SUBSTITUTED PYRIDINE COMPOUNDS AS KINASES INHIBITORS

CROSS REFERENCE

[0001] This invention claims the benefit of U.S. Provisional Patent Application No.

61/851,545, filed on March 11, 2013, which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION

[0002] The present invention is directed to inhibitors of kinase and pharmaceutically acceptable salts, solvates, hydrates, prodrugs and metabolites thereof, the preparation method thereof, and the use of such compounds to treat kinase mediated diseases and conditions such as cancer.

BACKGROUND OF THE INVENTION

[0003] Protein kinases represent a large family of enzymes, which catalyze the

phosphorylation of target protein substrates. The phosphorylation is usually a transfer reaction of a phosphate group from ATP to the protein substrate. Common points of attachment for the phosphate group to the protein substrate include, for example, a tyrosine, serine or threonine residue. Examples of kinases in the protein kinase family include, without limitation, Abll (v-Abl Abelson murine leukemia viral oncogene homo log 1), Akt, Alk, Bcr- Abll, Blk, Brk, Btk, c-Kit, c-Met, c-Src, c-Fms, CDKl-10, b-Raf, c-Rafl, CSF1R, CSK, EGFR, ErbB2, ErbB3, ErbB4, Erk, FGFR1, FGFR2, FGFR3, FGFR4, FGFR5, Flt-1, Fps, Frk, Jak, KDR, MEK, PDGFR, PIK, PKC, PYK2, Ros, Tie, Tie2, and Zap70. Due to their activity in numerous cellular processes, protein kinases have emerged as important

therapeutic targets.

[0004] ALK (Anaplastic Lymphoma Kinase ) is a 1620 amino acid transmembrane protein, consisting of extracellular domain with amino-terminal signal peptide, intracellular domain with a juxtamembranous segment harboring a binding site for insulin receptor substrate- 1, and a carboxy-terminal kinase domain. ALK is a member of the insulin receptor tyrosine kinases, Echinoderm microtubule-associated protein- like 4 (EML4) is a 120 KDa

cytoplasmic protein, which involves in the formation of microtubules and microtubule binding protein . EML4-ALK is a novel fusion gene arising from an inversion on the short arm of chromosome 2 that joined exons 1-13 of EML4 to exons 20-29 of ALK . The presence of EML4-ALK fusion is identified in approximately 3-13% of NSCLC (non-small cell lung cancer) patients.

[0005] To this end, attempts have been made to identify small molecules which act as PK inhibitors. For example, Amino heteroaryl compounds diaryl ureas (PCT WO2004/076412) have been described as ALK/c-MET inhibitors. Azaindole derivatives (PCT WO2010/068292) have been described as ALK/EGFR kinase inhibitors.

[0006] Thus, the compounds that can inhibit protein kinases such as ALK and other kinases activity either independently or together can be used to treat human diseases such as Cancers.

SUMMARY OF THE INVENTION

[0007] The present invention provides compounds of Formula I:

I

or a pharmaceutically acceptable salt, solvate or a prodrug or an enantiomer, or a metabolite thereof, wherein

R ¹ is hydrogen or Ci_ ₆ alkyl;

R ² is a residue of amino acid;

and with the proviso that when R ² is 2-aminopropionyl, R ¹ is not hydrogen.

[0008] The present invention further provides pharmaceutical compositions comprising a compound of Formula I described above and a pharmaceutically acceptable carrier.

[0009] The present invention further provides methods for treating or preventing a kinase mediated disorder comprising administrating to a mammalian subject a therapeutically effective amount of any of the compounds of Formula I described above.

DETAILED DESCRIPTION OF THE INVENTION In some embodiments of the present invention, there are provided compounds of

I

or a pharmaceutically acceptable salt, solvate or an enantiomer, or a prodrug or a metabolite thereof, wherein

R ¹ is hydrogen or Ci_ ₆ alkyl;

R ² is a residue of amino acid;

and with the proviso that when R ² is 2-aminopropionyl (or alanyl), R ¹ is not hydrogen; when R ² is (S)-2-aminopropionyl (or L-alanyl), R ¹ is hydrogen or Ci_ ₆ alkyl; and when R ² is (R)-2-aminopropionyl (or D-alanyl), R ¹ is hydrogen or Ci_ ₆ alkyl.

