CN104230816B - Levosimendan potential genotoxic impurity as well as preparation and detection methods thereof - Google Patents
Levosimendan potential genotoxic impurity as well as preparation and detection methods thereof Download PDFInfo
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Abstract
The invention discloses a levosimendan potential genotoxic impurity adopting a diazoamino genotoxic warning structure and further provides a preparation method and a detection method of the impurity as well as applications of the impurity to detection of potential genotoxic impurities in a levosimendan bulk drug and preparations of the levosimendan bulk drug.
Description
Technical field
The invention belongs to pharmaceutical synthesis field and in particular to a kind of levosimendan latent gene toxic impurities and its preparation,
The research of detection method.
Background technology
Levosimendan（Levosimendan）, (R)-[[4- (1,4,5,6- tetrahydrochysene -4- methyl -6- oxo -3- pyridazinyl)
Phenyl]-hydrazono-] Cyanoacetyl-Cyacetazid（Formula II）Be ORION company of Finland develop pyridazinone, there is positive inotropic action
Calcium sensitizer, it is directly combined with troponin, makes the cardiac muscle fiber albumen necessary to myocardial contraction of calcium ion induction
Steric configuration is stabilized, so that myocardial contraction increases, can effectively treatment heart failure there is significant calcium to troponin
Rely on sexual reaction it is adaptable to need to increase the acute decompensation heart failure of myocardial contraction（ADHF）Short term therapy；
Meanwhile, WO9321921 also describes good result in treatment myocardial ischaemia for the levosimendan.
Levosimendan as a kind of calcium sensitizer of effective positive inotropic action at home and abroad extensively concerned while, face
Bed researcher the drug safety of levosimendan has been also carried out deeper into research.WO9965888A2 points out, in bacterial mutagenesis
Property test in find that the levosimendan sample synthesizing accidentally can show potential genotoxicity, and describe the latent of levosimendan
It is by [[4- (2- nitrine -3- methyl -5- oxo-tetrahydrofuran -2- base) phenyl] hydrazono-] Cyanoacetyl-Cyacetazid in genotoxicity（Formula III）
Cause, be effective mutagenic agent；Also indicate that simultaneously, can generate in levosimendan building-up process enough in bacterial mutagenesis examination
The amount of positive findingses is drawn, this result of study causes extensive concern both domestic and external in testing.
Formula II.
Formula III.
Genotoxicity refers to, in the internal/in vitro tests with DNA reacting substance as main study subject, DNA is had latent
Destructiveness.Genotoxicity impurity, as a class of process contaminants, is subject to great attention both domestic and external in recent years.European Union's medicine
Management board（EMA）Issue《The genotoxicity limit of impurities is guided》, and promulgated 36 kinds of chemistry knots with latent gene toxicity
Structure, it is intended to the abundant research to latent gene toxic impurities and effective control, ensures drug safety.
At present, in addition to the latent gene toxic impurities that WO9965888A2 describes levosimendan, both at home and abroad to left western Meng
In denier, the document report of potential genotoxicity impurity is very few.Therefore, genotoxicity impurity potential in levosimendan is carried out
More in-depth study, has become as an extremely important problem with the safety ensureing levosimendan medication.
Content of the invention
Present invention aim at providing a kind of levosimendan latent gene toxic impurities（Formula I）.
.
Another object of the present invention is to providing a kind of formula I pharmaceutically acceptable salt, solvate.Described salt can be
Organic salt or inorganic salt, including but not limited to hydrochlorate, nitrate, phosphate, formates, acetate, oxalate, benzoic acid
Salt, tartrate, malate, maleate.
It is a further object of the present invention to provide the preparation method of said gene toxic impurities, including by a certain temperature,
6- (4- aminophenyl) -4,5- dihydro -5- methyl -3 (2H) -2H-Pyridazin-3-one（Formula IV）Carry out in appropriate solvent instead with nitrous acid
Should.
.
Described reaction equation is as follows：
；
Wherein, described formula IV compound can be reacted in the form of salt, unhindered amina, solvate；Described Asia
Nitric acid can be nitrite and sour reaction in-situ preparation or nitrous acid solution.
