WO2004069829A1 - (2s)-2-((pyrimidin-4-yl)amino)-4-methylpentanoic acid aminoethylamid derivatives as il-8 receptor modulators for the treatment of atherosclerosis and rheumatoid arthritis - Google Patents

Abstract

The invention relates to compounds of the formula: (I); wherein R1 is a basic moiety having the structure -NH(CH2)2NR5R6, (Formula II) or (Formula II); wherein R5, R6 and R7 are independently selected from H, (1-6C)alkyl, (2-6C)alkenyl; (3-8C)cycloalkyl, and (1-6C)alkyl substituted with furanyl, (1-6C)alkoxy, (3-8C)cycloalkyl, trifluoromethyl or amido; or R5 and R6 together with N to which they are attached are a (4-8) membered heterocycle, which heterocycle may be substituted with (1-6C)alkoxy, trifluoromethyl or amido and which heterocycle may be fused with another (5-6) membered heterocycle; n is 1, 2 or 3; and the ring structure A represents a (4-6) membered heterocycle; R2 is (1-6C)alkyl, optionally substituted with (1-6C)alkoxy; R3 is aryl, heterocyclyl, -X-(CH2)maryl or -X-(CH2)m-heterocyclyl, wherein X is O, S or NR8, R8 being H or (1-6C)alkyl, and m is 0, 1, 2 or 3; and R4 is H, (1-8C)akyl, (1-8C)alkoxy or (2-8C)alkenyl, the alkyl moiety of each optionally being substituted with hydroxy, halogen, cyano, nitro, oxo, amino, alkylamino, dialkylamino, amido, alkylamido, carboxy, (1-8C)alkoxy, (1-8C)alkylthio, perfluoro (1-4C)alkyl, (3-8C)cycloalky, aryl, aryloxy, heterocyclyl or-0-heterocyclyl; or a pharmaceutically acceptable salt thereof. The compounds of the invention are Il-8 receptor modulators, in particular inhibitors thereof, and can be used for treating or preventing Il-8 receptor mediated disorders, such as atherosclerosis, inflammation, rheumatoid arthritis and related disorders.

Description

( 2S ) -2- ( (PYRIMIDIN-4-YL) MINO) -4-METHYLPENTANOIC ACID AM I NO ETHYL AM ID DERIVATIVES AS IL-8 RECEPTOR MODULATORS FOR THE TREATMENT OF ATHEROSCLEROSIS AND RHEUMATOID

ARTHRITIS

Cross Reference to Related Applications

This application claims priority from US provisional application 60/439,362, filed

January 10, 2003, and is related in subject matter to US non-provisional application

10/340,398, filed January 10, 2003, and to US provisional application 60/439,357, filed January 10, 2003 . The entire disclosures of all are incorporated herein by reference.

Field of the Invention

The invention relates to chemical compounds which are α-amino acid amides substituted on the α-amino group with a 4-pyrimidinyl residue and on the amide nitrogen with a basic residue, to a process for the preparation of the compounds, to pharmaceutical compositions containing the same, as well as to the use of the compounds for the manufacture of a medicament for treating or preventing IL-8 (CXCL8) receptor mediated disorders.

Background of the Invention

Chemokines are pro-inflammatory mediators that primarily control leukocyte migration into selected tissues and upregulation of adhesion receptors. By interaction with their respective G protein-coupled receptor (GPCR) chemokines have a profound influence on the selective recruitment of specific cell types in several inflammatory diseases.

Chemokines are members of the cytokine superfamily. They generally have low molecular weights ranging from 7 to 15 kDa and stimulate the recruitment of well defined leukocyte subtypes. Chemokines are secondary pro-inflammatory mediators that are induced by primary pro-inflammatory mediators like IL-I and TNF. Chemokines are divided into two large families (CXC and CC) according to the organisation of the first two of four conserved cysteines in the primary molecular structure. Actions of CXC chemokines are mediated through four cell surface receptors, CXCR1 to CXCR4, which are G protein-coupled receptors (GPCRs). IL-8 signals through CXCR1 and CXCR2 (IL-8 receptors). Pharmacological distinction between both receptors is possible since GRO, ENA78, and NAP-2 only bind CXCR2, whereas IL-8 binds both CXCR1 and CXCR2. This two-receptor system does not exist in rodents. Rodents appear to possess only the CXCR2 homologue, which is designated IL-8Rh (IL-8 receptor homologue).

The phenotype of mice that lack the murine IL-8Rh resembles that of patients with leukocyte adhesion deficiency, i.e. neutrophils that fail to infiltrate tissues (Cacalano G et al, Science 265;682-684, 1994). Until recently, CXCR2 was especially regarded as a chemokine receptor inducing neutrophil activation. More recently it became clear that CXCR2 also functions on monocytes, macrophages, and T cells. Strong, recently collected, data suggest a crucial role of CXCR2 in both atherosclerosis and rheumatoid arthritis.

The process of vascular transformation that ultimately leads to atherosclerosis involves the influx of monocytes into the vascular wall. These monocytes transfer to macrophages and become loaded with large amounts of lipids. A vast amount of evidence points to a crucial role of monocytes/macrophages in the development and progress of the disease.

It was shown only recently that IL-8 and CXCR2 are important for monocyte infiltration into the vascular wall. Expression of IL-8 is induced by Ox-LDL in monocytes and is expressed in atherosclerotic plaques. IL-8 and CXCR2 signalling have been shown to be associated with atherosclerosis in vivo. In 1998 Boisvert et al. (JCI 101;353-363, 1998) showed that atherosclerosis susceptible mice (LDL-R- deficient) that were lethally irradiated and transplanted with bone marrow from mice that lack the mIL-8Rh develop less severe lesions compared to mice that receive normal bone marrow. Lesion area from these mice were reduced by a factor 2.5 and contained significantly less macrophages as compared to control recipients (Boisvert WA et al, JCI 101;353-363, 1998); Thus, there seems to be a role for a CXCR2 homologue in the recruitment of monocytes/macrophages. In mice, KC/GRO-α and MIP-2 are endogenous ligands for mIL-8Rh. KC/GRO-α expression was generally abundant in atherosclerotic lesions of LDLR knock-out mice (Boisvert WA et al, JCI 101;353-363, 1998). The role of CXCR2 in atherosclerosis was further underscored by researchers from SmithKline Beecham. They reported a 40% reduction in plaque density in LDLR knock-out mice compared to placebo treated animals. Using human cells, Gerszten et al. (Nature 398, 718-723, 1999) showed that IL-8 mediates firm ^" adhesion of human monocytes on endothelial cells (HUNECs). Furthermore, IL-8 was identified as an inducer of monocyte/macrophage migration. Thus, convincing data links IL-8 and CXCR2 through their activity on monocytes/macrophages with atherosclerosis.

The current concept of rheumatoid arthritis deals with the assumption that inflammation and joint destruction are the net results of complex cell-cell interactions. The driving force is supposed to be the (false) recognition of self-peptides by CD4⁺ T cells on the surface of various types of antigen presenting cells. Released lymphokines activate monocytes/macrophages, which respond with an increase in their cytokine (e.g. TΝFα, IL.l), chemokine (e.g IL-8) and enzyme (e.g. MMPs) output. These secreted products maintain and amplify the aforementioned process and further stimulate fibroblasts and chondrocytes to release cartilage degrading enzymes (e.g. MMPs), adhesion molecules and chemotactic factors (e.g. IL-8) for new recruitment of T cells, monocytes and neutrophils to the afflicted joints. In recent years, especially the role of macrophages in the immunopathogenesis of rheumatoid arthritis has gained more and more attention, because macrophage-secreted chemokines and cytokines, together with fibroblast-derived cytokines, constitute the major part of the cytokine profile in synovial fluid and because the number of macrophages is increased considerably in the synovial tissue of rheumatoid arthritis patients at sites where cartilage degration is fulminate.

The secretion pattern of monocytes/macrophages includes IL-8. In vitro stimulation of IL-8 production can be achieved by simple contact with synovial fluid taken from rheumatoid arthritis patients and, without the interference of soluble mediators, by direct contact with activated T cells (Koch et al., J. Immunol., 147, 2187-2195, 1991; Rodenburg et al., Ann Rheum. Dis., 58, 648-652, 1999). Pro-inflammatory cytokines, like TΝFα, IL-1 and IL-17, which are all prominantly present in synovial fluid of arthritis patients, induce high levels of IL-8 as well as GROα production by synovial chondrocytes and fibroblasts (Pulsatelli et al., J. Rheumatol.,26, 1992-2000, 1999).

