HK1090639B - Immunosuppressant compounds and compositions - Google Patents

Description

Immunosuppressant compounds and compositions

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional patent application No.60/471,931 (filed on 19/5/2003) and U.S. provisional patent application No.60/562,183 (filed on 14/4/2004). The entire disclosures of these applications are hereby incorporated by reference in their entirety for all purposes.

Background

Technical Field

The present invention provides a novel class of immunosuppressant compounds useful in the treatment or prevention of diseases or disorders mediated by lymphocyte interactions, particularly diseases associated with EDG receptor mediated signal transduction.

Background

EDG receptors belong to a family of closely related, lipid-activated G-protein coupled receptors. EDG-1, EDG-3, EDG-5, EDG-6 and EDG-8 (also referred to as S1P1, S1P3, S1P2, S1P4 and S1P5, respectively) were identified as receptors specific for sphingosine-1-phosphate (S1P). EDG2, EDG4 and EDG7 (also referred to as LPA1, LPA2 and LPA3, respectively) are receptors specific for lysophosphatidic acid (LPA). Among S1P receptor isoforms, EDG-1, EDG-3 and EDG-5 are widely expressed in various tissues, whereas the expression of EDG-6 is largely limited to lymphoid tissues and platelets, and the expression of EDG-8 is limited to the central nervous system. EDG receptors are responsible for signal transduction and are thought to play a major role in cellular processes involving cell development, proliferation, feeding, migration, differentiation, plasticity, and apoptosis. Certain EDG receptors are associated with diseases mediated by lymphocyte interactions, for example in transplant rejection, autoimmune diseases, inflammatory diseases, infectious diseases, and cancer. Alterations in EDG receptor activity contribute to the pathology and/or symptomology of these diseases. Thus, molecules that themselves alter EDG receptor activity may be useful as therapeutic agents in the treatment of such diseases.

Summary of The Invention

The present invention relates to compounds of formula (I) and N-oxide derivatives, prodrug derivatives, protected derivatives, individual isomers and mixtures of isomers thereof; and pharmaceutically acceptable salts and solvates (e.g. hydrates) of such compounds,

wherein:

n is 1 or 2;

a is selected from-C (O) OR₉、-OP(O)(OR₉)₂、-P(O)(OR₉)₂、-S(O)₂OR₉、-P(O)(R₉)OR₉And 1H-tetrazol-5-yl; r₉Selected from hydrogen and C_1-6An alkyl group;

x is a bond or is selected from C_1-4Alkylene radical, -X₁OX₂-、-X₁NR₁₀X₂-、-X₁C(O)NR₁₀X₂-、-X₁NR₁₀C(O)X₂-、-X₁S(O)X₂-、-X₁S(O)₂X₂-、-X₁SX₂-and C_2-9A heteroarylene group; wherein X₁And X₂Independently selected from the group consisting of a bond and C_1-3An alkylene group; r₁₀Selected from hydrogen and C_1-6An alkyl group; any heteroarylene group of X is optionally selected from halo and C_1-6Radical substitution of alkyl;

y is a fused 5, 6 or 6, 6 heterobicyclic ring system comprising at least one aromatic ring, wherein said fused bicyclic ring system of Y may optionally be substituted with 1 to 3 groupsSubstituted by a group selected from halo, hydroxy, cyano, nitro, C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkyl and halo C_1-6An alkoxy group;

R₁is selected from C_6-10Aryl and C_2-9A heteroaryl group; wherein R is₁Any aryl or heteroaryl of (a) is optionally substituted with a group selected from: c_6-10Aryl radical C_0-4Alkyl radical, C_2-9Heteroaryl C_0-4Alkyl radical, C_3-8Cycloalkyl radical C_0-4Alkyl radical, C_3-8Heterocycloalkyl radical C_0-4Alkyl or C_1-6An alkyl group; wherein R is₁Any aryl, heteroaryl, cycloalkyl or heterocycloalkyl of (a) may be optionally substituted with 1 to 3 groups selected from halo, C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkyl and halo C_1-6An alkoxy group; r₁Any alkyl group of (a) may optionally have a methylene group replaced by an atom or group selected from: -S-, -S (O)₂-、-NR₁₀-and-O-; wherein R is₁₀Selected from hydrogen or C_1-6An alkyl group;

R₂、R₃、R₅、R₆、R₇and R₈Independently selected from hydrogen, C_1-6Alkyl, halo, hydroxy, C_1-6Alkoxy, halo C_1-6Alkyl and halo C_1-6An alkoxy group;

R₄selected from hydrogen and C_1-6An alkyl group; or R₇And R₂、R₄Or R₅One and R₂、R₄、R₅And R₇The atoms to which they are attached together form a4 to 7 membered ring; wherein the 4 to 7 membered ring is saturated or partially unsaturated.

A second aspect of the invention is a pharmaceutical composition comprising a compound of formula I or an N-oxide derivative, an individual isomer or a mixture of isomers thereof or a pharmaceutically acceptable salt thereof, in admixture with one or more suitable excipients.

A third aspect of the invention is a method of treating a disease in an animal in which alteration of EDG receptor mediated signal transduction is capable of preventing, inhibiting or ameliorating the pathology and/or symptomology of the disease, which method comprises administering to the animal a therapeutically effective amount of a compound of formula I or an N-oxide derivative, an individual isomer or mixture of isomers thereof or a pharmaceutically acceptable salt thereof.

A fourth aspect of the invention is the use of a compound of formula I in the manufacture of a medicament for the treatment of a disease in an animal, wherein an alteration in EDG receptor mediated signal transduction contributes to the pathology and/or symptomology of the disease.

A fifth aspect of the invention is a process for the preparation of compounds of formula I and N-oxide derivatives, prodrug derivatives, protected derivatives, individual isomers and mixtures of isomers thereof and pharmaceutically acceptable salts thereof.

Description of the preferred embodiments

The present invention provides compounds useful for the treatment and/or prevention of diseases or disorders mediated by lymphocyte interactions. Methods of treating such diseases or disorders are also provided.

Definition of

In this specification, unless defined otherwise:

"alkyl" as a group and structural element of other groups such as haloalkyl, alkoxy, acyl, alkylthio, alkylsulfonyl and alkylsulfinyl may be straight-chain or branched. "alkenyl" as a group and a structural element of another group contains one or more carbon-carbon double bonds and may be straight-chain or branched. Any double bond may be in either the cis-or trans-configuration. "alkynyl" as a group and other groups and structural elements of compounds contains at least one C.ident.C triple bond, and may also contain one or more than oneC ═ C double bonds, and may be straight chain or branched, as far as possible. Any cycloalkyl group, alone or as a structural element of another group, may contain 3 to 8 carbon atoms, preferably 3 to 6 carbon atoms. "alkylene" and "alkenylene" are divalent radicals derived from "alkyl" and "alkenyl", respectively. In this application, R¹Any alkyl of (a) may optionally be selected from-S-, -S (O)₂-、-NR²⁰-and-O- (wherein R²⁰Is hydrogen or C_1-6Alkyl) is interrupted. These groups include-CH₂-O-CH₂-、-CH₂-S(O)₂-CH₂-、-(CH₂)₂-NR²⁰-CH₂-、-CH₂-O-(CH₂)₂-and the like.

"aryl" means a monocyclic or fused bicyclic aromatic ring combination containing from six to ten ring carbon atoms. E.g. C_6-12Aryl may be phenyl, biphenyl or naphthyl, preferably phenyl. The fused bicyclic ring can be partially saturated, such as 1, 2, 3, 4-tetrahydronaphthalene, and the like. "arylene" refers to a divalent group derived from an aryl group. For example, arylene as used herein may be phenylene, biphenylene, naphthylene, and the like.

"halo" or "halogen" means F, Cl, Br or I, preferably F or Cl. Haloalkyl groups and compounds may be partially halogenated or perhalogenated, wherein in the case of polyhalogenation, the halogen substituents may be the same or different. Preferred perhalogenated alkyl groups are, for example, trifluoromethyl or trifluoromethoxy.

"heteroaryl" means aryl as defined herein, with the addition of at least one heteroatom moiety selected from N, O or S, each ring consisting of 5 to 6 ring atoms, unless otherwise specified. E.g. C₂Heteroaryl groups include oxadiazole, triazole, and the like. C₉Heteroaryl groups include quinoline, 1, 2, 3, 4-tetrahydroquinoline, and the like. C for use in the present application_2-9Heteroaryl includes thienyl, pyridyl, furyl, isoxazolyl, benzoxazolyl or benzo [1, 3]]Dioxolyl, preferably thienyl, furyl or pyridyl. "A", "AHeteroaryl "means heteroaryl as defined herein, with the proviso that the ring combination comprises a divalent group. Fused bicyclic heteroaryl ring systems may be partially saturated, such as 2, 3-dihydro-1H-isoindole, 1, 2, 3, 4-tetrahydroquinoline, and the like.

The EDG-1 selective compounds (reagents or modulators) used in the present invention have a specificity that is selective for EDG-1 over EDG-3 and one or more of EDG-5, EDG-6 and EDG-8. As used herein, selectivity for one EDG receptor ("selective receptor") over another EDG receptor ("non-selective receptor") means that the compound is much more potent in inducing activity mediated by a selective EDG receptor (e.g., EDG-1) than non-selective S1P-specific EDG receptor. EC of EDG-1 selective compounds for Selective receptor (EDG-1) if measured in a GTP- γ S binding assay (described in the examples below)₅₀(effective concentration resulting in 50% maximal response) is generally greater than its EC for a non-selective receptor (e.g., one or more of EDG-3, EDG-5, EDG-6, and EDG-8)₅₀At least 5, 10, 25, 50, 100, 500, or 1000 times lower.

Detailed Description

The present invention provides compounds useful for treating or preventing diseases or disorders mediated by lymphocyte interactions. In one embodiment, for compounds of formula I, R₁Is phenyl, naphthyl, furyl or thienyl, optionally substituted by C_6-10Aryl radical C_0-4Alkyl radical, C_2-9Heteroaryl C_0-4Alkyl radical, C_3-8Cycloalkyl radical C_0-4Alkyl radical, C_3-8Heterocycloalkyl radical C_0-4Alkyl or C_1-6Alkyl substitution; wherein R is₁Any aryl, heteroaryl, cycloalkyl or heterocycloalkyl of (a) may be optionally substituted with one to five groups selected from halo, C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkyl and halo C_1-6An alkoxy group; r₁Any alkyl group of (a) may optionally have a methylene group replaced by an atom or group selected from: -S-、-S(O)-、-S(O)₂-、-NR₁₀-and-O-; wherein R is₁₀Is hydrogen or C_1-6An alkyl group.

In another embodiment, Y is selected from:

and

wherein R is₁₁Is hydrogen or C_1-6An alkyl group; the left and right asterisks of Y indicate-C (R) of formula I₂)(R₃) Between-and X or X and-C (R) of formula I₂)(R₃) -a connection point therebetween; y may be optionally substituted with 1 to 3 groups selected from halo, hydroxy, cyano, nitro, C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkyl and halo C_1-6An alkoxy group.

In a further embodiment, R₁Selected from:

and

wherein the asterisk is R₁The point of attachment to X; m is selected from 1 and 2; r₁₂Is hydrogen, C_6-10Aryl radical C_0-4Alkyl radical, C_2-9Heteroaryl C_0-4Alkyl radical, C_3-8Cycloalkyl radical C_0-4Alkyl radical, C_3-8Heterocycloalkyl radical C_0-4Alkyl or C_1-6An alkyl group; wherein R is₁₂Any aryl, heteroaryl, cycloalkyl or heterocycloalkyl of (a) may be optionally substituted with one to three groups selected from halo, C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkyl and halo C_1-6An alkoxy group; r₁₂Any alkyl group of (a) may optionally have a methylene group replaced by an atom or group selected from: -S-, -S (O)₂-、-NR₁₀-and-O-; wherein R is₁₀Is hydrogen or C_1-6An alkyl group; r₁₃Selected from halo, C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkyl and halo C_1-6An alkoxy group.

