HK1126121B - Method of treating abnormal cell growth - Google Patents

Description

Method for treating abnormal cell growth

The benefit of U.S. provisional application No.60/742,766, filed on 5.12.2005 and U.S. provisional application No.60/864,637, filed on 7.11.2006, are claimed herein and are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to the use of c-Met/HGFR inhibitors for the treatment of abnormal cell growth in mammals. More specifically, the invention provides methods of treating a mammal having cancer.

Background

Formula (II)1The compound (R) -3- [1- (2, 6-dichloro-3-fluoro-phenyl) -ethoxy]-5- (1-piperidin-4-yl-1H-pyrazol-4-yl) -pyridin-2-ylamine

Is a effective small molecule inhibitor of c-Met/HGFR (hepatocyte growth factor receptor) kinase and ALK (anaplastic lymphoma kinase) activity. Compound (I)1Has pharmacological mediated antitumor properties through inhibition of c-Met/HGFR (involved in regulating the growth and metastatic progression of various tumor types) and ALK (associated with the pathogenesis of ALCL (anaplastic large cell lymphoma)). Compound (I)1Disclosed in international patent application No. PCT/IB2005/002837 and U.S. patent application No. 11/212,331, both of which are incorporated herein by reference in their entirety. Furthermore, the compounds1Are disclosed in international patent application No. PCT/IB05/002695 and U.S. patent application No. 11/213,039, both of which are incorporated herein by reference in their entirety.

Human cancers include a variety of diseases that collectively contribute to one of the leading causes of death in developed countries worldwide (American Cancer Society, Cancer Facts and regulations 2005. Atlanta: American Cancer Society; 2005). The development of cancer is caused by a complex series of multiple genetic and molecular events, including genetic mutations, chromosomal ectopic and karyotypic abnormalities (Hanahan D, Weinberg ra. thehallmarks of cancer. cell 2000; 100: 57-70). Although the underlying genetic causes of cancer are diverse and complex, it has been observed that each cancer type may exhibit common characteristics and require the ability to facilitate their development. Such desirable capabilities include deregulated cell growth, the ability to continue to recruit blood vessels (i.e., angiogenesis), and the ability of tumor cells to spread locally and metastasize to secondary organ sites (Hanahan D, Weinberg RA. the hallmarks of cancer. cell 2000; 100: 57-70). Thus, the ability to identify novel therapeutic agents that 1) inhibit molecular targets that can change during cancer development, or 2) target multiple processes common to cancer development in multiple tumors is an important unmet need.

A large body of literature suggests that c-Met/HGFR is one of the RTKs that are most frequently mutated or otherwise aberrantly activated in various human cancers (Christensen JG, Burrows J, Salgia R.c-Met as a target in human cancers and mutation of inhibition for therapeutic intervention. cancer Letters 2005; 225: 1-26). The types of tumors in which genetic engineering of c-Met/HGFR by mutation or gene amplification is reported include, but are not limited to, oncological indications with important, unmet medical needs, such as renal cancer, metastatic colorectal cancer, glioma, non-small cell lung cancer, gastric cancer, and head and neck cancer (Christensen JG, Burrows J, Salgia R.c-Met a target in human cancer and mutation of the cancer bit for therapeutic intervention. cancer Letters 2005; 225: 1-26).

HGFR mutations have been found to be associated with kidney Cancer (see, for example, L.Schmidt, K.junker, N.Nakaigawa, T.Kinjierski, G.Weirich, M.Miller et al, Novelmutation of the MET pro-Oncogene in platelet recalecanics, Oncogene 1999; 18: 2343. sup. 2350; L.Schmidt, F.M.Duh, F.Chen, T.Kishida, G.Glenn, P.Choyke et al, Germin and molecular dynamics in the tyrosine kinase of the protein pro-Oncogene in platelet recalecanic, Nat.Genet al, 16: Schmitt.1997; 16: 68-73; L.K.junker, G.Wei. promoter, G.japonica, G.9. promoter et al; Yeast protein kinase, protein 1729. mu.58. promoter et al; Yeast et al, Cancer promoter, protein 1729. see, Cancer # 2. wild et al). HGFR mutations are associated with head and neck cancers (see, for example, M.F.Di Renzo, M.Olivero, T.Martone, A.Maffe, P.Maggiora, A.D.Stefani et al, colloidal mutations of the MET on gene area selected reduced quantitative peptides of human HNSC cartomans, Oncogene 2000; 19: 1547. minus 1555; D.M.Aebrosold, O.Landt, S.Berthou, G.Gruber, K.T.beer, R.H.Greiner, Y.Zimmer, preliminary and clinical genes Y1253D-activating point mutation of tissue culture medium of 8523; the M.F.Di Renzo, M.Olivero, T.Martone, A.Maffe, P.Mag.P.Mag, A.D.Stefani et al, materials of human and clinical genes, Met 125853-activating point mutation of tissue culture medium 8523; 19: 8523. Ongkuwann et al). HGFR mutations are also associated with lung Cancer (see, e.g., P.C.Ma, T.Kijima, G.Maulik, E.A.Fox, M.Sattler, J.D.Griffin et al, c-methyl analysis in small cell size Cancer: noveljjjjjjxambremberambose registration, Cancer Res.2003; 63: 6272-.

In addition, HGFR mutations have been associated with other indications, including, but not limited to, childhood hepatocellular carcinoma, human gastric cancer, gastric cancer of the hard-cancerous type, colorectal cancer, and malignant melanoma. (see, for example, W.S. park, S.M.Dong, S.Y.Kim, E.Y.Na, M.S.shin, J.H.Pi et al, colloidal microorganisms in the enzyme domain of the Met/cellulose growth promoter vector in Cancer Res.1999; 59: 307. 310; J.H.Lee, S.U.Han, H.Cho, B.J.Di, B.Gerrard, M.Decan et al, alpha. promoter in molecular polypeptide in molecular promoter, colloidal gene 2000; 4947. 4953; A.Lorenza to, M.O.Ocular promoter, S.M.S.M.M.S.M.Dong, S.M.M.S.M.J.S.M.S.M.M.S.M.M.S.M.M.S.M.M.S.J.M.M.S.M.M.J.M.P.S.S.S.J.P.S.S.S.J.S.S.M.S.S.M.J.P.S.S.S.S.S.S.S.S.S.S.M.J.J.1.P.P.P.P.P.P.P.A.P.P.A.P.P.P.A.P.P.A.A.P.P.P.P.A.A.P.A.A.A.A.A.P.P.P.A.A.P.P.P.A.A.P.P.A.A.P.A.P.P.P.P.P.P.A.A.P.P.P.P.P.P.P.P.P.P.P.P.P.P.A.P.P.P.P.P.P.P.P.P.P.P.P.P.A.A.No. No. 1.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.P.No. No. P.P.P.P.P.P.P.P.P.P, cancer res.1995; 1: 147-; T.Hara, A.Ooi, M.Kobayashi, M.Mai, K.Yanagihara, I.Nakanishi, Amplification of c-myc, K-sam, adc-met in gastic cans: detection by fluorescence in localization, lab. invest.1998; 78: 1143-1153).

The relationship between The function of HGFR mutations and The oncogenic potential has also been determined (see, for example, M.Jeffers, L.Schmidt, N.Nakaigawa, C.P.Webb, G.Weirich, T.Kishida et al, Activating mutations for The tyrosine kinase in human cancer, Proc.Natl Acad.Sci.USA 1997; 94: 11445-.

Finally, HGFR mutations are associated with and studied in mouse tumors (see, for example, H.Takayama, W.J.LaRochelle, R.Sharp, T.Otsuka, P.Kriebel, M.Anver et al, variant situated in associated with expression vector expression, C.Natl Act.Sci. USA 1997; 94: 701 and 706; T.Otsuka, H.Takayama, R.Sharp, G.Celli, W.J.Larotalle, D.P.2004 Bottaro et al, c-expression vector of expression vector, Met strain of protein, Met strain of strain, strain of strain.

NPM-ALK is an oncogenic fusion protein variant of anaplastic lymphoma kinase, which is produced by chromosomal ectopic events and is implicated in the pathogenesis of human anaplastic large Cell lymphoma (Pulford K, Morris SW, Turturo F. anaplastic lymphoma kinase proteins ingowth control and cancer r.J Cell physiology 2004; 199: 330-58). The role of aberrant expression of constitutively active ALK chimeric proteins in the pathogenesis of ALCL is well established (Weihua Wan et al, anticancer enzyme activity infection for the promotion and survival of anticancer cell lymphoma cells, blood First Edition Paper, prepublished online October 27, 2005; DOI 10.1182/blood-2005-08-3254).

Inappropriate activation of c-Met/HGFR (including wild-type c-Met) is also associated with dysregulation of various tumor oncogenic processes, such as mitosis, survival, angiogenesis, invasive growth and especially metastatic processes (Christensen et al, 2005). Furthermore, expression of c-Met and HGF (the only high affinity ligands thereof) was shown to be associated with poor prognosis or metastatic development in a variety of major human cancers (Christensen et al, 2005). NPM-ALK is associated with dysregulation of cell proliferation and apoptosis in ALCL lymphoma cells (Pulford et al, 2004).

Disclosure of Invention

In one embodiment, the present invention provides a method of treating abnormal cell growth in a mammal in need of such treatment, comprising: administering to said mammal a therapeutically effective amount of a compound of formula (la)1The compound of (1):

or a pharmaceutically acceptable salt thereof.

In another embodiment, the mammal is a human. In another embodiment, the mammal is a dog.

In another embodiment, the abnormal cell growth is mediated by at least one genetically engineered tyrosine kinase. In another embodiment, the abnormal cell growth is mediated by hepatocyte growth factor receptor (c-Met/HGFR) kinase or Anaplastic Lymphoma Kinase (ALK). In another embodiment, the abnormal cell growth is mediated by hepatocyte growth factor receptor (c-Met/HGFR) kinase. In another embodiment, the abnormal cell growth is mediated by Anaplastic Lymphoma Kinase (ALK).

In another embodiment, the abnormal cell growth is cancer. In another embodiment, the cancer is selected from the group consisting of lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, epidermal or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, tumors of the Central Nervous System (CNS), primary CNS lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, and combinations thereof.

In yet another embodiment, the cancer is selected from the group consisting of: non-small cell lung cancer (NSCLC), squamous cell carcinoma, hormone refractory prostate cancer, papillary renal cell carcinoma, colorectal adenocarcinoma, neuroblastoma, Anaplastic Large Cell Lymphoma (ALCL), and gastric carcinoma.

In yet another embodiment, the compound or pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition comprising the formula1And at least one pharmaceutically acceptable carrier.

In yet another embodiment, the present invention provides a method of treating a disease or disorder by administering a compound of formula 1:

or a pharmaceutically acceptable salt thereof, inhibits c-Met/HGFR kinase activity in cells.

In yet another embodiment, the cell is selected from the group consisting of: a549 human lung carcinoma, GTL-16 human gastric carcinoma, HT29 human colon carcinoma, Co1O205 human colon carcinoma, A498 human kidney carcinoma, 786-O human kidney carcinoma, MBA-MD-231 human breast carcinoma, Madlin-Darby canine kidney (MDCK) epithelial cells, MDCK cells engineered to express P-glycoprotein (MDCK-MDR1), mIMCD3 mouse kidney epithelium, HUVEC (human umbilical vein endothelial cells), Caki-1 kidney carcinoma, and NIH-3T3 cells engineered to express human wild-type c-Met/HGFR and mutant c-Met/HGFR including HGFR-V1092I, HGFR-H109 1094R, HGFR-Y1230C, and HGFR-M1250T.

Drawings

FIG. 1: compound (I)1ATP-competitive inhibition of recombinant human c-Met/HGFR kinase activity.

