CN104086564B - A kind of preparation method of high-purity tamiros - Google Patents
A kind of preparation method of high-purity tamiros Download PDFInfo
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Abstract
The invention discloses high-purity tamiros shown in a kind of formula (I), and HPLC purity is up to 99.5% or more.The present invention also provides the methods for preparing above-mentioned high-purity tamiros by preparative high performance liquid chromatography separation system, including unlimited order carry out step: A) Normal-phase HPLC and B) reversed-phase high performance liquid chromatography separation, wherein step A) stationary phase is silicon ball filler, mobile phase is that 60~100v/v% of ethyl acetate and n-hexane or 40~0v/v% of normal heptane carry out gradient elution；Step B) stationary phase is C4, C6, C8 or C18 filler, mobile phase is acetonitrile and pH is 3.5~6.0 aqueous solution carries out gradient elution；System temperature is 10~30 DEG C.This method simple process, it is environmentally friendly, high-purity, the tamiros of high quality can be made.
Description
Technical field
The present invention relates to field of pharmaceutical chemistry technology, and in particular to a kind of preparation method of high-purity tamiros.
Background technique
Tamiros (temsirolimus) also known as tesirolimus, trade name Torisel, entitled 42- [the 3- hydroxyl of chemistry
Base -2- (methylol) -2 Methylpropionic acid ester]-rapamycin, molecular formula C56H87NO16, structural formula is as follows:
Tamiros is a kind of targeting mTOR kinases inhibitor, is the thunder generated by Streptomyces hygroscopicus FC904 fermentation
The derivative of pa mycin (sirolimus).In May, 2007, U.S. FDA ratify tamiros and are used for intractable advanced renal cell carcinoma
First-line treatment, November in the same year, Europe EMBA also ratified its listing.Tamiros is cut as molecular targeted therapy for operation
It removes, radiotherapy, chemotherapy, the undesirable advanced metastatic patients with renal cell carcinoma of immunization therapy and other effects bring dawn.
Since the commercial dosage forms of tamiros are injection with small volume, to the quality requirement of tamiros bulk pharmaceutical chemicals
Height, especially to remaining sirolimus raw material, isomers and other related degradation impurity, clarity of solution, the side such as loss on drying
Face control is tighter.During preparing tamiros, due to tamiros structural instability itself, especially itself is non-
It often is easy to be converted to isomer impurities, causes the related substance detection in bulk pharmaceutical chemicals finished product to be difficult up to standard.If storage is improper,
Such as high temperature and humidity condition or strong illumination, it is easy to which open loop of degrading generates impurity.
The purification process of existing tamiros (i.e. tesirolimus) is typically implemented in the post-processing that crude product is prepared
In.CN103664999A discloses a kind of method of purification: tesirolimus precursor is placed in 5mL THF, 2N HCl is added dropwise, 0 DEG C is stirred
12h is mixed, TLC judges reaction end.The dilution of 10mL ethyl acetate is added after the reaction was completed, 10mL water is added, with 20mL acetic acid second
Ester extracts organic phase three times, merges organic phase.Organic phase saturated sodium carbonate solution washing one time, water washing three times, saturated common salt
Water washing one time, anhydrous magnesium sulfate is dry.Silica gel column chromatography after vacuum distillation, ethyl acetate: n-hexane (2:1~4:1) is quick
Elution, obtains tesirolimus.
CN103421023A discloses a kind of method from tesirolimus precursor preparation tesirolimus, in 1L round bottom
Investment 75g tesirolimus precursor, 1g p-methyl benzenesulfonic acid are dissolved in the tetrahydrofuran of 400mL in flask, and 5mL is added dropwise at 0~5 DEG C
Ethylene glycol drips off in 10min, and after stirring 1h, reaction terminates, and the water of 1L is added, and (500mL*3) three times is extracted with ethyl acetate,
Gained ethyl acetate layer washes (200mL*3) three times with saturated sodium-chloride again, and after being dried over anhydrous sodium sulfate, evaporated under reduced pressure obtains crude product
72g, for gained crude product through silica gel column chromatography, PE:AC=4:1 elutes to obtain 45g tesirolimus.
Above-mentioned method of purification is all silica gel column chromatography, and product purity obtained is not high, poor to impurity removal effect, and
Starting material sirolimus content is not controlled, is difficult the quality requirement for meeting injection type to bulk pharmaceutical chemicals.Therefore, it is
Effective removing impurity, obtains the other tamiros of injection stage of higher purity, need to develop effectively reliable method to its into
Row purifying preparation.
