CN103483352A

CN103483352A - Medicinal bulk drug for resisting tumors

Info

Publication number: CN103483352A
Application number: CN201310493317.9A
Authority: CN
Inventors: 李友香; 孙俊
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-10-18
Filing date: 2013-10-18
Publication date: 2014-01-01

Abstract

The invention relates to a medicinal bulk drug for resisting tumors, and in particular relates to a bulk drug for medicines. The active component of the bulk drug is a compound in the formula I or pharmaceutical salt thereof, wherein each substituent is described as the specification. The invention also relates to a medicinal composition containing the compound in the formula I or salt thereof, a preparation method of the medicinal composition and pharmaceutical application thereof. The bulk drug has excellent characteristics, such as stability and safety, when serving as an anti-tumor drug.

Description

Antineoplastic medicinal raw material medicine

Technical field

The present invention relates to medical technical field, relate to a kind of Macrocyclolactone lactone kind medicine compound with anti-tumor activity.

Background technology

Halichondrin B is a kind of effective anticancer agent, from the sponge Halichondria okadai of ocean, isolate at first, subsequently little axle sponge (Axinella sp.) undetermined, Phakellia carteri and plant discovery in name flat lance sponge (Lissondendryxsp.) undetermined in kind of name.Within 1992, announced halichondrin B complete synthesis (Aicher, T, D. etc., J.Am.Chem.Soc.114:3162-3164).Proved that halichondrin B suppresses the assembling of tubulin polymerization, microtubule, β in vitro ^s-tubulin is crosslinked, GTP and vincaleucoblastine and tubulin in conjunction with and the GTP hydrolysis that depends on tubulin, and demonstrated the in vitro and in vivo anticancer property.

Known have with the halichondrins analogue of following formula I structure or its pharmacologically acceptable salts for example with its mesylate shown in following formula Ia:

For example antitumour activity or antimitotic (blocking-up mitotic division) are active to have clear and definite medical active.

It is believed that formula I compound or pharmaceutically acceptable salt thereof can suppress the vegetative period of microtubule and can not affect the shortening phase, and tubulin is separated into without producing active polymkeric substance.Formula I compound or pharmaceutically acceptable salt thereof is by the antimitotic mechanism retardance G of tubulin ₂/ M the cell cycle, the cracking mitotic spindle, final, cell is because of the mitotic division apoptosis that is obstructed.For clinical formula I compound or pharmaceutically acceptable salt thereof, can be used as a kind of microtubule inhibitors, be applicable to metastatic breast cancer patient etc.

Its molecular formula of the mesylate of formula I compound (being formula Ia compound) is C ₄₀h ₅₉nO ₁₁cH ₄o ₃s, molecular weight is 826.0 (free alkali is that the molecular weight of formula I compound is 729.9).Formula Ia compound is a kind of white powder material, in soluble in water, ethanol equal solvent.

Prior art discloses the illustrative methods of preparation I compound.For example CN1216051C discloses a kind of synthetic compound of formula i (compound B-11 939,62 pages, specification sheets) method, it is to start to make compd B 2318 (36 pages, specification sheets) through polystep reaction with L-arabinose, then through polystep reaction, make compd B 2304 (44 pages, specification sheets), then through polystep reaction, make the compound B-11 793 (47 pages, specification sheets) with two oh groups.Stage in the end, this compound B-11 793 with two free hydroxyl groups reacts to obtain single methanesulfonates compd B 2294 (49 pages, specification sheets) with one of them hydroxy esterification with methylsulfonyl chloride, finally compd B 2294 azides and amination being obtained to compound B-11 939 (62 pages, specification sheets) is formula I compound.Then optionally, can make the further salify of formula I compound for example form mesylate, the method for this salify is that those skilled in the art are according to there being experience easily to realize.At above-mentioned final stage B1793, carry out in the process of methylsulfonic acid esterification reaction, may carry out esterification with the another one hydroxyl, follow-up azide and ammoxidation and possible configuration reversal that then this esterification products is carried out to be similar to B2294, final formation has the present invention's compound of the formula Ix as impurity hereinafter described.

As everyone knows, the single contaminant in medicine material or its preparation need to be controlled in certain level, for example below 5%, below 2.5%, below 2.0%, below 1.5%, below 1.0%, below 0.5%, below 0.25%, below 0.2% or 0.1% with inferior.After having stipulated its limit, that medicine amount of this impurity in its storage validity period allows trace but the growth that can not go beyond the limit.Have been found that, the raw material of formula I compound or pharmaceutically acceptable salt thereof or its preparation are in the long-term storage process, have certain specific impurity to show the trend that increase is arranged with the time lengthening that keeps sample, this phenomenon is very disadvantageous for the pharmaceuticals of being strict with " quality controllable ".

Therefore, this area still expects to have new method to have the formula I compound or pharmaceutically acceptable salt thereof of Good Pharmacy performance with acquisition.

Summary of the invention

The object of the invention is to provides a kind of new method to have the formula I compound or pharmaceutically acceptable salt thereof of Good Pharmacy performance with acquisition for clinical.Inventor's discovery, the formula I compound or pharmaceutically acceptable salt thereof that art methods obtains can effectively make impurity Ix be reduced to significant limit after the inventive method is processed; And after dropping to below this limit, for example impurity Iy unlikely remarkable increase with the prolongation of drug storage time of other impurity in raw material and preparation prepared therefrom.Also have been surprisingly found that, the formula I compound or pharmaceutically acceptable salt thereof of the present invention with low levels impurity Iy has beat all biological property and causes that such as it probability of the side reactions such as neutrophil leucocyte minimizing reduces.The present invention is based on this discovery and be accomplished.

For this reason, first aspect present invention provides a kind of preparation method of bulk drug of hyoscine, and the activeconstituents of this bulk drug is with the following formula I compound:

Or its pharmaceutical salts, the method comprises with an organic solvent carries out refining step to the bulk drug that comprises formula I compound or pharmaceutically acceptable salt thereof.

Formula I compound of the present invention also is called eribulin (eribulin) usually, and formula Ia compound also is called methylsulfonic acid eribulin (eribulin mesylate) usually.In the methods of the invention, in above-mentioned " bulk drug that comprises formula I compound or pharmaceutically acceptable salt thereof is carried out to refining step ", it can be rough bulk drug or crude product for described term " bulk drug that comprises formula I compound or pharmaceutically acceptable salt thereof ", it can be for example the bulk drug prepared according to art methods, although the bulk drug that art methods prepares equally can the preparation as injection liquid for formulation example, but by the inventive method, the bulk drug of the prior art is further processed, so that it and the preparation that is prepared into by it are given better performance, better stability for example, better security.

It can be used as for example bulk drug of pharmaceutical preparation production use of pharmaceutical composition " bulk drug of hyoscine " of the present invention or " bulk drug ", thus its also to can be regarded as formula I compound or pharmaceutically acceptable salt thereof be the bulk drug of eribulin or its pharmaceutical salts.Well-known, as the bulk drug of the hyoscine of useful in preparing drug formulations, they comprise the formula I compound or pharmaceutically acceptable salt thereof as main ingredient, can also comprise the trace impurity of limit up to specification.

According to the method for first aspect present invention, wherein said bulk drug comprises with following formula Ia compound (be the mesylate of formula I compound, usually also can be described as the methylsulfonic acid eribulin):

According to the method for first aspect present invention, it comprises that the bulk drug to comprising formula I compound or pharmaceutically acceptable salt thereof carries out recrystallization to reach the step of refining purpose.

According to the method for first aspect present invention, described recrystallization is that bulk drug is dissolved in the first solvent, then in this solution, adds the second solvent, so that formula I compound or pharmaceutically acceptable salt thereof is settled out crystallization from solvent.

According to the method for first aspect present invention, described the first solvent is selected from: water, methyl alcohol, ethanol, 1-octanol, phenylcarbinol, methyl-sulphoxide, methylene dichloride, ethyl acetate and combination thereof.In one embodiment, described the first solvent is selected from: ethanol, 1-octanol, methyl-sulphoxide, methylene dichloride, ethyl acetate and combination thereof.In one embodiment, the mixture that described the first solvent is ethanol and ethyl acetate.In one embodiment, described the first solvent is the mixture of ethanol and ethyl acetate volume ratio 1:2～8, preferably the mixture of ethanol and ethyl acetate volume ratio 1:3～7.

According to the method for first aspect present invention, described the second solvent is selected from: t-butyl methyl ether, normal heptane, normal hexane, Skellysolve A and combination thereof.In one embodiment, described the second solvent is selected from: normal heptane, normal hexane, Skellysolve A and combination thereof.In one embodiment, described the second solvent is normal hexane.

According to the method for first aspect present invention, the volume ratio of described the first solvent and the second solvent is 1:5～20, for example 1:6～15, for example 1:6～12.

According to the method for first aspect present invention, described recrystallization is to carry out under 20～50 ℃ of conditions, is for example to carry out under 30～40 ℃ of conditions.

Method according to first aspect present invention, while being dissolved in the first solvent by bulk drug, those skilled in the art understand that now the solvent for use amount is more low better usually, as long as normally reach the volume of a little higher than meltage, for example reach the solvent volume of 2～4 times of meltages, in order to create conditions for follow-up crystallization and aftertreatment.Therefore needn't do special stipulation to this first solvent load in the present invention.But, in order to obtain good separating effect, in one embodiment of the invention, the ratio that can make bulk drug be dissolved in the first solvent with the concentration of 0.1～0.2g/ml is processed.

Have been found that and use crystallization method of the present invention can effectively remove impurity Ix (also can be described as the similar titles such as Ix impurity, formula Ix compound), impurity Ix derives from final stage prepared by formula I compound.This impurity Ix has following chemical structural formula:

Method according to first aspect present invention, its prepared bulk drug comprises the formula I compound or pharmaceutically acceptable salt thereof as main ingredient, and the optional impurity of the Ix as impurity, the Ix impurity phase for formula I compound content lower than 5% (for example, lower than 2.5%, for example, lower than 1.7%).

In bulk drug of the present invention (wherein comprising formula I compound or pharmaceutically acceptable salt thereof), the content of impurity Ix is with respect to being free form but not the percentage amounts of the formula I compound of pharmaceutical salts form in itself, that is: for certain a collection of bulk drug of the present invention or for example material of preparation of its pharmaceutical composition of making as raw material of take, while calculating the content of impurity Ix wherein, can calculate with following formula:

Impurity Ix content=(quality of this material Chinese style of the quality ÷ of impurity Ix I compound in this material) * 100%

According to above definition, above-mentioned material can be both to take formula I compound or pharmaceutically acceptable salt thereof to be the main bulk drug that becomes to form, can also be take formula I compound or pharmaceutically acceptable salt thereof as activeconstituents the formulation example that is added with other pharmaceutical excipient as injection liquid.Thus, the calculating of impurity Ix content not can because of other composition for example the existence of pharmaceutical excipient be affected.Other impurity the present invention relates to for example during the calculating of impurity Iy, also has analogue.

