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WO2002017900A2 - Modulateurs de l'us28 - Google Patents

Modulateurs de l'us28 Download PDF

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Publication number
WO2002017900A2
WO2002017900A2 PCT/US2001/027363 US0127363W WO0217900A2 WO 2002017900 A2 WO2002017900 A2 WO 2002017900A2 US 0127363 W US0127363 W US 0127363W WO 0217900 A2 WO0217900 A2 WO 0217900A2
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group
accordance
alkyl
haloalkyl
halogen
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WO2002017900A9 (fr
WO2002017900A3 (fr
Inventor
Thomas J. Schall
Brian E. Mcmaster
Daniel J. Dairaghi
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Chemocentryx Inc
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Chemocentryx Inc
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Publication of WO2002017900A9 publication Critical patent/WO2002017900A9/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • Cytomegalovirus is an important human pathogen and a major opportunist which emerges to cause disease in the immuno-compromised such as AIDS patients, neonates, and individuals who have been given immunosuppressive drugs as part of a transplantation regimen.
  • CMN Cytomegalovirus
  • the consequences of CMN in acute or re-emerging infections can be dire, including retinitis, encephalitis, and pneumocystis, among other pathologies.
  • CMN establishes a persistent lifelong infection through which it has been linked to a variety of inflammatory conditions including coronary artery occlusion following heart transplant and arthrectomy and restenosis following angioplasty.
  • CMN interacts with leukocytes during acute infection of the host as well as during lifelong latency.
  • leukocytes are important players in CMN-induced disease and have been implicated in the acute phase of infection as vehicles for dissemination of virus and as sites of residence during lifelong latency.
  • the present invention provides an assay for identifying a compound useful for blocking CMN dissemination in a host by determining whether the compound inhibits the binding of a chemokine to US28 or a US28 fragment.
  • the assay will be run as a competitive binding assay using a labeled chemokine.
  • chemokines are known to bind to US28 and are useful in this aspect of the invention.
  • the chemokine is fractalkine and the assay is a radioligand binding assay.
  • the present invention provides methods for blocking CMN dissemination in a host by administering to the host an effective amount of a compound which blocks the binding of a chemokine to US28.
  • the compound is one which was identified using an assay of the present invention.
  • the present invention provides pharmaceutical compositions for the treatment of CMN comprising compounds identified in the present assays and further described below.
  • Figure 1 illustrates the specific displacement of chemokine (fractalkine) binding to the US28 chemokine receptor.
  • Figure 2 illustrates the signaling profile and cross desensitization between methiothepin and a chemokine ligand (fractalkine) for US28.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. -Cio means one to ten carbons).
  • saturated hydrocarbon radicals include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.
  • An unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • alkyl groups examples include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • alkyl refers to unsubstituted versions of the radicals indicated above. Substituted forms of "alkyl" are defined in more detail below.
  • alkylene by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified by -CH CH 2 CH 2 CH 2 -, and further includes those groups described below as “heteroalkylene.”
  • an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention.
  • a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • alkoxy alkylamino and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • the heteroatom(s) O, N and S may be placed at any interior position of the heteroalkyl group.
  • the heteroatom Si may be placed at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule.
  • heteroalkylene by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified by -CH 2 - CH 2 -S-CH 2 CH 2 - and -CH 2 -S-CH 2 -CH 2 -NB-CH 2 -.
  • heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenedia ino, and the like).
  • cycloalkyl and “heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl”, respectively.
  • heterocycloalkyl or heterocyclyl a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule.
  • examples of cycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.
  • heterocycloalkyl examples include 1 -(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4- morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien- 2-yl, tetrahydrothien-3-yl, 1 -piperazinyl, 2-piperazinyl, and the like.
  • halo or halogen
  • haloalkyl by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl.
  • ( -C- haloalkyl) is mean to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
  • acyl is used in its conventional sense and refers to an organic radical derived from an organic acid by the removal of the hydroxyl group.
  • acyl groups include acetyl, propionyl, butanoyl, hexanoyl, isobutyryl, octanoyl, and the like.
  • aryl means, unless otherwise stated, a polyunsaturated, typically aromatic, hydrocarbon substituent which can be a single ring or multiple rings (up to three rings) which are fused together or linked covalently.
  • heteroaryl refers to aryl groups (or rings) that contain from zero to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quateraized.
