CN101117330A

CN101117330A - Tartrate of isofagomine and use thereof

Info

Publication number: CN101117330A
Application number: CNA2007101388103A
Authority: CN
Inventors: B·穆格拉格; K·A·谢特; D·帕林; P·J·雷布琴斯基
Original assignee: Amicus Therapeutics Inc
Current assignee: Amicus Therapeutics Inc
Priority date: 2006-05-24
Filing date: 2007-05-24
Publication date: 2008-02-06
Anticipated expiration: 2027-05-24
Also published as: CN101117330B; ES2364586T3

Abstract

A novel tartaric acid salt of Isafagomine (Isofagomine tartrate) that can be used for the treatment of Gaucher disease is provided. The invention also provides a crystalline form of isofagomine tartrate, method for preparing the salt, a pharmaceutical composition containing the salt, and a method of treating Gaucher disease.

Description

Tartrate of isofagomine and application method thereof

The application requires to enjoy the right of priority of following application: application number is 60/808020, the applying date is purpose U.S. Provisional Patent Application and application number is 60/890719, the applying date is the U.S. Provisional Patent Application on February 20th, 2007 Mays 24 in 2006, and more than application is incorporated herein by reference in full.

Background of invention

Gaucher disease (Gaucher disease) be with patient's cell in, the relevant lysosomal storage disease of glycosphingolipid (GSL) accumulation in monocyte and the scavenger cell especially.The accumulation of this unusual sphingoglycolipid results from coding lysosomal enzyme acid the beta-glucosidase enzyme ((glucocerebrosidase of acid β-glucosidase), GCase) hereditary defect in the gene (sudden change), sour beta-glucosidase enzyme are a kind of lysosomal hydrolases that can decompose GSL glucosylceramide (GluCer).Most of glucocerebrosidase gene (Gba) sudden change has caused GCase folding mistake in endoplasmic reticulum (ER).The GCase of this folding mistake is discerned by the endoplasmic reticulum quality control system, is degraded subsequently, rather than processed and be transported to lysosome.

Gaucher disease is that the total man plants disease, and total incidence is about 1/50,000-100,000 births.The sickness rate of specific crowd is than higher.For example, in Ashkenazim group, it is the carrier of Gba sudden change that 1/15th people is arranged approximately.According to the data of national Gaucher disease foundation, there are 2500 Americans to suffer from Gaucher disease approximately.

Gaucher disease is the autosomal recessive disease, also is modal lysosomal storage disease.According to the situation of involving of nerve and the severity of disease, this disease can be divided into 3 kinds of Clinical types.1 type is the most common, and feature is not involve nerve.The severity scope of patient's performance is very wide; Some people is asymptomatic throughout one's life.Great majority 1 type patient shows as spleen and liver increases, bone is unusual, bone damages and lasting inflammatory reaction.1 type Gaucher disease patient's liver glucose cerebroside ester level is higher than normal level 23-389 doubly.

2 type Gaucher diseases are the rarest and the most serious types.It is relevant with the early stage morbidity of acute nervous system disease.The characteristic feature of nervosa Gaucher disease is that level is watched attentively unusually.This kind of patient develops into and carries out encephalopathy (HIE) and extrapyramidal symptoms, for example tetanic and Parkinson's sample motion (parkinson's syndrome).Great majority 2 type Gaucher disease patients die from breathlessness or the mistake suction that nerve degeneration causes in early days in childhood.

3 type Gaucher diseases also involve nerve, and are just light than the degree of 2 types.These patients also have the splenauxe and skeletal defect symptom of the liver in 1 type, also have the central nervous system symptom simultaneously, comprise ataxia (ataxia), convulsive seizure, eye muscle paralysis, epilepsy and dementia.3 type Gaucher disease patients can live to and grow up, but probable life is shortened.Had report 3 type Gaucher diseases that 3 subclass are arranged: the 3a type, with the significant splenauxe and bone marrow disease of liver; The 3b type is with limited constitutional symptom (limited systemic symptoms); The 3c type,, corneal clouding splenauxe, the ataxia (progressive ataxia) of carrying out property and dementia, heart valve and aortic root calcification with liver.

The methods of treatment of Gaucher disease comprise enzyme replacement treatment (ERT), bone marrow transplantation (BMT), substrate reduce therapy (substrate reduction therapy) (SRT), gene therapy and pharmacology chaperone treatment (chaperone treatment).Isofagomine (isofagomine) is effective inhibitor of recombinant human acid beta-glucosidase enzyme (GCase).Improve the United States Patent (USP) 6 that the active pharmacology chaperone treatment of mutant enzyme is being owned together in the lysosomal storage disease by enzyme inhibitorss such as application examples such as isofagomines, 916,829,6,599,919,6,589,964,6,583,158 and 7, have in 141,582 openly, they are incorporated herein by reference in full.For example, show, the GCase inhibitor is joined to cause GCase transportation in the fibroblast cell cultures and lysosome is active improves, point out this class inhibitor having the treatment meaning aspect the Gaucher disease treating.

Recently have been found that between Gba transgenation and the Parkinson's disease relevant.In a research, find, one group suffer from rare, early onset thereof, to treatment have resistivity 17 patients of parkinson's syndrome have the allelotrope of at least one Gba missense mutation, comprise isozygotying and heterozygous individual of N370S, N370S is typically relevant with 1 type Gaucher disease of non-neuropathic sudden change (Tayebi etc., Mol.Genet.Metab.2003; 79; 104-109).In another research, 99 6 Gba sudden changes (N370S, L444P, 84GG, V394L and R496H) of suffering from the parkinsonian Ashkenazim group of the special property sent out have been studied.31 Parkinson's disease patients have the Gba allelotrope of 1 or 2 sudden change: wherein 23 people's N370S is a heterozygosis; 3 people's N370S isozygotys; 4 people's 84GG is a heterozygosis; 1 people's R496H is heterozygosis (people such as Aharon-Peretz, New Eng.J.Med.2004; 351:1972-77).The allelic incidence of mutant N370S is 5 times of incidence among 1573 normal subjectses, is among the normal subjects 21 times for the incidence of 84GG.In the parkinsonian, also the patient age than non-carrier is little to carry the patient of Gba sudden change.This studies show that the heterozygosis of Gba sudden change may make the Ashkenazim easily suffer from Parkinson's disease.Shown that isofagomine can pass the hemato encephalic barrier of animal and improve this two kinds of mutant wild-type GCase activity, therefore it can be used for treating the Parkinson patient with GCase heterozygous mutant, or treatment is owing to other factors has the patient who develops into parkinsonian risk but can benefit from the raising of wild-type GCase level.

Though the isofagomine compound is effectively and has optionally recombinant human acid beta-glucosidase enzyme (GCase) inhibitor, its application as medicine is faced with challenge.For example; the hydrochloride of isofagomine (isofagomine-HCl) at United States Patent (USP) 5; 844; 102 is open; yet; isofagomine-HCI and isofagomine free alkali are not easy by large scale purification, and they have bad solid property to the application of industrial scale production method and medicine preparation.

Summary of the invention

In one embodiment, the invention provides the tartrate compound of isofagomine, it has following chemical structure:

Wherein, n is 1 or 2.The present invention also provides highly purified and has been the tartrate of isofagomine of crystallized form.

In another embodiment, the invention provides the composition that comprises tartrate of isofagomine, be preferably at least 50%, preferred 90%, even more preferably 99%.The present invention also provides and has comprised at least 90% or the composition of above tartrate of isofagomine, and wherein the particle diameter of 90% tartrate of isofagomine is 1200 μ m.

In another embodiment, the invention provides the pharmaceutical composition that comprises tartrate of isofagomine and one or more pharmaceutically acceptable vehicle.

In another embodiment, the invention provides the method for preparing tartrate of isofagomine.The present invention also provides the method for preparing the high purity tartrate of isofagomine.

And, in another embodiment, the invention provides the method for the mammiferous Gaucher disease of treatment, thereby tartrate of isofagomine or its pharmaceutical composition of this method by giving medicine effective quantity improves that mammiferous GCase is active to be realized.

In another embodiment, the invention provides L-(+)-tartrate of isofagomine.The present invention also provides the mixture of tartrate and isofagomine.

In another embodiment, the invention provides the crystallized form of tartrate of isofagomine.Preferred this crystallized form has the X-ray powder diffraction pattern that comprises 5 or more a plurality of following peaks: (2 θ) 9.29 ± 0.009,14.17 ± 0.009,16.34 ± 0.009,18.07 ± 0.009,18.72 ± 0.009,19.44 ± 0.009,20.56 ± 0.009,22.13 ± 0.009,23.01 ± 0.009,24.54 ± 0.009 and 27.12 ± 0.009.More preferably X-thanks to line chart and comprises following peak: (2 θ) 9.29,14.17,16.34,18.07,18.72,19.44,20.56,22.13,23.01,24.54 and 27.12.Even more preferably this crystallized form has X-ray powder diffraction pattern same as shown in Figure 5 substantially.

The accompanying drawing summary

Fig. 1 has shown the positive ESI mass spectrum according to the tartrate of isofagomine of one embodiment of the invention preparation.

Fig. 2 has shown that the tartrate of isofagomine for preparing according to one embodiment of the invention is at D ₂Among the O ¹H NMR figure.

Fig. 3 has shown that the tartrate of isofagomine for preparing according to one embodiment of the invention is at D ₂Among the O ¹³C NMR figure.

Fig. 4 has shown that the tartrate of isofagomine for preparing according to one embodiment of the invention is at D ₂Spin echo among the O ¹³C NMR figure.

Fig. 5 has shown the X-ray powder diffraction pattern according to the tartrate of isofagomine of one embodiment of the invention preparation.

Fig. 6 has shown thermogravimetric analysis (TGA) figure according to the tartrate of isofagomine of one embodiment of the invention preparation.

Fig. 7 has shown the infared spectrum according to the tartrate of isofagomine of one embodiment of the invention preparation.

Fig. 8 A has shown according to isofagomine L-(+)-tartrate (2: 1) of one embodiment of the invention preparations ¹H NMR (D ₂O) figure.

Fig. 8 B has shown according to isofagomine D-(-)-tartrate (2: 1) of one embodiment of the invention preparations ¹H NMR (D ₂O) figure.

Fig. 8 C has shown according to isofagomine D-(-)-tartrate (1: 1) of one embodiment of the invention preparations ¹H NMR (D ₂O) figure.

Fig. 9 has shown that the IFG tartrate is to the active exercising result of healthy individual GCase.

Figure 10 has shown the size distribution analytical results according to the isofagomine of one embodiment of the invention preparation.

Detailed Description Of The Invention

In general, term used herein has the common definition of this area in the context of the present invention with in the used specific context of each term. Describing the compositions and methods of the invention and how to prepare and use in them, hereinafter or the other parts of this specification some term has been discussed, in order to provide other guidance to the practitioner.