Beside a role in atherosclerosis and rheumatoid arthritis, elevated production and local concentrations of IL-8 in affected tissues have been found to be associated with a number of disease states. Chemokine (such as, but not limited to IL-8, Gro-alpha (CXCLl), Gro-beta (CXCL2), Gro-gamma (CXCL3), NAP-2 or ENA-78 (CXCL5)) mediated diseases include psoriasis, atopic dermatitis, asthma, chrome obstructive pulmonary disease, ulcerative colitis, idiopathic pulmonary fibrosis, inflammatory bowel disease, adult respiratory distress syndrome (ARDS), Crohn's disease, stroke, meconium aspiration syndrome, cardiac and renal reperfusion injury, glomerulonephritis, graft vs. host reaction, alzheimers disease, allograft rejections, malaria, restenosis, angiogenesis, undersired hematopoietic stem cell release, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, and cancer.

As described above several diseases are associated with an increase in IL-8 production and other ligands of CXC chemokine receptors leading to chemotaxis of cells involved in the disease process. Therefore, these diseases would benefit by compounds that are inhibitors of chemokine receptor binding.

It has now been found that compounds of the formula I

wherein

R¹ is a basic moiety having the structure -NH(CH₂)₂NH⁵R⁶,

or

wherein R , R and R are independently selected from H, (l-6C)alkyl, (2-6C)alkenyl, (3-8C)cycloalkyl, and (l-6C)alkyl substituted with furanyl, (l-6C)alkoxy, (3-8C)cycloalkyl, trifluoromethyl or amido; or R⁵ and R⁶ together with N to which they are attached are a (4-8) membered heterocycle, which heterocycle may be substituted with (l-6C)alkoxy, trifluoromethyl or amido and which heterocycle may be fused with another (5-6). membered heterocycle; n is 1, 2 or 3; and the ring structure A represents a (4-6) membered heterocycle; R² is (l-6C)alkyl, optionally substituted with (l-6C)alkoxy;

R³ is aryl, heterocyclyl,-X-(CH₂)_m-aryl or -X-(CH₂)_m-heterocyclyl, wherein X is O, S or NR⁸, R⁸ being H or (l-6C)alkyl, and m is 0, 1, 2 or 3; and R⁴ is H, (l-8C)alkyl, (l-8C)alkoxy or (2-8C)alkenyl, the alkyl moiety of each optionally being substituted with hydroxy, halogen, cyano, nitro, oxo, amino, alkylamino, dialkylamino, amido, alkylamido, carboxy, (l-SC)alkoxy, (l-SC)alkylthio, perfluoro(I-4C)alkyl, (3-8C)cycloalkyl, aryl, aryloxy, heterocyclyl or -O- heterocyclyl; or a pharmaceutically acceptable salt thereof are potent IL-8 receptor modulators, in particular inhibitors thereof, having anti- inflammatory activity, which display advantageous solubility properties.

The compounds of the present invention are useful for treating or preventing IL-8 receptor mediated disorders, such as atherosclerosis, inflammation, rheumatoid arthritis and related disorders. Further, the term 'IL-8 receptor mediated disorders' refers to any and all disease states in which chemokines play a role (vide supra). Preferred compounds according to the invention have the formula I, wherein R³ is imidazolyl or imidazolyl substituted with (1 -6C)alkyl, (substituted) phenyl, (substituted)benzofuranyl or (substituted) benzothiophenyl, wherein the optional substituents are selected from (l-6C)alkyl, halogen, trifluoromethyl and trifluoromethoxy, or R³ is imidazolyl substitued with [(l-6C)alkyl substituted] thiophenyl or [(l-6C)alkyl substituted] furanyl wherein the furanyl group optionally is subtituted with halogen, or R³ is furanyl, thiophenyl, benzofuranyl, benzothiophenyl or benzimidazolyl, each optionally substituted with (l-6C)alkyl, halogen, trifluoromethyl or trifluoromethoxy, or R³ is phenyl, -O-(CH₂)_p-phenyl, S-(CH₂)_P- phenyl, -NR⁹-(CH₂)_p-phenyl, pyridinyl, -O-(CH₂)_p-pyridinyl, -S-(CH₂)_p-pyridinyl, - NR⁹-(CH )p-pyridinyl wherein R⁹ is H or (l-6C)alkyl, p being 0, 1,2 or 3, and the phenyl and pyridinyl moieties optionally being substituted with (l-6C)alkyl; and R⁴ is H, (l-8C)alkyl, (l-8C)alkoxy, trifluoromethyl, trifluoromethoxy, (l-8C)alkyl substituted with (l-8C)alkoxy, (3-8C)cycloalkyl, amido, -C(O)NHR¹⁰ wherein R¹⁰ is (l-SC)alkyl, or trifluoromethyl, or R⁴ is (l-8C)alkoxy substituted with (3- 8C)cycloalkyl or trifluoromethyl or R⁴ is -(CH₂)_q-phenyl or -(CH₂)_q-phenoxy, q being 0, 1, 2 or 3 and the phenyl group optionally being substituted with (l-8C)alkyl, (1- 8C)alkoxy, hydroxy, halogen, cyano, nitro or trifluoromethyl. Other preferred compounds of formula I are those, wherein R³ is imidazolyl or imidazolyl substituted with (l-6C)alkyl, (substituted) phenyl, (substituted) benzofuranyl or (substituted) benzothiophenyl, wherein the optional substituents are selected from halogen, trifluoromethyl and trifluoromethoxy, or R is benzimidazolyl or benzofuranyl, each of which optionally substituted with halogen, trifluoromethyl or trifluoromethoxy; and R⁴ is H, (l-SC)alkyl, (l-8C)alkoxy, trifluoromethyl, trifluoromethoxy, (l-8C)alkyl substituted with (3-8C)cycloalkyl or trifluoromethyl, or R⁴ is (l-8C)alkoxy substituted with (3-8C)cycloalkyl or trifluoromethyl. In particular preferred are compounds of

9 1 formula I, wherein R is -CH₂CH(CH₃)₂. Most preferred are compounds, wherein R is -NH(CH₂)₂NR⁵R⁶.

Further preferred are compounds of formula I, wherein R² is (l-6C)alkyl; and R⁴ is H, (l-6C)alkyl or (l-6C)alkoxy, wherein the alkyl moiety may be substituted with trifluoromethyl or (3-8C)cycloalkyl, and in particular preferred are the compounds wherein R is imidazolyl, benzimidazolyl, halogen substituted benzimidazolyl, phenyl substituted imidazolyl, phenyl substituted pyrazinyl, wherein the phenyl group is substituted with halogen, trifluoromethyl or trifluromefhoxy. Most preferred are such compounds, wherein R is -CH2CH(CH₃)₂.

The terms (l-6C)alkyl and (l-8C)alkyl mean a branched or unbranched alkyl group having 1 to 6 or 1 to 8 carbon atoms, respectively, such as methyl, ethyl, isopropyl, t- butyl and the like. The terms (l-6C)alkoxy and (l-8C)alkoxy mean a alkoxy groups having 1 to 6 or 1 to 8 carbon atoms, respectively, the alkyl moiety of which having the meaning as previously defined. The terms (2-6C)alkenyl and (2-8C)alkenyl mean a branched or unbranched unsaturated hydrocarbon group having 2 to 6 or 2 to 8 carbon atoms, respectively. Examples are ethenyl, propenyl, allyl, and the like.

The term (3-8C)cycloalkyl means a cycloalkyl group having 3-8 carbon atoms, being cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclo-octyl. Cyclopentyl and cyclohexyl are preferred cycloalkyl groups.

The terms aryl and heteroaryl mean a 5- or 6-membered aromatic or heteroaromatic ring containing 0-3 heteroatoms selected from O, N, and S; a bicyclic 9- or 10- membered aromatic or heteroaromatic ring system containing 0-3 heteroatoms selected from O, N, and S; or tricyclic 13- or 14-membered aromatic or heteroaromatic ring system containing 0-3 heteroatoms selected from 0, N, and S; each of which rings is optionally substituted with up to three substituents chosen independently from lower alkyl, oxo, nitro, halogen, hydroxy, alkoxy, alkylsulfonyl; methylenedioxy, alkoxyethoxy, cyano, amino, alkylamino, dialkylamino, acylamino, aminosulfonyl, (l-6C)-alkoxycarbonyl, carboxy, methylsulfonamido, perfluoroalkyl, phenyl, benzyl, trityl, and phenoxy. 6- to 14-Membered aryl residues include, for example, benzene and naphthalene, and the 5- to 10-membered heteroaryl residues include, for example, imidazole, pyridine, indole, oxazole, thiophene, benzopyranone, benzodioxan, benzodioxole, thiazole, furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine, pyrimidinone, pyridazine, tetrazole, and pyrazole.

