A kind of isomer impurities PY3 of Tacalcitol and its preparation method and applicationTechnical field
The invention belongs to technical field of pharmaceutical chemistry, and in particular to the isomer impurities PY3 and its system of a kind of TacalcitolPreparation Method and by the use of the impurity as impurity reference substance, detection in the bulk pharmaceutical chemicals and preparation of Tacalcitol and analysis etc.Purposes in terms of quality control.
Background technology
Psoriasis, also known as psoriasis are a kind of chronic inflammatory skins, and sick time is long, are easy to recur, some casesAlmost it cannot be cured all one's life；It is fallen ill based on person between twenty and fifty, and health and the state of mind on patient are influenced；Clinically oftenFor symptoms such as erythema and the scales of skin that peel offs, whole body can fall ill, and with scalp, it is more that four limbs stretch side, easily aggravates in winter；Treatment silver bits at presentSick external used medicine mainly has vitamin D analog derivative, cortical steroid, tretinoin, anthraline, cutin to take off stripping agent etc.；Cortex classSterol is the steroid substance generated by adrenal cortex, is mostly steroids, such as glucocorticosteroid and mineralocorticoidDeng；In psoriasis treatment, it is known to use combination therapy, wherein involving a kind of sterid, such as corticosteroidClose object and a kind of novel vitamin D analogues.
Vitamin D (vitamin D) is sterol analog derivative, has anti-rachitic effect, also known as antirachitic vitamin.MeshBefore think that vitamin D is also a kind of steroid hormone, most important member is VD2 (calciferols in vitamin D family memberAlcohol) and VD3 (Vitamin D3).Vitamin D is derivative of the different provitamin Ds after ultraviolet irradiation.Be deficient in vitamin DIt may be extremely harmful to body.Vitamin D derivative activity is higher, therefore its formulation content is all very low, usually 0.0001 ~Between 0.0005%.
Tacalcitol (Tacalcitol) is the analog of vitamine D3 active metabolite, chemical entitled (+)-(5Z,7E, 24R) -9,10- open chain cholesterics -5,7,10 (19)-triolefin -1a, 3b, 24- triol, chemical formula C27H44O3, CASAccession number 57333-96-7；It is a kind of drug for treating psoriasis (psoriasis).In Supreme Being people's pharmacy strain formula by Japan in 1993(Teijin) biological medicine research institute of commercial firm releases listing, trade name Bonalfa.Due to Tacalcitol good drug efficacy and can useIn the treatment of the psoriasis of facial infection, therefore as the main liniment for the treatment of psoriasis (psoriasis).
Tacalcitol property is unstable, very sensitive to light, heat, air, especially easily generates one during the reactionA little isomer impurities and other related substances, it is difficult to it isolates and purifies, thus the more difficult assurance of synthesis condition, synthesis technology thresholdHeight, difficulty are big.Meanwhile better requirement also proposed to the quality control of bulk pharmaceutical chemicals and formulation products.
So far, since the content of Tacalcitol in the formulation is extremely low, the physicochemical property of main ingredient in itself has extremely unstable.Therefore, in open source literature, the impurity research and quality control of Tacalcitol are rarely reported.
The isomer impurities PY3 of Tacalcitol, i.e. (1 β, 3 β -5Z, 7E) -9,10- open loop cholesterics -5,7,10 (19)-threeAlkene -1,3,24- triols, be finished product Tacalcitol 1 beta isomer, in the presence of product quality, structural formula such as following formula 1 can be influencedShown in compound,
。
Through retrieval, there has been no the document report synthesized about the impurity, therefore it provides a kind of isomers of Tacalcitol is miscellaneousThe synthetic method of matter PY3, the preparation for impurity reference substance have important practical significance.
Invention content
The shortcomings that it is an object of the invention to overcome the prior art, provides a kind of isomer impurities PY3 of Tacalcitol, i.e.,(1 β, 3 β -5Z, 7E) -9,10- open loop cholesterics -5,7,10 (19)-triolefin -1,3, the synthetic method of 24- triols, the synthetic methodWith the advantages that easy to operate, raw material is cheap and easily-available, high income, purity is high.
The purpose of the present invention is what is be achieved through the following technical solutions.
The present invention provides a kind of isomer impurities PY3 of Tacalcitol, i.e. (1 β, 3 β -5Z, 7E) -9,10- open loop couragesSteroid -5,7,10 (19)-triolefin -1,3,24- triols, structure as shown in 1 compound of formula,
。
Preferably, the efficient liquid phase purity of the impurity be more than or equal to 98.5%, it is more excellent preferably greater than equal to 99.0%It is selected as being more than or equal to 99.5%；Purification process is can to recrystallize by column chromatography or preparative liquid chromatography is purified；This is miscellaneousThe purposes of matter is the related substance reference substance for Tacalcitol or the impurity identification for Tacalcitol.
Above-mentioned impurity PY3 provided by the invention is as impurity reference substance, in the quality control of Tacalcitol bulk pharmaceutical chemicals and preparationPurposes in system.
