CN114716391A - Methazolamide impurity and preparation method and application thereof - Google Patents
Methazolamide impurity and preparation method and application thereof Download PDFInfo
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- CN114716391A CN114716391A CN202210450074.XA CN202210450074A CN114716391A CN 114716391 A CN114716391 A CN 114716391A CN 202210450074 A CN202210450074 A CN 202210450074A CN 114716391 A CN114716391 A CN 114716391A
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- methazolamide
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- 239000012535 impurity Substances 0.000 title claims abstract description 119
- FLOSMHQXBMRNHR-DAXSKMNVSA-N methazolamide Chemical compound CC(=O)\N=C1/SC(S(N)(=O)=O)=NN1C FLOSMHQXBMRNHR-DAXSKMNVSA-N 0.000 title claims abstract description 46
- 229960004083 methazolamide Drugs 0.000 title claims abstract description 46
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 150000001875 compounds Chemical class 0.000 claims abstract description 39
- 238000006243 chemical reaction Methods 0.000 claims abstract description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 39
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 27
- 239000002904 solvent Substances 0.000 claims description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 14
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 12
- 239000000047 product Substances 0.000 claims description 12
- 238000000926 separation method Methods 0.000 claims description 11
- WGQKYBSKWIADBV-UHFFFAOYSA-N benzylamine Chemical compound NCC1=CC=CC=C1 WGQKYBSKWIADBV-UHFFFAOYSA-N 0.000 claims description 10
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- YBBRCQOCSYXUOC-UHFFFAOYSA-N sulfuryl dichloride Chemical compound ClS(Cl)(=O)=O YBBRCQOCSYXUOC-UHFFFAOYSA-N 0.000 claims description 9
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 8
- 239000000523 sample Substances 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- 239000012295 chemical reaction liquid Substances 0.000 claims description 6
- 239000012044 organic layer Substances 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 5
- 238000003908 quality control method Methods 0.000 claims description 5
- 239000012488 sample solution Substances 0.000 claims description 5
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 5
- 235000011152 sodium sulphate Nutrition 0.000 claims description 5
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 238000010898 silica gel chromatography Methods 0.000 claims description 3
- 239000012086 standard solution Substances 0.000 claims description 3
- 239000012043 crude product Substances 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 9
- 238000004458 analytical method Methods 0.000 abstract description 5
- 239000013558 reference substance Substances 0.000 abstract description 2
- 239000012071 phase Substances 0.000 description 20
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 8
- 239000000543 intermediate Substances 0.000 description 8
- 229940079593 drug Drugs 0.000 description 7
- 238000001228 spectrum Methods 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000012512 characterization method Methods 0.000 description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 5
- 229910052739 hydrogen Inorganic materials 0.000 description 5
- 239000001257 hydrogen Substances 0.000 description 5
- 238000001819 mass spectrum Methods 0.000 description 5
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 238000005576 amination reaction Methods 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 239000004215 Carbon black (E152) Substances 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 229930195733 hydrocarbon Natural products 0.000 description 2
- 150000002430 hydrocarbons Chemical class 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 238000004611 spectroscopical analysis Methods 0.000 description 2
- 238000005728 strengthening Methods 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- GDGIVSREGUOIJZ-UHFFFAOYSA-N 5-amino-3h-1,3,4-thiadiazole-2-thione Chemical compound NC1=NN=C(S)S1 GDGIVSREGUOIJZ-UHFFFAOYSA-N 0.000 description 1
- 201000002862 Angle-Closure Glaucoma Diseases 0.000 description 1
- 239000004156 Azodicarbonamide Substances 0.000 description 1
- 102000003846 Carbonic anhydrases Human genes 0.000 description 1
- 108090000209 Carbonic anhydrases Proteins 0.000 description 1
- 208000010412 Glaucoma Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- FLOSMHQXBMRNHR-UHFFFAOYSA-N N-(3-methyl-5-sulfamoyl-1,3,4-thiadiazol-2-ylidene)acetamide Chemical compound CC(=O)N=C1SC(S(N)(=O)=O)=NN1C FLOSMHQXBMRNHR-UHFFFAOYSA-N 0.000 description 1
- 206010030348 Open-Angle Glaucoma Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 201000001326 acute closed-angle glaucoma Diseases 0.000 description 1