  • a heteroaryl group can be attached to the remainder of the molecule through a heteroatom.
  • Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1 -pyrrolyl, 2- pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4- oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2- thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3- pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl
  • aryl when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above.
  • arylalkyl is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3- (l-naphthyloxy)propyl, and the like).
  • alkyl e.g., benzyl, phenethyl, pyridylmethyl and the like
  • oxygen atom e.g., phenoxymethyl, 2-pyridyloxymethyl, 3- (l-naphthy
  • heteroaryl are meant to include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.
  • R', R" and R"' each independently refer to hydrogen, unsubstituted (d-C ⁇ alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C 1 -C )alkyl groups.
  • R' and R" When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7- membered ring.
  • -NR'R is meant to include 1-pyrrolidinyl and 4- mornholinvl. From the above discussion of s ⁇ hstit ⁇ ents.
  • R', R" and R'" are independently selected from hydrogen, (d-C ⁇ alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted arylH - C 4 )alkyl, and (unsubstituted aryl)oxy-(C 1 -C 4 )alkyl.
  • Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(O)-(CH 2 ) q -U-, wherein T and U are independently -NH-, -O-, -CH 2 - or a single bond, and q is an integer of from 0 to 2.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH 2 ) r -B-, wherein A and B are independently -CH 2 -, -O-, -NH-, -S-, -S(O)-, -S(O) 2 -, -S(O) 2 NR'- or a single bond, and r is an integer of from 1 to 3.
  • One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CH ) s -X-(CH 2 ) r , where s and t are independently integers of from 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O) 2 -, or -S(O) 2 NR'-.
  • the substituent R' in -NR'- and -S(O) 2 NR'- is selected from hydrogen or unsubstituted (Ci- C 6 )alkyl.
  • heteroatom is meant to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).
  • salts are meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
  • inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
  • salts of arnino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge, S.M., et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19).
  • Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
  • the neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
  • the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.
  • the present invention provides compounds which are in a prodrug form.
  • Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention.
  • prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.
  • Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
  • Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the present invention.
  • the compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds.
  • the compounds may be radiolabeled with radioactive isotopes, such as for example tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.
  • CMN harbors in its genome an open reading frame (ORF), designated US28, which encodes a protein that acts as a functional receptor for certain human and viral chemokines.
  • ORF open reading frame
  • US28 Upon infection of a cell by CMN, US28 is expressed on the surface of the infected cell and becomes capable of responding to chemokines in the environment. Because the virus on its own is inherently non-motile, and because chemokines and their receptors encoded by human cells are known to regulate the migration of leukocytes and other cells through the body, CMN US28 is now thought to be encoded by the virus to facilitate the dissemination of CMN through the body during and after infection.
  • CMN US28 has been shown to bind a variety of human, murine , and virus-encoded CC chemokines in a variety of assay formats.
  • CX3C chemokine, Fractalkine binds with a very high affinity (K ⁇ 50 pM) to US28.
  • Fractalkine is expressed on certain endothelial cell surfaces and on populations of dendritic cells (DC), and may thus define a portal through which CMN infected cells go from the circulation to the tissue space, as well as find residence in the DC.
  • the present invention provides a novel mechanism for control of cytomegalovirus induced disease.
  • the compounds described herein can limit the viral spread to secondary organs and so limit viral replication.
  • the compounds described herein do not act at the stage of viral D ⁇ A replication and so are less prone to problems with toxicity and the development of viral resistance.
  • Other GPCR targeted therapeutics have demonstrated high efficacy and been well tolerated for a number of indications. Description of the Embodiments
  • the present invention provides assays for identifying a compound capable of blocking CMN dissemination in a host, by determining whether the compound inhibits the binding of a chemokine to US28 or a US28 fragment.
  • the assays provided herein are typically cell-based assays in which a cell which stably expresses US28 is treated with a candidate compound and a chemokine in a competitive binding format.
  • a variety of other assay formats are also useful in the present invention.
  • substrate-bound or support-bound chemokines (or ligands) can be contacted with a labeled cell or liposome having an associated US28 or US28 fragment
  • the cell line is a mouse cell line (e.g., ⁇ SO cells from R&D Systems, Minneapolis, Minnesota, USA).
  • the cell line is a human cell line (e.g., primary human lung and foreskin fibroblasts from Clonetics, San Diego California, USA, or human diploid lung fibroblasts (MRC-5 and WI-38), or HUNECs). Additionally, human embryonic kidney 293 cells ("HEK293" from American Tissue
  • the cell line is a primary rhesus monkey dermal fibroblast (from University of California at Davis Primate Center).
