Compounds and compositions as hedgehog pathway modulators

Background of the invention

During embryonic development, the hedgehog signaling pathway is important for many processes such as control of cell proliferation, cell differentiation and cell pattern formation. The abnormal activity of the hedgehog signaling pathway may be, for example, the result of increased activation, but may have pathological consequences. Accordingly, activation of the hedgehog signaling pathway in adult tissues can cause specific types of cancer, in particular, but not limited to, brain, muscle and skin cancer, prostate, spinal cord, pancreatic adenoma and small cell lung cancer. Increased activation of the hedgehog signaling pathway affects the pathological picture and / or symptoms of many diseases. Accordingly, molecules that modulate the activity of the hedgehog signaling pathway are useful as therapeutic agents in the treatment of such diseases.

Summary of the Invention

In one aspect, the present invention provides a compound of Formula I for use in the treatment of cancer by modulating the hedgehog signaling pathway:

<img file="PL2363393T3_D0001.tif" />

wherein:

Y1 and Y2 are independently selected from N and CH;

R1 is selected from cyano, C1-6alkyl substituted with C1-6 halogen, alkyl, C1-6alkoxy, halogen substituted C1-6alkoxy, dimethylamino, C1-6alkylsulfanyl and C3-8heterocycloalkyl optionally substituted with up to 2 C1-6alkyl radicals;

-2- EP 2363393

R2 and R5 are independently selected from hydrogen, cyano, halogen, C1-6alkyl substituted with C1-6alkyl, C1-6alkoxy substituted with C1-6alkoxy and dimethylamino;

R3 and R4 are independently selected from hydrogen, halogen, cyano, C1-6alkyl substituted with C1-6alkyl, C1-6alkoxy and halogen substituted C1-6alkoxy;

R6 and R7 are independently selected from hydrogen, methyl, chlorine, fluorine, bromine, trifluoromethyl and methoxy; provided that the R6 and R7 groups are not both hydrogen; and

R8 is selected from hydrogen, fluoro, chloro, methyl and trifluoromethyl; and

R9 is selected from -S (O) 2R11, -C (O) R11, -NR12aR12b and -R11; where R11 is selected from thiomorpholine, sulfonomorpholine, sulfanomorpholine, morpholine, cyclohexyl, phenyl, azepan-1-yl, 2-oxopiperazin-1-yl, 1,4-oxazepan-4-yl, piperidin-1-yl, tetrahydro-2H- pyran-4-yl, piperidin-3-yl, piperazinyl, pyrrolidinyl and

1,4-diazepan-1-yl; and R12a and R12b are independently selected from isobutyl, hydroxy-ethyl, wherein said thiomorpholine, sulfonomorpholine, sulfanomorpholine, morpholine, cyclohexyl, phenyl, azepan-1-yl, 2-oxopiperazin-1-yl, 1,4-oxazepan-4-yl groups , piperidin-1-yl, tetrahydro-2H-pyran-4-yl, piperidin-3-yl, piperazinyl, pyrrolidinyl and 1,4-diazepan-1-yl groups R9 may be optionally substituted with 1 to 3 radicals independently selected from methyl , ethyl, methoxy, benzyl, thienyl-methyl and pyridinyl-methyl, benzo [d] [1,3] dioxol-6-yl and 2,3-dihydrobenzo [b] [1,4] dioxin-7-yl, wherein said phenyl or benzyl substituent group R9 is optionally substituted with 1 to 3 radicals independently selected from methoxy, ethoxy, methylpiperazinyl, methyl, trifluoromethoxy, chlorine, fluorine and trifluoromethyl;

or a pharmaceutically acceptable salt or stereoisomer thereof;

wherein said cancer is selected from pancreatic cancer, prostate cancer, breast cancer, medulloblastoma, basal cell skin cancer and small cell lung cancer.

Also described is a pharmaceutical composition comprising a compound of Formula I or a Peroxide derivative thereof, individual isomers and mixtures of isomers; or a pharmaceutically acceptable salt thereof, in a mixture with one or more suitable excipients.

In another aspect, there is also disclosed a method of using a compound of Formula I for the manufacture of a medicament for treating a disease in an animal in which activation of the hedgehog signaling pathway affects the pathological picture and / or symptoms of the disease as claimed.

Definitions "Alkyl" as a group and as a structural element of other groups, for example alkyl and alkoxy substituted with halogen, may be straight or branched. C 1-4 alkoxy includes methoxy, ethoxy and the like. Halo-substituted alkyl includes trifluoromethyl, pentafluoroethyl and the like.

"Aryl" means a monocyclic or fused bicyclic aromatic ring system containing from six to ten ring carbon atoms. For example, aryl may be phenyl or XXX, preferably phenyl. "Arylene" means a divalent radical derived from an aryl group.

EP 2363393 "Heteroaryl" has the designation specified above for aryl where one or more ring atoms are heteroatoms. For example, C5-10heteroaryl is at least 5 membered, as indicated by the number of carbon atoms given, but said carbon atoms can be replaced by a heteroatom. Accordingly, C5-10heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, benzofuranyl, benzopyranyl, benzothiopyranyl, benzo [1,3] dioxol, imidazolyl, benzimidazolyl, pyrimidinyl, furanyl, oxazolyl, trisazolyl, pyrazolyl, thienyl, etc.

"Cycloalkyl" means a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring system containing the number of ring atoms given. For example, C3-10 cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

"Heterocycloalkyl" means cycloalkyl as defined herein, in which one or more ring carbon atoms have been replaced by a group selected from -O-, -N =, -NR-, -C (O) -, -S-, -S (O ) - or -S (O) 2-, wherein R is hydrogen, C 1-4 alkyl or a nitrogen protecting group. For example, C 3-8 heterocycloalkyl as used in this application describes compounds of the invention including morpholino, pyrrolidinyl, pyrrolidinyl-2-one, piperazinyl, piperidinyl, piperidinyl, 1,4-dioxa-8-aza-spiro [4.5] dec-8 -yl, thiomorpholine, sulfanomorpholine, sulfonomorpholine, etc.

"Halo" (or halo) preferably means chloro or fluoro, but can also be bromo or iodo.

"Acquisition of the hedgehog function" refers to an incorrect modification or mutation of the Ptc gene, hedgehog gene or smoothened gene or reduction (loss) of the expression level of such gene, resulting in a phenotype that resembles contact of the cell with the hedgehog protein, for example incorrect pathway activation hedgehog. The acquisition of function may include a loss of the ability of the Ptc gene product to regulate the expression of Gli genes, e.g., Gli1, Gli2, and Gli3. The term "acquisition of hedgehog function" is also used herein to refer to any similar phenotype of cells (e.g., excessive proliferation) that occurs due to changes anywhere in the hedgehog signal transduction pathway, in particular, but not exclusively hedgehog modification or mutation. For example, tumor cells with an extremely high degree of proliferation due to the activation of the hedgehog signaling pathway would have the "hedgehog acquisition" phenotype even if the hedgehog gene was not mutated in that cell.

"Loss of patched" refers to an incorrect modification or mutation of a Ptc gene or a reduced level of gene expression, resulting in a phenotype that resembles contacting a cell with a hedgehog protein, for example, incorrect activation of the hedgehog pathway. Loss of function may include loss of the ability of the Ptc gene product to regulate expression of Gli genes, e.g., Gli1, Gli2, and Gli3.

"Acquisition of Gli function" refers to an abnormal modification or mutation of the Gli gene or an increased level of gene expression, resulting in a phenotype that resembles contacting a cell with a hedgehog protein, for example, incorrect activation of the hedgehog pathway.

"Acquiring Smoothened" refers to an incorrect modification or mutation of the Smo gene or an increased level of gene expression, resulting in a phenotype,

Which resembles contacting a cell with a hedgehog protein, for example incorrect activation of the hedgehog pathway.

"Treat", "treatment" and "therapy" refer to a method of alleviating or reducing the disease and / or its accompanying symptoms.

The present invention relates to the finding that signal transduction pathways regulated by hedgehog, patched (Ptc), gli and / or smoothened can be modulated by compounds of Formula I.

