unless indicated otherwise , all references herein to the inventive compounds include references to salts , solvates , hydrates and complexes thereof , and to solvates , hydrates and complexes of salts thereof , including polymorphs , stereoisomers , and isotopically labeled versions thereof . as used herein , unless otherwise indicated , the term “ abnormal cell growth ” refers to cell growth that is independent of normal regulatory mechanisms ( e . g ., loss of contact inhibition ). as used herein , unless otherwise indicated , the term “ treating ”, means reversing , alleviating , inhibiting the progress of , or preventing the disorder or condition to which such term applies , or one or more symptoms of such disorder or condition . the term “ treatment ”, as used herein , unless otherwise indicated , refers to the act of treating as “ treating ” is defined immediately above . as used herein the term “ pharmaceutically acceptable salts ” includes acid addition and base salts ( including disalts ). suitable acid addition salts are formed from acids which form non - toxic salts . examples include the acetate , aspartate , benzoate , besylate , bicarbonate / carbonate , bisulphate / sulfate , borate , camsylate , citrate , edisylate , esylate , formate , fumarate , gluceptate , gluconate , glucuronate , hexafluorophosphate , hibenzate , hydrochloride / chloride , hydrobromide / bromide , hydroiodide / iodide , isethionate , lactate , malate , maleate , malonate , mesylate , methylsulfate , naphthylate , 2 - napsylate , nicotinate , nitrate , orotate , oxalate , palmitate , pamoate , phosphate / hydrogen phosphate / dihydrogen phosphate , saccharate , stearate , succinate , tartrate , tosylate and trifluoroacetate salts . suitable base salts are formed from bases which form non - toxic salts . examples include the aluminum , arginine , benzathine , calcium , choline , diethylamine , diolamine , glycine , lysine , magnesium , meglumine , olamine , potassium , sodium , tromethamine and zinc salts . for a review on suitable pharmaceutically acceptable salts , see “ handbook of pharmaceutical salts properties , selection , and use ” by stahl and wermuth ( wiley - vch , weinheim , germany , 2002 ), the disclosure of which is incorporated herein by reference in its entirety . a pharmaceutically acceptable salt of the inventive compounds can be readily prepared by mixing together solutions of the compound and the desired acid or base , as appropriate . the salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent . the degree of ionization in the salt may vary from completely ionized to almost non - ionized . the compounds of the invention may exist in both unsolvated and solvated forms . the term ‘ solvate ’ is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules , for example , ethanol . the term ‘ hydrate ’ is employed when the solvent is water . pharmaceutically acceptable solvates in accordance with the invention include hydrates and solvates wherein the solvent of crystallization may be isotopically substituted , e . g . d 2 o , d 6 - acetone , d 6 - dmso . the invention also includes isotopically - labeled compounds , which are identical to compound 1 , except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature . examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen , carbon , nitrogen , oxygen , phosphorus , sulfur , fluorine and chlorine , such as 2 h , 3 h , 13 c , 14 c , 15 n , 18 o , 17 o , 31 p , 32 p , 35 s , 18 f , and 36 cl , respectively . compounds of the present invention and pharmaceutically acceptable salts of said compounds , which contain the aforementioned isotopes and / or other isotopes of other atoms , are within the scope of this invention . certain isotopically - labeled compounds of the present invention , for example those into which radioactive isotopes such as 3 h and 14 c are incorporated , are useful in drug and / or substrate tissue distribution assays . tritiated , i . e ., 3 h , and carbon - 14 , i . e ., 14 c , isotopes are particularly preferred for their ease of preparation and detectability . further , substitution with heavier isotopes such as deuterium , i . e ., 2 h , can afford certain therapeutic advantages resulting from greater metabolic stability , for example increased in vivo half - life or reduced dosage requirements and , hence , may be preferred in some circumstances . an isotopically labeled compound 1 of this invention can generally be prepared by carrying out the procedures described for the non - labeled compound , substituting a readily available isotopically labeled reagent for a non - isotopically labeled reagent . also included within the scope of the invention are complexes such as clathrates , drug - host inclusion complexes wherein , in contrast to the aforementioned solvates , the drug and host are present in stoichiometric or non - stoichiometric amounts . also included are complexes of the drug containing two or more organic and / or inorganic components which may be in stoichiometric or non - stoichiometric amounts . the resulting complexes may be ionized , partially ionized , or non - ionized . for a review of such complexes , see j pharm sci , 64 ( 8 ), 1269 - 1288 by haleblian ( august 1975 ), the disclosure of which is incorporated herein by reference in its entirety . the compounds of the invention may be administered orally . oral administration may involve swallowing , so that the compound enters the gastrointestinal tract , or buccal or sublingual administration may be employed by which the compound enters the blood stream directly from the mouth . formulations suitable for oral administration include solid formulations such as tablets , capsules containing particulates , liquids , or powders , lozenges ( including liquid - filled ), chews , multi - and nano - particulates , gels , solid solution , liposome , films ( including muco - adhesive ), ovules , sprays and liquid formulations . liquid formulations include suspensions , solutions , syrups and elixirs . such formulations may be used as fillers in soft or hard capsules and typically include a pharmaceutically acceptable carrier , for example , water , ethanol , polyethylene glycol , propylene glycol , methylcellulose , or a suitable oil , and one or more emulsifying agents and / or suspending agents . liquid formulations may also be prepared by the reconstitution of a solid , for example , from a sachet . the compounds of the invention may also be used in fast - dissolving , fast - disintegrating dosage forms such as those described in expert opinion in therapeutic patents , 11 ( 6 ), 981 - 986 by liang and chen ( 2001 ), the disclosure of which is incorporated herein by reference in its entirety . for tablet dosage forms , depending on dose , the drug may make up from 1 wt % to 80 wt % of the dosage form , more typically from 5 wt % to 60 wt % of the dosage form . in addition to the drug , tablets generally contain a disintegrant . examples of disintegrants include sodium starch glycolate , sodium carboxymethyl cellulose , calcium carboxymethyl cellulose , croscarmellose sodium , crospovidone , polyvinylpyrrolidone , methyl cellulose , microcrystalline cellulose , lower alkyl - substituted hydroxypropyl cellulose , starch , pregelatinized starch and sodium alginate . generally , the disintegrant will comprise from 1 wt % to 25 wt %, preferably from 5 wt % to 20 wt % of the dosage form . binders are generally used to impart cohesive qualities to a tablet formulation . suitable binders include microcrystalline cellulose , gelatin , sugars , polyethylene glycol , natural and synthetic gums , polyvinylpyrrolidone , pregelatinized starch , hydroxypropyl cellulose and hydroxypropyl methylcellulose . tablets may also contain diluents , such as lactose ( monohydrate , spray - dried monohydrate , anhydrous and the like ), mannitol , xylitol , dextrose , sucrose , sorbitol , microcrystalline cellulose , starch and dibasic calcium phosphate dihydrate . tablets may also optionally include surface active agents , such as sodium lauryl sulfate and polysorbate 80 , and glidants such as silicon dioxide and talc . when present , surface active agents are typically in amounts of from 0 . 2 wt % to 5 wt % of the tablet , and glidants typically from 0 . 2 wt % to 1 wt % of the tablet . tablets also generally contain lubricants such as magnesium stearate , calcium stearate , zinc stearate , sodium stearyl fumarate , and mixtures of magnesium stearate with sodium lauryl sulphate . lubricants generally are present in amounts from 0 . 25 wt % to 10 wt %, preferably from 0 . 5 wt % to 3 wt % of the tablet . other conventional ingredients include anti - oxidants , colorants , flavoring agents , preservatives and taste - masking agents . exemplary tablets contain up to about 80 wt % drug , from about 10 wt % to about 90 wt % binder , from about 0 wt % to about 85 wt % diluent , from about 2 wt % to about 10 wt % disintegrant , and from about 0 . 25 wt % to about 10 wt % lubricant . tablet blends may be compressed directly or by roller to form tablets . tablet blends or portions of blends may alternatively be wet -, dry -, or melt - granulated , melt congealed , or extruded before tableting . the final formulation may include one or more layers and may be coated or uncoated ; or encapsulated . the formulation of tablets is discussed in detail in “ pharmaceutical dosage forms : tablets , vol . 1 ”, by h . lieberman and l . lachman , marcel dekker , n . y ., n . y ., 1980 ( isbn 0 - 8247 - 6918 - x ), the disclosure of which is incorporated herein by reference in its entirety . solid formulations for oral administration may be formulated to be immediate and / or modified release . modified release formulations include delayed -, sustained -, pulsed -, controlled -, targeted and programmed release . suitable modified release formulations are described in u . s . pat . no . 