CN110590737B - Tritium-labeled zaltoprofen with high specific activity and preparation method thereof - Google Patents
Tritium-labeled zaltoprofen with high specific activity and preparation method thereof Download PDFInfo
- Publication number
- CN110590737B CN110590737B CN201910912425.2A CN201910912425A CN110590737B CN 110590737 B CN110590737 B CN 110590737B CN 201910912425 A CN201910912425 A CN 201910912425A CN 110590737 B CN110590737 B CN 110590737B
- Authority
- CN
- China
- Prior art keywords
- zaltoprofen
- tritium
- methyl
- labeled
- propionate
- 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
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
- C07B59/002—Heterocyclic compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D337/00—Heterocyclic compounds containing rings of more than six members having one sulfur atom as the only ring hetero atom
- C07D337/02—Seven-membered rings
- C07D337/06—Seven-membered rings condensed with carbocyclic rings or ring systems
- C07D337/10—Seven-membered rings condensed with carbocyclic rings or ring systems condensed with two six-membered rings
- C07D337/14—[b,f]-condensed
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/05—Isotopically modified compounds, e.g. labelled
Abstract
The invention discloses tritium-labeled zaltoprofen with high specific activity and a preparation method thereof, belonging to the technical field of medicine preparation. The invention uses 2-(3-carboxymethyl-4-phenylthiophenyl) methyl propionate is used as a raw material, and is subjected to bromine substitution, dehydration condensation and hydrolysis to generate 4-bromozaltoprofen, and then is subjected to tritium-halogen exchange with tritium gas under the action of a palladium-carbon catalyst and an alkali acceptor to prepare 4-propanoic acid3H-zaltoprofen. The synthesized product is purified by a preparation liquid phase to obtain tritium-labeled zaltoprofen with high specific activity (29.30ci/g), high radiochemical purity (more than or equal to 98%) and high chemical purity (more than or equal to 99%). 4-prepared by the invention3The H-zaltoprofen provides a material basis for research on absorption, distribution, metabolism and excretion of the zaltoprofen in animals.
Description
Technical Field
The invention belongs to the technical field of medicine preparation, and particularly relates to tritium-labeled zaltoprofen with high specific activity and a preparation method thereof.
Background
Zaltoprofen (Zaltoprofen i), chemically known as 10, 11-dihydro-alpha-methyl-10-oxo-dibenzo [ b, f ] thiopin-2-acetic acid, is a nonsteroidal anti-inflammatory drug developed by the company chemiphar, japan and first marketed in japan on 9/1 of 1993.
Zaltoprofen is a non-steroidal anti-inflammatory drug with the functions of diminishing inflammation, easing pain and relieving fever, and has strong effect on acute inflammation. Zaltoprofen is well absorbed orally, has no drug accumulation after repeated administration, and is widely used for controlling infection or non-infectious inflammation and pain clinically. Research shows that the antipyretic analgesic and anti-inflammatory effects of the zaltoprofen on rats and mice are equal to or stronger than those of other non-steroidal anti-inflammatory drugs, and the zaltoprofen has the prospect of being developed into veterinary antipyretic analgesic drugs. If the new veterinary drug is used for food animals, the absorption, distribution, metabolism and excretion of the new veterinary drug in the animal body and the rule of residue elimination must be clearly researched, and residue markers and residue target tissues are determined, so that a reasonable drug holiday is formulated, and the food safety of animal origin is ensured.
The radioactive isotope tracer technology is used for the research of absorption, distribution, metabolism and excretion of the medicine in vivo, has the advantages of good specificity, high sensitivity, wide application range and the like, and is an irreplaceable tool for researching the new medicine in the in vivo process. At present, the research on the process of the zaltoprofen labeled by radioactive isotopes in animals, particularly food animals, is not seen. If the radio isotope labeled zaltoprofen can be provided, the method has important significance for researching the absorption, distribution, metabolism and excretion of the zaltoprofen in the animal body and the rule of residue elimination, determining a residue marker and a residue target tissue and ensuring the food safety of animal sources.
Disclosure of Invention
The invention aims to provide tritium-labeled zaltoprofen with high specific activity and a preparation method thereof. According to the invention, through the chemical structure analysis of the zaltoprofen, tritium labeling is carried out on a benzene ring, so that the radioactive labeled drug with definite labeling site and high specific activity can be obtained, the radionuclide can be prevented from being lost in the in-vivo metabolic process, and the method is suitable for the tracing research of the drug in the target animal body.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides tritium-labeled zaltoprofen with high specific activity, which has a chemical structural formula shown as a formula I:
the invention also provides a preparation method of the tritium-labeled zaltoprofen with high specific activity, which comprises the following steps: using 2- (3-methoxycarbonyl methyl-4-thiophenyl phenyl) methyl propionate as raw material, making bromine substitution, dehydration condensation and hydrolysis three-step reaction to produce 4-bromine zhuton ibuprofen, then making it and tritium gas produce tritium-halogen exchange under the action of palladium-carbon catalyst and alkali acceptor so as to obtain 4-substituted benzene3H-zaltoprofen.
