CN106632221B - macrolide compound and application thereof in preparation of antitumor drugs - Google Patents
macrolide compound and application thereof in preparation of antitumor drugs Download PDFInfo
- Publication number
- CN106632221B CN106632221B CN201611201008.XA CN201611201008A CN106632221B CN 106632221 B CN106632221 B CN 106632221B CN 201611201008 A CN201611201008 A CN 201611201008A CN 106632221 B CN106632221 B CN 106632221B
- Authority
- CN
- China
- Prior art keywords
- compound
- human
- formula
- cancer
- antitumor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D313/00—Heterocyclic compounds containing rings of more than six members having one oxygen atom as the only ring hetero atom
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/08—Oxygen as only ring hetero atoms containing a hetero ring of at least seven ring members, e.g. zearalenone, macrolide aglycons
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses macrolide compounds and application thereof in preparing antitumor drugs, wherein the macrolide compounds have the structure shown in formula (I), and have the structure shown in formula (I), namely compound 1: β -OH, or compound 2: α -OH, or compound 3: β -OH, or compound 4: α -OH in formula (II), or compound 5 in formula (III), and the macrolide compounds-compound 2, compound 3, compound 4 and compound 5 are novel compounds, have obvious inhibition effect on tumor cells or test bacteria, can be used for preparing antitumor or antibacterial drugs and treating tumor or bacterial infection, thus the invention provides alternative compounds for developing novel antitumor or antibacterial drugs and has important significance for developing Chinese marine drug resources.
Description
The technical field is as follows:
the invention belongs to the field of natural products, and particularly relates to five macrolide compounds and application thereof in preparation of antitumor drugs.
Background art:
the world health organization predicts that by 2020, there will be 2000 ten thousand new cases of malignant tumors, of which the number of deaths reaches 1200 ten thousands and most of which will occur in developing countries, and natural products and derivatives thereof play an important role in the treatment of malignant tumors, and the number of antineoplastic drugs derived from natural products accounts for more than 60% of the number of antineoplastic drugs on the market from 1981 to 2008, and the number of novel natural products and derivatives thereof as new antineoplastic drugs is increasing.
The invention content is as follows:
th object of the present invention is to provide five macrolide compounds having antitumor activity and pharmaceutically acceptable salts thereof.
The structure of the macrolide compound or the medicinal salt thereof is shown as the formulas (I), (II) and (III):
in the formula (I), the compound 1: β -OH, or the compound 2: α -OH, or in the formula (II), the compound 3: β -OH, or the compound 4: α -OH, or in the formula (III), the compound 5.
The inventor finds that the ultraviolet absorption characteristics of secondary metabolites produced by Streptomyces sp.SCSIO ZJ89 are distinct through HPLC-DAD map analysis of fermentation extracts of actinomyces marine, and obtains 5 similar compounds basically caused by ultraviolet absorption through shaking table amplification fermentation and extraction purification.
Through the evaluation of the antitumor activities of the compound 1, the compound 2, the compound 3, the compound 4 and the compound 5, the compound 1-5 is found to have obvious inhibition effects on human lung cancer (A549), human nasopharyngeal carcinoma (CNE2), human cervical carcinoma (HeLa), human liver cancer (HepG2) and human breast cancer (MCF-7).
Therefore, the second object of the present invention is to provide the use of compound 1, compound 2, compound 3, compound 4 or compound 5 for preparing an antitumor drug.
Preferably, the anti-tumor drug is a drug for resisting human lung cancer, human nasopharyngeal cancer, human cervical cancer, human liver cancer or human breast cancer.
The third purpose of the invention is to provide kinds of antitumor drugs, which is characterized by comprising an effective amount of compound 1, compound 2, compound 3, compound 4 or compound 5 shown in formulas (I), (II) and (III) or pharmaceutically acceptable carriers thereof as active ingredients.
Preferably, the anti-tumor drug is a drug for resisting human lung cancer, human nasopharyngeal cancer, human cervical cancer, human liver cancer or human breast cancer.
The fourth purpose of the invention is to provide methods for preparing compound 1, compound 2, compound 3, compound 4 and compound 5, which is characterized in that the compound 1, compound 2, compound 3, compound 4 and compound 5 are prepared and separated from fermentation products of Streptomyces sp.SCSIO ZJ 89.