The term heterocyclyl means a cycloalkyl where one to three carbon atoms is replaced with a heteroatom, such as O, NR (R= H, alkyl), N→O, S, SO, S0₂ and the like. The term includes residues in which one or more rings is optionally substituted with up to three substituents chosen independently from lower alkyl, oxo, halogen, hydroxy, alkoxy, amino, alkylamino, alkylthio, dialkylamino, acylamino, aminosulfonyl, (1- 6C)-alkoxycarbonyl, carboxy, mefhylsulfonamido, perfluoroalkyl, phenyl, benzyl, trityl; and phenoxy. When two heteroatoms are separated by a single carbon, the resulting heterocycloalkyls tend to be unstable in aqueous solutions and are therefore not preferred. Heterocyclyl includes heteroaryl, which is a subset of heterocyclyl. Examples of heterocycloalkyls include: pyrrolidirie, morpholine, tetrahydrofuran, tetrahydropyran, piperidine, piperazine, pyridine-N-oxide, 2-methyl-l,3-dithiane, dioxane, and the like.

Substituted aryl, heteroaryl or heterocyclyl means aryl, heteroaryl or heterocyclyl, wherein hydrogen atoms are replaced by halogen, hydroxy, carboxy, carboalkoxy, carboxamido, cyano, alkyl carbonyl (acyl), nitro, alkoxy, methylenedioxy, alkoxymethyl, alkoxyethoxy, amino, alkylamino, dialkylamino, acylamino, aminosulfonyl, (l-6C)-alkoxycarbonyl, (l-6C)alkylaminocarbonyl, methylsulfonamido, methylsulfonyl, alkylthio, perfluoroalkyl, phenyl, benzyl, trityl, phenoxy, alkylphenoxy, amidino, guanidino, ureido, and benzyloxy. When a carbon of an aryl, heteroaryl or heterocyclyl bears two hydrogen atoms, the two hydrogens may be replaced by =O, =S or =NH.

The terms (4-6), (5-6) and (4-8) membered heterocycle in the definitions of R⁵ and R⁶ and for the ring structure A mean heterocyclic groups containing 4 or 5 up to 6 to 8 atoms, in the case of ring structure A one of which is the heteroatom N attached to R⁷, and the heterocycle may optionally contain more than one heteroatom selected from O, S or N. The heterocycle A may be substituted with (l-6C)alkyl or (l-6C)alkoxy.

The term halogen means fluorine, chlorine, bromine or iodine. Preferred halogens are fluorine and chlorine.

In the definitions, the term substituted means substituted by one or more substituent.

The compounds of the present invention may suitably be prepared as outlined in the synthetic schemes described for the examples. The compounds of the invention, which can be in the form of a free base, may be isolated from the reaction mixture in the form of a pharmaceutically acceptable salt. The pharmaceutically acceptable salts may also be obtained by treating the free base of formula I with an organic or inorganic acid such as, but not limited to, hydrogen chloride; hydrogen bromide, hydrogen iodide, sulfuric acid, phosphoric acid, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, maleic acid, malonic acid, methanesulphonic acid, fumaric acid., succinic acid, tartaric . acid, citric acid, benzoic acid, and ascorbic acid.

Compounds of the invention may exist in solvated as well as in unsolvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Compounds of the present invention may exist as amorphous forms, but also multiple crystalline forms may be possible. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of this invention.

The compounds of this invention possess one or more chiral carbon atoms, and may therefore be obtained as a pure enantiomer, or as a mixture of enantiomers, or as a mixture containing diastereomers. Methods for obtaining the pure enantiomers are well known in the art, e.g. crystallization of salts which are obtained from optically active acids and the racemic mixture; or chromatography using chiral columns. For diastereomers straight phase or reversed phase columns may be used.

The compounds of the invention may be administered enterally or parenterally, and for humans preferably in a daily dosage of 0.001-100 mg per kg body weight, preferably 0.01-10 mg per kg body weight. The oral administration is generally preferred. Appropriate dosage forms for such administration may be prepared by convention techniques. Mixed with pharmaceutically suitable auxiliaries, e.g. as described in the standard reference, Gennaro et al., Remington's Pharmaceutical Sciences, (20th ed., Lippincott Williams & Wilkins, 2000, see especially Part 5: Pharmaceutical Manufacturing) the compounds may be compressed into solid dosage units, such as pills, tablets, or be processed into capsules or suppositories. By means of pharmaceutically suitable liquids the compounds can also be applied in the form of a solution, suspension, emulsion, e.g. for use as an injection preparation, or as a spray, e.g. for use as a nasal spray.

For making dosage units, e.g. tablets, the use of conventional additives such as fillers, colorants, polymeric binders and the like is contemplated. In general any pharmaceutically acceptable additive that does not interfere with the function of the active compounds can be used.

Suitable carriers with which the compositions can be administered include lactose, starch, cellulose derivatives and the like, or mixtures thereof, used in suitable amounts.

The invention is further illustrated by -but not limited to- the following examples.

EXAMPLES

Abbreviations :

Formula I Synthetic Overview

All non commercially available alkyl substituents on R4 are introduced on . 2,4 dichloropyrimidine using the alkyl bromine in the presence of lithium (J. Org. Chem.,1988, 53, 4137-4140, Scheme 1).

The synthesis of analogs with the R3 group (e.g. arylimidazolyl, benzimidazolyl, benzofuranylimidazolyl and furanylimidazolyl) linked to the pyrimidine ring via a N-C^'bond, can be performed using the method outlined in Scheme 1. For example, an aminoalcohol was reacted with 2,4-dichloro-6-methylpyrimidine and the resulting regioisomers were separated. Subsequent reaction of the 2-chloropyrimidine isomer with an arylimidazole, oxidation and amide formation, afforded the final product. Other nitrogen containing nucleophiles such as benzimidazoles can be used to replace the arylimidazole in these procedures to obtain the corresponding analogs.

- .CO,

R3 D SO

TBTU DIEA R1 DCM

Scheme 1

The synthetic methods for some reagents incorporated as the R3 or Rl groups (Formula I) are outlined in Schemes 2 to 5.

Several arylimidazoles used in the invention were synthesized from the reaction of aryl aldehydes (Scheme 2) with p-tolylsulfonyl isocyanide (TosMIC) followed by heating in methanolic ammonia (Heterocycles, 1994, 39, 139).

Scheme 2

Alternatively, several (aryl)imidazoles were synthesized from the reaction of 2- bromomethylketones with formamide (Scheme 3). This synthetic procedure is known from the literature (J. Am. Chem. Soc, 1994, 116, 11030). In some cases, the bromomethylketones used in Scheme 3 were obtained from the corresponding ketones via a known bromination procedure (Aust. J. Chem., 1989, 42, 1735).

II . for amide a _a .N.

• Br — ^reflux - ^{• "}Q H

Scheme 3

Several benzimidazoles used in the invention were obtained by the reaction of 2-aminoanilines with formic acid and aqueous HC1 (Scheme 4) using a literature procedure (Org. Syn., 1943, Coll. Vol. 2, 65). Alternatively, these compounds were obtained by refluxing 2-aminoanilines with ethoxymethylenemalononitrile in isopropyl alcohol (Scheme 5) following a literature procedure (Tetrahedron Lett, 1993, 34, 1897).

Scheme 4

iPrOH reflux

Scheme 5

Furanyl-imidazoles were prepared by the TosMIC reaction from aldehydes, which can be obtained either by reduction of acids, esters or amides; or by oxidation of primary alcohols. These procedures are well known in the literature. For example, ethyl 2-trifluoromethyl-furan- 3-carboxylate (Scheme 6) prepared from literature procedures (U.S. Patent 5,405,865) was converted to an aldehyde by reduction of a Weinreb amide. The aldehyde was then reacted with the TosMIC and methanolic ammonia to yield 4-(2-trifluoromethylfuran-3-yl)imidazole. 0 0 (T — CF₃ 1. Na0H, Et0H -CF3

COOEt 2. HNMe(OMe), EDCI, HOBt, DIEA -H

3. LiAIH₄, Et₂0

Scheme 6

Benzofiiranylimidazoles used in the current invention were prepared from the corresponding phenols (Scheme 7). The phenol was converted to a benzofuran (J Med. Chem., 1997, 40, 322) which was subsequently formylated. The aldehyde was reacted with the TosMIC reagent, followed by heating in methanolic ammonia to give the imidazole.

