Patent Abstract:
selective antagonists of a 2a adenosine receptors like those of formula i are provided , wherein y forms a ring . the novel a 2a blockers are useful for the treatment of parkinsons disease and other diseases .

Detailed Description:
unless specifically noted otherwise herein , the definition of the terms used are standard definitions used in the art of organic synthesis and pharmaceutical sciences . where a carbonyl group or a carbonyl derivative such as a thio carbonyl or an imine and the like , is represented by a group such as — c (═ o ) o — or — c (═ o ) nr a —, for example , it is intended that the corresponding isomeric group that is — oc (═ o )— or — nr a c (═ o )— is also included . an “ alkyl ” group is a straight , branched , saturated or unsaturated , aliphatic group having a chain of carbon atoms , optionally with oxygen , nitrogen or sulfur atoms inserted between the carbon atoms in the chain or as indicated . a ( c 1 – c 20 ) alkyl , for example , includes alkyl groups that have a chain of between 1 and 20 carbon atoms , and include , for example , the groups methyl , ethyl , propyl , isopropyl , vinyl , allyl , 1 - propenyl , isopropenyl , ethynyl , 1 - propynyl , 2 - propynyl , 1 , 3 - butadienyl , penta - 1 , 3 - dienyl , penta - 1 , 4 - dienyl , hexa - 1 , 3 - dienyl , hexa - 1 , 3 , 5 - trienyl , and the like . an alkyl group may also be represented , for example , as a —( cr 1 r 2 ) m — group where r 1 and r 2 are independently hydrogen or are independently absent , and for example , m is 1 to 8 , and such representation is also intended to cover both saturated and unsaturated alkyl groups . an alkyl as noted with another group such as an aryl group , represented as “ arylalkyl ” for example , is intended to be a straight , branched , saturated or unsaturated aliphatic divalent group with the number of atoms indicated in the alkyl group ( as in ( c 1 – c 20 ) alkyl , for example ) and / or aryl group ( as in ( c 5 – c 14 ) aryl , for example ) or when no atoms are indicated means a bond between the aryl and the alkyl group . nonexclusive examples of such group include benzyl , phenethyl and the like . an “ alkylene ” group is a straight , branched , saturated or unsaturated aliphatic divalent group with the number of atoms indicated in the alkyl group ( as in —( c 1 – c 20 ) alkylene - or —( c 1 – c 20 ) alkylenyl -, for example ), optionally with one or more oxygen , nitrogen or sulfur atoms inserted ( or “ interrupted ”) between the carbon atoms in the chain or as indicated . a “ cyclyl ” such as a monocyclyl or polycyclyl group includes monocyclic , or linearly fused , angularly fused or bridged polycycloalkyl , or combinations thereof . such cyclyl group is intended to include the heterocyclyl analogs . a cyclyl group may be saturated , partically saturated or aromatic . a “ heterocyclyl ” or “ heterocycle ” is a cycloalkyl wherein one or more of the atoms forming the ring is a heteroatom that is a n , o , or s . non - exclusive examples of heterocyclyl include piperidyl , 4 - morpholyl , 4 - piperazinyl , pyrrolidinyl , 1 , 4 - diazaperhydroepinyl , 1 , 3 - dioxanyl , and the like . “ pharmaceutically acceptable salts ” means salt compositions that is generally considered to have the desired pharmacological activity , is considered to be safe , non - toxic and is acceptable for veterinary and human pharmaceutical applications . such salts include acid addition salts formed with inorganic acids such as hydrochloric acid , hydrobromic acid , sulfuric acid , phosphoric acid , and the like ; or with organic acids such as acetic acid , propionic acid , hexanoic acid , malonic acid , succinic acid , malic acid , citric acid , gluconic acid , salicylic acid and the like . “ substituted or unsubstituted ” means that a group such as , for example , alkyl , aryl , heterocyclyl , ( c 1 – c 8 ) cycloalkyl , ( c 6 – c 12 ) bicycloalkyl , hetrocyclyl ( c 1 – c 8 ) alkyl , aryl ( c 1 – c 8 ) alkyl , heteroaryl , heteroaryl ( c 1 – c 8 ) alkyl , the mono -, bi - or polycyclic rings that define the z group , and the like , unless specifically noted otherwise , may be unsubstituted or , may substituted by 1 , 2 , 3 , 4 or 5 substitutents selected from the group such as halo , nitro , trifluoromethyl , trifluoromethoxy , methoxy , carboxy , — nh 2 , — oh , — sh , — nhch 3 , — n ( ch 3 ) 2 , — sme , propargyl , cyano , — nnh , — nnch 3 , — opo 2 h , — opo 2 ch 3 , — s ( so 2 ) h , — s ( so 2 ) oh , — s ( so 2 ) ch 3 and — s ( so 2 ) och 3 , and the like . the ability of compounds to stimulate motor activity in normal mice was measured using a simple , computer - assisted locomotor activity cage system . c57bl / 6 mice ( n ═ 6 – 8 purchased from the jackson &# 39 ; s lab ) were habituated for the testing environment for 120 minute prior to drug treatment . the test compounds were dissolved in vehicle ( 10 % dmso , 10 % castrol oil el - 620 and 80 % saline ). the drug was administrated intraperitoneally at a dose of 15 mg / kg , and locomotor activity was recorded for 2 hours before and after drug administration . fig1 shows that atl - 2 produced strong motor stimulation , reaching peak within 20 minutes and lasting for about 60 minutes ( arrow marks the injection ). from our previous experience with other a 2a r antagonists , the motor stimulant effect of atl2 is comparable or stronger than other a 2a r antagonists such as sch58261 and kw6002 . absence of motor stimulant effect of atl - 2 in mice lacking the a 2a receptor we validated that atl - 2 acts on the a 2a receptor to stimulate motor activity by using a 2 ar ko mice ( in both mixed ( 129sv x c57bl / 6 ) as well as congenic ( c57bl / 6 genetic background ) developed over the last several years . we evaluated the motor stimulant effect of atl - 2 in a 2a receptor ko and their wt littermates . wt and a 2a ko mice ( n = 4 ) were habituated for 60 minutes and treated with atl - 2 ( 15 mg / kg ) and recorded for motor activity for 120 minutes . fig2 shows that atl - 2 produced motor activity in wt mice ( relatively high basal locomotion is likely due to short habituation time ( 60 minute instead of 120 minutes ) and demonstrates relatively higher basal locomotion in wt compared to ko mice as we noted previously , 35 but this motor stimulation was absent in a 2a receptor ko mice . the following procedures may be employed for the preparation of the compounds of the present invention . the starting materials and reagents used in preparing these compounds are either available from commercial suppliers such as the aldrich chemical company ( milwaukee , wis . ), bachem ( torrance , calif . ), sigma ( st . louis , mo . ), or are prepared by methods well known to a person of ordinary skill in the art , following procedures described in such references as fieser and fieser &# 39 ; s reagents for organic synthesis , vols . 1 – 17 , john wiley and sons , new york , n . y ., 1991 ; rodd &# 39 ; s chemistry of carbon compounds , vols . 1 – 5 and supps ., elsevier science publishers , 1989 ; organic reactions , vols . 