Patent Application: US-201414911138-A

Abstract:
the present invention relates to diphenyloxyalkylamine derivatives and aryloxyalkylamine derivatives that are structurally analogous to mexiletine , said derivatives having important biological activity and not causing the undesired side effects observed with the prototype , as well as with other drugs from the same therapeutic class as the prototype . the derivatives of the present invention have formulas ii and iii and are used for treating , preventing or inhibiting pulmonary inflammatory diseases , for example , asthma and chronic obstructive pulmonary disease .

Description:
it has been observed by the inventors that suitable structural modifications in the mexiletine molecule result in obtaining analogues with anti - inflammatory and bronchodilator properties . an important aspect is that such derivatives have low activity on the sodium channel , unlike the prototype , as seen in electrophysiological assays using the “ patch clamp ” technique in gh3 cells . based on these data , the present invention proposes a new therapeutic method for the treatment of diseases related to obstruction and inflammation of airways , such as asthma and copd , by topical or systemic administration of diphenyloxyalkylamine derivatives and aryloxyalkylamine derivatives , devoid of local anesthetic and antiarrhythmic activity , such as diphenyloxyalkylamine derivatives and aryloxyalkylamine derivatives disclosed by this invention . the diphenyloxyalkylamine derivatives and aryloxyalkylamine derivatives of the invention are characterized in that the compounds , or one of its salts formed by organic or mineral acids , are represented by the formulas ( ii ) and ( iii ) below : the substituents r1 , r2 , r3 , r4 , r5 , r6 , r7 , r8 , r9 and r12 may be characterized by one ( or more ) h , ch3 , oh , cf 3 , alkoxides , halogens , linear or branched and / or cyclic alkyl radicals , benzyl groups , phenyl , alkenes or alkynes , hydroxyl , hydroxyalkyl , thioalkyl or oxygen functions in acyclic or cyclic systems , forming the heterocyclic ring . include electron donor and remover groups , as acetamide and the nitro group ; the substituents r10 and r11 may be represented by h , ch3 , linear or branched and / or cyclic alkyl radicals , benzyl groups , phenyl , alkenes or alkynes , or oxygen functions in acyclic or cyclic systems , forming the heterocyclic ring . “ n ” can be formed of 1 to 4 carbon atoms as spacer ; the substituents r1 and r2 may be characterized by one ( or more ) h , ch3 , linear or branched and / or cyclic alkyl radicals , benzyl groups , phenyl , alkenes or alkynes , oxygen functions in acyclic or cyclic systems , forming the heterocyclic ring ; the substituent r3 may be characterized by ch3 , linear or branched and / or cyclic alkyl radicals , benzyl groups , phenyl , alkenes or alkynes ; r4 , r5 , r6 , r7 and r8 may be represented by h , ch3 , oh , linear or branched and / or cyclic alkyl radicals , benzyl groups , phenyl , alkenes or alkynes , ethers , thioethers , halogens , amines , and alkylamines . include electron donor and remover groups . electron donor and remover groups are defined in the description of preferred embodiments . “ n ” can be formed of 1 to 4 carbon atoms as a spacer ; the examples shown herein are intended only to exemplify , but without limiting the scope of the invention . as used herein , the term alkyl means an alkyl group of linear , branched or cyclic chain , of up to eight ( 8 ) carbon atoms . examples of alkyl groups used in the present invention are methyl , ethyl , propyl , butyl , “ alkyl ether ”, i . e . alkoxy can be interpreted herein as alkyl group , e . g ., methoxy , ethoxy . as used herein , the term alkene means an alkene group of linear , branched or cyclic chain , of up to eight ( 8 ) carbon atoms . examples of alkene groups used in the present invention are methylene , ethylene , propylene . as used herein , the term cyclic alkyl means a cycle alkane , alkene or containing heteroatoms , e . g ., oxygen or sulfur . as used herein , “ room temperature ” includes a range of 20 to 35 ° c . ; as used herein , the term electron remover grouping includes nitro , cyano , azide , carbonyl , carboxyl , amidine , halogen groups ; as used herein , the term electron donor grouping includes methoxyl , ethoxyl , hydroxyl , alkylamine , amine groups . salts of compounds of formula ii or iii include acid salts , such as hcl and hbr . preferred salts are those pharmaceutically acceptable . salts of compounds of formula ii or iii correspond to pharmaceutically acceptable salts , including acid salts , such as hcl and hbr . the preferred compounds of the present invention are defined by the following structures — table i , among others : examples contemplating the synthesis of the compounds structurally analogous to mexiletine , according to the present invention , will be shown below . the examples shown herein are intended only to exemplify , but without limiting the scope of the invention . the starting material substituted phenyl - phenol ( 29 . 38 mmol ) was dissolved in acetone along with sodium carbonate ( 1 - 5 eq .) and catalytic amount of potassium iodide . after previous reflux , a solution of chloroacetone ( 1 - 3 eq .) was added over 0 . 5 to 2 hours , remaining under reflux for 2 to 5 hours . the medium was evaporated to dryness , to follow with the addition of water ( 30 ml ) and extraction with ethyl acetate . the organic phase was dried and evaporated to give the first intermediate in the form of a dark oil ( 70 - 90 %). the propanone , obtained as above ( 29 . 