CN115057804A

CN115057804A - Purification method of calcitriol

Info

Publication number: CN115057804A
Application number: CN202210592338.5A
Authority: CN
Inventors: 丁伯祥; 胡永康; 肖昌琴; 王雪萌; 李聚江; 石若鹏
Original assignee: Nanjing Hairong Pharmaceutical Co ltd
Current assignee: Nanjing Hairong Pharmaceutical Co ltd
Priority date: 2022-05-27
Filing date: 2022-05-27
Publication date: 2022-09-16
Anticipated expiration: 2042-05-27
Also published as: CN115057804B

Abstract

The invention relates to a method for purifying calcitriol. The calcitriol preparation method is characterized in that fermentation liquor containing a calcitriol crude product prepared by microbial fermentation is subjected to separation and purification, TBS protection, deprotection, refining and purification, the production process is mature, the conditions are mild, and the method is suitable for industrial production. The purity of the calcitriol pure product prepared by the method is more than 98%, preferably more than 99%, and most preferably more than 99.8%.

Description

Purification method of calcitriol

Technical Field

The invention belongs to the technical field of chemical pharmacy, and particularly relates to a purification method of calcitriol.

Background

Calcitriol (Calcitriol) with the chemical name: 1, 25-dihydroxycholecalciferol or 1, 25-dihydroxyvitamin D ₃ The structural formula is as follows:

calcitriol is a metabolite of vitamin D3 which is metabolized by liver and kidney hydroxylase to be the most active in resisting rickets. Can promote the absorption of calcium in intestinal tract and regulate the calcification of bone. Calcitriol was first produced by roche, switzerland, marketed in 1978 under the trade designation "Rocaltrol", and was used for the treatment of postmenopausal osteoporosis, chronic renal dysfunction, postoperative hypothyroidism, idiopathic parathyroidism, pseudohypothyroidism, vitamin D-dependent rickets, hypophosphatemic vitamin D-resistant rickets, and the like. Because calcitriol is an endogenous substance, the curative effect is definite, and the calcitriol is safe and stable, the calcitriol is the first choice medicine for osteoporosis.

Calcitriol has high physiological activity, and the dosage of the calcitriol taken at a single time is very small, and the common treatment dosage is only 0.25 mu g/day to 1.0 mu g/day. Calcitriol is unstable in property, sensitive to light and heat, contains a plurality of chiral centers, is long in the traditional photochemical synthesis route, inevitably generates isomer impurities and other related substances easily in the synthesis process, and is difficult to separate and purify, so that the synthesis process technology threshold is high and the difficulty is high.

The preparation process of the calcitriol is different from the traditional photochemical synthesis method and is prepared by adopting a microbial fermentation method, two extremely high impurities PZA and PZB are unexpectedly found in the process of extracting and purifying fermentation liquor, the total content of the two extremely high impurities is more than 10%, the polar position of the impurity is found to be close to that of the calcitriol by a thin-layer point plate, and the two extremely high impurities are difficult to remove by adopting purification methods such as column chromatography, DAC dynamic preparation and the like, so that higher difficulty is brought to the purification work of final products.

Disclosure of Invention

In order to solve the problems, the invention provides a method for purifying calcitriol, which is obtained by separating and purifying fermentation liquor containing a calcitriol crude product obtained by microbial fermentation, TBS protection, deprotection, refining and purification, has mature production process and mild conditions, and is suitable for industrial production.

The technical scheme for realizing the invention is as follows:

the invention provides a purification method of calcitriol, which comprises the following steps:

(1) the fermentation process comprises the following steps: taking alfacalcidol as a starting material, converting the alfacalcidol into calcitriol through one-step microbial fermentation, centrifuging the obtained fermentation liquor to remove fungus residues, and collecting supernatant;

(2) and (3) crude product extraction and purification: extracting the fermented supernatant by using an organic solvent, collecting an organic phase, and separating by using column chromatography to obtain a calcitriol crude product 1;

(3) TBS protection of hydroxyl groups: introducing a protecting group TBS into hydroxyl groups at 1 and 3 positions of a calcitriol crude product 1, and separating by column chromatography to obtain a calcitriol TBS protecting substance;

(4) TBS removal: and (3) carrying out TBS removal protection on the TBS protection substance of calcitriol, carrying out column chromatography separation, crystallizing and filtering to obtain the calcitriol.

