WO2002030876A2

WO2002030876A2 - Cyclic carboxylic acids as integrin antagonists

Info

Publication number: WO2002030876A2
Application number: PCT/EP2001/011585
Authority: WO
Inventors: Thomas Lehmann; Thomas RÖLLE; Markus Albers; Gerhard Müller; Gerhard Hessler; Rüdiger Fischer; Masaomi Tajimi; Karl Ziegelbauer; Kevin Bacon; Haruki Hasegawa; Hiromi Okigami
Original assignee: Bayer Aktiengesellschaft
Priority date: 2000-10-09
Filing date: 2001-10-08
Publication date: 2002-04-18
Also published as: GB0024695D0; AU2002218214A1; GB2367817A; WO2002030876A3

Abstract

The present invention relates to compounds of the general formula (I), process for their preparation, pharmaceutical compositions containing them as well as their use for the production of pharmaceutical compositions for the treatment of inflammatory diseases.

Description

Cyclic carboxylic acids as integrin antagonists

The present invention relates to compounds of formula (I),

R⁶— -X— A— Cyc — Y— R¹ _(I

their preparation and use as pharmaceutical compositions as integrin antagonists, especially as α βι and/or α₄β and or α₉βι integrin antagonists and in particular for the production of pharmaceutical compositions suitable for the inhibition or the pre- vention 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.

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 vascu- lar 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 num- ber 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 (NCAM-1) and very late antigen 4 (NLA-4, α βι integrin), as well as the interactig.n between mucosal addressin cell adhesion molecule 1 (MAdCAM-1) and α₄β 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 NCAM-1 / NLA-4 and/or MAdCAM-1 / α₄β₇ 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. Al- lergy Clin. Immunol. 1999, 104, 917-26).

NCAM-1 is a member of immunoglobulin (Ig) superfamily and is one of the key regulators of leukocyte trafficking to sites of inflammation. NCAM-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 α (TΝF-α), as well as by lipopolysaccharide (LPS), via nuclear factor KB (ΝF- B) dependent pathway. However, these molecules are not expressed on resting endothelium. Cell adhesion mediated by NCAM-1 may be involved in numerous physiological and pathological processes including myogenesis, hematopoiesis, in- flammatoiy reactions, and the development of autoimmune disorders. Integrins NLA-

4 and α β₇ both function as leukocyte receptors for NCAM-1.

The integrin α₄βi is a heterodimeric protein expressed in substantial levels on all circulating leukocytes except mature neutrophils. It regulates cell migration into tis- sues during inflammatory responses and normal lymphocyte trafficking. NLA-4 binds to different primary sequence determinants, such as a QIDSP motif of NCAM- 1 and an ILDNP 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 α₄ integrins interaction in leukocyte-mediated inflammation. Blocking of NLA-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 α₄βi integrin receptor antagonists: WO 96/22966, WO 97/03094, WO 99/33789, WO 99/37605. However, no aminobenzoic acids or aminocycloalkyl- carboxylic acids or homologues thereof or heterocyclics analogues thereof with c_₄βι integrin receptor antagonists activity have been described.

Further to their a _\ integrin antagonistic activity, the compounds of the present invention may also be used as α₄β₇ or α₉βi 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):

R— X— A— Cyc — Y— R¹

(I)

wherein

Cyc represents a 5- or 6-membered carbocycle, which can optionally be substituted with up to two residues R^cyc, wherein the residues R^cyc can independently be selected from the group consisting of halogen, trifluoromethyl, amino, nitro, cyano,