Summary of the invention
The purpose of the present invention is to provide a kind of high-purity tamiros and preparation method thereof.The method passes through efficient liquid
Phase chromatographic system removes remaining sirolimus and other organic impurities in crude product, simple and safe, environmentally friendly, effectively improves
The purity of product meets ejection preparation to the quality requirement of bulk pharmaceutical chemicals.
To achieve the above object, the present invention the following technical schemes are provided:
According to the first aspect of the invention, a kind of high-purity tamiros is provided, chemical structure is shown in formula I, institute
The HPLC purity for stating high-purity tamiros is greater than 98%,
In a preferred embodiment, the HPLC purity of the high-purity tamiros is greater than 99%.
In further preferred embodiment, the HPLC purity of the high-purity tamiros is greater than 99.5%, even
99.9%.
According to the second aspect of the invention, a kind of method for preparing above-mentioned high-purity tamiros is provided, comprising steps of
A) Normal-phase HPLC: by tamiros crude product through performance liquid chromatographic column purification system, with positive phase filling
For stationary phase, is eluted, obtained as mobile phase using the mixture of ethyl acetate and normal heptane or ethyl acetate and n-hexane
Tamiros semi-finished product；
B it) reversed-phase high performance liquid chromatography: by tamiros semi-finished product through performance liquid chromatographic column purification system, is filled out with reverse phase
Material is stationary phase, is eluted using the mixture of acetonitrile and the aqueous solution of pH3.5~6.0 as mobile phase, obtains smooth sieve of high-purity
Mo Si.
The step A) in, positive phase filling is silicon ball filler.The partial size of silicon ball filler can be within the scope of 5~45 μm
Any number or section, such as 5,10,15,20,25,20~45 μm, etc., preferably 5 μm, 10 μm, more preferable 10 μm.
The step A) in, tamiros crude product, which can be dissolved in ethyl acetate, carries out upper prop.Tamiros crude product with
The w/v of ethyl acetate can be 1g/3~10ml, preferably 1g/5ml.
The step A) in, with the total volume meter of the mobile phase, contain 60~100v/ of ethyl acetate in mobile phase
V% contains 40~0v/v% of n-hexane or normal heptane.
The step A) in, type of elution is preferably gradient elution, and in 0~10min of elution, mobile phase contains acetic acid second
Ester 60v/v%, containing n-hexane or normal heptane 40v/v%, the content of ethyl acetate in 10~100min of elution, mobile phase
100v/v% is changed to from 60v/v%, while the content of normal heptane or n-hexane changes to 0v/v% from 40v/v%.
The step A) in, the system temperature of performance liquid chromatographic column is 10~30 DEG C, such as 10,15,20,25,30
DEG C, preferably 15~25 DEG C, more preferable 20 DEG C.
The step A) in, gradient elution Fractional Collections obtain the eluent containing tamiros, by the eluent of collection
It is concentrated under reduced pressure at 30 DEG C or less, obtains tamiros semi-finished product.It is preferably concentrated under reduced pressure and is carried out at 20~30 DEG C.
The step B) in, reverse phase filler is selected from C4, C6, C8 or C18 filler, preferably C18 filler.The partial size of reverse phase filler
It can be any number in 5~45 μ ms or section, such as 5,10,15,20,25,20~45 μm etc., preferably 5 μm, 10 μ
M, more preferable 10 μm.
The step B) in, tamiros semi-finished product, which can be dissolved in acetonitrile, carries out upper prop.Tamiros semi-finished product with
The w/v of acetonitrile is 1g/3~10ml, preferably 1g/5ml.
The step B) in, aqueous solution is that acidic materials are added in water to be formulated.Acidic materials can be this field
Any acid or ackd salt that technical staff knows etc., including organic acid, inorganic acid, disulfate, hydrophosphate etc., such as preferably
Have formic acid, acetic acid, propionic acid, phosphoric acid, sulfuric acid, potassium acid sulfate, sodium bisulfate, ammonium hydrogen sulfate, potassium dihydrogen phosphate, biphosphate
Sodium, ammonium dihydrogen phosphate, potassium phosphate etc..Those skilled in the art can easily adjust the amount of acidic materials addition, make the water
The pH of solution is 3.5~6.0, and preferably pH is 4~5.5.
The step B) in, with the total volume meter of the mobile phase, contains 50~80v/v% of acetonitrile in mobile phase, contain
50~20v/v% of aqueous solution of pH3.5~6.0.
The step B) in, type of elution is preferably gradient elution, and in 0~10min of elution, mobile phase contains acetonitrile
50v/v%, the aqueous solution 50v/v% containing pH3.5~6.0, in 10~100min of elution, mobile phase the content of acetonitrile from
50v/v% changes to 80v/v%, while the content of the aqueous solution of pH3.5~6.0 changes to 20v/v% from 50v/v%.
