WO2006022234A1

WO2006022234A1 - NFATc1 EXPRESSION INHIBITOR, OSTEOCLAST DIFFERENTIATION OR ACTIVATION INHIBITOR, AND PHARMACEUTICAL COMPOSITION

Info

Publication number: WO2006022234A1
Application number: PCT/JP2005/015223
Authority: WO
Inventors: Kazuo Umezawa; Hiroshi Takatsuna
Original assignee: Signal Creation Inc.
Priority date: 2004-08-23
Filing date: 2005-08-22
Publication date: 2006-03-02
Also published as: JPWO2006022234A1

Abstract

[PROBLEMS] To provide an inhibitor capable of inhibiting any expression of NFATc1; a differentiation inhibitor capable of inhibiting any differentiation of osteoclast attributed to the expression of NFATc1, or an activation inhibitor capable of inhibiting any activation thereof; and a remedial composition capable of remedy of diseases attributed to the expression of NFATc1. [MEANS FOR SOLVING PROBLEMS] The compound of the formula: (1) (wherein R1 is a hydrogen atom or C2-C4 alkanoyl) is capable of inhibiting any expression of NFATc1. Consequently, substances comprising as an active ingredient the compound of the above formula (1) or a pharmacologically acceptable salt thereof are capable of inhibiting any differentiation or activation of osteoclast attributed to the expression of NFATc1 and are capable of remedy of diseases attributed to the expression of NFATc1.

Description

Specification

NFATcl expression inhibitor, osteoclast differentiation or activation inhibitor, and pharmaceutical composition

Technical field

[0001] The present invention relates to an inhibitor of NFATcl expression, an inhibitor of osteoclast differentiation or activity, and a pharmaceutical composition.

Background art

[0002] Diseases such as autoimmune arthritis (such as rheumatoid arthritis), periodontal disease, osteoporosis, Paget's disease, and bone tumors (such as multiple myeloma) may cause bone destruction due to excessive differentiation and activation of osteoclasts. What happens is known to develop more than this.

[0003] Osteoclasts become multinucleated at the final stage of differentiation. This multinucleation occurs when osteoclast precursor cells (such as monocytes and macrophages) separated from hematopoietic stem cells are activated by the action of RA NKL (receptor activator of NF-κB ligand) produced from osteoblasts. It is considered. In recent years, NFATcl (Nuclear Factor Activated T-cell cl) downstream of RANKL has been identified as a transcription factor that determines osteoclast differentiation. In hematopoietic stem cells deficient in this gene, osteoclasts are stimulated by RANKL stimulation. It is clear that differentiation into cells does not occur (see Dev. Cell 3, 889-901, 2002). Therefore, if it is possible to suppress the expression of NFATcl, it is thought that osteoclast differentiation, proliferation and activity can be suppressed, leading to the improvement of the above-mentioned diseases, and the expression of NFATcl is suppressed. There is a need for material development.

Disclosure of the invention

Problems to be solved by the invention

[0004] Therefore, the present invention provides an expression inhibitor that suppresses NFATcl expression, an inhibitory substance that suppresses osteoclast differentiation caused by NFATcl expression, or an activity of osteoclasts. An object of the present invention is to provide a pharmaceutical composition capable of ameliorating a disease caused by the expression of NFATcl.

Means for solving the problem [0005] During the osteoclast differentiation process, RANKL expressed on osteoblasts interacts with RANK on osteoclast progenitor cells, transmitting signals to osteoclast progenitor cells and NF -It is known to induce activation of κB, and if the transcriptional activity of NF-κB can be suppressed, the activity of osteoclasts can be suppressed. It is considered. In recent years, it has been clarified that NF-κB gene-deficient mouse force shows the pathology of marble bone disease, and NF-κB is considered to be an essential factor for osteoclast differentiation. It has been.

[0006] Therefore, the inventors of the present invention are compounds represented by the following formula (2), which are NF-κB activity inhibitors

(Hereinafter sometimes referred to as “(-)-DHMEQ”) to examine whether or not the division into osteoclasts can be suppressed, the mouse macrophage-like cell line RAW264. When differentiating 7 into osteoclasts by RANKL or when bone marrow-derived macrophages induced with M-CSF (macrophage colony stimulating factor) are differentiated into osteoclasts by RANKL (-)- The effect of (-)-DHMEQ on osteoclast differentiation was examined by treatment with DHMEQ.

[Chemical 1]

[0007] As a result, (-)-DHMEQ was found to suppress osteoclast differentiation in a concentration-dependent manner. In addition, when the effect of (-)-DHMEQ on cell proliferation was examined, it was difficult to inhibit cell proliferation at a concentration that suppressed osteoclast fractionation.

[0008] In recent years, it is known that the increase in the expression of proteins such as c-Fos, TRAF6, and NFA Tel is important for the differentiation of osteoclasts induced by RANKL. Therefore, the present inventors examined whether or not the expression of these proteins is changed by treating osteoclasts with (−)-DHMEQ. As a result, it was found that the expression of NFATcl by RANKL decreases depending on the concentration of (-) -DHMEQ by treatment with (-) -DHMEQ.