Scheme 7

Most of the Rl groups used at the current invention were prepared starting with primairy amines. The amines were converted to the corresponding ethylcarbamates. The carbamates were reduced with LiAlFL_t and the obtained amines were alkylated with chloro acetonitrile and subsequently reduced with LiAlH-v (Scheme 8 A). Alternatively commercially available secondary amines were alkylated with (2-bromoethyl)carbamic acid tert-butylester. After treating the amines with acetyl chloride in MeOH the desired Rl groups were obtained (Scheme 8 B).

CH,CN

THF LiAlH₄ R

Scheme 8

Some of the rigid Rl groups were prepared via a reductive amination (Scheme 9).

Scheme '9

Representative procedures for reactions shown in Schemes 1 to 9 are as follows:

4-(4-Chloro-3-trifIuoromethylphenyl)imidazole.

To a round-bottom flask containing 4-chloro-3-(trifluoromethyl)benzaldehyde (1.83 gram) and TosMIC (1.56 gram) was added EtOH (15 mL, absolute) and KCN (48 mg) The mixture was stirred at room temperature for 3 hours and the resulting suspension was filtered. The solid residue was washed with cold EtOH and dried in vacuo. The tosyloxazoline intermediate was added to a glass pressure vessel containing NH₃ in methanol (50 ml of a 7.0 N solution). The vessel was capped and heated to 100 °C for 14 hours. The contents were carefully cooled to 0 °C and the vessel was opened. Upon warming slowly to room temperature, most of the ammonia had dissipated. The mixture was transferred to a round-bottom flask and concentrated in vacuo. The title compound (0.334 g, 17%) was isolated from the crude mixture by flash chromatography (dichloromethane/methanol 10/1). ESI-MS (m/z) 247 [M+H]⁺.

4-(3-Trifluoromethoxyphenyl)imidazole.

Bromine (0.76 mL) was added dropwise to a 0°C solution of 3'- (trifluoromethoxy)acetophenone (3.0 g) and hydrobromic acid (48 % aqueous, 1 mL) in acetic acid (15 mL). The solution was allowed to stir at room temperature for 45 minutes, then heated to 40°C for 90 minutes. The volatiles were removed in vacuo to give a mixture of 2- bromo- 1 -[3 -(trifluorome thoxy)phenyl] -ethan- 1 -one and 2,2-dibromo- 1 - [3 -

(trifluoromethoxy)phenyl]ethan-l-one.

The mixture (2.6 g) and formamide (35 mL) were heated to reflux for 2.5 hours. The resulting solution was allowed to cool to room temperature, pH was adjusted to 10 with 10% potassium carbonate, and extracted three times with chloroform. The chloroform extracts were combined, washed with brine, dried over sodium sulfate, filtered, and the volatiles were removed in vacuo. The resulting residue was purified on silica gel using ethyl acetate as the mobile phase to yield 4-(3-trifluoromethoxy-phenyl)imidazole (0.3 g). ESI-MS (m/z) 229 [M4Η]⁺.

4-fert-ButyI-LH^r-imidazoIe. A solution of l-bromo-3,3-dimethylbutan-2-one (33.1 gram) in formamide (120 ml) was stirred at 150°C for 6 hours. At room temperature a solution of K₂CO₃ (10 % in water) was added until pH = 9. The resulting solution was extracted three times with dichloromethane. The combined dichloromethane layers were washed with a solution of K₂CO₃ (10 % in water), brine, dried over magnesium sulfate, filtered, and the volatiles were removed in vacuo. The title compound (18.0 gram) was used without fiither purification.

^rfixχ H

5-Trifluoromethylbenzimidazole.

A mixture of 4-(trifluoromethyl)-l,2-phenylenediamine (3.0 g), formic acid (1.3 mL), and hydrochloric acid (4 M, 17 mL) was heated to reflux for 45 minutes. The mixture was allowed to cool to room temperature and neutralized with concentrated ammonium hydroxide. The aqueous layer was removed off to give 5-trifluoromethyl-lH-benzimidazole (2.56 g) as a black solid. ESI-MS (m/z) 187 [M+H]⁺.

^'X } H

5-Fluorobenzimidazole.

A solution of 4-fluoro-l,2-phenylenediamine (2.0 g) and ethoxymethylenemalononitrile (2.9 g) in isopropanol (80 mL) was heated to reflux for 17 hours. The volatiles were removed in vacuo to yield 5-fluoro-lH-benzimidazole (1.8 g). ESI-MS (m/z) 137 [M+H]⁺.

l-(2,2-Diethoxyethoxy)-4-trifluoromethyIbenzene.

4-Hydroxybenzotrifluoride (6.48 gram), cesium carbonate (18.25 gram) and bromoacetaldehyde diethyl acetal (11.82 gram) were suspended in DMF (100 mL) in a round- bottom flask under argon and heated to 65 °C for 60 h. The mixture was cooled to room temperature and submitted to an aqueous workup with diethyl ether and water. The combined organics were dried over MgSO₄, filtered and concentrated to a crude residue (15.6 g) from which the title compound (8.25 gram) was isolated by flash chromatography (n- hexanes/dichloromethane 2/1). F₃C J^C XO

5-Trifluoromethylbenzofuran.

To a round-bottom flask containing l-(2,2-diethoxy-ethoxy)-4-trifluoromethylbenzene (8.25 g, 29.6 mmol, 1 eq) and benzene (80 mL, anhydrous) was added polyphosphoric acid (8.2 g). The mixture was heated to 90 °C for 6 hours. The solvent was removed carefully in vacuo and the crude mixture was applied to a silica gel column. Elution with «-hexanes gave pure fractions of product which were carefully concentrated to give 1.32 g of a colourless, oily mixture of title compound and «-hexanes (suspected volatility of title compound), which was used without fiither purification.

5-Trifluoromethylbenzofuran-2-carbaIdehyde.

A round-bottom flask under argon was charged with the above mixture of 5- trifluoromethylbenzofuran and w-hexanes (1.32 g). THF (130 mL, anhydrous) was added and the flask was cooled to -78 °C. A solution of n-butyllithium in π-hexanes (6.88 mL of a 1.6 M solution) was added. After stirring for 5 min., the mixture, N,N-dimethylformamide (27 mL) was added dropwise. Stirring continued for 2 hours at -78 °C. The mixture was quenched through slow addition of a saturated solution of ΝH₄C1 and submitted to extractive workup with diethyl ether. The combined organics were dried on MgSO₄, filtered and concentrated in vacuo to give a crude residue (1.51 g) which was used immediately, without purification, in the subsequent protocol.

-Q

F₃C J^ 1

4-(5-Trifluoromethylbenzofuran-2-yl)imidazole.

To a round-bottom flask containing 5-trifluoromethylbenzofuran-2-carbaldehyde (1.51 gram) was added ethanol (20 mL), followed by TosMIC (1.38 gram). The mixture stirred at .room temperature for 10 min and NaCN (34 mg) was added. After an additional 30 min stirring, the solid/suspension was filtered. The solid cake was washed with cold ethanol and dried in vacuo to give the tosyloxazoline intermediate (2.16 g) as a white solid. The intermediate was added to a glass pressure vessel containing NH₃ in methanol (120 ml of a 7.0 N solution). The vessel was capped and heated to 90 °C for 14 hours. The contents were carefully cooled to 0 °C and the vessel was opened. Upon warming slowly to room temperature, most of the ammonia had dissipated. The mixture was transferred to a round-bottom flask and concentrated in vacuo. The title compound (0.56 g, 31%) was isolated from the crude mixture by flash chromatography (dichloromethane/methanol 10/1).

Cyclopenten-3-ylcarbamic acid ethyl ester.

To a solution of l-amino-3-cyclopentene hydrochloride (2 gram) and ethylchloroformate (1.6 ml) in dichloro- methane (25 ml) at 0°C was slowly added DIEA (5.8 ml). The reaction was stirred for 2 hours at room temperature. The solution was taken up in water, and extracted three times with ethyl acetate. The ethyl acetate layers were combined, washed with brine, dried over magnesium sulfate, filtered,, and removal of the solvents provided 2.5 g of the title compound. ESI-MS(m/z) 156[M+H]⁺.

Cyclopenten-3-yl-π-ιethylamine.

