CN101522690B - Crystalline forms of rapamycin analogs - Google Patents

Abstract

A rapamycin analog composition includes a crystalline form of a rapamycin analog. The crystal can be a hydrate, dehydrate, solvate, or desolvate. The rapamycin analog can have a structure of Formula (I), which is optionally a prodrug, salt, derivative, or combination thereof. The formula (I) is shown in the specifications.

Description

Crystalline forms of rapamycin analogs

the cross reference of related application

The application requires the U.S. Provisional Patent Application No.60/820 submitting on July 25th, 2006, title is " crystalline forms of rapamycin analogs ", submission on July 23rd, 317 and 2007, number of patent application are that title is the U.S. Patent application No.11/781 of " crystalline forms of rapamycin analogs ", submission on July 23rd, 804 and 2007, title are the U.S. Patent application No.11/781 of " preparation method of crystalline forms of rapamycin analogs ", 807 right of priority; The invention people of described patent is Shekhar Viswanath, Larry Bartelt, Robert Leanna, Michael Rasmussen, Madhup Dhaon, Rodger Henry, Thomas Borchardt, Shuang Chen, and Geoff Zhang, the content of described patent is incorporated herein by specifically quoting.

Invention field

The present invention relates to rapamycin (RAPAMYCIN) analogue crystal and composition thereof, purposes with and manufacture method.More particularly, the present invention relates to forms of rapamycin analogs zotarolimus (being ABT-578) crystal.

Background of invention

In pharmaceuticals, conventionally between its drug solubility, stability, absorptivity and bioavailability, there is compromise balance, such balance can regulate by the form of medicine.The active compound solvability of some form is very low, or soluble in water, produces the first hepatic metabolism of crossing widely.The active compound of some form is because it is compared with the low water-soluble lower absorptivity that has.A kind of attribute of solid-state active compound (such as its crystalline habit and morphological structure) can affect its performance significantly.Therefore, a kind of form of activeconstituents selects to change significantly the usefulness of medicine and other chemical.In general, prepared rapamycin and the forms of rapamycin analogs existing with unformed (amorphous) form in composite medicine.

Although developed crystallization method, still limited to the ability of the control of crystallization and design crystal and other solids according to Structure Understanding.For the control of nucleation, growth, dissolving and the molecular crystal form still stage (Weissbuch in " mix and attempt ", I., Lahav, M. and Leiserowitz, L, Molecular Modelling Applications in Crystallization (the molecular model application in crystallization), 166,1999).Because many variablees affect crystallization, precipitation, phase shift and consequent solid-state, and because can there is many reagent and process variable, the test of single solid molding and crystalline structure modification is a very loaded down with trivial details process.Although crystalline structure is made a difference in pharmacy industry, there is no fully actively systematically to seek best crystalline structure or best amorphous solid thing.Therefore, a kind of selection of forms of rapamycin analogs form, such as crystalline form, can change its usefulness in concrete application significantly; Such form still needs to be sought.

Therefore, useful is the crystalline forms of rapamycin analogs that can have in a kind of being used for the treatment of.In addition, useful is composition, using method and the manufacture method can with described crystalline forms of rapamycin analogs.

Summary of the invention

The present invention relates to crystalline forms of rapamycin analogs, particularly, is zotarolimus crystal (being ABT-578), its composition, purposes and manufacture method.

One embodiment of the invention has comprised crystalline forms of rapamycin analogs.Crystalline forms of rapamycin analogs can be prepared by several different methods, and these class methods have description in this article.Can prepare such crystalline substance, make suitable crystalline form can be used for a certain specific end use.Described forms of rapamycin analogs can have the structure of formula 1 as follows, formula 2 or formula 3.Equally, crystal forms of rapamycin analogs can be its prodrug, salt, Xing is biological or its composition.

Formula 1

Formula 2

Formula 3

In one embodiment, described crystal is solvate.Therefore, described crystal can comprise the organic solvent wherein containing, and wherein said solvent is for the preparation of crystal.Described organic solvent can be selected from the solvent that can be used to prepare forms of rapamycin analogs, comprise acetone, ethyl acetate, methyl alcohol, ethanol, n-propyl alcohol, Virahol, isopropylcarbinol, the trimethyl carbinol, 2-butanols, acetonitrile, tetrahydrofuran (THF), isobutyl acetate, n-butyl acetate, ethyl formate, n-propyl acetate, isopropyl acetate, methyl ethyl ketone, toluene, N, dinethylformamide, methyl-phenoxide, Methyl isobutyl ketone, Nitromethane 99Min., propionitrile, 2-butanone (being methyl ethyl ketone or MEK), tetrahydrofuran (THF), 1, 2-glycol dimethyl ether, isopropyl acetate and arbitrary composition etc. thereof.

In one embodiment, described crystal is desolventizing thing.Therefore, described crystal can be selected from acetone desolventizing thing, toluene desolventizing thing, acetonitrile desolventizing thing, ethyl formate desolventizing thing, isobutyl acetate desolventizing thing, DMF and arbitrary composition thereof.

In one embodiment, the present invention includes the method for preparing crystalline forms of rapamycin analogs.The method comprises the following steps: forms of rapamycin analogs and the combination of at least one organic medium are made to mixture; Gained mixture is incubated until forms of rapamycin analogs crystallization; From organic medium, reclaim crystal forms of rapamycin analogs.

In one embodiment, described organic medium can be by least one solvent composition, to form mixture.Therefore, the method for preparing crystalline forms of rapamycin analogs comprises is dissolved in solvent forms of rapamycin analogs, and described solvent is incubated until forms of rapamycin analogs crystallization.

In one embodiment, described method is included in and in solution, forms crystal forms of rapamycin analogs slurry.In one embodiment, described method comprises that stirring forms of rapamycin analogs mixture is until forms of rapamycin analogs crystallization.In one embodiment, described method comprises that to make forms of rapamycin analogs solution saturated.This comprises formation supersaturation forms of rapamycin analogs solution.

In one embodiment, described method comprises and uses anti-solvent to help form crystal forms of rapamycin analogs.Described method comprises at least one anti-solvent and forms of rapamycin analogs and solvent combination, forms two-phase mixture; By the insulation of this two-phase mixture, make liquid-liquid phase separation, most of forms of rapamycin analogs in solvent and small portion forms of rapamycin analogs in anti-solvent.Optionally, before crystal separation, can be by solution and anti-separated from solvent.

It should be understood that above general remark and following detailed description are all only for demonstration with explain, can not regard as is limitation of the invention.After reading the detailed description of following discloses embodiment, will find advantage how of the present invention.These embodiments are systematically discussed in accompanying drawing and claims.

Accompanying drawing summary

In order further to illustrate above and other advantage and feature of the present invention, with reference to the specific embodiments illustrating in accompanying drawing, provide more specific description of the present invention.Understand, these accompanying drawings have only been described exemplary embodiments of the present invention, so its scope is not limited to this.To use accompanying drawing, clear and definite at length present invention is described and explain, wherein:

Fig. 1 has shown an embodiment preparing forms of rapamycin analogs method.

Fig. 2 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs acetone solvent compound crystal.

Fig. 2 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs acetone solvent compound crystal.

Fig. 3 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs acetone desolventizing thing crystal.

Fig. 3 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs acetone desolventizing thing crystal.

Fig. 4 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs toluene solvate crystal.

Fig. 4 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs toluene solvate crystal.

Fig. 4 C is the schematic diagram of an embodiment of the single x-ray crystal structure of Fig. 4 B forms of rapamycin analogs toluene solvate.

Fig. 4 D shows along the crystalline structure embodiment schematic diagram of the solvent channel of " b " axle for Fig. 4 B forms of rapamycin analogs toluene solvate.

Fig. 4 E is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs toluene desolventizing crystal.

Fig. 4 F is the figure that has shown that x-ray diffractogram of powder changes in Fig. 4 B forms of rapamycin analogs toluene solvate desolventizing process.

Fig. 5 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs acetonitrile solvent compound crystal.

Fig. 5 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs acetonitrile solvent compound crystal.

Fig. 6 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs acetonitrile desolventizing thing crystal.

Fig. 6 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs acetonitrile desolventizing thing crystal.

Fig. 6 C is the thermogravimetric analysis of an embodiment of forms of rapamycin analogs acetonitrile desolventizing thing crystal.

Fig. 7 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs ethyl formate solvate crystals.

Fig. 7 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs ethyl formate solvate crystals.

Fig. 7 C is the thermogravimetric analysis of an embodiment of forms of rapamycin analogs ethyl formate solvate crystals.

Fig. 8 is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs ethyl formate desolventizing thing crystal.

Fig. 9 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs isobutyl acetate solvate crystals.

Fig. 9 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs isobutyl acetate solvate crystals.

Fig. 9 C is the thermogravimetric analysis of an embodiment of forms of rapamycin analogs isobutyl acetate solvate crystals.

Figure 10 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs DMF solvate crystals.

Figure 10 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs DMF solvate crystals.

Figure 10 C is the thermogravimetric analysis of an embodiment of forms of rapamycin analogs solvent dimethylformamide compound crystal.

Figure 11 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs methyl-phenoxide solvate crystals.

Figure 11 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs methyl-phenoxide solvate crystals.

Figure 11 C is the thermogravimetric analysis of an embodiment of forms of rapamycin analogs methyl-phenoxide solvate crystals.

Figure 12 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs alcohol solvent compound crystal.

Figure 12 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs ethanol desolventizing crystal.

Figure 13 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs methanol solvate compound crystal.

Figure 13 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs methyl alcohol desolventizing thing crystal.

Figure 14 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs ethyl acetate solvent compound crystal.

Figure 14 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs ethyl acetate desolventizing thing crystal.

Figure 15 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs Methyl isobutyl ketone solvate crystals.

Figure 15 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs Methyl isobutyl ketone desolventizing thing crystal.

Figure 16 is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs Nitromethane 99Min. solvate crystals.

Figure 17 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs isopropyl acetate solvate crystals.

Figure 17 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs isopropyl acetate desolventizing thing crystal.

Figure 18 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs propionitrile solvate crystals.

Figure 18 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs propionitrile desolventizing thing crystal.

Figure 19 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs methyl ethyl ketone solvate crystals.

Figure 19 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs methyl ethyl ketone desolventizing thing crystal.

Figure 20 A is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs tetrahydrofuran solvent compound crystal.

Figure 20 B is the x-ray diffractogram of powder of an embodiment of forms of rapamycin analogs tetrahydrofuran (THF) desolventizing thing crystal.

Figure 21 A is forms of rapamycin analogs 1, the x-ray diffractogram of powder of an embodiment of 2-glycol dimethyl ether solvate crystals.

Figure 21 B is forms of rapamycin analogs 1, the x-ray diffractogram of powder of an embodiment of 2-glycol dimethyl ether desolventizing thing crystal.

dESCRIPTION OF THE PREFERRED

Generally speaking, the present invention relates to crystalline forms of rapamycin analogs for example crystalline habit, composition, purposes and the preparation method of forms of rapamycin analogs zotarolimus (being ABT-587).Can prepare crystalline forms of rapamycin analogs by described different methods herein.Can prepare such crystalline form, thereby suitable crystalline form can be used for specific end use.

I. crystal forms of rapamycin analogs

In one embodiment, forms of rapamycin analogs can have the structure of formula 1, formula 2, formula 3 or their unitized construction.

Formula 1

Formula 2

Formula 3

The forms of rapamycin analogs of formula 2 can be described as zotarolimus or ABT-578.In addition, this medicine can be any pharmacy acceptable salt or the prodrug of forms of rapamycin analogs.Pharmaceutically acceptable biologically active agent salt and/or prodrug are as well known in the art in the technology of preparing of zotarolimus.

In addition, the forms of rapamycin analogs of formula 1-3 can coexist in solution with another analogue balance shown in formula 4.The forms of rapamycin analogs of formula 4 can also be the corresponding analogue of formula 2-3.Similarly, the forms of rapamycin analogs of formula 4 (with the equivalent of formula 2-3) also can form crystal or be attached in the crystalline forms of rapamycin analogs of formula 1-3.

Formula 4

In one embodiment, forms of rapamycin analogs can be the derivative of analogue shown in formula 1-4.Can be by less replacement as hydroxylation, methylate, ethylation reaction or change substituting group by minimum degree and prepare derivative.

In some examples, if possible, forms of rapamycin analogs can be made into salt, comprises pharmaceutically acceptable negatively charged ion, comprises acetate, benzene sulfonate, benzoate, hydrocarbonate, bitartrate, bromide, Ca-EDTA, camsilate, carbonate, muriate, bromide, iodide, Citrate trianion, dihydrochloride, edetate, ethanedisulphonate, estolate, esilate, fumarate, gluceptate, gluconate, glutaminate, to hydroxyl kharophen phenyl-arsonate, Sucrets salt, breathe out amine (hydrabamine), Hydroxynaphthoate, isethionate, lactic acid salt, Lactobionate, malate, maleate, mandelate, mesylate, Methylsulfate, muscat (muscate), naphthalenesulfonate, nitrate, panthotlienate, phosphoric acid salt/diphosphate, Polygalacturonate, salicylate, stearate, succinate, vitriol, tannate, tartrate, teoclate (teoclate), triethiodide compound (triethiodide) and embonate (that is: 1,1 '-methylene-bis (2-hydroxyl-3-naphthoate)).

A. crystalline form

Different crystal forms of rapamycin analogs of the present invention can have different characteristics.Be that crystal can have different structures, physics, pharmacy or chemical property.Structural performance includes but not limited to the description of crystal polymorphic and crystalline habit.Whether structural performance also comprises that it forms, such as solid, be hydrate, dehydrate, solvate, desolventizing thing, salt and composition thereof etc.

The physical condition of crystal forms of rapamycin analogs also can be further divided into: whether (1) crystal matrix comprises additional compound (coadduct); (2) form (as: crystalline habit) and (3) internal structure (as: polycrystalline).In auxiliary affixture, crystal matrix can comprise this chemistry or non-stoichiometric adducts, for example recrystallisation solvent or water (as: solvate or hydrate).Non-stoichiometric solvate or hydrate comprise inclusion compound, and wherein solvent or water are distributed in crystal lattice matrix with random interval.Stoichiometric solvate or hydrate are that wherein crystal matrix comprises solvent or water with specified proportion at specific position.That is to say, this solvent or water molecule energy are with a part that is arranged as crystal matrix for appointment.In addition, the physical condition of crystal matrix can be present at first additional compound in crystal matrix and changes by isolating one.For example, if solvent or water are isolated from solvate or hydrate, will in crystal matrix, form hole, thereby form new physical condition.These physical conditions are in this article referred to as the hydrate (being dehydrate) of dehydration or the solvate (being desolventizing thing) of desolventizing.

Crystalline habit is that the profile of single crystal is described.For example, crystal can have cubes, quadrangle, quadrature, monocline, three tiltedly, the shape such as rhomboid or hexagon.

The internal structure of crystal refers to crystal shape or polymorph.A given compound, forms of rapamycin analogs for example, polymorphic form that can be different (being different crystal seeds) exists.Conventionally, a kind of different polymorphic forms of given compound can be variant on structure and characteristics as the crystal of two kinds of different compounds.Solubleness, fusing point, density, hardness, crystalline form, photoelectric characteristic, the meetings such as vapour pressure, stability change along with polycrystalline form.

The crystalline structure of compound (as forms of rapamycin analogs), the photographic fixing sound of fighting to the finish plays an important role as the Purificatiou of medicine and the performance of effect.The performance of chemical compound lot can change by structural changes.For example, the crystal of different polycrystal or same pharmaceutical compounds may have different treatment activities.Understand structure-performance relation for making desired properties (as by the curative effect of the medicine) optimization of forms of rapamycin analogs there is vital role.

B. crystallization

Crystallisation process is a process that forms of rapamycin analogs is sorted with solid-state crystalline network.In this course, the molecule of the random organization in solution, solution or gas phase occupies the rotine positioning in crystalline network.The conventional organization of this kind of lattice is responsible for forming many unique properties of crystal, comprises the diffraction of x ray, definite fusing point and brightly analyses clear definite crystal face.Precipitation is commonly referred to as without symmetry or order arrangement and the habit definition of can not usining to be determined or forms as multicrystal amorphous substance, but it also can refer to the process that forms crystal by Precipitation.Crystallization and Precipitation all cause because solution can not dissolve forms of rapamycin analogs completely, and can cause by changing in some way the state (such as change parameter) of composition.