A represents an amide moiety of the structure

-NR^A-1C(O)- or-C^N ^¹-,

wherein R^A_1 represents hydrogen or C _\ -C 1 o alkyl,

R¹ represents a 4- to 9-membered saturated, unsarurated or aromatic cyclic residue,

which can contain 0 to 3 heteroatoms selected independently from the group N, S and O,

and wherein R¹ is substituted by -R -Z, wherein

R^1"1 represents a bond, -O-, -S-, NR^1"2, C_rC₁₀ alkyl, C₂-C₁₀ alkenyl,

C₂-Ci₀ 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,

wherein R .1-1 can optionally be substituted by 1 to 2 substituents selected from the group R^1"3,

wherein R^1"2 can optionally be hydrogen, CrC₁₀ alkyl, C₂-C₁₀ alkenyl or C₂-C₁₀ alkynyl, and wherein R^1"3 represents hydrogen, -Cio alkyl, C₂-C₁₀ 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^z_1, -C(O)NR^z"2R^z"3, -SO₂NR^z-²R^z-³, -SO(OR^z-¹), -SO₂(OR^z"1), -P(O)R^z_1(OR^z-³) or -POCOR^XOR^2"3),

wherein R^z"2 is hydrogen, C1-C4 alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₆ or C₁₀ aryl, -C(O)R^z"4 or -SO₂R^z"4,

wherein R^z"4 is C1-C4 alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₆ or C₁₀ aryl,

R^z_1 and R^z"3 are independently selected from the group hydrogen, Cι-C₄ alkyl, C₂ -C₆ alkenyl, C₂ -C₆ alkynyl, C -C₆ cycloalkyl, C₆ or do aryl or benzyl,

wherein R^z_1 and R^z"3 can optionally be substituted by 1 to 3 substitu- ents selected from the group Cι-C alkyl, Q-C₄ alkyloxy, halogen, ni- tro, cyano,

and wherein R¹ can optionally be substituted by 0 to 2 substituents R^1"4, halogen, nitro, amino, cyano and oxo,

wherein

R^1"4 is selected from the group Cι-C alkyl, Cι-C₄ alkyloxy, phenyl, phenoxy, phenylamino, C₃-C₆ cycloalkyl, R 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 -NR^0"2C(O)NR^6"3R^6"4 and can furthermore optionally be substituted by halogen,

wherein R ^" and R ^" are independently selected from the group hydrogen or C C₄ alkyl, or together form a group

and wherein R^6"4 represents phenyl,

wherein R^6"4 can optionally be substituted by 1-2 substituents selected from the group -C₄ alkyl, -C₄ alkyloxy, halogen, nitro, trifluoromethyl, trifluoromethoxy or cyano,

X represents bond or -CR^X_1R^X"2-

wherein R^x_1 and R^x"2 can be independently selected from the group hydrogen, C1-C4 alkyl, C - C₄ alkenyl, C2-C4 alkynyl,

Y represents an amide moiety of the structure

-NR^Y"1C(O)- or -C(O)NR^Y"1-

wherein R^γ_1 represents hydrogen or C₁-C4 alkyl, wherein the moiety A-Cyc-Y represents a γ-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 R¹ represents a 1,4-substituted phenyl ring.

In another preferred embodiment, the present invention relates to compounds of general formula (I), wherein wherein R ^" represents a bond and Z represents COOR ^" , wherein R^z_1 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) HR^6"4, wherein R^6"4 is substituted with methyl or trifluoromethoxy.

A preferred process for preparation of compounds of general formula (Nil) has also been found, which comprises reaction of compounds of general formula (F)

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, 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 -Cio alkyl, very preferred is Cι-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 -C₁₀ alkenyl or alkinyl, very preferred is Ci-Cβ alkenyl or alkinyl.

Cycloalkyl stands for a cyclic alkyl group such as cyclopropyl, cyclobutyl, cyclo- pentyl, 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. tetra- hydrofur-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- 1-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 O, S, N or P.

In the context of the present invention, the moiety A-Cyc-Y represents a γ-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 α₄βi and or α β₇ and/or α₉βι 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 admini- stration 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 admini- stration 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 speci- tied 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 disper- sants.