In another embodiment, A is-C (O) OH; r₂、R₃、R₅、R₆And R₈Is hydrogen; r₇Selected from hydrogen and fluorine; r₄Selected from hydrogen and C_1-6An alkyl group; or R₇And R₄And R₇And R₄The atoms to which they are attached together form an azetidine.

In further embodiments, Y is selected from:

and

wherein R is₁₁Is hydrogen or C_1-6An alkyl group; the left and right asterisks of Y indicate-C (R) of formula I₂)(R₃) Between-and X or X and-C (R) of formula I₂)(R₃) -a connection point therebetween; y may be optionally substituted with 1 to 3 groups selected from chloro, fluoro, methyl, ethyl, cyano and bromo.

In another embodiment, X is selected from the group consisting of a bond, -NH-, and-N (CH)₃)-；R₁Selected from:

and

wherein m is selected from 1 and 2; r₁₂Is hydrogen, phenyl, piperidinyl, 2-methyl-butyl, 3-methyl-butyl, cyclohexyl, cyclohexyloxy, cyclopentyloxy, sec-butoxy, tetrahydropyranyl, phenoxy, benzo [1, 3]]Dioxolyl, naphthyl, 2-dimethyl-pentyl, butyl, benzo [ b ]]Furyl, benzyl, phenethyl, phenyl-vinyl, 1-phenyl-ethyl and cyclopropyl; wherein R is₁₂Any aryl, heteroaryl, cycloalkyl or heterocycloalkyl of (a) may be optionally substituted with one to three groups selected from fluoro, isobutyl, 2-methyl-butyl, trifluoromethyl, chloro, methyl, trifluoromethoxy and methoxy; r₁₃Selected from trifluoromethyl, trifluoromethoxy, methyl, fluoro, chloro and methoxy.

Preferred compounds of the invention are selected from the group consisting of 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-piperidin-1-yl-3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -thieno [2, 3-b ] pyridin-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-6-ylmethyl ] -amino } -propane-propionic acid Acids, 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -2, 3-dihydro-1H-isoindol-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzoxazol-5-ylmethyl ] -amino } -propionic acid, 1- [2- (4-isobutyl-3-trifluoromethyl-phenyl) -benzoxazol-6-ylmethyl ] -azetidine-3-carboxylic acid, 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzofuran-5-ylmethyl ] -amino } -propionic acid, acetic acid, propionic acid, acetic acid, propionic acid, 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzothiazol-6-ylmethyl ] -amino } -propionic acid, 3- { [ 3-chloro-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 1- [2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-5-ylmethyl ] -azetidine-3-carboxylic acid, 3- { [2- (2' -fluoro-2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof, 3- { [2- (5-fluoro-2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-6-ylmethyl ] -amino } -propionic acid, 3- { [ 3-fluoro-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-cyclohexyl-3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [ 4-chloro-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid 1- [2- (4-cyclohexyl-3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -azetidine-3-carboxylic acid, 3- { [ 6-methoxy-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [ 6-chloro-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-cyclopentyloxy-3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } - Propionic acid, 3- { [2- (4-sec-butoxy-3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-sec-butyl-3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-isobutyl-3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-cyclohexyloxy-3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, a salt thereof, a pharmaceutically acceptable salt thereof, and, 3- ({2- [4- (tetrahydro-pyran-4-yl) -3-trifluoromethyl-phenyl ] -benzo [ b ] thiophen-5-ylmethyl } -amino) -propionic acid, 3- { [ 3-methyl-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [ 3-cyano-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [ 3-bromo-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophene-5- Ylmethyl ] -amino } -propionic acid, 3- { [2- (3-fluoro-5-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-fluoro-3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 1- [2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-4-ylmethyl ] -azetidine-3-carboxylic acid, and pharmaceutically acceptable salts thereof, 3- { [2- (4-chloro-3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-4-ylmethyl ] -amino } -propionic acid, 3- { [2- (2, 5-bis-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-methyl-5-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, a salt thereof, a pharmaceutically acceptable salt thereof, 3- [ (2-phenyl-benzo [ b ] thiophen-5-ylmethyl) -amino ] -propionic acid, 3- { [2- (4-methyl-3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 1- [2- (3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -azetidine-3-carboxylic acid, 3- { [2- (4-fluoro-3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 2-fluoro-3- { [2- (3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-yl ] -thiophene-5-carboxylic acid -ylmethyl ] -amino } -propionic acid, 3- { [2- (3, 5-bis-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-trifluoromethoxy-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 1- [2- (2-fluoro-5-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -azetidine-3-carboxylic acid, 3- { [2- (2-chloro-5-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, a pharmaceutically acceptable salt thereof, and a pharmaceutically, 3- { [2- (3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 1- [2- (3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -pyrrolidine-3-carboxylic acid, 3- { [2- (2-fluoro-5-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-methoxy-3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-e -5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-methoxy-5-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [3- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [5- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-2-ylmethyl ] -amino } -propionic acid, 3- { [5- (4-cyclohexyl-3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-2-ylmethyl ] -amino } -propionic acid 3- { [ 3-chloro-5- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-2-ylmethyl ] -amino } -propionic acid, 1- [5- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-2-ylmethyl ] -azetidine-3-carboxylic acid, 3- { [ 3-bromo-5- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-2-ylmethyl ] -amino } -propionic acid, 3- { [2- (2' -fluoro-2-trifluoromethyl-biphenyl-4-yl) -benzoxazol-5-ylmethyl ] -amino } - Propionic acid, 3- { [2- (3 ' -fluoro-2-trifluoromethyl-biphenyl-4-yl) -benzoxazol-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2 ' -chloro-2-trifluoromethyl-biphenyl-4-yl) -benzoxazol-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-phenoxy-3-trifluoromethyl-phenyl) -benzoxazol-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2 ' -fluoro-2-trifluoromethyl-biphenyl-4-yl) -benzoxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-cyclohexyl-3-trifluoromethyl-phenyl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (5 ' -fluoro-2 ' -methyl-2-trifluoromethyl-biphenyl-4-yl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 2-fluoro-3- { [2- (2 ' -fluoro-2-trifluoromethyl-biphenyl-4-yl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid 3- { [5, 7-dichloro-2- (2-trifluoromethyl-biphenyl-4-yl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (3' -chloro-2-trifluoromethyl-biphenyl-4-yl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [ 5-chloro-2- (2-trifluoromethyl-biphenyl-4-yl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [ 5-bromo-2- (2-trifluoromethyl-biphenyl-4-yl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, a salt thereof, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, 3- { [2- (4-isobutyl-3-trifluoromethyl-phenyl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-benzo [1, 3] dioxol-5-yl-3-trifluoromethyl-phenyl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-cyclohexyl-3-fluoro-phenyl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-fluoro-biphenyl-4-yl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, a pharmaceutically acceptable salt thereof, 3- { [2- (3 '-chloro-4' -fluoro-2-trifluoromethyl-biphenyl-4-yl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-sec-butyl-3-trifluoromethyl-phenyl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [ 5-ethyl-2- (2-trifluoromethyl-biphenyl-4-yl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-naphthalen-2-yl-3-trifluoromethyl-phenyl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, a, 1- {2- [4- (2, 2-dimethyl-propyl) -3-trifluoromethyl-phenyl ] -benzooxazol-6-ylmethyl } -azetidine-3-carboxylic acid, 3- { [2- (4-butyl-3-trifluoromethyl-phenyl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-benzofuran-2-yl-3-trifluoromethyl-phenyl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- ({2- [4- (2, 6-difluoro-benzyl) -3-trifluoromethyl-phenyl ] -benzooxazol-6-ylmethyl } - Amino) -propionic acid, 3- { [2- (4-phenethyl-3-trifluoromethyl-phenyl) -benzoxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-styryl-3-trifluoromethyl-phenyl) -benzoxazol-6-ylmethyl ] -amino } -propionic acid, 3- ({2- [4- (1-phenyl-ethyl) -3-trifluoromethyl-phenyl ] -benzoxazol-6-ylmethyl } -amino) -propionic acid, 3- { [2- (5 '-fluoro-2' -methoxy-2-trifluoromethyl-biphenyl-4-yl) -benzoxazol-6-ylmethyl ] - Methyl-amino } -propionic acid, 3- { [2- (5 '-fluoro-2' -methoxy-2-trifluoromethyl-biphenyl-4-yl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (3-trifluoromethyl-phenyl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-tert-butyl-phenyl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 1- [2- (2-fluoro-5-trifluoromethyl-phenyl) -benzooxazol-6-ylmethyl ] -azetidine-3-carboxylic acid, water-soluble salts thereof, and pharmaceutically acceptable salts thereof, 3- { [ 5-chloro-2- (3-trifluoromethyl-phenyl) -benzoxazol-6-ylmethyl ] -amino } -propionic acid, 1- [2- (2-fluoro-5-trifluoromethyl-phenyl) -benzoxazol-5-ylmethyl ] -azetidine-3-carboxylic acid, 1- [2- (2-fluoro-5-trifluoromethyl-phenyl) -benzofuran-5-ylmethyl ] -azetidine-3-carboxylic acid, 3- { [2- (4-chloro-3-trifluoromethyl-phenyl) -benzoxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-cyclopropyl-3-trifluoromethyl-phenyl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-fluoro-phenyl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (3-fluoro-phenyl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-fluoro-phenyl) -benzooxazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-cyclohexyl-3-trifluoromethyl-phenyl) -benzofuran-5-ylmethyl ] -amino } -propionic acid, water-soluble salt thereof, 3- { [2- (4-cyclohexyl-3-trifluoromethyl-phenyl) -2, 3-dihydro-1H-isoindol-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-cyclohexyl-3-trifluoromethyl-phenyl) -2H-isoindol-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzothiazol-7-ylmethyl ] -amino } -propionic acid, a pharmaceutically acceptable salt thereof, 3- { [2- (3-trifluoromethyl-phenyl) -benzothiazol-7-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -2H-indazol-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (5-fluoro-2-trifluoromethyl-biphenyl-4-yl) -2H-indazol-6-ylmethyl ] -amino } -propionic acid, 1- [2- (5-fluoro-2-trifluoromethyl-biphenyl-4-yl) -2H-indazol-6-ylmethyl ] -azetidine-3-carboxylic acid, and, 1- [2- (2-trifluoromethyl-biphenyl-4-yl) -1H-benzimidazol-5-ylmethyl ] -azetidine-3-carboxylic acid, 3- { [ 3-methyl-2- (2-trifluoromethyl-biphenyl-4-yl) -3H-benzimidazol-5-ylmethyl ] -amino } -propionic acid, 3- { [ 1-methyl-2- (2-trifluoromethyl-biphenyl-4-yl) -1H-benzimidazol-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-trifluoromethyl-biphenyl-4-ylmethyl) -2, 3-dihydro-1H-isoindol-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-trifluoromethyl-biphenyl-4-ylamino) -benzoxazol-5-ylmethyl ] -amino } -propionic acid, 3- ({2- [ methyl- (2-trifluoromethyl-biphenyl-4-yl) -amino ] -benzoxazol-5-ylmethyl } -amino) -propionic acid, 3- { [ 4-oxo-2- (2-trifluoromethyl-biphenyl-4-yl) -4H-benzopyran-7-ylmethyl ] -amino } -propionic acid, 3- { [ 4-oxo-2- (2-trifluoromethyl-biphenyl-4-yl) -4H-chromen-6-ylmethyl ] -amino } -propionic acid and 1- [ 4-oxo-2- (2-trifluoromethyl-biphenyl-4-yl) -4H-chromen-6-ylmethyl ] -azetidine-3-carboxylic acid. Further preferred compounds are also shown in the examples and in table 1 below.

