CN112480203B - Withanolide compound and preparation method and application thereof - Google Patents
Withanolide compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses withanolide compounds and a preparation method and application thereof. The withanolide compound has a structure shown as a formula (I) or (Ia), has a good effect of inhibiting human triple negative breast cancer cell proliferation in vitro, has no cytotoxicity to normal human mammary epithelial cells, and has good development and application prospects. The invention also provides a preparation method of the withanolide compound, and the preparation method is simple to operate and low in production cost.
Description
Technical Field
The invention relates to the technical field of natural pharmaceutical chemistry, and particularly relates to withanolide compounds and a preparation method and application thereof.
Background
According to the statistics of 2018 Global cancer reports (Global cancer statistics 2018) issued by the WHO, the highest incidence rate of female cancers is breast cancer and the incidence rate is second to lung cancer in the Global range. According to the latest statistics of the Cone Jie team: breast cancer is the cancer with the highest incidence in Chinese women. Triple-negative breast cancer (TNBC) accounts for 12-17% of breast cancer, and has high malignancy, strong invasiveness, extremely poor prognosis and mortality rate close to morbidity; and the onset of the disease is also in a very significant youthful trend.
At present, no molecular targeted chemotherapeutic drug for TNBC exists. After conventional chemotherapy, TNBC relapse rates are very high. The screening of the anti-TNBC precursor with high efficiency, low toxicity and strong selectivity has important significance in developing novel molecular targeted chemotherapeutic drugs.
The Chinese medicinal dragon bead is dry whole plant of dragon bead (Tubocapsacicum anomalum (Franch. et Sav.) Makino) of Solanaceae (Solanaceae) genus dragon bead, and is mainly distributed in Zhejiang, Jiangxi, Fujian, Taiwan, Guangdong, Guangxi, Guizhou and Yunnan provinces. Has the effects of clearing away heat and toxic material and promoting urination, and is commonly used for treating stranguria with urine, dysentery, furuncle and the like.
Withanolides (withanolides) are a representative active ingredient in solanaceae plants, and longzhu is also rich in withanolides. Withanolides are a class of natural C28 steroids with an ergostane backbone structure having: anti-inflammatory, immunosuppressive, cytotoxic, neurodegenerative disease treating activities, and the like, and has wide application prospects in the field of biological medicine.
The following compounds have been reported in the literature (Withapurvin H, a without of Physicis perviana roots [ J ]. Journal of Chemical Society Chemical Communications, 1989 (10): 628-629.), but their pharmacological activities have not been investigated.
In addition, the antitumor activity of the compounds shown below has been reported in the literature (cytoxic microorganisms from Tubocapssicum anomalum [ J ]. Journal of Natural Products, 2007.).
Although a series of withanolides have been isolated from solanaceae plants so far, no report has been made on the antitumor activity and toxicity to normal cells of withanolides containing a seven-membered S, O heterocyclic ring.
Disclosure of Invention
The invention aims to provide a withanolide compound containing a seven-membered S, O heterocyclic ring, a preparation method of the withanolide compound and application of the withanolide compound in preparation of anti-tumor drugs, particularly anti-triple negative breast cancer drugs.
In order to achieve the purpose, the technical scheme adopted by the invention comprises
A withanolide compound shown as a formula (I), an isomer thereof or a pharmaceutically acceptable salt thereof:
the withanolide compound is shown as a formula (Ia):
the withanolide compound has a unique seven-membered S, O heterocyclic ring, is very rare, has a strong cytotoxic effect on human triple-negative breast cancer cells, and does not have a cytotoxic effect on normal human mammary epithelial cells under the administration dosage. The withanolide compound has multiple potential application prospects in the field of biological medicines, can be a novel single or multiple target inhibitor such as STAT3 and NF-kappa B, Hsp90, and is expected to be used for prevention and treatment of various diseases. The withanolide reported by the invention is used as a precursor, and various derivatives with similar structures can be prepared, and the derivatives possibly have good application prospects.
The invention provides an application of the withanolide compound, the isomer of the withanolide compound or the pharmaceutically acceptable salt of the withanolide compound in preparing a medicine for treating or preventing tumor.
The tumor is triple negative breast cancer.
