Classifications

• • 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
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to methods and compounds useful for inhibiting T helper cell differentiation. Methods and compounds for decreasing IL- 12 signaling in T helper cells are also provided.
• T helper (Th) cells are a sub-group of white blood cells (i.e., lymphocytes) that play an important role in establishing and maximizing the capabilities of the immune system. T helper cells are involved in activating and directing the activities and functions of other immune cells. For example, T helper cells are essential in determining B cell antibody class switching, in the activation and growth of cytotoxic T cells, and in maximizing bactericidal activity of phagocytic cells, such as macrophages.
• na ⁇ ve T helper cells i.e., ThO cells
• HLA class II molecules antigen stimulation
• ThI or Th2 cell a decision that is primarily influenced by cytokines within the local environment. If IL-12 is the major cytokine present during antigen stimulation, a ThI response occurs and a Th2 response is inhibited; if EL-4 is the major cytokine present during antigen stimulation, a Th2 response occurs and a ThI response is inhibited. Therefore, IL-12 and IL-4 play decisive roles in the differentiation of na ⁇ ve T helper cells into ThI cells or Th2 cells.
• ThI cells also referred to as pro-inflammatory T cells
• cytokines include IL-2, TNF- ⁇ , TNF- ⁇ , IL-I, and IFN- ⁇ and are associated with cell- mediated immune responses
• Th2 cells produce predominantly anti-inflammatory cytokines, including IL-4, IL-5, IL-6, IL-IO, and IL-13 and are associated with humoral immune responses.
• ThI responses are associated with a number of organ-specific and systemic autoimmune conditions, including chronic inflammatory diseases and delayed type hypersensitivity reactions.
• Current methods used to reduce ThI responses including various non-specific immunosuppressive agents, such as, for example, cyclosporine and azathioprine, often require administration of therapeutic agents in high doses and are thus associated with toxicity and adverse side effects.
• it would be desirable to reduce cell-mediated immune responses i.e., reduce ThI -mediated responses
• humoral immune responses i.e., Th2 -mediated responses.
• the present invention meets this need by providing methods and compounds effective at inhibiting the differentiation of T helper cells, including na ⁇ ve T helper cells, into ThI cells.
• the present invention provides methods for inhibiting the differentiation of T helper cells into ThI cells.
• the invention provides a method for inhibiting the differentiation of a T helper cell into a ThI cell, the method comprising contacting the T helper cell with an effective amount of a compound that inhibits the activity of a hypoxia-inducible factor (HIF) prolyl hydroxylase enzyme, thereby inhibiting the differentiation of the T helper cell into the ThI cell.
• HIF hypoxia-inducible factor
• the invention provides a method for inhibiting the differentiation of a T helper cell into a ThI cell in a subject, the method comprising administering to the subject an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby inhibiting the differentiation of the T helper cell into a ThI cell in the subject.
• the T helper cell is a naive T helper cell.
• the T helper cell is a T cell receptor (TCR)-stimulated T helper cell.
• TCR T cell receptor
• the differentiation of a T helper cell into a ThI cell is induced by IL-12, and the methods of the present invention specifically provide for inhibiting IL-12-induced differentiation of a T helper cell into a ThI cell.
• the present invention also provides for the use of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme in the manufacture of a medicament for inhibiting the differentiation of a T helper cell into a ThI cell in a subject.
• the invention provides a method for inhibiting EL- 12 signaling in a T helper cell, the method comprising contacting the T helper cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby inhibiting EL- 12 signaling in the T helper cell.
• the present invention provides methods for inhibiting IL-12-mediated T helper cell expression of IL12R ⁇ 2, IL18R.1, or IL18RAP, the method comprising contacting a T helper cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby inhibiting IL-12-mediated gene expression of IL12R ⁇ 2, IL18R1, or IL18RAP in the T helper cell.
• the T helper cell is a naive T helper cell.
• the T helper cell is a T cell receptor (TCR)-stimulated T helper cell.
• Methods for inhibiting IL-12 signaling in a subject comprising administering to the subject an effective amount of a compound that inhibits HIF prolyl hydroxylase activity, are also provided herein.
• Methods for inhibiting T helper cell secretion of pro-inflammatory cytokines are also provided by the present invention.
• the present invention provides a method for inhibiting IFN- ⁇ secretion from a T helper cell, the method comprising contacting the T helper cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby inhibiting the IFN- ⁇ secretion from the T helper cell.
• the present invention provides a method for inhibiting TNF- ⁇ secretion from a T helper cell, the method comprising contacting the T helper cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby inhibiting the TNF- ⁇ secretion from the T helper cell.
• the T helper cell is a naive T helper cell.
• the T helper cell is a T cell receptor (TCR)-stimulated T helper cell.
• the secretion of IFN- ⁇ or TNF- ⁇ by a T helper cell is induced by IL- 12, and the methods of the present invention specifically provide for inhibiting IL-12-induced IFN- ⁇ and TNF- ⁇ secretion from a T helper cell.
• the invention further provides methods for inhibiting T helper cell secretion of IFN- ⁇ and TNF- ⁇ in a subject, the methods comprising administering to the subject an effective amount of a compound that inhibits HIF prolyl hydroxylase activity.
• a compound used in the present methods is a structural mimetic of 2-oxoglutarate, wherein the compound inhibits the target HIF prolyl hydroxylase enzyme competitively with respect to 2-oxoglutarate and noncompetitively with respect to iron.
• compounds of the present invention include heterocyclic carboxamides, phenanthrolines, and hydroxamates.
• a heterocyclic carboxamide of the present invention is a pyridine carboxamide, a quinoline carboxamide, a quinolone carboxamide, an isoquinoline carboxamide, a cinnoline carboxamide, or a beta-carboline carboxamide.
• compounds of the present invention include variously substituted 3-hydroxy-pyridine-2-carbonyl-glycines, 4-hydroxy- pyridazine-3-carbonyl-glycines, 3-hydroxy-quinoline-2-carbonyl-glycines, 4-hydroxy-2-oxo-l,2-dihydro- quinoline-3 -carbonyl-glycines, 4-hydroxy-2-oxo- 1 ,2-dihydro-naphthyridine-3 -carbonyl-glycines, ⁇ -hydroxy- ⁇ -oxo ⁇ -dihydro-pyridopyrazine-T-carbonyl-glycines, 4-hydroxy-isoquinoline-3-carbonyl- glycines, 4-hydroxy-cinnoline-3-carbonyl-glycines, 7-hydroxy-thienopyridine-6-carbonyl-glycines, 4-hydroxy-thienopyridine-5-carbonyl-glycines, V-hydroxy-thi
• the compound is [(4- Hydroxy-l-methyl-T-phenoxy-isoquinoline-S-carbony ⁇ -aminoJ-acetic acid (Compound A), [(4-Hydroxy- 7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid (Compound B), [(I -Benzoyl -4-hydroxy- isoquinoline-3-carbonyl)-amino]-acetic acid (Compound C), [(8-Chloro-4-hydroxy-5-methyl- isoquinoline-3-carbonyl)-amino] -acetic acid (Compound D), ⁇ [4-Hydroxy-8-(4-methoxy-phenoxy)-l- methyl-isoquinoline-3-carbonyl]-amino ⁇ -acetic acid (Compound E), (S)-2-[(l-Cyano-4-hydroxy-8- phenoxy-isoquinoline-3-carbonyl)-amino]-
• Compounds for use in the present invention are compounds that inhibit HIF prolyl hydroxylase activity.
• a compound that inhibits HIF prolyl hydroxylase activity is any compound that reduces or otherwise inhibits the activity of at least one HIF prolyl hydroxylase enzyme.
• Various compounds that inhibit HIF prolyl hydroxylase have been identified and are suitable for use in the methods and medicaments as claimed in the present invention.
• a compound for use in the present methods and medicaments is a pyridine-2- carboxamide, a pyridazine-3-carboxamide, a quinoline-2-carboxamide, an isoquinoline-3-carboxamide or ester thereof as described in European Patent Nos. EP0650960 and EP0650961.
• a compound for use in the present methods and medicaments is a pyridine-2-carboxamide as described in U.S. Patent Application Publication No. 2007/0299086.
• a compound for use in the present methods and medicaments is a pyridine-2-carboxamidoester, a pyridazine-3- carboxamidoester, or an isoquinoline-3-carboxamidoester as described in U.S. Patent No. 5,658,933.
• a compound for use in the present methods and medicaments is a pyridine-2- carboxamide, a pyridizine-3-carboxamide, or a quinoline-2-carboxamide as described in U.S. Patent No. 5,620,995.
• a compound for use in the methods and medicaments of the present invention is a 3-hydroxypyridine-2-carboxamidoester as described in U.S. Patent No. 6,020,350; a sulfonamidocarbonylpyridine-2-carboxamide as described in U.S. Patent No.
• a compound for use in the present methods and medicaments is a quinoline-2-carboxamide as described in U.S. Patent Nos. 5,719,164 and 5,726,305.
• a compound for use in the present methods and medicaments is an isoquinoline-3- carboxamide as described in U.S. Patent No. 6,093,730 and 7,323,475.
• a compound for use in the present methods and medicaments is an isoquinoline-3-carboxamide as described in U.S. Patent Application Publication No. 2007/0298104.
• a compound for use in the present methods and medicaments is a beta-carboline-3-carboxamide, a pyrrolo[3,2-c]pyridine- 6-carboxamide, a pyrrolo[2,3-c]pyridine-5-carboxamide, a thiazolo[4,5-c]pyridine-6-carboxamide, or a thiazolo[5,4-c]pyridine-6-carboxamide as described in U.S. Patent Application Publication No. 2008/0004309.
• a compound for use in the present methods and medicaments is a thieno[3,2- c]pyridine-6-carboxamide or a thieno[2,3-c]pyridine-5-carboxamide as described in U.S. Patent Application Publication No. 2006/0199836.
• a compound for use in the present methods and medicaments is a 2,4-dioxo-l,2,3,4-tetrahydro-pyrimidine-5-carboxamide or a 4-oxo-2- thioxo-l,2,3,4-tetrahydro-pyrimidine-5-carboxamide as described in International Publication No. WO 2007/150011.
• a compound for use in the present methods and medicaments is a 6-oxo-l,6-dihydro-pyrimidine-5-carboxamide as described in U.S. Patent Application Publication No. 2008/0171756.
• a compound for use in the present methods and medicaments is a 2-oxo-l,2- dihydro-quinoline-3-carboxamide as described in International Publication No. WO 2007/038571 and U.S. Patent Application Publication No. 2007/0249605.
• a compound for use in the present methods and medicaments is a 2-oxo-l,2-dihydro-[l,8]naphthyridine-3-carboxamide, a 2-oxo-l,2- dihydro-[l,6]naphthyridine-3-carboxamide, or a 6-oxo-5,6-dihydro-pyrido[2,3-b]pyrazine-7-carboxamide as described in International Publication Nos. WO 2007/103905, WO 2008/076425, and WO 2008/130527.
• a compound for use in the present methods and medicaments is a 6-oxo-6,7-dihydro-thieno[2,3-b]pyridine-5-carboxamide, a 5-oxo-4,5-dihydro-thieno[3,2-b]pyridine- 6-carboxamide, or a 6-oxo-6,7-dihydro-pyrazolo[3,4-b]pyridine-5-carboxamide as described in International Publication No. WO 2007/136990.
• a compound for use in the present methods and medicaments is a 3-oxo-2,3-dihydro- pyridazine-4-carboxamide as described in U.S. Patent Application Publication No. 2008/0214549.
• a compound for use in the present methods and medicaments is a 3-oxo-3,4-dihydro- naphthalene-2-carboxamide, a 7-oxo-7,8-dihydro-quinoline-6-carboxamide, or a 7-oxo-7,8-dihydro- isoquinoline-6-carboxamide as described in International Publication No. WO 2008/076427.
• a compound for use in the present methods and medicaments is a 3-hydroxy-l-oxo-lH- indene-2-carboxamide as described in International Publication No. WO 2008/130508.
• a compound for use in the present methods and medicaments is a 4-oxo-[l,10]- phenanthroline as described in U.S. Patent Nos. 5,916,898 and 6,200,974, and International Publication No. WO 99/21860.
• a 4-oxo-[l,10]-phenanthroline is 4-oxo-l,4-dihydro- [l,10]phenanthroline-3-carboxylic acid (see, e.g., Seki et al. (1974) Chem Abstracts 81:424, No. 21).
• a compound for use in the present methods and medicaments is a hydrozone as described in U.S. Patent No. 6,660,737.