32 mmol ), was dissolved in methanol and the medium cooled in ice bath , to follow with the addition of excess sodium borohydride ( 2 - 5 eq .). the reaction medium was stirred at room temperature for 2 - 5 hours . after addition of water , the reaction medium remained under stirring for another 30 - 60 minutes . the medium was concentrated at reduced pressure and extracted with ethyl acetate . a colorless oil was obtained after drying and evaporating the organic phase . the above oil was dissolved in pyridine ( 10 - 30 ml ) and the medium cooled in ice bath . excess tosyl chloride ( 1 to 5 eq .) was added for up to 15 minutes . after 12 - 24 hours of reaction , the medium was added a solution of hcl until reaching ph 2 to 5 . after stirring at room temperature , a white solid was precipitated in the reaction medium , which was removed by filtration . after drying , the solid was dissolved in methanol and reaction was performed with sodium azide ( 3 eq .) under reflux for 2 - 6 hours . after evaporation of the solvent , water was added and extracted with ethyl acetate . the organic phase was dried and evaporated to obtain the second intermediate , in the form of a yellowish oil ( 50 - 70 %). the azide , obtained as above ( 16 . 6 mmol ), was dissolved in methanol and catalyst pd / c was added to this solution . the medium was bubbled with h 2 for up to 10 minutes and then allowed to stir in the presence of this gas for 2 - 10 hours . after filtering the palladium , the filtrate was evaporated to obtain an oil which was subsequently dissolved in acetone and filtered again . this solution was cooled in ice bath and treated with hcl gas flow until reaching ph 1 - 5 . the precipitate was isolated by filtration followed by washing with cold acetone . the final product was obtained after drying , as a white solid , which spectral data are listed below : jme 209 [ obtained from 4 - phenyl - phenol according to the synthesis described in the example 1 ]: m . p . : 252 - 254 ° c . ; 1 h nmr ( meod , 500 mhz ) 7 . 1 - 7 . 6 ( m , 9h , ar h ), ( 4 . 0 - 4 . 2 , m , 2h , — o — c h 2 —), 3 . 75 ( m , 1h , — c h —), 1 . 44 ( d , 3h , c h 3 ), 13 c nmr ( meod , 125 mhz ): 159 . 13 , 141 . 97 , 136 . 18 , 130 . 60 , 129 . 94 , 129 . 34 , 128 . 68 , 128 . 38 , 127 . 39 , 127 . 10 , 116 . 83 , 115 . 55 , 69 . 98 , 47 . 87 , 15 . 11 ; ir ( kbr ): 4368 , 3047 , 1924 , 1608 , 1049 , 883 , 812 , 437 ; gc - ms ( 100 %): m / z 227 ( free base ). jme 257 [ obtained from 4 -( 4 ′- bromo - phenyl )- phenol according to the synthesis described in the example 1 ] m . p . :& gt ; 300 ° c . ; 1 h nmr ( meod , 400 mhz ) 7 . 1 - 7 . 6 ( m , 8h , ar h ), 4 . 0 - 4 . 3 ( m , 2h , — o — c h 2 —), 3 . 75 ( m , 1h , — c h —), 1 . 44 ( d , 3h , c h 2 , j = 6 . 76 hz ); 13 c nmr ( meod , 100 mhz ): 159 . 45 , 141 . 06 , 134 . 86 , 130 . 60 , 133 . 07 , 129 . 55 , 129 . 26 , 122 . 01 , 116 . 36 , 70 . 17 , 48 . 56 , 15 . 59 ; ir ( kbr ): 3000 , 2989 , 1603 , 1485 , 1247 , 1041 , 810 , 734 ; gc - ms ( 99 %): m / z 305 ( free base ). jme 260 [ obtained from 4 -( 4 ′- fluoro - phenyl )- phenol according to the synthesis described in the example 1 ] m . p . : 225 - 227 ° c . ; 1 h nmr ( meod , 400 mhz ): 7 . 0 - 7 . 6 ( m , 8h , ar h ), ( 4 . 0 - 4 . 3 , m , 2h , — o — c h 2 —), 3 . 75 ( m , 1h , — ch —), 1 . 45 ( d , 3h , c h 3 , j = 6 . 80 hz ); 13 c nmr ( meod , 100 mhz ): 164 . 94 , 162 . 51 , 159 . 14 , 138 . 31 , 135 . 16 , 129 . 54 , 129 . 46 , 129 . 25 , 116 . 69 , 116 . 48 , 116 . 30 , 70 . 16 , 48 . 56 , 15 . 61 ; ir ( kbr ): 2968 , 2879 , 1597 , 1498 , 1230 , 1041 , 815 , 559 , 513 ; gc - ms ( 100 %): m / z 245 ( free base ). the appropriately substituted phenol derivative ( 24 . 88 mmol ) was dissolved in acetone along with potassium carbonate ( 1 to 5 eq .) and catalytic amount of potassium iodide . under reflux , a solution of chloroacetone ( 1 to 3 eq .) was added in acetone for a period of 0 . 5 - 2 hours , remaining in this condition for a further period of 2 - 5 hours . then , water was added and extracted with ethyl acetate . the organic phase was dried and evaporated to give the first intermediate in the form of a dark oil ( 70 - 95 %). the propanone , obtained as above ( 24 . 51 mmol ), was dissolved in methanol and the medium cooled in ice bath , to follow with the addition of excess sodium borohydride ( 2 - 5 eq .). the reaction medium was stirred at room temperature for 2 - 5 hours . after addition of water , the reaction medium remained under stirring for another 30 - 60 minutes . the medium was concentrated at reduced pressure and extracted with ethyl acetate . a colorless oil was obtained after drying and evaporating the organic phase . the above oil was dissolved in pyridine ( 20 - 50 ml ) and the solution formed was cooled in ice bath . excess tosyl chloride ( 1 to 5 eq .) was added for up to 15 minutes . after 12 - 24 hours of reaction , the medium was added a solution of hcl until reaching ph 2 to 5 . after stirring at room temperature , a white solid was precipitated in the reaction medium , which was removed by filtration . after drying , the solid was dissolved in methanol and reaction was performed with sodium azide ( 3 - 7 eq .) under reflux for up to 20 hours . after evaporation of the solvent , water was added and extracted with ethyl acetate . the organic phase was dried and evaporated to obtain the second intermediate , in the form of a yellowish oil ( 50 - 70 %). the azide , obtained as above ( 12 . 32 mmol ), was dissolved in tetrahydrofuran and this solution was added triphenylphosphine ( 1 - 2 eq ). the medium was then stirred at room temperature for up to 20 hours . then , water was added and the reaction medium was heated until reflux , after which it was maintained for up to 3 hours . the organic solvent was removed by evaporation to form an oil which was dissolved again in acetone ( 30 ml ). after cooling , the solution was subjected to hcl gas flow until ph 2 - 3 , water was added and extracted with ethyl ether . the aqueous phase was basified until ph 10 - 12 and extracted with ethyl acetate . the organic phase was dried and concentrated to give an oil which was subsequently dissolved in acetone . the solution was cooled in ice bath and subjected to hcl gas flow until the medium ph is between 1 and 5 , leading to the formation of a precipitate . the precipitate was isolated by filtration followed by washing with cold acetone . the final product was obtained after drying , as a white solid , which spectral data are listed below : jme 141 ( obtained from 3 - iodine - phenol according to the synthesis described in the example 2 ): m . p . : 204 - 206 ° c . ; 1 h nmr ( d 2 o , 500 mhz ): 7 . 