Some important steps in crystallisation process are nucleation, growth kinetics, surface phenomenon, assemble and break.After having overcome phase transformation energy barrier, can produce nucleogenesis, so just allow to form in supersaturated solution particle.Crystal growth is the expansion of crystal grain, by forms of rapamycin analogs, on the existing surface of crystal, is deposited and causes.The speed of relative movement of nucleation and growth has determined the size-grade distribution of the crystal that forms.The thermodynamic driving force of nucleation and growth is supersaturation effect, and this effect is defined as the deviation of thermodynamic equilibrium.Gathering is by two or more particles (such as crystal), to be bonded together and to form larger particle to form larger crystalline structure.

Forms of rapamycin analogs can form multiple different crystal habit and size, and this depends on rule and the condition that forms crystalline form.The crystal characteristic (such as polymorphic, crystalline size, crystalline habit and crystal size distribution etc.) that needs lay special stress on medicine industry, because crystalline structure and size may affect manufacture, formulation and pharmacokinetics etc., comprise and affect bioavailability.A kind of given mixture may have four kinds of different pan class: form; Habit; Polymorphic and crystalline size.

It is single compound (as: pure forms of rapamycin analogs) or compound that crystal composition is described solids conventionally.For example, solids can its neutral form exist, such as free alkali or the class saloid (as: containing the hydrochloride of alkaline nitrogen compound) as having alkaline nitrogen compound.Crystal composition also can be described the crystal that comprises adducts molecule.In crystallization and precipitation process, adducts molecule (as: solvent or water) can be incorporated into lattice matrix, absorbs from the teeth outwards or is trapped in crystal lattices.This constituents is called inclusion compound, as hydrate (as: being attached to the water molecules in lattice) and solvent (as: being trapped in intracell solvent).Whether crystal forms inclusion compound can for example, produce impact greatly to attribute (the simple and easy degree of the bioavailability of forms of rapamycin analogs and processing or production).For example, inclusion compound can more or less dissolve than being easier to, or has mechanical properties or the intensity different from the corresponding non-clathrate crystal structure of same compound.

Therefore, forms of rapamycin analogs can crystallize into different shapes, except other factors, depends on composition and the temperature of crystallization medium.The shape description of crystal face is crystalline habit.This information is very important, because crystalline habit has a great impact the S/V value tool of crystal.Although different crystalline habits may have similar internal structure and identical single crystal pattern, they still may demonstrate different pharmaceutical properties (1975, Haleblian, J.Pharm.Sci. (pharmaceutical journal), 64:1269).Crystalline habit may affect several drugs feature, for example: mechanical factor (as: syringeability, compressing tablet behavior, filtration, dry and with the mixing of other material (as: vehicle)) and on-mechanical factor (as: solubleness).

In addition, same forms of rapamycin analogs can crystallize into multiple different crystal seed (such as, there is different internal crystal framework structures) or convert another kind of crystal seed to from a kind of crystal seed.This phenomenon is called heteromorphism, and different crystal seeds is called polymorphs body.Polymorphs body can represent different optical properties, fusing point, solvability, chemically reactive, dissolution rate and different biological usabilities.As everyone knows, the different polymorphs bodies of same medical compounds can have different pharmacokinetics.Such as, a polymorphs body can more easily be absorbed than its counterpart.When extreme, only have the polymorph of a known drug may be applicable to the treatment of disease.But different polymorphs may have can together or separate the different properties using.For example, the polymorphs body with different dissolving properties can be used together, so that customization discharges or wash-out collection of illustrative plates, maybe can use in different preparations or treatment.Therefore, research and development novel or useful forms of rapamycin analogs are extremely important, especially at pharmaceutical field.

Amorphous solid, as traditional rapamycin and forms of rapamycin analogs do not have crystalline form, can not characterize according to habit or polymorphs body form.Common noncrystal be glass, its Atom and molecule exist with non-unified form of arranging.Noncrystal normally quick-setting product, and can by x-ray powder diffraction, determine easily, this is because these solids provide lines or the noncrystalline diffractogram disperseing very much.Although noncrystal, may often have desirable pharmaceutical properties, as quick dissolution rate, because their physics and chemistry unstable, it is not preferred conventionally.Xln, noncrystal in high-energy structural state relatively, therefore, storing or the crystallization of In transit possibility.And amorphous solid may be more responsive to oxygenizement (1997, Pikal etc., J.Pharm.Sci. (pharmaceutical journal), 66:1312).Amorphous solid can obtain by a kind of curing of the preferred crystallisation process of thermodynamics of avoiding.They can be prepared by upsetting existing crystalline structure equally.

Crystallization and precipitating action are phase transformations, cause forming xln or amorphous solid from solution.Crystallization also comprises the polycrystalline conversion that converts another kind of crystal seed from a kind of crystal seed to.Modal crystallization form is exactly crystallization from solution, and wherein, material is dissolved in solvent under proper temperature, then system is processed and is reached supersaturation, produces subsequently nucleation and crystal growth.

C. crystallographic component

As mentioned above, solvent effect crystallization and generation crystalline forms of rapamycin analogs.In general, most crystal compositions comprise the solvent as a kind of component.Solvent can pass through the formation of polarity, viscosity, boiling point, volatility, charge distribution and shape of molecule impact and guiding crystal.Solvent species and concentration are to control a kind of saturated method.In fact, a kind of material can be realized crystallization by the simple a kind of non-solvent (being anti-solvent) that adds in a kind of initial not exclusively saturated solution under isothermal condition.Equally, the forms of rapamycin analogs solution that has added the anti-solvent of different amounts can change crystallization and generate crystal, because surpassed the solubility limit of forms of rapamycin analogs while adding the anti-solvent of critical amount.Increase more anti-solvent and strengthen the supersaturation of solution, therefore, the rate of increase of forms of rapamycin analogs crystallization also increases thereupon.

Mixed solvent has increased the handiness that is independent of temperature and changes a kind of thermodynamic activity of solvent equally.Therefore, can prepare hydrate or solvate by simply a series of solvent compositions are carried out to crystallization at the temperature of appointment.For example, from methyl alcohol very the methanol-water solution crystallization of enrichment can generate the crystalline hydrate that only has a small amount of water xln (as: dihydrate right/and semihydrate), and the solution of Shuifu County's collection will generate the hydrate that contains more water in xln.When the concentration of solvent composition is variable, generates the unit that the accurate boundary of hydrate is arranged by detection separately and usually determine.

In one embodiment, the crystallization of the solvent of pharmacy for forms of rapamycin analogs will be widely used in pharmaceutical industries.Also can use the various mixtures of these solvents.The solvability of forms of rapamycin analogs in some solvent is compared with high and lower in other solvent.Solution can be mixed, the solvent solution lower with solvability that wherein solvability is higher mixes, until guide crystal formation.Solvent includes but not limited to group water solution, as: water and aqueous acids, alkali, salt, buffer reagent or its mixture and organic solvent, as: protonic solvent, aprotic solvent, polar organic solvent or non-polar organic solvent.

The application-specific of crystalline compounds may produce extra requirement.As: with regard to medicine, such as forms of rapamycin analogs, according to their biological usability and solvability selective solvent.For example, forms of rapamycin analogs is dissolved in the easiness of solvent and solution is the Consideration of selecting solution to the minimizing of the side effect of analogue.Preferred organic solvent has volatility or has compared with lower boiling or can under vacuum state, remove, and trace is acceptable while giving human body administration, as methylene dichloride.Also can use other solvent, as: ethyl acetate, ethanol, methyl alcohol, dimethyl formamide, acetone, acetonitrile, tetrahydrofuran (THF), acetic acid, methyl-sulphoxide and chloroform and composition thereof.Preferred solvent is for be decided to be those solvents of 3 grades of residual solvents by FDA, as Federal Register (federal register) the 62nd volume, No. 85, disclosed in 24301-24309 page (in May, 1997).For enteron aisle external administration or general more common as the solvent of the forms of rapamycin analogs of solution or suspension be distilled water, buffer saline, lactated ringer's inj or other some pharmaceutically acceptable carriers.

The specific examples that can be used for preparing the solvent of forms of rapamycin analogs comprises acetone, ethanol ethyl ester, methyl alcohol, ethanol, n-propyl alcohol, Virahol, isopropylcarbinol, the trimethyl carbinol, 2-butanols, acetonitrile, tetrahydrofuran (THF), isobutyl acetate, n-butyl acetate, ethyl formate, n-propyl acetate, isopropyl acetate, methyl ethyl ketone, toluene, N, dinethylformamide, methyl-phenoxide, methyl isopropyl Ketone, Nitromethane 99Min., propionitrile, 2-butanone (being methyl ethyl ketone or MEK), tetrahydrofuran (THF), 1,2-glycol dimethyl ether, isopropyl acetate and arbitrary composition thereof etc.

The specific examples that can be used for preparing the anti-solvent of forms of rapamycin analogs comprises hexanaphthene, heptane, hexane, octane, octane-iso, methylcyclohexane and arbitrary composition thereof etc.

The specific examples that can be used for preparing the solvent/anti-solvent system of forms of rapamycin analogs comprises acetone/normal heptane.

Also other material can be added to the generation that affects crystal in crystallization reaction.These crystallization additives can be any other different additive of finding in reaction by-product, associated molecule, the compound (as: those that exist in small molecules storehouse) arbitrarily screening or drug ingredient.They can be used for promoting or control nucleogenesis, to guide the growth of actual crystal or crystal group or growth velocity and any other to affect other parameter of crystallization.The impact of crystallization additive may be depended on its relative concentration, therefore, the invention provides the method for assessment crystallization additive and concentration range.The example of crystallization additive include but not limited to promote and/or control nucleation additive, affect the additive of crystalline habit, and affect polymorphous additive.

The specific examples that can be used for preparing the crystallization additive of forms of rapamycin analogs comprises rapamycin solvate, rapamycin desolventizing thing, rapamycin hydrate and rapamycin dehydrate.

In another embodiment, can use other material, comprise the small molecules storehouse (as existed with solid phase) of solid phase GRAS compound or equivalent substitution.

In crystallisation vessel, the existence of tensio-active agent quasi-molecule may affect crystal coring, and can optionally facilitate the growth of different polymorphs bodies.Therefore, can tensio-active agent quasi-molecule be incorporated in crystallisation vessel by pre-treatment or the method being added directly in crystallization medium.Surfactant molecule can be according to it impact in instructing crystallisation process and selecting especially or random screening.In addition, the effect of surfactant molecule depends on its concentration in crystallisation vessel, therefore, and the concentration of care should be used to control surface bioactive molecule.

In some cases, the direct introducing crystal seed of crystallization reaction will cause the diversity of the crystal habit of generation to increase.In one embodiment, particle is added in crystallization reaction.In another embodiment, the crystal of nano-scale (such as nanoparticle) is added in crystallization reaction.These particles can be nano-scale or larger.

II. crystal forms of rapamycin analogs

In one embodiment, the present invention includes crystalline forms of rapamycin analogs.This crystalline forms of rapamycin analogs can be prepared by several different methods described herein.Can prepare described crystalline form makes suitable crystalline form can be used for special purpose.Forms of rapamycin analogs can have the structure of formula 1, formula 2 or formula 3 as shown above.Equally, crystal forms of rapamycin analogs can be its prodrug, salt, derivative or its composition.

In one embodiment, described crystallization is solvate.Like this, described crystal can comprise the organic solvent being included.Described organic solvent can be selected from acetone, ethyl acetate, methyl alcohol, ethanol, n-propyl alcohol, Virahol, isopropylcarbinol, the trimethyl carbinol, 2-butanols, acetonitrile, tetrahydrofuran (THF), isobutyl acetate, n-butyl acetate, ethyl formate, n-propyl acetate, isopropyl acetate, methyl ethyl ketone, toluene, DMF, methyl-phenoxide and arbitrary composition thereof.

In one embodiment, described crystallization is desolventizing thing.Like this, described crystal can be selected from acetone desolventizing thing, toluene desolventizing thing, acetonitrile desolventizing thing, ethyl formate desolventizing thing, isobutyl acetate desolventizing thing, DMF desolventizing thing and arbitrary composition thereof.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.2,9.1 and/or 13.2 places.And described x-ray diffractogram of powder substantially as shown in Figure 2 A.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.3,10.2,10.6,13.3 and/or 16.0 places.And described x-ray diffractogram of powder substantially as shown in Figure 2 B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.3,10.2,10.5 and/or 13.3 places.And described x-ray diffractogram of powder is basic as shown in Fig. 3 A.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 6.3 and/or 12.6 places.And described x-ray diffractogram of powder substantially as shown in Figure 3 B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.4,5.9,9.9,13.8 and/or 15.5 places.And described x-ray diffractogram of powder is basic as shown in Fig. 4 A and/or Fig. 4 B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.9,6.2,9.1,9.8,12.5,13.6,16.4,17.7,17.9 and/or 21.8 places.And described x-ray diffractogram of powder is basic as shown in Fig. 4 E.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.2,5.6,6.0,7.3,10.0 and/or 21.5 places.And described x-ray diffractogram of powder substantially as shown in Figure 5 A.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.3,10.6,12.8,13.3,15.9,16.7,21.3 and/or 21.9 places.And described x-ray diffractogram of powder substantially as shown in Figure 5 B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 3.9,8.7,9.5,13.8,15.7 and/or 16.9 places.And described x-ray diffractogram of powder is basic as shown in Fig. 6 A.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 6.2,10.4,11.9,12.5,15.4,18.5 and/or 21.5 places.And described x-ray diffractogram of powder is basic as shown in Fig. 6 B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.9,7.7,9.1,10.0 and/or 10.5 places.And described x-ray diffractogram of powder substantially as shown in Figure 7A.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.3,5.5,10.6,15.9,16.5 and/or 19.2 places.And described x-ray diffractogram of powder is basic as shown in Fig. 7 B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 6.2,12.5 and/or 15.4 places.And described x-ray diffractogram of powder substantially as shown in Figure 8.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.0,7.0,9.1,10.1,15.4 and/or 16.0 places.And described x-ray diffractogram of powder is basic as shown in Fig. 9 A and/or Fig. 9 B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.1,7.2,9.0,9.2,10.3,11.5,15.7 and/or 16.3 places.And described x-ray diffractogram of powder is basic as shown in Figure 10 A and/or Figure 10 B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 6.1,8.9,9.4,10.0,10.2 and/or 12.2 places.And described x-ray diffractogram of powder is basic as shown in Figure 11 A and/or Figure 11 B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.3,7.2,10.5,15.8,16.6,19.1 and/or 21.2 places.And described x-ray diffractogram of powder is basic as shown in Figure 12 A.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 6.3,9.2,12.7,13.8 and/or 16.1 places.And described x-ray diffractogram of powder substantially as shown in Figure 12B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.4,6.0,8.8,10.0,12.1,14.1,17.6,18.4 and/or 19.0 places.And described x-ray diffractogram of powder substantially as shown in FIG. 13A.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 6.2,9.1,10.5,12.5,14.3,16.5,18.0,20.1,21.8 and/or 22.2 places.And described x-ray diffractogram of powder substantially as shown in Figure 13 B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.4,10.8,11.8,16.9 and/or 17.9 places.And described x-ray diffractogram of powder substantially as shown in Figure 16.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.8,9.6,11.7,13.6,15.9,17.4,20.6 and/or 23.5 places.And described x-ray diffractogram of powder is basic as shown in Figure 18 A.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 6.4,6.8,9.3,13.8 and/or 16.8 places.And described x-ray diffractogram of powder substantially as shown in Figure 18 B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.2,10.5,13.3,15.8,16.5 and/or 19.1 places.And described x-ray diffractogram of powder is basic as shown in Figure 14 A.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 6.6,7.1,8.6,9.1,12.6,14.5 and/or 15.0 places.And described x-ray diffractogram of powder substantially as shown in Figure 14 B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.2,10.5,10.8,15.7,16.5 and/or 19.0 places.And described x-ray diffractogram of powder is basic as shown in Figure 17 A.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.5,6.1,8.0,10.5,12.6,13.6,16.6 and/or 19.5 places.And described x-ray diffractogram of powder substantially as shown in Figure 17 B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.3,10.5,13.3,15.8 and/or 16.6 places.And described x-ray diffractogram of powder is basic as shown in Figure 19 A.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 6.3,8.1,12.7 and/or 16.5 places.And described x-ray diffractogram of powder is basic as shown in Figure 19 B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.1,10.2,16.3,17.1,19.2,20.1 and/or 20.5 places.And described x-ray diffractogram of powder is basic as shown in Figure 15 A.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.1,6.2,10.2,12.4,16.4 and/or 17.2 places.And described x-ray diffractogram of powder substantially as shown in Figure 15 B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 4.6,5.2,9.3,16.5,17.0 and/or 18.6 places.And described x-ray diffractogram of powder is basic as shown in Figure 20 A.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 3.8,6.0,9.2,9.9,11.8,12.4 and/or 13.7 places.And described x-ray diffractogram of powder is basic as shown in Figure 20 B.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 5.3,10.1,10.5,15.8,16.5,19.1,19.6 and/or 21.1 places.And described x-ray diffractogram of powder is basic as shown in Figure 21 A.

