HK1240924B - A deuterated triazolopyridazine as a kinase modulator - Google Patents

Description

Deuterated triazolopyridazines as kinase modulators

Technical Field

The present invention relates to novel compounds useful as modulators of protein tyrosine kinases. More particularly, the present invention relates to novel compounds which are inhibitors of c-Met.

Background of the invention

The present invention relates to triazolopyridazines as tyrosine kinases including c-Met inhibitors. Triazolopyridazines having useful therapeutic properties have been reported and are included in WO 2007/075567.

Protein kinases are enzymatic components of signal transduction pathways that catalyze the transfer of a terminal phosphate from ATP to the hydroxyl group of tyrosine, serine and/or threonine residues of proteins. Thus, compounds that inhibit protein kinase function are valuable tools for assessing the physiological consequences of protein kinase activation. Overexpression or inappropriate expression of normal or mutant protein kinases in mammals has been the subject of extensive research and has been shown to play an important role in the development of a number of diseases, including diabetes, angiogenesis, psoriasis, restenosis, ocular diseases, schizophrenia, rheumatoid arthritis, atherosclerosis, cardiovascular diseases and cancer. The cardiotonic benefit of kinase inhibition was also investigated. In summary, inhibitors of protein kinases have particular utility in the treatment of human and animal diseases.

The Hepatocyte Growth Factor (HGF), also known as Scattering Factor (SF), receptor, c-Met, is a receptor tyrosine kinase that regulates cell proliferation, morphogenesis and motility. The c-Met gene is translated into a 170kD protein, which is processed into a cell surface receptor consisting of a 140kD beta transmembrane subunit and a 50kD glycosylated extracellular alpha subunit.

Mutations in c-Met, overexpression of c-Met and/or HGF/SF, expression of c-Met and HGF/SF from the same cells, and overexpression and/or aberrant c-Met signaling are present in a variety of human solid tumors and are thought to be involved in angiogenesis, tumorigenesis, invasion and metastasis.

For example, cell lines with uncontrolled c-Met activation are both highly invasive and metastatic. A significant difference between normal and transformed cells expressing the c-Met receptor is that phosphorylation of the tyrosine kinase domain in tumor cells is generally independent of the presence of ligand.

Mutations/alterations in C-Met have been identified in a variety of human diseases including tumors and cancers-such as hereditary and sporadic human papillary renal cancers, breast cancer, colorectal cancer, gastric cancer, glioma, ovarian cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, testicular cancer, basal cell carcinoma, liver cancer, sarcoma, malignant pleural mesothelioma, melanoma, multiple myeloma, osteosarcoma, pancreatic cancer, prostate cancer, synovial sarcoma, thyroid cancer, non-small cell lung cancer (NSCLC) and small cell lung cancer, transitional cell carcinoma of the bladder, testicular cancer, basal cell carcinoma, liver cancer and leukemia, lymphomas and myelomas such as Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Acute Promyelocytic Leukemia (APL), Chronic Lymphocytic Leukemia (CLL), Chronic Myeloid Leukemia (CML), chronic Neutrophilic Leukemia (CNL), Acute Undifferentiated Leukemia (AUL), Anaplastic Large Cell Lymphoma (ALCL), prolymphocytic leukemia (PML), juvenile myelomonocytic leukemia (JMML), adult T-cell ALL, AML with trilineage myelodysplasia (trilineage myelodysplasia) (AML/TMDS), Mixed Lineage Leukemia (MLL), myelodysplastic syndrome (MDS), myeloproliferative disorders (MPD), multiple myeloma, (MM), myeloid sarcoma, non-hodgkin lymphoma and hodgkin's disease (also known as hodgkin lymphoma).

Due to the role of aberrant HGF/SF-Met signaling in the pathogenesis of various human cancers, inhibitors of the c-Met receptor tyrosine kinase have broad applications in the treatment of cancers in which Met activity contributes to the invasive/metastatic phenotype, including those cancers in which c-Met is not overexpressed or otherwise altered. Inhibitors of c-Met also inhibit angiogenesis and are therefore considered useful in the treatment of diseases associated with neovasculature formation, such as rheumatoid arthritis, retinopathy.

Overexpression of c-Met is also considered a potentially useful predictor of prognosis in certain diseases such as breast cancer, non-small cell lung cancer, pancreatic endocrine tumors, prostate cancer, esophageal adenocarcinoma, colorectal cancer, salivary gland cancer, diffuse large B-cell lymphoma and endometrial cancer.

Many strategies have been devised to attenuate aberrant Met signaling in human tumors. Some of these strategies include the use of HGF antagonists and small molecule inhibitors.

Safety, pharmacokinetics, pharmacodynamics and initial efficacy of potent and selective c-Met inhibitors having the structure

(hereinafter referred to as Compound A)

The study was conducted in phase I, the first in vivo test. This results in the detection of accidental nephrotoxicity. These data are in conflict with preclinical testing that shows the non-toxic profile in rats and dogs. Numerous additional preclinical experiments were performed to understand the nature of the renal effect. The metabolic data point in the direction that rabbits become the appropriate toxicological species. Toxicology studies in rabbits show that compound a does affect kidney function, and histological analysis shows crystal formation, followed by degenerative and inflammatory changes in the kidney. Further studies have shown the production of aldehyde oxidase-dependent species-specific insoluble metabolites that cause kidney injury through crystal formation in the renal tubules. The following metabolites were found to form crystals:

metabolite 1:

6- { difluoro [6- (1H-pyrazol-4-yl) [1,2,4] triazolo [4,3-b ] pyridazin-3-yl ] methyl } quinolin-2 (1H) -one.

Metabolite 2:

6- { difluoro [6- (1-methyl-1H-pyrazol-4-yl) [1,2,4] triazolo [4,3-b ] pyridazin-3-yl ] methyl } quinolin-2 (1H) -one.

Solubility of metabolite 2:

solubility at pH 4.84, 0.001mg/ml

Solubility at pH 7.33, 0.002 mg/ml.

Since no feasible strategy was identified to circumvent renal toxicity, further clinical development of compound a was abandoned.

Summary of The Invention

The present invention provides a novel triazolopyridazine as protein tyrosine kinase modulator, especially c-Met inhibitor, and the use of such compounds to reduce or inhibit the kinase activity of c-Met and modulate the expression of c-Met in a cell or subject, and the use of such compounds to prevent or treat cell proliferative disorders and/or disorders associated with c-Met in a subject. In particular, the invention relates to said compounds for use as medicaments for the treatment of cell proliferative disorders and/or disorders related to c-Met. The invention relates to the use of said compounds for the prophylaxis or treatment, in particular for the treatment of cancer, cell proliferative disorders and/or c-Met-related disorders, or to the use of said compounds for the preparation of a medicament for the prophylaxis or treatment, in particular for the treatment of cancer, cell proliferative disorders and/or c-Met-related disorders.

The invention also relates to pharmaceutical compositions comprising a compound of the invention and a pharmaceutically acceptable carrier. Another aspect of the invention is a pharmaceutical composition prepared by mixing a compound of the invention and a pharmaceutically acceptable carrier.

Other features and advantages of the invention will be apparent from the following detailed description of the invention and from the claims.

Drawings

FIG. 1: A) western blot of EBC-1; B) pMet protein levels normalized to actin in EBC-1 cells; C) Snu-5B Western blot: D) pMet protein levels normalized to actin in Snu-5 cells.

Detailed Description

The present invention relates to compounds of the following formula (I):

and N-oxides, pharmaceutically acceptable salts and solvates thereof, wherein D represents deuterium.

In one aspect, the present invention relates to compounds of the following formula (I):

and pharmaceutically acceptable salts and solvates thereof, wherein D represents deuterium.

In one aspect, the present invention relates to compounds of the following formula (I):

and pharmaceutically acceptable salts thereof, wherein D represents deuterium.

In one aspect, the present invention relates to compounds of the following formula (I):

wherein D represents deuterium.

It will be appreciated that certain variations in natural isotopic abundance occur in the synthesized compounds, depending on the source of the chemical materials used in the synthesis. Thus, the preparation of compound a will inherently contain a small amount of deuterium. The concentration of such naturally occurring deuterium is small (natural abundance of 0.015%) and is unimportant compared to the deuterium content of the compounds of the present invention.

The compounds of the present invention differ from this naturally occurring minor form in that the term "compound" as used herein refers to a composition of matter in which the abundance of deuterium is much higher (e.g., at least 1000 times (15%) higher) than the natural abundance (0.015%).

In one aspect of the invention, the compounds of formula (I) have a deuterium content (D) at the 2-position of the quinoline of at least 50% (D/H ratio at least 1:1), at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. Preferably, the deuterium content (D) in the 2-position of quinoline is at least 93%, more preferably the deuterium content (D) in the 2-position of quinoline is at least 97% or 98%.

When a position is specifically designated as "H" or "hydrogen", or its chemical representation means hydrogen, it is to be understood as hydrogen having a natural abundance isotopic composition.

It was found that by the compounds of formula (I) according to the invention, the formation of insoluble/less soluble aldehyde oxidase mediated metabolites is down-regulated. This may reduce renal toxicity.

Furthermore, it was found that the compounds of formula (I) according to the invention have a metabolic shift compared to the metabolism of compound a (up-regulation of CYP450 mediated metabolite formation). In the case of the compounds of the formula (I) according to the invention, the administration of Compound A is compared with the formation of the N-demethylated metabolite having the structure

More N-demethylated metabolites with the following structure

(active metabolite). This may reduce the therapeutically effective dose of the compound of formula (I) compared to compound a.

Furthermore, it was found that the compound of formula (I) was also shown to be active in OCT2 cells¹⁴Inhibition of C-metformin uptake.

As used hereinafter, the terms "compound of formula (I)" and "compound of formula (I)" also include N-oxides, pharmaceutically acceptable salts and solvates thereof.

Pharmaceutically acceptable salts

The compounds of the invention may also be present in the form of pharmaceutically acceptable salts, in particular pharmaceutically acceptable acid addition salts.

For use in medicine, salts of the compounds of the invention are referred to as non-toxic "pharmaceutically acceptable salts". FDA approval of pharmaceutically acceptable salt forms (cf. International J.Pharm.1986, 33, 201-217; J.Pharm.Sci., 1977, Jan, 66(1), p1) includes pharmaceutically acceptable acidic/anionic or basic/cationic salts.

Pharmaceutically acceptable acid addition salts include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camphorsulfonate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, etolate (estolate), ethanesulfonate, fumarate, glyceptate, gluconate, glutamate, glycollylalkylarsenate (glycollylsanilate), hexylresorcinate (hexyrexresorcinate), hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, maleate, mandelate, methanesulfonate, methyl bromide, methyl nitrate, methylsulfate, mucate, naphthalenesulfonate, nitrate, pamoate, pantothenate, phosphate/diphosphate (hydrogen phosphate), polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, 8-chlorotheyl salt, tosylate and triethyliodide. Organic or inorganic acids also include, but are not limited to, hydriodic acid, perchloric acid, sulfuric acid, phosphoric acid, propionic acid, glycolic acid, methanesulfonic acid, hydroxyethanesulfonic acid, oxalic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, cyclamic acid, caproic acid (saccharanic) or trifluoroacetic acid. The pharmaceutically acceptable salts of the present invention also include stereochemically isomeric forms thereof.

Stereoisomeric forms

One skilled in the art will recognize that the compounds of formula (I), particularly in the case of salts, may have one or more asymmetric carbon atoms in their structure. It is intended that the present invention includes within its scope single enantiomeric forms, racemic mixtures of the compounds, and mixtures of enantiomers in which an enantiomeric excess exists.

The term "single enantiomer" as used herein defines all the possible homochiral forms which the compounds of formula (I) may possess.