• a compound for sue in the present methods and medicaments is a dihydropyrazole or a dihydropyrozolone as described in U.S. Patent No. 6,878,729 and International Publication No. WO 2008/049539.
• a compound for use in the present methods and medicaments is a dipyridyl dihyropyrazones as described in International Publication No. WO 2006/114213.
• a compound for use in the present methods and medicaments is a spiroindalone as described in International Publication No. WO 2008/144266.
• compounds for use in the present invention are selected from the group consisting of 2-oxoglutarate mimetics, iron chelators, and proline analogs.
• the compound used in the methods and medicaments of the present invention is a 2-oxoglutarate structural mimetic.
• the compound used in the methods and medicaments of the present invention is a 2-oxoglutarate structural mimetic that inhibits HIF prolyl hydroxylase competitively with respect to 2-oxoglutarate and noncompetitively with respect to iron.
• a compound for use in the methods and medicaments of the present invention is, in various embodiments, a cyclic carboxamide.
• the cyclic carboxamide is a carbonyl glycine.
• the carboxamide is replaced by a carbonyl proprionic acid.
• the compound used in the methods and medicaments of the present invention is a carbocyclic carboxamide.
• cyclic carboxamides suitable for use in the present invention are heterocyclic carboxamides.
• a compound of the present invention is a heterocyclic carboxamide having a heterocyclic group selected from the group consisting of: azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, furan, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,
• the heterocyclic group is a single ring selected from the group consisting of a pyridine, a pyridinone, a pyradizine, a pyridazinone, a pyrimidine, and a pyrimidinone ring.
• the heterocyclic group is a multiple condensed ring selected from the group consisting of an isoquinoline, an isoquinolone, a naphthyridinone, a pyrrolopyridine, a pyrrolopyridinone, a pyrozolopyridinone, a pyrrolopyridizinone, a quinoline, a quinolone, a chromenone, a thiochromenone, a thienopyridine, a thienopyridinone, a thiazolopyridine, and a thiazolopyridinone.
• a particularly preferred compound of the present invention is a heterocyclic carbonyl glycine.
• the heterocyclic carbonyl glycine suitable for use in the present invention is a heterocyclic carbonyl glycine having a heterocyclic group that is selected from the following list: azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, furan, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazin
• the heterocyclic carbonyl glycine suitable for use in the present invention is a heterocyclic carbonyl glycine having a heterocyclic group, wherein the heterocyclic group is a single ring selected from the following list: a pyridine, a pyridinone, a pyradizine, a pyridazinone, a pyrimidine, and a pyrimidinone ring.
• the heterocyclic carbonyl glycine suitable for use in the present invention is a heterocyclic carbonyl glycine having a heterocyclic group, wherein the heterocyclic group is a multiple condensed ring selected from the group consisting of an isoquinoline, an isoquinolone, a naphthyridinone, a pyrrolopyridine, a pyrrolopyridinone, a pyrozolopyridinone, a pyrrolopyridizinone, a quinoline, a quinolone, a chromenone, a thiochromenone, a thienopyridine, a thienopyridinone, a thiazolopyridine, and a thiazolopyridinone.
• the heterocyclic group is a multiple condensed ring selected from the group consisting of an isoquinoline, an isoquinolone, a naphthyridinone, a pyrrolopyridine,
• a compound for use in the present methods and medicaments is an isoquinoline carbonyl glycine; preferably, an isoquinoline-S-carbonyl-glycine or a 4-hydroxy-isoquinoline-3-carbonyl glycine.
• a compound for use in the present methods and medicaments is ⁇ [4- Hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carbonyl]-amino ⁇ -acetic acid (Compound A); [(4- Hydroxy-l-methyl-V-phenoxy-isoquinoline-S-carbony ⁇ -aminoJ-acetic acid (Compound B); or ⁇ [1- Cyano-6-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino ⁇ -acetic acid (Compound C).
• a compound for use in the present methods and medicaments is a HIF prolyl hydroxylase inhibitor compound of Formula I:
• HIF prolyl hydroyxlase inhibitors include, but are not limited to, variously substituted pyridine-2-carbonyl-glycines, pyridazine-3- carbonyl-glycines, quinoline-2-carbonyl-glycines, 2-oxo-l ,2-dihydro-quinoline-3-carbonyl-glycines, 2- oxo-1, 2-dihydro-naphthyridine-3-carbonyl-glycines, 6-oxo-4,6-dihydro-pyridopyrazine-7-carbonyl- glycines, isoquinoline-3-carbonyl-glycines, cinnoline-3-carbonyl-glycines,
• a compound for use in the methods and medicaments of the present invention is a compound of Formula II:
• R 1 is selected from hydrogen and (CVC 4 )-alkyl
• R 1 , R 2 , R 3 , R 4 and R 5 are identical or different and are selected from the group consisting of hydrogen, hydroxyl, halogen, cyano, trifiuoromethyl, nitro, carboxyl; (C 1 -C 2 O)- alkyl, (C 3 -C 8 )-cycloalkyl, (C 3 -C 8 )-cycloalkoxy, (C 6 -C 12 )-aryl, (C 7 -C 16 )-aralkyl, (C 7 -C, 6 )-aralkenyl, (C 7 -C 16 )-aralkynyl, (C 2 -C 20 )-alkenyl, (C 2 -C 20 )-alkynyl, (C,- C 20 )-alkoxy, (C 2 -C 20 )-alkenyloxy, (C 2 -C 20 )-alkynyloxy, retinyloxy, (C 6
• a compound for use in the present invention is a compound of Formula II wherein: R' is hydrogen or (C,-C 3 )-alkyl;
• R 1 is selected from hydrogen, halo, (Ci-C 3 )-alkyl, cyano, or arylacyl
• R 2 is hydrogen or (Ci-C 3 )-alkyl
• R 3 is hydrogen or arylsulfanyl
• R 4 is hydrogen or aryloxy
• R 5 is selected from hydrogen, halo, aryloxy, or aryloxy substituted with (d-C 3 )-alkoxy.
• a compound for use in the present invention is a compound of Formula II wherein: R' is hydrogen or methyl;
• R 1 is selected from hydrogen, chloro, methyl, cyano, or benzoyl
• R 2 is selected from hydrogen or methyl
• R 3 is hydrogen or phenylsulfanyl
• R 4 is selected from hydrogen or phenoxy
• R 5 is selected from hydrogen, chloro, phenoxy, or 4-methoxy-phenoxy.
• a compound for use in the methods and medicaments of the present invention is a compound of Formula III:
• R 6 is selected from the group consisting of hydrogen, halo, cyano, (C r C 3 )-alkyl, and aryl
• R 7 is selected from the group consisting of hydrogen, halo, cyano, (Ci-C ⁇ )-alkyl, and aryl, wherein the aryl is optionally substituted by one or two halo;
• R 8 is selected from the group consisting of hydrogen, halo, cyano, (Ci-C 6 )-alkyl, trifluoromethyl, and aryl optionally substituted with halo;
• R 9 is selected from the group consisting of hydrogen, (Ci-Cio)-alkyl, (C ] -C 3 )-alkyl-(C 1 -Cio)-alkyl, (C 1 -C 3 )-alkoxy-(C 1 -C 6 )-alkyl, (C 4 -C 6 )-cycloalkyl, (C 4 -C 6 )-cycloalkyl-(C,-C 3 )-alkyl, aryl, (C 7 -C 12 )-aralkyl, aryl-aralkyl, and heteroaralkyl; where in each case an aryl or heteroaryl may be optionally substituted by one or two halo, trifluoromethyl, or (Ci-C 4 )-alkoxy; or pharmaceutically acceptable salts, single stereoisomers, mixtures of stereoisomers, esters, or prodrugs thereof.
• a compound for use in the present invention is a compound of Formula III wherein:
• A C(R 7 )-
• B is -N(R 9 )-
• R 6 is selected from hydrogen or cyano
• R 7 is selected from hydrogen or halo
• R 8 is selected from hydrogen or aryl optionally substituted with halo
• R 9 is selected from (C,-C 3 )-alkoxy-(Ci-C 3 )-alkyl.
• a compound for use in the present invention is a compound of Formula III wherein:
• A C(R 7 )-
• B is -N(R 9 )-
• R 6 is selected from hydrogen or cyano
• R 7 is selected from hydrogen or bromo
• R 8 is selected from hydrogen or 4-chloro-phenyl
• R 9 is selected from methoxymethyl or phenyl.
• a compound suitable for use in the claimed methods and medicaments is a compound of Formula III wherein:
• A is -N(R 9 )-;
• B C(R 7 )-
• R 6 is IC 11 C 3 ]LaIlCyI
• R 7 is halogen
• R 8 is halogen; and R 9 is aralkoxy.
• a compound for use in the methods and medicaments of the present invention is a compound of Formula IH wherein:
• R 6 is methyl
• R 7 is chloro
• R 8 is chloro
• R 9 is benzyl
• a compound for use in the methods and medicaments of the invention is a compound represented by Formula IV,
• R 15 is selected from the group consisting of hydrogen, cyano, acetyl, (C r C 6 )-alkyl, (CV C 4 )-alkynyl, (C 7 -C 12 )-aralkyl, (C 7 -Ci 2 )-aralkenyl, (C 1 -C 6 )-alkyl-sulfanyl-(C 1 -C 3 )- alkyl, aryl, heterocyclyl, and heteroaryl; wherein each substituent is optionally substituted with halo or cyano; and
• R 16 is selected from the group consisting of (Ci-C 6 )-alkyl, (C 6 -C )2 )-aryl optionally substituted with one or two substituents each independently selected from cyano, (C 1 -C 4 )-alkyl, trifluoromethyl, halo, (C]-C 4 )-alkoxy, aryl, or aryloxy; aryloxy, heteocyclyl, and heteroaryl optionally substituted with halo, (C]-C 4 )-alkoxy, aryloxy, or arylsulfanyl; or pharmaceutically acceptable salts, single stereoisomers, mixtures of stereoisomers, esters, or prodrugs thereof.
• a compound for use in the methods and medicaments of the present invention is a compound of Formula IV wherein:
• R 15 is (Ci-C 3 )-alkyl or (C 7 -C 12 )-aralkyl
• R 16 is selected from (C r C 6 )-alkyl or (C 6 -C, 2 )-aryl.
• a compound for use in the methods and medicaments of the present invention is a compound of Formula IV, wherein:
• R 15 is selected from methyl or phenethyl
• R 16 is selected from /-butyl or phenyl.
• R 17 is is selected from the group consisting of hydrogen, (C r C 10 )-alkyl, (C 2 -Ci 0 )-alkenyl, (C 2 -Cio)-alkynyl, wherein alkenyl or alkynyl contains one or two C-C multiple bonds; unsubstituted fluoroalkyl radical of the formula -[CH 2 ] x -C f H (2f+1 _ g) -F g , aryl, heteroaryl, and (C 7 -Ci i)-aralkyl;
• R 18 , R 19 , R 20 , and R 21 are identical or different and are selected from the group consisting of hydrogen, hydroxyl, halogen, cyano, trifluoromethyl, nitro, carboxyl; (C 1 -C 20 )- alkyl, (C 3 -C 8 )-cycloalkyl, (C 3 -C 8 )-cycloalkoxy, (C 6 -C 12 )-aryl, (C 7 -C 16 )-aralkyl, (C 7 -C 16 )-aralkenyl, (C 7 -C 16 )-aralkynyl, (C 2 -C 20 )-alkenyl, (C 2 -C 20 )-alkynyl, (C 1 - C 20 )-alkoxy, (C 2 -C 20 )-alkenyloxy, (C 2 -C 20 )-alkynyloxy, retinyloxy, (C 6 -C 12
• a compound for use in the present invention is a compound of Formula V wherein:
• R 17 is selected from hydrogen or aralkoxy
• R 18 and R 21 are hydrogen
• R 19 is selected from hydrogen or aryloxy
• R 20 is selected from hydrogen or halo.
• a compound for use in the present invention is a compound of Formula V wherein: R 17 is selected from hydrogen or benzyloxy; R 18 and R 21 are hydrogen; R 19 is selected from hydrogen or phenoxy; and R 20 is selected from hydrogen or chloro.
• R 21 is selected from hydrogen or aryl
• R 22 is selected from hydrogen or aryl
• R 23 is selected from (C r Ci 0 )-alkyl or aralkyl.
• a compound for use in the present invention is a compound of Formula VI wherein:
• R 21 is selected from hydrogen or phenyl
• R 22 is selected from hydrogen or phenyl
• R 23 is selected from hexyl or benzyl.
