OA12231A - IL-8 receptor antagonists. - Google Patents

Abstract

This invention relates to novel compounds of Formula (I) to (VII), and compositions thereof, useful in the treatment of disease states mediated by the chemokine, Interleukin-8 (IL-8).

Description

IL-8 RECEPTOR ANTAGONISTS 012231

FIELD OF THE INVENTION

This invention relates to novel sulfonamide substituted diphenyl ureacompounds, pharmaceutical compositions, processes for their préparation, and use 5 thereof in treating IL-8, GROa, GROp, GROy, NAP-2, and ENA-78 mediated diseases.

BACKGROUND OF THE INVENTION

Many different names hâve been applied to Interleukin-8 (IL-8), such asneutrophil attractant/activation protein-1 (NAP-1), monocyte derived neutrophil 10 chemotactic factor (MDNCF), neutrophil activating factor (NAF), and T-cell lymphocyte chemotactic factor. Interleukin-8 is a chemoattractant for neutrophils,basophils, and a subset of T-cells. It is produced by a majority of nucleated cellsincluding macrophages, fibroblasts, endothélial and épithélial cells exposed toTNF, IL-la, IL-Ιβ or LPS, and by neutrophils themselves when exposed to LPS or 15 chemotactic factors such as FMLP. M. Baggiolini et al., J. Clin. Invest. 84,1045(1989); J. Schroder et al, J. Immunol. 139, 3474 (1987) and J. Immunol. 144, 2223 (1990) ; Strieter, et al., Science 243,1467 (1989) and J. Biol. Chem. 264,10621 (1989); Cassatella et al., J. Immunol. 148. 3216 (1992). . GROa, GROP, GROy and NAP-2 also belong to the chemokine family. 20 Like IL-8 these chemokines hâve also been referred to by different names. Forinstance GROa, β, y hâve been referred to as MGSAa, β and yrespectively(Melanoma Growth Stimulating Activity), see Richmond et al., J. Cell Physiology129,375 (1986) and Chang et al., J. Immunol 148,451 (1992). AU of thechemokines of the α-family which possess the ELR motif directly preceding the 25 CXC motif bind to the IL-8 B receptor (CXCR2). IL-8, GROa, GROp, GROy, NAP-2, and ENA-78 stimulate a number of fonctions in vitro. They hâve aU been shown to hâve chemoattractant propertiesfor neutrophils, while IL-8 and GROa hâve demonstrated T-lymphocytes, andbasophilie chemotactic activity. In addition IL-8 can induce histamine release 30 from basophils ffom both normal and atopie individuals. GRO-α and IL-8 can inaddition, induce lysozomal enzyme release and respiratory burst from neutrophils. 012231 IL-8 has also been shown to increase the surface expression of Mac-1 (CD1 lb/CD18) on neutrophils without de novo protein synthesis. This maycontribute to increased adhesion of the neutrophils to vascular endothélial cells.Many known diseases are characterized by massive neutrophil infiltration. As EL-8, GROa, GROP, GROy and NAP-2 promote the accumulation and activation ofneutrophils, these chemokines hâve been implicated in a wide range of acute andchronic inflammatory disorders including psoriasis and rheumatoid arthritis,Baggioiini et al., FEBS Lett. 307. 97 (1992); Miller et al., Crit. Rev. Immunol, 12.17 (1992); Oppenheim et al., Annu. Rev. Immunol. 9, 617 (1991); Seitz et al., J.Clin. Invest. 87,463 (1991); Miller et al., Am. Rev. Respir. Dis. 146.427 (1992);Donnely et al., Lancet 341. 643 (1993). In addition the ELR chemokines (thosecontaining the amino acids ELR motif just prior to the CXC motif) hâve also beenimplicated in angiostasis, Strieter et al., Science 258, 1798 (1992).

In vitro, IL-8, GROa, GROp, GROyand NAP-2 induce neutrophil shapechange, chemotaxis, granule release, and respiratory burst, by binding to andactivating receptors of the seven-transmembrane, G-protein-linked family, inparticular by binding to IL-8 receptors, most notably the IL-8P receptor (CXCR2).Thomas et al., J. Biol. Chem. 266. 14839 (1991); and Holmes et al., Science 253.1278 (1991). The development of non-peptide small molécule antagoniste formembers of this receptor family has precedent. For a review see R. Freidinger inProgress in Drug Research, Vol. 40, pp. 33-98, Birkhauser Verlag, Basel 1993.Hence, the IL-8 receptor represents a promising target for the development ofnovel anti-inflammatory agents.

Two high affinity human IL-8 receptors (77% homology) hâve beencharacterized: IL-8Ra, which binds only IL-8 with high affinity, and IL-8RP,which has high affinity for EL-8 as well as for GROa, GROp, GROyand NAP-2.See Holmes et al., supra; Murphy et al., Science 253, 1280 (1991); Lee et al., J.Biol. Chem. 267,16283 (1992); LaRosa et al., J. Biol. Chem. 267.25402 (1992);and Gayle et al., J. Biol. Chem. 268.7283 (1993).

There remains a need for treatment, in this fîeld, for compounds, which are capable of binding to the IL-8 a or P receptor. Therefore, conditions associated with an increase in IL-8 production (which is responsible for chemotaxis of -2- 012231 , neutrophil and T-cells subsets into the inflammatory site) would benefit by compounds, which are inhibitors of IL-8 receptor binding.

« SUMMARY OF THE INVENTION -

This invention provides for a method of treating a chemokine mediated5 disease, wherein the chemokine is one which binds to an IL-8 a or b receptor and which method comprises administering an effective amount of a compound ofFormula (I) or a pharmaceutically acceptable sait thereof. In particular thechemokine is IL-8.

This invention also relates to a method of inhibiting the binding of IL-8 to10 its receptors in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I).

The présent invention also provides for the novel compounds of Formula (I), and pharmaceutical compositions comprising a compound of Formula· (I), and apharmaceutical carrier or diluent. 15 Compounds of Formula (I) useful in the présent invention are represented by the structure:

20 wherein

Rb is independently selected rom the group consisting of hydrogen, NR6R7, OH, ORa,Ci-5alkyl, aryl, arylCi-4alkyl, aryl C2-4alkenyl; cycloalkyl, cycloalkyl Cj_5alkyl, heteroaryl, heteroarylCj^alkyi, heteroarylC2-4 alkenyl, heterocyclic,heterocyclic Ci_4alkyl, and a heterocyclic C2-4alkenyl, ail of which moieties may 25 be optionally substituted one to three times independently by a substituent selectedfrom the group consisting of halogen, nitro, halosubstituted C1-4 alkyl, C1-4 alkyl, -3- 012231 amino, mono or di-Ci.4 alkyl substituted amine, ORa, C(O)Ra, NRaC(O)ORa, JOC(O)NR6R7, hydroxy, NR9C(O)Ra, S(O)m’Ra, C(O)NR6R7, C(O)OH, C(O)ORa, S(O)2NRôR7, NHS(O)2Ra, or, the two R5 substituents join to form a 3-10 membered ring, optionally substituted and containing, in addition to carbon, 5 independently, 1 to 3 moieties selected from the group consisting of NRa, O, S, S O, or SO2; and wherein the substituent can be optionally unsaturated;

Ra is selected from the group consisting of alkyl, aryl, arylCi_4alkyl, heteroaryl, heteroaryl Ci_4alkyl, heterocyclic, COOR13, and a heterocycîic Ci_4alkyl moiety,ail of which moieties may be optionally substituted; 10 m is an integer having a value of 1 to 3; m’is 0, or an integer having a value of 1 or 2;n is an integer having a value of 1 to 3;q is 0, or an integer having a value of 1 to 10;t is 0, or an integer having a value of 1 or 2; 15 s is an integer having a value of 1 to 3;

Rl is independently selected from the froup consisting of hydrogen, halogen, nitro, cyano, Ci-io alkyl, halosubstituted Cl-io alkyl, C2-10 alkenyl, Cl-ίο alkoxy,halosubstituted Ci-ioalkoxy, azide, S(O)tR4, (CRgRg)q S(O)tR4, hydroxy, hydroxysubstituted Ci_4alkyl, aryl, aryl Ci_4 alkyl, aryl C2-10 alkenyl, aryloxy, aryl Ci_4 20 alkyloxy, heteroaryl, heteroarylalkyl, heteroaryl C2-IO alkenyl, heteroaryl Ci-4alkyloxy, heterocyclic, heterocyclic Ci-4alkyl, heterocyclicCi-4alkyloxy,heterocyclicC2-10 alkenyl, (CRgRg)q NR4R5, (CRgRg)qÇ(O)NR4R5, C2-10alkenyl C(O)NR4R5, (CRgRg)q C(O)NR4Rl0, S(O)3R8, (CRgRg)q C(O)Rl 1, C2-IO alkenyl C(O)Rn, C2-10 alkenyl C(O)ORn, (CRgRg)q C(O)ORn, 25 (CRgRg)q OC(O)Ri 1 ? (CR8Rg)qNR4C(O)Ri 1, (CRgRg)q C(NR4)NR4R5, (CRgRg)q NR4C(NR5)R1 b (CRgRg)q NHS(O)2Ri3, and (CRgRg)q S(O)2NR4R5;or two Ri moieties together may form O-(CH2)sO or a 5 to 6 membered saturated orunsaturated ring, and wherein the alkyl, aryl, arylalkyl, heteroaryl, heterocyclicmoieties may be optionally substituted; -4- 012231 R4 and R5 are independently selected from the group consisting of hydrogen, optionally substituted Cl.4 alkyl, optionally substituted aryl, optionally substitutedaryl Ci_4alkyl, optionally substituted heteroaryl, optionally substituted heteroarylCi-4alkyl, heterocyclic, and heterocyclicCi-4 alkyl; or R4 and R5 together with 5 the nitrogen to which they are attached form a 5 to 7 member ring which mayoptionally comprise an additional heteroatom selected from O, N and S; R.6 and R7 are independently selected from the group consisting of hydrogen, Cl.4alkyl, heteroaryl, aryl, aklyl aryl, and alkyl Ci.4 heteroalkyl; or Rô and R7together with the nitrogen to which they are attached form a 5 to 7 member ring 10 which ring may optionally contain an additional heteroatom is selected fromoxygen, nitrogen and sulfur; wherein the ring may be optionally substituted; Y is selected from the group conisting of CR14C15, NR14, O, CO, and S (O)tRg is hydrogen or C1-4 alkyl; R9 is Ci-4 alkyl; 15 Rio isCi-10 alkyl C(O)2R8;

Rl 1 is selected from the group consisting of hydrogen, optionally substituted Ci.4alkyl, optionally substituted aryl, optionally substituted aryl Ci_4alkyl, optionallysubstituted heteroaryl, optionally substituted heteroaryICi-4alkyl, optionallysubstituted heterocyclic, and optionally substituted heterocyclicCi_4alkyl; and 20 R12 is selected from the group consisting of hydrogen Ci.4 alkyl, aryl, aryl Ci-4alkyl, heteroaryl, heteroarylCi-4alkyl, heterocyclic, and heterocyclicCi-4alkyl;

Rl3 is selected from the group consisting of Ci_4 alkyl, aryl, aryl Ci-4alkyl,heteroaryl, heteroarylCi_4alkyl, heterocyclic, and heterocyclicCi-4alkyl; R14 and R15 are, independently, selected from the group consisting of hydrogen, 25 optionally substituted Cl-4 alkyl group, ORa, and NR4R5; or R14 and R15 together with the atom (s) to which they are attached may form a 4 to 7 memberring which may optionally contain an additional heteroatom which heteroatom isselected from the group consisting of oxygen, nitrogen and sulfur; wherein the ringmaybe optionally substituted; 30 or a pharmaceutically acceptable sait thereof. -5- 012231

DETAILED DESCRIPTION OF THE INVENTION

The compounds of Formula (I), may also be used in association with theveterinary treatment of mammals, other than humans, in need of inhibition of IL-8or other chemokines which bind to the IL-8 a and β receptors. Chemokinemediated diseases for treatment, therapeutically or prophylactically, in animaisinclude disease States such as those noted herein in the Methods of Treatmentsection.

