CCR8 Antagonists Scope of the Invention This invention relates to certain substituted benzenesulfonamides and their use for inhibiting the chemokine receptor nominated CCR8, [also known as ChemRl (1), TER1 (2) and CMKBR8&num (systematic Human Genome nomenclature)], resulting in treatment of respiratory diseases such as asthmatic conditions and the like.

Area of the Invention Recruitment of activated T lymphocytes to sites of inflammation is a tightly controlled process regulated by a multiplicity of molecules, in particular, chemokines.

Chemokines are small molecular weight secreted proteins that direct leukocyte trafficking through interaction with G-protein coupled seven-transmembrane receptors.

In asthma, T lymphocytes of the Th2 subtype are involved with the initiation and the maintenance of airway inflammation and obstruction following antigen challenge.

Activated Th2 CD4+ lymphocytes are recruited to the airways where they produce the cytokines IL-4, IL-5 and IL-13. These cytokines recruit eosinophils as well as additional Th2 cells to the lungs and ensure their survival upon their arrival. Large numbers of Th2 lymphocytes and their concomitant cytokine secretions have been recovered in bronchoalveolar lavage fluid as well as in bronchial biopsies from asthmatic patients and in animal models of asthma.

Recently, bronchial biopsies and bronchoalveolar lavage fluid were analysed for the presence of chemokines and their cognate receptors before and following antigen challenge of atopic asthmatic patients (Panina-Bordignon, et al. 2001. J. Cliva. Invest 107 (11): 1357).

These data demonstrated that Th2 cells expressing CCR8 were increased in the lung of challenged asthmatics, but were undetectable in healthy controls. It was found that the number of CCR8+ Th2 lymphocytes in the airway after challenge correlated with the degree of bronchoconstriction during the late airway response (LAR). Furthermore TARC (thymus-and activation-regulated chemokine) which may activate CCR8 was highly upregulated in the airway epithelia after challenge. These data suggest that expression of CCR8 by activated Th2 lymphocytes in asthmatic airways is important for the specific recruitment of these cells to the site of inflammation during the LAR. Thus, inhibition of CCR8 could decrease the ability of activated CCR8+ Th2 lymphocytes to home to the lungs during the asthmatic inflammatory response.

Further support for the hypothesis that CCR8 blockade would inhibit asthmatic inflammation and hyperreactivity is provided by data reported by Chensue, et al. (2001. J.

Exp. Med. 193: 573). They demonstrated that in mice, deletion of CCR8 significantly attenuated Th2 cytokine production and associated eosinophil accumulation following antigen challenge of these animals. Furthermore, the development of antigen-induced airways hyperreactivity was inhibited in these animals. Thus, these data support the idea that blockade of CCR8 might provide significant therapeutic value for the treatment of asthma.

Accordingly, there is provided herein compounds and methods for treating an asthmatic condition and other conditions mediated by CCR8 where inhibition of this chemokine receptor would effect a positive outcome in a patient suffering from or pre- disposed to suffer from such a malady.

Summary of the Invention In a first aspect, this invention relates to compounds of Formula I wherein n is 0, to 6 ; m is l, to 4 ; pis l, to4 ; Ar is unsubstituted phenyl, thiophenyl, furanyl, or pyridinyl; or phenyl, thiophenyl, furanyl, or pyridinyl substituted with one or more radicals selected from the group consisting of Cl-Cg alkoxy, CI-Cg alkyl, halo, cyano, hydroxy, amino, N-Cl-Cg alkyl amino, N, N-di-Cl-Cg alkyl amino, carboxyl, Cl-C8 alkyl carbamoyl, Cl-Cg alkoxy carbonyl and trihalomethyl; R1 and R6 are independently selected from the group consisting of hydrogen, C1- C8 branched or unbranched alkyl, C3-Cg cycloalkyl lower alkyl, and unsubstituted or substituted phenyl lower alkyl ; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Cl-Cg alkoxy, Cl-C8 alkyl, halo, cyano and trihalomethyl; R2, R3, R4, and R5 are independently selected from the group consisting of hydrogen, Cl-Cg branched or unbranched alkyl, Cg-Cg cycloalkyl lower alkyl, Cl-Cg

alkoxy, halo, cyano, trihalomethyl, hydroxy, amino, N-Cl-C8 alkyl amino, N, N-di-Cl-Cg alkyl amino, carboxyl, Cl-Cg alkyl carbamoyl, Cl-C8 alkoxy carbonyl and unsubstituted or substituted phenyl lower alkyl ; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Cl-C8 alkoxy, Cl-Cg alkyl, halo, cyano and trihalomethyl; R7 is hydrogen, unsubstituted or substituted Cl-calo branched or unbranched alkyl, unsubstituted or substituted C3-C8 cycloalkyl lower alkyl, or unsubstituted or substituted phenyl lower alkyl ; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Cl-Cg alkoxy, Cl-C8 alkyl, halo, cyano, hydroxy, amino, N-Cl-Cg alkyl amino, N, N-di-Cl-Cg alkyl amino, carboxyl, Ci-Cg alkyl carbamoyl, Cl-C8 alkoxy carbonyl and trihalomethyl; or a pharmaceutically acceptable salt thereof.

Another aspect of this invention is that of a pharmaceutical formulation comprising a compound of Formula I in admixture with a pharmaceutically acceptable excipient.

In a further aspect, this invention relates to a method for inhibiting CCR8 to a degree which effects prevention of a condition or treatment of a condition associated with the inhibition of CCR8,, which methdo comprises administering a compound of formula 1 along or in admixture with a pharmaceutically acceptable excipient to mammal in need thereof in an amount effective for inhibiting said enzyme.

A further object of this invention is the use of a compound of Formula I in the manufacture of a medicament for treating or preventing a condition associated with the inhibition of CCR8.

In a particular aspect, the methods of this invention are especially useful for treatment of respiratory condition such as an asthmatic condition.

Detailed Description Definitions The meaning of any substituent at any one occurrence in Formula I or any sub- formula thereof is independent of its meaning, or any other substituent's meaning, at any other occurrence, unless specified otherwise.

Abbreviations and symbols commonly used in the peptide and chemical arts are used herein to describe the compounds of the present invention. In general, the amino acid abbreviations follow the IUPAC-IUB Joint Commission on Biochemical Nomenclature as described in Eur. J. Biochem. , 158,9 (1984).

The term"Cl Cg alkyl"and""Cl-Clo alkyl"is used herein includes both straight or branched chain radicals of 1 to 8 or 10 carbon atoms. By example this term includes, but is not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl and the like."Lower alkyl"has the same meaning as C1 Cg alkyl.