Impurity PY3 provided by the invention, the impurity analysis method that when quality control uses for high performance liquid chromatography,Computational methods are selected from external standard method, and the Self-control method of the correction up factor is not added with the Self-control method of correction factor, peak area normalizingOne kind in change method.
The present invention also provides a kind of methods for preparing above-mentioned impurity PY3, comprise the following steps：
（1）Paranitrobenzoic acid, triphenylphosphine, tetrahydrofuran and 6 compound of formula are added in into reaction bulb, stirring and dissolving, ice salt bathCooling, and diethyl azodiformate solution is added dropwise at low temperature, 0 DEG C of reaction is dripped off, the reaction was complete for TLC monitoring, and concentration is remainingObject is dissolved with ethyl acetate, and saturated salt solution washes twice, liquid separation, and organic phase is concentrated under reduced pressure；Add into the residue after concentrationEnter methanol and potash solid, 30 DEG C are stirred to react, TLC monitoring the reaction was complete, be concentrated under reduced pressure, residue add in ethyl acetate andWater dissolution, liquid separation, organic phase are washed twice, and dark oil object is done to obtain in anhydrous sodium sulfate drying, concentration；Column chromatography silica gel purifies,Eluant, eluent is n-hexane：Ethyl acetate=30:1, it is concentrated under reduced pressure and does, dichloromethane, imidazoles are added in gained grease, stirring is moltenTert-butyl chloro-silicane is added dropwise in Xie Hou, 0-5 DEG C of temperature control；It is warmed to room temperature after being added dropwise and reacts 2h, TLC is monitored to raw materialThe reaction was complete, washing, and anhydrous sodium sulfate drying, concentration obtains dark brown oil, i.e. 7 compound of formula；
（2）7 compound of formula is dissolved in dichloromethane, is cooled down, -20 ~ -10 DEG C are passed through sulfur dioxide gas into reaction solution, lead toFinish, be warming up to temperature control stirring at -15 ~ -10 DEG C and react 1 h, the reaction was complete for TLC monitorings, and removing solvent obtains yellow oil, i.e. formula8 compounds；
（3）8 compound of formula is dissolved in isometric dichloromethane and tetrahydrofuran in the mixed solvent, after being cooled to -65 ~ -80 DEG CBe passed through ozone, be stirred to react 6 ~ 8 h, TLC monitorings add in triphenylphosphine after the reaction was complete, and 0.5h is stirred at room temperature, by reaction solution according toSecondary respectively to washed once with 5% sodium bicarbonate solution, water, saturated brine, anhydrous sodium sulfate drying, filtrate is concentrated to dryness to obtain yellowSolid, i.e. 9 compound of formula；
（4）9 compound of formula is dissolved in 95% ethyl alcohol, adds in sodium bicarbonate, stirring is warming up to reaction 1 ~ 2 h, TLC at 75~85 DEG CThe reaction was complete for monitoring, adds in ethyl acetate and purifying water dissolution liquid separation, organic phase washed with water, saturated common salt washing are primary, nothingAqueous sodium persulfate, filtrate are concentrated to dryness to obtain brown-red solid, i.e. 10 compound of formula；
（5）By 10 compound of formula and 3- methyl-1s-(triphen methylene phosphine) -2- butanone, it is added in toluene, 110 ~ 115 DEG CThe reaction was complete for lower 30 ~ 35 h of back flow reaction, TLC monitoring, is cooled to room temperature, is concentrated to dryness rear pillar chromatographic purifying, obtains light yellow oilShape object, i.e. 11 compound of formula；
（6）11 compound of formula, sodium bicarbonate, sodium dithionate, hexadecyltrimethylammonium chloride are added to toluene and purifyingIt in water, is heated at 60 ~ 65 DEG C, reacts 1 ~ 1.5 h, the reaction was complete for TLC monitorings, is cooled to room temperature, the rear ethyl acetate and pure of adding inChange moisture liquid, organic layer is successively with water, saturated common salt water washing, and anhydrous sodium sulfate drying, filtrate is concentrated to dryness to obtain light yellowGrease, 12 compound of formula；
（7）12 compound of formula is dissolved in tetrahydrofuran, adds in (1S, 2R)-(-) -1- amino -2- indanols, under nitrogen protection, dropTo 0 DEG C hereinafter, the tetrahydrofuran solution of three sec-butyl potassium borohydrides is added dropwise, drop finishes temperature, and 1 h, TLC monitorings are stirred at -5 ~ 0 DEG CIt adds in ice water after the reaction was complete to be quenched, rear to add in ethyl acetate and purified water liquid separation, organic phase is primary with saturated salt washing, anhydrousSodium sulphate is dried, and filtrate is concentrated to dryness column chromatography purifying, obtains light yellow oil, i.e. 13 compound of formula；
（8）13 compound of formula, 9- acetyl group anthracene, pyridine and toluene are added in photochemical reactor, under nitrogen protection intoRow illumination reaction, the reaction was complete for TLC monitorings, removes solvent, obtains light yellow oil, i.e. 14 compound of formula；
（9）14 compound of formula is dissolved in the tetrahydrofuran solution of tetrabutyl ammonium fluoride, 50-55 DEG C of reaction 2h, TLC monitoring reactionCompletely, solvent is removed, ethyl acetate and moisture liquid are added in residue, organic phase saturated common salt water washing concentrates after dryTo dry, addition maleic anhydride and ethyl acetate in residue, it is stirred to react for 24 hours at 30-35 DEG C, is concentrated to dryness rear column chromatography and obtainsThe white solid of high-purity, i.e. 1 compound of formula.