Description of preferred projects

In one embodiment, with respect to compounds of Formula I, Y1 and Y2 are independently selected from N and CH;

R6 and R7 are independently selected from hydrogen, methyl, chlorine, fluorine, bromine, trifluoromethyl and methoxy; provided that the R6 and R7 groups are not both hydrogen; R8 is selected from hydrogen, fluorine, chlorine, methyl and trifluoromethyl.

In another embodiment, R1 is selected from the cyano, methyl, ethyl, t-butyl, propyl, isobutyl, isopropyl, isopropyloxy, butoxyl, methoxy, dimethylamino, ethoxy, methylsulfanyl, trifluoromethyl, trifluoromethoxy and piperazinyl groups, optionally substituted with a maximum of 2 methyl radicals; R2 and R5 are independently selected from hydrogen, chlorine, fluorine, cyano, methyl, trifloromethyl, isopropyloxy, methoxy, ethoxy, trifluoromethoxy and dimethylamino; R3 and R4 are independently selected from hydrogen, chlorine, methyl, methoxy and cyano.

In another embodiment, R9 is selected from -S (O) 2R11, -C (O) R11, -NR12aR12b and -R11; where R11 is selected from thiomorpholine, sulfonomorpholine, sulfanomorpholine, morpholine, cyclohexyl, phenyl, azepan-1-yl, 2-oxopiperazin-1-yl, 1,4-oxazepan-4-yl, piperidin-1-yl, tetrahydro-2H- pyran-4-yl, piperidin-3-yl, piperazinyl, pyrrolidinyl and 1,4-diazepan-1-yl; and R12a and R12b are independently selected from isobutyl and hydroxyethyl; wherein said thiomorpholine, sulfonomorpholine, sulfanomorpholine, morpholine, cyclohexyl, phenyl, azepan-1-yl, 2-oxopiperazin-1-yl, 1,4-oxazepan-4-yl, piperidin-1-yl, tetrahydro-2H-pyran-4 groups -yl, piperidin-3-yl, piperazinyl, pyrrolidinyl and 1,4-diazepan-1-yl groups R9 may be optionally substituted with 1 to 3 radicals independently selected from methyl, ethyl, methoxy, benzyl, thienylmethyl and pyridinylmethyl, benzo [d] [1,3] dioxol-6-yl and 2,3-dihydrobenzo [b] [1,4] dioxin-7-yl, wherein said phenyl or benzyl substituent of the R9 group is optionally substituted with 1 to 3 radicals independently selected from methoxy, ethoxy, methyl piperazinyl, methyl, trifluoromethoxy, chloro, fluoro and trifluoromethyl.

