WO2009084737A1

WO2009084737A1 - Compound having activity to suppress degranulation and cytokine production and use thereof

Info

Publication number: WO2009084737A1
Application number: PCT/JP2008/073977
Authority: WO
Inventors: Masakazu Hase; Miyuki Nakashima
Original assignee: Interprotein Corporation
Priority date: 2007-12-28
Filing date: 2008-12-26
Publication date: 2009-07-09

Abstract

The present invention provides a compound represented by the formula (I): wherein L is an alkylene group;X is a bond, -O-, -S- or -CO-;Y is a bond, -NR3- or -CO-;Z is an organic group or OH;R1 and R2 are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom; and R3 is an optionally substituted alkyl group or a hydrogen atom, or a salt thereof.

Description

DESCRIPTION

COMPOUND HAVING ACTIVITY TO SUPPRESS DEGRANULATION AND CYTOKINE

PRODUCTION AND USE THEREOF

Technical Field [0001]

The present invention realtes to a novel compound having an activity to suppress degranulation and cytokine production, which is useful as an agent for the prophylaxis or treatment of diseases such as allergic diseases and the like.

Background Art [0002]

As current therapeutic drugs for allergy, antihistamines, steroids having immunosuppressive effect and the like can be mentioned. The antihistamines suppress binding of histamine released from effector cells (e.g., mast cell etc.) to a specific receptor expressed on a target cell. However, since chemical transmitters produced by effector cells contain not only histamine but also serotonin, prostaglandin and the like, antihistamines are not expected to show effect on such chemical transmitters. On the other hand, steroids have a strong immunosuppressive or anti-inflammatory action, and are frequently used as therapeutic drugs for allergy. However, steroids are known to cause many side effects such as opportunistic infection and the like. [0003]

It has been clarified that mast cells have an ability to produce cytokine or granules containing a chemical transmitter such as histamine and the like, and degranulation and cytokine production from mast cell plays an important role in allergic diseases such as pollinosis, bronchial asthma, atopic dermatitis and the like, and in various inflammatory diseases including autoimmune diseases. Therefore, suppression of degranulation is effective as a method for the treatment of such diseases. [0004]

As pharmaceutical agents that suppress release of a chemical transmitter through degranulation, by stabilizing the membrane of mast cells, Intal (sodium cromoglycate) , Rizaben (Tranilast) and the like can be mentioned. These pharmaceutical agents are already used as antiallergic agents in clinical practice. Degranulation occurs by signal transduction via IgE receptor. Since the process is calcium ion-dependent, an attempt has also been made to control degranulation by controlling the intracellular calcium concentration. However, since calcium ion has an important role in any site in the body, controlled intracellular calcium concentration has a risk of affecting other biological functions. [0005]

See non-patent references 1 and 2 for the prior art relating to degranulation and antiallergic agents. See non- patent references 3 and 4 for prior art relating to cytokine production from mast cells . [0006]

Hirano et al . have found that degranulation and cytokine production can be regulated by controlling the intracellular zinc ion concentration, and reported that ^'TPEN (N,N,N'₇N'- tetrakis (2-pyridylmethyl) ethylenediamine) , a zinc ion chelator, suppresses degranulation of mast cells and is useful as an agent for the prophylaxis or treatment of allergic diseases and the like (patent reference 1) . However, since TPEN is a compound developed as a strong chelator of zinc ion, there is always a risk of causing unexpected side effects when administered to living organisms. [0007]

On the other hand, as medical or pharmaceutical products, ibuprofen piconol and Ufenamate, which are non-steroidal antiinflammatory drugs applied to atopic dermatitis are known (non- patent references 5 and 6) [0008]

[0009]

[0010]

In addition, ibuprofen and flufenamic acid have an inhibitory action on COX-I and 2, and are widely used as antiinflammatory drugs (non-patent references 7 and 8) . [0011]

[0012]

[0013] patent reference 1: WO 2006/080581 non-patent reference 1: Turner H. & Kinet JP. Signalling through the high-affinity IgE receptor Fc epsilonRI. Nature 402, B24-30 (1999) non-patent reference 2: Blank U, Rivera J. The ins and outs of IgE-dependent mast-cell exocytosis. TRENDS in Immunology 25, 266-273 (2004) non-patent reference 3: Burd PR, Rogers HW, Gordon JR, Martin CA, Jayaraman S, Wilson SD, Dvorak AM, Galli SJ, Dorf ME. Interleukin 3-dependent and -independent mast cells stimulated with IgE and antigen express multiple cytokines. J. Exp. Med. 170, 245-257 (1989) non-patent reference 4: Song JS, Haleem-Smith H, Arudchandran R, Gomez J, Scott PM, Mill JF, Tan TH, Rivera J. Tyrosine phosphorylation of Vav stimulates IL-6 production in mast cells by a Rac/c-Jun N-terminal kinase-dependent pathway. J. Immunology 163, 802-810' (1999) non-patent reference 5: "STADERM OINTMENT" pharmaceutical product package insert, Torii Pharmaceutical Co., Ltd., revised February 2004 (2nd edition) non-patent reference 6: "FENAZOL OINTMENT" pharmaceutical productpackage insert, ABBOTT JAPAN CO., LTD., revised June 2005 (3rd edition) non-patent reference 7: "Brufen tablet 100" pharmaceutical product package insert, Kaken Pharmaceutical Co., Ltd., revised December 2005 (9th edition) non-patent reference 8: "OPYRIN tablet 125 mg" pharmaceutical product package insert, Taisho Toyama Pharmaceutical Co., Ltd., revised July 2007 (8th edition)

Disclosure of the Invention Problems to be Solved by the Invention [0014]

An object of the present invention is to provide a novel compound having a strong degranulation suppressive effect and a cytokine production suppressive effect, which is low toxic and useful for the prophylaxis or treatment of allergic diseases and inflammatory diseases .

Means of Solving the Problems [0015]

The present inventors have conducted intensive studies of a compound having degranulation suppressive effect and cytokine production suppressive effect and found that a compound represented by the formula (I) : [0016]

[0017] wherein

L is an alkylene group; X is a bond, -O-, -S- or -CO-;

Y is a bond, -NR³- or -CO-;

Z is an organic group or OH; and

R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted alkyl group or a hydrogen atom, has a superior degranulation suppressive effect and a cytokine production suppressive effect and low toxicity, which resulted in the completion of the present invention. [0018]

Accordingly, the present invention relates to the following.

[1] A compound represented by the formula (I) :

[0019]

[0020] wherein

L is an alkylene group; Z is an optionally substituted hydrocarbon group or an optionally substituted heterocyclic group; and R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, or a salt thereof. [2] The compound of [1], wherein L is a Ci-₆ alkylene group.

[3] The compound of [1] , wherein Z is an optionally substituted alkyl group; an optionally substituted aryl group; an optionally substituted aralkyl group; or an optionally substituted heterocyclic group containing at least one hetero atom selected from an oxygen atom and a nitrogen atom. [4] The compound of [1] , which is 2-[N,N-bis (2-pyridylmethyl) amino] ethyl 2- (4- isobutylphenyl) propionate, 2-[N,N-bis (2-pyridylmethyl) amino] ethyl 2-(3- trifluoromethylphenylamino) benzoate,

2-[N,N-bis (2-pyridylmethyl) amino] ethyl acetate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl isobutyrate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl phenylacetate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl 3-phenylpropionate, 2- [N, N-bis (2-pyridylmethyl) amino] ethyl benzoate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl isonicotinate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl furan-2-carboxylate, or a salt thereof.

[5] A degranulation suppressant comprising a compound represented by the formula (I) : [0021]

[0022] wherein

L is an alkylene group;

X is a bond, -O-, -S- or -CO-;

Y is a bond, -NR³- or -CO-;

Z is an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group or OH; and

R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein^' R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N,N, N' ,N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof.

[6] The suppressant of [5] , wherein X is -0-, Y is -CO-, and Z is an optionally substituted hydrocarbon group or an optionally substituted heterocyclic group. [7] The suppressant of [5], wherein X is -0-, Y is a bond, and Z is an optionally substituted hydrocarbon group.

[8] The suppressant of [5] , wherein the compound represented by the formula (I) is

N,N-bis (2-pyridylmethyl) glycine, N,N-bis (2-pyridylmethyl) glycylglycine tert-butyl ester,

N,N-bis (2-pyridylmethyl) glycylglycine,

N,N-bis (2-pyridylmethyl) glycyl-L-phenylalanine tert-butyl ester,

N,N-bis (2-pyridylmethyl) glycyl-L-phenylalanine,

2- [N,N-bis (2-pyridylmethyl) amino] -1-morpholinoethanone, 2- [N,N-bis (2-pyridylmethyl) amino] -N' -phenethylacetamide, 2- [N,N-bis (2-pyridylmethyl) amino] ethanol, 2-[N,N-bis (2-pyridylmethyl) amino] ethyl 2- (4- isobutylphenyl) propionate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl 2- (3- trifluoromethylphenylamino) benzoate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl acetate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl isobutyrate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl phenylacetate,

2- [N, N-bis (2-pyridylmethyl) amino] ethyl isonicotinate, or 2- [N,N-bis (2-pyridylmethyl) amino] ethyl furan-2-carboxylate. [9] The suppressant of [5], wherein the compound represented by the formula (I) is N,N-bis (2-pyridylmethyl) -beta-alanine,

N-{2- [2-N, N-bis (2-pyridylmethyl) amino-ethoxymethyl] phenyl} -3- trifluoromethylphenylamine,

N- (2-benzyloxyethyl) -N, N-bis (2-pyridylmethyl) amine,

3- [N, N-bis (2-pyridylmethyl) amino] -N-phenyl-propionamide, N- (2-methoxyethyl) -N, N-bis (2-pyridylmethyl) amine,

2-{2- [N, N-bis (2-pyridylmethyl) amino] ethoxy}ethanol, or N- (3-methoxypropyl) -N, N-bis (2-pyridylmethyl) amine. [10] An agent for the prophylaxis or treatment of an allergic disease, comprising a compound represented by the formula (I) : [0023]

[0024] wherein

L is an alkylene group; X is a bond, -O-, -S- or -CO-; Y is a bond, -NR³- or -CO-;

R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N,N, N' ,N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof. [11] The agent of [10], wherein X is -0-, Y is -CO-, and Z is an optionally substituted hydrocarbon group or an optionally substituted heterocyclic group.

[12] The agent of [10], wherein X is -0-, Y is a bond, and Z is an optionally substituted hydrocarbon group. [13] The agent of [10] , wherein the compound represented by the formula (I) is

N,N-bis (2-pyridylmethyl) glycine,

N,N-bis (2-pyridylmethyl) glycylglycine tert-butyl ester,

N,N-bis (2-pyridylmethyl) glycylglycine, N, N-bis (2-pyridylmethyl) glycyl-L-phenylalanine tert-butyl ester,

N,N-bis (2-pyridylmethyl) glycyl-L-phenylalanine,

2- [N,N-bis (2-pyridylmethyl) amino] -1-morpholinoethanone,

2- [N, N-bis (2-pyridylmethyl) amino] -N' -phenethylacetamide,

2- [N, N-bis (2-pyridylmethyl) amino]ethanol, 2-[N,N-bis (2-pyridylmethyl) amino] ethyl 2- [A- isobutylphenyl) propionate,

2- [N, N-bis (2-pyridylmethyl) amino] ethyl 2- (3- trifluoromethylphenylamino) benzoate,

2- [N, N-bis (2-pyridylmethyl) amino] ethyl acetate, 2- [N, N-bis (2-pyridylmethyl) amino] ethyl isobutyrate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl phenylacetate,

2- [N, N-bis (2-pyridylmethyl) amino] ethyl 3-phenylpropionate,

2- [N, N-bis (2-pyridylmethyl) amino] ethyl benzoate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl isonicotinate, or 2- [N,N-bis (2-pyridylmethyl) amino] ethyl furan-2-carboxylate.

[14] The agent of [10] , wherein the compound represented by the formula (I) is

N,N-bis (2-pyridylmethyl) -beta-alanine, N-{2-[2-N,N-bis (2-pyridylmethyl) amino-ethoxymethyl] phenyl} -3- trifluoromethylphenylamine,

N- (2-benzyloxyethyl) -N,N-bis (2-pyridylmethyl) amine,

3- [N,N-bis (2-pyridylmethyl) amino] -N-phenyl-propionamide,

N- (2-methoxyethyl) -N,N-bis (2-pyridylmethyl) amine, 2-{2- [N, N-bis (2-pyridylmethyl) amino] ethoxyjethanol, or

N- (3-methoxypropyl) -N,N-bis (2-pyridylmethyl) amine.

[15] The agent of [10] , wherein the allergic disease is allergic dermatitis .

[16] A cytokine production suppressant comprising a compound represented by the formula (I) :

[0025]

[0026] wherein L is an alkylene group;

X is a bond, -O-, -S- or -CO-;

Y is a bond, -NR³- or -CO-;

Z is an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group or OH; and R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N, N, N' ,N'-tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof. [17] An agent for the prophylaxis or treatment of an inflammatory disease, comprising a compound represented by the formula (I) : [0027]

[0028] wherein

L is an alkylene group;

X is a bond, -O-, -S- or -CO-; Y is a bond, -NR³- or -CO-;

R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N, N, N' ,N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof.

[18] A compound represented by the formula (I) : [0029]

[0030] wherein

L is an alkylene group; X is a bond, -O-, -S- or -CO-; Y is a bond, -NR³- or -CO-; Z is an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group or OH; and

R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N,N, N' ,N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof, for use for suppressing degranulation. [19] A compound represented by the formula (I) : [0031]

[0032] wherein

L is an alkylene group; X is a bond, -0-, -S- or -CO-;

Y is a bond, -NR³- or -CO-;

R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N, N, N' ,N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof, for use for prophylaxis or treatment of an allergic disease.

[20] A compound represented by the formula (I) :

[0033]

[0034] wherein

L is an alkylene group; 5 X is a bond, -0-, -S- or -CO-;

Y is a bond, -NR³- or -CO-;

R¹ and R² are the same or different and each is an optionallyo substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N, N,N' ,N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof, for use for suppressing cytokine production.s [21] A compound represented by the formula (I) :

[0035]

[0036] wherein o L is an alkylene group;

X is a bond, -0-, -S- or -CO-;

Y is a bond, -NR³- or -CO-;

Z is an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group or OH; and 5 R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N,N,N' ,N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof, for use for prophylaxis or treatment of an inflammatory disease.

[22] A method of suppressing degranulation in a mammal, comprising administering, to said mammal, an effective amount of a compound represented by the formula (I) : [0037]

[0038] wherein

L is an alkylene group;

X is a bond, -0-, -S- or -CO-; Y is a bond, -NR³- or -CO-;

R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom; wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N,N, N' ,N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof.

[23] A method of preventing or treating an allergic disease in a mammal, comprising administering, to said mammal, an effective amount of a compound represented by the formula (I) :

[0039]

[0040] wherein

L is an alkylene group; X is a bond, -0-, -S- or -CO-;

Y is a bond, -NR³- or -CO-;

R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted al'kyl group or a hydrogen atom, (excluding N, N,N' , N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof. [24] A method of suppressing cytokine production in a mammal, comprising administering, to said mammal, an effective amount of a compound represented by the formula (I) :

[0041]

[0042] wherein

L is an alkylene group; X is a bond, -0-, -S- or -CO-;

Y is a bond, -NR³- or -CO-; Z is an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group or OH; and R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom; wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N,N,N' ,N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof.

[25] A method of preventing or treating an inflammatory disease in a mammal, comprising administering, to said mammal, an effective amount of a compound represented by the formula (I) : [0043]

[0044] wherein

L is an alkylene group; X is a bond, -O-, -S- or -CO-;

Y is a bond, -NR³- or -CO-;

R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N, N,N' , N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof. [26] A compound represented by the formula (I) :

[0045]

[0046] wherein

L is an alkylene group; X is -0-;

Y is -CO- or a bond;

Z is a Ci-₆ alkyl group, a phenyl group optionally substituted by an amino group [the amino group may be substituted by a phenyl group (the phenyl group may be substituted by a Ci-₆ alkyl group optionally substituted by halogen) ] , a O₇-1₃ aralkyl group optionally substituted by a C^₆ alkyl group or an amino group [the amino group may be substituted by a phenyl group (the phenyl group may be substituted by a Ci_₆ alkyl group optionally substituted by halogen) ] , a pyridyl group or a furyl group; and R¹ and R² are hydrogen atoms, or a salt thereof.

Effect of the Invention [0047] Since compound (I) has an activity to suppress degranulation in mast cells and the like, as well as cytokine production, the compound is highly useful as an agent for the prophylaxis or treatment of allergic diseases such as allergic dermatitis and the like and inflammatory diseases .

Brief Description of the Drawings [0048]

FIG. 1 shows representative NMR spectrum of compound 13. FIG. 2 shows representative NMR spectrum of compound 14. FIG. 3 shows representative NMR spectrum of compound 15. FIG. 4 shows representative NMR spectrum of compound 16.

FIG. 5 shows representative NMR spectrum of compound 17.

FIG. 6 shows representative NMR spectrum of compound 18.

FIG. 7 shows representative NMR spectrum of compound 19. FIG. 8 shows representative NMR spectrum of compound 20.

FIG. 9 shows representative NMR spectrum of compound 21.

FIG. 10 shows representative NMR spectrum of compound 22.

FIG. 11 shows representative NMR spectrum of compound 22.

FIG. 12 shows representative MS spectrum of compound 22. FIG. 13 shows representative NMR spectrum of compound 23.

FIG. 14 shows representative NMR spectrum of compound 23.

FIG. 15 shows representative NMR spectrum of compound 24.

FIG. 16 shows representative NMR spectrum of compound 24.

FIG. 17 shows degranulation inhibitory activity of TPEN derivatives (compounds 1 - 6) .

FIG. 18 shows- degranulation inhibitory activity of TPEN derivatives (compounds 7 - 10) .

FIG. 19 shows comparison (release rate) of degranulation inhibitory activity of TPEN, compound 9 and compound 10. FIG. 20 shows comparison (degranulation rate) of degranulation inhibitory activity of TPEN, compound 9 and compound 10.

FIG. 21 shows degranulation inhibitory activity (using aqueous sodium hydrogen carbonate solution as a solvent) of ibuprofen and flufenamic acid.

FIG. 22 shows degranulation inhibitory activity (using DMSO as a solvent) of ibuprofen and flufenamic acid.

FIG. 23 shows calculation of IC5₀ of ibuprofen and flufenamic acid. FIG. 24 shows degranulation inhibitory activity of compounds 14 - 17.

FIG. 25 shows the results of zinc ion rescue test.

FIG. 26 shows degranulation inhibitory activity of the Example compound on human mast cell . FIG. 27 shows degranulation inhibitory activity of the Example compound on human mast cell .

FIG. 28 shows degranulation inhibitory activity of the test substance on human mast cell. FIG. 29 shows degranulation inhibitory activity of the test substance on human mast cell.

FIG. 30 shows the effect of compound 10 on the number of scratches.

FIG. 31 shows the effect of compound 9 on the number of scratches.

FIG. 32 shows time course changes in the thickness of ear coated with a test substance.

FIG. 33 shows the effect of Compound 10 on histamine release from human basophil. FIG. 34 shows the experiment schedule of atopic dermatitis model.