Suitably, Rb is independently hydrogen, NR6R7, OH, ORa, Ci-4alkyl, aryl,aiylCi-4alkyl, aryl C2-4alkenyl, heteroaryl, heteroarylCi_4alkyl, heteroarylC2-4alkenyl, heterocyclic, heterocyclic Ci_4alkyl, or a heterocyclic C2-4alkenyl moiety, ailof which moieties may be optionally substituted one to three times independently byhalogen, nitro, halosubstituted Cl-4 alkyl, Cl-4 alkyl, amino, mono or di-Ci_4 alkylsubstituted amine, cycloalkyl, cycloalkyl Ci.5 alkyl, ORa, C(O)Ra, NRaC(O)ORa,OC(O)NRgR7, aryloxy, aryl C}_4 oxy, hydroxy, Ci_4 alkoxy, NR9C(O)Ra,S(O)m’Ra, C(O)NR6R7, C(O)OH, C(O)ORa, S(O)2NR6R7, NHS(O)2Ra.Altematively, the two Rb substituents can join to form a 3-10 membered ring,optionally substituted and containing, in addition to carbon, independently, 1 to 3NR9, O, S, S O, or S O2 moitiés which can be optionally substituted.

Suitably, Rais an alkyl, aryl, arylCi_4alkyl, heteroaryl, heteroaryl Ci_4alkyl,heterocyclic, or a heterocyclic Ci-4alkyl moiety, ail of which moieties may beoptionally substituted.

Suitably, Ri is independently selected from hydrogen; halogen; nitro; cyano;halosubstituted Ci_io alkyl, such as CF3, Ci-io alkyl, such as methyl, ethyl,isopropyl, or n-propyl, C2-io alkenyl, Ci-io alkoxy, such as methoxy, or ethoxy;halosubstituted Ci_io alkoxy, such as trifluoromethoxy, azide, (CRgRg)q S(0)^4,wherein t is 0, 1 or 2, hydroxy, hydroxy Ci-4alkyl, such as methanol or éthanol,aryl, such as phenyl or naphthyl, aryl Ci.4 alkyl, such as benzyl, aryloxy, such asphenoxy, aryl Cl-4 alkyloxy, such as benzyloxy; heteroaryl, heteroarylalkyl,heteroaryl C1-4 alkyloxy; aryl C2-l0 alkenyl, heteroaryl C2-IO alkenyl, -6- 012231 heterocyclic C2-10 alkenyl, (CR8R8)qNR4R5, C2-10 alkenyl C(O)NR4R5,(CR8R8)qC(O)NR4R5, (CR8R8)qC(O)NR4Rl0, S(O)3H, S(O)3R8,(CR8R8)qC(O)Rn, C2-IO alkenyl C(O)Ri 1, C2-10 alkenyl C(O)ORn, (CR8R8)qC(O)Rn, (CR8R8)qC(O)ORn, (CR8R8)q OC(O)Rn, (CR8R8)qNR4C(O)Rn, 5 (CR8R8)qC(NR4)NR4R5, (CR8R8)q NR4C(NR5)R! b (CR8R8)qNHS(O)2R13,(CR8R8)qS(O)2NR4R5- AU of the aryl, heteroaryl, and heterocyclic-containingmoieties may be optionally substituted as defîned herein below.

For use herein the term "the aryl, heteroaryl, and heterocyclic containingmoieties" refers to both the ring and thé alkyl, or if included, the alkenyl rings, 10 such as aryl, arylalkyl, and aryl alkenyl rings. The term "moieties" and "rings"may be interchangeably used throughout.

Suitably, R4 and R5 are independently hydrogen, optionally substituted Cb4 alkyl, optionally substituted aryl, optionaUy substituted aryl Cb4alkyl,optionally substituted heteroaryl, optionally substituted heteroaryl Ci-4alkyl, 15 heterocyclic, heterocyclicCb4 alkyl, or Rzj. and R5 together with the nitrogen towhich they are attached form a 5 to 7 member ring which may optionaUy comprisean additional heteroatom selected from O, N and S.

Suitably Rô and R7 are hydrogen, or a Cb4 alkyl, heteroaryl, alkyl Cb4heteroalkyl or Rô and R7 together with the nitrogen to which they are attached 20 form a 5 to 7-member ring which ring may optionaUy contain an additional heteroatom that is selected from oxygen, nitrogen or sulfur, and which ring may beoptionally substituted;

Suitably, R8 is independently hydrogen or Ci_4 alkyl.

Suitably, R9 is hydrogen or a Cl-4 alkyl; 25 Suitably, q is 0 or an integer having a value of 1 to 10.

Suitably, Rio is Ci-ίο alkyl C(O)2R8, such as CH2C(O)2H or CH2C(O)2CH3.

Suitably, R11 is hydrogen, Ci4 alkyl, aryl, aryl Ci-4 alkyl, heteroaryl,heteroaryl Cb4alkyl, heterocycüc, or heterocycüc Cb4alkyl.

Suitably, R12 is hydrogen, Cbio alkyl, aryl or arylalkyl. 30 -7- WO 01/6808 9,3 223 1

Suitably, R13 is Ci-4alkyl, aryl, arylalkyl, heteroaryl, heteroarylCi-4alkyl, J heterocyclic, or heterocyclicCi_4aIkyl, wherein ail of the aryl, heteroaryl and * heterocyclic containing moieties may ail be optionally substituted. · R14 and R15 are suitably hydrogen, optionally substituted Ci-4 alkyl group, 5 ORa, NR4R5 or R14 and R15 together with the atom (s) to which they are attachedmay form a 4 to 7 member ring which may optionally contain an additional heteroatomwhich is selected from oxygen, nitrogen or sulfur; this ring maybe optionallysubstituted.

Suitably, Y is CR14R15, NR14, O 10 Suitably, Ra is an alkyl, aryl C1.4 alkyl, heteroaryl, heteroaryl-C^alkyl, heterocyclic, or a heterocyclicCi.4 alkyl, wherein ail of these moieties may ail beoptionally substituted. ’

As used herein, "optionally substituted" unless specifically defxned shallmean such groups as halogen, such as fluorine, chlorine, bromine or iodine, 15 hydroxy; hydroxy substituted Ci-ioalkyl, Ci-io alkoxy, such as methoxy orethoxy, S(O)m’ Cl-ίο alkyl, wherein m’is 0,1 or 2, such as methyl thio, methylsulfinyl or methyl sulfonyl; amino, mono & di-substituted amino, such as in theNR8Rl2 group, NHC(O)Ri3, C(O)NRsRl2, C(O)OH, S(O)2NR8Rl2, NHS(O)2Rl3, Cl-ίο alkyl, such as methyl, ethyl, propyl, isopropyl, or t-butyl, 20 halosubstituted Cl-10 alkyl, such CF3, an optionally substituted aryl, such asphenyl, or an optionally substituted arylalkyl, such as benzyl or phenethyl,optionally substituted heterocylic, optionally substituted heterocyclicalkyl,optionally substituted heteroaryl, optionally substituted heteroaryl alkyl, whereinthese atyl, heteroaryl, or heterocyclic moieties may be substituted one to two times 25 by halogen; hydroxy; hydroxy substituted alkyl, Cj-io alkoxy; S(0)mCi-io alkyl;amino, mono & di-substituted alkyl amino, such as in the NR6R7 group; Cl-10alkyl, or halosubstituted Cl-10 alkyl, such as CF3.

Suitable phannaceutically acceptable salts are well known to those skilled in the art and include basic salts of inorganic and organic acids, such as 30 hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methane sulphonic acid, ethane sulphonic acid, acetic acid, malic acid, tartaric acid, citric -8- 1 012231 » acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid and mandelic acid. In addition, pharmaceutically f acceptable salts of compounds of Formula (I) may also be formed with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations 5 are well known to those skilled in the art and include alkaline, alkaline earth,ammonium and quatemary ammonium cations.

The following terms, as used herein, refer to: • "halo" - ail halogens, that is chloro, fluoro, bromo and iodo. • "Ci-ioalkyl" °r "alkyl" - both straight and branched chain moieties of 110 to 10 carbon atoms, unless the chain length is otherwise limited, including, but not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl and the like. • "cycloalkyl" is used herein to mean cyclic moiety, preferably of 3 to Scarbons, including but not limited to cyclopropyl, cyclopentyl, cyclohexyl, and the 15 like. • "alkenyl" is used'herein at ail occurrences to mean straight or branchedchain moiety of 2-10 carbon atoms, unless the chain length is limited thereto,including, but not limited to ethenyl, 1-propenyI, 2-propenyI, 2-methyl-l-propenyl,1-butenyî, 2-butenyl and the like. 20 · "aryl" - phenyl and naphthyl; • "heteroaryl" (on its own or in any combination, such as "heteroaryloxy",or "heteroaryl alkyl") - a 5-10 membered aromatic ring System in which one ormore rings contain one or more heteroatoms selected from the group consisting ofN, O or S, such as, but not limited, to pyrrole, pyrazole, furan, thiophene, 25 quinoline, isoquinoline, quinazolinyl, pyridine, pyrimidine, oxazole, tetrazole,thiazole, thiadiazole, triazole, imidazole, or benzimidazole. • "heterocyclic" (on its own or in any combination, such as"heterocyclicalkyl") - a saturated or partially unsaturated 4-10 membered ringSystem in which one or more rings contain one or more heteroatoms selected from 30 the group consisting of N, O, or S; such as, but not limited to, pyrrolidine,piperidine, piperazine, morpholine, tetrahydropyran, thiomorpholine, or -9- 012231 imidazolidine. Furthermore, sulfur may.be optionally oxidized to the sulfone orthe sulfoxide. • "arylalkyl" or "heteroarylalkyl" or "heterocyclicalkyl" is used herein to · mean Cj-jo alkyl, as defined above, attached to an aryl, heteroaryl or heterocyclicmoiety, as also defined herein, unless otherwise indicated. • "sulfmyl" - the oxide S (O) of the corresponding sulfide, the terni "thio"refers to the sulfide, and the term "sulfonyl" refers to the fully oxidized S(O)2moiety. • "wherein two Ri moieties may together form a 5 or 6 memberedsaturated or unsaturàted ring" is used herein to mean the formation of an aromaticring System, such as naphthalene, or is a phenyl moiety having attached a 6membered partially saturated or unsaturated ring such as a C6 cycloalkenyl, i.e.hexene, or a C5 cycloalkenyl moiety, such as cyclopentene.

