New 2,6,9-trisubstituted adenines as adenosine receptor antagonists : a preliminary SAR profile A new series of 2,6,9-trisubstituted adenines -LRB- 5 -- 14 -RRB- have been prepared and evaluated in radioligand binding studies for their affinity at the human A1 , A2A and A3 adenosine receptors and in adenylyl cyclase experiments for their potency at the human A2B subtype . From this preliminary study the conclusion can be drawn that introduction of bulky chains at the N6 position of 9-propyladenine significantly increased binding affinity at the human A1 and A3 adenosine receptors , while the presence of a chlorine atom at the 2 position resulted in a not univocal effect , depending on the receptor subtype and/or on the substituent present in the N6 position . However , in all cases , the presence in the 2 position of a chlorine atom favoured the interaction with the A2A subtype . These results demonstrated that , although the synthesized compounds were found to be quite inactive at the human A2B subtype , adenine is a useful template for further development of simplified adenosine receptor antagonists with distinct receptor selectivity profiles . Introduction Adenosine , a naturally occurring nucleoside , is involved in a wide variety of physiological and pathophysiological processes -LSB- 1 -RSB- . Adenosine mediates these effects through the activation of at least four human receptor subtypes -LRB- P1 -RRB- , belonging to the superfamily of G protein-coupled receptors , which have been recently cloned -LSB- 2 -RSB- and classified as A1 , A2A , A2B and A3 -LSB- 3 -RSB- . The subtypes are classified on the bases of coupling to second messengers and pharmacological profiles for agonists and antagonists . In fact , A1 and A3 adenosine receptor subtypes are linked to inhibition of adenylyl cyclase and A2A and A2B subtypes are linked to stimulation of the same enzyme -LSB- 4 -RSB- . In particular , A2B receptors have been implicated in several physiological functions such as the regulation of mast cell secretion -LSB- 5 , 6 -RSB- , gene expression -LSB- 5 , 7 , 8 -RSB- , cell growth -LSB- 9 -RSB- and intestinal functions . A2B receptors may also play a role in asthma , since they mediate mast cell degranulation from human mast cells and are present in high density in human blood eosinophils -LSB- 10 , 11 -RSB- . For this reason A2B antagonists could be considered potential antiasthmatic agents -LSB- 10 -- 12 -RSB- . While the A1 , A2A and A3 adenosine receptors have been pharmacologically characterized through the use of highly potent and selective agonists and/or antagonists , an accurate investigation of the pathophysiological role of A2B receptors is precluded due to the lack of very selective ligands -LSB- 13 -RSB- . On the other hand , only recently radiolabelled adenosine antagonists have been used for binding assays at the A2B receptor subtype -LSB- 1 -RSB- . Recently , xanthine derivatives , such as compounds 1 and 2 in Fig. 1 , have been proposed as potent and selective adenosine receptor antagonists -LSB- 14 , 15 -RSB- . On the other hand , in the non-xanthine family poor results have been obtained in recent years . However , mention should be made