Herein"C1 Cg alkoxy"includes straight and branched chain radicals of the likes of -O-CH3,-0-CH2CH3, and the n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, pentoxy, and hexoxy, and the like.

"C3-Cg-cycloalkyl"as applied herein is meant to, include substituted and unsubstituted cyclopropane, cyclobutane, cyclopentane and cyclohexane.

"Halogen"or"halo"means F, Cl, Br, and I.

Here and throughout this application the term Co denotes the absence of the substituent group immediately following; for instance, in the moiety ArCO-6alkyl, when C is 0, the substituent is Ar, e. g. , phenyl. Conversely, when the moiety ArCo 6alkyl is identified as a specific aromatic group, e. g. , phenyl, it is understood that the value of C is 0.

The present invention includes all hydrates, solvates, complexes and prodrugs of the compounds of this invention. Prodrugs are any covalently bonded compounds which release the active parent drug according to Formula I in vivo. If a chiral center or another form of an isomeric center is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereomers, are intended to be covered herein. Inventive compounds containing a chiral center may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone. In cases in which compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.

Preferred Embodiments The preferred compounds of Formula I include those compounds wherein n is 0, 1, or 2 ; m is 1 or 2 ; pis 1 or 2 ;

Ar is unsubstituted phenyl, thiophenyl, or furanyl; or phenyl, thiophenyl, or furanyl substituted with one or more radicals selected from the group consisting of Cl-C8 alkoxy, Cl-C8 alkyl, halo, cyano, and trihalomethyl; R1 andR6 are independently selected from the group consisting of hydrogen, Cl-C8 branched or unbranched alkyl, Cg-Cg cycloalkyl lower alkyl, and unsubstituted or substituted phenyl lower alkyl ; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Cl-Cg alkoxy, C-Cg alkyl, halo, cyano and trihalomethyl; R2, R3, R4, and Rs are independentlyselected from the group consisting of hydrogen, Cl-Cg branched or unbranched alkyl, Cg-Cg cycloalkyl lower alkyl, Cl-Cg alkoxy, halo, cyano, trihalomethyl, and unsubstituted or substituted phenyl lower alkyl ; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Cl-C8 alkoxy, Cl-C8 alkyl, halo, cyano and trihalomethyl ; and R7 is hydrogen, unsubstituted or substituted Cl-Clo branched or unbranched alkyl, unsubstituted or substituted C3-Cg cycloalkyl lower alkyl, or unsubstituted or substituted phenyl lower alkyl ; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Cl-C8 alkoxy, Cl-C8 alkyl, hydroxy, halo, cyano, and trihalomethyl.

More preferred are the compound of Formula I wherein: n is 0 ; m is 1; pis 1 or2 ; Ar is unsubstituted phenyl, thiophenyl, or furanyl; or phenyl, thiophenyl, or furanyl substituted with one or more radicals selected from the group consisting of Cl-C8 alkoxy, Cl-Cg alkyl, halo, cyano, and trihalomethyl; R1 is hydrogen; R2, R3, R4, and Rs are independently selected from the group consisting of hydrogen, Cl-C8 branched or unbranched alkyl, C3-Cg cycloalkyl lower alkyl, Cl-Cg alkoxy, halo, cyano, trihalomethyl, and unsubstituted or substituted phenyl lower alkyl ; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Cl-Cg alkoxy, Cl-C8 alkyl, halo, cyano and trihalomethyl; and R7 is hydrogen, unsubstituted or substituted Cl-Clo branched or unbranched alkyl, unsubstituted or substituted C3-Cg cycloalkyl lower alkyl, or unsubstituted or substituted phenyl lower alkyl ; wherein, when substituted, a group is substituted by one or more

radicals selected from the group consisting of Cl-Cg alkoxy, Cl-C8 alkyl, hydroxy, halo, cyano, and trihalomethyl.

Even more preferred are those compounds where n is 0 ; m is 1 ; pis 1 or 2 ; Ar is phenyl, thiophenyl, or furanyl substituted with one or more radicals selected from the group consisting of C1-C8 alkoxy, Cl-C8 alkyl, halo, cyano, and trihalomethyl; Rl is hydrogen; R2, R3, R4, and Rs are independently selected from the group consisting of hydrogen, Cl-Cg branched or unbranched alkyl, C3-Cg cycloalkyl lower alkyl, Cl-Cg alkoxy, and halo; and R7 is unsubstituted or substituted C1-C10 branched or unbranched alkyl, unsubstituted or substituted C3-Cg cycloalkyl lower alkyl, or unsubstituted or substituted phenyl lower alkyl ; wherein, when substituted, a group is substituted by one or more radicals selected from the group consisting of Cl-Cg alkoxy, Cl-Cg alkyl, hydroxy, halo, cyano, and trihalomethyl.

The exemplified compounds are: N- {3- [1- (4-butyl- [ 1, 4] diazepan-1-yl)-methanoyl]-phenyl}-3, 4-dichloro- benzenesulfonamide; 3, 4-dichloro-N-{3-[1-(4-propyl- [1,4] diazepan-1-yl)-methanoyl]-phenyl}- benzenesulfonamide; N-{3-[1-(4-butyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}-3-chloro- benzenesulfonamide; 3, 4-dibromo-N- {3- [l- (4-propyl- [l, 4] diazepan-l-yl)-methanoyl]-phenyl}- benzenesulfonamide; 3-bromo-N-{3-[1-(4-butyl-[1, 4] diazepan-l-yl)-methanoyl]-phenyl}- benzenesulfonamide; 3-bromo-N- {3- [l- (4-propyl- [1, 4] diazepan-1-yl)-methanoyl]-phenyl}- benzenesulfonamide; 4-methoxy-N- {3-11-(4-propyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}- benzenesulfonamide; 4-methoxy-N- {3- [1- (4-pentyl- [1, 4] diazepan-1-yl)-methanoyl]-phenyl}- benzenesulfonamide;

