extracellular adenosine 
 acts as a signaling molecule with a generally 
 cytoprotective function in the body . adenosine mediates cell signaling 
 through binding to four known subtypes ( a1 , a2a , a2b , and a3 ) of adenosine receptors ( ars ) . a1 , a2a , and a3ars are activated 
 by low levels of adenosine ( ec50 = 0.011.0 m ) 
 similar to physiological levels of adenosine , whereas a2bar is activated by high levels of adenosine ( ec50 = 24 
 m ) . a1 and a3ars are gi - coupled g protein - coupled receptors ( gpcrs ) , 
 and a2a and a2bars are gs - coupled 
 gpcrs . binding of adenosine to the ars modulates second messengers 
 such as adenosine 3,5-cyclic phosphate ( camp ) , inositol 
 triphosphate ( ip3 ) , and diacylglycerol ( dag ) . for example , the gi - coupled a3ar inhibits 
 adenylate cyclase ( ac ) , resulting in camp down - regulation , while it 
 stimulates phospholipase c ( plc ) , which increases the levels of ip3 and dag . therefore , ars have been attractive targets for 
 the development of new therapeutic agents related to cell signaling . chronic kidney disease ( ckd ) is characterized by kidney fibrosis 
 and is becoming a major health problem worldwide , and the use of renin  angiotensin aldosterone 
 system ( raas ) inhibitors is one of a few therapeutic 
 options for the treatment of ckd . however , the efficacy of raas inhibitors 
 is limited ; it is , therefore , highly desirable 
 to develop new therapeutic agents to improve the prognosis of ckd 
 patients . extracellular adenosine in the kidney dramatically increases 
 in response to renal hypoxia and ischemia , and increased adenosine 
 has been reported to be associated with ckd . ars were upregulated in unilateral ureteral obstructed rat kidneys , 
 which is a well - characterized model of ckd , and a3ar knockout mice were protected against ischemia- 
 and myoglobinuria - induced kidney failure . therefore , a3ar antagonists may become effective renoprotective 
 agents for the treatment of ckd . adenosine as a natural ligand 
 has served as a good lead for the 
 development of new ar ligands . extensive 
 modifications on the n and/or 4-ch2oh of adenosine have been explored , giving several potent and selective 
 a3ar agonists such as n-(3-iodobenzyl)-5-n - methylcarbamoyladenosine 
 ( ib - meca ) , 2-chloro - n-(3-iodobenzyl)-5-n - methylcarbamoyladenosine 
 ( cl - ib - meca),n-(3-iodobenzyl)-5-n - methylcarbamoyl-4-thioadenosine 
 ( thio - ib - meca ) , 2-chloro - n-(3-iodobenzyl)-5-n - methylcarbamoyl-4-thioadenosine 
 ( thio - cl - ib - meca ) , and 3-amino - n-{5-chloro-2-(3-methylisoxazol-5-ylmethoxy)benzyl}-5-n - methylcarbamoyladenosine ( cp-608039 ) . these compounds contain the potency- and efficacy - enhancing 
 5-methyluronamide moiety and the n - hydrophobic 
 moiety . also , ar agonists that combined n - alkyl and 2-alkynyl substitutions proved useful in the identification 
 of a3 or a2b ar agonists with various selectivity 
 profiles , depending on the type of 2-alkynyl substitution . on the other hand , the truncated nucleosides 
 where the 5-methyluronamide of the a3ar agonists 
 was deleted were converted into potent and selective a3ar antagonists , because there was no 5-uronamide , which serves 
 as the hydrogen bonding donor required for receptor activation . among these , introduction of the 2-hexynyl group on the c2-position 
 of 1 but no substitution on the n - position 
 converted 1 into dually acting a2aar agonist 
 and a3ar antagonist 2 . molecular modeling and empirical structure activity studies 
 in both the ribose and the 4-thioribose series indicated that 
 the c2 binding sites of a2aar and a3ar were 
 spacious enough to accommodate the bulky substituent . aki values ( nm ) or % inhibition at 10 m in binding to human a1 , a2a , and a3 adenosine receptors . ki values ( nm ) or % inhibition at 10 m in binding to human a1 , a2a , and a3 adenosine receptors . truncated ( n)-methanocarba - nucleosides 3(20a ) were also reported to be selective 
 and potent a3ar antagonists , indicating that compound 3 can also serve as a good template for the development of 
 a3ar ligands . thus , we designed and synthesized the truncated 
 c2-hexynyl-(n)-methanocarba - nucleosides 4 , which hybridize the structure of c2-hexynyl derivative 2 with that of ( n)-methanocarba - nucleoside 3 to determine if similar biological trends between 2 and 4 were observed . for the synthesis of the 
 target nucleoside 4 , copper - catalyzed and palladium - catalyzed cross - coupling 
 reactions were employed as key steps for functionalization of the 
 c2-position of 6-chloropurine nucleosides . herein , we report the synthesis 
 of truncated c2-hexynyl - n - substituted-(n)-methanocarba - nucleosides 4 as potent and 
 selective a3ar antagonists and their renoprotective effects 
 using tgf-1-stimulated mprox cells , a cell culture model for 
 kidney fibrosis . the desired c2-hexynyl - methanocarba - adenosine 
 derivatives 4a4i were synthesized 
 from our known cyclopentenone intermediate 5(26 ) using a palladium - catalyzed cross - coupling reaction 
 as a key step ( scheme 1 ) . reagents and conditions : ( a ) 
 nabh4 , cecl37h2o , methanol , 
 0 c , 2 h ; ( b ) et2zn , ch2i2 , 
 ch2cl2 , rt , 5 h ; ( c ) 2-iodo-6-chloropurine , 
 ph3p , diad , thf , rt , 18 h ; ( d ) 1-hexyne , ( ph3p)4pd , cs2co3 , cui , dmf , 50 c , 
 6 h ; ( e ) 2 n hcl / thf ( 1/1 ) , 40 c , 18 h ; ( f ) r nh2 , et3n , ethanol , 90 c , 18 h. the cyclopentenone derivative 5 was converted 
 to the 
 glycosyl donor 7 according to the reported procedure developed by our laboratory . direct condensation 
 of 7 with 6-chloro-2-iodopurine under the standard mitsunobu conditions in thf afforded the -anomer 8 in 67% yield , similar to a literature report . the anomeric -configuration of 8 was readily assigned by the diagnostic coupling constants 
 typical of the boat conformation of the bicyclo[3.1.0]hexane system , 
 which has been extensively confirmed by x - ray crystallography and 
 nmr analysis . the coupling constants 
