the serotonin transporter 
 ( sert ) is a member of the solute carrier 
 6 ( slc6 ) family of transporters that functions to regulate serotonin 
 neurotransmission and homeostasis . in particular , selective 
 serotonin reuptake inhibitors ( ssris ) and tricyclic antidepressants 
 ( tcas ) , widely prescribed medications for treatment of anxiety and 
 major depressive disorders , notably , ( s)-citalopram ( 1 , escitalopram , figure 1 ) binds with high 
 affinity and selectivity to hsert . however , despite its well - documented 
 clinical success , the molecular interactions between ( s)-citalopram and hsert that determine its reuptake inhibition potency 
 and selectivity over the norepinephrine transporter ( net ) and dopamine 
 transporter ( dat ) remain poorly understood . chemical structures of 
 ( s)-citalopram ( 1 ) and known dat and/or 
 sert inhibitor pals ( 27 ) . the 3d structure of sert as well 
 as its structure , function , and 
 regulation , including oligomerization and distribution in cells or 
 brain tissue , has been studied with small molecular probes containing 
 electrophiles ( e.g. , n3 , ncs ) and radiolabels ( e.g. , h , i ) . in particular , the well - known technique 
 of photoaffinity labeling utilizes molecular probes that contain a 
 functional group capable of forming a covalent bond to a biological 
 target upon photoactivation . of the numerous photoreactive 
 functional groups that can be employed in the design of photoaffinity 
 ligands ( pals ) ( e.g. , benzophenones , aliphatic and aromatic diazirines , 
 etc . ) , aryl azides are frequently used given their relatively small 
 size , ease of synthetic incorporation , and chemical stability . numerous [ i]-radiolabeled - arylazido 
 analogues of 
 dopamine transporter ( dat ) inhibitors including cocaine , gbr12909 ( 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine ) , benztropine , pyrovalerone , bupropion , and methylphenidate have been developed to elucidate molecular components of the ligand - binding 
 site and mechanisms underlying uptake inhibition . the 4-azido , 
 3-iodo - substituted phenyl ring is a common structural motif 
 found in many pals that bind dat irreversibly upon photoactivation 
 ( e.g. , rti 82 ( 2 ) , mfz 2 - 24 ( 3 ) , and jhc 
 2 - 48 ( 4 ) ; figure 1 ) . specifically , tropane - based pals 2 and 3 have been instrumental in defining drug  protein interactions 
 at the molecular level and thus further defining the structural components 
 of dat that are critical for transport inhibition by cocaine . in contrast to dat pals , the development of sert - selective 
 pals 
 has been more limited . to the best of our knowledge , only a few pals 
 have been synthesized and experimentally validated to label sert ( figure 1 ) . although tropane - based 
 pals 24 bind to sert as well as 
 dat , to date , only probes 3 and 4 have been 
 used to photolabel hsert . other inhibitor - based 
 ligands that photolabel sert include an aryl - azido derivative of the 
 ssri paroxetine ( 5 ) and 
 a tricyclic antidepressant - based pal , [ h]-2-nitroimipramine 
 ( 6 ) , that showed covalent incorporation into sert present 
 in membrane homogenates of rat brain and liver and human platelets . finally , photoinactivation of serotonin uptake has been demonstrated 
 by an azidobenzamidine derivative of serotonin ( 7 ) . however , to date , none of these sert pals have 
 been used to define the structural basis of their binding interactions 
 at hsert . binding 
 site interactions at 
 dat , our concept for the present study was that appending 
 the 4-azido , 3-iodo aryl moiety onto different positions 
 of the ( s)-citalopram base might reveal different 
 sites of adduction on hsert that would enable a clearer understanding 
 of the ( s)-citalopram binding mechanism . extensive 
 structure  activity relationships have been described with analogues 
 of citalopram that suggest positions c-1 and c-5 can accommodate significant 
 steric bulk without appreciable loss in hsert binding affinity . herein , we describe the design , synthesis , pharmacological evaluation , 
 and radiosynthesis of three novel c-1- or c-5-substituted 4-azido , 
 3-iodo analogues of ( s)-citalopram and demonstrate 
 their ability to covalently label hsert upon photoactivation . phthalimide derivative 9(35 ) was synthesized by stirring 2-(4-aminophenyl)ethan-1-ol 
 ( 8) with phthalic anhydride in pyridine , at reflux , and 
 oxidizing to aldehyde 10 using a dess ( s)-n - desmethylcitalopram 
 ( 11 ) was then coupled to aldehyde 10 using standard reductive amination conditions to provide 
 tertiary amine 12 in 80% yield , followed by phthalimide 
 deprotection to give aniline 13 . iodination with iodine 
 monochloride ( icl ) gave the intermediate 14 , which was 
 then treated with nano2 followed by nan3 to 
 give 4-azido , 3-iodo product 15 . reagents 
 and conditions : ( a ) 
 phthalic anhydride , pyridine , reflux , 16 h ; ( b ) dess martin 
 periodinane , ch2cl2 , 78 c to rt , 
 3 h ; ( c ) nabh(oac)3 , hoac , dce , rt 18 h ; ( d ) hydrazine , 
 etoh , reflux , 3 h ; ( e ) icl ( 1 m in ch2cl2 ) , 
 05 c to rt ; ( f ) nano2 , nan3 , tfa , 
 05 c to rt . the synthesis of 
 c-5-substituted ( s)-citalopram 
 pals 22 and 26 is shown in scheme 2 . 3-(4-aminophenyl)propanoic 
 acid ( 16 ) was initially stirred at reflux with phthalic 
 anhydride to afford the corresponding phthalimide carboxylic acid 17 . subsequent coupling of 17 to primary amine 18(31,32 ) via the acid chloride provided amide 19 in 97% yield . deprotection of 19 using hydrazine provided aniline intermediate 20 , which was reacted with icl to give 3-iodo - substituted 
 product 21 . conversion to azido pal 22 was 
 then performed as previously described for 15 via diazotization 
 and azide displacement of aniline 21 . this final step 
 went in poor yield ( 18% ) ; thus , a second strategy was also developed 
 that featured a decrease in the number of synthetic steps and an increase 
 in the overall yield . this alternative strategy began with electrophilic 
 iodination of aniline 16ortho to the 
 amine to provide the corresponding iodo - substituted aniline 23 in 67% yield , which was subsequently treated with nano2 followed by nan3 in tfa to give iodo - azide 24 . primary amine 18 was then coupled to azido - iodo carboxylic acid 24 using 