• a compound for use in the present methods and medicaments is a compound of Formula VII:
• R 24 , R 25 , and R 26 are each independently selected from hydrogen or halogen
• R 27 is (C 7 -Ci 2 )-aralkyl optionally substituted on the aryl with one or two substituents selected from the group consisting of halo, trifluoromethyl, and (Ci-C 3 )-alkoxy.
• a compound for use in the present invention is a compound of Formula VII wherein:
• R 24 is selected from hydrogen or halo
• R 25 is selected from hydrogen or halo
• R 26 is selected from hydrogen or halo
• R 27 is selected from (C 1 -Ci 0 )-alkyl or (C 7 -Ci 2 )-aralkyl, wherein the aryl is optionally substituted with trifluoromethyl.
• a compound for use in the present invention is a compound of Formula VII wherein:
• R 24 is selected from hydrogen or chloro
• R 25 is selected from hydrogen or chloro
• R 26 is selected from hydrogen or chloro
• R 27 is selected from hexyl, benzyl, or 2-trifluoromethyl-benzyl.
• compositions or medicaments effective for use in any of the present methods are provided herein.
• the compositions comprise an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase and an acceptable carrier.
• Figures IA and IB set forth data showing a compound of the present invention decreased EFN- ⁇ and TNF- ⁇ secretion from T cells.
• Figures 2A and 2B set forth data showing a compound of the present invention did not reduce T cell number or viability.
• Figures 3 A, 3B, and 3C set forth data showing a compound of the present invention decreased IL-12-mediated expression of DL12Rb2, DL18R1, and IL18RAP in T cells.
• Figures 4A and 4B set forth data showing a compound of the present invention decreased IL-12-mediated IFN- ⁇ secretion, but not IL-4-mediated EL-5 secretion, from T cells.
• Figures 5 A and 5B set forth data showing a compound of the present invention inhibited ThI cell differentiation and IL-12-mediated IFN- ⁇ secretion from T cells.
• Figures 6A and 6B set forth data showing a compound of the present invention inhibited IL-12-mediated IFN- ⁇ and TNF- ⁇ secretion ex vivo from T cells activated in vivo.
• a HIF prolyl hydroxylase enzyme may include a plurality of such enzymes; a reference to a “compound that inhibits the activity of a hypoxia-inducible factor prolyl hydroxylase enzyme” may be a reference to one or more compounds that inhibits the activity of a hypoxia-inducible factor prolyl hydroxylase enzyme; and so forth.
• the present invention relates to the discovery that inhibiting hypoxia-inducible factor (HIF) hydroxylase is effective at inhibiting the differentiation of T helper cells into ThI cells.
• the present invention provides a method for inhibiting the differentiation of T helper cells into ThI cells, the method comprising contacting a T helper cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby inhibiting the differentiation of the T helper cell into a ThI cell.
• the T helper cell in these methods is a naive T helper cell.
• the present invention provides a method for inhibiting the differentiation of a T helper cell into a ThI cell in a subject, the method comprising administering to the subject an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby inhibiting the differentiation of the T helper cell into a ThI cell in the subject.
• the invention also provides compounds for use in manufacturing a medicament for inhibiting the differentiation of a T helper cell into a ThI cell in a subject, wherein the compound inhibits the activity of a HIF prolyl hydroxylase enzyme.
• the T helper cell in these methods is a na ⁇ ve T helper cell.
• ThI cells and Th2 cells are subsets of CD4+ T cells derived from a common CD4+ na ⁇ ve T helper cell (i.e., a ThO cell).
• a common CD4+ na ⁇ ve T helper cell i.e., a ThO cell.
• IL- 12 and IL-4 two key cytokines, which induce the differentiation of na ⁇ ve T helper cells into ThI cells and Th2 cells, respectively.
• ThI cells secrete various cytokines associated with inflammation, such as, for example, IFN- ⁇ , TNF- ⁇ , TNF- ⁇ , and IL-2.
• IFN- ⁇ is an important component to the inflammatory response, contributing to phagocytic cell activation, up-regulation of MHC expression of antigen presenting cells, and generally associated with inflammatory and immune responses.
• Lymphokine Research 8:329-333. See, e.g., Heremann et al. (1989) Lymphokine Research 8:329-333.
• the present invention shows that compounds effective at inhibiting HIF prolyl hydroxylase enzyme activity inhibit IL-12-induced T helper cell production of IFN- ⁇ . Therefore, in one embodiment, the present invention provides a method for inhibiting IL-12-mediated induction of DFN- ⁇ synthesis in a subject, the method comprising administering to the subject an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby inhibiting IL-12-mediated induction of IFN- ⁇ synthesis in the subject.
• the present invention provides a method for inhibiting IL-12-mediated induction of IFN- ⁇ synthesis in a T helper cell, the method comprising contacting a T helper cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby inhibiting IL-12-mediated induction of IFN- ⁇ synthesis in the T helper cell.
• Methods for inhibiting the induction of IFN- ⁇ synthesis in a T helper cell the method comprising contacting the T helper cell, in an amount sufficient to inhibit IL-12-induced production of IFN- ⁇ , with a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, are specifically contemplated.
• the T helper cell is a ThI cell.
• the T helper cell is a na ⁇ ve T helper cell.
• the present invention also shows that compounds effective at inhibiting HIF prolyl hydroxylase enzyme activity inhibit IL-12-induced T helper cell production of TNF- ⁇ . Therefore, in one embodiment, the present invention provides a method for inhibiting IL-12-mediated induction of TNF- ⁇ synthesis in a subject, the method comprising administering to the subject an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby inhibiting IL-12-mediated induction of TNF- ⁇ synthesis in the subject.
• the present invention provides a method for inhibiting IL-12-mediated induction of TNF- ⁇ synthesis in a T helper cell, the method comprising contacting a T helper cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby inhibiting IL-12-mediated induction of TNF- ⁇ synthesis in the T helper cell.
• Methods for inhibiting the induction of TNF- ⁇ synthesis in a T helper cell the method comprising contacting the T helper cell, in an amount sufficient to inhibit IL-12-induced production of TNF- ⁇ , with a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, are specifically contemplated.
• the T helper cell is a ThI cell.
• the T helper cell is a naive T helper cell.
• the present invention also provides methods and compounds useful for inhibiting IL- 12 signaling in T helper cells.
• the present invention provides a method for inhibiting IL- 12 signaling in a T helper cell, the method comprising contacting a T helper cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby inhibiting IL- 12 signaling in the T helper cell.
• the T helper cell is a ThI cell.
• the T helper cell is a naive T helper cell.
• the present methods and compounds are useful for inhibiting IL-12-mediated gene expression in these cells.
• the present invention provides a method for inhibiting IL-12-mediated gene expression in a T helper cell, the method comprising contacting the T helper cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby inhibiting IL-12-mediated gene expression in the T helper cell.
• the present invention shows that compounds effective at inhibiting HIF prolyl hydroxylase enzyme activity inhibit the expression of three EL-12-induced genes: interleukin-18 receptor beta 2 (IL-18R ⁇ 2); interleukin-18 receptor 1 (IL-18R1), also known as interleukin-18 receptor alpha chain (IL-18RA, IL-18Ra, IL-18R ⁇ ) and interleukin-1 receptor-related protein (IL-IRRP); and interleukin-18 receptor- associated protein (IL-18RAP), also known as interleukin-18 receptor beta chain (IL-18RB, IL-18R ⁇ ) and accessory protein-like (ACPL).
• IL-18R ⁇ 2 interleukin-18 receptor beta 2
• IL-18R1 interleukin-18 receptor 1
• IL-18RAP interleukin-18 receptor-associated protein
• ACPL accessory protein-like
• the methods and compounds of the present invention are useful for inhibiting T helper cell expression of IL12R ⁇ 2, IL18R1, and IL18RAP, the method comprising contacting a T helper cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby inhibiting IL-12-mediated gene expression of IL12R ⁇ 2, EL18R1, and EL18RAP in the T helper cell.
• the T helper cell is a ThI cell.
• the T helper cell is a naive T helper cell.
• the present methods and compounds are useful for and effective at inhibiting T helper cell expression of IL12R ⁇ 2, IL18R1, and IL18RAP in a subject, the method comprising administering to the subject an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby inhibiting T helper cell expression of IL12R ⁇ 2, IL18R1, and IL18RAP in the subject.
• the inhibition of T helper cell expression of IL12R ⁇ 2, IL18R1, and IL18RAP is inhibition of IL-12-induced gene expression of IL12R ⁇ 2, IL18R1, and IL18RAP expression in T helper cells in the subject.
• the T helper cell is a ThI cell.
• the T helper cell is a naive T helper cell.
• the present invention relates to methods and compounds useful for inhibiting T helper cell differentiation in a subject by administering to the subject an effective amount of a compound that inhibits the activity of a hypoxia-inducible factor (HIF) prolyl hydroxylase.
• HIF hypoxia-inducible factor
• the invention is applicable to a variety of different organisms, including, for example, vertebrates, large animals, and primates.
• the subject is a mammalian subject, and in a most preferred embodiment, the subject is a human subject.
• medical applications with humans are clearly foreseen, veterinary applications are also envisaged herein.
• the methods and compounds of the present invention are particularly suitable for a subject who would benefit from inhibition of T helper cell differentiation, in particular, from inhibition of T helper cell differentiation into ThI cells, such as, for example, a subject having or at risk for having a cell-mediated immune response.
• a subject suitable for treatment with the present methods and compounds is a subject having or is at risk for having a cell-mediated immune response.
• a suitable subject for the present methods is one in which the subject would benefit from a reduction in cell-mediated immune responses (i.e., Thl-mediated responses) without affecting humoral immune responses (i.e., Th2-mediated responses).
• a subject suitable for treatment with the present methods and compounds is a subject having or at risk for having a pathological ThI cell response, such as, for example, a subject having or at risk for having an organ-specific or systemic autoimmune condition, including a chronic inflammatory disease or a delayed type hypersensitivity reaction.
• HIF prolyl hydroxylase refers to any enzyme that is capable of hydroxylating a proline residue within an alpha subunit of HIF.
• HIF prolyl hydroxylases include protein members of the EGL-9 (EGLN) 2-oxoglutarate- and iron-dependent dioxygenase family described by Taylor (2001) Gene 275:125-132; and characterized by Aravind and Koonin (2001) Genome Biol 2:RESEARCH0007; Epstein et al.
• HIF prolyl hydroxylases include human SM-20 (EGLNl) (GenBank Accession No. AAG33965; Dupuy et al. (2000) Genomics 69:348-54), EGLN2 isoform 1 (GenBank Accession No. CAC42510), EGLN2 isoform 2 (GenBank Accession No. NP_060025), and EGLN3 (GenBank Accession No. CAC42511; Taylor, supra); mouse EGLNl (GenBank Accession No. CAC42515), EGLN2 (GenBank Accession No.
• HIF prolyl hydroxylase may include Caenorhabditis elegans EGL-9 (GenBank Accession No. AAD56365) and Drosophila melanogaster CGl 114 gene product (GenBank Accession No. AAF52050).
• the term "HIF prolyl hydroxylase” also includes any active fragment of the foregoing full-length proteins.
• a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme refers to any compound that reduces or otherwise modulates the activity of at least one HIF prolyl hydroxylase enzyme.
• a compound may additionally show inhibitory activity toward one or more other 2-oxoglutarate- and iron-dependent dioxygenase enzymes, e.g. factor inhibiting HIF (FIH; GenBank Accession No. AAL27308), procollagen prolyl 4-hydroxylase (cP4H), etc.
• compounds used in the present methods and medicaments provided herein are structural mimetics of 2-oxoglutarate, wherein the compound inhibits the target HIF prolyl hydroxylase enzyme competitively with respect to 2-oxoglutarate and noncompetitively with respect to iron.
• Examples of compounds that may be used in the methods and medicaments provided herein can be found, e.g., in Majamaa et al. (1984) Eur. J. Biochem. 138:239-245; Majamaa et al. (1985) Biochem. J. 229:127-133; Kivirikko, and Myllyharju (1998) Matrix Biol. 16:357- 368; Bickel et al.
• WO 03/049686 WO 02/074981, WO 03/080566, WO 2004/108681, WO 2006/094292, WO 2007/038571, WO 2007/090068, WO 2007/070359, WO 2007/103905, and WO 2007/115315.
