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  • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
  • C07C279/28—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to cyano groups, e.g. cyanoguanidines, dicyandiamides
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  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
  • C07C279/18—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to carbon atoms of six-membered aromatic rings
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  • C07C335/04—Derivatives of thiourea
  • C07C335/16—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton

Definitions

• This invention relates to novel guanidine substituted compounds, pharmaceutical compositions, processes for their preparation, and use thereof in treating IL-8, GRO ⁇ , GRO ⁇ , GRO ⁇ and NAP-2 mediated diseases
• Interleuk ⁇ n-8 Interleuk ⁇ n-8
• NAP-1 neutrophil attractant/activation protein- 1
• MDNCF monocyte derived neutrophil chemotactic factor
• NAF neutrophil activating factor
• Interleuk ⁇ n-8 is a chemoattractant for neutrophils, basophils, and a subset of T-cells It is produced by a majority of nucleated cells including macrophages, fibroblasts, endothelial and epithelial cells exposed to TNF, IL-l ⁇ , IL-l ⁇ or LPS, and by neutrophils themselves when exposed to LPS or chemotactic factors such as FMLP.
• GRO ⁇ , GRO ⁇ , GRO ⁇ , ENA-78 and NAP-2 also belong to the chemokine a family Like IL-8 these chemokmes have also been referred to by different names For instance Gro ⁇ , GRO ⁇ , and GRO ⁇ have been referred to as MGSA ⁇ , ⁇ and ⁇ respectively (Melanoma Growth Stimulating Activity), see Richmond et ai, J Cell Physiology 129, 375 (1986) and Chang et al, J Immunol 148, 451 (1992) All of the chemokmes of the a-family which possess the ELR motif directly preceding the CXC motif bind to the IL-8 B receptor.
• IL-8, GRO ⁇ , GRO ⁇ , GRO ⁇ and NAP-2 stimulate a number of functions in vitro They have all been shown to have chemoattractant properties for neutrophils, while IL-8 and GROa have demonstrated T-lymphocytes, and basophiles chemotactic activity
• IL-8 can induce histamine release from basophils from both normal and atopic individuals
• GRO- ⁇ and IL-8 can in addition, induce lysozomal enzyme release and respiratory burst from neutrophils
• IL-8 has also been shown to increase the surface expression of Mac- 1 (CD1 lb/CD 18) on neutrophils without de novo protein synthesis This may contribute to increased adhesion of the neutrophils to vascular endothelial cells
• Many known diseases are characterized by massive neutrophil infiltration As IL-8.
• IL-8, GRO ⁇ , GRO ⁇ , GRO ⁇ and NAP-2 induce neutrophil shape change, chemotaxis, granule release, and respiratory burst, by binding to and activating receptors of the seven-transmembrane, G-protein-hnked family, in particular by binding to IL-8 receptors, most notably the B-receptor Thomas et al , J Biol Chem 266. 14839 ( 1991 ). and Holmes et al . Science 253. 1278 ( 1991 )
• the development of non-peptide small molecule antagonists for members of this receptor family has precedent For a review see R Freidinger in Progress in Drug Research.
• IL-8Ra which binds only IL-8 with high affinity
• IL-8Rb which has high affinity for IL-8 as well as for GRO ⁇ , GRO ⁇ , GRO ⁇ and NAP-2
• IL-8Ra which binds only IL-8 with high affinity
• IL-8Rb which has high affinity for IL-8 as well as for GRO ⁇ , GRO ⁇ , GRO ⁇ and NAP-2
• This invention provides for a method of treating a chemokine mediated 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 of Formula (I) or a pharmaceutically acceptable salt thereof.
• the chemokine is IL-8.
• This invention also relates to a method of inhibiting the binding of IL-8 to its receptors in a mammal m need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I).
• R is any functional moiety having an lomzable hydrogen and a pKa of 10 or less
• R is independently selected from hydrogen; halogen; nitro; cyano: halosub.stituted Ci- io alkyl; C ⁇ _ ⁇ o alkyl; C2-10 alkenyl; C i- io alkoxy; halosubstituted C i- io alkoxy; azide; (CR R 8 )q S(O)tR4; hydroxy; hydroxy C j-4alkyl; aryl; aryl C j-4 alkyl: aryloxy; aryl C
• R6 and R7 are independently hydrogen or a C j -4 alkyl group, or R and R7 together with the nitrogen to which they are attached form a 5 to 7 member ring which ring may optionally contain an additional heteroatom selected from oxygen, nitrogen or sulfur, R is independently selected from hydrogen or C j-4 alkyl,
• R l 1 is hydrogen, C 1-4 alkyl, optionally substituted aryl, optionally substituted aryl C i -4alkyl, optionally substituted heteroaryl, optionally substituted heteroarylCi -4aikyl, optionally substituted heterocychc, or optionally substituted heterocychcC ⁇ -4alkyl
• R l2 is hydrogen, C j - io alkyl, optionally substituted aryl or optionally substituted arylalkyl
• R 13 and R 14 are independently hydrogen or C ] -4 alkyl
• R l5 and R j ⁇ are independently hydrogen, optionally substituted C ) -4 alkyl, optionally substituted aryl, optionally substituted aryl C i -4alkyl, optionally substituted heteroaryl, optionally substituted heteroarylCi -4alkyl, optionally substituted heterocychc, optionally substituted heterocychcC j -4alkyl, 01 R 55 and R 16 may together with the nitrogen to which they are attached form a 5 to 7 member ring optionally containing an additional heteroatom selected from oxygen, nitrogen, or sulfur, R 17 is C 1 -4 alkyl, NR ⁇ 5R 1 (., OR ⁇ ⁇ , optionally substituted aryl, optionally substituted aryl C ] -4alkyl, optionally substituted heteroaryl, optionally substituted heteroarylC i-4alkyl, optionally substituted heterocychc.
