HK1073842A

HK1073842A - Metabolites of (3 - {[4 - tert - butyl - benzyl) - (pyridine - 3 - sulfonyl) - amino] -methyl} - phenoxy) - acetic acid

Info

Publication number: HK1073842A
Application number: HK05106375.7A
Authority: HK
Inventors: 金伯利．奥基夫．卡梅伦; 金．A．约翰森; 布鲁斯．A．莱夫克; 钱德拉．A．普拉卡什
Original assignee: 辉瑞产品公司
Priority date: 2002-01-31
Filing date: 2003-01-20
Publication date: 2005-10-21

Description

Metabolites of (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid

no marking

Technical Field

The present invention relates to metabolites of (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid. The invention also relates to methods of using metabolites of (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid for the treatment of osteoporosis and for aiding in fracture healing. Furthermore, the present invention relates to a method for determining whether a patient has been administered (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy ] -acetic acid.

Background

Compound (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl)) -amino group]-methyl } -phenoxy) -acetic acid is a selective EP that can be used for the treatment of osteoporosis, for the assistance of fracture healing and for the treatment of other diseases associated with bone loss₂An agonist. The compound (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid is disclosed in WO 99/19300(PCT/IB 98/01540). The compounds may also be used in open wedge osteotomies, treatment of primary bone loss in children, bone loss associated with periodontitis, glucocorticoid-induced osteoporosis, hyperthyroidism-induced osteoporosis, immobilization-induced osteoporosis, heparin-induced osteoporosis, immunosuppressant-induced osteoporosis and for increasing and maintaining bone mass, healing of bones after cosmetic surgery, healing of bones after maxillofacial reconstruction, healing of bones after mandible reconstruction, induction of vertebral bony associations, increased long bone elongation, increased bone graft healing ratio, and increased prosthesis ingrowth.

The present invention provides a metabolite of (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid or a pharmaceutically acceptable salt or prodrug of said metabolite or a salt of the prodrug. These metabolites can be used to treat the same diseases as (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid or can be used to determine whether a patient is administered the compound (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid.

Summary of The Invention

The present invention provides the following compounds or pharmaceutically acceptable salts thereof:

2- (4- { [ (3-carboxymethoxy-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenyl) -2-methyl-propionic acid;

(3- { [ [4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid;

(3- { [ (4-tert-butyl-benzyl) - (pyridine-N-oxide-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid;

(5- { [ (4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -hydroxy-phenoxy) -acetic acid;

2- {4- [ pyridine-N-oxide-3-sulfonylamino) -methyl } -phenyl } -2-methyl-propionic acid;

(3- { [ [4- (2-hydroxy-1, 2-dimethyl-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid glucuronide;

sulfate conjugates of pyridine-N-oxide-3-sulfonic acid 4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzamide;

sulfate conjugates of (3- { [ [4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid;

sulfate conjugates of pyridine-3-sulfonic acid 4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzamide;

2-methyl-2- {4- [ (pyridine-3-sulfonylamino) -methyl ] -phenyl } -propionic acid;

(3- { [ [4- (1, 2-dihydroxy-1-methyl-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid;

(3- { [ [4- (1-hydroxy-1-methyl-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid;

(3- { [ (4- (1, 1-dimethyl-2-oxo-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid, and

(3- { [ (4-isopropenyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid.

The invention also provides a method of determining whether a patient has been administered (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid, which method comprises determining whether a sample of plasma, urine, bile or faeces obtained from said patient shows the presence of one or more compounds selected from the group consisting of:

(3- { [ (4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid glucuronide;

sulfate conjugates of (3- { [ [4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino) -methyl } -phenoxy) -acetic acid;

(3- { [ [4- (1, 1-dimethyl-2-oxo-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid; and

The present invention also provides a method of treating osteoporosis or assisting in the healing of bone fractures, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound selected from the group consisting of:

(3- { [ [4- (1, 1-dimethyl-2-oxo-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid; or

The present invention also provides a pharmaceutical composition comprising one or more compounds selected from the group consisting of:

(3- { [ [4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid glucuronide;

Brief Description of Drawings

FIG. 1 is a representative HPLC radiochromatogram of metabolites of (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid in rat, dog, monkey and human liver microsomes.

FIG. 2 representative HPLC radiochromatograms of metabolites of (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid in rat, dog, monkey and human hepatocytes.

FIG. 3 ion spectrum (m/z 469) of CID product of (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid.

FIG. 4 ion spectrum (M/z 321) of CID product of metabolite M21.

FIG. 5 ion spectrum (M/z 335) of CID product of metabolite M11.

FIG. 6 ion spectrum (M/z 501) of CID product of metabolite M2.

FIG. 7 ion spectrum (M/z 499) of CID product of metabolite M3.

FIG. 8 ion spectrum (M/z 485) of CID product of metabolite M4.

FIG. 9 ion spectrum (M/z 321) of CID product of metabolite M19.

FIG. 10 ion spectrum (M/z 565) of CID product of metabolite M6.

FIG. 11 ion spectrum (M/z 485) of CID product of metabolite M5.

FIG. 12 ion spectrum (M/z 485) of CID product of metabolite M12.

FIG. 13 ion spectrum (M/z 305) of CID product of metabolite M20.

FIG. 14 ion spectrum and 1H NMR (M/z 487) of CID product of metabolite M22.

FIG. 15 ion spectrum and 1H NMR (M/z 471) of CID product of metabolite M23.

FIG. 16 ion spectrum and 1H NMR (M/z 483) of CID product of metabolite M24.

FIG. 17 ion spectrum and 1H NMR (M/z 453) of CID product of metabolite M26.

Detailed Description

The following compound is a metabolite of (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid: 2- (4- { [ (3-carboxymethoxy-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenyl) -2-methyl-propionic acid; (3- { [ [4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid; 2- {4- [ pyridine-N-oxide-3-sulfonylamino) -methyl ] -phenyl } -2-methyl-propionic acid; (3- { [ [4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid glucuronide; sulfate conjugates of pyridine-N-oxide-3-sulfonic acid 4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzamide; sulfate conjugates of (3- { [ [4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzyl ] - - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid; sulfate conjugates of pyridine-3-sulfonic acid 4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzamide; 2-methyl-2- {4- [ (pyridine-3-sulfonylamino) -methyl ] -phenyl } -propionic acid; (3- { [ (4-tert-butyl-benzyl) - (pyridine-N-oxide-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid; (5- { [ (4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -hydroxy-phenoxy) -acetic acid; (3- { [ [4- (1, 2-dihydroxy-1-methyl-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid; (3- { [ [4- (1-hydroxy-1-methyl-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino) -methyl } -phenoxy) -acetic acid; (3- { [ [4- (1, 1-dimethyl-2-oxo-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid; and (3- { [ (4-isopropenyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid.

These metabolites or pharmaceutically acceptable salts thereof or prodrugs thereof or salts of these prodrugs can be used in therapy with other EP' s₂Agonists and in particular (3- { [ 4-tert-butyl-benzyl]- (pyridine-3-sulfonyl) -amino } -methyl) -phenoxy) -acetic acid for the treatment of the same diseases and disorders. Can use EP₂Examples of agonist-treated diseases and conditions are disclosed in international patent application publication No. WO 99/19300 and include open wedge osteotomy, primary bone loss in children, bone loss associated with periodontitis, glucocorticoid-induced osteoporosis, nailOsteoporosis induced by hyperfunction of the gonadal glands, osteoporosis induced by immobilization, osteoporosis induced by heparin, osteoporosis induced by immunosuppressants and used for increasing and maintaining bone mass, healing of bone after cosmetic surgery, healing of bone after maxillofacial reconstruction, healing of bone after mandible reconstruction, induction of vertebral bony union, increase of long bone elongation, increase of bone graft healing ratio and increase of prosthesis ingrowth. Preferred treatments include the treatment of osteoporosis and the assistance of fracture healing.

Those skilled in the art recognize anti-bone resorption agents, such as pregnenes, polyphosphonates, diphosphonates, estrogen agonists/antagonists [ also known as SERMs (selective estrogen receptor modulators) ]]Estrogens, estrogen/progesterone combinations, and promatrilin^*(conjugated estrogens), estrone, estriol or 17 α -or 17 β -ethinylestradiol may be combined with the compounds of the invention.

Typical pregnones are obtained from commercial sources and include: progestengender, altrenogest, amadenone acetate, anagesterone acetate, chlormadinone acetate, dienogest, chlorprogestin acetate, megestrol acetate, demegestone acetate, desogestrel, demegestone, dydrogesterone, chlornorethindrone, norethindrone diacetate, etonogestrel, fluogestrel acetate, gestodene caproate, gestrinone caproate, haloprogesterone, hydroxyprogesterone caproate, levonorgestrel, lynestrenol, medrogesterone acetate, medroxyprogesterone acetate, medrenol diacetate, norethindrone acetate, norethindrone, norgestimate, norgestrel, norethindrone phenylpropionate, progesterone, quinigestrol acetate, quinigestrel and tengestrol. Preferred progestones are medroxyprogesterone, norethindrone and norethindrone.

Typical bone resorption inhibiting polyphosphonates include polyphosphonates of the type disclosed in US patent US3,683,080. Preferably the polyphosphonic acid is a bisphosphonate (also known as a diphosphonate). Sodium tiludronate is a particularly preferred polyphosphonate. Ibandronic acid is a particularly preferred polyphosphonic acid. Alendronate is a particularly preferred polyphosphonate. Zoledronic acid is a particularly preferred polyphosphonic acid. Other preferred polyphosphonic acids are 6-amino-1-hydroxy-hexylidene-diphosphonic acid and 1-hydroxy-3 (methylpentylamino) -propylidene-diphosphonic acid. The polyphosphonic acid may be administered in the form of an acid, a soluble alkali metal salt or an alkaline earth metal salt. Also included are hydrolyzable esters of polyphosphonic acids. Specific examples include ethane-1-hydroxy-1, 1-diphosphonic acid, methane diphosphonic acid, pentane-1-hydroxy-1, 1-diphosphonic acid, methane dichlorodiphosphonic acid, methane hydroxydiphosphonic acid, ethane-1-amino-1, 1-diphosphonic acid, ethane-2-amino-1, 1-diphosphonic acid, propane-3-amino-1-hydroxy-1, 1-diphosphonic acid, propane-N, N-dimethyl-3-amino-1-hydroxy-1, 1-diphosphonic acid, propane-3, 3-dimethyl-3-amino-1-hydroxy-1, 1-diphosphonic acid, phenylaminomethanediphosphonic acid, N-dimethylaminomethyldiphosphonic acid, N-1-diphosphonic acid, N (2-hydroxyethyl) aminomethane diphosphonic acid, butane-4-amino-1-hydroxy-1, 1-diphosphonic acid, alkane-5-amino-1-hydroxy-1, 1-diphosphonic acid, hexane-6-amino-1-hydroxy-1, 1-diphosphonic acid and pharmaceutically acceptable esters and salts thereof.

In another embodiment, the compounds of the invention may be used in combination with estrogen agonists/antagonists. Preferred estrogen agonists/antagonists are droloxifene ((E) -3- (1- (4- (2- (dimethylamino) ethoxy) phenyl) -2-phenyl-1-butenyl) -phenol) and related compounds disclosed in US5,047,431.

Another preferred estrogen agonist/antagonist is 3- (4- (1, 2-diphenyl-but-1-enyl) -phenyl) -acrylic acid disclosed in Endocrinology, 1997, 138, 3901-one 3911, Willson et al.

Another preferred estrogen agonist/antagonist is tamoxifen (2- (-4- (1, 2-diphenyl-1-butenyl) phenoxy) -N, N-dimethyl-ethylamine, (Z) -2-hydroxy-1, 2, 3-propanetricarboxylate (1: 1)) and related compounds disclosed in US patent 4,536,516.

Another related compound is 4-hydroxy tamoxifen disclosed in US patent US4,623,660.

A preferred estrogen agonist/antagonist is raloxifene ((6-hydroxy-2- (4-hydroxyphenyl) benzo [ b ] thiophen-3-yl) (4- (2- (1-piperidinyl) ethoxy) phenyl) -methanone hydrochloride) disclosed in U.S. patent No. 4,418,068.

Another preferred estrogen agonist/antagonist is toremifene (2- (4- (4-chloro-1, 2-diphenyl-1-butenyl) phenoxy) -N, N-dimethyl-ethylamine, (Z) -2-hydroxy-1, 2, 3-propanetricarboxylate (1: 1)) disclosed in US4,996,225.

Another preferred estrogen agonist/antagonist is chroman disclosed in US3,382,287: 1- (2- ((4- (-methoxy-2, 2, dimethyl-3-phenyl-chroman-4-yl) -phenoxy) -ethyl) -pyrrolidine, levomexifene is also preferred.

Another preferred estrogen agonist/antagonist is idoxifene disclosed in US3,382,287: (E) -1- (2- (4- (1- (4-iodo-phenyl) -2-phenyl-but-1-enyl) -phenoxy) -ethyl) -pyrrolidone.

Another preferred estrogen agonist/antagonist is 2- (4-methoxy-phenyl) -3- [4- (2-piperidin-1-yl-ethoxy) -phenoxy ] -benzo [ b ] thiophen-6-ol disclosed in US5,488,058.

Another preferred estrogen agonist/antagonist is 6- (4-hydroxy-phenyl) -5- (4- (2-piperidin-1-yl-ethoxy) -benzyl) -naphthalen-2-ol disclosed in US patent No. 5,484,795.

Another preferred estrogen agonist/antagonist is (4- (2- (2-aza-bicyclo [2.2.1] hept-2-yl) -ethoxy) -phenyl) - (6-hydroxy-2- (4-hydroxy-phenyl) -benzo [ b ] thiophen-3-yl) -methanone, disclosed in PCT publication No. WO 95/10513, along with methods of preparation.