Illustrative compounds of Formula (I) include:N-(3-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N'-cyclohexylurea; N-(3 -aminosulfony l-4-chloro-2-hy droxypheny 1)-N' -( 1 -adamantyl)urea; N-(3-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N'-(tetrahydro-2-pyranyl)urea; N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N'-(3-tetrahydrofuryl)urea; 6-Ch]oro-2-hydjroxy-3-[3-(2-methyl-cyclopropyl)-ureido]-benzenesulfonamide; N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N'-cyclohexylurea; 6-Chloro-2-hydroxy-3-[3-(2,2,3,3-tetrmethyl-cyclopropyl)-ureido]~ benzenesulfonamide ; 6-Chloro-2-hydroxy-3-(3-piperidin-4-yl-ureido)-benzenesulfonamide; N-[4-chloro-2-hydroxy-3-sulfamylphenyl]~N’-(4-methyl-cyclohexyI) urea; 6-Chloro-2-hydroxy-3-[3-(3-methoxy-cyclohexyl)-ureido]-benzenesulfonamide; N-[4-chIoro-2-hydroxy-3-sulfamylphenyl]-N’-cyclopentylurea; N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N'-cyclobutylurea; N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N'-cyclopropylurea; 4-[6-Chloro-3-(3-cyclopentyl-ureido)-2-hydroxy-benzenesulfonyl]-piperazine-l- carboxylic acid tert butyl ester; 1 -[4-Chloro-2-hydroxy-3-(piperazine-1 -sulfonyl)-phenyl]-3-cyclopentyl-urea; -10- 012231 4-[6-Chloro-3-(3-cyclobutyl-ureido)-2-hydroxy-benzenesulfonyl]-piperazine-l-carboxylic acid tert-butyl ester; 3-[3-((lS,2S)-2-Benzyloxy-cyclohexyl)-ureido]-6-chloro-2-hydroxy-benzenesuifonamide; and 5 6-Chloro-3-(3-cyclobutyl-ureido)-2-hydroxy-N,N’-dimethyl-benzenesulfonamide.

METHODS OF PREPARATION

The compounds of Formulas (I) may be obtained by applying syntheticprocedures, some of which are illustrated in the Schemes below. The synthesisprovided for in these Schemes is applicable for the producing compounds of 10 Formulas (I), having a variety of different R, Rb, and Y groups which are reacted,employing optional substituents which are suitably protected, to achievecompatibility with the reactions outlined herein. Subséquent deprotection, in thosecases, then affords compounds of the nature generally disclosed. Once the ureanucléus has been established, further compounds of these formulas may be 15 prepared by applying standard techniques for functional group interconversion,well known in the art.

Scheme 1

11- Q1 2231 *» a)i)NCS, AcOH, H2O, ii NR’R’TI, pyr b)H2SO4, HNO3 c)NaOAc, 18-crown-6 d)H2SO4, MeOH e) Pd/C, H2 f)RCNO, DMF ,

The desired 4-chloro N-(3-sulfonamido-2-hydroxy phenyl)-N"-cycloalkylurea can synthesized from the commercially available 2, 6-dichloro thiophenolusing the procedure elaborated above in Scheme 1. The thiol can be oxidized tothe corresponding sulfonyl halide using a halogenating agent, such as NCS, NBS,Cl2 or Br2, in the presence of a protic solvent, such as water, acetic acid, or analcohol or combination. The yield may be increâsed if a buffering agent, such assodium or potassium acetate is included in the reaction mixture, and the reaction isconducted at or below room température. The corresponding sulfonyl halide canthen be condensed with an amine in presence of a base such as pyridine, triethylamine, potassium carbonate or sodium hydride to form the analogous sulfonamide 2-scheme 1. The dichlorosulfonamide 2-scheme 1 can be nitrated using strongnitrating conditions such as nitric acid in sulfuric acid to form the aromatic nitrocompound 3-scheme 1. The chlorine ortho to the nitro group can be selectivelyhydrolyzed using acetate sait such as sodium acetate in the presence of a crownether, such as 18-crown-6, to form the acetate 4-scheme 1. The acetate group canbe hydrolyzed under acidic conditions in an alcohol solvent such as methanol oréthanol with a catalytic amount of acid to form the phénol 5-scheme 1. The nitrocan be reduced by conditions well known in the art such as hydrogen andpalladium on carbon, tin chloride in methanol, zinc in acetic acid or thiol to formthe corresponding aniline 5-scheme 1. The aniline can then be çoupled with acommercially available isocyanate or thioisocyanate to form the desired urea orthio urea. Altematively the desired isocyanates can be made by condensing theamine with triphosgene in the presence of base (such as potassium carbonate) or byreacting the carboxylic acid with diphenyl phosphoryl azide in the presence of abase (such as triethyl amine). - 12- ♦

Scheme 2 012231

a)NaH, RX b)NaH R"X

If the sulfonamide 1-scheme 2 (3-scheme 1) is unfunctionalized R’=R"=H5 then it can be functionalized as required herein, by alkylation. The sulfonamide is . deprotonated using a base such as sodium hydride and then alkylated using an alkylhalide such as benzyl bromide or methyl iodide form 2-scheme 2. The sulfonamidecan then be alkylated a second time using sodium hydride and another alkyl halideto form 3-scheme 2. This compound can then be converted to the desired urea 10 using the process elaborated in scheme 1.

Scheme 3

15 a)i)NCS, AcOH, H2O ii)NaOH MeOH b)H2SO4, HNO3 c)NaOH MeOH d) PCI5,POCI3 e)NHRR", Et3N

An alternative route to 5-scheme 3 (3-scheme 1) is outlined above, inscheme 3 wherein the commercially available 2,6-dichloro thiol can be oxidized tothe sulfonyl halide using a halogenating agent such as NCS, NBS, chlorine or 20 bromine in the presence of a protic solvent such as alcohol, acetic acid or water. -13- 012231 »»

The sulfonyl halide can be hydrolyzed by using a métal hydroxide such as sodiumor potassium hydroxide to form lhe corresponding sulfonic acid sait. The sulfonicacid sait can then be nitrated under nitration conditions such as nitric acid in a · solvent of strong acid such as sulfuric acid to form the nitro phenyl sulfonic acid 3-scheme 3. The sulfonic acid 3-scheme 3 can be converted to the sulfonamide 5-scheme 3 using a three step procedure involving the formation of the métal saitusing a base such as sodium hydroxide, sodium hydride or sodium carbonate toform 4-scheme 3. The sulfonic acid sait is then converted to the sulfonyl chlorideusing PCI5 with POCI3 as a solvent. The sulfonyl chloride can then be convertedto the corresponding sulfonamide using the desired amine HNRR" in triethylamine at températures ranging from -78 °C to 60 °C to form the correspondingsulfonamide 5-scheme 3 ( 3-scheme 1). The sulfonamide 5-scheme 3 can befurther elaborated by the methods contained in scheme 1. This method is notlimited to the 2,6-dichloro thiol it can also be applied to the 2,6-diflouro thiol, 2,6-dibromo thiol and the 2,6-diiodo thiol. The halogens in these compounds can beconverted to the corresponding cyano, amino, thiol, or alkoxy compounds bynucleophilic displacement reactions using nucleophiles such as alkyl thiolates,alkoxides, amine and cyanides. The halogens can also be further functionalized bypalladium coupling and carbonylation reactions, well known in the art, to form thecorresponding amido, carbonyl, alkenyl, alkyl, phenyl and heterocyclic substitutedproducts as required by Formula (I).

SYNTHETIC EXAMPLES

The invention will now be described by reference to the following exampleswhich are merely illustrative and are not to be construed as a limitation of the scope ofthe présent invention. Ail températures are given in degrees centigrade, ail solventsare highest available purity and ail reactions run under anhydrous conditions in anargon atmosphère unless otherwise indicated.

In the Examples, ail températures are in degrees Centigrade (°C). Mass spectrawere performed upon a VG Zab mass spectrometer using fast atom bombardment, unlessotherwise indicated. iH-NMR (hereinafter "NMR") spectra were recorded at 250 MHzusing a Bruker AM 250 or Am 400 spectrometer. Multiplicities indicated are: s=singlet, - 14- 012231 «* d=doublet, t=triplet, q=quartet, m=multiplet and br indicates a broad signal. Sat.indicates a saturated solution, eq indicates the proportion of a molar équivalent ofIl reagent relative to the principal reactant. 5 General synthesis of 2-hvdroxv-3-amino-6-chlorobenzenesulfonamide a) 2,6-dichlorobenzenesulfonyl chloride

Into a mixture of 200 milliliters (hereinafter "mL") of acetic acid, water anddichloromethane (3/1/4, v/v/v), 2,6 dichlorobenzenethiol (10.0 grams (hereinafter 10 "g”), 55.8 millimoles (hereinafter "mmol"), N-chlorosuccinimide (37.28 g, 279 mmol) and potassium acetate (2.29 g, 27.9 mmol) were added. The resultingmixture was stirred at 0°C, then warmed to room température ovemight. Themixture was then diluted with 200 mL of dichloromethane, and washed with water(100 mL x 3). The organic layer was dried (NajSO4) and concentrated to give the 15 desired product (11g, 80%). ‘H NMR (CDC13): δ 7.57 (d, 2H), 7.47 (t, 1H). b) 2,6-dichlorobenzenesulfonamide A solution of 2,6-dichlorobenzenesulfonyl chloride (10.50 g, 42.77 mmol) in 100mL of pyridine was added into 100 mL of pyridine dropwise while anhydrous 20 ammonia gas was passing through the solution simultaneously for 4 hours at 0°C.The mixture was acidified to pH >1 with 6N aq. HCl, then extracted with ethylacetate. The combined organic layer was then dried (NajSOJ and concentrated togive the desired product (8.69 g, 90%). EI-MS (m/z)225.0,227.1 (M). 25 c) 2,6-dichloro-3-nitrobenzenesulfonamide

Into a solution of 2,6-dichlorobenzenesulfonamide (7.8 g, 34.5 mmol) in 30 mL ofconcentrated sulfuric acid at 0°, nitric acid (1.74 mL, 41.4 mmol) was addeddropwise. The mixture was stirred at 0°C for 2 hours, then 200 mL of water wasadded to produce a precipitate. The resulting mixture was filtered. The white solid 30 was collected, washed with water and dried in vacuo to give the desired product(7.17 g, 76%). ’H NMR (DMSO-d6): δ 8.25 (s, 2H), 8.20 (d, 1H), 7.92 (d, 1H). -15- 4 d) 2-acetoxy-3-nitro-6-chlorobenzesulfonamide 012231 A solution of 2,6-dichloro-3-nitrobenzenesulfonamide (2.04 g, 7.5 mmol),potassium acetate (2.21 g, 22.5 mmol) and 18-crown-ô (5.95 g, 22.5 mmol) in 50mL of dimethyl sulfoxide was heated to 45°C for 7 days. The mixture wasacidified with IN aq. HCl, and extracted with ethyl acetate. The organic layer wasconcentrated to give the crude. Column chromatography on silica gel, eluting withethyl acetate/hexane/acetic acid (50/49/1, v/v/v), gave the desired product (1.67 g, 76%). EI-MS (m/z) 293.1,295.1 (M'). e) 2-hydroxy-3-nitro-6-chlorobenzesulfonamide A solution of 2-acetoxy-3-nitro-6-chlorobenzesulfonamide (1.72 g, 5.83 mmol),chlorotrimethylsilane (2 mL) and fuming sulfuric acid (0.5 mL) in methanol washeated to reflux for 20 hours. The solvent was evaporated. The residue wasdiluted with ethyl acetate and washed with water. The organic layer was then dried(NajSO4) and concentrated to give the desired product (1.0 g, 68%). EI-MS (m/z)251.1,253.2 (M'). f) 2-hydroxy-3-axnino-6-chlorobenzenesulfonamide

To a solution of 2-hydroxy-3-nitro-6-chlorobenzenesulfonamide (1.1 g, 4.36mmol) in ethyl acetate, was added 10 % Pd/C (500 mg). The mixture was flushedwith hydrogen, and then stirred under a hydrogen atmosphère at balloon pressurefor 4 hours at room température. The mixture was filtered through celite and thecelite was washed with methanol. The solvent was evaporated to give the desiredproduct (91%). EI-MS (m/z) 221.1,223.1 (M‘).