[0009] Further, the present inventors suppress bone resorption activity by osteoclasts with (-)-DHMEQ. In order to investigate whether or not this is possible, the effect of (−)-DHMEQ on osteoclasts induced to differentiate into bone marrow-derived macrophages by RANKL was examined. As a result, (-)-DHME Q was found to inhibit osteoclast activity in a concentration-dependent manner. These results suggest that (-)-DHMEQ suppresses osteoclast differentiation and activity by suppressing NFATcl expression. Therefore, (-)-DHMEQ is useful for improving diseases caused by NFATcl expression, osteoclasts separated by NFATcl expression, and diseases caused by osteoclast activity. It is thought that.

[0010] Further, the present inventors have found that (-)-DHME Q suppresses osteoclast differentiation and suppresses bone destruction in vertebrates in which osteoclast destruction occurs. It was. Thus, the present inventors have completed the present invention.

That is, the expression inhibitor that suppresses the expression of NFATcl according to the present invention contains, as an active ingredient, a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof.

[Chemical 2]

In the formula (1), R ¹ is a hydrogen atom or a C2-4 alkanoyl group. Examples of the alkanoyl group include an acetyl group, a propionyl group, a butanol group, or an anisotropic group thereof. It is. In addition, it is preferable that the compound is a compound represented by the above formula (2), and it is particularly preferable that the compound does not substantially contain an L-form (+) compound.

[0013] Further, the differentiation inhibitor according to the present invention is a separation inhibitor that suppresses the differentiation of osteoclasts by suppressing the expression of NFATcl, and is represented by the above formula (1). Or a pharmacologically acceptable salt thereof as an active ingredient. In the formula (1), R ¹ is a hydrogen atom or a C2-4 alkanoyl group, and examples of the alkanoyl group include an acetyl group, a propionyl group, a butanol group, or an isomer group thereof. It is preferable that the compound is a compound represented by the above formula (2), which is substantially a compound of L form (+). Things are especially preferred, not containing things.

[0014] Furthermore, the activation inhibitor according to the present invention is an activity inhibitor that suppresses the activity of osteoclasts by suppressing the expression of NFATcl, and is represented by the above formula (1). A compound represented by the formula or a pharmacologically acceptable salt thereof is contained as an active ingredient. In the formula (1), R ¹ is a hydrogen atom or a C2-4 alkanol group, and examples of the alkanol group include an acetyl group, a propiol group, a butanol group, or an isomer group thereof. It is particularly preferable that the compound does not contain a substantially L-form (+) compound, which is preferably a compound represented by the above formula (2).

[0015] The pharmaceutical composition according to the present invention is a pharmaceutical composition for improving a disease caused by NFATcl expression (including prevention, treatment, inhibition of progression, etc.) by suppressing NFATcl expression. And the compound represented by the above formula (1) or a pharmacologically acceptable salt thereof as an active ingredient. In the formula (1), R ¹ is a hydrogen atom or a C2-4 alkanol group, and the alkanol group is, for example, an acetyl group, a propiol group, a butanol group, or an isomer group thereof. . The compound is preferably a compound represented by the above formula (2), and it is particularly preferable that the compound does not substantially contain the L-form (+) compound. Examples of the disease caused by the expression of NFATcl include a disease caused by osteoclast differentiation and a disease caused by osteoclast activity. More specifically, rheumatism, bone tumor, periodontal disease, osteoporosis and the like can be mentioned.

[0016] The method for suppressing the expression of NFATcl according to the present invention comprises a composition containing the compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. Contains contact with progenitor cells (eg, monocytes, macrophages, etc.) or osteoclasts. In the formula (1), R ¹ is a hydrogen atom or a C2-4 alkanoyl group, and examples of the alkanoyl group include an acetyl group, a propionyl group, a butanol group, or an anisotropic group thereof. In addition, it is preferable that the compound is a compound represented by the above formula (2), and it is particularly preferable that the compound does not substantially contain an L-form (+) compound.

[0017] Further, the differentiation inhibiting method according to the present invention is a method for inhibiting osteoclast differentiation by inhibiting the expression of NFATcl, wherein the compound represented by the above formula (1) or A composition containing the pharmacologically acceptable salt as an active ingredient is an osteoclast precursor cell (for example, , Monocytes, macrophages, etc.). In the formula (1), R ¹ is a hydrogen atom or a C2-4 alkanoyl group. Examples of the alkanoyl group include an acetyl group, a propiol group, a butanol group, or an isomer group thereof. is there. The compound is particularly preferably a compound that does not contain a substantially L-form (+) compound, which is preferably a compound represented by the above formula (2).

[0018] Further, the method for inhibiting activation according to the present invention is a method for inhibiting the activity of osteoclasts by inhibiting the expression of NFATcl, comprising the compound represented by the above formula (1) Alternatively, the method comprises contacting osteoclasts with a composition containing a pharmacologically acceptable salt thereof as an active ingredient. In the formula (1), R ¹ is a hydrogen atom or a C2-4 alkanol group. Examples of the alkanol group include an acetyl group, a propiol group, a butanol group, or an isomer group thereof. is there. In addition, it is preferable that the compound is a compound represented by the above formula (2), and it is particularly preferable that the compound does not substantially contain an L-form (+) compound.

