CN116102479A - Preparation method and application of calcipotriol related isomer impurities - Google Patents
Preparation method and application of calcipotriol related isomer impurities Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 105
- LWQQLNNNIPYSNX-UROSTWAQSA-N calcipotriol Chemical compound C1([C@H](O)/C=C/[C@@H](C)[C@@H]2[C@]3(CCCC(/[C@@H]3CC2)=C\C=C\2C([C@@H](O)C[C@H](O)C/2)=C)C)CC1 LWQQLNNNIPYSNX-UROSTWAQSA-N 0.000 title claims abstract description 48
- 229960002882 calcipotriol Drugs 0.000 title claims abstract description 48
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 238000001514 detection method Methods 0.000 claims abstract description 12
- 239000003814 drug Substances 0.000 claims abstract description 10
- 229940079593 drug Drugs 0.000 claims abstract description 9
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- 238000006243 chemical reaction Methods 0.000 claims description 41
- 150000001875 compounds Chemical class 0.000 claims description 40
- 239000000243 solution Substances 0.000 claims description 32
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- 239000000203 mixture Substances 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 22
- 239000000047 product Substances 0.000 claims description 21
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 18
- 239000012043 crude product Substances 0.000 claims description 17
- RAHZWNYVWXNFOC-UHFFFAOYSA-N Sulphur dioxide Chemical compound O=S=O RAHZWNYVWXNFOC-UHFFFAOYSA-N 0.000 claims description 16
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 16
- 239000013558 reference substance Substances 0.000 claims description 16
- 230000002829 reductive effect Effects 0.000 claims description 15
- 238000004440 column chromatography Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical group [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 12
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 12
- 239000003085 diluting agent Substances 0.000 claims description 12
- 239000000523 sample Substances 0.000 claims description 12
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 238000005286 illumination Methods 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 238000012544 monitoring process Methods 0.000 claims description 6
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 6
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 6
- 239000011550 stock solution Substances 0.000 claims description 6
- 229910052786 argon Inorganic materials 0.000 claims description 5
- UWTDFICHZKXYAC-UHFFFAOYSA-N boron;oxolane Chemical compound [B].C1CCOC1 UWTDFICHZKXYAC-UHFFFAOYSA-N 0.000 claims description 5
- 230000006835 compression Effects 0.000 claims description 5
- 238000007906 compression Methods 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- YWWARDMVSMPOLR-UHFFFAOYSA-M oxolane;tetrabutylazanium;fluoride Chemical compound [F-].C1CCOC1.CCCC[N+](CCCC)(CCCC)CCCC YWWARDMVSMPOLR-UHFFFAOYSA-M 0.000 claims description 5
- 238000006552 photochemical reaction Methods 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims description 5
- 238000011160 research Methods 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- 239000002585 base Substances 0.000 claims description 4
- 238000006482 condensation reaction Methods 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 4
- NXXNVJDXUHMAHU-UHFFFAOYSA-N 1-anthracen-9-ylethanone Chemical compound C1=CC=C2C(C(=O)C)=C(C=CC=C3)C3=CC2=C1 NXXNVJDXUHMAHU-UHFFFAOYSA-N 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 239000003513 alkali Substances 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 239000005457 ice water Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000010606 normalization Methods 0.000 claims description 3
- FPLYNRPOIZEADP-UHFFFAOYSA-N octylsilane Chemical group CCCCCCCC[SiH3] FPLYNRPOIZEADP-UHFFFAOYSA-N 0.000 claims description 3
- 238000010791 quenching Methods 0.000 claims description 3
- 230000000171 quenching effect Effects 0.000 claims description 3
- 238000006722 reduction reaction Methods 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 239000012085 test solution Substances 0.000 claims description 3
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-diisopropylethylamine Substances CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 2
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- 238000001228 spectrum Methods 0.000 description 10
- 239000000126 substance Substances 0.000 description 9
- 201000004681 Psoriasis Diseases 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 4
- UORVGPXVDQYIDP-UHFFFAOYSA-N borane Chemical compound B UORVGPXVDQYIDP-UHFFFAOYSA-N 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 238000001953 recrystallisation Methods 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 229910000085 borane Inorganic materials 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000013067 intermediate product Substances 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 239000002674 ointment Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 238000007239 Wittig reaction Methods 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