N-{3-[1-(4-isopropyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}-3, 4-dimethoxy- benzenesulfonamide; N- {3- [1- (4-hexyl- [1, 4] diazepan-l-yl)-methanoyl]-phenyl}-3, 4-dimethoxy- benzenesulfonamide; 3, 4-dimethoxy-N- {3-11-(4-phenethyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}- benzenesulfonamide; 4-chloro-N- {3-[1-(4-cyclopropylmethyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}- 2,5-dimethyl-benzenesulfonamide ; 4-chloro-N-{3-[1-(4-heptyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}-2, 5-dimethyl- benzenesulfonamide; 5-bromo-N-[3-(4-isobutyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}-2-methoxy- benzenesulfonamide ; 4-chloro-2, 5-dimethyl-N- {3- [1- (4-phenethyl- [1, 4] diazepan-1-yl)-methanoyl]- phenyl}-benzenesulfonamide ; N-{3-[3-[1-(4-butyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}-3, 4-dimethoxy- benzenesulfonamide ; 3, 4-dimethoxy-N-{3-[1-(4-propyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl)- benzenesulfonamide ; N-13- [I- (4-cyclopropylmethyl- [1, 4) diazepan-1-yl)-methanoyll-phenyl)-3, 4- dimethoxy-benzenesulfonamide; 3, 4-dimethoxy-N- {3- [l- (4-ethyI- [l, 4] diazepan-l-yl)-methanoyl]-phenyl}- benzenesulfonamide; 3, 4-dimethoxy-N- { 3- [1- (4-pentyl- [ 1, 4] diazepan-l-yl)-methanoyl]-phenyl}- benzenesulfonamide ; 3, 4-dimethoxy-N- {2-methyl-5- [1- (4-propyl- [ 1, 4] diazepan-l-yl)-methanoyl]- phenyl}-benzenesulfonamide ; N- {2-chloro-5-[1-(4-propyl-[1, 4] diazepan-l-yl)-methanoyl]-phenyl}-3, 4- dimethoxy-benzenesulfonamide; N- {5- [1- (4-butyl- [ 1, 4] diazepan-1-yl)-methanoyl]-2-methyl-phenyl}-3, 4-dimethoxy- benzenesulfonamide; N- {5- [1- (4-butyl- [ 1, 4] diazepan- l-yl)-methanoyl]-2-chloro-phenyl}-3, 4-dimethoxy- benzenesulfonamide; 3, 4-dimethoxy-N- {2-methyl-5- [l- (4-pentyl- [1, 4] diazepan-1-yl)-methanoyl]- phenyl}-benzenesulfonamide ;

N- {2-chloro-S-[1-(4-pentyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}-3, 4- dimethoxy-benzenesulfonamide; N- {5- [1- (4-cyclopropylmethyl- [l, 4] diazepan-1-yl)-methanoyl]-2-methyl-phenyl}- 3,4-dimethoxy-benzenesulfonamide ; N- 2-chloro-5- [l- (4-cyclopropylmethyl- [1, 4] diazepan-1-yl)-methanoyl]-phenyl}- 3, 4-dimethoxy-benzenesulfonamide ; 3,4-dimethoxy-N- {2-methyl-3-[1-(4-propyl-[l, 41diazepan-l-yl)-methanoyl]- phenyl}-benzenesulfonamide ; N- {4-chloro-3- [1- (4-propyl- [1, 4] diazepan-1-yl)-methanoyl]-phenyl}-3, 4- dimethoxy-benzenesulfonamide; N- {4-bromo-3- [1- (4-propyl- [1, 4] diazepan-1-yl)-methanoyl]-phenyl}-3, 4- dimethoxy-benzenesulfonamide ; N- {3-[1-(4-butyl-[1, 4] diazepan-1-yl)-methanoyl]-2-methyl-phenyl}-3, 4-dimethoxy- benzenesulfonamide; N- {3-[1-(4-butyl-[1, 4] diazepan-l-yl)-methanoyl]-4-chloro-phenyl}-3, 4-dimethoxy- benzenesulfonamide ; N- {4-bromo-3- [1- (4-butyl- [1,4] diazepan-1-yl)-methanoyl]-phenyl}-3, 4-dimethoxy-, benzenesulfonamide; 3, 4-dimethoxy-N- {2-methyl-3-11-(4-pentyl-l1, 4Jdiazepan-l-yl)-methanoyl]- phenyl}-benzenesulfonamide ; N- {4-chloro-3- [ 1- (4-pentyl- [1,4] diazepan-1-yl)-methanoyl]-phenyl}-3, 4- dimethoxy-benzenesulfonamide ; N- {4-bromo-3- [1- (4-pentyl- [1, 4] diazepan-l-yl)-methanoyl]-phenyl}-3, 4- dimethoxy-benzenesulfonamide; N- 3- [l- (4-cyclopropylmethyl- [1, 4] diazepan-1-yl)-methanoyl]-2-methyl-phenyl}- 3,4-dimethoxy-benzenesulfonamide ; N- {4-chloro-3- [1- (4-cyclopropylmethyl- [1,4] diazepan-1-yl)-methanoyl]-phenyl}- 3, 4-dimethoxy-benzenesulfonamide ; N- {4-bromo-3- [1-(4-cyclopropylmethyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}- 3,4-dimethoxy-benzenesulfonamide ; 4-chloro-2, 5-dimethyl-N-{3-[1-(4-propyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}- benzenesulfonamide ; N- {3- [1- (4-isobutyl- [1, 4] diazepan-1-yl)-methanoyl]-phenyl}-3, 4-dimethoxy- benzenesulfonamide;

3,4-dimethoxy-N- {3-[1-(4-octyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}- benzenesulfonamide ; N- {3- [1- (4-heptyl- [ 1, 4] diazepan-l-yl)-methanoyl]-phenyl}-3, 4-dimethoxy- benzenesulfonamide ; N- [3-[1-(4-ethyl-[1,4]diazepan-1-yl)-methanoyl]-phenyl}-3, 4-dimethoxy- benzenesulfonamide ; 3,4-dibromo-N- {3-[1-(4-butyl-[1, 4] diazepan-l-yl)-methanoyl]-phenyl}- benzenesulfonamide; 4, 5-dibromo-thiophene-2-sulfonic acid {3- [1- (4-butyl- [1, 4] diazepan-l-yl)- methanoyl]-phenyl}-amide ; N- {3- [l- (4-butyl- [l, 4] diazepan-l-yl)-methanoyl]-phenyl}-4-chloro-2, 5-dimethyl- benzenesulfonamide ; 3, 4-dimethoxy-N- (3-{1-[4-(4-methoxy-benzyl)-[1, 4] diazepan-1-yl]-methanoyl}- phenyl) -benzenesulfonamide; 3-chloro-N-{3-[1-(4-cycloproylmethyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}- benzenesulfonamide ; 3, 4-dichloro-N-{3-[1-(4-cyclopropylmethyl-[1, 4] diazepan-1-yl)-methanoyl]- phenyl} -benzenesulfonamide; 3, 4-dichloro-N- {3- [l- (4-ethyl- [l, 4] diazepan-1-yl)-methanoyl]-phenyl}- benzenesulfonamide ; 3-chloro-N-{3-[1-(4-propyl- [1,4] diazepan-1-yl)-methanoyl]-phenyl}- benzenesulfonamide; 5-bromo-2-methoxy-N- {3- [1- (4-propyl- [1, 4] diazepan-1-yl)-methanoyl]-phenyl}- benzenesulfonamide; 2,5-dichloro-thiophene-3-sulfonic acid {3- [l- (4-propyl- [l, 4] diazepan-1-yl)- methanoyl]-phenyl}-amide ; biphenyl-4-sulfonic acid {3- [1-(4-propyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}- amide; 4-isopropyl-N- {3- [1- (4-propyl- [1, 4] diazepan-1-yl)-methanoyl]-phenyl}- benzenesulfonamide; N- [l- (4-propyl- [1, 4] diazepan-l-yl)-methanoyl]-phenyl}-4-trifluoromethoxy- benzenesulfonamide; N-{3-[1-(4-propyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}-trifluoromethyl- benzenesulfonamide ;