In the present invention, the method for mensuration impurity Ix content can have many, for example, according to well known to a person skilled in the art the measuring method that mode is definite.A kind of typical method is high performance liquid chromatography.In addition, can separate and obtain highly purified for example chromatographic purity and reach impurity reference substance more than 98% (impurity Ix for example by preparative high performance liquid chromatography, and impurity Iy mentioned below for example), take this impurity reference substance is contrast, measures the relative content of this kind of impurity in the bulk drug that the present invention comprises formula I compound or pharmaceutically acceptable salt thereof and the pharmaceutical preparation prepared by them by the known feasible method of those skilled in the art.A kind of typical high performance liquid chromatography is high performance liquid chromatography A (can be called for short [HPLC-A] in the present invention) hereinafter described, its can be used for measuring various impurity in the various materials of the present invention (comprising bulk drug and preparation) for example impurity Ix (and for example impurity Iy mentioned below) in this material with respect to the amount of formula I compound.Certainly, it will be appreciated by those skilled in the art that analytical procedure is easily improved, the method for the various impurity in any available and the various materials of potential mensuration the present invention all can be used for the present invention; In view of the content of the various impurity in the various materials of the present invention can be because improving of analytical procedure changes, so the measuring method of the foreign matter content in the various materials of the present invention or relative content can be not particularly limited.Certainly, in the present invention, if not otherwise indicated, for example the content of impurity Ix (and such as but not limited to impurity Iy mentioned below) or the method for relative content are all to adopt [HPLC-A] described herein to measure various impurity in various materials (comprising bulk drug and preparation).

Have been found that, for the bulk drug of the present invention that comprises formula I compound or pharmaceutically acceptable salt thereof or the pharmaceutical preparation of being made by them, along with the prolongation of storage time there will be a kind of special degradation impurity, it is called impurity Iy (also can be described as the similar titles such as Iy impurity, formula Iy compound) in the present invention, and this impurity Iy has following chemical structural formula:

In the present invention, have been found that, the bulk drug of the present invention that comprises formula I compound or pharmaceutically acceptable salt thereof by purifying, after making the impurity Ix that comes from technique wherein comprised be reduced to a certain degree, the speed that the bulk drug of the present invention that this comprises formula I compound or pharmaceutically acceptable salt thereof or its preparation generate degradation impurity Iy is significantly suppressed, and this is fully beat all.

Although the present invention has confirmed the chemical structure of impurity Ix and impurity Iy by multiple means, but as well known to those skilled in the art, these impurity can also come qualitative or characterize by the high performance liquid chromatography of regulation, for example, by the high performance liquid chromatography operation of regulation, (be relative retention time, RRT) determine this impurity by measuring the retention behavior of impurity in this regulation high performance liquid chromatography.Particularly, the present invention has been found that in [HPLC-A] of the present invention system, for formula I compound, the relative retention time of impurity Ix is in 1.06～1.09 scopes, thereby the present invention also can be called RRT1.07 impurity, impurity RRT1.07 or RRT1.07 by this impurity Ix; For formula I compound, the relative retention time of impurity Iy is in 1.27～1.31 scopes, thereby the present invention also can be called RRT1.29 impurity, impurity RRT1.29 or RRT1.29 by this impurity Iy.In the present invention, while mentioning RRT1.07 impurity, all refer to that, in [HPLC-A] system, for the retention time of formula I compound, relative retention time impurity occurs in 1.06～1.09 scopes, this impurity is impurity Ix; Similarly, in the present invention, while mentioning RRT1.29 impurity, all refer to that, in [HPLC-A] system, for the retention time of formula I compound, relative retention time impurity occurs in 1.27～1.31 scopes, this impurity is impurity Iy.

The implication of relative retention time is being known in the art; for example; the relative retention time of impurity Ix (being abbreviated as RRT) refers in color atlas; the retention time of impurity Ix chromatographic peak is divided by the value of the retention time gained of formula I compound chromatographic peak; be the RRT=impurity Ix retention time ÷ formula I compound guard time of Ix, this algorithm is well known to a person skilled in the art.

According to the method for first aspect present invention, in its prepared bulk drug of the present invention that comprises formula I compound or pharmaceutically acceptable salt thereof, the Ix impurity phase for formula I compound content lower than 5% (for example, lower than 2.5%, for example, lower than 1.7%).The calculating of this limit can be used high performance liquid chromatography to calculate.

In the present invention, the measuring method of [HPLC-A] is as follows:

(1) according to two contained high performance liquid chromatography of appendix V D of Chinese Pharmacopoeia version in 2010, carry out;

(2) chromatographic condition and system suitability:

Chromatographic column: the chromatographic column that the octadecylsilane chemically bonded silica of take is weighting agent, filler aperture

packing material size 3 μ m, the pillar size: internal diameter 3.0mm * column length 100～250mm (can select suitable length pillar so that flow rate of mobile phase in 0.5ml/min condition following formula I compound retention time in 26～32min scope, for example can use the long pillar of 150mm);

[in the present invention, in test hereinafter, if not otherwise indicated, use purchased from Britain Advanced Chromatography

The ACE-C18-300 chromatographic column of Technologies Ltd company is tested, this chromatographic column aperture packing material size 3 μ m, pillar size: internal diameter 3.0mm * column length 150mm, product article No. ACE-211-1503]

Column temperature: 40 ℃;

Mobile phase A: 760mL water and 240mL acetonitrile are mixed, add the 7.0g trifluoromethanesulfacid acid ammonium, biphosphate TBuA (tetrabutylammonium dihydrogenphosphate) aqueous solution 3.0mL that adds again 1.0M, mix, measure the pH value of solution, by the pH value to 7.0 of 5.6% ammonium hydroxide aqueous solution or 1mol/L hydrochloric acid conditioning solution, obtain mobile phase A in case of necessity;

Mobile phase B: the 2-propyl alcohol of 300mL water, 700mL acetonitrile and 20mL is mixed, add the 7.0g trifluoromethanesulfacid acid ammonium, the biphosphate TBuA aqueous solution 3.0mL that adds again 1.0M, mix, measure the pH value of solution, by the pH value to 7.0 of 5.6% ammonium hydroxide aqueous solution or 1mol/L hydrochloric acid conditioning solution, obtain Mobile phase B in case of necessity;

The gradient elution program:

Time (min)	Mobile phase A ratio (%)	Mobile phase B ratio (%)
			0	100	0
55	100	0
			75	0	100
85	0	100
			86	100	0
115	100	0

Flow velocity: during 0～55min, with the first flow velocity, carry out wash-out, when 55min, flow velocity changes 0.63ml/min into and continues to 115min, described the first flow velocity be a constant flow rate in 0.47～0.52ml/min scope carry out so that formula I compound retention time in 26～32min scope;

Sampling volume: 5 μ L; Ultraviolet detection: wavelength 200nm; Working time and data acquisition time: 115min and 55min;

(3) preparation of test fluid:

Dosing solvent: water 650mL, acetonitrile 350mL, 85% phosphoric acid solution 0.7mL are mixed, measure the pH value of solution, by the pH value to 7.0 of 5.6% ammonium hydroxide aqueous solution or 1mol/L hydrochloric acid conditioning solution, obtain in case of necessity;

Impurity solution 1: take 2mg impurity Ix and be placed in the 50mL measuring bottle, by the dosing dissolution with solvents and be diluted to scale, shake up, obtain (impurity Ix solution, concentration is 40 μ g/ml approximately);

Impurity solution 2: take 2mg impurity Iy and be placed in the 5mL measuring bottle, by the dosing dissolution with solvents and be diluted to scale, shake up, obtain (impurity Iy solution, concentration is 400 μ g/ml approximately);

System suitability test soln: take 20mg formula I compound standard substance (chromatographic purity is greater than 99.0%) and be placed in the 5mL measuring bottle, add 1mL impurity solution 1 and 0.5mL impurity solution 2, by the dosing dissolution with solvents and be diluted to scale, shake up, obtain (comprising the approximately about about 40 μ g/ml of 8 μ g/ml, impurity Iy of 4000 μ g/ml, impurity Ix of formula I compound);

For sample solution: precision takes or measures that (it can be the bulk drug of the present invention that comprises formula I compound or pharmaceutically acceptable salt thereof containing the about 100mg trial-product of formula I compound, it can be also the pharmaceutical preparation that comprises auxiliary material, the about 100mg of formula I compound that comprises free alkali form in the material that takes or measure), be placed in the 25mL measuring bottle, by the dosing dissolution with solvents and be diluted to scale, shake up, obtain (containing approximately 4000 μ g/ml of formula I compound concentration);

Contrast solution: precision measures above-mentioned need testing solution 5ml and puts in the 500ml measuring bottle, with the dosing solvent cut, to scale, shakes up, and obtains (containing approximately 40 μ g/ml of formula I compound);

(4) system suitability test, requirement meets following limit: in system suitability test soln gained color atlas, the resolution at formula I compound and impurity Ix peak is not less than 1.5, the tailing factor of formula I compound peaks is not less than 0.5 and not higher than 1.5, the theoretical plate number of formula I compound peaks is not less than 13000, and the relative standard deviation that the system suitability test soln repeats the formula I compound peaks area of 5 calculating of sample introduction is no more than 1.0%;

(5) relative retention time: the RRT=1.00 of formula I compound, the RRT of impurity Ix is in 1.06～1.09 scopes, and the RRT of impurity Iy is in 1.27～1.31 scopes;

(6) test: get contrast solution 5 μ l injection liquid chromatographies, regulate detection sensitivity, make the peak height of principal constituent chromatographic peak be about 20% of full range, then precision measures need testing solution and each 5 μ l of contrast solution, the injection liquid chromatography, record color atlas respectively;

(7) read the peak area at principal constituent in the contrast solution color atlas (be formula I compound, also there is identical meanings while mentioning at other place of the present invention) peak; Read the peak area at principal constituent peak in the need testing solution color atlas, and calculate through area normalization method the peak area that peak area percentage ratio is greater than 0.01% impurity peaks in this color atlas, calculate the relative retention time of these impurity peaks with respect to the principal constituent peak;

Calculate in this trial-product its content with respect to formula I compound of any one single contaminant with following formula:

Method according to first aspect present invention, its prepared bulk drug of the present invention that comprises formula I compound or pharmaceutically acceptable salt thereof is measured according to the present invention [HPLC-A], wherein relative retention time (also can be described as in the present invention RRT1.07 impurity or is called RRT1.07 at the impurity at 1.06～1.09 places, it is essentially impurity Ix) for formula I compound content lower than 5% (for example, lower than 2.5%, for example, lower than 1.7%).

The present invention finds, impurity Ix content is controlled at lower than 5% (for example, lower than 2.5%, for example, lower than 1.7%) time, can be so that in bulk drug of the present invention and the pharmaceutical composition for preparing thereof, impurity Iy have significant increase along with the prolongation of storage time.There is in the present invention the discovery of clinical meaning to be, impurity Iy is a kind of material that can obviously cause that neutrophil leucocyte reduces, and in medical compounds of the present invention and the pharmaceutical composition for preparing thereof impurity Iy with respect to the content of formula I compound lower than 10% (for example, lower than 5%, for example, lower than 2.8%) time, this bulk drug or its pharmaceutical composition prepared can not cause that obvious neutrophil leucocyte reduces to animal subject, but in this bulk drug or its pharmaceutical composition prepared impurity Iy relative content higher than 2.8% (particularly higher than 5%, particularly higher than 10%) time, this bulk drug or its pharmaceutical composition prepared still can cause that the obvious neutrophil leucocyte of animal subject reduces this side effect.

Therefore, according to the method for first aspect present invention, in its prepared bulk drug of the present invention that comprises formula I compound or pharmaceutically acceptable salt thereof, impurity Iy with respect to the content of formula I compound lower than 10% (for example, lower than 5%, for example, lower than 2.8%).

