-adrenoceptor ligands comprise 
 one of the most commonly 
 used classes of drugs in clinical practice . -agonists have 
 been used since the 1940s for their beneficial effects in respiratory 
 disease and -antagonists ( -blockers ) 
 since the 1960s for the control of cardiovascular disease , and both classes remain among the most commonly prescribed drugs 
 today . the major therapeutic aim of -blockade is antagonism 
 of 
 endogenous catecholamines at the cardiac 1-adrenoceptors 
 to reduce myocardial demand and workload . this reduces mortality following myocardial infarction , and therefore -blockers are used for both 
 symptomatic control and life prolongation in ischemic heart disease 
 and acute coronary syndrome and are increasingly being used earlier 
 in the disease process . -blockers 
 also reduce mortality in patients with heart failure and are used in the management of arrhythmias , hypertension , portal 
 hypertension , benign essential tremor , thyrotoxicosis , anxiety , migraine , 
 and glaucoma . the major therapeutic 
 aim of -agonist administration is 
 to mimic adrenaline and stimulate the 2-adrenoceptors 
 in bronchial smooth muscle , thus increasing airway caliber and relieving 
 the breathlessness and wheeze of bronchospasm . -agonists are used in the emergency setting for 
 short - term rescue of bronchospasm in asthma and copd ( e.g. , salbutamol 
 inhalers and nebulizers ) as well as in the daily prevention of bronchospasm , 
 particularly using longer acting molecules ( labas , e.g. , salmeterol , 
 formoterol , indacaterol , and vilanterol ) . as -ligands have been used in clinical practice for around 
 for 70 years , and a great many different agonist and antagonist molecules 
 have been generated , it would be expected that good tool agonist and 
 antagonist compounds for each of the human -adrenoceptor subtypes 
 are available . however , it is surprising that there are actually very 
 few truly selective molecules , especially for the 1-adrenoceptor . drug discovery efforts driven by the clinical need 
 have yielded several highly selective 2-adrenoceptor 
 agonists for the treatment of asthma and copd ( e.g. , salmeterol , formoterol , 
 indacaterol , and vilanterol ) and one highly selective 2-antagonist ( ici 118551 ) that has become a very useful tool 
 in pharmacological studies of -adrenoceptors . for the 1-adrenoceptor , despite the fact that more 1-selective -blockers would be preferable to minimize the 2-mediated respiratory side effect of bronchospasm in the clinical 
 setting , the selectivity of the clinically available -blockers 
 for the human 1-adrenoceptor over the human 2-adrenoceptor is poor . for this reason , -blockers remain absolutely contraindicated 
 in patients with asthma and relatively contraindicated in people with 
 copd . our desire to address this significant unmet clinical need initially 
 led us to assess reported ligands that displayed very high 1-adrenoceptor versus 2-adrenoceptor selectivity . 
 from the literature , one of the most highly 1-selective 
 antagonists described to date was 1 ( lk 204 - 545 ) . although this ligand provided an ideal structural 
 template to investigate further , there was no published synthetic 
 route and , in reality , very little pharmacological data available 
 for this compound . furthermore , it was not commercially available 
 and therefore not readily accessible for pharmacological and other 
 studies . as we also observed , there were also very few 1-selective -agonists , with xamoterol and ici 89406 
 being the most selective 1-partial agonists reported 
 to date . here , we report a synthesis and the in vitro pharmacological 
 characterization 
 of 1 and several novel , highly 1-selective 
 -adrenoceptor ligands , most of which display significant agonist 
 activity . whereas the synthesis 
 of 1 is 
 not described in the literature , a synthetic route to the related 
 compound , 1-(2-cyano-4-(2-cyclopropylmethoxyethoxy)phenoxy)-3-(2-(3-phenylureido)ethylamino)-2-propanol , 
 is outlined as an example of this class of compound in a patent . the introduction of the cyano group employed 
 an undesirable cyanation step , alongside several protection and deprotection 
 steps , complicating the synthesis and compromising overall yield . conscious that we would require appreciable quantities of this ligand 
 to benchmark any newer ligands against , we embarked upon a new and 
 more expeditious route to its synthesis ( scheme 1 ) . reagents 
 and conditions : ( a ) 
 et3n , pivaloyl chloride , dmf , 0 c  rt , 72% ; 
 ( b ) ( i ) nah , dmf ; ( ii ) p - methoxybenzyl bromide , 0 
 c  rt , 33% ; ( c ) 37% nh3 ( aq ) , i2 , thf , 98% ; ( d ) sodium tert - butoxide , meoh , 34% ; 
 ( e ) allyloxyethanol , diad , pph3 , dcm , 100% ; ( f ) et2zn , ch2i2 , toluene 0 c  
 rt , 25% 8 and 29% 9 ; ( g ) can , h2o , mecn ; ( h ) et3n , rac - epichlorohydrin , 
 80 c , 100% ; ( i ) 4-(benzyloxy)phenylisocyanate , dcm , 94% ; ( j ) 
 ( i ) concd hcl ; ( ii ) 2 m naoh ( aq ) , neutralization , 73% ; ( k ) 13 , propan-2-ol , reflux , 15% ; ( l ) allyloxyethanol , diad , ph3p , dcm , rt , 71% ; ( m ) et2zn , ch2i2 , toluene , 0 c  rt , 97% ; ( n ) h2 , 
 10% pd / c , etoh , 100% ; ( o ) ( i ) 2 m naoh(aq ) ; ( ii ) rac - epichlorohydrin , 60 c , 62% ; ( p ) 13 , propan-2-ol , reflux , 21% . the new synthesis 
 of 1 relied upon an alternative 
 synthesis of epoxide 10 , with subsequent aminolysis of 10 using amine 13 . the availability of methods 
 to easily convert an aryl aldehyde to the corresponding nitrile allowed 2,5-dihydroxybenzaldehyde ( 2 ) to be selected as an appropriate starting material in the synthesis 
 of epoxide 10 . a procedure reported by chen et 
 al . facilitated selective monoprotection 
 of 2 using pivaloyl 
 chloride to generate pivaloate ester 3 in good yield . the remaining hydroxy group then required protection orthogonal with 
 respect to the pivaloate ester ; hence , the p - methoxybenzyl 
 ( pmb ) group was selected for this purpose . the ability to cleave a 
 pmb ether under oxidative conditions , using reagents such as ceric(iv ) ammonium nitrate ( can ) , was anticipated , because this would allow selective removal of the pmb group in the 
 presence of the nitrile later in the synthesis . conversion of 
 aldehyde 4 to the desired nitrile was 
 achieved cleanly by oxidative amination in nearly 
 quantitative yield to give 5 . alkoxide - mediated removal 
 of the pivaloate ester then revealed phenol 6 , which 
 was subsequently submitted to mitsunobu coupling with 2-(allyloxy)ethanol 
 to generate ether 7 . the desired pmb - protected product 8 was isolated and subjected to can - mediated oxidative pmb group cleavage 
 conditions . unfortunately , this did not yield the desired phenol 8 , possibly due to instability of the molecule to can , for 
 example , through dealkylation of the aralkyl ether . re - examination of crude reaction products from the cyclopropanation 
 step revealed that apart from the expected pmb - protected product 9 , the oxidative conditions of the simmons smith procedure 
 had caused partial removal of the pmb group to afford phenol 8 directly , although in modest yield . consequently , phenol 8 was recovered , albeit as a side product and in relatively 
