chronic neuropathic pain ( np ) is a widespread 
 condition that is often associated with diabetes , cancer , injury , 
 exposure to toxic substances , and a variety of other diseases . when it occurs subsequent to cancer chemotherapy , it often necessitates 
 the discontinuation of a life - saving treatment . the currently used 
 therapies for np are poorly efficacious and suffer from serious side 
 effects , ranging from liver toxicity to addiction and personality 
 changes . in many cases , the therapy involves drugs developed for a 
 different condition that were incidentally found to reduce np , e.g. , 
 biogenic amine reuptake inhibitors such as the antidepressant amitriptyline 
 or anticonvulsant drugs such as gabapentin . opioids , which are effective 
 against acute pain , are not the first line of treatment for chronic 
 np , both because of addiction liability and low efficacy . thus , there is an unsolved medical need for chronic 
 neuropathic pain treatment that necessitates the exploration of novel 
 mechanisms , such as purinergic therapy by agonists of adenosine receptors 
 ( ars ) , inhibitors of adenosine kinase , and modulators of nucleotidases . the a3 subtype of the ars ( a3ar ) is expressed 
 in low levels in many tissues and cell types , including neurons and 
 glial cells , and has recently become a promising target for treating 
 np . a3ar agonists , such as nucleosides 15 ( chart 1 ) , are efficacious 
 in models of inflammatory disease , cancer , stroke , cardiac ischemia , 
 and other conditions . prototypical a3ar agonists , ribosides n-(3-iodobenzyl)-5-n - methylcarboxamidoadenosine ( ib - meca , 1 ) and 
 its 2-chloro analogue 2 and thioriboside 3 are efficacious in vivo . prototypical agonists 1 and 2 are advancing to phase ii and iii clinical trials 
 for psoriasis , rheumatoid arthritis , and hepatocellular carcinoma . a3ar agonists 4 and 5 additionally 
 have a conformationally rigid ribose substitution consisting of a 
 bicyclo[3.1.0]hexane ( methanocarba ) ring system that maintains a receptor - preferred 
 north ( n ) conformation . a3ar agonists of general structure 6 furthermore contain a rigid c2-phenylethynyl 
 extension that enhances selectivity , especially in the ( n)-methanocarba 
 series . compounds 1 , 2 , and 4 were shown to be active in reducing or preventing the development 
 of mechanically- and chemotherapy - induced np in mice and rats . the specific example of 6a in which 
 ar is 3,4-difluorophenyl was also shown to be effective in in vivo 
 models of np . these selective nucleoside 
 a3ar agonists potently and dose dependently reduce chronic 
 np and also augment the effects of commonly used agents for this condition , 
 such as opiates . a3ar agonists reduce the np associated 
 with administration of various chemotherapeutic agents representing 
 three different mechanisms of anticancer action . although agonists of the a1 and a2aars also have analgesic properties , they additionally have potent 
 cardiovascular side effects such as changes in heart rate and blood 
 pressure , which are not as strongly 
 associated with the activation of the a3ar . thus , we seek orally bioavailable and highly selective a3ar agonists that have activity against chronic np of distinct etiologies . we have approached this problem by designing 
 and synthesizing new analogues of our previously reported chemical 
 class of 2-alkynyl-(n)-methanocarba adenosine 5-methyluronamides that might display improved and more drug - like 
 physicochemical properties than previous agents . there are both peripheral 
 and central mechanistic components to the observed protection against 
 np by a3ar agonists . thus , 
 the ability to cross the blood  brain barrier as well as oral 
 bioavailability would be desirable in a clinical candidate . the 2-phenylethynyl groups that we previously reported to enhance 
 the selectivity of ( n)-methanocarba adenosine derivatives as a3ar agonists are the focus of this structure the current 
 strategy is to expand structurally the family of a3ar agonists 
 for treatment of np and other diseases . as shown in chart 1 , in this study ring structures appended to the 
 2-ethynyl group were varied to encompass diverse heterocyclic and 
 other aryl groups and cycloalkyl groups . the reference compound 6a is highly efficacious in reversing chronic np , but it has nonoptimal physicochemical properties ( lipophilicity 
 and mw ) for drug development . therefore , a group frequently included 
 at the n position of a3ar 
 agonists , n-(3-halobenzyl ) , was replaced 
 with a smaller n - methyl group , which 
 we already demonstrated to maintain human ( h ) a3ar binding 
 affinity in this chemical series . a beneficial 
 effect of the n - methyl substitution was 
 to reduce the molecular weight to below 500 , which brings the analogues 
 in closer compliance to lipinski s guidelines for bioavailable 
 compounds . there was a cost associated 
 with this structural shift , i.e. , a species difference in receptor 
 affinity , which was decreased at the mouse ( m ) a3ar in 
 comparison to the ha3ar . nevertheless , 
 when administered po in mice , these derivatives consistently displayed 
 in vivo activity reflective of a3ar activation , with varying 
 degrees of efficacy at a single dose . we have used an in vivo 
 phenotypic drug discovery ( pdd ) screen that reflects both pharmacokinetic 
 and pharmacodynamics factors to demonstrate oral bioavailability and 
 high efficacy of several of the newly synthesized nucleoside analogues . the pharmaceutical industry is rediscovering the predictive value 
 of physiology - based pdd strategies . the 
 initial screening used was the bennett mouse model of chronic neuropathic 
 pain resulting from constriction injury ( cci ) of the sciatic nerve , in which the compound was administered by oral 
 gavage ( po ) rather than intraperitoneally ( ip ) , as we reported in 
 earlier studies of a3ar agonists in the same model . although the expectation of a3ar 
 selectivity of the newly synthesized analogues was high based on existing 
 sar , they were additionally subjected to measurement of their ar interactions . binding and functional assays at the heterologously expressed h and 
 m ars were performed , as well as screening of selected compounds at 
 off - target sites . the a3ar 
 is a g protein - coupled receptor ( gpcr ) of the rhodopsin - like family 
 a and as such is amenable to molecular modeling based on homology 
 to closely related gpcrs , specifically the structures 
 of the agonist - bound active - like a2aar . novel structures that were efficacious in vivo and bound potently 
 to the receptor were docked to an a3ar homology model . thus , in vitro pharmacology and molecular modeling were used to help 
 interpret the positive in vivo results obtained for promising analogues . the novel ( n)-methanocarba adenosine analogues 1418 and 2038 ( table 1 ) were synthesized and tested in a phenotypic screen 
 as described below . charged compounds were not included because they 
 might be impaired in the ability to cross the blood  brain barrier . related previously reported reference compounds included in table 1 are n-(3-chlorobenzyl)-3,4-difluorophenyl 6a , n - methyl - c2- halophenylethynyl 813 and simple 
 phenyl 7a , biphenyl 7b , and 2-pyridyl 19 analogues . the new nucleosides are all n - methyl compounds , for which the c2 
 substituent consists of the following substituted ethynyl groups : 