In some embodiments, the microbial fermentation conversion method in step (1) is to perform fermentation culture and centrifugal separation on the Pseudonocardia HRW001 to obtain the thallus of the Pseudonocardia HRW001, and apply the thallus to the starting material alfacalcidol for microbial conversion.

In some embodiments, the specific method for extracting the crude product in step (2) is as follows: transferring the fermentation supernatant into a reaction kettle, adding ethyl acetate, stirring, standing for 3-5 min, separating liquid, collecting an organic layer, introducing a water layer into the reaction kettle, adding ethyl acetate, stirring, standing for 3-5 min, separating liquid, collecting the organic layer, removing the water layer, combining the organic layers, stirring and drying with anhydrous sodium sulfate, filtering, concentrating the filtrate at 40-50 ℃ under reduced pressure until the filtrate is dry, and dissolving a distillation substrate with n-hexane at 40-50 ℃ under normal pressure for later use.

In some embodiments, the eluent used for column chromatography separation in step (2) is ethyl acetate-n-hexane solution by the following specific method: eluting with n-hexane/ethyl acetate (5/1), changing to the n-hexane/ethyl acetate (1/1) to elute the main product, monitoring by TLC, and after elution is finished, concentrating the main point eluent at 40-50 ℃ under reduced pressure to obtain a calcitriol crude product 1.

In some embodiments, the specific method for protecting hydroxyl groups with TBS in step (3) is: dissolving TBSCl in tetrahydrofuran for later use, dissolving a calcitriol crude product 1 in tetrahydrofuran, putting the mixture into a reaction bottle, adding imidazole, controlling the temperature to be below 30 ℃, dropwise adding a TBSCl tetrahydrofuran solution for later use, keeping the temperature to be 25 +/-5 ℃ for reaction for 2-3 h, monitoring the reaction by TLC, after the reaction is completed, adding purified water, stirring, standing, layering, collecting an organic layer, stirring and drying with anhydrous sodium sulfate, filtering, concentrating the filtrate at 40-50 ℃ under reduced pressure until the filtrate is dry, and dissolving a distillation substrate in normal pressure at 40-50 ℃ with normal hexane for later use.

In some embodiments, the eluent used for column chromatography separation in step (3) is ethyl acetate-n-hexane solution by the following specific method: eluting the precursor impurities by using normal hexane/ethyl acetate (100/1), then eluting the main product by using normal hexane/ethyl acetate (1/1), monitoring by using TLC (thin layer chromatography), and after the elution is finished, concentrating the main point eluent at 40-50 ℃ under reduced pressure to obtain the calcitriol TBS protective substance.

In some embodiments, in the step (4), the calcitriol TBS protective substance is dissolved in tetrahydrofuran and transferred to a reaction flask, 1M TBAF-THF is added, nitrogen is replaced three times and protected, the temperature is raised to 50-60 ℃ for reaction for 3-4 h, TLC monitors the reaction, purified water is added after the reaction is completed, the mixture is stirred, the mixture is stood still and layered, an organic layer is collected, the mixture is stirred and dried with anhydrous sodium sulfate, the filtrate is filtered, the filtrate is concentrated to dryness at 40-50 ℃ under reduced pressure, and a distillation substrate is dissolved in normal pressure at 40-50 ℃ by normal hexane for standby.

In some embodiments, the eluent used for column chromatography separation in step (4) is ethyl acetate-n-hexane solution by the following specific method: eluting with normal hexane/ethyl acetate (5/1), eluting a main product with normal hexane/ethyl acetate (1/1), monitoring by TLC (thin layer chromatography), concentrating the main point eluent at 40-50 ℃ under reduced pressure, dissolving a distillation substrate with 3-5 times of ethyl acetate at 40 ℃, dropwise adding 3-5 times of ethyl acetate, heating to 50 +/-5 ℃, slowly cooling to room temperature, cooling to 0 +/-5 ℃, stirring, crystallizing for 0.5-3 h, filtering, and drying a filter cake to obtain calcitriol.

