Patent Application: US-201113885098-A

Abstract:
the present invention relates to the use of quinolinone derivatives of general formula , ligands of the smo receptor or of related receptors which target a binding site of the receptor which is different from the known ligand binding sites , as research tools for identifying modulators of the smo receptor or of related receptors , characterizing the hedgehog signalling pathway and diagnosis ; the invention also relates to kits containing said derivatives of general formula .

Description:
in a round - bottomed flask equipped with a condenser , n - hexylaniline ( 355 mg , 2 mmol ) is added to a solution of methanetricarboxylic acid triethyl ester ( 1 . 35 ml , 6 . 4 mmol ). the resulting reaction mixture is placed in a cem discovery microwave oven and irradiated in the open round - bottomed flask , and then the ethanol formed is distilled off ( the parameters are the following : power = 250 w , temperature = 225 ° c ., execution time = 5 min , hold time = 15 min ). after the microwave heating , the reaction crude is purified on a chromatography column , with petroleum ether : ethyl acetate at 4 : 1 . the crystalline product obtained is dried to give the compound : yield = 430 mg ( 69 %). 1 h nmr ( cdcl 3 ): δ 8 . 16 ( d , j = 8 hz , 1h ), 7 . 64 ( m , 1h ), 7 . 27 - 7 . 19 ( m , 2h ), 4 . 48 ( q , j = 8 hz , 2h ), 4 . 18 ( t , j = 8 hz , 2h ), 1 . 72 - 0 . 86 ( m , 14h ). ethyl 1 - hexyl - 4 - hydroxy - 2 - oxo - 1 , 2 - dihydroquinoline - 3 - carboxylate ( 159 mg , 0 . 5 mmol ) and tert - butyl 4 - amino - benzoate ( 193 mg , 1 mmol ) are suspended in anhydrous toluene ( 4 ml ) and microwave - heated with the container open ( the parameters are the following : power = 200 w , temperature = 120 ° c ., execution time = 7 min , hold time = 10 min ). at the end of the reaction , ⅔ of the solvent are evaporated off without a condenser . the reaction crude is purified by means of a chromatographic column , using 4 : 1 petroleum ether : ethyl acetate . the crystalline product obtained is dried to give the title compound : yield = 195 mg ( 84 %), 1 h nmr ( dmso ): δ 8 . 16 ( d , j = 8 hz , 1h ), 7 . 90 - 7 . 75 ( m , 6h ), 7 . 38 ( m , 1h ), 4 . 25 ( bs , 2h ), 1 . 60 ( bs , 2h ), 1 . 52 ( s , 9h ), 1 . 39 ( bs , 2h ), 1 . 28 ( bs , 5h ), 0 . 84 ( bs , 2h ). tert - butyl 4 -( 1 - hexyl - 4 - hydroxy - 2 - oxo - 1 , 2 - dihydroquinoline - 3 - carboxamido ) benzoate ( 83 mg , 0 . 18 mmol ) is added , in fractions , to a solution of trifluoroacetic acid ( tfa ) cooled to 0 ° c . ; the mixture is left to react for 3 hours . the crude product obtained is washed with ethyl ether until a white crystalline solid forms . the product is dried to give a yield of 61 mg ( 83 %). 1 h nmr ( dmso ): δ 8 . 16 ( d , j = 8 hz , 1h ), 7 . 97 - 7 . 65 ( m , 6h ), 7 . 38 ( m , 1h ), 4 . 27 ( bs , 2h ), 1 . 59 ( bs , 2h ), 1 . 39 ( bs , 2h ), 1 . 28 ( bs , 5h ), 0 . 84 ( bs , 2h ). a solution of 4 -( 1 - hexyl - 4 - hydroxy - 2 - oxo - 1 , 2 - dihydroquinoline - 3 - carboxamido ) benzoic acid ( 41 mg , 0 . 1 mmol ) in thf is cooled to 0 ° c ., and benzyl alcohol ( 21 mg , 20 μl , 0 . 2 mmol ) is added , along with edci ( 19 mg , 0 . 1 mmol ) and dimethylaminopyridine ( 3 mg , 0 . 02 mmol ). the reaction mixture is left to stir overnight . the organic phase is washed with water and evaporated under reduced pressure . the crude product is purified by column chromatography , using 4 : 1 petroleum ether : ethyl acetate , with a yield of 30 mg ( 60 %). 1h nmr ( dmso ): δ 8 . 16 ( d , j = 8 hz , 1h ), 8 . 04 ( d , j = 8 hz , 2h ), 7 . 83 ( d , j = 8 hz , 3h ), 7 . 68 ( d , j = 8 hz , 1h ), 7 . 46 - 7 . 25 ( m , 6h ), 7 . 38 ( m , 1h ), 5 . 30 ( s , 2h ), 4 . 28 ( bs , 2h ), 1 . 59 ( bs , 2h ), 1 . 41 - 1 . 15 ( m , 6h ), 0 . 84 ( bs , 3h ). demonstration of the binding of the compounds of general formula ( i ) to a specific binding site ( smo2 ) different than that on which the sag compound acts ( smo1 ) the effect of the compounds of general formula ( i ) according to the invention on the stimulation of differentiation was determined in vitro by analyzing the pluripotent fibroblast cell line c3h10t1 / 2 . this activation can be inhibited by compounds previously described as hh pathway antagonist and more specifically smo receptor antagonist . moreover , these pharmacological agents have a signal transmission mode distinct from that used by sag , which is an hh pathway agonist ( chen et al . 