J. Med. Chem. 1 9 8 8 , 3 1 , 745-751
745
lz51-Labeled%PhenylxanthineDerivatives: Antagonist Radioligands for Adenosine Al Receptorst Joel Linden,* Amrat Patel, Craig Q. Earl, Rebecca H. Craig,$ and Susan M. Dalugef Departments of Physiology and Internal Medicine (Cardiology), University of Virginia School of Medicine, Charlottesuille, Virginia 22908, and The Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709. Received July 22, 1987 A series of 8-phenylxanthine derivatives has been synthesized with oxyacetic acid on the para phenyl position to increase aqueous solubility and minimize nonspecific binding and iodinatable groups on the 1-or 3-position of the xanthine ring. The structure-activity relationship for binding of these compounds to A, adenosine receptors of bovine and rat brain and A, receptors of human platelets was examined. The addition of arylamine or photosensitive aryl azide groups to the 3-position of xanthine had little effect on Al binding affinity with or without iodination, whereas substitutions at the 1-position caused greatly reduced A, binding affinity. The addition of an aminobenzyl group to the 3-position of the xanthine had little effect on A2 binding affinity, but 3-aminophenethyl substitution decreased A, binding affinity. Two acidic 3-(arylamino)-B-phenylxanthinederivatives were labeled with lZ5I and evaluated as A, receptor radioligands. The new radioligands bound to A, receptors with KDvalues of 1-1.25 nM. Specific binding represented over 80% of total binding. High concentrations of NaCl or other salts increased the binding affinity of acidic but not neutral antagonists, suggesting that interactions between ionized xanthines and receptors may be affected significantly by changes in ionic strength. On the basis of binding studies with these multiple agonist affinity antagonists and isotope dilution with the agonist [1251]N6-(4-amino-3-iodobenzy1)adenosine, states of AI receptors have been identified.
Adenosine regulates cellular function in a number of peripheral tissues and brain, at least partially through recept,ors on the extracellular surface of cell membranes.l Two classes of adenosine receptors have been extensively characterized, Al and A2 receptors, which inhibit and stimulate adenylate cyclase activity, respectively.2 The A, receptor is thought to be involved in the mediation of central depressant effects of adenosine agonist^.^ Alkylxanthines such as theophylline and caffeine are adenosine receptor antagonists that bind to A, receptors of rat brain with KD values of 5.9 and 24 pM,re~pectively.~ Increased antagonist potency for the Al subclass of receptors is achieved by adding an &phenyl group to the xanthine ring.5 Such compounds are very poorly soluble in water, but we found that solubility can be increased without greatly reducing binding affinity by adding acidic side chains to the para position of the &phenyl group.6 For the purpose of synthesizing radioligands, we reasoned that acidic compounds might be preferable to nonpolar compounds since polar compounds are likely to exhibit minimal nonspecific binding. Although a number of tritiated7 and iodinated4v8agonist radioligands for adenosine A, receptors are available, the addition of GTP converts receptors in brain membranesg or receptors solubilized in cholatelO or digitoninll into a state of low affinity for agonists. N-Ethylmaleimide (NEM) has a similar effect, apparently by interacting with Gi.12 In contrast, the binding of antagonist radioligands is not diminished by uncoupling of receptors from G p r o t e i n ~ ; ~ hence, J ~ J ~ they may be preferable to agonists for following the various conformational states of adenosine Al