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- 230000000670 limiting effect Effects 0.000 description 1
- 239000000865 liniment Substances 0.000 description 1
- 229940040145 liniment Drugs 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
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- 238000012827 research and development Methods 0.000 description 1
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- 238000005070 sampling Methods 0.000 description 1
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- 230000001225 therapeutic effect Effects 0.000 description 1
- 150000005671 trienes Chemical group 0.000 description 1
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C401/00—Irradiation products of cholesterol or its derivatives; Vitamin D derivatives, 9,10-seco cyclopenta[a]phenanthrene or analogues obtained by chemical preparation without irradiation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
- C07F7/02—Silicon compounds
- C07F7/08—Compounds having one or more C—Si linkages
- C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
- C07F7/1804—Compounds having Si-O-C linkages
- C07F7/1872—Preparation; Treatments not provided for in C07F7/20
- C07F7/1892—Preparation; Treatments not provided for in C07F7/20 by reactions not provided for in C07F7/1876 - C07F7/1888
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/02—Systems containing only non-condensed rings with a three-membered ring
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/14—The ring being saturated
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2602/00—Systems containing two condensed rings
- C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
- C07C2602/14—All rings being cycloaliphatic
- C07C2602/24—All rings being cycloaliphatic the ring system containing nine carbon atoms, e.g. perhydroindane
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
The invention discloses a preparation method and application of calcipotriol related isomer impurities. In particular to a preparation method and application of calcipotriol impurities L and M. The invention aims to protect the preparation methods of the impurities L and M, establish a detection method, analyze the impurity content and determine reasonable impurity limit so as to ensure the product quality and the medication safety of calcipotriol.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry synthesis, and particularly relates to a preparation method and application of calcipotriol related isomer impurities.
Background
Calcipotriol (Calcipotriol), chemical name: (5Z, 7E,22E, 24S) -24-cyclopropyl-9, 10-ring-opened cholest-5, 7,10 (19), 22-tetraene-1α,3β, 24-triol having the structural formula:
calcipotriol is active vitamin D 3 The preparation has the effects of inhibiting DNA synthesis and inhibiting cell proliferation, and is developed by Danish Liao pharmaceutical Co. The single ointment is used for the local treatment of psoriasis vulgaris (commonly known as psoriasis), and the compound ointment is mainly used for the adult stable plaque psoriasis suitable for the local treatment. The single liniment suitable for head psoriasis and the compound gel for external treatment of adult head psoriasis are developed later, and the research and development of the preparation can meet the requirements of patients suffering from psoriasis to a great extent.
The calcipotriol has high physiological activity, the single administration dosage is extremely small, and the common therapeutic dosage is only 0.25-1.0 mug/day. The calcipotriol has unstable property, is sensitive to light and heat, contains a plurality of chiral centers, has a longer traditional synthesis route, is inevitably easy to produce some isomer impurities and other related substances in the synthesis process, and is difficult to separate and purify, so that the technical threshold of the synthesis process is high and the difficulty is high. In the process of developing a calcipotriol bulk drug, two 20S-isomer impurities are inevitably introduced, and 24-bit configurations of the two 20S-isomer impurities are 24S and 24R respectively. The content of the impurities in the crude calcipotriol product exceeds 0.5%, and the impurities are difficult to remove by the traditional purification modes such as column chromatography, recrystallization and the like, so that hidden danger can be brought to the safety of the product.
Disclosure of Invention
The invention aims to provide a preparation method and application of calcipotriol related isomer impurities. In particular to a preparation method and application of calcipotriol impurities L and M. The invention aims to protect the preparation methods of the impurities L and M, establish a detection method, analyze the impurity content and determine reasonable impurity limit so as to ensure the product quality and the medication safety of calcipotriol.