 low yield . alkylation of phenol 8 with rac - epichlorohydrin in the presence of tea afforded epoxide 10 in quantitative yield , with no further purification required . in 
 this preliminary study , although it was well understood that the s - form was the more pharmacologically active of the two 
 aryloxypropanolamine enantiomers , we elected to synthesize racemic 
 derivatives throughout , as a more expeditious and cost - effective route 
 to explore early sar , with the intention that favorable pharmacology 
 would prompt synthesis of individual stereoisomers if warranted . construction of aminophenol 13 required initial addition 
 of 1,2-ethanediamine ( 11 ) to 4-(benzyloxy)phenyl isocyanate . dropwise introduction of a solution of the isocyanate into a solution 
 containing an excess of 11 allowed selective mono - addition 
 to occur , as the product urea 12 was found to precipitate 
 on formation , allowing facile isolation and purification by filtration . conversely , the poor solubility of 12 in a variety of 
 appropriate solvents meant that attempted hydrogenolysis progressed 
 slowly . acidolytic o - benzyl deprotection was therefore 
 chosen as an alternative to give aminophenol 13 in good 
 yield . finally , aminolysis of epoxide 10 with 13 under neutral conditions , by reflux in propan-2-ol , afforded 1 after purification by flash column chromatography . table 1 and pharmacology discussion ) indicated a slightly 
 lower 1/2-selectivity than previously 
 reported . in addition to this , we noted 
 with interest that previous studies indicated that the presence of 
 substituents of the phenyl ring ortho to the oxypropanolamine 
 moiety did not improve 1/2-selectivity 
 but led to either increased or decreased affinity at both receptors simultaneously . because our aim was to devise 1-selective ligands , 
 we were therefore encouraged to explore whether removal of the cyano 
 group was a worthwhile modification because , if successful , it would 
 allow a more expeditious route into a range of new analogues . we therefore 
 embarked upon the more efficient synthesis of the decyanated analogue 
 of 1 ( 19 ) , which had not previously been 
 described , to assess the impact of the nitrile group on 1/2-receptor pharmacology ( scheme 1 ) . mitsunobu coupling of 2-(allyloxy)ethanol 
 with commercially available 
 4-(benzyloxy)phenol ( 14 ) furnished the desired phenylether 15 , whose terminal allyl ether function was efficiently converted 
 to the corresponding cyclopropylmethyl ether 16 using 
 simmons subsequent benzyl ether hydrogenolysis of 16 over pd(0 ) 
 on charcoal , using standard conditions , afforded phenol 17 in quantitative yield . alkylation of 17 was achieved 
 in moderate yield by initial deprotonation in aqueous 2 m naoh solution , 
 followed by heating in excess rac - epichlorohydrin 
 to afford oxirane 18 . finally , oxirane 18 was subjected to aminolysis with 13 under basic conditions 
 by refluxing in propan-2-ol . the desired aryloxypropanolamine 19 was obtained as the hydroformate salt , after plc and subsequent 
 preparative rp - hplc purification . pharmacological and in vivo 
 analysis of our early lead ligand 19 gave mixed results . 
 whereas its selectivity was somewhat 
 diminished compared to that of 1 ( cf . pharmacology results ) , 
 both compounds possessed significant partial agonism , rendering them 
 unsuitable for future therapeutic development as a selective 1-adrenoceptor antagonist . therefore , to explore the sars of 
 this class of molecule further and to ascertain if selectivity could 
 be enhanced while decreasing partial agonism , the more facile synthesis 
 of 19 now allowed access to new analogues in a more expeditious 
 manner . initially we tried to ascertain the effect of the alkoxyalkylether 
 and the phenylurea termini on affinity , selectivity , and partial agonism . 
 to explore the influence of the alkoxyalkoxy terminus on pharmacology , 
 and on the basis of established literature precedent , we synthesized 
 oxiranes 28a  c in moderate yield 
 ( scheme 2 ) , essentially 
 in the same manner as for 10 . although 2-ethoxyethanol 
 ( 25 ) was commercially available , the analogous 2-(cyclopentyloxy)ethanol 
 ( 21 ) and 2-(4-fluorophenethyloxy)ethanol ( 24 ) starting materials required independent synthesis ( scheme 2 ) . reagents and conditions : 
 ( a ) 
 nabh4 , zrcl4 , thf 05 c , 81% ; ( b ) 
 ( i ) nah , dmf 60 c ; ( ii ) chloroacetic acid , 60 c , 50% ; 
 ( c ) lialh4 , thf , 0 c , 67% ; ( d ) ph3p , 14 , di - tert - butyl azodicarboxylate or diethyl 
 azodicarboxylate , dcm , 3585% ; ( e ) h2 , 10% pd / c , 
 etoh , 68100% ; ( f ) ( i ) nah , dmf , 0 c  rt ; ( ii ) rac - epichlorohydrin , 7184% ; ( g ) ( i ) 2 m naoh(aq ) ; ( ii ) rac - epichlorohydrin , 60 c , 
 84% ; ( h ) 13 , propan-2-ol , reflux , 2129% . 2-(cyclopentyloxy)ethanol ( 21 ) was obtained 
 in good 
 yield from ketal 20 , according to the nabh4/zrcl4 reductive cleavage conditions reported by chary 
 et al . in the case of 2-(4-fluorophenethyloxy)ethanol 
 ( 24 ) , this entailed initial alkylation 
 of 4-fluorophenethyl alcohol ( 22 ) with chloroacetic acid 
 to give alkoxyacetic acid 23 . subsequent reduction was 
 achieved with lialh4 , to give alcohol 24 in 
 acceptable yield . the alcohols 21 , 24 , and 25 were subjected to mitsunobu coupling with 4-(benzyloxyphenol ) 
 ( 14 ) in a similar manner as previously described , to 
 give o - benzyl ethers 26a  c . subsequent alkylation of phenol 27c was carried out using the previously described method to give oxirane 28c . in the case of phenols 27a , b , nah was used as a base in anhydrous dmf , with subsequent alkylation 
 proceeding at room temperature to furnish oxiranes 28a , b . the target aryloxypropanolamines 29a  c were obtained in the free base form , from 
 oxiranes 28a  c through aminolysis 
 with 13 as previously detailed . encouraged by the 
 pharmacological data of the novel compounds possessing 
 the 2-(cyclopentyloxy)ethoxyphenyl terminus ( cf . table 4 and pharmacology discussion ) , 
 we kept this group constant to investigate the pharmacological effects 
 of modifying the phenylurea substituent at the opposite pole of the 
 molecule . this required synthesis of a small set of phenyl - substituted 
 1-(2-aminoethyl)-3-phenylureas ( scheme 3 ) . reagents and conditions : ( a ) o - tolylisocyanate ( 30 ) , dcm , 0 c  
 rt , 64% ; ( b ) di - tert - butyl dicarboxylate , dcm , 87% ; 
 ( c ) substituted phenylisocyanate , dcm , 0 c  rt , 46  
 95% ; ( d ) meoh / concd hcl , 80100% ; ( e ) phthalic anhydride , 150 
 c , 94% ; ( f ) ( i ) diphenylphosphoryl azide , tea , toluene ; ( ii ) 
 reflux ; ( iii ) 2-aminophenol or 3-aminophenol , 7576% ; ( g ) hydrazine 
 monohydrate , etoh , reflux ; ( ii ) acidic workup , 5160% ; ( h ) 33a  r , 10 m naoh(aq ) , propan-2-ol , 
 reflux , 731% ; ( i ) 33t  u , 
 et3n , propan-2-ol , reflux , 2125% . in accordance with the synthesis of 12 , attempted 
 condensation of o - tolylisocyanate ( 30 ) with an excess of ethylenediamine ( 11 ) afforded urea 33b in only modest yield , alongside the undesirable bis - urea 
