the 
 opioid receptors (  ,  ,  , and the opioid - like 
 receptor orl-1 ) belong to the super family of g - protein coupled receptors 
 ( gpcrs ) that possess seven helical trans - membrane spanning domains 
 in their architecture . the majority of 
 research efforts focused upon this group of proteins has been directed 
 toward the  receptor because it mediates the analgesic actions 
 of opiates such as morphine ( chart 1 ) . over the years , however , it has become increasingly 
 clear that the entire family of opioid proteins are actively involved 
 in a host of important physiological processes . studies with selective  
 opioid receptor antagonists have 
 shown that this system is intimately involved in brain processes that 
 relate to stress , fear , and anxiety as well as reward - seeking behavior . studies have shown that ( 3r)-1,2,3,4-tetrahydro-7-hydroxy - n-[(1s)-1-[[(3r,4r)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl]-2-methylpropyl]-3-isoquinolinecarboxamide 
 ( jdtic ) and nor - binaltorphimine ( nor - bni ) , another  opioid 
 selective antagonist , dose - dependently reduce fear and stress - induced 
 responses in multiple behavioral paradigms with rodents ( immobility 
 in the forced - swim assay , reduction of exploratory 
 behavior in the elevated plus maze , and fear - potentiated startle ) . further , selective  opioid receptor antagonists 
 have been shown to reduce stress - induced reinstatement of cocaine 
 self - administration in rats to block the 
 stress - induced potentiation of cocaine place preference conditioning , to decrease dependence - induced ethanol self - administration , to attenuate the expression of both the physical 
 ( somatic signs hyperalgesia ) and effective ( anxiety - related behavior 
 conditional place aversion ) signs of nicotine - induced withdrawal in 
 mice , to diminish deprivation - induced eating in rats , and to prevent prepulse inhibition mediated by u50,488 . these observations regarding the behavioral 
 consequences of receptor blockade in several animal tests suggest 
 that  opioid receptor antagonists could be useful for treating 
 anxiety , depression , schizophrenia , addiction , and eating disorders . compounds 1 ( az - mtab),2 ( pf-4455242 ) , and 3 ( ly2456302 ) have been reported as newer selective  opioid receptor antagonists 
 ( chart 1 ) . see also ref ( 3 ) for a review of these studies . these newer  opioid receptor antagonists show activity in various 
 animal models similar to those reported for norbni and jdtic . in addition , 
 jdtic and compounds 2 and 3 have undergone 
 phase 1 and/or phase 2 studies directed toward various cns disorders . no drugs for the treatment of cocaine and methamphetamine 
 abuse , 
 however , are currently available . further , 
 nicotine replacement therapy ( nrt ) , bupropion , and varenicline are 
 used to treat nicotine addiction , but no more than 25% of patients 
 respond to these treatments . naltrexone 
 is used to treat alcoholism but has limited efficacy . a number of antidepressants are on the market , but many 
 patients do not respond to any of them . , we report the synthesis and in vitro efficacy as determined 
 by [ s]gtps assay of jdtic analogues 415 ( see table 2 for structures ) . these compounds have the hydroxyl group on the 4-(3-hydroxylphenyl ) 
 or 7-hydroxy - tetrahydroisoquinoline parts of jdtic replaced with other 
 functional groups . a comparison of their in vitro efficacy properties 
 to those of jdtic show that several of the analogues were potent and 
 selective  opioid receptor antagonists . preclinical adme studies 
 show that some of the antagonists have better drug - like properties 
 than jdtic . bis - triflate 17 was 
 prepared by treating 16 with an excess of triflic anhydride 
 at 78 c . subjection of 17 to palladium - catalyzed transfer hydrogenation 
 in dmf at 80 c afforded intermediate 18 . reduction 
 of 18 with lithium aluminum hydride in toluene and tetrahydrofuran 
 mixture cleaved the triflamide to give ( 3r,4r)-3,4-dimethyl-4-phenylpiperidine ( 19 ) . reductive 
 amination of 19 with boc - l - valinal , prepared 
 according to the procedure reported by skiles et al . , followed by t - butoxycarbonyl ( boc ) deprotection 
 with trifluoroacetic acid in dichloromethane afforded 20 . coupling of 20 with ( 3r)-2-(tert - butoxycarbonyl)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic 
 acid ( boc-7-hydroxy - d - tic ) using n , n , n,n-tetramethyl - o-(1h - benzotriazol-1-yl)uronium hexafluorophosphate 
 ( hbtu ) in dichloromethane followed by boc deprotection with trifluoroacetic 
 acid in dichloromethane afforded 4 . scheme 2 shows the synthesis of 8 via intermediate 25 , which was also used to synthesize 5 , 6 , and 7 ( scheme 3 ) . treatment of 22 with n - phenyl(bis - trifluoromethanesulfonimide ) 
 afforded the triflate 23 . amide coupling 
 of 26 with boc-7-hydroxy - d - tic , followed by 
 removal of the boc protecting group with hydrochloric acid in aqueous 
 methanol , afforded the desired final product 8 . the compounds 5 , 6 , and 7 where the phenolic group 
 of the 4-(3-hydroxyphenyl ) group in jdtic 
 has been replaced by a fluoro , chloro , and bromo substituent , respectively , 
 were prepared as described in scheme 3 . alternatively , sandmeyer halogenation of 
 the diazo intermediate afforded the chloro and bromo intermediates 28 and 29 . subsequent deprotection of the phthaloyl 
 protected amines present in 27 , 28 , and 29 using hydrazine in ethanol afforded 30 , 31 , and 32 , respectively . these intermediates 
 were coupled with boc-7-hydroxy - d - tic using hbtu in acetonitrile 
 or 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride ( edchcl ) 
 and a catalytic amount of n - hydroxybenzotriazole 
 ( hobt ) in dichloromethane followed by treatment with hydrogen chloride 
 to afford 5 , 6 , and 7 . coupling of amines 20 and 21(30 ) with commercially 
 available boc-(3r)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic 
 acid ( boc - d - tic ) , followed by treatment with trifluoroacetic 
 acid in dichloromethane , gave 11 and 9 , 
 respectively . coupling of 21 and 30 with 
 ( 3r)-2-[tert - butoxy)carbonyl]-7-fluoro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic 
 acid ( 7-fluoro - boc - d - tic ) , followed by boc deprotection using 
 aqueous methanolic hydrogen chloride , afforded 10 and 12 , respectively . the methyl ester 
 of boc-7-hydroxy - d - tic ( 33 ) prepared using trimethylsilyldiazomethane 
 in methanol and toluene was converted to the intermediate aryl triflate 
 with triflic anhydride , which was transformed 
 to the benzonitrile ( 35 ) via palladium - catalyzed cyanation . careful hydrolysis of the methyl ester using 
 lithium hydroxide in aqueous dioxane , followed by addition of hydrogen 
 peroxide to the cooled solution , resulted in a very rapid hydrolysis 
 of the benzonitrile to the benzamide 36 . the appropriate 
 amine ( 21 , 3-{(3r,4r)-1-[(2s)-2-amino-3-methylbutyl]-3,4-dimethylpiperidin-4-yl}benzamide , or 20 ) could then be coupled with 36 using hbtu or edchcl to afford the intermediate amides 
 which yielded the desired compounds 13 , 14 , and 15 upon deprotection of the boc group with trifluoroacetic 
 acid in dichloromethane or hydrochloric acid in aqueous methanol . because [ s]gtps binding 
 strongly correlates with animal behavior studies of previously reported 
  antagonists , measures of opioid receptor antagonism and specificity 
 for the compounds in the study were obtained by monitoring the ability 
 of selected test compounds to inhibit stimulation of [ s]gtps binding produced by the selective agonists ( d - ala , mephe , gly - ol)encephalin 
 ( damgo ,  receptor ) cyclo[d - pen , d - pen]encephalin ( dpdpe ,  ) and 5,7,8-()-n - methyl - n-[7-(1-pyrrolidinyl)-1-oxaspirodec-8-yl]benzeneacetamide 
 ( u69,593 ,  ) in cloned human receptors using previously reported 
 methods.ke values were calculated as previously reported . several in 
 vitro studies were 
 conducted to characterize  opioid receptor antagonists 4 , 5 , 13 , and 14 and 
 compared to the results from jdtic and previously reported 37(33 ) ( chart 2 ) , which 
 like compounds 13 and 14 has its phenol 
 groups replaced by a carboxamido group . an in vitro model using mdck - mdr1 
 cells was used to predict brain penetration . plasma and s9 stability 
 of each compound was determined using procedures similar to those 
 previously reported . compounds that interact with 
 the human ether - a - go - go gene herg product ( which is a potassium channel ) 
 are cardiotoxic . thus , the affinity of synthesized  opioid 
 receptor antagonists toward the herg channel was determined . the interaction 
 of these test compounds with the herg channel was analyzed using a 
 radioligand displacement assay based on a protocol developed by chiu 
 et al . for these studies , [ h]astemizole was used as the high - affinity herg radioligand ( ki  20 nm ) . see experimental 
 section for details . solubility of the compounds 
 was determined using a kinetic 96-well 
 plate assay essentially as described by zhu et al . the parallel artificial membrane permeability assay ( pampa ) was 
 used to predict oral absorption in a 96-well format as has been described 
 previously and detailed in the experimental section . in the late 1970s , zimmerman 
 and co - workers reported that n - methyl - trans-4-phenylpiperidine 38 ( ly83577 ) ( chart 2 ) was an opioid 
 receptor pure antagonist whose potency was significantly increased 
 by adding a phenolic group to the aromatic ring to give the n - methyl - trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine 39 ( ly99335 ) ( chart 2 ) . in a study directed toward determining if the 3-hydroxy group 
 present 
 on the aromatic ring in 40 ( ly255582 ) was required for 
 potent antagonist efficacy , it was found that removal of the 3-hydroxy 
 group led to 40- , 135- , and 39-fold reduction in the kb value at the  ,  , and  receptors 
 relative to 40 using [ s]gtps assays 
 ( table 1 ) . in addition , replacement of the 3-hydroxy group 
 in 40 with 10 other functional groups all led to much 
 reduced in vitro antagonist efficacy relative to 40 , 
 suggesting that the phenolic group was essential for the high potency 
 of 40 . , we demonstrate that 4 , which 
 has the hydroxyl group of the 4-(3-hydroxyphenyl ) group in jdtic replaced 
 by a hydrogen ( table 2 ) , is a potent and selective  opioid receptor antagonist . compound 4 has ke values 
 of 8.9 , 442 , and 0.024 nm at the  ,  , and  receptors , 
 respectively , compared to ke values of 
 25 , 74 , and 0.02 nm for jdtic . compound 4 with 370- and 18400-fold selectivity for the  receptor relative to the  
 and  receptors was more selective than jdtic for the  
 receptor but a little less selective than jdtic for the  receptor . 