 of the jh1,h2 and jh1,h5 should be zero , because 
 both h1c  c  h2 and h1c  c  h5 dihedral angles with trans relationships are close to 90 , indicating that 1-h of 8 should appear 
 as a singlet . indeed , h nmr of 8 showed that 
 1-h appeared as a singlet at 5.03 ppm , confirming the structure 
 of 8 . sonogashira coupling 
 of 8 with 1-hexyne in the presence of palladium catalyst 
 yielded the c2-hexynyl derivative 9 ( 56% ) . substitution of the 6-position of 10 with 
 ammonia and various primary alkyl- , cycloalkyl- , and arylalkyl - amines 
 afforded the final nucleosides 4a4i . the target nucleosides were also synthesized using a lithiation - mediated 
 stannyl transfer reaction and a copper - catalyzed 
 cross - coupling reaction as key steps 
 for functionalization of the c2-position ( scheme 2 ) . the glycosyl donor 7 was condensed with 6-chloropurine 
 under the same conditions used in scheme 1 to 
 give 6-chloropurine derivative 11 . treatment of 11 with litmp followed by reacting the resulting anion with 
 tri - n - butyltin chloride afforded the c2-stannyl derivative 12 exclusively . copper - catalyzed 
 coupling of 12 with 1-iodohexyne 
 yielded the 2-hexynyl derivative 9 , which was converted 
 to the same final nucleosides 4a4i according the same procedure used in scheme 1 . reagents and conditions : ( a ) 
 6-chloropurine , ph3p , diad , thf , rt , 18 h ; ( b ) litmp , bu3sncl , thf , 78 c , 5 h ; ( c ) 1-iodohexyne , cui , 
 dmf , 50 c , 16 h. binding assays were carried out using 
 standard radioligands and membrane preparations from chinese hamster 
 ovary ( cho ) cells stably expressing the human ( h ) a1 or 
 a3ar , rbl-2h3 basophilic leukemia cells expressing rat 
 ( r ) a3ar , or human embryonic kidney ( hek)-293 cells expressing 
 the ha2aar . binding at the 
 ha3ar or ra3ar in this study was carried out 
 using [ i]n-(3-iodo-4-aminobenzyl)-5-n - methylcarboxamidoadenosine ( i - ab - meca , 13 ) as a radioligand . binding at the ha1ar using 
 [ h ] ( -)-n-2-phenylisopropyl 
 adenosine ( r - pia , 14 ) or ha2aar using [ h]cgs21680 ( 2-[p-(2-carboxyethyl)phenylethylamino]-5-n - ethylcarboxamidoadenosine , 15 ) was carried out . in cases of weak binding , the percent inhibition 
 of radioligand binding to the ha1ar and ha2aar was determined at 10 m . nonspecific binding was defined 
 using 10 m of 5-n - ethylcarboxamidoadenosine 
 ( neca , 16 ) . because binding affinity of similar 
 ( n)-methanocarba compounds was reported to be very 
 weak or absent at the ha2bar subtype , we did not include this receptor in the radioligand binding 
 assays . to confirm that activity of the present chemical series is 
 weak at the a2bar , we performed a functional assay in cho 
 cells expressing the ha2bar . compound 4b at 
 10 m produced only 15.7  12.6% of the activation of camp 
 production seen with full agonist 16 . as shown 
 in table 1 , a variety of n - alkyl , cycloalkyl , and arylalkyl substituents 
 in truncated ( n)-methanocarba - nucleoside derivatives 
 have produced nanomolar binding affinity at the ha3ar subtype , 
 indicating that bulky c2 and n substituents could be tolerable 
 in the binding site of a3ar . however , a hydrophobic substituent 
 at the n - position reduced the binding affinity greatly 
 at the ha2a ar subtype in the presence of a hydrophobic 
 c2-hexynyl group , and only an unsubstituted 6-amino group showed good 
 binding affinity ( ki = 100 nm ) at the 
 ha2aar , indicating that the n binding site 
 of ha2aar is small . this trend is similar to that of truncated 
 2-hexynyl-4-thioadenosine ( 2 ) , but truncated carbanucleoside derivative 4a was 14-fold less potent than truncated 4-thioadenosine derivative 2 . this result may be due to the fixed conformation of ( n)-methanocarba - nucleosides unlike the flexible conformation 
 of 4-thioadenosine derivatives , hindering them from forming 
 a favorable hydrophobic interaction in the binding site of a2aar . however , all compounds showed very weak binding affinity at the 
 ha1ar , suggesting that the binding sites may not be large 
 enough to accommodate the bulky c2 and/or n substituent . 
 among compounds tested , 4b ( r = ch3 ) exhibited 
 the highest binding affinity ( ki = 4.9 
 nm ) at the ha3ar subtype with high selectivity for the 
 ha1 and ha2aars . the primary amine - substituted n - alkyl- and n - cycloalkyl- 
 derivatives ( 4b4e ) generally exhibited 
 better binding affinity at the ha3ar than the free amino 
 derivative 4a , except cyclopentyl derivative 4e . the order of compounds showing high binding affinity at the ha3ar is as follows : 4b ( r = ch3 , ki = 4.6 nm ) > 4c ( r = ethyl , ki = 6.7 nm ) > 4d ( r = cyclopropyl , ki = 9.2 nm ) > 4a ( r = h , ki = 16.2 nm ) . the binding affinities of 3-halobenzylamine 
 derivatives 4f4i at the ha3ar did not differ significantly , with ki values of 7.816.0 nm . the binding affinity at the ha3ar in this series decreased in the following order : 3-cl derivative 4h , 3-br derivative 4 g > 3-i derivative 4f > 3-f derivative 4i . all synthesized compounds 4a4i have also produced nanomolar binding 
 affinity at the ra3ar , but they showed weaker binding affinity 
 than that at the ha3ar . the n - alkyl derivatives 4b and 4c exhibited 
 lower binding affinity at the ra3ar than the free amino 
 derivative 4a , the n - cycloalkyl 
 derivatives 4d and 4e , and the 3-halobenzylamine 
 derivatives 4f4i , which showed similar 
 binding affinities at the ra3ar , with ki values in the range of 10.765 nm . the 3-chlorobenzyl 
 derivative 4h exhibited the highest binding affinity 
 ( ki = 10.7 nm ) at the ra3ar , 
 unlike the n - methyl derivative 4b showing the highest affinity ( ki = 4.9 nm ) at the ha3ar . all binding experiments were performed 
 using adherent mammalian cells stably transfected with cdna encoding 
 the appropriate har ( a1ar and a3ar in cho cells 
 and a2aar in hek-293 cells ) or ra3ar expressed 
 endogenously in rbl-2h3 cells . binding was carried out using 1 nm 
 [ h]14 , 10 nm [ h]15 , or 0.5 nm [ i]13 as radioligands for 
 a1 , a2a , and a3ars , respectively . 
