Compounds and compositions as hedgehog pathway modulators

[0001] The present invention relates to a method of modulating the activity of the hedgehog signaling pathway. In particular, a method of inhibiting growth abnormalities resulting from phenotypes, such as loss of Ptc function, acquisition of hedgehog function, acquisition of smoothened function or acquisition of Gli function has been described, involving contacting the cell with sufficient N- (6 - ((2R, 6S) -2 6-dimethylmorpholino) pyridin-3-yl) -2-methyl-4 '- (trifluoromethoxy) biphenyl-3-carboxamide).

Background of the Invention [0002] During embryonic development, the hedgehog signaling pathway is important for many processes such as control of cell proliferation, cell differentiation and tissue 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 pathway in adult tissues may cause some types of cancer, which include, but are not limited to, brain, muscle and skin cancer, prostate, spinal cord, pancreatic adenocarcinoma, 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 hedgehog signaling pathway are useful as therapeutic agents in the treatment of such diseases.

Summary of the Invention [0003] In one aspect, the present invention provides the compound, N- (6 ((2R, 6S) -2,6-dimethylmorpholino) pyridin-3-yl) -2-methyl-4 '- (trifluoromethoxy) biphenyl Carboxamide), with the formula:

<img file="PL2021328T3_D0001.tif" />

or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0004] In another aspect, the present invention provides a pharmaceutical composition comprising the compound, N- (6 - ((2R, 6S) -2,6-dimethylmorpholino) pyridin-35-yl) -2-methyl-4 '- (trifluoromethoxy) biphenyl -3-carboxamide), with the formula:

<img file="PL2021328T3_D0002.tif" />

in free form or in the form of a pharmaceutically acceptable salt in combination with at least one pharmaceutically acceptable carrier or diluent.

[0005] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound, N- (6 - ((2R, 6S) -2,6-dimethylmorpholino) pyridin-3-yl) -2-methyl-4 '- ( trifluoromethoxy) biphenyl-3-carboxamide), with the formula:

<img file="PL2021328T3_D0003.tif" />

or a pharmaceutically acceptable salt thereof, in combination with one or more therapeutic agents.

[0006] In another aspect, the present invention provides the compound, N- (6 ((2R, 6S) -2,6-dimethylmorpholino) pyridin-3-yl) -2-methyl-4 '- (trifluoromethoxy) biphenyl-3-carboxamide) , with the formula:

F

<img file="PL2021328T3_D0004.tif" />

or a pharmaceutically acceptable salt thereof for use in the treatment of pancreatic cancer, prostate cancer, medullary carcinoma; basal cell carcinoma and small cell lung cancer.

[0007] The present disclosure describes formula I:

R /

Re

<img file="PL2021328T3_D0005.tif" />

Q

Ra [0008] wherein [0009] Y1 and Y2 are independently selected from N and CR10; wherein R10 is selected from hydrogen, halogen, C1-6alkyl, halogen substituted C1-6alkyl, C1-6alkoxy, halogen substituted C1-4alkoxy and -OXNR10aR10b; wherein R10a and R10b are independently selected from hydrogen and C1-4alkyl;

[0010] R1 is selected from the cyano group, halogen, C1-6alkyl, halogen substituted C1-4alkyl, C1-6alkoxy, halogen substituted C1-6alkoxy, C6-10aryl, dimethylamino, C1-6alkylsulfanyl and

C 3-8 heterocycloalkyl optionally substituted with up to 2 C 1-6 alkyl radicals;

[0011] R2 and R5 are independently selected from hydrogen, cyano, halogen, C1-6alkyl, halogen substituted C1-6alkyl, C1-4alkoxy, halogen substituted C1-6alkoxy and dimethylamino;

[0012] R3 and R4 are independently selected from hydrogen, halogen, cyano, C1-6alkyl, halogen substituted C1-6alkyl, C1-6alkoxy and halogen substituted C1-6alkoxy; or R1 and R2, or R1 and R5 together with the phenyl to which they are attached form C5-10 heteroaryl;

[0013] R6 and R7 are independently selected from hydrogen, C1-6alkyl, halogen substituted C1-6alkyl, C1-4alkoxy and halogen substituted C1-6alkoxy; provided that the R6 and R7 groups are not both hydrogen;

[0014] R8 is selected from halogen, C1-6alkyl, halogen substituted C1-6alkyl, C1-6alkoxy and halogen substituted C1-6alkoxy;

[0015] R9 is selected from -S (O) 2R11, -C (O) R11, -OR11, -NR12aR12b and -R11; wherein R11 is selected from aryl, heteroaryl, cycloalkyl and heterocycloalkyl; R12 and R12b are independently selected from C1-6alkyl and hydroxyl substituted C1-6alkyl;