• heterocyclic carboxamides including pyridine carboxamides, quinoline carboxamides, isoquinoline carboxamides, a quinolone carboxamide, cinnoline carboxamides, or beta- carboline carboxamides
• phenanthrolines hydroxamates
• 3-hydroxy-pyridine- 2-carbonyl -glycines 4-hydroxy-pyridazine-3 -carbonyl-glycines, 3 -hydroxy-quinoline-2-carbonyl- glycines, 4-hydroxy-2-oxo-l ,2-dihydro-quinoline-3-carbonyl-glycines, 4-hydroxy-2-oxo-l ,2-dihydro- naphthyridine-3-carbonyl-glycines, 8-hydroxy-6-oxo-4,6-dihydro-pyridopyrazine-7
• the compound is [(4-Hydroxy-l-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid (Compound A), [(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid (Compound B), [(l-Benzoyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]-acetic acid (Compound C), [(8-Chloro-4- hydroxy-5-methyl-isoquinoline-3-carbonyl)-amino]-acetic acid (Compound D), ⁇ [4-Hydroxy-8-(4- methoxy-phenoxy)-l-methyl-isoquinoline-3-carbonyl]-amino ⁇ -acetic acid (Compound E), (S)-2-[(l- Cyano-4-hydroxy-8-phenoxy-isoquinoline-3-carbonyl)-amino]-propionic acid (
• Compounds for use in the present invention are compounds that inhibit HIF prolyl hydroxylase activity.
• a compound that inhibits HIF prolyl hydroxylase activity is any compound that reduces or otherwise inhibits the activity of at least one HIF prolyl hydroxylase enzyme.
• Various compounds that inhibit HIF prolyl hydroxylase have been identified and are suitable for use in the methods and medicaments as claimed in the present invention.
• Exemplary pyridine-2-carboxamides, pyridazine-3-carboxamides, quinoline-2-carboxamides, isoquinoline-3-carboxamides and esters thereof are described in European Patent Nos. EP0650960 and EP0650961. All compounds listed in EP0650960 and EP0650961, in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated into the present application by reference herein. Additional pyridine-2-carboxamides are described in U.S. Patent Application Publication No. 2007/0299086. All compounds listed in U.S. Patent Application Publication No.
• 2007/0299086 in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated into the present application by reference herein.
• exemplary pyridine-2-carboxamidoesters, pyridazine-3-carboxamidoesters, and isoquinoline-3- carboxamidoesters are described in U.S. Patent No. 5,658,933. All pyridine-2-carboxamidoesters, pyridazine-3-carboxamidoesters, and quinoline-2-carboxamidesters are listed in U.S. Patent No. 5,658,933, in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated into the present application by reference herein.
• Exemplary isoquinoline-3-carboxamides are described in U.S. Patent No. 6,093,730 and 7,323,475. All compounds listed in U.S. Patent No. 6,093,730 and 7,323,475, in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated into the present application by reference herein. Particularly exemplary embodiments of isoquinoline-3- carboxamides are described in U.S. Patent Application Publication No. 2007/0298104. All compounds listed in U.S. Patent Application Publication No. 2007/0298104, in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated into the present application by reference herein.
• Exemplary beta-carboline-3-carboxamides, pyrrolo[3,2-c]pyridine-6-carboxamides, pyrrolo[2,3- c]pyridine-5-carboxamides, thiazolo[4,5-c]pyridine-6-carboxamides, and thiazolo[5,4-c]pyridine-6- carboxamides are described in U.S. Patent Application Publication No. 2008/0004309. All compounds listed in U.S. Patent Application Publication No. 2008/0004309, in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated into the present application by reference herein.
• Exemplary 2,4-dioxo-l,2,3,4-tetrahydro-pyrirnidine-5-carboxamides and 4-oxo-2-thioxo-l,2,3,4- tetrahydro-pyrimidine-5-carboxamides are described in International Publication No. WO 2007/150011. All compounds listed in the foregoing publication, in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated into the present application by reference herein. Exemplary 6-oxo-l,6-dihydro-pyrimidine-5-carboxamides are described in U.S. Patent Application Publication No. 2008/0171756. All compounds listed in U.S. Patent Application Publication No. 2008/0171756, in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated into the present application by reference herein.
• Exemplary 2-oxo-l ,2-dihydro-[ 1 ,8]naphthyridine-3-carboxamides, 2-oxo-l ,2-dihydro-[l ,6]naphthyridine- 3-carboxamides, and 6-oxo-5,6-dihydro-pyrido[2,3-b]pyrazine-7-carboxamides are described in International Publication Nos. WO 2007/103905, WO 2008/076425, and WO 2008/130527. All compounds listed in the foregoing publications, in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated into the present application by reference herein.
• Exemplary 6-oxo-6,7-dihydro-thieno[2,3-b]pyridine-5-carboxamides, 5-oxo-4,5-dihydro-thieno[3,2- b]pyridine-6-carboxamides, 6-oxo-6,7-dihydro-pyrazolo[3,4-b]pyridine-5-carboxamides are described in International Publication No. WO 2007/136990. All compounds listed in the foregoing publications, in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated into the present application by reference herein. Exemplary 3-oxo-2,3-dihydro-pyridazine-4-carboxamides are described in U.S. Patent Application Publication No. 2008/0214549. All compounds listed in U.S. Patent Application Publication No. 2008/0214549, in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated into the present application by reference herein.
• Exemplary 4-oxo-[l,10]-phenanthrolines are described in U.S. Patent Nos. 5,916,898 and 6,200,974, and International Publication No. WO 99/21860. All compounds listed in the foregoing patents and publication, in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated into the present application by reference herein.
• An exemplary 4-oxo- [l,10]-phenanthroline is 4-oxo-l,4-dihydro-[l,10]phenanthroline-3-carboxylic acid (see, e.g., Seki et al. (1974) Chem Abstracts 81:424, No. 21).
• Exemplary dihydropyrazoles and dihydropyrozolones are described in U.S. Patent No. 6,878,729 and International Publication No. WO 2008/049539, respectively. All compounds listed in U.S. Patent No. 6,878,729, in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated into the present application by reference herein. Exemplary dipyridyl dihyropyrazones are described in International Publication No. WO 2006/114213. All compounds listed in International Publication No. WO 2006/114213, in particular, those listed in the compound claims and the final products of the working examples, are hereby incorporated into the present application by reference herein.
• compounds suitable for use in the present invention are selected from the group consisting of 2-oxoglutarate mimetics, iron chelators, and proline analogs.
• the compound is a 2-oxoglutarate structural mimetic.
• 2-oxoglutarate structural mimetics suitable for use in the claimed methods include structural mimetics of 2-oxoglutarate that inhibit HIF prolyl hydroxylase activity competitively with respect to 2-oxoglutarate.
• the compound is a 2-oxoglutarate structural mimetic that inhibits HIF prolyl hydroxylase competitively with respect to 2-oxoglutarate and noncompetitively with respect to iron.
• a compound of the present invention is, in various embodiments, a cyclic carboxamide.
• the cyclic carboxamide is a carbonyl glycine.
• the carboxamide is replaced by a carbonyl proprionic acid.
• the compound of the present invention is a carbocyclic carboxamide.
• Preferred cyclic carboxamides suitable for use in the present invention are heterocyclic carboxamides.
• Such heterocyclic carboxamide compounds include heterocyclic carboxamides previously identified as inhibitors of HIF prolyl hydroxylase activity, and known and available to those of skill in the art.
• a compound of the present invention is a heterocyclic carboxamide having a heterocyclic group selected from the group consisting of: azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, furan, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimi
• the heterocyclic group is a single ring selected from the group consisting of a pyridine, a pyridinone, a pyradizine, a pyridazinone, a pyrimidine, and a pyrimidinone ring.
• the heterocyclic group is a multiple condensed ring selected from the group consisting of an isoquinoline, an isoquinolone, a naphthyridinone, a pyrrolopyridine, a pyrrolopyridinone, a pyrozolopyridinone, a pyrrolopyridizinone, a quinoline, a quinolone, a chromenone, a thiochromenone, a thienopyridine, a thienopyridinone, a thiazolopyridine, and a thiazolopyridinone.
• a particularly preferred compound of the present invention is selected from the group consisting of an isoquinoline, a pyrrolopyridine, a thiazolopyridine, a quinoline, a pyridinone, and a pyrrolopyridazinone carboxamide.
• heterocyclic carboxamides suitable for use in the claimed methods are heterocyclic carbonyl glycines.
• Preferred such heterocyclic carbonyl glycines include those represented by Formula I, infra.
• the heterocyclic carbonyl glycine suitable for use in the present invention is a heterocyclic carbonyl glycine having a heterocyclic group that is selected from the following list: azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, furan, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, pipe
• the heterocyclic carbonyl glycine suitable for use in the present invention is a heterocyclic carbonyl glycine having a heterocyclic group, wherein the heterocyclic group is a single ring selected from the following list: a pyridine, a pyridinone, a pyradizine, a pyridazinone, a pyrimidine, and a pyrimidinone ring.
• the heterocyclic carbonyl glycine suitable for use in the present invention is a heterocyclic carbonyl glycine having a heterocyclic group, wherein the heterocyclic group is a multiple condensed ring selected from the group consisting of an isoquinoline, an isoquinolone, a naphthyridinone, a pyrrolopyridine, a pyrrolopyridinone, a pyrozolopyridinone, a pyrrolopyridizinone, a quinoline, a quinolone, a chromenone, a thiochromenone, a thienopyridine, a thienopyridinone, a thiazolopyridine, and a thiazolopyridinone.
• the heterocyclic group is a multiple condensed ring selected from the group consisting of an isoquinoline, an isoquinolone, a naphthyridinone, a pyrrolopyridine,
• heterocyclic carbonyl glycines suitable for use in the claimed methods are heterocyclic carbonyl glycines having a heterocyclic moiety selected from the group consisting of an isoquinoline, a pyrrolopyridine, a thiazolopyridine, a quinoline, a pyridinone, and a pyrrolopyridazinone moiety.
• Isoquinoline carbonyl glycines suitable for use in the present invention include isoquinoline-3-carbonyl- glycines; more preferably, 4-hydroxy-isoquinoline-3-carbonyl-glycines.
• Exemplary 4-hydroxy- isoquinoline-3-carbonyl-glycines include, but are not limited to: [(l-Chloro-4-hydroxy-6-phenylsulfanyl- isoquinoline-3-carbonyl)-amino]-acetic acid (Compound T); [(4-Hydroxy-7-phenoxy-isoquinoline-3- carbonyl)-amino] -acetic acid (Compound B); [(4-Hydroxy-l -methyl -7 -phenoxy-isoquinoline-3- carbonyl)-amino] -acetic acid (Compound A); [(l-Benzoyl-4-hydroxy-isoquinoline-3-carbonyl)-amino]- acetic acid (Compound C); (S)-2-[(l-Cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]- propionic acid (Compound G); ⁇ [4-Hydroxy-8-(4
• Preferred pyrrolopyridine carbonyl glycines include pyrrolo[2,3-c]pyridine-5-carbonyl-glycines; in particular, 4-hydroxy-pyrrolo[2,3-c]pyridine-5-carbonyl-glycines.
• Preferred pyrrolopyridine carbonyl glycines include pyrrolo[3,2-c]pyridine-6-carbonyl-glycines; in particular 7-hydroxy- pyrrolo[3,2-c]pyridine-6-carbonyl-glycines.
• Exemplary pyrrolopyridine carbonyl glycines include, but are not limited to: ⁇ 2-[3-Bromo-2-(4-chloro-phenyl)-4-hydroxy-l-phenyl-lH-pyrrolo[2,3-c]pyridine-5- carbonyl)-amino] -acetic acid (Compound H); [(7-Cyano-4-hydroxy-l-methoxymethyl-lH-pyrrolo[2,3- c]pyridine-5-carbonyl)-amino]-acetic acid (Compound I); and [(I -Benzyl -2,3-dichloro-7-hydroxy-4- methyl-lH-pyrrolo[3,2-c]pyridine-6-carbonyl)-amino]-acetic acid (Compound U), and other compounds of Formula III, infra.
• thiazolopyridine carbonyl glycines are thiazolopyridine carbonyl glycines.
• Exemplary thiazolopyridine carbonyl glycines include thiazolo[4,5-c]pyridine-6-carboxamides.
• Particularly preferred thiazolo[4,5-c]pyridine-6-carboxamides include, but are not limited to, 7-hydroxy-thiazolo[4,5- c]pyridine-6-carboxamides.
• Representative such compounds include (S)-2-[(7-Hydroxy-4-methyl-2- phenyl-thiazolo[4,5-c]pyridine-6-carbonyl)-amino]-propionic acid (Compound S) and [(2-tert-Butyl-7- hydroxy-4-phenethyl-thiazolo[4,5-c]pyridine-6-carbonyl)-amino]-acetic acid (Compound R), and other such compounds encompassed by Formula IV, infra.
