GB2367817A

GB2367817A - Cyclic carboxylic acids as integrin antagonists

Info

Publication number: GB2367817A
Application number: GB0024695A
Authority: GB
Inventors: Thomas Roelle; Thomas Lehmann; Markus Albers; Gerhard Hessler; Gerhard Mueller; Ruediger Fischer; Masaomi Tajimi; Karl Ziegelbauer; Kevin Bacon; Haruki Hasegawa; Hiromi Okigami
Original assignee: Bayer AG
Current assignee: Bayer AG
Priority date: 2000-10-09
Filing date: 2000-10-09
Publication date: 2002-04-17
Also published as: WO2002030876A3; AU2002218214A1; GB0024695D0; WO2002030876A2

Abstract

Compounds of the general formula (I),<BR> <BR> R<SP>6</SP> G X G A G Cyc G Y G R<SP>1</SP> (I)<BR> <BR> wherein A, Cyc, R<SP>1</SP>, R<SP>6</SP>, X and Y are defined herein, processes for their preparation, pharmaceutical compositions containing them as well as their use for the production of pharmaceutical compositions for the treatment of inflammatory, autoimmune and immune disorders.

Description

Cyclic carboxylic acids as integrin antagonists The present invention relates to compounds of formula (I),

R6 X-A-Cyc-Y-Rl (1)

their preparation and use as pharmaceutical compositions as integrin antagonists, especially as Ct4ssl and/or 0. 47 and/or agpi integrin antagonists and in particular for the production of pharmaceutical compositions suitable for the inhibition or the prevention of cell adhesion and cell-adhesion mediated disorders. Examples are the treatment and the prophylaxis of atherosclerosis, asthma, chronic obstructive pulmonary disease (COPD), allergies, diabetes, inflammatory bowel disease, multiple sclerosis, myocardial ischemia, rheumatoid arthritis, transplant rejection and other in flammatory, autoimmune and immune disorders.

Adhesive interactions between the leukocytes and endothelial cells play a critical role in leukocyte trafficking to sites of inflammation. These events are essential for normal host defense against pathogens and repair of tissue damage, but can also contribute to the pathology of a variety of inflammatory and autoimmune disorders. Indeed, eosinophil and T cell infiltration into the tissue is known as a cardinal feature of allergic inflammation such as asthma.

The interaction of circulating leukocytes with adhesion molecules on the luminal surface of blood vessels appears to modulate leukocyte transmigration. These vascular cell adhesion molecules arrest circulating leukocytes, thereby serving as the first step in their recruitment to infected or inflamed tissue sites. Subsequently, the leukocytes reaching the extravascular space interact with connective tissue cells such as fibroblasts as well as extracellular matrix proteins such as fibronectin, laminin, and collagen. Adhesion molecules on the leukocytes and on the vascular endothelium are hence essential to leukocyte migration and attractive therapeutic targets for intervention in many inflammatory disorders.

Leukocyte recruitment to sites of inflammation occurs in a stepwise fashion beginning with leukocyte tethering to the endothelial cells lining the blood vessels. This is followed by leukocyte rolling, activation, firm adhesion, and transmigration. A number of cell adhesion molecules involved in those four recruitment steps have been identified and characterized to date. Among them, the interaction between vascular cell adhesion molecule 1 (VCAM-1) and very late antigen 4 (VLA-4, cpi integrin), as well as the interaction between mucosal addressin cell adhesion molecule 1 (MAdCAM-1) and a4ss7 integrin, has been shown to mediate the tethering, rolling, and adhesion of lymphocytes and eosinophils, but not neutrophils, to endothelial cells under a physiologic flow condition. This suggests that the VCAM-1/VLA-4 and/or MAdCAM-1/a4p7 integrin mediated interactions could predominantly mediate a selective recruitment of leukocyte subpopulations in vivo. The inhibition of this interaction is a point of departure for therapeutic intervention (A. J. Wardlaw, J. AI. lergy Clin. Immunol. 1999,104, 917-26).

VCAM-1 is a member of immunoglobulin (Ig) superfamily and is one of the key regulators of leukocyte trafficking to sites of inflammation. VCAM-1, along with intracellular adhesion molecule 1 (ICAM-1) and E-selectin, is expressed on inflamed endothelium activated by such cytokines as interleukin 1 (IL-1) and tumor necrosis factor a (TNF-a), as well as by lipopolysaccharide (LPS), via nuclear factor KB (NF KB) dependent pathway. However, these molecules are not expressed on resting endothelium. Cell adhesion mediated by VCAM-1 may be involved in numerous physiological and pathological processes including myogenesis, hematopoiesis, inflammatory reactions, and the development of autoimmune disorders. Integrins VLA

4 and a4ss7 both function as leukocyte receptors for VCAM-1.

The integrin a4ss 1 is a heterodimeric protein expressed in substantial levels on all circulating leukocytes except mature neutrophils. It regulates cell migration into tissues during inflammatory responses and normal lymphocyte trafficking. VLA-4 binds to different primary sequence determinants, such as a QIDSP motif of VCAM

1 and an ILDVP sequence of the major cell type-specific adhesion site of the alternatively spliced type III connecting segment domain (CS-1) of fibronectin. In vivo studies with neutralizing monoclonal antibodies and inhibitor peptides have demonstrated a critical role for a4 integrins interaction in leukocyte-mediated inflammation. Blocking of VLA-4/ligand interactions, thus, holds promise for therapeutic intervention in a variety of inflammatory, autoimmune and immune diseases

(Zimmerman, C. ; Exp. Opin. Ther. Patents 1999, 9, 129-133).

Furthermore, compounds containing a bisarylurea moiety as a substituent were disclosed as a. integrin receptor antagonists : WO 96/22966, WO 97/03094, WO 99/33789, WO 99/37605. However, no aminobenzoic acids or aminocycloalkylcarboxylic acids or homologues thereof or heterocyclics analogues thereof with a4pl integrin receptor antagonists activity have been described.

Further to their a4pi integrin antagonistic activity, the compounds of the present invention may also be used as α4ss7 or ccgpl integrin antagonists.

An object of the present invention is to provide new, alternative, cyclic carboxylic acids or homologues thereof derived integrin antagonists for the treatment of inflammatory, autoimmune and immune diseases.

The present invention therefore relates to compounds of the general formula (I) :

R6 X-A-Cyc-Y-Rl (1)

wherein Cyc represents a 5-or 6-membered carbocycle, which can optionally be substituted with up to two residues Rcyc,

wherein the residues Rcyc can independently be selected from the group consisting of halogen, trifluoromethyl, amino, nitro, cyano, A represents an amide moiety of the structure - C (O)- or-C (O) NRA-, wherein RA l represents hydrogen or Ci-CIo alkyl, R I represents a 4-to 9-membered saturated, unsaturated or aromatic cyclic residue, which can contain 0 to 3 heteroatoms selected independently from the group N, S and 0, and wherein RI is substituted by -R1-1-Z, wherein R1-1 represents a bond,-0-,-S-, NRI-2, C1-C10 alkyl, Cz-Cjo alkenyl, C2-C10 alkynyl, C6 or Clo aryl, C3-C7 cycloalkyl or a 4-9-membered saturated or unsaturated heterocyclic residue containing up to 3 het eroatoms selected from the group oxygen, nitrogen or sulfur, wherein R can optionally be substituted by I to 2 substituents se lected from the group RI-3, wherein Rl-2 can optionally be hydrogen, Cl-cl alkyl, C2-C10 alkenyl or C2-C10 alkynyl, and

wherein Rl-3 represents hydrogen, Cl-Clo alkyl, C2-CIO alkenyl, Cz-Cio alkynyl, C6 or CIO aryl, C3-C7 cycloalkyl or a 4-9-membered saturated or unsaturated heterocyclic residue containing up to 3 heteroatoms selected from the group oxygen, nitrogen or sulfur,