[0016] wherein said aryl, heteroaryl, cycloalkyl and heterocycloalkyl of the R9 group may be optionally substituted with 1 to 3 radicals independently selected from C1-6alkyl, halogen substituted C1-6alkyl, C1-6alkoxy, halogen substituted C1-6alkoxy, C6-10aryl-C0 -4alkyl, C5-10heteroaryl-C0-4alkyl, C3-12cycloalkyl and C3-8heterocycloalkyl;

[0017] wherein said aryl-alkyl substituent of the R9 group is optionally substituted with 1 to 3 radicals independently selected from halogen, C16alkyl, halogen substituted C1-6alkyl, C1-6alkoxy, halogen substituted C1-6alkoxy and methylpiperazinyl; and N-oxide derivatives, prodrug derivatives, protected derivatives, single isomers and mixtures of isomers; and pharmaceutically acceptable salts and solvates (e.g. hydrates) of these compounds.

[0018] Also described is a pharmaceutical composition comprising a compound of formula I or an N-oxide derivative thereof, individual isomers and mixtures of isomers; or a pharmaceutically acceptable salt thereof, in a mixture with one or more suitable excipients.

[0019] Also described is a method of treating a disease in an animal in which modulation of the signaling pathway activity may prevent, inhibit or ameliorate the pathological picture and / or symptoms of the diseases, comprising administering to the animal a therapeutically effective amount of a compound of formula I or its N-oxide derivative, single isomers and mixtures of isomers, or a pharmaceutically acceptable salt thereof.

[0020] Also disclosed is the use of 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.

[0021] Also disclosed is a process for the preparation of compounds of formula I and their Peroxide derivatives, prodrug derivatives, protected derivatives, single isomers and mixtures of isomers, as well as pharmaceutically acceptable salts thereof.

Definitions [0022] "Alkyl" as a group and as a structural element of other groups, for example halo-substituted-alkyl and alkoxy, can be straight or branched. C 1-4 alkoxy includes methoxy, ethoxy and the like. Halo-substituted alkyl includes trifluoromethyl, pentafluoroethyl and the like.

[0023] "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 naphthyl, preferably phenyl. "Arylene" means a divalent radical derived from an aryl group.

[0024] "Heteroaryl" is as defined 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. In this regard, C5-10heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, benzofuranyl, benzopyranyl, benzothiopyranyl, benzo [1,3] dioxol, imidazolyl, benzimidazolyl, pyrimidinyl, furanyl, isoxazolyl, tetazolazolyl , pyrazolyl, thienyl etc.

[0025] "Cycloalkyl" means a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring system containing the number of ring atoms given. For example, C 3-10 cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0026] "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, C38heterocycloalkyl, as used herein, 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.

[0027] "Halo" (or halo) preferably means chloro or fluoro, but can also be bromo or iodo.

[0028] "Acquisition of hedgehog function" refers to the incorrect modification or mutation of a Ptc gene, Hedgehog gene or smoothened gene or reduction (loss) of expression of such gene, resulting in a phenotype that resembles contact of a cell with a hedgehog protein, for example incorrect activation of the hedgehog pathway. The acquisition of function may include loss of the ability of the Ptc gene product to regulate the expression level of the Gli genes, for example, 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, including, but not limited to, modifications or hedgehog mutation. For example, tumor cells with an extremely high degree of proliferation in connection with activation of the hedgehog signal pathway would have the "hedgehog acquisition" phenotype even if the hedgehog gene was not mutated in that cell.

[0029] "Loss of patched function" refers to an incorrect modification or mutation of a Ptc gene or a reduced level of gene expression, which leads to a phenotype that resembles cell contact with a hedgehog protein, for example incorrect activation of the hedgehog pathway. Functionalities may include loss of the ability of the Ptc gene product to regulate the level of gene expression, for example Gli Gli1, Gli2 and Gli3.

[0030] "Acquisition of Gli function" refers to an incorrect modification or mutation of the Gli gene or an increased level of gene expression, which leads to a phenotype that resembles the contact of a cell with a hedgehog protein, e.g., incorrect activation of the hedgehog pathway.

[0031] "Acquisition of Smoothened function" refers to an incorrect modification or mutation of the Smo gene or an increased level of gene expression, which leads to a phenotype that resembles cell contact with a hedgehog protein, for example, incorrect activation of the hedgehog pathway.

[0032] "Treat", "treatment" and "therapy" refer to a method of alleviating or reducing the disease and / or its accompanying symptoms.