Method according to first aspect present invention, its prepared bulk drug of the present invention that comprises formula I compound or pharmaceutically acceptable salt thereof is measured according to the present invention [HPLC-A], wherein relative retention time (also can be described as in the present invention RRT1.29 impurity or is called RRT1.29 at the impurity at 1.27～1.31 places, it is essentially impurity Iy) for formula I compound content lower than 10% (for example, lower than 5%, for example, lower than 2.8%).

Method according to first aspect present invention, its prepared bulk drug of the present invention that comprises formula I compound or pharmaceutically acceptable salt thereof is measured according to the present invention [HPLC-A], wherein relative retention time the impurity phase at 1.06～1.09 places for formula I compound content lower than 5% (for example, lower than 2.5%, for example, lower than 1.7%); Relative retention time the impurity phase at 1.27～1.31 places for formula I compound content lower than 10% (for example, lower than 5%, for example, lower than 2.8%).

Method according to first aspect present invention, its prepared bulk drug of the present invention that comprises formula I compound or pharmaceutically acceptable salt thereof is placed 6 months in 38 ℃ of condition lower seals, lucifuge, calculate the content of disposing with this understanding after 6 months when certain impurity phase was for 0 month and increase percentage ratio, wherein the foreign matter content of relative retention time at 1.27～1.31 places increases percentage ratio lower than 200%, for example, lower than 150%, for example, lower than 100%, for example, lower than 75%, for example, lower than 50%, for example, lower than 40%.This test method in the present invention can be referred to as investigating 38 ℃-June.Described term " foreign matter content increase percentage ratio " is to calculate according to following formula:

In above calculating formula, 0 month content of impurity or impurity content in June refer to this impurity relative content with respect to the formula I compound of free alkali form in material.

Further, second aspect present invention provides a kind of bulk drug of hyoscine, and the activeconstituents of this bulk drug is with the following formula I compound:

Or its pharmaceutical salts.

According to the bulk drug of second aspect present invention, comprising the formula I compound or pharmaceutically acceptable salt thereof as main ingredient, and the optional impurity of the Ix as impurity.

According to the bulk drug of second aspect present invention, wherein the Ix impurity phase for formula I compound content lower than 5% (for example, lower than 2.5%, for example, lower than 1.7%).

According to the bulk drug of second aspect present invention, comprising the formula I compound or pharmaceutically acceptable salt thereof as main ingredient, and the optional impurity of the Iy as impurity.

According to the bulk drug of second aspect present invention, wherein impurity Iy with respect to the content of formula I compound lower than 10% (for example, lower than 5%, for example, lower than 2.8%).

Bulk drug according to second aspect present invention, it is measured according to the present invention [HPLC-A], wherein relative retention time (also can be described as in the present invention RRT1.07 impurity or is called RRT1.07 at the impurity at 1.06～1.09 places, it is essentially impurity Ix) for formula I compound content lower than 5% (for example, lower than 2.5%, for example, lower than 1.7%).

Bulk drug according to second aspect present invention, it is measured according to the present invention [HPLC-A], wherein relative retention time (also can be described as in the present invention RRT1.29 impurity or is called RRT1.29 at the impurity at 1.27～1.31 places, it is essentially impurity Iy) for formula I compound content lower than 10% (for example, lower than 5%, for example, lower than 2.8%).

According to the bulk drug of second aspect present invention, it is measured according to the present invention [HPLC-A], wherein relative retention time the impurity phase at 1.06～1.09 places for formula I compound content lower than 5% (for example, lower than 2.5%, for example, lower than 1.7%); Relative retention time the impurity phase at 1.27～1.31 places for formula I compound content lower than 10% (for example, lower than 5%, for example, lower than 2.8%).

Bulk drug according to second aspect present invention, it is placed 6 months in 38 ℃ of condition lower seals, lucifuge, calculate the content of disposing with this understanding after 6 months when certain impurity phase was for 0 month and increase percentage ratio, wherein the foreign matter content of relative retention time at 1.27～1.31 places increases percentage ratio lower than 200%, for example, lower than 150%, for example, lower than 100%, for example, lower than 75%, for example, lower than 50%, for example, lower than 40%.

According to the bulk drug of second aspect present invention, it is to carry out recrystallization by the bulk drug to comprising formula I compound or pharmaceutically acceptable salt thereof to obtain to reach refining purpose.

According to the bulk drug of second aspect present invention, wherein said recrystallization is that bulk drug is dissolved in the first solvent, then in this solution, adds the second solvent, so that formula I compound or pharmaceutically acceptable salt thereof is settled out crystallization from solvent.

According to the bulk drug of second aspect present invention, wherein said the first solvent is selected from: water, methyl alcohol, ethanol, 1-octanol, phenylcarbinol, methyl-sulphoxide, methylene dichloride, ethyl acetate and combination thereof.In one embodiment, described the first solvent is selected from: ethanol, 1-octanol, methyl-sulphoxide, methylene dichloride, ethyl acetate and combination thereof.In one embodiment, the mixture that described the first solvent is ethanol and ethyl acetate.In one embodiment, described the first solvent is the mixture of ethanol and ethyl acetate volume ratio 1:2～8, preferably the mixture of ethanol and ethyl acetate volume ratio 1:3～7.

According to the bulk drug of second aspect present invention, wherein said the second solvent is selected from: t-butyl methyl ether, normal heptane, normal hexane, Skellysolve A and combination thereof.In one embodiment, described the second solvent is selected from: normal heptane, normal hexane, Skellysolve A and combination thereof.In one embodiment, described the second solvent is normal hexane.

According to the bulk drug of second aspect present invention, the volume ratio of wherein said the first solvent and the second solvent is 1:5～20, for example 1:6～15, for example 1:6～12.

According to the bulk drug of second aspect present invention, wherein said recrystallization is to carry out under 20～50 ℃ of conditions, is for example to carry out under 30～40 ℃ of conditions.

According to the bulk drug of second aspect present invention, in wherein said recrystallization, the ratio that bulk drug is dissolved in the first solvent with the concentration of 0.1～0.2g/ml is processed.

Further, third aspect present invention provides a kind of pharmaceutical composition (for example pharmaceutical preparation), wherein comprises the described bulk drug of second aspect present invention any one, and optional pharmaceutically acceptable carrier or auxiliary material.In one embodiment, pharmaceutical composition of the present invention is to use the described bulk drug of second aspect present invention any one for example, to prepare through pharmaceutical composition (pharmaceutical preparation) preparation technology.

According to the pharmaceutical composition of third aspect present invention, it is oral preparations or injection formulations.

According to the pharmaceutical composition of third aspect present invention, it is tablet, capsule, granule, injection (comprising injection liquid and lyophilize powder injection), suspensoid, pill.

According to the pharmaceutical composition of third aspect present invention, it is injection liquid.

According to the pharmaceutical composition of third aspect present invention, it is injection liquid, in the every 1ml of this injection liquid, comprises: the formula I compound or pharmaceutically acceptable salt thereof of 0.25～0.75mg, dehydrated alcohol 0.025～0.075ml and add to the water for injection of full dose.

Pharmaceutical composition according to third aspect present invention, it is injection liquid, in the every 1ml of this injection liquid, comprises: 0.25～0.75mg formula I compound or pharmaceutically acceptable salt thereof, dehydrated alcohol 0.025～0.075ml, optional pH adjusting agent is appropriate so that the final pH value of injection liquid is adjusted to 6.5～7.5 and add to the water for injection of full dose.In one embodiment, described pH adjusting agent is hydrochloric acid, sodium hydroxide or its combination.

Pharmaceutical composition according to third aspect present invention, it is injection liquid, in the every 1ml of this injection liquid, comprises: the formula Ia compound of 0.25～0.75mg (being the mesylate of formula I compound), dehydrated alcohol 0.025～0.075ml, optional pH adjusting agent is appropriate so that the final pH value of injection liquid is adjusted to 6.5～7.5 and add to the water for injection of full dose.

Pharmaceutical composition according to third aspect present invention, it is injection liquid, in the every 1ml of this injection liquid, comprises: the formula Ia compound of 0.4～0.6mg (being the mesylate of formula I compound), dehydrated alcohol 0.04～0.06ml, optional pH adjusting agent is appropriate so that the final pH value of injection liquid is adjusted to 6.5～7.5 and add to the water for injection of full dose.

According to the pharmaceutical composition of third aspect present invention, it is the lyophilize powder injection, in this powder injection, comprises: the formula I compound or pharmaceutically acceptable salt thereof of 1 weight part, the freeze-dried excipient of 40～400 weight parts.

Pharmaceutical composition according to third aspect present invention, it is the lyophilize powder injection, in this powder injection, comprises: the formula I compound or pharmaceutically acceptable salt thereof of 1 weight part, the freeze-dried excipient of 40～400 weight parts, optional pH adjusting agent is appropriate so that the pH value of this powder injection solution when with water for injection, being diluted to formula I compound concentration and being 0.5mg/ml in 6.5～7.5 scopes.

Pharmaceutical composition according to third aspect present invention, it is the lyophilize powder injection, in this powder injection, comprises: the formula I compound or pharmaceutically acceptable salt thereof of 1 weight part, the freeze-dried excipient of 50～250 weight parts, optional pH adjusting agent is appropriate so that the pH value of this powder injection solution when with water for injection, being diluted to formula I compound concentration and being 0.5mg/ml in 6.5～7.5 scopes.

In one embodiment, described freeze-dried excipient can be selected from N.F,USP MANNITOL, lactose, maltose, sucrose, sodium-chlor etc.

According to the pharmaceutical composition of third aspect present invention, comprising the formula I compound or pharmaceutically acceptable salt thereof as active ingredient, and the optional impurity of the Ix as impurity.

According to the pharmaceutical composition of third aspect present invention, wherein the Ix impurity phase for formula I compound content lower than 5% (for example, lower than 2.5%, for example, lower than 1.7%).

According to the pharmaceutical composition of third aspect present invention, comprising the formula I compound or pharmaceutically acceptable salt thereof as active ingredient, and the optional impurity of the Iy as impurity.

According to the pharmaceutical composition of third aspect present invention, wherein impurity Iy with respect to the content of formula I compound lower than 10% (for example, lower than 5%, for example, lower than 2.8%).

Pharmaceutical composition according to third aspect present invention, it is measured according to the present invention [HPLC-A], wherein relative retention time (also can be described as in the present invention RRT1.07 impurity or is called RRT1.07 at the impurity at 1.06～1.09 places, it is essentially impurity Ix) for formula I compound content lower than 5% (for example, lower than 2.5%, for example, lower than 1.7%).

Pharmaceutical composition according to third aspect present invention, it is measured according to the present invention [HPLC-A], wherein relative retention time (also can be described as in the present invention RRT1.29 impurity or is called RRT1.29 at the impurity at 1.27～1.31 places, it is essentially impurity Iy) for formula I compound content lower than 10% (for example, lower than 5%, for example, lower than 2.8%).

According to the pharmaceutical composition of third aspect present invention, it is measured according to the present invention [HPLC-A], wherein relative retention time the impurity phase at 1.06～1.09 places for formula I compound content lower than 5% (for example, lower than 2.5%, for example, lower than 1.7%); Relative retention time the impurity phase at 1.27～1.31 places for formula I compound content lower than 10% (for example, lower than 5%, for example, lower than 2.8%).

