364
Bloconjugate Chem. 1994, 5, 364-369
%SubstitutedThioadenine Nucleoside and Nucleotide Analogues: Synthesis and Receptor Subtype Binding Affinities (1) Ahmad Hasan,**+ Tahir Hussain,* S. Jamal Mustafa,* and Prem C. Srivastavas Health Sciences Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6229, and Department of Pharmacology, School of Medicine, East Carolina University, Greenville, North Carolina 27858. Received February 4, 1994"
The design, synthesis, and receptor subtype binding affinities of several 2-substituted thioadenosine nucleoside and nucleotide analogues are described. Alkylation of 2-thioadenosine (1) with iodopen(9). tenylboronic acid followed by iododeboronation gave 2-((E)-1-iodo-1-penten-5-y1)thioadenosine Compound 1 on treatment with 4-nitrobenzyl bromide and propargyl bromide furnished compounds 3 and 5, respectively. The 5'-monophosphate analogues of compounds 3, 5, 7, and 9 were prepared similarly using 24hioadenosine 5'-monophosphate (2). Treatment of 1 with bromoethylamine hydrobromide provided 2-[(aminoethyl)thiol adenosine (11) which on coupling with N-succinimidyl 3-(4-hydroxyphenyl)propionategave 2-[ [[3-(4-hydroxyphenyl)propionamido]ethyl]thioladenosine (12). Iodination of 12 gave 2-[[[3-(4-hydroxy-3-iodophenyl)propionamido]ethyllthio]adenosine(13). Compounds 3-13 were evaluated for their affinities toward AI and A2 adenosine receptors in rat brain cortex and striatum, respectively using [3HlDPCPXand l3H1CGS 21680 as ligands. The nucleotide analogues 4 , 6 , 8 , and 10 inhibited binding of [3HlDPCPX by 10-20% and of r3H]CGS 21680 by 40-50% at a concentration of 100 pM suggesting weak affinity toward adenosine receptors. The nucleoside analogues 3,5,7,9,12, and 13 inhibited the A2 receptor binding of PHICGS 21680 with Ki values of 1.2-3.67 pM, while A1 receptor binding of [3HlDPCPX was inhibited with Ki values 10-17 pM. The AdA2 ratios suggest 4-8-fold A2 receptor selectivity.
INTRODUCTION Adenosine is an effective mediator of a wide variety of physiological functions including vasodilation, cardiac depression, inhibition of platelet aggregation, inhibition of lymphocyte functions, inhibition of insulin release and potentiation of glucagon release in the pancreas, and inhibition of lipolysis (2). Many of these effects are mediated by extracellular receptor subtypes, A1 and A2, linked to adenylate cyclase in an inhibitory and stimulatory manner, respectively. These two receptor subtypes can be distinguished on the basis of structure-activity relationships (3-5) using receptor binding assays (6, 7 ) . Substitutions on the adenine base have been found to alter the affinity of adenosine for its receptors (8-14).For example, W-cyclopentyladenosine (8, 9) is a potent and selective agonist a t the A1 adenosine receptor (7,lO).The availability of W-substituted adenosine analogues has aided in the development of models for the A1 adenosine receptor (10,15),and also for the A2 adenosine receptor (16, 17), and a large number of W-substituted purine nucleosideshave been prepared. Even though several early studies of adenosine receptor of the platelet (18-20) and the coronary artery (21,22)indicate greater selectivity of 2-substituted adenosine analogues as agonist at the A2 subtype, the synthesis and biological evaluation of this class of compounds has been less vigorously pursued.
* Author to whom correspondenceshould be addressed.
+ Current address: Department of Chemistry, Box 90349, Duke University, Durham, NC 27708-0349. Phone (919)-660-1553;Fax
(919)-660-1605. East Carolina University. Work was done during the tenure at ORNL. Current address: Medical Applicationsand Biophysical Research Division, ER-73; US Department of Energy, Washington, D.C. Abstract published in Advance ACSAbstracts, June 1,1994.