Example 1

Standard procedure for the condensation of an aniline with an isocyante.

Préparation of N-(3-ammosulfonvI-4-chIoro-2-hydroxyphenvl)-N,«cvelohexvl urea A solution of 3-amino-6-chloro-2-hydroxybenzenesulfonamide (150 mg, 0.67 mmol) and cyclohexyl isocyanate (95 pL, 0.74 mmol) in 1 mL of N,N- dimethyl-formamide was stirred at room température for 20 hours. The mixture -16- ·» 012231 was diluted with ethyl acetate and washed with water to give the crade.

Purification upon column chromatograph on silica gel, eluting with ethyl• acetate/hexane (50/50, v/v), followed by recrystallization from acetone and hexane, gave the desired product (121 mg, 52%). LC-MS (m/z) 348.0 (M+). 5

Example 2

Préparation of N-(3-aminosulfonyl-4-chloro-2-hydroxyphenvl)-N>-(l- adamantvl) urea A solution of 3-amino-6-chloro-2-hydroxybenzenesulfonamide (120 mg, 10 0.54 mmol) and 1-adamantyl isocyanate (115 mg, 0.64 mmol) in 1 mL of N,N- dimethyl-formamide was stirred at room température for 20 hours. The mixturewas diluted with ethyl acetate and washed with water to give the crade.

Purification upon column chromatograph on silica gel, eluting with ethylacetate/hexane (50/50, v/v), gave the desired product (122 mg, 56%). LC-MS (m/z) 15 400.0 (M+).

Example 3

Préparation of N-(3-aminos^lfonvl-4-chloro-2-hvdroxvphenvi)-N,- (tetrahydro-2-pvranvl) nrea 20 A solution of 3-amino-6-chloro-2-hydroxybenzenesulfonamide (170 mg, 0.76 mmol) and tetrahydro-2-pyranyl isocyanate (117 mg, 0.92 mmol) in 1.5 mL ofΝ,Ν-dimethyl-formamide was stirred at room température for 20 hours. Themixture was diluted with ethyl acetate and washed with water to give the crade.Purification upon column chromatograph on silica gel, eluting with ethyl 25 acetate/hexane (60/40, v/v), followed by séparation with Gilson HPLC, gave thedesired product (28 mg, 10%). LC-MS (m/z) 350.2 (M+).

Example 4

Standard procedure for the svnthesis of ureas bv coupling carboxylic acids 30 with an aniline. Svnthesis of N-[4-chloro-2-hvdroxy-3-sulfamvlphenvn-N’-(3- tetrahydrofnryl) urea - 17- 012231 Το a solution of Tetrahydro-furan-3-carboxylic acid (0.093 mL, 1.0 mmol)in DMF (0.5 mL) was added DPPA (0.25 mL, 1.2 mmol) and TEA (0.25 mL, 1.8mmol) and the reaction heated at 70°C. After 18hrs, 3-Amino-6-chloro-2-hydroxy- ·benzenesulfonamide (1.0 mmol) was added and the reaction heated at 70°C. After18hrs, the reaction mixture was quenched with water and extracted with ethylacetate. The organic layers were dried over anhydrous magnésium sulfate, andconcentraed under reduced pressure. The crude residue was purified via HPLC tpgive 18 mg (5%) of N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N’-(3-tetrahydrofuryl)urea. LC-MS (m/z) 336 (M+).

Example 5

Svnthesis of 6-Chloro-2-hydroxy-3-f3-(2-methyl"cycIopropyI)-ureidol- benzenesulfonamide

To a solution of 2-Methyl-cyclopropanecarboxylic acid (0.097 mL, 1.0mmol) in DMF (0.5 mL) was added DPPA (0.25 mL, 1.2 mmol) and TEA (0.25mL, 1.8 mmol) and the reaction heated at 90°C. After 18hrs, 3-Amino-6-chloro-2-hydroxy-benzenesulfonamide (1.0 mmol) was added and the reaction heated at90°C. After 18hrs, the reaction mixture was quenched with water and extractedwith ethyl acetate. The organic layers were dried over anhydrous magnésiumsulfate, and concentraed under reduced pressure. The crude residue was purifiedvia HPLC to give 29 mg (11%) of 6-Chloro-2-hydroxy-3-[3-(2-methyl-cyclopropyl)-ureido]-benzenesulfonamide. LC-MS (m/z) 320 (M+).

Example 6

Synthesis of 6-Chloro-2-hydroxy-3-f3-(2,2,3,3-tetrmethyl-cyclopropyl)- ureidol-benzenesulfonamide

To a solution of 2,2,3,3-tetramethyI-cyclopropanecarboxylic acid (0.142mL, 1.0 mmol) in DMF (0.5 mL) was added DPPA (0.25 mL, 1.2 mmol) and TEA(0.25 mL, 1.8 mmol) and the reaction heated at 80°C. After 18hrs, 3-Amino-6-chloro-2-hydroxy-benzenesulfonamide (1.0 mmol) was added and the reactionheated at 80°C. After 18hrs, the reaction mixture was quenched with water and - 18- 012231 J extracted with ethyl acetate. The organic layers were dried over anhydrous magnésium sulfate, and concentraed under reduced pressure. The crude residue * « was purified via HPLC to give 96 mg of 6-Chloro-2-hydroxy-3-[3-(2,2,3,3- tetrmethyl-cyclopropyl)-ureido]-benzenesulfonamide. LC-MS (m/z) 347 (M+). 5

Example 7

Synthesis of 6-Chloro-2-hvdroxv-3-(3-pîperidin-4-vI-ureido)- benzenesulfonamide

To a solution of piperidine-l,4-dicarboxylic acid mono-tert-butyl ester 10 (0.23g, l.o mmol) in DMF (0.5 mL) was added DPPA (0.215 mL, 1.0 mmol) and TEA (0.139 mL, 1.0 mmol) and the reaction heated at 80°C. After 18hrs, 3-Amino-6-chloro-2-hydroxy-benzenesulfonamide (1.0 mmol) was added and thereaction heated at 80°C. After 18hrs, the reaction mixture was quenched withwater and extracted with ethyl acetate. The organic layers were dried over 15 anhydrous magnésium sulfate, and concentraed under reduced pressure. The cruderesidue was purified via HPLC to give 12 mg of 6-Chloro-2-hydroxy-3-(3-piperidin-4-yl-ureido)-benzenesulfonamide. LC-MS (m/z) 349 (M+).

Example 8 20 Synthesis of N-i4-chloro-2-hvdroxv-3-sulfamvlphenvll-N’-(4-methvl- cyclohexvl) urea

To a solution of 4-Methyl-cyclohexanecarboxylic acid (0.141 mL) in DMF(0.5 mL) was added DPPA (0.215 mL) and TEA (0.139 mL) and the reactionheated at 80°C. After 18hrs, 3-Amino-6-chloro-2-hydroxy-benzenesulfonamide 25 (0.150 g) was added and the reaction heated at 80°C. After 18hrs, the reaction mixture was quenched with water and extracted with ethyl acetate. The organiclayers were dried over anhydrous magnésium sulfate, and concentraed underreduced pressure. The crude residue was purified via HPLC to give 15 mg of N-[4chloro-2-hydroxy-3-sulfamylphenyl]-N’-(4-methyl-cyclohexyl) urea. LC-MS (m/z) 30 347 (M+). -19- 01 2231 ’

Example 9 *

Synthesis of 6-Chloro-2-hvdroxv-3-|3-(3-methoxv-cvclohexvl)-ureidol- · benzenesulfonamide

To a solution of 3-Methoxy-cyclohexanecarboxylic acid (0.143 mL) inDMF (0.5 mL) was added DPPA (0.25 mL) and TEA (0.25 mL) and the reactionheated at 70°C. After 18hrs, 3-Amino-6-chloro-2-hydroxy-benzenesulfonamide(0.222 g) was added and the reaction heated at 70°C. After 18hrs, the reactionmixture was quenched with water and extracted with ethyl acetate. The organiclayers were dried over anhydrous magnésium sulfate, and concentraed underreduced pressure. The crude residue was purified via HPLC to give 3 mg of 6-Chloro-2-hydroxy-3-[3-(3-methoxy-cyclohexyl)-ureido]-benzenesulfonamide. LC-MS (m/z) 378 (M+).

Example 10 N-(3-aminosuïfonyl-4-chloro-2-hvdroxyphenyl)-N>-cyclopentyl urea A solution of 3-amino-6-chloro-2-hydroxybenzenesulfonamide (200 mg,0.90 mmol) and cyclopentyl isocyanate (100 mg, 0.90 mmol) in 1.5 mL of N,N-dimethyl-formamide was stirred at room température for 20 hours. Purificationupon column chromatograph on silica gel, eluting with ethyl acetate/hexane (20/80,v/v), followed by recrystallization from diethyl ether and hexane, gave the desiredproduct (130 mg, 43%). LC-MS (m/z) 334.2 (M+).

Example 11 N-(3-aminosulfonvi-4-chloro-2-hvdroxvphenvl)-N’-cvclobutvï urea

Under Ar, the mixture of cyclobutanecarboxylic acid (250 mg, 2.50 mmol),diphenylphosphoryl azide (756 mg, 2.74mmol), and triethyl amine (0.38mL,2.74mmol) in 3mL of Ν,Ν-dimethyl-formamide was heated to 80°C for 2 hours.The mixture was cooled to room température, 3-amino-6-chloro-2-hydroxybenzenesulfonamide (556mg, 2.50mmol) was added. The resultingmixture was stirred at room température for 20 hours. Purification upon Gilson 20- 012231 » HPLC, eluting with acetonitrile/water (10/90, v/v to 90/10, v/v, over 10min), gave * the desired product (137mg, 17%). LC-MS (m/z) 320.0 (M+). •

Example 12 5 N-fS-aminosulfonyl^-chloro^-hvdroxyphenvD-N’-cyclopropanyl urea

Under Ar, the mixture of cyclopropanecarboxylic acid (500 mg, 5.81mmol), diphenylphosphoryl azide (1.58g, 5.81mmol), and triethyl amine (0.81mL,

I 5.8Immol) in 3mL of Ν,Ν-dimethyl-formamide was heated to 80°C for 3 hours.