of the pyrazolo-triazolo-pyrimidine derivative 3 , which showed promising binding affinity at the A2B adenosine receptor although the level of selectivity vs the human -LRB- h -RRB- A3 subtype was still poor -LSB- 16 -RSB- . Very recently , a bipyrimidyl derivative 4 has been proposed as an A2B adenosine receptor antagonist , with affinity in the same range of compound 3 , while the selectivity vs the other receptor subtypes was found to be significantly better -LSB- 17 -RSB- -LRB- Fig. 1 -RRB- . Fig. 1Structures and binding profile -LRB- Ki nM -RRB- of some A2B adenosine receptor antagonists A structural analysis of the derivatives 1 and 3 clearly shows the presence of bulky substituents such as aryloxyacetylamino-phenyl groups at the 8 position -LRB- compound 1 -RRB- and arylacetyl moiety at the N5 position -LRB- compound 3 -RRB- . On the other hand , in recent years a number of substituted adenines have been synthesized and tested at the four adenosine receptor subtypes , demonstrating that the introduction of different substituents at the 2 , 8 and 9 positions of the adenine core resulted in high-affinity antagonists with distinct receptor selectivity profile -LSB- 18 -- 22 -RSB- . At the A2B receptor the derivatives bearing an ethyl in the 9 position and linear chains in the 2 position showed potency in the μM range , while the presence of sterically hindered substituents in the same positions was detrimental for the potency . Furthermore , substitution of the 9-ethyl group with a propyl chain seems to favour the interaction with human A2B receptors -LSB- 21 -RSB- . Hence , on the basis of the results obtained with compound 3 , introduction of a bulky substituent on the N6 amino group of the adenine and 2-chloroadenine moiety could increase potency and selectivity for the human A2B adenosine receptor subtypes . Hence , bulky substituents such as arylacetyl or aryloxyphenylacetyl moieties were introduced on the N6 amino group of compounds 5 and 6 to obtain derivatives 7 -- 14 , with the aim of finding a new class of A2B antagonists -LRB- Fig. 2 -RRB- . Fig. 2Structures of designed compounds Chemistry The designed compounds -LRB- 5 -- 14 -RRB- have been synthesized as summarized in Schemes 1 and 2 . The starting 9-propyladenine -LRB- 5 -RRB- -LSB- 23 -RSB- was obtained by alkylation of commercially available adenine -LRB- 15 -RRB- with propyliodide in the presence of potassium carbonate . Flash chromatography led to the desired 9-substituted isomer 5 as the major product -LRB- yield 79 % -RRB- together with the 7-isomer 5a -LRB- yield 9 % -RRB- . Isomeric structure of compounds 5 and 5a was assigned on the bases of 1D-1H-NOE difference spectra . In fact , irradiation of both CH2 groups of the propylic chain in compound 5a gave a NOE at both H-C -LRB- 8 -RRB- and 6-NH2 groups , demonstrating the 7 position as the alkylation site . On the contrary , a NOE at both H-C -LRB- 8 -RRB- and H-C -LRB- 2 -RRB- , and not at the 6-NH2 group in compound 5 , upon saturation of CH2 groups of the propylic chain , confirmed the 9 position as the alkylation site . Scheme 1a Synthesis of 9-propyladenine ; b Synthesis of 2-chloro-9-propyladenine . Reagents : i : DMF , K2 CO3 , propyliodide , RT ; ii : liq . NH3 , sealed tube , RTScheme 2Reagents : i : CDI , dry THF , reflux The 2-chloro derivative 6 was obtained by alkylation of commercially available 2,6-dichloropurine -LRB- 16 -RRB- with propyliodide , using the same procedure utilized for compound 5 , to afford the 9-substituted isomer 17 as the major product -LRB- yield 75 % -RRB- along with the 7-isomer 17a -LRB- yield 10 % -RRB- -LSB- 24 -RSB- . -LSB- 1H -RSB- - NMR spectra of 17 and 17a in CDCl3 are in agreement with those reported in the literature -LSB- 24 -RSB- ; in the experimental part -LSB- 1H -RSB- - NMR spectra of the same compounds are reported using dimethyl sulfoxide -LRB- DMSO -RRB- as the solvent . Compound 17 was reacted with liquid ammonia in a sealed tube at room temperature -LRB- RT -RRB- overnight to give the 2-chloro-9-propyladenine -LRB- 6 -RRB- -LSB- 24 -RSB- -LRB- Scheme 1 -RRB- . Final compounds 7 -- 14 were obtained by condensation of amino compounds 5 or 6 with the appropriate acid 18 -- 22 -LSB- 25 -RSB- in the presence of carbonyldiimidazole in tetrahydrofurane -LRB- THF -RRB- at reflux for 18 h -LRB- Scheme 2 -RRB- . Results and discussion All the compounds were evaluated at the human recombinant adenosine receptors , stably transfected into Chinese hamster ovary -LRB- CHO -RRB- cells , utilizing radioligand binding studies -LRB- A1 , A2A , A3 -RRB- or adenylyl cyclase activity assay -LRB- A2B -RRB- . Receptor binding affinity was determined using -LSB- 3H -RSB- CCPA -LRB- 2-chloro-N6-cyclopentyladenosine -RRB- as the radioligand for A1 receptors , whereas -LSB- 3H -RSB- NECA -LRB- 5 ' - N-ethylcarboxamidoadenosine -RRB- was used for the A2A and A3 subtypes . In the case of A2B receptors Ki values were calculated from IC50 values determined by inhibition of NECA-stimulated adenylyl cyclase activity . Ki values are in μM , with 95 % confidence intervals in parentheses -LSB- 26 -RSB- . The results of binding and cyclase activity studies are reported in Table 1 . Table 1Biological profile of synthesized compounds 5 -- 14aDisplacement of specific -LSB- 3H -RSB- - CCPA binding at human A1 receptors expressed in CHO cells . bDisplacement of specific -LSB- 3H -RSB- - NECA binding at human A2A receptors expressed in CHO cells . cKi values of the inhibition of NECA-stimulated adenylyl cyclase activity in CHO cells expressing human A2B receptors . dDisplacement of specific -LSB- 3H -RSB- - NECA binding at human A3 receptors expressed in CHO cells . All the tested compounds 5 -- 14 showed affinities at the human A1 , A2A and A3 adenosine receptors in the μM range without significant levels of selectivity . At A2B receptors most compounds were found to be inactive when tested at a concentration up to 100 μM -LRB- Ki values > 30 μM -RRB- . It is quite evident that the introduction of phenylacetic or aryloxyphenylacetic moieties at the N6 position of 9-propyladenine -LRB- 5 -RRB- or of 2-chloro-9-propyladenine -LRB- 6 -RRB- to give compounds 7 -- 14 modifies the binding profile of the derivatives , although without significantly increasing binding affinity -LRB- Table 1 -RRB- . On the other hand , the same substitutions were found to be detrimental