6 , 106 , 864 . details of other suitable release technologies such as high energy dispersions and osmotic and coated particles can be found in verma et al , pharmaceutical technology on - line , 25 ( 2 ), 1 - 14 ( 2001 ). the use of chewing gum to achieve controlled release is described in wo 00 / 35298 . the disclosures of these references are incorporated herein by reference in their entireties . the compounds of the invention may also be administered directly into the blood stream , into muscle , or into an internal organ . suitable means for parenteral administration include intravenous , intraarterial , intraperitoneal , intrathecal , intraventricular , intraurethral , intrasternal , intracranial , intramuscular and subcutaneous . suitable devices for parenteral administration include needle ( including micro needle ) injectors , needle - free injectors and infusion techniques . parenteral formulations are typically aqueous solutions which may contain excipients such as salts , carbohydrates and buffering agents ( preferably to a ph of from 3 to 9 ), but , for some applications , they may be more suitably formulated as a sterile non - aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile , pyrogen - free water . the preparation of parenteral formulations under sterile conditions , for example , by lyophilization , may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art . the solubility of compounds of the invention used in the preparation of parenteral solutions may be increased by the use of appropriate formulation techniques , such as the incorporation of solubility - enhancing agents . formulations for parenteral administration may be formulated to be immediate and / or modified release . modified release formulations include delayed -, sustained -, pulsed -, controlled -, targeted and programmed release . thus compounds of the invention may be formulated as a solid , semi - solid , or thixotropic liquid for administration as an implanted depot providing modified release of the active compound . examples of such formulations include drug - coated stents and pgla microspheres . the compounds of the invention may also be administered topically to the skin or mucosa , that is , dermally or transdermally . typical formulations for this purpose include gels , hydrogels , lotions , solutions , creams , ointments , dusting powders , dressings , foams , films , skin patches , wafers , implants , sponges , fibers , bandages and microemulsions . liposomes may also be used . typical carriers include alcohol , water , mineral oil , liquid petrolatum , white petrolatum , glycerin , polyethylene glycol and propylene glycol . penetration enhancers may be incorporated ; see , for example , j pharm sci , 88 ( 10 ), 955 - 958 by finnin and morgan ( october 1999 ). other means of topical administration include delivery by electroporation , iontophoresis , phonophoresis , sonophoresis and micro needle or needle - free ( e . g . powderject ™, bioject ™, etc .) injection . the disclosures of these references are incorporated herein by reference in their entireties . formulations for topical administration may be formulated to be immediate and / or modified release . modified release formulations include delayed -, sustained -, pulsed -, controlled -, targeted and programmed release . the compounds of the invention can also be administered intranasally or by inhalation , typically in the form of a dry powder ( either alone , as a mixture , for example , in a dry blend with lactose , or as a mixed component particle , for example , mixed with phospholipids , such as phosphatidylcholine ) from a dry powder inhaler or as an aerosol spray from a pressurized container , pump , spray , atomizer ( preferably an atomizer using electrohydrodynamics to produce a fine mist ), or nebulizer , with or without the use of a suitable propellant , such as 1 , 1 , 1 , 2 - tetrafluoroethane or 1 , 1 , 1 , 2 , 3 , 3 , 3 - heptafluoropropane . for intranasal use , the powder may include a bioadhesive agent , for example , chitosan or cyclodextrin . the pressurized container , pump , spray , atomizer , or nebulizer contains a solution or suspension of the compound ( s ) of the invention comprising , for example , ethanol , aqueous ethanol , or a suitable alternative agent for dispersing , solubilizing , or extending release of the active , a propellant ( s ) as solvent and an optional surfactant , such as sorbitan trioleate , oleic acid , or an oligolactic acid . prior to use in a dry powder or suspension formulation , the drug product is micronized to a size suitable for delivery by inhalation ( typically less than 5 microns ). this may be achieved by any appropriate comminuting method , such as spiral jet milling , fluid bed jet milling , supercritical fluid processing to form nanoparticles , high pressure homogenization , or spray drying . capsules ( made , for example , from gelatin or hpmc ), blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the invention , a suitable powder base such as lactose or starch and a performance modifier such as l - leucine , mannitol , or magnesium stearate . the lactose may be anhydrous or in the form of the monohydrate , preferably the latter . other suitable excipients include dextran , glucose , maltose , sorbitol , xylitol , fructose , sucrose and trehalose . a suitable solution formulation for use in an atomizer using electrohydrodynamics to produce a fine mist may contain from 1 μg to 20 mg of the compound of the invention per actuation and the actuation volume may vary from 1 μl to 100 μl . a typical formulation includes a compound of the invention , propylene glycol , sterile water , ethanol and sodium chloride . alternative solvents which may be used instead of propylene glycol include glycerol and polyethylene glycol . suitable flavors , such as menthol and levomenthol , or sweeteners , such as saccharin or saccharin sodium , may be added to those formulations of the invention intended for inhaled / intranasal administration . formulations for inhaled / intranasal administration may be formulated to be immediate and / or modified release using , for example , poly ( dl - lactic - coglycolic acid ( pgla ). modified release formulations include delayed -, sustained -, pulsed -, controlled -, targeted and programmed release . in the case of dry powder inhalers and aerosols , the dosage unit is determined by means of a valve which delivers a metered amount . units in accordance with the invention are typically arranged to administer a metered dose or “ puff ” containing a desired mount of the compound of the invention . the overall daily dose may be administered in a single dose or , more usually , as divided doses throughout the day . compounds of the invention may be administered rectally or vaginally , for example , in the form of a suppository , pessary , or enema . cocoa butter is a traditional suppository base , but various alternatives may be used as appropriate . formulations for rectal / vaginal administration may be formulated to be immediate and / or modified release . modified release formulations include delayed -, sustained -, pulsed -, controlled -, targeted and programmed release . compounds of the invention may also be administered directly to the eye or ear , typically in the form of drops of a micronized suspension or solution in isotonic , ph - adjusted , sterile saline . other formulations suitable for ocular and aural administration include ointments , biodegradable ( e . g . absorbable gel sponges , collagen ) and non - biodegradable ( e . g . silicone ) implants , wafers , lenses and particulate or vesicular systems , such as niosomes or liposomes . a polymer such as crossed - linked polyacrylic acid , polyvinylalcohol , hyaluronic acid , a cellulosic polymer , for example , hydroxypropylmethylcellulose , hydroxyethylcellulose , or methyl cellulose , or a heteropolysaccharide polymer , for example , gelan gum , may be incorporated together with a preservative , such as benzalkonium chloride . such formulations may also be delivered by iontophoresis . formulations for ocular / aural administration may be formulated to be immediate and / or modified release . modified release formulations include delayed -, sustained -, pulsed -, controlled -, targeted , or programmed release . compounds of the invention may be combined with soluble macromolecular entities , such as cyclodextrin and suitable derivatives thereof or polyethylene glycol - containing polymers , in order to improve their solubility , dissolution rate , taste - masking , bioavailability and / or stability for use in any of the aforementioned modes of administration . drug - cyclodextrin complexes , for example , are found to be generally useful for most dosage forms and administration routes . both inclusion and non - inclusion complexes may be used . as an alternative to direct complexation with the drug , the cyclodextrin may be used as an auxiliary additive , i . e . as a carrier , diluent , or solubilizer . most commonly used for these purposes are alpha -, beta - and gamma - cyclodextrins , examples of which may be found in pct publication nos . wo 91 / 11172 , wo 94 / 02518 and wo 98 / 55148 , the disclosures of which are incorporated herein by reference in their entireties . the amount of the active compound administered will be dependent on the subject being treated , the severity of the disorder or condition , the rate of administration , the disposition of the compound and the discretion of the prescribing physician . however , an effective dosage is typically in the range of about 0 . 001 to about 100 mg per kg body weight per day , preferably about 0 . 01 to about 35 mg / kg / day , in single or divided doses . for a 70 kg human , this would amount to about 0 . 07 to about 7000 mg / day , preferably about 0 . 