CN112239439A - Preparation method and application of four kinds of crispy diterpenoids with anticancer activity - Google Patents
Preparation method and application of four kinds of crispy diterpenoids with anticancer activity Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/94—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom spiro-condensed with carbocyclic rings or ring systems, e.g. griseofulvins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- General Chemical & Material Sciences (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
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- Public Health (AREA)
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a preparation method and application of four fury crusted tetter diterpenoids with anticancer activity, wherein the novel compounds 1-4 have the following structures. The four new compounds of the invention have cytotoxic activity on human lung cancer cells A549 and human hepatoma cells HepG2, and can be used for development and application of anti-cancer drugs.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a novel clerodane diterpenoid compound in strong foot bone crispness as well as a preparation method and application thereof.
Background
Because the occurrence and development of tumors cannot be distinguished from the proliferation of cancer cells, most of the current clinical anticancer drugs exert anticancer efficacy by interfering the proliferation of tumor cells and then inducing apoptosis.
Tumor cells can escape from the proliferative senescence mechanism and gain the ability to proliferate indefinitely. Cell proliferation depends on cell division, which requires a complete cycle of cycles including interphase and division phases including G1, S and G2, and the action of drugs on any one of the cell cycles interrupts cell division, breaks its immortal state, and exerts anticancer effects by inducing apoptosis.
The plants serve as an abundant natural product resource library to provide a large number of lead compounds for people, and in order to search for new high-efficiency anti-tumor compounds, a human lung cancer cell line A549 and a human liver cancer cell line HepG2 cell screening model is established. Adding tested drugs with different concentrations in the exponential growth phase of the cells, processing for a certain time, adding MTT to determine the optical density value to judge the cytotoxicity of the compound, thereby screening out the compound with stronger cytotoxic activity and determining the cell strain most sensitive to the compound, further analyzing the induced apoptosis and cycle retardation effects of the compound on the most sensitive cell strain through flow cytometry, and finally discovering possible anticancer lead compounds to provide a basis for further development of the compound.
Disclosure of Invention
The invention aims to provide 4 novel clerodane diterpenoid compounds in the strong foot bone crispness as well as a preparation method and application thereof.
The novel compounds 1-4 provided by the invention belong to clerodane diterpenes, and the structures of the compounds are shown in figure 1.
The present invention also provides a process for the preparation of said novel compounds 1-4, which process comprises the steps of:
(1) strong foot bone crisp (Casearia graveolensDalzell) is extracted with a solvent, and the extract is recovered to obtain a crude extract;
(2) dissolving the crude extract obtained in the step (1) in water, extracting by adopting an organic solvent immiscible with water, and recovering the solvent to obtain an extract;
(3) separating the extract obtained in the step (2) by silica gel column chromatography, and performing gradient elution by using a mixed solvent of petroleum ether/acetone or petroleum ether/ethyl acetate;
(4) separating the fraction obtained in the step (3) by MPLC (medium pressure liquid chromatography, ODS is chromatographic packing), and performing gradient elution by using methanol/water or acetonitrile/water mixed solvent as a mobile phase;
(5) and (3) carrying out HPLC-RI (high performance liquid-differential detection) chromatographic separation on the fractions obtained in the step (4), and eluting by taking methanol/water as a mobile phase or acetonitrile/water as a mobile phase to obtain the compounds 1-4.
The invention provides a preparation method of a novel compound 1-4, wherein the gristle is gristle of the gristle genus of the chafer family (Flavourtiaceae) ((Casearia graveolensDalzell) stem and leaf extract.
The invention provides a preparation method of a new compound 1-4, the extraction method in the step (1) is heating reflux extraction or ultrasonic extraction, the used solvent is at least one of dichloromethane, chloroform, ethyl acetate, methanol and ethanol, and the medicinal materials: the weight-volume ratio of the solvent is 1: 5-1: 15.
The preparation method of the new compound 1-4 provided by the invention is the extraction method in the step (2), the used organic solvent is any one of petroleum ether, dichloromethane, chloroform and ethyl acetate, and the volume ratio of the aqueous solution to the organic solvent is 1: 1-1: 2.
According to the preparation method of the new compound 1-4, in the step (3), an elution solvent is a petroleum ether/acetone or petroleum ether/ethyl acetate mixed solvent, and the ratio of the petroleum ether/acetone to the petroleum ether/ethyl acetate mixed solvent is 100: 2-100: 30.
