CN101842356A - Polymorphs of 3- ( ( (4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino) -methyl) -phenoxy) -acetic acid sodium salt or a hydrate thereof and methods for making the same - Google Patents

Polymorphs of 3- ( ( (4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino) -methyl) -phenoxy) -acetic acid sodium salt or a hydrate thereof and methods for making the same Download PDF

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CN101842356A
CN101842356A CN200880113737A CN200880113737A CN101842356A CN 101842356 A CN101842356 A CN 101842356A CN 200880113737 A CN200880113737 A CN 200880113737A CN 200880113737 A CN200880113737 A CN 200880113737A CN 101842356 A CN101842356 A CN 101842356A
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crystalline form
amino
methyl
pyridine
alkylsulfonyl
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戴维·B·戴蒙
洪金阳
约瑟夫·F·克齐扎尼亚克
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Pfizer Products Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/70—Sulfur atoms
    • C07D213/71—Sulfur atoms to which a second hetero atom is attached
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4406—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 3, e.g. zimeldine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Abstract

The present invention relates to polymorphic crystalline forms or a non- crystalline form or amorphous of the compound (3-(((4-tert-butyl-benzyl)-(pyridine- 3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt or a hydrate thereof together with processes for preparing, methods for using, and pharmaceutical compositions containing the same. The invention also relates to substantially pure polymorphic crystalline forms or a non-crystalline form or amorphous form of (3- (((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt or a hydrate thereof.

Description

Polymorphs body of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt or its hydrate and preparation method thereof
Technical field
The present invention relates to the therapeutic activity of prostaglandin(PG) and the polymorphs body of selective modulator, especially EP 2The polymorphs body of agonist, comprise the pharmaceutical composition of these compounds, the preparation method of these compounds and these compounds are used for the treatment of the application of the patient's condition relevant with the adjusting of prostaglandin(PG) (for example, bone disorders, glaucoma and high intraocular pressure).More specifically; the present invention relates to the polymorphs body of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt; the pharmaceutical composition that comprises the polymorphs body of this compound, the preparation method of these polymorphs bodies and the polymorphs body of this compound are used for the treatment of the application of the patient's condition relevant with the adjusting of prostaglandin(PG).
Background technology
In drug discovery process, it has been generally acknowledged that the stable crystal form of finding medicine is extremely important.This stable crystal form is possible have best chemical stability and thereby have a form of preservation period the longest in preparation.Yet, further advantageously have the multiple form of medicine, for example, salt, hydrate, polymorphs body, crystalline form and amorphous form.Because different physical form provides different advantages, thereby does not have a kind of ideal medicine physical form.The result is difficult to expect to the searching of stable form and described other form is very difficult.
Successful drug development needs it to satisfy the particular requirement for the treatment of as the treatment validity that is used for the patient.These requirements fall into two categories: (1) successfully makes the requirement of formulation and (2) successfully carried and settled medicine after pharmaceutical preparation was applied to the patient requirement.
The pharmaceutical preparation that exists many kinds to be used for using by all means, and different probably for the optimal drug form of different preparations.As mentioned above, pharmaceutical preparation must have enough preservation perives so that can successfully be distributed to the patient who needs treatment.In addition, oral drug preparation must be provided as medicine the form in the gi tract that can be dissolved in the patient when oral administration.For the oral administration of the formulation that discharges at once with tablet, capsule, suspension agent or bag agent etc., thereby it is desirable to have the complete solvent of higher drug salts of solvability or medicament forms assurance dosage and the bioavailability of the best usually as release at once.For the relatively poor medicine of some drugs, particularly low-solubility drug or wettability, maybe advantageously utilize noncrystal medicament forms, noncrystal medicament forms has higher initial dissolution degree than crystalline form usually when being applied to gi tract.The amorphous form of medicine is often poorer than the chemical stability of crystalline form.Therefore, thus advantageously identify and have the noncrystal medicament forms that sufficient chemical stability can provide following practical product: the stability of this product is enough to enough allowing formulation manufacturing, packing, storing and be distributed in time of patient all over the world and keep its effectiveness.
On the other hand, there is the formulation that can work better during than indissoluble when medicament forms.For example, chewable tablets or suspension agent or bag agent formulation make tongue directly contact with medicine.For this class formulation, it is solid-state to it is desirable to make the solvability of medicine in mouth to minimize so that keep the part of medicine to be in, thereby farthest reduces halitosis.For this class formulation, it is desirable to use low-solubility salt or crystalline form usually.
For oral or injectable (for example, the subcutaneous or intramuscular) formulation of controlled release, the drug solubility of expectation is the complicated function that transport way, dosage, dosage form design and expectation discharge duration.For medicine, may it is desirable to utilize than the crystalline salt of low-solubility or polymorphs body being used for the controlled release formulation, so that helps to realize slowly release by slow dissolving with high resolution.For medicine, may must utilize higher deliquescent crystalline salt or polymorphs body or amorphous form so that obtain enough dissolution rate to support required drug release rate from the controlled release type with low-solubility.
In soft gelatin capsule formulation (" soft capsule "), medicine dissolution is in a spot of solvent or supporting agent (as triglyceride oil or polyoxyethylene glycol) and be packaged in the gelatine capsule.Optimal drug form for this formulation is the higher deliquescent form that has in suitable soft capsule supporting agent.Usually, more easily molten medicament forms is understood more indissoluble in water in triglyceride oil.Evaluation to the suitable medicament forms that is used for soft capsule dosage form need be studied various salt, polymorphs body, crystal and amorphous form.
Therefore as seen, the expectation solvability of medicament forms depends on desired use, and is not that all medicament forms all are equal to.
For the medicament forms that can be actually used in the mankind or treatment of animals, it is desirable to this medicament forms and show minimum water absorbability.The formulation that contains height water absorbability medicine needs the protective packing, and if may demonstrate the dissolving situation of change may be stored in the wet environment time.Therefore, it is desirable to identify the non-hygroscopic crystalline salt and the polymorphs body of medicine.If medicine is noncrystal, perhaps amorphous form is improved solvability and dissolution rate if desired, then it is desirable to identify the noncrystal salt or the form that have for other noncrystal salt or form than agent of low hygroscopicity.
Crystal or non-crystal medicine may be with anhydrous forms or as hydrate or solvate or hydrate/solvate and exist.The hydration status of medicine and solvation state influence its solvability and solubility behavior.
For different salt, polymorphs body, crystal and amorphous form, the fusing point of medicine may be different.In order to make it possible on commercial tabletting machine, to make tablet, the fusing point that it is desirable to medicine greater than about 60 ℃, be preferably greater than about 100 ℃ to avoid medicine in the tablet manufacturing processed, to melt.In this case, preferred medicament forms is the form with peak melting point.In addition, it is desirable to have high-melting-point to guarantee the chemical stability of the solid pharmaceutical in the solid dosage of higher ambient storage temperature (for example, sunlight direct radiation and as near geographic area such as the equator).Soft capsule dosage form preferably has the lower medicament forms of fusing point so that the crystallization of formulation Chinese traditional medicine is minimized if desired.Therefore as seen, the expectation fusing point of medicament forms depends on desired use, and is not that all medicament forms all are equal to.
When the dosage of medicine is higher, or during if desired little formulation, the selection of salt, hydrate or solvate can influence the effectiveness of per unit weight.For example, the drug salts with gegenion of higher molecular weight will have the pharmaceutical efficacy of lower every gram than the drug salts of the gegenion with lower molecular weight.It is desirable to select have the medicament forms of the effectiveness of the highest per unit weight.
The preparation method of different crystal crystal formation and amorphous form difference between different pharmaceutical is very big.It is desirable in these methods to use the solvent of toxicity minimum, particularly for last synthesis step, and if particularly medicine tend to the synthetic final step in used solvent exist with solvate.The preferred agents form is to utilize the form of the less solvent of toxicity in it is synthetic.
Medicine forms good tablet on commercial size ability depends on various medicine physical propertiess, for example, and Hiestand H, Smith D.Indices of tableting performance.Powder Technology, 1984; Compressing tablet index described in the 38:145-159 (Tableting Indices).These indexs can be used to identify the medicament forms (for example) with excellent compressing tablet performance.A this index is a reflection brittle embrittlement index (BFI), and scope is 0 (good-low fragility)～1 (poor-high fragility).Other mechanical properties, the useful indicators or the standard of measurement of flowing property and compressing tablet performance comprise stress under compression, absolute density, solid fraction, dynamic penetration hardness, ductility, Young's modulus, the reduction Young's modulus, the quasistatic penetration hardness, shearing modulus, tensile strength, impaired tensile strength (compromised tensile strength), the optimum adhesion index, the poorest bonding index, the bonding index of fragility/visco-elasticity, the strain index, the viscoelastic number, effective angle of inner friction (from shear cell (shear cell) test), cohesion (from powder avalanche test) and rheological.Many these standard of measurements obtain on the medicine compressing tablet, and the medicine compressing tablet preferably adopts three water pressure engine preparations.Many these standard of measurements are further described in Hancock B, Carlson G, Ladipo D, Langdon B and Mullamey M.Comparison of the Mechanical Properties of theCrystalline and Amorphous Forms of a Drug Substance.International Journal ofPharmaceutics, 2002; Among the 241:73-85.
It is not only very important for the manufacturing of Tabules to influence mobile medicament forms character, and also very important for the manufacturing of capsule, suspension agent and bag agent.
The size distribution of drug powder also may make a big impact to manufacturing processed, particularly the influence by flow of powder.Different taking on a different character property of medicament forms size distribution.
Apparent from above-mentioned discussion, do not exist and a kind ofly use all ideal medicament forms for all treatments.Therefore, importantly seek multiple unique medicament forms that can be used for various preparations, for example, salt, polymorphs body, amorphous form.The selection of the medicament forms of using for concrete formulation or treatment need be considered aforesaid various character, and may be to have a kind of good character of particularly important and other character can be accepted or inadequate acceptable form for the optimised form of application-specific.
United States Patent (USP) the 6th, 498 discloses a kind of for example compound of bone disorders (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate that can be used for treating 172B1 number.This application is being mentioned pharmacologically acceptable salt in general sense and is being disclosed the preparation of sodium salt.Yet this application had not both been described any polymorphs body form that any crystallisation step is not discussed this compound yet.
International Patent Application WO of having announced 99/19300 and WO 98/28264 disclose prostaglandin agonists and they in order to treat by topical application (for example, being applied to fracture or osteotomy site) and to promote to fracture and the purposes of osteotomy healing.
S.C.Miller and S.C.Marks, Jr., Bone 14, and 143-151 (1993) has studied and has passed through PGE 1(PGE 1) to the lip-deep new osteoplastic local excitation of the periosteum of dog mandibular bone, and the conveying of having compared the controlled release pill that passes through the osmotic pressure Micropump and pass through to implant at the subperiosteum of contiguous mandibular bone outside cortex.
S.C.Marks, Jr. and S.C.Miler, J.Oral Pathol.17:500-505 (1988) have reported that the dosage with 500 μ g/ week～2000 μ g/ weeks forms produced surprising locality alveolar bone in 3 weeks of PGEl local infusion in the lower jaw of dog.
At M-S.Shih and R.W.Norrdin, among the Am.J.Vet.Res.48:828-830 (1986), in the rib of beasle dog of growing up, perform a surgical operation and cause transverse fracture, and every day twice 10% ethanol Tris damping fluid supporting agent or 0.5ml PGE with 0.5ml 1(in 10% ethanol Tris damping fluid, contain 0.2mgPGE 1) direct injection is to fracture site, continues 10 days.According to inferring, PGE 1Use the formation that has caused the ground substance of bone on the periosteum envelope adjacent with fracture site and offside thereof coupling site.
M-S.Shih and R.W.Norrdin, Calcif.Tissue Int. (1986) 39:191-197 have studied the PGE that performs the operation in back 10 days with in the defect sites of double injection every day to the shin bone of beasle dog 1The effect of (0.2mg/kg, 10% ethanolic soln).It is found that, accept PGE in the part 1Dog have more periosteum and cortex bone inner membrance bone forming, and the amount that shows osteoid increases.
R.Yang, T.Liu and S.Lin-Shiau, Calcif.Tissue Int., 52:57-61 (1993) studied in 14 days every day by bone in approach be injected to the effect of the prostaglandin E2 in the metaphysis of left tibia.According to this reference, this dosage regimen causes the remarkable increase of trabecular bone in the metaphysis.
K.Notoya etc., The Journal of Pharmacology and Experimental Therapeutics, 290:1054-1064 (1999) investigated a kind of novel osteoblast differentiation promote compound TAK-778 when continuing to discharge the microcapsule topical application for the effect of regeneration of body endoskeleton and bone reparation.
Summary of the invention
According to another implementation of the invention, the invention provides the crystalline form of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt or its hydrate.
According to another embodiment of the present invention, the invention provides and comprise (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt or the crystalline form of its hydrate and the pharmaceutical composition of pharmaceutically acceptable diluent or carrier for the treatment of significant quantity.
According to another implementation of the invention; the invention provides the mammiferous method that treatment suffers from glaucoma, high intraocular pressure or shows the patient's condition of low bone amount, described method comprises (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-the phenoxy group)-acetic acid sodium salt of this administration treatment significant quantity or the crystalline form of its hydrate.
According to another embodiment of the present invention; the invention provides the method that improves and keep the bone amount in the Mammals, described method comprises (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-the phenoxy group)-acetic acid sodium salt of this administration treatment significant quantity or the crystalline form of its hydrate.
According to another embodiment of the present invention, the invention provides the preparation method of the crystalline form A of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt semihydrate, described method comprises that (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-the phenoxy group)-acetate that makes as free acid contacts to form reaction mixture with sodium hydroxide in organic solvent; This reaction mixture is heated to about 50 ℃～about 90 ℃ first temperature and keeps time of at least 1 hour in this first temperature; Described reaction mixture is cooled to the about 15 ℃～second about 25 ℃ temperature; And from this reaction mixture, collect solid.Used organic solvent can be the isopropyl acetate that exists with about 8ml～about 12ml/mmol (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate in this method.The used sodium hydroxide of this method can be 50% the aqueous sodium hydroxide solution that exists with about 1mmol～about 1.3mmol/mmol (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate.
According to another implementation of the invention, the invention provides the amorphous form or the amorphous form of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt or its hydrate.
According to another embodiment of the present invention, the invention provides and comprise (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt or the amorphous form of its hydrate or the pharmaceutical composition of amorphous form and pharmaceutically acceptable diluent or carrier for the treatment of significant quantity.
According to another embodiment of the present invention; the invention provides the mammiferous method that treatment suffers from glaucoma, high intraocular pressure or shows the patient's condition of low bone amount, described method comprises (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-the phenoxy group)-acetic acid sodium salt of this administration treatment significant quantity or the amorphous form or the amorphous form of its hydrate.
According to another implementation of the invention; the invention provides the method that improves and keep the bone amount in the Mammals, described method comprises (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-the phenoxy group)-acetic acid sodium salt of this administration treatment significant quantity or the amorphous form or the amorphous form of its hydrate.
Compound " (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate " is meant the free acid of this compound.
Term " Mammals " is meant and comprises for example dog, cat, ox, sheep, horse and the mankind's etc. animal.Preferred mammal comprises the mankind.
Phrase " substantially pure " is meant the required polymorphs body of total amount for compound (that is the amount of required polymorphs body and/or salt and any other polymorphs body and amorphous form, salt and free acid) and/or the relative purity of salt.The polymorphic forms of " substantially pure " should contain with respect to total amount of compound at least about 90% required polymorphs body.Preferably, the polymorphic forms of " substantially pure " should contain the required polymorphs body at least about 95%.In some embodiments of the present invention, the polymorphic forms of " substantially pure " can contain the required polymorphs body at least about 99%.