1 – 40 , john wiley and sons , new york , n . y ., 1991 ; march j . : advanced organic chemistry , 4th ed ., john wiley and sons , new york , n . y . ; and larock : comprehensive organic transformations , vch publishers , new york , 1989 . in some cases , protective groups may be introduced and finally removed . suitable protective groups for amino , hydroxy , and carboxy groups are described in greene et al ., protective groups in organic synthesis , second edition , john wiley and sons , new york , 1991 . standard organic chemical reactions can be achieved by using a number of different reagents , for examples , as described in larock : comprehensive organic transformations , vch publishers , new york , 1989 . in one variation , the compounds of this invention can be synthesized by the steps outlined in scheme 1 . guanosine , a ′, is acetylated to protect the ribose during reductive chlorination by pocl 3 / diethylaniline to form 6 - chloroguanosine , c ′. non - aqueous diazotization in the presence of elemental iodine in diiodomethane is a standard route to the protected 6 - chloro - 2 - iodonebularine , d ′. heating in methanolic ammonia deprotects the sugar and displaces the 6 - chloro substituent to form 2 - iodoadenosine , e ′. palladium - catalyzed coupling of e ′ with a terminal alkyne generates 2 - alkynyladenosine f ′, which undergoes acid hydrolysis to form 9h - adenine g ′. alkylation with an appropriate halide ( alkyl , cycloalkyl or heterocyclic ) completes the synthesis of target 2 , 9 - disubstituted adenine h ′. preparation of the terminal alkynes ( s )- 1 - ethynyl - 1 - hydroxy -( r )- 3 - methylcyclohexane and 2 - ethynyladamantan - 2 - ol is achieved by treatment of the corresponding ketone with ethynylmagnesium bromide . preparation of the substituted piperidine - carboxylate terminal alkynes ( scheme 2 ) starts with 4 - carboxypiperidine ( isonipecotic acid ) i ′ in anticipation of acylating the methyl ester , j ′, with the appropriate alkyl chloroformate to form the n - carbamoyl ester k ′. borohydride reduction of the ester generates the 4 - hydroxymethylpiperidine , l ′, which undergoes tosylation to m ′ in preparation for condensation with lithium acetylide to form terminal alkyne n ′. + k i & lt ; 10 , 000 nm ; ++ k i & lt ; 1 , 000 nm ; +++ k i & lt ; 500 nm ; ++++ k i & lt ; 100 nm . + k i & lt ; 10 , 000 nm ; ++ k i & lt ; 1 , 000 nm ; +++ k i & lt ; 500 nm ; ++++ k i & lt ; 100 nm . compound no . ( cr 9 r 10 ) n — y ( cr 1 r 2 ) m - z human k i ( nm ) 60 propargyl ++++ 61 propargyl ++++ 63 propargyl ++++ + k i & lt ; 10 , 000 nm ; ++ k i & lt ; 1 , 000 nm ; +++ k i & lt ; 500 nm ; ++++ k i & lt ; 100 nm . ( s )- 1 , 1 - ethynyl - hydroxy -( r )- 3 - methylcyclohexane . a solution of 0 . 5 m ethynylmagnesium bromide in thf ( 150 . 0 ml , 0 . 0750 mol ) was added to an ice cold solution of ( r )-(+)- 3 - methylcyclohexanone ( 2 . 77 g , 0 . 02469 mol ) in anhydrous thf ( 100 ml ). the ice bath was removed and the mixture stirred at room temperature 24 h . the mixture was cooled over ice and quenched with water ( 15 . 0 ml ). the volume of thf was reduced to approximately 50 ml and the mixture filtered through a bed of celite / sand , washing with ether . the solution is then evaporated to dryness and the crude purified by column chromatography , eluting with a gradient of hexanes to hexanes / ethyl acetate ( 10 %) to afford the pure product as a white crystalline solid : yield 1 . 412 g , 41 %. 1 h nmr ( cdcl 3 ) δ 0 . 73 – 0 . 95 , 1 . 10 – 1 . 19 , 1 . 35 – 1 . 45 , 1 . 51 – 1 . 84 , 1 . 93 – 2 . 03 ( 5 × m , 9h , cyclohexyl ), 0 . 93 ( d , 3h , — ch 3 ), 2 . 48 ( s , 1h , alkyne ). 2 - ethynyl - adamantan - 2 - ol . a solution of 0 . 5 m ethynylmagnesium bromide in thf ( 400 . 0 ml , 0 . 2000 mol ) was added to an ice cold solution of 2 - adamantone ( 7 . 706 g , 0 . 05130 mol ) in anhydrous thf ( 250 ml ). the mixture was stirred over ice 0 . 5 h and then at room temperature 21 h . the volume was reduced to half and the solution cooled over ice . the reaction was quenched with water ( 5 . 0 ml ), filtered through a bed of celite / sand and evaporated to dryness . the crude was taken up in ether ( 400 ml ) and washed with water ( 2 × 40 ml ) and brine ( 50 ml ), dried over mgso 4 , filtered , and evaporated to dryness to afford the pure product as a crystalline white solid : yield 8 . 961 g , 99 %. 1 h nmr ( cdcl 3 ) δ 1 . 54 – 1 . 61 , 1 . 68 – 1 . 72 , 1 . 76 – 1 . 99 , 2 . 11 – 2 . 21 ( 4 × m , 14h , adamantly ), 2 . 53 ( s , 1h , alkyne ). representative procedure for n6 - amino substitution : 2 - iodoadenosine . a suspension of 6 - chloro - 2 - iodo - 9 -( 2 ′, 3 ′, 5 ′- o - triacetylfuranosyl )- 9h - purine ( 14 . 70 g , 0 . 02729 mol ) in meoh ( 300 ml ) was cooled over an ice bath . ammonia gas was then bubbled through the mixture until it was saturated . the reaction vessel was sealed and heated at 40 ° c . for 18 h and at 60 ° c . for 5 days . the mixture was cooled over ice and nitrogen gas bubbled through the solution , the mixture being allowed to warm to room temperature . the solvent was then removed under reduced pressure and the crude recrystallized from water containing 3 – 4 drops of glacial acetic acid . the resulting precipitate was filtered and washed with water and ether to afford a white solid : yield 7 . 167 g , 67 %. representative procedure for n6 - alkylamino substitution : 2 - iodo - 6 -( 3 - pentyl ) adenosine . 6 - chloro - 2 - iodo - 9 -( 2 ′, 3 ′, 5 ′- o - triacetylfuranosyl )- 9h - purine ( 6 . 723 g , 0 . 01248 mol ), 3 - aminopentane ( 1 . 673 ml , 0 . 01436 mol ) and diisopropylethylamine ( 2 . 725 ml , 0 . 01560 mol ) were stirred in denatured ethanol ( 150 ml ) at 90 ° c . in a pressure apparatus for 21 h . the reaction was then cooled over ice and ammonia gas bubbled through the mixture until it was saturated . the reaction vessel was closed and the mixture stirred at room temperature 21 h . the mixture was cooled over ice and nitrogen gas bubbled through the solution , the mixture being allowed to warm to room temperature . the solvent was removed under reduced pressure and the crude purified by column chromatography , eluting with a gradient of dcm / meoh ( 0 – 4 %) to afford the pure product as an off white solid : yield 4 . 838 g , 84 %. representative procedure for c2 coupling : 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenosine . to a solution of 2 - iodoadenosine ( 3 . 105 g , 7 . 898 mmol ) in freshly degassed acetonitrile / dmf ( 100 ml , 1 : 1 ) was added degassed triethylamine ( 11 . 0 ml , 78 . 9 mmol ), pd ( pph 3 ) 4 ( 113 mg , 0 . 09779 mmol ), cui ( catalytic ), and 2 , 2 - ethynyl - hydroxy - adamantanyl ( 1 . 516 g , 8 . 601 mmol ). the mixture was stirred at room temperature under and inert atmosphere for 71 h . silica bound pd ( ii ) scavenger si - thiol ( 561 mg ) and pd ( 0 ) scavenger si - taacoh ( 541 mg ) were added and stirring continued a further 4 . 5 h . the suspension was filtered through celite and the resulting solution evaporated to dryness . the crude was purified by column chromatography , eluting with a gradient of dcm / meoh ( 0 – 15 %) to afford the pure product as a white solid : yield 3 . 476 g , 100 %. representative procedure for ribose cleavage : 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine . a solution of 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenosine ( 3 . 486 g , 7 . 896 mmol ) in methanol ( 100 ml ) and 1 . 0 m hcl ( 10 . 0 ml ) was stirred at 90 ° c . in a pressure apparatus for 18 – 50 h . the ph was adjusted to 4 . 2 with 5 . 0 m naoh and the volume was reduced to half under reduced pressure . after cooling the resulting precipitate was filtered and washed with methanol to afford the pure product as a white solid : yield 2 . 153 g , 88 %. 1 h nmr ( dmso - d 6 ) δ 8 . 13 ( s , 1h ), 7 . 25 ( br s , 2h ), 5 . 56 ( s , 1h ), 2 . 18 – 2 . 09 , 1 . 94 – 1 . 89 , 1 . 82 – 1 . 64 , 1 . 52 – 1 . 45 ( 4 × m , 12h ). lrms esi ( m + h + ) 310 . 1 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine . using the representative procedure for ribose cleavage above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenosine ( 1 . 03 g ) gave the product as a white solid : yield 0 . 725 g , 98 %. lrms esi ( m + h + ) 342 . 2 . an appropriate 9 - unsubstituted adenine ( 1 . 649 mmol ) was dissolved in dmf ( 80 ml ). anhydrous potassium carbonate ( 358 mg , 2 . 590 mmol ) and an appropriate alkyl halide ( 3 . 295 mmol ) were added and the mix stirred at between 25 – 100 ° c . for 5 – 100 h . the reaction mixture was adhered to silica and purified by column chromatography , eluting with a gradient of dcm / meoh ( 0 – 10 %) to afford the pure product . 9 - cyclopropylmethyl - 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl } adenine ( 1 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl } adenine ( 29 mg ) gave 1 as a white solid : yield 19 mg , 55 %. 1 h nmr ( cd 3 od ) δ 8 . 21 ( s , 1h ), 4 . 06 ( d , 2h , j ═ 7 . 3 hz ), 2 . 13 – 2 . 05 , 1 . 94 – 1 . 66 , 1 . 49 – 1 . 28 , 0 . 92 – 0 . 80 , 0 . 66 – 0 . 59 , 0 . 49 – 0 . 44 ( 6 × m , 14h ), 1 . 17 ( t , 1h , j = 12 . 3 hz ), 0 . 96 ( d , 2h , j = 6 . 6 hz ). lrms esi ( m + h + ) 326 . 1 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- 9 - propargyladenine ( 2 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl } adenine ( 112 mg ) gave 2 as an off white solid : yield 88 mg , 69 %. 1 h nmr ( cd 3 od ) δ 8 . 23 ( s , 1h ), 5 . 04 ( s , 2h ), 2 . 98 ( t , 1h , j = 2 . 6 hz ), 2 . 13 – 2 . 05 , 1 . 