Patent Application: US-74600808-A

Abstract:
there is a need for pharmaceutical compounds which have activity at , at least one of a ppar and a cannabinoid receptor . thus there are provided such compounds , wherein the compound comprises : a ppar pharmacophore and a cannabinoid pharmacophore linked together by a moiety comprising a fused bicyclic ring comprising a five membered ring fused with a six membered ring or a six membered ring fused with a six membered ring ; wherein the cannabinoid pharmacophore comprises the fused bicyclic ring ; and the ppar pharmacophore comprises a salicylic acid , alkoxybenzylacetic acid or a alkoxyphenylacetic acid functionality ; and wherein the ppar pharmacophore is linked to the bicyclic ring of the cannabinoid pharmacophore through a linker comprising an amine or an amide functional group .

Description:
during the course of the studies into the dual active compounds of the present invention of the mtl approach , it was surprising discovered that a number of the compounds have surprisingly advantageous utility at , at least a single receptor , rather than a balanced activity at both receptors concurrently . in particular , it was surprisingly found that a number of the compounds of the invention were particularly potent at a ppar receptor when compared to normal control compounds known to have reasonable activity for a particular given dose . these compounds when used at comparable doses appear to be substantially more potent at ppar - γ receptors in particular . the results show that the compounds were surprisingly more active at the ppar receptor than was initially indicated by the goldscore docking results initially carried out . compound structural modifications involved introducing the 4 - amino ( 4 - asa ) or 5 - aminosalicylate ( 5 - asa ) groups , which were known to activate the pparα and γ receptor , into the cb 2 agonists ligands . the compound win 55 , 212 - 2 is an example of a potent non - classical cannabinoid receptor agonist , and acts as a potent analgesic in a rat model of neuropathic pain . win 55 , 212 - 2 is a member of the aminoalkylindole family and is a weaker partial agonist than thc , but displays a higher affinity towards the cb 1 receptor . another compound , jte - 907 , a 2 - oxoquinoline family member , has been found to be a highly selective cb 2 ligand which behaves as an inverse agonist in vitro , but has an anti - inflammatory effect in vivo . it is known to possess a potent analgesic and anti - inflammatory activity and does not exhibit undesirable psychotropic effects . jte - 907 binds in vitro with high affinity at human cb 1 and cb 2 receptors and exerts an agonist activity . moreover , aja binds to pparγ and activates the receptor . its anti - inflammatory activity is certainly mediated by this mechanism . 8 , 21 , 22 thus aminoalkylindoles and 2 - oxoquinolines were chosen as starting points in the design of non - cleavable conjugated pharmacophores . in the aminoalkylindoles family , the morpholine group of win - 55212 - 2 derivatives was replaced by the 4 - amino ( 4 - asa ) or 5 - aminosalicylate ( 5 - asa ) group . sar data indicated that exchange at the r 1 and r 3 substituents on the aminoalkylindole should lead to retention of target activity . 19 in the 2 - oxoquinoline family , the benzodioxole group of jte - 907 was replaced by salicylate groups . 19 , 20 the structure of the human ppars ligand - binding domain was obtained from its complexed tesaglitazar ( az 242 ) x - ray crystal structure which is available in the rcsb protein data bank ( http :// www . rcsb . org / pdb / home / home . do ) ( pdb id : 117i ). 16 , 17 since the experimental determination of the g - protein coupled receptors ( gpcrs ) structures has not yet been realised , a theoretical model of the cb 2 receptor was constructed by homology modelling using the x - ray structure of the gpcr bovine rhodopsin as a template . 18 structurally modified cb 2 selective agonist compounds and their ppars and cb 2 active sites binding modes were investigated ( see tables 1 and 2 ). the retained compounds were found to belong to the classical and non - classical cannabinoids , i . e ., the aminoalkylindoles and 2 - oxoquinolines families respectively . docking simulations were carried out in order to predict the binding mode of these compounds in the ppars and cb 2 active sites . automated docking of the ligands into the receptors active sites provided multiple docking solutions . among the best scored solutions , a visual inspection was performed to retain the conformations forming the interactions considered to be essential for the pparγ activity , including hydrogen bonding with his323 , his449 , and tyr473 ( fig2 ), those for the pparα activity , including hydrogen bonding with tyr314 , his440 , and tyr464 ( fig3 ), and also those for the cb 2 agonist activity , i . e ., multiple hydrophobic contacts and hydrogen bonding with lys109 and / or ser285 ( fig4 and 5 ). molecular modelling studies were performed using sybyl software version 6 . 9 . 1 25 running on silicon graphics octane 2 workstations . as the pk a of compounds are unknown , the sparc online calculator was used to determine the species occurring at physiological ph ( 7 . 4 ) ( http :// ibmlc2 . chem . uga . edu / sparc / index . cfm ) 26 . three - dimensional model of ionized compounds were built from a standard fragments library , and their geometry was subsequently optimized using the tripos force field 27 including the electrostatic term calculated from gasteiger and hückel atomic charges . the method of powell available in the maximin2 procedure was used for energy minimization until the gradient value was smaller than 0 . 001 kcal / mol . å . the structure of the human ppars ligand - binding domain was obtained from its complexed x - ray crystal structure with the tesaglitazar ( az 242 ) available in the rcsb protein data bank ( http :// www . rcsb . org / pdb / home / home . do ) 17 ( pdb id : 117i ) 16 , 17 . an homology model of the cb 2 receptor was constructed by aligning its sequence ( uniprotkb entry : p34972 ) 28 on the bovine rhodopsine ( uniprotkb entry : po2699 ) 29 with clustalw 30 then transferring the 3d coordinates of the bovine rhodopsine crystallographic structure ( pdb id : 1u19 ) 31 with jackal . 32 in order to create a model in a putative activated conformation , transmembrane domains 3 and 6 ( tm3 and tm6 ) were rotated by 20 ° and 30 ° respectively as described for cb 1 by mcallister and coworkers . 33 flexible docking of the compounds into the receptors active sites was performed using gold 3 . 1 . 1 software . the most stable docking models were selected according to the best scored conformation predicted by the goldscore scoring function . 34 the complexes were energy - minimized using the powell method available in maximin2 procedure with the tripos force field and a dielectric constant of 4 . 0 until the gradient value reached 0 . 