Other preferred estrogen agonists/antagonists include compounds as described in commonly assigned U.S. patent No. US5,552,412. Particularly preferred compounds described therein are:

cis-6- (4-fluoro-phenyl) -5- (4- (2-piperidin-1-yl-ethoxy) -phenyl) -5, 6, 7, 8-tetrahydro-naphthalen-2-ol;

(-) -cis-6-phenyl-5- (4- (2-pyrrolidin-1-yl-ethoxy) -phenyl) -5, 6, 7, 8-tetrahydro-naphthalen-2-ol;

cis-6-phenyl-5- (4- (2-pyrrolidin-1-yl-ethoxy) -phenyl) -5, 6, 7, 8-tetrahydro-naphthalen-2-ol;

cis-1- (6 '-pyrrolidinoethoxy-3' -pyridyl) -2-phenyl-6-hydroxy-1, 2,3, 4-tetrahydronaphthalene;

1- (4' -pyrrolidinoethoxyphenyl) -2- (4 "-fluorophenyl) -6-hydroxy-1, 2,3, 4-tetrahydroisoquinoline;

cis-6- (4-hydroxyphenyl) -5- (4- (2-piperidin-1-yl-ethoxy) -phenyl) -5, 6, 7, 8-tetrahydro-naphthalen-2-ol; and

1- (4' -pyrrolidine ethoxy phenyl) -2-phenyl-6-hydroxy-1, 2,3, 4-four hydrogen isoquinoline.

A particularly preferred compound is (-) -cis-6-phenyl-5- (4- (2-pyrrolidin-1-yl-ethoxy) -phenyl) -5, 6, 7, 8-tetrahydro-naphthalen-2-ol D-tartrate disclosed in US patent US5,948,809.

Other estrogen agonists/antagonists are described in U.S. Pat. No. 4,133,814. Derivatives of 2-phenyl-3-aroyl-benzothiophene and 2-phenyl-3-aroyl-benzothiophene-1-oxide are disclosed in U.S. Pat. No. 4,133,814.

Other preferred estrogen agonists/antagonists include TSE-424 (U.S. Pat. No. 5,998,402), arozoxifene (U.S. Pat. No. 5,723,474), EM-652, EM-800, GW 5638, and GW 7604 or optical or geometric isomers thereof, pharmaceutically acceptable salts, N-oxides, esters, quaternary ammonium salts, or prodrugs thereof.

Other preferred estrogen agonists/antagonists include compounds of formula V or VI or optical or geometric isomers thereof or pharmaceutically acceptable salts, N-oxides, esters, quaternary ammonium salts or prodrugs thereof:

wherein:

R_1Bselected from H, OH, -O-C (O) -C₁-C₁₂Alkyl (straight or branched chain), -O-C₁-C₁₂Alkyl (linear or branched or cyclic) or halogen or C₁-C₄Halogenated ethers;

R_2B、R_3B、R_4B、R_5Band R_6BIndependently selected from H, OH, -O-C (O) -C₁-C₁₂(straight or branched chain), -O-C₁-C₁₂(linear or branched or cyclic), halogen or C₁-C₄Halogenated ethers, cyano, C₁-C₆Alkyl (linear or branched) or trifluoromethyl;

X_Aselected from H, C₁-C₆Alkyl, cyano, nitro, trifluoromethyl and halogen;

s is 2 or 3;

Y_Ais the following group:

wherein:

a)R_7Band R_8BIndependently selected from H, C₁-C₆Alkyl or optionally substituted by CN, C₁-C₆Alkyl (straight or branched), C₁-C₆Alkoxy (straight or branched), halogen, -OH, -CF₃or-OCF₃Substituted phenyl;

or

b)R_7BAnd R_8BA 5-membered saturated heterocyclic ring attached to contain one nitrogen heteroatom, the heterocyclic ring being optionally substituted with 1-3 substituents independently selected from hydrogen, hydroxy, halogen, C₁-C₄Alkyl, trihalomethyl, C₁-C₄Alkoxy, trihalomethoxy, C₁-C₄Acyloxy, C₁-C₄Alkylthio radical, C₁-C₄Alkylsulfinyl radical, C₁-C₄Alkylsulfonyl and hydroxy (C)₁-C₄) Alkyl, -CO₂H、-CN、-CONHR_1B、-NH₂、-NH(C₁-C₄Alkyl), -N (C)₁-C₄Alkyl radical)₂、-NHSO₂R_1B、NHCOR_1B、-NO₂Or optionally 1-3 (C)₁-C₄) Alkyl-substituted phenyl; or

c)R_7BAnd R_8BTo form a 6-membered saturated heterocyclic ring containing one nitrogen heteroatom, which heterocyclic ring is optionally substituted with 1-3 substituents independently selected from hydrogen, hydroxy, halogen, C₁-C₄Alkyl, trihalomethyl, C₁-C₄Alkoxy, trihalomethoxy, C₁-C₄Acyloxy, C₁-C₄Alkylthio radical, C₁-C₄Alkylsulfinyl radical, C₁-C₄Alkylsulfonyl and hydroxy (C)₁-C₄) Alkyl, -CO₂H、-CN、-CONHR_1B、-NH₂、-NH(C₁-C₄Alkyl), -N (C)₁-C₄Alkyl radical)₂、-NHSO₂R_1B、NHCOR_1B、-NO₂Or optionally 1-3 (C)₁-C₄) Alkyl-substituted phenyl; or

d)R_7BAnd R_8BAttached to a 7-membered saturated heterocyclic ring containing one nitrogen heteroatom, which heterocyclic ring is optionally substituted with 1-3 substituents independently selected from hydrogen, hydroxy, halogen, C₁-C₄Alkyl, trihalomethyl, C₁-C₄Alkoxy, trihalomethoxy, C₁-C₄Acyloxy, C₁-C₄Alkylthio radical, C₁-C₄Alkylsulfinyl radical, C₁-C₄Alkylsulfonyl and hydroxy (C)₁-C₄) Alkyl, -CO₂H、-CN、-CONHR_1B、-NH₂、-NH(C₁-C₄Alkyl), -N (C)₁-C₄Alkyl radical)₂、-NHSO₂R_1B、NHCOR_1B、-NO₂Or optionally 1-3 (C)₁-C₄) Alkyl-substituted phenyl; or

e)R_7BAnd R_8BTo form an 8-membered saturated heterocyclic ring containing one nitrogen heteroatom, which heterocyclic ring is optionally substituted with 1-3 substituents independently selected from hydrogen, hydroxy, halogen, C₁-C₄Alkyl, trihalomethyl, C₁-C₄Alkoxy, trihalomethoxy, C₁-C₄Acyloxy, C₁-C₄Alkylthio radical, C₁-C₄Alkylsulfinyl radical, C₁-C₄Alkylsulfonyl and hydroxy (C)₁-C₄) Alkyl, -CO₂H、-CN、-CONHR_1B、-NH₂、-NH(C₁-C₄Alkyl), -N (C)₁-C₄Alkyl radical)₂、-NHSO₂R_1B、NHCOR_1B、-NO₂Or optionally 1-3 (C)₁-C₄) Alkyl-substituted phenyl; or

f)R_7BAnd R_8BA saturated bicyclic heterocycle containing 6 to 12 bridging or fused carbon atoms and containing one nitrogen heteroatom, which heterocycle is optionally substituted with 1 to 3 substituents independently selected from hydrogen, hydroxy, halogen, C₁-C₄Alkyl, trihalomethyl, C₁-C₄Alkoxy, trihalomethoxy, C₁-C₄Acyloxy, C₁-C₄Alkylthio radical, C₁-C₄Alkylsulfinyl radical, C₁-C₄Alkylsulfonyl and hydroxy (C)₁-C₄) Alkyl, -CO₂H、-CN、-CONHR_1B、-NH₂、-NH(C₁-C₄Alkyl), -N (C)₁-C₄Alkyl radical)₂、-NHSO₂R_1B、NHCOR_1B、-NO₂Or optionally 1-3 (C)₁-C₄) Alkyl-substituted phenyl groups.

Another preferred estrogen agonist/antagonist is a compound of formula Va or an optical or geometric isomer thereof or a pharmaceutically acceptable salt, N-oxide, ester, quaternary ammonium salt or prodrug thereof:

other preferred estrogen agonists/antagonists include compounds of formula III (EM-652) or formula IV (EM-800) or an optical or geometric isomer thereof or a pharmaceutically acceptable salt, N-oxide, ester, quaternary ammonium salt or prodrug thereof:

it is considered by those skilled in the art that other bone anabolic agents, also known as bone mass increasing agents, may be combined with the compounds of the present invention. Bone mass increasing agents are agents that increase bone mass to a level above the fracture threshold as specifically described by the world health organization research group in "assessment of fracture hazards and their use in screening for postmenopausal osteoporosis (1994), world health organization research group-world health organization technology series 843 report".

The compounds of the invention can also be used in combination with other EP' s₂The agonists are used in combination. Preferred EP₂Agonists include 7- [ (4-butyl-benzyl) -methanesulfonyl-amino]Monosodium heptanoate or other compounds disclosed in US patent 6,288,120.

One skilled in the art would recognize that IGF-1, sodium fluoride, parathyroid hormone (PTH), active fragments of parathyroid hormone, growth hormone or growth hormone secretagogues may also be combined with the compounds of the present invention.

The compounds of the present invention may also be used in combination with prostaglandins. Various prostaglandins are described and referred to below. However, other prostateGlandins are also well known to those skilled in the art. Typical prostaglandins are disclosed in US patents US4,171,331 and US3,927,197. Norrdin et al in prostagladins Leukotriene Essential Fatty Acids 41, 139-150, 1990Prostate gland Action of hormone in bone in vivoIs an overview of bone anabolic prostaglandins.

Other compounds that may be used in combination with the compounds of the present invention include those disclosed in the following references: 2-decarboxylated-2- (tetrazol-5-yl) -11-deoxy-15-substituted-omega-pentanorgladins for osteogenic activity are disclosed in US3,932,389; U.S. Pat. No. 6, 4,018,892 discloses 16-aryl-13, 14-dihydro-PGE for osteogenic activity₂P-biphenylesters; 2,3, 6-substituted-4-pyrones for osteogenic activity are disclosed in US4,219,483; US patent US4,132,847 discloses 2,3, 6-substituted-4-pyrones for osteogenic activity; U.S. Pat. No. 6, 4,000,309 discloses 16-aryl-13, 14-dihydro-PGE for osteogenic activity₂P-biphenylesters; U.S. Pat. No. 6, 3,982,016 discloses 16-aryl-13, 14-dihydro-PGE for osteogenic activity₂P-biphenylesters; substituted cyclopentanes for osteogenetic activity are disclosed in US patent US4,621,100; and cyclopentanones for osteogenic activity are disclosed in US5,216,183.

Sodium fluoride may also be used in combination with the compounds of the present invention. The term "sodium fluoride" refers to all forms of sodium fluoride (e.g., slow release sodium fluoride, sustained release sodium fluoride). Sustained release sodium fluoride is disclosed in US patent No. 4,904,478, the contents of which are incorporated herein by reference. The activity of sodium fluoride is readily determined by the skilled worker according to biotechnological protocols (see, for example, Eriksen E.F. et al, Bone Histomorphometry, Raven Press, New York, 1994, pages 1-74; Grier S.J. et al, use of dual energy X-ray analysis in animals-Inv.Radiol., 1996, 31 (1): 50-62; Wahner H.W. and Fogelman I., evaluation of osteoporosis: use of dual energy X-ray analysis in clinical practice, Martin Dunitz Ltd., London 1994, pages 1-296).

Bone morphogenetic proteins can also be used in combination with the compounds of the invention (see, e.g., Ono et al, prostaglandin E1 for promotion of osteogenic Activity of recombinant human bone morphogenetic proteins-Bone，1996，19(6)，581-588)。

In addition, any parathyroid hormone (PTH) may be used in combination with the compounds of the present invention. The term "parathyroid hormone" refers to parathyroid hormone, fragments or metabolites thereof, and structural analogs thereof that can stimulate bone production and increase bone mass. Also included are parathyroid hormone-related peptides and active fragments and analogs of parathyroid hormone-related peptides (see PCT publication No. WO 94/01460). Such bone anabolic functional activity is readily determined by one skilled in the art according to standard assays (see, e.g., Eriksen E.F. et al, BoneHistomorphometry, Raven Press, New York, 1994, pages 1-74; Grier S.J. et al, use of Dual energy X-ray analysis in animals-Inv.Radiol., 1996, 31 (1): 50-62; Wahner H.W. and Fogelman I., evaluation of osteoporosis: use of Dual energy X-ray analysis in clinical practice, MartinDunitz Ltd., London 1994, pages 1-296). Various types of these compounds are described and referred to below. However, parathyroid hormone is well known to those skilled in the art. Typical parathyroid hormones are disclosed in the following references.

"human parathyroid peptide therapy for spinal Osteoporosis" -osteoporotosis iut, 3, (Supp 1): 199-203.

"PTH 1-34 therapy in combination with hormone replacement therapy for the treatment of osteoporosis: biochemical, kinetic and histological reactions "-osteoporotosis iht.1: 162-170.

In addition, any growth hormone or growth hormone secretagogue may be used in combination with the compounds of the present invention. The term growth hormone secretagogue refers to compounds that stimulate growth hormone release or mimic the action of growth hormone (e.g., increase osteogenesis, and thus bone mass). Such effects are readily determined by those skilled in the art according to standard assays well known to those skilled in the art. Various types of these compounds are disclosed in the following published PCT patent applications: WO 95/14666; WO 95/13069; WO 94/19367; WO 94/13696; and WO 95/34311. However, other growth hormones or growth hormone secretagogues are known to those skilled in the art.

Particularly preferred growth hormone secretagogues are N- [1(R) - [1, 2-dihydro-1-methanesulfonylspiro [ 3H-indole-3, 4 '-piperidin ] -1' -yl ] carbonyl ] -2- (phenylmethoxy) ethyl ] -2-amino-2-methylpropanamide: MK-677.

Other preferred growth hormone secretagogues include:

2-amino-N- (2- (3a- (R) -benzyl-2-methyl-3-oxo-2, 3, 3a, 4,6, 7-hexahydro-pyrazolo- [4, 3-c ] pyridin-5-yl) -1- (R) -benzyloxymethyl-2-oxo-ethyl) -isobutyramide (U.S. patent US6,107,306) or the L-tartrate salt thereof;

2-amino-N- {1(R) -benzyloxymethyl-2- [1, 3-dioxo-8 a- (S) -pyridin-2-ylmethyl-2- (2, 2,2, -trifluoro-ethyl) -hexahydro-imidazo- [1, 5a ] pyrazin-7-yl ] -2-oxo-ethyl } -2-methyl-propionamide (US patent 6,251,902) or its hydrochloride salt;

2-amino-N- (1- (R) -benzyloxymethyl-2- (3a- (R) - (4-fluoro-benzyl) -2-methyl-3-oxo-2, 3, 3a, 4,6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -2-oxo-ethyl) isobutyramide;

2-amino-N- (2- (3a- (R) -benzyl-3-oxo-2, 3, 3a, 4,6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -1- (R) benzyloxymethyl-2-oxo-ethyl) isobutyramide; and

2-amino-N- (1- (2, 4-difluoro-benzyloxymethyl) -2-oxo-2- (3-oxo-3 a-pyridin-2-ylmethyl-2- (2, 2, 2-trifluoro-ethyl) -2, 3, 3a, 4,6, 7-hexahydro-pyrazolo [4, 3-c ] pyridin-5-yl) -ethyl) -2-methyl-propionamide (US patent 6,110,932) or the L-tartrate salt thereof.