In the preparation method of the novel compound 1-4 provided by the invention, in the step (4), the ratio of the methanol/water mixed solvent is 6: 4-9: 1, preferably 7: 3-8: 2, or the ratio of the acetonitrile/water mixed solvent is 6: 4-8: 1, preferably 7: 3-8: 2.
According to the preparation method of the novel compound 1-4, the volume ratio of the mobile phase methanol and water mixed solvent or the acetonitrile and water mixed solvent in the step (5) is 6: 4-9: 1, and preferably 7: 3-9: 1.
The four new terpenoids provided by the invention have cytotoxic activity on human lung cancer cells A549 and human hepatoma cells HepG 2. Used for preparing anticancer drugs or lead compounds of the anticancer drugs.
Drawings
FIG. 1 structural formulas of compounds 1-4 of the present invention;
FIG. 2 Process for preparation of Compound 1 of the present invention1H NMR spectrum;
FIG. 3 Compounds of the invention1 of13C NMR spectrum;
FIG. 4 DEPT135 spectrum of inventive compound 1;
FIG. 5 HMQC spectra of Compound 1 of the invention;
FIG. 6 HMBC spectra of Compound 1 of the present invention;
FIG. 7 preparation of Compound 1 of the present invention1H-1H COSY spectrum;
FIG. 8 preparation of Compound 2 of the present invention1H NMR spectrum;
FIG. 9 preparation of Compound 2 of the present invention13C NMR spectrum;
FIG. 10 HMQC spectra of Compound 2 of the invention;
FIG. 11 HMBC spectra of Compound 2 of the present invention;
FIG. 12 preparation of Compound 3 of the present invention1H NMR spectrum;
FIG. 13 preparation of Compound 3 of the present invention13C NMR spectrum;
FIG. 14 HMQC spectra of Compound 3 of the invention;
FIG. 15 HMBC spectra of compound 3 of the present invention;
FIG. 16 preparation of Compound 4 of the present invention1H NMR spectrum;
FIG. 17 preparation of Compound 4 of the present invention13C NMR spectrum;
FIG. 18 HMQC spectra of Compound 4 of the invention;
FIG. 19 HMBC spectra of Compound 4 of the present invention;
FIG. 20 HMBC and HMBC of compounds 1-4 of the present invention1H-1H COSY correlation signal diagram;
FIG. 21 flow cytometry detection of A549 cell apoptosis induced by Compound 1 of the present invention;
figure 22 flow cytometry examined the effect of compound 1 of the present invention on the a549 cell cycle.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto.
Example 1
(1) Extracting 14.0 kg of crispy stem and leaf of radix Seu folium Cayratiae Oligocarpae with methanol for 3 times (3 × 140L), and recovering the extractive solution under reduced pressure to obtain crude extract;
(2) adding water into the methanol extract obtained in the step (1) to prepare suspension, and extracting with ethyl acetate and petroleum ether respectively to obtain ethyl acetate extract and petroleum ether extract;
(3) respectively separating the petroleum ether extract and the ethyl acetate extract by silica gel column chromatography, and sequentially eluting with petroleum ether, acetone 100: 0, 100: 2, 100: 4, 100: 6, 100: 8, 100: 11, 100: 16, 100: 23 and 100: 30;
(3) separating 100: 2-100: 30 parts of the petroleum ether and acetone obtained in the step (2) by using medium-pressure liquid chromatography (MPLC), and performing gradient elution by using methanol/water 6: 4-9: 1 as a mobile phase;
(4) separating 6: 4-9: 1 parts of methanol/water obtained in the step (3) by HPLC-RI separation, and eluting with 60: 40-90: 10 parts of methanol/water as a mobile phase to obtain the new compounds 1 (yield 0.002%), 2 (yield 0.001%), 3 (yield 0.001%), and 4 (yield 0.001%).
The structures of the compounds 1-4 are identified according to the physicochemical properties and spectral data of the compounds 1-4 (the structures of the compounds 1-4 are shown in FIG. 1; and the spectra of the compounds 1-4 are shown in FIGS. 2-19).