In one embodiment, crystal forms of rapamycin analogs has the x-ray diffractogram of powder that has peak at approximately 6.6,7.1,9.2,14.6 and/or 15.2 places.And described x-ray diffractogram of powder is basic as shown in Figure 21 B.

As all types of surveying instruments, the type of equipment and operational condition will affect data.Therefore, powder x-ray diffraction data can be changed a little according to equipment and condition.Correspondingly, powder x-ray diffraction data can be as accurate as in 0.5, and more preferably 0.2, most preferably 0.1.Equally, crystal structure characteristic can comprise at least 2 peaks, 3 peaks, 4 peaks, 5 peaks, 6 peaks, 7 peaks, 8 peaks, 9 peaks and/or 10 peaks according to x-ray diffractogram of powder.

Equally, use is approximately copper-potassium α 1 (Cu K α 1) radiation measure x-ray diffractogram of powder.Equally, wavelength available is cu K α 1 and Cu K α 2 radiations measure x-ray diffractogram of powder, this is for unclarified.Single crystal unit can be used without the graphite monochromator of paper tinsel strainer and measure.For single crystal, wavelength can be equally, can be for unclarified molybdenum radiation or for special K α 1.

In one embodiment, crystal forms of rapamycin analogs exists with treatment significant quantity.

III. prepare crystal forms of rapamycin analogs

Crystal forms of rapamycin analogs can be prepared by method described herein.Like this, can control various conventional parameters to promote crystallization.This common process parameter includes but not limited to adjust temperature, adjustment time, adjusts pH value, amount or concentration, the component unicity (for example adding one or more other components) of the amount of adjusting related compound or concentration, adjustment component, adjust that solvent is removed speed, introduces into nuclear activity, introduced the activity of separating out, control solvent evaporation (for example adjust force value or adjust evaporation surface) and adjustment solvent composition.Other crystallization method comprises desolventizing and the grinding (Guilloroy, J.K.Polymorphism in Pharmaceutical Solids (heteromorphism of pharmacy solid), 186,1999) of distillation, vapor diffusion, crystal solvent compound.

In one embodiment, the crystallization of forms of rapamycin analogs can be undertaken by adjusting temperature or circulating temperature, to cause crystallization.Like this, crystallisation process comprises forms of rapamycin analogs is dissolved in one or more solvents, and described solvent can comprise or not comprise one or more anti-solvents.Solvability for example, is controlled by composition (component kind) and/or temperature conventionally.Temperature is controlled in industry crystallizer the most common, and wherein, the solution of material is cooled to and surpasses deliquescent state from complete solvable state, therefore reaches supersaturation state.For example, crystal forms of rapamycin analogs can be prepared by being heated to a certain temperature (T1), is preferably heated to all solids and is dissolved in the temperature in solution completely.Then composition cools is arrived to a certain lower temperature (T2).Can determine the existence of solid afterwards.Temperature when usable temp sensor record detects first crystal or throw out.

In one embodiment, consider temperature, a large amount of or a series of crystal forms of rapamycin analogs of individual curing simultaneously, and should also provide and control microheater, cooling worm and the temperature sensor of each sample.If each sample has identical component, and experiment be designed to gather a large amount of temperature curves to find the curve of the required solid shape of those outputs, so this method is of great use.In one embodiment, the composition of each sample is controlled and whole composition series is carried out to heating and cooling as a unit.

In general, in crystal nucleation and growth phase process, some different temperature and/or temperature curve have been arrived in test.Temperature can be controlled by static state or dynamical fashion.Static temperature refers to and in whole crystallisation procedure, uses the holding temperature of setting.Or, can use temperature gradient.For example, can in crystallisation procedure, with a certain special speed, reduce temperature.In addition, can also control temperature according to the mode with Static and dynamic composition.For example, in crystallization reagent mix process, maintain constant temperature (for example 60 ℃).After reagent mix completes, start to control temperature decline (for example in several minutes from 60 ℃ to 25 ℃).

In one embodiment, forms of rapamycin analogs crystallization can carry out the time of composition insulation by adjusting, to cause crystallization.Correspondingly, different forms of rapamycin analogs compositions can be incubated different time span (for example, 5 minutes, 60 minutes, 48 hours etc.), thereby causes and complete crystallization.Because phase change is by Time dependent, the crystallization of monitoring forms of rapamycin analogs by the function of time has advantage.

Under many circumstances, time control is very important, and for example, the first solid shape of crystallization may not be the most stable, but metastable setting, it can change into and stablize shape subsequently within for some time.This process is called " aging ".The aging change that also can follow crystallographic dimension and/or habit.This type aging manifesting is called Ostwald ripening.Like this, just can adopt and crystal composition is incubated to different time guides crystallization and promotion to crystallize into needed crystalline product.

In one embodiment, the crystallization of forms of rapamycin analogs can be undertaken by adjusting the pH value of synthetics, to cause or to promote crystallization.The pH value of forms of rapamycin analogs synthetics can be measured physical condition and the performance of generated crystal.PH value can be controlled by adding inorganic and organic bronsted lowry acids and bases bronsted lowry, for example in this area as the well-known buffer reagent of standard substance.Sample pH value value can be monitored by standard pH meter, and this pH meter is changed according to the capacity of sample.

In one embodiment, the crystallization of forms of rapamycin analogs can be controlled by adjusting the concentration of forms of rapamycin analogs, to cause or to promote crystallization.Supersaturation is the thermodynamic driving force of crystal nucleation and growth, in preparing crystal forms of rapamycin analogs, is therefore key variables.Supersaturation is by caused with the deviation of heat power solvability balance, thereby more solute (for example forms of rapamycin analogs) is suspended in solution.Therefore, saturation ratio just can be controlled as the amount of adducts or concentration by temperature, forms of rapamycin analogs and other component.In general, in meta region, can control saturation ratio, when surpassing meta limit, nucleation will occur.

The amount of forms of rapamycin analogs and/or other component or concentration can greatly affect physical condition and the performance of gained solid.Like this, for a given temperature, according to the composition of starting soln, can issue product nucleus and growth in the supersaturation of difference amount.The speed of nucleation and growth increases with the increase of saturation ratio, and this will affect crystalline habit.For example, growth fast must adapt to the release of crystallization heat.In crystallisation procedure, this heat effect can form dendrite crystal.The macroshape of crystal is subject to the dendrite crystal impact that even secondary dendrite crystal exists to a great extent.

Second chemical composition that impact is gained solid of relative quantity forms of rapamycin analogs and solvent.For example, the first crystal forming from strong solution forms at the higher temperature of the temperature than form crystal from diluting soln.Balance solid phase is to obtain from the high temperature of phasor, and when at high temperature separating out from the aqueous solution, first strong solution can form semihydrate crystal.But, when starting from diluting soln, may first form semihydrate.In this case, forms of rapamycin analogs/solvent phasor is exactly the phasor that dihydrate is at high temperature decomposed into semihydrate.This is normal situation and to common being seen solvate, is also like this conventionally.

In one embodiment, the crystallization of forms of rapamycin analogs can for example, be undertaken by adjusting component kind (solvent and/or adducts) in composition, to cause or to promote crystallization.In composition, the kind of component can produce great impact to nearly all aspect of crystallization.Component kind can promote or suppress physical condition and the performance of crystal nucleation and growth and the crystal that generates.Like this, a kind of composition can be just a kind of material, and the desired result of this material is exactly the formation that causes, suppresses, prevents or reverse crystalline forms of rapamycin analogs.

A kind of composition can guide the formation of crystal, amorphous solid, hydrate, solvate or the salt of forms of rapamycin analogs.Component can also affect the inside and outside bilge construction of formed crystal, such as polymorphic form and crystal habit.The example of component includes but not limited to vehicle, solvent, salt, acids, bases, gas; Small molecules such as hormone, steroid, Nucleotide, nucleosides and amino acid, macromole such as oligonucleotide, polynucleotide, oligonucleotide and polynucleotide conjugate, protein, peptide, polypeptide thing and polysaccharide; Other medicines; Crystallization additive such as promote and/or control nucleation additive, affect crystalline habit additive, affect polymorphic form additive, affect the additive of particle or crystallographic dimension, structurally stable crystal or the additive of amorphous solid be, the additive of the additive of dissolved solids, inhibition crystallization or solid formation, optically active solvent, optically active reagent and optically active catalyzer.

In one embodiment, can by adjusting, solvent be removed speed and/or anti-solvent is removed the crystallization that speed is carried out forms of rapamycin analogs, to cause or to promote crystallization.The control that solvent is removed is combined with saturated control.Be accompanied by removing of solvent, the concentration of forms of rapamycin analogs and more non-volatile component becomes higher.According to remaining composition, saturation ratio changes the various factorss such as the polarity with remaining component and stickiness.For example, because solvent is removed, the concentration of forms of rapamycin analogs will rise, until reach the meta limit and nucleation occurs and crystal growth.

Solvent remove speed can by temperature, pressure and can occur evaporation surface-area be controlled.For example, solvent can be removed by distillation under preset temperature and pressure, or solvent also can at room temperature evaporate simply and remove.Some time, can use solvent absorbent.

In one embodiment, the crystallization of forms of rapamycin analogs can be undertaken by introducing nucleation or the activity of separating out.In general, this relates to and makes supersaturation forms of rapamycin analogs solution accept certain form energy, such as ultrasonic or mechanical stimulus, or by adding other component to cause supersaturation.

Crystal nucleation is from liquid, amorphous phase, gas or from different crystal solid phases, forms the process of crystal solid phase.Nucleation has been set the characteristic of crystallisation procedure, therefore and become one of composition (the The Encyclopedia of Chemical Technology (encyclopedia of chemical technology) of most critical in design business crystallisation procedure, 7Kirk-Othomer (the 4th edition, 692 pages) (1993)).

Elementary nucleation can be undertaken by out-phase or homophase mechanism, and these two kinds of mechanism all relate to and are combined to form continuously crystal by crystalline component.Elementary nucleation does not relate to already present crystalline forms of rapamycin analogs, but obtain from the self-assembling formation of crystal.Elementary nucleation can be brought out by increase saturation ratio on the meta limit, or by nucleation, brings out during lower than the meta limit when saturation ratio.Nucleation activity comprises mechanical stimulus, such as the stirring rotor by crystallizer, contact crystallization medium and be exposed to the energy, such as sound wave (ultrasonic), electric power or laser energy (Garetz etc., 1996Physical review Letters (physical comment bulletin) 77:3475).Elementary nucleation can be brought out by adding elementary nucleation accelerating agent, such as the material that is different from forms of rapamycin analogs solid shape.The additive that reduces forms of rapamycin analogs surface energy can bring out nucleation.Surface can reduction be conducive to nucleation because the obstacle of nucleation while being formed by solid-liquid surface the increase of energy cause.Therefore, can control nucleation by adjusting the interfacial tension of crystallization medium, and this adjustment realizes by introducing tensio-active agent quasi-molecule, can pre-treatment crystallisation chamber also can directly add.The one-tenth nuclear effect of surfactant molecule depends on their concentration, therefore should carefully control this parameter.Described tension adjustment additive is not limited to surfactant.A lot of compounds relevant to forms of rapamycin analogs all have significant surfactivity.Other heterogeneous nucleation is brought out the solid particulate that additive comprises various materials, the impurity staying after the synthetic or course of processing such as solid excipients or forms of rapamycin analogs.

Secondary nucleation relates to secondary nucleation promotor processes crystallization medium, and described promotor is solid, preferably has the crystal of the required feature of crystal forms of rapamycin analogs.Required crystal forms of rapamycin analogs minicrystal can be used as the promotor of secondary nucleation.Under various physical conditions, directly add a plurality of forms of rapamycin analogs nucleation crystal seeds that a kind of means of the formation of bringing out different crystal forms in different syntheticss are provided.In one embodiment, the particle that is different from forms of rapamycin analogs has been added in crystal composition.In another embodiment, the nano-scale crystal of forms of rapamycin analogs (for example nanoparticle) has been added in sample.

In organic solvent, found crystalline form forms of rapamycin analogs, as ABT-578 (being zotarolimus), described organic solvent comprises acetone, toluene, acetonitrile, ethyl formate, isopropyl acetate, isobutyl acetate, ethanol, DMF and methyl-phenoxide.Acetonitrile solvent can be used for making the crystallization of rapamycin acetonitrile solvent compound, after described solvate dry (as suitable pressure, temperature and evaporitic environment drying conditions), form crystal desolventizing thing (being the acetonitrile solvent compound of desolventizing), thereby remove the acetonitrile in crystal.The acetonitrile solvent compound of crystal desolventizing may have chemically stable characteristic, can exempt the process of adding amorphous ABT-578 in current practice using BHT as antioxidant to.In addition, as described herein, can be by greatly improve the impurity situation of ABT-578 in conjunction with crystallisation step in preparation process.In some powder x-ray diffractions (PXRD) figure in the accompanying drawings, at two peak values at～38 and 44 places from X ray anchor.

There is several different methods to prepare crystal forms of rapamycin analogs.In one embodiment, the method for preparing crystal forms of rapamycin analogs medicine comprises makes forms of rapamycin analogs from pharmacology in acceptable solvent or crystallization in solvent mixture.In other embodiments, the method for preparing crystal forms of rapamycin analogs medicine also comprises makes forms of rapamycin analogs crystallization from organic solvent or solvent mixture.Certainly, those skilled in the art can differentiate all solvents that can be used in embodiment of the present invention, and the solvent that is not limited to list herein.

In one embodiment, the process that the present invention comprises the forms of rapamycin analogs of preparing crystalline form.This process comprises the following steps: forms of rapamycin analogs to be combined with more than one organic medium to form mixture; Mixture is incubated until forms of rapamycin analogs crystallization; With from organic medium, reclaim crystal forms of rapamycin analogs.

In one embodiment, described organic medium comprises at least one solvent, to form mixture.Therefore, the method for preparing crystalline form forms of rapamycin analogs comprises is dissolved in solvent forms of rapamycin analogs, and solvent is incubated until forms of rapamycin analogs crystallization.

In one embodiment, described method is included in and in solution, forms crystal forms of rapamycin analogs slurry.In one embodiment, described method comprises that stirring forms of rapamycin analogs mixture is until forms of rapamycin analogs crystallization.In one embodiment, described method comprises that to make forms of rapamycin analogs solution saturated.This may comprise a supersaturation forms of rapamycin analogs solution of formation.

In one embodiment, described method comprises and comprises that the anti-solvent of use helps to form crystal forms of rapamycin analogs.This method comprises more than one anti-solvent and forms of rapamycin analogs and solvent, forms biphase mixture.By the insulation of gained biphase mixture, cause liquid-liquid phase position separated, wherein most of forms of rapamycin analogs is in solvent, and small part forms of rapamycin analogs is in anti-solvent.Optionally, can be by solvent and anti-separated from solvent before crystal is separated out.

In one embodiment, described organic medium is toluene, acetonitrile, ethanol, isobutyl acetate, ethyl formate, isopropyl acetate, ethanol, DMF and composition thereof.

In one embodiment, described solvent is organic solvent.Like this, organic solvent can be polar organic solvent.The example of polar organic solvent comprises acetone, ethyl acetate, methyl alcohol, ethanol, n-propyl alcohol, Virahol, isopropylcarbinol, the trimethyl carbinol, 2-butanols, acetonitrile, tetrahydrofuran (THF), isobutyl acetate, n-butyl acetate, ethyl formate, n-propyl acetate, isopropyl acetate, methyl ethyl ketone or and arbitrary composition.Preferably, polar organic solvent is acetone.The example of anti-solvent comprises hexanaphthene, heptane, hexane, octane, octane-iso, methylcyclohexane or its arbitrary composition.Preferably, anti-solvent is heptane.Preferably, organic medium is acceptable on pharmacology with regard to medicine preparation.For example, organic medium can be acceptable solvent on pharmacology, is suitable for preparing the composition of pharmaceutical grade.