Pharmaceutical composition according to third aspect present invention, it is placed 4 months in 38 ℃ of condition lower seals, lucifuge, calculate the content of disposing with this understanding after 4 months when certain impurity phase was for 0 month and increase percentage ratio, wherein the foreign matter content of relative retention time at 1.27～1.31 places increases percentage ratio lower than 200%, for example, lower than 150%, for example, lower than 100%, for example, lower than 75%, for example, lower than 50%, for example, lower than 40%.This test method in the present invention can be referred to as investigating 38 ℃-April.

Further, fourth aspect present invention provides the purposes of the first aspect present invention bulk drug that described method prepares, the described bulk drug of second aspect or the described pharmaceutical composition of the third aspect, for the preparation of the medicine as microtubule inhibitors or for example, for the preparation for the treatment of tumour (optimum or malignant tumour, for example cancer, for example mammary cancer) medicine.

According to the purposes of fourth aspect present invention, the side reaction that neutrophil leucocyte reduces does not appear in wherein said medicine when microtubule suppresses or treats tumour.

According to the purposes of fourth aspect present invention, wherein said medicine when being applied to the experimenter, in this medicine for formula I compound the content of RRT1.29 impurity lower than 10% (for example, lower than 5%, for example, lower than 2.8%).According to the purposes of fourth aspect present invention, wherein said medicine is measured according to the present invention [HPLC-A], wherein relative retention time the impurity phase at 1.27～1.31 places for formula I compound content lower than 10% (for example, lower than 5%, for example, lower than 2.8%).According to the purposes of fourth aspect present invention, wherein said medicine when being applied to the experimenter, in this medicine for formula I compound the content of RRT1.07 impurity lower than 5% (for example, lower than 2.5%, for example, lower than 1.7%).According to the purposes of fourth aspect present invention, wherein said medicine is measured according to the present invention [HPLC-A], wherein relative retention time the impurity phase at 1.06～1.09 places for formula I compound content lower than 5% (for example, lower than 2.5%, for example, lower than 1.7%).

According to either side of the present invention, the content of wherein said medicinal raw material medicine Chinese style I compound or formula Ia compound is greater than 95%, for example, for example, in 95.0%～105.0% scope, in 97.0%～102.0% scope.

According to either side of the present invention, the chromatographic purity of wherein said medicinal raw material medicine is in 97.0%～99.9% scope, for example, in 98.0%～99.9% scope, for example, in 98.5%～99.9% scope.

According to either side of the present invention, in wherein said medicinal raw material medicine or described pharmaceutical composition, for formula I compound, RRT1.07 content is in 0.10～1.70% scope, for example, in 0.10～1.5% scope, for example, in 0.10～1.0% scope.

According to either side of the present invention, in wherein said medicinal raw material medicine or described pharmaceutical composition, for formula I compound, RRT1.29 content is in 0.05～2.80% scope, for example, in 0.05～2.0% scope, for example, in 0.5～1.5% scope, for example, in 0.5～1.0% scope.

Arbitrary embodiment of either side of the present invention, can be combined with other embodiment, as long as they not there will be contradiction.In addition, in arbitrary embodiment of either side of the present invention, arbitrary technical characterictic goes for this technical characterictic in other embodiment, as long as they not there will be contradiction.

In the present invention, the implication of " % " can be determined according to concrete environment for use, and particularly it has as implication as described in " metering " lower the 28 (4) money in two notes on the use of version Chinese Pharmacopoeia in 2010.

In the present invention, while mentioning separately, " ethanol " refers to dehydrated alcohol.

At the formula I compound the present invention relates to, take its mesylate as example, its exemplary English chemistry is by name:

11,15:18,21:24,28-Triepoxy-7,9-ethano-12,15-methano-9H,15H-furo[3,2-i]furo[2',3':5,6]pyrano[4,3-b][1,4]dioxacyclopentacosin-5(4H)-one,2-[(2S)-3-amino-2-hydroxypropyl]hexacosahydro-3-methoxy-26-methyl-20,27bis(methylene)(2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13aR,13bS,15S,18S,21S,24S,26R,28R,29aS),methanesulfonate(salt)；

Molecular formula: C ₄₀h ₅₉nO ₁₁cH ₃sO ₃h,

Molecular weight: 826.0 (free alkali is that the molecular weight of formula I compound is 729.9),

Take formula I compound mesylate as each carbon atom of example can number indicate as follows:

Above-mentioned exemplary numbering can adopt in this article.Although from corresponding carbon in the disclosed compound B-11 939 of CN1216051C (62 pages, specification sheets), to paint method different on the method for painting for the 12-position carbon in above structure, the two chiral configuration is identical, is the R type.

In the present invention, although the inventor has known the particular chemical of RRT1.07 and two specific impurities of RRT1.29, but these impurity also can carry out qualitative sign in [HPLC-A] of specific, concrete of the present invention by it, particularly use the RRT parameter presented under specific HPLC condition to characterize.As well known to those skilled in the art, can fully independently use RRT to characterize and needn't consider its particular chemical fully, reason is that RRT is a constant parameter under regulation HPLC condition, and RRT characterizes with structural characterization and can be separate or use in combination with one another.And this characterizing method is pharmaceutical field, bulk drug or preparation quality standard are allowed, possess industrial applicability fully.

In the present invention, while determining the content of various materials (such as various as bulk drug, pharmaceutical composition such as pharmaceutical preparation etc. for pharmaceutical preparation) Chinese style I compound or pharmaceutically acceptable salt thereof or other impurity, and while determining the chromatographic purity in these materials, can adopt following [HPLC-A] method to be measured.The embodiment part hereinafter in the present invention, while as needs, measuring content, the related substance of some material, if not otherwise indicated, is all according to should [HPLC-A] method carrying out.

The measuring method of [HPLC-A] is as follows:

(2) chromatographic condition and system suitability:

packing material size 3 μ m, the pillar size: internal diameter 3.0mm * this chromatographic column of column length 150mm[is the ACE-C18-300 chromatographic column purchased from Britain Advanced ChromatographyTechnologies Ltd company, product article No. ACE-211-1503];

[this chromatographic column is the ACE-C18-300 chromatographic column purchased from Britain Advanced Chromatography Technologies Ltd company, product article No. ACE-211-1503]

Column temperature: 40 ℃;

The gradient elution program:

(3) preparation of test fluid:

Above [HPLC-A] can be advantageously used in measuring the related substance in various materials.When measuring the content of the formula I compound or pharmaceutically acceptable salt thereof in various materials, can use the chromatographic purity of formula I compound or pharmaceutically acceptable salt thereof to be greater than 99.0% reference substance in contrast, with external standard method, carry out quantitatively.When needs are measured the absolute magnitude of impurity in these materials, also can use the chromatographic purity of this impurity to be greater than 99.0% reference substance in contrast, with external standard method, carry out quantitatively; But while typically characterizing the amount of impurity, with this impurity phase, the relative percentage composition for formula I compound means.

The present invention hereinafter tests in above [HPLC-A] of use, the first flow velocity is 0.50ml/min, formula I compound retention time in 26～32min scope, formula I compound peaks tailing factor 0.97, theoretical plate number 19310, the resolution 2.5 of formula I compound and RRT1.07.

The chromatographic purity of mentioning in the present invention refers in the present invention [HPLC-A] method measures relevant arriving.As well known to those skilled in the art, this chromatographic purity is normally measured in the related substance checking process, and deduction solvent peak, methanesulfonate, auxiliary material isochromatic spectrum peak calculate and obtain through area normalization method.

In addition, in arbitrary embodiment of the present invention, when with respect to formula I compound, characterizing the relative percentage composition of each impurity, no matter be the material of formula I compound free alkali form, or the material of its pharmaceutical salts form, all convert accepted way of doing sth I compound free alkali form meter.Reason is that this free alkali form or pharmaceutical salts form are when injecting high performance liquid chromatography, and at 26～32min wash-out, main peak out is all free alkali forms, and the salt root for example the methanesulfonate ion and this chromatographic peak not overlapping.

In the present invention, can be by well known to a person skilled in the art method, in the peak area by each impurity peaks and contrast solution color atlas, the main peak Area comparison calculates the content of this impurity in trial-product; For example in the contrast solution color atlas, the main peak area is 1000 (its be equivalent to need testing solution concentration 1%), if the peak area of certain impurity is 5000 the content of this impurity in trial-product is 5% in the need testing solution color atlas, if the peak area of certain impurity is 2000 the content of this impurity in trial-product is 2% in the need testing solution color atlas; The content, specific impurities that these calculating can obtain the maximum single contaminant in this trial-product is RRT1.07 and RRT1.29 content for example; The content of all dirt adds and can obtain total impurities content.Above when measuring related substance, for the pharmaceutical composition that comprises pharmaceutical excipient, the chromatographic peak of auxiliary material in the subtractive color spectrogram when calculating.

" pharmaceutically acceptable carrier " used in pharmaceutical composition of the present invention can be the carrier of any routine in field of pharmaceutical preparations.The selection of specific support will be depended on administering mode or disease type and the state that is used for the treatment of particular patient.For the preparation method of the suitable drug composition of specific administration pattern fully in pharmaceutical field technician's ken.For example, can be used as thinner, carrier, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier and the lubricant etc. that pharmaceutically acceptable carrier comprises the pharmaceutical field routine.In case of necessity, can also in pharmaceutical composition, add flavouring agent, preservative and sweetener etc.

Pharmaceutical composition of the present invention can be made the various ways such as tablet, pulvis, granule, capsule, oral liquid, paste, creme, injectable emulsion (aseptic powder needle for injection).The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.