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, dis- persants (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 en- hancers 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 indivi- dual 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., aluminum, 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 methylamine, t- butylamine, procaine, lower alkylpiperidines such as N-ethylpiperidine, cycloalkyl- amines such as cyclohexylamine 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 γ-amino acids. They can be prepared employing standard amide coupling procedures. In case A stands for -

NR C(O)- and Y stands for -NR C(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 PG¹ the amides (V) can be obtained. Coupling with the carboxylic acids or activated derivatives (VI) affords carboxylic acids of type (Nil). If an protecting group PG² is used to protect an carboxylic acid functionality on R , removal of the protecting group PG follows.

In the above scheme cyc in formulas (III) - (V) and (Nil) as well as in scheme 2 represents a cyclic moiety. The depicted ring in formulas (NI) - (NIII) 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 Nerlag, 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 = wo-butyl-carbonate or by a coupling agents such as, for example dicyclohexylcarbodiimid (DCC), l-ethyl-3-(3'-dimethylamino- proρyl)carbodiimidexHCl (EDCI), 2-(7-aza-3-oxido- 1H- 1 ,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 Ν-hydroxybenzotriazole esters may also be employed.

In the above scheme PG¹ 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, 3^rd ed., John Wiley, New York,

1999. The amino group is preferably protected by carbamates, PG¹ being for example tert-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (FMOC) or benzyloxy- carbonyl (Cbz- / Z-) or other oxycarbonyl derivatives.

PG² 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, 3 ed., John Wiley, New York, 1999. The carboxyl group is preferably esterified, PG² being C_1-6-alkyl such as, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, a C_3-7- cycloalkyl such as, for example, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclo- pentyl, 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 wo-butylcarbonate or N-hydroxybenzo- triazole ester - with the desired amine (II) or an acceptable salt thereof.

Zϊo-butylcarbonates can be prepared in situ by reaction of the N-protected amino acid (III) with tsO-butylchloroformate as described below. Activated derivatives of the acids (III) such as other anhydrides, halides, esters e.g. succinyl, N-hydroxybenzo- triazole or pentafluorophenyl esters or activated carboxylic acids obtained by the reaction with coupling agents such as, for example dicyclohexylcarbodiimid (DCC), l-ethyl-3-(3 '-dimethylaminopropyl)carbodiimideχHCl (EDCI), 2-(7-aza-3-oxido- 1 H- 1 ,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 (III) in presence of an coupling agents such as, for example, dicyclohexylcarbodiimid (DCC), 1-ethyl- 3-(3 '-dimethylaminopropyl)carbodiimidexHCl (EDCI), 2-(7-aza-3-oxido-lH-l ,2,3- benzotriazol-l-yl)-l,l,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 (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 -15°C and iso-b\ιty\ 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 further purification.

2) 1-Hydroxy-lH-benzotriazol ester procedure

A solution of carboxylic acid, l-hydroxy-lH-benzotriazol (HOBt) and l-ethyl-3-(3'- dimethylaminopropyl)carbodiimidexHCl (EDCI) in an inert solvent is stirred at r. . After addition of the amine and a non-nucleophilic base such as ethyldiisopropyl- amine 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 R^1"1 is a methylen group, the carbon chain can be elongated by Arndt-Eistert- reaction and optionally be derivatized by common methods for α-derivatization of carboxylic acids such as nucleophilic substitution.

Step B

The removal of protecting group PG¹ can be performed, depending on the nature of PG¹, either by an acid such as trifluoroacetic acid for example in the case PG is tert- butyloxycarbonyl (Boc), a base such as piperidine for example in the case PG¹ is 9- fluorenyhnethyloxycarbonyl (FMOC) or by catalytic hydrogenation for example in the case PG¹ 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 ethyldiisopropyl- amine 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 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. dimethylfbrmamide. If necessary, mixtures of the above solvents may be used.

In case A stands for -C(O)NR²- and Y stands for -C(O)NR⁸-, 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 PG¹, 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 R¹, removal of the protecting group PG² follows. In the above scheme the depicted ring in formulas (III) - (V), (VII) and (VIII) 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 respective carboxylic acid (III), such as an /rø-butylcarbonate or N-hydroxybenzo- triazole ester - with the desired amine (II) or an acceptable salt thereof.