 values expressed as a percentage in italics refer to percent inhibition 
 of specific radioligand binding at 10 m for 3  5 duplicate 
 determinations , with nonspecific binding defined using 10 m 16 . concentration 
 to inhibit the tgf-1-induced 
 collagen i mrna expression by 50% . in a 
 camp functional assay at the 
 ha3ar expressed in cho cells , the most potent compound 4b behaved as a partial agonist , in contrast to full antagonists 2 and 3 ( figure 1 ) . compound 4b at 10 m displayed an ec50 of 45.8 nm 
 and a maximal stimulation of camp formation of 29.1  5.0% relative 
 to the full agonist 16 (= 100% ) . similarly , other compounds 
 proved to be partial agonists of the ha3ar ( % activation 
 relative to 16 , triplicate determination ) : 4c , 15.5  6.7 ; 4d , 19.8  4.6 ; 4e , 27.1  14.6 ; 4i , 18.9  7.5 . compounds 4f , 4 g , and 4h induced < 5% of 
 the activation seen with 16 and were therefore antagonists . effect 
 of compound 4b on forskolin - induced stimulation 
 of camp production at the ha3ar expressed in cho cells , 
 compared to 16 as reference full agonist (= 100% ) . all synthesized 
 compounds were 
 tested for an antifibrotic effect in murine proximal ( mprox ) cells , 
 a cell line of mouse proximal tubular epithelial cells . as shown in table 1 , 
 most of the tested compounds strongly inhibited transforming growth 
 factor ( tgf)-1-induced collagen i upregulation . compound 4b showed the most potent inhibitory activity ( ic50 = 0.83 m ) against tgf-1-induced collagen i mrna expression 
 ( figure 2 ) . the binding affinity at the a3ar was almost proportional to the antifibrotic activity , which 
 indicates that the small n - hydrophobic substituent is 
 also favored for renoprotective effects . inhibition of tgf-1-induced col1a1 gene 
 expression in mprox24 cells , a cell line of mouse proximal tubular 
 epithelial cells , by 4b . data are mean  se of three 
 experiments . * p < 0.05 vs tgf-1-stimulated 
 mprox24 cells : relative increase in col1a1 gene expression ( 1.0 is the effect of 5 ng / ml tgf-1 ) , at the concentration of 4b in m indicated 
 in column 1 . the truncated c2-substituted 
 thio - ribose compound 2 ( a2aki = 7.19 nm ) exhibited excellent binding affinity , and 
 the methanocarba analogue 4a ( a2aki = 100 nm ) showed 14-fold less binding 
 affinity at the ha2a ar . in addition , the presence of the 
 3-iodobenzyl group at the n - position in 4f led to a substantial decrease in its binding affinity at the ha2aar with a ki of 2530 nm . in view 
 of the observed variations in the ha2aar binding affinities 
 among these compounds , molecular docking and binding free energy calculations 
 were carried out considering the x - ray structure of the ha2aar complexed with an agonist , 16 ( pdb code 2ydv ) . the common interactions among n - unsubstituted compounds 2 and 4a at the 
 ha2aar includes : ( i ) the adenine ring stabilized through 
  stacking interaction with phe168 ( extracellular 
 loop 2 ) and a h - bonding interaction with asn253 , ( ii ) 
 the exocyclic 6-amino group h - bonded with asn253 and 
 glu169 , and ( iii ) the projection of c2-hexynyl group toward the extracellular 
 side exhibiting hydrophobic interaction with phe168 , ile66 , leu267 , met270 , ile274 , and tyr271 residues ( figure 3 ) . predicted binding modes of n - unsubstituted nucleosides 2 ( a ) and 4a ( b ) in the ha2aar agonist - bound 
 crystal structure . compounds 2 and 4a are 
 depicted in ball - and - stick , with carbon atoms in magenta and purple , 
 respectively . the key amino acid residues are shown as capped - stick , 
 with carbon atoms in white . the connolly surface of the receptor was 
 generated by molcad with green color and z - clipped for visual convenience . the hydrogen bonds are shown as black dashed lines , and the nonpolar 
 hydrogen atoms are not displayed for clarity . in contrast , they exhibited different binding modes at the 
 ribose 
 binding site formed by val84 , leu85 , 
 trp246 , leu249 and ile274 , ser277 , and his278 . the 2- 
 and 3-hydroxyl groups of 2 formed h - bonds with 
 the two key residues his278 and ser277 , respectively ( figure 3a ) , whereas 4a lost one of the key h - bond interactions with ser277 ( figure 3b ) . this residue ser277 
 is a key residue reported to be important for ha2aar agonistic 
 activity and potency using site - directed mutagenesis . it appears that a decrease in the binding affinity of 4a at the ha2aar could be due to the loss of h - bonding with 
 ser277 at the ribose binding site . the loss of h - bonding 
 with ser277 may particularly be attributed to the methanocarba 
 ring ( 4a ) , being less flexible than the thio - ribose ring 
 ( 2 ) . furthermore , the calculated prime mm - gbsa binding 
 free energies ( gbind ) for 2 and 4a were 104.16 and 90.37 
 kcal / mol , respectively , which are in good agreement with their observed 
 binding affinities at the ha2aar . however , 4f with a bulky group at the n - position did not fit well 
 at the binding site of the ha2a ar . these results show 
 that the h - bond interactions with both ser277 and his278 at the ribose binding site are important for high affinity 
 and potency , and the bulky group at the n - position is 
 unfavorable toward high binding affinity at the ha2aar . in addition , we also performed the molecular docking studies of 
 the analogues 2 and 4a to ha3ar ( see figure 1s in supporting information ) . because the x - ray crystal structure of ha3ar is not 
 available yet , the homology model available in the protein data bank 
 ( pdb code 1oea ) was used . the docking results showed that the binding 
 modes of the analogues in ha3ar are flipped compared to 
 those in ha2aar . in ha3ar , the bulky c2-hexynyl 
 group positions toward the middle of the trans - membrane region exhibited 
 hydrophobic interactions . however , in ha2aar , there is 
 limited space at the bottom of the pocket , making the bulky hexynyl 
 group face toward the extracellular side . the nh2 group 
 at n - position forms the hydrogen bonding with asn6.55 
 in both ha2aar ( asn253 ) and ha3ar ( asn250 ) . interestingly , there is a relatively bigger space near this region 
 in ha3ar , whereas the nh2 group binds tightly 
 in the pocket of ha2aar . it appears according to this docking 
 mode that this is why the n - substituted derivatives ( 4b4i ) maintained their binding affinity 
 at the ha3ar , but not at the ha2aar . the series of truncated n - substituted-(n)-methanocarba - adenosine derivatives , 4a4i with 2-hexynyl group were synthesized in order to examine 
 if this class of nucleosides behaves as the corresponding 4-thioadenosine 
 derivatives . the functionalization at the c2-position of 6-chloropurine 
 derivatives was achieved using lithiation - mediated stannyl transfer 
 and copper- or palladium - catalyzed cross - coupling reactions . it was 
 revealed that all synthesized nucleosides showed very high binding 
 affinity at the ha3ar as well as at the ra3ar , 
 as in the case of the corresponding 4-thioadenosine derivatives , 
 indicating that the hydrophobic n and/or c2 substituent 
 could be tolerable in the binding site of the a3ar . however , 
 only an unsubstituted 6-amino group in the presence of a bulky c2-hexynyl 
 group was associated with high binding affinity at the ha2aar ( compound 4a ) . this trend is similar to that of the 
 corresponding 4-thioadenosine derivatives , serving as dual 
 acting a2a and a3ar ligands . however , the binding 
 affinity at the ha2aar of the truncated ( n)-methanocarba - nucleoside 4a is 14-fold less potent 
 than the truncated 4-thioadenosine derivative 2 . it is attributed to the loss of key hydrogen bonding due to the 
 rigid structure , which was confirmed by a ha2aar molecular 
 docking study . the specific structure activity relationship 
 for this series 
 of conformationally constrained nucleosides might arise from the molecule 
 of lacking in the flexibility required for optimal interaction in 
 the binding site because of the rigidity of ( n)-methanocarba - nucleosides . 