 1,1-carbonyldiimidazole ( cdi ) in thf to generate amide 22 . although this route using 24 is preferred 
 for generating nonradioactive 22 in high yield , the initial 
 route that provided 20 was utilized in the synthesis 
 of [ i]-labeled 22 . similarly , 18 was coupled to 4-azido-3-iodobenzoic acid ( 25 ) using edc and hobt to provide pal 26 in 67% yield . reagents and conditions : ( a ) 
 phthalic anhydride , pyridine , reflux , 16 h ; ( b ) socl2 , 
 3 h ; ( c ) hydrazine , etoh , reflux , 6 h ; ( d ) icl , ch2cl2 , 05 c to rt ; ( e ) nano2 , nan3 , hoac , h2o ; ( f ) icl , hoac , rt ; ( g ) nano2 , nan3 , tfa ; ( h ) 24 , cdi , thf , 0 c 
 to rt ; ( i ) edc , hobt , et3n , dmf , 0 c to rt . radioiodinated pals [ i]15 , [ i]22 , and [ i]26 were prepared 
 under no - carrier - added conditions as shown in scheme 3 . electrophilic radioiodination of anilines 13 and 20 was accomplished by treatment with 
 [ i]nai and chloramine - t at ambient temperature for 30 
 min . acidification with hoac followed by treatment with nano2 at 5 c generated the diazonium salts in situ . decomposition upon warming , in the presence of nan3 , 
 gave [ i]-labeled aryl azides [ i]15 and [ i]22 . this sequence was 
 accomplished in one flask , as previously described for other radioiodinated 
 pals . electrophilic 
 radioiododestannylation of tri - n - butylstannyl azide 27 using chloramine - t as the oxidant in methanolic acetic 
 acid for 3 min was employed to prepare [ i]26 . the organostannane precursor was obtained in 52% yield by treating 
 nonradioactive iodide 26 with bis(tri - n - butyltin ) and bis(triphenylphosphine)palladium(ii ) dibromide . since 
 carryover of trace amounts of 26 would dramatically lower 
 the specific radioactivity of [ i]26 prepared 
 from 27 , this material was 
 purified by reversed - phase hplc prior to radiolabeling . reagents 
 and conditions : ( a ) 
 [ i]nai , chloramine - t , naoac ( 0.2 m , ph 4.0 ) , rt , 30 
 min ; ( b ) hoac ( 3.0 m ) , nano2 ( 0.5 m ) , 5 c , 
 20 min ; ( c ) nan3 ( 0.5 m ) , rt , 30 min ; ( d ) na2s2o5 ( 50 mm ) , rt ; ( e ) pd(pph3)2br2 , ( ( n - bu)3sn)2 , toluene , 105 c , 4 h ; ( f ) [ i]nai , chloramine - t , 
 meoh , 3% hoac , rt , 3 min . all three radioligands 
 were purified by reversed - phase hplc and 
 then formulated as concentrated solutions in meoh using solid - phase 
 extraction . specific 
 radioactivities were determined as 2045 to 2098 mci/mol , near 
 the theoretical value of 2175 mci/mol , using hplc to determine 
 the mass associated with the uv absorbance peak in samples of known 
 radioactivity . radiochemical purities were 98% by hplc ( figure 2 ) , and the materials 
 were stable for at least 5 weeks when stored at 20 c 
 in the dark . the three radioligands proved to be lipophilic , with 
 [ i]22 ( tr = 
 13.2 min , k = 10.4 ) and [ i]26 ( tr 13.4 min , k = 10.6 ) being substantially less hydrophobic than [ i]15 ( tr = 22.4 
 min , k = 18.3 ) . these findings are in accord 
 with their relative clogp values ( 15 , 7.02 ; 22 , 6.39 ; 26 , 6.35 ; generated in chemdraw ) . reversed - phase 
 hplc chromatograms of isolated radioiodinated ligands 
 illustrating purity and relative lipophilicity . chromatography performed 
 using 45% meoh / ch3cn ( 1:1 , v / v ) and 55% water containing 
 et3n ( 2.1% v / v ) and hoac ( 2.8% v / v ) at a flow rate of 3.0 
 ml / min on a c-18 column . inhibition 
 of [ h]5-ht 
 uptake by wt hsert and the hsert ser438thr mutant was evaluated to 
 determine inhibitory constants for pals 15 , 22 , 26 and parent compound ( s)-citalopram 
 ( 1 ) . when tested against wt hsert , c-5-substituted pals 26 and 22 showed affinities of 24 and 38 nm , 
 respectively , values that were reduced by 11- and 17-fold compared 
 to that of ( s)-citalopram ( ki = 2.2 nm ) ( figure 3 and table 1 ) . in contrast , c-1 substituted pal 15 showed a greater 
 reduction in affinity ( ki = 227 nm , 100-fold ) 
 compared to that of ( s)-citalopram , which is in accord 
 with structure  activity relationships previously described . when tested against the hsert ser438thr mutant , 
 all four compounds showed dramatic rightward shifts in inhibitory 
 potency ( figure 3 , 
 dashed lines ) with ki values ranging from 
 3.8 to 9.9 m ( table 1 ) . these findings are consistent with previous reports that 
 ser438 is a key residue for high - affinity binding of many hsert antagonists 
 and that its mutation to thr results in a significant loss in potency 
 for ( s)-citalopram and many other hsert inhibitors . interestingly , c-1-substituted pal 15 demonstrated lower affinity than ( s)-citalopram 
 or the other pals for wt hsert but higher affinity for the s438 t mutant . moreover , the difference in binding affinities between the mutant 
 and wt hsert for 15 was only 17-fold , as compared to 
 a more dramatic shift for parent ligand 1 ( 3600-fold ) . [ h]5-ht uptake inhibition analysis for ( s)-citalopram , 15 , 22 , and 26 . hek-293 griptite 
 cells expressing hsert ( solid lines ) or hsert 
 s438 t ( dashed lines ) were assayed for [ h]5-ht uptake in 
 the presence of the indicated concentrations of ( s)-citalopram ( 1 ) (  ) , 15 (  ) , 22 (  ) , or 26 (  ) . data shown are 
 the mean  sem of transport activity for each form in the absence 
 of competitor , set to 100% . data are ki values ( nm ) ( mean  
 sem ) for the ability 
 of the ( s)-citalopram - based pals to inhibit uptake 
 of [ h]5-ht , as shown in figure 3 . data were analyzed by paired t - tests for statistical significance , where * indicates that the ki value obtained with the hsert s438 t mutant 
 is significantly different than the ki of that compound for wt hsert ( p < 0.001 ) and 
 # indicates that the ki value for the 
 analogue is significantly different than the ki for ( s)-citalopram obtained with wt hsert 
 ( p < 0.001 ) . the ki values obtained with hsert s438 t for the analogues and ( s)-citalopram were not statistically different from one 
 another . next , 
 we tested [ i]15 , [ i]22 , and [ i]26 for irreversible 
 labeling of ha - hsert ( figure 4 ) . for these studies , ha - hsert llcpk1 cells were 
 incubated with the pals in the presence or absence of ( s)-citalopram and irradiated with uv light to cross - link the ligand 
 to the protein . lysates were immunoprecipitated with anti - ha or anti - hsert 
 antibodies , and samples were analyzed by sds - page and autoradiography . 