ZsO-butylcarbonates can be prepared in situ by reaction of the N-protected amino acid (III) with wo-butylchloroformate as described below. Activated derivatives of the acids (III) such as other anhydrides, halides, esters e.g. succinyl, N-hydroxybenzo- triazole or pentafluorophenyl esters or activated carboxylic acids obtained by the re- action with coupling agents such as, for example dicyclohexylcarbodiimid (DCC), l-ethyl-3-(3'-dimethylaminopropyl)carbodiimidexHCl (EDCI), 2-(7-aza-3-oxido- 1 H- 1 ,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 (III) in presence of an coupling agents such as, for example, dicyclohexylcarbodiimid (DCC), 1-ethyl- 3-(3'-dimethylaminopropyl)carbodiimidexHCl (EDCI), 2-(7-aza-3-oxido-lH-l,2,3- benzotriazol-l-yl)-l,l,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 (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 -15°C and iso-butyl chloro formate 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 further purification.

2) 1 -Hydroxy- 1 H-benzotriazol ester procedure A solution of carboxylic acid, 1 -hydroxy- lH-benzotriazol (HOBt) and l-ethyl-3-(3'- dimethylaminopropyl)carbodiimidexHCl (EDCI) in an inert solvent is stirred at r.t.. After addition of the amine and a non-nucleophilic base such as ethyldiisopropyl- amine 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.

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 ace- tic acid ester and a phenol derivative followed by saponification as described in Step

D.

Step B

The removal of protecting group PG¹ can be performed, depending on the nature of PG¹, either by an acid such as trifluoroacetic acid for example in the case PG¹ is tert- butyloxycarbonyl (Boc), a base such as piperidine for example in the case PG¹ is 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 de- rivatives 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:

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. 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 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 1 -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,l,3,3-tetramethylurθ' 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-κB nuclear factor KB

NMR nuclear magnetic resonance n.d. not determined

PE light petroleum (b.p. 40-60 °C) r.t. room temperature R_f TLC: R_f value = distance spot traveled / distance solvent front traveled

TFA trifluoroacetic acid

THF tetrahydrofurane

TLC thin layer chromatography TNF-α tumor necrosis factor α t_R retention time determined by HPLC

VCAM-1 vascular cell adhesion molecule 1

VLA-4 very late antigen 4 (α₄βι 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, 40-63 μm (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- Methylphenyhsocyanate (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) R_f 0.32; 1H-NMR (400 MHz, D₆-DMSO) δ 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, 1H); 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. Chem. 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 °C;

TLC (PE/EtOAc 1/1) R_f 0.65; 1H NMR (400 MHz, D₆-DMSO) δ 1.39 (s, 9H); 2.24 (s, 3H); 4.06 (d, J-6 Hz, 2H); 6.93 (mc, 1H); 7.12-7.17 (m, 4); 7.32 (mc, 1H); 7.40 (mc, 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 °C; TLC (PE/EtOAc 3/2) R_f 0.14; 1H NMR (400 MHz, D₆-DMSO) δ 2.24 (s, 3H); 3.97 (q, J=5 Hz, 2H); 6.96 (mc, 1H); 7.13-7.19 (m, 2); 7.36 (mc, 2H); 7.51 (mc, 2H); 7.81 (mc, 2H); 8.06 (s, 1H); 8.08 (s, 3H); 9.23 (s, 1H).

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

To a solution of 2-(4-aminophenyl)acetic acid (108.8 g, 0.72 mol) in CH₂C1₂ (1.0 1) and triethylarnine (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 HCI. 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. 205-206°C; TLC (CH₂Cl₂/MeOH 9:1): R_f 0.14. 1H-ΝMR (400 MHz, D₆- DMSO): 12.21 (br s, 1H), 9.11 (s, 1H), 8.00 (s, 1H), 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 N: Methyl 4-[({(lS^*,3R^*)-3 - [(tert-butoxycarbonyl) amino]cyclopentyl} carbonyl)amino]benzoate