[0011] In some embodiments of the present invention, there are provided compounds of Formula II:

II

or a pharmaceutically acceptable salt, solvate or an enantiomer, or a prodrug or a metabolite thereof, wherein

R ¹ is hydrogen or Ci_ ₆ alkyl;

R ² is a residue of amino acid. [0012] In certain embodiments, the invention provides for compounds of Formula I or Formula II wherein R ¹ is hydrogen.

[0013] In other embodiments, the invention provides for compounds of Formula I or Formula II wherein R ¹ is a methyl or ethyl.

[0014] In other embodiments, the invention provides for compounds of Formula I or Formula II wherein R ¹ is CD ₃ or CD ₂ CD ₃ .

[0015] In certain embodiments, the invention provides for compounds of Formula I or Formula II wherein R ¹ is hydrogen and R ² is 2-aminoacetyl.

[0016] In certain embodiments, the invention provides for compounds of Formula I or Formula II wherein R ¹ is hydrogen and R ² is (S)-2-aminopropionyl (or L-alanyl) or (R)-2- aminopropionyl (or D-alanyl).

[0017] In certain embodiments, the invention provides the following compounds:

2-amino-N-(3-(l-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(l-(l -methylpiperidin-4-yl)-lH- pyrazol-4-yl)pyridin-2-yl)acetamide;

(R)-2-amino-N-(3-(l-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-( l-(piperidin-4-yl)-lH-pyrazol- 4-yl)pyridin-2-yl)acetamide;

2-amino-N-(3-(l-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(l-(p iperidin-4-yl)-lH-pyrazol-4- yl)pyridin-2-yl)acetamide;

2-amino-N-(3-((R)-l-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-( l-(piperidin-4-yl)-lH-pyrazol- 4-yl)pyridin-2-yl)propanamide;

(R)-2-amino-N-(3-((R)-l-(2,6-dichloro-3-fluorophenyl)ethoxy) -5-(l-(piperidin-4-yl)-lH- pyrazol-4-yl)pyridin-2-yl)propanamide;

(S)-2-amino-N-(3-((R)- 1 -(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(l -(piperidin-4-yl)- 1H- pyrazol-4-yl)pyridin-2-yl)propanamide;

(R)-2-amino-N-(3-((R)-l-(2,6-dichloro-3-fluorophenyl)ethoxy) -5-(l-(piperidin-4-yl)-lH- pyrazol-4-yl)pyridin-2-yl)-3-methylbutanamide;

(S)-2-amino-N-(3-((R)- 1 -(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(l -(piperidin-4-yl)- 1H- pyrazol-4-yl)pyridin-2-yl)-3-methylbutanamide;

(S)-2-amino-N-(3-((R)- 1 -(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(l -(piperidin-4-yl)- 1H- pyrazol-4-yl)pyridin-2-yl)-3-phenylpropanamide;

(R)-2-amino-N-(3-((R)-l-(2,6-dichloro-3-fluorophenyl)ethoxy) -5-(l-(piperidin-4-yl)-lH- pyrazol-4-yl)pyridin-2-yl)-3-phenylpropanamide;

(R)-2-amino-N-(3-((R)-l-(2,6-dichloro-3-fluorophenyl)ethoxy) -5-(l-(piperidin-4-yl)-lH- pyrazol-4-yl)pyridin-2-yl)-2-phenylacetamide; (S)-2-amino-N-(3-((R)- 1 -(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(l -(piperidin-4-yl)- 1H- pyrazol-4-yl)pyridin-2-yl)-2-phenylacetamide.

[0018] In other embodiments, the compound of this invention is in the form of

pharmaceutically acceptable salt. In some embodiments, the compound of this invention is in the form of a solvate. In other embodiments, the compound of this invention is in the form of a metabolite. In other embodiments, the compound of this invention is in the form of a prodrug. In some embodiments, the compound of this invention is an enantiomer. In other embodiments, the compound of this invention is a diastereomer. In another embodiment, the deuterium enrichment in compounds of this invention is at least about 1%.