 these methods ( described in detail in the experimental 
 methods ) have been used by our laboratories to verify photolabeling 
 of dat and sert by numerous structurally diverse tropane- and non - tropane - based 
 ligands . the results ( figure 4 ) show that when using ha - hsert expressing cells , [ i]15 , [ i]22 , and [ i]26 all covalently label a protein of the expected 
 hsert molecular mass of 100 kda , whereas photolabeled bands 
 were not obtained from nontransfected parent cells ( upper panels ) , 
 strongly supporting the identity of the photolabeled bands as hsert . immunoblotting ( lower panels ) verified the presence and absence of 
 hsert protein in transfected and untransfected cells , respectively . the photoaffinity labeled proteins were immunoprecipitated using anti - ha 
 antibody ( figure 4 ) 
 as well as with the anti - hsert antibody st51 ( not shown ) , but they 
 were not precipitated with nonimmune igg ( not shown ) , also supporting 
 the identity of the bands as hsert . similar irreversible labeling 
 and immunoprecipitation results were obtained using tropane pal 3 , which was previously demonstrated to photolabel hsert . together , these results demonstrate that hsert 
 protein is irreversibly labeled with [ i]15 , [ i]22 , and [ i]26 upon photoactivation , providing the first demonstration of photoaffinity 
 labeling of hsert by analogues of ( s)-citalopram . ha - hsert llcpk1 cells 
 or nontransfected ( parent ) llcpk1 cells were incubated 
 with the indicated i - labeled ligands ( 26 , 22 , 15 , 3 ) in the absence 
 (  ) or presence ( + ) of 10 m ( s)-citalopram 
 ( s - cit ) followed by cross - linking to sert with uv light . cell lysates 
 were immunoprecipitated with anti - ha antibody followed by sds - page 
 and autoradiography to detect [ i ] radiolabeled proteins 
 ( upper panels ) or were analyzed by immunblotting ( ib ) with anti - ha 
 antibody to detect total hsert ( lower panels ) . , 
 ha - hsert - llcpk1 cells were incubated with vehicle or 10 
 m ( s)-citalopram prior to addition of the 
 radioiodinated pals . incorporation of [ i]22 and [ i]26 was blocked by > 99% , demonstrating 
 that ( s)-citalopram fully displaces these c-5 substituted 
 radioligands , similar to displacement of hsert labeling by [ i]3 . in contrast , 10 m 
 ( s)-citalopram inhibited [ i]15 labeling of hsert by only 30% , suggesting that this c-1-substituted 
 pal exhibits a different binding mode compared to that of the two 
 c-5-substituted ligands . in a previous sar study , wherein ( s)-citalopram was modified by n - substitution at the c-5 
 position with a second molecule of ( s)-citalopram 
 to create a homobifunctional ligand , binding data suggested simultaneous 
 interaction of the ligand with the s1 and s2 binding sites . moreover , the affinity of ( s)-citalopram for s2 is much lower than that for s1 . thus , it is conceivable that [ i]15 may be binding to both the s1 and s2 sites and that the latter might 
 not be fully inhibited by 10 m ( s)-citalopram . in summary , three novel pals , [ i]15 , 
 [ i]22 , and [ i]26 , were designed by substituting the parent ssri , ( s)-citalopram , with a 4-azido , 3-iodophenyl moiety 
 in either the c-1 or c-5 position , with variable - length linker chains . our results showed that the nonradioiodinated compounds were able 
 to inhibit [ h]5-ht uptake with high to moderate potency 
 and that the radioiodinated pals displayed requisite characteristics 
 as irreversible probes for molecular characterization of the hsert 
 ( s)-citalopram binding site(s ) . preincubation of 
 hsert with ( s)-citalopram completely eliminated irreversible 
 labeling by c-5-substituted [ i]22 and [ i]26 , but it reduced irreversible labeling 
 by c-1-substituted [ i]15 by only 30% . the inability of ( s)-citalopram to completely block 
 [ i]15 hsert photolabeling indicates that 
 this pal has complex hsert binding properties compared to those of 
 c-5-substituted pals [ i]22 and [ i]26 that will require further investigation . anhydrous solvents were purchased from aldrich and were used without 
 further purification , except for tetrahydrofuran , which was freshly 
 distilled from sodium benzophenone ketyl . all other chemicals and 
 reagents were purchased from sigma - aldrich co. llc , combi - blocks , 
 tci america , acros organics , and alfa aesar . unless otherwise stated , 
 final amine products were converted into oxalate salts , typically 
 by treating the free base in chcl3 with a 1:1 molar ratio 
 of oxalic acid in acetone . as described , some of the oxalate salts 
 were recrystallized from hot methanol or a methanol h and c nmr spectra were acquired using a varian mercury 
 plus 400 spectrometer at 400 and 100 mhz , respectively . chemical shifts 
 are reported in parts per million ( ppm ) and referenced according to 
 deuterated solvent for h spectra ( cdcl3 , 7.26 
 ppm , or cd3od - d4 , 3.31 ppm ) 
 and c spectra ( cdcl3 , 77.2 ppm , or cd3od - d4 , 49.00 ppm ) . infrared ( ir ) 
 spectra were obtained ( neat ) on a perkinelmer spectrum two ftir spectrometer . gas chromatography / mass spectrometry ( gc / ms ) data were acquired ( where 
 obtainable ) using an agilent technologies ( santa clara , ca ) 6890n 
 gas chromatograph equipped with an hp-5ms column ( cross - linked 5% 
 ph me siloxane , 30 m  0.25 mm i.d . 0.25 m film 
 thickness ) and a 5973 mass - selective ion detector in electron - impact 
 mode . ultrapure grade helium was used as the carrier gas at a flow 
 rate of 1.2 ml / min . the injection port and transfer line temperatures 
 were 250 and 280 c , respectively , and the oven temperature gradient 
 used was as follows : the initial temperature ( 100 c ) was held 
 for 3 min , increased to 295 c at 15 c / min over 13 min , 
 and finally maintained at 295 c for 10 min . to acquire high - resolution 
 mass spectrometry ( hrms ) data , 1 l of sample was mixed with 
 1 l of matrix ( saturated solution of 2,4,6-trihydroxyacetophenone/2,5-dihydroxybenzoic 
 acid in 50:50 ethanol / water ) ; then , 1 l was deposited on a 
 stainless steel plate and analyzed in positive ion mode in a maldi 
 ltq - xl orbitrap ( thermo - scientific , san jose , ca ) using a laser energy 
 fixed at 6 j and a mass resolution of 60  000 at an m / z of 400 th . ( norcross , ga ) , and the results 
 agree within 0.4% of the calculated values , unless indicated 
 otherwise ( s.i . ) . melting point determination was conducted using 
 a thomas - hoover melting point apparatus ; the melting points are uncorrected . 