(IS ,3R )-3-[(tert-Butoxycarbonyl)amino]cyclopentanecarboxylic acid (Lit.: Marco-

Contelles, Jose; Bemabe, Manuel; Tetrahedron Lett. 1994; 35, 6361-6364) (4.59 g, 20.00 mmol) was dissolved in THF (50 mL), Ν-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 Ν HCI, sat. soda, brine and dried (MgSO₄). FC (PE/EE 9:1) yielded methyl 4-[({(lS^*,3R^*)-3-[(tert-butoxycarbonyl)- amino]cyclopentyl}-carbonyl)amino]benzoate (5.95 g, 16.42 mmol). TLC (PE/EtOAc 6/4) R_f 0.26; 1H-ΝMR (400 MHz, D₆-DMSO) δ 1.38 (s, 9H); 1.47-1.64

(m, 2H); 1.82 (mc, 3H); 2.08-2.15 (m, 1H); 2.80-2.88 (m, 1H); 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-({[(lS ,3R )-3-aminocyclopentyl]carbonyl}amino)benzoate

To a solution of methyl 4-[({(lS ,3R )-3-[(tert-butoxycarbonyl)amino]cyclopentyl}- carbonyl)amino]benzoate (2.70 g, 7.45 mmol) in CH₂C1₂ (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) δ 1.70 (mc, IH); 1.80-2.03 (m, 4H); 2.25 (mc, IH); 2.98 (mc, IH); 3.60 (mc, IH); 3.83 (s, 3H); 7.73-7.76 (m, 2H); 7.88-7.96 (m, 5H); 10.16 (s, IH).

Step C:

Example 1: Methyl 4-({[(lS^*,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 (10 mL), HOBt (880 mg, 5.73 mmol), EDCI (1.10 g, 5.73 mmol), DIPEA (6.06 g, 46.89 mmol) was added and stirring was continued for 2 h at r.t. Methyl 4-({[(lS ,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-({[(lS^*,3R^*)-3-({[4-({[(2-methylphenyl)amino]carbonyl}amino)phenyl]acetyl}- amino)cyclopentyl]carbonyl}amino)benzoate (2.10 g, 3.97 mmol). M.p. 208-210 °C.

ESI-MS: 529 [M+H]⁺

Step D:

Example 2: 4-({[(lS^*,3R^*)-3-({[4-({[(2-Methylphenyl)amino]carbonyl}amino)- phenyljacetyl} 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 °C 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-( {[(lS^*,3R^*)-3-( {[4-( {[(2-Methylρhenyl)amino]carbonyl} amino)phenyl]acetyl} - amino)cyclopentyl]carbonyl}amino)benzoic acid (1.18 g, 2.29 mmol). M.p. 270- 273 °C. ESI-MS: 513 [M-H]⁺

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

StepB

StepD

Scheme 4 Step A:

Example VII: tert-Butyl-(lS^*,3R^*)-3-({[4-({[(2-methylphenyl)amino]carbonyl}- amino)phenyl]amino}carbonyl)cyclopentylcarbamate

Contelles, Jose; Bernabe, Manuel; Tetrahedron Lett. 1994, 35, 6361-6364) (2.00 g, 8.72 mmol) was dissolved in DMF (15 mL), HOBT (1.47 g, 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 2 h. N-(4-Aminophenyl)-N'-(2-methylphenyl)urea (2.18 g, 9.60 mmol dissolved in 25 L DMSO) was added and stirring was confined for 12 h. The reaction mixture was hydrolysed with ice, tert-butyl-(lS ,3R )-3-({[4-({[(2-methylphenyl)amino]carb- onyl}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. (°C)

amino} carbonyl)cyclopentyl -carbamate

Step B: Example IX: (lR^*,3S^*)-3-amino-N-[4-({[(2-methylphenyl)amino]carbonyl}amino)- phenyl]cyclopentanecarboxamid

tert-Butyl-(lS^*,3R^*)-3-({[4-({[(2-methylphenyl)amino]carbonyl}amino)phenyl] was added to TFA (616 mL) at -5 °C 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 (1R ,3S^*)-3-amino-N-[4-({[(2-methylphenyl)amino]carbonyl}amino)phenyl]cyclo- pentanecarboxamid (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