The withanolide disclosed by the invention has a relatively obvious selective inhibition effect on human triple negative breast cancer cell strains. The MTT method is used for researching the proliferation activity of the novel withanolide compound in the dragon bead for inhibiting the human triple negative breast cancer cell strain. The results show that the novel withanolide has a remarkable inhibiting effect on MDA-MB-231 and MDA-MB-468 two human triple-negative breast cancer cell strains, and half of inhibitory concentration IC is 48 hours after administration50About: 29.88-45.42 μmol/L.
The withanolide compound can be combined with a commercially available or commonly used carrier to prepare a medicament for preventing or/and treating or/and synergistically treating cancer. The medicament can be: fat emulsion, injection oil, powder for injection, tablet, capsule, etc.
The invention also provides a preparation method of the withanolide compound, the isomer of the withanolide compound or the pharmaceutically acceptable salt of the withanolide compound, which comprises the following steps:
(1) extraction: pulverizing whole plant of genus Vermilion of family Solanaceae (Tubocapsis anomalum (Franch. et Sav.) Makino) into coarse powder, soaking in extraction solvent, percolating, and collecting percolate;
(2) and (3) extraction: concentrating the percolate obtained in the step (1), and suspending the obtained extract by using water; extracting the suspension with extractant, mixing extractive solutions, and concentrating to obtain folium Dracaenae Fordii extract;
(3) normal phase column chromatography: performing column chromatography on the normal phase column of the longzhu extract obtained in the step (2), performing gradient elution by using a mixed solvent of petroleum ether and ethyl acetate as an eluent, collecting a fraction rich in the withanolide compound, and concentrating to obtain a crude product;
(4) medium-pressure column chromatography: carrying out medium-pressure column chromatography on the crude product obtained in the step (3), and further collecting a target fraction rich in the withanolide compound;
(5) reversed-phase preparative high-pressure column chromatography: and (4) carrying out reversed-phase preparative high performance liquid column chromatography on the target fraction collected in the step (4) to obtain the withanolide compound.
The withanolide compound has a plurality of chiral carbons, the semisynthesis or the total synthesis of the withanolide needs a day, the total synthesis and the mass production have great technical difficulty, so that the preparation method which is simple, convenient, feasible, scientific and reasonable is provided, and the preparation method has important significance for the deep research and the application of the compound.
In the step (1), more medicinal materials are extracted, including a percolation method, the percolation method is simple to operate, the extraction efficiency is high, the dosage of an extraction solvent is small, normal-temperature extraction is realized, the energy consumption is low, particularly thermosensitive effective components can be protected, and the method can be also suitable for ensuring full extraction when the content of the effective components in the medicinal materials is low. The longzhu has the effects of clearing away heat and toxic materials and promoting urination. It is commonly used for stranguria with urine, dysentery, furuncle, etc., and has a long history of medication.
In the step (1), the extraction solvent is one or a mixture of water and alcohol; the soaking time is 24-60 h, and the extraction solvent is safe to use, low in price and good in extraction efficiency.
Preferably, the extraction solvent is an ethanol water solution containing 70-95% of ethanol by volume, and the dosage ratio of the extraction solvent to the longzhu medicinal material is 15-25L: 1 kg.
More preferably, the extraction solvent is an ethanol water solution containing 90-95% by volume of ethanol. The property of the extraction solvent is very suitable for extracting withanolide components in the dry aerial parts and/or the whole grass of the longzhu, so that the withanolide active components in the medicinal parts can be more efficiently and completely obtained, and a large amount of non-withanolide impurities with larger polarity can be removed.
The steps of soaking and percolating extraction by using an extraction solvent are as follows: wetting the coarse powder, performing column loading, adding 90-95% ethanol, performing cold leaching for 24h, and performing percolation, wherein the dosage ratio of the extraction solvent to the longzhu medicinal material is 20L: 1 kg.
The percolation method has the advantages of high extraction efficiency, small using amount of extraction solvent, low energy consumption for normal-temperature extraction, simple percolation equipment and capability of greatly saving the production cost, and can avoid the degradation of thermosensitive active ingredients.
In the step (2), the extraction solvent is more than two of petroleum ether, diethyl ether, dichloromethane, chloroform, ethyl acetate, n-butanol and water. During extraction, single solvents are sequentially used for extraction according to the polarity from small to large.