• R l 8 is optionally substituted C i-4 alkyl, optionally substituted aryl, optionally substituted aryl C i -4alkyl, optionally substituted heteroaryl, optionally substituted heteroarylC i -4alkyl, optionally substituted heterocychc, or optionally substituted heterocychcC i -4alkyl,
• R l9 is C [ -4alkyI, aryl, arylalkyl, heteroaryl, heteroarylC] _4alkyl, heterocychc, or heterocychcC i-4alkyl, wherein the all of these moieties may be optionally substituted, is NR6R7, alkyl, arylC j_4 alkyl, arylC 2-4 alkenyl. heteroaryl, hetroaryl-Cj _4alkyl, heteroarylC2-4 alkenyl. heterocychc, heterocychcC 1.4 alkyl, wherein the alkyl. aryl, arylalkyl.
• R20 is an optionally substituted C5_g cycloalkyl, optionally substituted C ⁇ . ⁇ Q alkyl, optionally substituted C2- 10 alkenyl, or optionally substituted C2-10
• the E' containing ring is optionally selected from
• the compounds of Formula (I) may also be used in association with the veterinary treatment of mammals, other than humans, in need of inhibition of IL-8 or other chemokmes which bind to the IL-8 a and b receptors
• Chemokine mediated diseases for treatment, therapeutically or prophylactically, in animals include disease states such as those noted herein in the Methods of Treatment section
• (*) may optionally be present If if it is not present the ring is a phenyl moiety which is substituted by the R ⁇ terms as shown
• the E' ring may be substituted by
• R is suitably any functional moiety which provides an lonizable hydrogen having a pKa of 10 or less, preferably from about 3 to 9, more preferably from about 3 to 7
• Such functional groups include, but are not limited to, hydroxy, carboxyhc
• sulfonamides 10 acid, thiol, SR2, OR2, NH-C(O)R a , C(O)NR6'R7 ⁇ a substituted sulfonamides of the formula- NHS(O)2Rb, S(O)2NHR c> NHC(X 2 )NHRb, or a tetrazolyl; wherein X 2 is oxygen or sulfur, preferably oxygen
• the functional group is other than a sulfonic acid, either directly or as a substituent group on the aryl, heteroaryl, or heterocychc moiety ring, such as in SR2 or OR2 More preferably R is OH. SH, or i s NHS ( O)2Rb
• R2 is a substituted aryl, heteroaryl, or heterocychc moiety which ring has the functional moiety providing the lonizable hydrogen having a pKa of 10 or less
• R6' and R7' are hydrogen, Ci-4 alkyl, aryl, arylC i -4alkyl, 0 arylC2-4alkenyl, heteroaryl, heteroarylC i-4alkyl, heteroarylC2-4 alkenyl, heterocychc, heterocychc Cj-4alkyl, heterocychc C2-4alkenyl moiety , all of which may be optionally substituted one to three times independently by halogen; nitro, halosubstituted C1-4 alkyl, such as CF3, C 1.4 alkyl, such as methyl, C j-4 alkoxy, such as methoxy.
• R6 and R7 are independently hydrogen or a C i -4 alkyl group, or R and R7 together with the nitrogen to which they are attached form a 5 to 7 member ring which ring may optionally contain an additional heteroatom which heteroatom is selected from oxygen, nitrogen or sulfur This hetero ⁇ ng may be 0 optionally substituted as defined herein
• R a is an aryl, arylC i-4alkyl, heteroaryl, heteroarylC ] -4alkyl, heterocychc, or a heterocychc C
• _4alkyl moiety all of which may be optionally substituted, as defined herein below
• Rb is a NR6R7 , alkyl, aryl, arylC
• R9 is hydrogen or a C 1 -4 alkyl, preferably hydrogen
• R a is preferably an alkyl group, such as methyl
• R c is hydrogen, alkyl, aryl, arylC ⁇ _4alkyl, arylCj-4alkenyl, heteroaryl, heteroarylCi-4alkyl, heteroarylC i-4alkenyl, heterocychc, heterocychc C i-4alkyl, or heterocychc C ) -4alkenyl moiety, all of which may be optionally substituted one to three times independently by halogen, nitro, halosubstituted C j-4 alkyl, C1.4 alkyl, C i -4 alkoxy.
• NR9C(0)R a C(O)NR6R7, S(O)3H. or C(O)OCi-4 alkyl
• R a is preferably an alkyl group, such as methyl
• R c is hydrogen, alkyl,
• R is an OR2 or SR2 moiety it is recognized by one of skill in the art that the aryl ring must, therefore, contain the required lonizable hydrogen
• the aryl ring may also be additionally substituted, independently, by one to three groups, which groups may also contain an additional lonizable group, and which include but are not limited to, halogen, nitro, halosubstituted C 1 -4 alkyl, C 1.4 alkyl, C 1.4 alkoxy, hydroxy, SH, C(O)NR6R7, NH-C(O)R a , NHS(O)2Rb- S(O)2NR6R7, C(O)OR 8) or a tetrazolyl ring
• R] is independently selected from hydrogen; halogen; nitro; cyano; halosubstituted C j- io alkyl, such as CF3, C i-io alkyl, such as methyl, ethyl, isopropyl, or n-propyl; C2-10 alkenyl.