The preparation method of the tritium-labeled zaltoprofen with high specific activity comprises the following steps:
(1) taking methyl 2- (3-methoxycarbonylmethyl-4-phenylthiophenyl) propionate, adding the methyl 2- (3-methoxycarbonylmethyl-4-phenylthiophenyl) propionate into 100-150 mL of acetonitrile, adding bromine after the methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate is dissolved, reacting, evaporating to remove the acetonitrile, adding 100mL of distilled water, extracting with ethyl acetate, combining ethyl acetate layers, and evaporating to remove a solvent to obtain a yellow oily intermediate methyl 12- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate, wherein the structural formula of the methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate is shown as a formula II:
(2) adding 4g of methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate obtained in the step (1) into 20 g-30 g of polyphosphoric acid, adding 0.5g of anhydrous sodium carbonate, stopping the reaction when a black sticky substance is generated, cooling to room temperature, adding 100mL of water and 200mL of ethyl acetate, stirring for 10 minutes, standing for layering, taking an upper organic phase, evaporating to remove ethyl acetate to obtain a brown solid substance, recrystallizing the brown solid substance with ethyl acetate to obtain a light yellow crystalline intermediate 210, 11-dihydro-a-methyl-10-oxo-dibenzo [ b, f ] thioza-2' -bromo-2-methyl acetate, 10, 11-dihydro-a-methyl-10-oxo-dibenzo [ b ], the structural formula of f ] thioxepin-2' -bromo-2-methyl acetate is shown as formula III:
(3) taking 3g of the 10, 11-dihydro-a-methyl-10-oxo-dibenzo [ b, f ] thiopine-2' -bromo-2-methyl acetate obtained in the step (2), adding 100 mL-150 mL of a mixed solvent consisting of methanol/tetrahydrofuran/water, wherein the volume ratio of methanol/tetrahydrofuran/water is (1.5: 1.5: 1) - (2: 2: 1), then adding 0.61g of sodium hydroxide, evaporating to remove the methanol, slowly adding 2M dilute hydrochloric acid to adjust the pH value to 2, precipitating a white precipitate, filtering to obtain a white solid, recrystallizing the solid by using ethyl acetate to obtain white crystalline powder 4-bromozatoprofen, wherein the structural formula of the 4-bromozatoprofen is shown as a formula IV:
(4) taking 35mg of 4-bromozatron obtained in the step (3), adding 3.68mg of sodium hydroxide, adding 2mL of ethanol to completely dissolve the 4-bromozatron, adding 20mg of 10% Pd/C mixture, wherein Pd in the Pd/C mixture accounts for 10 wt% of the mixture, introducing high-purity tritium gas into a tritiation device, adjusting the reaction pressure to 220-262 mmHg, stirring the mixture to react at the reaction temperature of 30 ℃ for 30-60 minutes, freezing the mixture by using liquid nitrogen to recover the tritium gas, centrifuging the mixture to remove Pd/C, and evaporating the reaction liquid under reduced pressure to remove ethanol to obtain a tritium-labeled zatron crude product;
(5) separating and purifying the tritium-labeled zaltoprofen crude product obtained in the step (4) by using high performance liquid chromatography to obtain 4-ion-doped medicaments with high specific activity, high radiochemical purity and high chemical purity3H-zaltoprofen.
As a further improvement of the invention, the feed-liquid ratio of the methyl 2- (3-methoxycarbonylmethyl-4-thiophenyl phenyl) propionate to bromine in the step (1) is 5 g: 0.74 mL.
As a further improvement of the invention, the reaction in the step (1) is carried out for 6-10 hours at 25-50 ℃.
As a further improvement of the method, after the anhydrous sodium carbonate is added in the step (2), the temperature is raised to 90-98 ℃, and the stirring reaction is carried out for 5-10 hours.
As a further improvement of the invention, after the sodium hydroxide is added in the step (3), the reaction is carried out for 8 to 12 hours at the temperature of 30 to 40 ℃.
As a further improvement of the invention, the high performance liquid chromatography in the step (5) is prepared according to the following technical parameters or steps: the chromatographic column adopts octadecylsilane chemically bonded silica as a filler, the size of the chromatographic column is 250 multiplied by 20mm, the particle size is 5 mu m, and the volume ratio of a mobile phase is 30: 70% acetonitrile and 0.1% phosphoric acid water; the flow rate is 10 mL/min; the ultraviolet detection wavelength is 228 nm; the detection time is 25 min; the column temperature is 30 ℃, the flow parts corresponding to the retention time and the zaltoprofen are collected, the solvent is removed after the combination, and the 4-substituted material with high specific activity, high radiochemical purity and high chemical purity is obtained3H-zaltoprofen.
As a further improvement of the invention, the prepared 4-3And adding 60mL of methanol into H-zaltoprofen for dissolution, and then adding 20mg of non-labeled zaltoprofen for dilution to obtain a tritium-labeled zaltoprofen stock solution. Transferring 10 mu L of tritium labeled zaltoprofen stock solution, diluting the solution to 10mL of methanol, separating the solution through a 0.2 mu m microporous filter membrane by high performance liquid chromatography, collecting fractions after a chromatographic column every 1min, placing the fractions in a scintillation bottle, and adding scintillation liquid to determine the radioactivity value. And (3) drawing a time-radioactivity value curve graph by taking time as an abscissa and a radioactivity value as an ordinate, and calculating the radioactive purity of the tritium-labeled zaltoprofen. The map of the radiochemical purity of tritium-labeled zaltoprofen is shown in figure 4, and the radiochemical purity is more than 98 percent through calculation.
As a further improvement of the invention, 10 mu L of tritium labeled zaltoprofen stock solution is removed, the diluted solution is diluted to 1mL by methanol to obtain a diluted solution a, and the diluted solution is removed from the diluted solution a to obtain 10 mu L of methanol to be diluted to 1mL by the diluted solution b. The radioactivity of the dilution b was determined by 10. mu.L static liquid flash to be 30414 dpm. Accurately weighing 5mg of the zaltoprofen raw material medicine in a 10mL volumetric flask, and performing constant volume with methanol to obtain the standard zaltoprofen stock solution. Dilution c was prepared by diluting 10. mu.L of Zaltoprofen standard stock solution with methanol to 1 mL. Respectively carrying out liquid phase detection on the diluent a and the diluent c, and obtaining that the chemical content of tritium labeled tiotropium contained in 10 mu L of the diluent a is 0.0466 mu g by calculation with an external standard method; the specific activity of the tritiated zaltoprofen is calculated to be 29.30Ci/g according to the ratio of the radioactive amount of the tritiated zaltoprofen to the chemical content of the tritiated zaltoprofen.