In one embodiment of the invention, described reaction is carried out as follows, and formula IV compound is dissolved in water
In, under stirring condition, in reactant liquor, Deca hydrochloric acid, reaction system is cooled to 0oC；Under stirring condition, by sodium nitrite water
Solution is slowly dropped in reactant liquor, separates out a large amount of solids in reaction system；Continue reaction, TLC detection reaction is completely；Post processing obtains
To final product.
In the preferred technical solution of the present invention, described reaction temperature is -78oC~100oC, preferably -10oC~30oC,
In a specific embodiment, described reaction temperature is 0oC.
In the preferred technical solution of the present invention, described reaction dissolvent is selected from water；Halogenated hydrocarbon solvent, including but not limited to
Chloroform, dichloromethane, carbon tetrachloride；Alcohols solvent, including but not limited to methanol, ethanol, isopropanol；Esters solvent include but not
It is limited to ethyl acetate；Aromatic hydrocarbon solvent, including but not limited to benzene, toluene；Ketones solvent, including but not limited to acetone, butanone；Ether
Class solvent, including but not limited to ether, oxolane；Nitrile solvents, including but not limited to acetonitrile, propionitrile；Amide solvent, bag
Include but be not limited to DMF；And any one or a combination thereof of organic acid, mineral acid and its solution, preferably water.
In the preferred technical solution of the present invention, described organic acid and its preferred acetic acid of solution.
In the preferred technical solution of the present invention, described mineral acid and its solution are preferably hydrochloric acid, hydrobromic acid, sulphuric acid.
36 kinds of levosimendan latent gene toxic impurities contrast European Union of the present invention promulgation have latent gene poison
The chemical constitution of property, has diazoamino genotoxicity caution structure.
Another object of the present invention is to providing one kind to be used for detecting potential genotoxicity impurity in levosimendan sample
Reference reagent, described reagent includes type I compound and/or its pharmaceutically acceptable salt, solvate.Further, institute
State reference reagent also to include analyzing acceptable carrier.
The invention reside in providing a kind of formula I for detecting latent gene toxic impurities in levosimendan sample and preparation
Application.
The present invention also provides a kind of detection method of the levosimendan genotoxicity impurity of formula I, including adopting efficient liquid phase
The content of this genotoxicity impurity in chromatography determination levosimendan.
Preferably, described high performance liquid chromatography is reverse-phase chromatography；Preferred column is C8, C18, phenyl bonded silica
Glue is the analysis chromatographic column of filler；Detector is UV-detector；With methanol and phosphate buffer as mobile phase, according to isocratic
Detected with gradient elution program.The pH value of described phosphate buffered solution controls in the range of 1 ~ 4.
In one instantiation of the present invention, by levosimendan is carried out efficient liquid phase chromatographic analysis.Wherein using color
Spectrum post：Agilent XDB-C18 4.6*250mm 5um, column temperature：30 DEG C, flow velocity：1.0ml/min, wavelength：380nm, flowing
Phase：Methanol-phosphate buffer（Take 1.8g disodium hydrogen phosphate to be dissolved in 1000ml water, adjust PH to 2.1 with phosphoric acid）=6:4.
The invention reside in providing the structural identification method of levosimendan latent gene toxic impurities, including using high-resolution matter
The technology such as spectrum, NMR (Nuclear Magnetic Resonance) spectrum carry out structural identification；Wherein, the nucleus magnetic hydrogen spectrum data parsing of formula I latent gene toxic impurities
As follows：1H-NMR (d 6-DMSO, 400 MHz):δ= 12.78 (s, 1H, NH), 11.02 (s, 1H, NH),
10.92(s, 1H, NH), 7.84 (m, 4H, Ar), 7.61 (d, 2H, Ar), 7.40 (d, 2H, Ar), 3.39
(m, 2H, CH2), 2.72 (m, 2H, CH2), 2.25 (m, 2H, CH), 1.10 (d, 6H, 2CH3) ppm.
Brief description
Fig. 1 is levosimendan latent gene toxic impurities（Formula I）High resolution mass spectrum figure.