Term " Gaucher disease " comprises 1 type, 2 types and 3 types (comprising 3a, 3b and 3c), and intermediate form and based on the subgroup of Phenotypic Expression.

Term " effective dose " and " effectively ... amount " refer to be enough to produce the amount for the treatment of response. Treatment response can be that user (such as the doctor) regards as is to be effectively any response of response for treatment, comprises aforementioned symptom and the improvement of the clinical marker that substitutes. Thereby treatment responds the normally improvement of one or more symptoms of Gaucher disease, such as the neurodegenerative improvement of carrying out property among 2 types and the 3 type Gaucher disease patients. " treatment effective dose " will change according to used prescription, Gaucher disease type and the order of severity thereof and mammiferous age, body weight, condition and the response for the treatment of. Treatment response also can be that sick (α-synucleinopathies) is such as the improvement of one or more symptoms of dementia with Lewy body for Parkinson's disease or other alpha-synapse nucleoprotein, for described disease, the expection tartrate of isofagomine can be used for the treatment of them.

Phrase " pharmaceutically acceptable " refer to when being applied to the people be can tolerate on the physiology and usually can not produce with unacceptable level molecular entity and the composition of undesirable reaction. Preferably, as used herein, term " pharmaceutically acceptable " refer to by the management organization of federation or state government approval or in American Pharmacopeia or other universally recognized pharmacopeia, list and can be used for animal, people more especially.

Used term " carrier " refers to diluent, excipient or the medium jointly used with tartrate of isofagomine in pharmaceutical composition of the present invention. The selection of carrier can be put into practice to select according to method of administration and the standard pharmaceutical of expection. Pharmaceutical composition can comprise adhesive, lubricant, suspending agent, coating agent, the solubilizer as---or comprising---any suitable of carrier except carrier. These pharmaceutical carriers can be aseptic liquid, and for example water, saline solution, D/W, glycerine water solution and oil comprise oil, animal, plant or synthetic oil of originating, for example peanut oil, soya-bean oil, mineral oil and sesame oil. Suitable pharmaceutical carrier has description in " Remington ' s Pharmaceutical Sciences " (the 18th edition) that E.W.Martin shows. In a particularly preferred embodiment of the present invention, carrier is suitable for quick-release, and for example most of or all active components are gone through short period, for example 60 minutes or shorter time release, and make the quick absorption of medicine become possibility.

" pharmaceutically suitable carrier " refers to can be used for to prepare safe, nontoxic, both also carrier of the pharmaceutical composition do not expected of non-other side of abiology, comprises for medicinal usage it being acceptable excipient. Used " pharmaceutically suitable carrier " comprises a kind of and more than one this class carrier among the application.

Term " hydroxyl protecting group " refers to protect hydroxyl with any conventional group of the reaction avoiding not expecting, such as but not limited to methoxy, 4-methoxy-benzyl, benzyl, dimethyl isopropyl silicyl, trimethyl silyl and alkyl-carbonyl.

" individuality " refers to mammal, and the preferred mankind, domestic animal, rodent or primate are most preferably human.

" need treatment individuality " refers to develop into and maybe may develop into Gaucher disease or a-synucleinopathies, parkinsonian individuality for example. In one embodiment, individual for being diagnosed with Gaucher disease or because the heredity in the Gba gene suddenlys change to be confirmed to be has excessive risk and develop into a member among the Ashkenazim group of Gaucher disease. Yet the individuality of suffering from arbitrarily in the world Gaucher disease contained in term " individuality ", or on science of heredity the risky individuality that develops into this disease, perhaps risky a-synucleinopathies, the parkinsonian individuality for example of developing into.

The activity of term " raising " GCase represents to make among the ER the proteic conformation of mutant GCase stable, so that i) to allow its conformation that withdraws from ER folding, cause GCase level raising among the ER, and/or ii) obtain its natural place in cell, and/or iii) demonstrate metabolic activity to its lipid substrates cerebroside.This term also refers to improve or prolong the proteic activity of the exogenous GCase that uses, thereby promptly prolongs its activity by stability and its transformation period of prolongation of improving GCase.

Term used herein " pure basically " refers to that isofagomine salt comprises and is no more than other compound of about 5%.Preferably " pure basically " isofagomine salt comprises about 2% or any other compound still less.Even more preferably " pure basically " isofagomine salt comprises about 1% or any other compound still less.

Term " about " and " approaching " should refer to usually in view of the character of measuring or precision for the acceptable error degree of the amount of being measured.Typical exemplary error degree be the value of giving or the value of giving scope 20% in, in preferred 10%, more preferably in 5%.Selectively and particularly in the biology system, term " about " and " approaching " can refer in the value rank of the value of giving, preferably in 10 times or 5 times, more preferably in 2 times.Unless otherwise indicated, the quantitative value that this paper gave all is approximations, this means that term " about " can be inferred with " approaching " when not offering some clarification on.

Singulative " one " " a kind of " and " being somebody's turn to do " comprise plural form as used herein, and context has except the clearly indication in addition.Thereby for example, appellation " a kind of " carrier comprises one or more carriers.

The invention provides the particular form of isofagomine---tartrate of isofagomine.Compare with the isofagomine form of former description, tartrate of isofagomine has many improved character.For example, compare with known other isofagomine salt form, the easier purifying of tartrate of isofagomine especially in solvent such as water and/or ethanol, and has higher stability.Tartrate of isofagomine is particularly suitable for the production of industrially scalable, for example greater than 1 kilogram products production.

Isofagomine be (5R)-5-(hydroxymethyl)-3,4-piperidines glycol has following chemical structure for 3R, 4R:

Or

Its molecular formula is C ₆H ₁₃NO ₃, molecular weight is 147.17g/mol.The synthetic of this compound described in people's such as people's such as Sierks United States Patent (USP) 5,844,102 and LundRren the United States Patent (USP) 5,863,903.Patent 5,844,102 disclose wherein described compound can form pharmacologically acceptable salt, comprises organic carboxylate, for example acetate, lactic acid, tartrate, oxysuccinic acid, different thionic acid (isothionic acid), lactobionic acid and succsinic acid.Yet unique salt of being given an example in this patent (and the document subsequently known to the applicant) is hydrochloride.As described herein, hydrochloride is not suitable for the preparation of suitability for industrialized production or dosage form.

Term tartrate of isofagomine used herein refers to the tartrate of isofagomine, can followingly represent:

Wherein n is 1 or 2.Tartrate can have different stereoisomeric forms in any ratio: D-or L-tartrate, or DL-or meso-tartrate.The present invention and embodiment are main, and employing L-(+)-tartrate is described as embodiment preferred and isofagomine salt thereof.Yet, term tartrate is intended to contain D and L isomer and DL mixture and meso-tartrate, thereby the term tartrate of isofagomine is intended to comprise single-or two-isofagomine-L-tartrate, list-or two-isofagomine-D-tartrate, list-or two-isofagomine-DL-tartrate and/or singly-or two-isofagomine-meso-tartrate.L-tartrate is that the enantiomer enriching quantity is 97% or above (2R, 3R)-(+)-tartrate, D-tartrate is that the enantiomer enriching quantity is 97% or above (2S, 3S)-(-)-and tartrate, DL-tartrate is the enantiomer enriching quantity less than 97% D-and the tartaric mixture of L-.

Tartrate of isofagomine can be prepared as follows: the isofagomine free alkali is dissolved in alcohol, the preferred alcohol; handle in alcohol, preferred alcohol with substituted carboxylic acid; simultaneously in stirring at room; wherein said substituted carboxylic acid comprises amino acid, di-carboxylic acid or tartrate, and tartrate comprises diacyl tartrate.Hydrochlorate (acid salt) is settled out from ethanolic soln.Filter and collect the tartrate of isofagomine crude product.Before adding acid, can be with the pre-filtering of isofagomine solution to remove any granule foreign.After adding acid, cooling gained suspension is so that the salt precipitation fully.

In another embodiment, isofagomine hydrochlorate of the present invention can be prepared as follows: substituted carboxylic acid or tartrate are added in the solution, and wherein isofagomine is separated by in-situ preparing.

Perhaps, isofagomine hydrochlorate of the present invention can prepare from the inorganic acid salt such as the isofagomine hydrochloride of isofagomine.This conversion can followingly be finished: the preparation isofagomine, then handle free alkali with substituted carboxylic acid.For example, the isofagomine free alkali can followingly form: handle hydrochloride with alkalescence source, for example mineral alkali, ammonia or ammonium hydroxide aqueous solution, perhaps be placed in the basic resin or basic resin post of solid support.When using basic resin, as the eluant solution among methyl alcohol, ethanol, the IPA etc., so that the isofagomine free alkali to be provided, it can be converted into isofagomine hydrochlorate of the present invention at alcohol for resin column used water, ammonium hydroxide aqueous solution or ammonium hydroxide.

Because tartrate is diprotic acid, so can finish by following soda acid ratio range to the conversion of tartrate: tartrate is the 0.5-1 molar equivalent with respect to the isofagomine free alkali.Tartrate can be one of racemic (D or L-form) or three kinds of stereoisomeric forms in any ratio---L-(+) form, D-(-) form and meso-form---.The optimum condition of preparation tartrate adopts solution of ammonium hydroxide to generate free alkali, adopt 9: 1 ethanol/ammonium hydroxide wash-out free alkali on silicagel column, evaporating solvent and excessive ammonium hydroxide form tartrate in water/ethanol, and crystallization goes out from water/ethanol.

Isofagomine and tartrate can combinations in stoichiometric range.Because tartrate is diprotic acid, mol ratio is that isofagomine/tartrate of 2: 1 to 1: 1 provides stable salt (seeing embodiment 3).Preferred molar ratio is 1: 1.Stoichiometric range can be applicable to all tartrate isomer.

Purification process, preferred recrystallization that the isofagomine hydrochlorate can adopt most conventional to use come purifying.For example, the tartrate of isofagomine crude product can by or recrystallization in water not by proton or non-proton cosolvent, described cosolvent is alcohols preferably, for example methyl alcohol, ethanol, propyl alcohol or butanols.Recrystallization not only can on a small scale but also can carry out for example inferior kilogram quantities with industrially scalable effectively.Table 1 has been summed up purity and the yield of some embodiment of and purifying prepared according to the present invention.

Table 1

Sample number	Purity (%)	Yield (g)
Sample number	Purity (%)	Yield (g)	1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16	＞98 ＞98 96.8 84.5 95.9 87.4 94.6 95.6 95.8 99.8 98.1 97.6 99.0 99.3 99.5 98.0	5 15 55 45 40 71 45 343 851 14 134 128 72 116 57 1368

In a preferred embodiment, the tartrate of isofagomine crude product is soluble in water, in gained solution, add equivalent ethanol, obtain the precipitation of compound.Then add ethanol (1 volume) and stirring in addition.The ethanol that adds other 2 parts of aliquots containigs makes the ratio of ethanol/water approach 4: 1 to repeat this process.Though most of tartrate of isofagomine go out crystallization after adding the 1st part of volume of ethanol, can adopt other aliquots containig to make the yield maximization.Behind the recrystallization, filter and the washing solid.Whole purge process can at room temperature be carried out.Tartrate of isofagomine can be purified to about 99% or higher purity with this method.Thereby the tartrate of isofagomine that obtains according to one embodiment of the invention has 95% or higher purity, preferred 98% or higher purity, or even more preferably 99% or higher purity.