 from this study , n - methyl derivative 4b was discovered as a preferred ha3ar ligand ( low 
 efficacy partial agonist ) with high selectivity , whereas 3-chlorobenzyl 
 derivative 4h was discovered as the most potent / selective 
 ra3ar ligand in this series . the nature of the n substituent in this chemical series 
 modulates the level of ha3ar agonist efficacy ( ranging 
 from nearly 0% to 29% of full agonist ) . for these assays , we used 
 a cho cell with a high level of stable expression of the ha3ar , which would tend to amplify partial agonist action . because even 
 these partial agonists have a relatively low efficacy , they can be 
 expected to behave similarly to full antagonists in some pharmacological 
 models , especially in cases of low receptor expression . a3ar antagonist 1 was recently shown to 
 inhibit unilateral ureteral obstruction - induced renal fibrosis and 
 collagen i upregulation . this suggests 
 that a3ar antagonists might be useful therapeutically to 
 block the development and attenuate the progression of renal fibrosis . tested , 4b exhibited the most 
 potent inhibitory activity ( ic50 = 0.83 m ) against 
 tgf-1-induced collagen i upregulation . these findings indicate 
 that this series of truncated ( n)-methanocarba - nucleoside 
 derivatives acting as partial agonists of low efficacy or as antagonists , 
 which show high binding affinity at the human a3ar , can 
 serve as a good lead for the development of antirenal fibrosis agents . h nmr spectra ( cdcl3 , cd3od , or dmso - d6 ) were recorded on a varian unity invoa 400 mhz instrument . the h nmr data are reported as peak multiplicities : s 
 for singlet , d for doublet , dd for doublet of doublets , t for triplet , 
 q for quartet , brs for broad singlet , and m for multiplet . coupling 
 constants are reported in hertz . c nmr spectra ( cdcl3 , cd3od , or dmso - d6 ) were recorded on a varian unity inova 100 mhz instrument . f nmr spectra ( cdcl3 , cd3od ) were recorded 
 on a varian unity inova 376 mhz instrument . the chemical shifts were 
 reported as parts per million (  ) relative to the solvent peak . infrared spectra were recorded on ft - ir ( fts-135 ) made by bio - rad . elemental analyses ( c , h , and n ) were used to determine 
 the purity of all synthesized compounds , and the results were within 
 0.4% of the calculated values , confirming 95% purity . flash 
 column chromatography was performed on silica gel 60 ( 230400 
 mesh , merck ) . all the anhydrous solvents used 
 were redistilled over cah , p2o5 or sodium / benzophenone 
 prior to the reaction . to a stirred solution of 2-iodo-6-chloropurine ( 1.23 g , 
 4.4 mmol ) and triphenylphosphine ( ph3p ) ( 1.90 g , 4.4 mmol ) 
 in anhydrous thf ( 20 ml ) was added diisopropyl azodicarboxylate ( diad ) 
 ( 1.44 ml , 9.16 mmol ) in thf ( 10 ml ) under n2 at 0 c , 
 and the mixture was stirred at the same temperature for 15 min . to 
 this solution was added a solution of compound 7(19 ) ( 0.5 g , 2.93 mmol ) in thf ( 10 ml ) at 0 c , 
 and the reaction mixture was stirred at room temperature for 16 h. 
 the reaction mixture was concentrated under reduced pressure , and 
 the crude residue was purified by flash silica gel column chromatography 
 ( hexane : etoac = 3:1 ) to give 8 ( 0.85 g , 67% ) as a white 
 solid : mp 9496 c ; uv ( meoh ) max 282 
 nm . ms ( esi ) : [ m + h ] calcd for c14h15clin4o2 , 432.9923 ; found , 432.9931 ; [ ]d = 10.4 ( c 0.2 , meoh ) ; h nmr ( cdcl3 )  0.951.01 ( m , 2 h ) , 
 1.26 ( s , 3 h ) , 1.55 ( s , 3 h ) , 1.631.68 ( m , 1 h ) , 2.122.18 
 ( m , 1 h ) , 4.654.68 ( m , 1 h ) , 5.03 ( s , 1 h ) , 5.355.38 
 ( m , 1 h ) , 8.12 ( s , 1 h ) ; c nmr ( cdcl3 )  
 9.4 , 24.4 , 25.6 , 26.1 , 26.5 , 61.5 , 81.6 , 89.1 , 112.8 , 116.9 , 132.1 , 
 143.9 , 150.9 , 152.1 . ( c14h14clin4o2 ) c , h , n. to a stirred solution of 8 ( 0.30 
 g , 0.69 mmol ) in anhydrous dmf ( 10 ml ) were added tetrakis(triphenylphosphine)palladium 
 ( ( ph3p)4pd ) ( 0.20 g , 0.17 mmol ) , copper iodide 
 ( 0.016 g , 0.08 mmol ) , cesium carbonate ( 0.226 g , 0.69 mmol ) , and 1-hexyne 
 ( 0.07 ml , 0.62 mmol ) at room temperature and the reaction mixture 
 was stirred at 50 c for 5 h. the reaction mixture was cooled 
 to room temperature , quenched with saturated nahco3 ( 5 
 ml ) solution , and diluted with etoac ( 10 ml ) . the organic layer was 
 separated and aqueous layer was further extracted with etoac ( 3  
 5 ml ) . the combined organic layers were washed with brine ( 10 ml ) 
 and water ( 10 ml ) , dried over anhydrous mgso4 , and filtered . the solvent was evaporated under reduced pressure and the crude residue 
 was purified by flash silica gel column chromatography ( hexane : etoac 
 = 3:1 ) to give 9 ( 0.15 g , 56% ) as a white foam : mp 105107 
 c ; uv ( meoh ) max 284 nm . ms ( esi ) : [ m + h]calcd for c20h24clin4o2 , 387.1582 ; found , 387.1586 ; [ ]d = + 2.5 ( c 0.2 , meoh ) . h nmr ( cdcl3 , 400 mhz )  : 0.941.03 
 ( m , 5 h ) , 1.25 ( s , 3 h ) , 1.471.54 ( m , 2 h ) , 1.55 ( s , 3 h ) , 
 1.631.70 ( m , 3 h ) , 2.122.17 ( m , 1 h ) , 2.482.51 