2,4, 5-trichloro-N-{3-[1-(4-propyl-[1, 4] diazepan-l-yl)-methanoyl]-phenyl}- benzenesulfonamide; N- {3- [l- (4-butyl- [l, 4] diazepan-l-yl)-methanoyl]-phenyl}-2, 4,5-trichloro- benzenesulfonamide; N- {3- [1- (4-butyl- [ 1, 4] diazepan-1-yl)-methanoyl]-phenyl}-2, 4-dichloro- benzenesulfonamide; N- {3- [1- (4-butyl- [1,4] diazepan-1-yl)-methanoyl]-phenyl}-trifluoromethyl- benzenesulfonamide; N- {3- [1-(4-butyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}-4-trifluoromethoxy- benzenesulfonamide; biphenyl-4-sulfonic acid {3- [1- (4-butyl- [1, 4] diazepan-1-yl)-methanoyl]-phenyl}- amide; 2,5-dichloro-thiophene-3-sulfonic acid {3- [1- (4-butyl- [1, 4] diazepan-l-yl)- methanoyl]-phenyl}-amide ; 4-bromo-N-{3-1-(4-butyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}-2, 5-difluoro- benzenesulfonamide; 5-bromo-N- {3- [1-(4-butyl-[1, 4] diazepan-1-yl)-methanoyl]-phenyl}-2-methoxy- benzenesulfonamide ; N- {3- [1- (4-cyclohexylmethyl- [1, 4] diazepan-1-yl)-methanoyl]-phenyl}-3, 4- dimethoxy-benzenesulfonamide 4,5-dibromo-thiophene-2-sulfonic acid {3- [1- (4-cyclopropylmethyl- [1, 4] diazepan- l-yl)-methanoyl]-phenyl}-amide ; and 3, 4-dichloro-N- { 3- [1- (4-methyl- [ 1, 4] diazepan-l-yl)-methanoyl]-phenyl}- benzenesulfonamide.

Formulations This invention also provides a pharmaceutical composition which comprises a compound according to Formula I and a pharmaceutically acceptable carrier, diluent or excipient. Accordingly, the compounds of Formula I may be used in the manufacture of a medicament. Pharmaceutical compositions of the compounds of Formula I prepared as hereinbefore described may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation may be a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution.

Such formulation is especially suitable for parenteral administration, but may also be used

for oral administration or contained in a metered dose inhaler or nebulizer for insufflation.

It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.

Alternately, these compounds may be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit. The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p. o. or filled into a soft gelatin capsule.

For rectal administration, the compounds of this invention may also be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.

Typical compositions for inhalation are in the form of a dry powder, solution, suspension or emulsion. Administration may for example be by dry powder inhaler (such as unit dose or multi-dose inhaler, e. g. as described in US Patent 5,590, 645 or by nebulisation or in the form of a pressurized aerosol. Dry powder compositions typically employ a carrier such as lactose, trehalose or starch. Compositions for nebulisation typically employ water as vehicle. Pressurized aerosols typically employ a propellant such as dichlorodifluoromethane, trichlorofluoromethane or, more preferably, 1,1, 1,2- tetrafluoroethane, 1,1, 1,2, 3,3, 3-heptafluoro-n-propane or mixtures thereof. Pressurized aerosol formulations may be in the form of a solution (perhaps employing a solubilising agent such as ethanol) or a suspension which may be excipient free or employ excipients including surfactants and/or co-solvents (e. g. ethanol). In dry powder compositions and suspension aerosol compositions the active ingredient will preferably be of a size suitable for inhalation (typically having mass median diameter (MMD) less than 20 microns e. g. 1- 10 especially 1-5 microns). Size reduction of the active ingredient may be necessary e. g. by micronisation.

Pressurized aerosol compositions will generally be filled into canisters fitted with a valve, especially a metering valve. Canisters may optionally be coated with a plastics material e. g. a fluorocarbon polymer as described in WO 96/32150. Canisters will be fitted into an actuator adapted for buccal delivery.

Typical compositions for nasal delivery include those mentioned above for inhalation and further include non-pressurized compositions in the form of a solution or suspension in an inert vehicle such as water optionally in combination with conventional excipients such as buffers, anti-microbials, tonicity modifying agents and viscosity modifying agents which may be administered by nasal pump.

Typical dermal and transdermal formulations comprise a conventional aqueous or non-aqueous vehicle, for example a cream, ointment, lotion or paste or are in the form of a medicated plaster, patch or membrane.

Preferably the composition is in unit dosage form, for example a tablet, capsule or metered aerosol dose, so that the patient may administer a single dose.

For acute therapy, parenteral administration of a compound of Formula I is preferred. An intravenous infusion of the compound in 5% dextrose in water or normal saline, or a similar formulation with suitable excipients, is most effective, although an intramuscular bolus injection is also useful. Typically, the parenteral dose will be about 0.001 to about 100 mg/kg ; preferably between 0.01 and 20 mg/kg, in a manner to maintain the concentration of drug in the plasma at a concentration effective to inhibit CCR8. The compounds are administered one to four times daily at a level to achieve a total daily dose of about 0.4 to about 400 mg/kg/day. The precise amount of an inventive compound which is therapeutically effective, and the route by which such compound is best administered, is readily determined by one of ordinary skill in the art by comparing the blood level of the agent to the concentration required to have a therapeutic effect.