Stereochemically pure isomeric forms may be obtained by applying principles known in the art. Diastereomers can be separated by physical separation methods such as fractional crystallization and chromatographic techniques, and enantiomers can be separated from one another by selective crystallization of diastereomeric salts with optically active acids or bases or by chiral chromatography. Pure stereoisomers may also be prepared synthetically from the appropriate stereochemically pure starting materials, or by using stereoselective reactions.

The term "isomers" refers to compounds having the same composition and molecular weight but differing in physical and/or chemical properties. These species have the same number and kind of atoms, but differ in structure. The structural difference may be the configuration (geometric isomers) or the ability to rotate the plane of polarized light (enantiomers).

The term "stereoisomer" refers to an isomer of the same composition that differs in the arrangement of atoms in space. Enantiomers and diastereomers are stereoisomers in which the asymmetrically substituted carbon atom serves as a chiral center.

The term "chiral" refers to a structural feature of a molecule that does not superimpose it on its mirror image.

The term "enantiomer" refers to one of a pair of molecular species that are mirror images of each other and do not overlap.

The term "diastereomer" refers to stereoisomers that are not mirror images.

The symbols "R" and "S" represent the configuration of the substituents around the chiral carbon atom.

The term "racemate" or "racemic mixture" refers to a composition consisting of equimolar amounts of two enantiomers, wherein the composition is optically inactive.

The term "homochiral" refers to a state of enantiomeric purity.

The term "optically active" refers to the degree to which a plane of polarized light is rotated by a homochiral molecule or a non-racemic optically active mixture of chiral molecules.

The term "geometric isomer" refers to isomers that differ in the orientation of the substituent atoms associated with a carbon-carbon double bond, cycloalkyl ring or bridged bicyclic ring system. The substituent atoms (other than H) on each side of the carbon-carbon double bond may be in the E or Z configuration. In the "E" (opposite side) configuration, the substituent is on the opposite side relative to the carbon-carbon double bond; in the "Z" (same side) configuration, the substituents are oriented on the same side relative to the carbon-carbon double bond. The substituent atoms (other than H) attached to the carbocyclic ring may be in either the cis or trans configuration. In the "cis" configuration, the substituents are on the same side relative to the plane of the ring; in the "trans" configuration, the substituents are on opposite sides relative to the plane of the ring. Compounds having a mixture of "cis" and "trans" species are referred to as "cis/trans".

It is to be understood that the various substitutable stereoisomers, geometric isomers and mixtures thereof used to prepare the compounds of the present invention are commercially available, can be prepared synthetically from commercially available starting materials, or can be prepared as mixtures of isomers and then obtained as resolved isomeric forms using techniques well known to those of ordinary skill in the art.

As used herein, the isomer descriptors "R", "S", "E", "Z", "cis" and "trans" are used to indicate the configuration of the atoms relative to the core molecule and are intended to be used as defined in the literature (IUPAC Recommendation for Fundamental Stereochemistry, section E, Pure appl. chem., 1976, 45: 13-30).

The compounds of the present invention may be prepared as individual isomers by isomer-specific synthesis or by resolution from a mixture of isomers. Conventional resolution techniques include the formation of the free base of each isomer of the isomer pair using optically active salts (followed by fractional crystallization and regeneration of the free base), the formation of esters or amides of the individual isomers of the isomer pair (followed by chromatographic separation and removal of the chiral auxiliary) or the resolution of the isomer mixture of the starting material or the final product using preparative TLC (thin layer chromatography) or chiral HPLC (high performance/high pressure liquid chromatography) columns.

Homoplasmic pleomorphic variants and solvates

In addition, the compounds of the present invention may have one or more polymorphic crystalline forms or may be amorphous. Accordingly, these forms are intended to be included within the scope of the present invention. In addition, the compounds may form solvates, for example with water (i.e. hydrates) or common organic solvents (e.g. alcohols). As used herein, the term "solvate" refers to a physical association of a compound of the present invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. The term "solvate" is intended to include both solution phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like. It is intended that the present invention include within its scope solvates of the compounds of the invention. Pharmaceutically acceptable salts and N-oxides of the compounds of the invention may also form solvates. Solvates of the pharmaceutically acceptable salts and N-oxides of the compounds of the invention are also included within the scope of the invention.

Thus, in the methods of treatment or prevention of the present invention, the term "administering" shall include means for treating, ameliorating or preventing the syndromes, disorders or diseases described herein with the compounds of the present invention or solvates thereof, which shall be expressly included within the scope of the present invention, although not specifically disclosed.

Preparation of the Compounds of the invention

The compounds of formula (I) may be prepared by: reduction of a deuterated intermediate of formula (II) (wherein W₁Representing chlorine, bromine or iodine, iodine being preferred), in the presence of deuterium gas and in the presence of a suitable catalyst (e.g. a palladium catalyst, such as palladium on carbon 10% (10% Pd/C) or a Pt catalyst, palladium catalysts, especially palladium on carbon being preferred), in a suitable solvent or solvent mixture (e.g. methanol, deuterated)Methanol (d1-MeOD, d4-MeOD), tetrahydrofuran, N-methyl-2-pyrrolidone (NMP) or mixtures thereof, e.g., mixtures of tetrahydrofuran and methanol, or mixtures of tetrahydrofuran and deuterated methanol, the latter being preferred, in the presence of a suitable base, e.g., triethylamine or sodium carbonate (Na)₂CO₃) The latter being preferred). It is preferred to dry the catalyst, since traces of water can act as a source of hydrogen. Furthermore, the catalyst is preferably pre-deuterated with deuterium gas to remove catalyst-bound hydrogen. Furthermore, the catalyst is preferably washed to remove hydrogen bound by the catalyst. The v: v ratio of deuterated methanol to tetrahydrofuran in the solvent mixture is preferably from 1:9 to 1:2, preferably 1: 4.

The compounds of formula (I) may also be prepared by: reacting an intermediate of formula (III) (wherein W₂Representing a suitable leaving group, e.g. halo, e.g. chloro etc.) with an intermediate of formula (IV) in the presence of a suitable solvent, such as an alcohol, e.g. n-butanol.

For the synthesis of intermediates of formula (III) reference is made to WO2007/075567, which is incorporated herein by reference.

The compounds of formula (I) may also be prepared by: reacting an intermediate of formula (V) (wherein W₃Representing a suitable leaving group, e.g. halo, with an intermediate of (VI), in a suitable catalyst (e.g. Pd)₂dba₃) In the presence of a suitable ligand (e.g., P (tBu)₃)BF₄) In the presence of a suitable base (e.g. Na)₂CO₃) In the presence of a suitable solvent, such as dioxane.

For the synthesis of intermediates of formula (VI), reference is made to WO2007/075567, which is incorporated herein by reference.

The intermediate of formula (v) may be prepared by: reacting an intermediate of formula (IV) with an intermediate of formula (VII) (wherein W₃As defined above) in the presence of a suitable solvent, e.g., an alcohol such as n-butanol.

For the synthesis of intermediates of formula (VII), reference is made to WO2007/075567, which is incorporated herein by reference.

Embodiments of the present invention relate to a process for preparing a compound of formula (I) characterized in that:

a) reducing the deuterated intermediate of formula (II) in the presence of deuterium and in the presence of a suitable catalyst, a suitable solvent or solvent mixture, and a suitable base, wherein W is₁Represents chlorine, bromine or iodine,

wherein D represents deuterium;

b) reacting an intermediate of formula (III) (wherein W₂Represents a suitable leaving group) with an intermediate of formula (IV) (wherein D represents deuterium) in the presence of a suitable solvent,

c) reacting an intermediate of formula (V) (wherein W is₃Represents a suitable leaving group, and wherein D represents deuterium) with an intermediate of formula (VI),

or, if desired, the compounds of formula (I) are converted into therapeutically active non-toxic acid addition salts by treatment with an acid, or conversely, the acid addition salt forms are converted into the free base by treatment with a base, or, if desired, the solvate or N-oxide forms thereof are prepared.

Examples of the synthesis of the individual compounds are shown below.

Example 1

a) Drying the catalyst: the catalyst 10% Pd/C (Escat 1931, BASF) was dried before use. The following conditions were applied

Cabinet dryer, applied at 85 ℃/<100mbar/24 hours, then at 85 ℃/<1mbar/24 hours

Diffusion of the wet catalyst in beaker glass (fill height <5mm, container covered with tissue paper (tissue))

b) Pre-deuteration of the catalyst: a shake flask (6L, glass) containing 19.3g of dry catalyst (10% Pd/C, Escat 1931, BASF), 40.6g of sodium carbonate (2 equivalents, 0.384mol, Aldrich 71347), 1.6L of Tetrahydrofuran (THF) (Aldrich 87371) and 200ml of d 1-methanol (Aldrich 151939) was flushed with nitrogen. The flask was sealed, purged with three cycles of deuterium/vacuum, and finally placed under deuterium atmosphere (1.05 bar absolute). The shaker was started and the catalyst was pre-deuterated at 25 ℃ for 1 hour.

The deuterium gas was replaced by nitrogen and the pre-deuteration was stopped. Finally, the solvent was removed by decantation.

c) A reductive deuteration method: a slurry of 96.5g of starting material 1(0.192mol) in 1.6l THF (Aldrich 87371) and 390ml d 1-methanol (Aldrich 151939) was added to the pre-deuterated catalyst/additive mixture. The flask was sealed, purged with three cycles of deuterium/vacuum, and finally placed under deuterium atmosphere (1.05 bar). The shaker was started and deuterium uptake was monitored. (deuterium uptake is at a very low level during the first hour of reaction time)

After a reaction time of 24 hours, the deuteration was interrupted by replacing the deuterium gas with nitrogen. Analytical samples were taken and analyzed by HPLC. According to HPLC analysis, the starting material was completely converted.

The reaction mixture was diluted with 1l Dichloromethane (DCM), the catalyst was filtered off and the filter cake was washed with 500ml DCM. To isolate the desired product 2, the solvent was removed by evaporation at 45 deg.C/vacuum. About 100g of crude product was isolated as a yellow solid (still containing inorganic salts).

Liquid-liquid extraction: the crude product was dissolved in 1.6l DCM/1l 1M NaOH and transferred to a separatory funnel. After mixing, the two layers were separated and the organic layer was washed with 1l of deionized water. All aqueous layers were extracted a second time with 1l DCM. The two DCM layers were combined and Na was added₂SO₄Drying and finally removing the solvent by evaporation (45 ℃/vacuum).

65.4g of product 2 (compound of formula (I)) were isolated as an off-white solid. According to HPLC analysis, the material was 97% pure. Based on¹The deuterium content at the 2-position of the quinoline moiety was 98.6% by H-NMR analysis

Starting material 1 was prepared according to the following reaction scheme:

step 1: in the presence of a suitable oxidizing agent, such as mCPBA (m-chloroperbenzoic acid), and a suitable solvent, such as dichloromethane. The reaction was carried out at room temperature.

Step 2: in the presence of TosCl (tosyl chloride; 4-methylbenzenesulfonyl chloride), a suitable base, such as NaOAc (sodium acetate), and a suitable solvent, such as dichloromethane, followed by reaction in the presence of LiOH and a suitable solvent, such as an alcohol, e.g., methanol.

And step 3: in NaI, (CF)₃SO₂)₂O (trifluoromethanesulfonic anhydride; trifluoromethanesulfonic anhydride) in the presence of a suitable solvent such as acetonitrile and pyridine.

Synthesis of starting Material 3

Starting materials 4(400g) (Compound A; WO2007/075567), mCBA (m-chloroperbenzoic acid) (1.2 equivalents) and dichloromethane (10V) (1V is per kg of starting materials1 liter) was mixed at room temperature for 17 hours. The mixture was saturated with Na₂CO₃Neutralizing the aqueous solution to pH>8. The mixture was stirred for 0.5 hour. The mixture was filtered and the solid was washed with water until the pH was about 7. The solid was dried under vacuum at room temperature. Yield: 410g of feed 3.

