Summary of the invention
In order to control the drug quality of CDB-2914 bulk drug and preparation thereof better, the present invention studies the impurity in bulk drug, obtains related impurities compound, and provides its preparation method and detection method.Impurity compound prepared by the present invention, as impurity reference substance, can be used for the mass analysis of this CDB-2914 bulk drug and preparation thereof, also can provide reference for the selection of processing condition simultaneously, be conducive to the control of production process Quality Evaluation of Chinese Medicinal amount.
Technical solution of the present invention is as follows:
The invention provides a kind of compound or its salt, the structure of described compound is as shown in the formula shown in compound F 17-hydroxy-corticosterone or compound K:
Compound or its salt described above, wherein said salt is inorganic acid salt or organic acid salt.
As another object of the present invention, a kind of preparation method of compound or its salt described above is also provided, it comprises and preparing in the reaction product of CDB-2914 through being separated the step preparing described compound F 17-hydroxy-corticosterone or compound K from by the two ketal (intermediate VI) of 3,20-.Preferably, the described reaction process preparing CDB-2914 by the two ketal (intermediate VI) of 3,20-is as follows:
Preparation method described above, wherein:
Described epoxidation reaction is:
Such as, compound VI is dissolved in methylene dichloride, under alkaline condition, reacts under adding oxygenant and perhalogeno acetone room temperature.Preferably, described alkali is selected from pyridine, dipotassium hydrogen phosphate, potassium primary phosphate, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC etc.; Oxygenant is selected from hydrogen peroxide, metachloroperbenzoic acid etc., preferred hydrogen peroxide; Temperature of reaction-10-10 DEG C.
Described Grignard reagent reaction is:
Such as, at THF, under cuprous chloride catalysis, carry out addition with 4-(N, N mono-dimethylamino) phenyl-magnesium-bromide Grignard reagent; Preferably, reaction raw materials and Grignard reagent mol ratio 1:1.5-5, temperature of reaction-10-40 DEG C, reaction times 2-8h.
Described hydrolysis reaction is:
Such as, be hydrolyzed under stirring in methylene dichloride and diluted acid reaction, and preferably, acid is selected from the mineral acids such as hydrochloric acid, sulfuric acid, sodium pyrosulfate, is preferably 0.2-4NHCl solution, temperature of reaction-10-50 DEG C, reaction times 1-5h.
Described acetylization reaction is:
Such as, carry out acylation reaction with in Glacial acetic acid, perchloric acid or aceticanhydride under any one or two or more acylating agents, in preferred perchloric acid and aceticanhydride, add Glacial acetic acid; Temperature of reaction-40-25 DEG C, preferably-10-25 DEG C; The 1-50%V/V of the acetylation reagent volume that the amount of Glacial acetic acid accounts for Glacial acetic acid, perchloric acid, aceticanhydride are formed, preferred 10-15%V/V.
As another embodiment of the present invention, the preparation method of another kind of compound or its salt described above is also provided, it comprises and preparing in the reaction product of CDB-2914 through being separated the step preparing described compound F 17-hydroxy-corticosterone or compound K from by the two ketal (intermediate VI) of 3,20-.The two ketal (intermediate VI) of 3,20-prepares the reaction of CDB-2914, such as can see can the method for suitability for industrialized production CDB-2914 disclosed in prior art CN102516345B.
Preparation method described above, wherein said separation is prepared as preparation high pressure liquid chromatography and is separated; Preferably, described preparation high pressure liquid chromatography condition is: stationary phase is C18 reverse phase filler, and moving phase is triethylamine phosphoric acid solution-acetonihile gradient elution, and determined wavelength is 302nm.
Preferably, preparation method described above, is preferably separated and prepares described compound F 17-hydroxy-corticosterone or compound K from the recrystallization mother liquor that CDB-2914 recrystallization obtains.
As another object of the present invention, also provide the detection method of a kind of compound or its salt described above or Compound D or E, wherein:
Wherein said method is HPLC method or HPLC-MS method;
Preferably, the testing conditions of described HPLC method is: stationary phase take octadecylsilane chemically bonded silica as weighting agent, and moving phase is triethylamine phosphoric acid solution-acetonihile gradient elution, and determined wavelength is 302nm; Preferably, moving phase is the triethylamine phosphoric acid solution-acetonitrile of pH2.5 ~ 3.5, and volume ratio is 30: 70 ~ 50: 50; More preferably, moving phase is the triethylamine phosphoric acid solution-acetonitrile of pH3.0, and volume ratio is 40: 60;
Preferably, the testing conditions of described HPLC-MS method is: the chromatographic condition of HPLC is stationary phase take octadecylsilane chemically bonded silica as weighting agent, moving phase is acetonitrile: the condition of 0.1% formic acid water=50:50, MS is for adopting selectivity HPLC-ION, detection compound D parent ion [M+H]
+m/z462.2, compd E parent ion [M+H]
+m/z434.0, compound F 17-hydroxy-corticosterone parent ion [M+H]
+m/z518.2, or compound K parent ion [M+H]
+m/z492.3.