The structural identification data for compound 1 is as follows:
colorless oil [ alpha ]α] 
+43.8 (c 0.2, CH2Cl2); ECD (CH3CN) 205 (Δε 20.3), 233 (Δε-5.4) nm; IR (film) v max 3479, 2960, 2928, 1749, 1371, 1227, 1001, 934, 890, 737 cm−1; ESIMS m/z 541 [M + Na]+; HRESIMS m/z 541.2780 [M + Na]+ (calcd for C29H42NaO8, 541.2777). 13C NMR (100 MHz, CDCl3) And1H NMR (400 MHz, CDCl3) The data are shown in tables 1 and 2, and the HMBC related signals of the compounds are shown in FIG. 20. The absolute configuration of the compound is determined by ECD (electronic circular dichroism) calculation by using a TDDFT (time density functional theory) method, and an ECD spectrogram measured by an experiment is calculatedComparing the obtained ECD spectra of the enantiomers, and determining that the absolute configuration of the compound is 2R, 5S, 6S, 8R, 9R, 10S, 18R, 19S。
The structural identification data for compound 2 is as follows:
colorless oil [ alpha ]α]
38.1 (c 0.2，CH2Cl2)；ECD(CH3CN) 200 (Δε8.7), 240 (Δε -0.6) nm; IR (film) v max 3460, 2958, 2927, 1727, 1373, 1195, 1110, 1007, 896, 735 cm−1;HRESIMS m/z 485.2878 [M + Na]+ (calcd for C27H42NaO6, 485.2879); 13C NMR (100 MHz, CDCl3) And1H NMR (400 MHz, CDCl3) The data are shown in tables 1 and 2, and the HMBC related signals of the compounds are shown in FIG. 20. The absolute configuration of the compound is determined by ECD calculation, and the absolute configuration of the compound is 2R, 5S, 6S, 8R, 9R, 10S, 18S, 19R。
The structural identification data for compound 3 is as follows:
colorless oil [ alpha ]α]
+39.6 (c 0.5, CH2Cl2); ECD(CH3CN) 202 (Δε23.5), 233 (Δε-6.0) nm; IR (film) v max 3479, 2960, 2932, 1727, 1371, 1225, 1005, 947, 890, 736 cm−1; ESIMS m/z 513 [M + Na]+; HRESIMS m/z 513.2824 [M + Na]+ (calcd for C28H42NaO7, 513.2828); 13C NMR (100 MHz, CDCl3) And1H NMR (400 MHz, CDCl3) The data are shown in tables 1 and 2, and the HMBC related signals of the compounds are shown in FIG. 20. The absolute configuration of the compound is determined by ECD calculation, and the absolute configuration of the compound is 2R, 5S, 6S, 8R, 9R, 10S, 18S, 19S。
The structural identification data for compound 4 is as follows:
colorless oil [ alpha ]α]
+35.8 (c 0.2, CH2Cl2); ECD(CH3CN) 203 (Δε10.2), 238 (Δε-0.9) nm; IR (film) v max 3469, 2964, 2930, 1728, 1374, 1226, 1006, 947, 892, 736 cm−1; ESIMS m/z 513 [M + Na]+; HRESIMS m/z 513.2828 [M + Na]+ (calcd for C28H42NaO7, 513.2828); 1H NMR (400 MHz, CDCl3) And13C NMR (100 MHz, CDCl3) The data are shown in tables 1 and 2, and the HMBC related signals of the compounds are shown in FIG. 20. The absolute configuration of the compound is determined by ECD calculation, and the absolute configuration of the compound is 2R, 5S, 6S, 8R, 9R, 10S, 18S, 19S。
Of compounds 1 to 4 of Table 113C NMR data
TABLE 2 of Compounds 1 to 41H NMR data
Example 2
(1) Extracting 10.0 kg of crispy stem and leaf of radix Seu folium Cayratiae Oligocarpae with ethanol for 3 times (3 × 30L), and recovering extractive solution under reduced pressure to obtain crude extract;
(2) adding water into the ethanol extract obtained in the step (1) to prepare suspension, and extracting with ethyl acetate to obtain an ethyl acetate extract;
(3) separating by silica gel column chromatography, eluting by petroleum ether, namely acetone 100: 4, 100: 6, 100: 8, 100: 11, 100: 16, 100: 23 and 100: 30 in sequence;
(3) separating the petroleum ether and ethyl acetate flow portions obtained in the step (2) by using medium-pressure liquid chromatography (MPLC) in a ratio of 100: 2-100: 30, and performing gradient elution by using methanol/water in a ratio of 7: 3-9: 1 as a mobile phase;
(4) separating the methanol/water (8: 2) flow obtained in the step (3) by HPLC-RI separation, and eluting by using methanol/water 70: 30-90: 10 as a mobile phase to obtain the new compounds 1 (yield 0.002%), 2 (yield 0.002%), 3 (yield 0.001%), and 4 (yield 0.001%).