- 230000001384 anti-glaucoma Effects 0.000 description 1
- XOZUGNYVDXMRKW-AATRIKPKSA-N azodicarbonamide Chemical compound NC(=O)\N=N\C(N)=O XOZUGNYVDXMRKW-AATRIKPKSA-N 0.000 description 1
- 235000019399 azodicarbonamide Nutrition 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000005100 correlation spectroscopy Methods 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000012156 elution solvent Substances 0.000 description 1
- JBTWLSYIZRCDFO-UHFFFAOYSA-N ethyl methyl carbonate Chemical compound CCOC(=O)OC JBTWLSYIZRCDFO-UHFFFAOYSA-N 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 1
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 1
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 231100000683 possible toxicity Toxicity 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D285/00—Heterocyclic compounds containing rings having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by groups C07D275/00 - C07D283/00
- C07D285/01—Five-membered rings
- C07D285/02—Thiadiazoles; Hydrogenated thiadiazoles
- C07D285/04—Thiadiazoles; Hydrogenated thiadiazoles not condensed with other rings
- C07D285/12—1,3,4-Thiadiazoles; Hydrogenated 1,3,4-thiadiazoles
- C07D285/125—1,3,4-Thiadiazoles; Hydrogenated 1,3,4-thiadiazoles with oxygen, sulfur or nitrogen atoms, directly attached to ring carbon atoms, the nitrogen atoms not forming part of a nitro radical
- C07D285/135—Nitrogen atoms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides unknown methazolamide impurities, and preparation, analysis methods and applications thereof, and belongs to the field of organic synthetic drug chemistry. The invention discloses two new impurity compounds in methazolamide and preparation and analysis methods thereof for the first time, and the impurity samples prepared by the preparation method have high purity, simple and efficient operation, moderate reaction conditions, strong safety and easy control, and are suitable for preparing impurities as reference substances to monitor the product quality.
Description
Technical Field
The invention relates to the field of organic synthetic pharmaceutical chemistry, and particularly relates to methazolamide impurity and a preparation method and application thereof.
Background
Methazolamide, synonym azodicarbonamide, foreign trade name: naptazane, english name: methazolamide, United states Pharmacopeia, has been previously documented. The product is a carbonic anhydrase inhibitor-type anti-glaucoma drug, and is mainly used for clinically treating chronic open-angle glaucoma, acute angle closure glaucoma, secondary glaucoma, ocular tension reduction in ophthalmic surgery, and the like.
For example, chinese patent application 200510050599.0 discloses a method for preparing methazolamide, which comprises using 5-amino-2-mercapto-1, 3, 4-thiadiazole as raw material, subjecting to condensation, acetylation, methylation, oxidation, amination reaction to obtain a crude methazolamide product, refining to obtain a refined methazolamide product, adding sodium hypochlorite in the oxidation and amination reaction process, reacting in the presence of iron chloride catalyst, and subjecting the intermediate obtained by the reaction to amination reaction to obtain a crude methazolamide product; adding a 5% NaOH solution into the crude methazolamide product, adjusting the pH value to 4-5 to obtain a precipitate, washing and drying to obtain a finished product; wherein the reaction temperature is controlled between 35 ℃ and 50 ℃ during the oxidation reaction. The reaction formula is as follows:
impurities are inevitably produced during the synthesis, processing and storage of the azomethine, and may cause potential toxicity during use. Therefore, it is necessary to investigate the impurities in the product of the methazolamide and to limit its content in the final product. In each large pharmacopoeia, the content of a single impurity is required to be controlled to be less than 0.10%, so that the content of part of impurities is small, and the impurities are difficult to separate and confirm, so that a standard product cannot be obtained to perform quality control on the impurities. Further improvement of methazolamide hybrid mass spectrum is required, and the quality control level of methazolamide is improved.
Disclosure of Invention
As mentioned above, the identification of the structure of the impurity is the basic requirement for drug registration, and in order to further improve the quality of methazolamide, the content of the impurity in the drug methazolamide is controlled.
One of the purposes of the invention is to provide a methazolamide impurity compound shown as the following formula (I),
the compound of the formula (I) can be used as a standard substance in methazolamide finished product detection and analysis, and is favorable for strengthening the safety control of medicines.