FIG. 35 shows the effect of Compound 10, TPEN and tacrolimus hydrate on the body weight of atopic dermatitis model mice. FIG. 36 shows the effect of Compound 10, TPEN and tacrolimus hydrate on ear thickening.

FIG. 37 shows the effect of Compound 10 and tacrolimus hydrate on the auriclar skin symptom scores.

FIG. 38 shows the effect of Compound 10 and tacrolimus hydrate on various auriclar skin symptoms.

FIG. 39 shows the effect of Compound 10 on GOT, GPT, ALP and BUN.

FIG. 40 shows the effect of Compound 10 on eotaxin production by BEAS-2B. FIG. 41 shows the effect of Compound 10 on the airway responsiveness to acetylcholine (left panel) and the numbers of neutrophils and eosinophils in BALF (right panel) .

FIG. 42 shows the effect of Compound 10 (oral administration) on PCA reaction. FIG. 43 shows the effect of Compound 10 (oral administration) on the enteric length of IBD model mice.

FIG. 44 shows the effect of TPEN and Compound 10 on the growth of conjunctiva-derived cell line. FIG. 45 shows the effect of Compound 22 on PCA reaction.

Best Mode for Carrying out the Invention [0049]

The present_, invention provides a compound represented by the formula (I) : [0050]

[0051] wherein L is an alkylene group;

X is a bond, -O-, -S- or -CO-;

Y is a bond, -NR³- or -CO-;

Z is an organic group or OH; and

R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted alkyl group or a hydrogen atom (hereinafter to be referred to as compound (I)), or a salt thereof.

[0052] In the formula (I) , the alkylene group for L may be linear or branched. The alkylene group is preferably a Ci-io alkylene group, more preferably a Ci_₆ alkylene group, further preferably a Ci-₃ alkylene group.

Preferable examples of the alkylene group include -CH₂-, - (CHs)₂-, -(CH₂)S-, -(CHs)₄-, -(CH₂)S-, -(CH₂J₆-, - (CH₂) ₇-, - (CH₂) ₈", - (CH₂ ) ₉- , - (CHs) ₁₀-, -CH (CH₃) -, -C (CH₃ ) 2", - (CH (CH₃) ) ₂- , " (CHa) ₂C (CH₃) Z-, - (CH₂ ) 3C (CH₃ ) 2- and the like .

L is preferably -CH₂-, -(CH₂J₂-, -(CH₂J₃-, -CH(CH₃)- or - C (CH₃) ₂-, more preferably -CH₂- or - (CH₂) ₂-. [0053]

In the formula (I), X is preferably -O- or -CO-, more preferably -0-. [0054]

In the formula (I) , Y is preferably -NR³- or -CO-, more preferably -CO-. [0055]

In another aspect, Y in the formula (I) is preferably a bond. [0056] In the optionally substituted alkyl group for R³, the alkyl group may be linear or branched. The alkyl group is preferably a Ci-₁₀ alkyl group, more preferably a C₁-₆ alkyl group, more preferably a C₁-₃ alkyl group.

Preferable examples of the alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1, 1-dimethylbutyl, 2, 2-dimethylbutyl, 3, 3-dimethylbutyl, 2- ethylbutyl and the like.

The alkyl group may have 1 to 3 substituents . Examples of such substituent include a halogen atom, Cχ_io alkoxy optionally substituted by 1 to 3 halogen atoms, hydroxy, nitro, amino and the like.

As the Ci-₁₀ alkoxy group, those exemplified in the below- mentioned "optionally substituted hydroxy group" can be mentioned.

Unless otherwise specified, the "halogen atom" in the present specification means a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like.

R³ is preferably a Ci_₆ alkyl group or a hydrogen atom, more preferably a hydrogen atom. [0057]

While the combination of X and Y is not particularly limited, preferable examples are as follows : 1) X is -0- and Y is -CO-;

2) X is -CO- and Y is -NR³- (preferably -NH-) . [0058]

In another aspect, a preferable example of the combination of X and Y is as follow: 3) X is -0- and Y is a bond. [0059]

In the optionally substituted alkyl group for R¹ or R², the alkyl group may be linear or branched. The alkyl group is preferably a Ci-io alkyl group, more preferably a Cχ-₆ alkyl group, still more preferably a C₁-₃ alkyl group.

Preferable examples of the alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2, 2-dimethylbutyl, 3, 3-dimethylbutyl, 2- ethylbutyl and the like.

The alkyl group may have 1 to 3 substituents . Examples of such substituent include a halogen atom, Ci-io alkoxy optionally substituted by 1 to 3 halogen atoms, hydroxy, nitro, amino and the like. As the Ci-io alkoxy group, those exemplified in the below- mentioned ^""optionally substituted hydroxy group" can be mentioned.

R¹ and R² are preferably the same or different and each is a Ci-₆ alkyl group, a halogen atom or a hydrogen atom, more preferably a halogen atom or a hydrogen atom. Particularly preferably, both are hydrogen atoms.

R¹ and R² are substituted at any substitutable position on the pyridine ring. [0060] In the formula (I), examples of the organic group for Z include an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group and the like. [0061] Examples of the "hydrocarbon group" of the aforementioned "optionally substituted hydrocarbon group" include an aliphatic hydrocarbon group, an alicyclic hydrocarbon group, an aromatic hydrocarbon group, an aromatic aliphatic hydrocarbon group, an alicyclic aliphatic hydrocarbon group and the like. [0062]

Examples of the aliphatic hydrocarbon group include a linear or branched Ci_₁₅ aliphatic hydrocarbon group, specifically, an alkyl group, an alkenyl group, an alkynyl group and the like.

The alkyl group is preferably a Ci_io alkyl group, more preferably a Ci-β alkyl group, still more preferably a C₁-₃ alkyl group .

Preferable examples of the alkenyl group include C2-1₀ alkenyl groups such as ethenyl, 1-propenyl, 2-propenyl, 2- methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 3-methyl-2- butenyl, 1-pentenyl, 2-pentenyl-, 3-pentenyl, 4-pentenyl, 4- methyl-3-pentenyl, 1-hexenyl, 3-hexenyl, 5-hexenyl, 1-heptenyl, 1-octenyl and the like.

Preferable examples of the alkynyl group include C₂-1₀ alkynyl groups such as ethynyl, 1-propynyl, 2-propynyl, 1- butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3- pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl,

5-hexynyl, 1-heptynyl, 1-octynyl and the like.

[0063]

Examples of the alicyclic hydrocarbon group include a saturated or unsaturated C₃-_I2 alicyclic hydrocarbon group, specifically, a cycloalkyl group, a cycloalkenyl group, a cycloalkadienyl group and the like.

Preferable examples of the cycloalkyl group include C₃-10 cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo [2.2. l]heptyl, bicyclo[2.2.2]octyl, bicyclo [3.2.1] octyl, bicyclo [3.2.2] nonyl, bicyclo[3.3.1]nonyl, bicyclo [4.2.1] nonyl, bicyclo [4.3.1] decyl and the like. Preferable examples of the cycloalkenyl group include C₃_io cycloalkenyl groups such as 2-cyclopenten-l-yl, 3-cyclopenten-l- yl, 2-cyclohexen-l-yl, 3-cyclohexen-l-yl and the like.

Preferable examples of the cycloalkadienyl group include C₄-₁₀ cycloalkadienyl groups such as 2, 4-cyclopentadien-l-yl, 2,4- cyclohexadien-1-yl, 2, 5-cyclohexadien-l-yl and the like. [0064]

Examples of the aromatic hydrocarbon group include a C6-14 aryl group .

Preferable examples of the aryl group include phenyl, naphthyl, anthryl, phenanthryl, acenaphthylenyl, biphenylyl, indenyl and the like. Of these, phenyl, naphthyl and the like are preferable.

The aryl group may be partially saturated, and examples of the partially saturated aryl group include dihydroindenyl and the like. [0065]

Examples of the aromatic aliphatic, hydrocarbon group include a C_7~i₃ aromatic aliphatic hydrocarbon group, specifically, an aralkyl group, an arylalkenyl group and the like. The aralkyl group is preferably a C₇-i₃ aralkyl group, more preferably a C₇-_U aralkyl group, more preferably a C₇-₉ aralkyl group.

As the aryl contained in the aralkyl group, .those exemplified as the aforementioned "aromatic hydrocarbon group" can be mentioned. The aryl is preferably phenyl.

As the alkylene contained in the aralkyl group, those exemplified as the alkylene group for L above can be mentioned.

Preferable examples of the aralkyl group include benzyl, phenethyl, phenylpropyl, 1-phenylethyl, l-methyl-2-phenylethyl, naphthylmethyl, benzhydryl and the like.

Preferable examples of the arylalkenyl group include Cs-_I3 arylalkenyl groups such as styryl and the like. [0066] Examples of the alicyclic aliphatic hydrocarbon group include a C₄_i₃ alicyclic aliphatic hydrocarbon group, specifically, a cycloalkylalkyl group, a cycloalkylalkenyl group and the like.

Preferable examples of the cycloalkylalkyl group include C₄-₁₃ cycloalkylalkyl groups such as cyclopropylmethyl, cyclopropylethyl, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl and the like.

Preferable examples of the cycloalkylalkenyl group include C₅-_I3 cycloalkylalkenyl groups such as cyclopropylethenyl, cyclopentylethenyl, cyclohexylethenyl and the like. [0067]

The "hydrocarbon group" is preferably an aliphatic hydrocarbon group, an aromatic hydrocarbon group or an aromatic aliphatic hydrocarbon group, more preferably an alkyl group (e.g., Ci-₁₀ alkyl group), an aryl group (e.g., C₆-₁₄ aryl group), an aralkyl group (e.g., C₇-_I3 aralkyl group) and the like. [0068]

The above-mentioned "hydrocarbon group" may have 1 to 3 substituents at substitutable position (s) . Examples of such sub^'stituent include a halogen atom, a nitro group, an optionally substituted amino group, an optionally substituted hydroxy group, an optionally substituted acyl group, an optionally substituted alkyl group and the like. [0069] Examples of the "optionally substituted amino group" include an amino group optionally mono- or di-substituted by Ci-I₀ alkyl group, C₂-₁₀ alkenyl group, C₃_i₀ cycloalkyl group, C3-10 cycloalkenyl group, C₆-_H aryl group, C₇-_I3 aralkyl group, C1-13 acyl group or a heteroaryl group, each of which optionally has substituent (s) . [0070]

Here, examples of the C1-10 alkyl group, C2-10 alkenyl group, C3-₁₀ cycloalkyl group, C₃-₁0 cycloalkenyl group, Ce-u aryl group and C₇-_I3 aralkyl group include those exemplified as the

"hydrocarbon group" in the "optionally substituted hydrocarbon group" for Z.

Here, preferable examples of the C₁-₁₀ alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1, 1-dimethylbutyl, 2,2-dimethylbutyl, 3,3- dimethylbutyl, 2-ethylbutyl and the like.

Preferable examples of the C₂-io alkenyl group include ethenyl, 1-propenyl, 2-propenyl, 2-methyl-l-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 3- hexenyl, 5-hexenyl, 1-heptenyl, 1-octenyl and the like.

Preferable examples of the C₃_io cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo [2.2. l]heptyl, bicyclo [2.2.2] octyl, bicyclo[3.2.1]octyl, bicyclo [3.2.2] nonyl, bicyclo [3.3. l]nonyl, bicyclo [4.2.1]nonyl, bicyclo [4.3.1] decyl and the like.

Preferable examples of the C₃-₁0 cycloalkenyl group include 2-cyclopenten-l-yl, 3-cyclopenten-l-yl, 2-cyclohexen-l-yl, 3- cyclohexen-1-yl and the like.

Preferable examples of the C₆-H aryl group include phenyl, naphthyl, anthryl, phenanthryl, acenaphthylenyl, biphenylyl, indenyl and the like.

Preferable examples of the C₇-I₃ aralkyl group include benzyl, phenethyl, phenylpropyl, 1-phenylethyl, l-methyl-2- phenylethyl, naphthylmethyl, benzhydryl and the like. [0071]

Examples of the aforementioned Ci-₁₃ acyl group include those exemplified as the acyl group of the below-mentioned "optionally substituted acyl group".

The acyl group is preferably formyl, Ci-₁₀ alkyl-carbonyl, Ci-₆ alkoxy-carbonyl, C₆-₁₄ aryl-carbonyl, C₇_i₃ aralkyl-carbonyl and the like. [0072]

Examples of the aforementioned heteroaryl group include the aromatic heterocyclic group exemplified as the heterocycle of the below-mentioned "optionally substituted heterocycle" for Z. Of these, pyridyl, imidazolyl, triazolyl, pyrimidinyl and the like are preferable. [0073]

These Ci-₁₀ alkyl group, C₂-₁₀ alkenyl group, C₃-₁0 cycloalkyl group, C₃-₁0 cycloalkenyl group, Cβ-₁₄, aryl group, C₇_i3 aralkyl group, Ci-₁₃ acyl group and heteroaryl group may have 1 to 6, preferably 1 to 2 substituents at substitutable position (s). Examples of such substituents include a halogen atom, a Ci-₁0 alkyl group (preferably Ci_₆ alkyl group) optionally substituted by 1 to 3 halogen atoms (e.g., fluorine atom), a Ci-₁₀ alkoxy group optionally substituted by 1 to 3 halogen aroms, hydroxy, nitro, amino, a Ci_₆ alkylsulfonyl group and the like.

Here, examples of the Ci-₁₀ alkyl group include those exemplified as the "hydrocarbon group" of the "optionally substituted hydrocarbon group" for Z. Preferable examples of the Ci-₁₀ alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1, 1-dimethylbutyl, 2,2- dimethylbutyl, 3, 3-dimethylbutyl, 2-ethylbutyl and the like.

Examples of the Ci-₁₀ alkoxy group include those exemplified in the below-mentioned "optionally substituted hydroxy group". Preferable examples of the alkoxy group include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, heptyloxy, nonyloxy and the like. [0074]

The "optionally substituted amino group" is preferably an amino group optionally mono- or di-substituted by a Cβ-₁4 aryl group (e.g., phenyl) optionally having substituent (s) , more preferably an amino group optionally mono-substituted by a C₆-_I4 aryl group optionally having substituent (s) .

The Cβ-14 aryl group is preferably a Cs-u aryl group optionally having 1 to 6 Ci-io alkyl groups (the alkyl group may be substituted by 1 to 3 halogen atoms (e.g., fluorine atom)) at substitutable position (s), more preferably phenyl optionally having 1 or 2 Ci_io alkyl groups (the alkyl group may be substituted by 1 to 3 halogen atoms) at substitutable position (s) . [0075]

As for the "optionally substituted hydroxy group", examples of the "substituted hydroxy group" include an alkoxy group, an alkenyloxy group, a cycloalkyloxy group, a cycloalkenyloxy group, an aryloxy group, an aralkyloxy group, an acyloxy group, a heteroaryloxy group and the like, each of which is optionally substituted. [0076]

The alkoxy group is preferably a Ci_₁₀ alkoxy group, more preferably a Cχ-ζ alkoxy group, still more preferably a Ci-₄ alkoxy group.

Preferable examples of the alkoxy group include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, heptyloxy, nonyloxy and the like.

Preferable examples of the alkenyloxy group include C2-10 alkenyloxy groups such as allyl (allyl) oxy, crotyloxy, 2- pentenyloxy, 3-hexenyloxy and the like.

Preferable examples of the cycloalkyloxy group include C3-10 cycloalkyloxy groups such as cyclobutoxy, cyclopentyloxy, cyclohexyloxy and the like. Preferable examples of the cycloalkenyloxy group include C₃-₁₀ cycloalkenyloxy groups such as 2-cyclopentenyloxy, 2- cyclohexenyloxy and the like.

Preferable examples of the aryloxy group include C_δ-u aryloxy groups such as phenoxy, naphthyloxy and the like. Preferable examples of the aralkyloxy group include C₇_i₃ aralkyloxy groups such as benzyloxy, phenethyloxy, naphthylmethyloxy and the like.

Preferable examples of the acyloxy group include C₂-1₃ acyloxy groups such as Ci-₆ alkyl-carbonyloxy groups (e.g., acetyloxy, propionyloxy, butyryloxy, isobutyryloxy) and the like,

Preferable examples of the heteroaryloxy group include 5- to 7-membered monocyclic heteroaryloxy groups such as 2- pyridyloxy, 3-pyridyloxy, 2-imidazolyloxy, 2-pyrimidinyloxy, 1,2, 4-triazol-5-yloxy and the like. [0077]

The above-mentioned alkoxy group, alkenyloxy group, cycloalkyloxy group, cycloalkenyloxy group, aryloxy group, aralkyloxy group, acyloxy group and heteroaryloxy group may have 1 to 3 substituents, preferably 1 or 2 substituents, at substitutable position (s). Examples of such substituents include a halogen atom, a Ci-₁₀ alkyl group optionally substituted by 1 to 3 halogen atoms, a Ci-₁₀ alkoxy group optionally substituted by 1 to 3 halogen atoms, hydroxy, nitro, amino and the like.

Here, examples of the Ci-1₀ alkyl group include those exemplified as the "hydrocarbon group" of the "optionally substituted hydrocarbon group" for Z. Preferable examples of the Ci-₁₀ alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1, 1-dimethylbutyl, 2,2- dimethylbutyl, 3, 3-dimethylbutyl, 2-ethylbutyl and the like.

Examples of the Ci-io alkoxy group include those exemplified in the aforementioned "optionally substituted hydroxy group". [0078] Examples of the acyl group of the "optionally substituted acyl group" include groups represented by the formulas: -COR⁴, - CO-OR⁴, -SO₂R⁴, -SOR⁴ wherein R⁴ is a hydrogen atom or a hydrocarbon group, and the like.

Examples of the "hydrocarbon group" for R⁴ include those exemplified as the "hydrocarbon group" of the "optionally substituted hydrocarbon group" for Z .

The hydrocarbon group is preferably an alkyl group (e.g., Ci-io alkyl group) , a cycloalkyl group (C₃-.₁₀ cycloalkyl group) , an aryl group (Cβ-w aryl group) , or an aralkyl group (C₇_i₃ aralkyl group) .

Here, examples of the alkyl group, cycloalkyl group, aryl group and aralkyl group include those exemplified as the "hydrocarbon group" of the "optionally substituted hydrocarbon group" for Z . Preferable examples of the alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2, 2-dimethylbutyl, 3, 3-dimethylbutyl, 2- ethylbutyl and the like. Preferable examples of the cycloalkyl group include C3-10 cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo [2.2. l]heptyl, bicyclo[2.2.2]octyl, bicyclo [3.2.1] octyl, bicyclo [3.2.2] nonyl, bicyclo[3.3.1]nonyl, bicyclo [4.2.1] nonyl, bicyclo [4.3.1] decyl and the like.

Preferable examples of the Cβ-u aryl group include phenyl, naphthyl, anthryl, phenanthryl, acenaphthylenyl, biphenylyl, indenyl and the like.