Z represents-C (O) OR,-C (O) NRz-2Rz-3,-SO2NRz-2Rz-3,-SO (ORz-1), - S02 (ORz-1),-P (0) Rz-1 (ORz-3) or-PO (ORZ-1) (OR), wherein R is hydrogen, Ci-C4 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C6 or C10 aryl, -C (O) RZ-4 or -SO2RZ-4, wherein RZ-4 is C1-C4 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C6 or C10 aryl, R-I and Rz-3 are independently selected from the group hydrogen, Cl-C4 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C6 or CIO aryl or benzyl, wherein RZ-1 and RZ-3 can optionally be substituted by 1 to 3 substitu ents selected from the group Cl-C4 alkyl, Cl-C4 alkyloxy, halogen, ni tro, cyano, and wherein R'can optionally be substituted by 0 to 2 substituents Rl-4, halogen, nitro, amino, cyano and oxo, wherein R1-4 is selected from the group C1-C4 alkyl, Cl-C4 alkyloxy, phenyl, phenoxy, phenylamino, C3-C6 cycloalkyl,

R6 represents phenyl or a 5-to 6-membered aromatic heterocyclic residue containing up to 3 heteroatoms independently selected from the group oxygen, nitrogen or sulfur, which is substituted by -NR6-2C (O) NR6-3R6-4 and can furthermore optionally be substituted by halogen,

wherein R and R 6 are independently selected from the group hydrogen or Ci-C4 alkyl, or together form a group

and wherein R6-4 represents phenyl, wherein R can optionally be substituted by 1-2 substituents selected from the group Cl-C4 alkyl, Cl-C4 alkyloxy, halogen, nitro, trifluoromethyl, trifluoromethoxy or cyano, X represents bond or -CRX-1RX-2-,

wherein RX' and Rx-2 can be independently selected from the group hydrogen, Ci-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, Y represents an amide moiety of the structure -NRY-1C(O)- or -C(O)NRY-1-, wherein R''represents hydrogen or Cl-C4 alkyl,

wherein the moiety A-Cyc-Y represents a y-amino acid, and pharmaceutically acceptable salts thereof.

In a preferred embodiment, the present invention relates to compounds of general formula (I), wherein Cyc represents a 5-membered carbocycle.

In another preferred embodiment, the present invention relates to compounds of general formula (I), wherein R'represents a 1, 4-substituted phenyl ring.

In another preferred embodiment, the present invention relates to compounds of general formula (I), wherein

wherein Rl-l represents a bond and Z represents COORZ-I, wherein R-'has the meaning indicated above. In another preferred embodiment, the present invention relates to compounds of general formula (I), wherein R6 represents phenyl, which is substituted by-NHC (O) NHR6-4,

wherein R is substituted with methyl or trifluoromethoxy.

A preferred process for preparation of compounds of general formula (VII) has also been found, which comprises reaction of compounds of general formula (I')

wherein Cyc, X, R5, R6 and R8 have the abovementioned meaning, with compounds of the general formula (1")

wherein R1 has the abovementioned meaning and AG represents an activating group, in inert solvents, which will be described in more detail in the descriptive part of the specification.

In the context of the present invention alkyl stands for a straight-chain or branched alkyl residue, such as methyl, ethyl, n-propyl, iso-propyl, n-pentyl. If not stated otherwise, preferred is Cl-Clo alkyl, very preferred is Cl-C6 alkyl.

Alkenyl and alkinyl stand for straight-chain or branched residues containing one or more double or triple bonds, e. g. vinyl, allyl, isopropinyl, ethinyl. If not stated otherwise, preferred is Cl-Clo alkenyl or alkinyl, very preferred is Cl-C6 alkenyl or alkinyl.

Cycloalkyl stands for a cyclic alkyl group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. Preferred is monocyclic C3-C7 cycloalkyl.

Halogen in the context of the present invention stands for fluorine, chlorine, bromine or iodine. If not specified otherwise, chlorine or fluorine are preferred.

A 4-to 9-membered saturated, unsaturated or aromatic cyclic residue stands for a monocyclic system containing 4 to 9 ring atoms and containing 0,1 or more double bonds, which can be attached via a carbon atom or eventually via a heteroatom within the ring, for example phenyl, thiazolyl, pyridyl, cyclopentyl.

Aryl stands for a monocyclic Hueckel-aromatic cyclic system containing 6 or 10 ring carbon atoms.

Heteroaryl or aromatic heterocyclic residue stands for a monocyclic heteroaromatic system containing 4 to 9 ring atoms, which can be attached via a carbon atom or eventually via a nitrogen atom within the ring, for example, furan-2-yl, furan-3-yl, pyrrol-1-yl, pyrrol-2-yl, pyrrol-3-yl, thienyl, thiazolyl, oxazolyl, imidazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidyl or pyridazinyl.

A saturated or unsaturated heterocyclic residue stands for a heterocyclic system containing 4 to 9 ring atoms, which can contain one or more double bonds and which can be attached via a ring carbon atom or eventually via a nitrogen atom, e. g. tetrahydrofur-2-yl, pyrrolidine-1-yl, piperidine-1-yl, piperidine-2-yl, piperidine-3-yl, piperidine-4-yl, piperazine-1-yl, piperazine-2-yl morpholine-1-yl, 1, 4-diazepine-l-yl or 1, 4-dihydropyridine-l-yl.

Carbocycle stands for a ring consisting of carbon atoms.

If not specified otherwise, in the context of the present invention heteroatom stands preferably for 0, S, N or P.

In the context of the present invention, the moiety A-Cyc-Y represents a y-amino acid. This group can therefore be represented as:

Surprisingly, the compounds of the present invention show good integrin antagonistic activity. They are therefore suitable for the treatment of diseases, especially as 0. 4 ? i and/or Ct. 4 7 and/or agi integrin antagonists and in the manufacture of a medicament for the treatment or the prevention of a condition mediated by integrins and in particular for the production of pharmaceutical compositions for the inhibition or the prevention of cell adhesion and cell-adhesion mediated disorders. Examples are the treatment and the prophylaxis of atherosclerosis, asthma, chronic obstructive pulmonary disease (COPD), allergies, diabetes, inflammatory bowel disease, multiple sclerosis, myocardial ischemia, rheumatoid arthritis, transplant rejection and other inflammatory, autoimmune and immune disorders.

The integrin antagonists of the invention are useful not only for treatment of the physiological conditions discussed above, but are also useful in such activities as purification of integrins and testing for activity.

For the treatment of the above-mentioned diseases, the compounds according to the invention can exhibit non-systemic or systemic activity, wherein the latter is preferred. To obtain systemic activity the active compounds can be administered, among other things, orally or parenterally, wherein oral administration is preferred.

For parenteral administration, forms of administration to the mucous membranes (i. e. buccal, lingual, sublingual, rectal, nasal, pulmonary, conjunctival or intravaginal) or into the interior of the body are particularly suitable. Administration can be carried out by avoiding absorption (i. e. intracardiac, intra-arterial, intravenous, intraspinal or

intralumbar administration) or by including absorption (i. e. intracutaneous, subcutaneous, percutaneous, intramuscular or intraperitoneal administration).

For the above purpose the active compounds can be administered per se or in administration forms.

Suitable administration forms for oral administration are, inter alia, normal and enteric-coated tablets, capsules, coated tablets, pills, granules, pellets, powders, solid and liquid aerosols, syrups, emulsions, suspensions and solutions. Suitable administation forms for parenteral administration are injection and infusion solutions.

The active compound can be present in the administration forms in concentrations of from 0.001-100 % by weight; preferably the concentration of the active compound should be 0.5-90% by weight, i. e. quantities which are sufficient to allow the specified range of dosage.