for the activity at the A2B receptor subtype . In fact , the N6-unsubstituted derivative 6 proved to be the most potent of the series with Ki A2B = 11 μM . The effect of the chlorine at the 2 position on binding affinity at the adenosine receptors it is not univocal , depending on the receptor subtype and/or on the substituent in N6 . Analysis of the binding profile of the N6-unsubstituted derivatives in more detail revealed that the presence of a chlorine atom at the 2 position -LRB- compound 6 -RRB- increased the affinity -LRB- A1 , A2A and A3 -RRB- and potency -LRB- A2B -RRB- at adenosine receptors two - to threefold compared with the unsubstituted analogue 5 . A quite similar profile could be observed when a 4-bromophenylacetic group was introduced at the N6 position -LRB- compare compound 8 with 7 -RRB- . An opposite effect of the chlorine atom was detected at A1 , A2B and A3 receptors when a bulkier substituent , such as the 4-aryloxyphenylacetic chain , was introduced at the N6 position . In fact , this kind of combination significantly reduced or did not modify the A1 and A3 affinity and the A2B potency -LRB- 9 : Ki A1 = 1.4 μM , Ki A2B > 30 μM , Ki A3 = 5.3 μM vs 10 : Ki A1 = 26 μM , Ki A2B > 30 μM , Ki A3 = 4.9 μM and 12 : Ki A1 = 13 μM , Ki A2B = 22 μM , Ki A3 = 10 μM vs 13 : Ki A1 = 22 μM , Ki A2B > 30 μM , Ki A3 = 19 μM -RRB- . However , in all cases , the presence of a chlorine atom in the 2 position favoured the interaction with the A2A subtype -LRB- compare A2A affinity of 5 , 7 , 9 and 12 with 6 , 8 , 10 and 13 , respectively -RRB- , while the presence of any substituent on the N6 position seems to somewhat reduce the affinity . In fact the compound endowed with the highest A2A affinity proved to be the 2-chloro-9-propyladenine -LRB- 6 : Ki A2A = 2.2 μM -RRB- . These findings are in agreement with previous observations related to adenosine analogues strongly suggesting that the introduction of substituents in the N6 position dramatically reduces the A2A affinity -LSB- 27 -- 32 -RSB- . Nevertheless , it should be underlined that the presence of a bulky chain at the N6 position significantly increased -LRB- 20 - to 70-fold -RRB- the affinity at the A1 and A3 subtypes in comparison with 9-propyladenine -LRB- 9 : Ki A1 = 1.4 μM and 11 : Ki A3 = 1.4 μM vs 5 : Ki A1 = 24 μM and Ki A3 > 100 μM -RRB- . This increase of affinity seems also to be modulated by the substituent on the aryloxyphenylacetic group ; in fact , substitution with a lipophilic bromine -LRB- 9 -RRB- or methyl group -LRB- 11 -RRB- at the para position is responsible for the increased A1 and A3 receptor affinity , respectively , while the presence of a hydrogen -LRB- 12 -RRB- or a methoxy group -LRB- 14 -RRB- did not positively influence the binding profile . Conclusions In conclusion the study herein presented , although it did not reach the proposed goal of obtaining A2B adenosine receptor antagonists , increased knowledge of the structure-activity relationships in adenine derivatives . Moreover , it was demonstrated that the introduction of bulky substituents at the N6 position of adenine derivatives significantly increased the affinity at the A1 and A3 adenosine receptors , while the presence of a chlorine atom in the 2 position favoured the interaction with the A2A subtype . These results demonstrated that , although the synthesized compounds were found to be quite inactive at the human A2B subtype , adenine is a useful template for further development of simplified adenosine receptor antagonists with distinct receptor selectivity profiles , opening up new chances to design structurally simplified A1 and A3 adenosine receptor antagonists . Experimental section Chemistry General : melting points were determined with a Büchi apparatus and are uncorrected . 1H NMR spectra were obtained with Varian VXR 300 MHz spectrometer ; δ in ppm , J in Hz . All exchangeable protons were confirmed by addition of D2O . Thin layer chromatography -LRB- TLC -RRB- was carried out on precoated TLC plates with silica gel 60 F-254 -LRB- Merck -RRB- . For column chromatography , silica gel 60 -LRB- Merck -RRB- was used . Elemental analyses were determined on Fisons Instruments Model EA 1108 CHNS-O model analyser and are within ± 0.4 % of theoretical values . 9-Propyladenine -LRB- 5 -RRB- and 7-propyladenine -LRB- 5a -RRB- To a solution of adenine -LRB- 15 -RRB- -LRB- 0.5 g , 3.7 mmol -RRB- in dry DMF -LRB- 10 ml -RRB- , under nitrogen , K2CO3 -LRB- 0.83 g , 5.97 mmol -RRB- and propyliodide -LRB- 0.433 ml , 4.44 mmol -RRB- were added . The mixture was stirred at RT for 16 h , then the solvent was removed under reduced pressure and the crude purified by flash chromatography -LRB- CHCl3-MeOH 98:2 -RRB- to afford 5 -LSB- 23 -RSB- and 5a -LRB- yield 79 and 9 % , respectively -RRB- as white solids , after crystallization from CH3OH . 5 : m.p. 173 -- 175 °C . 1H-NMR -LRB- DMSO-d6 -RRB- δ 0.85 -LRB- t , 3H , J = 7.4 , CH3 -RRB- ; 1.82 -LRB- m , 2H , CH2-CH3 -RRB- ; 4.11 -LRB- t , 2H , J = 7.0 , CH2-N -RRB- ; 7.21 -LRB- bs , 2H , NH2 -RRB- ; 8.15 -LRB- s , 2H , H-2 and H-8 -RRB- . Anal . Calcd . for C5H5N5 -LRB- 177.2 -RRB- C , 54.22 ; H , 6.26 ; N , 39.52 ; found : C , 54.54 ; H , 6.71 ; N , 39.33 . 5a : m.p. > 250 °C . 1H-NMR -LRB- DMSO-d6 -RRB- δ 0.86 -LRB- t , 3H , J = 7.3 , CH3 -RRB- ; 1.91 -LRB- m , 2H , CH2-CH3 -RRB- ; 4.26 -LRB- t , 2H , J = 7.0 , CH2-N -RRB- ; 7.75 -LRB- s , 1H , H-2 -RRB- ; 7.85 -LRB- bs , 2H , NH2 -RRB- ; 8.34 -LRB- s , 1H , H-8 -RRB- . Anal . Calcd . for C5H5N5 -LRB- 177.2 -RRB- C , 54.22 ; H , 6.26 ; N , 39.52 ; found : C , 54.45 ; H , 6.45 ; N , 39.45 . 2,6-Dichloro-9-propyl-9H-purine -LRB- 17 -RRB- and 2,6-dichloro-7-propyl-9H-purine -LRB- 17a -RRB- To a solution of 2,6-dichloropurine -LRB- 16 -RRB- -LRB- 1 g , 5.29 mmol -RRB- in dry DMF -LRB- 14 ml -RRB- , under nitrogen , K2CO3 -LRB- 1.18 g , 6.61 mmol -RRB- and propyliodide -LRB- 0.59 ml , 6.08 mmol -RRB- were added . The mixture was stirred at RT overnight , then the solvent was removed under reduced pressure and the crude purified by flash chromatography -LRB- cC6H12-EtOAc 75:25 -RRB- to afford 17 and 17a as white solids -LRB- yield 75 and 10 % , respectively -RRB- -LSB- 24 -RSB- . 17 : m.p. 58 -- 59 °C ; 1H-NMR -LRB- DMSO - d6 -RRB- δ 0.86 -LRB- t , 3H , J = 7.5 Hz , CH2CH3 -RRB- , 1.85 -LRB- m , 2H , CH2CH3 -RRB- , 4.21 -LRB- t , 2H , J = 7.0 Hz , N-CH2 -RRB- , 8.76 -LRB- s , 1H , H-8 -RRB- . Anal . Calcd . for C8H8Cl2N4 -LRB- 231.1 -RRB- C , 41.58 ; H , 3.49 ; N , 24.25 . Found : C , 41.85 ; H , 3.70 ; N , 24.10 . 