The inventors have found that the compound of formula (I) is an unknown impurity which has not been found so far in the production of methazolamide, and is generated by reacting a sulfonyl chloride intermediate with an impurity, benzylamine.
It is therefore another object of the present invention to provide a process for the preparation of the impurity compound of formula (I), said process comprising the steps of:
(1) mixing sulfonyl chloride intermediate, benzylamine, alkali and solvent, and reacting to obtain reaction liquid;
(2) and (2) concentrating the reaction liquid obtained in the step (1) under reduced pressure until the reaction liquid is dried, and eluting the reaction liquid through a column to obtain the impurity compound shown in the formula (I).
According to a specific embodiment of the present invention, in the step (1), the reaction conditions are: stirring at room temperature for 2-5 hr, heating to 40-70 deg.C, and stirring for 0.5-3 hr. Preferably, the reaction conditions are: after stirring at room temperature for 3.5 hours, the temperature was raised to 50 ℃ and the mixture was stirred for 1.5 hours.
According to a specific embodiment of the present invention, in step (1), the base includes, but is not limited to, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, triethylamine and the like, preferably sodium carbonate. The solvent may be a solvent conventional in the art, such as water, an alcohol solvent, an ester solvent, an ether solvent, etc., and preferably a non-hydroxyl solvent, such as tetrahydrofuran, dichloromethane, etc.
According to one embodiment of the invention, in step (2), the column used is a C18 chromatography column.
According to a specific embodiment of the invention, in the step (2), the eluent is a mixed solution of n-hexane and ethyl acetate, preferably a mixed solution of n-hexane and ethyl acetate in a volume ratio of 1: 1.
The sulfonyl chloride intermediate has a structural formula:
the structural formula of the benzylamine is as follows:
still another object of the present invention is to provide a methazolamide impurity compound represented by the following formula (II),
the compound shown in the formula (II) can be used as a standard substance in methazolamide finished product detection and analysis, and is favorable for strengthening the safety control of medicines.
The inventors have found that the compound of formula (II) is an unknown impurity which has not been found so far in the production of methazolamide, and is generated by the reaction of methazolamide with sulfonyl chloride intermediates.
It is therefore a further object of the present invention to provide a process for the preparation of an impurity compound of formula (II), said process comprising the steps of:
methazolamide is dissolved in a solvent, and the impurity compound (II) is separated by a DAC50 system.
According to a specific embodiment of the present invention, the solvent is at least one selected from methanol, acetonitrile, and water, preferably methanol, and more preferably 80% methanol.
According to a specific embodiment of the present invention, the specific steps of the separation are: separating for the first time, collecting the part containing impurity compound (II), concentrating under reduced pressure to obtain crude product, separating for the second time, and collecting impurity compound (II).
Further, according to a specific embodiment of the present invention, the conditions of the first separation are:
a chromatographic column: a reversed phase C18 column;
flow rate: 45.0-55.0 mL/min;
ultraviolet wavelength: 285 and 295 nm;
mobile phase: mixed mobile phase of sodium sulfate and acetonitrile.
Preferably:
and (3) chromatographic column: a reversed phase C18 column;
flow rate: 50.0 mL/min;
ultraviolet wavelength: 290 nm;
mobile phase: the volume ratio is 75: 25 mM 20mM sodium sulfate and acetonitrile.
Further, according to a specific embodiment of the present invention, the conditions of the second separation are:
a chromatographic column: a reversed phase C18 column;
flow rate: 45.0-55.0 mL/min;
ultraviolet wavelength: 285 and 295 nm;
mobile phase: formic acid-water and acetonitrile.
Preferably:
a chromatographic column: a reversed phase C18 column;
flow rate: 50.0 mL/min;
ultraviolet wavelength: 290 nm;
mobile phase: the volume ratio is 75: 25 of mixed mobile phase of 0.1% formic acid-water and acetonitrile.
Another preparation method of the impurity compound shown in the structure of the formula (II) comprises the following steps: mixing methazolamide, triethylamine and a solvent, stirring, adding a sulfonyl chloride intermediate for reaction, adding water and dichloromethane for extraction, concentrating an organic layer, performing silica gel column chromatography on the obtained organic layer, eluting the solvent by ethyl acetate and ethanol in a volume ratio of 5-15:1, and collecting an impurity compound (II).