7 to about 2500 mg / day . in some instances , dosage levels below the lower limit of the aforesaid range may be more than adequate , while in other cases still larger doses may be used without causing any harmful side effect , with such larger doses typically divided into several smaller doses for administration throughout the day . inasmuch as it may desirable to administer a combination of active compounds , for example , for the purpose of treating a particular disease or condition , it is within the scope of the present invention that two or more pharmaceutical compositions , at least one of which contains a compound in accordance with the invention , may conveniently be combined in the form of a kit suitable for coadministration of the compositions . thus the kit of the invention includes two or more separate pharmaceutical compositions , at least one of which contains a compound of the invention , and means for separately retaining said compositions , such as a container , divided bottle , or divided foil packet . an example of such a kit is the familiar blister pack used for the packaging of tablets , capsules and the like . the kit of the invention is particularly suitable for administering different dosage forms , for example , oral and parenteral , for administering the separate compositions at different dosage intervals , or for titrating the separate compositions against one another . to assist compliance , the kit typically includes directions for administration and may be provided with a memory aid . the biochemical k i values of compound 1 for the inhibition of the c - met / hgfr kinase were determined utilizing a general procedure to monitor nadh oxidation that is coupled to atp turnover as follows . compounds and kinase assay reagents are introduced into test wells and incubated for 10 minutes at 37 ° c . the assay is initiated by addition of the c - met / hgfr enzyme . enzyme inhibitors reduce the measured activity of the enzyme . in the continuous - coupled spectrophotometric assay the time - dependent production of adp by the kinase is determined by analysis of the rate of consumption of nadh by measurement of the decrease in absorbance at 340 nm . as the kinase enzyme produces adp it is re - converted to atp by reaction with phosphoenol pyruvate and pyruvate kinase . pyruvate is also produced in this reaction . pyruvate is subsequently converted to lactate by reaction with lactate dehydrogenase , which simultaneously converts nadh to nad . nadh has a measurable absorbance at 340 nm whereas nad does not . therefore , the assay endpoint is measured by spectrophotometry at 340 nm at the designated time points . kinases were pre - diluted to a 10 × working concentration prior to addition into the assay . briefly , substrates were dissolved and diluted to working stocks in deionizer water , apart from histone h1 ( 10 × working stock in 20 mm mops ph 7 . 4 ), pdktide ( 10 × working stock in 50 mm tris ph 7 . 0 ) and atf2 ( which is typically stored at a 20 × working stock in 50 mm tris ph 7 . 5 , 150 mm nacl , 0 . 1 mm egta , 0 . 03 % brij - 35 , 50 % glycerol , 1 mm benzamidine , 0 . 2 mm pmsf and 0 . 1 % □- mercaptoethanol ). the biochemical enzyme of interest was then assayed in a final reaction volume of 25 μl containing 5 - 10 mu of the enzyme of choice incubated with 8 mm mops ph 7 . 0 , 0 . 2 mm edta , 50 μm eaiyaapfakkk , 10 mm mgacetate and 32 p - atp ( specific activity approx . 500 cpm / μmol , concentration as required ). the reaction is initiated by the addition of the mgatp mix . after incubation for 40 minutes at room temperature , the reaction is stopped by the addition of 5 μl of a 3 % phosphoric acid solution . 10 μl of the reaction is then spotted onto a p30 filtermat and washed three times for 5 minutes in 75 mm phosphoric acid and once in methanol prior to drying and scintillation counting . cell lines utilized to evaluate compound 1 in in - vitro studies were as follows : a549 human lung carcinoma , gtl - 16 human gastric carcinoma , ht29 human colon carcinoma , colo205 human colon carcinoma , a498 human renal carcinoma , 786 - o human renal carcinoma , mba - md - 231 human breast carcinoma , madlin - darby canine kidney ( mdck ) epithelial cells , mdck cells engineered to express p - glycoprotein ( mdck - mdr1 ), mlmcd3 mouse kidney epithelial , huvec ( human umbilical vein endothelial cells ); nih - 3t3 cells engineered to express human wild type c - met / hgfr and mutated c - met / hgfrs including hgfr - v1092i , hgfr - h1094r , hgfr - y1230c , and hgfr - m1250t . cell lines utilized to evaluate inhibition of phosphorylation of other tyrosine kinases were as follows : karpas 299 , su - dhl - 1 , and jurkat human lymphoma cells , human umbilical vein endothelial cells ( huvec ), human macro vascular endothelial cells ( hmvec ), porcine aorta endothelial ( pae ) cells engineered to express human vegfr2 , pdgfrβ , trka and trkb ; nih - 3t3 cells engineered to express human ron , axl , sky and egfr / tie - 2 chimera ; hek293 cells engineered to express human irk , chinese hamster ovary ( cho - b ) cells engineered to express human ron , baf3 cells engineered to express human bcr - abl . all the engineered cell lines were generated at pfizer , gtl - 16 gastric carcinoma cells were a gift from dr . paolo comoglio ( university torino medical school , candiolo , italy ), huvec and hmvec ( human macrovascular endothelial cell ) were purchased from clonetics , inc . ( walkersville , md . ), and the others were from atcc ( manassas , va .). unless otherwise indicated , cell culture reagents were obtained from life technologies , inc . ( gaithersburg , md .). cells were maintained at 37 ° c . in a humidified atmosphere with 5 - 10 % co 2 and maintained using standard cell culture techniques . antibodies were utilized to evaluate compound 1 in in vitro elisa and immunoblotting studies were as follows : anti - total human c - met / hgfr and anti - phospho zap70 were from zymed / invitrogen , carlsbad , calif . ; anti - total ron , anti - total fgfr1 , anti - total pdgfrβ , anti - total trk , anti - total tie - 2 , and anti - phospho tyrosine ( py - 20 ) were from santa cruz biotechnology , santa cruz , calif . ; anti - total axl and anti - total mouse c - met / hgfr were form r & amp ; d systems , minneapolis , minn . ; anti - total irk from bd pharmingen , san diego , calif . ; anti - vegfr2 from novus biologicals , littleton , colo . ; anti - phospho - c - met / hgfr , anti - total and - phospho alk , anti - total c - abl , anti - total and phosphor gab1 , anti - total and phospho akt , anti - total and phosphor - mapk44 / 42 , anti - phospho raf , mek1 / 2 , p90rsk , and stat5 were from cell signaling technologies , boston , mass . most growth factors utilized in cell assays were from r & amp ; d systems , minneapolis , minn ., except bdgf from gibcobrl / invitrogen , carlsbad , calif ., and egf from roche applied science , indianapolis , ind . cellular assays ( i . e ., elisa or immunoblot ) used to directly determine the ability of compound 1 to inhibit ligand - dependent or constitutive kinase phosphorylation were performed using a variety of serum - starved cells . cells were seeded in 96 well plates in growth media ( media supplemented with 10 % fetal bovine serum - fbs ) and cultured overnight at 37 ° c . to facilitate attachment to assay plates . after attachment , growth media was removed and cells were cultured in serum - free media ( with 0 . 04 % bsa ). serial dilutions of compound 1 were performed , appropriate controls or designated concentrations of compound 1 were added to each well , and cells were incubated at 37 ° c . for 1 hour . in experiments investigating ligand - dependent rtk phosphorylation , corresponding growth factors ( e . g ., hgf , msp , gas6 , egf , ngf , bdnf , insulin , vegf , or pdgf bb ) were added to cells for 8 to 20 minutes . h 2 o 2 was used to stimulate human axl phosphorylation in huvec cells as described ( konishi , a ., aizawa , t . mohan , a ., korshunov , v . a . and berk , b . c ., hydrogen peroxide activates the gas6 - axl pathway in vascular smooth muscle cells . the journal of biological chemistry , 279 : 28766 - 28770 ( 2004 )). constitutive kinase phosphorylation was measured in absence of addition of exogenous ligand for cell lines with constitutively active kinase activity ( e . g ., c - met / hgfr in gtl - 16 cells , npm - alk in karpas 299 cells , ron in ron - cho - b cells , and bcr - abl in bcr - abl baf3 cells ). after incubation of cells with compound 1 and / or appropriate ligands for the designated times , cells were washed once with 1 mm na 3 vo 4 in hbss and then lysed using lysis buffer ( cell signaling technologies , boston mass .). phosphorylation of protein kinases of interest was assessed by a sandwich elisa method utilizing capture antibodies specific for each protein and a detection antibody specific for phosphorylated tyrosine residues . in each elisa assay , protein lysates generated from various cell lines treated with appropriate rtk ligands and / or compound 1 were transferred to a 96 well plate that was pre - coated with the corresponding antibodies including anti - c - met / hgfr , - ron , - axl , - sky , - ir , - tie2 , - kdr , - pdgfr β , - zap70 , and etc . antibody - coated plates were incubated in presence of protein lysates at 4 ° c . overnight and washed with 1 % tween 20 in pbs seven times . hrp - py20 ( horseradish peroxidase - conjugated anti - total - phosphotyrosine , santa cruz biotechnology , santa cruz , calif .) was diluted 1 : 500 in blocking buffer ( pierce , rockford , ill .) and added to each plate for 30 minutes . plates were then washed again and tmb peroxidase substrate ( bio - rad laboratories , hercules calif .) was added to initiate the hrp - dependent colorimetric reaction . the reaction was stopped by adding 0 . 09nh 2 so 4 . plates were measured at od - 450 nm using a spectrophotometer . ic 50 values were calculated by concentration - response curve fitting utilizing a four - parameter analytical method in an excel - based template . the ability of compound 1 to inhibit cellular kinase phosphorylation was also measured by immunoblotting method . cells were treated with dilutions of compound 1 in serum - free media and lysed for protein extraction as described above . cell lysates were normalized for protein concentration by bsa assay ( pierce , rockford , ill .) and specific antibodies were used immunoprecipitate proteins of interest . immunoprecipitated proteins were separated by sds - page and immunoblotting with anti - phosphotyrosine antibodies was performed to determine relative levels of phosphorylated proteins at each drug concentration . this immunoblotting method was also used to determine total protein levels for the molecules of interest . tumor cells were seeded in 96 well plates at low density in growth media ( media supplemented with 10 % fetal bovine serum - fbs ) and cultured overnight at 37 ° c . the following day , growth media was removed and cells were cultured in serum - free media ( 0 % fbs and 0 . 