In conclusion, the tritium labeled zaltoprofen crude product is separated and purified by the preparative high performance liquid chromatography to obtain the 4-valent oil with high specific activity (29.30ci/g), high radiochemical purity (more than or equal to 98 percent) and high chemical purity (more than or equal to 99 percent)3H-zaltoprofen.
The tritium-halogen exchange method is adopted to carry out tritium labeling on the C-4 position of the benzene ring of the zaltoprofen. Considering the safety of radioactive experiments and simultaneously reducing the waste of radioactive nuclides, the tritium-halogen exchange close to the final product as possible is considered when designing the radioactive labeling synthetic route, and the yield is higher. Therefore, the inventor introduces bromine atoms from the beginning of the synthesis of zaltoprofen, then refers to the synthetic method of zaltoprofen to obtain brominated zaltoprofen (brave, chenve, 2015), and finally performs tritium-bromine exchange to obtain tritium-labeled zaltoprofen.
The invention firstly takes 2- (3-methoxycarbonyl methyl-4-phenylthiophenyl) methyl propionate as raw material to react with bromine at 30 ℃ to obtain the 2- (3-methoxycarbonyl methyl-4- (4-bromophenyl thio) phenyl) methyl propionate, but in the following ring closing reaction, the reaction is difficult to be completely carried out and more impurities are generated due to the influence of bromine atoms on benzene rings, and the ring closing reaction conditions are improved by changing the reaction conditions such as reaction time, reaction temperature, reaction solvent and the like for many times as follows: adding a proper amount of anhydrous sodium carbonate in the process of ring closure of 2- (3-methoxycarbonyl methyl-4- (4-bromophenylthio) phenyl) methyl propionate, and increasing the reaction time to carry out dehydration condensation reaction with PPA for 8 hours at 98 ℃; intermediate 2 was obtained with a reaction yield of 90%.
On the other hand, after obtaining the brominated intermediate 2, if the direct hydrolysis according to the synthetic method of zaltoprofen leads to the replacement of bromine atoms, which results in the test yield being lower than 50%, therefore, the invention firstly separates and purifies the intermediate 2, and then hydrolyzes the intermediate in a mixed solvent of water, tetrahydrofuran and methanol by adopting sodium hydroxide with the mass being 2 times that of the intermediate, and as a result, the yield of the brominated zaltoprofen reaches more than 90%, and the purity is more than 98% after one-time recrystallization.
The invention discloses the following technical effects:
the tritium-labeled zaltoprofen is prepared by adopting radioactive isotope tritium to carry out positioning labeling on the non-steroidal anti-inflammatory drug zaltoprofen, and the radioactive nuclide of the tritium-labeled zaltoprofen obtained by the invention is labeled on a benzene ring, has definite site and good chemical stability and metabolic stability, meets the in vivo metabolic research requirement, and is suitable for the tracing research of the drug in the target animal body.3H is relatively cheap, has short half-life and is relatively easy to radioprotect and decontaminate. The invention thus provides a process for the preparation of a composition using the radioactive isotope tritium (A)3H) The labeled zaltoprofen and the preparation method thereof provide a material basis for research on metabolism and residue elimination of the labeled zaltoprofen in animals.
The invention takes 2- (3-methoxycarbonyl methyl-4-thiophenyl phenyl) methyl propionate as raw material, generates 4-bromine Zaltoprofen through three steps of bromine substitution, dehydration condensation and hydrolysis, and then carries out tritium-halogen exchange with tritium gas under the action of palladium-carbon catalyst and alkali acceptor to prepare 4-3H-zaltoprofen. The tritium-labeled zaltoprofen prepared by the preparation method of the tritium-labeled zaltoprofen has high chemical purity, high radiochemical purity and high specific activity, the specific activity of the synthesized tritium-labeled zaltoprofen can reach 29.30ci/g after liquid phase purification, the radiochemical purity is more than or equal to 98 percent, and the chemical purity is more than or equal to 99 percent. 4-prepared by the invention3The H-zaltoprofen provides a material basis for research on absorption, distribution, metabolism and excretion of the zaltoprofen in animals.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 is a synthetic scheme for tritium labeled zaltoprofen in accordance with the present invention;
FIG. 2 is a high performance liquid chromatogram of a zaltoprofen standard;
FIG. 3 shows the 4-3H-zaltoprofen liquid chromatogram;
FIG. 4 shows the 4-3H-zaltoprofen radiochemical purity map.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.