The mixture was cooled to room température, 3-amino-6-chloro-2- 10 hydroxybenzenesulfonamide (200mg, 0.89mmol) was added. The resultingmixture was stirred at room température for 20 hours. Purification upon GilsonHPLC, eluting with acetonitrile/water (10/90, v/v to 90/10, v/v, over 10min), gavethe desired product (74mg, 27%). LC-MS (m/z) 306.2 (M+). 15 Example 13 N-(3-(N’-Boc-piperazine)-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N"- cyclopentyl urea A solution of 3-N-(Boc-piperazine)amino-6-chloro-2-hydroxybenzenesulfonamide (200 mg, 0.51 mmol) and cyclopentyl isocyanate (57 fiL, 0.51 20 mmol) in 2 mL of Ν,Ν-dimethyl-formamide was stirred at room température for 20hours. Purification upon column chromatograph on silica gel, eluting with ethylacetate/hexane (30/70, v/v), followed by recrystallization from dichloromethaneand hexane, gave the desired product (180 mg, 70%). LC-MS (m/z) 503.2 (M+). 25 Example 14 N-(3-(N>-piperazipe)-aminosulfonyl-4-chlorO“2-hydroxvphepyl)-N,>· cyclopentyl urea hydrochloride

The solution of N-(3-(N’-Boc-piperazine)-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N’’-cyclopentylurea (140mg, 0.27mmol) in 2mL of 4N HCl in 1,4- 30 dioxane was stirred at room température for 1 hour. The mixture was concentrated. -21 - 012231

Recrystallization from acetonitrile gave the desired product (105mg, 88%). LC-MS * (m/z) 403.2 (M+). ,

Example 15 N-(3-(N>-Boc-piperazine)-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N,,- cyclobutyl urea

Under Ar, the mixture of cyclobutanecarboxylic acid (500mg, 4.99mmol),diphenylphosphoryi azide (1.18mL, 5.49mmol), and triethyl amine (0.76mL, 5.49mmol) in 3mL of Ν,Ν-dimethyl-formamide was heated to 80°C for 2, hours.

The mixture was cooled to room température, 3-N-(Boc-piperazine)amino-6-chloro-2-hydroxybenzenesulfonamide (1.95g, 4.97mmol) was added. Theresulting mixture was stirred at room température for 20 hours. Purification uponcolumn chromatograph on silica gel, eluting with ethyl acetate/hexane (30/70-50/50, v/v), gave the desired product (500 mg, 21%). LC-MS (m/z) 489.2 (M+).

Example 16

Svnthesis of N-(3-aminosulfonvl-4-chloro-2-hvdroxyphenvl)-N,-i(lS<2S)-(-)-2- benzyloxycvclohexyll urea (lS,2S)-(-)-2-benzyloxycyclohexyl isocynate

Under Ar, the solution of (1 S,2S)-(-)-2-benzyloxycyclohexylamine (100mg, 0.49mmol) in 5 mL of dichloromethane was added to a solution of di-tert-butyl dicarbonate (148mg, 0.68mmol) and 4-dimethylaminopyridine (87mg, 0.49mmol) in 5 mL of dichloromethane. The mixture was stirred for 15 min atroom température, then concentrated to give the crude material. FT-IR: 2237.92cm-k N-(3-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N’-r(lS,2S)-(-)“2- benzvloxycvclohexvn urea A solution of 3-amino-6-chloro-2-hydroxybenzenesulfonamide (109 mg, 0.49mmol) and the crude (lS,2S)-(-)-2-benzyloxycyclohexyl isocynate in 2 mL ofΝ,Ν-dimethyl-formamide was stirred at room température for 20 hours.

Purification upon Gilson HPLC, eluting with acetonitrile/ water (10/90, v/v to -22- 012231 90/10, v/v, over 10min), gave the desired product (60mg, 27% two steps). LC-MS(m/z) 454.0 (M+).

Example 17

Synthesis ofN-f3-(N’-dimethvI)-aminosulfonvI-4-chIoro-2-hvdroxvphenvl)- N”-cyclobutyl urea hydrochloride N-(3-(N’-dhnethyl)-aminosulfonyl>4-chloro-2-hydroxyphenyI)-N-Boc-N"-cyclobutyl urea

Under Ar, the mixture of cyclobutylamine (14|iL, O.lômmol), trimethylaluminum (80pL, 0.16mmol) in 3mL of dichloromethane was stirred at roomtempérature for 10 min. The solution of 7-(N-dimethyl)aminosulfonyl-3-Boc-5- .chloro-2-benzoxazolinone (60mg, 0.16mmol) in 3 mL of dichloromethane wasadded. The resulting mixture was stirred at room température for 20 hours.Purification upon column chromatograph on silica gel, eluting with ethylacetate/hexane (20/80, v/v), gave the desired product (36 mg, 51%). LC-MS (m/z)448.2 (M+). N-(3-(NMimethvl)-aminosuIfonvl-4-chloro-2-hvdroxyphenyl)-N,,-cyclobutvl urea hydrochloride

The solution of N-(3-(N’-dimethyl)-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N-Boc-N"-cyclobutylurea (36mg, 0.08mmol) in 2mL of 4N HClin 1,4-dioxane was stirred at room température for 1 hour. The mixture wasconcentrated. Recrystallization from acetone and hexane gave the desired product(20mg, 65%). Elemental analysis (%) theory: C 44.89, H 5.22, N 12.08,experiment C44.85, H 5.00, NI 1.91.

METHOD OF TREÀTMENT

The compounds of Formula (I), or a pharmaceutically acceptable saitthereof can be used in the manufacture of a medicine for the prophylactic ortherapeutic treatment of any disease State in a human, or other mammal, which isexacerbated or caused by excessive or unregulated IL-8 cytokine production bysuch mammal’s cell, such as but not limited to monocytes and/or macrophages, or -23- 012231 other chemokines which bind to the IL-8 a or β receptor, also referred to as the * type I or type Π receptor. *

Accordingly, the présent invention provides a method of treating a · chemokine mediated disease, wherein the chemokine is one which binds to an IL-8a or β receptor and which method comprises administering an effective amount ofa compound of Formula (I) or a pharmaceutically acceptable sait thereof. Inparticular, the chemokines are IL-8, GROa, GROp, GROy, NAP-2 or ENA-78.

The compounds of Formula (I) are administered in an amount sufficient toinhibit cytokine function, in particular IL-8, GROa, GROβ, GROy, NAP-2 orENA-78, such that they are biologically regulated down to normal levels of >physiological function, or in some case to subnormal levels, so as to ameliorate thedisease State. Abnormal levels of IL-8, GROa, GROβ, GROy, NAP-2 or ENA-78for instance in the context of the présent invention, constitute: (i) levels of free IL-8greater than or equal to 1 picogram per mL; (ii) any cell associated IL-8, GROa, GROβ, GROy, NAP-2 or ENA-78 above normal physiological levels; or (iii) thepresence of IL-8, GROa, GROp, GROy, NAP-2 or ENA-78 above basal levels incells or tissues in which EL-8, GROa, GROβ, GROy, NAP-2 or ENA-78respectively, is produced.

The compounds of Formula (I), in generally hâve been shown to hâve alonger t j/2 and improved oral bioavailabilty over the compounds disclosed in WO96/25157 and WO 97/29743 whose disclosures are incorporated herein by reference.

There are many disease States in which excessive or unregulated IL-8production is implicated in exacerbating and/or causing the disease. Chemokinemediated diseases include psoriasis, atopie dermatitis, osteo arthritis, rheumatoidarthritis, asthma, chronic obstructive pulmonary disease, adult respiratory distresssyndrome, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, stroke,septic shock, multiple sclerosis, endotoxic shock, gram négative sepsis, toxic shocksyndrome, cardiac and rénal reperfusion injury, glomerulonephritis, thrombosis,graft vs. host reaction, Alzheimer’s disease, allograft rejections, malaria, restenosis,angiogenesis, atherosclerosis, osteoporosis, gingivitis and undesired hematopoieticstem cells release and diseases caused by respiratory viruses, herpes viruses, andhepatitis viruses, meningitis, cystic fibrosis, pre-term labor, cough, pruritus, multi- -24- 012231 • organ dysfunction, trauma, strains, sprains, contusions, psoriatic arthritis, herpes, encephalitis, CNS vasculitis, traumatic brain injury, CNS tumors, subarachnoid • « Φ hemorrhage, post surgical trauma, interstitial pneumonitis, hypersensitivity, crystal induced arthritis, acute and chronic pancreatitis, acute alcoholic hepatitis, 5 necrotizing enterocolitis, chronic sinusitis, uveitis, polymyositis, vasculitis, acné,gastric and duodenal ulcers, celiac disease, esophagitis, glossitis, airflowobstruction, airway hyperresponsiveness, bronchiolitis obliterans organizingpneumonia, bronchiectasis, bronchiolitis, bronchiolitis obliterans, chronicbronchitis, cor pulmonae, dyspnea, emphysema, hypercapnea, hyperinflation, 10 hypoxemia, hyperoxia-induced inflammations, hypoxia, surgical lung volumeréduction, pulmonary fibrosis, pulmonary hypertension, right ventricularhypertropy, sarcoidosis, small airway disease, ventilation-perfusion mismatching,wheeze, colds and lupus.

These diseases are primarily characterized by massive neutrophil 15 infiltration, T-cell infiltration, or neovascular growth, and are associated withincreased IL-8, GROa, GROP, GROy, NAP-2 or ENA-78 production which isresponsible for the chemotaxis of neutrophils into the inflanunatory site or thedirectional growth of endothélial cells. In contrast to other inflammatory cytokines(IL-1, TNF, and IL-6), IL-8, GROa, GROP, GROy, NAP-2 or ENA-78 hâve the 20 unique property of promoting neutrophil chemotaxis, enzyme release including butnot limited to elastase release as well as superoxide production and activation. Theα-chemokines but particularly, GROa, GROP, GROy, NAP-2 or ENA-78, workingthrough the IL-8 type I or II receptor can promote the neovascularization of tumorsby promoting the directional growth of endothélial cells. Therefore, the inhibition 25 of IL-8 induced chemotaxis or activation would lead to a direct réduction in the neutrophil infiltration.

Recent evidence also implicates the rôle of chemokines in the treatment ofHIV infections, Littleman et al., Nature 381, pp. 661 (1996) and Koup et al.,

Nature 381, pp. 667(1996). 30 Présent evidence also indicates the use of IL-8 inhibitors in the treatment of atherosclerosis. The first reference, Boisvert et al., J. Clin. Invest, 1998, 101:353-363 shows, through bone marrow transplantation, that the absence of IL-8 -25- 012231 receptors on stem cells (and, therefore, on monocytes/macrophages) leads to a * réduction in the development of atherosclerotic plaques in LDL receptor déficient a mice. Additional supporting references are: Apostolopoulos, et al., Arterioscler, ·

Thromb. Vase. Biol. 1996,16:1007-1012; Liu, et al., Arterioscler. Thromb. Vase,

Biol, 1997, 17:317-323; Rus, et al., Atherosclerosis. 1996, 127:263-271.; Wang etal·, J- Biol. Chem. 1996, 271:8837-8842; Yue, et al., Eut. J. Pharmacol. 1993, 240:81-84; Koch, et al., Am. J. Pathol., 1993, 142:1423-1431.; Lee, et al.,

Immunol. Lett., 1996, 53, 109-113.; and Terkeltaub et al., Arterioscler. Thromb., 1994, 14:47-53.