More preferably, the extraction solvent is ethyl acetate.
According to the principle of similar phase and solvent extraction, the suspension obtained by adding water into the longzhu extract obtained in the step (1) is separated from small to large according to polarity, and the longzhu ethyl acetate extract is rich in withanolide components through Thin Layer Chromatography (TLC) and liquid-mass spectrometry (LC-MS). Thus, the ethyl acetate extract was concentrated and the ethyl acetate extract was further purified on the normal phase column. The distribution of withanolides is more concentrated by extracting with ethyl acetate, and a large amount of non-withanolides components are removed; subsequent experiments prove that the purification of the withanolide is not influenced by only using ethyl acetate for extraction, so that the step is simplified and the cost is saved by only using a single solvent for extraction.
In step (2), the extractant may be chosen from a wide variety of reagents, but at least includes ethyl acetate. The polarity of the ethyl acetate is larger than that of petroleum ether, diethyl ether, dichloromethane, chloroform and the like in the prior art, so that the withanolide component in the ethanol extract of the dragon ball can be completely extracted; the polarity of the ethyl acetate is smaller than that of n-butyl alcohol and the like in the prior art, so that the withanolide components can be completely extracted, and some glucoside impurities which are easily dissolved in the n-butyl alcohol and have larger polarity can be removed, so that the withanolide active components in the longzhu crude extract have higher content and higher purity; the toxicity of the ethyl acetate is low, and the industrial production also reflects the concept of green and environmental protection.
In the step (3), the filler used in the normal phase column is silica gel or alumina for column chromatography with 100-200 meshes.
The elution mode is gradient elution, and the eluent consists of petroleum ether and ethyl acetate in a volume ratio of 10-0: 1.
The eluate containing the target product is detected by TLC or HPLC or LC-MS. The eluent comprises a mixed solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 10: 1, a mixed solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 5: 1, a mixed solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 3: 1, a mixed solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 1: 2, a mixed solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 2: 5, a mixed solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 1: 4, and a mixed solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 0:1, and gradient elution is sequentially carried out according to the order of polarity from small to large. The novel withanolide is almost completely positioned in eluent after the elution of the eluent with the volume ratio of petroleum ether to ethyl acetate being 1: 2, so that the content of the novel withanolide in the crude extract of longzhu is higher, and the distribution is more concentrated.
In the step (3), the selection of the eluent has strict requirements, including a proper solvent system, a proper solvent ratio and the limitation of the volume of the eluted column. The petroleum ether-ethyl acetate system is more suitable for eluting and purifying the withanolide; the petroleum ether-ethyl acetate in the eluent is set in different proportions, and gradient elution is carried out according to the set proportion in a sequence of polarity from small to large, so that the full separation of the withanolide and other withanolide components is ensured; the limitation of the column volume eluted by each eluent also ensures the sufficient elution of the withanolide, so that the target compound is more intensively distributed.
In the step (4), the filler used for the medium-pressure column chromatography is as follows: octadecyl bonded phase silica gel. The filler has strong selectivity and good separation effect on withanolides compounds.
The medium-pressure column chromatography has the advantages of large sample loading amount, high separation efficiency and the like. The mobile phase of the medium-pressure column chromatography is 40-75% methanol water solution, and gradient elution is performed in sequence from large to small according to polarity during elution.
And (3) dissolving the effective fraction rich in the novel withanolide with methanol, mixing the effective fraction with silica gel, separating by using an octadecyl bonded phase silica gel-filled medium-pressure column, and eluting with 50%, 55%, 60% and 65% methanol water solutions respectively, wherein the mobile phase has strong elution capacity, and the novel withanolide is almost completely concentrated in a part of the fraction eluted by using 65% methanol water.
In the step (5), the reversed-phase preparative high performance liquid column chromatography uses the following fillers: octadecyl bonded phase silica gel; the filler has strong selectivity and good separation effect on withanolides compounds.
The mobile phase of the reversed-phase preparative high-performance liquid column chromatography at least comprises 30-60% acetonitrile water solution.
The preparative high-efficiency liquid phase also has the advantages of large sample loading amount, high separation efficiency and the like.
