Journal ofMedicina1 Chemistry 0 Copyright 1985 by the American Chemical Society
Volume 28, Number 10
October 1985
Communications to the Editor “Wycloalkyladenosines. Potent, A,-Selective Adenosine Agonists
Sir: Adenosine and adenosine agonists have numerous physiological effects in the nervous system, including inhibition of neurotransmitter release, anticonvulsant activity, analgesia, respiratory depression, hypothermia, and profound decreases in locomotor activity.12 The receptors that mediate these responses have been studied indirectly through the responses that they elicit8J4and directly by receptor binding methods.’J5 There are two main classes of extracellular adenosine receptors: A, receptors whose activation leads to inhibition of adenylate cyclase and A2 receptors whose activation leads to stimulation of adenylate ~yc1ase.l~ These receptors are designated Ri and R,, respectively, in an alternative nomenclature.8 Although N6-substituted adenosines, especially P-cyclohexyladenosine (CHA), are known to be selective Al agonists1J3J5and to possess activity as inhibitors of platelet aggregation7 and neurotransmis~ion,~ structure-activity relationships for lower and higher homologues of CHA have not been explored to date. The present study reports the discovery of Ns-cyclopentyladenosine (CPA) as a potent, A,-selective adenosine agonist, a finding that has allowed the development of an A2receptor binding assay. Additionally, this study reports the binding affinities for a series of Ns-cycloalkyladenosines a t both A, and A2 adenosine receptors. Chemistry. All adenosine analogues were synthesized a t Warner-Lambert/Parke-Davisaccording to standard chemical p r o c e d u r e ~ ~ 9except ~ J ~ 2-chloroadenosine, which was obtained from the Sigma Chemical Co. Physical properties (‘Hnuclear magnetic resonance, infrared, and (1) Bruns, R. F.; Daly, J. W.; Snyder, S. H. Proc. Natl. Acad. Sci. U.S.A.1980, 77,5547-5551. (2) Bruns, R. F.; Lu, G. H.; Pugsley, T. A., manuscript in preparation. Neurosci. Abstr. (3) Bruns, R. F.; Lu, G. H.; Pugsley, T. A. SOC. 1984,IO,957. (4) Cheng, Y.-C.; Prusoff, W. H. Biochem. Pharmacol. 1973,22, 3099-3108. (5) Cook, A. F.; Bartlett, R. T.; Gregson, R. P.; Quinn, R. J. J. Org. Chem. 1980,45, 4020-4025. (6) Haky, J. E.; Young, A. M. J. Liq. Chromatogr. 1984, 7, 675-689. (7) Kikugawa, K.; Iizuka, K.; Ichino, M. J . Med. Chem. 1973,16, 358-364. ( 8 ) Londos, C.; Cooper, D. M. F.; Wolff, J. Proc. Natl. Acad. Sci. U.S.A. 1980,77, 2551-2554. (9) Okada, Y.; Kuroda, Y. Eur. J. Pharmacol. 1980,61,137-146. (10) Prasad, R. N.; Bariana, D. S.; Fung, A.; Savic, M.; Tietje, K.; Stein, H. H.; Brondyk, H.; Egan, R. S. J. Med. Chem. 1980,23, 313-319.
