Compounds and compositions as hedgehog pathway modulators

TECHNICAL MEMORY

COMPOUNDS AND COMPOSITIONS AS MODULATORS

OF THE PATH OF HEDGEHOG

CROSS REFERENCE TO RELATED REQUESTS

This application is a United States national phase application for International Application Number PCT/US2007/068292 filed May 04, 2007, which application claims priority to United States Provisional Patent Application Number 60/797,949, filed on May 5, 2006. The full disclosure of these applications is incorporated herein by reference in its entirety and for all purposes.

Field of Invention

The invention provides a method for modulating the activity of the Hedgehog signaling pathway. In particular, the invention provides a method for inhibiting aberrant growth states resulting from phenotypes such as Ptc loss-of-function, Hedgehog gain-of-function, Smoothened gain-of-function, or Gli gain-of-function, which comprises contacting a cell with a sufficient amount of a compound of Formula I.

Background of the Invention

During embryonic development, the Hedgehog signaling pathway is essential for numerous processes, such as the control of cell proliferation, differentiation, and tissue patterning. However, aberrant activity of the Hedgehog signaling pathway, eg as a result of enhanced activation, may have pathological consequences. In this regard, activation of the Hedgehog pathway in adult tissues can result in specific types of cancer including, but not limited to, cancers of the brain, muscle and skin, prostate, medulloblastoma, pancreatic adenocarcinomas, and carcinomas. small cell lungs. Enhanced activation of the Hedgehog signaling pathway contributes to the pathology and/or symptomatology of a number of diseases. Accordingly, molecules that modulate the activity of the Hedgehog signaling pathway are useful as therapeutic agents in the treatment of these diseases.

Brief Description of the Invention

In one aspect, the present invention provides compounds of Formula I:

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where:

Hey₂ are independently selected from N and CR₁₀; where R₁₀ is selected from hydrogen, halogen, alkyl of 1-6 carbon atoms, alkyl of 1-6 carbon atoms substituted by halogen, alkoxy of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms substituted by halogen , and -OXNR_10thlaughed_0b; where R_10th and Rwb are independently selected from hydrogen and alkyl of 1 to 6 carbon atoms;

Ri is selected from cyano, halogen, alkyl of 1-6 carbon atoms, alkyl of 1-6 carbon atoms substituted by halogen, alkoxy of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms substituted by halogen, C6-C10-aryl, dimethyl-amino, C1-C6-alkyl-sulfanyl, and C3-C8-heterocycloalkyl optionally substituted with up to 2 C1-C6-alkyl radicals carbon;

R.₂ and R₅ are independently selected from hydrogen, cyano, halogen, alkyl of 1-6 carbon atoms, alkyl of 1-6 carbon atoms substituted by halogen, alkoxy of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms substituted by halogen, and dimethyl-amino;

R.₃ and R₄ are independently selected from hydrogen, halogen, cyano, alkyl of 1-6 carbon atoms, alkyl of 1-6 carbon atoms substituted by halogen, alkoxy of 1-6 carbon atoms, and alkoxy of 1-6 carbon atoms. carbon substituted by halogen; or Rt and R₂, or R₄ and R₅, together with the phenyl with which they are both attached, form heteroaryl of 5 to 10 carbon atoms;

R.₆ and R₇ are independently selected from hydrogen, alkyl of 1-6 carbon atoms, alkyl of 1-6 carbon atoms substituted by halogen, alkoxy of 1-6 carbon atoms, and alkoxy of 1-6 carbon atoms substituted by halogen ; with the condition that R₆ and R₇ they are not both hydrogen;

R.₈ is selected from hydrogen, halogen, alkyl of 1-6 carbon atoms, alkyl of 1-6 carbon atoms substituted by halogen, alkoxy of 1-6 carbon atoms, and alkoxy of 1-6 carbon atoms substituted by halogen;

Rg is selected from -S(O)₂Rn, -C(O)Rn, -ORn, -NRi_2alaughed_2b and -Rn¡ where R_r is selected from aryl, heteroaryl, cycloalkyl, and hetero-cycloalkyl; laughed_2a and R_12b are independently selected from alkyl of 1 to 6 carbon atoms and alkyl of 1 to 6 carbon atoms substituted by hydroxyl;

where the aryl, heteroaryl, cycloalkyl, and hetero-cycloalkyl of R_g may be optionally substituted with 1 to 3 radicals independently selected from alkyl of 1 to 6 carbon atoms, alkyl of 1 to 6 carbon atoms substituted by halogen, alkoxy of 1 to 6 carbon atoms, alkoxy of 1 to 6 atoms substituted by halogen, aryl of 6 to 10 carbon atoms-alkyl of 0 to 4 carbon atoms, hetero-aryl of 5 to 10 carbon atoms-alkyl of 0 to 4 carbon atoms, cycloalkyl of 3 to 12 atoms carbon, and hetero-cycloalkyl of 3 to 8 carbon atoms;

where the aryl-alkyl substituent of R_g is optionally substituted with 1 to 3 radicals independently selected from halogen, alkyl of 1 to 6 carbon atoms, alkyl of 1 to 6 carbon atoms substituted by halogen, alkoxy of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms substituted by halogen, and methyl-piperazinyl; and N-oxide derivatives, prodrug derivatives, protected derivatives, individual isomers, and isomer mixtures thereof; and the pharmaceutically acceptable salts and solvates (eg, hydrates) of these compounds.

In a second aspect, the present invention provides a pharmaceutical composition, which contains a compound of Formula I or an N-oxide derivative, individual isomers and mixtures of isomers thereof; or a pharmaceutically acceptable salt thereof, mixed with one or more suitable excipients.

In a third aspect, the present invention provides a method for the treatment of a disease in an animal where the modulation of the activity of the Hedgehog pathway can prevent, inhibit, or alleviate the pathology and/or symptomatology of the diseases, whose The method comprises administering to the animal a therapeutically effective amount of a compound of Formula I, or an N-oxide derivative, individual isomers and mixtures of isomers thereof, or a pharmaceutically acceptable salt thereof.

In a fourth aspect, the present invention provides the use of a compound of Formula I in the manufacture of a medicament for the treatment of a disease in an animal in which Hedgehog pathway activity contributes to the pathology and/or symptomatology. of the illness.

In a fifth aspect, the present invention provides a process for the preparation of compounds of Formula I, and the N-oxide derivatives, prodrug derivatives, protected derivatives, individual isomers and isomer mixtures thereof, and the pharmaceutically acceptable salts thereof.

Definitions

"Alkyl", as a group and as a structural element of other groups, for example halogen and alkoxy substituted alkyl and alkoxy, may be straight chain or branched. C1-C4-alkoxy includes methoxy, ethoxy, and the like. Halogen-substituted alkyl includes trifluoromethyl, pentafluoro-ethyl, and the like.

"Aryl" means a fused monocyclic or bicyclic aromatic ring assembly containing from 6 to 10 ring carbon atoms. For example, aryl can be phenyl or naphthyl, preferably phenyl.

"Arylene" means a divalent radical derived from an aryl group.

"Heteroaryl" is as defined for aryl above, wherein one or more of the ring members is a heteroatom. For example, heteroaryl of 5 to 10 carbon atoms is a minimum of five members, as indicated by the carbon atoms, but these carbon atoms may be replaced by a heteroatom. Accordingly, heteroaryl of 5 to 10 carbon atoms includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, benzo-furanyl, benzo-pyranyl, benzothiopyranyl, benzo-[1,3]-dioxole, imidazolyl, benzo-imidazolyl, pyrimidinyl, furanyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, thienyl, etc.

"Cycloalkyl" means a saturated or partially unsaturated monocyclic, fused bicyclic, or bridged polycyclic ring assembly containing the indicated number of ring atoms. For example, cycloalkyl of 3 to 10 carbon atoms includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

"Hetero-cycloalkyl" means cycloalkyl, as defined in this application, provided that one or more of the indicated ring carbon atoms is replaced by a moiety selected from -O-, -N=, -NR- , -C(O)-, -S-, -S(O)- or -S(O)₂-, wherein R is hydrogen, alkyl of 1 to 4 carbon atoms, or a nitrogen protecting group. For example, hetero-cycloalkyl of 3 to 8 carbon atoms, as used in this application to describe the compounds of the invention, includes morpholino, pyrrolidinyl, pyrrolidinyl-2-one, piperazinyl, piperidinyl, piperidinylone, 1,4-dioxa -8-aza-spiro[4.5]-dec-8-yl, thiomorpholino, sulfane-morpholino, sulfono-morpholino, etc.

Halogen” (or halo), preferably represents chlorine or fluorine, but it can also be bromine or iodine.

“Hedgehog gain of function” means an aberrant modification or mutation of a Ptc gene, Hedgehog gene, or Smoothened gene, or a decrease (or loss) in the expression level of this gene, resulting in a phenotype that appears to make contact with a cell with a Hedgehog protein, for example an aberrant activation of a Hedgehog pathway. Gain-of-function may include a loss of the ability of the Ptc gene product to regulate the expression level of the Gli genes, eg, GIH, GIÍ2, and GIÍ3. The term "Hedgehog gain of function" is also used herein to refer to any similar cellular phenotype (eg, exhibiting excess proliferation), which arises due to an alteration in any part of the transduction pathway. of Hedgehog signal, including, but not limited to, a modification or mutation of the Hedgehog itself. For example, a tumor cell with an abnormally high rate of proliferation due to activation of the Hedgehog signaling pathway would have a "Hedgehog gain-of-function" phenotype, even when the Hedgehog is not mutated in that cell.

"Patched loss of function" refers to an aberrant modification or mutation of a Ptc gene, or a reduced level of expression of the gene, resulting in a phenotype that appears to contact a cell with a Hedgehog protein, for example the aberrant activation of a Hedgehog lane. Loss of function may include a loss of the ability of the Ptc gene product to regulate the expression of Gli genes, eg, Gl¡1, GH2, and GH3.

"Gli gain-of-function" refers to an aberrant modification or mutation of a Gli gene, or an increased level of expression of the gene, which results in a phenotype that appears to contact a cell with a Hedgehog protein, for example the aberrant activation of a Hedgehog lane.