Fig. 2 is levosimendan latent gene toxic impurities（Formula I）Hydrogen nuclear magnetic resonance spectrogram.
Fig. 3 is levosimendan need testing solution genotoxicity impurity（Formula I）High-efficient liquid phase chromatogram, 1 be levosimendan,
2 is type I compound.
Specific embodiment
Detailed description below can make more detailed description to present disclosure, but subject of the present invention model
Enclose and be not limited to specific examples below, every technology realized based on present invention, technique belong to the model of the present invention
Enclose.
Embodiment 1Levosimendan latent gene toxic impurities（Formula I）Preparation（One）
2.04g 6- (4- aminophenyl) -4,5- dihydro -5- methyl -3 (2H) -2H-Pyridazin-3-one is dissolved in 30 mL water, stirring
Under the conditions of, in reactant liquor, Deca hydrochloric acid, reaction system is cooled to 0oC.Under stirring condition, by sodium nitrite in aqueous solution
（25mL）It is slowly dropped in reactant liquor, in reaction system, separate out a large amount of solids.Continue reaction 3 hours, TLC detection reaction is completely.
Filter, filter cake uses water, washing with alcohol, drying under reduced pressure successively, obtains yellow solid about 1.6g, yield：76%.1H-NMR (d 6-
DMSO, 400 MHz):δ= 12.78 (s, 1H, NH), 11.02 (s, 1H, NH), 10.92(s, 1H, NH),
7.84 (m, 4H, Ar), 7.61 (d, 2H, Ar), 7.40 (d, 2H, Ar), 3.39 (m, 2H, CH2), 2.72
(m, 2H, CH2), 2.25 (m, 2H, CH), 1.10 (d, 6H, 2CH3) ppm; HRMS-ESI (m/z):
440.1807 (M +Na+).
Embodiment 2Levosimendan latent gene toxic impurities（Formula I）Preparation（Two）
1.0 g 6- (4- aminophenyl) -4,5- dihydro -5- methyl -3 (2H) -2H-Pyridazin-3-one is dissolved in 5 mL dilute hydrochloric acid,
Reaction system is cooled to 0oC.Under stirring condition, by sodium nitrite in aqueous solution（25mL）It is slowly dropped in reactant liquor, after dripping off,
Continue stirring reaction 1 hour.Then, by 1.0g6- (4- aminophenyl) -4,5- dihydro -5- methyl -3 (2H) -2H-Pyridazin-3-one in batches
Add in reaction system, in reaction system, separate out a large amount of solids.Continue reaction 3 hours, TLC detection reaction is completely.Filter, filter cake
Use water, washing with alcohol, drying under reduced pressure successively, obtain yellow solid about 1.8 g, yield：86%.
Embodiment 3Levosimendan latent gene toxic impurities（Formula I）Preparation（Three）
2.04g 6- (4- aminophenyl) -4,5- dihydro -5- methyl -3 (2H) -2H-Pyridazin-3-one is dissolved in 10 mL acetic acid, stirs
Under the conditions of mixing, in reactant liquor, Deca hydrochloric acid, reaction system is cooled to 0oC.Under stirring condition, by sodium nitrite in aqueous solution
（25mL）It is slowly dropped in reactant liquor, in reaction system, separate out solid.Continue reaction 2 hours, TLC detection reaction is completely.Filter,
Filter cake uses water, washing with alcohol, drying under reduced pressure successively, obtains yellow solid about 1.5 g, yield：72%.
Embodiment 4Levosimendan latent gene toxic impurities（Formula I）Application as the comparison of high performance liquid chromatography impurity
For analyzing the detection being related to this impurity in levosimendan crude drug and its preparation.High performance liquid chromatography uses color
Spectrum post：Agilent XDB-C18 4.6*250mm 5um, column temperature：30 DEG C, flow velocity：1.0ml/min, wavelength：380nm, flowing
Phase：Methanol-phosphate buffer（Take 1.8g disodium hydrogen phosphate to be dissolved in 1000ml water, adjust pH to 2.1 with phosphoric acid）=6:4.