(1R ,3S )-3-Amino-N-[4-({[(2-methyl-

X phenyl)amino]carbonyl}amino)benzyl]-

cyclopentanecarboxamide

Step C:

Example 3: Methyl 4-({[(lS^*,3R^*)-3-({[4-({[(2-methylphenyl)amino]carbonyl}- amino)phenyl]amino}carbonyl)cyclopentyl]amino}carbonyl)benzoate

Monomethylterephthate (540 mg, 3.00 mmol) was dissolved in DMF (15 mL), 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^*,3S^*)-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]carb- onyl}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. 314- 316 °C. ESI-MS: 515 [M+H

Table 3: The following examples were prepared according to the general procedure

No Structure Name M.p.(°C) ESI-MS

lopentyl]amino}carbonyl)beπzoate

amino}carbonyl)cyclopeιιtyl]- amino} carbonyl)benzoate

a___ino}carbonyl)cyGlope_ιtyl]- amino} carbonyl)benzoate

Step D:

Example 7: 4-({[(lS^*,3i_^*)-3-({[4-({[(2-Methylphenyl)amino]carbonyl}amino)- phenyl]amino} carbonyl)cyclopentyl]amino}carbonyl)benzoic acid

Methyl 4-( { [(lS^*,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 °C for 5 h, MTBE was added, the aqueous phase was acidified (pH 2), 4-({[(lS^*,3R^*)-3-({[4-({[(2-methylphenyl)- aminojcarbonyl} amino)phenyl] amino} carbonyl)cyclopentyl]amino} carbonyl)benz- oic 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. (°C)

onyl)benzoic acid

amino} carbonyl)benzoic acid

H ^H aamm nnooj]ccaarrbboonnyyll aammiinnoo))pphheennyyll]] aammiinneo} carbonyl)cyclo- pentyl]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 NCAM-1 (GenBank ac- cession #M60335) was obtained using Rapid-ScreenTM cDΝA 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 NCAM-1 cDΝA was perform using Pfu DΝA polymerase (Stratagene) with the fol- lowing sets of primers: (U-NCAMdl-3) 5'-CCA TAT GGT ACC TGA TCA ATT

TAA AAT CGA GAC CAC CCC AGA A-3'; (L-NCAMdl-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 °C for 45 sec, 72 °C for 2 min, repeating 15 cycles. After the purification of the PCR product, the fragment was digested with Kpnl-Avrll. The digested fragment was ligated into pBluescript IISK(-) (Strategene), which was linearized by digesting with Kpnl-Xhol. 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 NCAM-1 molecule into the insect cell secretory pathway, the NCAM-1 coding sequence was fused to signal peptide sequence of honeybee melittin. The resulting melittin-NCAM fusion was placed in correct orientation to the baculovirus polyhedrin promoter. Baculovirus transfer vector containing first 3-domain form NCAM-1 (pHIO) was constructed by ligation of 0.9 kb fragment from Avrll/Klenow/Bcll digests of pH7 into Sall/Klenow/BamHI digests of pMelBacB (Invitrogen). Recombinant baculovirus was generated by using Bac-Ν-Blue™ Trans- fection kit (Invitrogen) according to the manufacture's instruction. The recombinant virus was amplified by infection to High-Five™ 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 °C, 130 rpm, for 48 hours before the culture supernatant was collected. The purification of 3-domain form of VCAM-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 μg/ml in PBS. Each well of the microtiter plates (Nalge Nunc International, Fluoro- nunc Cert, 437958) was coated with 100 μl 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 μl 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 NaCl, 27 mM KC1 and 2 mM MnCl₂ just 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 μg/ml streptomycin (Gibco BRL, 15140-122) in a humidified incubator at 37 °C with 5% CO₂.