• q is 0, or an integer having a value of 1 to 10
• s is preferably 1
• R[ forms an additional saturated or unsaturated ring it is preferably 6 membered ring resulting in a naphthylene ring system
• R4 and R5 are independently hydrogen, optionally substituted Ci-4 alkyl, optionally substituted aryl, optionally substituted aryl C i-4alkyl, optionally substituted heteroaryl, optionally substituted heteroaryl C ]-4alkyl, heterocychc, heterocychcC 1-4 alkyl, or R4 and R5 together with the nitrogen to which they are attached form a 5 to 7 member ring which may optionally comprise an additional heteroatom selected from O/N/S.
• Rg is suitably independently selected from hydrogen or C ( -4 alkyl Rio i suitably C ⁇ . ⁇ () alkyl C(0)2Rg, such as CH2C(0)2H or
• Rl 1 is suitably hydrogen, Ci-4 alkyl, aryl, aryl C [ -4 alkyl. heteroaryl, heteroaryl C
• R l2 is suitably hydrogen, C l- 10 alkyl, optionally substituted aryl or optionally substituted arylalkyl
• Rj9 is C j-4alkyl, aryl, arylalkyl, heteroaryl, heteroarylCi-4alkyl, heterocychc, or heterocychcC i-4alkyl, wherein the all of these moieties may be optionally substituted.
• Ri is halogen, cyano, nitro, CF3, C(O)NR4R5, alkenyl C(O)NR4R5, C(O) R4R 10. alkenyl C(0)OR ]2.
• R4 and R5 are both hydrogen or one is phenyl
• a preferred ring substitution for R i is in the 4-pos ⁇ t ⁇ on of the phenyl ring.
• R is OH, SH or NSO2Rb than Rj is preferably mono-substituted in the 3-pos ⁇ t ⁇ on, the 4- position or is di substituted in the 3,4- position
• the substituent group is suitably an electron withdrawing moiety
• R is OH, SH or NSO2Rb. than Rl is nitro, halogen, cyano, t ⁇ fluoromethyl group, C(O)NR4R5
• R is carboxyhc acid
• R i is preferably hydrogen, or R ] is preferably substituted in the 4-pos ⁇ t ⁇ on, more preferably substituted by t ⁇ fluoromethyl or chloro
• R13 and R)4 are independently hydrogen or Ci-4 alkyl which may be straight or branched as defined herein, v is 0, or an integer having a value of 1 to 4.
• Z is suitably cyano, OR j •* , C(O)NR ⁇ 5Ri ,
• R15 and R 15 are independently hydrogen, optionally substituted C i -4 alkyl, optionally substituted aryl, optionally substituted aryl C ⁇ _4alkyl, optionally substituted heteroaryl, optionally substituted heteroarylC i-4alkyl, optionally substituted heterocychc, optionally substituted heterocychcC ] _4alkyl, or 10 Rj5 and R ⁇ may together with the nitrogen to which they are attached form a 5 to 7 member ring optionally containing an additional heteroatom selected from oxygen, nitrogen, or sulfur
• R 17 is C j -4 alkyl, NR 15R16, OR i j , optionally substituted aryl, optionally substituted aryl C 1 _4alkyl, optionally substituted heteroaryl. optionally i s substituted heteroarylC i_4alkyl, optionally substituted heterocychc, or optionally substituted heterocychcC 1 -4alkyl
• R] g is suitably hydrogen, halogen, cyano, optionally substituted C i -4 alkyl, halo substituted -4 alkyl, C(O)NR
• R13 and R 14 are independently hydrogen, optionally substituted C i -4 alkyl which may be straight or branched as defined herein, or one of R 13 and R 14 are an optionally substituted aryl, v is 0, or an 5 integer having a value of 1 to 4
• Rj3 or R 14 are an optionally substituted alkyl
• the alkyl moiety may be substituted one to three times independently by halogen; halosubstituted Ci-4 alkyl such as t ⁇ fluromethyl, hydroxy, hydroxy Ci-4alkyl, Ci-4 alkoxy; such as methoxy, or ethoxy, halosubstituted Cj -io alkoxy, S(O)tR4, aryl, NR4R5; 0 NHC(O)R4; C(O)NR4Rs; or C(O)OR 8 .
• R20 suitably an optionally substituted C5_ cycloalkyl, optionally substituted C I _ IQ alkyl, optionally substituted C2- 10 alkenyl, or an optionally substituted C2-10 alkynyl moiety
• R20 i an optionally substituted C5_ 8 cycloalkyl ring
• the ring may be 5 substituted by (Y) n as defined below.