The present invention provides a form of a compound having a hydroxy or amino group in a protected form; they act as prodrugs. Prodrugs are compounds which are converted to the active pharmaceutical form by one or more chemical or biochemical transformations after administration. Forms of the compounds of the invention that are readily converted to the claimed compounds under physiological conditions are prodrugs of the claimed compounds and are also within the scope of the invention. Examples of prodrugs include forms in which the hydroxyl group is acylated to form a relatively labile ester, e.g., an acetate, and forms in which the amino group is acylated with a carboxylic acid group of glycine or an L-amino acid, e.g., serine, to form an amide bond that is particularly susceptible to hydrolysis by common metabolic enzymes.

The compounds of formula I may exist in free form or in salt form, for example as addition salts with inorganic or organic acids. If hydroxyl groups are present, these groups may also be present in the form of salts, for example ammonium salts or salts with metals such as lithium, sodium, potassium, calcium, zinc or magnesium, or mixtures thereof. Hydrate or solvate forms of the compounds of formula I and their salts are also part of the invention.

When the compounds of formula I have asymmetric centers in the molecule, various optical isomers are obtained. Enantiomers, racemates, diastereoisomers and mixtures thereof are also encompassed by the present invention. Furthermore, when the compounds of formula I include geometric isomers, the present invention encompasses cis-compounds, trans-compounds, and mixtures thereof. Similar considerations apply to starting materials having asymmetric carbon atoms or unsaturated bonds as described above.

Methods and pharmaceutical compositions for treating immune modulating disorders

The compounds of formula I, in free form or in pharmaceutically acceptable salt form, exhibit valuable pharmacological properties, such as lymphocyte recirculation modulating properties, and are therefore suitable for use in therapy, as shown, for example, in vitro and in vivo tests in example 6. The compounds of formula I preferably exhibit a size of 1X 10^-11To 1X 10^-5EC of M₅₀Preferably less than 50 nM. These compounds exhibit selectivity for one or more EDG/S1P receptors, preferably EDG-1/S1P-1. EDG-1/S1P-1 selective modulators of the invention may be identified by determining the binding of a compound to EDG-1/S1P-1 and one or more other EDG/S1P receptors (e.g., EDG-3/S1P-3, EDG-5/S1P-2, EDG-6/S1P-4, and EDG-8/S1P-5). EDG-1/S1P-1 selective modulators EC, generally at the EDG-1/S1P-1 receptor₅₀Is 1 × 10^-11To 1X 10^-5M, preferably less than 50nM, more preferably less than 5 nM. Its EC for one or more other EDG/S1P receptors₅₀EC over EDG-1/S1P-1₅₀At least 5, 10, 25, 50, 100, 500, or 1000 times higher. Thus, EC of some EDG-1/S1P-1 modulating compounds on EDG-1/S1P-1₅₀Less than 5nM, and their EC for one or more other EDG/S1P receptors₅₀At least 100nM or higher. In addition to measuring the binding activity to the EDG/S1P receptor, an EDG-1/S1P-1 selective agent can also be identified by examining the ability of a test agent to alter a cellular process or activity mediated by the EDG/S1P receptor.

The compounds of formula I are therefore useful in the treatment and/or prevention of diseases or disorders mediated by lymphocyte interactions, for example in transplantation, such as acute or chronic rejection of cell, tissue or organ allo-or xenografts or delayed graft function; graft versus host disease; autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I or II diabetes and disorders associated therewith, vasculitis, pernicious anemia, sjogren's syndrome, uveitis, psoriasis, Graves ' eye disease, alopecia areata, and others; allergic diseases such as allergic asthma, atopic dermatitis, allergic rhinitis/conjunctivitis, allergic contact dermatitis; optionally inflammatory diseases accompanied by abnormal reactions such as inflammatory bowel disease, crohn's disease or ulcerative colitis, intrinsic asthma, inflammatory lung injury, inflammatory liver injury, inflammatory glomerular injury, atherosclerosis, osteoarthritis, irritant contact dermatitis and other eczematous dermatitis, seborrheic dermatitis, skin manifestations of immune-mediated disorders, inflammatory eye diseases, keratoconjunctivitis, myocarditis or hepatitis; ischemia/reperfusion injury, such as myocardial infarction, stroke, intestinal ischemia, renal failure or hemorrhagic shock, traumatic shock, T cell lymphoma or T cell leukemia; infectious diseases, such as toxic shock (e.g. superantigen induced), septic shock, adult respiratory distress syndrome or viral infections, such as AIDS, viral hepatitis, chronic bacterial infections; or senile dementia. Examples of cell, tissue or solid organ transplants include, for example, pancreatic islets, stem cells, bone marrow, corneal tissue, neuronal tissue, heart, lung, combined heart-lung, kidney, liver, intestine, pancreas, trachea or esophagus. For the above uses, the required dosage will, of course, vary with the mode of administration, the particular condition being treated and the effect desired.

Furthermore, the compounds of formula I may be used in cancer chemotherapy, in particular of solid tumors, such as breast cancer, or as anti-angiogenic agents.

The desired dosage will, of course, vary with the mode of administration, the particular condition being treated and the effect desired. Generally, satisfactory results are obtained systemically at a daily dose of about 0.03 to 2.5mg/kg body weight. Daily dosages suitable for use in large mammals, such as humans, will range from about 0.5mg to about 100mg, for example conveniently in divided doses up to four times a day, or in a delayed manner. Unit dosage forms suitable for oral administration contain from about 1 to 50mg of the active ingredient.

The compounds of formula I may be administered by any conventional route, in particular enterally, e.g. orally, in dosage forms such as tablets or capsules; or parenterally, in dosage forms such as injectable solutions or suspensions; topical means, in the form of, for example, lotions, gels, ointments or creams, or nasal or suppository forms. Pharmaceutical compositions comprising a compound of formula I in free form or in pharmaceutically acceptable salt form in association with at least one pharmaceutically acceptable carrier or diluent may be prepared in conventional manner by mixing with a pharmaceutically acceptable carrier or diluent.

The compounds of formula I may be administered in free form or in the form of a pharmaceutically acceptable salt, for example as indicated above. Such salts can be prepared in a conventional manner and exhibit activity on the same scale as the free compound.

According to the above, the present invention further provides:

1.1 a method of preventing or treating a disorder or disease mediated by lymphocytes (e.g. as indicated above) in a subject in need of such treatment, which method comprises administering to said subject an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof;

1.2 a method of preventing or treating acute or chronic transplant rejection or a T-cell mediated inflammatory or autoimmune disease (e.g. as indicated above) in a subject in need of such treatment, which method comprises administering to said subject an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof;

1.3 a method of inhibiting or controlling an angiogenic disorder, such as sphingosine-1-phosphate (S1P) -mediated angiogenesis, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof;

1.4 a method for preventing or treating a disease mediated by a neo-angiogenesis process or associated with deregulated angiogenesis in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

2. A compound of formula I in free form or in pharmaceutically acceptable salt form for use as a medicament, for example in any of the methods as set out under 1.1 to 1.4 above.

3. A pharmaceutical composition, for example for use in any of the methods as described above in 1.1 to 1.4, comprising a compound of formula I in free form or in pharmaceutically acceptable salt form, in association with a pharmaceutically acceptable diluent or carrier.

4. A compound of formula I or a pharmaceutically acceptable salt thereof for use in the preparation of a pharmaceutical composition for use in any of the methods as described above in 1.1 to 1.4.

The compounds of formula I may be administered as the sole active ingredient or, for example, as an adjuvant in combination with other drugs, for example immunosuppressants or immunomodulators or other anti-inflammatory agents, for example for the treatment or prevention of allo-or xeno-inhibitor acute or chronic rejection or inflammatory or autoimmune disorders, or chemotherapeutic agents, for example, malignant cell antiproliferative agents. For example, the compounds of formula I may be used in combination with: calcineurin inhibitors, such as cyclosporin a or FK 506; mTOR inhibitors, such as rapamycin, 40-O- (2-hydroxyethyl) -rapamycin, CCI779, ABT578, or AP 23573; ascomycins with immunosuppressive properties, such as ABT-281, ASM981, and the like; a corticosteroid; cyclophosphamide; azathioprine; methotrexate; leflunom; mizoribine; mycophenolic acid; mycophenolate mofetil (mycophenolate mofetil); 15-deoxyspergualin (spergualine) or an immunosuppressive homolog, analog or derivative thereof; immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies to leukemia receptors, e.g., MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD58, CD80, CD86 or ligands thereof; other immunomodulatory compounds, such as recombinant binding molecules having at least a portion of an extracellular CTLA4 domain or mutants thereof, e.g., at least an extracellular CTLA4 portion or mutants thereof linked to a non-CTLA 4 protein sequence, e.g., CTLA4Ig (e.g., designated ATCC 68629) or mutants thereof, e.g., LEA 29Y; adhesion molecule inhibitors, such as LFA-1 antagonists, ICAM-1 or-3 antagonists, VCAM-4 antagonists or VLA-4 antagonists; or a chemotherapeutic agent.

The term "chemotherapeutic agent" means any chemotherapeutic agent, including but not limited to:

i. an aromatase inhibitor which is capable of inhibiting aromatase,

anti-estrogenic agents, anti-androgens (especially in the case of prostate cancer) or gonadotropin releasing factor agonists,

a topoisomerase I or II inhibitor,

a microtubule active agent, an alkylating agent, an anti-tumor anti-metabolite, or a platinum compound,

v. a compound targeting/decreasing a protein or lipid kinase activity or a protein or lipid phosphatase activity, a further anti-angiogenic compound or a compound inducing a cell differentiation process,

a bradykinin 1 receptor or angiotensin II antagonist,

cyclooxygenase inhibitors, bisphosphonates, histone deacetylase inhibitors, heparanase inhibitors (to prevent heparan sulfate degradation), such as PI-88; biological response modifiers, preferably lymphokines or interferons, such as interferon □; inhibitors of ubiquitination, or inhibitors that block anti-apoptotic pathways,

inhibitors of Ras oncogene isoforms, e.g., H-Ras, K-Ras or N-Ras, or farnesyl transferase inhibitors, e.g., L-744,832 or DK8G557,

telomerase inhibitors, such as telomestatin,

x. a protease inhibitor, a matrix metalloproteinase inhibitor, a methionine aminopeptidase inhibitor (e.g. bengamide or a derivative thereof) or a proteosome inhibitor (e.g. PS-341), and/or

An mtor inhibitor.

The term "aromatase inhibitor" as used herein relates to a compound which inhibits the production of estrogen, i.e. a compound which inhibits the conversion of the substrates androstenedione and testosterone to estrone and estradiol, respectively. The term includes, but is not limited to steroids, especially atamestane, exemestane and formestane, especially non-steroids, especially aminoglutethimide, roglethimide, pirglutethimide, trostane, testolactone, ketoconazole (ketoconazole), vorozole, fadrozole, anastrozole and letrozole. The combinations of the invention comprising a chemotherapeutic agent aromatase inhibitor are particularly useful for the treatment of hormone receptor positive tumors, such as breast tumors.

The term "antiestrogen" as used herein relates to compounds that antagonize the effects of estrogen at the estrogen receptor level. The term includes, but is not limited to, tamoxifen, fulvestrant, raloxifene and raloxifene hydrochloride. The combination of the invention comprising the chemotherapeutic agent antiestrogen is particularly useful for the treatment of estrogen receptor positive tumors, such as breast tumors.

The term "antiandrogen" as used herein relates to any substance capable of inhibiting the biological effects of androgens, including but not limited to bicalutamide.

The term "gonadotropin releasing factor agonist" as used herein includes, but is not limited to abarelix (abarelix), sexual relin and acetate sexual relin.