"Smoothened gain-of-function" refers to an aberrant modification or mutation of a Smo gene, or an increased level of expression of the gene, which results in a phenotype that appears to contact a cell with a Hedgehog protein, for example the aberrant activation of a Hedgehog lane.

“Treat”, treating”, and “treatment”, refer to a method of alleviating or abating a disease and/or its associated symptoms.

The present invention relates to the discovery that Hedgehog-regulated, (Ptc) Patched, Gli, and/or Smoothened signal transduction pathways can be modulated by compounds of Formula I.

Description of Preferred Modalities

In one embodiment, with respect to the compounds of Formula I, Υ_Ί and and₂ are selected from N and 01%; wherein Rm is selected from hydrogen, methyl, fluoro, chloro, bromo, dimethyl-aminoethoxy, and trifluoro-methyl; R.₆ and R₇ are independently selected from hydrogen, methyl, chloro, fluoro, bromo, trifluoro-methyl, and methoxy; with the condition of R₆ and R? they are not both hydrogen; and R₈ is selected from hydrogen, fluoro, chloro, methyl, and trifluoro-methyl.

In another modality, R! is selected from cyano, chloro, fluoro, methyl, ethyl, tertiary-butyl, propyl, isobutyl, isopropyl, isopropyloxy, butoxy, methoxy, dimethyl-amino, ethoxy, methyl-sulfanyl, phenyl, trifluoro-methyl, trifluoro-methoxy , and piperazinyl, optionally substituted with up to 2 methyl radicals; R.₂ and R₅ are independently selected from hydrogen, chloro, fluoro, cyano, methyl, trifluoromethyl, isopropyloxy, methoxy, ethoxy, trifluoro-methoxy, and dimethyl-amino; and R₃ and R₄ are independently selected from hydrogen, chloro, methyl, methoxy, and cyano; or R-ι and R₂, or Rt and R₅, together with the phenyl to which they are both attached, form quinoxalinyl.

In another modality, R₉ is selected from -S(O)₂Rn, -ORn, -C(O)Rn, -NR_12thR._12b and -Rn; wherein Rn is selected from thiomorpholino, sulfono-morpholino, sulfano-morpholino, morpholino, cyclohexyl, phenyl, azepan-1-yl, 2-oxopiperazin-1-yl, 1,4-oxazepan-4-yl, piperidin- 1-yl, tetrahydro-2Hpyran-4-yl, pipedin-3-yl, piperazinyl, pyrrolidinyl, and 1,4-diazepan-1-yl; R._12th and Ri_2b are independently selected from isobutyl and hydroxy-ethyl; wherein this thiomorpholino, sulfono-morpholino, sulfano-morpholino, morpholino, cyclohexyl, phenyl, azepan-1-yl, 2-oxopiperazin-1-yl, 1,4-oxazepan-4-yl, piperidin-1-yl, tetrahydro R -2H-pyran-4-yl, piperidin-3-yl, piperazinyl, pyrrolidinyl, or 1,4-diazepan-1-yl₉may be optionally substituted with 1 to 3 radicals independently selected from methyl, ethyl, methoxy, benzyl, thienyl-methyl, pyridinyl-methyl, benzo-[d]-[1,3]-dioxol-6-yl, and 2 ,3-dihydro-benzo[b]-[1,4]-dioxin-7-yl; wherein the phenyl or benzyl substituent of R₉ is optionally substituted with 1 to 3 radicals independently selected from methoxy, ethoxy, methyl-piperazinyl, methyl, trifluoro-methoxy, chloro, fluoro, and trifluoro-methyl.

Preferred compounds of Formula I are selected from:

4'-cyano-6-methyl-biphenyl-3-carboxylic acid [4-(morpholine-4-sulfonyl)-phenyl]-amide, [6-(2,6-dimethylmorpholine- 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid 4-yl)-pyridin-3-yl]-amide, 4'-(6-azepan-1-yl-pyridin-3-yl)-amide 4'-Methoxy-acid -cyano-2-methyl-bif en i l-3-carboxylic acid, (6-azepan-1 -yl-pyridin-3-yl)-amide

2-Methyl-biphenyl-3-carboxylic acid, 4'-methoxy-2-methyl-biphenyl-3-carboxylic acid (4-cyclohexyl-phenyl)-amide, [6-(2methyl-morpholin-4-yl) 4'-Methoxy-2-methyl-biphenyl-3-carboxylic acid -pyridin-3-l]-amide, 4'-dimethyl-amino-2- acid (4-cyclohexyl-phenyl)amide 4'dimethyl-amino-2-methyl-biphenyl-3-carboxylic acid (4-morpholin-4-yl-phenyl)-amide, (6- 6-Chloro-4'-dimethyl-amino-biphenyl-3-carboxylic acid [1,4]-oxazepan-4-yl-pyridin-3-yl)-amide, 6-Chloro-4'-dimethyl-amino- bif en i l-3-carboxylic acid (6-morpholin-4-yl-pyridin-3-yl)-amide, (6-azepan 6-Chloro-4'-dimethyl-am i non-biphenyl I-3-carboxylic acid -1 -yl-pyridin-3-yl)-amide, [6-(2-methyl-morpholin-4-yl)-p¡ 6-chloro-4'-methoxy-biphenyl-3-carboxylic acid ridin-3-yl]-amide, (6[1,4]-oxazepan-4-yl-pyridin-3-yl)-amide 6-chloro-4'-methoxy-biphenyl-3-carboxylic acid, (6-azepan-1 -yl-pyridin-

6-Chloro-4'-methoxy-biphenyl-3-carboxylic acid 3- yl)-amide, 6-chloro-4' (6-morpholin-4-¡l-pyridin-3-¡l)-amide 4'-Methoxy-6-methyl acid -methoxy-b¡phenyl-3-carboxylic, (6-morpholin-4-¡lp¡r¡d¡n-3-¡l)-amide 4'-Methoxy¡-6-methyl-biphenyl-3-carboxylic acid (6-[1,4]-oxazepan-4-yl-pyridin-3-yl)-amide, [6-( 4'-methoxy-6-methyl-biphenyl-3-carboxylic acid 2methyl-morpholin-4-yl)-pyridin-3-¡l]-amide, 4'-Dimethyl-amino-6-methyl-biphenyl-3-carboxylic acid [6-(2-methyl-morpholin-4-yl)-pyridin-3-yl]-amide, (6-[1,4]-oxazepan 4'-Dimethyl-amino-6-methyl-biphenyl-3-carboxylic acid -4-yl-pyridin-3yl)-amide, (6-morpholin-4-yl-pyridin-3-yl 4'-Dimethyl-amino-6-methyl-biphenyl-3-carboxylic acid )-amide, 4'-methoxy-6-(6-azepan-1-yl-pyridin-3-yl)-amide methylbiphenyl-3-carboxylic acid, 4'-methoxy-6-methyl-biphenyl-3-carboxylic acid (6-azepan-1-yl-pyridin-3-yl)-amide, 6-Methyl-4'-methyl-sulfanyl-biphenyl-3-carboxylic acid (6azepan-1-yl-pyridin-3-yl)-amide, (6-azepan-1-yl-pyridin3-yl)- 4'-Dimethyl-amino-6-methyl-biphenyl-3-carboxylic acid amide, 6-methyl-[1,1';4-(6-azepan-1-yl-pyridin-3-yl)-amide 3'-Chloro-6-methylbiphenyl-3-carboxylic acid ',1”]-terphenyl-3-carboxylic acid (6-azepan-1-yl-pyridin-3-1)-amide, (6-azepan- 2',4'-dichloro-6-methyl-b¡phenyl-3-carboxylic acid 1-yl-pyridin-3-yl)-amide, 2'-chloro-6-methyl-biphenyl-3-carboxylic acid (6azepan-1-yl-pyridin-3-yl)-amide, (6-azepan-1-yl-pyridin-3-yl)amide 3'-chloro-6-methyl-biphenyl-3-carboxylic acid, 3',4'd¡chloro- (6-azepan-1 -yl-pyridin-3-¡l)-amide 3'-chloro-6-methyl-4-acid 6-methyl-b¡phenyl-3-carboxylic, (6-azepan-1-¡l-pyridin-3-yl)-amide 6,4'-Dimethyl-biphenyl-3'-trifluoromethyl-biphenyl-3-carboxylic acid (6-azepan-1 -yl-p¡ridin-3-¡l)-amide -carboxylic, 4'-ethyl-6-methyl-lb¡phenyl-3-carboxylic acid (6azepan-1-yl-pyrid¡n-3-yl)-amide, (6-azepan-1 4'-tert-butyl-6-methyl-biphenyl-3-carboxylic acid -yl-pyridin-3-yl)-amide, (6-azepan-1-yl-pyridin-3-yl) 6-methyl-4'-propylbiphenyl-3-carboxylic acid-amide, 4'-isobutyl-6 (6-azepan-1 -yl-pyridin-3-yl)-amide 4'-¡sopropyl-6-methyl-biphenyl-3-carboxylic acid -methyl-biphenyl-3-carboxylic, (6azepan-1-yl-pyridin-3-yl)-amide, 6,2',6'-Trimethyl-biphenyl-3-carboxylic acid (6-azepan-1-yl-pyridin-3-yl)amide, (6-azepan-1-yl-pyridin-3 6,2',3'-trimethyl-biphenyl-3-carboxylic acid -¡l)-amide, 6-methyl-4'-(6-azepan-1-yl-pyridin-3-yl)-amide 6-Methyl-3'-trifluoro-methyl-biphenyl-3-carboxylic acid-(6-azepan-1-yl-pyridin-3-yl)-amide, (6azepan-1- 6-methyl-3',5'-b¡s-trifluoro-methyl-lb¡phenyl-3-carboxylic acid yl-pyridin-3-yl)-amide, 3'-Isopropoxy-6-methyl-biphenyl-3-carboxylic acid (6-azepan-1-ylpyridin-3-yl)-amide, (6-azepan-1-yl-pyridin-3 3'-ethoxy¡-6-methyl-biphenyl-3-carboxylic acid-yl)-amide, 2'-(6-azepan-1-yl-pyridin-3-yl)-amide₁6'-dimethoxy-6-methylbiphenyl-3-carboxylic acid, 6-methyl-4'-trifluoro-methoxy-b¡-(6-azepan-1-yl-pyrid¡n-3-yl)-amide phenyl-3-carboxylic acid, 6-methyl-3'-trifluoro-methoxy-bphenyl-3-carboxylic acid (6-azepan-1-l-pyridin-3-yl)-amide, 6-Methyl-biphenyl-3-carboxylic acid (4-morpholin-4-yl-phenyl)-amide, 4'methoxy acid (4-morpholin-4-yl-phenyl)-amide 3'-methoxy-6-methyl-biphenyl-3-carboxylic acid -6-methyl-biphenyl-3-carboxylic, (4-morpholin-4-yl-phenyl)-amide, 4'-(2-dimethyl-am¡no-ethoxy)-6-methyl-biphenyl-3-carboxylic acid (4-morpholin-4-l-phenyl)-amide, (4-morpholin- 3'-Dimethyl-amino-6-methyl-biphenyl-3-carboxylic acid 4-yl-phenyl)-amide, 4'-fluoro-6-(4-morpholin-4-yl-phenyl)-amide 3'-Fluoro-6-methylbiphenyl-3-carboxylic acid, (4-morpholine-4-morpholine-4-methyl-biphenyl-3-carboxylic)-methyl-biphenyl-3-carboxylic 2'-Fluoro-6-methyl-biphenyl-3-carboxylic acid, 4-methyl-N(4-morpholin-4-yl-phenyl)-3-quinoxalin-6-yl-yl-phenyl)-amide -benzamida, 6-Methyl-4'-(4-methylpiperazin-1-yl)-biphenyl-3-carboxylic acid (4-morpholin-4-yl-phenyl)-amide, (4-morpholin-4 2'-Cyano-6-methyl-b¡phenyl-3-carboxylic acid -yl-phenyl)-amide, 3'-cyano-6-methyl-biphenyl-(4-morpholin-4-yl-phenyl)-amide 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid 3-carboxylic, (6-[1,4]-oxazepan-4yl-pyridin-3-yl)-amide, (6-azepan-1 4'-cyano-6-methyl-b¡phenyl-3-carboxylic acid -yl-pyridin-3-yl)-amide, 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid [6-(2-methyl-morpholin-4-yl)-pyridin-3-¡l]-amide, (3,4,5,6-tetrahydro- 4'-Cyano-6-methylbiphenyl-3-carboxylic acid 2H-[1,2']-bipyridinyl-5'-yl)-amide, (6-morpholin-4-yl-pyridin-3-yl)-amide 4'-cyano-6-methyl-biphenyl-3-carboxylic acid, 4'-cyano-6-acid [6-(4methyl-piperazin-1-yl)-pyridin-3-yl]-amide -methyl-biphenyl-3-carboxylic acid, 4'-cyano-6-methyl-biphenyl-3-carboxylic acid (4-morpholin-4-yl-phenyl)amide, 4'Cyano-6-methyllb¡phenyl-3-carboxylic acid (3-fluoro-4-morpholin-4-yl-phenyl)-amide, (3-chloro-4-morpholin-4- ¡l-phenyl)-amide of 4'-cyano-6-methyl-b¡phenyl-3-carboxylic acid, (3-bromo-4-morpholin-4-yl-phenyl)-amide of 4'-cyano-6-methyl-biphenyl-3-carboxylic acid, 4'-cyano-6-methyl-biphenyl- (3-methyl-4-morpholin-4-yl-phenyl)-amide 3-carboxylic acid, 4'-cyano-6-methyl-biphenyl-3-carboxylic acid (4-morpholin-4-yl-3-trifluoro-methylphenyl)-amide, 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid (4-cyclohexyl-phenyl)-arnide, 4-cyano-6-methyl-biphenyl-3-carboxylic acid-biphenyl-4-yl-amide, (4' -methoxy-biphenyl-