94 – 1 . 65 , 1 . 49 – 1 . 38 , 0 . 91 – 0 . 80 , ( 4 × m , 7h ), 1 . 17 ( t , 1h , j = 12 . 3 hz ), 0 . 95 ( d , 2h , j = 6 . 6 hz ). lrms esi ( m + h + ) 310 . 1 . 9 - cyclopentyl - 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl } adenine ( 3 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl } adenine ( 34 mg ) gave 3 as a white solid : yield 21 mg , 50 %. 1 h nmr ( cd 3 od ) δ 8 . 21 ( s , 1h ), 4 . 91 ( tt , 1h , j = 7 . 0 hz ), 2 . 31 – 2 . 19 , 2 . 13 – 1 . 65 , 1 . 49 – 1 . 38 , 0 . 92 – 0 . 79 ( 4 × m , 17h ), 1 . 15 ( t , 1h , j = 12 . 3 hz ), 0 . 96 ( d , 2h , j = 6 . 6 hz ). lrms esi ( m + h + ) 340 . 2 . 9 - acetonitrile - 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl } adenine ( 4 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl } adenine ( 43 mg ) gave 4 as a white solid : yield 25 mg , 51 %. 1 h nmr ( cd 3 od ) δ 8 . 20 ( s , 1h ), 5 . 35 ( s , 1h ), 2 . 33 – 2 . 05 , 1 . 93 – 1 . 66 , 1 . 49 – 1 . 38 , 0 . 92 – 0 . 80 ( 4 × m , 9h ), 1 . 18 ( t , 1h , j = 12 . 3 hz ), 0 . 96 ( d , 2h , j = 6 . 6 hz ). lrms esi ( m + h + ) 311 . 1 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- 9 -( 4 - methoxybenzyl ) adenine ( 5 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl } adenine ( 24 mg ) gave 5 as a white solid : yield 29 mg , 84 %. 1 h nmr ( cd 3 od ) δ 8 . 10 ( s , 1h ), 7 . 26 ( d , 2h , j = 8 . 8 hz ), 6 . 88 ( d , 2h , j = 8 . 8 hz ), 5 . 32 ( s , 2h ), 3 . 75 ( s , 3h ), 2 . 33 – 2 . 05 , 1 . 92 – 1 . 66 , 1 . 49 – 1 . 38 , 0 . 92 – 0 . 80 ( 4 × m , 9h ), 1 . 17 ( t , 1h , j = 12 . 3 hz ), 0 . 95 ( d , 2h , j = 6 . 6 hz ). lrms esi ( m + h + ) 392 . 2 . 9 -( 3 , 4 - dichlorobenzyl )- 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl } adenine ( 6 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl } adenine ( 26 mg ) gave 6 as a white solid : yield 28 mg , 68 %. 1 h nmr ( cd 3 od ) δ 8 . 20 ( s , 1h ), 7 . 52 ( d , 1h , j = 2 . 1 hz ), 7 . 48 ( d , 1h , j = 8 . 3 hz ), 7 . 19 ( dd , 1h , j = 2 . 1 hz , j = 8 . 3 hz ), 5 . 40 ( s , 2h ), 2 . 11 – 2 . 04 , 1 . 92 – 1 . 64 , 1 . 48 – 1 . 37 , 0 . 92 – 0 . 79 ( 4 × m , 9h ), 1 . 16 ( t , 1h , j = 12 . 3 hz ), 0 . 94 ( d , 2h , j = 6 . 6 hz ). lrms esi ( m + h + ) 430 . 2 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- 9 -( 4 - trifluoromethylbenzyl ) adenine ( 7 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl } adenine ( 30 mg ) gave 7 as a white solid : yield 33 mg , 70 %. 1 h nmr ( cd 3 od ) δ 8 . 20 ( s , 1h ), 7 . 64 ( d , 2h , j = 8 . 2 hz ), 7 . 44 ( d , 2h , j = 8 . 1 hz ), 5 . 52 ( s , 2h ), 2 . 10 – 2 . 03 , 1 . 90 – 1 . 63 , 1 . 47 – 1 . 36 , 0 . 90 – 0 . 78 ( 4 × m , 9h ), 1 . 15 ( t , 1h , j = 12 . 3 hz ), 0 . 93 ( d , 2h , j = 6 . 6 hz ). lrms esi ( m + h + ) 430 . 1 . 9 -( 3 , 5 - dimethyl - isoxazol - 4 - ylmethyl )- 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl } adenine ( 8 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl } adenine ( 32 mg ) gave 8 as a white solid : yield 36 mg , 80 %. 1 h nmr ( cd 3 od ) δ 8 . 15 ( s , 1h ), 5 . 20 ( s , 2h ), 2 . 50 ( s , 3h ), 2 . 22 ( s , 3h ), 2 . 11 – 2 . 03 , 1 . 92 – 1 . 65 , 1 . 49 – 1 . 38 , 0 . 92 – 0 . 81 ( 4 × m , 9h ), 1 . 18 ( t , 1h , j = 12 . 2 hz ), 0 . 96 ( d , 2h , j = 6 . 6 hz ). lrms esi ( m + h + ) 381 . 1 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- 9 -[ 2 -( trifluoromethylphenyl ) thiazol - 4 - ylmethyl ] adenine ( 9 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl } adenine ( 26 mg ) gave 9 as a white solid : yield 30 mg , 61 %. 1 h nmr ( cd 3 od ) δ 8 . 31 ( s , 1h ), 8 . 09 ( d , 2h , j = 8 . 8 hz ), 7 . 74 ( d , 2h , j = 8 . 3 hz ), 7 . 55 ( s , 1h ), 5 . 58 ( s , 2h ), 2 . 12 – 2 . 04 1 . 91 – 1 . 64 , 1 . 48 – 1 . 37 , 0 . 91 – 0 . 78 ( 4 × m , 9h ), 1 . 16 ( t , 1h , j = 12 . 4 hz ), 0 . 94 ( d , 2h , j = 6 . 6 hz ). lrms esi ( m + h + ) 513 . 1 . 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl }- 9 -( 3 -( thiophen - 2 - yl ) prop - 2 - ynyl ) adenine ( 10 ). to a solution of 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- 9 - propargyladenine ( 30 mg , 0 . 09697 mmol ) in freshly degassed acetonitrile / dmf ( 15 ml , 2 : 1 ) was added degassed triethylamine ( 1 . 0 ml , 7 . 0175 mmol ), pd ( pph 3 ) 4 ( 30 mg , 0 . 02596 mmol ), cui ( catalytic ), and 98 +% 2 - bromothiophene ( 13 . 7 μl , 0 . 1161 mmol ). the mixture was stirred at room temperature under and inert atmosphere for 28 h . silica bound pd ( ii ) scavenger si - thiol ( 240 mg ) and pd ( 0 ) scavenger si - taacoh ( 155 mg ) were added and stirring continued a further 72 h . the suspension was filtered through celite and the resulting solution evaporated to dryness . the crude was purified by column chromatography , eluting with a gradient of dcm / meoh ( 0 – 6 %) to afford the impure product ( 27 mg ). the product was further purified by reverse phase column chromatography , eluting with a gradient of h 2 / meoh ( 50 – 75 %) to afford the pure product 10 as a white solid : yield 8 . 5 mg , 22 %. 1 h nmr ( cd 3 od ) δ 8 . 27 ( s , 1h ), 7 . 41 ( dd , 1h , j = 1 . 1 hz , j = 5 . 3 hz ), 7 . 25 ( dd , 2h , j = 1 . 1 hz , j = 3 . 5 hz ), 6 . 99 ( dd , 1h , j = 3 . 7 hz , j = 5 . 0 hz ), 5 . 29 ( s , 2h ), 2 . 14 – 2 . 05 , 1 . 94 – 1 . 65 , 1 . 49 – 1 . 37 , 0 . 99 – 0 . 79 ( 4 × m , 12h ), 1 . 17 ( t , 1h , j = 12 . 2 hz ). lrms esi ( m + h + ) 392 . 1 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- 9 - methyl - n6 -( 3 - pentyl ) adenine ( 11 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 20 mg ) gave 11 as a white solid : yield 9 mg , 43 %. 1 h nmr ( cd 3 od ) δ 8 . 02 ( s , 1h ), 4 . 23 ( m , 1h ), 3 . 79 ( s , 3h ), 2 . 14 – 2 . 06 , 1 . 96 – 1 . 38 , 0 . 99 – 0 . 80 ( 3 × m , 20h ), 1 . 17 ( t , 1h , j = 12 . 3 hz ). lrms esi ( m + h +) 356 . 3 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl )- 9 - propyladenine ( 12 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 29 mg ) gave 12 as a white solid : yield 26 mg , 80 %. 1 h nmr ( cd 3 od ) δ 8 . 08 ( s , 1h ), 4 . 28 – 4 . 13 ( m , 3h ), 2 . 15 – 2 . 06 , 1 . 95 – 1 . 38 , 0 . 99 – 0 . 80 ( 3 × m , 26h ), 1 . 17 ( t , 1h , j = 12 . 3 hz ). lrms esi ( m + h + ) 384 . 3 . 9 - isobutyl - 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 13 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 27 mg ) gave 13 as a white solid : yield 23 mg , 73 %. 1 h nmr ( cd 3 od ) δ 8 . 06 ( s , 1h ), 4 . 21 ( m , 1h ), 4 . 01 ( d , 2h , j = 7 . 4 hz ), 2 . 21 ( septet , 1h , j = 6 . 8 hz ), 2 . 12 – 2 . 05 , 1 . 96 – 1 . 38 , 0 . 98 – 0 . 81 ( 3 × m , 27h ), 1 . 17 ( t , 1h , j = 12 . 2 hz ). lrms esi ( m + h + ) 398 . 2 . 9 - cyclopropylmethyl - 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 14 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 30 mg ) gave 14 as a white solid : yield 17 mg , 57 %. 1 h nmr ( cd 3 od ) δ 8 . 15 ( s , 1h ), 4 . 22 ( m , 1h ), 4 . 05 ( d , 2h , j = 7 . 3 hz ), 2 . 14 – 2 . 05 , 1 . 96 – 1 . 28 , 0 . 98 – 0 . 79 ( 3 × m , 22h ), 1 . 17 ( t , 1h , j = 12 . 2 hz ), 0 . 65 – 0 . 58 ( m , 2h ), 0 . 48 – 0 . 44 ( m , 2h ). lrms esi ( m + h + ) 396 . 3 . 9 - cyclopentyl - 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 15 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 37 mg ) gave 15 as a white solid : yield 29 mg , 65 %. 1 h nmr ( cd 3 od ) δ 8 . 15 ( s , 1h ), 4 . 91 ( tt , 1h , j = 7 . 0 hz ), 4 . 21 ( m , 1h ), 2 . 31 – 2 . 17 , 2 . 14 – 1 . 35 , 0 . 98 – 0 . 79 ( 3 × m , 29h ), 1 . 17 ( t , 1h , j = 12 . 2 hz ). lrms esi ( m + h + ) 410 . 3 . 9 - allyl - 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 16 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 33 mg ) gave 16 as a white solid : yield 26 mg , 71 %. 1 h nmr ( cdcl 3 ) δ 7 . 77 ( br s , 1h ), 6 . 09 – 5 . 95 ( m , 1h ), 5 . 34 – 5 . 20 ( m , 2h ), 4 . 79 ( dt , 2h , j = 5 . 8 hz , j = 1 . 5 hz ), 4 . 28 ( m , 1h ), 2 . 21 – 2 . 11 , 1 . 96 – 1 . 42 , 0 . 96 – 0 . 78 ( 3 × m , 21h ), 1 . 23 ( t , 1h , j = 12 . 2 hz ). lrms esi ( m + h + ) 382 . 3 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl )- 9 -( propargyl ) adenine ( 17 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 134 mg ) gave 17 as a white solid : yield 79 mg , 53 %. 1 h nmr ( cd 3 od ) δ 8 . 17 ( s , 1h ), 5 . 03 ( d , 2h , j = 2 . 6 hz ), 4 . 22 ( m , 1h ), 2 . 97 ( t , 1h , j = 2 . 6 ), 2 . 14 – 2 . 06 , 1 . 95 – 1 . 38 , 0 . 