The technical scheme for realizing the aim of the invention is as follows:
in a first aspect, the present invention provides calcipotriol impurities L and M, having the following structural formulas:
in a second aspect, the invention provides a preparation method of calcipotriol impurities L and M, wherein the reaction route is as follows:
the preparation method comprises the following steps:
(1) Taking a compound of formula 1 as a raw material, and carrying out sulfur dioxide removal reaction under the action of alkali in ethanol to obtain a compound of formula 2;
(2) The compound of the formula 2 and the compound of the formula 3 are subjected to Wittig condensation reaction to obtain a compound of the formula 4;
(3) Carrying out reduction reaction on the compound of the formula 4 under the action of borane tetrahydrofuran complex and R-2-methyl-CBS-oxazaborolidine to obtain a mixture containing the formulas 5 and 6;
(4) Carrying out photochemical reaction on the mixture containing the formula 5 and the formula 6 to obtain a mixture containing the formula 7 and the formula 8;
(5) Adding TBAF-THF into the mixed solution in the step (4), and obtaining a mixture containing impurity L and impurity M crude products through TBS removal protection;
(1) And (3) after the crude product prepared in the step (5) is dissolved, a DAC-50 dynamic axial compression preparation column chromatography system is adopted to prepare, separate and purify the crude product, and finally the impurity L and the impurity M are obtained.
According to an embodiment of the present invention, the base in the step (1) is at least one selected from triethanolamine, N-diisopropylethylamine and sodium bicarbonate, the reaction temperature is 70-80 ℃, and the reaction time is 2-4 hours.
According to an embodiment of the invention, the base in step (1) is selected from sodium bicarbonate, the molar ratio of the compound of formula 1 to sodium bicarbonate being 1:8.
According to the embodiment of the invention, in the step (2), the Wittig condensation reaction is to dissolve the compound of the formula 2 and the compound of the formula 3 in toluene, heat reflux reaction is carried out for 3-4 hours, after-treatment is carried out when TLC monitors that the target product is 40%, and the compound of the formula 4 is obtained after decompression concentration to dry column chromatography; wherein the molar ratio of the compound of formula 2 to the compound of formula 3 is 1:3.
According to an embodiment of the present invention, in step (3), the borane tetrahydrofuran complex and tetrahydrofuran are added into a reaction bottle at room temperature, R-2-methyl-CBS-oxazol borane is stirred for 1h at room temperature, the temperature is reduced to below-15 ℃, tetrahydrofuran solution of formula 4 is added dropwise, TLC monitoring is carried out, about 1/3 reaction is carried out, and post treatment: cooling to below-20deg.C, quenching with ice water, washing with ethyl acetate twice, washing with saturated saline once, concentrating under reduced pressure to dry column chromatography to obtain mixture containing formula 5 and formula 6.
According to an embodiment of the present invention, the photochemical reaction of step (4) is carried out by dissolving a mixture comprising formulas 5 and 6 and then placing the mixture in a photochemical reactor, adding 9-acetylanthracene, et 3 N, replacing argon, cooling in an ice bath, controlling the temperature to be 0-20 ℃ for illumination reaction, and concentrating under reduced pressure until the mixture is dried after the completion of the illumination reaction to obtain a mixture containing the compounds of the formulas 7 and 8.
According to the embodiment of the invention, the TBS removal protection reaction in the step (5) is to dissolve the compounds in the formula 7 and the formula 8, then add 1.0M tetrabutylammonium fluoride tetrahydrofuran solution for reaction at 55-65 ℃, after the TLC monitoring reaction is finished, decompress and concentrate to dryness to obtain a mixture containing the impurity L and the impurity M crude product.
According to the embodiment of the invention, in the step (6), a DAC-50 dynamic axial compression preparation column chromatography system is adopted to prepare, separate and purify the crude product, a crude product solution is taken for sample injection, an eluent is a mixed solution of acetonitrile and water, wherein the volume ratio of the acetonitrile to the water is as follows: acetonitrile/water=55/45, flow rate: 80ml/min, wavelength: 265nm.
In a third aspect, the invention also provides application of the preparation method in pharmaceutical technology research, which can be used for calcipotriol impurity research.
