Abstract:
The present invention provides compounds of formula (I): 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:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is the national phase of International Application PCT/EP2007/005835 filed on Jul. 2, 2007 which, in turn, claims priority to European Patent Application 06014302.1 filed on Jul. 10, 2006. 
     FIELD OF THE INVENTION 
     The present invention relates to modulators of the vanilloid receptor, in particular to TRPV1 antagonists. 
     STATE OF THE ART 
     The Transient Receptor Potential Vanilloid 1 (TRPV1) is strongly involved in the genesis of thermal and mechanical hyperalgesia and has been proposed to play a key role in different pathological conditions including neuropathic pain and urological disorders. 
     Vanilloid receptor antagonists containing a biaryl moiety are known; WO 2004/056774 1  discloses, among others, the following substituted biaryl-4-carboxylic acid arylamides: 
     
       
                 
         
             
             
         
      
     
    
    
     DESCRIPTION OF THE INVENTION 
     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) 
     
       
                 
         
             
             
         
      
         
         
           
             wherein: 
             A is CH or N; 
             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) 
                                
in which Z is as defined above.
 
     A second group of preferred compounds according to the invention is that of formula (Ib) 
                                
in which Z is as defined above.
 
     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
 
with a compound of formula (III)
 
Z—NH 2   (III)
 
           
         
       
    
     wherein Z is as defined above. 
     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
 
and boronic acid (V)
 
           
         
       
    
     
       
                 
         
             
             
         
      
     
     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. 
     EXAMPLE 
     Materials and Methods 
     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. 
     EXAMPLE 
     N-(4-chlorophenyl)-6-(-isoquinolin-5-yl)pyridine-3-carboxamide (V394) 
     Step a)—6-Chloro-N-(4-chlorophenyl)pyridine-3-carboxamide 
     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) 
     Step b)—Isoquinolin-5-yl-5-boronic acid 
     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) 
     Step c)—N-(4-chlorophenyl)-6-(isoquinolin-5-yl)pyridine-3-carboxamide (Suzuki Reaction) 
     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). 
     Biological Assay 
     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. 
     Radioligand Binding Assay 
     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-Induced Secondary Allodynia in Rat 
     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 
     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. 
     Results 
     Radioligand Binding Assay 
     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. 
     Ca 2+  Fluorescence 
     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. 
     Capsaicin-Induced Secondary Allodynia in Rat 
     90 min. after the capsaicin challenge, compound V394 produced a significant preventive effect (55%) against the pro-allodinic effect of capsaicin. 
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