Synthesis of starting Material 2

Starting material 3(410g), TosCl (tosyl chloride; 4-methylbenzenesulfonyl chloride) (2 equivalents) and dichloromethane (20V) (1V 1 l/kg starting material) were combined. NaOAc (4 equivalents) was added and the mixture was stirred for 2 hours. The solvent was removed in vacuo. Methanol (20V) was added. The mixture was stirred. Adding LiOH H₂O (5 equivalents), the mixture was stirred at room temperature for 17 hours. The mixture was concentrated to remove 16V of methanol and 20V of water was added. The mixture was neutralized with concentrated HCl until the pH was about 6. The mixture was stirred for 0.5 hour and filtered. The solid was dried under vacuum at 50 ℃. The solid was slurried with water (10V) for 0.5 hour. The mixture was filtered. The solid was dried under vacuum at 50 ℃. The pulping step and the drying step are repeated once. Yield: 375g of feed 2.

Synthesis of starting Material 1

Starting material 2(190g), pyridine (1 eq) and acetonitrile (10 eq) were mixed and the mixture was cooled to below 0 ℃. (CF)₃SO₂)₂O (4 equivalents) was slowly added dropwise, and the reaction temperature was controlled to 5 ℃ or lower. After the addition, the mixture was heated to 20 ℃ and stirred for 1 hour. The reaction mixture was cooled to below 0 ℃. (CF)₃SO₂)₂O (1 eq) was added slowly dropwise. NaI (7V) (1V is 1 liter/kg of starting material) was slowly added to the reaction mixture, and the reaction temperature was controlled to 5 ℃ or lower. After the addition, the reaction mixture was heated to 50 ℃ and stirred at 50 ℃ for 17 hours. Ethyl acetate was added and the mixture was taken up in water and 10% Na₂S₂O₃Solution, brine wash. Anhydrous Na for organic layer₂SO₄Drying and purifying by gel chromatography. Yield: 98.5g of feed 1.

Example 2:

synthesis of intermediate 1:

3-Chloroperoxybenzoic acid (13.5g, 78.4mmol) was added portionwise at room temperature to the solution in CHCl₃6-iodoquinoline (CAS 13327-31-6) (10g, 39.2mmol) in (300 mL). The reaction mixture was stirred for 2 days and then poured into K₂CO₃10% aqueous solution. The organic layer was extracted with Dichloromethane (DCM). The organic layer was dried (MgSO₄) Filtered and evaporated to dryness to give 10.5g of intermediate 1 (99%).

Synthesis of intermediate 2:

intermediate 1(5.2g, 19.2mmol) and NaOD (40% D)₂O) solution (3.4mL, 48.4mmol) in D₂The mixture in O (100mL) was heated to 100 ℃ for 2 days. The mixture was cooled to room temperature. Addition of D₂O, filtering the precipitate with D₂O wash and dry to yield 4.9g of intermediate 2 (96%).

Synthesis of intermediate 3:

intermediate 2(4.8g, 17.64mmol), HCOO-NH₄ ⁺A mixture of (6.68g, 0.106mol) and Raney nickel (6.2g, 0.106mol) in MeOH (methanol) (130mL) was heated to 60 deg.C for 1.5 hours. The reaction mixture was cooled to room temperature and poured into D₂In O, with K₂CO₃Basified and extracted with EtOAc (ethyl acetate). The organic layer was dried (MgSO₄) Filtered and evaporated to dryness.

The residue (4g) was purified by chromatography on silica gel (80g of irregular SiOH35-40 μm, mobile phase: 100% DCM to 95% DCM 5% CH₃OH 0.1％NH₄Gradient of OH). The pure fractions were collected and evaporated to dryness to yield 2.9g of intermediate 3 (83%).

Synthesis of intermediate 4:

(+) -L-ascorbic acid sodium salt (2.32g, 11.7mmol) in N₂Adding CuSO at room temperature under atmosphere₄·5H₂To a solution of O (1.95g, 7.8mmol) in dimethyl sulfoxide (DMSO) (25mL) the mixture was stirred for 2 hours. Ethyl bromodifluoroacetate (0.55mL, 4.3mmol) was added and the reaction mixture was stirred for 1.5 hours before intermediate 3(1g, 3.9mmol) was added. After heating at 50 ℃ for 15 hours, the mixture was cooled to 10 ℃ and NH was added₂-NH₂.H₂O (4.76mL, 78.1 mmol). Dropwise addition of H₂O (12mL) (exothermic), the mixture was stirred at room temperature for 20 min. Adding EtOAc and passing the mixtureShort pad filtration. The organic layer was extracted and dried (MgSO)₄) Filtered and evaporated to dryness.

The residue (1g) was purified by chromatography on silica gel (40g silica gel 30 μm, mobile phase: 100% DCM to 90% DCM 10% CH₃OH 0.1％NH₄Gradient of OH). The pure fractions were collected and evaporated to dryness to yield 0.42g of intermediate 4 (45%).

Synthesis of intermediate 5:

3, 6-dichloropyridazine (4.57g, 0.0031mol), (1-methyl-1H-pyrazol-4-yl) boronic acid pinacol ester (3.82g, 0.0184mol) and Na₂CO₃A2M (18.3mL) solution in dioxane (18.4mL) was stirred for 1 min. Adding PdCl₂(PPh₃)₂(1.29g, 0.0018mol), the solution was heated at 80 ℃ for 15 hours. The mixture was cooled to room temperature and poured into water. Adding K₂CO₃The mixture is passed throughShort pad filtration. The organic layer was dried (MgSO₄) Filtered and evaporated to dryness. Will be provided withBy CH₂Cl₂Washing, drying the filtrate (MgSO)₄) And evaporated. The residue is derived from CH₂Cl₂And (4) medium crystallization. The precipitate was filtered and dried to yield 1.5g of first intermediate 5 (42%). The filtrate was purified by chromatography on silica gel (30g SiOH15-40 μm, mobile phase: CH)₂Cl₂Gradient of 100%, CH₂Cl₂ 95％/CH₃OH 5%). The pure fractions were collected and evaporated to dryness to yield 1.58g of the second crop of intermediate 5 (44%).

The total yield is 86%

Synthesis of a compound of formula (I):

intermediate 4(0.41g, 1.7mmol) and intermediate 5[943541-20-6 ]]A mixture of (0.335g, 1.7mmol) in n-butanol (30mL) was heated at 125 deg.C for 15 hours. The reaction mixture was cooled to room temperature and evaporated to dryness. The residue was purified by chromatography on silica gel (40g of irregular SiOH35-40 μm, mobile phase: 100% DCM to 90% DCM 10% CH₃OH 0.1％NH₄Gradient of OH). The pure fractions were collected and evaporated to dryness. The residue (0.41g) was purified by achiral SFC (supercritical fluid chromatography) (stationary phase: 2ETHYLPYRIDINE 6 μm 150X 21.2mm, mobile phase: 85% CO₂15% MeOH). The pure fractions were collected and evaporated to dryness. The residue (0.387g) was crystallized from diisopropyl ether. The precipitate was filtered and dried to yield 0.315g of the compound of formula (I) (48%, deuterium content at position 2 of quinoline moiety 93-94%). Melting point 201.6 ℃ (DSC).

Example 3

Synthesis of intermediate 6:

a mixture of intermediate 4(0.42g, 1.76mmol) and 3, 6-dichloropyridazine (0.788g, 5.3mmol) in n-butanol (12mL) was heated at 130 deg.CFor 2 hours. The mixture was cooled to room temperature and evaporated to dryness. DCM was added and the mixture was taken up with 10% K₂CO₃The aqueous solution was stirred together. The organic layer was extracted and dried (MgSO)₄) Filtered and evaporated to dryness. The residue (0.9g) was purified by chromatography on silica gel (40g of irregular SiOH35-40 μm, mobile phase: 100% DCM to 95% DCM 5% CH₃OH 0.1％NH₄Gradient of OH). The pure fractions were collected and evaporated to dryness to yield 0.385g of intermediate 6 (66%).

Synthesis of Compounds of formula (I)

In a sealed tube, intermediate 6(183mg, 0.55mmol), (1-methyl-1H-pyrazol-4-yl) boronic acid pinacol ester (343mg, 1.65mmol), P (tBu)₃)BF₄(47.9mg, 0.165mmol) and Na₂CO₃2M (1.65mL, 3.3mmol) in dioxane (4mL) with N₂Purge for 10 minutes. Adding Pd₂dba₃(101mg, 0.11mmol) and the mixture was purged again for 5 minutes. The mixture was heated at 85 ℃ for 15 hours and cooled to room temperature. (1-methyl-1H-pyrazol-4-yl) boronic acid pinacol ester (343mg, 1.65mmol), P (tBu)₃)BF₄(47.9mg，0.165mmol)，Pd₂dba₃(101mg, 0.11mmol) and Na₂CO₃2M aqueous solution (1.65mL, 3.3mmol) was added and the mixture was heated to 85 ℃ for 5 hours. The mixture was cooled to room temperature and poured into H₂O+K₂CO₃Neutralized and extracted with EtOAc. The organic layer was dried (MgSO₄) Filtered and evaporated to dryness. The residue was purified by chromatography on silica gel (24g of irregular SiOH35-40 μm, mobile phase: 100% DCM to 95% DCM 5% CH₃OH 0.1％NH₄Gradient of OH). The pure fractions were collected and evaporated to dryness to give 58mg of the compound of formula (I) (28%, deuterium content in position 2 of quinoline moiety 93-94%).

NMR method for determining deuterium/hydrogen content in example 1

The instrument Bruker Avance 300

Solvent CDCl₃

Sample preparation 10-25mg in 0.7ml CDCl₃In the middle, filtered

Probe 5mm QNP 1H/13

Pulse program zg30

Number of scans 16 or 254

The temperature is 29 DEG C

Relaxation time 4.6sec.

Chemical shift according to¹H-NMR predicted that a chemical shift of 8.84ppm was expected (ChemOffice). The hydrogen signal was assigned to the corresponding position in the range of 8.9 to 9.0ppm based on the integration and the expected chemical shift.

NMR method for determining deuterium/hydrogen content in examples 2 and 3

The instrument Bruker Avance III 500

DMSO or CDCl solvent₃

Sample preparation 4mg in 0.7ml CDCl₃Or in DMSO

Probe 5mm TXI Z-GRD (¹H/¹³C/¹⁵N)

Pulse program zg30

Number of scans 16

The temperature is 22 DEG C

Relaxation time 1sec.

Chemical shifts are based on an integral of 9.02ppm and the deuterium/hydrogen ratio determined by chemical shift.

Analytical HPLC method for determining product purity in example 1

Residence time product 2 (compound of formula (I)) 5.1 minutes.

Biological activity

The following representative assays may be performed in determining the biological activity of compounds within the scope of the present invention. They are administered to illustrate the invention in a non-limiting manner.

Inhibition of the proliferation of cancer cells carrying Met-amplification and dependent on Met signaling by compounds of formula (I)

Alama (Alamar) blue proliferation assay

Cells were seeded in 96-well plates in 180 μ l growth medium. The number of cells per well was different for each cell line according to the growth curve test. Cells were incubated at 37 ℃ in an incubator at 5% CO in the presence of moisture₂Incubate under atmosphere overnight. The next day: compound plates were prepared and 4. mu.l of compound was added to 196. mu.l of pre-warmed medium. Mu.l of the aforementioned was added to 180. mu.l of the cells. This was humidified at 37 ℃ with 5% CO₂After addition of the compound in the atmosphere, incubation was carried out for 4 days. After 4 days, 40. mu.l of Alamar (Alamar) blue solution was added. It was dried at 37 ℃ in moist 5% CO₂Incubate for 4 hours in atmosphere (depending on the cell line, this was tested before incubation for different hours during the growth curve experiment). After 4 hours, fluorescence was measured at excitation 530nm, emission 590 nm. The fluorescence of the control (DMSO treatment) was taken as 100%, and the fluorescence of cells incubated with compound was calculated relative to the control value, in%. Thus, dose response curves can be made, and the IC can be calculated₅₀。

Growth medium, cell culture medium it was used:

for Snu-5 Medium (Medium)

IMDM	500ml
		20％FCS	120ml
2mM L-Glutamine	6ml
		50 ug/ml gentamicin	6ml

For EBC-1 Medium (Medium)

EMEM	500ml
		10％FCS	57ml
2mM L-Glutamine	5.7ml
		1％PenStrep	5.7ml

As a result:

cell lines	IC50Compound A [ M ]]	IC50A compound of formula (I) [ M]
			SNU-5	1.38E-8	1.32E-8
EBC1	1.34E-08	1.2E-08

Inhibition of Met phosphorylation in a dose-responsive manner by compounds of formula (I)

Western blot

Cell line: EBC-1 and Sun-5

Samples were run on SDS-PAGE. Thereafter, the gel was run on an I-Blot machine (Invitrogen). The principle is as follows: proteins are transferred to PDVF membranes electrically.

