CN109851626B

CN109851626B - Method for separating and purifying temsirolimus

Info

Publication number: CN109851626B
Application number: CN201910017298.XA
Authority: CN
Inventors: 曹康平; 孙建; 谢义鹏; 刘艳霞
Original assignee: Sichuan Hairong Pharmaceutical Co Ltd Yangtze River Pharmaceutical Group
Current assignee: Sichuan Hairong Pharmaceutical Co Ltd Yangtze River Pharmaceutical Group
Priority date: 2019-01-08
Filing date: 2019-01-08
Publication date: 2021-11-02
Anticipated expiration: 2039-01-08
Also published as: CN109851626A

Abstract

The invention discloses a separation and purification method of temsirolimus, which is characterized by comprising the following steps: it comprises the following steps: (1) synthesizing a crude product; (2) and (5) separating and purifying. The method greatly improves the purity and yield of the temsirolimus by selecting a specific separation and purification method, reduces the cost and has wide market application prospect.

Description

Method for separating and purifying temsirolimus

Technical Field

The invention belongs to the field of separation and purification of a simulated drug, and particularly relates to a method for separating and purifying temsirolimus.

Background

Temsirolimus (Temsirolimus), an intravenous mTOR inhibitor, approved for the treatment of advanced renal cell carcinoma, is a derivative and prodrug of sirolimus. The temsirolimus is prepared from rapamycin (sirolimus) serving as a raw material by synthesis processes such as esterification and deprotection, and the finished temsirolimus product has low purity and high isomer content. Most of the research reports currently include methods for separating and purifying rapamycin (sirolimus) (CN104844620A, CN107619413A, and the like). The isolation and purification of temsirolimus are found to be less reported by a reaxys search. Zhengqi Son et al at the thirteenth national academy of antibiotics (pp. 196-201) published a refinement study of temsirolimus: purifying semi-synthesized crude temsirolimus by a silica gel column and a reversed phase HPLC, dissolving the crude temsirolimus in diethyl ether, stirring the mixture at the temperature of 20 ℃ for crystallization, refining the crude temsirolimus, wherein the yield is 70 percent, and the isomer ratio in the finished temsirolimus product is less than 0.5 percent.

According to reported documents, the research on the separation and purification of temsirolimus is less, the isomer content can reach below 1.0% by adopting a recrystallization mode, but the diethyl ether has a low boiling point, is inflammable and explosive, and is not suitable for industrial mass production.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a method for separating and purifying temsirolimus aiming at the existing synthesis process, which comprises the following steps:

(1) and (3) synthesis of a crude product:

a. preparation of acid chloride: dissolving 2,2, 5-trimethyl-5-carboxylic acid-1, 3-dioxane in n-hexane, adding thionyl chloride at 25 +/-5 ℃, stirring, and concentrating under reduced pressure to dryness to obtain acyl chloride;

b. ester forming reaction: mixing rapamycin, DIPEA and DCM, dripping acyl chloride obtained in the step a at 0-5 ℃, stirring at 25 +/-5 ℃, concentrating and evaporating to dryness, carrying out silica gel column chromatography on a crude product, and eluting to obtain an

esterified compound

2,2, 5-trimethyl [1, 3-dioxan ] -5-carboxylic acid-42-ester-rapamycin;

c. and (c) dissolving the esterified product in the step (b) in THF, dripping sulfuric acid aqueous solution at the temperature of 25 +/-5 ℃, reacting, adjusting the pH value to 7-8, extracting with EA, drying with a drying agent, and concentrating under reduced pressure to obtain a crude product of temsirolimus.

(2) Separation and purification: dissolving the crude product in the step c in ethanol-n-heptane, injecting into a forward preparative chromatographic column, carrying out gradient elution by using mobile phase ethanol-n-heptane, collecting the eluent by sections, combining the eluents with the purity of temsirolimus being more than or equal to 98%, and drying under reduced pressure to obtain a pure temsirolimus product;

the gradient elution procedure was: 0-25min, 100% → 50% n-heptane; 25-70min, 50% → 50% n-heptane; 70-90min, 50% → 20% n-heptane.

Further, the mass volume ratio of the 2,2, 5-trimethyl-5-carboxylic acid-1, 3-dioxane, n-hexane and thionyl chloride in the step a is 1 g: 14 ml: 1.37 g; the stirring time is 12-15 h; the acid chloride is a colorless oily liquid.

Further, in step b, the mass-to-volume ratio of rapamycin, DIPEA and DCM is 1 g: 0.72 ml: 2.83 ml; dripping acyl chloride until the concentration of acyl chloride is 0.75-1 g/ml; stopping stirring until the rapamycin content is less than 0.5%; the mesh number of the silica gel in the silica gel column is 200-300 meshes; the eluent used for the chromatography is petroleum ether: and (3) ethyl acetate.

Still further, the petroleum ether: the volume ratio of ethyl acetate is 1: 1.