 phenyl 1418 , nitrogen heterocycle 1926 , furan 2730 , thiophene 3135 , ferrocene 
 ( fe(c5h5)2 ) 36 , and 
 cycloalkyl 37 and 38 . for comparison with 
 the new analogues , structures and binding affinities of previously 
 reported potent a3ar agonists are provided . binding in membranes 
 prepared from cho or hek293 ( a2a only ) cells stably expressing 
 one of three har subtypes . the binding affinity for ha1 , a2a , and a3ars was expressed as ki values ( n = 34 ) , measured using 
 agonist radioligands [ h]n - r - phenylisopropyladenosine 65 , [ h]2-[p-(2-carboxyethyl)phenyl - ethylamino]-5-n - ethylcarboxamido - adenosine 66 , or [ i]n-(4-amino-3-iodobenzyl)adenosine-5-n - methyl - uronamide 67 , respectively . nonspecific 
 binding was determined using 68 ( 10 m at hars , 
 100 m at mars ) . the synthetic methods ( scheme 1 ) followed the route reported earlier starting with l - ribose . 6-chloro 5-ethyl ester intermediate 39 was 
 treated with five equivalents of methylamine hydrochloride in the presence of triethylamine 
 followed by a 40% methylamine solution ( aqueous ) at room temperature 
 to provide intermediate 2-iodo 5-methylamide 40 for the n - methyl derivatives . then , 40 was subjected to sonogashira coupling with the appropriate 
 aryl- or cycloalkylacetylene in the presence of pdcl2(ph3p)2 , cui , and triethylamine to give protected intermediates 4163 . finally , hydrolysis of the 2,3-isopropylidene 
 protecting group afforded the nucleoside target compounds 1418 and 2038 for biological testing . reagents : ( i ) menh2hcl , et3n , meoh ; ( ii ) 40% menh2 , meoh , rt ; ( iii ) hccr , pd(pph3)2cl2 , cui , et3n , dmf , rt ; ( iv ) 10% tfa , meoh , 70 c . the n - methyl ( n)-methanocarba 
 derivatives were tested in mice for the ability to reverse the development 
 of neuropathic pain in a standard model of cci . each nucleoside was administered orally ( po ) at the time 
 peak pain was reached , i.e. , on day 7 following constriction of the 
 sciatic nerve . measurements of paw withdrawl threshold ( value in g 
 increases with pain protection ) were made between 30 min and 5 h post 
 drug . the results indicating maximal protective effect ( emax as a percentage of complete reversal at peak protection ) 
 at a standard dose ( 3 mol / kg , roughly 1.21.6 mg / kg ) 
 and its duration ( based on the efficacy remaining at 3 h ) are presented 
 in table 2 , and time curves at three doses 
 for three selected compounds that achieved near maximal reversal of 
 np are shown in figure 1 . however , previously 
 reported halogenated c2-phenylethynyl derivatives812 did not 
 reach full reversal of np ( 4072% efficacy ) , but a 3,4-difluoro 
 analogue 13 was fully efficacious . methoxyphenyl- ( 1416 , with the o - isomer 14 having the highest efficacy ) , 3-trifluoromethyl- 17 , and 4-hydroxymethyl - phenyl 18 derivatives 
 did not exceed that range . all of the pyridyl derivatives ( 1921 ) were of low efficacy ( 2941% ) . a 
 2-(pyrimidin-2-yl)ethynyl analogue 22 , 2-(pyrazin-2-yl)ethynyl 
 analogue 23 , 2-(n - methylpyrazol-2-yl)ethynyl 
 analogue 26 , 2-(fur-2-yl)ethynyl analogue 27 , 2-(benzofur-2-yl)ethynyl analogue 30 , 2-(thien-2-yl)ethynyl 
 analogue 32 , 2-(5-chlorothien-2-yl)ethynyl derivative 33 and its 5-br analogue 34 , and an unusual organometallic 
 ( ferrocene ) derivative 36 were among the most efficacious 
 in the cci model with emax in the range 
 of 87100% . this protection exceeded that produced by a previously 
 characterized a3ar agonist , 2-(3,4-difluorophenylethynyl)-n-3-chlorobenzyl derivative 6a administered 
 po . thiazole analogue 35 was much less effective in vivo 
 than the structurally similar thiophene 32 . protection 
 against hind paw allodynia in mice by n - methyl derivatives 23 , 27 , and 33 at three doses ( po ) following cci of the sciatic nerve . there was 
 no effect on the ipsilateral side ( figure s1 , 
 supporting information ) . effect 
 shown for ipsilateral hind paw ; there is no effect on the contralateral 
 side . calculated using chembiodraw , 
 version 13.0 . the duration was indicated 
 by the % protection remaining at the 3 h time point . the duration 
 of effect of 5-chlorothienyl derivative 33 and ferrocene 
 derivative 36 appeared to be longer than for the other 
 analogues , with approximately 80% protection remaining at 3 h. the 
 effects of compounds 30 and 34 were also 
 long lasting , with roughly half of the peak efficacy remaining at 
 3 h. thus , on the basis of either in vivo emax or duration , the most favorable of the new compounds for in vivo 
 application were 23 ( ki at 
 ha3ar , nm 1.8 ) , 27 ( 0.6 ) , 32 ( 0.6 ) , 33 ( 0.7 ) , 34 ( 0.4 ) , and 36 ( 2.7 ) . 
 according to the dose response relationship in figure 1 , 33 ( intermediate dose of 0.46 mg / kg 
 producing 80% reversal ) was more potent in vivo than 23 and 27 . the nucleoside derivatives were 
 secondarily tested in standard radioligand binding assays at three 
 har subtypes ( table 1 ) to confirm a3ar selectivity . the ha1ar and ha3ar were stably expressed in cho cells , and the ha2aar was stably expressed in hek293 cells . the ki values at the ha3ar were in most cases in the 
 low nm range . at the ha1ar and ha2aar , only 
 1040% of binding inhibition was seen at 10 m for all 
 of the tested ( n)-methanocarba nucleosides . thus , there was high ha3ar selectivity for the entire structural family . the most 
 efficacious derivatives in the in vivo assay , such as long - acting 33 and 36 , were confirmed to display high selectivity 
 in binding with ki values at the ha3ar of 0.70 and 2.68 nm , respectively . however , high a3ar affinity and selectivity alone were not sufficient to provide 
 full protection in the cci assay ; other analogues that were less efficacious 
 such as o - cl - phenyl derivative 11 were 
 equally a3ar selective . nevertheless , a comparison 
 of ar binding sar was useful . a comparison of the binding affinities 
 of compounds 6a and 13 suggests that the 
 presence of an n - methyl group in 13 is associated with preserved or increased selectivity for 
 the ha3ar . compounds 1416 are regioisomers , in which a methoxy group is moved to different 
 positions on the phenylethynyl ring , but there is little effect on 
 the subnanomolar ha3ar affinity and selectivity . compounds 1921 are regioisomers at the pyridylethynyl 
 group , but there is little effect on the ha3ar affinity . pyrazine derivatives 2224 similarly 
 display at most a 2-fold difference in ha3ar binding affinity , 
 and pyrazole derivatives 25 and 26 are similar 
 in ha3ar affinity . furyl 2730 and thienyl 3135 derivatives , 
 ferrocene derivative 36 , and cycloalkyl analogues 37 and 38 also displayed only minor variation 
 of nanomolar ha3ar affinity . the ma3ar affinity of this series of closely related n - methyl congeners was typically > 30 nm , i.e. , reduced > 30-fold 
 in comparison to the ha3ar affinity . in general , variation 
 of the affinity at ma3ar was not parallel to changes at 
 the ha3ar ( figure s1 , supporting information ) . the unusual ferrocene derivative 36 was the most 
 potent among these analogues at the ma3ar with a ki of 5.46 nm , while many other analogues had ki values of 3050 nm . nevertheless , affinity 
 at the ma1ar and ma2aar ( single point determination 
 at 10 m ) was very weak , indicating that high a3ar 
 selectivity ( > 100-fold for 23 , 27 , 32 , and 33 ; > 1000-fold for 6a and 36 ) was still present in the mouse . selected 