*
@
1043-1002/94/ 29Q!i-O364$O4SO/O
Several 2-CH-, 2-0-, 2-NH- , and 2-S-alkyl-, and arylmodified adenine nucleosides have been reported (1830). The aralkoxy substitution at the 2 position of adenosine appears to enhance the A2 receptor affinity. The A2 receptor affinity of phenethoxy adenosine has been further optimized by substitution of the phenyl ring with F, C1, CH3 and CH30- functional groups (26). This led us to synthesize the new 2-substituted thioadenine nucleoside and nucleotide analogues for biological evaluation described in this paper. EXPERIMENTAL PROCEDURES General Methods. All solvents, chemicals, and reagents were analytical grade and were used without further purification unless otherwise indicated. The melting points (mp) were determined on a Thomas-Hoover apparatus in open capillary tubes and were uncorrected. 'H spectra were recorded on a Varian Gemini-200 spectrometer and reported in ppm downfield from the internal tetramethylsilane (TMS = 0) standard. The signals are expressed as s (singlet), d (doublet), t (triplet), m (multiplet), or br (broad). The presence of exchangeable protons was confirmed by treatment with deuterium oxide followed by reintegration of the NMR spectrum. UV spectra were recorded on Beckman DU-64 spectrophotometer. Baker analyzed silica gel (60-200 mesh) was used for column chromatography. Thin-layer chromatography (TLC) was performed using 250-pm layers of silica gel GF precoated glass plates (Analtech, Inc.). Spots on the TLC plates were detected by visualization under short wave ultraviolet (UV) light, exposure to iodine vapors, or heating the chromatogram at 100 "C after spraying with a solution of 5 96 sulfuric acid in methanol. The TLC solvent systems used were (A) isobutyric acid:water:concd ammonium hydroxide (33:17:0.5 v/v), (B) methano1:chloroform (1:5 v/v), (C) ethyl acetate:2-propanol:water (7:1:2 v/v, top layer), and (D) methano1:chloroform (2:8v/v). The paper 0 1994 American Chemical Society
Thioadenine Nucleoside and Nucleotide Analogues
chromatography was performed using Whatman no. 1 cellulose paper in an ascending technique with solvent system A. The elemental analyses were determined by Galbraith Laboratories (Knoxville, TN), and the results are within f 4 % of the theoretical value except where noted. 2-[ (4-Nitrobenzyl)thio]adenosine(3). Compound 3 was prepared by slight modification (NaH and DMF) of literature procedure (18)in 46 % yield, mp 152"C [(shrinks at 126-127 "C (lit. (18)130.5-132 "C)]. 'H NMR (DMSO&): 6 8.27 (s, lH, H-8), 8.15 (d, J = 8.5 Hz, 2H, Ar-H), 7.78 (d, J = 8.8 Hz, 2H, Ar-H), 7.46 (s,2H, NHd, 5.87 (d, J = 5.6 Hz, lH, H-l'), 5.45 (d, J = 5.86 Hz, lH, OH), 5.20 (d, J = 5.9 Hz, l H , OH), 5.06 (t, J = 5.5 Hz, l H , OH), 4.5 (m, l H , H-2'),4.12 (m, lH, H-3'),3.96 (d, J = 3.66 Hz, l H , H-4'),3.60 (m, 2H, H-5'), 3.14 (s,2H,-CH2-). Anal. Calcd for (C17H18N606S-CH30H): C, 46.35; H, 4.72; N, 18.03. Found: C, 46.66; H, 4.89; N, 17.78. 