Tartrate of isofagomine also can adopt other solvent or solvent systems to come purifying, for example 1: 1 ethanol/water, 1: 1 acetone, 2: 1 ethanol/waters, 2: 1 acetone or 3: 1 ethanol/waters.Also can use gac to remove any foreign pigment.These solvents can provide separately and be higher than 95% purity, and great majority provide and have been higher than 98% purity.

The purifying easiness of tartrate of isofagomine can by with the aqueous solution in the purifying of isofagomine hydrochloride compare and illustrate, for the latter, need lyophilize.Filter the material that the isofagomine hydrochloride can produce yellow and gluey viscosity.Simultaneously, tartrate of isofagomine of the present invention has good characters powder, for example, crystalline size, density and flowability, these features are suitable for medicine production process.Table 2 and 3 has been summed up the powder characteristics of tartrate of isofagomine sample prepared in accordance with the present invention.Tartrate of isofagomine prepared in accordance with the present invention is not thin powder, but be medium sized particle mostly, its bulk density is about 0.44g/ml, and Ka Er index (Carr Index) is 15%, and it has good fluidity and the easily processing property that is suitable for medicine production process thus.The consistence of size distribution between tartrate of isofagomine by the preparation of above-mentioned recrystallization process also demonstrates batch, baseline fluctuation scope thereby have been avoided in the preparation process the disadvantageous oarse-grained existence of the accurate measurement of salt between 0.7-1.5.Most of batch can so that more than 98% or the particle diameter of at least 90% tartrate of isofagomine be about 1200 μ m or lower.

Table 2

Screen analysis	Keep %
Screen analysis	Keep %	40	0.6
60	15.5	40	0.6
60	15.5	80	49.2
120	28.4	80	49.2
120	28.4	200	5.9
325	0.4	200	5.9
325	0.4	＞325	0.0

Table 3

Bulk density	0.44g/ml
Bulk density	0.44g/ml	Tap density	0.52g/ml
The Ka Er index	15％	Tap density	0.52g/ml
The Ka Er index	15％	11 batches baseline span range of variation (average span)	0.79-1.53(1.18)

And tartrate of isofagomine does not have water absorbability, and moisture absorption only is about 0.08% after being exposed to 75%RH and reaching 8 days.The sucting wet experiment of 6 different tartrate of isofagomine samples prepared in accordance with the present invention the results are summarized in table 4 and 5.

The Study on Hygroscopicity that table 4. adopts the Sodium Bromide saturated solution to carry out

	0 hour	24 hours	48 hours	8 days
	0 hour	24 hours	48 hours	8 days	Relative humidity (RH)	60％	59％	59％	59％
Temperature (℃)	19.4	20.5	20.8	20.2	Relative humidity (RH)	60％	59％	59％	59％
Temperature (℃)	19.4	20.5	20.8	20.2	Sample number	Weightening finish (%)	Weightening finish (%)	Weightening finish (%)	Weightening finish (%)
1	NA	0.06	0.04	0.06	Sample number	Weightening finish (%)	Weightening finish (%)	Weightening finish (%)	Weightening finish (%)
1	NA	0.06	0.04	0.06	2	NA	0.00	0.02	0.07
3	NA	0.05	0.10	0.10	2	NA	0.00	0.02	0.07
3	NA	0.05	0.10	0.10	Average weight gain (%)	NA	0.04	0.05	0.08

The Study on Hygroscopicity that table 5. adopts sodium chloride saturated solution to carry out

	0 hour	24 hours	48 hours	8 days
	0 hour	24 hours	48 hours	8 days	Relative humidity (RH)	72％	72％	71％	72％
Temperature (℃)	19.5	20.5	20.8	20.2	Relative humidity (RH)	72％	72％	71％	72％
Temperature (℃)	19.5	20.5	20.8	20.2	Sample number	Weightening finish (%)	Weightening finish (%)	Weightening finish (%)	Weightening finish (%)
1	NA	0.02	0.05	0.10	Sample number	Weightening finish (%)	Weightening finish (%)	Weightening finish (%)	Weightening finish (%)
1	NA	0.02	0.05	0.10	2	NA	0.00	0.00	0.00
3	NA	0.08	0.8	0.15	2	NA	0.00	0.00	0.00
3	NA	0.08	0.8	0.15	Average weight gain (%)	NA	0.04	0.05	0.08

Therefore, the present invention's method of preparing tartrate of isofagomine is suitable for preparing the large batch of tartrate of isofagomine that can be used for pharmaceutical composition.According to the preparation purpose, the tartrate of isofagomine in enormous quantities that makes according to the present invention can be made into the thick slightly form of purity about 80%.Yet, also tartrate of isofagomine can be prepared into purity and be 90% or higher, preferred at least 99% and 90% tartrate of isofagomine have about 1200 μ m or littler particle diameter.

HPLC can be used for measuring the existence of the content and the organic impurity of isofagomine hydrochlorate of the present invention.Short wavelength UV detection is suitable for ining contrast to reference standard and calculates content.Those skilled in the art's concentration, chromatographic column type, solvent per sample wait to determine the conditions suitable that HPLC analyzes.However, provide the example of following condition as the mensuration tartrate of isofagomine: moving phase can be 10mM volatile salt (NH ₄HCO ₃)/acetonitrile (CAN) 30/70, the isoconcentration pattern, flow velocity is 0.5mL/min; The HPLC post can be Alltech Prevail carbohydrate post (4.6 * 150mm, 5 μ m particle diameters), 50 ℃ of service temperatures; Detect wavelength and can be set to 210nm, 15 minutes working times; Drug sample dissolves in moving phase, and sampling volume is 10 μ L.

Electricity mist formula detector (CAD) can be used for measuring impurity.Sample also can adopt Evaporative Light-Scattering Detection Technology (ELSD) and UV to detect and analyze.The CAD detector adopts evaporation technique to dissolve analyte in the presence of nitrogen carrier gas.The corona spark is given nitrogen with charge transfer, and nitrogen is given analyte with charge transfer.At analyte its charge transfer is detected analyte during to electrometer, measure with electric current.When adopting the CAD detector, moving phase can be 5mM ammonium acetate/CAN 50/50, the isoconcentration pattern, and flow velocity is 1.0mL/min, the HPLC post is Primesep 100 (4.6 * 150mm, 5 μ m particle diameters), 25 ℃ of service temperatures.Drug sample prepares in moving phase, and sampling volume can be 10 μ L.Be about 70 minutes the working time of this method.Impurity adopts high/low sample introduction order (high/low injectionsequence) to measure, and sample carries out quantitatively with respect to the standard substance of 1% nominal sample concentration.

Can adopt FT-IR to carry out to the evaluation of isofagomine hydrochlorate of the present invention, perhaps further pass through ¹H NMR and ¹³C NMR confirms.Fig. 2-5 has shown according to the tartrate of isofagomine of one embodiment of the invention preparations ¹H, ¹³C NMR and IR collection of illustrative plates.Residual solvent can adopt headspace gas chromatography (GC) to monitor.The resistates of water, igniting and heavy metal can be monitored by the standard official method.Palladium is monitored by the ICP spectrography, because it is the used catalyzer of last step of hydrogenation.

Fig. 6 has shown the X-ray powder diffraction pattern according to the tartrate of isofagomine of one embodiment of the invention preparation.This figure adopts Bruker D8Advance diffractometer to obtain, and analyzes and adopts following condition to carry out under 2-45 ° of 2 θ:

Divergent slit	0.6mm	The anti-scatter slit	0.6mm
Divergent slit	0.6mm	The anti-scatter slit	0.6mm	Receive slit	0.1mm	The detector slit	0.6mm
Step-length	0.02°	The level time (step time) in step	5 seconds	Receive slit	0.1mm	The detector slit	0.6mm

Though the X-ray powder diffraction pattern can be used for the particular solid form of authenticating compound, i.e. polymorphic forms,, because specimen preparation or obtain the difference that is caused in the collection of illustrative plates process, its 2 θ value and intensity and d-spacing can change.And, when distributing 2 θ and d-spacing, may there be some margin of error.2 θ values have ± 0.009 deviation.Thereby in order to identify concrete crystallized form, a kind of preferred comparison X-ray powder diffraction pattern method is with the X-ray powder diffraction pattern of unknown form and overlapping and their characteristic peak of comparison of X-ray powder diffraction pattern of form known.However, table 6 has been summed up 2 θ, d-spacing, intensity and the % intensity level of Fig. 6.When in determining composition, whether having crystallized form of the present invention, can identify that the peak of in those the peak 5 or tool feature compares with table 6.The peak of tool feature comprises 9.29,14.17,16.34,18.07,18.72,19.44,20.56,22.13,23.01,24.54 and 27.12.

Table 6

Angle (2-θ °)	D value ()	Density (counting)	% density (%)
Angle (2-θ °)	D value ()	Density (counting)	% density (%)	9.29	9.5093	131	23.3
14.17	6.24684	129	22.8	9.29	9.5093	131	23.3
14.17	6.24684	129	22.8	16.34	5.42037	155	27.6
18.07	4.90414	330	58.5	16.34	5.42037	155	27.6
18.07	4.90414	330	58.5	18.72	4.73704	563	100
19.44	4.56252	165	29.3	18.72	4.73704	563	100
19.44	4.56252	165	29.3	20.56	4.31573	212	37.5
22.13	4.01417	338	60	20.56	4.31573	212	37.5
22.13	4.01417	338	60	23.01	3.86164	111	19.8
24.54	3.62444	210	37.2	23.01	3.86164	111	19.8
24.54	3.62444	210	37.2	27.57	3.23301	276	49

Isofagomine hydrochlorate of the present invention can be used with the form that is suitable for any route of administration, for example comprises with tablet, capsule or liquid form dosage forms for oral administration, or uses with the injection of aseptic aqueous solution form.It can be with following form dosage forms for oral administration: tablet, capsule, globule (ovules), elixir, solution or suspension, gel, syrup, mouth wash shua, perhaps at the dry powder that faces with preceding water or other suitable solvent reconstruct, choose wantonly and contain correctives and tinting material, be used for quick-release, postpone to discharge, change release, slowly-releasing, pulse release or controlled release application.Also can use solids composition, for example tablet, capsule, lozenge, pastille, pill, bolus, powder agent, paste, granule, bolus (bullet) or premixed type preparation.Solid or liquid oral composition can prepare according to method well known in the art.These compositions also can comprise the pharmaceutically acceptable carrier and the vehicle of one or more solids or liquid form.In a specific embodiment, tartrate of isofagomine is used with the form of powder filled capsules.When compound is formulated as oral dosage form, can make tablet or capsule by ordinary method, the pharmaceutically useful vehicle of employing, described vehicle for example has tackiness agent (for example pregelatinized corn starch, polyvinylpyrrolidone or Vltra tears); Weighting agent (for example lactose, Microcrystalline Cellulose or secondary calcium phosphate); Lubricant (for example Magnesium Stearate, talcum powder or silicon-dioxide); Disintegrating agent (for example potato starch or primojel); Perhaps wetting agent (for example Sulfuric acid,monododecyl ester, sodium salt).Tablet can adopt method well known in the art to carry out dressing.

