times, and concentrate under reduced pressure to dryness to obtain 8.6g of chloroform extract.

[0053] 3. The chloroform extract was crudely separated by MCI column chromatography. After gradient elution with MeOH-H2O (20%–100%, v / v) (20%→30%→40%→50%→60%→70%→80%→90%→100% methanol), 12 fractions were obtained, named Fr.1 to Fr.12.

[0054] 4. The Fr.7 fraction was purified by semi-preparative HPLC. The chromatographic column was X-Bridge C18, and the mobile phase was acetonitrile-water, 35:65, v / v. 0.05% (v / v) diethylamine (DEA) was added to the aqueous phase of the mobile phase, and fractions with tR = 15.5-18.2 min were collected.

[0055] 5. The components collected in step 4 were subjected to solvent removal by rotary evaporation and then dried under reduced pressure at room temperature to finally obtain 1.9 mg of a new compound as a brown amorphous powder.

[0056] Example 2: Spectroscopic and physicochemical data of β-carboline alkaloids

[0057] The structure of the new compound prepared in Example 1 of this invention is identified as follows:

[0058] Hydrogen spectrum data: 1 H NMR (CD3OD, 600MHz): δ H8.25 / 8.30(1H,s,N-CHO),7.40(1H,br d,J=7.7Hz,H-5),7.29(1H,br d,J=8.0Hz,H-8),7.07(1H,br dd,J=8.0,7.3Hz,H-7),6.99(1H,br dd,J=7.7,7.3Hz,H-6),4.71(4.68)(2H,br s,H-1),3.813.90(2H,t,J=5.82Hz,H-3),2.86(2.80)(2H,br t,J=5.83Hz,H-4).

[0059] Carbon spectrum data: 13 C NMR (CD3OD, 125MHz): δ C 164.4 / 164.0(C-10),138.2 / 138.1(C-8a),130.0 / 130.6(C-9a),128.2(C-5a),122.4(C-7),120.0(C-6),1 18.6(C-5),111.9 / 112.0(C-8),108.1 / 108.6(C-4a),45.6 / 45.0(C-3),39.4 / 39.7(C-1),23.1 / 21.8(C-4).

[0060] Mass spectrometry data include: (+)-ESI-MS: m / z 201 [M+H] + ;(-)-ESI-MS:m / z 199[MH] - ;(+)-HR-ESI-MS:m / z 201.1021[M+H] + (calcd.for 201.1022,C 12 H 12 N2O).

[0061] new compounds 1 The H-NMR spectrum is shown in [reference]. Figure 1 , 13 The C-NMR spectrum is shown below. Figure 2 HMBC spectrum can be found Figure 3 Based on the above information, the novel compound described in this invention can be identified as a β-carboline alkaloid (N-formyl-1,2,3,4-tetrahydro-β-carboline), with the chemical formula C. 12 H 12 N2O, structural formula as follows:

[0062]

[0063] Example 3: In vitro antitumor activity of β-carboline alkaloids

[0064] 1. Cells used for testing: HeLa (human cervical cancer cells) and HGC27 (human undifferentiated gastric cancer cells), purchased from the Chinese Academy of Sciences Cell Bank and Shanghai Rongmin Biotechnology Center, respectively.

[0065] 2. β-Carboline alkaloid concentrations (μM): 200, 100, 50, 25, 12.5.

[0066] 3. Reagents and Instruments: DMEM / F12 medium (TBD, TBD1056S), fetal bovine serum (FBS, F0193) (Biosharp, BL205A), 100 U / mL penicillin and 100 μg / mL streptomycin (Gibco, 15140-122); trypsin (Sigma, T4799); CCK-8 assay solution (Dojindo, CK04); MTT (Beyotime, ST316); CO2 incubator (Thermo, Forma3111); biosafety cabinet (Heal Force, HFsafe-1200); -80℃ freezer (Thermo, Forma 702); microplate reader (BioTek, Epoch); high-speed refrigerated centrifuge (Hitachi, CF15RN); inverted fluorescence microscope (Leica, DMI3000B).