Preferred compounds of Formula I are selected from the following: 4'-cyano-6-methyl-biphenyl-3-carboxylic acid [4- (morpholine-4-sulfonyl) phenyl] -amide, [6- (2,6-dimethylmorpholin-4-yl) -pyridin-3-yl] - 4'-cyano-6-methyl-biphenyl-3-carboxylic acid amide (6azepan-1-yl-pyridin-3-yl) -amide 4'-cyano-2-methyl-biphenyl-3-carboxylic acid, (6azepan- 4'-methoxy-2-methyl-biphenyl-3-carboxylic acid 1-yl-pyridin-3-yl) -amide, 4'-methoxy-2-methyl-biphenyl-3-carboxylic acid (4-cyclohexylphenyl) -amide, 4'-methoxy-2-methyl-biphenyl-3-carboxylic acid [6- (2-methylmorpholin-4-yl) amide, 4'-dimethylamino-2-methyl-biphenyl-3-carboxylic acid (4-cyclohexylphenyl) -amide , 4'-dimethylamino-2-methyl-biphenyl-3-carboxylic acid, (4-morpholin-4-yl-phenyl) -amide, (6-5-EP 2363393 [1,4] oxazepan-4-yl-pyridin-3- 6-chloro-4'-dimethylaminobiphenyl-3-carboxylic acid, (6-morpholin-4-yl-pyridin-3-yl) -amide, 6-chloro-4-dimethylaminobiphenyl-3-carboxylic acid, 6-chloro-4-dimethylaminobiphenyl-3-carboxylic acid (6-azepan-1-yl-pyridin-3-yl) -amide, [6- (2-methyl-morpholin-4-yl) -pyridin-3-yl ] 6-chloro-4'-methoxy-biphenyl-3-carboxylic acid, 6-chloro-4'-methoxy- (6- [1,4] oxazepan-4-yl-pyridin-3-yl) amide biphenyl-3-carboxylic acid, 6-chloro-4'-methoxy-biphenyl-3-carboxylic acid (6-azepan-1-yl-pyridin-3-yl) amide, (6-morpholin-4-yl-pyridin-3 6-chloro-4'-methoxy-biphenyl-3-carboxylic acid -yl) amide, 4'-methoxy-6-methyl-biphenyl-3-carboxylic acid (6-morpholin-4-yl-pyridin-3-yl) amide, (6- [1,4] oxazepan-4-yl-pyridin-3-yl) 4'-methoxy-6-methyl-biphenyl-3-carboxylic acid, [6- (2-methyl-morpholin-4-yl) pyridin-3-yl] -amide of 4'-methoxy-2-methyl biphenyl-3-carboxylic acid, [6- (2-methylmorpholin-4-yl) -pyridin-3-yl] -amide, 4'-dimethylamino-6-methyl-biphenyl-3-carboxylic acid, (6- [1,4] oxazepane 4'-dimethylamino-6-methyl-biphenyl-3-carboxylic acid -4-yl-pyridin-3-yl) -amide, 4-dimethylamino-6-methyl-biphenyl-3-carboxylic acid (6-morpholin-4-yl-pyridin-3-yl) -amide, (6-azepan-1-yl-pyridin-3-yl) -amide 4'-methoxy-6-methyl-biphenyl-3-carboxylic acid, (4-ethoxy-6-methyl-biphenyl-3-carboxylic acid (6-azepan-1-yl-pyridin-3-yl) -amide, (6- 6-methyl-4'-methylsulfanyl-biphenyl-3-carboxylic acid azepin-1-yl-pyridin-3-yl) -amide (6-azepan-1-yl-pyridin-3-yl) -amide of 4'-dimethylamino acid -6-methyl-biphenyl-3-carboxylic acid 4'-chloro-6-methyl-4'-trifluoromethyl-biphenyl-3-carboxylic acid (6-azepan-1-yl-pyridin-3-yl) -amide, (6-azepan-1-yl-pyridin-3-yl) ) 6,4'-dimethyl-biphenyl-3-carboxylic acidamide, (6-azepin-1-pyridin-1-yl) -amide of 4'-ethyl-6-methyl-biphenyl-3-carboxylic acid, (6-azepan-1 4'-tert-butyl-6-methyl-biphenyl-3-carboxylic acid -yl-pyridin-3-yl) -amide (6-methyl-4-azepan-1-yl-pyridin-3-yl) -amide '-propyl-biphenyl-3-carboxylic acid, 4'-isobutyl-6-methyl-biphenyl-3-carboxylic acid (6-azepan-1-yl-pyridin-3-yl) -amide, acid (4-azepan-1-yl-pyridin-3-yl) -amide '-isopropyl-6-methyl-biphenyl-3-carboxylic acid, (6-azepan-1-yl-pyridin-3-yl) -amide 6-methyl-4'-trifluoromethyl-biphenyl-3-carboxylic acid, (6-azepan-1- 6-methyl-4'-trifluoromethoxy-biphenyl-3-carboxylic acid, 6-morpholin-4-yl-phenyl) -amide of 4'-methoxy-6-methyl-biphenyl-3-yl-pyridin-3-yl) -amide carboxylic acid, 6-methyl-4 '- (4-methyl-piperazin-1-yl) -biphenyl-3-carboxylic acid (4-morpholin-4-yl-phenyl) -amide, (6- [1,4] oxazepan-4-yl 4'-cyano-6-methyl-biphenyl-3-carboxylic acid-pyridin-3-yl) -amide, 4'-cyano-6-methyl- (6-azepan-1-yl-pyridin-3-yl) -amide biphenyl-3-carboxylic acid, [6- (2-methylmorpholin-4-yl) -pyridin-3-yl] -amide, 4'-cyano-6-methyl-biphenyl-3-carboxylic acid, (3,4,5,6-tetrahydro- 4'-cyano-6-methyl-biphenyl-3-carboxylic acid 2H- [1,2 '] bipyridinyl-5'-yl) -amide, 4'-cyano-6-methyl-biphenyl-3-carboxylic acid (6-morpholin-4-yl-pyridin-3-yl) -amide, [6- (4-methyl-piperazin-1-yl) -pyridin-3 4'-cyano-6-methyl-biphenyl-3-carboxylic acid -yl] -amide, (4-cyano-6-methyl-biphenyl-3-carboxylic acid (6-morpholin-4-yl-phenyl) -amide, ( 4'-cyano-6-methyl-biphenyl-3-carboxylic acid 4-cyclohexylphenyl) -amide, 4'-cyano-6-methyl-biphenyl-3-carboxylic acid biphenyl-4-amide, 4'-cyano-6-methyl-biphenyl-3-carboxylic acid (4'-methoxy-biphenyl-4-6-yl) -amide; [4- (4-benzyl-piperazin-1-yl) -phenyl] -4'-cyano-6-methyl-biphenyl-3-carboxylic acid, [4- (piperidine-1-sulfonyl) phenyl] -amide of 4'-cyano-6-methyl-biphenyl-3-carboxylic acid, [4 4'-cyano-6-methyl-biphenyl-3-carboxylic acid (pyrrolidine-1-sulfonyl) phenyl] -amide 4'-cyano-6 (6-azepan-1-yl-pyridin-3-yl) -amide methoxy-biphenyl-3-carboxylic acid 4'-cyano-2-methoxy-biphenyl-3-carboxylic acid (6-azepan-1-yl-pyridin-3-yl) -amide 4'- (6-azepan-1-yl-pyridin-3-yl) -amide 3'-fluoro-4'-methoxy-6-methyl-biphenyl-3-carboxylic acid cyano-2-methyl-biphenyl-3-carboxylic acid, (6-azepan-1-yl-pyridin-3-yl) amide, ( 4'-isopropoxy-6-methyl-biphenyl-3-carboxylic acid 6-azepan-1-yl-pyridin-3-yl) -amide, 4'-butoxy acid (6-azepan-1-yl-pyridin-3-yl) -amide -6-methyl-biphenyl-3-carboxylic acid 3'-chloro-4'-methoxy-6-methyl-biphenyl-3-carboxylic acid (6-azepan-1-yl-pyridin-3-yl) -amide, (6-azepan-1-yl-pyridin-3-yl) - 4'-methoxy-6,3'-dimethyl-biphenyl-3-carboxylic acid amide, 4'-cyano-2-methyl-biphenyl-3-carboxylic acid [4- (piperidine-1-sulfonyl) -phenyl] -amide, [ 4'-cyano-6-fluorobiphenyl-3-carboxylic acid 4- (piperidine-1-sulfonyl) -phenyl] -amide, 6-bromo-4'-cyano-biphenyl acid [4- (piperidine-1-sulfonyl) -phenyl] -amide -3-carboxylic acid, 4'-cyano-6-methyl-biphenyl-3-carboxylic acid [6- (4-benzyl [1,4] diazepan-1-yl) -pyridin-3-yl] -amide, [6- {4-thiophene -3-cyano-6-methyl-biphenyl-3-carboxylic acid, [6- (2,6-dimethyl) -3-yl-methyl- [1,4] diazepan-1-yl) -pyridin-3-yl] -amide -morpholin-4-yl) -pyridin-3-yl] -amide, 4'-cyano-2-methyl-biphenyl-3-carboxylic acid, [6- (2,6-dimethyl-morpholin-4-yl) -pyridine- 4'-methoxy-2-methyl-biphenyl-3-carboxylic acid 3-amide, 2'-methyl-4-trifluoromethyl-biphenyl-3-carboxylic acid [6- (2,6-dimethyl-morpholin-4-yl) pyridin-3-yl] -amide, [6- (2-methyl-morpholin) 4'-cyano-2-methyl-biphenyl-3-carboxylic acid, 4-yl) -pyridin-3-yl] -amide 4'-cyano-2- [4- (piperidine-1-sulfonyl) -phenyl] -amide 4'-cyano-6-trifluoromethyl-biphenyl-3-carboxylic acid fluorobiphenyl-3-carboxylic acid, [4- (piperidine-1-sulfonyl) -phenyl] -amide, 4-cyano-6-methyl-biphenyl-3-carboxylic acid [6- (4-pyridin-4-yl-methyl- [1,4] diazepan-1-yl) -pyridin-3-yl] -amide, [ 4'-cyano-6-methyl-biphenyl-3-carboxylic acid 6- (4-pyridin-3-yl-methyl- [1,4] diazepan-1-yl) -pyridin-3-yl] -amide, {6- 4'-cyano-6-methyl-biphenyl-3-carboxylic acid [4- (2,6-dimethoxy-benzyl) - [1,4] diazepan-1-yl] -pyridin-3-yl} -amide, {6- [4 - 4'-cyano-6-methyl-biphenyl-3-carboxylic acid - (2-ethoxy-benzyl) - [1,4] diazepan-1-yl] pyridin-3-yl} -amide, 4'-cyano-6 (4- {4- [2- (4-methyl-piperazin-1-yl) -benzyl] [1,4] diazepan-1-yl} -pyridin-3-yl) -amide -methyl-biphenyl-3-carboxylic acid, {6- [4- (4-methoxy-2,3-dimethyl-benzyl) - [1,4] diazepan-1-yl] -pyridin-3-yl} -amide 4'-cyano-6-methyl-biphenyl-3-carboxyl, {6- [4- (2,3-dihydro-benzo [1,4] dioxin-6-ylmethyl) - [1,4] diazepan-1-yl ] pyridin-3-yl} -amide 4'-cyano-6-methyl-biphenyl-3-carboxylic acid, 4'-cyano-6-methyl-biphenyl-3-carboxylic acid [6- (4-pyridin-2-yl-methyl- [1,4] diazepan-1-yl) -pyridin-3-yl] -amide, [ 4'-cyano-6-methyl-biphenyl-3 - 6- (4-benzo [1,3] dioxo-4-yl-methyl- [1,4] diazepan-1-yl) -pyridin-3-yl] -amide -carboxylic, {6- [4- (2-trifluoromethoxy-benzyl) - [1,4] diazepan-1-yl] -pyridin-3-yl} -amide, 4'-cyano-6-methyl-biphenyl-3-carboxylic acid, 4'-cyano-6-methyl-biphenyl-3- acid {6- [4- (2-chloro-5-trifluoromethyl-benzyl) - [1,4] diazepan-1-yl] -pyridin-3-yl} -amide carboxylic acid, {6- [4- (2,3-difluoro-benzyl) [1,4] diazepan-1-yl] -pyridin-3-yl} -amide 4'-cyano-6-methyl-biphenyl-3 acid -carboxylic acid, {6- [4- (2-chloro-4-fluoro-benzyl) - [1,4] diazepan-1-yl] -pyridin-3-yl} -amide, 4'-cyano-6-methyl-biphenyl acid -3-carboxylic acid, 4'-cyano-6-methyl-biphenyl-3-carboxylic acid {6- (4- (2,6-difluoro-benzyl) - [1,4] diazepan-1-yl] -pyridin-3-yl} -amide, [4- (piperidine-1-sulfonyl) -7-2-chloro-4'-cyano-biphenyl-3-carboxylic acid, [6- (2,6-dimethyl-morpholin-4-yl) - 4'-cyano-6-trifluoromethyl-biphenyl-3-carboxylic acid, pyridin-3-yl] -amide [2- (2,6-dimethyl-morpholin-4-yl) -pyridin-3-yl] -amide 2- chloro-4'-cyano-biphenyl-3-carboxylic acid, 4'-cyano-6-ethyl-biphenyl-3-carboxylic acid [6- (2,6-dimethyl-morpholin-4-yl) -pyridin-3-yl] -amide, {6- [4- (3-fluoro- benzyl) -piperazin-1-yl] -pyridin-3-yl} -amide 4'-cyano-6-methyl-biphenyl-3-carboxylic acid, {6- [4- (2-trifluoromethoxy-benzyl) -piperazin 4'-cyano-6-methyl-biphenyl-3-carboxylic acid, 1-yl] -pyridin-3-yl} -amide, {6-4- (3-chlorobenzyl) -piperazin-1-yl] -pyridin-3-yl } 4'-cyano-6-methyl-biphenyl-3-carboxylic acid amide, [4-Cyano-6-methyl-biphenyl-3-carboxylic acid [6- (4-benzyl-piperazin-1-yl) -pyridin-3-yl] -amide, [6- (4-pyridin-3-yl-methyl) -piperazin-1-yl) -pyridin-3-yl] -amide, 4'-cyano-6-methyl-biphenyl-3-carboxylic acid, [6- (4-pyridin-4-yl-methyl-piperazin-1-yl) ) 4'-cyano-6-methyl-biphenyl-3-carboxylic acid, pyridin-3-yl] -amide [6- {4-pyridin-2-yl-methyl-piperazin-1-yl) -pyridin-3-yl ] 4'-cyano-6-methyl-biphenyl-3-carboxylic acid amide, (R) -2-methyl-N- (6- (2-methylmorpholino) pyridin-3-yl) -4 '- (trifluoromethoxy) biphenyl-3-carboxamide, 4'-cyano-2-methyl-N- (6-sulfonylmorpholinopyridine -3-yl) biphenyl-3-carboxamide, (S) -4'-cyano-2-methyl-N- (6- (2-methylmorpholino) pyridin-3-yl) biphenyl-3-carboxamide, (R) - 6-chloro-N- (6- (2-methylmorpholino) pyridin-3-yl) -4 '- (trifluoromethoxy) biphenyl-3-carboxamide,