FIG. 2: to tumors with established GTL-16Athymic mice were orally administered compounds at the indicated doses1Or vehicle alone for 11 days. FIG. 2A: studies investigating the inhibition of c-Met/HGFR phosphorylation in GTL-16. FIG. 2B: studies on the inhibition of GTL-16 tumor growth were explored.

FIG. 3: to a tumor (150 mm) with a defined U87MG tumor³) The athymic mice administered the compound orally at the indicated dose1Or vehicle alone for 9 days. FIG. 3A: study on tumor growth inhibition was explored. FIG. 3B: studies investigating the inhibition of c-Met/HGFR phosphorylation.

FIG. 4: daily oral administration of Compounds to athymic mice with Large definitive GTL-16 tumor xenografts1. FIG. 4A: degeneration of large established GTL-16 tumor xenografts in athymic mice. FIG. 4B: daily oral administration of compounds1Body weight of the mice.

FIG. 5: daily oral administration of Compounds to athymic mice with defined NCI-H441 or PC-3 tumor xenografts1. FIG. 5A: tumor regression in athymic mice with established NCI-H441. FIG. 5B: tumor regression in athymic mice with established PC-3 tumor xenografts.

FIG. 6: compound (I)1Antitumor efficacy in the NPM-ALK-dependent lymphoma model (Karpas 299 ALCL model). FIG. 6A: study on tumor growth inhibition was explored. FIG. 6B: studies on inhibition of NPM-ALK phosphorylation were explored.

Detailed Description

All references herein to the compounds of the invention include references to salts, solvates, hydrates and complexes thereof and solvates, hydrates and complexes of salts thereof, including polymorphs, stereoisomers and isotopically labeled forms thereof, unless otherwise indicated.

Definition of

The term "abnormal cell growth" as used herein, unless otherwise indicated, refers to cell growth that is not dependent on normal regulatory mechanisms (e.g., loss of contact inhibition).

As used herein, unless otherwise indicated, the term "treating" means reversing, alleviating, inhibiting the exacerbation of, or preventing the disease or condition to which such term applies or one or more symptoms of such disease or condition. The term "treatment" as used herein refers to the therapeutic activity of "treating" as defined immediately above, unless otherwise indicated.

The term "pharmaceutically acceptable salts" as used herein includes both acid addition salts and base addition salts (including disalts).

Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include: acetate, aspartate, benzoate, benzenesulfonate, bicarbonate/carbonate, bisulfate/sulfate, borate, camphorsulfonate, citrate, edisylate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, hydroxybenzoylbenzoate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthenate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/biphosphate/dihydrogenphosphate, saccharate, stearate, succinate, tartrate, dihydrogenphosphate, saccharate, fumarate, bromate, fumarate, bromate, and bromate, Tosylate, and trifluoroacetate.

Suitable base addition salts are formed from bases which form non-toxic salts. Examples include aluminum, arginine, benzathine (benzathine), calcium, choline, diethylamine, dialcohol, glycinate, lysine, magnesium, meglumine, alkanolamine (olamine), potassium, sodium, tromethamine and zinc salts.

A review of suitable pharmaceutically acceptable Salts is provided in Stahl and Wermuth, "Handbook of Pharmaceutical Salts: properties, Selection, and Use "(Wiley-VCH, Weinheim, Germany, 2002), the disclosure of which is incorporated herein by reference in its entirety.

Pharmaceutically acceptable salts of the compounds of the invention can be readily prepared by mixing together solutions of the compounds and the desired acid or base, as appropriate. The salt may be precipitated from the solution and collected by filtration or may be recovered by evaporation of the solvent. The degree of ionization of the salt can vary from fully ionized to almost non-ionized.

The compounds of the present invention may exist in both unsolvated and solvated forms. The term "solvate" as used herein refers to a molecular complex comprising a compound of the present invention and one or more pharmaceutically acceptable solvent molecules (e.g., ethanol). When the solvent is water, the term "hydrate" is used. Pharmaceutically acceptable solvates of the invention include those wherein the crystallization solvent may be isotopically substituted (e.g., D)₂O、d₆-acetone, d₆-DMSO) and solvates.

The invention also includes isotopically-labeled compounds, which are compounds and compounds of formula (I) except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature1The same is true. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, for example each²H、³H、¹³C、¹⁴C、¹⁵N、¹⁸O、¹⁷O、³¹P、³²P、³⁵S、¹⁸F and³⁶and (4) Cl. Compounds of the present invention containing the aforementioned isotopes and/or other isotopes of other atoms and pharmaceutically acceptable salts of such compounds are encompassed within the scope of the present invention. Certain isotopically-labelled compounds of the present invention, for example those into which³H and¹⁴compounds of the invention that are radioisotopes such as C may be used in drug and/or substrate tissue distribution assays. Containing tritium³H and carbon-14 i.e¹⁴The C isotope is particularly preferred for its ease of preparation and detectability. In addition, with, for example, deuterium²Heavier isotopic substitution of H may provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and thus may be preferred in certain circumstances. Isotopically-labelled compounds of the invention1Generally, they can be prepared by performing the procedures described for unlabeled compounds and substituting readily available isotopically labeled reagents for the non-isotopically labeled reagents.

Also included within the scope of the invention are complexes, such as clathrate compounds, drug-matrix inclusion complexes, wherein the drug and matrix are present in stoichiometric or non-stoichiometric amounts, as opposed to the solvates described above. The invention also encompasses complexes of drugs containing two or more organic and/or inorganic components, which may be in stoichiometric or non-stoichiometric amounts. The resulting complex may be ionized, partially ionized or unionized. For a review of this complex see J Pharm Sci by Haleblian,64(8) 1269-1288(1975, 8), the disclosure of which is incorporated herein by reference in its entirety.

Oral administration

The compounds of the invention may be administered orally. Oral administration may include swallowing (so the compound can enter the gastrointestinal tract) and/or buccal or sublingual administration (so the compound can enter the blood stream directly from the mouth).

Formulations suitable for oral administration include solid formulations (e.g., tablets), capsules containing granules, liquids, or powders, lozenges (including liquid filled), chewables, multi-and nanoparticles, gels, solid solutions, liposomes, films (including viscous binders), vaginal lozenges, sprays, and liquid formulations.

Liquid preparations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as a fill in soft or hard capsules and typically comprise a pharmaceutically acceptable carrier (for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil) and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by reconstitution of a solid contained, for example, in a sachet.

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

For tablet dosage forms, depending on the dosage, the drug may comprise from 1% to 80% by weight of the dosage form, more typically from 5% to 60% by weight of the dosage form. In addition to the drug, a disintegrant is typically included in the tablet. Examples of disintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropylcellulose, starch, pregelatinized starch, and sodium alginate. Generally, the disintegrant will comprise from 1 to 25 weight percent, preferably from 5 to 20 weight percent of the dosage form.

Binders are generally used to impart cohesive properties to the tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose and hydroxypropyl methyl cellulose. Tablets may also contain diluents such as lactose (monohydrate, spray-dried monohydrate, anhydrate and the like), mannitol, xylitol, glucose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.

Tablets may also contain surfactants (e.g., sodium lauryl sulfate and polysorbate 80) and glidants (e.g., silicon dioxide and talc), as appropriate. When included, the amount of surfactant may typically be from 0.2% to 5% by weight of the tablet, and the glidant may be from 0.2% to 1% by weight of the tablet.

Tablets also typically contain lubricants, for example, magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate and sodium lauryl sulfate. The lubricant is generally present in an amount of 0.25 to 10% by weight of the tablet, preferably 0.5 to 3% by weight.

Other conventional ingredients include antioxidants, coloring agents, flavoring agents, preservatives, and taste-masking agents.

Exemplary tablets comprise up to about 80% by weight of a drug, about 10% to about 90% by weight of a binder, about 0% to about 85% by weight of a diluent, about 2% to about 10% by weight of a disintegrant, and about 0.25% to about 10% by weight of a lubricant.

The tablet blend may be formed into tablets directly or by roller compaction. Alternatively, the tablet blend or portions of the blend may be wet-, dry-, or melt-granulated, melt congealed, or extruded prior to tableting. The final formulation may comprise one or more layers and may be coated or uncoated; or encapsulating.

Tablet formulations are detailed in "pharmaceutical dosage Forms" by h.lieberman and l.lachman: tablets, Vol.1 ", Marcel Dekker, N.Y., N.Y., 1980(ISBN 0-8247-6918-X), the disclosure of which is incorporated herein by reference in its entirety.

Solid formulations for oral administration may be formulated to be directly released and/or released in a modified manner. Modified release formulations include delayed release, sustained release, pulsatile release, controlled release, targeted release and programmed release.

Suitable modified release formulations are described in U.S. Pat. No.6,106,864. Details of other suitable delivery techniques (e.g., high energy dispersions and osmotic coated particles) are described in Verma et al, Pharmaceutical Technology On-line, 25(2), 1-14, (2001). The use of chewing gum to achieve controlled release is described in WO 00/35298. The disclosures of these references are all incorporated herein by reference in their entirety.

Parenteral administration

The compounds of the invention may also be administered directly into the bloodstream, muscle or internal organs. Suitable modes for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, and subcutaneous administration. Devices suitable for parenteral administration include needle (including microneedle) syringes, needle-free syringes, and infusion techniques.

Parenteral formulations are typically aqueous solutions which may contain excipients (e.g. salts, carbohydrates) and buffers (preferably to a pH of 3 to 9), but for some applications it may be more suitable to formulate them as sterile non-aqueous solutions or in dry form for use in combination with a suitable excipient (e.g. sterile, pyrogen-free water).

Preparation of parenteral formulations under sterile conditions (e.g., by freeze-drying) can be readily achieved using standard pharmaceutical techniques well known to those skilled in the art.

The solubility of the compounds of the invention for use in the preparation of parenteral solutions may be enhanced by means of suitable formulation techniques, for example the addition of solubilisers.

Formulations for parenteral administration may be formulated to be directly released and/or released in a modified manner. Modified release formulations include delayed release, sustained release, pulsatile release, controlled release, targeted release and programmed release. Thus, the compounds of the present invention may be formulated as solids, semi-solids, or thixotropic liquids for administration in an implanted depot form to provide modified release of the active compound. Examples of such formulations include drug-coated vascular stents and PGLA microspheres.

Topical application

The compounds of the invention may also be administered topically, i.e. transdermally or through the skin, to the skin or mucosa. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implantsSponges, fibers, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohols, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol, and propylene glycol. Optionally adding penetration enhancer; see, for example, J Pharm Sci by Finnin and Morgan,88(10) 955- "958 (10 months 1999). Other topical administration modes include by electroporation, iontophoresis, sonophoresis and microneedles or needle-free (e.g., Powderject)^TM、Bioject^TMEtc.) injection. The disclosures of these references are all incorporated herein by reference in their entirety.

Formulations for topical application may be formulated to be directly released and/or released in a modified manner. Modified release formulations include delayed release, sustained release, pulsatile release, controlled release, targeted release and programmed release.

Inhalation/intranasal administration

The compounds of the invention may also be administered intranasally or by inhalation, such administration being in the typical form: dry powders from dry powder inhalers (either alone, as a mixture (e.g. with lactose in dry blends), or as particles of a mixed component (e.g. with a phospholipid such as phosphatidylcholine)), as aerosol sprays from pressurised containers, pumps, sprayers, atomisers (preferably using electrohydrodynamic forces to generate a fine mist) or nebulisers (with or without the use of a suitable propellant, such as 1, 1, 1, 2-tetrafluoroethane or 1, 1, 1, 2,3, 3, 3-heptafluoropropane). For intranasal use, the powder may comprise a bioadhesive, for example, chitosan or cyclodextrin.

Pressurized containers, pumps, sprayers, atomizers or sprayers contain solutions or suspensions of the compounds of the invention consisting, for example, of ethanol, aqueous ethanol, or a suitable agent for dispersion, dissolution or extended release of the active substance, a propellant in the form of a solvent, and, as the case may be, a surfactant such as sorbitan trioleate, oleic acid, or polylactic acid.