receptors, particularly during receptor purification. In this paper we describe the synthesis of new acidic high-affinity lZ5I-labeled antagonist radioligands and photoaffinity labels. These compounds and the agonist [1251]P-(4-amino-3-iodobenzyl)adenosine have been used to characterize multiple conformational states of adenosine A, receptors. Chemistry In order to evaluate the effeds of arylamines, aryl azides, and iodination on the affinity of acidic a-phenylxanthine +Supported by NIH Grant HL31432 and the Burroughs Welcome Co. J.L. is the recipient of an Established Investigator Award from the American Heart Association. * T h e Wellcome Research Laboratories. 0022-262318811831-0745$01.50/0
derivatives for adenosine AI and Az receptors, 19 derivatives were synthesized (Figure 1). Novel ester precursors Ia-e (Figure 1) were prepared from the corresponding N-(4-nitroaralkyl)-5,6-diaminouracils by oxidative xanthine ring formation with the appropriate aldehyde in nitrobenzene, by use of methods derived from ref 23-25 and catalytic reduction of the nitro group to the amine. Iodinations of amino xanthines IIa-d were carried out with molecular iodine in aqueous sodium bicarbonate. The resulting iodoamines, IIIa-d, cochromatographed with the corresponding lZ5I-labeledxanthines prepared with chloramine T and [1251]sodiumiodide. This confirmation of the structure of [1251]-IIIa-dwas required since the chloramine T iodinations of these xanthines resulted in other iodinated products (see the Experimental Section). Iodo azides IVa-d were also characterized and compared by HPLC to the corresponding 1251-labeledxanthines to confirm the structures of the photoaffinity ligands. 6-(Aminobenzy1)adenosine (VIa) was synthesized in improved yield over previous methodss by preparing the triacetyl ribonucleoside Vb, which has much greater solubility than the unblocked ribonucleoside Va, allowing for (1) Londos, C.; Wolff, J. Proc. Natl. Acad. Sci. U.S.A. 1977, 74,
5482. (2) Londos, C.; Cooper, D. M. F.; Wolff, J. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 2551 and 2554.
(3) Phyllis, J. W.; Edstrom, J. P.; Kostopoulos, G. K.; Kirkpatrick, J. R. Can. J . Physiol. Pharrnacol. 1979, 57, 1289. (4) Linden, J. Mol. Pharrnacol. 1984, 26, 414. (5) Bruns, R. F.; Daly, J. W.; Snyder, S. H. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 2077. (6) The aqueous solubility of 8-phenylxanthines is imreased by substituting oxyacetate or other acidic groups onto the phenyl in the para position with only a moderate decrease in the binding affinity of the resultant compounds for adenosine AI receptors (Daluge, S. M.; Leighton, H. J. Eur. Pat. 203721, Dec 6, 1986). (7) Daly, J. W. J. Med. Chern. 1982, 25, 197. (8) Linden, J.; Patel, A.; Sadek, S. Circ. Res. 1985, 56, 279. (9) Lohse, M. J.; Lenschow, V.; Schwabe, U. Mol. Pharrnacol. '
1984, 26, 1. (lo) Gavish, M.; Goodman, R. R.; Synder, S. H. Science (Washington D.C.) 1982, 215, 1633. (11) Stiles, G. L. J. Riol. Chern. 1984, 260, 6728. (12) Asano, T.; Ogasawara, N. Mol. Pharrnacol. 1986, 29, 244. (13) Yeung, S.-M. H.; Green, R. D. J. Biol. Chem. 1983,258, 2334. (14) Linden, J.; Earl, C. 4.; Patel, A.; Craig, R. H.; Daluge, S. M.
In Topics and Perspectives in Adenosine Research; Gerlach, E., Becker, B. F., Eds.; Springer-Verlag: Berlin, 1987; p 3. 0 1988 American Chemical Society
746 Journal of Medicinal Chemistry, 1988, Vol. 31, No. 4
n = ILX =r;ll1.Y = I 1 . H = L 1 Ib: n i. 2. X = Fill2, Y = H. R = E l 110: n = I , X = hH2, Y = H . R = H Ilb: 11 =?. X = SH?.Y = tl, R = H IIIP: n = 1.1=VIl!, Y = I. R = I 1 Illb: n = 2, X = NH2, Y = I. R = H IVs: n = I,X = N,, Y = I. R = H I V b n = 2 . X = N l , Y = I.R = H 14:
I,
Linden et al.