And (4) carrying out reversed-phase preparative high performance liquid column chromatography on the crude product rich in the novel withanolide obtained in the step (4), eluting with 30-60% methanol water/acetonitrile water, preferably 42% acetonitrile water in isocratic elution, collecting fractions according to chromatographic peaks, and evaporating the obtained fractions to dryness to obtain the novel withanolide.
The novel withanolide has very similar polarity with other withanolides, and the two substances are difficult to separate by common silica gel column chromatography and reverse phase medium pressure column chromatography. The preparative high performance liquid chromatography has the advantages of large sample loading amount, high column efficiency, high separation efficiency and the like. In the experiment, firstly, 53% methanol water is used as a mobile phase for preparation and separation, but two kinds of withanolides with similar polarity cannot be effectively separated, the purity of the obtained novel withanolides is low, the separation efficiency and the yield are also very low, and in addition, the high-concentration methanol is used for enabling alpha-beta unsaturated carbonyl contained in the compound to generate addition reaction with the methanol in the purification process; subsequently, 42% acetonitrile water is used as a mobile phase, and a proper flow rate of the mobile phase is explored, so that two kinds of withanolides compounds with very similar polarity are well separated.
Compared with the prior art, the invention has the beneficial effects that:
the withanolide disclosed by the invention is novel in structure, strong in biological activity and good in anti-tumor activity, has a relatively remarkable inhibitory activity on two humanized triple-negative breast cancer cell lines in vitro, and an MTT (methyl thiazolyl tetrazolium) test shows that the withanolide has a relatively remarkable inhibitory effect on the two humanized triple-negative breast cancer cell lines, and after the withanolide is administered for 48 hours, the half Inhibitory Concentration (IC) of the withanolide on the two humanized triple-negative breast cancer cell lines is half that of the withanolide50) Comprises the following steps: 29.88-45.42 mu mol/L, and has more obvious antitumor activity in vitro; and has no obvious cytotoxic effect on normal human mammary epithelial cells MCF-10A, which shows that the compound has stronger selective inhibitory activity on triple negative breast cancer cells.
The withanolide compound with novel structure is obtained by separation and purification by using a system solvent method, normal phase chromatography, reverse phase chromatography, medium pressure chromatography and high pressure chromatography. The used solvent is cheap and easy to obtain, and has low toxicity. The preparation method of the novel withanolide in the longzhu plants is scientific and reasonable, simple to operate, low in production cost and good in application prospect. The preparation method of the withanolide compound reported by the invention is mature in process, simple in operation, scientific and reasonable, and the novel withanolide with stable physicochemical properties and higher purity can be obtained.
Drawings
FIG. 1 shows the growth inhibition curves of the compound of formula (Ia) on two human triple negative breast cancer cell lines and normal human mammary epithelial cells; wherein, MDA-MB-231 and MDA-MB-468 are two human triple negative breast cancer cells, MCF-10A is normal human mammary epithelial cell.
FIG. 2 shows the NMR spectrum of a compound of formula (Ia) according to the invention.
FIG. 3 is a nuclear magnetic resonance carbon spectrum of a compound having a structure of formula (Ia) according to the present invention.
FIG. 4 is a DEPT spectrum of a compound of the structure of formula (Ia) of the present invention.
FIG. 5 shows a compound of formula (Ia) according to the invention1H-1HCSOY spectra.
FIG. 6 is an HSQC spectrum of a compound of the structure of formula (Ia) of the present invention.
FIG. 7 shows an HMBC spectrum of a compound of formula (Ia) according to the present invention.
FIG. 8 is a NOESY spectrum of a compound of formula (Ia) according to the invention.
FIG. 9 is a high resolution mass spectrum of a compound of formula (Ia) according to the invention.
FIG. 10 is an IR spectrum of a compound of formula (Ia) according to the invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the present invention should not be construed as being limited thereto. Various alternatives and modifications can be devised by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Example 1: preparation of Compounds of formula (Ia)
(1) Extraction: pulverizing herba Draconis Vermilion (dried whole plant of herba Draconis Vermilion) into coarse powder. Taking 4kg of longzhu medicinal material coarse powder, adding a proper amount of 95% ethanol, cold-soaking for 24h, percolating and extracting, and collecting percolate. The total volume of the solvent is 20 times of the medicinal materials (80L of 95% ethanol is used).