  • CPN whole virus
  • US28 cD ⁇ A typically under the control of a CMN promoter
  • cell-free systems can also be employed wherein a fragment of US28 (e.g., ⁇ H 2 -terminal peptide, extracellular loops and the like) can be used alone (or in combinations of US28 fragments) to assay binding levels of a chemokine in the presence of a candidate agent.
  • expressed or synthesized receptor proteins of US28 can be embedded in artificial membrane systems to assay for chemokine binding in the presence of a candidate agent (see for example, systems described in Kitaguchi, et al, Biochem. Biophys. Res. Commun. 261(3):784-789 (1999) and Myung, et al., Anal. Biochem. 270(2):303-313 (1999)).
  • the cells are cultured in a suitable buffer (e.g., IMDM-5% FBS, DMEM 1885-10% FCS, HUVEC complete medium, and the like) then centrifuged and resuspended in assay buffer (e.g., HEPES with NaCl, CaCl 2 , MgCl 2 , and BSA) to a concentration of from about 5 x 10 5 to about 5 x 10 7 , preferably from about 2 to about 8 x 10 6 . Aliquots of the cells are then contacted with the candidate compounds and labeled chemokine.
  • a suitable buffer e.g., IMDM-5% FBS, DMEM 1885-10% FCS, HUVEC complete medium, and the like
  • assay buffer e.g., HEPES with NaCl, CaCl 2 , MgCl 2 , and BSA
  • chemokines can be used in this aspect of the invention, including, for example, fractalkine, RANTES, MCP-3, MBP-l ⁇ and MCP-1.
  • a number of the chemokines are commercially available from sources such as R&D Systems or Peprotech, Inc., New Jersey, USA.
  • the labeled chemokine is labeled fractalkine.
  • a variety of labels can also be used with the chemokines described above.
  • the label will be a fluorescence label, a phosphorescence label, a radiolabel, a colorimetric label, or the like.
  • the labeled chemokine is a radiolabeled fractalkine, more preferably, 125 I-fractalkine.
  • the assay mixture is typically incubated for a period of time of from about 1 to about 6 hours at a temperature of from about 1 to about 10°C. Preferably the mixture is incubated for a period of from about 2 to about 4 hours at a temperature of about 4°C.
  • assay conditions can be employed, depending on the cell line used, the concentrations of the compounds and chemokine and the concentration of the cells themselves.
  • the assay wells can be harvested under vacuum using filter plates, pre-soaked with PEI solution (for those embodiments carried out on 96-, 384-, 1536-well or larger plates). Scintillation fluid (for radiolabel assays) is added, the plates are sealed and the wells are counted. Alternatively, other quantitative methods are employed when, for example, fluorescent labels are used.
  • X 1 , X 2 , X 3 and X 4 are each independently N or C-R 1 , wherein R 1 is H, halogen, (d-C 4 )alkyL (C ⁇ -C )alkoxy, (d-C ⁇ alkylthio, (d-C ⁇ haloalkyl, (d-C ⁇ haloalkoxy, nitro, cyano, (C ⁇ -C )acyl, amino, (CrC ⁇ alkylamino, or di(C 1 -C 4 )alkylamino.
  • Y 1 , Y 2 , Y 3 and Y 4 are each independently N or C-R 2 , wherein R 2 is H, halogen, (C C- alkyl, (CrC- ⁇ alkoxy, ( -C ⁇ alkylthio, ( -C- haloalkyl, (C 1 -C 4 )haloalkoxy, nitro, cyano, (Q- C 4 )acyl, amino, (d-C ⁇ alkylamino, or di(C 1 -C 4 )alkylamino.
  • R 2 is H, halogen, (C C- alkyl, (CrC- ⁇ alkoxy, ( -C ⁇ alkylthio, ( -C- haloalkyl, (C 1 -C 4 )haloalkoxy, nitro, cyano, (Q- C 4 )acyl, amino, (d-C ⁇ alkylamino, or di(C 1 -C 4 )alkylamin
  • the symbol Z 1 represents a substituted or unsubstituted (C 1 -C 3 )alkylene.