PDVF membranes were first blocked with Blocking buffer (Odyssey Blocking buffer (PBS); Licor) for 1 hour at room temperature. After blocking, the membrane was incubated with the primary antibody overnight at 4 ℃. The next day, the blot was washed 3 times with TBS-tween 0.1% for 5 minutes and secondary antibodies were placed on the blot for 1 hour at room temperature. After incubation, the blot was washed 3 times with TBS-tween 0.1% for 5 minutes and scanned for signal.

The antibodies used were:

a first antibody:

signaling technology (Cell Signaling technology) #3077, anti- (anti-) pMet (Tyr1234/1235) rabbit mAb, 1/2,000

Signaling technology (Cell Signaling technology) #3127, anti- (anti-) Met (25H2) mouse mAb, 1/1,000

Sigma A1978, anti- (anti-) b-Act mouse mAb 1/30,000

Secondary antibody:

Invitrogen#A21076，Alexa 680Goat Anti-Rabbit IgG(H+L)，1/4,000

Rockland#610-732-124, Mouse (Mouse) IgG (H)&L) antibodies (antibodies)Conjugated Pre-adsorbed (Conjugated Pre-adsorbed), 1/4,000

The results are shown in FIG. 1

In vivo pharmacokinetic determination of Compound (I), Compound A and metabolites thereof in New Zealand white rabbits.

Male New Zealand rabbits (Crl: KBL (NZW), Charles River, France) and New Zealand female rabbits (NZW INRA A1077, Center Lago) were used. One male and two female rabbits per compound (I) and compound a) were used, with an average weight of 2.6 ± 0.2 kg. A complete plasma concentration time curve was obtained from each animal. Standard diet and tap water were available ad libitum. Both compound of formula (I) and compound A were dissolved at a final concentration of 1mg/ml in a 10% (w/v) SBE-B-CD (sulfobutyl ether-beta-cyclodextrin) research grade (Captisol) solution. HCl and PVP K30 were added to facilitate dissolution of the compound. After total dissolution, the pH was raised to 2.6/2.7 with NaOH. The formulations were stored at room temperature, protected from light, and analyzed quantitatively by LC-MS/MS on the day of preparation. The stability of the formulations was checked on the day of application. Animals were orally administered at 10ml/kg by gavage to obtain a final dose of 10 mg/kg. Blood samples were taken from animals to which each individual was administered at 30 minutes, 1,2,4, 7 and 24 hours after oral administration. Collecting blood by sampling from external auditory vein600K3E tube (Sarstedt). The samples were immediately placed on melted ice and plasma was obtained after centrifugation at about 1900 Xg for 10 minutes at 4 ℃. All samples were shielded from sunlight and stored at ≦ 18 ℃ before analysis. Plasma was analyzed for compound (I), compound a, metabolite 1, metabolite 2, N-desmethyl metabolite 3 (which was calculated on the curve for N-desmethyl metabolite 4) and N-desmethyl metabolite 4 using a qualified research LC-MS/MS method. Key analytical properties (linearity, upper and lower quantification limits, accuracy and precision) and plasma concentrations of the method are reported. For all compoundsThe lower limit of plasma quantification (LLOQ) was 1.00 ng/ml. Using Phoenix^TMProfessional (version 6.2.1) performed limited pharmacokinetic analyses. For all data, non-partitioned analysis using the lin/log trapezoidal rule of lin/log interpolation was used.

Results

Basic pharmacokinetic parameters of the compound of formula (I) and its metabolites after a single oral administration of 10mg/kg of compound (I) in male and female rabbits. Compound A (impurity) was also detected

ND not determined

MRT mean residence time (hours)

Basic pharmacokinetic parameters of Compound A and its metabolites after a single oral administration of 10mg/kg Compound A in male and female rabbits.

ND not determined

MRT mean residence time (hours)

Metabolite 1:

6- { difluoro [6- (1H-pyrazol-4-yl) [1,2,4] triazolo [4,3-b ] pyridazin-3-yl ] methyl } quinolin-2 (1H) -one

Metabolite 2:

6- { difluoro [6- (1-methyl-1H-pyrazol-4-yl) [1,2,4] triazolo [4,3-b ] pyridazin-3-yl ] methyl } quinolin-2 (1H) -one

N-demethylated metabolite 3;

n-demethylated metabolite 4;

6- { difluoro [6- (1H-pyrazol-4-yl) [1,2,4] triazolo [4,3-b ] pyridazin-3-yl ] methyl } quinoline

In vitro study of inhibition of OCT2(SLC22A2) transport by Compounds of formula (I)

This was tested using Chinese Hamster Ovary (CHO) cells, either parental or stably transfected with OCT 2.¹⁴C-metformin was used as OCT2 substrate.

The parental and CHO cell line stably transfected with OCT2 were obtained from Solvo Biotechnology (hungarian).

CHO cells were cultured in DMEM-F12 (Dulbecco's Modified Eagle medium supplemented with 0.03mg/mL L-proline, 1% L-glutamine, penicillin (50-100U/mL), streptomycin (50-100. mu.g/mL)) and 10% (v/v) fetal calf serum or calf serum (FCS), further referred to as "CHO medium".

OCT2 inhibition assay

Compound preparation

If desired, non-radiolabeled and radiolabeled compounds are mixed to obtain the appropriate chemical and radioactive concentrations. Stock solutions (200x) were prepared using the solvents shown in the table below. Appropriate solvent controls were included. The required test items and all reference and inhibitory compounds are shown in the table below.

For parental and OCT2 transfected CHO cell lines:

incubation procedure

T-24 hours

In CHO media, both CHO parental and OCT2 cells were seeded into 24-well plates (1 mL/well, 400000 cells/well).

Day of experiment

Transport experiments in Hank Balanced salt solution supplemented with 10mM HEPES pH7.4^+Ca，+Mg(HBSS^+/+) Is carried out in (1). All media added to the cells and plates was maintained at 37 ℃.

Prior to incubation, cells in each well were incubated with 1mL HBSS at 37 ℃^+/++10mM Hepes pH 7.4. Next, medium (250. mu.L/well) containing the reference substrate and the inhibitor (or inhibitor solvent) was added.

At the time of administration (0 min), 150 μ L of the administration solution was sampled in triplicate to determine the initial concentration by Liquid Scintillation Counting (LSC). During incubation, the plate was kept at 37 ℃.

To stop the reaction, 1.5mL of ice-cold HBSS was added to each well^+/+And the liquid is pumped. Again, 2mL of ice-cold HBSS were added to each well^+/+Add and aspirate while maintaining the angle of the plate. After aspiration of the last well, all wells were aspirated again, taking care not to touch the cells.

To lyse the cells, 250 μ L of mammalian protein extraction reagent (M-PER) lysis buffer was added to each well and the plates were shaken for at least 10 minutes (400 rpm). For LSC, 150. mu.L of sample/well was taken, and for protein, 25. mu.L of sample/well was taken. Protein analysis was performed according to bicinchoninic acid (BCA) method.

Data analysis

Data are expressed as picomoles per ml of protein, and as a percentage of control (solvent control ═ DMSO). Sigmaplot for computing IC₅₀The value is obtained.

Results and discussion

In OCT2 transfected CHO cells compared to parental cells¹⁴The uptake of C-metformin (OCT2 substrate) was much higher (7.39 and 17.2 fold). This uptake was inhibited by a positive control inhibitor, 300 μ M quinidine (85.5%, 100%). These data indicate that the assay conditions used are effective for studiesTest compounds were tested for inhibition of OCT 2-dependent transport.

Compounds of formula (I) are shown in OCT2 cells¹⁴Inhibition of C-metformin uptake, IC₅₀The concentration was 0.67. + -. 0.02. mu.M.

Cytotoxicity assays

The cytotoxicity of the compounds of formula (I) was determined at 100. mu.M and in the CHO parental and OCT2 cells. The 1% Triton-X100 condition was also included as a positive control cytotoxic agent. After 1 minute incubation, the supernatant was aspirated and the stem cells were incubated with 1mL HBSS^+/++10mM Hepes pH7.4 (37 ℃ C.) were washed twice. After aspirating the buffer, HBSS was added^+/++10mM Hepes PrestoBlue in pH7.4^TMThe plates were incubated at 37 ℃ for 60 minutes in 1/10 dilutions of viability reagents (Life Technologies) and stored protected from light. Each well was sampled (150. mu.L) in a black 96-well plate and fluorescence was measured (excitation: 560nm/12nm bandwidth, emission: 590nm/12nm bandwidth).

Results and discussion

No cytotoxic effect was observed for 100. mu.M of the compound of formula (I). Viability declined dramatically using a positive control cytotoxic agent, a 1% solution of Triton X-100 (see table below). This indicates that the possible inhibitory effect is not associated with loss of cell viability.

A method of treatment/prevention; use of the Compounds

In another aspect of the invention, the compounds of the invention are useful for inhibiting tyrosine kinase activity or expression, including c-Met activity, reducing kinase activity or expression, including c-Met activity, and modulating expression of c-Met in a cell or subject (subject), or treating a disorder associated with c-Met kinase activity or expression in a subject (subject). Inhibition of c-Met activity is thought to indirectly modulate c-Met expression.

In one embodiment of this aspect, the invention provides a method of reducing or inhibiting kinase activity and modulating c-Met expression of c-Met in a cell comprising the step of contacting the cell with a compound of formula (I). The present invention also provides a method of reducing or inhibiting kinase activity and modulating c-Met expression of c-Met in a subject comprising the step of administering to the subject. The invention also provides a method of inhibiting cell proliferation in a cell comprising the step of contacting the cell with a compound of formula (I). The invention also provides compounds of formula (I) for use in reducing or inhibiting the kinase activity of c-Met and modulating c-Met expression.

The term "subject" as used herein refers to an animal, preferably a mammal, most preferably a human, who is the object of treatment, observation or experiment.

The term "contacting" as used herein refers to adding a compound to a cell such that the compound is taken up by the cell.

In other embodiments of this aspect, the invention provides prophylactic and therapeutic methods for treating a subject at risk of developing a cell proliferative disorder or a disorder associated with c-Met (or susceptible to developing a cell proliferative disorder or a disorder associated with c-Met). These disorders include pre-existing conditions associated with c-Met expression (or overexpression) and/or c-Met mutation.

In one example, the present invention provides a method of preventing a cell proliferative disorder or a disorder associated with c-Met in a subject comprising administering to the subject a prophylactically effective amount of a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutically acceptable carrier. Administration of the prophylactic agent may occur prior to manifestation of symptoms characteristic of a cell proliferative disorder or a disorder associated with c-Met, such that the disease or disorder is prevented or its progression is delayed. The present invention provides compounds of formula (I) for use in the prevention of cell proliferative disorders or disorders associated with c-Met. The invention provides the use of a compound of formula (I) in the manufacture of a medicament for the prevention of a cell proliferative disorder or a disorder associated with c-Met.