Each dosage unit for oral administration contains suitably from 10 micrograms to 60 mg/Kg, and preferably from 0.1 mg to 30 mg/Kg of a compound or a pharmaceutically acceptable salt thereof. Each dosage unit for parenteral administration contains suitably from 0.1 mg to 100 mg/Kg, of the compound or a pharmaceutically acceptable salt thereof.

Each dosage unit for intranasal administration contains suitably 1-400 mcg and preferably 10 to 200 mcg per activation. A dry powder inhalation dose could contain 1-1000 micrograms per dose unit. A topical formulation contains suitably 0.001 to 5.0% of a present compound.

The active ingredient may be administered from 1 to 6 times a day, sufficient to exhibit the desired activity. Preferably, the active ingredient is administered once or twice a

day. The compounds of this invention may also be administered orally to the patient, in a manner such that the concentration of drug is sufficient to inhibit bone resorption or to achieve any other therapeutic indication as disclosed herein. Typically, a pharmaceutical composition containing the compound is administered at an oral dose of between about 0.1 to about 50 mg/kg in a manner consistent with the condition of the patient. Preferably the oral dose would be about 10 micrograms to about 20 mg/kg.

No unacceptable toxicological effects are expected when compounds of the present invention are administered in accordance with the present invention.

Utility of the Present Invention The compounds of Formula I are useful as inhibitors of the chemokine receptor known as CCR8. The present invention provides methods of treatment of diseases caused by pathological levels of CCR8, or the CCR8 chemokine ligand, I-309, which methods comprise administering to an animal in need thereof a therapeutically effective amount of a compound of the present invention. This course of treatment is particularly useful in treating mammals, most particularly humans.

Examples of disease states in which the compound of the invention has potentially beneficial anti-inflammatory effects include diseases of the respiratory tract such as bronchitis (including chronic bronchitis), bronchiectasis, asthma (including allergen- induced asthmatic reactions), chronic obstructive pulmonary disease (COPD), cystic fibrosis, sinusitis and rhinitis. Other relevant disease states include diseases of the gastrointestinal tract such as intestinal inflammatory diseases including inflammatory bowel disease (e. g. Crohn's disease or ulcerative colitis) and intestinal inflammatory diseases secondary to radiation exposure or allergen exposure.

Furthermore, the compound of the invention may be used to treat nephritis, skin diseases such as psoriasis, eczema, allergic dermatitis and hypersensitivity reactions and diseases of the central nervous system which have an inflammatory component (e. g.

Alzheimer's disease, meningitis, multiple sclerosis) HIV and AIDS dementia.

Compounds of the present invention may also be of use in the treatment of nasal polyposis, conjunctivitis or pruritis.

Further examples of disease states in which the compound of the invention have potentially beneficial effects include cardiovascular conditions such as atherosclerosis, peripheral vascular disease and idiopathic hypereosinophilic syndrome. Other diseases for which the compound of the present invention may be beneficial are other hypereosinophilic diseases such as Churg-strauss syndrome. Additionally, eosinophilia is commonly found in

parasitic diseases, especially helminth infections, and thus the compound of the present invention may be useful in treating inflammation arising from hyper-eosinophilic states of diseases such as hydatid cyst (Echinococcus sp. ), tapeworm infections (Taenia sp. ), blood flukes (schistosomiasis), and nematode (round worms) infections such as :- Hookworm (Ancylostoma sp. ), Ascaris, Strongyloides, Trichinella, and particularly lymphatic filariasis including Onchocerca, Brugia, Wucheria (Elephantiasis).

The compound of the invention may be useful as an immunosuppressive agent and so have use in the treatment of auto-immune diseases such as allograft tissue rejection after transplantation, rheumatoid arthritis and diabetes.

Compounds of the invention may also be useful in inhibiting metastasis.

Diseases of principal interest include asthma, COPD and inflammatory diseases of the upper respiratory tract involving seasonal and perennial rhinitis.

Preferred diseases of principal interest include asthma and inflammatory diseases of the upper respiratory tract involving seasonal and perennial rhinitis.

Further diseases also of principle interest include inflammatory diseases of the gastrointestinal tract such as inflammatory bowel disease.

It will be appreciated by those skilled in the art that references herein to treatment or therapy extend to prophylaxis as well as the treatment of established conditions.

No unacceptable toxicological effects are expected when compounds of Formula I are administered in accordance with the present methods.

Synthetic. Methods Synthetic methods to prepare the compounds of this invention frequently employ protective groups to mask a reactive functionality or minimize unwanted side reactions.

Such protective groups are described generally in Green, T. W, Protective Groups in Organic Synthesis, John Wiley & Sons, New York (1981).

Methyl-3-aminobenzoates (1) were loaded onto 2,6-dimethoxy-4- polystyrenebenzyloxy-benzaldehyde (DMHB resin) via reductive amination. Sulfonylation of the resulting resin-bound anilines with various sulfonyl chlorides gave sulfonamides 2.

After hydrolysis, the resulting resin-bound acids were coupled with cyclic diamines to afford intermediates 3. Reductive amination of 3 with various aldehydes, followed by cleavage from resin, afforded the targeted compounds 4.

Scheme 1

Conditions: a) DMHB resin, Na (OAc) 3BH, AcOH, NMP, rt; b) RISO2Cl, pyridine, DMAP, DCE, rt; c) TMSOK, THF, rt; d) cyclic diamines, PyBOP, DIEA, NMP, rt; e) R2CHO, Na (OAc) 3BH, AcOH, NMP, room temp.; f) 20% TFA in DCM, rt.

Experimentals Example 1 Preparation of3-chloro-N- {3-fl- (4-cYclopropylmethyl-fl. 41diazepan-l-yl)-methanovl1- phenyl T-benzenesulfonamide Resin loading : To a 250 mL shaker vessel was added 5. 0g of 2,6-dimethoxy-4- polystyrenebenzyloxy-benzaldehyde (DMHB resin, loading 1.45 mmol/g) and 145 mL of anhydrous N-methyl pyrrolidinone (NMP). 9.9 mL of methyl 3-aminobenzoate (72.5 mmol, 10 eq. ) was then added, followed by 14.5 mL of acetic acid (HOAc). After shaking for 5 min, 16.9g of sodium triacetoxyborohydride (Na (OAc) 3BH, 79.8 mmol, 11 eq. ) was added.