Claims (4)
1. a kind of levosimendan latent gene toxic impurities are used for detecting latent gene poison in levosimendan crude drug and its preparation
The application of property impurity；Described preparation is oral agents or injection, and described levosimendan latent gene toxic impurities have described in formula I
Structure,
2. according to claim 1 application it is characterised in that：The preparation side of described levosimendan latent gene toxic impurities
Method comprises the following steps：Under uniform temperature, 6- (4- aminophenyl) -4,5- dihydro -5- methyl -3 (2H)-pyridazine as shown in formula IV
Ketone is reacted in a solvent with nitrous acid：
Described solvent is water, halogenated hydrocarbon solvent, alcohols solvent, esters solvent, aromatic hydrocarbons
Any one in solvent, ketones solvent, ether solvent, nitrile solvents, amide solvent or organic acid, mineral acid or a combination thereof；
Wherein said organic acid is acetic acid；Described mineral acid is hydrochloric acid, hydrobromic acid, sulphuric acid；This impurity has following nuclear magnetic data：
H-NMR(d6- DMSO, 400MHz)：δ=12.78 (s, 1H, NH), 11.02 (s, 1H, NH), 10.92 (s, 1H, NH), 7.84 (m,
4H, Ar), 7.61 (d, 2H, Ar), 7.40 (d, 2H, Ar), 3.39 (m, 2H, CH2),2.72(m,2H,CH2),2.25(m,2H,
CH),1.10(d,6H,2CH3)ppm；Wherein each peak shift ± 0.1ppm.
3. a kind of detection method of levosimendan latent gene toxic impurities, this impurity has structure described in formula I
It is characterized in that this detection method includes：Using efficient liquid
Phase chromatography measures the content of this genotoxicity impurity in levosimendan；Described high performance liquid chromatography is reverse-phase chromatography；Color
Spectrum post is C8, C18 or phenyl bonded silica is the analysis chromatographic column of filler；Detector is UV-detector；With methanol and phosphoric acid
Salt buffer is mobile phase, is detected according to isocratic and gradient elution program；The pH value control of described phosphate buffered solution
System is in the range of 1～4.
4. method as claimed in claim 3 it is characterised in that：Described chromatographic column is Agilent XDB-C18 post；Column temperature is 30
℃；Flow velocity is 1.0ml/min；Detection wavelength is 380nm；Mobile phase methanol：Volume ratio=6 of phosphate buffer:4.
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| CN107037153B (en) * | 2017-04-21 | 2019-05-14 | 常州佳德医药科技有限公司 | The method that high performance liquid chromatography detects genotoxicity impurity in AL58805 bulk pharmaceutical chemicals or pharmaceutical preparation |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999065888A2 (en) * | 1998-06-18 | 1999-12-23 | Orion Corporation | A reference compound for use in the analysis of levosimendan batches |
| CN1616437A (en) * | 2004-09-27 | 2005-05-18 | 广西大学 | Racemization method of 6-4-(4-amino pheny)-4, 5-dihydro-5-methyl-3-(2H)-pyridazinone |
| CN102206202A (en) * | 2010-03-31 | 2011-10-05 | 河北医科大学 | Pyridazinone derivative and synthetic method thereof |
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2014
- 2014-09-19 CN CN201410479758.8A patent/CN104230816B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999065888A2 (en) * | 1998-06-18 | 1999-12-23 | Orion Corporation | A reference compound for use in the analysis of levosimendan batches |
| CN1616437A (en) * | 2004-09-27 | 2005-05-18 | 广西大学 | Racemization method of 6-4-(4-amino pheny)-4, 5-dihydro-5-methyl-3-(2H)-pyridazinone |
| CN102206202A (en) * | 2010-03-31 | 2011-10-05 | 河北医科大学 | Pyridazinone derivative and synthetic method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 左西孟旦和类似物的合成及其抗心衰活性研究;周红响;《广西大学硕士学位论文》;20040131;第29-30页第3.5节 * |
| 杨明华等.三氮烯试剂合成的新方法.《化学世界》.1998,第478-479页. * |
| 陈桂荣.钙增敏剂左西孟旦的合成.《沈阳药科大学硕士学位论文》.2007,第30页第3.5节. * |
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