4'-Cyano-6-methyl-b¡phenyl-3-carboxylic acid 4-¡l)-amide, [4-(4-benzyl-piperaz¡n-1 -yl)-phenyl]-amide 4'-cyano-6-methyl-biphenyl-3-carboxylic acid, 4'-cyano-6-methyl-biphenyl-3-carboxylic acid [4-(piperidine-1-sulfonyl)-phenyl]-amide, [4 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid -(pyrrolidin-1-sulfonyl)-phenyl]-amide, (6-azepan-1-yl-pyridin-3-yl)-acid 4-amide '-cyano-6-methoxy-biphenyl-3-carboxylic, 4'-Cyano-2-methoxy-biphenyl-3-carboxylic acid (6-azepan-1-yl-pyridin-3-yl)-amide, (6-azepan-1-yl-pyridin-3-yl)- 4'-Cyano-2-methylbif en i l-3-carboxylic acid amide, 3'-fluoro-4'- (6-azepan-1 -yl-pyridin-3-yl)-amide 4'-Isopropoxy-6-methyl-biphenyl-3-carboxyl acid methoxy-6-methyl-biphenyl I-3-carboxylic acid (6-azepan-1-yl-pyridin-3-I)-amide ¡co, 4'-butoxy-6-methyl-lb¡phenyl-3-carboxylic acid (6azepan-1-yl-pyrid¡n-3-yl)-amide, 3'-chloro-4'-methoxy-6-methyl-biphenyl-3-carboxylic acid (6-azepan-1-yl-pyridin-3-yl)amide, (6-azepan-1-yl-pyridin 4'-Methoxy¡-6,3'-dimethyl-biphenyl-3-carboxylic acid-3-yl)-amide, 4'-cyano-[4-(piperidine-1-sulfonyl)-phenyl]-amide 4'-Cyano-6-fluoro-biphenyl-3-carboxylic acid [4-(piperidine-1-sulfonyl)-phenyl]-amide, 2-methylbiphenyl-3-carboxylic acid, [4-piperidine-1-sulfonyl 6-bromo-4'-cyano-biphenyl-3-carboxylic acid )-phenyl]-amide, 4'-Cyano-6-methyl-biphenyl-3- acid [6-(4-benzyl-[1,4]diazepan-1 -yl)-p¡r¡d¡n-3-¡l]-amide 4'-cyano-6-methyl-biphenyl-carboxylic acid [6-(4-thiophen-3-yl-methyl-[1,4]diazepan-1-yl)-pyridin-3-yl]-amide 4'-Cyano-2-methyl-biphenyl-3-carboxylic acid 3-carboxylic, [6-(2,4-dimethyl-morpholin-4¡l)-pyridin-3-yl]-amide, [6 4'-Methoxy-2-methyl-lb¡phenyl-3-carboxylic acid -(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]amide, 2-Methyl-4'-trifluoro-methyl-biphenyl-3-carboxylic acid [6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-amide, 2-Methyl-4'-trifluoro-methoxy-biphenyl-3-carboxylic acid [6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-amide, [6-( 4'-cyano acid 2-methyl-morpholin-4-yl)-pyridin-3-yl]-amide