4'-cyano-N- (6- (diisobutylamino) pyridin-3-yl) -2-methyl-biphenyl-3-carboxamide, 4'-cyano-N- (2- (2S, 6R) 2,6-dimethylmorpholine) pyrimidin-5-yl) -2-methyl-biphenyl-3-carboxamide, N (2 - ((2S, 6R) -2,6-dimethylmorpholino) pyrimidin-5-yl) -2-methyl-4 '- (trifluoromethyl ) biphenyl-3-carboxamide, N- (2 - ((2S, 6R) -2,6-dimethylmorpholino) pyrimidin-5-yl) -2-methyl-4 '(trifluromethoxy) biphenyl-3-carboxamide, N- (2- (bis (2-hydroxyethyl) amino) pyrimidin-5-yl) -2-methyl-4 '- (trifluoromethoxy) biphenyl-3-carboxamide, 2-methyl-N- (6- (tetrahydro-2H-pyran-4-yloxy) pyridin-3-yl) -4 '- (trifluoromethoxy) biphenyl-3-carboxamide, N- (5-chloro-6 - ((2S, 6R) -2,6dimetylomorfolino) pyridin-3-yl) -2-methyl-4 '- (trifluoromethoxy) biphenyl-3-carboxamide,

N- (6- (4-ethylpiperazine-1-carbonyl) pyridin-3-yl) -2-methyl-4 '(trifluoromethoxy) biphenyl-3-carboxamide, 2-methyl-N- (6- (2-oxopiperazine) 1-yl) pyridin-3-yl) 4 '- (trifluoromethoxy) biphenyl-3-carboxamide, 2-methyl-N- (6- (1- (pyridin-4-ylmethyl) piperidin-4-yl) pyridin-3 -yl) -4 '- (trifluoromethoxy) biphenyl-3-carboxamide, 2-methyl-N- (6- (2-oxo-4- (pyridin-4-ylmethyl) piperazin-1-yl) pyridin-3- yl) -4 '- (trifluoromethoxy) biphenyl-3karboksamid. 2-methyl-N- (6- (1- (pyridin-4-ylmethyl) piperidin-3-yl) pyridin-3-yl) -4 '(trifluoromethoxy) biphenyl-3-carboxamide, N- (6- (1-ethylpiperidin-3-yl) pyridin-3-yl) -2-methyl4 '- (trifluoromethoxy) biphenyl-3-carboxamide and N- (6 - ((2R, 6S) -2,6-dimethylmorpholine) pyridin-3- yl) -2-methyl-4 '- (trifluoromethoxy) biphenyl-3-carboxamide.

In this regard, it is specifically contemplated that compounds of Formula I that interfere with aspects of hedgehog, Ptc or smoothened signal transduction pathway activity will also be able to inhibit proliferation (or other biological effects) in normal cells and / or cells with a loss phenotype patched functions, hedgehog functions, smoothed functions, or Gli functions. Therefore, it is believed that these compounds may be useful for inhibiting hedgehog activity in normal cells that, for example, do not have the genetic mutation that activates the hedgehog pathway. According to the invention, these compounds are

EP 2363393 capable of inhibiting at least some biological activities of hedgehog proteins, preferably especially in target cells.

Thus, the present disclosure describes the use of compounds of Formula I that agonize Ptc inhibition of hedgehog signaling, for example by inhibiting the activation of smoothened elements or further elements of the signaling pathway in the regulation of repair and / or functioning of a wide range of cells, tissues and organs, including healthy cells , tissues and organs, as well as those with a phenotype of loss of Ptc function, acquisition of hedgehog function, acquisition of smoothened function or acquisition of Gli function. For example, the present disclosure describes a method of therapeutic and cosmetic use, for regulating nerve tissues, bone and cartilage production and repair, regulation of spermatogenesis, regulation of smooth muscles, regulation of lungs, liver and other organs arising from the primary intestine, regulation of hematopoietic functions, growth regulation hair and skin, etc. In addition, the methods of the invention can be carried out in cells provided in culture (in vitro) or cells in the animal body (in vivo).

The present invention describes a use that targets epithelial cells having a phenotype of loss of patched function, acquisition of smoothened function or acquisition of Gli function. For example, the method of the present invention may be used to treat or prevent basal cell carcinoma of the skin or other disorders associated with the hedgehog pathway.

In some aspects, a compound of Formula I may inhibit activation of the hedgehog pathway by binding to smoothened protein or proteins downstream of this pathway. In certain aspects, an antagonist of the invention can inhibit activation of the hedgehog pathway by binding to patched proteins.

In another preferred aspect, the method of the present invention may be used to treat medullary and other primary CNS neuroectodermal malignancies.

In another aspect, the present disclosure relates to pharmaceutical preparations containing as an active ingredient a hedgehog signaling modulator, such as a compound of Formula I, a Ptc agonist, smoothened antagonist, antagonist of proteins downstream of the hedgehog pathway as described herein, prepared in an amount sufficient to in vivo inhibition of proliferation or other biological consequences of loss of patched function, acquisition of hedgehog function, acquisition of the smoothened function or acquisition of the Gli function.

The treatment object of the present invention using a compound of Formula I, Ptc agonists, smoothened antagonists or protein antagonists downstream on the hedgehog pathway can be effective for both humans and animals. The animals to which the present invention applies include both domestic and farm animals, raised either as pets or for commercial purposes. Examples are dogs, cats, cattle, horses, sheep, pigs and goats.

Pharmacology and application

The present invention describes compounds for inhibiting activation of the hedgehog signaling pathway, e.g., inhibition of abnormal growth signals resulting from phenotypes such as loss of patched function, acquisition of hedgehog function, acquisition of smoothened function or acquisition of Gli function, including contacting cells with a compound of Formula I, in an amount sufficient to agonize normal Ptc activity, antagonize normal

Hedgehog activity, antagonizing smoothened activity or antagonizing Gli activity, e.g., reversing or controlling abnormal growth.