IC:
n=I.X=NH~,Y=H.R=Et
Id
n = 2. X = NHz. Y = 11, R = El il NHz, Y = H. R = H n = 2. X = "2. Y = H. R = H n=l,X=NH2.Y=l,R=H n = 2. X = NH2. Y = I, R I H n = I, X = N,, Y = I, R H n = 2. X = L3.Y I I, R = H
IIC: n = I,X
Ild: Ilk:
Illd: IVC: IVd:
I1M) of salts (NaC1, NH,Cl, or KCl), which increased the binding affinity of these antagonists by over 10-fold. When assayed in the presence of 1 M NaCl, the KD values of two (3-iodoary1)amines ([1261]-IIIaand [1z51]-IIIb)were 1and 1.5 nM, respectively, and binding to rat brain Al receptors was 80% receptor specific. In contrast to its effect on acidic antagonist binding, 1M NaCl had little effect on the binding affinity of neutral 8-phenylxanthine derivatives. The most likely explanation for the selective effect of salts on the binding affinity of negatively charged 8-phenylxanthines is that charge repulsion between them and the negative charges in the phospholipid environment of receptors is diminished by raising the ionic strength of the solvent.16 This may account for the fact that xanthines that bind to adenosine receptors with the highest affinity in the absence of high salt are either neutral or positively charged. High-affinity neutral compounds include 8-(2-amino-4chlorophenyl)-l,3-dipropylxanthine,which binds to rat brain membranes with a KD of 2.5 nM,17 and S-cyclopentyl-l,3-dipropylxanthine, which binds with a KD of 0.42 nM.18 Positively charged compounds include xanthine (15) Earl, C. Q.; Patel, A.; Craig, R. H.; Daluge, S. M.; Linden, J. J. Med. Chem., adjoining paper in this issue. (16) Burke, T. G.; Tritton, T. R. Biochemistry 1995, 24, 5972. (17) Daly, J. W.; Padgett, W.; Shamin, M. T.; Butts-Lamb, P.; Waters, J. J.Med. Chem. 1985, 28, 487.
amino conjugates, which bind with KDas low as 1.2 nM.l9pZ0 The effect of NaCl to enhance specific acidic antagonist binding, which apparently results from masking of charge repulsion between the ligands and sites on the membranes, should not be confused with an effect of NaCl on agonist binding, which occurs at much lower concentrations, apparently by modifying the conformational state of the receptor. We examined the effects of NaC1, MgClZ,Gpp(NH)p, and NEM on agonist binding to adenosine A, receptors in order to better understand the various conformational states of the receptor. There has been disagreement about whether there are two or three agonist binding conformations of adenosine r e c e p t ~ r s ? J ~ In $~~ membranes pretreated with 10 mM EDTA to chelate tightly bound divalent cations, multiple adenosine Al receptor conformations can be demonstrated. A high-affinity (KD for [1251]-VII= 0.39 nM) binding state is demonstrable in the presence of MgC1, (without other salts). Guanine nucleotides or NEM convert the receptor into a low affinity state(s) presumably due to their effects on G proteins. At physiological concentrations, MgClz and NaCl have reciprocal effects on agonist binding. Maguire et alaz2have suggested that intracellular [Mg2+]may act as a physiological regulator of adenylate cyclase activity. Our data are consistent with the possibility that Mgz+concentration in a physiologically important pool could regulate the affmity of Al receptors for adenosine. NaCl and other salts a t concentrations between 100 and 500 mM decrease high affinity agonist binding by converting the adenosine AI receptor from a high affinity to a lower affinity conformation (KD for VI1 = 25 nM). NaCl does not act entirely by uncoupling receptors from G proteins since Gpp(NH)p and NEM could reduce binding affinity further when added to 1 M NaCl (KDfor VI1 = 1.27 pM). On the basis of competition studies with antagonist radioligands, it