[0019] In addition, the method for improving a disease caused by NFATcl expression (including prevention, treatment, progression suppression, etc.) is a method for improving a disease caused by NFATcl expression by suppressing NFATcl expression. In addition, a composition containing the compound represented by the above formula (1) or a pharmacologically acceptable salt thereof as an active ingredient is applied to a human or non-human vertebrate (eg, mouse, rat, etc.). Contains to administer. In the formula (1), R ¹ is a hydrogen atom or a C2-4 alkyl group, and examples of the alkanol group include an acetyl group, a propionyl group, a butanol group, or an isomer group thereof. It is. In addition, it is preferable that the compound is a compound represented by the above formula (2), and it is particularly preferable that the compound does not substantially contain an L-form (+) compound. Examples of the disease caused by the expression of NFATcl include a disease caused by osteoclast fraction and a disease caused by osteoclast activity. More specifically, rheumatism, bone tumor, periodontal disease, osteoporosis and the like can be mentioned.

[0020] Cross-reference with related literature

This application claims priority based on Japanese Patent Application No. 2004-242866 filed on August 23, 2004. This document is incorporated herein by reference.

Brief Description of Drawings

[0021] [FIG. 1] In one embodiment of the present invention, (-)-DHMEQ is RAW induced by RANKL. It is a figure which shows the result which suppresses the division from 264.7 cells to a multinucleated cell (osteoclast).

[FIG. 2] In one embodiment of the present invention, (-)-DHMEQ is RAW induced by RANKL.

It is a figure which shows the result which suppresses the division from 264.7 cells to a multinucleated cell (osteoclast).

FIG. 3 is a graph showing a result of (−)-DHMEQ suppressing the differentiation of bone marrow-derived macrophage force multinucleated cells (osteoclasts) induced by RANKL in one example of the present invention. .

FIG. 4 is a graph showing the results of (-)-DHMEQ suppressing the differentiation of bone marrow-derived macrophage force multinucleated cells (osteoclasts) induced by RANKL in one example of the present invention. .

[FIG. 5] In one embodiment of the present invention, the effect of (−)-DHMEQ on c-Fos, TRAF6, NFATcl and other proteins expressed in osteoclasts induced by RANKL was analyzed by Western plotting. It is a figure which shows the result of having investigated.

[Fig. 6] In one embodiment of the present invention, the immunofluorescence assay shows the effect of (-)-DHMEQ on proteins such as c-Fos, TRAF6, and NFATcl expressed in osteoclasts induced by RANKL. It is a figure which shows the result investigated by (1).

FIG. 7 is a graph showing the results of (−)-DHMEQ suppressing the bone resorption activity of sorted osteoclasts in one example of the present invention.

FIG. 8 shows the results of examining the effect of (−)-DHMEQ on vertebrates in which bone destruction by osteoclasts occurs in one example of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention completed based on the above knowledge will be described in detail with reference to examples. Unless otherwise stated in the embodiments and examples, J. Sambrook, E. F. Frits ch & T. Maniatis (Εα., Molecular cloning, a laboratory manual \ 0ra edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd. Use the method described in the standard protocol collection, or a modified or modified method. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained Use the attached protocol.

[0023] The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily read the description from the description of the present specification. The invention can be reproduced. The embodiments and specific examples of the present invention described below are preferred and embodiments of the present invention, and are shown for illustration or explanation. The invention is not limited to them. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

[0024] As described above, it has become clear that (-)-DHMEQ has an action of suppressing the expression of NFATcl. In addition, (-)-DHMEQ suppresses osteoclast differentiation induced by NFATcl expression, suppresses osteoclast activity caused by NFATcl expression, and osteoclast differentiation. It has been shown that vertebrates causing bone destruction suppress osteoclast differentiation and suppress bone destruction.

[0025] From the above, by using (-)-DHMEQ, diseases caused by NFATcl expression, diseases caused by osteoclast differentiation induced by NFATcl expression, caused by NFATcl expression It is considered possible to improve diseases (including prevention, treatment, and progression inhibition) caused by osteoclast activation. Examples of the disease include rheumatoid arthritis (rheumatoid arthritis), bone tumors (eg, multiple myeloma and bone metastasis due to cancer), periodontal disease, and osteoporosis.

[0026] In addition, a compound represented by the following formula (la) which is a racemic form of (-)-DHMEQ (hereinafter sometimes referred to as "(±) -DHMEQ") or a compound similar thereto: Including the compounds represented by the general formula (I) and salts thereof having the same action as (-)-DHMEQ, diseases caused by NFATcl expression, osteoclasts induced by NFATcl expression It is considered useful for the improvement of diseases caused by differentiation and diseases caused by osteoclast activity caused by the expression of N FATcl. The NFATcl expression inhibitor, the osteoclast differentiation or activity inhibitor, and the compound to be contained as an active ingredient in the pharmaceutical composition according to the present invention is preferably (shi) -DHMEQ ( -) -DHMEQ is particularly preferred, and most preferred is substantially free of (+)-DHMEQ. In addition, the compound represented by the general formula (I) is For example, a form having an organic salt such as a quaternary ammonium salt or a metal salt such as an alkali metal may be used. Such a salt of the compound can be produced by a known method.

[Chemical 3]

[Chemical 4]

In the formula (I), R ¹ is a hydrogen atom or a C2-4 alkanol group, and examples of the alkanol group include an acetyl group, a propiol group, a butanol group, and isomers thereof. R ² is a group represented by the following formulas (A) to (G)!

[Chemical 5]

(G)

[0028] formula (D), (E) and (G), R ³ is an alkyl group of Cl～4, the alkyl group, methylcarbamoyl group, Echiru group, propyl group, butyl group and their isomers Body groups.

