Respiratory formulations and compounds for use therein
10358445 ยท 2019-07-23
Assignee
Inventors
- Peter John Murray (London, GB)
- Stuart Thomas Onions (Nottingham, GB)
- Jonathan Gareth Williams (Nottingham, GB)
- Kevin Joly (Nottingham, GB)
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61K31/437
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
C07D475/02
CHEMISTRY; METALLURGY
A61K31/4985
HUMAN NECESSITIES
Y10T428/2982
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K9/0075
HUMAN NECESSITIES
International classification
A61K31/4985
HUMAN NECESSITIES
A61K31/437
HUMAN NECESSITIES
C07D475/02
CHEMISTRY; METALLURGY
Abstract
The present invention relates to respiratory formulations comprising a compound of formula (I): ##STR00001##
and use of said compounds and compositions in treatment, for example in the treatment of an inflammatory disease or a respiratory disorder, in particular an inflammatory mediated and/or virally mediated respiratory disorder such as asthma and COPD or the treatment or prevention of viral infection, for example infection by influenza virus, rhinovirus or RSV. The invention also extends to certain novel compounds of formula (I).
Claims
1. An inhaler device comprising a formulation of compound of formula (I): ##STR00108## J represents: ##STR00109## Q is N or CH; X is O or OCH.sub.2; Y is CH or N; Z is O, NR.sup.4, CR.sup.4aN or NCR.sup.4a whereby the latter two groups, together with the atoms to which they are attached, form a 6 membered ring; R.sup.1 is C.sub.1-6 alkyl, branched or unbranched, or C.sub.3-6 cycloalkyl, each optionally substituted by a hydroxyl group; R.sup.2a is H, halo, saturated or unsaturated branched or unbranched C.sub.1-8 alkylene chain, wherein one or more carbons are optionally replaced by a heteroatom(s) independently selected from O, N and/or S(O).sub.m and the chain is optionally substituted by one or more halogen atoms; R.sup.2b is H, halo, C.sub.1-6 alkoxy or C.sub.1-6 alkyl optionally substituted by OH; R.sup.2 and R.sup.3 each independently represent H, C.sub.1-4 alkyl, halogen; C.sub.1-4 haloalkyl, O(C.sub.1-4 alkyl), O(C.sub.1-4haloalkyl), CN, SO.sub.2R.sup.5, C(O)OR.sup.6 with the proviso that R.sup.2 and R.sup.3 do not both represent H concomitantly, or R.sup.2 and R.sup.3 together with the carbons to which they are attached form a six membered aromatic ring or a heteroaromatic ring comprising a nitrogen atom; R.sup.4 is H, C.sub.1-5 alkyl, C.sub.2-5 alkenyl, C.sub.2-5 alkynyl, C.sub.3-6 cycloalkyl, C.sub.3-6 cycloalkenyl, C.sub.6 aryl, a 5 or 6 membered heteroaryl or a 5 or 6 membered heterocyclic group, wherein said aryl, heteroaryl or heterocyclic group is substituted with 0 to 3 substituents selected from halogen, hydroxyl, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl, amino, C.sub.1-4 mono or di-alkyl amino, C.sub.1-4 mono or di-acyl amino, S(O).sub.qC.sub.1-6 alkyl, C.sub.0-6 alkylC(O)C.sub.1-6 alkyl or C.sub.0-6 alkylC(O)C.sub.1-6 heteroalkyl; R.sup.4a is H or C.sub.1-3 alkyl; R.sup.5 is H or C.sub.1-4 alkyl; R.sup.6 is H or C.sub.1-4 alkyl; m is 0, 1 or 2; q is 0, 1 or 2; a pharmaceutically acceptable salt thereof, including all stereoisomers, tautomers and isotopic derivatives thereof in particulate form, wherein the mass mean aerodynamic diameter (MMAD) of the population of particles is in the range 1 to 20 microns.
2. A compound selected from: 1-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)-3-(4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)naphthalen-1-yl)urea; 1-(3-tert-butyl-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)naphthalen-1-yl)urea; 1-(3-tert-butyl-1-phenyl-1H-pyrazol-5-yl)-3-(4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)naphthalen-1-yl)urea; 1-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)-3-(4-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)naphthalen-1-yl)urea; 1-(3-tert-butyl-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)naphthalen-1-yl)urea; 1-(3-tert-butyl-1-phenyl-1H-pyrazol-5-yl)-3-(8-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)quinolin-5-)urea; 1-(3-tent-butyl-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(8-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)quinolin-5-yl)urea; 1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((1-ethyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((1-isopropyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(2,3-dichloro-4-((1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)pheny)urea; 1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(8-((2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)quinolin-5-yl)urea; 1-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(4-((8-oxo-8,9-dihydro-7H-purin-6-yl)oxy) naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-hydroxyphenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-(hydroxymethyl)phenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-(hydroxymethyl)-3-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(2,3-dichloro-4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)phenyl)urea; 1-(3-(tert-Butyl)-1-(6-methoxypyridin-3-yl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(5-methylthiophen-2-yl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((7-oxo-7,8-dihydropteridin-4-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-butyl)-1-(6-methoxypyridin-3-yl)-1H-pyrazol-5-yl)-3-(4-((1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(4-(3-(tert-butyl)-5-(3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)ureido)-1H-pyrazol-1-yl)phenyl)-N-methylmethanesulfonamide; 1-(3-(tert-butyl)-1-(4-chlorophenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-cyclopropyl-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(1-methylcyclopropyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-ylurea; 1-(3-isopropyl-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-butyl)-1-(4-(methylsulfonyl)phenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-butyl)-1-(3,4-dichlorophenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-butyl)-1-(4-(methylthio)phenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; and pharmaceutically acceptable salts of any one thereof, including all stereoisomers, tautomers and isotopic derivatives thereof.
3. A compound of claim 2 selected from: 1-(3-tert-butyl-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)naphthalen-1-yl)urea; 1-(3-tert-butyl-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(8-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yloxy)quinolin-5-yl)urea; 1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((1-ethyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((1-isopropyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(2,3-dichloro-4-((1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)phenyl)urea; 1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(8-((2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)quinolin-5-yl)urea; 1-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(4-((8-oxo-8,9-dihydro-7H-purin-6-yl)oxy) naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-hydroxyphenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-(hydroxymethyl)phenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-(hydroxymethyl)-3-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(2,3-dichloro-4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)phenyl)urea; 1-(3-(tert-Butyl)-1-(6-methoxypyridin-3-yl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(5-methylthiophen-2-yl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((7-oxo-7,8-dihydropteridin-4-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-butyl)-1-(6-methoxypyridin-3-yl)-1H-pyrazol-5-yl)-3-(4-((1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(4-(3-(tert-butyl)-5-(3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)ureido)-1H-pyrazol-1-yl)phenyl)-N-methylmethanesulfonamide; 1-(3-(tert-butyl)-1-(4-chlorophenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-cyclopropyl-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(1-methylcycyclopropyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-ylurea; 1-(3-isopropyl-1-(p-tolyl)-H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-butyl)-1-(4-(methylsulfonyl)phenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-butyl)-1-(3,4-dichlorophenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; 1-(3-(tert-butyl)-1-(4-(methylthio)phenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea; and pharmaceutically acceptable salts of any one thereof, including all stereoisomers, tautomers and isotopic derivatives thereof.
Description
EXPERIMENTAL SECTION
(1) Abbreviations used herein are defined below (Table 1). Any abbreviations not defined are intended to convey their generally accepted meaning.
(2) TABLE-US-00001 TABLE 1 Abbreviations AcOH glacial acetic acid aq aqueous Ac acetyl ATP adenosine-5-triphosphate BALF bronchoalveolae lavage fluid Boc tert-butoxycarbonyl br broad BSA bovine serum albumin CatCart catalytic cartridge CDI 1,1-carbonyl-diimidazole COPD chronic obstructive pulmonary disease d doublet DCM dichloromethane DIAD diisopropylazadicarboxylate DIBAL-H diisobutylaluminium hydride DIPEA N,N-diisopropylethylamine DMF N,N-dimethylformamide DMSO dimethyl sulfoxide d-U937 cells PMA differentiated U-937 cells (ES.sup.+) electrospray ionization, positive mode Et ethyl EtOAc ethyl acetate FCS foetal calf serum FRET fluorescence resonance energy transfer HOBt 1-hydroxybenzotriazole hr hour(s) HRP horseradish peroxidise HRV human rhinovirus ICAM-1 inter-cellular adhesion molecule 1 JNK c-Jun N-terminal kinase LPS lipopolysaccharide (M + H).sup.+ protonated molecular ion MAPK mitogen protein activated protein kinase MAPKAP-K2 mitogen-activated protein kinase-activated protein kinase-2 Me methyl MeCN acetonitrile MeOH methanol MHz megahertz MMAD mass median aerodynamic diameter min minute(s) MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide m/z: mass-to-charge ratio NMM N-methylmorpholine; (4-methylmorpholine) NMP 1-methylpyrrolidin-2-one (N-methyl-2-pyrrolidone) NMR nuclear magnetic resonance (spectroscopy) Ph phenyl PBS phosphate buffered saline PMA phorbol myristate acetate PPh.sub.3 triphenylphosphine q quartet RT room temperature RP HPLC reverse phase high performance liquid chromatography RSV Respiratory syncytical virus s singlet sat saturated SCX solid supported cation exchange (resin) SDS sodium dodecyl sulphate S.sub.NAr nucleophilic aromatic substitution t triplet TBAF tetrabutylammonium fluoride TBDMS-Cl tert-butyldimethylchlorosilane TCID.sub.50 50% tissue culture infectious dose TFA trifluoroacetic acid THF tetrahydrofuran TIPS-Cl chlorotriisopropylsilane TMB 3,3,5,5-tetramethylbenzidine TNF tumor necrosis factor alpha XantPhos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
General Procedures
(3) All starting materials and solvents were obtained either from commercial sources or prepared according to the literature citation. Unless otherwise stated all reactions were stirred. Organic solutions were routinely dried over anhydrous magnesium sulfate. Hydrogenations were preformed on a Thales H-cube flow reactor under the conditions stated.
(4) Column chromatography was performed on pre-packed silica (230-400 mesh, 40-63 m) cartridges using the amount indicated. SCX was purchased from Supelco and treated with 1M hydrochloric acid prior to use. Unless stated otherwise the reaction mixture to be purified was first diluted with MeOH and made acidic with a few drops of AcOH. This solution was loaded directly onto the SCX and washed with MeOH. The desired material was then eluted by washing with 1% NH.sub.3 in MeOH.
(5) Preparative Reverse Phase High Performance Liquid Chromatography: Agilent Scalar column C18, 5 m (21.250 mm), flow rate 28 mL min.sup.1 eluting with a H.sub.2O-MeCN gradient containing 0.1% v/v formic acid over 10 min using UV detection at 215 and 254 nm. Gradient information: 0.0-0.5 min; 95% H.sub.2O-5% MeCN; 0.5-7.0 min; ramped from 95% H.sub.2O-5% MeCN to 5% H.sub.2O-95% MeCN; 7.0-7.9 min; held at 5% H.sub.2O-95% MeCN; 7.9-8.0 min; returned to 95% H.sub.2O-5% MeCN; 8.0-10.0 min; held at 95% H.sub.2O-5% MeCN.