98 – 0 . 80 ( 3 × m , 21h ), 1 . 17 ( t , 1h , j = 12 . 4 hz ). lrms esi ( m + h + ) 380 . 2 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl )- 9 -( pent - 4 - yne ) adenine ( 18 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 35 mg ) gave 18 as a white solid : yield 31 mg , 74 %. 1 h nmr ( cd 3 od ) δ 8 . 07 ( s , 1h ), 4 . 32 ( t , 2h , j = 6 . 9 hz ), 4 . 21 ( m , 1h ), 2 . 31 – 2 . 19 , 2 . 14 – 2 . 00 , 1 . 95 – 1 . 38 , 1 . 00 – 0 . 79 ( 4 × m , 21h ), 1 . 17 ( t , 1h , j = 12 . 3 hz ). lrms esi ( m + h + ) 408 . 1 . 9 -( 2 - hydroxyethyl )- 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 19 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 103 mg ) gave 19 as a white solid : yield 60 mg , 52 %. 1 h nmr ( cd 3 od ) δ 8 . 06 ( s , 1h ), 4 . 30 ( t , 2h , j = 5 . 1 hz ), 4 . 22 ( m , 1h ), 3 . 86 ( t , 2h , j = 5 . 1 hz ), 2 . 14 – 2 . 05 , 1 . 95 – 1 . 38 , 0 . 99 – 0 . 80 ( 3 × m , 21h ), 1 . 17 ( t , 1h , j = 12 . 2 hz ). lrms esi ( m + h + ) 386 . 2 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- 9 -( 3 - hydroxypropyl )- n6 -( 3 - pentyl ) adenine ( 20 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 59 mg ) gave 20 as a white solid : yield 35 mg , 51 %. 1 h nmr ( cd 3 od ) δ 8 . 11 ( s , 1h ), 4 . 32 ( t , 2h , j = 7 . 0 hz ), 4 . 20 ( m , 1h ), 3 . 56 ( t , 2h , j = 5 . 9 hz ), 2 . 14 – 2 . 00 , 1 . 93 – 1 . 38 , 0 . 99 – 0 . 80 ( 3 × m , 23h ), 1 . 17 ( t , 1h , j = 12 . 2 hz ). lrms esi ( m + h + ) 400 . 3 . 9 -( 2 - chloroethyl )- 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 21 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 38 mg ) gave 21 as a white solid : yield 28 mg , 62 %. 1 h nmr ( cd 3 od ) δ 8 . 11 ( s , 1h ), 4 . 55 ( t , 2h , j = 5 . 7 hz ), 4 . 22 ( m , 1h ), 3 . 96 ( t , 2h , j = 5 . 7 hz ), 2 . 14 – 2 . 05 , 1 . 94 – 1 . 38 , 0 . 99 – 0 . 79 ( 3 × m , 21h ), 1 . 17 ( t , 1h , j = 12 . 1 hz ). lrms esi ( m + h + ) 404 . 2 . 9 -([ 1 , 3 ]- dioxolan - 2 - ylmethyl )- 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 22 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 37 mg ) gave 22 as a white solid : yield 32 mg , 69 %. 1 h nmr ( cd 3 od ) δ 8 . 04 ( s , 1h ), 5 . 20 ( t , 1h , j = 3 . 3 hz ), 4 . 40 ( d , 2h , j = 3 . 3 hz ), 4 . 21 ( m , 1h ), 3 . 88 – 3 . 76 ( m , 4h ), 2 . 14 – 2 . 06 , 1 . 92 – 1 . 38 , 0 . 98 – 0 . 79 ( 3 × m , 21h ), 1 . 17 ( t , 1h , j = 12 . 2 hz ). lrms esi ( m + h + ) 428 . 3 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl )- 9 -( tetrahydro - pyran - 2 - ylmethyl ) adenine ( 23 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 32 mg ) gave 23 as a white solid : yield 27 mg , 66 %. 1 h nmr ( cd 3 od ) δ 8 . 01 ( s , 1h ), 4 . 31 – 4 . 07 ( m , 3h ), 3 . 96 – 3 . 87 ( m , 1h ), 3 . 67 – 3 . 58 ( m , 1h ), 3 . 93 – 3 . 32 ( m , 1h ), 2 . 14 – 2 . 06 , 1 . 95 – 1 . 38 , 1 . 31 – 1 . 12 , 0 . 99 – 0 . 80 ( 4 × m , 28h ). lrms esi ( m + h + ) 440 . 4 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl )- 9 -( isopropylcarboxylate ) adenine ( 24 ). a 1 . 0 m solution of isopropyl chloroformate in toluene ( 150 μl , 0 . 1500 mmol ) was added to an ice cold solution of 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 34 mg ) in anhydrous pyridine ( 2 . 0 ml ). after stirring over ice 1 . 5 h the solvent was removed under reduced pressure and the crude purified by column chromatography , eluting with a gradient of dcm / meoh ( 0 – 5 %) to afford the pure product 24 as an off white solid : yield 18 mg , 42 %. 1 h nmr ( cd 3 od ) δ 8 . 44 ( s , 1h ), 5 . 29 ( septet , 1h , j = 6 . 4 , j = 6 . 2 hz ), 4 . 21 ( m , 1h ), 2 . 14 – 2 . 05 , 1 . 97 – 1 . 37 , 1 . 00 – 0 . 79 ( 3 × m , 27h ), 1 . 16 ( t , 1h , j = 12 . 2 hz ). lrms esi ( m + h + ) 427 . 9 . 9 -( acetic acid ethyl ester )- 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 25 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 38 mg ) gave 25 as a white solid : yield 9 mg , 19 %. 1 h nmr ( cd 3 od ) δ 8 . 06 ( s , 1h ), 5 . 06 ( s , 2h ), 4 . 24 ( br q , 3h , j = 7 . 0 , 7 . 3 hz ), 2 . 15 – 2 . 05 , 1 . 95 – 1 . 37 , 1 . 05 – 0 . 79 ( 3 × m , 22h ), 1 . 29 ( t , 3h , j = 7 . 0 , 7 . 3 hz ), 1 . 17 ( t , 1h , j = 12 . 2 hz ). lrms esi ( m + h + ) 428 . 2 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- 9 -( 2 - oxo - oxazolidin - 5 - ylmethyl )- n6 -( 3 - pentyl ) adenine ( 26 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 56 mg ) gave 26 as a white solid : yield 43 mg , 60 %. 1 h nmr ( cd 3 od ) δ 8 . 09 ( s , 1h ), 5 . 04 ( m , 1h ), 4 . 52 ( d , 2h , j = 4 . 8 hz ), 4 . 21 ( m , 1h ), 3 . 79 – 3 . 70 ( m , 1h ), 3 . 47 – 3 . 39 ( m , 1h ), 2 . 13 – 2 . 04 , 1 . 95 – 1 . 38 , 0 . 99 – 0 . 79 ( 3 × m , 21h ), 1 . 17 ( t , 1h , j = 12 . 2 hz ). lrms esi ( m + h + ) 441 . 3 . 9 - benzyl - 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 27 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 37 mg ) gave 27 as a white solid : yield 31 mg , 66 %. 1 h nmr ( cd 3 od ) δ 8 . 06 ( s , 1h ), 7 . 37 – 7 . 25 ( m , 5h ), 5 . 40 ( s , 2h ), 4 . 22 ( m , 1h ), 2 . 14 – 2 . 05 , 1 . 95 – 1 . 37 , 0 . 99 – 0 . 79 ( 3 × m , 21h ), 1 . 16 ( t , 1h , j = 12 . 3 hz ). lrms esi ( m + h + ) 432 . 3 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl )- 9 -( pyridin - 3 - ylmethyl ) adenine ( 28 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 34 mg ) gave 28 as a white solid : yield 8 mg , 19 %. 1 h nmr ( cd 3 od ) δ 8 . 57 ( m , 1h ), 8 . 48 ( m , 1h ), 8 . 17 ( s , 1h ), 7 . 77 ( m , 1h ), 7 . 41 ( m , 1h ), 5 . 49 ( s , 2h ), 4 . 22 ( m , 1h ), 2 . 13 – 2 . 04 , 1 . 93 – 1 . 37 , 0 . 99 – 0 . 79 ( 3 × m , 21h ), 1 . 16 ( t , 1h , j = 12 . 3 hz ). lrms esi ( m + h + ) 433 . 3 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- 9 -( 4 - nitrobenzyl )- n6 -( 3 - pentyl ) adenine ( 29 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 33 mg ) gave 29 as a white solid : yield 36 mg , 78 %. 1 h nmr ( cd 3 od ) δ 8 . 19 ( d , 2h , j = 8 . 8 hz ), 8 . 16 ( s , 1h ), 7 . 46 ( d , 2h , j = 8 . 8 hz ), 5 . 55 ( s , 2h ), 4 . 22 ( m , 1h ), 2 . 12 – 2 . 02 , 1 . 92 – 1 . 36 , 1 . 00 – 0 . 78 ( 3 × m , 21h ), 1 . 15 ( t , 1h , j = 12 . 2 hz ). lrms esi ( m + h + ) 477 . 3 . 9 -( 3 , 5 - dimethyl - isoxazol - 4 - ylmethyl )- 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 30 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 50 mg ) gave 30 as a white crystalline solid : yield 50 mg , 76 %. 1 h nmr ( cd 3 od ) δ 8 . 08 ( s , 1h ), 5 . 18 ( s , 2h ), 4 . 19 ( m , 1h ), 2 . 50 ( s , 3h ), 2 . 23 ( s , 3h ), 2 . 13 – 2 . 04 , 1 . 94 – 1 . 38 , 0 . 99 – 0 . 80 ( 3 × m , 21h ), 1 . 18 ( t , 1h , j = 12 . 1 hz ). lrms esi ( m + h + ) 451 . 3 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- 9 -( 2 - methyl - thiazol - 5 - ylmethyl )- n6 -( 3 - pentyl ) adenine ( 31 ). 2 -{ 2 -[ 1 -( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 56 mg , 0 . 1640 mmol ) and 4 - chloromethyl - 2 - methylthiazole hydrochloride ( 127 mg , 0 . 6899 mmol ) were stirred in dmf ( 8 ml ) at 150 ° c . for 22 h . the reaction mixture was adhered to silica and purified by column chromatography , eluting with a gradient of dcm / meoh ( 0 – 6 %) to afford pure 31 as an off white solid : yield 2 mg , 3 %. 1 h nmr ( cd 3 od ) δ 8 . 12 ( s , 1h ), 7 . 29 ( s , 1h ), 5 . 44 ( s , 2h ), 4 . 22 ( m , 1h ), 2 . 65 ( s , 3h ), 2 . 14 – 2 . 05 , 1 . 94 – 1 . 37 , 0 . 99 – 0 . 80 ( 3 × m , 21h ), 1 . 18 ( t , 1h , j = 12 . 2 hz ). lrms esi ( m + h + ) 453 . 2 . n6 -[( s )-(+)- sec - butyl ]- 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- 9 - propargyl - adenine ( 32 ). using the representative procedure for n9 - alkylation above n6 -[( s )-(+)- sec - butyl ]- 2 -{ 2 -[ 1 -( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 29 mg ) gave 32 as a white solid : yield 20 mg , 62 %. 1 h nmr ( cd 3 od ) δ 8 . 17 ( s , 1h ), 5 . 03 ( d , 2h , j = 2 . 6 hz ), 4 . 32 ( m , 1h ), 2 . 97 ( t , 1h , j = 2 . 6 hz ), 2 . 14 – 2 . 06 , 1 . 95 – 1 . 58 , 1 . 49 – 1 . 38 , 1 . 00 – 0 . 79 ( 4 × m , 16h ), 1 . 25 ( d , 3h , j = 6 . 4 hz ), 1 . 17 ( t , 1h , j = 12 . 2 hz ). lrms esi ( m + h + ) 366 . 1 . n6 -[( s )-(+)- sec - butyl ]- 9 -( 3 , 5 - dimethyl - isoxazol - 4 - ylmethyl )- 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl } adenine ( 33 ). using the representative procedure for n9 - alkylation above n6 -[( s )-(+)- sec - butyl ]- 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - pentyl ) adenine ( 42 mg ) gave 33 as a white solid : yield 31 mg , 55 %. 