As another object of the present invention, also provide compound or its salt described above purposes as impurity reference substance in the related substance of CDB-2914 bulk drug or preparation detects.
As another object of the present invention, the quality determining method of a kind of CDB-2914 bulk drug or preparation is also provided, it comprises the step measuring wherein related substance, described related substance is selected from compound F 17-hydroxy-corticosterone described above, K, D or E, it is characterized in that described mensuration adopts HPLC method or HPLC-MS method to detect; Preferably, described HPLC method or HPLC-MS method as described above; More preferably, described detection adopts HPLC method to detect; More preferably, the testing conditions of described HPLC method is: stationary phase is octadecylsilane chemically bonded silica, triethylamine phosphoric acid solution-the acetonitrile of moving phase to be volume ratio be pH2.5 ~ 3.5 of 30: 70 ~ 50: 50, determined wavelength is DAD (diode-array detector) 302nm, and number of theoretical plate calculates should be not less than 2000 by CDB-2914 peak; More preferably, during described related substance detects, if any impurity peaks in the color atlas of need testing solution, with calculated by peak area, the peak corresponding with each impurity phase respectively must not be greater than 0.15 times (namely foreign matter content is no more than 0.15%) of impurity reference substance solution.
Purposes described above, wherein said detection adopts HPLC method or HPLC-MS method to detect; Preferably, described detection adopts HPLC method to detect; More preferably, described HPLC method testing conditions is: stationary phase is octadecylsilane chemically bonded silica, triethylamine phosphoric acid solution-the acetonitrile of moving phase to be volume ratio be pH2.5 ~ 3.5 of 30: 70 ~ 50: 50, more preferably moving phase is 0.2% triethylamine (phosphoric acid adjusts pH3.0) damping fluid: acetonitrile=40:60, determined wavelength is DAD (diode-array detector) 302nm, and number of theoretical plate calculates should be not less than 2000 by CDB-2914 peak; More preferably, during described related substance detects, if any impurity peaks in the color atlas of need testing solution, with calculated by peak area, the peak corresponding with each impurity phase respectively must not be greater than 0.15 times (namely foreign matter content is no more than 0.15%) of impurity reference substance solution.Total impurities content must not more than 1.0%.
As the present invention's embodiment, impurity compound D, E, F or K described above specifically prepare by following methods.
1, the preparation process of CDB-2914
By two for 3,20-ketal (compound VI 1mo1), methylene dichloride, pyridine, three water Perfluoroacetones, be cooled to-10-0 DEG C, drip 50% hydrogen peroxide, in-5-5 DEG C of reaction 2-3h, separate organic phase, aqueous phase dichloromethane extraction twice merging, with 10% sodium thiosulfate solution washing, organic phase washing twice, anhydrous magnesium sulfate drying, filters, is evaporated to constant weight.
By Mg (1.6mo1), 1, 2 one ethylene dibromides and THF add there-necked flask, drip the bromo-N of 4-, accelerine and THF, after adding thermal booster reaction, backflow drips, finish, Grignard reagent is obtained in 50-60 DEG C of stirring reaction 3h, be cooled to 25 DEG C, add cuprous chloride, 25 DEG C are stirred after 30 minutes, the lower dichloromethane solution dripping above-mentioned epoxide of cooling, keep temperature 10-20 DEG C, finish insulated and stirred 2h, pour in the saturated NH4Cl aqueous solution of ice, stir 10 minutes, separate organic phase, aqueous phase dichloromethane extraction, organic phase merges, add 2NHCl solution.2h is stirred at 25 DEG C, separate organic phase, aqueous phase dichloromethane extraction, merge, use saturated sodium bicarbonate aqueous solution successively, water is respectively washed once, anhydrous magnesium sulfate drying, filter, filtrate proceeds in reaction flask, add Glacial acetic acid (1.25mol), be cooled to-10-0 DEG C, add 70% perchloric acid (1.18mo1), then aceticanhydride (5.96mo1) is dripped in-10-0 DEG C under stirring, insulated and stirred is after 30 minutes, aqueous phase dichloromethane extraction, merge, use saturated sodium bicarbonate aqueous solution successively, water is respectively washed once, anhydrous magnesium sulfate drying, filter, be evaporated to dry about 110g, through with 10 times amount Virahols: ethanol (95:5) recrystallization obtains Light yellow crystals 244.8g, recrystallization mother liquor is concentrated does to obtain solid, stand-by.Product uses Virahol again: ethanol (95:5) recrystallization, and 60 DEG C of oven dry obtain the product of Light yellow crystals, and recrystallization mother liquor is concentrated does to obtain solid, stand-by.