Phrase " shows the patient's condition of low bone amount " and is meant the wherein flat patient's condition that is lower than as the World Health Organization " Assessment of Fracture Risk and its Application to Screening forPostmenopausal Osteoporosis (1994); Report of a World Health OrganizationStudy Group, World Health Organization Technical Series 843 " the age specificity normal value that standard limited of bone water gaging.Comprise primary and insecondary osteoporosis in " showing the patient's condition of low bone amount ".Secondary osteoporosis comprises that osteoporosis, hyperthyroidism inductive osteoporosis, the ligamentopexis of glucocorticoid inducible induce the osteoporosis of (immobilization-induced), heparin-induced osteoporosis and immunosuppressor inductive osteoporosis, comprise in addition that periodontopathy, alveolar bone are lost, behind the osteotomy and the Childhood the special property bone loss of sending out.Phrase " shows the patient's condition of low bone amount " and also comprises osteoporotic long-term complications, for example, and rachiocamposis, height reduction and prosthese operation.
The phrase patient's condition of bone amount " show low " also refers to chance that known its development comprises osteoporotic above-mentioned disease apparently higher than average Mammals (for example, Mammals), and for example, women after climacteric and age surpass 60 the male sex.
Other bone amount raising or enhanced purposes comprise that knitting, upper jaw reconstruction, mandibular reconstructure, the reconstruction of cranium face, prosthese interior grow (prosthetic ingrowth), vertebra synostosis, the long bone after bone reparation, increase union of fracture speed, wholly replace bone grafting, raising bone transplanting succeed rate, the facial reconstruction prolongs and spinal fusion.
Pharmaceutical composition of the present invention can also be united use as orthopaedics devices such as spinal fusion cage, spinal fusion hardware, inside and outside bone anchoring device, screw and pins with well known by persons skilled in the art.
Those of skill in the art will recognize that in fact term bone amount is meant the bone amount of per unit area, this sometimes (but being not correct on the stricti jurise) be called bmd (BMD).
That term used herein " treatment " comprises is preventative (for example, anti-pre-property), appeasing property (palliative) and curative therapy.
Term " significant quantity " is meant improvement, weakens or eliminates the symptom of specified disease or the patient's condition or the specified disease or the patient's condition or avoids or postpone the compound of morbidity of symptom of specified disease or the patient's condition or the specified disease or the patient's condition or the amount of compound combination.
Term " patient " is meant animal (for example, the people), companion animals (for example, dog, cat or horse) and domestic animal (for example, ox, pig and sheep).Particularly preferred patient comprises male and female Mammals, and then more preferably human.
Term used herein " pharmaceutically acceptable " is meant must compatible with other composition of preparation and can not be to the deleterious carrier of its recipient, supporting agent, thinner, vehicle and/or salt.
Expression " prodrug " is meant the compound as prodrug, and this compound discharges medicine (for example, a kind of prodrug is reaching physiology pH or is being converted into required medicament forms by enzyme as the time spent) in vivo by some chemistry or physiological processes after using.Exemplary prodrug discharges corresponding medical compounds through cutting the time.
Express " pharmacologically acceptable salt " and for example be meant anion salts such as (but being not limited to) chlorate, Bromide, iodized salt, vitriol, hydrosulfate, phosphoric acid salt, acetate, maleate, fumarate, oxalate, lactic acid salt, tartrate, Citrate trianion, gluconate, mesylate and 4-tosylate.This expression for example also refers to (but being not limited to) sodium salt, sylvite, calcium salt, magnesium salts, ammonium salt or protonated benzyl star (N, N '-dibenzyl ethylene diamine), choline, thanomin, diethanolamine, quadrol, meglumine (N-methyl glucoside amine), Benethamine diacetale (N-benzyl-1-phenylethylamine), piperazine and Trometamol cationic salts such as (2-amino-2 methylol-1, ammediols).
With reference to the following drawings, description and claim, these characteristics of the present invention, aspect and advantage and other characteristics, aspect and advantage will become and be easier to understand.
Description of drawings
Fig. 1 is the diffractogram of the form A of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt of the present invention;
Fig. 2 is the diffractogram of the form B of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt of the present invention;
Fig. 3 is the diffractogram of the form A of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt of the present invention;
Fig. 4 is the diffractogram of the form E of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt of the present invention;
Fig. 5 is the diffractogram of the form D of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt of the present invention;
Fig. 6 is the diffractogram of the form G of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt of the present invention;
Fig. 6 B is the diffractogram of the form H of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt of the present invention;
Fig. 6 C is the diffractogram of amorphous (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt of the present invention;
Fig. 7 is the solid-state of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt form A 13The C nuclear magnetic resonance spectrum; With
Fig. 8 is the solid-state of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt form H 13The C nuclear magnetic resonance spectrum.
Embodiment
Usually, can pass through United States Patent (USP) the 6th, 498, disclosed method preparation (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt in 172B1 number, this paper is by with reference to incorporating its whole subject contents into.Alternatively, can prepare (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt by the described novel method of following reaction formula I.Some manufacture method of the specific crystal formation of these compounds has been described in experimental section.All initial compounds can or come Sigma-Aldrich Corporation freely by the literature method acquisition, St.Louis, general commercial source such as MO.
In discussion, example and preparation, used following abbreviation: 2B EtOH-Denatured alcohol, br-broad peak, ℃-degree centigrade, d-doublet, DMSO-d 6-deuterated dimethyl sulfoxide, EtOAc-ethyl acetate, equiv-equivalent, g-gram, 1H NMR-proton magnetic resonance (PMR), H 2O-water, HCl-hydrogenchloride, Hz-hertz, iPr 2Net-diisopropylethylamine (HunigShi alkali), iPrOAc-isopropyl acetate, J-coupling constant (spacing of multiplet), kg-kilogram, L-liter, m-multiplet, M-volumetric molar concentration, MeCl 2-methylene dichloride, mg (s)-milligram, min-minute, mL-milliliter, mmol-mmole, mp-fusing point, MPa-MPa, MS-mass spectrum, N-equivalent concentration, NaHCO 3Platinum on-sodium bicarbonate, NaOH-sodium hydroxide, psi-pound/square inch, the Pt/C-carbon, RT-room temperature, s-are unimodal, and the t-triplet.In addition, in following discussion, embodiment and preparation, mention that Bruker is meant Bruker AXS, Inc.k Madison, the product of WI mentions that Kevex is meant Thermo Electron Corporation, Waltham, the product of Ma.
Reaction scheme I
Figure GPA00001118637200101
Step 1:{3-[(4-tertiary butyl benzylamino)-methyl]-phenoxy group } ethyl acetate succinate (II) Preparation
In 500mL Parr bottle (Parr bottle), add 2B ethanol (180mL) and platinum (2.00g) on 5% carbon of 50% water-wet.Add 3-formyl radical ethyl phenoxyacetate (20.0g, 96.06mmol, 2B ethanolic soln 1.0equiv), then add the 4-tert-butyl benzyl amine (16.86mL, 96.06mmol, 1.0equiv).Mixture was stirred 5 hours under room temperature and nitrogen atmosphere, at this moment the formation of the intermediate of perfect I.Reaction vessel placed under 50psi (0.3447MPa) hydrogen atmosphere and room temperature vibration 18 hours.Mixture is passed through diatomite filtration, and with 2B ethanol (40mL) washing leaching cake once.Filtrate is transferred in adaptive 1 liter of flask that dropping funnel and mechanical stirrer arranged.In 15 minutes, add succsinic acid (11.34g, 96.06mmol, 1.0equiv) hot solution in 2B ethanol (120mL).The gained slurries were filtered in stirring at room in 19 hours then.Solid is washed once also dry and generation II (33.10g, 73% productive rate) with 2B ethanol (50mL).mp＝138℃～139℃。C 22H 29NO 3C 4H 6O 4The analytical calculation value: C 65.94; H 7.45; N 2.96.Measured value: C 65.74; H 7.62; N 2.99. 1HNMR(400MHz，CD 3OD)：1.28(t，3H，J＝7.0Hz)，1.32(s，9H)，2.50(s，4H)，4.08(s，2H)，4.10(s，2H)，4.23(q，2H，J＝7.0Hz)，4.73(s，2H)，6.96-6.98(m，1H)，7.04-7.06(m，2H)，7.33-7.38(m，3H)，7.46-7.48(m，2H)。
Step 2 and 3:(3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)- The preparation of acetate (V)
(48.00g, 101mmol is 1.0equiv) by the baseization (free-based) of dissociating of partition between methylene dichloride (360mL) and 1M sodium bicarbonate aqueous solution (240mL) with Compound I I.Separate each layer and once with organic layer water (240mL) washing.Separate each layer and with N, (53.4mL, 304mmol 3.0equiv) are added into organic layer to the N-diisopropylethylamine.With pyridine hydrochloride-3-SULPHURYL CHLORIDE (III) (26.4g, 123mmol, 1.2equiv) and methylene dichloride (240mL) add independent reaction vessel and under nitrogen atmosphere, be cooled to 0 ℃.With free alkali/N, N-diisopropylethylamine solution added in 1.8 hours.Reaction mixture be heated to room temperature and kept 19 hours, this moment perfect IV intermediate ((3-{[(4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino]-methyl)-phenoxy group-ethyl acetate) formation.Reaction mixture with 1N hydrochloric acid (336mL) washing, is used 1M sodium bicarbonate aqueous solution (336mL) washing then.Add 2B ethanol (384mL) to organic layer, and mixture is about 400mL at air distillation volume in bottle.(19.9mL, 119mmol 1.2equiv) and with mixture kept 19 hours in room temperature to add 6N sodium hydroxide.Successively add concentrated hydrochloric acid (11.0mL, 134mmol, 1.3equiv) and water (192mL).Make slurries in room temperature granulation 5 hours and filtration.With solid with 50: 502B ethanol: water (144mL), water (144mL) and 2B ethanol (144mL) washing, dry then to generate compound V (34.78g, 73% productive rate).mp＝159℃～160℃。C 25H 28N 2O 5The analytical calculation value of S: C 64.08; H 6.02; N5.98.Measured value: C 64.13; H 6.11; N 5.99. 1H?NMR(400MHz，DMSO-d 6)：1.21(s，9H)，4.31(s，2H)，4.33(s，2H)，4.54(s，2H)，6.65-6.69(m，1H)，6.71-6.74(m，1H)，6.75-6.76(m，1H)，7.01(d，2H，J＝8.1Hz)，7.13(t，1H，J＝7.9Hz)，7.21(d，2H，J＝8.3Hz)，7.56-7.59(m，1H)，8.16-8.19(m，1H)，8.80-8.82(m，1H)，8.97-8.98(m，1H)，13.04(br?s，1H)。
Step 3R:(3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-second The recrystallization of acid (V)
(20.0g, 42.7mmol 1.0equiv) mix and are heated to 60 ℃ with methylethylketone (300mL) and all dissolve up to all solids with (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate (V).Solution is cooled to 35 ℃ of nylon filters that also pass through 0.45 micron to be filtered.With methylethylketone (20mL) washing filter.Filtrate merged then by air distillation volume to the bottle be about 160mL.Mixture is cooled to room temperature, and crystallization this moment begins.Solid is also dry to produce (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate (VI) (15.30g, 77% productive rate) with methylethylketone washing (using the 20mL washed twice, then with the 40mL washing once).mp＝159.0℃～159.5℃。C 25H 28N 2O 5The analytical calculation value of S: C 64.08; H 6.02; N 5.98.Measured value: C 63.89; H 5.82; N 5.91. 1H NMR (400MHz, DMSO-d 6): identical with above step 2 and 3. 1H NMR spectrum also indicates the existence of about 1.4% methylethylketone.
Step 4:(3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate The preparation of hemihydrate form A (VII)
(20.0g, 42.7mmol 1.0equiv) add reaction vessel with isopropyl acetate (400mL) with (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate (VI).Successively add 50% sodium hydroxide (3.520g, 44.0mmol, 1.03equiv) and deionized water (4.165g).(3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-(VII is 200mg) as seed crystal material for acetate hemihydrate form A in interpolation.Mixture was kept 5 hours at 30 minutes internal heating to 70 ℃ and in this temperature.Mixture was cooled to 40 ℃ in 4 hours, was cooled to 20 ℃ then in 1 hour, granulation 19 hours is filtered then.With solid with twice of 1% isopropyl acetate solution washing (each 20mL); dry then to obtain (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate hemihydrate form A (VII; 18.22g, 85% productive rate).The dehydration temperaturre of this semihydrate is about 120 ℃ and do not have fusing point.C 25H 27N 2O 5SNa1/2H 2The analytical calculation value of O: C61.21; H 5.55; N 5.71.Measured value: C 60.19; H 5.55; N 5.56.
1H?NMR(400MHz，DMSO-d 6)：1.21(s，9H)，4.07(s，2H)，4.31(s，4H)，6.59-6.61(m，1H)，6.64(m，1H)，6.71-6.74(m，1H)，7.01-7.09(m，3H)，7.22(d，2H，J＝8.4Hz)，7.56-7.59(m，1H)，8.14-8.17(m，1H)，8.79-8.80(m，1H)，8.94(m，1H)。
Alternatively, formation of the sodium salt of step 4 and recrystallization can use the step shown in the following reaction formula II to carry out.
The substituting sodium salt of scheme II-VII forms and recrystallization
(3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate (VI) (47.7g) is mixed with methyl alcohol (500mL).Slow interpolation 1N sodium hydroxide under stirring at room (1.01equiv, 103mL).Stir after 2 hours, mixture is concentrated and, use twice of methylene dichloride azeotropic (200mL at every turn) then to obtain amorphous foam with acetone azeotropic 5 times (each 300mL).To wherein adding acetone (500mL) and water (15mL), then with mixture heating up to 50 ℃.The material very thickness that becomes after 20 minutes, and add extra acetone (200mL).Mixture is cooled to room temperature and filtration.Filter cake is also under high pressure dry to obtain (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate hemihydrate form A (VII, 77% productive rate) of 39g with the washing of 2-propyl alcohol.
Powder x-ray diffraction
The X-ray powder diffraction collection of illustrative plates of crystalline form A, B, C, E, F and G by (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt comes it is characterized.Once, using copper radiation (wavelength: 1.54056
Figure GPA00001118637200132
) Bruker D5000 diffractometer on carried out the X-ray diffractogram spectrum analysis of form A, B, C, E, F and G.Tube voltage and strength of current are set at 40kV and 50mA respectively.Disperse with scatter slit and be set at 1mm, be set at 0.6mm and receive slit.By the radiation of Kevex PSI detector detection of diffracted.Use is with 2.4 °/minute (1 second/0.04 ° steps) θ-2 θ continuous sweep from 3.0 ° of 2 θ～40 ° 2 θ.Analyzed of the calibration of aluminum oxide standard substance with inspection apparatus.Collecting data also analyzes with Bruker axis software 7.0 editions.By sample being placed quartzy retainer prepare sample.