 both compounds , however , are highly selective for the  receptor 
 relative to both the  and  receptors . this discovery 
 is in contrast to previously reported structural activity studies 
 of 40 as well as other compounds in this class of compounds . the data represents the mean ( se ) 
 from at least three independent experiments . data taken from ref ( 33 ) . compound 5 , which has a fluoro group in place of the 
 hydrogen present in 4 or the hydroxyl group in jdtic , 
 with ke values of 14.8 , 249 , and 0.01 
 nm at the  ,  , and  receptors , respectively , and 
 1480- and 24900-fold  selectivity relative to the  and 
  receptors , is both a more potent and more selective  
 opioid receptor antagonist than jdtic or 4 . compounds 6 and 7 have a chloro and bromo group , respectively , 
 in place of the hydroxyl group in jdtic . compound 6 has 
 a ke = 0.039 nm at the  receptor , 
 with 175- and 17600-fold selectivity for the  relative to the 
  and  receptors and thus has good  potency and 
 selectivity . compound 7 , which has the larger bromo group 
 in place of the hydroxyl in jdtic , has a weaker ke value of 0.268 nm at the  receptor and only a 
 25-fold selectivity for the  receptor relative to the  
 receptor . replacement of the hydroxyl group in jdtic with the amino 
 electron donating amino group to give 8 results in a 
 decrease in potency at the  receptor ( ke = 0.25 nm ) and increased potency at the  receptor 
 ( ke = 10.6 nm ) . this results in only 42-fold 
 selectivity for the  relative to the  receptor . compound 8 has 7600-fold selectivity for the  relative to the 
  receptor and thus is more selective than jdtic for the  
 relative to the  receptor . in a previous report , we compared 
 the opioid receptor antagonist 
 efficacy of compounds 4145 ( chart 2 ) using the same conditions as that used in this 
 study . the nitro ( 41 ) , acetylamino 
 ( 42 ) , methanesulfonylamino ( 43 ) , and amino 
 ( 44 ) analogues were 320- , 70- , 200- , and 10-times less 
 potent as a  antagonist than jdtic and were not as selective 
 for the  receptor relative to the  and  receptors 
 as jdtic . the methoxy ( 45 ) analogue was only 3-fold less 
 potent than jdtic as a  antagonist . compound 45 was selective for the  receptor relative to the  and 
  but was not as selective as jdtic . the critical importance 
 of a methoxy or hydroxyl group in the tetrahydroisoquinoline 
 carboxamide ( tic ) part of jdtic is further shown by the results with 9 and 10 , where the tic hydroxyl group in jdtic 
 is replaced by a hydrogen or fluoro substituent , respectively . compound 9 has ke values of 16 , 158 , and 
 4.3 nm at the  ,  , and  receptors , respectively , 
 with only 3.7- and 37-fold selectivity for the  relative to 
 the  and  receptors . thus , 9 is much less 
 potent and selective as a  opioid antagonist than jdtic or 4 ( compound 9 was previously characterized as 
 the free base but was not evaluated for opioid antagonst efficacy 
 under conditions used in this study ) . compound 10 with ke values 
 of 7.7 and 2.20 nm at the  and  receptors , respectively , 
 is also much less potent and selective as a  opioid receptor 
 antagonist . compound 11 can 
 be viewed as a compound having both 
 hydroxyl groups in jdtic replaced by a hydrogen or by replacement 
 of the tic hydroxyl group in 4 with a hydrogen . viewing 
 the change either way again shows the importance of the tic hydroxyl 
 group to the high  potency and selectivity . compound 11 with a ke value of 16 nm at 
 the  receptor has low potency for this receptor . compound 12 can be viewed as a compound having both 
 hydroxyl groups in jdtic replaced by fluoro groups or by replacement 
 of the tic hydroxyl group in 5 with a fluoro group . regardless 
 of how 12 is viewed , its low  potency ( ke = 2.22 nm compared to 0.02 nm for jdtic ) shows 
 the importance of tic hydroxyl to the  potency and selectivity 
 of jdtic , 4 , and 5 . similar to 10 , 12 also behaved as a weak inverse agonist in the  
 opioid receptor assay . of all the jdtic analogues synthesized and 
 evaluated herein , these are the only two compounds that are inverse 
 agonists in the  receptor assay . one interesting finding 
 from the x - ray crystallographic structure 
 of the human  opioid receptor is that the interaction of ligand 
 hydroxyls with the receptor is mediated by intervening structured 
 water molecules ( figure 1a , receptor pocket 
 waters indicated by blue green spheres ) . as illustrated in the two - dimensional kor - jdtic interaction 
 diagram of the 7-hydroxy - d - tic , hydroxyl participates in 
 hydrogen bonds with two structured water molecules which in turn interact 
 with residues lys227 and tyr139 ( figure 1b ) . this binding arrangement suggested that replacing the ligand hydroxyls 
 with a substituent , which could replace the structured water in the 
 x - ray structure , both in location and hydrogen bonding capacity , would 
 result in direct ligand - to - receptor hydrogen bonding interactions . 
 removing the dependency on water molecules in the receptor pocket 
 might result in enhanced or altered properties . three of the compounds , 13 , 14 , and 15 , were prepared to 
 test this hypothesis by replacing one or both of the jdtic hydroxyls 
 with carboxamide substituents . water pair with a carboxamide group 
 was tested by computational docking studies of compound 13 ( in which the 7-hydroxyl of 7-hydroxy - d - tic is replaced 
 by a carboxamide ) . as anticipated , the overall binding pose of compound 13 is identical to that observed for jdtic , with the 13 carboxamide group directly providing a hydrogen - bond interaction 
 with lys227 ( figure 2a ) . the two - dimensional 
 kor-13 interaction diagram of the docking result ( figure 2b ) illustrates that 13 carboxamide 
 interaction with lys227 may not require an intervening water molecule . ( a ) three - dimensional 
 view of the hydrogen - bonding interactions 
 between jdtic and the kor ( pdb 4djh ) . ( b ) two - dimensional diagram of the 
 hydrogen - bond and hydrophobic interactions between the tic moiety 
 of jdtic and the kor ( pdb 4djh ) . the origin 
 of hydrophobic interactions are indicated by the direction of red 
 line segments around the receptor residues and ligand atoms . ( a ) three - dimensional 
 view of the hydrogen - bonding interactions 
 between compound 13 and the kor ( docking calculation ) . ( b ) two - dimensional diagram of the hydrogen - bond and hydrophobic interactions 
 between the tetrahydroisoquinoline-7-carboxamide group of compound 13 and the kor ( docking calculation ) . the origin of hydrophobic interactions are 
 indicated by the direction of red line segments around the receptor 
 residues and ligand atoms . the his291 nitrogen - to - carboxamide nitrogen 
 distance ( d1 ) is 3.89  and the lys227 carbonyl oxygen - to - carboxamide 
 nitrogen distance ( d2 ) is 2.83  . in this study , we 
 found that 13 had a ke = 
 0.02 nm and thus was as potent a  opioid receptor 
 antagonist as jdtic ( table 2 ) . compound 14 , which has both phenolic groups in jdtic replaced by a 
 carboxamide group , has a ke = 0.11 nm 
 at the  opioid receptor . all three compounds are highly selective 
 for the  relative to the  and  receptors . compound 15 can be viewed as an analogue of 4 , where the 
 hydroxyl group in the tic portion of 7 has been replaced 
 by a carboxamide group . this compound with a ke = 0.041 nm at the  receptor and 163- and 2700-fold 
 selectivity for the  receptor relative to the  and  
 receptors , respectively , is slightly less  potent and selective 
 than compound 4 . in previous studies , we reported that 37 , which has a carboxamide group replacing the hydroxyl of 
 the 4-(3-hydroxyphenyl ) group , has a ke = 0.12 nm at the  receptor , which was only 6 times less potent 
 than jdtic as a  opioid receptor ( kor ) antagonist ( table 2 ) . calculated 
 physiochemical properties such as topological polar 