Preferable examples of the C₇-I₃ aralkyl group include benzyl, phenethyl, phenylpropyl, 1-phenylethyl, l-methyl-2- phenylethyl, naphthylmethyl, benzhydryl and the like. [0079]

Preferable examples of the acyl group include formyl, carboxy, Ci_i₀ alkyl-carbonyl (e.g., acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, heptanoyl, octanoyl) , C₂-₁₀ alkenyl-carbonyl (e.g., crotonoyl) , C3-10 cycloalkyl-carbonyl (e.g., cyclobutanecarbonyl, cyclopentanecarbonyl, cyclohexanecarbonyl, cycloheptanecarbonyl) , C₃-₁₀ cycloalkenyl-carbonyl (e.g., 2-cyclohexenecarbonyl) , C₆-i4 aryl-carbonyl (e.g., benzoyl, 1-naphthoyl, 2-naphthoyl) , C₇_i3 aralkyl-carbonyl (e.g., benzylcarbonyl, phenethylcarbonyl) , aromatic heterocyclylcarbonyl (e.g., nicotinoyl, isonicotinoyl) , non-aromatic heterocyclylcarbonyl (e.g., pyrrolidinylcarbonyl, piperidinocarbonyl) , Ci-₁₀ alkoxy-carbonyl (e.g., methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, tert- bύtoxycarbonyl) , Cβ-u aryloxy-carbonyl (e.g., phenyloxycarbonyl, naphthyloxycarbonyl) , C₇_i₃ aralkyloxy-carbonyl (e.g., benzyloxycarbonyl, phenethyloxycarbonyl) , a Ci-₁₀ alkylsulfinyl group (e.g., methylsulfinyl, ethylsulfinyl) , a Ci-₁₀ alkylsulfonyl group (e.g., methylsulfonyl, ethylsulfonyl) , C₆-₁₄ arylsulfonyl (e.g., phenylsulfonyl) and the like. [0080]

The acyl group may have 1 to 3 substituents at substitutable position (s). Examples of such substituents include a Ci-₁₀ alkyl group optionally substituted by 1 to 3 halogen atoms, a Ci-₁₀ alkoxy group optionally substituted by 1 to 3 halogen atoms, a halogen atom, nitro, hydroxy, amino and the like. Here, examples of the Ci-₁₀ alkyl group include those exemplified as the "hydrocarbon group" of the "optionally substituted hydrocarbon group" for Z. Examples of the Ci-10 alkoxy group include those exemplified in the aforementioned "optionally substituted hydroxy group". [0081] The "optionally substituted acyl group" is preferably formyl, carboxy or optionally substituted alkoxy-carbonyl, more preferably carboxy or Ci-io alkoxy-carbonyl. [0082] Examples of the alkyl group of the "optionally substituted alkyl group" which the aforementioned "hydrocarbon group" may have as a substituent include those exemplified as the"hydrocarbon group" of the "optionally substituted hydrocarbon group" for Z . The alkyl group is preferably a Ci_io alkyl group, more preferably a Ci_₆ alkyl group.

The alkyl group may have 1 to 6 substituents, preferably 1 to 3 substituents, at substitutable position (s). Examples of such substituents include a halogen atom, hydroxy, nitro, amino and the like.

The "optionally substituted alkyl group" is preferably an optionally substituted Ci_io alkyl group, more preferably a Ci_₆ alkyl group (e.g., isobutyl). [0083] Examples of the heterocyclic group of the "optionally substituted heterocyclic group" for Z include a heterocyclic group containing, as a ring-constituting atom besides carbon atom, at least one (e.g., 1 to 4) hetero atom selected from an oxygen atom, a sulfur atom and a nitrogen atom. The heterocyclic group includes an aromatic heterocyclic group and a nonaromatic heterocyclic group. [0084]

Examples of the "aromatic heterocyclic group" include a 5- to 7-membered monocyclic aromatic heterocyclic group or condensed aromatic heterocyclic group containing, as ring- constituting atom besides carbon atom, 1 to 4 hetero atoms selected from an oxygen atom, a sulfur atom and a nitrogen atom. Examples of the condensed aromatic heterocyclic group include a group wherein such 5- to 7-membered monocyclic aromatic heterocyclic group is condensed with a β-membered ring containing 1 or 2 nitrogen atoms, a benzene ring, or a 5- membered ring containing one sulfur atom and the like. [0085] Preferable examples of the "aromatic heterocyclic group" include furyl (e.g., 2-furyl, 3-furyl) , thienyl (e.g., 2-thienyl, 3-thienyl) , pyridyl (e.g., 2-pyridyl, 3-pyridyl, 4-pyridyl) , pyrimidinyl (e.g., 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl) , pyridazinyl (e.g., 3-pyridazinyl, 4-pyridazinyl) , pyrazinyl (e.g., 2-pyrazinyl) , pyrrolyl (e.g., 1-pyrrolyl, 2- pyrrolyl, 3-pyrrolyl) , imidazolyl (e.g., 1-imidazolyl, 2- imidazolyl, 4-imidazolyl, 5-imidazolyl) , pyrazolyl (e.g., 1- pyrazolyl, 3-pyrazolyl, 4-pyrazolyl) , oxazolyl (e.g., 2-oxazolyl, 4-oxazolyl, 5-oxazolyl) , isoxazolyl, thiazolyl (e.g., 2- thiazolyl, 4-thiazolyl, 5-thiazolyl) , isothiazolyl, oxadiazolyl (e.g., 1, 2, 4-oxadiazol-5-yl, 1, 3, 4-oxadiazol-2-yl) , thiadiazolyl (e.g., l,3,4-thiadiazol-2-yl) , triazolyl (e.g., 1, 2, 4-triazol-l- yl, l,2,4-triazol-3-yl, 1, 2, 4-triazol-5-yl, 1, 2, 3-triazol-l-yl, l,2,3-triazol-2-yl, 1,2, 3-triazol-4-yl) , tetrazolyl (e.g., tetrazol-1-yl, tetrazol-5-yl) , quinolyl (e.g., 2-quinolyl, 3- quinolyl, 4-quinolyl), quinazolyl (e.g., 2-quinazolyl, 4- quinazolyl) , quinoxalyl (e.g., 2-quinoxalyl) , benzofuryl (e.g., 2-benzofuryl, 3-benzofuryl) , benzothienyl (e.g., 2-benzothienyl, 3-benzothienyl) , benzoxazolyl (e.g., 2-benzoxazolyl) , benzothiazolyl (e.g., 2-benzothiazolyl) , benzimidazolyl (e.g., benzimidazol-1-yl, benzimidazol-2-yl) , indolyl (e.g., indol-1-yl, indol-3-yl) , lH-indazolyl (e.g., lH-indazol-3-yl) , IH- pyrrolo [2, 3-b] pyrazinyl (e.g., lH-pyrrolo [2, 3-b]pyrazin-2-yl) , lH-pyrrolopyridinyl (e.g., lH-pyrrolo [2, 3-b]pyridin-β-yl) , IH- imidazopyridinyl (e.g., IH-imidazo [4, 5-b]pyridin-2-yl, IH- imidazo [4, 5-c]pyridin-2-yl) , lH-imidazopyrazinyl (e.g., IH- imidazo [4, 5-b]pyrazin-2-yl) , triazinyl, isoquinolyl, benzooxadiazolyl, benzothiadiazolyl, benztriazolyl and the like. [0086]

Examples of the "nonaromatic heterocyclic group" include a 5- to 7-membered monocyclic nonaromatic heterocyclic group or condensed nonaromatic heterocyclic group containing, as ring- constituting atom besides carbon atom, 1 to 4 hetero aroms selected from an oxygen atom, a sulfur atom and a nitrogen atom. Examples of the condensed nonaromatic heterocyclic group include a group wherein such 5- to 7-membered monocyclic nonaromatic heterocyclic group is condensed with a β-membered ring containing 1 or 2 nitrogen atoms, a benzene ring, or a 5- membered ring containing one sulfur atom and the like. [0087]

Preferable examples of the "nonaromatic heterocyclic group" include morpholinyl (e.g., morpholino) , pyrrolidinyl (e.g., 1-pyrrolidinyl) , piperidinyl (e.g., piperidino) , thiomorpholinyl (e.g., thiomorpholino) , piperazinyl (e.g., 1- piperazinyl) , hexamethyleneiminyl (e.g., hexamethyleneimin-1-yl) , oxazolidinyl (e.g., oxazolidin-3-yl) , thiazolidinyl (e.g., thiazolidin-3-yl) , imidazolidinyl (e.g., imidazolidin-3-yl) , imidazolinyl (e.g., imidazolin-1-yl, imidazolin-2-yl) , oxazolinyl (e.g., oxazolin-2-yl) , thiazolinyl (e.g., thiazolin- 2-yl) , oxazinyl (e.g., oxazin-2-yl) , tetrahydrofuranyl, azepanyl, tetrahydropyridinyl (e.g., 1,2,3, 6-tetrahydropyridin-l-yl) , dihydrobenzofuranyl, dioxolanyl, dithiolanyl, dioxothiazolidinyl, dioxooxazolidinyl and the like. [0088]

The nonaromatic heterocyclic group is preferably a nitrogen-containing heterocyclic group. Examples of the "nitrogen-containing heterocycle" include a 5-, 6- or 7-membered nitrogen-containing heterocycle containing at least one nitrogen atom as a ring-constituting atom besides carbon atom, and further optionally having 1 or 2 hetero aroms selected from an oxygen atom, a sulfur atom and a nitrogen atom. The nitrogen- containing heterocycle is more preferably a β-membered nitrogen- containing heterocycle containing one nitrogen atom besides carbon atom, and further, one oxygen atom (e.g., morpholine, [1, 3] oxazinan, [1,2] oxazinan) . [0089]

The above-mentioned heterocyclic group may have 1 to 3 substituents at substitutable position (s). Examples of such substituent include nitro, hydroxy, amino, oxo, a halogen atom, a Ci-io alkyl group optionally substituted by 1 to 3 halogen atoms, a C₁-_IO alkoxy group optionally substituted by 1 to 3 halogen atoms, a Cδ-i4 aryl group and the like. Here, examples of the Ci_io alkyl group and C_δ-i₄ aryl group include those exemplified as the "hydrocarbon group" of the "optionally substituted hydrocarbon group" for Z. Examples of the Ci-io alkoxy group include those exemplified in the aforementioned "optionally substituted hydroxy group". [0090]

The "optionally substituted heterocyclic group" is preferably an optionally substituted 5- or β-membered heterocyclic group containing nitrogen atom and/or one oxygen atom (e.g., furyl, pyridyl, morpholino) . [0091]

In the formula (I) , the organic group for Z may be a residue of a compound which is a non-steroidal anti-inflammatory agent (NSAID) . In other words, Z-COOH, Z-NH₂, Z-OH, Z-H and the like may be NSAIDs. Examples of NSAID include flufenamic acid, ibuprofen, acetylsalicylic acid and the like. Using, as Z, a residue of known NSAID having confirmed safety, the risk of side effects caused by the compound of the present invention can be minimized. [0092] As a first preferable embodiment, the invention provides a compound of the formula (I) , wherein Z is an optionally- substituted Ci-₆ alkyl group; an optionally substituted Ce-i4 aryl group; an optionally substituted C₇-i₃ aralkyl group; or an optionally substituted heterocyclic group containing at least one hetero atom selected from an oxygen atom and a nitrogen atom, or a salt thereof.

[0093]

In the first embodiment, preferably, L is a Ci-₃ alkylene group; X is -O-; Y is -CO-;

Z is a Ci_₆ alkyl group, a phenyl group optionally substituted by an amino group [the amino group may be substituted by a phenyl group (the phenyl group may be substituted by a Ci_₆ alkyl group optionally substituted by halogen) ] , a C₇-_I3 aralkyl group optionally substituted by a Ci_₆ alkyl group, a pyridyl group or a furyl group; and R¹ and R² are hydrogen atoms . [0094]

In another aspect of the first embodiment, preferably, L is an alkylene group (preferably Cχ-₆ alkylene group, more preferably C₁-₃ alkylene group) ; X is -O-; Y is -CO- or a bond;

Z is a Ci-₆ alkyl group, a phenyl group optionally substituted by an amino group [the amino group may be substituted by a phenyl group (the phenyl group may be substituted by a Ci_₆ alkyl group optionally substituted by halogen) ] , a C₇_₁₃ aralkyl group optionally substituted by a Ci_₆ alkyl group or an amino group [the amino group may be substituted by a phenyl group (the phenyl group may be substituted by a Ci_₆ alkyl group optionally substituted by halogen) ] , a pyridyl group or a furyl group; and R¹ and R² are hydrogen atoms. [ 0095]

As a second preferable embodiment, the invention provides a compound of the formula (I) , wherein Z is a Ci_i₀ alkyl group optionally substituted by a carboxy group or a Ci-io alkoxy- carbonyl group, or a C₇-i₃ aralkyl group optionally substituted by a carboxy group or a Ci-io alkoxy-carbonyl group, or a salt thereof.

In this embodiment, preferably, L is a C1-₃ alkylene group, X is -CO-, Y is -NH-, and R¹ and R² are hydrogen atoms. [0096]

As a third preferable embodiment, the invention provides a compound of the formula (I) , wherein Z is an optionally substituted β-membered nitrogen-containing nonaromatic heterocyclic group, or a salt thereof. In this embodiment, preferably, X is a bond, Y is -CO-, and R¹ and R² are hydrogen atoms. [0097]

In the fourth embodiment, preferably, L is a C₁-.₃ alkylene group; X is -0-; Y is a bond;

Z is an optionally substituted C_±s alkyl group, an optionally substituted Ce-io aryl group or an optionally substituted C₇-i₃ aralkyl group; and R¹ and R² are hydrogen atoms . [0098]

In the fourth embodiment, more preferably, Z is an optionally substituted Ci_₆ alkyl group or a C₇-₁₃ aralkyl group optionally substituted by an amino group [the amino group may be substituted by a phenyl group (the phenyl group may be substituted by a Ci_₆ alkyl group optionally substituted by halogen) ] .

[0099]

Preferable examples of the compound (I) include the following compounds :

N, N-bis (2-pyridylmethyl) glycine,

N,N-bis (2-pyridylmethyl) glycylglycine tert-butyl ester,

N, N-bis (2-pyridylmethyl) glycylglycine, N, N-bis (2-pyridylmethyl) glycyl-L-phenylalanine tert-butyl ester,

N,N-bis (2-pyridylmethyl) glycyl-L-phenylalanine,

2- [N, N-bis (2-pyridylmethyl) amino] -1-morpholinoethanone,

2- [N,N-bis (2-pyridylmethyl) amino] -N' -phenethylacetamide,

2- [N,N-bis (2-pyridylmethyl) amino] ethanol, 2- [N,N-bis (2-pyridylmethyl) amino] ethyl 2- (4- isobutylphenyl) propionate,

2- [N, N-bis (2-pyridylmethyl) amino] ethyl 2- (3- trifluoromethylphenylamino)benzoate,

2- [N, N-bis (2-pyridylmethyl) amino] ethyl phenylacetate,

2- [N, N-bis (2-pyridylmethyl) amino] ethyl 3-phenylpropionate,

2- [N, N-bis (2-pyridylmethyl) amino] ethyl benzoate,

2- [N, N-bis (2-pyridylmethyl) amino] ethyl isonicotinate, and 2- [N, N-bis (2-pyridylmethyl) amino] ethyl furan-2-carboxylate.

[0100]

In another aspect, preferable examples of the compound (I) include the following compounds :

N, N-bis (2-pyridylmethyl) -beta-alanine, N- {2- [2-N,N-bis (2-pyridylmethyl) amino-ethoxymethyl] phenyl} -3- trifluoromethylphenylamine, ^{■ •}

N- (2-benzyloxyethyl) -N,N-bis (2-pyridylmethyl) amine,

3- [N, N-bis (2-pyridylmethyl) amino] -N-phenyl-propionamide,

N- (2-methoxyethyl) -N, N-bis (2-pyridylmethyl) amine, 2-{2- [N, N-bis (2-pyridylmethyl) amino] ethoxy} ethanol, and

N- (3-methoxypropyl) -N, N-bis (2-pyridylmethyl) amine.

[0101]

Of these, the following compounds are superior in that they have a strong degranulation suppressive effect and a cytokine production inhibitory effect, and are low toxic:

2- [N, N-bis (2-pyridylmethyl) amino] ethyl 2- (4- isobutylphenyl) propionate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl isobutyrate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl phenylacetate,

2- [N, N-bis (2-pyridylmethyl) amino] ethyl 3-phenylpropionate, and

2- [N,N-bis (2-pyridylmethyl) amino] ethyl benzoate. '[0102]

In another aspect, the following compounds are superior in that they have a strong degranulation suppressive effect and a cytokine production inhibitory effect, and are low toxic:

N-{2- [2-N, N-bis (2-pyridylmethyl) amino-ethoxymethyl] phenyl} -3- trifluoromethylphenylamine, and

N- (2-benzyloxyethyl) -N, N-bis (2-pyridylmethyl) amine.

[0103]

As a salt of compound (I) , preferred is a pharmacologically acceptable salt. Examples of such salt include salts with inorganic base, salts with organic base, salts with inorganic acid, salts with organic acid, salts with basic or acidic amino acid and the like.

[0104]

Preferable examples of the salt with inorganic base include alkali metal salts such as sodium salt, potassium salt and the like_/ alkaline earth metal salts such as calcium salt, magnesium salt and the like; and aluminum salt, ammonium salt and the like.

[0105] Preferable examples of the salt with organic base include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine,

N,N-dibenzylethylenediamine and the like.

[0106] Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. [0107] Preferable examples of the salt with organic acid include salts with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like. [0108]

Preferable examples of the salt with basic amino acid include salts with arginine, lysine, ornithine and the like. [0109]

Preferable examples of the salt with acidic amino acid include salts with aspartic acid, glutamic acid and the like. [0110]

Compound (I) may be either anhydride or hydrate. [0111]

Furthermore, compound (I) may be labeled with an isotope (e.g., ³H, ¹⁴C, ³⁵S) . [0112]

When compound (I) contains an optical isomer, a stereoisomer, a positional isomer or a totamer, these are also encompassed as compound (I), and can be obtained as single products by a synthesis method and a separation method known per se. For example, when an optical isomer is present in compound (I), an optical isomer resolved from the compound is also encompassed in compound (I) . The optical isomer can be produced by a method known per se. [0113]

Compound (I) can be produced by [production method 1], [production method 2] described in detail below or a method analogous thereto.

The compounds to be used as starting compounds may be each used in the form of a salt. Examples of such salt include those exemplified as the aforementioned salts of compound (I) . [0114]

When alkylation reaction, hydrolysis, amination reaction, esterification reaction, amidation reaction, esterification reaction, etherification reaction, oxidation reaction, reduction reaction and the like are performed in the following [production method 1] and [production method 2] , these reactions are performed according to a method known per se. Examples of such method include the methods described in Organic Functional Group Preparations, 2nd edition, Academic Press, Inc. (1989); Comprehensive Organic Transformations, VCH Publishers Inc., (1989) and the like. [0115] [Production method 1]

Compound (I) wherein X is -CO- and Y is -NH- (hereinafter to be referred to as compound (Ia) ) can be produced, for example, by the following amidation reaction of compound (II) and compound (III) . (Amidation reaction)

[ 0116]

[0117] wherein the symbols in the formula are as defined above. [0118]

In the "amidation reaction", a dehydration condensation agent is preferably used. In this case, compound (III), 1-5 equivalents of compound (II) , and 1 to 3 equivalents of dehydration condensation agent relative to compound (III) are reacted in an inert solvent. Where necessary, the reaction may be performed in the co-presence of 1 to 3 equivalents of alcohol relative to compound (III) and/or a catalytic amount to 1-5 equivalents of a base relative to compound (III) . [0119]

Examples of the "dehydration condensation agent" include dicyclohexylcarbodiimide, l-ethyl-3- (3- dimethylaminopropyl) carbodiimide (EDC) hydrochloride and the like. [0120]

Examples of the "inert solvent" include nitrile solvents, amide solvents, halogenated hydrocarbon solvents, ether solvents, water and the like. These may be used in a mixture of two or more kinds in an appropriate proportion. Of these, acetonitrile, ^' DMF, dichloromethane, THF and the like are preferable. [0121]

Examples of the "nitrile solvent" include acetonitrile and propionitrile . Examples of the "amide solvent" include N, N- dimethylformamide (DMF) , N,N-dimethylacetamide and l-methyl-2- Pyrrolidione (NMP) .