Measure in order to carry out X-ray diffraction on Prague-Franz Brentano instrument (for example, the Bruker system) that in the measurement of this paper report, uses, usually sample is placed in the retainer with cavity.Push sample powder to guarantee random surface and correct height of specimen by slide glass or Equivalent.Then sample retainer is placed in the instrument.At first with the angle guiding sample less with respect to the retainer plane, allow the incident X-rays bundle move along circular arc then the incident X-rays bundle, this circular arc can be added to the angle between irradiating light beam and the retainer plane continuously.The measurement difference relevant with this x-ray powder analysis is produced by the multiple factor that comprises following factor: (a) error in the specimen preparation (for example, height of specimen), (b) instrumental error (for example, the planar sample error), (c) alignment error, (d) operator error (comprising the error that presents when determining the peak position) and (e) material character (for example, preferred orientation and transparency error).Alignment error and height of specimen error often cause all peaks to same direction displacement.Less height of specimen difference will cause the displacement of XRPD peak position when using flat holder.Systematic Study shows, when using the Shimadzu XRD-6000 of typical Prague-Franz Brentano setting, the height of specimen difference of 1mm has caused peak shift (Chen etc., J Pharmaceutical and BiomedicalAhalysis, 2001 up to 1 ° of 2 θ; 26,63).These displacements can be obtained identifying and by this displacement being compensated (the system compensation factor is applied to all peak position values) or the recalibration instrument is eliminated by X-ray diffractogram.As mentioned above, thus can correct the measurement of being undertaken by different machines by the application system correction factor makes the peak position consistent.Usually, the peak position that can be measured by Bruker of this correction factor may be consistent in the scope of 0～0.2 ° of 2 θ with the prediction peak position.
X-ray powder diffraction collection of illustrative plates by (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt amorphous form H characterizes it.Use the Siemens D5000 diffractometer that adopts copper radiation to produce the X-ray powder diffraction collection of illustrative plates of form H.This instrument is equipped with line-focus tube.Tube voltage and strength of current are set at 38kV and 38mA respectively.Disperse with scatter slit and be set at 1mm, be set at 0.6mm and receive slit.By Kevex PSI detector detection of diffracted Cu K α 1Radiation (λ=1.54056
Figure GPA00001118637200141
).Use is with the θ-2 θ continuous sweep of 2.4 ° of 2 θ/minute (1 second/0.04 ° 2 θ step) from 3.0 ° of 2 θ～40 ° 2 θ.Analyzed the calibration of aluminum oxide standard substance (NIST standard reference material 1976) with inspection apparatus.Collecting data also analyzes with BRUKER AXS DIFFRAC PLUS software 2.0 editions.By sample being placed quartzy retainer prepare sample.Use the high PXRD peak of manually selecting of maximum peak.
With reference to figure 1, wherein shown the diffractogram of form A of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-the phenoxy group)-acetic acid sodium salt of an embodiment of the invention.The form A of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt is by (adopting CuK from ° 2 θ, d spacing and relative intensity αRelative intensity 〉=7% of measuring on the radiating Bruker D5000 diffractometer) the X-ray powder diffraction collection of illustrative plates of aspect expression characterizes:
The X-ray powder diffraction collection of illustrative plates of table 1-sodium-salt form A
Figure GPA00001118637200142
Figure GPA00001118637200151
* relative intensity may change according to crystalline size and form
With reference to figure 2, wherein shown the diffractogram of form B of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-the phenoxy group)-acetic acid sodium salt of an embodiment of the invention.The form B of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt is by (adopting CuK from ° 2 θ, d spacing and relative intensity αRelative intensity 〉=3% of measuring on the radiating Bruker D5000 diffractometer) the X-ray powder diffraction collection of illustrative plates of aspect expression characterizes:
The X-ray powder diffraction collection of illustrative plates of table 2-sodium-salt form B
Figure GPA00001118637200152
Figure GPA00001118637200161
* relative intensity may change according to crystalline size and form
With reference to figure 3, wherein shown the diffractogram of form A of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-the phenoxy group)-acetic acid sodium salt of an embodiment of the invention.The form A of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt is by (adopting CuK from ° 2 θ, d spacing and relative intensity αRelative intensity 〉=5% of measuring on the radiating Bruker D5000 diffractometer) the X-ray powder diffraction collection of illustrative plates of aspect expression characterizes:
The X-ray powder diffraction collection of illustrative plates of table 3-sodium-salt form C
Figure GPA00001118637200171
* relative intensity may change according to crystalline size and form
With reference to figure 4, wherein shown the diffractogram of form E of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-the phenoxy group)-acetic acid sodium salt of an embodiment of the invention.The form E of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt is by (adopting CuK from ° 2 θ, d spacing and relative intensity αRelative intensity 〉=4% of measuring on the radiating Bruker D5000 diffractometer) the X-ray powder diffraction collection of illustrative plates of aspect expression characterizes:
The X-ray powder diffraction collection of illustrative plates of table 4-sodium-salt form E
Figure GPA00001118637200181
* relative intensity may change according to crystalline size and form
With reference to figure 5, wherein shown the diffractogram of form D of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-the phenoxy group)-acetic acid sodium salt of an embodiment of the invention.The form D of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt is by (adopting CuK from ° 2 θ, d spacing and relative intensity αRelative intensity 〉=5% of measuring on the radiating Bruker D5000 diffractometer) the X-ray powder diffraction collection of illustrative plates of aspect expression characterizes:
The X-ray powder diffraction collection of illustrative plates of table 5-sodium-salt form F
Figure GPA00001118637200182
* relative intensity may change according to crystalline size and form
Refer now to Fig. 6, wherein shown the diffractogram of form G of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-the phenoxy group)-acetic acid sodium salt of an embodiment of the invention.The form G of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt is by (adopting CuK from ° 2 θ, d spacing and relative intensity αRelative intensity 〉=6% of measuring on the radiating Bruker D5000 diffractometer) the X-ray powder diffraction collection of illustrative plates of aspect expression characterizes:
The X-ray powder diffraction collection of illustrative plates of table 6-sodium-salt form G
Figure GPA00001118637200192
Figure GPA00001118637200201
* relative intensity may change according to crystalline size and form
Conclusion is got up, and table 7 has been listed ° 2 θ and the relative intensity of the diffracted ray in the sample of crystalline form A, B, C, E, F and G of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt:
Table 7:(3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-the cutting edge of a knife or a sword position and the relative intensity of diffracted ray in the sample of form A, B, C, E, F and the G of acetic acid sodium salt
Figure GPA00001118637200202
Because (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt only has 6 kinds of crystalline forms known, combination that can be by single X-ray powder diffraction line, line or the pattern that is different from the X-ray powder diffraction of other form are identified every kind of crystalline form and with its and other crystalline form differentiation.
For example; table 8 has been listed the unique peak of form A, B, C, E, F and G of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt and the combination at 2 θ peaks, promptly for one group of x ray diffraction line of every kind of form.
Table 8:(3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-unique peak of form A, B, C, E, F and the G of acetic acid sodium salt and the combination at 2 θ peaks
Form A (° 2 θ) Form B (° 2 θ) Form A (° 2 θ) Form E (° 2 θ) Form D (° 2 θ) Form G (° 2 θ)
??3.2 ??3.3 ??3.6 ??7.0 ??5.5 ??3.2
??4.1 ??3.6 ??4.0 ??14.1 ??8.2 ??4.0
??16.1 ??7.1 ??5.9 ??21.2 ??11.0 ??7.9
??20.2 ??8.4 ??13.3 ??22.3 ??19.2 ??10.0
??20.8 ??13.8 ??16.9 ??28.5 ??27.5 ??13.1
Refer now to Fig. 6 B, wherein shown the diffractogram of amorphous form H of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-the phenoxy group)-acetic acid sodium salt of an embodiment of the invention.The form H of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt is by (adopting CuK from ° 2 θ, d spacing and relative intensity αRelative intensity 〉=6% of measuring on the radiating Bruker D5000 diffractometer) the X-ray powder diffraction collection of illustrative plates of aspect expression characterizes:
Figure GPA00001118637200212
Figure GPA00001118637200221
Refer now to Fig. 6 C, wherein shown the diffractogram of amorphous form I of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-the phenoxy group)-acetic acid sodium salt of an embodiment of the invention.The form I of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt is by being characterized as X-ray powder diffraction collection of illustrative plates shown in this Fig.
Solid state nmr
Refer now to Fig. 7, the form A of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt also can be characterized by its solid state nmr spectrum (SSNMR).Therefore, (Buker-Biospin, Billerica MA) have carried out the solid state nmr spectrum analysis of form A on the Avance DSX 500MHz NMR spectrograph at Bruker-Biospin.
13C?SSNMR
With about 70mg compound close packing in the 4mm ZrO turner that is used for being subjected to analytic sample.At 293K and normal pressure, on the Bruker 4mm BL CPMAS probe that is positioned at wide footpath Bruker-Biospin Avance DSX 500MHz NMR spectrograph, use 1H- 13One dimension is collected in C cross polarization evil spirit angle rotation (CPMAS) 13The C spectrum.Sample is in the 15.0kHz rotation of specifying speed of rotation corresponding to the maximum of this 4mm turner.Speed of rotation minimizes the intensity at rotation peak, limit (spinning side band) faster.In order to optimize signal sensitivity, cross polarization is adjusted to 2.0ms duration of contact, and is 85kHz the decoupling power setting.Compose to carbon with 2,820 scanning collection that adopt 3 seconds circulation delays.These carbon spectrums adopt the diamantane external sample as reference, and its High-Field resonance is set at 29.5ppm.
The form A of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt is by following solid-state 13C nucleus magnetic resonance (SSNMR) stave is levied, and wherein chemical shift is expressed with 1,000,000/a (ppm):
Table 9: form A's is solid-state 13The C nuclear magnetic resonance spectrum
??# ?? 13C displacement [ppm]
??1 ??178.2
??2 ??176.3
??3 ??173.2
??4 ??159.3
??5 ??158.3
??6 ??155.7
??7 ??153.3
??# ?? 13C displacement [ppm]
??8 ??152.2
??9 ??150.6
??10 ??149.6
??11 ??148.8
??12 ??148.0
??13 ??139.8
??14 ??138.3
??15 ??137.4
??16 ??135.3
??17 ??134.6
??18 ??133.7
??19 ??131.2
??20 ??130.7
??21 ??129.6
??22 ??128.0
??23 ??125.1
??24 ??124.0
??25 ??122.7
??26 ??121.3
??27 ??120.5
??28 ??119.9
??29 ??118.4
??# ?? 13C displacement [ppm]
??30 ??114.1
??31 ??113.2
??32 ??67.8
??33 ??57.5
??34 ??55.4
??35 ??53.9
??36 ??52.1
??37 ??51.3
??38 ??49.1
??39 ??34.5
??40 ??33.7
??41 ??32.1
??42 ??31.2
* with respect to value in ppm at the trimethyl silane (TMS) of 0ppm; Use the diamantane external sample as reference, set its High-Field resonance and be 29.5ppm.
Refer now to Fig. 8, the form H of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt also can be characterized by its solid state nmr spectrum (SSNMR).Therefore, (Buker-Biospin, Billerica MA) have carried out the solid-state nuclear magnetic resonance spectrum analysis of form A on the Avance DSX 500MHz NMR spectrograph at Bruker-Biospin.
Be used for form H's 13C SSNMR method
Will about 70mg compound close packing in 4mm ZrO rotor.At 294K and normal pressure, on the Bruker4mm BL CPMAS probe that is positioned at wide footpath Bruker-Biospin Avance DSX 500MHz NMR spectrograph, use 1H- 13One dimension is collected in C cross polarization evil spirit angle rotation (CPMAS) 13The C spectrum.Sample is in the 15.0kHz rotation of specifying speed of rotation corresponding to the maximum of this 4mm rotor.Speed of rotation minimizes the intensity at rotation peak, limit faster.In order to optimize signal sensitivity, cross polarization is adjusted to 2.0ms duration of contact, and is 85kHz the decoupling power setting.Compose to carbon with 5400 scanning collection that adopt 5 seconds circulation delays.These carbon spectrums adopt the diamantane external sample as reference, and its High-Field resonance is set at 29.5ppm.
Table 10: form H's is solid-state 13The C nuclear magnetic resonance spectrum
?? 13C chemical shift [ppm] Intensity
??175.8 ??1.0
??158.6 ??2.0
??151.2 ??2.4
??148.7 * ??1.2
??136.9 ??3.0
??130.0 ??3.7
??125.7 ??4.3
??118.8 ??1.5
??111.0 ??0.8
??68.0 ??1.4
??53.9 * ??0.7
??49.1 ??1.9
??34.9 ??3.2
??31.9 ??12.0
* the peak is takeed on
The crystalline form of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt of the present invention or amorphous form or amorphous form may exist with anhydrous form and aquation and solvation form.Usually, hydrated form is equal to not hydrated form and expection is contained within the scope of the invention.Crystalline form A, the B of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt and C preferably occur as hydrate, and form E, F and the G of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt preferably occur as anhydrous form.
No matter aquation and/or solvation degree how, have the crystalline form of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt of the present invention of the X-ray powder diffraction figure that is equal to or solid state NMR spectrum or amorphous form or amorphous form all within the scope of the invention.
Following non-limiting example is illustrated the preferred preparation method of compound of the present invention.
Embodiment
Form A:(3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt
Figure GPA00001118637200251
Step: isopropyl acetate (400mL), 50% aqueous sodium hydroxide solution (3.520g) and water (4.165g) are added in the 500mL jacketed reactor.(3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate (VI) is added this mixture (20.0g), then add (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt form A (by the method preparation of for example above-mentioned reaction formula II) as crystal seed (200mg).Mixture was stirred 5 hours at 30 minutes internal heating to 70 ℃ and in this temperature.Mixture is cooled to 40 ℃ in 4 hours, in 1 hour, is cooled to 20 ℃ then.Make slurries 20 ℃ of granulations 12 hours then.Slurries are filtered by B (filter paper).With filter cake with twice of the 1% isopropyl acetate solution washing of 20mL; then 40 ℃～45 ℃ vacuum-dryings spend the night and with nitrogen venting to obtain (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt hemihydrate form A (VIII, 79% productive rate) of 16.84g.
Form B:(3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt
Figure GPA00001118637200252
Step: with (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate (VI) (4.98g), ethyl acetate (59.8mL) and water (1.3mL) mixes and is heated to 55 ℃.Interpolation 2 ethyl hexanoic acid sodium (97%, ethyl acetate solution 2.025g) (20mL) and water (0.44mL), thus produce settled solution.Solution was handled 15 minutes and passed through diatomite filtration at 55 ℃～60 ℃ with gac.Ethyl acetate/2.2% water washing once with 5mL with filter cake.Filtrate is 40 ℃.(3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-the phenoxy group)-acetic acid sodium salt (VIII) that adds 50mg is as crystal seed, allows mixture be cooled to room temperature and stirs 18 hours.Dope filtration is obtained 3.1g (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt.
Filtrate is concentrated into 20mL;, also filtered in 3 hours as crystal seed with (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt (VIII) in stirring at room to obtain 0.1g (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt.
Second filtrate is concentrated into dried, granulation in the 40mL of room temperature water, filter and dry air to obtain the 2.4g solid.(97%, 1g) solution in the 10mL ethyl acetate mixes with these solids and 30mL ethyl acetate and 2 ethyl hexanoic acid sodium.Mixture stirring at room 18 hours, is filtered and dry to obtain noncrystal solid.With this material dissolves in 40mL ethyl acetate and 1mL water, and stirring at room 18 hours.Add hexane until reaching haze point (haze point), then mixture was stirred 24 hours.Formation thickness precipitation, with its filtration, with dry ethyl acetate washing, and dry to obtain (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt monohydrate form B (IX) of 1.27g.
Form A: (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt
Figure GPA00001118637200261
Step: mixed being incorporated in of the wet THF (2.2% water) of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt (501mg) and 5.0mL was heated to 50 ℃～55 ℃ in 1 hour under the nitrogen., make it be cooled to room temperature then and stirred 66 hours as the crystal seed in the above-mentioned suspension with (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt of 4mg.Solvent evaporates and remaining white solid fully.Add the wet THF (2.2% water) of extra 5mL and mixture was stirred 5 hours.With solid filtering and 45 ℃～50 ℃ vacuum-dryings to obtain (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt monohydrate form A (X, 65.3% productive rate) of 333mg.