Preferably, the elution solvent is ethyl acetate and ethanol in a volume ratio of 10: 1.
It is a further object of the present invention to provide the use of the impurity compounds of formula (I) and formula (II) for the quality control of methazolamide.
The final object of the invention is to provide a method for detecting methazolamide impurities, which is characterized in that the impurity compound of formula (I) and/or the impurity compound of formula (II) are/is used as standard substances, and the detection is carried out according to the following steps:
preparation of a sample solution: taking solution of mobile phase A and B at a ratio of 18:82 as solvent, dissolving sample at concentration of 0.1-10mg/ml, preferably 5mg/ml
Preparation of a standard solution: solvent the same sample solution, with concentration of 0.1-10 μ g/ml, preferably 5 μ g/ml
A chromatographic column: phenyl column
Mobile phase A acetonitrile
Mobile phase B potassium dihydrogen phosphate (6.8g/L)
Gradient elution:
| Time | B(％) |
| 0 | 80-85 |
| 10 | 80-85 |
| 30 | 38-42 |
| 35 | 38-42 |
| 35.1 | 80-85 |
| 45 | 80-85 |
wavelength: 290/250
Flow rate: 0.8-1.2mL/min
Sample introduction amount: 5 μ L.
The invention achieves the following beneficial effects:
1) the invention discloses the structures of the unknown impurity compounds of the methazolamide formulas (I) and (II) for the first time, and has important significance for the quality research of the methazolamide;
2) the invention discloses a preparation method of methazolamide unknown impurity compounds of formula (I) and formula (II) for the first time, and the impurity samples prepared by the preparation method have high purity, simple and efficient operation, moderate reaction conditions, strong safety and easy control, and are suitable for impurity preparation as reference substances to monitor the product quality.
Drawings
FIG. 1 mass spectrum of impurity (I);
FIG. 2 hydrogen spectrum of impurity (I);
FIG. 3 impurity (I) C13A spectrogram;
FIG. 4 Infrared Spectrum of impurity (I);
FIG. 5 UV spectrum of impurity (I);
FIG. 6 liquid phase spectrum of impurity (II);
fig. 7 mass spectrum (positive Ion) of Impurity (II);
FIG. 8 mass spectrum of impurity (II) (negative ions);
FIG. 9 Infrared Spectrum of impurity (II);
FIG. 10 UV spectrum of impurity (II);
FIG. 11 hydrogen spectrum of impurity (II);
FIG. 12 impurity (II) C13A spectrogram;
FIG. 13 DEPT-135 carbon spectrum of impurity (II);
FIG. 14 is a two-dimensional hydrogen spectrum (H-H COSY) diagram of impurity (II);
FIG. 15 two-dimensional hydrocarbon correlation (HSQC) plot of impurity (II);
FIG. 16 impurity (II) two-dimensional hydrocarbon correlation (HMBC) plot.
Detailed Description
The following exemplary reactions serve to illustrate the invention. The invention is protected by the technical scheme that simple replacement or improvement and the like of the invention are made by those skilled in the art. The reagents used in the present invention are either commercially available or can be prepared by the methods described herein.
EXAMPLE 1 preparation of the impurity Compound of formula (I)
(1) 2.05g of sulfonyl chloride intermediate, 0.87g of benzylamine, 0.86g of sodium carbonate and 20mL of tetrahydrofuran were added to the reaction flask, and the mixture was stirred at room temperature for 3.5 hours, then heated to 50 ℃ and stirred for 1.5 hours.
(2) Concentrating the dried reaction solution under reduced pressure, purifying with silica gel column to obtain eluent of n-hexane and ethyl acetate (1:1), concentrating, and removing the solution to obtain white solid impurity compound (I)0.8g with purity of 98% and yield of 70%.