It is further preferred that specific experimental implementation is as follows：
（1）Paranitrobenzoic acid 10g, triphenylphosphine 20g, tetrahydrofuran 400mL and 6 compound 15g of formula are added in into reaction bulb, stirredDissolving, ice salt bath cooling are mixed, and the solution that diethyl azodiformate 12g is dissolved in 50mL tetrahydrofurans is added dropwise at low temperature, dropComplete 0 DEG C of reaction, the reaction was complete for TLC monitoring, and concentration, residue is dissolved with ethyl acetate, and saturated salt solution washes twice, liquid separation,Organic phase is concentrated under reduced pressure；Methanol 400mL and potash solid 10g is added in into the residue after concentration, 30 DEG C are stirred to react,The reaction was complete for TLC monitoring, is concentrated under reduced pressure, and residue adds in ethyl acetate 500mL and water 500mL dissolvings, liquid separation, organic phase washingTwice, anhydrous sodium sulfate is dried, and dark oil object is done to obtain in concentration；Column chromatography silica gel purifies, and eluant, eluent is n-hexane：Ethyl acetate=30:1, reduced pressure is dry, addition dichloromethane, imidazoles in gained grease, and after stirring and dissolving, tertiary fourth is added dropwise in 0-5 DEG C of temperature controlBase dimethylchlorosilane；Reaction 2h, TLC are warmed to room temperature after being added dropwise and monitors that the reaction was complete to raw material, washing, anhydrous sodium sulfateDry, concentration obtains dark brown oil, i.e. 7 compound 8.4g of formula；
（2）7 compound 8.4g of formula is dissolved in dichloromethane 150mL, is cooled down, -20 ~ -10 DEG C are passed through titanium dioxide into reaction solutionSulphur gas leads to and finishes, and is warming up to temperature control stirring at -15 ~ -10 DEG C and reacts 1 h, the reaction was complete for TLC monitorings, and removing solvent obtains yellow oilShape object 9.0g, i.e. 8 compound of formula；
（3）8 compound 9.0g of formula is dissolved in isometric dichloromethane and tetrahydrofuran mixed solvent 120mL, is cooled to -65Ozone is passed through after ~ -80 DEG C, is stirred to react 6 ~ 8 h, TLC is monitored after the reaction was complete and is added in triphenylphosphine 10g, and 0.5h is stirred at room temperature,Reaction solution respectively washed once successively with 5% sodium bicarbonate solution, water, saturated brine, anhydrous sodium sulfate drying, filtrate is concentrated intoIt is dry to obtain yellow solid, i.e. 9 compound of formula；
（4）9 compound of formula is dissolved in 70mL95% ethyl alcohol, adds in sodium bicarbonate 8.6g, stirring be warming up to reaction 1 at 75~85 DEG C ~The reaction was complete for 2 h, TLC monitoring, adds in ethyl acetate and purifying water dissolution liquid separation, organic phase washed with water, saturated common salt washingOnce, anhydrous sodium sulfate, filtrate are concentrated to dryness to obtain brown-red solid 11g, i.e. 10 compound of formula；
（5）By 10 compound 11g of formula and 3- methyl-1s-(triphen methylene phosphine) -2- butanone 14g, it is added to toluene 100mLIn, the reaction was complete for 30 ~ 35 h of back flow reaction at 110 ~ 115 DEG C, TLC monitoring, is cooled to room temperature, is concentrated to dryness rear pillar chromatographic purifying,Obtain light yellow oil 2.1g, i.e. 11 compound of formula；
（6）By 11 compound 2.1g of formula, sodium bicarbonate 5g, sodium dithionate 6g, hexadecyltrimethylammonium chloride 0.7g, add inIt into 80mL toluene and 80mL purified waters, is heated at 60 ~ 65 DEG C, reacts 1 ~ 1.5 h, the reaction was complete for TLC monitorings, is cooled to roomTemperature, rear to add in ethyl acetate and purified water liquid separation, organic layer is successively with water, saturated common salt water washing, anhydrous sodium sulfate drying, filterLiquid is concentrated to dryness to obtain light yellow oil, 12 compound of formula；
（7）12 compound of formula is dissolved in tetrahydrofuran 20mL, adds in (1S, 2R)-(-) -1- amino -2- indanol 1g, nitrogen is protectedUnder shield, 0 DEG C is cooled to hereinafter, the tetrahydrofuran solution 10mL of three sec-butyl potassium borohydrides is added dropwise, drop finishes, and 1 is stirred at -5 ~ 0 DEG CH, TLC monitoring add in ice water after the reaction was complete and are quenched, rear to add in ethyl acetate 300mL and purified water 300mL liquid separations, organic phasePrimary with saturated salt washing, anhydrous sodium sulfate drying, filtrate is concentrated to dryness column chromatography purifying, obtains light yellow oil 1g, i.e.,13 compound of formula；
（8）13 compound of formula, 9- acetyl group anthracenes 0.05g, pyridine 0.1mL and toluene 70mL are added in photochemical reactor,Illumination reaction is carried out under nitrogen protection, and the reaction was complete for TLC monitorings, removes solvent, obtains light yellow oil 1g, i.e. formula 14 is changedClose object；