In some embodiments, the method comprises a further refining process, specifically, dissolving calcitriol prepared in the step (4) in ethyl formate, heating to 40-50 ℃ for dissolving under the protection of nitrogen, slowly cooling to room temperature, then cooling to-20 +/-5 ℃, stirring for crystallization for 2-3 hours, heating, cooling, crystallizing and filtering to obtain a pure calcitriol product.

The purity of the calcitriol purified by the method is more than 98%, preferably more than 99%, and most preferably more than 99.8%.

Drawings

FIG. 1 is a HPLC chromatogram for detecting related substances in fermentation supernatant.

FIG. 2 is a HPLC chromatogram for detecting related substances in crude calcitriol 1.

FIG. 3 is a HPLC chromatogram for detecting related substances in the crude calcitriol 2.

FIG. 4 is HPLC chromatogram for detecting related substances of calcitriol pure product.

Detailed Description

The present invention will be described in further detail with reference to the following examples and figures, which are illustrative only and not intended to be limiting, and the scope of the present invention is not limited thereto, and the materials used are commercially available or may be obtained by the self-made method unless otherwise specified.

EXAMPLE 1 Synthesis (fermentation Process) of calcitriol crude 1

The calcitriol is prepared by a microbial fermentation method, and the specific preparation method refers to the following patent publication numbers: CN110527650A, obtained by the method described in examples 1-5, using alfacalcidol as a starting material, and converting the alfacalcidol into a calcitriol crude product through one-step microbial fermentation. Pseudonocardia HRW001 for microbial fermentation. Identified by China general microbiological culture Collection center, the strain is classified and named as Pseudonocardia Pseudonocardia sp.HRW001, the strain is preserved in the China general microbiological culture Collection center in 2019 at 03.04.17524, and the preservation number is CGMCC No. 17524. HPLC detection results of fermentation supernatant related substances after the fermentation broth is subjected to centrifugal separation are shown in table 1, corresponding to figure 1.

TABLE 1

	Calcitriol producens	Impurity PY1	Calcitriol	Impurity PZA	Impurity PZB	Alfacalcidol
							Time to peak min	17.430	18.870	19.913	23.753	26.153	32.513
Peak area ratio%	2.48	0.64	72.75	4.31	7.82	2.00

The above results show that, in the process of extraction and purification of fermentation liquor, besides the impurity preosteviol and the impurity PY1 (trans-calcitriol) of the traditional process, two impurities PZA and PZB with extremely high content are also discovered unexpectedly, and the total content is more than 10%.

The two impurities are obtained by derivatization, separation and purification, and the structural formula and the nomenclature are as follows after the structure confirmation:

impurity PZA:

chinese culture name: (5Z,7E) -9, 10-Ring-opened cholest-5, 7,10(19) -triene-1 alpha, 3 beta, 26-triol

Chemical structural formula:

impurity PZB:

chinese culture name: (5Z,7E) -9, 10-Ring-opened cholest-5, 7,10(19) -triene-1 alpha, 3 beta, 24-triol

Chemical structural formula:

EXAMPLE 2 extraction and purification of crude calcitriol 1

Transferring 160L (calculated by 90g of substrate) of the centrifuged fermentation liquor into a 500L reaction kettle, adding 25L of ethyl acetate, stirring for 3-5 min, standing for 3-5 min, separating liquid, collecting an organic layer, introducing a water layer into the reaction kettle, adding 25L of ethyl acetate, stirring for 3-5 min, standing for 3-5 min, separating liquid, collecting the organic layer, and removing the water layer. And combining the two organic layers, drying the organic layer by using 1.8kg of anhydrous sodium sulfate, filtering, concentrating the filtrate at 40-50 ℃ under reduced pressure until the filtrate is dry, and adding 0.45L of n-hexane into the distillation substrate to dissolve the distillation substrate at 40 ℃ under normal pressure for later use.

Preparing a column with the diameter of 12cm and the height of about 120cm, weighing 1.8kg of 100-200-mesh silica gel, loading a standby n-hexane dissolving solution into the column at normal pressure, using 8.0-9.0L of n-hexane/ethyl acetate 5/1 for eluting the precursor impurity, using n-hexane/ethyl acetate 1/1 for eluting a main product, monitoring by TLC (a developing agent: n-hexane/ethyl acetate 1/2, a color development method: ultraviolet 254nm + phosphomolybdic acid heating), after the elution is finished, concentrating the main point eluent at 40-50 ℃ under reduced pressure, and drying to obtain 57.0g of oily matter, namely calcitriol crude product 1(GC-C1).