Example 2 structural characterization of impurity Compound (I)
2.1 Pop data:
UV-Visλ(CH3OH)nm:258,295(λmax)；IR(KBr)νmax cm-1:3070(s,br),2865(m),2801(m,sh),1590(s),1490(s),1455(s,sh),1400(s),1359(s),1323(s)；1H NMR(500MHz；DMSO-d6；TMS)ppm:δH 2.96(3H,s,H-7),3.84(3H,s,H-8),4.24(2H,d,H-10),7.218-7.302(5H,m,H-12/12’/13/13’/14),9.30(1H,t,9-NH)；13C NMR(125MHz；DMSO-d6；TMS)ppm:δC 26.38(C-7),38.23(C-8),46.33(C-11),127.37(C-14),127.80and 128.19(C-12/12’/13/13’),155.33(C-5),164.28(C-2),179.74(C-6)；ms([M+H]+)m/z:327.0590(Calc.327.0580).
2.2 Mass Spectroscopy, Hydrogen Spectroscopy, carbon Spectroscopy, Infrared characterization and ultraviolet characterization plots are shown in FIGS. 1-5.
EXAMPLE 3 preparation of the impurity Compound of formula (II)
A sample of methazolamide was dissolved in 80% methanol and isolated as impurity C by the DAC50 manufacturing system. The method specifically comprises the following steps:
(1) first separation: the separation preparation conditions are reverse phase C18 column (10um), flow rate is 50.0mL/min, ultraviolet wavelength is 290nm, mobile phase is a mixed mobile phase of 20mM sodium sulfate and acetonitrile, and the volume ratio is 75: sodium sulfate improves the peak profile of the chromatographic separation. The fraction containing impurity C was collected (RT ═ 45 min) and then concentrated under reduced pressure to give crude impurity C.
(2) And (3) second separation: again through the same C18 column, flow rate 50.0mL/min, uv wavelength 290nm, in a volume ratio of 75: the mixed mobile phase of 25.1% formic acid-water and acetonitrile was further purified for desalting, and the impurity C fraction was collected (RT ═ 45 min) and lyophilized to give a pure sample (172 mg).
EXAMPLE 4 preparation of the impurity Compound of formula (II)
15g of methazolamide, 7.78g of triethylamine and 50ml of DMF are added, the mixture is stirred at-20 ℃, 20.32g of sulfonyl chloride intermediate is added to react for 5 hours, 500ml of water and 300ml of dichloromethane are added, extraction is carried out, and an organic layer is concentrated. The obtained organic layer was subjected to silica gel column chromatography using ethyl acetate as an eluting solvent: the impurity compound (II) is collected with the ethanol ratio of 10:1, the purity of 98 percent and the yield of 60 percent.
Example 5 structural characterization of impurity Compound (II)
2.1 Pop data:
UV-Visλ(CH3OH)nm:202,255,292(λmax)；IR(KBr)νmax cm-1:3455(m,br),2931(m),2853(m),1731(s),1495(s),1379(s),1308(s)；1H NMR(500MHz；DMSO-d6；TMS)ppm:δH 2.25(6H,s,H-7/7’),3.86(6H,s,H-8/8’)；13C NMR(125MHz；DMSO-d6；TMS)ppm:δC26.38(C-7/7’),38.00(C-8/8’),158.88(C-5/5’),164.42(C-2/2’),179.52(C-6/6’)；ms([M+H]+)m/z:455.9884(Calc.455.9883)；ms([2M+H]+)m/z:910.9678(Calc.910.9693)；ms([M-H]-)m/z:453.9790(Calc.453.9737)；([M-CH3CO]-)m/z:411.9683(Calc.411.9632).
2.2 liquid phase spectrogram, mass spectrum, infrared characterization, ultraviolet characterization, hydrogen spectrum and carbon spectrum are shown in FIGS. 6-16.
Example 6
And (3) detecting the content of impurities in the methazolamide medicine by taking the impurities prepared in the examples 1 and 4 as standard substances.
