Patent Application: US-201113885089-A

Abstract:
the present invention relates to compounds which are quinolinone derivatives of general formula capable of modulating the activity , in particular of inducing the differentiation , of stem and progenitor cells ; these compounds are of use in the treatment of disorders related to a stem differentiation defect ; the invention also relates to novel compounds among these quinolinone derivatives and to pharmaceutical compositions containing the same .

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 microwave heating , the reaction crude is purified on a chromatographic column , with petroleum ether , ethyl acetate at 4 : 1 . the crystalline product obtained is dried to give the compound : yield = 430 mg ( 69 %). 1h 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 - aminobenzoate ( 193 mg , 1 mmol ) are suspended in anhydrous toluene ( 4 ml ) and are 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 , two thirds of the solvent is 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 %). 1h 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 allowed to react for 3 hours . the crude product obtained is washed with ethyl ether until a white crystalline solid is formed . the product is dried to give a yield of 61 mg ( 83 %). 1h nmr ( dmso ): δ 8 . 16 ( d , j = 8 hz , 1h ), 7 . 96 - 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 ). the effect of the compounds of general formula ( i ) in accordance with the invention on the stimulation of osteogenesis was determined in vitro by analyzing the differentiation of the c3h10t1 / 2 pluripotent mesenchymal cell line . the test compounds of formula ( i ) were dissolved in dimethyl sulfoxide ( dmso ) to a concentration of 2 . 5 mm , and then stored at a temperature of − 20 ° c . until use . the c3h10t1 / 2 pluripotent fibroblast cell line ( atcc ccl 226 ) was cultured under the conditions recommended by the atcc in dmem culture medium supplemented with 10 % of fetal calf serum at a temperature of 37 ° c . under an atmosphere at 5 % co 2 . 24 hours after seeding , the cells were stimulated for 6 days in the presence of the compounds of general formula ( i ) directly diluted in the culture medium . the activation by these compounds causes differentiation of the cell line to the osteoblast lineage and enables the latter to express alkaline phosphatase . said alkaline phosphatase is then detected by histochemistry or enzymatic assay . for this experiment , the cells are cultured in 6 - well plates containing a glass cover slip treated for 1h with 0 . 05 mg / ml poly - d - lysine . the c3h10t1 / 2 cells are seeded at a density of 150 000 cells per well . the compounds were applied at a concentration of 10 μm . the sigma staining kit ( 85l - 3r ) was used according to the protocol described . briefly , after fixing the cells for 30 seconds with a solution of citrate / acetone ( 2 - 3 ) and then rinsing with double - distilled water , the staining is carried out for 30 min in the presence of a solution of fast - violet / naphthol in the dark . the cover slips are then rinsed thoroughly with double - distilled water and then mounted in aqueous medium before being photographed on a dmrxa2 microscope ( leica microsystems ). the cells expressing alkaline phosphatase are stained red . the c3h10t1 / 2 cells were seeded onto 96 - well plates at a density of 5 × 10 3 cells per well . the compounds were applied at increasing concentrations ranging from 10 nm to 10 μm . the plates were then incubated for 6 days and the assays were then carried out in quadruplicate according to the methods previously described ( chen et al . 2002 ; frank - kamenetsky et al . 2002 ). the cells were washed in cold phosphate buffer ( pbs ) and then lysed by sonication at 4 ° c . in 50 μl of solution containing 0 . 9 % of nacl and 0 . 2 % of triton x - 100 . the alkaline phosphatase activity 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 μl 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 60 min , and then the optical density ( od ) was measured at a wavelength of 415 nm . by way of comparison , the activity of the sag compound , which activates osteogenesis by acting on the hedgehog pathway , was tested under the same conditions . the graphpad prism 4 ° software was used to plot the curves and to determine the effective concentrations 50 ( ec 50 ). ii . 2 - 1 . histochemical demonstration of the differentiation of the mesenchymal stem cells to the osteoblast lineage by the compounds of formula ( i ) the stimulation of osteogenesis in the c3h10t1 / 2 pluripotent cells was assessed by observing the induction of alkaline phosphatase ( ap ). after 6 days of differentiation in the presence of 10 μm of compounds of formula ( i ), the expression of ap was detected by histochemical staining . by way of example , an increase in the number of cells labeled is observed in the presence of compounds 1 , 4 , 6 and 8 with more or less strong intensities ( compound 4 & gt ; compound 1 & gt ; compound 8 & gt ; compound 6 ). no cell treated with the solvent ( dmso ) expresses ap at a level detectable by this method . ii . 2 - 2 . differentiation of the mesenchymal stem cells by the compounds of formula ( i ): assaying of the alkaline phosphatase activity the dose - response curves of the compounds of formula ( i ) were constructed between 10 nm and 10 μm , and the affinities of the compounds with respect to the activation of c3h10t1 / 2 cell differentiation were determined . fig3 shows a curve representative of compound 1 , produced in parallel with that of the smoothened receptor agonist sag . the latter gives a lower maximum stimulation than compound 1 , although their affinities are similar . the curves obtained for compounds 2 to 9 are shown in fig4 to 11 . all these compounds make it possible to stimulate differentiation with characteristic differences for each compound . the maximum activity is observed at 10 μm for most of them . the results of the exploitation of these curves are reported in table i hereinafter . the affinities of the compounds are of the order of 1 micromolar . compound 2 is the least active , with a maximum of 4 % of compound 1 . conversely , compound 4 is the one which exhibits the best activity , with an affinity of 0 . 6 μm and a maximum stimulation which is 20 % greater than that of compound 1 . the effective concentration 50 ( ec 50 ) of the compounds on the differentiation is expressed in μm . the maximum stimulation is expressed as a percentage of that obtained with compound 1 in the same experiment . the data correspond to the mean of 2 to 5 independent experiments . aghaloo , t . l ., c . m . amantea , et al . 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