Form E and F:(3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt
Step: (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt is dissolved in ethylene glycol, allows solution slowly evaporate then in room temperature.After 1 week, tiny spicule forms, with its filtration, dry air and be characterized by (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt anhydrous form E (XI).
Filtrate is evaporated to less volume and filtration.Separated products is characterized as being (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt anhydrous form F (XII).
Form G:(3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt
Step: (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt (50mg) was heated to 155 ℃ to produce (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt form G in 1 hour.
6 kinds of crystalline forms of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt of having identified and illustrated except this paper are (the form A～G); also identify a kind of amorphous form of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt, be assigned therein as form H.
The preparation of noncrystal (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt (form H)
Can use following two kinds of methods to prepare the amorphous form H of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt.
Form H, method 1: by medicine dissolution is made (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt solution of 20mg/mL～100mg/mL in water for injection.Solution is filtered with 0.22 μ sterilizing filter.Solution is packed in the bottle/syringe.Bottle/syringe is freezing and kept 2 hours in this temperature at-45 ℃.By then to finish lyophilize in 150m τ in that (20 ℃～25 ℃) are dry first at (20 ℃～30 ℃) after drying.Store the gained solid.
Form H, method 2: 1 gram (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt is dissolved in the 10mL water for injection to make 100mg/mL solution.Solution is filtered with 0.22 μ sterilizing filter.The solution aliquots containig of 0.5mL is packed in the vial.Vial is written in the Freeze Drying Equipment.With shelve temperature with 0.5 ℃/minute speed alternating temperature to-45 ℃.The temperature of shelving at-45 ℃ kept 20 hours.To shelve temperature with 0.5 ℃/minute speed alternating temperature to 30 ℃.Kept 10 hours at 30 ℃.The vacuum that in the whole freezing drying process, keeps 150m τ.
Six kinds of crystalline forms of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt of having identified and illustrated except this paper (form A～G) and the amorphous form H; also identify the amorphous form of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt, be appointed as form I.
Can use the amorphous form I of following two kinds of methods preparation (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt.
Form I, method 1: prepared amorphous (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt in 72 hours by at first excessive crystalline form A being added in the organic solvent (acetonitrile or methylene dichloride) and in stirring at room.With sample filtering, evaporating solns is analyzed by PXRD and is determined that it is amorphous solid to produce white solid under the envrionment conditions of room temperature then.
Form I; method 2: by at first excessive crystalline form A being added into one of following solvent system (isopropyl ether, methyl tertiary butyl ether, or 97% ethyl acetate/3% water) and preparing amorphous (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt in 72 hours 40 ℃ of stirrings.Then with sample filtering, with solution 40 ℃ of evaporations to produce white solid, analyze by PXRD and determine that it is amorphous solid.
Above-mentioned 3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-polymorphic forms of acetic acid sodium salt can be used as prostaglandin agonists and therefore can be used for using the method for this prostanoid agonist and contain in the pharmaceutical composition of this prostanoid agonist.3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-polymorphic forms of acetic acid sodium salt can be used for treating and/or preventing the patient's condition of regulating mediation by prostaglandin(PG), particularly by EP 2The patient's condition of the agonist mediation of acceptor.
Can recognize; the polymorphic forms of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt of the present invention can exist with the radio-labeling form, and promptly described compound can contain one or more comprising and the atomic mass or different atomic mass or the total mass numbers of total mass number that see usually in the nature.The radio isotope of hydrogen, carbon, phosphorus, fluorine and aluminium comprises respectively 3H, 14C, 32P, 35S, 18F and 36Cl.Other the radioisotopic The compounds of this invention that comprises these radio isotope and/or other atom belongs in the scope of the present invention.Tritium is for (promptly 3H) and carbon-14 (promptly 14C) radio isotope is easy to preparation because of it and detectability is preferred especially.The polymorphic forms of radiolabeled (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt of the present invention can prepare with the method for well known to a person skilled in the art usually.These radiolabeled compounds can prepare by replacing nonradioactive labeling's reagent with the radio-labeling reagent that is easy to obtain by the step of carrying out among above-mentioned reaction formula and/or the embodiment easily.
One skilled in the art will recognize that anti-absorption agent (for example, progestogen, polyphosphonic acid salt, diphosphonate, estrogen agonist/antagonist, oestrogenic hormon, estrogenic/progestogenic combination,
Figure GPA00001118637200291
Oestrone, trihydroxy-oestrin or 17 α-lynoral or 17 β-lynoral) can unite use with compound of the present invention.
Exemplary progestogen is commercially available and comprise: algestone acetophenide, altrenogest, amadinone acetate, anagestone acetate, chlormadinone acetate, cingestol, clogestone acetate, clomegestone acetate, the acetic acid delmadinone, desogestrel, dimethisterone, Dydrogesterone, ethynerone, the oxalic acid Norethindrone, Org 3236, Synchronate, gestaclone, gestodene, gestonorone caproate, gestrinone, haloprogesterone, Hydroxyprogesterone caproate bp 98, Levonorgestrel, Lynestrenol, medrogestone, medroxyprogesterone acetate, melengestrol acetate, oxalic acid metynodion (methynodiol diacetate), Norethisterone, Norethisterone Acetate, norethynodrel, norgestimate, norgestomet, norgestrel, oxogestone phenpropionate, progesterone, quingestanol acetate, quingestrone and tigestol.
Preferred progestogen is medroxyprogesterone, Norethisterone and norethynodrel.
The example of bone resorption inhibition polyphosphonic acid salt comprises United States Patent (USP) the 3rd, 683, disclosed polyphosphonic acid salt type in No. 080, and this paper is by with reference to incorporating its disclosure into.Preferred polyphosphonic acid salt is together with diphosphonate (being also referred to as diphosphonate).The tiludronic acid disodium is a kind of especially preferred polyphosphonic acid salt.Ibandronic acid is a kind of especially preferred polyphosphonic acid salt.Alendronate is a kind of especially preferred polyphosphonic acid salt.Zoledronic acid is a kind of especially preferred polyphosphonic acid salt.Other preferred polyphosphonic acid salt is two phosphonic acids of 6-amino-1-hydroxyl hexylidene and the two phosphonic acids of 1-hydroxyl-3 (methyl amyl amino)-propylidene.Polyphosphonic acid salt can be used with the form of acid or with the form of soluble alkali metal salts or alkaline earth salt.The hydrolyzable ester that similarly also comprises polyphosphonic acid salt.Specific examples comprises ethane-1-hydroxyl-1, the 1-di 2 ethylhexyl phosphonic acid, methanebisphosphonic acid, pentane-1-hydroxyl-1, the 1-di 2 ethylhexyl phosphonic acid, methane dichloro di 2 ethylhexyl phosphonic acid, methane hydroxyl bisphosphate, ethane-1-amino-1, the 1-bisphosphate, ethane-2-amino-1, the 1-di 2 ethylhexyl phosphonic acid, propane-3-amino-1-hydroxyl-1, the 1-di 2 ethylhexyl phosphonic acid, propane-N, N-dimethyl-3-amino-1-hydroxyl-1, the 1-di 2 ethylhexyl phosphonic acid, propane-3,3-dimethyl-3-amino-1-hydroxyl-1, the 1-di 2 ethylhexyl phosphonic acid, the phenyl amino methanebisphosphonic acid, N, N-dimethylamino methanebisphosphonic acid, N (2-hydroxyethyl) aminomethane di 2 ethylhexyl phosphonic acid, butane-4-amino-1-hydroxyl-1, the 1-di 2 ethylhexyl phosphonic acid, pentane-5-amino-1-hydroxyl-1, the 1-di 2 ethylhexyl phosphonic acid, hexane-6-amino-1-hydroxyl-1,1-di 2 ethylhexyl phosphonic acid and pharmaceutically acceptable ester and salt.
Especially, compound of the present invention can make up with the Mammals estrogen agonist/antagonist.Can use any estrogen agonist/antagonist as second compound of the present invention.The term estrogen agonist/antagonist is meant the compound that combines, suppresses the bone conversion with estrogen receptor and/or avoid bone loss.Especially, this paper is defined as estrogen agonist the compound that can combine and simulate the effect of oestrogenic hormon in one or more tissues with the estrogen receptor site in the mammalian tissues.This paper is defined as estrogen antagonist the compound that can combine and block the effect of oestrogenic hormon in one or more tissues with the estrogen receptor site in the mammalian tissues.These activity can be carried out easily determining in conjunction with test, standard bone histomorphometry and densimeter method and below with reference to the standard testing of document by comprising estrogen receptor by those skilled in the art: Eriksen E.F. etc., BoneHistomorphometry, Raven Press, New York, 1994, the 1-74 pages or leaves; Grier S.J. etc., The Use of Dual-Energy X-Ray Absorptiometry In Animals, Inv.Radiol., 1996,31 (1): 50-62; Wahner H.W. and Fogelman I., The Evaluation of Osteoporosis:DualEnergy X-Ray Absorptiometry in Clinical Practice., Martin Dunitz Ltd., London1994,1-296 page or leaf.Below describe and quoted multiple this compounds.
A kind of preferred estrogen agonist/antagonist is a United States Patent (USP) the 5th, 047, No. 431 disclosed droloxifene: (phenol, 3-(1-(4-(2-(dimethylamino) oxyethyl group) phenyl)-2-phenyl-1-butylene base)-, (E)-) and related compound, this paper incorporates its disclosure into by reference.
Another kind of preferred estrogen agonist/antagonist is Willson etc., Endocrinology, 1997,138, disclosed 3-among the 3901-3911 (4-(1,2-phenylbenzene-but-1-ene)-phenyl)-vinylformic acid.
Another kind of preferred estrogen agonist/antagonist is a United States Patent (USP) the 4th, 536, disclosed tamoxifen in No. 516: (ethamine, 2--4-(1,2-phenylbenzene-1-butylene base) phenoxy group)-N, the N-dimethyl, (Z)-and 2-, 2-hydroxyl-1,2,3-propane front three acid esters (1: 1)) and related compound, this paper is by with reference to incorporating its disclosure into.
Another kind of related compound is a United States Patent (USP) the 4th, 623, disclosed 4-hydroxyl tamoxifen in No. 660, and this paper is by with reference to incorporating its disclosure into.
A kind of preferred estrogen agonist/antagonist is a United States Patent (USP) the 4th, 418, No. 068 disclosed raloxifene: (ketone, (6-hydroxyl-2-(4-hydroxy phenyl) benzo [b] thiene-3-yl-) (4-(2-(piperidino) oxyethyl group) phenyl)-hydrochloride), this paper incorporates its disclosure into by reference.
Another kind of preferred estrogen agonist/antagonist is a United States Patent (USP) the 4th, 996, No. 225 disclosed toremifenes: (ethamine, 2-(4-(4-chloro-1,2-phenylbenzene-1-butylene base) phenoxy group)-N, the N-dimethyl-, (Z)-, 2-hydroxyl-1,2,3-propane front three acid esters (1: 1), this paper incorporates its disclosure into by reference.
Another kind of preferred estrogen agonist/antagonist is a United States Patent (USP) the 3rd, 822, No. 287 disclosed ormeloxifenes (centchroman): 1-(2-((4-(methoxyl group-2,2,-dimethyl-3-phenyl-chroman-4-yl)-phenoxy group)-ethyl)-tetramethyleneimine, this paper incorporates its disclosure into by reference.Levormeloxifene preferably in addition.
Another kind of preferred estrogen agonist/antagonist is a United States Patent (USP) the 4th, 839, No. 155 disclosed idoxifene: (E)-1-(2-(4-(1-(4-iodophenyl)-2-phenyl-but-1-ene base)-phenoxy group)-ethyl)-pyrrolidone, this paper incorporates its disclosure into by reference.
Another kind of preferred estrogen agonist/antagonist is a United States Patent (USP) the 5th, 488, No. 058 disclosed 2-(4-p-methoxy-phenyl)-3-[4-(2-piperidines-1-base oxethyl)-phenoxy group]-benzo [b] benzene sulphur-6-phenol, this paper incorporates its disclosure into by reference.
Another kind of preferred estrogen agonist/antagonist is a United States Patent (USP) the 5th, 484, No. 795 disclosed 6-(4-hydroxy phenyl)-5-(4-(2-piperidines-1-base oxethyl)-benzyl)-Betanaphthol, and this paper incorporates its disclosure into by reference.
Another kind of preferred estrogen agonist/antagonist is that the PCT that is assigned to Pfizer announces in WO95/10513 number disclosed (4-(2-(2-azabicyclo [2.2.1] heptan-2-yl)-oxyethyl group)-phenyl)-(6-hydroxyl-2-(4-hydroxy phenyl)-benzo [b] thiene-3-yl-)-ketone together with the preparation method.
Other preferred estrogen agonist/antagonist comprises as the 5th, 552, No. 412 described compounds of common transfer United States Patent (USP).Wherein described especially preferred compound is:
Suitable-6-(4-fluorophenyl)-5-(4-(2-piperidines-1-base oxethyl)-phenyl)-5,6,7,8-tetrahydrochysene Betanaphthol;
(-)-suitable-6-phenyl-5-(4-(2-tetramethyleneimine-1-base oxethyl)-phenyl)-5,6,7,8-tetrahydrochysene Betanaphthol;
Suitable-6-phenyl-5-(4-(2-tetramethyleneimine-1-base oxethyl)-phenyl)-5,6,7,8-tetrahydrochysene Betanaphthol;
Suitable-1-(6 '-tetramethyleneimine and oxyethyl group-3 '-pyridyl)-2-phenyl-6-hydroxyl-1,2,3, the 4-naphthane;
1-(4 '-tetramethyleneimine and ethoxyl phenenyl)-2-(4 " fluorophenyl)-6-hydroxyl-1,2,3, the 4-tetrahydroisoquinoline;
Suitable-6-(4-hydroxy phenyl)-5-(4-(2-piperidines-1-base-oxyethyl group)-phenyl)-5,6,7,8-tetrahydrochysene Betanaphthol; With
1-(4 '-tetramethyleneimine and ethoxyl phenenyl)-2-phenyl-6-hydroxyl-1,2,3, the 4-tetrahydroisoquinoline.
United States Patent (USP) the 4th, 133 has been described other estrogen agonist/antagonist in No. 814 (this paper incorporates its disclosure into by reference).United States Patent (USP) the 4th, 133 discloses the derivative of 2-phenyl-3-aroyl thionaphthene and 2-phenyl-3-aroyl thionaphthene-1-oxide compound No. 814.
One skilled in the art will recognize that other bone anabolic agent (being also referred to as bone amount rising agent) can unite use with compound of the present invention.Bone amount dose is the compound that the bone amount is increased to the level that is higher than fracture threshold, described fracture threshold is specified in World Health Organization's research (World Health Organization, " Assessment of Fracture Risk and its Application to Screening forPostmenopausal Osteoporosis (1994) Report of a WHO Study Group.WorldHealth Organization Technical Series 843 ").
Can be with any prostaglandin(PG) or prostaglandin agonists/antagonist as second compound in some aspect of the present invention.One skilled in the art will recognize that active fragments, tethelin or the growth hormone cinogenic agent that can also use IGF-1, Sodium Fluoride, Rat parathyroid hormone 1-34 (PTH), Rat parathyroid hormone 1-34.Following paragraph has carried out more detailed description to exemplary second compound of the present invention.
Any prostaglandin(PG) all can be used as second compound in some aspect of the present invention.The term prostaglandin(PG) is meant and can be used for treating osteoporotic natural prostaglandins PGD 1, PGD 2, PGE 2, PGE 1And PGF 2Analogue.These compounds combine with prostaglandin receptor.This combination can be by those skilled in the art with standard testing (for example, An S. etc., Cloning and Expression of the EP 2Subtype of Human Receptors for Prostaglandin E 2, Biochemical and BiophysicalResearch Communications, 1993,197 (1): 263-270) carry out easily determining.