 side product . to avoid this complication with the remaining analogues , tert - butyl-2-aminoethylcarbamate ( 31 ) selective mono - boc protection of ethylenediamine 
 ( 11 ) was accomplished in excellent yield using a literature 
 method to give amine 31 ( scheme 3 ) . the stoichiometric condensation of 31 with corresponding substituted phenylisocyanates permitted preparation 
 of the boc - protected intermediates 32a and 32c  r in moderate to good yield , with the pure products 
 being easily isolated by filtration following precipitation with hexanes . boc - deprotection of these intermediates with concentrated hcl in methanol 
 proceeded smoothly and in excellent yields , affording the desired 
 amines 33a and 33c  r as their respective hydrochloride salts . the remaining 1-(2-aminoethyl)-3-(hydroxyphenyl)urea 
 analogues ( 33t  u ) required an alternative 
 synthetic strategy , due to the lack of commercially available phenylisocyanate 
 starting materials . however , the availability of 2-amino and 3-aminophenol 
 allowed urea synthesis via condensation with the appropriate alkylisocyanates 
 ( scheme 3 ) . using -alanine ( 34 ) as a starting material , 
 the conversion of the primary amine to the phthalimide eliminated 
 possible side reactions during isocyanate formation ; monoacylated 
 amine protecting groups still have the propensity to attack the isocyanate 
 functionality . phthalimide protection 
 of 34 was effected through a solvent - free method , by 
 heating directly with phthalic anhydride to give acid 35 , which was converted to the corresponding 
 acyl azide with the aid of diphenylphosphoryl azide in the presence 
 of tea ; careful reflux of this intermediate promoted curtius rearrangement 
 to the isocyanate . the isocyanate solution was divided into two equal 
 portions , prior to addition of either 2- or 3-aminophenol , producing 
 protected intermediates 36a and 36b . the 
 desired amines 33 t and 33u were once again 
 isolated as the hydrochloride salts after standard hydrazinolytic 
 cleavage of the phthalimide group and subsequent acidic workup . finally , 
 aminolysis of oxirane 28a with amines 33a  u and subsequent plc purification afforded the 
 target aryloxypropanolamine compounds 37a  u ( scheme 3 ) . we initially evaluated and compared the affinity 
 of 1 with that of the decyanated analogue 19 at the human 1- , 2- , and 3-adrenoceptors , each stably expressed in chinese hamster ovary 
 cells . the kd values for -adrenoceptor 
 binding of h - cgp 12177 in these cell lines have previously 
 been established and are 0.42 , 0.17 , and 109.2 nm for the 1- , 2- , and 3-adrenoceptors , 
 respectively.1 completely 
 inhibited specific binding to the human 1-adrenoceptor 
 to yield a log kd of 8.04  
 0.03 , n = 9 , and 5.29  0.04 , n = 11 , for the human 2-adrenoceptor , 
 thus giving a selectivity for the 1-adrenoceptor 
 of 562-fold ( figure 1 ; table 1 ) . likewise , 19 was also shown to inhibit specific 
 binding to the human 1-adrenoceptor to yield a log kd of 7.49  0.03 . however , its 
 affinity for the human 2-adrenoceptor was so low 
 that measurement of complete inhibition of specific h - cgp 
 12177 binding was not possible ( figure 1d ) , 
 so that affinity ( kd values ) could not 
 be calculated by this method . the affinity of both ligands for the 
 human 3-adrenoceptor was also too low to establish 
 reliable kd values from this method . inhibition 
 of h - cgp 12177 binding to whole cells by 
 ( a , b ) 1 and ( c , d ) 19 in ( a , c ) cho 1 cells and ( b , d ) cho 2 cells . bars represent 
 total h - cgp 12177 binding , and nonspecific binding was 
 determined in the presence of 10 m propranolol . the concentration 
 of h - cgp 12177 present in each case was 0.73 nm . these single 
 experiments are representative of ( a ) 9 and ( b  d ) 11 separate 
 experiments . log kd values were obtained from h - cgp 12177 whole 
 cell binding studies in cho cells stably expressing the human 1 or 2-adrenoceptors . incomplete inhibition of specific h - cgp12177 
 binding meant that generation of an affinity value 
 ( log kd value ) by this method was not 
 possible . because 
 quantifiable antagonist affinity was difficult to determine 
 for 19 at the human 2- and 3-adrenoceptors , an alternative assay was employed to establish 
 accurate values via measurement from the shift of an agonist response 
 ( figure 2 ; table 2 ) . h - camp accumulation in response to cimaterol in ( a ) 
 cho 1 cells , ( b ) cho 2 cells , 
 and ( c ) cho 3 cells in the absence and presence 
 of 19 . bars show basal h - camp accumulation , 
 that in response to 10 m isoprenaline , and that in response 
 to 30 nm , 100 nm , 300 nm , 1 m , 3 m , and 10 m 19 alone . data points are the mean  sem of triplicate 
 values from single experiments that are representative of seven separate 
 experiments in each case . cimaterol was chosen as the agonist as it is a chemically 
 stable 
 nonselective agonist and thus could be used across all three human 
 -adrenoceptors and because cimaterol agonist responses are 
 also readily inhibited by classical -blockers , suggesting that 
 its primary action is agonism of the catecholamine conformation of 
 the human 1-adrenoceptor . cimaterol stimulated responses were 96.9  1.1% ( log ec50 = 8.13  0.03 , n = 9 ) , 98.9 
  1.7% ( log ec50 = 8.78  0.06 , n = 9 ) , and 89.6  1.9% ( log ec50 = 6.62 
  0.06 , n = 9 ) of the isoprenaline maximum response 
 at the human 1- , 2- , and 3-adrenoceptors , respectively ( figure 2 ) . these agonist responses were antagonized by both 1 and 19 ( table 2 ) . as both ligands 
 examined demonstrated clear partial agonist stimulatory effects ( e.g. , 
 figure 2 ) , the data were analyzed by the partial 
 agonist method of stephenson . log kd values were obtained from h - camp accumulation 
 assays following inhibition of the cimaterol agonist response in cho 
 cells stably expressing the human 1- , 2- , or 3-adrenoceptors . given the significant degree 
 of agonist activity seen above , full 
 concentration response relationships were investigated for the ligands . 1 caused an agonist response at the human 1-adrenoceptor that was 37.1  2.2% of the maximum response to 
 isoprenaline ( n = 5 ) . agonist responses were 
 also seen at the human 2- and 3-adrenoceptors ; however , the top of the concentration response curve 
 was not reached in each case . at the maximum concentration of 10 m , 1 stimulated responses that were 15.2  0.7% ( 2 ) and 8.6  0.4% ( 3 ) of the isoprenaline 
 maximum at each receptor . log ec50 values and % isoprenaline maximal responses were obtained from h - camp accumulation for cells expressing the human 1-adrenoceptor . for 
 the human 2- and 3-adrenoceptors , the top of the agonist concentration 
 response curve was not obtained even with the maximum concentration 
 of ligand . in these instances , the percentage of isoprenaline response 
 is given for the response at 10 m of ligand or , in the case 
 of xamoterol , 100 m . to assess the potential clinical effect of this degree of selectivity 
 and partial agonism , these parameters were investigated in a freely 
 moving conscious rat model . previous studies with this model have 