0 - 7 . 5 ( m , 4h , ar h ), 4 . 0 - 4 . 3 ( m , 2h , — o — c h 2 —), 3 . 8 ( m , 1h , — c h —), 1 . 4 ( d , 3h , — c h 3 j = 6 . 5 hz ); 13 c nmr ( meod , 125 mhz ): 158 . 17 , 131 . 88 , 130 . 58 , 123 . 84 , 114 . 39 , 94 . 56 , 73 . 06 , 55 . 76 , 16 . 28 ; ir ( kbr ): 3109 , 2985 , 1585 , 1502 , 1465 , 1290 , 1008 , 763 , 682 ; gc - ms ( 1000 ): m / z 277 ( free base ). jme 170 ( obtained from 2 - chloro - 5 - methyl - phenol according to the synthesis described in this example 2 ): m . p . : 220 - 222 ° c . ; 1 h nmr ( d 2 o , 400 mhz ): 7 . 22 ( m , 1h , ar h 6 ,), 7 . 05 ( m , 2h , ar h 2 , 4 ) 4 . 0 - 4 . 3 ( m , 2h , — o — c h 2 —), 3 . 90 ( m , 1h , — c h —), 2 . 27 ( s , 3h , arc h 3 ), 1 . 51 ( d , 3h , — c h 3 , j = 6 . 8 hz ); 13 c nmr ( meod , 100 mhz ): 156 . 32 , 131 . 63 , 131 . 33 , 125 . 88 , 121 . 31 , 112 . 37 , 68 . 98 , 47 . 09 , 30 . 26 , 14 . 90 , 14 . 39 ; ir ( kbr ): 3028 , 1593 , 1492 , 1246 , 1049 , 854 , 655 ; ms ( es ): 200 ( m + h ) jme - 173 ( obtained from 3 , 5 - dimethyl - 4 - bromo - phenol according to the synthesis described in the example 2 ): m . p . : 220 - 222 ° c . ; 1 h nmr ( d 2 o , 500 mhz ): 6 . 8 ( m , 2h , ar h ), 4 . 0 - 4 . 2 ( m , 2h , — o — c h 2 —), 3 . 80 ( m , 1h , — c h —), 2 . 3 ( s , 6h , arc h 3 ), 1 . 4 ( d , 3h , — c h 3 j = 10 hz ); 13 c nmr ( meod , 125 mhz ): 156 . 28 , 139 . 78 , 118 . 54 , 114 . 27 , 68 . 52 , 47 . 00 , 23 . 06 , 14 . 16 ; ir ( kbr ): 3066 , 2978 , 2120 , 1739 , 1585 , 1468 , 1319 , 1172 , 1018 , 856 , 812 , 663 ; cg - ms ( 100 %): m / z 227 ( free base ). jme 207 ( obtained from 2 - methyl - 4 - iodide - phenol according to the synthesis described in the example 2 ): m . p . : 233 - 235 ° c . ; 1 h nmr ( meod , 500 mhz ): 7 . 4 ( m , 3h , ar h ), 4 . 0 - 4 . 2 ( m , 2h , — o — c h 2 —), 3 . 7 ( m , 1h , — c h —), 2 . 2 ( s , 3h , arc h 3 ), 1 . 4 ( d , 3h , — c h 3 j = 6 . 8 hz ); 13 c nmr ( meod , 125 mhz ): 157 . 59 , 139 . 84 , 136 . 44 , 130 . 71 , 114 . 40 , 84 . 66 , 47 . 79 , 15 . 67 ir ( kbr ): 3066 , 2978 , 2120 , 1739 , 1585 , 1468 , 1319 , 1172 , 1018 , 856 , 812 , 663 ; cg - ms ( 100 %): m / z 291 ( free base ). melting points were determined in 130 fisatom apparatus and are uncorrected . analyses of proton magnetic resonance ( 1h nmr ) were determined in bruker ac 400 spectrometer at 400 mhz or 500 mhz . multiplicities were designated as : s , singlet ; d , doublet ; t , triplet ; dd , double doublet ; m , multiplet ; bs , broad signal . analyses of carbon magnetic resonance ( 13c nmr ) were determined at 100 mhz or 125 mhz . infrared spectra were obtained in a perkin - elmer 467 ftir spectrometer using potash bromide pellets . mass spectra were obtained in gc / ms column 122 5532 apparatus agilent by electron impact . the progress of all reactions was monitored by thin layer chromatography , using aluminum chromate films ( 2 . 0 × 6 . 0 cm , 0 . 25 mm ; silica gel 60 , hf - 254 , merck ) with the aid of ultraviolet light at 264 nm . for purification by chromatography column , silica gel was used ( 230 - 400 mesh ). the following examples illustrate the pharmacological properties of the compounds of the present invention in comparison to prototype compound mexiletine . they also illustrate the potential of these analogues on the inhibition of pulmonary inflammatory diseases , such as asthma and copd . evaluation of the blocking potency of the sodium current exhibited by mexiletine compared with the analogues jme - 141 , jme - 173 , jme - 188 , jme - 207 , jme - 209 , jme - 257 and jme - 260 pituitary gh3 cells obtained from mice were grown in rpmi 1640 medium containing 10 % fetal bovine serum , penicillin ( 100 u / ml ) and streptomycin ( 100 μg / ml ). the cells were kept at 37 ° c . in a humidified atmosphere with 5 % co 2 and grown in slides for 1 - 2 before use . the ion channel currents in gh3 cells were recorded according to the “ patch clamp ” technique , as previously described ( neper et al ., 1992 ). the slides containing adhered cells were placed in a chamber attached to a microscope , and continuously infused with saline with the following composition ( mm ): nacl ( 150 ), kcl ( 5 ), mgcl 2 ( 1 ), cacl 2 ( 0 . 01 ) egta ( 1 ), hepes ( 10 ), bacl 2 ( 2 ), and cdcl 2 ( 0 . 1 ). the solution ph was adjusted to 7 . 4 at room temperature with the aid of a naoh solution . the cells were observed in inverted microscope in phase contrast mode ( axiovert 100 , carl zeiss , oberkochem , germany ). the voltage clamp records in the “ whole cell ” configuration with gigaohm sealing (& gt ; 10 gω ) were obtained using an axopatch - 1d amplifier ( axon instruments , san mateo , calif .). sodium currents were recorded in saline with or without the tested compounds . the series resistance was 6 - 10 ms ) for all experiments , when the pipette was filled with intracellular saline solution with the following composition ( mm ): kcl ( 150 ), nacl ( 5 ), mgcl 2 ( 1 ), hepes ( 10 ), and egta ( 0 . 1 ). the saline solution ph was adjusted to 7 . 4 at room temperature with the aid of a naoh solution . fifteen minutes after the rupture of the membrane patch , the records of the ionic currents were started . the pulse protocols and data acquisition were controlled by an interface ( axon instruments , palo alto , calif .) and acquired using clampex 9 software . the records of sodium currents were filtered at 1 khz and sampled at 8 khz . around 26 % of series resistance was compensated electronically . the drugs were applied to the chamber by gravity . the infusion rate was maintained at 0 . 8 to 1 . 1 ml / minute and the bath volume was approximately 50 μl . the results were expressed as mean ± standard error of the mean . statistical differences were determined by using tests of analysis of variance , followed by the student - newman - keuls test . p - values lower than or equal to 0 . 