(2) And (3) extraction: and (2) concentrating the percolate obtained in the step (1) under reduced pressure until no alcohol smell exists, adding water into the obtained extract for suspension until the obtained extract is about 1.6L, extracting with 1.6L of ethyl acetate for 4 times, and combining the extract liquor to obtain the ethyl acetate extract of the longzhu. The solvent was recovered under reduced pressure, and concentrated to obtain 116.15g of an ethyl acetate extract of longzhu.
(3) Normal phase column chromatography: taking about 110g of the longzhu ethyl acetate extract, carrying out elution separation on a normal phase silica gel column with 100-200 meshes, and carrying out gradient elution by using petroleum ether-ethyl acetate mixed solvents with different proportions, wherein each gradient elution is about 10L (equivalent to 4 column volumes). In the elution solvent system, the volume ratio of petroleum ether to ethyl acetate is respectively 10: 1, 5: 1, 3: 1, 1: 2, 2: 5, 1: 4 and 0:1, and gradient elution is carried out in sequence from small to large in polarity. The resulting fractions were identified by Thin Layer Chromatography (TLC), liquid chromatography-mass spectrometry (LC-MS), and the fraction enriched in the novel withanolide was mainly concentrated in the petroleum ether and ethyl acetate 1: 2 eluent, which was collected and the solvent was recovered by a rotary evaporator under reduced pressure at 45 ℃ to obtain 7.5g of the novel withanolide-enriched extract.
(4) Medium-pressure column chromatography and preparative high-performance liquid-phase column chromatography: collecting about 3g of the above extract, performing medium pressure column chromatography, mixing sample with silica gel, separating with column, gradient eluting with 50%, 55%, 60%, 65% methanol water solution, each gradient eluting with about 600mL, collecting part of fraction corresponding to 65% methanol water eluate, recovering solvent under reduced pressure to obtain about 144.1mg of the extract, dissolving the extract with mobile phase, performing reversed phase preparative high performance liquid column chromatography, isocratically eluting with 42% acetonitrile water solution, collecting the fraction corresponding to corresponding absorption peak, and recovering solvent under reduced pressure to obtain compound (C) of formula (Ia)30H40O7S)7.8mg。
Example 2: structural characterization of the compound of formula (Ia) prepared in example 1:
taking about 7.8mg of the compound, with CDCl3Dissolving, testing it1An H NMR spectrum of the sample was obtained,13c NMR spectrum, 2D NMR spectrum; taking a proper amount of the compound to test the HR-MS spectrum and the IR spectrum of the compound, and testing the specific optical rotation of the compound.
FIG. 2 shows the NMR spectrum of a compound of formula (Ia) according to the invention.
FIG. 3 is a nuclear magnetic resonance carbon spectrum of a compound having a structure of formula (Ia) according to the present invention.
FIG. 4 is a DEPT spectrum of a compound of the structure of formula (Ia) of the present invention.
FIG. 5 shows a compound of formula (Ia) according to the invention1H-1H CSOY spectra.
FIG. 6 is an HSQC spectrum of a compound of the structure of formula (Ia) of the present invention.
FIG. 7 shows an HMBC spectrum of a compound of formula (Ia) according to the present invention.
FIG. 8 is a NOESY spectrum of a compound of formula (Ia) according to the invention.
FIG. 9 is a high resolution mass spectrum of a compound of formula (Ia) according to the invention.
FIG. 10 is an IR spectrum of a compound of formula (Ia) according to the invention.
According to the characterization of each map data, the obtained new compound is shown as a compound (C) shown as a formula 130H40O7S) about 7.8mg (i.e. a compound of the invention having the structure of formula (Ia).
The novel withanolide is a light yellow oily substance.