The mixture was shaken at room temperature (rt) for 16 h. The resin was washed with NMP (2x50 mL), methanol (MeOH, 2x50 mL), DCM (dichloromethane, 2x50 mL), MeOH (2x25 mL) and DCM (2x25 mL), and dried in a vacuum oven at 35°C for 16 h. Resin loading: 97% based on N-analysis: 1.17 mmol/g.

Sulfonylation : To the above resin was added 120 mL of dichloroethane (DCE), followed by 40 mL of pyridine (3. 0M) and 11.8g of 3-chlorobenzene sulfonyl chloride (0.35M). After shaking for 5 min, 1.17g of dimethylaminopyridine (DMAP, 0.06M) was added. The mixture was shaken at rt for 16 h. The resin was washed with DCM (4x50 mL), MeOH (2x 50 mL) and DCM (2x25 mL), and dried in a vacuum oven at 35°C for 16 h. An analytical amount of the resin was cleaved with 20% of trifluoroacetic acid (TFA) in DCM for 10 min. The resulting solution was concentrated in vacuo. MS (ESI): 326 [M+H] +.

Hydrolysis : To the above resin was added 100 mL of 0.7 M potassium trimethylsilanolate (TMSOK) in tetrahydrofuran (THF) and shaken at rt for 16 h. The resin

was washed with THF (4x50 mL), MeOH (2x50 mL), DCM (2x50 mL), MeOH (2x25 mL) and DCM (2x25 mL), and dried in a vacuum oven at 35°C for 16 h. An analytical amount of the resin was cleaved with 20% of TFA in DCM for 10 min. The resulting solution was concentrated in vacuo. MS (ESI): 312 [M+H] +.

Amide formation : To the above resin was added 100 mL of NMP, followed by 10.5 mL of diisopropylethyl amine (DIEA, 0.6M) and 12.0 g of homopiperazine (1.2M). After shaking for 2 h (ensuring that all homopiperazine was dissolved), 15.6g of PyBOP (purchased from Novabiochem) (0.3M) was added to the mixture. The resulting mixture was shaken at rt for 16 h. The resin was washed with NMP (4x50 mL), DCM (2x50 mL), MeOH (2x50 mL), DCM (2x25 mL), dried in a vacuum oven at 35°C for 16 h. An analytical amount of the resin was cleaved with 20% of TFA in DCM for 10 min. The resulting solution was concentrated in vacuo. MS (ESI): 394 [M+H] +.

Reductive amination : To 200 mg of the above resin in 3.7 mL NMP was added 0.18 mL of cyclopropanecarboxaldehyde (0.6 M) followed by 0.37 mL of HOAc. After shaking for 30 min, 0. 5 Ig of Na (OAc) 3BH (0.6 M) was added. The mixture was shaken at rt for 16 h, and the resin was washed with NMP (4x5 mL), DCM (2x5 mL), MeOH (2x5 mL), DCM (2x5 mL), MeOH (2x5 mL), DCM (2x5 mL) and dried over vacuum.

Cleavage : To 200 mg of the above resin was added 4 mL of 20% TFA in DCM.

The mixture was shaken at rt for 2h. The TFA solution was concentrated. The residue was purified using a Gilson semi-preparative HPLC system with a YMC ODS-A (C-18) column 50 mm by 20 mm ID, eluting with 10% B to 90% B in 3.2 min, hold for 1 min where A = H20 (0. 1% trifluoroacetic acid) and B = CH3CN (0. 1% trifluoroacetic acid) pumped at 25 mL/min, to produce 3-chloro-N- {3- [1- (4-cyclopropylmethyl- [1, 4] diazepan-l-yl)- methanoyl]-phenyl}-benzenesulfonamide as a mono-trifluoroacetic acid salt (white powder, 89 mg, 91% over 4 steps): MS (ESI) 448 [M+H] +.

Proceeding in a similar manner, but replacing 3-chlorobenzenesulfonyl chloride with the appropriate sulfonyl chlorides and replacing cyclopropanecarboxaldehyde with the appropriate aldehydes, the compounds listed in Table 1 were prepared.