04 % bsa ). serial dilutions of compound 1 were performed , appropriate controls or designated concentrations of compound 1 were added to each well , and cells were incubated at 37 ° c . for 24 to 72 hours . a mtt assay ( promega , madison , wis .) was then performed to determine the relative cell numbers . ic 50 values were calculated by concentration - response curve fitting utilizing a four - parameter analytical method . gtl - 16 cells were seeded in 96 well plates at 40 , 000 cells / well . designated concentrations of pf - 02341066 or vehicle were added to the wells in serum free media . cells were incubated in 37 ° c ., 5 % co 2 for 48 hours . the ssdna apoptosis elisa kit ( chemicon international ) was used to detect apoptosis . huvec cells ( passage 3 ) were grown to confluence in egm2 media ( walkersville , md .). cells were seeded in 96 well plates in egm2 media at high density ( 20 , 000 to 30 , 000 / well ) and incubated for 5 to 6 hours to allow for cell attachment . after attachment , cells were cultured in serum - free media ( cell applications , san diego , calif .) overnight at 37 ° c . at 5 % co 2 . the following day , cells were exposed to the starvation media ( cell applications , san diego , calif .) for 5 hours . compound 1 was serially diluted in serum - free media and appropriate controls or designated concentrations of compound 1 were added to each well . after 1 hour , human recombinant hgf ( r & amp ; d systems , minneapolis , minn .) was added to designated wells to achieve a final concentration of 100 ng / ml . a mtt assay ( promega ) was then performed after 48 to 72 hours to determine the relative cell numbers . ic 50 values were calculated by concentration - response curve fitting utilizing a four - parameter analytical method . the effect of compound 1 on hgf - stimulated nci - h441 human non - small cell lung carcinoma cell migration and matrigel invasion was determined utilizing a commercially available cell migration and invasion system ( bd biosciences , san jose , calif .). cells in log growth phase were trypsinized and suspended in serum - free media ( with 0 . 04 % bsa ) at a density of 400 , 000 cells / ml . compound 1 was serially diluted in serum - free media , designated concentrations were added to suspended cells , and cells were incubated at room temperature for 30 minutes . designated control or treated suspended cells ( 0 . 5 ml ) were added to each migration or invasion chamber ( i . e ., plate inserts ). in addition , 25 ng / ml hgf ( 0 . 75 ml ) was added to the lower well of each companion plate as a chemotractant to attract cells from migration or invasion chamber plate inserts inserted at the top of the companion plate and cells were incubated at 37 ° c . for 22 hours . cells that invaded or migrated to the lower well of the plate were then fixed and nuclei were stained ( 1 ug / mldap1 in 100 % meoh ) for 15 minutes at 37 ° c . cells were subsequently washed twice using tbs solution . five microscopic images were taken from each well and the cell number for migration or invasion was determined under each condition using image - pro plus software ( media cybernetics , silver spring , md .). ic 50 values were calculated by concentration - response curve fitting utilizing a four - parameter analytical method . an acea rt - ces system ( acea biologicals , san diego , calif .) was used to determine the effect of compound 1 on huvec matrigel invasion in vitro . acea electrosensing 96 - well plates were coated with 50 μl of 0 . 001 % fibronectin and 100 ng / ml hgf in pbs and incubated at 37 ° c . for 1 hour and at 4 ° c . for 30 minutes . after washing each plate with pbs at 4 ° c ., matrigel ( bd biosciences , san jose , calif .) was diluted 1 : 40 in starvation media ( sm , cell applications , san diego , calif . ), supplemented with hgf ( 100 ng / ml ) and / or different concentrations of compound 1 , added ( 50 μl ) to designated wells , and allowed to solidify at 37 ° c . for 2 hours . huvec cells were cultured in serum - free media ( cell applications , san diego , calif .) for 5 hours and then in sm for 2 hours . cells were subsequently collected in sm at 60 , 000 cells / ml and treated with 100 ng / ml hgf and / or appropriate of compound 1 for 30 minutes at 37 ° c . the huvec cell suspension ( 100 μl ) under designated conditions was transferred to the top of the matrigel layer in designated wells of the coated acea plate . the acea plate was then connected to the acea device station at 37 ° c ., 95 % air : 5 % co 2 and monitored in real time by an acea sensor analyzer for 48 hours . electronic sensors embedded on the bottom of the acea plates detected huvec cells that invaded through matrigel . the relative number ( cell index ) of invaded huvec cells was determined utilizing acea rt - ces ™ integrated software . ic 50 values were calculated by concentration - response curve fitting utilizing a four - parameter analytical method . mdck cells were seeded at low density ( 25 cells / well ) in a 96 - well plate in media supplemented with 10 % fbs and grown until small colonies of 10 - 15 cells appeared . cells were then stimulated with hgf ( 50 ng / ml ) in the presence of various concentrations of compound 1 diluted in growth medium . after overnight incubation , colonies were fixed and stained with 0 . 2 % crystal violet in 10 % buffered formalin and assessed for scattering at each concentration visually . five hundred hmvec were added to egm - 2 medium containing 0 . 24 % methylcellulose and transferred to u - bottom 96 - well plates to form spheroids overnight . spheroids were collected and mixed into 2 mg / ml fibrinogen solution containing 4 - 8 % fbs ± compound in 48 - well plates coated with thrombin ( 2 ml of 5 , 000 u / ml ). the resulting 3 - d fibrin gel was covered with egm - 2 containing 4 - 8 % fbs and incubated at 37 ° c ., 95 % air / 5 % co 2 . endothelial tube formation was observed daily under an inverted microscope . images were captured on day 7 by a digital camera ( olympus bx60 ) connected to the microscope . compound 1 was added at several concentrations and vascular sprouting was visually assessed . the effect of compound 1 on npm - alk - dependent cell cycle distribution and apoptosis of human lymphoma cells was evaluated by flow cytometric analysis ( facscalibur , bd biosciences , san jose , calif .). karpas 299 and su - dhl - 1 human lymphoma cells were treated with compound 1 for 24 to 48 hours in growth media ( rpmi + 10 % fbs ). cells were washed with pbs twice , fix and permeabilized with bdcytofix / cytoperm solution for 20 minutes at 4 ° c . cell cycle distribution and apoptosis of lymphoma cells was assessed utilizing the cyclotest plus dna reagent kit ( bd biosciences ). using this kit cells were washed two times with 1 × bd perm / wash buffer , non - ionic detergent and trypsin were added to isolate nuclei , propidium iodide was added to visualize dna content , and cells were analyzed by flow cytometry . dna content ( ploidy analysis to determine percent cell number in each cell cycle ) was assessed using cell quest pro and analyzed with modfit lt software ( bd biosciences ). the apoptotic peak ( a 0 ) was defined as the peak that occurs in channel numbers lower than g 0 / g 1 peak as described ( darzynkiewicz , z ., bruno , s ., del - bino , g ., gorczyca , w ., hotz , m . s ., lassota , p ., and traganos , f ., cytometry 13 : 795 - 808 ( 1992 )). apoptosis was also determined by flow cytometric analysis using annexin v - fitc staining ( bd biosciences , san jose , calif .) and also analyzed using facscalibur . unless otherwise indicated , cell culture reagents were obtained from life technologies , inc . ( gaithersburg , md .). cells were maintained at 37 ° c . in a humidified atmosphere with 5 - 10 % co 2 and passaged using standard cell culture techniques . u87mg ( human glioblastoma ), nci - h441 ( human nom - small cell lung adenocarcinoma ), pc - 3 ( human prostate adenocarcinoma ) cells were obtained from and cultured as recommended by the american type culture collection ( bethesda , md .). female or male nu / nu or scid / beige mice ( 5 - 8 weeks old ) were obtained from harlan ( madison , wis .) or charles river ( wilmington , mass .). animals were maintained under clean room conditions in sterile filter top cages with alpha - dri / bed - o - cob comb bedding housed on hepa - filtered ventilated racks . animals received sterile rodent chow and water ad libitum . cells for implantation into athymic mice were harvested and pelleted by centrifugation at 450 × g for 5 - 10 minutes . the cell pellets were washed once and re - suspended in sterile phosphate buffered saline or serum - free medium . tumor cells were supplemented with 30 - 50 % matrigel ( bd biosciences , san jose calif .) to facilitate tumor take and growth of selected tumor cells as xenografts . cells ( 2 - 5 × 10 6 in 100 μl ) were implanted sc into the hindflank region of the mouse and allowed to grow to the designated size prior to the administration of compound for each experiment . tumor size was determined by measurement with electronic calipers and tumor volume was calculated as the product of its length × width 2 × 0 . 4 . tumor cells expressing constitutively phosphorylated c - met / hgfr or alk were implanted subcutaneously in nude mice and allowed to grow untreated to a size of 300 - 800 mm 3 . mice were administered compound 1 as a single oral dose ( for acute pk / pd studies ) or multiple oral doses ( for steady state pk / pd studies ) at the designated dose levels . at the indicated times following compound 1 administration , individual mice were humanely euthanized , a blood sample was isolated from the cardiac left ventricle using a syringe primed with heparin sulfate , and tumors were resected . plasma samples were analyzed for compound 1 concentration using lcms analysis . resected tumors were snap frozen on dry ice , pulverized using a liquid nitrogen - cooled cryomortar and pestle , and lysed in cold 1 × cell lysis buffer ( cell signaling technologies , boston mont .)). proteins were extracted from tumor lysate and protein concentrations were determined using a bsa assay ( pierce , rockford , ill .). the level of total or / and phosphorylated proteins of interest in each tumor sample was determined using a capture elisa method below . in each elisa assay , protein lysates that were generated from vehicle - or compound 1 - treated tumors were transferred to a 96 well plate that was pre - coated with either anti - c - met / hgfr ( zymed lab / invitrogen , carlsbad , calif .) or anti - alk capture antibodies ( cell signaling technologies , boston mont .). antibody - coated plates were incubated in presence of tumor lysates at 4 ° c . overnight and washed with 1 % tween 20 in pbs seven times . hrp - py20 ( horseradish peroxidase - conjugated anti - total - phosphotyrosine , santa cruz biotechnology , santa cruz , calif .) was diluted 1 : 500 in blocking buffer ( pierce , rockford , ill .) and added to each plate for 30 minutes . plates were washed again and tmb peroxidase substrate ( bio - rad laboratories , hercules calif .) was added to initiate the hrp - dependent colorimetric reaction . reactions were stopped by adding 0 . 