1 h nmr ( cd 3 od ) δ 8 . 08 ( s , 1h ), 5 . 19 ( s , 2h ), 4 . 29 ( m , 1h ), 2 . 50 ( s , 3h ), 2 . 22 ( s , 3h ), 2 . 14 – 2 . 04 , 1 . 95 – 1 . 57 , 1 . 49 – 1 . 38 , 0 . 99 – 0 . 82 ( 4 × m , 16h ), 1 . 24 ( d , 3h , j = 6 . 4 hz ), 1 . 18 ( t , 1h , j = 12 . 2 hz ). lrms esi ( m + h + ) 437 . 2 . n6 -( 2 - diphenylethyl )- 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- 9 - methyladenine ( 34 ). using the representative procedure for n9 - alkylation above n6 -( 2 - diphenylethyl )- 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl } adenine ( 14 mg ) gave 34 as a white solid : yield 5 mg , 35 %. lrms esi ( m + h + ) 466 . 3 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -[( s )-(−)- alpha - napthalen - 1 - yl - ethyl ]- 9 -( propargyl ) adenine ( 35 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 1 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -[( s )-(−)- alpha - napthalen - 1 - yl - ethyl ] adenine ( 29 mg ) gave 35 as a white solid : yield 22 mg , 70 %. 1 h nmr ( cd 3 od ) δ 8 . 23 ( m , 1h ), 8 . 15 ( s , 1h ), 7 . 88 – 7 . 84 ( m , 1h ), 7 . 76 ( d , 1h , j = 8 . 4 hz ), 7 . 64 ( d , 1h , j = 7 . 0 hz ), 7 . 53 – 7 . 40 ( m , 3h ), 6 . 33 ( br s , 1h ), 5 . 00 ( d , 2h , j = 2 . 6 hz ), 2 . 96 ( t , 1h , j = 2 . 6 hz ), 2 . 08 – 1 . 99 , 1 . 87 – 1 . 59 , 1 . 45 – 1 . 33 , 1 . 18 – 1 . 08 , 0 . 95 – 0 . 70 ( 3 × m , 12h ). lrms esi ( m + h + ) 464 . 1 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - methoxybenzyl )- 9 -( propargyl ) adenine ( 36 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( 3 - methoxybenzyl ) adenine ( 48 mg ) gave 36 as a white solid : yield 40 mg , 76 %. 1 h nmr ( cd 3 od ) δ 8 . 14 ( s , 1h ), 7 . 18 ( t , 1h , j = 7 . 9 hz ), 6 . 96 – 6 . 90 ( m , 2h ), 6 . 77 ( m , 1h ), 5 . 01 ( d , 2h , j = 2 . 6 hz ), 4 . 75 ( br s , 2h ), 3 . 74 ( s , 3h ), 2 . 97 ( t , 1h , j = 2 . 6 hz ), 2 . 13 – 2 . 04 , 1 . 94 – 1 . 64 , 1 . 48 – 1 . 37 , 1 . 21 – 1 . 11 , 0 . 96 – 0 . 77 ( 5 × m , 12h ). lrms esi ( m + h + ) 430 . 2 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- 9 -( propargyl )- n6 -( pyridin - 2 - ylmethyl ) adenine ( 37 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -( pyridin - 2 - ylmethyl ) adenine ( 29 mg ) gave 37 as a white solid : yield 27 mg , 84 %. 1 h nmr ( cd 3 od ) δ 8 . 49 ( d , 1h , j = 4 . 4 hz ), 8 . 20 ( s , 1h ), 7 . 76 ( dt , 1h , j = 1 . 8 hz , j = 7 . 7 hz ), 7 . 43 ( d , 1h , j = 7 . 9 hz ), 7 . 29 ( m , 1h ), 5 . 04 ( d , 2h , j = 2 . 6 hz ), 4 . 91 ( br s , 2h ), 2 . 98 ( t , 1h , j = 2 . 6 hz ), 2 . 10 – 2 . 03 , 1 . 90 – 1 . 63 , 1 . 47 – 1 . 36 , 1 . 20 – 1 . 10 , 0 . 96 – 0 . 73 ( 5 × m , 12h ). lrms esi ( m + h + ) 401 . 2 . 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -[( methyl )( 2 - phenethyl )]- 9 -( propargyl ) adenine ( 38 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ 1 ( s )- hydroxy - 3 ( r )- methyl - 1 - cyclohexyl ] ethyn - 1 - yl }- n6 -[( methyl )( 2 - phenethyl )] adenine ( 54 mg ) gave 38 as a white solid : yield 47 mg , 79 %. 1 h nmr ( cd 3 od ) δ 8 . 09 ( s , 1h ), 7 . 30 – 7 . 11 ( m , 5h ), 5 . 00 ( d , 2h , j = 2 . 6 hz ), 4 . 19 ( br s , 2h ), 3 . 35 ( br s , 3h ), 2 . 98 – 2 . 91 ( m , 3h ), 2 . 14 – 2 . 06 , 1 . 96 – 1 . 66 , 1 . 50 – 1 . 39 , 1 . 27 – 1 . 13 , 0 . 98 – 0 . 80 ( 5 × m , 12h ). lrms esi ( m + h + ) 428 . 3 . 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl }- 9 - methyladenine ( 39 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 58 mg ) gave 39 as a white solid : yield 39 mg , 64 %. 1 h nmr ( cd 3 od ) δ 8 . 08 ( s , 1h ), 3 . 80 ( s , 3h ), 2 . 37 – 2 . 22 , 2 . 08 – 2 . 03 , 1 . 89 – 1 . 74 , 1 . 64 – 1 . 57 ( 4 × m , 14h ). lrms esi ( m + h + ) 324 . 2 . 9 - ethyl - 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 40 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 59 mg ) gave 40 as a white solid : yield 42 mg , 65 %. 1 h nmr ( cd 3 od ) δ 8 . 15 ( s , 1h ), 4 . 25 ( t , 2h , j = 7 . 4 hz ), 2 . 36 – 2 . 22 , 2 . 08 – 2 . 03 , 1 . 89 – 1 . 74 , 1 . 64 – 1 . 57 ( 4 × m , 14h ), 1 . 47 ( t , 3h , j = 7 . 3 hz ). lrms esi ( m + h + ) 338 . 2 . 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl }- 9 - propyladenine ( 41 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 56 mg ) gave 41 as a white solid : yield 46 mg , 72 %. 1 h nmr ( cd 3 od ) δ 8 . 13 ( s , 1h ), 4 . 17 ( t , 2h , j = 7 . 2 hz ), 2 . 36 – 2 . 22 , 2 . 08 – 2 . 03 , 1 . 92 – 1 . 74 , 1 . 64 – 1 . 57 ( 4 × m , 16h ), 0 . 93 ( t , 3h , j = 7 . 5 hz ). lrms esi ( m + h + ) 352 . 2 . 9 - hexyl - 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 42 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 50 mg ) gave 42 as a white solid : yield 43 mg , 68 %. 1 h nmr ( cd 3 od ) δ 8 . 13 ( s , 1h ), 4 . 21 ( t , 2h , j = 7 . 0 , 7 . 5 hz ), 2 . 37 – 2 . 22 , 2 . 08 – 2 . 03 , 1 . 91 – 1 . 74 , 1 . 65 – 1 . 57 , 1 . 37 – 1 . 27 ( 5 × m , 22h ), 0 . 88 ( t , 3h ). lrms esi ( m + h + ) 394 . 1 . 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl }- 9 - nonyladenine ( 43 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 43 mg ) gave 43 as a white solid : yield 47 mg , 78 %. 1 h nmr ( cd 3 od ) δ 8 . 13 ( s , 1h ), 4 . 20 ( t , 2h , j = 7 . 3 hz ), 2 . 36 – 2 . 22 , 2 . 08 – 2 . 03 , 1 . 90 – 1 . 74 , 1 . 64 – 1 . 56 , 1 . 35 – 1 . 23 ( 5 × m , 28h ), 0 . 87 ( t , 3h , j = 7 . 0 hz ). lrms esi ( m + h + ) 436 . 1 . 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl }- 9 - isobutyl - adenine ( 44 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 52 mg ) gave 44 as a white solid : yield 46 mg , 75 %. 1 h nmr ( cd 3 od ) δ 8 . 11 ( s , 1h ), 4 . 02 ( d , 2h , j = 7 . 5 hz ), 2 . 36 – 2 . 16 , 2 . 08 – 2 . 03 , 1 . 88 – 1 . 73 , 1 . 64 – 1 . 56 , ( 4 × m , 15h ), 0 . 91 ( d , 6h , j = 6 . 6 hz ). lrms esi ( m + h + ) 366 . 2 . 9 - cyclopropylmethyl - 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 45 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 33 mg ) gave 45 as a white solid : yield 11 mg , 28 %. 1 h nmr ( cd 3 od ) δ 8 . 21 ( s , 1h ), 4 . 06 ( d , 2h , j = 7 . 3 hz ), 2 . 36 – 2 . 22 , 2 . 08 – 2 . 03 , 1 . 89 – 1 . 74 , 1 . 65 – 1 . 57 , ( 4 × m , 14h ), 1 . 42 – 1 . 27 ( m , 1h ), 0 . 65 – 0 . 58 , 0 . 49 – 0 . 43 ( 2 × m , 4h ). lrms esi ( m + h + ) 364 . 2 . 9 - cyclobutylmethyl - 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 46 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 54 mg ) gave 46 as a white crystalline solid : yield 21 mg , 32 %. 1 h nmr ( cd 3 od ) δ 8 . 12 ( s , 1h ), 4 . 22 ( d , 2h , j = 7 . 5 hz ), 2 . 87 ( quintet , 1h , j = 7 . 7 hz ), 2 . 37 – 2 . 22 , 2 . 09 – 1 . 74 , 1 . 65 – 1 . 57 , ( 3 × m , 20h ). lrms esi ( m + h + ) 378 . 2 . 9 - cyclopentylmethyl - 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 47 ). 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 44 mg , 0 . 1422 mmol ) was dissolved in dmf ( 20 ml ) with heating . anhydrous potassium carbonate ( 51 mg , 0 . 3690 mmol ) and cyclopentylmethyl 4 - methylbenzenesulfonate ( 54 mg , 0 . 2123 mmol ) were added and the mixture stirred at 70 ° c . for 72 h . extra cyclopentylmethyl 4 - methylbenzenesulfonate ( 82 mg , 0 . 3224 mmol ) was added and stirring continued at 100 ° c . a further 4 . 5 h . the reaction mixture was adhered to silica and purified by column chromatography , eluting with a gradient of dcm / meoh ( 0 – 6 %) to afford the pure 47 as a white solid : yield 38 mg , 68 %. 1h nmr ( cd 3 od ) δ 8 . 15 ( s , 1h ), 4 . 14 ( d , 2h , j = 7 . 7 hz ), 2 . 48 ( quintet , 1h , j = 7 . 5 hz ), 2 . 37 – 2 . 22 , 2 . 08 – 2 . 02 , 1 . 89 – 1 . 58 , 1 . 37 – 1 . 24 ( 4 × m , 22h ). lrms esi ( m + h + ) 392 . 0 . 9 - cyclohexylmethyl - 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 48 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 49 mg ) gave 48 as a white solid : yield 45 mg , 70 %. 1 h nmr ( cd 3 od ) δ 8 . 10 ( s , 1h ), 4 . 04 ( d , 2h , j = 7 . 3hz ), 2 . 37 – 2 . 22 , 2 . 09 – 2 . 03 , 1 . 92 – 1 . 54 , 1 . 31 – 0 . 96 , ( 4 × m , 25h ). lrms esi ( m + h + ) 406 . 3 . 9 - cyclobutyl - 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 49 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 34 mg ) gave 49 as a white solid : yield 18 mg , 45 %. 