The CDB-2914 of above-mentioned gained is analyzed through LC-MS, and containing multiple impurity, the MS detected result comprising impurity F (i.e. compound F 17-hydroxy-corticosterone) and impurity K (i.e. compound K) is: impurity A: [M+H]
+397.16, impurity B: [M+H]
+415.1, impurity C:[M+H]
+373.1, impurity D:[M+H]
+462.2, impurity E: [M+H]
+434.0, impurity F: [M+H]
+518.2, impurity G:[M+H]
+476.2, impurity H:[M+H]
+441.2, impurity I:[M+H]
+516.2, impurity J:[M+H]
+474.2, impurity K:[M+H]
+492.3.
LC-MS condition used is:
HPLC: moving phase is Yi Jing ︰ 0.1% formic acid water=50 ︰ 50
Column temperature 25 ~ 35 DEG C
Flow velocity 0.3ml/min
Determined wavelength 302nm
Chromatographic column: AgilentPoroshe11120EC-C182.7um4.6X50mm
MS: condition
ESI,fg=25,MS2scan。
2, the preparation process of impurity D, E, F, K
Analyze through HPLC, the content of CDB-2914 lmpurities is lower than 0.1%, but through analyzing the mother liquor in building-up process, wherein the content of impurity is higher, therefore adopts preparative separation impurity in the mother liquor processed from building-up process.
Be concentrated into by gained recrystallization mother liquor in the preparation process of CDB-2914 and dryly obtain solid, containing higher proportion impurity in this solid, this solid is prepared liquid phase purifying through high pressure, process can obtain high-purity impurity reference substance, concrete operations are as follows:
Purification condition:
Gradient elution ratio:
Concrete operation step:
Mother liquor concentrations is obtained solid to dry, gets about 1g, dissolve by 40% acetonitrile/water, upper prop, gradient elution, according to going out peak situation, Fractional Collections elutriant, after each purifying completes, purification column, with after 5% acetonitrile solution rebalancing, can repeat sample introduction purifying.The elutriant containing each component purity >98% obtained by multiple batches of purifying merges respectively.The most of acetonitrile of concentrated removing, aqueous layer with ethyl acetate extracts, and concentrated ethyl acetate, to dry, obtains impurity solid respectively.Every Batch purification can obtain impurity A and be about 5mg, and impurity B is about 10mg, and impurity C is about 5mg, and impurity D is about 15mg, impurity E is about 5mg, and impurity F is about 5mg, and impurity G is about 5mg, and impurity H is about 15mg, impurity I is about 5mg, and impurity J is about 10mg, and impurity K is about 5mg.
Analyzing and testing condition in above-mentioned purge process is:
Moving phase is pH3.0 triethylamine phosphoric acid buffer: acetonitrile=40: 60
Determined wavelength 302nm
Column temperature: 30 DEG C
Flow velocity 1.0mL/min
As the present invention's embodiment, additionally provide impurity F, the purposes of K in CDB-2914 raw material and formulation products quality determining method thereof.Preferably use HPLC and/or HPLC-MS checked for impurities F or K and content thereof.
More preferably, the condition that described HPLC adopts is,
Moving phase: the triethylamine phosphoric acid solution-acetonitrile of pH2.5 ~ 3.5, volume ratio is 30: 70 ~ 50: 50; Wherein the triethylamine phosphoric acid solution of pH2.5 ~ 3.5 is by triethylamine-phosphoric acid buffer to forming, and 1000 parts of water add 1 ~ 3 part of triethylamine and obtain triethylamine solution, with phosphorus acid for adjusting pH for 2.5 ~ 3.5;
Flow velocity: 0.8 ~ 1.2mL/min (preferable flow rate 1.0mL/min);
Column temperature: 25 ~ 35 degrees Celsius, preferably 30 DEG C;
Determined wavelength: adopt multi-wavelength detection, select the maximum absorption wavelength of each impurity to carry out related substance detection, checking respectively.CDB-2914: 303nm; Impurity A: 259nm; Impurity B: 234nm; Impurity C:241nm; Impurity D:303nm; Impurity E: 304nm; Impurity F: 328nm; Impurity G:253nm; Impurity H:262nm; Impurity I:256nm; Impurity J:261nm; Impurity K:299nm.