Preferably, the specific steps are as follows:
A. preparing a fermentation product of Streptomyces sp.SCSIO ZJ 89;
B. carrying out centrifugal separation on a fermentation product of Streptomyces sp.SCSIO ZJ89 to obtain a fermentation liquid and mycelia, extracting the fermentation liquid with butanone, and concentrating butanone extract to obtain a fermentation liquid extract; extracting mycelia with acetone, concentrating the acetone extractive solution to obtain thallus extract, separating the extract with silica gel, and gradient eluting with chloroform/methanol from 100/0,98/2,96/4,94/6,92/8,90/10,80/20,50/50,0/100, and v/v to obtain 9 components A1-A9;
eluting with chloroform/methanol 94/6 to obtain fraction A4, eluting with chloroform/methanol 100/0,98/2,96/4,94/6,92/8,90/10, and v/v gradient elution to obtain 6 fractions B1-B6, eluting with chloroform/methanol 96/4 to obtain fraction B3, eluting with chloroform/methanol 100/0,98/2,96/4,94/6,92/8, and v/v to obtain 5 fractions C1-C5;
chloroform/methanol 92/8 to obtain fraction A5, chloroform/methanol 94/6 to obtain fraction B4, mixing, gradient eluting with petroleum ether/ethyl acetate P/E, 80/20,70/30,60/40,55/45,50/50,40/60,30/70,20/80, v/v to obtain fraction D1-D8, mixing chloroform/methanol 94/6 to obtain fraction C4, eluting with petroleum ether/ethyl acetate 40/60 to obtain fraction D6, eluting with petroleum ether/ethyl acetate 30/70 to obtain fraction D7, purifying with high performance liquid phase to obtain compound 5, mixing chloroform/methanol 98/2 to obtain fraction C2, eluting with chloroform/methanol 96/4 to obtain fraction C3, purifying with high performance liquid phase to obtain compound 1, compound 4, compound 3 and compound 2.
The above-mentionedThe fermentation product for preparing the marine actinomycete Streptomyces sp.SCSIO ZJ89 is obtained by culturing the marine actinomycete Streptomyces sp.SCSIO ZJ89 serving as a fermentation strain in an amplified fermentation culture medium; the amplification fermentation culture medium comprises 0.5 percent of soluble starch, 0.5 percent of soybean meal, 2 percent of glucose, 0.2 percent of yeast extract, 0.2 percent of peptone and K according to the total mass fraction of 100 percent2HPO40.05％，MgSO4·7H2O 0.05％，NaCl 0.4％，CaCO30.2 percent, 3 percent of crude sea salt, 7.2 to 7.4 percent of pH value and the balance of water.
The macrolide compound-compound 1, compound 2, compound 3, compound 4 and compound 5 have obvious inhibition effect on tumor cells, can be used for preparing antitumor drugs and treating tumors, thus providing alternative compounds for developing new antitumor drugs and having important significance for developing Chinese marine drug resources.
The marine actinomycete Streptomyces sp.SCSIO ZJ89 is preserved in China general microbiological culture Collection center (CGMCC) in 2016, 11 and 22, and has the address: the microbial research institute of department of China, Xilu No.1 Hospital, Beijing, Chaoyang, has a deposit number of: CGMCC NO. 13322.
Description of the drawings:
FIG. 1 is a schematic representation of compounds 2 to 51H-1Information related to H COSY and partial HMBC, wherein 2 represents compound 2, 3 represents compound 3, 4 represents compound 4, and 5 represents compound 5.
The specific implementation mode is as follows:
the following examples are illustrative of the present invention by way of further and are not intended to be limiting.
Example 1:
preparation and structural characterization of Compound 1, Compound 2, Compound 3, Compound 4 or Compound 5, as shown in formulas (I), (II) and (III)
preparation of Compound 1, Compound 2, Compound 3, Compound 4 or Compound 5 represented by formulas (I), (II) and (III)
1. Seed culture:
(1) the seed culture medium formula comprises: comprises 100% by weight of soluble starch 0.5%, soybean powder 0.5%, glucose 2%, yeast extract 0.2%, peptone 0.2%, and K2HPO40.05％，MgSO4·7H2O 0.05％，NaCl 0.4％，CaCO30.2 percent of crude sea salt, 3 percent of crude sea salt, 7.2 to 7.4 percent of pH and the balance of water, the substances are mixed with according to the formula, the prepared substances are subpackaged in 250mL conical bottles, 50mL of the prepared substances are contained in each bottle, and the prepared substances are sterilized for 20 minutes at 121 ℃ to be used as a seed culture medium for later use.
(2) Culturing seeds: inoculating mycelium or spore of Streptomyces sp.SCSIO ZJ89 into the seed culture medium, and shake culturing at 28 deg.C at 200rpm for 36 hr to obtain seed culture solution.