4'-Cyano-2-fluoro-biphenyl-3-carboxylic acid 2-methyl-biphenyl-3-carboxylic, [4-(piperidin-1-yl-sulfonyl)-phenyl]-amide, [4-( 4'-cyano-6-trifluoro-methyl-biphenyl-3-carboxylic acid piperidin-1-yl-sulfonyl)-phenyl]-amide, [6(4-pyridin-4-yl-methyl 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid -[ 1,4]-diazepan-1 -yl)-pyridin-3-yl]-amide, [6(4-pyridin- 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid 3-l-methyl-[1,4]-diazepan-1-yl)-pyridin-3-yl]-amide , 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid {6[4-(2,6-dimethoxy-benzyl)-[1,4]-diazepan-1-yl]-pyridin-3-yl}-amide Acid 4', {6-[4-(2-ethoxy-benzyl)-[1,4]-diazepan-1-¡l]-p¡r¡d¡n-3-¡l}-amide -cyano-6-methyl-biphenyl-3-carboxylic, (6-{4-[2-(4-methyl-piperazin-1-yl)-benzyl]-[1,4]-diazepan-1-yl}-p¡ 4'-Cyano6-methyl-bifen-yl-3-carboxylic acid ridin-3-yl)-amide, {6-[4-(4-methoxy-2,3-dimethyl-benzyl)-[ 1 4'-cyano-6-methyl-biphenyl-3-carboxylic acid ,4]-diazepan-1 -yl]-pyr¡d¡n-3-¡l}-am ¡ida, {6-[4-(2,3-dihydro-benzo-[1,4]-dioxin-6-yl-methyl)-[1,4]-diazepan-1-yl]-pyridin-3- 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid, [6-(4-pyridin-2-yl-methyl-[1,4]-diazepan-1-yl) yl}-amide 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid -pyridin-3-yl]-amide, [6-(4-benzo-[1,3]-dioxol-4-yl- 4'-cyano-6-methyl-b¡phenyl-3-carboxylic acid methyl-[1,4]diazepan-1-yl)-pyridin-3-yl]-amide, 4'-Cyano-6-methyl-biphenyl-acid {6-[4-(2-trifluoro-methoxybenzyl)-[1,4]-diazepan-1 -yl]-pyridin-3-yl}-amide 4'-Cyanο-6 3-carboxylic acid, {6-[4-(2-dimethylamino-benzyl)-[1,4]-diazepan-1-¡I]-pyridin-3-yl}-amide -methyl-biphenyl-3-carboxylic, {6-(4-(2chloro-5-trifluoro-methyl-benzyl)-[1,4]-diazepan-1-yl]-pyridin-3-yl}-am 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid, {6-(4-(2,3-difluoro-benzyl)-[1,4]-diazepan-1-yl]-pyridin-3- 4'-cyano-6-methylbiphenyl-3-carboxylic acid ¡l}-amide, 4'-Cyano6-methyl-acid {6-[4-(2-chloro-4-fluoro-benzyl)-[1,4]-diazepan-1-yl]-pyridin-3-yl}-amide biphenyl-3-carboxylic, {6-(4-(2,6-difluoro-benzyl)-[1,4]-diazepan-1 -yl]-pyr¡d¡n-3-¡l}-am¡da 4'-cyano-6-methyl-b¡phenyl-3-carboxylic acid, 2-chloro-4'-cyano-biphenyl-3-carboxylic acid [4-(piperid¡n-1-sulfonyl)-phenyl]-amide, 4'-Cyano-6-trifluoro-methyl-biphenyl-3-carboxylic acid [6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-amide, 2-chloro-4'-cyano-biphenyl-3-carboxylic acid [6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-amide, 4'-Cyano-6-ethyl-bphenyl-3-carboxylic acid [6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-amide, 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid {6-[4-(3-fluorobenzyl)-piperaz¡n-1 -yl]-pyridin-3-yl}-amide, {6-[ 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid 4-(2-trifluoromethoxy-benzyl)-piperaz¡n-1 -yl]-pyridin-3-yl}-amide, 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid {6-(4-(3-chloro-benzyl)-piperazin-1-yl]-pyridin-3-yl}-amide, {6-(4- 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid (4-isobutyl-benzyl)-piperazin-1-yl]-pyridin-3-yl}-amide, {6-(4-(4-ter-butyl-benzyl)- 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid piperazin-1-yl]-pyridin-3-yl}-amide, {6-(4-(7-methoxy-benzo-[1,3] 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid -dioxol-5-yl-methyl)-piperazin-1-yl]-pyridin-3-yl}-amide, 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid [6-(4-benzyl-piperazin-1 -yl)-pyridin-3-yl]-amide, [6(4-pyridine 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid -3-¡l-methyl-piperazin-1 -yl)-pyridin-3-¡l]-amide, {6-(4-(4-difluoro- 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid methoxy-benzyl)-piperazin-1-yl]-pyridin-3-yl}-amide, {6[4-(4-cyano-benzyl)- 4'-Cyano-6-methyl-b¡phenyl-3-carboxylic acid p¡peraz¡n-1 -yl]-pyridin-3-¡l}-amide, 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid [6-(4quinolin-5-yl-methyl-piperazin-1-yl)-pyridin-3-¡l]-amide, [6-(4p 4'-Cyano-6-methyl-b¡phenyl-3-carboxylic acid ¡ridin-4-yl-methyl-piperazin-1-yl)-pyridin-3-yl]-amide, [6-(4-pyridine 4'-Cyano-6-methyl-b¡phenyl-3-carboxylic acid -2-yl-methyl-piperazin-1-yl)-pyridin-3-yl]-amide, {6-(4-(4-imidazole- 4'-Cyano-6-methyl-b¡phenyl-3-carboxylic acid 1-yl-benzyl)-piperazin-1-yl]-pyridin-3-yl}-amide, 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid {6-[4(3-cyano-benzyl)-piperazin-1-yl]-pyridin-3-yl}-amide, [6- 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid (4-isoquinolin-5-yl-methyl-piperazin-1-yl)-pyridin-3-yl]-amide, (R)-2methyl -N-(6-(2-methyl-morphol¡no)-pyridin-3-yl)-4'-(trifluoro-methox¡)-biphenyl-3-carboxamide, 4'-cyano- 2-methyl-N-(6sulfon¡l-morpholino-pyrid¡n-3-¡l)-b¡fen¡l-3-carboxam¡da, (S)-4'-cyano-2-methylN -(6-(2-methyl-morpholino)-pyridin-3¡l)-biphenyl-3-carboxamide, (R)-6-chloro-N-(6-(2-methyl-moiíol¡no)-p¡ridin-3-yl)-4'-(trifluoro-methoxy)-b¡phenyl-3-carboxamide , 4'-cyano-2-methyl-N-(6-sulfinyl-morpholino-pyridin-3-yl)-biphenyl-3-carboxamide, 4'-cyano-N-(6-(di¡¡sobut¡l- am¡no)-p¡r¡d¡n-3-¡l)-2-methyllb¡phenyl-3-carboxam¡da, 4'-cyano-N-(2-((2S,6R )-2,6-dimethyl-morphol¡no)pyrimidin-5-yl)-2-methyl-biphenyl-3-carboxamide, N-(2-((2S,6R)-2,6 -dimethyl-morpholino)-pyrimidin-5-yl)-2-methyl-4'(trifluoro-methyl)-biphenyl-3-carboxamide, N-(2-((2S,6R)-2,6-dimethyl-morpholino)-p¡r¡m¡d¡n-5-¡l)-2-methyl-4'( trifluoro-methoxy)-biphenyl-3-carboxamide, N-(2-(bis(2-hydroxy-ethyl)-am¡no)-pyrimidin-5-yl)-2-methyl-4'-( trifluoromethoxy)-biphenyl-3-carboxamide, 2-methylN-(6-(tetrahydro-2H-pyran-4-yloxy)-pyridin-3-yl)-4'-( trifluoro-methoxy)biphenyl-3-carboxamide, N-(5-chloro-6-((2S_!6R)-2,6-dimethyl-morpholino)-pyridin-3-yl)-2-methyl-4'-(trifluoro-methoxy)biphenyl-3-carboxamide, N-(6-((2R,6S) -2,6-dimethyl-tetrahydro-2H-pyran-4-yl)-pyridin-3-yl)-2-methyl-4'-(trifluoromethox¡)-biphenyl-3-carboxamide, N -(6-(4-ethyl-piperazine-1-carbonyl)-pyridin-3-yl)-2-methyl-4'-(trifluoro-methoxy)biphenyl-3-carboxamide, 2-methyl- N-(6-(2-oxopiperazin-1 -¡l)-p¡r¡d¡n-3-¡l)-4'-(trifluoro-methox¡)-b¡fen¡l-3carboxamide, 2-methyl-N-(6-(1 -(pyridin-4-ylmethyl)-piperidin-4-yl)-pyridin-3-yl)-4'-(trifluoro-methox¡)-biphen¡ 1-3carboxamide, 2-methyl-N-(6-(2-oxo-4-(pyridin-4-ylmethyl)-piperazin-1-yl)-pyridin-3-yl)-4'-(trifluoro- methoxy¡)-b¡phenyl-3-carboxamide, 2-methyl-N-(6-(1 -(pyridin-4-ylmethyl)-piperidin-3-yl)-pyridin-3-yl)-4'-(trifluoro- methoxy)-biphenyl-3-carboxamide, N-(6-(1-ethyl-piperidin-3-i I)-pyridin-3-yl)-2-methyl-4'-(trifluoro-methoxy) -bif in i l-3-carboxam ¡da, and N(6-((2R,6S)-2,6-dimethyl-morpholino)-pyridin-3-¡l)-2-methyl-4'-(trifluoro-methoxy)-biphenyl-3- carboxamide.

Accordingly, it is specifically contemplated that compounds of Formula I that interfere with aspects of Hedgehog, Ptc, or Smoothened signal transduction activity, will similarly be capable of inhibiting proliferation (or other biological consequences) in cells. normal cells, and/or in cells having a Patched loss-of-function phenotype, a Hedgehog gain-of-function phenotype, a Smoothened gain-of-function phenotype, or a Gli gain-of-function phenotype. Accordingly, it is contemplated that, in certain embodiments, these compounds may be useful for inhibiting Hedgehog activity in normal cells, eg, that do not have a genetic mutation that activates the Hedgehog pathway. In preferred embodiments, the compounds are capable of inhibiting at least some of the biological activities of the Hedgehog proteins, preferably in a specific manner in target cells.

Therefore, the methods of the present invention include the use of compounds of Formula I that agonize Ptc inhibition of Hedgehog signaling, such as by inhibiting the activation of Smoothened or downstream components of the pathway. signaling, in the regulation of repair and/or functional performance of a wide number of cells, tissues, and organs, including normal cells, tissues, and organs, as well as those with the Ptc loss-of-function, Hedgehog gain-of-function, Smoothened gain-of-function, or Gli gain-of-function phenotype. For example, the present method has therapeutic and cosmetic applications from the regulation of neural tissues, the formation and repair of bone and cartilage, the regulation of spermatogenesis, the regulation of smooth muscle, the regulation of the lung, liver and other organs. that arise from the primitive intestine, the regulation of hematopoietic function, the regulation of skin and hair growth, etc. Furthermore, the present methods can be carried out on cells that are provided in culture (in vitro), or on cells in a whole animal (in vivo).

In another embodiment, the present method may be for treating epithelial cells having a Ptc loss-of-function, Hedgehog gain-of-function, Smoothened gain-of-function, or Gli gain-of-function phenotype. For example, the present method can be used for the treatment or prevention of basal cell carcinoma or other disorders related to the Hedgehog pathway.

In certain embodiments, a compound of Formula I can inhibit the activation of a Hedgehog pathway by binding to Smoothened or downstream proteins. In certain modalities, an object antagonist can inhibit the activation of a Hedgehog lane by linking with a Patched.

In another preferred embodiment, the present method can be employed as part of a treatment regimen for malignant medulloblastomas and other primary malignant neuroectodermal tumors of the central nervous system.

In another aspect, the present invention provides pharmaceutical preparations comprising, as an active ingredient, a modulator of Hedgehog signaling, such as a compound of Formula I, a Ptc agonist, a Smoothened antagonist, or a Ptc antagonist. downstream Hedgehog pathway protein, as described herein, formulated in an amount sufficient to inhibit, in vivo, proliferation or other biological consequences of Ptc loss-of-function, Hedgehog gain-of-function, Smoothened gain-of-function, or Gli gain-of-function.

The present treatments using a compound of Formula I, Patched agonists, Smoothened antagonists, or downstream Hedgehog pathway protein antagonists, can be effective for both human and animal subjects. Animal subjects to which the present invention is applicable extend to both domestic animals and livestock, or those kept as pets or for commercial purposes. Examples are dogs, cats, cattle, horses, sheep, pigs, and goats.

Pharmacology v Utility

The present invention provides methods and compounds for inhibiting Hedgehog signaling pathway activation, for example to inhibit aberrant growth states resulting from phenotypes such as Ptc loss-of-function, Hedgehog gain-of-function, gain-of-function of Smoothened, or Gli gain of function, which comprise contacting the cell with a compound of Formula I, in an amount sufficient to agonize normal Ptc activity, antagonize normal Hedgehog activity, antagonize Smoothened activity, or antagonize Gli activity, for example to reverse or control the aberrant growth state.