The step B) in, the system temperature of performance liquid chromatographic column is 10~30 DEG C, such as 10,15,20,25,30
DEG C, preferably 15~25 DEG C, more preferable 20 DEG C.
The step B) in, gradient elution Fractional Collections obtain the eluent containing tamiros.By the eluent of collection
It is concentrated under reduced pressure at 30 DEG C or less, it is dry, obtain high-purity tamiros.Preferably, the eluent of collection can be first concentrated under reduced pressure into
On a small quantity, it is extracted with ethyl acetate, saturated sodium chloride solution washing dries, filters, is concentrated to dryness, obtains high-purity Tan Luomo
Department.It is preferably concentrated under reduced pressure and is carried out at 20~30 DEG C.
In addition, the present inventor is also the study found that the sequencing of Normal-phase HPLC and reversed-phase high performance liquid chromatography
It can be interchanged, high-purity tamiros is equally prepared, i.e., the present invention also provides another preparation high-purity tamiros
Method, comprising steps of
1) reversed-phase high performance liquid chromatography: by tamiros crude product loading to performance liquid chromatographic column, it is with reverse phase filler
Stationary phase is eluted using the mixture of acetonitrile and the aqueous solution of pH3.5~6.0 as mobile phase, and tamiros semi-finished product are obtained；
2) Normal-phase HPLC: by tamiros semi-finished product loading to performance liquid chromatographic column, with positive phase filling
For stationary phase, is eluted, obtained as mobile phase using the mixture of ethyl acetate and normal heptane or ethyl acetate and n-hexane
High-purity tamiros.
Wherein, step 1) and carrying out respectively with the step B in the above method) and A) unanimously 2), it is right in a kind of upper method
Step A) and step B) description can quote and be respectively seen as herein to step 1) and restriction 2).
The preparation method provided according to the present invention, tamiros crude product is by two step high performance liquid chromatography of positive and reverse phase
After reason, the HPLC purity of resulting tamiros can reach 99.5% or more, and single impurity is less than 0.1%, and (isomers removes
Outside), the standard for meeting European Union ICH meets injection to the quality requirement of bulk pharmaceutical chemicals.
Method of the invention can carry out in full-automatic preparative high performance liquid chromatography column separating purification system, technique letter
Single, repeatability is high, and product purity is high, and quality is stablized, and preparation process does not use toxic reagent, on operator and environment influence compared with
It is small, there is good prospects for commercial application.
Specific embodiment
The features and advantages of the invention are further illustrated below by preferred embodiment.It should be understood that following realities
It applies the method in example and is merely to illustrate the present invention, rather than limiting the invention.
The purity of tamiros of the present invention, which refers to, carries out the Tan Luomo that area normalization method obtains according to HPLC detection data
The percentage that the peak area of department's compound occupies in all peak area summations.The content of sirolimus etc. refers to be examined according to HPLC
The peak area that measured data carries out the respective substances such as the sirolimus that area normalization method obtains occupies in all peak area summations
Percentage.
Embodiment 1
1) preparation of tamiros semi-finished product
By 8.3g tamiros crude product (purity 75.68%, sirolimus 3.47%, isomers 16.48%, area normalization
Change method, similarly hereinafter) with after the dissolution of 25mL ethyl acetate, being splined on positive silicon ball filled column, (it is limited that biological chromatography technology is matched in Nanjing hundred
Company, similarly hereinafter, 5 μm of partial size), gradient elution, 0~10min, acetic acid second are carried out with ethyl acetate: normal heptane mixed solvent system
Ester/normal heptane (v/v, similarly hereinafter)=60:40, in 10~100min, ethyl acetate/normal heptane tapers to 100 from 60:40:
0, system temperature is 20 DEG C, collects component, is concentrated to dryness at 30 DEG C, obtains 6.8g tamiros semi-finished product (purity
84.78%, sirolimus 0.02%, isomers 13.23%).
2) preparation of high-purity tamiros
After above-mentioned 6.8g tamiros semi-finished product are dissolved with 21mL acetonitrile, being splined on reverse phase C18 filled column, (Japan is big
Cao Co., Ltd., similarly hereinafter, 5 μm of partial size), ladder is carried out with acetonitrile: aqueous formic acid (pH 3.5) mixed solvent system of 1mmol/L
Degree elution, 0~10min, acetonitrile/aqueous formic acid=50:50,10~100min, acetonitrile/aqueous formic acid from 50:50 gradually
80:20 is changed to, system temperature is 20 DEG C, collects component, and 30 DEG C are concentrated under reduced pressure on a small quantity, are extracted with ethyl acetate, and is saturated chlorine
Change sodium solution washing, separates organic phase, dry, filter, be concentrated to dryness at 30 DEG C, obtain 4.9g high-purity tamiros
(purity 99.94%, sirolimus is not detected, isomers 0.08%, other single impurity are < 0.1%).