[0033] The present invention relates to the finding that signal transduction pathways regulated by hedgehog, patched (Ptc), gli / or smoothened can be modulated by compounds of formula I. The compound of the invention is N- (6 ((2R, 6S ) -2,6-dimethylmorpholine) pyridin-3-yl) -2-methyl-4 '- (trifluoromethoxy) biphenyl-3-carboxamide) or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0034] In this connection, it is specifically contemplated that compounds of formula I that interfere with aspects of hedgehog, Ptc or smoothened signal transduction activity will also be able to inhibit proliferation (or other biological effects) in normal cells and / or cells with the phenotype of loss of patched function, acquisition of hedgehog function, acquisition of smoothened function or acquisition of Gli function. 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 capable of inhibiting at least some biological activities of hedgehog proteins, particularly preferably 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 the phenotype of loss of patched 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, regulation hair and skin growth etc. In addition, the methods of the invention can be carried out on cells provided in culture (in vitro) or cells in the body of an animal (in vivo).

[0035] The present invention describes a use that targets epithelial cells having the phenotype of loss of patched function, acquisition of hedgehog function, acquisition of smoothened function or acquisition of Gli function. For example, the method object of the present invention can be used to treat or prevent basal cell carcinoma or other disorders associated with the hedgehog pathway.

[0036] The compound of formula I may inhibit the activation of the hedgehog pathway by binding to smoothened or downstream proteins. The antagonist of the invention may inhibit the activation of the hedgehog pathway by binding to patched proteins.

[0037] The use may be part of a treatment regimen for medulla and other primary CNS neuroectodermal malignancies.

[0038] In another aspect, the present disclosure relates to pharmaceutical preparations containing, as active ingredient, a Hedgehog signaling modulator, such as a compound of formula I, a Ptc agonist, a smoothened antagonist, an antagonist of proteins downstream on the hedgehog pathway as described herein , made in an amount sufficient to inhibit, in vivo, proliferate or other biological consequences of loss of patched function, acquisition of hedgehog function, acquisition of the smoothened function or acquisition of the Gli function.

[0039] Treatment will be the subject of the present invention using a compound of formula I, Ptc agonists, smoothened antagonists or protein antagonists downstream on the hedgehog pathway, may 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 Use [0040] The present invention describes methods and compounds for inhibiting activation of the hedgehog signaling pathway, e.g., inhibition of abnormal growth conditions resulting from phenotypes such as loss of patched function, acquisition of hedgehog function, acquisition of smoothened function or acquisition of Gli function, including cell contact 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.

[0041] 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 intracellular 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 cells 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 themselves (homeogenetic induction), and in other cases, cells inhibit the process of differentiation, which they undergo, of neighboring cells. 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 act to establish and maintain morphogenetic patterns as well as inducing differentiation.

[0042] The family of vertebrate hedgehog genes includes three members found in mammals, 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 autoproteolytic cleavage of the Hh precursor protein occurs via a 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. The biological effects are profound. As a result of attachment, a high local concentration of N-terminal Hedgehog peptide is generated on the surface of Hedgehog producing cells. This N-terminal peptide is both necessary and sufficient for short and long range Hedgehog signaling activity.

[0043] An inactive signal pathway occurs when the Patched (Ptc) transmembrane protein receptor 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 by interactions with cytoplasmic proteins, including Fused and Suppressor of fused (Sufu). Consequently, transcriptional activation of Hedgehog target genes is suppressed. Pathway activation is initiated by binding one of three mammalian ligands (Dhh, Shh or Ihh) to Ptc. Ligand binding causes reversal of Smo repression, thereby activating the cascade that leads to translocation of the active form of Gli transcription factor in the nucleus. Nuclear Gli activates expression of target genes, including Ptc and Gli itself.

[0044] Elevated levels of Hedgehog signaling are sufficient to initiate the tumor formation process and are required for tumor survival. These cancers include, but are 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. 7 October 2004; 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 US A. 2004 Aug 24; 101 (34): 13561-6), 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. August 1, 2004; 64 (17): 6071-4), medullary ("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. thirty August 2002; 297 (5586): 1559-61), '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 US A. 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. 1 January 1998; 391 (6662): 90-3), 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. 23 October 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 signalling within airway epithelial progenitors and in small-cell lung cancer", Watkins DN, Berman DM, Burkholder SG, Wang B, Beachy PA, Baylin SB ., Nature. March 20, 2003; 422 (6929): 313-7).

[0045] 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 And or a pharmaceutically acceptable salt thereof. For each of the above uses, the required dose will vary depending on the mode of administration, the particular condition being treated, and the effect desired.