In one embodiment, forms of rapamycin analogs solution (as biphase mixture etc.) forms, is incubated, stirs, mixes, sizes mixing, saturated and/or at approximately-20 ℃ to approximately 20 ℃, more preferably-10 ℃ to 10 ℃, even more preferably-5 ℃ to approximately 5 ℃, most preferably from about 0 ℃ is carried out crystallization.

In one embodiment, forms of rapamycin analogs solution (as biphase mixture etc.) forms, is incubated, stirs, mixes, sizes mixing, saturated and/or at approximately 10 ℃ to approximately 40 ℃, more preferably from about 12 ℃ to approximately 32 ℃, even more preferably from about 20 ℃ to approximately 25 ℃, most preferably from about 22 ℃ are carried out crystallization.

In one embodiment, forms of rapamycin analogs solution (as biphase mixture etc.) forms, is incubated, stirs, mixes, sizes mixing into suspensoid, saturated and/or crystallization approximately 0.1 hour to approximately 35 hours, more preferably from about 1 hour to approximately 30 hours, even more preferably from about 5 hours to approximately 25 hours, more preferably from about 10 hours to approximately 20 hours, most preferably from about 15 hours.

The forms of rapamycin analogs of being combined with organic medium in one embodiment, is crystal.For example, crystal forms of rapamycin analogs can be the acetonitrile solvent compound (being acetonitrile desolventizing thing) of acetonitrile solvent compound, desolventizing.Or crystalline form forms of rapamycin analogs also can be non-crystal state.

In one embodiment, the rapamycin mixture in organic medium and the second organic medium combination, the mixture of wherein, further processing (as being incubated, stirring, mix, size mixing, saturated and/or crystallization) comprises the second organic medium.For example, the first organic medium can be acetonitrile, toluene, ethanol, isobutyl acetate, methyl-phenoxide or its composition.The second organic medium can be ethyl formate, isopropyl acetate, ethanol, DMF, methyl-phenoxide with and composition.

In one embodiment, by the biphase mixture insulation of the forms of rapamycin analogs of dissolving, acetone and heptane being obtained to the crystal forms of rapamycin analogs of acetonitrile solvent compound form at 0 ℃.Correspondingly, forms of rapamycin analogs can join in the cast bottle that acetone and heptane are housed, thereby makes liquid phase saturated.When forms of rapamycin analogs is dissolved in solution, produce phase and a rich heptane top phase time at the bottom of a rich forms of rapamycin analogs-acetone, at this moment there will be liquid-liquid phase.For example, biphase mixture can be approximately 0 ℃ of insulation approximately 0.1 to approximately 10 day or more of a specified duration, until crystal can be observed in cast bottle bottom.Then by powder x-ray diffraction analysis crystal, as shown in Figure 2 A.Crystal is balance at ambient temperature, then further dry at approximately 30 ℃ under vacuum state (about 3 inches of high mercurys).Dried crystal is analyzed by powder x-ray diffraction, as shown in Figure 3A.

In one embodiment, at ambient temperature noncrystal forms of rapamycin analogs is dissolved in acetone, then at approximately 5 ℃, the solution obtaining is incubated, until can observe crystalline solids thing, can obtain the crystal forms of rapamycin analogs of acetone solvent compound form.Then by X-ray diffraction analysis crystal, as shown in Figure 2 B.Crystal is balance at ambient temperature, then further dry at approximately 30 ℃ under vacuum state (about 3 inches of high mercurys).Dried crystal is analyzed by powder x-ray diffraction, as shown in Figure 3 B.

In one embodiment, the crystal forms of rapamycin analogs that can prepare toluene solvate form.That is, noncrystal forms of rapamycin analogs is dissolved in toluene, forms solution, can obtain forms of rapamycin analogs toluene solvate crystal.Solution is approximately 22 ℃ of stir abouts 15 hours, or until can observe thick crystalline solids thing underflow.Can also use the crystal forms of rapamycin analogs such as acetonitrile solvent compound to obtain composition, impel crystallization, thereby prepare crystal.Then crystal is analyzed by powder x-ray diffraction, as shown in Figure 4 A.

In one embodiment, the crystal forms of rapamycin analogs that can prepare another kind of acetonitrile solvent compound form.That is,, by using noncrystal forms of rapamycin analogs that acetonitrile is saturated when 22 ℃ of left and right, then, about 0 ℃ insulation approximately 2 hours or until crystal formation, can obtain acetonitrile solvent compound crystal.Then crystal is analyzed by powder x-ray diffraction, as shown in Figure 5A.Dry acetonitrile solvent compound can obtain forms of rapamycin analogs acetonitrile desolventizing thing, and its x-ray diffractogram of powder analytical data as shown in Figure 6A.

In one embodiment, the crystal forms of rapamycin analogs that can prepare ethyl formate form.That is, during 0 ℃ of left and right in ethyl formate by the wet cake furnishing slurry of acetonitrile solvent compound, obtain forms of rapamycin analogs ethyl formate solvate crystals.Then crystal is analyzed by powder x-ray diffraction, as shown in Figure 7 A.

In one embodiment, the crystal forms of rapamycin analogs that can prepare isopropyl acetate solvate form thereof.That is, during 0 ℃ of left and right in isopropyl acetate by the wet cake furnishing slurry of acetonitrile solvent compound, obtain forms of rapamycin analogs isopropyl acetate solvate.

In one embodiment, the crystal forms of rapamycin analogs that can prepare isobutyl acetate solvate form thereof.That is, in cast bottle, add noncrystal forms of rapamycin analogs, then add isobutyl acetate to make it to dissolve.Can obtain like this forms of rapamycin analogs isobutyl acetate crystal.Solution was about 0 ℃ insulation approximately 16 hours, or until acquisition crystal slurry.Then crystal is analyzed by powder x-ray diffraction, as shown in Figure 9 A.

In one embodiment, the crystal forms of rapamycin analogs that can prepare alcohol solvent compound form.That is, in cast bottle, add noncrystal forms of rapamycin analogs, then add ethanol (200 intensity (proof)) to make it to dissolve.Can obtain like this forms of rapamycin analogs alcohol solvent compound crystal.Approximately, after 15 hours, use acetonitrile desolventizing thing to plant solution, then 0 ℃ of left and right, be incubated again approximately 16 hours, until obtain crystal slurry.

In one embodiment, the crystal forms of rapamycin analogs that can prepare DMF solvate form thereof.That is, during 0 ℃ of left and right in DMF by the wet cake furnishing slurry of acetonitrile solvent compound, obtain forms of rapamycin analogs solvent dimethylformamide compound crystal.Then crystal is analyzed by powder x-ray diffraction, as shown in Figure 10 A.

In one embodiment, the crystal forms of rapamycin analogs that can prepare methyl-phenoxide solvate form thereof.That is, during 0 ℃ of left and right in methyl-phenoxide by the wet cake furnishing slurry of acetonitrile solvent compound, obtain forms of rapamycin analogs methyl-phenoxide solvate crystals.Then crystal is analyzed by powder x-ray diffraction, as shown in Figure 11 A.

IV. crystal forms of rapamycin analogs composition

Crystal forms of rapamycin analogs of the present invention can be prepared into any medical composition, as the common composition being used in conjunction with noncrystal forms of rapamycin analogs.Thereby crystal forms of rapamycin analogs can be mixed with polymer composition, as bracket coating etc.Polymer composition can comprise hydrophilic, hydrophobic, biodegradable, nonbiodegradable polymkeric substance and any composition thereof.Polymkeric substance optional mixture and multipolymer from polyacrylic ester, polymethacrylate, polycarboxylic acid, cellulose polymer compound, gelatin, polyvinylpyrrolidone, maleic anhydride polymer, polymeric amide, polyvinyl alcohol, polyethylene oxide, mucopolysaccharide, saccharan, polyester, urethane, siloxanes, poe, poly-acid anhydrides, polycarbonate, polypropylene, poly(lactic acid), polyglycolic acid, polycaprolactone, PHBV, polyacrylamide, polyethers and above-mentioned substance.Can also use polymeric dispersions, as polyurethane dispersions (BAYHYDROL etc.) and acrylic latex dispersion etc.

Spendable biodegradable polymer comprises poly (l-lactic acid), PDLLA, polycaprolactone, poly butyric ester, PGA, PPDO (polydiaxanone), poly(hydroamyl ester), poe, multipolymer, PLGA (poly(lactide-co-glycolide)), polyhydroxybutyrate valeric acid copolyesters (polyhydroxy(butyrate-co-valerate)), PGA-trimethylene carbonate multipolymer, poly-acid anhydrides, poly phosphate, poly phosphate-urethane, polyamino acid, paracyanogen base propionic ester, protoplasma, scleroproein, Fibrinogen, Mierocrystalline cellulose, starch, collagen/hyaluronic acid and any composition thereof.Also can use the polymkeric substance of Biostatic, as urethane, siloxanes, polyester, polyolefine, polymeric amide, polycaprolactam, polyimide, polyvinyl chloride, polyvinyl methyl ether, polyvinyl alcohol, acrylate copolymer and multipolymer, polyacrylonitrile, the polystyrene copolymer of vinyl monomer and alkene is (as styrene-acrylonitrile copolymer, ethylene-methyl methacrylate methyl terpolymer, ethane-acetic acid ethyenyl ester), polyethers, regenerated fiber, Mierocrystalline cellulose is (as cellulose acetate, nitrocellulose, cellulose propionate etc.), polyphenylene ethyl and derivative thereof and its any composition.

Other can use polymkeric substance to have MPC subunit, comprises poly-(MPC _w; LAM _x: HPMA _y: TSMA _z) polymkeric substance, wherein w, x, y, z represent the mol ratio for the preparation of monomer used in the charging of polymkeric substance, MPC represent methylidene acrylyl oxy-ethyl phosphorylcholine (methacryoyloxyethylphosphorylcholine) unit, LMA represent methylidene lauryl acrylate unit, HPMA represent methylidene vinylformic acid 2-hydroxypropyl ester unit, TSMA represent methylidene vinylformic acid 3-trimethoxy-silylpropyl ester units.

In addition, crystal forms of rapamycin analogs can be prepared into any pharmaceutical composition.This pharmaceutical composition can comprise pharmacology acceptable carrier or vehicle, can be oral, rectal application, intestines externally applied agent, brain pond innerlich anwenden, intravaginal medication, intraperitoneal administration, local application's (pasting by powder, ointment, drops or transdermal medicine), oral cavity medicine (as oral cavity hydrojet agent or nasal spray) or local application, as be placed on the support in vascular system.Phrase " pharmacology acceptable carrier " refers to the auxiliary material that makes up a prescription of nontoxic solid, semisolid or liquid filler, thinner, packaged material or any type.Term " intestines are outer " refers to application method, comprises intravenous, endarterial, intramuscular, Intraabdominal, intrasternal, subcutaneous, IA injection, transfusion and placement etc., for example, in blood vessel.

Pharmaceutical composition can comprise the acceptable aseptic aqueous solution of pharmacology or non-aqueous solution, dispersion liquid, suspension or emulsion and before fast use for the sterilized powder of recombinate aseptic injectable solution or dispersion liquid.The example of suitable water carrier and nonaqueous carrier, thinner, solvent or medium has water, ethanol, polyvalent alcohol (as glycerine, propylene glycol, polyoxyethylene glycol etc.), carboxymethyl cellulose and suitable mixture, vegetables oil (as sweet oil) and injectable organic ester (as ethyl oleate).By using the coated materials such as Yelkin TTS, for dispersion, by granularity being maintained to the size of requirement, and by using tensio-active agent, can keep suitable mobility.

Pharmaceutical composition also can comprise the auxiliarys such as sanitas, wetting agent, emulsifying agent, dispersion agent.Add multiple antiseptic-germicide and anti-mycotic agent can suppress the activity of microorganism.Antiseptic-germicide and anti-mycotic agent have p-Hydroxybenzoate, Trichloroisobutyl Alcohol, phenol Sorbic Acid etc.Add the isotonic agents such as sugar, sodium-chlor also can reach desirable effect.The reagent that adds aluminum monostearate or colloid etc. can postpone to absorb can extend the soak time of injectable drug.

In some cases, in order to extend the effect of crystal forms of rapamycin analogs, the speed of absorbing crystal forms of rapamycin analogs from subcutaneous injection or intramuscular injection need to be slowed down.Use the crystal of poorly water-soluble or the liquid suspension that amorphous material is made can reach the effect slowing down.Then crystal forms of rapamycin analogs uptake rate just depends on the dissolution rate of himself, and it depends on size and the crystal formation of crystal conversely.Also having a kind of method is that medicine is dissolved or is suspended in oil base carrier, thereby postpones the absorption of intestines externally applied agent.

By form the micro-capsule matrix of medicine in the Biodegradable polymerics such as polylactide-PGA, thereby form injectable drug carriers.According to the character of the concrete polymkeric substance of the ratio of medicine and polymkeric substance and use, controlled pharmacy rate of release.The example of other Biodegradable polymeric has poe and poly-acid anhydrides.Also can by medicine is packed into micro-ester capsule of bodily tissue compatibility or microemulsion in obtain pharmaceutical carrier injectable formulation.

By the filtration of biofilter, or by before use in conjunction with being dissolved in or being scattered in the sterilizing agent of the aseptic solid composite form of sterilized water or other sterile injectable medium, injectable preparation is carried out disinfection.

Oral solid dosage comprises capsule, tablet, pill, medicinal powder and powder.In these solid dosages, crystal forms of rapamycin analogs and following material mix: the inertia of at least one, the acceptable vehicle of pharmacology or carrier (as Trisodium Citrate or secondary calcium phosphate), and/or (a) filler or extender: starch, lactose, sucrose, glucose, N.F,USP MANNITOL and silicic acid etc.; (b) tackiness agent: carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic etc.; (c) wetting agent: glycerine etc.; (d) disintegrating agent: agar, calcium carbonate, potato or tapioca (flour), Lalgine, some silicate and sodium carbonate etc.; (e) solution resistance Stagnation agent: paraffin etc.; (f) absorption enhancer: quaternary ammonium compound etc.; (g) wetting agent: cetyl alcohol and glyceryl monostearate etc.; (h) absorption agent: kaolin and wilkinite etc.; (i) lubricant: talcum, calcium stearate, Magnesium Stearate, solid polyethylene glycol, sodium lauryl sulfate and composition thereof etc.If capsule, tablet and pill, formulation also can comprise buffer reagent.

The similar solids composition of type also can be used as filler for soft, semi-solid and sandwich capsule or liquid filling capsule, and uses the vehicle such as polyoxyethylene glycol of lactose and high molecular.

While preparing the solid dosages such as tablet, dragee, capsule, pill and powder, can adopt coating and the shell such as well-known enteric coating in pharmacy field.Optional the selecting of these solid Types of Medicine comprises opacifying agent, also can be the only compound of release of active ingredients, or the preferred a certain section of optional delayed release of active elements at enteron aisle.The example of available embedding composition has polymeric material and wax.The embedding composition that contains medicine can be placed in the medical devices such as support, graft, conduit and air bag.Crystal forms of rapamycin analogs can be put into micro-capsule, if suitable, can add one or more above-mentioned vehicle.

Oral liquid dosage form has the acceptable emulsion of pharmacology, solution, suspension agent, syrup and elixir.Except crystal forms of rapamycin analogs, liquid preparation can comprise inert diluent conventional in this area, as water or other solvent, also comprise solubilizing agent and emulsifying agent, as ethanol, Virahol, ethyl-carbonate, ethyl acetate, phenylcarbinol, peruscabin, propylene glycol, 1,3-butyleneglycol, dimethyl formamide, oil (being especially Oleum Gossypii semen, peanut oil, Semen Maydis oil, germ oil, sweet oil, Viscotrol C and sesame oil), glycerine, tetrahydrofurfuryl alcohol, polyoxyethylene glycol and lipid acid sorbitol ester and composition thereof.Except inert diluent, oral compositions also can comprise auxiliary, as wetting agent, emulsifying agent, suspension agent, sweeting agent, odorant and flavouring agent.