is apparent that almost all adenosine Al receptors are converted to the very low affinity conformation by a combination of NaCl and Gpp(NH)p or NEM (Figure 7). The low affinity agonist binding site may represent receptors disscciated from guanine nucleotide binding proteins and influenced by NaC1. Such uncoupled receptors can be detected by antagonist but not by agonist radioligands. Greenz1concluded that there are three affinity states of adenosine receptors on the basis of studies on the binding of [3H]fl-(phenylisopropyl)adenosine( [3H]PIA) to rat hippocampal membranes. He found that at 37 O C NaCl decreases the affinity of binding sites for [3H]PIA,but Scatchard plots remain linear, whereas we found that at 21 OC addition of NaCl results in curvilinear Scatchard plots, which can be fit to a two-site binding model. The explanation for the difference between our results and those of Greenz1may be that at 37 "C binding reflects an equilibrium between high- and low-affinity receptor states Bruns, R. F.; Fergus, J. H.; Badger, E. W.; Bristol, J. A.; Santay, L. A.; Kartman, J. D.; Hays, s. J.; Huang, C. C. NaunynSchmiedeberg's Arch. Pharmacol. 1987, 335, 59. Jacobson, K. A,; Ukena, D.; Kirk, K. L.; Daly, J. W. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 4089. Bruns, R. F.; Fergus, J. H.; Barger, E. W.; Bristol, J. A.; Santay, L. A.; Hays, S. J. Nauyn-Schemideberg's Arch. Pharmacol. 1987, 335, 64. Green, R. D. J . Neurosci. 1984,4,2474. Grubbs, R. D.; Collins, S. D.; Maguire, M. E. J. Biol. Chem. 1984,259, 12184. Papesch, V.; Schroeder, E. F. J. Org. Chem. 1951, 16, 1879. Papesch, V.; Grove, M.; Schroeder, E. F. U.S. Pat. 2602795, 1952. Jerchel, D.; Kracht, M.; Krucker, K. Justus Liebigs Ann. Chem. 1954,590, 232.
750 Journal of Medicinal Chemistry, 1988, Vol. 31, No. 4
whereas at 21 "C receptors may not readily interconvert between these two states. Lohse and co-workers9found one or two affinity states for [3H]PIA binding to rat brain synaptosomal membranes assayed in the absence or presence, respectively, of GTP. Their assays were conducted in the absence of added monovalent cations, hence they did not detect the lower affinity binding states, which only occur in the presence of high salt or combinations of salt and guanine nucleotides or salt and N-ethylmaleimide (Figure 7). The availability of high affinity lZ51-labeled antagonist radioligands should simplify the process of studying conformations of the adenosine A, receptor, which bind agonists with low affinity. Experimental Section
Linden et al.
and dried in vacuo to give IVb as a beige solid (0.052 g, 45%), which was shielded from light, mp 198-200 OC dec. Anal. ( C Z ~ H ~ Z I NC,~ H, O ~N, ) I. The following were prepared by procedures analogous to that used in the synthesis of IVb: IVa, in 15% yield, mp slow dec starting a t 185 "C. Anal. (Cz,HzoIN705)C, H, N. IVc, in 28% yield, mp slow dec starting a t 170 "C. Anal. (C23H,oIN,05)C, H, N, I. IVd, in 56% yield, mp >250 "C. Anal. Calcd for CwHzzIN705: C, 46.84; H, 3.60; N, 15.93; I, 20.62. Found: C, 47.80, H, 3.78; N, 15.13; I, 19.49. MS of IVd indicated possible contamination by diiodinated derivative Cz4Hz1IZN7O6. 