The active compounds can be converted in the known manner into the abovementioned administration forms using inert non-toxic pharmaceutically suitable auxiliaries, such as for example excipients, solvents, vehicles, emulsifiers and/or dispersants.

The following auxiliaries can be mentioned as examples: water, solid excipients such as ground natural or synthetic minerals (e. g. talcum or silicates), sugar (e. g. lactose), non-toxic organic solvents such as paraffins, vegetable oils (e. g. sesame oil), alcohols (e. g. ethanol, glycerol), glycols (e. g. polyethylene glycol), emulsifying agents, dispersants (e. g. polyvinylpyrrolidone) and lubricants (e. g. magnesium sulphate).

In the case of oral administration tablets can of course also contain additives such as sodium citrate as well as additives such as starch, gelatin and the like. Flavour enhancers or colorants can also be added to aqueous preparations for oral administration.

For the obtainment of effective results in the case of parenteral administration it has generally proven advantageous to administer quantities of about 0. 001 to 100 mg/kg, preferably about 0.01 to 1 mg/kg of body weight. In the case of oral administration the quantity is about 0.01 to 100 mg/kg, preferably about 0. 1 to 10 mg/kg of body weight.

It may nevertheless be necessary to use quantities other than those mentioned above, depending on the body weight concerned, the method of administration, the individual response to the active compound, the type of preparation and the time or interval of administration.

Pharmaceutically acceptable salts of the compounds of the present invention that contain an acidic moiety include addition salts formed with organic or inorganic bases. The salt forming ion derived from such bases can be metal ions, e. g. , alumi- num, alkali metal ions, such as sodium of potassium, alkaline earth metal ions such as calcium or magnesium, or an amine salt ion, of which a number are known for this purpose. Examples include ammonium salts, arylalkylamines such as dibenzylamine and N, N-dibenzylethylenediamine, lower alkylamines such as methylamin, t-

butylamine, procaine, lower alkylpiperidines such as N-ethylpiperidine, cycloalkylamines such as cyclohexylamin or dicyclohexylamine, 1-adamantylamine, benza thine, or salts derived from amino acids like arginine, lysine or the like. The physiologically acceptable salts such as the sodium or potassium salts and the amino acid salts can be used medicinally as described below and are preferred.

Pharmaceutically acceptable salts of the compounds of the present invention that contain a basic moiety include addition salts formed with organic or inorganic acids. The salt forming ion derived from such acids can be halide ions or ions of natural or unnatural carboxylic or sulfonic acids, of which a number are known for this purpose. Examples include chlorides, acetates, trifluoroacetates, tartrates, or salts derived from amino acids like glycine or the like. The physiologically acceptable

salts such as the chloride salts, the trifluoroacetic acid salts and the amino acid salts can be used medicinally as described below and are preferred.

These and other salts which are not necessarily physiologically acceptable are useful in isolating or purifying a product acceptable for the purposes described below.

The compounds according to the invention can exist in different stereoisomeric forms, which relate to each other in an enantiomeric way (image and mirror image) or in a diastereomeric way (image different from mirror image). The invention relates to the enantiomers and the diastereomers as well as their mixtures. They can be separated according to customary methods.

The compounds according to the invention can exist in tautomeric forms. This is known to the artisan and such compounds are also object of the present invention.

General compound synthesis The compounds according to the present invention are y-amino acids. They can be prepared employing standard amide coupling procedures. In case A stands for NR2C (O)- and Y stands for-NR8C (O)-, the synthesis of compounds according to general formula (I) can be illustrated by the following scheme 1:

Scheme 1 By coupling of the amines (II) with the carboxylic acids or activated derivatives (III), followed by removal of the protecting group PG'the amides (V) can be obtained. Coupling with the carboxylic acids or activated derivatives (VI) affords carboxylic acids of type (VII). If an protecting group PG is used to protect an carboxylic acid functionality on R', removal of the protecting group PG2 follows.

In the above scheme cyc in formulas (III)- (V) and (VII) as well as in scheme 2 represents a cyclic moiety. The depicted ring in formulas (VI)- (VIM as well as in scheme 2 and 3 represents a cyclic moiety. AG stands for hydroxyl or a suitable activating group forming an activated carboxylic acid derivative. Activated carboxylic acids derivatives of this type are known to the person skilled in the art and are described in detail in standard textbooks such as, for example in (i) Houben

Weyl, Methoden der organischen Chemie [Methods of Organic Chemistry], Georg Thieme Verlag, Stuttgart or (ii) Comprehensive Organic Synthesis, Ed. B. M. Trost, Pergamon Press, Oxford, 1991. The carboxylic acid is preferably activated as mixed anhydride, such as, for example, AG = iso-butyl-carbonate or by a coupling agents such as, for example dicyclohexylcarbodiimid (DCC), l-ethyl-3- (3'-dimethylamino- propyl) carbodiimidexHCI (EDCI), 2- (7-aza-3-oxido-lH-l, 2, 3-benzotriazol-1-yl)

1, 1, 3, 3-tetramethyluronium hexafluorophosphate. Other activated carboxylic acid derivatives such as, for example symmetric anhydrides, halides, or activated esters e. g. succinyl, pentafluorophenyl or N-hydroxybenzotriazole esters may also be employed.

In the above scheme Pal stands for a suitable protecting group of the amino group that is stable under the respective reaction conditions. Protecting groups of this type are known to the person skilled in the art and are described in detail in T. W. Greene, P. G. Wuts, Protective Groups in Organic Synthesis, 3rd ed., John Wiley, New York, 1999. The amino group is preferably protected by carbamate, Pal being for example tert-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (FMOC) or benzyloxycarbonyl (Cbz-/Z-) or other oxycarbonyl derivatives.

PG stands for a suitable protecting group of the carboxyl group or COOPG2 stands for the carboxylic group attached to a polymeric resin suitable for solid phase synthesis. Protecting groups of this type are known to the person skilled in the art and are described in detail in T. W. Greene, P. G. Wuts, Protective Groups in Organic Synthesis, 3d ed. , John Wiley, New York, 1999. The carboxyl group is preferably esterified, PG2 being C1. 6-alkyl such as, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, a C3-7- cycloalkyl such as, for example, cyclopropyl, cyclopropyhnethyl, cyclobutyl, cyclopentyl, cyclohexyl, an aryl such as, for example, phenyl, benzyl, tolyl or a substituted derivative thereof.

Step A Formation of the amides (IV) can take place by reacting an activated form of the respective carboxylic acid (III), such as an iso-butylcarbonate or N-hydroxybenzotriazole ester-with the desired amine (II) or an acceptable salt thereof.

Iso-butylcarbonates can be prepared in situ by reaction of the N-protected amino acid (ill) with iso-butylchloroformate as described below. Activated derivatives of the acids (III) such as other anhydrides, halides, esters e. g. succinyl, N-hydroxybenzotriazole or pentafluorophenyl esters or activated carboxylic acids obtained by the reaction with coupling agents such as, for example dicyclohexylcarbodiimid (DCC), 1-ethyl-3- (3'-dimethylaminopropyl) carbodiimidexHCI (EDCI), 2- (7-aza-3-oxido- IH-1, 2, 3-benzotriazol-1-yl)-1, 1, 3,3-tetramethyluronium hexafluorophosphate may also be employed.

1-Hydroxy-1H-benzotriazol ester of (III) can be prepared, for example, by the reaction of the 1-hydroxy-lH-benzotriazol with the carboxylic acids (III) in presence

of an coupling agents such as, for example, dicyclohexylcarbodiimid (DCC), 1-ethyl3- (3'-dimethylaminopropyl) carbodiimidexHCI (EDCI), 2- (7-aza-3-oxido-1H-l, 2, 3 benzotriazol-1-yl)-1, 1, 3, 3-tetramethyluronium hexafluorophosphate. Further activated derivatives of the acids (III) such as other anhydrides, halides, esters e. g. succinyl or pentafluorophenyl esters or activated carboxylic acids obtained by the reaction with may also be employed.