 surface area ( tpsa ) , lipophilicity ( clogp ) , and derived values such 
 as logbb are useful indicators of a compound s potential to 
 penetrate the brain . these molecular descriptors were calculated for 
 jdtic , previously reported 37 , as well as 4 , 5 , 13 , and 14 ( table 3 ) . in general , cns drugs have clogp in the range 24 , tpsa less than 76  , and logbb greater than 1 . the lead compound , jdtic , which proceeded through 
 phase 1 clinical studies , has a tpsa = 84.83 , which is larger than 
 76  . compounds 13 , 14 , 
 and 37 , with tpsa values of 107.69 , 130.55 , and 107.69 , 
 respectively , are also above the 76  . compounds 4 and 5 both have tpsa values of 64.6 , which 
 is less than 76  . jdtic and all of the analogues 
 except 5 had clogp values of less than 4 . jdtic , 4 , and 5 have logbb values of 0.57 , 
 0.23 , and 0.19 and thus are greater than 1 , 
 predicting good brain penetration . compounds 13 , 14 , and 37 with logbb values of 0.98 , 
 1.39 , and 1.02 , are predicted to have poorer brain 
 penetration . compounds that interact with the human ether - a - go - go gene ( herg ) 
 product , which is a potassium channel , can produce qt prolongation 
 and cardiotoxic effects . compounds 13 , 14 , and 37 have ki values 
 of > 10 m ( table 4 ) . compounds 4 and 5 have ki values of 7.05 and 6.25 m , similar 
 to the 8.82 m value for jdtic ( table 4 ) . compounds that have > 5% permeability in the mdck assay , > 50% 
 stability 
 in the plasma and s9 stability assay , > 25% transported in the pampa 
 assay , and > 20 m solubility are considered desirable . all 
 of 
 the jdtic analogues except previously reported 37 have 
 > 5% permeability in the mdck assay . compounds 4 , 13 , and 14 , with 27 , 17 , and 14% , were more permeable 
 than jdtic , which had 11% permeability ( table 4 ) . all of the compounds showed > 20 m solubility at ph 3 , 
 and 
 all compounds were more soluble than jdtic , which was considered to 
 be highly soluble . compounds 13 , 14 , and 37 all also had > 20% solubility at ph 7.4 . compounds 4 and 5 had values of 11 and 10 m , which 
 are similar to the 11 m for jdtic . jdtic and the synthesized 
 analogues showed good stability in both the plasma and s9 stability 
 assays . jdtic , 4 , 13 , and 14 had > 25% transported at both ph 5.5 and 7.4 in the pampa assay . on the basis of the mdck , solubility , plasma and s9 stability , and 
 pampa results combined with the calculated tpsa , clogp , and logbb 
 data , 4 appears to have the best overall profile . however , 
 the ki = 7.05 m in the herg could 
 be of concern . compounds 13 and 14 show 
 very favorable mdck , solubility , plasma and s9 stability , and pampa 
 results as well as ki values of > 10 
 m 
 for binding in the herg assay . however , the calculated tpsa and logbb 
 suggest that brain penetration could be a concern . previously 
 reported 37 was evaluated for its ability 
 to block  agonist u50,488-induced diuresis at 330 mg / kg 
 ig and 130 mg / kg ip in rats . compound 37 blocked the u50,488-induced diuresis at 24 h and 8 days 
 at 1 , 10 , and 30 mg / kg , 15 days at 10 and 30 mg / kg , and 22 and 29 
 days at 30 mg / kg following ip administration . compound 37 was ineffective in blocking the u50,488-induced diuresis when given 
 ig . because  opioid receptor agonist - induced diuresis is mediated 
 by the central nervous system , the high potency of 37 in this assay after ip administration suggest that the high tpsa 
 valve ( 107.69 ) and logbb ( 1.02 ) are misleading for this jdtic 
 analogue having an aromatic carboxamido substituent . similarly , the 
 high tpsa and logbb values for 13 and 14 , both of which have aromatic carboxamido substituents , could also 
 be misleading . the lack of efficacy after oral ( ig ) administration 
 of 37 could be due to its poor pampa values ( 1.2 and 
 3.2% at ph 7.4 and 5.5 , respectively ) and its poor mdck value ( 2% ) . in summary , these studies provide the unexpected finding that replacement 
 of the 3-hydroxyl substituent of the 4-(3-hydroxphenol ) group of jdtic 
 with either a hydrogen , fluoro , or chloro group leads to  opioid 
 receptor antagonists that are as highly potent and selective as jdtic . this finding is in contrast to what would have been predicted based 
 on structure  activity relationship studies of other n - substituted 
 3,4-dimethyl-4-(3-hydroxyphenyl)piperidines such as 40 ( ly255582 ) , as well as much of the 
 sar studies reported for opioid ligands in general . the high 
  opioid receptor potency and selectivity relative 
 to the  and  opioid receptors of 4 , 5 , and 14 combined with their favorable herg , 
 mdck , pampa , solubility , and plasma and s9 stability in vitro preclinical 
 studies and calculated tpsa , clogp , and logbb values suggest that 
 the compounds should be considered for further development as potential 
 drugs for treating depression , anxiety , schizophrenia , and addiction 
 ( cocaine , nicotine , methamphetamine , alcohol , and eating disorders ) . melting 
 points were determined using a mel - temp ii capillary melting 
 point apparatus and are uncorrected . nuclear magnetic resonance ( h nmr and c nmr ) spectra were obtained on a varian 
 avance dpx-500 mhz nmr spectrometer or a bruker unity inova 300 mhz 
 nmr spectrometer . chemical shifts are reported in parts per million 
 ( ppm ) with reference to internal solvent . mass spectra ( ms ) were conducted 
 on a perkinelmer sciex ap1 150 ex mass spectrometer equipped with 
 esi ( turbospray ) source . atlanta , ga . the purity of the compounds ( > 95% ) 
 was 
 established by elemental analysis . optical rotations were measured 
 on an autopol iii polarimeter , purchased from rudolf research . analytical 
 thin - layer chromatography ( tlc ) was carried out using emd silica gel 
 60 f254 tlc plates . flash column chromatography was 
 done on a combiflash companion system using isco prepacked silica 
 gel columns or using em science silica gel 60a ( 230400 mesh ) . unless otherwise stated , reagent - grade chemicals were obtained 
 from commercial sources and were used without further purification . all moisture- and air - sensitive reactions and reagent transfers were 
 carried out under dry nitrogen . the amine 20 ( 137 mg , 0.50 mmol ) , boc-7-hydroxy - d - tic ( 161 mg , 0.55 
 mmol ) , hbtu ( 208 mg , 0.55 mmol ) , and net3 ( 280 l , 
 2.2 mmol ) were stirred in ch2cl2 ( 11 ml ) for 
 12 h. the concentrated residue was subjected to chromatography on 
 silica gel using a step gradient up to 50% cma80 in ch2cl2 to afford the boc - protected intermediate . this boc - protected 
 compound was stirred in tfa / ch2cl2 ( 1:1 , 10 
 ml ) for 12 h , concentrated , and the residue subjected to chromatography 
 on silica gel ( 12 g ) using a gradient up to 50% cma80 in ch2cl2 as the eluent to afford the free base of 4 . h nmr ( 300 mhz , cdcl3 )  7.087.34 
 ( m , 6h ) , 6.836.95 ( m , 1h ) , 6.586.68 ( m , 1h ) , 6.436.54 
 ( m , 1h ) , 3.954.12 ( m , 1h ) , 3.82 ( s , 2h ) , 3.333.49 
 ( m , 1h ) , 2.953.15 ( m , 1h ) , 2.442.91 ( m , 5h ) , 2.202.43 
 ( m , 2h ) , 1.842.03 ( m , 2h ) , 1.60 ( d , j = 13.0 
 hz , 1h ) , 1.29 ( s , 3h ) , 0.820.98 ( m , 6h ) , 0.640.79 
 ( m , 3h ) . c nmr ( 75 mhz , cdcl3 )  173.3 , 
 154.9 , 136.6 , 130.2 , 128.1 , 125.7 , 125.7 , 125.4 , 125.2 , 114.2 , 112.3 , 
 59.6 , 57.1 , 55.5 , 51.2 , 50.6 , 47.9 , 38.8 , 38.4 , 30.9 , 30.7 , 30.4 , 
 27.7 , 19.1 , 17.8 , 16.3 . ms ( esi ) m / z 450.7 ( m + h ) . the free base was converted to 67.7 mg 
 ( 24% ) of the dihydrochloride salt ( over two steps ) as a white powder : 
 mp 195199 c , [ ]d + 101 
 ( c 0.17 , ch3oh ) . ( c28h41cl2n3o22h2o ) c , h , n. the amine 30 ( 146 mg , 0.50 mmol ) was combined with boc-7-hydroxy - d - tic 
 ( 150 mg , 0.54 mmol ) and hbtu ( 200 mg , 0.53 mmol ) and dissolved in 
 ch3cn ( 10 ml ) before net3 ( 0.2 ml , 1.4 mmol ) 