01 kcal / mol . å . the anneal function was used to define a 10 å hot region and a 15 å region of interest around the ligand . the best docking results for both ppars and cb 2 receptors were obtained with pharmacophores derivatives , according to their goldscore values ( tables 1 and 2 ). the goldscore fitness function has been optimised for the prediction of ligand binding positions and takes into account factors such as h - bonding energy , van der waals energy and ligand torsion strain . goldscore give fitness scores that are dimensionless however , the scale of the score gives a guide to how good the pose is ; the higher the score , the better the docking result is likely to be . goldscore represents strength of binding interaction . results for examples of win - 55212 - 2 derivatives ( dwin ) and jte - 907 derivatives ( djte ) are presented in tables 1 and 2 respectively . docking results of dwin and djte compounds into the pparγ active site are presented in fig6 and 7 . docking results of dwin and djte compounds into the pparα active site are presented in fig8 and 9 . docking results of dwin and djte compounds into the cb 2 active site are presented in fig1 and 11 respectively . generally speaking , the new designed compounds scoring values are higher than reference ligands for pparγ ( 4 - asa , 5 - asa ) and are in the same range for cb 2 ( win - 55212 - 2 , jte - 907 ). the goldscore fitness function reflects the theoretical energy necessary to the position the ligand in the ligand binding domain of the receptor . it has been optimised for the prediction of ligand binding positions rather than the prediction of binding affinities , although some correlation with the latter has been found . it was designed to discriminate between different binding modes of the same molecule . extra terms are probably required to compare different molecules . for example , a term is probably required to account for the entropic loss associated with freezing rotatable bonds when the ligand binds . it is expected that molecules having the best goldscores for pparγ and cb 2 will have a synergistic anti - inflammatory and analgesic effect mediated by ppars and cb 2 . the preferred compounds of the invention are those having docking goldscore greater than that of win - 55212 - 2 or jte - 907 for the cb receptor or greater than the score of 5 - asa for ppar γ receptor . the highest ranking compounds , indicated from modelling studies , all show an activity similar / superior to that of mesalazine and jte - 907 . all chemically feasible variations were evaluated in order to achieve the best score ( affinity and activation of the receptor ) in computer docking experiments . consequently , it is believed that the compounds of the present invention show comparable function and / or activity to mesalazine and aja and do so through similar biological pathways . commercial chemicals were purchased from aldrich unless stated otherwise and were used as received . flash column chromatography was carried out using merck silica gel 60 ( 0 . 040 - 0 . 063 mm ). thin layer chromatography was performed on pre - coated plastic plates ( merck silica 60f254 ), and visualised using uv light and were developed with either aqueous kmno4 or cerric ammonium molybdate ( cam ). proton ( 1h ) and carbon ( 13c ) nmr spectra were recorded on varian inova 300 , 400 and 500 spectrometers . chemical shifts are quoted relative to tetramethylsilane and referenced to residual solvent peaks as appropriate . infrared spectra were recorded on a varian 3100 ft - ir excalibur series spectrophotometer as neat liquids or evaporated films using nacl plates . lr - ms were acquired using a waters separations module linked to a micromass quattro micro electrospray mass spectrometer . hplc analysis was performed using a thermo separation products system ( chromsoft software ) with 20 μl injections . intermediate 5 was prepared using the literature procedure of raitio et al . [ 1 ] and the yields and spectroscopic data for compounds 1 , 2 , 3 , 4 and 5 were consistent with the data given in this reference . acid 5 ( 0 . 4 g , 1 . 31 mmol , 1 eq ), 5 - aminomethyl salicylic acid methyl ester hcl ( 0 . 26 g , 1 . 43 mmol , 1 . 095 eq ), 1 - hydroxybenzotriazole ( 0 . 196 g , 1 . 44 mmol , 1 . 102 eq ) and n -( 3 - dimethylaminopropyl )- n ′- ethylcarbodiimide . hcl ( 0 . 276 g , 2 . 176 mmol , 1 . 66 eq ) were dissolved in dcm ( 2 ml ) and were stirred at ambient temperature for 18 h . the reaction mixture was poured into water ( 10 ml ) and dcm ( 10 ml ) was added , the ph was adjusted to 7 with dil . aq . naoh and the organic layer was poured off . the aqueous layer was then extracted with dcm ( 2 × 10 ml ) and the combined organic layers were washed with water ( 2 × 10 ml ), were washed with brine ( 10 ml ), were dried over na2so4 , filtered and the solvent was removed in vacuo . the product was purified via column chromatography eluted with a gradient from 1 : 1 to 1 : 3 cyh : etoac ( rf product = 0 . 7 , rf acid 5 = 0 . 4 in dcm / 5 % meoh , uv , cam ). this gave 0 . 558 g ( 91 %) of the product as a white solid . 1h - nmr ( cdcl3 ) 500 mhz : δ ( ppm )= 0 . 94 ( 3h , t , j = 7 . 1 hz , ch2ch2ch3 ), 1 . 35 - 1 . 50 ( 4h , m , ch2ch2ch3 ), 1 . 81 ( 2h , quin , j = 7 . 8 hz , ch2ch2ch2ch3 ), 3 . 93 ( 3h , s , cooch3 ), 3 . 97 ( 3h , s , coch3 ), 4 . 13 ( 2h , t , j = 6 . 9 hz , och2ch2 ), 4 . 58 ( 2h , d , j = 5 . 9 hz , nhch2c ), 6 . 93 ( 1h , d , j = 8 . 9 hz , chcoch3 ), 6 . 95 ( 1h , d , j = 8 . 8 hz , chcoh ), 7 . 45 ( 1h , d , j = 8 . 5 hz , chchcoch3 ), 7 . 50 ( 1h , dd , j = 2 . 3 hz , j = 8 . 5 hz , chchcoh ), 7 . 84 ( 1h , d , j = 2 . 3 hz , chccoh ), 8 . 90 ( 1h , s , ccchcconh ), 9 . 12 ( 1h , br . s , cnhcoc ), 9 . 97 ( 1h , br . t , j = 5 . 5 hz , nhch2c ), 10 . 69 ( 1h , s , coh ). 13c - nmr ( cdcl3 ) 125 mhz : δ ( ppm )= 14 . 0 ( ch3 ), 22 . 4 ( ch2 ), 28 . 0 ( ch2 ), 29 . 9 ( ch2 ), 42 . 8 ( nch2 ), 52 . 2 ( cooch3 ), 56 . 3 ( och3 ), 73 . 8 ( och2 ), 109 . 1 ( ch ), 112 . 2 ( c ), 114 . 2 ( c ), 117 . 9 ( ch ), 119 . 4 ( c ), 125 . 3 ( ch ), 129 . 1 ( ch ), 129 . 6 ( c ), 132 . 4 ( c ), 133 . 5 ( c ), 135 . 5 ( ch ), 145 . 1 ( ch ), 154 . 4 ( ch ), 160 . 8 ( c ), 162 . 1 ( co ), 163 . 6 ( co ), 170 . 4 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 32 . 45 , 29 . 53 , 1672 , 1621 , 1534 , 1495 , 1355 , 1288 , 1213 , 1110 . ms - es ( negative ): 467 . 7 ( m − h +). ms - es ( positive ): 469 . 8 ( m + h +). hplc : 14 . 615 min . methylester 6 ( 0 . 558 g , 1 . 19 mmol , 1 eq ) and naoh ( 0 . 189 g , 4 . 72 mmol , 4 eq ) was stirred in methanol ( 15 ml ) and water ( 5 ml ) at reflux temperature . hydrolysis was followed by hplc ( sm = 14 . 615 min , product = 10 . 857 min ) and was complete in 3 h . the reaction mixture was then cooled and the ph was adjusted to 4 with dil . aq . hcl , which caused the product to precipitate out of solution as a white solid which was washed with water ( 20 ml ) and ether ( 20 ml ), collected and dried in vacuo to give 0 . 493 g ( 91 %) of a white powder . 1h - nmr ( dmso d6 ) 500 mhz : δ ( ppm )= 0 . 89 ( 3h , t , j = 7 . 1 hz , ch2ch2ch3 ), 1 . 35 - 1 . 45 ( 4h , m , ch2ch2ch3 ), 1 . 78 ( 2h , quin , j = 7 . 2 hz , ch2ch2ch2ch3 ), 3 . 93 ( 3h , s , coch3 ), 3 . 99 ( 2h , t , j = 6 . 9 hz , och2ch2 ), 4 . 50 ( 2h , d , j = 6 . 0 hz , nhch2c ), 6 . 92 ( 1h , d , j = 8 . 5 hz , chcoh ), 7 . 13 ( 1h , d , j = 8 . 9 hz , chcoch3 ), 7 . 50 ( 1h , dd , j = 2 . 2 hz , j = 8 . 5 hz , chchcoh ), 7 . 69 ( 1h , d , j = 8 . 9 hz , chchcoch3 ), 7 . 78 ( 1h , d , j = 2 . 2 hz , chccoh ), 8 . 79 ( 1h , s , ccchcconh ), 10 . 08 ( 1h , br . t , j = 6 . 0 hz , nhch2c ), 11 . 27 ( 1h , br . s , coh ), 11 . 51 ( 1h , s , cnhcoc ), 13 . 77 ( 1h , br . s , cooh ). 13c - nmr ( dmso d6 ) 125 mhz : δ ( ppm )= 13 . 8 ( ch3 ), 21 . 8 ( ch2 ), 27 . 3 ( ch2 ), 28 . 6 ( ch2 ), 41 . 4 ( nch2 ), 56 . 3 ( och3 ), 72 . 7 ( och2 ), 109 . 2 ( ch ), 113 . 1 ( c ), 113 . 7 ( c ), 117 . 0 ( ch ), 118 . 7 ( c ), 125 . 7 ( ch ), 129 . 0 ( ch ), 130 . 0 ( c ), 132 . 2 ( c ), 133 . 9 ( c ), 134 . 9 ( ch ), 144 . 1 ( ch ), 154 . 1 ( c ), 160 . 0 ( c ), 162 . 1 ( co ), 162 . 9 ( co ), 171 . 6 ( co ). ir spectrum ; solid state : v ˜( cm - 1 )= 3584 , 3325 , 3164 , 3033 , 2930 , 2861 , 1670 , 1593 , 1539 , 1465 , 1333 , 1284 , 1228 , 1113 . ms - es ( negative ): 453 . 6 ( m − h +). ms - es ( positive ): 455 . 7 ( m + h +). hplc : 10 . 857 min , & gt ; 99 . 