Members of the hedgehog family of signaling molecules mediate many important short and long range pattern creation processes during vertebrate development. Pattern creation is an activity that allows embryonic cells to form ordered spatial systems of differentiated tissues. The physical complexity of higher organisms arises during embryogenesis, through the interaction of intracellular connections and extracellular signaling. Inductive interactions are important for the process of creating embryonic patterns in the process of vertebrate development from the earliest definition of the organism's plan to creating organ system patterns, to the production of different types of cells during tissue differentiation. The effects of developmental cell interaction are varied: responding cells are transferred from one cell differentiation pathway to another by inducing cells that differ from uninduced and induced responding cell states (induction). Sometimes cells induce neighboring cells to differentiate by their own pattern (homeogenetic induction), and in other cases cells inhibit the process of differentiation of neighboring cells, which they undergo. Early cell interaction can be sequential, such that the initial induction of two cell types leads to progressive amplification of diversity. In addition, induction interactions occur not only in embryos, but in adult cells and can interact by establishing and maintaining morphogenetic patterns as well as inducing differentiation.

The vertebrate hedgehog family of genes includes three mammalian members known as Desert (Dhh), Sonic (Shh) and Indian (Ihh) hedgehog, all of which encode secreted proteins. These different hedgehog proteins consist of a signal peptide, a highly conserved N-terminal region and a C-terminal domain with greater diversity. Biochemical studies have shown that the autoproteolytic cleavage of the Hh precursor protein occurs through an internal thioester intermediate, which is then cleaved in a nucleophilic substitution reaction. It is likely that the nucleophile is a small lipophilic molecule that binds covalently to the C-terminus of the N-peptide, attaching it to the cell surface. Biological effects are essential. Attachment produces a high local concentration of N-terminal hedgehog peptide on the surface of hedgehog producing cells. This N-terminal peptide is both necessary and sufficient for short and long-range hedgehog signaling activity.

An inactive signal pathway occurs when the patched receptor (Ptc) of a transmembrane protein inhibits the activity of the smoothened (Smo) protein with seven transmembrane domains. The Gli transcription factor, the downstream element of Hh signaling, is protected from entry into the nucleus through interactions with cytoplasmic proteins, including Fused and Supressor of fused (Sufu). Consequently, transcriptional activation of target hedgehog genes is suppressed. Pathway activation is initiated by binding one of three mammalian ligands (Dhh, Shh or Ihh) to Ptc. Ligand binding induces reversal of Smo repression, thus activating a cascade that leads to translocation of the active form of the Gli transcription factor in the nucleus. Nuclear Gli activates expression of target genes, including Ptc and Gli itself.

EP 2363393

Elevated levels of hedgehog signaling are sufficient to initiate the formation of tumors and are required for tumor survival. These cancers include in particular but not limited to prostate cancer ("Hedgehog signalling 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. 2004 Oct 7; 431 (7009): 707-12; "Inhibition of prostate cancer proliferation by interference with SONIC HEDGEHOG-GLI1 signaling", Sanchez P, Hernandez AM, Stecca B, Kahler AJ, DeGueme AM, Barrett A, Beyna M, Datta MW, Datta S, Ruiz and Altaba A., Proc Natl Acad Sci USA. 2004 Aug 24; 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 Sep 1; 64 (17): 6071-4), spinal cord ("Medulloblastoma growth inhibition by hedgehog pathway blockade", Berman DM, Karhadkar SS, Hallahan AR, Pritchard JI, Eberhart CG, Watkins DN, Chen JK, Cooper MK, Taipale J, Olson JM, Beachy PA., Science. 2002 Aug 30; 297 (5586): 155961), "Identification 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, Rubin LL, Wang FY., Proc Natl Acad Sci USA. 2003 Apr 15: 100 (8): 4616-21; "Activating Smoothened mutations in sporadic basal-cell carcinoma", Xie J, Murone M, Luoh SM, Ryan A, Gu Q, Zhang C, Bonifas JM, Lam CW, Hynes M, Goddard A, Rosenthal A, Epstein EH Jr, de Sauvage FJ., Nature. 1998 Jan 1; 391 (6662): 90-2), pancreatic cancer ("Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis", Thayer SP, di Magliano MP, Heiser PW, Nielsen CM, Roberts DJ, Lauwers GY, Qi YP, Gysin S, Fernandez-del Castillo C, Yiarka V, Antoniu B, McMahon M, Warshaw AL, Hebrok M., Nature. 2003 Oct 23; 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. 2003 Oct 23; 425 (6960): 846-51) and small cell lung cancer ("Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer", Watkins DN, Berman DM, Burkholder SG, Wang B, Beachy PA, Baylin SB., Nature. 2003 Mar 20; 422 (6929): 313-7).

Accordingly, the present invention provides a method of preventing or treating any of the diseases or disorders described above in patients in need of such treatment, comprising administering to said patient a therapeutically effective amount (see "Pharmaceutical Administration and Compositions", infra) of a compound of Formula I or a pharmaceutically-active acceptable salt. For each of the above uses, the required dose will vary depending on the mode of administration, the condition being treated, and the effect desired.

Pharmaceutical administration and compositions:

In general, the compounds of the invention will be administered in therapeutically effective amounts by any of the conventional and acceptable methods known in the art, either alone or in combination with one or more pharmaceutical agents. The therapeutically effective amount may vary depending on the severity of the disease, the age and relative health of the patient, the effectiveness of the compound used, and other factors. In general, satisfactory results can be obtained systemically at daily doses from about 0.03 to 2.5 mg / kg body weight. The indicated daily dose in most mammals, e.g. humans, ranges from 0.5 mg to about 100 mg, with convenient administration, e.g. in divided doses up to four times a day or in sustained release form. suitable

Unit dosage forms for oral administration contain from about 1 to 50 mg of active ingredient.

The compounds of the invention may be administered as pharmaceutical compositions by any conventional route, in particular enterally, e.g. orally, e.g. in the form of tablets or capsules, or parenterally, e.g. in the form of solutions or suspensions, topically, e.g. in the form of liquids, gels, ointments or creams, or in nasal form or suppositories. Pharmaceutical compositions containing a compound of the invention in free form or in the form of a pharmaceutically acceptable salt in combination with at least one pharmaceutically acceptable carrier or diluent can be prepared in a conventional manner by mixing, granulating or coating. For example, oral compositions may be tablets or gelatin capsules containing the active ingredient together with: a) diluents, for example lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, b) lubricants, e.g. silica, talc, acid stearic acid, its magnesium or calcium salt and / or polyethylene glycol; in the case of tablets, also c) binding agents, for example magnesium aluminum silicate, starch paste, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose and / or polyvinylpyrrolidone; if necessary d) disintegrants, for example starch, agar, alginic acid or its sodium salt, or effervescent mixtures; and / or e) absorbents, dyes, aromas and sweeteners. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories can be prepared from fatty emulsions or suspensions. The compositions may be sterilized and / or may contain auxiliary agents such as preserving, stabilizing, wetting or emulsifying agents, solubilizing agents, salts for regulating the osmotic pressure and / or buffers. In addition, they may also contain other therapeutically valuable substances. Suitable transdermal preparations contain an effective amount of the compound of the present invention together with the carrier. The carrier may include absorbable pharmacologically acceptable solvents to facilitate passage through the skin of the host. For example, transdermal devices may consist of a bandage containing a suitable pad, a reservoir containing the compound optionally with carriers, optionally barriers to regulate the delivery of the compound through the skin from control and a predetermined speed over a prolonged period of time, and elements fixing the article to the skin. Percutaneous matrix preparations may also be used. Suitable formulations for topical application, e.g. to the skin and eyes, are preferred aqueous solutions, ointments, creams or gels well known in the art. They may contain solubilizing agents, stabilizers, tonicity enhancing agents, buffers and preservatives.

In general, the compounds of the invention may be administered in therapeutically effective amounts in combination with one or more therapeutic agents (pharmaceutical combinations). For example, a synergistic effect may occur with immunomodulators or anti-inflammatory substances or other therapeutic anti-cancer drugs. Where the compounds of the invention are administered in combination with other therapies, the dosage of the compounds administered in combination will of course depend on the type of drug used together, the particular drug used, the condition being treated, etc.