The HPLC detection results of related substances of the calcitriol crude product 1 are shown in Table 2 and correspond to the attached figure 2.

TABLE 2

	Calcitriol producens	Impurity PY1	Calcitriol	Impurity PZA	Impurity PZB
						Time to peak min	17.430	18.647	19.890	23.733	26.147
Peak area ratio%	1.14	0.47	89.56	4.7	1.6

The results show that the calcitriol crude product obtained by extracting and purifying the fermentation liquor by silica gel column chromatography still contains high-content impurities PZA and PZB, and the impurities cannot be effectively removed by the traditional purification mode.

From the structural comparison of the two impurities PZA and PZB with calcitriol, it is not difficult to find the difference of the main structural difference in the position of the side chain introduced hydroxyl group, wherein, the hydroxyl at the 26 and 24 positions of the side chains of the impurities PZA and PZB is easy to introduce TBS protecting groups, and the steric hindrance of 25-hydroxyl of calcitriol is large, and TBS protecting groups are difficult to introduce, so that a crude product obtained after fermentation, by introducing TBS protecting group, only 1, 3-hydroxy is protected by TBS by calcitriol, and three hydroxyls of the impurities PZA and PZB can be protected by TBS, so that the impurities PZA and PZB can be separated by column chromatography through the difference of the polarity of the three after the TBS protection, the hydroxylTBS protectors of the impurities PZA and PZB can be directly removed, the purpose of separating and removing the impurities PZA and PZB can be indirectly achieved, and the obtained calcitriol 1 and 3-position hydroxylTBS protectors are subjected to TBS removal protection, column chromatography purification and recrystallization to obtain the calcitriol with higher purity.

EXAMPLE 3 calcitriol 1, 3-dihydroxy TBS protection

The reaction formula is as follows:

weighing 92.9g of TBSCl, dissolving in 114ml of tetrahydrofuran for later use, dissolving the crude product 1 in 570ml of tetrahydrofuran at room temperature, putting into a 2.0L three-mouth reaction bottle, adding 83.8g of imidazole, dropwise adding a TBSCl THF solution for later use at the temperature of below 30 ℃, keeping the temperature for 2-3 h at 25 +/-5 ℃, reacting for 2-3 h, monitoring the reaction by TLC, adding 285ml of purified water after the reaction is completed, stirring for 3-5 min, standing for 3-5 min, layering, collecting an organic layer, stirring and extracting a water layer with 285ml of ethyl acetate for 3-5 min, standing for 3-5 min, layering, discarding the water layer, combining the organic layers, stirring and drying with 171g of anhydrous sodium sulfate, filtering, concentrating the filtrate at 40-50 ℃ under reduced pressure until no liquid drops drop occurs, concentrating for 10-20 min again to obtain a light yellow substance, and dissolving in 513ml of n-hexane at 40 ℃ under normal pressure for later use.

Preparing a column with the diameter of 12cm and the height of about 120cm, weighing 1.8kg of 100-200-mesh silica gel, loading an n-hexane solution in which oily substances are dissolved into the column at normal pressure, eluting with n-hexane/ethyl acetate (100/1), eluting after the loading is finished (thin layer development is adopted, developing agent: n-hexane/ethyl acetate (10/1)), eluting main product points with the n-hexane/ethyl acetate (1/1), and concentrating the main point eluent at 40-50 ℃ under reduced pressure to obtain 70g of calcitriol TBS protector.

EXAMPLE 4 preparation of crude calcitriol 2

The reaction formula is as follows:

dissolving 70g of calcitriol TBS protector by 700ml of tetrahydrofuran, transferring the dissolved calcitriol TBS protector into a 2.0L three-mouth reaction bottle, adding 553ml of 1M TBAF-THF, replacing nitrogen for three times and protecting, heating to 50-60 ℃ for reaction for 3-4 h, monitoring by TLC, after the reaction is completed, concentrating to be dry at 40-50 ℃ under reduced pressure, adding 700ml of ethyl acetate and 700ml of purified water into a distillation substrate, stirring for 5-10 min, standing for 3-5 min, layering, discarding a water layer, adding 140g of anhydrous sodium sulfate into an organic phase, stirring and drying for 30 +/-5 min, filtering, concentrating a filtrate at 40-50 ℃ under reduced pressure to be dry, and dissolving the distillation substrate by 350ml of ethyl acetate at 40-50 ℃ under normal pressure for later use.