 was added . the concentrated residue was subjected to chromatography 
 on silica gel using a gradient of etoac in hexanes to afford an oil 
 ( 225 mg ) which was dissolved in ch3oh ( 3 ml ) and 6 n hcl 
 ( 3 ml ) and stirred 12 h. the concentrated residue was dissolved in 
 dilute nh4oh and extracted with ch2cl2 . the residue was subjected to chromatography on silica 
 gel using etoac then a gradient of cma80 in ch2cl2 as the eluent to afford the free base of 5 . h nmr ( cdcl3 )  7.177.26 ( m , 1h ) , 7.11 ( d , j = 9.0 hz , 1h ) , 7.01 ( d , j = 8.1 hz , 1h ) , 
 6.786.97 ( m , 3h ) , 6.65 ( d , j = 8.3 hz , 1h ) , 
 6.51 ( s , 1h ) , 4.03 ( dd , j = 4.9 , 9.8 hz , 1h ) , 3.87 
 ( s , 2h ) , 3.45 ( dd , j = 4.7,10.9 hz , 1h ) , 3.07 ( dd , j = 4.5 , 16.0 hz , 1h ) , 2.542.80 ( m , 3h ) , 2.382.52 
 ( m , 3h ) , 2.142.37 ( m , 2h ) , 1.842.01 ( m , 2h ) , 1.54 
 ( d , j = 12.6 hz , 1h ) , 1.221.33 ( m , 4h ) , 0.92 
 ( dd , j = 7.0 , 8.9 hz , 6h ) , 0.68 ( d , j = 6.8 hz , 3h ) . c nmr ( 75 mhz , cdcl3 )  
 173.2 , 163.0 ( d , j = 244 hz ) , 155.1 , 153.1 ( d , j = 6.5 hz ) , 136.7 , 130.1 , 129.4 ( d , j = 
 8.4 hz ) , 125.2 , 121.3 ( d , j = 2.3 hz ) , 114.2 , 112.8 
 ( d , j = 21.8 hz ) , 112.3 , 112.1 ( d , j = 19.5 hz ) , 59.5 , 57.1 , 55.4 , 51.1 , 50.8 , 47.9 , 38.8 , 30.8 , 30.5 , 
 30.4 , 27.5 , 19.2 , 17.7 , 16.2 . ms ( esi ) m / z 468.1 ( m + h ) . the free base was converted 
 to the dihydrochloride salt ( 100 mg , 34% over two steps ) as a white 
 powder . f nmr ( 282 mhz , dmso - d6 )  112.97 ; mp 219223 c ( fusion ) ; [ ]d = + 174 ( c 0.4 , ch3oh ) . ( c28h40cl2fn3o23h2o ) c , h , n. to a solution of 31 ( 56 mg , 0.18 mmol ) and boc-7-hydroxy - d - tic ( 59 mg , 0.20 
 mmol ) in ch2cl2 ( 10 ml ) was added edchcl 
 ( 77 mg , 0.40 mmol ) , hobt ( 3 mg , 0.02 mmol ) , and net3 ( 115 
 l , 0.82 mmol ) . the reaction mixture was stirred at ambient 
 temperature for 12 h then was diluted with ch2cl2 ( 20 ml ) and washed with aq nahco3 ( 10 ml ) . the 
 residue was subjected to chromatography on silica gel using a gradient 
 up to 50% cma80 in chcl3 to afford the 76 mg ( 72% ) of the 
 boc - protected intermediate as a white solid . the intermediate was 
 dissolved in acetonitrile ( 5 ml ) and treated with hcl in dioxane ( 4.0 
 m , 0.33 ml ) . the resulting residue was subjected to chromatography on silica using 
 a gradient up to 50% cma80 in chcl3 as the eluent to afford 
 35 mg ( 48% ) of the free base 6 as white solid . h nmr ( cd3od )  7.137.45 ( m , 4h ) , 7.01 ( d , j = 8.1 hz , 1h ) , 6.68 ( d , j = 7.5 hz , 1h ) , 
 6.58 ( br s , 1h ) , 4.93 ( br s , 2h ) , 4.044.43 ( m , 3h ) , 3.96 ( br 
 s , 1h ) , 3.23 ( br s , 1h ) , 3.073.19 ( m , 3h ) , 3.02 ( br s , 2h ) , 
 2.53 ( br s , 1h ) , 2.30 ( br s , 1h ) , 1.90 ( br s , 1h ) , 1.661.86 
 ( m , 1h ) , 1.41 ( s , 3h ) , 0.99 ( br s , 6h ) , 0.640.88 ( m , 3h ) . c nmr ( cd3od )  175.3 , 162.1 , 155.3 , 139.7 , 
 135.1 , 133.6 , 131.4 , 130.6 , 128.9 , 125.7 , 120.9 , 117.6 , 66.3 , 61.4 , 
 58.8 , 56.9 , 55.0 , 49.4 , 42.8 , 42.3 , 36.0 , 33.8 , 32.4 , 30.8 , 23.7 , 
 22.2 , 19.6 . ms ( esi ) the product was converted to the dihydrochloride 
 salt : [ ]d = + 92.0 ( c 0.52 , meoh ) . ( c28h40cl3n3o22.5h2o ) c , h , n ) . to a solution of 32 ( 94 mg , 0.26 mmol ) and boc-7-hydroxy - d - tic ( 89 mg , 0.30 
 mmol ) in ch2cl2 ( 10 ml ) was added edchcl 
 ( 116 mg , 0.60 mmol ) , hobt ( 5 mg , 0.03 mmol ) , and triethylamine ( 173 
 l , 1.24 mmol ) . the reaction mixture was stirred at ambient 
 temperature for 12 h. the reaction mixture was diluted with ch2cl2 ( 20 ml ) and washed with aq nahco3 ( 10 ml ) . the organic layer was dried ( na2so4 ) and concentrated to a residue which was subjected to chromatography 
 on silica gel using a gradient up to 50% cma80 in chcl3 as the eluent to afford 79 mg ( 46% ) of the boc - protected intermediate 
 as a white solid . h nmr ( cdcl3 )  7.137.27 
 ( m , 2h ) , 7.09 ( d , j = 4.90 hz , 2h ) , 6.91 ( d , j = 8.3 hz , 1h ) , 6.506.75 ( m , 1h ) , 6.45 ( br s , 1h ) , 
 4.384.54 ( m , 1h ) , 4.34 ( br s , 1h ) , 3.78 ( br s , 1h ) , 3.11 ( dd , j = 3.0 , 15.1 hz , 1h ) , 2.90 ( dd , j = 5.8 , 
 15.3 hz , 1h ) , 2.50 ( br s , 1h ) , 2.272.42 ( m , 1h ) , 2.24 ( br 
 s , 1h ) , 1.932.14 ( m , 3h ) , 1.591.93 ( m , 2h ) , 1.43 ( s , 
 9h ) , 1.35 ( br s , 1h ) , 1.14 ( s , 3h ) , 0.78 ( d , j = 
 6.6 hz , 3h ) , 0.69 ( d , j = 6.8 hz , 3h ) , 0.52 ( d , j = 6.8 hz , 3h ) . c nmr ( cdcl3 )  
 171.3 , 155.7 , 152.9 , 134.0 , 129.7 , 129.3 , 128.8 , 128.4 , 124.4 , 122.5 , 
 114.8 , 112.9 , 81.6 , 59.9 , 56.1 , 51.3 , 50.0 , 44.8 , 38.8 , 38.7 , 30.5 , 
 29.8 , 28.4 , 27.4 , 19.2 , 16.9 , 16.0 . ms ( esi ) m / z 628.6 ( m + h ) . the intermediate was dissolved 
 in acetonitrile ( 5 ml ) and treated with hcl in dioxane ( 4.0 m , 0.3 
 ml ) and stirred for 12 h. concentration of the reaction mixture afforded 
 a white solid which was chromatographed on silica using a gradient 
 up to 50% cma80 in chcl3 to afford 56 mg ( 88% ) of 7 free base as white solid . h nmr ( cdcl3 )  7.147.27 ( m , 2h ) , 6.957.14 ( m , 2h ) , 6.84 
 ( d , j = 8.3 hz , 1h ) , 6.486.66 ( m , 1h ) , 6.43 
 ( d , j = 2.1 hz , 1h ) , 3.944.23 ( m , 1h ) , 3.80 
 ( br s , 2h ) , 3.38 ( dd , j = 4.7 , 10.7 hz , 1h ) , 3.00 
 ( dd , j = 4.2 , 16.3 hz , 1h ) , 2.70 ( br s , 1h ) , 2.57 
 ( dd , j = 11.0 , 14.9 hz , 2h ) , 2.43 ( br s , 2h ) , 2.27 
 ( d , j = 8.10 hz , 1h ) , 2.17 ( br s , 1h ) , 1.98 ( s , 1h ) , 
 1.87 ( br s , 2h ) , 1.49 ( d , j = 12.2 hz , 1h ) , 0.971.28 
 ( m , 3h ) , 0.700.91 ( m , 6h ) , 0.62 ( d , j = 6.8 
 hz , 3h ) . c nmr ( cdcl3 )  173.1 , 154.5 , 
 152.5 , 136.8 , 130.2 , 129.7 , 128.9 , 128.5 , 125.6 , 124.4 , 122.6 , 114.0 , 
 112.2 , 59.4 , 57.0 , 55.2 , 51.1 , 50.6 , 47.9 , 38.7 , 38.6 , 30.6 , 30.3 , 
 27.5 , 19.2 , 17.8 , 16.2 . the free base was converted to the dihydrochloride 
 salt : [ ]d = + 98.0 ( c 0.61 , meoh ) . ( c28h40brcl2n3o20.5h2o ) c , h , n. to a solution 26 ( 125 mg , 0.43 mmol ) in ch2cl2 ( 10 ml ) was 
 added boc-7-hydroxy - d - tic ( 125 mg , 0.43 mmol ) , hobt ( 10 mg , 
 0.1 mmol ) , and edchcl ( 191 mg , 1.0 mmol ) , followed by the addition 
 of diisopropylethylamine ( 0.15 ml , 0.86 mmol ) . the resulting solution 
 was stirred at room temperature for 12 h then washed with saturated 
 aqueous nahco3 ( 5 ml ) . the combined organic layers were washed with 
 brine ( 5 ml ) , dried ( na2so4 ) , and concentrated . the resulting residue was purified by chromatography on silica gel 
 using a gradient up to 50% cma80 in ch2cl2 as 
 the eluent . the product containing fractions were combined and concentrated 
 to afford 174 mg ( 72% ) of the boc - protected intermediate . the intermediate 
 was then dissolved in ch3oh ( 10 ml ) to which aq hcl ( 6 
 n , 10 ml ) was added . the resulting residue was subjected to chromatography 
 on silica gel using a gradient up to 50% cma80 in ch2cl2 as the eluent to afford 8 free base . h nmr ( 300 mhz , cdcl3 )  6.997.18 ( m , 2h ) , 
 6.85 ( d , j = 8.3 hz , 1h ) , 6.416.72 ( m , 5h ) , 
 4.02 ( dt , j = 4.6 , 9.1 hz , 1h ) , 3.82 ( s , 2h ) , 3.383.48 
 ( m , 2h ) , 3.02 ( dd , j = 4.7 , 16.2 hz , 1h ) , 2.512.79 
 ( m , 3h ) , 2.362.51 ( m , 3h ) , 2.102.35 ( m , 2h ) , 1.792.00 
 ( m , 2h ) , 1.50 ( d , j = 12.6 hz , 1h ) , 1.161.31 
 ( m , 3h ) , 0.830.98 ( m , 6h ) , 0.66 ( d , j = 6.8 
 hz , 3h ) . c nmr ( 75 mhz , cdcl3 )  173.1 , 
 154.9 , 151.3 , 145.8 , 136.4 , 130.0 , 128.8 , 124.9 , 116.4 , 114.2 , 112.9 , 
 112.4 , 112.2 , 59.4 , 56.8 , 55.3 , 51.2 , 50.7 , 47.6 , 38.7 , 38.2 , 30.7 , 
 30.5 , 30.1 , 27.3 , 19.0 , 17.7 , 16.2 . the free base was converted to 
 the trihydrochloride salt , affording 132.4 mg ( 50% over two steps ) 
 of a white powder . ms ( esi ) m / z 465.5 
 ( m + h ) , mp 241243 c ( fusion ) , [ ]d + 98.7 ( c 0.38 , ch3oh ) . ( c28h43cl3n4o22.5h2o ) c , h , n. the amine 21 ( 145 mg , 0.50 mmol ) , boc - d - tic ( 152 mg , 0.55 mmol ) , hbtu 
 ( 208 mg , 0.55 mmol ) , and net3 ( 280 l , 2.2 mmol ) 