The term "topoisomerase I inhibitor" as used herein includes, but is not limited to, topotecan, irinotecan, 9-nitrocamptothecin and the macromolecular camptothecin conjugate PNU-166148 (compound a1 in WO 99/17804).

The term "topoisomerase II inhibitor" as used herein includes, but is not limited to, anthracyclines such as doxorubicin, daunorubicin, epirubicin, idarubicin and nemobicin (nemorubicin), the anthraquinones mitoxantrone and losoxantrone (losoxantrone); and etoposide (podophillilotoxine) and teniposide.

The term "microtubule active agent" relates to microtubule stabilizing and microtubule destabilizing agents, including but not limited to taxanes, such as paclitaxel and docetaxel; vinca alkaloids, such as vinblastine, especially vinblastine sulfate, vincristine, especially vincristine sulfate, and vinorelbine; discodermolide (discodermolide) and epothilones and derivatives thereof, such as epothilone B or derivatives thereof.

The term "alkylating agent" as used herein includes, but is not limited to, busulfan, chlorambucil, cyclophosphamide, ifosfamide, melphalan, or nitrosoureas (BCNU or Gliadel).

The term "antineoplastic antimetabolite" includes, but is not limited to, 5-fluorouracil, capecitabine, gemcitabine, cytarabine, fludarabine, thioguanine, methotrexate and edatrexate.

The term "platinum compound" as used herein includes, but is not limited to, carboplatin, cisplatin and oxaliplatin.

The term "compound targeting/reducing the activity of a protein or lipid kinase or further anti-angiogenic compound" as used herein includes, but is not limited to, protein tyrosine kinase and/or serine and/or threonine kinase inhibitors or lipid kinase inhibitors, such as compounds which target, reduce or inhibit the activity of: epidermal growth factor receptor tyrosine kinase family (homo-or heterodimers of EGFR, ErbB2, ErbB3, ErbB 4), vascular endothelial growth factor receptor tyrosine kinase family (VEGFR), platelet-derived growth factor receptor (PDGFR), Fibroblast Growth Factor Receptor (FGFR), insulin-like growth factor receptor 1(IGF-1R), Trk receptor tyrosine kinase family, Axl receptor tyrosine kinase family, Ret receptor tyrosine kinase, Kit/SCFR receptor tyrosine kinase, c-Abl family members with their gene fusion products (e.g., BCR-Abl), Protein Kinase C (PKC), (PKC) and serine/threonine kinase members of the Raf family, MEK, JAK, FAK, PDK or PI (3) kinase family members, and/or cell-dependent protein kinase family (CDK) members, and anti-angiogenic compounds whose activity has another mechanism (e.g., not associated with protein or lipid kinase inhibition).

Compounds which target, reduce or inhibit VEGFR activity are especially compounds, proteins or antibodies which inhibit VEGF receptor tyrosine kinases, inhibit VEGF receptors or bind to VEGF, especially those compounds, proteins or monoclonal antibodies generally and specifically disclosed in the following documents: WO98/35958, for example 1- (4-chloroanilino) -4- (4-pyridylmethyl) phthalazine or a pharmaceutically acceptable salt thereof, for example the succinate salt; WO 00/27820, e.g. N-aryl (thio) anthranilamide derivatives, e.g. 2- [ (4-pyridyl) methyl]amino-N- [ 3-methoxy-5- (trifluoromethyl) phenyl]Benzamide or 2- [ (1-oxo-4-pyridyl) methyl group]amino-N- [ 3-trifluoromethylphenyl group]A benzamide; or WO00/09495, WO 00/59509, WO 98/11223, WO 00/27819 and EP 0769947; prewett et al in Cancer Research59(1999) 5209-; WO 00/37502 and WO 94/10202; angiostatin^TMAs described in m.s.o' Reilly et al, Cell 79, 1994, 315-; endostatin^TMAs described in M.S.O' Reilly et al, Cell 88, 1997, 277-285; anthranilamides; ZD 4190; ZD 6474; SU 5416; SU 6668; or an anti-VEGF antibody or an anti-VEGF receptor antibody, e.g., RhuMab.

Antibodies mean intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies composed of at least 2 intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.

Compounds which target, reduce or inhibit the activity of the epidermal growth factor receptor family are in particular compounds, proteins or antibodies which inhibit members of the EGF receptor tyrosine kinase family, such as the EGF receptor, ErbB2, ErbB3 and ErbB4, or bind to EGF or EGF-related ligands, or which have a dual inhibitory effect on ErbB and VEGF receptor kinases, in particular those compounds, proteins or monoclonal antibodies which are generally specifically disclosed in the following documents: WO 97/02266, for example the compound of example 39, or EP 0564409, WO 99/03854, EP 0520722, EP 0566226, EP 0787722, EP 0837063, US 5,747,498, WO 98/10767, WO 97/30034, WO 97/49688, WO 97/38983, in particular WO 96/30347 (for example the compound known as CP 358774), WO 96/33980 (for example the compound ZD 1839) and WO95/03283 (for example the compound ZM105180) or PCT/EP 02/08780; such as Sinomenobu (HerpetinR), cetuximab, Iressa, OSI-774, CI-1033, EKB-569, GW-2016, E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, or E7.6.3.

Compounds which target, decrease or inhibit the activity of PDGFR are especially compounds which inhibit the PDGF receptor, for example N-phenyl-2-pyrimidine-amine derivatives, for example imatinib (imatinib).

Compounds which target, reduce or inhibit c-AbI family members and their gene fusion products are for example N-phenyl-2-pyrimidine-amine derivatives, such as imatinib; PD 180970; AG957 or NSC 680410.

Compounds which target, reduce or inhibit the activity of protein kinase C, Raf, MEK, SRC, JAK, FAK and PDK family members or PI (3) kinases or PI (3) kinase related family members and/or cyclin dependent kinase family (CDK) members are in particular those staurosporine derivatives disclosed in EP 0296110, for example, midostaurin; further examples of compounds include, for example, UCN-01, safingol, BAY 43-9006, bryodin 1, Perifosine; ilmofosine; RO 318220 and RO 320432; GO 6976; isis 3521; or LY333531/LY 379196.

Further anti-angiogenic compounds are for example Thalidomide (THALOMID) and TNP-470.

Compounds which target, reduce or inhibit the activity of protein or lipid phosphatases are for example inhibitors of phosphatase 1, phosphatase 2A, PTEN or CDC25, such as okadaic acid (okadaic acid) or derivatives thereof.

Compounds which induce a cell differentiation process are, for example, retinoic acid, alpha-, beta-or gamma-tocopherol or alpha-, beta-or gamma-tocotrienol.

The term cyclooxygenase inhibitor as used herein includes, but is not limited to, celecoxib (Celebrex), for example^R) Rofen coxib (Vioxx)^R) Etoricoxib, valdecoxib or 5-alkyl-2-arylaminophenylacetic acids, for example 5-methyl-2- (2 '-chloro-6' -fluoroanilino) phenylacetic acid.

The term "histone deacetylase inhibitor" as used herein includes, but is not limited to, MS-27-275, SAHA, pyroxamide, FR-901228 or valproic acid.

The term "bisphosphonates" as used herein includes, but is not limited to, itration, clodronic acid, tiludronic acid, pamidronic acid, alendronic acid, ibandronic acid, risedronic acid and zoledronic acid.

The term "matrix metalloproteinase inhibitor" as used herein includes, but is not limited to, collagen peptidomimetics (peptidomimetics) and non-peptidomimetics inhibitors, tetracycline derivatives, such as the hydroxamic acid peptidomimetics inhibitor batimastat and its orally bioavailable analogue, marimastat, prinomastat, BMS-279251, BAY 12-9566, TAA211 or AAJ 996.

The term "mTOR inhibitor" as used herein includes, but is not limited to, rapamycin (sirolimus) or its derivatives, such as 32-deoxorapamycin, 16-pent-2-ynyloxy-32 (S) -dihydrorapamycin, 16-pent-2-ynyloxy-32 (S) -dihydro-40-O- (2-hydroxyethyl) rapamycin, and more preferably 40-O- (2-hydroxyethyl) rapamycin. Further examples of rapamycin derivatives include, for example, CCI779 or 40- [ 3-hydroxy-2- (hydroxymethyl) -2-methylpropionate) ] rapamycin or pharmaceutically acceptable salts thereof, as disclosed in USP 5,362,718; ABT578 or 40- (tetrazolyl) rapamycin, in particular 40-epi- (tetrazolyl) rapamycin, for example as disclosed in WO 99/15530; or rapamycin analogues (rapalogs), as disclosed for example in WO 98/02441 and WO 01/14387, such as AP 23573.

If a compound of formula I is administered in combination with other immunosuppressive/immunomodulatory, anti-inflammatory or chemotherapeutic therapies, the dosage of the immunosuppressive, immunomodulatory, anti-inflammatory or chemotherapeutic compound co-administered will, of course, vary depending on the type of co-drug employed (e.g., whether it is a steroid or calcineurin inhibitor), the particular drug employed, the condition being treated, and the like.

In accordance with the foregoing, the present invention provides in a further aspect:

5. a method as defined above, comprising co-administration, e.g. simultaneous or sequential administration, of a therapeutically effective non-toxic amount of a compound of formula I with at least one second drug substance, e.g. an immunosuppressive, immunomodulatory, anti-inflammatory or chemotherapeutic agent, e.g. as indicated above.

6. A pharmaceutical combination, e.g. a kit, comprising a) a first active agent which is a compound of formula I as disclosed herein, in free form or in pharmaceutically acceptable salt form, and b) at least one co-agent, e.g. an immunosuppressive, immunomodulatory, anti-inflammatory or chemotherapeutic agent, e.g. as disclosed above. The kit may contain instructions for its use.

The terms "co-administration" or "co-administration" and the like as used herein are meant to encompass administration of the selected therapeutic agents to a single patient and shall include treatment regimens in which the individual active agents are not necessarily administered by the same route of administration or at the same time.

The term "pharmaceutical combination" as used herein means a product resulting from mixing or combining more than one active ingredient, including fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredients, e.g. a compound of formula I and a co-agent, are both administered to a patient simultaneously in a single entity or dosage form. The term "non-fixed combination" means that the active ingredients, e.g. a compound of formula I and a co-agent, are all administered to a patient as separate entities simultaneously, concurrently or sequentially with no specific time limitation, wherein such administration provides therapeutically effective levels of the 2 compounds in the patient. The latter is also applicable to cocktail therapy, e.g. the administration of 3 or more than 3 active ingredients.

Process for preparing the Compounds of the invention

The invention also includes methods of making the immunomodulatory compounds of the invention. In such reactions, if a reactive functional group is desired in the final product, it may be necessary to protect, for example, a hydroxyl group, an amino group, an imino group, a thio group, or a carboxyl group, to avoid them from undesirably participating in the reaction. Conventional protecting groups may be used in accordance with standard practice, see for example t.w.greene and p.g.m.wuts, "protecting groups in organic chemistry", John Wiley and Sons, 1991.