[0029] When the pharmaceutical composition according to the present invention is administered to vertebrates (particularly humans), the compound represented by the general formula (I) or a pharmacologically acceptable salt thereof is, for example, 2 to It may be possible to administer at a dose of 12 mg / kg (body weight), but the dose and the number of doses may be appropriately changed depending on symptoms, age, body weight, therapeutic effect, and the like.

[0030] == Production Method of Compound Represented by General Formula (I) ==

Hereinafter, a method for producing the compound represented by the general formula (I) will be described. The compound represented by the general formula (I) can be produced according to the synthesis method of wipl¾ (Synthesis, No. 12, pages 1549-561, 1995).

[0031] The compound represented by the general formula (I) can be produced, for example, based on the following reaction process formula.

[0032] In the following reaction scheme, R ¹ is a hydrogen atom or a C2-4 alkanoyl group. Examples of the alkanoyl group include an acetyl group, a propiol group, a butanol group, and an isomer group thereof. And particularly preferred is an acetyl group. R ³ is an alkyl group of Cl to 4, and examples of the alkyl group include a methyl group, an ethyl group, a propyl group, a butyl group, and the like. These isomer groups are exemplified, and a methyl group, in which a methyl group or an ethyl group is preferred, is particularly preferred. X is a halogen atom. Examples of the halogen atom include fluorine, chlorine, bromine, and iodine atoms, and a chlorine atom that is preferably a chlorine atom or a bromine atom is particularly preferable.

[Chemical 6]

(3)

Step (a): N— (2 Alkanoyloxybenzoyl) 1,5 Preparation of dimethoxy-line

2,5 Dimethoxy-phosphorus is dissolved in a solvent (for example, pyridine, etc.), and an ethyl acetate solution of O alkanoylsalicyloyl halide of formula (3) is added at 78-50 ° C, preferably under ice cooling. In addition, the reaction is carried out with stirring. After stopping the reaction by adding water, ethyl acetate is added, washed successively with hydrochloric acid, water, aqueous sodium bicarbonate, and water, dried, concentrated under reduced pressure, and dried under vacuum to give N— (2 Alkanoyloxybenzoyl) 2, 5 Dimethyoxyline is obtained. This compound can be used in the next step without purification. [0034] Step (b): Preparation of 3— (O alkanoylsalicyloylamide) -4,4-dialkoxy-2,5-cyclohexagenone

The compound of the formula (4) obtained above is dissolved in R ³ OH (for example, methanol, ethanol, etc.), and diacetoxybenzene is calcined at 20 to 50 ° C., preferably under ice cooling. The reaction is carried out with stirring at room temperature. After concentration under reduced pressure, ethyl acetate was added, washed sequentially with aqueous sodium bicarbonate and brine, and the residue obtained by concentrating the ethyl acetate layer under reduced pressure was purified by column chromatography to obtain the compound represented by formula (5) 3 — (O alkanoylsalicyloylamide) -4,4-dialkoxy 2,5 cyclohexagenone is obtained.

[0035] Step (c): 5, 6 Epoxy-4,4-dialkoxy 3 Preparation of alkanoylsalicyloylamide 2-cyclohexenone

The compound of formula (5) is dissolved in a solvent (for example, tetrahydrofuran, methanol, etc.), and at −20 to 50 ° C., preferably under ice cooling, hydrogen peroxide solution and sodium hydroxide solution are added, React with stirring. Ethyl acetate is added to the reaction solution, washed successively with hydrochloric acid solution, aqueous sodium thiosulfate solution and brine, dried in air and then vacuum dried. To remove the remaining raw material compound, dissolve the residue in acetone, add p-toluenesulfonic acid, and stir at room temperature to decompose the raw material compound. To the residue obtained by distilling off the solvent under reduced pressure, add ethyl acetate and wash with water. The residue obtained by drying the ethyl acetate layer was purified by column chromatography, and the 5, 6-epoxy-4, 4-dialkoxy-3-alkaneolsali represented by formula (6) was used. Tyroylamide 2-cyclohexenone is obtained.

[0036] Step (d): 5, 6 Epoxy-2 Alkanoylsalicyloylamide-2 Cyclohexene

-1, 4 Preparation

Dissolve the compound of formula (6) in a solvent (for example, methylene chloride), add an inorganic acid or an organic acid (for example, boron trifluoride jetyl ether complex) under ice-cooling, and apply stirring force. React. A solvent (for example, ethyl acetate) is added to the reaction solution, washed with water, the solvent layer is concentrated, and the resulting residue is washed with methanol to obtain a 5,6-epoxy compound represented by formula (7). 2 Alkanoylsalicyloylamide 2 Cyclohexene 1,4 dione is obtained.

[0037] Step (e): 5, 6 Epoxy 1-4 Hydroxy 1-3 Alkanoylsalicyloylamide-2 Preparation of cyclohexenone

The compound of formula (7) is suspended in a solvent (eg, methanol, ethanol, THF, etc.), and a reducing agent (eg, sodium borohydride, etc.) is added at −78 to 50 ° C., preferably under ice cooling. React and react. A solvent (for example, ethyl acetate, methylene chloride, etc.) is added to the reaction solution, washed successively with hydrochloric acid and water, the solvent layer is dried, concentrated under reduced pressure, suspended in methanol, stirred, washed, and washed with formula (1 5, 6 epoxy 1-4 hydroxy 1-3 alkanoyl salicyloyl amide 2-cyclohexenone is obtained.