The compounds of the present invention are generally administered in the form of a pharmaceutical composition comprising at least one compound of the present invention together with a pharmaceutically acceptable excipient or diluent. For oral administration, the pharmaceutical compositions may take the form of solutions, suspensions, tablets, pills, powders, and the like. Tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate, together with various disintegrants such as starch and preferably potato or tapioca starch and certain complex silicates, and binders such as polyvinylpyrrolidone, sucrose, gelatin and acacia may be used. Additionally, lubricants such as magnesium stearate, sodium lauryl sulfate and talc are commonly used for tableting purposes. Solid compositions of a similar type may also be used as fillers for soft and hard capsules; preferred materials in this regard also include lactose and high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the compounds of the invention can be combined with various sweetening agents, flavoring agents, coloring agents, emulsifying agents and/or suspending agents and such diluents as water, ethanol, propylene glycol, glycerin and various combinations thereof.

For parenteral administration, solutions of sesame or peanut oil or aqueous propylene glycol, as well as sterile aqueous solutions of the corresponding water-soluble salts, may be used. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient salt or glucose. These aqueous solutions are suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. In this regard, the sterile aqueous medium employed is readily obtained by standard techniques well known to those skilled in the art.

For transdermal (e.g., topical) administration purposes, sterile dilute or partial aqueous solutions (typically at concentrations of about 0.1% to 5%) are prepared, otherwise solutions similar to the parenteral solutions described above are prepared.

Capsules are prepared by mixing the compound with a suitable diluent and filling the appropriate amount of the mixture into capsules. Common diluents include inert powdered substances such as many different types of starch; powdered cellulose, especially crystalline cellulose and microcrystalline cellulose; sugars such as fructose, mannitol, and sucrose; cereal flour and similar edible powders.

Tablets are prepared by direct compression, by wet granulation or by dry granulation. Diluents, binders, lubricants and disintegrants and the compounds are generally mixed in the formulation. Typical diluents include: for example, various types of starch, sucrose, mannitol, kaolin, calcium phosphate or calcium sulfate; inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives may also be used. Typical tablet binders are substances such as starch, gelatin and sugars, such as lactose, fructose, glucose and the like. Natural and synthetic gums are also commonly used and include acacia, alginate, methylcellulose, polyvinylpyrrolidone, and the like. Polyethylene glycol, ethyl cellulose and waxes may also be used as binders.

A lubricant is essential in the tablet to prevent sticking of the tablet to the die. The lubricant is selected from the group of lubricating solids such as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.

Tablet disintegrants are substances that facilitate tablet disintegration when a tablet becomes moist to release a compound. They include starch, clay, cellulose, sodium alginate and gums, corn and potato starch, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponges, cation exchange resins, alginic acid, guar gum, citrus pulp, and carboxymethyl cellulose, which may be used, for example, in combination with sodium lauryl sulfate.

Tablets are usually sugar coated as a flavoring and sealing agent or film protecting agent to modify the dissolution characteristics of the tablet. The compounds can also be formulated into chewable tablets by using large amounts of taste-pleasing substances, such as mannitol, in the formulation and are currently well established in the art.

Typical bases may be used when it is desired to administer the compounds as suppositories. Cocoa butter is a traditional suppository base that may be modified by the addition of waxes to raise its melting point moderately. A wide range of applications specifically include water-miscible suppository bases of various molecular weight polyethylene glycols.

The action of the compounds can be delayed or prolonged by appropriate formulation. For example, slow dissolving granules of the compound may be prepared and incorporated into tablets or capsules. This technique can be improved by preparing several different dissolution rate particles and filling the capsules with a mixture of the particles. The tablets or capsules may be coated with a film coating that prevents dissolution for a predictable period of time. Even parenteral formulations can be made to exert a long-lasting effect by dissolving or suspending the compound in an emulsifying excipient which allows it to disperse only slowly in the serum.

The pharmaceutical compositions of the invention may contain from 0.1% to 95% of a compound of the invention, preferably from 1% to 70%. In any case, the administered composition or formulation contains some compound in an amount effective to treat the disease/condition.

The following paragraphs describe typical formulations and dosages for non-human animals. For example, the compounds of the present invention may be administered orally or parenterally by injection. The compounds of the invention may be administered in amounts such that an effective amount, i.e. a typical daily dose of usually 0.01-100mg/kg body weight, preferably 0.1-50mg/kg body weight, is received when orally administered to an animal. It is noted that more than one dose per day may be required and that the veterinarian can determine the effective amount based on considerations of a particular situation.

Conveniently, the compound is brought into the drinking water so that a therapeutic dose of the compound is taken through the daily drinking water. The compounds can be metered directly into the drinking water, preferably in the form of a liquid, water-soluble concentrate (such as an aqueous solution of a water-soluble salt). It may also be convenient to add the compounds as such directly to the feed or as an animal feed additive form, also known as a premix or concentrate. Premixes or concentrates of the compounds in a carrier are more commonly used to contain active agents in feed. Suitable carriers are liquids or solids, such as water; various dietary powders such as alfalfa meal, soybean meal, cottonseed oil meal, linseed oil meal, corn cob meal and corn meal, molasses, urea, bone meal; whereas mixtures such as minerals are commonly used in poultry feed. Particularly effective carriers are the corresponding animal feed itself; i.e. a small portion of such feed. The carrier facilitates uniform distribution of the compound in the final feed mixed with the premix. It is important that the compound is thoroughly mixed into the premix and subsequently the feed. In this regard, the compounds can be dispersed or dissolved in a suitable oil carrier, such as soybean oil, corn oil, cottonseed oil, and the like, or a volatile organic solvent and then mixed with the carrier. It will be appreciated that the proportion of the compound in the concentrate can vary widely, as the amount of active compound in the final feed can be adjusted to give the desired concentration of compound by mixing an appropriate proportion of the premix with the feed.

The high efficiency concentrate can be mixed with a protein carrier, such as soy flour and other dietary powders as described above, by a feed producer to produce a concentrated supplement that is suitable for direct feeding to an animal. In such cases, the animal is allowed to consume the usual diet. Alternatively, such concentrated additives may be added directly to the feed to produce a nutritionally balanced finished feed containing a therapeutically effective concentration of a compound of the invention. The mixture is thoroughly mixed by standard procedures, such as using a double-wall mixer to ensure homogeneity.

If the additive is used as a top dressing for a feed, it also helps to ensure that the compound is evenly distributed through the top dressing.

For parenteral administration to an animal, the compounds of the invention may be prepared in the form of a paste or pill and administered as an implant, typically under the skin of the animal's head or ear.

Pastes may be prepared by dispersing a compound of the invention in a pharmaceutically acceptable oil, such as peanut oil, sesame oil, corn oil, and the like.

Pills containing an effective amount of a compound of the present invention may be prepared by mixing the compound with a diluent, such as polyethylene glycol, carnauba wax, and the like. Lubricants such as magnesium stearate or calcium stearate may be added to improve the granulation process.

Of course, it is contemplated that more than one bolus may be administered to an animal in order to achieve the desired concentration. In addition, it has been found that the implant can also be periodically embedded during treatment of the animal in order to maintain an appropriate concentration of active agent in the animal.

"treatment" as used herein includes both prophylactic (e.g., prophylactic) and palliative treatment and "treatment" as used herein refers to the effect of treatment by prophylaxis and/or palliative treatment.

The term "therapeutically effective amount" refers to an amount of a compound of the invention or a combination of a compound of the invention and another compound that ameliorates one or more symptoms of a disease or condition or prevents or delays the onset of one or more symptoms of a disease or condition.

As used herein, "patient" refers to a mammal, particularly a human.

"glucuronic acid" is a substituent that is converted to a metabolite or to the parent compound to produce the metabolite from a phase II glucuronidation conjugation reaction. Glucuronic acid reacts with an acid or alcohol or phenol moiety on the metabolite or parent compound to produce a "glucuronide". Glucuronide substituents are abbreviated herein in the general formula as "Glu" or "glucuronide".

"sulfuric acid" is a substituent that is converted to a metabolite or to the parent compound to produce the metabolite from a sulfation conjugation reaction. The sulfuric acid reacts with alcohol or phenol groups on the metabolite or parent compound to form a "sulfate ester" or "sulfate ester conjugate".

Administration of a compound of the invention in combination with another compound or other compounds means that the compounds can be administered together, simultaneously or at different times, as a composition or as part of the same unit dosage form or separate dosage forms.

Chemists of ordinary skill in the art recognize that the compounds of the present invention contain one or more atoms that may be in a particular stereochemical, tautomeric or geometric configuration, thereby giving rise to stereoisomers, tautomers, regions and configurational isomers. All such isomers and mixtures thereof are included in the present invention. Also included are hydrates and solvates of the compounds of the present invention.

The present invention also includes isotopically-labeled compounds, which are identical to those of the present invention, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in practice. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as²H、³H、¹³C、¹⁴C、¹⁵N、¹⁸O、¹⁷O、³¹P、³²P、³⁵S、¹⁸F and⁶and (4) Cl. Compounds of the present invention, prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labelled compounds of the present invention, for example those into which are incorporated³H and¹⁴c such compounds of radioisotopes are useful in drug and/or substrate tissue distribution assays. Tritiation, i.e. tritiation, is particularly preferred³H and carbon-14 isotopes to facilitate preparation and detection. Furthermore, with heavier isotopes such as deuterium, i.e.²H substitution may provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements and may thus be preferred for use in certain circumstances. Isotopically-labeled compounds of the present invention and prodrugs thereof can generally be prepared by carrying out the following typical procedures or procedures well known in the art.

The compounds of the invention may be used as analytical standards for in vitro or in vivo metabolic studies or as intermediates in the chemical or biological synthesis of new chemical entities. The metabolites may be isolated as solids or as solutions. The compounds of the invention may also be used to identify patients who have been administered (3- { [ 4-tert-butyl-benzyl ] - (pyridine-3-sulfonyl) -amino } -methyl } -phenoxy) -acetic acid or a pharmaceutically acceptable salt or prodrug or salt of a prodrug thereof. To identify patients who have been administered (3- { [ 4-tert-butyl-benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid or a pharmaceutically acceptable salt or prodrug or salt of a prodrug thereof, serum, urine, faeces or bile samples are taken from the patients and the samples are analysed for the presence of one or more compounds of the invention. One method of analyzing the compounds of the present invention is by using chromatography and mass spectrometry. Other methods of analysis are well known to those skilled in the art. The presence of one or more compounds of the invention in a serum, urine, feces, or bile sample indicates that the patient has been administered (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid, or a pharmaceutically acceptable salt or prodrug or salt of a prodrug thereof.

In the methods of treatment of the present invention, the compounds of the present invention may be administered directly to the patient, such as in tablet form; or the compound produced by metabolism of the patient's body may be administered. In addition, if desired, the route of administration and the dosage of the compounds of the invention produced by metabolism may be varied to obtain the desired in vivo concentration and production ratio of the compounds of the invention.

Pharmaceutically acceptable acid addition salts of the compounds of the present invention may be formed from the compounds themselves or any of their esters and include pharmaceutically acceptable salts commonly used in pharmaceutical chemistry. For example, salts may be formed with inorganic or organic acids such as: hydrochloric acid; hydrobromic acid; hydriodic acid; sulfonic acids including agents such as naphthalenesulfonic acid, methanesulfonic acid, and toluenesulfonic acid; sulfuric acid; nitric acid; phosphoric acid; tartaric acid; pyrosulfuric acid; metaphosphoric acid; succinic acid; formic acid; phthalic acid; lactic acid and the like; most preferably hydrochloric, citric, benzoic, maleic, acetic and propionic acid.

When used as a medicament, the dosage of the compounds of the present invention administered to a human or other patient varies considerably and is at the discretion of the attendant clinician or veterinarian. It should be noted that when administered in the form of a salt having a salt-forming moiety of considerable molecular weight, such as laurate (laureate), it may be necessary to adjust the dose of the compound. Effective administration rates of the compounds generally range from about 0.001 mg/day to about 200 mg/day. A preferred range is about 0.01 mg/day to about 100 mg/day. Of course, in practice, daily doses of the compounds will generally be administered at different times of the day. However, in any given case, the amount of compound administered will depend on such factors as the solubility of the active ingredient, the formulation used and the route of administration.

Prodrugs are compounds that are converted in vivo to the compounds of the present invention. This conversion can occur by various mechanisms, such as by hydrolysis in blood. Higuchi and W.stella inA.C.S. Symposium SeriesVol.14 "as prodrug of the novel transport System" andBioreversible Carriers in Drug Designa detailed discussion of the use of prodrugs is provided in ed ward b.roche, American pharmaceutical association and Pergamon Press, 1987.

For example, if a compound of the invention contains a carboxylic acid functional group, a prodrug may include an ester formed by replacing a hydrogen atom on the acid group with: such as (C)₁-C₈) Alkyl, (C)₂-C₁₂) Alkanoyloxymethyl, 1- (alkanoyloxy) ethyl having 4 to 9 carbon atoms, 1-methyl-1- (alkanoyloxy) -ethyl having 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having 3 to 6 carbon atoms, 1- (alkoxycarbonyloxy) ethyl having 4 to 7 carbon atoms, 1-methyl-1- (alkoxycarbonyloxy) ethyl having 5 to 8 carbon atoms, N- (alkoxycarbonyloxy) aminomethyl having 3 to 9 carbon atoms, 1- (N- (alkoxycarbonyl) amino) ethyl having 4 to 10 carbon atoms, 3-benzo [ c ] c]Furanone group, 4-crotonolactone group, gamma-butyrolactone-4-yl group, di-N, N- (C)₁-C₂) Alkylamino radical (C)₂-C₃) Alkyl (such as alpha-dimethylaminoethyl), aminoFormyl radical (C)₁-C₂) Alkyl, N-di (C)₁-C₂) Alkylcarbamoyl- (C)₁-C₂) Alkyl, and piperidino, pyrrolidin-1-yl-or morpholino (C)₂-C₃) An alkyl group.