Example 1
(1) Taking 5g of methyl 2- (3-methoxycarbonylmethyl-4-phenylthiophenyl) propionate, adding 100mL of acetonitrile, adding 0.74mL of bromine after the methyl 2- (3-methoxycarbonylmethyl-4-phenylthiophenyl) propionate is dissolved, reacting for 10 hours at 50 ℃, then evaporating to remove the acetonitrile, adding 100mL of distilled water, extracting with ethyl acetate (100mL multiplied by 3), combining ethyl acetate layers, evaporating to remove a solvent, and obtaining a yellow oily methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate with the yield of 95%;
(2) taking 4g of methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate obtained in the step (1), adding 30g of polyphosphoric acid (PPA), adding 0.5g of anhydrous sodium carbonate, heating to 98 ℃, stirring for reaction for 10 hours, observing generation of black sticky matters, stopping the reaction, cooling to room temperature, adding 100mL of water and 200mL of ethyl acetate, stirring for 10 minutes, standing for layering, taking an upper organic phase, evaporating ethyl acetate to obtain a brown solid matter, recrystallizing the brown solid matter with ethyl acetate to obtain light yellow crystals of 10, 11-dihydro-a-methyl-10-oxo-dibenzo [ b, f ] thiopine-2' -bromo-2-methyl acetate with the yield of 90%;
(3) taking 4g of the 10, 11-dihydro-a-methyl-10-oxo-dibenzo [ b, f ] thiopine-2' -bromo-2-methyl acetate obtained in the step (2), and adding the mixture into a reactor with the volume ratio of 2: 2: 150mL of 1 methanol/tetrahydrofuran/water, then adding 0.82g of sodium hydroxide, reacting at 40 ℃ for 10 hours, evaporating to remove the methanol, slowly adding 2M dilute hydrochloric acid to adjust the pH to 2, separating out a white precipitate, filtering to obtain a white solid, and recrystallizing the solid by using ethyl acetate to obtain white crystalline powder 4-bromozaltoprofen, wherein the yield is 95%;
(4) taking 35mg of 4-bromozatron obtained in the step (3), adding 3.68mg of sodium hydroxide, adding 2mL of ethanol to completely dissolve the 4-bromozatron, adding 35mg of Pd/C mixture, wherein Pd in the Pd/C mixture accounts for 10 wt% of the mixture, introducing high-purity tritium gas into a tritiation device, adjusting the reaction pressure to 262mmHg, reacting at the temperature of 30 ℃, stirring for 30 minutes, freezing with liquid nitrogen to recover the tritium gas, centrifuging to remove Pd/C, and evaporating the reaction liquid under reduced pressure to remove ethanol to obtain a tritium-labeled zatron ibuprofen crude product, wherein the yield is 95%;
(5) separating and purifying the tritium-labeled zaltoprofen crude product obtained in the step (4) by using a preparative high performance liquid chromatography, and separating and preparing according to the following technical parameters or steps: the chromatographic column is a big Liriot sinochrom 250 multiplied by 20mm, 5 mu m, and the mobile phase adopts a volume ratio of 30: 70% acetonitrile and 0.1% phosphoric acid water; the flow rate is 10 mL/min; the ultraviolet detection wavelength is 228 nm; collecting mobile phase components with retention time corresponding to zaltoprofen, combining, removing the solvent to obtain tritium-labeled zaltoprofen, namely 4-3H-zaltoprofen.
4-prepared in this example3And adding 60mL of methanol into H-zaltoprofen for dissolution, and then adding 20mg of non-labeled zaltoprofen for dilution to obtain a tritium-labeled zaltoprofen stock solution. Transferring 10 mu L of tritium labeled zaltoprofen stock solution, diluting the solution to 10mL of methanol, separating the solution through a 0.2 mu m microporous filter membrane by high performance liquid chromatography, collecting fractions after a chromatographic column every 1min, placing the fractions in a scintillation bottle, and adding scintillation liquid to determine the radioactivity value. And (3) drawing a time-radioactivity value curve graph by taking time as an abscissa and a radioactivity value as an ordinate, and calculating the radioactive purity of the tritium-labeled zaltoprofen. The map of the radiochemical purity of tritiated zaltoprofen is shown in fig. 4, and the radiochemical purity is 99.4% by calculation.
10 mu L of tritium labeled zaltoprofen stock solution is removed, the diluted methanol solution is diluted to 1mL to be a diluted solution a, and the diluted methanol solution is 10 mu L of methanol solution to 1mL to be a diluted solution b. The radioactivity of the dilution b was determined by 10. mu.L static liquid flash to be 30414 dpm. Accurately weighing 5mg of the zaltoprofen raw material medicine in a 10mL volumetric flask, and performing constant volume with methanol to obtain the standard zaltoprofen stock solution. Dilution c was prepared by diluting 10. mu.L of Zaltoprofen standard stock solution with methanol to 1 mL. Respectively carrying out liquid phase detection on the diluent a and the diluent c, and obtaining that the chemical content of tritium labeled tiotropium contained in 10 mu L of the diluent a is 0.0466 mu g by calculation with an external standard method; the specific activity of tritiated zaltoprofen in the present example was calculated as 29.30Ci/g, based on the ratio of the amount of radioactivity to the chemical content of tritiated zaltoprofen.
Example 2
(1) Taking 5g of methyl 2- (3-methoxycarbonylmethyl-4-phenylthiophenyl) propionate, adding 120mL of acetonitrile, adding 0.74mL of bromine after the methyl 2- (3-methoxycarbonylmethyl-4-phenylthiophenyl) propionate is dissolved, reacting for 10 hours at 35 ℃, then evaporating to remove the acetonitrile, adding 100mL of distilled water, extracting with ethyl acetate (100mL multiplied by 3), combining ethyl acetate layers, evaporating to remove a solvent, and obtaining a yellow oily methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate with the yield of 94%;
(2) taking 4g of methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate obtained in the step (1), adding 25g of polyphosphoric acid (PPA), adding 0.5g of anhydrous sodium carbonate, heating to 95 ℃, stirring for reaction for 10 hours, observing generation of black sticky matters, stopping the reaction, cooling to room temperature, adding 100mL of water and 200mL of ethyl acetate, stirring for 10 minutes, standing for layering, taking an upper organic phase, evaporating ethyl acetate to obtain a brown solid matter, recrystallizing the brown solid matter with ethyl acetate to obtain pale yellow crystals of 10, 11-dihydro-a-methyl-10-oxo-dibenzo [ b, f ] thiopine-2' -bromo-2-methyl acetate, wherein the yield is 89%;
(3) taking 4g of the 10, 11-dihydro-a-methyl-10-oxo-dibenzo [ b, f ] thiopine-2' -bromo-2-methyl acetate obtained in the step (2), and adding the mixture into a reactor with the volume ratio of 2: 2: 1, 100mL of methanol/tetrahydrofuran/water, adding 0.82g of sodium hydroxide, reacting at 40 ℃ for 10 hours, evaporating to remove the methanol, slowly adding 2M dilute hydrochloric acid to adjust the pH value to 2, separating out a white precipitate, filtering to obtain a white solid, and recrystallizing the solid by using ethyl acetate to obtain white crystalline powder 4-bromozaltoprofen, wherein the yield is 93%;
(4) taking 35mg of 4-bromo-zated ibuprofen obtained in the step (3), adding 3.68mg of sodium hydroxide, adding 2mL of ethanol to completely dissolve the 4-bromo-zated ibuprofen, adding 35mg of Pd/C mixture, wherein Pd in the Pd/C mixture accounts for 10 wt% of the mixture, introducing high-purity tritium gas into a tritiation device, adjusting the reaction pressure to 240mmHg, the reaction temperature to 30 ℃, stirring for reaction for 30 minutes, freezing with liquid nitrogen to recover the tritium gas, centrifuging to remove Pd/C, and evaporating the reaction liquid under reduced pressure to remove ethanol to obtain a tritium-labeled zated ibuprofen crude product, wherein the yield is 91%;
(5) separating and purifying the tritium-labeled zaltoprofen crude product obtained in the step (4) by using a preparative high performance liquid chromatography, and separating and preparing according to the following technical parameters or steps: the chromatographic column is a big Liriot sinochrom 250 multiplied by 20mm, 5 mu m, and the mobile phase adopts a volume ratio of 30: 70% acetonitrile and 0.1% phosphoric acid water; the flow rate is 10 mL/min; the ultraviolet detection wavelength is 228 nm; collecting mobile phase components with retention time corresponding to zaltoprofen, combining, removing the solvent to obtain tritium-labeled zaltoprofen, namely 4-3H-zaltoprofen.