Preparing a column with the diameter of 5cm and the height of about 150cm, weighing 700g of 100-200 mesh silica gel, loading a standby ethyl acetate solution into the column at normal pressure, using the column after loading, eluting the previous impurities with 8.0-9.0L of n-hexane/ethyl acetate (5/1), then using n-hexane/ethyl acetate (1/1) to elute a main product (eluent can be repeatedly used when the product does not appear), monitoring impurity points (a developing agent: n-hexane/ethyl acetate (1/2)) behind the main product through a thin layer during the process, heating and developing through phosphomolybdic acid, collecting main point eluent which is confirmed to be not crossed with the impurity points, then concentrating to be dry at 40-50 ℃ under reduced pressure, dissolving a distillation substrate at 40 ℃ by using ethyl acetate with the volume of 3-5 times of the weight of the distillation substrate, then dropwise adding n-hexane with the volume of 3-5 times of the ethyl acetate, heating to 50 ℃ and maintaining for 0.5h after dropwise adding, cooling to 0 ℃, crystallizing for 0.5h, filtering, drying a filter cake at 30-40 ℃ for 2h to obtain 25.80g of calcitriol crude product 2 (GC-C2).

The HPLC detection results of related substances of the calcitriol crude product 2 are shown in Table 3 and correspond to the attached figure 3.

TABLE 3

	Calcitriol producens	Impurity PY1	Calcitriol	Impurity PZB
					Time to peak min	17.457	18.847	19.940	26.193
Peak area ratio%	0.46	0.17	99.22	0.04

EXAMPLE 5 calcitriol purification

Adding 25.80g of calcitriol crude product 2 and 516ml of ethyl formate into a 1000ml three-mouth reaction bottle, replacing nitrogen for three times and protecting, heating to 40-50 ℃ to dissolve, slowly cooling to room temperature, then cooling to-20 +/-5 ℃, stirring to crystallize for 2-3 h, filtering, and drying a filter cake in vacuum at 40-50 ℃ for 30-40 h to obtain 20.60g of calcitriol pure product (GC), wherein the yield is 79.84%.

The HPLC detection results of the related substances of the calcitriol refined product are shown in Table 4, corresponding to figure 4.

TABLE 4

	Calcitriol producens	Impurity PY1	Calcitriol
				Time to peak min	17.573	18.933	20.040
Peak area ratio%	0.07	0.05	99.81

The above results show that the purified calcitriol has a purity as high as 99.81%, and two fermentation impurities PZA and PZB are effectively removed.

Claims

1. A method for purifying calcitriol, which is characterized by comprising the following steps:

the fermentation process comprises the following steps: taking alfacalcidol as a starting material, converting the alfacalcidol into calcitriol through one-step microbial fermentation, centrifuging the obtained fermentation liquor to remove fungus residues, and collecting supernatant;

and (3) crude product extraction and purification: extracting the fermented supernatant by using an organic solvent, collecting an organic phase, and separating by using column chromatography to obtain a calcitriol crude product 1;

TBS protection of hydroxyl groups: introducing a protecting group TBS into hydroxyl groups at 1 and 3 positions of a calcitriol crude product 1, and separating by column chromatography to obtain a calcitriol TBS protecting substance;

TBS removal: and (3) carrying out TBS removal protection on the TBS protection substance of calcitriol, carrying out column chromatography separation, crystallizing and filtering to obtain the calcitriol.

2. The purification method according to claim 2, wherein the microbial fermentation conversion method in step (1) comprises subjecting the Pseudonocardia HRW001 to fermentation culture and centrifugal separation to obtain Pseudonocardia HRW001 cells, and applying the cells to the starting material alfacalcidol for microbial conversion.