6-[(4-Aminobenzyl)amino]-9-~-~-ribofuranosyl-9~-purine (VIa). T o an ice-cold solution of 6-[(p-nitrobenzyl)amino]-9-PD-ribofuranosylpurine (VaF6 11.65 g, 30.0 mmol) in anhydrous pyridine (50 mL) under nitrogen atmosphere was added acetic anhydride (16.0 mL, 170 mmol). The pale yellow solution was stirred a t 0 OC for 20 min and then at ambient temperature for 'H NMR spectra for all compounds, recorded with a Varian 3 h. After addition of 5 mL of methanol, the mixture was x1,-200 or XI,-300 instrument in DMSO-d, solution, were conevaporated to dryness, evaporated twice with toluene (100 mL) sistent with the proposed structures. Chemical ionization (CH,) plus methanol (10 mL) to remove pyridine, dissolved in CHzClz mass spectra, provided by Oneida Research Services with a (250 mL), washed twice with dilute aqueous sodium bicarbonate, Finnegan triple quadrupole, Model 4500 TSQ, gave parent moHzO, and saturated NaC1, dried over MgS04, and evaporated to lecular ions for all compounds. Elemental analyses were provided dryness. The residue was flash chromatographed (CHZClz/ by Atlantic Microlabs, Inc., and in all cases agreed with predicted MeOH). Fractions containing product were combined, evaporated values within f0.4% except where noted. Melting points are to dryness, and evaporated again with toluene (2 x 200 mL) and uncorrected and were determined with a Meltemp apparatus. then absolute EtOH (2 X 200 mL) to give the triacetyl riboXanthine products were homogeneous by thin-layer chromatognucleoside Vb as a solid foam (13.35 g, 84%). A solution of this raphy (silica gel, Whatman type MK6F) when developed with material in methanol (500 mL) was divided equally between two 7:3:0.1 CHCl3/MeOH/H20. Adenosine derivatives were homo500-mL Parr bottles, and each was hydrogenated a t 540 psi over geneous by thin-layer chromatography with 7:3 CHC13/MeOH. 2-[ 4-[3-(4-Aminophenethyl)-l,2,3,6-tetrahydro-2,6-dioxo- palladium on carbon (5%, 0.80 g) for 4 h. Catalyst was removed by filtration. The nearly colorless filtrate was placed under 1-propyl-9H-purin-8-yl]phenoxy]acetic Acid (ID). A 3-mL nitrogen, shielded from light, and treated with methanolic amportion of 1 N NaOH was added to a suspension of Ib (0.44 g, monia (ca. 4 N, 400 mL) added in four portions over 1.5 h. After 0.9 mmol) in 4% EtOH (25 mL). The solution that formed on crystallization on stirring overnight, the product (8.36 g) was warming to 70 "C was filtered, cooled, and brought to pH 5 by recrystallizedf i t from 90% methanol and then from 90% ethanol addition of 1 N HC1. The precipitate was filtered and washed to give VIa as a colorless solid (7.41 g, 87%),mp 205-206 "C. Anal. with HZO,then triturated in boiling EtOH (20 mL), cooled, filtered, and washed with EhO. The solid (0.31 g) was recrystalliied (C17HZON604) C, H, N. 6 4 (4-Am~no-3-iodobenzyI)amino]-9-~-~-ribofranosyI-9~from 1:l EtOH/DMF (40 mL) to give IIb as a pale yellow solid purine (VII). A 4.7-mL portion of 1 N HC1 was added to a (0.13 g, 31%), mp slow dec starting at 240 "C. Anal. (C.&&"O5) suspension of VIa (4.25 g, 11.4 mmol) in 50% EtOH (250 mL) C, 13, N. at 55 "C. The resulting solution was cooled to ambient temThe following were prepared by procedures analogues to that perature and treated with solid iodine (3.61 g, 14.2 mmol) in ca. used in the synthesis of IIb. IIa, in 24% yield, mp 261 "C dec. 0.5-g portions over 24 h with stirring to allow solvation of I2 Anal (Cz3H,N605.5/4Hz0) C, H, N. IIc, in 74% yield, mp >300 between additions. A total of 10 mL of 1 N NaOH was added "C. Anal. (Cz3Hz3N505.3/4Hz0) C, H, N. IId, in 77% yield, mp between 5 and 34 h as stirring a t ambient temperature was >280 "C. Anal. (Cz4Hz5N,05-3/4Hz0) C, H, N. IIe, in 57% yield, continued for a total of 5 days. The mixture was diluted with m p >300 "C. Anal. (CzsHz8N605-3/zHz0) C, H, N. treated , with sodium bisulfite (13.8 g), and brought 2 4 4-[3-(4-Amino-3-iodophenethyl)-1,2,3,6-tetrahydro-2,6- HzO (50 d) to p H 7 with 1 N NaOH (17 mL). The volume was reduced to dioxo~l~propyl-9H-purin-8-yl]phenoxy]acetic Acid (IIIb). 