Prior to use in dry powder or suspension formulations, the drug product is micronised to a size suitable for delivery by inhalation (typically less than 5 microns). This may be achieved by any suitable powderization method (e.g. spiral jet milling, fluidized bed jet milling, supercritical fluid processing) to form nanoparticles, high pressure homogenization, or spray drying.

Capsules (e.g., made from gelatin or HPMC), blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention, a suitable powder base (e.g., lactose or starch) and a performance-modifying agent (e.g., 1-leucine, mannitol, or magnesium stearate). Lactose may be anhydrous or in the form of a monohydrate, the latter being preferred. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

Solution formulations suitable for use in nebulizers using electrohydrodynamics to generate fine mist may contain from 1 μ g to 20mg of a compound of the invention per spray and the spray volume may vary from 1 μ l to 100 μ l. A typical formulation comprises a compound of the invention, propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents that may be used in place of propylene glycol include glycerol and polyethylene glycol.

Suitable flavoring agents (e.g., menthol and levomenthol), or sweetening agents (e.g., saccharin or saccharin sodium) may be added to those formulations of the invention intended for administration by inhalation/intranasal means.

Formulations for inhalation/intranasal administration may be formulated with, for example, poly (DL-lactic-co-glycolic acid) (PGLA) for direct release and/or modified release. Modified release formulations include delayed release, sustained release, pulsatile release, controlled release, targeted release and programmed release.

When dry powder inhalers and aerosols are used, the dosage unit is determined by means of a valve which can deliver a metered number. The device of the invention is typically set to administer a metered dose or "spray volume" containing a desired amount of a compound of the invention. The entire daily dose may be administered throughout the day in a single dose or, more usually, in divided doses.

Rectal/intravaginal administration

The compounds of the invention may be administered rectally or vaginally (e.g., in the form of suppositories, pessaries, or enemas). Cocoa butter is a conventional suppository base, but various alternatives may be used as appropriate.

Formulations for rectal or vaginal administration may be formulated to be directly releasable and/or releasable in a modified manner. Modified release formulations include delayed release, sustained release, pulsatile release, controlled release, targeted release and programmed release.

Eye (A)Topical application

The compounds of the invention may also be administered directly into the eye or ear, usually in the form of drops of micronized suspension or solution in isotonic, pH-adjusted sterile saline. Other formulations suitable for ocular or otic administration include ointments, biodegradable (e.g., absorbable gel sponges, collagen) and non-biodegradable (e.g., silicone) implants, wafers, lenses, and microparticles or porous systems (e.g., nonionic surfactant vesicles (niosomes) or liposomes). Polymers such as crosslinked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, cellulosic polymers (e.g., hydroxypropyl methylcellulose, hydroxyethyl cellulose, or methyl cellulose), or heteropolysaccharide polymers (e.g., gellan gum) may be incorporated with preservatives (e.g., benzalkonium chloride). Such formulations may also be delivered by iontophoresis.

Formulations for ocular/otic administration may be formulated to be released in real time and/or in a modified manner. Modified release formulations include delayed release, sustained release, pulsatile release, controlled release, targeted release or programmed release.

Other techniques

The compounds of the invention may be used in combination with soluble macromolecules (e.g., cyclodextrins and suitable derivatives thereof or polyethylene glycol containing polymers) to improve their solubility, dissolution rate, taste masking, bioavailability and/or stability when used in any of the above modes of administration.

For example, drug-cyclodextrin complexes are found to be generally useful in most dosage forms and routes of administration. Both inclusion and non-inclusion complexes may be used. As an alternative to direct complexation with the drug, cyclodextrins may be used as an auxiliary additive, i.e. as a carrier, diluent or solubiliser. Among the most commonly used for such purposes are alpha-, beta-, and gamma-cyclodextrins, examples of which are found in PCT publications WO 91/11172, WO 94/02518, and WO 98/55148, the disclosures of which are incorporated herein by reference in their entirety.

Dosage form

The amount of active compound administered will depend on the severity of the subject, disorder or condition being treated, the rate of administration, the disposition of the compound, and the judgment of the prescribing physician. However, an effective dose is between about 0.001mg/kg body weight/day to about 100mg/kg body weight/day, preferably about 0.01 mg/kg/day to about 35 mg/kg/day, administered in single or divided doses. For a 70kg human, this dose may be reduced to about 0.07 mg/day to about 7000 mg/day, preferably about 0.7 mg/day to about 2500 mg/day. In some cases, dosage levels below the lower limit of the aforesaid range may be more suitable, while in other cases still larger doses may be employed without causing any harmful side effects, wherein such larger doses are usually divided into several smaller doses for administration throughout the day.

Component type medicine box

Since it is desirable to administer a combination of active compounds, for example, for the purpose of treating a particular disease or condition, the scope of the present invention encompasses: two or more pharmaceutical compositions, at least one of which comprises a compound of the invention, may conveniently be combined in kit form suitable for the simultaneous administration of such compositions. Thus, the kit of the invention comprises two or more separate pharmaceutical compositions, at least one of which comprises a compound of the invention, and means for separately storing such compositions (e.g. a container, a split bottle or a split foil pack). Examples of such kits are the common blister packs used for tablets, capsules and packages such as these.

The kits of the invention are particularly suitable for administering different dosage forms (e.g., oral and parenteral), for administering separate compositions at different dosage intervals, or for titrating separate compositions from one another. To aid compliance, the kit will typically contain instructions for administration and may provide means to assist with memory.

Examples of the invention

In vitro assay

Materials and methods

In vitro methods

Biochemical kinase analysis method

Compounds were identified using the general procedure for monitoring NADH oxidation coupled with ATP conversion as follows1Biochemical K for inhibition of c-Met/HGFR kinase_iThe value is obtained. Compounds and kinase assay reagents were added to the test wells and incubated at 37 ℃ for 10 minutes. The assay was initiated by addition of the c-Met/HGFR enzyme. Enzyme inhibitors can reduce the activity of the enzyme measured. In a continuously coupled spectrophotometric assay, the ADP time-dependent production of the kinase was determined by NADH consumption rate analysis via measurement of the decrease in absorbance at 340 nm. When the kinase produces ADP, it can be reconverted to ATP by reaction with phosphoenolpyruvate and pyruvate kinase. Pyruvate is also produced in this reaction. Pyruvate is subsequently converted to lactate by reaction with lactate dehydrogenase, while NADH is converted to NAD. NADH had a measurable absorbance at 340nm while NAD did not. Thus, the analytical endpoint was measured spectrophotometrically at 340nm at the indicated time point.

Biochemical kinase analysis of cell signaling (Upstate)

The kinase was pre-diluted to 10 × working concentration before the analyte was added. Briefly, the substrates were dissolved in deionized water and diluted into working stock solutions except for histone H1(10 × working stock solution, in 20mM MOPS pH 7.4), PDKtide (10 × working stock solution, in 50mM Tris pH 7.0), and ATF2 (which is typically stored as 20 × working stock solution, in 50mM Tris pH 7.5, 150mM NaCl, 0.1mM EGTA, 0.03% Brij-35, 50% glycerol, 1mM benzamidine, 0.2mM PMSF, and 0.1% □ -mercaptoethanol). The biochemical enzyme of interest was then assayed in a 25. mu.l final reaction volume containing a mixture of 8mM OPS pH 7.0, 0.2mM EDTA, 50. mu. M EAIYAAPFAKKK, 10mM magnesium acetate and³²P-ATP (specific activity about 500cpm/pmol, if necessary concentrated) was incubated with 5 to 10mU of the selected enzyme. The reaction is initiated by adding a MgATP mixture. After incubation at room temperature for 40 minutes, the reaction was stopped by adding 5. mu.l of 3% phosphoric acid solution. 10 μ l of the reaction was then spotted on P30 filermat and washed three times for 5 minutes each in 75mM phosphoric acid and once in methanol, then dried and subjected to scintillation counting.

Cell lines

For evaluating compounds in vitro studies1The cell lines of (a) are as follows: a549 human lung cancer, GTL-16 human gastric cancer, HT29 human colon cancer, Colo205 human colon cancer, a498 human kidney cancer, 786-O human kidney cancer, MBA-MD-231 human breast cancer, Madlin-Darby canine kidney (MDCK) epithelial cells, MDCK cells engineered to express P-glycoprotein (MDCK-MDR1), mimmcd 3 mouse kidney epithelium, HUVEC (human umbilical vein endothelial cells); NIH-3T3 cells which are modified to express human wild type c-Met/HGFR and mutation c-Met/HGFR including HGFR-V1092I, HGFR-H1094R, HGFR-Y1230C and HGFR-M1250T. Cell lines used to assess inhibition of phosphorylation of other tyrosine kinases are as follows: KARPAS299, SU-DHL-1 and Jurkat human lymphoma cells, Human Umbilical Vein Endothelial Cells (HUVEC), human large vessel endothelial cells (HMVEC), Porcine Arterial Endothelial (PAE) cells engineered to express human VEGFR2, PDGFR β, TrkA and TrkB; engineered to express human Ron, Axl, SkyAnd NIH-3T3 cells of the EGFR/Tie-2 chimera; HEK293 cells engineered to express human IRK, Chinese hamster ovary (CHO-B) cells engineered to express human Ron, BaF3 cells engineered to express human BCR-Abl. All processed cell lines were formed at Pfizer; GTL-16 gastric cancer cells were donations from doctor Paolo Comoglio (University Torino Medical School, Candiolo, Italy); HUVEC and HMVEC (human large vessel endothelial cells) were purchased from Clonetics (Walkersville, Md.), and the other cells were from ATCC (Manassas, Va.). Unless otherwise indicated, cell culture reagents were purchased from Life technologies, Inc. (Gaithersburg, Md.). The cells were kept in a humid atmosphere at 37 ℃ with 5% to 10% CO₂And maintained using standard cell culture techniques.

Antibodies and growth factors

For evaluation of Compounds in vitro ELISA and immunoblot Studies1The antibodies of (a) are as follows: anti-total human c-Met/HGFR and anti-phosphorylated Zap70 antibodies were purchased from Zymed/Invitrogen, Carlsbad, CA; anti-total Ron, anti-total FGFR1, anti-total PDGFR β, anti-total Trk, anti-total Tie-2, and anti-phosphotyrosine (PY-20) antibodies were purchased from Santa Cruz biotechnology, Santa Cruz, CA; anti-Total Axl and anti-Total mouse c-Met/HGFR antibodies were purchased from R&D Systems, Minneapolis, MN; anti-total IRK antibodies were purchased from BDPharmingen, San Diego, CA; anti-VEGFR 2 antibodies were purchased from Novus Biologicals, Littleton, CO; anti-phospho-c-Met/HGFR, anti-total and-phospho ALK, anti-total c-ABL, anti-total and phospho Gab1, anti-total and phospho AKT, anti-total and phospho-MAPK 44/42, anti-phospho Raf, Mek1/2, P90RSK, and STAT5 antibodies were purchased from CellSignaling Technologies, Boston, MA.

Most growth factors used IN cell assays were purchased from R & D Systems, Minneapolis, MN, BDGF only from GibcoBRL/Invitrogen, Carlsbad, CA, and EGF from Roche Applied Science, Indiana polis, IN.

Cellular kinase phosphorylation assay

Starvation with various seraCell implementation for direct determination of Compounds1Cell assays (i.e., ELISA or immunoblot) that inhibit ligand-dependent or constitutive kinase phosphorylation.

