CN117820314A - Compound or pharmaceutically acceptable salt thereof, and preparation method and application thereof - Google Patents
Compound or pharmaceutically acceptable salt thereof, and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 14
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Landscapes
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Abstract
The application discloses a compound or pharmaceutically acceptable salt thereof, and a preparation method and application thereof, and belongs to the technical field of natural compounds. The compound is beta-carboline alkaloid, and has a structural formula of C 12 H 12 N 2 O has anti-tumor activity and lays a foundation for preparing anti-tumor products. The compound is derived from Phlebopus portentosus which has been cultivated in a large scale, has sufficient raw materials, simple and rapid separation and extraction steps, strong operability and stable results, and can realize large-scale industrial production.
Description
Technical Field
The invention relates to the technical field of natural compounds, in particular to a beta-carboline alkaloid derived from Phlebopus portentosus or pharmaceutically acceptable salt thereof, and a preparation method and application thereof.
Background
Boletus portentosus (Phlebopus portentosus), also known as Boletus portentosus, commonly known as Boletus nigrum, belongs to the order Boletales, calletaceae (Boletinellaceae), and Boletinium (Phlebopus). The Phlebopus portentosus has been successfully cultivated artificially at present, and large-scale mass production of industrialization can be performed.
Tumor (tumor) refers to a new growth (newgrowth) formed by local tissue cell proliferation of an organism under the action of various tumorigenic factors, because the new organism is often in the form of a occupying massive protrusion, which is also called neoplasm (neoplasm). Tumors are abnormal lesions formed by the fact that a certain cell of local tissues loses normal regulation of growth of the cells at the gene level under the action of various cancerogenic factors, so that the cells are clonally abnormally proliferated, and the tumors are one of main diseases which are harmful to human health at present and seriously threaten human lives. At present, more than 50 antitumor drugs are commonly used clinically, but most of the drugs only can relieve the illness state and cannot achieve the aim of complete cure. The development of new antitumor drugs has been a major aspect of the field of drug research.
Disclosure of Invention
Based on the above, the invention aims to provide the beta-carboline alkaloid compound which has anti-tumor activity and lays a foundation for the development of related health-care products or medicines.
To solve the above problems, a first aspect of the present invention provides a β -carboline alkaloid compound having the formula C 12 H 12 N 2 O has the chemical structural formula as follows:
the term "compound" as used in the present invention is intended to include all stereoisomers (e.g., enantiomers and diastereomers), geometric isomers, tautomers and isotopes of the structures shown. Unless otherwise indicated, a compound identified by name or structure as one particular tautomeric form in the invention is intended to include other tautomeric forms.
In one embodiment of the invention, the compound is isolated from Boletus fulgidus.
The compound is a novel compound, is beta-carboline alkaloid, is derived from Phlebopus portentosus, has anti-tumor activity, and can inhibit proliferation of tumor cells.
The specific name of the compound is N-formamyl-1, 2,3, 4-tetrahydroo-beta-carboline (N-formyl-1, 2,3, 4-tetrahydroo-beta-carboline).
In another aspect, the invention provides a pharmaceutical composition comprising a compound as described above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Wherein the salt can be potassium salt, sodium salt and the like.
The pharmaceutical compositions of the present invention may be prepared by methods well known in the pharmaceutical arts. In certain embodiments, the active substances of the present invention may be mixed with a pharmaceutically acceptable carrier for the preparation of a pharmaceutical composition. The active substances comprise the beta-carboline alkaloid or pharmaceutically acceptable salts thereof and the like.
The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In certain embodiments, pharmaceutically acceptable carriers, materials, compositions and/or dosage forms refer to those approved by a regulatory agency (e.g., the U.S. food and drug administration, the chinese food and drug administration, or the european pharmaceutical administration) or listed in a generally recognized pharmacopeia (e.g., the U.S. pharmacopeia, chinese pharmacopeia, or european pharmacopeia) for use in animals, and more particularly for use in humans.
Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, for example, pharmaceutically acceptable liquids, gels, or solid carriers, aqueous media (e.g., sodium chloride injection, ringer's solution injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated ringer's solution injection), non-aqueous media (e.g., fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil), antimicrobial substances, isotonic substances (e.g., sodium chloride or dextrose), buffers (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 methylcellulose, or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediamine tetraacetic acid) or EGTA (ethylene glycol bis (2-aminoethylether) tetraacetic acid)), emulsifying agents (e.g., polysorbate 80 (tween-80)), diluents, adjuvants, or non-toxic substances, other components known in the art, or combinations of various combinations thereof. Suitable components may include, for example, fillers, binders, disintegrants, buffers, preservatives, lubricants, taste-agitating agents, thickening agents, colorants or emulsifiers.