[0019] In some embodiments, there are provided pharmaceutical compositions comprising a compound of the invention and a pharmaceutically acceptable carrier. In certain

embodiments, the compositions are for the treatment of a disease regulated by a protein kinase. In certain embodiments, the compositions are for the prevention or the treatment of a hyper-proliferative disorder and/or angiogenesis disorder. In some embodiments, the pharmaceutical compositions further comprise an anti-neoplastic agent, an

immunosuppressant, an immunostimulant, or combination thereof. In other embodiments, the pharmaceutical compositions are suitable for oral, parenteral, or intravenous administration.

[0020] In some embodiments, the present invention provides methods for regulating the kinase signaling transduction comprising administrating to a mammalian subject a therapeutically effective amount of any of the inventive compounds described herein.

[0021] In other embodiments provide herein methods for treating or preventing a ALK (including all fusion and/or mutant kinases), c-Met mediated disorder, said method comprises administrating to a mammalian subject a therapeutically effective amount of any of the inventive compounds described herein.

[0022] In other embodiments, there are provided herein methods for treating neoplasia comprising administrating to a mammalian subject in need thereof, a therapeutically effective amount of any of the inventive compounds described herein. In certain embodiments, the neoplasia is selected from skin cancer, leukemias, colon carcinoma, renal cell carcinoma, gastrointestinal stromal cancer, solid tumor cancer, myeloma, breast cancer, pancreatic carcinoma, non-small cell lung cancer, non-Hodgkin's lymphoma, hepatocellular carcinoma, thyroid cancer, bladder cancer, colorectal cancer, and prostate cancer. In some embodiments, the methods further comprise administering one or more anti-cancer agents.

[0023] The following definitions should assist in understanding the invention described herein. [0024] The term "alkyl" is intended to include straight, branched, and cyclic hydrocarbon groups, which contain only single carbon-carbon bonds and which may be unsubstituted or optionally substituted with one or more functional groups. The preferred chain length of an alkyl group is from 1 to 6 carbon atoms. Ci-C ₆ alkyl is intended to include Ci (methyl), C ₂ (ethyl), C ₃ (n-propyl, isopropyl), C ₄ (e.g. n-butyl, isobutyl, sec-butyl, tert-butyl), C ₅ (e.g. n- pentyl)and C ₆ alkyl groups. Alkyl may be substituted or unsubstituted. Illustrative substituted alkyl group include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, aminomethyl, aminoethyl, hydoxymethyl, methoxymethyl, 2-fluoroethyl, and 2- methoxy ethyl, etc.

[0025] "Amino acid" means organic compounds composed of amine (-NH ₂ ) and

carboxylic acid (-COOH) functional groups, along with a side-chain specific to each amino acid. Examples include, but are not limited to, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine, 3-aminopropanoic acid, and the like.

[0026] The term "pharmaceutically acceptable" when used with reference to a compound of the invention is intended to refer to a form of the compound that is safe for administration to a subject. For example, a free base, a salt form, a solvate, a hydrate, a prodrug or derivative form of a compound of this invention, which has been approved for mammalian use, via oral ingestion or any other route of administration, by a governing authority or regulatory agency, such as the Food and Drug Administration (FDA) of the United States, is pharmaceutically acceptable.

[0027] The phrase "effective amount" is intended to quantify the amount of each agent, which will achieve the goal of improvement in disorder severity and the frequency of incidence over treatment of each agent by itself, while avoiding adverse side effects typically associated with alternative therapies. The effective amount, in one embodiment, is

administered in a single dosage form or in multiple dosage forms.

[0028] Starting materials of the invention, are either known, commercially available, or can be synthesized in analogy to or according to methods that are known in the art. Many starting materials may be prepared according to known processes and, in particular, can be prepared using processes described in the examples. In synthesizing starting materials, functional groups in some cases are protected with suitable protecting groups when necessary. Protecting groups, their introduction and removal are described above. [0029] In synthesizing a compound of formulas I according to a desired procedure, the steps in some embodiment, are performed in an order suitable to prepare the compound, including a procedure described herein or by an alternate order of steps described herein, and in one embodiment, be preceded, or followed, by additional protection/deprotection steps as necessary. The intermediates in some embodiments are isolated or carried on in situ, with or without purification. Synthetic chemistry transformations and protecting group

methodologies (protection and deprotection) useful in synthesizing the inhibitor compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3 ^rd edition, John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); A. Katritzky and A. Pozharski, Handbook of Heterocyclic Chemistry, 2 ^nd edition (2001); M. Bodanszky, A. Bodanszky, The Practice of Peptide Synthesis,

Springer- Verlag, Berlin Heidelberg (1984); J. Seyden-Penne, Reductions by the Alumino- and Borohydrides in Organic Synthesis, 2 ^nd edition, Wiley-VCH, (1997); and L. Paquette, editor, Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995).