Cell preparation

Cells were seeded in 96-well plates in growth medium (medium supplemented with 10% fetal bovine serum-FBS) and cultured overnight at 37 ℃ to promote attachment to assay plates. After attachment, the growth medium was removed and the cells were cultured in serum-free medium (containing 0.04% BSA). To the compound1Performing serial dilution, and adding appropriate control or specified concentration of compound1Added to each well and the cells were incubated at 37 ℃ for 1 hour. In experiments to study ligand-dependent phosphorylation of RTKs, the corresponding growth factors (e.g., HGF, MSP, Gas6, EGF, NGF, BDNF, insulin, VEGF, or PDGF BB) were added to the cells for 8 to 20 minutes. H.sub.handbay of Biological Chemistry 279: 28766-28770(2004)) as described in (Konishi, A., Aizawa, T.Mohan, A., Korshunov, V.A., and Berk, B.C., Hydrogenooxide activities the Gas6-Axl pathway in cellular tissue the Journal of Biological Chemistry 2004)₂O₂To stimulate human Axl phosphorylation in HUVEC cells. Constitutive kinase phosphorylation was measured on cell lines with constitutively active kinase activity without the addition of exogenous ligands (e.g., c-Met/HGFR in GTL-16 cells, NPM-ALK in Karpas299 cells, Ron in Ron-CHO-B cells, and BCR-Abl in BCR-Abl BaF3 cells). Contacting a cell with a compound1And/or the appropriate ligand for a specified period of time, the cells were incubated with 1mM Na in HBSS₃VO₄Once washed and then lysed using lysis buffer (Cell Signaling Technologies, Boston MA).

ELISA assay

Phosphorylation of the protein kinase of interest was assessed by sandwich ELISA using a capture antibody specific for each protein and a detection antibody specific for phosphorylated tyrosine residues. In each ELISA assay, the assay will be performed using the appropriate RTK ligand and/or compound1Various cell lines treatedThe resulting protein lysate was transferred to a 96-well plate precoated with the corresponding antibody (including anti-c-Met/HGFR, -Ron, -Axl, -Sky, -IR, -Tie2, -KDR, -PDGFR β, -Zap70, etc.). Antibody-coated plates were incubated overnight at 4 ℃ in the presence of protein lysates and washed seven times with 1% Tween 20 in PBS. HRP-PY20 (horseradish peroxidase-conjugated anti-total-phosphotyrosine, Santa Cruz Biotechnology, Santa Cruz, Calif.) was diluted 1: 500 in blocking buffer (Pierce, Rockford, Ill.) and added to each plate for 30 min. Plates were then washed and TMB peroxidase substrate (Bio-Rad laboratories, Hercules CA) was added to initiate the HRP-dependent colorimetric reaction. The reaction was carried out by adding 0.09N H₂SO₄And (6) terminating. Each plate was measured at OD-450nm using a spectrophotometer. IC calculation in Excel-based templates by concentration-response curve fitting Using four-parameter analysis₅₀The value is obtained.

Immunoblotting

Compounds were also measured by immunoblotting1The ability to inhibit cellular kinase phosphorylation. Compounds for use as described above1The cells were treated and lysed in a dilution of serum-free medium to obtain the protein extract. Protein concentrations of cell lysates were normalized by BSA assay (Pierce, Rockford, IL) and immunoprecipitated with specific antibodies for proteins of interest. The immunoprecipitated proteins were separated by SDS-PAGE and immunoblotting with anti-phosphotyrosine antibody was performed to determine the relative amount of phosphorylated protein at each drug concentration. This immunoblotting method is also used to determine the total protein content of the molecule of interest.

Cell proliferation and survival assays

Cell proliferation assay

Tumor cells were seeded at low density in 96-well plates in growth medium (medium supplemented with 10% fetal bovine serum-FBS) and cultured overnight at 37 ℃. The next day, growth medium was removed and cells were cultured in serum-free medium (0% FBS and 0.04% BSA). Carrying out the compound1The serial dilution of (1) is to be properAt the given or indicated concentration1Added to each well and the cells were incubated at 37 ℃ for 24 to 72 hours. MTT assays (Promega, Madison, Wis.) were then performed to determine relative cell numbers. IC calculation by concentration-response curve fitting Using four-parameter analysis₅₀The value is obtained.

Apoptosis/cell survival assay

GTL-16 cells were seeded at 40,000 cells/well in 96-well plates. PF-02341066 or vehicle was added to the serum-free medium at the indicated concentration in each well. Cells were incubated at 37 ℃ with 5% CO₂The cells were incubated for 48 hours. Apoptosis was detected using ssDNA apoptosis ELISA kit (Chemicon International).

HGF stimulated HUVEC survival assay

HUVEC cells (passage 3) were grown to confluence in EGM2 medium (Walkersville, MD). Cells were seeded at high density (20,000 to 30,000 per well) in 96-well plates in EGM2 medium and incubated for 5 to 6 hours to allow cells to attach. After attachment, cells were cultured in serum-free medium (Cell Applications, San Diego, Calif.) at 37 ℃ with 5% CO₂Incubate overnight. The following day, cells were exposed to starvation medium (Cell Applications, San Diego, CA) for 5 hours. The compound1Serial dilutions were made in serum-free medium and appropriate controls or indicated concentrations of compounds were added1Added to each well. After 1 hour, human recombinant HGF (R)&DSystems, Minneapolis, MN) were added to the indicated wells to achieve a final concentration of 100 ng/ml. MTT assay (Promega) was then performed after 48 to 72 hours to determine relative cell numbers. IC calculation by concentration-response curve fitting Using four-parameter analysis₅₀The value is obtained.

HGF-dependent cell migration and invasion assay

NCI-H441 cell migration and matrigel invasion assay

Compounds were identified using a commercially available cell migration and invasion System (BD Biosciences, San Jose, Calif.)1Effect on HGF-stimulated NCI-H441 human non-small cell lung cancer cell migration and matrigel invasionAnd (6) sounding. Cells in the logarithmic growth phase were trypsinized and suspended at a density of 400,000 cells/ml in serum-free medium (containing 0.04% BSA). Compound (I)1Serial dilutions were performed in serum-free medium to give the indicated concentrations of the compounds1Add to the suspension cells and incubate the cells at room temperature for 30 minutes. Designated control or treated suspension cells (0.5ml) were added to each migration or invasion chamber (i.e., plate insert). In addition, 25ng/ml HGF (0.75ml) was added to the lower well of each companion plate as a chemical attractant to attract cells from the migrating or invading chamber plate insert inserted on top of the companion plate and the cells were incubated at 37 ℃ for 22 hours. Cells invading or migrating to the wells in the lower layer of the plate were then fixed and stained for nuclei (1. mu.g/ml DAP1 in 100% MeOH) for 15 min at 37 ℃. The cells were then washed twice with TBS solution. Microscope images were taken from each well and the number of migrated or invaded cells was determined under each condition using Image-Pro Plus software (MediaCybernetics, Silver Spring, MD). IC calculation by concentration-response curve fitting Using four-parameter analysis₅₀The value is obtained.

HUVEC matrigel (matrigel) invasion assay

Compounds were identified using the ACEA RT-CES System (ACEA biologicals, San Diego, Calif.)1Effect on in vitro HUVEC matrigel invasion. ACEA electrosensitive 96-well plates were coated with 50. mu.l of 10.001% fibronectin and 100ng/ml HGF (in PBS) and incubated at 37 ℃ for 1 hour and 4 ℃ for 30 minutes. After washing each plate with PBS at 4 ℃, matrigel (BDbiosciences, San Jose, Calif.) was diluted 1: 40 in compounds supplemented with HGF (100ng/ml) and/or different concentrations1To starvation medium (SM, Cell Applications, san diego, CA), add it (50 μ l) to the designated wells and allow it to solidify at 37 ℃ for 2 hours. HUVEC cells were cultured in serum-free medium (Cell Applications, San Diego, CA) for 5 hours and then in SM for 2 hours. The cells were subsequently collected in SM at 60,000 cells/ml and used with 100ng/ml HGF and/or the appropriate compound1The treatment was carried out at 37 ℃ for 30 minutes. HUVEC cell suspension (100. mu.l) was transferred to matrigel in designated wells of coated ACEA plates under designated conditionsOn top of the layer. Then at 37 ℃, 95% air: 5% CO₂The ACEA plates were next collected into an ACEA Device Station and monitored by an ACEA Sensor Analyzer for 48 hours in real time. Electronic sensors embedded in the bottom of the ACEA plate detect HUVEC cells invading through the matrigel. Using ACEA RT-CES^TMThe assembly software determined the relative number of invading HUVEC cells (cell index). IC calculation by concentration-response curve fitting Using four-parameter analysis₅₀The value is obtained.

MDCK cell dispersion assay

MDCK cells were seeded at low density (25 cells/well) in 96-well plates in media supplemented with 10% FBS and grown until small colonies of 10 to 15 cells appeared. The compounds were then added to HGF (50ng/ml) at various concentrations diluted in growth medium1Stimulating the cells in the presence. After overnight incubation, colonies were fixed and stained with 0.2% crystal violet in 10% buffered formalin and visually assessed for dispersion at each concentration.

HMVEC angiogenic assay

500 HMVECs were added to EGM-2 medium containing 0.24% methylcellulose and transferred to U-bottom 96-well plates to form spheroids overnight. Spheroids were collected and mixed into a 2mg/ml fibrinogen solution containing 4% to 8% FBS + -compound in 48-well plates coated with thrombin (2 ml 5,000U/ml). The resulting 3-D fibrin gel was covered with EGM-2 containing 4% to 8% FBS and heated at 37 deg.C with 95% air/5% CO₂The following incubation was performed. Endothelial tube formation was observed daily under an inverted microscope. Images were acquired on day 7 with a digital camera (olympus bx60) attached to the microscope. Adding compounds at several concentrations1And the angiogenesis was assessed visually.

Cell cycle and apoptosis analysis by flow cytometry

Evaluation of compounds by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA)1Effects on NPM-ALK-dependent cell cycle distribution and apoptosis of human lymphoma cells. In the growth cultureCompounds for use in nutrient (RPMI + 10% FBS)1Karpas299 and SU-DHL-1 human lymphoma cells were treated for 24 to 48 hours. The cells were washed twice with PBS, fixed and permeabilized with BDCytofix/Cytoperm solution for 20 min at 4 ℃. Cell cycle distribution and apoptosis of lymphoma cells were assessed using the CycloTest Plus DNA reagent kit (BD Biosciences). Using this kit, cells were washed twice with 1 × BD Perm/Wash buffer, nuclei were separated by adding non-ionic detergent and trypsin, DNA content was visualized by adding propidium iodide, and cells were analyzed by flow cytometry. The DNA content was assessed with Cell Quest Pro (ploidy analysis to determine% of cells in each Cell cycle) and analyzed with ModFitLT software (BD Biosciences). An apoptosis peak (A) is detected as described in (Darzynkiewicz, Z., Bruno, S., Del-Bino, G., Gorczyca, W., Hotz, M.S., Lassota, P., and Trganos, F., cytometric 13: 795-₀) Is defined as being lower than G₀/G₁Peak number of channels of the peak. Apoptosis was also determined by flow cytometry using Annexin V-FITC staining (BD Biosciences, San Jose, Calif.) and also analyzed using FACSCalibur.

In vivo methods

Cell lines

Unless otherwise indicated, cell culture reagents were purchased from Life Technologies, Inc. (Gaithersburg, Md.). Cells were maintained at 37 ℃ with 5% to 10% CO₂And passaged using standard cell culture techniques. U87MG (human glioblastoma), NCI-H441 (human non-small cell lung adenocarcinoma), PC-3 (human prostate adenocarcinoma) cells were obtained from and cultured as recommended by the American type culture Collection (Bethesda, Md.).