For example, amides of type (IV) can be prepared as follows: 1) Mixed anhydride procedure A solution of the carboxylic acid derivative (III) and of N-methylmorpholine in an inert solvent was cooled to-1 5*C and iso-butyl chloroformate was added and stirred at 0 C. The amine (II) in an inert solvent was added at-15 C. The solution was stirred at 0 C, and at r. t. and was evaporated. The residue was redissolved in ethyl

acetate, washed with aqueous acid and base, dried and evaporated. If necessary the product was purified by trituration or by flash-chromatography or used without fur ther purification.

2) 1-Hydroxy-lH-benzotriazol ester procedure A solution of carboxylic acid, 1-hydroxy-lH-benzotriazol (HOBt) and l-ethyl-3- (3'- dimethylaminopropyl) carbodiimidexHCI (EDCI) in an inert solvent is stirred at r. t..

After addition of the amine and a non-nucleophilic base such as ethyldiisopropylamine or potassium carbonate stirring is continued at r. t. or elevated temperature.

After evaporation, the residue was redissolved in ethyl acetate, washed with aqueous acid and base, dried and evaporated. If necessary the product was purified by trituration or by flash-chromatography or used without further purification.

The above reactions and their implementation are well known to the person skilled in the art and are described in detail in standard textbooks such as, for example, in (i) Houben-Weyl, Methoden der organischen Chemie [Methods of Organic Chemistry], Georg Thieme Verlag, Stuttgart or Stuttgart or (ii) Comprehensive Organic Synthesis, Ed. B. M. Trost, Pergamon Press, Oxford, 1991.

Compounds of general formula (II) are commercially available, known or can be prepared by customary methods starting from known carboxylic acid derivatives.

In case Rl-l is a methylen group, the carbon chain can be elongated by Amdt-Eistert- reaction and optionally be derivatized by common methods for a-derivatization of carboxylic acids such as nucleophilic substitution.

Step B The removal of protecting group PG'can be performed, depending on the nature of PAGI, either by an acid such as trifluoroacetic acid for example in the case PGI is tertbutyloxycarbonyl (Boc), a base such as piperidine for example in the case Put ils 9

fluorenylmethyloxycarbonyl (FMOC) or by catalytic hydrogenation for example in the case PG'is benzyloxycarbonyl (Cbz-/Z-).

Step C Formation of the amides (VII) can take place by reacting the respective carboxylic acids (VI) -activated by a coupling agent such as DCC and HOBt ; EDCI and HOBt or HATU-with the desired amines (V) or an acceptable salt thereof. Activated derivatives of the acids (VI) such as anhydrides, halides, and esters e. g. succinyl or pentafluorophenyl esters may also be employed.

For example, amides (VII) can be prepared as follows: A solution of carboxylic acid, HOBt and EDCI in an inert solvent is stirred at r. t..

After addition of the amine and a non-nucleophilic base such as ethyldiisopropylamine stirring is continued at r. t. or elevated temperature. The reaction mixture is poured into water and worked up by standard procedures.

Compounds of general formula (VI) are commercially available, known or can be prepared by customary methods starting from known carboxylic acid derivatives.

Bisarylureas can be prepared by coupling of an amino phenyl acetic acid derivative and a phenylisocyanate.

Step D If necessary, a removal of the protecting group PG can be performed either by an acid such as trifluoroacetic acid or an base such as potassium hydroxide or lithium hydroxide, depending on the nature of Pug2. Reactions are carried out in aqueous, inert organic solvents such as alcohols e. g. methanol or ethanol, ethers e. g.

tetrahydrofurane or dioxane or polar aprotic solvents e. g. dimethylfbrmamidc. If necessary, mixtures of the above solvents may be used.

In case A stands for-C (O) NR2- and Y stands for-C (O) NR\ the synthesis of compounds according to general formula (I) can be illustrated by the following scheme 2:

Scheme 2 By coupling of the carboxylic acids or activated derivatives (II) with the amines (III), followed by removal of the protecting group PG', the amides (V) can be obtained.

Coupling with the carboxylic acids or activated derivatives (VI) affords carboxylic acids of type (VII), If an protecting group PG2 is used to protect an carboxylic acid functionality on R\ removal of the protecting group PG2 follows.

In the above scheme the depicted ring in formulas (III)- (V), (VII) and (VITS) as well as in scheme 1 represents a cyclic moiety. AG stands for hydroxyl or a suitable activating group forming an activated carboxylic acid derivative.

Step A Formation of the amides (IV) can take place by reacting an activated form of the re spective carboxylic acid (III), such as an iso-butylcarbonate or N-hydroxybenzotriazole ester-with the desired amine (II) or an acceptable salt thereof.

Iso-butylcarbonates can be prepared in situ by reaction of the N-protected amino acid (III) with iso-butylchloroformate as described below. Activated derivatives of the acids (III) such as other anhydrides, halides, esters e. g. succinyl, N-hydroxybenzotriazole or pentafluorophenyl esters or activated carboxylic acids obtained by the reaction with coupling agents such as, for example dicyclohexylcarbodiimid (DCC),

1-ethyl-3- (3'-dimethylaminopropyl) carbodiimidexHCl (EDCI), 2- (7-aza-3-oxido1 H-l, 2, 3-benzotriazol-1-yl)-1, 1, 3, 3-tetramethyluronium hexafluorophosphate may also be employed. 1-Hydroxy-lH-benzotriazol ester of (III) can be prepared, for example, by the

reaction of the 1-hydroxy-lH-benzotriazol with the carboxylic acids (ici) in presence of an coupling agents such as, for example, dicyclohexylcarbodiimid (DCC), 1-ethyl3- (3'-dimethylaminopropyl) carbodiimidexHCl (EDCI), 2- (7-aza-3-oxido-lH-l, 2, 3benzotriazol-1-yl)-1, 1, 3, 3-tetramethyluronium hexafluorophosphate. Further activat ed derivatives of the acids (III) such as other anhydrides, halides, esters e. g. succinyl or pentafluorophenyl esters or activated carboxylic acids obtained by the reaction with may also be employed.

For example, amides of type (fiv) can be prepared as follows: 1) Mixed anhydride procedure A solution of the carboxylic acid derivative (III) and of N-methylmorpholine in an inert solvent was cooled to-15 C and iso-butyl chlorofonnate was added and stirred at 0 C. The amine (II) in an inert solvent was added at-15 C. The solution was stirred at 0 C, and at r. t. and was evaporated. The residue was redissolved in ethyl acetate, washed with aqueous acid and base, dried and evaporated. If necessary the

product was purified by trituration or by flash-chromatography or used without fur ther purification.

2) I-Hydroxy-lH-benzotriazol ester procedure A solution of carboxylic acid, 1-hydroxy-lH-benzotriazol (HOBt) and l-ethyl-3- (3'- dimethylaminopropyl) carbodiimidexHCI (EDCI) in an inert solvent is stirred at r. t..

Compounds of general formulas (II) are commercially available, known or can be prepared by customary methods starting from known carboxylic acid derivatives.

Bisarylureas can be prepared by coupling of an amino phenyl acetic acid derivative and a phenylisocyanate. Bisarylamides can be prepared by coupling of an amino phenyl acetic acid and an activated benzoic acid derivative such as described under Step A. Bisarylcarbamates can be prepared by coupling of an isocyanato phenyl acetic acid ester and a phenol derivative followed by saponification as described in Step D.

Step B

The removal of protecting group PGI can be performed, depending on the nature of PG\ either by an acid such as trifluoroacetic acid for example in the case PG is tertbutyloxycarbonyl (Boc), a base such as piperidin for example in the case Pal ils 9 fluorenyhnethyloxycarbonyl (FMOC) or by catalytic hydrogenation for example in the case Psi ils benzyloxycarbonyl (Cbz-/Z-).