The compounds of formula I can be prepared as follows:

reaction scheme 1

Wherein n and R⁴、R⁹、R¹²And R¹³W is halogen, triflate (triflate), and the like, as defined in the summary of the invention. Initially, the reaction is carried out in the presence of a catalyst (e.g., palladium acetate, palladium chloride, palladium bromide, palladium cyanide, palladium acetylacetonate, bis (benzonitrile) palladium dichloride, tris (dibenzylideneacetone) dipalladium, etc.) and a ligand (e.g., a phosphorus ligand, e.g., triphenylphosphine, tri-tert-butylphosphine, 2- (di-tert-butylphosphino) biphenyl, dicyclohexylphosphino biphenyl, etc.), in a solvent (e.g., triphenylphosphine, tri-tert-butylphosphine, 2- (di-tert-butylphosphino) biphenyl, etc.Such as tetrahydrofuran, 1, 4-dioxane, benzene, toluene, xylene, N-dimethylformamide, N-methylpyrrolidone, etc.) at a temperature of about 20 to about 140 c, possibly over a period of up to about 48 hours. The bromination reaction is carried out in the presence of a brominating agent (e.g., N-bromosuccinimide, bromine, etc.) and a free radical initiator (e.g., 2' -azobisisobutyronitrile, benzoyl peroxide, etc.). Amination with an aminocarboxylate is carried out in the presence of a base (e.g., sodium hydride, triethylamine, diisopropylethylamine, potassium carbonate, sodium carbonate, etc.). The subsequent ester hydrolysis is carried out in the presence of an acid (e.g., trifluoroacetic acid, hydrochloric acid, etc.) or a base (e.g., lithium hydroxide, sodium hydroxide, potassium hydroxide, sodium carbonate, etc.).

Reaction scheme 2

Wherein n and R⁴、R⁹、R¹²And R¹³W is halogen, triflate, etc., as defined in the summary of the invention. The benzoxazole nucleus is formed by a condensation reaction of the appropriate aminophenol with an aldehyde, followed by oxidative cyclization. The coupling reaction is carried out in the presence of a catalyst (e.g., palladium acetate, palladium chloride, palladium bromide, palladium cyanide, palladium acetylacetonate, bis (benzonitrile) palladium dichloride, tris (dibenzylideneacetone) dipalladium, and the like) and a ligand (e.g., a phosphorus ligand, such as triphenylphosphine, tri-tert-butylphosphine, 2- (di-tert-butylphosphino) biphenyl, dicyclohexylphosphino biphenyl, and the like), in a solvent (e.g., tetrahydrofuran, 1, 4-dioxane, benzene, toluene, xylene, N-dimethylformamide, N-methylpyrrolidinone, and the like), at a temperature of about 20 to about 140 ℃, possibly over a period of up to about 48 hours. Similar transformations as in scheme 1 give the final compounds of formula I.

Some of the compounds of the present invention can be prepared as follows:

reaction scheme 3

Reaction scheme 4

Reaction scheme 5

Reaction scheme 6

Wherein n and R⁴、R⁹、R¹²And R¹³As defined in the summary of the invention.

Additional Process for preparing Compounds of the invention

The compounds of the present invention may be prepared as pharmaceutically acceptable acid addition salts by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid. Alternatively, pharmaceutically acceptable base addition salts of the compounds of the present invention may be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base. Alternatively, salts of the starting materials or intermediates may be used to prepare salt forms of the compounds of the present invention.

The free acid or free base forms of the compounds of the invention may be prepared from the corresponding base addition salts or acid addition salt forms, respectively. For example, a compound of the invention in acid addition salt form can be converted to the corresponding free base by treatment with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, etc.). The compounds of the present invention in base addition salt form can be converted to the corresponding free acids by treatment with a suitable acid, such as hydrochloric acid and the like.

The compounds of the invention in their unoxidized form may be prepared from the N-oxides of the compounds of the invention by treatment with a reducing agent (e.g., sulfur dioxide, triphenylphosphine, lithium borohydride, sodium borohydride, phosphorus trichloride, phosphorus tribromide, etc.) in a suitable inert organic solvent (e.g., acetonitrile, ethanol, aqueous dioxane, etc.) at 0 to 80 ℃.

Prodrug derivatives of the compounds of the invention may be prepared by methods known to those of ordinary skill in the art (see, e.g., Saulnier et al (1994), Bioorganic and medicinal Chemistry Letters, Vol. 4, p.1985 for further details). For example, suitable prodrugs can be prepared by reacting a non-derivatized compound of the invention with a suitable carbamoylation reagent (e.g., 1-acyloxyalkyl carbonyl chloride, p-nitrophenyl carbonate, etc.).

Protected derivatives of the compounds of the present invention may be prepared by means known to those of ordinary skill in the art. A detailed description of techniques that can be used to create protecting groups and their removal can be found in T w.greene, "protecting groups in organic chemistry", 3 rd edition, John Wiley and Sons, inc.

The compounds of the invention may suitably be prepared or solvates (e.g. hydrates) formed during the process of the invention. Hydrates of the compounds of the present invention can suitably be prepared by recrystallisation from water/organic solvent mixtures using organic solvents such as dioxin, tetrahydrofuran or methanol.

The compounds of the present invention may be prepared as their individual stereoisomers by reacting a racemic mixture of the compounds with an optically active resolving agent to produce a pair of diastereomeric compounds, separating the diastereomers, and recovering the optically pure enantiomers. Although resolution of enantiomers can be carried out using covalent diastereomeric derivatives of the compounds of the invention, dissociable complexes are preferred (e.g., crystalline diastereomeric salts). Diastereomers have different physical properties (e.g., melting points, boiling points, solubilities, reactivities, etc.) and can be readily separated by exploiting these dissimilarities. Diastereomers can be separated by means of chromatography or, preferably, by means of separation/resolution techniques based on differences in solubility. The optically pure enantiomer is then recovered, together with the resolving agent, by any practical means that does not lead to racemization. A more detailed description of techniques that can be used to resolve stereoisomers of compounds from their racemic mixtures can be found in jean jacques, Andre Collet, Samuel h.wilen, "enantiomers, racemates and resolution", John Wiley and Sons, inc., 1981.

In summary, compounds of formula I can be prepared by a process involving:

(a) reaction scheme 1, 2, 3, 4, 5 or 6; and

(b) optionally converting a compound of the invention into a pharmaceutically acceptable salt;

(c) optionally converting a salt form of a compound of the invention to a non-salt form;

(d) optionally converting the unoxidized form of the compound of the invention to a pharmaceutically acceptable N-oxide;

(e) optionally converting the N-oxide form of the compound of the invention to its unoxidized form;

(f) optionally resolving individual isomers of the compounds of the invention from a mixture of isomers;

(g) optionally converting a non-derivatized compound of the invention into a pharmaceutically acceptable prodrug derivative; and

(h) prodrug derivatives of the compounds of the invention are optionally converted to their underivatized forms.

Where the preparation of the starting materials is not specifically described, these compounds are known or may be prepared analogously to methods known in the art or as disclosed in the examples below.

It will be appreciated by those skilled in the art that the above transformations are merely representative of the methods of preparation of the compounds of the present invention and that other well known methods may be similarly employed.

Examples

The following examples provide detailed illustrations of the preparation of representative compounds, which are intended to illustrate, but not limit, the invention.

Example 1

3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b]Thien-5-ylmethyl]-amino } -propionic acid

To-78 ℃ of 5-methylbenzo [ b]Thiophene (1.0g, 6.75mmol) in anhydrous ether (17mL) was added n-BuLi (5.1mL of 1.52M in hexane, 7.75 mmol). The reaction flask was then moved to a 0 ℃ bath and stirred for 2.5 hours. The mixture was cooled back to-78 deg.C and neat trimethyl borate (1.51mL, 13.5mmol) was added. The mixture was allowed to warm to room temperature overnight and then treated with 2N HCl (10 mL). After 2 hours, the mixture was extracted with ether (5 ×), the organic solutions were combined and dried (MgSO)₄) And (4) concentrating. Crude product 5-methylbenzo [ b]The thienylboronic acid was used without further purification.

To a solution of 5-methylbenzo [ b ] thiophenylboronic acid (0.84g, 4.4mmol) in ethanol (2mL) and toluene (8mL) was added 2-chloro-5-bromobenzene trifluoride (1.14g, 4.4mmol) and tetrakis (triphenylphosphine) palladium (0) (0.254g, 0.22mmol), followed by the addition of a solution of sodium carbonate (1.86g, 17.6mmol) in water (8 mL). The mixture was stirred vigorously at 80 ℃ for 4 hours, then filtered through a pad of celite, rinsing with hexane. The filtrate was concentrated and purified by column chromatography (100% hexane) to give 1.15g (80%) of 2- (4-chloro-3-trifluoromethylphenyl) -5-methylbenzo [ b ] thiophene as a white solid.

To a solution of 2- (4-chloro-3-trifluoromethylphenyl) -5-methylbenzo [ b ] thiophene (0.746g, 2.28mmol) in carbon tetrachloride (23mL) were added N-bromosuccinimide (0.447g, 2.51mmol) and 2, 2' -azobisisobutyronitrile (AIBN, 0.075g, 0.46 mmol). The mixture was stirred at 90 ℃ overnight and then concentrated. The residue was passed through a pad of silica gel and further rinsed with hexane. The combined organic solutions were concentrated in vacuo. The crude product, 5-bromomethyl-2- (4-chloro-3-trifluoromethylphenyl) benzo [ b ] thiophene, was dissolved in DMF (2mL) and added to a previously stirred suspension of beta-alanine tert-butyl ester hydrochloride (0.828g, 4.56mmol) and sodium hydride (0.365g, 60% dispersion in mineral oil, 9.12mmol) in DMF (4 mL). The mixture was stirred at room temperature for 4 hours, quenched with water (1mL) and concentrated in vacuo. The resulting residue was purified by column chromatography (50% to 70% EtOAc/hexanes) to give 0.79g (73%) of tert-butyl 3- { [2- (4-chloro-3-trifluoromethylphenyl) benzo [ b ] thiophen-5-ylmethyl ] amino } propanoate as a yellow solid.

To a solution of the above chloride (0.79g, 1.68mmol) in THF (20mL) was added phenylboronic acid (0.41g, 3.36mmol), potassium fluoride (0.39g, 6.72mmol), 2- (dicyclohexylphosphino) biphenyl (59mg, 0.17mmol), and palladium (II) acetate (19mg, 0.084 mmol). The mixture was stirred at 60 ℃ for 24 hours under argon. After concentration, the residue was purified by column chromatography (50% to 70% EtOAc/hexanes) to give 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) benzo [ b ]]Thien-5-ylmethyl]Amino } propionic acid tert-butyl ester. In TFA-CH₂Cl₂(1: 2 v%, 20mL) the ester was hydrolyzed. The crude product was purified by preparative LCMS to give 0.55g (67%) of 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) benzo [ b ]]Thien-5-ylmethyl]Amino groupPropionic acid, conversion to the corresponding HCl salt:¹H NMR(400MHz，CD₃OD)δ8.03(d，1H)，7.94(dd，2H)，7.92(s，1H)，7.42-7.30(m，5H)，7.29-7.21(m，2H)，4.29(s，2H)，3.25(t，2H)，2.70(t，2H)；MS(ES)456.1(M+H⁺)。

example 2

3- { [2- (4-piperidin-1-yl-3-trifluoromethyl-phenyl) -benzo [ b]Thien-5-ylmethyl]-amino } -propionic acid

Piperidine (30. mu.L, 2.0mmol), Pd₂dba₃(2.7mg, 0.003mmol), potassium tert-butoxide (59mg, 0.53mmol) and 1, 3-bis- (2, 6-diisopropylphenyl) -4, 5-dihydroimidazol-2-ylideneHCl salt (2.6mg, 0.006mmol) were added successively to 3- { [2- (4-chloro-3-trifluoromethyl-phenyl) -benzo [ b ] b]Thien-5-ylmethyl]-amino } -propionic acid tert-butyl ester (72mg, 0.15mmol) in 1, 4-dioxane (0.8 mL). The mixture was irradiated in a microwave at 100 ℃ for 1 hour. The reaction was quenched with water and the mixture was concentrated in vacuo. The residue was dissolved in THF and 2N aqueous NaOH (4mL, 1: 1v/v) and stirred at 60 ℃ for 3 hours. Concentration and purification by preparative LCMS gave 20mg of 3- { [2- (4-piperidin-1-yl-3-trifluoromethyl-phenyl) -benzo [ b]Thien-5-ylmethyl]-amino } -propionic acid, which is converted into the HCl salt:¹HNMR(400MHz，CD₃OD)δ8.15-8.00(m，4H)，7.87(s，1H)，7.63(d，1H)，7.57(d，1H)，4.48(s，2H)，3.44(t，2H)，3.03(t，4H)，2.89(t，2H)，1.90-1.80(m，4H)，1.75-1.65(m，2H)；MS(ES)463.4(M+H⁺)。

example 3

3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -thieno [2, 3-b]Pyridin-5-ylmethyl]-amino } -propionic acid