Pharmaceutically acceptable vehicle also comprises Microcrystalline Cellulose, lactose, Trisodium Citrate, lime carbonate, secondary calcium phosphate and glycine, disintegrating agent such as starch (preferred W-Gum, potato starch or tapioca (flour)), primojel, croscarmellose sodium and some complex silicate, and particle binders such as polyvinylpyrrolidone, Hydroxypropyl ethyl cellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and gum arabic.In addition, can comprise lubricant such as Magnesium Stearate, stearic acid, glyceryl behenate and talcum powder.

Also can adopt the weighting material of the solids composition of similar type as gelatine capsule.In this regard, preferred vehicle comprises lactose, starch, Mierocrystalline cellulose, lactose (milk sugar) or high molecular weight polyethylene glycol.For aqueous suspension and/or elixir, can merge with material and various emulsifying agent and/or suspending agent and with thinner such as water, ethanol, propylene glycol, glycerine and their combination.

The liquid preparation of oral administration can be taked for example form of solution, syrup or suspensoid, perhaps they can be made and face with preceding water or other suitable solvent (for example ethanol or polyvalent alcohol such as glycerol, propylene glycol, polyoxyethylene glycol etc., the mixture that they are suitable, and vegetables oil) drying products of reconstruct.This class I liquid I preparation can adopt pharmaceutically acceptable additive to prepare by ordinary method, for example suspending agent (for example water, sorbitol syrups, derivatived cellulose or edible hydrogenated fat); Emulsifying agent (for example Yelkin TTS or gum arabic); Non-water-soluble matchmaker (for example Prunus amygdalus oil, oily ester, ethanol or fractionated vegetable oil); And sanitas (for example methyl p-hydroxybenzoate and propyl ester or Sorbic Acid).Oral Preparation can be made the preparation of controlled release or slowly-releasing isofagomine hydrochlorate of the present invention aptly.

Suitable flowability can by for example use dressing such as Yelkin TTS, by keep under the dispersion state required particle diameter and by using tensio-active agent to keep.Stop action of microorganisms to realize, for example parabens, trichloro-butyl alcohol, phenol, benzylalcohol, Sorbic Acid etc. by using various antiseptic-germicides and anti-mycotic agent.Under many circumstances, can reasonably comprise isotonic agent such as sugar or sodium-chlor.The prolongation of Injectable composition absorb can be by in composition, using delayed absorption material such as aluminum monostearate and gelatin realize.

Be suitable for gi tract outer/the tartrate of isofagomine pharmaceutical preparation of injectable (for example by vein single fast injection or infusion, or by intramuscular, subcutaneous or intrathecal route) purposes generally includes aseptic aqueous solution, dispersion liquid and is used for making the sterilized powder of sterile injectable solution or dispersion liquid temporarily.Tartrate of isofagomine can if necessary, can add sanitas with unit dosage form in ampoule or other unit-dose container or provide in multi-dose container.Composition for injection can be suspensoid, solution or the emulsion in oiliness or aqueous vehicles, and can comprise preparation material such as suspending agent, stablizer, solubilizing agent and/or dispersion agent.Perhaps, activeconstituents can be the sterilized powder form that is used to face with suitable solvent of preceding usefulness such as aseptic, pyrogen-free water reconstruct.In all cases, each form must be aseptic, and the mobile degree that must reach easy injection.It must be stable under production and condition of storage, and must be by anticorrosion pollution with opposing microorganism such as bacterium and fungi.The suitable parenteral formulations of preparation can easily be realized by standard pharmaceutical technology well known to those skilled in the art under aseptic condition.

Aseptic parenteral solution is prepared as follows: tartrate of isofagomine aequum, in appropriate solvent is mixed with above-named various other compositions, take the circumstances into consideration succeeded by filtering or final sterilization.Usually, dispersion liquid is prepared as follows: the activeconstituents of various sterilizations is mixed with sterile media, and described medium comprises basic dispersion medium and from those required other composition of above enumerating.For the sterilized powder that is used to prepare sterile injectable solution, preferred manufacturing procedure is vacuum-drying and Freeze Drying Technique, and it is produced activeconstituents and adds powder from the required composition of any interpolation of the solution of previous sterile filtration.

Pharmaceutical composition can provide sanitas, stablizer, dyestuff and or even perfume compound.The example of sanitas comprises Sodium Benzoate, xitix, p-Hydroxybenzoate.Also can use antioxidant and suspending agent.

Other pharmaceutically acceptable carrier that can comprise in the preparation has buffer reagent, for example citrate buffer agent, phosphate buffer, acetate buffer and bicarbonate buffer agent, amino acid, urea, alcohol, xitix, phosphatide, protein such as serum albumin, collagen, and gelatin; Salt, for example EDTA or EGTA and sodium-chlor; The liposome polyvinylpyrrolidone; Sugar, dextran for example, N.F,USP MANNITOL, sorbyl alcohol and glycerine; Propylene glycol and polyoxyethylene glycol (as PEG-4000, PEG-6000); Glycerine, glycine or other amino acid and lipid.The buffer system of using with preparation comprises Citrate trianion, acetate, supercarbonate and phosphate buffer.Phosphate buffer is for preferred embodiment.

Preparation also can comprise nonionogenic tenside.Preferred nonionic comprises Polysorbate 20, Polysorbate 80, Triton X-100, Triton X-114, Nonidet P-40, octyl group α-glucoside, octyl group β-glucoside, Brij 35, Pluronic and polysorbas20.

(transmission) approach of using includes but not limited to following one or more: oral (for example as tablet, capsule maybe can absorb solution), local, mucous membrane (for example as nose spraying or inhalation aerosol), intranasal, gi tract outer (for example passing through injectable forms), gi tract, in the backbone, intraperitoneal, intramuscular, intravenously, intrauterine, intraocular, intradermal, encephalic, in the tracheae, intravaginal, Intraventricular, in the brain, subcutaneous, eye (comprising in the vitreum or intracameral), through skin, rectum, the oral cavity, epidural and hypogloeeis.

Tartrate of isofagomine parenteral formulations described above can be used by periodically injecting quick single intravenous injection preparation, perhaps can use outside (for example IV bag) or inside (but for example the implant of bioerodable) by intravenously or intraperitoneal and store the storehouse and use.Referring to for example U.S. Patent number 4,407,957 and 5,798,113, each document is incorporated herein by reference.The method and apparatus of intrapulmonary delivery medicine is for example at U.S. Patent number 5,654, description arranged in 007,5,780,014 and 5,814,607, is equipped with document and is incorporated herein by reference.The outer delivery system of other useful gi tract comprises ethylene-vinyl acetate copolymer particulate, osmotic pump, implantable infusion system, pump transmission, seals cell transmission (encapsulated celldelivery), medicine injection, Needleless injection, atomizer, spraying gun, electroporation and transdermal patch are passed in liposome transmission, syringe needle.U.S. Patent number 5,879,327,5,520,639,5,846,233 and 5,704,911 have described needleless syringe devices, and the specification sheets of above document is incorporated herein by reference.Above-mentioned any preparation can adopt these methods to use.

And, for the convenient various devices that design of patient can be used for preparation of the present invention as herein described as injection pen and the Needleless injection device that can heavily fill.

Usually, the doctor will determine to be suitable for most individual curee's actual dose, and will provide effectively amount of treatment to the curee.Concrete dosage level for any concrete individuality can be different with administration frequency, it depends on multiple factor, comprises type, age, body weight, general health, sex, diet, administering mode and time, discharge rate, drug combination situation, disease severity, the individual treatment of just carrying out of compound activity, the Gaucher disease for the treatment of.For oral and gi tract external administration, the dosage that the dosage level of material can be single dose or separates.The significant quantity of preferred isofagomine hydrochlorate of the present invention or dosage are enough to improve mutant glucocerebrosidase expression levels, for example improve about 3-5% of this level of being found in the normal cell cell of the individuality of not suffering from Gaucher disease (promptly from), preferred about 10%, more preferably from about 30%, and/or can improve or stop the GCase that has clinical meaning among the curee active not enough.

Significant quantity can determine by normal experiment usually, but expection is to produce 0.01 to 100 μ M, preferred 0.01-10 μ M, the amount of the blood plasma level of 0.05-2 μ M most preferably.The effective dose of expection tartrate of isofagomine is the 0.5-1000mg/kg body weight/day, preferred 0.5-100,1-50mg/kg body weight/day most preferably.In a specific embodiment, dosage is about 1-600mg/ days, more especially 5-300mg/ days, is more particularly 10-150mg/ days.The mode of non-administration every day of focus attentions equally on.The applying date is that the U.S. Provisional Patent Application 60/914,288 on April 24th, 2007 has been described other dosage that uses tartrate of isofagomine treatment Gaucher disease to consider, document integral body is incorporated herein by reference.

Treatment monitoring of the present invention is applicable after the patient adopts the combined therapy of tartrate of isofagomine and other therapies such as ERT or gene therapy equally.This combined therapy has description in the U.S. Patent Application Publication No. of owning together 2004/0180419 and 2004/0219132, these two pieces of document integral body are incorporated herein by reference.

When isofagomine hydrochlorate of the present invention and second kind of therapeutical agent applied in any combination, the dosage of each compound can be different from the dosage of this compound list time spent.Those skilled in the art will easily estimate appropriate dosage.Being appreciated that compound of the present invention is used for the treatment of required amount will be according to institute sanatory character and patient's age with situation and different, and will finally be determined by the doctor.

When administration successively, compound of the present invention or second kind of therapeutical agent all can at first be used.When the while administration, can in same or different pharmaceutical composition, use said composition.

Be appreciated that when in same preparation, merging two kinds of compounds must be stable, each other and and other component of preparation between be compatible.When preparing respectively, they can with arbitrarily easily preparation, for example this compounds for this area is that known preparation provides.

Isofagomine can by the D-pectinose by in the past in the literature by people such as Danishefski at Tetrahedron Letters, 1990; 31 (16), 2229 intermediates of being reported synthesize.Yet this intermediate synthesis step of being reported in the past is uneconomic for extensive synthesizing.Method disclosed herein can be produced those intermediates (and new intermediate) and final product with technical scale and high purity reliably and predictably.This is because all intermediates all separate by crystallization, thereby the method that makes is suitable for scale operation.