Subcutaneous xenograft model in athymic mice

Female or male nu/nu mice or SCID/Beige mice (5-8 weeks old) were purchased from Harlan (Madison, Wis.) or Charles River (Wilmington, MA). Maintaining animals in clean room conditions in sterile filter cage containing HEPA-filtersAlpha-Dri/bed-o-cob comb bedding on the wind cage. Animals received sterile rodent chow and water ad libitum. Cells for implantation into athymic mice were collected and pelleted by centrifugation at 450 × g for 5 to 10 minutes. The cell pellet was washed once and resuspended in sterile phosphate buffered saline or serum free medium. Tumor cells were supplemented with 30% to 50% matrigel (BDBiosciences, San Jose CA) to promote tumor formation and growth of selected tumor cells as xenografts. Mixing cells (2X 10)⁶To 5X 10⁶100 μ l) were implanted in the mouse posterior costal region in the SC manner and allowed to grow to a designated size, and then the compound was administered for each experiment. Tumor size was determined by electronic caliper measurements and tumor length x width²X 0.4 scores to calculate tumor volume.

In vivo target mediated (PK/PD) studies

Tumor and plasma treatment for in vivo pharmacokinetic studies

Tumor cells expressing constitutively phosphorylated c-Met/HGFR or ALK were implanted subcutaneously in nude mice and allowed to grow to 300 to 800mm without treatment³. Administration of Compounds to mice in Single oral dose form (for acute PK/PD studies) or multiple oral dose form (for Steady State PK/PD studies) at the indicated dose levels1. In the administration of compounds1At the designated time later, each mouse was subjected to painless lethality in a humane manner, and blood samples were isolated from the left ventricle of the heart using syringes pretreated with heparin sulfate. Analysis of Compounds in plasma samples by LCMS1The concentration was analyzed. Excised tumors were snap frozen on dry ice, crushed with a liquid nitrogen cooled cryomortar and pestle, and lysed in cold 1 × cell lysis buffer (CellSignaling Technologies, Boston MT). Proteins were extracted from tumor lysates and protein concentrations were determined using BSA assay (Pierce, Rockford, IL). The total or/and phosphorylated protein of interest content in each tumor sample was determined using the capture ELISA method described below.

ELISA assays to assess pharmacokinetic inhibition of kinase targets

In each ELISA assay, a free-standing excipient or compound is used1Treated tumor-forming protein lysates were transferred to 96-well plates pre-coated with anti-c-Met/HGFR (Zymed Lab/Invitrogen, Carlsbad, Calif.) or anti-ALK capture antibody (Cell Signaling Technologies, Boston MT). Antibody-coated plates were incubated overnight at 4 ℃ in the presence of tumor lysates and washed seven times with 1% Tween 20 in PBS. HRP-PY20 (horseradish peroxidase-conjugated anti-total-phosphotyrosine, Santa Cruz Biotechnology, Santa Cruz, Calif.) was diluted 1: 500 in blocking buffer (Pierce, Rockford, Ill.) and added to each plate for 30 min. Plates were washed once more and TMB peroxidase substrate (Bio-Rad laboratories, Hercules CA) was added to initiate the HRP-dependent color-specific reaction. By adding 0.09N H₂SO₄To terminate the reaction. The Optical Density (OD) of each excipient or treated well was measured using a spectrophotometer at 450 nm. Pairing of compounds at the same time point according to OD readings1The total phosphorylation of c-Met/HGFR or ALK in tumors excised from treated animals was compared to the total phosphorylation of c-Met/HGFR or ALK in tumors excised from vehicle-treated animals. In this evaluation, the compounds in the tumor were calculated using the following equation1Inhibition of kinase target phosphorylation: % inhibition 100- [ (mean OD value treated/mean OD value untreated) × 100]。

Immunoblotting

Immunoblotting was also used to determine the relative kinase phosphorylation status and total protein content of the protein of interest in tumor samples. With different doses of the compound1Tumor bearing mice were treated and tumor lysates and protein samples were prepared as described above. Proteins of interest are immunoprecipitated using specific antibodies. Immunoprecipitated proteins were separated by SDS-PAGE and immunoblotting with anti-phosphotyrosine or total antibodies.

The antibodies used in the immunoblot study were as follows: anti-total human c-Met/HGFR antibodies from Zymed/Invitrogen, Carlsbad, CA; anti-phospho-c-Met/HGFR, anti-total and-phospho ALK, anti-total and phospho Gab1, anti-total and phospho AKT, anti-total and phospho-MAPK 44/42, STAT5 antibodies, available from Cell signaling technologies, Boston, MA.

Mini osmotic pump implant for in vivo infusion studies

Alzet 1003D and 1007D mini-osmotic pumps were purchased from Durect, Inc. (Cupertino, CA). Loading of a micropump with a defined concentration of a compound1The solution was pretreated with sterile saline solution at 37 ℃ until it reached equilibrium at 4 to 5 hours. The pump was surgically implanted subcutaneously in the left dorsal chest area of mice bearing tumors in the right intercostal area following the manufacturer's instructions. The incision is closed with a surgical clip, which is removed after 5 to 7 days, at which time the skin incision has completely healed. For studies in which the required infusion time and drug volume exceeded the pump capacity, a pump replacement procedure was performed at the indicated time.

Tumor histology and Immunohistochemistry (IHC) analysis

Tumor specimens for evaluation of immunohistochemical endpoints were collected and fixed in 10% buffered formalin containing protease and phosphatase inhibitors for 24 hours before transferring them to 70% ethanol. Tumor samples were then embedded in paraffin and cut into 4 micron sections and baked onto microscope slides. Deparaffinization and antigen recovery (based on EDTA) were performed using a commercially available uncovered chamber (biochare Medical, catalog number DC2001) according to the manufacturer's instructions. Tumor OCT frozen samples were also collected and sectioned for CD-31 staining. For immunostaining, slides were incubated with primary antibodies followed by secondary antibodies and visualized using colorimetry (DAO Envisi on-HARP, DAB kit, DAO, Carpentaria, CA) or fluorimetry (Alexa 488 or Alexa 635, molecular probes/Invitrogen, Carlsbad CA). All immunostained sections were counterstained with hematoxylin. Histological staining was also performed using an Automated Ventana Discovery xtstating Module (Ventana Medical Systems, Tucson, AZ) according to the manufacturer's instructions. The stained sections were analyzed with an Olympus microscope and quantitative analysis of section staining was performed using the ACIS system (Automated Cellular Imaging, Clarient, irvine ca). Slides were also analyzed by an internal pathologist using standard clinical methods.

Antibodies used in immunohistochemical studies included anti-phosphorylation-c-Met/HGFR from biosource internationals/Invitrogen, Carlbad, CA; anti-Ki 67 from Santa Cruz Biotechnology, Santa Cruz, CA; anti-CD 31 from Dacocytomation, Carpenteria, Calif.

Data and results

Enzymatic potency of Compound 1 against c-Met/HGFR RTK

Proven Compounds1Are potent ATP-competitive inhibitors of recombinant, human c-Met/HGFR kinase activity, wherein the average K is_iWas 4 nM.

In biochemical enzyme assays, compounds1Inhibit the kinase activity of c-Met/HGFR, with a Ki of 3 nM. To investigate kinase selectivity relative to c-Met/HGFR, compounds were further evaluated in biochemical kinase screening assays against a panel of > 120 recombinant kinases1. In these preliminary biochemical kinase selective screens, compounds were identified1A subgroup of kinases for which activity is exhibited, thereby estimating 100-fold lower selectivity for c-Met/HGFR than for c-Met/HGFR. Further on compounds in defined cell-based kinase selectivity assays in follow-up studies1The activity of these potential kinase candidates was evaluated (table 1). Compounds for the inhibition of c-Met/HGFR kinase by monitoring NADH oxidation coupled to ATP conversion1Biochemical Ki values. Compounds and kinase assay reagents were added to the test wells and incubated at 37 ℃ for 10 minutes and the reaction was initiated by the addition of c-Met/HGFR. NADH was measured by spectrophotometry at 340nm at the indicated time point.

Kinase selectivity of Compound 1 in cell-based assays

Evaluation in a set of cell-based kinase Activity assaysCompound (I)1Selectivity for selected kinases, such kinases are potential candidates in biochemical assays and other related RTKs (e.g., RON, SKY, IR). Compounds compared to c-Met/HGFR in cell-based studies1VEGFR2 and PDGFR β split-RTK were 1000-fold more selective, IR and Lck were 200-fold more selective, and Axl, Tie2, TrkA and TrkB were about 40-60-fold more selective (A549 IC)₅₀8.6nM) (error! The reference source did not find 1). To investigate whether a 50-fold window is sufficient for in vivo c-Met/HGFR selectivity, compounds were evaluated1The ability to inhibit Tie-2 phosphorylation in C6 xenografts in nude mice. In this study, the inhibition of c-Met/HGFR within 24 hours was expressed as IC at 50mg/kg of administration₉₉) Or a single PO dose of 100mg/kg, no significant inhibition of Tie-2 phosphorylation was observed at any time point. This indicates that inhibition of Tie-2, Axl or TrkA and B is unlikely to occur at doses up to 2-fold higher than those associated with complete inhibition of c-Met/HGFR within 24 hours. Compound (I)1Selectivity for RON kinase is 20 to 30 fold, a potential beneficial oncology target because it 1) over-expression and mutation in selected cancers, and 2) lacks the undesirable phenotype in RON-free mice. Compared with the RTK mentioned above, the compound1Shows nearly equal IC to the oncogenic form of the ALK RTK (anaplastic lymphoma kinase) fusion protein, NPM-ALK₅₀Value (24nM), NPM-ALK is an oncogenic fusion protein variant of ALK RTK (anaplastic lymphoma kinase) resulting from chromosomal ectopic events associated with the pathogenesis of human anaplastic large cell lymphoma (ALCL, present in human lymphoma cell line (wrong | reference source not found 1)).

Pharmacokinetic inhibition of c-Met/HGFR RTK Activity in cells

To confirm the effective enzyme activity for inhibition of conversion to c-Met/HGFR in cells, compounds were evaluated1The ability to inhibit cMet/HGFR phosphorylation in a group of tumor and endothelial cell lines. Compounds in the entire group of human tumor and endothelial cell lines1Inhibiting HGF-stimulated or constitutive total tyrosine autophosphorylation of wild-type c-Met/HGFR, wherein the average IC₅₀The value was 11nM (Table 1). Compound (I)1Similar values were shown in mIMCD3 mouse epithelial cells (IC)₅₀5nM) (table 1).

Effect of Compound 1 against cMet/HGFR active site mutation in cells

The c-Met/HGFR activating mutations have been identified in several human cancers and provide a powerful rationale for proof of experimental evidence based on other RTK targets and for conceptual clinical studies of preclinical experiments. Although c-Met/HGFR mutations in the extracellular or membrane-proximal domain are not expected to affect compound binding to the active site, there is a possibility that kinase domain mutations will cause loss of activity. To solve this problem, compounds are used1Assessment of RTK phosphorylation IC in treated NIH3T3 cells engineered to express wild-type c-Met/HGFR or a series of representative c-Met/HGFR active site mutations₅₀. In these studies, the compounds were compared to the wild-type receptor (12.6nM)1Improved or similar activity was shown for the ATP binding site mutant (V1092I, 19nM and H1094R, 2.2nM) or the P-loop mutant (M1250T, 15nM) (Table 1). Compound (I)1Also effectively exhibit inhibition of NCI-H69 (IC)₅₀: 13nM) and HOP92 (IC)₅₀: 16nM) comparable potency of c-Met phosphorylation in cells expressing the endogenous c-Met membrane proximal variants R988C and T1010I, respectively (table 1). In contrast, a significant change (10-fold) in potency was observed for the activation loop mutants (Y1230C, 127nM and Y1235D, 92nM) compared to the wild-type receptor (table 1).