Examples of the "halogenated hydrocarbon solvent" include dichloromethane, chloroform, 1,2-dichloroethane and carbon tetrachloride.

Examples of the "ether solvents" include diethyl ether, tetrahydrofuran (THF), 1,4-dioxane and 1, 2-dimethoxyethane. [0122]

Examples of the "alcohol" include Ci-₆ alkyl alcohol (preferably, methanol, ethanol, isopropanol, tert-butanol) . [0123]

Examples of the "base" include

1) strong base such as hydride of alkali metal or alkaline earth metal (e.g., lithium hydride, sodium hydride, potassium hydride, calcium hydride) , amide of alkali metal or alkaline earth metal (e.g., lithium amide, sodium amide, lithiumdiisopropylamide, lithiumdicyclohexylamide, lithiumhexamethyl disilazide, sodiumhexamethyl disilazide, potassium hexamethyl disilazide) , Ci-₆ alkoxide of alkali metal or alkaline earth metal (e.g., sodium methoxide, sodium ethoxide, potassium tert-butoxide) and the like;

2) inorganic base such as hydroxide of alkali metal or alkaline earth metal (e.g., sodium hydroxide, potassium hydroxide, lithium hydroxide, barium hydroxide) , carbonate of alkali metal or alkaline earth metal (e.g., sodium carbonate, potassium carbonate, cesium carbonate) , alkali metal hydrogencarbonate (e.g., sodium hydrogen carbonate, potassium hydrogen carbonate) and the like; 3) organic base such as amines (e.g., triethylamine, N₇N- diisopropylethylamine, N-methylmorpholine, 4- dimethylaminopyridine, DBU (1, 8-diazabicyclo [5.4.0] undeca-7-ene) ,

DBN (l,5-diazabicyclo[4.3.0] nona-5-ene) ) , basic heterocyclic compounds (e.g., pyridine, imidazole, 2, β-lutidine) and the like; and the like.

Of the above-mentioned bases, triethylamine, N,N- diisopropylethylamine, pyridine and the like are preferable.

[0124]

The reaction temperature is generally -20°C to 150°C, preferably, 0°C to 100⁰C. The reaction time is generally 1 hr to

90 hr, preferably 5 hr to 24 hr.

[0125]

Here, compound (II) can be synthesized, for example, according to the method described in D. D. Cox, S.J. Benkovic, L.M. Bloom, F. C. Bradley, M.J. Nelson, L. Que, Jr., D. E.

Wallick., J. Am. Chem. Soc, 1988, 110, 2026.

[0126]

[Production method 2]

Compound (I) wherein X is -O- and Y is -CO- (hereinafter to be referred to as compound (Ib) ) can be produced, for example, by the following esterification reaction of compound (IV) and compound (V) . (Esterification reaction) [0127]

[0128] wherein the symbols in the formula are as defined above. [0129] The "esterification reaction" includes the following "method using a dehydration condensation agent" and "a method using a reactive derivative of carboxylic acid". [0130] i) Method using a dehydration condensation agent Compound (IV) , 1-5 equivalents of compound (V) , and 1 to 3 equivalents of dehydration condensation agent relative to compound (IV) are reacted in an inert solvent. Where necessary, the reaction may be performed in the co-presence of 1 to 3 equivalents of alcohol relative to compound (IV) and/or catalytic amount to 1-5 equivalents of base relative to compound (IV) . [0131]

Examples of the "dehydration condensation agent" include dicyclohexylcarbodiimide, l-ethyl-3- (3- dimethylaminopropyl) carbodiimide (EDC) hydrochloride and the like. [0132]

Examples of the "inert solvent" include the aforementioned nitrile solvents, amide solvents, halogenated hydrocarbon solvents, ether solvents, water and the like. These may be used in a mixture of two or more kinds in an appropriate proportion.

Of these, acetonitrile, DMF, dichloromethane, THF and the like are preferable.

[0133]

Examples of ^■ the "alcohol" include Ci-₆ alkyl alcohol

(preferably methanol, ethanol, isopropanol, tert-butanol) .

[0134] Examples of the "base" include those exemplified for the aforementioned "amidation reaction". Of the above-mentioned bases, triethylamine, N,N-diisopropylethylamine, pyridine and the like are preferable.

[0135] The reaction temperature is generally -2O⁰C to 150°C, preferably O⁰C to 100°C. The reaction time is generally 1 hr to

90 hr, preferably, 5 hr to 24 hr.

[0136] ii) Method using a reactive derivative of carboxylic acid Compound (IV) and 1-5 equivalents (preferably, 1 to 3 equivalents) of a reactive derivative of compound (V) are reacted in an inert solvent. Where necessary, the reaction may be performed in the co-presence of 1 to 10 equivalents of base relative to compound (IV) . [0137]

Examples of the "reactive derivative" of compound (V) include acid halides (e.g., acid chloride, acid bromide), mixed acid anhydrides (e.g., Ci_₆ alkyl-carboxylic acid, acid anhydride with C_δ-io aryl-carboxylic acid or Ci_6 alkyl carbonate) , active esters (e.g., ester with 1-hydroxybenzotriazole or N- hydroxysuccinimide) , active amides (e.g., amide with N, N'- carbonyldiimidazole) and the like.

The reactive derivative is preferably active amide.

[0138] Examples of the ^Λλinert solvent" include ether solvents, halogenated hydrocarbon solvents, aromatic solvents, nitrile solvents, amide solvents, ketone solvents, sulfoxide solvents, water and the like. These may be used in a mixture of two or more kinds in an appropriate proportion. Of these, acetonitrile,

N,N-dimethylacetamide, THF, pyridine, chloroform and the like are preferable.

[0139]

Examples of the "base" include those exemplified for the aforementioned "amidation reaction". The base is preferably sodium hydride, potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate, potassium hydrogen carbonate, triethylamine, N, N- diisopropylethylamine, pyridine and the like. [0140]

The reaction temperature is generally -^'20⁰C to 60°C, preferably at room temperature. The reaction time is generally 1 hr to 40 hr, preferably, 5 hr to 24 hr.

[0141] Compound (IV) can be produced, for example, by the following amination reaction according to a method analogous to the method described in JP-A-2006-8790.

(Amination reaction)

[ 0142 ]

[ 0143 ] wherein A is a leaving group, and other symbols are as defined above . [0144] Examples of the "leaving group" for A include a halogen atom, an optionally halogenated Ci_₆ alkylsulfonyloxy group (e.g., methanesulfonyloxy, ethanesulfonyloxy, trifluoromethanesulfonyloxy) , a hydroxy group and the like. The "leaving group" is preferably a halogen atom. [0145]

This reaction is generally performed in an inert solvent. Examples of the "inert solvent" include alcohol solvents, ether solvents, halogenated hydrocarbon solvents, aromatic solvents, nitrile solvents, amide solvents, ketone solvents, sulfoxide solvents, water and the like. These may be used in a mixture of two or more kinds in an appropriate proportion. Of these, acetonitrile, N,N-dimethylformamide (DMF) , acetone, ethanol, pyridine, water and the like are preferable. [0146] The amount of compound (VIb) to be used is generally 0.5 to 2 equivalents (e.g., 1 equivalent) relative to compound (Via). The amount of compound (VII) to be used is generally 0.5 equivalent to 10 equivalents relative to the total of compound (Via) and compound (VIb) . In addition, an excess amount of compound (III) may be used as a reaction solvent. [0147]

The reaction temperature is generally about -20°C to 200°C, preferably room temperature to 100°C. The reaction time is, for example, about 0.5 hr to 1 day. [0148]

Where necessary, this reaction may be performed in the co- presence of a base.

Examples of the "base" include those exemplified for the aforementioned "amidation reaction". The base is preferably sodium hydride, potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate, potassium hydrogen carbonate, triethylamine, N, N- diisopropylethylamine, pyridine and the like. The amount of the base to be used is generally 0.1 to 100 equivalents, preferably, 1 to 10 equivalents, relative to compound (VII) . [0149]

In the above-mentioned production methods, when the starting compound has an amino group, a carboxy group, a hydroxy group or a carbonyl group as a substituent, these groups may be protected by a protecting group generally used in the peptide chemistry and the like, where the protecting group is removed as necessary after the reaction to give the object compound. [0150] Examples of the amino-protecting group include a formyl group, Ci-₆ alkyl-carbonyl groups (e.g., acetyl, propionyl) , Ci-₆ alkoxy-carbonyl groups (e.g., methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl) , a benzoyl group, C₇-_I0 aralkyl-carbonyl groups (e.g., benzylcarbonyl) , C₇-_I4 aralkyloxy-carbonyl groups (e.g., benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl) , a trityl group, a phthaloyl group, an N,N-dimethylaminomethylene group, substituted silyl groups (e.g., trimethylsilyl, triethylsilyl, dimethylphenylsilyl, tert-butyldimethylsilyl, tert- butyldiethylsilyl) , C₂-₆ alkenyl groups (e.g., 1-allyl) and the like. These groups may be substituted by 1 to 3 substituents selected from a halogen atom, Ci_₆ alkoxy groups (e.g., methoxy, ethoxy, propoxy) and a nitro group. [0151]

Examples of the carboxy-protecting group include Cχ-β alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, butyl, tert- butyl) , C₇-_H aralkyl groups (e.g., benzyl), a phenyl group, a trityl group, substituted silyl groups (e.g., trimethylsilyl, triethylsilyl, dimethylphenylsilyl, tert-butyldimethylsilyl, tert-butyldiethylsilyl) , C₂-₆ alkenyl groups (e.g., 1-allyl) and the like. These groups may be substituted by 1 to 3 substituents selected from a halogen atom, Ci_₆ alkoxy groups (e.g., methoxy, ethoxy, propoxy) and a nitro group. [0152]

Examples of the hydroxy-protecting group include Ci_₆ alkyl groups (e.g., methyl, ethyl, propyl, isopropyl, butyl, tert- butyl) , a phenyl group, a trityl group, C₇_₁₀ aralkyl groups (e.g., benzyl), a fomayl group, Ci-₆ alkyl-carbonyl groups (e.g., acetyl, propionyl) , a benzoyl group, C₇-io aralkyl-carbonyl groups (e.g., benzylcarbonyl) , a 2-tetrahydropyranyl group, a 2- tetrahydrofuranyl group, substituted silyl (e.g., trimethylsilyl, triethylsilyl, dimethylphenylsilyl, tert-butyldimethylsilyl, tert-butyldiethylsilyl) , C₂-β alkenyl groups (e.g., 1-allyl) and the like. These groups may be substituted by 1 to 3 substituents selected from a halogen atom, Ci-6 alkyl (e.g., methyl, ethyl, n- propyl) , Ci-₆ alkoxy (e.g., methoxy, ethoxy, propoxy) and a nitro group. [0153]

Examples of the carbonyl-protecting group include cyclic acetal (e.g., 1, 3-dioxane) , non-cyclic acetal (e.g., di-Ci_₆ alkylacetal) and the like. [0154]

The above-mentioned protecting groups can be removed by a method known per se, for example, the method described in Protective Groups in Organic Synthesis, John Wiley and Sons (1980) and the like. Specifically, methods using acid, base, ultraviolet rays, hydrazine, phenylhydrazine, sodium N- methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, trialkylsilyl halides (e.g., trimethylsilyl iodide, trimethylsilyl bromide and the like) and the like, reduction method and the like can be used. [0155]

Compound (I) obtained by the above-mentioned production methods can be isolated and purified by a known method, for example, solvent extraction, liquid conversion, phase transfer, crystallization, recrystallization, chromatography and the like. [0156]

Compound (I) or a salt thereof (hereinafter to be referred to as the compound of the present invention) has a superior degranulation suppressive effect and a cytokine production suppressive effect. Accordingly, it is useful as a degranulation suppressant, a cytokine production suppressant, or an agent for the prophylaxis or treatment of various allergic diseases, autoimmune diseases, inflammatory diseases and the like, in which a cell capable of degranulation of chemical transmitters (e.g., histamine) and the like (e.g., mast cell) or cell having a cytokine production capability is involved. [0157] The "cytokine" in the present invention is a humoral protein which can generally induce various allergic diseases and various inflammatory diseases including autoimmune diseases when it is produced^" or released in excess. Examples of cytokine include interleukin (IL) -1, IL-2, IL-3, IL-4, IL-5, IL-β, IL-IO, IL-12, IL-13, granulocyte colony stimulating factor (G-CSF) , granulocyte macrophage colony stimulating agent (GM-CSF) , tumor necrosis factor-α (TNF-α) , transforming growth factor-β (TGF-β) , interferon-γ (IFN-γ) , macrophage colony stimulating factor (M- CSF) , monocyte chemotactic promoting factor-1 (MCP-I) , MlPIβ, MIPIa, leukemia inhibitory factor (LIF), eotaxin and the like. [0158]

The compound of the present invention can suppress expression of various symptoms such as promoted vascular permeability, smooth muscle contraction, promoted gland secretion, vasodilation and the like due to Type I allergic reaction, and can improve various symptoms by suppressing degranulation and histamine secretion from mast cell and the like. In addition, the compound of the present invention can control cytokine production and release in mast cells, and is effective for various allergic diseases and inflammatory diseases including autoimmune diseases. Furthermore, the compound of the present invention has low toxicity (e.g., acute toxicity, chronic toxicity) . [0159] Therefore, the compound of the present invention can be safely administered to mammals (e.g., rat, mouse, guinea pig, rabbit, sheep, horse, swine, bovine, monkey, human) as an agent for the prophylaxis or treatment of diseases, in which degranulation or cytokine production in a cell (e.g., mast cell) are involved, and the like. [0160]

Examples of the diseases, in which degranulation or cytokine production are involved, include allergic diseases (particularly disease involving Type I allergic reaction) such as allergic dermatitis (e.g., atopic dermatitis), allergic rhinitis, allergic conjunctivitis, bronchial asthma, urticaria, allergic inflammation, anaphylactic shock and the like. Other examples thereof include inflammatory diseases including autoimmune diseases such as systemic lupus erythematosus, mixed connective-tissue disease, rheumatoid arthritis, Sjogren's syndrome, rheumatic fever, Goodpasture's syndrome, graves disease, Hashimoto's disease, Addison' s disease, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, myasthenia gravis, ulcerative colitis, Crohn's disease, sympathetic ophthalmia, multiple sclerosis, psoriasis, hepatitis and the like.

[0161]

The compound of the present invention can be used as it is or in the form of a pharmaceutical composition also containing a pharmacologically acceptable carrier, which is obtained by preparation making according to a method known per se, for example, the method described in the Japanese Pharmacopoeia. [0162] Examples of the pharmacologically acceptable carrier include various organic or inorganic carrier substances conventionally used as preparation materials, such as excipient, lubricant, binder and disintegrant for solid preparations; solvent, solubilizing agent, suspending agent, isotonicity agent, buffering agent and soothing agent for liquid preparations . In addition, additives such as preservative, antioxidant, colorant, sweetening agent, adsorbent, wetting agent and the. like can also be used as necessary during production of preparation. [0163]

Examples of the excipient include lactose, sucrose, D- mannitol, starch, cornstarch, crystalline cellulose and light anhydrous silicic acid. Examples of the lubricant include magnesium stearate, calcium stearate, talc and colloidal silica. Examples of the binder include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose and sodium carboxymethylcellulose. Examples of the disintegrant include starch, carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, sodium carboxymethyl starch and low- substituted hydroxypropylcellulose (L-HPC) . [0164]

Examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil and corn oil. Examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate and sodium citrate. Examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerol monostearate and the like; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose and the like. [0165]

Examples of the isotonicity agent include glucose, D- sorbitol, sodium chloride, glycerol and D-mannitol. Examples of the buffering agent include buffers of phosphate, acetate, carbonate, citrate and the like. Examples of the soothing agent include benzyl alcohol. Examples of the preservative include paraoxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid and sorbic acid.

Examples of the antioxidant include sulfite and ascorbic acid. [0166]

Examples of the colorant include aqueous food tar colors (e.g., food colors such as Food Color Red Nos. 2 and 3, Food Color Yellow Nos. 4 and 5, Food Color Blue Nos. 1 and 2 etc.), water insoluble lake dye (e.g., aluminium salt with the aforementioned aqueous food tar color), natural dye (e.g., β- carotene, chlorophyll, red iron oxide) . Examples of the sweetening agent include saccharin sodium, dipotassium glycyrrhizinate, aspartame and stevia. [0167]

Examples of the adsorbent include porous starch, calcium silicate (trade name: FloriteRE) , magnesium alumino metasilicate (trade name: Neusilin) and light anhydrous silicic acid (trade name: Sylysia) . Examples of the wetting agent include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelauryl ether. [0168] Examples of the dosage form of the aforementioned pharmaceutical composition include oral preparations such as tablets (including sublingual tablet, orally disintegrating tablet), powder, granule, drop, capsules (including soft capsule, microcapsule) , sachet, troche, syrup, emulsion, suspension and the like; parenteral preparations such as injections (e.g., subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection) , external preparations (e.g., dermal preparation, nasal preparation, ointment, cream, aerosol, spray, powder material, lotion etc.), suppositories (e.. g., rectal suppository, vaginal suppository), pellet, pulmonary preparation (inhalant) , drip infusion and the like. These can be safely administered orally or parenterally (e.g., topical, rectal, intravenous administrations) . The content of the compound of the present invention in the pharmaceutical composition is, for example, about 0.1 to 100 wt% in the total pharmaceutical composition. [0169]

Where necessary, tablets can be formed into general coated tablets such as sugar-coated tablet, enteric-coated tablet and film-coated tablet, or two-layer tablet or multi-layer tablet. Powder is prepared together with a base for pharmaceutically acceptable powder. Examples of the base include talc, lactose, starch and the like. Drop can be prepared together with an aqueous or non-aqueous base and one or more kinds of pharmaceutically acceptable diffusing agent, suspending agent, dissolution agent and the like. Capsule can be produced by filling a compound to be an active ingredient together with a pharmaceutically acceptable carrier. The compound of the present invention can be mixed with a pharmaceutically acceptable excipient and filled in a capsule or filled in a capsule without an excipient. Sachet can also be produced in the same manner. [0170]

Examples of the liquid for injection include solution, suspension, emulsion and the like. For example, an aqueous solution, a water-propylene glycol solution and the like can be used. The liquid may contain water, and can be produced in the form of a solution of polyethylene glycol and/or propylene glycol. [0171]

External preparation can be produced by mixing the compound of the present invention with a pharmaceutically acceptable diluent and a carrier. Ointment and cream are prepared, for example, by adding a thickener and/or a gelling agent to an aqueous or oily base. Examples of the base include water, liquid paraffin, vegetable oil and the like. Examples of the thickener include soft paraffin, aluminum stearate, cetostearyl alcohol, propylene glycol, polyethylene glycol, lanolin, hydrogenated lanolin, bee wax and the like. Topical agents may contain, where necessary, preservatives and bacterial growth preventive agents such as methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chloride and the like. Lotion can be prepared by adding one or more kinds of pharmaceutically acceptable stabilizer, suspending agent, emulsifier, diffusing agent, thickener, colorant, flavor and the like to an aqueous or oily base.