To a solution of LiAfflU (3 gram) in THF (81 ml) was added a solution of cyclopenten-3- ylcarbamic acid ethyl ester (2.5 gram) iti THF (40 ml) at 0°C. The reaction mixture was stirred at reflux for 2 hours. At 0°C, a saturated aqueous sodium sufate solution (15 ml) was added carefully. After stirring for 4 hours the precipitate was filtered off. To the fitrate was added concentrated HC1 (1.5 ml) and dioxane (100 ml). The solvents were removed in vacuo to furnish 2.3 gram of the title compound as its HC1 salt.

(CycIopenten-3-ylmethylamino)acetonitrile. A solution of cyclopenten-3-yl-methylamine (2.2 gram), chloroacetonitrile (5.4 ml) and K₂CO₃ (9.3 gram) in acetonitrile (100 ml) was stirred for 4 hours at reflux. The solution was taken up in water and extracted three times with ethyl acetate. The ethyl acetate layers were combined, washed with brine, dried over magnesium sulfate, filtered and the solvents were removed in vacuo to yield 2.2 gram of the title compound.

N^Cy openten-S-yl-N'-methylethane-l^-diamine.

To a solution of LiAlH-_t (950 mg) in THF (25 ml) was added a solution of (cyclopent-3-enyl- methylamino)acetonitrile (1.7 gram) in THF (13 ml) at 0°C. The reaction mixture was stirred at room temperature for 2 hours. At 0°C, a saturated aqueous sodium sulfate solution (5 ml) was added carefully. After stirring for 4 hours the precipitate was filtered off To the fitrate was added concentrated HCl (2.4 ml) and dioxane (100 ml). The solvents were removed in vacuo to yield 2.6 gram of the title compound as its HCl salt. ESI-MS(m/z) 141[M+H]⁺.

[2-(2-(-S)-CarbamoylpyrroIidin-l-yl)ethyI]carbamic acid tert-butyl ester.

A solution of (2-bromoethyl)carbamic acid tert-butyl ester (500 mg), L-proline amide hydr-ochloride (1 gram) and DIEA (1.95 ml) in acetonitrile (5 ml) was stirred at 80°C for 48 hours. The solvent was removed in vacuo. Silica gel chromatography (DCM/MeOH 4/1) provided 600 mg of the title compound. ESI-MS(m/z) 258[M+H]⁺.

l-(2-AminoethyI)pyrroIidine-2-(S)-carboxylic acid amide.

To a solution of acetyl chloride (1.25 ml) in MeOH (15 ml) was added [2-(2-S- carbamoylpyrrolidin-l-yl)ethyl]carbamic acid tert-butyl ester (600 mg) at 0°C. The reaction mixture was stirred for 17 hours at room temperature. The solvents were removed in vacuo to give 380 mg of the title compound as its HCl salt.

2-(3-Methoxypyrrolidin-l-yl)ethylamine.

(S)-(+)-N-(tert-Butoxycarbonyl)-3-hydroxypyrrolidine (936 mg) was dissolved in DMF (40 mL) under an argon atmosphere, cooled to 0 °C and treated with NaH (220 mg of a 60 % dispersion in mineral oil). After 15 min., the mixture was allowed to warm to room temperature and treated with iodomethane (740 uL) for 3 hours. The mixture was diluted with ethyl acetate and shaken with water. The aqueous phase was extracted with ethyl acetate three times and the combined organic phases were dried on MgSO₄, filtered and concentrated in vacuo to give a crude oil from which (S)-(+)-N-(tert-Butoxycarbonyl)-3-methoxypyrrolidine (750 mg) was isolated by flash chromatography (hexane/ethyl acetate 1/1). (S)-(+)-N-(tert-butoxycarbonyl)-3-methoxypyrrolidine (750 mg) was dissolved in dichloromethane (35 mL) and treated with TFA (15 mL) at room temperature for 30 min.. The solvents were removed in vacuo to give the TFA salt of (S)-3-methoxypyrrolidine (700 mg), used without further manipulation in the construction of 2-(3-methoxy-pyrrolidin-l-yl)- ethylamine according to the general method outlined above .

(l-Furan-2-yImethylpyrrolidin-3-(R)-yl)carbamic acid tert-butyl ester. A solution of pyrrolidin-3-(S)-yl-carbamic acid tert-butyl ester (2 gram), 2-furaldehyde (1.3 ml) and BH₃.pyridin complex (1.08 ml) in EtOH (40 ml) was stirred at room temperature for 6 hours. The solution was taken up in water, and extracted three times with ethyl acetate. The ethyl acetate layers were combined, washed with brine, dried over magnesium sulfate, filtered, and the solvents were removed in vacuo. Silica gel chromatography (heptane/acetone 9/1) provided 1.14 g of the title compound. ESI-MS(m/z) 267[M+H]⁺.

H,N ΓJLQ l-Furan-2-yImethyIpyrrolidm-3-(R)-ylamine.

To a solution of acetyl chloride (2.1 ml) in MeOH (20 ml) was added (l-furan-2- ylmethylpyrrolidin-3-(R)-yl)carbamic acid tert-butyl ester (1.1 gram) at 0°C. The reaction mixture was stirred for 17 hours at room temperature. The solvents were removed in vacuo. 1.0 gram of the title compound was obtained as its HCl salt. ESI-MS(m/z) 167[M+H]⁺.

(2-S)-2-([2-chloro-6-methylpyrimidin-4-yl]amino)-4-methylpentan-l-oI.

A solution of 2,4-dichloro-6-methylpyrimidine (5 gram), L-leucinol (4.1 ml) and D EA (11.9 gram) in dimethyl sulfoxide (90 ml) was stirred at room temperature for 17 hours. The solution was taken up in water, and extracted three times with ethyl acetate. The ethyl acetate layers were combined, washed with brine, dried over magnesium sulfate, filtered, and the solvents were removed in vacuo. Silica gel chromatography (heptane/ethyl acetate 4/1) provided 3.3 g of the title compound. ESI-MS(m/z) 244[M+H]⁺.

(2-S)-2-{[2-(4-[4-trifluoromethoxyphenyl]imidazol-l-yl)-6-methyIpyrimidin-4-yl]amino}- 4-methylpentan-l-ol.

A solution of (2S)-2-([2-chloro-6-methylpyrimidin-4-yl]amino)-4-methylpentan-l-ol (2.0 gram), 4-(3-trifluoromethoxyphenyl)-lH-imidazole (3.74 g), KF (480 mg) and K₂CO₃ (2.83 g) in dimethyl sulfoxide (30 ml) was stirred at 110°C for 17 hours. The solution was taken up in water, and extracted three times with ethyl acetate. The ethyl acetate layers were combined, washed with brine, dried over magnesium sulfate, filtered, and the solvents were removed in vacuo. Silica gel chromatography (toluene/acetone 9/1) provided 2.0 g of the title compound. ESI-MS(m/z) 436[M+H]⁺.

(2iS)-2-{[2-(4-[4-trifluoromethoxyphenyl]imidazol-l-yl)-6-methyIpyrimidin-4-yl]amino}- 4-methyIpentanoic acid.

A solution of (2S)-2-{[2-(4-[4-trifluoromethoxyphenyl]imidazol-l-yl)-6-methylpyrimidin-4- yl]amino}-4-methylpentan-l-ol (2.0 gram), iodobenzene diacetate (2.98 gram) and TEMPO (70 mg) in tetrahydrofurane/water (50 ml, 4/1) was stirred at room temperature for 17 hours. The solution was taken up in water, and extracted three times with dichloromethane. The dichloromethane layers were combined, washed with brine, dried over magnesium sulfate, filtered, and the solvents were removed in vacuo. Silica gel chromatography (toluene/acetone 3/1) provided 1.2 g of the title compound. ESI-MS(m/z) 450[M+H]⁺.

(2-S)-2-{[2-(4-[4-trifluoromethoxyphenyI]imidazol-l-yl)-6-methyIpyrimidin-4-yl]amino}- 4-methylpentanoic acid 2-(N-methyl-N-2-methoxyethyl)ethaneamide.

A solution of (2S)-2-{[2-(4-[4-trifluoromethoxyphenyl]imidazol-l-yl)-6-methylpyrimidin-4- yl]amino}-4-methylpentanoic acid (81 mg), 2-(lH-benzotriazole-l-yl)-l, 1,3,3- tetramethyluronium tetrafluoroborate (70 mg) and DIEA (42μL) in dichloromethane (5 ml) was stirred for 15 min at room temperature. A solution of N¹-(2-methoxy-ethyl)-N¹ -methyl- ethane- 1,2-diamine (82 mg) and DIEA (142 μL) in dichloromethane (3 ml) was added. The reaction mixture was stirred for 1 hour. The solution was taken up in water, and extracted three times with dichloromethane. The dichloromethane layers were combined, washed with brine, dried over magnesium sulfate, filtered, and the solvents were removed in vacuo. Silica gel chromatography (toluene/acetone 3/1) provided 35 mg of the title compound. ESI- MS(m/z)564[M+H]⁺. The following 1H-NMR data are representatives of compounds prepared using the above described methodes.