TABLE 1

Effect of Compound 1 on the c-Met/HGFR-or NPM-ALK-dependent oncogenic phenotype in cells

Phenotypic analysis

c-Met/HGFR has been found to interact with a variety of tumor cells and tumor endothelial cellsWhile NPM-ALK is associated with dysregulation of cell proliferation and apoptosis in ALCL lymphoma cells. In a series of cell-based functional assays, compounds1Effectively inhibits the growth of human GTL-16 gastric cancer cells, induces GTL-16 apoptosis, inhibits the migration and invasion of HGF-stimulated human NCI-H441 lung cancer cells through a matrigel matrix and inhibits the motility/dispersion of HGF-stimulated MDCK cells. (Table 2) Compounds1Also inhibits factor t 2; 5 proliferation of Karpa s299 or SU-DHL-1 ALCL cells ectopically expressing NPM-ALK fusion protein. Compounds in these NPM-ALK positive lymphoma cells1Inhibition of growth with G₀/G₁Cell cycle arrest and induction of apoptosis. Also to investigate potential anti-angiogenic activity, compounds1Inhibits HGF-mediated HUVEC endothelial cell survival and matrigel invasion and HMVEC endothelial cell tube generation in fibrin gel. These data demonstrate the compounds1Has the ability to inhibit both c-Met/HGFR-and NPM-ALK-dependent functions in cells expressing activated c-Met/HGFR or NPM-ALK, respectively. Furthermore, these data indicate that the compounds1The anti-tumor efficacy of (a) can be mediated by directly affecting tumor cell growth or survival and anti-angiogenic mechanisms.

TABLE 2

In vivo studies

Data and results

In vivo kinase target inhibition and tumor growth inhibition

Tumor model selection

Evaluation of c-Met/HGFR target inhibition, tumor growth inhibition and Compounds in vivo using a c-Met/HGFR dependent tumor xenograft model1Plasma exposure relationship of (a). Due to lack of expression by mouse mesenchymal cellsMouse HGF paracrine activation of tumor xenograft-expressed human c-Met/HGFR, a human xenograft model showing constitutive c-Met/HGFR activity was used as follows: 1) GTL-16 human gastric or Caki-1 renal cancer models expressing high levels of constitutively active c-Met/HGFR, 2) U87MG human glioblastoma or PC-3 human prostate cancer models expressing both HGF and c-Met/HGFR containing autocrine loops or 3) co-implantation of human tumor cells (e.g., NCI-H441NSCLC) with human MRC5 fibroblasts to provide a source of biologically active human HGF from the tumor interstitium to restore species-specific paracrine activation of c-Met/HGFR.

Correlation of c-Met/HGFR inhibition with anti-tumor efficacy following oral administration

GTL-16 tumors

To a tumor with established GTL-16 (250 mm)³) The athymic mice administered the compound orally at the indicated dose1Or vehicle alone for 11 days. For a study investigating the inhibition of c-Met/HGFR phosphorylation in GTL-16 (fig. 2A), mice were subjected to painless lethality at the end of the study at the indicated time point after administration in a humane manner, tumors were excised and frozen, and phosphorylation in vehicle and treatment groups was quantified by ELISA. Compounds in tumors1Inhibition of kinase target phosphorylation was calculated as follows: % inhibition 100- [ (mean OD value treated/mean OD value untreated) × 100]. In the study that studied the inhibition of GTL-16 tumor growth (FIG. 2B), the tumor volume was measured by caliper on the indicated days for each group of 15 mice, expressed as median tumor volume + -SEM. The percent (%) values shown are% tumor growth inhibition measured on day 20 for drug-treated mice compared to tumor growth inhibition for vehicle-treated mice and are calculated as follows: 100 {1- [ (treated day 20-treated day 10)/(control day 20-control day 10) })]}. Mean median tumor volume was significantly less in the treated group than the control group (P < 0.001), as determined using one-way anova (see fig. 2B).

To evaluate the compound1c-Met/HGFR PD reaction in a single administrationAnd repeated administration (steady state) of the compound in a study1At several time points later, GTL-16 tumors were collected. The c-Met/HGFR phosphorylation status in tumors was quantified by ELISA over a range of doses. When focusing attention on steady state PD studies (11 days) to map the relationship with tumor growth inhibition, the compounds as shown in figures 2A and 2B1The characteristics are as follows:

at 50 mg/kg/day: in GTL-16 tumors, 100% tumor growth inhibition was accompanied by complete inhibition of c-Met/HGFR phosphorylation for 24 hours (25mg/kg- -nearly complete inhibition of both phosphorylation and tumor growth). At 12.5 mg/kg/day: at 1-8 hours, 60% tumor growth inhibition was accompanied by 80% to 90% inhibition of c-Met/HGFR phosphorylation, which decreased to 50% to 60% inhibition at 16-24 hours.

At 6.25 mg/kg/day: at 1-8 hours, non-significant tumor growth inhibition tended to be accompanied by 30% to 50% inhibition of c-Met/HGFR phosphorylation and complete recovery at 16 hours.

U87MG tumor

To a tumor (150 mm) with a defined U87MG tumor³) The athymic mice administered the compound orally at the indicated dose1Or vehicle alone for 9 days. For the study to study tumor growth inhibition (fig. 3A), tumor volumes were measured by caliper on the indicated days for each group of 10-12 mice, expressed as median tumor volume ± SEM. The percent (%) values shown are% tumor growth inhibition of drug-treated mice compared to vehicle-treated mice measured at day 14 and calculated as follows: 100 {1- [ (treated day 14-treated day 6)/(control day 14-control day 6) }]}. Mean median tumor volume was significantly lower in the treated group than the control group (P < 0.001) as determined using one-way anova (see fig. 3A). Study for the inhibition of c-Met/HGFR phosphorylation (FIG. 3B), at the end of which the compound was administered1Mice were euthanized in a humane manner 4 hours later, tumors were excised and frozen, and phosphorylation in vehicle and treatment groups was quantified by ELISA.Compounds in tumors1Inhibition of kinase target phosphorylation was calculated as follows: % inhibition 100- [ (mean OD value treated/mean OD value untreated) × 100]。

No pharmacologically relevant inhibition of Tie-2 phosphorylation was observed in U87MG xenografts at dose levels up to 100mg/kg, indicating that the compounds1Selective for its intended target at similar dose levels.

Antitumor efficacy of Compound 1 in human xenograft models

Evaluation of compounds in multiple human tumor xenograft models representing cancer indications1Such cancer indications involve c-Met/HGFR disorders including GTL-16 gastric cancer, U87MG glioblastoma, NCI-H441NSCLC and PC-3 prostate cancer (Table 4).

GTL-16 gastric cancer model

When using the GTL-16 gastric cancer model, the compounds1Show large established tumors (> 600 mm)³) Clearly degraded ability (fig. 4). In this study, 50 mg/kg/day and 75 mg/kg/day of the compound1The treatment groups showed equal average tumor regression over the 43 day dosing schedule, further demonstrating that 50 mg/kg/day is the maximum effective dose level. As shown in FIG. 4, the mean tumor at 50 mg/kg/day or 75 mg/kg/day was the compound administered160% of the cells were degenerated after 43 days. In this study, the compound was present at day 431The mass of each tumor decreased at each dose level during the administration period, with 9 out of 14 mice showing > 30% tumor mass reduction (partial response) and one animal showing complete response with no tumor signs even after 10 days of treatment discontinuation.

Daily oral administration of compounds1Regression of large established GTL-16 tumor xenografts (FIG. 4A) and mouse body weight (FIG. 4B) in post-athymic mice. To a tumor with established GTL-16 (620 mm)³) The athymic mice administered the compound orally at the indicated dose levels1Or vehicle alone for 43 days. For the study ofAntitumor efficacy (fig. 4A), tumor volumes were measured by caliper on the indicated days for each group of 6 to 8 mice, expressed as median tumor volume ± SEM. (FIG. 4B) Compounds1The average mouse body of the treated and vehicle control groups is shown in the right hand panel.

NCI-H441NSCLC model/Caki-1/PC-3 tumor xenograft I

Oral administration of Compounds at indicated doses to athymic mice bearing established NCI-H441(100mm3) (FIG. 5A), Caki-1 (Table 3A, Table 3B), or PC-3 tumor xenografts (FIG. 5B), respectively1Or vehicle alone for 38, 40 or 20 days. Tumor volumes were measured by caliper on the indicated days, where expressed as median tumor volume ± SEM. Mean tumor volume was significantly lower in the treated group than the control group (P < 0.001), as determined using one-way anova. (see FIG. 5).

In the NCI-H441NSCLC model, the compound was administered at 50 mg/kg/day1After 38 days, a 43% average regression of the established tumors was observed (fig. 5). In this study, the compound was present at day 331The mass of each tumor decreased at 50 mg/kg/day during the administration cycle, with 3 of 11 mice showing > 30% tumor mass reduction (partial response) and 3 animals showing complete response with no tumor signs (FIG. 5). Compound (I)1Has a dose-dependence, wherein a defined regression of the NCI-H441 tumor was observed at 50 mg/kg/day and a partial inhibition of tumor growth (57% tumor growth inhibition) was observed at 15 mg/kg/day (fig. 5). Compounds observed in the NCI-H441 model in the Compound-treated group compared to vehicle-treated controls1The antitumor efficacy of the compounds is consistent with the inhibition of c-Met/HGFR phosphorylation in tumors. In Caki-1 renal carcinoma model, the compound is present at day 331A 53% reduction in mean tumor volume was observed at 50 mg/kg/day during the administration cycle (figure 5B). In Caki-1 studies, the compound was present in 33 days per tumor volume1At least 30% reduction during the application cycle (table 3B, table 4). Compounds also investigated in PC-3 prostate cancer xenograft models1And observed in this modelTo near complete inhibition of tumor growth (84% growth inhibition).

TABLE 3A

TABLE 3B

Correlation of antitumor efficacy with c-Met/HGFR inhibition

A series of dose-response antitumor efficacy and pharmacokinetic studies were performed to elucidate the relationship between c-Met/HGFR target inhibition and antitumor efficacy. To evaluate the compounds1Pharmacokinetic inhibition of c-Met/HGFR, orally administered compounds1At several later time points, GTL-16 gastric carcinoma tumors were collected. The c-Met/HGFR phosphorylation status in tumors was quantified by ELISA over a range of doses. In these studies, the compounds1A close relationship between dose-and time-dependent inhibition of c-Met/HGFR and tumor growth inhibition was shown. When defining the relationship between target PD and efficacy in the GTL-16 model, the following conclusions are evident: 1) complete inhibition of c-Met/HGFR activity within 24 hours was consistent with complete inhibition of tumor growth (50mg/kg, 100% TGI), 2) effective inhibition of c-Met/HGFR activity within only a portion of the schedule was consistent with suboptimal efficacy (12.5mg/kg, 60% TGI), 3) inability to achieve > 50% inhibition of c-Met/HGFR activity (3.125, 6.25mg/kg) was consistent with lack of significant Tumor Growth Inhibition (TGI) (FIGS. 2A and 2B). Additional GTL-16 studies showed 50 mg/kg/day and 75 mg/kg/day compounds1The control group showed equal average tumor regression, which further demonstrated that 50 mg/kg/day was the maximum effective dose level (fig. 4 and table 4). The results of these studies indicate the duration of c-Met/HGFR inhibition and the compounds1The antitumor efficacy of (A) is directly related.

In addition, similar compounds were observed with all tumor models (GTL-16, U87MG and NCI-H441) and schedule of administration1Dose-dose dependent effects on tumor growth and c-Met/HGFR phosphorylation, which further support the observations (table 4). In each of these studies, a 50 mg/kg/day dose level caused complete tumor growth inhibition or tumor regression (Table 4). In addition, a dose-dependent relationship was observed between inhibition of c-Met/HGFR phosphorylation and antitumor efficacy in each model, further supporting the concept of maximizing the extent and duration of c-Met/HGFR inhibition to achieve full efficacy. Taken together, these studies indicate that near complete inhibition of c-Met/HGFR phosphorylation over the duration of the schedule of administration for maximum therapeutic benefit is necessary and that the extent and duration of inhibition of c-Met/HGFR activity is directly related to the amount of antitumor efficacy. This data support compound1The correlation between inhibition of the intended pharmacological target of c-Met/HGFR and the degree of anti-tumor efficacy.