 shown that the heart rate response to isoprenaline is solely a 1-mediated response and that the hindquarters vascular conductance 
 is solely a 2-mediated response . we employed the hydrochloride salt of 19 ( 2 
 mg / kg iv bolus , 1 mg / kg / h iv infusion ) and observed that it significantly 
 inhibited the 1-mediated heart rate responses to 
 isoprenaline ( at 40 and 120 ng / kg / min , 3090 min following 
 administration ) while having no effect on the 2-mediated 
 hindquarters response ( n = 4 rats ) , consistent with 
 this compound s 1-adrenoceptor selectivity 
 ( figure 3 ) . in addition , the ligand caused 
 an increase in basal heart rate ( from 424  9 to 465  3 
 beats / min , an increase that corresponds to 44% of the response to 
 120 ng / kg / min isoprenaline , n = 4 animals ) , in keeping 
 with the partial agonist actions observed in the in vitro cell studies 
 with 19 . absolute values for heart rate ( a ) and hindquarters conductance 
 ( b ) in conscious freely moving rats ( n = 4 per group ) 
 before and at the end of 3 min infusions of isoprenaline ( 12 , 40 , 
 and 120 ng / kg / min ) . isoprenaline responses were measured before and 
 at intervals after saline or 19 ( hcl salt ) administration . 19 ( hcl salt ) was given as a 2 mg / kg bolus followed by 1 mg / kg / h 
 infusion for 90 min , and isoprenaline responses were measured during 
 the infusion ( 3090 min ) and after the 19 ( hcl 
 salt ) infusion was turned off ( 45 and 2425 h ) . isoprenaline 
 responses measured in saline - treated animals at 3090 min , 
 45 h , and 2425 h were highly reproducible , but for 
 the sake of clarity only the 3090 min time point is shown 
 here . to investigate whether the structural extremities 
 of our lead compounds could be modified to improve affinity and selectivity 
 while attenuating partial agonism , we employed an iterative screening 
 cascade approach . initially we used a h - cgp 12177 whole 
 cell binding assay to ascertain antagonist affinity measurements ( table 4 ) . log kd values were obtained from h - cgp 12177 whole 
 cell binding studies in cho cells stably expressing the human 1- or 2-adrenoceptors . incomplete inhibition of specific h - cgp12177 
 binding meant that generation of an affinity value 
 ( log kd value ) by this method was not 
 possible . informed from 
 previously established sars , we explored 
 three variants of the p - alkoxyalkoxy 
 side chain present in 1 and 19 , namely a para - positioned 2-(cyclopentyloxy)ethoxy- ( 29a ) , a 2-(4-fluorophenethyloxy)ethoxy- ( 29b ) , and an 2-(ethyloxy)ethoxy- 
 ( 29c ) moiety . because the 2-(cyclopentyloxy)ethoxy side 
 chain displayed the best combination of an increase in both binding 
 affinity and 1/2-selectivity when 
 compared to 19 , we retained this moiety to undertake 
 a sar evaluation of the phenylurea portion of the molecule ( table 5 ) . the unsubstituted phenylurea ( 37a ) displayed a loss in affinity at both the 1- and 
 2-adrenoceptors yet maintained good 1-selectivity . insertion onto the phenyl ring of individual 
 electron - withdrawing or electron - donating groups at the ortho- , meta- , or para - positions displayed 
 a set of measurable trends . in general , the ligand binding affinity 
 remained within an order of magnitude for the respective adrenoceptor 
 subtypes ( 1 log kd between 
 6.94 and 8.17 m ; 2 log kd between 5.52 and 6.54 m ) with all ligands 
 showing selectivity for the 1-subtype . in most cases 
 ( 37b , 37e , 37k , 37n , 37q , and 37 t ) an ortho - positioned substituent resulted in the weakest 1-adrenoceptor binding affinity within each three - ligand subset alongside 
 the smallest 1/2-selectivity . 
 the only exception to this observation was that of the o - fluoro derivative ( 37h ) , which , while displaying the 
 second highest affinity of the three ligands ( 37h  j ) for the 1-adrenoceptor , did possess the 
 greatest , albeit modest , 1/2-selectivity 
 ( 76-fold ) . log kd values were obtained from h - cgp 12177 whole 
 cell binding studies in cho cells stably expressing the human 1- or 2-adrenoceptors . the optimal ligands in terms of 
 both affinity and selectivity were 
 substituted in the meta- or para - positions with either a chlorine atom ( 37l and 37 m ) or a hydroxyl group ( 29a and 37u ) or , as mentioned above , were unsubstituted ( 37a ) . to cross - validate these results and attempt to determine 3-adrenoceptor affinity , we repeated the alternative assay 
 for determining affinity whereby the shift of a cimaterol - mediated 
 dose  response curve is measured , focusing upon the best ligands 
 from the phenylurea sar study and re - evaluating the original alkoxyalkoxy 
 compounds ( table 6 ) . log kd values were obtained from h - camp accumulation 
 assays following inhibition of the cimaterol agonist response in cho 
 cells stably expressing the human 1- , 2- , or 3-adrenoceptors . interestingly , this assay gave values 
 for ligand affinity ( and 
 selectivity ) slightly higher than that of the binding assay , for example , 
 with 29a 1-selectivity 478-fold ( over 
 2 ) in the binding assay but 513-fold in the camp 
 assay and with 37l 331-fold in the binding assay and 
 537-fold in the camp assay , similar to what was observed with previous 
 ligands ( tables 1 and 2 ) . this method therefore provides a similar pattern of 1- versus 2-selectivity , allowing comparison 
 between different chemical analogues . even using this method , the 
 affinity at the human 3-adrenoceptor once again 
 could not be assessed , because the maximum concentration of test compounds 
 did not cause a sufficient shift of the cimaterol concentration response 
 curve ( e.g. , tables 2 and 6 ; figure 2 ) . although the affinity and selectivity 
 profiles for many of the ligands 
 were extremely encouraging , we were also aware that the original lead 
 compounds ( 19 and 1 ) also displayed significant 
 partial agonism at the -adrenoceptors . we therefore investigated 
 the full concentration response relationships for the best ligands 
 ( table 7 ) . log ec50 values and % isoprenaline maximal responses were obtained from h - camp accumulation for cells expressing the human 1-adrenoceptor . for 
 the human 2- and 3-adrenoceptors , the top of the agonist concentration 
 response curve was not obtained even with the maximum concentration 
 of ligand . in these instances , the percentage of isoprenaline response 
 is given for the response at 10 m of ligand . whereas the affinities of the second- 
 and third - generation ligands 
 were comparable to those of 1 and 19 , there 
 was a modest drop in efficacy at the 1-adrenoceptor , 
 suggesting a marginal attenuation of partial agonism with these compounds . interestingly , the one exception to this observation was 29c , which contained the linear 2-(ethyloxy)ethoxy side chain . in this 
 example , the affinity at the 1-adrenoceptor was 
 an order of magnitude less than that of 1 and the 2-(4-fluorophenethyloxy)ethoxy 
 derivative ( 29b ) , yet this compound displayed a similar 
 efficacy profile . as the 1-adrenoceptor 
 is now accepted to exist in at least two agonist conformational states , it was important to assess at which state of the 1-adrenoceptor these novel -adrenoceptor ligands were exerting 