 compounds were examined for functional potency and efficacy at the 
 ma3ar in an assay of a3ar - induced inhibition 
 of the production of cyclic amp in stably transfected hek293 cells 
 ( figure 2 ) . the 
 tested compounds 23 , 32 , 33 , and 36 were all full agonists at the ma3ar , with ic50 values ranging from 3.14 nm ( 36 ) to 60.1 nm ( 32 ) . activity in the inhibition of cyclic 
 amp formation at the ha1ar and ha3ar was evaluated 
 for representative compounds 23 and 32 , 
 which were shown to be potent , full agonists with functional selectivity 
 of roughly 10000-fold for the a3ar . ( a  d ) activity of 
 compounds 23 , 32 , 33 , and 36 in an assay of inhibition of forskolin - stimulated cyclic 
 amp accumulation with hek293 cells expressing the ma3ar . ( e , f ) activity of compounds 23 and 32 in an assay of inhibition of forskolin - stimulated 
 cyclic amp accumulation with cho cells expressing the ha3ar . ec50 values are 23 , 1.46  0.37 nm ; 32 , 0.42  0.18 
 nm . molecular modeling was used to 
 analyze the putative interactions of the distal cyclic groups appended 
 to the c2-ethynyl substitutent with the a3ar . the environment of receptor - bound c2-arylethynyl 
 and cycloalkylethynyl groups was explored by docking to a3ar homology models . we used our previously reported homology models 
 of the h and m a3ars , based on a 
 hybrid a2aar-2 adrenergic receptor template . the hybrid template strategy that determined the outward shift of 
 the extracellular tip of tm2 with the creation of a larger pocket 
 was required to accommodate the rigid and extended c2 substituent of these derivatives , as previously described . in docking selected compounds of the present series , a common binding 
 mode was obtained at both the h and m a3ar models , and 
 this mode featured all the key interactions found to anchor the adenine 
 and pseudosugar moieties of similar derivatives . as an example , figure 3 shows the docking poses of compound 33 , which displayed high affinity at both the ha3ar ( 0.70 
 nm ) and ma3ar ( 36 nm ) , at the two receptors . the planar 
 bicyclic core formed a  stacking interaction 
 with a phenylalanine in the second extracellular loop ( el2 ) and two 
 hydrogen bonds with n6.55 , while the methanocarba region formed a 
 hydrogen bonding network with t3.36 , s7.42 , and h7.43 . the c2 terminal cyclic group was found to occupy a region close 
 to the extracellular environment in proximity of tm2 , with tolerance 
 for many substitutions and steric bulk , consistent with previous findings . even though the residues in proximity of the terminal cyclic group 
 are different between the h and m a3ars , a good accommodation 
 of all the different c2 substituents was found in 
 both cases . in the case of compound 33 , the aryl group 
 at the c2 position is almost coplanar with the adenine 
 core . however , for other compounds bearing heterocyclic rings with 
 h - bonding donor or acceptor groups , the dihedral angle between the 
 rings can vary to allow formation of h - bonds with residues in el2 , 
 in particular with the backbone nh of phe168 or with the side chain 
 of gln167 at the ha3ar and with the backbone nh of phe169 
 or with the side chain of his168 at the ma3ar . putative binding 
 modes of 2-(2-chlorothienylethynyl)-n - methyl ( n)-methanocarba derivative 33 ( green carbons ) 
 obtained after docking simulations : ( a ) at the ha3ar , ( b ) 
 at the ma3ar . weinstein 
 notation or locations are v72 , 2.64 ; 
 t94 , 3.36 ; q167 , el2 ; f168 , el2 ; w243 , 6.48 ; n250 , 6.55 ; y265 , 7.36 ; 
 s271 , 7.42 ; h272 , 7.43 . interactions with potential off - target receptor sites , which 
 could lead to side effects , were measured for the most promising leads 
 from the in vivo screen . binding of seven of the most promising n - methyl compounds , 23 , 26 , 27 , 3234 , and 36 , to various possible off - target sites ( mostly gpcrs ) was 
 assayed in a broad screen of binding activity using cloned human or 
 rodent cdnas for cns receptors and ion channels . this screening was 
 provided by the national institute of mental health psychoactive drug 
 screening program ( nimh - pdsp ) . data for 
 compounds 1 , 6a , and 13 , reported 
 elsewhere , are also provided for comparison . screening results indicated that various analogues displayed significant 
 in vitro binding to other sites in the micromolar range , including 
 biogenic amine receptors . in 44 assays of off - target sites ( supporting information ) , compounds 13 , 23 , and 32 at 10 m inhibited binding 
 by > 30% at only a few sites . various analogues showed significant 
 binding in the m range at m2 , 5ht2a , 3 , 5ht2b , 5ht2c ( except for dif - phenylethynyl ) , 
 and 2c receptors and at the translocator protein 
 ( tspo ) , also known as the peripheral benzodiazepine receptor ( pbr ) . the reference agonist 1 showed interactions ( ki in m ) with 5ht2b ( 1.08 ) 
 and 5ht2c ( 5.42 ) receptors . thus , some of the derivatives 
 have potential off - target activities that could present a liability 
 for drug development . more off - target interaction was observed in 
 the ( n)-methanocarba series when an n-3-chlorobenzyl group was present , i.e. , 6a ; thus , the switch to n - methyl was beneficial from this perspective . compounds 26 and 27 were found to be relatively free of off - target 
 receptor effects in the pdsp screening . compound 33 was 
 largely free of off - target receptor effects , except at tspo and 1 
 and 2 receptors . the sar patterns of the broader chemical series 
 at other gpcrs has been analyzed with the aid of molecular modeling . in vitro stability measurements in physiological 
 solutions were performed on selected compounds for comparison to the 
 same parameters determined for 6a to be reported elsewhere 
 ( table 3 ) . compound 6a was orally 
 active in vivo in reducing chronic np , although its physicochemical 
 properties are not optimal ( clogp of 4.15 , total polar surface area 
 of 122  , and molecular weight of 565 d ) . thus , it 
 was considered advantageous to find other smaller and less hydrophobic 
 a3ar agonists that were fully efficacious in vivo against 
 np . the physicochemical properties of several analogues are predictive 
 of greater drug - like properties in vivo . for example , compound 33 that has a prolonged duration of action displays a more 
 favorable clogp of 2.17 and molecular weight of 459 d , but the total 
 polar surface area was similar to 6a . nevertheless , some 
 compounds clearly in the favored ranges of physicochemical parameters 
 were not among the most efficacious in vivo . thus , the in vivo effects 
 are not ascribable to a simple combination of parameters . the solubility 
 of pyrazinyl 23 and furyl 27 derivatives 
 was greatly increased ( > 0.2 mg / ml ) and plasma protein binding in 
 three species diminished ( 2461% free ) while the in vivo efficacy 
 of this analogue was maintained . % inhibition at 10 m 34 : 1a2 , 15.0 ; 2c9 , 24.2 ; 2c19 , 34.0 ; 2d6 , 57.5 ( ic50 7.4 
 m ) ; 3a4 , 3.1 ( average of n = 2 ) . % inhibition at 10 m 36 : 1a2 , 30.5 ; 2c9 , 17.2 ; 2c19 , 18.1 ; 2d6 , 20.5 ; 3a4 , 51.6 ( ic50 9.4 m ) , ( average of n = 2 ) . clint values ( l / min / mg 
 protein ) in human liver microsomes : 23 , 2.99 ; 27 , 3.09 ; 33 , 3.69 ; 34 , 3.38 . stability tests ( table 3 ) indicated that 23 , 27 , 33 , and 34 are stable in liver microsomes 