Prostaglandin(PG) is the alicyclic compound relevant with the basic cpd prostanoic acid.The carbon atom of alkaline prostaglandin(PG) is numbered through cyclopentyl ring to the terminal carbon on the adjacent side chain successively from the carboxyl carbon atom.This adjacent side chain is in trans orientation usually.The existence of the oxo group at cyclopentyl partial C-9 place indicates the prostaglandin(PG) that is in the E type, and PGE 2At C 13-C 14Trans unsaturated double-bond is contained and at C in the place 5-C 6Cis-double bonds is contained in the position.
Below describe and quoted multiple prostaglandin(PG).Yet other prostaglandin(PG) also is that those skilled in the art are known.United States Patent (USP) the 4th, 171, No. 331 and the 3rd, 927, the example that discloses prostaglandin(PG) No. 197, this paper is by with reference to incorporating its disclosure into.
Norrdin etc., The Role of Prostaglandins in Bone In Vivo, ProstaglandinsLeukotriene Essential Fatty Acids 41,139-150, the 1990th, the summary of bone anabolism prostaglandin(PG).
Any prostaglandin agonists/antagonist can be as second compound in some aspect of the present invention.Term prostaglandin agonists/antagonist is meant with prostaglandin receptor and combines (for example, An S. etc., Cloning and Expression of the EP 2Subtype of Human Receptors forProstaglandin E 2, Biochemical and Biophysical Research Communications, 1993,197 (1): 263-270) and simulate the compound of in vivo effect of prostaglandin(PG) (for example, stimulate bone forming and increase the bone amount).This effect can by those skilled in the art with standard testing carry out easily determining (Eriksen E.F. etc., Bone Histomorphometry, Raven Press, New York, 1994, the 1-74 pages or leaves; Grier S.J. etc., The Use of Dual-Energy X-Ray Absorptiometry InAnimals, Inv.Radiol., 1996,31 (1): 50-62; Wahner H.W. and Fogelman I., TheEvaluation of Osteoporosis:Dual Energy X-Ray Absorptiometry in ClinicalPractice., Martin Dunitz Ltd., London 1994, the 1-296 pages or leaves).Below describe and quoted multiple this compounds.Yet other prostaglandin agonists/antagonist also is that those skilled in the art are known.Exemplary prostaglandin agonists/antagonist is described below.
Common transfer United States Patent (USP) the 3rd, 932, No. 389 (this paper incorporates its disclosure into by reference) discloses for movable useful 2-decarboxylation-2-(tetrazolium-5-yl)-11-deoxidation-15-replacement-ω-penta prostacyclin of bone forming.
Common transfer United States Patent (USP) the 4th, 018, No. 892 (this paper incorporates its disclosure into by reference) discloses for the movable useful 16-aryl-13 of bone forming, 14-dihydro-PGE 2To biphenyl ester.
Common transfer the possession of No. the 4th, 219,483, United States Patent (USP) (this paper by with reference to incorporating its disclosure into) disclose for bone forming movable useful 2,3,6-replacement-pyrokomane.
Common transfer the possession of No. the 4th, 132,847, United States Patent (USP) (this paper by with reference to incorporating its disclosure into) disclose for bone forming movable useful 2,3,6-replacement-pyrokomane.
United States Patent (USP) the 4th, 000, No. 309 (this paper incorporates its disclosure into by reference) disclose for the movable useful 16-aryl-13 of bone forming, 14-dihydro-PGE 2To biphenyl ester.
United States Patent (USP) the 3rd, 982, No. 016 (this paper incorporates its disclosure into by reference) discloses for the movable useful 16-aryl-13 of bone forming, 14-dihydro-PGE 2To biphenyl ester.
United States Patent (USP) the 4th, 621, No. 100 (this paper incorporates its disclosure into by reference) disclose for the movable useful substituted cyclopentane of bone forming.
United States Patent (USP) the 5th, 216, No. 183 (this paper incorporates its disclosure into by reference) disclose for the movable useful cyclopentanone of bone forming.
Sodium Fluoride can be as second compound in some aspect of the present invention.The term Sodium Fluoride is meant the Sodium Fluoride (for example, slow fluorideium fluoride time-release, the lasting Sodium Fluoride that discharges) of form of ownership.United States Patent (USP) the 4th, 904 discloses the Sodium Fluoride of lasting release in No. 478, and this paper is by with reference to incorporating its disclosure into.The activity of Sodium Fluoride can by those skilled in the art by biological scheme carry out easily determining (for example, referring to Eriksen E.F. etc., Bone Histomorphometry, Raven Press, New York, 1994, the 1-74 pages or leaves; Grier S.J. etc., The Use of Dual-Energy X-Ray Absorptiometry InAnimals, Inv.Radiol., 1996,31 (1): 50-62; Wahner H.W. and Fogelman I., TheEvaluation of Osteoporosis:Dual Energy X-Ray Absorptiometry in ClinicalPractice., Martin Dunitz Ltd., London 1994, the 1-296 pages or leaves).
Delicious peptide can be as second compound of the present invention (for example, referring to Ono etc., Promotion of the Osteogenetic Activity of Recombinant Human BoneMorphogenetic Protein by Prostaglandin E1 Bone, 1996,19 (6), 581-588).
Any Rat parathyroid hormone 1-34 (PTH) can be as second compound in some aspect of the present invention.The term Rat parathyroid hormone 1-34 be meant can stimulate bone forming and increase the Rat parathyroid hormone 1-34 of bone amount and fragment or metabolite with and analog.The activeconstituents and the analogue (announcing WO No. 94/01460) that comprise parathyroid hormone-related peptide and parathyroid gland related peptides in addition referring to PCT.This class bone anabolism functional activity can by those skilled in the art by standard testing carry out easily determining (for example, referring to Eriksen E.F. etc., Bone Histomorphometry, Raven Press, New York, 1994, the 1-74 pages or leaves; Grier S.J. etc., The Use of Dual-Energy X-Ray AbsorptiometryIn Animals, Inv.Radiol., 1996,31 (1): 50-62; Wahner H.W. and Fogelman I., TheEvaluation of Osteoporosis:Dual Energy X-Ray Absorptiometry in ClinicalPractice., Martin Dunitz Ltd., London 1994, the 1-296 pages or leaves).Below describe and quoted multiple this compounds.Yet other Rat parathyroid hormone 1-34 also is that those skilled in the art are known.The example of Rat parathyroid hormone 1-34 is disclosed in below with reference to document.
" Human Parathyroid Peptide Treatment of Vertebral Osteoporosis ", Osteoporosis Int., 3, (supplementary issue 1): 199-203.
″PTH?1-34?Treatment?of?Osteoporosis?with?Added?Hormone?ReplacementTherapy：Biochemical，Kinetic?and?Histological?Responses″Osteoporosis?Int.1：162-170.
Any tethelin or growth hormone cinogenic agent can be as second compounds of some aspect of the present invention.The term growth hormone cinogenic agent is meant the compound of the release of stimulating growth hormone or simulate growth functions of hormones (for example, thereby increasing bone forming causes the bone amount to increase).This effect can be undertaken easily determining by its known standard testing by those skilled in the art.Disclose multiple this compounds in the PCT of following announcement patent application: WO 95/14666; WO 95/13069; WO94/19367; WO 94/13696; With WO 95/34311.Yet other tethelin or growth hormone cinogenic agent also are that those skilled in the art are known.
Particularly, preferred growth hormone cinogenic agent be N-[1 (R)-[1,2-dihydro-1-methylsulfonyl spiral shell [3H-indoles-3,4 '-piperidines]-1 '-yl) glycosyl]-2-(phenyl methoxyl group) ethyl]-2-amino-2-methyl propionic acid amide: MK-677.
Other preferred growth hormone cinogenic agent comprises:
2-amino-N-(2-(3a-(R)-benzyl-2-methyl-3-oxo-2,3,3a, 4,6,7-six hydrogen pyrazolos [4,3-c] pyridine-5-yl)-1-(R)-benzyloxymethyl-2-oxoethyl) isobutyryl or its L-tartrate;
2-amino-N-(1-(R)-benzyloxymethyl-2-(3a-(R)-(4-luorobenzyl)-2-methyl-3-oxo-2,3,3a, 4,6,7-six hydrogen pyrazolos [4,3-c] pyridine-5-yl)-2-oxoethyl) isobutyramide;
2-amino-N-(2-(3a-(R)-benzyl-3-oxo-2,3,3a, 4,6,7-six hydrogen pyrazolos [4,3-c] pyridine-5-yl)-1-(R) benzyloxymethyl-2-oxoethyl) isobutyramide; With
2-amino-N-(1-(2,4-difluoro benzyloxymethyl)-2-oxo-2-(3-oxo-3a-pyridine-2-ylmethyl-2-(2,2, the 2-trifluoroethyl)-2,3,3a, 4,6,7-six hydrogen pyrazolos [4,3-c] pyridine-5-yl)-ethyl)-2-methyl propanamide.
Some preparation method who can be used for preparing compound as herein described far-end functional group (for example, primary amine, secondary amine, the carboxyl in the formula I precursor) that may need protection.Will be according to the character of far-end functional group and preparation method's condition and different to the needs of this protection.Needs to this protection can easily be determined by those skilled in the art.This class protection/deprotection method also belongs to art technology.Generality for blocking group and use thereof is described, referring to T.W.Greene, Protective Groups in Organic Synthesis, John Wiley﹠amp; Sons, New York, 1991.
Make the pharmacologically acceptable salt of compound of the present invention, its prodrug and described compound and prodrug be applicable to the therapeutic use of conduct at the reagent of particularly human moderate stimulation bone forming of vertebrates (for example, Mammals) and increase bone amount.Because the development of bone forming and osteoporosis and bone photo related disorders is closely related, the pharmacologically acceptable salt of these compounds, its prodrug and described compound and described prodrug by means of its on bone effect and osteoporosis stopped or falling back.
Compound of the present invention as medicinal agents to (for example vertebrates such as Mammals, human, women particularly) etc. (for example show low bone amount in the vertebrates, function in the treatment of patient's condition osteoporosis) is by active show of compound of the present invention in conventionally test, and described conventionally test comprises body build-in test, receptors bind test, ring AMP test and union of fracture test (all describing hereinafter).Body build-in test (when carrying out suitable variation in the art technology scope) can be used for determining the activity of other anabolic agent and prostaglandin agonists of the present invention.The estrogen agonist/antagonist scheme can be used for determining the particularly activity of estrogen agonist/antagonist, and the activity (when carrying out suitable variation in the art technology scope) that is used for determining other anti-absorption agent.Combination hereinafter described and order treatment plan can be used for showing the function of the combination of anabolic agent as herein described (for example, compound of the present invention) and anti-absorption agent (for example, estrogen agonist/antagonist).These tests also provide the mode that the activity of compound of the present invention (or other anabolic agent as herein described and anti-absorption agent) can be compared mutually and compare with the activity of other known compound.These results relatively can be used for being identified for treating the dosage level of the vertebrates (for example, comprising human Mammals) of described disease.
The body build-in test of anabolic agent
The bone anabolic agent can be tested in harmless male or female rats, sex hormone deficiency male rat (male castration) or female rats (oophorectomize) in the activity that stimulates bone forming and increase in the bone amount.
In this research, can use the different ages male or female rats at (for example, 3 monthly ages).Described rat is (spay or excision testis) harmless or castrating, and with the prostaglandin agonists subcutaneous injection of various dose (for example, 1mg/kg/ day, 3mg/kg/ day or 10mg/kg/ day) or tube feed 30 days.In the rat of castrating, treatment begins (in order to prevent the purpose of bone loss) or begin (in order to recover the purpose of bone amount) when bone loss taking place in postoperative next day.During studying, allow all rats can freely obtain water and contain 1.46% calcium, 0.99% phosphorus and 4.96IU/g vitamins D 3The particle commercial feed (Teklad Rodent Diet#8064, Harlan Teklad, Madison, WI).12 days and 2 days give the subcutaneous injection of 10mg/kg fluorexon to all rats before putting to death.Put to death rat.Determine following terminal point (endpoint):
Femur bone mineral are measured:
When necrotomy, the right femur of every rat removed and with the dual-energy x-ray absorption apparatus that is equipped with " regional high resolution scanning " software (DXA, QDR 1000/W, Hologic Inc., Waltham MA) scans.Scan field dimension is 5.08cm * 1.902cm, and resolving power is 0.0254cm * 0.0127cm, and sweep velocity is 7.25mm/ second.Analysis Unit's bone scanning image and the surface of bone of determining whole femur (WF), distal femoral metaphysis (DFM), femoral shaft (FS) and near end of thighbone (PF) amass, bone mineral content (BMC) and bmd (BMD).
The quantitative analysis of shin bone tissue morphology:
When necrotomy, right shin bone is removed, cut muscle, and be cut into 3 parts.With proximal tibia with shin bone is dried is fixed in 70% ethanol, in the ethanol of gradient concentration, dewater, degreasing in acetone, be embedded in then methyl methacrylate (Eastman Organic Chemicals, Rochester, NY) in.
Using Reichert-Jung Polycut S slicing machine cutting thickness is the metaphyseal front end section of proximal tibia of 4 μ m and 10 μ m.4 μ m section is dyeed with improvement Masson trichrome staining, and 10 μ m section keeps no dyeing.To be used for the cancellous bone tissue morphometry from a slice 4 μ m section and a slice 10 μ m section of every rat.
Using Reichert-Jung Polycut S slicing machine cutting thickness is the dried transverse section of shin bone of 10 μ m.These sections are used for the quantitative analysis of cortex of bone tissue morphology.
The cancellous bone tissue morphometry:
Use the Bioquant OS/2 tissue morphology metrology (R﹠amp of system; M Biometrics, Inc., Nashville TN) comes the metaphyseal secondary spongy bone of proximal tibia of distance growth plate-epiphysis joint 1.2mm～3.6mm is carried out static state and dynamic organization's norphometry mensuration.For limit of determination being formed on secondary spongy bone, need omit the preceding 1.2mm in tibial metaphysis district.Use 4 μ m section to determine the index relevant, and 10 μ m section is used for determining the index relevant with the bone conversion with bone forming with bone volume, bone structure and bone resorption.
I) with the mensuration and the calculating of bone volume and structurally associated: (1) total metaphysis area (TV, mm 2): apart from the metaphysis area of growth plate-epiphysis joint 1.2mm～3.6mm.(2) bone trabecula area (BV, mm 2): the girder total area in the TV.(3) the bone trabecula girth (BS, mm): the length of the overall circumference of girder.(4) Trabecula Bone Volume (BV/TV, %): BV/TV * 100.(5) the bone trabecula number (TBN, #/mm): 1.199/2 * BS/TV.(6) bone trabecula thickness (TBT, μ m): (2000/1.199) * (BV/BS).(7) bone trabecula separates (TBS, μ m): (2000 * 1.199) * (TV-BV).
II) mensuration and the calculating relevant with bone resorption: (1) osteoclast number (OCN, #): the osteoclast sum in the metaphyseal region.(2) the osteoclast girth (OCP, mm): the length of the girder girth that is covered by osteoclast.(3) osteoclast number/mm (OCN/mm, #/mm): OCN/BS.(4) osteoclast girth per-cent (%OCP, %): OCP/BS * 100.
III) with bone forming and relevant mensuration and the calculating of conversion: the girth of (1) single fluorexon mark (SLS, mm): the total length that carries out the girder girth of mark with a fluorexon mark.(2) girth of two fluorexon marks (DLS, mm): the total length that carries out the girder girth of mark with two fluorexon marks.(3) width (ILW, μ m) between mark: the mean distance between two fluorexon marks.(4) mineralize girth per-cent (PMS, %): (SLS/2+DLS)/BS * 100.(5) mineral apposition speed (MAR, μ m/ day): ILW/ mark spacing.(6) bone forming speed/surface is with reference to (BFR/BS, μ m 2/ d/ μ m): (SLS/2+DLS) * MAR/BS.(7) bone turnover rate (BTR, %/y): (SLS/2+DLS) * MAR/BV * 100.