[0030] The compounds of this invention in some embodiments also are represented in multiple tautomeric forms. The invention expressly includes all tautomeric forms of the compounds described herein.

[0031] The compounds in one embodiment also occur in cis- or trans- or E- or Z- double bond isomeric forms. All such isomeric forms of such compounds are expressly included in the present invention.

INDICATION

[0032] The present invention provides compounds which are capable of modulating one or more signal transduction pathways comprising, but not limited to ALK.

[0033] By the term "modulating," it is meant that the functional activity of the pathway (or a component of it) is changed in comparison to its normal activity in the absence of the compound. This effect includes any quality or degree of modulation, including, increasing, agonizing, augmenting, enhancing, facilitating, stimulating, decreasing, blocking, inhibiting, reducing, diminishing, antagonizing, etc.

[0034] The compounds of the present invention can also modulate one or more of the following processes, including, but not limited to, e.g., cell growth (including, e.g., differentiation, cell survival, and/or proliferation), tumor cell growth (including, e.g., differentiation, cell survival, and/or proliferation), tumor regression, endothelial cell growth (including, e.g., differentiation, cell survival, and/or proliferation), angiogenesis (blood vessel growth), lymphangiogenesis (lymphatic vessel growth), and/or hematopoiesis (e.g., T- and B- cell development, dendritic cell development, etc.).

[0035] While not wishing to be bound by any theory or mechanism of action, it has been found that compounds of the present invention possess the ability to modulate kinase activity. The methods of the present invention, however, are not limited to any particular mechanism or how the compounds achieve their therapeutic effect. By the phrase "kinase activity," it is meant a catalytic activity in which a gamma-phosphate from adenosine triphosphate (ATP) is transferred to an amino acid residue (e.g., serine, threonine, or tyrosine) in a protein substrate. A compound can modulate kinase activity, e.g., inhibiting it by directly competing with ATP for the ATP -binding pocket of the kinase, by producing a conformational change in the enzyme's structure that affects its activity (e.g., by disrupting the biologically-active three- dimensional structure), by binding to and locking the kinase in an inactive conformation, etc.

FORMULATIONS AND METHOD OF USE

[0036] The amount of compound(s) which is/are administered and the dosage regimen for treating cancer with the compounds and/or compositions of this invention depends on a variety of factors, including the age, weight, sex and medical condition of the subject, the type of disease, the severity of the disease, the route and frequency of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. A daily dose of about 0.01 to 500 mg/kg, advantageously between about 0.01 and about 50 mg/kg, more advantageously about 0.01 and about 30 mg/kg, even more advantageously between about 0.1 and about 10 mg/kg may be appropriate, and should be useful for all methods of use disclosed herein. The daily dose can be administered in one to four doses per day.

[0037] While it may be possible to administer a compound of the invention alone, in the methods described, the compound administered normally will be present as an active ingredient in a pharmaceutical composition. Thus, in another embodiment of the invention, there is provided a pharmaceutical composition comprising a compound of this invention in combination with a pharmaceutically acceptable carrier, which includes diluents, excipients, adjuvants and the like (collectively referred to herein as "carrier" materials) as described herein, and, if desired, other active ingredients. A pharmaceutical composition of the invention may comprise an effective amount of a compound of the invention or an effective dosage amount of a compound of the invention. An effective dosage amount of a compound of the invention includes an amount less than, equal to or greater than an effective amount of the compound; for example, a pharmaceutical composition in which two or more unit dosages, such as in tablets, capsules and the like, are required to administer an effective amount of the compound, or alternatively, a multi-dose pharmaceutical composition, such as powders, liquids and the like, in which an effective amount of the compound is administered by administering a portion of the composition.

ROUTES of ADMINSTRATION

[0038] Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary, transmucosal, transdermal, vaginal, otic, nasal, and topical administration. In addition, by way of example only, parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injections.