 of three species ( human , rat , and mouse ) and in simulated body fluids 
 ( gastric , intestinal ) , but 36 is rapidly degraded ( t1/2 in microsomes = 27 min ) . therefore , 
 it is likely that the long - lasting protective effects of 36 in the phenotypic screen are from an unidentified metabolite that 
 forms in vivo . there was an unfavorable cyp450 inhibition profile 
 for bromothienyl derivative 34 , with a measurable ic50 value of 7.4 m for the 2d6 isozyme . compounds 23 , 27 , and 33 were shown not to be substrates for p - glycoprotein in a 
 study of bidirectional permeability in a caco-2 cell monolayer ( supporting information ) . 27 and 33 displayed moderate apical to basal permeability , and 23 displayed 
 low permeability . selected compounds were examined by the pdsp for 
 functional inhibition of herg k channels ( % inhibition 
 at 10 m or ic50 ) : 6a ( 12.2 m ) , 13 ( 27 m ) , 26 ( 28% ) , 27 ( 21% ) , 32 ( 11.5 m ) , and 33 ( 12.9 m ) . thus , 
 the ha3ar binding affinity of these analogues exceeds herg 
 inhibition by typically 10,000-fold . although our approach is target - based at the 
 outset , i.e. , we have narrowed the structural scope based on new compounds 
 consistent with a well developed sar at the a3ar , we have 
 subjected the next stage of refinement to an in vivo screen that combines 
 multiple components of pharmacokinetics and possible interaction with 
 multiple mechanisms as well as the main mechanism of ar activation . we have administered the nucleosides orally , which is the preferred 
 route for a chronic pain drug . we have recently reported that 
 a3ar agonists including 6a are more potent 
 than currently used analgesics ( gabapentin , amitriptyline , morphine ) ; in contrast to opioids , these do not cause tolerance 
 upon repeated administration and are not rewarding . the 
 mechanisms of action whereby a3ar agonists block and reverse 
 neuropathic pain states do not rely upon an endogenous opioid or endocannabinoid 
 pathway but do rely upon activation of a3ar found in spinal cord dorsal horn and at supraspinal sites ( i.e. , 
 in the rvm ) to engage descending inhibitory noradrenergic and serotonergic 
 bulbospinal systems , leading to reduced spinal neuronal hyperexcitability . it is noteworthy that a3ar attenuates 
 neuropathic pain at least in part by reducing neuro - glia dysfunction 
 and downstream neuroinflammatory events that are critical to the development 
 of neuronal excitability associated with central sensitization , inhibition 
 of spinal glial cell activation and redox - sensitive signaling transduction 
 pathways ( mapk kinases and nfb ) leading to overall reduction 
 in proinflammatory cytokines ( tnf and il1 but increased levels 
 of the potent anti - inflammatory and neuroprotective cytokine , il-10 ) . the discovery of highly selective a3ar agonist such as 
 those described in this paper provides a necessary pharmacological 
 tool to enable our mechanistic understanding of the roles of the a3ar in chronic pain states and therefore the impact of targeting 
 this receptor to alleviate chronic pain and human suffering , thus 
 addressing a huge medical need with major socioeconomic consequences . the in vivo activities of various n - methyl derivatives were compared and correlated with structure , 
 and several preferred candidates have been identified . the unsubstituted c2-phenylethynyl analogue 7a displayed low 
 efficacy ( 44% of full reversal at peak effect ) in the cci model of 
 chronic np . aryl halogenation is a possible 
 approach to lengthening the in vivo duration of action by preventing 
 oxidation by cytochrome p450 enzymes in the liver . however , the monohalophenylethynyl analogues 812 were less than fully efficacious , and the 
 3,4-difluorophenyl group of the more efficacious 13 resulted 
 in a shorter peak duration in comparison to reference compound 6a . substitution of the phenyl ring with methoxy improved 
 the maximal effect ( 64% ) but only in the ortho position in 14 . all positions of nitrogen in the pyridyl analogues 19 , 20 , and 21 resulted in the same or lower 
 efficacy than the phenyl analogue 7a . however , dinitrogen 
 substitution in pyrazine derivative 23 increased the 
 efficacy to 100% , but the efficacy of isomers containing nitrogen 
 at different positions in 22 and 24 resulted 
 in less than full efficacy . a pyrazine moiety is found in many natural 
 products and drug - like molecules such as the diuretic amiloride . 2-furyl 27 and 2-thienyl 32 analogues were fully efficacious , with a 3 h duration 
 of action . addition of 5-chloro to the 2-thienyl 
 analogue 32 , resulting in compound 33 , prolonged 
 the duration of action while maintaining full efficacy , and 5-bromothienyl 
 substitution in 34 was also well tolerated in vivo and 
 in binding ( ki 0.4 nm ) . a similar 5-chlorothiophene-2-carboxamide 
 moiety was found to be relatively metabolically stable in a widely 
 used anticoagulant rivaroxaban . the ferrocene derivative 36 was fully efficacious , but 
 the toxicological properties of this organometallic compound were 
 not tested . other ferrocene compounds , such as anticancer derivatives 
 of tamoxifen , were found to be biocompatible and not highly toxic . a class of organometallic 
 ( ruthenium ) heterocyclic derivatives has already been reported as 
 a3ar antagonists . cycloalkyl 
 analogues 37 and 38 displayed less than 
 full efficacy in vivo ( 6070% ) . among mono- and dihalo - substituted 
 analogues , the 3,4-difluoro analogue 13 , bearing the 
 same c2 substituent as reference compound 6a , was the most efficacious . thus , the effects of introducing 
 a 2-alkynyl group in adenosine ( riboside ) derivatives on their ar 
 affinity and selectivity have been variable . in some cases , selectivity for the a3ar has been achieved . 
 in the present series of nucleoside derivatives that contain several 
 sources of conformational rigidity , the nm affinity and nearly complete 
 specificity for the a3ar is maintained for a wide variety 
 of cyclic groups attached to the 2-ethynyl moiety . docking studies 
 confirmed that the a3ar has sufficient plasticity in the 
 region around the extracellular tip of tm2 to accommodate a wide range 
 of steric and electronic character , allowing the rest of the ligand 
 molecule to strongly interact with all the key residues important 
 for ar binding . thus , we have extended previous 2-phenylethynyl series to provide a great breadth of substitution that 
 preserves target specificity . in summary , the analogues that reached 
 nearly full efficacy in reversing np in vivo were 13 , 22 , 23 , 26 , 27 , 32 , 33 , 34 , and 36 . thus , both 2-thienyl and 2-furyl derivatives are among the most efficacious 
 analogues in vivo . one of the major reasons for failure of a 
 variety of ar ligands on a clinical path was low bioavailabily . therefore , there was a need to optimize physicochemical 
 characteristics that are predictive of bioavailability . the smaller 
 molecular weight and lower hydrophobicity of the n - methyl analogues in comparison to the reference a3ar agonist 6a are consistent with drug - like properties 
 in vivo . pyrazine derivative 23 is much more polar than 6a and consequently more water - soluble 
 and less bound to plasma proteins , and it displays good efficacy in 
 the cci model . furan 27 and 2-chlorothiophene 33 derivatives are fully efficacious in cci model and display a 5-fold 