In another example, the present invention relates to a method of treating a cell proliferative disorder or a disorder associated with c-Met in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutically acceptable carrier. Administration of the therapeutic agent may occur concurrently with manifestation of symptoms characteristic of the disorder, such that the therapeutic agent acts as a therapeutic agent to compensate for a cell proliferative disorder or a disorder associated with c-Met. The present invention provides compounds of formula (I) for use in the treatment of cell proliferative disorders or disorders associated with c-Met. The present invention provides the use of a compound of formula (I) for the manufacture of a medicament for the treatment of a cell proliferative disorder or a disorder associated with c-Met.

In another example, the present invention relates to a method of modulating a cell proliferative disorder or a disorder associated with c-Met in a subject (subject) such that modulation of c-Met expression or c-Met activity level can be used to ameliorate the cell proliferative disorder or the disorder associated with c-Met, comprising administering to the subject (subject) a therapeutically effective amount of a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutically acceptable carrier. The present invention provides that compounds of formula (I) are useful for modulating cell proliferative disorders or disorders associated with c-Met, such that modulation of c-Met expression or levels of c-Met activity may be useful for ameliorating cell proliferative disorders or disorders associated with c-Met. The present invention provides the use of a compound of formula (I) in the manufacture of a medicament for modulating a cell proliferative disorder or a disorder associated with c-Met such that modulation of c-Met expression or c-Met activity level may be used to ameliorate a cell proliferative disorder or a disorder associated with c-Met.

The term "prophylactically effective amount" refers to an amount of an active compound or pharmaceutical agent that inhibits or delays the onset of a disorder in a subject (subject) that is sought by a researcher, veterinarian, medical doctor (doctor's doctor) or other clinician.

The term "therapeutically effective amount" as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject (subject) that is being sought by a researcher, veterinarian, medical doctor (doctor's doctor) or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.

Methods for determining therapeutically and prophylactically effective doses of the pharmaceutical compositions of the present invention are known in the art.

Methods for determining therapeutically and prophylactically effective amounts of the compounds of the present invention are known in the art.

As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

As used herein, the term "c-Met related disorder" or "c-Met receptor tyrosine kinase related disorder" shall include diseases associated with or implicated in c-Met activity, e.g., excessive activity of c-Met, and conditions associated therewith. The term "overactivity of c-Met" refers to the expression of c-Met in 1) cells that do not normally express c-Met; 2) c-Met activity of cells not normally having active c-Met; 3) increased c-Met expression, which leads to undesired cell proliferation; or 4) a mutation leading to constitutive activation of c-Met. Examples of "disorders related to c-Met" include disorders due to an abnormally high amount of c-Met or overstimulation of c-Met by mutations in c-Met, or disorders due to an abnormally high amount of c-Met or an abnormally high amount of activity of c-Met by mutations in c-Met.

It is known that excessive activity of c-Met has been implicated in the pathogenesis of a number of diseases such as cell proliferative disorders, neoplastic diseases and cancer.

The term "cell proliferative disorder" refers to unwanted cell proliferation of one or more subpopulations of cells in a multicellular organism that results in harm (i.e., discomfort or reduced life expectancy) to the multicellular organism. Cell proliferative disorders can occur in different types of animals and humans. Cell proliferative disorders include neoplastic diseases (as used herein, "neoplastic disease" refers to a tumor caused by abnormal or uncontrolled cell growth) and other cell proliferative disorders.

Examples of cell proliferative disorders associated with c-Met include tumors and cancers- -such as hereditary and sporadic human papillary renal cancers, breast cancer, colorectal cancer, gastric cancer, glioma, ovarian cancer, hepatocellular cancer, head and neck squamous cell carcinoma, testicular cancer, basal cell carcinoma, liver cancer, sarcoma, malignant pleural mesothelioma, melanoma, multiple myeloma, osteosarcoma, pancreatic cancer, prostate cancer, synovial sarcoma, thyroid cancer, non-small cell lung cancer (NSCLC) and small cell lung cancer, transitional cell carcinoma of the bladder, testicular cancer, basal cell carcinoma, liver cancer- -including leukemias, lymphomas and myelomas- -such as Acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML), Acute Promyelocytic Leukemia (APL), Chronic Lymphocytic Leukemia (CLL), Chronic Myeloid Leukemia (CML), chronic Neutrophilic Leukemia (CNL), Acute Undifferentiated Leukemia (AUL), Anaplastic Large Cell Lymphoma (ALCL), prolymphocytic leukemia (PML), juvenile myelomonocytic leukemia (JMML), adult T-cell ALL, AML with trilineage myelodysplasia (AML/TMDS), mixed lineage (mixlinkage) leukemia (MLL), myelodysplastic syndromes (MDSs), myeloproliferative disorders (MPD), multiple myeloma, (MM), myeloid sarcoma, non-hodgkin lymphoma and hodgkin's disease (also known as hodgkin lymphoma) and diseases associated with the formation of new blood vessels, such as rheumatoid, arthritis, and retinopathy.

Other cell proliferative disorders in which excessive activity of c-Met is implicated in its pathogenesis include cancers in which c-Met activity contributes to the invasive/metastatic phenotype, including cancers in which c-Met is not overexpressed or otherwise altered.

In another embodiment of this aspect, the invention encompasses a combination therapy (combination therapy) for treating or inhibiting the onset of a cell proliferative disorder or a disorder associated with c-Met in a subject (subject). Combination therapy (combination therapy) includes administering to a subject (subject) a therapeutically or prophylactically effective amount of a compound of formula (I) and one or more other anti-cell proliferation therapies, including chemotherapy, radiation therapy, gene therapy, and immunotherapy.

In one embodiment of the invention, the compounds of the invention may be administered in combination with chemotherapy. As used herein, chemotherapy refers to treatments involving chemotherapeutic agents. The present invention therefore relates to the combination of a compound of formula (I) with other chemotherapeutic agents. Various chemotherapeutic agents may be used in the combination therapy methods disclosed herein. Exemplary chemotherapeutic agents contemplated include, but are not limited to: platinum compounds (platinum-containing anti-cancer drugs) (e.g., cisplatin, carboplatin, oxaliplatin); taxane compounds (e.g., paclitaxel, docetaxel); camptothecin compounds (camptothecins compound) (irinotecan, topotecan); vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine); anti-tumor nucleoside derivatives (e.g., 5-fluorouracil, leucovorin, gemcitabine, capecitabine); alkylating agents (e.g., cyclophosphamide, carmustine, lomustine, thiotepa); epipodophyllotoxin/podophyllotoxin (e.g., etoposide, teniposide); aromatase inhibitors (e.g. anastrozole, letrozole, exemestane); anti-estrogenic compounds (e.g., tamoxifen, fulvestrant), antifolates (e.g., pemetrexed disodium); hypomethylating agents (e.g., azacitidine); biological agents (e.g., gemtuzumab ozogamicin, cetuximab, rituximab, pertuzumab, trastuzumab, bevacizumab, erlotinib); antibiotics/anthracyclines (e.g., idarubicin, actinomycin D, bleomycin, daunorubicin, doxorubicin, mitomycin C, dactinomycin, carminomycin); antimetabolites (e.g., clofarabine, aminopterin, cytosine arabinoside, methotrexate); tubulin-binding agents (e.g., combretastatin, colchicine, nocodazole); topoisomerase inhibitors (e.g., camptothecin); differentiating agents (e.g., retinoids, vitamin D, and retinoic acid); a Retinoic Acid Metabolism Blocker (RAMBA) (e.g., isotretinoin (isocutane)); kinase inhibitors (e.g., flavoperidol (flavoperidol), imatinib mesylate, gefitinib); farnesyl transferase inhibitors (e.g., tipifarnib); (ii) a histone deacetylase inhibitor; inhibitors of the ubiquitin-proteasome pathway (e.g., bortezomib, Yondelis); FGFR (fibroblast growth factor receptor) inhibitors.

In one embodiment, the chemotherapeutic agents that may be used in particular in the combinations (combinations) described herein are platinum compounds (platinum-containing anti-cancer drugs) (e.g. cisplatin, carboplatin, oxaliplatin), especially in view of the OCT2 inhibitory activity of the compound (I). Such a combination may reduce the side effects of the platinum compound and thus may provide a longer treatment time than the platinum compound. The present invention therefore relates to the combination of a compound of formula (I) and a platinum containing anticancer drug such as cisplatin, carboplatin, oxaliplatin. In one aspect, the invention relates to a product containing as a first active ingredient a platinum-containing anti-cancer drug (e.g. cisplatin, carboplatin, oxaliplatin) and as a second active ingredient a compound of formula (I) as a combined preparation for simultaneous, separate or sequential use in the treatment of a patient suffering from cancer.

In the combinations of the invention, the platinum-containing anti-cancer drugs, e.g. cisplatin, carboplatin, oxaliplatin and the compound of formula (I) may be formulated in separate pharmaceutical dosage forms which may be sold separately from each other but with instructions or instructions for their use in combination. The instructions or instructions may be in the form of a patient leaflet or the like, or in any form of communication, such as written or oral form.

In one embodiment, the chemotherapeutic agent which may be used in particular in the combinations described herein is an FGFR inhibitor. These combinations may be of particular interest, as cMet inhibitors of formula (I) may be used to prevent resistance, delay resistance, prevent the development of resistance or delay the development of resistance of a tumor or cancer to FGFR inhibitors, in particular FGFR inhibitors as described herein.

In one aspect, the invention relates to a product containing an FGFR inhibitor as first active ingredient and a compound of formula (I) as second active ingredient, as a combined preparation for simultaneous, separate or sequential use in the treatment of a patient suffering from cancer.

The FGFR inhibitor and the compound of formula (I) can be administered simultaneously (e.g. in separate or single compositions (components)) or sequentially in any order. In the latter case, the two compounds will be administered in a time and in an amount and manner sufficient to ensure that a beneficial or synergistic effect is achieved. It will be appreciated that the preferred method and order of administration of each component of the combination, as well as the respective dosages and schedules, will depend on the particular other drug(s) (agent (s)) and compound(s) of the COMBINATION OF THE INVENTION being administered, its route of administration, the particular tumour being treated and the particular host being treated. The optimal method and sequence of administration, as well as the dosages and formulations, can be readily determined by those skilled in the art using routine methods and in view of the information described herein.

The weight ratio of the combined compounds can be determined by one skilled in the art. The ratio and the exact dose and frequency of administration will depend on the particular compound combined, the particular condition being treated, the severity of the condition being treated, the age, weight, sex, diet, time and general physical condition of the particular patient, the mode of administration and other drugs the individual may take, as is well known to those skilled in the art. Furthermore, it is clear that the effective daily amount may be reduced or increased depending on the response of the treated subject (subject) and/or depending on the evaluation of the physician prescribing the combination of the instant invention. The weight ratio of FGFR inhibitor and compound of formula (I) may be from 1/10 to 10/1, more particularly from 1/5 to 5/1, even more particularly from 1/3 to 3/1.

In one embodiment, the FGFR inhibitor and the compound of formula (I) of the COMBINATION OF THE INVENTION are administered sequentially, in any order, in separate administration schedules. In this case, the two compounds will be administered in a time and in an amount and manner sufficient to ensure that a beneficial or synergistic effect is achieved.

In the combinations of the invention, the FGFR inhibitor and the compound of formula (I) can be formulated as separate pharmaceutical dosage forms, which can be sold independently of one another, but with instructions or instructions (manual) for using them in combination. The instructions or instructions may be in the form of a patient leaflet or the like, or in any form of communication, such as written or oral form.

In the combinations of the invention, the FGFR inhibitor and the compound of formula (I) can be administered by the same route of administration or via different routes of administration.

In one embodiment, the FGFR inhibitor and the compound of formula (I) of the COMBINATION OF THE INVENTION are administered by the same route of administration, in particular by the oral route.