100 mL by evaporation, and the precipitated solid was filtered, A 4-mL sample of 1 N NaOH was added to a suspension of Ib washed with water, and dried (5.26 g). The crude product thus (0.49 g, 1.0 mmol) in H20 (100 mL). The solution, which formed obtained was purifed by flash chromatography (CHZClz/MeOH) on heating to near reflux (indicating saponification of Ib to IIb), and recrystallized from 25% EtOH several times to give VI1 as was cooled and neutralized to pH 7 with 1N HC1 (slightly more a nearly white solid (0.3 g, 5.3%), mp 187-194 "C dec. Anal. T o the slightly hazy solution were added alternatively than 3 d,). in 1-mL portions over 4 h a 0.1 M ethanolic solution of iodine (C~'IH~JNBOI) C, H, N, 1. Radioiodination of 8-Phenylxanthine Derivatives. lZ5I(10 mI,) and a 0.1 M aqueous solution of sodium bicarbonate (10 labeled derivatives were prepared from corresponding arylamines mL). The hazy solution was stirred a t ambient temperature by the method of Hunter and Green~ood.'~Two millicuries of overnight and brought to p H 2 with 1 N HCI, and the dark NalZ5Iand 50 nmol of 8-phenylxanthine derivative were mixed precipitate was filtered, washed with HzO, and extracted twice in 0.1 mL of 0.3 M KP04, pH 7.5, with 5 fiL of chloramine T (1 with refluxing EtOH (150 mL). Concentration of the ethanol mg/mL in H20)for 60 s. Reactions were quenched by the addition extract to 15 mL gave IIIb as a pinkish beige solid (0.2 g, 34%), of 50 NL of sodium metabisulfite ( 5 mg/mL in HzO). Products nip 258 "C dec. Anal. (CZ4Hz4IN5O5) C, H, N, I. were extracted four times into 200 pL of ethyl acetate. The pooled The following were prepared by procedures analogous to that extracts, which contained 5@-60% of the starting lZ5I,were used in the synthesis of IIIb: IIIa, in 50% yield, mp 254-256 "C. evaporated to dryness under nitrogen, and the resulting residues Anal. (C23HzzIN605.Hz0) C, H, N, I. IIIc, in 44% yield, mp >300 were dissolved in chromatography buffer (MeOH/5 mM K P 0 4 "C. Anal. (Cz3Hz2IN5O5) C, H, N, I. IIId, in 4% yield, mp slow (pH 6), 1:l) and purified by isocractic HPLC over a 4.5 X 250 dec beginning a t 205 "C. Anal. (C,H,IN50,) C, H, N. mm, 5.0 pm C 18 column a t a flow rate of 1 mL/min. UV ab2-[4-[ 3-(4-Azido-3-iodophenethyl)1,2,3,6-tetrahydro-2,6sorption (310 nm) and y radiation (Beckman Model 170 radioxo-l-propyl-9H-purin-8-yl]phenoxy]acetic Acid (IVb). dioisotope detector) were continuously monitored in the eluate. A clear solution of IIIb (0.111 g, 0.188 mmol) in glacial acetic acid Figure 8 illustrates chromatographic purification of a typical (30 mI ,, warmed nearly to reflux to effect solution and then cooled to ambient temperature) plus HzO (15 mL added gradually) was treated first with a solution of sodium nitrite (0.020 g, 0.29 mmol) in HzO (0.5 mL) and then with a solution of sodium azide (0.015 (26) Dutta, S. P.; Tritsch, G. L.; Cox, C.; Chheda, G . B. J. Med. g, 0.23 mmol) in HzO (0.5 mL). The resulting fine suspension Chem. 1975,18, 780. was diluted gradually with H,O (40 mL), allowed to stand for (27) Hunter, W. M.; Greenwood, F. C. Nature (London) 1962,194, 495. several minutes, filtered, washed well with HzO and then EtOH,
Journal of Medicinal Chemistry, 1988, Vol. 31, No.4 751
I=Z-Labeled 8-Phenylxanthine Derivatives
Equilibrium radioligand binding was fit to the equation
2IL n 4
-
2
L
,
4
where i denotes the number of different affinity states of the receptor, KD denotes the dissociation constant(s) of the radioligand, B denotes bound radioligand, f is the fraction of receptors in each state, and [L] denotes the free radioligand concentration. Inhibition of the binding of a radioligand, L, by a competing compound, I, was fit to the equation A
I
6
8
I
\
.'A
I
10
-.