2002 ). the compounds of general formula ( i ) tested are compounds 1 and 6 : they were dissolved in dimethyl sulfoxide to a concentration of 2 . 5 mm , and then stored at a temperature of − 20 ° c . until used . the pluripotent fibroblast cell line c3h10t1 / 2 ( atcc ) was cultured under the conditions recommended by the atcc . the stimulation of the differentiation of these cells was carried out using increasing concentrations of compounds according to the methods described by chen et al . and frank - kamenetsky et al . ( chen et al . 2002 ; frank - kamenetsky et al . 2002 ). the activation by the compounds of general formula ( i ) causes differentiation of the cell line and enables the latter to express alkaline phosphatase . it was thus possible to measure the activity of the compounds via the measurement of alkaline phosphatase activity . by way of comparison , the activity of the sag compound ( chen et al . 2002 ), an smo activator , was tested under the same conditions as those used to test the compounds of general formula ( i ). the c3h10t1 / 2 cells were seeded onto 96 - well plates at a density of 5 × 10 3 cells per well , 24 hours before the addition of the test compounds at a concentration ranging from 10 nm to 10 μm in dmem culture medium supplemented with 10 % of fetal calf serum . the tests were carried out in quadruplicate . the plates were then incubated for 5 - 6 days at a temperature of 37 ° c . under an atmosphere of 5 % co 2 . the cells were then washed in cold phosphate buffer ( phosphate buffered saline : pbs ) and then lysed by sonication at 4 ° c . in 50 μl of a solution containing 0 . 9 % of nacl and 0 . 2 % of triton x - 100 . the alkaline phosphatase in the resulting lysates was then measured according to the method described by pepinsky et al . ( pepinsky et al . 1998 ). after the addition of 100 μm of reaction buffer ( 200 mm tris - hcl ; ph 10 . 5 ; 0 . 4 m of 2 - amino - 2 - methylpropanol and 8 mm of mgcl 2 ) and of 50 μl of substrate ( 4 mm of disodium p - nitrophenyl phosphate ), the lysates were incubated at 37 ° c . for 30 - 60 min and then the optical density was read at a wavelength of 415 nm . for the inhibition experiments , compound 1 ( 1 μm ), compound 6 ( 3 μm ) or sag ( 0 . 1 μm ) was used to stimulate differentiation in the presence of increasing concentrations ( from 0 . 1 nm to 30 μm ) of smo inhibitors previously described , such as cur61414 ( frank - kamenetsky et al . 2002 ), cyclopamine ( incardona et al . 1998 ), gdc - 0449 ( romer et al . 2004 ) and also the molecules mrt - 14 , mrt - 79 , mrt - 80 and mrt - 81 ( patent application fr 08 / 02302 ). these compounds were dissolved in 100 % ethanol ( cur61414 , cyclopamine ) or dimethyl sulfoxide ( compounds mrt - 14 , mrt - 79 , mrt - 80 , mrt - 81 and gdc - 0449 ) and then stored at − 20 ° c . for the experiments in the presence of forskolin , compound 1 ( 1 μm ) and sag ( 0 . 3 μm ) were used to stimulate differentiation in the presence of increasing concentrations ( 1 , 3 and 10 μm ) of forskolin ( sigma ). the iwr1 compound ( sigma ), for its part , was used at 10 μm on an activation by compound 1 at 1 μm or sag at 0 . 1 μm . the alkaline phosphatase activity was measured as previously . it was demonstrated that compounds 1 and 6 have the capacity to stimulate osteogenesis in c3h10t1 / 2 pluripotent cells . this differentiation is linked to the induction of alkaline phosphatase ( ap ) after 6 days of differentiation . fig2 represents the dose - response curves of compound 1 and of sag produced in parallel with respect to the stimulation of c3h10t1 / 2 cells . compound 1 makes it possible to obtain a maximum stimulation greater than that of the smo agonist sag , with a similar affinity . 