Isofagomine can synthesize by the synthetic route shown in the flow process 1.

Flow process 1.PG is a protecting group, and LG is a leaving group

Have or solvent-free (clean reaction) in the presence of, the D-pectinose can adopt suitable pure and mild activator to be converted into corresponding protected glucosides (A).For example, the scope of alcohol can comprise the benzylalcohol of benzylalcohol or replacement, as methoxyl group benzylalcohol, chlorobenzyl alcohol, methyl alcohol, ethanol, Virahol, cyclohexyl methyl alcohol etc., in solvent such as methylene dichloride, chloroform, THF, two  alkane, DMF, DMA or NMP, activator for example has HCl, HBr, H ₂SO ₄Or other acyl chlorides of some other mineral acids or Acetyl Chloride 98Min., propionyl chloride or carboxylic acid.Reaction can be carried out 2-48 hour to about 100 ℃ temperature in envrionment temperature.For the present invention, preferred alcohol is the benzylalcohol of benzylalcohol or replacement, more preferably benzylalcohol.Preferred solvent comprises two  alkane, THF or clean reaction, is more preferably clean reaction.Preferred activator comprises Acetyl Chloride 98Min. and H ₂SO ₄, more preferably Acetyl Chloride 98Min..Can easily isolate pure products by precipitating with non-polar solvent.For this product, preferred solvent and temperature are methyl tertiary butyl ether and envrionment temperature.

By adopt ketone or dimethyl ketal or its enol ether in the presence of the acid and have or does not have (clean reaction) polar co-solvent down (A) is converted into ketal (B), can be acetone solvate (acetonide) at 3-and further protection of 4-position hydroxyl with the general formula A glucosides of gained.For example, aliphatics or aromatic ketone, for example acetone, 2-butanone, benzophenone, pimelinketone, methyl phenyl ketone or their corresponding dialkyl group ketals, can be with the ortho position glycol in acid as H ₂SO ₄, tosic acid, camphorsulfonic acid or TMS triflate existence reaction down.Cosolvent comprises methylene dichloride, DMSO, DMF, DMA and NMP.In some cases, ketone also can be solvent, for example acetone.Temperature of reaction is an envrionment temperature to 100 ℃.For this reaction, preferred condition is to adopt acetone and 2, and 2-Propanal dimethyl acetal and tosic acid carry out at 40 ℃.Can easily isolate pure products by carrying out crystallization with the two-component system that comprises polarity and non-polar component.Preferred purification condition is ethyl acetate and heptane.

By being converted into corresponding alkoxide, subsequently with alkylation reactions so that general formula C to be provided compound, acetone solvate (acetonide) (B) can be on the 2-hydroxyl be ether by further protection.Expensive bromotoluene and expensive silver suboxide are adopted in the protective reaction of being reported in the past.The formation of alkoxide easily adopt highly basic as and alkali (metal) hydride in polar aprotic solvent such as dialkyl ether or THF, DMF, DMA, NMP or DMSO, finish, corresponding to PG2.Alkylating agent comprises the benzyl of benzyl chloride or replacement.Temperature of reaction can be-20 ℃ to 20 ℃.For this reaction, preferred condition is then carried out alkylation with benzyl chloride for adopting sodium hydride to generate alkoxide in DMF at 0 ℃-10 ℃.Wash to remove excessive water by aqueous precipitation with non-polar solvent, can easily isolate pure products.The non-polar solvent that is preferred for this purifying is a heptane.

At ambient temperature, adopt to dilute mineral acid such as HCl, HBr, H in as methyl alcohol, ethanol, Virahol at alcohol ₂SO ₄From general formula C compound, remove acetone solvate (acetonide), obtain the glycol of general formula D.For this reaction, preferred condition is to adopt HCl to carry out in methyl alcohol in envrionment temperature.Wash to remove excessive water by aqueous precipitation with non-polar solvent, can easily isolate pure products (D).The non-polar solvent that is preferred for this purifying is a heptane.

In order to change into target molecule, glycol need carry out Additional Protection.In the water azeotropic solvent, adopt the directed approach of tin in reflux temperature, carry out etherificate in mild temperature subsequently, can finish the selective etherification (E) of 3-hydroxyl.Adopt dialkyl group or aryl oxidized tin (IV) to form tin ether as phenylbenzene, dimethyl, dibutyl, diisobutyl or hexadecyl stannic oxide, in aprotic solvent such as benzene, toluene or dimethylbenzene.Alkylation subsequently can adopt alkyl or alkylaryl halogenide such as bromotoluene or benzyl chloride to finish.Reaction can be quickened by adopting the material such as CsF or etamon chloride, and temperature of reaction can be envrionment temperature to 100 ℃.For this reaction, preferable methods is in the presence of tetrabutylammonium chloride, uses Dibutyltin oxide in toluene and benzyl chloride.Purifying can easily be settled out tin reagent by water and finish.End product can obtain by crystallization from the solvent pairs system.The recrystallisation solvent that is preferred for this reaction is ethanol and heptane.

Intermediate pectinose derivative by three protections can be converted into corresponding wood sugar derivative (G) by activation system (F).Though the Mitsunobu conversion is generally used for changing the configuration of alcohol, and for the existing report of this specific conversion, these conditions are expensive for industrial scale.Route for choosing comprises that with the activation of pectinose hydroxyl be careful (discreet), separable activation system (G), then adopts cheap oxygen source to change displacement.Activation can be adopted ester such as p-toluenesulfonic esters, methanesulfonates, triflate etc., it is formed by corresponding acid anhydride and SULPHURYL CHLORIDE, in the presence of organic bases such as pyridine, trimethylpyridine, Hunig ' alkali, triethylamine, in non-polar solvent such as methylene dichloride, chloroform and toluene, in-20 ℃ to envrionment temperature.Configuration conversion displacement can adopt oxygen affinity nuclear reagent, preferred as alkali or alkaline earth metal nitrite be used for the type reaction in routine solvent to finish in 0 ℃ to 40 ℃, and described solvent for example has methylene dichloride, acetone, THF, DMF, DMA, NMP etc.Preferred condition be in-10 ℃ of use trifluoromethanesulfanhydride anhydride and pyridines in methylene dichloride, need not purifying subsequently and isolates triflate.Preferred triflate permutizer condition is to adopt Sodium Nitrite or potassium in envrionment temperature in DMF.Pure products can easily obtain by crystallization from the solvent pairs system that adopts polarity and non-polar component.The recrystallisation solvent that is preferred for this reaction is Virahol and heptane.

Can be carried out configuration by activation system by the wood sugar derivative of the general formula (H) of three protections and be converted into nitrile (I).Similar with aforesaid method, this route comprises that with the activation of wood sugar hydroxyl be careful (discreet), separable activation system (H), then replaces with cyano sources.Activation can followingly once more be carried out: the ester that adopts alkyl or aryl sulphonate, preferred p-toluenesulfonic esters, methanesulfonates, triflate etc., described ester is formed by corresponding acid anhydride or SULPHURYL CHLORIDE, in the presence of the organic bases of gentleness such as pyridine, trimethylpyridine, Hunig ' alkali, triethylamine etc., in non-polar solvent such as methylene dichloride, chloroform or toluene, in-20 ℃ to envrionment temperature.Configuration conversion displacement is preferred adopts reagent such as basic metal or alkaline-earth metal prussiate or tetraethyl-ammonium cyanide to finish in 0 ℃ to 40 ℃ in polar aprotic solvent such as THF, DMF, DMA, NMP, DMSO etc.Preferred condition adopts trifluoromethanesulfanhydride anhydride and pyridine to carry out in-10 ℃ in methylene dichloride.For the displacement of triflate, preferred condition is to adopt the tetraethyl-ammonium cyanide to carry out in envrionment temperature in THF.Pure products can by the extraction, crystallization obtains from the alcohol solvent then.Preferred solvent is an ethanol.

According to selected protecting group, the nitrile intermediate can be converted into the isofagomine hydrochloride by a step.Under hydrogenation conditions, nitrile reduces, takes off three protections, ring hydrogenations closed, epimino can be finished by a step, obtains the isofagomine of high yield.Catalytic hydrogenation can adopt various routines be used for this hydrogenant catalyzer, under the hydrogen-pressure of 14psi-100psi, carry out at proton or aprotic polar solvent, preferred alcohols such as methyl alcohol, ethanol, Virahol or ester or acetate, wherein said catalyzer comprises Pd/C, Pd (OH) ₂The combination of/C, PtO, Degussa catalyzer or catalyzer, carrying capacity 1%-20%.Hydrogenation acid as HCl, HBr, HClO ₄, H ₃PO ₄, H ₂SO ₄, carry out under acetate, trifluoroacetic acid or the tartaric existence.Hydrogenation carries out short or time expand and does not have the risk of product degradation.Preferred condition is that the employing carrying capacity is 5% to 20%d Pd/C and Pd (OH) ₂The mixture of/C, under the pressure of 40-100psi, containing in the alcohol solvent of HCl and reacting.Preferred condition is the Pd/C of 10% carrying capacity and the Pd (OH) of 10% carrying capacity ₂/ C, under the 80psi hydrogen pressure, containing in the ethanol of HCl and carrying out.This hydrochloride can be converted into isofagomine hydrochlorate of the present invention.

In the recent period, develop the another kind of improved synthetic method of preparation isofagomine and tartrate of isofagomine, and therefore submitted a independent patent application to.This novel method adopts D-(-)-pectinose or L-(-)-wood sugar to carry out through two ketal intermediates.

For the present invention that further explains, hereinafter provide embodiment.These embodiment only are illustrative, and limit the scope of the present invention or any term of giving an example by no means by any way.Equally, the present invention also is not limited to any specific preferred embodiment described herein.Really, after reading this specification sheets, to those skilled in the art, multiple accommodation of the present invention and change are conspicuous.

Comparative Example A An: the purifying of isofagomine free alkali:

The isofagomine free alkali uses Amberlite CG 50 resin column (NH ₄ ⁺2.5cm ID X 100.0cm L, volume 450mL) carries out chromatography.Adopt 0.5N solution of ammonium hydroxide (3 times are adopted deionized water (DI, 5 times, 2250mL) washing pillar 1200mL), then.Isofagomine crude product (1.0g) is dissolved in the 4.0mL water, then upper prop.Use 0.1N NH ₄OH/ water (1.36: 1) wash-out pillar.Collect the 10mL fraction.

Different fractions is carried out TLC (silica gel, Virahol: water: ammonium hydroxide (7: 2: 1)), detect by imines sugar (imino sugar) and triketohydrindene hydrate spraying.Analysis is through the fraction of imines sugar spraying test positive, and to determine its purity, then merging and lyophilize are 72 hours.