05 were considered significant . table 1 shows the concentrations of substances capable of inhibiting by 50 % ( ic 50 ) the sodium current in the target cells . by the patch clamp technique , it was found that the depolarization (− 90 mv to 60 mv ) of rat pituitary cells ( gh3 cell line ) generated sodium currents that were inhibited , on a concentration - dependent form by pre - treatment with tetrodotoxin ( ic 50 = 304 nm ) ( data not shown ), while the prototype substance mexiletine showed an ic 50 for blocking the sodium current of the order of 278 μm . table 1 also shows that the analogous compounds showed ic 50 values between 178 and 1208 times higher than that shown by co - incubation with mexiletine . since the main undesirable effects of mexiletine ( including cardiovascular depression ) result directly from the suppressing activity of sodium currents , it is possible to hypothesize that the analogous compounds have a lower toxicity potential compared to the prototype mexiletine . inhibitory activity of the contraction of tracheal smooth muscle of rat presented by the compounds mexiletine , jme - 173 and jme - 207 all experimental procedures involving animals regarding this patent application were approved by the ethics committee on animal use of oswaldo cruz foundation ( ceua license — lw - 23 / 10 ). in this study , wistar rats of both sexes were used , weighing between 200 and 250 g , coming from the laboratory animal breeding center of fundacdo oswaldo cruz . as previously described ( coelho et al ., 2008 ), the animals were sacrificed by exposure to air atmosphere enriched with co 2 , then , the anterior cervical region was opened so that the trachea could be located and removed . it was then transferred to a petri dish containing krebs solution with the following composition ( mm ): nacl ( 118 ), kcl ( 4 . 8 ), cacl 2 ( 2 . 5 ), mgso 4 ( 1 . 2 ), kh 2 po 4 ( 1 . 2 ), nahco 3 ( 24 ), and glucose ( 11 ). the total segment was divided into fragments of about 3 to 4 rings , which were kept in another petri dish containing krebs solution . each fragment was mounted vertically on a 10 ml cuvette with krebs solution maintained at 37 ° c . and aerated with carbogen mixture ( 95 % o 2 and 5 % co 2 ). the lower rod is fixed to the cuvette base and the top portion was attached to the isometric transducer for measuring the voltage variation of the fragment . the transducer was connected to a device which transforms the voltage variation in digital record . the fragments were subjected to a basal voltage of 1 g and were calibrated , so that the subsequent contractions could be expressed as a percentage of this 1 g voltage . the solutions were introduced inside the cuvettes with the aid of an automatic pipette . the end of the tip was always placed at the same height and position , without touching the muscle . the tracheal rings were initially contracted with 2 . 5 μm of carbachol . when the contractions reached the plateau , each segment was washed until the total relaxation of the smooth muscle . the compounds mexiletine ( 30 - 1000 μm ) jme - 173 ( 30 - 100 μm ) and jme - 207 ( 10 - 100 μm ) were added 10 minutes before the addition of increasing concentrations of carbachol ( 10 − 8 - 10 − 4 m ). all results were expressed as percentage of the contraction produced by 2 . 5 μm of carbachol ( coelho et al ., 2008 ). the values of the mean ± standard error of the mean of the groups investigated were statistically analyzed using the test of analysis of variance ( anova ), followed by student - newman - keuls test . p - values lower than or equal to 0 . 05 were considered significant . table 2 shows that the compounds jme - 173 and jme - 207 were equieffective in blocking the contractile response of rat tracheal rings induced by the muscarinic agonist carbachol ( 10 μm ), with ic 50 values of 44 . 4 and 40 . 9 μm , respectively . pre - treatment for 10 minutes with jme - 173 and jme - 207 ( 100 μm ) reached levels of inhibition of the muscarinic contraction of the order of 98 % and 93 %, respectively . the analogues were about 10 times more potent than the prototype mexiletine , which under the same conditions inhibited 50 % of the response ( ic 50 ) at the concentration of 466 . 4 μm , reaching the blocking of about 88 % of the muscarinic contraction at the concentration of 1000 μm . to assess the potential relaxing effect of the respiratory smooth muscle , rat tracheas were obtained and maintained in an isolated organ bath , as previously described ( coelho et al ., 2008 ). the tracheal segments were then pre - contracted with carbachol at the concentration of 2 . 5 μm and subjected to increasing concentrations of the tested compounds . all results were expressed as percentage of the contraction produced by carbachol ( coelho et al ., 2008 ). the values of the mean ± standard error of the mean of the groups investigated were statistically analyzed using the test of analysis of variance ( anova ), followed by student - newman - keuls test . p - values lower than or equal to 0 . 05 were considered significant . fig1 shows the relaxing effect of the compounds jme - 173 and jme - 207 in comparison to the prototype compound mexiletine . it was observed that at conditions of pre - contraction by carbachol , the addition of mexiletine ( 10 μm - 100 mm ), in isolated tracheal ring system , caused a relaxation which increased with increasing concentration of the prototype mexiletine , to achieve a maximum relaxation effect of 40 %± 3 % ( n = 10 ). on the other hand , treatments with jme - 173 and jme - 207 ( 10 μm - 10 mm ) resulted in maximum relaxation of 118 %± 21 % ( n = 11 ) ( mean ± sem ) and 111 %± 8 % ( n = 9 ), respectively . under these conditions , the ec 50 values of relaxation for mexiletine , jme - 173 and jme - 207 were 146 . 4 mm ± 39 . 2 mm ( mean ± sem ; n = 10 ), 3 . 1 mm ± 0 . 