1 % purity . the synthesis of these two compounds was undertaken using the procedure of kang et al . [ 5 ] however , changes were made and the actual procedures used are given in elsewhere herein . the spectroscopic data acquired on the products was consistent with the data given by kang et al . trifluoroacetic acid ( 50 ml ) and acetone ( 12 ml ) were added to the 4 - methysalicylic acid ( 10 g , 65 . 72 mmol , 1 eq ). reaction mixture was cooled to 0 ° c . and trifluoroacetic anhydride ( 30 ml ) was added dropwise over 2 min . reaction mixture was stirred for 3 days at room temperature and then the volatiles were removed in vacuo . the residues were purified through a dry - flash silica plug eluted with dcm (˜ 800 ml ). the oil was then additionally purified through another dry - flash silica gel plug eluted with toluene (˜ 1 l ). this gave the product as a yellow waxy solid ( 10 . 475 g , 83 %). acetonide 11 ( 6 . 0 g , 31 mmol , 1 eq ), n - bromo succinimide ( 6 . 4 g , 36 mmol , 1 . 16 eq ) and benzoyl peroxide ( 2 . 25 g , 7 mmol , 0 . 22 eq ) were dissolved in carbontetrachloride ( 20 ml ). the reaction mixture was stirred at 75 ° c . for 2 h and was then allowed to cool to ambient temperature . the white precipitate was filtered out and was washed with a small amount of cyclohexane . the filtrate was concentrated in vacuo and the residues were purified via a dry - flash silica gel plug eluted with dcm (˜ 300 ml ). dcm was evaporated . this gave bromide 12 at about 80 % conversion by 1h - nmr and this material was used directly in the next step . bromide 12 ( 0 . 574 g , 2 . 12 mmol , 1 eq ) was dissolved in chloroform ( 10 ml ), hexamethylenetetramine ( 0 . 44 g , 3 . 18 mmol , 1 . 5 eq ) was added and the mixture was heated to reflux temperature for 15 min . the reaction mixture was cooled and the resulting white solid was removed via filtration and washed with chloroform . this white solid was then heated to reflux in dil . aq . 1m hcl ( 10 ml ) for 1 h . the volatiles were then removed in vacuo and the residues were azeotropically dried with meoh . the residues were taken up in methanol ( 20 ml ), conc . h2so4 ( 3 ml ) was added and the mixture was heated to reflux temperature overnight . the reaction mixture was allowed to cool to ambient temperature and was then poured into a separating funnel , water ( 10 ml ) and dcm ( 50 ml ) were added . the layers were shaken and separated and the organic layer was discarded . then dcm ( 50 ml ) was added and the ph was adjusted to 7 and the organic layer was poured off . the aqueous layer was then extracted with dcm ( 2 × 50 ml ) and the combined organic layers were washed with water ( 2 × 10 ml ), were washed with brine ( 10 ml ), were dried over na2so4 , filtered and the solvent was removed in vacuo . this gave 0 . 263 g ( 68 %) of an off white solid . 1h - nmr ( cdcl3 ) 500 mhz : δ ( ppm )= 1 . 56 ( 2h , br . s , nh2 ), 3 . 85 ( 2h , s , nch2c ), 3 . 93 ( 3h , s , cooch3 ), 6 . 83 ( 1h , d , j = 8 . 2 hz , ch2cchchc ), 6 . 93 ( 1h , s , cchc ), 7 . 78 ( 1h , d , j = 8 . 2 hz , ch2cchchc ), 10 . 72 ( 1h , br . s , coh ). 13c - nmr ( cdcl3 ) 125 mhz : δ ( ppm )= 46 . 2 ( ch2 ), 52 . 3 ( ch3 ), 110 . 8 ( c ), 115 . 4 ( ch ), 117 . 9 ( ch ), 130 . 1 ( ch ), 151 . 9 ( c ), 161 . 8 ( c ), 170 . 4 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3585 , 3288 , 3170 , 2960 , 1675 , 1622 , 1575 , 1441 , 1341 , 1259 , 1092 . ms - es ( negative ): 180 . 1 ( m − h +). ms - es ( positive ): 182 . 1 ( m + h +). prepared on 0 . 328 mmol scale using the same procedure as for 7 ( section 5 . 4 . 1 ). the product was purified via column chromatography eluted with a gradient from 1 : 1 to 1 : 3 cyh : etoac ( rf product = 0 . 7 , rf acid 5 = 0 . 4 in dcm / 5 % meoh , uv , cam ). this gave 0 . 341 g ( 68 %) of the product as a white solid . 1h - nmr ( cdcl3 ) 500 mhz : δ ( ppm )= 0 . 94 ( 3h , t , j = 7 . 1 hz , ch2ch2ch3 ), 1 . 35 - 1 . 50 ( 4h , m , ch2ch2ch3 ), 1 . 82 ( 2h , quin , j = 7 . 7 hz , ch2ch2ch2ch3 ), 3 . 93 ( 3h , s , cooch3 ), 3 . 98 ( 3h , s , coch3 ), 4 . 14 ( 2h , t , j = 6 . 9 hz , och2ch2 ), 4 . 67 ( 2h , d , j = 6 . 0 hz , nhch2c ), 6 . 88 ( 1h , d , j = 8 . 2 hz , nhch2cchchc ), 6 . 94 ( 1h , d , j = 8 . 9 hz , chchcoch3 ), 6 . 99 ( 1h , s , cchcoh ), 7 . 45 ( 1h , d , j = 8 . 9 hz , chchcoch3 ), 7 . 78 ( 1h , d , j = 8 . 2 hz , nhch2cchchc ), 8 . 89 ( 1h , s , ccchcconh ), 9 . 15 ( 1h , br . s , cnhcoc ), 10 . 06 ( 1h , br . t , j = 5 . 7 hz , nhch2c ), 10 . 72 ( 1h , s , coh ). 13c - nmr ( cdcl3 ) 125 mhz : δ ( ppm )= 14 . 0 ( ch3 ), 22 . 4 ( ch2 ), 28 . 0 ( ch2 ), 29 . 9 ( ch2 ), 43 . 0 ( nch2 ), 52 . 2 ( cooch3 ), 56 . 3 ( och3 ), 73 . 9 ( och2 ), 109 . 1 ( ch ), 111 . 2 ( c ), 114 . 3 ( c ), 116 . 0 ( ch ), 118 . 2 ( ch ), 119 . 3 ( c ), 125 . 2 ( ch ), 130 . 2 ( ch ), 132 . 4 ( c ), 133 . 5 ( c ), 145 . 2 ( ch ), 147 . 3 ( c ), 154 . 4 ( c ), 161 . 8 ( c ), 162 . 1 ( co ), 163 . 8 ( co ), 170 . 4 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3242 , 3189 , 2954 , 2864 , 1671 , 1622 , 1534 , 1342 , 1260 , 1214 , 1110 . ms - es ( negative ): 467 . 2 ( m − h +). ms - es ( positive ): 469 . 3 ( m + h +). hplc : 14 . 730 min . prepared on 1 . 25 mmol scale using the same procedure as for djte3 ( section 5 . 4 . 2 ). hydrolysis was followed by hplc ( sm = 14 . 730 min , product = 10 . 997 min ) and was complete in 3 h . the reaction mixture was then cooled and the ph was adjusted to 4 with dil . aq . hcl , which caused the product to precipitate out of solution as a white solid which was collected and washed with water ( 20 ml ), then ether ( 20 ml ) and was dried in vacuo to give 0 . 499 g ( 88 %) of a white powder . 1h - nmr ( dmso d6 ) 500 mhz : δ ( ppm )= 0 . 89 ( 3h , t , j = 7 . 0 hz , ch2ch2ch3 ), 1 . 30 - 1 . 45 ( 4h , m , ch2ch2ch3 ), 1 . 78 ( 2h , quin , j = 7 . 2 hz , ch2ch2ch2ch3 ), 3 . 93 ( 3h , s , coch3 ), 4 . 00 ( 2h , t , j = 6 . 9 hz , och2ch2 ), 4 . 58 ( 2h , d , j = 6 . 0 hz , nhch2c ), 6 . 80 - 6 . 95 ( 2h , m , cchchcchcoh ), 7 . 14 ( 1h , d , j = 8 . 9 hz , chcoch3 ), 7 . 69 ( 1h , d , j = 8 . 9 hz , chchcoch3 ), 7 . 75 ( 1h , d , j = 8 . 5 hz , cchchcchcoh ), 8 . 79 ( 1h , s , ccchcconh ), 10 . 15 ( 1h , br . t , j = 6 . 0 hz , nhch2c ), 11 . 26 ( 1h , br . s , coh ), 11 . 44 ( 1h , s , cnhcoc ), 13 . 77 ( 1h , br . s , cooh ). 13c - nmr ( dmso d6 ) 125 mhz : δ ( ppm )= 13 . 8 ( ch3 ), 21 . 8 ( ch2 ), 27 . 3 ( ch2 ), 28 . 7 ( ch2 ), 42 . 0 ( nch2 ), 56 . 3 ( och3 ), 72 . 8 ( och2 ), 109 . 2 ( ch ), 111 . 3 ( c ), 113 . 7 ( c ), 115 . 1 ( ch ), 117 . 8 ( ch ), 118 . 6 ( c ), 125 . 7 ( ch ), 130 . 3 ( ch ), 132 . 2 ( c ), 133 . 9 ( c ), 144 . 2 ( ch ), 147 . 7 ( c ), 154 . 7 ( c ), 161 . 1 ( c ), 162 . 1 ( co ), 163 . 1 ( co ), 171 . 6 ( co ). ir spectrum ; solid state : v ˜( cm - 1 )= 3270 , 3070 , 2947 , 1671 , 1626 , 1530 , 1467 , 1269 , 1214 . ms - es ( negative ): 453 . 2 ( m − h +). hplc : 10 . 997 min , & gt ; 97 . 2 % purity . 2 - methylindole ( 6 . 88 g , 52 . 46 mmol , 1 eq ) was dissolved in ether ( 30 ml ) and the solution was cooled to 0 ° c . memgbr ( 3m in ether , 62 . 95 ml , 62 . 95 mmol , 1 . 2 eq ) was then added dropwise over 30 min and after the addition , the mixture was allowed to warm to ambient temperature . 1 - naphthoyl chloride ( 10 g , 52 . 46 mmol , 1 eq ) in ether ( 15 ml ) was added dropwise over 30 min and then the mixture was refluxed for 1 h , cooled and sat . aq . nh4cl ( 200 ml ) was added slowly to quench the reaction . the mixture was stirred until it was a pink slurry and the solids were then removed via filtration and were washed with water ( 50 ml ). the solids were suspended in methanol ( 200 ml ), a solution of naoh ( 3 g ) in water ( 100 ml ) was added and the mixture was refluxed overnight . the solids were then filtered , washed with water ( 500 ml ), washed with ether ( 250 ml ) and were dried in vacuo . the solids were dissolved in dcm and were dry loaded onto silica and were then chromatographed in 1 : 1 cyh / etoac ( rf sm = 0 . 