 their partial agonist actions . cgp 20712a ( neutral high - affinity 1-adrenoceptor antagonist , capable of inhibiting both conformational 
 states of the 1-adrenoceptor but with different 
 affinities ) measured in the presence 
 of cimaterol yielded a high affinity value of log kd = 9.48 ( supporting information table 1 ) compatible with the agonist activity occurring via the 
 primary catecholamine site of the receptor . cgp 12177 ( log ec50 = 7.91  0.04 , 58.2  2.6% isoprenaline 
 maximum , n = 5 ) was antagonized by cgp 20712a to 
 yield a much lower affinity ( log kd = 
 7.22  0.03 , supporting information table 1 ) , demonstrating that a far higher concentration of cgp 20712a 
 is required to block the cgp 12177 agonist response . the agonist activity 
 of cgp 12177 was therefore occurring via the secondary site of the 
 1-adrenocptor ( supporting information figure 1 ) . the agonist response of 
 our compounds was therefore inhibited by cgp 20712a to determine the 
 site of their agonist actions . the affinity values for cgp 20712a 
 were all high ( log kd values between 9.51 
 and 9.09 , supporting information table 1 ) . suggesting that , like the partial agonist xamoterol and 
 ici 89406 ( and unlike cgp 12177 ) , our new partial agonists were indeed 
 achieving their agonist actions via the catecholamine conformation 
 of the receptor . the human 3-adrenoceptor , similar 
 to the human 1-adrenoceptor , has also been demonstrated 
 to exist in two conformational states . however , given the low affinity and efficacy of our novel compounds 
 for the human 3-adrenoceptor , it was not possible 
 to determine at which receptor conformational state the 3-agonist responses were occurring . despite 70 years of experience and the availability of many adrenoceptor 
 ligands , there remain very few truly selective ligands for the 1-adrenoceptor . to date , cgp 20712a is a widely known and truly 
 selective 1-antagonist , whereas ici 118551 is a 
 2-selective antagonist . however , although 
 several very highly 2-selective agonists exist ( e.g. , 
 salmeterol and formoterol ) , there are no selective 1-agonist compounds  noradrenaline , dobutamine , xamoterol , 
 and ici 89406 all having relatively poor selectivity . here we have characterized the pharmacological properties of 1 and some novel derivatives and thus present the most selective 
 1-partial agonists reported to date . 1 was confirmed as being a truly selective 1-adrenoceptor ligand with a 1 over 2 selectivity of 562-fold as measured by whole - cell binding . the first analogue of 1 made was 19 , a decyanated 
 version of the molecule , and the pharmacological effect of this maneuver 
 was to reduce affinity at both human 1- and 2-adrenoceptors . it was clear from the antagonist studies 
 that 1 and 
 its derivative 19 had substantial agonist activity at 
 the human -adrenoceptors . given this observation , we investigated 
 the potential clinical impact of this degree of partial agonism in 
 a fully conscious rat model . infusion of the hydrochloride salt of 19 resulted in a substantial increase in the basal ( 1-mediated ) heart rate , in keeping with its 1-partial agonist effects , followed by inhibition of the 1-heart rate response to the isoprenaline ( compare figure 2a in cho 1 cells and figure 3a in conscious rats ) . no effect on basal hindquarters 
 vascular conductance or on the 2-mediated hindquarters 
 response to isoprenaline was observed , consistent with high 1-selectivity , thereby demonstrating the 1-selective partial agonist effects in vivo . this agonist effect is 
 significant because -ligands with agonist properties ( e.g. , 
 xamoterol ) have been shown to be less beneficial and , at times , even 
 detrimental if used long - term in cardiovascular disease . thus , although 19 maintained its 1-selectivity in vivo , its partial 
 agonist properties were too great for it to be pursued as a potential 
 clinical compound . given 19 had reduced affinity 
 and selectivity compared 
 to 1 , we designed a series of analogues of the molecule 
 looking for evidence of structure  activity relationships related 
 to affinity , selectivity , and partial agonism . substitution of the 
 alkoxyalkoxy side chain afforded ligands with increased 1-adrenoceptor affinity ( 29a and 29b ) . because all molecules remained partial agonists at the human 1-adrenoceptor ( table 7 ) , we therefore 
 examined the phenylurea portion of the molecule in an attempt to attenuate 
 this pharmacology . generally , regardless of the substituent , substitution 
 in the meta- and para - positions 
 of the phenyl ring afforded higher 1-affinity ( and 
 therefore higher 1-selectivity ) than substitutions 
 at the ortho - position . once again , however , these 
 ligands retained their 1-selective partial agonist 
 activity , and the agonist actions were demonstrated to occur via the 
 catecholamine conformation . in summary , 1 and the 
 new family of ligands that we 
 have generated are 1-selective partial agonists . 
 compared with currently published ligands , for example , xamoterol 
 and ici 89406 , 1 and some of our novel analogues are 
 the most 1-selective agonists to date and will prove 
 to be useful pharmacological tools ; however , the observed clinically 
 deleterious effects of significant partial agonism in -blockers preclude any of these ligands 
 from further clinical development . however , this preliminary body 
 of work clearly demonstrates that it is possible to generate more 
 1-selective molecules than those currently available 
 and in clinical use . molecules of higher selectivity , but devoid of 
 partial agonist effects , should prove to be a very powerful treatment 
 for people with concomitant cardiovascular and respiratory disease 
 and is the focus of ongoing work within our laboratories . chemicals 
 and solvents were 
 purchased from standard suppliers and used without further purification . merck kieselgel 60 , 230400 mesh , for flash column chromatography 
 ( fcc ) , was supplied by merck kgaa ( darmstadt , germany ) , and deuterated 
 solvents were purchased from goss international limited ( england ) 
 and sigma - aldrich co. ltd . reactions were 
 monitored by thin layer chromatography on commercially available precoated 
 aluminum - backed plates ( merck kieselgel 60 f254 ) . a solution 
 of ninhydrin ( in ethanol ) was used to visualize primary and secondary 
 amines . all organic extracts collected after aqueous workup procedures 
 were dried over anhydrous mgso4 or na2so4 before gravity filtration and evaporation to dryness . organic 
 solvents were evaporated in vacuo at 40 c ( water bath 
 temperature ) . purification using preparative layer chromatography 
 ( plc ) was carried out using fluka silica gel 60 pf254 containing 
 gypsum ( 200 mm  200 mm  1 mm ) . melting points ( mp ) were recorded on a reichert 7905 apparatus 
 or perkin - elmer pyris 1 differential scanning calorimeter and were 
 uncorrected . fourier transform infrared ( ft - ir ) spectra were recorded 
 as thin films or kbr disks in the range of 4000500 cm using an avatar 360 nicolet ft - ir spectrophotometer . hig- resolution mass spectra ( hrms)time - of - flight electrospray 