The molecular formula of the high-resolution mass spectrum is shown as follows: c30H40O7S, theoretical value [ M + Na]+Is m/z: 567.2397, measurement: [ M + Na ]]+Is m/z: 567.2399.
the IR spectrum shows that: 3449cm-1There is a strong and broad absorption peak,indicating that the compound contains a hydroxyl group; 1677cm-1The existence of a strong absorption peak indicates that the compound contains carbonyl, and the peak position of the carbonyl absorption peak is shifted to a low wave number, which indicates that the carbonyl is conjugated with the double bond, namely: the presence of an "alpha, beta unsaturated carbonyl". The compound has 11 unsaturations and DEPT bonding13C NMR spectrum showed that the compound had 5 CH36 CH 210 CH, 9 quaternary carbons.1In the H NMR spectrum,. delta.H 5.99(dd，J＝10.3，2.3Hz，1H，H-2)，δH6.38(dd, J ═ 10.3, 2.4Hz, 1H, H-3) set of signals;13in the C NMR spectrum,. delta.C 201.3(C-1)，δC 127.9(C-2)，δC144.2(C-3) A series of signals, combined with literature, indicate the presence of an "α, β unsaturated carbonyl" group at C-1 and a pair of double bonds conjugated to the carbonyl groups at C-2 and C-3. DeltaH4.79(s, 1H, H-4) and δC96.5 (C-1') HMBC correlation; in HSQC spectrum, δH5.33-5.24(m, 1H, H-1') and δC96.5 (C-1') is correlated; deltaH5.33-5.24(m, 1H, H-1') and δH2.82(dd, J ═ 16.3, 5.5Hz, 1H, H-2 ') and 3.09(dd, J ═ 16.3, 9.0Hz, 1H, H-2') (see above for clarity) were present1H-1H COSY correlation, deltaH2.97-2.91(m, 1H, H-6) and deltaC30.9 (C-2') HMBC correlation exists; binding to HR-MS indicated: there being a special O, S-substituted seven-membered heterocycle, delta, between the C-4 position and the C-6 positionC96.5 (C-1') signal sum deltaHSignals from 5.33 to 5.24(m, 1H, H-1 ') indicating a substitution of the hydroxyl group at the C-1' position, i.e.: c-1' is a hemiacetal carbon.13In the C NMR spectrum,. delta.C 138.2(C-13)，δC134.8(C-14) set of signals, combined1H NMR spectrum and HMBC spectrum, and determining that a pair of double bonds exist between C-13 and C-14 positions.13In the C NMR spectrum,. delta.C167.2(C-26), indicating the presence of an ester carbonyl at the C-26 position; deltaC 151.1(C-24)，δC121.6(C-25) two alkene carbon signals, indicating: a pair of double bonds are present at the C-24 and C-25 positions; bonding of1H NMR，13C NMR spectrum and literature indicate that the compound has a six-membered unsaturated lactone ring. NOESY correlation between H-4 and H-9 in the NOESY spectrum indicates that: h-4 is in the alpha configuration; me-19And H-6, H-8, H-1', indicating that: h-6 and H-8 are beta configuration, OH-1' is alpha configuration; the presence of Me-19 in relation to Me-18 and Me-18 in relation to H-16 indicates that: OH-16 is in the alpha configuration; me-18 correlates with H-20, Me-21 correlates with H-22, indicating that: h-22 is in the alpha configuration. The novel withanolides were finally deduced to be:
(3aS, 3a1R, 5R, 7aS, 8aR, 10R, 11S, 13aS, 13bR) -11- ((S) -1- ((R) -4, 5-dimethyl-6-oxo-3, 6-dimethyl-2H-pyran-2-yl) ethyl) -3a1, 5, 10-trihydroxy-11, 13b-dimethyl-3a, 3a1, 5, 6, 7a, 8, 8a, 9, 10, 11, 12, 13, 13a, 13 b-tetrahydroxy-1H-cyclopenta [7, 8] phenothrato [10, 1-ef ] [1, 4] oxathiophin-1-one. The structural formula is shown as a formula (Ia).
The compound is characterized by a nuclear magnetic resonance hydrogen spectrum, a nuclear magnetic resonance carbon spectrum, a two-dimensional nuclear magnetic resonance spectrum, a high-resolution mass spectrum, an infrared spectrum and the like, and has a chemical structure shown as a formula (Ia). The data of the nuclear magnetic resonance spectrum are shown in Table 1.