• R20 1 an optionally substituted C [ . ⁇ Q alkyl, an optionally substituted - C2-10 alkenyl, or an optionally substituted C2-10 alkynyl
• these moieties may be optionally substituted one or more times independently by halogen; nitro; cyano; halosubstituted Ci -io alkyl, such as t ⁇ fluoromethyl; Cj-io alkoxy; halosubstituted Ci-io alkoxy; S(O) ⁇ R4; hydroxy; hydroxy Ci -4alkyl; aryloxy; arylCi-4 alkyloxy; heteroaryloxy; heteroaryl Cj-4 alkyloxy; heterocychc; heterocychc C ⁇ _4alkyl; heterocychc-oxy; heterocychc C1.4
• R20 ⁇ an optionally substituted C2-10 alkenyl, or an optionally substituted C2-10 alkynyl
• these moieties may also, in addition to those moieties noted above, may also be optionally substituted with aryl, aryl C 1.4 alkyl, heteroaryl, and heteroarylalkyl.
• Y is independently selected from hydrogen; halogen; nitro, cyano. halosubstituted C] .
• s is preferably 1
• Y forms an additional saturated or unsaturated ring, it is preferably 6 membered ring resulting in a naphthylene ring system
• These rings may be substituted 1 to 3 times by other Y moieties as defined above.
• Rj is a NRgR7, alkyl, aryl C1.4 alklyl, arylC 2-4 alkenyl, heteroaryl, hetroaryl-Ci-4 lkyl, heteroarylC2-4 alkenyl, heterocychc, heterocychcC ⁇ _4 alkyl, or heterocychc C 2-4 alkenyl moiety, wherein the aryl, arylalkyl, arylalkenyl, heteroaryl, heteroarylalkyl, heteroarylalkenyl, heterocychc, and heterocychcalkyl, and heterocychcalkenyl moieties noted above may all be optionally substituted as defined herein.
• Y is preferably a halogen, C
• Y is more preferably mono-substituted halogen, disubstituted halogen, mono-substituted alkoxy, disubstituted alkoxy, methylenedioxy, aryl, or alkyl, more preferably these groups are mono or di-substituted in the 2'- position or 2'-, 3 -position
• Y may be substituted in any of the 5 ring positions when the ring is a cyclohexyl ring, Y is preferably mono-substituted in the 2'-pos ⁇ t ⁇ on or 3'- position, with the 4'- preferably being unsubstituted
• R20 is a cycloalkyl moiety
• both R ] and Y can both be hydrogen, it is prefered that at least one of the rings be substituted, preferably both rings are substituted
• halogen such as fluorine, chlorine, bromine or iodine, hydroxy, hydroxy substituted C [ -ioalkyl.
• R21 is suitably C i-4 alkyl, aryl, aryl C i -4alkyl, heteroaryl, heteroarylC 1. 4alkyl, heterocychc, or heterocychcC ⁇ _4alkyl.
• Suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of inorganic and organic acids, such as hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methane sulphonic acid, ethane sulphonic acid, acetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fuma ⁇ c acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid and mandehc acid
• pharmaceutically acceptable salts of compounds of Formula (I) may also be lormed with a pharmaceutically acceptable cation, for instance, if a substituent group comp ⁇ ses a carboxy moiety
• Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations .
• cycloalkyl is used herein to mean cyclic radicals, preferably of 3 to 8 carbons, including but not limited to cyclopropyl, cyclopentyl, cyclohexyl,
• alkenyl is used herein at all occurrences to mean straight or branched chain radical of 2- 10 carbon atoms, unless the chain length is limited thereto, including, but not limited to ethenyl, 1 -propenyl, 2-propenyl, 2-methyl- l - propenyl, 1-butenyl, 2-butenyl and the like
• heteroaryl alkyl - a 5- 10 membered aromatic ring system in which one or more rings contain one or more heteroatoms selected from the group consisting of N, O or S, such as, but not limited, to pyrrole, pyrazole, furan. thiophene, quinohne, isoquinoline, quinazohnyl, pyndine, py ⁇ midine, oxazole, thiazole, thiadiazole, 5 t ⁇ azole, imidazole, or benzimidazole
• heteroatoms selected from the group consisting of N, O, or S, such as, but not limited to, pyrrolidine, 0 piperidine, piperazine, morphohne, tetrahydropyran, or lmidazohdine
• arylalkyl or “heteroarylalkyl” or “heterocychcalkyl” is used herein to mean C i-io alkyl, as defined above, attached to an aryl, heteroaryl or heterocychc moiety, as also defined herein, unless otherwise indicated
• the compounds of the present invention may exist as stereoisomers, regioisomers, or diasterciomers These compounds may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms. 10 All of these compounds are included within the scope of the present invention.
• the compounds of Formula (I) may be obtained by applying synthetic procedures, some of which are illustrated in the Schemes below The synthesis I s provided for in these Schemes is applicable for the producing compounds of
• R' represents the -(R 13R j 4) v -R20 linkage as defined in compounds of
• the thiouronium salt (2_ ⁇ Scheme 1) can be synthesized by reacting sodium cyanamide with a commercially available isothiocyanate 1 (if the isothiocyanate is not commercially available it can be synthesized by reacting the desired amine with thiophosgene in the presence of a base like sodium bicarbonate).
• the thiouronium salt (2) can then be condensed with the appropriate substituted aniline in the presence of a coupling reagent like EDC-HCl or acidified to form the cvanothiourea and then reacted.