The invention also relates to pharmaceutical combinations, for example a kit comprising a) a first agent which is a compound according to the invention in the form

EP 2363393 as claimed herein, in free form or in the form of a pharmaceutically acceptable salt, and b) at least one agent used together. The kit may contain instructions for administration.

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

The term "pharmaceutical combination" as used herein means a product resulting from the mixing or combining of more than one active substance and includes both fixed and transient combinations of active ingredients. The term "fixed combination" means that the active ingredients, for example the compound of Formula I and the second drug, are administered to the patient simultaneously as a single unit or dose. The term "unsteady combination" means that the active ingredients, for example the compound of Formula I and the second drug, are administered to the patient as separate units simultaneously, simultaneously or sequentially without defined time limits, each such administration providing a therapeutically effective level 2 compounds in the body the patient. The latter also applies to cocktail therapy, for example the administration of 3 or more active substances.

Methods for making compounds of the invention

The present disclosure also includes processes for preparing compounds of the present invention. In the reactions described, it may be necessary to protect functional groups, for example, hydroxyl, amino, imino, thio or carboxyl groups, as they are desired in the final product to avoid their unwanted participation in the reactions. Typical protecting groups may be used in accordance with standard practice, see for example TW Greene and PGM Wuts in "Protective Groups in Organic

Chemistry ", John Wiley and Sons, 1991.

Compounds of Formula I can be obtained by following the scheme shown in the following Reaction Scheme I:

Reaction scheme I:

<img file="PL2363393T3_D0002.tif" />

2 '3 And where Y1, Y2, R1, R2, R3, R4, R5, R6, R7, R8 and R9 are as defined above for Formula I in the summary of the invention. The compound of Formula I can be obtained by reacting a compound of formula 2 (or 2 ') with a compound of formula 3 in the presence of a suitable solvent (e.g. dichloromethane, N, N-dimethylformamide or the like) in a temperature range from about -20 to about 100 ° C. The reaction may take up to about 20 hours to complete.

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

-13EP 2363393

Additional methods for preparing compounds of the invention

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

Alternatively, salts of the compounds of the invention may be prepared using salts of starting materials or intermediates.

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

Compounds of the invention in an anoxidized form can be obtained from the N-oxides of compounds of the invention by treatment with a reducing agent (e.g., sulfur, sulfur dioxide, triphenylphosphine, lithium borohydride, sodium borohydride, phosphorus trichloride, phosphorus tribromide, tribromide or the like) in a suitable inert organic solvent (for example acetonitrile, ethanol, aqueous dioxane or the like) at 0 to 80 ° C.

Prodrug derivatives of the compounds of the invention can be prepared by methods known to those skilled in the art (e.g., further information can be found in Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. 1985). For example, suitable prodrugs can be prepared by reacting the non-derivatized compound of the invention with a suitable carbamylating agent (e.g., 1,1-acyloxyalkyl chloroformate, para-nitrophenyl carbonate or the like).

Protected derivatives of the compounds of the invention can be prepared by methods known to those skilled 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", 3rd edition, John Wiley and Sons, Inc., 1999.

The compounds of the present invention may conveniently be synthesized or prepared by the process of the invention as solvates (e.g. hydrates). Hydrates of the compounds of the present invention can conveniently be obtained by recrystallization from an aqueous / organic solvent mixture, using organic solvents such as dioxins, tetrahydrofuran or methanol.

The compounds of the invention can be obtained as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereomeric compounds, separating the diastereomers and isolating the optically pure enantiomers. While the separation of enantiomers can be carried out using covalent diastereomeric derivatives of compounds of the invention, dissociable complexes (e.g., crystalline diastereomeric salts) are preferred. Diastereomers have different physical properties (e.g.

EP 2363393 melting points, boiling points, solubility, reactivity, etc.) and can be easily separated based on these differences. Diastereomers may be separated by chromatography or, preferably, separation / resolution techniques based on differences in solubility. The optically pure enantiomer is then recovered along with the resolving agent by any method that does not result in racemization. A more detailed description of the techniques applicable to the separation 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, compounds of Formula I can be obtained by a process involving:

(a) the procedure outlined in Reaction Scheme I; and (b) optionally converting the compound of the invention into a pharmaceutically acceptable salt;

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

(d) optionally converting the non-oxygenated form of the compound of the invention to a pharmaceutically acceptable N-oxide;

(d) optionally converting the N-oxide of a compound of the invention to an anoxidized form;

(f) optionally separating a single isomer of a compound of the invention from a mixture of isomers;

(g) optionally converting the non-derivatized compound of the invention into a pharmaceutically acceptable prodrug derivative, and (h) optionally converting the prodrug derivative of the compound of the invention into its non-derivatized form.

The synthesis of starting materials has not been previously described, and therefore these compounds should be considered known or can be obtained by methods analogous to those known in the art or by the methods disclosed in the examples below.

It will be apparent to those skilled in the art that the above transformations are only representative of the methods for preparing the compounds of the present invention and that other well known methods can be similarly used.

Examples:

The present invention is illustrated in particular, but not exclusively, by the following example, which illustrates the process for preparing compounds of Formula I according to the invention.

Example 1 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid [4- (morpholine-4-sulfonyl) phenyl] amide

EP 2363393

CN and

<img file="PL2363393T3_D0003.tif" />

H₂SO₄

MeOH ftfe

OMe

NC — 4 \ —B (OH)

Pd (OAc) 2, KF ligand, dioxane

Me-.

And oxalyl chloride, OMF, CHjCi ^

<img file="PL2363393T3_D0004.tif" />

NaOH

Dioxane-H2O

<img file="PL2363393T3_D0005.tif" />

Ζ · ^ '\ #

2) FUN —- 4 Λ— 5 — NO

V · &lt;/ «\ /

EUN, CH-.CU &lt;-I / 'O

Example 1 S &lt;-⁰

Step 1: To a solution of 3-iodo-4-methyl-benzoic acid (10.0 g, 38.2 mmol) in methanol (70 ml) is added concentrated sulfuric acid (0.5 ml). The reaction mixture is heated at 70 ° C for 48 hours, cooled to room temperature and then concentrated. Then ethyl acetate (100 mL) and aqueous NaHCO3 (saturated, 100 mL) are added to the residue. The organic layer is separated and washed once with aqueous NaHCO3 (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 ester 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, 3H), 3.35 (s, 3H); LC-MS m / z: 277.0 (M + 1).

Step 2: Into a round bottom flask containing 3-iodo-4-methylbenzoic acid methyl ester (1.38 g, 5.00 mmol), 4-cyanophenylboronic acid (1.10 g, 7.48 mmol), palladium acetate (168 mg, 0.748 mmol), 2- ( dicyclohexylphosphino) biphenyl (0.526 g, 1.50 mmol) and potassium fluoride (0.870 g, 15.0 mmol) anhydrous 1,4-dioxane (15 ml) is added. The flask is flushed with argon and sealed. The mixture is stirred at 130 ° C for 18 hours, cooled to ambient temperature, and then water (20 ml) and ethyl acetate (20 ml) are added. The precipitate is removed by filtration under vacuum. The filtrate is extracted with ethyl acetate EtOAc (20 mL x

2). The organic layers are combined, washed with aqueous HCl (5%, 20 mL) and saturated NaHCO3 (20 mL). Dried over MgSO4 and concentrated. The residue is purified by silica gel chromatography (EtOAc / hexane gradient) to give 4'-cyano-6-methyl-biphenyl-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 methyl ester 2 (2.56 g, 10.3 mmol) in a 1,4-dioxane-H2O mixture (1: 1 mixture, 20 ml) is added NaOH ( 1.22 g, 30.2 mmol)). The reaction mixture is stirred at ambient temperature for 24 hours. HCl (1 N, 36 mL) is added to this mixture, followed by extraction with ethyl acetate (40 mL x 3). The organic layers are combined and dried over anhydrous Na2SO4. The solvent is removed. The resulting precipitate is washed with a small amount of acetonitrile and air dried to give 4'-cyano-6-methyl-biphenyl-3-carboxylic acid 3: ¹H NMR (DMSOd6) δ 7.94 (d, 2 H, J = 8.0 Hz), 7.84 (dd, 1 H, J1 = 8.4 Hz, J2 = 1.2 Hz), 7.75 (d, 1 HJ = 1.2 Hz), 7.61 ( d, 2 H, J = 8.0 Hz), 7.48 (d, 1 H, J = 8.4 Hz), 2.29 (s, 3 H); LC-MS m / z 238.1 (M + 1).