The structural identification of compounds 1-4 is shown in example 1.
Example 3
(1) Extracting 10.0 kg of crispy stem and leaf of radix Seu folium Cayratiae Oligocarpae with acetone for 3 times (3 × 30L), and recovering extractive solution under reduced pressure to obtain crude extract;
(2) adding water into the acetone extract obtained in the step (1) to prepare a suspension, and extracting with dichloromethane to obtain a dichloromethane extract;
(3) separating by silica gel column chromatography, eluting by petroleum ether, namely acetone 100: 4, 100: 6, 100: 8, 100: 11, 100: 16, 100: 23 and 100: 30 in sequence;
(4) separating 100: 2-100: 30 parts of the petroleum ether and acetone obtained in the step (3) by using medium-pressure liquid chromatography (MPLC), and performing gradient elution by using 7: 3-9: 1 methanol/water as a mobile phase;
(5) separating methanol/water (8: 2) fractions obtained in the step (4) by HPLC-RI, and eluting with acetonitrile/water 70: 30-90: 10 as a mobile phase to obtain new compounds 1 (yield 0.002%), 2 (yield 0.001%), 3 (yield 0.001%), and 4 (yield 0.001%).
The structural identification of compounds 1-4 is shown in example 1.
Example 4
Testing the cytotoxic activity of the novel compounds 1-4 in the crunchy bones of the intense foot.
(1) Principle of experiment
Tetramethylazoazolium salt (MTT), a yellow dye that can accept hydrogen ions, can be bound into the respiratory chain of mitochondria of living cells and reduced by succinate dehydrogenase and cytochrome to produce blue-purple Formazan crystals. The amount of Formazan crystal generated is only positively correlated with the number of living cells, and related enzymes in dead cells are degraded and MTT cannot be reduced. The formed Formazan crystal can be dissolved by dimethyl sulfoxide (DMSO), the optical density OD value of the solution is measured at 490 nm by a microplate reader, and the OD value is in direct proportion to the amount of the formed Formazan crystal, so that the influence of the compound on cell proliferation is reflected.
(2) Experimental methods
(ii) culture of tumor cells
Preparing cell culture solution containing 10% fetal calf serum and 1% double antibody (penicillin: streptomycin =1: 1) based on 1640 culture medium (human lung cancer cell A549) or DMEM high-sugar culture medium (human liver cancer cell HepG 2), and culturing at 37 deg.C with 5% CO2Culturing in an incubator until the cells are basically paved at the bottom of a culture bottle, and carrying out passage or experimental treatment.
Process for preparation of compounds
The test compound was dissolved in DMSO to prepare a stock solution at a concentration of 30 mM and stored at-20 ℃. It is diluted with a culture medium at the time of use to 60. mu.M, 30. mu.M, 6. mu.M in this order, or further lower concentrations depending on the case.
Testing the cytotoxic activity of the test compound
Taking cells in exponential growth phase, regulating cell density to 1 × 104 Inoculating 100 μ L of the extract into 96-well plate, culturing at 37 deg.C in 5% incubator for 24 hr, adding compounds to be tested with different concentrations, and adding 20/well after 48 hr
MTT solution with the concentration of 5 mg/mL, discarding the upper layer culture solution after 4 h, adding 150 mu L DMSO, shaking for 7 min, measuring the absorbance of each hole at 490 nm by using a microplate reader, and calculating the inhibition rate of each compound on the proliferation of tumor cells according to the absorbance.
④ IC50Is calculated by
Calculating the IC of the compound for inhibiting tumor cell proliferation by nonlinear regression fitting of each dose and inhibition rate parameter50The value is obtained.
(3) The experimental results are as follows: cytotoxic Activity of Compounds 1-4 IC50See table 3.
TABLE 3 cytotoxic Activity of novel Compounds 1-4 IC50Value of
a Etoposide is a positive control drug.
Example 5
Experiment for inducing A549 cell apoptosis by using novel compound 1 in the crunchy foot.