 ( t , 2 h , j = 7.2 hz ) , 4.634.65 ( d , 1 h , j = 5.1 hz ) , 5.13 ( s , 1 h ) , 5.355.38 ( t , 1 h , j = 6.00 hz ) , 8.13 ( s , 1 h ) . c nmr ( cdcl3 , 100 mhz )  : 9.3 , 13.7 , 19.2 , 22.3 , 24.4 , 25.3 , 26.0 , 
 26.5 , 30.2 , 60.7 , 79.7 , 81.4 , 89.1 , 90.9 , 112.6 , 130.8 , 144.2 , 146.3 , 
 151.1 , 151.3 . anal . ( c20h23clin4o2 ) c , h , n. to a stirred ice - cooled solution of 9 ( 0.30 g , 0.77 mmol ) in thf ( 3 ml ) was added 2 n hcl ( 3 ml ) , 
 and the mixture was stirred at 40 c for 16 h. the reaction mixture 
 was neutralized with saturated nahco3 ( 2 ml ) solution and 
 then diluted with etoac ( 10 ml ) . the organic layer was separated , 
 and the aqueous layer was further extracted with etoac ( 2  5 
 ml ) . the combined organic layers were washed with brine ( 10 ml ) , dried 
 over anhydrous mgso4 , filtered , and evaporated under reduced 
 pressure . the crude residue was purified by flash silica gel column 
 chromatography ( hexane : etoac = 2:1 ) to give 10 ( 0.22 
 g , 73% ) as a white solid : mp 120122 c ; uv ( meoh ) max 285 nm . ms ( esi ) : [ m + h ] calcd 
 for c17h20cln4o2 , 347.1269 ; 
 found , 347.1274 ; [ ]d = + 27.0 ( c 0.2 , meoh ) . h nmr ( cdcl3 , 400 mhz ) 
  : 0.840.87 ( m , 1 h ) , 0.940.97 ( t , 3 h , j = 7.2 hz ) , 1.291.32 ( m , 1 h ) , 1.471.53 
 ( m , 2 h ) , 1.621.70 ( m , 3 h ) , 2.102.14 ( m , 1 h ) , 2.472.51 
 ( t , 2 h , j = 7.2 hz ) , 4.054.06 ( d , 1 h , j = 6.0 hz ) , 4.864.89 ( t , 1 h , j = 6.0 hz ) , 5.04 ( s , 1 h ) , 8.20 ( s , 1 h ) . c nmr ( cdcl3 , 100 mhz )  : 7.8 , 14.1 , 19.6 , 19.9 , 22.7 , 24.6 , 30.6 , 
 63.4 , 72.3 , 77.1 , 79.9 , 91.8 , 131.3 , 144.4 , 146.5 , 151.6 , 151.7 . anal . ( c17h19cln4o2 ) c , h , n. a solution of 10 ( 0.05 
 g , 0.42 mmol ) in saturated nh3 in t - buoh 
 ( 5 ml ) was stirred at 110 c for 16 h. the reaction mixture was 
 evaporated , and the residue was purified by flash silica gel column 
 chromatography ( ch2cl2 : meoh = 9 : 1 ) to give 4a ( 0.103 g , 87% ) as a white solid : mp 122124 c ; 
 uv ( meoh ) max 271 nm . ms ( esi ) : [ m + 
 h ] calcd for c17h22n5o2 , 329.1796 ; found , 329.1797 ; [ ]d = + 14.7 ( c 1.75 , meoh ) . h nmr 
 ( cd3od , 400 mhz )  : 0.760.78 ( m , 1 h ) , 0.961.00 
 ( t , 3 h , j = 7.2 hz ) , 1.341.37 ( m , 1 h ) , 
 1.501.70 ( m , 5 h ) , 1.982.01 ( m , 1 h ) , 2.452.48 
 ( t , 2 h , j = 7.2 hz ) , 3.863.88 ( d , 1 h , j = 6.8 hz ) , 4.664.69 ( t , 1 h , j = 5.6 hz ) , 4.83 ( s , 1 h ) , 8.24 ( s , 1 h ) . c nmr ( cd3od )  : 8.2 , 14.1 , 19.6 , 19.7 , 23.2 , 24.7 , 31.6 , 64.0 , 
 73.0 , 77.4 , 81.3 , 88.6 , 120.3 , 141.4 , 147.9 , 157.2 , 167.2 . anal . ( c17h21n5o2 ) c , h , n. to a solution of 10 ( 1 equiv ) in 
 etoh ( 10 ml ) were added et3n ( 3 equiv ) and the appropriate 
 amine ( 1.5 equiv ) at room temperature , and the mixture was stirred 
 at 90 c for 18 h in a steel bomb . the reaction mixture was evaporated 
 and the residue was purified by flash silica gel column chromatography 
 ( ch2cl2/meoh = 12:1 ) to give 4b4i . yield : 75% ; white solid ; mp 118120 
 c ; uv ( meoh ) max 273 nm . ms ( esi ) : [ m + h]calcd for c18h24n5o2 ,  342.1925 ; found , 342.1925 ; [ ]d = + 35.5 ( c 0.2 , meoh ) . h nmr ( cd3od , 400 mhz )  : 0.730.79 
 ( m , 1 h ) , 0.961.00 ( t , 3 h , j = 7.2 hz ) , 
 1.341.38 ( m , 1 h ) , 1.491.71 ( m , 5 h ) , 1.952.01 
 ( m , 1 h ) , 2.452.49 ( t , 2 h , j = 7.2 hz ) , 
 3.11 ( brs , 3 h ) , 3.843.86 ( d , 1 h , j = 6.8 
 hz ) , 4.644.67 ( t , 1 h , j = 5.6 hz ) , 4.82 
 ( s , 1 h ) , 8.16 ( s , 1 h ) . c nmr ( cd3od , 100 
 mhz )  : 7.9 , 13.9 , 19.6 , 19.6 , 23.2 , 24.6 , 27.8 , 31.6 , 63.8 , 
 73.0 , 77.2 , 81.6 , 87.9 , 120.2 , 140.3 , 148.1 , 149.4 , 156.6 . anal . ( c18h23n5o2 ) c , h , n. yield : 76% ; white solid ; mp 98100 
 c ; uv ( meoh ) max 273 nm . ms ( esi ) : [ m + h]calcd for c19h26n5o2 , 356.2081 ; found , 356.2083 ; [ ]d = + 8.5 ( c 0.2 , meoh ) ; h nmr ( cd3od , 400 mhz )  : 0.740.77 
 ( m , 1 h ) , 0.961.00 ( t , 3 h , j = 7.2 hz ) , 
 1.271.31 ( t , 3 h , j = 7.2 hz ) , 1.341.37 
 ( m , 1 h ) , 1.501.70 ( m , 5 h ) , 1.962.00 ( m , 1 h ) , 2.452.50 
 ( t , 2 h , j = 7.2 hz ) , 3.62 ( brs , 2 h ) , 3.843.85 
 ( d , 1 h , j = 6.8 hz ) , 4.644.67 ( t , 1 h , j = 5.6 hz ) , 4.81 ( s , 1 h ) , 8.16 ( s , 1 h ) . c nmr ( cd3od , 100 mhz )  : 7.9 , 14.0 , 15.1 , 19.5 , 
 19.2 , 23.2 , 24.6 , 31.6 , 36.6 , 63.8 , 73.1 , 77.1 , 81.6 , 87.9 , 119.9 , 
 140.3 , 148.1 , 149.5 , 155.9 . anal . ( c19h25n5o2 ) c , h , n. yield : 68% ; white solid ; mp 9496 
 c ; uv ( meoh ) max 275.0 nm . ms ( esi ) : [ m + h]calcd for c20h26n5o2 , 356.2081 ; found , 368.2078 ; [ ]d = + 20.2 ( c 0.2 , meoh ) . h nmr ( cd3od , 400 mhz )  : 0.610.65 
 ( m , 2 h ) , 0.730.79 ( m , 1 h ) , 0.860.91 ( m , 2 h ) , 0.961.00 
 ( t , 3 h , j = 7.2 hz ) , 1.331.38 ( m , 1 h ) , 
 1.481.71 ( m , 5 h ) , 1.952.01 ( m , 1 h ) , 2.462.50 
 ( t , 2 h , j = 7.2 hz ) , 3.05 ( brs , 1 h ) , 3.843.86 
 ( d , 1 h , j = 6.8 hz ) , 4.654.67 ( t , 1 h , j = 5.6 hz ) , 4.83 ( s , 1 h ) , 8.18 ( s , 1 h ) . c nmr ( cd3od , 100 mhz )  : 7.7 , 7.9 , 14.0 , 19.6 , 
 19.7 , 23.2 , 24.6 , 24.8 , 31.6 , 63.8 , 73.0 , 77.2 , 81.6 , 88.2 , 120.1 , 
 140.7 , 148.0 , 149.8 , 157.1 . anal . ( c20h25n5o2 ) c , h , n. yield : 65% ; white solid ; mp 9597 