Table 1: process for preparing compounds1H NMR spectrum (600MHz),13C NMR spectra (150MHz) (delta, ppm TMS, CDCl3)
Test example 1
The novel withanolide compound is used for in vitro antitumor experiments:
human triple-negative breast cancer cell strains MDA-MB-231, MDA-MB-468 and a normal human mammary epithelial cell strain MFC-10A are purchased from cell banks of Chinese academy of sciences. After the cells in the logarithmic growth phase are digested and counted, the number of cells is 3X 103Inoculating each cell at 100 μ L/well in 96-well cell culture plate, and culturing for 24 hrAfter cell attachment, four wells per well were treated with different concentrations (0, 1, 10, 20, 50 μ M) of the novel withanolide prepared in example 1. After the drug and the tumor cells are incubated for 48h, MTT is added, incubation is continued for 4h at 37 ℃, the culture is stopped, the culture solution is slightly sucked off by a pipette gun, DMSO (150 mu L/hole) is added, after shaking is uniform, the optical density OD value of each hole is measured at 570nm by an enzyme labeling instrument, the OD value of each parallel hole is averaged, the cell activity is calculated, the background OD value is subtracted from the OD value of each test hole, and the inhibition rate of the drug on the tumor cells is calculated.
The cell viability was defined as the average optical density value of the administration well/the average optical density value of the control group × 100%, and the inhibition was defined as (1-the average optical density value of the administration well/the average optical density value of the control group) × 100%. Drawing growth inhibition curves of the compound on two human triple-negative breast cancer cell lines and normal human mammary epithelial cells according to cell viability, wherein the results are shown in figure 1; calculating half Inhibitory Concentration (IC) of drug on tumor cells according to inhibition rate50)，IC50Calculated using GraphPad Prism 5.0 software. The experiment was repeated 3 times, IC50Mean. + -. SD was taken and the results are shown in Table 2. The growth inhibition curves of fig. 1 show that the novel withanolide has selective inhibitory activity against two human triple negative breast cancer cell lines, and is free from cytotoxicity to normal human breast epithelial cells at the administration dose. The results in table 2 show that the novel withanolide has a more significant inhibitory effect on two human triple negative breast cancer cells in vitro.
Table 2: novel withanolide half inhibition concentration IC for human triple negative breast cancer cell line50Value (mean ± SD, n ═ 3)
The novel withanolide compound in the plant of the longzhu genus prepared by the invention can be combined with a carrier which is commercially available or commonly used, and can be used for preparing a medicament for preventing, treating and synergistically treating cancers. The medicine can be in the form of fat emulsion, injection, powder injection, tablet, capsule, etc.
The withanolide compound in the plants of the genus longzhu prepared by the present invention can provide different effects when applied (administered) therapeutically. Generally, withania lactone compounds prepared according to the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium. The formulated drug may be administered by conventional routes including (but not limited to): intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal or topical administration. Carrier media used include (but are not limited to): physiological saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The novel withanolide compounds of the present invention can be prepared in the form of fat emulsion, for example, with an aqueous solution of soybean oil for injection, lecithin, glycerin and other adjuvants, by a conventional method. Medicaments such as tablets and capsules may also be prepared by conventional methods. The amount of the pharmaceutically active ingredient administered is a therapeutically effective amount, for example, from 1. mu.g/kg body weight to 2000mg/kg body weight per day. In addition, the novel withanolide compound prepared by the invention can also be used in a synergistic way with other anti-tumor drugs.
When the novel withanolide compound of the present invention is used as a medicament in plants of the genus lobelia, a therapeutically effective amount of the withanolide compound can be administered to a mammal, wherein the therapeutically effective amount is usually at least 10 μ g/kg body weight, preferably about 10 μ g/kg body weight to about 30mg/kg body weight. The particular dosage will depend upon such factors as the mode of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Although terms are used more often herein, the possibility of using other terms is not excluded. These terms are used merely to more conveniently describe and explain the nature of the present invention; they are to be construed as being without limitation to any additional limitations that may be imposed by the spirit of the present invention.
Claims (9)
1. A withanolide compound represented by formula (Ia) or a pharmaceutically acceptable salt thereof:
2. the withanolide compound or pharmaceutically acceptable salt thereof according to claim 1, for use in the preparation of a medicament for treating or preventing a tumor.