09nh 2 so 4 . the optical density ( od ) of each vehicle or treatment well was measured at 450 nm using a spectrophotometer . total phosphorylation of c - met / hgfr or alk in tumors resected from compound 1 - treated animals was compared with that in tumors resected from vehicle - treated animals at the same time point based on the od readings . in this evaluation , the inhibition of kinase target phosphorylation by compound 1 in tumors was calculated using the following equation : % inhibition = 100 −[( mean od treated / mean od untreated )× 100 ]. immunoblotting method was also used to determine relative kinase phosphorylation status and total protein levels in tumor samples for the protein of interest . tumor bearing mice were treated with different doses of compound 1 , tumor lysates and protein samples were prepared as described above . specific antibodies were used immunoprecipitate proteins of interest . immunoprecipitated proteins were separated by sds - page and immunoblotting with anti - phosphotyrosine or total antibodies . antibodies utilized in immunoblotting studies were as follows : anti - total human c - met / hgfr from zymed / invitrogen , carlsbad , calif . ; anti - phospho - c - met / hgfr , anti - total and - phospho alk , anti - total and phosphor gab1 , anti - total and phospho akt , anti - total and phosphor - mapk44 / 42 , stat5 were from cell signaling technologies , boston , mass . alzet 1003d and 1007d osmotic mini - pumps were purchased from durect corporation ( cupertino , calif .). mini pumps were loaded with solutions of designated concentrations of compound 1 , and primed in sterile saline solution at 37 ° c . until they reached equilibrium at 4 to 5 hours . pumps were surgically implanted subcutaneously per manufacturer &# 39 ; s instructions into left dorsal thoracic area of mice bearing tumors in their right flank region . the incision was closed using surgical clips which were removed after 5 - 7 days when the skin incision was completely healed . pump replacement surgery was conducted at the designated time for studies that required infusion time and drug volume that exceeded pump capacity . tumor specimens to be evaluated for immunohistochemical endpoints were harvested and fixed in 10 % buffered formalin with protease and phosphotase inhibitors for 24 hours before being transferred to 70 % ethanol . tumor specimens were subsequently paraffin - embedded and 4 μm sections were cut and baked on to microscope slides . deparaffinization and antigen retrieval ( edta - based ) was performed following manufacturer instructions using a commercially available decloaking chamber ( biocare medical , cat # dc2001 ). tumor oct frozen samples were also collected and sectioned for cd - 31 staining . for immunostaining , slides were incubated with the primary antibodies then secondary antibodies , and visualized using either a calorimetric method ( dao envision - harp , dab kit , dao , carpentaria , calif . ), or a fluoremetric method ( alexa 488 or alexa 635 , molecular probes / invitrogen , carlsbad calif .). all of the immunostained sections were counterstained using hematoxylin . automated ventana discovery xt staining module ( ventana medical systems , tucson , ariz .) was also used to conduct histological staining following manufacturer instructions . stained sections were analyzed using an olympus microscope and quantitative analysis of section staining was performed utilizing the acis system ( automated cellular imaging , clarient , irvine calif .). the slides were also analyzed by in house pathologists using standard clinical methods . antibodies utilized in immunohistochemistry studies included anti - phospho - c - met / hgfr from biosource internationals / invitrogen , carlbad , calif . ; anti - ki67 from dacocytomation , carpenteria , calif . ; anti - cd31 from santa cruz biotechnology , santa cruz , calif . compound 1 was demonstrated to be a potent atp - competitive inhibitor of recombinant , human c - met / hgfr kinase activity with a mean k i of 4 nm . compound 1 inhibited kinase activity of c - met / hgfr in a biochemical enzyme assay with a ki of 3 nm . to investigate kinase selectivity relative to c - met / hgfr , compound 1 was further evaluated in biochemical kinase screening assays against a panel of & gt ; 120 recombinant kinases . in these preliminary biochemical kinase selectivity screens , a subset of kinases were identified against which compound 1 exhibited activity such that selectivity for c - met / hgfr was estimated to be less than 100 - fold compared with c - met / hgfr . the activity of compound 1 against these potential kinase hits was further evaluated in follow - up studies in definitive cell - based kinase selectivity assays ( table 1 ). the biochemical ki values of compound 1 for the inhibition of the c - met / hgfr kinase were determined by monitoring nadh oxidation which is coupled to atp turnover . compounds and kinase assay reagents were introduced into test wells and incubated for 10 minutes at 37 ° c . and the reaction was initiated by adding the c - met / hgfr . nadh was measured by spectrophotometry at 340 nm at the designated time points . the selectivity of compound 1 was evaluated in a panel of cell based kinase activity assays for the selected kinases that were potential hits in biochemical assays and other related rtks ( e . g ., ron , sky , ir ). in cell - based studies , compound 1 was greater than 1000 - fold selective for vegfr2 and pdgfrβ split - rtks , greater than 200 - fold selective for ir and lck , and approximately 40 - 60 - fold selective for axl , tie2 , trka , and trkb , all compared with c - met / hgfr ( a549 ic 50 = 8 . 6 nm ). to investigate whether a 50 - fold window was sufficient for c - met / hgfr selectivity in vivo , compound 1 was evaluated for its ability to inhibit tie - 2 phosphorylation in c6 xenografts in nude mice . in this study , no significant inhibition of tie - 2 phosphorylation was observed at any time point following a single po dose of 50 mg / kg ( representing ic 99 for c - met / hgfr inhibition over 24 hrs ) or 100 mg / kg . this indicates that inhibition of tie - 2 , axl , or trka & amp ; b would be unlikely at doses up to 2 - fold higher than dose levels associated with full inhibition of c - met / hgfr over 24 h . compound 1 was 20 - 30 - fold selective for ron kinase which represents a potentially beneficial oncology target due to 1 ) overexpression and mutation in selected cancers and 2 ) lack of adverse phenotype in ron - null mice . in contrast to the aforementioned rtks , compound 1 demonstrated a near equivalent ic 50 value ( 24 nm ) against an oncogenic form of the alk rtk ( anaplastic lymphoma kinase ) fusion protein , npm - alk an oncogenic fusion protein variant of the alk rtk ( anaplastic lymphoma kinase ), that results from a chromosomal translocation which is implicated in the pathogenesis of human anaplastic large cell lymphoma ( alcl , in a human lymphoma cell line . to confirm that the potent enzymatic activity translated into inhibition of c - met / hgfr in cells , the ability of compound 1 to inhibit cmet / hgfr phosphorylation in a panel of tumor and endothelial cell lines was evaluated . compound 1 inhibited hgf - stimulated or constitutive total tyrosine autophosphorylation of wild type c - met / hgfr with a mean ic 50 value of 11 nm across a panel of human tumor and endothelial cell lines ( table 1 ). compound 1 demonstrated a similar value in mlmcd3 mouse epithelial cells ( ic 50 = 5 nm ) ( table 1 ). potency of compound 1 against cmet / hgfr active site mutations in cells c - met / hgfr activating mutations have been identified in several human cancers and provide a strong rationale for proof of concept clinical studies based on both experimental evidence and clinical precedence with other rtk targets . although c - met / hgfr mutations in the extracellular or juxtamembrane domain are not predicted to affect compound binding to active site , it is possible that kinase domain mutations will cause loss of activity . to address this issue , rtk phosphorylation ic 50 was evaluated in compound 1 treated nih3t3 cells engineered to express wild - type c - met / hgfr or a series of representative c - met / hgfr active - site mutations . in these studies , compound 1 exhibited improved or similar activity against atp binding site mutants ( v1092i , 19 nm and h1094r , 2 . 2 nm ) or p - loop mutants ( m1250t , 15 nm ) compared with wild - type receptor ( 12 . 6 nm ) ( table 1 ). compound 1 also potently exhibited comparable potency inhibiting c - met phosphorylation in nci - h69 ( ic 50 : 13 nm ) and hop92 ( ic 50 : 16 nm ) cells which express the endogenous c - met juxtamembrane variants r988c and t1010i , respectively ( table 1 ). in contrast , a significant shift in potency ( 10 - fold ) was observed against the activation loop mutants ( y1230c , 127 nm and y1235d , 92 nm ) compared with wild type receptor ( table 1 ). c - met / hgfr has been implicated in the dysregulation of cell growth , migration , and invasion of a variety of tumor cells and tumor endothelial cells while npm - alk is implicated in the dysregulation of cell proliferation and apoptosis in alcl lymphoma cells . in a series of cell - based functional assays , compound 1 potently inhibited human gtl - 16 gastric carcinoma cell growth , induced gtl - 16 cell apoptosis , inhibited hgf - stimulated human nci - h441 lung carcinoma cell migration and invasion through a matrigel matrix , and inhibited hgf - stimulated mdck cell motility / scattering . ( table 2 ) compound 1 also inhibited proliferation of karpas 299 or su - dhl - 1 alcl cells that express an npm - alk fusion protein due to a t2 ; 5 chromosomal translocation . growth inhibition by compound 1 in these npm - alk positive lymphoma cells was associated with g 0 / g 1 cell cycle arrest and induction of apoptosis . to also investigate potential anti - angiogenic activity , compound 1 inhibited hgf - mediated huvec endothelial cell survival and matrigel invasion as well as hmvec endothelial