2-[(4-Nitrobenzyl)thio]adenosine5'-Monophosphate (4). Sodium methoxide (540 mg, 10 mmol) was added to a suspension of 2-thioadenosine 5'-monophosphate (32) (1.0 g, 2.6 mmol) in methanol (200 mL) under an argon atmosphere. The reaction mixture was stirred at room temperature for 30 min after which 4-nitrobenzyl bromide (570 mg, 2.6 mmol) was added, and the stirring was continued for 10 h at room temperature. Solvents was removed under reduced pressure, and the residue was dissolved in water. The solution was adjusted to pH 2 with 2 N hydrochloric acid. The crude product was collected by filtration, washed withcold water, and purified by dissolution in 1 N sodium hydroxide followed by adjusting the solution to pH 2 with 1N hydrochloric acid. The mixture was kept at room temperature overnight, and the crystalline solid that separated was collected by filtration to yield 4 (570 mg, 42%), mp 192-194 "C. lH NMR (DMSO-&): 6 8.4 (s, lH, H-8), 8.17 (d, J =7.86 Hz, 2H, Ar-H), 7.79 (d, J = 7.89 Hz, 2H, Ar-H), 6.02 (d, J = 4.48 Hz, lH, H-1'1, 4.42 (m, 2H, H-2', H-3'),4.15 (bs, 2H, -CH2-), 4.01 (bm, 3H, H-4', H-5'). UV (pH 1): Am, 269. Anal. Calcd for (C17H&&kW2H20): C, 37.09; H, 4.18; N, 15.27. Found: C, 36.94; H, 3.90; N, 15.06. 2-[(Propargyl)thio]adenosine (5). Sodium hydride (40 mg, 1 mmol, 60% suspension in oil) was added to a suspension of compound 1 (299 mg, 1mmol) in anhydrous DMF (10 mL) under an argon atmosphere. The mixture was stirred at room temperature until evaluation of hydrogen gas ceased (-25 min). A solution of propargyl bromide (2 mL, 80% solution in toluene) in anhydrous DMF (5 mL) was added to the reaction mixture, and stirring was continued overnight. Solvent was removed under reduced pressure, and the residue was triturated with water, filtered, and washed with cold water. The crude product was purified by silica gel column chromatography. The column was eluted with ethyl acetate followed by 5 % methanol in ethyl acetate, and the fractions (Rf = 0.46, silica gel TLC in solvent C) containing the major product were combined, and solvent was removed to yield 5 (277 mg, 82%),mp 121 "C (78-80 "C shrinks). 'H NMR (CDCl3 + CD3OD): 6 7.94 (s, l H , H-8), 5.8 (d, J = 6.06 Hz, lH, H-l'), 4.79 (t, J = 5.5 Hz, l H , H-2'), 4.37 (m, l H , H-39, 4.2 (m, lH, H-49, 3.79 (m, 2H, H-59, 3.40 (s, 2H, -CH2-), 2.25 (t, J = 2.6 Hz, l H , C=CH). Anal. Calcd for (C13H15N504SCH30H): C, 45.53; H, 5.15; N, 18.97. Found: C, 45.82; H, 4.90; N, 19.12. 2-[(Propargy1)thioladenosine5'-Monophosphate (6). Compound 6 was prepared following the procedure described for 4, using 2 (230 mg, 0.6 mmol), sodium methoxide (125 mg, 2.3 mmol), and propargyl bromide (250 mg, 80% wt solution in toluene) in methanol at 60
Bloconjugate Chem., Vol. 5, No. 4, 1994 365