8 mm ( n = 9 ) and 1 . 0 mm ± 0 . 3 mm ( n = 8 ), respectively . the findings show that the relaxing potency of the analogues is significantly higher than that evidenced by the prototype in this preparation . more specifically , the compounds jme - 173 and jme - 207 were , in this order , 47 and 146 times more potent as relaxation inducers than mexiletine . the results reinforce the interpretation that these analogues have therapeutic application in the control of spasm of airways . antispasmodic activity of the compound jme - 173 evaluated in the system of anaphylactic contraction of tracheal ring in this study , wistar rats of both sexes were used , weighing between 200 and 250 g , coming from the laboratory animal breeding center of fundacdo oswaldo cruz . as described previously ( da costa et al ., 2007 ), the animals were sensitized by injection into the dorsal subcutaneous tissue with mixture containing 50 μg of ovalbumin and 5 mg of aluminum hydroxide on days 0 and 7 . on the 14 th day after the first sensitization , the animals were sacrificed for the removal of the trachea . after a stabilization period of 30 minutes , the tracheal rings were contracted initially with carbachol ( 2 . 5 μm ) for testing the feasibility and reproducibility of the responses of the preparation . the tracheal rings were exposed to increasing concentrations of jme - 173 ( 3 - 30 μm ), or carrier ( 0 . 9 % nacl ) for 30 minutes before the triggering of the contractile response triggered by ovalbumin ( 100 μg / ml ). salmeterol ( 30 μm ) was used as reference treatment . the responses were expressed as mean ± standard error of the mean of at least 5 tracheal segments . all results were expressed as percentage of the contraction produced by 2 . 5 μm of carbachol . the values of the mean ± standard error of the mean of the groups investigated were statistically analyzed using the test of analysis of variance ( anova ), followed by student - newman - keuls test . the statistical evaluation of the data obtained for treatment with salmeterol was performed using the student &# 39 ; s t - test . p - values lower than or equal to 0 . 05 were considered significant . fig2 a shows the antispasmodic effect , concentration - dependent , of the compound jme - 173 on the contractile response induced by the addition of the allergen . it was observed that jme - 173 inhibited 50 % of the anaphylactic contraction response ( ec 50 ) at the concentration of 8 . 3 μm . the response was completely inhibited after treatment with 30 μm of jme - 173 . importantly , at the same concentration ( 30 μm ), the blocking exhibited by salmeterol was only 52 % ( fig2 b ). the findings demonstrate that jme - 173 was more potent than salmeterol in blocking anaphylactic contraction . it was also evident the greater antispasmodic activity of jme - 173 in the anaphylactic contraction system , in comparison to the blocking exhibited by this compound on carbachol - induced contraction . anti - anaphylactic activity of the compounds mexiletine , jme - 173 , jme - 207 and jme - 209 , evaluated in the system of degranulation of sensitized mast cells induced by antigen for this study , mast cells of the rbl - 2h3 line were used , as previously reported ( beaven et al ., 1987 ). the cells were maintained in d - mem medium supplemented with 15 % fetal bovine serum , penicillin ( 100 iu / ml ) and streptomycin ( 0 . 1 mg / ml ), and placed in an oven at 37 ° c . and atmosphere of 5 % co 2 until reaching confluence . the cells were then dissociated from the plate using trypsin , centrifuged at 1000 rpm for 5 minutes and distributed in 48 - well plates at a density of 125 , 000 cells per well . the cells were sensitized with monoclonal dnp - specific igm ( 1 μg / ml ) diluted in the same medium used for the cultivation and maintained in the oven for 20 hours . after this period , the cells were washed with tyrode - gelatin and subjected to treatment with increasing concentrations of jme - 173 , jme - 207 , jme - 209 or mexiletine for 60 minutes . then , incubation was performed with dnp - bsa ( 10 ng / ml ) for a further period of 60 minutes . after this period , 10 μl of supernatant were collected from each well and added to a 96 - well plate . the cells were lysed with 200 μl of 0 . 1 % triton x - 100 and 10 μl of the lysate of each plate were added to the 96 - well plate . then , 40 μl of substrate for the β - hexosaminidase enzyme were added to the samples . after 40 minutes of reaction , reaction stopping solution ( 0 . 2 m glycine ) was added , generating a colorimetric response , which was measured by spectrophotometer ( λ = 405 nm ). the compounds jme - 173 , jme - 207 , jme - 209 and mexiletine were also evaluated for their cytotoxic potential , based on the alamar blue assay , as previously reported ( czekanska , 2011 ). in this test , the compound terfenadine was used as positive control . the results were expressed as inhibition percentage . statistical differences were determined by using tests of analysis of variance , followed by the student - newman - keuls test . p - values lower than or equal to 0 . 05 were considered significant . several local anesthetic agents , such as lidocaine , inhibit mast cell degranulation induced by mechanisms mediated or not mediated by ige , by blocking calcium channels ( yanagi et al ., 1996 ). our results showed that the compound mexiletine also inhibited the anaphylactic degranulation of mast cells at concentrations ranging from 100 μm to 1000 μm ( fig3 ). the same figure shows that the analogues studied jme - 173 , jme - 207 and jme - 209 were equally effective in blocking mast cell degranulation caused by exposure to the allergen , evidencing , however , greater potency of the analogues studied when compared to mexiletine . table 3 shows the comparative potency values ( ic 50 ) and efficacy ( emax ) of the compounds studied . all of them inhibited by about 100 % the degranulation response , whereas ic 50 values decreased from 381 . 