 higher ha3ar affinity than 6a . compound 33 had a favorable combination of physicochemical parameters 
 within this group of nucleosides . moreover , compound 27 completely lacks off - target interactions at 10 m and has 
 a relatively low molecular weight ( 408 ) , predictive of increased bioavailability , 
 and possibly cns entry . thus , we have expanded the structural range 
 of orally active adenosine derivatives for the treatment of chronic 
 pain . the most efficacious compounds in vivo had a clogp of 2.3 
 and a tpsa of 131 . however , there appears 
 to be no direct correlation between the in vivo efficacy against np 
 and the three physicochemical parameters listed in table 3 . a molecular weight of 526 in potentially short - lived 
 ferrocene derivative 36 still allowed full efficacy , 
 as did a tpsa of > 120 ( e.g. , 23 ) , which is considered 
 the upper limit for molecules expected to readily cross the blood 
 brain barrier . three 2-thienyl 
 derivatives were among the most efficacious and long lasting in vivo . cytochrome p450s may catalyze the oxidation 
 of thiophene compounds with the simultaneous formation of two reactive 
 intermediates , a thiophene - s - oxide and a thiophene 
 epoxide . however , some 5-halothiophene derivatives demonstrate greater 
 stability in vivo . strikingly , in this 
 study , the in vivo pharmacological effects of 5-substituted thiophene 
 derivatives 33 and 34 were among the most 
 prolonged . these compounds were sufficiently stable in vivo to provide 
 an extended duration of action compared to the majority of other analogues , 
 suggesting that rapid degradation is not occurring in these derivatives . both compounds were stable in liver microsomes , but compound 34 inhibited a cyp450 isozyme in the m range ; therefore , 33 appears to be the leading candidate molecule arising from 
 this study . the translation of ar agonists into therapeutic 
 products has been impeded by efficacy , bioavailability , and side effect 
 issues . this study progresses beyond 
 the most common initial screen of binding selectivity alone to some 
 of parameters that are predictive of derisking in pharmaceutical development 
 such as oral bioactivity . we have modified the c2-arylethynyl group of our previously reported a3ar agonists 
 with different rings , including both substituted benzene rings and 
 small heterocyclic rings like thiophene . these are mostly in the n - methyl series , which has reduced affinity 
 at the mouse a3ar but may be advantageous because of favorable 
 pharmacokinetics over the n - benzyl series 
 due to a lower mw and lower log p. five - membered 
 heteroring derivatives 27 and 3234 were fully efficacious in reducing neuropathic pain in 
 vivo , suggesting structural consistency . an unusual organometallic 
 derivative 36 was among those compounds showing a prolonged 
 duration of action that would not have been predicted from the binding 
 affinity alone , but this compound was unstable under physiological 
 conditions . the most promising fully efficacious analogues in this 
 study , with respect to aqueous solubility and absence of off - target 
 sites , were pyrazinyl 23 and furyl 27 derivatives . 5-chlorothienyl derivative 33 had a favorable balance 
 of high and prolonged efficacy , predicted in vivo stability , and few 
 off - target interactions . measuring the potential off - target effects , 
 such as interaction with -adrenergic receptors , provides another 
 means of identifying possible liabilities early in the drug discovery 
 process . the lead compounds discovered here are now suitable for more 
 extensive in vivo testing , including pharmacokinetic and toxicity 
 evaluation . on the basis of these leads , we hope to identify other 
 highly efficacious analogues with in vivo activity using phenotypic 
 screening . all reagents and solvents were purchased 
 from sigma - aldrich ( st . louis , mo ) . h nmr spectra were 
 obtained with a bruker 400 spectrometer using cdcl3 and 
 cd3od as solvents . chemical shifts are expressed in  
 values ( ppm ) with tetramethylsilane (  0.00 ) for cdcl3 and water (  3.30 ) for cd3od . tlc analysis was 
 carried out on glass sheets precoated with silica gel f254 ( 0.2 mm ) 
 from aldrich . all the final sulfonate nucleoside compounds were purified 
 by hplc with a luna 5 mrp - c18(2 ) semipreparative column ( 250 
 mm  10.0 mm ; phenomenex , torrance , ca ) and using the following 
 conditions : flow rate of 2 ml / min , 10 mm trifluoroacetic acid  water 
 ( tfa  h2o)ch3cn from 80:20 to 
 30:70 in 2040 min . packard 1100 hplc equipped with 
 a zorbax sb - aq 5 m analytical column ( 50 mm  4.6 mm ; 
 agilent technologies inc . , mobile phase : linear gradient 
 solvent system , 5 mm tbap ( tetrabutylammonium dihydrogen phosphate)ch3cn from 80:20 to 0:100 in 13 min ; the flow rate was 0.5 ml / min . peaks were detected by uv absorption with a diode array detector at 
 230 , 254 , and 280 nm . all derivatives tested for biological activity 
 showed > 95% purity by hplc analysis ( detection at 254 nm ) . low - resolution 
 mass spectrometry was performed with a jeol sx102 spectrometer with 
 6 kv xe atoms , following desorption from a glycerol matrix or on an 
 agilent lc / ms 1100 msd with a waters ( milford , ma ) atlantis c18 column . high resolution mass spectroscopic ( hrms ) measurements were performed 
 on a proteomics optimized q - tof-2 ( micromass - waters ) using external 
 calibration with polyalanine , unless noted . observed mass accuracies 
 are those expected based on known performance of the instrument as 
 well as trends in masses of standard compounds observed at intervals 
 during the series of measurements . reported masses are observed masses 
 uncorrected for this time - dependent drift in mass accuracy . all of 
 the monosubsituted alkyne intermediates were purchased from sigma - aldrich 
 ( st . louis , mo ) , small molecules , inc . ( hoboken , nj ) , anichem ( north 
 brunswick , nj ) , pharmablock , inc . ( sunnyvale , ca ) , frontier scientific 
 ( logan , ut ) , and tractus ( perrineville , nj ) . compound 14 ( 69% ) was prepared 
 from compound 40 following the same method for compound 22 . h nmr ( cd3od , 600 mhz )  
 8.10 ( s , 1h ) , 7.59 ( d , j = 6.0 hz , 1h ) , 7.45 ( t , j = 7.2 hz , 1h ) , 7.10 ( d , j = 8.4 hz , 1h ) , 
 7.01 ( t , j = 7.2 hz , 1h ) , 5.15 ( d , j = 6.0 hz , 1h ) , 4.90 ( s , 1h ) , 4.04 ( d , j = 6.0 hz , 
 1h ) , 3.96 ( s , 3h ) , 3.15 ( br s , 3h ) , 2.82 ( s , 3h ) , 2.092.07 
 ( m , 1h ) , 1.86 ( t , j = 4.8 hz , 1h ) , 1.431.41 
 ( m , 1h ) . hrms calculated for c23h25n6o4 ( m + h ) , 449.1937 ; found , 449.1932 . compound 15 ( 69% ) was prepared 
 from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  
 8.10 ( s , 1h ) , 7.35 ( t , j = 6.0 hz , 1h ) , 7.247.21 
 ( m , 2h ) , 7.02 ( d , j = 8.4 hz , 1h ) , 5.06 ( d , j = 6.8 hz , 1h ) , 4.92 ( s , 1h ) , 4.02 ( d , j = 6.4 hz , 1h ) , 3.85 ( s , 3h ) , 3.15 ( br s , 3h ) , 2.84 ( s , 3h ) , 2.132.09 
 ( m , 1h ) , 1.88 ( t , j = 4.4 hz , 1h ) , 1.411.38 
 ( m , 1h ) . hrms calculated for c23h25n6o4 ( m + h ) , 449.1937 ; found , 449.1944 . compound 16 ( 68% ) was prepared 
 from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  
 8.08 ( s , 1h ) , 7.60 ( d , j = 9.2 hz , 1h ) , 7.01 ( d , j = 9.2 hz , 1h ) , 5.05 ( d , j = 5.2 hz , 1h ) , 
 4.91 ( s , 1h ) , 4.02 ( d , j = 6.0 hz , 1h ) , 3.86 ( s , 
 3h ) , 3.15 ( br s , 3h ) , 2.85 ( s , 3h ) , 2.132.09 ( m , 1h ) , 1.88 