[0038] Step (f): Preparation of 3, 3 dialkoxy 4, 5 epoxy 6 hydroxy 2 alkanoyl salicyloylamide cyclohexene

Dissolve the compound of formula (6) in a solvent such as methanol and a sodium bicarbonate solution, add a reducing agent (for example, sodium borohydride) at -78 to 50 ° C, preferably under ice cooling, and stir. React with a strong force. Add a solvent (eg, ethyl acetate) to the reaction solution, wash with hydrochloric acid and water sequentially, dry the solvent layer, concentrate under reduced pressure, vacuum dry, purify by column chromatography, etc. The 3,3 dialkoxy 4,5 epoxy-1 6 hydroxy-1-2-alkanoylsalicyloylamide cyclohexene shown is obtained.

[0039] Step (g): 5, 6 Preparation of epoxy-4-hydroxy-2 alkanoylsalicyloylamide-2 cyclohexenone

Dissolve the compound of formula (8) in a solvent (for example, acetone), add p-toluenesulfonic acid, and react at room temperature with stirring. A solvent (for example, ethyl acetate) is added to the reaction solution, washed with water, the solvent layer is dried, concentrated under reduced pressure, and purified to give a 5, 6-epoxy 4 hydroxy 2 alkanoyl of formula (9). Rusalicyloylamide 2 cyclohexenone can be obtained.

[0040] In addition, as the O alkanoylsalicyloyl halide of the formula (3), O acetylsalicyloyl chloride is used, and methanol is used as a solvent for dissolving the compound of the formula (4) in the step (b). As a result, the above-mentioned (shi) -DHMEQ and the compound represented by the following formula (lb) can be produced.

[Chemical 7]

[0041] = = Preparation of compound represented by formula (2) ==

In recent years, the present inventors have found that (-)-DHMEQ has a superior activity inhibition activity of NF-κΒ than (shi) -DHMEQ. Therefore, (-)-DHMEQ is considered to have superior NFATcl expression-suppressing action and osteoclast differentiation and activation-suppressing action superior to (shi) -DHMEQ. It can also be expected that (-) -DHMEQ has fewer side effects than (shi) -DHMEQ. The preparation of (-)-DHMEQ (formula (2)) will be described below.

[0042] As shown in the following reaction formula, the compound represented by the formula (2) is a compound obtained by protecting the phenolic hydroxyl group of (shi) -DHMEQ (formula (la)) with a silyl group, and (10) is divided into a compound represented by the formula (11) and a compound represented by the formula (12) by a chiral column, and then the compound represented by the formula (11) is deprotected to give the formula (2 ) ((-)-DHMEQ) can be obtained (Bioorg. Med. Chem. Lett. 10, 865-869, 2000). It is also possible to obtain the compound represented by formula (13) ((+)-DHMEQ) by deprotecting the compound represented by formula (12) in the same manner as described above.

[Chemical 8]

(2) (13) In addition, the compound represented by the formula (2) ((-)-DHMEQ: D DHMEQ) and the compound represented by the formula (13) ((+)-DHMEQ: L DHMEQ) is a general formula (14)

[Chemical 9]

[0044] (Shi)-DHMEQ can also be obtained by direct optical resolution using a separating agent comprising a polysaccharide aromatic force rubamate derivative substituted with a group represented by the formula:

[0045] The aromatic aromatic rubamate derivative of the polysaccharide includes a substituent (one R ⁴ , —R ⁵ , —R ⁶ , —R ⁷ , and —R ⁸ ) in the general formula (14), a hydrogen atom, carbon Ability of mentioning aromatic power rubamate derivatives of polysaccharides composed of alkyl groups having 1 to 8 carbon atoms, alkoxy groups having 1 to 8 carbon atoms, aromatic groups having 6 to 14 carbon atoms, halogen atoms, etc. Amylose tris (3,5-dimethylphenylcarbamate), particularly preferred is cellulose tris (3,5-dimethylphenol carbamate), and cellulose tris (3,5-dimethylphenol carbamate). Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0046] The optical resolution of the (shi) -DHMEQ is an optical solution filled with a separating agent containing a polysaccharide aromatic force rubamate derivative substituted with a group represented by the general formula (14) as an active ingredient. It is preferably carried out by high performance liquid chromatography using an active column such as DAICEL CHIRALPAK AD (10 mm id × 250 mm). As the mobile phase, for example, methanol supplemented with 0.1 to 0.9 v / v% acetic acid can be used.

[0047] The optical purity of (-)-DHMEQ and (+)-DHMEQ obtained in this way can be calculated by measuring the light intensity with a JASC 0 DIP-360 polarimeter ( (-) -DHM EQ is [α] ² ° —241. (CO. L MeOH), (+) — DHMEQ is [α] ² ° + 239. (cO. L MeO

D D

H). ).

Example

[0048] Hereinafter, this will be described in more detail with reference to examples and drawings.

[0049] [Example 1]

Macrophage-derived RAW264.7 cells differentiate into osteoclasts when treated with RANKL. RANKL / RANK signals are also known to induce NF-κΒ activation. NF-KB is considered to be an essential factor for osteoclast differentiation. Therefore, using (-) -DHMEQ, an inhibitor of NF-κB activation, (-) -DHMEQ against osteoclast differentiation induced when RAW264.7 cells derived from macrophages were treated with RANKL. B and J were examined for the effects of.