  • the symbol Z 2 represents a divalent moiety selected from -O-, -S- and -N(R)- wherein R is H, halogen, (C 1 -C )alkyl, (Cj-C )alkoxy, (d-C- haloalkyl, (C 1 -C 4 )haloalkoxy, nitro, cyano, (C 1 -C 4 )acyl, amino, (CrC ⁇ alkylamino, or d ⁇ Q-C ⁇ aU ylammo.
  • N Het represents a substituted or unsubstituted 4-, 5-, 6-, or 7- membered nitrogen heterocycle.
  • at least two of X 1 , X 2 , X 3 and X 4 are CH, more preferably three of X 1 , X 2 , X 3 and X 4 are CH and the fourth is C-R 1 , wherein R 1 is halogen, ( -C ⁇ alkylthio, (d-C ⁇ haloalkyl, (d-C ⁇ haloalkoxy, nitro, cyano, or (Q- C 4 )acyl.
  • Y 1 , Y 2 , Y 3 and Y 4 are each independently C-R 2 , wherein R 2 is H, halogen, (d-C- alkyl, (C 1 -C 4 )alkoxy, ( - C 4 )alkylthio, (d-G haloalkyl, (d-C 4 )haloalkoxy, nitro, cyano, (d-C 4 )acyl, amino, (C ⁇ - C 4 )alkylamino, di(d-C 4 )alkylamino.
  • each of Y 1 , Y 2 , Y 3 and Y 4 are independently C-R 2 , wherein R 2 is H, halogen, (d-C 4 )alkylthio, or (CrC ⁇ haloalkyl.
  • Z 1 represents an ethylene or propylene group, more preferably an ethylene group in which N Het is attached at the position adjacent to the ring defined by Y 1 , Y 2 , Y 3 and Y 4 .
  • Z 2 is -O- or -S-, more preferably -S-.
  • N Het are the substituted or unsubstituted 5- or 6-membered nitrogen heterocycles.
  • Particularly preferred heterocycles include piperidine, piperazine, pyrrolidine, oxazoline, imidazoline, pyrazine and morpholine.
  • N Het is a substituted or unsubstituted 6-membered nitrogen heterocycle.
  • N Het is a substituted or unsubstituted piperazine which is attached to Z 1 through a nitrogen atom of the piperazine ring.
  • Preferred substituents for the piperazine ring are (d-C- alkyl, (d-C ⁇ haloalkyl, or (d-C 4 )acyl. Further preferred substitutents are (d-C )alkyl, with methyl, ethyl and propyl substituents being the most preferred.
  • the compounds are substituted 10- piperazino-10,1 l-dihydrodibenzo(b,f)thiepins having the formula:
  • R 1 and R 2 are substituents independently selected from the group of halogen, (d-C 4 )alkyl, (d-C 4 )alkoxy, (d-C )alkylthio, (d-C 4 )haloalkyl, (d- C 4 )haloalkoxy, nitro, cyano, (d-C 4 )acyl, amino, (CrC ⁇ alkylamino, and di(d- C 4 )alkylamino.
  • R 3 represents (d-C ⁇ alkyl, (d-C 4 )haloalkyl, or (d-C 4 )acyl.
  • m is 0 and n is 1. More preferably, m is 0, n is 1 and R 2 is selected from the group of halogen, (d-C 4 )alkyl, (d-G alkoxy, (d-C 4 )alkylthio and (d-G haloalkyl. Still further preferred are those embodiments in which m is 0, n is 1 and R 2 is selected from the group of halogen and (d-C 4 )alkylthio. Most preferably, the R 2 substituent is at the 8-position of the dihydrodibenzo(b,f)thiepin ring system.
  • Particularly preferred compounds for use in the present invention are methiothepin (free base or salt, CAS No. 20229-30-5) and octoclothepin (free base or salt, CAS No. 4789-68-8, for the maleate salt).
  • compositions and methods are described in U.S. Patent No. 3,379,729 "Piperazinyldibenzothiepins" April 23, 1968. See also U.S. Patent No. 4,444,778. Still other related and useful dihydrodibenzo(b,f)thiepins are described in Jilek, et al., Collect. Czech. Chem. Commun. 33(6):1831-1845 (1968).
  • compositions useful in the treatment ofCMV infection are described in U.S. Patent No. 3,379,729 "Piperazinyldibenzothiepins" April 23, 1968. See also U.S. Patent No. 4,444,778. Still other related and useful dihydrodibenzo(b,f)thiepins are described in Jilek, et al., Collect. Czech. Chem. Commun. 33(6):1831-1845 (1968).