 c ; uv ( meoh ) max 275 nm . ms ( esi ) : [ m+h ] calcd for c22h30n5o2 , 396.2394 ; found , 396.2400 ; [ ]d = + 21.4 ( c 0.2 , meoh ) . h nmr ( cd3od , 400 mhz )  : 0.740.78 ( m , 1 
 h ) , 0.961.00 ( t , 3 h , j = 7.2 hz ) , 1.341.37 
 ( m , 1 h ) , 1.501.83 ( m , 10 h ) , 1.961.99 ( m , 2 h ) , 2.062.12 
 ( m , 2 h ) , 2.452.48 ( t , 2 h , j = 7.2 hz ) , 
 3.833.85 ( d , 1 h , j = 6.4 hz ) , 4.60 ( brs , 
 1 h ) , 4.644.67 ( t , 1 h , j = 5.6 hz ) , 4.81(s , 
 1 h ) , 8.18 ( s , 1 h ) . c nmr ( cd3od , 100 mhz ) 
  : 7.9 , 13.9 , 19.55 , 19.60 , 23.2 , 24.6 , 24.7 , 31.6 , 34.0 , 53.5 , 
 63.8 , 73.0 , 77.1 , 81.7 , 87.9 , 119.8 , 140.3 , 148.1 , 149.5 , 155.5 . anal . ( c22h29n5o2 ) c , h , n. yield : 88% ; white solid ; mp 128130 
 c ; uv ( meoh ) max 274 nm . ms ( esi ) : [ m + h ] calcd for c24h27in5o2 , 544.1204 ; found , 544.1212 ; [ ]d = + 30.3 ( c 0.2 , meoh ) . h nmr ( cd3od , 400 mhz )  : 0.730.79 ( m , 1 
 h ) , 0.961.00 ( t , 3 h , j = 7.2 hz ) , 1.341.37 
 ( m , 1 h ) , 1.501.58 ( m , 2 h ) , 1.601.72 ( m , 3 h ) , 1.962.01 
 ( m , 1 h ) , 2.452.48 ( t , 2 h , j = 7.2 hz ) , 
 3.853.87 ( d , 1 h , j = 6.4 hz ) , 4.654.67 
 ( t , 1 h , j = 5.6 hz ) , 4.76 ( brs , 2 h ) , 4.83 ( s , 1 
 h ) , 7.077.11 ( t , 1 h , j = 7.6 hz ) , 7.397.41 
 ( m , 1 h ) , 7.597.61 ( m , 1 h ) , 7.80 ( s , 1 h ) , 8.19 ( s , 1 h ) . c nmr ( cd3od , 100 mhz )  : 7.9 , 14.0 , 19.5 , 
 19.6 , 23.2 , 24.6 , 31.6 , 44.4 , 63.8 , 73.1 , 77.1 , 81.7 , 88.1 , 94.9 , 
 120.1 , 128.2 , 131.4 , 137.4 , 137.9 , 140.7 , 143.1 , 147.9 , 149.9 , 155.7 . 
 anal . ( c24h26in5o2 ) c , 
 h , n. yield : 90% ; white solid ; mp 124126 
 c ; uv ( meoh ) max 275 nm . ms ( esi ) : [ m + h]calcd for c24h27brn5o2 , 496.1343 ; found , 496.1350 ; [ ]d = + 21.2 ( c 0.2 , meoh ) . h nmr ( cd3od , 400 mhz )  : 0.750.77 
 ( m , 1 h ) , 0.961.00 ( t , 3 h , j = 7.2 hz ) , 
 1.341.37 ( m , 1 h ) , 1.501.56 ( m , 2 h ) , 1.601.71 
 ( m , 3 h ) , 1.971.99 ( m , 1 h ) , 2.452.48 ( t , 2 h , j = 7.2 hz ) , 3.853.87 ( d , 1 h , j = 6.8 hz ) , 4.654.68 ( t , 1 h , j = 5.6 hz ) , 
 4.80 ( brs , 2 h ) , 4.83 ( s , 1 h ) , 7.227.26 ( m , 1 h ) , 7.367.41 
 ( m , 2 h ) , 7.59 ( s , 1 h ) , 8.19 ( s , 1 h ) . c nmr ( cd3od , 100 mhz )  : 7.9 , 14.0 , 19.2 , 19.6 , 23.2 , 24.6 , 31.6 , 
 44.5 , 63.9 , 73.1 , 77.2 , 81.6 , 88.0 , 120.1 , 123.5 , 127.6 , 131.3 , 131.4 , 
 131.8 , 140.7 , 143.2 , 148.0 , 150.0 , 155.8 . anal . ( c24h26brn5o2 ) c , h , n. yield : 90% ; white solid ; 
 mp 109111 c ; uv ( meoh ) ms ( esi ) : [ m + h ] calcd for c24h27cln5o2 , 452.1848 ; found , 452.1842 ; 
 [ ]d = + 13.1 ( c 0.2 , 
 meoh ) . h nmr ( cd3od , 400 mhz )  : 0.730.79 
 ( m , 1 h ) , 0.901.00 ( t , 3 h , j = 7.2 hz ) , 
 1.341.37 ( m , 1 h ) , 1.501.72 ( m , 5 h ) , 1.962.04 
 ( m , 1 h ) , 2.442.48 ( t , 2 h , j = 7.2 hz ) , 
 3.853.87 ( d , 1 h , j = 6.8 hz ) , 4.654.68 
 ( t , 1 h , j = 5.6 hz ) , 4.80 ( brs , 2 h ) , 4.83 ( s , 1 
 h ) , 7.247.34 ( m , 3 h ) , 7.427.43 ( m , 1 h ) , 8.19 ( s , 
 1 h ) . c nmr ( cd3od , 100 mhz )  : 7.9 , 
 14.0 , 19.5 , 19.6 , 23.1 , 24.6 , 31.6 , 44.6 , 63.8 , 73.0 , 77.1 , 81.7 , 
 88.1 , 120.5 , 127.1 , 128.3 , 128.8 , 131.1 , 135.4 , 140.7 , 142.9 , 147.9 , 
 149.9 , 155.8 . anal . ( c24h26cln5o2 ) c , h , n. yield : 70% ; white solid ; 
 mp 99101 c ; uv ( meoh ) max 273 nm . ms ( esi ) : [ m + h]calcd for c24h27fn5o2 , 436.2143 ; found , 436.2141 ; 
 [ ]d = + 2.5 ( c 0.2 , 
 meoh ) . h nmr ( cd3od , 400 mhz )  : 0.730.79 
 ( m , 1 h ) , 0.961.00 ( t , 3 h , j = 7.2 hz ) , 
 1.351.37 ( m , 1 h ) , 1.491.71 ( m , 5 h ) , 1.952.01 
 ( m , 1 h ) , 2.442.47 ( t , 2 h , j = 7.2 hz ) , 
 3.853.87 ( d , 1 h , j = 6.4 hz ) , 4.654.67 
 ( t , 1 h , j = 5.6 hz ) , 4.80 ( brs , 2 h ) , 4.83 ( s , 1 
 h ) , 6.946.99 ( m , 1 h ) , 7.127.15 ( m , 1 h ) , 7.207.22 
 ( m , 1 h ) , 7.307.35 ( m , 1 h ) , 8.18 ( s , 1 h ) . c 
 nmr ( cd3od , 100 mhz )  : 7.9 , 13.9 , 19.5 , 19.6 , 23.1 , 
 24.6 , 31.6 , 44.6 , 63.8 , 73.0 , 77.1 , 81.7 , 88.0 , 115.3 , 115.5 , 120.0 , 
 124.5 , 131.2 , 131.3 , 140.7 , 143.5 , 148.0 , 150.0 , 155.8 ( c24h26fn5o2 ) c , h , n. compound 6 ( 0.50 g , 2.93 mmol ) was converted to 11 ( 0.63 
 g , 70% ) as a white solid according to the same procedure used in the 
 preparation of 8 : mp 8486 c ; uv(ch2cl2 ) max 265 nm . ms ( esi ) : [ m + h ] calcd for c14h16cln4o2 , 307.0956 ; found , 307.0951 ; [ ]d = 40.5 ( c 0.2 , meoh ) . h nmr ( cdcl3 , 400 mhz )  : 0.961.04 
 ( m , 2 h ) , 1.24 ( s , 3 h ) , 1.56 ( s , 3 h ) , 1.701.75 ( m , 1 h ) , 
 2.132.19 ( m , 1 h ) , 4.684.70 ( m , 1 h ) , 5.08 ( s , 1 h ) , 
 5.365.39 ( t , 1 h , j = 6.8 hz ) , 8.18 ( s , 1 
 h ) , 8.78 ( s , 1 h ) . c nmr ( cdcl3 , 100 mhz ) 
  : 9.3 , 24.3 , 25.5 , 26.0 , 26.1 , 61.4 , 81.4 , 89.1 , 112.6 , 132.1 , 
 143.9 , 151.3 , 151.4 , 152.3 . anal . ( c14h15cln4o2 ) c , h , n. to a stirred solution of 2,2,6,6-tetramethylpiperidine 
 ( tmp , 1.36 ml , 8.00 mmol ) in dry hexane ( 5 ml ) and dry thf ( 10 ml ) 
 was added n - butyllithium ( 5.6 ml , 1.5 m solution 
 in hexanes , 8.47 mmol ) dropwise at 78 c over 30 min , 
 and the mixture was stirred at the same temperature for 1 h. to this 