The présent invention also provides for a means of treating, in an acute setting, aswell as preventing, in those individuals deemed susceptible to, CNS injuries by thechemokine receptor antagonist compounds of Formula (I). CNS injuries as defined herein include both open or penetrating headtrauma, such as by surgery, or a closed head trauma injury, such as by an injury tothe head région. Also included within this définition is ischémie stroke,particularly to the brain area.

Ischémie stroke may be defined as a focal neurologie disorder that resultsfrom insufficient blood supply to a particular brain area, usually as a conséquenceof an embolus, thrombi, or local atheromatous closure of the blood vessel. Therôle of inflammatory cytokines in this area has been emerging and the présentinvention provides a mean for the potential treatment of these injuries. Relativelylittle treatment, for an acute injury such as these has been available. TNF-α is a cytokine with proinflammatory actions, including endothélialleukocyte adhesion molécule expression. Leukocytes infiltrate into ischémie brainlésions and hence compounds which inhibit or decrease levels of TNF would beuseful for treatment of ischémie brain injury. See Liu et al., Stroke, Vol. 25., No. 7, pp. 1481-88 (1994) whose disclosure is incorporated herein by reference.

Models of closed head injuries and treatment with mixed 5-LO/CO agentsis discussed in Shohami et al., J. of Vaisc &amp; Clinical Phvsiology andPharmacology, Vol. 3, No. 2, pp. 99-107 (1992) whose disclosure is incorporatedherein by reference. Treatment, which reduced edema formation, was found toimprove functional outcome in those animais treated. -26- 012231

The compounds of Formula (I) are administered in an amount sufficient toinhibit IL-8, binding to the IL-8 alpha or beta receptors, from binding to thesereceptors, such as evidenced by a réduction in neutrophil chemotaxis andactivation. The discovery that the compounds of Formula (I) are inhibitors of IL-8binding is based upon the effects of the compounds of Formulas (I) in the in vitroreceptor binding assays which are described herein. The compounds of Formula(I) hâve been shown to be inhibitors of type II IL-8 receptors.

As used herein, the term "IL-8 mediated disease or disease State" refers toany and ail disease States in which IL-8, GROa, GROp, GROy, NAP-2 orENA-78plays a rôle, either by production of IL-8, GROa, GROP, GROy, NAP-2 or ENA-78 themselves, or by IL-8, GROa, GROp, GROy, NAP-2 or ENA-78 causinganother monokine to be released, such as but not limited to IL-1, IL-6 or TNF. Adisease State in which, for instance, IL-1 is a major component, and whoseproduction or action, is exacerbated or secreted in response to IL-8, wouldtherefore be considered a disease State mediated by IL-8.

As used herein, the term "chemokine mediated disease or disease State"refers to any and ail disease States in which a chemokine which binds to an IL-8 aor P receptor plays a rôle, such as but not limited to IL-8, GRO-a, GRO-β, GROy,NAP-2 or ENA-78. This would include a disease State in which, IL-8 plays a rôle,either by production of IL-8 itself, or by IL-8 causing another monokine to bereleased, such as but not limited to IL-1, IL-6 or TNF. A disease State in which, forinstance, IL-1 is a major component, and whose production or action, isexacerbated or secreted in response to IL-8, would therefore be considered adisease stated mediated by IL-8.

As used herein, the term "cytokine" refers to any secreted polypeptide thataffects the functions of cells and is a molécule, which modulâtes interactionsbetween cells in the immune, inflammatory or hematopoietic response. A cytokineincludes, but is not limited to, monokines and lymphokines, regardless of whichcells produce them. For instance, a monokine is generally referred to as beingproduced and secreted by a mononuclear cell, such as a macrophage and/ormonocyte. Many other cells however also produce monokines, such as naturalkiller cells, fibroblasts, basophils, neutrophils, endothélial cells, brain astrocytes, -27- 012231 bone marrow stromal cells, epideral kératinocytes and B-lymphocytes. *

Lymphokines are generally referred to as being produced by lymphocyte cells. *

Examples of cytokines include, but are not limited to, Interleukin-1 (IL-1), ·

Interleukin-6 (EL-6), Interleukin-8 (IL-8), Tumor Necrosis Factor-alpha (TNF-a)and Tumor Necrosis Factor beta (TNF-β).

As used herein, the term "chemokine" refers to any secreted polypeptidethat affects the functions of cells and is a molécule which modulâtes interactionsbetween cells in the immune, inflammatory or hematopoietic response, similar tothe term "cytokine" above. A chemokine is primarily secreted through celltransmembranes and causes chemotaxis and activation of spécifie white blood cellsand leukocytes, neutrophils, monocytes, macrophages, T-cells, B-cells, endothélialcells and smooth muscle cells. Examples of chemokines include, but are notlimited to IL-8, GRO-a, GRO-β, GRO-γ, NAP-2, ENA-78, IP-10, ΜΙΡ-Ια, MIP-β, PF4, andMCP 1,2, and3.

In order to use a compound of Formula (I) or a pharmaceutically acceptablesait thereof in therapy, it will normally be formulated into a pharmaceuticalcomposition in accordance with standard pharmaceutical practice. This invention,therefore, also relates to a pharmaceutical composition comprising an effective,non-toxic amount of a compound of Formula (I) and a pharmaceutically acceptablecarrier or diluent.

Compounds of Formula (I), pharmaceutically acceptable salts thereof andpharmaceutical compositions incorporating such may conveniently be administeredby any of the routes conventionally used for drug administration, for instance,orally, topically, parenterally or by inhalation. The compounds of Formula (I) maybe administered in conventional dosage forms prepared by combining a compoundof Formula (I) with standard pharmaceutical carriers according to conventionalprocedures. The compounds of Formula (I) may also be administered inconventional dosages in combination with a known, second therapeutically activecompound. These procedures may involve mixing, granulating and compressing ordissolving the ingrédients as appropriate to the desired préparation. It will beappreciated that the form and character of the pharmaceutically acceptablecharacter or diluent is dictated by the amount of active ingrédient with which it is -28- 012231 to be combined, the route of administration and other well-known variables. Thecarrier(s) must be "acceptable" in the sense of being compatible with the otheringrédients of the formulation and not deleterious to the récipient thereof.

The pharmaceutical carrier employed may be, for example, either a solid or 5 liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin,agar, pectin, acacia, magnésium stéarate, stearic acid and the like. Exemplary ofliquid carriers are syrup, peanut oil, olive oil, water and the like. Similarly, thecarrier or diluent may include time delay material well known to the art, such asglyceryl mono-stearate or glyceryl distearate alone or with a wax. 10 A wide variety of pharmaceutical fonns can be employed. Thus, if a solid carrier is used, the préparation can be tableted, placed in a hard gelatin capsule inpowder or pellet form or in the form of a troche or lozenge. The amount of solidcarrier will vary widely but preferably will be from about 25mg to about lg. Whena liquid carrier is used, the préparation will be in the form of a syrup, émulsion, 15 soft gelatin capsule, stérile injectable liquid such as an arnpule or nonaqueousliquid suspension.

Compounds of Formula (I) may be administered topically, that is by non-systemic administration. This includes the application of a compound of Formula.(I) extemally to the epidermis or the buccal cavity and the instillation of such a 20 compound into the ear, eye and nose, such that the compound does not significantly enter the blood stream. In contrast, systemic administration refers tooral, intravenous, intraperitoneal and intramuscular administration.

Formulations suitable for topical administration include liquid or semi-liquid préparations suitable for pénétration through the skin to'the site of 25 inflammation such as liniments, lotions, creams, ointments or pastes, and dropssuitable for administration to the eye, ear or nose. The active ingrédient maycomprise, for topical administration, from 0.001% to 10% w/w, for instance from1 % to 2% by weight of the Formulation. It may however comprise as much as10% w/w but preferably will comprise less than 5% w/w, more preferably from 30 0.1 % to 1 % w/w of the Formulation.

Lotions according to the présent invention include those suitable for application to the skin or eye. An eye lotion may comprise a stérile aqueous -29- 012231 solution optionally containing a bactéricide and may be prepared by methods ‘ * similar to those for the préparation of drops. Lotions or liniments for application to *the skin may also include an agent to hasten drying and to cool the skin, such as an < alcohol or acetone, and/or a moisturizer such as giycerol or an oil such as castor oilor arachis oil.

Creams, ointments or pastes according to the présent invention are semi-solid formulations of the active ingrédient for extemal application. They may bemade by mixing the active ingrédient in finely-divided or powdered form, alone orin solution or suspension in an aqueous or non-aqueous fluid, with the aid ofsuitable machinery, with a greasy or non-greasy base. The base may comprisehydrocarbons such as hard, soft or liquid paraffin, giycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor orolive oil; wool fat or its dérivatives or a fatty acid such as steric or oleic acidtogether with an alcohol such as propylene glycol or a macrogel. The formulationmay incorporate any suitable surface active agent such as an anionic, cationic ornon-ionic surfactant such as a sorbitan ester or a polyoxyethylene dérivativethereof. Suspending agents such as natural gums, cellulose dérivatives or inorganicmaterials such as silicaceous silicas, and other ingrédients such as lanolin, may alsobe included.

Drops according to the présent invention may comprise stérile aqueous oroily solutions or suspensions and may be prepared by dissolving the activeingrédient in a suitable aqueous solution of a bactericidal and/or fungicidal agentand/or any other suitable preservative, and preferably including a surface activeagent. The resulting solution may then be clarified by filtration, transferred to asuitable container which is then sealed and sterilized by autoclaving or maintaining at98-100°C for half an hour. Altematively, the solution may be sterilized byfiltration and transferred to the container by an aseptie technique. Examples ofbactericidal and fungicidal agents suitable for inclusion in the drops arephenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) andchlorhexidine acetate (0.01%). Suitable solvents for the préparation of an oilysolution include giycerol, diluted alcohol and propylene glycol. -30- 012231 r Compounds of formula (I) may be administered parenterally, that is by intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal or < * intraperitoneal administration. The subcutaneous and intramuscular forms of parentéral administration are generally preferred. Appropriate dosage forms for 5 such administration may be prepared by conventional techniques. Compounds ofFormula (I) may also be administered by inhalation that is by intranasal and oralinhalation administration. Appropriate dosage forms for such administration, suchas an aérosol formulation or a metered dose inhaler, may be prepared byconventional techniques. 10 For ail methods of use disclosed herein for the compounds of Formula (I) the daily oral dosage regimen will preferably be from about 0.01 to about 80 mg/kgof total body weight. The daily parentéral dosage regimen about 0.001 to about 80mg/kg of total body weight. The daily topical dosage regimen will preferably befrom 0.1 mg to 150 mg, administered one to four, preferably two or three times 15 daily. The daily inhalation dosage regimen will preferably be from about 0.01mg/kg to about 1 mg/kg per day. It will also be recognized by one of skill in theart that the optimal quantity and spacing of individual dosages of a compound ofFormula (I) or a pharmaceutically acceptable sait thereof will be determined by thenature and extent of the condition being treated, the form, route and site of 20 administration, and the particular patient being treated, and that such optimums canbe determined by conventional techniques. It will also be appreciated by one ofskill in the art that the optimal course of treatment, i.e., the number of doses of acompound of Formula (Γ) or a pharmaceutically acceptable sait thereof given perday for a defîned number of days, can be ascertained by those Skilled in the art 25 using conventional course of treatment détermination tests.