[0172]

When the compound of the present invention is prepared as a suppository, it is prepared according to a conventional method together with a base such as vegetable oil (castor oil, olive oil, peanut oil etc.), mineral oil (vaseline, white petrolatum etc.), wax, partially synthesized or totally synthesized glycerol fatty acid ester and the like. [0173]

The pharmaceutical composition thus obtained is administered orally or parenterally as a degranulation suppressant or an agent for the prophylaxis or treatment of the above-mentioned diseases . [0174]

The dose of the compound of the present invention is appropriately determined according to the subject of administration, administration route, disease and the like.

For example, the daily dose of the compound of the present invention by parenteral administration to an adult patient (body weight about 60 kg) with allergic dermatitis is about 0.001 - about 100,000 mg, which can be administered in one to several portions a day. [0175] The present invention is explained in more detail in the following by referring to Examples below, which are not to be construed as limitative .

Examples [ 0176] (Example 1)

Production of N, N-bis (2-pyridylmethyl) glycine (compound 1 ) [0177 ]

[0178]

The title compound was synthesized according to the method described in D. D. Cox, S.J. Benkovic, L.M. Bloom, F. C. Bradley,

M.J. Nelson, L. Que, Jr., D. E. Wallick., J. Am. Chem. Soc, 1988, 110, 2026.

[0179]

(Example 2)

Production of N, N-bis (2-pyridylmethyl) glycylglycine tert-butyl ester (compound 2) [0180]

[ 0181 ]

. To the compound (4 g, 15.55 iranol) of Example 1 were added CH₂Cl₂ (60 ml) and H-GIy-OtBu-HCl (2.61 g, 15.55 mmol) , and the mixture was cooled to 0°C. To the mixture were added EDC*HCl (3.28 g, 17.11 πnnol) , HOBt (2.52 g, 18.66 irimol) and NEt₃ (3.30 g, 32.66 mmol) , and the mixture was allowed to warm to room temperature and stirred for 13 hr. Tap water was added to the reaction solution and the mixture was extracted with chloroform. The organic layer was washed twice with tap water and once with saturated brine. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The concentrated residue was purified by silica gel column chromatography (chloroform-κ:hloroform:methanol=200:l-»100:l-»50:l->30:l) to give the title compound, yield 4.90 g, 85%, yellow solid, melting point 94-95⁰C.

¹H NMR (200 MHz, CDCl₃): δ = 1.45 (s, 9 H), 3.36 (s, 2 H), 3.90 (s, 4 H), 3.98(d, 2 H, J = 5.7 Hz), 7.16 (ddd, 2 H, J = 1.1, 4.8, 7.5 Hz), 7.37 (dm, 2 H, J =7.9 Hz), 7.63 (ddd, 2 H, J = 1.8, 7.5, 7.6 Hz), 8.55 (ddd, 2 H, J = 0.9, 1.8, 4.8 Hz), 8.89 (t, 1 H, J = 5.6 Hz) . [0182] (Example 3) Production of N,N-bis (2-pyridylmethyl) glycylglycine (compound 3) [0183]

[0184]

The title compound was synthesized according to the description in the following documents.

T. Kobayashi, T. Okuno, T. Suzuki, M. Kunita, S. Ohba, Y.

Nishida, Polyhedron, 1998, 17, 1553.

S. Ito, T. Okuno, H. Matsushima, T. Tokii, Y. Nishida, J. Chem.

Soc, Dalton Trans ., 1996, 4479. N. Niklas, O. Walter, R. Alsfasser, Eur. J. Inorg. Chem., 2000, 1723. [0185] (Example 4)

Production of N, N-bis (2-pyridylmethyl) glycyl-L-phenylalanine tert-butyl ester (compound 4 ) [0186]

[ 0187 ]

To the compound (3 g, 11.66 mmol) of Example 1 were added CH₂Cl₂ (120 ml) and H-Phe-OtBu-HCl (2.78 g, 10.79 mmol), and the mixture was cooled to O⁰C. To the mixture were added EDC*HCl (2.46 g, 12.83 mmol), HOBt (1.89 g, 13.99 mmol) and NEt₃ (2.48 g, 24.49 mmol), and the mixture was allowed to warm to room temperature and stirred for 63 hr. Tap water was added to the reaction solution and the mixture was extracted with chloroform. The organic layer was washed twice with tap water and once with saturated brine. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The concentrated residue was purified by silica gel column chromatography

(chloroform-»chloroform:methanol=200:lH>100:l-»50:l) to give the title compound, yield 4.50 g, 92%, yellow oil.

¹H NMR (200 MHz, CDCl₃): δ = 1.38 (s, 9 H), 3.15 (dd, 2 H, J =

3.2, 7.1 Hz), 3.26 (s, 2 H), 3.79 (d, 4 H, J = 14.1 Hz), 4.79 (ddm, 1 H, J = 7.1, 8.1 Hz), 7.11-7.25 (m, 9 H), 7.56 (ddd, 2 H,

J = 1.8, 7.5, 7.7 Hz), 8.52 (ddd, 2 H, J = 0.9, 1.8, 4.9 Hz),

8.84 (d, 1 H, J = 8.1 Hz) .

[0188]

(Example 5) Production of N, N-bis (2-pyridylmethyl) glycyl-L-phenylalanine (compound 5) [0189]

[0190 ] The title compound was synthesized according to the description in the following documents .

N. Niklas, F. Hampel, R. Alsfasser, Chem. • Commun. , 2003, 1586. N. Niklas, O. Walter, R. Alsfasser, Eur. J. Inorg. Chem., 2000, 1723. [0191]

(Example 6)

Production of 2- [N, N-bis (2-pyridylmethyl) amino] -1- morpholinoethanone (compound 6) [0192]

[ 0193 ]

To the compound (1 g, 3.89 rranol) of Example 1 were added CH₂Cl₂ (10 ml, 10 v/w) and morpholine (0.34 g, 3.89 mmol) , and the mixture was cooled to 0°C. To the mixture were added EDC*HCl (0.82 g, 4.28 mmol), HOBt (0.63 g, 4.67 mmol) and NEt₃ (0.43 g, 4.28 mmol), and the mixture was allowed to warm to room temperature and stirred for 18 hr. Tap water was added to the reaction solution and the mixture was extracted with chloroform. The organic layer was washed with tap water and saturated brine. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography

(chloroform→chloroform:methanol=200:l→100:l-->50:l--»30:l) and the crude product was recrystallized from ethyl acetate-heptane to give the title compound, yield 0.41 g, 32%, white solid, melting point 81-82°C.

¹H NMR (200 MHz, CDCl₃): δ = 3.61 - 3.44 (m, 10 H), 3.91 (s, 4 H), 7.17 (ddd, 2 H, J = 1.3, 4.9, 7.5 Hz), 7.50 (dm, 2 H, J = 7.7 Hz), 7.66 (ddd, 2 H, J = 1.8, 7.5, 7.7 Hz), 8.55 (ddd, 2 H, J = 0.9, 1.8, 4.9 Hz) . [0194]

(Example 7)

Production of 2- [N, N-bis (2-pyridylmethyl) amino] -N' - phenethylacetamide ( compound 7 ) [ 0195 ]

[ 0196]

To the compound (1 g, 3.89 mmol) of Example 1 were added CH₂Cl₂ (10 ml, 10 v/w) and 2-phenylethylamine (0.47 g, 3.89 mmol), and the mixture was cooled to 0°C. To the mixture were added EDC-HCl (0.82 g, 4.28 mmol), HOBt (0.63 g, 4.67 mmol) and NEt₃ (0.43 g, 4.28 mmol), and the mixture was allowed to warm to room temperature and stirred for 18 hr. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction solution and the mixture was extracted with chloroform. The organic layer was washed twice with each of 5% aqueous citric acid solution and tap water, and once with saturated brine. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column ■chromatography (chloroform—»chloroform:methanol=100: l->50: 1->3O:1) to give the title compound, yield 0.78 g, 56%, yellow oil.

¹H NMR (200 MHz, CDCl₃): δ = 2.88 (t, 2 H, J= 7.0 Hz), 3.28 (s, 2 H), 3.58 (dt, 2 H, J = 7.0, 7.0 Hz), 3.77 (s, 4 H), 7.11-7.25 (m, 9 H), 7.56 (ddd, 2 H, J = 1.8, 7.5, 7.7 Hz), 8.49 (mdd, 2 H, J = 5 1.8, 5.5 Hz) , 8.66 (t, I H, J = 6.6 Hz) . [0197] (Example 8)

Production of 2- [N,N-bis (2-pyridylmethyl) amino] ethanol trihydrochloric acid (compound 8) io [0198]

[0199]

Aminoethanol (2 g, 32.74 mmol) and picolyl chloride hydrochloride (11.2 g, 68.28 mmol) were dissolved in EtOH (40 ml,

15 20 v/w) and tap water (60 ml, 30 v/w) . K₂CO₃ (9.44 g, 68.28 mmol) was added, and the mixture was stirred for 25 hr. The reaction solution was allowed to warm to room temperature and extracted with saturated aqueous sodium hydrogen carbonate solution and ethyl acetate. The aqueous layer was extracted with

20 ethyl acetate again. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The concentrated residue was purified by silica gel column chromatography. Ethyl acetate and 4N HCl-AcOEt (25 ml, 12.5 v/w) were added to the purified product, and the mixture was stirred

25 at room temperature. The precipitated crystals were filtered, and dried under reduced pressure to give the title compound, yield 5.26 g, 46%, white solid (hygroscopic), melting point 143- 145⁰C (reference document: JP-A-2006-8790) . 1H NMR (200 MHz, D₂O): δ = 2.92 (t, 2 H, J= 4.9 Hz), 3.74 (t, 2 H, J = 4.9 Hz), 4.41 (s_f 4 H), 8.00 (ddd, 2 H, J = 1.3, 5.9, 7.5 Hz), 8.09 (md, 2 H, J = 8.2 Hz), 8.57 (ddd, 2 H, J = 1.6, 7.9, 8.1 Hz), 8.77 (ddd, 2 H, J = 0.7, 1.6, 5.9 Hz) . [0200] (Example 9)

Production of 2- [N, N-bis (2-pyridylmethyl) amino] ethyl 2- (4- isobutylphenyl) propionate (compound 9)

[0201]

[ 0202 ]

To the compound (0.5 g, 2.06 mmol) of Example 8 were added CH₂Cl₂ (5 ml, 10 v/w) and IBP (0.42 g, 2.06 mmol), and the mixture was cooled to 0°C. To the mixture were added EDC*HCl (0.44 g, 2.27 mmol), HOBt (0.33 g, 2.47 mmol) and NEt₃ (0.23 g, 2.27 mmol), and the mixture was allowed to warm to room temperature and stirred for 19 hr. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction solution and the mixture was extracted with chloroform. The organic layer was washed twice with each of saturated aqueous sodium hydrogen carbonate solution and tap water, and once with saturated brine. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform->chloroform:methanol=200:l->-100:l→-50: l-»30:l) to give the title compound, yield 0.57 g, 64%, yellow oil.

¹H NMR (200 MHz, CDCl₃) : δ = 0.87 (d, 6 H, J= 6.6 Hz), 1.47 (d, 3 H, J = 7.1 Hz), 1.82 (tq, 1 H, J = 6.6, 7.0 Hz), 2.42 (d, 2 H, J = 7.1 Hz), 2.83 (t, 2 H, J = 5.7 Hz), 3.67 (q, 1 H, J = 7.1 Hz), 3.82 (s, 4 H), 4.19 (t, 2 H, J = 5.7 Hz), 7.04-7.21 (m, 6 H), 7.41 (md, 2 H, J= 7.8 Hz), 7.59 (ddd, 2 H, J = 1.8, 7.7, 7.7 Hz) , 8.50 (ddd, 2 H, J = 0.9, 1.8, 4.9 Hz) . [0203]

(Example 10)

Production of 2- [N,N-bis (2-pyridylmethyl) amino] ethyl 2-(3- trifluoromethylphenylamino)benzoate (compound 10) [0204]

[ 0205 ]

THF (3.5 ml, 5 v/w) was added to flufenamic acid (0.81 g, 2.88 iranol) , and the mixture was cooled to 0°C. CDI (0.47 g, 2.88 mmol) was added thereto, and the mixture was stirred for 30 min. A solution of the compound (0.70 g, 2.88 mmol) of Example 8 in THF (1.4 ml, 2.v/w) was added to the mixture. The mixture was allowed to warm to room temperature and stirred for 18^' hr. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction solution and the mixture was extracted with ethyl acetate. The organic layer was washed twice with each of saturated aqueous sodium hydrogen carbonate and tap water, and once with saturated brine. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform-»chloroform:methanol=100:l->50:l) to give the title compound, yield 0.71 g, 49%, yellow oil. 1H NMR (200 MHz, CDCl₃): δ = 3.02 (t, 2 H, J= 5.7 Hz), 4.45 (t, 2 H, J = 5.7 Hz), 6.82 (ddd, 1 H, J = 1.3, 6.8, 8.1 Hz), 7.13 (ddd, 2 H, J = 2.2, 4.9, 6.6 Hz), 7.27-7.61 (m, 10 H), 7.95 (dd, 1 H, J = 1.6, 8.1 Hz), 8.52 (ddd, 2 H, J = 1.0, 1.3, 4.9 Hz), 9.58 (s, 1 H). [0206] (Example 11)

Production of N,N-bis (2-pyridylmethyl) glycylglycine trihydrochloride (compound 11)

[0207]

The compound (1 g, 2.70 mmol) of Example 2 was dissolved in THF (5 ml) , 4N HCl-AcOEt (10 ml) was added, and the mixture was stirred at room temperature for 41 hr. The reaction solution was concentrated. Then, ethyl acetate was added to the solution and the mixture was concentrated. Ethyl acetate was further added to the resultant product, and the mixture was concentrated to dryness. After decantation with diethyl ether several times, the mixture was concentrated under reduced pressure and dried under reduced pressure to give the title compound, yield 0.51 g, 45%, white solid, hygroscopic.

¹H NMR (200 MHz, D₂O): δ = 3.67 (s, 2 H), 3.94 (s, 2 H), 4.44 (s, 4 H) 7.97 (ddd, 2 H, J = 1.3, 6.0, 7.7 Hz), 8.05 (dm, 2 H, J = 8.1 Hz), 8.53 (ddd, 2 H, J = 1.4, 7.9, 8.1 Hz), 8.75 (ddd, 2 H, J = 0.7, 1.5, 5.9 Hz). [0209]

(Example 12)

Production of N,N-bis (2-pyridylmethyl) glycyl-L-phenylalanine trihydrochloride (compound 12) [0210]

The compound (2 g, 4.34 mmol) of Example 4 was dissolved in ethyl acetate (4 ml), 4N HCl-AcOEt (10 ml) was added, and the mixture was stirred at room temperature for 19 hr. The reaction solution was concentrated. Then, ethyl acetate was added to the solution and the mixture was concentrated. Ethyl acetate was further added to the resultant product, and the mixture was concentrated to dryness. After decantation with diethyl ether several times, the mixture was concentrated under reduced pressure and dried under reduced pressure to give the title compound, white solid, hygroscopic. 1H NMR (200 MHz, D₂O): δ = 2.89 (dd, 1 H, J= 9.9, 14.1 Hz), 3.29 (dd, 1 H, J = 4.8, 14.1 Hz), 3.46 (d, 2 H, J = 16.3 Hz), 4.13 (d, 4 H, J = 15.7 Hz), 4.64 (dd, 1 H, J = 4.8, 9.9 Hz), 7.09 (dd, 1 H, J = 4.0, 8.6 Hz), 7.21 (md, 4 H, J = 4.4 Hz), 7.86 (dm, 2 H, J = 7.7 Hz), 7.92 (dm, 2 H, J = 6.0 Hz), 8.44 (ddd, 2 H, J = 1.6, 7.8, 8.1 Hz), 8.66 (ddd, 2 H, J = 0.7, 2.1, 6.0 Hz). [0212]

(Example 13)

Production of 2- [N, N-bis (2-pyridylmethyl) amino] ethyl acetate (compound 13) [0213]

[0214]

(1) Picolylation

Picolyl chloride (23.49 g, 143 mmol) was dissolved in ethanol (84 ml), and water (126 ml) and ethanolamine (4.20 ml, 68.8 mmol) were added thereto in this order at room temperature. Furthermore, potassium carbonate (19.73 g, 143 mmol) was added, and the mixture was reacted at 80°C for 28 hr. The reaction mixture was cooled to room temperature, sodium hydrogen carbonate (7.0 g) was added to adjust the reaction mixture to pH 8, and the mixture was extracted 3 times with ethyl acetate (300 ml) . The ethyl acetate layer was dried over anhydrous magnesium sulfate, and the filtrate obtained by filtration was concentrated under reduced pressure. The residue was purified by silica gel column to give the object product (9.56 g) . yield: 57%. [0215] (2) Acetylation

The alcohol form (0.5O g, 2.1 mmol) obtained in (1) was dissolved in chloroform (5 ml), and triethylamine (0.32 g, 3.15 mmol) was added at room temperature. The reaction mixture was cooled to 5⁰C with ice water, and acetyl chloride (0.20 g, 2.52 mmol) was added dropwise at 15°C or below. After dropwise addition, the mixture was reacted at room temperature for 1 hr. The reaction mixture was poured into water (20 ml) and partitioned. The aqueous layer was extracted with chloroform (10 ml) , and the chloroform layer was combined and dried over magnesium sulfate. The filtrate obtained by filtration was concentrated under reduced pressure to give a crude oil. This was purified by silica gel column chromatography to give an acetate form (0.49 g, R=methyl) . yield: 82%. [0216] (Example 14 - Example 19)

In the same manner as in Example 13, the compounds of the following Examples 14 - 19 were synthesized.

Example 14 : 2- [N>N-bis (2-pyridylmethyl) amino] ethyl isobutyrate (R= isopropyl) (compound 14) , Example 15: 2- [N,N-bis (2-pyridylmethyl) amino] ethyl phenylacetate (R= benzyl) (compound 15) ,

Example 16: 2- [N, N-bis (2-pyridylmethyl) amino] ethyl 3-phenyl- propionate (R= phenylethyl) (compound 16) , Example 17: 2- [N, N-bis (2-pyridylmethyl) amino] ethyl benzoate (R= phenyl) (compound 17) ,

Example 18 : 2- [N,N-bis (2-pyridylmethyl) amino] ethyl isonicotinate (R= 4-pyridyl) (compound 18) ,

Example 19: 2- [N,N-bis (2-pyridylmethyl) amino] ethyl furan-2- carboxylate (R=2-furyl) (compound 19) . [0217]

The amount charged for production of each compound and the yield are shown in the following Table. [0218] Table 1

[0219] The representative NMR spectra of the compounds of Examples 13 - 19 are shown in FIGs. 1 - 7, respectively.