（9）14 compound of formula is dissolved in the tetrahydrofuran solution 100mL of tetrabutyl ammonium fluoride, 50-55 DEG C of reaction 2h, TLC prisonThe reaction was complete for survey, removes solvent, ethyl acetate 300mL and water 300mL liquid separations, organic phase saturated salt solution are added in residueWashing is concentrated to dryness after dry, and maleic anhydride 0.1g and ethyl acetate 50mL is added in residue, is stirred to react at 30-35 DEG CFor 24 hours, it is concentrated to dryness rear column chromatography and obtains the white solid 300mg of high-purity, i.e. 1 compound of formula, HPLC purity：99.5%；
6 compound of starting materials of formula that the present invention prepares impurity PY3 can be with bibliography Org. Process Res. Dev.The preparation method of the compound 14 of experimental section obtains in 2012,16,967-975, is as follows：
（a）By 2 compounds of formula, that is, calciferol 18.97g, in a low temperature of about -10 DEG C, sulfur dioxide gas is slowly introducing,It is allowed to be dissolved in the sulfur dioxide of liquid, the reaction was complete for TLC monitoring, and decompression pumps sulfur dioxide gas, obtains grease formula 3Compound, about 24.30g；
（b）By above-mentioned 3 compound of formula under nitrogen protection, 7.81g imidazoles is added in, then 132mL dichloromethane adds in tertiary butylAdding tert-butyl chloro-silicane 1.44g, TLC monitoring after dimethylchlorosilane 8.65g, 80min again, the reaction was complete, and removing is moltenAgent, residue ethyl acetate 200mL are dissolved, are washed successively with water 100mL, saturated salt solution 100mL, and anhydrous magnesium sulfate is doneDry, filtrate is concentrated to dryness, and obtains 4 compound of formula, about 27.91g；
（c）95% ethyl alcohol of 250mL will be added in above-mentioned 4 compound of formula, sodium bicarbonate 2.5g adds carbon after being heated to reflux 140minSour hydrogen sodium 5g, the 2h that finishes that the reaction was continued, remove solvent, ethyl acetate are added in, successively with water, saturated common salt water washing, anhydrous sulphurSour magnesium drying, filtrate are concentrated to dryness, and obtain 5 compound of formula, about 21.01g；
（d）Above-mentioned 5 compound of formula is dissolved in 210mL dichloromethane, N-methyl morpholine oxide 19.3g is added in, is heated to 42 DEG CLeft and right is allowed to clarify, and adds the solution that selenium dioxide 3.19g is dissolved in methanol 210mL, the reaction was continued 2h, removes solvent, remainsExcess is dissolved in ethyl acetate 300mL, and successively with water, saturated common salt water washing, anhydrous magnesium sulfate drying, filtrate is concentrated to give formula 6Then the crude product of compound by column chromatography or prepares liquid phase and isolates and purifies, obtains 6 compound fine work 15g of formula.
Because of above-mentioned 6 compound of formula, vitamin D synthesis field belongs to relatively conventional intermediate again, can be by the art ripeThe method known prepares gained.If the above method reported for the present invention optimizes, for example replace reaction reagent, becomeMore reaction sequence, also all within protection scope of the present invention.
It is another object of the present invention to the impurity PY3 using Tacalcitol, as impurity reference substance, detection and analysisMethod in relation to substance in Tacalcitol bulk pharmaceutical chemicals, which is characterized in that specifically comprise the following steps：
Operation is protected from light, this product about 2mg is taken, puts in 20ml brown volumetric flasks, mobile phase is added in right amount to dissolve sample and is diluted to quarterDegree, shakes up, as test solution；Precision measures test solution 1ml and puts in 200ml measuring bottles, and scale is diluted to mobile phase,It shakes up, obtains contrast solution.It takes Tacalcitol and impurity PY3 each appropriate, is configured to every ml Tacalcitols containing 0.1mg and 1 μ g impurityThe solution of PY3 as system suitability solution, is surveyed according to high performance liquid chromatography (four general rules 0512 of Chinese Pharmacopoeia version in 2015)It is fixed, it is filler with straight chain starch derivative chiral chromatographic column（CHIRALCEL AD-H 250*4.6mm 5μm）；With normal heptane-Absolute ethyl alcohol（86：14）For mobile phase；30 DEG C of column temperature；Detection wavelength is 265nm；50 μ l of system suitability solution is taken to inject liquid phaseChromatograph adjusts flow velocity, and the appearance time for making Tacalcitol is about 12min, and the separating degree of PY3 and Tacalcitol should meet ruleIt is fixed；It measures each 50 μ l injections liquid chromatograph of test solution, contrast solution respectively again, records chromatogram；Test solutionIf any PY3 peaks in chromatogram, PY3 areas are not greater than 2 times of contrast solution main peak area（1.0%）.