Comparative Examples B: the purifying of isofagomine hydrochloride:

Isofagomine free alkali (30mg) is dissolved in methyl alcohol (5mL) and 4N HCl (0.5mL) in Virahol and acetone (4.0mL).The sample freezer storage is spent the night, and filters then.In solution, observe crystallization.Yet after filtering, product is the yellow substance with gluey viscosity.

By adopting ion exchange chromatography, water and ammoniacal liquor wash-out, purifying isofagomine hydrochloride crude product.

To concentrate from the fraction of ion exchange column by lyophilize, obtain the semi-solid material of viscosity.

Embodiment 1: From the synthetic tartrate of isofagomine of D-(-)-pectinose

Reaction is monitored by TLC, adopts 5%H ₂SO ₄/ methyl alcohol, phosphorus molybdenum acid solution or 254nm UV colour developing.

Step 1: with the D-pectinose (50kg, 330.04moles) and benzylalcohol (132.2kg, 4.33 equivalents) stir and be heated to 35 ℃.Add Acetyl Chloride 98Min. (10.9kg, 0.42 equivalent), keep temperature＜45 ℃, spend the night in 50 ℃ of stirrings then.Mixture is cooled to 20 ℃, with MTBE (600kg) dilution.Mixture was stirred 0.5-5 hour.Solid collected by filtration is with MTBE (2 * 40kg) washings.Product is dry in the filter moisture eliminator.Obtain 2-phenyl-D-pectinose, be pale solid, 70.9kg (88.6%).

¹H?NMR(300MHz，DMSO-d ₆)：δ7.32(m，5H)，4.76(s，1H)，4.66(d，J＝12Hz，1H)，4.59(m，3H)，4.45(d，J＝12Hz，1H)，3.70(m，4H)，3.47(dd，J＝12，3Hz，1H).

Step 2: (73.5kg 305.92moles) mixes with acetone (522kg) with 2-benzyl-D-pectinose.Disposable adding 2,2-Propanal dimethyl acetal (26.6kg, 1.9 equivalents) adds tosic acid monohydrate (39.3g, 0.0007 equivalent) then.Mixture was stirred 18 hours in 40 ℃.After reacting completely, add triethylamine (193mL, 0.0046 equivalent).In 30 ℃ of removal of solvent under reduced pressure, until obtaining dense thick oil.With resistates and ethyl acetate (2 * 20kg) coevaporations.Add ethyl acetate (19.2kg) to form solution.Disposable adding heptane (145.8kg) in solution, spending the night is cooled to-10 ℃ to 0 ℃.Solid collected by filtration is with heptane (2 * 51.5kg) washings.Product is the nitrogen blowing drying in the filter moisture eliminator.Obtain ketal derivatives (3aR, 6R, 7S, 7aS)-6-(benzyloxy)-2,2-dimethyl tetrahydro-3aH-[1,3] dioxole [4,5-c] pyrans-7-alcohol also, be pale solid, 70.4kg (82%).Fusing point 58-59 ℃.

¹H?NMR(400MHz，CDCl ₃)：δ7.34(m，5H)，4.92(d，J＝4Hz，1H)，4.79(d，J＝12Hz，1H)，4.54(d，J＝12Hz，1H)，4.20(m，2H)，4.00(dd，J＝13，3Hz，1H)，3.92(dd，J＝13，2Hz，1H)，3.80(m，1H)，2.24(d，J＝7Hz，1H)，1.52(s，3H)，1.35(s，3H).

Step 3: (78.2kg 278.97moles) mixes and is cooled to 5 ℃ with DMF (295kg, 3.77kg/kg raw material) with ketal derivatives.Divide 3 to 4 batches to add in the reactor sodium hydride (13.4kg, 1.2 equivalents), keep reaction mixture to be lower than 10 ℃, stirred then 1.5 hours.Go through 1 hour adding benzyl chloride (45.9kg, 1.3 equivalents) in 2 ℃.Reactant was stirred 12 hours in 10 ℃ to 15 ℃.After reacting completely, mixture is cooled to 2 ℃, goes through and added entry (20kg) in 1 hour.Go through and added the water (570kg) of amount in addition in 4 hours.Mixture was stirred 10 hours under this temperature.Collect product by centrifuging, water (2 * 10kg) and heptane (2 * 15kg) wash, and Rotary drying spends the night.Obtain the dibenzyl derivative (3aR, 6R, 7S, 7aR)-6,7-two (benzyloxy)-2,2-dimethyl tetrahydro-3aH-[1,3] dioxa cyclopentenyl [4,5-c] pyrans, be white solid, 74.0kg (71.6%).

Step 4: (37.6kg 101.50moles) adds methyl alcohol, among the AR (259kg, 8.7kg/kg raw material), mixture is cooled to 15 ℃ with the dibenzyl derivative.Go through and added 2.5N HCl solution (76.2kg, 1.8 equivalents) in 1 hour.Add entry (20kg) in addition, mixture was stirred 12 hours in 15 ℃.(1035kg, 4 times of volumes methanol AR) add in the reactor, stir at least 0.5 hour with water.Product is through centrifuging, water (2 * 10kg) and heptane (2 * 15kg) washings, Rotary drying spends the night.Obtain glycol (3R, 4R, 5S, 6R)-5,6-two (benzyloxy) tetrahydrochysene-2H-pyrans-3, the 4-glycol is white solid, 31.5kg (94%).

Step 5: (37.5kg 113.51moles) mixes with toluene (207.6kg, 5.5kg/kg glycol) and Dibutyltin oxide (31.1kg, 1.1 equivalents) with diol, derivatives.To reactor assembling Dean-Stark device, reactor content is heated to backflow (about 110 ℃), be collected removal (8-12 hour) up to no longer including water.Reactor content is cooled to 35 ℃, disposable adding tetrabutylammonium chloride (18.3kg, 0.5 equivalent).Add benzyl chloride (15.8kg, 1.1 equivalents) with the speed that keeps temperature＜40 ℃, continue down to stir 12 hours at 35 ℃.Repeat every day reinforced and stirring in 12 hours, totally 4 days, up to reacting completely.After reacting completely, mixture is cooled to 25 ℃, disposablely adds entry (150kg), contents stirred is spent the night.Reaction mixture is filtered through bed of diatomaceous earth (1kg/kg glycol), with toluene (10kg) flushing bed.Leave standstill filtrate (1 hour), layering.Repeat to add water, stirring, filtration and separate.Combining water layer is with ethyl acetate (25kg) extraction, layering.Merge organic layer, long-pending in 45 ℃ of vacuum concentration to minimum whippable body.Add heptane (102.6kg).Mixture stirred 20 minutes, was cooled to 0 ℃, stirred 8-12 hour.Solid collected by filtration is with heptane (10kg) washing.Solid crude product is dissolved in 6: 1 heptane/200pf ethanol (7kg/kg solid crude product) in 35 ℃, is cooled to-5 ℃ to 0 ℃, stirring is spent the night.Solid collected by filtration is with heptane (10kg) washing.Product purity adopts the TLC monitoring.Usually needs 2 times or more times recrystallization are to remove impurity.Tribenzyl derivative behind the purifying is dry in vacuum drying oven in 30 ℃.Obtain (3R, 4R, 5S, 6R)-4,5,6-three (benzyloxy) tetrahydrochysene-2H-pyrans-3-phenol is white solid, 17.5kg (37%).Fusing point 59-60 ℃.

¹H?NMR(400MHz，CDCl ₃)：δ7.38(m，15H)，4.89(d，J＝4Hz，1H)，4.82(d，J＝12Hz，1H)，4.71(m，3H)，4.57(d，J＝12Hz，1H)，4.55(d，J＝12Hz，1H)，4.01(br?s，1H)，3.95(dd，J＝10，3Hz，1H)，3.83(m，2H)，3.71(dd，J＝12.2Hz，1H)，2.56(br?s，1H).

Step 6: (12.0kg 28.54mol) mixes with methylene dichloride (79.2kg, 6.6kg/kg raw material) and pyridine (11.3kg, 5 equivalents), is cooled to-10 ℃ with tribenzyl pectinose derivative.Add trifluoromethanesulfanhydride anhydride (10.1kg, 1.25 equivalents) with the speed that keeps temperature to be lower than 0 ℃.Reaction mixture-10 ℃ to 0 ℃ stirrings, is exhausted up to raw material.After reacting completely, reaction mixture is washed with 7.5%HCl solution (3 * 68kg, 17 equivalents) and water (48kg).Temperature＜5 ℃ that keep reaction mixture in the washing process.With 7.5% sodium hydrogen carbonate solution (55.0kg) purging compound, to regulate its pH 〉=6.Add triethylamine (0.4kg, 0.3kg/kg raw material), with the dry organic phase of Anhydrous potassium carbonate (1.2kg, 0.1 equivalent).Filtering mixt is extremely done in 20 ℃ to 35 ℃ vacuum concentration, obtain (3S, 4R, 5S, 6R)-4,5,6-three (benzyloxy) tetrahydrochysene-2H-pyrans-3-phenol.Not purified this triflate of use.

¹H?NMR(300MHz，CDCl ₃)：δ7.31-7.16(m，15H)，5.12(br?s，1H)，4.83(d，J＝4Hz，1H)，4.76(d，J＝11Hz，1H)，4.64(m，2H)，4.50(d，J＝9Hz，1H)，4.46(d，J＝8Hz，1H)，3.97(dd，J＝10，3Hz，1H)，3.86(d，J＝14Hz，1H)，3.77-3.72(m，2H).

Step 7: triflate is dissolved in DMF (36.2kg, 3.02kg/kg raw material) and is cooled to 10 ℃.Add Sodium Nitrite (5.9kg, 3.0 equivalents), the solution heat release is cooled to reaction mixture 15 ℃ to 25 ℃ then to about 30 ℃, stirs 12-16 hour.Mixture is cooled to 5 ℃, with the speed that keeps temperature＜15 ℃ add entry (152kg, 4.2kg/kgDMF).The gained mixture was stirred 2 hours in 10 ℃.Cross filter solid, water (2 * 12kg) washings.The filtering solid of institute is dissolved in the ethyl acetate (21.6kg, 1.8kg/kg raw material).Solution, filters through sal epsom (2.5kg) drying with salt solution (15.5kg) washing, and filtrate is extremely done in 35 ℃ of vacuum concentration.Add Virahol (9.5kg), be heated to 75 ℃ with the dissolving crude product.Heptane (24.6kg) is added in the solution, mixture is cooled to 15 ℃ to 25 ℃.Mixture further is cooled to 0 ℃, and stirring is spent the night.Cross filter solid, with heptane (2 * 8.2kg) washings.Product is dry in vacuum drying oven.Obtain yellow solid (3S, 4R, 5S, 6R)-4,5,6-three (benzyloxy) tetrahydrochysene-2H-pyrans-3-phenol, 5.3kg (44%).