Pharmaceutical administration and compositions:

[0046] 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 therapeutic 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 of from about 0.03 to 2.5 mg / kg body weight. The indicated daily dose in larger mammals, e.g. humans, ranges from about 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 substance.

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 the form of nasal 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) binders, 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 emollient, 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 control the delivery of the compound through the skin at a controlled and predetermined speed over a prolonged period of time, and elements fixing the product to the skin. Transdermal matrix preparations may also be used. Suitable formulations for topical application, e.g. to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well known in the art. They may contain solubilizing agents, stabilizers, tonicity enhancing agents, buffers and preservatives.

[0048] 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.

[0049] The invention also relates to pharmaceutical combinations, for example a kit comprising a) a first agent which is a compound of the invention 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 its administration.

[0050] The terms "combined administration", "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 in the same way. time.

[0051] The term "pharmaceutical combination" as used herein means a product resulting from the mixing or combining of more than one active ingredient and includes both fixed and transient combinations of active ingredients. The term "fixed combination" means that the active ingredients, for example the compound of the invention and the second drug, are administered to the patient simultaneously in the form of a single unit or dose. The term "transient combination" means that the active ingredients, for example the compound of the invention and the second drug, are administered to the patient as separate units simultaneously, simultaneously or sequentially without specific time limits, such administration providing therapeutically effective levels of 2 compounds in the patient's body. The latter also applies to cocktail therapy, for example the administration of 3 or more active substances.

Methods for making compounds of formula I [0052] The present invention also includes methods for preparing compounds of formula I. In the reactions described, it may be necessary to protect reactive functional groups, for example, hydroxyl, amino, imino, thio or carboxyl groups, when these are desired in the 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 PG M. Wuts in Protective Groups in Organic Chemistry, John Wiley and Sons, 1991.

[0053] Compounds of formula I can be obtained by following the scheme shown in the following reaction scheme I:

Reaction scheme 1

<img file="PL2021328T3_D0006.tif" />

2 '3 And where Y1, Y2, R1, R2, R3, R4, R5, R6, R7, R8 and R9 are as defined above for formula I. A 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.

[0054] Detailed examples of the synthesis of the compound of formula I can be found in the examples below.

Additional methods for preparing compounds of the invention [0055] The compound of the invention may be prepared as 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 compound in the form of the free acid with a pharmaceutically acceptable inorganic or organic base.

[0056] Alternatively, salts of the compounds of the invention may be prepared using salts of starting materials or intermediates.

[0057] The compound of the invention in the form of 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 in the form of an acid addition salt can be converted into the corresponding free base by treatment with a suitable base (e.g., ammonium hydroxide, 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.).

[0058] 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 or the like) in a suitable inert solvent organic (e.g. acetonitrile, ethanol, aqueous dioxane or similar) at 0 to 80 ° C.

[0059] Prodrug derivatives of the compounds of the invention may be prepared by methods known to those skilled in the art (for example, 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 obtained 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).

[0060] Protected derivatives of the compounds of the invention may be prepared by methods known to those skilled in the art. A detailed description of the techniques used to create and remove protecting groups can be found in TW Greene, "Protective Groups in Organic Chemistry," ed. 3, John Wiley and Sons, Inc, 1999.

[0061] The compounds of the present invention may conveniently be synthesized or prepared by the process of the invention as solvates (for example 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.

[0062] 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., 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.

[0063] In summary, compounds of formula I can be obtained by a process including:

(a) the procedure outlined in reaction scheme I; and (b) optionally converting a 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;

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

(f) optionally separating a single isomer of a compound of the invention from the 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.

[0064] 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.

[0065] 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 [0066] The following example illustrates a process for preparing compounds of formula I. Reference Example 1 4'-Cyano-6-methyl-biphenyl-3-carboxylic acid [4- (Morpholine-4-sulfonyl) -phenyl] -amide

<img file="PL2021328T3_D0007.tif" />

[0068] 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-methylbenzoic acid methyl ester 1. This ester is used without further purification in the next step. ¹H NMR (400 MHz, MSO-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- (dicyclohexylphosphine) 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 (20 mL × 2). The organic layers are combined, washed with aqueous hydrochloric acid (5%, 20 mL) and saturated NaHCO3 (20 mL), dried over MgSO4 and concentrated. The residue is purified by silica gel chromatography (ethyl acetate / hexane gradient) to give 4'-cyano-6-methyl-biphenyl-3-carboxylic acid methyl ester 2, LC-MS m / z: 252.1 (M + 1 ).