Hedgehog signaling members mediate many short- and long-range patterning processes during vertebrate development. Pattern formation is the activity by which embryonic cells form ordered spatial arrangements of differentiated tissues. The physical complexity of higher organisms arises during embryogenesis through the interplay of intrinsic cell lineage and extrinsic cell signaling. Inductive interactions are essential for the formation of embryonic patterns in vertebrate development from the earliest establishment of the body plan, to the patterning of organ systems, and to the generation of various cell types during differentiation. of the tissues. The effects of developmental cellular interactions are varied: responding cells are shunted from one cellular differentiation pathway to another by inducing cells that differ from both the uninduced and induced states of responding cells (inductions). Sometimes cells induce their neighbors to differentiate like themselves (homeogenetic induction); in other cases, a cell inhibits its neighbors from differentiating like itself. Cellular interactions in early development can be sequential such that an initial induction between two cell types leads to a progressive amplification of diversity. Furthermore, inductive interactions occur not only in embryos, but also in adult cells, and may act to establish and maintain morphogenetic patterns, as well as induce differentiation.

The vertebrate Hedgehog gene family includes three members that exist in mammals, known as the Desert (Dhh), Sonic (Shh), and Indian (Ihh) Hedgehogs, all of which encode the secreted proteins. These different Hedgehog proteins consist of a signal peptide, a highly conserved N-terminal region, and a more divergent C-terminal domain. Biochemical studies have shown that the auto-proteolytic cleavage of the Hh precursor protein proceeds via an internal thioester intermediate that subsequently cleaves in a nucleophilic substitution. The nucleophile is likely to be a small lipophilic molecule that becomes covalently bound. to the C-terminus of the N-peptide, tethering it to the cell surface. The biological implications are profound. As a result of this binding, a local high concentration of the N-terminal Hedgehog peptide is generated on the surface of the Hedgehog producer cells. It is this N-terminal peptide that is both necessary and sufficient for short- and long-range Hedgehog signaling activities.

One inactive Hedgehog signaling pathway is where the Patched transmembrane protein (Ptc) receptor inhibits the activity of Smoothened (Smo), a seven-transmembrane protein. The transcription factor Gli, a downstream component of Hh signaling, is prevented from entering the nucleus through interactions with cytoplasmic proteins, including Fused and Supressor de Fused (Sufu). As a consequence, the transcriptional activation of Hedgehog target genes is repressed. Activation of the pathway is initiated through the binding of any of the three mammalian ligands (Dhh, Shh, or Ihh) to Ptc. Ligand binding results in reversal of Smo repression, thus activating a cascade leading to translocation of the active form of the transcription factor Gli to the nucleus. Nuclear Gli activates the expression of the target gene, including Ptc and Gli themselves.

Higher levels of Hedgehog signaling are sufficient to initiate cancer formation, and are required for tumor survival. These cancers include, but are not limited to, prostate cancer (“Hedgehog signaling in prostate regeneration, neoplasia and metastasis”, Karhadkar SS, Bova GS, Abdallah N, Dhara S, Gardner D, Maitra A, Isaacs JT, Berman DM, Beachy PA, Nature. 7 October 2004; 431(7009):707-12; “Inhibition of prostate cancer proliferation by interference with SONIC HEDGEHOG-GLI1 signaling”, Sánchez P, Hernández AM, Stecca B, Kahler AJ, DeGueme AM, Barrett A, Beyna M, Datta MW, Datta S., Ruiz i Altaba A., Proc. nati. Acad. Sci. USA. 24 August 2004; 101 (34):12561 -6), breast cancer (“Hedgehog signaling pathway is a new therapeutic target for patients with breast cancer”, Kubo M, Nakamura M, Tasaki A, Yamanaka N, Nakashima H, Nomura M, Kuroki S , Katano M., Cancer Res. 2004 Sept 1;64(17):6071-4), medulloblastoma (“Medulloblastoma growth inhibition by hedgehog pathway blockade”, Berman DM, Karhadkar SS, Hallahan AR, Pñtchard JI, Eberhart CG , Watkins DN, Chen JK, Cooper Μ. K., Taipale J, Olson JM, Beachy PA, Science. August 30, 2002; 297(5586):1559-61), basal cell carcinoma (“Identif¡cation of a small molecule inhibitor of the hedgehog signaling pathway: effects on basal cell carcinoma-like lesions”, Williams JA, Guicherit OM, Zaharian BI, Xu Y, Chai L, Wichterle H, Kon C, Gatchalian C, Porter JA, Rubín LL, Wang FY, Proc. Nati. Acad. Sel. USA. fifteen April 2003; 100(8):4616-21; “Activating Smoothened mutations in sporadic basal-cell carcinoma”, Xie J, Murone M, Luoh SM, Ryan A, Gu Q, Zhang C, Bonitas JM, Lam C. W, Hynes M, Goddard A, Rosenthal A, Epstein EH Jr ., de Sauvage FJ, Nature. January 1, 1998; 391 (6662):90-2), pancreatic cancer (“Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis”, Thayer SP, di Magliano Μ. P., Heiser PW, Nielsen CM, Roberts DJ, Lauwers GY, Qi AND. P., Gysin S, Fernandez-del Castillo C, Yajnik V, Antoniu B, McMahon M, Warshaw AL, Hebrok M., Nature. October 23, 2003; 425(6960):851-6; “Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumors”, Berman DM, Karhadkar SS, Maitra A, Montes De Oca R, Gerstenblith MR, Briggs K, Parker AR, Shimada Y, Eshleman JR, Watkins DN, Beachy PA, Nature. 23 October 2003; 425(6960):846-51), and small-cell lung cancer (“Hedgehog signaling within airway epithelial progenitors and in small-cell lung cancer”, Watkins D. N, Berman DM, Burkholder SG, Wang B, Beachy PA, Baylin SB, Nature 2003 Mar 20;422(6929):313-7).

In accordance with the foregoing, the present invention further provides a method for preventing or treating any of the diseases or disorders described above, in a subject in need of said treatment, which method comprises administering to this subject a therapeutically effective amount ("Administration and Pharmaceutical Compositions", infra) of a compound of Formula I, or a pharmaceutically acceptable salt thereof. For any of the above uses, the required dosage will vary depending on the mode of administration, the particular condition to be treated, and the effect desired.

Administration and Pharmaceutical Compositions:

In general, the compounds of the invention will be administered in therapeutically effective amounts by any of the usual and acceptable ways known in the art, either alone or in combination with one or more therapeutic agents. A therapeutically effective amount can vary widely, depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors. In general, satisfactory results are indicated to be obtained systemically with daily dosages of about 0.03 to 2.5 milligrams/kilogram of body weight. An indicated daily dosage in the higher mammal, for example in humans, is in the range of about 0.5 milligrams to about 100 milligrams, conveniently administered, for example, in divided doses up to four times daily, or in a retard form. Unit dosage forms suitable for oral administration comprise from about 1 to 50 milligrams of active ingredient.

The compounds of the invention may be administered as pharmaceutical compositions by any conventional route, in particular enterally, for example orally, for example in the form of tablets or capsules, or parenterally, for example in the form of injectable solutions or suspensions, topically, for example in the form of lotions, gels, ointments or creams, or in a nasal or suppository form. Pharmaceutical compositions comprising a compound of the present invention in free form or in pharmaceutically acceptable salt form, in association with at least one pharmaceutically acceptable carrier or diluent, may be manufactured in a conventional manner by mixing, granulating, or coating methods. . For example, oral compositions may be tablets or gelatin capsules comprising the active ingredient together with: a) diluents, for example lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and/or glycine; b) lubricants, for example silica, talc, stearic acid, its magnesium or calcium salt, and/or polyethylene glycol; for tablets also c) binders, eg magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone; if desired d) disintegrants, for example starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) adsorbents, colorants, flavorants, and sweeteners. Injectable compositions can be isotonic aqueous solutions or suspensions, and suppositories can be prepared from fat emulsions or suspensions. The compositions may be sterilized and/or may contain adjuvants, such as preservatives, stabilizers, wetting, or emulsifying agents, solution promoters, salts to regulate osmotic pressure, and/or pH regulators. In addition, they may also contain other therapeutically valuable substances. Formulations suitable for transdermal applications include an effective amount of a compound of the present invention with a carrier. A vehicle may include absorbable pharmacologically acceptable solvents to aid passage through the skin of the host. For example, transdermal devices are in the form of a patch comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate control barrier for delivering the compound to the host skin at a controlled rate and previously determined for an extended period of time, and elements to secure the device to the skin. Matrix transdermal formulations can also be used. Formulations suitable for topical administration, eg to the skin and eyes, are preferably aqueous solutions, ointments, creams, or gels, well known in the art. These may contain solubilizers, stabilizers, tonicity-enhancing agents, pH adjusters, and preservatives.

The compounds of the invention may be administered in therapeutically effective amounts in combination with one or more therapeutic agents (pharmaceutical combinations). For example, synergistic effects may occur with immunomodulatory or anti-inflammatory substances or other anti-tumor therapeutic agents. When the compounds of the invention are administered in conjunction with other therapies, the dosages of the co-administered compounds will, of course, vary depending on the type of co-drug employed, the specific drug employed, the condition being treated, etc. .

The invention also provides pharmaceutical combinations, for example a kit, which comprises: a) a first agent which is a compound of the invention as disclosed herein, in free form or in pharmaceutically acceptable salt form, and b) at least one co-agent. The kit may comprise instructions for its administration.

The terms "co-administration" or "combined administration", or the like, as used herein, are intended to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens where the agents are not necessarily interrelated. administered by the same route of administration or at the same time.

The term "pharmaceutical combination", as used herein, means a product that results from mixing or combining more than one active ingredient, and includes both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredients, for example a compound of Formula I and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, for example a compound of Formula I and a co-agent, are both administered to a patient as separate entities, either simultaneously, concurrently, or in sequence, without specific time limits, whereby this administration provides therapeutically effective levels of the two compounds in the patient's body. The latter also applies to cocktail therapy, for example the administration of three or more active ingredients.