2 - 2 . inhibition of the activity of compounds 1 and 6 by smoothened antagonists the activity of compound 1 can be inhibited by smo receptor antagonists ( fig3 ). it is reflected by a gradual inhibition of the alkaline phosphatase activity induced by compound 1 in the presence of increasing concentrations of known antagonists such as cyclopamine , cur61414 , gdc - 0449 or the compounds mrt - 14 and mrt - 81 . compound 6 also has the characteristic of being able to be inhibited by smo antagonists ( fig4 ). when the activities of the antagonists tested on the two compounds 1 and 6 are compared , great pharmacological similarity can be noted for these two osteogenesis - stimulating agents . this suggests one and the same mode of action for these compounds . the affinities of the smo antagonists for the two compounds 1 and 6 were determined and are reported in table i . they are expressed as ic 50 , which corresponds to the concentration that makes it possible to inhibit 50 % of the biological response induced by the agonist compound . the compounds mrt - 14 and mrt - 81 exhibit good affinity for the response , with extremely close values for the two compounds . the affinities of mrt - 14 are , respectively , 0 . 32 and 0 . 4 μl for compounds 1 and 6 , whereas that of mrt - 81 is 0 . 06 μm for both compounds . conversely , cyclopamine , cur61414 and gdc - 0449 have little affinity for these compounds ( ic 50 = 2 - 10 μm ). via the alkaline phosphatase activity induced by compounds 1 ( 1 μm ) and 6 ( 3 μm ). the ic 50 values are derived from the inhibition curves as is shown in fig3 and 4 . mean ± sen of 2 to compounds 1 and 6 bind to smo2 binding sites and their binding is blocked by hedgehog pathway antagonists with the same affinity . 2 - 3 . comparison of the activities of the smoothened antagonists with respect to the smo1 and smo2 sites dose - response curves with respect to the antagonists were also produced in the presence of 0 . 1 μm of sag ( fig5 ) in parallel to those performed on compound 1 . the sag compound acts on a binding site called smo1 . the comparison of the pharmacology of the antagonists with respect to these two compounds is given in table ii . when the activities of the smo antagonists with respect to the stimulation by sag or by compound 1 are compared , important differences exist both in terms of the maximum inhibitions obtained and the affinities ( ic 50 ). the first notable difference is the weaker inhibition by cyclopamine , cur61414 and gdc - 0449 observed at 10 μm for compound 1 , whereas these compounds enable complete inhibition of the presence of sag . with respect to the smo1 and smo2 sites . the response measuring by compound 1 and the sag compound on c3h10t1 / 2 cells is measured via the alkaline phosphatase activity induced by compound 1 ( 1 μm ) and the sag compound ( 0 . 1 μm ). the ic 50 values are derived from the inhibition curves as shown in fig3 and 5 ( mean ± sem the sag compound and compound 1 bind , respectively , to the smo1 and smo2 binding sites . their binding is blocked by hedgehog pathway antagonists ( mean ± sem of 2 to 6 independent experiments in quadruplicate ). the compounds mrt - 14 and mrt - 81 and also the compound sant - 1 exhibit comparable affinities for the smo1 site ( stimulated by sag ) and the smo2 site ( stimulated by compound 1 ) with similar values . on the other hand , other hedgehog pathway antagonists are much more selective with respect to smo1 sites than smo2 sites ( cur61414 , gdc - 0449 ). conversely , the compounds mrt - 81 and mrt - 79 are more selective with respect to smo2 sites than the smo1 sites . this is explained when the ratio between the ic 50 values of the compounds obtained with sag and compound 1 is calculated ( table iii ). to smo1 and smo2 sites . the ic 50 values of the antagonists with respect to the smo1 sites ( stimulated by sag , 0 . 1 μm ) and the smo2 sites ( stimulated by compound 1 , 1 μm ) were evaluated as in tables i and ii ( ratio of the means of the ic 50 values a selectivity of more than 300 - fold is observed with gdc - 0449 and of 17 - fold with cur61414 for the smo1 site compared with the smo2 site , whereas sant - 1 maintains a similar activity for the smo1 and smo2 sites . conversely , the compounds mrt - 81 , mrt - 55 and mrt - 79 are , respectively , 1 . 3 , 2 . 5 and 12 . 