What the present invention obtained beneficial has the technical effect that：
（1）Condition of the present invention is easily-controllable, and route is simple, and solvent is easy to get；
（2）The isomer impurities PY3 of Tacalcitol prepared by the present invention can be used up to more than 99% purity as reference substance；
（3）The isomer impurities PY3 of Tacalcitol is synthesized, not the only impurity identification of Alfacalcidol and defects inspecting offer pairAccording to product, so as to be promoted in the analysis of Tacalcitol finished product detection to the accurate positionin of impurity PY3 and qualitative, be conducive to strengthen miscellaneous to thisThe control of matter, so raising to the quality standards of Tacalcitol bulk pharmaceutical chemicals and its preparation and the quality control of product provide it is beneficialWith reference to, also for similar compound synthesis offer reference；
（4）The optimization of chiral reduction condition：During 13 compound of formula, in order to further improve the choosing of chiral reductionSelecting property, we attempt a variety of reaction conditions, finally select three sec-butyl potassium borohydrides in chiral auxiliary (1S, 2R)-(-) -1- ammoniaThe existence of base -2- indanols carries out chiral reduction, and using simple column chromatography preliminary purification, chiral purity can meet willIt asks, if wanting to further improve chiral purity, the slow crystallization of n-hexane-ethyl acetate system can be used.Such post processingThe tedious steps for preparing liquid phase separation chiral isomer are eliminated, while reduce entreprise cost, also meet environment protection requirement.
Specific embodiment
With reference to preferred embodiment, the present invention is further described, but the present invention is not limited to following embodiments.
Agents useful for same and material of the present invention are commercially available.
Synthetic route is as follows.
.
The fragment compound wherein used during 11 compound of synthesis formula, i.e. 3- methyl-1s-(triphen methylene phosphine)-2- butanone can purchase commercially available intermediate, and gained can also be prepared by a conventional method, including but not limited to the following method, i.e., withMethyl isopropyl Ketone for starting, first through the bromo-reaction in methanol solvate, then in toluene solution add in triphenylphosphine condensation andInto.
The preparation of 1 formula of embodiment, 7 compound
Paranitrobenzoic acid 10g, triphenylphosphine 20g, tetrahydrofuran 400mL and 6 compound 15g of formula are added in into reaction bulb, stirringDissolving, ice salt bath cooling, and the solution that diethyl azodiformate 12g is dissolved in 50mL tetrahydrofurans is added dropwise at low temperature, drip off 0DEG C reaction, the reaction was complete for TLC monitoring, and concentration, residue dissolves with ethyl acetate, and saturated salt solution washes twice, liquid separation, organicMutually it is concentrated under reduced pressure；Methanol 400mL and potash solid 10g is added in into the residue after concentration, 30 DEG C are stirred to react, TLC prisonsThe reaction was complete for control, is concentrated under reduced pressure, and residue adds in ethyl acetate 500mL and water 500mL dissolvings, liquid separation, and organic phase is washed twice,Anhydrous sodium sulfate is dried, and dark oil object is done to obtain in concentration；Column chromatography silica gel purifies, and eluant, eluent is n-hexane：Ethyl acetate=30:1, reduced pressure is dry, addition dichloromethane, imidazoles in gained grease, and after stirring and dissolving, tertiary butyl two is added dropwise in 0-5 DEG C of temperature controlMethylchlorosilane；Reaction 2h, TLC are warmed to room temperature after being added dropwise and monitors that the reaction was complete to raw material, washing, anhydrous sodium sulfate is doneDry, concentration obtains dark brown oil, i.e. 7 compound 8.4g of formula.
The preparation of 2 formula of embodiment, 8 compound
7 compound 8.4g of formula is dissolved in dichloromethane 150mL, is cooled down, -20 ~ -10 DEG C are passed through sulfur dioxide gas into reaction solutionBody leads to and finishes, and is warming up to temperature control stirring at -15 ~ -10 DEG C and reacts 1 h, the reaction was complete for TLC monitorings, and removing solvent obtains yellow oil9.0g, i.e. 8 compound of formula.