[0050] Dulbecco's modification containing 10% fetal bovine serum (JRH Biosciences, 200 μg / ml Rikicho, non, 100units / ml penicillin G, 600 μg / ml L-glutamine, and 2.25 g / l NaHCO

Three

Seed RAW264.7 cells (4 x 10 ⁴ cells / well) in a 24 well plate containing 0.5 ml of Eagle's medium, 100 ng / ml RANKL (recombinant human RANKL; Wako Chemical) and 0.3, 1, or 3 μg / ml DHMEQ supplemented and cultured for 5 days (In addition, cells cultured without supplementing RANKL and DHMEQ and cells cultured with only 100 ng / ml RANKL supplemented were prepared. O) After incubation, the cells were washed with PBS containing 5% noraformaldehyde (phosphate buffer; 8g / l NaCl, 0.2g / l KC1, 0.916g / l Na HPO, 0.2g / l KHPO) for 10 minutes.

2 4 2 4

After fixing at room temperature and rinsing with PBS, it was treated with acetone / ethanol (1: 1 v / v) for 30 seconds at room temperature.

[0051] Subsequently, the fixed cells were treated with TRAP staining solution (0.01% naphtol AS-MX phosphate (ICN biomedicals), 0.05% fast red violet LB salt (Sigma), 50 mM sodium tartrate, and 90 mM sodium acetate (pH 5.0)), and rinsed with water and observed with a microscope. The result is shown in Fig. 1. In addition, the number of multinucleated cells (MNC) specialized in 4 or more nuclei stained with TRAP staining solution was counted. The result is shown in Fig.2.

[0052] As shown in FIG. 1 and FIG. 2, it was revealed that DHMEQ inhibits the RAN26-induced differentiation of RAW264.7 cells into osteoclasts in a concentration-dependent manner. Although no data was shown, inhibition of cell growth by DHMEQ could not be confirmed, suggesting that the above-described inhibitory effect of DHMEQ was not due to nonspecific growth inhibition.

[0053] [Example 2]

We also investigated the effect of (-)-DHMEQ on the osteoclast differentiation induced by RANKL treatment of bone marrow-derived macrophages induced by M-CSF.

[0054] 10% FBS, 200 μg / ml kanamycin, 100 units / ml penicillin G, 2.25 g / l NaHCO,

3 and 10ng / ml M-CSF (recombinant human M-CSF; including GenzymeJ a-minimum es 24-well plates containing essential medium (Invitrogen) were seeded with 8-week-old ICR mice (Charles River Japan) -derived bone marrow cells (5 X 10 ⁵ cells / well) and cultured for 2 days. Cells were washed away and adherent cells were used as bone marrow derived macrophages. The bone marrow-derived macrophage culture medium was supplemented with 100 ng / ml RANKL and 0.3, 1, or 3 μg / ml DHMEQ, and cultured for 2 days (note that RANKL and DHMEQ were not added as controls). 2) Prepared after culturing with 100 ng / ml RANKL only.) O After culture, bone marrow-derived macrophages were fixed and stained with TRAP staining solution as in Example 1. And observed with a microscope. In addition, multinucleated cells having 4 or more nuclei were selected from the cells stained with TRAP staining solution, and the number of the cells was counted. The results are shown in Fig. 3 and Fig. 4, respectively.

[0055] As shown in FIGS. 3 and 4, DHMEQ was found to inhibit the differentiation of bone marrow-derived macrophages into osteoclasts induced by RANKL in a concentration-dependent manner. Although data are not shown, inhibition of cell growth by DHMEQ was not confirmed, indicating that this differentiation inhibitory effect is not due to growth inhibition.

[0056] [Example 3]

In osteoclast differentiation induced by RANKL, it is known that increased expression of proteins such as c_Fos, TRAF6, and NFATcl is important. Therefore, the effect of (-)-DHMEQ on these proteins was examined.

[0057] 10% FBS, 200 μg / ml kanamycin, 100 units / ml penicillin G, 2.25 g / l NaHCO,

3 and 10 ng / ml M-CSF in a 60 mm dish containing 'a -minimum essential medium (Invitrogen) and seeded with bone marrow cells (5 X 10 ⁶ cells / well) derived from 8-week-old ICR mice Cultured for days, non-adherent cells were washed away and the adherent cells were used as bone marrow-derived macrophages. This bone marrow-derived macrophage culture medium was supplemented with lOOng / ml RANKL and 0.3, 1, or 3 μg / ml DHMEQ, and cultured for 24 hours (note that RANK L and DHMEQ were not added as controls) 2) Prepared by culturing with only 100 ng / ml RANKL and cultivating bone marrow-derived macrophages and lysing solution (20 mM Tris-HCl (pH 8.0)) , 150 mM NaCl, 2 mM EDTA, 100 μM NaF, 400 M Na V

Three

O, 1% Nonidet P- 40, 1 μ g / ml Roipe leptin, 1 _mu g / ml antipain, and 1 MP Cells were lysed with MSF) and subjected to SDS-PAGE.