(1) Principle of experiment
Annexin V can be selectively combined with Phosphatidylserine (PS), the phosphatidylserine is mainly distributed on the inner side of a cell membrane, the PS on the inner side of the cell membrane can be everted in the early stage of apoptosis, and the PS can promote blood coagulation and inflammation reaction after being exposed to the surface of a cell. The Annexin V labeled cells with a green fluorescent probe FITC can directly detect the important characteristic of the cell apoptosis of PS eversion by a flow cytometer. Propidium Iodide (abbreviated as PI) can penetrate through a cell membrane structure with necrotic cells or cell membrane integrity loss at the late apoptosis stage, enter into nuclei to be combined with DNA, and is detected by a flow cytometer to show red fluorescence. The apoptosis effect strength of the drug-induced cells can be reflected by comparing the proportion of the apoptotic cells in different treatment groups.
(2) Experimental methods
Taking cells in exponential growth phase, adjusting cell density to 1 × 105one/mL, 500. mu.L of the suspension was inoculated into a 12-well plate and placed at 37 ℃ in 5% CO2After culturing for 24 h, adding compounds to be tested with different concentrations, setting three parallel controls for each group, after 48 h, sucking cell culture solution into a centrifugal tube, washing adherent cells once with PBS, adding 150 mul pancreatin, collecting cells after digestion, transferring the cells into the centrifugal tube, centrifuging for 3 min at 1000 rpm,the supernatant was discarded, the cells were collected, and the cells were gently resuspended in PBS and counted. 5-10 ten thousand of the resuspended cells were centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and 195. mu.L of Annexin V-FITC conjugate was added to resuspend the cells. Add 5. mu.L Annexin V-FITC and 10. mu.L propidium iodide staining solution and mix gently. After incubation for 20 min at room temperature in the dark, the cells were placed in an ice bath and protected from light with aluminum foil, and then the apoptotic status of the cells was detected by flow cytometry.
(3) The experimental results are as follows: the apoptosis-inducing activity of compound 1 is shown in figure 21.
Example 6
Experiment for blocking cell cycle by the novel compound 1 in the crispy crunchy feet with strong flavor.
(1) Principle of experiment
Propidium Iodide (PI) can generate fluorescence after being combined with double-stranded DNA, and the fluorescence intensity is in direct proportion to the content of the double-stranded DNA. After the DNA in the cells is stained by PI, the DNA can be detected by a flow cytometer, and then cell cycle analysis is carried out according to the distribution of the DNA content. After PI staining, if the fluorescence intensity of G0/G1 phase cells is 1, then the theoretical fluorescence intensity of G2/M phase cells containing double DNA is 2, and the fluorescence intensity of S phase cells undergoing DNA replication is between 1 and 2.
(2) Experimental methods
Taking cells in exponential growth phase, adjusting cell density to 1 × 105one/mL, 500. mu.L of the suspension was inoculated into a 12-well plate and placed at 37 ℃ in 5% CO2After culturing for 24 h, adding compounds to be detected with different concentrations for treatment, arranging three parallel controls in each group, after 48 h, sucking cell culture solution into a centrifugal tube, washing adherent cells once by PBS, adding 150 mu L of pancreatin for digestion, collecting cells, transferring the cells into the centrifugal tube, centrifuging at 1000 rpm for 3 min, discarding supernatant, collecting cells, gently suspending the cells by PBS, centrifuging again, discarding supernatant, adding 1 mL of 70% ethanol precooled by ice bath, gently blowing uniformly, and fixing at 4 ℃ for 24 h. Centrifuging at 1000 rpm for 3 min, carefully removing supernatant, adding 1 mL of precooled PBS, centrifuging at 1000 rpm for 3 min, carefully removing supernatant, flicking the bottom of the tube, adding 0.5 mL of prepared PI staining solution, slowly and sufficiently resuspending cells, bathing in a dark place at 37 ℃ for 30 min, and then allowing to attachAnd (4) machine detection.
(3) The experimental results are as follows: the results of the compounds blocking the cell cycle are shown in figure 22.
The results show that the novel compound 1 prepared in the examples has stronger cytotoxicity on A549 cells, and the compound 1 plays a cytotoxic role by inducing apoptosis by blocking the A549 cell cycle in the G0/G1 phase.
Claims (10)
1. Four novel clerodane diterpenoid compounds (1-4) in the strong foot bone crispness are characterized in that: has the following structure.