(CDCI₃): δ 8.6(s,Ha), 8.1(s,Hb), 2.4(s,Hc), 1.0(dd,Hd), 2.2(s,He), 3.3(s,Hf).

(CDCb): δ 1.3(s,Ha), 8.4(s,Hb), 7.5(s,Hc), 2.2(s,Hd), 1.0(dd,He).

(CDC1₃): δ 9.0(s,Ha), 2.6(t,Hb), 1.0(m,Hc), 2.2(s,Hd), 3.5(m,He).

(DMSO-de): δ 7.4(s,Ha), 8.9(s,Hb), 8.5(s,Hc), 2.8(t,Hd), 1.3(m,He), 3.0(s,Hf), 3.5(s,Hg).

Tables 1 through 5 list representative compounds prepared according to the above described methods.

Table 2

Table 3

able 4

Table 5

The oxyalkyl substituents (R4) were introduced using the potassium salt of the corresponding alcohol and 2-(R3)-4,6-dichloropyrimidine as the starting compound, followed by aminoalcohol introduction, oxidation of the alcohol to the corresponding carboxylic acid and amide formation using TBTU. (Scheme 9)

THF R₄bOH KOtBU

1) aminoalcohol introduction 2) alcohol oxidation

3) a ide formation (introduction R1)

Scheme 9

Representative procedures for reactions shown in Scheme 9 are as follows:

l-(4,6-Dichloropyrimidin-2-yI)-5,6-difluoro-lH-benzimidazole.

A solution of 2,4,6-trichloropyrimidin (4.2 gram), 5,6-difluorobenzimidazole (3.6 gram) and K₂CO₃ (6.4 gram) in dimethyl sulfoxide (150 ml) was stirred at 40°C for 17 hours. The solution was taken up in water, and extracted three times with ethyl acetate. The ethyl acetate layers were combined, washed with brine, dried over magnesium sulfate, filtered, and the solvents were removed in vacuo. Silica gel chromatography (heptane/ethyl acetate 9/1) provided 1.2 g of the title compound.

l-(4-Chloro-6-methox pyrimidin-2-yI)-5,6-difluoro-lH-benzimidazole.

To a solution of KOtBu (41 mg) in THF (3 ml) and MeOH (66 μL) was added l-(4,6- dichloropyrimidin-2-yl)-5,6-difluoro-lH-benzimida2;ole (100 mg). The reaction mixture was stirred for 30 min. at room temperature. The solution was taken up in water, and extracted three times with ethyl acetate. The ethyl acetate layers were combined, washed with brine, dried over magnesium sulfate, filtered and the solvents were removed in vacuo. Silica gel chromatography (heptane/ethyl acetate 9/1) provided 50 mg of the title compound. ESI- MS(m/z) 297[M+Η]⁺.

2-[2-(5,6-Difluorobenzimidazol-l-yl)-6-methoxypyrimidin-4-ylamino]-4-methyIpentan-l- ol.

A solution of l-(4-chloro-6-methoxypyrimidin-2-yl)-5,6-difluoro-lH-benzimidazole (370 mg), L-leucinol (162 μL), K₂CU₃ (431 mg) and KF (5 mg) in dimethyl sulfoxide (10 ml) was stirred at 110°C for 3 hours. The solution was taken up in water, and extracted three times with ethyl acetate. The ethyl acetate layers were combined, washed with brine, dried over magnesium sulfate, filtered and the solvents were removed in vacuo. Silica gel chromatography (toluene/acetone 9/1) provided 141 mg of the title compound. ESI-MS(m/z) 378[M+Η]⁺.

2-[2-(5,6-Difluorobenzimidazol-l-yl)-6-methoxypyrimidin-4-ylamino]-4-methylpentdnoic acid.

A solution of 2-[2-(5,6-difluorobenzimidazol-l-yl)-6-methoxypyrimidin-4-ylamino]-4- methylpentan-1-ol (145 mg), iodobenzene diacetate (253 mg) and TEMPO (10 mg) in dioxane/water (6 ml, 5/1) was stirred at room temperature for 17 hours. The solvents were removed in vacuo. Silica gel chromatography (toluene/acetone 4/1) provided 87 mg of the title compound. ESI-MS(m/z) 392[M+H]⁺.

2-[2-(5,6-DifluorobenzoimidazoI-l-yl)-6-methoxypyrimidin-4-ylamino]-4-methyl- pentanoic acid {2-[(2-methoxyethyl)methylamino]ethyl}amide.

A solution . of 2-[2-(5,6-difluorobenzimidazol-l-yl)-6-methoxypyrimidin-4-ylamino]-4- methylpentanoic acid (87 mg), 2-(lH-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate (86 mg) and DIEA (78 μL) in dichloromethane (5 ml) was stirred for 15 min at room temperature. A solution of N¹-(2-methoxy-ethyl)-N^I -methyl-ethane- 1,2-diamiήe (92 mg) and DIEA_. (234 μL) in dichloromethane (3 ml) was added. The reaction mixture was stirred for 2 hour. The solution was taken up in water, and extracted three times with dichloromethane. The dichloromethane layers were combined, washed with brine, dried over magnesium sulfate, filtered and the solvents were removed in vacuo. Silica gel chromatography (DCM/MeOH 9/1) provided 35 mg of the title compound. ESI-MS(m/z) 506[M+H]⁺.

The following 1H-ΝMR data are representatives of compounds prepared using the above described methods.

(CDCl₃):δ 9.0(s,Ha), 4.0(s,Hb), 1.0(dd,Hc), 2.3(s,Hd), 3.3(s,He).

(CDCl₃):δ 8.0(s,Ha), 8.5(s,Hb), 4.8(m,Hc), 1.0(dd,Hd), 2.2(s,He), 3.5(s,Hf).

Table 6 list representative compounds prepared according to the above described methods.

Table 6

Arylpiperazines (R3; Scheme 10) used in the invention were synthesized via a palladium mediated coupling of an arylbromide with piperazine (Tetrahedron Lett, 1998, 39, 617). The arylpiperazine was reacted with a chloropyrimidine derivative to obtain the desired product. Similarly, arylpiperidines can be used to replace the arylpiperazine in Scheme 10.

Scheme 10

The synthesis shown in Scheme 10 can be carried out as follow:

2-(2-Chloro-6-methylpyridin-4-ylamino)-4-methylpentanoic acid [2-(furan-2-yImethyI- methy!amino)ethyl] amid e.

A solution of 2,4-dichloro-6-methylpyrimidin (1.12 gram), 2-amino-4-methylpentanoic acid [2-(&ran-2-ylmethyl-methylamino)ethyl]amide (2.6 gram) and DIEA (4.9 ml) in DMSO (100 ml) was stirred at 55°C for 17 hours. The solution was taken up in water, and extracted three times with ethyl acetate. The ethyl acetate layers were combined, washed with brine, dried over magnesium sulfate, filtered and the solvents were removed in vacuo. Silica gel chromatography (DCM MeOH 99/1) provided 768 mg of the title compound.

2-{2-[4-(4-Chlorophenyl)piperazin-l-yl]-6-methylpyrimidin-4-γIamino}-4- methylpentanoic acid [2-(furan-2-ylmethyl-methylamino)ethyl]amide.

A solution of 2-(2-Chloro-6-methylpyridin-4-ylamino)-4-methylpentanoic acid [2-(ftιran-2- ylmethyl-methylamino)ethyl]amide (52 mg), l-(4-chlorophenyl)piperazine (82 mg), K₂CO₃ (69 mg) and KF (30 mg) in DMSO (3 ml) was stirred for 17 hours at 120°C. The solution was taken up in water, and extracted three times with ethyl acetate. The ethyl acetate layers were combined, washed with brine, dried over magnesium sulfate, filtered and the solvents were removed in vacuo. Silica gel chromatography (touene/acetone 1/1) provided 70 mg of the title compound. ESI-MS(m/z) 554[M+H]⁺

Example 99

Η-NMR CDCls): δ 3.2(m,Ha), 3.9(m,Hb), 2.18(s,Hc), 1.0(dd,Hd), 2.2(s,He), 3.5(s,Hf).