Neutrophil leucocyte (Neutrophil or Neutrophil granulocyte, can be abbreviated as NG in the present invention) is a kind of of blood leucocyte, is also topmost a kind of white corpuscle in mammalian.Neutrophil leucocyte plays a very important role in the non-specific cell immunity system of blood, it resists microbial pathogen in body, particularly, at the First Line of suppurative bacterium invasion, when inflammation occurs, they are attracted inflammation part by the chemotaxis material.Because they are to borrow glycolysis-to obtain energy, therefore under the anaerobic conditions of swelling thrombosis, still can survive, they form cell toxicant here and have the cytolemma that destroys bacterium and near tissue.Because neutrophil leucocyte contains a large amount of lysosomal enzymes, therefore can will engulf into intracellular bacterium and fragment of tissue and decompose, like this, the bacterium of invasion is enclosed in a part, and eliminates, and prevents that pathogenic micro-organism from spreading in vivo.When neutral granulocyte itself disintegrates, disengage each lysosomal enzymes and can dissolve surrounding tissue and form abscess.The cytolemma of neutrophil leucocyte can discharge a kind of unsaturated fatty acids---arachidonic acid, under the effect of enzyme, further generate again one group of paracrine hormone substance by it, as thromboxane and prostaglandin(PG) etc., this class material has obvious effect to regulating external caliber and permeability, can also cause inflammation and react and pain, and affect blood coagulation.Neutrophil leucocyte includes the peculiar particle of the tiny pale red or lilac of many disperses distributions, contains myeloperoxidase, acid phosphatase, phagocytin, N,O-Diacetylmuramidase etc. in particle.Myeloperoxidase is that neutrophil leucocyte institute is peculiar, even also seldom or fully there is no this kind of enzyme in strong phagocytotic scavenger cell is arranged.On cytochemistry, the general sign using this myeloperoxidase as neutrophil leucocyte.Neutrophil leucocyte has very strong chemotaxis.So-called chemotaxis is exactly that the direction that stimulates towards a certain chemical substance of cell moves.The centering granulocyte plays the material of chemotaxis, is called neutrophilic chemotactic factor.On neutral thickness after birth, Chemokine Receptors is arranged, acceptor is combined with chemokine, activates the calcium pump on after birth, and cell forwards stretches out the sheet foot, makes cell shift to the position that produces chemokine.After the sufficient foreign matter with producing chemokine of the sheet of neutrophil leucocyte contacts, it is pseudopodium that the kytoplasm around contact position forms protuberance, and the cytolemma of contact site is recessed, by the foreign matter encirclement, forms the phagosome or the phagocytic vacuole that contain foreign matter.There are IgGFc acceptor and complement C3 acceptor in the Polymorphonuclear Leukocytes Membrane surface, can accelerate phagolysis.When the foreign matter of being engulfed is wrapped with antibody and complement, with the corresponding receptors bind on Polymorphonuclear Leukocytes Membrane, and strengthen the phagolysis of cell to it, be called opsonization.Cell is along with phagocytotic beginning, causes cytolemma disorderly and cause that respiratory burst, cell oxygen-consumption increase, and produces the cytotoxic effect molecules such as a large amount of superoxide and super-oxide, and parasite is had to killing activity.Under IFN-γ and TNF stimulation, can produce more peroxide metabolizable anions, kill the born of the same parents epizoa.Neutrophil leucocyte is after killing the foreign matters such as bacterium of engulfing, and itself is also dead, and dead neutrophil leucocyte is called pyocyte.Neutrophil leucocyte is subject to bacterial product, immune complex etc. to do the used time, and the granular contents of cell discharges to extracellular.The aspartic protease disengaged and neutral protease, can decompose collagen protein and complement C5, C15 in elastin and blood plasma and the prokinin etc. of vascular basement membrane, glomerular basement membrane, reticular tissue.What its degradation production had is again the thin chemokine that runs of neutral grain, can attract more neutrophil leucocyte.In the material that neutrophil leucocyte discharges, also have eotaxin, neutrophil immobilizing factor(NIF) (NIF), kininogenase, profibr(in)olysin, thrombin, leukotriene etc.Except play important defense reaction in anti-infective, neutrophil leucocyte can cause the inflammatory reaction of infection site and participate in the transformation reactions that parasitic infection causes, thereby cause the immunopathogenesis infringement.Antibody directly acts on the antigen on tissue or cell, and neutrophil leucocyte is combined with the IgGFc on target cell surface section by its Fc acceptor, performance ADCC effect, thus cause the cytotoxic type hypersensitivity infringement; When the antigen-antibody ratio is applicable to and the immunocomplex of formation 19S size, be difficult for being engulfed, be deposited on capillary wall, activating complement, attract neutrophil leucocyte to local.Neutrophil leucocyte is combined with immunocomplex and engulfs it by Fc acceptor and C3b receptor.In phagocytosis, de-particle, discharge a series of lysosomal enzymeses, causes the damage of blood vessel and surrounding tissue; , also there is the gathering of neutrophil leucocyte at the position of the immediate allergy mediated at IgE, illustrates that neutrophil leucocyte has also participated in the pathological lesion that immediate allergy causes.Thus, the performance that in blood of human body, neutrophil leucocyte reduces for the normal physiological function of human body is disadvantageous.

The existing eribulin medicament of list marketing (such as injection liquid etc.), it is used for the treatment of metastatic breast cancer patient etc. as a kind of microtubule inhibitors.The invention provides a kind of new eribulin bulk drug, it has good characteristics aspect stability, and demonstrate good biological safety, therefore material medicine of the present invention can be used for equally treating metastatic breast cancer and has low rate of side effects, and for example low neutrophil leucocyte reduces this rate of side effects.

The accompanying drawing explanation

Fig. 1: a kind of bulk drug of the present invention is measured the HPLC figure of related substance.

Fig. 2: a kind of pharmaceutical composition of the present invention is measured the HPLC figure of related substance.

Embodiment

By the following examples, can conduct further description the present invention, yet scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and under the prerequisite that does not deviate from the spirit and scope of the present invention, can carry out various variations and modification to the present invention.The present invention carries out generality and/or concrete description to the material and the test method that use in test.Although, for to realize that many materials and working method that the object of the invention is used are well known in the art, the present invention still does to describe in detail as far as possible at this.

embodiment 1: prepare eribulin/methylsulfonic acid eribulin bulk drug

Step (1)---synthetic compound of formula i: shine 28 page of 11 row of CN1216051C (Chinese patent ZL99809658.X) specification sheets from the synthetic triol 1 of L-arabinose, obtain the method that compound B-11 939 is formula I compound of the present invention (free alkali form) to 62 page of 7 row of specification sheets, making product is formula I compound (crude product).It is measured through [HPLC-A], RRT1.07 content=4.17%, RRT1.29 content=0.73%.

Step (2)---salify: at the temperature of 20～25 ℃, to the methylsulfonic acid that adds step (1) products therefrom 50 grams and 70mmol in the mixing solutions of 1400ml water and 1300ml acetonitrile, stir and make reaction, by the reactant concentrating under reduced pressure; The gained resistates is dissolved in the 400ml methylene dichloride, filters, in filtrate, add the 3000ml Skellysolve A; Leach precipitation, with the Skellysolve A washing, in vacuum-drying, making product is formula Ia compound.It is measured through [HPLC-A], RRT1.07 content=3.36%, RRT1.29 content=0.61%.

Step (3)---recrystallizing and refining: at the temperature of 33～36 ℃, step (2) products therefrom 1.5g is dissolved in to dehydrated alcohol-ethyl acetate mixed solvent (1:5, v/v) in 10ml, slowly drip wherein normal hexane 90ml, the standing abundant precipitation (approximately 24 hours) that makes, leach precipitation, with normal hexane, washs, vacuum-drying, obtain (recrystallization yield 93.3%).It is measured through [HPLC-A], RRT1.07 content=0.83%, RRT1.29 content=0.37%.Through a recrystallization purifying, the content of RRT1.07 has reduced i.e. (3.36-0.83) ÷ 3.36 * 100% of 75.3%[].It can be used as methylsulfonic acid eribulin bulk drug of the present invention.Fig. 1 has shown typical HPLC figure when this methylsulfonic acid eribulin bulk drug is measured related substance, in figure, display type Ix compound goes out peak (this peak is that formula I compound is the free alkali peak at the 27.684min place, formula Ia salt dissociates in moving phase, methanesulfonate does not go out peak herein), show Ix impurity (occurrence of RRT is 1.073) at the 29.717min place, show Iy impurity (occurrence of RRT is 1.284) at the 35.552min place.

embodiment 2: prepare eribulin/methylsulfonic acid eribulin bulk drug

Get the resulting product of embodiment 1 step (2), reference example 1 step (3) operating method is carried out recrystallizing and refining.

At the temperature of 30～35 ℃, embodiment 1 step (2) products therefrom 1.0g is dissolved in to dehydrated alcohol-ethyl acetate mixed solvent (1:3, v/v) in 10ml, slowly drip wherein normal hexane 60ml, the standing abundant precipitation (approximately 6 hours) that makes, leach precipitation, with normal hexane, washs, vacuum-drying, obtain (recrystallization yield 91.4%).It is measured through [HPLC-A], RRT1.07 content=0.76%, RRT1.29 content=0.33%.Through a recrystallization purifying, the content of RRT1.07 has reduced by 77.4%.It can be used as methylsulfonic acid eribulin bulk drug of the present invention.

embodiment 3: prepare eribulin/methylsulfonic acid eribulin bulk drug

At the temperature of 35～40 ℃, embodiment 1 step (2) products therefrom 2.0g is dissolved in to dehydrated alcohol-ethyl acetate mixed solvent (1:7, v/v) in 10ml, slowly drip wherein normal hexane 120ml, the standing abundant precipitation (approximately 36 hours) that makes, leach precipitation, with normal hexane, washs, vacuum-drying, obtain (recrystallization yield 95.2%).It is measured through [HPLC-A], RRT1.07 content=0.91%, RRT1.29 content=0.44%.Through a recrystallization purifying, the content of RRT1.07 has reduced by 72.9%.It can be used as methylsulfonic acid eribulin bulk drug of the present invention.

embodiment 4: prepare eribulin/methylsulfonic acid eribulin bulk drug

Get the resulting product of embodiment 1 step (3), reference example 1 step (3) operating method is carried out recrystallizing and refining again.This recrystallization yield 93.3%.This product is measured through [HPLC-A], RRT1.07 content=0.24%, RRT1.29 content=0.14%.This recrystallization purifying, the content of RRT1.07 has reduced by 71.1%.It can be used as methylsulfonic acid eribulin bulk drug of the present invention.

embodiment 5: prepare eribulin/methylsulfonic acid eribulin bulk drug

Step (1)---with embodiment 1 step (1).

Step (2)---salify: at the temperature of 20～25 ℃, to the methylsulfonic acid that adds step (1) products therefrom 60 grams and 80mmol in the mixing solutions of 1400ml water and 1300ml acetonitrile, stirring makes reaction, adds 4ml ammonium hydroxide, by the reactant concentrating under reduced pressure; The gained resistates is dissolved in the 400ml methylene dichloride, filters, in filtrate, add the 3000ml Skellysolve A; Leach precipitation, with the Skellysolve A washing, in vacuum-drying, making product is formula Ia compound.It is measured through [HPLC-A], RRT1.07 content=3.52%, RRT1.29 content=0.69%.

Step (3)---recrystallizing and refining: at the temperature of 30～40 ℃, step (2) products therefrom 1.5g is dissolved in to dehydrated alcohol-ethyl acetate mixed solvent (1:6, v/v) in 10ml, slowly drip wherein normal hexane 100ml, the standing abundant precipitation (approximately 24 hours) that makes, leach precipitation, with normal hexane, washs, vacuum-drying, obtain (recrystallization yield 91.6%).It is measured through [HPLC-A], RRT1.07 content=0.87%, RRT1.29 content=0.43%.This recrystallization purifying, the content of RRT1.07 has reduced by 75.3%.It can be used as methylsulfonic acid eribulin bulk drug of the present invention.

embodiment 6: prepare eribulin/methylsulfonic acid eribulin bulk drug

Step (1)---preparation I compound:

The compound B-11 793 (47 pages, specification sheets, the C that comprise two hydroxyls with the CN1216051C gained ₄₀h ₅₈o ₁₂, Mr730.88) be initial substance.At the temperature lower than-10 ℃, in the 30ml methylene dichloride, add the pyridine of the 2，4，6-trimethylpyridine of tosic acid acid anhydride, 4mmol of 731mg (1mmol) B1793 and 1.05mmol and 0.05mmol to form the tosilate of following formula:

Reactant is suitably concentrated, add 100ml Virahol, 120ml ammonium hydroxide, the temperature of 20～25 ℃ under stir and make reaction, below forming in reaction process as the epoxide of reaction intermediate:

TLC monitoring reaction process is so that above-mentioned epoxide disappearance in reactant forms formula I compound of the present invention; At the temperature of<30 ℃, that reactant is concentrated, add methylene dichloride 30ml, with 5% sodium bicarbonate and the extraction of 5% sodium carbonate solution, divide and get organic layer successively, concentrated at the temperature of<30 ℃, obtain resistates; The gained resistates is loaded on silicagel column, use successively acetonitrile/water/0.25M ammonium acetate gradient elution: first use the acetonitrile/water of 20 column volumes/0.25M ammonium acetate (100/0/0, v/v) wash-out, then with the acetonitrile/water of 30 column volumes/0.25M ammonium acetate (90/7/3, v/v) wash-out, then with the acetonitrile/water of 30 column volumes/0.25M ammonium acetate (85/12/3, v/v) wash-out, then with the acetonitrile/water of 20 column volumes/0.25M ammonium acetate (80/18/3, v/v) wash-out; Elutriant is concentrated at the temperature of<40 ℃, obtain resistates, add methylene dichloride 20ml, with 5% sodium bicarbonate and the extraction of 5% sodium carbonate solution, divide and get organic layer successively, concentrated at the temperature of<30 ℃, obtain resistates; Above resistates is dissolved in the mixed solution (1:3, v/v) of methylene dichloride and Skellysolve A, filters, add acetonitrile at the temperature of<30 ℃, standingly make to separate out precipitation, filter, with acetonitrile washing, vacuum-drying, obtain being the formula I compound of the present invention of free alkali form.It is measured through [HPLC-A], RRT1.07 content=2.74%, RRT1.29 content=0.42%.