 on the basis of nmr , hrms , ( s)-n - desmethylcitalopram ( 11 , 0.164 g , 0.53 
 mmol ) and 2-(4-(1,3-dioxoisoindolin-2-yl)phenyl)acetaldehyde ( 10 , 0.140 g , 0.53 mmol ) were mixed 
 in 1,2-dichloroethane ( 10 ml ) and then treated with sodium triacetoxyborohydride 
 ( 0.167 g , 0.79 mmol ) and hoac ( 0.2 ml ) . the mixture was then stirred 
 at rt for 6 h under an inert atmosphere . the reaction mixture was 
 quenched with 2 n aq naoh ( 10 ml ) and extracted with etoac ( 3  
 20 ml ) . purification by flash column 
 chromatography eluting with 2:8 acetone / chcl3 provided 
 0.235 g of tertiary amine 12 ( 80% ) . h nmr 
 ( 400 mhz , cdcl3 )  7.89 ( dd , j = 
 5.2 , 3.2 hz , 2h ) , 7.73 ( dd , j = 5.2 , 3.2 hz , 2h ) , 
 7.54 ( d , j = 7.6 hz , 1h ) , 7.457.28 ( m , 8h ) , 
 6.986.94 ( m , 2h ) , 5.16 ( d , j = 12.8 hz , 1h ) , 
 5.11 ( d , j = 12.4 hz , 1h ) , 2.762.72 ( m , 2h ) , 
 2.552.51 ( m , 2h ) , 2.35 ( t , j = 6.8 hz , 2h ) , 
 2.192.06 ( m , 5h ) , 1.481.30 ( m , 2h ) . c 
 nmr ( 100 mhz , cdcl3 )  167.2 , 161.9 ( d , j = 244.8 hz ) , 149.4 , 140.6 , 140.2 , 139.7 , 139.6 , 134.3 , 131.8 , 131.7 , 
 129.5 , 129.3 , 126.7 ( d , j = 7.6 hz ) , 126.4 , 125.1 , 
 123.6 , 122.8 , 118.6 , 115.2 ( d , j = 21.2 hz ) , 111.5 , 
 91.0 , 71.2 , 58.9 , 57.2 , 41.9 , 38.9 , 33.2 , 21.8 . hydrazine ( 0.092 g , 2.88 mmol ) was added 
 to a solution of phthalimide 12 ( 0.538 g , 0.96 mmol ) 
 in etoh ( 20 ml ) . the reaction mixture was subsequently stirred at 
 reflux for 3 h , concentrated , and diluted with 30% aq k2co3 . the crude mixture was extracted ( 3  20 ml ) 
 with chcl3 , concentrated , and purified by flash column 
 chromatography using 2.5:7.5 acetone / chcl3 to provide 0.395 
 g of aniline 13 ( 96% ) . h nmr of the oxalate 
 salt ( 400 mhz , cd3od )  7.707.55 ( m , 5h ) , 
 7.137.05 ( m , 5h ) , 6.93 ( d , j = 6.8 hz , 1h ) , 
 5.24 ( d , j = 13.2 hz , 1h ) , 5.17 ( d , j = 13.6 hz , 1h ) , 3.233.21 ( m , 4h ) , 2.922.88 ( m , 2h ) , 
 2.81 ( s , 3h ) , 2.352.20 ( m , 2h ) , 1.711.62 ( m , 2h ) . c nmr of oxalate salt ( 100 mhz , cd3od )  
 164.6 ( d , j = 243.3 hz ) , 150.3 , 141.7 , 140.7 , 133.24 , 
 130.9 , 128.2 ( d , j = 8.3 hz ) , 126.7 , 124.3 , 119.5 , 
 116.4 ( d , j = 24.3 hz ) , 113.1 , 92.0 , 72.5 , 58.2 , 
 57.1 , 40.5 , 38.5 , 30.6 , 20.5 . the oxalate salt was precipitated from 
 acetone ; mp 122123 c . ( c27h28fn3o2c2h2o4h2o ) c , h , n. a 1 m solution of icl in ch2cl2 ( 0.085 g , 0.64 mmol ) was added dropwise to a solution 
 of aniline 13 ( 0.230 g , 0.53 mmol ) in ch2cl2 at 05 c . the organic layer was collected , dried , 
 concentrated , and purified by flash chromatography using 2:8 acetone / chcl3 to give 0.095 g of iodide 14 ( 32% ) . h nmr ( 400 mhz , cdcl3 )  7.59 ( d , j = 8.4 hz , 1h ) , 7.497.41 ( m , 5h ) , 7.046.99 ( m , 2h ) , 
 6.92 ( dd , j = 8.0 , 2 hz , 1h ) , 6.67 ( d , j = 8.0 hz , 1h ) , 5.20 ( d , j = 13.6 hz , 1h ) , 5.14 
 ( d , j = 12.8 hz , 1h ) , 2.752.69 ( m , 6h ) , 2.40 
 ( s , 3h ) , 2.282.15 ( m , 2h ) , 1.611.48 ( m , 2h ) . c nmr ( 100 mhz , cdcl3 )  162.2 ( d , j = 245.6 hz ) , 149.3 , 145.5 , 140.3 , 139.3 , 139.3 , 138.9 , 
 132.2 , 129.9 , 126.9 ( d , j = 8.3 hz ) , 125.4 , 123.0 , 
 118.8 , 115.6 ( d , j = 21.3 hz ) , 114.9 , 112.0 , 91.1 , 
 84.3 , 71.5 , 58.4 , 56.6 , 41.2 , 38.8 , 30.9 , 20.8 . hrms calcd for 
 c27h27fin3o [ m + h ] , 
 556.1251 ; found , 556.1252 . to a solution of aniline 14 ( 0.095 g , 0.17 mmol ) in tfa ( 3 ml ) was added nano2 ( 0.023 
 g , 0.34 mmol ) at 0 c , in the dark . nan3 ( 0.111 
 g , 1.71 mmol ) and anhydrous ether ( 3 ml ) was then added to the reaction 
 mixture , followed by stirring for 1 h at 05 c , and the 
 mixture was then diluted with h2o and ether ( 10 ml each ) . the organic layer was separated , dried , concentrated , and purified 
 by flash chromatography using 3:7 acetone / chcl3 to provide 
 0.086 g of azide 15 as an oil ( 87% ) . h nmr 
 ( 400 mhz , cdcl3 )  7.59 ( d , j = 
 7.2 hz , 2h ) , 7.49 ( s , 1h ) , 7.37 ( dd , j = 8.4 , 5.6 
 hz , 2h ) , 7.33 ( d , j = 8.4 hz , 1h ) , 7.16 ( d , j = 8.4 hz , 1h ) , 7.026.98 ( m , 3h ) , 5.18 ( d , j = 12.8 hz , 1h ) , 5.13 ( d , j = 13.2 hz , 
 1h ) , 2.672.63 ( m , 2h ) , 2.532.49 ( m , 2h ) , 2.392.35 
 ( m , 2h ) , 2.202.02 ( m , 5h ) , 1.511.26 ( m , 2h ) . c nmr ( 100 mhz , cdcl3 )  162.3 ( d , j = 245.7 hz ) , 148.7 , 141.2 , 140.0 , 138.6 , 132.3 , 130.2 , 
 126.7 ( d , j = 8.3 hz ) , 125.5 , 122.8 , 118.9 , 118.6 , 
 115.8 ( d , j = 21.2 hz ) , 112.3 , 90.7 , 88.2 , 71.4 , 
 57.0 , 56.0 , 53.9 , 39.8 , 37.9 , 29.4 , 19.4 . ir : azide , 2112 cm ; hrms calcd for c27h25fin5o [ m 
 + h ] , 582.1160 ; found , 582.1159 . ( c27h25fin5o2.5h2o ) : c , h , n. a mixture of carboxylic acid 17(38 ) ( 0.257 g , 0.87 mmol ) and thionyl chloride 
