CN109851626A - A kind of isolation and purification method of tesirolimus - Google Patents
A kind of isolation and purification method of tesirolimus Download PDFInfo
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- CN109851626A CN109851626A CN201910017298.XA CN201910017298A CN109851626A CN 109851626 A CN109851626 A CN 109851626A CN 201910017298 A CN201910017298 A CN 201910017298A CN 109851626 A CN109851626 A CN 109851626A
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- tesirolimus
- normal heptane
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Abstract
The invention discloses a kind of isolation and purification methods of tesirolimus, it is characterised in that: it the following steps are included: (1) crude product synthesis;(2) it isolates and purifies.The present invention reduces costs by selecting specific isolation and purification method to substantially increase the purity and yield of tesirolimus, has a vast market application prospect.
Description
Technical field
The invention belongs to imitation medicines to isolate and purify field, specially a kind of isolation and purification method of tesirolimus.
Background technique
Tesirolimus (tamsimos) (Temsirolimus) is a kind of mTOR inhibitors of intravenous injection, granted to be used to
Advanced renal cell carcinoma is treated, is the derivative and prodrug of sirolimus.Tesirolimus is original with rapamycin (sirolimus)
Material, by esterification, the synthesis technologies generation tesirolimus finished product purity such as deprotection is low, and content of isomer is higher.Research at present
The isolation and purification method (CN104844620A, CN107619413A etc.) of mostly rapamycin (sirolimus) of report.Through
Reaxys retrieval discovery tesirolimus isolate and purify report it is less.Zheng is from Son etc. in " the 13rd national antibiotic science
Meeting (page 196 page -201) " discloses a kind of purification research of tesirolimus: will be thick by the tesirolimus of semi-synthetic acquisition
Product are purified through silica gel column purification, reversed-phase HPLC, then use ether dissolved clarification, stirring and crystallizing at 20 DEG C, to tesirolimus crude product into
Row refines, yield 70%, isomer proportion < 0.5% in tesirolimus finished product.
It is less to can be seen that the research isolated and purified at present to tesirolimus from the document of report, using recrystallization
Mode can make content of isomer reach 1.0% hereinafter, still ether low boiling point and inflammable and explosive, it is big raw to should not be used in industrialization
It produces.
Summary of the invention
In order to solve the above-mentioned technical problem, it is an object of the invention to be directed to existing synthesis technology, provides a kind of for western sieve
The isolation and purification method that do not take charge of, it the following steps are included:
(1) synthesis of crude product:
A. the preparation of acyl chlorides: 2,2,5- trimethyl -5- carboxylic acid -1,3- dioxy, six alkane is dissolved in n-hexane, at 25 ± 5 DEG C
Thionyl chloride is added, stirring is concentrated to dryness, obtains acyl chlorides;
B. it is reacted at ester: rapamycin, DIPEA is mixed with DCM, acyl chlorides obtained by instillation step a at 0-5 DEG C, 25 ± 5 DEG C
Lower stirring, concentration are evaporated, crude product silica gel column chromatography, elute to obtain 2,2,5- trimethyl of carboxylate [1,3- dioxy, six alkane] -5- carboxylic
Acid -42- ester-rapamycin;
C. step b carboxylate is dissolved in THF, aqueous sulfuric acid is instilled at 25 ± 5 DEG C, reacted, adjust pH to 7-8, EA
Extraction is concentrated under reduced pressure with desiccant dryness, is obtained tesirolimus crude product.
(2) it isolates and purifies: step c crude product is dissolved in ethyl alcohol-normal heptane, inject positive preparation chromatographic column, use mobile phase
Ethyl alcohol-normal heptane gradient elution, Fractional Collections eluent merge the eluent of tesirolimus purity >=98%, are dried under reduced pressure,
Obtain tesirolimus sterling;
The gradient elution program are as follows: 0~25min, 100% → 50% normal heptane;25~70min, 50% → 50% just
Heptane;70-90min, 50% → 20% normal heptane.
Further, 2,2,5- trimethyl -5- carboxylic acid -1,3- dioxy, six alkane, n-hexane described in step a and thionyl chloride
Mass volume ratio is 1g:14ml:1.37g;The mixing time is 12~15h;The acyl chlorides is colourless oil liquid.
Further, the mass volume ratio of rapamycin, DIPEA and DCM described in step b is 1g:0.72ml:2.83ml;
The instillation acyl chlorides to acyl chlorides concentration is 0.75~1g/ml;;It is described stirring to rapamycin content less than 0.5% when stop;It is described
The mesh number of silica gel is 200-300 mesh in silicagel column;The eluant, eluent used that chromatographs is petroleum ether: ethyl acetate.
Further, the petroleum ether: ethyl acetate volume ratio 1:1.
Further, the mass volume ratio 1g:7.95ml:1.88ml of carboxylate, THF and aqueous sulfuric acid described in step c;
The aqueous sulfuric acid concentration is 2mol/L;The reaction time is 14h;The reagent for adjusting pH is that saturated sodium carbonate is water-soluble
Liquid;The desiccant is anhydrous sodium sulfate.