• quinoline carbonyl glycines suitable for use in the present methods and medicaments include quinoline-2-carbonyl-glycones, such as [(3-Benzyloxy-7-chloro-quinoline-2-carbonyl)-amino]-acetic acid (Compound P); [(7-Chloro-3-hydroxy-quinoline-2-carbonyl)-amino]-acetic acid (Compound O); and [(3- Hydroxy-6-phenoxy-quinoline-2-carbonyl)-amino]-acetic acid (Compound Q); and other compounds of Formula V, infra.
• quinoline-2-carbonyl-glycones such as [(3-Benzyloxy-7-chloro-quinoline-2-carbonyl)-amino]-acetic acid (Compound P); [(7-Chloro-3-hydroxy-quinoline-2-carbonyl)-amino]-acetic acid (Compound O); and [(3- Hydroxy-6-phenoxy-quino
• pyridinone carbonyl glycines in particular, 2-oxo-l,2-dihydro-pyridine-3-carbonyl- glycines. More preferred are 4-hydroxy-2-oxo-l,2-dihydro-pyridine-3-carbonyl-glycines.
• Exemplary such compounds are represented by Formula VI, infra, and include, but are not limited to, [(I -Benzyl -4- hydroxy-2-oxo-6-phenyl-l,2-dihydro-pyridine-3-carbonyl)-amino]-acetic acid (Compound J); [(1-Hexyl- 4-hydroxy-2-oxo-6-phenyl-l,2-dihydro-pyridine-3-carbonyl)-amino]-acetic acid (Compound K); and (1- BenzyM-hydroxy ⁇ -oxo-S-phenyl-l ⁇ -dihydro-pyridine-S-carbony ⁇ -aminoJ-acetic acid (Compound L); and other compounds of formula IV, infra.
• Formula VI infra
• pyrrolopyridazinone carbonyl glycines preferably, 2-oxo-pyrrolo[l,2-b]pyridazine-3-carboxamides; more preferably, 4-hydroxy-2-oxo- pyrrolo[l,2-b]pyridazine-3-carboxamides.
• Exemplary such compounds include, but are not limited to: [( 1 -Benzyl-5 ,6,7-trichloro-4-hydroxy-2-oxo- 1 ,2-dihydro-pyrrolo[ 1 ,2-b]pyridazine-3 -carbonyl)-amino]- acetic acid (Compound V); ⁇ [4-Hydroxy-2-oxo-l-(2-trifluoromethyl-benzyl)-l,2-dihydro-pyrrolo[l,2- b]pyridazme-3-carbonyl]-amino ⁇ -acetic acid (Compound M); and [(l-Hexyl-4-hydroxy-2-oxo-l,2- dihydro-pyrrolo[l,2-b]pyridazine-3-carbonyl)-amino]-acetic acid (Compound N); and other compounds of Formula VII, infra.
• a compound of the present invention is a HIF prolyl hydroxylase inhibitor compound of Formula I:
• X is an optionally substituted cyclic moiety; and R' is hydrogen or (Q-C ⁇ -alkyl.
• the cyclic moiety is a hetercyclic moiety and R' is hydrogen.
• HIF prolyl hydroyxlase inhibitors include, but are not limited to, variously substituted pyridine-2-carbonyl-glycines, pyridazine-3 -carbonyl-glycines, quinoline-2-carbonyl-glycines, 2-oxo- 1 ,2-dihydro-quinoline-3 -carbonyl- glycines, 2-oxo-l ,2 -dihydro-naphthyridine-3 -carbonyl-glycines, 6-oxo-4,6-dihydro-pyridopyrazine-7- carbonyl-glycines, isoquinoline-S-carbonyl-glycines, cinnoline-S-carbonyl-glycines, thienopyridine-6- carbonyl-glycines, thienopyridine-5 -
• R' is selected from hydrogen and (C r C 4 )-alkyl
• R 1 , R 2 , R 3 , R 4 and R 5 are identical or different and are selected from the group consisting of hydrogen, hydroxyl, halogen, cyano, trifluoromethyl, nitro, carboxyl; (Ci-C 20 )- alkyl, (C 3 -C 8 )-cycloalkyl, (C 3 -C 8 )-cycloalkoxy, (C 6 -Ci 2 )-aryl, (C 7 -C 16 )-aralkyl, (C 7 -C, 6 )-aralkenyl, (C 7 -C 16 )-aralkynyl, (C 2 -C 20 )-alkenyl, (C 2 -C 20 )-alkynyl, (C 1 - C 20 )-alkoxy, (C 2 -C 20 )-alkenyloxy, (C 2 -C 20 )-alkynyloxy, retinyloxy, (C 6
• the compound of the present invention is a compound of Formula II wherein: R' is hydrogen or (Ci-C 3 )-alkyl;
• R 1 is selected from hydrogen, halo, (C r C 3 )-alkyl, cyano, or arylacyl
• R 2 is hydrogen or (C 1 -C 3 )-alkyl
• R 3 is hydrogen or arylsulfanyl
• R 4 is hydrogen or aryloxy
• R 5 is selected from hydrogen, halo, aryloxy, or aryloxy substituted with (C]-C 3 )-alkoxy.
• a compound of the present invention is a compound of Formula II wherein: R' is hydrogen or methyl;
• R 1 is selected from hydrogen, chloro, methyl, cyano, or benzoyl
• R 2 is selected from hydrogen or methyl
• R 3 is hydrogen or phenylsulfanyl
• R 4 is selected from hydrogen or phenoxy
• R 5 is selected from hydrogen, chloro, phenoxy, or 4-methoxy-phenoxy.
• a compound of the present invention is a compound of Formula III:
• R 6 is selected from the group consisting of hydrogen, halo, cyano, (Ci-C 3 )-alkyl, and aryl;
• R 7 is selected from the group consisting of hydrogen, halo, cyano, (Ci-C 6 )-alkyl, and aryl, wherein the aryl is optionally substituted by one or two halo;
• R 8 is selected from the group consisting of hydrogen, halo, cyano, trifiuoromethyl, and aryl optionally substituted with halo;
• R 9 is selected from the group consisting of hydrogen, (Q-CnO-alkyl, (C 1 -C 3 )-alkyl-(C ) -Cio)-alkyl, (Ci-C 3 )-alkoxy-(C,-C 6 )-alkyl, (C 4 -C 6 )-cycloalkyl, (C 4 -C 6 )-cycloalkyl-(Ci-C 3 )-alkyl, aryl, (C 7 -Ci 2 )-aralkyl, aryl-aralkyl, and heteroaralkyl; where in each case an aryl or heteroaryl may be optionally substituted by one or two halo, trifiuoromethyl, or (Ci-C 4 )-alkoxy; or pharmaceutically acceptable salts, single stereoisomers, mixtures of stereoisomers, esters, or prodrugs thereof.
• a compound of the present invention is a compound of Formula III wherein:
• A C(R 7 )-
• B is -N(R 9 )-
• R 6 is selected from hydrogen or cyano
• R 7 is selected from hydrogen or halo
• R 8 is selected from hydrogen or aryl optionally substituted with halo
• R 9 is selected from (Ci-C 3 )-alkoxy-(Ci-C 3 )-alkyl.
• R 6 is selected from hydrogen or cyano; R 7 is selected from hydrogen or bromo; R 8 is selected from hydrogen or 4-chloro-phenyl; and R 9 is selected from methoxymethyl or phenyl.
• a compound suitable for use in the claimed methods and medicaments is a compound of Formula III wherein:
• A is -N(R 9 )-;
• B C(R 7 )-
• R 6 is ⁇ CizCjJbalkyl
• R 7 is halogen
• R 8 is halogen
• R 9 is aralkoxy.
• a compound of the present invention is a compound of Formula III wherein: R 6 is methyl; R 7 is chloro; R 8 is chloro; and R 9 is benzyl.
• a compound of the invention is a compound represented by Formula IV,
• R 15 is selected from the group consisting of hydrogen, cyano, acetyl, (Ci-C 6 )-alkyl, (C 1 - C 4 )-alkynyl, (C 7 -Ci 2 )-aralkyl, (C 7 -Ci 2 )-aralkenyl, (C,-C 6 )-alkyl-sulfanyl-(C,-C 3 )- alkyl, aryl, heterocyclyl, and heteroaryl; wherein each substituent is optionally substituted with halo or cyano; and
• R 16 is selected from the group consisting of (d-C 6 )-alkyl, (C 6 -Ci 2 )-aryl optionally substituted with one or two substituents each independently selected from cyano, (Ci-C 4 )-alkyl, trifluoromethyl, halo, (Ci-C 4 )-alkoxy, aryl, or aryloxy; aryloxy, heteocyclyl, and heteroaryl optionally substituted with halo, (Ci-C 4 )-alkoxy, aryloxy, or arylsulfanyl; or pharmaceutically acceptable salts, single stereoisomers, mixtures of stereoisomers, esters, or prodrugs thereof.
• a compound of the present invention is a compound of Formula IV wherein: R 15 is (C r C 3 )-alkyl or (C 7 -C 12 )-aralkyl; and R 16 is selected from (C,-C 6 )-alkyl or (C 6 -C ]2 )-aryl.
• a compound of the present invention is a compound of Formula IV, wherein: R 15 is selected from methyl or phenethyl; and R 16 is selected from /-butyl or phenyl.
• R 17 is is selected from the group consisting of hydrogen, (Ci-Cio)-alkyl, (C 2 -C )0 )-alkenyl, (C 2 -C 10 )-alkynyl, wherein alkenyl or alkynyl contains one or two C-C multiple bonds; unsubstituted fluoroalkyl radical of the formula -[CH 2 ] x -C f H (2f+1 . g) -F g , aryl, heteroaryl, and (C 7 -Ci i)-aralkyl;
• R 18 , R 19 , R 20 , and R 21 are identical or different and are selected from the group consisting of hydrogen, hydroxyl, halogen, cyano, trifluoromethyl, nitro, carboxyl; (Ci-C 20 )- alkyl, (C 3 -C 8 )-cycloalkyl, (C 3 -C 8 )-cycloalkoxy, (C 6 -Ci 2 )-aryl, (C 7 -C 16 )-aralkyl, (C 7 -C 16 )-aralkenyl, (C 7 -C 16 )-aralkynyl, (C 2 -C 20 )-alkenyl, (C 2 -C 20 )-alkynyl, (C r C 20 )-alkoxy, (C 2 -C 20 )-alkenyloxy, (C 2 -C 20 )-alkynyloxy, retinyloxy, (C 6 -C 12
• a compound of the present invention is a compound of Formula V wherein: R 17 is selected from hydrogen or aralkoxy; R 18 and R 21 are hydrogen; R 19 is selected from hydrogen or aryloxy; and R 20 is selected from hydrogen or halo.
• a compound of the present invention is a compound of Formula V wherein: R 17 is selected from hydrogen or benzyloxy; R 18 and R 21 are hydrogen; R 19 is selected from hydrogen or phenoxy; and R 20 is selected from hydrogen or chloro.
• R 21 is selected from hydrogen or aryl
• R 22 is selected from hydrogen or aryl
• R 23 is selected from (C r Ci 0 )-alkyl or aralkyl.
• the compound of the present invention is a compound of Formula VI wherein: R 21 is selected from hydrogen or phenyl; R 22 is selected from hydrogen or phenyl; and R 23 is selected from hexyl or benzyl.
• a compound for use in the present methods and medicaments is a compound of Formula VII:
• R 24 , R 25 , and R 26 are each independently selected from hydrogen or halogen
• R 27 is (C 7 -C 12 )-aralkyl optionally substituted on the aryl with one or two substituents selected from the group consisting of halo, trifluoromethyl, and (C ! -C 3 )-alkoxy.
• the compound of the present invention is a compound of Formula VII wherein: R 24 is selected from hydrogen or halo; R 25 is selected from hydrogen or halo; R 26 is selected from hydrogen or halo; and
• R 27 is selected from (Ci-Cio)-alkyl or (C 7 -Ci 2 )-aralkyl, wherein the aryl is optionally substituted with trifluoromethyl.
• the compound of the present invention is a compound of Formula VII wherein: R 24 is selected from hydrogen or chloro; R 25 is selected from hydrogen or chloro; R 26 is selected from hydrogen or chloro; and R 27 is selected from hexyl, benzyl, or 2 -trifluoromethyl -benzyl.
• hydroxy or "hydroxyl” refer to the group -OH.
• halo or halogen refers to fluoro, chloro, bromo, and iodo.
• cyano refers to the group -CN.
• nitro refers to the group — NO ⁇ .
• carboxyl refers to -COOH or salts thereof.
• alkyl refers to saturated monovalent hydrocarbyl groups having from 1 to 10 carbon atoms; more particularly, from 1 to 5 carbon atoms; and, even more particularly, 1 to 3 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, wo-propyl, n-butyl, f-butyl, n-pentyl, and the like.