 ( 2 ml ) was stirred at reflux for 3 h and then concentrated in vacuo . the crude solid was dissolved in the chcl3 and added dropwise to a vigorously stirred mixture of amine 18(31,32 ) ( 0.395 g , 0.73 mmol ) , chcl3 ( 10 ml ) , and 0.5 m aq naoh ( 10 ml ) at 0 c , followed 
 by stirring for 3 h at rt . the organic layer was separated , dried , 
 and purified by flash chromatography using 6:94 meoh / chcl3 eluent to provide 0.425 g of amide 19 as a white solid 
 ( 97% ) , mp 114115 c . h nmr ( 400 mhz , cdcl3 )  7.927.88 ( m , 2h ) , 7.787.74 ( m , 2h ) , 
 7.457.40 ( m , 2h ) , 7.317.21 ( m , 5h ) , 7.087.04 
 ( m , 2h ) , 6.966.90 ( m , 2h ) , 6.35 ( d , j = 6.0 
 hz , 1h ) , 5.10 ( d , j = 12.4 hz , 1h ) , 5.06 ( d , j = 12.4 hz , 1h ) , 4.35 ( d , j = 5.6 hz , 
 2h ) , 3.022.98 ( m , 2h ) , 2.512.47 ( m , 2h ) , 2.252.21 
 ( m , 2h ) , 2.192.06 ( m , 8h ) , 1.481.29 ( m , 2h ) . c nmr ( 100 mhz , cdcl3 )  171.8 , 167.3 , 161.7 
 ( d , j = 243.3 hz ) , 143.4 , 141.1 , 141.0 , 139.6 , 138.2 , 
 134.5 , 131.7 , 129.8 , 129.2 , 127.2 , 126.8 ( d , j = 
 7.6 hz ) , 126.7 , 123.7 , 122.0 , 120.6 , 114.9 ( d , j = 
 21.2 hz ) , 90.7 , 71.7 , 59.5 , 45.1 , 43.3 , 39.3 , 39.0 , 31.2 , 22.1 . hydrazine ( 0.069 g , 2.15 mmol ) was added 
 to a solution of phthalimide 19 ( 0.425 g , 0.72 mmol ) 
 in etoh ( 15 ml ) . the reaction mixture was stirred at reflux for 6 
 h , concentrated , and diluted with a 30% aq k2co3 solution . the organic layer was extracted , dried , and purified by 
 flash chromatography using 8:92 meoh / chcl3 to provide 0.301 
 g of aniline 20 ( 88% ) . h nmr ( 400 mhz , cdcl3 )  7.467.42 ( m , 2h ) , 7.20 ( d , j = 7.6 hz , 1h ) , 7.06 ( d , j = 8.0 hz , 1h ) , 7.006.94 
 ( m , 5h ) , 6.586.54 ( m , 2h ) , 5.62 ( m , 1h ) , 5.12 ( d , j = 12.4 hz , 1h ) , 5.08 ( d , j = 12.4 hz , 
 1h ) , 4.37 ( d , j = 5.6 hz , 2h ) , 3.58 ( bs , 2h ) , 2.882.85 
 ( m , 2h ) , 2.472.43 ( m , 2h ) , 2.262.06 ( 10h ) , 1.491.29 
 ( m , 2h ) . c nmr ( 100 mhz , cdcl3 )  172.3 , 
 161.9 ( d , j = 244.1 hz ) , 144.8 , 143.5 , 141.2 , 141.2 , 
 139.8 , 138.2 , 130.6 , 129.4 , 127.3 , 126.9 ( d , j = 
 8.3 hz ) , 122.1 , 120.7 , 115.4 , 115.1 ( d , j = 21.2 
 hz ) , 90.9 , 71.9 , 59.8 , 50.9 , 45.5 , 43.4 , 39.5 , 39.0 , 31.0 , 22.4 . the 
 hygroscopic oxalate salt was precipitated from acetone . ( c29h34cl2fn3o22c2h2o45h2o ) : c , h , n. a 1.0 m solution of icl in ch2cl2 ( 0.083 g , 0.63 mmol ) was added dropwise to a solution 
 of aniline 20 ( 0.231 g , 0.48 mmol ) in ch2cl2 ( 15 ml ) at 05 c over 30 min . the reaction mixture 
 was allowed to warm to rt and stirred overnight . the reaction mixture was then diluted with 10% aq na2s2o3 ( 25 ml ) , stirred for 10 min , and extracted in ch2cl2 . after discarding the aqueous layer , the black residue was 
 dissolved in meoh and combined with the previous ch2cl2 solution . the organic layer was concentrated and purified 
 by flash chromatography using 7:93 meoh / chcl3 to provide 
 0.292 g of iodide 21 as an oil ( 62% ) . h nmr 
 ( 400 mhz , cdcl3 )  7.567.51 ( m , 2h ) , 7.46 
 ( d , j = 1.6 hz , 1h ) , 7.32 ( d , j = 
 7.6 hz , 1h ) , 7.076.95 ( m , 5h ) , 6.70 ( d , j = 8.4 hz , 1h ) , 5.14 ( d , j = 12.8 hz , 1h ) , 5.09 
 ( d , j = 13.2 hz , 1h ) , 4.30 ( s , 2h ) , 2.962.92 
 ( m , 2h ) , 2.792.75 ( m , 2h ) , 2.64 ( s , 6h ) , 2.482.44 
 ( m , 2h ) , 2.292.13 ( m , 2h ) , 1.661.51 ( m , 2h ) . c nmr ( 100 mhz , cdcl3 )  175.0 , 163.3 ( d , j = 242.6 hz ) , 147.6 , 143.8 , 142.2 , 142.2 , 140.6 , 140.2 , 
 139.7 , 133.0 , 130.7 , 128.1 ( d , j = 7.6 hz ) , 128.0 , 
 123.0 , 121.1 , 116.0 ( d , j = 21.2 hz ) , 115.9 , 91.7 , 
 84.4 , 73.0 , 59.4 , 43.9 , 43.7 , 39.1 , 38.8 , 31.3 , 21.6 . hrms calcd for 
 c29h33fin3o2 [ m + h ] , 602.1666 ; found , 602.1667 . nano2 ( 9.6 mg , 
 0.14 mmol ) was added 
 to a suspension of aniline 21 ( 60 mg , 0.10 mmol ) in a 
 mixture of glacial hoac ( 3 ml ) and h2o ( 1 ml ) at 0 c , 
 in the dark . the mixture was stirred for 30 min ; then , nan3 ( 9.7 mg , 0.15 mmol ) was added to the reaction mixture . after stirring 
 at 05 c for 30 min , the reaction was diluted with water 
 ( 10 ml ) . the mixture was then carefully quenched with saturated aq 
 nahco3 to a ph of 910 . the crude product was extracted 
 with chcl3 , dried over na2so4 , concentrated , 
 and purified by preparative tlc using 15:85 meoh / chcl3 to 
 provide 20 mg of azide 22 as a yellow oil ( 18% ) . h nmr ( 400 mhz , cdcl3 )  7.63 ( d , j = 2.0 hz , 1h ) , 7.467.41 ( m , 2h ) , 7.23 ( d , j = 6.8 hz , 2h ) , 7.09 ( d , j = 8.0 hz , 1h ) , 
 7.046.95 ( m , 4h ) , 6.02 ( bt , j = 5.0 hz , 1h ) , 