Cortex bone tissue morphology metrology:
Use Bioquant OS/2 tissue morphology metering system (R﹠amp; M Biometrics, Inc., Nashville TN) does cortex bone to shin bone and carries out static state and dynamic organization's norphometry mensuration.Measured the total tissue area on periosteum surface and inner cortex surface, the medullary space area, the periosteum girth, the inner cortex girth, single mark girth, width between double-tagging girth and mark, and calculated cortex bone area (total tissue area-medullary space area), cortex bone area percentage (cortex bone area/total tissue area * 100), marrow area percentage (medullary space area/total tissue area * 100), periosteum and intracortical mark girth per-cent [(single mark girth/2+ double-tagging girth)/overall circumference * 100], mineral apposition speed (width/spacing between mark) and bone forming speed [mineral apposition speed * [(single mark girth/2+ double-tagging girth)/overall circumference].
Statistics
(Berkeley CA) comes counting statistics information for Abacus Concepts, Inc. can to use StatView 4.0 bags.User's difference analysis (ANOVA) test then uses Fisher ' s PLSD (Stat View, Abacus Concepts Inc., 1918 Bonita Ave, Berkeley, CA 94704-1014) to come the comparative group differences.
Stablized express recombinant people EP 2 And EP 4 What cAMP raise in the 293-S clone of acceptor determines
Use based on the Oligonucleolide primers of the sequence of having announced and as template from just for human kidney cells (EP 2) or just for people's cell (EP 4) RNA, produce representative EP by the reversed transcriptive enzyme polymerase chain reaction 2And EP 4CDNA (the Regan of the complete open reading frame of acceptor, J.W.Bailey, T.J.Pepperl, D.J.Pierce, K.L.Bogardus, A.M.Donello, J.E.Fairbaim, C.E.Kedzie, K.M.Woodward, D.F. and Gil, D.W.1994 Cloning of a Novel Human ProstaglandinReceptor with Characteristics of the Pharmaclogically Defined EP 2Subtype.Mol.Pharmacology 46:213-220; And Bastien, L., Sawyer, N., Grygorczyk, R., Metters, K. and Adam, M.1994 Cloning, Functional Expression, andCharacterization of the Human Prostaglandin E2 Receptor EP2 Subtype.J.Biol.Chem.Vol 269,16:11873-11877).CDNA is cloned in the multiple cloning site of pcDNA3 (San Diego, CA 92121 for InvitrogenCorporation, 3985B Sorrento Valley Blvd.), and is used for coming transfection 293-S human embryonic kidney cell by the coprecipitation of calcium phosphate method.With G418 resistivity colony amplification and test its specificity [ 3H] PGE 2In conjunction with.To show high-caliber specificity [ 3H] PGE 2Bonded transfection thing is analyzed by Scatchard and is further characterized to determine PGE 2Bmax and Kds.The clone that is selected to screening compound is for PGE 2(EP 2) have about 338,400 acceptors and Kd=12nM, for PGE 2(EP 4) have about 256,400 acceptors and a Kd=2.9nM.The constitutive expression of two kinds of acceptors in parent 293-S cell can be ignored.Cell is remained among the RPMI that is supplemented with foetal calf serum (10% is final) and G418 (700 μ g/ml are final).
By via acutely bouncing, add serum-free RPMI to 1 * 10 6The ultimate density of cell/ml and add 3-sec.-propyl-1-methyl xanthine (IBMX) to the 1mM ultimate density so that cell from culturing bottle, break away among the PBS that lacks to Ca++ and the Mg++ of 1ml, thereby determine 293-S/EP 2And 293-S/EP 4CAMP response in the clone.Immediately with 1 ml cells suspension five equilibrium to the little whizzer of single 2ml screw cap and when the open-ended at 37 ℃, 5%CO 2With 95% relative humidity incubation 10 minutes.Then with testing mixture with 1: thereby 100 thinning ratio is added into the final DMSO of cell or alcohol concn is 1%.Add behind the compound immediately with channel closure, by reversing twice mixing, and 37 ℃ of incubations 12 minutes.Then by 100 ℃ of incubations 10 minutes with sample dissociation and immediately cooled on ice 5 minutes.By centrifugal 5 minutes of 1000 * g with the cell debris granulating, and the lysate after will clarifying is transferred in the new pipe.Clarifying lysate was diluted in the cAMP RIA assay buffer (being included in the following test kit) with 1: 10 after, use is purchased cAMP radioimmunity test kit RIA (NEK-033, DuPont/NEN Research Products, 549 Albany St., Boston, MA 02118) determine cAMP concentration.Usually, handle cell with the testing compound of 6～8 times of concentration with the increment of 1log.The linear regression analysis of use on the linear portion of dose response curve carried out EC50 and calculated on counter.
PGE 2 The combination test of acceptor
Membrane prepare: all operations all carries out at 4 ℃.To express prostaglandin(PG) 1 receptor (EP 1), 2 receptor (EP 2), 3 receptor (EP 3) or 4 receptor (EP 4) transfectional cell collect and at buffer A [50mM Tris-HCl (pH 7.4), 10mM MgCl 2, 1mM EDTA, 1mM Pefabloc peptide (Boehringer Mannheim Corp., Indianapolis, IN), 10 μ M phosphoramidons (Sigma, St.Louis, MO), 1 μ M Pepstatin A peptide (Sigma, St.Louis, MO), 10 μ M elastase inhibitor peptides (Sigma, St.Louis, MO), 100 μ M antipain peptide (Sigma, suspending St.Louis, MO)] is 2000000 cells/ml.(Danbury CT) passes through ultrasonic degradation with burst in 2 times 15 seconds with cell for 250 model, Branson UltrasonicsCorporation with the ultrasonic device of Branson.Uncracked cell and fragment by removing at 100 * g in centrifugal 10 minutes.Then by coming collection membrane in centrifugal 30 minutes at 45,000 * g.Granular film is resuspended to 3mg albumen/ml～10mg albumen/ml, and protein concentration is determined by Bradford method [Bradford, M., Anal.Biochem., 72,248 (1976)].Then will be through resuspended film before use in-80 ℃ of refrigerated storage.
In conjunction with test: the frozen film that will as above prepare melts and dilutes in above-mentioned buffer A is 1mg albumen/ml.With the 3nM's of the test compounds of the membrane prepare thing of 1 volume and 0.05 volume or damping fluid and 1 volume 3The H-PGE 2(#TRK 431, Amersham, and Arlington Heights, IL) solution in buffer A mixes.With mixture (cumulative volume 205 μ L) 25 ℃ of incubations 1 hour.Then by through GF/C type glass fibre filter (#1205-401, Wallac, Gaithersburg, (Orange CT) comes reclamation film for Mach II/96 type, Tomtec MD) filter to use the Tomtec collector.Be combined with 3The H-PGE 2Film be filtered device and catch, and damping fluid and unconjugated 3The H-PGE 2Pass strainer and enter waste liquid.Then with [50mM Tris-HCl (pH 7.4), the 10mM MgCl of each sample with 3ml 2, 1mM EDTA] wash 3 times.Then by in microwave oven, heating with the strainer drying.In order to determine with membrane-bound 3The H-PGE 2Amount, with dried strainer place the plastics bag that has scintillation solution and LKB 1205 Betaplate readers (Wallac, Gaithersburg, MD) in counting.Specificity bonded by displacement 50% 3The H-PGE 2The concentration of required test compounds is determined IC50.
As Funk etc., Journal of Biological Chemistry, 1993,268, disclosed such total length EP for preparing among the 26767-26772 1Acceptor.As Regan etc., Molecular Pharmacology, 1994,46, disclosed such total length EP for preparing among the 213-220 2Acceptor.As Regan etc., British Journalof Pharmacology, 1994,112, disclosed such total length EP for preparing among the 377-385 3Acceptor.As Bastien, Journal of Biological Chemistry, 1994,269, disclosed such total length EP for preparing among the 11873-11877 4Acceptor.These total length acceptors are used for preparation express EP 1, EP 2, EP 3And EP 4The 293S cell of acceptor.
Produce expressing human EP according to method known to those skilled in the art 1, EP 2, EP 3Or EP 4The 293S cell of acceptor.Usually, according to disclosed known method preparation above corresponding to the 5 ' end of the total length acceptor of being announced and PCR (polymerase chain reaction) primer of 3 ' end, and with these primers be used for use from people's kidney (for EP 1), people's lung is (for EP 2), people's lung is (for EP 3) or human lymphocyte (for EP 4) total RNA as in the RT-PCR of the resource reaction.With the PCR product by the outstanding method of TA be cloned into pCR2.1 (Invitrogen, Carlsbad, CA) in and confirm the identity of the acceptor of being cloned by dna sequencing.
By electroporation 293S cell (Mayo, department of biochemistry of Northwest University) is carried out transfection with the clone's acceptor among the pcDNA3.After selecting transfectional cell, establish the stable cell lines of expressing described acceptor with G418.
At the PGE that uses un-marked 2Carry out full cell as competitor 3H-PGE 2After test, select the cloned cell line of the acceptor of having expressed maximum quantity.
The union of fracture test
Test for the union of fracture effect after the systemic administration
The fracture technology: the Sprage-Dawley rat at 3 monthly ages is used Patients Under Ketamine Anesthesia.Side direction is produced the 1cm otch in the proximal part of right shin bone or right femur preceding.The shin bone surgical technic has below been described.Otch is through to bone, and gets out the 1mm hole at far-end 4mm place apart from the tibial tubercle of preceding keel side 2mm.With the 0.8mm stainless steel tube carry out drift bolt in the marrow (overall loading 36.3N, maximum stiffness 61.8N/mm, with bone photo with condition under test).Do not carry out the pulp cavity reaming.Carry out 3 bendings by the tweezer regulated that has blunt pincers that uses particular design and come the standardized closed fracture of 2mm place generation above the tibiofibula joint.For soft tissue injury is minimized, do not make displacement fracture carefully.With monofilament nylon suture with skin closure.Operation is carried out under aseptic condition.Behind drift bolt, obtain the radiograph of all fracture immediately, get rid of and to have the rat of the fracture of specified backbone area outside or have the rat of the steel nail of displacement.Each subgroup that remaining animal is divided into each time point at random has following group of 10～12 animals to carry out the union of fracture test.(water: 100% ethanol=95: 5), and all the other groups are accepted the testing compound (1ml/ rat) of tube feed 0.01mg/kg/ day every day to first group of supporting agent of accepting tube feed 1ml/ rat every day in 10 days, 20 days, 40 days and 80 days.
At the 10th day, 20 days, 40 days and 80 days, will be from 10～12 rats of each group with Patients Under Ketamine Anesthesia and pass through sacrificed by exsanguination.Tibiofibula is removed and is peelled off all soft tissues together by dissection.To be stored in from the bone of 5～6 rats of each group in 70% ethanol being used for histologic analysis, and will be stored in the buffering Ringer's solution (+4 ℃, pH 7.4) to carry out the test of radiograph and biomechanics from the bone of other 5～6 rats of each group.
Histologic analysis: the histologic analysis method of knochenbruch is delivered (The Effects of Growth Hormone on Fracture Healing in Rats:A HistologicalDescription.Bone, 14:19-27,1993) by Mosekilde and Bak before this.In brief, under the fracture side saw with astragalus broken line each 8mm of both sides, be embedded in the methyl methacrylate, and on Reichert-Jung Polycut slicing machine, be cut to the front end section of thick 8 μ m without decalcification.Use makes for the cellular response and the tissue response of the union of fracture of adopting and do not adopt treatment visual through the middle front end section (comprising shin bone and fibula) of Masson trichrome stain.Be used to woven bone and the lamellar bone showing the feature of poroma structure and distinguish the fracture site place through the section of Sirius red colouring.Carry out following mensuration: (1) fracture gap-be determined as shortest distance between the cortex bone end in the fracture, (2) poroma length and poroma diameter, (3) total bone volume area of poroma, (4) per unit is organized the osseous tissue of area in the poroma zone, (5) fibrous tissue in the poroma and the cartilage area in (6) poroma.
Biomechanical analysis: the biomechanical analysis method is delivered (TheEffects of Aging on Fracture Healing in Rats.Calcif Tissue Int 45:292-297,1989) by Bak and Andreassen before this.In brief, before carrying out the biomechanics test, obtain the radiograph of all fracture.Analyze the mechanical property of healing fracture at 3 by destructive or 4 bending programs.Distortion and maximum stress when the energy when having determined overall loading, rigidity, overall loading, overall loading.
Test for the union of fracture effect after the topical application
The fracture technology: the female or male beasle dog in about 2 years old age is used for this research after anesthesia.By (Lenehan, T.M. as described in Lenehan etc.; Balligand, M.; Nunamaker, D.M.; Wood, F.E.:Effects of EHDP on Fracture Healing in Dogs.J Orthop Res3:499-507; 1985) slowly continuous loading produces and laterally scratches the bone fracture in 3 bendings.The wire pulling is passed fracture site to guarantee the complete anatomy fracture of bone.Thereafter, the slow release of the compound of carrying by slow releasing pill or by realizing that with suitable preparation (for example, sticking with paste sol solution or suspension) administered compound the part of the prostaglandin agonists in 10 weeks, 15 weeks or 20 weeks to fracture site carries.
Histologic analysis: the histologic analysis method of knochenbruch is before this by (Peter, C.P. such as Peter; Cook, W.O.; Nunamaker, D.M.; Provost, M.T.; Seedor, J.G.; Rodan, G.A.Effects ofalendronate on fracture healing and bone remodeling in dogs.J.Orthop.Res.14:74-70,1996) and Mosekilde and Bak deliver (The Effects ofGrowth Hormone onFracture Healing in Rats:AHistological Description.Bone, 14:19-27,1993).In brief, after the execution, under the fracture side saw with astragalus broken line each 3mm of both sides, be embedded in the methyl methacrylate, and on Reichert-Jung Polycut slicing machine, be cut to the front end section of thick 8 μ m without decalcification.Use makes for the cellular response and the tissue response of the union of fracture of adopting and do not adopt treatment visual through the middle front end section (comprising shin bone and fibula) of Masson trichrome stain.Be used to woven bone and the lamellar bone showing the feature of poroma structure and distinguish the fracture site place through the section of Sirius red colouring.Carry out following mensuration: (1) fracture gap-be determined as shortest distance between the cortex bone end in the fracture, (2) poroma length and poroma diameter, (3) total bone volume area of poroma, (4) per unit is organized the osseous tissue of area in the poroma zone, (5) fibrous tissue in the poroma and the cartilage area in (6) poroma.
Biomechanical analysis: the biomechanical analysis method is before this by (Peter, C.P. such as Bak and Andreassen (TheEffects of Aging on Fracture Healing in Rats.Calcif Tissue Int 45:292-297,1989) and Peter; Cook, W.O.; Nunamaker, D.M.; Provost, M.T.; Seedor, J.G.; Rodan, G.A.Effects of Alendronate On Fracture Healing And BoneRemodeling In Dogs.J.Orthop.Res.14:74-70,1996) deliver.In brief, before carrying out the biomechanics test, obtain the radiograph of all fracture.Analyze the mechanical property of healing fracture at 3 by destructive or 4 bending programs.Distortion and maximum stress when the energy when having determined overall loading, rigidity, overall loading, overall loading.
