CN116410173A - Pyran naphthoquinone compound, preparation method, composition and application thereof - Google Patents
Pyran naphthoquinone compound, preparation method, composition and application thereof Download PDFInfo
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- -1 Pyran naphthoquinone compound Chemical class 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
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- 150000001875 compounds Chemical class 0.000 claims abstract description 107
- 239000003814 drug Substances 0.000 claims abstract description 16
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- 208000029742 colonic neoplasm Diseases 0.000 claims abstract description 9
- 201000007270 liver cancer Diseases 0.000 claims abstract description 8
- 208000014018 liver neoplasm Diseases 0.000 claims abstract description 8
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 6
- 229940041181 antineoplastic drug Drugs 0.000 claims abstract description 6
- 201000006585 gastric adenocarcinoma Diseases 0.000 claims abstract description 3
- 239000003208 petroleum Substances 0.000 claims description 60
- 239000011259 mixed solution Substances 0.000 claims description 53
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 31
- HWJHWSBFPPPIPD-UHFFFAOYSA-N ethoxyethane;propan-2-one Chemical compound CC(C)=O.CCOCC HWJHWSBFPPPIPD-UHFFFAOYSA-N 0.000 claims description 31
- 239000000284 extract Substances 0.000 claims description 29
- 239000002904 solvent Substances 0.000 claims description 28
- 238000004440 column chromatography Methods 0.000 claims description 24
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 20
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
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- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 11
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- HTSGKJQDMSTCGS-UHFFFAOYSA-N 1,4-bis(4-chlorophenyl)-2-(4-methylphenyl)sulfonylbutane-1,4-dione Chemical compound C1=CC(C)=CC=C1S(=O)(=O)C(C(=O)C=1C=CC(Cl)=CC=1)CC(=O)C1=CC=C(Cl)C=C1 HTSGKJQDMSTCGS-UHFFFAOYSA-N 0.000 claims description 7
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- KBKBQWDPOVRZID-UHFFFAOYSA-N naphthalene-1,4-dione;2h-pyran Chemical class C1OC=CC=C1.C1=CC=C2C(=O)C=CC(=O)C2=C1 KBKBQWDPOVRZID-UHFFFAOYSA-N 0.000 abstract description 17
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 125000000182 1,4-naphthoquinonyl group Chemical group C1(C(=CC(C2=CC=CC=C12)=O)*)=O 0.000 description 1
- XILIYVSXLSWUAI-UHFFFAOYSA-N 2-(diethylamino)ethyl n'-phenylcarbamimidothioate;dihydrobromide Chemical compound Br.Br.CCN(CC)CCSC(N)=NC1=CC=CC=C1 XILIYVSXLSWUAI-UHFFFAOYSA-N 0.000 description 1
- WRMNZCZEMHIOCP-UHFFFAOYSA-N 2-Phenylethanol Natural products OCCC1=CC=CC=C1 WRMNZCZEMHIOCP-UHFFFAOYSA-N 0.000 description 1
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- KIWBPDUYBMNFTB-UHFFFAOYSA-N Ethyl hydrogen sulfate Chemical compound CCOS(O)(=O)=O KIWBPDUYBMNFTB-UHFFFAOYSA-N 0.000 description 1
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- ZUKLFFYDSALIQW-MSUKCBDUSA-N Iridoid glycoside Chemical compound [H][C@]12CC[C@H](C(O)=O)[C@@]1([H])[C@H](OC1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O)OC=C2 ZUKLFFYDSALIQW-MSUKCBDUSA-N 0.000 description 1
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- FRASJONUBLZVQX-UHFFFAOYSA-N naphthoquinone group Chemical group C1(C=CC(C2=CC=CC=C12)=O)=O FRASJONUBLZVQX-UHFFFAOYSA-N 0.000 description 1
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-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/92—Naphthopyrans; Hydrogenated naphthopyrans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to pyran naphthoquinone compounds, a preparation method, a composition and application thereof, and relates to the technical field of medicines, 3 pyran naphthoquinone compounds are obtained by separating verbena, and the structural formula of the pyran naphthoquinone compounds is shown as the formula I-III:
the 3 pyranoquinone compounds have good inhibition effects on tumor cells such as colon cancer cells HCT116, brain glioma cells LN229, gastric adenocarcinoma cells SGC-7901, liver cancer cells HepG2 and the like. The pyran naphthoquinone compound has good application prospect in preparing anti-tumor drugs.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to pyran naphthoquinone compounds, a preparation method, a composition and application thereof.
Background
Tumors, especially malignant tumors, are severely threatening the life health of humans. Tumor statistics by WHO are the major causative causes of death worldwide, accounting for about 1000 tens of thousands of deaths each year, accounting for one sixth of the deaths worldwide. Clinical strategies for drug treatment of tumors mainly include chemotherapy, hormone treatment, targeted therapy, tumor immunotherapy and the like, and although great progress has been made, the overall efficacy is still not ideal. The natural medicine is an important source for the discovery of the lead compound of the new medicine because of the characteristics of rich chemical structure, various pharmacological activities and the like. The discovery of antitumor lead compounds from traditional Chinese medicines or natural medicines is a hot spot field of research in the current scientific community.