Except activeconstituents, suspension can comprise suspension agent, as oxyethyl group i-octadecanol, polyoxyethylene sorbitol, sorbitan ester, Microcrystalline Cellulose, oxygen (hydrogen-oxygen) are changed aluminium (aluminum metahydroxide), wilkinite, agar, yellow Stilbene glue and composition thereof.

Topical comprises by skin or mucosa delivery, comprises the surface of lung and eyes.The composition of topical comprises composition for inhalation, can be made into pressurization or non-pressurized dry powder.Do not pressurizeing in dry powder composite, finely powdered activeconstituents can mix and use with the acceptable inert support of large size pharmacology.Described inert support comprises that diameter reaches the particle of 100 microns.Suitable inert support comprises the sugar such as lactose.Wish that the effective size of grain size of at least 95% weight active ingredient particle is between 0.01 to 10 micron.The composition that part on skin is used also comprises ointment, face cream, washing lotion and gelifying agent.

Or described composition also can pressurize, and comprises pressurized gas, as the gaseous propellant of nitrogen or liquefaction.Described liquefaction propulsive medium and the preferred activeconstituents of whole composition do not dissolve.Pressurized compositions can comprise tensio-active agent.Tensio-active agent can be liquid or solid nonionic surface active agent, or solid aniorfic surfactant.Preferably use the solid aniorfic surfactant of sodium-salt form.

The another kind of form of topical is by ophthalmic administration, and the immunologically mediated disease for the treatment of eyes, as autoimmune disease, allergy or inflammatory disease and corneal transplantation.Use the carrier of the acceptable applicable eyes of pharmacology to smear crystal forms of rapamycin analogs, make synthetics and eye Surface Contact time enough, so that synthetics can infiltrate the inside of cornea and eyes, as anterior chamber of the eye, posterior chamber of the eye, vitreum, aqueous humor, vitreous humor, cornea, iris/ciliary body, lens, choroid/nethike embrane and sclera.The carrier of the acceptable applicable eyes of pharmacology can be ointment, vegetables oil or packaged material.

The composition of rectal administration or vagina administration is preferably suppository or enema.Crystal forms of rapamycin analogs and suitable non-irritating vehicle or carrier mixing can be obtained to suppository or enema.Vehicle or carrier comprise theobroma oil, polyoxyethylene glycol or suppository wax etc., are solid under room temperature state, but can become liquid after entering human body, melt release of active ingredients at internal rectum or intravaginal.

Crystal forms of rapamycin analogs equally can be with the form administration of liposome.As known in the art, liposome is conventionally from phosphatide or other lipid matter.Liposome is crystal formation by the single or multiple lift aqua liquid being scattered in water medium.Acceptable and can prepare can using by metabolism lipid of liposome on any nontoxic, physiology.The present composition in liposome form, except compound of the present invention, can comprise stablizer, sanitas, vehicle etc.Preferred lipid is phosphatide and phosphatidylcholine (Yelkin TTS), can be natural also can synthesizing.The method of making liposome is well known in the art.(see the Methods in Cell Biology (cell biology method) of Prescott Ed, the 14 volume, the 33rd page of new york academic press (1976) and following.)

Crystal forms of rapamycin analogs can be applied in and carry out on the support of coating with polymkeric substance.The polymeric coating that described compound or medicine is attached to support can be implemented by following: the support that scribbles polymkeric substance is immersed in the solution that contains compound or medicine, long enough (for example 5 minutes), then the support of coating is dried up, preferably use air-dry mode, long enough (for example 30 minutes).Containing support compound or medicine, that have polymeric coating just can be sent to coronary vasodilator by configuration from balloon catheter.Except support, be used for introducing medicine of the present invention to the miscellaneous equipment of vascular system including, but not limited to graft, conduit and air bag.In addition, can be used for replacing other compound or medicine that medicine of the present invention is used to include but not limited to A-94507 and SDZ RAD (also claiming everolimus).

Crystal forms of rapamycin analogs can mix and use with other medicines preparation.At effective medicament aspect prevention (postoperative) restenosis, can be divided into the classes such as antiproliferative, anti-platelet agents, antiphlogistic drug, antithrombotic reagent and thrombolytic agent.These classification can further be segmented.For example, antiproliferative can be divided into antimitotic.Antimitotic agent suppresses or affects cell fission, conventionally so the process relevant with cell fission can not occur.A subclass of antimitotic agent comprises catharanthus alkaloid.The exemplary of catharanthus alkaloid is including, but not limited to vincristine(VCR), taxol, etoposide, R 17934, Indirubin and anthracycline derivative, for example zhengdingmeisu, daunomycin and Plicamycin.Other subclass of antimitotic agent comprises antimitotic alkylating agent; for example cow-bezoar Mo Siting, bofumustine and fotemustine; with antimitotic meta-bolites, for example methotrexate, Fluracil, 5-bromouracil deoxyribose, 6-nitrogen cytidine and cytosine arabinoside.Antimitotic alkylating agent changes DNA by covalency, and therefore RNA or protein influence cell fission suppress DNA replication dna, rna transcription, RNA conversion, protein synthesis or above-mentioned combination.

Anti-platelet agents is treatment entity, by following manner, works: (1) suppresses on surface the particularly platelet adhesion reaction on thrombus surface, (2) anticoagulant, and (3) suppress platelet activation, or (4) above-mentioned combination.Hematoblastic activation is such a process, and thrombocyte is converted into experience because of the state of the surperficial some metamorphosis that bring out of contact thrombus from a static inactive state.These variations comprise the change in thrombocyte shape, are accompanied by the formation of pseudopodium, with the combination of membrane receptor, and for example secretion of adenosine diphosphate (ADP) (ADP) and platelet factor 4 of small molecules and protein.As Eptifibatide, Tirofiban, RGD (arginine-glycine-aspartic acid) the base peptide including, but not limited to the combination of inhibition and gpIIbIIIa or α v β 3 to the anti-platelet agents of the inhibitor of platelet adhesion reaction, the antibody of prevention and gpIIbIIIa or α v β 3 combinations, anti-palatelet-selectin antibody, anti-E-Selectin antibody and prevention palatelet-selectin or E-Selectin are attached to compound, vitamin B13 and the temperature resistance Wei Baishi factor antibody on aglucon separately.The medicine of the platelet aggregation of inhibition ADP mediation is including, but not limited to depolymerization peptide (disagregin) and Cilostazole.

Can use anti-inflammation drugs equally.These medicine for example, including, but not limited to prednisone, dexamethasone, hydrocortisone, estradiol, fluticasone propionate, Clobetasol and non-steroid antiphlogistic drug, acetaminophen, Ibuprofen BP/EP, Naproxen Base and sulindac.Other example of these medicaments comprises that those suppress cytokine or chemokine is attached to homoreceptor, with this, suppresses the medicament that inspires scorching signal of cytokine or chemokine transduction.The exemplary of these medicaments is including, but not limited to anti-IL1, anti-IL2, anti-IL3, anti-IL4, anti-IL8, anti-IL15, anti-IL18, anti-GM-CSF and anti-TNF (tumour necrosis factor) antibody.

Antithrombotic preparation comprises chemistry and the biological entities that can intervene in any stage of the approach that condenses.The example of concrete entity includes but not limited to the small molecules of anticoagulant factor xa activity.In addition, can directly or indirectly suppress the heparin class medicine of FXa and zymoplasm, for example heparin, heparin sulfate, low molecular weight heparin, for example, have the compound of trade mark and synthetic oligose, for example, have the compound of (injection) trade mark.Direct thrombin inhibitor, for example peptide medicament on the phenylalanine-Pro-Arg of Melagatran, ximelagatran, argatroban, Inogatran and zymoplasm (Phe-Pro-Arg) Fibrinogen matrix combining site in addition that comprise equally.The antithrombotic preparation that an other class can be transmitted is the inhibitor of proconvertin/VIIa, for example anti-proconvertin/VIIa antibody, rNAPc2 and tissue factor pathway inhibitor (TFPI).

Thrombolytics, may be defined as the medicament that helps to reduce thrombi (grumeleuse), because the solubility behavior of grumeleuse can help the thrombocyte of scatter-gather in thrombus fiber parent, so it can be used as auxiliary equally.The exemplary of thrombolytics is including, but not limited to urokinase or recombinaton urokinase, pro-urokinase or restructuring pro-urokinase, tissue plasminogen activator or its recombinant forms and streptokinase.

The other medicines that can be combined with crystal forms of rapamycin analogs are cytotoxic drugs, for example cell death inducer, for example transforming growth factor (TGF) and Topoisomerase inhibitors, for example 10-hydroxycamptothecine, irinotecan and Zorubicin.The other medicines type that can be combined with crystal forms of rapamycin analogs is dedifferented medicine and the cytostatic medicine of (anti-differentiation) for suppressing cell.Other medicament that can be combined with crystal forms of rapamycin analogs comprises the antagonist of fenofibrate, batimistat and endothelin-A receptor, for example, reach the antagonistic of Lu Shengtan and α v β 3 integrin receptors.

Crystal forms of rapamycin analogs equally can with administration together with the medicine of one or more inhibitive abilities of immunity.Inhibitive ability of immunity medicine in the scope of the invention including, but not limited to azathioprine sodium, brequinar sodium, gusperimus trihydrochloride (having another name called deoxidation spergualin), mizoribine (having another name called mine-laying enlightening peaceful), mycophenolate mofetile ciclosporin-A ( under trade mark, as ciclosporin-A different dosage form, go on the market), tacrolimus (having another name called FK-506), sirolimus and leflunomide (having another name called HWA-486), glucocorticosteroid, for example hydrogenation Bo Nisong and derived product thereof, antibody therapy is as orthoclone (OKT ₃) and with antithymocyte globulin as rabbit antithymocyte globulin.

the treatment of V crystal forms of rapamycin analogs

Crystal forms of rapamycin analogs (particularly mankind) in Mammals has the activity of immunomodulator.As immunosuppressor, crystal forms of rapamycin analogs can be used for treatment and prevents following disease: immune-mediated disease after one's own heart, the organ or tissue such as kidney, liver, marrow, skin, cornea, lung, pancreas, ileum, limbs, muscle, nerve, duodenum, small intestine, islet cells transplants the repulsion producing; The graft versus host disease (GVH disease) that bone marrow transplantation produces; Autoimmune disease, such as rheumatic arthritis, systemic lupus erythematosus, struma lymphomatosa, multiple sclerosis, myasthenia gravis, type i diabetes, uveitis, allergic encephalomyelitis, glomerulonephritis etc.More purposes also comprises the immunologically mediated disease for the treatment of and preventing inflammatory and proliferative skin disorders, skin sex expression, for example psoriasis, atopic dermatitis, contact dermatitis and further eczematoid dermatitis, seborrheic dermatitis, lichen planus, pemphigus, spherical pemphigoid, epidermolysis bullosa, urticaria, angioedema, vasculitis, erythema, skin eosinophilia, lupus erythematosus, acne and alopecia areata; Various eye diseases (autoimmunization and other), for example relevant uveitis, keratitis, herpetic keratitis, keratoconus, dystrophia epithelialis corneae, walleye and the ocular pemphigus of keratoconjunctivitis, vernal conjunctivitis and Behcet's disease.Reversible obstructive airway diseases in addition, comprise these illnesss, for example, such as asthma (bronchial asthma, atopic asthma, intrinsic asthma, extrinsic asthma and dust asthma), asthma especially long-term or that root is dark (for example late-onset asthma and airway hyperreactivity), bronchitis, allergic rhinitis etc. all by crystal forms of rapamycin analogs as target.Mucous membrane and vascular inflammatory, blood vessel injury and thrombosis that for example stomach ulcer, ischemic disease cause.In addition, higher proliferation vascular disease, such as smooth muscle cell proliferation, restenosis and vascular occlusion, especially, after mediation blood vessel injury biology or machinery, all can be used crystal forms of rapamycin analogs treat and prevent.

Other can treat illness including, but not limited to ischemic enteropathy, enteritis, necrotic enteritis, enteritis/allergy such as celiac disease, rectitis, eosinophilic gastroenteritis, mastocytosis, Crohn disease and ulcerative colitis; Sacred disease such as polyneuritis, mononeuritis and radiculopathy; Endocrinopathy is such as hyperthyroidism and Basedow's disease; Hematologic disease such as pure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia and red corpuscle occur can not; Osteopathy is such as osteoporosis; Respiratory tract disease such as sarcoidosis, fibroid lung and spontaneous interstitial pneumonia; Tetter such as dermatomyositis, ordinary leukodermia, ordinary ichthyosis, photoallergy sensitivity and epidermis t cell lymphoma; Circulation system disease such as arteriosclerosis, atherosclerosis, aortitis syndrome, polyarteritis nodosa and myocardosis; Collagen disease such as scleroderma, wegener granulomatosis and xerosis; Obesity; Eosinophilic fasciitis; Periodontopathy such as gum infringement, periodontium, alveolar bone and black substance bone dental pulp; Such as brightic nephrotic syndrome; By preventing epilation or providing hair to produce and/or promote male pattern alopecia disease or the senile alopecia disease of hair metabolism and natural on-off cycles of hair growth; Muscular dystrophy; Pyoderma and Sezary syndrome; A Disenshi is sick; The disease of active oxygen mediation for example organ is dampened, for example, such as occurring in preservation, transplanting previous organ (as heart, liver, kidney and digestive tube) ischemical reperfusion injury or ischemic disease (thrombosis and myocardial infarction); Enteropathy is such as medicine or radioactive endomycin shock, pseudomembranous colitis and colitis; Ephrosis is such as ischemic acute renal insufficiency and chronic renal insufficiency; Toxonosis, lung cancer and pulmonary emphysema that tuberculosis such as lung oxygen or medicine (for example prednisone (paracort) and bleomycin) cause; Illness in eye such as cataract, siderosis, the retinitis, retinochrome pathology, senile macular degeneration SMD, vitreum scar and corneal alkali burn; The spherical dermatitis of IgA (immunoglobulin A) and the cement dermatitis of dermatitis such as erythema multiforme, linearity; With other disease for example, causing such as gingivitis, periodontitis, septicemia, pancreatitis, environmental pollution (atmospheric pollution), aging, the formation of cancer, the transfer of cancer and hypobaropathy; The disease that the release of histamine or leukotriene-C4 causes; Behcet's disease such as colon, vascular or nervosa behcet's disease, and affect equally the behcet's disease of oral cavity, skin, eyes, vulva, joint, epididymis, lung, kidney etc.

In addition, crystal forms of rapamycin analogs can be used for treatment and prevention of liver disease, and such as immunogenic disease, (chronic autoimmunization hepatopathy for example, such as autoimmunization hepatitis, Primary Hepatic cirrhosis and sclerosing cholangitis), partially hepatectomized, acute severe hepatitis (the necrosis for example being caused by toxin, viral hepatitis, shock or anoxia), hepatitis B, non-A type/non-B hepatitis, sclerosis (such as alcoholic cirrhosis) and liver failure, such as explosive liver failure, the liver failure of late coming and " urgent slowly " liver failure (acute hepatic failure on chronic hepatopathy), in addition due to its useful activity, increase such as chemotherapy effect, cytomegalovirus infection, particularly cytomegalovirus (HCMV) infects and anti-inflammatory action, it can be used for various diseases, and for example hardening disease and fiber disease, such as ephrosis, scleroderma, pulmonary fibrosis, arteriosclerosis, congestive heart failure, ventricular hypertrophy, postoperative bonding and scab, apoplexy, myocardial infarction and with local anemia with pour into again relevant damage etc.

In addition, crystal forms of rapamycin analogs has FK-506 Antagonism.Therefore crystal forms of rapamycin analogs can be used for the treatment of to immunosuppression or relate to immunosuppressant disorders.The example that relates to immunosuppression disorders comprises acquired immune deficiency syndrome (AIDS), cancer, fungal infection, senile dementia, wound (comprising wound healing, surgical operation and shock), chronic bacterial infection and some central nervous system disorder.Immunosuppression to be treated can cause by immunosuppressant macrocylc compound excessive, 12-(2-cyclohexyl-1-methyl ethylene)-13,19,21 for example, 27-tetramethyl--11,28-dioxy-4-aza-tricycle [22.3.1.0 ^4,9] 28 carbon-18-alkene) and Xing biology such as FK-506 or rapamycin.Once patient recognizes and forget at specific time and take medicine, clothes for patients is very common with overdose of medicine thing and can causes severe side effect.