9 , rf product = 0 . 51 , uv , kmno4 ). this gave 10 . 847 g ( 70 %) of the product as a pink solid . 1h - nmr ( dmso d6 ) 400 mhz : δ ( ppm )= 2 . 17 ( 3h , s , ch3 ), 3 . 34 ( 1h , s , nh ), 6 . 94 - 6 . 99 ( 1h , m , ncchchchchc ), 7 . 08 - 7 . 14 ( 1h , m , ncchchchchc ), 7 . 25 ( 1h , br . d , j = 8 . 0 hz , ncchchchchc ), 7 . 37 ( 1h , br . d , j = 8 . 0 hz , ncchchchchc ), 7 . 44 - 7 . 58 ( 3h , m , cchchchcco , cchchchchccco ), 7 . 61 ( 1h , dd , j = 7 . 0 hz , j = 8 . 1 hz , cchchchchccco ), 7 . 83 ( 1h , br . d , j = 8 . 3 hz , cchchchcco ), 8 . 03 ( 1h , br . d , j = 8 . 2 hz , cchchchchccco ), 8 . 07 ( 1h , br . d , j = 8 . 2 hz , cchchchchccco ). 13c - nmr ( dmso d6 ) 100 mhz : δ ( ppm )= 14 . 1 ( ch3 ), 111 . 2 ( ch ), 113 . 7 ( c ), 120 . 1 ( ch ), 121 . 3 ( ch ), 122 . 0 ( ch ), 124 . 2 ( ch ), 124 . 7 ( ch ), 125 . 4 ( ch ), 126 . 2 ( ch ), 126 . 7 ( ch ), 126 . 9 ( c ), 128 . 2 ( ch ), 129 . 1 ( ch ), 129 . 3 ( c ), 133 . 1 ( c ), 134 . 9 ( c ), 140 . 5 ( c ), 145 . 7 ( c ), 191 . 9 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3173 , 1720 , 1569 , 1433 , 1237 , 1099 , 1043 . ms - es ( negative ): 284 . 1 ( m − h +). ms - es ( positive ): 308 . 0 ( m + na +). methyl 4 - amino - 2 - methoylbenzoate ( 2 g , 11 . 04 mmol , 1 eq ) was dissolved in methanol ( 30 ml ) and a 1 : 1 mixture ( 2 ml ) of 6m aq . hcl and methanol was added . chloroacetaldehyde ( 50 % in water , 2 . 08 ml , 13 . 27 mmol , 1 . 2 eq ) was added and the mixture was cooled to 0 ° c . nabh3cn ( 0 . 78 g , 12 . 37 mmol , 1 . 12 eq ) was added in portions over 2 min and the mixture was stirred for 5 days at ambient temperature . the mixture was poured into sat . aq . nahco3 ( 100 ml ) and dcm ( 100 ml ) was added , the ph was adjusted to 7 - 8 with dil . aq . hcl and the organic layer was poured off . the aqueous layer was then extracted with dcm ( 2 × 50 ml ) and the combined organic layers were washed with water ( 2 × 100 ml ), were washed with brine ( 50 ml ), were dried over na2so4 , filtered and the solvent was removed in vacuo . the product was purified via column chromatography eluted with a gradient from 1 : 1 to 1 : 3 cyh : etoac ( rf product = 0 . 5 , rf sm = 0 . 35 in 1 : 3 cyh : etoac , uv , kmno4 ). this gave 1 . 968 g ( 73 %) of white solid . 1h - nmr ( cdcl3 ) 500 mhz : δ ( ppm )= 3 . 55 ( 2h , br . quart , j = 5 . 1 hz , clch2ch2 ), 3 . 71 ( 2h , t , j = 5 . 9 hz , clch2ch2 ), 3 . 82 ( 3h , s , coch3 ), 3 . 86 ( 3h , s , cooch3 ), 4 . 48 ( 1h , br . s , clch2ch2nh ), 6 . 13 ( 1h , d , j = 1 . 9 hz , cchcn ), 6 . 19 ( 1h , dd , j = 2 . 0 hz , j = 8 . 6 hz , cchchcn ), 7 . 77 ( 1h , d , j = 8 . 6 hz , cchchcn ). 13c - nmr ( cdcl3 ) 125 mhz : δ ( ppm )= 43 . 1 ( ch2 ), 44 . 8 ( ch2 ), 51 . 4 ( ch3 ), 55 . 8 ( ch3 ), 96 . 1 ( ch ), 104 . 1 ( ch ), 108 . 8 ( c ), 134 . 3 ( ch ), 152 . 2 ( c ), 161 . 8 ( c ), 166 . 1 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3361 , 2950 , 2840 , 1700 , 1607 , 1526 , 1346 , 1255 , 1182 , 1085 . ms - es ( negative ): 242 . 1 ( m − h +), 244 . 1 ( m − h +). ms - es ( positive ): 244 . 1 ( m + h +), 246 . 1 ( m + h +). indole 15 ( 2 . 303 g , 8 . 07 mmol , 1 eq ) and nbu4nbr ( 50 mg ) were dissolved in dmf ( 8 ml ). sodium hydride ( 60 % dispersion in mineral oil , 0 . 339 g , 8 . 47 mmol , 1 . 05 eq ) was added and the mixture was stirred for 15 min . chloride 16 ( 1 . 967 g , 8 . 07 mmol , 1 eq ) was dissolved in dmf ( 8 ml ) and was then added rapidly to the reaction mixture and the reaction was heated to 50 ° c . overnight . after cooling , the reaction mixture was poured into water ( 100 ml ) and dcm ( 100 ml ) was added and the organic layer was poured off . the aqueous layer was then extracted with dcm ( 2 × 50 ml ) and the combined organic layers were washed with water ( 2 × 100 ml ), were washed with brine ( 50 ml ), were dried over na2so4 , filtered and the solvent was removed in vacuo . the product was purified via column chromatography eluted with a gradient from 1 : 1 to 1 : 1 . 3 cyh : etoac ( rf indole sm = 0 . 5 , rf chloride 16 = 0 . 4 , rf product = 0 . 2 in 1 : 1 cyh : etoac , uv , cam ). this gave 1 . 825 g ( 46 %) of a foamy white solid . 1hnmr ( cdcl3 ) 500 mhz : δ ( ppm )= 2 . 34 ( 3h , s , cch3 ), 3 . 63 ( 3h , s , coch3 ), 3 . 66 ( 2h , quart , j = 5 . 8 hz , nch2ch2nhc ), 3 . 82 ( 3h , s , cooch3 ), 4 . 27 ( 1h , t , j = 6 . 5 hz , nch2ch2nhc ), 4 . 34 ( 2h , t , j = 5 . 8 hz , nch2ch2nhc ), 5 . 86 ( 1h , d , j = 1 . 8 hz , cchcn ), 6 . 10 ( 1h , dd , j = 2 . 0 hz , j = 8 . 6 hz , cchchcn ), 7 . 04 ( 1h , t , j = 7 . 6 hz , ncchchchchc ), 7 . 18 ( 1h , t , j = 7 . 2 hz , ncchchchchc ), 7 . 25 - 7 . 30 ( 2h , m , ncchchchchc ), 7 . 40 - 7 . 53 ( 4h , m , cchchchcco , cchchchchccco ), 7 . 75 ( 1h , d , j = 8 . 6 hz , cchchcn ), 7 . 91 ( 1h , br . d , j = 8 . 2 hz , cchchchccco ), 7 . 96 ( 1h , br . d , j = 8 . 0 hz , cchchchchccco ), 8 . 08 ( 1h , br . d , j = 8 . 4 hz , cchchchchccco ). 13c - nmr ( cdcl3 ) 125 mhz : δ ( ppm )= 12 . 6 ( ch3 ), 42 . 3 ( ch2 ), 42 . 8 ( ch2 ), 51 . 4 ( ch3 ), 55 . 5 ( ch3 ), 95 . 2 ( ch ), 103 . 8 ( ch ), 108 . 8 ( c ), 109 . 0 ( ch ), 115 . 5 ( c ), 121 . 6 ( ch ), 122 . 4 ( ch ), 122 . 6 ( ch ), 125 . 0 ( ch ), 125 . 4 ( ch ), 125 . 9 ( ch ), 126 . 3 ( ch ), 126 . 9 ( ch ), 127 . 2 ( c ), 128 . 3 ( ch ), 130 . 2 ( ch ), 130 . 3 ( c ), 133 . 8 ( c ), 134 . 3 ( ch ), 135 . 9 ( c ), 140 . 1 ( c ), 145 . 4 ( c ), 151 . 9 ( c ), 161 . 9 ( c ), 166 . 1 ( co ), 193 . 5 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3352 , 3053 , 2946 , 1696 , 1606 , 1513 , 1413 , 1250 , 1090 . ms - es ( negative ): 491 . 3 ( m − h +). ms - es ( positive ): 493 . 3 ( m + h +). methyl ether 17 ( 3 . 31 g , 6 . 72 mmol , 1 eq ) was dissolved in dcm ( 50 ml ) and the solution was cooled to − 78 ° c . bbr3 ( 2 . 54 ml , 26 . 88 mmol , 4 eq ) dissolved in dcm ( 50 ml ) was then added dropwise over 2 min to the reaction and the reaction was stirred for 2 h at − 78 ° c . the mixture was then warmed to ambient temperature and poured into sat . aq . nahco3 ( 100 ml ) and the organic layer was poured off . the aqueous layer was then extracted with dcm ( 2 × 50 ml ) and the combined organic layers were washed with water ( 2 × 100 ml ), were washed with brine ( 50 ml ), were dried over na2so4 , filtered and the solvent was removed in vacuo . the product was purified via column chromatography eluted with 1 : 1 cyh : etoac ( rf product = 0 . 78 , rf sm = 0 . 33 , uv , cam ). this gave 2 . 0 g ( 62 %) of a foamy white solid . 1h - nmr ( cdcl3 ) 500 mhz : δ ( ppm )= 2 . 31 ( 3h , s , cch3 ), 3 . 59 ( 2h , quart , j = 5 . 7 hz , nch2ch2nhc ), 3 . 88 ( 3h , s , cooch3 ), 4 . 30 ( 2h , t , j = 5 . 9 hz , nch2ch2nhc ), 4 . 40 ( 1h , t , j = 6 . 4 hz , nch2ch2nhc ), 5 . 93 ( 1h , dd , j = 2 . 3 hz , j = 8 . 8 hz , cchchcn ), 6 . 04 ( 1h , d , j = 2 . 2 hz , cchcn ), 7 . 03 ( 1h , t , j = 7 . 2 hz , ncchchchchc ), 7 . 17 ( 1h , t , j = 7 . 2 hz , ncchchchchc ), 7 . 24 ( 1h , d , j = 8 . 2 hz , ncchchchchc ), 7 . 26 ( 1h , d , j = 7 . 5 hz , ncchchchchc ), 7 . 40 - 7 . 52 ( 4h , m , cchchchcco , cchchchchccco ), 7 . 57 ( 1h , d , j = 8 . 7 hz , cchchcn ), 7 . 91 ( 1h , d , j = 8 . 2 hz , cchchchccco ), 7 . 96 ( 1h , dd , j = 2 . 5 hz , j = 6 . 8 hz , cchchchchccco ), 8 . 09 ( 1h , d , j = 8 . 5 hz , cchchchchccco ), 11 . 03 ( 1h , s , coh ). 13c - nmr ( cdcl3 ) 125 mhz : δ ( ppm )= 12 . 5 ( ch3 ), 42 . 0 ( ch2 ), 42 . 1 ( ch2 ), 51 . 6 ( ch3 ), 97 . 5 ( ch ), 102 . 5 ( c ), 105 . 4 ( ch ), 109 . 1 ( ch ), 115 . 4 ( c ), 121 . 4 ( ch ), 122 . 3 ( ch ), 122 . 6 ( ch ), 125 . 0 ( ch ), 125 . 4 ( ch ), 125 . 7 ( ch ), 126 . 3 ( ch ), 126 . 9 ( ch ), 127 . 1 ( c ), 128 . 3 ( ch ), 130 . 1 ( ch ), 130 . 2 ( c ), 131 . 5 ( ch ), 133 . 7 ( c ), 135 . 9 ( c ), 140 . 1 ( c ), 145 . 7 ( c ), 153 . 0 ( c ), 163 . 