2. A process for the preparation of a compound according to claim 1, characterized in that: the method comprises the following steps:
(1) strong foot bone crisp (Casearia graveolensDalzell) is extracted by solvent, and the extracting solution is recycled to obtain crude extract;
(2) dissolving the crude extract obtained in the step (1) in water, extracting by adopting an organic solvent immiscible with water, and recovering the solvent under reduced pressure to obtain an extract;
(3) separating the extract obtained in the step (2) by silica gel column chromatography, and performing gradient elution by using a mixed solvent of petroleum ether/acetone or petroleum ether/ethyl acetate;
(4) separating the fraction obtained in the step (3) by MPLC (medium pressure liquid chromatography, ODS is chromatographic packing), and performing gradient elution by using methanol/water or acetonitrile/water mixed solvent as a mobile phase;
(5) and (3) carrying out HPLC-RI (high performance liquid-differential detection) chromatographic separation on the fractions obtained in the step (4), and eluting by taking methanol/water as a mobile phase or acetonitrile/water as a mobile phase to obtain the compounds 1-4.
3. A process for the preparation of a compound according to claim 2, characterized in that: what is needed isThe above-mentioned strong-flavor foot bone crisp is the strong-flavor foot bone crisp of the genus of the family Hydrangeaceae (Flavourtiaceae) (ii)Casearia graveolensDalzell) stem and leaf extract.
4. A process for the preparation of a compound according to claim 2, characterized in that: the extraction method in the step (1) is heating reflux extraction or ultrasonic extraction for 1-3 times, the used solvent is at least one of petroleum ether, cyclohexane, dichloromethane, chloroform, ethyl acetate, acetone, methanol and ethanol, and the medicinal materials are as follows: the weight-volume ratio of the solvent is 1: 5-1: 15.
5. A process for the preparation of a compound according to claim 2, characterized in that: according to the extraction method in the step (2), the volume ratio of the aqueous solution to the organic solvent is 1: 1-1: 2, and the used extraction solvent is one of petroleum ether, dichloromethane, chloroform and ethyl acetate.
6. A process for the preparation of a compound according to claim 2, characterized in that: the ratio of the petroleum ether/acetone or petroleum ether/ethyl acetate mixed solvent used as the elution solvent in the step (3) is 100: 2-100: 30.
7. A process for the preparation of a compound according to claim 2, characterized in that: the ratio of the methanol/water or acetonitrile/water mixed solvent in the step (4) is 3: 2-9: 1.
8. A process for the preparation of a compound according to claim 2, characterized in that: the mobile phase methanol/water or acetonitrile/water mixed solvent in the step (5) is a mobile phase, and the proportion of the mixed solvent in the mobile phase is 3: 2-9: 1 to obtain a compound 1; the proportion of the mixed solvent in the mobile phase is 3: 2-9: 1 to obtain a compound 2; the proportion of the mixed solvent in the mobile phase is 3: 2-9: 1 to obtain a compound 3; the ratio of the mixed solvent in the mobile phase is 3: 2-9: 1 to obtain a compound 4.
9. A pharmaceutical formulation comprising a compound or pharmaceutically acceptable salt as claimed in claim 1 and pharmaceutically acceptable adjuvants, diluents and carriers.
10. The use of a novel compound as claimed in claim 1 for the preparation of a medicament for the treatment of malignant tumours.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6107335A (en) * | 1997-11-18 | 2000-08-22 | Hoechst Marion Roussel Deutschland Gmbh | Esculentin A and esculentin B, a process for their preparation, and their use in the manufacture of medicaments |
| BRPI0805322A2 (en) * | 2008-11-28 | 2010-08-17 | Univ Estadual Paulista Julio D | compounds with cytomodulatory action, formulations containing them and process for their preparation |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6107335A (en) * | 1997-11-18 | 2000-08-22 | Hoechst Marion Roussel Deutschland Gmbh | Esculentin A and esculentin B, a process for their preparation, and their use in the manufacture of medicaments |
| BRPI0805322A2 (en) * | 2008-11-28 | 2010-08-17 | Univ Estadual Paulista Julio D | compounds with cytomodulatory action, formulations containing them and process for their preparation |
Non-Patent Citations (2)
| Title |
|---|
| NICHOLAS H. OBERLIES ET AL.: "Novel Bioactive Clerodane Diterpenoids from the Leaves and Twigs of Casearia sylvestris", JOURNAL OF NATURAL PRODUCTS * |
| THANESUAN NUANYAI ET AL.: "Cytotoxicity of clerodane diterpenoids from fresh ripe fruits of Casearia grewiifolia", SONGKLANAKARIN J. SCI. TECHNOL. * |
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