 ( t , j = 5.2 hz , 1h ) , 1.411.39 ( m , 1h ) . hrms 
 calculated for c23h25n6o4 ( m + h ) , 449.1937 ; found , 449.1944 . compound 17 ( 70% ) was prepared from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  8.14 ( s , 1h ) , 7.97 
 ( s , 1h ) , 7.92 ( d , j = 7.6 hz , 1h ) , 7.78 ( d , j = 7.6 hz , 1h ) , 7.68 ( t , j = 8.0 hz , 1h ) , 
 5.08 ( d , j = 4.8 hz , 1h ) , 4.90 ( s , 1h ) , 4.04 ( d , j = 5.2 hz , 1h ) , 3.15 ( br s , 3h ) , 2.84 ( s , 3h ) , 2.132.10 
 ( m , 1h ) , 1.88 ( t , j = 5.2 hz , 1h ) , 1.421.39 
 ( m , 1h ) . hrms calculated for c23h22n6o3f3 ( m + h ) , 487.1705 ; found , 
 487.1716 . compound 18 ( 66% ) was prepared 
 from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  
 8.13 ( s , 1h ) , 7.63 ( d , j = 8.0 hz , 2h ) , 7.43 ( d , j = 8.0 hz , 2h ) , 5.06 ( d , j = 6.4 hz , 1h ) , 
 4.89 ( s , 1h ) , 4.67 ( s , 2h ) , 4.02 ( d , j = 6.4 hz , 
 1h ) , 3.15 ( br s , 3h ) , 2.84 ( s , 3h ) , 2.132.09 ( m , 1h ) , 1.88 
 ( d , j = 4.8 hz , 1h ) , 1.411.39 ( m , 1h ) . hrms 
 calculated for c23h25n6o4 ( m + h ) , 449.1937 ; found , 449.1942 . compound 20 ( 60% ) was prepared from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  8.87 ( s , 1h ) , 8.64 
 ( s , 1h ) , 8.18 ( d , j = 6.4 hz , 1h ) , 8.15 ( s , 1h ) , 
 7.617.58 ( m , 1h ) , 5.05 ( d , j = 5.2 hz , 1h ) , 
 4.90 ( s , 1h ) , 4.02 ( d , j = 5.2 hz , 1h ) , 3.13 ( br 
 s , 3h ) , 2.82 ( s , 3h ) , 2.112.09 ( m , 1h ) , 1.87 ( t , j = 4.0 hz , 1h ) , 1.401.37 ( m , 1h ) . hrms calculated for c21h22n7o3 ( m + h ) , 420.1784 ; found , 420.1785 . compound 21 ( 63% ) was prepared from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  8.63 ( d , j = 6.0 hz , 2h ) , 8.14 ( s , 1h ) , 7.65 ( d , j = 6.0 hz , 1h ) , 5.06 ( d , j = 6.0 hz , 1h ) , 4.90 ( s , 
 1h ) , 4.03 ( d , j = 6.8 hz , 1h ) , 3.14 ( br s , 3h ) , 2.132.10 
 ( m , 1h ) , 1.88 ( d , j = 4.8 hz , 1h ) , 1.421.40 
 ( m , 1h ) . hrms calculated for c21h22n7o3 ( m + h ) , 420.1784 ; found , 420.1786 . pdcl2(pph3)2 ( 6.08 mg , 
 0.02 mmol ) , cui ( 1.0 mg , 0.005 mmol ) , 2-ethynylpyrimidine ( 27.1 mg , 
 0.26 mmol ) , and triethylamine ( 0.06 ml , 0.43 mmol ) were added to a 
 solution of compound 40 ( 21 mg , 0.04 mmol ) in anhydrous 
 dmf ( 1 ml ) , and the mixture stirred at room temperature overnight . solvent was evaporated under vacuum , and the residue was roughly purified 
 on flash silica gel column chromatography . the resulting compound 
 was dissolved in meoh ( 2 ml ) and 10% trifluoroacetic acid ( 2 ml ) and 
 heated at 70 c for 5 h. solvent was evaporated under vacuum , 
 and the residue was purified on flash silica gel column chromatography 
 ( ch2cl2:meoh = 25:1 ) to give the compound 22 ( 12.2 mg , 67% ) as syrup . h nmr ( cd3od , 400 mhz )  8.86 ( d , j = 5.2 hz , 2h ) , 8.14 
 ( s , 1h ) , 7.53 ( t , j = 5.2 hz , 1h ) , 5.17 ( d , j = 6.8 hz , 1h ) , 4.92 ( s , 1h ) , 4.6 ( d , j = 6.4 hz , 1h ) , 3.14 ( br s , 3h ) , 2.86 ( s , 3h ) , 2.112.07 ( m , 
 1h ) , 1.84 ( t , j = 5.2 hz , 1h ) , 1.421.39 ( m , 
 1h ) . hrms calculated for c20h21n8o3 ( m + h ) , 421.1737 ; found , 421.1732 . compound 23 ( 65% ) was prepared from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  8.92 ( s , 1h ) , 8.69 
 ( d , j = 2.4 hz , 1h ) , 8.64 ( d , j = 
 2.4 hz , 1h ) , 8.14 ( s , 1h ) , 5.12 ( d , j = 5.2 hz , 1h ) , 
 4.89 ( s , 1h ) , 4.05 ( d , j = 6.4 hz , 1h ) , 3.15 ( br 
 s , 3h ) , 2.85 ( s , 3h ) , 2.122.08 ( m , 1h ) , 1.86 ( t , j = 5.2 hz , 1h ) , 1.421.40 ( m , 1h ) . hrms calculated for c20h21n8o3 ( m + h ) , 421.1737 ; found , 421.1725 . compound 24 ( 65% ) was prepared from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  9.23 ( d , j = 3.2 hz , 1h ) , 8.15 ( s , 1h ) , 8.03 ( d , j = 6.8 hz , 1h ) , 7.837.81 ( m , 1h ) , 5.14 ( d , j = 5.2 hz , 1h ) , 4.90 ( s , 1h ) , 4.07 ( d , j = 5.2 hz , 
 1h ) , 3.15 ( br s , 3h ) , 2.84 ( s , 3h ) , 2.112.08 ( m , 1h ) , 1.86 
 ( t , j = 4.8 hz , 1h ) , 1.421.39 ( m , 1h ) . hrms 
 calculated for c20h21n8o3 ( m + h ) , 421.1737 ; found , 421.1734 . compound 25 ( 64% ) was prepared 
 from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  
 8.10 ( s , 1h ) , 7.73 ( s , 1h ) , 6.66 ( s , 1h ) , 5.12 ( d , j = 6.8 hz , 1h ) , 4.87 ( s , 1h ) , 4.04 ( d , j = 6.8 hz , 
 1h ) , 3.13 ( br s , 3h ) , 2.85 ( s , 3h ) , 2.102.07 ( m , 1h ) , 1.85 
 ( t , j = 4.8 hz , 1h ) , 1.411.38 ( m , 1h ) . hrms 
 calculated for c19h21n8o3 ( m + h ) , 409.1737 ; found , 409.1731 . compound 26 ( 63% ) was prepared from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  8.08 ( s , 1h ) , 7.98 
 ( s , 1h ) , 7.75 ( s , 1h ) , 5.05 ( d , j = 5.6 hz , 1h ) , 
 4.88 ( s , 1h ) , 4.02 ( d , j = 6.4 hz , 1h ) , 3.94 ( s , 
 3h ) , 3.13 ( br s , 3h ) , 2.86 ( d , j = 4.4 hz , 1h ) , 2.122.08 
 ( m , 1h ) , 1.87 ( t , j = 4.8 hz , 1h ) , 1.411.37 
 ( m , 1h ) . hrms calculated for c20h23n8o3 ( m + h ) , 423.1888 ; found , 423.1888 . compound 27 ( 58% ) was prepared 
 from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  
 8.16 ( s , 1h ) , 7.66 ( d , j = 1.2 hz , 1h ) , 6.95 ( d , j = 2.8 hz , 1h ) , 6.58 ( dd , j1 = 1.6 , j2 = 2.0 hz , 1h ) , 5.07 ( d , j = 5.2 hz , 1h ) , 4.89 ( s , 1h ) , 4.03 ( d , j = 6.4 hz , 1h ) , 3.14 ( br s , 3h ) 2.87 ( s , 3h ) , 2.112.09 ( m , 
 1h ) , 1.86 ( t , j = 4.8 hz , 1h ) , 1.411.40 ( m , 
 1h ) . hrms calculated for c20h21n6o4 ( m + h ) , 409.1624 ; found , 409.1611 . compound 28 ( 63% ) was prepared 
 from compound 40 following the same method for compound 35 . h nmr ( cd3od , 400 mhz )  
 8.09 ( s , 1h ) , 6.69 ( s , 1h ) , 5.06 ( d , j = 6.4 hz , 
 1h ) , 4.85 ( s , 1h ) , 4.01 ( d , j = 6.4 hz , 1h ) , 3.12 
 ( br s , 3h ) , 2.87 ( s , 3h ) , 2.26 ( s , 3h ) , 2.122.08 ( m , 1h ) , 
 1.99 ( s , 3h ) , 1.85 ( t , j = 4.8 hz , 1h ) , 1.401.38 
 ( m , 1h ) . hrms calculated for c22h25n6o4 ( m + h ) , 437.1932 ; found , 437.1934 . compound 29 ( 53% ) was prepared 
 from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  
 8.11 ( s , 1h ) , 6.82 ( d , j = 7.2 hz , 1h ) , 6.18 ( d , j = 7.6 hz , 1h ) , 5.08 ( d , j = 6.4 hz , 1h ) , 
 4.93 ( s , 1h ) , 4.02 ( d , j = 6.4 hz , 1h ) , 3.14 ( br 
 s , 3h ) , 2.752.69 ( m , 2h ) , 2.112.08 ( m , 1h ) , 1.86 ( t , j = 5.2 hz , 1h ) , 1.411.38 ( m , 1h ) , 1.29 ( t , j = 7.6 hz , 3h ) . hrms calculated for c22h25n6o4 ( m + h ) , 437.1932 ; 
 found , 437.1932 . compound 30 ( 62% ) was prepared 
 from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  
 8.12 ( s , 1h ) , 7.67 ( d , j = 7.6 hz , 1h ) , 7.5 ( d , j = 7.6 hz , 1h ) , 7.43 ( t , j = 6.0 hz , 1h ) , 
 7.337.31 ( m , 2h ) , 5.11 ( d , j = 5.2 hz , 1h ) , 
 4.89 ( s , 1h ) , 4.04 ( d , j = 6.4 hz , 1h ) , 3.15 ( br 
 s , 3h ) , 2.89 ( s , 3h ) , 2.132.10 ( m , 1h ) , 1.87 ( t , j = 4.8 hz , 1h ) , 1.421.39 ( m , 1h ) . hrms calculated for c24h23n6o4 ( m + h ) , 459.1775 ; found , 459.1777 . compound 31 ( 59% ) was prepared from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  8.13 ( s , 1h ) , 7.88 
 ( dd , j1 = 1.2 , j2 = 2.0 hz , 1h ) , 7.54 ( dd , j1 = 
 2.0 , j2 = 3.2 hz , 1h ) , 7.33 ( dd , j1 = 1.2 , j2 = 4.0 
 hz , 1h ) , 5.07 ( d , j = 5.2 hz , 1h ) , 4.89 ( s , 1h ) , 
 4.04 ( d , j = 5.2 hz , 1h ) , 3.15 ( br s , 3h ) , 2.85 ( s , 
 3h ) , 2.132.09 ( m , 1h ) , 1.87 ( t , j = 4.8 hz , 
 1h ) , 1.421.39 ( m , 1h ) . hrms calculated for c20h21n6o3s ( m + h ) , 425.1396 ; 
 found , 425.1403 . compound 32 ( 61% ) was prepared from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  8.10 ( s , 1h ) , 7.57 