3. The use of claim 2, wherein the tumor is triple negative breast cancer.
4. The process for preparing withanolide compound or pharmaceutically acceptable salt thereof according to claim 1, comprising the steps of:
(1) extraction: pulverizing whole plant of genus Vermilion of family Solanaceae into coarse powder, soaking in extraction solvent, percolating, and collecting percolate;
(2) and (3) extraction: concentrating the percolate obtained in the step (1), and suspending the obtained extract by using water; extracting the suspension with an extraction solvent, mixing the extracts, and concentrating to obtain folium Draconis extract;
(3) normal phase column chromatography: performing column chromatography on the normal phase column of the longzhu extract obtained in the step (2), performing gradient elution by using a mixed solvent of petroleum ether and ethyl acetate as an eluent, collecting a fraction rich in the withanolide compound, and concentrating to obtain a crude product;
(4) medium-pressure column chromatography: carrying out medium-pressure column chromatography on the crude product obtained in the step (3), and further collecting a target fraction rich in the withanolide compound;
(5) reversed-phase preparative high-pressure column chromatography: and (4) carrying out reversed-phase preparative high performance liquid column chromatography on the target fraction collected in the step (4) to obtain the withanolide compound.
5. The preparation method according to claim 4, wherein in the step (1), the extraction solvent is one or a mixture of water and alcohol; the soaking time is 24-60 hours.
6. The preparation method according to claim 5, wherein in the step (1), the extraction solvent is an ethanol aqueous solution containing 70-95% by volume of ethanol, and the ratio of the extraction solvent to the dragon pearl medicinal material is 15-25L/1 kg.
7. The preparation method of withanolide compounds according to claim 4, wherein in the step (3), the filler used in the positive phase column is silica gel or alumina for column chromatography with 100-200 meshes; the eluent consists of petroleum ether and ethyl acetate in a volume ratio of 10-0: 1.
8. The preparation method of withanolide compounds as claimed in claim 4, wherein in step (4), the filler used in the medium-pressure column chromatography is octadecyl bonded silica gel, the mobile phase of the medium-pressure column chromatography is 40-75% methanol aqueous solution, and gradient elution is performed sequentially from large to small according to polarity during elution.
9. The preparation method of withanolide compounds as claimed in claim 4, wherein in step (5), the filler used in the reversed-phase preparative high performance liquid column chromatography is octadecyl bonded silica gel, and the mobile phase of the reversed-phase preparative high performance liquid column chromatography is 30-60% acetonitrile water solution.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012111713A (en) * | 2010-11-25 | 2012-06-14 | National Institute Of Advanced Industrial Science & Technology | Anticancer agent obtained by combining withanolide ingredient |
| CN103396470A (en) * | 2013-08-15 | 2013-11-20 | 中国药科大学 | Withanolides type compounds and antitumor use thereof |
| CN103432144A (en) * | 2013-09-12 | 2013-12-11 | 山东大学 | Application of withanolide compound in preparation of drug or healthcare product for treating or preventing tumors and neurodegenerative diseases |
| CN111377994A (en) * | 2018-12-28 | 2020-07-07 | 南开大学 | Seven withanolides compounds from cape gooseberry and preparation method and application thereof |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012111713A (en) * | 2010-11-25 | 2012-06-14 | National Institute Of Advanced Industrial Science & Technology | Anticancer agent obtained by combining withanolide ingredient |
| CN103396470A (en) * | 2013-08-15 | 2013-11-20 | 中国药科大学 | Withanolides type compounds and antitumor use thereof |
| CN103432144A (en) * | 2013-09-12 | 2013-12-11 | 山东大学 | Application of withanolide compound in preparation of drug or healthcare product for treating or preventing tumors and neurodegenerative diseases |
| CN111377994A (en) * | 2018-12-28 | 2020-07-07 | 南开大学 | Seven withanolides compounds from cape gooseberry and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Withaperuvin H, a Withanolide of Physalis peruviana Roots;Yoshiteru Oshima等;《J. CHEM. SOC., CHEM. COMMUN.》;19891231;628-629 * |
| 醉茄内酯类化合物的研究进展;张光华等;《中草药》;19981231;第29卷(第10期);708-711 * |
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