• cycloalkyl refers to a saturated or an unsaturated, but nonaromatic, cyclic alkyl groups of from 3 to 10, 3 to 8, or 3 to 6 carbon atoms having single or multiple cyclic rings including, by way of example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, cyclohexenyl, and the like.
• cycloalkoxy refers to an -O-cycloalkyl group.
• aryl refers to a monovalent aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl), which condensed rings may or may not be aromatic (e.g., 2-benzoxazolinone, 2H-l,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the point of attachment is the aryl group.
• Preferred aryls include phenyl and naphthyl.
• heterocyclic or “heterocyclyl” refer to a saturated or unsaturated ring system having a single ring or multiple condensed rings, from 1 to 10 carbon atoms, and from 1 to 4 hetero atoms selected from the group consisting of nitrogen, sulfur, or oxygen within the ring.
• heteroaryl refers to an aromatic heterocyclic group of from 1 to 15 carbon atoms, preferably from 1 to 10 carbon atoms, and 1 to 4 heteroatoms within the ring selected from the group consisting of oxygen, nitrogen, and sulfur.
• Such heteroaryl groups can have a single ring (e.g., pyridinyl, furyl, or thienyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl), which condensed rings may or may not be aromatic provided the point of attachment is through a ring containing the heteroatom and that ring is aromatic.
• the nitrogen can optionally be oxidized to provide for the N-oxide
• the sulfur ring atoms can optionally be oxidized to provide for the sulfoxide and sulfone derivatives.
• heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, furan, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, phthal
• groups are exemplified by vinyl (ethen-1-yl), allyl, but-3-enyl, and the like.
• alkynyl refers to acetylinic unsaturated monovalent hydrocarbyl groups having from 2 to 6, preferably from 2 to 3, carbon atoms and having at least 1, preferably from 1 to 2, sites of acetylenic (-C ⁇ C-) unsaturation. This group is exemplified by ethyn-1-yl, propyn-1-yl, propyn-2-yl, and the like.
• alkoxy refers to the group “alkyl-O-,” which includes, by way of example, methoxy, ethoxy, n-propoxy, /s ⁇ -propoxy, n-butoxy, ?-butoxy, sec-butoxy, n-pentoxy, and the like.
• alkenyloxy refers to the group “alkenyl-O-.”
• alkynyloxy refers to the group “alkynyl-O-.”
• aryloxy refers to the group aryl-O- that includes, by way of example, phenoxy, naphthoxy, and the like.
• aralkyloxy refers to the group aralkyl-O- that includes, by way of example, benzyloxy, and the like.
• aminoacyl or “amide”, or the prefixes “carbamoyl” or “carboxamide,” refer to the group -C(O)NR q R q where each R q is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, and heterocyclic; or where each R q is joined to form together with the nitrogen atom a heterocyclic wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.
• amino refers to the group -NH 2 .
• alkylsulfanyl refers to the groups -S-alkyl where alkyl is as defined above.
• sulfinyl refers to the group -S(O)-.
• sulfonyl refers to the group -S(O) 2 -.
• heterocyclyloxy refers to the group -O-heterocyclic.
• cycloalkylene refers to divalent cycloalkyl groups as defined above.
• cycloalkylthio or “cycloalkylsulfanyl” refer to the groups -S-cycloalkyl where cycloalkyl is as defined herein.
• arylthio or "arylsulfanyl” refer to the group -S-aryl, where aryl is as defined herein.
• heteroarylthio or “heteroarylsulfanyl” refer to the group -S-heteroaryl, where heteroaryl is as defined herein.
• heterocyclicthio or “heterocyclicsulfanyl” refer to the group -S-heterocyclic, where heterocyclic is as defined herein.
• alkyl alcohol refers to the group “alkyl-OH”. "Alkyl alcohol” is meant to include methanol, ethanol, 2-propanol, 2-butanol, butanol, etc.
• acyl refers to the groups H-C(O)-, alkyl-C(O)-, alkenyl-C(O)-, alkynyl-C(O)-, cycloalkyl- C(O)-, aryl -C(O)-, heteroaryl-C(O)-, and heterocyclic-C(O)-, provided that a nitrogen atom of the heterocyclic is not bound to the -C(O)- group, wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.
• acyloxy refers to the groups alkyl-C(O)O-, alkenyl-C(O)O-, alkynyl-C(O)O-, aryl-C(O)O-, cycloalkyl-C(O)O-, heteroaryl-C(0)0-, and heterocyclic-C(O)O-, wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.
• groups are exemplified by vinyl (ethen-1-yl), allyl, but-3-enyl and the like.
• alkynyl refers to acetylinic unsaturated monovalent hydrocarbyl groups having from 2 to 6, preferably from 2 to 3, carbon atoms and having at least 1, preferably from 1 to 2, sites of acetylenic (-C ⁇ C-) unsaturation. This group is exemplified by ethyn-1-yl, propyn-1-yl, propyn-2-yl, and the like.
• acylamino refers to the groups -NR 4 C(O)alkyl, -NR'C(O)cycloalkyl, -NR'C(O)alkenyl, -NR t C(O)alkynyl,-NR t C(O)aryl, -NR'C(O)heteroaryl, and -NR l C(O)heterocyclic where R 1 is hydrogen or alkyl, and wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are defined herein.
• carbonyloxyamino refers to the groups -NR u C(O)O-alkyl, -NR u C(O)O-alkenyl, -NR U C(O)O- alkynyl, -NR u C(O)O-cycloalkyl, -NR u C(O)O-aryl, -NR u C(O)O-heteroaryl, and -NR u C(O)O-heterocyclic, where R u is hydrogen or alkyl and wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.
• oxycarbonylamino refers to the groups -NR u C(O)O-alkyl, -NR u C(O)O-alkenyl, -NR U C(O)O- alkynyl, -NR u C(O)O-cycloalkyl, -NR u C(O)O-aryl, -NR u C(O)O-heteroaryl, and -NR u C(O)O-heterocyclic, where R u is hydrogen or alkyl, and wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.
• oxythiocarbonylamino refers to the groups -NR u C(S)O-alkyl,-NR u C(S)O-alkenyl, -NR u C(S)O-alkynyl, -NR u C(S)O-cycloalkyl, -NR u C(S)O-aryl, -NR u C(S)O-heteroaryl, and -NR U C(S)O- heterocyclic, where R u is hydrogen or alkyl, and wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.
• aminocarbonyloxy or the prefix “carbamoyloxy” refer to the groups -OC(O)NR V R V where each R v is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic; or where each R v is joined to form, together with the nitrogen atom, a heterocyclic, and wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, substituted heteroaryl, and heterocyclic are as defined herein.
• aminocarbonylamino refers to the group -NR W C(O)N(R W ) 2 where each R w is independently selected from the group consisting of hydrogen and alkyl.
• aminothiocarbonylamino refers to the group -NR W C(S)N(R W ) 2 where each R w is independently selected from the group consisting of hydrogen and alkyl.
• aryloxyaryl refers to the group -aryl-O-aryl.
• carboxyl ester refers to the groups -C(O)O-alkyl, -C(O)O-alkenyl, -C(O)O-alkynyl, -C(O)O- cycloalkyl, -C(O)O-aryl, -C(O)O-substituted aryl, -C(O)O-heteroaryl, -C(O)O-substituted heeteroaryl, -C(O)O-heterocyclic, and -C(O)O-substituted heterocyclic.
• cycloalkylene refers to divalent cycloalkyl groups as defined above.
• heteroaryl oxy refers to the group -O-heteroaryl.
• sulfonyl refers to the group -S(O) 2 -, and may be included in the groups -S(O) 2 H, -SO 2 -alkyl, -SO 2 -alkenyl, -SO 2 -alkynyl, -SO 2 -cycloalkyl, -SO 2 -cycloalkenyl, -SO 2 -aryl, -SO 2 -substituted aryl, -SO 2 -heteroaryl, and -SO 2 -heterocyclic, wherein alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocyclic are as defined herein.
• heterocyclyloxy refers to the group -O-heterocyclic.
• arylthio or "arylsulfanyl” refer to the group -S-aryl.
• heteroarylthio or “heteroarylsulfanyl” refer to the group -S-heteroaryl.
• heterocyclicthio or “heterocyclicsulfanyl” refer to the group -S-heterocyclic.
• Conjugated terms refer to a linear arrangement of the separate substituents as each separate term is defined herein.
• aralkyl refers to an aryl-alkyl group and includes, by way of example, benzyl;
• aralkylcarbamoyl refers to an aryl-alkyl-carbomoyl substituent wherein each term is as defined herein, etc.
• impermissible substitution patterns e.g., methyl substituted with 5 fluoro groups or a hydroxyl group alpha to ethenylic or acetylenic unsaturation.
• impermissible substitution patterns are well known to the skilled artisan.
• pharmaceutically acceptable salt refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art, and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and, when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and the like.
• stereoisomer or “stereoisomers” refer to compounds that differ in the chirality of one or more stereocenters.
• Stereoisomers include enantiomers (compounds are non-superimposable mirror images) and diastereomers (compounds having more than one stereogenic center that are non-mirror images of each other and wherein one or more stereogenic center differs between the two stereoisomers).
• the compounds of the invention can be present as a mixture of stereoisomers or as a single stereoisomer.
• prodrug refers to compounds that include chemical groups which, in vivo, can be converted into the carboxylate group and/or can be split off from the amide N-atom and/or can be split off from the R atom to provide for the active drug, a pharmaceutically acceptable salt thereof, or a biologically active metabolite thereof.
• Suitable groups are well known in the art and particularly include: for the carboxylic acid moiety, a prodrug selected from, e.g., esters including, but not limited to, those derived from alkyl alcohols, substituted alkyl alcohols, hydroxy substituted aryls and heteroaryls and the like; amides, particularly amides derived from amines of the Formula HNR 200 R 210 where R 200 and R 210 are independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and the like; hydroxymethyl, aldehyde and derivatives thereof.
• esters refers to compounds that include the group -COOR where R is alkyl, substituted alkyl, alkoxy, or substituted alkoxy.
• excipient means an inert or inactive substance used in the production of pharmaceutical products or other tablets, including without limitation any substance used as a binder, disintegrant, coating, compression/encapsulation aid, cream or lotion, lubricant, parenteral, sweetener or flavoring, suspending/gelling agent, or wet granulation agent.
• Binders include, e.g.
• coatings include, e.g., cellulose acetate phthalate, ethylcellulose, gellan gum, maltodextrin, etc.
• compression/encapsulation aids include, e.g., calcium carbonate, dextrose, fructose dc, honey dc, lactose (anhydrate or monohydrate; optionally in combination with aspartame, cellulose, or microcrystalline cellulose), starch dc, sucrose, etc.
• disintegrants include, e.g., croscarmellose sodium, gellan gum, sodium starch glycolate, etc.
• creams and lotions include, e.g., maltodextrin, carrageenans, etc.
• lubricants include, e.g., magnesium stearate, stearic acid, sodium stearyl fumarate, etc.
• materials for chewable tablets include, e.g.
• parenterals include, e.g., mannitol, povidone, etc.
• plasticizers include, e.g., dibutyl sebacate, polyvinylacetate phthalate, etc.
• suspending/gelling agents include, e.g., carrageenan, sodium starch glycolate, xanthan gum, etc.
• sweeteners include, e.g., aspartame, dextrose, fructose dc, sorbitol, sucrose dc, etc.
• wet granulation agents include, e.g., calcium carbonate, maltodextrin, microcrystalline cellulose, etc.
• Assays for hydroxylase activity are standard in the art. Such assays can directly or indirectly measure hydroxylase activity.
• an assay can measure hydroxylated residues, e.g., proline, asparagine, etc., present in the enzyme substrate, e.g., a target protein, a synthetic peptide mimetic, or a fragment thereof. (See, e.g., Palmerini et al. (1985) J Chromatogr 339:285-292.)
• a reduction in hydroxylated residue, e.g., proline or asparagine, in the presence of a compound is indicative of a compound that inhibits hydroxylase activity.
• assays can measure other products of the hydroxylation reaction, e.g., formation of succinate from 2-oxoglutarate.
• assays can measure other products of the hydroxylation reaction, e.g., formation of succinate from 2-oxoglutarate.
• Target protein may include HIF ⁇ or a fragment thereof, e.g., HIF(556-575).
• Enzyme may include, e.g., HIF prolyl hydroxylase or active fragments thereof (see, e.g., GenBank Accession No. AAG33965, etc.) or HIF asparaginyl hydroxylase or active fragments thereof(see, e.g., GenBank Accession No. AAL27308, etc.), obtained from any source.