The invention will now be described by reference to the following biological examples which are merely illustrative and are not to be construed as alimitation of the scope of the présent invention.

BIOLOGICAL EXAMPLES 30 The EL-8, and GRO-α chemokine inhibitory effects of compounds of the présent invention are determined by the following in vitro assay: -31 - 012231

Receptor Binding Assays: jL_g (human recombinant) is obtained from Amersham Corp., *

Arlington Heights, IL, with spécifie activity 2000 Ci/mmoi. GRO-α is obtained * from NEN- New England Nuclear. Ail other Chemicals are of analytical grade.

High levels of recombinant human IL-8 type a and β receptors were individuallyexpressed in Chinese hamster ovaiy cells as described previously (Holmes, et al.,

Science, 1991,253,1278). The Chinese hamster ovary membranes were homogenized according to a previously described protocol (Haour, et al., J, Biol,

Chem., 249 pp 2195-2205 (1974)). Except that the homogenization buffer ischanged to lOmM Tris-HCL, ImM MgS04, 0.5mM EDTA (ethylene-diaminetetra-acetic acid), ImM PMSF (α-toluenesulphonyl fluoride), 0.5 mg/L Leupeptin, pH7.5. Membrane protein concentration is determined using Pierce Co. micro-assaykit using bovine sérum albumin as a standard. Ail assays are performed in a 96-well micro plate format. Each reaction mixture contains 125j jl-8 (0.25 nM) or125j GRO-α and 0.5 pg/mL of IL-8Ra or 1.0 pg/mL of IL-8Rp membranes in 20mM Bis-Trispropane and 0.4 mM Tris HCl buffers, pH 8.0, containing 1.2 mMMgSO4, 0.1 mM EDTA, 25 mM Na and 0.03% CHAPS. In addition, drug orcompound of interest is added which has been pre-dissolved in DMSO so as toreach a final concentration of between 0.0 lnM and 100 uM. The assay is initiatedby addition of 125j_il_8 After 1 hour at room température the plate is harvestedusing a Tomtec 96-well harvester onto a glass fiber filtermat blocked with 1%polyethyîenimineZ 0.5% BSA and washed 3 times with 25 mM NaCl, 10 mMTrisHCI, 1 mM MgSO4, 0.5 mM EDTA, 0.03 % CHAPS, pH 7.4. The fîlter isthen dried and counted on the Betaplate liquid scintillation counter. Therecombinant IL-8 Roc, or Type I, receptor is also referred to herein as the non-permissive receptor and the recombinant IL-8 RP, or Type Π, receptor is referred toas the permissive receptor.

Représentative compounds of Formula (I), Examples 1 to 106 hâve exhibited positive inhibitory activity in this assay at IC50 levels < 30 uM. -32- 01 2231 • Chemotaxis Assay: * The in vitro inhibitory properties of these compounds are determined in the « r neutrophil chemotaxis assay as described in Current Protocols in Immunology, vol. I, Suppl 1, Unit 6.12.3., whose disclosure is incoiporated herein by reference in its 5 entirety. Neutrophils where isolated from human blood as described in CurrentProtocols in Immunology Vol. I, Suppl 1 Unit 7.23.1, whose disclosure isincorporated herein by reference in its entirety. The chemoattractants IL-8, GRO-cx, GRO-β, GRO-γ and NAP-2 are placed in the bottom chamber of a 48 multiwellchamber (Neuro Probe, Cabin John, MD) at a concentration between 0.1 and 100 10 nM. The two chambers are separated by a 5 uM polycarbonate filter. When compounds of this invention are tested, they are mixed with the cells (0.001 - 1000nM) just prior to the addition of the cells to the upper chamber. Incubation is allowed to proceed for between about 45 and 90 min at about 37°C in a humidifiedincubator with 5% CO2. At the end of the incubation period, the polycarbonate 15 membrane is removed and the top side washed, the membrane then stained usingthe Diff Quick staining protocol (Baxter Products, McGaw Park, IL, USA). Cellswhich hâve chemotaxed to the chemokine are visually counted using a microscope.

Generally, four fields are counted for each sarnple, these numbers are averaged to 1 give the average number of cells which had migrated. Each sample is tested in 20 triplicate and each compound repeated at least four times. To certain cells (positive control cells) no compound is added, these cells represent the maximumchemotactic response of the cells. In the case where a négative control(unstimulated) is desired, no chemokine is added to the bottom chamber. Thedifférence between the positive control and the négative control represents the 25 chemotactic activity of the cells.

Elastase Release Assay:

The compounds of this invention axe tested for their ability to preventElastase release from human neutrophils. Neutrophils are isolated from humanblood as described in Current Protocols in Immunology Vol. I, Suppl 1 Unit 7.23.1 30 PMNs 0.88 x 106 cells suspended in Ringer’s Solution (NaCl 118, KC14.56,NaHCO3 25, KH2PO4 1.03, Glucose 11.1, HEPES 5 mM, pH 7.4) are placed in -33- 012231 each well of a 96 weil plate in a volume of 50 ul. To this plate is added the testcompound (0.001 - 1000 nM) in a volume of 50 ul, Cytochalasin B in a volume of50 ul (20ug/ml) and Ringers buffer in a volume of 50 ul. These cells are allowed towarm (37 °C, 5% CO2,95% RH) for 5 min before IL-8, GROa, GROp, GROyorNAP-2 at a final concentration of 0.01 - 1000 nM was added. The reaction isallowed to proceed for 45 min before the 96 well plate is centrifuged (800 xg 5min.) and 100 ul of the supematant removed. This supematant is added to a second96 well plate followed by an artificial elastase substrate (MeOSuc-Ala-Ala-Pro-Val-AMC, Nova Biochem, La Jolla, CA) to a final concentration of 6 ug/ml dissolved inphosphate buffered saline. Immediately, the plate is placed in a fluorescent 96 wellplate reader (Cytofluor 2350, Millipore, Bedford, MA) and data collected at 3 minintervals according to the method of Nakajima et al J. Biol. Chem. 254 4027 (1979).The amount of Elastase released from the PMNs is calculated by measuring the rateof MeOSuc-Ala-Ala-Pro-Val-AMC dégradation. TNF-α in Traumatic Brain Injury Assay

The présent assay provides for examination of the expression of tumor

necrosis factor mRNA in spécifie brain régions, which follow experimentally,induced latéral fluid-percussion traumatic brain injury (TBI) in rats. AdultSprague-Dawley rats (n=42) were anesthetized with sodium pentobarbital (60mg/kg, i.p.) and subjected to latéral fluid-percussion brain injury of moderateseverity (2.4 atm.) centered over the left temporaparietal cortex (n=18), or "sham"treatment (anesthésia and surgery without injury, n=18). Animais are sacrificed bydécapitation at 1, 6 and 24 hr. post injury, brains removed, and tissue samples ofleft (injured) pariétal cortex (LC), corresponding area in the contralatéral rightcortex (RC), cortex adjacent to injured pariétal cortex (LA), corresponding adjacentarea in the right cortex (RA), left hippocampus (LH) and right hippocampus (RH)are prepared. Total RNA are isolated and Northern blot hybridization is performedand quantitated relative to an TNF-α positive control RNA (macrophage = 100%).A marked increase of TNF- a mRNA expression is observed in LH (104±17% ofpositive control, p < 0.05 compared with sham), LC (105±21%, p< 0.05) and LA(69±8%, p < 0.01) in the traumatized hemisphere 1 hr. following injury. Anincreased TNF- a mRNA expression is also observed in LH (46+8%, p < 0.05), LC -34- 012231 (30±3%, p < 0.01) and LA (32±3%, p < 0.01) at 6 hr which résolves by 24 hrfollowing injury. In the contralatéral hemisphere, expression of TNF- a mRNA isincreased in RH (46±2%, p < 0.01), RC (4±3%) and RA (22±8%) at 1 hr and in RH(28±11%), RC (7±5%) and RA (26±6%, p < 0.05) at 6 hr but not at 24 hr following 5 injury. In sham (surgery without injury) or naive animais, no consistent changes inexpression of TNF- a mRNA are observed in any of the 6 brain areas in eitherhemisphere at any times. These results indicate that following parasagittal fluid-percussion brain injury, the temporal expression of TNF-α mRNA is altered inspécifie brain régions, including those of the non-traumatized hemisphere. Since 10 TNF-α is able to induce nerve growth factor (NGF) and stimulate the release ofother cytokines from activated astrocytes, this post-traumatic alteration in geneexpression of TNF-α plays an important rôle in both the acute and regenerativeresponse to CNS trauma.

CNS Injury model for IL-Ιβ mRNA 15 This assay characterizes the régional expression of interleukin-1B (IL-IB) mRNA in spécifie brain régions following experimental latéral fluid-percussiontraumatic brain injury (TBI) in rats. Adult Sprague-Dawley rats (n=42) areanesthetized with sodium pentobarbital (60 mg/kg, i.p.) and subjected to latéralfluid-percussion brain injury of moderate severity (2.4 atm.) centered over the left 20 temporaparietal cortex (n=18), or "sham" treatment (anesthésia and surgery withoutinjury). Animais are sacrificed at 1, 6 and 24 hr. post injury, brains removed, andtissue samples of left (injured) pariétal cortex (LC), corresponding area in thecontralatéral right cortex (RC), cortex adjacent to injured pariétal cortex (LA),corresponding adjacent area in the right cortex (RA), left hippocampus (LH) and 25 right hippocampus (RH) are prepared. Total RNA is isolated and Northern blothybridization was performed and the quantity of brain tissue IL-ΙΒ mRNA ispresented as percent relative radioactivity of IL-ΙΒ positive macrophage RNAwhich was loaded on the same gel. At 1 hr following brain injury, a marked andsignificant increase in expression of IL-ΙΒ mRNA is observed in LC (20.0±0.7% of 30 positive control, n=6, p < 0.05 compared with sham animal), LH (24.5±0.9%, p < 0.05) and LA (21.5±3.1%, p < 0.05) in the injured hemisphere, which remained

elevated up to 6 hr. post injury in the LC (4.0±0.4%, n=6, p < 0.05) and LH -35- 012231 (5.0±1.3%, p < 0.05). In sham or naïve animais, no expression of IL-Ιβ mRNA isobserved in any of the respective brain areas. These results indicate that followingTBI, the temporal expression of IL-Ιβ mRNA is regionally stimulated in spécifiebrain régions. These régional changes in cytokines, such as IL-Ιβ play a rôle in the 5 post-traumatic.