The invention also relates to a pharmaceutical product or a commercial package comprising a combination according to the invention, in particular together with instructions (instructions) for simultaneous, separate or sequential use thereof in the treatment of a FGFR tyrosine kinase activity mediated disease, in particular cancer.

In one embodiment, in the combination of the invention, the FGFR inhibitor and the compound of formula (I) are administered simultaneously.

In case of the combination of the invention comprising compound X or a pharmaceutically acceptable salt or solvate thereof as FGFR inhibitor, it is advantageous to administer said compound less frequently than the compound of formula (I) because compound X shows a lysogenic property and a long target shutdown (target shutdown).

The FGFR inhibitor and the compound of formula (I) of the COMBINATION OF THE INVENTION may also be co-formulated in a single formulation.

In one embodiment, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an FGFR inhibitor as first active ingredient, in particular selected from N- (3, 5-dimethoxyphenyl) -N' - (1-methylethyl) -N- [3- (1-methyl-1H-pyrazol-4-yl) quinoxalin-6-yl ] ethane-1, 2-diamine or a pharmaceutically acceptable salt thereof or a solvate thereof, and N- (2-fluoro-3, 5-dimethoxyphenyl) -N- (1H-imidazol-2-ylmethyl) -3- (1-methyl-1H-pyrazol-4-yl) pyrido [2,3-b ] pyrazin-6-amine, or a pharmaceutically acceptable salt thereof, or a solvate thereof; and comprises as a second active ingredient a compound of formula (I).

Examples of FGFR inhibitors

N- (3, 5-Dimethoxyphenyl) -N' - (1-methylethyl) -N- [3- (1-methyl-1H-pyrazol-4-yl) quinoxalin-6-yl ] ethane-1, 2-diamine (Compound X) is represented by the following formula

N- (2-fluoro-3, 5-dimethoxyphenyl) -N- (1H-imidazol-2-ylmethyl) -3- (1-methyl-1H-pyrazol-4-yl) pyrido [2,3-b ] pyrazin-6-amine (Compound Y) is represented by the following formula

The compounds N- (3, 5-dimethoxyphenyl) -N' - (1-methylethyl) -N- [3- (1-methyl-1H-pyrazol-4-yl) quinoxalin-6-yl ] ethane-1, 2-diamine (compound X) or a pharmaceutically acceptable salt or solvate thereof, and N- (2-fluoro-3, 5-dimethoxyphenyl) -N- (1H-imidazol-2-ylmethyl) -3- (1-methyl-1H-pyrazol-4-yl) pyrido [2,3-b ] pyrazin-6-amine (compound Y) or a pharmaceutically acceptable salt or solvate thereof, and their chemical syntheses are described in WO2011/135376 and WO2013/061080, which is incorporated herein by reference. They are described as inhibitors or modulators of the activity of certain protein tyrosine kinases, particularly FGFR, and the compounds are therefore useful in the treatment or prophylaxis, particularly treatment, of disease states or conditions mediated by these tyrosine kinases, particularly FGFR. The compounds are useful for the treatment or prevention of cancer, in particular for the treatment of cancer.

In WO2011/135376, compound X is also cited as the hydrochloride salt. In WO2013/061080, the present compounds Y are also listed as sulfates, hydrochlorides, phosphates, lactates, fumarates.

FGFR kinase inhibitor compounds X and Y described herein have differential selectivity profiles, which provide new opportunities for using these targeted agents in a subset of patients with diseases driven by FGFR deregulation. The FGFR kinase inhibitor compounds X and Y described herein exhibit reduced inhibition of additional kinases, particularly VEGFR, more particularly VEGFR2 and PDGFR, particularly PDGFR- β, and provide the opportunity to have differentiated side effects or toxicity profiles, thus allowing more effective treatment of these indications. Inhibitors of VEGFR2 and PDGFR-beta have been associated with toxicity, respectively, such as hypertension or edema. In the case of VEGFR2 inhibitors, this hypertensive effect is often dose-limiting and may be contraindicated in certain patient populations, requiring clinical management. FGFR kinase inhibitor compounds X and Y described herein are FGFR1, 2,3, and 4 inhibitors.

Vascular Endothelial Growth Factor (VEGFR)

Vascular Endothelial Growth Factor (VEGF), a polypeptide, is mitogenic for endothelial cells in vivo and stimulates angiogenic responses in vivo. VEGF is also associated with inappropriate angiogenesis. VEGFR is a Protein Tyrosine Kinase (PTKs). PTKs catalyze the phosphorylation of specific tyrosine residues in proteins involved in cellular function, thereby regulating cell growth, survival and differentiation.

Three PTK receptors have been identified for VEGF: VEGFR-1 (Flt-1); VEGFR-2(Flk-1 or KDR) and VEGFR-3 (Flt-4). These receptors are involved in angiogenesis and in signal transduction. Of particular interest is VEGFR-2, which is a transmembrane receptor PTK expressed primarily in endothelial cells. Activation of VEGFR-2 by VEGF is a key step in the signal transduction pathway that initiates tumor angiogenesis. VEGF expression may be constitutive to tumor cells and may also be upregulated in response to certain stimuli. One such stimulus is hypoxia, where VEGF expression is upregulated in tumors and associated host tissues. The VEGF ligand activates VEGFR-2 by binding to its extracellular VEGF binding site. This results in receptor dimerization of VEGFR and autophosphorylation of tyrosine residues at the intracellular kinase domain of VEGFR-2. The kinase domain serves to transfer phosphate from ATP to tyrosine residues, thereby providing a binding site for signaling proteins downstream of VEGFR-2, ultimately initiating angiogenesis.

PDGFR

Malignant tumors are the product of uncontrolled cellular proliferation. Cell growth is controlled by a delicate balance between growth promoting and growth inhibiting factors. In normal tissues, the production and activity of these factors results in the growth of differentiated cells in a controlled and regulated manner that maintain the normal integrity and function of the organ. Malignant cells evade this control; the natural balance is disturbed (by various mechanisms) and irregular abnormal cell growth occurs. A growth factor important in tumor development is Platelet Derived Growth Factor (PDGF), which comprises a family of peptide growth factors that signal through cell surface tyrosine kinase receptors (PDGFR) and stimulate various cellular functions including growth, proliferation and differentiation.

BGJ398(3- (2, 6-dichloro-3, 5-dimethoxyphenyl) -1- [6- [4- (4-ethylpiperazin-1-yl) anilino ] pyrimidin-4-yl ] -1-methylurea) has the following formula

AZD-4547(N- (5- (3, 5-dimethoxyphenethyl) -1H-pyrazolyl-3-yl) -4- ((3S,5R) -3, 5-dimethylpiperazin-1-yl) benzamide) has the following formula

PD 173074(N- [2- [ [4- (diethylamino) butyl ] amino ] -6- (3, 5-dimethoxyphenyl) pyrido [2,3-d ] pyrimidin-7-yl ] -N' - (1, 1-dimethylethyl) urea) has the formula

(R, E) -2- (4- (2- (5- (1- (3, 5-dichloropyridin-4-yl) ethoxy) -1H-indazol-3-yl) vinyl) -1H-pyrazolyl-1-yl) ethanol) LY-2874455 having the formula

Brianib (Brivanib) (alanine ester) (S) - (R) -1- ((4- ((4-fluoro-2-methyl-1H-indol-5-yl) oxy) -5-methylpyrrolo [2,1-f ] [1,2,4] triazin-6-yl) oxy) propan-2-yl 2-aminopropionate.

City) Indanib (Intedanib)

One degree) victinib (dovidinib)

Cediranib (Cediranib)

Masitinib (Masitinib)

Olanetib (Oratinib)

Punatinib (Ponatinib) (AP 245634)

E-7080 (lenvatinib)

*)E-3810(lucitanib)

*)BAY1163877，TAS-120，ARQ087，ASP5878，FF284，

Antibodies or related compounds, e.g., HGS 1036/FP-1039; MFGR 1877S; AV-370; GP369/AV-396 b; HuGAL-FR 21; monoclonal antibody (BAY1179470, RG-7444)

Other useful agents for the combinations described herein include verapamil, calcium antagonists, which have been found to be useful in combination with antineoplastic agents for establishing chemosensitivity in tumor cells resistant to the chemotherapeutic agent received, and enhancing the efficacy of these compounds in drug-sensitive malignancies. See Simpson WG, The calcium channel blocker verapamil and cancer chemotherapeutics, cell calcium, 1985 Dec; 6(6):449-67. In addition, no chemotherapeutic agents have emerged that are believed to be useful in combination with the compounds of the present invention.

In another embodiment of the invention, the compounds of the invention may be administered in combination with radiation therapy. As used herein, "radiation therapy" refers to a treatment that includes exposing a subject (object) in need thereof to radiation. Such treatments are known to those skilled in the art. A suitable protocol for radiation therapy would be similar to radiation therapy already used in clinical therapy, where radiation therapy is used alone or in combination with other chemotherapeutic agents.

In another embodiment of the invention, the compounds of the invention may be administered in combination with gene therapy. As used herein, "gene therapy" refers to therapy that targets specific genes involved in tumor development. Possible gene therapy strategies include the restoration of defective cancer suppressor genes, cell transduction or transfection with antisense DNA corresponding to genes encoding growth factors and their receptors, RNA-based strategies such as ribozymes, RNA decoys, antisense messenger RNA and small interfering RNA (sirna) molecules and so-called "suicide genes".

In other embodiments of the invention, the compounds of the invention may be administered in combination with immunotherapy. As used herein, "immunotherapy" refers to a treatment that targets a particular protein involved in tumor development by an antibody specific for such protein. For example, monoclonal antibodies against vascular endothelial growth factor have been used to treat cancer.

When a second drug is used in addition to the compound of the present invention, the two drugs may be administered as follows: simultaneously (e.g., in separate or single compositions), sequentially in any order, at about the same time, or using separate administration regimens. In the latter case, the two compounds will be administered in a time and in an amount and manner sufficient to ensure that a beneficial or synergistic effect is achieved. It will be understood that the preferred method and order of administration, as well as the respective dosages and schedules of each of the components of the combination will depend on the particular chemotherapeutic agent being administered in combination with the compounds of the invention, their route of administration, the particular tumor being treated and the particular host being treated.

As will be understood by those of ordinary skill in the art, appropriate dosages of chemotherapeutic agents are generally comparable to or smaller than those already used in clinical therapy, where the chemotherapeutic agents are administered alone or in combination with other chemotherapeutic agents.

The optimal method and order of administration as well as the dosages and schedules can be readily determined by those skilled in the art using routine methods and in view of the information described herein.

By way of example only, the platinum compound is advantageously present at 1 to 500mg per square meter (mg/m)²) E.g. 50 to 400mg/m²Is administered, particularly for cisplatin, at a dose of about 75mg/m²And about 300mg/m for a carboplatin dose²Each course of treatment. Cisplatin is not absorbed orally and must therefore be delivered by intravenous, subcutaneous, intratumoral or intraperitoneal injection.

Merely by way of example, the taxane compound is advantageously present at 50 to 400mg per square meter (mg/m)²) (e.g., 75 to 250 mg/m)²) Is administered, particularly at a paclitaxel dose of about 175 to 250mg/m²And for docetaxel areEach course of treatment.

By way of example only, camptothecin compounds are advantageously present at 0.1 to 400mg per square meter (mg/m)²) E.g. 1 to 300mg/m²Is administered, in particular for irinotecan at a dose of about 100 to 350mg/m²And about 1 to 2mg/m for topotecan dose²Each course of treatment.

By way of example only, vinca alkaloids may advantageously be present at 2 to 30mg per square meter (mg/m)²) Is administered, in particular for vinblastine in a dose of about 3 to 12mg/m²For vincristine the dosage is about 1-2mg/m²And for vinorelbine dosage of about 10-30mg/m²Each course of treatment.