  • compositions useful for preventing CMN dissemination in a host which comprises a pharmaceutically acceptable carrier or adjuvant and an effective amount of a compound identified using the assays described herein.
  • the compound is a compound of formula I, more preferably a compound of formula la.
  • Other preferred compounds are those described in Provisional
  • compositions typically contain from about 0.1% to about 99% by weight of active compound, and preferably from about 10% to about 60% by weight depending on which method of administration is employed.
  • a CMN dissemination-inhibiting amount is that amount of active compound required to slow the progression of viral dissemination or reduce the amount of viral dissemination from that which would otherwise occur without administration of the compound. Or, it is an amount of active compound required to slow the progression or reduce the intensity of symptoms resulting from CMN infection or elimination thereof.
  • CMN dissemination-inhibiting activity of compounds of the invention can be determined according to the assays described herein. The assays provide an indication of chemokine binding to US28, more typically fractalkine binding to US28.
  • the compounds provided herein inhibit the binding of fractalkine to US28 with activity expressed as IC50 (that amount of compound that reduces fractalkine binding by 50%).
  • IC50 that amount of compound that reduces fractalkine binding by 50%.
  • the compounds provided herein will typically exhibit an IC50 of approximately 50 ⁇ g/mL or less, preferably 25 ⁇ g/mL or less, more preferably 10 ⁇ g/mL or less, and most preferably less than 1 ⁇ g/mL.
  • compositions of the invention the proportion of each carrier, diluent or adjuvant is determined by the solubility and chemical nature of the compound and the route of administration according to standard pharmaceutical practice. In order to obtain consistency of administration, however, it is preferred that a composition of the invention is in the form of a unit dose.
  • the unit dose presentation forms for oral administration may be tablets and capsules and may contain conventional excipients such as binding agents (e.g., acacia, gelatin, sorbitol, or polyvinylpyrrolidone), fillers (e.g., lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine), tableting lubricants (e.g., magnesium stearate), disintegrants (e.g., starch, polyvinylpyrrolidone, sodium starch glycoallate or microcrystalline cellulose), or pharmaceutically acceptable wetting agents (e.g., sodium lauryl sulfate).
  • binding agents e.g., acacia, gelatin, sorbitol, or polyvinylpyrrolidone
  • fillers e.g., lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine
  • tableting lubricants e.g.
  • the compounds may be injected parenterally; this being intramuscularly, intravenously, or subcutaneously.
  • the compound may be used in the form of sterile solutions containing other solutes, for example, sufficient saline or glucose to make the solution isotonic.
  • the amount of active ingredient administered parenterally will be approximately 0.01 to 250 mg/kg/day, preferably about 1 to 10 mg/kg/day, more preferably about 0.5 to 30 mg/kg/day, and more most preferably about 1-20 mg/kg/day.
  • the compounds may be administered orally in the form of tablets, capsules, or granules containing suitable excipients such as starch, lactose, white sugar and the like.
  • the compounds may be administered orally in the form of solutions which may contain coloring and/or flavoring agents.
  • the compounds may also be administered sublingually in the form of tracheas or lozenges in which each active ingredient is mixed with sugar or corn syrups, flavoring agents and dyes, and then dehydrated sufficiently to make the mixture suitable for pressing into solid form.
  • the amount of active ingredient administered orally will depend on bioavailability of the specific compound.
  • the solid oral compositions may be prepared by conventional methods of blending, filling, tableting, or the like. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of tillers. Such operations are, of course, conventional in the art.
  • the tablets may be coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating.
  • Oral liquid preparations may be in the form of emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may or may not contain conventional additives.
  • suspending agents such as sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, or hydrogenated edible fats
  • emulsifying agents such as sorbitan monooleate or acaci
  • non- aqueous vehicles which may include edible oils), such as almond oil, fractionated coconut oil, oily esters selected from the group consisting of glycerin, propylene glycol, ethylene glycol, and ethyl alcohol
  • preservatives for instance methyl para- hydroxybenzoate, ethyl para-hydroxybenzoate, n-propyl parahydroxybenzoate, or n-butyl parahydroxybenzoate of sorbic
  • the compounds of the present invention may also be administered in the form ofsuppositori.es for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials are cocoa butter and polyethylene glycols.
  • topical application For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compounds of the present invention are employed. As used herein, topical application is also meant to include the use of mouth washes and gargles.
