Patent Application: US-30792107-A

Abstract:
the present invention provides compounds of formula : wherein a and z are as defined in the description , along with methods for preparing such derivatives and their use for the treatment of inflammatory diseases such as neuropathic pain .

Description:
it has now been found that by replacing the pyridyl moiety in the above compounds with a isoquinolin - 5 - yl ring , trpv1 antagonists with improved properties can be obtained accordingly , the invention provides improved vanilloid - 1 receptor modulators of general formula ( i ) z is a phenyl or a pyridyl ring , optionally substituted with one or two r groups , which can be the same or different from one another and are selected from c 1 - c 4 alkyl , preferably methyl , isopropyl or tert - butyl , c 1 - c 4 haloalkyl , preferably trifluromethyl , or halogen . for the purposes of the present description , halogen means a halogen atom selected from fluorine , chlorine , bromine and iodine , preferably chlorine . a first group of preferred compounds of the invention is that of formula ( ia ) a second group of preferred compounds according to the invention is in the compounds of formula ( ia ) and ( ib ) z is preferably a phenyl ring substituted at the para - position with an r group other than hydrogen , preferably chlorine . the most preferred compound according to the invention is n -( 4 - chlorophenyl )- 6 -( isoquinolin - 5 - yl ) pyridine - 3 - carboxamide ( herein after referred to as v394 ) the compounds of formula ( i ) can be prepared by means of conventional methods , such as the reaction of a compound of formula ( ii ) wherein a is as defined above and the carboxy group is activated as chloride preferably , the compounds of formula ( i ) can be obtained by suzuki reaction 2 between a compound of formula ( iv ) wherein a and z are as defined above and x is a halogen selected from iodine and bromine for example , compound v394 is conveniently prepared by suzuki reaction between 6 - bromo - or 6 - chloro - n -( 4 - chlorophenyl ) pyridine - 3 - carboxamide and isoquinolin - 5 - yl - 5 - boronic acid . the compounds of formula ( i ) modulate the vanilloid trpv1 receptor ; the preferred compound v394 showed a k i value of 15 nm ( 13 - 17 ) in rat spinal chord and an ic 50 value of 0 . 83 nm ( 0 . 74 - 0 . 93 ) in cultured rat dorsal root ganglia neurons . accordingly , the compounds of the invention can be used for the preparation of pharmaceutical compositions for the treatment of inflammatory states , such as chronic pain and inflammatory hyperalgesia . these formulations can be prepared by conventional methods and excipients , such as those disclosed in remington &# 39 ; s pharmaceutical science handbook , xvii ed . mack pub ., n . y ., u . s . a . the invention will be herein after illustrated by means of the following example . all commercially available compounds were purchased from aldrich and were used without further purification . reaction courses were monitored by thin - layer chromatography on silica gel ( precoated f 254 merck plates ), the spots were examined with uv light and visualized with aqueous kmno 4 . flash chromatography was performed using merck silica gel ( 230 - 240 mesh ). 1 h - nmr spectra were recorded on a varian 400 mhz spectrometer using tms as internal standard . mass spectra were obtained with a waters - micromass zmd spectrometer . melting points were determined on a buchi - tottoli apparatus and are uncorrected . commercially available 6 - chloro - nicotinic chloride ( 56 . 8 mmol , 10 g ) was dissolved in 50 ml of anhydrous ch 2 cl 2 and added dropwise to a solution of diisopropylethylamine ( diea ) ( 1 . 2 equivalents , 68 . 2 mmol , 11 . 67 ml ) and 4 - chloroaniline ( 1 . 2 equiv ., 68 . 2 mmol , 8 . 70 g ) in 50 ml ch 2 cl 2 at o ° c . the mixture was stirred at room temperature for 20 h , then diluted with ch 2 cl 2 ( 200 ml ) and washed with water ( 1 × 200 ml ) and brine ( 1 × 100 ml ). the organic layer was dried over sodium sulphate and concentrated . the crude was crystallized from diethyl ether to give 13 g of a white solid . yield = 86 %. 1 h nmr ( cdcl 3 , 200 mhz ) δ 7 . 35 ( 2h , d , j = 8 . 8 hz ), 7 . 47 ( 1h , d , j = 8 . 2 hz ), 7 . 58 ( 2h , d , j = 8 . 8 hz ), 7 . 88 ( 1h , bs ), 8 . 16 ( 1h , dd , j = 8 . 4 hz , j ′= 2 . 8 hz ), 8 . 84 ( 1h , d , j = 2 . 4 hz ) a 2 . 5 m solution of n - buli ( 1 . 2 equiv ., 3 mmol , 1 . 2 ml ) in 20 ml of freshly distilled thf , cooled to − 78 ° c ., was added with a solution of 5 - bromoisoquinoline ( 2 . 