(6) Analytical Methods
(7) Reverse Phase High Performance Liquid Chromatography: Method 1): Agilent Scalar column C18, 5 m (4.650 mm) or Waters XBridge C18, 5 m (4.650 mm) flow rate 2.5 mL min.sup.1 eluting with a H.sub.2O-MeCN gradient containing either 0.1% v/v formic acid (Method 1 acidic) or NH.sub.3 (Method 1 basic) over 7 min employing UV detection at 215 and 254 nm. Gradient information: 0.0-0.1 min, 95% H.sub.2O-5% MeCN; 0.1-5.0 min, ramped from 95% H.sub.2O-5% MeCN to 5% H.sub.2O-95% MeCN; 5.0-5.5 min, held at 5% H.sub.2O-95% MeCN; 5.5-5.6 min, held at 5% H.sub.2O-95% MeCN, flow rate increased to 3.5 mL min.sup.1; 5.6-6.6 min, held at 5% H.sub.2O-95% MeCN, flow rate 3.5 mL min-; 6.6-6.75 min, returned to 95% H.sub.2O-5% MeCN, flow rate 3.5 mL min.sup.1; 6.75-6.9 min, held at 95% H.sub.2O-5% MeCN, flow rate 3.5 mL.Math.min.sup.1; 6.9-7.0 min, held at 95% H.sub.2O-5% MeCN, flow rate reduced to 2.5 mL min.sup.1.
(8) Reverse Phase High Performance Liquid Chromatography: Method 2: Agilent Extend C18 column, 1.8 m (4.630 mm) at 40 C.; flow rate 2.5-4.5 mL min.sup.1 eluting with a H.sub.2O-MeCN gradient containing 0.1% v/v formic acid over 4 min employing UV detection at 254 nm. Gradient information: 0-3.00 min, ramped from 95% H.sub.2O-5% MeCN to 5% H.sub.2O-95% MeCN; 3.00-3.01 min, held at 5% H.sub.2O-95% MeCN, flow rate increased to 4.5 mL min.sup.; 3.01 3.50 min, held at 5% H.sub.2O-95% MeCN; 3.50-3.60 min, returned to 95% H.sub.2O-5% MeCN, flow rate reduced to 3.50 mL min.sup.; 3.60-3.90 min, held at 95% H.sub.2O-5% MeCN; 3.90-4.00 min, held at 95% H.sub.2O-5% MeCN, flow rate reduced to 2.5 mL min.sup..
(9) .sup.1H NMR Spectroscopy: .sup.1H NMR spectra were acquired on a Bruker Avance III spectrometer at 400 MHz using residual undeuterated solvent as reference.
(10) Generic Routes to Compound Examples.
(11) 1. Compound Examples that Contain a 5-6 Ring System (ZNR.sup.4)
(12) Compounds examples that comprise of a naphthalene core (R.sup.2 and R.sup.3 and the carbon atoms to which they are attached comprise a fused phenyl ring) or dichlorophenyl core (R.sup.2 and R.sup.3Cl) were prepared by a final coupling reaction, to generate the central urea, between compounds represented by Intermediate A with compounds represented by Intermediate B.
(13) ##STR00029##
(14) For quinoline analogues, (R.sup.2 and R.sup.3 and the carbon atoms to which they are attached comprise a fused pyridine ring) compound examples were prepared by the formation of the terminal imidazopyridinone ring system (YCH) from compounds represented by Intermediate C by reduction of the nitroarene, followed by cyclisation of the resulting diamine.
(15) ##STR00030##
2. Compound Examples that Contain a 6-6 Ring System (ZNCR.sup.4)
(16) Compounds examples that comprise of a naphthalene core (R.sup.2 and R.sup.3 and the carbon atoms to which they are attached comprise a fused phenyl ring) or a dichlorophenyl core (R.sup.2 and R.sup.3 Cl) were prepared by a final coupling reaction, to generate the central urea, between compounds represented by Intermediate A with compounds represented by Intermediate D.
(17) ##STR00031##
3. The Preparation of Generic Compound Intermediates
(18) Compounds represented by Intermediate B (ZNR.sup.4) were obtained from compounds represented by Intermediate E by chemoselective N-alkylation of the carbamate nitrogen followed by base induced cyclisation to generate the imidazolone ring system. Examples of Intermediate B that contain an unsubstituted imidazolone ring (R.sup.4H), were prepared using an alternative method, from compounds represented by Intermediate F, by cyclisation of the aryl 1,2-diamine with an electrophilic carbonyl equivalent such as CDI.
(19) ##STR00032##
(20) Compounds represented by Intermediate F were transformed into compounds represented by Intermediate E by selective acylation with an alkyl chloroformate, followed by removal of the protective group [P.sup.1]. An example of a suitable protective group for this series of transformations is a tert-butyloxycarbonyl (Boc) group which may be removed by acidolysis For those cases in which Intermediate B comprises of an electron poor aniline (for example when R.sup.2R.sup.3Cl), the requisite precursors represented by Intermediate E were prepared by the direct regioselctive acylation of a diaryl triamine represented by Intermediate G.
(21) Compounds represented by Intermediate C are accessible from precursors represented by Intermediate H by a urea coupling reaction with an compound represented by Intermediate A.
(22) ##STR00033##
(23) Structures represented by Intermediate D, required for the preparation of compound examples of the disclosure that incorporate a pyridopyrazinone (YCH) or a pteridinone (YN) ring system were derived from compounds represented by Intermediate F by condensation with an -carbonyl ester, followed by deprotection of the anilino nitrogen. For example, in those cases wherein R.sup.4a=Me the -carbonyl ester is a pyruvate ester such as ethyl pyruvate. Alternatively, those examples of Intermediate D that comprise of an electron
(24) ##STR00034##
poor aniline (for example when R.sup.2R.sup.3Cl), and for those examples of Intermediate D for which X is represented by OCH.sub.2 may be prepared by the condensation of an -carbonyl ester with compounds represented by Intermediate G in which selective protection/deprotection of the anilino nitrogen is not required. For example, in those cases wherein R.sup.4aH the -carbonyl ester is a glyoxalate ester such as ethyl glyoxalate. An example of a suitable protective group for this series of transformations is the Boc group which may be removed by exposure to fluoride, for example by treatment with TBAF.
(25) Compounds represented by Intermediate F and Intermediate G were obtained by reduction of nitroarenes represented by Intermediate J. In the former case a nitrogen protective group [P.sup.1] is selected that is orthogonal, that is, one which is stable and not removed under the conditions used to affect the reduction step. An example of a reductive process that is suitable for this transformation is catalytic hydrogenation and an example of a suitable protective group which is orthogonal to these conditions and may be conveniently selected is a Boc group. In the second instance it may be advantageous to employ a protective group [P.sup.1] that is labile under the conditions used to reduce the nitroarene. An example of a
(26) ##STR00035##
reductive procedure that is suitable for this conversion is a dissolving metal reduction, such as iron in hydrochloric acid and an example of a protective group which is labile and may be conveniently selected for this process is an aryldiazene such as a phenyl diazene. [i.e. P.sup.1PhN]. In some instances (for example when R.sup.2R.sup.3Cl) no protective group may be required [i.e. P.sup.1H]
(27) Compounds represented by Intermediate J were assembled from suitably protected aryl amine derivatives by S.sub.NAr reactions with activated halopyridines and halopyrimidines under basic conditions. These starting materials are either commercially available or are known compounds or may be prepared from readily available precursors by standard procedures, known in the art. In some instances, for example, where the aniline component is electron deficient (for example when R.sup.2R.sup.3Cl) no nitrogen protective group may be required [i.e. P.sup.1H] to maintain chemoselectivity during the S.sub.NAr coupling step.
(28) ##STR00036##
(29) Compounds represented by Intermediates H were similarly obtained by SNAr condensation of 4-chloro-3-nitropyridin-2-amine with an aminoquinoline nucleophile under basic conditions.
(30) ##STR00037##
INTERMEDIATES TO COMPOUND EXAMPLES
(31) The following Intermediates used to prepare compound examples of the disclosure have been previously described or are available from commercial sources and were either purchased or prepared using the procedures in the references cited below (Table 2).
(32) TABLE-US-00002 TABLE 2 Commercially Available and Previously Described Intermediates. Intermediate Structure Name, LCMS Data and Reference A1
(33) Additional intermediates that were required for the synthesis of compound examples of the disclosure were prepared as described below.
Intermediate A12
3-(tert-Butyl)-1-(4-(((triisopropylsilyl)oxy)methyl)phenyl)-1H-pyrazol-5-amine
(34) ##STR00053##
(35) To a solution of (4-(5-amino-3-tert-butyl-1H-pyrazol-1-yl)phenyl)methanol (Ito, K. et al., WO 2010/067131) (8.50 g, 35.0 mmol) and 1H-imidazole (2.71 g, 39.8 mmol) in dry THF (100 mL) under N.sub.2 was added chlorotriisopropylsilane (8.16 mL, 38.1 mmol) and the reaction mixture maintained at RT for 5 days. The resulting mixture was evaporated in vacuo and the residue was partitioned between EtOAc (250 mL) and water (100 mL). The organic layer was separated and was washed with (water (100 mL) and brine (80 mL) and then dried and evaporated in vacuo to afford the title compound, Intermediate A12 as a dark red solid (14.6 g, 97%); R.sup.t 3.26 min (Method 2); m/z 402 (M+H).sup.+, (ES.sup.+).
Intermediate A13
1-(4-(5-Amino-3-tert-butyl-1H-pyrazol-1-yl)phenyl)-N-methyl methanesulfonamide
(36) ##STR00054##
(37) To a suspension of 1-(4-aminophenyl)-N-methylmethanesulfonamide (500 mg, 2.50 mmol) in hydrochloric acid (6 M, 10 mL) at 0 C. was added a solution of sodium nitrite (181 mg, 2.62 mmol) in water (3.0 mL) over 5 min. The reaction mixture was maintained at 0 C. for 2.5 hr, was treated with tin(II) chloride (1.33 g, 6.99 mmol) in hydrochloric acid (6M, 15 mL) and was then warmed to RT for 64 hr. The resulting mixture was basified to pH 12 with aq NaOH (6.0 M) and was extracted with EtOAc (2100 mL). The combined organic extracts were washed with water and then dried and evaporated in vacuo to give a residue that was suspended in ethanol (10 mL) and treated with 4,4-dimethyl-3-oxopentanenitrile (160 mg, 1.3 mmol) and conc hydrochloric acid (12 M, 106 L, 1.27 mmol). The resulting mixture was heated at reflux for 5 hr and after a further 23 hr at RT was evaporated in vacuo, to remove the ethanol. The resulting aq mixture was basified with aq NaOH (2.0 M) and was then extracted with DCM (230 mL). The combined organic extracts were dried and evaporated in vacuo to afford the title compound, Intermediate A13, as an orange solid (146 mg, 85% purity, 15%); R.sup.t 1.34 min (Method 2); m/z 323 (M+H).sup.+, (ES.sup.+). This material was used in subsequent steps without further purification.
Intermediate A14
3-tert-Butyl-1-(4-methylthiophen-2-yl)-1H-pyrazol-5-amine
(38) ##STR00055##
(39) To a solution of 2-iodo-4-methylthiophene (WO 2008/121666) (1.00 g, 4.50 mmol) in anhydrous toluene (15.0 mL) was added 3-tert-butyl-1H-pyrazol-5-amine (683 mg, 4.91 mmol) followed by (1R,2R)N.sup.1,N.sup.2-dimethylcyclohexane-1,2-diamine (140 L, 0.89 mmol) and potassium carbonate (1.30 g, 9.37 mmol). The mixture was purged with N.sub.2, copper(I) iodide (42 mg, 0.22 mmol) was added and the reaction mixture was heated at reflux under N.sub.2 for 16 hr during which time most of the solvent was lost. The resulting mixture was partitioned between ethyl acetate (150 mL) and water (150 mL) and the organic layer was separated and extracted with aq. citric acid solution (10% w/v, 150 mL) followed by brine (50 mL), and then dried and evaporated in vacuo. The residue was purified by flash column chromatography (SiO.sub.2, 120 g, DCM, isocratic elution) to afford the title compound, Intermediate A14, as a brown solid (340 mg, 32%); R.sup.t 1.94 min (Method 2); m/z 236 (M+H).sup.+ (ES.sup.+).