By way of example only, the anti-tumor nucleoside derivative may advantageously be present at 200 to 2500mg per square meter (mg/m)²) Volumetric surface area, e.g. 700 to 1500mg/m²The dosage of (a). 5-Fluorouracil (5FU) is generally administered intravenously in a dose ranging from 200 to 500mg/m²(preferably 3 to 15 mg/kg/day). Gemcitabine is advantageously present at about 800 to 1200mg/m²And capecitabine is advantageously administered at about 1000-²And (4) application.

Merely by way of example, the alkylating agent may advantageously be present at 100 to 500mg per square meter (mg/m)²) Of body surface area, e.g. 120 to 200mg/m²Is administered, in particular, at a cyclophosphamide dose of about 100 to 500mg/m²About 0.1 to 0.2mg/kg body weight for chlorambucil and about 150 to 200mg/m for carmustine²And about 100 to 150mg/m for lomustine²Each course of treatment.

Merely by way of example, the podophyllotoxin derivative may advantageously be present at 30 to 300 mg/square meter (mg/m)²) Body surface area (e.g., 50 to 250 mg/m)²) Is administered, in particular, at a dose of about 35 to 100mg/m for etoposide²And for teniposide doses from about 50 to 250mg/m²Each course of treatment.

Merely by way of example, the anthracycline derivative may advantageously be present at 10 to 75mg per square meter (mg/m)²) Body surface area, e.g. 15 to 60mg/m²Is about 40 to 75mg/m, in particular for doxorubicin doses²For daunorubicin, the dosage is about 25 to 45mg/m²And for idarubicin dosage from about 10 to 15mg/m²Each course of treatment.

By way of example only, anti-estrogen compounds may advantageously be administered at a dosage of about 1 to 100mg per day, depending on the particular agent and condition to be treated. Tamoxifen is advantageously administered orally at a dose of 5 to 50mg, preferably 10 to 20mg, twice daily, continuing the treatment for a sufficient time to reach and maintain the therapeutic effect. Toremifene is advantageously administered orally once a day at a dose of about 60mg, and treatment is continued for a sufficient time to achieve and maintain the therapeutic effect. Anastrozole is advantageously administered orally once a day at a dose of about 1 mg. Droloxifene is advantageously administered orally once daily at a dose of about 20-100 mg. Raloxifene is advantageously administered orally once a day in a dose of about 60 mg. Exemestane is advantageously administered orally once a day in a dose of about 25 mg.

By way of example only, the biological agent may advantageously be present at about 1 to 5mg per square meter (mg/m)²) The dosage of the body surface area is administered, or as known in the art, if different. For example, trastuzumab advantageously is present at 1 to 5mg/m²In particular in an amount of 2 to 4mg/m²The dose per course of treatment is administered.

The dose may be administered, for example, once, twice or more per course of treatment, which may be repeated, for example, every 7, 14, 21 or 28 days.

The compounds of the invention may be administered to a subject (subject) systemically, e.g., intravenously, orally, subcutaneously, intramuscularly, intradermally, or parenterally. The compounds of the invention may also be administered topically to a subject (subject). Non-limiting examples of local delivery systems include the use of intraluminal medical devices including intravascular drug delivery catheters, guidewires (wires), pharmacological stents and endoluminal paving. In particular, the compounds of the invention are administered orally.

The compounds of the present invention may further be administered to a subject (subject) in combination with a targeting agent to achieve high local concentrations of the compounds at the target site. In addition, the compounds of the present invention may be formulated for rapid or slow release with the aim of contacting the drug or agent with the target tissue for a period ranging from hours to weeks.

The invention also provides pharmaceutical compositions comprising a compound of formula (I) in combination with a pharmaceutically acceptable carrier. The pharmaceutical composition may contain from about 0.1mg to 1000mg, preferably from about 100 to 500mg of the compound and may be constituted in any form suitable for the mode of administration chosen.

The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when properly administered to an animal or human. Veterinary uses are also included in the present invention, and "pharmaceutically acceptable" compositions include compositions for clinical and/or veterinary use.

Carriers include necessary and inert pharmaceutical excipients, including, but not limited to, binders, suspending agents, lubricants, flavoring agents, sweetening agents, preservatives, dyes and coatings. Compositions suitable for oral administration include solid forms such as pills, tablets, caplets, capsules (each including immediate release, timed release and sustained release formulations), granules and powders, and liquid forms such as solutions, syrups, elixirs, emulsions, and suspensions. Forms for parenteral administration include sterile solutions, emulsions and suspensions.

The pharmaceutical compositions of the invention also include pharmaceutical compositions for the sustained release of the compounds of the invention. The compositions include a sustained release carrier (typically a polymeric carrier) and a compound of the invention.

Sustained release biodegradable carriers are well known in the art. These are materials that can be formed into particles that capture the active compound therein and slowly degrade/dissolve in a suitable environment (e.g., aqueous, acidic, basic, etc.) to degrade/dissolve in body fluids and release the active compound. The particles are preferably nanoparticles (i.e., ranging from about 1 to 500nm in diameter, preferably about 50-200nm in diameter, and most preferably about 100nm in diameter).

The invention also provides a method for preparing the pharmaceutical composition of the invention. The compound of formula (I) as active ingredient is intimately admixed with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques, which carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. oral or parenteral such as intramuscular. In preparing the oral dosage form compositions, any of the usual pharmaceutical media may be employed. Thus, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like; for solid oral preparations such as powders, capsules, caplets, soft capsules and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, the tablets may be (sugar) coated or enteric coated by standard techniques. For parenteral administration, the carrier will typically comprise sterile water, although other ingredients may be included, for example, for solubility assistance or for preservation purposes. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. In formulations for slow release, for example, a slow release carrier (typically a polymeric carrier) and a compound of the invention are first dissolved or dispersed in an organic solvent. The resulting organic solution is then added to an aqueous solution to obtain an oil-in-water emulsion. Preferably, the aqueous solution includes a surfactant. Subsequently, the organic solvent is evaporated from the oil-in-water emulsion to give a colloidal suspension of particles containing the slow-release carrier and the compound of the invention.

The pharmaceutical compositions herein will contain per dosage unit (e.g., tablet, capsule, powder, injection, teaspoonful, etc.) an amount of the active ingredient required to deliver an effective dose as described above. The pharmaceutical compositions herein will contain from about 0.01mg to 200mg per kg body weight per day per unit dosage unit (e.g., tablet, capsule, powder, injection, suppository, teaspoonful, and the like). Preferably, the range is from about 0.03 to about 100mg/kg body weight/day, most preferably from about 0.05 to about 10mg/kg body weight/day. The compounds may be administered from 1 to 5 times per day. However, the dosage may vary depending on the requirements of the patient, the severity of the condition being treated and the compound being used. Daily administration or post periodic (post periodic) administration may be used.

Preferably, these compositions are in unit dosage forms, such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampoules, autoinjector devices or suppositories; for oral parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. Alternatively, the composition may be in a form suitable for once weekly or once monthly administration; for example, insoluble salts of the active compound such as the decanoate salt may be suitable to provide a depot formulation for intramuscular injection. To prepare solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier (e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water) to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention or a pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition can be readily divided into equivalent dosage forms such as tablets, pills and capsules. The solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500mg of the active ingredient of the invention. The tablets or pills of the novel composition may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, a tablet or pill may comprise an inner dose and an outer dose component, the latter being in the form of a capsule on the former. The two components may be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials may be used for such enteric layers or coatings, including a number of polymeric acids with materials such as shellac (shellac), acetyl alcohol and cellulose acetate.

Liquid forms in which the compounds of formula (I) may be incorporated for oral or injectable administration include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions (suspensions) and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate (alginate), dextran, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or gelatin. Liquid forms in suitable flavored suspending or dispersing agents may also include synthetic and natural gums such as tragacanth, acacia, methylcellulose and the like. For parenteral administration, sterile suspensions and solutions are required. When intravenous administration is desired, isotonic formulations, which usually contain suitable preservatives, are used.

Advantageously, the compound of formula (I) may be administered in a single daily dose, or the total daily dose may be administered in divided doses of two, three or four times daily. In addition, the compounds of the present invention may be administered in intranasal form via topical use of suitable intranasal vehicles or via transdermal patches well known to those of ordinary skill in the art. For administration in the form of a transdermal delivery system, of course, the dosage administration is continuous rather than intermittent throughout the administration regimen.

For example, for oral administration in the form of a tablet or capsule, the active pharmaceutical ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Further, when desired or required, a suitable binder; lubricants, disintegrants and colorants may also be incorporated into the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like.

The daily dose of the compounds of the invention may vary within a wide range of 1 to 5000mg per adult human per day. For oral administration, the compositions are preferably provided in the form of tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150, 200, 250 and 500 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. The effective amount of the drug is typically provided at a dosage level of about 0.01mg/kg to about 200mg/kg of body weight per day. In particular, the range is from about 0.03 to about 100mg/kg or from about 0.03 to about 15mg/kg body weight/day, more particularly from about 0.05 to about 10mg/kg body weight/day. The compounds of the invention may be administered up to four or more times per day, preferably from 1 to 2 times per day.

The optimal dosage to be administered can be readily determined by one skilled in the art and will vary with the particular compound used, the mode of administration, the strength of the formulation, the mode of administration and the advancement of the disease condition. In addition, factors associated with the particular patient being treated, including patient age, weight, diet and time of administration, will result in the need to adjust the dosage.

The compounds of the invention may also be administered in the form of liposome delivery systems such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of lipids including, but not limited to, amphiphilic lipids such as phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphorylcholine, cardiolipin, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, diacyltrimethylammonium propane, diacyldimethylammonium propane and stearamide, neutral lipids such as triglycerides, and combinations thereof. They may or may not contain cholesterol.

The compounds of the present invention may also be administered topically. Any delivery device may be used, such as intravascular drug delivery catheters, guide wires (wires), pharmacological stents and intraluminal membranes. The delivery system for such devices may include a local infusion catheter that delivers the compound at a rate controlled by the administrator.

The present invention provides a drug delivery device comprising an intraluminal medical device, preferably a stent, and a therapeutic dose of a chemical substance of the invention.

The term "stent" refers to any device capable of being delivered by a catheter. Stents are routinely used to prevent vascular occlusion due to physical abnormalities such as undesirable ingrowth of vascular tissue from surgical trauma, etc. It typically has a tubular, expanded lattice type structure adapted to reside within the lumen of a conduit to alleviate obstruction. The stent has a lumen wall contacting surface and a lumen exposed surface. The lumen wall-contacting surface is the outer surface of the tube and the lumen-exposed surface is the inner surface of the tube. The scaffold may be polymeric, metallic or both polymeric and metallic, and it may optionally be biodegradable.

Typically, stents are inserted into a lumen in a non-expanded form and then expanded in situ, either autonomously or with the aid of a second device. Typical expansion methods occur through the use of catheter-mounted angioplasty balloons, which are inflated within a stenotic blood vessel or body passageway in order to shear and disrupt obstructions associated with wall members of the blood vessel and to obtain an enlarged lumen. Self-expanding stents as described in U.S 6,776,796(Falotico et al) may also be used. The combination of a stent with a drug, agent or compound that prevents inflammation and proliferation may provide the most effective treatment for post-vascular restenosis.

The compound of formula (I) may be incorporated into or immobilized on the stent in a variety of ways and using any number of biocompatible materials. In one exemplary embodiment, the compound is incorporated directly into a polymer matrix, such as the polymer polypyrrole, and subsequently coated onto the outer surface of the stent. The compound is eluted from the matrix by polymer diffusion. Stents and methods of coating a stent with a drug are discussed in detail in the art. In another exemplary embodiment, a stent is first coated with a substrate comprising a solution of a compound, ethyl ene-co-ethyl acetate, and polybutyl methacrylate. The stent is then further coated with an outer layer comprising only polybutylmethacrylate. The outer layer acts as a diffusion barrier to prevent the compound from eluting too quickly and into the surrounding tissue. The thickness of the outer or facing layer determines the rate at which the chemical elutes from the matrix. WIPO publication No. WO9632907, U.S. publication No. 2002/0016625 and references disclosed therein discuss the method of stent and coating in detail.