4-prepared in this example3And adding 60mL of methanol into H-zaltoprofen for dissolution, and then adding 20mg of non-labeled zaltoprofen for dilution to obtain a tritium-labeled zaltoprofen stock solution. Transferring 10 mu L of tritium labeled zaltoprofen stock solution, diluting the solution to 10mL of methanol, separating the solution through a 0.2 mu m microporous filter membrane by high performance liquid chromatography, collecting fractions after a chromatographic column every 1min, placing the fractions in a scintillation bottle, and adding scintillation liquid to determine the radioactivity value. And (3) drawing a time-radioactivity value curve graph by taking time as an abscissa and a radioactivity value as an ordinate, and calculating the radioactive purity of the tritium-labeled zaltoprofen. The radiochemical purity was found to be 98.5% by calculation.
mu.L of tritium-labeled zaltoprofen stock solution in the present example was removed, diluted with methanol to 1mL to obtain a diluted solution a, and diluted with 10. mu.L of methanol to 1mL to obtain a diluted solution b. The radioactivity of 10. mu.L of the dilution b was determined by static liquid flash to be 29550 dpm. Accurately weighing 5mg of the zaltoprofen raw material medicine in a 10mL volumetric flask, and performing constant volume with methanol to obtain the standard zaltoprofen stock solution. Dilution c was prepared by diluting 10. mu.L of Zaltoprofen standard stock solution with methanol to 1 mL. Respectively carrying out liquid phase detection on the diluent a and the diluent c, and obtaining that the chemical content of tritium labeled tiotropium contained in 10 mu L of the diluent a is 0.0460 mu g by calculation with an external standard method; the specific activity of the tritiated zaltoprofen of the present example was calculated as 28.90Ci/g, based on the ratio of the amount of radioactivity to the chemical content of the tritiated zaltoprofen.
Example 3
(1) Taking 5g of methyl 2- (3-methoxycarbonylmethyl-4-phenylthiophenyl) propionate, adding 100mL of acetonitrile, adding 0.74mL of bromine after the methyl 2- (3-methoxycarbonylmethyl-4-phenylthiophenyl) propionate is dissolved, reacting for 10 hours at 50 ℃, then evaporating to remove the acetonitrile, adding 100mL of distilled water, extracting with ethyl acetate (100mL multiplied by 3), combining ethyl acetate layers, evaporating to remove a solvent, and obtaining a yellow oily methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate with the yield of 95%;
(2) taking 4g of methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate obtained in the step (1), adding 25g of polyphosphoric acid (PPA), adding 0.5g of anhydrous sodium carbonate, heating to 98 ℃, stirring for reacting for 8 hours, observing the generation of black sticky matters, stopping the reaction, cooling to room temperature, adding 100mL of water and 200mL of ethyl acetate, stirring for 10 minutes, standing for layering, taking an upper organic phase, evaporating ethyl acetate to obtain a brown solid matter, recrystallizing the brown solid matter with ethyl acetate to obtain light yellow crystals of 10, 11-dihydro-a-methyl-10-oxo-dibenzo [ b, f ] thiopine-2' -bromo-2-methyl acetate with the yield of 90%;
(3) taking 4g of the 10, 11-dihydro-a-methyl-10-oxo-dibenzo [ b, f ] thiopine-2' -bromo-2-methyl acetate obtained in the step (2), and adding the mixture into a reactor with the volume ratio of 2: 2: 150mL of 1 methanol/tetrahydrofuran/water, then adding 0.82g of sodium hydroxide, reacting at 38 ℃ for 10 hours, evaporating to remove the methanol, slowly adding 2M dilute hydrochloric acid to adjust the pH to 2, separating out a white precipitate, filtering to obtain a white solid, and recrystallizing the solid by using ethyl acetate to obtain white crystalline powder 4-bromozaltoprofen, wherein the yield is 94%;
(4) taking 35mg of 4-bromozatron obtained in the step (3), adding 3.68mg of sodium hydroxide, adding 2mL of ethanol to completely dissolve the 4-bromozatron, adding 35mg of Pd/C mixture, wherein Pd in the Pd/C mixture accounts for 10 wt% of the mixture, introducing high-purity tritium gas into a tritiation device, adjusting the reaction pressure to 230mmHg, the reaction temperature to 30 ℃, stirring for 40 minutes, freezing with liquid nitrogen to recover the tritium gas, centrifuging to remove Pd/C, and evaporating the reaction liquid under reduced pressure to remove ethanol to obtain a tritium-labeled zatron ibuprofen crude product, wherein the yield is 91%;
(5) separating and purifying the tritium-labeled zaltoprofen crude product obtained in the step (4) by using a preparative high performance liquid chromatography, and separating and preparing according to the following technical parameters or steps: the chromatographic column is a big Liriot sinochrom 250 multiplied by 20mm, 5 mu m, and the mobile phase adopts a volume ratio of 30: 70% acetonitrile and 0.1% phosphoric acid water; the flow rate is 10 mL/min; the ultraviolet detection wavelength is 228 nm; collecting mobile phase components with retention time corresponding to zaltoprofen, combining, removing the solvent to obtain tritium-labeled zaltoprofen, namely 4-3H-zaltoprofen.