In certain embodiments, the pharmaceutical composition may be used in combination with one or more other drugs. In certain embodiments, the pharmaceutical composition comprises at least one additional drug.
In another aspect, the present invention provides a method for preparing the above compound, comprising the steps of:
s1: leaching: dissolving Phlebopus portentosus in an organic solvent for leaching to obtain an extract;
s2: extraction: extracting the extract with an organic solvent to obtain an extract;
s3: separating: separating the extract by column chromatography to obtain a separate product;
s4: purifying: purifying the isolate to obtain the compound.
The compound is derived from Phlebopus portentosus, the Phlebopus portentosus has been artificially cultivated, raw materials can be provided in a large scale, and the preparation method has the advantages of simple and rapid steps and strong operability, and can realize large-scale industrial production.
In one specific embodiment of the present application, step S1 specifically includes:
adding Phlebopus portentosus into methanol solution, mixing well, leaching, concentrating the leaching solution under reduced pressure, evaporating to dryness to obtain crude extract.
In a specific embodiment of the present application, step S1 further includes:
and adding the fruiting bodies or mycelia of the Phlebopus portentosus into 70-85% methanol solution, uniformly mixing, and leaching.
In a specific embodiment of the invention, the leaching time is 20-28 hours. For example 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h or 28h.
In a specific embodiment of the invention, the number of leaches is 3-6. For example 3 times, 4 times, 5 times or 6 times.
In a specific embodiment of the present invention, step S2 specifically includes:
suspending the crude extract with water, adjusting pH to 9-10, extracting with chloroform, concentrating under reduced pressure, and evaporating to dryness to obtain extract.
In a specific embodiment of the present invention, step S3 specifically includes:
the extract was separated by MCI column chromatography to obtain 12 fractions, fr.1 to fr.12 in sequence.
In one embodiment of the present invention, the MCI column chromatography separation process is specifically: the extract was eluted through a gradient of 20% -100% methanol-water over an MCI column.
In one embodiment of the present invention, step S4 specifically includes:
fr.7 was purified by HPLC to give the compound.
In a specific embodiment of the invention, the specific steps of the HPLC purification are: an X-Bridge C18 chromatographic column is selected, and the mobile phase is acetonitrile-water with the volume ratio of 35:65.
In a specific embodiment of the invention, diethylamine is added in a volume ratio of 0.05% to the aqueous phase of the mobile phase. Diethylamine was added to alter the peak shape and degree of separation of the compounds, making them easier to separate.
In one embodiment of the invention, the preparation method of the compound specifically comprises the following steps:
s1: taking a Phlebopus portentosus fruiting body or mycelium sample, adding 80% methanol solution at room temperature, mixing well, leaching for 24h, leaching for 5 times, combining the extracting solutions, concentrating under reduced pressure, and evaporating to obtain crude extract;
s2: suspending the above crude extract with water, and adding Na 2 CO 3 Regulating pH to 9-10, extracting with equal volume of chloroform for 5 times, concentrating under reduced pressure, and evaporating to dryness to obtain extract;
s3: the extract was separated by MCI column chromatography, followed by MeOH-H 2 O (20% -100%, v/v) gradient elution to obtain 12 components, which are Fr.1-Fr.12 in sequence;
s4: purification of Fr.7 by HPLC, column X-Bridge C18, mobile phase acetonitrile-water, 35:65, v/v, addition of 0.05% (v/v) Diethylamine (DEA) to the aqueous phase, collection of t R ＝15.5～18.2min。
According to a further aspect of the present invention there is provided the use of a compound as described above for the preparation of an anti-tumour agent for the prophylaxis and/or treatment of cancer. The compound has anti-tumor activity and lays a foundation for preparing anti-tumor products.
In a first embodiment of the invention, the tumor is caused by cervical cancer cells or gastric cancer cells.
The technical scheme of the invention has at least one of the following beneficial technical effects: the invention provides a novel compound which is a beta-carboline alkaloid compound and has anti-tumor activity, and lays a foundation for preparing anti-tumor products. The compound is derived from Phlebopus portentosus which has been cultivated in a large scale, has sufficient raw materials, simple and rapid separation and extraction steps, strong operability and stable results, and can realize large-scale industrial production.