1 h nmr ( cd 3 od ) δ 8 . 32 ( s , 1h ), 5 . 03 ( m , 1h ), 2 . 72 – 2 . 49 , 2 . 38 – 2 . 22 , 2 . 04 – 1 . 74 , 1 . 64 – 1 . 57 ( 4 × m , 20h ). lrms esi ( m + h + ) 364 . 2 . 9 - cyclopentyl - 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 50 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 473 mg ) gave 50 as a white solid : yield 371 mg , 64 %. 1 h nmr ( cd 3 od ) δ 8 . 21 ( s , 1h ), 4 . 90 ( m , 1h ), 2 . 37 – 2 . 20 , 2 . 08 – 1 . 73 , 1 . 64 – 1 . 57 ( 4 × m , 22h ). lrms esi ( m + h + ) 378 . 2 . 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl }- 9 - propargyl - adenine ( 51 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 510 mg ) gave 51 as a white solid : yield 416 mg , 73 %. 1 h nmr ( cd 3 od ) δ 8 . 22 ( s , 1h ), 5 . 04 ( d , 2h , j = 2 . 6 hz ), 2 . 98 ( t , 1h , j = 2 . 5 hz ), 2 . 36 – 2 . 22 , 2 . 08 – 2 . 03 , 1 . 89 – 1 . 75 , 1 . 65 – 1 . 57 ( 4 × m , 14h ). lrms esi ( m + h + ) 348 . 2 . 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl }- 9 -( 2 - hydroxyethyl ) adenine ( 52 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 49 mg ) gave 52 as a white solid : yield 21 mg , 38 %. 1 h nmr ( cd 3 od ) δ 8 . 12 ( s , 1h ), 4 . 31 ( t , 2h , j = 5 . 2 hz ), 3 . 87 ( t , 2h , j = 4 . 7 hz ), 2 . 36 – 2 . 21 , 2 . 08 – 2 . 01 , 1 . 89 – 1 . 73 , 1 . 65 – 1 . 57 ( 4 × m , 14h ). lrms esi ( m + h + ) 354 . 2 . 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl }- 9 -( 2 - hydroxypropyl ) adenine ( 53 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 55 mg ) gave 53 as a white solid : yield 37 mg , 57 %. 1 h nmr ( cd 3 od ) δ 8 . 13 ( s , 1h ), 4 . 32 ( t , 2h , j = 7 . 0 hz ), 3 . 55 ( t , 2h , j = 5 . 9 , 6 . 2 hz ), 2 . 36 – 2 . 21 , 2 . 11 – 2 . 00 , 1 . 89 – 1 . 73 , 1 . 65 – 1 . 57 ( 4 × m , 16h ). lrms esi ( m + h + ) 368 . 2 . 2 -{ 2 -[ hydroxy - norbornan - 2 - yl ] ethyn - 1 - yl }- 9 - propargyladenine : isomer a ( 60 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - norbornan - 2 - yl ] ethyn - 1 - yl } adenine ( 42 mg ) gave 60 as a white solid : yield 18 mg , 38 %. 1 h nmr ( cd 3 od ) δ 8 . 22 ( s , 1h ), 5 . 03 ( s , 2h ), 2 . 47 – 2 . 51 ( m , 1h ), 2 . 19 – 2 . 30 ( m , 2h ), 1 . 90 – 2 . 08 ( m , 2h ), 1 . 53 – 1 . 66 ( m , 1h ), 1 . 27 – 1 . 47 ( m , 4h ). lrms esi ( m + h + ) 308 . 1 . 2 -{ 2 -[ hydroxy - norbornan - 2 - yl ] ethyn - 1 - yl }- 9 - propargyladenine : isomer b ( 61 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - norbornan - 2 - yl ] ethyn - 1 - yl } adenine ( 31 mg ) gave 61 as a white solid : yield 10 mg , 29 %. 1 h nmr ( cd 3 od ) δ 8 . 22 ( s , 1h ), 5 . 03 ( s , 2h ), 2 . 47 – 2 . 51 ( m , 1h ), 2 . 19 – 2 . 30 ( m , 2h ), 1 . 90 – 2 . 08 ( m , 2h ), 1 . 53 – 1 . 66 ( m , 1h ), 1 . 27 – 1 . 47 ( m , 4h ). lrms esi ( m + h + ) 308 . 1 . 9 -( but - 3 - ynyl )- 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 62 ). using the representative procedure for n9 - alkylation above 2 -{ 2 -[ hydroxy - adamantan - 2 - yl ] ethyn - 1 - yl } adenine ( 52 mg ) gave 62 as a white solid : yield 14 mg , 24 %. 1 h nmr ( cd 3 od ) δ 8 . 18 ( s , 1h ), 4 . 36 ( t , 2h , j = 6 . 6 hz ), 2 . 77 ( dt , 2h , j = 2 . 6 , 6 . 6 hz ), 2 . 36 ( t , 1h , j = 2 . 6 hz ), 2 . 36 – 2 . 22 , 2 . 08 – 2 . 03 , 1 . 89 – 1 . 74 , 1 . 64 – 1 . 57 ( 4 × m , 16h ). lrms esi ( m + h + ) 362 . 0 . 2 -{ 3 -[ 1 -( methoxycarbanoyl ) piperidin - 4 - yl ] propyn - 1 - yl }- 9 - propargyladenine ( 63 ). using the representative procedure for n9 - alkylation above 2 -{ 3 -[ 1 -( methoxycarbanoyl ) piperidin - 4 - yl ] propyn - 1 - yl } adenine ( 53 mg ) gave 63 as a white solid : yield 18 mg , 30 %. 1 h nmr ( cd 3 od ) δ 8 . 21 ( s , 1h ), 5 . 02 ( s , 2h ), 4 . 21 – 4 . 09 ( m , 2h ), 3 . 66 ( s , 3h ), 2 . 94 – 2 . 74 ( m , 2h ), 2 . 47 – 2 . 40 ( m , 3h ), 1 . 95 – 1 . 74 ( m , 3h ), 1 . 38 – 1 . 20 ( m , 2h ). lrms esi ( m + h + ) 353 . 1 . the compounds in tables 1 to 7 , or their pharmaceutically acceptable salts either as single stereoisomers or mixtures are representative examples of the invention . table 7 r 7 , r 8 = h compound ( cr 1 r 2 ) m - z w w ′ r 6 nc265 propargyl ch ch co 2 me nc266 c - pentyl ch n co 2 me nc267 propargyl n ch co 2 me nc268 c - pentyl n n co 2 me nc269 propargyl ch ch co 2 me nc270 c - pentyl ch n co 2 me nc271 propargyl n ch co 2 me nc272 c - pentyl n n co 2 me nc273 propargyl ch ch ch 2 oh nc274 c - pentyl ch n ch 2 oh nc275 propargyl n ch ch 2 oh nc276 c - pentyl n n ch 2 oh nc277 propargyl ch ch ch 2 oh nc278 c - pentyl ch n ch 2 oh nc279 propargyl n ch ch 2 oh nc280 c - pentyl n n ch 2 oh nc281 propargyl ch ch co 2 h nc282 c - pentyl ch n co 2 h nc283 propargyl n ch co 2 h nc284 c - pentyl n n co 2 h nc285 propargyl ch ch co 2 h nc286 c - pentyl ch n co 2 h nc287 propargyl n ch co 2 h nc288 c - pentyl n n co 2 h nc289 propargyl ch ch ch 2 oac nc290 c - pentyl ch n ch 2 oac nc291 propargyl n ch ch 2 oac nc292 c - pentyl n n ch 2 oac nc293 propargyl ch ch ch 2 oac nc294 c - pentyl ch n ch 2 oac nc295 propargyl n ch ch 2 oac nc296 c - pentyl n n ch 2 oac nc297 propargyl ch ch conh 2 nc298 c - pentyl ch n conh 2 nc299 propargyl n ch conh 2 nc300 c - pentyl n n conh 2 nc301 propargyl ch ch conh 2 nc302 c - pentyl ch n conh 2 nc303 propargyl n ch conh 2 nc304 c - pentyl n n conh 2 nc305 propargyl ch ch conhme nc306 c - pentyl ch n conhme nc307 propargyl n ch conhme nc308 c - pentyl n n conhme nc309 propargyl ch ch conhme nc310 c - pentyl ch n conhme nc311 propargyl n ch conhme nc312 c - pentyl n n conhme nc313 propargyl ch ch co 2 tbu nc314 c - pentyl ch n co 2 tbu nc315 propargyl n ch co 2 tbu nc316 c - pentyl n n co 2 tbu nc317 propargyl ch ch co 2 tbu nc318 c - pentyl ch n co 2 tbu nc319 propargyl n ch co 2 tbu nc320 c - pentyl n n co 2 tbu nc321 propargyl ch ch co 2 et nc322 c - pentyl ch n co 2 et nc323 propargyl n ch co 2 et nc324 c - pentyl n n co 2 et nc325 propargyl ch ch co 2 et nc326 c - pentyl ch n co 2 et nc327 propargyl n ch co 2 et nc328 c - pentyl n n co 2 et nc329 propargyl ch ch co 2 ibu nc330 c - pentyl ch n co 2 ibu nc331 propargyl n ch co 2 ibu nc332 c - pentyl n n co 2 ibu nc333 propargyl ch ch co 2 ibu nc334 c - pentyl ch n co 2 ibu nc335 propargyl n ch co 2 ibu nc336 c - pentyl n n co 2 ibu nc337 propargyl ch ch co 2 ipr nc338 c - pentyl ch n co 2 ipr nc339 propargyl n ch co 2 ipr nc340 c - pentyl n n co 2 ipr nc341 propargyl ch ch co 2 ipr nc342 c - pentyl ch n co 2 ipr nc343 propargyl n ch co 2 ipr nc344 c - pentyl n n co 2 ipr nc345 propargyl ch ch come nc346 c - pentyl ch n come nc347 propargyl n ch come nc348 c - pentyl n n come nc349 propargyl ch ch come nc350 c - pentyl ch n come nc351 propargyl n ch come nc352 c - pentyl n n come nc353 propargyl ch ch coc ( ch 3 ) 3 nc354 c - pentyl ch n coc ( ch 3 ) 3 nc355 propargyl n ch coc ( ch 3 ) 3 nc356 c - pentyl n n coc ( ch 3 ) 3 nc357 propargyl ch ch coc ( ch 3 ) 3 nc358 c - pentyl ch n coc ( ch 3 ) 3 nc359 propargyl n ch coc ( ch 3 ) 3 nc360 c - pentyl n n coc ( ch 3 ) 3 nc361 propargyl ch ch coch 2 ( ch 3 ) 3 nc362 c - pentyl ch n coch 2 ( ch 3 ) 3 nc363 propargyl n ch coch 2 ( ch 3 ) 3 nc364 c - pentyl n n coch 2 ( ch 3 ) 3 nc365 propargyl ch ch coch 2 ( ch 3 ) 3 nc366 c - pentyl ch n coch 2 ( ch 3 ) 3 nc367 propargyl n ch coch 2 ( ch 3 ) 3 nc368 c - pentyl n n coch 2 ( ch 3 ) 3 nc369 propargyl ch ch c ( o ) n ( ch 3 ) 2 nc370 c - pentyl ch n c ( o ) n ( ch 3 ) 2 nc371 propargyl n ch c ( o ) n ( ch 3 ) 2 nc372 c - pentyl n n c ( o ) n ( ch 3 ) 2 nc373 propargyl ch ch c ( o ) n ( ch 3 ) 2 nc374 c - pentyl ch n c ( o ) n ( ch 3 ) 2 nc375 propargyl n ch c ( o ) n ( ch 3 ) 2 nc376 c - pentyl n n c ( o ) n ( ch 3 ) 2 nc377 propargyl ch ch c ( o ) n ( ch 3 ) et nc378 c - pentyl ch n c ( o ) n ( ch 3 ) et nc379 propargyl n ch c ( o ) n ( ch 3 ) et nc380 c - pentyl n n c ( o ) n ( ch 3 ) et nc381 propargyl ch ch c ( o ) n ( ch 3 ) et nc382 c - pentyl ch n c ( o ) n ( ch 3 ) et nc383 propargyl n ch c ( o ) n ( ch 3 ) et nc384 c - pentyl n n c ( o ) n ( ch 3 ) et nc385 propargyl ch ch c ( o ) n ( ch 3 ) ipr nc386 c - pentyl ch n c ( o ) n ( ch 3 ) ipr nc387 propargyl n ch c ( o ) n ( ch 3 ) ipr nc388 c - pentyl n n c ( o ) n ( ch 3 ) ipr nc389 propargyl ch ch c ( o ) n ( ch 3 ) ipr nc390 c - pentyl ch n c ( o ) n ( ch 3 ) ipr nc391 propargyl n ch c ( o ) n ( ch 3 ) ipr nc392 c - pentyl n n c ( o ) n ( ch 3 ) ipr nc393 propargyl