2. Amplification fermentation culture:
(1) the formula of the amplified fermentation medium is as follows: comprises 0.5 percent of soluble starch, 0.5 percent of soybean meal, 2 percent of glucose, 0.2 percent of yeast extract, 0.2 percent of peptone and K according to the total mass fraction of 100 percent2HPO40.05％，MgSO4·7H2O 0.05％，NaCl0.4％，CaCO30.2 percent of crude sea salt, 3 percent of pH 7.2-7.4 and the balance of water, mixing the materials in according to the formula, preparing the materials in batches, then counting to 21.8L of amplified fermentation culture medium, preparing the prepared materials, then subpackaging the prepared materials in conical flasks of 1000mL, packaging 200mL of each flask, and sterilizing at 121 ℃ for 20 minutes to obtain the standby amplified fermentation culture medium.
(2) Fermentation culture:
respectively inoculating the cultured seed culture solution into an amplification fermentation culture medium under aseptic operation, inoculating bottles of seed culture solution (50mL) into each 1000mL conical flask (containing 200mL of amplification fermentation culture medium), inoculating each 50mL seed culture solution into each 1000mL conical flask filled with 200mL of amplification fermentation culture medium, and performing shake culture at 28 ℃ for 7 days at the rotation speed of 200rpm to obtain a fermentation product of Streptomyces sp.
3. Extraction and separation:
centrifuging the fermentation product of Streptomyces sp.SCSIO ZJ89 at 3600rpm to obtain supernatant fermentation liquid and precipitate mycelium. Extracting supernatant fermentation liquor with butanone for 3 times in equal volume, and concentrating butanone extract at a temperature lower than 40 deg.C under reduced pressure to obtain fermentation liquor extract; repeatedly extracting the precipitated mycelium with 3L acetone for three times, and concentrating the acetone extractive solution at a temperature below 40 deg.C under reduced pressure to obtain thallus extract; after HPLC-DAD detection, about 31.22g of extract is obtained by combining the fermentation liquid extract and the thallus extract. The extract is separated by 100-mesh silica gel with 200 meshes, and 9 components (A1-A9) are obtained by adopting chloroform/methanol (100/0,98/2,96/4,94/6,92/8,90/10,80/20,50/50,0/100 and v/v) gradient elution after sample mixing and column filling by a dry method. Fraction A4 (fraction eluted with chloroform/methanol 94/6) was eluted with chloroform/methanol (100/0,98/2,96/4,94/6,92/8,90/10, v/v) in a gradient order to give 6 fractions (B1-B6). Fraction B3 (fraction eluted with chloroform/methanol 96/4) was purified with chloroform/methanol (100/0,98/2,96/4,94/6,92/8, v/v) to give 5 fractions (C1-C5). Fractions A5 (chloroform/methanol 92/8 fractions) and B4 (chloroform/methanol 94/6 fractions) were combined and then eluted with a petroleum ether/ethyl acetate (P/E, 80/20,70/30,60/40,55/45,50/50,40/60,30/70,20/80, v/v) gradient to give fractions D1-D8. The fractions C4 (fraction eluted with chloroform/methanol 94/6), D6 (fraction eluted with petroleum ether/ethyl acetate 40/60) and D7 (fraction eluted with petroleum ether/ethyl acetate 30/70) were combined, and the mixture was subjected to detection at A wavelength of 254nm, A flow rate of 2.5ml/min, and semi-preparative high pressure liquid phase separation (SP-HPLC) using reverse phase column YMC-Pack ODS-A column (250X 10mm,5 μm) at 42/58 (water/acetonitrile, v/v), to give compound 5(39.9mg) at A retention time of 26min, and similarly, the fractions C2 (fraction eluted with chloroform/methanol 98/2) -C3 (fraction eluted with chloroform/methanol 96/4) were combined, A flow rate of 2.5ml/min, A flow rate of 40/60 (water/acetonitrile, v/v) elution was performed by semi-preparative high pressure liquid chromatography (SP-HPLC) using reverse phase column YMC-Pack ODS-A column (250X 10mm,5 μm), and Compound 1(139.1mg), Compound 4(3.7mg), Compound 3(3.6mg) and Compound 2(14.3mg) were obtained at 17.8min, 20.4min, 22.7min and 24.7 min.