Embodiment 2
1) preparation of tamiros semi-finished product
56g tamiros crude product (purity 79.03%, sirolimus 2.54%, isomers 15.63%) is used into 560mL second
After acetoacetic ester dissolution, positive silicon ball filled column (10 μm) are splined on, are carried out with the mixed solvent system of ethyl acetate and n-hexane
Gradient elution, 0~10min, ethyl acetate/n-hexane=60:40,10~100min, ethyl acetate/n-hexane from 60:40 by
Gradually change to 100:0, system temperature is 10 DEG C, collects component, is concentrated to dryness at 20 DEG C, obtain 46g tamiros half at
Product (purity 86.17%, sirolimus is not detected, isomers 11.65%).
2) preparation of high-purity tamiros
It after above-mentioned 46g tamiros semi-finished product are dissolved with 460mL acetonitrile, is splined on reverse phase C6 filled column (10 μm), uses
The mixed solvent system of the potassium dihydrogen phosphate aqueous solution (pH5) of acetonitrile and 20mmol/L carries out gradient elution, 0~10min, second
Nitrile/potassium dihydrogen phosphate aqueous solution=50:50,10~100min, acetonitrile/potassium dihydrogen phosphate aqueous solution are tapered to from 50:50
80:20, system temperature are 10 DEG C, collect component, and 25 DEG C are concentrated under reduced pressure on a small quantity, are extracted with ethyl acetate, saturated sodium-chloride is molten
Liquid washing, separates organic phase, dries, filters, be concentrated to dryness at 20 DEG C, obtain 35g high-purity tamiros (purity
99.81%, sirolimus is not detected, isomers 0.12%, other single impurity are < 0.1%).
Embodiment 3
1) preparation of tamiros semi-finished product
100g tamiros crude product (purity 76.89%, sirolimus 1.95%, isomers 15.92%) is used into 500mL
After ethyl acetate dissolution, be splined on positive silicon ball filled column (20-45 μm), with ethyl acetate: normal heptane mixed solvent system into
Row gradient elution, 0~10min, ethyl acetate/normal heptane=60:40,10~100min, ethyl acetate/normal heptane is from 60:40
100:0 is tapered to, system temperature is 15 DEG C, collects component, is concentrated to dryness at 30 DEG C, obtains 84.4g tamiros
Semi-finished product (purity 84.23%, sirolimus 0.01%, isomers: 13.47%).
2) preparation of tamiros finished product
After 84.4g tamiros semi-finished product are dissolved with 422mL acetonitrile, it is splined on reverse phase C8 filled column (20-45 μm),
Gradient elution, 0~10min, second are carried out with acetonitrile: sodium bisulphate solution (pH5.5) mixed solvent system of 3.5 μm of ol/L
Nitrile/sodium bisulphate solution=50:50,10~100min, acetonitrile/sodium bisulphate solution taper to 80 from 50:50:
20, system temperature is 15 DEG C, collects component, and 30 DEG C are concentrated under reduced pressure on a small quantity, is extracted with ethyl acetate, saturated sodium chloride solution is washed
It washs, separates organic phase, dry, filter, be concentrated to dryness at 30 DEG C, obtain 59.4g high-purity tamiros (purity
99.75%, sirolimus 0.01%, isomers 0.16%, other single impurity are < 0.1%).
Embodiment 4
1) preparation of tamiros semi-finished product
230g tamiros crude product (purity 75.32%, isomers 11.23%, sirolimus 2.14%) is used into 690mL
After acetonitrile dissolution, reverse phase C18 filled column (5 μm) are splined on, are mixed with acetonitrile: the acetic acid aqueous solution (pH4.5) of 0.1mmol/L
Dicyandiamide solution progress gradient elution, 0~10min, acetonitrile/acetic acid aqueous solution=50:50,10~100min, acetonitrile/containing acetic acid
Aqueous solution=80:20, system temperature are 20 DEG C, collect component, and 30 DEG C are concentrated under reduced pressure on a small quantity, are extracted with ethyl acetate, and are saturated
Sodium chloride solution washing, separates organic phase, dries, filters, be concentrated to dryness at 30 DEG C, obtain 158g tamiros semi-finished product
(purity 98.23%, isomers 0.15%, sirolimus 1.46%).