 mixture , a solution of 11 ( 0.50 g , 1.60 mmol ) in dry 
 thf ( 10 ml ) was added dropwise , and the mixture was stirred at 78 
 c for 30 min . tributyltin chloride ( 1.74 ml , 8.0 mmol ) was successively 
 added dropwise to the dark reaction mixture , and the mixture was stirred 
 at the same temperature for another 2 h. the resulting dark solution 
 was quenched by dropwise addition of a saturated aqueous nh4cl solution ( 15 ml ) . after the mixture was stirred at room temperature 
 for 15 h , the mixture was diluted with ch2cl2 ( 15 ml ) . the organic layer was washed with saturated nahco3 solution , dried over anhydrous mgso4 , and filtered . the crude syrup was 
 purified by flash silica gel column chromatography ( hexane / etoac = 
 5:1 ) to give 12 ( 0.68 g , 70% ) as a colorless syrup : uv 
 ( meoh ) max 269 nm . ms ( esi ) : [ m + h ] calcd for c26h42cln4o2sn , 597.2011 ; found , 595.2020 ; [ ]d = 36.5 ( c 0.2 , meoh ) . h nmr ( cdcl3 )  : 0.850.97 ( m , 11 h ) , 1.131.38 
 ( m , 15 h ) , 1.521.67 ( m , 10 h ) , 2.032.13(m , 1 h ) , 4.794.81 
 ( d , 1 h , j = 7.2 hz ) , 4.96 ( s , 1 h ) , 5.43- 5.47 ( m , 
 1 h ) , 8.01 ( s , 1 h ) . c nmr ( cd3od )  : 
 9.4 , 10.9 , 24.3 , 25.8 , 26.1 , 26.7 , 27.4 , 29.1 , 62.1 , 81.9 , 89.1 , 112.4 , 
 131.0 , 142.9 , 149.8 , 150.5 , 182.1 . to 
 a stirred solution of 12 ( 0.40 g , 0.60 mmol ) and copper 
 iodide ( 0.013 g , 0.06 mmol ) in anhydrous dmf ( 5 ml ) was added 1-iodohexyne 
 ( 0.124 ml , 0.6 mmol ) in dmf ( 4 ml ) dropwise via syringe pump over 
 a period of 1 h at room temperature , and the reaction mixture was 
 stirred at 50 c for 5 h. the reaction mixture was cooled to 
 room temperature , quenched with saturated nahco3 ( 5 ml ) 
 solution , and diluted with etoac ( 10 ml ) . the organic layer was separated , 
 and the aqueous layer was further extracted with etoac ( 3  5 
 ml ) . the combined organic layers were washed with brine ( 5 ml ) and 
 water ( 5 ml ) , dried over anhydrous mgso4 , and filtered . the solvent was evaporated under reduced pressure , and the residue 
 was purified by flash silica gel column chromatography ( hexane / etoac 
 = 3 : 1 ) to give 9 ( 0.155 g , 60% ) as a white foam , whose 
 spectral data were identical to those of authentic sample . cho cells expressing 
 the recombinant ha1 or a3r and hek-293 cells 
 expressing the ha2aar were cultured in dulbecco s 
 modified eagle s medium ( dmem ) and f12 ( 1:1 ) supplemented with 
 10% fetal bovine serum , 100 u / ml penicillin , 100 g / ml streptomycin , 
 and 2 mol / ml glutamine . after homogenization and 
 suspension , cells were centrifuged at 500 g for 10 
 min , and the pellet was resuspended in 50 mm trishcl buffer 
 ( ph 7.4 ) containing 10 mm mgcl2 . the suspension was homogenized 
 with an electric homogenizer for 10 s and was then recentrifuged at 
 20 000 g for 20 min at 4 c . the resultant pellets 
 were resuspended in buffer containing 3 u / ml adenosine deaminase , 
 and the suspension was stored at 80 c until the binding 
 experiments . the protein concentration was measured using the bradford 
 assay . for binding to the ha1ar , 50 l of increasing 
 concentrations of a test ligand and 50 l of [ h]14 ( 2 nm , perkinelmer , boston , ma ) were incubated with membranes 
 ( 40 g / tube ) from cho cells stably expressing the ha1 ar at 25 c for 60 min in 50 mm trishcl buffer ( ph 7.4 ; 
 mgcl2 , 10 mm ) in a total assay volume of 200 l . nonspecific binding was determined using 10 
 m of n - cyclopentyladenosine 
 ( cpa , 17 ) . for ha2aar binding , membranes ( 20 
 g / tube ) from hek-293 cells stably expressing the ha2aar were incubated at 25 c for 60 min with a final concentration 
 of 15 nm [ h]15 ( american radiolabeled chemicals , 
 inc . , st . louis , mo ) in a mixture containing 50 l of increasing 
 concentrations of a test ligand and 200 l of 50 mm trishcl , 
 ph 7.4 , containing 10 mm mgcl2 . compound 16 ( 10 m ) was used to define nonspecific binding . the reaction 
 was terminated by filtration with gf / b filters . filters for a1 and a2aar binding were placed in scintillation 
 vials containing 5 ml of hydrofluor scintillation buffer and counted 
 using a perkinelmer tricarb 2810tr liquid scintillation analyzer . each tube in the competitive binding assay contained 100 l 
 membrane suspension ( 20 g protein ) , 50 l [ i]13 ( 1.0 nm , perkinelmer , boston , ma ) , and 50 l 
 of increasing concentrations of the test ligands in trishcl 
 buffer ( 50 mm , ph 8.0 ) containing 10 mm mgcl2 , 1 mm edta . binding reactions were terminated by filtration 
 through whatman gf / b filters under reduced pressure using a mt-24 
 cell harvester ( brandell , gaithersburgh , md , usa ) . after centrifugation and resuspension in medium , cells were plated 
 in 24-well plates in 0.5 ml medium . after 24 h , the medium was removed , 
 and cells were washed three times with 1 ml dmem , containing 50 mm n-(2-hydroxyethyl)piperazine - n-2-ethanesulfonic 
 acid ( hepes ) , ph 7.4 . cells were then treated with agonists and/or 
 test compounds in the presence of rolipram ( 10 m ) and adenosine 
 deaminase ( 3 units / ml ) . for assay of the ha3ar but not 