OC for 12 h. The residue was dissolved in water (2 mL), and the solution was adjusted to pH 2 with dilute hydrochloric acid. The crude product was collected by filtration, washed with water, dried under vacuum, and crystallized from water to yield 6 (75 mg, 30%),mp 218220 "C. 'H NMR (DMSO-&): 6 8.44 (9, lH, H-8), 5.9 (d, J = 5.49 Hz, lH, H-l'), 4.65 (t, J = 4.8 Hz, lH, H-2'), 4.18-4.0 (m, 5H, H-3', H-4', and H-5'),3.9 (s,2H, -CH2-), 2.5 (t, J = 1.75 Hz, lH, HCEC). UV (pH 1): ,A, 269.6. Anal. Calcd for ( C ~ ~ H ~ ~ N ~ O ~ S PC,- H 35.86; Z O )H, : 4.14; N, 16.09. Found: C, 35.85; H, 3.96; N, 16.07. 2-[ ((E)-l-Borono-l-penten-5-yl)thio]adenosine (7). Compound 7 was prepared following the procedure described for 5,using 1 (299 mg, 1mmol), sodium hydride (40 mg, 1 mmol, 60% dispersion in oil), and (1-iodo-5penten-5-y1)boronicacid (242 mg, 1mmol) in DMF. The residue was dissolved in hot water and filtered. The solution was adjusted to pH 5.5 with glacial acetic acid. The solvent was evaporated to dryness, and the residue was purified by silica gel column chromatography. The column was eluted with 8-12 % methanol in ethyl acetate, the fractions (Rf= 0.27, silica gel TLC in solvent C) were collected, and solvent was removed to yield 7 (238 mg, 58%), mp 229-233 "C. 'H NMR (DMSO-&): 6 8.19 (s, l H , H-8), 7.4 (bs, 2H, NHz),6.65 (bm, l H , HC=CH), 6.12 (d, J = 14.1 Hz, l H , HC=CH), 5.64 (d, J = 5.2 Hz, l H , H-l'), 3.5 (m, 2H, CHz), 2.55 (m, 2H, CH2), 1.89 (m, 2H, CH2). UV (pH 1): Am, 268. Anal. Calcd for (C15H22BN*506S): C, 43.80; H, 5.35; N, 17.03. Found: C, 44.16; H, 5.22; N 17.44. *N: calcd 17.03, found 17.44. 2-[((E)-1-Iodo-1-penten-5-yl)thio]adenosine (9). Compound 7 (155 mg, 0.37 mmol) was dissolved in 50% aqueous THF (4.0 mL). Sodium iodide (84 mg, 1.5 equiv) was added followed by addition of a solution of chloramine-T (126 mg, 0.55 mmol) in 50% aqueous THF (2 mL). The reaction mixture was stirred in the dark at room temperature for 1hand quenched with a saturated solution of sodium thiosulfate (50mg in 0.5 mL water). The solvent was evaporated, and the residue was dissolved in water (10 mL) and extracted with ethyl acetate (5 X 20 mL). The ethyl acetate portion was dried (sodium sulfate), filtered, and evaporated. The crude product was crystallized from hot methanol to yield 9 (145 mg, 78%), mp 89-91 "C (methanol and water). TLC: Rf = 0.68, silica gel, solvent C. lH NMR (DMSO-&): 6 8.23 (8, lH, H-8), 7.38 (bs, 2H, NHz), 6.66 (dt, J = 14.2 and 7.1 Hz, l H , HC=CHI),6.2 ( d , J = 14.3 Hz, lH,HC=C), 5 . 8 1 ( d , J = 5.5 Hz, l H , H-l'), 4.61 (d, J = 6.3 Hz, l H , H-2'), 4.1 (d, J = 4.8 Hz, l H , H-3'), 3.9 (d, J = 3.1 Hz, l H , H-4'), 3.5 (m, 2H, H-5'1, 3.18 (d, J = 5.1 Hz, 2H, CHz), 2.15 (m, 2H, CH2), 1.75 (m, 2H, CH2). UV (pH 1): Am, 268.8. Anal. Calcd for (C15HzoIN504S.O.5HzO): C, 35.85; H, 4.18; N, 13.94. Found: C, 35.93; H, 4.30; N, 13.91. 2-[((E)-l-Borono-l-penten-5-yl)thio]adenosine 5'Monophosphate (8). Iodopentenylboronic acid (37.6mg, 0.16 mmol) was added to a mixture of 2 (50mg 0.13 mmol) and sodium methoxide (27 mg, 0.5 mmol) in DMF (2 mL) under an argon atmosphere. The mixture was stirred at room temperature for 18 h. The solvent was removed under reduced pressure and the residue was dissolved in water (-2 mL). The solution was adjusted to pH 2 with 2 N hydrochloric acid. The mixture was passed through a column (1X 10 cm) packed with AG50 WX1 resin. The column was eluted with water (75 mL) followed by 45% acetic acid in water. The fractions (Rj = 0.44, paper chromatography in solvent A) were combined, and the solvent was evaporated to yield 8 (27 mg, 42 % 1, mp >300 "C. The compound was highly insoluble in deuteriosol-