Similarly, if the compounds of the invention contain alcohol functional groups, it is possible to use compounds such as (C)₁-C₆) Alkanoyloxymethyl, 1- ((C)₁-C₆) Alkanoyloxy) ethyl, 1-methyl-1- ((C)₁-C₆) Alkanoyloxy) ethyl group, (C)₁-C₆) Alkoxycarbonyloxymethyl, N- (C)₁-C₆) Alkoxycarbonylaminomethyl, succinyl, (C)₁-C₆) Alkanoyl, alpha-amino (C)₁-C₄) Alkanoyl, aroyl and alpha-aminoacyl or alpha-aminoacyl-alpha-aminoacyl groups each independently selected from naturally occurring L-amino acids, P (O) (OH) to form a prodrug by substituting a hydrogen atom on an alcohol group₂、-P(O)(O(C₁-C₆) Alkyl radical)₂Or a glycosyl (a group derived from a carbohydrate by removal of the hydroxyl group from its hemiacetal form).

If the compounds of the invention contain amine functional groups, it is possible to use, for example, R^X-carbonyl, R^XO-carbonyl, NR^XR^X1A radical of carbonyl substituting a hydrogen atom on an amine group to form a prodrug, wherein R^XAnd R^X1Each independently is ((C)₁-C₁₀) Alkyl, (C)₃-C₇) Cycloalkyl, benzyl or R^XCarbonyl as a natural alpha-aminoacyl or natural alpha-aminoacyl-natural alpha-aminoacyl, -C (OH) C (O) OY^XWherein Y is^XIs H, (C)₁-C₆) Alkyl or benzyl, -C (OY)^X0)Y^X1Wherein Y is^X0Is (C)₁-C₄) Alkyl and Y^X1Is ((C)₁-C₆) Alkyl, carboxyl (C)₁-C₆) Alkyl, amino (C)₁-C₄) Alkyl or mono-N-or di-N, N- (C)₁-C₆) Alkylaminoalkyl, -C (Y)^X2)Y^X3Wherein Y is^X2Is H or methyl and Y^X3Is mono-N-or di-N, N- (C)₁-C₆) Alkylamino, morpholino, piperidin-1-yl or pyrrolidin-1-yl.

Advantageously, the invention also provides kits for use by a consumer for treating a disease. The kit comprises: a) pharmaceutical compositions comprising a compound of the invention and a pharmaceutically acceptable carrier, excipient or diluent; and b) instructions describing a method of use of the pharmaceutical composition for treating a particular disease.

As used herein, a "kit" includes a container containing individual unit dosage forms, such as a separate vial or a separate foil package. The container may be of any conventional shape or form known in the art made of pharmaceutically acceptable materials, such as paper or tissue cartons, glass or plastic vials or jars, re-sealable bags (e.g., different containers made for "refilling" the placed tablets), or blister packs containing individual doses that are extrusion packaged according to a treatment protocol. The container used may depend on the exact dosage form involved, e.g. commonly used tissue cartons are not generally used for containing liquid suspensions. It is possible to use more than one container in a single package with each other for the purpose of marketing a single dosage form. For example, tablets may be filled into vials, which are then boxed.

An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are widely used for packaging unit pharmaceutical dosage forms (tablets, capsules, etc.). The blister pack is generally composed of a relatively rigid material covered with a foil, preferably of transparent plastic. During packaging, pockets are formed in the plastic foil. Such pockets may be of the size and shape of the individual tablets or capsules packaged or may be of the size and shape to accommodate multiple pieces and/or capsules packaged. The next step is to place the tablet or capsule into the recess and thereby seal the sheet of relatively rigid material to the foil forming the plastic foil surface in the opposite direction to the recess. The result is that the tablets or capsules are sealed separately or together, if desired, in the recesses between the plastic foil and the plate. Preferably the strength of the panel is such that the tablet or capsule can be removed from the blister pack by pressing by hand on the depression thereby forming an opening in the panel at the surface of the depression. The tablet or capsule can then be removed through the opening.

There is a need to provide a written storage means of the type containing information and/or instructions for use by a clinician, pharmacist or subject, for example in the form of the number of tablets or capsules to be used next, wherein said number corresponds to the number of days of the regimen that a particular tablet or capsule should be ingested or a card containing the same type of information. Another example of such a storage appliance is a calendar printed on a card, for example as follows: "first week, monday, tuesday,". et al. "second week, monday, tuesday,.", et al. Other variations of storage appliances are readily known. The "daily dose" may be a single or single capsule or several tablets or capsules administered on the indicated day.

Another embodiment is actually a dispenser for dispensing daily doses each time. The dispenser is preferably fitted with a memory to further facilitate compliance with the protocol. An example of such a memory is a mechanical counter which indicates the number of doses per day that have been dispensed. Another example of such a memory is a battery powered microchip memory connected to a liquid crystal reader or an audible alert signal, for example, to read the date the last daily dose has been taken and/or to alert the patient when the next dose is taken.

In another embodiment of the kit, the pharmaceutical composition may further comprise another compound that may be used in combination with a compound of the present invention; or the kit may comprise two pharmaceutical compositions: one containing a compound of the invention and the other containing another compound that may be used in combination with the compound of the invention.

The references cited herein, including any patents and patent applications, are incorporated herein by reference.

The examples provided herein are intended to explain specific embodiments of the invention and are not intended to limit the specification or claims in any way.

Examples

¹⁴C- (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid radiolabel mass balance and metabolic profile in Sprague Dawley rats

Purpose(s) to

To determine the single dose of 15mg/kg administered intravenously¹⁴C- (3- { [ 4-tert-butyl-benzyl]- (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid post- (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid radioactivity balance and metabolic profile in Sprague-Dawley rat urine, feces, bile and plasma.

Materials and methods

Radiolabelled compounds

¹⁴C- (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid (specific activity 4.36mCi/mmol) showed a radioactive purity > 99%.

*¹⁴C-mark position

Can be prepared according to the following scheme¹⁴C- (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid.

Radiolabelled aldehydes (ChemSyn Laboratories, Lenexa, KS) and amine A were stirred overnight in dichloromethane at room temperature (NaBH added from an aliquot)₄Inhibition by neutralization and monitoring of imine formation by HPLC analysis) and NaBH₄Is processed to obtainTo compound B, which is then treated with excess pyridine-3-sulfonyl chloride and Hunig's base (diisopropylethylamine) in dichloromethane to form compound C, which is reacted with trifluoroacetic acid in dichloromethane to form¹⁴C- (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid.

Compound a can be prepared as follows:

step 1

1eq (eq) of aldehyde, 1eq of carbonate, 1.25eq of tert-butyl bromoacetate in acetone

Reflux for 20-24 hours

Inhibition with water

Extracted into ethyl acetate, substituted with 2-propanol (IPO) and used in the next step

Step 2

1eq IPO solution of Compound D, 2eq hydroxylamine HCl, 2eq pyridine (Py)

Heating under reflux for 3 to 4 hours

Treatment with Ethyl acetate and HCl

Replacement with 2B ethanol (denatured with toluene) for the next step

Step 3

2B ethanol solution of Compound E, 30% Raney Nickel loading, 28% ammonium

Isopropyl ether extraction; concentrating to obtain oily substance

Dissolving the oil in IPO and adding a solution of maleic acid in IPO

Addition of isopropyl ether to precipitate out the salt

Animal model

A group of 3 male and 3 female jugular vein cannulated Sprague-Dawley rats (230-240g) were each housed in a stainless steel metabolic cage for urine and feces collection and administered intravenously with a single 15mg/kg dose¹⁴C- (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid.

For pharmacokinetic and plasma characterization, two groups of animals were dosed intravenously with a single 15mg/kg dose¹⁴C- (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid (3 males and 3 females for pharmacokinetic characterization, and 2 males and 2 females for plasma characterization).

Animals of the fourth group (bile duct and jugular vein cannulation, 2 males and 2 females) were dosed for bile and urine collection for evaluation of the secretory pathway and bile metabolites.

Sample collection

Urine and feces were collected from group I animals for 7 days at 0-24, 24-48, 48-72, 72-96, 96-120, 120-144 and 144-168 hours post dose administration. Whole blood was collected at 5 minutes, 15 minutes, 1, 2,4, 6, 8, 24 and 48 hours post dose for pharmacokinetic analysis and at 1 and 4 hours post dose for identification of circulating metabolites. Plasma was separated from whole blood by centrifugation. Bile and urine were collected from cannulated animals at 0-8, 8-24 and 24-48 hours after dosing.

Sample analysis

Radioactivity determination

Radioactivity in urine, bile and plasma was determined by liquid scintillation counting. Aliquots of urine (0.1g), bile (0.025g) or plasma (0.025g) were mixed with 5ml of the Ecolite (+) scintillation cocktail and counted using a Wallac #1409 liquid scintillation counter (Gaithersburg, Md.). Stool samples collected at each time point were homogenized with water and the total weight of the stool homogenate was recorded. Aliquots (0.1-0.3g) of the homogenate were oxidized with Packard oxidizer (Packard instruments co., down's Grove, IL) and scintillation counting was performed thereafter.

The radioactivity in the dose was taken as 100% of the total radioactivity. Radioactivity in urine and feces collected at each time point was defined as the percentage of dose secreted in the corresponding matrix.

Radioactivity measured in plasma was converted to ng equivalents of (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid based on the specific activity of the dosing vehicle of 19.75 dpm/ng.

Quantitative evaluation of metabolite secretion

The metabolites were quantified by measuring the HPLC peak areas of the respective isolates using beta-RAM (IN/US, Win-flow). beta-RAM provides a printout of typical integration peaks and the percentage of radioactive material in CPM. The beta-RAM was operated using a solid scintillation element.

Extraction of metabolites from biological samples

The urine sample (about 10ml) was evaporated overnight under nitrogen. The sample residue was redissolved with 1ml of 0.1% formic acid/acetonitrile (50: 50). These solutions were vortexed for about 1 minute, transferred to a 1.5ml eppendoff tube and then centrifuged at 14,000rpm for about 2 minutes. An aliquot of the supernatant (10-20 μ l) was injected onto the HPLC column without further purification.

Fecal homogenates containing the highest levels of secreted radioactivity were collected (0-48 hours). An aliquot (about 5g) from the collected sample (about 80-135g) was suspended in 15ml of acetonitrile. The suspension was sonicated (about 30 minutes), vortexed and centrifuged at 3200rpm for 10 minutes. After transferring the supernatant to a clean 15ml conical tube, the residue was further extracted 2 times with 15ml acetonitrile as described above. Aliquots (200ul) from each extraction were counted in a liquid scintillation counter. The recovery rate of extracted radioactivity is in the range of 92-96%. The supernatant was evaporated to dryness in a Turbo Vap LV evaporator (Zymark, Hopkinton, MA) under nitrogen and the residue redissolved with 2ml of mobile phase. An aliquot (10-20ul) of concentrated fecal extract was injected onto the HPLC column.

Plasma was precipitated using 2 volumes of acetonitrile for identification of circulating metabolites. The suspension was sonicated (about 30 minutes), vortexed and centrifuged at 3200rpm for 10 minutes. After transfer of the supernatant to a clean 15ml conical tube, the residue was further extracted with 2 × 15ml acetonitrile as described above. The supernatants were combined and evaporated to dryness in a Turbo Vap LV evaporator under nitrogen and the residue redissolved with 0.5ml mobile phase. An aliquot (50-100ul) of the concentrated plasma extract was injected onto the HPLC column.

Bile was injected directly into the HPLC/MS system for analysis without further purification.

High performance liquid chromatography

The HPLC system consisted of an HP-1100 solvent delivery system, an HP-1100 membrane degasser, an HP-1100 auto-injector (Hewlett Packard) and an IN/US radioactivity monitor (. beta. -RAM). Chromatography was performed using a YMCAQ (C-18) column (4.6 mm. times.150 mm, 3um) (Waters, Milford, Mass.). The mobile phase initially consisted of 10mM ammonium formate (solvent A) and acetonitrile (solvent B) at pH 5.0. The solvent transport step gradient program was performed as follows:

time (minutes)	% solvent A	% solvent B
time (minutes)	% solvent A	% solvent B	0-33-2525-2626-2929-3131-35	903510109090	106590901010

The system was allowed to equilibrate for 10 minutes, after which the next injection was made. The flow rate was maintained at 1.0 ml/min throughout the analysis. For quantification of plasma metabolites, the HPLC effluent was passed into the flow cell of a β -ram radioactivity detector. Beta-ram and HPLC were controlled externally using an ARC (accurate radioisotope counting) system for low level radioactive counting.

Mass spectrometry

Metabolites were identified using a Finnigan TSQ7000 triple quadrupole mass spectrometer equipped with an API-2 electrospray interface (Finnigan, San Jose, Calif.). The HPLC column effluent was separated to introduce about 50. mu.l/min into the API interface. The remaining effluent was passed to the flow cell of beta-RAM. The beta-RAM response was recorded as a real-time analog signal with the MS data acquisition system. The data collected from the radioactivity and MS detectors was separated according to the residence volume flowing from the MS into the radioactivity detector (corresponding to a residence time of about 0.2 minutes). Electrospray voltage was run at-4.5 eV as mass spectrometer data collected in positive ion mode. Collision Induced Dissociation (CID) studies were performed in Q2(Q2 is the second quadrupole) using argon, applying a collision energy of 30-35eV and a collision gas consistency of about 2.1 mTorr.

Results and discussion

Metabolite identification

Metabolite M8

Metabolite M8 had retention times of 10:26-11:35 (min: s) and showed protonated molecular ions at M/z 351. It was detected in urine (male animals) and bile. The CID fragmentation pattern of M8 has primary fragments at M/z 305, 146 and 131. The loss of an ion at m/z 305 of 46amu from the molecular ion suggests the presence of a carboxylic acid moiety. The ions at m/z 131 and 146 are due to carboxyisopropylbenzyl and carboxyisopropylbenzylamine moieties, respectively, where there is a contaminant of loss of formic acid. M8 is believed to be due to fragmentation pattern and molecular ionsPhenoxyacetic acid moieties are formed by N-debenzylation followed by oxidation of the tert-butyl moiety to the carboxylic acid and hydroxylation of the pyridine ring. Reaction of the metabolite with titanium trichloride (20% phosphoric acid solution) increased the retention time of the metabolite by about 1 minute and the resulting M + H⁺The ion was reduced by 16amu, suggesting that the metabolite is an N-oxide. Based on this data, M8 was identified as 2- {4- [ pyridine-N-oxide-3-sulfonamido]-methyl } -phenyl } -2-methyl-propionic acid.