[0220] (Example 20)

Production of 2- [N, N-bis (2-pyridylmethyl) amino] ethanol (compound 20)

2-Chloromethylpyridine hydrochloride (6.56 g, 40.0 mmol) , 2-aminoethanol (1.22 g, 20.0 mmol), powderized K₂CO₃ (16.6 g, 120 mmol) and acetonitrile (70 mL) were mixed at room temperature, and the mixture was heated under reflux for 2 days. The reaction mixture was cooled to room temperature, the solvent was evaporated under reduced pressure, water (30 mL) was added to the residue, and the mixture was extracted 3 times with dichloromethane (30 mL) . The organic layer was dried over sodium sulfate, concentrated under reduced pressure and evaporated under reduced pressure to give the title compound (yield 3.35 g, 13.8 mmol, 69%, orange oil).

The representative NMR spectra (¹H NMR(300 MHz, CDCl₃)) of the compound 20 are shown in FIG. 8. [0221]

(Example 21)

Production of N,N-bis (2-pyridylmethyl) -beta-alanine (compound

21)

2-Chloromethylpyridine hydrochloride (9.50 g, 58.0 mmol), β-alanine (2.50 g, 28.1 mmol) and water (30 mL) were mixed at room temperature, the mixture was adjusted to pH 10 by dropwise addition of 5 mol/L aqueous NaOH solution, and the mixture was stirred as it was at room temperature. The reaction solution was stirred for 5 days while sometimes adding dropwise 5 mol/L aqueous NaOH solution to maintain the pH at 9-10, since the pH of the reaction solution changes toward neutral with the progress of the reaction. Water (20 mL) was added, and the mixture was washed 3 times with ethyl acetate (60 mL) . The aqueous layer was adjusted to pH 3 by adding 3 mol/L hydrochloric acid, and the mixture was extracted 3 times with chloroform (40 mL) . The organic layer was dried over sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (chloroform:methanol=20: 1 —» 10:1 -» 5:1) to give the title compound (yield 497 mg, 1.83 mmol, 6.5%, brown solid).

The representative NMR spectra (¹H NMR(300 MHz, CDCl₃)) of compound 21 are shown in FIG. 9.

[0222]

(Example 22 ) Production of N- { 2- [2-N, N-bis (2-pyridylmethyl) amino- ethoxymethyl] phenyl } -3-trifluoromethylphenylamine (compound 22 )

( 1) Step 1

[0223]

[0224]

Compound 20 (258 mg, 1.0 mol) was dissolved in 10 ml of dry THF, and a solution of 1.6 M BuLi in hexane (0.94 mL, 1.5 mmol) was added dropwise under an argon atmosphere in an ice bath. After stirring for 20 rain, compound (a) (324 mg, 1.5 mmol) dissolved in 10 ml of dried THF was added dropwise to the mixture. The reaction solution was stirred as it was for 2 hr in an ice bath, reacted at room temperature for 2 days, and heated under reflux for 2 days. The solvent was evaporated under reduced pressure, and the residue was dissolved in chloroform, washed with water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure to give a crude product, which was purified using an alumina column (eluted with chloroform containing 0.5 % methanol) to give the object product (b) (58 mg, 0.15 mmol, yield 15%). [0225] (2) Step 2 [0226]

[0227]

Compound (b) (45 mg) was dissolved in methanol (20 ml) and subjected to a reduction reaction with platinum oxide (17 mg) in a hydrogen gas at normal pressure. The catalyst was filtered off, and the filtrate was concentrated under reduced pressure to quantitatively give the object product (c) . [0228]

[0230] Dry THF (1 ml) was added to a mixture of Compound (c) (100 mg, 0.3 mmol) , compound (d) (134 mg, 0.6 mmol) , potassium t- butoxide (34 mg, 0.3 mmol), palladium acetate (3.4 mg, 0.015 mmol) and DPEphos (9.7 mg, 0.018 mmol), and the mixture was heated using a microwave synthesizer (400 W) at 100°C for 30 min. Methylene chloride was added to the reaction mixture, and the mixture was washed with water, dried over sodium sulfate, and concentrated under reduced pressure to give a crude product, which was purified using an alumina column (eluted with chloroform containing 0.5 % methanol) to give the title compound (16 mg, 0.033 mmol, yield 11%).

[0231]

The representative NMR spectra (¹H NMR(300 MHz, CDCl₃)) of compound 22 are shown in FIGs. 10 and 11.

In addition, the representative mass spetrum of compound 22 is shown in FIG. 12. The measurement conditions of mass spectrometry were as follows, ionization mode: ESI+ orifice 1 voltage sweep : 85V range of mass-to-charge ratio : 100 . 0-1000. 0 spectrum recording interval : 1. 0 s representative spectrum measurement time : 0 . 033 min [0232]

(Example 23)

Production of N- (2-benzyloxyethyl) -N, N-bis (2-pyridylmethyl) amine

(compound 23 )

A solution of 1.6 M butyl lithium in hexane (2 mL, 3.2 mrtiol) was added to an argon-substituted solution of compound 20 (500 mg, 2.05 mmol) in dry THF (10 mL) under ice-cooling, and the mixture was stirred for 20 min. Benzyl chloride (0.39 g, 3.08 mmol) dissolved in dry THF (5 mL) was added to the red mixture under ice-cooling conditions. The mixture was allowed to warm to room temperature under an argon atmosphere over 1 hr, and sequentially heated under reflux overnight. Septum came off during heating under reflux and the solvent was dried. Water (30 mL) was added to the residue, and the mixture was extracted with chloroform (30 mL) . The organic layer was dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel chromatography

(chloroform: acetone=10 : 1 —» 2:1 —> chloroform:methanol=50 : 1 -> 10:1) to give the title compound (yield 635 mg, 1.90 mmol, 93%, brown oil) . The representative NMR spectra (¹H NMR(300 MHz, CDCl₃)) of compound 23_' are shown in FIGs. 13 and 14.

[0233]

(Example 24)

Production of 3- [N, N-bis (2-pyridylmethyl) amino] -N-phenyl- propionamide (compound 24)

Compound 21 (200 mg, 737 μmol) , aniline (68.6 mg, 737 μmol) and triethylamine (157 mg, 1.55 mmol) were dissolved in dichloromethane, and the mixture was stirred and ice-cooled. To this solution were added HOBt (120 mg, 888 μmol) and EDCHCl (155 mg, 809 μmol) , and the mixture was warmed to room temperature and stirred for 21 hr. The reaction solution was concentrated under reduced pressure, water (30 mL) was added to the residue, and the mixture was extracted 3 times with chloroform (30 mL) . The organic layer was dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (chloroform: acetone=10:l -> 5:1 —> 2:1 -» 1:1 -» chloroform:methanol=50: 1 -> 10:1) to give the title compound (yield 251 mg, 725 μmmol, 98%, brown oil) .

The representative NMR spectra (¹H NMR(300 MHz, CDCl₃)) of compound 24 are shown in FIGs. 15 and 16. [0234] (Example 25 - Example 27) N- (2-Methoxyethyl) -N,N-bis (2-pyridylmethyl) amine (compound 25) and N- (3-methoxypropyl) -N,N-bis (2- pyridylmethyl) amine (compound 27) were produced according to the method described in Inorganica Chimica Acta, 357 (2004) 2694- 2702. 2-{2-[N,N-bis (2-Pyridylmethyl) amino] ethoxy}ethanol (compound 26) was produced according to the method described in Dalton Trans. (2004) 1201-1207. Each compound was kindly provided by associate professor Yuji Mikata of Nara Women's University. [0235] The structures of the compounds produced in the above- mentioned Examples are shown in Tables 2-6.

[ 0241 ]

(Test Example 1)

Effects of example compounds on mouse mast cell degranulation Preparation of reagents The following reagents were used for the test:

Dimethyl sulfoxide CHROMASOLV^R Plus (DMSO, Sigma-Aldrich) , RPMI1640 (Wako), Fatal Bovine serum (FBS, Lot# SFB30-1540, EQUITECH-BIO Inc.), 10,000 Units/ml penicillin-streptomycin solution (Invitrogen) , 2-mercaptoethanol (Invitrogen) . As recombinant mouse Interleukin-3 (rmIL-3) , mIL-3 secreted in a culture supernatant of CHO cells incorporating the mouse IL-3 gene was used (supplied by the Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology) . Tyrode's buffer solution was prepared with the composition shown below:

140 mM NaCl, 2.7 πM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 12 itiM NaHCO₃, 0.4 mM NaH₂PO₄, 5.6 mM Glucose, 10 mM HEPES (pH 7.4).

A solution of bovine serum albumin (Sigma-Aldrich) was prepared to 10% (w/v) using Tyrode's buffer solution. This solution was further diluted to prepare a 1% BSA solution.

By diluting 1 mg/ml anti-DNP antibody clone SPE-7 (SPE-7, Sigma-Aldrich) with a medium to test concentrations, sensitizers were prepared. By dissolving DNP-human serum albumin (DNP-HSA, Sigma- Aldrich) to 10 mg/ml in Tyrode's buffer solution, and further diluting the solution to test concentrations using Tyrode's buffer solution containing 1% BSA before measurements, inducers were prepared. The buffer solution for cell extraction used was Tyrode's buffer solution containing 1% (v/v) Triton-XIOO.

The enzyme reaction solution used was a 100 mM citrate buffer solution (pH 4.5) containing 1.3 mg/ml p-nitrophenyl-N- acetyl-s-D-glucopyranoside (Sigma-Aldrich) . The reaction stopper used was 0.2 M glycine-NaOH (pH 10.2) . [0242]

Each compound produced according to Examples above (supplied by Hamari Chemicals Ltd. and Takahashi Gosei

Kenkyusho) was dissolved to 100 mM in DMSO, and this solution was used after being diluted before measurement.

The control substance used was N,N,N' ,N' -tetrakis (2- pyridylmethyl) ethylenediamine (TPEN, Sigma-Aldrich) . TPEN was dissolved to 10 mM in DMSO, and this solution was used after being diluted before measurement.

Ibuprofen (supplied by Hamari Chemicals Ltd.), to 400 mM, and flufenamic acid (supplied by Hamari Chemicals Ltd.), to 100 mM, were dissolved in a 500 mM aqueous solution of sodium hydrogen carbonate. Taking into account the influence of the solvent, the ibuprofen and flufenamic acid solutions were adjusted to 500 mM using DMSO. [0243]

Mouse bone marrow-derived mast cells (BMMC, supplied by the Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology) were used. The cells were maintained with the addition of a culture supernatant containing rmIL-3 as a growth factor to a prepared medium.

By adding FCS (final concentration: 10% (v/v) ) , penicillin-streptomycin (final concentration: 100 Units/ml) and mercaptoethanol (final concentration: 55 μM) to RPMI1640, the medium was prepared. [0244]

Test to measure the amount of degranulation The cells were seeded to a 24-well plate (Corning Inc.) at 3 x 10⁵ cells/well using a prepared medium containing 1.0 μg/ml SPE-7. After cultivation at 37⁰C in a 5% CO₂ incubator for 4 hours, TPEN and each example compound (0.2% (v/v) DMSO), previously diluted to assay concentrations, were added, and a reaction was further carried out for 2 hours. After the cells were washed twice using Tyrode's buffer solution, they were transferred to a V-bottomed 96-well plate (Nunc) at 1 x 10⁵ cells/well, and the plate was allowed to stand at 37°C for 5 minutes. DNP-HSA, an antigen, was added at the optimum concentration, and a reaction was carried out at 37°C for 30 minutes to induce degranulation. A portion of the supernatant (released fraction) was transferred to a 96-well plate (Corning Inc.) for absorbance determination, the remaining portion of the supernatant was removed, the buffer solution for cell extraction was added, and the supernatant of the cell extract obtained (intracellular fraction) was transferred to the 96-well plate in the same manner. A freshly prepared enzyme reaction solution was added, and a reaction was carried out at 37⁰C for 1 hour. After the reaction was stopped using the reaction stopper solution, a measurement was made using a plate reader (Apollo LB911, BERTHOLD Technologies) .

Release rates were determined by the equation shown below, and IC₅₀ was calculated using KaleidaGraph (Synergy Software) . Release rate (%) = supernatant fraction / (supernatant fraction + intracellular fraction) x 100 [0245] Results 1. Measurement of degranulation inhibitory activity of each example compound

Using TPEN as the control, the inhibitory activity of each example compound was examined in the concentration range of 1 to 200 μM. The amount of degranulation was measured using β-hexosaminidase activity; the degree of degranulation is shown with the β-hexosaminidase release rate without stimulation being 0%, and the antigen-induced release rate being 100%. From FIGs. 17 and 18, compounds 1, 2, 4, 5, 6, 7 and 8 were found to have inhibitory activity. Particularly, compound 4 exhibited potent inhibitory activity at 200 μM. On the other hand, compounds 9 and 10 did not permit measurements because of turbidity when added at 200 μM (solution in 0.2% DMSO). However, both compounds exhibited inhibitory activity- equivalent to, or more than, that of TPEN, at 20 μM. [0246]

2. Comparison of the inhibitory activities of TPEN, compound 9 and compound 10

By performing simultaneous measurements using the same lot of mBMMC, the inhibitory activities of TPEN, compound 9 and compound 10 were compared in detail. FIG. 10 shows degranulation levels in terms of β-hexosaminidase release rate; FIG. 11, like FIGs. 8 and 9, shows relative degrees of degranulation. The results of calculations of the IC₅₀ and ICgo of TPEN, compound 9 and compound 10 from FIGs. 19 and 20 are shown in Table 7. [0247] Table 7

0248]

The IC₅₀ of TPEN, compound 9 and compound 10 were 4.8, 3.8, and 6.3 μM, respectively, with almost no difference in IC₅₀ observed. However, TPEN did not show an inhibitory potential of 80% or more at 20 μM, whereas the ICso of compound 9 and compound 10 were 7.4 and 11.5 μM, respectively, which were confirmed to exceed TPEN in terms of inhibitory potential.

From these results, it was suggested that compound 9 and compound 10 might be degranulation inhibitors exceeding TPEN, and promising as anti-allergic agents. [0249] 3. Inhibitory activities of ibuprofen and flufenamic acid The degranulation inhibitory activities of ibuprofen and flufenamic acid on mouse mast cells were examined. Making use of the property of being readily soluble in aqueous solutions of sodium hydrogen carbonate, the inhibitory activities of ibuprofen and flufenamic acid were examined in the absence of DMSO (FIG. 21) . At relatively high concentrations, both ibuprofen and flufenamic acid exhibited inhibitory activity. Next, taking into account the influence of the vehicle, the inhibitory activities were examined with the use of a DMSO- containing vehicle (FIG. 22) . As with the results shown in FIG. 12, both ibuprofen and flufenamic acid exhibited potent inhibitory activity at 1 inM. From these results, it was found that ibuprofen and flufenamic acid had potent degranulation inhibitory activity at high concentrations. Hence, the degranulation inhibitory activities of ibuprofen and flufenamic acid were examined in detail (FIG. 14). The IC₅₀ of ibuprofen and flufenamic acid for mouse mast cell degranulation were 107 and 95.9 μM, respectively, being about 20 times as weak as the IC₅₀ of compound 9 and compound 10.

From the above, it was shown that the degranulation inhibitory activities of compound 9 and compound 10 were much more potent than those of partial structures thereof (compound 8 (IC₅₀>100 μM) , ibuprofen and flufenamic acid per se) . [0250]

(Test Example 2)

Test to measure the amount of degranulation

As in Test Example 1, the effects of compounds 14 to 17 on mouse mast cell degranulation were examined. Compound 10 was used as the positive control. For each compound, IC₅₀ and ICgo were calculated. [0251] Results

The results are shown in FIGs. 23 and 24 and Table 8. [0252]

[0253]

All of compounds 14 to 17 dose-dependently suppressed mouse mast cell degranulation (FIGs. 23 and 24) . The IC₅₀ of compound 16 was nearly equivalent to that of compound 10 or

TPEN (Table 8) . The IC₅₀ of the other compounds were higher than that of compound 10. As stated above, TPEN did not show an inhibitory activity of 80% or more at concentrations of 20 μM or more, whereas all of compounds 14 to 17 were capable of inhibiting mouse mast cell degranulation by 80% or more.

From these results, it was suggested that compounds 14 to 17, like compound 9 and compound 10, might be potent degranulation inhibitors, and promising as anti-allergic agents.

[0254]

(Test Example 3) Influence of addition of zinc ions on the degranulation suppressive effects of compound 9 and compound 10

Preparation of reagents

By dissolving zinc sulfate to 0.1 M in purified water, previously sterilized with high-pressure steam, and diluting the concentrate solution obtained to test concentrations before measurement, zinc sulfate solutions were prepared.

That is, by dissolving l-hydroxypyridine-2-thione zinc salt to 0.1 M in DMSO, a pyrithione solution (Zn²⁺ ionophore) was prepared. The other reagents were prepared in the same manner as Test Example 1. [0255] Test to measure the amount of degranulation

As in Test Example 1, a test to measure mouse mast cell degranulation was performed. Just before adding DNP-HSA, an antigen, the zinc sulfate solution and pyrithione solution were added, and the mixture was allowed to stand at 37°C for 10 minutes .

[ 0256] Results

To determine whether or not the inhibition of mouse mast cell degranulation by compounds 9 and 10 is attributable to the chelating of Zn²⁺, the influences of addition of Zn²⁺ and the ionophore (pyrithione) on the degranulation inhibitory activities of TPEN, compound 9 and compound 10 were examined. As a result, it was confirmed that for all of TPEN, compound 9 and compound 10, the degranulation inhibitory activity decreased proportionally to the amounts of Zn²⁺ and • , ionophore added (FIG. 25) . From this, it was found that all of TPEN, compound 9 and compound 10 inhibited mast cell degranulation by chelating Zn²⁺.

[0257]

(Test Example 4)

Effects of test substances on human mast cell degranulation Preparation of reagents

By diluting 1 mg/ml human IgE (YAMASA CORPORATION) with a medium to test concentrations, sensitizers were prepared. By diluting goat IgG fraction to human IgE

(epsilon__chain) (Cappel) with Tyrode's buffer solution before measurement, an inducer was prepared.

The other reagents were prepared in the same manner as Test Example 1. [0258] Human peripheral blood-derived mast cells, previously differentiated by adding SCF, IL-3 and IL-β to human monocytes, were used for the test.