In a fourth aspect, the invention provides a method for detecting impurities L and M in a calcipotriol bulk drug, which specifically comprises the following steps:
(1) Sample preparation:
system applicability solution: taking a proper amount of each of the impurity L reference substance and the impurity M reference substance, placing the impurity L reference substance and the impurity M reference substance into different measuring flasks, adding methanol, shaking to dissolve and quantitatively dilute the mixture to prepare solutions containing 0.1mg of each 1ml, shaking uniformly, and respectively serving as each impurity stock solution; taking a proper amount of calcipotriol reference substance, precisely weighing, placing into a measuring flask, adding methanol accounting for 20% of the volume of the measuring flask, shaking for dissolution, adding a certain amount of each impurity stock solution, and quantitatively diluting with a diluent to prepare a solution containing 0.4mg of calcipotriol, 0.4 mug of impurity L and 0.4 mug of impurity M in each 1ml as a system applicability solution.
Test solution: about 10mg of the product is taken, precisely weighed, placed in a 25ml brown measuring flask, added with 5ml of methanol, shaken to dissolve, diluted to a scale with a diluent, and shaken uniformly to obtain the product.
Control solution: precisely measuring 1ml of the sample solution, placing in a 100ml measuring flask, adding a diluent to dilute to a scale, and shaking uniformly; and precisely measuring 1ml, placing in a 10ml measuring flask, adding a diluent to dilute to a scale, and shaking uniformly to obtain the product.
(2) Setting chromatographic conditions:
chromatographic column: octyl silane bonded silica gel (Kromasil 100-5-C8.6mm.times.250 mm,5 μm);
mobile phase a: water, mobile phase B: acetonitrile, gradient elution is carried out according to the following table, and the initial concentration proportion of acetonitrile is 38% -42%;
wavelength: 260-270nm;
sample injection amount: 10-30 μl;
flow rate: 1.3-1.7ml/min;
column temperature: 30-40 ℃;
run time: 55-70min.
(3) And (3) detection:
precisely measuring 20 mu L of each solution, injecting the solution into a liquid chromatograph, recording the chromatograms, and calculating the contents of the impurity L and the impurity M in the sample according to an area normalization method.
According to an embodiment of the invention, chromatographic conditions are set:
wavelength: 264nm;
sample injection amount: 20 μl;
flow rate: 1.5ml/min;
column temperature: 35 ℃;
run time: 65min.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention provides calcipotriol impurities L and M with brand new structures, which are 20S-isomer impurities generated in the calcipotriol synthesis process through preparation and structure confirmation, wherein the content of the impurities in a calcipotriol crude product exceeds 0.5%, and the impurities are difficult to remove through a traditional purification mode, so that hidden danger is brought to the safety of the product. The invention provides a novel method for preparing impurities L and M, the purity of liquid phase is higher than 96.0%, and the method has important practical significance for preparing impurity reference substances.
(2) The invention obtains the two impurities through preparation, and confirms the structure of the compound through structure confirmation, and discloses a method for detecting impurities L and M in calcipotriol bulk drug by using an HPLC detection method. The implementation of the invention is helpful for better controlling the quality of calcipotriol, and has important significance for researching and controlling the production quality of medicines and researching adverse reactions of medicine receptors.
Drawings
FIG. 1 is a TLC control chart of sulfur dioxide removal reactions.
FIG. 2 is a HPLC chart of the detection of substances related to the crude calcipotriol product.
Fig. 3 is an HPLC profile of calcipotriol impurity L.
Fig. 4 is a hydrogen spectrum of calcipotriol impurity L.
Fig. 5 is a carbon spectrum of calcipotriol impurity L.
Fig. 6 is an HPLC profile of calcipotriol impurity M.
Fig. 7 is a hydrogen spectrum of calcipotriol impurity M.
Fig. 8 is a carbon spectrum of calcipotriol impurity M.
FIG. 9 is a HPLC chart of calcipotriol pure related substance detection.
Detailed Description
The invention will now be described in further detail with reference to the following specific examples and figures, which are intended to be illustrative only and not limiting in any way, and the raw materials used, unless otherwise specified, may be either commercially available or self-made.