8 ( c ), 170 . 4 ( co ), 193 . 4 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3399 , 3335 , 3054 , 2950 , 1656 , 1624 , 1516 , 1439 , 1412 , 1348 , 1270 , 1197 , 1159 . ms - es ( negative ): 477 . 3 ( m − h +). ms - es ( positive ): 479 . 2 ( m + h +). hplc : 15 . 012 min . methyl ester 18 ( 2 g , 4 . 18 mmol , 1 eq ) and naoh ( 0 . 67 g , 16 . 72 mmol , 4 eq ) was stirred in methanol ( 50 ml ) and water ( 17 ml ), the mixture was heated to reflux temperature . hydrolysis was followed by hplc ( sm = 15 . 012 min , product = 10 . 698 min ) and once completed ( overnight ) the ph of the mixture was adjusted to 7 with dil . aq . hcl and the volatiles were removed in vacuo . the residues were azeotroped dry with meoh and were then dry loaded onto silica and the product was purified via column chromatography eluted with a gradient from etoac to etoac / 10 % meoh ( rf product = 0 . 3 , uv , cam ). this gave 1 . 5 g ( 77 %) of a foamy yellow solid . 1h - nmr ( dmso d6 ) 500 mhz : δ ( ppm )= 2 . 08 ( 1h , s , coh ). 2 . 21 ( 3h , s , cch3 ), 3 . 48 ( 2h , quart , j = 5 . 7 hz , nch2ch2nhc ), 4 . 35 ( 2h , t , j = 5 . 6 hz , nch2ch2nhc ), 5 . 86 ( 1h , s , cchcn ), 5 . 92 ( 1h , d , j = 8 . 7 hz , cchchcn ), 6 . 38 ( 1h , br . s , nch2ch2nhc ), 6 . 98 ( 1h , t , j = 7 . 7 hz , ncchchchchc ), 7 . 11 - 7 . 20 ( 2h , m , ncchchchchc ), 7 . 38 - 7 . 42 ( 2h , m , ncchchchchc , cchchcn ), 7 . 46 - 7 . 51 ( 1h , m , cchchchcco ), 7 . 51 - 7 . 58 ( 1h , m , cchchchcco , cchchchchccco ), 7 . 87 ( 1h , d , j = 8 . 5 hz , cchchchccco ), 8 . 03 ( 1h , d , j = 8 . 1 hz , cchchchchccco ), 8 . 07 ( 1h , d , j = 8 . 1 hz , cchchchchccco ), 13 . 08 ( 1h , s , cooh ). 13c - nmr ( dmso d6 ) 125 mhz : δ ( ppm )= 12 . 1 ( ch3 ), 41 . 1 ( ch2 ), 42 . 2 ( ch2 ), 96 . 5 ( ch ), 102 . 8 ( c ), 103 . 5 ( ch ), 110 . 1 ( ch ), 113 . 9 ( c ), 120 . 1 ( ch ), 121 . 6 ( ch ), 122 . 0 ( ch ), 124 . 8 ( ch ), 124 . 9 ( ch ), 125 . 2 ( ch ), 126 . 2 ( ch ), 126 . 5 ( c ), 126 . 8 ( ch ), 128 . 1 ( ch ), 129 . 4 ( c ), 129 . 4 ( ch ), 131 . 0 ( ch ), 133 . 1 ( c ), 135 . 8 ( c ), 140 . 2 ( c ), 146 . 3 ( c ), 153 . 1 ( c ), 163 . 8 ( c ), 172 . 5 ( co ), 191 . 9 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3361 , 1923 , 1701 , 1576 , 1498 , 1348 , 1227 , 1085 . ms - es ( negative ): 463 . 2 ( m − h +). ms - es ( positive ): 465 . 2 ( m + h +). hplc : 10 . 698 min , 97 . 0 % purity . 5 - methyl salicylic acid ( 10 g , 65 . 3 mmol , 1 eq ) was dissolved in methanol ( 80 ml ) and conc . h2so4 ( 10 ml ) was added carefully . the mixture was heated to reflux temperature overnight and was then allowed to cool to ambient temperature and was then poured into a separating funnel and water ( 100 ml ) and dcm ( 100 ml ) were added . the ph was adjusted to 7 with dil . aq . naoh and the organic layer was poured off . the aqueous layer was then extracted with dcm ( 2 × 50 ml ) and the combined organic layers were washed with water ( 2 × 100 ml ), were washed with brine ( 50 ml ), were dried over na2so4 , filtered and the solvent was removed in vacuo . this gave 9 . 778 g ( 62 %) of an off white solid . 1h - nmr ( dmso d6 ) 500 mhz : δ ( ppm )= 3 . 85 ( 3h , s , ch3 ), 4 . 78 ( 2h , br . s , nh2 ), 6 . 70 ( 1h , d , j = 8 . 7 hz , cchchcn ), 6 . 82 ( 1h , dd , j = 2 . 9 hz , j = 8 . 7 hz , cchchcn ), 7 . 01 ( 1h , d , j = 2 . 9 hz , cchcn ), 9 . 74 ( 1h , s , coh ). 13cnmr ( dmso d6 ) 125 mhz : δ ( ppm )= 52 . 1 ( ch3 ), 112 . 1 ( c ), 112 . 8 ( ch ), 117 . 5 ( ch ), 123 . 0 ( ch ), 141 . 0 ( c ), 151 . 5 ( c ), 169 . 6 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3408 , 3328 , 3220 , 3082 , 2958 , 1675 , 1616 , 1485 , 1441 , 1303 , 1231 , 1083 . ms - es ( positive ): 168 . 06 ( m + h +). phenol 19 ( 5 g , 29 . 9 mmol , 1 eq ) and tbuok ( 3 . 35 g , 29 . 9 mmol , 1 eq ) were stirred in dmso ( 70 ml ) for 2 h at ambient temperature . dimethylsulphate ( 3 ml , 3 . 17 mmol , 1 . 06 eq ) was added and the mixture was stirred for 5 min before being poured into water ( 100 ml ) and etoac ( 100 ml ). the ph was adjusted to 7 with dil . aq . hcl and the organic layer was poured off . the aqueous layer was then extracted with etoac ( 2 × 50 ml ) and the combined organic layers were washed with water ( 2 × 100 ml ), were ashed with brine ( 50 ml ), were dried over na2so4 , filtered and the solvent was removed in vacuo . the product was purified via column chromatography eluted with 1 : 1 etoac : cyh ( rf sm = 0 . 4 , rf product = 0 . 2 , uv , cam ). this gave 3 . 152 g ( 53 %) of a brown oil . 1h - nmr ( cdcl3 ) 500 mhz : δ ( ppm )= 3 . 50 ( 2h , br . s , nh2 ), 3 . 83 ( 3h , s , coch3 ), 3 . 87 ( 3h , s , cooch3 ), 6 . 80 - 6 . 85 ( 2h , m , cchchcn ), 7 . 15 ( 1h , br . s , cchcn ). 13c - nmr ( cdcl3 ) 125 mhz : δ ( ppm )= 51 . 9 ( ch3 ), 56 . 8 ( ch3 ), 114 . 2 ( ch ), 117 . 9 ( ch ), 120 . 2 ( ch ), 120 . 6 ( c ), 139 . 6 ( c ), 152 . 3 ( c ), 166 . 7 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3432 , 3360 , 3230 , 2951 , 2837 , 1717 , 1627 , 1501 , 1441 , 1313 , 1227 , 1081 , 1023 . ms - es ( positive ): 182 . 07 ( m + h +). prepared on 3 . 53 mmol scale using the same procedure as for 16 ( section 5 . 6 . 2 ). the product was purified via column chromatography eluted with a gradient from 1 : 1 to 1 : 3 cyh : etoac ( rf product = 0 . 77 , rf sm = 0 . 4 in 1 : 3 cyh : etoac , uv , kmno4 ). this gave 0 . 348 g ( 40 %) of a white solid . 1hnmr ( cdcl3 ) 500 mhz : δ ( ppm )= 3 . 47 ( 2h , t , j = 5 . 8 hz , clch2ch2 ), 3 . 70 ( 2h , t , j = 5 . 9 hz , clch2ch2 ), 3 . 83 ( 3h , s , coch3 ), 3 . 88 ( 3h , s , cooch3 ), 3 . 80 - 4 . 00 ( 1h , br . s , clch2ch2nh ), 6 . 68 ( 1h , dd , j = 3 . 0 hz , j = 8 . 8 hz , cchchcn ), 6 . 87 ( 1h , d , j = 8 . 9 hz , cchchcn ), 7 . 11 ( 1h , d , j = 3 . 0 hz , cchcn ). 13c - nmr ( cdcl3 ) 125 mhz : δ ( ppm )= 43 . 5 ( ch2 ), 46 . 3 ( ch2 ), 52 . 0 ( ch3 ), 56 . 9 ( ch3 ), 114 . 4 ( ch ), 116 . 2 ( ch ), 118 . 9 ( ch ), 120 . 9 ( c ), 140 . 6 ( c ), 152 . 3 ( c ), 166 . 8 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3381 , 1951 , 2838 , 1720 , 1617 , 1584 , 1505 , 1437 , 1235 , 1081 . ms - es ( positive ): 244 . 1 ( m + h +), 246 . 1 ( m + h +). prepared on 0 . 82 mmol scale using the same procedure as for 17 ( section 5 . 6 . 3 ). the product was purified via column chromatography eluted with a gradient from 1 : 1 to 1 : 1 . 3 cyh : etoac ( rf indole sm = 0 . 5 , rf cl sm = 0 . 4 , rf product = 0 . 36 in 1 : 1 cyh : etoac , uv , cam ). this gave 0 . 209 g ( 52 %) of a foamy white solid . 1h - nmr ( cdcl3 ) 500 mhz : 6 ( ppm )= 2 . 37 ( 3h , s , cch3 ), 3 . 56 ( 2h , t , j = 6 . 0 hz , nch2ch2nhc ), 3 . 65 ( 1h , br . s , nch2ch2nhc ), 3 . 83 ( 3h , s , coch3 ), 3 . 86 ( 3h , s , cooch3 ), 4 . 32 ( 2h , t , j = 5 . 8 hz , nch2ch2nhc ), 6 . 63 ( 1h , dd , j = 3 . 0 hz , j = 8 . 9 hz , cchchcn ), 6 . 83 ( 1h , d , j = 8 . 9 hz , cchchcn ), 7 . 02 ( 1h , d , j = 3 . 1 hz , cchcn ), 7 . 03 ( 1h , t , j = 8 . 0 hz , ncchchchchc ), 7 . 18 ( 1h , t , j = 8 . 1 hz , ncchchchchc ), 7 . 26 ( 1h , br . d , j = 8 . 0 hz , ncchchchchc ), 7 . 29 ( 1h , br . d , j = 8 . 2 hz , ncchchchchc ), 7 . 41 - 7 . 45 ( 1h , m , cchchchcco ), 7 . 46 - 7 . 54 ( 3h , m , cchchchcco , cchchchchccco ), 7 . 91 ( 1h , br . d , j = 8 . 2 hz , cchchchccco ), 7 . 96 ( 1h , br . d , j = 7 . 9 hz , cchchchchccco ), 8 . 10 ( 1h , br . d , j = 8 . 4 hz , cchchchchccco ). 13c - nmr ( cdcl3 ) 125 mhz : 6 ( ppm )= 12 . 6 ( ch3 ), 42 . 6 ( ch2 ), 43 . 4 ( ch2 ), 52 . 0 ( ch3 ), 56 . 9 ( ch3 ), 109 . 2 ( ch ), 114 . 5 ( ch ), 115 . 0 ( ch ), 115 . 3 ( c ), 118 . 2 ( ch ), 120 . 8 ( c ), 121 . 4 ( ch ), 122 . 2 ( ch ), 122 . 5 ( ch ), 125 . 0 ( ch ), 125 . 5 ( ch ), 125 . 7 ( ch ), 126 . 2 ( ch ), 126 . 9 ( ch ), 127 . 2 ( c ), 128 . 2 ( ch ), 130 . 0 ( ch ), 130 . 3 ( c ), 133 . 8 ( c ), 136 . 0 ( c ), 140 . 