To 2- (2-trifluoromethyl-biphenyl-4-yl) -thieno [2, 3-b]Pyridine-5-carboxaldehyde (32mg, 0.083mmol) in MeOH (2mL) was added β -alanine HCl salt (37mg, 0.42mmol) and triethylamine (23uL, 0.16 mmol). The mixture was stirred at 50 ℃ for 20 minutes. Sodium borohydride (30mg, 0.8mmol) was added at room temperature and stirred for 10 min. The mixture was purified by preparative LCMS to give 22mg of 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -thieno [2, 3-b]Pyridin-5-ylmethyl]-amino } -propionic acid, which is converted into the HCl salt:¹H NMR(400MHz，CD₃OD)δ8.56(d，1H)，8.30(d，1H)，8.08(d，1H)，7.98(dd，1H)，7.84(s，1H)，7.42(d，1H)，7.40-7.34(m，3H)，7.30-7.24(m，2H)；MS(ES)457.1(M+H⁺)。

example 4

3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b]Thien-6-ylmethyl]-amino } -propionic acid

Bromoacetaldehyde dimethyl acetal (1.6mL, 0.01mol) was added dropwise to m-methylthiophenol (1.5mL, 0.01mol) and K at room temperature₂CO₃(1.66mg, 0.01mol) in 20mL of acetone. The reaction mixture was stirred for 16 hours and then filtered. The solid was washed with acetone, and the filtrate and washings were combined and concentrated in vacuo. The residue was diluted with water and extracted with ether. The ether layer was washed with 0.5M KOH, water and brine, dried, filtered and concentrated in vacuo to give 2g of a yellow oil.

At room temperature, the CH of the above yellow oil₂Cl₂The solution (20mL) was added dropwise to BF-containing solution₃In a solution of ether (0.7mL, 0.005mol) in dichloromethane (100 mL). The reaction mixture was stirred for 3 hours with NaHCO₃The aqueous solution was treated and stirred until the two phases were clear. Separation of CH₂Cl₂Layer, dried, filtered and concentrated in vacuo to give 0.45g of 4-and 6-methylbenzo [ b ]]An approximately 1: 3 mixture of thiophenes as a dark brown oil. Major isomers:¹H NMR(DMSO-d₆)：δ7.78(d，1H)，7.76(d，1H)，7.64(d，1H)，7.39(m，1H)，7.17(m，1H)，2.43(s，3H)。

6-methylbenzo [ b ] at-60 ℃ via syringe]A solution of thiophene (0.16g, 0.001mol) in 10mL of anhydrous THF was added dropwise n-BuLi (0.8mL, 0.0012 mol). After stirring for 30 minutes, triisopropyl borate (0.3mL, 0.0012mol) was added dropwise. The reaction mixture was warmed to 0 ℃ and then partitioned between 1.0N HCl and EtOAc. The organic layer was separated, dried, filtered and concentrated to give a white solid which was triturated from ether/hexane. Filtration gave 0.17g of 6-methyl-benzo [ b ]]Thiophene-2-boronic acid as a white solid. MS M/z 193[ M +1 ]]⁺.

To 10mL of a benzene slurry of 6-methyl-benzo [ b ] thiophen-2-boronic acid (0.19g, 0.001mol) was added 4-bromo-2-trifluoromethyl-biphenyl (0.3g, 0.001 mol). The reaction flask was then covered with aluminum foil and protected from light. 58mg of tetrakis (triphenylphosphine) palladium (O) was added followed by 1mL of 2.0N sodium carbonate solution. The two-phase mixture was heated at 85 ℃ for 3 hours with vigorous stirring. The mixture was cooled to room temperature and 10mL of brine solution was added. The organic layer was separated, dried and concentrated in vacuo to give 0.2g of 6-methyl-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophene.

The above product (0.2g, 0.54mmol) was dissolved in 10mL CCl₄Followed by addition of NBS (86mg, 0.54mmol) and benzoyl peroxide (24mg, 0.1 mmol). The reaction mixture was heated to reflux for 5 hours. Is cooled down andafter removal of the solvent, the residue was applied to a column (hexane: EtOAc 95: 5). After elution of the column, 180mg of 6-bromomethyl-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] are obtained]Thiophene.

To 6-bromomethyl-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b]Thiophene (180mg, 0.4mmol) in 5mL DMSO was added to Ag₂CO₃(330mg, 1.2 mmol). The suspension was heated to 100 ℃ for 3 hours. After treatment, the organic layer was dried and concentrated. The residue was applied to a column (hexane: EtOAc 9: 1) to give 100mg of 2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b]Thiophene-6-carbaldehyde, a beige solid. MS M/z 383[ M +1 ]]⁺。

To 2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b]Thiophene-6-carbaldehyde (23mg, 0.05mmol) in 3mL MeOH was added beta-alanine (8.7mg, 0.1mmol) and a catalytic amount of Et₃And N is added. The suspension was heated to 50 ℃ for 0.5 h, followed by the addition of 2mg NaBH₄. After preparation of-LC-MS, 10mg of 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] are obtained]Thien-6-ylmethyl]-amino } -propionic acid as white solid. MS M/z 456[ M +1 ]]⁺。¹H NMR(MeOD-d₄)：δ8.22(d，1H)，7.93(m，2H)，7.86(d，1H)，7.81(s，1H)，7.40(m，1H)，7.34(d，1H)，7.28(m，3H)，7.24(m，2H)，4.26(s，2H)，3.15(t，2H)，2.52(t，2H)。

Example 5

3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -2, 3-dihydro-1H-isoindol-5-ylmethyl]-amino } -propionic acid

3, 4-bis-bromomethyl-benzoic acid methyl ester (0.32g, 0.001mol) and 4-bromo-3-trifluoromethyl-phenylamine (0.72mL, 0.003mol) were dissolved in 30mL of anhydrous EtOH. Mixing the reactionThe compound was heated to reflux for 3 hours and then cooled to room temperature. After filtration, the product was obtained as a white solid. MS M/z 400[ M +1 ]]⁺。

Methyl 2- (4-bromo-3-trifluoromethyl-phenyl) -2, 3-dihydro-1H-isoindole-5-carboxylate (0.2g, 0.0005mol) was dissolved in 20mL of anhydrous toluene, followed by the addition of 40mg of tetrakis (triphenylphosphine) palladium (O). Introducing N into the solution₂After 3 minutes, tributyl-phenyl-stannane (0.22g, 0.0006mol) was added to the solution. The reaction mixture was heated to reflux for 16 hours. After column elution (9: 1 hexanes: EtOAc), the product was obtained as a white solid (0.18 g). MS M/z 398[ M +1 ]]⁺。

Will be provided with2- (2-trifluoromethyl-biphenyl-4-yl) -2, 3-dihydro-1H-isoindole-5-carboxylic acid methyl ester(0.18g.0.0005mol) was dissolved in 10mL of anhydrous THF followed by 1.5mL of LAH (0.0015 mol). The reaction mixture was stirred at room temperature for 5 minutes and quenched with 5% NaOH solution. After treatment, the organic layer was dried under vacuum and the residue was dissolved in 50mL of CHCl₃Followed by the addition of 500mg MnO₂. The suspension was stirred at room temperature for 3 hours, followed by filtration. The organic solution was dried and applied to the column (4: 1 hexane: EtOAc). After elution of the column, the product was obtained as a white solid (40 mg). MS M/z 368[ M +1 ]]⁺。

To a solution of 2- (2-trifluoromethyl-biphenyl-4-yl) -2, 3-dihydro-1H-isoindole-5-carbaldehyde (19mg, 0.05mmol) in 3mL MeOH was added β -alanine (8.7mg, 0.1mmol) and a catalytic amount of Et₃And N is added. The suspension was heated to 50 ℃ for 0.5 h, followed by the addition of 2mg NaBH₄. After preparative-LC-MS 8mg of 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -2, 3-dihydro-1H-isoindol-5-ylmethyl are obtained]-amino } -propionic acid as white solid. MS M/z 441[ M +1 ]]⁺。¹H NMR(DMSO-d₆)：δ7.08-7.26(m，9H)，6.80(brs，2H)，4.53(s，4H)，3.68(s，2H)，2.62(t，2H)，2.16(t，2H)。

Example 6

3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzooxazol-5-yl-methyl]-amino } -propionic acid

A solution of 2-amino-4-methylphenol (1eq) and 4-chloro-3-trifluoromethyl-benzaldehyde (1eq) in methanol (0.1M) was heated at 50 ℃ for 30 minutes. After concentration, the residue was dissolved in CH₂Cl₂(0.1M), treated with DDQ (1.05 eq). The resulting mixture was stirred at room temperature for 10 minutes. Then using CH₂Cl₂Diluting with NaHCO₃And a brine wash. The organic layer was washed with Na₂SO₄And (5) drying. After concentration, the desired product was purified by column chromatography (5% EtOAc/hexanes) to give a white solid. MS: (ES)⁺)：312.0(M+1)⁺。

2- (4-chloro-3-trifluoromethyl-phenyl) -5-methyl-benzoxazole (1eq), phenylboronic acid (1.5eq), Pd (OAc)₂A mixture of (0.03eq), phosphine ligand (0.06eq) and KF (3eq) in anhydrous THF (0.5M) was heated in a microwave at 100 ℃ for 30 minutes. The resulting mixture was diluted with EtOAc and washed with brine. The organic layer was washed with Na₂SO₄And (5) drying. After concentration, the residue was purified by column chromatography (5% EtOAc in hexanes) to afford the desired product as a white solid. MS: (ES)⁺)：354.1(M+1)⁺。

5-methyl-2- (2-trifluoromethyl-biphenyl-4-yl) -benzoxazole (1eq), NBS (1eq) and AIBN (0.1eq) in CCl₄The mixture in (0.1M) was refluxed for 5 hours. After concentration, the desired product was purified by column chromatography (10% EtOAc/hexanes). MS: (ES)⁺)：432.0(M+1)⁺。

To a solution of beta-alanine methyl ester hydrochloride (2eq) in anhydrous DMF (0.5M) was added NaH (3.5 eq). After stirring at room temperature for 10 min, a solution of 5-bromomethyl-2- (2-trifluoromethyl-biphenyl-4-yl) -benzoxazole (1eq) in anhydrous DMF (1M) was added. The resulting mixture was allowed to stand at room temperatureStirred for 2 hours. By H₂Diluted with O and extracted with EtOAc. The organic solution was washed with brine, over Na₂SO₄And (5) drying. After concentration, the residue was dissolved in MeOH (0.2M) and treated with 2N LiOH solution (3eq) for 10 hours. The final product was purified by preparative LCMS to give 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzooxazol-5-ylmethyl]-amino } -propionic acid;¹H NMR(400MHz，CD₃OD)δ2.74(t，J＝6.7Hz，2H)，3.30(t，J＝6.8Hz，2H)，4.39(s，2H)，7.34(m，2H)，7.43(m，3H)，7.57(m，2H)，7.78(d，J＝8.3Hz，1H)，7.94(s，1H)，8.43(d，J＝8.0Hz，1H)，8.59(s，1H)。MS：(ES⁺)：441.3(M+1)⁺。

example 7

1- [2- (4-isobutyl-3-trifluoromethyl-phenyl) -benzooxazol-6-ylmethyl]-azetidine-3-carboxylic acid