A detection step: methazolamide analysis method
Preparation of a sample solution: taking solution with mobile phase A and B at a ratio of 18:82 as solvent, dissolving sample at concentration of 0.5mg/ml
Preparation of a standard solution: solvent same sample solution with concentration of 5 μ g/ml
A chromatographic column: agilent phenyl column
Mobile phase A acetonitrile
Mobile phase B potassium dihydrogen phosphate (6.8g/L)
Gradient elution:
| Time | B(％) |
| 0 | 82 |
| 10 | 82 |
| 30 | 40 |
| 35 | 40 |
| 35.1 | 82 |
| 45 | 82 |
wavelength: 290/250
Flow rate: 1mL/min
Sample introduction amount: 5 μ L
And (3) detection results: the content of impurity I is about 0.03-0.05%, and the content of impurity II is about 0.06-0.12%. The impurity preparation method provided by the invention is simple, can be used as a standard substance to realize effective detection of corresponding impurities in methazolamide medicines, and further enhances the quality control of methazolamide medicines.
Example 6 impurity toxicity test
Performing mouse LD50 determination of oral route on impurity I and impurity II, and determining that oral LD50 of methazolamide impurity I mouse is 624 mg/kg; the oral LD50 of the methazolamide impurity II mice is 383mg/kg, which is obviously lower than that of the methazolamide.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (10)
1. A methazolamide impurity compound shown as the following formula (I),
2. the preparation method of the impurity compound shown in the structure of the formula (I) is characterized by comprising the following steps:
(1) mixing sulfonyl chloride intermediate, benzylamine, alkali and solvent, and reacting to obtain reaction liquid;
(2) and (2) concentrating the reaction liquid obtained in the step (1) under reduced pressure to dryness, and eluting through a column to obtain the impurity compound shown in the formula (I).
3. The method according to claim 2, wherein in the step (1), the reaction conditions are as follows: stirring at room temperature for 2-5 hours, heating to 40-70 ℃, and stirring for 0.5-3 hours; the alkali is sodium carbonate; the solvent is a non-hydroxyl solvent.
4. The preparation method according to claim 2, wherein in the step (2), the eluent is a mixed solution of n-hexane and ethyl acetate, preferably a mixed solution of n-hexane and ethyl acetate in a volume ratio of 1: 1.
5. A methazolamide impurity compound shown as the following formula (II),
6. a method for preparing an impurity compound represented by the structure of formula (II), comprising the steps of:
dissolving methazolamide in a solvent, and separating by using a DAC50 system to obtain an impurity compound (II); preferably, the specific steps of the separation are as follows: performing first separation, collecting the part containing the impurity compound (II), concentrating under reduced pressure to obtain crude product, performing second separation, and collecting the impurity compound (II); preferably, the solvent is selected from at least one of methanol, acetonitrile, water.
7. The method according to claim 6, wherein the conditions for the first separation are:
a chromatographic column: a reversed phase C18 column;
flow rate: 45.0-55.0 mL/min;
ultraviolet wavelength: 285 and 295 nm;
mobile phase: a mixed mobile phase of sodium sulfate and acetonitrile,
the conditions of the second separation are as follows:
a chromatographic column: a reversed phase C18 column;
flow rate: 45.0-55.0 mL/min;
ultraviolet wavelength: 285 and 295 nm;
mobile phase: formic acid-water and acetonitrile.
8. A method for preparing an impurity compound represented by the structure of formula (II), comprising the steps of: mixing methazolamide, triethylamine and a solvent, stirring, adding a sulfonyl chloride intermediate for reaction, adding water and dichloromethane for extraction, concentrating an organic layer, performing silica gel column chromatography on the obtained organic layer, eluting the solvents, namely ethyl acetate and ethanol in a volume ratio of 5-15:1, and collecting an impurity compound (II).
9. The impurity compound shown in the formula (I) and the impurity compound shown in the formula (II) are applied to the quality control of methazolamide.
10. A method for detecting methazolamide impurities, which is characterized in that the impurities in claim 1 and/or claim 5 are used as standard substances, and the detection is carried out according to the following steps:
preparation of a sample solution: taking solution of mobile phase A and B at a ratio of 18:82 as solvent, dissolving sample at concentration of 0.1-10mg/ml, preferably 5mg/ml
Preparation of a standard solution: solvent same product solution with concentration of 0.1-10 μ g/ml, preferably 5 μ g/ml
And (3) chromatographic column: phenyl column
Mobile phase A acetonitrile
Mobile phase B potassium dihydrogen phosphate (6.8g/L)
Gradient elution:
wavelength: 290/250
Flow rate: 0.8-1.2mL/min
Sample introduction amount: 5 μ L.
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