The vervain (Verbena officinalis l.) is a plant of the genus vervain family Verbenaceae. Is recorded in Ming Yi Bie Lu, has the effects of clearing away heat and toxic materials, promoting blood circulation, dispelling blood stasis, inducing diuresis and relieving edema; it is mainly used for treating exogenous fever, damp-heat jaundice, edema, abdominal mass, carbuncle, swelling, and sore. The chemical components of the vervain mainly comprise iridoid glycoside, phenethyl alcohol glycoside, flavone, triterpene, volatile oil and the like. The research shows that the verbena extract has obvious inhibition effect on a mouse ascites tumor model established by an H22 liver cancer cell line. In addition, the two cell lines of the rat colon cancer cell DHD/K12/PRO and the human colon cancer cell HCT-116 prove that the verbena water extract has obvious anti-tumor effect. The invention carries out systematic research on the antitumor active ingredients of the vervain, and has important significance for developing low-toxicity and high-efficiency antitumor drugs.
Pyranoquinone compounds are secondary metabolites with important biological activities which widely exist in nature, and the compounds are reported to have various pharmacological effects of anticancer, analgesic, anti-inflammatory, antimalarial, antifungal, antiviral and the like, for example, patent CN 103980285B. Research shows that the compound containing 1, 4-naphthoquinone ring has excellent effect of inhibiting tumor cell proliferation, and the change of the substituent group on the naphthoquinone ring has great influence on pharmacological activity. Therefore, the discovery of new naphthoquinone compounds has become a hotspot in the development of new antitumor drugs.
Although eighty compounds with various biological activities have been isolated and identified from Verbena in recent years, pyran naphthoquinone compounds with antitumor activity have not been reported to date from the secondary metabolites of Verbena. In view of this, the present invention provides pyran naphthoquinone compounds, methods of preparation, compositions and uses thereof.
Disclosure of Invention
The invention aims to solve the technical problem of providing pyran naphthoquinone compounds, a preparation method, a composition and application thereof. Aims to extract pyran naphthoquinone compounds with anti-tumor activity from vervain.
The first object of the present invention is to provide pyran naphthoquinone compounds, wherein the structural formula of the pyran naphthoquinone compounds is selected from formula I, ii or I:
wherein the general formula of the formula I and the general formula of the formula II can be written as shown in I':
The beneficial effects of the invention are as follows: (1) The 3 pyran naphthoquinone compounds are separated from vervain; in-vitro anti-tumor experiments show that the pyranoquinone compounds have strong inhibition activities on colon cancer cells HCT116, brain glioblastoma cells LN229, gastric cancer cells SGC-7901 and liver cancer cells HepG2, and can provide lead compounds for developing new anti-tumor drugs.
On the basis of the technical scheme, the invention can be improved as follows.
The second object of the present invention is to provide a process for preparing the pyran naphthoquinone compound, comprising the steps of:
(1) Adding alcohol solvent into stems and leaves of herba Verbenae cut into small sections, heating and reflux-extracting, and concentrating the obtained extractive solution containing the alcohol solvent under reduced pressure to obtain the alcohol solvent extract;
(2) Dissolving the extract of the alcohol solvent in water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and concentrating the obtained ethyl acetate extract layer under reduced pressure to obtain the ethyl acetate extract;
(3) Subjecting the ethyl acetate extract to normal phase silica gel column A chromatography, and gradient eluting with petroleum ether-acetone mixed solution a; subjecting the collected eluate of petroleum ether-acetone mixed solution a containing the pyran naphthoquinone compound to normal phase silica gel column B chromatography, and gradient eluting with petroleum ether-ethyl acetate mixed solution; subjecting the collected eluate of the petroleum ether-ethyl acetate mixed solution containing the pyranoquinone compounds to sephadex column chromatography, performing isocratic elution by using dichloromethane-methanol mixed solution, and collecting the extract after sephadex column purification; aims at removing pigment impurities such as chlorophyll
(4) Subjecting the extract purified by the sephadex column to normal phase silica gel column C chromatography, and gradient eluting by petroleum ether-acetone mixed solution b; collecting eluate of petroleum ether-acetone mixed solution b containing the pyran naphthoquinone compound I, subjecting to sephadex column chromatography by using petroleum ether-dichloromethane-methanol mixed solution as mobile phase to obtain compound I shown in formula I, subjecting to sephadex column chromatography by using petroleum ether-dichloromethane-methanol mixed solution as mobile phase to obtain eluate of petroleum ether-acetone mixed solution b containing the pyran naphthoquinone compound I, subjecting to sephadex column chromatography by using petroleum ether-dichloromethane-methanol mixed solution as mobile phase to obtain compound I shown in formula II;
wherein the chemical name of formula I is 3,4-dehydro-3, 9-dihydroxy-alpha-lapachone; the chemical name of formula I is 3, 4-dehydro-3-hydroxy-alpha-lapachone (3, 4-dehydro-3-hydroxy-alpha-lapachone), and the chemical name of formula I is 4R-4, 9-dihydroxy-alpha-lapachone (4R-4, 9-dihydroxy-alpha-lapachone).