Step (2)---salify: carry out according to the embodiment of the present invention 1 step (2), obtain formula Ia compound.It is measured through [HPLC-A], RRT1.07 content=2.13%, RRT1.29 content=0.34%.

Step (3)---recrystallizing and refining: carry out purifying according to the embodiment of the present invention 1 step (3), obtain can be used as the formula Ia product (recrystallization yield 92.4%) of bulk drug of the present invention.It is measured through [HPLC-A], RRT1.07 content=0.51%, RRT1.29 content=0.18%.This recrystallization purifying, the content of RRT1.07 has reduced by 76.6%.It can be used as methylsulfonic acid eribulin bulk drug of the present invention.

Step (4)---free alkali is refining: step (1) gained formula I free alkali is dissolved in the mixed solution (1:3, v/v) of methylene dichloride and Skellysolve A, filters, add acetonitrile at the temperature of<30 ℃, standingly make to separate out precipitation, filter, with acetonitrile washing, vacuum-drying; Repeat again this precipitation crystallization operation 3 times, obtain being the formula I compound of the present invention of free alkali form.It is measured through [HPLC-A], RRT1.07 content=0.94%, RRT1.29 content=0.31%.

comparing embodiment 1: with reference to the operation of this paper embodiment 1 step (3), different is only by the first solvent wherein is that dehydrated alcohol-ethyl acetate mixed solvent replaces with ethanol and ethyl acetate volume ratio 1:3～7 water, methyl alcohol, ethanol, 1-octanol, methylene dichloride, ethyl acetate, makes 6 batches of products.Calculate these 6 batches of products and reduce degree through the content of RRT1.07 after recrystallization purifying, the content of RRT1.07 reduces all in 14.2～32.3% scopes as a result, when using dehydrated alcohol of the present invention-ethyl acetate mixed solvent content reduce up to 70% low.

comparing embodiment 2: with reference to the operation of this paper embodiment 1 step (3), different is only by the first solvent wherein is that dehydrated alcohol-ethyl acetate mixed solvent 1:5 volume ratio changes 1:1,1:10 or 1:15 into, makes 3 batches of products.Calculate these 3 batches of products and reduce degree through the content of RRT1.07 after recrystallization purifying, the content of RRT1.07 reduces all in 26.5～42.8% scopes as a result, more than use specified proportion of the present invention dehydrated alcohol-the ethyl acetate mixed solvent is low.

comparing embodiment 3: with reference to the operation of this paper embodiment 1 step (3), different is only by the second solvent wherein is that normal hexane replaces with t-butyl methyl ether, normal heptane or Skellysolve A, makes 3 batches of products.Calculate these 3 batches of products and reduce degree through the content of RRT1.07 after recrystallization purifying, the content of RRT1.07 reduces all in 19.6～30.5% scopes as a result, and more than using, the present invention is low with normal hexane.

preparation example 1: prepare methylsulfonic acid eribulin reference substance

At the temperature of 33～36 ℃, above embodiment 4 gained methylsulfonic acid eribulin bulk drug 1.5g are dissolved in methylene dichloride 10ml, slowly drip wherein Skellysolve A 100ml, the standing abundant precipitation (approximately 24 hours) that makes, leach precipitation, with the Skellysolve A washing, vacuum-drying, obtain (recrystallization yield 65.4%).It is measured through [HPLC-A], RRT1.07 content=0.11%, RRT1.29 content=0.06%, HPLC purity 99.73%.It can be used as the methylsulfonic acid eribulin reference substance that the present invention tests use.

preparation example 2: preparation RRT1.07 impurity and RRT1.29 impurity

Get above embodiment 1 step (1) gained formula I compound (crude product), C18 post (Pinnacle II C18HPLC preparative column with preparative, pillar size: internal diameter 50mm * column length 250mm, article No. 9214570, purchased from Ming Nike company), the middle mobile phase A of the present invention [HPLC-A] of take is eluting solvent, collects respectively the elution peak of RRT1.07 impurity and RRT1.29 impurity, and two parts of elutriants reduce pressure and desolventize respectively; The resistates that obtains is carried out to crystalline deposit with purifying with reference to the mode of preparation example 1 above respectively, and purifying 2 times, obtain respectively RRT1.07 impurity and RRT1.29 impurity.Two kinds of impurity are measured according to the present invention [HPLC-A], and chromatographic purity is respectively 98.2% and 98.5%.They can be used as RRT1.07 impurity and the RRT1.29 impurity that the present invention tests use.

test example 1: the performance test of bulk drug of the present invention

The physicochemical data of test implementation example 1 step (3), embodiment 2, embodiment 3, embodiment 4, embodiment 5 steps (3), embodiment 6 steps (3) and preparation example 1 gained methylsulfonic acid eribulin T etc., result is as follows:

(1) appearance character: be white powder.

(2) TG-DTA: all decompose in 193～208 ℃ of scopes.

(3) X-ray powder diffraction: scanning between 2 θ angle 0～40 degree all is shown as typical amorphous diffraction spectra.

(4) specific optical rotation: under 20 ℃, measure, concentration 5mg/mL, solvent DMSO, the specific optical rotation of preparation example 1 product is-192.1 °.

(5) Advances in crystal X-ray diffraction: the Advances in crystal X-ray diffraction of test implementation example 1 step (1) gained formula I compound (with reference to preparation example 1 method, carrying out recrystallization purifying so that chromatographic purity reaches more than 98.0%) shows that its configuration conforms to following formula:

(6) NMR spectrum: the methylsulfonic acid eribulin reference substance of preparation example 1 preparation ¹h NMR spectrum and ¹³the demonstration of C NMR spectrum conforms to formula Ia structure, and concrete data are as follows:

Position	¹H(J=Hz)	¹³C	Position	¹H(J=Hz)	¹³C
						0a	2.46(m)	48.5	19’a	5.01(brd)	105.1
0b	2.66(m)	48.5	19’b	5.11(brd,2)	105.1
						1	-	208.0	20	4.47(brd,11)	75.4
2a	2.30(brd,17)	49.5	21a	1.42(m)	31.1
						2b	2.73(dd,17,10)	49.5	21b	2.02(m)	31.1
3	3.97(m)	74.3	22a	1.46(m)	32.8
						4ax	1.34(m)	32.2	22b	1.77(m)	32.8
4eq	1.60(m)	32.2	23	3.83(brdd,11,11)	74.5
						5ax	1.38(m)	31.2	24ax	1.02(ddd,12,12,11)	45.1
5eq	2.01(m)	31.2	24eq	1.76(m)	45.1
						6	4.28(m)	69.9	25	2.35(m)	36.7
7	2.93(dd,10,2)	78.5	25’	1.12(3H,d,6)	18.5
						8	4.29(m)	75.4	26	-	153.3
9	4.12(dd,7,4)	74.5	26’a	4.82(brs)	105.1
						10	4.16(dd,7,4)	77.7	26’b	4.85(brs)	105.1
11	4.61(dd,5,4)	84.0	27	3.74(brd,11)	76.1
						12	4.71(dd,5,5)	82.6	28a	1.86(m)	39.5
13a	1.96(dd,13,5)	49.4	28b	2.07(m)	39.5
						13b	2.09(d,13)	49.4	29	3.86(m)	82.3
14	-	111.0	30	2.46(m)	44.6
						15a	1.55(m)	36.4	31	3.37(d,3)	88.5
15b	2.19(m)	36.4	31’	3.42(3H,s)	57.4
						16a	1.45(m)	29.8	32	3.89(m)	78.4
16b	2.18(m)	29.8	33a	1.84(m)	34.7
						17	4.10(m)	78.1	33b	1.92(m)	34.7
18a	2.36(m)	40.1	34	3.96(m)	66.8
						18b	2.86(m)	40.1	35a	2.89(dd,13,9)	45.8
19	-	154.3	35b	3.06(dd,13,3)	45.8
						?	?	?	Methanesulfonate	2.68(3H,s)	39.5

the physicochemical property test of test example 2:RRT1.07 impurity and RRT1.29 impurity

Tested the physicochemical data of preparation example 2 gained RRT1.07 impurity and RRT1.29 impurity in this test example, result is as follows:

(1) through methods such as mass spectrum, ultimate analyses, measure, the molecular formula of determining RRT1.07 impurity is C ₄₀h ₅₉nO ₁₁, molecular weight is 729.90.

Comprehensive this paper test result, can reasonably infer that according to the chemosynthesis principle this impurity is the final stage in CN1216051C, B1793 carries out in the process of methylsulfonic acid esterification reaction, may carry out esterification with the another one hydroxyl, follow-up azide and ammoxidation and possible configuration reversal that then this esterification products is carried out to be similar to B2294, final formation has the compound of the formula Ix as impurity of the present invention.In addition, not through azide in the embodiment of the present invention 6 but form epoxide intermediates, can reasonably infer that according to the chemosynthesis principle can and configuration reversal occur with ammonia react at this epoxide C34 position carbon forms the formula Ix compound as impurity equally.The test result of the context of the invention has confirmed the consistence of above-mentioned supposition and RRT1.07 impurity chemical structure.

Through methods such as mass spectrum, ultimate analyses, measure, the molecular formula of determining RRT1.29 impurity is C ₄₀h ₆₁nO ₁₂, molecular weight is 747.91.Can reasonably infer that according to the chemosynthesis principle this impurity is the degraded product for bulk drug and preparation, and the final stage of reaction particularly for example this paper from the compound B-11 793 that comprises two hydroxyls, start also can produce this impurity follow-up synthesis step, this material be formula I compound in the C14 position product through the ketal hydrolysis.The test result of the context of the invention has confirmed the consistence of above-mentioned supposition and RRT1.29 impurity chemical structure.

(2) NMR spectrum: RRT1.07 impurity ¹h NMR spectrum and ¹³the demonstration of C NMR spectrum conforms to formula Ix structure, RRT1.29 impurity ¹h NMR spectrum and ¹³c NMR spectrum shows and conforms to formula Iy structure.