In the process of developing a calcipotriol bulk drug, we find that in the process of one-step reaction, namely, removing sulfur dioxide from a compound KC-M04 under the action of alkali to obtain an intermediate product KC-M05, two 20S-isomer impurities are inevitably introduced due to the fact that the reaction temperature and time are difficult to control, and 24-site configurations of the two 20S-isomer impurities are 24S and 24R respectively. The content of the impurity in the crude calcipotriol product is more than 0.5%, the HPLC detection results of related substances of the crude calcipotriol product are shown in Table 1, and the corresponding detection patterns are shown in FIG. 2.
The impurities are difficult to remove by the traditional purification modes such as column chromatography, recrystallization and the like, and cannot be completely removed in the reaction process, so that hidden danger is brought to the safety of the product. The specific process research and investigation process is as follows:
the reaction formula:
in the process of technological investigation, we finally select sodium bicarbonate to examine the reaction time of the compound KC-M04 in ethanol (95%) solution at 75-85 ℃, and from the TLC result, the KC-M04 has basically reacted completely at 1.5h, and the impurity point becomes obviously large at 2h, so that the reaction time is initially set to be 1.5h in the normal process, the reaction time is set to be 2-3h when the impurity is synthesized, and the TLC diagram for reaction control is shown in figure 1.
The reaction is to remove sulfur dioxide by sodium bicarbonate to recover conjugated triene structure, and then to obtain intermediate KC-M05 by extraction, concentration, drying and other operations after the reaction is finished. We have also considered further purification of KC-M05 to remove the two 20S-isomer impurities mentioned above. However, since the product is a viscous oily substance, the operations such as recrystallization and drying cannot be performed, and the product contains partial residual solvents such as ethanol and ethyl acetate, the actual yield is larger than the theoretical yield, and the impurities cannot be completely removed by the traditional column chromatography and recrystallization methods.
The invention obtains the two impurities through the sulfur dioxide removal reaction of the calcipotriol intermediate product KC-M04, the Wittig reaction with the intermediate KC-M02, the borane reduction reaction, the photochemical reaction and the TBS removal protection reaction, and the DAC preparation, separation and purification, and the structural formula and the naming are as follows:
impurity L (20 s,24 s-isomer), named: (5Z, 7E,20S,22E, 24S) -24-cyclopropyl-9, 10-seco-cholest-5, 7,10 (19), 22-tetraen-1α,3β, 24-triol
The chemical structural formula:
impurity M (20 s,24 r-isomer), named: (5Z, 7E,20S,22E, 24R) -24-cyclopropyl-9, 10-ring-opened cholest-5, 7,10 (19), 22-tetraene-1α,3β, 24-triol, chemical formula:
the invention aims to protect the preparation methods of the impurities L and M, establish a detection method, analyze the impurity content and determine reasonable impurity limit so as to ensure the product quality and the medication safety of calcipotriol.
EXAMPLE 1 Process for the preparation of impurity L and M
The reaction formula:
step one: 125g of the compound of formula 1 is dissolved in 1.8L of 95% ethanol, 131g of sodium bicarbonate is added, sulfur dioxide removal reaction is carried out for 2-3 hours at 70-80 ℃, then the reaction solution is concentrated under reduced pressure, extracted by ethyl acetate, separated, dried by anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 119g of the compound of formula 2.
Step two: 119g of the compound of formula 2 and 215g of the compound of formula 3 are taken and dissolved in 1.2L of toluene, reflux reaction is carried out for 3-4 hours at the temperature of 110-115 ℃ after argon replacement, after-treatment is carried out when the target product is monitored by TLC to be 40%, and the mixture is concentrated to be dry under reduced pressure, so that 30g of the compound of formula 4 is obtained.