4-prepared in this example3And adding 60mL of methanol into H-zaltoprofen for dissolution, and then adding 20mg of non-labeled zaltoprofen for dilution to obtain a tritium-labeled zaltoprofen stock solution. Transferring 10 mu L of tritium labeled zaltoprofen stock solution, diluting the solution to 10mL of methanol, separating the solution through a 0.2 mu m microporous filter membrane by high performance liquid chromatography, collecting fractions after a chromatographic column every 1min, placing the fractions in a scintillation bottle, and adding scintillation liquid to determine the radioactivity value. And (3) drawing a time-radioactivity value curve graph by taking time as an abscissa and a radioactivity value as an ordinate, and calculating the radioactive purity of the tritium-labeled zaltoprofen. The radiochemical purity was found to be 99.2% by calculation.
mu.L of tritium-labeled zaltoprofen stock solution in the present example was removed, diluted with methanol to 1mL to obtain a diluted solution a, and diluted with 10. mu.L of methanol to 1mL to obtain a diluted solution b. The radioactivity of 10. mu.L of the dilution b was determined by static liquid scintillation to be 29737 dpm. Accurately weighing 5mg of the zaltoprofen raw material medicine in a 10mL volumetric flask, and performing constant volume with methanol to obtain the standard zaltoprofen stock solution. Dilution c was prepared by diluting 10. mu.L of Zaltoprofen standard stock solution with methanol to 1 mL. Respectively carrying out liquid phase detection on the diluent a and the diluent c, and calculating by an external standard method to obtain that the chemical content of tritium labeled zaltoprofen contained in 10 mu L of the diluent a is 0.0470 mu g; the specific activity of the tritiated zaltoprofen of the present example was calculated as 28.50Ci/g, based on the ratio of the amount of radioactivity to the chemical content of the tritiated zaltoprofen.
Comparative example 1
(1) Taking 5g of methyl 2- (3-methoxycarbonylmethyl-4-phenylthiophenyl) propionate, adding 120mL of acetonitrile, adding 0.74mL of bromine after the methyl 2- (3-methoxycarbonylmethyl-4-phenylthiophenyl) propionate is dissolved, reacting for 6 hours at 25 ℃, then evaporating to remove the acetonitrile, adding 100mL of distilled water, extracting for 3 times by using 100mL of ethyl acetate, combining ethyl acetate layers, and evaporating to remove a solvent to obtain a yellow oily methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate with the yield of 92%;
(2) taking 4g of methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate obtained in the step (1), adding 20g of polyphosphoric acid (PPA), heating to 90 ℃, stirring for reacting for 1 hour, observing that a black sticky substance is generated, stopping the reaction, cooling to room temperature, adding 100mL of water and 200mL of ethyl acetate, stirring for 10 minutes, standing for layering, taking an upper organic phase, evaporating to remove ethyl acetate to obtain a brown solid substance, recrystallizing the brown solid substance with ethyl acetate to obtain light yellow crystalline 10, 11-dihydro-a-methyl-10-oxo-dibenzo [ b, f ] thioza-2' -bromo-2-methyl acetate with the yield of 45.5%;
(3) taking 3g of the 10, 11-dihydro-a-methyl-10-oxo-dibenzo [ b, f ] thiopine-2' -bromo-2-methyl acetate obtained in the step (2), and adding the mixture into a reactor according to a volume ratio of 2: 1, adding 0.61g of sodium hydroxide into 300mL of methanol/water mixed solution, reacting at 30 ℃ for 8 hours, evaporating to remove methanol, slowly adding 2M dilute hydrochloric acid to adjust the pH value to 2, separating out a white precipitate, filtering to obtain a white solid, and recrystallizing the solid by using ethyl acetate to obtain white crystalline powder 4-bromozaltoprofen, wherein the yield is 90%;
(4) taking 35mg of 4-bromozatroprofen obtained in the step (3), adding 3.68mg of sodium hydroxide, adding 2mL of ethanol to completely dissolve the 4-bromozatroprofen, adding 10mg of Pd/C mixture, wherein the Pd accounts for 10 wt% of the mixture, introducing high-purity hydrogen on a hydrogenation device, adjusting the reaction pressure to 225mmHg, reacting at the temperature of 30 ℃, stirring for 1 hour, freezing by using liquid nitrogen to recover hydrogen, centrifuging to remove Pd/C, and decompressing and distilling the reaction liquid to remove ethanol to obtain the zatroprofen with the yield of 70%.