[0070] 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) NaOH (1.22 g, 30.2 mmol) is added. The reaction mixture is stirred at ambient temperature for 24 hours. Hydrochloric acid (1 N, 36 ml) is added to this mixture and then extracted with ethyl acetate (40 ml x 3). The organic layers are combined, 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 (DMSO-d6) 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 H, J = 1.2 Hz), 7.61 (d, 2 H, J = 8.0 Hz), 7.48 (d, 1 H, 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 mg, 0.17 mmol) in anhydrous methylene chloride (5 mL) is added DMF drops, followed by oxalyl chloride (32 mg, 22 μΐ, 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 μ ^ 0.33 mmol ) in methylene chloride (2 ml). The mixture is stirred for 2 hours, concentrated and the residue is purified by preparative HPLC with mass spectroscopy detection (C18 column, elution with CH3CN-H2O containing 0.05% TFA) to give [4- (morpholine-4-sulfonyl) - 4'-cyano-6-methyl-biphenyl-3-carboxylic acid phenyl] -amide: ¹H NMR (DMSO-d6) δ 10.64 (s, 1H), 8.07 (d, 2 H, 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, 4 H), 2.84 (m, 4 H) 2, 32 (s, 3H); LCMS m / z: 462.1 (M + 1).

Reference Example 2 [4- (2,6-Dimethyl-morpholin-4-yl) -pyridin-3-yl] -amide 4'-cyano-6-methyl-biphenyl-3-carboxylic acid [0072]

<img file="PL2021328T3_D0008.tif" />

2,6-dimethylmorpholine (1.73 g, 15 mmol) K2CO3 (4.14 g, 30 mmol) is added. The mixture is heated at 50 ° C overnight. After concentration, the residue is partitioned between ethyl acetate and water. The ethyl acetate layer is dried with 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. LCMS m / z: 238.1 (M + 1).

[0074] 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 concentrated filtrate to give crude product 7 as a dark brown oil which is used directly in the next step without further purification. LCMS m / z: 208.1 (M + 1).

[0075] Step 3: To a solution of 3-bromo-4-methylbenzoic acid (108 mg, 0.5 15 mmol), 6- (2,6-dimethyl morpholin-4-yl) pyridin-3-ylamine 7 (104 mg, 0.5 mmol) and

HATU (190 mg, 0.5 mmol) in dry DMF (5 ml) is added triethylamine (139 μΐ, 1.0 mmol) dropwise. The resulting mixture is stirred at room temperature for 2 hours. After concentration, the residue is partitioned between ethyl acetate and water. The organic layer is dried and concentrated to give a crude product. The final compound is purified by flash column chromatography using 50% ethyl acetate in hexane as the eluent to give 8 as a white solid. LC-MS m / z: 404.1 (M + 1).

[0076] Step 4: A 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 solvent system: toluene (0 , 2 ml), water (0.2 ml) and ethanol (0.05 ml) are heated at 140 ° C under microwave irradiation for 30 minutes. The reaction mixture is diluted with ethyl acetate and water. The aqueous layer is extracted with ethyl acetate. 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, CH3CN-H2O elution with 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).

[0077] By repeating the procedures described in the above examples, using appropriate substrates, compound 153 is obtained.

<img file="PL2021328T3_D0009.tif" />

[0078] The compounds of the present invention are tested to determine their ability to inhibit the Hedgehog signaling pathway.

Assay with Gli-Luc reporter for inhibition of the Hh pathway [0079] Mouse TM3 cells (obtained from 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 the air. 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 values ​​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 Smo agonists. Test compounds are added to the plates at various concentrations. After 48 hours, 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 M3-Luc cell line (TM3 cell stably transfected with the vector for constitutive luciferase expression).

[0080] Compounds of formula I typically have an EC50 of less than 500 nM, more preferably less than 200 nM.

Cytotoxicity study [0081] A cytotoxicity study was conducted to compare the effect of a compound of the invention on medullary cells (Daoy cells), basal cell carcinoma cells (TE354.T cells) and control cells (normal human fibroblasts) according to the following procedure:

[0082] Daoy cells (spinal cord cell line) are purchased from ATCC and cultured in Minimum Essential (Eagle) medium with 2 mM L-glutamine and Earle'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 the atmosphere of air.

[0083] TE354.T cells (from ATCC) are cultured in modified Eagle's medium

Dulbecco with 4 mM L-glutamine, fetal bovine serum and 10% FBS.

[0084] Normal human skin fibroblasts (Clonetics) are cultured in fibroblast culture medium (Clonetics).

[0085] Each of the above cell lines is seeded independently in 9620 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 50% inhibited.

[0086] Compounds of formula I typically have an EC50 of less than 500 nM, more typically less than 200 nM.

IRM LLC, Bermuda Representative:

Z-10589/13

EP 2 021 328 B1