The beneficial effects of adopting above-mentioned scheme are: the pyran naphthoquinone compound is obtained by separating the verbena, and the method is simple and convenient to operate, high in product purity and suitable for large-scale production.
Further, the alcohol solvent in the step (1) comprises any one or a mixture of at least two of ethanol, methanol and propanol;
the step (1) comprises the following steps: adding alcohol solvent into stems and leaves of the vervain which are cut into small sections, heating and reflux-extracting, wherein the volume of the added alcohol solvent is 5-10 times of the weight of the stems and leaves of the vervain, the reflux-extracting times are 2-5, the temperature of each reflux-extracting is 85-100 ℃, the time of each reflux-extracting is 1-4 hours, and merging the obtained extracting solutions containing the alcohol solvent, and then concentrating under reduced pressure to obtain the alcohol solvent extract.
Further, in the step (2), the weight-to-volume ratio of the extract of the alcohol solvent to the water is 1: (1-3); the weight-volume ratio of the extract of the alcohol solvent to the petroleum ether is 1: (1-3); the weight volume ratio of the extract of the alcohol solvent to the ethyl acetate is 1: (1-3); the weight volume ratio of the extract of the alcohol solvent to the n-butanol is 1: (1-3).
Further, the step (3) specifically comprises: subjecting the ethyl acetate extract to normal phase silica gel column A chromatography, and gradient eluting with petroleum ether-acetone mixed solution a with the volume ratio of 50:1-1:1; subjecting the collected eluent of the petroleum ether-acetone mixed solution a with the volume ratio of 20:1 to normal phase silica gel column B chromatography, and carrying out gradient elution by adopting petroleum ether-ethyl acetate mixed solution with the volume ratio of 20:1-5:1; eluting the collected eluent of the petroleum ether-ethyl acetate mixed solution with the volume ratio of 10:1 by using sephadex LH-20 column chromatography, eluting by using dichloromethane-methanol mixed solution with the volume ratio of 1:1, and collecting the extract after sephadex LH-20 column purification.
Further, the step (4) specifically comprises: subjecting the extract purified by the Sephadex LH-20 column to normal phase silica gel column C chromatography, carrying out gradient elution by using petroleum ether-acetone mixed solution b with the volume ratio of 30:1-10:1, subjecting the collected eluate of the petroleum ether-acetone mixed solution b with the volume ratio of 30:1 to the Sephadex LH-20 column chromatography, using petroleum ether-dichloromethane-methanol mixed solution with the volume ratio of 2:1 to the Sephadex LH-20 column chromatography as a mobile phase, obtaining a compound I shown in formula I by using the petroleum ether-acetone mixed solution b with the volume ratio of 20:1, subjecting the collected eluate of the petroleum ether-acetone mixed solution b with the volume ratio of 20:1 to the Sephadex LH-20 column chromatography, using petroleum ether-dichloromethane-methanol mixed solution with the volume ratio of 2:1 to the mobile phase, obtaining a compound I shown in formula I by using the petroleum ether-acetone mixed solution b with the volume ratio of 10:1 to the Sephadex LH-20 column chromatography, and obtaining a compound II shown in formula II by using the petroleum ether-methanol mixed solution with the volume ratio of 2:1 to the Sephadex LH-20 column chromatography.
The third object of the invention is to provide a pharmaceutical composition comprising the pyran naphthoquinone compound and pharmaceutically acceptable carriers and/or excipients.
The actual dosage of the active ingredient (pyran naphthoquinone compound of the present invention) in the pharmaceutical composition should be determined according to various relevant factors, including the severity of the disease to be treated, the administration route, the age, sex, weight of the patient, and thus, the above dosage should not limit the scope of the present invention in any way. The pharmaceutically acceptable carriers and/or excipients include pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the dosage form designed.
Further, the dosage forms of the pharmaceutical composition comprise tablets, capsules, oral liquid, buccal tablets, granules, suspension, suppositories, injections, powder injections, dripping pills, sustained release agents, controlled release agents or targeted preparations.
The pharmaceutical composition can be any pharmaceutically acceptable dosage form. The appropriate dosage of the pharmaceutical composition may be formulated according to the method of preparation, the mode of administration, the age, weight, sex, condition, diet, time of administration, route of administration and sensitivity of response of the patient, and the like, and the skilled doctor can easily determine the prescription and the dosage of the prescription effective for the desired treatment.
The fourth object of the invention is to provide the application of the pyran naphthoquinone compound in preparing antitumor drugs.
Further, the pyranoquinone compound is applied to the preparation of medicines for resisting colon cancer, brain glioma, gastric adenocarcinoma or liver cancer.