Intermediate A15
3-Cyclopropyl-1-p-tolyl-1H-pyrazol-5-amine
(40) ##STR00056##
(41) To a solution of acetonitrile (500 L, 390 mg, 9.6 mmol) in THF (30 mL) at RT was added a solution of potassium 2-methylbutan-2-olate in toluene (1.7 M, 17.0 mL, 29.0 mmol) followed by the dropwise addition of ethyl cyclopropanecarboxylate (4.56 mL, 4.37 g, 38.0 mmol) and the reaction mixture maintained at RT for 16 hr. The resulting mixture was concentrated in vacuo to a volume of 15 mL and was then diluted with EtOH (20 mL) and p-tolylhydrazine hydrochloride (1.52 g, 9.57 mmol) was added. The mixture was acidified to pH 1 by the addition of conc hydrochloric acid and the resulting heterogeneous mixture was heated to 70 C. for 2 hr, then cooled to RT and concentrated in vacuo to a volume of 20 mL. The mixture was diluted with water (30 mL) and was adjusted to pH 12 by the addition of 2M aq NaOH and extracted with diethyl ether (220 mL). The combined organic extracts were washed with brine (30 mL) and then dried and evaporated in vacuo. The residue was triturated with isohexane (20 mL) to afford the title compound, Intermediate A15, as a beige solid (1.60 g, 77%); R.sup.t 3.35 min (Method 1 basic); m/z 214 (M+H).sup.+, (ES.sup.+).
Intermediate A16
3-tert-Butyl-1-(3-methoxy-4-((triisopropylsilyloxy)methyl)phenyl)-1H-pyrazol-5-amine
(42) ##STR00057##
(43) To a suspension of 4-amino-2-methoxybenzoic acid (3.00 g, 18.0 mmol) in hydrochoric acid (1.0 M, 30 mL) at 0 C. was added a solution of sodium nitrite (1.28 g, 18.5 mmol) in water (5.0 mL) over 5 min. The reaction mixture was maintained at 0 C. for 5 hr and was then treated with tin(II) chloride (9.53 g, 50.3 mmol) in hydrochloric acid (1M, 60 mL) and warmed to RT for 64 hr. A thick precipitate formed which was isolated by filtration, washed with water (50 mL) and ether (15 mL) and dried in vacuo to afford 2-methoxy-4-hydrazinylbenzoic acid hydrochloride as a beige solid (1.05 g, 90% purity, 24%); R.sup.t 0.17 min (Method 2); m/z 181 (M+H).sup.+, (ES.sup.+). This material was used in the subsequent step without further purification.
(44) A suspension of 2-methoxy-4-hydrazinylbenzoic acid hydrochloride (1.05 g, 90% purity, 4.34 mmol) and 4,4-dimethyl-3-oxopentanenitrile (664 mg, 5.31 mmol) in ethanol (20 mL) containing conc hydrochloric acid (12 M, 0.44 mL, 5.0 mmol) was heated at reflux for 28 hr and then cooled to RT. The reaction mixture was treated with aq NaOH (2.0 M, 8.6 mL, 17 mmol) and was maintained at RT for 20 hr and then concentrated to half its original volume in vacuo. Then residue was washed with DCM (215 mL) and the aq layer was acidified to pH 5 with conc hydrochloric acid (12 M, 1.0 mL) and re-extracted with DCM (530 mL). The combined organic extracts were washed with brine (10 mL) and then dried and evaporated in vacuo to afford 4-(5-amino-3-tert-butyl-1H-pyrazol-1-yl)-2-methoxybenzoic acid as a brown solid (900 mg, 61%); R.sup.t 1.47 min (Method 2); m/z 290 (M+H).sup.+, (ES.sup.+).
(45) To a solution of 4-(5-amino-3-(tert-butyl)-1H-pyrazol-1-yl)-2-methoxybenzoic acid (900 mg, 3.11 mmol) in THF (6.0 mL) at 0 C. under N.sub.2 was added a solution of borane in THF (1.0 M 8.0 mL, 8.0 mmol) over 5 min and the reaction mixture warmed to RT. Additional aliquots of the borane solution were added after 22 hr (1.0 mL, 1.0 mmol) and 27 hr (1.7 mL, 1.7 mmol) and after 46 hr the reaction was quenched with MeOH (30 mL), kept at RT for 10 min and then evaporated in vacuo. The residue was partitioned between DCM (20 mL) and brine (10 mL) and the aq layer was separated and extracted with DCM (10 mL). The combined organic layers were dried and evaporated in vacuo to afford 3-tert-butyl-1-(3-chloro-4-(hydroxy methyl)phenyl)-1H-pyrazol-5-amine as a dark orange oil (840 mg, 90% purity, 88%); R.sup.t 3.62 min (Method 1 basic); m/z 276 (M+H).sup.+, (ES.sup.+). This material was used in the subsequent step without further purification
(46) To a solution of 3-tert-butyl-1-(3-chloro-4-(hydroxymethyl)phenyl)-1H-pyrazol-5-amine (840 mg, 90% purity, 2.75 mmol) in dry THF (10 mL) under N.sub.2 was added imidazole (240 mg, 3.5 mmol) and chlorotriisopropylsilane (720 L, 650 mg, 3.40 mmol) and the reaction mixture kept at RT for 18 hr. The resulting mixture was diluted with DCM (5.0 mL) and was treated with a second aliquot of chlorotriisopropylsilane (720 L, 650 mg, 3.40 mmol) and after a further 5 hr with additional imidazole (240 mg, 3.50 mmol). After 20 hr the reaction mixture was treated for a third time with chlorotriisopropylsilane (360 L, 330 mg, 1.70 mmol) and imidazole (120 mg, 1.8 mmol) and after 3 days was evaporated in vacuo. The residue was taken up into EtOAc (50 mL) and was washed with water (20 mL) and brine (10 mL) and then dried and evaporated in vacuo. The residue was purified by flash column chromatography (SiO.sub.2, 40 g, EtOAc in isohexane, 0-100%, gradient elution) and then by SCX capture and release to afford a brown oil which comprised of a mixture of starting material and the desired product. This mixture was taken up into THF (1.0 mL) and was treated with chlorotriisopropylsilane (200 L, 200 mg, 0.30 mmol) and imidazole (75 mg, 1.1 mmol) and was maintained at RT for 24 hr and then evaporated in vacuo. The residue was partitioned between EtOAc (10 mL) and water (10 mL) and the organic phase was separated and extracted brine (10 mL) and then dried and evaporated in vacuo. The residue was purified by flash column chromatography (SiO.sub.2, 40 g, EtOAc in isohexane, 0-100%, gradient elution) to afford the title compound, Intermediate A16 as a yellow oil (510 mg, 37%); R.sup.t 5.74 min (Method 1 basic); m/z 432 (M+H).sup.+, (ES.sup.+).
Intermediate B3
7-((4-Aminonaphthalen-1-yl)oxy)-1-ethyl-1H-imidazo[4,5-b]pyridin-2(3H)-one
(47) ##STR00058##
(48) To a solution of Intermediate E1 (500 mg, 1.48 mmol) in DMF (10 mL) at 0 C. under N.sub.2 was added sodium hydride (60% wt in mineral oil, 59 mg, 1.5 mmol) and after 1 hr the mixture was treated drop-wise, over 1 hr, with a solution of iodoethane (120 L, 230 mg, 1.5 mmol) in DMF (1.5 mL). The resulting mixture was warmed to RT and after 2 hr was re-cooled to 0 C., and sodium hydride (60% wt in mineral oil, 120 mg, 3.0 mmol) was added. The reaction mixture was warmed to RT for 1 hr and was then poured onto ice-water (100 mL). The mixture was extracted with EtOAc (360 mL) and the combined organic extracts were washed with brine (350 mL) and then dried and evaporated in vacuo to afford a brown oil. This residue was triturated with ether and the resulting solid was isolated by filtration and washed with ether to afford Intermediate B3 as a pale brown solid (332 mg, 89% purity by HPLC, 63%); R.sup.t 1.50 min (Method 2, 89% pure); m/z 321 (M+H).sup.+ (ES.sup.+).
Intermediate B4
7-((4-Aminonaphthalen-1-yl)oxy)-1-isopropyl-1H-imidazo[4,5-b]pyridin-2(3H)-one
(49) ##STR00059##
(50) To a solution of Intermediate E1 (500 mg, 1.48 mmol) in DMF (10 mL) at 0 C. under N.sub.2 was added sodium hydride (60% wt in mineral oil, 59 mg, 1.5 mmol) and after 1 hr the resulting mixture was treated drop-wise over 1 hr with a solution of 2-bromopropane (140 L, 180 mg, 1.5 mmol) in DMF (1.5 mL). The mixture was warmed to RT for 48 hr and was then heated to 80 C. and treated with a second aliquot of 2-bromopropane (33 L, 42 mg, 0.35 mmol) in DMF (0.5 mL), followed immediately by sodium hydride (60% wt in mineral oil, 14 mg, 0.35 mmol). The reaction mixture was maintained at 80 C. for 1 hr and was then cooled to RT. After a further 4 days sodium hydride (60% wt in mineral oil, 120 mg, 3.0 mmol) was added and the reaction mixture was maintained at RT for 1 hr and then poured onto ice-water (100 mL). The mixture was extracted with EtOAc (360 mL) and the combined organic extracts were washed with brine (350 mL), dried and evaporated in vacuo to afford a brown oil. This residue was triturated with ether and the resulting solid was isolated by filtration and washed with ether to afford Intermediate B4 as a pale brown solid (223 mg, 81% purity by HPLC, 36%); R.sup.t 1.66 min (Method 2, 81% pure); m/z 335 (M+H).sup.+ (ES.sup.+).
Intermediate B5
7-(4-Amino-2,3-dichlorophenoxy)-1-methyl-1H-imidazo[4,5-b]pyridin-2(3H)-one
(51) ##STR00060##
(52) To a solution of Intermediate E2 (230 mg, 0.640 mmol) in dry DMF (4.0 mL) at 0 C. under N.sub.2 was added sodium hydride (60% dispersion in mineral oil, 26 mg, 0.64 mmol) portionwise, over 2 min. After 45 min at 0 C. the reaction mixture was treated dropwise, over 2 min, with iodomethane (40 L, 0.64 mmol) in DMF (1.0 mL) and was then warmed to RT for 6.5 hr and treated with a second aliquot of iodomethane (8 L, 0.1 mmol) in dry DMF (0.1 mL). After 3 days the reaction mixture was re-cooled to 0 C. and sodium hydride (60% dispersion in mineral oil, 52 mg, 1.3 mmol) was added portionwise over 2 min. The resulting mixture was warmed to RT for 22 hr and was then partitioned between EtOAc (30 mL), and a mixture of water (30 mL) and sat brine (15 mL). The aq layer was separated and was extracted with EtOAc (325 mL) and the combined organic extracts were dried and evaporated in vacuo. The residue was purified by flash column chromatography (SiO.sub.2, 40 g, MeOH in DCM, 0-8%, gradient elution) to afford Intermediate B5 as a brown solid (11 mg, 5%); R.sup.t 1.64 min (Method 2); m/z 325/327 (M+H).sup.+ (ES.sup.+).