2 ( c ), 140 . 4 ( c ), 145 . 7 ( c ), 152 . 7 ( c ), 166 . 7 ( co ), 193 . 3 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3378 , 3051 , 2998 , 2838 , 1719 , 1609 , 1507 , 1412 , 1234 , 1085 . ms - es ( negative ): 491 . 3 ( m − h +). ms - es ( positive ): 493 . 3 ( m + h +). prepared on 2 . 03 mmol scale using the same procedure as for 16 ( section 5 . 6 . 4 ). the product was purified via column chromatography eluted with 1 : 1 cyh : etoac ( rf product = 0 . 45 , rf sm = 0 . 33 in 4 : 6 cyh : etoac , uv , cam ). this gave 0 . 534 g ( 55 %) of a foamy off white solid . 1h - nmr ( cdcl3 ) 500 mhz : δ ( ppm )= 1 . 55 ( 1h , br . s , nch2ch2nhc ), 2 . 42 ( 3h , s , cch3 ), 3 . 58 ( 2h , t , j = 6 . 2 hz , nch2ch2nhc ), 3 . 89 ( 3h , s , cooch3 ), 4 . 38 ( 2h , t , j = 6 . 1 hz , nch2ch2nhc ), 6 . 76 ( 1h , dd , j = 2 . 7 hz , j = 8 . 9 hz , cchchcn ), 6 . 85 ( 1h , d , j = 8 . 9 hz , cchchcn ), 6 . 69 ( 1h , d , j = 2 . 6 hz , cchcn ), 7 . 03 ( 1h , t , j = 7 . 3 hz , ncchchchchc ), 7 . 19 ( 1h , t , j = 7 . 2 hz , ncchchchchc ), 7 . 22 ( 1h , d , j = 8 . 0 hz , ncchchchchc ), 7 . 32 ( 1h , d , j = 8 . 2 hz , ncchchchchc ), 7 . 44 ( 1h , t , j = 8 . 2 hz , cchchchcco ), 7 . 47 - 7 . 53 ( 3h , m , cchchchcco , cchchchchccco ), 7 . 91 ( 1h , br . d , j = 8 . 2 hz , cchchchccco ), 7 . 97 ( 1h , br . d , j = 7 . 5 hz , cchchchchccco ), 8 . 10 ( 1h , br . d , j = 8 . 4 hz , cchchchchccco ), 10 . 22 ( 1h , s , coh ). 13c - nmr ( cdcl3 ) 125 mhz : δ ( ppm )= 12 . 6 ( ch3 ), 42 . 6 ( ch2 ), 43 . 8 ( ch2 ), 52 . 3 ( ch3 ), 109 . 2 ( ch ), 111 . 4 ( ch ), 112 . 3 ( c ), 115 . 4 ( c ), 118 . 7 ( ch ), 121 . 5 ( ch ), 122 . 2 ( ch ), 122 . 5 ( ch ), 123 . 4 ( ch ), 125 . 0 ( ch ), 125 . 5 ( ch ), 125 . 8 ( ch ), 126 . 3 ( ch ), 126 . 9 ( ch ), 127 . 2 ( c ), 128 . 3 ( ch ), 130 . 1 ( ch ), 130 . 3 ( c ), 133 . 8 ( c ), 136 . 1 ( c ), 138 . 8 ( c ), 140 . 2 ( c ), 145 . 6 ( c ), 155 . 0 ( c ), 170 . 2 ( co ), 193 . 4 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3584 , 3348 , 3053 , 2951 , 1678 , 1613 , 1503 , 1440 , 1411 , 1290 , 1207 , 1088 . ms es ( negative ): 477 . 3 ( m − h +). ms - es ( positive ): 479 . 2 ( m + h +). hplc : 14 . 462 min . prepared on 1 . 78 mmol scale using the same procedure as for dwin1 ( section 5 . 6 . 4 ). hydrolysis was followed by hplc ( sm = 14 . 462 min , product = 10 . 120 min ) and was completed in 1 h . the product was purified via column chromatography eluted with a gradient from etoac to etoac / 10 % meoh ( rf product = 0 . 25 , uv , cam ). this gave 290 mg ( 35 %) of a foamy yellow solid . 1h - nmr ( cdcl3 ) 500 mhz : δ ( ppm )= 1 . 91 ( 1h , s , coh ), 2 . 28 ( 3h , s , cch3 ), 3 . 35 ( 2h , t , j = 5 . 9 hz , nch2ch2nhc ), 4 . 33 ( 2h , t , j = 6 . 2 hz , nch2ch2nhc ), 5 . 08 ( 1h , br . s , nch2ch2nhc ), 6 . 48 ( 1h , d , j = 8 . 6 hz , cchchcn ), 6 . 52 ( 1h , dd , j = 2 . 9 hz , j = 8 . 6 hz , cchchcn ), 6 . 96 ( 1h , t , j = 7 . 3 hz , ncchchchchc ), 7 . 06 ( 1h , d , j = 5 . 7 hz , ncchchchchc ), 7 . 07 ( 1h , s , cchcn ), 7 . 15 ( 1h , t , j = 8 . 2 hz , ncchchchchc ), 7 . 46 - 7 . 50 ( 2h , m , cchchchchccco ), 7 . 53 - 7 . 64 ( 3h , m , cchchchcco , ncchchchchc ), 7 . 87 ( 1h , d , j = 8 . 4 hz , cchchchccco ), 8 . 03 ( 1h , d , j = 8 . 2 hz , cchchchchccco ), 8 . 08 ( 1h , d , j = 8 . 2 hz , cchchchchccco ), 13 . 36 ( 3h , br . s , cooch ). 13c - nmr ( cdcl3 ) 125 mhz : δ ( ppm )= 12 . 2 ( ch3 ), 42 . 3 ( ch2 ), 43 . 0 ( ch2 ), 110 . 2 ( ch ), 113 . 1 ( ch ), 113 . 7 ( c ), 115 . 9 ( ch ), 117 . 6 ( ch ), 119 . 6 ( c ), 120 . 0 ( ch ), 121 . 6 ( ch ), 122 . 0 ( ch ), 124 . 8 ( 2 × ch ), 125 . 3 ( ch ), 126 . 2 ( ch ), 126 . 4 ( c ), 126 . 8 ( ch ), 128 . 2 ( ch ), 129 . 4 ( ch , c ), 133 . 1 ( c ), 135 . 9 ( c ), 138 . 8 ( c ), 140 . 3 ( c ), 146 . 4 ( c ), 153 . 7 ( c ), 172 . 4 ( co ), 191 . 9 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3407 , 3045 , 2919 , 1701 , 1565 , 1486 , 1408 , 1353 , 1227 , 1085 . ms - es ( negative ): 463 . 3 ( m − h +). ms - es ( positive ): 465 . 3 ( m + h +). hplc : 10 . 120 min , 96 . 3 % purity . 2 - methylindole ( 15 . 1 g , 0 . 115 mol , 1 eq ) was dried under high vacuum and then dissolved in dry thf ( 100 ml ) and cooled to 0 ° c . nbutyllithium ( 1 . 6 m in hexanes , 77 ml , 0 . 115 mol , 1 eq ) was added at a rate of 80 ml / h via a syringe pump . reaction mixture was stirred at 0 ° c . for 15 min then a solution of anhydrous zncl2 ( 15 . 7 g , 0 . 115 mol , 1 eq ) in thf ( 100 ml ) was added to the reaction mixture . reaction mixture was stirred at ambient temperature for 20 h then the thf was removed in vacuo . the residue was redissolved in dry toluene ( 50 ml ) and bromoacetic acid ethyl ester ( 19 ml , 0 . 172 mol , 1 . 5 eq ) was added and the reaction was stirred for 2 days . the mixture was then poured into water ( 200 ml ) and was extracted with etoac ( 3 × 100 ml ), the combined organic layers were then washed with water ( 100 ml ), sat . aq . nahco3 ( 100 ml ), brine ( 50 ml ), were dried over na2so4 , filtered and the solvent was removed in vacuo . the residues were then dry - flash chromatographed through a silica plug eluted with a gradient from toluene to 1 : 1 toluene : dcm to dcm to elute the product ( rf prod = 0 . 27 in 1 : 1 cyh : etoac , uv , cam ). this gave 18 . 5 g ( 74 %) of a yellowbrown oil . 1h - nmr ( cdcl3 ) 500 mhz : δ ( ppm )= 1 . 26 ( 3h , t , j = 7 . 1 hz , ch2ch3 ), 2 . 41 ( 3h , s , ch3 ), 3 . 71 ( 2h , s , cch2co ), 4 . 16 ( 2h , quart , j = 7 . 1 hz , ch2ch3 ), 7 . 10 - 7 . 17 ( 1h , m , nhcchchchchc ), 7 . 27 ( 1h , d , j = 6 . 0 hz , nhcchchchchc ), 7 . 57 ( 1h , d , j = 7 . 0 hz , nhcchchchchc ), 7 . 89 ( 1h , br . s , nh ). 13c - nmr ( cdcl3 ) 125 mhz : δ ( ppm )= 11 . 6 ( ch3 ), 14 . 2 ( ch3 ), 30 . 5 ( ch2 ), 60 . 6 ( ch2 ), 104 . 7 ( c ), 110 . 2 ( ch ), 118 . 1 ( ch ), 119 . 5 ( ch ), 121 . 2 ( ch ), 128 . 5 ( c ), 132 . 6 ( c ), 135 . 1 ( c ), 172 . 0 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3393 , 3053 , 2980 , 2927 , 1724 , 1463 , 1304 , 1172 , 1031 . ms - es ( negative ): 216 . 1 ( m − h +). ms - es ( positive ): 218 . 2 ( m + h +). indole 30 ( 5 g , 23 . 0 mmol , 1 eq ) was dissolved in dmf ( 50 ml ) and was cooled to 0 ° c . sodium hydride ( 60 % dispersion in mineral oil , 1 . 01 g , 25 . 31 mmol , 1 . 1 eq ) was added and the mixture was stirred for 30 min . 2 , 3 - dichlorobenzoyl chloride ( 5 . 06 g , 24 . 16 mmol , 1 . 05 eq ) was dissolved in dmf ( 25 ml ) and this solution was added to the reaction over 2 min and the mixture was stirred overnight at ambient temperature . the mixture was poured into water ( 100 ml ) and dcm ( 100 ml ) and the organic layer was poured off . the aqueous layer was then extracted with dcm ( 2 × 50 ml ) and the combined organic layers were washed with water ( 2 × 100 ml ), were washed with brine ( 50 ml ), were dried over na2so4 , filtered and the solvent was removed in vacuo . the product was purified via column chromatography eluted with a gradient from 4 : 1 to 1 : 1 cyh : etoac ( rf product = 0 . 4 , rf sm = 0 . 27 , uv , cam ). this gave 7 . 224 g ( 80 %) of a yellow - green oil . 1h - nmr ( cdcl3 ) 500 mhz : δ ( ppm )= 1 . 24 ( 3h , t , j = 7 . 1 hz , ch2ch3 ), 2 . 26 ( 3h , s , ch3 ), 3 . 66 ( 2h , s , cch2co ), 4 . 14 ( 2h , quart , j = 7 . 1 hz , ch2ch3 ), 7 . 13 ( 1h , t , j = 7 . 3 hz ncchchchchc ), 7 . 24 ( 1h , t , j = 7 . 6 hz ncchchchchc ), 7 . 32 ( 1h , d , j = 8 . 3 hz , ncchchchchc ), 7 . 37 ( 1h , dd , j = 7 . 6 hz , j = 7 . 6 hz , cchchchccl ), 7 . 39 ( 1h , dd , j = 2 . 0 hz , j = 7 . 6 hz , cchchchccl ), 7 . 50 ( 1h , d , j = 7 . 8 hz ncchchchchc ), 7 . 60 ( 1h , dd , j = 2 . 0 hz , j = 7 . 6 hz , cchchchccl ). 13c - nmr ( cdcl3 ) 125 mhz : δ ( ppm )= 13 . 5 ( ch3 ), 14 . 2 ( ch3 ), 30 . 3 ( ch2 ), 61 . 0 ( ch2 ), 114 . 3 ( c ), 114 . 6 ( ch ), 118 . 