• Alternatively 4 can be synthesized by reacting the commercially available (Aldrich_Chem. Co.), diphenyl cyanocarboimidate (5, Scheme 2) with an amine to form an intermediate O-phenylisourea 6_which then reacts with the appropriately substituted aniline in the presence of trimethyl aluminum by the method of Atwal. (Atwal, K. S., Tetrahedron Lett, 35, 8085 ( 1994).).
• Compounds wherein R' is alkyl may be prepared by heating with the appropriately substituted alkylamine, but without the presence of catalyst.
• the title compound can be synthesized using a protected ortho- substituted aniline (8, Scheme 3, See synthesis as described in US provisional application USSN 60/020655 filed June 27, 1996, Attorney Docket No.: P50467P, WO96/25157 filed 22 August 1996, Widdowson et al (Attorney Docket No P50324- 1 ), and USSN 08/701.299 filed 21 August 1996 (Attorney Docket No . P50324-2) whose disclosures are incorporated herein by reference in their entireties
• An ortho-substituted aniline (7, Scheme 3) is first protected Oe.
• tert-butyl dimethyl silyl, allyl, benzyl, mom or other suitable protecting group by reacting the ortho- substituted aniline with the appropriate alkyl or silyl ha de in the presence of a suitable base (ic cesium carbonate, potassium carbonate or lmidazole) in an aprotic solvent
• a suitable base ic cesium carbonate, potassium carbonate or lmidazole
• the protected ortho-substituted aniline may also be synthesized from an ortho-substituted nitrobenzene (9 ) by reacting it with a protecting group under conditions well known m the art (see Greene, T Protecting Groups in Organic Synthesis.
• This protected ortho-substituted aniline (8) can then be converted into an isothiocyanate using thiophosgene and then reacted with the anion ZNH" (formed from reaction of ZNH2 with a base such as NaH).
• Z is as defined in compounds of
• the thioimidate ( Q, Scheme 5) can be converted to the title compound 4 by reaction with the amine R'NH2- This reaction can be accelerated by the addition of metal salt with a high affinity for sulfur such as mercuric oxide or silver acetate or by oxidation of the sulfur with dimethyloxirane to form a better leaving group. Finally the phenol protection is removed by standard methods to form the title compound 4
• Suitable bases for use herein include, secondary amine, such as py ⁇ dine, and amino substituted py ⁇ dine derivatives
• suitable solvents for use herein when Z is cyano include various aprotic solvents, such as 0 acetonitnle, halogenated solvents, such as chloroform and methylene chloride; ethyl gylcol-dimethyl ether (monoGLYME), dioxane.
• solvents herein will be the solubility of the cyano de ⁇ vatived compound.
• Z is other than cyano
• suitable solvents such as protic solvents, i.e. alcohols, may be used.
• the reaction (when Z is cyano) temperature is from about - 10° to about 100°, preferably about 10° to about 50°, more preferably around room temperature, i.e. 20 to 30 °C. 0
• the protected R" moiety may be suitably deprotected using art recognized techniques
• the deprotection is by deallylation catalyzed by palladium (O) when the protecting group is an allyl derivative.
• the carbodiimide JJ_ is prepared from the thiourea ( 12a, Scheme 7) by treatment with phosgene and a tertiary amine base or from the thiourea ( 12a) or urea ( 12b) by reaction with tnphenylphosphine, carbon tetrachlonde and triethylamine
• the carbodiimide may also be prepared by reaction of the thiourea ( 12b) with an excess, such as 2 or more equivalents of methanesulfonyl chloride and a tertiary amine base, such as Hunig's base (diisopropylethyiamine), triethylamine, tn- lsopropylethylamine, N,N-d ⁇ methylbenzylam ⁇ ne, or N,N-d ⁇ methyhsopropylam ⁇ ne, preferably triethylamine.
• reaction temperatures are from about -30°C to about 80°C, preferably - 10°C to about 50°C, more preferably from about 0°C to about room temperature. See Fell and Coppola (Fell, J. B , Coppola, J B , Syn Communications 25, 43, ( 1995)
• the thiourea ( 12a. Scheme 8) may also be prepared by reaction of the protected ortho-substituted aniline and two equivalents of an appropriate base such as NaH, KH, calcium hydride, and reacting this anion with a commercially available isothiocyanate (Wj-NCS, wherein W - is as defined for compounds of Formula (I))
• Wj-NCS isothiocyanate
• the reaction make take place in any suitable aprotic solvent or halogenated solvent, preferably dimethyl formamide. Suitable reaction temperatures for this reaction are from about - 10° to about 50°.
• the desired isothiocyanate is not commercially available, it may be prepared by reaction of a corresponding aniline with thiophosgene and a suitable base such as sodium bicarbonate.