Intermediate B6
6-((4-Aminonaphthalen-1-yl)oxy)-7H-purin-8(9H)-one
(53) ##STR00061##
(54) To a solution of Intermediate F1 (330 mg, 56% pure, 0.50 mmol) in DMF (6.0 mL) at RT under N.sub.2 was added CDI (168 mg, 1.04 mmol) and the reaction mixture maintained at RT and treated with additional portions of CDI (168 mg, 1.04 mmol) after 4.5 hr, after 22.5 hr and after 27 hr. After a total of 48 hr the resulting mixture was partitioned between EtOAc (35 mL) and sat aq. NaHCO.sub.3 (35 mL). The aq layer was separated and extracted with EtOAc (435 mL) and the combined organic extracts were washed with brine (30 mL) and then dried and evaporated in vacuo. The residue was purified by flash column chromatography (SiO.sub.2, 40 g, [5% MeOH in EtOAc] in isohexane, 0-100%, gradient elution) to afford tert-butyl (4-((8-oxo-8,9-dihydro-7H-purin-6-yl)oxy)naphthalen-1-yl)carbamate as a pale brown solid (119 mg, 59%); R.sup.t 1.81 min (Method 2); m/z 394 (M+H).sup.+ (ES.sup.+).
(55) To a solution of the product obtained above (119 mg, 0.302 mmol) in DCM (6.0 mL) at 0 C. under N.sub.2 was added TFA (2.0 mL, 30 mmol) and the reaction mixture warmed to RT for 3 hr and then evaporated in vacuo. The residue was purified by SCX capture and release to afford Intermediate B6; R.sup.t 0.94 min (Method 2, 89% pure); m/z 294 (M+H).sup.+ (ES.sup.+). This material was used as obtained, without further purification.
Intermediate C1
1-(8-((2-Amino-3-nitropyridin-4-yl)oxy)quinolin-5-yl)-3-(3-(tert-butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)urea
(56) ##STR00062##
(57) To a solution of CDI (500 mg, 3.00 mmol) in dry DCM (6.0 mL) at RT was added Intermediate A2 (760 mg, 3.10 mmol) and the mixture kept at RT for 4 hr and then treated with a solution of Intermediate H1 (600 mg, 2.00 mmol) in dry THF (5.0 mL). After 2 hr the reaction was quenched with MeOH (3.0 mL) for 5 min at RT and was then evaporated in vacuo. The residue was triturated with water (50 mL) and then purified by flash column chromatography (SiO.sub.2, 80 g, MeOH in DCM, 0-5%, gradient elution) to afford Intermediate C1 as a yellow solid (520 g, 45%); R.sup.t 2.12 min (Method 2); m/z 569 (M+H).sup.+, (ES.sup.+);
Intermediate D2
8-(4-Amino-2,3-dichlorophenoxy)pyrido[2,3-b]pyrazin-3(4H)-one
(58) ##STR00063##
(59) To a solution of Intermediate G1 (440 mg, 1.54 mmol) in EtOH (10 mL) under N.sub.2 at reflux was added ethyl glyoxolate (0.5 M in toluene, 400 L, 2.0 mmol). The reaction mixture was maintained at reflux for 1.5 hr and was then cooled to RT, during which time a precipitate formed. This precipitate was isolated by filtration and washed with isohexane to afford the title compound, Intermediate D2 as a brick red solid (128 mg, 92% purity, 24%); R.sup.t 1.72 min (Method 2, 92% pure); m/z 323 (M+H).sup.+ (ES.sup.+).
Intermediate D3
4-((4-aminonaphthalen-1-yl)oxy)pteridin-7(8H)-one
(60) ##STR00064##
(61) To a solution of the Intermediate F1 (6.9 g, 19 mmol) in EtOH (6.0 mL) at RT under N.sub.2 was added ethyl 2-oxoacetate (50% solution in toluene, 5.6 mL, 28 mmol) and the reaction mixture maintained at RT for 16 hr and then heated at reflux for 5 hr, during which time a precipitate formed. The reaction mixture was cooled to RT and the precipitate was isolated by filtration to afford tert-butyl 4-(7-oxo-7,8-dihydropteridin-4-yloxy)naphthalen-1-ylcarbamate as a pale pink solid (3.5 g, 42%); R.sup.t 1.90 min (Method 2); m/z 406 (M+H).sup.+ (ES.sup.+).
(62) To a solution of the product obtained above (50 mg, 0.12 mmol) in DCM (1.0 mL) at RT was added TFA (0.47 mL, 0.70 g, 6.2 mmol). After 30 min at RT the reaction mixture was evaporated in vacuo to afford Intermediate D3 as a dark brown oil (90 mg, 80% purity by HPLC, >100%); R.sup.t 1.10 min (Method 2); m/z 306 (M+H).sup.+ (ES.sup.+). This material was used in subsequent steps without further purification.
Intermediate D4
8-((4-aminonaphthalen-1-yloxy)methyl)pyrido[2,3-b]pyrazin-3(4H)-one
(63) ##STR00065##
(64) To a solution of Intermediate G2 (170 mg, 0.56 mmol) in EtOH (5.0 mL) at reflux was added ethyl 2-oxoacetate (50% v/v in toluene, 190 L, 0.97 mmol) and the reaction mixture was maintained at 80 C. for 30 min during which time a brown precipitate formed. The reaction mixture was cooled to RT and the solid was isolated by filtration, triturated with methanol and purified by flash column chromatography (SiO.sub.2, 40 g, 5% MeOH in DCM, isocratic elution). The crude product so obtained was triturated with MeOH, washed with ether and then suspended in THF (3.0 mL). Hydrochloric acid (1M, 0.4 mL, 0.4 mmol) was added and the mixture was sonicated for 5 min during which time a white precipitate formed. The solid which was collected by filtration and was washed with THF (21.0 mL) to afford the title compound, Intermediate D4 as a pale yellow solid (7.5 mg, 85% purity by HPLC, 4%); R.sup.t1.14 min (Method 2 basic); m/z 317 (MH).sup., (ES.sup.).
Intermediate E2
Ethyl (2-amino-4-(4-amino-2,3-dichlorophenoxy)pyridin-3-yl) carbamate
(65) ##STR00066##
(66) To a solution of Intermediate G1 (440 mg, 1.54 mmol) and pyridine (250 L, 3.00 mmol) in THF (20 mL) at 0 C. under N.sub.2 was added ethyl chloroformate (1 M solution in THF, 1.62 mL, 1.62 mmol) over 5 min. The reaction mixture was maintained at 0 C. for 1.5 hr and was then partitioned between EtOAc (50 mL) and sat aq. NaHCO.sub.3 (40 mL). The aq layer was separated and was extracted with EtOAc (50 mL) and the combined organic extracts were dried and evaporated in vacuo. The residue was purified by flash column chromatography (SiO.sub.2, 40 g, MeOH in DCM, 0-10%, gradient elution) to afford Intermediate E2 as a pale purple solid (235 mg, 90% purity by HPLC, 38%); R.sup.t 1.36 min (Method 2); m/z 357 (M+H).sup.+(ES.sup.+)
Intermediate F1
6-((4-Aminonaphthalen-1-yl)oxy)-7H-purin-8(9H)-one
(67) ##STR00067##
(68) To a solution of Intermediate J1 (8.02 g, 21.8 mmol) in AcOH (100 mL). was added iron powder (6.75 g, 121 mmol) and the resulting mixture was heated to 50 C. for 1 hr and was then filtered through a pad of celite. The pad was washed with EtOAc/THF (3:1 v/v, 500 mL), and the filtrate and washings were combined and evaporated in vacuo. The residue was partitioned between EtOAc/THF (3:1 v/v, 400 mL) and water (300 mL). The aq layer was separated and adjusted to pH8 with solid Na.sub.2CO.sub.3) and was then extracted with EtOAc/THF (3:1 v/v, 200 mL). The combined organic extracts were washed with water (300 mL) and brine (300 mL) and then dried (Na.sub.2SO.sub.4) and evaporated in vacuo to afford Intermediate F1 as a brown solid (6.9 g, 93%); R.sup.t 1.59 min (Method 2); m/z 368 (M+H).sup.+ (ES.sup.+).
Intermediate G1
4-(4-Amino-2,3-dichlorophenoxy)pyridine-2,3-diamine
(69) ##STR00068##
(70) To a stirred solution of 4-amino-2,3-dichlorophenol (1.00 g, 5.62 mmol) in dry DMF (20 mL) under N.sub.2 was added K.sub.2CO.sub.3 (854 mg, 6.18 mmol) and the mixture kept at RT for 10 min and then treated with a solution of 4-chloro-3-nitropyridin-2-amine (1.024 g, 5.90 mmol) in dry DMF (5.0 mL) in a single portion. The reaction mixture was maintained at RT for 19 hr and was then partitioned between EtOAc (100 mL) and water (100 mL). The aq layer was separated and was extracted with EtOAc (470 mL) and the combined organic extracts were dried and evaporated in vacuo. The residue was purified by flash column chromatography (SiO.sub.2, 80 g, EtOAc in isohexane, 0-70%, gradient elution) to afford 4-(4-amino-2,3-dichlorophenoxy)-3-nitropyridin-2-amine as a dark solid (1.28 g, 70%); R.sup.t 1.85 min (Method 2); m/z 315 (M+H).sup.+ (ES.sup.+).
(71) A suspension of the nitroarene obtained above (1.28 g, 4.06 mmol) and iron powder (1.36 g, 24.4 mmol) in AcOH (200 mL) was heated to 55 C. for 1.5 hr and was then cooled to RT and diluted with EtOAc (20 mL). The mixture was filtered through a pad of celite, which was then washed with EtOAc and with THF. The filtrate and washings were combined and evaporated in vacuo and the residue was partitioned between sat aq NaHCO.sub.3 and EtOAc/THF (3:1 v/v, 100 mL). The aq layer was separated and extracted with EtOAc/THF (3:1 v/v, 280 mL) and the combined organic extracts were dried and evaporated in vacuo to afford Intermediate G1 as a dark purple oil (886 mg, 75%); R.sup.t 0.99 min (Method 2); m/z 285 (M+H).sup.+ (ES.sup.+).
Intermediate G2
4-((4-aminonaphthalen-1-yloxy)methyl)pyridine-2,3-diamine
(72) ##STR00069##
(73) To a solution of 4-methyl-3-nitropyridin-2-amine (10.0 g, 65.0 mmol) in a mixture of dioxane (400 mL) and water (50 mL) was added selenium dioxide (29.0 g, 260 mmol) and the reaction mixture was heated to 100 C. for 16 hr and then cooled to RT and filtered through a pad of celite. The pad was washed with a mixture of EtOAc/THF (3:1 v/v, 100 mL) and the combined filtrate and washings were evaporated in vacuo. The residue was partitioned between EtOAc/THF (3:1 v/v, 100 mL) and sat aq. NaHCO.sub.3 (150 mL) diluted with water (150 mL). The aq layer was separated and was extracted with EtOAc/THF (3:1 v/v, 4100 mL) and the combined organic extracts were dried and evaporated in vacuo to afford 2-amino-3-nitroisonicotinaldehyde as a brown solid (2.73 g, 25%); R.sup.t 0.75 min (Method 2); m/z 168 (M+H).sup.+, (ES.sup.+).
(74) To a suspension of the aldehyde obtained above (850 mg, 5.1 mmol) in MeOH (25 mL) at 0 C. was added sodium tetrahydroborate (190 mg, 5.10 mmol) and the reaction mixture warmed to RT for 2 hr and then quenched by the addition of hydrochloric acid (1 M, 5.0 mL) The mixture was diluted with sat aq. NaHCO.sub.3 (40 mL) and was extracted with DCM (80 mL, 6100 mL). The combined organic extracts were dried and evaporated in vacuo to afford (2-amino-3-nitropyridin-4-yl)methanol as an orange solid (630 mg, 69%); R.sup.t 0.40 min (Method 2); m/z 170 (M+H).sup.+, (ES.sup.+).