Further, the mass-to-volume ratio of the esterified product, THF and the sulfuric acid aqueous solution in the step c is 1 g: 7.95 ml: 1.88 ml; the concentration of the sulfuric acid aqueous solution is 2 mol/L; the reaction time is 14 h; the pH adjusting agent is saturated sodium carbonate aqueous solution; the drying agent is anhydrous sodium sulfate.

Further, the mass-to-volume ratio of the crude product in the step (2) to ethanol-n-heptane is 0.2: 1 g/ml; the volume ratio of ethanol to n-heptane: 0-8: 1 to 2.

Further, the forward preparative chromatographic column in the step (2) is: x2 (4.6X 250mm, 10 μm, 14011407Y).

Further, the flow rate of the flowing phase in the forward preparative chromatographic column in the step (2) is 1mL/min, the column temperature is 30 ℃, and the wavelength is 277 nm.

Further, the step (2) of collecting the segments starts from 45min, and the collection is carried out 1 time every 1 min.

According to the temsirolimus separation and purification method, silica gel purification is performed on the key intermediate, namely the

esterified compound

2,2, 5-trimethyl [1, 3-dioxahexane ] -5-carboxylic acid-42-ester-rapamycin, under the condition of ensuring high conversion rate in the temsirolimus synthesis process, so that the subsequent purification difficulty of temsirolimus is reduced; the forward preparative chromatographic column is easier to concentrate and dry for separation and purification, and the content of isomers in the temsirolimus product can be effectively reduced; the n-heptane/ethanol is used for gradient elution, so that impurities with high polarity and low polarity in a sample can be fully separated, and the purity of the temsirolimus product is improved.

The temsirolimus product generated by the temsirolimus separation and purification method has the purity as high as 99 percent, the yield higher than 80 percent and the isomer content lower than 0.2 percent. Various indexes of the temsirolimus obtained by the separation and purification method are superior to those of products obtained in the prior art, and the method is simple and convenient to operate and suitable for industrial production.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 HPLC chromatogram for purification

FIG. 2 shows the product purity detection pattern and data (combined eluent with purity not less than 98%)

Detailed Description

The invention is further illustrated and described below by way of examples.

Example 1 isolation and purification of temsirolimus of the present invention

a. Dissolving 2,2, 5-trimethyl-5-carboxylic acid-1, 3-dioxane in n-hexane, adding thionyl chloride at 25 + -5 deg.C, mixing, and mixing at mass volume ratio of m_{2,2, 5-trimethyl-5-carboxylic acid-1, 3-dioxahexaalkane}：V_N-hexane：m_{Thionyl chloride}1 g: 14 ml: 1.37g, stirred for 15 h. Concentrating under reduced pressure to dryness to obtain colorless oily liquid, i.e. acyl chloride.

b. Rapamycin, N-Diisopropylethylamine (DIPEA) and Dichloromethane (DCM) are mixed in a mass-to-volume ratio of m_Rapamycin：V_DIPEA：V_DCM1 g: 0.72 ml: 2.83ml, dripping the acyl chloride prepared in the step a at 0-5 ℃ to ensure that the concentration of the acyl chloride is 1g/ml, stirring and reacting at 25 +/-5 ℃ for 22 hours, sampling, concentrating and evaporating to dryness after the content of rapamycin is detected to be less than 0.5 percent by HPLC, and obtaining a crude product by petroleum ether: ethyl acetate 1:1 as eluent, purifying by 200-300 mesh chromatographic column silica gel, isocratic eluting to obtain esterified 2,2, 5-trimethyl [1, 3-dioxane]-5-carboxylic acid-42-ester-rapamycin.

c. 2,2, 5-trimethyl [1, 3-dioxane)]Dissolving-5-carboxylic acid-42-ester-rapamycin in Tetrahydrofuran (THF), and adding dropwise 2mol/L sulfuric acid aqueous solution at 25 + -5 deg.C to obtain mass volume ratio of each component in the system_{2,2, 5-trimethyl [1, 3-dioxahexa-decane]-5-Carboxylic acid-42-ester-rapamycin}：V_THF：V_{Sulfuric acid water}1 g: 7.95 ml: 1.88ml, reacted for 14 h. Adjusting the pH value of a saturated sodium carbonate aqueous solution to 7-8, extracting with Ethyl Acetate (EA), drying with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain a crude temsirolimus product.

(2) Dissolving crude temsirolimus product in ethanol-n-heptane (volume ratio of 1:1) to obtain crude product with concentration of 0.2g/mL, and adopting a forward preparative chromatographic column X2(4.6 × 250mm, 10 μm, 14011407Y) with flow rate of 1mL/min, column temperature of 30 deg.C and wavelength of 277 nm. Eluting with ethanol-n-heptane (gradient elution with n-heptane/ethanol volume ratio of 1:0 to 1:1 within 0-25min, isocratic elution with n-heptane/ethanol volume ratio of 1:1 within 25-70min, gradient elution with n-heptane/ethanol volume ratio of 1:1 to 1:4 within 70-90 min), collecting eluate by stages (see fig. 1 and table 1), detecting by HPLC, mixing eluates with temsirolimus purity of not less than 98% (see fig. 2 and table 2), and drying under reduced pressure to obtain temsirolimus pure product.