8 μm , obtained after treatment with mexiletine , to 28 . 6 μm , 3 . 4 μm and 2 . 3 μm after jme - 209 , jme - 173 and jme - 207 , respectively . these results , obtained with mast cells passively sensitized with ige , indicate that the analogues jme - 173 , jme - 207 and jme - 209 were capable of inhibiting the anaphylactic activation of mast cells with higher potency ( up to two orders of magnitude ) when compared to the prototype . a / j mice of both sexes were used , weighing between 18 and 20 g , coming from the laboratory animal breeding center of oswaldo cruz foundation . using barometric whole - body plethysmography ( buxco research system , wilmington , n . c . ), bronchospasm responses caused by subsequent inhalations of methacholine ( 12 , 25 and 50 mg / ml for 2 . 5 minutes , 5 - minute intervals ) were measured in standard a / j mice , awake , not immobilized , as previously reported ( coelho et al ., 2008 ; hamelmann et al ., 1997 ). penh measures in response to methacholine challenge were performed 1 hour and 3 hours after treatment with jme - 207 and jme - 209 ( 30 and 100 mg / kg ) administered orally ( gavage ). the results were expressed as mean ± standard error of the mean . statistical differences were determined by using tests of analysis of variance , followed by the student - newman - keuls test . p - values lower than or equal to 0 . 05 were considered significant . fig4 shows the effect of treatment with jme - 209 ( 30 and 100 mg / kg , orally ) or carrier ( 0 . 9 % nacl ) on the response of increase of penh ( indicative of increase in lung resistance ) induced by methacholine challenge ( 12 - 50 mg / ml ) in the times 1 hour and 3 hours after treatment . there was a slight blocking of cholinergic bronchospasm with both doses used ( 30 and 100 mg / kg ) in the analysis conducted 1 hour after treatment . the blocking shown to be active only at the highest dose , when the tests of stimulation response with methacholine was repeated 3 hours after treatment , suggesting that the compound has an action time of at least 3 hours when the substance is administered orally at a dose of 100 mg / kg . similar results were obtained when the animals were treated with jme - 207 . administered orally , at doses of 30 and 100 mg / kg , the compound significantly inhibited the response of methacholine - induced bronchoconstriction 3 hours after treatment ( fig5 ). the results together demonstrate the antispasmodic activity of the compounds jme - 209 and jme - 207 , in vivo , confirming our data obtained in isolated organ system ( in vitro ). therapeutic effect of the compounds jme - 141 , jme - 173 , jme - 207 and jme - 188 on lung inflammation and hyperreactivity in asthma model in mice male a / j mice ( 18 - 20 g ), coming from the laboratory animal breeding center of oswaldo cruz foundation , were used in the experiments . the sensitizing and antigen challenge procedures used in this study followed the experimental protocol shown in fig6 . the animals were previously sensitized with a mixture of ovalbumin ( ova ) ( 50 μg ) ( grade v ; sigma , st . louis , mo ., usa ) and aluminum hydroxide ( 5 mg ) administered subcutaneously on day 0 with boost on day 14 ( equal suspension administered intraperitoneally ). nasal instillations of ova ( 25 μg / 25 μl in sterile 0 . 9 % nacl ) were administered on days 14 , 21 , 28 and 35 , with the hyperresponsiveness analysis performed 24 hours after the last challenge . as shown in fig6 , the treatments with jme - 141 , jme - 173 , jme - 207 , and jme - 188 were carried out only on days 28 and 35 , 1 hour before the challenge with ova , by nebulization for 30 minutes . that is , the treatments took place after the installation of asthma framework , reflecting a therapeutic action of the respective compounds analyzed . the effect of the treatments on bronchial hyperreactivity was investigated by measuring the resistance changes and pulmonary elastance , using invasive barometric plethysmography whole - body system ( buxco , usa ), as previously described ( olsen et al ., 2012 ). the results were expressed as mean ± standard error of the mean . statistical differences were determined by using tests of analysis of variance , followed by the student - newman - keuls test . p - values lower than or equal to 0 . 05 were considered significant . as shown in fig7 , the nebulization of the animals for 30 minutes with the compounds jme - 141 , jme - 173 , jme - 207 and jme - 188 ( 2 %), starting from the third week of allergen challenge , abolished the framework of hyperreactivity of airways , observed in animals antigenically sensitized and challenged , treated only with carrier ( tween - 80 , 0 . 2 %). the inhibition was found to be effective both in lung resistance increasing response , as in the increase of lung elastance observed after exposure of methacholine . all compounds were equally active in blocking leukocyte infiltration , evaluated by bronchoalveolar lavage , especially for the inhibition of eosinophilic infiltration , inhibited by 50 % by all compounds tested ( fig8 ). analyses of cellularity were also carried out 24 hours after the last antigen challenge . our results indicated that the inhibition of hyperresponsiveness and cellular recruitment responses , as evidenced by treatment with jme - 173 , was shown to be associated to the blocking of pro - inflammatory cytokine generation including eotaxin - 2 , il - 5 and il - 13 , without change in the increased levels of the anti - inflammatory cytokine il - 10 ( fig9 ). the production of cytokines il - 5 and il - 13 was equally sensitive to the treatment with jme - 207 or jme - 188 , but only the latter inhibited eotaxin - 2 , while both failed to modify the increased production of eotaxin - 1 . these data indicate that , with minor particularities , the compounds jme - 141 , jme - 173 , jme - 207 and jme - 188 , administered via nebulization ( 2 %), are active in blocking lung inflammation and hyperreactivity associated with the