  • the invention provides the subject compounds in the form of a pro-drug, which can be metabolically or chemically converted to the subject compound by the recipient host.
  • a pro-drug which can be metabolically or chemically converted to the subject compound by the recipient host.
  • pro-drug derivatives are known in the art such as those that rely on hydrolytic cleavage or oxidative activation of the prodrug.
  • compositions may be advantageously combined and/or used in combination with other antiviral agents which are either therapeutic or prophylactic agents, and different from the subject compounds.
  • the compositions may also be advantageously combined and/or used in combination with agents that treat or induce conditions often associated with the viral infections that are sensitive to the present compounds, such as anti-HIN agents or immunosuppressive agents.
  • agents that treat or induce conditions often associated with the viral infections that are sensitive to the present compounds such as anti-HIN agents or immunosuppressive agents.
  • admimstration in conjunction with the subject compositions enhances the efficacy of such agents.
  • Exemplary antiviral agents include ganciclovir, foscarnet and cidofovir.
  • Exemplary anti-HIN agents include indinavir, ritonavir, AZT, lamivudine and saquinavir.
  • immunosuppressive agents include cyclosporin and FK-506.
  • the compositions may also be advantageously used as antiviral prophylactic treatment in
  • the present invention provides novel methods for the use of the foregoing compounds and compositions.
  • the invention provides novel methods for treating or preventing viral dissemination from CMN infection. The methods typically involve administering to a patient an effective formulation of one or more of the subject compositions.
  • the invention provides methods of using the subject compounds and compositions to treat disease or provide medicinal prophylaxis to individuals who possess a compromised immune system or are expected to suffer immunosuppressed conditions, such as patients prior to undergoing immunosuppressive therapy in connection with organ transplantation or anticancer chemotherapy. These methods generally involve administering to the host an effective amount of the subject compounds or pharmaceutically acceptable compositions.
  • the compositions and compounds of the invention and the pharmaceutically acceptable salts thereof can be administered in any effective way such as via oral, parenteral or topical routes. Generally, the compounds are administered in dosages ranging from about 2 mg up to about 2,000 mg per day, although variations will necessarily occur depending on the disease target, the patient, and the route of administration.
  • Preferred dosages are administered orally in the range of about 0.05 mg/kg to about 20 mg/kg, more preferably in the range of about 0.05 mg/kg to about 2 mg/kg, most preferably in the range of about 0.05 mg/kg to about 0.2 mg per kg of body weight per day.
  • Therapeutic and prophylactic methods of this invention comprise the step of treating patients in a pharmaceutically acceptable manner with those compounds or compositions.
  • Such compositions may be in the form of tablets, capsules, caplets, powders, granules, lozenges, suppositories, reconstitutable powders, or liquid preparations, such as oral or sterile parenteral solutions or suspensions.
  • Compounds of the invention may also be administered via an intraocular implant for treating retinitis as a result of CMN infection.
  • compounds may be embedded in a polymer based implant which will be release into the eye over an extended period of time.
  • Dosages may vary with the mode of administration and the particular compound chosen.
  • the dosage may vary with the particular patient under treatment.
  • the dosage of the compoimd used in the treatment will vary, depending on viral load, the weight of the patient, the relative efficacy of the compound and the judgment of the treating physician.
  • Such therapy may extend for several weeks or months, in an intermittent or uninterrupted manner.
  • the US28 expressing cells used in most assays consist of a mouse cell line (NSO cells from ATCC) stably expressing transfected US28 cDNA under the control of a CMV promoter (from R & D Systems). These cells were cultured in IMDM-5% FBS, and harvested when the concentration was between 0.5-1.0 x 10 ⁇ cells/mL. Some assays were performed with adherent human 293 cells (US28-293 cells) or membranes.
  • the cells were centrifuged and resuspended in assay buffer (20 mM HEPES, 140 mM NaCl, lmM CaC-2, 5mM MgCl2, and with 0.2% bovine serum albumin) to a concentration of 5.6 x l ⁇ 6 cells/mL.