4 ( ch ), 123 . 8 ( ch ), 124 . 3 ( ch ), 127 . 2 ( ch ), 128 . 1 ( ch ), 130 . 2 ( c ), 130 . 3 ( c ), 132 . 5 ( ch ), 134 . 3 ( c ), 134 . 4 ( c ), 135 . 8 ( c ), 138 . 4 ( c ), 165 . 8 ( co ), 170 . 6 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3068 , 2980 , 2931 , 1734 , 1687 , 1456 , 1358 , 1320 , 1160 . ms - es ( positive ): 390 . 1 ( m + h +), 392 . 1 ( m + h +). ester 31 ( 3 . 784 g , 9 . 70 mmol , 1 eq ) was dissolved in toluene ( 20 ml ) and was cooled to − 78 ° c . dibal - h ( 1 . 5m in toluene , 9 . 70 ml , 14 . 54 mmol , 1 . 5 eq ) was added at a rate of 3 ml / min via a syringe pump , after the addition was complete the mixture was stirred for a further 30 min . methanol ( 10 ml ) was added at − 78 ° c . at 6 ml / min via a syringe pump , and then as the mixture warmed to ambient temperature , dil . aq . hcl ( 2m , 50 ml ) was added . once the solution had cleared , the organic layer was poured off . the aqueous layer was then extracted with etoac ( 2 × 50 ml ) and the combined organic layers were washed with water ( 2 × 100 ml ), were washed with brine ( 50 ml ), were dried over na2so4 , filtered and the solvent was removed in vacuo . the product was not isolated and was used directly in the next step . aldehyde 32 ( ca . 3 . 36 g , 9 . 70 mmol , 1 eq ) and amine 35 ( was dissolved in methanol ( 20 ml ), glacial acetic acid ( 2 . 1 ml ) was added and the mixture was cooled to 0 ° c . nabh3cn ( 1 . 34 g , 21 . 33 mmol , 2 . 2 eq ) was added in portions and the mixture was stirred overnight at ambient temperature . the mixture was poured into sat . aq . nahco3 ( 100 ml ) and dcm ( 100 ml ) was added , the ph was adjusted to 7 - 8 with dil . aq . naoh and the organic layer was poured off . the aqueous layer was then extracted with dcm ( 2 × 50 ml ) and the combined organic layers were washed with water ( 2 × 100 ml ), were washed with brine ( 50 ml ), were dried over na2so4 , filtered and the solvent was removed in vacuo . the product was purified via column chromatography eluted with a gradient from 4 : 1 to 1 : 1 cyh : diethylether ( rf 31 = 0 . 5 , rf product = 0 . 35 , rf 33 & amp ; 35 = 0 . 3 in 1 : 1 cyh : diethylether , uv , cam ) and was rechromatographed eluted with a gradient from toluene to toluene / 3 % diethylether ( rf 31 = 0 . 7 , rf product = 0 . 63 , rf 33 & amp ; 35 = 0 . 5 in 9 : 1 toluene : diethylether , uv , cam ). this gave 1 . 037 g ( 19 %) of a foamy yellow solid . 1h - nmr ( cdcl3 ) 500 mhz : δ ( ppm )= 2 . 10 ( 3h , s , ch3 ), 2 . 97 ( 2h , t , j = 6 . 6 hz , ch2ch2nh ), 3 . 46 ( 2h , br . t , j = 6 . 3 hz , ch2ch2nh ), 4 . 21 ( 1h , br . s , ch2ch2nh ), 5 . 32 ( 2h , s , ch2ph ), 6 . 01 ( 1h , dd , j = 2 . 3 hz , j = 8 . 8 hz , cchchcn ), 6 . 09 ( 1h , d , j = 2 . 3 hz , cchcn ), 7 . 15 - 7 . 20 ( 1h , m , ncchchchchc ), 7 . 23 - 7 . 27 ( 1h , m , ncchchchchc ), 7 . 34 - 7 . 47 ( 9h , m , ncchchchchc , cchchchccl , ph ), 7 . 63 ( 1h , d , j = 8 . 9 hz , cchchcn ), 7 . 64 ( 1h , dd , j = 2 . 3 hz , j = 6 . 9 hz , cchchchccl ), 10 . 97 ( 1h , s , coh ). 13cnmr ( cdcl3 ) 125 mhz : δ ( ppm )= 13 . 5 ( ch3 ), 23 . 7 ( ch2 ), 42 . 4 ( ch2 ), 66 . 1 ( ch2 ), 97 . 7 ( ch ), 101 . 9 ( c ), 105 . 6 ( ch ), 114 . 9 ( ch ), 117 . 9 ( ch & amp ; c ), 123 . 9 ( ch ), 124 . 4 ( ch ), 127 . 3 ( ch ), 128 . 1 ( ch ), 128 . 2 ( ch ), 128 . 2 ( ch ), 128 . 6 ( ch ), 130 . 2 ( c ), 130 . 2 ( c ), 131 . 4 ( ch ), 132 . 6 ( ch ), 133 . 5 ( c ), 134 . 3 ( c ), 136 . 0 ( c × 2 ), 138 . 4 ( c ), 153 . 9 ( c ), 164 . 0 ( c ), 165 . 8 ( co ), 169 . 8 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3408 , 3071 , 2930 , 1651 , 1527 , 1455 , 1378 , 1268 , 1155 . ms - es ( negative ): 571 . 2 ( m − h +), 573 . 1 ( m − h +). ms - es ( positive ): 573 . 2 ( m + h +), 575 . 1 ( m + h +). 4 - aminosalicylic acid ( 3 g , 19 . 6 mmol , 1 eq ), pyridinium ptoluenesulphonic acid ( 0 . 5 g , 1 . 96 mmol , 0 . 1 eq ) and n -( 3 - dimethylaminopropyl )- n ′- ethylcarbodiimide . hcl ( 6 . 57 g , 34 . 3 mmol , 1 . 75 eq ) were dissolved in dcm ( 15 ml ) and benzyl alcohol ( 3 . 05 ml , 29 . 4 mmol , 1 . 5 eq ) was added . the reaction was stirred overnight and then was poured into water ( 50 ml ) and dcm ( 50 ml ). the ph was adjusted to 7 with dil . aq . naoh and the organic layer was poured off . the aqueous layer was then extracted with dcm ( 2 × 50 ml ) and the combined organic layers were washed with water ( 2 × 100 ml ), were washed with brine ( 50 ml ), were dried over na2so4 , filtered and the solvent was removed in vacuo . the product was purified via column chromatography eluted with 2 : 3 etoac : cyh ( rf product = 0 . 61 , rf bnoh = 0 . 52 , uv , cam ). benzyl alcohol that coeluted with the product was later removed via trituration with cyclohexane ( 10 ml ), the product was filtered off as a white powder ( 1 . 851 g , 39 %). 1h - nmr ( cdcl3 ) 500 mhz : δ ( ppm )= 4 . 09 ( 2h , br . s , nh2 ), 5 . 33 ( 2h , s , ch2 ), 6 . 13 ( 1h , dd , j = 2 . 2 hz , j = 8 . 6 hz , cchchcn ), 6 . 16 ( 1h , d , j = 2 . 2 hz , cchchcn ), 7 . 32 - 7 . 45 ( 5h , m , ph ), 7 . 67 ( 1h , d , j = 8 . 6 hz cchcn ), 10 . 92 ( 1h , s , coh ). 13c - nmr ( cdcl3 ) 125 mhz : δ ( ppm )= 66 . 2 ( ch2 ), 100 . 7 ( ch ), 103 . 0 ( c ), 106 . 8 ( ch ), 128 . 1 ( ch ph ), 128 . 2 ( ch ph ), 128 . 6 ( ch ph ), 131 . 7 ( ch ), 135 . 9 ( c ), 153 . 4 ( c ), 163 . 7 ( c ), 169 . 8 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3460 , 3370 , 1637 , 1511 , 1385 , 1275 , 1152 . ms - es ( positive ): 242 . 1 ( m + h +), 244 . 2 ( m + h +). hplc : 21 . 067 min . benzylester 34 ( 1 g , 1 . 74 mmol , 1 eq ) was dissolved in methanol ( 160 ml ) and raney - ni ( slurry in water , ˜ 200 mg washed twice with meoh ) was added . the mixture was purged with nitrogen and then with hydrogen and then was left stirring for 2 h with a hydrogen balloon attached . hydrogenolysis was followed by hplc ( sm = 21 . 098 min , product = 14 . 405 min ). the reaction was purged with nitrogen and then filtered through a celite plug , the plug was washed with meoh ( 100 ml ) and the solvent was removed in vacuo to give 0 . 52 g ( 62 %) of a foamy yellow solid . 1h - nmr ( dmso d6 ) 500 mhz : δ ( ppm )= 1 . 97 ( 3h , s , ch3 ), 2 . 87 ( 2h , t , j = 6 . 9 hz , ch2ch2nh ), 3 . 29 ( 2h , t , j = 6 . 8 hz , ch2ch2nh ), 5 . 90 ( 1h , d , j = 2 . 1 hz , cchcn ), 6 . 04 ( 1h , dd , j = 2 . 1 hz , j = 8 . 8 hz , cchchcn ), 6 . 44 ( 1h , j = 6 . 6 hz , ch2ch2nh ), 7 . 19 ( 1h , t , j = 7 . 2 hz , ncchchchchc ), 7 . 27 ( 1h , t , j = 7 . 2 hz , ncchchchchc ), 7 . 41 ( 1h , d , j = 8 . 7 hz , cchchcn ), 7 . 43 ( 1h , br . d , j = 8 . 2 hz , ncchchchchc ), 7 . 56 ( 1h , dd , j = 7 . 8 hz , j = 7 . 8 hz , cchchchccl ), 7 . 57 ( 1h , br . d , j = 7 . 6 hz , ncchchchchc ), 7 . 61 ( 1h , dd , j = 1 . 6 hz , j = 7 . 6 hz , cchchchccl ), 7 . 90 ( 1h , dd , j = 1 . 6 hz , j = 8 . 0 hz , cchchchccl ), 12 . 05 ( 1h , br . s , coh ), acid signal not obvious . 13c - nmr ( dmso d6 ) 125 mhz : δ ( ppm )= 12 . 9 ( ch3 ), 23 . 1 ( ch2 ), 41 . 6 ( ch2 ), 96 . 3 ( ch ), 102 . 0 ( c ), 104 . 4 ( ch ), 114 . 4 ( ch ), 118 . 2 ( ch ), 118 . 3 ( c ), 123 . 7 ( ch ), 124 . 0 ( ch ), 127 . 8 ( ch ), 128 . 2 ( c ), 129 . 3 ( ch ), 130 . 1 ( c ), 130 . 9 ( ch ), 132 . 5 ( c ), 132 . 6 ( ch ), 132 . 8 ( c ), 135 . 3 ( c ), 138 . 0 ( c ), 154 . 1 ( c ), 163 . 6 ( c ), 165 . 0 ( co ), 172 . 1 ( co ). ir spectrum ; evaporated film : v ˜( cm - 1 )= 3628 , 3422 , 3057 , 2920 , 1676 , 1623 , 1532 , 1446 , 1359 , 1320 , 1260 , 1130 . mses ( negative ): 481 . 2 ( m − h +), 483 . 1 ( m − h +). ms - es ( positive ): 483 . 1 ( m + h +), 485 . 1 ( m + h +). hplc : 14 . 313 min , 98 . 