Processes to Prepare the Compounds of the Invention

The present invention also includes processes for the preparation of the compounds of the invention. In the reactions described, it may be necessary to protect reactive functional groups, for example hydroxyl, amino, imino, thio, or carboxyl groups, where these are desired in the final product, in order to avoid their undesired participation in the reaction. The reactions. Conventional protecting groups can be used in accordance with standard practice, eg see TW Greene and PGM Wuts in “Protective Groups in Organic Chemistry”, John Wiley and Sons, 1991.

Compounds of Formula I can be prepared by proceeding as in the following Reaction Scheme I:

Reaction Scheme I r₂

<img file="ECSP088860A_D0002.tif" />

2'

I where Yi, Y₂,R_b R.₂,R₃,R₄,R₅,R₅,R₇,R_yes, and Rg are as defined for Formula I in the Brief Description of the Invention. A compound of Formula I can be prepared by reacting a compound of Formula 2 (or 2') with a compound of Formula 3 in the presence of a suitable solvent (for example, dichloromethane, Ν,Ν- dimethyl-formamide, or the like), in a temperature range of about -20^yesC to about 100²C. The reaction can take up to approximately 20 hours to complete.

Detailed examples of the synthesis of the compounds of Formula I can be found in the Examples below.

Additional Processes for the Elaboration of the Compounds of the Invention

A compound of the invention may be prepared as a pharmaceutically acceptable acid addition salt by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid. Alternatively, a pharmaceutically acceptable basic addition salt of a compound of the invention may be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base.

Alternatively, salt forms of the compounds of the invention can be prepared using the salts of the starting materials or intermediates.

The free acid or free base forms of the compounds of the invention may be prepared from the corresponding acid addition salt or base addition salt form, respectively. For example, a compound of the invention in acid addition salt form can be converted to the corresponding free base by treatment with a suitable base (eg, ammonium hydroxide solution, sodium hydroxide, and the like). A compound of the invention in base addition salt form can be converted to the corresponding free acid by treatment with a suitable acid (eg hydrochloric acid, etc.).

The compounds of the invention in non-oxidized form can be prepared from the dioxides of the compounds of the invention by treating them with a reducing agent (e.g. sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride , sodium borohydride, phosphorus trichloride, tribromide, or the like) in a suitable inert organic solvent (for example, acetonitrile, ethanol, aqueous dioxane, or the like), of 0⁹C to 80⁹c.

Prodrug derivatives of the compounds of the invention can be prepared by methods known to those of ordinary skill in the art (eg, for further details, see Saulnier et al. (1994), Bioorganic and Medicinal Chemistry Letters, Volume 4 , page 1985). For example, appropriate prodrugs can be prepared by reacting a non-derivative compound of the invention with a suitable carbamylating agent (for example, 1,1-acyloxy-alkyl carbanochlorhidate, para-nitro-phenyl carbonate, or Similar).

Protected derivatives of the compounds of the invention can be made by means known to those of ordinary skill in the art. A detailed description of the techniques applicable to the creation of protecting groups and their removal can be found in TW Greene, "Protecting Groups in Organic Chemistry", 3^S Edition, John Wiley and Sons, Inc., 1999.

The compounds of the present invention may conveniently be prepared or formed during the process of the invention, as solvates (eg, hydrates). Hydrates of the compounds of the present invention may be conveniently prepared by recrystallization from a mixed aqueous/organic solvent, using organic solvents such as dioxin, tetrahydrofuran, or methanol.

The compounds of the invention can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separate the diastereomers, and recover the optically pure enantiomers. Although resolution of the enantiomers can be carried out using divalent diastereomeric derivatives of the compounds of the invention, dissociable complexes (eg, crystalline diastereomeric salts) are preferred. Diastereomers have different physical properties (for example, melting points, boiling points, solubilities, reactivity, etc.), and can be easily separated by taking advantage of these differences. Diastereomers can be separated by chromatography, or preferably by separation/resolution techniques based on differences in solubility. The optically pure enantiomer is then recovered, together with the resolving agent, by any practical means that does not result in racemization. A more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixtures can be found in Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and Resolutions", John Wiley And Sons, Inc., 1981.

In summary, the compounds of Formula I can be made by a process that involves:

(a) those of Reaction Scheme I; and (b) optionally converting a compound of the invention to a pharmaceutically acceptable salt;

(c) optionally converting a salt form of a compound of the invention to a non-salt form;

(d) optionally converting a non-oxidized form of a compound of the invention to a pharmaceutically acceptable dioxide;

(e) optionally converting an N-oxide form of a compound of the invention to its non-oxidized form;

(f) optionally resolving an individual isomer of a compound of the invention from a mixture of isomers;

(g) optionally converting a non-derivative compound of the invention to a pharmaceutically acceptable prodrug derivative; and (h) optionally converting a prodrug derivative of a compound of the invention to its underivatized form.

Insofar as the production of the starting materials is not particularly described, the compounds are known or can be prepared in a manner analogous to methods known in the art, or as disclosed in the Examples found herein below. .

One skilled in the art will appreciate that the above transformations are only representative of the methods for the preparation of the compounds of the present invention, and that other well-known methods may be similarly employed.

EXAMPLES

The present invention is further exemplified, but not limited, by the following Example which illustrates the preparation of the compounds of Formula I according to the invention.

Example 1 f4-(morphol¡n-4-sulfon¡l)-fen¡ll-amide of acid 4^!-cyano-4-methyl-biphenyl-3-carboxylic

<img file="ECSP088860A_D0003.tif" />

h₂SW₄

MeOH

<img file="ECSP088860A_D0004.tif" />

<img file="ECSP088860A_D0005.tif" />

B(OH)₂

Pd(OAc)2, ligand

KF, dioxane

<img file="ECSP088860A_D0006.tif" />

<img file="ECSP088860A_D0007.tif" />

<img file="ECSP088860A_D0008.tif" />

Step 1: To a solution of 3-iodo-4-methyl-benzoic acid (10.0 grams, 38.2 mmol) in methanol (70 milliliters), concentrated sulfuric acid (0.5 milliliters) is added. The reaction mixture is heated to 70^QC for 48 hours, cool to room temperature, and then concentrate. Thereafter, ethyl acetate (100 mL), and aqueous NaHCO3 solution (saturated, 100 mL) are added to the residue. The organic layer is separated and washed again with aqueous NaHCO3 solution (saturated, 100 mL). The organic layer is separated, dried over anhydrous Na2SO4, and concentrated to give 3-iodo-4-methyl-benzoic acid methyl ester 1. This is used without further purification in the next step.¹H NMR (400 MHz, DMSO-d6) δ 8.31 (s, 1H), 7.87 (d, 1H, J = 8.4 Hz), 7.48 (d, 1H, J = 8.4 Hz), 3.85 (s, 3 H), 3.35 (s, 3H); LC-MS m/z: 277.0 (M+1).

Step 2: To a round bottom flask containing 3-iodo-4-methylbenzoic acid methyl ester (1.38 grams, 5.00 mmol), 4-cyano-phenyl-boronic acid (1.10 grams, 7.48 mmol), palladium (168 milligrams, 0.748 mmol), 2-(dicyclo-hexyl-phosphino)-biphenyl (0.526 grams, 1.50 mmol), and potassium fluoride (0.870 grams, 15.0 mmol), 1,4-dioxane is added anhydrous (15 milliliters). The flask is purged with argon and sealed. The mixture is stirred at 130-C for 18 hours, cooled to room temperature, and then water (20 milliliters) and ethyl acetate (20 milliliters) are added. The solid is removed by vacuum filtration. The filtrate is extracted with EtOAc (20 mL, twice). The organic layers are combined, washed with aqueous HCI (5 percent, 20 mL), and NaHCO₃ saturated (20 milliliters). dried over MgSO₄and they concentrate. The residue is purified by column chromatography on silica gel (EtOAc/hexane, gradient) to give 4'-cyano-6-methylbiphenyl-3-carboxylic acid methyl ester 2: LC-MS m/z: 252.1 (M+1).

Step 3: To a solution of 4'-cyano-6-methyl-biphenyl-3-carboxylic acid 2-methyl ester (2.56 grams, 10.3 mmol) in 1,4-dioxane-H₂0 (1:1 mix, 20 milliliters), NaOH (1.22 grams, 30.2 millimoles) is added. The reaction is stirred at room temperature for 24 hours. To this mixture is added aqueous HCI (1N, 36 mL), and then extracted with ethyl acetate (140 mL, three times). The organic layers are combined, and dried over Na₂SW₄ anhydrous. The solvent is removed. The obtained solid is washed with a small amount of acetonitrile, and air-dried, to give 4'-cyano-6-methyl-biphenyl-3-carboxylic acid 3:¹H NMR (DMSO-d₆) δ 7.94 (d, 2H, J = 8.0 Hz), 7.84 (dd, 1H, J₄ = 8.4Hz, J₂ =

1.2 Hz), 7.75 (d, 1H, J = 1.2 Hz), 7.61 (d, 2H, J = 8.0 Hz), 7.48 (d, 1H, J = 8.4 Hz), 2.29 (s, 3H) ; LC-MS m/z 238.1 (M+1).

Step 4: To a suspension of 4'-cyano-6-methyl-biphenyl-3-carboxylic acid 3 (40 milligrams, 0.17 mmol) in anhydrous methylene chloride (5 milliliters), add 2 drops of dimethyl-formamide. Oxalyl chloride (32 milligrams, 22 microliters, 0.25 mmol) is then added. The mixture is stirred at room temperature until it becomes clear. After that, it is concentrated, redissolved in anhydrous methylene chloride (3 milliliters), and added to a solution of 4-(morpholin-4-sulfon¡I)phenyl-amine (61 milligrams, 0.25 mmol) and triethylamine (34 milligrams, 47 microliters, 0.33 mmol) in methylene chloride (2 milliliters). The mixture is stirred for 2 hours, concentrated, and the residue is purified by mass-directed prep HPLC (C18 column, eluted with CH₃CN-H₂O containing 0.05 percent trifluoro-acetic acid), to give 4'-cyano6-methyl-biphenyl-3-carboxylic acid [4-(morpholine-4-sulfonyl)-phenyl]-amide:¹H NMR (DMSO-d₆) δ 10.64 (s, 1H), 8.07(d, 2H, J = 8.8 Hz), 7.97 (d, 2H, J =

8.4 Hz), 7.95 (d, 1H, J = 8.8 Hz), 7.89 (s, 1H), 7.43 (d, 2H, J = 8.4 Hz), 7.67 (d, 2H, J = 8.8 Hz) , 7.53 (d,

H, J = 8.8 Hz), 3.63 (m, 4H), 2.84 (m, 4H) 2.32 (s, 3H); LC-MS m/z: 462.1 (M+1).