Metabolite M13

Metabolite M13 had a retention time on HPLC of about 10:47 (min: sec) and it was detected only in bile (male animals). It shows protonated molecular ions at m/z 661, higher than the parent compound 192 amu. The CID product ion spectra show the major fragment ions at m/z 485, 467, 342, 324, 165 and 145. The ion at m/z 485 is generated by the loss of glucuronic acid from the molecular ion. Ions at m/z 467 are generated by losing water from ions at m/z 485. Ions at m/z 342 and 324 are produced by the subsequent loss of the sulfonylpyridine moiety and water. Similar to the ion of the parent compound, the ion at m/z 165 is generated from a protonated methylphenoxyacetic moiety. The ion at m/z 145 indicates the loss of water from the hydroxy t-butylbenzyl moiety. Based on these data, M13 was identified as (3- { [ [4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino } -methyl) -phenoxy) -acetic acid glucuronide.

Metabolite M9

Metabolite M9 has a protonated molecular ion at M/z 417 and it is only detected in the urine of female rats. It has a retention time on HPLC of about 11:35 (min: sec). Protonated molecule at m/z 417The result was that the ion was lower than the parent compound 52amu, suggesting that M9 is a cleavage product. The CID product ion spectrum of M9 shows the primary ions at M/z 319, 160, and 145. The result that the ion at m/z 319 was 98amu lower than the protonated molecule ion suggests that water and sulphuric acid molecules were lost from the molecule. The result that the ion at m/z 145 is lower than the parent compound 2amu at m/z 147 suggests that the tert-butylbenzyl group has been hydroxylated and that water molecules are lost during fragmentation. In addition, the reaction of the metabolite with titanium trichloride (20% phosphoric acid solution) increased the retention time of the metabolite by about 2 minutes and the resulting M + H⁺The ion was reduced by 16amu, suggesting that the metabolite is an N-oxide. Based on these data, the structure of M9 is believed to be the sulfate conjugate of pyridine-N-oxide-3-sulfonic acid 4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzylamide.

Metabolite M6

Metabolite M6 has a retention time on HPLC of about 11:23 to 12:37 (min: s) it is found only in feces, bile and plasma. It shows protonated molecular ions at m/z 565 (96 amu higher than the parent compound), suggesting the addition of an oxygen atom and a sulfate group. The ion spectrum of the CID product of M6 shows dominant fragments at M/z 485, 467, 342, 324, 165, and 145. The ions at m/z 485 and 467 are generated by the subsequent loss of sulfuric acid and water from the protonated molecular ions. The ion at m/z 165 is similar to that of the parent compound, indicating that the methylphenoxyacetic moiety is unchanged. The ion at m/z 342 (higher than the 16amu observed in the parent compound) indicates that an oxygen atom has been added to the tert-butylbenzyl moiety. The ions at m/z324 and 145 were below the corresponding values of 2amu in the spectrum of the parent compound, again suggesting that oxygen was added to the tert-butylbenzyl moiety and water molecules were lost upon fragmentation. Based on this data, M6 was identified as a sulfate conjugate of (3- { [ [4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid.

Metabolite M10

Metabolite M10 has a protonated molecular ion at M/z 401 and it is found only in feces and in female urine. It has a retention time on HPLC of about 13:26 (min: sec). The result that the molecular ion at m/z 401 was 68 daltons below the parent compound suggests that it is a cleavage product. The CID product ion spectrum of M10 shows the primary ions at M/z 303, 160, and 145. The result that the ion at m/z 303 is 98amu lower than the protonated molecule ion suggests that sulfuric acid is lost from the molecule. The result that the ion at m/z 145 is lower than the ion 2amu of the parent compound at m/z 147 indicates that the tert-butylbenzyl group has been hydroxylated and that a water molecule has been lost during fragmentation. Based on these data, the structure of M10 is believed to be the sulfate conjugate of pyridine-3-sulfonic acid 4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzylamide.

Metabolite M11

Metabolite M11 has a retention time of about 12:04-14:20 (min: s) and it is found in urine and bile and shows protonated molecular ions at M/z 335. The result that the molecular ion at m/z335 is lower than 134 daltons for the parent compound indicates that it is a cleavage product. The CID fragmentation pattern of M11 has primary fragments at M/z 289, 146 and 131. The result of the loss of 46amu of ion from the molecule at m/z 289 suggests the presence of a carboxylic acid moiety. The ions at m/z 131 and 146 may result from carboxyisopropylbenzyl and carboxyisopropylbenzylamine moieties, respectively, with contaminants that lose formic acid. Based on these data, the structure of M11 was considered to be 2-methyl-2- {4- [ (pyridine-3-sulfonylamino) -methyl ] -phenyl } -propionic acid.

Metabolite M3

Metabolite M3 had a retention time on HPLC of approximately 12:33 to 14:21 (min: s) and was found in (male animals) and feces. It shows protonated molecular ions at m/z499 (30 amu above the parent compound). The CID product ion spectrum shows major fragments at m/z453, 356, 310, 165 and 131. The ion at m/z 165 is similar to that of the parent compound, suggesting that the methylphenoxyacetic moiety is unchanged. The ion at m/z356 was higher than the 30amu observed in the parent compound, suggesting that two oxygen atoms were added and two hydrogen atoms were lost. The ions at m/z 310 and 131 were generated by the loss of 46amu from the ions at m/z356 and 177 respectively, suggesting the presence of carboxylic acid moieties. Based on these data, metabolite M3 was identified as 2- (4- { [ (3-carboxymethoxy-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenyl) -2-methyl-propionic acid.

Metabolite M4

Metabolite M4 has a retention time on HPLC of about 13:20-15:00 (min: sec) and it is found in feces, bile, plasma and urine of female animals. It shows a protonated molecular ion at m/z 485 (16 amu above the parent compound). The CID product ion spectrum at m/z 485 shows the dominant fragment ions at m/z 467, 342, 324, 165 and 145. The ion at m/z 467 is generated by the loss of water from the molecule. The ion at m/z 342 is generated by the loss of the sulfonylpyridine moiety and subsequent loss of water to form the ion at m/z 324. The ion at m/z 165, which is similar to the parent compound ion, is generated from a protonated methylphenoxyacetic moiety. The ion at m/z 145 indicates the loss of water from the hydroxy tert-butylbenzyl moiety. Based on these data, M4 was identified as (3- { [ [4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid.

Metabolite M5

Metabolite M5 had a retention time on HPLC of approximately 15:23 (min: sec) and was found in plasma and bile. It shows a protonated molecular ion at m/z 485 (16 amu above the parent compound). The CID product ion spectrum of M5 shows the major fragment ions at M/z 439, 326, 165 and 147. The ion at m/z 439 is generated by the loss of formic acid from the molecule. The ions at m/z 326, 165 and 147 are similar to those of the parent compound, suggesting that the methylphenoxyacetic acid and tert-butyl benzyl moieties are unchanged. Based on these data, M5 was identified as (3- { [ (4-tert-butyl-benzyl) - (pyridine-N-oxide-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid.

Metabolite M14

Metabolite M14 had a retention time on HPLC of approximately 16:90 and was found only in bile. It shows a protonated molecular ion at m/z 645 of 176 daltons above the parent compound, suggesting that it is a glucuronide conjugate. The CID product ion spectrum at m/z 645 shows the major fragment ions at m/z 469, 423, 413, 326, 165 and 147. The ion at m/z 469 is generated by the loss of the glucuronic acid moiety from the molecule. The ion at m/z 423 is generated by the loss of formic acid from the molecule. Loss of the tert-butyl group produces an ion at m/z 413. The ion at m/z 326 is generated by the loss of the sulfonylpyridine moiety. The ion at m/z 165 is generated from a protonated methylphenoxyacetic acid moiety. Based on these data, the metabolite was identified as (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid glucuronide.

Metabolite M12

Metabolite M12 had a retention time on HPLC of 15:50-17:50 (min: s) and was found in feces and bile. It shows a protonated molecular ion at m/z 485 (16 amu above the parent compound). The CID product ion spectrum of M12 shows major fragments at M/z 485, 342, 305, 181, 162, and 147. The ions at m/z 162 and 147 are similar to those observed in the parent compound, suggesting that the t-butylbenzyl moiety is unchanged. The ions at m/z 181 and 342 are 16amu higher than those observed in the parent compound (at m/z 165 and m/z 326, respectively), indicating the addition of an oxygen atom to the methylphenoxyacetic moiety. The ion at m/z 305 is generated by the loss of the hydroxyphenoxyacetic acid moiety. Based on these data, M12 was identified as (5- { [ (4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -hydroxy-phenoxy) -acetic acid. Note that when this chemical name is used in this application, the position of the hydroxyl group on the phenyl ring is not specifically designated and the name is intended to include every possible position of the hydroxyl group.

A summary of the metabolic pathways of (3- { [ 4-tert-butyl-benzyl ] - (pyridine-3-sulfonyl) -amino } -methyl) -phenoxy) -acetic acid is given in FIG. 1. The main oxidation pathway is due to oxidation of the tert-butyl moiety to yield the hydroxymethyl metabolite M4 (19.7% in males; 6.5% in females). M4 was further oxidized to the carboxylic acid metabolite M3 (male 32.8%; female 1.66%) or conjugated with sulfuric acid to the metabolite M6 (male 12.7%; female 36.2%). Other minor metabolites are produced by N-oxidation of the pyridine ring (M5) and partial hydroxylation and N-dealkylation of methyl-phenoxyacetic acid followed by biphasic conjugation. In addition to the parent compound, M4, M5 and M6 are also included in the circulating metabolites.

Synthesis protocol 1. intravenous administration of a single dose of 15mg/kg¹⁴C- (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid post- (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid metabolic pathway in Sprague-Dawley rats.

Identification of in vitro metabolites of (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid in rat, dog, monkey and human liver microsomes and hepatocytes

(3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid is extensively metabolized in liver microsomes and hepatocytes in rats, dogs, monkeys and humans. The major metabolic pathways result from oxidation of the tert-butyl moiety to the alcohol, oxidation of the pyridine moiety and/or N-dealkylation of the methylphenoxyacetic acid moiety. The alcohol metabolite M4 was further oxidized to the corresponding carboxylic acid M3. In hepatocytes, M4 was conjugated with sulfuric acid. In the hepatocytes of dogs, one of the metabolites M12 was produced by aromatic oxidation of methylphenoxyacetic acid moieties.

Purpose(s) to

To determine the metabolic pathway of (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid in liver microsomes and hepatocytes in humans, rats, dogs and monkeys.

Material

Radiolabelled compounds

¹⁴C- (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid (specific activity 4.36mCi/mmol) showed a radioactive purity > 99% and was synthesized as described above.

Microsomal culture

Liver microsomes were prepared from human (HL-pool 12), rat, monkey and dog by differential centrifugation using standard methods. Before use, liver microsomes were thawed on ice and re-solubilized using 100mM potassium phosphate ph 7.4. Will 2¹⁴C]- (3- { [ 4-tert-butyl-benzyl]- (pyridine-3-sulfonyl) -amino } -methyl) -phenoxy) -acetic acid was dissolved in 100mM potassium phosphate pH7.4 to give a final substrate concentration of 20. mu.M. The samples were pre-incubated with microsomes (CYP 450; 0.5uM) for 3 minutes at 37 ℃ in a shaking water bath. By adding 100. mu.l of cofactor (1.1mM NADPH, 10mM MgCl)₂) The incubation was started with 1ml of the incubation mixture. After 30 minutes the culture was stopped by adding an equal volume of cold acetonitrile.

Hepatocyte culture

Human hepatocytes were obtained from a mixture of 3 livers. Rats and monkeys were obtained from sprague dawley rats (12 livers), macaques (1 liver) and beagle dogs (2 livers). Cryopreserved hepatocytes were suspended in William E medium containing 10% FBS to a viable count of 2 million cells/ml and started to pass 95/5O₂/CO₂Gas and incubate every 1 hour. In a 25ml Erlenmeyer flask was added 20uM (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid 2.5ml of hepatocyte suspension was added and incubated for 4 hours at 37 ℃ in a shaking water bath.

Sample analysis

Quantitative evaluation

The metabolites were quantified by measuring the HPLC peak area for each separation using beta-RAM (IN/US, Win-flow). beta-RAM provides a printout of typical integration peaks and the percentage of radioactive material in CPM. beta-RAM was operated using a solid scintillation cell.

Extraction of metabolites from in vitro matrices

The culture was terminated by the addition of an equal volume of cold acetonitrile, sonicated and centrifuged at 3000rpm for 10 minutes. The supernatant was removed and evaporated to dryness under nitrogen. The residue was redissolved with 50: 50 (acetonitrile: water) and an aliquot (50-90. mu.l) was injected onto an HPLC column for analysis.

High performance liquid chromatography

The HPLC system consisted of an HP-1100 solvent delivery system, an HP-1100 membrane degasser, an HP-1100 auto-injector (Hewlett Packard) and an IN/US radioactivity monitor (. beta. -RAM). Chromatography was performed using a YMCAQ (C-18) column (4.6 mm. times.150 mm, 3um) (Waters, Milford, Mass.). The mobile phase initially consisted of 10mM ammonium acetate containing formic acid (solvent A) and acetonitrile (solvent B) at pH 3.5.

The solvent transport step gradient program was performed as follows:

time (min)% solvent A% solvent B

0-3 90 10

3-25 35 65

25-26 10 90

26-29 10 90

29-31 90 10

31-35 90 10

The system was allowed to equilibrate for 10 minutes, after which the next injection was made. The flow rate was maintained at 1.0 ml/min throughout the analysis.

Mass spectrometry

Metabolites were identified using a Finnigan TSQ7000 triple quadrupole mass spectrometer equipped with an API-2 electrospray interface. The HPLC column effluent was separated to introduce about 50. mu.l/min into the API interface. The remaining effluent was passed to the flow cell of beta-RAM. The beta-RAM response was recorded as a real-time analog signal with the MS data acquisition system. The data collected from the radioactivity and MS detectors was separated according to the residence volume flowing from the MS into the radioactivity detector (corresponding to a residence time of about 0.2 minutes). Electrospray voltage was run at-4.5 eV into mass spectrometer data collected as positive ions. Collision Induced Dissociation (CID) studies were performed in Q2 using argon, applying a collision energy of 30-35eV and a collision gas consistency of about 2.1 mTorr.