366 Bioconjugate Chem., Vol. 5, No. 4, 1994
vents at a suitable concentration for NMR; hence, NMR data is not included for this compound. Anal. Calcd for (C15Hp,BN509SP-H20): C, 35.36; H, 4.91; N, 13.75. Found: C, 35.60; H, 4.81; N, 13.69. 24((E)-l-Iodo-l-pentend-yl)thio]adenosine5’-Mon0phosphate (10). A solution of chloramine-T (17.3 mg, 76 pmol) in aqueous THF (1mL) was added to a mixture of 8 (25 mg, 50 pmol) and sodium iodide (11.4 mg, 76 pmol) in 50% aqueous THF (2 mL). The reaction mixture was stirred at room temperature for 24 h in the dark and quenched by addition of a saturated solution of sodium thiosulfate ( 1 mL). The solvent was removed under reduced pressure, and the residue was dissolved in water (2 mL) and filtered. The filtrate was adjusted to pH 2 with 2 N hydrochloric acid. The crude product separated as a solid was filtered and washed with cold water. The crude product was crystallized from a mixture of water and ethanol to yield 10 (18 mg, 62%), mp 169-171 OC. TLC: Rf = 0.76, paper chromatography, solvent A. lH NMR (DMSO-de): 6 8.28 (s, l H , H-8), 6.55 (dt, J = 14.2 and 7.0 Hz, lH, HC=CHI), 6.3 (d, J = 14.33 Hz, lH, HC=CHI),5.9 ( d , J = 5.2Hz71H,H-l’),2.5 (m,2H,CH2), 268. 2.2 (m, CHd, 1.82 (m, 2H, CH2). UV (pH 1): A, Anal. Calcd for ( C ~ ~ H ~ ~ I N S P O + ~C,. ~29.56; H ~ OH,) :4.11; N, 11.49. Found: C, 29.42; H, 3.99; N, 11.26. 24(Aminoethy1)thioladenosine(1 1). Compound 11 was prepared following the procedure described for 5,using 1 (448.5 mg, 1.5 mmol), sodium hydride (60 mg, 1.5 mmol, 60 % dispersion in oil), and 2-bromoethylamine hydrobromide (307 mg, 1.5 mmol) in DMF. The residue was dissolved in water and adjusted to pH 8 with 1N sodium hydroxide. The solvent was evaporated to dryness, and the residue was purified by silica gel column chromatography. The column was eluted with 6-10% methanol in chloroform and the fractions (Rf = 0.46, TLC in solvent B) were combined. The solvent on evaporation gave 11 (380 mg, 74%), mp 198-205 OC dec. ‘H NMR (CDBOD): 6 8.25 (s, lH, H-8), 6.0 (d, J = 6.2 Hz, l H , H-l’), 4.7 (t, J = 5.4Hz7lH, H-2’),4.38 (m, lH, H-3’), 4.15 (m, lH, H-49, 3.85 (m, 2H, H-5’1, 3.4 (bm, 4H,(CH2)2). 