[0067] 4. Experimental method: Tumor cells (0.8*10⁶) were seeded in 96-well plates. 4 Cells / well were incubated at 37°C in a 5% CO2 incubator for 24 hours. The culture medium was then removed, and 100 μL / well of the test drug (β-carboline alkaloid) solution was added, with three replicates for each concentration. The treatment without the drug served as a negative control. After 48 hours of incubation, the liquid in the wells was removed, and 100 μL of glucose-free, serum-free DMEM medium containing 5 mg / mL MTT was added to each well. Incubation continued for 2-4 hours. The supernatant was removed, and 100 μL of DMSO was added to each well. The cells were dissolved by low-speed shaking, and the OD value was measured at 490 nm. The cell proliferation inhibition rate was calculated as: [(mean OD value of negative control group - blank OD value) - (mean OD value of drug group - blank OD value)] / (mean OD value of negative control group - blank OD value) × 100%. The experimental results are shown in Table 1.

[0068] Table 1. In vitro antitumor activity of β-carboline alkaloids

[0069]

[0070] As shown in Table 1, the inhibition rate of cell growth increases with increasing concentration of β-carboline alkaloids. The β-carboline alkaloids provided in this invention exhibit inhibitory effects on HeLa229 cells at concentrations above 100 μM and on HGC27 cells at concentrations above 12.5 μM. Therefore, the novel compounds screened in this application possess certain inhibitory activity against human cervical cancer cells and human undifferentiated gastric cancer cells, demonstrating broad-spectrum activity. This provides a foundation for the research of broad-spectrum antitumor drugs and their combination with other drugs for antitumor therapy.

[0071] The β-carboline alkaloids provided by this invention exhibit certain anti-tumor cell proliferation activity and can be used in the development of related health products or pharmaceuticals.

[0072] In summary, the β-carboline alkaloid isolated from Boletus thunbergii in this invention has antitumor activity and can be used to develop related health products or pharmaceuticals. Moreover, the separation and extraction process is simple to operate, the raw materials are widely available, and large-scale industrial production can be achieved.

[0073] It should be understood that the specific embodiments described above are merely illustrative or explanatory of the principles of the invention and do not constitute a limitation thereof. Therefore, any modifications, equivalent substitutions, improvements, etc., made without departing from the spirit and scope of the invention should be included within the protection scope of the invention. Furthermore, the appended claims are intended to cover all variations and modifications falling within the scope and boundaries of the appended claims, or equivalent forms of such scope and boundaries.

Claims

1. A method for preparing a compound, characterized in that, The molecular formula of the compound is C 12 H 12 N2O, chemical structural formula as follows: ， The method for preparing the compound includes the following steps: S1: Extraction: The dark brown stalked Boletus was dissolved in methanol solution for extraction to obtain the extract; S2: Extraction: The above extract is suspended in water, the pH is adjusted to 9-10 with Na2CO3, and then the extract is extracted with an equal volume of chloroform. The extract is then concentrated under reduced pressure and evaporated to dryness to obtain the extract. S3: Separation: The extract was separated by MCI column chromatography to obtain the isolate. After elution with a methanol-water gradient of 20%-100% (v / v) in the order of 20%→30%→40%→50%→60%→70%→80%→90%→100% methanol, 12 fractions were obtained, which were named Fr.1~Fr.12 in sequence. S4: Purification: The Fr.7 fraction was purified by semi-preparative HPLC using an X-Bridge C18 column and an acetonitrile-water mixture with a volume ratio of 35:

65. Fractions were collected at tR = 15.5 ~ 18.2 min. The collected fractions were then dried under reduced pressure at room temperature after solvent removal using a rotary evaporator to obtain a brown amorphous powder.

2. The preparation method according to claim 1, characterized in that, Step S1 specifically includes: The dark brown stalked Boletus was mixed with methanol solution and extracted. The extract was then concentrated under reduced pressure and evaporated to dryness to obtain a crude extract.

3. The preparation method according to claim 2, characterized in that, Step S1 further includes: The fruiting bodies of Boletus glomeratus are added to a 70-85% methanol solution, mixed thoroughly, and then extracted.

4. The preparation method according to claim 2, characterized in that, The extraction time is 20-28 hours, and optionally, the number of extractions is 3-6 times.

5. The preparation method according to claim 1, characterized in that, Diethylamine at a volume ratio of 0.05% was added to the aqueous phase of the mobile phase.

6. The use of a compound in the preparation of an antitumor drug for prevention and / or treatment, said compound having the molecular formula C 12 H 12 N2O, chemical structural formula as follows: 。

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