¹H?NMR(300MHz，CDCl ₃)：δ7.37(m，15H)，4.96(d，J＝11Hz，1H)，4.80(m，2H)，4.68(d，J＝12Hz，1H)，4.61(m，2H)，4.53(d，J＝12Hz，1H)，3.78(m，1H)，3.67(m，3H)，3.50(dd，J＝9，3Hz，1H)，2.42(br?s，1H).

Step 8: (10.4kg 24.73moles) mixes with methylene dichloride (68.6kg, 6.6kg/kg raw material) and pyridine (9.8kg, 5 equivalents), is cooled to-10 ℃ with tribenzyl wood sugar derivative.Add trifluoromethanesulfanhydride anhydride (8.7kg, 1.25 equivalents) with the speed that keeps temperature to be lower than 0 ℃.Reaction mixture-10 ℃ to 0 ℃ stirrings, is exhausted up to raw material.After reacting completely, with 7.5%HCl solution (3 * 58.9kg, 17 equivalents) and water (41.6kg) washing reaction mixture.In the washing process, keep temperature＜5 ℃ of reaction mixture.With saturated sodium bicarbonate solution (44.6kg) purging compound, with its pH regulator to 〉=6.Add triethylamine (0.4kg, 0.3kg/kg raw material), with the dry organic phase of Anhydrous potassium carbonate (1.2kg, 0.1 equivalent).Filtering mixt is extremely done in 20 ℃ to 35 ℃ vacuum concentration.NMR？

Step 9: be dissolved in triflate among the THF (29kg, 2.8kg/kg raw material) and be cooled to 10 ℃.Add cyaniding Tetrylammonium (4.6kg, 1.2 equivalents), the solution heat release is cooled to reaction mixture 20 ℃ then, stirs 12 hours.Add ethyl acetate (21.8kg), with 10% sodium chloride solution (3 * 14.3kg) washing organic phases.Extract the water layer that is merged with ethyl acetate (21.8kg).Merge organic layer,, filter, be concentrated into dried with sal epsom (2kg) drying.Add ethanol (200pf, 3.23kg/kg raw material) and be heated to 70 ℃ with the dissolving crude product.Solution is cooled to 20 ℃, further is cooled to 5 ℃ then, stirring is spent the night.Cross filter solid, with heptane (2 * 10.4kg) washings.Repeat to carry out crystallization with 200pf ethanol (7mL/g solid).Cross filter solid, with heptane (2 * 10.4kg) washings.Product is dry in vacuum drying oven.Obtain (3R, 4R, 5S, 6S)-4,5,6-three (benzyloxy) tetrahydrochysene-2H-pyrans-3-formonitrile HCN is the light brown solid, 6.3kg (59%).

¹H NMR (300MHz, CDCl ₃): δ 7.31 (m, 15H), 4.90 (d, J=3Hz, 1H), 4.81-4.73 (complexity, 3H), 4.70 (d, J=12Hz, 1H), 4.62 (d, J=12Hz, 1H), 4.55 (d, J=12Hz, 1H), 3.99 (dd, J=9,5Hz, 1H), 3.91 (dd, J=12,3Hz, 1H), 3.82-3.74 (overlapped signal, 2H), 3.13m, 1H).

Step 10: (2.5kg 5.8moles) is dissolved in the absolute ethanol (138.1kg), is heated in 35 ℃ and obtains settled solution with carbonitrile derivatives.Add wetting palladium carbon (250g; 10%w/w), then add palladium hydroxide (250g; 20%w/w) and hydrochloric acid (0.6L).Solution with purging with nitrogen gas twice, is used the hydrogen purge once.With hydrogen solution is pressurized to 80psi, stirs, in 35 ℃ of heating 72 hours, pressurization once more as required.Mixture is filtered, in 30-35 ℃ of vacuum concentration.With isofagomine hydrochloride crude product and NH ₄The OH aqueous solution (3L) mixes.Solution is filtered, with 9: 1 ethanol/NH ₄OH aqueous solution solvent systems, by silicagel column (about 20kg) purifying.Product is at 35 ℃ to 40 ℃ vacuum concentration.The isofagomine free alkali is dissolved in absolute ethanol (7.7mL/g resistates), filters.L-(+)-tartrate (1185g, 1g/g resistates) is dissolved in absolute ethanol (7.7mL/g resistates), filters, slowly add in the ethanolic soln of isofagomine.With this solution stirring 45 minutes, filter, with ethanol (2.5L, 1mL/g raw material) washing.Product is dried to constant weight in 44 ℃ in vacuum drying oven.Obtain tartrate of isofagomine, be white solid, 1.2kg (purity is 97.5%).

¹HNMR(300MHz，D2O)：δ4.40(s，2H)，3.70(dd，J＝12，4Hz，1H)，3.66-3.58(m，2H)，3.38(m，3H)，2.83(t，J＝13Hz，1H)，2，79(t，J＝13Hz，1H)，1.88-1.77(m，1H).

Embodiment 2: The recrystallization of tartrate of isofagomine

At ambient temperature, with tartrate of isofagomine (1,767g) water-soluble (1.767L).Add absolute ethanol (1.767L), stirred 30 minutes.Drip the absolute ethanol (1.767L) of another aliquots containig and stirred 30 minutes with slow flow velocity.Repeat twice of this process (comprising 30 minutes stirring), obtain the solution of 4: 1 ethanol/waters.Cross filter solid, with ethanol/water (4: 1) washing, in vacuum drying oven in 43 ℃ of dried overnight to constant weight (be after drying clean weightless after other 2 hours＜1%).The productive rate of recrystallization is 91%.Find that according to HPLC sample contains 1.3% impurity.The NMR spectrum of recrystallized product is shown in Fig. 3 and Fig. 4.Fusing point 168-169 ℃.

Embodiment 3: Synthesizing of tartrate of isofagomine

Isofagomine L (+)-tartrate (2: 1)

(102mg, 0.679mmol) solution in deionized water (1.0mL) adds in the solution of isofagomine (200mg, 2.0 equivalents) in deionized water (2.0mL) in room temperature with L (+)-tartrate.With solution stirring 10 minutes, lyophilized overnight.With resistates in 45 ℃ further dry 3 days in a vacuum, obtain required salt (275.6mg, 91%).Fusing point 92-93 ℃,

¹H NMR (300MHz, D ₂O): δ 4.22 (s, 2H), 3.71 (dd, J=12,3.6Hz, 1H), 3.67-3.59 (m, 2H), 3.44-3.37 (m, 3H), 2.85 (t, J=12Hz, 1H), 2.75 (t, J=12Hz, 1H), 1.85 (m, 1H) (Fig. 8 A)

Isofagomine D-(-)-tartrate (2: 1)

(102mg, 0.679mmol) solution in deionized water (1.0mL) adds in the solution of isofagomine (200mg, 2.0 equivalents) in deionized water (2.0mL) in room temperature with D-(-)-tartrate.With solution stirring 10 minutes, lyophilized overnight.With resistates in 45 ℃ further dry 3 days in a vacuum, obtain required salt (287.2mg, 95%).Fusing point 94-95 ℃,

¹H NMR (300MHz, D ₂O): δ 4.22 (s, 2H), 3.71 (dd, J=12,3.6Hz, 1H), 3.67-3.59 (m, 2H), 3.44-3.36 (m, 3H), 2.85 (t, J=12Hz, 1H), 2.75 (t, J=12Hz, 1H), 1.84 (m, 1H) (Fig. 8 B).

Isofagomine D-(-)-tartrate (1: 1)

(204mg, 1.359mmol) solution in deionized water (2.0mL) adds in the solution of isofagomine (200mg, 2.0 equivalents) in deionized water (2.0mL) in room temperature with D-(-)-tartrate.With solution stirring 10 minutes, lyophilized overnight.With resistates in 45 ℃ further dry 3 days in a vacuum, obtain required salt (396.9mg, 98%).Fusing point 73-74 ℃,

¹H NMR (300MHz, D ₂O): δ 4.41 (s, 2H), 3.71 (dd, J=12,3.3Hz, 1H), 3.66-3.59 (m, 2H), 3.44-3.36 (m, 3H), 2.84 (t, J=12Hz, 1H), 2.75 (t, J=12Hz, 1H), 1.84 (m, 1H) (Fig. 8 C)

Embodiment 4: the capsular preparation of tartrate of isofagomine

	The 10mg capsule, prototype 1			The 100mg capsule, prototype 2
	The 10mg capsule, prototype 1			The 100mg capsule, prototype 2				％w/w	The mg/ capsule	G/ criticizes	％w/w	The mg/ capsule	G/ criticizes
Tartrate of isofagomine	5.50	10.00	15.00	50.00	100.00	40.00		％w/w	The mg/ capsule	G/ criticizes	％w/w	The mg/ capsule	G/ criticizes
Tartrate of isofagomine	5.50	10.00	15.00	50.00	100.00	40.00	Avicel PH102  (Microcrystalline Cellulose)	94.00	170.79	256.19	49.50	99.00	39.60
Magnesium Stearate	.050	.091	1.36	0.50	1.00	.040	Avicel PH102  (Microcrystalline Cellulose)	94.00	170.79	256.19	49.50	99.00	39.60
Magnesium Stearate	.050	.091	1.36	0.50	1.00	.040	Total amount	100.00	181.70	272.55	100.00	200.00	80.00
Opaque white color capsule shell size tbd	1500 capsules (estimating capsule shell size=2 or 3)			400 capsules (estimating capsule shell size=2 or 3)			Total amount	100.00	181.70	272.55	100.00	200.00	80.00

	The 10mg capsule, prototype 3			The 100mg capsule, prototype 4
	The 10mg capsule, prototype 3			The 100mg capsule, prototype 4				％w/w	The mg/ capsule	G/ criticizes	％w/w	The mg/ capsule	G/ criticizes
Tartrate of isofagomine	4.35	10.00	15.00	40.00	100.00	40.00		％w/w	The mg/ capsule	G/ criticizes	％w/w	The mg/ capsule	G/ criticizes
Tartrate of isofagomine	4.35	10.00	15.00	40.00	100.00	40.00	Emcompress ^(secondary calcium phosphate)	47.43	109.08	163.62	29.60	74.00	29.60
Avicel?PH102 ^(Microcrystalline Cellulose)	47.43	109.08	163.62	29.60	74.00	29.60	Emcompress ^(secondary calcium phosphate)	47.43	109.08	163.62	29.60	74.00	29.60
Avicel?PH102 ^(Microcrystalline Cellulose)	47.43	109.08	163.62	29.60	74.00	29.60	Cab-O-Sil ^(colloid (gas phase) silicon-dioxide)	.30	.69	1.04	.30	.75	.30
Magnesium Stearate	0.50	1.15	1.73	.050	1.25	.050	Cab-O-Sil ^(colloid (gas phase) silicon-dioxide)	.30	.69	1.04	.30	.75	.30
Magnesium Stearate	0.50	1.15	1.73	.050	1.25	.050	Total amount	100.00	230.00	345.00	100.00	250.00	100.00
Opaque white color capsule shell size tbd	1500 capsules (estimating capsule shell size=2 or 3)			400 capsules (estimating capsule shell size=2 or 3)			Total amount	100.00	230.00	345.00	100.00	250.00	100.00

Embodiment 5: improve in the active cell of GCase in Gaucher disease patient's the inoblast

Adopt the interior activity that increases of cell of inoblast research isofagomine L-(+)-tartrate of Gaucher disease patient.In order to estimate the effect of IFG to mutant GCase level, employing has been carried out external response investigations from the scavenger cell and the EBV-conversion lymphoblast of 40 patients' peripheral leukocytes.