2) preparation of tamiros finished product
After above-mentioned 158g tamiros semi-finished product are dissolved with 475mL ethyl acetate, it is splined on positive silicon ball filled column (5 μ
M), with ethyl acetate: normal heptane mixed solvent system carries out gradient elution, 0~10min, ethyl acetate/normal heptane=60:40,
10~100min, ethyl acetate/normal heptane taper to 100:0 from 60:40, and system temperature is 20 DEG C, collection component, and 30
It is concentrated to dryness at DEG C, obtains 128g high-purity tamiros (purity 99.80%, isomers 0.15%, sirolimus
0.01%, other single impurity are < 0.1%).
Embodiment 5
1) preparation of tamiros semi-finished product
Tamiros crude product 75g (purity 76.93%, isomers 12.01%, sirolimus 1.89%) is used into 750mL second
After nitrile dissolution, reverse phase C8 filled column (10 μm) are splined on, it is molten with acetonitrile: aqueous potassium hydrogen sulfate (pH5) mixing of 15 μm of ol/L
Agent system carries out gradient elution, 0~10min, acetonitrile/aqueous potassium hydrogen sulfate=50:50,10~100min, acetonitrile/hydrogen sulfate
Aqueous solutions of potassium tapers to 80:20 from 50:50, and system temperature is 10 DEG C, Fractional Collections, liquid phase monitoring, merging component, and 30 DEG C
It is concentrated under reduced pressure on a small quantity, is extracted with ethyl acetate, saturated sodium chloride solution washing is layered, dry filter, is concentrated under reduced pressure at 30 DEG C
To doing, 53g tamiros semi-finished product (purity 97.64%, isomers 0.14%, sirolimus 1.37%) is obtained.
(2) preparation of high-purity tamiros
After above-mentioned 53g tamiros semi-finished product are dissolved with 530mL ethyl acetate, it is splined on positive silicon ball filled column (20-
45 μm), with ethyl acetate: normal heptane mixed solvent system carries out gradient elution, 0~10min, ethyl acetate/normal heptane=60:
40,10~100min, ethyl acetate/normal heptane taper to 100:0 from 60:40, and system temperature is 10 DEG C, collect component,
It is concentrated to dryness at 30 DEG C, obtaining 43g high-purity tamiros, (purity 99.81%, isomers 0.16%, sirolimus is not
Detection, other single impurity are < 0.1%).
Embodiment 6
1) preparation of tamiros semi-finished product
Tamiros crude product 320g (purity 78.34%, isomers 11.05%, sirolimus 2.52%) is used into 1600mL
After acetonitrile dissolution, above enter reverse phase C6 filled column (20-45 μm), with acetonitrile: disodium hydrogen phosphate/biphosphate sodium water solution is (with phosphorus
Acid group meter 0.03mol/L, pH6) mixed solvent system progress gradient elution, 0~10min, acetonitrile/water solution=50:50,10
~100min, acetonitrile/water solution taper to 80:20 from 50:50, and system temperature is 15 DEG C, collect component, 30 DEG C of decompressions are dense
It is reduced on a small quantity, is extracted with ethyl acetate, saturated sodium chloride solution washing separates organic phase, dries, filters, depressurize at 30 DEG C dense
It is reduced to dry, obtains 228g tamiros semi-finished product (purity 97.50%, isomers 0.18%, sirolimus 2.06%, other lists
A impurity is < 0.1%).
2) preparation of high-purity tamiros
After above-mentioned tamiros semi-finished product 228g is dissolved with 1140mL ethyl acetate, above enter positive silicon ball filled column (10 μ
M), with ethyl acetate: n-hexane mixed solvent system carries out gradient elution, 0~10min, ethyl acetate/n-hexane=60:40,
10~100min, ethyl acetate/n-hexane change to 100:0 from 60:40, and system temperature is 20 DEG C, collection component, at 30 DEG C
Be concentrated to dryness, obtain 182g high-purity tamiros (purity 99.76%, isomers 0.20%, sirolimus: 0.01%,
Other single impurity are < 0.1%).
Reference examples 1
It is tested with reference to the tamiros method of purification of CN103664999A: tamiros precursor (5g, 4.38mmol)
Be placed in the tetrahydrofuran of 25mL, be added dropwise 2N HCl22ml (44mmol), 0 DEG C stir about 12 hours, TLC contact plate reaction raw materials are
Fundamental reaction is complete.The dilution of 200mL ethyl acetate is added after the reaction was completed, 200mL water is added, is extracted with 100mL ethyl acetate
Organic phase three times, merges organic phase.Organic phase is washed one time with saturated sodium carbonate solution, and water washing three times, saturated common salt washing
It washs one time, anhydrous magnesium sulfate is dry.It is evaporated under reduced pressure at 30 DEG C after doing and obtains crude product, crude product is through 200-300 mesh silica gel column chromatography, acetic acid
Ethyl ester: n-hexane (4:1 → 2:1) quickly elutes, and obtains tamiros 2.4g (purity 88.75%, sirolimus 2.5%, isomery
Body 8.56%).