Table 1 Example R1 R2 MS [M+H] + 2 3, 4-dichloro-phenyl 4-hydroxybenzyl 534 3 3,4-dichloro-phenyl ethyl 456 3, 4-dichloro-phenyl isopropyl 470 5 3,4-dichloro-phenyl n-butyl 484 6 3,4-dichloro-phenyl cyclopropylmethyl 482 7 3, 4-dichloro-phenyl n-heptyl 526 8 3, 4-dichloro-phenyl n-propyl 470 9 3, 4-dichloro-phenyl n-pentyl 498 10 3,4-dichloro-phenyl phenethyl 532 11 3, 4-dichloro-phenyl methyl 442 12 3,4-dichloro-phenyl n-octyl 540 13 2,5-dimethoxy-phenyl n-octyl 532 14 3-chloro-phenyl 4-hydroxy-benzyl 500 15 3-chloro-phenyl ethyl 422 16 3-chloro-phenyl isopropyl 436 17 3-chloro-phenyl n-butyl 450 18 3-chloro-phenyl n-hexyl 478 19 3-chloro-phenyl isobutyl 450 20 3-chloro-phenyl n-octyl 506 21 3-chloro-phenyl n-propyl 436 22 3-chloro-phenyl n-pentyl 464 23 3-chloro-phenyl cyclohexylmethyl 490 24 2,4, 5-trichloro-phenyl isopropyl 504 25 2,4, 5-trichloro-phenyl n-butyl 518 26 2,4, 5-trichloro-phenyl cyclopropylmethyl 516 27 2,4, 5-trichloro-phenyl n-pentyl 532 28 2,4, 5-trichloro-phenyl cyclohexylmethyl 558 29 2,4-dichloro-phenyl isopropyl 470 30 2,4-dichloro-phenyl cyclopropylmethyl 482 31 2,4-dichloro-phenyl n-propyl 470 32 3, 4-dibromo-phenyl 4-hydroxybenzyl 622 33 3, 4-dibromo-phenyl ethyl 544 34 3, 4-dibromo-phenyl isopropyl 558 35 3,4-dibromo-phenyl n-butyl 572 36 3,4-dibromo-phenyl cyclopropylmethyl 570 37 3,4-dibromo-phenyl n-propyl 558 38 3,4-dibromo-phenyl n-pentyl 586 39 3-bromo-phenyl 4-hydroxybenzyl 545 40 3-bromo-phenyl ethyl 466 41 3-bromo-phenyl isopropyl 480 42 3-bromo-phenyl n-butyl 494 43 3-bromo-phenyl cyclopropylmethyl 492 44 3-bromo-phenyl n-heptyl 536 45 3-bromo-phenyl n-hexyl 522 46 3-bromo-phenyl isobutyl 494 47 3-bromo-phenyl n-octyl 550 48 3-bromo-phenyl n-propyl 480 3-bromo-phenyl n-pentyl 508 50 4-methoxy-phenyl isopropyl 432 51 4-methoxy-phenyl n-butyl 446 52 4-methoxy-phenyl cyclopropylmethyl 444 53 4-methoxy-phenyl n-hexyl 474 54 4-methoxy-phenyl phenethyl 494 55 4-methoxy-phenyl n-propyl 432 56 4-methoxy-phenyl n-pentyl 460 57 4-trifluoromethyl-phenyl ethyl 456 58 4-trifluoromethyl-phenyl n-butyl 484 59 4-trifluoromethy-lphenyl phenethyl 532 60 3,4-dimethoxy-phenyl 4-hydroxybenzyl 526 61 3,4-dimethoxy-phenyl ethyl 448 62 3,4-dimethoxy-phenyl isopropyl 462 63 3, 4-dimethoxy-phenyl n-butyl 476 64 3,4-dimethoxy-phenyl cyclohexylmethyl 516 65 3,4-dimethoxy-phenyl cyclopropylmethyl 474 66 3,4-dimethoxy-phenyl n-heptyl 518 67 3,4-dimethoxy-phenyl n-hexyl 504 68 3,4-dimethoxy-phenyl isobutyl 476 69 3,4-dimethoxy-phenyl 4-methoxybenzyl 540 70 3,4-dimethoxy-phenyl phenethyl 524 71 3,4-dimethoxy-phenyl n-propyl 462 72 3, 4-dimethoxy-phenyl n-pentyl 490 73 4-propyl-phenyl n-propyl 444 74 4-biphenyl isopropyl 478 75 4-biphenyl cyclopropylmethyl 490 76 4-biphenyl isobutyl 492 77 4-biphenyl phenethyl 540 78 4-biphenyl n-propyl 478 79 4-biphenyl n-pentyl 506 80 4-isopropyl-phenyl n-propyl 444 81 2, 3-dibromothiophen-4-yl n-butyl 578 82 2,3-dibromothiophen-4-yl n-octyl 534 83 2,5-dichlorothiophen-3-yl 4-trifluoromethylbenzyl 592 84 2,5-dichlorothiophen-3-yl cyclohexylmethyl 530 85 2, 5-dichlorothiophen-3-yl n-octyl 546 86 2-bromo-phenyl isopropyl 480 87 2-bromo-phenyl n-butyl 494 88 2-bromo-phenyl n-hexyl 522 89 2,6-dichloro-phenyl 4-trifluoromethylbenzyl 586 90 2,6-dichloro-phenyl 4-hydroxybenzyl 534 91 4-chloro-2,5-dimethyl-phenyl 4-hydroxybenzyl 528 92 4-chloro-2,5-dimethyl-phenyl ethyl 450 93 4-chloro-2,5-dimethyl-phenyl isopropyl 464 94 4-chloro-2,5-dimethyl-phenyl n-butyl 478 95 4-chloro-2,5-dimethyl-phenyl cyclohexylmethyl 518 96 4-chloro-2, 5-dimethyl-phenyl cyclopropylmethyl 476 97 4-chloro-2,5-dimethyl-phenyl n-heptyl 520 98 4-chloro-2, 5-dimethyl-phenyl n-hexyl 506 99 4-chloro-2,5-dimethyl-phenyl n-octyl 534 100 4-chloro-2,5-dimethyl-phenyl n-propyl 464 101 4-chloro-2,5-dimethyl-phenyl phenethyl 526 102 4-chloro-2,5-dimethyl-phenyl n-pentyl 492 103 5-bromo-2-methoxy-phenyl 4-hydroxybenzyl 574 104 5-bromo-2-methoxy-phenyl cyclopropylmethyl 522 105 5-bromo-2-methoxy-phenyl n-hexyl 552 106 5-bromo-2-methoxy-phenyl isobutyl 524 107 5-bromo-2-methoxy-phenyl n-octyl 580 108 5-bromo-2-methoxy-phenyl n-propyl 510 109 3,4-dichloro-phenyl n-hexyl 512 110 5-bromothiophen-2-yl n-propyl 486 111 2,5-dichlorothiophen-3-yl n-propyl 476 112 4-trifluoromethoxy-phenyl n-propyl 486 113 3,4-dimethoxy-phenyl n-octyl 532 114 4-trifluoromethyl-phenyl n-propyl 470 115 2,4, 5-trichloro-phenyl n-propyl 504 116 2,4-dichloro-phenyl n-butyl 484 117 4-trifluoromethoxy-phenyl n-butyl 500 118 4-n-propyl-phenyl n-butyl 458 119 4-biphenyl n-butyl 492 120 4-isopropyl-phenyl n-butyl-458 121 5-chlorothiophen-2-yl n-butyl 456 122 5-bromothiophen-2-yl n-butyl 500 123 2, 5-dichlorothiophen-2-yl n-butyl 500 124 4-bromo-2,5-difluoro-phenyl n-butyl 530 125 2, 5-dimethoxy-phenyl n-butyl 476 126 2-fluoro-phenyl n-butyl 434 127 5-bromo-2-methoxy-phenyl n-butyl 524 128 4,5-dibromothiophen-2-yl cyclopropylmethyl 576

Proceeding in a similar manner, but replacing methyl 3-aminobenzoate with the appropriate substituted methyl-3-aminobenzoates, replacing 3-chlorobenzenesulfonyl chloride with the appropriate sulfonyl chlorides and replacing cyclopropanecarboxaldehyde with the appropriate aldehydes, and using either homopiperazine or piperazine, the compounds listed in Table 2 were prepared.