[0058] Subsequently, Western blotting was performed using a PVDF membrane (polyviniliaene difluonde membrane; Amersnam Biosciences). Primary antibodies are anti-NFATcl antibody (Santa Cruz Biotechnolgy; diluted 200-fold), anti-c-Fos antibody (Santa Cruz Biotechnology; diluted 500-fold), anti-TRAF6 antibody (Santa Cruz Biotechnology; diluted 500-fold), anti-tubulin antibody (Sigma Secondary antibody was horseradish peroxidase-conjugated Hedge anti-rabbit Ig antibody or Hedge anti-mouse Ig antibody (Amersham Biosciences; diluted 1000 times), and detection was performed using ECL (Enhanced Chemiluminescence). ). The results are shown in Fig. 5.

[0059] Further, the influence of (-)-DHMEQ on proteins such as c-Fos, TRAF6, and NFATcl was confirmed by an immunofluorescence assay.

[0060] Bone marrow-derived macrophage culture solution obtained in the same manner as described in Example 2 was supplemented with 100 ng / ml RANKL and 3 μg / ml DHMEQ and cultured for 24 or 48 hours (note that As a control, a culture prepared by adding only lOOng / ml RANKL was prepared. After culturing, the cells were washed twice with ice-cold PBS, fixed with a 5% formaldehyde solution at room temperature for 15 minutes, and then treated with PBS containing 1% Triton X for 5 minutes. The cells were then washed 3 times with PBS and incubated with PBS containing 5% BSA for 30 minutes at room temperature.

[0061] Next, the cells were reacted with anti-NFATcl antibody, anti-c-Fos antibody, or anti-TRAF6 antibody for 60 minutes at room temperature, then washed 3 times with PBS, and Alexa Fluor 546 goat anti-rabbit IgG antibody or Staining was performed using Alexa F1 uor 488 goat anti-mouse IgG antibody (Molecular Probe). In order to confirm whether apoptosis was induced in the cells, the cells were stained with Hoechest 33258 for 40 minutes at room temperature. Each of the cells thus stained was washed 3 times with PBS and observed with a microscope. The result is shown in Fig. 6.

[0062] As shown in FIGS. 5 and 6, (-)-DHMEQ is induced by RANKL, although it does not affect the expression levels of TRAF6 and c-Fos that are induced by RANKL. It was revealed that the expression level of NFATcl decreased in a concentration-dependent manner. This suggests that (-)-DHMEQ suppresses the osteoclast differentiation by suppressing the expression of NFATcl.

[0063] [Example 4] In addition, the effect of (-)-DHMEQ on bone resorption by disseminated osteoclasts was examined.

[0064] Bone marrow-derived macrophages (5 × 10 ⁴ cells / well) obtained by the same method as in Example 3 were seeded on OAAS plates (Osteogenic Core Technology) coded with calcium phosphate crystals, and 10 ng / Incubation was performed for 3 days with ml M-CSF and 100 ng / ml RANKL. The cells were then treated with 3 g / ml (-)-DHMEQ and cultured for an additional 2 days (as a control, incubated with 10 ng / ml M-CSF alone, and not further treated with (-)-DHM EQ And cultivated with 10 ng / ml M-CSF and 100 ng / ml RANKL, and further cultured without treatment with (-)-DHMEQ. The plate was treated with sodium chlorite for 5 minutes, washed with water, dried and observed with a microscope. As a result, as shown in FIG. 7, it was revealed that (-)-DHMEQ suppresses the bone resorption ability of the separated osteoclasts.

[0065] From the above, it was revealed that (-) -DHMEQ suppresses osteoclast differentiation and activation (bone resorption ability). By using (-) -DHMEQ, NFATcl expression It was suggested that the disease caused by these phenomena can be effectively ameliorated by suppressing the overdifferentiation and activation of osteoclasts.

[Example 5]

Next, in order to confirm whether (-) -DHMEQ is useful for vertebrates in which bone destruction by osteoclasts occurs, a bone destruction model mouse was prepared, and (-) -DHMEQ The effect was investigated.

[0067] Using a 1 ml syringe (24G needle), 7 weeks old C57BL mice (female; n = 3) raised for 1 week were injected intraperitoneally with (-)-DHMEQ solution at 8 mg / kg-body weight. Administered. The (-)-DHMEQ solution was prepared by dissolving in DMSO (dimethyl sulfoxide) to a concentration of 10 mg / ml and then diluting to a concentration of 270 g / ml in RPMI1640 medium. The same amount of RPMI 1640 medium was administered to 6 mice in the control group.

[0068] One hour after administration of each solution, 5 mg / ml LPS (lipopolysaccharide; Sigma) dissolved in PBS was subcutaneously injected into the top of the head at 25 mg / kg-body weight using a syringe (Hamilton). . In addition, 3 out of 6 mice in the control group were subcutaneously injected with PBS alone at the top of the head. Thereafter, an equal volume of (-)-DHMEQ solution (experimental group) or RPMI1640 medium (control group) every 24 hours Was administered intraperitoneally for 5 consecutive days.

[0069] On day 6, mice were sacrificed by cervical dislocation, decapitated, and only the head was immersed in 45 ml of fixative (PBS containing 10% formaldehyde and 0.5% glutaraldehyde) for 2 days at 4 ° C. Shake to fix. Thereafter, the plate was shaken and washed with PBS all day and night, and only the head calvaria was collected using a cutter. Then, use tweezers to peel only the scalp of the calvaria, wrap it in nylon mesh, immerse it in demineralized liquid (10% EDTA-NaOH pH 7.0), shake at 4 ° C for 2 weeks, and decalcify it. went. The decalcification liquid was changed every 5 days.