RRT1.07 impurity: at CD ₃test RRT1.07 impurity in OD, result is as following table (600MHz, 30 ℃, the position mark of proton and carbon is referring to following structure):

Position	¹H(J=Hz)	¹³C	Position	¹H(J=Hz)	¹³C
						0a	2.44(m)	48.5	19’a	5.01(brd)	105.2
0b	2.68(m)	48.5	19’b	5.12(brd,2)	105.2
						1	-	208.2	20	4.48(brd,11)	75.5
2a	2.32(brd,17)	49.3	21a	1.41(m)	31.1
						2b	2.71(dd,17,10)	49.3	21b	2.01(m)	31.1
3	3.95(m)	74.6	22a	1.47(m)	32.6

4ax	1.31(m)	32.2	22b	1.78(m)	32.6
						4eq	1.72(m)	32.1	23	3.84(brdd,11,11)	74.6
5ax	1.37(m)	31.3	24ax	1.03(ddd,12,12,11)	45.0
						5eq	2.01(m)	31.3	24eq	1.77(m)	45.0
6	4.30(m)	69.7	25	2.34(m)	36.6
						7	2.93(dd,10,2)	78.5	25’	1.12(3H,d,6)	18.5
8	4.29(m)	75.5	26	-	153.1
						9	4.12(dd,7,4)	74.5	26’a	4.83(brs)	105.1
10	4.18(dd,7,4)	77.6	26’b	4.84(brs)	105.1
						11	4.61(dd,5,4)	84.1	27	3.74(brd,11)	76.2
12	4.70(dd,5,5)	82.4	28a	1.86(m)	39.5
						13a	1.98(dd,13,5)	49.4	28b	2.08(m)	39.5
13b	2.08(d,13)	49.4	29	3.86(m)	82.4
						14	-	111.0	30	2.47(m)	44.7
15a	1.55(m)	36.4	31	3.36(d,3)	88.4
						15b	2.19(m)	36.4	31’	3.43(3H,s)	57.5
16a	1.45(m)	29.7	32	3.88(m)	78.5
						16b	2.18(m)	29.7	33a	1.89(m)	30.7
17	4.08(m)	78.1	33b	1.96(m)	30.7
						18a	2.36(m)	40.1	34	2.97(m)	45.3
18b	2.88(m)	40.1	35a	3.98(m)	70.3
						19	-	154.3	35b	4.02(m)	70.3

RRT1.29 impurity: at CD ₃test RRT1.29 impurity in OD, result is as following table (600MHz, 30 ℃, the position mark of proton and carbon can be similar to the structure of top Ix):

Position	¹H(J=Hz)	¹³C	Position	¹H(J=Hz)	¹³C
						0a	2.44(m)	48.5	19’b	5.11(brd,2)	105.2
0b	2.67(m)	48.5	20	4.48(brd,11)	75.5
						1	-	208.2	21a	1.41(m)	31.1
2a	2.32(brd,17)	49.3	21b	2.01(m)	31.1
						2b	2.71(dd,17,10)	49.3	22a	1.47(m)	32.6
3	3.94(m)	74.6	22b	1.78(m)	32.6
						4ax	1.31(m)	32.2	23	3.84(brdd,11,11)	74.6
4eq	1.72(m)	32.1	24ax	1.03(ddd,12,12,11)	45.0
						5ax	1.37(m)	31.3	24eq	1.77(m)	45.0

5eq	2.01(m)	31.3	25	2.34(m)	36.6
						6	4.30(m)	69.7	25’	1.12(3H,d,6)	18.5
7	2.93(dd,10,2)	78.5	26	-	153.1
						8	4.26(m)	71.5	26’a	4.83(brs)	105.1
9	4.15(dd,7,4)	81.3	26’b	4.84(brs)	105.1
						10	4.28(dd,7,4)	84.4	27	3.74(brd,11)	76.2
11	4.61(dd,5,4)	80.2	28a	1.86(m)	39.5
						12	4.73(dd,5,5)	86.8	28b	2.08(m)	39.5
13	2.37(2H,d)	43.4	29	3.86(m)	82.4
						14	-	201.2	30	2.47(m)	44.7
15	2.66(2H,m)	36.9	31	3.36(d,3)	88.4
						16a	1.45(m)	29.7	31’	3.43(3H,s)	57.5
16b	2.18(m)	29.7	32	3.88(m)	78.5
						17	4.08(m)	78.1	33a	1.84(m)	34.7
18a	2.36(m)	40.1	33b	1.90(m)	34.7
						18b	2.88(m)	40.1	34	3.95(m)	66.9
19	-	154.3	35a	2.88(dd,13,9)	45.8
						19’a	5.02(brd)	105.2	35b	3.06(dd,13,3)	45.8

(3) Advances in crystal X-ray diffraction: test respectively the Advances in crystal X-ray diffraction of RRT1.07 impurity and RRT1.29 impurity, show that its configuration conforms to formula Iy with formula Ix above.

test example 3: the study on the stability of bulk drug

Get the various materials of above preparation, and the RRT1.07 impurity (and/or impurity RRT1.29) that the methylsulfonic acid eribulin reference substance made by preparation example 1 makes with preparation example 2 mixes (with a little anhydrous alcohol solution with certain proportion, vacuum-drying immediately so that they fully mix, measure Ix wherein and/or the Iy content with respect to formula I) make the assembly thing, by these material sealings, in 38 ℃ of condition lower seals, lucifuge, place 6 months, calculating the content of disposing with this understanding after 6 months when certain impurity phase was for 0 month increases percentage ratio.The results are shown in Table 1.

Table 1:

Above result shows, no matter be for formula I free alkali, or its pharmaceutical salts mesylate for example, when Ix content wherein lower than/while equaling 1.7%, after 6 months, the content of this impurity of Iy increases percentage ratio all lower than 75%, particularly all lower than 50%, and when wherein Ix content is greater than 1.7%, after 6 months, the content of this impurity of Iy increases percentage ratio all higher than 75%, particularly all higher than in 100%, particularly all higher than in 130%, significantly be greater than Ix content lower than/situation while equaling 1.7%.

Composition example 1: embodiment 1 step (3) product of take prepares injection liquid as raw material, and filling a prescription is: bulk drug 0.5mg, dehydrated alcohol 0.05ml, water for injection add to 1ml in right amount.Preparation method's (with preparation of 1000ml amount): by the bulk drug dissolve with ethanol, then add water to full dose, measure the pH value of solution, by the pH value to 7.0 of 1M sodium hydroxide or 1M hydrochloric acid conditioning solution, filtration sterilization, divide and install in vial in case of necessity, the 2ml/ bottle, sealing, obtain.The HPLC figure of its determination of related substances is shown in Fig. 2.Through 38 ℃-April, investigate, the percentage ratio of the content of Iy impurity increase as a result is 37.3%.

Composition example 2: embodiment 1 step (3) product of take prepares powder injection as raw material, and filling a prescription is: bulk drug 1mg, N.F,USP MANNITOL 150mg, water for injection add to 2ml in right amount.Preparation method's (with preparation of 1000ml amount): bulk drug is dissolved with appropriate water for injection, add N.F,USP MANNITOL to make to dissolve, then add water to full dose, measure the pH value of solution, use in case of necessity the pH value to 7.0 of 1M sodium hydroxide or 1M hydrochloric acid conditioning solution, filtration sterilization, divide and install in vial, 2ml/ bottle, lyophilize, sealing, obtain.Through 38 ℃-April, investigate, the percentage ratio of the content of Iy impurity increase as a result is 32.8%.

Composition example 3: embodiment 6 steps (4) product of take prepares tablet as raw material, formula is: bulk drug 5mg, lactose 50mg, Microcrystalline Cellulose 60mg, Vltra tears 2mg (water is made 5% solution and used as tackiness agent), micropowder silica gel 1mg, method for preparing tablet thereof routinely is pressed into tablet, obtains.Through 38 ℃-April, investigate, the percentage ratio of the content of Iy impurity increase as a result is 31.3%.Fig. 2 has shown typical HPLC figure when this tablet is measured related substance, in figure, display type I compound goes out peak at the 28.707min place, show Ix impurity (occurrence of RRT is 1.075) at the 30.850min place, show Iy impurity (occurrence of RRT is 1.300) at the 37.331min place.

Composition example 4: according to formula and the method for composition example 1, No13 to the No29 sample used of table 1 of take respectively is raw material, prepares 17 kinds of injection liquids.The content of investigating Iy impurity through 38 ℃-April increases percentage ratio, and the Iy foreign matter content that result: No13 to No20, No26 sample used is raw material gained pharmaceutical composition increases percentage ratio all in 11～38% scopes; But the Iy foreign matter content that sample used is raw material gained pharmaceutical composition for No21 to No25, No27 to No29 increases percentage ratio all in 136～364% scopes.

Composition example 5: according to formula and the method for composition example 2, No13 to the No29 sample used of table 1 of take respectively is raw material, prepares 17 kinds of powder injection.The content of investigating Iy impurity through 38 ℃-April increases percentage ratio, and the Iy foreign matter content that result: No13 to No20, No26 sample used is raw material gained pharmaceutical composition increases percentage ratio all in 9～36% scopes; But the Iy foreign matter content that sample used is raw material gained pharmaceutical composition for No21 to No25, No27 to No29 increases percentage ratio all in 127～337% scopes.

Composition example 6: according to formula and the method for composition example 3, the described assembly thing 13 of No26 to No29 to the sample of assembly thing 16 of table 1 of take respectively is raw material, prepares 4 kinds of tablets.The content of investigating Iy impurity through 38 ℃-April increases percentage ratio, result: it is 36.3% that the Iy foreign matter content that the No26 sample is raw material gained pharmaceutical composition increases percentage ratio; But the Iy foreign matter content that is raw material gained pharmaceutical composition for No27 to No29 sample increases percentage ratio all in 143～365% scopes.

The result of above each composition example shows, formula I compound or the formula Ia compound of the Ix impurity that use contains different amounts are bulk drug, the pharmaceutical composition of making (preparation that comprises solution-type and solid type), they all show that composition have satisfactory stability for the content of formula I compound lower than 1.7% the time at the Ix impurity phase, impurity Iy increment is low, but significantly increases when Ix foreign matter content impurity Iy increment in composition higher than 1.7% time.

test example 4: biological test

This test example is investigated the impact of related substances on the neutrophil leucocyte in mouse blood.

(1) animal: BALB/c mouse, female 6～8 week age, purchased from experimentation on animals center, Guangdong Province.

(2) reagent: use the formula I compound of preparation example 1 gained formula Ia reference substance of the present invention, preparation example 2 gained Ix impurity, Iy impurity, embodiment 6 steps (4), their mixed thing to be tested, use physiological saline to be mixed with the sterile solution of suitable concentration.

(3) grouping: mouse random packet, 10 every group; Concrete group is as follows:

A) control group: injection and the isopyknic physiological saline of each administration group; B) Compound I a group: preparation example 1 gained formula Ia reference substance; C) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=0.52%, (by appropriate Iy and appropriate Ia, be mixed, this 0.52% is measured with [HPLC-A], the result that IyIy calculates with respect to the amount of I; Lower same); D) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=1.51%; E) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=2.15%; F) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=2.75%; G) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=3.35%; H) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=4.25%; I) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=6.5%; J) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=10.5%; K) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=22.5%; L) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=45.3%; M) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=83.5%; N) impurity Iy group: preparation example 2 gained Iy impurity; O) impurity Ix group: preparation example 2 gained Ix impurity; P) the I/Iy group that is mixed: the I/Iy assembly, containing Iy=1.53%; Q) the I/Iy group that is mixed: the I/Iy assembly, containing Iy=2.72%; R) the I/Iy group that is mixed: the I/Iy assembly, containing Iy=3.35%; S) the I/Iy group that is mixed: the I/Iy assembly, containing Iy=6.55%; T) the I/Iy group that is mixed: the I/Iy assembly, containing Iy=23.1%; U) the composition example is 1 group.