II, structural identification of Compound 1, Compound 2, Compound 3, Compound 4 and Compound 5
The following physicochemical property data were obtained by performing structural analysis tests on compound 1, compound 2, compound 3, compound 4 and compound 5, and the nuclear magnetic data thereof are shown in tables 1 and 2:
compound 1 white amorphous powder [ α ]]25 D-18.0(c12.1,ethanol)；(+)-HR-ESI-MS m/z490.3171[M+H]+(calcd for C28H44NO6,490.3163)and 512.2986[M+Na]+(calcd forC28H43NNaO6,512.2983)。
Compound 2 white amorphous powder [ α ]]25 D+14.1(c 3.0,ethanol)；(+)-HR-ESI-MS m/z490.3161[M+H]+(calcd for C28H44NO6,490.3169)and 512.2987[M+Na]+(calcd forC28H43NNaO6,512.2988)
Compound 3 white amorphous powder [ α ]]25 D+41.1(c 2.6,ethanol)；(+)-HR-ESI-MS m/z490.3162[M+H]+(calcd for C28H44NO6,490.3163)and 512.2980[M+Na]+(calcd forC28H43NNaO6,512.2983)
Compound 4 white amorphous powder [ α ]]25 D-35.8(c 3.1,ethanol)；(+)-HR-ESI-MS m/z490.3161[M+H]+(calcd for C28H44NO6,490.3163)and 512.2986[M+Na]+(calcd forC28H43NNaO6,512.2983)
Compound 5 white amorphous powder [ α ]]25 D-4.7(c 8.8,ethanol)；(+)-HR-ESI-MS m/z536.3566[M+H]+(calcd for C30H50NO7,536.3582)and 558.3389[M+Na]+(calcd forC30H49NNaO7,558.3401)
Table 1: compounds 1-3 in CD3OD.1H (500MHz) and13c NMR (125HMz) data
*Resonances overlapped
Table 2: compounds 4-5 in CD3OD.1H (500MHz) and13c NMR (125HMz) data
The 2D NMR-related information for compound 2, compound 3, compound 4 and compound 5 is shown in figure 1:
according to the analysis of the physicochemical data, the specific structures of the compound 1, the compound 2, the compound 3, the compound 4 and the compound 5 are shown as the formulas (I) and (II).
In the formula (I), the compound 1: β -OH, or the compound 2: α -OH, or in the formula (II), the compound 3: β -OH, or the compound 4: α -OH, or in the formula (III), the compound 5.
Example 2:
antitumor cell experiments with macrolide compound-compound 1, compound 2, compound 3, compound 4 and compound 5 of example 1.
Adopting an international universal tumor cell strain, namely: human lung cancer cell strain (A549), human nasopharyngeal carcinoma cell strain (CNE2), human cervical cancer cell strain (HeLa), human hepatoma cell strain (HepG2), human breast cancer cell strain (MCF-7), normal human liver cell (L02) and normal human umbilical vein endothelial cell (Huvect-12). The test method is a CCK-8 method:
1) cell culture: tumor cells and normal cells in logarithmic growth phase were seeded at 100. mu.L/well in 96-well plates and grown adherent for 24 hours, depending on the cell growth rate.
2) Add sample (drug product): mu.L of medium dilution solutions of Compound 1, Compound 2, Compound 3, Compound 4 and Compound 5 at different concentrations were added to each well, and medium control and cell-free wells at corresponding concentrations were set.
3) Adding medicine to culture cells: tumor cells 5% CO at 37 ℃2Incubated under conditions for 48 hours.
4) And (3) activity test: after the cells are cultured, 10 mu L of CCK-8 solution is added into each hole, the cells are continuously cultured for 2 hours, and the OD value is measured by a microplate reader at the wavelength of 450 nm. Doxorubicin and cisplatin were used as positive controls.
5) And (6) reporting the activity. Five parallel experiments were performed for each cell line, and the results are shown in table 3:
table 3: inhibition of tumor cell lines and Normal cells by Compounds 1, 2, 3, 4 and 5 (IC)50,μM)
aPositive control
The experimental results show that the compound 1, the compound 2, the compound 3 and the compound 4 have obvious inhibition effect on tumor cells, and particularly the compound 4 has obvious stronger inhibition activity on the tumor cells, obviously weaker inhibition activity on normal cells and smaller toxicity on the normal cells compared with the tumor cells.
In conclusion, the invention provides a new lead compound for developing new antitumor drugs, and has important significance for developing marine drug resources in China.
Claims (5)
1. Macrolide compounds or medicinal salts thereof have a structure shown in a formula (II):
in the formula (II), the compound 3: β -OH or the compound 4: α -OH.
2. The use of compound 3 or compound 4, or a pharmaceutically acceptable salt thereof, as claimed in claim 1, in the manufacture of an anti-neoplastic agent.