[0259]

Test to measure the amount of degranulation The cells were seeded to a 24-well plate (Corning Inc.) at 1.5 x 10⁵ cells/well using a medium containing 1.0 μg/ml hlgE, a sensitizer. After cultivation at 37⁰C in a 5% CO₂ incubator for 4 hours, TPEN or each example compound (0.2% (v/v) DMSO) , previously diluted to test concentrations, was added, and a reaction was further carried out for 2 hours. After the cells were washed twice using Tyrode's buffer solution, they were transferred to a V-bottomed 9β-well plate (Nunc) at 0.5 x 10⁵ cells/well, and the plate was allowed to stand at 37°C for 5 minutes. Anti-IgE, an antigen, was added at the optimum concentration, and a reaction was carried out at 37⁰C for 30 minutes to induce degranulation. A portion of the supernatant (released fraction) was transferred to a 96- well plate for absorbance determination (Corning Inc.), the remaining portion of the supernatant was removed, the buffer solution for cell extraction was added, and the supernatant of the cell extract obtained (intracellular fraction) was transferred to the 9β-well plate in the same manner. The enzyme reaction liquid was added, and a reaction was carried out at 37⁰C for 1 hour. After the reaction was stopped using the reaction stopper solution, a measurement was made using a plate reader (Apollo LB911, BERTHOLD Technologies) .

Release rates were determined by the equation shown below, and IC₅₀ was calculated using KaleidaGraph (Synergy Software) . Release rate (%) = supernatant fraction / (supernatant fraction + intracellular fraction) x 100

[0260]

Results

An investigation was performed to determine whether example compounds could inhibit human mast cell degranulation, like that of mouse mast cells (FIG. 26) . As a result, it was found that all of TPEN, compound 9 and compound 10 had degranulation inhibitory activity on human mast cells. At 20 μM, compound 10 exhibited the highest inhibitory activity, inhibiting degranulation by about 50%; the inhibitory activity thereof exceeded that of TPEN. On the other hand, the inhibitory activity of compound 9 was about 20%.

From the results shown above, it was suggested that compound 9 and compound 10 might be degranulation suppressants that are also effective on human mast cells.

[0261]

(Test Example 5)

Effects of example compounds on human mast cell degranulation

As in Test Example 4, the effects of example compounds on human mast cell degranulation were investigated. The effects of prednisolone, tranilast, and DSCG (cromolyn) were also investigated.

[0262]

Results 1. Degranulation suppressive effects of compound 9, compound

10 and compound 17

All of compound 9, compound 10 and compound 17 dose- dependently suppressed human mast cell degranulation (FIG. 27) .

In particular, compound 9 and compound 10 exhibited the highest activity, the IC₅₀ thereof being about 10 μM (Table 9) .

TPEN exhibited only weak activity. [ 0263 ] Table 9

[0264] 2. Comparison of degranulation suppressive effects of various drugs

For prednisolone and DSCG (cromolyn) , no suppressive activity on human mast cell degranulation was observed (FIG.

28). Tranilast exhibited weak suppressive activity, the IC₅₀ thereof being about 1000 μM (FIGs. 28 and 29, Tables 10 and 11)

Compared to tranilast, compound 9 and compound 10 were about

100 times more active; it was shown that compound 9 and compound 10 were highly promising as degranulation suppressants . [0265]

Table 10

[ 0266 ] Table 11

[0267]

(Test Example 6)

Investigation of anti-allergic action of test substance on IgE-dependent DNFB-induced mouse pruritis model (1) Methods

Fifty female Balb/cAnNCrlCrlj mice at 5 weeks of age were purchased and acclimated for 7 days, after which they were grouped on the basis of measurements of auricle skin thickness (Table 12) . Anti-DNP-mouse IgE antibody (10 g/animal) was administered intravenously to the caudal vein to achieve passive sensitization; 24 hours after the sensitization, auricle scratching behavior was induced by applying DNFB to the right auricle (0.75% DNFB applied at 5 μL/animal) . 24 hours before, 3 hours before and 1 hour before inductive administration, a test substance was applied to the right auricle (5 μL/animal) . The test substance used was compound 10, By counting auricle scratches, the anti-allergic action of the test substance was evaluated. Scratches were counted over 1 hour from 30 minutes after the induction. For data on scratches, a two-group comparison was made between the control group and the 1% prednisolone acetate group; next, multiple comparisons were made among the control group, the 0.3% compound 10 group, the 1% compound 10 group, and the 3% compound 10 group. That is, with respect to the data on scratches, Mann-Whitney U-test was performed for the 2-group comparison. Kruskal Wallis H-test was performed for the multiple comparisons. For both analytical procedures, the level of significance was set at 5% (P<0.05) for one side. [ 0268 ] Table 12

[0269] Results Auricle scratching behavior was induced by applying DNBF to the right auricle, and scratches were counted over 1 hour from 30 minutes thereafter; as a result, the number of scratches was 129 for the control group, 120 for the 0.3% compound 10 group, 86 for the 1% compound 10 group, 117 for the 3% compound 10 group, and 100 for the 1% prednisolone acetate group; the 0.3% compound 10 group and the 3% compound 10 group showed values equivalent to the value for the control group. By contrast, in the 1% compound 10 group and the 1% prednisolone acetate group, the number of scratches tended to decrease (FIG. 30) . [0270]

(Test Example 7)

Investigation of anti-allergic action of test substance on IgE-dependent DNFB-induced mouse pruritis model (2) Methods

An experiment was performed in the same manner as Test Example 6, except that the animals were grouped as shown in the Table below; on the basis of the number of auricle scratches, the anti-allergic action of a test substance was evaluated. The test substance used was compound 9. [0271] Table 13

[0272] Results

Auricle scratching behavior was induced by applying DNBF to the right auricle, and scratches were counted over 1 hour from 30 minutes thereafter; as a result, the number of scratches was 127 for the control group, 105 for the 1% compound 9 group, 101 for the 3% compound 9 group, 143 for the 10% N-acetylcarnosine group, and 122 for the 1% prednisolone acetate group; the 10% N-acetylcarnosine group showed a higher value than the value for the control group, and the 1% prednisolone acetate group showed a value equivalent to the value for the control group. By contrast, in the 1% compound 9 group and the 3% compound 9 group, the number of scratches tended to decrease (FIG. 31) . [0273]

(Test Example 8)

Investigation of influence of repeated application of test substance to auricle skin using BALB/c mice Methods

Twenty female Balb/cAnNCrlCrlj mice at 5 weeks of age were purchased and acclimated for 7 days, after which they were grouped on the basis of measurements of auricle skin thickness (Table 14) . A test substance was repeatedly applied to the right auricle for 4 weeks. During this period, auricle skin thickness was measured at a frequency of once/week, and the presence or absence of the influence of repeated application of the test substance on the skin was investigated.

After completion of the measurement of auricle skin ' thickness following the repeated administration for 4 weeks (Day 28), right auricles of two animals in each group were collected, immersed and fixed in 10% neutrally buffered formalin. Using the collected auricles, histopathological specimens (HE stained, toluidine blue-stained and non-stained) were prepared and examined pathologically. [0274] Table 14

[0275] Results

In the compound 10 application group and the prednisolone acetate application group, auricle skin thickness showed almost no change, as in the control group. By contrast, in the TPEN application group, remarkable thickening of the auricle skin was observed (FIG. 32) .

In the pathologic examination, in the TPEN application group, crust formation, epidermal thickening, and infiltration of inflammatory cells, mainly neutrophils, and epidermal ulceration were remarkably observed in the center and sides of the auricle. These changes were more severely observed on the outer surface of the ear. In the prednisolone acetate application group, crust formation in a small area was observed at one site in each animal. By contrast, in the compound 10 application group, like in the control group-, no abnormalities were observed in the center and sides of the auricle (Table 15) .

From these results, it was shown that TPEN had an inflammatory adverse reaction on the skin, and that compound 10 did not have this adverse reaction.

[ 0277 ]

(Test Example 9)

Effects of example compounds on cytokine production by mouse mast cells Mouse bone marrow-derived mast cells (BMMC, supplied by the Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology) were used. The cells were seeded to a 96-well plate at 1 x 10⁵ cells/well using a prepared medium containing 10.0 μg/ml SPE-7. After overnight cultivation at 37°C in a 5% CO₂ incubator, TPEN and each example compound (0.2% (v/v) DMSO), previously diluted to assay concentrations, were added, and a reaction was further carried out for 2 hours. After the plate was washed twice using Tyrode's buffer solution, DNP-HSA (5 ng/ml) or PMA (50 ng/ml) , an antigen, was added at the optimum concentration, and the cells were incubated at 37°C for 3 hours. The supernatant was recovered, and mIL-β concentrations were measured by ELISA (Biosource) . [0278] Results

All of TPEN, compound 10 and compound 17 dose-dependently suppressed IL-6 production by mouse mast cells upon stimulation with the antigen (DNP-HSA) or PMA- (Table 16) . [0279]

[0280]

(Test Example 10 ) Effect on histamine release from human basophil Method

Red blood cell was removed from human peripheral blood by density gradient centrifigation using a methylcellulose solution to separate leukocytes. The obtained leukocytes were washed with PBS to remove contaminating plasma components. Compound 10 was added to the leukocytes and, ^'after incubation for 2 hr, anti-IgE antibody was added and the cells were incubated for 40 min. Since basophil alone has IgE receptors from among human leukocytes, addition of IgE induces activation of basophil and histamine release by degranulation. After incubation, blood cell was separated from the supernatant by centrifugation, and each fraction was stabilized by addition of 0.2N HClO₄. The supernatant of each sample was applied to a histamine separation column (Shimpack VP-ODS, Shimazu) and the released histamine was quantified. [0281] Results

As shown in FIG. 33, compound 10 suppressed histamine release from basophil in a dose-dependent manner. Since the histamine release from basophil is known to contribute to the third phase of allergy reaction (very late response) , the compound of the present invention is suggested to suppress the third phase of the allergy reaction. [0282] (Test Example 11)

Effect of test compounds on atopic dermatitis model using NC mouse

Method

Seven-week-old female NC mice were reared for acclimation for 7 days, and then used for the experiment. The thickness of the ear of each animal was measured before sensitization on the day of the start of dermatophagoides antigen sensitization (Day 0), and the mice were divided into 4 groups such that the average value of the thickness was equivalent. [ 0283] Table 17

[0284] Following the sensitization schedule (FIG. 34), NC mice were sensitized by intradermal administration of a Dermatophagoides antigen solution to the right auricle twice a week for 2 weeks. Dermatophagoides antigen solution (1 mg/mL) was prepared by adding injectable physiological saline (10 iuL) to Dermatophagoides antigen (Mite Extract-Dp: 10 mg protein) . This was dispensed to a tube by 500 μL and cryopreserved (- 4O⁰C), and used after warming to room temperature when in use. For sensitization, Dermatophagoides antigen solution (5 μL) was intradermally administered to the right auricle of the NC mice under an ether anesthesia. [0285]

A vehicle control and a test substance were intraperitoneally administered twice a day for 15 consecutive days, and tacrolimus hydrate (Protopic) ointment (positive control substance) was coated on the right auricle according to the same administration schedule. The vehicle control was obtained by adding injectable physiological saline such that the final concentrations of DMSO and Cremophor were 10% and 2%, respectively. Compound 10 was dissolved in DMSO to a final concentration of 10 mg/mL, and TPEN was dissolved in DMSO to a final concentration of 1 mg/mL. Cremophor and injectable physiological saline were added to give each test substance solution. The test substance solution was prepared such that the vehicle composition was the same as that of the vehicle control liquid. The control vehicle solution and each test compound solution were intraperitoneally administered to the mice twice a day for 15 days from the day of the start of the sensitization to the day of the completion of the sensitization (Day 0 - Day 14) at a dose of 10 mL/kg body weight. The dose was calculated from the measurement data of each body weight measurement day (Days 0, 4, 7, 11 and Day 14) Tacrolimus hydrate ointment was taken in a given amount (lO±l mg equivalent amount of white petrolatum) using a spatula and administered by coating on the right auricle according to a similar administration schedule. [0286]

The effect of the test coumpounds on the chronic dermatitis reaction (atopic dermatitis-like symptom) induced with the Dermatophagoides antigen was evaluated by measuring the ear thickness and scoring auricular skin symptoms at 24 hr after the intradermal administration of Dermatophagoides antigen and comparing them with those of the vehicle control group. The measurement method of each evaluation item was as ^J follows .

(1) Measurement of ear thickness

The thickness of the right auricle of the mice was measured under ether anesthesia during grouping and at about 24 hr after Dermatophagoides antigen sensitization. For measurement, R-I type Upright gauge (PEACOCK) was used, and the measurement was performed on Days 0, 1, 5, 8, 12 and Day 15 (FIG. 34) . In addition, the ear thickening value was calculated by dividing the ear thickness value measured on each measurement day by the ear thickness value measured on Day 0 (day of grouping) .

(2) Scoring of auricular skin symptom

The right auricular skin symptoms were macroscopically observed at 24 hours after sensitization with Dermatophagoides antigen, and the intensity of the symptoms was evaluated for ^■ the following 5 items by scoring into no symptom (0 point) , mild (1 point) , moderate (2 points) and severe (3 points) . The scoring was performed according to the schedule of Days 1, 5, 8, 12 and Day 15 (FIG. 34) . A: flare -flush

B: crust -epidermis detachment C: hemorrhage -clot D: hardening E: ear swelling [0287]

After measurement of the evaluation items on the final day of experiment, the blood (about 75 μL) was collected from the tail vein of the mice of the vehicle control group, compound 10 group and Tacrolimus hydrate ointment group. Then, the plasma was separated from the collected blood, and preserved at -40⁰C. From among the preserved plasma, the plasma of the vehicle control group and compound 10 group was measured for the plasma GOT, GPT, ALP and BUN according to the following method and using Fuji Dri-Chem 3500V for animal (Fuji Film) .

1) GOT: oxaloacetic acid decarboxylase-POP-POD-leuco dye method

2) GPT: POP-POD-leuco dye method

3) ALP: p-nitrophenyl phosphate substrate method

4) BUN: urease-BCG indicator method [0288]

On the final day of experiment, two individual mice showing average skin symptoms were selected from each of the vehicle control group, compound 10 group and Tacrolimus hydrate ointment group, and sacrificed by exsanguination by abdominal aorta ablation under ether anesthesia. The right auricle was removed, fixed with 10% neutrally bufferized aqueous formalin solution, and embedded in paraffin. Slices (4 μm) were subjected to hematoxylin-eosin (HE) staining, toluidine blue (TB) staining for mast cell detection, and Luna staining for eosinophil detection, and microscopically examined. The grades of pathological observation were as follows : Grade 0 : no abnormality Grade 1: minor Grade 2: mild Grade 3 : medium Grade 4 : high [0289] Using the body weight and ear thickening value measured on each measurement day, F test was performed between the vehicle control group and each test substance group or Tacrolimus hydrate ointment group. When a significant difference was not observed, the difference in the average values between two groups was determined by Student's t-test, and when a significant difference was observed, the difference was determined by Aspin-Welch' s t-test. As for the auricular skin symptom score, the difference in the average values between the two groups of vehicle control group and each test substance group or Tacrolimus hydrate ointment group was determined by Mann-Whitney's U test. As for the plasma GOT, GPT, ALP and BUN after the completion of the experiment, F test was performed between the vehicle control group and compound 10 group. When a significant difference was not observed, the difference in the average values between two groups was determined by Student's t-test, and when a significant difference was observed, the difference was determined by Aspin-Welch' s t-test. In any analysis, P < 0.05 was evaluated as the presence of a significant difference. No statistical analysis was performed for the measured auricular thickness. [0290] Results (1) Body weight

In all measurement days, no significant difference was observed in the body weights of compound 10 group and Tacrolimus hydrate ointment group as compared to the vehicle control group. On the other hand, average body weight of the TPEN group continued to decrease from the start of the administration, and the difference became significant from Day 7 as compared to the vehicle control group (FIG. 35) . Throughout the test, 5 animals in total of the TPEN group died (one on Day 11, three on Day 12, one on Day 15) . [0291]

(2) Ear thickness

In the compound 10 group, TPEN group and Tacrolimus ^■ hydrate ointment group, ear thickening was significantly suppressed as compared to the vehicle control group at all measurement points from Day 5 to the final measurement day (Day 15) (FIG. 36) . [0292]

(3) Auricular skin symptom

In the compound 10 group, TPEN group and Tacrolimus hydrate ointment group, the total value of all 5 items of auricular skin symptom score (hereinafter: score) was significantly suppressed as compared to the vehicle control group at Day 5 and Day 8 (FIG. 37) . By comparison of each scoring item, compound 10 group (Day 5) and Tacrolimus hydrate ointment group (Day 5 and Day 8) showed a significant suppression in "A: flare -flush", Tacrolimus hydrate ointment group (Day 12) showed a significant suppression in ^λλB : crust- epidermis detach", and compound 10 group (Day 15) showed a significant suppression in ^ΛλD: hardening", as compared to the vehicle control group (FIG. 38) . Comparison of each scoring item was not performed for the TPEN group. Particularly, since compound 10 showed a superior effect on hardening, it was suggested that the compound of the present invention can treat skin hardening associated with atopic dermatitis. [0293]

(4) Blood biochemistry

On the final experiment day (Day 15) , using the plasma collected from the animals of the vehicle control group and compound 10 group, GOT, GPT, ALP and BUN were measured (ALP was unmeasurable for one animal of the vehicle control group due to hemolysis) . As a result, a significant difference was not observed between the vehicle control group and compound 10 group in all measurement items (FIG. 39) . [0294]

(5) Pathological finding

The results are shown in the following Table. [0295] Table 18

[0296]

A) Vehicle control group

Severe epidermal erosion, crust formation and medium level of hyperplasia were observed in the center of the lateral surface of the auricle. Severe inflammatory cell infiltration mainly by eosinophils (Luna positive cells) , and severe hyperplasia of the connective tissue were observed in the lamina propria of the same region. Mild or medium infiltration of mast cells (metachromasia by TB staining) was observed in the upper and lower part of the auricle, with tendency toward decrease in the erosion site. While cell debris was found in the erosion site, eosinophils infiltration was not observed. A medium level of inflammatory cell infiltration mainly of eosinophils (Luna stain positive) , infiltration of mast cells, mild hyperplasia in the connective tissue, and mild hyperplasia in epidermis were observed in lamina propria of the medial surface of the auricle. Besides these, brown dye deposition considered to show hemosiderin was observed in the lamina propria. In addition, mild atrophy of musculus cutaneus was found occasionally. [0297] ^■

B) Compound 10 group

Mild or medium epidermal erosion, crust formation and hyperplasia were observed in the center of the lateral surface of the auricle. Medium level of inflammatory cell infiltration mainly of eosinophils (Luna stain positive) , and medium hyperplasia of the connective tissue were observed in the lamina propria of the same region. Mild or medium infiltration of mast cells (metachromasia by TB staining) was observed in the upper and lower part of the auricle, with tendency toward decrease in the erosion site. While cell debris was found in the erosion site, eosinophils infiltration was not observed. A mild level of inflammatory cell infiltration mainly of eosinophils (Luna stain positive) , mild hyperplasia in the connective tissue, and mild hyperplasia in epidermis were observed in lamina propria of the medial surface of the auricle. Besides these, brown dye deposition considered to show hemosiderin was observed in the lamina propria in the upper part of the auricle. In addition, mild atrophy of musculus cutaneus was found occasionally. By comparison with the changes in the vehicle control group, qualitatively same changes were observed; however, the level was mostly weaker.