2-[[[3-(4-Hydroxyphenyl)propionamido]ethyl]thio]adenosine (12). A solution of N-succinimidyl 344hydroxypheny1)propionate (65.3 mg, 0.25 mmol) in DMF (1mL) was slowly added to a solution of 11 (85.5 mg, 0.25 mmol) in DMF (2 mL). The reaction mixture was stirred at room temperature for 24 h. The solvent was removed under reduced pressure, and the mixture was purified using preparative TLC (solvent D). The UV-vis band (Rf = 0.46, silica gel TLC in solvent D) was scrapped and eluted with a solution of chloroform and methanol. The solvent was evaporated to yield 12 (35 mg, 29%), mp 110 “C (methanol). ‘H NMR (CDzOD): 6 8.23 (s, l H , H-8),7.01 (d, J = 8.4 Hz, 2H, Ar-H), 6.68 (d, J = 8.4 Hz, 2H, Ar-H), 6.0 (d, J = 5.7 Hz, l H , H-l’), 4.7 (t, J = 5.33 Hz, l H , OH), 4.4 (t, J = 3.58 Hz,lH, H-2’), 4.18 (t, J = 2.55 Hz, lH, H-37, 3.85 (m, 2H, H-4’ and OH), 3.5 (m, 2H, H-5’), 3.55 (m,2H,CH2),3.2(t7J=6.6Hz,2H7CH2),2.75(t,J=7.24 Hz, CH2), 2.46 (t, J =7.02 Hz, 2H, CH3. UV (pH 1): A, 268.6. The compound was analyzed as the corresponding iodo derivative 13. 24[ [3-(4-Hydroxy-3-iodophenyl)propionamido]ethyl]thio]adenosine (13). Sodium iodide (53 mg, 0.35 mmol) was added to a solution of 12 (172 mg, 0.35 mmol) in 50% aqueous THF (5 mL). Subsequently, a solution of chloramine-?’ (80 mg, 0.35 mmol) in THF (1mL) was added, and the reaction mixture was stirred in the dark for 1h. A solution of sodium thiosulfate (50 mg in 0.5 mL water) was added, and the solvent was removed under N
Hasan et al.
reduced pressure. The resulting residue was extracted with chloroform and washed with water. The organic layer was dried (sodium sulfate) and filtered, and the residue obtained after evaporation of the solvent was purified using preparative TLC (solvent C). The major band visible under UV light was scrapped and eluted with a solution of chloroform and methanol (1:l v/v). The solvent was removed to yield 13 (150 mg, 56%1. A portion of this was crystallized from a mixture of water and ethanol to yield an analytical sample, mp 190-192 OC. lH NMR (CD3OD): 6 8.23 (s, lH, H-8), 7.53 (d, J = 2.2 Hz, l H , Ar-H), 6.99 (dd, J = 8.2 and 2.1 Hz, lH, Ar-H), 6.77 (d, J = 8.2 Hz, l H , Ar-H), 6.04 (d, J = 5.6 Hz, l H , H-1’1, 4.42 (t, J = 3.58 Hz, lH, H-2), 4.19 (d, J = 3.11 Hz, l H , H-3’),3.86 (m, 3H, H-4’ and H-5’),3.53 (m, 2H, CH2),3.23 (t, J = 5.5 Hz, 2H, CH2),2.7 (m, 2H, CH2), 2.39 (m, 2H, CH2). Anal. Calcd for (C21H25IN60&-2H20): C, 38.65; H, 4.45; I, 19.48; N, 12.88. Found: C, 38.48; H, 4.33; I, 19.41; N, 12.76. Biological Studies. Membrane Preparation. The membranes from male Wistar rat (170-200 g) cerebral cortex and striatum were prepared as described (33). Protein was measured according to the method described by Bradford (34). Radioligand Binding Assays. Radioligand binding of A1 receptors from rat cortical membranes was measured as described (35)for the AI antagonist r3H1DPCPX. [3HlCGS 21680 was used to measure A2 receptor binding in rat striatal membranes (36)containing approximately 100 pg of protein in a total volume of 250 pL at 25 OC. Cold (R)-PIA (10 pM) and CGS 21680 (10 pM) were used to define the non specific binding for AI and A2 receptor, respectively. The values of inhibition constant (Ki)were calculated using EBDA computer program. This program uses the IC5,, values for calculating Ki according to the equation of Cheng and Prusoff (37).