(75) To a suspension of the alcohol obtained above (500 mg, 2.70 mmol), 4-nitronaphthalen-1-ol (500 mg, 2.70 mmol) and triphenylphosphine (980 mg, 3.70 mmol) in THF (5.0 mL) at 78 C. was added DIAD (780 L, 810 mg, 4.00 mmol). Upon completion of the addition the reaction mixture was warmed to RT for 3 hr and was then quenched by the addition of methanol and evaporated in vacuo. The residue was triturated with MeOH to afford 3-nitro-4-(((4-nitronaphthalen-1-yl)oxy)methyl)pyridin-2-amine as a yellow solid (170 mg, 19%); R.sup.t 2.30 min (Method 2); m/z 341 (M+H).sup.+, (ES.sup.+).
(76) A suspension of the nitroarene obtained above (170 mg, 0.51 mmol) and iron powder (170 mg, 3.0 mmol) in AcOH (3.0 mL) was heated to 60 C. for 3 hr. An additional portion of iron powder (0.85 g, 1.5 mmol) was added and the mixture heated for a further 2 hr and then filtered through a pad of celite. The celite pad was washed with EtOAc/THF (3:1 v/v, 50 mL) and the filtrate and washings were combined and evaporated in vacuo. The residue was co-evaporated with toluene in vacuo and the residue was partitioned between EtOAc/THF (3:1 v/v, 100 mL) and water. Solid Na.sub.2CO.sub.3 was added to the mixture until the aq layer was basic (universal indicator paper). The layers were separated and the aq layer was extracted with EtOAc/THF (3:1 v/v, 250 mL). The organic extracts were combined, washed with brine (250 mL) and then dried and evaporated in vacuo to afford Intermediate G2 as a brown/black solid (140 mg, 85% purity by HPLC, 85%). R.sup.t 0.81 min (Method 2); m/z 281 (M+H).sup.+, (ES.sup.+).
Intermediate H1
8-((2-Amino-3-nitropyridin-4-yl)oxy)quinolin-5-amine
(77) ##STR00070##
(78) To a stirred solution of 5-aminoquinolin-8-ol dihydrochloride (2.00 g, 8.60 mmol) in DMSO (30 mL) under N.sub.2 was added K.sub.2CO.sub.3 (3.79 g, 27.5 mmol) and after 1 hr at RT the mixture was treated with 4-chloro-3-nitropyridin-2-amine (1.34 g, 7.72 mmol) in a single portion. The reaction mixture was heated to 70 C. for 2 hr and was then cooled to RT and diluted with water (500 mL). A precipitate formed which was isolated by filtration, washed with water (220 mL) and then dried in vacuo at 40 C. to afford the title compound, Intermediate H1 as a dark brown solid (1.50 g, 53%); R.sup.t 0.99 min (Method 2); m/z 298 (M+H).sup.+ (ES.sup.+).
Intermediate J1
tert-Butyl (4-((6-amino-5-nitropyrimidin-4-yl)oxy)naphthalen-1-yl) carbamate
(79) ##STR00071##
(80) To a stirred solution of tert-butyl (4-hydroxynaphthalen-1-yl)carbamate (20.0 g, 73.0 mmol) in MeCN (200 mL) under N.sub.2 was added DBU (13 mL, 87 mmol) and after 1 hr at RT the resulting mixture was treated dropwise with a solution of 4,6-dichloro-5-nitropyrimidine (15.5 g, 80.0 mmol) in MeCN (150 mL). After 2.5 hr at RT the mixture was evaporated in vacuo and the residue was triturated with DCM (150 mL) and isohexane (200 mL). The insoluble residue was separated and set aside and the triturant was evaporated in vacuo. The residue so obtained was partitioned between DCM (200 mL) and sat aq NaHCO.sub.3 (200 mL) and the organic layer was separated and evaporated in vacuo. The residue was triturated with EtOAc to give an off white solid (batch 1). The EtOAc triturant was concentrated in vacuo to 20 mL and the precipitate which formed was isolated by filtration and washed with EtOAc to afford a second batch of same material, also as an off white solid. The first and second batches of product were combined to afford tert-butyl (4-((6-chloro-5-nitropyrimidin-4-yl)oxy)naphthalen-1-yl)carbamate (10.4 g, 34%); R.sup.t2.69 min (Method 2); m/z 439/441 (M+Na).sup.+ (ES.sup.+).
(81) The original insoluble residue was suspended in DCM (150 mL) and isohexane (200 mL) and the mixture was stirred vigorously for 16 hr. The supernatant liquor was decanted from the mixture and filtered. The filtrate was evaporated in vacuo and the residue was triturated with isohexane/EtOAc (2:1 v/v) to afford a third batch of the same product, as a pale pink solid (6.3 g, 21%).
(82) To a solution of the di-aryl ether obtained above (15.7 g, 37.7 mmol) in THF (160 mL) at RT was added NH.sub.3 (2M solution in IPA, 75 mL, 150 mmol) and after 1 hr at RT the reaction mixture was evaporated in vacuo. The residue was partitioned between a mixture of CHCl.sub.3/MeOH (9:1 v/v, 350 mL) and water (350 mL). The organic layer was separated, dried and evaporated in vacuo and the residue was triturated with ether (200 mL) to afford Intermediate J1 as a yellow solid (8.2 g, 55%); R.sup.t 2.17 min (Method 2); m/z 398 (M+H).sup.+ (ES.sup.+).
COMPOUND EXAMPLES
(83) Those reference compounds employed as biological screening standards and compound examples of the invention that have been previously disclosed were prepared according to the procedures described in the literature sources cited (Table 3).
(84) TABLE-US-00003 TABLE 3 Screening Standards and Compound Examples Example No. Structure Compound Name & Lit. Reference BIRB 796
Example 9
1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)naphthalen-1-yl)urea
(85) ##STR00083##
(86) To a solution of CDI (63 mg, 0.39 mmol) in dry DCM (1.0 mL) at RT was added Intermediate A2 (95 mg, 0.39 mmol), the reaction mixture kept at RT for 4 days and then treated with Intermediate B1 (70 mg, 0.24 mmol) and diluted with DCM (2.0 mL). After a further 16 hr the reaction was quenched with MeOH (1.0 mL) for 5 min at RT and evaporated in vacuo. The residue was triturated with water (50 mL) and with ether (10 mL) to afford the title compound, Example 9 as a dark brown solid (72 mg, 52%); R.sup.t 2.02 min (Method 2); m/z 564 (M+H).sup.+, (ES.sup.+); .sup.1H NMR (400 MHz, DMSO-d.sub.6) : 1.27 (9H, s), 3.83 (3H, s), 6.20 (1H, d), 6.38 (1H, s), 7.12 (2H, d), 7.29 (1H, d), 7.48 (2H, d), 7.59 (1H, m), 7.65 (1H, m), 7.69 (1H, d), 7.90 (1H, d), 7.94 (1H, dd), 8.06 (1H, d), 8.71 (1H, s), 9.09 (1H, s), 11.36 (1H, br s), 11.42 (1H, br s).
Example 10
1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)naphthalen-1-yl)urea
(87) ##STR00084##
(88) To a solution of CDI (500 mg, 3.00 mmol) in dry DCM (6.0 mL) at RT was added Intermediate A2 (760 mg, 3.1 mmol) and the reaction mixture kept at RT for 4 hr. An aliquot of the resulting solution (0.70 mL, 0.40 mmol) was added to a suspension of Intermediate B2 (60 mg, 20 mol) in dry THF (1.5 mL) and the reaction mixture was maintained at RT for 16 hr and was then quenched with MeOH (1.0 mL). After a further 5 min at RT the mixture was evaporated in vacuo and the residue was purified by flash column chromatography (SiO.sub.2, 12 g, EtOAc in isohexane, 0-100%, gradient elution) to afford the title compound, Example 10 as a pale brown solid (99 mg, 86%); R.sup.t 2.16 min (method 2); m/z 578 (M+H).sup.+, (ES.sup.+); .sup.1H NMR (400 MHz, DMSO-d.sub.6) : 1.28 (9H, s), 3.53 (3H, s), 3.83 (3H, s), 6.29 (1H, d), 6.38 (1H, s), 7.12 (2H, d), 7.25 (1H, d), 7.48 (2H, d), 7.60 (1H, m), 7.66 (1H, m), 7.74 (1H, d), 7.89 (1H, d), 8.07-8.09 (2H, overlapping m), 8.69 (1H, br s), 9.07 (1H, br s), 11.67 (1H, br s).
Example 11
1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((1-ethyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)naphthalen-1-yl)urea
(89) ##STR00085##
(90) To a solution of CDI (140 mg, 0.84 mmol) in dry DCM (1.0 mL) at RT was added Intermediate A2 (210 mg, 0.84 mmol) and the reaction mixture maintained at RT for 2 hr. An aliquot of the resulting solution (0.90 mL, 0.70 mmol) was added dropwise to a solution of Intermediate B3 (100 mg, 0.31 mmol) in dry THF (2.0 mL) and the reaction mixture was kept at RT for 3 days and was then partitioned between DCM (5.0 mL) and sat aq NaHCO.sub.3 (5.0 mL). The organic layer was separated and dried and was treated with activated charcoal and evaporated in vacuo. The residue was purified by flash column chromatography (SiO.sub.2, 12 g, MeOH in DCM, 0-5%, gradient elution) to afford the title compound, Example 11 as a pale brown solid (112 mg, 58%); R.sup.t 2.24 min (Method 2); m/z 592 (M+H).sup.+, (ES.sup.+); .sup.1H NMR (400 MHz, DMSO-d.sub.6) : 1.28 (9H, s), 1.32 (3H, t), 3.84 (3H, s), 4.03 (2H, q), 6.28 (1H, d), 6.39 (1H, s), 7.13 (2H, d), 7.29 (1H, d), 7.49 (2H, d), 7.62 (1H, ddd), 7.68 (1H, ddd), 7.75 (1H, d), 7.91 (1H, d), 8.04 (1H, dd), 8.08 (1H, d), 8.72 (1H, brs), 9.11 (1H, brs), 11.71 (1H, br s).
Example 12
1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((1-isopropyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)naphthalen-1-yl)urea
(91) ##STR00086##
(92) To a solution of CDI (130 mg, 0.81 mmol) in dry DCM (1.0 mL) at RT was added Intermediate A2 (0.20 mg, 0.81 mmol) and the reaction mixture kept at RT for 2 hr. An aliquot of the resulting solution (0.90 mL, 0.70 mmol) was added dropwise to a solution of Intermediate B4 (100 mg, 0.30 mmol) in dry THF (1.0 mL) and the reaction mixture was maintained at RT and after 1 hr was treated with an additional portion of the preformed CDI adduct (25 L, 20 mol). After 3 days at RT the resulting mixture was partitioned between DCM (5.0 mL) and sat aq. NaHCO.sub.3 (5.0 mL). The organic layer was separated and dried and then treated with activated charcoal and evaporated in vacuo. The residue was purified by flash column chromatography (SiO.sub.2, 12 g, MeOH in DCM, 0-5%, gradient elution) to afford the title compound, Example 12 as a pale brown solid (95 mg, 51%); R.sup.t 2.34 min (Method 2); m/z 606 (M+H).sup.+, (ES.sup.+); .sup.1H NMR (400 MHz, DMSO-d.sub.6) : 1.27 (9H, s), 1.52 (6H, d), 3.83 (3H, s), 4.92 (1H, m), 6.22 (1H, d), 6.38 (1H, s), 7.12 (2H, d), 7.30 (1H, d), 7.48 (2H, d), 7.62 (1H, ddd), 7.67 (1H, ddd), 7.73 (1H, d), 7.92 (1H, d), 7.97 (1H, dd), 8.09 (1H, d), 8.72 (1H, br s), 9.12 (1H, br s), 11.71 (1H, br s).