Antitumor efficacy of Compound 1 in NPM-ALK-dependent lymphoma model

Karpas299 ALCL model

To a tumor with established Karpas299 (220 mm)³) SCID-beige mice administered the compounds orally at the indicated doses1Or vehicle alone for the indicated time. For the study to study tumor growth inhibition (fig. 6A), tumor volumes were measured by caliper on the indicated days for each group of 8 to 12 mice, expressed as median tumor volume ± SEM. The percent (%) values shown are% tumor growth inhibition of drug-treated mice compared to vehicle-treated mice measured at day 23 and are calculated as follows: 100 {1- [ (treated day 23-treated day 12)/(control day 23-control day 12) })]}. Mean tumor volume was significantly lower in the treated group than the control group (P < 0.001), as determined using one-way anova. Study for the study of the inhibition of NPM-ALK phosphorylation (FIG. 6B), at the end of which compound was administered1Mice were euthanized 4 hours later by a humane procedure, tumors were excised and frozen, and ALK phosphorylation in vehicle and treated tumors was quantified by ELISA. Compounds in tumors1Inhibition of kinase target phosphorylation was calculated as follows: % inhibition 100- [ (mean OD value treated/mean OD value untreated) × 100]。

When using the Karpas299 ALCL model, the compounds1Appear to cause established tumors (> 200 mm)³) Clearly degraded ability (fig. 6A). In this study, compounds were administered at 100 mg/kg/day1Complete regression of tumors was induced in all mice in this administration group within 15 days after the start of compound administration (fig. 6A). After 17 days, the compounds were stopped1Treatment, resulting in tumor regrowth. Growth of tumors to larger size (> 600 mm)³) When it is time to restart the compound1Treatment, continued for 13 days and again showed complete regression of the tumor (fig. 6A, table 4). Compound (I)1Consistent with its observation of anti-proliferative and apoptotic effects on ALCL cells in vitro. The relationship between inhibition of tumor NPM-ALK phosphorylation and anti-tumor efficacy was also determined at various dose levels and time points. Similar to the results observed in the c-Met/HGFR-dependent tumor model, near complete inhibition (> 90% inhibition) of NPM-ALK activity within the complete dosing interval (24 hours) was consistent with maximum anti-tumor efficacy at 100mg/kg (complete regression) (fig. 6A and 6B). Incomplete inhibition of NPM-ALK phosphorylation (< 90% inhibition at 25mg/kg or 50 mg/kg) was consistent with sub-maximal antitumor efficacy (FIGS. 6A and 6B). Similar to the study in the c-Met/HGFR-dependent tumor model, this data supports compounds in the NPM-ALK-dependent tumor model1The extent of inhibition of NPM-ALK and the degree of antitumor efficacy.

TABLE 4

Synthesis of Compound 1

PLE is an enzyme produced by Roche and sold by the company biocatalysts in the form of a crude esterase preparation from porcine liver, often referred to AS PLE-AS (purchased AS ICR-123 from biocatalysts, sold AS an ammonium sulfate suspension). This enzyme is classified under CAS registry number as "carboxylic ester hydrolase, CAS No. 9016-18-6". The corresponding enzyme classification number is ec 3.1.1.1. This enzyme is known to have a wide range of substrate specificities for the hydrolysis of various esters. The lipase activity was determined in a pH titrator using a method based on hydrolysis of ethyl butyrate. 1LU (lipase unit) is the amount of enzyme that releases 1. mu. mol of titratable butyric acid at 22 ℃ and pH 8.2. The formulations reported herein (PLE-AS, in suspension form) are typically transported AS an opaque brown-green liquid with a repopulating activity of > 45LU/mg (protein content about 40 mg/ml).

(1S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol

(1S) -1- (2, 6-dichloro-3-fluorophenyl) ethanol (shown as compound (S-1) in the lower scheme) was prepared according to scheme B by a combination of enzymatic hydrolysis, esterification, and chemical hydrolysis with translocation of racemic 1- (2, 6-dichloro-3-fluorophenyl) ethyl acetate. Racemic ethyl 1- (2, 6-dichloro-3-fluorophenyl) acetate (compound a2) was prepared according to reaction scheme a.

Reaction scheme A

1- (2, 6-dichloro-3-fluorophenyl) ethanol (a 1): sodium borohydride (90mg, 2.4mmol) was added to 2 ', 6 ' -dichloro-3 ' -fluoro-acetophenone (Aldrich, Cat. No. 52, 294-5) (207mg, 1mmol) dissolved in 2ml of anhydrous CH₃Solution of OH. The mixture was stirred at room temperature for 1 hour and then evaporated to give a colorless oily residue. The residue was purified by flash chromatography (eluting with 0 → 10% EtOAc in hexane) to give Compound A1(180 mg; 0) as a colorless oil88 mmol; yield 86.5%); MS (APCI) (M-H)^-208; 1H NMR (400MHz, chloroform-D) δ ppm 1.64(D, J ═ 6.82Hz, 3H)3.02(D, J ═ 9.85Hz, 1H)6.97-7.07(m, 1H)7.19-7.33(m, 1H).

Ethyl 1- (2, 6-dichloro-3-fluorophenyl) acetate (a 2): acetic anhydride (1.42ml, 15mmol) and pyridine (1.7ml, 21mmol) were added sequentially to compound A1(2.2 g, 10.5mmol) dissolved in 20ml CH₂Cl₂In the solution of (1). The mixture was stirred at room temperature for 12 hours and then evaporated to give a pale yellow oily residue. The residue was purified by flash chromatography (eluting with 7 → 9% EtOAc in hexane) to give compound A2 as a colorless oil (2.26 g; 9.0 mmol; yield 85.6%); 1H NMR (400MHz, chloroform-D) δ ppm 1.88(D, J ═ 6.82Hz, 3H)2.31(s, 3H)6.62(q, J ═ 6.82Hz, 1H)7.25(t, J ═ 8.46Hz, 1H)7.49(dd, J ═ 8.84, 5.05Hz, 1H).

Reaction scheme B

A50 ml jacketed flask equipped with a pH electrode, an overhead stirrer and a base addition line (1M NaOH) was charged with 1.2ml of 100mM potassium phosphate buffer (pH 7.0) and 0.13ml of PLEAS suspension. Then, compound a2(0.13 g, 0.5mmol, 1.00 eq) was added dropwise and the resulting mixture was stirred at room temperature for 20 hours, keeping the pH of the reaction constant at 7.0 with 1M NaOH. The conversion and enantiomeric excess (ee) values of the reaction were monitored by RP-HPLC and terminated after 50% of the starting material had been consumed (about 17 hours under these conditions). The mixture was then extracted three times with 10ml of ethyl acetate to recover both the ester and the alcohol as a mixture of R-1 and S-2.

Methanesulfonyl chloride (0.06ml, 0.6mmol) was added to a mixture of R-1 and S-2(0.48mmol) dissolved in 4ml pyridine under a nitrogen atmosphere. The mixture was stirred at room temperature for 3 hours and then evaporated to give an oil. Water (20ml) was added to the mixture and thenEtOAc (20 ml. times.2) was added to extract the aqueous solution. The organic layers were combined, dried, filtered and evaporated to give a mixture of R-3 and S-2. The mixture was used for the next reaction without further purification.¹H NMR (400MHz, chloroform-D) δ ppm 1.66(D, J ═ 7.1Hz, 3H)1.84(D, J ═ 7.1Hz, 3H)2.09(s, 3H)2.92(s, 3H)6.39(q, J ═ 7.0Hz, 1H)6.46(q, J ═ 6.8Hz, 1H)6.98-7.07(m, 1H)7.07-7.17(m, 1H)7.23-7.30(m, 1H)7.34(dd, J ═ 8.8, 4.80Hz, 1H).

Potassium acetate (0.027 g, 0.26mmol) was added to a mixture of R-3 and S-2(0.48mmol) in 4ml DMF under a nitrogen atmosphere. The reaction mixture was heated at 100 ℃ for 12 hours. Water (20ml) was added to the reaction mixture and the aqueous solution was extracted by adding EtOAc (20 ml. times.2). The combined organic layers were dried, filtered and evaporated to give S-2 as an oil (72mg, 61% yield over two steps). Chiral ee: 97.6 percent.¹H NMR (400MHz, chloroform-D) δ ppm 1.66(D, J ═ 7.1Hz, 3H)2.09(s, 3H)6.39(q, J ═ 6.8Hz, 1H)7.02(t, J ═ 8.5Hz, 1H)7.22-7.30(m, 1H).

Sodium methoxide (19 mmol; 0.5M in methanol) was slowly added to compound S-2(4.64 g, 18.8mmol) at 0 ℃ under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 4 hours. Evaporation of the solvent and addition of H₂O (100 ml). The cooled reaction mixture was neutralized to pH 7 with sodium acetate-acetic acid buffer solution. The aqueous solution was extracted by adding ethyl acetate (100 ml. times.2). The combined organic layers were passed over Na₂SO₄Drying, filtration and evaporation gave S-1 as a white solid (4.36 g, 94.9% yield); SFC-MS: 97% ee.¹H NMR (400MHz, chloroform-D) δ ppm 1.65(D, J ═ 6.8Hz, 3H)5.58(q, J ═ 6.9Hz, 1H)6.96-7.10(m, 1H)7.22-7.36(m, 1H).

5-bromo-3- [1- (2, 6-dichloro-3-fluoro-phenyl) -ethoxy ] -pyridin-2-ylamine (racemate):

1.2, 6-dichloro-3-fluoroacetophenone (15 g, 0.072mol) was stirred in THF (150ml, 0.5M) at 0 ℃ for 10 minutes with the aid of an ice bath. Lithium aluminum hydride (2.75 g, 0.072mol) was added slowly. The reaction was stirred at ambient temperature for 3 hours. The reaction was cooled in an ice bath and water (3ml) was added dropwise followed by slow addition of 15% NaOH (3 ml). The mixture was stirred at ambient temperature for 30 minutes. 15% NaOH (9ml), MgSO was added₄And the mixture was filtered to remove solids. Such solid was washed with THF (50ml) and the filtrate was concentrated to give 1- (2, 6-dichloro-3-fluoro-phenyl) -ethanol (14.8 g, 95% yield) as a yellow oil.¹HNMR(400MHz，DMSO-d₆)δ1.45(d，3H)，5.42(m，2H)，7.32(m，1H)，7.42(m，1H)。

2. To a stirred solution of triphenylphosphine (8.2 g, 0.03mol) and DEAD (13.65ml, 40% in toluene) in THF (200ml) was added a solution of 1- (2, 6-dichloro-3-fluoro-phenyl) -ethanol (4.55 g, 0.021mol) and 3-hydroxy-nitropyridine (3.35 g, 0.023mol) in THF (200ml) at 0 ℃. The resulting bright orange solution was stirred at ambient temperature for 4 hours under a nitrogen atmosphere, at which time all starting material had been consumed. The solvent was removed and the dry crude material was loaded onto silica gel and eluted with ethyl acetate-hexanes (20: 80) to give 3- (2, 6-dichloro-3-fluoro-benzyloxy) -2-nitro-pyridine (6.21 g, 0.021mol, 98%) as a pink solid.¹H NMR(CDCl₃，300MHz)δ1.8-1.85(d，3H)，6.0-6.15(q，1H)，7.0-7.1(t，1H)，7.2-7.21(d，1H)，7.25-7.5(m，2H)，8.0-8.05(d，1H)。