The beneficial effects of adopting above-mentioned scheme are: the research of the invention shows that the pyranoquinone compound separated from the verbena by the method has stronger anti-tumor activity. For colon cancer cell HCT116, compound I IC 50 IC of compound I with a value of 7.89+ -0.54 μM 50 IC with a value of 12.31+ -3.14 μM for Compound I 50 The value is 4.74 plus or minus 0.24 mu M; IC for brain glioblastoma cell LN229, compound I 50 IC of compound I with a value of 17.40+ -2.26 μM 50 The value is 8.97+ -1.02 μm; IC of compound I 50 The value is 13.28+/-1.36 mu M; IC of compound I for gastric cancer cell SGC-7901 50 IC of compound I with a value of 7.51+ -0.26 μM 50 The value is 8.07+ -0.51 μM; IC of compound I 50 The value is 6.09+/-0.48 mu M; IC for liver cancer cell HepG2, compound I 50 IC of compound I with a value of 13.60+ -2.12 μM 50 The value is 12.29+/-1.73 mu M; IC of compound I 50 The value was 25.58.+ -. 5.16. Mu.M.
Drawings
FIG. 1 is a diagram of a vervain plant and medicinal material of the present invention;
FIG. 2 is a spectrum of HR-ESI-MS of compound I of the present invention;
FIG. 3 shows Compound I of the present invention 1 H NMR spectrum;
FIG. 4 shows Compound I of the present invention 13 C NMR and DEPT-135 spectra;
FIG. 5 is a spectrum of HSQC of Compound I of the present invention;
FIG. 6 is a HMBC spectrum of Compound I of the present invention;
FIG. 7 shows Compound I of the present invention 1 H- 1 H COSY profile;
FIG. 8 is a spectrum of HR-ESI-MS of compound I of the present invention;
FIG. 9 is the presentCompound I of the invention 1 H NMR spectrum;
FIG. 10 shows the compound I of the present invention 13 C NMR and DEPT-135 spectra;
FIG. 11 is a spectrum of HSQC of compound I of the present invention;
FIG. 12 is a HMBC spectrum of compound I of the present invention;
FIG. 13 shows the compound I of the present invention 1 H- 1 H COSY profile;
FIG. 14 shows the compound I of the present invention 1 H NMR spectrum;
FIG. 15 shows the compound I of the present invention 13 C NMR spectrum;
FIG. 16 is a HMBC spectrum of compound I of the present invention;
FIG. 17 is a schematic diagram showing critical HMBC and HMBC related to Compound I 1 H- 1 H COSY correlation diagram;
FIG. 18 is a critical HMBC and for Compound II of the present invention 1 H- 1 H COSY correlation diagram.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention.
Example 1: preparation and structural identification of pyran naphthoquinone compounds I-I
1. Preparation of pyran naphthoquinone compounds I-I
The preparation of pyran naphthoquinone compounds I-I specifically comprises the following steps:
(1) Pulverizing aerial parts of herba Verbenae 25.0kg (figure 1), soaking in 95% ethanol 6 times of volume fraction for 1 hr, reflux extracting under heating for 3 times (3 hr, 2 hr), concentrating the extractive solution under reduced pressure to obtain ethyl acetate extract;
(2) The extract was dissolved and dispersed in 20.0L deionized water, and extracted with petroleum ether, ethyl acetate, and n-butanol in this order for 4 times (20.0L each time). Combining the obtained ethyl acetate extraction layers, and recovering the solvent under reduced pressure to obtain 172.5g of ethyl acetate extract;
(3) Extraction of ethyl acetateSubjecting the obtained product to normal phase silica gel column A chromatography
200-300 meshes, 700-900 g) and the petroleum ether-acetone mixed solution a with the volume ratio of mobile phases of (50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 8:1, 4:1, 2:1 and 1:1) is subjected to gradient elution. The eluate was collected every 500.0mL, and the fractions distilled under reduced pressure were subjected to TLC (thin layer chromatography) analysis, and the same components were combined. Collecting petroleum ether-acetone 20:1 eluate, and subjecting to normal phase silica gel column B chromatography (++L)>
200-300 meshes, 250-300 g) and the mobile phase is eluted by petroleum ether-ethyl acetate gradient, wherein the volume ratio of petroleum ether-ethyl acetate is (20:1, 15:1, 10:1, 5:1). Collecting petroleum ether-ethyl acetate 10:1 fraction, separating by Sephadex LH-20 column chromatography, eluting with dichloromethane-methanol at volume ratio of 1:1, collecting eluent once every 100.0mL, inspecting by TLC thin layer chromatography, and collecting fraction containing compounds I-I15-35; combining the fractions containing the compounds I to I15 to 35, recovering the solvent under reduced pressure, and collecting the extract purified by the sephadex column;
(4) And (3) subjecting the extract after the sephadex column purification to normal phase silica gel column C chromatography, and sequentially performing gradient elution with petroleum ether-acetone mixed solution b with the volume ratio of (30:1, 20:1, 10:1). Collecting eluate of normal phase silica gel chromatography 30:1 petroleum ether-acetone mixed solution b, taking petroleum ether-dichloromethane-methanol with volume ratio of 2:1:1 as mobile phase, purifying by Sephadex LH-20 column chromatography to obtain compound I (23.2 mg, accounting for 0.013% of total extract); purifying the eluent of the normal phase silica gel chromatography 20:1 petroleum ether-acetone mixed solution b by Sephadex LH-20 column chromatography under the same eluting conditions to obtain a compound I (19.0 mg, accounting for 0.011% of the total extract). Purifying the eluent of the normal phase silica gel chromatography 10:1 petroleum ether-acetone mixed solution b by Sephadex LH-20 column chromatography under the same eluting conditions to obtain a compound I (74.0 mg, accounting for 0.042% of the total extract).