Reference examples 2
It is tested with reference to the method for the tesirolimus precursor preparation tesirolimus of CN103421023A: in the circle of 1L
Tamiros precursor (7.5g, 6.56mmol) is put into the flask of bottom and p-methyl benzenesulfonic acid (0.1g, 0.58mmol) is dissolved in 400mL
Tetrahydrofuran, at 0 DEG C be added dropwise 5mL ethylene glycol, drip off within 5 minutes or so, drop finish, stirring 1 hour after, TLC detection discovery
Raw material fully reacting, is added the water of 1L, is extracted with ethyl acetate (500mL*3) three times, and gained ethyl acetate layer is again with saturation
Sodium chloride washes (200mL*3) three times, and after being dried over anhydrous sodium sulfate, evaporated under reduced pressure obtains about 7g crude product, and gained crude product is through 200-
300 mesh silica gel column chromatographies, are quickly eluted using mixed solvent PE:AC=4:1, obtain 4.2g tamiros (purity 85.43%,
Sirolimus 1.2%, isomers 12.24%).
Reference examples 3
1) preparation of tamiros semi-finished product
Tamiros crude product 85.1g (purity 74.65%, sirolimus 3.04%, isomers 16.88%) is used into 255mL
After acetone solution, it is splined on positive silicon ball filled column (20-45 μm), gradient is carried out with acetone: normal hexane mixed solvent system and is washed
It taking off, 0~10min, acetone/n-hexane=90:10,10~100min, acetone/n-hexane tapers to 100:0 from 90:10,
System temperature is 20 DEG C, collects component, is concentrated to dryness at 30 DEG C, obtains 60.4g tamiros semi-finished product (purity
84.78%, sirolimus 0.84%, isomers 11.89%).
2) preparation of tamiros finished product
After above-mentioned 60.4g tamiros semi-finished product are dissolved with 286mL methanol, it is splined on reverse phase C18 filled column (20-45 μ
M), with methanol: 1 μm of ol/L aqueous sulfuric acid (pH5) mixed solvent system carries out gradient elution, 0~10min, methanol/sulfuric acid water
Solution=60:40,10~100min, methanol/aqueous sulfuric acid taper to 90:10 from 60:40, and system temperature is 20 DEG C,
Component is collected, 30 DEG C are concentrated under reduced pressure on a small quantity, are extracted with ethyl acetate, and saturated sodium chloride solution washing separates organic phase, drying
It filters, is concentrated to dryness at 30 DEG C, obtains 47.7g tamiros (purity 97.25%, sirolimus 0.83%, isomers
1.24%).
Reference examples 4
1) preparation of tamiros semi-finished product
Tamiros crude product 117g (purity 77.63%, sirolimus 1.58%, isomers 15.69%) is used into 585mL
After ethyl acetate dissolution, above enters positive silicon ball filled column (10 μm), carry out gradient with ethyl acetate: normal heptane mixed solvent system
Elution, 0~10min, ethyl acetate/normal heptane=60:40,10~100min, ethyl acetate/normal heptane=100:0, system temperature
Degree is 20 DEG C, collects component, is concentrated to dryness at 30 DEG C, obtains tamiros semi-finished product (purity 82.43%, the west of 89.6g
Luo Mosi 0.65%, isomers 12.45%).
2) preparation of high-purity tamiros
Above-mentioned 89.6g tamiros semi-finished product 473mL acetonitrile is dissolved, reverse phase C18 filled column (10 μm) are splined on,
Gradient elution, 0~10min, acetonitrile/phosphoric acid water are carried out with acetonitrile: 0.05mol/L acetic acid aqueous solution (pH3) mixed solvent system
Solution=50:50,10~100min, acetonitrile/phosphate aqueous solution taper to 80:20 from 50:50, and system temperature is 20 DEG C,
Component is collected, 30 DEG C are concentrated under reduced pressure on a small quantity, are extracted with ethyl acetate, and saturated sodium chloride solution washing separates organic phase, does
It is dry, it filters, is concentrated to dryness at 30 DEG C, obtains 56.8g tamiros (purity 97.50%, sirolimus 0.64%, isomery
Body 1.58%).