Results and discussion

Transformation in microsomes

The conversion of (3- { [ 4-tert-butyl-benzyl ] - (pyridine-3-sulfonyl) -amino } -methyl } -phenoxy) -acetic acid in liver microsomes was highest in rats (94.3%), followed by humans (92.8%), monkeys (74.9%) and dogs (41.6%). A representative HPLC chromatogram for microsomal culture is shown in fig. 1. Metabolites were quantified by on-line radioactive counting and the relative percentages are listed in table 1.

Transformation in hepatocytes

(3- { [ 4-tert-butyl-benzyl ] - (pyridine-3-sulfonyl) -amino } -methyl) -phenoxy) -acetic acid was incubated with rat, dog, monkey and human hepatocytes. Relative conversion rates were rat ═ monkey > human > dog for 4 species. The relative percentage of metabolites in hepatocyte culture is listed in table 2. A representative HPLC chromatogram of the hepatocyte culture is shown in fig. 2.

Fragmentation of (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid by LC/MS/MS

(3- { [ 4-tert-butyl-benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid has a retention time on HPLC of 17-17:28 (min: s) and shows the protonated molecular ion at m/z 469. CID product ion spectra of m/z 469 show the major fragment ions at m/z 423, 413, 326, 165 and 147 (FIG. 3). The ion at m/z 423 is generated by the loss of formic acid from the molecule. Loss of the tert-butyl group produces an ion at m/z 413. The ion at m/z 326 results from the loss of the sulfonylpyridine moiety. The ion at m/z 165 results from a protonated methylphenoxyacetic acid moiety, while the ion at m/z 147 results from t-butyl *. The proposed fragmentation of (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid is shown below:

identification of metabolites

Metabolite M21

Metabolite M21 has protonated molecular ions at M/z 321 and a retention time on HPLC of 10:58 (min: sec). It is found in rat, dog and monkey hepatocytes. The molecular ion at m/z 321 suggests N-debenzylation and hydroxylation of the parent compound. The CID product ion spectrum of M21 shows the dominant ions at M/z 178, 160, 145, 133, and 78 (fig. 4). The ion at m/z 178 is generated by cleavage of the sulfonamide linkage. Water is lost from the ions at m/z 178 to give ions at m/z 160. The ion at m/z 145 is 2amu lower than the ion of the parent compound at m/z 147, suggesting hydroxylation on the t-butyl portion of the molecule. The ion at m/z 78 results from the stabilization of the charge on the pyridine ring. Based on these data, the structure of M21 was identified as pyridine-3-sulfonic acid 4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzylamide.

Metabolite M11

Metabolite M11 had a retention time on HPLC of 9:48-10:48 (min: sec) and the protonated molecular ion is shown at M/z 335. It is found in liver microsomes of rats and monkeys. The molecular ion at m/z335 is lower than the parent compound 134 daltons, suggesting that it is a cleavage product. The CID product ion spectrum of M11 shows fragmentation at M/z 289, 146 and 131 (fig. 5). The ion at m/z 289 lost 46amu from the molecular ion, suggesting the presence of a carboxylic acid moiety. The ions at m/z 131 and 146 may result from modified tert-butylbenzyl and tert-butylbenzyl amine moieties with loss of formic acid contaminants. Based on the fragmentation pattern and molecular ion, it was suggested that M11 was formed as a result of N-debenzylation of phenoxyacetic acid moieties followed by oxidation of the tert-butyl moiety to a carboxylic acid. It was therefore identified as 2-methyl-2- {4- [ (pyridine-3-sulfonylamino) -methyl ] -phenyl } -propionic acid.

Metabolite M2

Metabolite M2 had a retention time on HPLC of 12:24 (min: s) and showed protonated molecular ion at M/z 501 (32 amu above the parent compound). M2 was found in rat and monkey hepatocytes. It shows the primary fragments at m/z 483, 342, 324, 165 and 145 (FIG. 6). The ion at m/z 483 is generated by the loss of water from the molecule. The ion at m/z 342 results from the loss of the sulfonylpyridine moiety, followed by the loss of water to form the ion at m/z 324. Similar ions at m/z 165 to the parent compound are generated from methylphenoxyacetic moieties. The ion at m/z 145 indicates the loss of water from the modified tert-butylbenzyl moiety. Based on these data, M2 was identified as dihydroxy (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid. Note that when this chemical name is used in this application, the position of the hydroxyl group on the pyridyl ring is not specifically designated and the name is intended to include every possible position of the hydroxyl group.

Metabolite M3

Metabolite M3 had a retention time on HPLC of 11:55-13:54 (min: sec) and showed protonated molecular ions at M/z499 (30 amu above the parent compound). It is found in liver microsomes and hepatocytes of all species in rats, monkeys, and humans. The CID product ion spectrum of M3 shows major fragments at M/z453, 356, 310, 177, 165 and 131 (fig. 7). The ion at m/z 165 is similar to that of the parent compound, suggesting that the methylphenoxyacetic moiety is unchanged. The ion at m/z356 was 30amu higher than that observed in the parent compound, suggesting that two oxygen atoms were added and two hydrogen atoms were lost. The ions at m/z 310 and 131 were generated by the loss of 46amu from the ions at m/z356 and 177 respectively, suggesting the presence of a carboxylic acid moiety. Based on these data, metabolite M3 was identified as carboxy (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid.

Metabolite M4

Metabolite M4 had a retention time on HPLC of 13:29 (min: s) and showed protonated molecular ion at M/z 485 (16 amu higher than the parent compound). It is found in rat, monkey and human liver microsomes and human hepatocytes. The CID product ion spectra show the major fragment ions at m/z 467, 342, 335, 165 and 145 (FIG. 8). The ion at m/z 467 results from the loss of water from the molecule. The ion at m/z 342 results from the loss of the sulfonylpyridine moiety, followed by the loss of water to form the ion at m/z 324. The ion at m/z 165, which is similar to the parent compound ion, results from a protonated methylphenoxyacetic moiety. The ion at m/z 145 indicates the loss of water from the modified tert-butylbenzyl moiety. Based on these data, M4 was identified as hydroxy (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid.

Metabolite M19

Metabolite M19 had a retention time on HPLC of 14:40 (min: s) and showed protonated molecular ion at M/z 321 (lower than the parent compound 148 amu). It was found in all kinds of microsomes analyzed. The CID product ion spectrum of M19 shows the major fragment ions at M/z 321, 265, 162, and 147 (fig. 9). Ions at m/z 162 and 147 were observed in the CID product ion spectrum of the parent compound, suggesting that the phenyl tert-butyl group was unchanged and that the molecule had undergone N-dealkylation. The ion at m/z 265 results from the loss of the tert-butyl moiety, suggesting that oxidation has occurred on the pyridine moiety. Based on this data, M19 was identified as 5-hydroxy-pyridine-3-sulfonic acid 4-tert-butyl-benzylamide. Note that when this chemical name is used in this application, the position of the hydroxyl group on the pyridyl ring is not specifically designated and the name is intended to include every possible position of the hydroxyl group.

Metabolite M6

Metabolite M6 had a retention time on HPLC of 11:52 (min: s) and showed protonated molecular ion at M/z 565 higher than the parent compound 96amu, suggesting the addition of an oxygen atom and a sulfuric acid moiety. It is found in rat, monkey and human hepatocytes. The CID product ion spectrum of M6 shows major fragments at M/z 485, 467, 342, 324, 165, and 145 (fig. 10). The ions at m/z 485 and 467 result from the loss of sulfuric acid and water from the protonated molecular ions. The ion at m/z 165 is similar to that of the parent compound, indicating that the methyl-phenoxyacetic acid moiety is unchanged. The ion at m/z 342 (higher than the 16amu observed in the parent compound) indicates that an oxygen atom has been added to the tert-butylbenzyl moiety. The ions at m/z324 and 145 were lower than the 2amu ion on the corresponding parent compound spectrum, again suggesting that the tert-butylbenzyl moiety is charged with oxygen and water molecules are lost upon fragmentation. Based on these data, M6 was identified as a sulfate conjugate of hydroxy- (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid.

Metabolite M5

Metabolite M5 had a retention time on HPLC of approximately 15:29 and showed protonated molecular ion at M/z 485 (16 amu higher than the parent compound). M5 was found to be present in liver microsomes of rats, dogs, monkeys and humans. CID product ion spectra at m/z 485 shows the major fragment ions at m/z 439, 326, 165 and 147 (FIG. 11). The ions at m/z 326, 147 and 165 are all similar to those observed in the CID spectrum of the parent compound and suggest that the phenyl tert-butyl and phenoxy acetic acid moieties are unchanged. The ion at m/z 439 was 16amu higher than the ion observed in the parent compound. This data suggests hydroxylation of the pyridine moiety. In addition, treatment of the metabolite with titanium trichloride resulted in disappearance of the peak at 15.3 minutes on the chromatogram and an increase in the relative area of (3- { [ 4-tert-butyl-benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid, suggesting that M5 is an N-oxide of the parent compound. Based on these data, M5 was identified as (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid N-oxide.

Metabolite M12

Metabolite M12 had a retention time on HPLC of approximately 16:01 (min: sec) and was found only in liver microsomes in dogs. It shows a protonated molecular ion at m/z 485 (16 amu above the parent compound). The CID product ion spectrum of metabolite M12 shows major fragments at M/z 485, 342, 305, 181, 162, and 147 (FIG. 12). The ions at m/z 162 and 147 are similar to those observed in the parent compound, suggesting that the t-butylbenzyl moiety is unchanged. The ions at m/z 181 and 342 are 16amu higher than those observed in the parent compound (at m/z 165 and 326, respectively), indicating the addition of an oxygen atom to the methyl-phenoxyacetic acid moiety. The ion at m/z 305 is generated by the loss of the phenoxyacetic acid moiety. Based on these data, M12 was identified as hydroxy (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid. Note again that when this chemical name is used in this application, the position of the hydroxyl group on the phenyl ring is not specifically designated and the name is intended to include every possible position of the hydroxyl group.

Metabolite M20

Metabolite M20 had a retention time on HPLC of approximately 19:45 minutes and showed protonated molecular ions at M/z 305 (16 amu below the parent compound). The CID product ion spectrum of metabolite M12 shows major fragments at M/z 485, 342, 305, 181, 162, and 147. The CID ion spectrum of M20 shows the major fragment ions at M/z 162 and 147 (FIG. 13). These ions were observed in the CID product ion spectrum of the parent compound and thus suggest that (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid had undergone N-dealkylation, producing a cleaved metabolite at m/z 305. Based on these data, M20 was identified as pyridine-3-sulfonic acid 4-tert-butyl-benzylamide.

(3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid is extensively metabolized in liver microsomes and hepatocytes in rats, dogs, monkeys and humans. The proposed metabolic pathway is shown in synthetic scheme 2. The major metabolic pathways result from oxidation of the tert-butyl moiety to an alcohol, oxidation of the pyridine moiety, and/or N-dealkylation of the methylphenoxyacetic acid moiety. The alcohol metabolite M4 was further oxidized to the corresponding carboxylic acid M3. In hepatocytes, M4 was conjugated with sulfuric acid. In the hepatocytes of dogs, one of the metabolites M12 was produced by aromatic oxidation of methylphenoxyacetic acid moieties.

TABLE 1 percentage of metabolites of (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid in rat, dog, monkey and human liver microsomes

Percent radioactivity of metabolite sequence number

RT M/z rat dog monkey human

M11 9:48 335 20.7 - 7.97 -

M3 11:55 499 56.2 - 8.80 50.0

M4 13:29 485 4.38 11.5 47.3 26.5

M5 14:40 321 3.26 3.61 4.37 7.06

M19 15:29 485 7.80 20.1 5.21 -

Parent body 17: 004695.7058.425.27.16

M20 19:45 305 - 1.96 - -

The parent compound is (3- { [ 4-tert-butyl-benzyl ] - (pyridine-3-sulfonyl) -amino } -methyl) -phenoxy) -acetic acid; RT is the retention time in minutes: seconds.

TABLE 2 The percentages of metabolites of (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid in rat, dog, monkey and human hepatocytes

Percent radioactivity of metabolite sequence number

RT M/z rat dog monkey^*Human being^*

M21 10:58 321 8.91 7.92 4.53 -

M6 11:52 565 9.55 - 52.9 29.6

M2 12:24 501 15.1 6.59 - -

M3 13:54 499 57.3 12.3 29.9 17.8

M4 13:29 485 - - - 41.2

M12 16:01 485 - 15.0 - -

Parent body 17: 28469-51.4-5.68

^*Monkey and human hepatocyte samples were analysed by HPLC using the same gradient and column, but the mobile phase contained ammonium acetate (10mM) at ph 5.0. The retention times given are also specific to the specific gradient described above.

Synthesis scheme 2Proposed¹⁴C- (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid is metabolized after in vitro culture in liver microsomes and hepatocytes.

Metabolites produced using recombinant human P-450 s

Recombinant human cytochrome P-450 isoforms rCYP3A4, rCYP3A5, and rCYP2C8 were purchased from Gentest (Woburn, Mass.). The amount of rCYP expressed per culture was about 1mg protein/ml culture. Before use, the microsomes were thawed on ice and 100mM pH7.4 containing (3- { [ 4-tert-butyl-benzyl)]Potassium phosphate of- (pyridine-3-sulfonyl) -amino } -methyl) -phenoxy) -acetic acid (50uM) was redissolved. The samples were pre-incubated with recombinant CYP for 3 minutes at 37 ℃ in a shaking water bath. By adding 100. mu.l of cofactor (1.1mM NADPH, 10mM MgCl)₂) The culture was started. After incubation for 60 minutes, the sample was acidified to pH3.0 with acetic acid and extracted with an equal volume of methyl tert-butyl ether (MTBE). The MTBE layer was evaporated to dryness under a nitrogen atmosphere and redissolved with 10mM ammonium acetate acetonitrile (1: 1) for LC/MS analysis.