Jurkat cells were incubated with phosphate balanced solution (PBS, Nissui, 05913) containing 25 μM 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 pellet 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-HCl (pH 7.4), 137 mM NaCl, 27 mM KCl, 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 μM, 10 μM or various concentrations ranging from 0.0001 μM to 10 μM 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 X- 100 (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 fluo- rescent 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 IC₅₀ value ranges are indicated in Table 5 according to the scheme B > 10 μM > A.

Table 5.

Claims

1. Compounds of the general formula (I),

R⁶— X— A— Cyc — Y— R¹

(I)

wherein

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

wherein the residues R^cyc can independently be selected from the group consisting of halogen, trifluoromethyl, amino, nitro, cyano,

A represents an amide moiety of the structure

- R^A_1C(O)- or-C(O)NR^A_1-,

wherein R^A-1 represents hydrogen or Ci-Cio alkyl,

R¹ represents a 4- to 9-membered saturated, unsaturated .or aromatic cyclic residue,

and wherein R¹ is substituted by -R^M-Z, wherein R^1"1 represents a bond, -O-, -S-, NR^1"2, C1-C10 alkyl, C₂-C₁₀ 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,

wherein R^1"1 can optionally be substituted by 1 to 2 substituents se- lected from the group R 1-3

wherein R ^" can optionally be hydrogen, Ci-Cio alkyl, C₂-C₁₀ alkenyl or C₂- ₀ alkynyl, and

wherein R^1"3 represents hydrogen, C1- ₀ alkyl, C₂-C₁₀ alkenyl, C₂-Cιo 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^z_1, -C(O)NR^z"2R^z"3, -SO₂NR^z"2R^z'3, -SO(OR^z_1), -SOztOR^2"1), -P(O)R^z-¹(OR^z"3) or -PO OR^XOR^2"3),

wherein R ,Z-2 is hydrogen, C1-C4 alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,

C₃-C₆ cycloalkyl, C₆ or C₁₀ aryl, -C(O)R^z"4 or-SO₂R^z"4,

R^z_1 and R^z"3 are independently selected from the group hydrogen, Q- alkyl, C₂ -C₆ alkenyl, C₂ -C₆ alkynyl, C₃ -C₆ cycloalkyl, C₆ or C₁₀ aryl or benzyl, wherein R^z_1 and R^z"3 can optionally be substituted by 1 to 3 substituents selected from the group - alkyl, - alkyloxy, halogen, ni- tro, cyano,

wherein

R^1"4 is selected from the group C₁-C₄ alkyl, -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 -NR^6"2C(O)NR^0"3R^6"4 and can furthermore optionally be substituted by halogen,

wherein R^6"2 and R^6"3 are independently selected from the group hydrogen or d-C₄ alkyl, or together form a group

and wherein R ,6-4 represents phenyl, wherein R^6"4 can optionally be substituted by 1-2 substituents selected from the group d-Q alkyl, C1-C₄ alkyloxy, halogen, nitro, trifluoromethyl, trifluoromethoxy or cyano,

X represents bond or -CR^X"!R ^"2-,

y 1 wherein R ^" and R ^" can be independently selected from the group hydrogen, C1-C4 alkyl, C₂ - C₄ alkenyl, C2-C4 alkynyl,

Y represents an amide moiety of the structure

-mi? C(0)- or -C(O)NR^Y_1-,

wherein R^γ_1 represents hydrogen or C₁-C₄ alkyl,

wherein the moiety A-Cyc-Y represents a γ-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 R¹ represents a 1,4-substituted phenyl ring.

4. Compounds of general formula (I) according to any one of claims 1 to 3,

1 1 7 1 wherein R ^" represents a bond and Z represents COOR ^" , wherein R ^" 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)NHR^6"4, wherein R^6"4 is substituted with methyl or trifluoromethoxy.

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 (!')

wherein

Cyc, X, R⁵, R⁶ and R^s have the abovementioned meaning,

with compounds of the general formula (F ')

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 manufacture 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.

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