 were stirred in ch2cl2 ( 11 ml ) for 12 h. the 
 concentrated residue was subjected to chromatography on silica gel 
 using a step gradient up to 50% cma80 in ch2cl2 as the eluent to afford the boc - protected intermediate . this boc - protected 
 compound was stirred in tfa / ch2cl2 ( 1:1 , 10 
 ml ) for 12 h , concentrated , and the residue subjected to chromatography 
 on silica gel ( 12 g ) using a gradient up to 50% cma80 in ch2cl2 as the eluent to afford the free base of 9 . h nmr ( 300 mhz , cdcl3 )  6.967.21 
 ( m , 5h ) , 6.736.82 ( m , 2h ) , 6.596.67 ( m , 1h ) , 4.07 
 ( dt , j = 4.9 , 9.3 hz , 1h ) , 4.00 ( s , 2h ) , 3.56 ( dd , j = 5.1 , 10.7 hz , 1h ) , 3.20 ( dd , j = 5.0 , 
 16.7 hz , 1h ) , 2.702.83 ( m , 2h ) , 2.64 ( d , j = 10.4 hz , 1h ) , 2.292.49 ( m , 4h ) , 2.20 ( dt , j = 4.3 , 12.4 hz , 1h ) , 1.802.01 ( m , 2h ) , 1.54 ( d , j = 13.0 hz , 1h ) , 1.231.30 ( m , 3h ) , 0.93 ( dd , j = 6.8 , 9.0 hz , 6h ) , 0.67 ( d , j = 6.8 
 hz , 3h ) . c nmr ( 75 mhz , cdcl3 )  172.9 , 
 155.9 , 152.1 , 135.8 , 134.4 , 129.3 , 129.1 , 126.5 , 126.1 , 125.5 , 117.7 , 
 113.1 , 112.4 , 59.6 , 56.7 , 55.1 , 51.1 , 50.6 , 47.8 , 38.9 , 38.5 , 31.1 , 
 30.6 , 27.5 , 19.2 , 17.8 , 16.2 . ms ( esi ) m / z 450.5 ( m + h ) . the free base was converted 
 to the dihydrochloride salt ( 50.3 mg , 18% over two steps ) as a white 
 powder : mp 197200 c ( fusion ) , [ ]d = + 108 ( c 0.10 , ch3oh ) . ( c28h41cl2n3o22h2o ) c , h , n. amine 21 ( 145 
 mg , 0.50 mmol ) and acid 7-fluoro - boc - d - tic ( 162 mg , 0.55 mmol ) were combined in ch2cl2 ( 10 ml ) and treated with edchcl ( 191 mg , 1.0 mmol ) 
 then net3 ( 0.35 ml , 2.5 mmol ) . after 12 h , the concentrated 
 residue was subjected to chromatography on silica gel using a gradient 
 up to 60% cma80 in ch2cl2 as the eluent . the 
 product containing fractions were concentrated then treated with meoh 
 ( 5 ml ) and aq hcl ( 6 n , 5 ml ) . after 1 h , the concentrated residue 
 was subjected to chromatography on silica gel using a gradient up 
 to 75% cma80 in ch2cl2 as the eluent to afford 10 free base . h nmr ( 300 mhz , cdcl3 )  7.067.22 ( m , 2h ) , 7.01 ( dd , j = 
 5.8 , 8.3 hz , 1h ) , 6.616.86 ( m , 5h ) , 4.09 ( tt , j = 4.7 , 9.4 hz , 1h ) , 3.95 ( s , 2h ) , 3.52 ( dd , j = 
 4.9 , 10.6 hz , 1h ) , 3.14 ( dd , j = 4.8 , 16.5 hz , 1h ) , 
 2.612.82 ( m , 3h ) , 2.272.55 ( m , 4h ) , 2.112.27 
 ( m , 1h ) , 1.801.98 ( m , 2h ) , 1.52 ( d , j = 12.8 
 hz , 1h ) , 1.25 ( s , 3h ) , 0.92 ( t , j = 7.7 hz , 6h ) , 
 0.66 ( d , j = 6.8 hz , 3h ) . c nmr ( 75 
 mhz , cdcl3 )  172.9 , 161.2 ( d , j = 245 hz ) , 156.4 , 151.9 , 137.4 ( d , j = 6.5 hz ) , 
 130.7 ( d , j = 7.8 hz ) , 129.7 ( d , j = 2.9 hz ) , 129.1 , 117.3 , 113.6 ( d , j = 21.2 hz ) , 
 113.1 , 112.6 , 112.1 ( d , j = 21.1 hz ) , 59.6 , 56.7 , 
 55.1 , 51.3 , 50.6 , 47.6 , 38.8 , 38.4 , 30.8 , 30.7 , 30.3 , 27.5 , 19.2 , 
 17.8 , 16.3 . the free base was converted to the dihydrochloride 
 salt ( 90.7 mg , 15% over two steps ) as a white powder : mp 202206 
 c ( fusion ) ; [ ]d = + 93 ( c 0.1 , ch3oh ) . ( c28h40cl2fn3o22.5h2o ) 
 c , h , n. the amine 20 ( 137 mg , 0.50 mmol ) , boc - d - tic ( 152 mg , 0.55 mmol ) , hbtu 
 ( 208 mg , 0.55 mmol ) , and net3 ( 280 l , 2.2 mmol ) 
 were stirred in ch2cl2 ( 11 ml ) for 12 h. the 
 concentrated residue was subjected to chromatography on silica gel 
 using a step gradient up to 50% cma80 in ch2cl2 as the eluent to afford the boc - protected intermediate . this boc - protected 
 compound was stirred in tfa / ch2cl2 ( 1:1 , 10 
 ml ) for 12 h , concentrated , and the residue subjected to chromatography 
 on silica gel ( 12 g ) using a gradient up to 50% cma80 in ch2cl2 as the eluent to afford the free base of 11 . h nmr ( 300 mhz , cdcl3 )  7.207.34 
 ( m , 4h ) , 7.067.19 ( m , 3h ) , 6.967.05 ( m , 1h ) , 4.034.10 
 ( m , 1h ) , 4.00 ( s , 2h ) , 3.53 ( dd , j = 4.9 , 10.9 hz , 
 1h ) , 3.133.25 ( m , 1h ) , 2.88 ( d , j = 15.1 
 hz , 1h ) , 2.672.84 ( m , 3h ) , 2.58 ( d , j = 10.4 
 hz , 3h ) , 2.262.48 ( m , 3h ) , 2.002.09 ( m , 1h ) , 1.852.01 
 ( m , 1h ) , 1.561.69 ( m , 1h ) , 1.31 ( s , 3h ) , 0.880.99 
 ( m , 6h ) , 0.70 ( d , j = 7.0 hz , 2h ) . c 
 nmr ( 75 mhz , cdcl3 )  172.9 , 135.9 , 134.4 , 129.3 , 
 128.2 , 126.4 , 126.1 , 125.6 , 125.5 , 59.2 , 56.8 , 55.1 , 51.2 , 50.3 . 47.9 , 
 38.6 , 38.3 , 31.2 , 30.7 , 27.5 , 19.2 , 17.9 , 16.1 . the free base was 
 converted to the dihydrochloride salt ( 21.9 mg , 8% over two steps ) 
 as a white powder : mp 162165 c ( fusion ) , [ ]d = + 96 ( c 0.10 , ch3oh ) . ( c28h41cl2n3o2h2o ) c , h , n. amine 30 ( 24.2 
 mg , 0.083 mmol ) and acid 7-fluoro - boc - d - tic ( 55.7 mg , 0.19 
 mmol ) were combined in ch2cl2 ( 8 ml ) and treated 
 with edchcl ( 40 mg , 0.2 mmol ) then net3 ( 0.10 ml , 
 0.72 mmol ) . after 12 h , the concentrated residue was subjected to 
 chromatography on silica gel using a gradient up to 50% cma80 in ch2cl2 . the product containing fractions were concentrated 
 then treated with meoh ( 5 ml ) and aq hcl ( 5 ml ) . after 1 h , the concentrated 
 residue was subjected to chromatography on silica gel using a gradient 
 up to 50% etoac in hexanes with 1% nh3 ( prepared by adding 
 1% concd nh4oh by volume as the eluent and drying over 
 na2so4 ) as the eluent to afford 12 free base . h nmr ( 300 mhz , cdcl3 )  
 7.197.29 ( m , 1h ) , 6.987.14 ( m , 3h ) , 6.93 ( td , j = 2.1 , 11.3 hz , 1h ) , 6.84 ( tt , j = 2.8 , 
 8.3 hz , 2h ) , 6.73 ( dd , j = 2.6 , 9.0 hz , 1h ) , 3.944.10 
 ( m , 3h ) , 3.51 ( dd , j = 4.9 , 10.6 hz , 1h ) , 3.15 ( dd , j = 5.0 , 16.5 hz , 1h ) , 2.582.85 ( m , 3h ) , 2.062.54 
 ( m , 5h ) , 1.852.02 ( m , 2h ) , 1.56 ( dd , j = 
 1.2 , 12.9 hz , 1h ) , 1.28 ( s , 3h ) , 0.880.99 ( m , 6h ) , 0.66 ( d , j = 7.0 hz , 3h ) . c nmr ( 75 mhz , cdcl3 )  172.5 , 163.2 ( d , j = 244.4 hz ) , 161.3 
 ( d , j = 244.7 hz ) , 153.3 ( d , j = 
 6.4 hz ) , 137.7 ( d , j = 6.5 hz ) , 130.8 ( d , j = 7.8 hz ) , 130.0 ( d , j = 2.9 hz ) , 129.6 
 ( d , j = 8.2 hz ) , 121.3 ( d , j = 2.6 
 hz ) , 113.7 ( d , j = 21.2 ) , 112.8 ( d , j = 21.4 hz ) , 112.3 ( d , j = 21.1 hz ) , 112.2 ( d , j = 21.1 hz ) , 59.5 , 56.7 , 55.2 , 51.2 , 50.6 , 47.7 , 38.7 , 
 38.7 , 30.6 , 30.5 , 30.4 , 27.4 , 19.2 , 17.8 , 16.2 . ms ( esi ) m / z 470.9 ( m + h ) . the free base was converted to the dihydrochloride salt ( 21.9 mg , 
 46% over two steps ) as a white powder : mp 152156 c ( fusion ) , 
 [ ]d = + 115 ( c 0.10 , 
 ch3oh ) . ( c28h39cl2f2n3o2h2o ) c , h , n. the amine 21 ( 153 mg , 0.53 mmol ) was added to a solution of the acid 36 ( 170 mg , 0.53 mmol ) , hbtu ( 209 mg , 0.55 mmol ) , and net3 ( 0.24 ml , 1.7 mmol ) in ch3cn ( 40 ml ) . after 12 h , the 
 residue obtained on concentration was subjected to chromatography 
 on silica gel using a gradient up to 70% cma80 in ch2cl2 as the eluent . the product containing fractions were dissolved 
 in ch2cl2 ( 5 ml ) and tfa ( 5 ml ) and stirred 
 12 h. the residue obtained on concentration was subjected to chromatography 
 on silica gel using a gradient of cma80 in ch2cl2 as the eluent . the product containing fractions were concentrated 
 and subjected to chromatography on c18-reverse phase using a gradient 
 from 40 to 60% aq ch3cn with 0.1% tfa . the product containing 
 fractions were concentrated and again subjected to chromatography 
 on silica gel using a gradient of cma80 in ch2cl2 to afford 13 free base . h nmr ( 300 mhz , 
 cdcl3 )  7.54 ( s , 1h ) , 7.45 ( d , j = 7.9 hz , 1h ) , 7.19 ( d , j = 9.5 hz , 1h ) , 7.137.01 
 ( m , 2h ) , 6.746.59 ( m , 3h ) , 6.49 ( bs , 1h ) , 6.02 ( bs , 1h ) , 4.123.86 
 ( m , 3h ) , 3.64 ( t , j = 6.7 hz , 1h ) , 3.142.94 
 ( m , 2h ) , 2.732.54 ( m , 2h ) , 2.502.22 ( m , 4h ) , 2.121.98 
 ( m , 1h ) , 1.901.70 ( m , 2h ) , 1.44 ( d , j = 12.5 
 hz , 1h ) , 1.21 ( s , 3h ) , 0.970.87 ( m , 6h ) , 0.38 ( d , j = 6.8 hz , 3h ) . c nmr ( 75 mhz , cdcl3 )  172.4 , 170.0 , 156.5 , 152.1 , 138.8 , 136.2 , 130.8 , 129.7 , 