Step 4: To the 4'-cyano-6-methyl-biphenyl-3-carboxylic acid 3 suspension (40 mg,

0.17 mmol) in anhydrous methylene chloride (5 ml) 2 drops of DMF are added. Then adds

-16EP 2363393 oxalyl chloride (32 mg, 22 μΙ, 0.25 mmol). The mixture is stirred at ambient temperature until clarification. The mixture is then concentrated, dissolved in anhydrous methylene chloride (3 mL) and added to a solution of 4- (morpholine-4-sulfonyl) -phenylamine (61 mg, 0.25 mmol) and triethylamine (34 mg, 47 μΙ, 0.33 mmol) in methylene chloride (2 ml). The mixture is stirred for 2 hours, concentrated and the residue is purified by preparative HPLC (C18 column, eluting with CH3CN-H2O containing 0.05% TFA) to give [4- (morpholine-4-sulfonyl) phenyl] -amide 4'- cyano-6-methyl-biphenyl-3-carboxylic acid: ¹H NMR (DMSO-d6) δ 10.64 (s, 1H), 8.07 (d, 2H, J = 8.8 Hz), 7.97 (d, 2 H, J = 8.4 Hz), 7.95 (d, 1 H, J = 8.8 Hz), 7.89 (s, 1 H), 7.43 (d, 2 H, J = 8.4 Hz), 7.67 (d, 2 H, J = 8.8 Hz), 7.53 (d, 2 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 [6- (2,6-dimethyl-morpholin-4-yl) -pyridin-3-yl] -amide

OiN, • 'N

AND,

K₂WHAT₃:

DMF oHj / PcFC

Aug &amp; H

HE

Br ______________ O ................

HATU, ESjN, DMF

Ptf {PPfi₃) 4, NajCCs PhCH3-ĘiÓH-H₂0 ABOUT

Example 2

Step 1: K2CO3 (4.14 g, 30 mmol) is added to a solution of 2-chloro-5-nitro-pyridine 4 (2.38 g, 15 mmol) and cis-2,6-dimethylmorpholine (1.73 g, 15 mmol). The mixture is heated at 50 ° C overnight. After concentration, the residue is partitioned between EtOAc and water. The EtOAc layer is dried over anhydrous Na2SO4 and concentrated to give crude product 6 as a yellow solid. The crude product is used directly in the next step without further purification. LC-MS m / z: 238.1 (M + 1).

Step 2: The above crude material 6 is hydrogenated in the presence of Pd-C (0.2 g) in MeOH (100 ml) under a hydrogen atmosphere 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-methylbenzoic acid (108 mg, 0.5 mmol), 6- (2,6-dimethyl-morpholin-4-yl) -pyridin-3-ylmine 7 (104 mg, 0.5 mmol) and HATU (190 mg, 0.5 mmol) in dry DMF (5 mL) is added triethylamine (139 µL, 1.0 mmol) 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 dry and concentrated to give a crude product. The final compound is purified by flash column chromatography using 50% EtOAc in hexane as the eluent to give 8 as a white solid. LC-MS m / z: 404.1 (M + 1).

-17EP 2363393

Step 4: Mixture of 4-cyanophenylboronic acid (18 mg, 0.12 mmol), 3-bromo-N [6- (2,6-dimethyl-morpholin-4-yl) -pyridin-3-yl] -4-methyl-benzamide 8 (40 mg, 0.1 mmol), Pd (PPh3) 4 (11 mg, 0.01 mmol) and Na2CO3 (42 mg, 0.4 mmol) in a mixed system of solvents (0.2 ml), water (0.2 ml) and ethanol (0.05 ml) heated at 140 ° C under microwave irradiation for 30 minutes. The reaction mixture is diluted with EtOAc and water. The aqueous layer is extracted with EtOAc. The combined organic layers are washed with brine and concentrated to give a crude product which is purified by preparative HPLC with mass spectroscopy detection (C18 column, elution with CH3CN-H2O containing 0.05% TFA) to give [6- (2,6-dimethyl -morpholin-4-yl) -pyridin-3-yl] -amide 4'-cyano-6-methyl-biphenyl-3-carboxylic acid. LC-MS m / z: 427.2 (M + 1).

By repeating the procedures described in the above examples, using appropriate starting materials, the following compounds of Formula I are obtained, which are listed in Table No. 1.

Table 1

Relationship Number

Structure

<img file="PL2363393T3_D0006.tif" />

Physical data MS (m / z)

LC-MS m / z 411.2 (M + 1).

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

<img file="PL2363393T3_D0007.tif" />

LC-MS m / z 400.2 (M + 1)

LC-MS m / z 418.2 (M + 1).

LC-MS m / z 413.2 (M + 1).

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

-18EP 2363393 /

<img file="PL2363393T3_D0008.tif" />

<img file="PL2363393T3_D0009.tif" />

<img file="PL2363393T3_D0010.tif" />

<img file="PL2363393T3_D0011.tif" />

LC-MS m / z 451.2 (M + 1)

LC-MS m / z 437.2 (M + 1).

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

LC-MS m / z 438.2 (M + 1).

LC-MS m / z 438.2 (M + 1).

LC-MS m / z 435.2 (M + 1).

LC-MS m / z 424.1 (M + 1).

LC-MS m / z 404.2 (M + 1).

-19EP 2363393

<img file="PL2363393T3_D0012.tif" />

LC-MS m / z 413.2 (M + 1).

LC-MS m / z 418.2 (M + 1).

LC-MS m / z 431.2 (M + 1).

LC-MS m / z 431.2 (M + 1).

LC-MS m / z 417.2 (M + 1).

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

LC-MS m / z 430.2 (M + 1).

LC-MS m / z 432.2 (M + 1).

-20EP 2363393

<img file="PL2363393T3_D0013.tif" />

LC-MS m / z 429.2 (M + 1).

LC-MS m / z 488.1 (M + 1).

LC-MS m / z 400.2 (M + 1).

LC-MS m / z 414.2 (M + 1).

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

LC-MS m / z 428.2 (M + 1).

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

LC-MS m / z 428.2 (M + 1).

LC-MS m / z 454.2 (M + 1).

-21EP 2363393

<img file="PL2363393T3_D0014.tif" />

LC-MS m / z 470.2 (M + 1).

LC-MS m / z 403.2 (M + 1).

LC-MS m / z 471.2 (M + 1).

<img file="PL2363393T3_D0015.tif" />

LC-MS m / z 413.2 (M + 1).

<img file="PL2363393T3_D0016.tif" />

LC-MS m / z 411.2 (M + 1).

<img file="PL2363393T3_D0017.tif" />

LC-MS m / z 413.2 (M + 1).

<img file="PL2363393T3_D0018.tif" />

LC-MS m / z 397.2 (M + 1).

<img file="PL2363393T3_D0019.tif" />

LC-MS m / z 399.2 (M + 1).

LC-MS m / z 412.2 (M + 1).

-22EP 2363393

LC-MS mlz 398.2 (M + 1).

<img file="PL2363393T3_D0020.tif" />

<img file="PL2363393T3_D0021.tif" />

<img file="PL2363393T3_D0022.tif" />

<img file="PL2363393T3_D0023.tif" />

<img file="PL2363393T3_D0024.tif" />

LC-MS m / z 385.2 (M + 1).

LC-MS m / z 389.1 (M + 1).

LC-MS m / z 419.2 (M + 1).

LC-MS m / z 487.2 (M + 1).

LC-MS m / z 460.2 (M + 1).

LC-MS m / z 446.1 (M + 1).

LC-MS m / z 427.2 (M + 1).