For example, amides (VII) can be prepared as follows : A solution of carboxylic acid, HOBt and EDCI in an inert solvent is stirred at r. t.. After addition of the amine and a non-nucleophilic base such as ethyldiisopropylamine stirring is continued at r. t. or elevated temperature. The reaction mixture is poured into water and worked up by standard procedures.

Step D The removal of the protecting group PG2 can be performed either by an acid such as trifluoroacetic acid or an base such as potassium hydroxide or lithium hydroxide, depending on the nature of PG2. Reactions are carried out in aqueous, inert organic solvents such as alcohols e. g. methanol or ethanol, ethers e. g. tetrahydrofurane or dioxane or polar aprotic solvents e. g. dimethylformamide. If necessary, mixtures of the above solvents may be used.

Examples Abbreviations AcOH acetic acid Boc tert-butyloxycarbonyl DCC dicyclohexylcarbodiimid DCM dichloromethane DIPEA diisopropylethylamine EDCI l-ethyl-3- (3'-dimethylaminopropyl) carbodiimidexHCl eq. equivalents EtOAc ethyl acetate FC flash chromatography GC gas chromatography HATU 2- (7-aza-3-oxido-lH-l, 2, 3-benzotriazol-l-yl)-l, 1, 3, 3-tetramethyluro nium hexafluorophosphate HOBt N-hydroxybenzotriazole monohydrate HPLC high performance liquid chromatography ICAM-1 intracellular adhesion molecule 1 IL-1 interleukin 1 LPS lipopolysaccharide MAdCAM-1 mucosal addressin cell adhesion molecule 1 MeOH methanol MeCN acetonitril min. minutes M. p. melting point NF-KB nuclear factor KB NMR nuclear magnetic resonance n. d. not determined

PE light petroleum (b. p. 40-60 OC) r. t. room temperature

Rf TLC : Rf value = distance spot traveled/distance solvent front traveled TFA trifluoroacetic acid THF tetrahydrofurane TLC thin layer chromatography TNF-a tumor necrosis factor a tR retention time determined by HPLC VCAM-1 vascular cell adhesion molecule 1 VLA-4 very late antigen 4 (azol integrin) General remarks In the examples below, all quantitative data, if not stated otherwise, relate to percentages by weight.

Flash chromatography was carried out on silica gel 60, 40-63gm (E. Merck, Darmstadt, Germany).

Thin layer chromatography was carried out, employing silica gel 60 F254 coated aluminum sheets (E. Merck, Darmstadt, Germany) with the mobile phase indicated.

Melting points were determined in open capillaries and are not corrected.

The mass determinations were carried out using the electron spray ionization (ESI) method employing loop injection or split injection via a HPLC system.

Precursor synthesis Example I : N- (4-aminophenyl)-N'- (2-methylphenyl) urea

2-Methylphenylisocyanate (24.6 g, 184. 9 mmol) was added dropwise at 0 C to a solution of 1, 4-diamino benzene (20. 00 g, 184. 9 mmol) in 1000 mL EtOAc. After stirring for 2 h at r. t. the product was collected by filtration (42.7 g, 177.0 mmol).

M. p. > 300 C ; TLC (PE/EtOAc 1/4) Rf 0. 32; IH-NMR (400 MHz, D6-DMSO) 8 2. 10

(s, 3H) ; 4. 76 (s, 2H) ; 6. 59 (mc, 2H) ; 6. 89 (mc, 1H) ; 7. 07-7. 15 (m, 4H) ; 7. 73 (s, IH) ; 7. 85 (mc, 2H) ; 8. 50 (s, 1H).

Example II : tert-Butyl 4- ( { [ (2-methylphenyl) amino] carbonyl} amino) benzyl carbamate

2-Methylphenylisocyanate (7.57 g, 59. 83 mmol) was added dropwise at 0 C to a solution of (4-amino-benzyl)-carbamic acid tert-butyl ester (Moloney, Gerard P.; Martin, Graeme R.; Mathews, Neil; Milne, Aynsley; Hobbs, Heather; et al. ; J Med. Chers. 1999, 42, 2504-2526; 13.30 g, 59.83 mmol) in 120 mL DCM. The reaction was heated under reflux for 16 h, cooled to r. t. and the precipitated product was

collected by filtration and dried in vacuum (19. 20 g, 54. 00 mmol). M. p. 200-202 oC ; TLC (PE/EtOAc 1/1) Rf 0. 65 ; IH NMR (400 MHz, D6-DMSO) 8 1. 39 (s, 9H) ; 2. 24 (s, 3H) ; 4. 06 (d, J=6 Hz, 2H) ; 6. 93 (me, 1H) ; 7. 12-7. 17 (m, 4) ; 7. 32 (mc, 1H) ; 7. 40 (me, 2H) ; 7. 85 (mc, 1H) ; 7. 90 (s, 1H) ; 8. 98 (s, 1H).

Example III : N- [4- (Aminomethyl) phenyl]-N'- (2-methylphenyl) urea

To a solution of tert-butyl 4- ( {[ (2-methylphenyl) amino]carbonyl} amino) benzylcarb amate (2.00 g, 5.63 mmol) in CH2C12 (120 mL) TFA (36 mL) was added at 0 C and stirred for 2 h at r. t.. The reaction mixture was evaporated and the product was collected (2. 72 g, TFA salt). M. p. 142-143 OC ; TLC (PE/EtOAc 3/2) Rf 0.14 ;'H

NMR (400 MHz, D6-DMSO) 8 2. 24 (s, 3H) ; 3. 97 (q, J=5 Hz, 2H) ; 6. 96 (me, 1H) ; 7.13-7. 19 (m, 2); 7.36 (me, 2H); 7.51 (me, 2H); 7.81 (me, 2H); 8.06 (s, 1H); 8.08 (s, 3H); 9.23 (s, 1H).

Example IV: [4- ( {[ (2-Methylphenyl) amino] carbonyl} amino) phenyl] acetic acid

To a solution of2- (4-aminophenyl) acetic acid (108.8 g, 0.72 mol) in CH2C12 (1.0 1) and triethylamine (120 ml) was added a solution of 2-methylphenyl isocyanate (90.5 ml, 0.72 mol) in CH2C12 (500 ml) dropwise at r. t.. After stirring for 18 h at r. t., water (2.5 1) and CH2C12 (2.0 1) were added and the layers were separated. The organic layer was extracted with water (3 x 400 ml). The combined aqueous layers were concentrated to 3.0 1 and acidified to pH 2 by the addition of concentrated aqueous Hic1. The precipitate was collected by filtration, washed with cold water and dried in an exsiccator over concentrated H2SO4 affording 166.5 g (82%) white solid.

M. p. 205-206OC ; TLC (CH2C2/MeOH 9: 1): Rf 0. 14.'H-NMR (400 MHz, D6 DMSO): 12.21 (br s, IH), 9.11 (s, 1H), 8. 00 (s, IH), 7.83 (d, 7.6 Hz, 1H), 7.40 (d, 8.5 Hz, 2H), 7.17-7. 12 (m, 4H), 6.96-6. 92 (m, 1H), 3.48 (s, 2H), 2.24 (s, 3H).

Compound synthesis

Examples 1 and 2 were prepared by the following general procedure. Schemes 3 and 4 shall illustrate the process in an exemplaric way :

Scheme 3

Step A : Example V : Methyl 4- [ ( { (15'*, 3*)-3- [ (tert-butoxycarbonyl) ammo] cyclopentyl}carbonyl) amino benzoate

(IS*, 3R)-3- [ (tert-Butoxycarbonyl) amino] cyclopentanecarboxylic acid (Lit. : Marco Contelles, Jose; Bernabe, Manuel; Tetrahedron Lett. 1994; 35, 6361-6364) (4.59 g, 20.00 mmol) was dissolved in THF (50 mL), N-methylmorpholin (2. 04 g, 20.00 mmol) and isobutylchloroformate (2. 73 g, 20. 00 mmol) were added at -15 C.