 5.13 ( d , j = 12.4 hz , 1h ) , 5.07 ( d , j = 12.4 hz , 1h ) , 4.37 ( d , j = 5.6 hz , 2h ) , 2.91 
 ( t , j = 7.2 hz , 2h ) , 2.642.57 ( m , 2h ) , 2.48 
 ( t , j = 7.2 hz , 2h ) , 2.39 ( s , 6h ) , 2.19 ( m , 2h),1.661.47 
 ( m , 2h ) . c nmr ( 100 mhz , cdcl3 )  171.6 , 
 161.9 ( d , j = 243.4 hz ) , 143.4 , 141.1 , 139.9 , 139.8 , 
 139.6 , 138.2 , 130.0 , 127.6 , 126.8 ( d , j = 10.6 hz ) , 
 122.1 , 120.9 , 118.4 , 115.1 ( d , j = 21.2 hz ) , 115.0 , 
 90.9 , 87.9 , 71.9 , 59.5 , 45.1 , 43.5 , 39.4 , 37.9 , 30.5 , 22.3 . ir : azide , 
 2114 cm ; hrms calcd for c29h31fin5o2 [ m + h ] , 628.1579 ; found , 
 628.1569 . ( c29h31fin5o22h2o0.5c3h6o ) : 
 c , h , n. cdi ( 0.064 g , 0.39 mmol ) 
 was added to a solution 
 of carboxylic acid 24(39 ) ( 0.125 
 g , 0.39 mmol ) in thf ( 15 ml ) under an argon atmosphere . the reaction 
 mixture was stirred for 6 h and then cooled to 05 c 
 using an ice bath . primary amine 18(31,32 ) ( 0.129 g , 0.39 mmol ) was then added dropwise after dilution with 
 thf ( 10 ml ) . the reaction mixture was allowed to warm to rt and stirred 
 overnight , followed by subsequent concentration , dilution with water 
 ( 20 ml ) , and extraction with chcl3 ( 2  20 ml ) . the 
 organic layer was dried , concentrated , and purified by flash chromatography 
 using 3:97 meoh / chcl3 as eluent to provide 0.054 g of amide 22 as a yellow oil ( 22% ) . a 1 m 
 solution of icl in hoac ( 0.240 
 g , 1.82 mmol ) was added dropwise slowly to a solution of 3-(4-aminophenyl)propanoic 
 acid ( 16 , 0.250 g , 1.51 mmol ) in glacial hoac ( 8 ml ) 
 at rt . the crude product was extracted 
 with ch2cl2 ( 2  20 ml ) , dried , concentrated , 
 and purified by flash chromatography using 3:97 meoh / chcl3 to provide 0.296 g of acid 23 as a pure white solid 
 ( 67% ) . azide 24 was prepared 
 using the same method as described for azide 22 ( method 
 a ) in tfa . the crude product was purified by flash 
 chromatography using 2:98 meoh / chcl3 and used directly 
 in the next step , method b , to synthesize 22 . to a solution of 4-azido-3-iodobenzoic 
 acid ( 25 , 87 mg , 0.30 mmol ) 
 in dmf ( 2 ml ) at 0 c were added edc ( 49 mg , 0.31 mmol ) and hobt 
 ( 43 mg , 0.31 mmol ) . the resulting mixture was stirred in the dark 
 for 1 h at 0 c . to this mixture were added a solution of 18(31,32 ) ( 99 mg , 0.30 mmol ) in dmf ( 1 
 ml ) and et3n ( 0.2 ml ) , followed by stirring in the dark 
 at rt overnight . the combined organic extracts were washed 
 with 1 m aq naoh and brine , dried ( mgso4 ) , filtered , concentrated , 
 and chromatographed using 95:5 etoac / et3n to provide 120 
 mg of amide 26 as a colorless semisolid ( 67% ) . h nmr ( 400 mhz , cdcl3 )  8.18 ( d , j = 1.9 hz , 1h ) , 7.81 ( dd , j = 8.3 , 1.9 hz , 1h ) , 
 7.507.40 ( m , 2h ) , 7.307.20 ( m , 2h ) , 7.16 ( s , 1h ) , 
 7.10 ( d , j = 8.3 hz , 1h ) , 6.97 ( t , j = 8.7 hz , 2h ) , 6.75 ( t , j = 5.5 hz , 1h ) , 5.11 ( dd , j = 17.1 , 12.6 hz , 2h ) , 4.56 ( d , j = 5.7 
 hz , 2h ) , 2.32.0 ( m , 10h ) , 1.51.2 ( m , 2h ) . c nmr ( 100 mhz , cdcl3 ) :  164.9 , 162.9 , 144.9 , 143.8 , 
 140.9 , 139.8 , 138.7 , 137.6 , 131.9 , 128.5 , 127.4 , 126.7 , 126.6 , 122.1 , 
 120.8 , 117.9 , 115.1 , 114.8 , 90.8 , 87.5 , 71.7 , 59.6 , 45.4 , 43.9 , 39.3 , 
 22.3 . hrms calcd for c27h26fin5o2 [ m + h ] , 600.1266 ; found , 600.1268 . a mixture 
 of iodide 26 ( 170 mg , 0.28 mmol ) , pd(pph3)2br2 ( 13.5 mg , 0.02 mmol ) , and bis(tri - n - butyltin ) ( 0.26 ml , 0.51 mmol ) in toluene ( 10 ml ) was heated at 
 105 c for 4 h. the mixture was then cooled to room temperature , 
 diluted with saturated aq k2co3 , and then extracted 
 with etoac . the organic layers were then washed with brine , dried 
 ( mgso4 ) , filtered , concentrated , and chromatographed ( etoac / et3n , 95:5 ) to give 110 mg of the requisite organostannane 27 as a brown oil ( 52% ) . final purification was achieved by 
 reversed - phase hplc of 35 mg portions on a waters c-18 nova - pak 
 column ( radial compression module , 8  100 mm , 6 m ) using 
 meoh ( 42.5% ) , ch3cn ( 42.5% ) , and an aqueous solution ( 15% ) 
 of et3n ( 2.1% v / v ) and hoac ( 2.8% v / v ) at a flow rate of 
 3 ml / min with uv detection ( 254 nm ) . material with retention time 
 ( tr ) 8.5 min was collected , water was 
 added to lower the organic component to 9% of the total volume , and 
 the mixture was passed through an activated ( meoh / water ) solid - phase 
 extraction cartridge ( waters sep - pak plus t - c-18 ) 
 that was flushed with water ( 5 ml ) and then with air . elution with 
 meoh ( 6 ml ) and concentration under a stream of argon at 60 c 