The preparation of 3 formula of embodiment, 10 compound
8 compound 9.0g of formula is dissolved in isometric dichloromethane and tetrahydrofuran mixed solvent 120mL, it is cooled to -65 ~ -Ozone is passed through after 80 DEG C, is stirred to react 6 ~ 8 h, TLC is monitored after the reaction was complete and added in triphenylphosphine 10g, and 0.5h is stirred at room temperature, willReaction solution respectively washed once successively with 5% sodium bicarbonate solution, water, saturated brine, and anhydrous sodium sulfate drying, filtrate is concentrated to drynessObtain yellow solid, i.e. 9 compound of formula；
9 compound of formula is dissolved in 70mL95% ethyl alcohol, adds in sodium bicarbonate 8.6g, stirring, which is warming up at 75~85 DEG C, reacts 1 ~ 2The reaction was complete for h, TLC monitoring, adds in ethyl acetate and purifying water dissolution liquid separation, organic phase washed with water, saturated common salt washing oneSecondary, anhydrous sodium sulfate, filtrate is concentrated to dryness to obtain brown-red solid 11g, i.e. 10 compound of formula.
The preparation of 4 formula of embodiment, 11 compound
10 compound 11g of formula and 3- methyl-1s-(triphen methylene phosphine) -2- butanone 14g are added in toluene 100mL,The reaction was complete for 30 ~ 35 h of back flow reaction at 110 ~ 115 DEG C, TLC monitoring, is cooled to room temperature, is concentrated to dryness rear pillar chromatographic purifying, obtainsTo light yellow oil 2.1g, i.e. 11 compound of formula.
The preparation of 5 formula of embodiment, 13 compound
By 11 compound 2.1g of formula, sodium bicarbonate 5g, sodium dithionate 6g, hexadecyltrimethylammonium chloride 0.7g, it is added toIt in 80mL toluene and 80mL purified waters, being heated at 60 ~ 65 DEG C, reacts 1 ~ 1.5 h, the reaction was complete for TLC monitorings, is cooled to room temperature,Ethyl acetate and purified water liquid separation are added in afterwards, and organic layer is successively with water, saturated common salt water washing, anhydrous sodium sulfate drying, filtrateIt is concentrated to dryness to obtain light yellow oil, 12 compound of formula；
12 compound of formula is dissolved in tetrahydrofuran 20mL, adds in (1S, 2R)-(-) -1- amino -2- indanol 1g, nitrogen protectionUnder, 0 DEG C is cooled to hereinafter, the tetrahydrofuran solution 10mL of three sec-butyl potassium borohydrides is added dropwise, drop finishes, and 1 h is stirred at -5 ~ 0 DEG C,TLC monitorings add in ice water after the reaction was complete and are quenched, rear to add in ethyl acetate 300mL and purified water 300mL liquid separations, and organic phase is with fullPrimary with salt washing, anhydrous sodium sulfate drying, filtrate is concentrated to dryness column chromatography and purifies to obtain light yellow oil 1g, i.e. formula 13 is changedClose object.
The preparation of 6 formula of embodiment, 14 compound
13 compound of formula, 9- acetyl group anthracenes 0.05g, pyridine 0.1mL and toluene 70mL are added in photochemical reactor,Nitrogen protection is lower to carry out illumination reaction, and the reaction was complete for TLC monitorings, removes solvent, obtains light yellow oil 1g, i.e. 14 chemical combination of formulaObject.
The preparation of 7 formula of embodiment, 1 compound
14 compound of formula is dissolved in the tetrahydrofuran solution 100mL of tetrabutyl ammonium fluoride, 50-55 DEG C of reaction 2h, TLC monitoring is anti-Should be complete, solvent is removed, ethyl acetate 300mL is added in residue and water 300mL liquid separations, organic phase is washed with saturated common saltIt washs, is concentrated to dryness after dry, maleic anhydride 0.1g and ethyl acetate 50mL is added in residue, is stirred to react at 30-35 DEG CFor 24 hours, it is concentrated to dryness rear column chromatography and obtains the white solid 300mg of high-purity, i.e. 1 compound of formula, HPLC purity：99.5%.