ch ch c ( o ) n ( ch 3 ) ibu nc394 c - pentyl ch n c ( o ) n ( ch 3 ) ibu nc395 propargyl n ch c ( o ) n ( ch 3 ) ibu nc396 c - pentyl n n c ( o ) n ( ch 3 ) ibu nc397 propargyl ch ch c ( o ) n ( ch 3 ) ibu nc398 c - pentyl ch n c ( o ) n ( ch 3 ) ibu nc399 propargyl n ch c ( o ) n ( ch 3 ) ibu nc400 c - pentyl n n c ( o ) n ( ch 3 ) ibu nc401 propargyl ch ch c ( o ) nh ( et ) nc402 c - pentyl ch n c ( o ) nh ( et ) nc403 propargyl n ch c ( o ) nh ( et ) nc404 c - pentyl n n c ( o ) nh ( et ) nc405 propargyl ch ch c ( o ) nh ( et ) nc406 c - pentyl ch n c ( o ) nh ( et ) nc407 propargyl n ch c ( o ) nh ( et ) nc408 c - pentyl n n c ( o ) nh ( et ) nc409 propargyl ch ch c ( o ) nh ( ipr ) nc410 c - pentyl ch n c ( o ) nh ( ipr ) nc411 propargyl n ch c ( o ) nh ( ipr ) nc412 c - pentyl n n c ( o ) nh ( ipr ) nc413 propargyl ch ch c ( o ) nh ( ipr ) nc414 c - pentyl ch n c ( o ) nh ( ipr ) nc415 propargyl n ch c ( o ) nh ( ipr ) nc416 c - pentyl n n c ( o ) nh ( ipr ) nc417 propargyl ch ch c ( o ) nh ( ibu ) nc418 c - pentyl ch n c ( o ) nh ( ibu ) nc419 propargyl n ch c ( o ) nh ( ibu ) nc420 c - pentyl n n c ( o ) nh ( ibu ) nc421 propargyl ch ch c ( o ) nh ( ibu ) nc422 c - pentyl ch n c ( o ) nh ( ibu ) nc423 propargyl n ch c ( o ) nh ( ibu ) nc424 c - pentyl n n c ( o ) nh ( ibu ) nc425 propargyl ch ch ch 2 ococh 3 nc426 c - pentyl ch n ch 2 ococh 3 nc427 propargyl n ch ch 2 ococh 3 nc428 c - pentyl n n ch 2 ococh 3 nc429 propargyl ch ch ch 2 ococh 3 nc430 c - pentyl ch n ch 2 ococh 3 nc431 propargyl n ch ch 2 ococh 3 nc432 c - pentyl n n ch 2 ococh 3 nc433 propargyl ch ch ch 2 ocoet nc434 c - pentyl ch n ch 2 ocoet nc435 propargyl n ch ch 2 ocoet nc436 c - pentyl n n ch 2 ocoet nc437 propargyl ch ch ch 2 ocoet nc438 c - pentyl ch n ch 2 ocoet nc439 propargyl n ch ch 2 ocoet nc440 c - pentyl n n ch 2 ocoet nc441 propargyl ch ch ch 2 ocoipr nc442 c - pentyl ch n ch 2 ocoipr nc443 propargyl n ch ch 2 ocoipr nc444 c - pentyl n n ch 2 ocoipr nc445 propargyl ch ch ch 2 ocoipr nc446 c - pentyl ch n ch 2 ocoipr nc447 propargyl n ch ch 2 ocoipr nc448 c - pentyl n n ch 2 ocoipr nc449 propargyl ch ch ch 2 ocoibu nc450 c - pentyl ch n ch 2 ocoibu nc451 propargyl n ch ch 2 ocoibu nc452 c - pentyl n n ch 2 ocoibu nc453 propargyl ch ch ch 2 ocoibu nc454 c - pentyl ch n ch 2 ocoibu nc455 propargyl n ch ch 2 ocoibu nc456 c - pentyl n n ch 2 ocoibu evaluation of novel a 2a antagonists in four mouse models of pd : the a 2a receptor antagonist atl - 2 enhances motor function in a dose - dependent manner in normal and dopamine - depleted mice . in the set of experiments , we perform a dose response study of atl - 2 in stimulating motor activity in normal mice , and then we further extend this to dopamine - depleted mice . adult male mice are habituated for 120 minutes and treated ( i . p .) with saline or varying doses of compound , and their locomotor activity recorded for 120 minutes . in a second set of experiments , we utilize the mptp treatment paradigm to create animal model of pd by severely depleting dopamine in mice . we use a single mptp treatment paradigm ( 40 mg / kg ) which has been reproducibly reducing dopamine to 30 – 40 % of normal dopamine contents in striatum in our previous studies . 35 , 74 adult male mice (˜ 25 mg / kg ) are treated with single dose of mptp ( 40 mg / kg ). thirty minutes after the mptp treatment , mice are injected ( i . p .) with vehicle or compounds of the invention at the same doses discussed above . their motor activity is recorded for 180 minutes . results : based on our previous experiments with other a 2a r antagonists and our pilot study , we observe the maximal stimulant dose as well as sub - threshold doses of atl compounds in normal and mptp - treated mice . without being bound by any theory , it is proposed that motor stimulant effect may manifest best in dopamine - depleted animals than normal animals , indicating that a 2a r antagonists preferentially act at the a 2a r in a pd condition to stimulate motor activity . a 2a receptor antagonists synergize with l - dopa to stimulate motor activity in dopamine - depleted mice . we further tested the ability of these compounds to synergistically enhance motor function in conjunction with l - dopa , the standard therapy . mice are injected ( i . p .) with mptp at a dose ( 1 – 2 . 5 mg / kg ) that markedly decreases striatal dopamine levels . thirty minutes later ( when the mice exhibit an immobility ), the mice are then randomly assigned to the following different treatment groups ( n = 10 ): ( 1 ) l - dopa ( 25 mg / kg ); ( 2 ) atl - 2 ( 0 . 3 , 1 , 3 , and 10 mg / kg ), and ( 3 ) l - dopa ( 25 mg / kg )+ atl - 2 ( 0 . 3 , 1 , 3 , and 10 mg / kg ). locomotor behavior is monitored for 120 min before and after the treatment . results : based on the preliminary results and on the known feature of other a 2a r antagonists , a synergistic effect of atl - 2 with l - dopa in stimulating locomotor activity in dopamine - depleted mice is observed . this synergistic effect of atl - 2 and l - dopa is exhibited in a left - shift of the dose - response curve . a 2a antagonists potently and specifically attenuate mptp - induced neurotoxicity by inhibiting mptp metabolism . c57bl / 6 mice ( n = 10 – 12 mice per group ) are pretreated with the a 2a antagonist atl - 2 ( 0 . 3 , 1 . 0 , 3 . 0 and 10 . 0 mg / kg , i . p ) 5 min prior to each of four mptp ( 40 mg / kg ) injections at 2 hr intervals . these doses are selected based on our preliminary results ( with csc ) and on motor and neuroprotective effects ( against ischemia ) by sch58261 and dpcpx . the specificities for the a 2a r in these dose ranges of atl - 2 have been confirmed using a 2a ko mice . seven days after the mptp (± csc , sch58261 or cpa ) treatment , the striatum from one side are dissected out and processed for hplc analysis of dopamine and dopac levels . the other half brain is quickly frozen for sectioning coronally through the striatum and substantial nigra . dat binding density in striatum may be determined by receptor autoradiography using 3 h - mazindol as a specific ligand . quantitation of dat ( 3 h - mazindol ) binding autoradiography may be performed by densitometry analysis . the numbers of dopaminergic neurons may be determined by th immunohistochemistry in the substantial nigra . stereological methods may be used to estimate the absolute reduction in th + nigral neurons in mptp - treated wt mice and any attenuation in those pretreated with atl - 2 . in the same sections , cell counts may also be performed for th + neurons in the more medial vta , which is less affected in mptp treated mice as well as in pd . results : guided by our preliminary results , neuroprotection in a dose - dependent manner ( at least from 0 . 5 to 5 mg / kg range ) may be observed . the potency of a 2a antagonists for neuroprotection may be observed with their potency for motor stimulation and for possible attenuation of behavioral sensitization ( see above ). similarly , the potency of csc or sch58261 for neuroprotection against mptp may be compared to that for neuroprotection against ischemic injury and excitoxicity . a significant difference in an a 2a antagonist &# 39 ; s potency in neuroprotection against mptp and against ischemia or excitoxicity may suggest different mechanisms and sites of action ( e . g . glial versus neuronal compartments which may have different g - protein coupling mechanisms ). on the other hand , the same potency of a 2a antagonists for motor stimulation , neuroprotection and possibly delayed sensitization to l - dopa would suggest that the same type of a 2a r is responsible for all these potential benefits of a 2a antagonists in different animal models of pd . a 2a antagonists delay and a 2a agonists accelerate l - dopa - induced locomotor sensitization in unilateral 6 - ohda - lesioned mice . the ability of atl - 2 to modify the development of l - dopa - induced locomotor sensitization in hemiparkinsonian mice are tested . c57bl / 6 mice ( from the jackson &# 39 ; s lab , bar harbor , mich .) are lesioned with 6 - ohda by unilateral intrastriatal using a standard lesioning protocol . seven days after the 6 - ohda ( or mpp + ) treatment , mice are injected with l - dopa ( 2 . 