The ability of crystal forms of rapamycin analogs treatment proliferative disease can be by Bunchman ET and CA Brookshire, Transplantation Proceed (transplanting program) the 23rd volume 967-968 page (1991); Mountain bank etc., Biochem.Res.Comm. (biological chemistry and biophysical research communication) the 191st volume 840-846 page (1993); And Shichiri etc., the method described in J.Clin.Invest. (clinical investigation magazine) 87 volume 1867-1871 pages (1991) is proved.Proliferative disease comprises that restenosis or other pathologic after proliferation of smooth muscle, systemic sclerosis, liver cirrhosis, adult respiratory distress syndrome, idiopathic cardiomyopathy, lupus erythematosus, diabetic retinopathy or other retinopathy, psoriasis, scleroderma, hyperplasia of prostate, cardiac hypertrophy, arterial injury is narrow.In addition, crystal forms of rapamycin analogs has Antagonism to the cellular response of these somatomedins, therefore has the characteristic of angiogenesis inhibitor, and this makes it become the beneficial agents of controlling or stoping the fiber disease of some knurl growth and lung, liver and kidney.

Aqueous liquid composition is particularly useful for treatment and prevents various illness in eye, for example, repulsion such as autoimmune disease (comprising, keratoconus, keratitis, dystrophia epithelialis corneae, walleye, Mooren's ulcer, scleritis and Ge Lafu illness in eye) and corneal transplantation.

While using, can adopt a kind of crystal forms of rapamycin analogs for the treatment of significant quantity, with the form of acceptable salt, ester and prodrug on pure (when such state exists), pharmacology in above-mentioned or other treatment.Or crystal forms of rapamycin analogs can be used as the medical composition that contains acceptable vehicle on target compound and one or more pharmacology and carries out administration." the treatment significant quantity " of term crystal forms of rapamycin analogs refers to the q.s that compound is treated illness with rational benefit/risk ratio in any pharmacological agent.Yet, it is to be understood that, use total amount in the scope of reliable medical judgment, to determine by curing mainly physician the day of the compounds of this invention and composition.Concrete treatment effective dose to the especial patient of any degree will depend on various factors, comprise the illness that is treated and the severity of illness; The activity of the particular compound of using; The concrete composition using; Age, body weight, healthy state, sex, patient's diet; Secretion/the discharge rate of the time of administration, dosage regimen and the particular compound used; The time length for the treatment of; The medicine that is combined with or is used in conjunction with used particular compound; And in medical field well-known similar factor.For example, well known to the skilled person, compound predose should be first lower than reaching the desired level of expectation result for the treatment of, more progressively increases dosage until reach and do the effect of expecting.

Administration was approximately 0.01 to 10 mg/kg/days to the scope of the TDD of people or zootic crystal forms of rapamycin analogs.For oral medication, more preferably dosage can be for approximately 0.001 to 3 mg/kg/days.In order to carry out part from support, carry, the per daily dose that patient receives is according to stent length.For example, the coronary artery pipe of one 15 millimeters can hold the medicine of approximately 1 to 120 micrograms, can to the time period in several weeks, carry this medicine at several hours.As needs, effectively per daily dose can be divided into a plurality of dosage according to administration object; Like this, the composition of single dosage can comprise that such amount or its approximate number are to form per daily dose.According to application site, the dosage range of topical was approximately 0.001 to 3% mg/kg/day.

Embodiment

embodiment 1

In conjunction with following synthetic schemes and the method and the method for preparing crystalline forms of rapamycin analogs of preparing forms of rapamycin analogs, more can understand forms of rapamycin analogs of the present invention and method, described synthetic schemes and preparation method have illustrated the method for preparing crystal forms of rapamycin analogs of the present invention.

Forms of rapamycin analogs of the present invention can be prepared by some synthetic routes.Representational step is shown in Fig. 1.As shown in Figure 1, the C-42 hydroxyl of rapamycin is converted into the leavings group of trifluoromethanesulfonic acid or fluosulfonic acid, has produced A structure.For example, under the non-nucleophilic base that is obstructed (2,6-lutidine or preferably diisopropylethylamine) exists, with tetrazolium, replace leavings group, obtain formula 2 and formula 3, by rapid column chromatography, it is carried out to separation and purification.

With reference to the embodiment that the following describes the method for the preparation of compound in the present invention, can better understand foregoing, this embodiment is not intended to the scope of the invention of definition in additional application to limit.

embodiment 1A

7.5 grams of rapamycins are dissolved in 30 grams of methylene dichloride (DCM).Add 1.76 gram 2,6-lutidine.In acetonitrile-the dry ice bath, solution is cooled to 30 ℃, in 10 minutes, slowly adds 2.89 grams of trifluoromethanesulfanhydride anhydrides.Reaction mixture is stirred 20 minutes, then detect the rapamycin of existence to determine the consumption in reaction.First add 1.44 grams of 1-H-tetrazoliums, and then add 5.29 grams of diisopropylethylamine (DIEA).At room temperature reactant is stirred 6 hours, then directly pack the tetrahydrofuran (THF) with 1:1 into: on 270 grams of silicagel columns that normal heptane (volume/volume) prepares.Use the tetrahydrofuran (THF) of 1:1: normal heptane is purified to crude product mixture.Collect and concentrate and contain after a while the cut of the product of wash-out (before this N-2 isomer, be then N-1 isomer).By concentrated dissolution of solid in minimum methylene dichloride (DCM) and pack the normal heptane that is encapsulated in 70:30 into: on 135 grams of silicagel columns in acetone.Use the normal heptane of 70:30: acetone carries out wash-out to silicagel column, the concentrated cut that contains pure products of being confirmed by thin-layer chromatography (TLC).

Pure products is dissolved in 9 grams of t-BME, is slowly added in 36 grams of normal heptanes, and carry out strong stirring at 10+/-10 ℃.The solid of precipitation is stirred at 5-10 ℃, filter, use n-normal heptane clean and use nitrogen to be dried up on funnel.0.006 gram of butylated hydroxytoluene (BHT) is added in solid.Make dissolution of solid in 20 grams of acetone, by strainer concentrated.With acetone, resistates is processed to (each 20 grams) 2 times, be all concentrated into dry at every turn.Product is carried out to vacuum-drying at least 18 hours being no more than at 50 ℃, obtain 2.5 grams of zotarolimus.

embodiment 1B

The solution in 0.3 milliliter of isopropyl acetate continues to process to embodiment 1A to use (87ul, 0.5 mmole) diisopropylethylamine and (35 milligrams, 0.5 mmole) 1H-TETRAZOLE, stirs afterwards 18 hours.The layering between 10 ml waters and 10 milliliters of ether of this mixture.By 10 mL of saline, organism is washed and is dried (Na ₂sO ₄).Concentrated organism, produced yellow viscous solid, on 3.5 grams, 70-230 object silicagel column, this yellow viscous solid is carried out to chromatography purification, with 10 ml n-hexanes, normal hexane: ether (4:1 (10 milliliters), 3:1 (10 milliliters), 2:1 (10 milliliters), 1:1 (10 milliliters)), 30 milliliters of ether, normal hexane: acetone (1:1 (30 milliliters)) wash-out.One of them isomer is collected (MS (ESI) m/e 966 (M) in ether cut ^-; The 42-of corresponding diagram 1 Chinese style 3 (2-tetrazyl)-rapamycin (isomer that polarity is less)).

embodiment 1C

In embodiment 1C, adopt normal hexane, acetone (1:1) moving phase to collect slow moving belt from chromatography column, obtain appointed compound (MS (ESI) m/e966 (M) ^-; The 42-of corresponding diagram 1 Chinese style 2 (1-tetrazyl)-rapamycin (isomer that polarity is larger)).

embodiment 2

By the immunosuppressive activity thing of the forms of rapamycin analogs obtaining from embodiment 1B and embodiment 1C and rapamycin and two forms of rapamycin analogs: 40-epi-N-[2 '-pyridone]-rapamycin and 40-epi-N-[4 '-pyridone]-rapamycin is (all at U.S. Patent number 5, open in 527,907) compare.Use Kino, T etc. are at Transplantation Proceedings (transplanting program) the 19 volume (5 joint) 36-39 page, and mankind's mixed lymphocyte reacion (MLR) analysis that the 6th supplementary issue (1987) is described is carried out activity and determined.Analytical results has been proved the compounds of this invention and under nanomolar concentration, has been effective immunomodulator, as shown in table 1.

Table 1

Example	Mankind's mixed lymphocyte reacion IC 50±S.EM.(nM)
		Rapamycin	0.91±0.36
2-pyridone	12.39±5.3
		4-pyridone	0.43±0.20
Embodiment 1B	1.70±0.48
		Embodiment 1C	0.66±0.19

The pharmacological kinetics feature of the forms of rapamycin analogs of embodiment 1B and embodiment 1C is determined after cynomolgus monkey being used to the intravenous injection dosage of single 2.5 mg/kg (every group of n=3).20% ethanol of each compound in water medium: 30% propylene glycol: 2% polyoxyethylenated castor oil: be prepared into 2.5 mg/ml solution in 48% glucose 5%.The intravenous injection dosage of 1 ml/kg is slowly injected to (~ 1-2 minute) to the saphena of monkey.6 minutes (only the 4th (positions)) before administration and afterwards, 15 minutes, half an hour, 1 hour, 1.5 hours, 2 hours, 4 hours, 6 hours, 9 hours, 12 hours, 24 hours and 30 hours the femoral artery from each animal or vein obtain blood sample.The sample that ethylenediamine tetraacetic acid (EDTA) is preserved mixes thoroughly and extracts, for subsequent analysis.

One equal portions blood (1.0mL) with comprise interior target 20% methanol/water (0.5mL) and carry out haemolysis.Mixture (1:1 (volume/volume (v/v)), 6.0mL) extraction of ethyl acetate and hexane for haemolysis sample.At room temperature use nitrogen gas stream that organic horizon is evaporated to dry.At methyl alcohol: reconstitute sample in water (1:1,150 μ L).The RPLC (instrument) by use with UV detection (HPLC) is opened title compound (50 μ L injection) with separated from contaminants.In operational process, sample should keep cooling (4 ℃).All samples of each research should be analyzed on HPLC as single batch.

By using Sciex MacQuan ^tMsoftware is determined forms of rapamycin analogs and interior target area under curve (AUC) measurement of embodiment 1B, embodiment 1C.Calibration curve comes from the linear peak area ratio that adds spirituous blood standard (parent drug/interior mark) returning of least square using with respect to the ratio of theoretical concentration.These methods are estimating that for two kinds of mixtures it is all linear quantizing the standard curve range (dependency >0.99) that limit is 0.1ng/mL.Maximum haemoconcentration (C _mAX) and reach the time (T of maximum haemoconcentration _mAX) can directly from the haemoconcentration-time data of observation, read.Haemoconcentration data are by being used CSTRIP to carry out multi index option fitting of a curve to obtain the appraisal of pharmacokinetic parameter.By using NONLIN84 further to determine the parameter of estimating.Adopt the linear trapezoidal rule of blood-time data chart to calculate the area (AUC from 0 to t hour (finally can test the time point of haemoconcentration) haemoconcentration-time curve after taking medicine _0-t).Residual area is presumed to infinity, according to the haemoconcentration (C of final measurement _t) divided by terminal elimination rate constant (β) and add AUC _0-tto produce total area under curve (AUC _0-t) determine.

As shown in Figure 2 and Table 1, when comparing with rapamycin, the forms of rapamycin analogs of embodiment 1B and embodiment 1C has shorter terminal and eliminates transformation period (t _1/2).Therefore, only have mixture of the present invention when enough effect (table 1) is provided, to provide the shorter terminal transformation period (table 2).

Table 2

embodiment 3

The object of the present embodiment is to determine the impact of forms of rapamycin analogs on Neointimal formation in the porcine coronary that comprises support.The present embodiment explanation, when the crown support of the BiodiviYsioPC from bio-compatible mixes and transmits, forms of rapamycin analogs A-179578 (that is, ABT-578; Corresponding with the formula 2 of Fig. 1) neointimal hyperplasia in porcine coronary and inner chamber size are had to Beneficial Effect.This discovery explanation, if be suitably applied to the mankind by restriction neointimal hyperplasia, this type of is in conjunction with having great clinical benefit.

The research and design of stating in the present embodiment reduces the ability of the crown support model of pig neointimal hyperplasia for evaluating forms of rapamycin analogs A-179578.The effect of A-179578 in this model will show the clinical potentiality of the crown restenosis in its restriction after skin revascularization with in processing support.In research, used house to raise pigs, because this model seems to obtain the result with other investigation that is intended to seek neointimal hyperplasia in human body to limit with comparability.

The present embodiment is tested from being placed on the A-179578 of the crown support wash-out in immature farm pig, and these results are compared with crt bracket.Crt bracket is polymer-coated, but there is no medicine.This is important, because polymer itself can not stimulate neointimal hyperplasia to sizable degree.When wash-out medicine disappears, polymeric inflammatory response can cause " the catching up with phenomenon " in late period, and in this phenomenon, restenosis process does not stop, but has slowed down speed.This phenomenon is by the restenosis causing in tested human body in late period.

Support is implanted in two blood vessels of each pig.The pig of using in this model normally 2 to 4 months large, weigh 30 to 40kg.Two crown supports pass through " conventionally " support at visual evaluation 1.1-1.2: artery is than implanting in each pig.

In program, start the same day, give the oral acetylsalicylic acid (325mg every day) of pig and continue remaining process, thereupon.By intramuscular injection then intravenous injection pig is carried out to the injection of ketamine (30mg/kg) and xylazine (3mg/kg), thereby realize general anesthesia.During introducing, other medicines treatments comprises the coromegine (1mg) of intramuscular injection and penicillin (flocillin) (1g).In support implant procedure, 10, the intra-arterial pill of the heparin of 000Ge unit is taken.

By the cutting on RECA and placement 8F sheath shape (coating)/crust, can obtain artery and enter.After this program, maintain the normal diet of animal, there is no cholesterol or other special supplementing.

Adopt BiodivYsio support, nominal blood vessel target size is 3.0mm.Two coronary artery random arrangements of each pig carry out support configuration.This support is bracket for eluting medicament (polymer adds drug stent), or only scribbles polymeric support (only having polymeric support).Utilize standard catheter and wire to carry these supports.Be less than in time of 30 seconds, making support airbag inflation to suitable size.

Each pig is furnished with only have polymeric support and a polymer being placed in different coronary arteries and adds drug stent, and accordingly, each pig will have a support for medicine, and a support is for contrast.Select altogether 20 pigs as sample, the expectation difference of the neointima thickness 0.2mm that when power for detection of 0.95 and beta0.02, standard deviation is 0.15mm.

After 28 days, make animal euthanasia to carry out histopathological examination and quantification.After perfusion pumped vacuum systems removes heart, left auricle of heart is removed to enter proximal coronal artery.Prejudicial coronary artery segment is dissected in the epicardial situation of disengaging.Part with damage is separated, and therefore allowing has enough tissues to comprise irrelevant blood vessel at two ends.Aforementioned each several part, the length of each is approximately 2.5cm, utilizes standard plastic embedded technology to embed and processes.These tissues are processed and dye by hematoxylin-eosin dyestuff and elasticity van Gieson technology subsequently.

Use low power and high power optical microscope, by the wire cross through calibration and the digital microscope system that is connected to the computer that uses calibration analyte software, carry out the linear measure in microcosmic plane.

The severity of vascular lesion and neointima reaction are measured by calibrated digital microscope.Those skilled in the art are familiar with the importance that inner elastomeric thin layer is complete.Through confirming, histopathology injury severity score and neointima thickness in the blood vessel that support is put are closely related.This severity is relevant to the degree of depth of damage, as follows: 0 represents that inner elastomeric thin layer is intact; Endothelial layer is denuded, and medium is compressed, but is not torn; 1 represents the inner elastic thin layer of tearing; Medium is obviously compressed, but is not torn; 2 represent what inner elastomeric was torn; Medium is obviously torn; Outside elastic thin layer is intact, but compressed; And 3 represent that outside elastic thin layer is torn; Typically, a large amount of breaches of medium are through outside elastic thin layer; Coil remains in adventitia sometimes.