 129.3 , 125.4 , 125.1 , 117.6 , 113.2 , 112.7 , 60.6 , 55.6 , 54.9 , 51.9 , 
 50.9 , 46.2 , 38.8 , 38.5 , 31.1 , 30.7 , 30.2 , 27.3 , 19.6 , 18.1 , 16.1 . the free base was converted to 41.9 mg of the dihydrochloride salt 
 ( 13% ) as a white powder : mp 195200 c ( fusion ) , [ ]d + 103 ( c 1.00 , ch3oh ) . ( c29h42cl2n4o33h2o ) c , h , n. the amine 3-{(3r,4r)-1-[(2s)-2-amino-3-methylbutyl]-3,4-dimethylpiperidin-4-yl}benzamide ( 24 mg , 0.08 mmol ) was added to a solution of 
 the acid 36 ( 25 mg , 0.08 mmol ) , hbtu ( 30 mg , 0.08 mmol ) , 
 and net3 ( 30 l , 0.2 mmol ) in ch2cl2 ( 10 ml ) . after 12 h , the concentrated residue was subjected 
 to chromatography on silica gel using a gradient up to 70% cma80 in 
 ch2cl2 as the eluent . the product containing 
 fractions were dissolved in ch2cl2 ( 5 ml ) and 
 tfa ( 5 ml ) and stirred 12 h. the concentrated residue was subjected 
 to chromatography on silica gel using a gradient of cma80 in ch2cl2 to afford 14 free base . h nmr ( 300 mhz , cdcl3 )  7.72 ( s , 1h ) , 7.54 ( dd , j = 6.59 , 14.69 hz , 3h ) , 7.297.46 ( m , 2h ) , 7.017.22 
 ( m , 2h ) , 5.906.78 ( m , 4h ) , 3.99 ( br s , 3h ) , 3.59 ( d , j = 2.83 hz , 1h ) , 3.12 ( d , j = 13.37 hz , 
 1h ) , 2.803.00 ( m , 1h ) , 2.72 ( d , j = 9.80 
 hz , 1h ) , 2.59 ( d , j = 10.93 hz , 1h ) , 2.122.51 
 ( m , 5h ) , 1.89 ( dd , j = 6.97 , 12.24 hz , 3h ) , 1.58 
 ( d , j = 12.06 hz , 1h ) , 1.191.33 ( m , 3h ) , 
 0.92 ( t , j = 7.72 hz , 6h ) , 0.53 ( d , j = 6.78 hz , 3h ) . c nmr ( 75 mhz , cdcl3 )  
 172.2 , 172.1 , 170.2 , 169.4 , 151.1 , 138.8 , 136.2 , 133.2 , 131.1 , 129.4 , 
 128.3 , 125.1 , 125.0 , 124.7 , 124.1 , 59.9 , 55.9 , 55.0 , 51.3 , 50.8 , 46.9 , 
 38.7 , 38.5 , 30.7 , 30.7 , 30.6 , 27.4 , 19.2 , 17.9 , 16.2 . ms ( esi ) m / z 521.0 ( m + h ) . the free 
 base was converted to 20.4 mg ( 39% ) of the dihydrochloride salt as 
 a pale - yellow powder : mp 210215 c ( fusion ) , [ ]d + 101 ( c 0.50 , ch3oh ) . ( c30h43cl2n5o33.25h2o ) c , h , n. to a solution of 20 ( 100 mg , 0.3 mmol ) in ch2cl2 ( 10 
 ml ) was added 36 ( 100 mg , 0.3 mmol ) , hobt ( 10 mg , 0.1 
 mmol ) , and edchcl ( 75 mg , 0.4 mmol ) , followed by the addition 
 of diisopropylethylamine ( 0.26 ml , 1.5 mmol ) . the resulting cloudy 
 solution remained cloudy upon the addition of nmp ( 0.1 ml ) . after 
 12 h , the mixture was washed with saturated aqueous nahco3 ( 10 ml ) . the aqueous layer was extracted with ch2cl2:thf ( 2:1 , 20 ml  2 ) . the combined organic layers were 
 washed with brine ( 5 ml ) , dried ( na2so4 ) , and 
 concentrated . the resulting residue was purified by chromatography 
 on silica gel using a gradient up to 40% cma80 in ch2cl2 as the eluent . the product containing fractions were combined 
 and concentrated to afford 134 mg of the boc - protected intermediate . the intermediate was then dissolved in ch3oh ( 10 ml ) to 
 which aq hcl ( 6 n , 10 ml ) was added . the resulting residue was subjected to chromatography 
 on silica gel using a gradient up to 75% cma80 in ch2cl2 to afford 15 free base . h nmr ( 300 
 mhz , cdcl3 )  7.387.51 ( m , 2h ) , 6.957.28 
 ( m , 9h ) , 5.936.34 ( m , 2h ) , 3.844.04 ( m , 2h ) , 3.46 
 ( dd , j = 5.0 , 10.3 hz , 1h ) , 3.11 ( dd , j = 4.8 , 17.1 hz , 1h ) , 2.622.83 ( m , 2h ) , 2.55 ( d , j = 10.7 hz , 1h ) , 2.302.46 ( m , 3h ) , 2.082.29 
 ( m , 2h ) , 1.751.99 ( m , 2h ) , 1.51 ( d , j = 12.2 
 hz , 1h ) , 1.131.28 ( m , 3h ) , 0.740.94 ( m , 6h ) , 0.56 
 ( d , j = 6.8 hz , 3h ) . c nmr ( 75 mhz , 
 cdcl3 )  172.2 , 169.3 , 150.1 , 138.8 , 136.2 , 131.1 , 
 129.4 , 128.0 , 125.4 , 125.3 , 125.0 , 59.5 , 56.3 , 55.2 , 51.3 , 50.6 , 47.4 , 
 38.6 , 38.4 , 31.0 , 30.5 , 27.5 , 19.1 , 17.7 , 16.3 . ms ( esi ) m / z 477.5 ( m + h ) . the free base was 
 converted to the dihydrochloride salt , which was sonicated in etoac . the solvent was decanted and the solids dried under nitrogen to afford 
 43 mg ( 24% over two steps ) as a white powder : mp 218222 c 
 ( fusion ) , [ ]d + 105 ( c 0.195 , ch3oh ) . ( c29h42cl2n4o22.75h2o ) c , h , 
 n. the title compound 
 was prepared by the addition of trifluoromethanesulfonic anhydride 
 ( 3.4 ml , 20 mmol ) to 3-[(3r,4r)-34-dimethylpiperidin-4-yl]phenol 
 ( 16 ) ( 1.0 g , 4.9 mmol ) and diisopropylethylamine ( 5.1 
 ml , 29 mmol ) in ch2cl2 ( 30 ml ) at 78 
 c . the solution was allowed to warm to room temperature , quenched 
 with a brine wash , and concentrated . the ether layer was washed with 1 m hcl , aq nahco3 , then brine . after drying ( na2so4 ) h nmr ( 300 mhz , cdcl3 )  7.417.48 
 ( m , 1h ) , 7.247.32 ( m , 1h ) , 7.117.19 ( m , 2h ) , 4.01 
 ( d , j = 13.2 hz , 1h ) , 3.543.71 ( m , 2h ) , 3.313.45 
 ( m , 1h ) , 2.36 ( dt , j = 5.0 , 13.1 hz , 1h ) , 2.062.20 
 ( m , 1h ) , 1.74 ( d , j = 13.6 hz , 1h ) , 1.42 ( s , 3h ) , 
 0.75 ( d , j = 7.0 hz , 3h ) . a solution of the triflate 17 ( 2.3 g , 4.9 mmol ) in dmf ( 10 ml ) was treated with nbu3 ( 3.5 ml , 15 mmol ) , pdcl2(pph3 ) ( 170 mg , 0.25 
 mmol ) , and formic acid ( 0.4 ml , 11 mmol ) . the solution was heated 
 to 80 c for 5 h , then concentrated and purified by rapid elution 
 of the product through silica gel using 20% etoac in hexanes as eluent 
 to afford 1.43 g ( 90% ) of 18 . h nmr ( 300 
 mhz , cdcl3 )  7.307.39 ( m , 2h ) , 7.207.28 
 ( m , 3h ) , 3.98 ( d , j = 13.0 hz , 1h ) , 3.62 ( bs , 2h ) , 
 3.303.46 ( m , 1h ) , 2.38 ( dt , j = 5.1 , 13.1 
 hz , 1h ) , 2.082.22 ( m , 1h ) , 1.681.79 ( m , 1h ) , 1.40 
 ( s , 3h ) , 0.75 ( d , j = 7.0 hz , 3h ) . a sample of triflamide 18 ( 520 
 mg , 1.6 mmol ) was dissolved in toluene ( 10 ml ) and thf ( 5 ml ) and 
 treated with lialh4 ( 320 mg , 8.3 mmol ) and heated with 
 a microwave to 150 c in a sealed tube for 10 min . the cooled 
 solution was diluted with ether , chilled in an ice bath , and quenched 
 with the sequential addition of water ( 0.3 ml ) , 15% naoh ( 0.3 ml ) , 
 then water ( 0.6 ml ) . the resulting suspension was filtered through 
 celite and concentrated to afford 226 mg of an oil . h nmr ( 300 mhz , cdcl3 )  7.087.38 ( m , 5h ) , 3.26 ( dd , j = 
 3.3 , 13.1 hz , 1h ) , 2.913.07 ( m , 2h ) , 2.672.78 ( m , 
 1h ) , 2.072.23 ( m , 1h ) , 1.832.00 ( m , 2h ) , 1.491.62 
 ( m , 1h ) , 1.39 ( s , 3h ) , 0.71 ( d , j = 7.2 hz , 3h ) . the amine ( 19 ) ( 226 mg , 0.92 
 mmol ) was combined with boc - l - valinal ( 355 mg , 1.8 mmol ) 
 in trifluoroethanol ( 5 ml ) and treated with na(cn)bh3 ( 3 
 ml , 1 m in thf ) . after 1 h , the solution was concentrated and subjected 
 to chromatography on silica gel using a gradient of etoac in hexanes 
 as the eluent to afford 367 mg ( 61% over two steps from 18 ) of the boc - protected product . the concentrated 
 residue was subjected to chromatography on silica gel eluting with 
 a gradient of cma80 in ch2cl2 as eluent to afford 20 in quantitative yield . h nmr ( 300 mhz , cdcl3 )  7.237.36 ( m , 4h ) , 7.127.21 ( m , 1h ) , 
 2.122.81 ( m , 7h ) , 2.02 ( d , j = 6.8 hz , 1h ) , 
 1.451.79 ( m , 5h ) , 1.32 ( s , 3h ) , 0.850.97 ( m , 6h ) , 
 0.690.79 ( m , 3h ) . this material was used without further purification . a solution of 3-{(3r,4r)-1-[(2s)-2-amino-3-methylbutyl]-3,4-dimethylpiperidin-4-yl}phenol 
 ( 21 ) ( 6.84 g , 23.6 mmol ) in chcl3 ( 230 ml ) was refluxed with phthalic anhydride ( 4.5 g , 30 mmol ) for 24 h. the 
 cooled solution was washed with aq nahco3 then concentrated . the residue was subjected to chromatography on silica gel using a 
 gradient of cma80 in ch2cl2 as the eluent to 
 afford 5.80 g ( 58% ) of 22 . h nmr ( 300 mhz , 
 cdcl3 )  7.74 ( d , j = 2.3 hz , 2h ) , 
 7.63 ( d , j = 2.6 hz , 2h ) , 7.08 ( t , j = 7.8 hz , 1h ) , 6.70 ( d , j = 8.1 hz , 1h ) , 6.64 ( bs , 
 1h ) , 6.58 ( d , j = 7.9 hz , 1h ) , 4.004.12 ( m , 
 1h ) , 3.26 ( 5 , j = 12.0 hz , 1h ) , 2.63 ( d , j = 10.0 hz , 2h ) , 2.232.57 ( m , 4h ) , 1.922.09 