Step three: taking 70ml of borane tetrahydrofuran complex and 207ml of tetrahydrofuran in a reaction bottle, dissolving a compound of formula 4 in 138ml of tetrahydrofuran, placing in a dropping funnel, setting up a reaction device, replacing by argon, adding R-2-methyl-CBS-oxazoloborane at room temperature, stirring for 1h at room temperature, cooling to below-15 ℃, dropwise adding a tetrahydrofuran solution of raw materials, dropwise adding Bi Kongwen-15 ℃ for reaction, monitoring by TLC, and carrying out 1/3 reaction, and carrying out aftertreatment: cooling to below-20deg.C, quenching with ice water, washing with ethyl acetate twice, washing with saturated saline once, concentrating under reduced pressure to dry column chromatography to obtain 30g mixture containing formula 5 and formula 6.
Step four: the mixture was dissolved in 2.1L of toluene, placed in a photochemical reactor, and 1.0g of 9-acetylanthracene, et, was added 3 N1.5 ml, replacing with argon, cooling in ice bath, controlling the temperature to be 0-20 ℃ for illumination reaction, and concentrating under reduced pressure to dryness after the completion of the illumination reaction to obtain the concentrate containing the formula 7 and the formula 8.
Step five: dissolving the concentrate in 619ml of tetrahydrofuran, adding 281ml of 1.0M tetrabutylammonium fluoride tetrahydrofuran solution, reacting at 55-65 ℃, and concentrating under reduced pressure to dry column chromatography after TLC monitoring reaction is finished to obtain 8.5g crude products containing impurities L and M.
Step six: dissolving the crude product in methanol, preparing, separating and purifying the crude product by adopting a DAC-50 dynamic axial compression preparation column chromatography system, wherein the eluent is a mixed solution of acetonitrile and water, and the volume ratio of acetonitrile to water is as follows: acetonitrile/water=55/45, flow rate: 80ml/min, wavelength: 265nm; taking crude product solution, sampling with about 1g each time, collecting
Segment and->
And (3) respectively combining the section target preparation solutions, concentrating under reduced pressure, extracting with ethyl acetate, washing with water, drying an organic layer by using anhydrous sodium sulfate, filtering, concentrating to dryness, preparing and separating twice, and finally obtaining the impurity L290 mg and the impurity M430 mg. />
HPLC purity of impurity L: 96.4% of the impurity L has an HPLC spectrum shown in FIG. 3, a nuclear magnetic resonance hydrogen spectrum shown in FIG. 4, a carbon spectrum shown in FIG. 5, [ M+Na ]] + Is 435.28731.
HPLC purity of impurity M: 97.0%, the HPLC spectrum of impurity M is shown in FIG. 6, the nuclear magnetic resonance hydrogen spectrum is shown in FIG. 7, the carbon spectrum is shown in FIG. 8, [ M+Na ]] + Is 435.28723.
Example 2
The detection method of calcipotriol related substances can effectively separate impurities L, M and other known impurities, and specifically comprises the following steps:
(1) Sample preparation:
system applicability solution: taking a proper amount of each of the impurity L reference substance and the impurity M reference substance, placing the impurity L reference substance and the impurity M reference substance into different measuring flasks, adding methanol, shaking to dissolve and quantitatively dilute the mixture to prepare solutions containing 0.1mg of each 1ml, shaking uniformly, and respectively serving as each impurity stock solution; taking a proper amount of calcipotriol reference substance, precisely weighing, placing into a measuring flask, adding methanol accounting for 20% of the volume of the measuring flask, shaking for dissolution, adding a certain amount of each impurity stock solution, and quantitatively diluting with a diluent to prepare a solution containing 0.4mg of calcipotriol, 0.4 mug of impurity L and 0.4 mug of impurity M in each 1ml as a system applicability solution.
Test solution: about 10mg of the product is taken, precisely weighed, placed in a 25ml brown measuring flask, added with 5ml of methanol, shaken to dissolve, diluted to a scale with a diluent, and shaken uniformly to obtain the product.
Control solution: precisely measuring 1ml of the sample solution, placing in a 100ml measuring flask, adding a diluent to dilute to a scale, and shaking uniformly; and precisely measuring 1ml, placing in a 10ml measuring flask, adding a diluent to dilute to a scale, and shaking uniformly to obtain the product.