[0298]

C) Tacrolimus hydrate group Although epidermal erosion was not observed in the center of the lateral surface of the auricle, mild crust formation was observed in one case out of two. Mild or medium epidermal hyperplasia was observed. Mild or medium inflammatory cell infiltration mainly by eosinophils (Luna stain positive) , medium level of infiltration of macrophage, and mild hyperplasia of the connective tissue were observed in the lamina propria of the same region. A mild level of inflammatory cell infiltration mainly of eosinophils (Luna stain positive) , mild hyperplasia in the connective tissue, and mild hyperplasia in epidermis were observed in lamina propria of the medial surface of the auricle. Besides these, brown dye deposition considered to show hemosiderin was observed in the lamina propria in the upper part of the auricle. In addition, mild atrophy of musculus cutaneus was found occasionally. By comparison with the changes in the vehicle control group or compound 10 group, qualitatively same changes were observed in the Tacrolimus hydrate treatment group; however, the level was mostly weaker.

[0299]

By this study, it was clarified that TPEN drastically decreases the body weight and shows toxicity as evidenced by the death of a part of the animals and the like. In contrast, compound 10 did not show such toxicity as shown by TPEN, even after continuous intraperitoneal administration, and showed a suppressive effect on ear thickening and auricular skin symptom scores.

From the foregoing results, the compound of the present invention showed a suppressive effect on chronic dermatitis reaction (atopic dermatitis-like symptoms) , and was suggested to be a safe compound. [0300]

(Test Example 11)

Effect on eotaxin production in human respiratory epithelial cells Method

It is known that chemokine is produced in excess from epithelial cells under allergic conditions, and human respiratory epithelial cell (e.g. BEAS-2B) produces eotaxin, one kind of chemokine, in vitro by IL-13 stimulation. Thus, the effect of the compound of the present invention on the eotaxin production was evaluated.

BEAS-2B was plated on a 24 well plate (IWAKI) at 2 x 10⁵ cells/well (300 μl) , and cultured overnight. Various concentrations of compound 10 were added, and the cells were cultured for 1 hr. Then, the supernatant was removed, the cells were washed twice with PBS, and 300 μl of a medium (DMEM/F12 containing 10% (v/v) FBS, 100 units/ml penicillin- streptomycin, 10 mM HEPES and 0.2% (w/v) sodium hydrogen carbonate) supplemented with rhIL-13 (50 ng/ml) was added to stimulate the cells. After culture in a CO2 incubator for 48 hr, the supernatant was recovered and centrifuged at 1000 rpm for 3 min, and eotaxin amount in the supernatant was quantified using Eotaxin ELISA Kit (R&D) . [0301] Results

As shown in FIG. 40, compound 10 inhibited the eotaxin production by BEAS-2B in a dose-dependent manner. Thus, it was suggested that the compound of the present invention can suppress eotaxin production in human respiratory epithelial cells. [0302]

(Test Example 12) Effect on asthma model Method

On Days 1, 3 and 5, 0.5 μg of recombinant human IL-13 (Sigma) or PBS (negative control) was intratracheally injected by dropwise addition to A/J mouse to induce asthma (Am. J. Respir. Crit. Care Med., vol. 173, pp. 1216-1221, 2006). Every day from Day 1 to Day 5, compound 10 (100 mg/kg) or vehicle was intraperitoneally administered twice a day.

On Day 6, airway responsiveness to acetylcholine and the number of neutrophils and eosinophils in the bronchoalveolar lavage fluid (BALF) were measured. [0303]

Results

As shown in FIG. 41, compound 10 suppressed the airway responsiveness to acetylcholine increased by IL-13, and decreased the number of neutrophils and eosinophils in BALF. From these results, it was suggested that the compound of the present invention can treat asthma.

[0304]

(Test Example 13)

Effect on PCA (oral administration) Method

0.5 mg of IgE was intradermally injected into the ear of a mouse to sensitize the mouse. After rearing for one night, various doses of compound 10 were orally administered and, 15 min or 120 nαin later, DNP-HAS was administered together with Evans blue to induce an allergy reaction. 15 min later, the mouse was sacrificed, the sensitized ear was separated, and the infiltrated Evans blue was quantified.

[0305] Results The oral administration of compound 10 suppressed PCA reaction in a dose-dependent manner (FIG. 42, left panel) . Both at 15 min and 120 min after the administration, compound 10 suppressed a PCA reaction (FIG. 42, right panel) . From these results, it was shown that the compound of the present invention can suppress PCA, and the compound of the present invention can exhibit a significant pharmacological effect by oral administration. [0306] (Test Example 14)

Effect on inflammatory bowel disease (IBD) (oral administration)

Method

5% DSS was added to drinking water to induce inflammatory enteritis in mouse (CD-I, five-week-old) . 3 mg/ml of compound 10, vehicle (0.5 % Cremophore in pure water) or 10 mg/ml Predsisolone was orally administered to the mouse once a day for 8 days . On the next day from the completion of the administration, the enteric length was measured. It is known that the enteric length becomes shorter due to IBD.

[0307]

Results

As shown in FIG. 43, oral administration of compound 10 suppressed a decrease in the enteric length. Prednisolone did not show a significant effect. From these results, it was suggested that the compound of the present invention can treat

IBD.

[0308]

(Test Example 15) Evaluation of toxicity on conjunctiva-derived cells

Method

Conjunctiva obtained from a 66-year-old male during conjunctiva squamous cell carcinoma resection was cultivated to give conjunctiva-derived cells. E6E7 gene of human papilloma virus 16 was introduced into the cells by a retrovirus vector method to establish two conjunctiva-derived cell lines (No. 63-1 and No. 63-2) .

80% Confluent cells were detached with trypsin, and prepared to IxIO⁵ cells/ml with a medium (DMEM/F-12 containing 15% FBS) . The cell suspension was passaged by 100 μl in a 96 well plate (Greiner) . The next day, the medium was completely suctioned with an aspirator, a medium containing TPEN (0.1 - 1 μM) or compound 10 (0.1 - 1 μM) was added by 100 μl, and the cells were cultured for 3 days. As the vehicle, a medium containing 0.2% DMSO was used.

3 days later, CellTiter 96^(R) AQueous One Solution Cell Proliferation Assay (Promega, Madison, U.S.A.) was added by 20 μl to the cultured medium, the mixture was reacted at 37⁰C, CO2 5% for 2 hr, and the absorbance at 495 ran was measured by microplate reader LS Plate manager 2001 (Wako, Japan) . [0309] Results

As shown in FIG. 44, even when two kinds of cell lines were treated with 0.1 μM or 1 μM TPEN, the absorbance did not change much from that of the medium. When treated with 10 μM TPEN, the absorbance decreased drastically. The absorbance of 10 μM TPEN treatment group was equal to that when an assay reagent was added to a medium free of cells. As a result of microscopic observation, cell adhesion was not observed.

Therefore, 10 μM of TPEN was considered to have induced cell death rather than the suppression of cell proliferation.

On the other hand, the absorbance did not vary much from that of the medium even when treated with 0.1 μM and 1 μM of compound 10. However, when treated with 10 μM of compound 10, the absorbance tended to decrease somewhat. Microscopic observation revealed cell adhesion unlike TPEN.

From these results, it was suggested that the cytotoxicity of the compound of the present invention was remarkably reduced as compared to TPEN, and that the compound can be formulated into safe eye drops. [0310]

(Test Example 16) Test to measure the amount of degranulation

As in Test Example 1, the effects of compounds 3, 11-13 and 18-27 on mouse mast cell degranulation were examined. For each compound, IC₅₀ were calculated. [0311] Results

The results are shown in Table 19. [0312] Table 19

[0313]

All of compounds 13 and 18-20, 22-27 dose-dependently suppressed mouse mast cell degranulation.

From these results, it was suggested that compounds 3, 11-13 and 18-27 might be potent degranulation inhibitors, and promising as anti-allergic agents.

[0314]

(Test Example 17)

Effect on PCA Method

0.5 mg of IgE was intradermally injected into the ear of a mouse to sensitize the mouse. After rearing for one night, compounds 1, 10, 17 and 20 were intravenously injected at a dose of 30 mg/kg and, 15 min later, DNP-HAS was administered together with Evans blue to induce an allergy reaction. 15 min later, the mouse was sacrificed, the sensitized ear was separated, and the infiltrated Evans blue was quantified. [0315] Results

The relative amounts of infiltrated Evans blue to that with administration of vehicle as 100 are shown in Table 20. [0316] Table 20

[0317]

As shown in Table 20, compounds 1, 10, 17 and 20 suppressed the PCA reaction. These results show that the compound of the present invention is useful as an anti-allergic agent.

[0318]

(Test Example 18)

Test to measure the amount of degranulation

As in Test Example 1, the effects of compound 22 on mouse mast cell degranulation were examined and IC50 of compound 22 was calculated.

[0319]

Results As is shown in FIG. 45, compound 22 potently suppressed mouse mast cell degranulation in a dose-dependent manner

(IC₅₀=8 (JJM)) .

From these results, it was suggested that compound 22 might be a potent degranulation inhibitor, and promising as an anti-allergic agent.

[0320]

(Test Example 19)

Effect on PCA Method

As in Test Example 17, the effects of compound 22 on PCA reaction were examined, and the infiltrated Evans blue was quantified.

[0321] Results

As shown in FIG. 45, compound 22 suppressed the PCA reaction. These results show that the compound of the present invention is useful as an anti-allergic agent.

Industrial Applicability

[0322]

The compound of the present invention is highly useful as an agent for the prophylaxis or treatment of allergic diseases such as allergic dermatitis and the like and inflammatory diseases, since the compound has an activity to suppress degranulation in mast cells and the like, as well as cytokine production.

This application is based on a patent application No.

2007-341361 filed in Japan (filing date: December 28, 2007), the contents of which are incorporated in full herein by this reference.

Claims

1. A compound represented by the formula (I) :

wherein

L is an alkylene group;

Z is an optionally substituted hydrocarbon group or an optionally substituted heterocyclic group; and R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, or a salt thereof.

2. The compound of claim 1, wherein L is a Ci_g alkylene group.

3. The compound of claim 1, wherein Z is an optionally substituted alkyl group; an optionally substituted aryl group; an optionally substituted aralkyl group; or an optionally substituted heterocyclic group containing at least one hetero atom selected from an oxygen atom and a nitrogen atom.

4. The compound of claim 1, which is 2-[N,N-bis (2-pyridylmethyl) amino] ethyl 2- (4- isobutylphenyl) propionate,

2-[N,N-bis (2-pyridylmethyl) amino] ethyl 2-(3- trifluoromethylphenylamino) benzoate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl acetate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl isobutyrate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl phenylacetate,

2- [N, N-bis (2-pyridylmethyl) amino] ethyl 3-phenylpropionate, 2-[N,N-bis (2-pyridylmethyl) amino] ethyl benzoate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl isonicotinate,

5. A degranulation suppressant comprising a compound represented by the formula (I) :

wherein

L is an alkylene group;

X is a bond, -O-, -S- or -CO-;

Y is a bond, -NR³- or -CO-;

R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N,N,N' ,N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof.

6. The suppressant of claim 5, wherein X is -0-, Y is -CO-, and Z is an optionally substituted hydrocarbon group or an optionally substituted heterocyclic group.

7. The suppressant of claim 5, wherein X is -0-, Y is a bond, and Z is an optionally substituted hydrocarbon group.

8. The suppressant of claim 5, wherein the compound represented by the formula (I) is

N,N-bis (2-pyridylmethyl) glycine,

N,N-bis (2-pyridylmethyl) glycylglycine tert-butyl ester,

N, N-bis (2-pyridylmethyl) glycylglycine, N,N-bis (2-pyridylmethyl) glycyl-L-phenylalanine tert-butyl ester,

N,N-bis (2-pyridylmethyl) glycyl-L-phenylalanine,

2- [N,N-bis (2-pyridylmethyl) amino] -1-morpholinoethanone,

2- [N, N-bis (2-pyridylmethyl) amino] -N' -phenethylacetamide,

2- [N,N-bis (2-pyridylmethyl) amino] ethanol, 2- [N, N-bis (2-pyridylmethyl) amino] ethyl 2- (4- isobutylphenyl) propionate,

2- [N, N-bis (2-pyridylmethyl) amino] ethyl acetate, 2- [N,N-bis (2-pyridylmethyl) amino] ethyl isobutyrate,

2- [N, N-bis (2-pyridylmethyl) amino] ethyl phenylacetate,

2- [N,N-bis (2-pyridylmethyl) amino] ethyl 3-phenylpropionate,

2- [N, N-bis (2-pyridylmethyl) amino] ethyl benzoate,

2- [N, N-bis (2-pyridylmethyl) amino] ethyl isonicotinate, or 2- [N,N-bis (2-pyridylmethyl) amino] ethyl furan-2-carboxylate.

9. The suppressant of claim 5, wherein the compound represented by the formula (I) is

N, N-bis (2-pyridylmethyl) -beta-alanine, N-{2- [2-N, N-bis (2-pyridylmethyl) amino-ethoxymethyl] phenyl} -3- trifluoromethylphenylamine,

N- (2-benzyloxyethyl) -N, N-bis (2-pyridylmethyl) amine, 3- [N, N-bis (2-pyridylmethyl) amino] -N-phenyl-propionamide, N- (2-methoxyethyl) -N, N-bis (2-pyridylmethyl) amine, 2-{2- [N, N-bis (2-pyridylmethyl) amino] ethoxy}ethanol, or N- (3-methoxypropyl) -N, N-bis (2-pyridylmethyl) amine.

10. An agent for the prophylaxis or treatment of an allergic disease, comprising a compound represented by the formula (I) :

wherein

L is an alkylene group/ X is a bond, -0-, -S- or -CO-; Y is a bond, -NR³- or -CO-;

11. The agent of claim 10, wherein X is -0-, Y is -CO-, and Z is an optionally substituted hydrocarbon group or an optionally substituted heterocyclic group.

12. The agent of claim 10, wherein X is -0-, Y is a bond, and Z is an optionally substituted hydrocarbon group.

13. The agent of claim 10, wherein the compound represented by the formula (I) is N,N-bis (2-pyridylmethyl) glycine,

N,N-bis (2-pyridylmethyl)glycylglycine tert-butyl ester,

N,N-bis (2-pyridylmethyl) glycylglycine,

N, N-bis (2-pyridylmethyl) glycyl-L-phenylalanine tert-butyl ester,

N,N-bis (2-pyridylmethyl) glycyl-L-phenylalanine, 2- [N, N-bis (2-pyridylmethyl) amino] -1-morpholinoethanone, 2- [N,N-bis (2-pyridylmethyl) amino] -N' -phenethylacetamide, 2- [N,N-bis (2-pyridylmethyl) amino] ethanol, 2-[N,N-bis (2-pyridylmethyl) amino] ethyl 2- (4- isobutylphenyl) propionate, 2- [N, N-bis(2-pyridylmethyl) amino] ethyl 2- (3- trifluoromethylphenylamino)benzoate, 2- [N, N-bis (2-pyridylmethyl) amino] ethyl acetate, 2- [N, N-bis (2-pyridylmethyl) amino] ethyl isobutyrate, 2- [N,N-bis (2-pyridylmethyl) amino] ethyl phenylacetate, 2- [N,N-bis (2-pyridylmethyl) amino] ethyl 3-phenylpropionate, 2-[N,N-bis (2-pyridylmethyl) amino] ethyl benzoate, 2- [N, N-bis (2-pyridylmethyl) amino] ethyl isonicotinate, or 2- [N, N-bis (2-pyridylmethyl) amino] ethyl furan-2-carboxylate.

14. The agent of claim 10, wherein the compound represented by the formula (I) is

N, N-bis (2-pyridylmethyl) -beta-alanine,

N-{2- [2-N,N-bis (2-pyridylmethyl) amino-ethoxymethyl] phenyl} -3- trifluoromethylphenylamine, N- (2-benzyloxyethyl) -N, N-bis (2-pyridylmethyl) amine,

3- [N, N-bis (2-pyridylmethyl) amino] -N-phenyl-propionamide, N- (2-methoxyethyl) -N, N-bis (2-pyridylmethyl) amine, 2-{ 2- [N, N-bis (2-pyridylmethyl) amino] ethoxy}ethanol, or N- (3-methoxypropyl) -N, N-bis (2-pyridylmethyl) amine.

15. The agent of claim 10, wherein the allergic disease is allergic dermatitis.

16. A cytokine production suppressant comprising a compound represented by the formula (I) :

wherein

L is an alkylene group; X is a bond, -0-, -S- or -CO-; Y is a bond, -NR³- or -CO-;

17. An agent for the prophylaxis or treatment of an inflammatory disease, comprising a compound represented by the formula (I) :

wherein L is an alkylene group;

X is a bond, -0-, -S- or -CO-;

Y is a bond, -NR³- or -CO-;

Z is an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group or OH; and R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N, N,N' ,N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof.

18. A compound represented by the formula (I) :

wherein L is an alkylene group;

X is a bond, -0-, -S- or -CO-;

Y is a bond, -NR³- or -CO-;

Z is an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group or OH; and R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N, N, N' , N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof, for use for suppressing degranulation.

19. A compound represented by the formula (I)

wherein

Z is an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group or OH; and R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N, N, N' , N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof, for use for prophylaxis or treatment of an allergic disease.

20. A compound represented by the formula (I) :

L is an alkylene group;

X is a bond, -0-, -S- or -CO-;

Y is a bond, -NR³- or -CO-; ' Z is an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group or OH; and

R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N, N, N' ,N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof, for use for suppressing cytokine production.

21. A compound represented by the formula (I) :

wherein

R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N, N,N' ,N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof, for use for prophylaxis or treatment of an inflammatory disease.

22. A method of suppressing degranulation in a mammal, comprising administering, to said mammal, an effective amount of a compound represented by the formula (I) :

wherein

L is an alkylene group; X is a bond, -0-, -S- or -CO-; Y is a bond, -NR³- or -CO-; Z is an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group or OH; and R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom, wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N, N, N' , N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof.

23. A method of preventing or treating an allergic disease in a mammal, comprising administering, to said mammal, an effective amount of a compound represented by the formula (I) :

wherein

L is an alkylene group; X is a bond, -0-, -S- or -CO-;

Y is a bond, -NR³- or -CO-;

24. A method of suppressing cytokine production in a mammal, comprising administering, to said mammal, an effective amount of a compound represented by the formula (I) :

wherein

R¹ and R² are the same or different and each is an optionally substituted alkyl group, a halogen atom or a hydrogen atom wherein R³ is an optionally substituted alkyl group or a hydrogen atom, (excluding N, N, N' ,N' -tetrakis (2- pyridylmethyl) ethylenediamine) , or a salt thereof.

25. A method of preventing or treating an inflammatory disease in a mammal, comprising administering, to said mammal, an effective amount of a compound represented by the formula (I) :

wherein

L is an alkylene group;

X is a bond, -0-, -S- or -CO-;

Y is a bond, -NR³- or -CO-;

26. A compound represented by the formula (I) :

wherein L is an alkylene group; X is -0- ;

Y is -CO- or a bond;

Z is a Ci_6 alkyl group, a phenyl group optionally substituted by an amino group [the amino group may be substituted by a phenyl group (the phenyl group may be substituted by a Ci_₆ alkyl group optionally substituted by halogen) ] , a C₇_i₃ aralkyl group optionally substituted by a Cis alkyl group or an amino group [the amino group may be substituted by a phenyl group (the phenyl group may be substituted by a Ci-6 alkyl group optionally substituted by halogen) ] , a pyridyl group or a furyl group; and R¹ and R² are hydrogen atoms, or a salt thereof.