Drawings
FIG. 1 shows N-formyl-1,2,3, 4-tetrahydro-beta-carboline provided by the invention 1 H-NMR spectrum;
FIG. 2 is a schematic diagram of N-formyl-1,2,3 provided by the invention4-tetrahydro-beta-carbolines 13 C-NMR spectrum;
FIG. 3 is a chart of HMBC spectra of N-formyl-1,2,3, 4-tetrahydro-beta-carboline provided by the invention.
Detailed Description
The objects, technical solutions and advantages of the present invention will become more apparent by the following detailed description of the present invention with reference to the accompanying drawings. It should be understood that the description is only illustrative and is not intended to limit the scope of the invention. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention.
The products or components herein, unless otherwise specified, are all commercially available products.
EXAMPLE 1 preparation of beta-carboline alkaloid
1. 14.6kg of fresh Phlebopus portentosus is chopped, placed in a barrel, added with 10.00L of 80% methanol solution at room temperature, uniformly mixed, immersed for 24 hours, immersed for 5 times, combined, concentrated under reduced pressure and evaporated to dryness to obtain 831.5g of tan crude extract.
2. Suspending the above crude extract with water, and adding Na 2 CO 3 The pH was adjusted to 9-10, extracted with an equal volume of chloroform for 5 times, concentrated under reduced pressure and evaporated to dryness to give 8.6g of chloroform extract.
3. The chloroform extract was separated roughly by MCI column chromatography, followed by MeOH-H 2 After O (20% -100%, v/v) gradient elution (20%. Fwdarw.30%. Fwdarw.40%. Fwdarw.50%. Fwdarw.60%. Fwdarw.70%. Fwdarw.80%. Fwdarw.90%. Fwdarw.100% methanol), 12 fractions were obtained, fr.1-Fr.12 in sequence.
4. The fr.7 fraction was purified by semi-preparative HPLC with column X-Bridge C18 and acetonitrile-water as mobile phase, 35:65, v/v, 0.05% (v/v) Diethylamine (DEA) was added to the aqueous phase of the mobile phase and the tr=15.5-18.2 min fraction was collected.
5. The fraction collected in step 4 was subjected to solvent removal by rotary evaporator and then dried under reduced pressure at room temperature to finally obtain 1.9mg of a novel compound as a brown amorphous powder.
Example 2 spectral and physicochemical data of beta-carboline alkaloids
The structure of the novel compounds prepared in example 1 of the present invention was identified as follows:
hydrogen spectrum data: 1 H NMR(CD 3 OD,600MHz):δ H 8.25/8.30(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)。
carbon spectrum data: 13 C NMR(CD 3 OD,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),118.6(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)。
the mass spectrum data includes: (+) -ESI-MS m/z 201[ M+H ]] + ；(-)-ESI-MS:m/z 199[M-H] - ；(+)-HR-ESI-MS:m/z 201.1021[M+H] + (calcd.for 201.1022,C 12 H 12 N 2 O)。
New compounds 1 The H-NMR spectrum is shown in FIG. 1, 13 the C-NMR spectrum is shown in FIG. 2, the HMBC spectrum is shown in FIG. 3, and the novel compound can be identified as beta-carboline alkaloid (N-formiyl-1, 2,3, 4-tetrahydroo-beta-carboline) with the chemical formula of C by combining the information 12 H 12 N 2 O has the following structural formula:
example 3 in vitro anti-tumor Activity of beta-carboline alkaloids
1. The cells used were tested: hela (human cervical cancer cell), HGC27 (human undifferentiated gastric cancer cell), purchased from China academy of sciences cell bank and Shanghai banyan Biotechnology center respectively.
2. Beta-carboline alkaloid concentration (μM): 200. 100, 50, 25, 12.5.
3. Reagents and instrumentation: DMEM/F12 medium (TBD, TBD 1056S), fetal bovine serum (FBS, F019)3) (Biosharp, BL 205A), 100U/mL penicillin and 100. Mu.g/mL streptomycin (Gibco, 15140-122); pancreatin (Sigma, T4799); CCK-8 detection solution (Dojindo, CK 04); MTT (Beyotime, ST 316); CO 2 Incubator (Thermo, forta 3111); biological safety cabinet (Heal Force, HFsafe-1200); -80 ℃ refrigerator (Thermo, forta 702); microplate reader (BioTek, epoch); high-speed refrigerated centrifuges (Hitachi, CF15 RN); inverted fluorescence microscopy (Leica, DMI 3000B).