3. 3- (2, 6-dichloro-3-fluoro-benzyloxy) -2-nitro-pyridine (9.43 g, 0.028mol) and iron filings (15.7 g, 0.28mol) were suspended in a stirred mixture of AcOH (650ml) and EtOH (500 ml). The reaction was slowly heated to reflux and stirred for 1 hour. The reaction was cooled to room temperature, and then diethyl ether (500ml) and water (500ml) were added. The solution was carefully neutralized by the addition of sodium carbonate. The combined organic extracts were extracted with saturated NaHCO₃(2×100ml)、H₂O (2X 100ml) and brine (1X 100ml) were washed and then dried (Na)₂SO₄) Filtered and concentrated to dryness in vacuo to afford 3- (2, 6-dichloro-3-fluoro-benzyloxy) -pyridin-2-ylamine (9.04 g, 0.027mol, 99%) as a pale pink solid.¹H NMR(CDCl₃，300MHz)δ1.8-1.85(d，3H)，4.9-5.2(brs，2H)，6.7-6.84(q，1H)，7.0-7.1(m，1H)，7.2-7.3(m，1H)，7.6-7.7(m，1H)。

4. A stirred solution of 3- (2, 6-dichloro-3-fluoro-benzyloxy) -pyridin-2-ylamine (9.07 g, 0.03mol) in acetonitrile was cooled to 0 ℃ with the aid of an ice bath. To this solution was added N-bromosuccinimide (NBS) (5.33 g, 0.03mol) in portions. The reaction was stirred at 0 ℃ for 15 minutes. The reaction was concentrated in vacuo to dryness. The resulting black oil was dissolved in EtOAc (500ml) and purified via silica gel chromatography. The solvent was then removed in vacuo to afford 5-bromo-3- (2, 6-dichloro-3-fluoro-benzyloxy) -pyridin-2-ylamine (5.8 g, 0.015 mol, 51%) as a white crystalline solid.¹H NMR(CDCl₃，300MHz)δ1.85-1.95(d，3H)，4.7-5.0(brs，2H)，5.9-6.01(q，1H)，6.8-6.95(d，1H)，7.01-7.2(t，1H)，7.4-7.45(m，1H)，7.8-7.85(d，1H)。

5-bromo-3- [1(R) - (2, 6-dichloro-3-fluoro-phenyl) -ethoxy ] -pyridin-2-ylamine:

the enantiomerically pure R isomer of the racemate was prepared as described above, but the enantiomerically pure starting material described above was used.¹H NMR(400MHz，DMSO-d₆)δ1.74(d，3H)，6.40(m，1H)，6.52(br s，2H)，7.30(m，1H)，7.48(m，1H)，7.56(s，1H)；MS m/z 382(M+1)。

4-Methanesulfonyloxy-piperidine-1-carboxylic acid tert-butyl ester (2)

Tert-butyl 4-hydroxy-piperidine-1-carboxylate (7.94 g, 39.45mmol) dissolved in CH after cooling to 0 deg.C₂Cl₂(100ml) to a stirred solution NEt was added slowly₃(5.54ml, 39.45mmol) followed by methanesulfonyl chloride (3.06ml, 39.45mmol) and DMAP (48mg, 0.39 mmol). The mixture was stirred at room temperature overnight. To the mixture was added water (30 ml). By CH₂Cl₂(3X 30ml) extraction followed by drying (Na)₂SO₄) And the solvent was removed in vacuo to give tert-butyl 4-methanesulfonyloxy-piperidine-1-carboxylate (11.00 g, > 99% yield) as a white solid.¹H NMR(CDCl₃，400MHz)δ4.89(m，1H)，3.69(m，2H)，3.31(m，2H)，3.04(s，3H)，1.95(m，2H)，1.83(m，2H)，1.46(s，9H)。

4- [4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-pyrazol-1-yl ] piperidine-1-carboxylic acid tert-butyl ester

4- (4-indol-1H-pyrazol-1-yl) piperidine-1-carboxylic acid tert-butyl ester(s) ((s))3)

NaH (1.2 eq, 0.68mmol) was added portionwise to a stirred solution of 4-indolpyrazole (0.57mmol) in DMF (2 l) at 4 ℃. The resulting mixture was stirred at 4 ℃ for 1 hour and then 4-methanesulfonyloxy-piperidine-1-carboxylic acid tert-butyl ester, i.e. compound2(1.1 eq, 0.63 mmol). The resulting mixture was heated at 100 ℃ for 12 hours. By H₂The reaction was quenched with EtOAc and extracted several times. The combined organic layers were dried, filtered and concentrated to give an orange oil. The residue was purified by silica gel chromatography (eluting with 5% EtOAc in pentane) to give the compound as a white solid3(140 g, 66%).

4- [4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-pyrazol-1-yl]Piperidine-1-carboxylic acid tert-butyl ester(s) ((R))4)

Bis (valeryl) diboron (1.4 eq, 134 g, 0.52mol) and potassium acetate (4 eq, 145 g, 1.48mol) were added to the compound in that order3(140 g, 0.37mol) in a solution of 1.5 l DMSO. The mixture was purged several times with nitrogen and then dichlorobis (triphenylphosphine) palladium (II) (0.05 eq, 12.9 g, 0.018mol) was added. The resulting mixture was heated at 80 ℃ for 2 hours. The reaction mixture was cooled to room temperature and filtered through a bed of celite and washed with EtOAc. The filtrate was washed with saturated NaCl (500 ml. times.2) over Na₂SO₄Dried, filtered and concentrated. The residue was purified by silica gel chromatography (eluting with 5% EtOAc in hexane) to give the compound as a white solid4(55 g, 40%).

3- [ (R) -1- (2, 6-dichloro-3-fluoro-phenyl) -ethoxy]-5- (1-piperidin-4-yl-1H-pyrazol-4-yl) -pyridin-2-ylamine (1)

In the presence of 3- [ (R) -1- (2, 6-dichloro-3-fluoro-phenyl) -ethoxy]-5- (4, 4, 5, 5-tetramethyl- [1, 3, 2)]Dioxopentaborane-2-yl) -pyridin-2-ylamine (15.22 g, 35.64mmol) and tert-butyl 4- (4-bromo-pyrazol-1-yl) -piperidine-1-carboxylate (14.12 g, 42.77mmol) in a stirred solution of DME (143ml) were added Na₂CO₃(11.33 g, 10692mmol) was dissolved in water (36 ml). The solution was degassed and flushed with nitrogen three times. To this solution was added Pd (PPh)₃)₂Cl₂(1.25mg, 1.782 mmol). The reaction solution was degassed and flushed with nitrogen three more times. The reaction solution was stirred in an 87 ℃ oil bath for about 16 hours (or until borane pinacol ester was consumed), cooled to ambient temperature and diluted with EtOAc (600 ml). The reaction mixture was filtered through a pad of celite and washed with EtOAc. The EtOAc solution was washed with brine, over Na₂SO₄Dried and concentrated. The crude product was purified using EtOAc/hexanes systemPurification on a conventional eluted silica gel column (Biotage 90+ column: equilibrated with 600ml 100% hexane, section 1: 2250ml 50% EtOAc/hexane, Linear; section 2: 4500ml 75% EtOAc/hexane Linear; section 3: 4500ml 100% EtOAc) afforded 4- (4- { 6-amino-5- [ (R) -1- (2, 6-dichloro-3-fluoro-phenyl) -ethoxy ] -ethyl]-pyridin-3-yl } -pyrazol-1-yl) -piperidine-1-carboxylic acid tert-butyl ester (11.8 g, 60% yield, purity approx. 95%) with an Rf of 0.15 (50% EtOAc/hexanes). MS M/e 550(M +1)⁺。

In the presence of 4- (4- { 6-amino-5- [ (R) -1- (2, 6-dichloro-3-fluoro-phenyl) -ethoxy]-pyridin-3-yl } -pyrazol-1-yl) -piperidine-1-carboxylic acid tert-butyl ester (11.8 g, 21.45mmol) was dissolved in CH₂Cl₂To a solution (59ml, 0.2M) was added 4N HCl/dioxane (21 ml). The solution was stirred overnight to form a solid. The solid was thoroughly crushed with a glass rod and sonicated to release the entrapped starting material in the solid. Additional 4N HCl/dioxane (21ml) was added and stirred at room temperature for an additional 2 hours, where LCMS showed no starting material present. The suspension was filtered in a buchner funnel lined with filter paper. The mother liquor was preserved as it contained < 5% product. The solid was transferred to a 500ml beaker and HPLC water was added until the solid was completely dissolved. By adding solid Na₂CO₃The pH was adjusted to 10. CH for the aqueous solution₂Cl₂(5X 200ml) extracted or until LCMS showed no product present in the aqueous layer. CH (CH)₂Cl₂The solution is passed through Na₂SO₄Dried and concentrated. Redissolving in CH₂Cl₂Crude product in (10ml) and MeOH (1ml) in CH₂Cl₂/MeoH/NEt₃Purification on silica gel column eluted systematically (Biotage 40+ column: with 600ml CH)₂Cl₂100% equilibrium to give by-product, zone 1: 1200ml 10% MeOH/CH₂Cl₂Linearity; section 2: 2400ml 10% MeOH/CH₂Cl₂A step of; section 3: 2400ml 9% MeOH/1% NEt₃/CH₂Cl₂). Collecting the desired fractions to give 3- [ (R) -1- (2, 6-dichloro-3-fluoro-phenyl) -ethoxy]-5- (1-piperidin-4-yl-1H-pyrazol-4-yl) -pyridin-2-ylamine (7.19 g, combinationYield 75%, white solid). MS M/e 450(M +1)⁺。¹H NMR(DMSO-d₆，400MHz)δ7.92(s，1H)，7.76(s，1H)，7.58(m，1H)，7.53(s，1H)，7.45(m，1H)，6.90(s，1H)，6.10(m，1H)，5.55(b s，2H)，4.14(m，1H)，3.05(m，2H)，2.58(m，2H)，1.94(m，2H)，1.80(d，3H)，1.76(m，2H)。

The solid product, 3- [ (R) -1- (2, 6-dichloro-3-fluoro-phenyl) -ethoxy ] -5- (1-piperidin-4-yl-1H-pyrazol-4-yl) -pyridin-2-ylamine, was dissolved in dichloromethane and the solvent was slowly evaporated to yield a fine crystalline solid. After drying under high vacuum, the sample was identified as single polymorph form a, having a melting point of 194 ℃.

Claims (7)

1. Use of (R) -3- [1- (2, 6-dichloro-3-fluoro-phenyl) -ethoxy ] -5- (1-piperidin-4-yl-1H-pyrazol-4-yl) -pyridin-2-ylamine, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating a cancer mediated by anaplastic lymphoma kinase in a mammal in need thereof.
2. 2. The use according to claim 1, wherein the mammal is a human.
3. 3. The use according to claim 1, wherein the mammal is a dog.
4. 4. The use according to claim 1, wherein the cancer is selected from the group consisting of lung cancer, bone cancer, pancreatic cancer, cancer of the head and neck, epidermal or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, hodgkin's disease, carcinoma of the esophagus, carcinoma of the small intestine, carcinoma of the thyroid gland, carcinoma of the parathyroid gland, carcinoma of the adrenal gland, sarcoma of soft tissue, carcinoma of the urethra, carcinoma of the penis, carcinoma of the prostate, chronic or acute leukemia, lymphocytic lymphomas, cancer of the bladder, renal cell carcinoma, carcinoma of the renal pelvis, primary central nervous system lymphoma, spinal tumors, brain stem glioma, pituitary adenoma, and.
5. 5. Use according to claim 1, wherein the cancer is selected from skin cancer, cancer of the endocrine system, cancer of the kidney or ureter, tumors of the central nervous system and combinations thereof.
6. 6. The use according to claim 1, wherein the cancer is selected from the group consisting of: non-small cell lung cancer, squamous cell carcinoma, hormone refractory prostate cancer, papillary renal cell carcinoma, colorectal adenocarcinoma, neuroblastoma, anaplastic large cell lymphoma, and gastric carcinoma.
7. 7. Use according to any one of claims 1 to 6, wherein the medicament is administered as a pharmaceutical composition comprising (R) -3- [1- (2, 6-dichloro-3-fluoro-phenyl) -ethoxy ] -5- (1-piperidin-4-yl-1H-pyrazol-4-yl) -pyridin-2-ylamine and at least one pharmaceutically acceptable carrier.