 ( m , 1h ) , 1.82 ( d , j = 6.2 hz , 1h ) , 1.42 ( d , j = 12.6 hz , 1h ) , 1.23 ( s , 3h ) , 1.05 ( d , j = 6.8 hz , 3h ) , 0.88 ( d , j = 6.6 hz , 3h ) , 0.29 ( d , j = 6.8 hz , 3h ) . ms ( esl ) m / z 421.7 ( m + h ) . the phthalimide - protected 22 ( 5.80 g , 13.8 mmol ) was dissolved in ch2cl2 ( 150 ml ) containing triethylamine ( 2.8 ml , 20 mmol ) and n - phenyl - bis(trifluoromethanesulfonimide ) ( 5.4 g , 15 mmol ) . after 12 h , the solution was washed with aq nahco3 , dried 
 ( na2so4 ) , and concentrated . the residue was 
 subjected to chromatography on silica gel using a gradient 020% 
 etoac in hexanes as the eluent to afford 4.81 g ( 63% ) of triflate 23 . h nmr ( 300 mhz , cdcl3 )  
 7.76 ( bs , 2h ) , 7.617.70 ( m , 2h ) , 7.247.35 ( m , 1h ) , 
 7.16 ( d , j = 7.9 hz , 1h ) , 6.977.05 ( m , 2h ) , 
 3.984.13 ( m , 1h ) , 3.26 ( t , j = 12.2 hz , 1h ) , 
 2.592.76 ( m , 2h ) , 2.242.57 ( m , 4h ) , 2.02 ( dt , j = 4.7 , 12.4 hz , 1h ) , 1.84 ( d , j = 5.8 
 hz , 1h ) , 1.25 ( s , 3h ) , 1.05 ( d , j = 6.6 hz , 3h ) , 
 0.89 ( d , j = 6.8 hz , 3h ) , 0.26 ( d , j = 7.0 hz , 3h ) . the triflate 23 ( 1.59 g , 2.9 mmol ) , benzylamine 
 ( 0.46 ml , 4.2 mmol ) , potassium tert - butoxide ( 550 
 mg , 4.9 mmol ) , pd(oac)2 ( 7.7 mg , 0.035 mmol ) , and ( 2-biphenyl)di - tert - butylphosphine ( 21 mg , 0.07 mmol ) were combined and 
 degassed in toluene ( 3.5 ml ) then stirred overnight at room temperature . the organic layer was washed with aq nh4cl , dried 
 ( na2so4 ) , and concentrated . the residue was 
 subjected to chromatography on silica gel using a gradient of etoac 
 in hexanes as the eluent to afford 352 mg ( 24% ) of 24 . h nmr ( 300 mhz , cdcl3 )  7.75 ( d , j = 2.6 hz , 2h ) , 7.64 ( dd , j = 3.0 , 5.3 
 hz , 2h ) , 7.207.37 ( m , 5h ) , 7.03 ( t , j = 7.8 
 hz , 1h ) , 6.52 ( d , j = 7.7 hz , 1 h ) , 6.356.46 
 ( m , 2h ) , 4.25 ( s , 2h ) , 3.984.10 ( m , 1h ) , 3.173.29 
 ( m , 1h ) , 2.552.70 ( m , 2h ) , 2.232.55 ( m , 5h ) , 1.98 
 ( dt , j = 4.5 , 12.6 hz , 1h ) , 1.79 ( d , j = 6.8 hz , 1h ) , 1.39 ( d , j = 13.0 hz , 1h ) , 1.20 
 ( s , 3h ) , 1.04 ( d , j = 6.6 hz , 3h ) , 0.89 ( d , j = 6.8 hz , 3h ) , 0.28 ( d , j = 7.0 hz , 3h ) . the benzyl aniline 24 ( 353 mg , 0.69 mmol ) 
 was dissolved in etoh ( 100 ml ) with 20% pd(oh)2/c ( 0.2 
 g ) and shaken under hydrogen ( 45 psi ) overnight . the filtered concentrate 
 was subjected to chromatography on silica gel using 20% etoac in hexanes 
 as the eluent to recover 202 mg of starting material , followed by 
 50% etoac in hexanes with 1% nh3 as the eluent to afford 
 55 mg ( 19% ) of the desired aniline 25 . h 
 nmr ( 300 mhz , cdcl3 )  7.75 ( d , j = 2.6 hz , 2h ) , 7.577.68 ( m , 2h ) , 7.00 ( t , j = 7.8 hz , 1h ) , 6.56 ( d , j = 8.1 hz , 1 h ) , 6.396.50 
 ( m , 2h ) , 4.06 ( ddd , j = 4.8 , 9.8 , 11.6 hz , 1h ) , 3.24 
 ( t , j = 12.1 hz , 1h ) , 2.562.70 ( m , 2h ) , 2.212.54 
 ( m , 4h ) , 1.922.03 ( m , 1h ) , 1.751.87 ( m , 1h ) , 1.40 
 ( dd , j = 1.2 , 12.9 hz , 1h ) , 1.21 ( s , 3h ) , 1.05 ( d , j = 6.6 hz , 3h ) , 0.850.94 ( m , 3h ) , 0.29 ( d , j = 67.0 hz , 3h ) . this material was used without further 
 purification . a solution of 25 ( 1.34 g , 
 3.2 mmol ) 
 in dioxane ( 6 ml ) and hcl ( 6 m , 6 ml ) was stirred at reflux for 18 
 h. the mixture was concentrated then partitioned between aq nahco3 and etoac . the organic 
 layer was separated , dried ( na2so4 ) , and concentrated . the resulting residue was subjected to chromatography on silica gel 
 with 50% etoac in cma80 as the eluent ( rf 0.5 ) to afford 0.41 g ( 44% ) of 26 as a light - orange 
 oil . h nmr ( cdcl3 )  7.10 ( t , j = 7.8 hz , 1h ) , 6.676.73 ( m , 1h ) , 6.62 ( t , j = 2.0 hz , 1h ) , 6.51 ( ddd , j = 0.9 , 2.3 , 
 7.8 hz , 1h ) , 3.61 ( br s , 1h ) , 2.572.78 ( m , 2h ) , 2.51 ( dt , j = 2.6 , 11.8 hz , 1h ) , 2.272.43 ( m , 3h ) , 2.25 ( d , j = 3.6 hz , 1h ) , 2.132.22 ( m , 1h ) , 1.892.03 
 ( m , 2h ) , 1.68 ( br s , 2h ) , 1.451.59 ( m , 2h ) , 1.271.31 
 ( m , 3h ) , 0.92 ( d , j = 7.0 hz , 6h ) , 0.78 ( d , j = 7.0 hz , 3h ) . ms ( esi ) m / z 290.3 ( m + h ) . this material was used without further 
 purification . the aniline 25 ( 55 mg , 0.13 mmol ) was dissolved 
 in ch2cl2 ( 1 ml ) and treated with bf3oet2 ( 32 l , 0.26 mmol ) then isoamyl nitrite 
 ( 26 l , 0.20 mmol ) . after stirring 15 min , the resulting crystalline solids 
 were collected by filtration , dried , and heated neat . the resulting 
 residue was subjected to chromatography on silica gel eluting with 
 etoac as the eluent to afford 36 mg ( 66% ) of 27 . h nmr ( cdcl3 )  7.797.90 ( m , 2h ) , 
 7.687.79 ( m , 2h ) , 7.117.34 ( m , 2h ) , 6.807.04 
 ( m , 3h ) , 4.144.26 ( m , 1h ) , 4.00 ( t , j = 11.9 
 hz , 1h ) , 3.243.78 ( m , 4h ) , 2.55 ( br s , 2h ) , 2.182.37 
 ( m , 2h ) , 1.41 ( s , 3h ) , 1.061.17 ( m , 3h ) , 0.770.89 
 ( m , 3h ) , 0.520.74 ( m , 3h ) . this material was used without 
 further purification . a solution of 25 ( 340 mg , 0.81 mmol ) in aq 
 hcl ( 37% , 5 ml ) was cooled to 5 c and stirred for 10 
 min . a solution of nano2 ( 63 mg , 0.89 mmol ) in water ( 1.5 
 ml ) was added dropwise to the reaction which then was stirred for 
 1 h. a cold solution of copper(i ) chloride ( 92 mg , 0.93 mmol ) in water 
 ( 1.5 ml ) was then added dropwise . the reaction mixture was stirred 
 for 30 min and allowed to warm to rt then was heated to 65 c 
 for 3 h. the resulting suspension was poured in to a mixture of concd 
 nh4oh ( 20 ml ) and ice ( 6 g ) . the resulting solution with 
 was extracted with ethyl acetate ( 2  40 ml ) . the combined organic 
 layers were dried ( na2so4 ) and evaporated to 
 a residue , which was subjected to chromatography on silica gel using 
 a gradient up to 50% etoac in hexanes as the eluent to afford 100 
 mg ( 28% ) of 28 as an oil that solidified to a white solid 
 upon standing . h nmr ( cdcl3 )  7.77 ( br 
 s , 2h ) , 7.65 ( br s , 2h ) , 6.957.21 ( m , 4h ) , 4.06 ( m , 1h ) , 3.26 
 ( t , j = 12.1 hz , 1h ) , 2.582.79 ( m , 2h ) , 2.182.56 
 ( m , 3h ) , 1.932.11 ( m , 1h ) , 1.761.91 ( m , 1h ) , 1.371.50 
 ( m , 1h ) , 1.24 ( s , 3h ) , 1.06 ( d , j = 6.8 hz , 3h ) , 
 0.90 ( d , j = 6.8 hz , 3h ) , 0.28 ( d , j = 6.9 hz , 3h ) . c nmr ( cdcl3 )  169.1 , 
 152.8 , 134.0 , 133.6 , 132.0 , 129.2 , 125.8 , 125.3 , 123.7 , 122.8 , 57.2 , 
 55.0 , 54.1 , 51.4 , 38.6 , 38.5 , 30.4 , 29.9 , 27.2 , 20.4 , 20.3 , 15.4 . a solution of 25 ( 250 mg , 0.6 mmol in aq 
 hbr ( 48% , 0.5 ml ) and water ( 1 ml ) was cooled to 5 c 
 and stirred for 10 min . a solution of nano2 ( 46 mg , 0.65 
 mmol ) in water ( 1 ml ) was added dropwise , and the reaction mixture 
 stirred for 2 h. urea was added to the reaction mixture to consume 
 any excess nitrous acid . copper(i ) bromide ( 103 mg , 0.72 mmol ) , aq 
 hbr ( 48% , 0.2 ml ) , and water ( 0.5 ml ) were added consecutively to 
 the reaction mixture . after stirring for 1 h , the resulting suspension was 
 poured in to a mixture of concd nh4oh ( 20 ml ) and ice ( 6 
 g ) . the resulting solution was extracted with ethyl acetate ( 2  
 40 ml ) . the combined organic layers were dried ( na2so4 ) and evaporated to a residue , which was subjected to chromatography 
 on silica gel using a gradient up to 50% etoac in hexanes as the eluent 
 to afford 110 mg ( 38% ) of 29 as a colorless oil . h nmr ( cdcl3 )  7.77 ( br s , 2h ) , 7.65 ( dd , j = 2.9 , 5.2 hz , 2h ) , 7.187.26 ( m , 2h ) , 6.947.18 
 ( m , 2h ) , 3.944.21 ( m , 1h ) , 3.26 ( t , j = 12.1 
 hz , 1h ) , 2.582.78 ( m , 2h ) , 2.202.57 ( m , 4h ) , 1.942.03 
 ( m , 1h ) , 1.771.93 ( m , 1h ) , 1.371.55 ( m , 1h ) , 1.23 
 ( s , 3h ) , 1.06 ( d , j = 6.6 hz , 3h ) , 0.91 ( d , j = 6.8 hz , 3h ) , 0.28 ( d , 3h , j = 7.2 hz , 
 3h ) . c nmr ( cdcl3 )  169.1 , 153.1 , 133.6 , 
 132.0 , 129.6 , 128.7 , 128.3 , 128.0 , 125.4 , 125.1 , 124.2 , 122.8 , 122.5 , 
 57.2 , 55.0 , 54.1 , 51.4 , 38.7 , 38.4 , 30.3 , 29.9 , 27.3 , 20.4 , 20.3 , 
 15.5 . a solution of 27 ( 36 mg , 0.085 
 mmol ) was heated at reflux in etoh ( 10 ml ) and hydrazine ( 1 ml ) overnight . 