The solution of the compound of the invention and the biocompatible material/polymer can be incorporated into or onto the stent in a variety of ways. For example, the solution may be sprayed onto the stent, or the stent may be immersed in the solution. In a preferred embodiment, the solution is sprayed onto the stent and then allowed to dry. In another exemplary embodiment, the solution may be charged to one polarity and the stent electrically changed to the opposite polarity. In this way, the solution and the scaffold will attract each other. When using this type of spray process, waste can be reduced and more control over the coating thickness can be achieved. The compound is preferably immobilized on the outer surface of the scaffold in contact with only one tissue. However, for some compounds, the entire scaffold may be coated. The combination of the dose of compound applied to the stent and the polymeric coating that controls the release of the drug is important in the effectiveness of the drug. The compound is preferably retained on the scaffold for at least three days, up to about six months or more, preferably seven to thirty days.

Any number of non-erodible biocompatible polymers may be used in combination with the compounds of the present invention. It is important to note that different polymers may be used for different scaffolds. For example, the above-described ethyl alkenyl-co-vinyl acetate and polybutyl methacrylate matrices work well with stainless steel stents. Other polymers may be more effective for stents formed from other materials, including materials that exhibit superelasticity, such as alloys of nickel and titanium.

Restenosis after coronary angioplasty results in significant morbidity and mortality. Restenosis occurs through a combination of four processes, including elastic recoil (elastic recoil), thrombosis, intimal hyperplasia, and extracellular matrix remodeling. Several growth factors have recently been identified that play an important role in these processes, leading to restenosis. See Schile TM et al, 2004, "Vascular relaxation-simulation for therapy," Expert Opin Pharmacother.5(11): 2221-32. Vascular Smooth Muscle Cells (VSMC) express the c-Met receptor. Exposure to ligands for hepatocyte growth factor, c-Met, stimulates these cells to exhibit a migratory phenotype. See Taher et al, Hepatocyte growth factor generators signaling catalysts mediating vascular cell migration biochem Biophys Res Commun (2002)298(1): 80-6; morishita R, Aoki M, Yo Y, Ogihara T.hepatocyte growth factor as a cardiovascular hormone, role of HGF in the pathogenesis of cardiovascular disease. Endocer J. (2002) Jun; 49(3):273-84. Since the migration of VSMC from the culture medium to the intima of arteries plays a role in the development of atherosclerosis and restenosis, antagonists of c-Met kinase activity are considered to represent a viable therapeutic strategy in the treatment of these diseases.

Accordingly, the present invention provides a method for the treatment of c-Met related diseases including restenosis, intimal hyperplasia or inflammation in the vessel wall comprising the controlled delivery of a therapeutically effective amount of a compound of the invention by release from an intraluminal medical device such as a stent. The invention also provides compounds of formula (I) for use in the treatment of diseases associated with c-Met including restenosis, intimal hyperplasia or inflammation in the vessel wall.

Methods of introducing stents into the lumen of a body organ are well known, and the compound-coated stents of the present invention are preferably introduced using a catheter. As one of ordinary skill in the art will appreciate, the method will vary slightly based on the location of the stent implantation. For coronary stent implantation, a balloon catheter carrying a stent is inserted into the coronary artery with the stent in the desired position. The balloon is inflated to expand the stent. When the stent is expanded, the stent comes into contact with the lumen wall. Once the stent is positioned, the balloon is deflated and removed. The stent is held in a position where the lumen-contacting surface carries the chemical substance in direct contact with the lumen wall surface. If desired, stent implantation may be accompanied by anticoagulation therapy.

The optimal conditions for delivering the compounds for use in the stents of the present invention may vary with the different local delivery systems used and the nature and concentration of the compounds used. Conditions that may be optimized include, for example, the concentration of the compound, the volume delivered, the rate of delivery, the depth of penetration of the vessel wall, the proximal inflation pressure, the amount and size of the perforations, and the fit of the drug delivery catheter balloon. Conditions can be optimized to inhibit smooth muscle cell proliferation at the site of injury so that significant arterial obstruction due to restenosis does not occur, as measured, for example, by the proliferative capacity of smooth muscle cells or by changes in vascular resistance or lumen diameter. The optimal conditions can be determined from data from animal model studies using conventional computational methods.

Another alternative method for administering the compounds of the present invention may be by conjugating the compound with a targeting agent that directs the conjugate to its intended site of action, i.e., vascular endothelial cells, or tumor cells. Antibodies and non-antibody targeting agents may be used. Due to the specific interaction between the targeting agent and its corresponding binding partner, the compounds of the invention can be administered at high local concentrations at or near the target site, thereby more effectively treating disorders at the target site.

Antibody targeting agents include antibodies or antigen binding fragments thereof that bind to a targetable or accessible component of tumor cells, tumor vasculature (vasculature) or tumor stroma (stroma). The "targetable or accessible component" of the tumor cells, tumor vessels or tumor stroma (stroma) is preferably a surface-expressed, surface-accessible or surface-located component. Antibody targeting agents also include antibodies or antigen binding fragments thereof that bind to intracellular components released from necrotic tumor cells. Preferably, such antibodies are monoclonal antibodies or antigen binding fragments thereof that bind to an insoluble intracellular antigen, which is present in a cell that may be induced to be permeable, or in a cellular host of substantially all tumor and normal cells, but which is absent or accessible outside of normal living cells of a mammal. In the present invention, the targetable or accessible component may be the c-Met receptor, as it is accessible and expressed on or near the target tissue.

As used herein, the term "antibody" is intended to broadly refer to any immunological binding agent, such as IgG, IgM, IgA, IgE, F (ab ')2, monovalent fragments, such as Fab', Fab, Dab, and engineered antibodies such as recombinant antibodies, humanized antibodies, bispecific antibodies, and the like. The antibody may be a polyclonal or monoclonal antibody, although monoclonal is preferred. A very broad range of antibodies are known in the art which have immunological specificity for the cell surface of virtually any solid tumor type (see the summary table of solid tumor monoclonal antibodies of Thorpe et al, U.S. patent No.5,855,866). Methods for producing and isolating antitumor antibodies are known to those skilled in the art (U.S. Pat. Nos. 5, 855, 866 to Thorpe et al and 6, 34, 2219 to Thorpe et al).

Techniques For conjugating therapeutic moieties to Antibodies are well known, see, e.g., Amon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", In Monoclonal Antibodies And Cancer Therapy, Reisfeld et al (eds.), pp.243-56(Alan R.Liss, Inc.1985); hellstrom et al, "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson et al (eds.), pp.623-53(Marcel Dekker, Inc.1987); thorpe, "Antibodies Of Cytotoxic Agents In Cancer Therapy: A Review", In Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al (eds.), pp.475-506 (1985). Similar techniques can also be used to link the compounds of the invention to non-antibody targeting agents. One skilled in the art will know of, or be able to determine, methods of forming conjugates with non-antibody targeting agents such as small molecules, oligopeptides, polysaccharides or other polyanionic compounds.

Although any linking moiety that is reasonably stable in blood may be used to link the compounds of the invention to the targeting agent, a biologically releasable bond and/or a selectively cleavable spacer or linker is preferred. "bioreleasable bonds" and "selectively cleavable spacers or linkers" still have reasonable stability in circulation, but are releasable, cleavable or hydrolysable only or preferably under certain conditions (i.e. in certain circumstances, or in contact with specific reagents). Such linkages include, for example, disulfide and trithio and acid labile linkages as described in U.S. patent nos. 5, 474, 765 and 5, 762, 918, and enzyme sensitive linkages including peptide, ester, amide, phosphodiester and glycoside linkages as described in U.S. patent nos. 5, 474, 765 and 5, 762, 918. This selective release design feature facilitates the sustained release of the compound from the conjugate at the intended target site.

The present invention provides a pharmaceutical composition comprising an effective amount of a compound of the present invention conjugated to a targeting agent and a pharmaceutically acceptable carrier.

The invention also provides a method of treating a c-Met related disorder, in particular a tumor, comprising administering to a subject (subject) a therapeutically effective amount of a compound of formula (I) conjugated to a targeting agent. The invention further provides compounds of formula (I) conjugated to targeting agents for use in the treatment of c-Met related disorders, in particular tumours. The invention also provides the use of a compound of formula (I) conjugated to a targeting agent for the manufacture of a medicament for the treatment of a c-Met related disorder, in particular a tumour.

When proteins such as antibodies or growth factors or polysaccharides are used as targeting agents, administration in the form of injectable compositions is preferred. The injectable antibody solution will be administered into a vein, artery or into spinal fluid over a period of 2 minutes to about 45 minutes, preferably 10 to 20 minutes. In certain instances, intradermal and intraluminal administration may be advantageous for tumors that are limited to areas near a particular region of the skin and/or areas of a particular body lumen. In addition, intrathecal administration may be used for tumors located in the brain.

The therapeutically effective dose of the compounds of the invention conjugated to the targeting agent depends on the individual, the type of disease, the disease state, the method of administration and other clinical variables. Effective dosages can be readily determined using data from animal models. Experimental animals bearing solid tumors are often used to optimize the appropriate therapeutic dose prior to conversion to a clinical setting. This model is known to be very reliable in predicting effective anti-cancer strategies. For example, solid tumor bearing mice are widely used in preclinical testing to determine the working range of therapeutic agents that achieve beneficial anti-tumor effects with minimal toxicity.

The HGF/MET pathway is involved in inducing more immunosuppressive tumor microenvironments directly by modulating T cell activity and indirectly by inducing enzymes responsible for T cell anergy. Thus, inhibition of the Met pathway by the compounds of formula (I) may elicit immune responses to checkpoint blockers (checkpoint blockers include blockers such as PD-1 and CTLA-4), as well as mitigate tumor-induced immune suppression and activate host immune responses.

While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptations and/or modifications as come within the scope of the following claims and their equivalents.

Claims (14)

1. A compound of formula (I)

Or a pharmaceutically acceptable salt thereof, wherein D represents deuterium, and wherein the deuterium content at the 2-position of the quinoline at the D position is at least 93%.

2. The compound of claim 1, wherein the compound is

3. The compound of claim 1 or 2, wherein the deuterium content at the 2-position of the quinoline at the D position is at least 95%.

4. The compound of claim 3, wherein the deuterium content at the 2-position of the quinoline at the D position is at least 98%.

5. Use of a compound according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of cancer.

6. Use of a compound according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of a cell proliferative disorder.

7. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as active ingredient, a therapeutically effective amount of a compound as claimed in any one of claims 1 to 4.

8. Use of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as active ingredient, a therapeutically effective amount of a compound according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of cancer.

9. Use of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as active ingredient, a therapeutically effective amount of a compound according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of a cell proliferative disorder.

10. Process for the preparation of the compounds as claimed in claim 1, characterized in that intermediates of the deuterated formula (II) are reduced in the presence of deuterium and in the presence of a suitable catalyst, a suitable solvent or solvent mixture, and a suitable base, wherein W is₁Represents chlorine, bromine or iodine,

wherein D represents deuterium;

alternatively, if desired, the compounds of formula (I) are converted into therapeutically active non-toxic acid addition salts by treatment with an acid, or conversely, the acid addition salt form is converted into the free base by treatment with a base.

11. A combination of a compound according to any one of claims 1 to 4 with another chemotherapeutic agent.

12. The combination according to claim 11 wherein said chemotherapeutic agent is a kinase inhibitor.

13. The combination according to claim 12, wherein the kinase inhibitor is an FGFR inhibitor.

14. The combination according to claim 11 wherein said chemotherapeutic agent is a platinum-containing anticancer drug.