4. The experimental method comprises the following steps: tumor cells were seeded in 96-well plates (0.8 x 10) 4 Individual cells/well), 37 ℃,5% CO 2 Incubator, culture for 24h. The culture solution was aspirated, 100. Mu.L/well of the drug to be tested (. Beta. -carboline alkaloid) solution was added, 3 duplicate wells were set for each concentration, and the treatment without drug was used as a negative control. After 48h incubation of the drug, the liquid in the wells was aspirated, 100. Mu.L of sugar-free serum-free DMEM medium containing 5mg/mL MTT was added to each well, and incubation was continued for 2-4h. The supernatant was aspirated, 100. Mu.L of DMSO was added to each well, the mixture was dissolved by low-speed shaking, OD value was measured at 490nm, and the cell proliferation inhibition ratio was calculated: [ (average OD value of negative control group-blank OD value) - (average OD value of drug group-blank OD value)]/(average OD value of negative control group-blank OD value) ×100%. The experimental results are shown in table 1.
Table 1 in vitro antitumor Activity of beta-carboline alkaloid compounds
As can be seen from table 1, as the β -carboline alkaloid concentration increases, the inhibition rate of cell growth is also higher. The beta-carboline alkaloid provided by the invention shows inhibition effect on Hela229 cells when the concentration of the beta-carboline alkaloid is more than 100 uM; above 12.5uM concentration, inhibition of HGC27 cells was shown. Therefore, the novel compounds screened by the application have certain inhibitory activity on human cervical cancer cells and human undifferentiated gastric cancer cells, show a certain broad spectrum, and provide a foundation for research of broad spectrum antitumor drugs and antitumor treatment by combining other drugs.
The beta-carboline alkaloid provided by the invention shows a certain activity of resisting tumor cell proliferation, and can be used for developing related health-care products or medicines.
In conclusion, the beta-carboline alkaloid separated from the Phlebopus portentosus has anti-tumor activity, can be used for developing related health-care products or medicines, has simple separation and extraction process operation and wide raw material sources, and can realize mass industrialized production.
It is to be understood that the above-described embodiments of the present invention are merely illustrative of or explanation of the principles of the present invention and are in no way limiting of the invention. Accordingly, any modification, equivalent replacement, improvement, etc. made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. Furthermore, the appended claims are intended to cover all such changes and modifications that fall within the scope and boundary of the appended claims, or equivalents of such scope and boundary.
Claims (13)
1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound has the formula C 12 H 12 N 2 O has the chemical structural formula as follows:
2. a pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
3. A process for the preparation of a compound as claimed in claim 1, comprising the steps of:
s1: leaching: dissolving Phlebopus portentosus in an organic solvent for leaching to obtain an extract;
s2: extraction: extracting the extract with an organic solvent to obtain an extract;
s3: separating: separating the extract by column chromatography to obtain a separate product;
s4: purifying: purifying the isolate to obtain the compound.
4. A method according to claim 3, wherein step S1 comprises:
adding Phlebopus portentosus into methanol solution, mixing well, leaching, concentrating the leaching solution under reduced pressure, evaporating to dryness to obtain crude extract.
5. The method of claim 4, wherein step S1 further comprises:
and adding the fruiting bodies or mycelia of the Phlebopus portentosus into 70-85% methanol solution, uniformly mixing, and leaching.
6. The method of claim 4, wherein the leaching time is 20-28h, and optionally, the number of leaches is 3-6.
7. The method according to claim 4, wherein step S2 specifically comprises:
suspending the crude extract with water, adjusting pH to 9-10, extracting with chloroform, concentrating under reduced pressure, and evaporating to dryness to obtain extract.
8. The method according to claim 3, wherein step S3 specifically comprises:
the extract was separated by MCI column chromatography to obtain 12 fractions, fr.1 to fr.12 in sequence.
9. The preparation method according to claim 8, wherein the MCI column chromatography separation process specifically comprises: the extract is eluted through an MCI chromatographic column by a methanol-water gradient with the volume ratio of 20-100%.
10. The preparation method according to claim 8, wherein step S4 specifically comprises:
fr.7 was purified by HPLC to give the compound.
11. The preparation method according to claim 10, wherein the specific steps of HPLC purification are: an X-Bridge C18 chromatographic column is selected, and the mobile phase is acetonitrile-water with the volume ratio of 35:65.
12. The method according to claim 11, wherein diethylamine is added to the aqueous phase of the mobile phase in a volume ratio of 0.05%.
13. Use of a compound according to claim 1 for the preparation of a product for the prophylaxis and/or treatment of antitumor.
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