• compositions of Formula (I) may be 0 obtained in known manner, for example by treatment thereof with an appropriate amount of acid or base in the presence of a suitable solvent
• the compounds of Formula (I), or a pharmaceutically acceptable salt thereof s can be used in the manufacture of a medicament tor the prophylactic or therapeutic treatment of any disease state in a human, or other mammal, which is exacerbated or caused by excessive or unregulated IL-8 cytokine production by such mammal's cell, such as but not limited to monocytes and or macrophages, or other chemokmes which bind to the IL-8 a or b receptor, also referred to as the type I or type II 0 receptor
• the present invention provides a method of treating a chemokine mediated disease, wherein the chemokine is one which binds to an IL-8 a or b receptor and which method comp ⁇ ses administering an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof
• the chemokmes are IL-8 GRO ⁇ , GRO ⁇ , GRO ⁇ . ENA-78 or NAP-2
• the compounds ol Formula (I) are administered in an amount sufficient to inhibit cytokine function, in particular IL-8, GRO ⁇ , GRO ⁇ , GRO ⁇ , ENA-78 or NAP-2 , such that they are biologically regulated down to normal levels of physiological function, or in some case to subnormal levels, so as to ameliorate the disease state
• Abnormal levels of IL-8, GRO ⁇ , GRO ⁇ , GRO ⁇ , ENA-78 or NAP-2 for instance in the context of the present invention constitute (1) levels of free IL-8 greater than or equal to 1 picogram per mL, (11) any cell associated IL-8, GRO ⁇ , GRO ⁇ , GRO ⁇ , ENA-78 or NAP-2 above normal physiological levels, or (111) the presence of IL-8, GRO ⁇ , GRO ⁇ , GRO ⁇ , ENA-78 or NAP-2 above basal levels in 5 cells or tissues in which IL-8, GRO ⁇ , GRO ⁇ , GRO ⁇ , ENA-78 or NAP-2 respectively, is produced There are many disease states in
• T-cell infiltration, or neovascular growth are associated with increased IL-8, GRO ⁇ , GRO ⁇ .
• GRO ⁇ or NAP-2 production which is responsible for the chemotaxis of neutrophils into the inflammatory site or the directional growth of endothelial cells In contrast to other inflammatory cytokines (IL- 1 , TNF, and IL-6), IL-8, GRO ⁇ .
• GRO ⁇ , GRO ⁇ or NAP-2 has the unique property of promoting neutrophil chemotaxis, enzyme release including but not limited to elastase release as well as superoxide production and activation
• the ⁇ -chemokmes but particularly GRO ⁇ , GRO ⁇ , GRO ⁇ or NAP-2, working through the IL-8 type I or II receptor can promote the neovascula ⁇ zation of tumors by promoting the directional growth of endothelial cells Therefore, the inhibition of IL-8 induced chemotaxis or activation would lead to a direct reduction in the neutrophil infiltration
• the present invention also provides for a means of treating, in an acute setting, as well as preventing, in those individuals deemed susceptible to, CNS injuries by the chemokine receptor antagonist compounds of Formula (I)
• CNS injuries as defined herein include both open or penetrating head trauma, such as by surgery, or a closed head trauma injury, such as by an injury to the head region Also included within this definition is ischemic stroke, particularly to the brain area
• Ischemic stroke may be defined as a focal neurologic disorder that results from insufficient blood supply to a particular brain area, usually as a consequence of an embolus, thrombi, or local atheromatous closure of the blood vessel
• embolus a focal neurologic disorder that results from insufficient blood supply to a particular brain area
• thrombi a progressive neurologic disorder that results from insufficient blood supply to a particular brain area
• local atheromatous closure of the blood vessel The role of inflammatory cytokines in this are has been emerging and the present invention provides a mean for the potential treatment of these injuries Relatively little treatment, for an acute injury such as these has been available
• TNF- ⁇ is a cytokine with proinflammatory actions, including endothelial leukocyte adhesion molecule expression
• Leukocytes infiltrate into ischemic brain lesions and hence compounds which inhibit or decrease levels of TNF would be useful for treatment of ischemic brain injury See Liu et al., Stoke. Vol. 25 , No. 7, pp 1481-88 ( 1994) whose disclosure is incorporated herein by reference
• the compounds of Formula (I) are administered in an amount sufficient to inhibit IL-8. binding to the IL-8 alpha or beta receptors, from binding to these receptors, such as evidenced by a reduction in neutrophil chemotaxis and activation
• the discovery that the compounds of Formula (I) are inhibitors of IL-8 binding is based upon the effects of the compounds of Formulas (I) in the in vitro receptor binding assays which are described herein
• the compounds of Formula (I) have been shown, in some instances, to be dual inhibitors of both recombinant type I and type II IL-8 receptors.
• the compounds are inhibitors of only one receptor, more preferably Type II
• IL-8 mediated disease or disease state refers to any and all disease states in which IL-8, , GRO ⁇ , GRO ⁇ , GRO ⁇ , ENA-78 or NAP-2 plays a role, either by production oi IL-8, GRO ⁇ , GRO ⁇ . GRO ⁇ , ENA-78 or NAP-2 themselves, or by IL-8, GRO ⁇ . GRO ⁇ .
• GRO ⁇ , ENA-78 or NAP-2 causing another monokine to be released such as but not limited to IL- 1 , IL-6 or TNF A disease state in which, for instance, IL- 1 is a major component, and whose production or action, is exacerbated or secreted in response to IL-8, would therefore be considered a disease stated mediated by IL-8
• chemokine mediated disease or disease state refers to any and all disease states in which a chemokine which binds to an IL-8 a or b receptor plays a role, such as but not limited to IL-8, GRO ⁇ , GRO ⁇ , GRO ⁇ , ENA-78 or NAP-2 This would include a disease state in which, IL-8 plays a role, either by production of IL-8 itself, or by IL-8 causing another monokine to be released, such as but not limited to IL-1 , IL-6 or TNF A disease state in which, tor instance.
• IL-1 is a ma
• cytokine refers to any secreted polypeptide that affects the functions of cells and is a molecule which modulates interactions between cells in the immune, inflammatory or hematopoietic response.