L C/MS structural characterization of P-450 metabolites

The HPLC system consisted of an HP-1100 solvent delivery system, an HP-1100 membrane degasser, an HP-1100 auto-injector (Hewlett Packard) and an IN/US radioactivity monitor (. beta. -RAM). Chromatography was carried out using a YMCODS AQ (C-18) column (4.6 mm. times.150 mm, 3 um). The mobile phase initially consisted of 10mM ammonium acetate containing acetic acid (solvent A) and acetonitrile (solvent B) at pH 5.0. Metabolites were identified using a Micromass Q-Tof 2(Beverly, MA) mass spectrometer equipped with an electrospray interface. The HPLC column effluent was separated to introduce about 50. mu.l/min into the API interface. The remaining effluent was passed to the flow cell of beta-RAM. The beta-RAM response was recorded as a real-time analog signal with the MS data acquisition system. The data collected from the radioactivity and MS detectors were separated according to the residence volume flowing from MS into the radioactivity detector (equivalent to a residence time of-0.1 min). Electrospray voltage was run at-3 eV into mass spectrometer data collected as positive ions. A Collision Induced Dissociation (CID) study was performed in Q2 using argon, with a collision energy of 20-30eV and a penning pressure of-5X 10-5 Torr. The internal lock mass (quinidine, m/z 325.1916) was used by the indexed Lockspray throughout the analysis, so that a calibration (calibrent) was introduced into the mass spectrometer every 5 seconds.

Metabolite M22: (3- { [ [4- (1, 2-dihydroxy-1-methyl-ethyl) -benzyl ] -ethyl } -methyl-ethyl) -benzyl]- (pyridine-3-sulfonyl) - Amino) -methyl) -phenoxy) -acetic acid

Metabolite M22 had a retention time on HPLC of 14 minutes and a protonated molecular ion at M/z 487 (higher than the parent compound 18 amu). The CID product ion spectrum of metabolite M2 shows the major fragment ions at M/z 469, 451, 423, 395, 335, 326, 165 and 147 (FIG. 14). The ion at m/z 165 was similar to that observed in the parent drug, suggesting that the phenoxyacetic acid moiety was not altered. The ions at m/z 469 and 451 lost two 18amu in succession from the protonated molecular ion, suggesting that two molecules of water have been lost. Empirical information obtained from empirical formula C suggested by high resolution mass spectrometry (Q-Tof)₂₄H₂₇N₂O₇And S. Based on these data and the LC-NMR data supported, M22 was identified as (3- { [ [4- (1, 2-dihydroxy-1-methyl-ethyl) -benzyl]- (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid.

Metabolite M23: (3- { [ [4- (1-hydroxy-1-methyl-ethyl) -benzyl ] -ethyl ester]- (pyridine-3-sulfonyl) -amino]- Methyl) -phenoxy) -acetic acid

Metabolite M23 had a retention time on HPLC of about 16.9 and a protonated molecular ion at M/z 471 (2 amu higher than the parent compound). The CID product ion spectrum of metabolite M23 shows the major fragment ions at M/z453, 335, 310, 165 and 131 (FIG. 15). The ion at m/z 165 was similar to that observed in the parent drug, suggesting that the phenoxyacetic acid moiety was not altered. The ion at m/z453 lost 18amu from the protonated molecular ion, suggesting that one water molecule had been lost. The ions at m/z 310 and 131 (16 amu lower than the ion observed in the parent compound) suggest that the methyl group is replaced by a water molecule and then lost during fragmentation. Based on these data and the supported LC-NMR data, M23 was identified as (3- { [ [4- (1-hydroxy-1-methyl-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid.

Metabolite M24: (3- { [ [4- { 1-dimethyl-2-oxo-ethyl) -benzyl]- (pyridine-3-sulfonyl) -ammonia Base of]-methyl } -phenoxy) -acetic acid

Metabolite M24 had a retention time on HPLC of approximately 18.6 minutes and a protonated molecular ion at M/z 483 (lower than the parent compound 14 amu). The CID product ion spectrum of metabolite M24 shows the major fragment ions at M/z 483, 437, 409, 340, 165 and 161 (fig. 16). The ions at m/z 423, 340 and 161 were all higher than the 14amu ion observed in the spectrum of the parent drug, suggesting the addition of an oxygen atom and the loss of a contaminant of two hydrogen atoms. The high resolution mass spectrum obtained for this metabolite gave a molecular weight of 483.1590 and C₂₅H₂₇N₂O₆Experimental formula of S. Based on these data and the LC-NMR data supported, M24 was identified as (3- { [ [4- (1, 1-dimethyl-2-oxo-ethyl) -benzyl]- (pyridine-3-sulfonyl) -amino) -methyl } -phenoxy) -acetic acid.

Metabolite M26: (3- { [ (4-isopropenyl-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy Phenyl) -acetic acid

Metabolite M26 has a retention time on HPLC of22 minutes and has protonated molecular ion at m/z453 (16 amu below the parent compound). CID product ion spectra show major fragment ions at m/z453, 407, 310, 165 and 131 (FIG. 17). The ion at m/z 165 was similar to that observed in the parent compound, suggesting that the phenoxyacetic acid moiety was not altered. The ions at 407, 310 and 131 were all 16amu lower than those observed in the spectrum of the parent compound, suggesting the loss of 16 mass units from the tert-butyl moiety. The high resolution mass spectrum obtained for this metabolite gave a molecular weight of 453.1487 and C₂₄H₂₅N₂O₅Experimental formula of S. The combination of these data for this metabolite with LC-NMR results in the formation of the amino group as (3- { [ (4-isopropenyl-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid has identical terminal olefin groups.

Synthetic schemes

The following preparative procedures relate to the synthesis of the intermediates used in the synthesis of the compounds of examples 1 and 2.

Preparation 1

{3- [ (pyridine-3-sulfonylamino) -methyl group]-phenoxy } -acetic acid tert-butyl ester

Step A

(3-formyl-phenoxy) -acetic acid tert-butyl ester

To a solution of 3-hydroxybenzaldehyde (5.00g, 40.9mmol) in DMF (40mL) was added a 1M solution of potassium tert-butoxide in tert-butanol (40.9mL, 40.9 mmol). The reaction was stirred for 2 min and tert-butyl bromoacetate (6.61mL, 40.9mmol) was added. The reaction was stirred for 1 hour and quenched with 200mL of water. The product was extracted into EtOAc and the organic solution was washed with water, MgSO₄Dried, filtered and concentrated in vacuo. Purification by flash chromatography on silica gel (9)1 hexanes: EtOAc) gave the title compound as a clear oil (3.53g).¹H NMR(400MHz，CDCl₃)δ9.94(s，1H)，7.48(m，2H)，7.32(s，1H)，7.21(m，1H)，4.56(s，2H)，1.45(s，9H)。

Step B

[3- (hydroxyimino-methyl) -phenoxy]-tert-butyl acetate

To a solution of (3-formyl-phenoxy) -acetic acid tert-butyl ester (2.05g, 8.68mmol) in MeOH (30mL) was added NH₂OH HCl (0.66g, 9.54mmol) and pyridine (3.5mL, 43.4mmol) and the reaction was stirred for 2 hours. MeOH was removed in vacuo and the residue was diluted with EtOAc and 1 NHCl. The layers were separated and the aqueous solution was washed with EtOAc. With MgSO₄The combined organic layers were dried, filtered and concentrated in vacuo to give the title compound (1.99 g).¹H NMR(400MHz，CDCl₃)δ8.07(s，1H)，7.23-7.28(m，2H)，7.12(m，1H)，6.93(d，1H)，4.51(s，2H)，1.46(s，9H)。

Step C

(3-aminomethyl-phenoxy) -acetic acid tert-butyl ester

To [3- (hydroxyimino-methyl) -phenoxy]To the solution of tert-butyl acetate (2.25g, 5.96mmol) in EtOH (10mL) was added Raney nickel in 100mL of ethanol (ca. 1g, washed with water followed by EtOH). Additional EtOH (90mL) was required for transfer. Ammonium hydroxide (10mL) was added and the mixture was washed with H at 45psi₂Shake for 4 hours. Through Celite^*The catalyst was removed by filtration (celite) and the solution was concentrated to a clear oil. Purification by flash chromatography on silica gel (96.5/3.5/0.1-9/1/0.1 CH)₂Cl₂/MeOH/NH₄OH) to yield the title compound as a yellow oil.¹H NMR(400MHz，CDCl₃)δ7.23(m，1H)，6.92(m，2H)，6.72(d，1H)，4.50(s，2H)，3.82(s，2H)，1.96(m，2H)，1.46(s，9H)；MS 238(M+1)。

Step D

To (3-aminomethyl-phenoxy) -acetic acid tert-butyl ester (296mg, 1.25mmol) was dissolved in CH at 0 deg.C₂Cl₂To the resulting solution was added pyridine-3-sulfonyl chloride hydrochloride (279mg, 1.31mmol), followed by Et₃N (0.36mL, 2.6 mmol). The reaction was stirred at room temperature for 24 hours and saturated NaHCO with a 1: 1 aqueous solution₃And (4) inhibiting by using an aqueous solution. By CH₂Cl₂(3 ×) wash the aqueous solution. The combined organic solutions were dried (MgSO)₄) Filtered and concentrated. Medium pressure chromatography (1: 1 hexanes: EtOAc) afforded the title compound as a white solid (369.5 mg).¹H NMR(400MHz，CDCl₃)δ9.04(s，1H)，8.75(m，1H)，8.09(d，1H)，7.44(m，1H)，7.15(m，1H)，6.76(m，3H)，5.23(bs，1H)，4.44(s，2H)，4.16(d，2H)，1.47(s，9H)；MS 379(M+1)。

Preparation 2

2- (4-Bromomethyl-phenyl) -2-methyl-propionic acid ethyl ester

Step A

2-methyl-2-p-tolyl-propionic acid ethyl ester

NaH (60% by weight oil dispersion, 3.9g, 98.2mmol) was washed with DMF and fresh DMF (175mL) was added. The mixture was cooled to 0 ℃ and Mel (6.1mL, 98.2mmol) was added followed by a solution of ethyl p-tolylacetate (5.0g, 28.05mmol) in DMF (15 mL). The reaction was stirred at room temperature for 48 hours. Water was added and the aqueous solution was washed with EtOAc (3 ×). With water (4X) and saturated NaHCO₃The combined organic solutions were washed with aqueous solution (1 ×). The organic solution was dried (MgSO₄) Filtered and concentrated. Medium pressure chromatography (95: 5 hexane: EtOAc) afforded 2-methyl-2-p-tolyl-propionic acid ethyl ester (1.2 g).¹H NMR(400MHz，CDCl₃)δ7.21(d，2H)，7.11(d，2H)，4.10(q，2H)，2.31(s，3H)，1.54(s，6H)，1.17(t，3H)。

Step B

2- (4-Bromomethyl-phenyl) -2-methyl-propionic acid ethyl ester

To ethyl 2-methyl-2-p-tolyl-propionate (263mg, 1.27mmol) and N-bromosuccinimide (272mg, 1.53mmol) were dissolved in CCl₄To the resulting solution (15mL) was added 1, 1' -azobis (cyclohexanecarbonitrile) (15.5mg, 0.06 mmol). The reaction system was heated under reflux for 1 hour and water and CH were used₂Cl₂And (6) diluting. Separate the layers and use CH₂Cl₂(3X) Wash the aqueous layer. The combined organic layers were dried (MgSO)₄) Filtered and concentrated. Medium pressure chromatography (95: 5 hexanes: EtOAc) afforded 2- (4-bromomethyl-phenyl) -2-methyl-propionic acid ethyl ester (354 mg).¹HNMR(400MHz，CDCl₃)δ7.31(m，4H)，4.47(s，2H)，4.10(q，2H)，1.54(s，6H)，1.17(t，3H)。

Preparation 3

[2- (4-bromomethyl-phenyl) -2-methyl-propoxy]-tert-butyl-dimethyl-silane

Step A

2-methyl-2-p-tolyl-propan-1-ol

To a solution of ethyl 2-methyl-2-p-tolyl-propionate (510mg, 2.47mmol) in THF (10mL) at 0 deg.C was added lithium aluminum hydride (1M Et₂O solution, 2.6mL, 2.6 mmol). The reaction was stirred for 0.5 h and the reaction was quenched by the sequential addition of water (0.1mL), 15% NaOH (0.1mL) and water (0.3 mL). The reaction was diluted with EtOAc and dried (MgSO)₄) Filtration and concentration gave 2-methyl-2-p-tolyl-propan-1-ol (405 mg).¹H NMR(400MHz，CDCl₃)δ7.27(d，2H)，7.15(d，2H)，3.58(s，2H)，2.32(s，3H)，1.31(s，6H)；MS 165(M+1)。

Step B

Tert-butyl-dimethyl- (2-methyl-2-p-tolyl-propoxy) -silane

To a solution of 2-methyl-2-p-tolyl-propan-1-ol (405mg, 2.46mmol) in DMF (5mL) was added imidazole (335mg, 4.92mmol) followed by tert-butyldimethylsilyl chloride (465mg, 3.08 mmol). The reaction was stirred for 24 hours and water was added. The aqueous solution was washed with EtOAc (3 ×). The combined organic solutions were washed with water (4 ×). The organic solution was dried (MgSO₄) Filtered and concentrated. Medium pressure chromatography (hexanes) gave tert-butyl-dimethyl- (2-methyl-2-p-tolyl-propoxy) -silane (619 mg).¹H NMR(400MHz，CDCl₃)δ7.26(d，2H)，7.10(d，2H)，3.49(s，2H)，2.31(s，3H)，1.27(s，6H)，0.85(s，9H)，-0.06(s，6H)。

Step C

[2- (4-bromomethyl-phenyl) -2-methyl-propoxy]-tert-butyl-dimethyl-silane

The title compound was prepared according to the procedure described in preparation 2, step B, using tert-butyl-dimethyl- (2-methyl-2-p-tolyl-propoxy) -silane (398mg, 1.42mmol) as the starting material. The reaction time was 24 hours and the compound was purified by medium pressure chromatography using hexane as eluent.¹H NMR(400MHz，CDCl₃)δ7.31(m，4H)，4.46(s，2H)，3.48(s，2H)，1.26(s，6H)，0.81(s，9H)，-0.10(s，6H)。

Example 1

2- (4- { [ (3-carboxymethoxy-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenyl) -2-methyl- Propionic acid

Step A

2- (4- { [ (3-tert-Butoxycarbonylmethoxy-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenyl) -2-methyl-propionic acid ethyl ester

NaH (60% by weight oil dispersion, 23mg, 0.55mmol) was washed with DMF (5mL) and fresh DMF was added. The reaction was cooled to 0 ℃ and {3- [ (pyridine-3-sulfonylamino) -methyl ] was added]-phenoxy } -acetic acid tert-butyl ester (184mg, 0.486mmol) in DMF (1 mL). The reaction was stirred for 1 hour and a solution of 2- (4-bromomethyl-phenyl) -2-methyl-propionic acid ethyl ester (145mg, 0.51mmol) in DMF (1mL) was added. The reaction was warmed to room temperature and heated at 100 ℃ for 2 hours. Water was added and the aqueous solution was washed with EtOAc (3 ×). The combined organic solutions were washed with water (5 ×), dried (MgSO)₄) Filtered and concentrated. Medium pressure chromatography (2: 1 hexane: EtOAc) afforded 2- (4- { [ (3-tert-butoxycarbonylmethoxy-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenyl) -2-methyl-propionic acid ethyl ester (138 mg).¹H NMR(400MHz，CDCl₃)δ9.01(s，1H)，8.76(m，1H)，7.97(m，1H)，7.40(m，1H)，7.19(d，2H)，7.13(m，1H)，7.02(d，2H)，6.76(m，1H)，6.67(m，2H)，4.41(s，2H)，4.33(s，4H)，4.10(q，2H)，1.52(s，6H)，1.49(s，9H)，1.17(t，3H)。

Step B

2- (4- { [ 3-carboxymethoxy-benzyl) - (pyridine-3-sulfonyl) -amino]-methyl } -phenyl) -2-methyl- Propionic acid ethyl ester

To the 2- (4- { [ (3-tert-Butoxycarbonylmethoxy-benzyl) - (pyridine-3-sulfonyl) -amino group]To a solution of ethyl (138mg, 0.237mmol) of-methyl } -phenyl) -2-methyl-propionate in MeOH (5mL) was added aqueous NaOH (2N, 0.36mL, 0.72 mmol). The reaction was heated at 100 ℃ for 1 hour and cooled to room temperature. The reaction was concentrated in vacuo and diluted with water and EtOAc. The pH was adjusted to about 5 with 1n hcl. The aqueous solution was washed with EtOAc (3 ×). The combined organic solutions were dried (MgSO)₄) Filtered and concentrated. To the aqueous solution was added sodium chloride and the solution was washed with EtOAc (3 ×). The combined organic solutions were dried (MgSO)₄) Filtered and concentrated. The combined title compound (104mg) was washed with brineFurther purified and used in the next step. MS 527(M + 1).