Among them, TLC (thin layer chromatography) analysis is specifically: thin-layer chromatography inspection is carried out by adopting petroleum ether-acetone with the volume ratio of 3:1 as developing agent, at the position of the Rf value of 0.3-0.5, the compounds I and I are yellow under the 254nm of an ultraviolet lamp, the compound I is yellow green under the 254nm of the ultraviolet lamp, and the three compounds are yellow under the heating of 10% sulfuric acid ethanol.
2. Structure identification of compounds I-I
2.1 structural identification of Compound I
Compound i was a yellow amorphous powder. The TLC plate showed yellow-brown color at 254nm, yellow fluorescence at 365nm under ultraviolet light, and yellow color by heating with 10% ethanol sulfate. From high resolution Mass Spectrometry HR-ESI-MS (FIG. 2) ([ M+H)] + Experimental value m/z 273.0753, calculated value 273.0757) speculates that the molecular formula of the compound is C 15 H 12 O 5 The degree of unsaturation was 10.
1 H NMR spectra (Table 1 and FIG. 3) show two active hydrogen proton signals [ delta ] H 12.16 (1H, brs, 9-OH) and 4.83 (1H, brs, 3-OH)]Aromatic proton signal [ delta ] at three ortho positions H 7.73(1H，t，J＝7.6Hz，H-7)，7.67(1H，dd，J＝7.2，0.8Hz，H-6)，7.28(1H，dd，J＝8.4，1.2Hz，H-8)]The method comprises the steps of carrying out a first treatment on the surface of the Proton signal delta of double bond H 6.85 (1H, s, H-4) and two methyl signals [ delta ] H 1.64(6H，s，H 3 -11 and H 3 -12)]。 13 C NMR and DEPT-135 spectra (Table 1 and FIG. 4) show that the compound has fifteen carbon signals, including two methyl groups, four methine groups and nine quaternary carbon signals, including two ketocarbonyl carbon signals [ delta ] C 188.0 (C-10) and 173.0 (C-5)]A quaternary carbon signal [ delta ] of oxygen C 69.3(C-2)]Two double bond carbon signals [ delta ] C 170.7 (C-3) and 102.7 (C-4)]It is presumed to be a pyranoquinone compound.
All hydrocarbon signals were attributed through the HSQC spectrum (figure 5), 1 H- 1 the H COSY spectrum (FIG. 7) shows that H-7 is related to H-6 and H-8, presumably to the presence of trisubstituted aromatic rings. HMBC spectra (FIG. 6) show that H-7 correlates with C-5a and C-9, H-8 correlates with C-6, C-9 and C-9a, and an A-loop fragment is deduced; the way in which the B-ring fragments are ligated is deduced from the correlation of H-6 with C-5, C-8, C-9a and C-10, and H-8 with C-10. H-4 and C-3The C-4a, C-5, C-10 and C-10a are related, and the way of ligating the C-ring fragments is deduced, see in detail FIG. 17.9-OH is associated with C-8, C-9 and C-9a, inferred to be attached to the carbon at the 9-position of the A ring; 3-OH is associated with C-2, C-3 and C-4, inferred to be attached to the carbon at the 3-position of the C ring; h 3 -11 is C-12 related, H 3 -11 and H 3 -12 is related to C-2, C-3, thereby deducing CH 3 -11 and CH 3 -12 is attached to the carbon at the 2-position of the C-ring.
1 H NMR 13 Compared with the compound I, the C NMR data shows that the spectrogram of the compound I is increased by a double bond proton signal [ delta ] H 6.84(1H，s，H-4)]The hydroxyl connection position is changed from C-4 position to C-3 position. The compound shown in the formula I is determined to be 3,4-dehydro-3, 9-dihydroxyl-alpha-lapachone through literature retrieval and database comparison, and is a novel structure compound which is not reported.
Table 1 compound i 1 H (400 MHz) and 13 c NMR (100 MHz) data [ deuterated reagent (CD) 3 ) 2 CO]
2.2 structural identification of Compound I
Compound i is a yellow amorphous powder. The TLC plate showed yellow-brown color at 254nm, green fluorescence at 365nm under UV light, and yellow color on heating by 10% sulfuric acid-ethanol TLC. From high resolution Mass Spectrometry HR-ESI-MS (FIG. 8) ([ M+H)] + Experimental value m/z 257.0805, calculated value 257.0808), supposedly the molecular formula of the compound is C 15 H 14 O 4 The unsaturation was 9.