Stirring was continued for 45 min at 0 C. 4-Aminomethylbenzoate (3.02 g, 20.00 mmol, dissolved in 10 mL THF) were added dropwise at -15 C. The reaction mixture was heated under reflux for 48 h. The solvent was evaporated, the residue was redissolved in EtOAc and washed with 1 N HCI, sat. soda, brine and dried (MgS04). FC (PE/EE 9: 1) yielded methyl 4-[({(1S*,3R*)-3[(tert-butoxycarbonyl)amino] cyclopentyl}-carbonyl) amino] benzoate (5.95 g, 16.42 mmol). TLC (PE/EtOAc 6/4) Rf 0. 26 ; 1H-NMR (400 MHz, D6-DMSO) 8 1.38 (s, 9H); 1.47-1. 64

(m, 2H) ; 1. 82 (me, 3H) ; 2. 08-2. 15 (m, 1H) ; 2. 80-2. 88 (m, IH) ; 3. 82 (s, 4H) ; 6. 82 (bs, 1H) ; 7. 72-7. 74 (m, 2H) ; 7. 89-7. 91 (m, 2H) ; 10. 10 (bs, 1H).

Step B : Example VI : Methyl 4- ( { [ (15'*, 3*)-3-aminocyclopentyl] carbonyl} amino) benzoate

To a solution of methyl 4-[ ( { (lS., 3R.)-3-[ (tert-butoxycarbonyl) amino]cyc1opentyl}carbonyl) amino] benzoate (2. 70 g, 7. 45 mmol) in CH2Clz (50 mL) TFA (10 mL) was added at 0 C and stirred for 2 h at r. t.. The reaction mixture was evaporated and the product was collected (4.10 g, TFA salt). 1H-NMR (400 MHz, D6-DMSO) 1.70

(me, IH) ; 1. 80-2. 03 (m, 4H) ; 2. 25 (me, 1H) ; 2. 98 (me, 1H) ; 3. 60 (me, 1H) ; 3. 83 (s, 3H) ; 7. 73-7. 76 (m, 2H) ; 7. 88-7. 96 (m, 5H) ; 10. 16 (s, 1H).

Step C : Example 1 : Methyl 4- ( { [ (15'', 3R*)-3- ( { [4- ( { [ (2-methylphenyl) amino) carbonyl} amino) phenyl] acetyl} amino) cyclopentyl] carbonyl} amino) benzoate

[4- ( { [ (2-Methylphenyl) amino] carbonyl} amino) phenyl] acetic acid (1.48 g, 5. 21 mmol) was dissolved in DMF (lOmL), HOBt (880 mg, 5.73 mmol), EDCI (1. lOg, 5. 73 mmol), DIPEA (6. 06 g, 46. 89 mmol) was added and stirring was continued for 2 h at r. t. Methyl 4-({[(1S*,3R*)-3-aminocyclopentyl]carbonyl} amino) - benzoate (1.96 g, 5. 21 mmol dissolved in 8 mL DMF) was added at r. t. After stirring

for 48 h, water (80 mL) was added and the precipitate was collected, yielding methyl 4- ( { [ (15'*, 3')-3- ( { [4- ( { [ (2-methylphenyl) amino] carbonyl} amino) phenyl] acetyl}amino) cyclopentyl] carbonyl} ammo) benzoate (2. 10 g, 3. 97 mmol). M. p. 208-210 oc, ESI-MS : 529 [M+Ht Step D : Example 2 : 4- ( { [ (l*, 3)-3- ( { [4- ( { [ (2-Methylphenyl) amino] carbonyl} amino)phenyl] acetyl} amino) cyclopentyl] carbonyl} amino) benzoic acid

Methyl 4- ( {[ (lS., 3R.)-3- ( {[4- ( {[ (2-methylphenyl) amino]carbonyl} amino) phenyl]acetyl} amino) cyclopentyl] carbonyl} amino) benzoate (1. 39 g, 2. 63 mmol) was dissolved in MeOH/water (1/1), potassium hydroxide (160 mg, 2. 89 mmol) was added and the reaction mixture was stirred at 30 oC for 12 h. MTBE was added, the aqueous layer was extracted once with MTBE, HCI was added to the organic layer (pH 2) and 4- ( { [ (1*, 3*)-3- ( { [4- ( { [ (2-Methylphenyl) amino] earbonyl} amino) phenyl] acetyl}amino) cyclopentyl] carbonyl} amino) benzoic acid (1. 18 g, 2. 29 mmol). M. p. 270273 oC. ESI-MS : 513 [M-H] +

Examples 3-10 were prepared by the following general procedure.

Scheme 4

Step A : Example VII : tert-Butyl- (19*, 3R*)-3- ( { [4- (I [ (2-methylphenyl) amino] carbonyl)amino) phenyl] amino} carbonyl) cyclopentylcarbamate

1, 3'*)-3- [ (tert-Butoxycarbonyl) amino] cyclopentanecarboxylic acid (Lit. : Marco Contelles, Jose ; Bemabe, Manuel ; Tetrahedron Lett. 1994, 35, 6361-6364) (2. 00 g, 8. 72 mmol) was dissolved in DMF (15mL), HOBT (1. 47g, 9. 60 mmol), EDCI (1. 84 g, 9. 60 mmol) and DIPEA (3. 38 g, 26. 17 mmol) were added at r. t. and stirred for 2h. N- (4-Aminophenyl)-N'- (2-methylphenyI) urea (2. 18 g, 9. 60 mmol dissolved in 25 mL DMSO) was added and stirring was contined for 12 h. The reaction mixture was hydrolysed with ice, tert-butyl- (15'*, 3R*)-3- ( { [4- ( { [ (2-methylphenyl) amino] carbonyl} amino) phenyl] amino} carbonyl) cyclopentylcarbamate (3. 32 g, 7. 34 mmol) was collected by filtration, washed with water and isolated. M. p. 188-190 C. ESI-MS : 453 [M+H] + Table 1 : The following compound was synthesized according to the same protocol

No Structure Name M. p. (OC) /-Butyl (l, 3R)-3- ( { [4 H ( {[ (2-methylphenyl) amino] VEII H carbonyl) amino) benzyl]- 180-182 amino} carbonyl) cyclopentyl - carbamate

Step B : Example IX : (il*, 39*)-3-amino-N- [4- ( { [ (2-methylphenyl) amino] carbonyl) amino)phenyljcyclopentanecarboxamid

tert-Butyl- (lS*, 3R*)-3- ( { [4- ( ( [ (2-methylphenyl) amino] carbonyl} amino) phenyl] was added to TFA (616 mL) at-5 OC and stirred for 0. 75 h at r. t. TFA was removed under vacuum, the residue was triturated with MTBE and DCM and dried, was collected (il*, 35'*)-3-amino-N- [4- ( { [ (2-methylphenyl) amino] carbonyl} amino) phenyl] cyclopentanecarboxamid (23. 87 g, TFA salt). ESI-MS : 353 [M+H] + Table 2 : The following compound was synthesized according to the same protocol

No Structure Name . x TFA (JR., 3S.)-3-Amino-N-[ 4- ( {[ (2-methyl N) % r N 0 N H phenyl) amino] carbonyl) amino) benzyl] X 9H I t 2 H cyclopentanecarboxamide

Step C : Example 3 : Methyl 4- ( { [ (15'*, 3*)-3- ( { [4- ( { [ (2-methylphenyl) amino] carbonyl}amino) phenyl] amino} carbonyl) cyclopentyl] amino} carbonyl) benzoate