Example 2

4'-Cyano-6-methyl-biphenyl-3-carboxylic acid 16-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-ill-amide

<img file="ECSP088860A_D0009.tif" />

<img file="ECSP088860A_D0010.tif" />

<img file="ECSP088860A_D0011.tif" />

h

No.

<img file="ECSP088860A_D0012.tif" />

Example 2

Step 1: To a solution of 2-chloro-5-nitro-pyridine 4 (2.38 grams, 15 mmol), and c/s-2,6-dimethylmorpholine (1.73 grams, 15 mmol), K is added₂CO₃ (4.14 grams, 30 millimoles). The mixture is heated to 50⁹C overnight. After concentration, the residue is partitioned between EtOAc and water. The EtOAc layer is dried over Na₂SW₄ anhydrous, and concentrate to give crude product 6 as a yellow solid. The crude product is used directly in the next step without further purification. LCMS m/z: 238.1 (M+1).

Step 2: Crude material 6 above is hydrogenated in the presence of Pd-C (0.2 grams) in MeOH (100 milliliters) under hydrogen for 10 hours. The suspension is filtered through Celite, and the filtrate is concentrated, to give crude product 7 as a dark brown oil, which is used directly in the next step without further purification. LC-MS m/z: 208.1 (M+1).

Step 3: To a solution of 3-bromo-4-methyl-benzoic acid (108 milligrams, 0.5 mmol), 6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl-amine 7 ( 104 mg, 0.5 mmol), and HATU (190 mg, 0.5 mmol) in dry dimethylformamide (5 mL), triethylamine (139 microliters, 1.0 mmol) is added dropwise. The resulting mixture is stirred at room temperature for 2 hours. After concentration, the residue is partitioned between EtOAc and water. The organic layer is dried and concentrated to give the crude product. The final compound is purified by flash column chromatography using 50 percent EtOAc in hexane as eluant to give 8 as a white solid. LC-MS m/z: 404.1 (M+1).

Step 4: A mixture of 4-cyano-phenyl-boronic acid (18 milligrams, 0.12 mmol), 3-bromo-N-[6(2,6-dimethyl-morphol¡n-4-¡l)-pyr¡din -3-yl]-4-methyl-benzamide 8 (40 milligrams, 0.1 mmol), Pd(PPh₃)₄ (11 milligrams, 0.01 mmol), and Na₂CO₃ (42 milligrams, 0.4 mmol) in a combined solvent system of toluene (0.2 milliliters) and water (0.2 milliliters) and ethanol (0.05 milliliters), heat to 140^yesC under microwave irradiation for 30 min. The reaction mixture is diluted with EtOAc and water. The aqueous layer is extracted with EtOAc. The combined organic layer is washed with brine and concentrated to give the crude product, which is purified by mass-directed prep HPLC (C18 column, eluted with CH₃CN-H₂O containing 0.05 percent trifluoro-acetic acid), to give 4'-cyano-6-methyl acid [6-(2,6dimethyl-morpholin-4-yl)-pyridin-3-yl]-amide -biphenyl-3-carboxylic. LC-MS m/z: 427.2 (M+1).

By repeating the procedures described in the Examples above, using the appropriate starting materials, the following compounds of Formula I are obtained, as identified in Table 1.

Table 1

Compound Number

Structure

<img file="ECSP088860A_D0013.tif" />

<img file="ECSP088860A_D0014.tif" />

MS Physical Data (m/z)

m/z LC-MS

411.2 (M+1).

m/z LC-MS

416.2 (M+1).

m/z LC-MS

400.2 (M+1).

m/z LC-MS

418.2 (M+1).

Data

physical

MS (m/z)

Compound

Number

Structure

<img file="ECSP088860A_D0015.tif" />

m/z LC-MS

413.2 (M+1).

<img file="ECSP088860A_D0016.tif" />

m/z LC-MS

416.2 (M+1).

<img file="ECSP088860A_D0017.tif" />

<img file="ECSP088860A_D0018.tif" />

-EITHER

<img file="ECSP088860A_D0019.tif" />

EITHER.

no 0

m/z LC-MS

451.2 (M+1).

m/z LC-MS

437.2 (M+1).

LC-MS m/z 449.2 (M+1).

m/z LC-MS

438.2 (M+1).

m/z LC-MS

Compound

Number

Structure

-EITHER

<img file="ECSP088860A_D0020.tif" />

<img file="ECSP088860A_D0021.tif" />

-EITHER

<img file="ECSP088860A_D0022.tif" />

MS Physical Data (m/z)

438.2 (M+1).

m/z LC-MS

436.2 (M+1).

m/z LC-MS

424.1 (M+1).

m/z LC-MS

404.2 (M+1).

m/z LC-MS

418.2 (M+1).

m/z LC-MS

418.2 (M+1).

Data

physical

MS (m/z)

Compound

Number

Structure

<img file="ECSP088860A_D0023.tif" />

m/z LC-MS

431.2 (M+1).

m/z LC-MS

431.2 (M+1).

<img file="ECSP088860A_D0024.tif" />

m/z LC-MS

417.2 (M+1).

LC-MS m/z 416.2 (M+1).

m/z LC-MS

430.2 (M+1).

m/z LC-MS

432.2 (M+1).

Data

physical

MS (m/z)

Structure

<img file="ECSP088860A_D0025.tif" />

m/z LC-MS

429.2 (M+1).

m/z LC-MS

462.2 (M+1).

m/z LC-MS

454.1 (M+1).

m/z LC-MS

420.2 (M+1).

m/z LC-MS

420.2 (M+1).

m/z LC-MS

420.2 (M+1).

m/z LC-MS

454.2 (M+1).

Data

physical

MS (m/z)

Compound

Number

Structure

<img file="ECSP088860A_D0026.tif" />

m/z LC-MS

488.1 (M+1).

m/z LC-MS

400.2 (M+1).

m/z LC-MS

414.2 (M+1).

LC-MS m/z 442.2 (M+1).

m/z LC-MS

428.2 (M+1).

<img file="ECSP088860A_D0027.tif" />

LC-MS m/z 442.2 (M+1).

<img file="ECSP088860A_D0028.tif" />

m/z LC-MS

428.2 (M+1).

Structure

<img file="ECSP088860A_D0029.tif" />

MS Physical Data (m/z)

m/z LC-MS

414.2 (M+1).

m/z LC-MS

414.2 (M+1).

m/z LC-MS

454.2 (M+1).

<img file="ECSP088860A_D0030.tif" />

m/z LC-MS

454.2 (M+1).

F

<img file="ECSP088860A_D0031.tif" />

m/z LC-MS

522.2 (M+1).

<img file="ECSP088860A_D0032.tif" />

m/z LC-MS

444.2 (M+1).

m/z LC-MS

430.2 (M+1).

m/z LC-MS

446.2 (M+1).

Data

physical

MS (m/z)

Compound

Number

Structure

<img file="ECSP088860A_D0033.tif" />

m/z LC-MS

470.2 (M+1).

m/z LC-MS

470.2 (M+1).

m/z LC-MS

373.2 (M+1).

m/z LC-MS

403.2 (M+1).

m/z LC-MS

403.2 (M+1).

<img file="ECSP088860A_D0034.tif" />

m/z LC-MS

460.2 (M+1).

^/NA7_

zhjrOO

m/z LC-MS

416.2 (M+1).

Data

physical

MS (m/z)

Compound

Number

Structure

<img file="ECSP088860A_D0035.tif" />

m/z LC-MS

391.2 (M+1).

<img file="ECSP088860A_D0036.tif" />

LC-MS m/z 391.2 (M+1).

<img file="ECSP088860A_D0037.tif" />

m/z LC-MS

391.2 (M+1).

<img file="ECSP088860A_D0038.tif" />

m/z LC-MS

425.2 (M+1).

m/z LC-MS

471.2 (M+1).

m/z LC-MS

398.2 (M+1).

m/z LC-MS

398.2 (M+1).

Data

physical

MS (m/z)

Compound

Number

Structure

<img file="ECSP088860A_D0039.tif" />

m/z LC-MS

413.2 (M+1).

m/z LC-MS

411.2 (M+1).

m/z LC-MS

413.2 (M+1).

m/z LC-MS

397.2 (M+1).

m/z LC-MS

399.2 (M+1).

m/z LC-MS

412.2 (M+1).

m/z LC-MS

398.2 (M+1).

Data

physical

MS (m/z)

Compound

Number

Structure

<img file="ECSP088860A_D0040.tif" />

m/z LC-MS

416.2 (M+1).

m/z LC-MS

432.1 (M+1).

m/z LC-MS

476.1 (M+1).

m/z LC-MS

412.2 (M+1).

m/z LC-MS

466.2 (M+1).

m/z LC-MS

385.2 (M+1).

m/z LC-MS

389.1 (M+1).

Data

physical

MS (m/z)

Structure

<img file="ECSP088860A_D0041.tif" />

m/z LC-MS

419.2 (M+1).

<img file="ECSP088860A_D0042.tif" />

m/z LC-MS

487.2 (M+1).

<img file="ECSP088860A_D0043.tif" />

m/z LC-MS

460.2 (M+1).

<img file="ECSP088860A_D0044.tif" />

m/z LC-MS

446.1 (M+1).

m/z LC-MS

427.2 (M+1).