This research comprises that 21 are suffered from the male sex of I type Gaucher disease, 1 male sex who suffers from III type Gaucher disease and 18 women that suffer from I type Gaucher disease.Patient age is 7 to 83 years old, has 38 to accept enzyme replacement treatment (ERT) among 40 patients.Successfully take out scavenger cell in 34 from 40 patients, wherein 32 show that when adopting tartrate of isofagomine treatment (5 days) the GCase level is the raising of dose-dependently (average=2.8 times).From other 5 lymph matricyte systems of taking from the patient, observe similar result.Isofagomine has significantly improved the level of GCase in different genotype patient's the cell, and described genotype comprises N370S/N370S (11), N370S/L444P (8), N370S/84insG (11), N370S/R163X, N370S/Y212H, L444P/del 136T, L444P/F216Y, L444P/L174F, G202R/R463C and the compound B exon 9/10 of K79N/ (III type GD).About 30 μ M isofagomines have obtained the maximum raising of GCase in the scavenger cell.

Embodiment 6: the I phase of security, pharmacokinetics and pharmacodynamics that is used for the treatment of the new pharmacology chaperone tartrate of isofagomine of Gaucher disease is studied

Tartrate of isofagomine is to be used for the treatment of the pharmacology chaperone that the lysosomal storage disease Gaucher disease is developed.Application is based on the model and the animal model of cell, and we are verified, and isofagomine has improved the cell levels of the glucocerebrosidase that is lacked in the Gaucher disease (GCase).In 72 healthy volunteers (39 male sex, 33 women), carried out randomized, double-blind I phase clinical study.Tartrate of isofagomine passes through oral administration with the form of the aqueous solution.In the research of first-in-human single ascending-dose, using (6 every group are active medicine, and 2 are placebo) dosage is 8,25,75,150 (two groups) and 300mg.In the research of ascending-dose repeatedly, use 25,75 and the dosage of 225mg every day, totally 7 days (6 every group is active medicine, and 2 is placebo).In two researchs, tartrate of isofagomine is all tolerated well at all dosage on the whole, and the urgent adverse events that the treatment that occurs in two researchs causes also great majority is gentle.Serious adverse events does not take place.

The tartrate of isofagomine oral route has shown good systemic drug exposure (systemicexposure).In single dose research, plasma A UC and C _MaxValue and dosage linear dependence.Average plasma levels was at 3.4 hours peakings (SEM:0.6 hour), and the plasma clearance transformation period is 14 hours (SEM:2 hour).In multiple doses research, oral administration found that pharmacokinetics behavior and dosage are linear dependence after 7 days, and the isofagomine of not expecting is accumulated.T _MaxSimilar with the transformation period with the observations of studying at single dose.

In multiple doses research, during using tartrate of isofagomine the 1st, 3,5 with 7 days and treatment back wash-out phase during the 9th, 14 with measured in 21 days separate GCase activity in the white corpuscle.Accept among the curee of tartrate of isofagomine at all, the GCase level significantly improves during the treatment, descends along with the removing of medicine subsequently, gets back to the level (Fig. 9) near baseline on the 21st day.The raising of enzyme level is that dosage is relevant, reaches to be higher than about 3.5 times of baseline values.The result of these relevant securities that obtain in the healthy volunteer, pharmacokinetics and preliminary pharmacodynamics effect provides support for further estimating tartrate of isofagomine to the therapeutic action of Gaucher disease.

The invention is not restricted to the scope of particular described herein.Really, except as herein described those, various accommodations of the present invention will become according to above stated specification and accompanying drawing and will be apparent to those skilled in the art.This class accommodation falls into the scope of claims.

Should be understood that further that all values are for being worth approximately, they are to be used for describing.

The application has quoted patent, patent application, publication, the description of product and scheme in the whole text, and their disclosure integral body is incorporated herein the reference that is used for all purposes.

Claims

1. the tartrate of isofagomine (tartrate of isofagomine) compound has following representational chemical structure:

Wherein n is 1 or 2.

2. the compound of claim 1, wherein tartrate of isofagomine is pure basically form.

3. the compound of claim 1, wherein the purity of tartrate of isofagomine is at least 95%.

4. the compound of claim 1, wherein the purity of tartrate of isofagomine is at least 98%.

5. the compound of claim 1, wherein the purity of tartrate of isofagomine is at least 99%.

6. the compound of claim 1, wherein n=1.

7. the compound of claim 1, wherein n=2.

8. the compound of claim 1, wherein tartrate of isofagomine is L-(+)-tartrate of isofagomine, and n=1.

9. the mixture of tartrate and isofagomine.

10. the composition that comprises tartrate of isofagomine.

11. the composition of claim 10, wherein said composition comprises the tartrate of isofagomine of at least 50% weight.

12. the composition of claim 10, wherein said composition comprises the tartrate of isofagomine of at least 90% weight.

13. the composition of claim 12, wherein 90% or above tartrate of isofagomine have 1200 μ m or littler particle diameter.

14. the composition of claim 10, said composition comprise the tartrate of isofagomine of at least 99% weight.

15. prepare the method for tartrate of isofagomine, comprising:

(a) isofagomine free alkali or its ionized form and tartrate are reacted in solvent; (b) separate tartrate of isofagomine.

16. the method for claim 15, its unresolvable tartaric acid are L-(+) tartrate.

17. the method for claim 15 wherein prepares one kilogram of tartrate of isofagomine at least.

18. the method for claim 15, wherein solvent is the alcohol of water-based.

19. the method for claim 15, wherein alcohol is selected from methyl alcohol, ethanol, propyl alcohol, butanols or their mixture.

20. the method for claim 15, wherein alcohol is ethanol.

21. the method for claim 15 is wherein separated the poly-recrystallization that comprises of step.

22. the method for claim 21, wherein 90% or above tartrate of isofagomine have 1200 μ m or littler particle diameter.

23. the method for claim 21, wherein recrystallization carries out in water by secondary solvent (secondary solvent).

24. the method for claim 22, wherein secondary solvent is a protic solvent.

25. the method for claim 22, wherein protic solvent is an alcohol.

26. prepare the method for high purity tartrate of isofagomine with industrially scalable, this method comprises:

(a) about 1kg or more isofagomine free alkali or its ionized form and tartrate are reacted in solvent;

(b) obtain the tartrate of isofagomine crude product;

(c) adopt aqueous solution to make tartrate of isofagomine crude product recrystallization; With

(d) tartrate of isofagomine of separating high-purity.

27. the method for claim 26, wherein aqueous solution is a water.

28. the method for claim 24, wherein aqueous solution is the mixture of water and alcohol, and wherein water and alcohol are used simultaneously or used with interval.

29. the method for claim 24, wherein high purity is at least 84% purity by weight.

30. the method for claim 24, wherein high purity is at least 98% purity by weight.

31. the method for claim 24, wherein high purity is at least 99% purity by weight.

32. the method for claim 24, wherein 90% or above high purity tartrate of isofagomine have 1200 μ m or littler particle diameter.

33. pharmaceutical composition comprises the tartrate of isofagomine and the pharmaceutically acceptable vehicle of medicine effective quantity.

34. the pharmaceutical composition of claim 33, wherein medicine effective quantity is about 5 to the 300mg/ agent.

35. the pharmaceutical composition of claim 33, wherein medicine effective quantity is about 10 to the 100mg/ agent.

36. the pharmaceutical composition of claim 33, wherein pharmaceutically acceptable vehicle is Microcrystalline Cellulose, Magnesium Stearate or secondary calcium phosphate.

37. the pharmaceutical composition of claim 33, wherein tartrate of isofagomine is a crystallized form.

38. compound is the tartrate of isofagomine crystallization.

39. the compound of claim 38, wherein tartrate of isofagomine crystalline X-ray powder diffraction pattern comprises 5 or more a plurality of peak in the following peak: (2 θ) 9.29 ± 0.009,14.17 ± 0.009,16.34 ± 0.009,18.07 ± 0.009,18.72 ± 0.009,19.44 ± 0.009,20.56 ± 0.009,22.13 ± 0.009,23.01 ± 0.009,24.54 ± 0.009 and 27.12 ± 0.009.

40. the compound of claim 38, wherein tartrate of isofagomine crystalline X-ray powder diffraction pattern comprises following peak: (2 θ) 9.29,14.17,16.34,18.07,18.72,19.44,20.56,22.13,23.01,24.54 and 27.12.

41. the compound of claim 38, wherein the tartrate of isofagomine crystallization has the X-ray powder diffraction pattern substantially the same with figure shown in Figure 5.

42. the method for treatment Gaucher disease, this method comprise that the individuality to the needs treatment gives the tartrate of isofagomine of significant quantity.

43. the method for claim 42, wherein tartrate of isofagomine is pure basically.

44. the method for claim 42, wherein treatment further comprises and gives functional glucocerebrosidase to individuality.

45. the method for claim 42, wherein the significant quantity of tartrate of isofagomine is about 10-100mg/ agent.

46. the method for glucocerebrosidase activity in the raising mammalian cell, described method comprise cell is contacted with the tartrate of isofagomine that effectively improves the amount of glucocerebrosidase activity.

47. the method for claim 46, wherein tartrate of isofagomine is a crystallized form.

48. the method for claim 46, wherein the X-ray powder diffraction pattern of this crystallized form comprises 5 or more a plurality of peak in the following peak: (2 θ) 9.29 ± 0.009,14.17 ± 0.009,16.34 ± 0.009,18.07 ± 0.009,18.72 ± 0.009,19.44 ± 0.009,20.56 ± 0.009,22.13 ± 0.009,23.01 ± 0.009,24.54 ± 0.009 and 27.12 ± 0.009.

49. the method for claim 46, wherein mammalian cell behaviour cell.

50. the method for claim 46, wherein tartrate of isofagomine combines with glucocerebrosidase is reversible with the amount of effectively stablizing glucocerebrosidase.

51. the pharmacologically acceptable salt of isofagomine, wherein pharmacologically acceptable salt easily adopts aqueous solution with 1kg or the more extensive recrystallization that carries out, and is tartrate of isofagomine.