[0039] The compounds of the invention may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the blood stream directly from the mouth. Formulations suitable for oral administration include solid formulations such as tablets, capsules containing particulates, liquids, or powders, lozenges (including liquid-filled), chews, multi- and nanoparticulates, gels, solid solution, liposome, films (including muco-adhesive), ovules, sprays and liquid formulations.

[0040] The compounds of the invention may also be used in fast-dissolving, fast- disintegrating dosage forms such as those described in Expert Opinion in Therapeutic Patents, 11 (6), 981-986 by Liang and Chen (2001), the disclosure of which is incorporated herein by reference in its entirety.

[0041] Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. Thus compounds of the invention may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active compound. Examples of such formulations include drug-coated stents and PGLA microspheres.

COMBINATIONS

[0042] While the compounds of the invention can be dosed or administered as the sole active pharmaceutical agent, they can also be used in combination with one or more compounds of the invention or in conjunction with other agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are administered simultaneously or sequentially at different times, or the therapeutic agents can be given as a single composition.

[0043] The synthesis of compounds in the invention was described in the following Scheme 1.

The amino group of A can be coupled with amino acid B (HO-R2 -protected) which amino- protected by BOC to generate C. The amino-protected group of C was deprotected to generate I.

Scheme 1

Proton NMR Spectra

[0044] Unless otherwise indicated, all 1H NMR spectra were run on a Varian series Mercury 300, 400, 500 MHz instrument or a Bruker series 400, 500 MHz instrument. Where so characterized, all observed protons are reported as parts-per-million (ppm) downfield from tetramethylsilane (TMS) or other internal reference in the appropriate solvent indicated. Abbreviations

DMF means N,N-dimethylformamide.

DCM means dichloromethane.

DIPEA means diisopropyl ethylamine.

DCC means Dicyclohexylcarbodiimide.

THF means tetrahydrofuran.

EA means ethyl acetate.

m-CPBA means meto-Chloroperoxybenzoic acid.

Boc-Gly-OH means N-(te/t-Butoxycarbonyl)glycine.

BOP means (Benzotriazol-l-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate. Pd(dppf)Cl ₂ means [1,1 ' -Bis(diphenylphosphino)ferrocene]dichloropalladium. SYNTHEIS OF COMPOUNDS

[0045] The synthesis of compounds in the invention was described in the Scheme 2.

Scheme 2

Example 1: The synthesis of (R)-2-amino-5-bromo-3-(l-(2,6-dichloro-3- fluorophenyl)ethoxy)pyridine 1-oxide (Compound 2).

[0046] To a solution of (R)-5-bromo-3-(l-(2,6-dichloro-3-fluorophenyl)ethoxy)pyridin -2- amine (1.0 g, 3.0 mmol, 1.0 eq) in DCM 50 mL at 0 °C was added m-CPBA (546.6 mg, 3.2 mmol, 1.05 eq). The reaction was stirred at 0 °C for 30 minutes, then concentrated under vacuum. The resulting dark oil was dissolved in ethyl acetate, and purified by flash column chromatography on silica gel to give (R)-2-amino-5-bromo-3-(l-(2,6-dichloro-3- fluorophenyl)ethoxy)pyridine 1 -oxide as an off white solid 850 mg.

Example 2: The synthesis of (R)-2-amino-5-(l-(l-(tert-butoxycarbonyl)piperidin-4-yl)- lH-pyrazol-4-yl)-3-(l-(2,6-dichloro-3-fluorophenyl)ethoxy)py ridine 1-oxide (Compound 4).