Reference examples 5
1) preparation of tamiros semi-finished product
Tamiros crude product 5.6g (purity 76.38%, isomers 12.39%, sirolimus 2.86%) is used into 22.4mL
After methanol dissolution, it is splined on reverse phase C18 filled column (20-45 μm), is mixed with methanol: 0.9 μm of ol/L phosphate aqueous solution (pH6)
Dicyandiamide solution progress gradient elution, 0~10min, methanol/phosphate aqueous solution=60:40,10~100min, methanol/phosphoric acid are water-soluble
Liquid tapers to 90:10 from 60:40, and system temperature is 20 DEG C, collects component, and 30 DEG C are concentrated under reduced pressure on a small quantity, with acetic acid second
Ester extraction, saturated sodium chloride solution washing, separates organic phase, dries, filters, be concentrated to dryness at 30 DEG C, it is smooth to obtain 3.89g
Luo Mosi semi-finished product (purity 96.12%, isomers 1.31%, sirolimus 2.38%).
(2) preparation of high-purity tamiros
After above-mentioned 3.89g tamiros semi-finished product 23.3mL acetone solution, it is splined on positive silicon ball filled column (20-
45 μm), with acetone: n-hexane mixed solvent system carries out gradient elution, 0~10min, acetone/n-hexane=70:30,10~
100min, acetone/n-hexane taper to 100:0 from 70:30, and system temperature is 20 DEG C, collect component, depressurize at 30 DEG C dense
It is reduced to dry, obtains the tamiros finished product (purity 97.48%, isomers 1.35%, sirolimus 0.42%) of 3.03g.
Reference examples 6
1) preparation of tamiros semi-finished product
Tamiros crude product 10.6g (purity 76.98%, isomers 12.15%, sirolimus 1.06%) is used into 85mL
After acetonitrile dissolution, reverse phase C18 filled column (10 μm) are splined on, with acetonitrile: 0.06mo/L propionic acid aqueous solution (pH3) mixed solvent body
System carries out gradient elution, 0~10min, acetonitrile/aqueous solution=50:50 containing propionic acid, 10~100min, the acetonitrile/water containing propionic acid
Solution=80:20, system temperature are 20 DEG C, collect component, and 30 DEG C are concentrated under reduced pressure on a small quantity, are extracted with ethyl acetate, and are saturated chlorine
Change sodium solution washing, be layered, dry filter, is concentrated to dryness at 30 DEG C, obtains the tamiros semi-finished product (purity of 7.3g
95.41%, isomers 1.30%, sirolimus 0.78%).
(2) preparation of high-purity tamiros
After above-mentioned tamiros semi-finished product 7.3g is dissolved with 58mL ethyl acetate, above enter positive silicon ball filled column (10 μ
M), with ethyl acetate: normal heptane mixed solvent system carries out gradient elution, 0~10min, ethyl acetate/normal heptane=60:40,
10~100min, ethyl acetate/normal heptane taper to 100:0 from 60:40, and system temperature is 20 DEG C, collection component, and 30
It is concentrated to dryness at DEG C, obtains 5.0g tamiros finished product (purity 96.69%, isomers 1.32%, sirolimus
0.32%).
It is above-mentioned to provide only a small number of embodiments, in which it can be seen that the group of mobile phase in positive and reversed-phase high performance liquid chromatography
It is crucial factor at type of elution.The sequence of Normal-phase HPLC and reversed-phase high performance liquid chromatography can be interchanged, first
For semi-finished product after inverted high performance liquid chromatography separation when by Normal-phase HPLC separation, the content of isomers may
It can have increased slightly, but the isomers does not influence the drug effect of tamiros.Two methods of the invention, which can be prepared, to be met
The high-purity tamiros that ejection preparation requires.Not specified operating condition can be according to this field routine side in the present invention
Method carries out.Certain operating process, for example, in mobile phase the aqueous solution of pH3.5~6.0 preparation, at the collection and concentration of eluent
Reason etc. can also be carried out according to other conventional methods known in the art, under the premise of essence and spirit of the invention
The improvement or equivalence changes made belong to the scope of protection of the invention.