• a cytokine includes, but is not limited to, monokines and lymphokines, regardless of which cells produce them.
• a monokine is generally referred to as being produced and secreted by a mononuclear cell, such as a macrophage and/or monocyte.
• Lymphokines are generally referred to as being produced by lymphocyte cells.
• cytokines include, but are not limited to, Interleukin- 1 (IL- 1 ), Interleuk ⁇ n-6 (IL-6), Interleuk ⁇ n-8 (IL-8), Tumor Necrosis Factor-alpha (TNF- ⁇ ) and Tumor Necrosis Factor beta (TNF- ⁇ )
• chemokine refers to any secreted polypeptide that affects the functions of cells and is a molecule which modulates interactions between cells in the immune, inflammatory or hematopoietic response, similar to the term "cytokine” above.
• a chemokine is primarily secreted through cell transmembranes and causes chemotaxis and activation of specific white blood cells and leukocytes, neutrophils, monocytes, macrophages, T-cells, B-cells, endothelial cells and smooth muscle cells.
• chemokmes include, but are not limited to, IL-8, GRO- ⁇ , GRO- ⁇ , GRO ⁇ , ENA-78, NAP-2, IP- 10, MlP- l a, MlP-b, PF4, and MCP 1 , 2, and 3
• a compound of Formula (I) or a pharmaceutically acceptable salt thereof in therapy it will normally be formulated into a pharmaceutical composition in accordance with standard pharmaceutical practice.
• This invention also relates to a pharmaceutical composition comprising an effective, non- toxic amount of a compound of Formula (I) and a pharmaceutically acceptable carrier or diluent.
• Compounds of Formula (I), pharmaceutically acceptable salts thereof and pharmaceutical compositions incorporating such may conveniently be administered by any of the routes conventionally used for drug administration, for instance, orally, topically, parenterally or by inhalation.
• the compounds of Formula (I) may be administered in conventional dosage forms prepared by combining a compound of Formula (I) with standard pharmaceutical carriers according to conventional procedures.
• the compounds of Formula (I) may also be administered in conventional dosages in combination with a known, second therapeutically active compound. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.
• the pharmaceutical carrier employed may be. for example, either a solid or liquid.
• solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, steanc acid and the like.
• liquid carriers are syrup, peanut oil, olive oil, water and the like.
• the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax.
• the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge.
• the amount of solid carrier will vary widely but preferably will be from about 25mg. to about lg
• the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous liquid 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) externally to the epidermis or the buccal cavity and the instillation of such a compound into the ear, eye and nose, such that the compound does not significantly enter the blood stream.
• systemic administration refers to oral, intravenous, intraperitoneal and intramuscular administration.
• Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of inflammation such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.
• the active ingredient may comprise, for topical administration, from 0.001% to 10% w/w, for instance from 1% to 2% by weight of the formulation. It may however comprise as much as 10% w/w but preferably will comprise less than 5% w/w, more preferably from 0.1 % to 1 % w/w of the formulation.
• Lotions according to the present invention include those suitable for application to the skin or eye.
• An eye lotion may comprise a sterile aqueous solution optionally containing a bacte ⁇ cide and may be prepared by methods similar to those for the preparation of drops
• Lotions or liniments for application to the skin may 5 also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil
• Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by l() mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy base
• the base may comprise hydrocarbons such as hard, solt or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage, an oil of natural origin such as almond, corn, arachis, castor or olive oil, wool fat oi
• the formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as a sorbitan ester or a polyoxyethylene derivative thereof
• suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as a sorbitan ester or a polyoxyethylene derivative thereof
• Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous 0 silicas, and other ingredients such as lanolin, may also be included
• Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the active ingredient in a suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active 5 agent.
• the resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed and sterilized by autoclaving or maintaining at 98-100 C for half an hour.
• the solution may be sterilized by filtration and transferred to the container by an aseptic technique
• bactericidal and fungicidal agents suitable for inclusion in the drops are 0 phenylmercunc nitrate or acetate (0.002%), benzalkonium chloride (0 01%) and chiorhexidine acetate (001 )
• Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol
• Compounds of formula (I) may be administered parenterally, that is by intravenous, intramuscular, subcutaneous intranasal, lntrarectal, intravaginal or 5 intraperitoneal administration.
• the subcutaneous and intramuscular forms of parenteral administration are generally preferred.
• Appropriate dosage forms for such administration may be prepared by conventional techniques
• Compounds of Formula (I) may also be administered by inhalation, that is by intranasal and oral inhalation administration.
• Appropriate dosage forms for such administration such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques
• the daily oral dosage regimen will preferably be from about 001 to about 80 mg/kg of total body weight.
• the daily parenteral dosage regimen about 0.001 to about 80 mg/kg of total body weight.
• the daily topical dosage regimen will preferably be from 0 1 mg to 150 mg, administered one to four, preferably two or three times daily.
• the daily inhalation dosage regimen will preferably be from about 0.01 mg/kg to about 1 mg/kg per day.