Further, step (2) crude product and ethyl alcohol-normal heptane mass volume ratio are 0.2:1g/ml;The second
Alcohol-normal heptane volume ratio: 0~8:1~2.
Further, step (2) the positive preparation chromatographic column are as follows: X2 (4.6 × 250mm, 10 μm, 14011407Y).
Further, flow rate of mobile phase is 1mL/min in step (2) the positive preparation chromatographic column, and column temperature is 30 DEG C, wave
A length of 277nm.
Further, step (2) described Fractional Collections are since 45min, and every 1min is collected 1 time.
Tesirolimus isolation and purification method of the invention guarantees high conversion in tesirolimus synthesis process
Under the conditions of, to key intermediate --- carboxylate 2,2,5- trimethyl [1,3- dioxy, six alkane] -5- carboxylic acid -42- ester-rapamycin
Silica gel purification is carried out, the purifying difficulty of subsequent tesirolimus is reduced;It is easier to using forward direction preparation chromatography column separating purification
It is concentrated and dried, the content of tesirolimus Isomers In Products can be effectively reduced;Carrying out gradient elution with normal heptane/ethyl alcohol can
It is sufficiently separated the big polarity in sample and low polar impurity, improves the purity of tesirolimus product.
The tesirolimus product purity that tesirolimus isolation and purification method of the invention generates is up to 99%, high income
In 80%, content of isomer is lower than 0.2%.The tesirolimus indices that isolation and purification method through the invention obtains are equal
Better than product obtained in the prior art, and the method for the present invention is easy to operate, is suitble to industrialized production.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
HPLC spectrogram when Fig. 1 is purified
Fig. 2 product purity detection map and data (merging purity >=98% eluent)
Specific embodiment
With embodiment, the present invention is further elaborated and explanation below.
The tesirolimus of the present invention of embodiment 1 isolates and purifies
A. 2,2,5- trimethyl -5- carboxylic acid -1,3- dioxy, six alkane is dissolved in n-hexane, is added two at 25 ± 5 DEG C
Chlorine sulfoxide, mass volume ratio m after mixingSix alkane of 2,2,5- trimethyl -5- carboxylic acid -1,3- dioxy: VN-hexane: mThionyl chloride=1g:14ml:1.37g stirs 15h.
It is concentrated to dryness, obtains colourless oil liquid, i.e. acyl chlorides.
B. rapamycin, n,N-diisopropylethylamine (DIPEA) are mixed with methylene chloride (DCM), mass volume ratio
mRapamycin: VDIPEA: VDCMThe acyl chlorides prepared in above-mentioned a is instilled at 0-5 DEG C, makes acyl chlorides concentration by=1g:0.72ml:2.83ml
1g/ml is sampled after being stirred to react 22h at 25 ± 5 DEG C, and after HPLC detects rapamycin content less than 0.5%, concentration is evaporated, institute
Crude product is obtained using petroleum ether: ethyl acetate 1:1 is eluant, eluent, and by 200-300 mesh chromatographic column silica gel purification, isocratic elution must be esterified
Object 2,2,5- trimethyl [six alkane of 1,3- dioxy] -5- carboxylic acid -42- ester-rapamycin.
C. 2,2,5- trimethyl [six alkane of 1,3- dioxy] -5- carboxylic acid -42- ester-rapamycin is dissolved in tetrahydrofuran
(THF) in, 2mol/L aqueous sulfuric acid is added dropwise at 25 ± 5 DEG C, makes the mass volume ratio of each ingredient in system
m2,2,5- trimethyl [six alkane of 1,3- dioxy] -5- carboxylic acid -42- ester-rapamycin: VTHF: VSulfuric acid water=1g:7.95ml:1.88ml reacts 14h.Saturated sodium carbonate water
Solution adjusts PH to 7-8, and ethyl acetate (EA) extraction, anhydrous sodium sulfate is dry, is concentrated under reduced pressure to give tesirolimus crude product.
(2) tesirolimus crude product is dissolved with ethyl alcohol-normal heptane (volume ratio 1:1), obtains crude product concentration 0.2g/ml, used
Forward direction preparation chromatographic column X2 (4.6 × 250mm, 10 μm, 14011407Y), flow velocity 1mL/min, column temperature are 30 DEG C, and wavelength is
277nm.With ethyl alcohol-, (normal heptane/ethyl alcohol volume ratio rises to 1:1 gradient elution, 25- by 1:0 in 0-25min for normal heptane elution
70min is that normal heptane/ethyl alcohol volume ratio is 1:1 isocratic elution, and normal heptane/ethyl alcohol volume ratio rises to 1:4 by 1:1 in 70-90min
Gradient elution), Fractional Collections eluent (see Fig. 1, table 1) merges washing for tesirolimus purity >=98% after HPLC is detected
De- liquid (see Fig. 2, table 2), is dried under reduced pressure to obtain tesirolimus sterling.