Claims (5)
1. a kind of method for preparing high-purity tamiros, comprising steps of
A) Normal-phase HPLC: being solid with positive phase filling by tamiros crude product through performance liquid chromatographic column purification system
Determine phase, eluted using the mixture of ethyl acetate and normal heptane or ethyl acetate and n-hexane as mobile phase, obtains smooth sieve
Do not take charge of semi-finished product；
B) reversed-phase high performance liquid chromatography: by tamiros semi-finished product through performance liquid chromatographic column purification system, it is with reverse phase filler
Stationary phase is eluted using the mixture of acetonitrile and the aqueous solution of pH 3.5~6.0 as mobile phase, and high-purity Tan Luomo is obtained
Department；
Wherein, the step A) in, with the total volume meter of the mobile phase, contain 60~100v/ of ethyl acetate in mobile phase
V% contains 40~0v/v% of n-hexane or normal heptane；
The step B) in, with the total volume meter of the mobile phase, contains 50~80v/v% of acetonitrile in mobile phase, contain pH
3.5~6.0 50~20v/v% of aqueous solution；Aqueous solution is that acidic materials are added in water to be formulated, the acidic materials choosing
From formic acid, acetic acid, propionic acid, phosphoric acid, sulfuric acid, potassium acid sulfate, sodium bisulfate, ammonium hydrogen sulfate, potassium dihydrogen phosphate, sodium dihydrogen phosphate,
One of ammonium dihydrogen phosphate is a variety of；
The step A) in, it elutes as gradient elution, in 0~10min of elution, mobile phase contains ethyl acetate 60v/v%, contains
There are n-hexane or normal heptane 40v/v%, the content of ethyl acetate becomes from 60v/v% in 10~100min of elution, mobile phase
Change to 100v/v%, while the content of normal heptane or n-hexane changes to 0v/v% from 40v/v%；
The step B) in, it elutes as gradient elution, in 0~10min of elution, mobile phase contains acetonitrile 50v/v%, contains pH
3.5~6.0 aqueous solution 50v/v%, the content of acetonitrile is changed to from 50v/v% in 10~100min of elution, mobile phase
80v/v%, while the content of the aqueous solution of pH 3.5~6.0 changes to 20v/v% from 50v/v%；
The positive phase filling is silicon ball filler, and the reverse phase filler is selected from C4, C6, C8 or C18 filler.
2. a kind of method for preparing high-purity tamiros, comprising steps of
B) reversed-phase high performance liquid chromatography: being solid with reverse phase filler by tamiros crude product through performance liquid chromatographic column purification system
Determine phase, eluted using the mixture of acetonitrile and the aqueous solution of pH 3.5~6.0 as mobile phase, obtains tamiros semi-finished product；
A) Normal-phase HPLC: by tamiros semi-finished product through performance liquid chromatographic column purification system, it is with positive phase filling
Stationary phase is eluted as mobile phase using the mixture of ethyl acetate and normal heptane or ethyl acetate and n-hexane, obtains height
Purity tamiros；
Wherein, the step A) in, with the total volume meter of the mobile phase, contain 60~100v/ of ethyl acetate in mobile phase
V% contains 40~0v/v% of n-hexane or normal heptane；
The step B) in, with the total volume meter of the mobile phase, contains 50~80v/v% of acetonitrile in mobile phase, contain pH
3.5~6.0 50~20v/v% of aqueous solution；Aqueous solution is that acidic materials are added in water to be formulated, the acidic materials choosing
From formic acid, acetic acid, propionic acid, phosphoric acid, sulfuric acid, potassium acid sulfate, sodium bisulfate, ammonium hydrogen sulfate, potassium dihydrogen phosphate, sodium dihydrogen phosphate,
One of ammonium dihydrogen phosphate is a variety of；
The step A) in, it elutes as gradient elution, in 0~10min of elution, mobile phase contains ethyl acetate 60v/v%, contains
There are n-hexane or normal heptane 40v/v%, the content of ethyl acetate becomes from 60v/v% in 10~100min of elution, mobile phase
Change to 100v/v%, while the content of normal heptane or n-hexane changes to 0v/v% from 40v/v%；
The step B) in, it elutes as gradient elution, in 0~10min of elution, mobile phase contains acetonitrile 50v/v%, contains pH
3.5~6.0 aqueous solution 50v/v%, the content of acetonitrile is changed to from 50v/v% in 10~100min of elution, mobile phase
80v/v%, while the content of the aqueous solution of pH 3.5~6.0 changes to 20v/v% from 50v/v%；
The positive phase filling is silicon ball filler, and the reverse phase filler is selected from C4, C6, C8 or C18 filler.
3. method according to claim 1 or 2, the HPLC purity of the high-purity tamiros is greater than 98%.
4. method according to claim 1 or 2, the HPLC purity of the high-purity tamiros is greater than 99%.
5. method according to claim 1 or 2, the HPLC purity of the high-purity tamiros is greater than 99.5%.
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| CN108948047A (en) * | 2017-05-20 | 2018-12-07 | 鲁南制药集团股份有限公司 | A kind of purification process of tesirolimus |
| CN109851626B (en) * | 2019-01-08 | 2021-11-02 | 扬子江药业集团四川海蓉药业有限公司 | Method for separating and purifying temsirolimus |
| JP2023028473A (en) * | 2021-08-19 | 2023-03-03 | 日本マイクロバイオファーマ株式会社 | Method for producing everolimus |
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