 ( tof es  ) were recorded on a waters 2795 separation module / micromass 
 lct platform . h nmr spectra were recorded on a bruker - av 
 400 at 400.13 mhz . chemical shifts (  ) are recorded in parts per million ( ppm ) 
 with reference to the chemical shift of the deuterated solvent / an 
 internal tetramethylsilane ( tms ) standard . coupling constants ( j ) and carbon  fluorine coupling constants ( jcf ) are recorded in hertz and the significant 
 multiplicities described by singlet ( s ) , doublet ( d ) , triplet ( t ) , 
 quadruplet ( q ) , broad ( br ) , multiplet ( m ) , doublet of doublets ( dd ) , 
 and doublet of triplets ( dt ) . spectra were assigned using appropriate 
 cosy , distortionless enhanced polarization transfer ( dept ) , hsqc , 
 and hmbc sequences . analytical reverse - phase high - performance 
 liquid chromatography 
 ( rp - hplc ) was performed on a waters millenium 995 lc using both system 
 1 and system 2 described below and was used to confirm that all final 
 products were 95% pure . phenomenex onyx monolithic 
 reverse phase 
 c18 column ( 100  4.6 mm ) , a flow rate of 3.00 ml / min 
 and uv detection at 287 nm . solvent a , 0.1% formic acid ( fa ) in water ; solvent b , 
 0.1% fa in mecn . waters symmetry reverse 
 phase c18 column ( 75  4.6 mm ) , a flow rate of 1.00 
 ml / min , and uv detection 
 at 287 nm . linear gradient 595% solvent b over 20 min . solvent 
 a , 0.1% fa in water ; solvent b , 0.1% fa in meoh . preparative hplc 
 was performed using a phenomenex onyx monolithic reverse phase c18 column ( 100  10 mm ) , a flow rate of 14.10 ml / min , 
 and uv detection at 287 nm . solvent a , 0.1% fa in water ; solvent b , 0.1% fa in 
 mecn . a solution 
 of 4-(benzyloxy ) phenylisocyanate ( 3.739 g , 16.61 mmol ) in anhydrous 
 dcm ( 30 ml ) was dripped into a flask containing vigorously stirred 
 1,2-ethanediamine ( 11 ) ( 6 ml , 89.80 mmol , 5.4 equiv ) 
 under nitrogen . instant precipitation of a white solid was noted , 
 and the reaction was allowed to stir for a further 3 h after addition 
 of isocyanate solution was complete . after removal of all volatiles 
 under reduced pressure , the crude solid was washed with et2o , before drying to give 4.472 g ( 94% ) of white solid : mp 147149 
 c ; ft - ir h , str ) , 2932 , 
 2864 ( alkyl c  h , str ) , 1642 ( urea c = o , str ) , 1604 ( 1 h , bend ) , 1111 ( c  o , str ) , 830 ( aryl c  h , 
 bend , para - disubstituted ring ) , 741 , 697 ( aryl c  h , 
 bend , phenyl ring ) ; h nmr ( dmso - d6 )  8.48 ( s , 1h , nhar ) , 7.317.44 ( m , 5h , aromatic 
 benzyl ch ) , 7.28 , 6.88 ( d , j = 9.0 hz , 2  2h , para - disubstituted ring ) , 6.24 ( t , j = 
 5.2 hz , 1h , nhconhar ) , 5.02 ( s , 2h , phch2o ) , 4.27 ( br s , 2h , nh2 ) , 3.103.17 
 ( m , 2h , ch2nh ) , 2.67 ( t , j = 6 hz , ch2nh2 ) ; c nmr ( dmso - d6 )  155.72 ( c = o ) , 
 152.88 , 137.37 , 133.97 ( 4 c ) , 128.37 , 127.71 , 127.63 ( benzyl 
 ch ) , 119.29 , 114.87 ( aryl ch ) , 69.37 ( benzyl ch2 ) , 40.87 
 ( ch2nh2 ) , 40.38 ( chnh ) ; m / z hrms ( tof es ) c16h20n3o2 [ mh ] calcd 286.1550 ; found 286.1547 . 1-(2-aminoethyl)-3-(4-(benzyloxy)phenyl ) 
 urea ( 12 ) ( 113 mg , 0.40 mmol ) was stirred overnight in 
 a solution of concd hcl ( 10 ml ) . the mixture was concentrated under 
 reduced pressure and the residue redissolved in water ( 10 ml ) before 
 neutralization with aqueous 0.5 m naoh . after reconcentration , the 
 residue was dissolved in the minimum amount of meoh and filtered ( gravity ) , 
 before purification by plc ( eluent 37% aqueous nh3/meoh / dcm 
 2:25:73 ) . this gave 56 mg ( 73% ) of a brown semisolid : ft - ir 3339 ( 1 h , str ) , 3118 ( br , o  h , str ) , 1643 ( urea c = o , 
 str ) , 1615 ( 1 amine n h , bend ) , 1570 ( aryl , str ) , 836 
 ( aryl c  h , bend , para - disubstituted ring ) ; h nmr ( meod - d4 )  7.17 ( d , j = 8.7 hz , 2h , aryl 3-h and 5-h ) , 6.73 ( d , j = 8.7 hz , 2h , aryl 2-h and 6-h ) , 3.45 ( t , j = 5.6 
 hz , 2h , ch2nh(c = o)nh ) , 3.05 ( t , j = 6.0 hz , 2h , ch2nh2 ) ; c nmr ( meod - d4 )  
 159.38 ( 4 aryl 4-c ) , 154.70 ( c = o ) , 132.02 ( 4 aryl 
 1-c ) , 123.61 , 116.32 ( aryl ch ) , 41.73 ( ch2nh2 ) , 38.79 ( ch2nh(c = o)nh ) ; m / z hrms ( tof es ) c9h14n3o2 [ mh ] calcd 196.1081 ; 
 found 196.1081 . 2-(cyclopentyloxy)ethanol ( 21 ) ( 3.751 
 g , 28.81 mmol ) , 
 triphenylphosphine ( 9.448 g , 36.02 mmol , 1.25 equiv ) , and 4-(benzyloxy)phenol 
 ( 14 ) ( 5.769 g , 28.81 mmol , 1 equiv ) were dissolved in 
 dcm ( 70 ml ) . di - tert - butyl azodicarboxylate ( 8.294 
 g , 36.02 mmol , 1.25 equiv ) in dcm ( 20 ml ) was added dropwise to the 
 reaction mixture , which was allowed to stir overnight . after removal 
 of approximately half of the solvent from the reaction mixture under 
 reduced pressure , the resulting slurry was diluted with hexanes ( 100 
 ml ) and washed with aqueous 1 m hcl ( 2  50 ml ) , aqueous 1 m 
 naoh ( 2  50 ml ) , water ( 2  50 ml ) , and brine ( 1  
 50 ml ) . the organic layer was concentrated and redissolved in dcm 
 ( 30 ml ) . the flask was left in the freezer for 1 h before 
 filtration of the precipitate and washing with hexanes and et2o . after concentration of the filtrate , purification was achieved 
 via fcc ( eluent et2o / hexanes 10:90 ) to give 6.75 g ( 75% ) 
 of a clear colorless oil : ft - ir 2870 , 2954 ( alkyl c  h , str ) , 
 1507 ( aryl , str ) , 1109 ( c  o  c , str ) , 824 ( aryl c  h , 
 bend , para - disubstituted ring ) , 738 , 696 ( aryl c  h 
 bend , phenyl ring ) ; h nmr  7.34  7.48 ( m , 
 5h , aromatic benzyl ch ) , 6.91 , 6.96 ( d , j = 9.2 hz , 
 2  2h , aryl - dioxy ring ) , 5.04 ( s , 2h , phch2o ) , 4.09 ( t , j = 4.9 hz , 2h , ch2oarobn ) , 4.024.06 ( m , 1h , pe ch ) , 
 3.76 ( t , j = 5.3 hz , 2h , ch2ch2oarobn ) , 1.731.84 ( m , 6h , pe ch2 ) , 1.561.63 ( m , 2h , pe ch2 ) ; c nmr  153.02 , 153.26 ( 4 c , aryl - dioxy 
 ring ) , 137.29 ( 4 benzyl c ) , 127.44 , 127.83 , 128.50 ( benzyl ch ) , 
 115.62 , 115.68 ( ch aryl - dioxy ring ) , 81.89 ( pe ch ) , 70.55 
 ( benzyl ch2 ) , 68.16 ( ch2oarobn ) , 
 67.28 ( ch2ch2oarobn ) , 32.27 
 ( 2-c and 5-c pe ring ) , 23.55 ( 3-c and 4-c pe ring ) ; m / z hrms ( tof es ) c20h25o3 [ mh ] calcd 313.1798 ; found 313.1766 . 1-(2-(cyclopentyloxy)ethoxy)-4-(benzyloxy)benzene 
 ( 26a ) ( 6.326 g , 20.25 mmol ) was hydrogenated according 
 to the general procedure for o - benzyl deprotection 
 to give the title compound in quantitative yield as a clear colorless 
 oil : ft - ir 3381 ( br , o  h , str ) , 2960 , 2871 ( alkyl c  h , 
 str ) , 1510 ( aryl , str ) , 1104 ( c  o  c , str ) , 827 ( aryl 
 c  h , bend , para - disubstituted ring ) ; h nmr  7.60 ( br s , 1h , oh ) , 6.69 , 6.73 ( d , j = 9.2 hz , 2  2h , para - disubstituted 
 phenol ) , 3.963.99 ( m , 3h , ch , ch2oar ) , 3.70 ( t , j = 5.0 hz , 2h , peoch2 ) , 1.621.78 ( m , 6h , pe 
 ch2 ) , 1.451.53 ( m , 2h , pe ch2 ) ; c nmr  150.05 , 
 152.01 ( 4 c ) , 115.53 , 115.88 ( ch phenolic ring ) , 82.05 ( pe ch ) , 67.82 ( ch2oar ) , 67.11 
 ( ch2ch2oar ) , 32.87 ( 2-c and 
 5-c pe ring ) , 23.20 ( 3-c and 4-c pe ring ) ; m / z hrms ( tof es ) c13h17o3 [ m  h ] calcd 221.1183 ; found 221.1191 . nah 60% suspension in mineral oil ( 863 mg , equivalent 