17a : m.p. 103 -- 105 °C ; 1H-NMR -LRB- DMSO-d6 -RRB- δ 0.87 -LRB- t , 3H , J = 7.4 Hz , CH2CH3 -RRB- , 1.84 -LRB- m , 2H , CH2CH3 -RRB- , 4.40 -LRB- t , 2H , J = 7.2 Hz , N-CH2 -RRB- , 8.89 -LRB- s , 1H , H-8 -RRB- . Anal . Calcd . for C8H8Cl2N4 -LRB- 231.1 -RRB- C , 41.58 ; H , 3.49 ; N , 24.25 . Found : C , 41.75 ; H , 3.55 ; N , 24.19 . 2-Chloro-9-propyladenine -LRB- 6 -RRB- Liquid ammonia -LRB- 5 ml -RRB- and compound 17 -LRB- 0.46 g , 1.97 mmol -RRB- were poured into a sealed tube and the resulting mixture was stirred at RT overnight . Ammonia was evaporated and the crude purified by flash chromatography -LRB- CHCl3-MeOH 99:1 -RRB- to give 6 -LSB- 24 -RSB- as a white solid -LRB- yield 75 % -RRB- m.p. 224 -- 226 °C . 1H-NMR -LRB- DMSO-d6 -RRB- δ 0.84 -LRB- t , 3H , J = 7.3 Hz , CH2CH3 -RRB- , 1.79 -LRB- m , 2H , CH2CH3 -RRB- , 4.05 -LRB- t , 2H , J = 7.2 Hz , N-CH2 -RRB- , 7.72 -LRB- s , 2H , NH2 -RRB- , 8.15 -LRB- s , 1H , H-8 -RRB- . Anal . Calcd . for C8H10ClN5 -LRB- 211.7 -RRB- C , 45.40 ; H , 4.76 ; N , 33.09 . Found : C , 45.75 ; H , 4.80 ; N , 32.87 . General procedure for the preparation of the N6-acylaminoadenine -LRB- 7 -- 14 -RRB- A solution in dry THF -LRB- 4 ml -RRB- of the appropriate acid -LRB- 18 -- 22 -RRB- -LRB- 0.46 mmol -RRB- and carbonyldiimidazole -LRB- 83 mg , 0.51 mmol -RRB- was poured at reflux under nitrogen for 1 h . Then the amino compound 5 or 6 -LRB- 0.46 mmol -RRB- was added and the resulting mixture was refluxed overnight . The solvent was removed under reduced pressure and the crude purified by flash chromatography to afford the desired final compounds 7 -- 14 . 6 - -LSB- -LRB- 4-Bromophenyl -RRB- acetyl -RSB- amino-9-propyladenine -LRB- 7 -RRB- Eluent for chromatography CHCl3-MeOH 95:5 ; yield 59 % , white solid ; m.p. 149 -- 151 °C -LRB- dec. -RRB- ; 1H-NMR -LRB- DMSO-d6 -RRB- : δ 0.83 -LRB- t , 3H , J = 7.2 Hz , CH2CH3 -RRB- , 1.84 -LRB- m , 2H , CH2CH3 -RRB- , 3.89 -LRB- s , 2H , CH2-CO -RRB- , 4.19 -LRB- t , 2H , J = 7.1 Hz , N-CH2 -RRB- , 7.30 -LRB- d , 2H , J = 8.4 Hz , H-Ph -RRB- , 7.51 -LRB- d , 2H , J = 8.4 Hz , H-Ph -RRB- , 8.47 -LRB- s , 1H , H-8 -RRB- , 8.62 -LRB- s , 1H , H-2 -RRB- , 10.91 -LRB- s , 1H , NH -RRB- . Anal . Calcd . for C16H16BrN5O -LRB- 374.2 -RRB- C , 51.35 ; H , 4.31 ; N , 18.71 . Found : C , 51.65 ; H , 4.80 ; N , 18.50 . 6 - -LSB- -LRB- 4-Bromophenyl -RRB- acetyl -RSB- amino-2-chloro-9-propyladenine -LRB- 8 -RRB- Eluent for chromatography CHCl3-cC6H12 80:20 ; yield 26 % , white solid ; m.p. 164 -- 166 °C ; 1H-NMR -LRB- DMSO-d6 -RRB- : δ 0.84 -LRB- t , 3H , J = 7.5 Hz , CH2CH3 -RRB- , 1.83 -LRB- m , 2H , CH2CH3 -RRB- , 3.88 -LRB- s , 2H , CH2-CO -RRB- , 4.15 -LRB- t , 2H , J = 6.9 Hz , N-CH2 -RRB- , 7.30 -LRB- d , 2H , J = 8.4 Hz , H-Ph -RRB- , 7.53 -LRB- d , 2H , J = 8.4 Hz , H-Ph -RRB- , 8.50 -LRB- s , 1H , H-8 -RRB- , 11.25 -LRB- s , 1H , NH -RRB- . Anal . Calcd . for C16H15BrClN5O -LRB- 408.7 -RRB- C , 47.02 ; H , 3.70 ; N , 17.14 . Found : C , 47.49 ; H , 3.83 ; N , 17.40 . 