The estrogen agonist/antagonist scheme
Estrogen agonist/antagonist is a compounds that suppresses the bone conversion and avoid estrogen deficiency inductive bone loss.Ovariectomized rat bone is lost the model that model is widely used as bone loss after the menopause.Use this model can test the estrogen agonist/antagonist compound in the effectiveness that prevents on bone loss and the inhibition bone resorption.
These research in adopt different ages (for example, 5 monthly ages) Sprague-Dawley female rats (Charles River, Wilmington, MA).Experimental session with rat independent stable breeding in 20cm * 32cm * 20cm cage.Allow all rats can freely obtain water and the vitamins D that contains 0.97% calcium, 0.85% phosphorus and 1.05IU/g 3The particulate state commercial feed (Agway ProLab 3000, Agway CountyFood, Inc., Syracuse, NY).
One group of rat (8～10) is carried out sham-operation and with supporting agent (10% ethanol and 90% salt solution, 1ml/ day) oral administration, excises (OVX) also with supporting agent (oral), 17 beta estradiols (Sigma, E-8876, E and the residue rat is carried out bilateral ovaries 2, 30 μ g/kg, subcutaneous injection every day) or estrogen agonist/antagonist (for example the droloxifene of 5mg/kg, 10mg/kg or 20mg/kg, every day is oral) the treatment one end time (for example, 4 weeks).Give (fluorescence dye bone seeker) subcutaneous injection of 10mg/kg fluorexon to all rats when before execution 12 days and 2 days so that check dynamic change in the osseous tissue.After treating for 4 weeks, rat put to death and carry out necrotomy.Determined with lower extreme point:
Weight increase: the body weight when body weight during necrotomy deducts operation.
Uterus weight and histology: in the necrotomy process, the uterus is removed from each rat and weigh immediately.Uterus carried out as Histological determinings such as uterus cross section tissue area, matter thickness and chamber epithelial thickness thereafter.
The total serum cholesterol: obtain blood and be allowed to condition at 4 ℃ by cardiac puncture and condense, then 2, centrifugal 10 minutes of 000g.Use efficient cholesterol calorimetric test (Boehringer MannheimBiochemicals, Indianapolis, IN) analyzing total serum cholesterol.
Femur bone mineral are measured: when necrotomy, will remove and use to be equipped with " regional high resolution scanning " software (Hologic Inc., Waltham, dual-energy x-ray absorptiometer (DEXA MA) from the right femur of each rat, QDR 1000/W, Hologic Inc., Waltham MA) scans.Scan field dimension is 5.08cm * 1.902cm, and resolving power is 0.0254cm * 0.0127cm, and sweep velocity is 7.25mm/ second.Analysis Unit's bone scanning image and the surface of bone of determining whole femur (WF), distal femoral metaphysis (DFM), femoral shaft (FS) and near end of thighbone (PF) amass, bone mineral content (BMC) and bmd (BMD).
The quantitative analysis of shin bone tissue morphology:
When necrotomy, right shin bone is removed, cut muscle, and be cut into 3 parts.With proximal tibia with shin bone is dried is fixed in 70% ethanol, in the ethanol of gradient concentration, dewater, degreasing in acetone, be embedded in then methyl methacrylate (Eastman Organic Chemicals, Rochester, NY) in.Using Reichert-Jung Polycut S slicing machine cutting thickness is the metaphyseal front end section of proximal tibia of 4 μ m and 10 μ m.To be used for the cancellous bone tissue morphometry from a slice 4 μ m section and a slice 10 μ m section of every rat.4 μ m section is dyeed with improvement Masson trichrome staining, and 10 μ m section keeps no dyeing.
Use the Bioquant OS/2 tissue morphology metrology (R﹠amp of system; M Biometrics, Inc., Nashville TN) comes the metaphyseal secondary spongy bone of proximal tibia of distance growth plate-epiphysis joint 1.2mm～3.6mm is carried out static state and dynamic organization's norphometry mensuration.For limit of determination being formed on secondary spongy bone, need omit the preceding 1.2mm in tibial metaphysis district.Use 4 μ m section to determine the index relevant, and 10 μ m section is used for determining the index relevant with the bone conversion with bone forming with bone volume, bone structure and bone resorption.
I. with the mensuration and the calculating of bone volume and structurally associated:
1. total metaphysis area (TV, mm 2): apart from the metaphysis area of growth plate-epiphysis joint 1.2mm～3.6mm.
2. bone trabecula area (BV, mm 2): the girder total area in the TV.
The bone trabecula girth (BS, mm): the length of the overall circumference of girder.
Trabecula Bone Volume (BV/TV, %): BV/TV * 100.
The bone trabecula number (TBN, #/mm): 1.199/2 * BS/TV.
6. bone trabecula thickness (TBT, μ m): (2000/1.199) * (BV/BS).
7. bone trabecula separates (TBS, μ m): (2000 * 1.199) * (TV-BV).
II. relevant with bone resorption mensuration and calculating:
The osteoclast number (OCN, #): the osteoclast sum in the metaphyseal region.
The osteoclast girth (OCP, mm): the length of the girder girth that is covered by osteoclast.
Osteoclast number/mm (OCN/mm, #/mm): OCN/BS.
Osteoclast girth per-cent (%OCP, %): OCP/BS * 100.
III. relevant with bone forming mensuration and calculating with conversion:
The girth of 1. single fluorexon mark (SLS, mm): the total length that carries out the girder girth of mark with a fluorexon mark.
The girth of 2. two fluorexon marks (DLS, mm): the total length that carries out the girder girth of mark with two fluorexon marks.
3. width (ILW, μ m) between mark: the mean distance between two fluorexon marks.
4. mineralize girth per-cent (PMS, %): (SLS/2+DLS)/BS * 100.
5. mineral apposition speed (MAR, μ m/ day): ILW/ mark spacing.
6. bone forming speed/surface is with reference to (BFR/BS, μ m 2/ d/ μ m): (SLS/2+DLS) * MAR/BS.
Bone turnover rate (BTR, %/y): (SLS/2+DLS) * MAR/BV * 100.
Statistics
(Berkeley CA) comes counting statistics information for Abacus Concepts, Inc. can to use StatView 4.0 bags.User's difference analysis (ANOVA) test then uses Fisher ' s PLSD (Stat View, Abacus Concepts Inc., 1918Bonita Ave, Berkeley, CA 94704-1014) to come the comparative group differences.
Combined therapy scheme and order treatment plan
Certainly, following scheme can be changed by those skilled in the art.For example, can use the male rat (male castration) or the female rats (oophorectomize) of harmless male or female rats, sex hormone deficiency.In addition, can use the male or female rats (for example, 12 monthly ages) of different ages under study for action.Rat can be (spay or excision testis) harmless or through castrating, and with various dose (for example to it, 1mg/kg/ day, 3mg/kg/ day or 6mg/kg/ day) (for example use anabolic agent, compound of the present invention) for some time (for example, 2 weeks～February), then (for example with various dose, 1mg/kg/ day, 5mg/kg/ day or 10mg/kg/ day) (for example use for some time as anti-absorption agents such as droloxifenes, 2 weeks～February), perhaps simultaneously with anabolism and and anti-absorption agent carry out for some time the combined therapy in (for example, 2 weeks～February) with various dose.In the rat of castrating, treatment begins (purpose is to avoid bone loss) after surgery next day or begin (purpose is to recover the bone amount) when bone loss takes place.
Rat is put to death under Patients Under Ketamine Anesthesia.Determine with lower extreme point:
Carrying out femur bone mineral as mentioned described in the estrogen agonist/antagonist scheme like that measures.
The lumbar vertebra mineral are measured: use to be equipped with " regional high resolution scanning " software (Hologic Inc., Waltham, MA) dual-energy x-ray absorptiometer (DEXA, QDR 1000/W, Hologic Inc., Waltham, (surface of bone of LV1～LV6) is long-pending, bone mineral content (BMC) and bmd (BMD) MA) come to determine each of whole lumbar vertebrae and six lumbar vertebrae vertebras in the rat of anesthesia.The mixture of ketamine/xylazine (ratio is 4: 3) by injection (intraperitoneal) 1ml/kg is placed on rat anesthesia on the rat platform then.Scan field dimension is 6cm * 1.9cm, and resolving power is 0.0254cm * 0.0127cm, and sweep velocity is 7.25mm/ second.Obtain the scan image of whole lumbar vertebrae and analyze.Each (LV1～LV6), determine that surface of bone amasss (BA) and bone mineral content (BMC), and calculating bmd (MBC is divided by BA) for whole lumbar vertebrae and six lumbar vertebrae vertebras.
Carry out proximal tibia metaphysis cancellous bone tissue morphometric and stereologic analysis as mentioned described in the estrogen agonist/antagonist scheme like that.
Carry out mensuration and the calculating relevant with structure as mentioned described in the estrogen agonist/antagonist scheme like that with the trabecular bone volume.In addition, also carry out mensuration relevant and calculating as mentioned described in the estrogen agonist/antagonist scheme like that with bone resorption.In addition, carry out mensuration and the calculating relevant with conversion as mentioned described in the estrogen agonist/antagonist scheme like that with bone forming.In addition, use the statistical operation described in the estrogen agonist/antagonist scheme above to analyze the data of acquisition.
Kidney regeneration test
Pass through PGE 2(PGE 2) or prostaglandin agonists to wild-type 293S cell with EP 2The inducibility of the expression of the bone morphogenetic protein 7 (BMP-7) in the 293S cell of transfection is studied the effect of prostaglandin agonists in kidney regeneration.
Method: make 293S cell and EP2293S cell grow in Dulbecco and improve Eagle substratum (DMEM, Gibco, BRL; Gaithersburg, MD) in.Use PGE 2Or the proxima luce (prox. luc) of prostaglandin agonists treatment, with cell with 1.5 * 10 6The density of cell/10cm ware is carried out plating.Usually after about 16～24 hours, with OptiMEM (Gibco, BRL; Gaithersburg, MD) the washed cell individual layer once then exists and does not exist supporting agent (DMSO), PGE 2(10 -6M) or prostate gland agonist (10 -6M) add 10ml OptiMEM/ ware the time.Collecting cell and extract RNA when 8 hours, 16 hours and 24 hours.By using 32The BMP-7 probe of P mark is surveyed the rna blot analysis (20mg/ swimming lane) that each trace carries out total RNA.With trace by with 32The 18s ribosome-RNA(rRNA) probe hybridization of P mark comes RNA loading carrying out normalization method.PGE 2Induce EP with prostaglandin agonists in the time-dependent manner mode 2BMP-7 in the 293S cell expresses.This inducing usually of expressing do not observed in parental cell line.In view of the known action of BMP-7 in kidney regeneration, the ability that prostaglandin agonists induces the BMP-7 in the 293S nephrocyte to express indicates the effect of prostaglandin agonists in kidney regeneration.
Using of compound of the present invention can carry any method of compound of the present invention to carry out by systematically and/or partly the site of fracture, osteotomy or bone surgery (for example).These methods comprise oral route, parenteral approach, intraduodenal route etc.Usually compound of the present invention is Orally administered, but for example use when being not suitable for for target or when the patient can't swallow medicine, can utilize parenteral to use (for example, in intravenously, intramuscular, transdermal, subcutaneous, rectum or the marrow) when oral.
Described compound can be used for treating and promoting to fracture and the healing of osteotomy by topical application (for example, being applied to the fracture site of osteotomy) compound of the present invention or its composition.For example, by will be (for example at appropriate solvent, as peanut wet goods oil-based solvent) in compound injection of the present invention to the chondrosin long slab, perhaps in the situation of open surgery by (for example with suitable carrier or thinner, bone wax, demineralization bone meal, polymerization bone cement, bone seal gum etc.) in described compound be applied topically to the chondrosin long slab, thereby compound of the present invention is applied to fracture or the site of osteotomy.Alternatively, can be by solution or the dispersion of described compound in suitable carriers or thinner be applied in the bone surgery surface of the conventional solid that uses or semi-solid implant (for example, terylene net, gelfoam and Kiel bone) or prosthese or incorporates into and wherein realize topical application.
In addition, compound of the present invention can also be applied topically to the combination of one or more anabolic agents mentioned above or fracture absorption agent to fracture in suitable carriers or thinner or the site of osteotomy.
This combination within the scope of the present invention can side by side or with any order in turn be used jointly, perhaps can aforesaid (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate with comprising and the single medicine composition and aforesaid second compound of the polymorphic forms of the drug salts of salt, its prodrug or described compound or described prodrug in pharmaceutically acceptable carrier or thinner, use.
For example, the bone anabolic agent can be used separately in the present invention or be used in combination March～3 year with anti-absorption agent, then will resist absorption agent use March separately～3 years, repeat whole treatment circulation in case of necessity.Alternatively, for example, the bone anabolic agent can be used separately or be used in combination March～3 year, then will resist absorption agent in the remaining time of patient's life, to use separately with anti-absorption agent.For example; in a preferred mode of administration; can be with the polymorphic forms once-a-day administration of the pharmacologically acceptable salt of aforesaid (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate and sodium salt or its prodrug or described prodrug; and aforesaid second compound (for example, estrogen agonist/antagonist) can be used with single or multiple dosage every day.Alternatively; for example; in another preferred mode of administration; two kinds of compounds can be used successively; wherein can be with aforesaid (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate and sodium salt compound thereof; the polymorphic forms of the pharmacologically acceptable salt of its prodrug or described prodrug is in the time interim once-a-day administration that is enough to the bone amount is increased to the level that is higher than fracture threshold (World Health Organization's research " Assessment of Fracture Risk and itsApplication to Screening for Postmenopausal Osteoporosis (1994) .Report of aWorld Health Organization Study Group.World Health Organization TechnicalSeries 843 "); aforesaid second compound (for example, estrogen agonist/antagonist) every day with single or multiple dosage used thereafter.Preferably with aforesaid first compound with as transporting pattern once-a-day administration fast such as oral delivery.
Certainly, under any circumstance, depend on seriousness, the method for application of subject study subject, sufferer on the amount of the compound of being used and opportunity and the doctor's that prescribes judgement.Therefore, because patient and inter-patient variation, the dosage that hereinafter provides is a guide, and the doctor can demarcate drug dose and thinks for the suitable treatment of patient (for example, bone amount raising) to obtain the doctor.When considering required treatment degree, the doctor must balance such as bone measures beginning level, patient age, the existence of the disease that is pre-existing in and other disease have various factorss such as (for example, cardiovascular disordeies).
Usually, the usage quantity of compound of the present invention is enough to the bone amount is increased to the level that is higher than fracture threshold (in World Health Organization's research of quoting before this as this paper detailed description).
Usually, the effective dose of used above-mentioned anabolic agent is 0.001mg/kg/ day～100mg/kg/ day among the present invention, is preferably 0.01mg/kg/ day～50mg/kg/ day.
Following paragraph provides the preferred dose scope of various anti-absorption agents.
Determine the amount to be used of this anti-absorption agent as the activity of bone loss inhibitor by anti-absorption agent.This activity is used as schemes such as above-mentioned scheme (for example, estrogen agonist/antagonist scheme) the mode of the relative maximum effective dose of minimum effective dose of inhibition bone loss with this compound by the pharmacokinetics of each compound and is determined.
Usually, the effective dose of anti-absorption agent is about 0.001mg/kg/ day～about 20mg/kg/ day.
Usually, the effective dose of progestogen is about 0.1mg/ day～10mg/ day; Preferred dose is about 0.25mg/ day～5mg/ day.
Usually, the effective dose of polyphosphonic acid salt is determined by its effectiveness as the bone resorption inhibitor of standard testing.
The scope of using the every day of some polyphosphonic acid salt is about 0.001mg/kg/ day～about 20mg/kg/ day.