All support wire of each holder part have been evaluated to the measures of quantization of damage.Also adopt calibrated digital picture to measure the neointima thickness of each support wire position.Region in cavity region, the region that comprises inner elastomeric thin layer and outside elastic thin layer is also measured.The neointima thickness of measuring each pillar in a given part, then averages, with the neointima thickness of determining that this is a part of.Use middle bracket is partly measured, analysis and comparison.Record the data (being included in the data division of this part of report) of near-end and distal portions simultaneously.The data analysing method of this research does not need to consider the variable arterial injury through processings/control group, because slight and moderate lesion has been enough to detection and has treated difference.Carried out paired t test relatively to pass and to only have polymeric support (control group) and polymer to add the variable of drug stent (treatment group).Before predetermined point of time, in this research, there is no animal dead.

Table 3 has shown pig and the artery using.In table 3, LCX means the circumflex branch of arteria coroaria sinistra, and LAD means left anterior descending coronary artery, and RCA means arteria coronaria dextra.

Table 3

	Object	The artery using
			1	2000-G-693	RCA-contrast
		LCX-test
			2	2000-G-698	RCA-test
		LAD-contrast
			3	2000-G-702	RCA-test
		LAD-contrast
			4	2000-G-709	RCA-contrast
		LAD-test
			5	2000-G-306	RCA-contrast
		LAD-test
					*LCX-test
6	2000-G-672	RCA-test
					LAD-contrast
7	2000-G-712	RCA-contrast
					LCX-test
8	2000-G-735	RCA-contrast
					LAD-test
9	2000-G-736	RCA-contrast
					LCX-test
10	2000-G-740	RCA-test
					LAD-contrast
11	2000-G-742	LAD-test
					OM (LCX)-contrast
12	2000-G-744	RCA-test
					LAD-contrast
13	2000-G-748	RCA-test
					LAD-contrast

14	2000-G-749	RCA-contrast
					LCX-test
15	2000-G-753	RCA-contrast
					LAD-test
16	2000-G-754	RCA-test
					LCX-contrast
17	2000-G-755	RCA-contrast
					LAD-test
18	2000-G-756	RCA-test
					LAD-contrast
19	2000-G-757	LAD-contrast
					LCX-test
20	2000-G-760	LAD-test
					LCX-contrast

Table 4 has shown the summarized results of the average damage of each support and all data of neointima thickness, comprises near-end, middle-end and distal portions.

Table 4 has also shown according to inner cavity size, constriction per-cent and the artery size of inner elastomeric thin layer (IEL) and outside elastic thin layer (EEL) measurement.

Table 4

Gather: all measurements (far-end, middle-end, near-end)

By statistics, neointima area or the thickness through test group (polymer adds drug stent) or the interior near-end of control group (only having polymeric support), middle-end or distal portions does not have obvious difference.This observations and previous research are in full accord, and allow only to use middle-end part with statistical testing installation (polymer adds drug stent) with contrast equipment (only having polymeric support).

Table 5 has shown that the statistics t of test group and control group tests relatively.By statistics, neointima thickness, neointima area, inner chamber size, and there is notable difference in the narrow per-cent of inner chamber, drug releasing stent is obviously favourable.On the contrary, by statistics, between test group (polymer adds drug stent) and control group (only having polymeric support), on average damaging severity, outside elastic thin layer or inner elastomeric thin layer area does not have obvious difference.

The statistics contrast of table 5 test and control parameters: data of middle part

Support being put to the reference artery of segment proximal and far-end observes and quantizes.These blood vessels are acted normally in all cases, in control group (only having polymeric support) and test group (polymer adds drug stent), are intac.The data of table 6 show, the size between the support in statistics in control group and the support of test group does not have obvious difference.

Table 6

Data show, have obvious difference in statistics, and these difference are supported the support of wash-out A-179578.Support of the present invention produces lower neointima area, lower neointima thickness and larger inner chamber area.With regard to neointima or impairment parameter, in test group (polymer adds drug stent) and control group (only having polymeric support), there is no obvious difference.Compare with test group, the artery size (comprising support) of control group does not have obvious difference.These results of study below show there is no marked difference in comprising the polymer coated Arterial Remodeling characteristic of medicine.

At polymer, add drug stent and only have and on polymeric support, all found slight inflammation at most.This discovery shows, even, in the situation that not having medicine to pack into, polymer has also presented gratifying biocompatibility.Other studies show that, when medicine departs from polymer completely, polymer itself causes sufficient inflammation so that neointima produces.This phenomenon may be the reason of " catching up with " phenomenon late period of appearance restenosis in clinical late period.Because the polymer in the present embodiment can not cause inflammation coronarius, after drug depletion, the later stage problem relevant to polymer is improbable.

Conclusion is, one comprises the support with polymer and A-179578 and shows, when its implanted coronary artery, the neointimal hyperplasia in pig model reduces.

embodiment 4

The object of the present embodiment is the speed of determining that A-179578 (ABT-578) medicine discharges from scribbling the polymeric 316L electropolishing stainless steel coupon of the biocompatible that comprises phosphorylcholine side group.

From bottle, remove the diaphragm of rubber of HPLC bottle cover, then put into vial, " tetrafluoroethylene " faced up.These barrier films serve as the support of sample.Test sample is the polymeric 316L stainless steel coupon that had previously scribbled the biocompatible that comprises phosphorylcholine side group (PC polymer).Crown support is made by 316L stainless steel conventionally, and can coat PC polymer and provide storage location as drug loading.To be placed on barrier film for simulating the coating sample that has of support.By using glass Hamilton syringe, the solution of A-179578 and ethanol (10 μ l) is applied to each specimen surface.This solution comprises the A-179578 (30.6mg) in the ethanol (3.0ml) that is dissolved in 100%.At each application interval, with ethanol, clean syringe.The lid of vial is placed on bottle by loosely, thereby guarantees suitable ventilation.Allow samples dried at least 1.5 hours.Pack by this way 12 samples into, 6 for determining the average quantity be loaded in the medicine on equipment, and 6 discharge the required time for measuring by medicine slave unit.

In order to determine the total amount that is loaded in an ABT-578 on sample, a sample is removed from bottle, then put it in 50/50 acetonitrile/0.01M phosphate buffered saline buffer (pH6.0,5.0ml).This sample is placed on 5210Branson sonicator and reaches one hour.Then this sample is removed from solution, and analyzed this solution by HPLC.

By submergence and in the fresh aliquot (10.0ml) of the 0.01M phosphate buffered saline buffer that is 6.0 from pH of following each timed interval, remove the individual sample releasing research of prescribing a time limit: 5,15,30 and 60 minutes.For some remaining time of 120,180,240,300,360 minutes, adopt 5.0ml damping fluid.In order to promote to mix in the drug release stage, sample is placed on the Eberbach vibrator that is set in lower velocity.After the test of last sample completes, by HPLC, all solution aliquots are analyzed.

The serial instrument of Hewlett-Packard 1100 that use has following setting carries out HPLC analysis: injection volume is 100 μ l; Acquisition time is 40 minutes; Flow rate is 1.0ml/min; Column temperature is 40 ℃; Wavelength is 278nm; Moving phase is 65% acetonitrile/35%H ₂o; And cylinder is YMC ODS-AS5 μ m, 4.6 * 250mm (parts number: A12052546WT).

The result of above-mentioned experiment has shown the release data that show in table 7.

Table 7

Time (minute)	Discharge per-cent	Standard deviation
			0.00	0.00	0.00
5.00	1.87	1.12
			15.00	2.97	1.47
30.00	3.24	1.28
			60.00	3.29	1.29
120.00	3.92	1.28

180.00	4.36	1.33
			240.00	4.37	1.35
300.00	6.34	2.07
			360.00	7.88	1.01

embodiment 5

The present embodiment is to load and release ABT-578 in order to determine from the BiodivYsio drug conveying support of 15mm.In order to give support drug loading, prepare concentration in ethanol, be the ABT-578 solution of 50mg/ml and be allocated in 12 vials.12 coated independent racks of polymer are positioned over and are designed in the stationary installation that keeps support vertical, and vertically immerse five minutes in liquid.From vial, remove support and stationary installation, and contact with support and sop up unnecessary liquid by absorption agent.Subsequently support reversing is vertically placed in to air air-dry 30 minutes.

Support is removed from stationary installation, every is placed on in acetonitrile/phosphate buffered saline buffer of 50/50, (pH value is 5.1,2.0ml) and to it carries out the ultrasonic wave of hour.From solution, remove support solution is carried out to drug level chemical examination, so just can calculate the original medication amount on support.Present method is shown separately, can remove from bracket coating at least 95% medicine.On an average, these supports can hold 60 microgram medicines, and tolerance is ± 20 micrograms.

Being placed in stationary installation and putting into the 0.01M phosphate buffered saline buffer of each vial (pH value is 6.0,1.9ml) drug loading.These samples are placed on the Eberhard vibrator of low speed, so that front and back vibration to be back and forth provided.Medicine in damping fluid is saturated, should on following point, regularly support be transferred in fresh buffer bottle: 15,30,45,60,120,135,150,165,180,240,390 minutes.At drug release cycle studies end, (high performance liquid chromatography carries out drug level chemical examination to dissolving damping fluid bottle to use HPLC.Data are expressed as the function of time per-cent that medicine accumulative total discharges, as shown in the table:

Table 8

Time (minute)	The per-cent that medicine accumulative total discharges
		15	0.3
30	1.1
		45	2.1
60	3.2
		120	4.3
135	5.9
		150	6.3
165	6.8
		180	7.4
240	10.8
		390	13.2

embodiment 6

The present embodiment is in order to assess different pharmaceutical dosage to the security of Neointimal formation and effectiveness.By BiodivYsio OC support (15mm) delivering medicament of coated ABT-578.Neointimal formation in the intracoronary stent of Ying Yisi the adult miniature pig of time interval measurement: 3 days, 1 month and 3 months.In per timed interval, study 40 (40) animals (every dosage needs 10 animals).The support of a coating medicine of every animals received and a crt bracket.This crt bracket is not containing any medicine.Table 9 has shown the dosage that carries out pig efficacy study.

Table 9

By checking stent area, adjacent crown section, all pathological changes of organizing and promoting myocardium tissue of blood vessel, evaluation is in the potential local organization toxicity in all timed intervals.Studied that mortality ratio, angiography are transplanted and the histopathology of data, tissue morphology data and backing positions again.

three days groups

Provide to the histopathology of scanning electronic microscope combination the short term response information that implant frame is relevant.In control group and all dosage groups, these responses are all similar, and the middle mould that these responses relate to without obvious necrocytosis contracts, mainly concentrates on the thrombus of stent strut and the early stage evidence that inflammatory cell is accumulated and endothelium is fully recovered and thin mural thrombus piece smooth muscle cell is invaded.Without any large-area thrombi or obvious intramural hemorrhage.The adventitia of some samples shows focus or disperses inflammatory infiltration, or occur that vasa vasorum stops up or extravasated blood.In any sample, there is no medial necrosis sign.

Implant coronary stent after three days, scanning electronic microscope shows: the surface of internal cavity in all dosage groups is all similar.Support shape is obviously imbedded in the thin layer of tissue.The endothelial layer of even crossing pillar between pillar is not under any damage; The fusion of endotheliocyte or almost fused layer are coated surface of internal cavity.Intact remaining endotheliocyte in support and pillar inside has the miniature thrombi of adhesivity thrombocyte, thrombocyte and the white cell of dispersion to cross pipe.There being more serious support to cause the intra-arterial of blood vessel injury, have more substantial tube wall thrombi, but the endothelium recovery from illness degree of stent strut seems not delay, no matter the dosage of ABT-578 is how many.

a month group:

The tissue morphology data presentation of a month series the obvious retarding effect of Neointimal formation in the porcine coronary of ABT-578 docking upper bracket of local elution.Compare with control group, in dosage group 3 and 4 (10 and 27ug/mm), the Intimal area that normality turns to wound marking scales significantly reduces; , compare with control group, also there is minimizing trend in dosage group 3 and 4 absolute Intimal area and inner film thickness meanwhile, the trend that dosage group 3 exists the narrow per-cent of weave construction to reduce.

Crt bracket shows the representative configuration structure of the implantation Yucatan miniature pig support coronarius of month.Compressed or the attenuation of middle film, under stent strut section, top layer is without necrosis; Between only existing or inflammatory infiltration; New intima size range is from relative thinness to medium thinness; Fusiformis and stellate cell in a large amount of extracellular matrixs form, and around stent strut section, only have few small-sized scleroproein material focus.The support of coating medicine demonstrates similar middle mould contracting, and any dosage does not have any substantial necrosis; As comparison device, almost NIP occurs.In dosage group 3 and 4, the obvious attenuation of inner membrance, only comprises several confluent monolayer cells sometimes.In all dosage groups, can in considerable sample deep layer new intima structure, observe median size fibrin deposition and concentrated thrombi.These are conventionally associated with stent strut, but sometimes also can between strut section, extend.Yet, because settling is encapsulated in fiber and cell tissue, and coated by the smooth inner chamber endothelium shape cell of one deck, have in no instance thrombus and be exposed on the face of chamber.

Scanning electronic microscope confirms: one deck amalgamation endotheliocyte or endothelium shape cell be coated whole standoff surface, simultaneously aspect blood ingredient adhesive power, the support that medicine is coated and contrast between between without any difference; In all groups, white blood cell count is roughly the same.These find that proof, when the inner membrance of ABT-578 and minimizing and lasting tube wall thrombi are associated, can, after implant frame in one month, fully cure the vessel wall that support damages.Vessel wall recovery from illness make chamber face aspect platelet adhesion reaction and thrombosis without any activity, at white cell, adhere to activity and minimize.In addition, because adherent bad without any middle film necrosis or support, even in maximum dose level (27ug/mm), also without any the sign of vessel wall toxicity.

three months groups:

During trimestral research, between each dosage group, connect the coronary artery Size dependence tissue morphology measuring parameter of support without any notable difference.Yet two initializaing variables, cross-sectional area and the narrow per-cent of official jargon area of describing Neointimal formation only have minimum minimizing trend.

The Histopathologic appearance of the crt bracket after implant frame in trimestral porcine coronary sample is similar in appearance to the crt bracket of a month group, simultaneously similar in appearance to other all groups during three months.All samples show: at new intima fibrocyte Neointimal formation, having majority is the unstriated muscle shape cell of fusiformis and one deck amalgamation inner chamber cellular layer.In new intima without any intramural hemorrhage and lasting fibrin deposition; Yet, there is previous thrombus accumulation and neovascularization tissue subsequently in new intima in the sample that some samples, especially new intima are thicker.Sometimes sample demonstration concentrates on the Inflammatory response from moderate to severity in stent strut, and it is relevant with the breaking-up of middle membrane structure.As a rule, these Inflammatory responses are also relevant with thicker new intima.Yet these Inflammatory responses seldom, all occur or find these Inflammatory responses in amount in control group and medicine covered stent group.According to inferring, these Inflammatory responses represent the specific general reaction of animal to implant frame, and support pollutes, or represents the combination of these two kinds of factors, and porcine coronary is being implanted in the research of support, conventionally has the Inflammatory response Probability of 10-15%.In any sample, there is no middle film necrosis or the media sign separated with support.The adventitia neovascularization of three months group implant frames of great majority seems all than one month, to organize the slightly high of implant frame, but crt bracket group or test bracket group do not have this phenomenon.In scanning electronic microscope proof control group and all dosage groups, there is the amalgamation endothelial layer that only has few adhesivity blood cell.

The support of coated ABT-578 reduces the Neointimal formation in porcine coronary inner tube, and obviously proves the bio-pharmaceutical curative effect (undecomposed thrombi/new intima fibrin deposition) in month.The support of coated ABT-578 slightly shows lasting retarding effect in the long period interval of three months groups.In any timed interval of testing, about any dosage group, comprise the maximal dose that is about 27ug/mm, there is not any media necrosis or adherent bad local coronary arterial wall toxicity of support of showing as.All supports all engage among tissue rightly, in a month interval and three months intervals, have the stable healing reaction evidence that shows as the merging of fibrocyte new intima and endothelium coating.Support insert in animal body after trimestral lasting retarding effect trend astonishing, prove the potential lasting effect that has the restenosis that prevents clinically the support generation because implanting.

embodiment 7

By making analogue crystalline crystalline forms of rapamycin analogs in biphase mixture.Briefly, ABT-578 is added in the bottle that contains 0.23g acetone and 0.82g normal heptane, and at 0 ℃, it is incubated, to make liquid phase saturated.By mixture insulation until as ABT-578, be dissolved in acetone soln always, ABT-578-acetone will be mostly in lower phase and normal heptane occurs liquid-liquid phase separation in upper phase time.At 0 ℃, by biphase mixture insulation 10 days, can observe crystalline forms of rapamycin analogs in bottle bottom like this.X-ray diffractogram of powder (PXRD) as shown in Figure 2 A.