 to 518 mg 
 of nah , 21.58 mmol , 1.1 equiv ) was suspended in anhydrous dmf ( 20 
 ml ) with stirring under a nitrogen atmosphere . after 5 min , 4-(2-(cyclopentyloxy)ethoxy)phenol 
 ( 27a ) ( 4.360 g , 19.61 mmol ) in anhydrous dmf ( 20 ml ) 
 was added dropwise , with the vessel cooled over an ice bath . this 
 mixture was then allowed to stir at rt for 20 min before addition 
 of rac - epichlorohydrin ( 15.34 ml , 196.10 mmol , 10 
 equiv ) . after removal of all volatiles under reduced pressure , 
 the crude residue was partitioned between water ( 30 ml ) and et2o ( 30 ml ) and the aqueous layer washed again with et2o ( 3  30 ml ) . the combined organic extracts were concentrated 
 before purification through a silica plug ( initial wash with hexanes , 
 followed by etoh / dcm 5:95 ) to give 4.558 g ( 84% ) of a clear yellow 
 oil : ft - ir 3052 ( epoxide c  h , str , weak ) , 2961 , 2870 ( alkyl 
 c  h , str ) , 1508 ( aryl , str ) , 1110 ( c  o  c , str ) , 
 828 ( aryl c  h , bend , para - disubstituted ring ) ; h nmr  6.80 ( s , 4h , aryl c  h ) , 4.11 ( dd , j = 11.1/3.1 hz , 1h , aroch2ch ) , 
 3.99 ( t , j = 4.9 hz , 2h , ch2oar ) , 3.923.96 ( m , 1h , pe ch ) , 3.82 ( dd , j = 11.1/5.7 , 1h , aroch2ch ) , 
 3.67 ( t , j = 5.3 hz , 2h , peoch2 ) , 3.253.29 ( m , 1h , epoxide ch ) , 2.82 ( d , j = 4.9/4.9 hz , 1h , epoxide 
 ch2 ) , 2.67 ( dd , j = 5.0/2.7 
 hz , 1h , epoxide ch2 ) , 1.58  1.77 
 ( m , 6h , pe ch2 ) , 1.41  
 1.56 ( m , 2h , pe ch2 ) ; c nmr  152.65 , 153.36 ( 4 c ) , 115.46 , 115.53 
 ( aryl ch ) , 69.35 ( aroch2ch ) , 68.08 ( ch2oar ) , 67.18 ( ch2ch2oar ) , 50.13 ( epoxide ch ) , 44.49 ( epoxide ch2 ) , 32.17 ( 2-c and 5-c pe ring ) , 23.45 ( 3-c and 4-c pe ring ) ; m / z hrms ( tof 
 es ) c16h22nao4 [ mna ] calcd 301.1410 ; found 301.1414 . 2-((4-(2-(cyclopentyloxy)ethoxy)phenoxy)methyl)oxirane 
 ( 28a ) was opened with 1-(2-aminoethyl)-3-(4-hydroxyphenyl)urea 
 ( 13 ) as described in the general procedure for aminolysis 
 of oxiranes ( cf . supporting information ) . yield : 29% , white amorphous solid ; mp 135138 c ; 
 ft - ir 3311 ( br , o  h , str ) , 2929 , 2869 ( alkyl c  h , str ) , 
 1636 ( urea c = o , str ) , 1509 , 1568 ( aryl , str ) , 1111 ( c  o  c , 
 str ) , 820 ( aryl c  h , bend , para - disubstituted 
 ring ) ; h nmr ( dmso - d6 )  
 8.91 ( br s , 1h , phenol ) , 8.20 ( s , 1h , nh(c = o)nhar ) , 7.13 ( d , j = 8.8 hz , 2h , aryl c  h ortho to urea ) , 6.84 ( s , 4h , aryl - dioxy ring ) , 6.62 ( d , j = 8.8 hz , 2h , aryl c  h ortho to 
 phenol ) , 6.02 ( t , j = 5.4 hz , 1h , nh(c = o)nhar ) , 4.96 ( br s , 1h , nh ) , 3.793.97 ( m , 6h , 
 ch2oar , pe ch , ch(oh ) , aroch2 ) , 3.61 
 ( t , j = 4.8 hz , 2h , peoch2 ) , 3.123.16 ( m , 2h , nhch2ch2 ) , 2.56  2.71 ( m , 4h , ch(oh)ch2nh , nhch2ch2 ) , 1.541.69 ( m , 6h , pe ch2 ) , 1.421.51 ( m , 2h , pe ch2 ) ; c nmr ( dmso - d6 )  152.74 , 152.49 , 151.85 , 132.14 ( aryl 4 c ) , 155.65 
 ( c = o ) , 119.72 ( aryl c  h ortho to urea ) , 
 115.33 ( ch aryl - dioxy ring ) , 115.04 ( aryl c  h ortho to phenol ) , 80.84 ( pe ch ) , 71.23 ( aroch2 ) , 68.08 ( ch(oh ) ) , 67.71 ( ch2oar ) , 66.72 ( peoch2 ) , 52.20 ( ch(oh)ch2nh ) , 49.38 ( nhch2ch2 ) , 39.15 ( nhch2ch2 ) , 
 31.78 ( 2-c and 5-c pe ring ) , 23.12 ( 3-c and 4-c pe ring ) ; m / z hrms ( tof es ) c25h36n3o6 [ mh ] calcd 474.2599 ; found 474.2578 ; rp - hplc rt 3.17 ( system 1 ) , 10.30 ( system 3 ) . fetal calf serum was from 
 paa laboratories ( teddington , middlesex , uk ) . white - sided view plates 
 were supplied by thermo fisher scientific ( basingstoke , uk ) . microscint 
 20 scintillation fluid and ultima gold xr scintillation fluid were 
 from perkinelmer . radioligands ( h - cgp 12177 , h - adenine , and c - camp ) were from amersham international 
 ( buckinghamshire , uk ) . ici 89406 , cimaterol , and cgp 20712a were from 
 tocris life sciences ( avonmouth , uk ) . all other reagents were from 
 sigma chemicals ( poole , dorset , uk ) . cho - k1 stably expressing either the 
 human 1 ( 1146fmol / mg protein ) , the human 2 ( 466fmol / mg protein ) , or the human 3-adrenoceptor 
 ( 790fmol / mg protein ) was used throughout this study . cells were grown in dulbecco s modified eagle s 
 medium nutrient mix f12 ( dmem / f12 ) containing 10% fetal calf serum 
 and 2 mm l - glutamine in a 37 c humidified 5% co2/95% air atmosphere . cells 
 were grown to confluence in white - sided , flat - bottom 96-well view 
 plates . briefly , the medium was removed 
 from each well , and 100 l of serum - free medium containing the 
 competing ligand at twice the final required concentration was added 
 to each well . a fixed concentration of 100 l h - cgp 
 12177 was then immediately added to each well ( 1:2 dilution in well ) , 
 and the cells were incubated for 2 h at 37 c , 5% co2 . the cells were washed twice by the addition and removal of 200 
 l of 4 c phosphate - buffered saline per well . one hundred 
 microliters of microscint 20 was added to each well , a white sticky 
 base was applied to the bottom of the plate , and a sealant top was 
 applied to the top of the plate . the plates were left at room temperature 
 overnight in the dark and then counted on a topcount at 21 c 
 for 2 min / well . the cells were prelabeled with h - adenine by incubation for 2 h with 2 ci / ml the h - adenine was removed , and each well washed by the addition and removal 
 of 1 ml of serum - free medium . one milliliter of serum - free medium 
 containing 1 mm ibmx was added to each well , and the cells were incubated 
 for 15 min . when used , antagonists were added at a final concentration 
 at this stage and thus had 15 min of incubation . agonist ( in 10 l 
 of serum - free medium ) was added to each well , and the plates were 
 incubated for 5 h. the reaction was terminated by the addition of 
 50 l of concentrated hcl per well . the plates were then frozen 
 and thawed , and h - camp was separated from other h - nucleotides by sequential dowex and alumina column chromatography , 
 as previously described . as measurements 
 of partial agonism depend on many factors , including particularly 
 the receptor expression level in the cells being examined , all camp 
 experiments were performed in cells at the same passage throughout 
 this entire study . in addition , whenever possible , experiments were 
 performed with all ligands being investigated in parallel studies 
 on the same day . thus , every ligand was examined on the same split 
 of cells on the same day , with each separate n number 
 being performed on different days . all data points on each binding 
 curve were performed in triplicate , and each 96-well plate also contained 
 three to six determinations of total and nonspecific binding . a one - site sigmoidal binding curve ( eq 1 ) 
 was then fitted to the data using graphpad prism 2.01 , and the ic50 was then determined as the concentration required to inhibit 
 50% of the specific binding.1 in eq 1 , a is the concentration 
 of the competing ligand , ic50 is the concentration at which 
 half of the specific binding of h - cgp 12177 has been inhibited , 
 and ns is the nonspecific binding . from the ic50 value 