Monomethylterephthate (540 mg, 3. 00 mmol) was dissolved in DMF (15mol), HOBT (510 mg, 3. 30 mmol), EDCI (630 mg, 3. 30 mmol) and DIPEA (3. 59 g, 27. 00 mmol) were added. After stirring for 2 h at r. t., (lR*, 35'*)-3-amino-N- [4- ( { [ (2-methylphenyl) amino]carbonyl} amino) phenyl] cyclopentanecarboxamid (2. 33 g, TFA salt dissolved in 2 mL DMF) was added and stirring was continued for 12 h. The reaction mixture was hydrolysed with ice, methyl 4- ( { [ (lS*, 3R*)-3- ( { [4- ( { [ (2-methylphenyl) amino] carbonyl} amino) phenyl] amino} carbonyl) cyclopentyl] amino} carbonyl) benzoate (1. 10 g,

2. 14 mmol) was collected by filtration, washed with water and isolated. M. p. 314316 oC. ESI-MS : 515 [M+H] + Table 3 : The following examples were prepared according to the general procedure

No Structure Name M. p. (OC) ESI-MS TMethyl 4- ( { [ (1, 36'')-3- ( { [4- ( { [ (2 N 4 0 CL JL JJ '' ethylphenyl) amino] carbonyl}- 217-219 H H 0 amino) benzyl] amino} carbonyl) cyclopentyl] amino} carbonyl) benzoate 5 Methyl 3, 5-dimethyl-4- ( { [ (17 ? *, 35'*)3- ( {[4- ( {[ (2-metbylphenyl)- 269 1) 6yv ! o anino] carbonyl} amino) phenzyl]amino} carbonyl) cyclopentyl]amino} carbonyl) benzoate H iMethyl 3, 5-dnnethyl-4- ( { [ (I', 3')3- ( {[ 4- ( {[ (2-methylphenyl)- n. d. 557 !"" amino] carbonyl} amino) benzyl]- [M+H] + amino} carbonyl) cyclopentyl]amino} carbonyl) benzoate

Step D : Example 7 : 4- ( {[ (1S., 3R*)-3- ( {[4- ( {[ (2-Methylphenyl) amino]carbonyl} amino)phenyl] amino} carbonyl) cyclopentyl] amino} carbonyl) benzoic acid

Methyl 4- ( {[ (15'*, 3R')-3- ( { [4- ( {[ (2-methylphenyl) amino] carbonyl} amino) phenyl]amino} carbonyl) cyclopentyl] amino} carbonyl) benzoate (800 mg, 1. 56 mmol) was dissolved in water/methanol/dioxane (1/1/4) and potassium hydroxide (870 mg, 15. 55 mmol) was added at r. t. After stirring at 50 oC for 5 h, MTBE was added, the aqueous phase was acidified (pH 2), 4- ( { [ (l, 3jR*)-3- ( { [4- ( { [ (2-methylphenyl)

amino] carbonyl} amino) phenyl] amino} carbonyl) cyclopentyl] amino} carbonyl) benzoic acid (564 mg, 1. 13 mmol) was collected by filtration, washed with water and isolated. M. p. > 270 C. ESI-MS : 501 [M+H] + Table 4: The following examples were prepared according to the general procedure

No Structure Name ESI-MS M. p. (Oc) H 8 J. ' I h OH 4- ( {[ (IR, 3S)-3- ( {[4- ( {[ (2-Methylphenyl) ammo]carbonyl}- 258-262 ) JL \/"' '' H H 0 amino) benzyl]amino} carbonyl) cyclopentyl] amino} carbonyl) benzoic acid H 9 ( ' I h OH 3, 5-Dlmethyl-4- ( {[ (1R, 38)-3- ( {[4- ( {[ (2-methylphenyl)- 543 n, d. amino] carbonyl} amino) benzyl] amino} carbonyl) cyclopentyl]- [M+H] + H H 0 amino} carbonyl) benzoic acid 0 HN 3, 5-Dimethyl-4- ( { [ (IS, 3R)-3- ( ( [4- ( ( [ (2-methylphenyl)- > 269 10 110 1 OH H 0 amino] carbonyl} amino) phenyl]amino} carbonyl) cyclopentyl] amino} carbonyl) benzoic acid

In vitro assay : adhesion of Jurkat cells to immobilized VCAM-1 (domains 1-3) Preparation of VCAM-1 (extracellular domains 1-3) Complementary DNA (cDNA) encoding 7-domain form of VCAM-1 (GenBank accession &num;M60335) was obtained using Rapid-ScreenTM cDNA library panels

(OriGene Technologies, Inc) at Takara Gene Analysis Center (Shiga, Japan). The primers used were 5'-CCA AGG CAG AGT ACG CAA AC-3' (sense) and 5'-TGG CAG GTA TTA TTA AGG AG-3' (antisense). PCR amplification of the 3-domain VCAM-1 cDNA was perform using Pfu DNA polymerase (Stratagene) with the following sets of primers: (U-VCAMdl-3) 5'-CCA TAT GGT ACC TGA TCA ATT TAA AAT CGA GAC CAC CCC AGA A-3' ; (L-VCAMdl-3) 5'-CCA TAT AGC AAT CCT AGG TCC AGG GGA GAT CTC AAC AGT AAA-3'. PCR cycle was 94

C for 45 sec, 55 oC for 45 sec, 72 C for 2 min, repeating 15 cycles. After the purifi cation of the PCR product, the fragment was digested with KpnI-Avrll. The digested fragment was ligated into pBluescript IISK (-) (Strategene), which was linearized by digesting with KpnI-XhoI. The ligation was followed by transformation to a Dam/Dcm methylase-free E. coli strain SCSI 10 (Strategene) to create the donor plasmid pHH7. To direct VCAM-1 molecule into the insect cell secretory pathway, the VCAM-1 coding sequence was fused to signal peptide sequence of honeybee melittin. The resulting melittin-VCAM fusion was placed in correct orientation to the baculovirus polyhedrin promoter. Baculovirus transfer vector containing first 3-domain form VCAM-1 (pH10) was constructed by ligation of 0. 9 kb fragment from AvrII/Klenow/BclI digests of pH7 into SalI/Klenow/BamHI digests of pMelBacB (Invitrogen). Recombinant baculovirus was generated by using Bac-N-Blue" Transfection kit (Invitrogen) according to the manufacture's instruction. The recombinant virus was amplified by infection to High-FiveTM insect cells for 5-6 days, and virus titer was determined by plaque assay.

High-Five insect cells were pelleted in a 225 ml conical tube by centrifugation at 1000 rpm for 5 min. After discarding the supernatant, the pellet was resuspended in 1.5 x 109 pfu (MOI = 5) of high-titer virus solution, followed by incubation for 1.5

hours at room temperature. The cells were pelleted again and washed once in fresh Express Five" serum free medium. The cells were pelleted again and finally, resus pended in 200 ml of fresh Express Five TM medium, transferred to a 1,000 ml shaker flask, and incubated in a shaker at 27 oC, 130 rpm, for 48 hours before the culture supernatant was collected. The purification of 3-domain form ofVCAM-1 from the culture supernatant was performed by one-step anion exchange chromatography. Protein concentration was determined by using Coomassie protein assay reagent (Pierce) according to the manufacture's instruction.

Preparation of VCAM-1 coated microtiter plates Recombinant human VCAM-1 (extracellular domains 1-3) was dissolved at 1.0 J. lglml in PBS. Each well of the microtiter plates (Nalge Nunc International, Fluoronunc Cert, 437958) was coated with 100 III of substrate or for background control with buffer alone for 15 hours at 4 C. After discarding the substrate solution, the wells were blocked using 150 III per well of block solution (Kirkegaard Perry Laboratories, 50-61-01) for 90 minutes. The plate was washed with wash buffer containing 24 mM Tris-HCl (pH 7.4), 137 mM NaCI, 27 mM KCI and 2 mM MnCIzjust before addition of the assay.