0 mg / kg , daily ) for 14 days . five min prior to each l - dopa treatment , the mice receive intraperitoneal pretreatment with : ( 1 ) vehicle , ( 2 ) atl - 2 ( 3 mg / kg ) or ( 3 ) atl - 2 ( 10 mg / kg ). in these dose ranges , the selective a 2a antagonists have been shown to produce motor stimulant effects ( see preliminary results ). rotational responses to l - dopa are recorded on the days 1 , 3 , 5 , 7 , 10 and 15 . following the behavioral measurement , mice may be sacrificed and their brains sectioned through striatum and substantia nigra . striatal enkephalin mrna levels are determined by in situ hybridization histochemistry . similarly , dat ( 3 h - mazindol ) binding is measured by receptor autoradiography to ensure successful and equivalent lesions among different experimental groups . results : based on our previous study with sch58261 in this repeated l - dopa - induced sensitization model , atl - 2 delays or prevent the development of locomotor sensitization . the prevention or delayed appearance with l - dopa locomotor sensitization by co - injection of an a 2a antagonist indicates an important role of the a 2a r in the development of l - dopa - induced behavioral sensitization . furthermore , this helps exclude the possibility that an attenuated behavioral sensitization to chronic l - dopa observed in a 2a ko mice results from a developmental effect of a 2a r deficiency . thus combined genetic and pharmacological approaches provide the clearest assessment of the a 2a r &# 39 ; s role in the development of behavioral sensitization to l - dopa , and provides insights into its role in l - dopa - induced dyskinesia . wt and a 2a ko mice ( generated as above ) as well as commercially procured c57bl / 6 mice ( taconic , n . y .) may be used for this study . since our pilot study and other reports [ 40 , 88 ] indicate that animal age is a critical factor in determining the extent of an mptp lesion , animal body weight around 25 – 30 grams ( corresponding to approximately 10 weeks of age ) is tightly controlled . the mice are housed in temperature and humidity - controlled rooms with a 24 - hour 1 : 1 light : dark cycle . adenosinergic and dopaminergic agents are injected at the volume of 0 . 1 ml / 10 gram body weight of mice . other adenosinergic and dopaminergic drugs are purchased from rbi ( natick , mass .). from our previous work , we have adapted a special solvent ( 15 % dmso , 15 % alkamuls - el 620 and 70 % saline ) for dissolving a 2a antagonists , including csc and sch58261 . catalepsy behavior may be induced by haloperidol ( 1 mg / kg , i . p .) or reserpine ( 5 mg / kg , i . p . see below ). catalepsy score may be determined by the bar and grid tests . for the bar test , both mouse forepaws are placed on a 6 cm - high horizontal bar ( diameter 0 . 7 cm ). in the grid test , mice are allowed to cling to a metal - framed vertical grid ( 1 . 3 cm squares ). the latency from paw placement until the first complete removal of one paw from the support is measured ( maximal test duration 180 sec ). upon the completion of behavioral assessment , mice are sacrificed and the brains are processed for neurochemical and histochemical analyses . a ) intraperitoneal injection of mptp : the mptp administration regimen ( 20 mg / kg × 4 at 2 hr interval ) has been shown to produce severe dopamine depletion ( consistently greater than ˜ 80 % in our preliminary results fig6 and 7 ). naive c57bl / 6 mice are pretreated with adenosine antagonists 5 min prior to mptp treatment . b ) intrastriatal injection of 6 - ohda : wild - type c57bl / 6 or a2ar mutant mice are anesthetized with avertin and positioned in a stereotaxic frame . three microliters of 6 - ohda ( 3 μg / μl ) are injected into the left striatum ( coordinates from bregma : ap + 0 . 0 , l + 2 . 5 . 0 , dv − 4 . 4 ) via a infusion minipump over a 4 min period . due to its photolability , 6 - ohda is dissolved in 0 . 01 % ascorbic acid and injected under a light - protected environment . c ) post - treatment care : dopamine - depleted mice may be continually monitored , and special care may be taken to maintain mouse body temperature with a heating blanket or warming lights . during the first 48 hours post - operation , mashed food pellets and water are provided to the mice inside the cage at floor - level for easy access . ( a ) measurements of catecholamines and indoleamines in striatum by hplc : to measure tissue catecholamine and indoleamine levels , mice are decapitated , their brains are removed rapidly , and striata are dissected out and frozen on dry ice . striata are weighed frozen and then homogenized in 200 μl of 150 mm trichloroacetic acid containing 0 . 1 mm edta and 1 μm epinephrine ( as an internal standard ). homogenates are centrifuged for 5 min at 15 , 000 g . the catecholamines in the supernatant are separated over a reverse - phase hydrophobic interaction c - 18 hplc column ( beckman , 5μ ods ) and measured using an electrochemical detector ( esa coulochem 5100a ) with electrodes set in series at oxidizing (+ 0 . 22 v ) and then reducing (− 0 . 35 v ) potentials . both the retention time and the ratio of oxidation to reduction currents for given sample peaks are compared against those for external standards to ensure proper identification of analytes . ( b ) stereologic quantitation of neuronal loss in substantia nigra : one week after lesioning , mice may be perfusion - fixed and their brains may be microtome - cut into 40 μm coronal free - floating sections . every sixth section may be processed for th immunohistochemistry using a 1 : 1 , 000 dilution of a polyclonal rabbit antiserum against rat th ( eugene tech . intl ., nj ). immunostaining is completed using standard avidin - biotin procedures described previously [ 18 , 130 ]. a non - biased stereological technique is employed to quantify the effect of treatment on total th + nigra ( pars compacta ) cell counts as described previously [ 81 ]. all counts are performed by a single observer who is unaware of the treatment group at the time of neuronal estimates . based on our pilot studies in wt mice mpp + at this dose ( 3 μg / striatum ) produced a ˜ 40 % loss of ipsilateral th + nigral neurons . ( c ) a2a receptor binding autoradiography : twenty micron striatal sections are preincubated for 5 minutes with ice - cold buffer ( 509 mm tris - hcl , 5 mm kcl and 300 mm nacl , ph 7 . 9 ) and then incubated for 60 minutes in the same buffer containing 6 nm 3h - sch58261 ( provided generously by dr . e . ongini ) [ 131 ]. the slides are washed twice and then air - dried before exposure to hyperfilm ( amersham , il ) for 2 – 4 weeks . the films are analyzed with a video - based image analysis system ( multianalyst ; biorad ), and total striatal 3h - sch58261 binding ( fmol / mg tissue ) is calculated using a tritium - labeled calibration standard [ 17 , 131 ]. single statistical comparisons of an a2ar ko group to its wt control are generally performed using a student &# 39 ; s t test , two - tailed . comparison of more than two factors ( e . g . genotype , drug treatment and time course ) and their interactions are made using 2 - way anova followed by newman - keuls post hoc analysis . if data are not normally distributed , non - parametric tests ( kruskal - wallis or mann - whitney u test ) are used . vertebrate animals . mice are the only animals that are be used in experiments . the mice are monitored daily ( co - investigators or technician ) under the supervision of a staff veterinarian . in the majority of the experiments the mice are kept under spf conditions with no more than 5 mice / cage of females and 4 mice / cage of males . all husbandry and veterinary care meets nih and aaalac standards for humane care for use of laboratory animals . in addition , because of daily observation of all animals , any moribund animal is humanely euthanized by co 2 . models of pd are used to investigate pre - clinical efficacy and pharmacokinetics of a2aar antagonist . because we have used these model in our laboratory , the model is now well characterized and the experimental manipulation of mice for these studies are well established . 1 . hauser r a , hubble j p , truong d d . randomized trial of the adenosine a ( 2a ) receptor antagonist istradefylline in advanced pd . neurology . 2003 ; 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