 ( dd , j1 = 1.2 , j2 = 4.0 hz , 1h ) , 7.50 ( dd , j1 = 
 1.2 , j2 = 4.0 hz , 1h ) , 7.13 ( dd , j1 = 1.6 , j2 = 3.6 
 hz , 1h ) , 5.05 ( d , j = 6.4 hz , 1h ) , 4.88 ( s , 1h ) , 
 4.02 ( d , j = 6.4 hz , 1h ) , 3.14 ( br s , 3h ) , 2.86 ( s , 
 3h ) , 2.132.09 ( m , 1h ) , 1.88 ( t , j = 5.2 hz , 
 1h ) , 1.411.39 ( m , 1h ) . hrms calculated for c20h21n6o3s ( m + h ) , 425.1396 ; 
 found , 425.1388 . compound 33 ( 59% ) was prepared 
 from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  
 8.11 ( s , 1h ) , 7.31(d , j = 4.0 hz , 1h ) , 7.03 ( d , j = 4.0 hz , 1h ) , 5.04 ( d , j = 7.2 hz , 1h ) , 
 4.86 ( s , 1h ) , 4.01 ( d , j = 6.8 hz , 1h ) , 3.13 ( br 
 s , 3h ) , 2.86 ( s , 3h ) , 2.122.09 ( m , 1h ) , 1.88 ( d , j = 4.8 hz , 1h ) , 1.411.38 ( m , 1h ) . hrms calculated for c20h20n6o3scl ( m + h ) , 459.1006 ; found , 459.1005 . compound 34 ( 48% ) was prepared 
 from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  
 8.10 ( s , 1h ) , 7.28 ( d , j = 4.0 hz , 1h ) , 7.15 ( d , j = 4.0 hz , 1h ) , 5.05 ( d , j = 5.2 hz , 1h ) , 
 4.91 ( s , 1h ) , 4.02 ( d , j = 5.6 hz , 1h ) , 3.14 ( br 
 s , 3h ) , 2.86 ( s , 3h ) , 2.122.09 ( m , 1h ) , 1.88 ( t , j = 4.8 hz , 1h ) , 1.411.37 ( m , 1h ) . hrms calculated for c20h20n6o3sbr ( m + h ) , 503.0495 ; found , 503.0498 . compound 35 ( 64% ) was prepared from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  8.18 ( s , 1h ) , 7.97 
 ( d , j = 3.2 hz , 1h ) , 7.82 ( d , j = 
 3.2 hz , 1h ) , 5.12 ( d , j = 5.2 hz , 1h ) , 4.90 ( s , 1h ) , 
 4.04 ( d , j = 6.4 hz , 1h ) , 3.14 ( br s , 3h ) , 2.86 ( s , 
 3h ) , 2.112.07 ( m , 1h ) , 1.86 ( t , j = 4.8 hz , 
 1h ) , 1.421.38 ( m , 1h ) . hrms calculated for c19h20n7o3s ( m + h ) , 426.1343 ; 
 found , 426.1342 . a solution of compound 40 ( 31 
 mg , 0.06 mmol ) in methanol ( 3 ml ) and 10% trifluoromethanesulfonic 
 acid ( 2 ml ) was heated at 70 c for 5 h. solvent was evaporated 
 under vacuum , and the residue was purified on flash silica gel column 
 chromatography ( ch2cl2:meoh = 25:1 ) to give 
 the isopropylidene - deblocked derivative ( 27 mg , 95% ) as syrup . pdcl2(pph3)2 ( 8.5 mg , 0.012 mmol ) , cui ( 1.1 
 mg , 0.006 mmol ) , ethynylferrocene ( 76.6 mg , 0.36 mmol ) , and triethylamine 
 ( 0.08 ml , 0.6 mmol ) were added to a solution of the obtained compound 
 ( 27 mg , 0.06 mmol ) in anhydrous dmf ( 1.2 ml ) , and the mixture stirred 
 at room temperature overnight . solvent was evaporated under vacuum , 
 and the residue was purified on flash silica gel column chromatography 
 ( ch2cl2:meoh = 30:1 ) to give the compound 36 ( 29 mg , 86% ) as light - yellow syrup . h nmr ( cd3od , 400 mhz )  8.10 ( s , 1h ) , 5.01 ( d , j = 6.4 hz , 1h ) , 4.89 ( s , 1h ) , 4.63 ( s , 2h ) , 4.40 ( s,2h ) , 4.31 ( s , 
 6h ) , 4.01(d , j = 6.4 hz , 1h ) , 3.15 ( br s , 3h ) , 2.88 
 ( s , 3h ) , 2.132.10 ( m , 1h ) , 1.91 ( t , j = 4.8 
 hz , 1h ) , 1.421.38 ( m , 1h ) . hrms calculated for c26h27n6o3fe ( m + h ) , 527.1489 ; 
 found , 527.1489 . compound 37 ( 68% ) was prepared 
 from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  
 8.06 ( s , 1h ) , 5.02 ( d , j = 6.0 hz , 1h ) , 4.83 ( s , 
 1h ) , 3.97 ( d , j = 6.4 hz , 1h ) , 3.10 ( br s , 3h ) , 2.88 
 ( s , 3h ) , 2.102.07 ( m , 1h ) , 1.86 ( t , j = 4.8 
 hz , 1h ) , 1.591.52 ( m , 1h ) , 1.401.37 ( m , 1h ) , 1.000.98 
 ( m , 2h ) , 0.940.86 ( m , 2h ) . hrms calculated for c19h23n6o3 ( m + h ) , 383.1832 ; 
 found , 383.1838 . compound 38 ( 71% ) was prepared 
 from compound 40 following the same method for compound 22 . h nmr ( cd3od , 400 mhz )  
 8.06 ( s , 1h ) , 4.98 ( d , j = 6.8 hz , 1h ) , 4.85 ( s , 
 1h ) , 3.95 ( d , j = 6.4 hz , 1h ) , 3.11 ( br s , 3h ) 2.86 
 ( s , 3h ) , 2.862.62 ( m , 1h ) , 2.112.08 ( m , 1h ) , 1.991.96 
 ( m , 2h ) , 1.89 ( t , j = 4.8 hz , 1h ) , 1.841.80 
 ( m , 2h ) , 1.631.58 ( m , 3h ) , 1.431.39 ( m , 4h ) . hrms 
 calculated for c22h29n6o3 ( m + h ) , 425.2301 ; found , 425.2308 . previously 
 published homology models of the ha3ar and ma3ar , built on the basis of a hybrid template 
 structure using the homology modeling tool implemented in the moe 
 suite , were used in this study . to build 
 these models , an agonist - bound ha2aar crystal structure 
 ( pdb code 3qak ) was used as a template for the entire 
 a3ar structure except for the extracellular terminus of 
 tm2 ( residues from val63 to ser73 at the ha3ar and from 
 val64 to ser74 at the ma3ar ) and el1 ( residues from leu74 
 to tyr81 at the ha3ar and from leu75 to tyr82 at the ma3ar ) . the x - ray structure of the h2 adrenergic 
 receptor in complex with the gs protein ( pdb code 3sn6 ) , after superimposition with the ha2aar crystal 
 structure , was used as template to build the extracellular terminus 
 of tm2 . structures of compounds were built and prepared 
 for docking using the build panel and the ligprep panel implemented 
 in the schrdinger suite . molecular 
 docking of the ligands at the a3ar models was performed 
 by means of the glide package part of 
 the schrdinger suite . the docking site was defined using key 
 residues in the binding pocket of the a3ar models , namely 
 phe ( el2 ) , asn ( 6.55 ) , trp ( 6.48 ) , and his ( 7.43 ) , and a 20  
  20   20  box was centered on these residues . docking of ligands was performed in the rigid binding site using the 
 xp ( extra precision ) procedure . the top scoring docking conformations 
 of each ligand were subjected to visual inspection and analysis of 
 protein  ligand interactions to select the final binding conformations . [ h]r - n - phenylisopropyladenosine ( 65 , [ h]r - pia , 63 ci / mmol ) , [ h](2-[p-(2-carboxyethyl)phenyl - ethylamino]-5-n - ethylcarboxamido - adenosine ) , 
 ( 66 , [ h]cgs21680 , 40.5 ci / mmol ) , and [ i]n-(4-amino-3-iodobenzyl)adenosine-5-n - methyluronamide ( 67 , [ i]i - ab - meca , 
 2200 ci / mmol ) were purchased from perkin test compounds were prepared as 5 mm stock solutions 
 in dmso and stored frozen . pharmacological standards 2 ( a3ar agonist ) , adenosine-5-n - ethylcarboxamide ( 68 , neca , nonselective ar agonist ) , 
 and 2-chloro - n - cyclopentyladenosine ( 69 , ccpa , a1ar agonist ) were purchased from tocris 
 r&d systems ( minneapolis , mn ) . cho cells stably expressing the recombinant ha1 and ha3ars and hek293 cells stably expressing 
 the ha2aar were cultured in dulbecco s modified 
 eagle medium ( dmem ) and f12 ( 1:1 ) supplemented with 10% fetal bovine 
 serum , 100 units / ml penicillin , 100 g / ml streptomycin , and 2 mol / ml glutamine . in addition , 800 
 g / ml geneticin was added to the a2a media , while 
 500 g / ml hygromycin was added to the a1 and a3 media . . the pellet was discarded , and the 
 suspension was recentrifuged at 20000 g for 60 min 
 at 4 c . the resultant pellets were resuspended in tris buffer , 
 incubated with adenosine deaminase ( 3 units / ml ) for 30 min at 37 c . the suspension was homogenized with an electric homogenizer for 10 
 s , pipetted into 1 ml vials , and then stored at 80 c 
 until the binding experiments . the protein concentration was measured 
 using the bca protein assay kit from pierce biotechnology , inc . ( rockford , 
 il ) . into each tube in the binding assay was added 50 l of increasing 
 concentrations of the test ligand in tris - hcl buffer ( 50 mm , ph 7.5 ) 
 containing 10 mm mgcl2 , 50 l of the appropriate 
 agonist radioligand , and finally 100 l of membrane suspension . 