3. The purification method according to claim 1, wherein the fermentation supernatant in the step (2) is transferred to a reaction kettle, ethyl acetate is added and stirred, the mixture is kept still for 3-5 min, the liquid is separated, the organic layer is collected, the water layer is introduced into the reaction kettle again, ethyl acetate is added and stirred, the mixture is kept still for 3-5 min, the liquid is separated, the organic layer is collected, the water layer is removed, the organic layers are combined, anhydrous sodium sulfate is used for stirring and drying, the filtration is carried out, the filtrate is concentrated to dryness under reduced pressure at 40-50 ℃, and the distillation substrate is dissolved by normal pressure by normal hexane at 40-50 ℃ for standby.

4. The purification method according to claim 1, wherein the eluent used in the column chromatography separation in step (2) is an ethyl acetate-n-hexane solution, and the specific method is as follows: eluting with n-hexane/ethyl acetate =5/1, then eluting with n-hexane/ethyl acetate =1/1 to obtain a main product, monitoring by TLC, and after elution is finished, concentrating the main point eluent at 40-50 ℃ under reduced pressure to obtain a calcitriol crude product 1.

5. The purification method of claim 1, wherein the TBS in step (3) protects hydroxyl groups by a specific method comprising: dissolving TBSCl in tetrahydrofuran for later use, dissolving a calcitriol crude product 1 in tetrahydrofuran, putting the solution into a reaction bottle, adding imidazole, dropwise adding a TBSCl tetrahydrofuran solution for later use at the temperature of below 30 ℃, keeping the temperature at 25 +/-5 ℃ for reaction for 2-3 h, monitoring the reaction by TLC, adding purified water after the reaction is completed, stirring, standing, layering, collecting an organic layer, stirring and drying with anhydrous sodium sulfate, filtering, concentrating the filtrate at 40-50 ℃ under reduced pressure to dryness, and dissolving a distillation substrate in normal pressure at 40-50 ℃ with normal hexane for later use.

6. The purification method according to claim 1, wherein the eluent used in the column chromatography separation in step (3) is an ethyl acetate-n-hexane solution, and the specific method is as follows: eluting the precursor impurities by using normal hexane/ethyl acetate =100/1, then eluting the main product by using normal hexane/ethyl acetate =1/1, monitoring by TLC, and after the elution is finished, concentrating the main point eluent at 40-50 ℃ under reduced pressure to obtain the calcitriol TBS protector.

7. The purification method as claimed in claim 1, wherein in the step (4), the calcitriol TBS protector is dissolved in tetrahydrofuran and transferred to a reaction bottle, 1M of TBAF-THF is added, nitrogen is replaced for three times and protected, the temperature is raised to 50-60 ℃ for reaction for 3-4 h, TLC is used for monitoring the reaction, after the reaction is completed, purified water is added, stirring is carried out, standing is carried out, layering is carried out, an organic layer is collected, anhydrous sodium sulfate is used for stirring and drying, filtering is carried out, the filtrate is concentrated to dryness under reduced pressure at 40-50 ℃, and a distilled substrate is dissolved in normal pressure at 40-50 ℃ by using normal hexane for standby.

8. The purification method according to claim 1, wherein the eluent used in the column chromatography separation in step (4) is an ethyl acetate-n-hexane solution, and the specific method is as follows: eluting with n-hexane/ethyl acetate =5/1, eluting a main product with n-hexane/ethyl acetate =1/1, monitoring by TLC, after elution is finished, concentrating the main point eluent at 40-50 ℃ under reduced pressure to dry, dissolving a distillation substrate with 3-5 times of ethyl acetate by weight at 40 ℃, dropwise adding 3-5 times of ethyl acetate by volume of n-hexane, after dropwise adding, heating to 50 +/-5 ℃, slowly cooling to room temperature, then cooling to 0 +/-5 ℃, stirring for crystallization for 0.5-3 h, filtering, and drying a filter cake to obtain calcitriol.

9. The purification method according to claim 1, wherein the method comprises a further refining process, and specifically comprises the steps of dissolving the calcitriol prepared in the step (4) in ethyl formate, heating to 40-50 ℃ for dissolution under the protection of nitrogen, slowly cooling to room temperature, then cooling to-20 +/-5 ℃, stirring for crystallization for 2-3 hours, heating for cooling, crystallizing and filtering to obtain a pure calcitriol product.

10. The purification method according to any one of claims 1 to 9, wherein the purity of the calcitriol pure product is greater than 98%, preferably greater than 99%, most preferably greater than 99.8%.