To the crystal information analysis of being correlated with of acetone solvent compound, as shown in table 10.Determined the solvate molecular separation ABT-578 molecule along C-axle.Solvate molecule is quite unordered, but it looks like every ABT-578 and has four acetone and two water moleculess.Between ABT-578 molecule, by Van der Waals, interact and interact along a-axle and b-axle.

Table 10: acetone solvent compound crystal information

embodiment 8

By following steps, generate ABT-578 toluene solvate crystal.By 100mg amorphous ABT-578 is dissolved in 300mg toluene and prepares clear solution.At 22 ℃, stirred solution 15 hours, so just can observe the dense thick slurries of crystalline solid.Use the solid of above-mentioned preparation to prepare toluene solvate crystal as crystal seed, its x-ray diffractogram of powder as shown in Figure 4 A.

embodiment 9

By at 22 ℃, with the saturated acetonitrile of amorphous ABT-578, prepare the acetonitrile solvent compound crystal of ABT-578 desolventizing, then at 0 ℃, by saturated solution insulation 2 hours.Crystal powder X-ray diffractogram as shown in Figure 5A.Can make subsequently crystal become dry and form acetonitrile desolventizing thing, desolventizing thermogravimetric analysis data as shown in Figure 6 C.

embodiment 10

By at 0 ℃, make the wet cake of acetonitrile solvent compound in ethyl formate, become slurries and prepare ABT-578 ethyl formate solvate crystals.This crystal powder X-ray diffractogram and thermogravimetric analysis are respectively as shown in Fig. 7 A and 7C.

embodiment 11

By at 0 ℃, make acetonitrile solvent compound wet cake in isopropyl acetate, become slurries and prepare ABT-578 isopropyl acetate solvate crystals.

embodiment 12

By 380mg amorphous ABT-578 is added into bottle, and then add 870mg isobutyl acetate to make it dissolve preparation ABT-578 crystal.0 ℃ of insulation 16 hours, so obtain slurry.Crystal powder X-ray diffractogram and thermogravimetric analysis are respectively as shown in Fig. 9 A and 9C.

embodiment 13

By the noncrystal ABT-578 of 417mg is added into bottle, and then add the 315mg ethanol of 200proof to make it dissolve preparation ABT-578 alcohol solvent compound crystal.After 15 hours, use the acetonitrile solvent compound of desolventizing to be planted, then 0 ℃ of insulation 16 hours, so obtain slurry.

embodiment 14

By at 0 ℃, make acetonitrile solvent compound wet cake in dimethyl formamide, become slurries and prepare ABT-578N, N solvent dimethylformamide compound crystal.Crystal powder X-ray diffractogram and thermogravimetric analysis are respectively as shown in Figure 10 A and 10B.

embodiment 15

By at 0 ℃, make acetonitrile solvent compound wet cake in methyl-phenoxide, become slurries and prepare ABT-578 methyl-phenoxide solvate crystals.Crystal powder X-ray diffractogram and thermogravimetric analysis are respectively as shown in Figure 11 A and 11C.

embodiment 16

By at room temperature, the amorphous rapamycin analogue of about 120mg is dissolved in 200uL acetone, preparation shows as the crystal forms of rapamycin analogs of acetone solvent compound, and at 5 ℃, generated solution is incubated to 14 hours, or until in slurry, observes crystalline solid.Use powder x-ray diffraction analysis crystal, as shown in Figure 2 B.At room temperature balanced crystal, subsequently, further carries out vacuum-drying (mercury is about 3 inches) to it at 30 ℃.Then use the dried crystal of powder x-ray diffraction analysis, as shown in Figure 3 B.

embodiment 17

By at 45 ℃, the amorphous rapamycin analogue of about 220mg is dissolved in about 400uL toluene to prepare solution, preparation shows as the crystal forms of rapamycin analogs of toluene solvate.At 5 ℃, prepared solution is incubated to 1 hour, or until observes crystalline solid.The diffractogram of toluene solvate as shown in Figure 4 B.The diffractogram of the toluene solvate of desolventizing as shown in Figure 4 E.Can pass through at room temperature balanced solvate crystals, at 30 ℃, further it be carried out to the crystal that desolventizing is obtained in vacuum-drying (mercury is about 3 inches) subsequently.

In heat-processed, toluene loss can be divided into 3 stages descriptions.The toluene loss of first stage is in temperature lower than 90 ℃ in the situation that.Subordinate phase temperature between 90 ℃ to 130 ℃, and the phase III after melting, be greater than 150 ℃.Therefore, by the dry crystal desolventizing toluene solvate obtaining, be, part desolventizing product.Measured the x-ray crystal structure of toluene solvate.Crystal information is as shown in table 11.

ABT-578 toluene solvate is at P ₂₁crystallization in chirality spacer, per unit structure cell is containing two ABT-578 molecules.As shown in Figure 4 C, it is by using molybdenum-K alpha-particle (0.070930) radiation to obtain to the x-ray crystal structure of ABT-578 toluene solvate.From structure, can find out, in each asymmetric unit of crystal, contain three toluene molecules and a Zotarolimus molecule, so toluene solvate be trimethylbenzene solvate.At these three toluene molecule (T _a, T _band T _c) in, T _aand T _bshort some contact ABT-578 molecule (is C-H ... π and C=O ... H-C=C interacts).T _cvia faint van der waals force and interaction of molecules around.What is interesting is, in toluene solvate crystalline structure, along b axle, have solvent channel, as shown in Figure 4 D.Toluene molecule T _aand T _bmore easily be exposed in passage, expection is easily gone it from crystal.On the other hand, toluene molecule T _bsemitight is in the chamber being surrounded by ABT-578 molecule.Therefore, for these three different toluene molecules, T _bmolecular binding is compact, T _amolecule appropriateness is solidifying tight, and T _cmolecule loose binding in ABT-578 molecule.This demonstrates interim toluene loss when being dried/heating with regard to soluble toluene solvate, and from crystal, removes the very difficult fact of toluene completely.Its simultaneously soluble when dry crystal lattice along a axle, shrink, can change and prove by the x-ray diffractogram of powder of desolventizing toluene solvate, as shown in Fig. 4 F.

embodiment 18

By at 45 ℃, the amorphous rapamycin analogue of about 100mg is dissolved in about 200uL acetonitrile, preparation shows as the crystal forms of rapamycin analogs of acetonitrile solvent compound.At-12 ℃, generated solution is incubated to approximately 30 hours, then uses micro-toluene solvate crystal to plant it.After-12 ℃ of further insulations, will generate crystalline solid.Then available X-ray diffraction analysis crystal, as shown in Figure 5 B.At room temperature balanced crystal, further carries out vacuum-drying (mercury is about 3 inches) to it at 30 ℃ subsequently.The dried crystal of available X-ray diffraction analysis, as shown in Figure 6B.

embodiment 19

By at 45 ℃, the amorphous rapamycin analogue of about 100mg is dissolved in about 200uL ethyl formate, preparation shows as the crystal forms of rapamycin analogs of ethyl formate solvate, and at approximately 5 ℃, generated solution is incubated to approximately 14 hours, or until generates crystal.The diffractogram of ethyl formate solvate as shown in Figure 7 B.The diffractogram of the ethyl formate solvate of desolventizing as shown in Figure 8.Can pass through at room temperature balanced solvate crystals, at 30 ℃, further it be carried out to the crystal that desolventizing is obtained in vacuum-drying (mercury is about 3 inches) subsequently.

embodiment 20

By at room temperature, the amorphous rapamycin analogue of about 100mg is dissolved in about 200uL isopropyl acetate, preparation shows as the crystal forms of rapamycin analogs of isopropyl acetate solvate.At 5 ℃, generated solution is incubated to 14 hours, or until observes crystalline solid.The diffractogram of isopropyl acetate solvate as shown in Figure 17 A.Can pass through at room temperature balanced solvate crystals, at 30 ℃, further it be carried out to the crystal that desolventizing is obtained in vacuum-drying (mercury is about 3 inches) subsequently.The X-ray diffractogram of the solvate of desolventizing is as shown in Figure 17 B.

embodiment 21

By at room temperature, 400mg amorphous ABT-578 is added into bottle, and then adds about 870mg isobutyl acetate that it is dissolved in bottle, preparation shows as the crystal forms of rapamycin analogs of isobutyl acetate solvate.Then at approximately 0 ℃, generated solution is incubated to approximately 16 hours, or until obtains crystallization suspension.Use subsequently X-ray diffractogram to analyze crystal, as shown in Figure 9 B.

embodiment 22

By at 45 ℃, the amorphous rapamycin analogue of about 100mg is dissolved in 400uL ethanol (200proof), preparation shows as the crystal forms of rapamycin analogs of alcohol solvent compound, and at approximately 5 ℃, generated solution is incubated to 14 hours, or until generates crystal.The diffractogram of solvate is as shown in Figure 12 A.Can pass through at room temperature balanced solvate crystals, at 30 ℃, further it be carried out to the crystal that desolventizing is obtained in vacuum-drying (mercury is about 3 inches) subsequently.The X-ray diffractogram of the solvate of desolventizing as shown in Figure 12 B.

embodiment 23

The crystal of ABT-578 methanol solvate compound is by room temperature, 93mg amorphousness ABT-578 is dissolved in the methyl alcohol of 200uL, and before using micro-toluene solvate crystal to plant, stores 30 hours and be prepared from the temperature of-12 ℃.After planting ,-12 ℃ of further insulations, will form crystalline solid.Figure 13 A and 13B have shown respectively the x-ray diffractogram of powder of crystal solvent compound and desolventizing solvate.Desolventizing crystal is by room temperature making crystal solvent compound reach balance, after vacuum-drying at 30 ℃ subsequently (about 3 inches of mercurys), obtains.

embodiment 24

The crystal of ABT-578 ethyl acetate solvent compound is by room temperature, 103mg amorphousness ABT-578 is dissolved in the ethyl acetate of 200uL, and before use micro-toluene solvate crystal to plant, at the temperature of-12 ℃, store and be prepared from for 30 hours., plant after, at-12 ℃ further insulation, will form crystalline solid.Figure 14 A and 14B have shown respectively the x-ray diffractogram of powder of crystal solvent compound and desolventizing solvate.Desolventizing crystal is by room temperature making crystal solvent compound reach balance, obtains subsequently after 30 ℃ of vacuum-dryings (about 3 inches of mercurys).

embodiment 25

The crystal of ABT-578 Methyl isobutyl ketone solvate is by room temperature, 96mg amorphousness ABT-578 is dissolved in the Methyl isobutyl ketone of 200uL, and before use micro-toluene solvate crystal to plant, at the temperature of-12 ℃, store and be prepared from for 30 hours.After planting, at-12 ℃, further insulation, will form crystalline solid.Figure 15 A and 15B have shown respectively the x-ray diffractogram of powder of crystal solvent compound and desolventizing solvate.Desolventizing crystal is by room temperature making crystal solvent compound reach balance, obtains subsequently after 30 ℃ of vacuum-dryings (about 3 inches of mercurys).

embodiment 26

The crystal of ABT-578 Nitromethane 99Min. solvate is by room temperature, 100mg amorphousness ABT-578 is dissolved in the Nitromethane 99Min. of 200uL, and before use micro-toluene solvate crystal to plant, at the temperature of-12 ℃, store and be prepared from for 30 hours.After planting, at-12 ℃, further insulation, will form crystalline solid.The Nitromethane 99Min. solvate of ABT-578 is at room temperature easy to desolventizing and with the form of semi-crystalline phase, occurs in x-ray diffractogram of powder analysis (Figure 16).

embodiment 27

The crystal of ABT-578 propionitrile solvate is by 45 ℃, 108mg amorphousness ABT-578 is dissolved in the propionitrile of 200uL, and before using micro-toluene solvate crystal to plant, and stores to be prepared from for 30 hours at the temperature of-12 ℃.After planting ,-12 ℃ of further insulations, will form crystalline solid.Figure 18 A has shown the x-ray diffractogram of powder of crystal solvent compound, and the desolventizing of crystal has produced semi-crystalline phase.Desolventizing crystal is by room temperature making crystal solvent compound reach balance, obtains subsequently after 30 ℃ of vacuum-dryings (about 3 inches of mercurys).Figure 18 B has shown the x-ray diffractogram of powder of desolventizing solvate.

embodiment 28

The crystal of ABT-578 methyl ethyl ketone solvate is by room temperature, 94mg amorphousness ABT-578 is dissolved in fourth (methylethyl) ketone of 200uL, and before use micro-toluene solvate crystal to plant, at the temperature of-12 ℃, store and be prepared from for 30 hours.After planting ,-12 ℃ of further insulations, will form crystalline solid.Figure 19 A has shown the x-ray diffractogram of powder of crystal solvent compound, and the desolventizing of crystal has produced semi-crystalline phase.Desolventizing crystal is by room temperature making crystal solvent compound reach balance, obtains subsequently after 30 ℃ of vacuum-dryings (about 3 inches of mercurys).Figure 19 B has shown the x-ray diffractogram of powder of desolventizing solvate.

embodiment 29

The crystal of ABT-578 tetrahydrofuran solvent compound is by room temperature, 107mg amorphousness ABT-578 is dissolved in the tetrahydrofuran (THF) of 200uL, and before use micro-toluene solvate crystal to plant, at the temperature of-12 ℃, store and be prepared from for 30 hours.After planting ,-12 ℃ of further insulations, will form crystalline solid.Figure 20 A has shown the x-ray diffractogram of powder of crystal solvent compound, and the desolventizing of crystal has produced semi-crystalline phase.Desolventizing crystal is by room temperature making crystal solvent compound reach balance, obtains subsequently after 30 ℃ of vacuum-dryings (about 3 inches of mercurys).Figure 20 B has shown the x-ray diffractogram of powder of desolventizing solvate.

embodiment 30

ABT-5781, the crystal of 2-glycol dimethyl ether solvate is by room temperature, 110mg amorphousness ABT-578 is dissolved in to 1 of 200uL, in 2-glycol dimethyl ether solvent, and before use micro-toluene solvate crystal to plant, at the temperature of-12 ℃, store and be prepared from for 30 hours.After planting ,-12 ℃ of further insulations, will form crystalline solid.Figure 21 A has shown the x-ray diffractogram of powder of crystal solvent compound, and the desolventizing of crystal has produced semi-crystalline phase.Desolventizing crystal is by room temperature making crystal solvent compound reach balance, obtains subsequently after 30 ℃ of vacuum-dryings (about 3 inches of mercurys).Figure 21 B has shown the x-ray diffractogram of powder of desolventizing solvate.

Although at some embodiment or in practice, in the improvement of inferring, present invention is described, open, explanation and showing, but scope of the present invention is not intended to or should not be considered and be confined to this, and retain especially other this type of improvement or embodiment that the teaching in presents proposes, because it belongs in the scope that is attached to the claims in presents.In addition, all publications of the narration in presents are all incorporated herein by specifically quoting.

Claims (2)

1. a composition that comprises the crystalline form of the compound shown in formula 2,

Wherein, described crystalline form is the desolventizing thing of toluene;

Wherein said desolventizing thing has substantially X-ray diffractogram as shown in Figure 4 E.

2. a method of preparing the crystalline form of the compound shown in claim 1 Chinese style 2,

Described method comprises:

At 45 ℃, the amorphous rapamycin analogue of 220mg is dissolved in 400 μ L toluene to prepare solution;

At 5 ℃, prepared solution is incubated to 1 hour, or until observe crystalline solid;

At room temperature balanced solvate crystals, then further carries out vacuum-drying to prepare the desolventizing thing of the toluene of crystal to it at 30 ℃, obtains the crystal of desolventizing; Wherein said vacuum pressure is 3 inches of mercury; And

The desolventizing thing of wherein said toluene has substantially X-ray diffractogram as shown in Figure 4 E.