 for the a1ar ( 22 g of protein / tube ) , the radioligand 
 used was [ h]65 ( final concentration of 3.5 
 nm ) . for the a2aar ( 20 g / tube ) , the radioligand 
 used was [ h]66 ( 10 nm ) . for the a3ar ( 21 g / tube ) , nonspecific binding was determined using a final 
 concentration of 10 m 68 diluted with the buffer . the mixtures were incubated at 25 c for 60 min in a shaking 
 water bath . binding reactions were terminated by filtration through 
 brandel gf / b filters under a reduced pressure using a m-24 cell harvester 
 ( brandel , gaithersburg , md ) . filters were washed three times with 
 3 ml of 50 mm ice - cold tris - hcl buffer ( ph 7.5 ) . filters for a1 and a2aar binding were placed in scintillation 
 vials containing 5 ml of hydrofluor scintillation buffer and counted 
 using a perkinelmer liquid scintillation analyzer ( tri - carb 2810tr ) . similar competition 
 binding assays were conducted using hek293 cell membranes expressing 
 mars using [ i]67 to label a1 or a3ars and [ h]65 to label 
 a2aars . hek293 cells were detached from cell culture plates , resuspended 
 in serum - free dmem containing 25 mm hepes ( ph 7.4 ) , 1 unit / ml adenosine 
 deaminase , 4-(3-butoxy-4-methoxyphenyl)methyl-2-imidazolidone ( tocris , ro 20,1724 , 20 m ) and 300 nm 8-[4-[4-(4-chlorophenzyl)piperazide-1-sulfonyl)phenyl]]-1-propylxanthine 
 ( tocris , psb603 , 300 nm , to inhibit a2bars expressed endogenously 
 in hek293 cells ) , and then transferred to polypropylene tubes ( 2  
 10 cells / tube ) . the cells were coincubated with forskolin 
 ( 10 m ) and ar ligands for 15 min at 37 c with shaking , 
 after which the assays were terminated by adding 500 l of 1 
 n hcl . the cyclic amp concentration was determined in the supernatants 
 using a competitive binding assay , as previously described . ec50 and emax values were calculated by fitting the data to e = emin + ( emax male cd-1 mice ( 2530 g ) from harlan ( indianapolis , in ) 
 were housed 45 ( for mice ) per cage in a controlled environment 
 ( 12 h light / dark cycles ) with food and water available ad libitum . experiments were performed in accordance with international association 
 for the study of pain , nih guidelines on laboratory animal welfare , 
 and saint louis university institutional animal care and use committee 
 recommendations . cci 
 to the sciatic nerve of the left hind leg in mice was performed under 
 general anesthesia using the well characterized bennett model . briefly , mice ( weighing 2530 g at the 
 time of surgery ) were anesthetized with 3% isoflurane/100% o2 inhalation and maintained on 2% isoflurane/100% o2 for 
 the duration of surgery . the left thigh was shaved and scrubbed with 
 nolvasan ( zoetis , madison , nj ) , and a small incision ( 11.5 
 cm in length ) was made in the middle of the lateral aspect of the 
 left thigh to expose the sciatic nerve . the nerve was loosely ligated 
 around the entire diameter of the nerve at three distinct sites ( spaced 
 1 mm apart ) using silk sutures ( 6.0 ) . the surgical site was closed 
 with a single muscle suture and a skin clip . pilot studies established 
 that under our experimental conditions peak mechano - allodynia develops 
 by d5d7 following cci . test substances or their vehicles were 
 administered as 3 mol / kg doses given by gavage ( 0.2 ml po ) 
 at peak mechano - allodynia ( d7 ) . the vehicle used consisted of 10% 
 dmso in 0.5% methylcellulose ( diluted from a 5 mm stock solution in 
 dmso ) . 021m0067v ) was obtained from sigma 
 viscosity 400 cp and prepared in sterile distilled water ( ups ) . significant 
 differences were defined at a p < 0.05 . all statistical 
 analysis was performed using graphpad prism ( v5.03 , graphpad software , 
 inc . , san diego , ca ) . ki determinations and binding profiles in a broad 
 screen of receptors and channels were generously provided by the national 
 institute of mental health s psychoactive drug screening program , 
 contract no . the nimh pdsp is directed 
 by bryan l. roth md , ph.d . at the university of north carolina at 
 chapel hill and project officer jamie driscol at nimh , bethesda md , 
 usa . for experimental details , please refer to the pdsp web site http://pdsp.med.unc.edu/ and click on  binding assay  
 or
( n)-methanocarba 
 adenosine 5-methyluronamides containing 2-arylethynyl groups 
 were synthesized as a3 adenosine receptor ( ar ) agonists 
 and screened in vivo ( po ) for reduction of neuropathic pain . 
 a small n6-methyl group maintained binding affinity , 
 with human > mouse a3ar and mw < 500 and other favorable 
 physicochemical properties . 
 emax ( maximal 
 efficacy in a mouse chronic constriction injury pain model ) of previously 
 characterized a3ar agonist , 2-(3,4-difluorophenylethynyl)-n6-(3-chlorobenzyl ) derivative 6a , mrs5698 , was surpassed . 
 more efficacious analogues ( in vivo ) contained 
 the following c2-arylethynyl groups : pyrazin-2-yl 23 ( binding ki , ha3ar , nm 1.8 ) , fur-2-yl 27 ( 0.6 ) , thien-2-yl 32 ( 0.6 ) and its 5-chloro 33 , mrs5980 ( 0.7 ) and 5-bromo 34 ( 0.4 ) equivalents , and physiologically unstable ferrocene 36 , mrs5979 ( 2.7 ) . 33 and 36 displayed 
 particularly long in vivo duration ( > 3 h ) . selected analogues were 
 docked to an a3ar homology model to explore the environment 
 of receptor - bound c2 and n6 groups . 
 various analogues bound with m affinity at off - target 
 biogenic amine ( m2 , 5ht2a , 3 , 5ht2b , 5ht2c , and 2c ) or other receptors . 
 thus , we have expanded the structural range of orally active a3ar agonists for chronic pain treatment .