 provided 27 for direct use in the radioiodination reaction . h nmr ( 400 mhz , cdcl3 ) :  7.83 ( d , j = 2.1 hz , 1h ) , 7.74 ( dd , j = 8.3 , 2.1 
 hz , 1h ) , 7.507.40 ( m , 2h ) , 7.25 ( s , 2h ) , 7.19 ( s , 1h ) , 7.13 
 ( d , j = 8.3 hz , 1h ) , 6.97 ( t , j = 
 8.7 , 2h ) , 6.42 ( t , j = 5.6 hz , 1h ) , 5.12 ( dd , j = 17.4 , 12.5 hz , 2h ) , 4.62 ( d , j = 5.7 
 hz , 2h ) , 2.302.0 ( m , 10h ) , 1.601.40 ( m , 7h ) , 1.41.2 
 ( m , 7h ) , 1.21.0 ( m , 6h ) , 0.90.8 ( m , 9h ) . c nmr ( 100 mhz , cdcl3 )  166.9 , 162.9 , 160.5 , 149.2 , 
 143.6 , 140.9 , 139.7 , 137.9 , 136.3 , 135.1 , 130.0 , 128.5 , 127.2 , 126.7 , 
 126.6 , 121.9 , 120.6 , 116.5 , 114.9 , 114.7 , 90.7 , 71.6 , 59.6 , 45.3 , 
 43.7 , 39.3 , 28.9 , 27.2 , 22.3,13.6 , 10.1 . ir : azide , 2109 cm ; hrms calcd for c39h54fn5o2snh , 764.3356 ; found , 764.3369 . radioactivity was measured with 
 a dose calibrator ( capintec crc-15w ; ramsey , nj ) employing similar 
 counting geometries and attenuation correction factors as necessary . 
 the radio - hplc system was equipped with flow - through uv absorbance 
 ( 254 nm ) and radioactivity detectors . ( milford , ma ) 
 c-18 nova - pak hplc column ( 8  100 mm , 6 m ; radial compression 
 module ) was used for preparative separations and for analytical work . hplc mobile phases were composed of varying proportions of an organic 
 component ( meoh / ch3cn ; 1:1 , v / v ) and an aqueous component 
 containing et3n ( 2.1% v / v ) and hoac ( 2.8% v / v ) . activated 
 sep - pak light t - c-18 cartridges ( waters corp . ) were 
 used for solid - phase extraction . specific radioactivities were calculated 
 by determining the carrier mass associated with the absorbance peak 
 for carrier in purified samples of known radioactivity . the carrier 
 mass in the radioactive samples was assessed by hplc in reference 
 to linear ( r = 1.0 ) six - point standard 
 curves ( 03000 or 06000 pmol ) generated using the corresponding 
 nonradioactive materials . a solution of aniline 13 as the oxalate salt ( 75 l , 4.0 mm ) in naoac buffer 
 ( ph 4.0 ; 0.2 m ) was treated with [ i]nai ( 2.42 mci ) in 
 naoh ( 20 l , 10 m ) followed by n - chloro-4-toluenesulfonamide 
 ( chloramine - t ) trihydrate ( 20 l , 5.0 mm ) . after 30 min at room 
 temperature , the mixture was chilled in an ice / meoh bath and treated 
 sequentially with ice - cold hoac ( 75 l , 3.0 m ) and nano2 ( 30 l , 0.5 m ) . after 20 min in the ice bath , nan3 ( 30 l , 0.5 m ) was added , and the mixture was allowed 
 to warm to room temperature . after 30 min , the reaction was quenched 
 with na2s2o5 ( 10 l , 50 mm ) . purification was achieved by reverse - phase hplc ( 45% organic/55% aqueous ; 
 3.0 ml / min ) . radioactive material with retention time ( tr ) 22.4 min and capacity factor ( k ) 
 18.3 corresponded to the chromatographic profile established for nonradioactive 15 and was collected in a 7.5 ml volume . this sample was diluted 
 to 32 ml with h2o and then passed through an activated 
 ( meoh / h2o ) solid - phase extraction cartridge that was flushed 
 with h2o ( 2.0 ml ) and then with air . elution with meoh 
 ( 1.5 ml ) gave [ i]15 ( 1.20 mci ) in 50% radiochemical 
 yield . a 98% radiochemical purity was determined by hplc , 
 the radioactive material coeluted with nonradioactive 15 , and the specific radioactivity was calculated as 2045 mci/mol . the major nonradioactive products observed during hplc purification 
 are likely to be the aryl azide ( tr = 
 10.2 min , k = 7.8 ) and chlorinated aryl azide 
 ( tr = 16.6 min , k 
 = 13.3 ) analogues . a solution of free - base 20 ( 75 l , 4.0 mm ) in naoac buffer ( ph 4.0 ; 0.2 m ) was 
 treated with [ i]nai ( 2.14 mci ) in naoh ( 20 l , 
 10 m ) and chloramine - t trihydrate ( 15 l , 5.0 mm ) for 
 30 min . further treatments with hoac / nano2 , nan3 , and na2s2o5 were performed as 
 described above for [ i]15 . purification 
 was accomplished by hplc ( 42% organic/58% aqueous ; 3.0 ml / min ) . radioactive 
 material ( tr = 20.0 min , k = 16.2 ) corresponding to 22 was collected in 
 a 12.5 ml volume , diluted to 50 ml with h2o , and processed 
 by solid - phase extraction as described above to give pure [ i]22 ( 1.17 mci ) in 55% radiochemical yield . this material 
 coeluted with nonradioactive 22 on hplc , and a specific 
 radioactivity of 2098 mci/mol was calculated . nonradioactive 
 products observed during hplc purification are likely to be the aryl 
 azide ( tr = 9.6 min , k = 7.3 ) and chlorinated aryl azide ( tr = 15.1 min , k = 12.0 ) analogues . a solution of organostannane 27 ( 0.11 mg , 0.15 mol ) 
 in meoh ( 25 l ) was treated with [ i]nai ( 2.07 
 mci ) in dilute naoh ( 20 l , 10 m ) followed by a mixture 
 of aqueous chloramine - t trihydrate ( 15 l , 5.0 mm ) and 3% glacial 