Reacting [2- (4-chloro-3-trifluoromethyl-phenyl) -benzoxazol-6-yl]Methanol (1eq) with Pd (PBu)^t ₃)₂(0.05eq) of the mixture was treated with a solution of isobutylzinc bromide in THF (0.5M, 3 eq). The resulting mixture was heated in a microwave at 100 ℃ for 30 minutes. The reaction mixture was diluted with aqueous HCl (5%) and extracted with EtOAc. The organic solution was washed with brine, over Na₂SO₄And (5) drying. After concentration, the residue was purified by flash column chromatography (30% EtOAc in hexanes) to give the desired intermediate [2- (4-isobutyl-3-trifluoromethyl-phenyl) -benzooxazol-6-yl]-methanol. MS: (ES)⁺)：350.1(M+1)⁺。

Reacting [2- (4-isobutyl-3-trifluoromethyl-phenyl) -benzoxazol-6-yl]Methanol (1eq) in dioxane (0.2M) with MnO₂(10eq) treatment. Subjecting the obtained product toThe mixture was refluxed for 20 minutes and filtered through celite. After concentration, the residue was redissolved in MeOH (0.2M) and azetidine-3-carboxylic acid (2eq) and Et were added₃N (1.8 eq). The resulting mixture was heated at 50 ℃ for 1 hour. After cooling to room temperature, NaBH is added in portions₃CN (3 eq). The final compound was purified by preparative LCMS.¹H NMR(400MHz，CD₃OD)δ8.49(s，1H)，8.38(d，J＝8.4Hz，1H)，7.88(s，1H)，7.84(d，J＝8.4Hz，1H)，7.66(d，J＝8.4Hz，1H)，7.52(d，J＝8.4Hz，1H)，4.57(s，2H)，4.34(m，4H)，3.70(m，1H)，2.76(d，J＝6.8Hz，2H)，2.03(m，1H)，0.95(d，J＝7.2Hz，6H)。MS：(ES⁺)：433.2(M+1)⁺。

Example 8

3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzofuran-5-ylmethyl]-amino } -propionic acid

To a solution of 4-hydroxy-3-iodobenzaldehyde (1eq) and 1-chloro-4-ethynyl-2-trifluoromethylbenzene (1eq) in DMF (0.2M) was added copper (I) iodide (0.1eq), dichlorobis (triphenylphosphine) palladium (II) (0.1eq) and diisopropylethylamine (3 eq). The mixture was irradiated with microwaves at 80 ℃ for 10 minutes. The product 2- (4-chloro-3-trifluoromethyl-phenyl) -benzofuran-5-carbaldehyde was purified by column chromatography.

To CH of 2- (4-chloro-3-trifluoromethyl-phenyl) -benzofuran-5-carbaldehyde (1eq)₃To the OH solution (0.2M) was added beta-alanine tert-butyl ester (2.5eq) and triethylamine (2 eq). The mixture was stirred at 50 ℃ for 30 minutes. Sodium borohydride (5eq) was then added at room temperature and the mixture was stirred for 10 min. Product 3- { [2- (4-chloro-3-trifluoromethyl-phenyl) -benzofuran-5-ylmethyl]-amino } -propionic acid tert-butyl ester was purified by column chromatography.

To 3- { [2- (4-chloro-3-trifluoromethyl-phenyl) -benzofuran-5-ylmethyl]Tert-butyl-amino } -propionate (1eq) and phenylboronic acid (1.5eq) in THF (0.5M) palladium (II) acetate (0.1eq), 2- (dicyclohexylphosphino) biphenyl (0.2eq) and potassium fluoride (4.0eq) were added. The mixture was irradiated with microwaves at 120 ℃ for 45 minutes. Product 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzofuran-5-ylmethyl]-amino } -propionic acid tert-butyl ester was purified by column chromatography. At room temperature with TFA-containing CH₂Cl₂(1: 2, v/v) hydrolysis of the ester. Purification by preparative LCMS gave 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzofuran-5-ylmethyl]-amino } -propionic acid, converted to HCl salt:¹H NMR(400MHz，CD₃OD)δ8.29(d，1H)，8.18(dd，1H)，7.82(d，1H)，7.71(d，1H)，7.55-7.30(m，8H)，4.36(s，2H)，3.32(t，2H)，2.77(t，2H)；MS(ES)440.2(M+H⁺)。

example 9

3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzothiazol-6-ylmethyl]-amino } -propionic acid

To p-toluidine (0.44g, 4.1mmol) in 10mL CH₂Cl₂The solution (pre-cooled to 0 ℃) was added Et₃5mL CH of N (1.14mL, 2eq.) and 4-chloro-3-trifluoromethyl-benzoyl chloride (1g, 4.1mmol)₂Cl₂And (3) solution. The mixture was allowed to warm slowly to room temperature and stirring was continued at room temperature for 1 hour. The mixture was quenched with 50mL CH₂Cl₂Dilute and wash with 1N HCl solution and brine. The organic layer was separated over MgSO₄Drying, filtering and concentrating. The residue was purified by column chromatography (EtOAc/hexane 2: 3) to give 1.25g (97%) of 4-chloro-N-p-tolyl-3-trifluoromethyl-benzamide.

In a microwave vial was mixed 4-chloro-N-p-tolyl-3-trifluoromethyl-benzamide (1.0g, 3.19mmol), Lawesson's reagent (774mg, 0.6eq.) and toluene (2.5 mL). The mixture was heated to 120 ℃ for 1000 seconds using microwave irradiation. The mixture became a clear solution. Ether (50mL) was added to dilute the reaction mixture. The solution was then washed with brine, over MgSO₄Drying, filtering and concentrating. The mixture was purified by column chromatography (EtOAc/hexanes ═ 5/95) to give 970mg (92%) of 4-chloro-N-p-tolyl-3-trifluoromethyl-thiobenzamide as a yellow solid.

To 2M K₃Fe(CN)₆Aqueous solution (4mL, 8mmol) (preheated to 90 ℃ C.) was added dropwise a suspension of 4-chloro-N-p-tolyl-3-trifluoromethyl-thiobenzamide (660mg, 2mmol) in 2M NaOH (9mL) and EtOH (3 mL). The mixture was heated at 90 ℃ overnight. The mixture was cooled to room temperature and extracted with EtOAc (50 mL. times.2). The combined organic layers were washed with brine and MgSO₄Drying, filtering and concentrating. The mixture was purified by ISCO system (EtOAc/hexanes: 20 min run 0 to 100% EtOAc). 2- (4-chloro-3-trifluoromethyl-phenyl) -6-methylbenzothiazole (170mg, 26%) was isolated, along with 380mg of starting material.

A microwave vial was charged with 2- (4-chloro-3-trifluoromethyl-phenyl) -6-methylbenzothiazole (170mg, 0.519mmol), phenylboronic acid (95mg, 1.5eq.), KF (90mg, 3eq.), Pd (OAc)₂(6mg, 5 mol%), (dicyclohexylphosphino) biphenyl (18mg, 10 mol%) and THF (0.5 mL). The mixture was heated to 120 ℃ for 30 minutes using microwave irradiation. The mixture was then filtered through celite, washing with EtOAc. The filtrate was concentrated and purified by column chromatography (EtOAc/hexane 5/95) to give 120mg (63%) of 6-methyl-2- (2-trifluoromethyl-biphenyl-4-yl) benzothiazole.

To 6-methyl-2- (2-trifluoromethyl-biphenyl-4-yl) benzothiazole (120mg, 0.325mmol) in CCl₄NBS (64mg, 1.1eq.) was added as a solution (3.5 mL). The mixture was heated to reflux for 15 minutes, then AIBN (5mg, 0.1eq.) was added. The reaction was refluxed overnight, filtered through celite, and washed with CCl₄And (6) washing. Concentrating the filtrate byPurification by column chromatography (EtOAc/hexanes: 9/95). 6-bromomethyl-2- (2-trifluoromethyl-biphenyl-4-yl) benzothiazole (105mg, 72%) was isolated.

To a solution of β -alanine tert-butyl ester hydrochloride (47mg, 1.1eq.) in DMF (2mL) was added NaH (60% mineral oil) (28mg, 3eq.) at room temperature. The mixture was stirred at room temperature for 15 minutes, then a solution of 6-bromomethyl-2- (2-trifluoromethyl-biphenyl-4-yl) benzothiazole (105mg, 0.234mmol) in DMF (1mL) was added. The mixture was stirred at room temperature overnight, diluted with EtOAc and taken up with 10% Na₂S₂O₃Washed with brine and over MgSO₄Drying, filtering and concentrating. Purification by column Chromatography (CH)₂Cl₂/CH₃OH, 95/5) to yield 31mg (26%) of 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzothiazol-6-ylmethyl]-amino } -propionic acid tert-butyl ester.

Reacting 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzothiazol-6-ylmethyl]-amino } -propionic acid tert-butyl ester (31mg, 0.0605mmol) dissolved in TFA/CH₂Cl₂(1/1) (1 mL). The solution was stirred at room temperature for 1 hour. The mixture was concentrated and purified by reverse phase preparative LC/MS to give 10mg of 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzothiazol-6-ylmethyl]-amino } -propionic acid:¹H NMR(CD₃OD，400MHz)δ8.54(s，1H)，8.35(d，1H)，8.22(s，1H)，8.18(d，2H)，7.68(d，1H)，7.45(m，3H)，7.37(m，2H)，4.44(s，2H)，3.36(m，2H)，2.80(m，2H)；MS(ES⁺)457.0(M+H⁺)。

example 10

3- { [ 3-chloro-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b]Thien-5-ylmethyl]-amino } -propionic acid

To 5-methyl-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b]Thiophene (184mg, 0.5mmol) in CHCl₃(2.5mL) solution SO was added₂Cl₂(44. mu.L, 1.1 eq.). The mixture was heated to reflux overnight (about 14 hours). All solvents were removed under reduced pressure. The residue is treated with CH₂Cl₂(50mL) extraction with saturated NaHCO₃Aqueous, brine, over MgSO₄Drying, filtering and concentrating to obtain oil. The mixture was purified by column chromatography (EtOAc/hexane gradient) to give 112mg of 3-chloro-5-methyl-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ]]Thiophene, yield 56%.

To 3-chloro-5-methyl-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b]CCl of thiophene (110mg, 0.273mmol)₄NBS (49mg, 1eq.) was added as a solution (3 mL). The mixture was heated at reflux for 15 min, then AIBN (4.5mg, 0.1eq.) was added. The reaction was further heated at reflux overnight. The mixture was filtered through celite and CCl was used₄And (6) washing. The filtrate was concentrated and purified by column chromatography (EtOAc/hexane gradient) to give 50mg of 5-bromomethyl-3-chloro-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ]]Thiophene, yield 38%.

To 5-bromomethyl-3-chloro-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b]Thiophene (50mg, 0.104mmol) in DMF (1mL) was added beta-alanine tert-butyl ester hydrochloride (19mg, 1eq.) and K₂CO₃(68mg, 5 eq.). The mixture was stirred at 50 ℃ overnight. The mixture was diluted with EtOAc (40mL) and 10% Na₂S₂O₃Aqueous, brine, over MgSO₄Drying, filtering and concentrating. The mixture was purified by column chromatography (EtOAc/hexane gradient) to give 43mg of 3- { [ 3-chloro-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ]]Thien-5-ylmethyl]-amino } -propionic acid tert-butyl ester, yield 76%.