Example 100 ESI-MS(m/z) 588CM+H]^"1 The synthesis of analogs with the R3 group linked to the pyrimidine ring via a C-C bond, can be performed using the method outline in Scheme 11. An aryl group can be introduced directly to the pyrimidine ring via a Stille coupling reaction. For example, (5-chlorobenzofuran-3- yl)tributylstannane was coupled with a 2-phlorόpyrimidine derivative to yield desired product.

Scheme 11

The aryl tin reagents are either commercially available or prepared by methods known in the literature. Reaction of an aryl lithium with trialkyltin chloride afforded the aryl tin reagents for the Stille coupling reaction. A specific example for the preparation of a trialkyltin reagent is shown in Scheme 12.

Br. DCM

Scheme 12

Representative procedures for carrying out syntheses shown in Schemes Hand 12 are as follows:

l-Chloro-4-(2,2-diethoxyethoxy)benzeπe.

To a solution of 4-chlorophenol (10.1 gram) in DMF (200 ml) was added K₂CO₃ (21.6 gram), DIEA (27.2 ml) and bromoacetaldehyde diethylacetate (30.8 gram). The solution was stirred for 17 hours at 100°C. The solution was taken up in water, and extracted three times with ethyl acetate. The ethyl acetate layers were combined, washed with brine, dried over magnesium sulfate, filtered and the solvents were removed in vacuo. Silica gel chromatography (heptane/ethyl acetate 6/1) provided 13.9 gram of the title compound.

5-Chlorobenzofuraπ.

To a solution of l-chloro-4-(2,2-diethoxyethoxy)benzene (13.1 gram) in toluene (375 ml) was added PPA (81.3 gram). The reaction mixture was stirred for 3 hours at reflux. The mixture was taken up in water, and extracted three times with toluene. The toluene layers were combined, washed with brine, dried over magnesium sulfate, filtered, and the solvents were removed carefully (suspected volatility of title compound) in vacuo. Silica gel chromatography (pentane) provided 3.7 gram of the title compound.

-o C - ■ Br cr

Br

2,3-Dibromo-5-chloro-2,3-dihydrobenzofuran.

To a solution of 5-chloro-benzofurane (3.7 gram) in DCM (70 ml) was added dropwise a solution of bromine (2.45 ml) in DCM (3 ml) at - 30°C. The solution was stirred for 17 hours at room temperature. Removal of the solvents provided 7.2 gram of the title compound.

3-Bromo-5-chlorobenzofuran. To a solution of 2,3-dibromo-5-chloro-2,3-dihydro-benzofuran (7.2 gram) in EtOH (110 ml) was added KOH (9.1 gram). The reaction mixture was stirred for 2 hours at 80°C. The solution was taken up in water, and extracted three times with ethyl acetate. The ethyl acetate layers were combined, washed with brine, dried over magnesium sulfate, filtered, and the solvents were removed in vacuo. Silica gel chromatography (heptane) provided 4.5 gram of the title compound.

Tributyl-(5-chlorobenzofurane-3-yl)stannane.

At -75 °C, n-butyllithium (1.6 M in hexane, 7.0 mL) was added to a solution of 3-bromo-5- chlorofuran (2 g) in diethyl ether (100 mL). After 1 min, tributyltin chloride (3.65 g) in diethyl ether was added and the reaction mixture was allowed to warm to room temperature over 2 h. Water was added to quench the reaction. The organic layer was washed three times with water, dried over magnesium sulfate and evaporated. The crude product was purified by column chromatography (heptane) to afford 2.25 gram of the desired product

(2S)-[2-(5-Chlorobenzofuran-3-yl)-6-methyIpyrimidin-4-ylamino]-4-methyl-pentanoic acid [2-(furan-2-yImethyl-methylamino)ethyl]amide.

A solution of 2-(2-chloro-6-methylpyrimidin-4-ylamino)-4-methylpentanoic acid [2-(furan-2- ylmethyl-methylamino]amide (106 mg), (5-chlorobenzofurane-3-yl)tributylstannane (335 mg), bis(triphenylphosphine) palladium (II) chloride (10 mg) and K₂CO₃ (186 mg) in DMSO (2.5 ml) was stirred at 160°C for 15 minutes in a microwave (300 W). The solution was taken up in water, and extracted three times with ethyl acetate. The ethyl acetate layers were combined, washed with brine, dried over magnesium sulfate, filtered, and the solvents were removed in vacuo. Silica gel chromatography (toluene/acetone 9/1) provided 120 mg of the title compound. ESI-MS(m/z) 510[M+H]⁺

Example 101

1H-NMR(MeOD): δ 8.8(s,Ha), 2.8(s,Hb), 1.0(dd,Hc), 2.6(s,Hd), 4.4(s,He).

Pharmacological assays

Receptor Binding Assay

Ki values of compounds of formula I were measured by means of IL-8 displacement. [^I25I]-IL8 (human recombinant) was obtained from NEN-New England Nuclear, Boston, Mass. with a specific activity of 2200 Ci/mmol. All other chemicals were of analytical grade. High levels of recombinant CXCR2 receptors were expressed in Chinese hamster ovary cells as described previously (Holmes, et al., Science 253; 1278 1991). The Chinese hamster ovary membranes were homogenized according to a previously described protocol (Haour et al, J. Biol. Chem. 249; 2195-2205 1974), using a homogenizafion buffer of 20 mM HEPES and 10 mM EDTA adjusted to pH 8.0.

Membrane protein concentration was determined using Pierce Co. micro-assay kit using bovine serum albumin as a standard. All assays were performed in a 96-well micro plate format. Each reaction contained [^l25I] IL-8 (0.1 nM) and 100 μg/mL of CXCR2 membranes in 1 % DMSO, 10 mM Tris, pH=8, 1.2 mM MgSO₄, 0.1 mM EDTA, 25 mM NaCl, 0.03 % CHAPS. In addition, drug or compound of interest was added which had been pre-dissolved in DMSO so as to reach a final concentration of between 0.01 nM and 100 μM. The assay was initiated by addition of CXCR2 membranes to the plate. After 1 hour at room temperature the plate was harvested using a Tomtech 96-well harvester onto a glass fiber filterplate (Millipore Corporation; Multiscreen-FC, Opaque plates, 1.2 μM type C filter; cat # MAFCNOB50) pre- wet with 0.3 % polyethyleneimine and washed 3 times with 10 mM Tris pH=8, 1 mM MgSO₄_ 0.5 mM EDTA, 25 mM NaCl, 0.03 % CHAPS, 0.5 % BSA using 100 μL/well. 50 μl/well of Microscint20 scintillation liquid (Packard Bioscience cat #3013621) was added to each well and the plate was counted on the Packard liquid scintillation counter.

Compounds of formula I were found to exhibit inhibitory activity of Ki<1000 nM in the receptor binding assay. Preferred compounds have a Ki<100nM.

Calcium influx assay

Calcium (Ca²⁺) influx inhibition with compounds of formula I have been tested in human Acute Monocyte Leukemia (AML-193) cells and human neutrophils. AML-193 cells (ATCC: CRL-9589, Rockwell MD) were maintained in suspension at a concentration of 3x10⁵ cells/mL in culture medium (Iscove modified Dulbecco medium (Life Technologies # 21980-065) containing 5 μg/mL transferrin and insulin, 1 % (v/v) of penicilline/streptomycine solution (Sigma), 5 μg/mL GM-CSF (R&D systems # 215-GM) and 10 % fetal calf serum (FCS) (v/v). AML-193 cells were stimulated to differentiate with all-trans-retinoic acid (ATRA) in the concentration of lxlO^"7 M during 3-4 days. Cells were harvested and washed in Krebs buffer (120 mMNaCl, 4.75 mM KCL, 1 mM KH₂PO₄, 1.44 mM MgSO₄.7H₂O, 1.1 mM CaCl₂.2H₂O, 5 mM NaHCO₃, 11 mM glucose, 25 mM Hepes adjusted to pH 7.4). The cell suspension was centrifiiged at 25000 N/kg at room temperature for 6 min. The supernatant was siphoned off and the pellet was resuspended in Krebs buffer in order to obtain a suspension containing lxl0⁷cells/mL.

Human neutrophils were obtained from free-flowing blood from healthy volunteers. Blood was collected in 0.1 volume sodium citrate dihydrate in water (38 g/L) and diluted 5 times with medium (RPMI 1640 solution (Life Technologies #32404-014), 2 mM L-glutamine (Sigma #G7513) and 0.01 volume of penicilline/streptomycine solution (Sigma #P0781) + 0.1 % bovine serum albumin (Sigma #A-9418)). The neutrophils were separated by the Ficoll Paque technique. In different tubes containing 20 ml Ficoll Paque (Amersham-Pharmacia #17-1440- 03) 30 ml bloodcells were pipetted. The tubes were centrifiiged at 25000 N/kg for 60 minutes at room temperature. The plasma and the lymphocytes layers were siphoned off. The red pellet was collected and adjusted to approximately 100 ml with medium in a sterile flask. An equal volume of Dextran solution (3 % w/w Dextran T500 (Amersham-Pharmacia #115977) in NaCl 0.9 %) was added and mixed with the cells. The mixture was divided into two graduated- glasses of 250 ml and the air bubles were removed. After an incubation of 30 minutes at room temperature the upper-layer containing neurophils was siphoned off and 30 ml was pipetted into Falcon tubes. The cells were centrifiiged at 20000 N/kg for 6 minutes at 4° C. The supernatant was siphoned off. The pellet was shaken free from the bottom and 20 ml ice-cold NaCl 0.2 % was added and the suspension was mixed for exactly 30 sec. The red cells were lysed and the cell suspension was neutralised by adding 20 ml ice-cold NaCl 1.6 %. The cells were centrifiiged again at 20000 N/kg for 6 min. at 4° C. The supernatant was siphoned off and the white pellet was resuspended- in medium in order to obtain a suspension containing lxl0⁷cells/ml.

Assay: Cells were labelled with Fura-2 AM. Fifty μg of Fura-2 AM (Molecular Probes #F- 1221) was dissolved in 50 μl DMSO and subsequently 25 μl 10% w/w Pluronic F127 (Sigma #P2443) in water was added. Three μL Fura-2 AM solution was added to 1 mL cell suspension and the mixture was incubated for 30 min at 37°C. The cell suspension was centrifuged at 25000 N/kg (1200 rpm) at roorntemperature for 6 min and the cells were washed in Krebs buffer and centrifuged again. The washstep was repeated once. The pellet was resuspended in Krebs buffer in order to obtain a suspension containing 1x10 cells/mL. The assay was performed in a 96 well plate. Each reaction contained 40 μL celsuspension in Krebs buffer (total volume 250 μL). In addition, drug or compound of interest was added which had been pre-dissolved in DMSO so as to reach a final concentration of between 0.1 nM and 10 μM. The assay was initiated by addition of 50 μl agonist solution (final concentrations 2 nM IL-8 (R&D systems # 618-IL) or 10 nM Gro-alpha (R&D systems # 275-GR)) to each well automatically by Victor² Wallac 1420 multilabel HTS counter (PerkinElmer) equipped with a dispenser. Subsequently the responses were recorded alternating at 380 nm (excitation) and 510 (emission) for determination of unbound Fura-2 and to 340 nm (excitation) and 510 (emission) for Ca²⁺ bound Fura-2. Fifty counts were recorded for each well which took about 40 seconds. The ratio of fluorescence at 340 and 380 nm was calculated and concentrations were calculated using the maximum (0.67% Triton X-100) and minimum (5.6 mM EGTA) calcium response.

Compounds of formula I were found to exhibit an IC50<1000 nM in the calcium influx assay. Preferred compounds have a Ki<100nM.

Claims

1. A compound of the formula I

(I), wherein

R¹ is a basic moiety having the structure -NH(CH₂)₂NH⁵R⁶,

or

wherein R⁵, R⁶ and R⁷ are independently selected from H, (l-6C)alkyl, (2-6C)alkenyl,

(3-8C)cycloalkyl, and )l-6C)alkyl substituted with furanyl, (l-6C)alkoxy,

(3-8C)cycloalkyl, trifluoromethyl or amido; or R⁵ and R⁶ together with N to which they are attached are a (4-8) membered heterocycle, which heterocycle may be substituted with (l-6C)alkoxy, trifluoromethyl or amido and which heterocycle may be fused with another (5-6) membered heterocycle; n is 1,2 or 3; and the ring structure A represents a (4-6) membered heterocycle;

R²is (l-6C)alkyl, optionally substituted with (l-6C)alkoxy;

R³ is aryl, heterocycle, -X(CH₂)_m-aryl or -X-(CH₂)_m-heterocyclyl, wherein X is O, S or

NR⁸, R⁸ being H or (l-6C)alkyl, and m is 0, 1, 2 or 3; and R⁴ is H, (l-8C)Alkyl, (l-SC)alkoxy or (2-8C)alkenyl, the alkyl moiety of each optionally being substituted with hydroxy, halogen, cyano, nitro, oxo, amino, alkylamino, dialkylamino, amido, alkylamido, carboxy, (l-8C)alkoxy, (l-8C)alkylthio, perfluoro(l-

4C)alkyl, (3-8C)cycloalky, aryl, aryloxy, heterocyclyl or -O-heterocyclyl; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein

R³ is imidazolyl or imidazolyl substituted with (l-6C)alkyl, (substituted) phenyl, (substituted) benzofuranyl or (substituted) benzothiophenyl, wherein the optional substituents are selected from (l-6C)alkyl, halogen, trifluoromethyl and trifluoromethoxy, or R is imidazolyl substituted with [(l-6C)alkyl substituted] thiophenyl or [(l-όC)alkyl substituted] furanyl wherein the furanyl group optionally is substituted with halogen, or R³ is furanyl, thiophenyl, benzofuranyl, benzothiophenyl or benzimidazolyl, each optionally substituted with (l-6C)alkyl, halogen, trifluoromethyl or trifluoromethoxy, or R³ is phenyl, -O-(CH₂)_p-phenyl, -S-(CH₂)-phenyl, -NR⁹ -(CH₂)- phenyl, pyridinyl, -O-(CH₂)_p-pyridinyl, -S-(CH )_p-pyridinyl, -NR9-(CH₂)_p-pyridinyl wherein R⁹ is H or (l-6C)alkyl, p being 0, 1, 2 or 3, and the phenyl and pyridinyl moieties optionally being substituted with (l-6C)alkyl; and R⁴ is H, (l-8C)alkyl, (l-SC)alkoxy, trifluoromethyl, trifluoromethoxy, (l-8C)alkyl substituted with (l-8C)alkoxy, (3-8C)cycloalkyl, amido, -C(O)NHR¹⁰ wherein R¹⁰ is (1- 8C)alkyl, or trifluoromethyl, or R⁴ is (l-8C)alkoxy substituted with (3-8C)cycloakyl or trifluoromethyl or R⁴ is -(CH₂)-phenyl or -(CH₂)_q-phenoxy, q being 0, 1, 2 or 3 and the phenyl group optionally being substituted with (l-8C)alkyl, (l-8C)alkoxy, hydroxy, halogen, cyano, nitro or trifluoromethyl.

3. The compound of claim 2, wherein R³ is imidazolyl or imidazolyl substituted with (1- 6C)alkyl, (substituted) phenyl, (substituted) benzofuranyl or (substituted) benzothiophenyl, wherein the optional substituents are selected from halogen, trifluoromethyl and trifluoromethoxy, or R³ is benzimidazolyl or benzofuranyl, each of which optionally substituted with halogen, trifluoromethyl or trifluoromethoxy; and R⁴ is H, (l-SC)alkyl, (l-SC)alkoxy, trifluoromethyl, trifluoromethoxy, (l-8C)alkyl substituted with (3-8C)cycloalkyl or trifluoromethyl or (l-8C)alkoxy substituted with (3- 8C)cycloalkyl or trifluoromethyl.

4. The compound of claim 1 , wherein R¹ is -NH(CH₂)₂NR⁵R⁶.

5. The compound of claim 4, wherein R² is (1 -6C)alkyl; and R R⁴⁴ iiss HH,, ((ll--66CC))aallkkyyll oorr ((ll--66CC))aallkkooxxjy, wherein the alkyl moiety may be substituted with trifluoromethyl or (3-8C)cycloalkyl.

6. The compound of claim 4 or 5, wherein R³ is imidazolyl, benzimidazolyl, halogen substituted benzimidazolyl, phenyl substituted imidazolyl, phenyl substituted pyrazinyl, wherein the phenyl group is substituted with halogen, trifluoromethyl or trifluromethoxy.

The compound of any one of claims 1 - 6, wherein R is -CH₂CH(CH₃)₂.

8. A pharmaceutical composition comprising the compound of any one of claims 1-7 and pharmaceutically suitable auxiliaries.

9. The compound of any one of claims 1 -7 for use in therapy.

10. Use of the compound of any one of claims 1 -7 for the manufacture of a medicament for treating or preventing IL-8 receptor mediated disorders.