In vitro assay using Jurkat cells Preparation of fluorescence labeled Jurkat cells: Jurkat cells (American Type Culture Collection, Clone E6-1, ATCC TIB-152) were cultured in RPMI 1640 medium (Nikken Bio Medical Laboratory, CM1101) supplemented with 10% fetal bovine serum (Hyclone, A-1119-L), 100 U/ml penicilin (Gibco BRL, 15140-122) and 100 ug/ml streptomycin (Gibco BRL, 15140-122) in a humidified incubator at 37 OC with 5% CO2.

Jurkat cells were incubated with phosphate balanced solution (PBS, Nissui, 05913) containing 25 uM of 5 (-and-6)-carboxyfluorescein diacetate, succinimidyle ester

(CFSE, Dojindo Laboratories, 345-06441) for 20 min at room temperature while gently swirling every 5 min. After centrifugation at 1000 rpm for 5 min, the cell pel let was resuspended with adhesion assay buffer at a cell density of 4 x 106 cells/ml. The adhesion assay buffer was composed of 24 mM Tris-cl (pH 7.4), 137 mM NaCI, 27 mM KC1, 4 mM glucose, 0.1 % bovine serum albumin (BSA, Sigma, A9647) and 2 mM MnCl2.

Assay procedure (Jurkat cells) The assay solution containing each test compounds was transferred to the VCAM-1 coated plates. The final concentration of each test compounds was 5 J. IM, 10 uM or various concentrations ranging from 0.0001 MM to 10 uM using a standard 5-point serial dilution. The assay solution containing the labeled Jurkat cells was transferred to the VCAM-1 coated plates at a cell density of 2 x 105 cells per well and incubated for 1 hour at 37 C. The non-adherent cells were removed by washing the plates 3 times with wash buffer. The adherent cells were broken by addition of 1 % Triton X100 (Nacalai Tesque, 355-01). Released CFSC was quantified fluorescence measurement in a fluorometer (Wallac, ARVO 1420 multilabel counter).

The adhesion of Jurkat cells to VCAM-1 was analyzed by percent binding calculated by the formula : 100 x (FTS-FBG)/ (FTB-FBG) = % binding, where FTB is the total fluorescent intensity from VCAM-1 coated wells without test compound; FBG is the fluorescent intensity from wells lacking VCAM-1 and FTS is the fluorescent intensity from wells containing the test compound of this invention.

In vitro activity: In the Jurkat-VCAM-1 assay the observed ICso value ranges are indicated in Table 5 according to the scheme B > 10 M # A.

Table 5.

Example No ICso 2 A 7 A 8 A 9 B 10 B

Claims

1. Compounds of the general formula (I), R'-X-A-Cyc-Y-R' wherein Cyc represents a 5-or 6-membered carbocycle, which can optionally be substituted with up to two residues Rcyc, wherein the residues Rcyc can independently be selected from the group consisting of halogen, trifluoromethyl, amino, nitro, cyano, A represents an amide moiety of the structure - NRC (O)- or-C (O) NR''-, wherein RA-l represents hydrogen or Cl-Clo alkyl, R1 represents a 4-to 9-membered saturated, unsaturated or aromatic cyclic residue, which can contain 0 to 3 heteroatoms selected independently from the group N, S and 0, and wherein R'is substituted by-R-Z, wherein

R'-'represent a bond,-0-,-S-, Nui-2, Ci-Cio alkyl, C2-CIO alkenyl, C2-Cio alkynyl, C6 or Cio aryl, C3-C7 cycloalkyl or a 4-9-membered saturated or unsaturated heterocyclic residue containing up to 3 heteroatoms selected from the group oxygen, nitrogen or sulfur, wherein Ri l can optionally be substituted by 1 to 2 substituents selected from the group R1-3, wherein Rl-2 can optionally be hydrogen, C1-C10 alkyl, C2-C alkenyl or C2-C10 alkynyl, and wherein RI-3 represents hydrogen, Ci-Clo alkyl, Cz-Cio alkenyl, C2-C10 alkynyl, C6 or C10 aryl, C3-C7 cycloalkyl or a 4-9-membered saturated or unsaturated heterocyclic residue containing up to 3 heteroatoms selected from the group oxygen, nitrogen or sulfur,

Z represents-C (O) OR,-C (O) NRR,-SO2NRR,-SO (OR), - S02 (OR-'),-P (O) R'(OR) or-PO (OR) (OR), wherein Rz-2 is hydrogen, Ci-C4 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C6 or C10 aryl, -C (O) RZ-4 or -SO2RZ-4, wherein RZ-4 is C1-C4 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C6 or C10 aryl, Rz-'and Rz-3 are independently selected from the group hydrogen, C1-C4 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C6 or C10 aryl or benzyl,

wherein RZ-l and Rz-3 can optionally be substituted by 1 to 3 substitu ents selected from the group Cl-C4 alkyl, Cl-C4 alkyloxy, halogen, ni tro, cyano, and wherein R1 can optionally be substituted by 0 to 2 substituents Rl-4, halogen, nitro, amino, cyano and oxo, wherein R1-4 is selected from the group Cl-C4 alkyl, Cl-C4 alkyloxy, phenyl, phenoxy, phenylamino, C3-C6 cycloalkyl, R6 represents phenyl or a 5-to 6-membered aromatic heterocyclic residue containing up to 3 heteroatoms independently selected from the group oxygen, nitrogen or sulfur, which is substituted by -NR6-2C (0) NR6-3R6-4 and can furthermore optionally be substituted by halogen, wherein R6-2 and R6-3 are independently selected from the group hydrogen or Ci-C4 alkyl, or together form a group

and wherein R represents phenyl,

wherein R can optionally be substituted by 1-2 substituents selected from the group Cl-C4 alkyl, Cl-C4 alkyloxy, halogen, nitro, trifluoromethyl, trifluoromethoxy or cyano, X represents bond or-CRx-'Rx-2-,

wherein Rx-I and RX-2 can be independently selected from the group hydrogen, Ci-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, Y represents an amide moiety of the structure -NRY-1C(O)- or -C(O)NRY-1-, wherein R represents hydrogen or Cl-C4 alkyl, wherein the moiety A-Cyc-Y represents a y-amino acid, and pharmaceutically acceptable salts thereof.

2. Compounds of general formula (I) according to claim 1, wherein Cyc represents a 5-membered carbocycle.

3. Compounds of general formula (I) according to any one of claims 1 or 2, wherein R'represents a 1,4-substituted phenyl ring.

4. Compounds of general formula (1) according to any one of claims 1 to 3, wherein R1-1 represents a bond and Z represents COOR', wherein RZ-1 has the meaning indicated above.

5. Compounds of general formula (I) according to any one of claims 1 to 4, wherein R6 represents phenyl, which is substituted by-NHC (O) NHR6-4, wherein R6-4 is substituted with methyl or trifluoromethoxy.

6. A process for preparation of compounds of general formula (I) according to any one of claims 1 to 5, which comprises reaction of compounds of general formula (I')

wherein Cyc, X, R5, R6 and R8 have the abovementioned meaning, with compounds of the general formula (I")

wherein R1 has the abovementioned meaning and AG represents an activating group, in inert solvents.

7. Compounds according to any one of claims 1 to 5 for the treatment of diseases.

8. The use of a compound according to any one of claims 1 to 5 in the manu facture of a medicament for the treatment or the prevention of a condition mediated by integrins.

9. The use according to claim 8. in the manufacture of a medicament for the treatment or the prevention of atherosclerosis, asthma, chronic obstructive pulmonary disease (COPD), allergies, diabetes, inflammatory bowel disease, multiple sclerosis, myocardial ischemia, rheumatoid arthritis, transplant rejection and other inflammatory, autoimmune and immune disorders.

10. Pharmaceutical composition, comprising compounds according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.