5 times more selective with respect to smo2 sites . this specificity of the compounds is significant and demonstrates an important pharmacological difference at the level of the smo1 and smo2 binding sites . the compounds of general formula ( i ) and sag act via distinct pathways . indeed , it is known that forskolin is an inhibitor of osteogenesis induced by the hh pathway in mesenchymal stem cells ( wu et al . 2004 ). the purpose of the tests which follow is to determine whether this characteristic also applies to the compounds of general formula ( i ). the differentiation of the c3ht10t1 / 2 cells was stimulated by compound 1 in the presence of forskolin . as demonstrated in fig6 , panel ( a ), it appears that forskolin has a potentiating effect on the response to compound 1 , whereas it inhibits the activity of the sag compound ( b ). these two osteogenesis - stimulating agents therefore act via distinct pathways . this effect of forskolin was found for other compounds of general formula ( i ). modulation of the activity of compound 1 and of sag by the iwr1 compound , a wnt pathway antagonist the difference in activity of forskolin on compound 1 and sag led to a search for other intracellular pathways involved in the activity of the compounds of general formula ( i ). in addition to the hh pathway , differentiation of the c3h10t1 / 2 cells can be stimulated by signaling pathways such as the wnt , nocht , tgf - β or bmp pathways , for example . with regard to the wnt pathway , activation of the canonical pathway induces activation of β - catenin which participates in the activation of the transcription of the tcf / lef genes . in the absence of wnt ligands , β - catenin is phosphorylated , and then degraded . recently , a molecule iwr1 which stabilizes axin2 and leads to the destruction of beta - catenins was discovered ( chen et al . 2009 ). this inhibitor was used to determine whether axin2 and / or β - catenins could be involved in the transduction of the signal induced by compound 1 . iwr1 makes it possible to reduce by 60 % the activity of compound 1 used at a concentration of 1 μm , whereas it does not modify that of sag ( fig7 ). a dose - response curve of compound 1 with the iwr1 compound made it possible to deduce an ic 50 = 2 . 5 μm with respect to alkaline phosphatase inhibition . this value is in agreement with the affinity of iwr1 determined by means of a test measuring its inhibition of the wnt morphogen pathway ( induction test using a tcf / luciferase reporter gene ) ( chen et al ., nat chem biol , 2009 ). this experiment indicates that the action of the compounds of general formula ( i ) involves the axin2 proteins . the role of the axin2 proteins in the control of the activity of β - catenin suggests that the biological activity induced by these compounds could involve the β - catenin pathway . this pathway is associated with multiple pathological conditions , such as colorectal cancers for example . more generally , it is involved in the maintaining of stem cells in tissues in the embryo and the adult , it modulates neurogenesis or is responsible for genetic diseases ( see review 2010 by freese et al .). the identification of selective compounds which make it possible to block the smo2 binding sites is therefore of great interest . these experiments demonstrate that the compounds of general formula ( i ) can be used in order to identify and select cell differentiation antagonists exhibiting particular structures acting on one or another of the smo1 or smo2 sites , or alternatively on both sites . the identification of other biological responses ( proliferation , migration , apoptosis , gene induction , etc .) induced by the compounds of the formula ( i ) will enable the development of tests for identifying compounds which modulate these smo2 binding sites . these novel compounds will make it possible to modulate these various responses in healthy individuals or individuals suffering from pathological conditions . ahn , s , and a . l . joyner ( 2005 ). “ in vivo analysis of quiescent adult neural stem cells responding to sonic hedgehog .” nature 437 ( 7060 ): 894 - 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