1 H NMR spectra (Table 2 and FIG. 9) show aromatic proton signals [ delta ] in four ortho positions H 7.72(1H，dd，J＝7.6，1.6Hz，H-6)，8.16(1H，td，J＝7.6，2.0Hz，H-7)，8.19(1H，td，J＝7.6，2.0Hz，H-8)]，7.76(1H，dd，J＝7.6，1.6Hz，H-9)]The method comprises the steps of carrying out a first treatment on the surface of the Proton signal delta of double bond H 6.82 (1H, s, H-4) and two methyl signals [ delta ] H 1.69(6H，s，H 3 -11 and H 3 -12)]。
13 C NMR and DEPT-135 spectra (Table 2 and FIG. 10) show that the compound has fifteen carbon signals, including two methyl groups, five methine groups and eight quaternary carbon signals, two of which are ketocarbonyl carbon signals [ delta ] C 173.6 (C-10) and 180.9 (C-5)]Two double bond carbon signals [ delta ] C 168.1 (C-3) and 102.8 (C-4)]. The skeleton of the compound is supposed to be consistent with the formula I, and the compound is a pyran naphthoquinone compound.
The hydrocarbon signal is attributed through the HSQC spectrum (figure 11), 1 H- 1 the H COSY spectrum (FIG. 13) shows that H-7 is associated with H-6 and H-8, and H-8 is associated with H-7 and H-9, thus determining the ligation fragment of H-6/H-7/H-8/H-9. HMBC spectra (FIG. 12) show that H-9 correlates with C-5a, C-7, C-8 and C-9a, thus inferring the A loop fragment; the other is the same as in the HMBC of compound I, see in particular fig. 18. By comparing the compounds of formula I and formula II 1 H NMR 13 C NMR data, which were found to be substantially identical, were only distinguished by the chemical shifts at the H-9 and C-9 positions, and combined with high resolution mass spectrometry, compound II was found to have a molecular weight 16 less than compound I.
By combining the analysis, the compound II is judged to lack a hydroxyl group at the C-9 position, so that the compound II is determined to be 3, 4-dehydro-3-hydroxy-alpha-lapachone, and the compound II is a novel structure compound which is not reported.
Table 2 compound i 1 H (400 MHz) and 13 c NMR (100 MHz) data [ deuterated reagent CDCl ] 3 ]
| NO | δ H ,mult(J in Hz) | δ C ,DEPT |
| 2 | - | 69.6,C |
| 3 | - | 168.1,C |
| 4 | 6.82,1H,s | 102.8, |
| 4a | - | 131.5,C |
| 5 | - | 180.9, |
| 5a | - | 133.2, |
| 6 | 7.72,1H,dd(7.6,1.6) | 133.9, |
| 7 | 8.16,1H,td(7.6,2.0) | 127.1, |
| 8 | 8.19,1H,td(7.6,2.0) | 127.0,CH |
| 9 | 7.76,1H,dd(7.6,1.6) | 134.1,CH |
| 9a | - | 132.7,CH |
| 10 | - | 173.6, |
| 10a | - | 151.9, |
| 11 | 1.69,3H,s | 28.9, |
| 12 | 1.69,3H,s | 28.9,CH 3 |
2.3 structural identification of Compound I
The compound I is yellow amorphous powder, [ alpha ]] 22 D 96.3 (c 0.5, meOH). The TLC plate showed yellow-brown color at 254nm, yellow fluorescence at 365nm under UV light, and yellow color by heating with 10% sulfuric acid-ethanol TLC.
1 The H NMR spectrum (Table 3 and FIG. 14) shows an active hydrogen proton signal delta H 11.59 (1H, s), three aromatic proton signals [ delta ] H 7.73(1H,t,J＝8.0Hz,H-7),7.51(1H,d,J＝8.0Hz,H-6),7.28(1H,d,J＝8.0Hz,H-8)]A methine proton signal [ delta ] H 4.75(1H,dd,J＝8.0,4.8Hz,H-4)]Two methine proton signals [ delta ] H 1.99(1H,dd,J＝14.4,3.2Hz,H-3a),1.89(1H,dd,J＝14.8,5.2Hz,H-3b)]And two unimodal methylhydrogen signals [ delta ] H 1.45(3H,s,H 3 -11),1.43(3H,s,H 3 -12)]。
According to 13 The presence of two ketocarbonyl carbon signals [ delta ] for this compound was known by C NMR (Table 3 and FIG. 15) C 184.4 (C-10) and 182.5 (C-5)]Five sp 2 Hybrid quaternary carbon signal [ delta ] C 121.3(C-4a),132.0(C-5a),160.3(C-9),114.2(C-9a),153.3(C-10a)]Three sp 2 Hybrid methine signal [ delta ] C 118.0(C-6),137.1(C-7),123.1(C-8)]The compound is presumed to have a pyranoquinone structure. The HMBC spectrum of compound I is shown in fig. 16. The compound I is 4R-4, 9-dihydroxy-alpha-lapachone (4R-4, 9-dihydroxy-alpha-lapachone) which is determined by comparison with literature.