(2) Setting chromatographic conditions:
chromatographic column: octyl silane bonded silica gel (Kromasil 100-5-C8.6mm.times.250 mm,5 μm);
mobile phase a: water, mobile phase B: acetonitrile, gradient elution was performed as follows;
wavelength: 264nm;
sample injection amount: 20 μl;
flow rate: 1.5ml/min;
column temperature: 35 ℃;
run time: 65min.
(1) And (3) detection:
precisely measuring 20 μl of each solution, injecting into a liquid chromatograph, recording the chromatograms, and calculating the content of each impurity in the sample according to an area normalization method.
Claims (10)
1. A method for preparing calcipotriol related isomer impurities L and M, which is characterized in that the structural formulas of the isomer impurities L and M are as follows:
the preparation method comprises the following steps:
(1) Taking a compound of formula 1 as a raw material, and carrying out sulfur dioxide removal reaction under the action of alkali in ethanol to obtain a compound of formula 2;
(2) The compound of the formula 2 and the compound of the formula 3 are subjected to Wittig condensation reaction to obtain a compound of the formula 4;
(3) Carrying out reduction reaction on the compound of the formula 4 under the action of borane tetrahydrofuran complex and R-2-methyl-CBS-oxazaborolidine to obtain a mixture containing the formulas 5 and 6;
(4) Carrying out photochemical reaction on the mixture containing the formula 5 and the formula 6 to obtain a mixture containing the formula 7 and the formula 8;
(5) Adding TBAF-THF into the mixed solution in the step (4), and obtaining a mixture containing impurity L and impurity M crude products through TBS removal protection;
(6) And (3) after the crude product prepared in the step (5) is dissolved, a DAC-50 dynamic axial compression preparation column chromatography system is adopted to prepare, separate and purify the crude product, and finally the impurity L and the impurity M are obtained.
2. The preparation method according to claim 1, wherein the base in the step (1) is at least one selected from triethanolamine, N-diisopropylethylamine and sodium bicarbonate, the reaction temperature of the sulfur dioxide removal reaction is 70-80 ℃, and the reaction time is 2-4 hours.
3. The process according to claim 1, wherein in step (1) the base is selected from sodium bicarbonate and the molar ratio of the compound of formula 1 to sodium bicarbonate is 1:8.
4. The preparation method of claim 1, wherein in the step (2), the Wittig condensation reaction is to dissolve the compound of formula 2 and the compound of formula 3 in toluene, heat reflux reaction is carried out for 3-4 hours, after-treatment is carried out when TLC monitors 40% of target product, and then the mixture is concentrated under reduced pressure to dry column chromatography to obtain the compound of formula 4; wherein the molar ratio of the compound of formula 2 to the compound of formula 3 is 1:3.
5. The process of claim 1, wherein in step (3), the borane tetrahydrofuran complex and tetrahydrofuran are added into a reaction flask, R-2-methyl-CBS-oxazoleboronide is added at room temperature, stirring is performed for 1h at room temperature, the temperature is reduced to below-15 ℃, tetrahydrofuran solution of formula 4 is added dropwise, TLC monitoring is performed, about 1/3 of reaction is performed, and post-treatment: cooling to below-20deg.C, quenching with ice water, washing with ethyl acetate twice, washing with saturated saline once, concentrating under reduced pressure to dry column chromatography to obtain mixture containing formula 5 and formula 6.
6. The process according to claim 1, wherein the photochemical reaction in step (4) is carried out by dissolving a mixture containing the compounds of the formulae 5 and 6, and then charging 9-acetylanthracene, et 3 N, replacing argon, cooling in an ice bath, controlling the temperature to be 0-20 ℃ for illumination reaction, and concentrating under reduced pressure until the mixture is dried after the completion of the illumination reaction to obtain a mixture containing the compounds of the formulas 7 and 8.
7. The preparation method of claim 1, wherein the TBS removal protection reaction in step (5) is carried out by dissolving the compounds of formula 7 and formula 8, adding 1.0M tetrabutylammonium fluoride tetrahydrofuran solution, reacting at 55-65 ℃, and concentrating under reduced pressure to dryness after TLC monitoring reaction is finished to obtain a mixture containing crude products of impurity L and impurity M.