[0070] The decalcified calvaria was washed by shaking overnight with PBS, then shaken with an 80% ethanol solution overnight at 4 ° C, then replaced with 90% ethanol and shaken overnight, and then, The dehydration treatment was performed by exchanging with 100% ethanol and shaking all day and night. Thereafter, the calvaria was soaked in 100% acetone and similarly shaken all day and night for degreasing.

[0071] The degreased calvaria was soaked in JB-4 greaves of JB-4 embedding Kit (Polysciences, Inc., Warrington, Pa.) And shaken at 4 ° C for 4 days. JB-4 succinate was changed once every two days. After shaking, it was immersed in JB-4 rosin containing a polymerization initiator and allowed to stand for 2 hours under a nitrogen stream to solidify. Thereafter, a 2 m thick section was prepared using a microtome, immersed in TRAP staining solution, stained at 37 ° C for 1 hour, washed with water and observed with a microscope (Fig. 8A).

[0072] The sections were stained with methyl green staining solution at room temperature for 10 minutes, washed with water, air-dried, encapsulated with Entray-Yu mounting medium (Sigma), and observed with a phase contrast microscope. Multinucleated cells (MNC) having 4 or more nuclei were counted as osteoclasts (FIG. 8B). Further, a micrograph of the above section was taken, the area of the bone marrow cavity was measured using the image analysis software NIH Image, LPS and (−)-DHMEQ were administered, and the bone marrow in the calvarial region of the mouse. Assuming that the area of the cavity was “1”, the increase rate of the bone marrow cavity in the calvarial region of the mice administered LPS and RPMI1640 and the mice administered LPS and (−)-DHMEQ was calculated (FIG. 8C).

) o

[0073] As shown in Fig. 8A, osteoclasts (darkly stained portions such as arrows in Fig. 8A) were remarkably induced in mice administered with LPS and RPMI1640, but LPS and (- )-In the DHMEQ-administered mice, osteoclasts were hardly induced. Furthermore, as shown in Fig.8B, the number of osteoclasts within the range of 500 m LPS injection point force Is approximately 30% lower in mice administered LPS and (-)-DHMEQ than in mice administered LPS and RPMI1640, with the same number of osteoclasts as in mice not administered LPS. there were. In addition, when the degree of bone destruction was examined using the increase rate of the bone marrow cavity as an index, as shown in Fig. 8C, the mice administered LPS and RPMI1640 increased about 70% compared to the mice not administered LPS. Despite this, the mice that received LPS and (-)-DHMEQ had an increase rate of about 20%.

[0074] Based on the above, (-)-DHMEQ suppresses osteoclast differentiation and bone destruction even in vivo! / Against vertebrates in which bone destruction by osteoclasts occurs. It was suggested to be useful. In addition, the toxicity of (-)-DHMEQ due to daily intraperitoneal administration could not be confirmed in this example Possibility of industrial use

[0075] According to the present invention, a suppressor that suppresses NFATcl expression, a differentiation suppressor that suppresses osteoclast differentiation caused by NFATcl expression, or an activation suppressor that suppresses activation, Furthermore, an ameliorating substance that can ameliorate a disease caused by the expression of NFATcl can be provided.

Claims

The scope of the claims

[1] An expression inhibitor that suppresses the expression of NFATcl, comprising as an active ingredient a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof.

(Wherein R ¹ is a hydrogen atom or a C2-4 alkanoyl group.)

[2] The expression inhibitor according to claim 1, wherein the compound is represented by the following formula (2):

[3] A protein inhibitor that suppresses osteoclast differentiation by suppressing the expression of NFATcl, and is a compound represented by the following formula (1) or a pharmacologically acceptable compound thereof: A differentiation inhibitor comprising a salt as an active ingredient.

(Wherein R ¹ is a hydrogen atom or a C2-4 alkanoyl group.)

[4] The separation inhibitor according to claim 3, wherein the compound is represented by the following formula (2).

[5] An activation inhibitor that suppresses the activation of osteoclasts by suppressing the expression of NFATcl, comprising a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof: An activation inhibitor characterized by containing as an active ingredient.

(Wherein R ¹ is a hydrogen atom or a C2-4 alkanoyl group.)

[6] The active ingredient inhibitor according to [5], wherein the compound is represented by the following formula (2):

[7] A pharmaceutical composition for ameliorating a disease caused by NFATcl expression by suppressing NFATcl expression, comprising a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof: A pharmaceutical composition comprising as an active ingredient.

(Wherein R ¹ is a hydrogen atom or a C2-4 alkanoyl group.)

8. The pharmaceutical composition according to claim 7, wherein the compound is represented by the following formula (2).

[9] The pharmaceutical composition according to claim 7, wherein the disease caused by the expression of NFATcl is a disease caused by osteoclast differentiation.

[10] The pharmaceutical composition according to claim 7, wherein the disease caused by the expression of NFATcl is a disease caused by osteoclast activity.

[11] The pharmaceutical composition according to claim 7, wherein the disease caused by the expression of NFATcl is any disease selected from the group power of rheumatism, bone tumor, periodontal disease, and osteoporosis. object.