Example 13
1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(2,3-dichloro-4-((1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)phenyl)urea
(93) ##STR00087##
(94) To a solution of CDI (170 mg, 1.0 mmol) in dry DCM (1.5 mL) at RT was added Intermediate A2 (260 g, 1.00 mmol) in DCM (2.0 mL) and the reaction mixture maintained at RT for 18 hr. An aliquot of the resulting solution (0.15 mL, 0.1 mmol) was added to a solution of Intermediate B5 (11 mg, 0.035 mmol) in dry THF (2.0 mL) and the reaction mixture was kept at RT for 7 hr and then treated with an additional aliquot of the preformed CDI adduct (30 L). After 24 hr the reaction was partitioned between DCM (25 mL) and sat aq. NaHCO.sub.3 (25 mL). The aq layer was separated and was extracted with DCM (20 mL) and the combined organic extracts were dried and evaporated in vacuo. The residue was purified by flash column chromatography (SiO.sub.2, 4.0 g, MeOH in DCM, 0-5%, gradient elution) to afford the title compound, Example 13 as a pale brown solid (11 mg, 48%); R.sup.t 2.26 min (Method 2); m/z 596/598 (M+H).sup.+ (ES.sup.+); .sup.1H NMR (400 MHz, DMSO-d.sub.6) : 1.27 (9H, s), 3.49 (3H, s), 3.82 (3H, s), 6.37 (2H, overlapping m), 7.10 (2H, m), 7.38 (1H, d), 7.42 (2H, m), 7.79 (1H, d), 8.14 (1H, d), 8.5 (1H, br s), 9.14 (1H, br s), 11.70 (1H, br s).
Example 14
1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(8-((2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-7-yl)oxy)quinolin-5-yl)urea
(95) ##STR00088##
(96) A mixture of Intermediate C1 (510 mg, 0.900 mmol) and iron powder (250 mg, 4.5 o mmol) in AcOH (15 mL) was heated to 50 C. for 1.5 hr and was then cooled to RT and poured onto solid K.sub.2CO.sub.3 (18 g). The resulting mixture was extracted with THF (250 mL) and the combined extracts were evaporated in vacuo. The residue was partitioned between a mixture of EtOAc (100 mL) and MeOH (30 mL) and sat aq NaHCO.sub.3 (30 mL). The organic layer was separated and was washed with water (30 mL) and brine (30 mL) and was then dried and evaporated in vacuo. The residue was triturated with ether (15 mL) to afford 1-(3-tert-butyl-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(8-(2,3-diaminopyridin-4-yloxy)quinolin-5-yl)urea as a grey solid (230 mg, 45%); R.sup.t 1.58 min (Method 2); m/z 539 (M+H).sup.+, (ES.sup.+).
(97) To a solution of CDI (38 mg, 0.23 mmol) in dry DMF (1.0 mL) was added the diamine obtained above (100 g, 0.20 mmol) in a single portion and the reaction mixture maintained at RT for 4 days. An additional portion of CDI (30 mg, 0.20 mmol) was then added and after further 24 hr the reaction mixture was diluted with water (40 mL). The resulting precipitate was collected by filtration and was purified by flash column chromatography (SiO.sub.2, 12 g, MeOH in DCM, 0-10%, gradient elution) to afford the title compound, Example 14, as a pale yellow solid (36 mg, 34%); R.sup.t 1.80 min (Method 2); m/z 565 (M+H).sup.+, (ES.sup.+); .sup.1H NMR (400 MHz, DMSO-d.sub.6) : 1.28 (9H, s), 3.83 (3H, s), 5.97 (1H, d), 6.38 (1H, s), 7.11 (2H, d), 7.48 (2H, d), 7.59 (1H, d), 7.61-7.63 (2H, overlapping m), 7.97 (1H, d), 8.43 (1H, dd), 8.72 (1H, br s), 8.85 (1H, dd), 9.24 (1H, br s), 11.30 (1H, br s), 11.34 (1H, br s)
Example 15
1-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(4-((8-oxo-8,9-dihydro-7H-purin-6-yl)oxy)naphthalen-1-yl)urea
(98) ##STR00089##
(99) To a solution of CDI (200 mg, 1.20 mmol) in dry DCM (2.5 mL) at RT was added Intermediate A1 (280 mg, 1.20 mmol) and the reaction mixture maintained at RT for 15 hr. An aliquot of the resulting solution (1.0 mL, 0.50 mmol) was added to a solution of Intermediate B6 (89 mg, 0.30 mmol) in dry THF (5.0 mL) and the reaction mixture was kept at RT for 3 days and was then partitioned between DCM (40 mL) and sat aq NaHCO.sub.3 (40 mL). The aq layer was separated and was extracted with EtOAc (240 mL) and the combined organic extracts were dried over MgSO.sub.4. The desiccant was removed by filtration and washed with THF/EtOAc (3:1 v/v, 3). These washes were combined and evaporated in vacuo and the residue was purified by flash column chromatography (SiO.sub.2, 12 g, MeOH in DCM, 0-8%, gradient elution). The impure product so obtained was triturated with ether to afford the title compound, Example 15 as a pale brown solid (57 mg, 33%); R.sup.t 1.80 min (Method 2); m/z 549 (M+H).sup.+, (ES.sup.+); .sup.1H NMR (400 MHz, DMSO-d.sub.6) : 1.28 (9H, s), 2.40 (3H, s), 6.40 (1H, s), 7.37-7.39 (3H, overlapping m), 7.46 (2H, d), 7.54 (1H, ddd), 7.62 (1H, ddd), 7.86 (1H, d), 7.88 (1H, d), 8.02 (1H, s), 8.05 (1H, d), 8.76 (1H, br s), 9.09 (1H, br s), 11.6 (1H, br s), 11.9 (1H, br s).
Example 16
1-(3-(tert-Butyl)-1-(4-hydroxyphenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea
(100) ##STR00090##
(101) To a solution of CDI (160 mg, 0.99 mmol) in dry DCM (2.0 mL) at RT was added Intermediate A3 (340 mg, 0.99 mmol) and the reaction mixture maintained at RT for 4 days. An aliquot of the resulting solution (1.2 mL, 0.60 mmol) was added dropwise to a solution of Intermediate D1 (100 mg, 0.33 mmol) in dry THF (2.5 mL) and the reaction mixture kept at RT for 24 hr and then evaporated in vacuo. The residue was triturated with MeOH to furnish 1-(3-(tert-butyl)-1-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-di hydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea as a grey solid (68 mg, 30%); R.sup.t2.84 min (method 2); m/z 676 (M+H).sup.+, (ES.sup.+).
(102) To a solution of the silyl protected intermediate obtained above (68 mg, 0.10 mmol) in THF (1.5 mL) was added TBAF (1 M in THF, 100 L, 0.1 mmol) and the reaction mixture kept at RT for 2.5 hr and then partitioned between EtOAc/THF (3:1 v/v, 50 mL) and sat aq NaHCO.sub.3. The organic layer was separated and was washed with brine (50 mL) and with water (50 mL) and then dried and evaporated in vacuo. The residue was triturated with MeOH to afford the title compound, Example 16 as a beige solid (26 mg, 46%); R.sup.t 1.83 min (Method 2); m/z 562 (M+H).sup.+ (ES.sup.+); .sup.1H NMR (400 MHz, DMSO-d.sub.6) : 1.28 (9H, s), 6.38-6.40 (2H, overlapping m), 6.94 (2H, m), 7.35 (2H, m), 7.40 (1H, d), 7.58 (1H, m), 7.67 (1H, m), 7.84 (1H, d), 7.99 (1H, d), 8.10 (1H, d), 8.26-8.28 (2H, overlapping m), 8.71 (1H, s), 9.17 (1H, br s), 9.83 (1H, br s), 12.98 (1H, br s).
Example 17
1-(3-(tert-Butyl)-1-(4-(hydroxymethyl)phenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea
(103) ##STR00091##
(104) To a solution of CDI (2.06 g, 12.7 mmol) in dry DCM (40 mL) at RT was added Intermediate A12 (5.10 g, 12.7 mmol) in DCM (10 mL) and the reaction mixture kept at RT for 4 hr. An aliquot of the resulting solution (2.1 mL, 0.53 mmol) was added to a solution of Intermediate D1 (89 mg, 0.29 mmol) in dry THF (4.0 mL) and the reaction mixture was maintained at RT and treated with additional aliquots of CDI adduct after 17 hr (1.1 mL, 0.28 mmol) and 23 hr (1.1 mL, 0.28 mmol). After 2 days the reaction mixture was partitioned between DCM (40 mL) and sat aq. NaHCO.sub.3 (40 mL). The aq layer was separated and was extracted with DCM (40 mL) and the combined organic extracts were dried and evaporated in vacuo. The residue was purified by flash column chromatography (SiO.sub.2, 12 g, MeOH in DCM, 0-4%, gradient elution) to afford 1-(3-(tert-butyl)-1-(4-(((triisopropylsilyl)oxy)methyl)phenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea as an off-white solid (52 mg, 22%); R.sup.t 3.21 min (Method 2); m/z 732 (M+H).sup.+, (ES.sup.+).
(105) To a solution of the silyl protected intermediate obtained above (51 mg, 0.070 mmol) in THF (1.75 mL) under N.sub.2 at 0 C. was added TBAF (1M solution in THF, 77 L, 0.077 mmol) and the reaction mixture then warmed to RT. After 2 hr the mixture was partitioned between water (25 mL) and 2-methyltetrahydrofuran (25 mL). The organic layer was separated and washed with brine (25 mL) and was then dried and evaporated in vacuo. The residue was triturated with ether and the residue was purified by flash column chromatography (SiO.sub.2, 12 g, MeOH in DCM, 0-10%, gradient elution) to afford the title compound, Example 17 as a pale yellow solid (24 mg, 58%); R.sup.t 1.87 min (Method 2); m/z 576 (M+H).sup.+, (ES.sup.+); .sup.1H NMR (400 MHz, DMSO-d.sub.6) : 1.31 (9H, s), 4.59 (2H, d), 5.09 (1H, br s), 6.39 (1H, s), 6.44 (1H, d), 7.32 (1H, d), 7.49 (2H, d), 7.55 (2H, d), 7.56 (1H, t), 7.65 (1H, t), 7.90 (1H, s), 7.92 (1H, s), 8.12 (1H, d), 8.21 (1H, s), 8.27 (1H, d), 8.66 (1H, br s), 9.00 (1H, brs), 12.7 (1H, br s).
Example 18
1-(3-(tert-Butyl)-1-(4-(hydroxymethyl)-3-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea
(106) ##STR00092##
(107) To a solution of CDI (96 mg, 0.59 mmol) in dry DCM (1.2 mL) at RT was added Intermediate A16 (240 mg, 0.59 mmol) and the reaction mixture maintained at RT for 24 hr. An aliquot of the resulting solution (0.60 mL, 0.30 mmol) was added to a solution of Intermediate D1 (60 mg, 0.20 mmol) in dry THF (2.0 mL) and the reaction mixture was kept at RT for 3 hr and was then partitioned between DCM (20 mL) and sat aq. NaHCO.sub.3 (20 mL). The organic layer was separated and was washed with brine (20 mL) and then dried and evaporated in vacuo. The residue was triturated with DCM to afford 1-(3-tert-butyl-1-(4-((tert-butyldimethylsilyloxy) methyl)-3-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-(3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl oxy)naphthalen-1-yl)urea as a yellow solid (113 mg, 78%); R.sup.t 2.95 min (method 2); m/z 720 (M+H).sup.+, (ES.sup.+).