Stationary phase: take octadecylsilane chemically bonded silica as weighting agent
More a step is preferred, and the condition that described HPLC-MS adopts is:
Chromatographic condition is: moving phase: 0.05% ~ 0.5%HAc/ water: acetonitrile=(75 ~ 80): (25 ~ 20); Preferred 0.1%HAc/ water, 78:22 preferred with acetonitrile ratio
Flow velocity: 0.1 ~ 0.5ml/min, preferred 0.3ml/min; Column temperature: 20 ~ 40 DEG C, preferably 30 DEG C
Mass Spectrometry Conditions is: adopt selected ion monitoring ((SIM) method
Dry gas temperature: 300 ~ 400 DEG C, preferably 350 DEG C
Flow rate of carrier gas: 8 ~ 12L/min, preferred 10L/min
Atomization gas pressure: 30 ~ 50psi, preferred 40psi
Capillary voltage: 4000V
As a preferred embodiment of the invention, provide above-mentioned impurity F or K as the application of impurity reference substance in the mass analysis of the Related substances separation of CDB-2914 and preparation thereof, wherein said CDB-2914 and the detection method of impurity thereof are preferably HPLC method, and HPLC testing conditions is as follows:
Stationary phase: take octadecylsilane chemically bonded silica as weighting agent
Triethylamine phosphoric acid solution-the acetonitrile of moving phase: pH3.0, volume ratio is 40: 60; Wherein the triethylamine phosphoric acid solution of pH3.0 is by triethylamine-phosphoric acid buffer to forming, and 1000 parts of water add 2 parts of triethylamines, with phosphorus acid for adjusting pH for 3.0;
Flow velocity: 0.8 ~ 1.2mL/min (preferable flow rate 1.0mL/min);
Column temperature: 25 ~ 35 degrees Celsius, preferably 30 DEG C;
Determined wavelength: preferably 302nm.
Number of theoretical plate calculates should be not less than 2000 by CDB-2914 peak
Determined wavelength: UV scanning show, each impurity and principal constituent maximum absorption wavelength as follows:
CDB-2914: 303nm; Impurity A: 259nm; Impurity B: 234nm; Impurity C:241nm; Impurity D:303nm; Impurity E: 304nm; Impurity F: 328nm; Impurity G:253nm; Impurity H:262nm; Impurity I:256nm; Impurity J:261nm; Impurity K:299nm, considers, and adopts multi-wavelength detection, selects the maximum absorption wavelength of each impurity to carry out related substance detection, checking respectively.
Experimental implementation:
Take CDB-2914 reference substance and impurity A respectively, B, C, D, E, F, G, H, I, J, K reference substance is in right amount each, add moving phase to dissolve and dilute and to make in every 1ml respectively containing the mixing solutions of each about 20ug of CDB-2914, impurity A, B, C, D, E, F, G, H, I, J, K, as system suitability solution.Precision measures system suitability solution 10ul injection liquid chromatography, record color atlas, and CDB-2914 and the mutual resolution of each impurity peaks should meet the requirements, and number of theoretical plate calculates should be not less than 2000 by CDB-2914 peak.
Assay method
Precision takes this product 10mg, puts in 20ml measuring bottle, dissolves, and is diluted to scale, shake up, as need testing solution by moving phase.Precision measures need testing solution 1ml and puts in 100ml measuring bottle, is diluted to scale by moving phase, shakes up, in contrast solution.Measure contrast solution 10 μ l, injection liquid chromatography, regulate detection sensitivity to make the peak height of principal constituent chromatographic peak be the 10-20% of full range.Precision measures need testing solution 10 μ l again, injection liquid chromatography, and record color atlas is to 3 times of principal constituent peak retention time.
Each impurity primary study determined wavelength:
Wavelength |
Impurity |
302nm |
Impurity D, E, F, K |
237nm |
Impurity B, C |
258nm |
Impurity A, G, H, I, J |
If any impurity peaks in the color atlas of need testing solution, with calculated by peak area, the peak corresponding with each impurity phase respectively must not be greater than 0.15 times (namely foreign matter content is no more than 0.15%) of contrast solution; Other arbitrary single impurity peaks all must not be greater than 0.15 times (namely foreign matter content is no more than 0.15%) of contrast solution, total impurities must not more than 1.0 (i.e. total impurities content must not more than 1.0%), ignore in the peak being less than contrast solution main peak area 0.05 times (namely content is less than 0.05%).
The advantage of the method for the detection of above-mentioned CDB-2914 related substance provided by the invention be measurement result accurately and reliably, specificity is strong, and practical, each impurity can effectively detect, and all reach good separation between CDB-2914 and each impurity, adopt this system to detect plurality of impurities.
Aforesaid method is suitable for the Related substance of CDB-2914 preparation equally.