LC-MS m / z 427.2 (M + 1)

-23EP 2363393

LC-MS m / z 411.2 (M + 1)

<img file="PL2363393T3_D0025.tif" />

<img file="PL2363393T3_D0026.tif" />

<img file="PL2363393T3_D0027.tif" />

LC-MS m / z 434.2 (M + 1)

LC-MS m / z 444.3 (M + 1)

LC-MS m / z 458.3 (M + 1)

LC-MS m / z 450.2 (M + 1).

LC-MS m / z 430.2 (M + 1)

LC-MS m / z 460.2 (M + 1)

LC-MS m / z 464.1 (M + 1)

-24EP 2363393

LC-MS m / z 524.1 (M + 1)

<img file="PL2363393T3_D0028.tif" />

LC-MS m / z 502.3 (M + 1)

LC-MS m / z 508.2 (M + 1)

<img file="PL2363393T3_D0029.tif" />

LC-MS m / z 427.2 (M + 1)

LC-MS m / z 432.2 (M + 1)

LC-MS m / z 470.2 (M + 1)

LC-MS m / z 413.2 (M + 1)

LC-MS m / z 464.1 (M + 1)

-25EP 2363393

LC-MS m / z 514.1 (M + 1)

100

101

102

103

<img file="PL2363393T3_D0030.tif" />

<img file="PL2363393T3_D0031.tif" />

LC-MS m / z 503.3 (M + 1)

LC-MS m / z 503.3 (M + 1)

LC-MS m / z 562.3 (M + 1)

LC-MS m / z 546.3 (M + 1)

<img file="PL2363393T3_D0032.tif" />

LC-MS m / z 600.3 (M + 1)

<img file="PL2363393T3_D0033.tif" />

LC-MS m / z 560.3 (M + 1)

104

-26EP 2363393

LC-MS m / z 560.3 (M + 1)

105

106

107

108

110

<img file="PL2363393T3_D0034.tif" />

<img file="PL2363393T3_D0035.tif" />

LC-MS m / z 503.3 (M + 1)

<img file="PL2363393T3_D0036.tif" />

LC-MS m / z 546.2 (M + 1)

LC-MS m / z 586.2 (M + 1)

<img file="PL2363393T3_D0037.tif" />

LC-MS m / z 604.2 (M + 1)

<img file="PL2363393T3_D0038.tif" />

LC-MS m / z 538.2 (M + 1)

111

-27EP 2363393

LC-MS m / z 554.2 (M + 1)

112

113

114

115

<img file="PL2363393T3_D0039.tif" />

LC-MS m / z 538.2 (M + 1)

LC-MS m / z 480.1 (M + 1)

<img file="PL2363393T3_D0040.tif" />

LC-MS m / z 481.2 (M + 1)

LC-MS m / z 447.2 (M + 1)

116

-28EP 2363393

LC-MS m / z 441.2 (M + 1)

117

118

119

120

124

125

<img file="PL2363393T3_D0041.tif" />

LC-MS m / z 506.2 (M + 1)

LC-MS m / z 572.2 (M + 1)

LC-MS m / z 522.2 (M + 1)

LC-MS m / z 488.2 (M + 1)

LC-MS m / z 489.2 (M + 1)

LC-MS m / z 489.2 (M + 1)

129

-29EP 2363393

LC-MS m / z 489.2 (M + 1)

130

134

135

136

137

139

<img file="PL2363393T3_D0042.tif" />

LC-MS m / z 472.1 (M + 1)

LC-MS m / z 447.1 (M + 1)

LC-MS m / z 413.1 (M + 1)

LC-MS m / z 492.1 (M + 1)

<img file="PL2363393T3_D0043.tif" />

LC-MS m / z 441.1 (M + 1)

<img file="PL2363393T3_D0044.tif" />

LC-MS m / z 428.2 (M + 1)

140

-30EP 2363393

LC-MS m / z 471.2 (M + 1)

141

142

143

144

145

147

<img file="PL2363393T3_D0045.tif" />

LC-MS m / z 487.2 (M + 1)

<img file="PL2363393T3_D0046.tif" />

LC-MS m / z 477.2 (M + 1)

LC-MS m / z 513.2 (M + 1)

LC-MS m / z 473.2 (M + 1)

LC-MS m / z 445.2 (M + 1)

LC-MS m / z 471.2 (M + 1)

148

-31EP 2363393

LC-MS m / z 547.2 (M + 1)

149

150

151

152

<img file="PL2363393T3_D0047.tif" />

<img file="PL2363393T3_D0048.tif" />

LC-MS m / z 562.2 (M + 1)

<img file="PL2363393T3_D0049.tif" />

LC-MS m / z 541.2 (M + 1)

LC-MS m / z 484.2 (M + 1)

The compounds of the present invention are tested to determine their ability to inhibit the hedgehog signaling pathway.

Test with Gli-Luc reporter for inhibition of the Hh pathway

TM3 mouse cells (obtained from the American Type Culture Collection, ATCC, Manassas,

VA) are cultured in DMEM / F12 medium (Gibco / Invitrogen, Carlsbad, CA) with the addition of 5% heat inactivated horse serum and 2.5% FBS (Gibco / Invitrogen. Carlsbad. CA), 50 units / ml penicillin and 50 μg / ml streptomycin (Gibco / Invitrogen, Carlsbad, CA) at 37 ° C with 5% CO2 in an air atmosphere. TM3 cells are transfected with pTA-8xGli-Luc reporter plasmid. A stably transfected clone called TMHh-12 is selected.

TMHh-12 clone shows a good response to Shh-N stimulation. To assess the IC50 value of antagonists, 8,000 TMHh-12 cells are plated in each well on 384 well plates with 50% DMEM / F12 medium supplemented with 2% FBS. After 12 hours, the Hh pathway is activated by adding recombinant mouse Shh protein (expressed in E. Coli, 8 μg / ml) or by adding Smni agni. Test compounds are added to the plates at various concentrations. After 48 hours, the firefly luciferase activity is determined using a Bright-Glo ™ luciferase test system (Promega, Madison, WI). IC 50 is measured when the effect of a compound reduces the luminescence signal by 50%. The toxicity of these compounds is assessed in TM3 cells using CellTiter Glo assays or using the TM3-Luc cell line (TM3 cell stably transfected with the vector for constitutive luciferase expression).

EP 2363393

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

200 nM.

Cytotoxicity study

A cytotoxicity study was carried out to compare the effect of the compound of the invention on germ cell cells (TE354.T cells) and control cells (normal human fibroblasts) according to the following procedure:

Daoy cells (spinal cord cell line) are purchased from ATCC and cultured in Minimum Essential (Eagle's) medium with 2 mM L-glutamine and Eagle's BSS to contain 1.5 g / L sodium bicarbonate, endogenous amino acids at a concentration of 0.1 mM and sodium pyruvate at a concentration of 1.0 mM and 10% FBS at 37 ° C with 5% CO2 in an air atmosphere.

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

Normal human skin fibroblasts (Clonetics) are grown in fibroblast culture medium (Clonetics).

Each of the above cell lines is seeded independently in 96-well plates and grown to a density of 5000-10000 cells / well. The compound of the invention is added to the cell culture at various concentrations. After 2 days, cell viability is assessed using the Cell Titer-Glo Luminescent Cell Viability Assay Kit (Promega) according to the manufacturer's instructions. Cell viability is measured directly based on luminescent signals and EC50 values ​​are determined when the signal is inhibited by 50%.

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

It should be assumed that the examples and implementation described herein are provided for illustrative purposes only, and that within the framework of the following claims various modifications and changes will be addressed to those skilled in the art.

"ATENTOWA" BELLEPAT "LAW OFFICE

Izabela Szych ulukc-Hawranek ul Słowackiego 44, 37-700 Prz * ';> uśl phone (016) 7j2-37-77 fax: (016) 675-02-87 mobile phone (0608) 503-081 e-maii <a href="mailto:fcellepat@op.pl">fcellepat@op.pl</a> NIP: 795-207-16-72 REGON: 1803505 (6

Proxy:

<img file="PL2363393T3_D0050.tif" />

-1EP 2363393