Step C

To 2- (4- { [ (3-carboxymethoxy-benzyl) - (pyridine-3-sulfonyl) -amino](iii) -methyl } -phenyl) -2-methyl-propionic acid ethyl ester (83mg, 0.157mmol) dissolved in THF (10mL) to a solution was added water (1mL) and LiOH. H₂O (66mg, 1.58 mmol). The reaction system was heated under reflux for 24 hours. Then LiOH. H is added₂O (66mg, 1.58mmol) in water (2mL) and the reaction was heated at reflux for 30 h. The mixture was concentrated in vacuo and THF (3mL) and water (1.5mL) were added to the residue. The reaction was heated under reflux for 24 hours and cooled to room temperature. The solution was diluted with water and the pH was adjusted to about 5 by the addition of 1N HCl. Sodium chloride was added and the aqueous solution was washed with EtOAc (3 ×). The combined organic solutions were dried (MgSO)₄) Filtered and concentrated to give the title compound (74 mg).¹H NMR(400MHz，CDCl₃)δ8.94(s，1H)，8.73(s，1H)，8.13(d，1H)，7.55(m，1H)，7.23(d，2H)，7.11(m，3H)，6.78(d，1H)，6.73(d，1H)，6.68(s，1H)，4.90(s，2H)，4.39(s，2H)，3.30(s，2H)，1.50(s，6H)；MS 497(M-1)。

Example 2

(3- { [ [4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzyl ] -ethyl } -methyl-ethyl-benzyl-amine]- (pyridine-3-sulfonyl) -amino]-methyl } -benzene Oxy) -acetic acid

Step A

(3- { [ {4- [2- (tert-butyl-dimethyl-silanyloxy) -1, 1-dimethyl-ethyl]-benzyl } - (pyridine-3- Sulfonyl) -amino]-methyl } -phenoxy) -acetic acid tert-butyl ester

The title compound was prepared by alkylating {3- [ (pyridine-3-sulfonylamino) -methyl ] -phenoxy } -acetic acid tert-butyl ester (122mg, 0.322mmol) with [2- (4-bromomethyl-phenyl) -2-methyl-propoxy ] -tert-butyl-dimethyl-silane (121mg, 0.339mmol) as described in example 1, step a. The reaction time was 1 hour. The crude product (238mg) was used in the next step without purification. MS 655(M + 1).

Step B

(3- { [ [4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzyl ] -ethyl } -methyl-ethyl-benzyl-amine]- (pyridine-3-sulfonyl) -amino]-methyl } -benzene Oxy) -acetic acid tert-butyl ester

To (3- { [ {4- [2- (tert-butyl-dimethyl-silanyloxy) -1, 1-dimethyl-ethyl]To a solution of-benzyl } - (pyridine-3-sulfonyl) -amino } -methyl) -phenoxy) -acetic acid tert-butyl ester (210mg, 0.321mmol) in THF (2mL) was added tetrabutylammonium fluoride (1mM in THF, 0.34mL, 0.34 mmol). The reaction was heated at reflux for 24 h and tetrabutylammonium fluoride (1mM in THF, 0.34mL, 0.34mmol) was added. The reaction was heated at reflux for 24 h and tetrabutylammonium fluoride (1mM in THF, 0.34mL, 0.34mmol) was added. The reaction was heated for 45 minutes and cooled to room temperature. Adding water and CH₂Cl₂And the layers were separated. By CH₂Cl₂(3 ×) wash the aqueous solution. The combined organic layers were dried (MgSO)₄) Filtered and concentrated. Medium pressure chromatographic separation (1: 1 hexane: EtOAc-3: 2 hexane: EtOAc) to give (3- { [ [4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzyl]- (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -acetic acid tert-butyl ester (108 mg). MS 541(M + 1).

Step C

A mixture of (3- { [ [4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzyl]- (pyridine-3-sulfonyl) -amino]-methyl } -phenoxy) -ethylTert-butyl ester (108mg, 0.199mmol) in CH₂Cl₂(2mL) the resulting solution was cooled to 0 ℃ and trifluoroacetic acid (1mL) was added. The reaction was stirred at room temperature for 1 hour. Passing with CH in vacuum₂Cl₂(3x) azeotropically concentrating the solution. The residue was dissolved in THF and 1N HCl (0.4mL) was added. Passing with CH in vacuum₂Cl₂(3x) azeotropically concentrating the solution. By using a solvent gradient (CH)₂Cl₂-20% MeOH CH₂Cl₂Solution) was purified by radial chromatography to give the title compound (34 mg).¹H NMR(400MHz，CDCl₃)δ8.79(s，1H)，8.69(s，1H)，8.11(d，1H)，7.52(m，1H)，7.21(d，2H)，7.09(m，1H)，7.02(d，2H)，6.76(d，1H)，6.70(m，2H)，4.86(s，2H)，4.32(m，4H)，3.48(s，2H)，1.22(s，6H)；MS 485.2(M+1)，483.4(M-1)。

The MR spectra were recorded for the proton nuclei using a Varian Unity 400 spectrometer (Varian Co., Palo Alto, Calif.) at about 23 ℃ and 400 MHz. Chemical shifts are expressed in parts per million. The peak shapes are shown below: s, singlet; d, double peak; t, triplet; q, quartet; m, multiplet; bs, broad singlet. Atmospheric Pressure Chemical Ionization (APCI) mass spectra were obtained using a fisonsplate II spectrometer (micromass inc., Beverly, Massachusetts). In describing the strength of chlorine or bromine containing ions, the expected strength ratio (to the content of chlorine or bromine) is observed³⁵Cl/³⁷About 3: 1 for Cl ions and for Cl ions⁷⁹Br/⁸¹Br-ions are 1: 1) and the only low mass ion strength is obtained.

Medium pressure chromatographic separation was performed using a Biotage purification system (Biotage, Dyax Corporation, Charlottesville, Virginia) under nitrogen pressure. Flash chromatography was performed on a glass column under nitrogen pressure using Baker Silica Gel (40 μm) (j.t. Baker, phillips burg, n.j.) or Silica Gel 60(EM Sciences, Gibbstown, n.j.). Radial chromatography separation was performed using Chromatotron (model 7924T, Harrison Research, Palo Alto, California). Dimethylformamide (DMF), Tetrahydrofuran (THF) and dichloromethane (CH) as reaction solvents₂Cl₂) Is a dry grade supplied by Aldrich chemical Company (Milwaukee, Wisconsin). The term "concentration" refers to the removal of solvent on a rotary evaporator under the suction pressure of water. The term "EtOAc" refers to ethyl acetate. The terms "methylene chloride" and "methylene chloride" are synonymous and are used interchangeably in the context and examples and preparations of this specification.

Rat EP₂Receptor binding assays

Prostaglandin E₂Receptor binding assays

Full-length EP's were prepared according to the method disclosed by Nemoto et al in Prostagladins and other Lipid Mediators, 1997, 54, 713-₂A receptor. The full-length receptor is used for preparing expression EP₂293S cells of the recipient.

Preparation of expression rat EP's according to methods known to those skilled in the art₂Prostaglandin E₂293S cells of the recipient. Generally, PCR (polymerase chain reaction) primers corresponding to the 5 'and 3' ends of the disclosed full-length receptor were prepared according to the well-known method disclosed above and used for RT-PCR reaction using total RNA from rat kidney as a source. The PCR products were cloned into PCR2.1(Invitrogen, Carlsbad, CA) by TA overhang method and the identity of the cloned receptor was confirmed by DNA sequencing. For expression, the confirmed cDNA was subcloned into the mammalian expression vector PURpCI, a vector produced by subcloning a puromycin-resistant selection marker into the mammalian expression vector pCl (Promega, Madison, Wis.).

293S cells were transfected by lipid-mediated transfection with the receptor cloned in PURpCi. Stable cell lines expressing the receptor were established by screening transfected cells with puromycin.

By intact cells³H-PGE₂Binding assays Using unlabeled PGE₂The clonal cell line expressing the greatest number of receptors was selected as the competitor.

Preparation of the film: all operations were carried out at 4 ℃. Collection of prostaglandin E expressing type 2₂(EP₂) Cells were transfected with the receptor and placed in buffer A [50mM Tris-HCI (pH7.4), 10mM MgCl₂1mM EDTA, 1mM Pefabloc peptide (Boehringer Mannheim Corp., Indianapolis, IN), 10uM Phosporamidon peptide (Sigma, St. Louis, MO), 1uM Isogastrin peptide A (Sigma, St. Louis, MO), 10uM Elastin inhibitor peptide (Sigma, St. Louis, MO), 100uM Antiproteinases peptide (Sigma, St. Louis, MO)]To 2 million cells/ml. Cells were lysed by sonication with a Branson Sonifier (Branson Ultrasonics Corporation, Danbury, CT) with 2 15 second shocks. Unlysed cells and debris were removed by centrifugation at 100Xg for 10 min. The membranes were then collected by centrifugation at 45,000Xg for 30 minutes. The precipitated membrane was resuspended to 3-10mg protein/ml, i.e.according to the method of Bradford [ Bradford, M., anal.biochem., 72,248(1976)]The protein concentration determined. The resuspended membrane was then frozen at-80 ℃ until use.

Binding assay: the frozen membrane prepared as described above was thawed and diluted to 1mg protein/ml with buffer a described above. 1 volume of membrane preparation was mixed with 0.05 volumes of test compound or buffer and 1 volume of 3nM³H-prostaglandin E₂(Amersham, Arlington Heights, IL) was mixed in buffer A. The mixture (205. mu.L total volume) was incubated at 25 ℃ for 1 hour. The membrane was then filtered through a glass fiber membrane of type GF/C (Wallac, Gaithersburg, Md.) and recovered using a Tomtec collector (Tomtec, Orange, CT). Capturing bound-containing material with said filter³H-prostaglandin E₂To allow buffer to bind to unbound³H-prostaglandin E₂Pass through the filter membrane and become waste. Then 3ml of [50mM Tris-HCl (pH7.4), 10mM MgCl₂、1mM EDTA]Each sample was washed 3 times. The filter membrane is subsequently dried by heating with a microwave oven. For measuring binding to membranes³Amount of H-prostaglandin, dry filters were placed in scintillation fluid-containing plastic bags and counted with LKB 1205 β plate reader (Wallac, Gaithersburg, Md.). According toShows 50%³H-prostaglandin E₂Determination of IC by concentration of test Compound required for specific binding₅₀。

Rat EP of example 1₂Bonding IC₅₀＝750nM

Rat EP of example 2₂Bonding IC₅₀＝170nM

The following abbreviations are used herein:

i.v. intravenous

HPLC high performance liquid chromatography

CPM counts per minute

min for

MS mass spectrometry

CID Collision induced dissociation

AMU atomic mass unit

FBS fetal bovine serum

LC liquid chromatography

DMF dimethyl formamide

EtOAc ethyl acetate

MeOH methanol

NMR nuclear magnetic resonance

Et₃N-Triethylamine

THF tetrahydrofuran

H hours

Claims

1. The compound 2- (4- { [ (3-carboxymethoxy-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenyl) -2-methyl-propionic acid, or a pharmaceutically acceptable salt thereof.

2. The compound (3- { [ [4- (2-hydroxy-1, 1-dimethyl-ethyl) -benzyl ] - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid, or a pharmaceutically acceptable salt thereof.

3. The compound (3- { [ (4-tert-butyl-benzyl) - (pyridine-N-oxide-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid, or a pharmaceutically acceptable salt thereof.

4. The compound (5- { [ (4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -hydroxy-phenoxy) -acetic acid, or a pharmaceutically acceptable salt thereof.

5. The following compounds or pharmaceutically acceptable salts thereof:

2- {4- [ pyridine-N-oxide-3-sulfonylamino) -methyl ] -phenyl } -2-methyl-propionic acid;

6. A method for determining whether a patient is administered (3- { [ 4-tert-butyl-benzyl) - (pyridine-3-sulfonyl) -amino ] -methyl } -phenoxy) -acetic acid, which method comprises the step of determining whether a sample of plasma, urine, bile or faeces obtained from said patient shows the presence of one or more compounds selected from the group consisting of:

7. A method of treating osteoporosis or assisting in the healing of a bone fracture, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound selected from the group consisting of:

8. A pharmaceutical composition comprising one or more compounds selected from the group consisting of:

2- {4- [ pyridine-N-oxide-3-sulfonylamino ] -methyl } -phenyl } -2-methyl-propionic acid;