8. The preparation method of claim 1, wherein in the step (6), a DAC-50 dynamic axial compression preparation column chromatography system is adopted to prepare, separate and purify the crude product, the crude product solution is taken for sample injection, the eluent is a mixed solution of acetonitrile and water, and the volume ratio of acetonitrile to water is: acetonitrile/water=55/45, flow rate: 80ml/min, wavelength: 265nm.
9. Use of the preparation method according to any one of claims 1-7 in pharmaceutical process research, for calcipotriol impurity research.
10. The method for detecting the impurities L and M in the calcipotriol bulk drug specifically comprises the following steps:
(1) Sample preparation:
system applicability solution: taking a proper amount of each of the impurity L reference substance and the impurity M reference substance, placing the impurity L reference substance and the impurity M reference substance into different measuring flasks, adding methanol, shaking to dissolve and quantitatively dilute the mixture to prepare solutions containing 0.1mg of each 1ml, shaking uniformly, and respectively serving as each impurity stock solution; taking a proper amount of calcipotriol reference substance, precisely weighing, placing into a measuring flask, adding methanol accounting for 20% of the volume of the measuring flask, shaking for dissolution, adding a certain amount of each impurity stock solution, and quantitatively diluting with a diluent to prepare a solution containing 0.4mg of calcipotriol, 0.4 mug of impurity L and 0.4 mug of impurity M in each 1ml as a system applicability solution;
test solution: taking about 10mg of the product, precisely weighing, placing into a 25ml brown measuring flask, adding 5ml methanol, shaking to dissolve, diluting to scale with diluent, and shaking to obtain the final product;
control solution: precisely measuring 1ml of the sample solution, placing in a 100ml measuring flask, adding a diluent to dilute to a scale, and shaking uniformly; precisely weighing 1ml, placing in a 10ml measuring flask, adding diluent, diluting to scale, and shaking to obtain the final product;
(2) Setting chromatographic conditions:
chromatographic column: octyl silane bonded silica gel (Kromasil 100-5-C8.6mm.times.250 mm,5 μm);
mobile phase a: water, mobile phase B: acetonitrile, gradient elution is carried out according to the following table, and the initial concentration proportion of acetonitrile is 38% -42%;
wavelength: 260-270nm;
sample injection amount: 10-30 μl;
flow rate: 1.3-1.7ml/min;
column temperature: 30-40 ℃;
(3) And (3) detection:
precisely measuring 20 mu L of each solution, injecting the solution into a liquid chromatograph, recording the chromatograms, and calculating the contents of the impurity L and the impurity M in the sample according to an area normalization method.
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| WO2008089093A2 (en) * | 2007-01-12 | 2008-07-24 | Quatrx Pharmaceuticals Company | Efficient processes for preparing steroids and vitamin d derivatives with the unnatural configuration at c20 (20 alpha-methyl) from pregnenolone |
| CN104478921A (en) * | 2014-12-12 | 2015-04-01 | 重庆华邦制药有限公司 | Intermediate Mb of calcipotriol and separation and detection method of impurities thereof |
| CN106908559A (en) * | 2015-12-23 | 2017-06-30 | 重庆华邦胜凯制药有限公司 | The separation of Calcipotriol intermediate L and related impurities and assay method |
| CN115290777A (en) * | 2022-07-27 | 2022-11-04 | 江苏知原药业股份有限公司 | Detection method of calcipotriol liniment related substances |
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| WO2008089093A2 (en) * | 2007-01-12 | 2008-07-24 | Quatrx Pharmaceuticals Company | Efficient processes for preparing steroids and vitamin d derivatives with the unnatural configuration at c20 (20 alpha-methyl) from pregnenolone |
| CN104478921A (en) * | 2014-12-12 | 2015-04-01 | 重庆华邦制药有限公司 | Intermediate Mb of calcipotriol and separation and detection method of impurities thereof |
| CN106908559A (en) * | 2015-12-23 | 2017-06-30 | 重庆华邦胜凯制药有限公司 | The separation of Calcipotriol intermediate L and related impurities and assay method |
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