6 - -LSB- -LRB- 4 - -LRB- 4-Bromobenzyloxy -RRB- phenyl -RRB- acetyl -RSB- amino-9-propyladenine -LRB- 9 -RRB- Eluent for chromatography CHCl3-MeOH 95:5 ; yield 58 % , white solid ; m.p. 154 -- 156 °C ; 1H-NMR -LRB- DMSO-d6 -RRB- : δ 0.85 -LRB- t , 3H , J = 7.5 Hz , CH2CH3 -RRB- , 1.85 -LRB- m , 2H , CH2CH3 -RRB- , 3.82 -LRB- s , 2H , CH2-CO -RRB- , 4.21 -LRB- t , 1H , J = 7.0 Hz , N-CH2 -RRB- , 5.07 -LRB- s , 2H , CH2-O -RRB- , 6.95 -LRB- d , 2H , J = 8.8 Hz , H-Ph -RRB- , 7.27 -LRB- d , 2H , J = 8.4 Hz , H-Ph -RRB- , 7.40 -LRB- d , 2H , J = 8.4 Hz , H-Ph -RRB- , 7.58 -LRB- d , 2H , J = 8.4 Hz , H-Ph -RRB- , 8.48 -LRB- s , 1H , H-8 -RRB- , 8.62 -LRB- s , 1H , H-2 -RRB- , 10.81 -LRB- s , 1H , NH -RRB- . Anal . Calcd . for C23H22BrN5O2 -LRB- 480.4 -RRB- C , 57.51 ; H , 4.62 ; N , 14.58 . Found : C , 57.99 ; H , 4.66 ; N , 14.55 . 6 - -LSB- -LRB- 4 - -LRB- 4-Bromobenzyloxy -RRB- phenyl -RRB- acetyl -RSB- amino-2-chloro-9-propyladenine -LRB- 10 -RRB- Eluent for chromatography CHCl3-cC6H12-MeOH 50:48:2 ; yield 14 % , white solid ; m.p. 176 -- 178 °C ; 1H-NMR -LRB- DMSO-d6 -RRB- : δ 0.81 -LRB- t , 3H , J = 7.5 Hz , CH2CH3 -RRB- , 1.79 -LRB- m , 2H , CH2CH3 -RRB- , 3.76 -LRB- s , 2H , CH2-CO -RRB- , 4.12 -LRB- t , 2H , J = 7.4 Hz , N-CH2 -RRB- , 5.03 -LRB- s , 2H , CH2-O -RRB- , 6.92 -LRB- d , 2H , J = 8.6 Hz , H-Ph -RRB- , 7.22 -LRB- d , 2H , J = 8.4 Hz , H-Ph -RRB- , 7.36 -LRB- d , 2H , J = 8.4 Hz , H-Ph -RRB- , 7.54 -LRB- d , 2H , J = 8.4 Hz , H-Ph -RRB- , 8.46 -LRB- s , 1H , H-8 -RRB- , 11.11 -LRB- s , 1H , NH -RRB- . Anal . Calcd . for C23H21BrClN5O2 -LRB- 514.8 -RRB- C , 53.66 ; H , 4.11 ; N , 13.60 . Found : C , 53.75 ; H , 4.25 ; N , 13.29 . 6 - -LSB- -LRB- 4 - -LRB- 4-Methylbenzyloxy -RRB- phenyl -RRB- acetyl -RSB- amino-9-propyladenine -LRB- 11 -RRB- Eluent for chromatography CHCl3-cC6H12-MeOH 70:28:2 ; yield 35 % , white solid ; m.p. 131 -- 132 °C ; 1H-NMR -LRB- DMSO-d6 -RRB- : δ 0.83 -LRB- t , 3H , J = 7.3 Hz , CH2CH3 -RRB- , 1.84 -LRB- m , 2H , CH2CH3 -RRB- , 2.28 -LRB- s , 3H , CH3-Ph -RRB- , 3.80 -LRB- s , 2H , CH2-CO -RRB- , 4.20 -LRB- t , 2H , J = 7.1 Hz , N-CH2 -RRB- , 5.02 -LRB- s , 2H , CH2-O -RRB- , 6.93 -LRB- d , 2H , J = 8.4 Hz , H-Ph -RRB- , 7.24 -LRB- m , 6H , H-Ph -RRB- , 8.47 -LRB- s , 1H , H-8 -RRB- , 8.62 -LRB- s , 1H , H-2 -RRB- , 10.83 -LRB- s , 1H , NH -RRB- . Anal . Calcd . for C24H25N5O2 -LRB- 415.5 -RRB- C , 69.38 ; H , 6.06 ; N , 16.86 . Found : C , 69.74 ; H , 6.35 ; N , 16.54 . 6 - -LSB- -LRB- 4-Benzyloxyphenyl -RRB- acetyl -RSB- amino-9-propyladenine -LRB- 12 -RRB- Eluent for chromatography CHCl3-MeOH 97:3 ; yield 52 % , white solid ; m.p. 121 -- 123 °C ; 1H-NMR -LRB- DMSO-d6 -RRB- : δ 0.83 -LRB- t , 3H , J = 7.5 Hz , CH2CH3 -RRB- , 1.84 -LRB- m , 2H , CH2CH3 -RRB- , 3.80 -LRB- s , 2H , CH2-CO -RRB- , 4.19 -LRB- t , 2H , J = 7.1 Hz , N-CH2 -RRB- , 5.07 -LRB- s , 2H , CH2-O -RRB- , 6.95 -LRB- d , 2H , J = 8.4 Hz , H-Ph -RRB- , 7.25 -LRB- d , 2H , J = 8.8 Hz , H-Ph -RRB- , 7.37 -LRB- m , 5H , H-Ph -RRB- , 8.47 -LRB- s , 1H , H-8 -RRB- , 8.62 -LRB- s , 1H , H-2 -RRB- , 10.82 -LRB- s , 1H , NH -RRB- . Anal . Calcd . for C23H23N5O2 -LRB- 401.5 -RRB- C , 68.81 ; H , 5.77 ; N , 17.44 . Found : C , 68.97 ; H , 5.89 ; N , 17.35 . 