 and the known concentration of radioligand [ h - cgp 12177 ] , a kd ( concentration 
 at which half the receptors are bound by the competing ligand ) value 
 was calculated using eq 2:2 agonist responses 
 were best described by a one - site sigmoidal concentration response 
 curve ( eq 3)3where emax is the maximum response , [ a ] 
 is the agonist concentration , 
 and ec50 is the concentration of agonist that produces 
 50% of the maximal response as several ligands have too low 
 affinity at the human 2 and 3 receptors 
 to assess accurately in the whole cell binding assay , the affinity 
 ( log kd value ) of these antagonists was 
 calculated in functional assays from the shift of the agonist concentration 
 responses in the presence of a fixed concentration of antagonist using 
 eq 44where dr ( dose ratio ) is the ratio of the 
 agonist concentration required to stimulate an identical response 
 in the presence and absence of a fixed concentration of antagonist 
 [ b ] . several of the compounds , however , displaced clear partial 
 agonist 
 activities ; that is , they antagonized the more efficacious agonist 
 cimaterol while stimulating an agonist response of their own . when 
 clear partial agonism was seen , the affinity ( log kd ) was calculated according to the method of stephenson 
 using eq 5:5 in eq 5 [ p ] is the concentration 
 of the partial agonist , [ a1 ] is the concentration of the 
 agonist at the point where the partial agonist alone causes the same 
 response , [ a2 ] is the concentration of agonist causing 
 a given response above that achieved by the partial agonist , and [ a3 ] is the concentration of the agonist in the presence the 
 partial agonist causing the same stimulation as [ a2 ] . isoprenaline ( 10 m ) was included in all experiments , and 
 therefore all maximal responses are expressed as a percentage of this 
 maximum . dawley 
 rats ( charles river , margate , kent , uk ) , weighing 300350 g , 
 were housed in groups in a temperature - controlled ( 2123 c ) 
 environment with a 12 h light  dark cycle ( lights on at 6:00 
 a.m. ) and free access to food ( teklad global 18% protein rodent diet , 
 bicester , oxon , u.k . ) and water for at least 7 days after arrival 
 from the supplier before any surgical intervention . surgery 
 was performed in two stages under general anesthesia ( fentanyl and 
 medetomidine , 300 g / kg of each ip , supplemented as required ) , 
 with reversal of anesthesia and postoperative analgesia provided by 
 atipamezole ( 1 mg / kg sc ) and buprenorphine ( 0.02 mg / kg sc ) . at the 
 first surgical stage , a miniature pulsed doppler flow probe was sutured 
 around the distal abdominal aorta to monitor hindquarters hemodynamics . the wires from the probe were taped and sutured at the nape of the 
 neck , and the animals were returned to the holding room . at the second 
 surgical stage , which took place at least 10 days after the surgery 
 for probe implantation , and following a satisfactory inspection from 
 the named veterinary surgeon , catheters were implanted in the distal 
 abdominal aorta via the caudal artery ( for arterial blood pressure 
 ( bp ) monitoring and the derivation of heart rate ( hr ) ) and in the 
 right jugular vein ( for drug administration ) . three separate intravenous 
 catheters were placed in the jugular vein to enable concurrent administration 
 of different substances . at this stage , the wires from the probe were 
 soldered into a miniature plug ( microtech inc . , boothwyn , pa , usa ) , 
 which was mounted onto a custom - designed harness worn by the rat . the catheters emerged from the same point as the probe wires and were 
 fed through a protective spring secured to the harness and attached 
 to a counterbalanced pivot system . the arterial catheter was connected 
 to a fluid - filled swivel for overnight infusion of heparinized ( 15 
 units / ml ) saline to maintain patency . experiments began 24 h 
 after surgery for catheter implantation , 
 with animals fully conscious and unrestrained in home cages , with 
 free access to food and water . all procedures were carried out with 
 approval of the university of nottingham local ethical review committee , 
 under home office project and personal license authority . cardiovascular variables 
 were recorded using a customized , computer - based system ( instrument 
 development engineering evaluation ( ideeq ) , maastrich instruments 
 bv , the netherlands ) connected to a transducer amplifier ( gould , usa ; 
 model 13 - 4615 - 50 ) and a doppler flowmeter ( crystal biotech ( holliston , 
 usa ) vf-1 mainframe ( pulse repetition frequency = 125 khz ) fitted 
 with high - velocity ( hvpd-20 ) modules ) . raw data were sampled by ideeq 
 every 2 ms , averaged , and stored to disc every cardiac cycle . hindquarters 
 vascular conductance ( hvc ) changes were calculated from the changes 
 in bp and doppler shift . in all 
 experiments , atropine 
 methyl nitrate ( 1 mg / kg / h ; 0.4 ml / h ) was infused continuously to remove 
 any parasympathetic influence on the control of heart rate . starting 
 2 h after the onset of the atropine infusion , rats were given 3 min 
 infusions ( 0.15 ml / min ) of isoprenaline ( 12 , 40 , and 120 ng / kg / min ) 
 in ascending order separated by at least 20 min . at least 45 min after 
 the last infusion of isoprenaline , saline or 19 was given 
 as an iv bolus ( 0.1 ml ) maintained by continuous infusion ( 0.4 ml / h ) , 
 and the isoprenaline infusions were repeated , starting 30 min thereafter . responses to isoprenaline were measured as the 
 difference between steady - state values immediately before the isoprenaline 
 infusion and during the third minute of infusion . changes in baseline 
 were measured as the difference between the control values for the 
 last dose of isoprenaline before , and the first dose of isoprenaline 
 after , saline or 19 administration . data were analyzed 
 by t test or anova with bonferroni correction as 
 appropriate ; p < 0.05 was taken as significant 
 ( graphpad prism version 5.02 ) .
-adrenoceptor 
 antagonists boast a 50-year use for symptomatic 
 control in numerous cardiovascular diseases . 
 one might expect highly 
 selective antagonists are available for the human -adrenoceptor 
 subtype involved in these diseases , yet few truly 1-selective molecules exist . to address this clinical need , 
 we re - evaluated 
 lk 204 - 545 ( 1),1 a selective 
 1-adrenoceptor antagonist , and discovered it possessed 
 significant partial agonism . 
 removal of 1 s aromatic 
 nitrile afforded 19 , a ligand with similar 1-adrenoceptor selectivity and partial agonism ( log kd of 7.75 and 5.15 as an antagonist 
 of functional 1- and 2-mediated 
 responses , respectively , and 34% of the maximal response of isoprenaline 
 ( 1 ) ) . in vitro -adrenoceptor selectivity 
 and partial agonism of 19 
 were mirrored in vivo . 
 we designed 
 analogues of 19 to improve affinity , selectivity , and 
 partial agonism . 
 although partial agonism could not be fully attenuated , 
 sar suggests that an extended alkoxyalkoxy side chain , alongside substituents 
 at the meta- or para - positions of 
 the phenylurea , increases ligand affinity and 1-selectivity .