• Enzyme may also be present in a crude cell lysate or in a partially purified form. For example, procedures that measure HIF hydroxylase activity are described in Ivan et al.
• compositions of the present invention can be delivered directly or in pharmaceutical compositions containing excipients, as is well known in the art.
• the present methods of treatment involve administration of an effective amount of a compound of the present invention to a subject in need, wherein the subject would benefit from inhibition of the differentiation of a T helper cell into a ThI cell.
• an effective amount, e.g., dose, of compound or drug can readily be determined by routine experimentation, as can an effective and convenient route of administration and an appropriate formulation.
• Various formulations and drug delivery systems are available in the art. (See, e.g., Gennaro, ed. (2000) Remington's Pharmaceutical Sciences, supra; and Hardman, Limbird, and Gilman, eds. (2001) The Pharmacological Basis of Therapeutics, supra.)
• Suitable routes of administration may, for example, include oral, rectal, topical, nasal, pulmonary, ocular, intestinal, and parenteral administration.
• Primary routes for parenteral administration include intravenous, intramuscular, and subcutaneous administration.
• Secondary routes of administration include intraperitoneal, intra-arterial, intra-articular, intracardiac, intracisternal, intradermal, intralesional, intraocular, intrapleural, intrathecal, intrauterine, and intraventricular administration.
• the indication to be treated, along with the physical, chemical, and biological properties of the drug dictate the type of formulation and the route of administration to be used, as well as whether local or systemic delivery would be preferred.
• the compounds of the present invention are administered orally.
• the invention provides for oral administration of [(4-Hydroxy-l-methyl-7- phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid (Compound A), [(4-Hydroxy-7-phenoxy- isoquinoline-3-carbonyl)-amino] -acetic acid (Compound B), [(I -Benzoyl -4-hydroxy-isoquinoline-3- carbonyl)-amino] -acetic acid (Compound C), [(8-Chloro-4-hydroxy-5-methyl-isoquinoline-3-carbonyl)- aminoj-acetic acid (Compound D), ⁇ [4-Hydroxy-8-(4-methoxy-phenoxy)-l-methyl-isoquinoline-3- carbonyl]-amino ⁇ -acetic acid (Compound E), (S)-2-[(l-Cyano-4-hydroxy-8-phenoxy-isoquinoline-3- carbonyl)
• Pharmaceutical dosage forms of a compound of the invention may be provided in an instant release, controlled release, sustained release, or target drug-delivery system.
• Commonly used dosage forms include, for example, solutions and suspensions, (micro-) emulsions, ointments, gels and patches, liposomes, tablets, dragees, soft or hard shell capsules, suppositories, ovules, implants, amorphous or crystalline powders, aerosols, and lyophilized formulations.
• special devices may be required for application or administration of the drug, such as, for example, syringes and needles, inhalers, pumps, injection pens, applicators, or special flasks.
• Pharmaceutical dosage forms are often composed of the drug, an excipient(s), and a container/closure system.
• One or multiple excipients also referred to as inactive ingredients, can be added to a compound of the invention to improve or facilitate manufacturing, stability, administration, and safety of the drug, and can provide a means to achieve a desired drug release profile. Therefore, the type of excipient(s) to be added to the drug can depend on various factors, such as, for example, the physical and chemical properties of the drug, the route of administration, and the manufacturing procedure.
• Pharmaceutically acceptable excipients are available in the art, and include those listed in various pharmacopoeias.
• compositions of the present invention can include one or more physiologically acceptable inactive ingredients that facilitate processing of active molecules into preparations for pharmaceutical use.
• the composition may be formulated in aqueous solution, if necessary using physiologically compatible buffers, including, for example, phosphate, histidine, or citrate for adjustment of the formulation pH, and a tonicity agent, such as, for example, sodium chloride or dextrose.
• physiologically compatible buffers including, for example, phosphate, histidine, or citrate for adjustment of the formulation pH
• a tonicity agent such as, for example, sodium chloride or dextrose.
• semisolid, liquid formulations, or patches may be preferred, possibly containing penetration enhancers.
• penetration enhancers are generally known in the art.
• the compounds can be formulated in liquid or solid dosage forms and as instant or controlled/sustained release formulations.
• Suitable dosage forms for oral ingestion by a subject include tablets, pills, dragees, hard and soft shell capsules, liquids, gels, syrups, slurries, suspensions, and emulsions.
• the compounds may also be formulated in rectal compositions, such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
• Solid oral dosage forms can be obtained using excipients, which may include, fillers, disintegrants, binders (dry and wet), dissolution retardants, lubricants, glidants, antiadherants, cationic exchange resins, wetting agents, antioxidants, preservatives, coloring, and flavoring agents.
• excipients may include, fillers, disintegrants, binders (dry and wet), dissolution retardants, lubricants, glidants, antiadherants, cationic exchange resins, wetting agents, antioxidants, preservatives, coloring, and flavoring agents.
• excipients can be of synthetic or natural source.
• excipients examples include cellulose derivatives, citric acid, dicalcium phosphate, gelatine, magnesium carbonate, magnesium/sodium lauryl sulfate, mannitol, polyethylene glycol, polyvinyl pyrrolidone, silicates, silicium dioxide, sodium benzoate, sorbitol, starches, stearic acid or a salt thereof, sugars (i.e. dextrose, sucrose, lactose, etc.), talc, tragacanth mucilage, vegetable oils (hydrogenated), and waxes. Ethanol and water may serve as granulation aides.
• coating of tablets with, for example, a taste-masking film, a stomach acid resistant film, or a release-retarding film is desirable.
• Natural and synthetic polymers, in combination with colorants, sugars, and organic solvents or water, are often used to coat tablets, resulting in dragees.
• the drug powder, suspension, or solution thereof can be delivered in a compatible hard or soft shell capsule.
• the compounds of the present invention can be administered topically, such as through a skin patch, a semi-solid or a liquid formulation, for example a gel, a (micro)-emulsion, an ointment, a solution, a (nano/micro)-suspension, or a foam.
• the penetration of the drug into the skin and underlying tissues can be regulated, for example, using penetration enhancers; the appropriate choice and combination of lipophilic, hydrophilic, and amphiphilic excipients, including water, organic solvents, waxes, oils, synthetic and natural polymers, surfactants, emulsif ⁇ ers; by pH adjustment; and use of complexing agents.
• Other techniques, such as iontophoresis may be used to regulate skin penetration of a compound of the invention. Transdermal or topical administration would be preferred, for example, in situations in which local delivery with minimal systemic exposure is desired.
• the compounds for use according to the present invention are conveniently delivered in the form of a solution, suspension, emulsion, or semisolid aerosol from pressurized packs, or a nebuliser, usually with the use of a propellant, e.g., halogenated carbons derived from methane and ethane, carbon dioxide, or any other suitable gas.
• a propellant e.g., halogenated carbons derived from methane and ethane, carbon dioxide, or any other suitable gas.
• hydrocarbons like butane, isobutene, and pentane are useful.
• the appropriate dosage unit may be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of, for example, gelatin, for use in an inhaler or insufflator may be formulated. These typically contain a powder mix of the compound and a suitable powder base such as lactose or starch.
• compositions formulated for parenteral administration by injection are usually sterile and, can be presented in unit dosage forms, e.g., in ampoules, syringes, injection pens, or in multi-dose containers, the latter usually containing a preservative.
• the compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents, such as buffers, tonicity agents, viscosity enhancing agents, surfactants, suspending and dispersing agents, antioxidants, biocompatible polymers, chelating agents, and preservatives.
• the vehicle may contain water, a synthetic or vegetable oil, and/or organic co-solvents.
• the parenteral formulation would be reconstituted or diluted prior to administration.
• Depot formulations providing controlled or sustained release of a compound of the invention, may include injectable suspensions of nano/micro particles or nano/micro or non- micronized crystals.
• Polymers such as poly(lactic acid), poly(glycolic acid), or copolymers thereof, can serve as controlled/sustained release matrices, in addition to others well known in the art.
• Other depot delivery systems may be presented in form of implants and pumps requiring incision.
• Suitable carriers for intravenous injection for the molecules of the invention are well-known in the art and include water-based solutions containing a base, such as, for example, sodium hydroxide, to form an ionized compound, sucrose or sodium chloride as a tonicity agent, for example, the buffer contains phosphate or histidine.
• a base such as, for example, sodium hydroxide
• sucrose or sodium chloride as a tonicity agent
• the buffer contains phosphate or histidine.
• Co-solvents such as, for example, polyethylene glycols, may be added.
• These water-based systems are effective at dissolving compounds of the invention and produce low toxicity upon systemic administration.
• the proportions of the components of a solution system may be varied considerably, without destroying solubility and toxicity characteristics.
• the identity of the components may be varied.
• low-toxicity surfactants such as polysorbates or poloxamers
• polyethylene glycol or other co-solvents polyethylene glycol or other co-solvents
• biocompatible polymers such as polyvinyl pyrrolidone may be added, and other sugars and polyols may substitute for dextrose.
• composition useful for the present methods of treatment a therapeutically effective dose can be estimated initially using a variety of techniques well-known in the art. Initial doses used in animal studies may be based on effective concentrations established in cell culture assays. Dosage ranges appropriate for human subjects can be determined, for example, using data obtained from animal studies and cell culture assays.
• a therapeutically effective dose or amount of a compound, agent, or drug of the present invention refers to an amount or dose of the compound, agent, or drug that results in amelioration of symptoms or a prolongation of survival in a subject.
• Toxicity and therapeutic efficacy of such molecules can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
• the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ ED50. Agents that exhibit high therapeutic indices are preferred.
• the effective amount or therapeutically effective amount is the amount of the compound or pharmaceutical composition that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by the researcher, veterinarian, medical doctor, or other clinician, e.g., treatment of cancer, including induction of anti-tumor effects, etc.
• Dosages preferably fall within a range of circulating concentrations that includes the ED50 with little or no toxicity. Dosages may vary within this range depending upon the dosage form employed and/or the route of administration utilized. The exact formulation, route of administration, dosage, and dosage interval should be chosen according to methods known in the art, in view of the specifics of a subject's condition. Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety that are sufficient to achieve the desired effects, i.e., minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from, for example, in vitro data and animal experiments. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
• MEC minimal effective concentration
• effective doses for compounds of the invention include doses of 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, and 30 mg/kg, respectively.
• effective treatment regimes for compounds of the invention include administration two or three times weekly.
• the amount of agent or composition administered may be dependent on a variety of factors, including the sex, age, and weight of the subject being treated, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician.
• compositions may, if desired, be presented in a pack or dispenser device containing one or more unit dosage forms containing the active ingredient.
• a pack or device may, for example, comprise metal or plastic foil, such as a blister pack, or glass and rubber stoppers such as in vials.
• the pack or dispenser device may be accompanied by instructions for administration.
• Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
• T helper cell secretion of proinflammatory cytokines was evaluated as follows.
• Naive T helper cells AllCells LLC, Emeryville, CA
• TCR T cell receptor
• 5 ⁇ g/ml anti-CD3 antibody clone UCHTl, R&D Systems, Minneapolis, MN
• cells were cultured with media alone or with media containing 2 ng/ml IL-12 (R&D Systems, Minneapolis, MN) (to stimulate differentiation of the T helper cells into ThI cells) and 10 ⁇ g/ml anti-IL-4 antibody (R&D Systems, Minneapolis, MN) (to block differentiation of the T helper cells into Th2 cells).
• Compound A or 1% DMSO (Veh) was added to the existing media and the cells were cultured for an additional seven days.
• the conditioned media was then collected from the cell cultures and analyzed for interferon-gamma (IFN- ⁇ ) and tumor necrosis factor-alpha (TNF- ⁇ ) levels using MS2400 Human Tissue Culture Cytokine Detection Kits (MesoScale Technologies, Gaithersburg, MD) according to the manufacturer's instructions.
• IFN- ⁇ interferon-gamma
• TNF- ⁇ tumor necrosis factor-alpha
• TCR-stimulated T helper cells treated with Compound A resulted in a reduction of IFN- ⁇ and TNF- ⁇ levels in the conditioned media of the cells, respectively.
• TCR-stimulated T helper cells treated with IL-12 in the absence of Compound A showed increased IFN- ⁇ levels and increased TNF- ⁇ levels compared to the IFN- ⁇ levels and TNF- ⁇ levels observed in vehicle-only control treated cells.
• IL-12 stimulates the differentiation of T helper cells into ThI cells, evidenced by the induction of IFN- ⁇ and TNF- ⁇ synthesis, these results indicated that the TCR-stimulated T helper cells differentiated into ThI cells in response to IL-12 addition.