TABLE 3 Compounds I 1 H (400 MHz) and 13 c NMR (100 MHz) data [ deuterated reagent CDCl ] 3 ]
Example 2: antitumor Activity study of pyran naphthoquinone Compounds
Four tumors, namely human colon cancer cell HCT116, brain glioblastoma cell LN229, gastric cancer cell SGC-7901 and liver cancer cell HepG2, were selected to evaluate the antitumor activity of pyranoquinone compounds I-III.
Wherein, cells HCT116, LN229, SGC-7901 and HepG2 used in the test are purchased from Shanghai cell institute of China academy of sciences; 5-Fu and doxorubicin were purchased from Merck sigma.
Cell viability was determined using the CCK-8 method. Taking logarithmic growth phase of cells HCT116 cells, LN229 cells, SGC-7901 cells and HepG2 cells at 5×10 4 cells/mL were inoculated into 96-well plates, 100. Mu.L of cell suspension was added to each well, and the wells were placed at 37℃in 5% CO 2 Incubating in an incubator, and discarding the original culture medium after the culture medium grows for 24 hours in an adherence way.
Adding 100 mu L of culture medium containing medicines with different concentrations of pyran naphthoquinone compounds I-III (6 compound holes are arranged in each concentration), wherein the concentrations of the pyran naphthoquinone compounds I-III are respectively 0, 3.125, 6.25, 12.5, 25, 50 and 100 mu mol/L; adding the blank group into an equal volume of culture medium; the concentration gradient of the positive control group [ 5-fluorouracil (5-Fu) and doxorubicin ] is the same as that of the drug to be tested. After each of the above groups was further cultured for 24 hours, 10. Mu.L of CCK-8 solution was added to each well, and incubation was continued in a cell incubator for 2 hours, and the absorbance OD value of each well was measured at 450nm using an ELISA reader. Cell viability was calculated according to the formula = [ (experimental well-blank well)/(control well-blank well) ]x100%.
Experimental data dose response regression curves were plotted using GraphPad Prism 9.0 treatment with drug concentration on the abscissa and cell viability on the ordinate to calculate IC 50 Values. The experimental results are shown in Table 3.
TABLE 3 IC of pyran naphthoquinones I-III for different tumor cells 50 Value (mu M)
| Compounds of formula (I) | HCT116 | LN229 | SGC-7901 | HepG2 |
| I | 7.89±0.54 | 17.40±2.26 | 7.51±0.26 | 13.60±2.12 |
| II | 12.31±3.14 | 8.97±1.02 | 8.07±0.51 | 12.29±1.73 |
| III | 4.74±0.24 | 13.28±1.36 | 6.09±0.48 | 25.58±5.16 |
| 5-Fu | 51.23±6.14 | — | 48.74±2.21 | 34.12±4.61 |
| Doxorubicin | 0.08±0.03 | 0.32±0.01 | 0.18±0.02 | 0.13±0.06 |
The results in table 3 show that: for colon cancer cell HCT116, compound I IC 50 IC of compound I with a value of 7.89+ -0.54 μM 50 IC with a value of 12.31+ -3.14 μM for Compound I 50 The value was 4.74.+ -. 0.24. Mu.M. IC for brain glioblastoma cell LN229, compound I 50 IC of compound I with a value of 17.40+ -2.26 μM 50 The value is 8.97+ -1.02 μm; IC of compound I 50 The value was 13.28.+ -. 1.36. Mu.M. IC of compound I for gastric cancer cell SGC-7901 50 IC of compound I with a value of 7.51+ -0.26 μM 50 The value is 8.07+ -0.51 μM; IC of compound I 50 The value was 6.09.+ -. 0.48. Mu.M. IC for liver cancer cell HepG2, compound I 50 IC of compound I with a value of 13.60+ -2.12 μM 50 The value is 12.29+/-1.73 mu M; IC of compound I 50 The value was 25.58.+ -. 5.16. Mu.M. IC of pyranoquinone compounds I-III for inhibiting HCT116, LN229, SGC-7901 and HepG2 tumor cells 50 The values are all better than that of the positive control medicine 5-Fu, which shows that the compounds have good anti-tumor activity and can be used for preparing anti-tumor medicines.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (10)
1. The pyran naphthoquinone compound is characterized in that the structural formula of the pyran naphthoquinone compound is selected from formulas I, II or I:
2. a process for the preparation of pyranoquinone compounds according to claim 1, characterized in that it comprises the following steps:
(1) Adding alcohol solvent into stems and leaves of herba Verbenae cut into small sections, heating and reflux-extracting, and concentrating the obtained extractive solution containing the alcohol solvent under reduced pressure to obtain the alcohol solvent extract;
(2) Dissolving the extract of the alcohol solvent in water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and concentrating the obtained ethyl acetate extract layer under reduced pressure to obtain the ethyl acetate extract;
(3) Subjecting the ethyl acetate extract to normal phase silica gel column A chromatography, and gradient eluting with petroleum ether-acetone mixed solution a; subjecting the collected eluate of petroleum ether-acetone mixed solution a containing the pyran naphthoquinone compound to normal phase silica gel column B chromatography, and gradient eluting with petroleum ether-ethyl acetate mixed solution; subjecting the collected eluate of the petroleum ether-ethyl acetate mixed solution containing the pyranoquinone compounds to sephadex column chromatography, performing isocratic elution by using dichloromethane-methanol mixed solution, and collecting the extract after sephadex column purification;