Usually, the effective dose that estrogen agonist/antagonist of the present invention is used for treatment of the present invention (for example, of the present invention bone resorption treatment) is 0.01mg/kg/ day～200mg/kg/ day, is preferably 0.5mg/kg/ day～100mg/kg/ day.
Particularly, the effective dose of droloxifene is 0.1mg/kg/ day～40mg/kg/ day, is preferably 0.1mg/kg/ day～5mg/kg/ day.
Particularly, the effective dose of raloxifene is 0.1mg/kg/ day～100mg/kg/ day, is preferably 0.1mg/kg/ day～10mg/kg/ day.
Particularly, the effective dose of tamoxifen is 0.1mg/kg/ day～100mg/kg/ day, is preferably 0.1mg/kg/ day～5mg/kg/ day.
Particularly, 2-(4-p-methoxy-phenyl)-3-[4-(2-piperidines-1-base oxethyl)-phenoxy group]-effective dose of benzo [b] benzene sulphur-6-phenol is 0.001mg/kg/ day～1mg/kg/ day.
Particularly, the effective dose of following compound is 0.0001mg/kg/ day～100mg/kg/ day, is preferably 0.001mg/kg/ day～10mg/kg/ day:
Suitable-6-(4-fluorophenyl)-5-(4-(2-piperidines-1-base oxethyl)-phenyl)-5,6,7,8-tetrahydrochysene Betanaphthol;
(-)-suitable-6-phenyl-5-(4-(2-tetramethyleneimine-1-base oxethyl)-phenyl)-5,6,7,8-tetrahydrochysene Betanaphthol;
Suitable-6-phenyl-5-(4-(2-tetramethyleneimine-1-base oxethyl)-phenyl)-5,6,7,8-tetrahydrochysene Betanaphthol;
Suitable-1-(6 '-tetramethyleneimine and oxyethyl group-3 '-pyridyl)-2-phenyl-6-hydroxyl-1,2,3, the 4-naphthane;
1-(4 '-tetramethyleneimine and ethoxyl phenenyl)-2-(4 " fluorophenyl)-6-hydroxyl-1,2,3, the 4-tetrahydroisoquinoline;
Suitable-6-(4-hydroxy phenyl)-5-(4-(2-piperidines-1-base-oxyethyl group)-phenyl)-5,6,7,8-tetrahydrochysene Betanaphthol; Or
1-(4 '-tetramethyleneimine and ethoxyl phenenyl)-2-phenyl-6-hydroxyl-1,2,3, the 4-tetrahydroisoquinoline.
Particularly, the effective dose of 4-trans-Hydroxytamoxifen is 0.0001mg/kg/ day～100mg/kg/ day, is preferably 0.001mg/kg/ day～10mg/kg/ day.
Usually, compound of the present invention is used with the form of the pharmaceutical composition that comprises at least a compound of the present invention and pharmaceutically acceptable supporting agent or thinner.Therefore, compound of the present invention can be used separately or is used jointly with conventional oral dosage form, parenteral dosage form, rectum formulation or transdermal formulation.
For Orally administered, pharmaceutical composition can be taked forms such as solution, suspension agent, tablet, pill, capsule and pulvis.Comprise as the tablet of various vehicle such as Trisodium Citrate, lime carbonate and calcium phosphate can with use jointly as various disintegrating agents such as starch (preferred potato starch or tapioca (flour)) and some composition silicate and as tackiness agents such as polyvinylpyrrolidone, sucrose, gel and gum arabics.In addition, usually very useful as lubricants such as Magnesium Stearate, sodium lauryl sulphate and talcums for the compressing tablet purpose.The solids composition that also can adopt similar type is as the filler in soft-filled gelatin capsule and the hard-filled gelatin capsule; Preferred material in this respect also comprises lactose or nougat and high molecular weight polyethylene glycol.When for Orally administered when needing aqueous suspension agent and/or elixir, can be with compound of the present invention and various sweeting agent, seasonings, tinting material, emulsifying agent and/or suspension agent and as thinner and their various combined hybrid such as water, ethanol, propylene glycol, glycerine.
For the purpose that parenteral is used, can adopt the solution in sesame oil or peanut oil or the aseptic aqueous solution of solution in aqueous propylene glycol and corresponding water-soluble salt.In case of necessity, this class aqueous solution suitably can be cushioned, and at first make liquid diluent etc. ooze with enough salt solution or glucose.These aqueous solution are particularly useful for the purpose of intravenously, intramuscular, subcutaneous and peritoneal injection.In this respect, the sterile aqueous media that is adopted all can easily obtain by well known to a person skilled in the art standard technique.
For the purpose that transdermal (for example, the surface) is used, prepare rare aseptic, water-based or part aqueous solution (concentration is about 0.1%～5% usually), otherwise similar to above-mentioned parenteral solution.
The preparation of drug combination method that has a certain amount of activeconstituents is known, perhaps will it will be apparent to those skilled in the art according to the disclosure.For the example of preparation of drug combination method, referring to Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easter, Pa., the 15th edition (1975).
Pharmaceutical composition of the present invention can comprise 0.1%～95%, preferred 1%～70% compound of the present invention.Under any circumstance, composition to be administered or preparation will contain a certain amount of compound of the present invention, and its amount can effectively be treated the disease/patient's condition of subject study subject, for example, and bone disorders.
Because one aspect of the present invention relates to treatment by the combination of adopting activeconstituents that can separate administration and improves and keep the bone amount, the invention still further relates to independent pharmaceutical composition is merged with kit form.Described test kit comprises the compound of two kinds of independent pharmaceutical composition: formula I and the pharmacologically acceptable salt and aforesaid second compound of prodrug or described compound or described prodrug thereof.Described test kit comprises the container instrument that is used to hold described independent composition, for example, bottle that separates or the paper tinsel bag that separates, however the described composition that separates also can be contained in the one undivided container.Usually, described test kit comprises the specification sheets that is used to use the described composition that separates.When preferably the composition that separates being used, when using at interval with various dose, perhaps ought be needed prescriber's each composition scale timing to described combination, the kit form particularly advantageous with different dosage form (for example, oral and parenteral dosage form).
An example of this class test kit is so-called blister packages (blister pack).Blister Package is known and be widely used in packaged pharmaceuticals unit dosage form (tablet and capsule etc.) in the packaging industry.Blister Package is made of the relative harder plate of material that a slice is coated with the paper tinsel that is preferably transparent plastic material usually.In wrapping process, in plastic foil, form recess.This recess has the tablet to be packaged or the size and dimension of capsule.Then, place recess also relative harder plate of material to be sealed on the plastic foil in tablet or capsule, sealing is formed on the plastic foil face opposite with the direction of recess formation.As a result, tablet or capsule are sealed in the recess between plastic foil and the plate.Preferably, thus the intensity of plate make and can form opening and tablet or capsule are taken out from Blister Package by the recess location place in plate of on recess, manually exerting pressure.Tablet or capsule through described opening can be taken out thereafter.
Thereby it is desirable to can be on test kit for example provide the fate of the auxiliary scheme that should numeral should be swallowed corresponding to specified formulation of memory with form in the numeral on tablet or capsule next door.Auxiliary another example of this memory is the calendar that is printed on the card, and is for example as follows: " first week, Monday, Tuesday ... etc.. second week, Monday, Tuesday ... " etc.It is very apparent to remember auxiliary other variation." dosage every day " can be single tablet or capsule or several tablets or the capsule taken in given one day.In addition, dosage every day of the pharmacologically acceptable salt of the compound of formula I and prodrug thereof or described compound or described prodrug can be made up of a tablet or capsule, and dosage every day of second compound can be made up of several tablets or capsule, and vice versa.Memory is auxiliary should to reflect this point.
In another embodiment of the present invention, provide design to come provide and deliver the successively dispensing device of dosage every day of expection application order with dosage every day.Preferably, dispensing device is equipped with memory auxiliary, thereby further helps to comply with scheme.This class remember an auxiliary example be indicate provided and delivered every day dosage the mechanical counter of number.Auxiliary another example of this class memory is to provide the microchip internal memory of power with liquid crystal reader coupled by battery, perhaps for example read took last time every day dosage date and/or remind the auditory tone cues signal of the time of the dosage that should take next time.
Usually separately or combination with one another or use with preparation easily with other compound combination with compound of the present invention.Following formulation examples only is not to be intended to limit the scope of the invention for illustrative.
In following preparation, " activeconstituents " is meant one or more compounds of the present invention.
Preparation 1: gelatine capsule agent
Use following material to prepare hard-gelatin capsules:
Figure GPA00001118637200521
Use following composition to prepare tablet formulation:
Preparation 2: tablet
Figure GPA00001118637200522
Composition mixed and compression to form tablet.
Alternatively, the following tablet that respectively contains 0.25mg～100mg activeconstituents of making:
Preparation 3: tablet
Figure GPA00001118637200523
Activeconstituents, starch and Mierocrystalline cellulose are sieved and thorough mixing by 45 order U.S. sieves.With polyvinylpyrrolidonesolution solution and gained powder mixes, sieve by 14 order U.S. sieves then.The particle of so making is also sieved by 18 order U.S. sieves then 50 ℃～60 ℃ dryings.Then, sodium starch glycolate, Magnesium Stearate and the talcum that sieves by 60 order U.S. sieves before this is added in the described particle, after the mixing it compressed on tabletting machine to produce tablet.
Be prepared as follows the suspension agent that every 5ml dosage contains 0.25mg～100mg activeconstituents:
Preparation 4: suspension agent
Figure GPA00001118637200524
Activeconstituents is sieved by 45 order U.S. sieves and mix to form even paste with Xylo-Mucine and syrup.With benzoic acid solution, seasonings and pigment with the dilution of certain water and when stirring, be added into said mixture.It is volume required to generate to add enough water then.
Preparation contains the aerosol solution of following composition:
Preparation 5: aerosol
Figure GPA00001118637200531
Activeconstituents is mixed with ethanol, and mixture is added in a part of propelling agent, be cooled to 30 ℃ and be transferred to filling device.Then aequum is fed to stainless steel vessel and dilutes with residual propellant.Then valve element is assembled to this container.
Be prepared as follows suppository:
Preparation 6: suppository
Figure GPA00001118637200532
Activeconstituents sieved by 60 order U.S. sieves and be suspended in the saturated fatty acid glyceride that uses minimum essential heat melts before this.Then mixture is poured in the nominal 2g volumetrical suppository mould and allowed its cold going.
Be prepared as follows iv formulation:
Preparation 7: iv formulation
Figure GPA00001118637200533
The solution of mentioned component is used through intravenously the patient with about 1mL/ minute speed.
Above-mentioned activeconstituents also can be an agent combination.
Certainly, should be appreciated that preamble relates to illustrative embodiments of the present invention, and can under the situation that does not deviate from the spirit and scope of the invention described in the following claim, make amendment.

Claims (28)

1. the crystalline form of one kind (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt or its hydrate.
2. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form A; And
The X-ray powder diffraction collection of illustrative plates contains use CuK αThe following 2 θ values of radiation detection: 3.2,4.1,20.2 and 20.8.
3. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form A; And
The X-ray powder diffraction collection of illustrative plates contains use CuK αThe following 2 θ values of radiation detection: 3.2,4.1,16.1,20.2 and 20.8.
4. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form A; And
Described crystalline form contains in every mole of crystalline form 0.5 mole water.
5. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form B; And
The X-ray powder diffraction collection of illustrative plates contains use CuK αThe following 2 θ values of radiation detection: 3.3 and 3.6.
6. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form B; And
The X-ray powder diffraction collection of illustrative plates contains use CuK αThe following 2 θ values of radiation detection: 3.3,3.6,7.1,8.4 and 13.8.
7. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form B; And
Described crystalline form contains in every mole of crystalline form 1.0 moles water.
8. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form C; And
The X-ray powder diffraction collection of illustrative plates contains use CuK αThe following 2 θ values of radiation detection: 3.6,4.0 and 13.3.
9. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form C; And
The X-ray powder diffraction collection of illustrative plates contains use CuK αThe following 2 θ values of radiation detection: 3.5,4.0,5.9,13.3 and 16.9.
10. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form C; And
Described crystalline form contains in every mole of crystalline form 1.0 moles water.
11. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form E; And
The X-ray powder diffraction collection of illustrative plates contains use CuK αThe following 2 θ values of radiation detection: 7.0 and 21.2.
12. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form E; And
The X-ray powder diffraction collection of illustrative plates contains use CuK αThe following 2 θ values of radiation detection: 7.0,14.1,21.2,22.3 and 28.5.
13. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form F; And
The X-ray powder diffraction collection of illustrative plates contains use CuK αThe following 2 θ values of radiation detection: 5.5,8.2 and 11.0.
14. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form F; And
The X-ray powder diffraction collection of illustrative plates contains use CuK αThe following 2 θ values of radiation detection: 5.5,8.2,11.0,19.2 and 27.5.
15. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form G; And
The X-ray powder diffraction collection of illustrative plates contains use CuK αThe following 2 θ values of radiation detection: 3.2,4.0 and 7.9.
16. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form G; And
The X-ray powder diffraction collection of illustrative plates contains use CuK αThe following 2 θ values of radiation detection: 3.2,4.0,7.9,10.0 and 13.1.
17. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form A; And
Described crystalline form solid-state 13The C nucleus magnetic resonance has the following chemical shift with 1,000,000/a expression: 31.2,32.1,33.7 and 34.5.
18. crystalline form as claimed in claim 17, wherein said crystalline form solid-state 13The C nucleus magnetic resonance has the following chemical shift with 1,000,000/a expression: 31.2,32.1,33.7,34.5,173.2,176.3 and 178.2.
19. crystalline form as claimed in claim 1, wherein:
Described crystalline form is crystalline form A; And
Described crystalline form solid-state 13The C nucleus magnetic resonance has the following chemical shift with 1,000,000/a expression: 173.2,176.3 and 178.2.
20. the crystalline form of the substantially pure of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt or its hydrate.
21. the isolation of crystalline form of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt or its hydrate.
The crystalline form A of (22. 3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt semihydrate.
23. the preparation method of the crystalline form A of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt semihydrate, described method comprises:
(3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate as free acid is contacted in organic solvent to form reaction mixture with sodium hydroxide;
Reaction mixture is heated to about 50 ℃～about 90 ℃ first temperature and keeps at least 1 hour time of this first temperature;
Reaction mixture is cooled to the about 15 ℃～second about 25 ℃ temperature; And
From reaction mixture, collect solid.
24. method as claimed in claim 23, wherein said organic solvent is an isopropyl acetate.
25. method as claimed in claim 24, wherein
Isopropyl acetate exists with about 8ml～about 12ml/mmol (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate;
Sodium hydroxide is 50% aqueous sodium hydroxide solution; And
Sodium hydroxide exists with about 1mmol～about 1.3mmol/mmol (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetate.
26. the amorphous form of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt or its hydrate, wherein
Described amorphous form is amorphous form H; And
The X-ray powder diffraction collection of illustrative plates contains use CuK αThe following 2 θ values of radiation detection: 3.1.
27. amorphous form as claimed in claim 26, wherein:
Described amorphous form solid-state 13The C nucleus magnetic resonance has the following chemical shift with 1,000,000/a expression: 125.7,151.2 and 175.8.
28. the amorphous form of (3-(((4-tertiary butyl benzyl)-(pyridine-3-alkylsulfonyl)-amino)-methyl)-phenoxy group)-acetic acid sodium salt or its hydrate, wherein
Described amorphous form is amorphous form I; And
The X-ray powder diffraction collection of illustrative plates is substantially shown in Fig. 6 C.
CN200880113737A 2007-08-29 2008-08-25 Polymorphs of 3- ( ( (4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino) -methyl) -phenoxy) -acetic acid sodium salt or a hydrate thereof and methods for making the same Pending CN101842356A (en)

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