oc

[0047] A mixture of tert-butyl 4-(4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-lH- pyrazol-1- yl) piperidine-l-carboxylate (Compound 3) (760 mg, 2.0 mmol, 1.0 eq), (R)-2- amino-5-bromo-3-(l-(2,6-dichloro-3-fiuorophenyl)ethoxy)pyrid ine 1-oxide (943 mg, 2.5 mmol, 1.25 eq), and K ₂ CO ₃ (636 mg, 6.0 mmol, 3.0 eq) were added with DMF (20 mL), water (5 mL), and Pd(dppf)Cl ₂ (73 mg, 0.1 mmol, 0.05 eq) under nitrogen. The reaction was purged with nitrogen for 2 minutes, and then heated at 100 °C overnight. The reaction was cooled to room temperature, poured into water (100 mL), and extracted with ethyl acetate (6 x 50 mL). The combined organic layers were washed with brine, dried (MgSC^), filtered, and concentrated. The crude product was purified by flash column chromatography on silica gel to give the (R)-2-amino-5-(l-(l-(tert-butoxycarbonyl)piperidin-4-yl)-lH- pyrazol-4-yl)-3-(l- (2,6-dichloro-3-fiuorophenyl)ethoxy)pyridine 1-oxide 800 mg. Example 3: The synthesis of (R)-5-(l-(l-(tert-butoxycarbonyl)piperidin-4-yl)-lH- pyrazol-4-yl)-2-(2-(tert-butoxycarbonylamino)acetamido)-3-(l -(2,6-dichloro-3- fluorophenyl)ethoxy)pyridine 1-oxide (Compound 5)

c

[0048] To a solution of (R)-2-amino-5-(l-(l-(tert-butoxycarbonyl)piperidin-4-yl)-lH- pyrazol-4-yl)-3-(l-(2,6-dichloro-3-fluorophenyl)ethoxy)pyrid ine 1-oxide (800 mg, 1.41 mmol, 1.0 eq) in DMF 20 mL were added Boc-Gly-OH (249 mg, 1.31 mmol, 1.0 eq), BOP (753 mg, 1.7 mmol, 1.2 eq), and DIEA (458 mg, 3.55 mmol, 2.5 eq). The reaction was stirred at 0 °C for 4 hours, and then extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with brine, dried over MgS0 ₄ , filtered, and concentrated. Purification by flash column chromatography on silica gel to give (R)-5-(l-(l-(tert- butoxycarbonyl)piperidin-4-yl)- 1 H-pyrazol-4-yl)-2-(2-(tert- butoxycarbonylamino)acetamido)-3-(l-(2,6-dichloro-3-fluoroph enyl)ethoxy)pyridine 1-oxide 750 mg.

Example 4: The synthesis of (R)-tert-butyl 4-(4-(6-(2-((tert- butoxycarbonyl)amino)acetamido)-5-(l-(2,6-dichloro-3-fluorop henyl)ethoxy)pyridin-3- yl)-lH-pyrazol-l-yl)piperidine-l-carboxylate (Compound 6) c

[0049] To a solution of H ₂ 0 (10 niL) and EtOH (20 mL) were added (R)-5-(l-(l-(tert- butoxycarbonyl)piperidin-4-yl)- 1 H-pyrazol-4-yl)-2-(2-(tert- butoxycarbonylamino)acetamido)-3-(l-(2,6-dichloro-3-fluoroph enyl)ethoxy)pyridine 1 -oxide (550 mg, 0.776 mmol, l .Oeq), iron chips (348 mg, 6.21 mmol, 10 eq) and NH ₄ Cl ( 83 mg, 1.55 mol, 2.0 eq). The reaction was heated slowly to reflux for lhour and then cooled to room temperature. Ethyl acetate and water were added and the reaction was carefully neutralized by sodium carbonate. The combined organic layers were washed with saturated sodium bicarbonate, water, brine, dried over Na ₂ S0 ₄ , filtered and concentrated under vacuum to yield (R)-tert-butyl 4-(4-(6-(2-((tert-butoxycarbonyl)amino)acetamido)-5-(l-(2,6- dichloro-3- fluorophenyl)ethoxy)pyridin-3-yl)- 1 H-pyrazol- 1 -yl)piperidine- 1 -carboxylate 260 mg.