Comparative example 2
(1) Taking 5g of methyl 2- (3-methoxycarbonylmethyl-4-phenylthiophenyl) propionate, adding 120mL of acetonitrile, adding 0.74mL of bromine after the methyl 2- (3-methoxycarbonylmethyl-4-phenylthiophenyl) propionate is dissolved, reacting for 10 hours at 25 ℃, then evaporating to remove the acetonitrile, adding 100mL of distilled water, extracting with ethyl acetate (100mL multiplied by 3), combining ethyl acetate layers, evaporating to remove a solvent, and obtaining a yellow oily methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate with the yield of 95%;
(2) taking 4g of methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate obtained in the step (1), adding 25g of polyphosphoric acid (PPA), adding 0.5g of anhydrous sodium carbonate, heating to 98 ℃, stirring for reacting for 8 hours, observing the generation of black sticky matters, stopping the reaction, cooling to room temperature, adding 100mL of water and 200mL of ethyl acetate, stirring for 10 minutes, standing for layering, taking an upper organic phase, evaporating ethyl acetate to obtain a brown solid matter, recrystallizing the brown solid matter with ethyl acetate to obtain light yellow crystals of 10, 11-dihydro-a-methyl-10-oxo-dibenzo [ b, f ] thiopine-2' -bromo-2-methyl acetate, wherein the yield is 85%;
(3) taking 3g of the 10, 11-dihydro-a-methyl-10-oxo-dibenzo [ b, f ] thiopine-2' -bromo-2-acetic acid methyl ester obtained in the step (2), and adding the mixture into a reactor with the volume ratio of 1.5: 1.5: 1, 100mL of methanol/tetrahydrofuran/water, adding 0.62g of sodium hydroxide, reacting at 30 ℃ for 12 hours, evaporating to remove the methanol, slowly adding 2M dilute hydrochloric acid to adjust the pH value to 2, separating out a white precipitate, filtering to obtain a white solid, and recrystallizing the solid by using ethyl acetate to obtain white crystalline powder 4-bromozaltoprofen, wherein the yield is 92%;
(4) taking 35mg of 4-bromozatroprofen obtained in the step (3), adding 3.68mg of sodium hydroxide, adding 2mL of ethanol to completely dissolve the 4-bromozatroprofen, adding 35mg of Pd/C mixture, wherein Pd accounts for 10 wt% of the Pd/C mixture, introducing high-purity hydrogen into a hydrogenation device, adjusting the reaction pressure to 262mmHg, reacting at the temperature of 30 ℃, stirring for 30 minutes, freezing by using liquid nitrogen to recover hydrogen, centrifuging to remove Pd/C, and evaporating the ethanol from the reaction liquid under reduced pressure to obtain the zatroprofen with the yield of 88%.
From the above, it can be seen that tritiated zaltoprofen is provided for in the present applicationAfter the crude product is separated and purified by high performance liquid chromatography, 4-ion-exchange resin with high specific activity (29.30ci/g), high radiochemical purity (more than or equal to 98 percent) and high chemical purity (more than or equal to 99 percent) can be obtained3H-zaltoprofen.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (6)
1. A preparation method of tritium-labeled zaltoprofen with high specific activity is characterized by comprising the following steps:
(1) taking methyl 2- (3-methoxycarbonylmethyl-4-phenylthiophenyl) propionate, adding the methyl 2- (3-methoxycarbonylmethyl-4-phenylthiophenyl) propionate into 100-150 mL of acetonitrile, adding bromine after the methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate is dissolved, reacting, evaporating to remove the acetonitrile, adding 100mL of distilled water, extracting with ethyl acetate, combining ethyl acetate layers, and evaporating to remove a solvent to obtain a yellow oily methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate, wherein the structural formula of the methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate is shown as a formula II:
(2) adding 4g of methyl 2- (3-methoxycarbonylmethyl-4- (4-bromophenylthio) phenyl) propionate obtained in the step (1) into 20 g-30 g of polyphosphoric acid, adding 0.5g of anhydrous sodium carbonate, stopping the reaction when a black sticky substance is generated, cooling to room temperature, adding 100mL of water and 200mL of ethyl acetate, stirring for 10 minutes, standing for layering, taking an upper organic phase, evaporating to remove ethyl acetate to obtain a brown solid substance, recrystallizing the brown solid substance with ethyl acetate to obtain light yellow crystals 10, 11-dihydro-a-methyl-10-oxo-dibenzo [ b, f ] thioza-2' -bromo-2-methyl acetate, wherein the 10, 11-dihydro-a-methyl-10-oxo-dibenzo [ b, the structural formula of f ] thioxepin-2' -bromo-2-methyl acetate is shown as formula III:
(3) taking 3g of the 10, 11-dihydro-a-methyl-10-oxo-dibenzo [ b, f ] thiopine-2' -bromo-2-methyl acetate obtained in the step (2), adding 100 mL-150 mL of a mixed solvent consisting of methanol/tetrahydrofuran/water, wherein the volume ratio of methanol/tetrahydrofuran/water is (1.5: 1.5: 1) - (2: 2: 1), then adding 0.61g of sodium hydroxide, evaporating to remove the methanol, slowly adding 2M dilute hydrochloric acid to adjust the pH value to 2, precipitating a white precipitate, filtering to obtain a white solid, recrystallizing the solid by using ethyl acetate to obtain white crystalline powder 4-bromozatoprofen, wherein the structural formula of the 4-bromozatoprofen is shown as a formula IV:
(4) taking 35mg of 4-bromozatron obtained in the step (3), adding 3.68mg of sodium hydroxide, adding 2mL of ethanol to completely dissolve the 4-bromozatron, adding 20mg of Pd/C mixture, wherein Pd in the Pd/C mixture accounts for 10 wt% of the mixture, introducing high-purity tritium gas into a tritiation device, adjusting the reaction pressure to 220-262 mmHg, reacting at 30 ℃, stirring for 30-60 minutes, freezing with liquid nitrogen to recover the tritium gas, centrifuging to remove Pd/C, and evaporating the reaction liquid under reduced pressure to remove ethanol to obtain a tritium-labeled zatron crude product;
(5) separating and purifying the tritium-labeled zaltoprofen crude product obtained in the step (4) by using high performance liquid chromatography to obtain 4-ion-doped medicaments with high specific activity, high radiochemical purity and high chemical purity3H-zaltoprofen;
said 4-3The chemical structural formula of the H-zaltoprofen is shown as a formula I:
2. the preparation method of tritium-labeled zaltoprofen according to claim 1, wherein the feed-liquid ratio of methyl 2- (3-methoxycarbonylmethyl-4-phenylthiophenyl) propionate to bromine in the step (1) is 5 g: 0.74 mL.