Ail publications, including but not limited to patents and patentapplications, cited in this spécification are herein incorporated by reference as ifeach individual publication were specifically and individually indicated to beincorporated by reference herein as though fully set forth. 10 The above description fully discloses the invention including preferred embodiments thereof. Modifications and improvements of the embodimentsspecifically disclosed herein are within the scope of the following daims. Withoutfurther élaboration, it is believed that one skilled in the art can, using the precedingdescription, utilize the présent invention to its fullest extent. Therefore the 15 Examples herein are to be construed as merely illustrative and not a limitation ofthe scope of the présent invention in any way. The embodiments of the inventionin which an exclusive property or privilège is claimed are defined as follows. -36-

Claims (12)

1. What is Claimed Is: 012231

   1. A compound of the formula (I):

   wherein Rb is independently selected rom the group consisting of hydrogen, NR6R7, OH, ORa, 10 Ci_5alkyl, aryl, arylCi-4alkyl, aryl C2~4alkenyl; cycloalkyl, cycloalkyl Cj_5alkyl, heteroaryl, heteroarylCi-4alkyl, heteroarylC2-4 alkenyl, heterocyclic,heterocyclic Ci-4alkyl, and a heterocyclic C2-4alkenyl, ail of which moieties maybe optionally substituted one to three times independently by a substituent selectedfrom the group consisting of halogen, nitro, halosubstituted C1-4 alkyl, Ci-4 alkyl, 15 amino, mono or di-Ci_4 alkyl substituted amine, ORa, C(O)Ra, NRaC(O)ORa,OC(O)NR6R7, hydroxy, NR9C(O)Ra, S(O)m>Ra, C(O)NR6R7, C(0)OH,C(O)ORa, S(O)2NR6R7> NHS(O)2Ra, or, the two Rb substituents join to form a 3-10 membered ring, optionally substituted and containing, in addition to carbon,independently, 1 to 3 moieties selected from the group consisting of NRa, O, S, 20 SO, or SO2; and wherein the substituent can be optionally unsaturated; Ra is selected from the group consisting of alkyl, aryl, arylCi_4alkyl, heteroaryl, heteroaryl Ci_4alkyl, heterocyclic, COOR13, and a heterocyclic Ci-4alkyl moiety,ail of which moieties may be optionally substituted; m is an integer having a value of 1 to 3; 25 m’is 0, or an integer having a value of 1 or 2;n is an integer having a value of 1 to 3; -37- 012231 q is 0, or an integer having a value of 1 to 10;t is 0, or an integer having a value of 1 or 2;s is an integer having a value of 1 to 3; Rl is independently selected from the froup consisting of hydrogen, halogen, nitro,cyano, Ci_io alkyl, halosubstituted Cj-io alkyl, C2-10 alkenyl, Cj-io alkoxy,halosubstituted Ci-ioalkoxy, azide, S(O)tR4, (CRgRg)q S(O)tR4, hydroxy, hydroxysubstituted Ci-4alkyl, aryl, aryl Ci_4 alkyl, aryl C2-10 alkenyl, aryloxy, aryl Ci_4alkyloxy, heteroaryl, heteroarylalkyl, heteroaryl C2-10 alkenyl, heteroaryl C1-4alkyloxy, heterocyclic, heterocyclic Ci_4alkyl, heterocyclicCi-4alkyloxy,heterocyclicC2-10 alkenyl, (CRgRg)q NR4R5, (CR8R8)qC(O)NR4R5, C2-10alkenyl C(O)NR4R5, (CRgR8)q C(O)NR4Ri0, S(O)3R8, (CR8R8)q C(O)Rl 1,C2-IO alkenyl C(O)Ri 1, C2-10 alkenyl C(O)ORi 1, (CRgRg)q C(O)ORi b(CRgRg)q OC(O)Rn, (CRgR8)qNR4C(O)Ru, (CRgRg)q C(NR4)NR4R5,(CR8Rg)q NR4C(NR5)Ri b (CR8R8)q NHS(O)2Ri3, and (CR8R8)q S(O)2NR4R5;or two Ri moieties together may form O-(CH2)sÛ or a 5 to 6 membered saturated orunsaturated ring, and wherein the alkyl, aryl, arylalkyl, heteroaryl, heterocyclicmoieties may be optionally substituted; R4 and R5 are independently selected from the group consisting of hydrogen, optionally substituted Ci-4 alkyl, optionally substituted aryl, optionally substitutedaryl Ci-4alkyl, optionally substituted heteroaryl, optionally substituted heteroarylCi_4alkyl, heterocyclic, and heterocyclicCi-4 alkyl; or R4 and R5 together withthe nitrogen to which they are attached form a 5 to 7 member ring which mayoptionally comprise an additional heteroatom selected from O, N and S ; R6 and R7 are independently selected from the group consisting of hydrogen, Cf-4alkyl, heteroaryl, aryl, aklyl aryl, and alkyl Cj_4 heteroalkyl; or Rb and R7together with the nitrogen to which they are attached form a 5 to 7 member ringwhich ring may optionally contain an additional heteroatom is selected fromoxygen, nitrogen and sulfur; wherein the ring may be optionally substituted; Y is selected from the group conisting of CR14C15, NR14, O, CO, and S(O\ R8 is hydrogen or Cf-4 alkyl; 38- R.9 is Cj _4 alkyl; 012231 Rio is Ci-10 alkyl C(O)2R8; Rl 1 is selected from the group consisting of hydrogen, optionally substituted Ci_4alkyl, optionally substituted aryl, optionally substituted aryl Ci_4alkyl, optionally 5 substituted heteroaryl, optionally substituted heteroarylCi_4alkyl, optionallysubstituted heterocyclic, and optionally substituted heterocyclicCi-4alkyl; and Rl2 is selected from the group consisting of hydrogen Ci_4 alkyl, aryl, aryl Ci_4alkyl,heteroaryl, heteroarylCi_4alkyl, heterocyclic, and heterocyclicCi-4alkyl; Rl3 is selected from the group consisting of Ci_4 alkyl, aryl, aryl Ci-4alkyl, 10 heteroaryl, heteroarylCi-4alkyl, heterocyclic, and heterocyclicCi _4alkyl; R14 and R15 are, independently, selected from the group consisting of hydrogen,optionally substituted Ci_4 alkyl group, ORa, and NR4R5; or R14 and R15together with the atom (s) to which they are attàched may form a 4 to 7 memberring which may optionally contain an additional heteroatom which heteroatom is 15 selected from the group consisting of oxygen, nitrogen and sulfur; wherein the ringmaybe optionally substituted; or a pharmaceutically acceptable sait thereof.
2. 2. The compound according to Claim 1 wherein Rl is substituted in the 4-position by an électron withdrawing moiety.
3. 3. The compound according to Claim 2 wherein Ri is halogen, cyano or nitro.
4. 4. The compound according to Claim 3 wherein Ri is halogen. 51 The compound according to Claim 4 wherein Rl is independently, fluorine, chlorine, or bromine.
5. 6. The compound according to Claim 1 wherein R^ is hydrogen, C 1,4 alkyl, or C 25 i_4 alkyl substituted with C(O)OH, or C(O)ORa.
6. 7. The compound according to Claim 1 which is selected from the groupconsisting of: N-(3-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N-cyclohexylurea; N-(3-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N’-(l-adamantyl)urea; 30 N-(3-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N-(tetrahydro-2-pyranyl)urea; -39- 012231 N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N’-(3-tetrahydrofuryl)urea;

   6-Chloro-2-hydroxy-3-[3-(2-methyl-cyciopropyl)-ureidoj-benzenesulfonamide:N-[4-chloro-2-hydroxy-3-suîfamylphenyl]-N’-cyclohexylurea;6-Chloro-2-hydroxy-3-[3-(2,2,33-tetnnethyl-cyclopropyl)-ureido]-benzenesulfonamide ;

   6-Chloro-2-hydroxy-3-(3-piperidin-4-yl-ureido)-benzenesulfonamide; N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N’-(4-methyl-cyclohexyl) urea;

   6-Chloro-2-hydroxy-3-[3-(3-methoxy-cyciohexyI)-ureido]-benzenesulfonamide; N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N’-cyclopentylurea; N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N’-cyclobutylurea; N-[4-chloro-2-hydroxy-3-sulfamylphenyl]-N’-cyclopropylurea; 4-[6-Chloro-3-(3-cyciopentyl-ureido)-2-hydroxy-benzenesulfonyl]-piperazine-l-carboxylic acid tert butyl ester; 1 -[4-Chloro-2-hy droxy-3-(piperazine-1 -sulfony l)-phenyl]-3-cy ciopenty i-urea; 4-[6-Chloro-3-(3-cyclobutyl-ureido)-2-hydroxy-benzenesulfonyl]-piperazine-l-carboxylic acid tert-butyl ester; 3-[3-((lS,2S)-2-Benzyloxy-cyclohexyl)-ureido]-6-chloro-2-hydroxy- benzenesulfonamide;

   6-Chloro-3-(3-cyclobutyl-ureido)-2-hydroxy-N,N’-dimethyl-benzenesulfonamide.
7. 8. A compound according to claim 7 which isN-(3-aminosulfonyl-4-chloro-2-hydroxyphenyl)-N’-cyclohexylurea
8. 9. A compound according to claim 7 wherein the compound is in its sodiumsait form.
9. 10. A compound according to claim 7 wherein the compound is in itspotassium sait form.
10. 11. A phannaceutical composition comprising a compound according to any ofClaims 1 to 10 and a pharmaceuticalîy acceptable carrier or diluent.
11. 12. Use of a compound of the formula according to any one of Claims 1 to 14 in the manufacture of a médicament for treating a chemokine mediated disease, wherein the chemokine binds to an IL-8 a or b receptor in a mammal. -40- 012231
12. 13. Use according to Claim 12 wherein the mammal is afflicted with achemokine mediated diseàse selected ftom the group consisting of:psoriasis, atopie dermatitis, osteo arthritis, rheumatoid arthritis, asthma, chronicobstructive pulmonary disease, adult respiratory distress syndrome, inflammatory 5 bowel disease, Crohn’s disease, ulcerative colitis, stroke, septic shock, multiplesclerosis, endotoxic shock, gram négative sepsis, toxic shock syndrome, cardiacand rénal reperfusion injury, glomerulonephritis, thrombosis, graft vs. hostreaction, Alzheimers disease, allograft rejections, malaria, restenosis,angiogenesis, atherosclerosis, osteoporosis, gingivitis and undesired hematopoietic 10 stem cells release and diseases caused by respiratory viruses, herpes virusès,, andhepatitis viruses, meningitis, cystic fibrosis, pre-term labor, cough, pruritus, multi-organ dysfunction, trauma, strains, sprains, contusions, psoriatic arthritis, herpes,encephalitis, CNS vasculitis, traumatic brain injury, CNS tumors, subarachnoidhemorrhage, post surgical trauma, interstitial pneumonitis, hypersensitivity, crvstal 15 induced arthritis, acute and chronic pancreatitis, acute alcoholic hepatitis, necrotizing enterocolitis, chronic sinusitis, uveitis, polymyositis, vasculitis, acné,gastric and duodenal ulcers, celiac disease, esophagitis, glossitis, airflowobstruction, airway hyperresponsiveness, bronchiolitis obliterans organizingpneumonia, bronchiectasis, bronchiolitis, bronchiolitis obliterans, chronic 20 bronchitis, cor pulmonae, dyspnea, emphysema, hypercapnea, hyperinflation,hypoxemia, hyperoxia-induced inflammations, hypoxia, surgerical lung volumeréduction, pulmonary fibrosis, pulmonary hypertension, right ventricularhypertropy, sarcoidosis, small airway disease, ventilation-perfusion mismatching,wheeze, colds and lupus. -41 - i