Example 5: The synthesis of (R)-2-amino-N-(3-(l-(2,6-dichloro-3-fluorophenyl)ethoxy)- 5-(l-(piperidin-4-yl)-lH-pyrazo -4-yl)pyridin-2-yl)acetamide (Compound 7)

To a solution of (R)-tert-butyl 4-(4-(6-(2-((tert-butoxycarbonyl)amino)acetamido)-5-(l- (2,6-dichloro-3 -fluorophenyl)ethoxy)pyridin-3 -yl)- 1 Et-pyrazol- 1 -yl)piperidine- 1 -carboxylate (230 mg, 0.84 mmol, 1.0 eq) in DCM (5 mL) was added 1,4-dioxane in HC1 (4M, 2ml) with an ice bath. The reaction mixture was stirred at room temperature for 4 hours and concentrated to dryness under vacuum to yield (R)-2-amino-N-(3-(l-(2,6-dichloro-3- fluorophenyl)ethoxy)-5 -( 1 -(piperidin-4-yl)- 1 H-pyrazol-4-yl)pyridin-2-yl)acetamide 180 mg as a HC1 salt. 1H-NMR(DMSO-d ₆ , 400Hz): 510.06 (br, 1H), 9.27-9.25 (br, 1H), 9.10-9.07 (br, 1H), 9.27-9.25 (m, 5H), 7.84 (s, 1H), 7.61-7.51 (br, 1H), 7.49-7.45 (br, 1H), 7.40 (s, 1H), 6.23-6.21 (br, 1H), 4.54-4.51 (br, 1H), 3.90-3.80 (br, 3H), 3.39-3.27 (m, 2H), 3.10-3.07 (m, 2H), 2.45-2.21 (m, 4H), 1.81(d, J=6.4Hz, 3H), MS m/z 507.6 [M+l].

[0050] Example 6: The synthesis of (R)-2-amino-N-(3-((R)-l-(2,6-dichloro-3- fluorophenyl)ethoxy)-5-(l-(piperidin-4-yl)-lH-pyrazol-4-yl)p yridin-2-yl)propanamide (Compound 8).

[0051] Compound 8 is prepared using (R)-2-aminopropanoic acid as the starting material following a similar procedure that described for the synthesis of Compound 7. MS m/z 521 [M+l].

Example 7: The synthesis of (R)-2-amino-N-(3-(l-(2,6-dichloro-3-fluorophenyl)ethoxy)- 5-(l-(piperidin-4-yl)-lH-pyrazol-4-yl)pyridin-2-yl)acetamide (Compound 9).

[0052] Compound 9 was prepared using (S)-2-aminopropanoic acid as the starting material following a similar procedure that described for the synthesis of Compound 7. MS m/z 521 [M+l]. IC ₅₀ < 0.2 μΜ for ALK inhibition.

Biological Assays:

[0053] As stated hereinbefore, the compounds defined in the present invention possess anti-proliferation activity. These properties may be assesses, for example, using one or more of the procedures set out below:

[0054] An in vitro assay which determines the ability of a test compound to inhibit ALK kinase.

[0055] kinase-tagged T7 phage strains were prepared in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage and incubated with shaking at 32°C until lysis. The lysates were centrifuged and filtered to remove cell debris. The remaining kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in lx binding buffer (20% SeaBlock, 0.17x PBS, 0.05% Tween 20, 6 mM DTT). All reactions were performed in polystyrene 96-well plates in a final volume of 0.135 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (lx PBS, 0.05%> Tween 20). The beads were then re-suspended in elution buffer (lx PBS, 0.05%> Tween 20, 0.5 μΜ non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR. Compound 7 has IC ₅₀ less than 0.2 μΜ for ALK inhibition.

[0056] A representative compound was assayed against different cancer cell lines such as NCIH460, SMMC7721, HCT116 or HEPG2 using the cell proliferation assays:

Cell proliferation assays:

1. 5x l0 ³ cells per well in 100 μΐ of medium were seeded in 96-well plate, here the medium contained 5% FBS 2. 24 hours later, 100 μΐ fresh medium was added with various concentrations of compounds into each well, while the medium here was free of FBS

3. After the cells were treated with compounds for 72 hours, 20 μΐ MTT (5mg/ml) was added into each well, and then the assay plate was incubated at 37 °C for 4 more hours.

4. The assay plate was centrifuged at 800 g for 10 min. The medium was aspirated, 150 μΐ DMSO was added into each well. The plate was gently shaked for 10 min.

5. The absorbance at 570 nm was measured on the plate reader.

6. IR% = (WC-WT)/WC * 100%.

[0057] The following Table A lists compound representative of the invention and its activity in cell assays.

[0058] Table A

It has been shown that the compound representative of the invention surprisingly possesses more potency on inhibiting cancer cells than Crizotinib by the results of Table A.