4 % purity . pharmacological activity experiments will enable selection of lead compounds for further development in animal models of acute ( e . g . stroke ) and / or chronic ( e . g . alzheimer &# 39 ; s disease ) neurodegenerative disorders . determination of the capability of the compound to bind to ppar - γ and cb2 receptors in vitro screening for ppar - γ activity of the compounds in cell - based assays ; comparative potencies and selectivity of the compounds in inducing ppar - γ activation in thp - 1 xderived macrophages employing a cell - based transcriptional factor assay . the prototypic activity of ppars is to activate transcription in a ligand - dependent manner following direct binding to dna response elements in the promoter or enhancer regions of target genes — the so called dr - 1 elements or ppar response elements ( ppres )— a process known as ligand dependant trans - activation . ppars , like other nuclear receptor family members , contain both a ligand binding domain , directing specific interaction with the cognate ligand , and a dna - binding domain that mediates binding to specific ppres in the regulatory / promoter domains . in response to ligand binding , ppars undergo a conformational change that facilitates : a ) the formation of a heterodimeric complex with another ligand - activated nuclear receptor retinoid x receptor ( rxr ); b ) high affinity interactions with co - activators ( i . e . the ncor - containing co - repressor complexes are dismissed and are replaced with co - activator complexes ) that remodel chromatin and activate the cellular transcription machinery inducing ppar transactivation of the target genes . thus , the rate of transcriptional activation of genes that contain ppres is increased and their mrna levels are elevated . as a consequence cell - based ppar transactivation assays were first performed to address : a ) whether the newly synthesized compounds bind / activate ppar - γ in biological systems ; b ) the biological potency and ppar selectivity of the compounds , in comparison to known ppar - γ ligands ; c ) their effects on cell viability at biologically active concentrations by determining , in addition to cell viability , ppar dna binding activity in nuclear extracts of thp - 1 human monocytic cells differentiated into macrophage - like cells exposed to different concentrations of the compounds . in addition , because ppar subtypes share a high level of sequence and structural similarity , the nuclear receptor selectivity of the compounds found to activate ppar - γ were tested for effects on ppar - α and - δ . selection to employ thp - 1 derived macrophages was based on the following criteria : a ) thp - 1 cells differentiated towards macrophages employing phorbol esters express high levels of ppar - γ ; c ) thp - 1 cells have been widely employed to assess biological effects of ppar - γ and ppar - α agonists in monocytes / macrophages ( see next step ); d ) thp - 1 derived macrophages have been employed for drug screening purposes of ppar - γ agonists employing immunoabsorbent ( elisa )- based transcriptional factor assays . briefly , thp - 1 monocytes ( atcc ) in culture were treated with pma ( 400 ng / ml ) for 72 hours to induce monocyte differentiation into macrophages . thereafter , test compounds at different concentrations ( 0 . 01 to 50 um ), selective ppar - γ agonists ( e . g . rosiglitazone , positive control ) or vehicle ( 0 . 1 % dmso ) with or without the ppar - γ antagonist gw9662 ( 5 μm , 1 h prior to the samples ), were added and incubated for 48 h in culture medium and nuclear extracts employed for assessment of ppar - γ activation . at all times , cell viability , employing mtt assay , were assessed . the activation of ppar - γ was determined by an immunosorbent assay ( elisa ) utilizing ppar - γ factor transcription factor assay kits ( e . g . cayman chemicals , usa ), whilst the ppar complete transcription factor assay kit ( cayman chemicals ) was employed for assessment of effects on pparα and δ , of the active compounds . comparative potencies were be determined in terms of fold activation at different concentrations . screening for cb2 receptor binding affinity , selectivity and potency of the newly synthesized compounds to assess the capability of the compounds to bind to cb2 receptors and to behave as agonists / inverse agonists at cb2 receptors , the following experimental in vitro paradigms will be employed : a ) in vitro binding assays to exploit cb2 receptor affinity and selectivity off the newly - synthesized compounds via testing of their ability to selectively displace binding of [ 3h ]- cp55 , 940 to membrane preparations expressing recombinant human cb2 receptor versus membrane preparations expressing recombinant human cb1 receptors . [ 3 h ] cp55940 is the most widely used radio - labelled cb1 / 2 receptor probe . it has approximately equal affinity for cb1 and cb2 binding sites and displacement assays with [ 3h ] cp55940 that are directed at characterizing the binding properties of novel unlabeled ligands are generally performed with membranes that are known to contain either cb1 or cb2 receptors but not both receptor types . these membranes are often obtained from cho cells transfected with cb1 or cb2 receptors ( hcb1 / 2 - cho ). b ) in vitro functional bioassays to exploit relative capability of selected compounds to inhibit forskolin - induced stimulation of cyclic amp production in cells transfected with cb2 receptors ( e . g . hcb2 - cho cells ). cb2 receptors are negatively coupled to adenylyl cyclase and the ability of cannabinoid cb1 / 2 receptor agonists to inhibit basal or forskolin - induced cyclic amp production is widely exploited for functional assessment of ligand receptor binding potency in vitro . assays will be performed utilizing existing procedures and different concentrations of the compounds . intracellular camp in cellular lysates will be measured by camp enzyme immunoassays techniques . c ) in vitro functional bioassays to exploit effects of selected compounds on the coupling of cb2 receptors to g proteins via assessment of their effects on the binding of [[ 35 s ] gtrγs to recombinant cell membranes expressing cb2 receptors ( e . g . hcb1 / 2 - cho ). although this assay is less sensitive than the cyclic amp assay , it provides a total measure of g protein - mediated cannabinoid receptor activation rather than a measure of the activation of just one particular cannabinoid receptor effector mechanism as in the cyclic amp assay . in general , it is expected that the binding of gtrγs to g proteins wouls be stimulated by agonists for g protein - coupled receptors and inhibited by inverse agonists for such receptors . in brief , in these experiments , membranes were incubated in the presence of absence of different concentrations of the compounds , [[ 35 s ] gtpγs will be assessed . the tables set out the results obtained from the initial dose - response curves shown in fig1 - 15 . the results in table 1 are the average ec50 determined in duplicate as shown in fig1 and 13 . fig1 and 15 show the results for tests in cell based systems for dwin1 and dwin2 versus rogiglitazone as control and the results for the cb2 control win 55212 - 2 . comparison of the half maximal effective concentration ( ec 50 ) shows that for the ppar - γ receptor the tested compounds are substantially more potent than the gw1929 high affinity agonist of ppar - γ γ sold by sigma aldrich . the potency is dramatically higher in the cell free and cell based tests . similarly , the goldscore docking studies for the cb2 receptor indicated that the affinity for the receptor was comparable to that of the control compound win 55212 - 2 . on this basis it is expected that the compounds of the invention tested will be at least as potent as the control compound in the cell free and cell based systems experiments to be conducted . 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