'--.
I
T
12 14 16 18 20 22 24
Elution Time, minutes
F i g u r e 8. Purification of [1251]-IIIa. IIa was iodinated with carrier-free lZ5Iand chromatographed isocratically by HPLC as described in the text. UV absorption was monitored a t 310 nm was monitored by an in-line y-detector. Fractions conand 1251 taining purified [1*51]-IIIawere pooled, diluted in methanol, and stored a t -20 "C. [1251]iodoarylamine,['251]-IIIa. In this and all cases, the 'Wabeled product cochromatographed with the corresponding lnI-containing compound. Iodination of this and other arylamine-containing 8-phenylxanthines resulted in additional unidentified iodinated products, which did not bind to adenosine receptors. These were eluted from the HPLC column with 100% MeOH and discarded. P r e p a r a t i o n of Brain Membranes. Cerebral cortices of bovine or Sprague-Dawley rat brains were homogenized (Brinkmann) for 10 s in 10 volumes of ice-cold buffer A (10 mM HEPES, 10 mM EDTA, 1mM dithiothreitol, 10% (w/v) sucrose, 10 pg/mL phenylmethanesulfonyl fluoride, and 0.1 mM benzamidine, pH 7.4). The homogenate was filtered through four layers of gauze, and the filtrate was centrifuged at 500g for 10 min. The supernatant and top layer of bifurcated pellets were collected and recentrifuged a t 40000g for 30 min. The resulting pellets were resuspended in 10 volumes of ice-cold buffer A lacking sucrose and stirred a t 4 "C for 30 min. After recentrifugation a t 40000g for 30 min, the pellet was resuspended in 5 volumes of ice-cold buffer B consisting of 10 mM HEPES, 1mM EDTA, and 0.1 mM benzamidine, pH 7.4, aliquoted in fractions containing 5 mg/mL of protein, and stored a t -70 "C. Radioligand Binding Assays. For equilibrium binding assays, membranes were diluted with buffer B so that the final protein content was 0.5 mg of protein/mL and incubated with radioligands in 100 pL of buffer B supplemented with 2.5 units/mL adenosine deaminase, 2.5 mM MgC12, and various concentrations of other salts for 2-3 h at 21 "C. Assays tubes generally contained 0.1-0.3 nCi of radioligands ( ['251]-VII, ['251]-IIIa,or ['261]-IIIb). In some cases radioligands were diluted with the corresponding 1271-containing compounds, 0.1-100 nM. Membranes were trapped on Whatman GF/C glass fiber filters with a modified cell harvester (Brandel) and washed three times with 4 mL of ice-cold 10 mM Tris-HC1, pH 7.4, with or without 0.5 M NaCl for antagonist and agonist radioligands, respectively. Nonspecific binding was determined by the addition of 100 pM PIA, l mM theophylline, or 1MMIIIa with equivalent results. The KD values of the antagonists were calcualted from their IC50values to inhibit [1251]-VII binding to bovine brain membranes with correction for the concentrations of radioligands and receptor.28 Radioligand binding parameters were estimated by the method of ScatchardZ9and then fit to one- or two-site nontransformed equations by Marquardt's method as described p r e v i o ~ s l y . ~ ~ (28) Linden, J. J . Cyclic Nucleotide Res. 1982, 8, 163. (29) Scatchard, G. Ann. N.Y. Acad. Sci. 1949, 51, 660.
(30) Linden, J.; Patel, A.; Spanier, A. M.; Weglicki, W. M. J. Biol. Chem. 1984, 259, 15115.