3- { [ 3-chloro-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b]Thien-5-ylmethyl]-amino } -propionic acid tert-butyl ester (43mg, 0.079mmol) in TFA/CH₂Cl₂(1/1) (1 mL). The solution was stirred at room temperature for 1 hour. Concentrating the mixture, and performing reverse phase preparationPreparative LC/MS purification gave 23mg3- { [ 3-chloro-2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b]Thien-5-ylmethyl]-amino } -propionic acid:¹HNMR(CD₃OD，400MHz)δ8.10(s，1H)，7.96-8.02(m，3H)，7.54(d，1H)，7.46(d，1H)，7.35-7.37(m，3H)，7.28-7.29(m，2H)，4.37(s，2H)，3.26(m，2H)，2.71(m，2H)，MS(ES⁺)490.3(M+H⁺)。

the procedures described in the above examples were repeated using the appropriate starting materials to give the following compounds of formula I, as identified in Table 1.

TABLE 1

Example 11

The compounds of formula I exhibit biological activity

A. In vitro: GPCR activation assay for measuring GTP [ gamma- ³⁵ S]And from expression of human EDG receptor Binding of membranes prepared from CHO cells

EDG-1(S1P1)GTP[γ-³⁵S]Binding assay: homogenized membranes were prepared from CHO cell clones stably expressing N-terminal c-myc-tagged human EDG-1. The cells were grown in suspension at two 850cm²Transferred into bottles for three or four days and then collected. The cells were centrifuged, washed once with cold PBS, resuspended in ≤ 20mL buffer A (20mM HEPES pH7.4, 10mM EDTA, complete protease inhibitor cocktail without EDTA [1 tablet/25 mL ]]). The cell suspension was homogenized on ice using a Polytron homogenizer at 30000rpm, three times each for 15 seconds. The homogenate was first centrifuged at low speed at 2000rpm for 10 minutes on a table top. After passing through the cell filter, the supernatant was recentrifuged at 50,000Xg for 25 minutes at 4 ℃. The pellet was resuspended in buffer B (15% glycerol, 20mM HEPES pH7.4, 0.1mM EDTA, meta EDTA complete protease inhibitor cocktail [1 tablet/10 mL)]). Protein concentration of the preparations was determined using BCA protein assay kit (Pierce) using BSA as standard. The membrane was divided into aliquots and stored frozen at-80 ℃.

Solutions of test compounds were prepared in DMSO at concentrations ranging from 10mM to 0.01 nM. S1P was diluted in 4% BSA solution as a positive control. The required amount of membrane preparation was diluted with ice-cold assay buffer (20mM HEPES pH7.4, 100mM NaCl, 10mM MgCl)₂0.1% fatty acid free BSA, 5 μ M GDP), vortexed well. 2 μ l or less of the compound was dispensed into each well of a round bottom 96 well polystyrene assay plate, followed by addition of 100 μ l of diluted membrane (3-10 μ g/well) and storage on ice until addition of hot GTP γ S. Will 2³⁵S]GTP γ S was diluted 1: 1000(v/v) with cold assay buffer and 100. mu.l was added to each well. The reaction was carried out at room temperature for 90 minutes, after which the membrane was collected in a Perkin-Elmer Unifilter using a Packard Filtermate harvesterGF/B-96 filter plates. Washing with washing buffer (20mM HEPES pH7.4, 100mM NaCl, 10mM MgCl)₂) After washing several times, the filter was rinsed with 95% ethanol and dried in an oven at 37 ℃ for 30 minutes. MicroScint-20 was added and the plates were sealed for TopCount scintillation counting. Activating GTP [ gamma-³⁵S]The binding curves (raw data) were fitted with a dose-response curve fitting tool from GraphPad Prism to yield EC₅₀The value is obtained. Concentration response curves were generated using six or twelve different concentrations (three data points for each concentration).

Using membranes from CHO cells stably expressing c-terminal c-myc-tagged or untagged receptors, following the interaction with EDG-1 GTP [ gamma-³⁵S]EDG-3, -5, -6 and-8 GTP [ gamma-³⁵S]Binding assays. For each membrane preparation, a titration experiment was first performed with the S1P control to determine the optimal amount of membrane to add to each assay well. The compounds of the present invention were tested according to the above assay and observed to exhibit EDG-1 receptor selectivity. For example, 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b]Thien-5-ylmethyl]-amino } -propionic acid (example 1) has an EC of 0.6nM in the above assay₅₀EDG-1 selectivity is at least 1000 fold compared to one or more other receptors, including EDG-3, EDG-5, EDG-6, and EDG-8.

B. In vitro: FLIPR calcium flux assay

The compounds of the invention were tested for agonist activity against EDG-1, EDG-3, EDG-5 and EDG-6 using the FLIPR calcium flux assay. Briefly, EDG receptor-expressing CHO cells were fed in F-12K medium (ATCC) containing 5% FBS and 500. mu.g/mL G418. Prior to assay, cells were plated in F-12K medium containing 1% FBS at a density of 10,000 cells/well/25 μ L in 384 black clear bottom plates. The following day, cells were washed three times with wash buffer (25 μ L each). Add approximately 25. mu.L of stain to each well at 37 ℃ and 5% CO₂The cells were incubated for 1 hour. The cells were then washed four times with washing buffer (25. mu.L each). Assay after adding 25. mu.L of SEQ2871 solution to each well of cellsCalcium flux. The same assay was performed using cells expressing each different EDG receptor. Titration values in the FLIPR calcium flux assay were recorded at 3 minute intervals and quantified as the maximum peak height percent response relative to EDG-1 activation.

C. In vivo: screening assays for measuring blood lymphocyte failure and assessing cardiac effects

Measurement of circulating lymphocytes:the compounds were dissolved in DMSO, diluted to a final concentration of 4% DMSO (v/v, final concentration), and further diluted in a constant volume of Tween 8025%/H₂In Ov/v. Respectively comprises Tween 8025%/H₂O (200. mu.L), 4% DMSO and FTY 720 (10. mu.g) were used as negative and positive controls. Under short-time isoflurane anesthesia, mice (C57bl/6 male, 6-10 weeks old) were orally administered 250-300. mu.L of compound solution by gavage.

Under short-time isoflurane anesthesia, blood was collected from the retro-orbital sinus 6 and 24 hours after drug administration. Hematological analysis was performed on whole blood samples. The number of peripheral lymphocytes was determined using an automated analyzer. Peripheral blood lymphocyte subpopulations were stained with specific antibodies conjugated to fluorochromes and analyzed using a fluorescence activated cell sorter (Facscalibur). Two mice were used for each compound screened to assess lymphocyte depletion activity. The result is ED₅₀It is defined as the effective dose required to show 50% blood lymphocyte depletion. The compounds of the invention are tested according to the above assay, preferably finding ED₅₀Less than 1mg/kg, more preferably ED₅₀Less than 0.5 mg/kg. For example, 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b]Thien-5-ylmethyl]-amino } -propionic acid (example 1) showed an ED of 0.2mg/kg₅₀。

Assessment of cardiac effects: the effect of compounds on cardiac function was monitored using the AnonyMOUSE ECG screening system. Electrocardiograms of conscious mice (C57bl/6 males, 6-10 weeks old) were recorded before and after compound administration. The ECG signal was then processed and analyzed using e-MOUSE software. A further 90. mu.g dilution in 200. mu.L water, 15% DMSO was madeCompound IP injection. Four mice were used for each compound to assess cardiac effects.

D: in vivo: anti-angiogenic activity

A porous chamber containing (i) sphingosine-1-phosphate (5. mu.M/chamber) or (ii) 0.5mL of 0.8% w/v agar (containing heparin 20U/mL) with human VEGF (1. mu.g/chamber) was implanted subcutaneously in the flank of mice. S1P or VEGF induces growth of vascularized tissue surrounding the porous chamber. This response is dose-dependent and can be quantified by measuring the weight of the tissue and the blood content. Mice were treated orally or intravenously once daily with a compound of formula I starting 4-6 hours prior to implantation into the porous chamber for 4 days. Animals were sacrificed and vascularized tissue was measured 24 hours after the last dose. The weight and blood content of the vascularized tissue surrounding the porous chamber is determined. Animals treated with the compound of formula I show a reduction in the weight and/or blood content of vascularized tissue compared to animals treated with the carrier alone. The compounds of formula I are antiangiogenic when administered at a dose of about 0.3 to about 3 mg/kg.

E: in vitro: antitumor activity

A mouse breast cancer cell line originally isolated from breast cancer, such as jygmc (a), is used. Prior to manipulation, the cell number was adjusted to 5X 10⁵For plating in fresh medium. Cells were incubated with fresh medium containing 2.5mM thymidine without FCS for 12 hours, then washed twice with PBS, followed by addition of fresh medium containing 10% FCS and incubation for another 12 hours. Then, the cells were incubated with fresh medium containing 2.5mM thymidine without FCS for 12 hours. To release cells from the block, cells were washed twice with PBS and replated in fresh medium containing 10% FCS. After synchronization, the cells are incubated with or without various concentrations of the compound of formula I for 3, 6, 9, 12, 18 or 24 hours. Cells were harvested after treatment with 0.2% EDTA, fixed with ice cold 70% ethanol solution, hydrolyzed with 250. mu.g/mL RNase A (type 1-A: SigmaChem. Co.) at 37 ℃ for 30 min, and stained with 10mg/mL propidium iodide for 20 min. After the incubation period, the culture medium is,the cell number was determined by counting in a Coulter counter and by means of an SRB colorimetric assay. Under these conditions, the compound of formula I is at 10^-12To 10^-6The proliferation of tumor cells is inhibited at the concentration of M.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.

Claims (8)

1. A compound of formula I:

wherein:

n is 1 or 2;

a is-C (O) OH;

x is a bond;

y is selected from:

wherein R is₁₁Is hydrogen; the left and right asterisks of Y indicate X and-C (R) of formula I₂)(R₃) -a connection point therebetween; y is optionally substituted with 1 to 3 groups, said groups being C_1-6An alkyl group;

R₁selected from phenyl, naphthyl, optionally substituted by C_6-10Aryl radical C_0-4Alkyl, one to three halogenated C_1-6Alkyl substitution;

R₂、R₃、R₅、R₆and R₈Is hydrogen; r₇Selected from hydrogen and fluorine; r₄Selected from hydrogen and C_1-6An alkyl group.

2. The compound of claim 1, wherein Y is:

y is optionally substituted with 1 to 3 groups selected from methyl, ethyl.

3. The compound of claim 2, wherein X is a bond; r₁Is phenyl, optionally substituted by C_6-10Aryl, one halo C_1-6Alkyl substitution.

4. A compound of claim 3 selected from: 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-6-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -2, 3-dihydro-1H-isoindol-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzofuran-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-4-ylmethyl ] -amino } -propionic acid, 3- { [2- (2, 5-bis-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- [ (2-phenyl-benzo [ b ] thiophen-5-ylmethyl) -amino ] -propionic acid, 3- { [2- (3, 5-bis-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid -propionic acid, 3- { [2- (4-trifluoromethoxy-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (3-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [2- (4-trifluoromethyl-phenyl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [3- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid, 3- { [5- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] b Thien-2-ylmethyl ] -amino } -propionic acid.

3- { [2- (2-trifluoromethyl-biphenyl-4-yl) -benzo [ b ] thiophen-5-ylmethyl ] -amino } -propionic acid and pharmaceutically acceptable salts thereof.

6. A pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of claims 1-5 and a pharmaceutically acceptable excipient.

7. Use of a compound according to any one of claims 1-5 in the manufacture of a medicament for preventing or treating a disorder or disease mediated by lymphocytes, acute or chronic graft rejection or a T-cell mediated inflammatory or autoimmune disease, inhibiting or controlling an angiogenic disorder, or preventing or treating a disease mediated by a neo-angiogenic process or associated with an angiogenic disorder in a subject.

8. Use of a compound according to any one of claims 1-5 in the manufacture of a medicament for the treatment of a disease in an animal, wherein alteration of EDG/S1P receptor mediated signal transduction contributes to the pathology and/or symptomology of the disease.