6 - -LSB- -LRB- 4-Benzyloxyphenyl -RRB- acetyl -RSB- amino-2-chloro-9-propyladenine -LRB- 13 -RRB- Eluent for chromatography CHCl3-MeOH 99:1 ; yield 17 % , white solid ; m.p. 150 -- 152 °C ; 1H-NMR -LRB- DMSO-d6 -RRB- : δ 0.84 -LRB- t , 3H , J = 7.3 Hz , CH2CH3 -RRB- , 1.82 -LRB- m , 2H , CH2CH3 -RRB- , 3.79 -LRB- s , 2H , CH2-CO -RRB- , 4.14 -LRB- t , 2H , J = 7.0 Hz , N-CH2 -RRB- , 5.07 -LRB- s , 2H , CH2-O -RRB- , 6.95 -LRB- d , 2H , J = 8.6 Hz , H-Ph -RRB- , 7.25 -LRB- d , 2H , J = 8.4 Hz , H-Ph -RRB- , 7.37 -LRB- m , 5H , H-Ph -RRB- , 8.49 -LRB- s , 1H , H-8 -RRB- , 11.16 -LRB- s , 1H , NH -RRB- . Anal . Calcd . for C23H22ClN5O2 -LRB- 435.9 -RRB- C , 63.37 ; H , 5.09 ; N , 16.07 . Found : C , 63.56 ; H , 5.14 ; N , 15.76 . 6 - -LCB- -LSB- -LRB- 4-Methoxybenzyloxy -RRB- phenyl -RSB- acetyl -RCB- amino-2-chloro-9-propyladenine -LRB- 14 -RRB- Eluent for chromatography CHCl3-MeOH 99:1 ; yield 29 % , white solid ; m.p. 174 -- 176 °C ; 1H-NMR -LRB- DMSO-d6 -RRB- : δ 0.84 -LRB- t , 3H , J = 7.4 Hz , CH2CH3 -RRB- , 1.82 -LRB- m , 2H , CH2CH3 -RRB- , 3.73 -LRB- s , 3H , CH3-O -RRB- , 3.79 -LRB- s , 3H , CH2-CO -RRB- , 4.14 -LRB- t , 2H , J = 7.0 Hz , N-CH2 -RRB- , 4.98 -LRB- s , 2H , CH2-O -RRB- , 6.92 -LRB- d , 2H , J = 8.0 Hz , H-Ph -RRB- , 6.93 -LRB- d , 2H , J = 8.4 Hz , H-Ph -RRB- , 7.24 -LRB- d , 2H , J = 8.4 Hz , H-Ph -RRB- , 7.35 -LRB- d , 2H , J = 8.4 Hz , H-Ph -RRB- , 8.49 -LRB- s , 1H , H-8 -RRB- , 11.15 -LRB- s , 1H , NH -RRB- . Anal . Calcd . for C24H24ClN5O3 -LRB- 465.9 -RRB- C , 61.87 ; H , 5.19 ; N , 15.03 . Found : C , 61.99 ; H , 5.33 ; N , 14.91 . Biology All pharmacological methods followed the procedures as described earlier -LSB- 26 -RSB- . In brief , membranes for radioligand binding were prepared from CHO cells stably transfected with human adenosine receptor subtypes in a two-step procedure . In a first low-speed step -LRB- 1,000 g -RRB- cell fragments and nuclei were removed . The crude membrane fraction was sedimented from the supernatant at 100,000 g . The membrane pellet was resuspended in the buffer used for the respective binding experiments , frozen in liquid nitrogen and stored at − 80 °C . For the measurement of adenylyl cyclase activity only one high speed centrifugation of the homogenate was used . The resulting crude membrane pellet was resuspended in 50 mM Tris/HCl , pH 7.4 and immediately used for the cyclase assay . For radioligand binding at A1 adenosine receptors 1 nM -LSB- 3H -RSB- CCPA was used , whereas 30 and 10 nM -LSB- 3H -RSB- NECA were used for A2A and A3 receptors , respectively . Non-specific binding of -LSB- 3H -RSB- CCPA was determined in the presence of 1 mM theophylline ; in the case of -LSB- 3H -RSB- NECA 100 pM R-PIA was used . Ki values from competition experiments were calculated with the program SCTFIT -LSB- 33 -RSB- . At A2B adenosine receptors inhibition of NECA-stimulated adenylyl cyclase activity was used as a measurement of potency of the new compounds . IC50 values from these experiments were converted to Ki values with the Cheng and Prusoff equation -LSB- 34 -RSB- .

O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

adenine derivatives
adenosine receptors
adenosine receptor antagonists

adenosine receptor ligands
g protein-coupled receptors