5 mmol , 520 mg ) in 5 ml of thf . the resulting mixture was allowed to react at this temperature over 45 ′. a solution of triisopropylborate ( 1 . 2 equiv ., 3 mmol , 0 . 7 ml ) was then added and the mixture was stirred at the same temperature for 5 ′ and then allowed to warm to room temperature and stirred for an additional hour . the mixture was quenched by slow addition of a 5 % naoh solution ( 30 ml ). the aqueous layer was separated and acidified to ph 5 / 6 by addition of 10 % hcl at o ° c . extraction with ethyl acetate , evaporation of the organic phase and crystallisation from diethyl ether gave 250 mg of a white solid . yield = 58 %. 1 h nmr ( d 6 - dmso , 200 mhz ) δ 7 . 66 ( 1h , t , j = 7 . 2 hz ), 8 . 07 ( 1h , d , j = 5 . 8 hz ), 8 . 13 ( 1h , d , j = 8 . 0 hz ), 8 . 34 ( 1h , d ), 8 . 47 ( 1h , d ), 8 . 50 ( 2h , bs ), 9 . 29 ( 1h , s ); [ m + 1 ] 174 . 1 ( c 9 h 8 bno 2 requires 172 . 98 ) a mixture of isoquinolin - 5 - yl - 5 - boronic acid ( 1 . 5 equiv ., 8 . 4 mmol , 1 . 46 g ), 6 - chloro - n -( 4 - chlorophenyl ) pyridine - 3 - carboxamide ( 5 . 6 mmol , 1 . 5 g ), palladium acetate ( 4 % mol , 48 mg ), triphenylphosphine ( 2 equiv ., 2 . 94 g ), 15 % na 2 co 3 ( 4 ml ), etoh ( 4 ml ) and toluene ( 50 ml ) was heated at 80 ° c . for 16 h . after evaporation , a saturated sodium bicarbonate solution was added and the precipitated solid was filtered and then washed with ethyl acetate . the residue was recrystallized from methanol to obtain 1 . 4 g of compound v394 as a white solid . m . p . ( diethyl ether )= 258 ° c . yield = 69 %. 1 h nmr ( d 6 - dmso , 400 mhz ) δ 7 . 471 ( 2h , d , j = 8 . 8 hz ), 7 . 838 ( 1h , m ), 7 . 852 ( 2h , d , j = 8 . 8 hz ), 7 . 953 ( 1h , d , j = 8 . 0 hz ), 8 . 049 ( 1h , dd , j = 7 . 2 hz , j ′= 0 . 8 hz ), 8 . 085 ( 1h , d , j = 6 . 0 hz ), 8 . 290 ( 1h , d , j = 8 . 4 hz ), 8 . 490 ( 1h , dd , j = 8 . 4 hz , j ′= 2 . 2 hz ), 8 . 543 ( 1h , d , j = 6 . 0 hz ), 9 . 300 ( 1h , d , j = 1 . 6 hz ), 9 . 438 ( 1h , s ), 10 . 695 ( 1h , s ); [ m + 1 ] 360 . 4 ( c 21 h 14 cln 3 o requires 359 . 81 ). newborn and adult sprague - dawley rats (˜ 250 g ) were used ( harlam , italy ). all experiments complied with the national guidelines and were approved by the regional ethics committee . male sprague - dawley rats with body weight between 250 to 350 g at the time for testing were used . for binding assays rats were sacrificed by decapitation under anesthesia and the spinal cord was removed and disrupted using a polytron tissue homogenizer in ice cold buffer containing 5 mm kcl , 5 . 8 mm nacl , 0 . 75 mm cacl 2 , 2 mm mgcl 2 , 320 mm sucrose , 10 mm hepes , ph 8 . 6 . 5 the homogenized tissue was centrifuged at 1000 × g for 10 min at 4 ° c . and the supernatant was centrifuged again at 35000 × g for 30 min at 4 ° c . ( beckman avanti j25 ). the pellet was resuspended in the same buffer as described above and used in binding experiments . in saturation experiments , 150 μg protein / sample from membrane suspensions were incubated with [ 3 h ]- resiniferatoxin ([ 3 h ]- rtx ) ( 0 . 003 - 3 nm ) in the assay buffer containing 0 . 25 mg / ml fatty acid - free bovine serum albumin at 37 ° c . for 60 min . in competition experiments , the membranes were incubated at 37 ° c . for 60 min with [ 3 h ] rtx ( 0 . 4 nm ) and with increasing concentrations ( from 0 . 1 nm to 3 μm ) of examined compounds . non specific binding was evaluated in the presence of 1 μm rtx . after incubation the reaction mixture was cooled at 0 ° c . and incubated with bovine α1 - acid glycoprotein ( 200 μg per tube ) for 15 min to reduce non - specific rtx binding . membrane - bound rtx was separated from free rtx by centrifuging the samples at 18500 × g for 15 min . the tip of the microcentrifuge tube containing the pellet was cut off and the radioactivity was determined by scintillation counting ( packard 2500 tr ). protein concentration was determined according to a bio - rad method with bovine serum albumin as a standard reference ( bradford , 1976 ). saturation and competition studies were analyzed with the ligand program . 