The HPLC detection method comprises the following steps: using octyl silane bonded silica gel as a packed column (Agilent Zorbax SB-C8, 4.6mm multiplied by 150mm,3.5 μm), using a mobile phase A as water and a mobile phase B as methanol, wherein the flow rate is 0.8ml/min, the column temperature is 35 ℃, and the detection wavelength is 277 nm; the gradient elution procedure was as follows:

TABLE 1 eluent Collection

Fraction (b) of	Time	Fraction (b) of	Time	Fraction (b) of	Time
						F1	45.7～46.7	F9	53.7～54.7	F17	61.7～62.7
F2	46.7～47.7	F10	54.7～55.7	F18	62.7～63.7
						F3	47.7～48.7	F11	55.7～56.7	F19	63.7～64.7
F4	48.7～49.7	F12	56.7～57.7	F20	64.7～65.7
						F5	49.7～50.7	F13	57.7～58.7	F21	65.7～66.7
F6	50.7～51.7	F14	58.7～59.7	F22	66.7～67.7
						F7	51.7～52.7	F15	59.7～60.7	F23	67.7～68.7
F8	52.7～53.7	F16	60.7～61.7

Table 2 eluent purity data

The yield of the pure temsirolimus product obtained by the separation and purification method is 80.1 percent, the single impurity of the pure temsirolimus product detected by the HPLC method is less than 0.5 percent, and the ratio of the main body to the isomer is 99.4: 0.2.

The method has the advantages of high conversion rate, cost reduction, purification difficulty of temsirolimus, low isomer content in the obtained temsirolimus product, less high-polarity and low-polarity impurities, high product purity, improved quality and wide market application prospect.

Claims

1. A method for separating and purifying temsirolimus is characterized by comprising the following steps:

(1) and (3) synthesis of a crude product:

b. ester forming reaction: mixing rapamycin, DIPEA and DCM, dripping acyl chloride obtained in the step a at 0-5 ℃, stirring at 25 +/-5 ℃, concentrating and evaporating to dryness, carrying out silica gel column chromatography on a crude product, and eluting to obtain an esterified compound 2,2, 5-trimethyl [1, 3-dioxan ] -5-carboxylic acid-42-ester-rapamycin; the mesh number of the silica gel in the silica gel column is 200-300 meshes; the eluent used for the chromatography is petroleum ether: ethyl acetate;

c. dissolving the esterified product in the step b in THF, dripping sulfuric acid aqueous solution at the temperature of 25 +/-5 ℃, reacting, adjusting the pH to 7-8, extracting EA, drying by using a drying agent, and concentrating under reduced pressure to obtain a crude product of temsirolimus;

the gradient elution procedure was: 0-25min, 100% → 50% n-heptane; 25-70min, 50% → 50% n-heptane; 70-90min, 50% → 20% n-heptane;

the forward preparative chromatographic column is as follows: x2, size 4.6X 250mm, 10 μm, model 14011407Y.

2. The separation and purification method according to claim 1, wherein the mass volume ratio of the 2,2, 5-trimethyl-5-carboxylic acid-1, 3-dioxane, n-hexane and thionyl chloride in step a is 1 g: 14 ml: 1.37 g; the stirring time is 12-15 h; the acid chloride is a colorless oily liquid.

3. The separation and purification method according to claim 1, wherein the mass-to-volume ratio of rapamycin, DIPEA and DCM in step b is 1 g: 0.72 ml: 2.83 ml; dripping acyl chloride until the concentration of acyl chloride is 0.75-1 g/ml; and stirring until the rapamycin content is less than 0.5%.

4. The separation and purification method according to claim 1, wherein the petroleum ether: the volume ratio of ethyl acetate is 1: 1.

5. The separation and purification method according to claim 1, wherein the mass-to-volume ratio of the esterified product, THF and aqueous sulfuric acid solution in step c is 1 g: 7.95 ml: 1.88 ml; the concentration of the sulfuric acid aqueous solution is 2 mol/L.

6. The separation and purification method according to claim 1, wherein the reaction time in step c is 14 h; the pH adjusting agent is saturated sodium carbonate aqueous solution; the drying agent is anhydrous sodium sulfate.

7. The separation and purification method according to claim 1, wherein the mass-to-volume ratio of the crude product in step (2) to ethanol-n-heptane is 0.2: 1 g/ml; the volume ratio of ethanol to n-heptane: 0-8: 1 to 2.

8. The separation and purification method according to claim 1, wherein the flow rate of the mobile phase in the forward preparative chromatography column of step (2) is 1mL/min, the column temperature is 30 ℃ and the wavelength is 277 nm.

9. The separation and purification method according to claim 1, wherein the step (2) of collecting the fractions is started from 45min and collecting the fractions 1 time every 1 min.