The preparation of 8 formula of embodiment, 1 compound
（1）Paranitrobenzoic acid 10g, triphenylphosphine 20g, tetrahydrofuran 400mL and 6 compound 15g of formula are added in into reaction bulb, stirredDissolving, ice salt bath cooling are mixed, and the solution that diethyl azodiformate 12g is dissolved in 50mL tetrahydrofurans is added dropwise at low temperature, dropComplete 0 DEG C of reaction, the reaction was complete for TLC monitoring, and concentration, residue is dissolved with ethyl acetate, and saturated salt solution washes twice, liquid separation,Organic phase is concentrated under reduced pressure；Methanol 400mL and potash solid 10g is added in into the residue after concentration, 30 DEG C are stirred to react,The reaction was complete for TLC monitoring, is concentrated under reduced pressure, and residue adds in ethyl acetate 500mL and water 500mL dissolvings, liquid separation, organic phase washingTwice, anhydrous sodium sulfate is dried, and dark oil object is done to obtain in concentration；Column chromatography silica gel purifies, and eluant, eluent is n-hexane：Ethyl acetate=30:1, reduced pressure is dry, addition dichloromethane, imidazoles in gained grease, and after stirring and dissolving, tertiary fourth is added dropwise in 0-5 DEG C of temperature controlBase dimethylchlorosilane；Reaction 2h, TLC are warmed to room temperature after being added dropwise and monitors that the reaction was complete to raw material, washing, anhydrous sodium sulfateDry, concentration obtains dark brown oil, i.e. 7 compound 8.4g of formula；
（2）7 compound 8.4g of formula is dissolved in dichloromethane 150mL, is cooled down, -20 ~ -10 DEG C are passed through titanium dioxide into reaction solutionSulphur gas leads to and finishes, and is warming up to temperature control stirring at -15 ~ -10 DEG C and reacts 1 h, the reaction was complete for TLC monitorings, and removing solvent obtains yellow oilShape object 9.0g, i.e. 8 compound of formula；
（3）8 compound 9.0g of formula is dissolved in isometric dichloromethane and tetrahydrofuran mixed solvent 120mL, is cooled to -65Ozone is passed through after ~ -80 DEG C, is stirred to react 6 ~ 8 h, TLC is monitored after the reaction was complete and is added in triphenylphosphine 10g, and 0.5h is stirred at room temperature,Reaction solution respectively washed once successively with 5% sodium bicarbonate solution, water, saturated brine, anhydrous sodium sulfate drying, filtrate is concentrated intoIt is dry to obtain yellow solid, i.e. 9 compound of formula；
（4）9 compound of formula is dissolved in 70mL95% ethyl alcohol, adds in sodium bicarbonate 8.6g, stirring be warming up to reaction 1 at 75~85 DEG C ~The reaction was complete for 2 h, TLC monitoring, adds in ethyl acetate and purifying water dissolution liquid separation, organic phase washed with water, saturated common salt washingOnce, anhydrous sodium sulfate, filtrate are concentrated to dryness to obtain brown-red solid 11g, i.e. 10 compound of formula；
（5）By 10 compound 11g of formula and 3- methyl-1s-(triphen methylene phosphine) -2- butanone 14g, it is added to toluene 100mLIn, the reaction was complete for 30 ~ 35 h of back flow reaction at 110 ~ 115 DEG C, TLC monitoring, is cooled to room temperature, is concentrated to dryness rear pillar chromatographic purifying,Obtain light yellow oil 2.1g, i.e. 11 compound of formula；
（6）By 11 compound 2.1g of formula, sodium bicarbonate 5g, sodium dithionate 6g, hexadecyltrimethylammonium chloride 0.7g, add inIt into 80mL toluene and 80mL purified waters, is heated at 60 ~ 65 DEG C, reacts 1 ~ 1.5 h, the reaction was complete for TLC monitorings, is cooled to roomTemperature, rear to add in ethyl acetate and purified water liquid separation, organic layer is successively with water, saturated common salt water washing, anhydrous sodium sulfate drying, filterLiquid is concentrated to dryness to obtain light yellow oil, 12 compound of formula；
（7）12 compound of formula is dissolved in tetrahydrofuran 20mL, adds in (1S, 2R)-(-) -1- amino -2- indanol 1g, nitrogen is protectedUnder shield, 0 DEG C is cooled to hereinafter, the tetrahydrofuran solution 10mL of three sec-butyl potassium borohydrides is added dropwise, drop finishes, and 1 is stirred at -5 ~ 0 DEG CH, TLC monitoring add in ice water after the reaction was complete and are quenched, rear to add in ethyl acetate 300mL and purified water 300mL liquid separations, organic phasePrimary with saturated salt washing, anhydrous sodium sulfate drying, filtrate is concentrated to dryness column chromatography and purifies to obtain light yellow oil 1g, i.e. formula13 compounds；
（8）13 compound of formula, 9- acetyl group anthracenes 0.05g, pyridine 0.1mL and toluene 70mL are added in photochemical reactor,Illumination reaction is carried out under nitrogen protection, and the reaction was complete for TLC monitorings, removes solvent, obtains light yellow oil 1g, i.e. formula 14 is changedClose object；
（9）14 compound of formula is dissolved in the tetrahydrofuran solution 100mL of tetrabutyl ammonium fluoride, 50-55 DEG C of reaction 2h, TLC prisonThe reaction was complete for survey, removes solvent, ethyl acetate 300mL and water 300mL liquid separations, organic phase saturated salt solution are added in residueWashing is concentrated to dryness after dry, and maleic anhydride 0.1g and ethyl acetate 50mL is added in residue, is stirred to react at 30-35 DEG CFor 24 hours, it is concentrated to dryness rear column chromatography and obtains the white solid 300mg of high-purity, i.e. 1 compound of formula, HPLC purity：99.5%.