(108) To a solution of the silyl protected intermediate obtained above (110 mg, 0.150 mmol) in THF (2.5 mL) under N.sub.2 at 00 C. was added TBAF (1M solution in THF, 0.19 mL, 0.19 mmol) and the mixture warmed to RT for 2.5 hr. An additional aliquot of TBAF (1M solution in THF, 0.19 mL, 0.19 mmol) was added and after 16 hr the mixture was partitioned between DCM (5.0 mL) and sat aq NaHCO.sub.3 (10 mL). The organic layer was separated and was washed with brine (10 mL) and then dried and evaporated in vacuo. The residue was triturated with MeOH to afford the title compound, Example 18 as a yellow solid (28 mg, 30%); R.sup.t 1.84 min (Method 2); m/z 606 (M+H).sup.+, (ES.sup.+); .sup.1H NMR (400 MHz, DMSO-d.sub.6) : 1.29 (9H, s), 3.85 (3H, s), 4.57 (2H, d), 5.15 (1H, t), 6.39 (1H, d), 6.43 (1H, s), 7.14-7.16 (2H, overlapping m), 7.39 (1H, d), 7.54 (1H, d), 7.57 (1H, ddd), 7.66 (1H, ddd), 7.85 (1H, dd), 7.97 (1H, d), 8.11 (1H, d), 8.25 (1H, d), 8.27 (1H, d), 8.81 (1H, br s), 9.18 (1H, br s), 12.95 (1H, br s).
Example 19
1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(2,3-dichloro-4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)phenyl)urea
(109) ##STR00093##
(110) To a solution of CDI (110 mg, 0.700 mmol) in dry DCM (1.5 mL) at RT was added Intermediate A2 (170 mg, 1.20 mmol) and the reaction mixture maintained at RT for 4 hr. An aliquot of the resulting solution (1.2 mL, 0.60 mmol) was added to a solution of Intermediate D2 (650 mg, 0.200 mmol) in dry THF (3.0 mL) and the reaction mixture was kept at RT for 3 days and was then partitioned between DCM (30 mL) and sat aq NaHCO.sub.3 (30 mL). The aq layer was separated and was extracted with DCM (20 mL) and the combined organic extracts were washed with brine (25 mL), dried and evaporated in vacuo. The residue was purified by flash column chromatography (SiO.sub.2, 12 g, MeOH in DCM, 0-7%, gradient elution) to afford the title compound, Example 19 as a pale brown solid (52 mg, 43%); R.sup.t 2.27 min (Method 2); m/z 594/596 (M+H).sup.+, (ES.sup.+); .sup.1H NMR (400 MHz, DMSO-d.sub.6) : 1.27 (9H, s), 3.82 (3H, s), 6.37 (1H, s), 6.55 (1H, d), 7.10 (2H, d), 7.40 (1H, d), 7.43 (1H, d), 8.17 (1H, d), 8.20 (1H, s), 8.36 (1H, d) 8.86 (1H, br s), 9.15 (1H, br s), 13.0 (1H, br s).
Example 20
1-(3-(tert-Butyl)-1-(6-methoxypyridin-3-yl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea
(111) ##STR00094##
(112) To a solution of CDI (160 mg, 0.98 mmol) in dry DCM (2.5 mL) at RT was added Intermediate A9 (270 mg, 0.99 mmol) and the reaction mixture kept at RT for 2 hr. The resulting solution was added dropwise to a solution of Intermediate D1 (100 mg, 0.33 mmol) in dry THF (2.5 mL) and the reaction mixture was maintained at RT for 18 hr and was then evaporated in vacuo. The residue was triturated with MeOH to furnish a lilac-coloured solid (63 mg). A portion of this solid (30 mg) was purified by preparative HPLC to afford the title compound, Example 20 as an off-white solid (15 mg, 16% pro rata); R.sup.t 1.97 min (Method 2); m/z 577 (M+H).sup.+, (ES.sup.+); .sup.1H NMR (400 MHz, DMSO-d.sub.6) : 1.29 (9H, s), 3.94 (3H, s), 6.39-6.42 (2H, overlapping m), 7.02 (1H, d), 7.39 (1H, d), 7.54 (1H, m), 7.66 (1H, m), 7.85 (1H, d), 7.89-7.95 (2H, overlapping m), 8.08 (1H, d), 8.25-8.27 (2H, overlapping m), 8.39 (1H, d), 8.80 (1H, br s), 9.10 (1H, br s), 12.95 (1H, br s).
Example 21
1-(3-(tert-Butyl)-1-(5-methylthiophen-2-yl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[2,3-b]pyrazin-8-yl)oxy)naphthalen-1-yl)urea
(113) ##STR00095##
(114) To a solution of CDI (100 mg, 0.590 mmol) in dry DCM (1.0 mL) at RT was added Intermediate A14 (140 mg, 0.590 mmol) and the reaction mixture was maintained at RT for 16 hr. A portion of the resulting solution (0.60 mL, 0.30 mmol) was added dropwise to a solution of Intermediate D1 (60 mg, 0.20 mmol) in dry THF (2.0 mL) and the reaction mixture was kept at RT. A precipitate formed after 5 min and after 1 hr this was collected by filtration and washed with MeOH to afford the title compound, Example 21 as a yellow solid (80 mg, 71%); R.sup.t 2.18 min (Method 2); m/z 564 (M+H).sup.+, (ES.sup.+); .sup.1H NMR (400 MHz, DMSO-d.sub.6) : 1.28 (9H, s), 2.28 (3H, s), 6.41-6.42 (2H, overlapping m), 7.10-7.12 (2H, overlapping m), 7.40 (1H, d), 7.59 (1H, m), 7.70 (1H, m), 7.88 (1H, d), 7.97 (1H, d), 8.17 (1H, d), 8.26-8.28 (2H, overlapping m), 8.87 (1H, s), 9.26 (1H, s), 12.96 (1H, s).
Example 22
1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((7-oxo-7,8-dihydropteridin-4-yl)oxy)naphthalen-1-yl)urea
(115) ##STR00096##
(116) To a solution of CDI (66 mg, 0.40 mmol) in dry DCM (1.5 mL) at RT was added Intermediate A2 (99 mg, 0.41 mmol) in DCM (1.5 mL) and the reaction mixture maintained at RT for 18 hr. The resulting solution was added to a solution of Intermediate D3 (90 mg, 0.12 mmol) in dry DCM (2.5 mL), after which DIPEA (47 L, 0.27 mmol) was added and the mixture kept at RT for 2 hr. The reaction was quenched with MeOH (6.0 mL) and the mixture was evaporated onto silica and then purified by flash column chromatography (SiO.sub.2, 12 g, MeOH in DCM, 0-100%, gradient elution). The impure material so obtained was purified by preparative HPLC to afford the title compound, Example 22 as an off white solid (1.5 mg, 2%); R.sup.t 2.10 min (Method 2); m/z 577 (M+H).sup.+ (ES.sup.+); .sup.1H NMR (400 MHz, DMSO-d.sub.6) : 1.28 (9H, s), 3.84 (3H, s), 6.40 (1H, s), 7.12 (2H, d), 7.37 (1H, d), 7.48 (2H, d), 7.52 (1H, ddd), 7.61 (1H, ddd), 7.77 (1H, d), 7.90 (1H, d), 8.05 (1H, d), 8.07 (1H, s), 8.26 (1H, d), 8.76 (1H, s), 9.13 (1H, s).
Example 23
1-(3-tert-butyl-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(4-((3-oxo-3,4-dihydropyrido[3,2-b]pyrazin-8-yl)methoxy)naphthalen-1-yl)urea
(117) ##STR00097##
(118) To a solution of CDI (77 mg, 0.47 mmol) in dry DCM (500 L) at RT was added Intermediate A2 (120 mg, 0.81 mmol) and the reaction mixture maintained at RT for 10 min, warmed to 40 C. for 5 min and then re-cooled to RT. After a further 1 hr the solution was diluted with DCM (500 L) and an aliquot of this solution (75 L, 0.06 mmol) was added to a suspension of Intermediate D4 (7.5 mg, 85% pure, 0.02 mmol) in dry THF (1.0 mL) The reaction mixture was kept at RT for 1.5 hr and was then quenched by the addition of MeOH (1.0 mL) and evaporated in vacuo. The residue was trituated with MeOH (1.5 mL) then washed with MeOH (21 mL) and dried in vacuo to afford the title compound, Example 23 as a yellow solid (8.5 mg, 68%); R.sup.t 2.26 min (Method 2); m/z 590 (M+H).sup.+, (ES.sup.+); .sup.1H NMR (400 MHz, DMSO-d.sub.6) : 1.26 (9H, s), 3.83 (3H, s), 5.81 (2H, s), 6.34 (1H, s), 7.06 (1H, d), 7.11 (2H, m), 7.46 (2H, m), 7.58-7.62 (4H, overlapping m), 7.64 (1H, d), 7.92 (1H, m), 8.30 (1H, s), 8.35 (1H, m), 8.55 (1H, s), 8.60 (1H, d), 8.80 (1H, s), 13.0 (1H, br s).
(119) Further compound examples of the invention were prepared employing the generic synthetic processes and the intermediates described herein above (Table 4).
(120) TABLE-US-00004 TABLE 4 Additional Compound Examples of the Invention. Ex. No. Structure Compound Name & Analytical Data 24
Example 34
(121) A dry powder formulation may be prepared as follows:
(122) A compound according to the invention such as 0.20 mg of a compound of formula (I) with a mass median diameter of 3 m is blended with 12 mg milled lactose (wherein not greater than 85% of the particles have a mass median diameter of 60-90 m and not less than 15% of particles have a mass median diameter of 15 m. The resulting melange can then be filled into blisters, for example a peelable blister strip containing 60 blisters.
Example 35
(123) An aerosol formulation containing a compound of formula (I) may be prepared as follows:
(124) A compound according to the invention such as 250 g of a compound of formula (I) with a mass median diameter of 3 m, is mixed with 1,1,1,2-tetrafluoroethane 50 L (amounts per actuation) in a total amount for 120 actuations. The formulation is then filled into a canister fitted with a metering valve adapted to dispense 50 L.
(125) Biological Profiling
(126) The known p38 MAPK inhibitor BIRB 796 was tested in the assay systems for comparison purposes. The inhibitory potencies observed for the compound against the isoform of the isolated p38 enzyme and against LPS-induced TNF release from THP-1 cells were consistent with published results. A description of these assays follows below.
(127) The inhibitory effects of compounds versus HRV-16 replication and poly I:C induced ICAM-1 expression were also determined.
(128) Enzyme Inhibition Assay
(129) The enzyme inhibitory activity of test compounds was determined by fluorescence resonance energy transfer (FRET) using synthetic peptides labelled with both donor and acceptor fluorophores (Z-LYTE, Invitrogen). Recombinant, phosphorylated p38 MAPK (MAPK12:Millipore) was diluted in HEPES buffer, mixed with compound at the desired final concentrations and incubated for 2 hr at RT. The FRET peptide (2 M) and ATP (100 M) were next added to the enzyme/compound mixture and incubated for 1 hr. Development reagent (protease) was added for 1 hr prior to detection in a fluorescence microplate reader.