• T helper cell secretion of pro-inflammatory cytokines was evaluated as follows.
• Naive T helper cells were stimulated substantially as described above with the following modification: cell cultures contained lOOU/ml penicillin- 100U/ml streptomycin.
• Various compounds of the present invention or 1% DMSO (Veh) was added to the existing media and the cells were cultured for an additional six days.
• IFN- ⁇ interferon-gamma
• TCR-stimulated T cells treated with IL- 12 in the absence of compound showed increased IFN- ⁇ levels compared to the IFN- ⁇ levels observed in vehicle-only control TCR-stimulated T cells.
• IL-12 stimulates the differentiation of naive T cells into ThI cells, evidenced by the induction of IFN- ⁇ synthesis, these results indicated that the TCR-stimulated T cells differentiated into ThI cells in response to IL- 12 addition.
• Addition of compounds of the invention to EL- 12 treated, TCR-stimulated T cells inhibited the increase in IFN- ⁇ levels compared to that observed in TCR-stimulated T cells treated with EL-12 in the absence of compounds of the invention (See Figure 2).
• T helper cell number and viability were studied.
• Human cord blood na ⁇ ve T helper cells (AllCells LLC, Emeryville, CA) were stimulated (i.e., T cell receptor (TCR) stimulation) by seeding into 96-well culture dishes coated with 5 ⁇ g/ml anti-CD3 antibody (clone UCHTl, R&D Systems, Minneapolis, MN), and 5 ⁇ g/ml anti-CD28 antibody (clone ANC28.1/D10, Ancell Corporation, Bayport, MN), and cultured at 37 0 C, 5% CO 2 in RPMI-1640 (Mediatech Inc., Herndon, VA) containing 1% FBS, lU/ml penicillin- 1 ⁇ g/ml streptomycin (Invitrogen Corporation, Carlsbad, California), and 50 U/ml IL-2 (Roche Diagnostics Corporation, Indianapolis, IN).
• TCR T cell receptor
• the cells were cultured with media alone or with media containing 2 ng/ml IL- 12 (R&D Systems, Minneapolis, MN) (to stimulate differentiation of the T helper cells into ThI cells) and 10 ⁇ g/ml anti-IL-4 antibody (R&D Systems, Minneapolis, MN) (to block differentiation of the T helper cells into Th2 cells).
• Compound B or 1% DMSO (Veh) was added to the culture media and the cells were cultured for an additional seven days.
• the T helper cells were then collected and cell number and cell viability were analyzed using Guava ViaCount reagents and the Guava PCA System (Guava Technologies, Hayward, CA).
• T helper cell viability was not reduced by addition of Compound A. Specifically, Compound A did not decrease the total number or the percent of viable T helper cells present at day 7 in these studies. These results showed that methods and compounds of the present invention do not decrease T helper cell number or compromise viability.
• T helper cell number and viability was determined.
• Na ⁇ ve T helper cells were stimulated substantially as described above with the following modification: cell cultures contained 100U/ml penicillin- lOOU/ml streptomycin.
• Various compounds of the present invention or 1% DMSO (Veh) was added to the existing media and the cells were cultured for an additional six days.
• the T helper cells were then collected and cell number and cell viability were analyzed using Guava ViaCount reagents and the Guava PCA System (Guava Technologies, Hayward, CA).
• T helper cells Human cord blood na ⁇ ve T helper cells (AllCells LLC, Emeryville, CA) were stimulated (i.e., T cell receptor (TCR) stimulation) by seeding into 96-well culture dishes coated with 5 ⁇ g/ml anti-CD3 antibody (clone UCHTl, R&D Systems, Minneapolis, MN), and 5 ⁇ g/ml anti-CD28 antibody (clone ANC28.1/D10, Ancell Corporation, Bayport, MN), and cultured at 37 0 C, 5% CO 2 in RPMI-1640 (Mediatech Inc., Herndon, VA) containing 1% FBS, lU/ml penicillin- 1 ⁇ g/ml streptomycin (Invitrogen Corporation, Carlsbad, California), and 50 U/ml IL-2 (Roche Diagnostics Corporation, Indianapolis, IN).
• TCR T cell receptor
• cells were cultured with media alone or with media containing 2 ng/ml IL- 12 (R&D Systems, Minneapolis, MN) (to stimulate differentiation of the T helper cells into ThI cells) and 10 ⁇ g/ml anti-IL-4 antibody (R&D Systems, Minneapolis, MN) (to block differentiation of the T helper cells into Th2 cells).
• Compound B or 1% DMSO (Veh) was added to the existing culture media and the cells cultured for an additional six days.
• the cells were then lysed using Cell-to-Signal lysis buffer (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions.
• the cell lysates were diluted with RNAse-free and DNAse- free water and used as templates in quantitative one-step duplex RT-PCR reactions using QuantiTect Multiplex RT-PCR Kit reagents (Qiagen Inc., Valencia, CA) and Taqman primers and probes (Applied Biosystems, Foster City, CA) as follows: IL18RAP Taqman primer/probe, catalog no. HS00187256M1; IL-12R ⁇ 2 Taqman primer/probe, catalog no.
• RT-PCR reactions were performed according to the manufacturers' instructions. Changes in gene expression of IL12R ⁇ 2, IL18R1, and IL18RAP following IL-12 addition in each sample were normalized relative to 18s ribosomal RNA gene expression measured in the same sample.
• T helper cell synthesis of pro-inflammatory and anti-inflammatory cytokines the following studies were performed.
• Human cord blood naive CD4+ T Cells (AllCells LLC, Emeryville, CA) were stimulated (i.e., T cell receptor (TCR) stimulation) by seeding into 96-well culture dishes coated with 5 ⁇ g/ml anti-CD3 antibody (clone UCHTl, R&D Systems, Minneapolis, MN), and 5 ⁇ g/ml anti-CD28 antibody (clone ANC28.1/D10, Ancell Corporation, Bayport, MN), and cultured at 37 0 C, 5% CO 2 in RPMI-1640 (Mediatech Inc., Herndon, VA) containing 1% FBS, lU/ml penicillin- 1 ⁇ g/ml streptomycin (Invitrogen Corporation, Carlsbad, California), and 50 U/ml IL-2 (Roche Diagnostics Corporation, Indianapolis, IN).
• TCR T cell receptor
• cells were treated according to one of the following conditions: 1) media alone (Con); 2) 10 ng/ml IL-4, 10 ⁇ g/ml anti-IFN- ⁇ antibody, and 10 ⁇ g/ml anti-IL-12 antibody (R&D Systems, Minneapolis, MN); or 3) 2 ng/ml BL- 12 and 10 ⁇ g/ml anti-IL-4 antibody.
• Compound A or 1% DMSO (Veh) was added to existing culture media and the cells were cultured for an additional seven days.
• the cell-conditioned media was collected and analyzed for EFN- ⁇ levels (a pro-inflammatory cytokine) and IL-5 levels (an anti-inflammatory cytokine) using MS2400 Human Tissue Culture Kits (MesoScale Technologies, Gaithersburg, MD) according to the manufacturer's instructions.
• EL-4 condition 2 above
• TCR-stimulated T helper cells increased EL-5 secretion compared to the secretion observed in vehicle-treated control T helper cells, indicative of the TCR-stimulated T helper cells having differentiated into Th2 cells in response to EL-4 stimulation.
• EL-12 had no effect on EL-5 secretion from these cells.
• Addition of Compound A to TCR-stimulated T helper cells treated with EL-4 resulted in increased EL-5 levels compared to that observed in TCR-stimulated T helper cells treated with EL-4 alone.
• ThI differentiation was induced by culturing anti-CD3 stimulated T cells in the presence of exogenous IL-12.
• Human cord blood naive T helper cells (AllCells LLC, Emeryville, CA) were stimulated (i.e., T cell receptor (TCR) stimulation) by seeding into 96-well culture dishes coated with 5 ⁇ g/ml anti-CD3 antibody (clone UCHTl, R&D Systems, Minneapolis, MN), and 5 ⁇ g/ml anti-CD28 antibody (clone ANC28.1/D10, Ancell Corporation, Bayport, MN), and cultured at 37 0 C, 5% CO 2 in RPMI-1640 (Mediatech Inc., Hemdon, VA) containing 1% FBS, lU/ml penicillin- 1 ⁇ g/ml streptomycin (Invitrogen Corporation, Carlsbad, California), and 50 U/ml IL-2 (Roche Diagnostics Corporation
• T helper cells were cultured with media alone or with media containing 2 ng/ml IL-12 and 10 ⁇ g/ml anti-IL4 to induce ThI cell differentiation.
• Compound A or 1% DMSO (Veh) was added to existing culture media and the cells cultured for an additional seven days. These steps lead to a first T cell receptor stimulation of the T helper cells.
• the cells were then collected, washed twice with RPMI-1640 media containing 1% FBS, and counted before equal numbers of cells per well were re-stimulated (second T cell receptor (TCR) stimulation following washout of all prior treatments) by re-seeding the cells into 96-well culture dishes coated with 5 ⁇ g/ml anti-CD3 antibody and 5 ⁇ g/ml anti-CD28 antibody.
• the cells were then cultured at 37°C, 5% CO 2 in RPMI-1640 media containing 1% FBS, lU/ml penicillin- 1 ⁇ g/ml streptomycin, and 50 U/ml IL-2. After 24 hours the conditioned media from the cells was collected and analyzed for IFN- ⁇ levels using a MS2400 Tissue Culture Kit (MesoScale Technologies, Gaithersburg, MD) according to the manufacturer's instructions.
• TCR-stimulated T helper cells stimulated in the presence of IL-12 (to induce the differentiation of the T helper cells into ThI cells) secreted higher levels of IFN- ⁇ following a second TCR stimulation in the absence of IL-12.
• IL-12 differentiation conditions Veh bar in Figure 7A.
• the presence of Compound A during the first TCR-stimulation in the presence of IL-12 inhibited IL- 12- induced T cell differentiation as evidenced by reduced IFN- ⁇ secretion following the second TCR stimulation.
• Cmp A bar in Figure 7 A. See IL-12 differentiation conditions, Cmp A bar in Figure 7 A.
• the cells were collected, washed twice with RPMI- 1640 media containing 1% FBS, and counted before equal numbers of cells per well were stimulated a third time (third TCR stimulation) by re-seeding into 96-well culture dishes coated with 5 ⁇ g/ml anti-CD3 antibody and 5 ⁇ g/ml anti-CD28 antibody.
• the cells were then cultured at 37 0 C, 5% CO 2 in RPMI-1640 containing 1% FBS and lU/ml penicillin- 1 ⁇ g/ml streptomycin. After 24 hours the conditioned media was collected and analyzed for IFN- ⁇ levels using a MS2400 Tissue Culture Kit (MesoScale Technologies, Gaithersburg, MD) according to the manufacturer's instructions.
• TCR-stimulated T helper cells stimulated in the presence of IL-12 (to induce the differentiation of the T helper cells into ThI cells) secreted higher levels of IFN- ⁇ following a second TCR stimulation in the absence of IL-12.
• IL-12 differentiation conditions See IL-12 differentiation conditions, Veh bar in Figure 8.
• the presence of compounds of the present invention during the first TCR-stimulation in the presence of IL-12 inhibited IL-12-induced T cell differentiation as evidenced by reduced IFN- ⁇ secretion following the second TCR stimulation.
• Example 6 Decreased IFN- ⁇ and TNF- ⁇ Levels ex vivo Following T Cell Activation in vivo
• T cell activation was first induced by eliciting an inflammatory reaction in rats by injection of peptidoglycan-polysaccharide polymers.
• An exemplary compound of the present invention was then administered to the animals.
• T cells were then isolated from the blood of the animals and analyzed ex vivo for IFN- ⁇ and TNF- ⁇ secretion.
• peptidoglycan-polysaccharide polymers 15 ⁇ g/gm body weight, Lee Laboratories, Grayson, GA. Animals were administered Compound A (40 mg/kg) via oral gavage on days 28, 30, 32, 35, 37, 39, 42, and 44 following peptidoglycan-polysaccharide polymer injection.
• Peripheral blood mononuclear cells PMBCs were isolated from the blood of the animals by Ficol gradient (BD Vacutainer CPT tubes) on day 44.
• Equal numbers of PMBCs (50,000 cells/well) were plated on culture dishes coated with anti-CD3 antibodies in RPMI- 1640 media containing 10% serum, and the cells were cultured for 72h. Supematants from the cell cultures were tested for IFN- ⁇ and TNF- ⁇ levels using commercially-available ELISA assays (R&D).

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