cell tubulogenesis in fibrin gels . these data demonstrate the ability of compound 1 to inhibit both c - met / hgfr - and npm - alk - dependent functions in cells expressing activated c - met / hgfr or npm - alk , respectively . in addition , these data suggest that anti - tumor efficacy of compound 1 may be mediated by both direct effects on tumor cell growth or survival as well as anti - angiogenic mechanisms . c - met / hgfr - dependent tumor xenograft models were utilized to evaluate the relationship of c - met / hgfr target inhibition , tumor growth inhibition , and plasma exposure of compound 1 in vivo . due to lack of paracrine activation of human c - met / hgfr expressed by tumor xenografts by mouse hgf expressed by mouse mesenchymal cells , human xenograft models exhibiting constitutive c - met / hgfr activity were utilized as follows : 1 ) the gtl - 16 human gastric carcinoma or caki - 1 renal carcinoma model that expresses elevated levels of constitutively active c - met / hgfr , 2 ) the u87mg human glioblastoma or pc - 3 human prostate carcinoma model that expresses both hgf and c - met / hgfr comprising an autocrine loop , or 3 ) co - implantation of human tumor cells ( e . g ., nci - h441 nsclc ) with human mrc5 fibroblasts to supply a source of bioactive human hgf from the tumor stromal compartment to restore species - specific paracrine activation of c - met / hgfr . athymic mice bearing established gtl - 16 tumors ( 250 mm 3 ) were administered compound 1 orally at the indicated dose or vehicle alone for 11 days . for studies investigating inhibition of c - met / hgfr phosphorylation in gtl - 16 ( fig2 a ), mice were humanely euthanized at the end of study at designated time points post - administration , tumors were resected and frozen , and phosphorylation in vehicle and treated groups was quantitated by elisa . inhibition of kinase target phosphorylation by compound 1 in tumors was calculated as : % inhibition = 100 −[( mean od treated / mean od untreated )× 100 ]. for studies investigating gtl - 16 tumor growth inhibition ( fig2 b ), tumor volume was measured by calipers on the indicated days with the median tumor volume ± sem indicated for groups of 15 mice . percent (%) values shown are the % of tumor growth inhibition measured on day 20 for drug - treated compared to vehicle - treated mice and are calculated as : 100 *{ 1 −[( treated day 20 − treated day 10 )/( controlday 20 − control day 10 )]}. an * denotes that the median tumor volumes are significantly less in the treated vs . the control group ( p & lt ; 0 . 001 ) as determined using one - way analysis of variance ( see fig2 b ). to evaluate the c - met / hgfr pd response to compound 1 , gtl - 16 tumors were harvested at several time points following oral administration of compound 1 in both single dose - and repeat - dose ( steady state ) studies . c - met / hgfr phosphorylation status in tumors was quantitated by elisa over a range of doses . with focus on steady - state pd studies ( 11 - day ) to draw a correlation with tumor growth inhibition , compound 1 demonstrated the following as shown in fig2 a and 2b : at 50 mg / kg / day : 100 % tumor growth inhibition correlated with complete inhibition of c - met / hgfr phosphorylation in gtl - 16 tumors sustained for 24 hours ( 25 mg / kg — near complete inhibition of both phosphorylation and tumor growth ). at 12 . 5 mg / kg / day : 60 % tumor growth inhibition correlated with 80 - 90 % inhibition of c - met / hgfr phosphorylation at 1 - 8 hours which decreased to 50 - 60 % inhibition by 16 - 24 hours . at 6 . 25 mg / kg / day : non - significant trend toward tumor growth inhibition correlated with 30 - 50 % inhibition of c - met / hgfr phosphorylation at 1 - 8 hours with full recovery by 16 hours . athymic mice bearing established u87mg tumors ( 150 mm 3 ) were administered compound 1 orally at the indicated dose or vehicle alone for 9 days . for studies investigating tumor growth inhibition ( fig3 a ), tumor volume was measured by calipers on the indicated days with the median tumor volume ± sem indicated for groups of 10 - 12 mice . percent (%) values shown are the % of tumor growth inhibition measured on day 14 for drug - treated compared to vehicle - treated mice and are calculated as : 100 *{ 1 −[( treated day 14 − treated day 6 )/( controlday 14 − control day 6 )]}. an * denotes that the median tumor volumes are significantly less in the treated vs . the control group ( p & lt ; 0 . 001 ) as determined using one - way analysis of variance ( see fig3 a ). for studies investigating inhibition of c - met / hgfr phosphorylation ( fig3 b ), mice were humanely euthanized at the end of study 4 hours post - administration of compound 1 , tumors were resected and frozen , and phosphorylation in vehicle and treated groups was quantitated by elisa . inhibition of kinase target phosphorylation by compound 1 in tumors was calculated as : % inhibition = 100 −[( mean od treated / mean od untreated )× 100 ]. pharmacologically relevant inhibition of tie - 2 phosphorylation was not observed in u87mg xenografts at dose levels up to 100 mg / kg suggesting that compound 1 was selective for its intended targets at similar dose levels . the antitumor efficacy of compound 1 was evaluated in a variety of human tumor xenograft models representative of cancer indications in which dysregulation of c - met / hgfr is implicated including gtl - 16 gastric carcinoma , u87mg glioblastoma , nci - h441 nsclc , and pc - 3 prostate carcinoma ( table 4 ). utilizing the gtl - 16 gastric carcinoma model , compound 1 demonstrated the ability to cause marked regression of large established tumors (& gt ; 600 mm 3 ) ( fig4 ). in this study , the 50 and 75 mg / kg / day compound 1 treatment cohorts exhibited equivalent mean tumor regression during the 43 - day administration schedule , which provided further evidence that 50 mg / kg / day represents the maximally efficacious dose level . as illustrated in fig4 , the mean tumor regression at 50 or 75 mg / kg / day was 60 % after 43 days of compound 1 administration . in the present study , each tumor decreased in mass during the 43 - day compound 1 administration cycle at each dose level , with 9 of 14 mice exhibiting a ≧ 30 % decrease in tumor mass ( partial response ), and one animal exhibiting a complete response with no evidence of tumor even after cessation of treatment for 10 days . regression of large established gtl - 16 tumor xenografts in athymic mice ( fig4 a ) and mouse body weight ( fig4 b ) following daily oral administration of compound 1 . athymic mice bearing established gtl - 16 tumors ( 620 mm3 ) were administered compound 1 orally at the indicated dose levels or vehicle alone for 43 days . to investigate antitumor efficacy ( fig4 a ), tumor volume was measured by calipers on the indicated days with the median tumor volume ± sem indicated for groups of 6 - 8 mice . ( fig4 b ) mean mouse weight in compound 1 treatment and vehicle control groups are illustrated in right hand panel . athymic mice bearing established nci - h441 ( 100 mm3 ) ( fig5 a ), caki - 1 ( table 3a , table 3b ) or pc - 3 tumor xenografts ( fig5 b ) were administered compound 1 orally at the indicated dose or vehicle alone for 38 , 40 or 20 days , respectively . tumor volume was measured by calipers on the indicated days with the median tumor volume ± sem . an * denotes that the median tumor volumes are significantly less in the treated vs . the control group ( p & lt ; 0 . 001 ) as determined using one - way analysis of variance . ( see fig5 ). in nci - h441 nsclc model , a 43 % mean regression of established tumors was observed at 50 mg / kg / day after 38 - day of compound 1 administration ( fig5 ). in this study , each tumor decreased in mass during the 33 - day compound 1 administration cycle at 50 mg / kg / day , with 3 of 11 mice exhibiting a ≧ 30 % decrease in tumor mass ( partial response ), and 3 animals exhibiting a complete response with no evidence of tumor ( fig5 ). the antitumor efficacy of compound 1 was dose - dependent with regression of established nci - h441 tumors observed at 50 mg / kg / day and partial inhibition of tumor growth ( 57 % tumor growth inhibition ) observed at 15 mg / kg / day ( fig5 ). the antitumor efficacy of compound 1 observed in the nci - h441 model was consistent with the inhibition of c - met / hgfr phosphorylation in tumors from compound treatment groups compared with vehicle treated controls . in the caki - 1 renal carcinoma model , a 53 % decrease in mean tumor volume was observed at 50 mg / kg / day during the 33 - day compound 1 administration cycle ( fig5 b ). in the caki - 1 study , each tumor decreased in volume by at least 30 % during the 33 - day compound 1 administration cycle ( table 3b , table 4 ). antitumor efficacy of compound 1 was also investigated in the pc - 3 prostate carcinoma xenograft model and near complete inhibition of tumor growth ( 84 % growth inhibition ) was observed in this model . a series of dose - response antitumor efficacy and pharmacodynamic studies were performed to demonstrate the relationship between c - met / hgfr target inhibition to antitumor efficacy . to evaluate the pharmacodynamic inhibition of c - met / hgfr by compound 1 , gtl - 16 gastric carcinoma tumors were harvested at several time points following oral administration of compound 1 . c - met / hgfr phosphorylation status in tumors was quantitated by elisa over a range of doses . in these studies , compound 1 demonstrated a strong correlation of dose - and time - dependent inhibition of c - met / hgfr to inhibition of tumor growth . when defining the relationship of target pd to efficacy in the gtl - 16 model the following conclusions were apparent 1 ) complete inhibition of c - met / hgfr activity for 24 hours is consistent with complete inhibition of tumor growth ( 50 mg / kg , 100 % tgi ), 2 ) potent inhibition of c - met / hgfr activity for only a portion of the schedule is consistent with suboptimal efficacy ( 12 . 5 mg / kg , 60 % tgi ), 3 ) inability to achieve & gt ; 50 % inhibition of c - met / hgfr activity ( 3 . 125 , 6 . 