asthmatic response . the joint results also suggest that the inhibition of the generation of the pro - inflammatory th 2 cytokines can be involved in the blocking of pathological features of asthma profile observed in this model . effect of nebulization with tme - 173 on inflammation , mucus production and lung remodeling in murine model of asthma male a / j mice ( 18 - 20 g ), coming from the laboratory animal breeding center of oswaldo cruz foundation , were used in the experiments . the sensitization procedures , antigen challenge and treatment used in this study followed the experimental protocol shown in fig6 . histological techniques were used for the quantification of mucus and peribronchial fibrosis , following previously reported and validated experimental protocols ( serra et al ., 2012 ). the results were expressed as mean ± standard error of the mean . statistical differences were determined by using tests of analysis of variance , followed by the student - newman - keuls test . p - values lower than or equal to 0 . 05 were considered significant . the treatment by nebulization with jme - 173 in the concentrations of 0 . 5 %, 1 % or 2 % for 30 minutes , started at the third week of allergen challenge ( fig1 ), confirmed the antiasthmatic effect of this compound . it was evident in the three aerosol concentrations tested that the compound jme - 173 was able to block the response of airway hyperreactivity even at the lowest concentration ( 0 . 5 %), as illustrated in fig1 . in this model , challenge with ovalbumin caused a significant increase in the amount of mucus present in the airways of the sensitized animals ( micrograph b of fig1 ) ( pas staining , arrowheads ), when compared with control animals challenged with 0 . 9 % saline ( micrography b ). it is noted in fig1 , part c , that the exacerbated mucus production observed in asthmatic mice was substantially inhibited by the treatment with jme - 173 ( 0 . 5 %). fig1 d shows the result of quantitative analysis , where it is evident that jme - 173 inhibited mucus production by about 70 %. the staining of histological sections of lung tissue with gömöri trichrome stain evidenced a marked accumulation of extracellular matrix in the peribronchial region ( indicated by the arrowhead ) in the animals challenged with ovalbumin ( fig1 , part b ), when compared to animals in the negative control group ( part a ). the treatment with jme - 173 clearly abolished the fibrotic response , as illustrated by the representative image , as well as by the quantitative analysis conducted based on the morphometry ( fig1 part c and d , respectively ). taken together , the data suggest that the nebulization treatment with jme - 173 is capable of reversing airways hyperreactivity , as well as inhibits mucus production in the lower airways and peribronchial fibrosis caused by intranasal instillation of allergen agent in sensitized mice . effect of the compound jme - 209 on lung inflammation and airways hyperreactivity caused by lps male a / j mice ( 18 - 20 g ), coming from the laboratory animal breeding center of oswaldo cruz foundation , were used in the experiments . the mice were anesthetized with halothane aerosol ( cristália , sp , brazil ) to receive intranasal administration of lps ( 25 μg / 25 μl 0 . 9 % nacl , instillation ) or 0 . 9 % nacl ( 25 μl ) ( negative control ). the animals were pretreated with jme - 209 ( 30 and 100 mg / kg , orally ) 1 hour before instillation of lps , and analysis of the impact of treatment on leukocyte recruitment in the airspace was performed 18 hour after challenge . obtaining of bronchoalveolar lavage , as well as the total and differential leukocyte counts carried out in this effluent , were made as previously described ( kummerle et al ., 2012 ). thus , after 18 hrs of lps instillation , the mice were sacrificed by terminal anesthesia with thiopental ( 500 mg / kg ). then , they had the trachea dissected and cannulated . bronchoalveolar lavage ( bal ) was performed by 3 consecutive lavages of 800 μl of pbs containing edta ( 10 mm ). the lavages were then subjected to centrifugation ( 1500 rpm - 10 minutes ) and the cell “ pellet ” resuspended in the volume of 0 . 5 ml of pbs / edta solution 10 mm . the total leukocyte count from the lavage was performed in a neubauer chamber by light microscopy ( 100 × magnification ), diluting an aliquot of the cell suspension from the lavage in tprk liquid ( 1 : 40 ). the differential counting was performed on cytocentrifuged , which were stained with may - grunwald - giemsa and assessed using oil immersion objective ( 1000 × magnification ) ( kummerle et al ., 2012 ). the airway hyperreactivity was also assessed 18 hours after lps , by exposing the animals to increasing concentrations of aerosolized methacholine ( 3 - 27 mg / ml ) in finepoint r / c buxco ® system ( buxco electronics , sharon , conn ., usa ). the mice were anesthetized with nembutal ( 60 mg / kg , i . p .) for the tracheostomy procedure and connection of the animal to mechanical ventilation and pneumotachograph of the finepoint platform . the neuromuscular activity was blocked with pancuronium bromide ( 1 mg / kg , i . v .) to enable the pulmonary resistance records ( cm h20 / ml / s ) and elastance ( cm h20 / ml ) in each respiratory cycle , as previously reported ( olsen et al ., 2011 ). the results were expressed as mean ± standard error of the mean . statistical differences were determined by using tests of analysis of variance , followed by the student - newman - keuls test . p - values lower than or equal to 0 . 