0022-2623/85/1828-1383$01.50/0
, C H i NH
HO &I
N6 -Cycloal kyladenosines
HO OH
R-PIA
NH,
NH,
HO OH
NECA
4 -Methylisoguanosine
mass (low- and/or high-resolution) spectra, elemental analyses, melting points) were consistent with the chemical structures. Lipophilicity (log k 9 was determined by using a high-performance liquid chromatography (HPLC) correlation method.6 Statistical analyses were performed by using the Statistical Analysis System (SAS).’l Receptor Binding. 13H]CHAbinding to A, receptors was performed essentially as previously described’ except that whole rat brain (minus brainstem and cerebellum) was used instead of guinea pig brain. A2 receptor binding was performed in exactly the same way as A, receptor binding with the following exceptions: 4 nM [3H]-l-(6-amin~-9H-purin-9-yl)-l-deoxy-N-ethyl-~D-ribofuronamide ( [3H]NECA)was used as radioligand, the tissue was 5 mg of tissue wet weight of rat striatal membranes, the incubation volume was 1 mL, 10 mM MgCl, was added to the buffer, and all incubations contained 50 nM CPA to eliminate A, receptor binding. Nonspecific binding was defined as binding in the presence of 100 pM CPA. This method is a variation of the [3H]NECA binding assay of Yeung and Green;15 a detailed characterization of the method will be reported elsehere.^,^
ICbovalues in A, and A2 binding were calculated from eight-point curves, including total binding, nonspecific (11) SAS Institute, Inc., Cary, NC. (12) Snyder, S. H. Ann. Rev. Neurosci. 1985,8, 103-124. (13) Trost, T.; Stock, K. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1977,299,33-40. (14) van Calker, D.; Muller, M.; Hamprecht, B. J. Neurochem. 1979,33,999-1005. (15) Yeung, S.-M. H.; Green, R. D. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1984,325,218-225.
0 1985 American Chemical Society
1384 J o u r n a l of Medicinal Chemistry, 1985, Vol. 28, No. 10
Communications to the Editor
Table I. Affinities of N6-Cycloalkyladenosines and Reference Agents in AI and A, Adenosine Receptor Binding Assays A, Ki2 nM, [ 3H]NECA iWCycloalkyladenosines 319 f 0.49 1240 f 130 0.777 f 0.028 263 f 5 0.590f 0.023 462 f 15 1.31 f 0.009 363 f 46 2.79 f 0.36 1690 f 190 4.03 f 0.16 2450 f 500 100 f 11 5190 f 50 5390 f 351 47700 f 8500 A, Ki; nM, [3H]CHA
NB-cyclopropylado' NB-cyclobutylado NG-cyclopentylado (CPA) NG-cyclohexylado (CHA) NG-cycloheptylado NG-cyclooctylado NG-cyclodecylado NG-cyclododecylado
Al selec ratiob
389 338 783 277 606 608 51.9 8.85
1%
k'
mp,f "C
0.53 1.22 1.56 1.60 2.40 2.90 3.62 4.52
184-187h 13@ g g g
L?
138-14oh 144-145'
Reference Adenosine Agonists 6.24 f 0.45 10.6 f 0.5 1.70 NECAd 8.74 f 0.11 63.1 f 7.5 7.22 2-chloroado 3250 f 92 22.6 l-methylisoguanosine 144 f 3 S-PIA' 47.6 f 2.3 1810 f 380 38.0 1.15 f 0.20 124 f 9 108 R-PIA' nValues are means f standard errors for three or four separate experiments for each compound. * A l selectivity ratio is the A, Ki divided " B - [ ( Ror S)-l-Methylby the A, Ki. Adenosine is abbreviated ado. 1-(6-Amino-9H-purin-9-yl)-l-deoxy-N-ethyl-~-~-ribofuronamide. 2-phenylethylladenosine. fMelting points are listed for new compounds only and are uncorrected. gSee ref 7. hC, H, N: f0.4%. 'C, H: f0.4%; N: +O.6%. Exact mass: f5 ppm.