(130) The site-specific protease only cleaves non-phosphorylated peptide and eliminates the FRET signal. Phosphorylation levels of each reaction were calculated using the ratio of coumarin emission (donor) over fluorescein emission (acceptor) with high ratios indicating high phosphorylation levels and low ratios indicating low phosphorylation levels. The percentage inhibition of each reaction was calculated relative to non-inhibited control, and the 50% inhibitory concentration (IC.sub.50 value) then calculated from the concentration-response curve.
(131) For p38 MAPK alpha (MAPK14: Invitrogen), enzyme activity was evaluated indirectly by determining activation/phosphorylation of the down-stream molecule, MAPKAP-K2. The p38 MAPK protein was mixed with compound for 2 hr at RT. The FRET peptide (2 M), which is a phosphorylation target for MAPKAP-K2, inactive MAPKAP-K2 (Invitrogen) and ATP (10 M) were then added to the enzymes/compound mixture and incubated for 1 hr. Development reagent was then added and the mixture incubated for 1 hr before detection by fluorescence completed the assay protocol.
(132) The enzyme inhibitory activity of compound was determined by fluorescence resonance energy transfer (FRET) using synthetic peptides labelled with both donor and acceptor fluorophores (Z-LYTE, Invitrogen).
(133) For HCK assay, HCK enzyme (Invitrogen) was incubated with compound for 2 hr at RT. The FRET peptides (Z-LYTE kit-Tyrosine 2 peptide) (2 M) and 15 M ATP solution were then added to the enzymes/compound mixtures and incubated for 1 hr. For the c-SRC assay, the FRET peptides (Z-LYTE kit-Tyrosine 2 peptide) (2 M) and 200 M ATP solutions were then added to the cSrc enzyme (invitrogen)/compound mixtures and incubated for 1 hr. After development reagent was added, the mixtures were incubated for 1 hr and the assay protocol was completed by detection of the fluorescence levels in a microplate reader (Varioskan Flash, ThermoFisher Scientific).
(134) As we have reported in WO2011/070369 (herein incorporated by reference in its entirety), HCK inhibitory activity is associated with activity against influenza virus. The inventors have also discovered that inhibition of c-SRC is associated with activity against rhinovirus (see unpublished international patent application based on GB1010196.2, herein incorporated by reference in its entirety.
(135) LPS-Induced TNF/IL-8 Release in d-U937Cells.
(136) U937 cells, a human monocytic cell line, were differentiated to macrophage-type cells by incubation with phorbol myristate acetate (PMA; 100 ng/mL) for 48 to 72 hr, whereupon the cells become adherent. Cells were pre-incubated with final concentrations of test compound for 2 hr and were then stimulated with 0.1 g/mL of LPS (from E. Coli: 0111:B4, Sigma) for 4 hr. The supernatant was collected for determination of TNF and IL-8 concentrations by sandwich ELISA (Duo-set, R&D systems). The inhibition of TNF production was calculated as a percentage of that achieved by 10 g/mL of BIRB796 at each concentration of test compound by comparison against vehicle control. The relative 50% effective concentration (REC.sub.50) was determined from the resultant concentration-response curve. The inhibition of IL-8 production was calculated at each concentration of test compound by comparison with vehicle control. The 50% inhibitory concentration (IC.sub.50) was determined from the resultant concentration-response curve.
(137) LPS-Induced TNF Release in THP-1 Cells.
(138) THP-1 cells, a human monocytic cell line, were stimulated with 3 g/mL of LPS (from E. Coli; 0111:B4, Sigma) for 4 hr and the supernatant collected for determination of the TNF concentration by sandwich ELISA (Duo-set, R&D systems). The inhibition of TNF production was calculated at each concentration by comparison with vehicle control. The 50% inhibitory concentration (IC.sub.50) was determined from the resultant concentration-response curve.
(139) Poly I:C-Induced ICAM-1 Induction in BEAS2B Cells.
(140) Poly I:C (1 g/mL) (Invivogene Ltd., San Diego, Calif.) was transfected into BEAS2B cells (human bronchial epithelial cells, ATCC) with Oligofectamine (Invitrogen, Carlsbad, Calif.). Cells were pre-incubated with final concentrations of test compounds for 2 hr and the level of ICAM1 expression on the cell surface was determined by cell-based ELISA. At a time point 18 hr after poly I:C transfection, cells were fixed with 4% formaldehyde in PBS and then endogenous peroxidase was quenched by the addition of 0.1% sodium azide and 1% hydrogen peroxide. Cells were washed with wash-buffer (0.1% Tween in PBS: PBS-Tween) and after blocking the wells with 5% milk in PBS-Tween for 1 hr, the cells were incubated with anti-human ICAM-1 antibody (Cell Signaling Technology, Danvers, Mass.) in 1% BSA PBS overnight at 4 C. The cells were washed with PBS-Tween and incubated with the secondary antibody (HRP-conjugated anti-rabbit IgG, Dako Ltd., Glostrup, Denmark). The ICAM-1 signal was detected by adding substrate and reading the absorbance at 450 nm against a reference wavelength of 655 nm using a spectrophotometer. The cells were then washed with PBS-Tween and total cell numbers in each well were determined by reading absorbance at 595 nm after Crystal Violet staining and elution by 1% SDS solution. The measured OD 450-655 readings were corrected for cell number by dividing with the OD595 reading in each well. The inhibition of ICAM-1 expression was calculated at each concentration of test compound by comparison with vehicle control. The 50% inhibitory concentration (IC.sub.50) was determined from the resultant concentration-response curve.
(141) We have used poly I:C challenge as a simple pharmacological challenge which mimics many of the consequences of virus infection.
(142) Rhinovirus-Titre Assay.
(143) MRC5 cells (human lung fibroblast, ATCC) were infected with 1 MOI (multiplicity of infection of 1.0) of HRV16 and incubated for 1 hr at 33 C. with gentle shaking to stimulate adsorption. The cells were then washed with PBS, fresh media was added and the cells were incubated for a further 96 hr. The supernatant was collected and 10-fold serial dilutions of virus containing supernatant were prepared. All titrations were performed by infecting confluent Hela cell monolayers with serially diluted supernatant (10.sup.1-10.sup.5) and assessing the cytopathic effect by visual inspection 4 days after infection. The amount of virus required to infect 50% of Hela cells was calculated in each treatment as TCID.sub.50. Test compounds (0.04 g/mL) were added 2 hr before HRV infection and 1 hr after infection when non-infected HRV was washed out. The anti-HRV activity of each test compound was determined as the difference of the log TCID.sub.50 of HRV16 in the presence of the compound with the log TCID.sub.50 of HRV16 in the absence of compound. The comparative potency of test compounds was then expressed as a ratio of their anti-viral activity, relative to the known anti-HRV agent Pleconaril, at a fixed dose (0.1 g/mL). Anti-HRV activities were also shown as inhibitory % of TCID.sub.50 vs. HRV16 infected control.
(144) MTT Assay: Cell Viability
(145) Differentiated U937 cells were pre-incubated with each test compound under two protocols: the first for 4 hr in 5% FCS and the second in 10% FCS for 24 h. The supernatant was replaced with 200 L of new media and 10 L of MTT stock solution (5 mg/mL) was added to each well. After incubation for 1 hr the media were removed, 200 L of DMSO was added to each well and the plates were shaken lightly for 1 hr prior to reading the absorbance at 550 nm. The percentage loss of cell viability was calculated for each well relative to vehicle (0.5% DMSO) treatment. Consequently an apparent increase in cell viability for drug treatment relative to vehicle is tabulated as a negative percentage.
(146) TABLE-US-00005 TABLE 1 Enzyme Inhibitory Profiles of BIRB796 and Compound Examples Test Compound Enzyme Profile: IC.sub.50 Values (nM) Example No p38 p38 HCK cSrc BIRB 796 12 296 >1894 >1894 Ref. 1 5 402 47 162 Ref 2 12 344 3 5 1 17 74 <2 7 2 5 67 <2 36 3 1 24 2 4 4 18 214 7 0.9 5 2 43 3 5 6 3 72 3 9 7 3 100 3 7 8 7 99 5 7 9 21 119 <2 7 10 6 45 3 7 11 2 348 5 9 12 11 495 8 12 13 7 23 4 5 14 5 180 <2 0.8 15 30 539 36 35 16 4 45 18 10 17 7 318 24 11 18 5 24 5 6 19 2 35 4 7 20 6 44 3 8 21 2 41 3 17 22 17 2620 458 406 23 12 193 22 35 24 7 126 5 16 25 4 50 5 12 26 7 29 3 13 27 3 237 918 16 28 2 >18000 9 22 29 5 19 4 10 30 2 39 4 8 31 5 47 3 6 32 4 89 4 6 33 5 74 4 7
(147) TABLE-US-00006 TABLE 2 Cellular Profiles of BIRB 796 and Compound Examples Test Compd THP1 d-U937 BEAS2B NBEC (LPS) (LPS) (polyIC) (HRV) d-U937 Example No HRV titre TNF TNF IL-8 ICAM1 Relative anti- IC.sub.50 REC.sub.50 IC.sub.50 IC.sub.50 viral activity.sup.a MTT Assay.sup.c (nM) (nM) (nM) (nM) (inhibitory %).sup.b 4 h 24 h BIRB 796 4.1 4.8 >1894 3530 NE (28.sup.b) .sup. .sup.d .sup. .sup.d Ref. 1 2.3 0.9 92 618 1 (96.sup.b) .sup. .sup.d .sup. .sup.d Ref. 2 13 0.13 1.3 2.1 100 (100.sup.b) 1 1.6 0.2 19 2.7 0.1 (94) 2 1.4 3.0 7.5 9.8 1 (60) 3 0.7 1.1 17 4.1 0.2 (36) 4 185 171 378 581 0.7 (60) 5 13 6.2 4.1 6.6 (36) 6 0.7 1.1 5.2 11 (80) 7 7.0 1.7 4.2 3.0 (60) 8 2.5 0.3 1.7 0.6 (91) 9 1.6 3.1 27 24 3.2 (36) 10 0.6 1.3 1.0 6.0 (91) 11 4.0 0.7 1.6 3.6 (60) 12 1.3 0.5 1.1 1.9 (94) 13 0.6 0.6 4.6 6.2 ND + 14 163 166 726 675 1.5 (8) 15 126 33 164 >182 (8) 16 116 55 47 172 (8) 17 14 12 24 14 (36) 18 17 12 7.4 15 (80) 19 2.1 1.3 1.6 0.6 (80) 20 13 3.4 16 21 (36) 21 11 7.1 3.3 17 (80) 22 >1733 >1733 >1733 >173 (8) 23 ND 30 90 23 ND 24 4.4 1.3 11 12 (91) 25 1.6 1.8 8.1 8.3 (36) 26 75 1.9 1.6 207 (300) 27 7.5 1.6 1.1 2.2 (94) 28 161 40 71 >184 (36) 29 17 5.6 6.7 13 (60) 30 34 1.1 4.3 27 (36) 31 62 60 68 178 (8) 32 8.8 5.5 7.0 10 (80) 33 13 8.2 9.4 3.2 (80) .sup.aRelative anti-viral activity at 0.04 g/mL vs. pleconaril at 0.1 g/mL. Figure in parentheses is the % inhibition observed vs. HRV16 infection control; .sup.bperformed at 0.2 g/mL; .sup.cCell viability screen: and + indicate the result is below and above the no significant effect threshold, respectively (<30% inhibition at a concentration of 1 g/ml) at the time point indicated; .sup.d performed at 10 g/mL ND: not determined; NE: no effect
(148) These data demonstrate the anti-inflammatory and anti-viral potential of the tested compounds.
(149) Throughout the specification and the claims which follow, unless the context requires otherwise, the word comprise, and variations such as comprises and comprising, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.
(150) All patents and patent applications referred to herein are incorporated by reference in their entirety.
(151) The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the claims.