25 mg / kg ) is consistent with lack of significant tumor growth inhibition ( tgi ) ( fig2 a and 2b ). an additional gtl - 16 study demonstrated that the 50 and 75 mg / kg / day compound 1 treatment cohorts exhibited equivalent mean tumor regression which provided further evidence that 50 mg / kg / day represents the maximally efficacious dose level ( fig4 and table 4 ). these findings suggest that the duration of c - met / hgfr inhibition is directly linked to anti - tumor efficacy of compound 1 . furthermore , a similar dose - dose dependent effect of compound 1 on tumor growth and c - met / hgfr phosphorylation was observed utilizing all tumor models ( gtl - 16 , u87mg , and nci - h441 ) and dosing schedules further these supporting observations ( table 4 ). in each of these studies the 50 mg / kg / day dose level resulted in either complete tumor growth inhibition or tumor regression ( table 4 ). in addition , a dose - dependent correlation was observed between inhibition of c - met / hgfr phosphorylation and antitumor efficacy in each model further supporting the concept of maximizing of the extent and duration of c - met / hgfr inhibition to achieve full efficacy . collectively , these studies suggest that near - complete inhibition of c - met / hgfr phosphorylation for the duration of the administration schedule is necessary to maximize therapeutic benefit and the extent and duration of the inhibition c - met / hgfr activity was directly linked to the level of anti - tumor efficacy . this data supports the link between inhibition of the intended pharmacologic target of compound 1 , c - met / hgfr , and the degree of antitumor efficacy . scid - beige mice bearing established karpas 299 tumors ( 220 mm 3 ) were administered compound 1 orally at the indicated dose or vehicle alone for designated time periods . for studies investigating tumor growth inhibition ( fig6 a ), tumor volume was measured by calipers on the indicated days with the median tumor volume ± sem indicated for groups of 8 - 12 mice . percent (%) values shown are the % of tumor growth inhibition measured on day 23 for drug - treated compared to vehicle - treated mice and are calculated as : 100 *{ 1 −[( treated day 23 − treated day 12 )/( control day 23 − control day 12 )]}. an * denotes that the median tumor volumes are significantly less in the treated vs . the control group ( p & lt ; 0 . 001 ) as determined using one - way analysis of variance . for studies investigating inhibition of npm - alk phosphorylation ( fig6 b ), mice were humanely euthanized at the end of study 4 hours post - administration of compound 1 , tumors were resected and frozen , and alk phosphorylation in vehicle and treated tumors was quantitated by elisa . inhibition of kinase target phosphorylation by compound 1 in tumors was calculated as : % inhibition = 100 −[( mean od treated / mean od untreated )× 100 ]. utilizing the karpas 299 alcl model , compound 1 demonstrated the ability to cause marked regression of established tumors (& gt ; 200 mm 3 ) ( fig6 a ). in this study , the administration of compound 1 at 100 mg / kg / day resulted in complete regression of tumors of all mice in this dosing cohort within 15 days of the initiation compound administration ( fig6 a ). after 17 days compound 1 treatment was stopped resulting in tumor re - growth . when tumors grew to a larger size (& gt ; 600 mm 3 ), compound 1 treatment was reinitiated for an additional 13 days and complete regression of tumors was demonstrated once again ( fig6 a , table 4 ). the cytoreductive effect of compound 1 is consistent with the observation of its anti - proliferative and apoptotic effects against alcl cells in - vitro . the relationship of inhibition of tumor npm - alk phosphorylation to antitumor efficacy was also determined at multiple dose levels and time points . similar to observations in c - met / hgfr - dependent tumor models , near complete inhibition (& gt ; 90 % inhibition ) of npm - alk activity for the full dosing interval ( 24 hours ) is consistent with maximal antitumor efficacy ( complete regression ) at 100 mg / kg ( fig6 a and 6b ). incomplete inhibition of npm - alk phosphorylation (& lt ; 90 % inhibition at 25 or 50 mg / kg ) is consistent with submaximal antitumor efficacy ( fig6 a and 6b ). similar to studies in c - met / hgfr - dependent tumor models , this data supports the link between inhibition of the other intended pharmacologic target of compound 1 , npm - alk , and the degree of antitumor efficacy in a npm - alk - dependent tumor model . ple is an enzyme produced by roche and sold through biocatalytics inc . as a crude esterase preparation from pig liver , commonly known as ple - as ( purchased from biocatalytics as icr - 123 , sold as an ammonium sulfate suspension ). the enzyme is classified in the cas registry as a “ carboxylic - ester hydrolase , cas no . 9016 - 18 - 6 ”. the corresponding enzyme classification number is ec 3 . 1 . 1 . 1 . the enzyme is known to have broad substrate specificity towards the hydrolysis of a wide range of esters . the lipase activity is determined using a method based on hydrolysis of ethyl butyrate in a ph titrator . 1 lu ( lipase unit ) is the amount of enzyme which liberates 1 μmol titratable butyric acid per minute at 22 ° c ., ph 8 . 2 . the preparation reported herein ( ple - as , as a suspension ) is usually shipped as an opaque brown - green liquid with a declared activity of & gt ; 45 lu / mg ( protein content around 40 mg / ml ). ( 1s )- 1 -( 2 , 6 - dichloro - 3 - fluorophenyl ) ethanol , shown as compound ( s - 1 ) in the schemes below , was prepared by a combination of enzymatic hydrolysis of racemic 1 -( 2 , 6 - dichloro - 3 - fluorophenyl ) ethyl acetate , esterification and chemical hydrolysis with inversion according to scheme b . racemic 1 -( 2 , 6 - dichloro - 3 - fluorophenyl ) ethyl acetate ( compound a2 ) was prepared according to scheme a . 1 -( 2 , 6 - dichloro - 3 - fluorophenyl ) ethanol ( a1 ): sodium borohydride ( 90 mg , 2 . 4 mmol ) was added to a solution of 2 ′, 6 ′- dichloro - 3 ′- fluoro - acetophenone ( aldrich , catalog # 52 , 294 - 5 ) ( 207 mg , 1 mmol ) in 2 ml of anhydrous ch 3 oh . the reaction mixture was stirred at room temperature for 1 h then was evaporated to give a colorless oil residue . the residue was purified by flash chromatography ( eluting with 0 → 10 % etoac in hexanes ) to give compound a1 as a colorless oil ( 180 mg ; 0 . 88 mmol ; 86 . 5 % yield ); ms ( apci ) ( m - h ) − 208 ; 1h nmr ( 400 mhz , chloroform - d ) δ ppm 1 . 64 ( d , j = 6 . 82 hz , 3h ) 3 . 02 ( d , j = 9 . 85 hz , 1h ) 6 . 97 - 7 . 07 ( m , 1h ) 7 . 19 - 7 . 33 ( m , 1h ). 1 -( 2 , 6 - dichloro - 3 - fluorophenyl ) ethyl acetate ( a2 ): acetic anhydride ( 1 . 42 ml , 15 mmol ) and pyridine ( 1 . 7 ml , 21 mmol ) were added sequentially to a solution of compound a1 ( 2 . 2 g , 10 . 5 mmol ) in 20 ml of ch 2 cl 2 . the reaction mixture was stirred at room temperature for 12 h and then evaporated to give a yellowish oil residue . the residue was purified by flash chromatography ( eluting with 7 → 9 % etoac in hexanes ) to give compound a2 as a colorless oil ( 2 . 26 g ; 9 . 0 mmol ; 85 . 6 % yield ); 1h nmr ( 400 mhz , chloroform - d ) δ ppm 1 . 88 ( d , j = 6 . 82 hz , 3 h ) 2 . 31 ( s , 3h ) 6 . 62 ( q , j = 6 . 82 hz , 1h ) 7 . 25 ( t , j = 8 . 46 hz , 1h ) 7 . 49 ( dd , j = 8 . 84 , 5 . 05 hz , 1h ). to a 50 ml jacketed flask equipped with a ph electrode , an overhead stirrer and a base addition line ( 1m naoh ), was added 1 . 2 ml of 100 mm potassium phosphate buffer ph 7 . 0 and 0 . 13 ml of ple as suspension . then , compound a2 ( 0 . 13 g , 0 . 5 mmol , 1 . 00 eq ) was added dropwise and the resulting mixture was stirred at room temperature for 20 h , maintaining the ph of the reaction constant at 7 . 0 using 1 m naoh . both the conversion and ee &# 39 ; s of the reaction were monitored by rp - hplc , and stopped after 50 % starting material was consumed ( approximately 17 hours under these conditions ). the mixture was then extracted three times with 10 ml of ethyl acetate to recover both ester and alcohol as a mixture of r - 1 and s - 2 . methanesulfonyl chloride ( 0 . 06 ml , 0 . 6 mmol ) was added to a solution of a mixture of r - 1 and s - 2 ( 0 . 48 mmol ) in 4 ml of pyridine under nitrogen atmosphere . the reaction mixture was stirred at room temperature for 3 h then evaporated to obtain an oil . water ( 20 ml ) was added to the mixture and then etoac ( 20 ml × 2 ) was added to extract the aqueous solution . the organic layers were combined , dried , filtered , and evaporated to give a mixture of r - 3 and s - 2 . this mixture was used in the next step reaction without further purification . 1 h nmr ( 400 mhz , chloroform - d ) δ ppm 1 . 66 ( d , j = 7 . 1 hz , 3h ) 1 . 84 ( d , j = 7 . 1 hz , 3h ) 2 . 09 ( s , 3h ) 2 . 92 ( s , 3h ) 6 . 39 ( q , j = 7 . 0 hz , 1h ) 6 . 46 ( q , j = 6 . 8 hz , 1h ) 6 . 98 - 7 . 07 ( m , 1h ) 7 . 07 - 7 . 17 ( m , 1h ) 7 . 23 - 7 . 30 ( m , 1h ) 7 . 34 ( dd , j = 8 . 8 , 4 . 80 hz , 1h ). potassium acetate ( 0 . 027 g , 0 . 26 mmol ) was added to a mixture of r - 3 and s - 2 ( 0 . 48 mmol ) in 4 ml of dmf under nitrogen atmosphere . the reaction mixture was heated to 100 ° c . for 12 h . water ( 20 ml ) was added to the reaction mixture and etoac ( 20 ml × 2 ) was added to extract the aqueous solution . the combined organic layer was dried , filtered , and evaporated to give an oil of s - 2 ( 72 mg , 61 % yield in two steps ). chirality ee : 97 . 6 %. 1 h nmr ( 400 mhz , chloroform - d ) δ ppm 1 . 66 ( d , j = 7 . 1 hz , 3h ) 2 . 09 ( s , 3h ) 6 . 39 ( q , j = 6 . 8 hz , 1h ) 7 . 02 ( t , j = 8 . 5 hz , 1h ) 7 . 22 - 7 . 30 ( m , 1h ). sodium methoxide ( 19 mmol ; 0 . 5 m in methanol ) was added slowly to compound s - 2 ( 4 . 64 g , 18 . 8 mmol ) under a nitrogen atmosphere at 0 ° c . the resulting mixture was stirred at room temperature for 4 hours . the solvent was evaporated and h 2 o ( 100 ml ) was added . the cooled reaction mixture was neutralized with sodium acetate - acetic acid buffer solution to ph 7 . ethyl acetate ( 100 ml × 2 ) was added to extract the aqueous solution . the combined organic layers were dried over na 2 so 4 , filtered , and evaporated to obtain s - 1 as a white solid ( 4 . 36 g , 94 . 9 % yield ); sfc - ms : 97 % ee . 1 h nmr ( 400 mhz , chloroform - d ) δ ppm 1 . 65 ( d , j = 6 . 8 hz , 3 h ) 5 . 58 ( q , j = 6 . 9 hz , 1h ) 6 . 96 - 7 . 10 ( m , 1h ) 7 . 22 - 7 . 36 ( m , 1h ). 1 . 2 , 6 - dichloro - 3 - fluoroacetophenone ( 15 g , 0 . 072 mol ) was stirred in thf ( 150 ml , 0 . 5m ) at 0 ° c . using an ice bath for 10 min . lithium aluminum hydride ( 2 . 75 g , 0 . 