(4) dosage, administration, method: each animal is at least raised 48 hours before administration, in the time of-24 hours, 0 hour, eye socket is got blood, cell counting (using the routine blood test automatic analyser that U.S. Coulter company produces to measure) centering granulocyte count routinely, calculating every treated animal counts average at the neutrophil leucocyte of each time point (this average is at the following PNG of being abbreviated as, this average of 0 hour can be abbreviated as PNG0), for every treated animal, within-24 hours, the PNG with 0 hour two time point differs and is no more than 3%.Gave respectively to organize reagent 3 times respectively at 0 hour, 24 hours, 48 hours through abdominal injection, give total compound amount (with formula I compound, formula Ix compound, formula Iy compound or its summation meter) 50 μ mol/kg the weight of animals at every turn, for example, for j) the Ia/Iy group that is mixed, the two the dosage of summation meter 50 μ mol/kg the weight of animals of Ia (in I) and Iy.Get blood at the 54th hour (after being the last administration 6 hours) each animal eye socket, cell counting centering granulocyte count, calculate the neutrophil leucocyte of every treated animal in the time of the 54th hour and count average (this average is abbreviated as PNG56) routinely.

(5) result is calculated: the neutrophil leucocyte relative number that is calculated as follows every treated animal:

Neutrophil leucocyte relative number (%)=(PNG56 ÷ PNG0) * 100%

The changing conditions of neutrophil leucocyte in above neutrophil leucocyte relative number reflection peripheral blood, this parameter more shows that close to 100% the neutrophil leucocyte in blood changes less, this parameter is lower than 100% the time, numerical value is lower shows that the neutrophil leucocyte minimizing is more, and this parameter shows while being greater than 100% that neutrophil leucocyte increases.The results are shown in Table 2.

Table 2

Group	The neutrophil leucocyte relative number	Group	The neutrophil leucocyte relative number
				A) control group	102.2%	L) the Ia/Iy group that is mixed	3.31%
B) Compound I a group	99.8%	M) the Ia/Iy group that is mixed	1.72%
				C) the Ia/Iy group that is mixed	103.6%	N) impurity Iy group	1.14%
D) the Ia/Iy group that is mixed	102.6%	O) impurity Ix group	95.8%
				E) the Ia/Iy group that is mixed	99.5%	P) the I/Iy group that is mixed	100.8%
F) the Ia/Iy group that is mixed	96.7%	Q) the I/Iy group that is mixed	97.1%
				G) the Ia/Iy group that is mixed	54.5%	R) the I/Iy group that is mixed	51.3%
H) the Ia/Iy group that is mixed	45.2%	S) the I/Iy group that is mixed	35.2%
				I) the Ia/Iy group that is mixed	37.5%	T) the I/Iy group that is mixed	13.1%

J) the Ia/Iy group that is mixed	23.2%	U) the composition example is 1 group	101.3%
				K) the Ia/Iy group that is mixed	11.4%	?	?

For n) group, the 4580/μ L of neutrophil count during by the 0th hour, the 52/μ L while being reduced to the 54th hour.For Iy content lower than 2.8% assembly thing, this does not change neutrophil leucocyte base in blood, but when Iy content is greater than 3%, particularly be greater than 5%, while more especially being greater than 10%, neutrophil leucocyte number in blood sharply reduces, this for the experimenter maintain normal body function particularly normal immunologic function be totally unfavorable.Formula I compound all shows the similar combined result with formula Ia.

test example 5: biological test

This test example is investigated the impact of related substances on the neutrophil leucocyte in rat blood.

(1) animal: 6 weeks male SD rats, the male and female dual-purpose, body weight 180～220g, be purchased from experimentation on animals center, Guangdong Province.

(3) grouping: mouse random packet, 3 every group; Concrete group is as follows:

A) control group: injection and the isopyknic physiological saline of each administration group; B) Compound I a group: preparation example 1 gained formula Ia reference substance; C) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=0.75%, (compounding method is with test example 4; Lower same); D) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=1.70%; E) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=2.75%; F) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=3.75%; G) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=5.15%; H) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=8.55%; I) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=20.3%; J) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=45.3%; K) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=80.7%; L) impurity Iy group: preparation example 2 gained Iy impurity; M) the composition example is 2 groups.

(4) dosage, administration, method: with test example 4.

(5) result is calculated: calculate the neutrophil leucocyte relative number of every treated animal in the time of the 54th hour, the results are shown in Table 3.

Table 3

Group	The neutrophil leucocyte relative number	Group	The neutrophil leucocyte relative number
				A) control group	101.5%	H) the Ia/Iy group that is mixed	54.3%
B) Compound I a group	99.3%	I) the Ia/Iy group that is mixed	41.2%
				C) the Ia/Iy group that is mixed	101.6%	J) the Ia/Iy group that is mixed	33.7%
D) the Ia/Iy group that is mixed	100.5%	K) the Ia/Iy group that is mixed	27.3%
				E) the Ia/Iy group that is mixed	98.7%	L) impurity Iy group	22.6%
F) the Ia/Iy group that is mixed	74.6%	M) the composition example is 2 groups	98.7%
				G) the Ia/Iy group that is mixed	62.2%	?	?

For l) group, the 3250/μ L of neutrophil count during by the 0th hour, the 735/μ L while being reduced to the 54th hour.

test example 6: pharmaceutical properties is investigated

This test example is investigated the impact of related substances on the neutrophil leucocyte in blood of human body.

(1) experimenter: healthy volunteer 9 people are divided into 3 groups, 3 people/group, the male sex, body weight 60～70kg at random.

(2) reagent and group: a) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=1.5%; B) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=5.1%; C) the Ia/Iy group that is mixed: the Ia/Iy assembly, containing Iy=25.0%.

(3) dosage and administration: mixed thing is made to injection liquid, and the dosage that gives Ia and Iy summation 20 μ mol with the every 1 square metre of body surface area of experimenter was administered once at the 1st day and the 7th day, intravenous injection.For every experimenter, measure the neutrophil count (N0) in its blood before first administration, measure the neutrophil count (N1) in its blood while following 48 hours after the last administration.Calculate every experimenter neutrophil leucocyte relative number in blood when off-test, calculating formula is:

Neutrophil leucocyte relative number (%)=(N1 ÷ N0) * 100%

Result: the neutrophil leucocyte relative number of a) organizing three experimenters all, in 96.3～102.6% scopes, shows that the neutrophil leucocyte number has no reduction; B) the neutrophil leucocyte relative number of organizing three experimenters all, in 37.7～51.3% scopes, shows that neutrophil leucocyte digital display work reduces; C) the neutrophil leucocyte relative number of organizing three experimenters is all in 12.4～26.7% scopes, and indivedual experimenter's neutrophil leucocyte numbers in blood when off-test are down to 450/μ L, shows that neutrophil leucocyte digital display work reduces.

test example 7: pharmaceutical properties is investigated

Get embodiment 1 step (3) product (Ia bulk drug), embodiment 2 products (Ia bulk drug), embodiment 3 products (Ia bulk drug), embodiment 4 products (Ia bulk drug), embodiment 5 steps (3) product (Ia bulk drug), embodiment 6 steps (3) product (Ia bulk drug), these 7 kinds of bulk drugs of embodiment 6 steps (4) product (I bulk drug), preparation example 1 product (Ia reference substance), composition example 1 injection liquid (Ia composition), composition example 2 powder injection (Ia composition), each material of these 3 kinds of compositions of composition example 3 tablets (I composition), measure them in the time of 0 month, and through 40 ℃ of encapsulation process the principal constituent chromatographic purity ([HPLC-A] after 6 months, area normalization method is calculated, the deduction solvent, methanesulfonate, auxiliary material isochromatic spectrum peak), RRT1.07 content, RRT1.29 content.

Result: all the chromatographic purity of material in the time of 0 month is all in 98.5%～99.9% scope, RRT1.07 content is all in 0.11～0.98% scope, RRT1.29 content all, in 0.06～0.47% scope, shows that these materials have good pharmaceutical property as bulk drug or the preparation of medicine.After 40 ℃-June, all the chromatographic purity of material is all in 97.3%～99.2% scope, RRT1.07 content is all in 0.16～1.43% scope, and RRT1.29 content all, in 0.18～0.86% scope, shows that these materials have good specificity as bulk drug or the preparation of medicine.

When with preparation example 1 product, be the Ia reference substance in contrast, while measuring this 7 kinds of bulk drugs, wherein the content of principal constituent (formula I or formula Ia compound) is in 97.0%～102.0% scope.

In above whole HPLC test, the sample that comprises reference examples and reference composition example, through repeatedly test (different personnel, different instrument, different experiments chamber), their RRT1.07 impurity relative retention time is all in 1.06～1.09 scopes, and RRT1.29 impurity relative retention time is all in 1.27～1.31 scopes.

Claims

1. the bulk drug of a hyoscine, the activeconstituents of this bulk drug is with the following formula I compound:

Or its pharmaceutical salts.

2. according to the bulk drug of claim 1, comprising the formula I compound or pharmaceutically acceptable salt thereof as main ingredient, and the optional impurity of the Ix as impurity, the structure of this Ix impurity is as follows:

3. according to the bulk drug of claim 1, wherein the Ix impurity phase for formula I compound content lower than 5%, lower than 2.5% or lower than 1.7%.

4. according to the bulk drug of claim 1, comprising the formula I compound or pharmaceutically acceptable salt thereof as main ingredient, and the optional impurity of the Iy as impurity, the structure of this Iy impurity is as follows:

5. according to the bulk drug of claim 1, wherein impurity Iy with respect to the content of formula I compound lower than 10%, lower than 5% or lower than 2.8%.

6. according to the bulk drug of claim 1, it is measured according to [HPLC-A], wherein the impurity of relative retention time at 1.06～1.09 places be the RRT1.07 impurity phase for formula I compound content lower than 5%, lower than 2.5% or lower than 1.7%.

7. according to the bulk drug of claim 1, it is measured according to [HPLC-A], wherein the impurity of relative retention time at 1.27～1.31 places be the RRT1.29 impurity phase for formula I compound content lower than 10%, lower than 5% or lower than 2.8%.

8. a pharmaceutical composition, wherein comprise the described bulk drug of claim 1～7 any one, and optional pharmaceutically acceptable carrier or auxiliary material.

9. the preparation method of the bulk drug of hyoscine, the activeconstituents of this bulk drug is with the following formula I compound:

Or its pharmaceutical salts for example mesylate be formula Ia compound, the method comprises with an organic solvent carries out refining step to the bulk drug that comprises formula I compound or pharmaceutically acceptable salt thereof.

10. the pharmaceutical applications of the bulk drug that the described bulk drug of claim 1～7 any one, the described pharmaceutical composition of claim 8 or the described method of claim 9 prepare, for the preparation of the medicine as microtubule inhibitors or for the preparation of the medicine for the treatment of tumour.