Aforementioned HPLC detection method are as follows: using octyl silane group silica gel as packed column (Agilent ZorbaxSB-C8,
4.6mm × 150mm, 3.5 μm), mobile phase A is water, and Mobile phase B is methanol, and flow velocity 0.8ml/min, detects wave by 35 DEG C of column temperature
A length of 277nm;Gradient elution program is as follows:
1 eluent of table is collected
Fraction | Time | Fraction | Time | Fraction | 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 |
2 eluent purity data of table
The tesirolimus sterling yield 80.1% that isolation and purification method obtains through the invention is detected through aforementioned HPLC method
Gained tesirolimus sterling list is miscellaneous less than 0.5%, and main body and ratios of the isomers are 99.4:0.2.
The method of the present invention has high conversion, reduces costs, also reduces the purifying difficulty of tesirolimus, obtain
Tesirolimus Isomers In Products content is low, and big polarity and low polar impurity are few, and product purity is high, improves quality, has
Wide market application prospect.
Claims (10)
1. a kind of isolation and purification method of tesirolimus, which is characterized in that it the following steps are included:
(1) synthesis of crude product:
A. the preparation of acyl chlorides: 2,2,5- trimethyl -5- carboxylic acid -1,3- dioxy, six alkane is dissolved in n-hexane, is added at 25 ± 5 DEG C
Thionyl chloride, stirring, is concentrated to dryness, obtains acyl chlorides;
B. it is reacted at ester: rapamycin, DIPEA is mixed with DCM, acyl chlorides obtained by step a is instilled at 0-5 DEG C, is stirred at 25 ± 5 DEG C
It mixes, concentration is evaporated, crude product silica gel column chromatography, elutes to obtain 2,2,5- trimethyl of carboxylate [1,3- dioxy, six alkane] -5- carboxylic acid -
42- ester-rapamycin;
C. step b carboxylate being dissolved in THF, aqueous sulfuric acid is instilled at 25 ± 5 DEG C, reacted, adjust pH to 7-8, EA is extracted,
With desiccant dryness, it is concentrated under reduced pressure, obtains tesirolimus crude product;
(2) it isolates and purifies: step c crude product is dissolved in ethyl alcohol-normal heptane, positive preparation chromatographic column is injected, with mobile phase ethyl alcohol-
Normal heptane gradient elution, Fractional Collections eluent merge the eluent of tesirolimus purity >=98%, are dried under reduced pressure, must replace
Sirolimus sterling;
The gradient elution program are as follows: 0~25min, 100% → 50% normal heptane;25~70min, 50% → 50% normal heptane;
70-90min, 50% → 20% normal heptane.
2. isolation and purification method according to claim 1, which is characterized in that 2,2,5- trimethyl -5- carboxylic acids-described in step a
The mass volume ratio of six alkane of 1,3- dioxy, n-hexane and thionyl chloride is 1g:14ml:1.37g;The mixing time be 12~
15h;The acyl chlorides is colourless oil liquid.
3. isolation and purification method according to claim 1, which is characterized in that rapamycin described in step b, DIPEA and DCM
Mass volume ratio is 1g:0.72ml:2.83ml;The instillation acyl chlorides to acyl chlorides concentration is 0.75~1g/ml;It is described to stir to thunder
Stop when pa mycin content is less than 0.5%;The mesh number of silica gel is 200-300 mesh in the silicagel column;It is described to chromatograph the elution used
Agent is petroleum ether: ethyl acetate.
4. isolation and purification method according to claim 3, which is characterized in that the petroleum ether: ethyl acetate volume ratio 1:
1。
5. isolation and purification method according to claim 1, which is characterized in that carboxylate described in step c, THF and sulfuric acid are water-soluble
The mass volume ratio 1g:7.95ml:1.88ml of liquid;The aqueous sulfuric acid concentration is 2mol/L.
6. isolation and purification method according to claim 1, which is characterized in that the reaction time described in step c is 14h;The tune
The reagent of pH is saturated aqueous sodium carbonate;The desiccant is anhydrous sodium sulfate.
7. isolation and purification method according to claim 1, which is characterized in that step (2) crude product and ethyl alcohol-normal heptane
Mass volume ratio is 0.2:1g/ml;The ethyl alcohol-normal heptane volume ratio: 0~8:1~2.
8. isolation and purification method according to claim 1, which is characterized in that step (2) the positive preparation chromatographic column are as follows: X2
(4.6 × 250mm, 10 μm, 14011407Y).
9. isolation and purification method according to claim 1, which is characterized in that flowed in step (2) the positive preparation chromatographic column
Dynamic phase flow velocity is 1mL/min, and column temperature is 30 DEG C, wavelength 277nm.
10. isolation and purification method according to claim 1, which is characterized in that step (2) described Fractional Collections are from 45min
Start, every 1min is collected 1 time.
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CN114814020A (en) * | 2022-04-20 | 2022-07-29 | 安康市农产品质量安全检验监测中心 | Method for analyzing residual organophosphorus pesticide in agricultural products |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN114814020A (en) * | 2022-04-20 | 2022-07-29 | 安康市农产品质量安全检验监测中心 | Method for analyzing residual organophosphorus pesticide in agricultural products |
CN114814020B (en) * | 2022-04-20 | 2023-08-29 | 安康市农产品质量安全检验监测中心 | Analysis method for residual organophosphorus pesticide in agricultural products |
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