3. The use of claim 2, wherein the anti-tumor drug is a drug against human lung cancer, human nasopharyngeal carcinoma, human cervical carcinoma, human liver cancer or human breast cancer.
antitumor agents comprising an effective amount of Compound 3 or Compound 4 of claim 1, or a pharmaceutically acceptable salt thereof, as an active ingredient, and a pharmaceutically acceptable carrier.
5. The antitumor drug as claimed in claim 4, wherein the antitumor drug is a drug for human lung cancer, human nasopharyngeal cancer, human cervical cancer, human liver cancer or human breast cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611201008.XA CN106632221B (en) | 2016-12-22 | 2016-12-22 | macrolide compound and application thereof in preparation of antitumor drugs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611201008.XA CN106632221B (en) | 2016-12-22 | 2016-12-22 | macrolide compound and application thereof in preparation of antitumor drugs |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106632221A CN106632221A (en) | 2017-05-10 |
| CN106632221B true CN106632221B (en) | 2020-01-31 |
Family
ID=58826453
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611201008.XA Active CN106632221B (en) | 2016-12-22 | 2016-12-22 | macrolide compound and application thereof in preparation of antitumor drugs |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106632221B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109651329B (en) * | 2019-01-15 | 2022-11-25 | 浙江海正药业股份有限公司 | Tetradecyl macrolide compound, preparation method and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10147594A (en) * | 1996-11-19 | 1998-06-02 | Banyu Pharmaceut Co Ltd | Antitumor substances be-43547s |
| JPH1135578A (en) * | 1997-07-15 | 1999-02-09 | Noyaku Bio Technol Kaihatsu Gijutsu Kenkyu Kumiai | Compound n-0285, its production and use |
-
2016
- 2016-12-22 CN CN201611201008.XA patent/CN106632221B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN106632221A (en) | 2017-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106834160B (en) | Streptomyces erythropolis for producing keratin compound | |
| CN103910708A (en) | Marine fungus-derived Azaphilones compound, and preparation method and application thereof | |
| CN103910746B (en) | The Berkeleyones compound in one class marine fungi source and its preparation method and application | |
| CN108358946A (en) | A kind of anthraquinone analog compound and preparation method thereof and the application in preparing treating cancer drug | |
| CN106432168B (en) | The anti-vibrios reactive compound and preparation method thereof in mangrove cusp sea lotus endogenetic fungus source | |
| CN102516368A (en) | Cyclopeptide-7 compounds and application thereof in preparation of anti-tumor medicines | |
| CN108315264A (en) | A kind of polyketide in sea paint endogenetic fungus source and its application in preparing anti-inflammatory drug | |
| CN106188084B (en) | The naphthalene Spiroketals class compound in marine fungi source and its preparation method and application | |
| CN106967024A (en) | A kind of α pyrone derivatives and its preparation method and application | |
| CN101735236A (en) | Dimer citrinin compounds and preparation and use thereof | |
| CN106632221B (en) | macrolide compound and application thereof in preparation of antitumor drugs | |
| CN103911407B (en) | The preparation method of the Azaphilone class dimer compound in a kind of marine fungi source and application | |
| CN107973769A (en) | A kind of benzodihydropyrone class compound and its preparation method and application | |
| CN108570025A (en) | The oxygen-containing pentacyclic pimarane diterpene-kind compound of one kind, preparation method and applications | |
| CN105017203A (en) | Azaphilones derivative compound from marine fungi as well as preparation method and application of azaphilones derivate compound | |
| CN110330544B (en) | 4,4, 1-bicyclic steroid compound and preparation method and application thereof | |
| CN103910701A (en) | Marine fungus-derived naphthoquinone compound, and preparation method and application thereof | |
| CN102492006B (en) | Canthaxanthin compound and application of compound to preparation of antitumor drugs | |
| CN103396389B (en) | Naphthoquinone sesquiterpenes compound and application thereof in preparation of anti-tumour or antibacterial medicine | |
| CN111689895B (en) | Two-branch chain isomerization piericins compound and application thereof in preparation of anti-renal cancer drugs | |
| CN103360329B (en) | One class compound phenazine and preparing the application in antitumor drug | |
| CN109384823B (en) | Two piericins glucoside and application thereof in anti-renal cancer drugs | |
| CN102304555B (en) | Preparation method and application of antibiotics lobophorin A and lobophorin B | |
| CN107226800A (en) | A kind of xanthone classes compound and its preparation method of monocrystalline and the application as anti-Mycobacterium marinum medicine | |
| CN110669103A (en) | Double demethyl actinomycin derivatives and application thereof in preparation of drug-resistant bacteria infection resisting drugs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |