WO2003007939A1 - Azetidine derivatives and their use as ccr3 receptor antagonists - Google Patents

Abstract

The invention relates to compounds of Formula (I) in free or salt form, where Ar?1, Ar2, R1¿, X and n are as hereinbefore defined. Compositions containing them, methods for their preparation and their use as pharmaceuticals are also described.

Description

A_ZET_IDINE DERIVATIVES AND THEIR USE AS CCR3 RECEPTOR ANTAGONISTS

This invention relates to organic compounds, their preparation and their use as pharmaceuticals.

In one aspect the invention provides compounds of formula I

in free or salt form, where

Ar¹ is phenyl substituted by one or more halogen atoms;

Ar² is phenyl optionally substituted by one or more substitutuents selected from halogen, cyano, hydroxy, nitro, Q-Q-alkyl, Q-Q-haloalkyl, Q-Q-alkoxy, Q-Q-alkoxycarbonyl or di(Q-Q-alkyl)aminocarbonylrnethoχy;

R¹ is hydrogen or Q-Q-alkyl optionally substituted by hydroxy, Q-Q-alkoxy, acyloxy, halogen, carboxy, Q-Q-alkoxycarbonyl, -N(R²)R³, -CON(R⁴)R⁵ or by a monovalent cyclic organic group having 3 to 15 atoms in the ring system;

R² and R³ are each independently hydrogen or Ci-Cg-alkyl, or R² is hydrogen and R³ is hydroxy-Q-Q-alkyl, acvl, -S0₂R⁶ or -CON(R⁴)R⁵, or R² and R³ together with the nitrogen atom to which they are attached denote a 5 -or 6-membered heterocyclic group;

R⁴ and R⁵ are each independently hydrogen or Q-Q-alkyl, or R⁴ and R⁵ together with the nitrogen atom to which they are attached denote a 5- or 6-membered heterocyclic group;

R⁶ is rQ-alkyl, Q-Q-haloalkyl, or phenyl optionally substituted by Q-Q-alkyl;

X is -C(-sO)-, -O- or -CH₂-; and n is 1, 2, 3 or 4.

Terms used in the specification have the following meanings:

"Q-Q-alkyl" as used herein denotes straight chain or branched Q-Cg-alkyl, which may be, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, straight or branched pentyl, straight or branched hexyl, straight or branched heptyl, or straight or branched octyl. Preferably, Q-Q-alkyl is Q-Q-alkyl. "Q-Q-alkoxy" as used herein denotes straight chain or branched Q-Q-alkoxy which may be, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, straight or branched pentoxy, straight or branched hexyloxy, straight or branched heptyloxy, or straight or branched octyloxy. Preferably, Q-Q-alkoxy is Q-Q-alkoxy.

"Q-Q-haloalkyl" as used herein denotes C Cg-alkyl as hereinbefore defined substituted by one or more halogen atoms, preferably one, two or three halogen atoms.

"Acyl" as used herein denotes alkylcarbonyl, for example Q-Q-alkylcarbonyl where Q-Cg- alkyl may be one of the Q-Q-alkyl groups hereinbefore mentioned, optionally substituted by one or more halogen atoms; cycloalkylcarbonyl, for example Q-Q-cycloalkylcarbonyl where Q-Q-cycloalkyl may be, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl; 5- or 6- membered heterocyclylcarbonyl having one or two hetero atoms selected from nitrogen, oxygen and sulfur in the ring, such as furylcarbonyl or pyridylcarbonyl; arylcarbonyl, for example Q-Qo-arylcarbonyl such as benzoyl; or aralkylcarbonyl, for example Q to Qo-aryl-Q-Q-alkylcarbonyl such as benzylcarbonyl or phenylethylcarbonyl. Preferably acyl is Q-Q-alkylcarbonyl.

"Acyloxy" as used herein denotes alkylcarbonyloxy, for example Q-Q-alkylcarbonyloxy where Q-Cg-alkyl may be one of the Q-Q-alkyl groups hereinbefore mentioned, optionally substituted by one or more halogen atoms; cycloalkylcarbonyloxy, for example Q-Q- cycloalkylcarbonyloxy where Q-Q-cycloalkyl may be, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl; 5- or 6- membered heterocyclylcarbonyloxy having one or two hetero atoms selected from nitrogen, oxygen and sulfur in the ring, such as furylcarbonyloxy or pyridylcarbonyloxy; arylcarbonyloxy, for example Q-Qo- arylcarbonyloxy such as benzoyloxy; or aralkylcarbonyloxy, for example Q to Qo-aryl-Q-Q- alkylcarbonyloxy such as benzylcarbonyloxy or phenylethylcarbonyloxy. Preferably acyloxy is Q-Q-alkylcarbonyloxy.

"Halogen" as used herein may be fluorine, chlorine, bromine or iodine; preferably it is fluorine, chlorine or bromine.

"Q-Q-alkoxycarbonyl" as used herein denotes Q-Q-alkoxy as hereinbefore defined attached through the oxygen atom to a carbonyl group. "Di-(Q-Q-alkyl)aminocarbonylmethoxy" as used herein denotes aminocarbonylmethoxy disubstituted on the amino nitrogen atom by Q-Q-alkyl as hereinbefore defined, the two Q-Q-alkyl groups being the same or different.

In Ar¹, the phenyl group may be substituted by one, two or three, preferably one or two halogen atoms, preferably selected from fluorine and chlorine atoms. When there is one halogen substituent, it is preferably para to the indicated group X. When there are two or three halogen substituents, preferably one is para to the indicated group X and at least one of the others is ortho to the para-halogen substituent.

Ar² as substituted phenyl may, for example, be substituted by one, two, three, four or five, preferably by one, two or three, of the abovementioned substituents. Ar² may be, for example, monosubstituted phenyl in which the substituent, preferably halogen, cyano, nitro, Q-Q- alkoxy or di(CrQ-alkyl)aminocarbonylmethoxy, is preferably ortho or meta to the indicated - CH=CH- group. Ar² may alternatively be, for example, disubstituted phenyl in which the substituents are preferably selected from halogen, cyano, hydroxy, nitro, Q-Q-alkoxy, di(Q- C₄-alkyl)aminocarbonylmethoxy, Q-Q-alkyl and Q-Q-haloalkyl, especially two halogen substituents (same or different halogen), two Q-Q-alkoxy groups, two Q-Q-alkyl groups, two Q-Q-haloalkyl groups, one halogen and one cyano, one halogen and one Q-Q-alkoxy, one halogen and one nitro, one halogen and one hydroxy, one halogen and one Q-Q- haloalkyl, one cyano and one Q-Q-alkoxy, one hydroxy and one CrQ-alkyl, or one hydroxy and one Q-Q-alkoxy group. Ar² may alternatively be, for example, trisubstituted phenyl in which the substituents are preferably selected from halogen, hydroxy, Q-Q-alkoxy and Q-Q- alkoxycarbonyl, especially three halogen substituents (same or two or three different halogens), or two Q-Q-alkoxy and one halogen, hydroxy or Q-Q-alkoxycarbonyl. Ar² may alternatively be, for example, penta-substituted phenyl in which the substituents are preferably halogen, especially fluorine. Preferably Ar² is phenyl substituted by one or more substitutents selected from cyano, halogen, Q-Q-alkoxy or di(Q-C₄-alkyl)aminocarbonylmethoxy. Especially preferred groups Ar² are cyanophenyl, particularly meta-cyanophenyl, and disubstituted phenyl where one substituent is Q-Q-alkoxy, or di(CrQ-alkyl)aminocarbonyl- methoxy, preferably ortho to the -CH=CH- group, and the other, preferably para to the Q-Q- alkoxy group, is Q-Q-alkoxy, halogen, cyano or Q-Q-alkyl.

R¹ as optionally substituted Q-Q-alkyl is preferably optionally substituted Q-Q-alkyl, especially Q-Q-alkyl or substituted methyl or ethyl. When R¹ is substituted by a cyclic organic group, the latter may be a carbocycUc or heterocyclic group, for example a Q-Q₅-carbocyclic group or a 5- to 7-membered heterocyclic group having one or more, preferably one, two or three, ring hetero atoms selected from nitrogen, oxygen and sulfur. The Q-Q₅-carbocyclic group may be, for example, a cycloaliphatic group having 3 to 8 carbon atoms, preferably Q - or Q - cycloalkyl such as cyclopentyl, methylcyclopentyl or cyclohexyl. The Q-Q₅-carbocyclic group may alternatively be, for example, a Q-Q5 aromatic group, such as phenyl, which is unsubstituted or substituted by CrCg-alkyl, Q-Q-alkoxy, halogen, cyano, -CON(R⁴)R⁵, - S0₂N(R⁴)R⁵ or CrQ-alkylsulfonylamino where R⁴ and R⁵ are as hereinbefore defined. The heterocyclic group may have one nitrogen, oxygen or sulfur atom in the ring or it may have two nitrogens, or one oxygen and one or two nitrogens, or one sulfur and one or two nitrogens in the ring. The heterocyclic group is preferably a heterocyclic aromatic group, especially a 5- or 6- membered heterocyclic group such as furyl, imidazolyl, thiazolyl or pyridyl. Preferred embodiments include those in which R¹ is hydrogen or Q-Q-alkyl substituted by hydroxy or Q-Q-alkoxy.

Preferred compounds of formula I in free or salt form include those in which Ar¹ is phenyl substituted by one or two substituents selected from fluorine and chlorine; Ar² is phenyl substituted by at least two substituents selected from halogen, cyano, Q-Q- alkoxy, and di(CrQ-alkyl)aminocarbonylmethoxy; R¹ is hydrogen or Q-Q-alkyl substituted by hydroxy or Q-Q-alkoxy; X is -C(=0)- or -O-; and n is 1 or 2.

Further preferred compounds of formula I in free or salt form include those in which

Ar¹ is phenyl substituted by fluorine or chlorine para to the indicated group X, and optionally further substituted by fluorine or chlorine meta to the group X;

Ar² is phenyl substituted ortho to the indicated -CH=CH- group by Q-Q-alkoxy or di(Q-Q- alkyl)aminocarbonylmethoxy and para to the Q-Q-alkoxy or di(Cι-C₄-alkyl)aminocarbonyl- methoxy group by cyano, halogen or Q-Q-alkoxy;

R¹ is hydrogen or Q-Q-alkyl substituted by hydroxy;

X is -C(=0) - or -O-; and n is 2.

The compounds represented by formula I are capable of forming acid addition salts, particularly pharmaceutically acceptable acid addition salts. Pharmaceutically acceptable acid addition salts of the compound of formula I include those of inorganic acids, for example, hydrohaUc acids such as hydrofluoric acid, hydrochloric acid, hydrobromic acid or hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid; and organic acids, for example aliphatic monocarboxyUc acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid and butyric acid, aliphatic hydroxy acids such as lactic acid, citric acid, tartaric acid or malic acid, dicarboxyUc acids such as maleic acid or succinic acid, aromatic carboxyUc acids such as benzoic acid, p-chlorobenzoic acid, diphenylacetic acid or triphenylacetic acid, aromatic hydroxy acids such as o-hydroxybenzoic acid, p-hydroxybenzoic acid, 1-hydroxynaphthalene- 2-carboxylic acid or 3-hydroxynaphthalene-2-carboxyUc acid, and sulfonic acids such as methanesulfonic acid or benzenesulfonic acid. These salts may be prepared from compounds of formula I by known salt-forming procedures.

Compounds of formula I which contain acidic, e.g. carboxyl, groups, are also capable of forming salts with bases, in particular pharmaceutically acceptable bases such as those well known in the art; suitable such salts include metal salts, particularly alkali metal or alkaline earth metal salts such as sodium, potassium, magnesium or calcium salts, or salts with ammonia or pharmaceutically acceptable organic amines or heterocyclic bases such as ethanolamines, benzylamines or pyridine. These salts may be prepared from compounds of formula I by known salt-forming procedures.

When R¹ is other than hydrogen, the carbon atom to which R¹ is attached in formula I is asymmetric, in which case the compounds exist in individual optically active isomeric forms or as mixtures thereof, e.g. as racemic or diastereomeric mixtures. The invention embraces both individual optically active R and S isomers as well as mixtures, e.g.racemic or diastereomeric mixtures, thereof.

Specific preferred compounds of the invention are those described hereinafter in the Examples.

The invention also provides a process for the preparation of compounds of formula I which comprises

(i) reacting a compound of formula

with a compound of formula

or an amide forming derivative thereof, where Ar¹, Ar², R¹, X and n are as hereinbefore defined, and

(ii) recovering the product in free or salt form.

Process step (i) may be effected using known methods, for example by reacting a compound of formula II with an acid halide, particularly acid chloride, of the acid of formula HI using known amide-forming procedures. Conveniently, the compound of formula II is reacted with a free carboxyUc acid of formula HI, for example using known procedures, such as reaction in the presence of a tertiary amine and a peptide coupUng agent such as a phosphonium salt, 2- (1H benzotriazol-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate or diisopropylcarbodiimide; this reaction may be carried out in an inert organic solvent, for example a halohydrocarbon such as dichloromethane; the reaction temperature is conveniently from 0 to 40°C, preferably ambient temperature.

Compounds of formula IE may be prepared by reacting a compound of formula

Ar¹ — X — ^ NH IV

with a compound of formula

where Ar¹, R¹, Y and n are as hereinbefore defined, with the proviso that when R¹ contains a reactive functional group such as a hydroxy group, the reactive group may be in protected form, for example a hydroxy group protected as a tert-butoxy group, R⁷ is hydrogen or an amine-protective group, for example a tert-butoxycarbonyl group, and Y is halogen and, where R⁷ is a protective group, replacing R⁷ in the product by hydrogen, and, where R¹ in the product contains a protected functional group, replacing the protecting group by hydrogen. When R⁷ is hydrogen, reaction between a compound of formula V and a salt of a compound of formula VI may be effected by the procedures described in US 4559349. When R⁷ is a protective group, reaction between compounds of formulae V and VI may be effected using known methods, for example in the presence of a tertiary organic base such as triethylamine or 1,8-diazabicyclo- [5.4.0]undec-7-ene (DBU), conveniently in an inert organic solvent, for example a polar solvent such as dimethylformamide, the reaction temperature suitably being from 0 to 40°C, preferably ambient temperature. Replacement of a protective group R⁷ by hydrogen may be effected using known procedures; for example, where R⁷ is tert-butoxycarbonyl, by treatment with a carboxylic acid such as trifluoroacetic acid. Replacement of a protecting group in R¹ may be affected using known procedures, for example, when R¹ contains a hydroxy group protected as an ether group, such as tert-butoxy, by treatment with HBr in a carboxylic acid such as acetic acid; when R⁷ is a protective group, this treatment also replaces R⁷ by hydrogen.

Where reference is made herein to protected functional groups or to protecting groups, the protecting groups may be chosen in accordance with the nature of the functional group, for example as described in Protective Groups in Organic Synthesis, T.W. Greene and P.G.M. Wuts, John Wiley & Sons Inc, Second Edition, 1991, which reference also describes procedures suitable for replacement of the protecting groups by hydrogen.

Compounds of formula IV where X is -O- may be prepared by reacting a compound of formula

CH₃SO₂O — < N— R⁹ VI

with a compound of formula A^-OH in the presence of sodium hydride, where Ar¹ is as hereinbefore defined and R⁹ is a protecting group, and replacing R⁹ in the product by hydrogen. The reaction may be carried out in an inert organic solvent such as DMF. Suitable reaction temperatures may be from 20°C to 150°C, conveniently from 50 to 70°C. The replacement of R⁹ by hydrogen may be affected using known procedures, for example where R⁹ is benzhydryl by reacting the product of the reaction of the compound of formula VI and Ar¹ -OH with 1-chloroethyl chloroformate, a suitable reaction tempterature being 10-30°C, conveniently at room temperature.

Compounds of formula V and VI are known or may be prepared using known procedures.

Compounds of formula TV where X is -C(=0)- may be prepared by reacting a compound of formula

with a compound of formula

Ar¹ — MgBr VIII

where Ar¹ and R⁹ are as hereinbefore defined, and replacing R⁹ in the product by hydrogen. Reaction of compounds of formulae Nπ amd VHI may be affected in an inert organic solvent, e.g. an ether such as THF and/or diethyl ether; suitable reaction temperatures may be from -10°C to 10°C, conveniently from -5 to 5°Q Replacement of R⁹ in the product by hydrogen may be effected as hereinbefore described. Compounds of formula VII may be prepared by reacting a compound of formula

with O, N-dimethylhydroxylamine hydrochloride in the presence of a peptide coupling agent such as di-imidazol-1-yl-methanone, conveniently in an inert organic solvent such as THF, suitably at reflux temperature.

Compounds of formulae VHI and IX are known or may be prepared using known procedures.

Compounds of formula IV where X is -CH₂- may be prepared by reduction of compounds of formula IV where X is -C(=0) -, for example using known reduction procedures.

Compounds of formula I in free form may be converted into salt form, and vice versa, in a conventional manner. The compounds in free or salt form can be obtained in the form of hydrates or solvates containing a solvent used for crystalUzation.Compounds of formula I can be recovered from reaction mixtures and purified in a conventional manner. Isomers, such as enantiomers, may be obtained in a conventional manner, e.g. by fractional crystallization or asymmetric synthesis from correspondingly asymmetrically substituted, e.g. optically active, starting materials. Compounds of formula I in free or pharmaceutically acceptable salt form, hereinafter referred to alternatively as agents of the invention, are useful as pharmaceuticals. Accordingly the invention also provides a compound of formula I in free or pharmaceutically acceptable salt form for use as a pharmaceutical. The agents of the invention act as CCR-3 receptor antagonists, thereby inhibiting the infiltration and activation of inflammatory cells, particularly eosinophils, and inhibiting allergic response. The inhibitory properties of agents of the invention can be demonstrated in the following assay:

CCR-3 Binding Assay

In this assay the effect of agents of the invention on the binding of human eotaxin to human CCR-3 is determined. Recombinant cells expressing human CCR-3 are captured by wheatgerm agglutinin (WGA) polyvinyltoluidene (PVT) SPA beads (available from Amersham), through a specific interaction between the WGA and carbohydrate residues of glycoproteins on the surface of the cells. [¹²⁵I]-human eotaxin (available from Amersham) binds specifically to CCR- 3 receptors bringing the [¹²⁵I]-human eotaxin in close proximity to the SPA beads. Emitted a- particles from the [¹²⁵IJ-human eotaxin excite, by its proximity, the fluorophore in the beads and produce light. Free [¹²⁵I]-human eotaxin in solution is not in close proximity to the scintillant and hence does not produce light. The scintillation count is therefore a measure of the extent to which the test compound inhibits binding of the eotaxin to the CCR-3.

Preparation of Assay Buffer: 5.96 g HEPES and 7.0 g sodium chloride are dissolved in distilled water and 1M aqueous CaCl₂ (1 mL) and 1M aqueous MgCl₂ (5 mL) are added. The pH is adjusted to 7.6 with NaOH and the solution made to a final volume of 1 L using distilled water. 5 g bovine serum albumin and 0.1 g sodium azide are then dissolved in the solution and the resulting buffer stored at 4°C. A Complete™ protease inhibitor cocktail tablet (available from Boehringer) is added per 50 mL of the buffer on the day of use.

Preparation of Homogenisation Buffer: Tris-base (2.42g) is dissolved in distilled water, the pH of the solution is adjusted to 7.6 with hydrochloric acid and the solution is diluted with distilled water to a final volume of IL. The resulting buffer is stored at 4°C. A Complete™ protease inhibitor cocktail tablet is added per 50 mL of the buffer on the day of use.

Preparation of membranes: Confluent rat basophil leukemia (RBL-2H3) cells stably expressing CCR3 are removed from tissue culture flasks using enzyme-free cell dissociation buffer and resuspended in phosphate-buffered saline. The cells are centrifuged (800 g, 5 minutes), the pellet resuspended in ice-cold homogenisation buffer using 1 mL homogenisation buffer per gram of cells and incubated on ice for 30 minutes.The cells are homogenised on ice with 10 strokes in a glass mortar and pestle.The homogenate is centrifuged (800 g, 5 minutes, 4°C), the supernatant further centrifuged (48,000 g, 30 minutes, 4°C) and the pellet redissolved in Homogenisation Buffer containing 10% (v/v) glycerol.The protein content of the membrane preparation is estimated by the method of Bradford (AnaLBiochem. (1976) 72:248) and aliquots are snap frozen and stored at -80°C.

The assay is performed in a final volume of 250 μL per well of an Optiplate (ex Canberra Packard). To selected wells of the Optiplate are added 50 μL of solutions of a test compound in Assay Buffer containing 5 % DMSO (concentrations from O.OlnM to 10 uM). To determine total binding, 50 μL of the Assay Buffer containing 5 % DMSO is added to other selected wells. To determine non-specific binding, 50 μL of lOOnM human eotaxin (ex R&D Systems) in Assay Buffer containing 5 % DMSO is added to further selected wells. To all wells are added 50 μL [¹²⁵I]-Human eotaxin (ex Amersham) in Assay Buffer containing 5 % DMSO at a concentration of 250 pM (to give a final concentration of 50 pM per well), 50 μL of WGA- PVT SPA beads in Assay Buffer (to give a final concentration of l.Omg beads per well) and 100 μL of the membrane preparation at a concentration of 100 μg protein in Assay Buffer (to give a final concentration of 10 μg protein per well). The plate is then incubated for 4 hours at room temperature. The plate is sealed using TopSeal-S (ex Canberra Packard) according to the manufacturer's instructions. The resulting scintillations are counted using a Canberra Packard TopCount, each well being counted for 1 minute. The concentration of test compound at which 50% inhibition occurs (IQo) is determined from concentration-inhibition curves in a conventional manner.

The compounds of the Examples hereinbelow have IQ₀ values below lμM in the above assay. For instance, the compounds of Examples 11, 16, 17 and 18 have IQ₀ values of 32nM, 7nM, InM, and 9nM respectively.

Most of the compounds of the Examples exhibit selectivity for inhibition of CCR-3 binding relative to inhibition of binding of the alpha-1 adrenergic receptor. The inhibitory properties of agents of the invention on binding of the alpha-1 adrenergic receptor can be determined in the following assay: Cerebral cortices from male Sprague-Dawley rats (175-200 g) are dissected and homogenised in 10 volumes of ice cold 0.32 M sucrose (containing ImM MgCl₂ dihydrate and ImM K₂HP0₄) with a glass/teflon homogeniser. The membranes are centrifuged at 1000 x g for 15 min., the pellet discarded and the centrifugation repeated. The supernatants are pooled and centrifuged at 18,000 x g for 15 min. The pellet is osmotically shocked in 10 volumes of water and kept on ice for 30 min. The suspension is centrifuged at 39,000 x g for 20 min., resuspended in Krebs-Henseleit buffer pH 7.4 (1.17mM MgS0₄ anhydrous, 4.69 mM KCl, 0.7mMK₂HPO₄ anhydrous, O.llM NaCl, 11 mM D-glucose and 25 mM NaHC0₃) containing 20mM Tris, and kept for 2 days at -20°C. The membranes are then thawed at 20- 23°C, washed three times with Krebs-Henseleit buffer by centrifugation at 18,000 x g for 15 min., left overnight at 4°C and washed again three times. The final pellet is resuspended with a glass/teflon homogeniser in 125mL/100 membranes in the same buffer. A sample is taken to determine the protein concentration (using the Bradford Assay with gamma globulin as the standard) and the remainder aliquoted and stored at -80°C.

The resulting membranes are subjected to a radioligand binding assay. The assay is conducted in triplicate using 96 well plates containing [¹²⁵Ij-HEAT (Amersham) (40pM, K_d: 58.9 ± 18.7 pM), unlabelled test compound and membrane (57.1μg mL) to yield a final volume of 250μL (assay buffer containing 50 mM Tris-base and 0.9% (w/v) NaCl, pH 7.4). The plates are incubated at 37°C for 60 min., after which rapid vacuum filtration over Whatman GF/C 96 well filter plates is carried out. Each plate is then washed three times with 10ml of ice cold assay buffer using a Brandel Cell harvester (Gaithersburg, MD). Following drying of the plates for 3 h. at 50°C, 40 μL of Microscint 20 is added to each well, the plates incubated at room temperature for a further 20 min. and the retained radioactivity quantified in a Packard Topcount NXT scintillation counter.

Stock solutions of test compounds are dissolved initially in 100 % DMSO and diluted with assay buffer to the required concentrations to yield 1 % (v/v) DMSO.

The concentration of test compound at which 50% inhibition occurs (IQo) is determined from concentration-inhibition curves in a conventional manner.

Having regard to their inhibition of binding of CCR-3, agents of the invention are useful in the treatment of conditions mediated by CCR-3, particularly inflammatory or allergic conditions. Treatment in accordance with the invention may be symptomatic or prophylactic. Accordingly, agents of the invention are useful in the treatment of inflammatory or obstructive airways diseases, resulting, for example, in reduction of tissue damage, bronchial hyperreactivity, remodelUng or disease progression. Inflammatory or obstructive airways diseases to which the present invention is appUcable include asthma of whatever type or genesis including both intrinsic (non-allergic) asthma and extrinsic (allergic) asthma, mild asthma, moderate asthma, severe asthma, bronchitic asthma, excercise-induced asthma, occupational asthma and asthma induced following bacterial or viral infection. Treatment of asthma is also to be understood as embracing treatment of subjects, e.g. of less than 4 or 5 years of age, exhibiting wheezing symptoms and diagnosed or diagnosable as "wheezy infants", an established patient category of major medical concern and now often identified as incipient or early-phase asthmatics. (For convenience this particular asthmatic condition is referred to as "wheezy-infant syndrome".)

Prophylactic efficacy in the treatment of asthma will be evidenced by reduced frequency or severity of symptomatic attack, e.g. of acute asthmatic or bronchoconstrictor attack, improvement in lung function or improved airways hyperreactivity. It may further be evidenced by reduced requirement for other, symptomatic therapy, i.e. therapy for or intended to restrict or abort symptomatic attack when it occurs, for example anti-inflammatory (e.g. corticosteroid) or bronchodilatory. Prophylactic benefit in asthma may in particular be apparent in subjects prone to "morning dipping". "Morning dipping" is a recognised asthmatic syndrome, common to a substantial percentage of asthmatics and characterised by asthma attack, e.g. between the hours of about 4 to 6 am, i.e. at a time normally substantially distant form any previously administered symptomatic asthma therapy.

Other inflammatory or obstructive airways diseases and conditions to which the present invention is appUcable include acute lung injury (ALI), acute/adult respiratory distress syndrome (ARDS), chronic obstructive pulmonary, airways or lung disease (COPD, COAD or COLD), including chronic bronchitis or dyspnea associated therewith, emphysema, as well as exacerbation of airways hyperreactivity consequent to other drug therapy, in particular other inhaled drug therapy. The invention is also applicable to the treatment of bronchitis of whatever type or genesis including, e.g., acute, arachidic, catarrhal, croupus, chronic or phthinoid bronchitis. Further inflammatory or obstructive airways diseases to which the present invention is applicable include pneumoconiosis (an inflammatory, commonly occupational, disease of the lungs, frequently accompanied by airways obstruction, whether chronic or acute, and occasioned by repeated inhalation of dusts) of whatever type or genesis, including, for example, aluminosis, anthracosis, asbestosis, chaUcosis, ptilosis, siderosis, silicosis, tabacosis and byssinosis.

Having regard to their anti-inflammatory activity, in particular in relation to inhibition of eosinophil activation, agents of the invention are also useful in the treatment of eosinophil related disorders, e.g. eosinophiUa, in particular eosinophil related disorders of the airways (e.g. involving morbid eosinophiUc infiltration of pulmonary tissues) including hyper- eosinophiUa as it effects the airways and/or lungs as well as, for example, eosinophil-related disorders of the airways consequential or concomitant to Lόffler's syndrome, eosinophiUc pneumonia, parasitic (in particular metazoan) infestation (including tropical eosinophiUa), bronchopulmonary aspergillosis, polyarteritis nodosa (including Churg-Strauss syndrome), eosinophiUc granuloma and eosinophil-related disorders affecting the airways occasioned by drug-reaction.

Agents of the invention are also useful in the treatment of inflammatory or allergic conditions of the skin, for example psoriasis, contact dermatitis, atopic dermatitis, alopecia areata, erythema multiforma, dermatitis herpetiformis, scleroderma, vitiligo, hypersensitivity angiitis, urticaria, bullous pemphigoid, lupus erythematosus, pemphisus, epidermolysis bullosa acquisita, and other inflammatory or allergic conditions of the skin.

Agents of the invention may also be used for the treatment of other diseases or conditions, in particular diseases or conditions having an inflammatory component, for example, treatment of diseases and conditions of the eye such as conjunctivitis, keratoconjunctivitis sicca, and vernal conjunctivitis, diseases affecting the nose including allergic rhinitis, e.g. atrophic, chronic, or seasonal rhinitis, inflammatory conditions of the gastrointestinal tract, for example inflammatory bowel disease such as ulcerative coUtis and Crohn's disease, diseases of the bone and joints including rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis and systemic sclerosis, and other diseases such as athersclerosis, multiple sclerosis, diabetes (type I), myasthenia gravis, hyper IgE syndrome and acute and chronic allograft rejection, e.g. following transplantation of heart, kidney, liver, lung or bone marrow.

The effectiveness of an agent of the invention in inhibiting inflammatory conditions, for example in inflammatory airways diseases, may be demonstrated in an animal model, e.g. a mouse or rat model, of airways inflammation or other inflammatory conditions, for example as described by Szarka et al, J. Immunol. Methods (1997) 202:49-57; Renzi et al, Am. Rev. Respir. Dis. (1993) 148:932-939; Tsuyuki et al., J. CUn. Invest. (1995) 96:2924-2931; and Cernadas et al (1999) Am. J. Respir. Cell Mol. Biol. 20:1-8.

The agents of the invention are also useful as co-therapeutic agents for use in combination with other drug substances such as anti-inflammatory, bronchodilatory or antihistamine drug substances, particularly in the treatment of obstructive or inflammatory airways diseases such as those mentioned hereinbefore, for example as potentiators of therapeutic activity of such drugs or as a means of reducing required dosaging or potential side effects of such drugs. An agent of the invention may be mixed with the other drug substance in a fixed pharmaceutical composition or it may be administered separately, before, simultaneously with or after the other drug substance. Such anti-inflammatory drugs include steroids, in particular glucocorticosteroids such as budesonide, beclamethasone, fluticasone, ciclesonide or mometasone, LTB4 antagonists such as those described in US 5451700, LTD4 antagonists such as montelukast and zafirlukast, and PDE4 inhibitors such as Ariflo^® (GlaxoSmith Khne), Roflumilast (Byk Gulden),V-11294A (Napp), BAY19-8004 (Bayer), SCH-351591 (Schering- Plough), Arofylline (Almirall Prodesfarma) and PD189659 (Parke-Davis). Such bronchodilatory drugs include anticholinergic or antimuscarinic agents, in particular ipratropium bromide, oxitropium bromide and tiotropium bromide, and beta-2 adrenoceptor agonists such as salbutamol, terbutaline, salmeterol and, especially, formoterol and pharmaceutically acceptable salts thereof, and compounds (in free or salt or solvate form) of formula I of PCT International Publication No. WO00/75114, which document is incorporated herein by reference, preferably compounds of the Examples thereof, especially a compound of formula

and pharmaceutically acceptable salts thereof. Co-therapeutic antihistamine drug substances include cetirizine hydrochloride, acetaminophen, clemastine fumarate, promethazine, loratidine, desloratidine, diphenhydramine and fexofenadine hydrochloride. Combinations of agents of the invention and steroids, beta-2 agonists, PDE4 inhibitors or LTD4 antagonists may be used, for example, in the treatment of COPD or, particularly, asthma. Combinations of agents of the invention and antichoUnergic or antimuscarinic agents, PDE4 inhibitors, dopamine receptor agonists or LTB4 antagonists may be used, for example, in the treatment of asthma or, particularly, COPD. Other useful combinations of agents of the invention with anti-inflammatory drugs are those with other anatagonists of chemokine receptors, e.g. CCR-1, CCR-2, CCR-3, CCR-4, CCR-5, CCR-6, CCR-7, CCR-8, CCR-9 and CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, particularly CCR-5 antagonists such as Schering-Plough antagonists SC-351125, SCH-55700 and SCH-D, Takeda antagonists such as N-[[4-[[[6,7-dihydro-2-(4-methylphenyl)-5H- benzocyclohepten-8-yl]carbonyl]amino]phenyl]-methyl]tetrahydro-N,N-dimethyl-2H-pyran-4- aminium chloride (TAK-770), CCR-5 antagonists described in US6166037 (particularly claims 18 and 19), WO00/66558 (particularly claim 8), and WO00/66559 (particularly claim 9).

In accordance with the foregoing, the invention also provides a method for the treatment of a condition mediated by CCR-3, for example an inflammatory or allergic condition, particularly an inflammatory or obstructive airways disease, which comprises administering to a subject, particularly a human subject, in need thereof an effective amount of a compound of formula I in a free or pharmaceutically acceptable salt form as hereinbefore described. In another aspect the invention provides the use of a compound of formula I, in free or pharmaceutically acceptable salt form, as hereinbefore described for the manufacture of a medicament for the treatment of a condition mediated by CCR-3, for example an inflammatory or allergic condition, particularly an inflammatory or obstructive airways disease.

The agents of the invention may be administered by any appropriate route, e.g. orally, for example in the form of a tablet or capsule; parenterally, for example intravenously; by inhalation, for example in the treatment of inflammatory or obstructive airways disease; intranasally, for example in the treatment of allergic rhinitis; topicaUy to the skin, for example in the treatment of atopic dermatitis; or rectally, for example in the treatment of inflammatory bowel disease.

In a further aspect, the invention also provides a pharmaceutical composition comprising as active ingredient a compound of formula I in free or pharmaceutically acceptable salt form, optionally together with a pharmaceutically acceptable diluent or carrier therefor. The composition may contain a co-therapeutic agent such as an anti-inflammatory bronchodilatory or antihistamine drug as hereinbefore described. Such compositions may be prepared using conventional diluents or excipients and techniques known in the galenic art. Thus oral dosage forms may include tablets and capsules. Formulations for topical administration may take the form of creams, ointments, gels or transdermal deUvery systems, e.g. patches. Compositions for inhalation may comprise aerosol (preferably with a HFA propellant such as HFA134a or HFA227) or other atomizable or dry powder formulations.

The invention includes (A) an agent of the invention in inhalable form, e.g. in an aerosol or other atomisable composition or in inhalable particulate, e.g. micronised form, (B) an inhalable medicament comprising an agent of the invention in inhalable form; (C) a pharmaceutical product comprising such an agent of the invention in inhalable form in association with an inhalation device; and (D) an inhalation device containing an agent of the invention in inhalable form.

Dosages of agents of the invention employed in practising the present invention will of course vary depending, for example, on the particular condition to be treated, the effect desired and the mode of administration. In general, suitable daily dosages for administration by inhalation are of the order of 0.01 to 30 mg/kg while for oral administration suitable daily doses are of the order of 0.01 to 100 mg/kg.

The invention is illustrated by the following Examples.

Examples 1-18

Compounds of formula I which are also of formula

are shown in the following table, the methods of preparation being described hereinafter. The value of n is 2 in all of the Examples except Examples 1, 5 and 6 where it is 1. The table also shows characterising mass spectometry data. The compounds are in free form, except Example 5, which is in the form of the trifluoroacetate salt.

(E)-3-(5-Cyano-2-methoxy-phenyl)-N-{3-[3-(4-fluoro-phenoxy)-azetidin-l-yl]-ρroρyl}- acrylamide - Example 13

Methanesulfonic acid l-benzhydryl-azetidin-3-yl ester

Anderson and Lok, J.Org.Chem., 37, 3953, 1972.

A cooled (-10°C) solution of l-benzhydryl-azetidin-3-ol (2.87g, 12.0mmol) in pyridine (30ml) is treated dropwise with methanesulfonyl chloride (1.38ml, 18mmol) and the reaction mixture stirred at -10°C for 1.5hr and then at 0-5°C overnight. The mixture is treated with iced water and then resulting precipitate filtered off and dried in vacuo to afford methanesulfonic acid 1- benzhydryl-azetidin-3-yl ester as pale yellow crystals [MH]⁺ 318.2.

l-Benzhydryl-3-(4-fluoro-phenoxy)-azetidine

A suspension of sodium hydride (60% dispersion, 0.228g, 5.7mmol) in dry DMF (20ml) is treated with 4-fluorophenol (0.70g, 6.29mmol). Effervescence is allowed to subside and the resultant mixture is treated with methanesulfonic acid l-benzhydryl-azetidin-3-yl ester (1.81g, 5.71mmol) and the reaction heated to 60°C for 2.5 days. The mixture is cooled and poured into iced water and extracted with diethylether. The organic phase is washed with brine, dried with magnesium sulfate and evaporated. The crude product is purified by flash siUca chromatography (ethyl acetate:hexane, 25:75 elution) to afford l-benzhydryl-3-(4-fluoro- phenoxy)-azetidine as a pale yellow crystalline solid ϊϊ NMR (400MHz, DMSO) δ 2.99 (m 2H), 3.60 (m 2H), 4.5 (s 1H), 4.8 (pj 5.6 1H), 6.83 (m 2H), 7.06 (t J 8.8 2H), 7.18 (t J 7.5 2H), 7.28 (t J 7.5 4H), 7.44 (d J 7.0 4H ).

3-(4-Fluoro-phenoxy)-azetidine hydrochloride

A solution of l-benzhydryl-3-(4-fluoro-phenoxy)-azetidine (0.15g, 0.45 mmol) in dichloromethane (5ml) is cooled (-7°C to 0°C) and treated with 1-chloroethyl chloroformate (0.063ml, 0.58mmol) added dropwise. The reaction mixture is warmed to room temperature, stirred for 2.5 hours and evaporated invacuo. The crude residue is dissolved in methanol (10ml), heated to reflux for 18 hours and evaporated. Diethylether is added and the resultant precipitate filtered off and dried invacuo to afford 3-(4-fluoro-phenoxy)-azetidine hydrochloride as an off-white crystalline solid [MH]⁺ 167.1.

3-[3-(4-Fluoro-phenoxy)-azetidin-l-yl]-propy nrine hydrobromide

A mixture of 3-(4-fluoro-phenoxy)-azetidine hydrochloride (O.lg, 0.6mmol), 3-bromopropylamine hydrobromide (0.157g, 0.71mmol), diisopropylethylamine (0.52ml, 3.03mmol) in acetonitrile (4ml) is stirred at room temperature overnight and then heated (50°C) for 1.5 hours. The solvents are removed in vacuo to afford crude 3-[3-(4-fluoro- phenoxy)-azetidin-l-yl]-propylamine hydrobromide [MH]⁺ 225.1.

(E)-3-(5-Cyano-2-methoxy-phenyl)-N-{3-[3-(4-fluoro-phenoxy)-azetidin-l-yl]-proρyl}- acrylamtde

(E)-3-(5-cyano-2-methoxy-phenyl)-acrylic acid (0.075g, 0.36mmol) is dissolved in dry DMF (5ml) and treated with 2-(lH-benzotriazol-l-yl)-l,l,3,3,-tetramethyluronium tetrafluoroborate (TBTU) (0.0987g, 0.307mmol) followed by dusopropylethylamine (0.15ml, 0.86mmol). The resultant solution is added to 3-[3-(4-fluoro-phenoxy)-azetidin-l-yl]-propylamine (0.3mmol) and stirred at room temperature for 2 hours. The reaction mixture is evaporated in vacuo and then dissolved in ethyl acetate which is washed with saturated aqueous Na₂C0₃, dried with MgS0₄ and evaporated. The crude residue is purified by preparative HPLC to afford (E)-3-(5- cyano-2-methoxy-phenyl)-N-{3-[3-(4-fluoro-phenoxy)-azetidin-l-yl]-propyl}-acrylamide [MH]⁺ 410.12.

Examples 2, 9, 10 and 12 are prepared analogously to Example 13.

(E)-N-{(S)-3-[3-(4-chloro-ρhenoxy)-azetidin-l-yl]-l-hydroxymethyl-ρropyl}-3-(5-cyano-2- methoxy-phenyl)-acrylamide - Example 1

((S)-l-tert-butoxymethyl-3-hydroxy-propyl)-carbamic acid benzyl ester

A cooled solution (0°C) of (S)-3-benzyloxycarbonylamino-4-tert-butoxy-butyric acid ethyl ester (11.2g, 33mmol), prepared according to the method of Podlech and Seebach, Liebigs Ann., 1995, 1712 for the methyl ester, in anhydrous diethylether (150ml) is treated with a solution of lithium borohydride in THF (2.0M, 33.1ml, 66.2mmol). The reaction mixture is allowed to warm to room temperature and stirred overnight. The reaction is diluted with water, acidified with aqueous citric acid (0.5M) and extracted with diethylether (3x). The combined ether extracts are dried (MgS0₄) and evaporated to afford the crude product as an oil which is purified by flash silica chromatography (ethyl acetate:hexane, 1:1 elution) to afford ((S)-l-tert- butoxymethyl-3-hydroxy-propyl)-carbamic acid benzyl ester as a colourless oil [MH]⁺ 296.1.

((S)-l-tert-Butoxymethyl-3-iodo-propyl)-carbamic acid benzyl ester

A suspension of polymer-bound triphenylphosphine (NovaBiochem)(3.68g, 4.4mmol) in anhydrous DCM (25ml) is treated with iodine (0.75g, 3mmol) and stirred for 15 minutes at room temperature. Imidazole (0.238g, 3.5 mmol) is added and the mixture stirred for 15 minutes and treated with ((S)-l-tert-butoxymethyl-3-hydroxy-propyl)-carbamic acid benzyl ester (0.40g, 1.38mmol) inDCM (3ml). The reaction mixture is heated to reflux for 2.5 hours, cooled to room temperature and filtered through Celite. The filtrate is washed with sodium thiosulfate (5%, 100ml), water (2x), dried (MgS04) and evaporated. The crude product is purified by flash silica chromatography (ethyl acetate:hexane, 7:3 elution) to afford ((S)-l-tert- butoxymethyl-3-iodo-propyl)-carbamic acid benzyl ester as an oil [M-tBu]⁺ 349.9.

{(S)-l-tert-Butoxymethyl-3-[3-(4-chloro-phenoxy)-azetidm-l-yl]-ρropyl}-carbamic acid benzyl ester

3-(4-chloro-phenoxy)-azetidine hydrochloride (0.068g, 0.37mmol), prepared analogously to 3- (4-fluorophenoxy)-azetidine hydrochloride, is dissolved in saturated aqueous sodium bicarbonate (10ml) and the solution extracted with dichloromethane (2x10ml). The combined organic phase is dried (MgS0₄) and evaporated and re-dissolved in DMF (3ml). The solution is treated with triethylamine (0.063ml, 0.45mmol) and ((S)-l-tert-butoxymethyl-3-iodo- propyl)-carbamic acid benzyl ester (0.164g, 0.40mmol) and stirred for 2 days at room temperature. The mixture is diluted with aqueous NaHC0₃ and ethyl acetate (10ml) and extracted with more ethyl acetate (2x10ml) which is dried (MgS0₄) and evaporated to afford the crude product as an oil. Purification by flash silica chromatography (ethyl acetate elution) affords {(S)-l-tert-butoxymethyl-3-[3-(4-chloro-phenoxy)-azetidin-l-yl]-propyl}-carbamic acid benzyl ester as a pale yellow oil [MH]⁺ 461.1.

(S)-2-Amino-4-[3-(4-chloro-phenoxy)-azetid-n-l-yl]-butan-l-olhydrobromide

{(S)-l-tert-butoxymethyl-3-[3-(4-chloro-phenoxy)-azetidin-l-yl]-propyl}-carbamic acid benzyl ester (0.062g, 0.134mmol) is treated with a solution of hydrogen bromide (45%) in acetic acid (2ml) followed by THF (1ml) and stirred at room temperature for 1 hour. The reaction mixture is evaporated and the residue co-evaporated from ethanol (x3). The crude residue is treated with diethylether and the resultant solid filtered off and then re-dissolved and co- evaporated from ethanol (x4). Drying in vacuo affords (S)-2-amino-4-[3-(4-chloro-phenoxy)- azetidin-l-yl]-butan-l-ol hydrobromide [MH]⁺ 271.0.

(E)-N-{(S)-3-[3-(4-cUoro-phenoxy)-azetidin-l-yl]-l-hydroxymethyl-ρropyl}-3-(5-cyano-2- methoxy-ρhenyl)-acrylamide

A solution of (E)-3-(5-cyano-2-methoxy-phenyl)-acryϋc acid (0.022g, 0.107mmol) in DCM (lml) is treated with diisopropylethylamine (0.045ml, 0.258mmol) and TBTU (0.043g, 0.135mmol) and stirred for 5 minutes. The resultant solution is treated with (S)-2-amino-4-[3- (4-chloro-phenoxy)-azetidin-l-yl]-butan-l-ol hydrobromide (0.0395g, 0.112mmol) in DMF (2ml) and stirred at room temperature overnight. The reaction mixture is evaporated in vacuo and the crude product is partitioned between ethyl acetate (20ml) and aqueous NaHC0₃ (20ml). The aqueous layer is extracted with ethyl acetate (20ml) and the combined organic layers washed with aqueous NaHC0₃ (40ml), brine (40ml), dried (MgS0₄) and evaporated. The crude residue is purified by flash silica chromatography (DCM:methanol, gradient 99:1 to 97:3 elution) to afford (E)-N-{(S)-3-[3-(4-chloro-phenoxy)-azetidin-l-yl]-l-hydroxymethyI- propyl}-3-(5-cyano-2-methoxy-phenyl)-acrylamide as a pale yellow oil [MH]⁺ 456.0.

Example 8 is prepared analogously to Example 16.

(E)-3-(5-Cyano-2-methoxy-ρhenyl)-N-{3-[3-(4-fluoro-benzoyl)-azetidin-l-yl]-propyl}- acrylamide - Example 15

l-Benzhydryl-azetidine-3-carboxylic acid methoxy-methyl-amide

A solution of l-benzhydryl-azetidine-3-carboxylic acid (3.0g, 11.2mmol) in dry THF is treated with di-imidazol-1-yl-methanone (CDI) (1.82g, 11.2mmol) and refluxed for 2 hours. 0,N- dimethylhydroxylamine hydrochloride (1.09g, 11.2mmol) is added and the reaction mixture heated to reflux for a further 18 hours. The reaction is evaporated in vacuo and the residue dissolved in ethyl acetate which is washed with saturated aqueous NaHC0₃, brine, dried with MgS04 and evaporated. The crude product is purified by flash silica chromatography (ethyl actetate:hexane gradient 1:1 to 1:0 elution) to afford l-benzhydryl-azetidine-3-carboxyϋc acid methoxy-methyl-amide [MH]⁺ 311.2.

(l-Benzhydryl-azetidm-3-yl)-(4-iluoro-phenyl)-methanone

A cooled (0-5°C) solution of l-benzhydryl-azetidine-3-carboxyUc acid methoxy-methyl-amide (3.78g, 12.2mmol) in dry THF is treated with a 2M solution of 4-fluorophenylrnagnesium bromide in diethylether (18.2ml, 36.4mmol) added dropwise. The reaction mixture is allowed to warm to room temperature overnight and evaporated in vacuo. The residue is dissolved in ethyl acetate which is washed with saturated aqueous NH4CI, brine, dried with MgS0₄ and evaporated. The crude product is purified by flash silica chromatography (ethyl acetate:hexane gradient, 5:95 to 10:90 elution) to afford (l-benzhydryl-azetidin-3-yl)-(4-fluoro-phenyl)- methanone [MH]⁺ 346.16. [l-(3-Amino-propyl)-azetidm-3-yl]-(4-ιluoro-ρhenyl)-methanone hydrobromide

A solution of azetidin-3-yl-(4-fluoro-phenyl)-methanone hydrochloride (0.5g, 2.84mmol), prepared from (l-benzhydryl-azetidin-3-yl)-(4-fluoro-phenyl)-methanone using a procedure analogous to that used for preparation of 3-(4-fluorophenoxy)azetidine hydrochloride, in dry acetonitrile (15ml) is treated with triethylamine (0.59ml, 4.25 mmol) followed by 3- bromopropylamine hydrobromide (0.75g, 3.40mmol). The resultant suspension is heated to 70°C for 1.5hours and the solvents removed in vacuo. The residue is co-evaporated from toluene three times to afford crude [l-(3-amino-propyl)-azetidin-3-yl]-(4-fluoro-phenyl)- methanone hydrobromide as a soUd [MH]⁺ 237.0.

(E)-3-(5-Cyano-2-methoxy-phenyl)-N-{3-[3-(4-fluoro-benzoyl)-azetidin-l-yl]-proρyl}- acrylamide

This is prepared from [l-(3-amino-propyl)-azetidin-3-yl]-(4-fluoro-ρhenyl)-methanone, as a white crystalline solid [MH]⁺ 422.1.

Examples 4, 5, 6, 7 and 11 are prepared analogously to Example 15.

(E)-3-(5-Cyano-2-methoxy-ρhenyl)-N-{(S)-3-[3-(4-fluoro-benzoyl)-azetidin-l-yl]-l- hydroxymethyl-propylj-acrylamide - Example 14

{(S)-l-tert-Butoxymethyl-3-[3-(4-fluoro-benzoyl)-azetidin-l-yl]-ρropyl}-carbamic acid benzyl ester

A solution of ((S)-l-tert-butoxymethyl-3-iodo-propyl)-carbamic acid benzyl ester (0.516mg, 1.28mmol) in dry acetonitrile (6ml) is treated with azetidin-3-yl-(4-fluoro-phenyl)-methanone hydrochloride followed by triethylamine (0.455ml, 3.26mmol). The resultant soution is stirred at room temperature for 18 hours and the solvents evaporated. The residue is treated with DCM:diethyl ether (1:1) and the mixture filtered . The filtrate is evaporated and the crude residue purified by flash silica chromatography (ethyl actetate:hexane, 1:1 followed by ethyl acetate:methanol, 4:1 gradient elution) to afford {(S)-l-tert-butoxymethyl-3-[3-(4-fluoro- benzoyl)-azetidin-l-yl]-propyl}-carbamic acid benzyl ester [MH]⁺ 457.0.

[l-((S)-3-Armno-4-hydroxy-butyl)-azetidin-3-yl]-(4-fluoro-phenyl)-methanone hydrobromide

{(S)-l-tert-butoxymethyl-3-[3-(4-fluoro-benzoyl)-azetidin-l-yl]-propyl}-carbamic acid benzyl ester (0.4g, 0.88mmol) is dissolved in HBr-CH₃COOH (45%, 3ml) at 0°C and stirred for 1 hour. The solvent is evaporated and the residue co-evaporated from toluene (x3) and ethanol (x5) to afford [l-((S)-3-amino-4-hydroxy-butyl)-azetidin-3-yl]-(4-fluoro-phenyl)-methanone hydrobromide as an oil which is used directly.

(E)-3-(5-Cyano-2-methoxy-phenyl)-N-{(S)-3-[3-(4-fluoro-benzoyl)-azetidin-l-yl]-l- hydroxymethyl-propylj-acrylamide

(E)-3-(5-cyano-2-methoxy-phenyl)-acryUc acid (0.116g, 0.56mmol) is dissolved in dry DMF (3ml) and treated with diisopropylethylamine (0.25ml, 1.4 mmol) followed by TBTU (0.183g, 0.56mmol). The resultant solution is stirred at room temperature for 0.5 hours, treated with a solution of [l-((S)-3-amino-4-hydroxy-butyl)-azetidin-3-yl]-(4-fluoro-phenyl)-methanone hydrobromide (0.152g, 0.56mmol) in DMF (2ml) and stirred at room temperature overnight. The reaction mixture is evaporated and the crude residue dissolved in ethyl acetate which is washed with aqueous NaHC0₃ (x3), brine (xl), dried (MgS0₄) and evaporated. The product is partly purified by flash silica chromatography (ethyl actetate:hexane, 1:1 followed by ethyl acetate:methanol, 4:1 gradient elution) to afford a mixture of acylated products. The mixture is dissolved in methanol (2ml) / water (0.5ml) and treated with K₂C0₃ (0.082g, 0.059mmol) and stirred at room temperature for 0.75 hours. The mixture is extracted with ethyl acetate which is dried (MgS0₄) and evaporated. The residue is purified by preparative TLC (ethyl acetate:methanol, 6:1 elution) to afford (E)-3-(5-cyano-2-methoxy-phenyl)-N-{(S)-3-[3-(4- fluoro-benzoyl)-azetidin-l-yl]-l-hydroxymethyl-propyl}-acrylamide [MH]⁺ 452.0.

Examples 1 and 3 are prepared analogously to Example 14.

(E)-3-(5-Cyano-2-methoxy-phenyl)-N-{(S)-3-[3-(4-fluoro-phenoxy)-azetidin-l-yl]-l- hydroxymethyl-propyl}-acrylamide - Example 18

(S)-l-tert-Butoxymethyl-3-iodo-proρyl)-carbamic acid benzyl ester

A solution of ((S)-l-tert-butoxymethyl-3-hydroxy-propyl)-carbamic acid benzyl ester (2.50g, 8.4mmol) in dry THF (40ml) at room temperature is treated with triphenylphosphine (2.2g, 8.4mmol) followed by imidazole (0.58g, 8.4mmol) and iodine (2.1g, 8.4mmol). The resultant suspension is stirred overnight with the exclusion of Ught. The mixture is filtered and the filtrate evaporated and re-dissolved in DCM which is washed with aqueous citric acid ( 0.5M, x4), brine (xl), dried (MgS0₄) and evaporated to give the crude product as an oil which is used directly. {(S)-l-tert-Butoxymethyl-3-[3-(4-fluoro-phenoxy)-azetidm-l-yl]-propyl}-carbamic acid benzyl ester

This is prepared analogously to {(S)-l-tert-butoxymethyl-3-[3-(4-fluoro-benzoyl)-azetidin-l-yl]- propyl}-carbamic acid benzyl ester ( intermediate in Example 14) but using 3-(4-fluoro- phenoxy)-azetidine hydrochloride ( intermediate in Example 13 ) as starting material.

Acetic acid (S)-2-amino-4-[3-(4-fluoro-phenoxy)-azetidin-l-yl]-butyl ester hydrobromide is prepared analogously to (S)-2-amino-4-[3-(4-chloro-phenoxy)-azetidin-l-yl]-butan-l-ol hydrobromide ( intermediate in Example 16). The product is isolated as the O-acetyl derivative (as characterised by *H NMR) and is used directly.

(E)-3-(5-Cyano-2-methoxy-phenyl)-N-{(S)-3-[3-(4-fluoro-phenoxy)-azetidin-l-yl]-l- hydroxymethyl-propylj-acrylamide

(E)-3-(5-cyano-2-methoxy-phenyl)-acrylic acid (1.2g, 5.83mmol) is dissolved in dry DMF (30ml) and treated with diisopropylethylamine (2.5ml, 14.6mmol) followed by TBTU (1.9g, 5.8mmol). The resultant solution is stirred at room temperature for 0.5 hours, treated with a solution of acetic acid (S)-2-amino-4-[3-(4-fluoro-phenoxy)-azetidin-l-yl]-butyl ester hydrobromide (2.2g, 0.56mmol) in DMF (10ml) and stirred at room temperature overnight. The reaction mixture is evaporated and the crude residue dissolved in ethyl acetate which is washed with aqueous NaHC0₃ (x3), brine (xl), dried (MgS0₄) and evaporated. The crude product is treated with methanol and filtered. The filtrate is treated with siUca, evaporated and the crude adsorbed product purified by flash silica chromatography (ethyl actetate:hexane, 1:1 followed by ethyl acetate and then ethylacetate:methanol, 9:1 gradient elution) to afford a mixture of acylated products as an oil. The mixture is dissolved in methanohwater, 3:1 (20ml) and treated with K₂C0₃ (one molar equivalent) and stirred at room temperature for 0.5 hours. The mixture is extracted with ethyl acetate which is dried (MgS0₄) and evaporated. The residue is purified by flash siUca chromatography (ethyl actetate:hexane, 1:1 followed by ethyl acetate and then ethyl acetate:methanol, 9:1 gradient elution) to afford (E)-3-(5-cyano-2- methoxy-phenyl)-N-{(S)-3-[3-(4-fluoro-phenoxy)-azetidin-l-yl]-l-hydroxymethyl-propyl}- acrylamide [MH]⁺ 440.0

(E)-N-{(S)-l-tert-Butoxymethyl-3-[3-(4-fluoro-phenoxy)-azetidin-l-yl]-ρropyl}-3-(5-cyano-2- methoxy-ρhenyl)-acrylamide - Example 17 (S)-l-tert-Butoxymethyl-3-[3-(4-fluoro-phenoxy)-azetidin-l-yl]-propylamine A solution of {(S)-l-tert-butoxymethyl-3-[3-(4-fluoro-phenoxy)-azetidin-l-yl]-propyl}-carbamic acid benzyl ester (intermediate in Example 18) (200mg, 0.45mmol) in MeOH (10ml) is treated with palladium on carbon (10% Pd, 66mg) under an inert atmosphere. The mixture is stirred at room temperature under an atmosphere of hydrogen for 3 hours. The mixture is filtered through CeUte and the filtrate evaporated. The crude product is dissolved in ethyl acetate which is washed with NaHC0₃ (x2), dried (MgS0₄) and evaporated to afford (S)-l-tert- butoxymethyl-3-[3-(4-fluoro-phenoxy)-azetidin-l-yl]-propylamine [MH]⁺ 311.3

(E)-N-{(S)-l-tert-Butoxymethyl-3-[3-(4-fluoro-phenoxy)-azetidin-l-yl]-propyl}-3-(5-cyano-2- methoxy-phenyl)-acrylamide

This is prepared from (S)-l-tert-butoxymethyl-3-[3-(4-fluoro-phenoxy)-azetidin-l-yl]- propylamine and (E)-3-(5-cyano-2-methoxy-phenyl)-acrylic acid by a procedure analogous to the last stage of Example 18. [MH]⁺ 496.4.

Claims

1. A compound of formula

in free or salt form, where

Ar¹ is phenyl substituted by one or more halogen atoms;

Ar² is phenyl optionally substituted by one or more substitutuents selected from halogen, cyano, hydroxy, nitro, CrCg-alkyl, Q-Q-haloalkyl, Q-Q-alkoxy, Q-Q-alkoxycarbonyl or di(CrQ-alkyl)aminocarbonylmethoxy;

R¹ is hydrogen or Q-Q-alkyl optionally substituted by hydroxy, Q-Q-alkoxy, acyloxy, halogen, carboxy, Q-Q-alkoxycarbonyl, -N(R²)R³, -CON(R⁴)R⁵ or by a monovalent cyclic organic group having 3 to 15 atoms in the ring system;

R² and R³ are each independently hydrogen or Q-Q-alkyl, or R² is hydrogen and R³ is hydroxy-Q-Q-alkyl, acyl, -S0₂R⁶ or -CON(R⁴)R⁵, or R² and R³ together with the nitrogen atom to which they are attached denote a 5-or 6-membered heterocycUc group;

R⁴ and R⁵ are each independently hydrogen or Q-Q-alkyl, or R⁴ and R⁵ together with the nitrogen atom to which they are attached denote a 5- or 6-membered heterocyclic group;

R⁶ is CrCg-alkyl, Q-Q-haloalkyl, or phenyl optionally substituted by CrCg-alkyl;

X is -C(=0)-, -O- or -CH₂-; and n is 1, 2, 3 or 4.

2. A compound according to claim 1, in which Ar¹ is phenyl substituted by one, two or three halogen atoms selected from fluorine and chlorine atoms.

3. A compound according to claim 1 or 2, in which Ar² is phenyl substituted by one or more substituents selected from cyano, halogen, Q-Q-alkoxy or di(Q-Q-alkyl)aminocarbonyl- methoxy.

4. A compound according to claim 1, 2 or 3, in which R¹ is hydrogen or Q-Q-alkyl substituted by hydroxy or Q-Q-alkoxy.

5. A compound according to claiml in which

Ar¹ is phenyl substituted by one or two substituents selected from fluorine and chlorine; Ar² is phenyl substituted by at least two substituents selected from halogen, cyano, Q-Q- alkoxy, and di(CrQ-alkyl)aminocarbonylmethoxy; R¹ is hydrogen or Q-Q-alkyl substituted by hydroxy or Q-Q-alkoxy; X is -C(=0)- or -0-; and n is 1 or 2.

6. A compound according to claim 1 in which

Ar¹ is phenyl substituted by fluorine or chlorine para to the indicated group X, and optionally further substituted by fluorine or chlorine meta to the group X;

Ar² is phenyl substituted ortho to the indicated -CH=CH- group by Q-Q-alkoxy or di(Q-Q- alkyl)aminocarbonylmethoxy and para to the Q-Q-alkoxy or di(Q-C₄-alkyl)aminocarbonyl- methoxy group by cyano or halogen;

R¹ is hydrogen or Q-Q-alkyl substituted by hydroxy or Q-Q-alkoxy;

X is -C(=0) - or -0-; and n is 2.

7. A compound according to claim 1, which is also a compound of formula X

wherein X, R_a, Rb, R¹, Re and Ra are as listed in the following table

8. A compound according to any one of the preceding claims for use as a pharmaceutical.

9. A pharmaceutical composition comprising as active ingredient a compound according to any one of claims 1 to 7.

10. The use of a compound according to any one of claims 1 to 7 for the manufacture of a medicament for the treatment of a condition mediated by CCR-3.

11. Use according to claim 10, in which the condition is an inflammatory or allergic condition, particularly an inflammatory or obstructive airways disease.

12. A process for the preparation of a compound of formula I which comprises

(i) reacting a compound of formula

with a compound of formula

or an amide forming derivative thereof, where Ar¹, Ar², R¹, X and n are as defined in claim 1, and

(ii) recovering the product in free or salt form.