  • assay buffer 20 mM HEPES, 140 mM NaCl, lmM CaC-2, 5mM MgCl2, and with 0.2% bovine serum albumin
  • cytoplasmic calcium mobilization experiments were done by loading US28-293 cells with INDO-1 dye (45 min. at room temperature), washing with PBS, and resuspending into Ca2+ 'flux' buffer (HBSS with 1% fetal bovine serum). For each test, 1 10 ⁇ cells were incubated at 37°C in the cuvette of a PTI spectrometer, and the ratio of 410/490 nm emission plotted over time (typically 2-3 minutes), with compounds added at 5 seconds, followed by fractalkine at 60 seconds.
  • a rise in intracellular Ca2+ is typically seen when US28-293 cells are challenged with fractalkine, an indication that the US28 receptor bound to the ligand, engaged a G-protein linked cascade which resulted in the mobilization of Ca2+ in the cytoplasm of the US28- bearing cells.
  • Compounds which inhibited fractalkine binding were tested in this assay for the effects on Ca2+ in this system.

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Abstract

L'invention concerne des essais, des compositions et des procédés de traitement permettant de moduler la liaison des chimiokines avec l'US28 à la surface des cellules.
PCT/US2001/027363 2000-08-30 2001-08-30 Modulateurs de l'us28 Ceased WO2002017900A2 (fr)

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AU2001287043A AU2001287043A1 (en) 2000-08-30 2001-08-30 Modulators of us28 (chemokines) for treating cytomegalovirus infections

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002017969A3 (fr) * 2000-08-30 2003-05-22 Chemocentryx Inc Reactifs et methodes permettant de diagnostiquer la dissemination du cmv
WO2002018954A3 (fr) * 2000-08-30 2003-07-24 Chemocentryx Inc Inhibition de l'infection par cmv et de la dissemination de ce dernier
EP1393728A1 (fr) * 2002-08-30 2004-03-03 Vrije Universiteit Agonistes inverses agissant sur les recepteurs couplés à une protéine G codées par un virus
US6740324B2 (en) 2001-02-02 2004-05-25 Chemocentryx, Inc. Methods and compositions useful for stimulating an immune response
US6821998B2 (en) 2001-08-30 2004-11-23 Chemocentryx, Inc. Arylamines as inhibitors of chemokine binding to US28
EP2462952A1 (fr) 2010-12-10 2012-06-13 Medizinische Universität Graz Thérapie génique du cancer utilisant des acides nucléiques codant US28

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FI46164B (fr) * 1965-06-22 1972-10-02 Spofa Vereinigte Pharma Werke
US6420121B1 (en) * 1998-08-31 2002-07-16 Oregon Health Sciences University Prevention of cell migration initiation with CMV US28 receptor antagonists
US20020193374A1 (en) * 2000-08-30 2002-12-19 Chemocentryx Reagents and methods for the diagnosis of CMV dissemination
EP1350113A2 (fr) * 2000-08-30 2003-10-08 Chemocentryx, Inc. Inhibition de l'infection par cmv et de la dissemination de ce dernier

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002017969A3 (fr) * 2000-08-30 2003-05-22 Chemocentryx Inc Reactifs et methodes permettant de diagnostiquer la dissemination du cmv
WO2002018954A3 (fr) * 2000-08-30 2003-07-24 Chemocentryx Inc Inhibition de l'infection par cmv et de la dissemination de ce dernier
US7025968B2 (en) 2000-08-30 2006-04-11 Chemocentryx, Inc. CMV vaccines
US6740324B2 (en) 2001-02-02 2004-05-25 Chemocentryx, Inc. Methods and compositions useful for stimulating an immune response
US6821998B2 (en) 2001-08-30 2004-11-23 Chemocentryx, Inc. Arylamines as inhibitors of chemokine binding to US28
US7101894B2 (en) 2001-08-30 2006-09-05 Chemocentryx, Inc. Bicyclic compounds as inhibitors of chemokine binding to US28
EP1393728A1 (fr) * 2002-08-30 2004-03-03 Vrije Universiteit Agonistes inverses agissant sur les recepteurs couplés à une protéine G codées par un virus
WO2004019942A1 (fr) * 2002-08-30 2004-03-11 Vrije Universiteit Van Amsterdam Agonistes inverses agissant au niveau des recepteurs couples a la proteine g virale
EP2462952A1 (fr) 2010-12-10 2012-06-13 Medizinische Universität Graz Thérapie génique du cancer utilisant des acides nucléiques codant US28
WO2012076694A1 (fr) 2010-12-10 2012-06-14 Medizinische Universität Graz Thérapie génique du cancer utilisant des acides nucléiques codant us28 et une protéine g

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