• the optimal quantity and spacing of individual dosages of a compound of Formula (I) or a pharmaceutically acceptable salt thereof will be determined by the nature and extent ot the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course ot treatment, i.e., the number of doses of a compound of Formula (I) or a pharmaceutically acceptable salt thereof given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests
• IL-8, and Gro- ⁇ chemokine inhibitiory effects of compounds of the present invention were determined by the following in vitro assay Receptor Binding Assays: [ 125 iL_g (human recombinant) is obtained from Amersham Corp.,
• 0.5mM EDTA ethylene-diaminetetra- acetic acid
• I mMPMSF ⁇ -toluenesulphonyl fluoride
• 0.5 mg/L Leupeptin pH 7.5.
• Membrane protein concentration is determined using Pierce Co. micro-assay kit using bovine serum albumin as a standard. All assays are performed in a 96-well micro plate format.
• Each reaction mixture contains 1-51 IL-8 (0.25 nM) or 125j Gro- ⁇ and 0.5 ⁇ g/mL of IL-8R ⁇ or 1.0 ⁇ g/mL of IL-8R ⁇ membranes in 20 mM Bis- Trispropane and 0.4 mM Tris HC1 buffers, pH 8.0, containing 1.2 mM MgSO4, 0.1 mM EDTA, 25 mM NaCl and 0.03% CHAPS.
• drug or compound of interest ss added which has been pre-dissoived in DMSO so as to reach a final concentration of between 0.0 InM and 100 uM.
• the assay is initiated by addition of ⁇ 25I-IL-8.
• the plate is harvested using a Tomtec 96-well harvester onto a glass fiber filtermat blocked with 1 % polyethylenimine/0.5% BSA and washed 3 times with 25 mM NaCl, 10 mM TrisHCl. 1 mM MgSOd, 0.5 mM EDTA, 0.03 % CHAPS. pH 7.4. The filter is then dried and counted on the Betaplate liquid scintillation counter.
• the recombinant IL- 8 R ⁇ , or Type I, receptor is also referred to herein as the non-permissive receptor and the recombinant IL-8 R ⁇ , or Type II, receptor is referred to as the permissive receptor.
• the in vitro inhibitory properties of these compounds are determined in the neutrophil chemotaxis assay as described in Current Protocols in Immunology, vol I, Suppl 1, Unit 6.12.3., whose disclosure is incorporated herein by reference in its entirety.
• Neutrophils where isolated from human blood as described in Current Protocols in Immunology Vol I, Suppl 1 Unit 7.23.1 , whose disclosure is incorporated herein by reference in its entirety.
• the chemoattractants IL-8, GRO- ⁇ , GRO- ⁇ , GRO- ⁇ and NAP-2 are placed in the bottom chamber of a 48 multiwell chamber (Neuro Probe, Cabin John, MD) at a concentration between 0.1 and 100 nM. The two chambers are separated by a 5um polycarbonate filter.
• the compounds of this invention are tested for their ability to prevent Elastase release from human neutrophils.
• Neutrophils are isolated from human blood as described in Current Protocols in Immunology Vol I, Suppl 1 Unit 7 23 1 PMNs 0 88 x IO 6 cells suspended in Ringer's Solution (NaCl 1 18. CI 4 56, NaHCO3 25. KH2PO4 1 03, Glucose 1 1 1 , HEPES 5 mM.
• TNF- ⁇ in Traumatic Brain Injury Assay 0 The present assay provides for examination of the expression of tumor necrosis factor mRNA in specfic brain regions which follow experimentally induced lateral fluid- percussion traumatic brain injury (TBI) in rats
• TBI experimentally induced lateral fluid- percussion traumatic brain injury
• a marked increase of TNF- ⁇ mRNA expression is observed in LH ( 104 ⁇ i 7% of positive control, p ⁇ 0 05 compared with sham), LC ( 105 ⁇ 21 %, p ⁇ 005) and LA (69 ⁇ 8%, p ⁇ 0 01) m the traumatized hemisphere 1 hr following injury
• An increased TNF- ⁇ mRNA expression is also observed in LH (46 ⁇ 8%, p ⁇ 005), LC (30 ⁇ 3%, p ⁇ 0 01
• TNF- ⁇ mRNA is increased in RH (46 ⁇ 2%, p ⁇ 0 01), RC (4 ⁇ 3%) and RA (22 ⁇ 8%) at 1 hr and in RH (28 ⁇ 1 1 %), RC (7 ⁇ 5%) and RA (26 ⁇ 6%, p ⁇ 0 05) at 6 hr but not at 24 hr following injury
• sham surgery without injury
• naive animals no consistent changes in expression of TNF- ⁇ mRNA are observed in any ot the 6 brain areas in either hemisphere at any times
• This assay characterizes the regional expression of interleukin- I B (IL- l ⁇ ) mRNA in specific brain regions following experimental lateral fluid-percussion traumatic brain injury (TBI) in rats
• TBI lateral fluid-percussion traumatic brain injury
• IL- l ⁇ interleukin- I B
• LC left (injured) parietal cortex
• RC contralateral right cortex
• LA left parietal cortex
• RA left hippocampus
• RH right hippocampus
• RNA Total RNA is isolated and Northern blot hybridization was performed and the quantity of brain tissue IL-l ⁇ mRNA is presented as percent relative radioactivity of IL-l ⁇ positive macrophage RNA which was loaded on same gel.
• LH 24 5 ⁇ 0 9%, p ⁇ 005) and LA (21 5 ⁇ 3 1 %, p ⁇ 0 05) in the injured hemisphere, which remained elevated up to 6 hr.

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