The structure elucidation of 9 formula of embodiment, 1 compound
From above-mentioned data it is found that sample hydrogen spectrum removes solvent C DCl319 groups of peaks are shared outside peak, corresponding ratio is：1:1:2:1:1:1:1:2:1:4:2:3:2:4:3:3:3:6:3, corresponding 44 hydrogen.Each hydrogen ownership is as follows：δ 6.6099-6.6328 (d, 1H, J=11.45Hz）, it is attributed to 6-H；δ 5.9130-5.9360 (d, 1H, J=11.5Hz）, it is attributed to 7-H；δ5.2381-5.2697（T,2H）, it is attributed to 22-H, 23-H；δ5.1660-5.1680（S, 1H）, it is attributed to 19（E）-H；δ5.0019-5.0058（S, 1H）,It is attributed to 19（Z）-H；δ4.5251-4.5443（T, 1H）, it is attributed to 1-H；δ4.2555-4.2893（M, 1H）, it is attributed to 3-H；δ2.8912-2.9179（D, 2H）, it is attributed to 4-H；δ2.2989-2.3434（M, 1H）, it is attributed to 9-H；δ2.0267-2.1232（M, 4H）, it is attributed to 2-H, 12-H, 17-H, 24-H；δ 1.8888-1.9463 (m, 2H), are attributed to 2-H, 20-H；δ1.7286-1.8004（M, 3H）, it is attributed to 9-H, 11-H, 16-H；δ1.5749-1.6781（S, 2H）, it is attributed to 1-OH, 3-OH；δ1.5077-1.6185（M, 4H）, it is attributed to 11-H, 15-H, 25-H；δ1.3312-1.4063（M, 3H）, it is attributed to 12-H, 14-H, 16-H；δ1.0660-1.0793（D, 3H）, it is attributed to 21-H；δ0.9671-0.9808（D, 3H）, it is attributed to 28-H；δ0.8712-0.9001（T, 6H）, it is attributed to 26-H, 27-H；δ0.6219（S, 3H）, it is attributed to 18-H.
It is removed in carbon-13 nmr spectra and 26 groups of peaks is shared outside solvent DMSO, corresponding 27 carbon, with sample structure molecular formulaC27H44O3There are 27 carbon phase symbols.δ12.3563；δ17.5892；δ19.6157；δ19.9154；δ21.0765；δ22.2282；δ23.5221；δ27.7028；δ29.0640；δ33.0739；δ36.7354；δ40.2887-40.3282；δ41.9843；δ42.8363；δ45.8567；δ56.5037-56.5771；δ65.8980；δ71.0874；δ109.5498；δ115.8708；δ123.3513；δ132.0557；δ132.6545；δ135.5253；δ145.2946；δ151.7430.
It can be seen that from mass spectral results [M+Na]+、[M-H]-M/z be respectively 439.3,415.3, can speculate sample pointSon amount is 416.3, with C27H44O3Theoretical molecular weight 416.6 is consistent.Meet the nitrogen rule of azo atom even molecule amount.
Know that molecular weight analyte is 416.6 according to the mass spectrometry results of sample, be consistent with structural theory molecular weight；Nuclear-magnetism is total toThe hydrogen that shakes is composed, hydrogen shown in carbon spectrum spectrogram, carbon number, and ownership is consistent with structure.In the preparation method of Tacalcitol impurity, pi-allylOxidation reaction may introduce 1 beta-hydroxy impurity, and the compound of the final generation is 1 β-isomers that compound is finished product,So sample structure is consistent with what is envisioned.
Embodiment 10 checks the related substance in Tacalcitol bulk pharmaceutical chemicals using impurity PY3 as impurity reference substance
The present invention provides a kind of impurity PY3 as impurity reference substance, check related substance in Alfacalcidol bulk pharmaceutical chemicalsMethod specifically comprises the following steps：
Operation is protected from light, this product about 2mg is taken, puts in 20ml brown volumetric flasks, mobile phase is added in right amount to dissolve sample and is diluted to quarterDegree, shakes up, as test solution；Precision measures test solution 1ml and puts in 200ml measuring bottles, and scale is diluted to mobile phase,It shakes up, obtains contrast solution.It takes Tacalcitol and impurity PY3 each appropriate, is configured to every ml Tacalcitols containing 0.1mg and 1 μ g impurityThe solution of PY3 as system suitability solution, is surveyed according to high performance liquid chromatography (four general rules 0512 of Chinese Pharmacopoeia version in 2015)It is fixed, it is filler with straight chain starch derivative chiral chromatographic column（CHIRALCEL AD-H 250*4.6mm 5μm）；With normal heptane-Absolute ethyl alcohol（86：14）For mobile phase；30 DEG C of column temperature；Detection wavelength is 265nm.50 μ l of system suitability solution is taken to inject liquidChromatography adjusts flow velocity, and the appearance time for making Tacalcitol is about 12min, and the separating degree of PY3 and Tacalcitol should meet ruleIt is fixed；It measures each 50 μ l injections liquid chromatograph of test solution, contrast solution respectively again, records chromatogram；Test solutionIf any PY3 peaks in chromatogram, PY3 areas are not greater than 2 times of contrast solution main peak area（1.0%）.
Disclosed above is only presently preferred embodiments of the present invention, cannot limit the right model of the present invention with this certainlyIt encloses, therefore, the equivalent variations that the claims in the present invention are made still fall within the range that the present invention is covered.