Table 2

Example R3 Rl R2 n MS [M+H] + 129 6-chloro 4-t-butyl-phenyl isobutyl 0 492 130 6-methyl 4-n-butyl-phenyl cyclopropylmethyl 0 470 131 6-methyl 4-n-butoxy-phenyl n-propyl 0 474 132 6-methyl 4-n-butoxy-phenyl cyclopropylmethyl 0 486 133 6-methyl 4-n-butoxy-phenyl n-butyl 0 488 134 6-methyl 4-n-butoxy-phenyl isobutyl 0 488 135 6-methyl 4-n-butoxy-phenyl methyl 0 446 136 6-methyl 4-n-butoxy-phenyl ethyl 0 460 137 6-methyl 4-biphenyl methyl 0 450 138 6-methyl 4-biphenyl ethyl 0 464 139 6-methyl 4-biphenyl n-propyl 0 478 140 6-methyl 4-biphenyl cyclopropylmethyl 0 490 141 6-methyl 4-biphenyl n-butyl 0 492 142 4-methoxy 3,4-dimethoxyphenyl n-propyl 1 492 143 6-methoxy 3,4-dimethoxyphenyl n-propyl 1 492 144 6-methyl 3,4-dimethoxyphenyl n-propyl 1 476 145 2-methyl 3,4-dimethoxyphenyl n-propyl 1 476 146 4-chloro 3,4-dimethoxyphenyl n-propyl 1 496 147 4-bromo 3, 4-dimethoxyphenyl n-propyl 1 540 148 6-chloro 3,4-dimethoxyphenyl n-propyl 1 496 149 5-nitro 3, 4-dimethoxyphenyl n-propyl 1 507 150 4-methoxy 3,4-dimethoxyphenyl n-butyl 1 506 151 6-methoxy 3, 4-dimethoxyphenyl n-butyl 1 506 152 6-methyl 3,4-dimethoxyphenyl n-butyl 1 490 153 2-methyl 3,4-dimethoxyphenyl n-butyl 1 490 154 4-chloro 3,4-dimethoxyphenyl n-butyl 1 510 155 4-bromo 3,4-dimethoxyphenyl n-butyl 1 554 156 6-chloro 3,4-dimethoxyphenyl n-butyl 1 510 157 5-nitro 3,4-dimethoxyphenyl n-butyl 1 521 158 4-methoxy 3,4-dimethoxyphenyl n-pentyl 1 520 159 6-methoxy 3,4-dimethoxyphenyl n-pentyl 1 520 160 6-methyl 3,4-dimethoxyphenyl n-pentyl 1 504 161 2-methyl 3,4-dimethoxyphenyl n-pentyl 1 504 162 4-chloro 3,4-dimethoxyphenyl n-pentyl 1 524 163 4-bromo 3,4-dimethoxyphenyl n-pentyl 1 568 164 6-chloro 3,4-dimethoxyphenyl n-pentyl 1 524 165 5-nitro 3,4-dimethoxyphenyl n-pentyl 1 535 166 4-methoxy 3,4-dimethoxyphenyl cyclopropylmethyl 1 504 167 6-methoxy 3,4-dimethoxyphenyl cyclopropylmethyl 1 504 168 6-methyl 3,4-dimethoxyphenyl cyclopropylmethyl 1 588 169 2-methyl 3,4-dimethoxyphenyl cyclopropylmethyl 1 588 170 4-chloro 3,4-dimethoxyphenyl cyclopropylmethyl 1 508 171 4-bromo 3,4-dimethoxyphenyl cyclopropylmethyl 1 552 172 6-chloro 3,4-dimethoxyphenyl cyclopropylmethyl I 508 173 4-chloro 3,4-dichloro-phenyl 3-methyl-butyl 1 532

Bioassays Biological Assay for the Determination of the Inhibition of I-309-mediated Intracellular Calcium Mobilization (FLIPR Assay) RBL-2H3 cells expressing the human CCR-8 receptor were grown in cell medium (EMEM medium with Earl's salts) containing 2mM L-Glutamine, 0.4 mg/ml G418 Sulfate from GIBCO BRL and 10% heat inactivated fetal calf serum from Hyclone Laboratories.

The cells were seeded 60,000 cells, /well into 96-well black clear bottom sterile plates from Costar (Corning Life Sciences, Acton, MA, USA). The seeded plate was incubated overnight at 37° C in 5% CO2. On the day of the assay the cell medium was aspirated before addition of calcium dye loading solution consisting of : 1 mg/mL bovine serum albumin (BSA), 1.5 mM sulfinpyrazone from SIGMA and 4 uM Fluo-3 AM dye from

Molecular Probes in cell medium, thereafter the 96-well plate was incubated for 1 hour at 37° C.

After aspirating the solution, 100 #Ls of fresh assay buffer (Kreb's Ringer Henseleit pH 7.4 containing 1 mM CaC12, 1. 1 mM MgC12, 1.5 mM sulfinpyrazone and 1.0 mg/mL gelatin) was added to all the wells and the plate was incubated for 10 minutes at 37 °C. before transferring to the Fluorescent Imaging Plate Reader (FLIPR) instrument. The assay and data acquisition were initiated by first addition of 50'Is of compound diluted to a relevant concentration in assay buffer. After 5 mins read 2nd addition of 50-ils human I-309 challenge, diluted to an appropriate concentration in assay buffer with 1 mg/mL BSA (no gelatin) was added to the plate and data was acquired from an additional 1.5 mins.

Concentration response data for compounds showing inhibition of calcium mobilization were performed in the presence of 35 nM human 1-309 to obtain the ICI values. IC50 is the concentration of compound needed to inhibit 50% of the human I-309 response.

Chemotaxis Assay The effect of compounds on I-309 mediated chemotaxis was evaluated by measuring the migration of a T cell line, HUT78 (ATCC, Manassas, Virginia, USA), through 3llm ChemoTX filter systems (Neuroprobe, Gaithersburg, Maryland, USA).

For the experimental procedure, cells were handled in RPMI/1% BSA. HUT78s were labelled with 2) 1 Calcein AM (Molecular Probes, Eugene, Oregon, USA, 5mg/ml stock solution in DMSO) per lx 107 cells/ml for 30 min at room temperature in the dark. 30 111 of medium was placed in each well of the lower compartment of the filter systems. 20 nM I-309 was usually the starting concentration for dose response curves. For inhibition experiments a constant concentration of 0.5 nM I-309 was used in combination with escalating concentrations of compound. 50 Ll of cells at a concentration of 2x 107/ml were incubated with 50 ul of a 2x concentration of compound in 96 well plates and pre-incubated for 5 min at room temperature. Therefore the equivalent concentration of compound was present in the upper and lower compartment of each well. 50 RI of cells were then placed on top of the filter system for each well. The filter system was incubated for 90 min at 37° C in the incubator and then the whole system was washed with one litre of phosphate buffered saline at a 45'angle. The membrane filter was measured using a fluorescence filter set at 485/535nM. Chequer board analysis and confocal microscopy confirmed that responses to I- 309 are chemotactic and are not chemokinetic or an adhesion event.