072 mol ) was slowly added . the reaction was stirred at ambient temperature for 3 hr . the reaction was cooled in ice bath , and water ( 3 ml ) was added drop wisely followed by adding 15 % naoh ( 3 ml ) slowly . the mixture was stirred at ambient temperature for 30 min . 15 % naoh ( 9 ml ), mgso 4 were added and the mixture filtered to remove solids . the solids were washed with thf ( 50 ml ) and the filtrate was concentrated to give 1 -( 2 , 6 - dichloro - 3 - fluoro - phenyl )- ethanol ( 14 . 8 gm , 95 % yield ) as a yellow oil . 1 h nmr ( 400 mhz , dmso - d 6 ) δ 1 . 45 ( d , 3h ), 5 . 42 ( m , 2h ), 7 . 32 ( m , 1h ), 7 . 42 ( m , 1h ). 2 . to a stirred solution of triphenyl phosphine ( 8 . 2 g , 0 . 03 mol ) and dead ( 13 . 65 ml of a 40 % solution in toluene ) in thf ( 200 ml ) at 0 ° c . was added a solution of 1 -( 2 , 6 - dichloro - 3 - fluoro - phenyl )- ethanol ( 4 . 55 g , 0 . 021 mol ) and 3 - hydroxy - nitropyridine ( 3 . 35 g , 0 . 023 mol ) in thf ( 200 ml ). the resulting bright orange solution was stirred under a nitrogen atmosphere at ambient temperature for 4 hours at which point all starting materials had been consumed . the solvent was removed , and the crude material was dry loaded onto silica gel , and eluted with ethyl acetate - hexanes ( 20 : 80 ) to yield 3 -( 2 , 6 - dichloro - 3 - fluoro - benzyloxy )- 2 - nitro - pyridine ( 6 . 21 g , 0 . 021 mol , 98 %) as a pink solid . 1 h nmr ( cdcl 3 , 300 mhz ) 1 . 8 - 1 . 85 ( d , 3h ), 6 . 0 - 6 . 15 ( q , 1h ), 7 . 0 - 7 . 1 ( t , 1h ), 7 . 2 - 7 . 21 ( d , 1h ), 7 . 25 - 7 . 5 ( m , 2h ), 8 . 0 - 8 . 05 ( d , 1h ). 3 . to a stirred mixture of acoh ( 650 ml ) and etoh ( 500 ml ) was suspended 3 -( 2 , 6 - dichloro - 3 - fluoro - benzyloxy )- 2 - nitro - pyridine ( 9 . 43 g , 0 . 028 mol ) and iron chips ( 15 . 7 g , 0 . 28 mol ). the reaction was heated slowly to reflux and allowed to stir for 1 hr . the reaction was cooled to room temperature then diethyl ether ( 500 ml ) and water ( 500 ml ) was added . the solution was carefully neutralized by the addition of sodium carbonate . the combined organic extracts were washed with sat &# 39 ; d nahco 3 ( 2 × 100 ml ), h 2 o ( 2 × 100 ml ) and brine ( 1 × 100 ml ) then dried ( na 2 so 4 ), filtered and concentrated to dryness under vacuum to yield 3 -( 2 , 6 - dichloro - 3 - fluoro - benzyloxy )- pyridin - 2 - ylamine ( 9 . 04 g , 0 . 027 mol , 99 %) as a light pink solid . 1 h nmr ( cdcl 3 , 300 mhz ) 1 . 8 - 1 . 85 ( d , 3h ), 4 . 9 - 5 . 2 ( brs , 2h ), 6 . 7 - 6 . 84 ( q , 1h ), 7 . 0 - 7 . 1 ( m , 1h ), 7 . 2 - 7 . 3 ( m , 1h ), 7 . 6 - 7 . 7 ( m , 1h ). 4 . a stirring solution of 3 -( 2 , 6 - dichloro - 3 - fluoro - benzyloxy )- pyridin - 2 - ylamine ( 9 . 07 g , 0 . 03 mol ) in acetonitrile was cooled to 0 ° c . using an ice bath . to this solution was added n - bromosuccinimide ( nbs ) ( 5 . 33 g , 0 . 03 mol ) portionwise . the reaction was stirred at 0 ° c . for 15 min . the reaction was concentrated to dryness under vacuum . the resulting dark oil was dissolved in etoac ( 500 ml ), and purified via silica gel chromatography . the solvents were then removed in vacuo to yield 5 - bromo - 3 -( 2 , 6 - dichloro - 3 - fluoro - benzyloxy )- pyridin - 2 - ylamine ( 5 . 8 g , 0 . 015 mol , 51 %) as a white crystalline solid . 1 h nmr ( cdcl 3 , 300 mhz ) 1 . 85 - 1 . 95 ( d , 3h ), 4 . 7 - 5 . 0 ( brs , 2h ), 5 . 9 - 6 . 01 ( q , 1h ), 6 . 8 - 6 . 95 ( d , 1h ), 7 . 01 - 7 . 2 ( t , 1h ), 7 . 4 - 7 . 45 ( m , 1h ), 7 . 8 - 7 . 85 ( d , 1h ). the enantiomerically pure r isomer was prepared as described above for the racemate , but using the enantiomerically pure starting materials described above . 1 h nmr ( 400 mhz , dmso - d 6 ) δ 1 . 74 ( d , 3h ), 6 . 40 ( m , 1h ), 6 . 52 ( br s , 2h ), 7 . 30 ( m , 1h ), 7 . 48 ( m , 1h ), 7 . 56 ( s , 1h ); ms m / z 382 ( m + 1 ). to a stirred solution of 4 - hydroxy - piperidine - 1 - carboxylic acid tert - butyl ester ( 7 . 94 g , 39 . 45 mmol ) in ch 2 cl 2 ( 100 ml ), cooled to 0 ° c ., was slowly added net 3 ( 5 . 54 ml , 39 . 45 mmol ) followed by methane sulfonyl chloride ( 3 . 06 ml , 39 . 45 mmol ) and dmap ( 48 mg , 0 . 39 mmol ). the mixture was stirred at room temperature overnight . to the mixture was added water ( 30 ml ). extraction with ch 2 cl 2 ( 3 × 30 ml ) followed by drying ( na 2 so 4 ) and removal of the solvent in vacuo afforded 4 - methanesulfonyloxy - piperidine - 1 - carboxylic acid tert - butyl ester as a white solid ( 11 . 00 g , & gt ; 99 % yield ). 1 h nmr ( cdcl 3 , 400 mhz ) δ 4 . 89 ( m , 1h ), 3 . 69 ( m , 2h ), 3 . 31 ( m , 2h ), 3 . 04 ( s , 3h ), 1 . 95 ( m , 2h ), 1 . 83 ( m , 2h ), 1 . 46 ( s , 9h ). nah ( 1 . 2 eq ., 0 . 68 mmol ) was added portionwise to a stirred solution of 4 - iodopyrazole ( 0 . 57 mmol ) in dmf ( 2 l ) at 4 ° c . the resulting mixture was stirred for 1 hour at 4 ° c . and 4 - methanesulfonyloxy - piperidine - 1 - carboxylic acid tert - butyl ester , compound 2 ( 1 . 1 eq ., 0 . 63 mmol ) was then added . the resulting mixture was heated to 100 ° c . for 12 h . the reaction was quenched with h 2 o and extracted with etoac several times . the combined organic layers were dried , filtered , and concentrated to afford an orange oil . the residue was purified by silica gel chromatography ( eluting with 5 % etoac in pentane ) to give compound 3 as a white solid ( 140 g , 66 %). bis ( pinacolato ) diboron ( 1 . 4 eq ., 134 g , 0 . 52 mol ) and potassium acetate ( 4 eq ., 145 g , 1 . 48 mol ) were added sequentially to a solution of compound 3 ( 140 g , 0 . 37 mol ) in 1 . 5 l of dmso . the mixture was purged with nitrogen several times and dichlorobis ( triphenylphosphino ) palladium ( ii ) ( 0 . 05 eq ., 12 . 9 g , 0 . 018 mol ) was then added . the resulting mixture was heated at 80 ° c . for 2 h . the reaction mixture was cooled to room temperature and filtered through a bed of celite and washed with etoac . the filtrate was washed with saturated nacl ( 500 ml × 2 ), dried over na 2 so 4 , filtered and concentrated . the residue was purified by silica gel chromatography ( eluting with 5 % etoac in hexanes ) to give compound 4 as a white solid ( 55 g , 40 %). to a stirred solution of 3 -[( r )- 1 -( 2 , 6 - dichloro - 3 - fluoro - phenyl )- ethoxy ]- 5 -( 4 , 4 , 5 , 5 - tetramethyl -[ 1 , 3 , 2 ] dioxaborolan - 2 - yl )- pyridin - 2 - ylamine ( 15 . 22 g , 35 . 64 mmol ) and 4 -( 4 - bromo - pyrazol - 1 - yl )- piperidine - 1 - carboxylic acid tert - butyl ester ( 14 . 12 g , 42 . 77 mmol ) in dme ( 143 ml ) was added a solution of na 2 co 3 ( 11 . 33 g , 10692 mmol ) in water ( 36 ml ). the solution was degassed and charged with nitrogen three times . to the solution was added pd ( pph 3 ) 2 cl 2 ( 1 . 25 mg , 1 . 782 mmol ). the reaction solution was degassed and charged with nitrogen again three times . the reaction solution was stirred at 87 ° c . oil bath for about 16 hours ( or until consumption of the borane pinacol ester ), cooled to ambient temperature and diluted with etoac ( 600 ml ). the reaction mixture was filtered through a pad of celite and washed with etoac . the etoac solution was washed with brine , dried over na 2 so 4 , and concentrated . the crude product was purified on a silica gel column eluting with etoac / hexane system ( biotage 90 + column : equilibrium 600 ml 100 % hexanes , segment 1 : 2250 ml 50 % etoac / hexanes linear , segment 2 : 4500 ml 75 % etoac / hexanes linear , segment 3 : 4500 ml 100 % etoac ) to afford 4 -( 4 -{ 6 - amino - 5 -[( r )- 1 -( 2 , 6 - dichloro - 3 - fluoro - phenyl )- ethoxy ]- pyridin - 3 - yl }- pyrazol - 1 - yl )- piperidine - 1 - carboxylic acid tert - butyl ester ( 11 . 8 g , 60 % yield , ˜ 95 % purity ) with a rf of 0 . 15 ( 50 % etoac / hexanes ). ms m / e 550 ( m + 1 ) + . to a solution of 4 -( 4 -{ 6 - amino - 5 -[( r )- 1 -( 2 , 6 - dichloro - 3 - fluoro - phenyl )- ethoxy ]- pyridin - 3 - yl }- pyrazol - 1 - yl )- piperidine - 1 - carboxylic acid tert - butyl ester ( 11 . 8 g , 21 . 45 mmol ) in ch 2 cl 2 ( 59 ml , 0 . 2m ) was added 4n hcl / dioxane ( 21 ml ). the solution was stirred overnight forming a solid . the solid was crushed thoroughly with a glass rod and sonicated to release starting material trapped in the solid . additional 4n hcl / dioxane ( 21 ml ) was added and stirred for another 2 hours at room temperature in which lcms showed no starting material . the suspension was filtered in a buchner funnel lined with filter paper . the mother liquor was saved because it contained & lt ; 5 % of product . the solid was transferred to a 500 ml beaker and hplc water was added until the solid dissolved completely . the ph was adjusted to 10 with the addition of solid na 2 co 3 . the water solution was extracted with ch 2 cl 2 ( 5 × 200 ml ) or until lcms showed no product in the aqueous layer . the ch 2 cl 2 solution was dried over na 2 so 4 and concentrated . the crude product , re - dissolved in ch 2 cl 2 ( 10 ml ) and meoh ( 1 ml ), was purified on a silica gel column eluting with ch 2 cl 2 / meoh / net 3 system ( biotage 40 + column : equilibrium 600 ml ch 2 cl 2 100 % giving byproduct , segment 1 : 1200 ml 10 % meoh / ch 2 cl 2 linear , segment 2 : 2400 ml 10 % meoh / ch 2 cl 2 step , segment 3 : 2400 ml 9 % meoh / 1 % net 3 / ch 2 cl 2 ). the desired fractions were collected to provide 3 -[( r )- 1 -( 2 , 6 - dichloro - 3 - fluoro - phenyl )- ethoxy ]- 5 -( 1 - piperidin - 4 - yl - 1h - pyrazol - 4 - yl )- pyridin - 2 - ylamine ( 7 . 19 g , 75 % combined yield , white solid ). ms m / e 450 ( m + 1 ) + . 1 h nmr ( dmso - d 6 , 400 mhz ) δ 7 . 92 ( s , 1h ), 7 . 76 ( s , 1h ), 7 . 58 ( m , 1h ), 7 . 53 ( s , 1h ), 7 . 45 ( m , 1h ), 6 . 90 ( s , 1h ), 6 . 10 ( m , 1h ), 5 . 55 ( bs , 2h ), 4 . 14 ( m , 1h ), 3 . 05 ( m , 2h ), 2 . 58 ( m , 2h ), 1 . 94 ( m , 2h ), 1 . 80 ( d , 3h ), 1 . 76 ( m , 2h ). the solid product 3 -[( r )- 1 -( 2 , 6 - dichloro - 3 - fluoro - phenyl )- ethoxy ]- 5 -( 1 - piperidin - 4 - yl - 1h - pyrazol - 4 - yl )- pyridin - 2 - ylamine was dissolved in dichloromethane , and the solvent was evaporated slowly to generate fine crystalline solid . after high vacuum dry , the sample was confirmed to be a single crystalline polymorph form a with a melting point of 194 ° c .
the present invention relates to the use of - 3 -- ethoxy ]- 5 -- pyridin - 2 - ylamine , a novel c - met / hgfr inhibitor , for treating abnormal cell growth in mammals . in particular , the invention provides methods of treating mammals suffering from cancer .