6 ca 2 + fluorescence measurements in cultured rat dorsal root ganglia neurones adults rats were terminally anaesthetized and decapitated . dorsal root ganglia were removed and placed in cold phosphate buffered solution ( pbs ) before being transferred to collagenase ( 10 mg / ml ), trypsin ( 5 mg / ml ) and dnase ( 1 mg / ml ) for 35 min at 37 ° c . the ganglia , placed in cold dmem supplemented with 10 % fetal bovine serum , 2 mm l - glutamine , 100 u / ml penicillin and 100 μg / ml streptomycin , were dissociated in single cells by several passages through a series of syringe needles ( 23 g down to 25 g ). the medium and the ganglia were filtered to remove debris , topped up with 8 ml of dmem medium and centrifuged ( 200 × g for 5 min ). the final cell pellet was re - suspended in dmem medium [ supplemented with 100 ng / ml mouse nerve growth factor ( mouse - ngf - 7s ) and cytosine - β - d - arabinofuranoside free base ( ara - c ) 2 . 5 μm ]. the cells were plated on poly - l - lysine ( 8 . 3 μm )- and laminin ( 5 μm )- coated 25 mm glass cover slips and kept for 5 to 8 days at 37 ° c . in a humidified incubator gassed with 5 % co 2 and air , then added with fura - 2 - am - ester ( 3 μm ) in a ca 2 + buffer solution having the following composition ( mm ): cacl 2 1 . 4 , kcl 5 . 4 , mgso 4 0 . 4 , nacl 135 , d - glucose 5 , hepes 10 with bsa ( 0 . 1 %), at ph 7 . 4 , for 40 min at 37 ° c . the cells were then washed twice with the ca 2 + buffer solution and transferred to a chamber on the stage of a nikon eclipse te300 microscope . fura - 2 - am - ester was excited at 340 nm and 380 nm to indicate relative [ ca 2 + ] i changes by the f 340 / f 380 ratio recorded with a dynamic image analysis system ( laboratory automation 2 . 0 , rcs , florence , italy ) and the cells were allowed ( at least 10 min ) to attain a stable fluorescence before beginning the experiment . a calibration curve was performed using buffer containing fura - 2 - am - ester and determinant concentrations of free ca 2 + . this curve was then used to convert the data obtained from the f 340 / f 380 ratio to [ ca 2 − ] i ( nm ). 8 the effects of pretreatments with capsazepine ( cpz ), sb366791 and v394 on the increase in [ ca 2 + ] i produced by 0 . 1 μm capsaicin were studied . capsaicin ( 20 nmols / 50 μl / paw ) was injected in the plantar surface of the glabrous skin of the right paw of rats anesthetized with diethyl ether ( chaplan et al ., 1994 ). compound v394 was orally administrated ( 30 μmol / kg ) 2 hours prior to capsaicin injection . tactile allodynia was evaluated 90 min after capsaicin challenge . drugs and reagents were obtained from the indicated companies : [ 3 h ]- resiniferatoxin ( perkin elmer , boston , mass . ), sb - 366791 ( tocris , uk ), capsaicin , capsazepine , ionomycin , laminin , poly - l - lysine , substance p ( sigma , italy ); mice ngf - 7s and collagenase / dispase ( roche diagnostics , italy ); dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ), foetal bovine serum ( fbs ) heat inactivated , l - glutamine ( 200 mm ), penicillin / streptomycin ( 10 , 000 iu / ml ± 10 , 000 ug / ml ), ( gibco , italy ); fura - 2 - am - ester ( società italiana chimici , italy ). the stock concentrations of capsaicin ( 10 mm ), capsazepine ( 10 mm ), ( e )- 3 -( 4 - chlorophenyl )- n -( 3 - methoxyphenyl ) acrylamide ( identified as sb - 366791 ) ( 1 mm ) and v394 were prepared in 50 % dmso and 50 % tween 80 . fura - 2 - am - ester and ionomycin were dissolved in 100 % dmso . all other drugs were dissolved in distilled water . appropriate dilutions were then prepared in krebs buffer solution . the saturation curve of [ 3 h ]- rtx to trpv1 expressed in rat spinal cord showed a k d value of 0 . 21 ( 0 . 16 - 0 . 27 ) and a b max value of 57 ( 53 - 62 ) fmol / mg protein . the scatchard plot was essentially linear and computer analysis of the data indicated that only one class of high affinity binding sites was present . competition binding experiments of [ 3 h ]- rtx revealed that v394 and the reference sb - 366791 had a k i value of 15 ( 13 - 17 ) nm and 36 ( 30 - 43 ) nm respectively . capsaicin ( 0 . 1 μm ) caused an increase in [ ca 2 + ] in the vast majority ( 95 %) of dorsal root ganglia neurons , which were thereby identified as trpv1 expressing neurons . the ic 50 value of v394 that inhibited capsaicin - evoked [ ca 2 + ] i mobilization was 0 . 83 nm ( 0 . 74 − 0 . 93 ). the reference trpv1 antagonists , capsazepine and sb - 366791 , inhibited the capsaicin response with an ic 50 of 948 ( 676 − 1330 ) nm and 8 . 7 ( 3 . 4 − 17 . 3 ) nm , respectively . the results are expressed as mean and 95 % fiducial limits . 90 min . after the capsaicin challenge , compound v394 produced a significant preventive effect ( 55 %) against the pro - allodinic effect of capsaicin . 1 . bakthavatchalam , r . ; 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