binding, and six drug concentrations that bracketed the IC50. Ki values for compounds in A, receptor binding were calculated from the Cheng-Prusoff equation4 using 1.31 nM as the Kd for [3H]CHA. The Kd for [3H]CHA was calculated from the ICm for unlabeled CHA of 2.31 nM. K, values in A, receptor binding were calculated on the basis of Kd values of 10.6 nM for [3H]NECAand 462 nM for CPA, which in turn were calculated from IC5,, values of 15.8 nM for NECA and 685 nM for CPA. SAR. Affinity of the P-cycloalkyladenosines in A, receptor binding varies as a smooth function of ring size, reaching a maximum with P-cyclopentyladenosine (Table I). CPA is approximately twice as potent as CHA a t the A, receptor, and with 0.59 nM affinity, CPA is the most potent adenosine agonist reported to date. For ring sizes n = 3-8, the adenosine analogues are all quite potent; only the two largest analogues ( n = 10,12) are substantially less potent than CHA. Preliminary attempts to correlate receptor binding in this series with physicochemical properties suggest a correlation with lipophilicity (log k ?, as indicated by the following equations, where n is the number of compounds included in the analysis, s is the root mean square error, r2 is the square of the correlation coefficient, F relates the variance of the null hypothesis to the correlation variance, p is the probability that a random set of data would yield a higher F value, and terms are given f their standard errors. log (A, K,) = [-1.33 (*0.23)] log 12' + [0.43 (*0.04)](log k q 2 + [0.99 (*0.25)] n = 8, s = 0.18, r2 = 0.99, F = 185.21, p < 0.0001
log (A, Ki) = [-0.50 (*0.31)] log lz' + [0.19 (fO.O6)](log k3* + [3.05 (fO.34)l n = 8, s = 0.25, r2 = 0.92, F = 28.87, p < 0.0018
Whether the correlations reflect whole molecule lipophilicity, side chain lipophilicity, size effects, or a combination of factors is currently under investigation. Finally, it is interesting to note that A, and A2 binding affinities are correlated in this series, as defined by the following equation: log (A, K,) = [1.74 (f0.21)] log (A2 K,) - [4.72 (*0.70)] n = 8, s = 0.42, rz = 0.92, F = 66.23, p < 0.0002
Because of its high a f f d t y for A, receptors, CPA proved useful in developing the A2 receptor binding assay used in the present study. A major problem in the use of [3H]NECAas an A, receptor ligand is its high affinity for Al receptors, so that even in favorable tissues such as rat striatum A, receptors account for only about half of specific binding.15 Reference agents including CHA give shallow dose-inhibition curves with incomplete separation between Al and A2 phases of receptor occupancy. In contrast, CPA shows a biphasic dose-inhibition curve with a clear plateau between the Al and A, phases., For this reason, 50 nM CPA is used routinely in our A2 receptor binding assay to eliminate the A, component of [3H]NECAbinding. The relative affmities of NECA and R-PIA in A, and A2 binding (Table I) are in good agreement with their affinities in A,-inhibited and A,-stimulated adenylate cyclase,8 respectively. CPA is the most Al selective of the P-cycloalkyladenosines (780-fold, Table I), but the cycloheptyl and cylcooctyl homologues are almost equally selective. All of the P-cycloalkyladenosines except the cyclodecyl and cyclododecyl homologues are more A, selective than the most selective reference agent, P[ (R)-l-methyl-2phenylethylladenosine (R-PIA). Studies exploring the biological properties of this homologous series and the use of these potent adenosine agonists as pharmacological tools are in progress.
Acknowledgment. We thank J. A. Bristol and T. A. Pugsley for helpful discussions, J. Fergus, K. Lawson, and G. Lu for Ki determinations, and J. E. Haky and A. M. Young for log k' determinations. R e g i s t r y No. CHA,36396-99-3; CPA,41552-82-3;N6-cycloNs-cyclobutylado, 97374-49-7; NG-cyclopropylado, 97374-48-6; N6-cyclOheptylado, 41552-83-4;N6-cycloocytyiado, 41552-84-5; N6-cyclododecy~ado,97374-51-1. decylado, 97374-50-0;
(16) Department of Chemistry. (17) Department of Pharmacology. W a l t e r H. Moos,*l6 Deedee S. Szotek16 R o b e r t F. Bruns" Departments of Chemistry a n d Pharmacology Warner-LambertfParke- Davis Pharmaceutical Research Ann Arbor, Michigan 48105 Received February 28, 1985