05 were considered significant . in this model of acute pulmonary inflammation caused by endotoxin , the treatments with jme - 209 ( 30 and 100 mg / kg , orally ) administered 1 hour before lps ( 25 μg / animal ), inhibited leukocyte infiltration in bronchoalveolar space , in particular reducing the levels of eosinophils and neutrophils , without significantly altering the increase in the number of mononuclear cells ( fig1 ). under these conditions , the treatment also inhibited the mechanical ventilation changes ( airway hyperreactivity ), represented by the significant increase in lung resistance and elastance values , which are indicators of air flow reduction in the central airways and reduction of expansion capacity of the lung parenchyma , respectively ( fig1 ) in conclusion , the results show that the pulmonary inflammation and airway hyperreactivity caused by lps were clearly inhibited by the oral treatment with jme - 209 , suggesting that this compound has potential for inhibiting chronic pulmonary inflammatory diseases , such as asthma and chronic obstructive pulmonary disease ( copd ). protective effect of tme - 209 on acute airway inflammation induced by tobacco smoke in mice male a / j mice ( 18 - 20 g ), coming from the laboratory animal breeding center of oswaldo cruz foundation , were used in the experiments . the animals were placed in a chamber and subjected to an atmosphere enriched with 100 ml of smoke from 4 filter cigarettes ( trade mark ) for 1 minute on four consecutive days . control animals were exposed to the condition in which cigarette smoke was replaced by equal volume of ambient air ( castro et al ., 2009 ). the treatments with dexamethasone ( 1 mg / kg ) or jme - 209 ( 30 and 100 mg / kg ) were carried out orally 1 hour before each exposure to smoke . the compounds were dissolved in 0 . 9 % nacl just prior to administration . obtaining of bronchoalveolar lavage , as well as the total and differential leukocyte counts carried out in this effluent , were made as previously described ( olsen et al ., 2011 ). thus , after 24 hrs of the last exposure to cigarette smoke , the mice were sacrificed by terminal anesthesia with thiopental ( 500 mg / kg ). then , they had the trachea dissected and cannulated bal was performed by 3 consecutive lavages of 800 μl of pbs containing edta ( 10 mm ). the lavages were then subjected to centrifugation ( 1500 rpm - 10 minutes ) and the cell “ pellet ” resuspended in the volume of 0 . 5 ml of pbs / edta solution 10 mm . the total leukocyte count from the lavage was performed in a neubauer chamber by light microscopy ( 100 × magnification ), diluting an aliquot of the cell suspension from the lavage in tprk liquid ( 1 : 40 ). the differential counting was performed on cytocentrifuged , which were stained with may - grunwald - giemsa and assessed using oil immersion objective ( 1000 × magnification ). the results were expressed as mean ± standard error of the mean . statistical differences were determined by using tests of analysis of variance , followed by the student - newman - keuls test . p - values lower than or equal to 0 . 05 were considered significant . in this model of acute pulmonary inflammation by cigarette smoke established in mice ( castro et al ., 2009 ), the treatment with jme - 209 ( 30 and 100 mg / kg , orally ) 1 hr prior to challenge with smoke significantly inhibited the accumulation of leukocytes in the bronchoalveolar space , while the treatment with dexamethasone ( 3 mg / kg , orally ) was ineffective ( fig1 ). the increase in total leukocytes resulted substantially from increases in the numbers of neutrophils , eosinophils and mononuclear cells in the bronchoalveolar effluent , which changes were blocked by jme - 209 . the treatment with the steroidal anti - inflammatory dexamethasone ( 1 mg / kg , orally ) also inhibited the slight increase in the number of eosinophils , but was unable to inhibit the accumulation of mononuclear cells and only partially inhibited neutrophil infiltration ( fig1 ). in conclusion , considering that cigarette smoke is a major cause of asthma and copd worsening , the results presented herein strongly suggest that the treatment with jme - 209 has the potential to prevent pulmonary inflammation associated with cigarette smoke , an important pathogenesis factor in these patients . the derivatives of the present invention , as described herein , are usually administered as a pharmaceutical composition . such compositions may be prepared by procedures well known in the pharmaceutical art and comprise at least one active compound of the invention . the compounds of this invention are usually administered in a pharmaceutically effective amount . the actual amount of compound administered will be typically determined by a physician , in the light of the relevant circumstances , including the condition to be treated , the chosen route of administration , the compound administered , the age , weight and response of the individual patient , the severity of the symptoms of the patient , and so forth . the derivatives and compositions described in this patent application can be administered to a subject , preferably a mammal , more preferably a human , to treat and / or prevent the disease by any suitable route . the compositions containing the derivatives of the present invention may be formulated as : the compositions of the present invention are typically formulated with suitable carriers and may be exemplified as follows . the carriers ( components ) described above for the compositions are merely representative . other materials , as well as processing techniques and the like , are set in specific literature , such as remington &# 39 ; s pharmaceutical sciences , 18th edition , 1990 , mack publishing company , easton , pa ., 18042 . although the present invention has been described with respect to specific embodiments , it is evident that many alternatives and variations are apparent to those skilled in the art . these alternatives and variations should be considered to be supported by the scope of the claims . documents belonging to the state of the art of the knowledge of the inventors and cited in the present descriptive report are listed below . 1 . u . s . pat . no . 3 , 659 , 019 2 . u . s . pat . no . 3 , 954 , 872 3 . u . s . pat . no . 4 , 031 , 244 4 . beaven m a , maeyama k , wolde - 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