<img file="ECSP088860A_D0045.tif" />

m/z LC-MS

427.2 (M+1)

m/z LC-MS

411.2 (M+1)

Data

physical

MS (m/z)

Compound

Number

Structure

-EITHER

<img file="ECSP088860A_D0046.tif" />

m/z LC-MS

434.2 (M+1)

m/z LC-MS

444.3 (M+1)

m/z LC-MS

458.3 (M+1)

<img file="ECSP088860A_D0047.tif" />

m/z LC-MS

450.2 (M+1)

m/z LC-MS

430.2 (M+1)

<img file="ECSP088860A_D0048.tif" />

HN

m/z LC-MS

460.2 (M+1)

Data

physical

MS (m/z)

Compound

Number

Structure

<img file="ECSP088860A_D0049.tif" />

m/z LC-MS

464.1 (M+1)

m/z LC-MS

524.1 (M+1)

<img file="ECSP088860A_D0050.tif" />

m/z LC-MS

502.3 (M+1)

<img file="ECSP088860A_D0051.tif" />

m/z LC-MS

508.2 (M+1)

m/z LC-MS

427.2 (M+1)

<img file="ECSP088860A_D0052.tif" />

EITHER

m/z LC-MS

432.2 (M+1)

Data

physical

MS (m/z)

Compound

Number

Structure

<img file="ECSP088860A_D0053.tif" />

m/z LC-MS

470.2 (M+1)

<img file="ECSP088860A_D0054.tif" />

m/z LC-MS

486.2 (M+1)

<img file="ECSP088860A_D0055.tif" />

No.

h

EITHER

m/z LC-MS

413.2 (M+1)

m/z LC-MS

464.1 (M+1)

m/z LC-MS

514.1 (M+1)

m/z LC-MS

503.3 (M+1)

Data

physical

MS (m/z)

Compound

Number

Structure

103

104

<img file="ECSP088860A_D0056.tif" />

<img file="ECSP088860A_D0057.tif" />

<img file="ECSP088860A_D0058.tif" />

<img file="ECSP088860A_D0059.tif" />

m/z LC-MS

503.3 (M+1)

m/z LC-MS

562.3 (M+1)

m/z LC-MS

546.3 (M+1)

m/z LC-MS

600.3 (M+1)

m/z LC-MS

560.3 (M+1)

m/z LC-MS

560.3 (M+1)

105

Data

physical

MS (m/z)

Compound

Number

Structure

106

107

108

109

<img file="ECSP088860A_D0060.tif" />

<img file="ECSP088860A_D0061.tif" />

<img file="ECSP088860A_D0062.tif" />

<img file="ECSP088860A_D0063.tif" />

EITHER

m/z LC-MS

503.3 (M+1)

m/z LC-MS

546.2 (M+1)

m/z LC-MS

586.2 (M+1)

m/z LC-MS

545.3 (M+1)

Data

physical

MS (m/z)

Compound

Number

Structure

110

111

<img file="ECSP088860A_D0064.tif" />

m/z LC-MS

604.2 (M+1)

m/z LC-MS

538.2 (M+1)

112

<img file="ECSP088860A_D0065.tif" />

m/z LC-MS

554.2 (M+1)

<img file="ECSP088860A_D0066.tif" />

No.

h

m/z LC-MS

538.2 (M+1)

113

Data

physical

MS (m/z)

Compound

Number

Structure

114

115

116

<img file="ECSP088860A_D0067.tif" />

m/z LC-MS

480.1 (M+1)

m/z LC-MS

481.2 (M+1)

m/z LC-MS

447.2 (M+1)

117

<img file="ECSP088860A_D0068.tif" />

<img file="ECSP088860A_D0069.tif" />

m/z LC-MS

441.2 (M+1)

Data

physical

MS (m/z)

Compound

Number

Structure

118

119

120

121

122

<img file="ECSP088860A_D0070.tif" />

m/z LC-MS

506.2 (M+1)

m/z LC-MS

572.2 (M+1)

m/z LC-MS

522.2 (M+1)

m/z LC-MS

544.3 (M+1)

m/z LC-MS

544.3 (M+1)

m/z LC-MS

562.2 (M+1)

123

Data

physical

MS (m/z)

Compound

Number

Structure

124

125

126

127

128

NC

<img file="ECSP088860A_D0071.tif" />

m/z LC-MS

488.2 (M+1)

m/z LC-MS

489.2 (M+1)

m/z LC-MS

554.2 (M+1)

m/z LC-MS

513.2 (M+1)

m/z LC-MS

539.3 (M+1)

NC

<img file="ECSP088860A_D0072.tif" />

m/z LC-MS

489.2 (M+1)

129

Data

physical

MS (m/z)

Compound

Number

Structure

130

<img file="ECSP088860A_D0073.tif" />

m/z LC-MS

489.2 (M+1)

131

132

133

NC

<img file="ECSP088860A_D0074.tif" />

<img file="ECSP088860A_D0075.tif" />

m/z LC-MS

554.3 (M+1)

<img file="ECSP088860A_D0076.tif" />

<img file="ECSP088860A_D0077.tif" />

m/z LC-MS

513.2 (M+1)

m/z LC-MS

539.3 (M+1)

<img file="ECSP088860A_D0078.tif" />

m/z LC-MS

472.1 (M+1)

134

Data

physical

MS (m/z)

Compound

Number

Structure

135

<img file="ECSP088860A_D0079.tif" />

m/z LC-MS

447.1 (M+1)

136

<img file="ECSP088860A_D0080.tif" />

m/z LC-MS

413.1 (M+1)

137

<img file="ECSP088860A_D0081.tif" />

<img file="ECSP088860A_D0082.tif" />

m/z LC-MS

492.1 (M+1)

138

<img file="ECSP088860A_D0083.tif" />

m/z LC-MS

431.1 (M+1)

139

<img file="ECSP088860A_D0084.tif" />

m/z LC-MS

441.1 (M+1)

m/z LC-MS

428.2 (M+1)

140

Compound_η

Structure Number

MS Physical Data (m/z)

<img file="ECSP088860A_D0085.tif" />

m/z LC-MS

471.2 (M+1)

<img file="ECSP088860A_D0086.tif" />

m/z LC-MS

487.2 (M+1)

<img file="ECSP088860A_D0087.tif" />

m/z LC-MS

477.2 (M+1)

<img file="ECSP088860A_D0088.tif" />

m/z LC-MS

513.2 (M+1)

145

m/z LC-MS

473.2 (M+1)

Data

physical

MS (m/z)

Compound

Number

Structure

146

147

148

149

<img file="ECSP088860A_D0089.tif" />

<img file="ECSP088860A_D0090.tif" />

<img file="ECSP088860A_D0091.tif" />

<img file="ECSP088860A_D0092.tif" />

<img file="ECSP088860A_D0093.tif" />

m/z LC-MS

520.2 (M+1)

m/z LC-MS

445.2 (M+1)

m/z LC-MS

471.2 (M+1)

m/z LC-MS

547.2 (M+1)

m/z LC-MS

562.2 (M+1)

150

Data

physical

MS (m/z)

Compound

Number

Structure

151

152

153

<img file="ECSP088860A_D0094.tif" />

<img file="ECSP088860A_D0095.tif" />

m/z LC-MS

547.2 (M+1)

m/z LC-MS

484.2 (M+1)

m/z LC-MS

486.2 (M+1)

Compounds of the present invention are tested for their ability to inhibit the Hedgehog signaling pathway.

Gli-Luc Reporter Assay for Hh Pathway Inhibition

Mouse TM3 cells (obtained from American Type Culture Collection, ATCC, Manassas, VA) are grown in DMEM/F12 medium (Gibco/lnvitrogen, Carlsbad, CA) supplemented with 5 percent heat-inactivated horse serum. and 2.5 percent fetal bovine serum (Gibco/lnvitrogen, Carlsbad, CA), penicillin 50 units/ml, and streptomycin 50 micrograms/ml (Gibco/lnvitrogen, Carlsbad, CA) at 37⁹C, with CO₂ at 5 percent in an air atmosphere. TM3 cells are transfected with the reporter plasmid pTA-8xGli-Luc. A stably transfected clone designated as TMHh-12 was selected. The TMHh-12 clone showed a good response to the stimulus of

Shh-N. In order to evaluate the CI₅₀For the antagonists, 8,000 TMHh-12 cells were applied in each well of 384-well plates with 50 percent DMEM/F12 medium supplemented with 2 percent fetal bovine serum. After 12 hours, the Hh pathway is activated by the addition of recombinant mouse Shh protein (expressed in E. coli, 8 micrograms/ml), or by the addition of Smo agonists. Test compounds are added to the plates at different concentrations. After 48 hours, firefly luciferase activities are assayed with the Bright-Glo Luciferase Assay System.^MR (Promega, Madison, Wl). The HF₅₀ it is measured when the effect of the compound reduces the luminescence signal by 50 percent. The toxicity of these compounds in TM3 cells is assessed using the CelITiter Glo assays, or by the TM3-Luc cell line (a TM3 cell stably transfected with a constitutive luciferase expression vector).

Compounds of Formula I preferably have an EC₅₀ less than 500 nM, more preferably less than 200 nM.

Cytotoxicity Assay

A cytotoxicity assay is performed to compare the effects of a compound of the invention on medulloblastoma cells (Daoy cells), basal cell carcinoma cells (TE354.T cells), and control cells (human fibroblasts). normal), according to the following procedure:

Daoy cells (medulloblastoma cell line) are purchased from ATCC, and cultured in minimal essential medium (Eagle) with 2 mM L-glutamine, and Earle's BSS adjusted to contain 1.5 grams/liter sodium bicarbonate, amino acids 0.1 mM nonessentials, and 1.0 mM sodium pyruvate, and 10 percent fetal bovine serum, at 37⁹C, with CO₂ at 5 percent in an air atmosphere.

TE354.T cells (from ATCC) are grown in Dulbecco's modified Eagle's medium, with 4 mM L-glutamine, and 10 percent fetal bovine serum.

Normal human dermal fibroblast cells (Clonetics) are grown in Fibroblast Growth Medium (Clonetics).

Each of the above cell lines is seeded independently in 96-well plates, and grown to a density of 5,000 to 10,000 cells/well. A compound of the invention is added, in different concentrations, to the cell cultures. After 2 days, cell viability is assessed with the Cell Titer-Glo Luminescent Cell Viability Assay Kit (Promega), following the manufacturer's protocol. Cell viability is directly measured by luminescent signaling, and EC₅₀s are measured when the signal is inhibited by 50 percent.

Compounds of Formula I preferably have an EC₅₀ less than 500 nM, more preferably less than 200 nM.

It is understood that the Examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to persons skilled in the art, and are to be included within the spirit and scope of this application, and within the scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference for all purposes.