 the concentrated residue was dissolved in etoac then washed with aq 
 nahco3 . the organic layer was dried ( na2so4 ) and concentrated to afford 24 mg ( 96% ) of the amine 30 . h nmr ( 300 mhz , cdcl3 )  
 7.127.26 ( m , 1h ) , 6.98 ( d , j = 8.1 hz , 1h ) , 
 6.90 ( dd , j = 1.9 , 11.3 hz , 1h ) , 6.79 ( dt , j = 2.1 , 8.3 hz , 1h ) , 2.072.77 ( m , 8h ) , 1.831.96 
 ( m , 1h ) , 1.421.58 ( m , 2h ) , 1.081.31 ( m , 5h ) , 0.740.95 
 ( m , 6h ) , 0.600.74 ( m , 3h ) . this material was used without 
 further purification . to a solution of 28 ( 100 mg , 
 0.23 mmol ) in ethanol ( 15 ml ) was added hydrazine monohydrate ( 115 
 mg , 2.3 mmol ) . the reaction mixture was stirred at reflux under nitrogen 
 for 12 h and then concentrated to obtain a white solid that was dissolved 
 in aq nahco3 ( 10 ml ) and extracted with etoac ( 2  
 20 ml ) . the combined organic layers were dried ( na2so4 ) and concentrated to afford 56 mg ( 79% ) of 31 as a colorless oil . h nmr ( 300 mhz , cdcl3 )  6.947.18 ( m , 4h ) , 2.502.73 ( m , 3h ) , 2.45 
 ( m , 1h ) , 2.042.38 ( m , 3h ) , 1.792.02 ( m , 2h ) , 1.371.56 
 ( m , 2h ) , 1.23 ( m , 3h ) , 0.85 ( d , j = 6.8 hz , 6h ) , 
 0.69 ( d , j = 6.9 hz , 3h ) . c nmr ( 75 
 mhz , cdcl3 )  151.7 , 133.1 , 128.3 , 124.9 , 124.5 , 
 122.8 , 61.7 , 53.5 , 51.9 , 51.0 , 37.9 , 37.6 , 31.1 , 29.6 , 26.4 , 18.3 , 
 17.2 , 15.2 . ms ( esi ) m / z 309.4 ( m 
 + h ) . this material was used without further purification . to a solution of 29 ( 110 mg , 
 0.23 mmol ) in ethanol ( 15 ml ) was added hydrazine monohydrate ( 115 
 mg , 2.3 mmol ) . the reaction mixture was stirred at reflux under nitrogen 
 for 12 h and then concentrated to obtain a white solid that was dissolved 
 in aq nahco3 ( 10 ml ) and extracted with etoac ( 2  
 20 ml ) . the combined organic layers were dried ( na2so4 ) and concentrated to obtain 94 mg ( 99% ) of 32 as a colorless oil . h nmr ( 300 mhz , cdcl3 )  7.33 ( s , 1h ) , 7.177.28 ( m , 1h ) , 7.037.17 
 ( m , 2h ) , 2.512.71 ( m , 3h ) , 2.102.49 ( m , 4h ) , 1.822.00 
 ( m , 1h ) , 1.381.65 ( m , 3h ) , 1.23 ( m , 3h ) , 0.85 ( d , j = 6.8 hz , 6h ) , 0.560.76 ( m , 3h ) . c nmr ( 75 mhz , cdcl3 )  152.1 , 128.7 , 127.8 , 127.4 , 
 127.1 , 124.5 , 124.3 , 123.2 , 121.6 , 61.8 , 53.5 , 51.9 , 51.0 , 37.9 , 37.6 , 
 31.2 , 29.6 , 26.4 , 18.3 , 17.2 , 15.3 . ms ( esi ) m / z 353.4 ( m + h ) . a solution of boc-7-hydroxy - d - tic 
 ( 1.47 g , 5 mmol ) in toluene ( 35 ml ) and ch3oh ( 10 ml ) was 
 treated with a solution of tmschn2 in ether ( 2.0 m , 2.5 
 ml ) until a slight yellow persisted . the residue was 
 dissolved in ch2cl2 ( 50 ml ) and net3 ( 0.9 ml , 6.5 mmol ) and treated with tf2o ( 0.85 ml , 5.0 
 mmol ) at 0 c . the reaction was allowed to warm to room temperature 
 and concentrated to a residue and subjected to a plug of silica gel , 
 eluting with 20% etoac in hexanes as the eluent . the fractions containing 
 product were concentrated and dissolved in dmf ( 6 ml ) with zn(cn)2 ( 1.0 g , 8.5 mmol ) . the mixture was degassed and kept under 
 nitrogen as pd(pph3)4 ( 200 mg , 0.2 mmol ) was 
 added . the mixture was heated to 100 c for 4 h , cooled , then 
 partitioned between etoac and aq nahco3 . the organic layer 
 was dried ( na2so4 ) , concentrated , and subjected 
 to chromatography on silica gel using 20% etoac in hexanes to afford 
 1.37 g of 35 ( 86% over 3 steps ) . h nmr ( 300 
 mhz , cdcl3 )  7.397.52 ( m , 2h ) , 7.227.31 
 ( m , 1h ) , 4.684.81 ( m , 1h ) , 4.424.60 ( m , 1h ) , 3.64 
 ( d , j = 4.9 hz , 3h ) , 3.30 ( d , j = 
 2.1 hz , 1h ) , 3.22 ( d , j = 5.8 hz , 2h ) , 1.50 ( d , j = 19 hz , 9h ) . this material was used without further purification . a sample of 35 ( 320 mg , 
 1.0 mmol ) was dissolved in dioxane ( 2 ml ) and thf ( 1 ml ) then treated 
 with aq lioh ( 1 m , 3 ml ) overnight . the resulting solution was cooled 
 in an ice bath and treated cautiously with h2o2 ( 30% , 1 ml ) . after warming , the reaction mixture was acidified with 
 hcl ( 2 m ) and diluted with water . the resulting solids were separated 
 by filtration and dried to afford 230 mg of 36 ( 72% ) . h nmr ( 300 mhz , dmso - d6 )  
 12.72 ( s , 1h ) , 7.90 ( br s , 1h ) , 7.617.74 ( m , 1h ) , 7.217.37 
 ( m , 1h ) , 4.89 ( br s , 1h ) , 4.68 ( s , 1h ) , 4.554.64 ( m , 1h ) , 
 4.52 ( d , j = 5.7 hz , 1h ) , 4.364.48 ( m , 1h ) , 
 3.063.26 ( m , 2h ) , 1.331.53 ( m , 9h ) . this material 
 was used without further purification . the binding assays were 
 performed for 60 min using 4 g herg expressing membranes , 3 
 nm [ h]astemizole , and various concentrations of the test 
 agent in a binding buffer ( 10 mm hepes , ph 7.4 , 130 mm nacl , 5 mm 
 kcl , 0.8 mm mgcl2 , 1 mm naedta , 10 mm glucose , 0.1% bsa ) . 
 binding was terminated by rapid filtration onto gf / b fiber filtermats , 
 presoaked in 0.3% polyethylenimine , followed by rapid washing 6 times 
 ( 2 ml ) with ice - cold solution containing 25 mm tris - hcl , ph 7.4 , 130 
 mm nacl , 5 mm kcl , 0.8 mm mgcl2 , 0.05 mm cacl2 , and 0.1% bsa using a brandel harvester . data were analyzed using 
 nonlinear regression ( graphpad prism ) , and ki values were determined as described before . all experiments were performed at least twice in duplicate , 
 and data reported are mean values . for these experiments , 10 
 mm dmso stocks of compounds were directly diluted into 10 mm phosphate 
 buffer at ph 7.4 or 3 and shaken for 90 min at room temperature . after the incubation , samples 
 were filtered through a 0.4 m filterplate ( millipore ) . analysis of compounds was performed by lc / ms 
 using previously available methods and concentrations determined . a commercially available pampa assay system 
 was used ( bd gentest precoated pampa system ) . assays were performed 
 in duplicate at 10 m final concentration at ph 7.4 and 5.5 
 as has been described previously in pbs buffer . the ligand preparation , 
 receptor preparation , and docking calculations were conducted under 
 our previously reported methods .
jdtic analogues 415 which have 
 the hydroxyl groups replaced with other groups were synthesized and 
 their in vitro efficacy at the  ,  , and  opioid 
 receptors determined and compared to jdtic using [ 35s]gtps 
 assays . 
 compounds 4 , 5 , 6 , 13 , 14 , and 15 had ke = 0.024 , 0.01 , 0.039 , 0.02 , 0.11 , and 0.041 nm compared 
 to the ke = 0.02 nm for jdtic at the  
 receptor and were highly selective for the  receptor relative 
 to the  and  opioid receptors . 
 unexpectedly , replacement 
 of the 3-hydroxyl substituent of the 4-(3-hydroxyphenyl ) group of 
 jdtic with a h , f , or cl substituent leads to potent and selective 
 kor antagonists . in vitro studies to determine various adme properties 
 combined with calculated tpsa , clogp , and logbb values suggests that 
 the potent and selective  opioid receptors 4 , 5 , 13 , and 14 deserve consideration 
 for further development toward potential drugs for cns disorders .