3. The preparation method of tritium-labeled zaltoprofen according to claim 1, wherein the reaction in the step (1) is performed at 25 to 50 ℃ for 6 to 10 hours.
4. The preparation method of tritium-labeled zaltoprofen according to claim 1, wherein the anhydrous sodium carbonate is added in the step (2), the temperature is raised to 90-98 ℃, and the reaction is stirred for 5-10 hours.
5. The preparation method of tritium-labeled zaltoprofen according to claim 1, wherein the sodium hydroxide is added in the step (3), and then the reaction is carried out at 30-40 ℃ for 8-12 hours.
6. The method for preparing high specific activity tritium-labeled zaltoprofen according to claim 1, wherein the high performance liquid chromatography in the step (5) is separately prepared according to the following technical parameters or steps: the chromatographic column adopts octadecylsilane chemically bonded silica as a filler, the size of the chromatographic column is 250 multiplied by 20mm, the particle size is 5 mu m, and the volume ratio of a mobile phase is 30: 70% acetonitrile and 0.1% phosphoric acid water; the flow rate is 10 mL/min; the ultraviolet detection wavelength is 228 nm; the detection time is 25 min; the column temperature is 30 ℃, the flow parts corresponding to the retention time and the zaltoprofen are collected, the solvent is removed after the combination, and the 4-substituted material with high specific activity, high radiochemical purity and high chemical purity is obtained3H-zaltoprofen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910912425.2A CN110590737B (en) | 2019-09-25 | 2019-09-25 | Tritium-labeled zaltoprofen with high specific activity and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910912425.2A CN110590737B (en) | 2019-09-25 | 2019-09-25 | Tritium-labeled zaltoprofen with high specific activity and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110590737A CN110590737A (en) | 2019-12-20 |
| CN110590737B true CN110590737B (en) | 2021-04-13 |
Family
ID=68863352
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910912425.2A Active CN110590737B (en) | 2019-09-25 | 2019-09-25 | Tritium-labeled zaltoprofen with high specific activity and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110590737B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111978267A (en) * | 2020-08-21 | 2020-11-24 | 华中农业大学 | Tritium-labeled sulfamethoxazole with high specific activity and preparation method thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101812049A (en) * | 2009-02-19 | 2010-08-25 | 严洁 | Method for preparing zaltoprofen |
| EP2842952A4 (en) * | 2012-04-24 | 2015-09-09 | Nippon Chemiphar Co | Method for producing zaltoprofen and derivative thereof |
| CN103420929B (en) * | 2012-12-17 | 2016-05-11 | 华中农业大学 | The preparation method of tritium or deuterium-labeled mequindox |
| CN103570631B (en) * | 2013-11-21 | 2016-05-25 | 华中农业大学 | The preparation method of tritiated NSC 408735 |
-
2019
- 2019-09-25 CN CN201910912425.2A patent/CN110590737B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN110590737A (en) | 2019-12-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106674123B (en) | A kind of tritiated metronidazole and preparation method thereof | |
| CN110590737B (en) | Tritium-labeled zaltoprofen with high specific activity and preparation method thereof | |
| CN101538249B (en) | Preparation method of tritium or deuterium-labeled cyadox | |
| CN102816085B (en) | Preparation method of iohexol impurity | |
| CN102863517B (en) | Goserelin purification method | |
| Jeffs et al. | Alkaloids of Sceletium species. III. Structures of four new alkaloids from S. strictum | |
| CN101891740A (en) | Method for extracting laburnine from upper part of thermopsis lanceolate | |
| CN103570631B (en) | The preparation method of tritiated NSC 408735 | |
| CN104311436B (en) | A kind of synthetic method of deuterium-labeled Ractopamine | |
| EP4148018A1 (en) | Simple astatine concentration method | |
| CN112409257A (en) | Preparation method of deuterium-labeled higenamine stable isotope compound | |
| US8624039B2 (en) | Method for preparing [18F]fallypride with low base concentration | |
| CN113105432B (en) | Carbon-11 (C)11C) Radiopharmaceutical, preparation method and application thereof | |
| CN104119281A (en) | Preparation method of tritium labeled diaveridine | |
| CN115216652A (en) | Method for separating and purifying lutetium from ytterbium-lutetium mixture | |
| CN106084004B (en) | 18F click-labeled transferrin receptor targeting polypeptide T7 as well as preparation method and application thereof | |
| CN105198734A (en) | Method of preparing gallic acid | |
| CN101613384B (en) | Dihydroxyphenethyl alcohol glycoside compound and preparation method thereof | |
| CN110818636A (en) | Compound or salt thereof, and application and synthesis method thereof | |
| CN104307002A (en) | Preparation method and applications of carrier-free [*I]MIBG suitable for clinical application | |
| CN112250658B (en) | Formylated bicyclol and preparation method thereof | |
| CN103923153B (en) | Oestrogenic hormon vitriol conjugate of a kind of mark and preparation method thereof | |
| CN203319892U (en) | Novel carbon-11 multifunctional automatic synthesis module | |
| CN103110966B (en) | Fluoride-18 marked dopamine D4 receptor developing agent and preparation method thereof | |
| CN113135899B (en) | Benzocycloheptapyridine compounds, process for their preparation and their use |
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 |