 the ha2bar , forskolin ( 10 m ) was added to the medium 
 after 45 min . after the addition of forskolin , the incubation was 
 continued an additional 15 min . the reaction was terminated upon removal 
 of the supernatant , and cells were lysed upon the addition of 200 
 l of 0.1 m ice - cold hcl . the cell lysate was resuspended and 
 stored at 20 c . for determination of camp production , protein kinase a ( pka ) was incubated with [ h]camp ( 2 nm ) 
 in k2hpo4/edta buffer ( k2hpo4 , 150 mm ; edta , 10 mm ) , 20 l of the cell lysate , and 
 30 l 0.1 m hcl or 50 l of camp solution ( 016 
 pmol/200 l for standard curve ) . bound radioactivity was separated 
 by rapid filtration through whatman gf / c filters and washed once with 
 cold buffer . binding and 
 functional parameters 
 were calculated using prism 5.0 software ( graphpad , san diego , ca , 
 usa ) . ic50 values obtained from competition curves were 
 converted to ki values using the cheng immortalized murine proximal tubular 
 cells ( mprox24 ) derived from microdissected proximal tubular segments 
 of c57bl6/j adult mouse kidneys were supplied from dr . mprox24 were 
 maintained in dmem supplemented with 10% fetal calf serum ( fcs ; gibco ) , 
 100 u / ml penicillin , 100 g / ml streptomycin , and 44 mm nahco3 under 5% co2 environment at 37 c . cells 
 were cultured in 6-well plate for mrna analysis . at next day after 
 seeding cell on 6-well plate , the cultured cells were growth - arrested 
 with a dmem medium containing 0.15% fcs for 24h . each synthesized 
 compound was dissolved in dmso to 50 mm and it was diluted to 20 mm , 
 10 mm , and 1 mm . after cells were pretreated with the synthesized 
 compound dissolved in dmem containing 0.15% fcs for 1 h , treated with 
 recombinant human transforming growth factor-1 ( htgf 1 , 
 r&d systems ) 5 ng / ml for 6 h. total rna was extracted from mprox24 
 using trizol ( invitrogen ) according to the standard protocol . mrna 
 expressions were measured by real - time pcr using steponeplus ( applied 
 biosystems ) with 20 l reaction volume consisting of cdna transcripts , 
 primer pairs , and sybr green pcr master mix ( applied biosystems ) . the sequences of mouse collagen 
 i1 primer pairs are 5-gaacatcacctacca ctgca-3 
 and 5-gttgggatggagggagttta-3. the x - ray crystal structure of the 
 human a2a ar in complex with an agonist , 16 ( pdb i d : 2ydv ) was retrieved from the 
 protein data bank ( pdb ) and prepared using the protein preparation 
 wizard in maestro v9.2 ( schrdinger , llc , ny , u.s.a . ) , where 
 water and ions were removed , hydrogen atoms were added and optimized , 
 and then the protein was minimized using the optimized potentials 
 for liquid simulations - all atom ( opls - aa ) 2005 force field . the structures 
 of the molecules were sketched in the maestro and energy minimized 
 using impact v5.7 ( schrdinger , llc , ny , u.s.a . ) considering 
 conjugant gradient algorithm with the maximum minimization cycles 
 of 1000 and convergence gradient of 0.001 kj / mol -  . the four 
 docking programs glide - sp ( standard precision ) , glide - xp ( extra precision ) , 
 gold , and surflex - dock showed consistent results , and the glide - xp 
 docking results are presented . the receptor grid box with 10  
 around the centroid of the cocrystallized neca was generated . the 
 best binding poses of 2 , 4a , and 4f were selected for the calculation of the receptor  ligand 
 binding free energy ( gbind ) using 
 prime molecular mechanics - generalized born surface area ( mm - gbsa ) 
 module ( schrdinger , llc , ny , u.s.a . ) . the ballesteros  weinstein 
 double - numbering system is used to describe 
 the transmembrane ( tm ) location of the amino acids . along with numbering 
 their positions in the primary amino acid sequence , the residues have 
 numbers in parentheses ( x.yz ) that indicate their position in each 
 transmembrane ( tm ) helix ( x ) , relative to a conserved reference residue 
 in that tm helix ( yz ) .
truncated n6-substituted-(n)-methanocarba - adenosine derivatives 
 with 2-hexynyl substitution 
 were synthesized to examine parallels with corresponding 4-thioadenosines . 
 hydrophobic n6 and/or c2 substituents were tolerated in 
 a3ar binding , but only an unsubstituted 6-amino group with 
 a c2-hexynyl group promoted high ha2aar affinity . a small 
 hydrophobic alkyl ( 4b and 4c ) or n6-cycloalkyl group ( 4d ) showed 
 excellent binding affinity at the ha3ar and was better 
 than an unsubstituted free amino group ( 4a ) . 
 a3ar affinities of 3-halobenzylamine derivatives 4f4i did not differ significantly , with ki values of 7.816.0 nm . 
 n6-methyl derivative 4b ( ki = 4.9 nm ) was a highly selective , low efficacy partial a3ar agonist . 
 all compounds were screened for renoprotective effects 
 in human tgf-1-stimulated mprox tubular cells , a kidney fibrosis 
 model . 
 most compounds strongly inhibited tgf-1-induced collagen 
 i upregulation , and their a3ar binding affinities were 
 proportional to antifibrotic effects ; 4b was most potent 
 ( ic50 = 0.83 m ) , indicating its potential as a good 
 therapeutic candidate for treating renal fibrosis .