(4) Subjecting the extract purified by the sephadex column to normal phase silica gel column C chromatography, and gradient eluting by petroleum ether-acetone mixed solution b; collecting eluate of petroleum ether-acetone mixed solution b containing the pyran naphthoquinone compound I, subjecting to sephadex column chromatography by using petroleum ether-dichloromethane-methanol mixed solution as mobile phase to obtain compound I shown in formula I, subjecting to sephadex column chromatography by using petroleum ether-dichloromethane-methanol mixed solution as mobile phase to obtain eluate of petroleum ether-acetone mixed solution b containing the pyran naphthoquinone compound I, subjecting to sephadex column chromatography by using petroleum ether-dichloromethane-methanol mixed solution as mobile phase to obtain compound I shown in formula II;
3. the method for producing a pyranoquinone compound according to claim 2, wherein the alcoholic solvent in step (1) comprises any one or a mixture of ethanol and methanol;
the step (1) comprises the following steps: adding alcohol solvent into stems and leaves of the vervain which are cut into small sections, heating and reflux-extracting, wherein the volume of the added alcohol solvent is 5-10 times of the weight of the stems and leaves of the vervain, the reflux-extracting times are 2-5, the temperature of each reflux-extracting is 85-100 ℃, the time of each reflux-extracting is 1-4 hours, and merging the obtained extracting solutions containing the alcohol solvent, and then concentrating under reduced pressure to obtain the alcohol solvent extract.
4. A process for the preparation of pyranoquinone compounds according to claim 2, characterized in that,
in the step (2), the weight-volume ratio of the alcohol solvent extract to the water is 1: (1-3); the weight-volume ratio of the extract of the alcohol solvent to the petroleum ether is 1: (1-3); the weight-volume ratio of the extract of the alcohol solvent to ethyl acetate is 1: (1-3); the weight volume ratio of the extract of the alcohol solvent to the n-butanol is 1: (1-3).
5. The method for producing a pyranoquinone compound according to claim 2, wherein step (3) is specifically: subjecting the ethyl acetate extract to normal phase silica gel column A chromatography, and gradient eluting with petroleum ether-acetone mixed solution a with the volume ratio of 50:1-1:1; subjecting the collected eluent of the petroleum ether-acetone mixed solution a with the volume ratio of 20:1 to normal phase silica gel column B chromatography, and carrying out gradient elution by using petroleum ether-ethyl acetate mixed solution with the volume ratio of 20:1-5:1; eluting the collected eluent of the petroleum ether-ethyl acetate mixed solution with the volume ratio of 10:1 by using sephadex LH-20 column chromatography, eluting by using dichloromethane-methanol mixed solution with the volume ratio of 1:1, and collecting the extract after sephadex LH-20 column purification.
6. The method for producing a pyranoquinone compound according to claim 2, wherein step (4) is specifically: subjecting the extract purified by the Sephadex LH-20 column to normal phase silica gel column C chromatography, carrying out gradient elution by using petroleum ether-acetone mixed solution b with the volume ratio of 30:1-10:1, subjecting the collected eluate of the petroleum ether-acetone mixed solution b with the volume ratio of 30:1 to the Sephadex LH-20 column chromatography, using petroleum ether-dichloromethane-methanol mixed solution with the volume ratio of 2:1 to the Sephadex LH-20 column chromatography as a mobile phase, obtaining a compound I shown in formula I by using the petroleum ether-acetone mixed solution b with the volume ratio of 20:1, subjecting the collected eluate of the petroleum ether-acetone mixed solution b with the volume ratio of 20:1 to the Sephadex LH-20 column chromatography, using petroleum ether-dichloromethane-methanol mixed solution with the volume ratio of 2:1 to the mobile phase, obtaining a compound I shown in formula I by using the petroleum ether-acetone mixed solution b with the volume ratio of 10:1 to the Sephadex LH-20 column chromatography, and obtaining a compound II shown in formula II by using the petroleum ether-methanol mixed solution with the volume ratio of 2:1 to the Sephadex LH-20 column chromatography.
7. A pharmaceutical composition comprising a pyranoquinone compound of claim 1 and a pharmaceutically acceptable carrier and/or adjuvant.
8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition comprises a tablet, capsule, oral liquid, buccal tablet, granule, suspension, suppository, injection, powder for injection, drop pill, sustained release agent, controlled release agent, or targeted formulation.
9. The application of the pyran naphthoquinone compound is characterized in that the pyran naphthoquinone compound is applied to the preparation of antitumor drugs according to claim 1.
10. The use of pyranoquinone compounds according to claim 9, characterized in that the pyranoquinone compounds are used in the preparation of a medicament against colon cancer, a medicament against glioma, a medicament against gastric adenocarcinoma or a medicament against liver cancer.
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