 hoac in meoh ( 85 l ) . after 3 min at room temperature , the reaction 
 was quenched with na2s2o5 ( 10 l , 
 50 mm ) . purification was accomplished by hplc ( 45% organic/55% aqueous ; 
 3.0 ml / min ) . the major radioactive product ( tr 13.4 min , k = 10.6 ) corresponded 
 to 26 and was resolved from minor nonradioactive and 
 radioactive side products . this material was collected in 7.5 ml of 
 mobile phase , diluted to 35 ml with water , and processed by solid - phase 
 extraction as described above for [ i]22 to give 0.96 mci ( 46% ) of pure [ i]26 that 
 coeluted with nonradioactive 26 on hplc and had a specific 
 radioactivity of 2096 mci/mol . hek-293 griptite 
 cells ( invitrogen ) were plated in 24-well culturplates ( perkinelmer ) 
 at 50  000 cells / well and transiently transfected with wild - type 
 hsert or hsert s438t - containing pcdna3-based plasmid constructs using 
 the trans - it lti transfection system ( mirus bio ) . assays were conducted 
 3648 h after transfection , where cells were washed with mkrhg 
 buffer ( 5 mm tris , 7.5 mm hepes , 120 mm nacl , 5.4 mm kcl , 1.2 mm cacl2 , 1.2 mm mgso4 , 10 mm dextrose , ph 7.4 ) and preincubated 
 with the competitor compounds for 5 min followed by addition of [ h]5-ht at a final concentration of 50 nm . uptake was allowed 
 to proceed for 10 min and then terminated by washing twice with ice - cold 
 mkrhg . [ h]5-ht uptake was quantified by dissolving cells 
 in microscint-20 ( perkinelmer ) and using the packard topcount nxt 
 ( perkinelmer ) to measure radioactivity ( cpm ) . data were normalized 
 to percent activity in the absence of a drug competitor for each form . photoaffinity 
 labeling 
 of hsert was performed using methods that have been verified previously 
 for assessing irreversible adduction of a variety of ligands for both 
 dat and sert . these procedures were performed using lewis lung carcinoma porcine 
 kidney ( llcpk1 ) cells stably expressing hemagluttinin ( ha)-hsert 
 ( generous gift of dr . cells were grown to 
 90% confluence in 12-well plates and washed with krh buffer . [ i]15 , [ i]22 , or 
 [ i]26 was added to a final concentration 
 of 10 nm and incubated for 1.5 h at 4 c . in some experiments , 
 hserts were irreversibly labeled in parallel with 10 nm [ i]3 as a positive control . for pharmacological displacement , 10 m ( s)-citalopram was added 30 min prior to addition of the radioligands . cells were irradiated with shortwave ultraviolet light ( 254 nm , fotodyne 
 uv lamp model 3 - 6000 ) for 5 min at a distance of 1520 mm to 
 photoactivate the radioligand , washed twice with 1 ml of ice - cold 
 krh buffer , and lysed by addition of 0.5 ml of ripa buffer ( 50 mm 
 naf , 2 mm edta , 125 mm na3po4 , 1.25% triton 
 x-100 , and 1.25% sodium deoxycholate ) containing protease inhibitors 
 for 30 min on ice . lysates were centrifuged at 20  000 g for 15 min at 4 c to remove insoluble material and 
 subjected to immunoprecipitation and immunoblotting using anti - ha 
 monoclonal antibody ( covance ) or anti - sert antibody st51 ( mab technologies ) 
 as previously described . immunoprecipitated 
 samples were separated on 420% sds - polyacrylamide gels followed 
 by autoradiography using hyperfilm mp film ( ge healthcare ) for 13 
 days at 80 c . for immunoblotting , cell lysates were 
 separated on 420% sds - polyacrylamide gels transferred to 0.45 
 m poly(vinylidene difluoride ) membranes and probed for hsert 
 with anti - ha antibody .
three photoaffinity ligands ( pals ) 
 for the human serotonin transporter 
 ( hsert ) were synthesized based on the selective serotonin reuptake 
 inhibitor ( ssri ) , ( s)-citalopram ( 1 ) . 
 the classic 4-azido-3-iodo - phenyl group was appended to either the 
 c-1 or c-5 position of the parent molecule , with variable - length linkers , 
 to generate ligands 15 , 22 , and 26 . 
 these ligands retained high to moderate affinity binding ( ki = 24227 nm ) for hsert , as assessed 
 by [ 3h]5-ht transport inhibition . when tested against ser438thr 
 hsert , all three pals showed dramatic rightward shifts in inhibitory 
 potency , with ki values ranging from 3.8 
 to 9.9 m , consistent with the role of ser438 as a key residue 
 for high - affinity binding of many ssris , including ( s)-citalopram . 
 photoactivation studies demonstrated irreversible adduction 
 to hsert by all ligands , but the reduced ( s)-citalopram 
 inhibition of labeling by [ 125i]15 compared 
 to that by [ 125i]22 and [ 125i]26 suggests differences in binding mode(s ) . 
 these radioligands 
 will be useful for characterizing the drug  protein binding 
 interactions for ( s)-citalopram at hsert .