B/Bmax = CfL[LI /[KD(l + I / K J + [LII r=l
where B denotes bound radioligand in the presence of a competitive compound, I , and Ki denotes the dissociation constant(s) for a competing compound. [L] was approximated by [LT]. Appropriate values for n were selected by F tests evaluated at p < 0.01. Proteins were determined from fluorescarnine f l u ~ r e s c e n c e .All ~ ~ figures are representative of a t least three experiments. Adenylate Cyclase Assays. The binding affinity of adenosine receptor antagonists to Az receptors was calculated on the basis of their ability to inhibit l-(6-amino-9H-purin-9-yl)-l-deoxy-Nethyl-P-D-ribofuranuronamide(NECA) stimulated adenylate cyclase activity in membranes prepared from human platelets. Three units of outdated platelet-rich plasma were centrifuged at 250g for 10 min. The pellet was discarded, and the platelets in the supernatant were pelleted by centrifugation at l6000g for 10 min. The cells were resuspended in 100 mL of 50 mM Tris.HC1 (pH 7.5), 150 mM NaCl, and 20 mM EGTA and washed a second time by centrifugation. The cells were resuspended in 100 mL of ice-cold lysing buffer ( 5 mM EGTA adjusted to pH 7.5 with Tris base), disrupted by sonication, and centrifuged at 40000g for 30 min. The pellet was resuspended in 30 mL of lysing buffer and frozen in 1-mL aliquots at -70 "C. Adenylate cyclase activity was assayed in 0.1 mL of buffer containing 25 pg of platelet membrane protein, 50 mM Na-HEPES (pH 7.6), 125 mM NaC1, 0.5 mM MgClZ, 0.5 mM dithiothreitol, 0.5 mM 4-(3,4-dimethoxybenzyl)-2'-imidazolidinone (Ro7-2956);l 10 pM Gpp(NH)p, 0.1 mM ATP, 2.5 mM phosphoenolpyruvate, 5 units/mL adenosine deaminase, and 50 units/mL pyruvate kinase. Assays were terminated after 5 min a t 37 "C by the addition of 0.4 mL of 50 mM HC1. The cyclic AMP that formed was acetylated by the addition of 22.5 fiL of 3 5 1 triethylamine/acetic anhydride. Following centrifugation a t 2000g for 10 min, acetyl cyclic AMP in the supernatant was detected by automated radioimmunoassay.32 NECA increased adenylate cyclase activity %fold with an EDb0of 0.4 pM. The potency of adenosine antagonists to inhibit the NECA response was calculated from the formula K, = [antagonist]/(dose ratio - l ) , where dose ratio refers to the ratio of ED5,'s of NECA in the presence and absence of antagonist.
Acknowledgment. We express our appreciation to Susie Wilson, Sheryl Christoferrson, and Pam Brett for their technical assistance and to Karen Bagby for helping with the preparation of this manuscript. We also thank Drs. Tom Zimmerman and Janet Rideout for their helpful comments. Registry No. Ia, 112533-60-5;Ib, 112533-54-7;IC,112533-61-6; Id, 112533-62-7;Ie, 112533-63-8;IIa, 112533-56-9;IIb, 112533-55-8; IIc, 112533-57-0; IId, 112533-58-1; IIe, 112533-59-2; IIIa, 112533-64-9; IIIa (lz5I), 112533-72-9; IIIb, 112548-91-1; IIIc, 112533-65-0; IIId, 112533-66-1;IVa, 112533-68-3;IVb, 112533-67-2; IVc, 112533-69-4;IVd, 112533-70-7;Va, 40297-54-9; Vb, 11253371-8; VIa, 95523-13-0; VII, 98866-49-0. (31) This non-xanthine cyclic AMP phosphodiesterase inhibitor was a kind gift of Dr. William E. Scott of Hoffman-La Roche. (32) Brooker, G.; Harper, J. F.; Terasaki, W. L.; Moylan, R. D. Adu. Cyclic Nucleotide Res. 1979, 10, 1.