RESULTS AND DISCUSSION Chemistry. The viable intermediate compounds, 2-thioadenosine (1)and 2-thioadenosine 5’-monophosphate (21, were prepared as reported (31,321,starting with adenosine and adenosine 5’-monophosphate, respectively. Boronovinyl (Scheme 1) and phenol (Scheme 2) moieties were introduced in 2-thioadenosine substrate sites for selective and easy iodination and potentially for radioiodination for receptor characterization studies. Alkylation of the sodium salt of 2-thioadenosinewith (iodopentenyl)boronic acid (38)in anhydrous DMF gave 7. The S-alkylation was supported by UV spectrometry (21). Iodination of 7 with sodium iodide and chloramine-T in anhydrous THF furnished 2-[ ((E)-l-iodo-l-penten-5-yl)thioladenosine (9) in 78 5% yield. Our attempts to directly phosphorylate (3942)the 5’-OH of either 7 or 9 were futile, and unchanged starting material was recovered in each case. It is possible that the steric nature of the 2-substituent obscures the nucleosideconformationmaking selective phosphorylation impossible. This led us to investigate an alternate route for the synthesis of compounds 8 and 10. Treatment of 2 with (iodopenteny1)boronicacid in the presence of 3.1 molar equiv of sodium methoxide in DMF gave 2-[((E)l-borono-l-penten-5-yl)thio] adenosine 5‘-monophosphate (8). Iododeboronation of 8 with sodium iodide and chloramine-T gave 2- [((E)-l-iodo-l-penten-5-yl)thioladenosine 5’-monophosphate (10). The E-isomeric configuration of the vinylic iodide moiety in compounds 9 and 10 was established on the basis of lH NMR spectroscopy. In the ‘H NMR spectrum of 9, the signals for the vinylic protons appeared as a doublet at 6 6.20 (J= 14.3 Hz) ppm and a set of triplets at 6 6.66 (J= 14.20 and 7.10 Hz) ppm,
Bioconjugate Chem., Vol. 5, No. 4, 1994
Thioadenlne Nucleoside and Nucleotide Analogues
307
Scheme 1
HO OH l,R=H 2, R I HZPO,
HO OH
7,RiH HZPO,
8, R
Scheme 2
HO
OH
-
11
OH
12 consistent with the literature report (38). Treatment of compounds 1 and 2 with 4-nitrobenzyl bromide under basic conditions (NaH) gave compounds 3 (18) and 4 in 46% (lit. (18) 36%) and 42% yield, respectively. Similarly, alkylation of 1 with propargyl bromide (80% solution in toluene) in the presence of sodium hydride gave 2-[(propargy1)thioladenosine(5). Compounds 3-6 were prepared as precursors for iodination. Our attempts to convert compound 5 to the corresponding tributyltin derivative following the literature procedure (43) resulted in desulfurization. The synthesis of a phenyl-substituted 2-thioadenosine analogue(13) containing a larger carbon chain interposition between the terminal phenyl group and the C-2 subregion of purine is outlined in Scheme 2. Reaction of 1 with 2-bromoethylamine hydrobromide in the presence of sodium hydride in DMF gave 2-[ (aminoethy1)thioladenosine (11) in 74 % yield. Coupling of 11 with N-suc-
13 cinimidyl 3- (4-hydroxypheny1)propionatein DMF gave 12. The incorporation of the phenolic moiety in compound 12 was confirmed by the lH NMR spectrum in which the signal for aromatic protons (phenyl ring) appeared as doublets at 6 7.01 (J = 8.40 Hz, 2H) and at 6 6.68 (J= 8.4 Hz, 2H) ppm. Iodination of 12 with sodium iodide and chloramine-?' in THF gave 2- [ [[3-(6hydroxy-&iodophenyl)propionamidolethyllthioladenosine (13) in 56% yield. In the lH NMR spectrum of 13 the signals for aromatic protons (phenyl ring) appeared as a doublet at 6 7.53 (J = 2.2 Hz, 1H) ppm, a set of doublets at 6 6.98 (J = 8.2 and 2.1 Hz, 1H) ppm, and another doublet at 6 6.77 (J = 8.2 Hz, 1H) ppm. Biological Evaluation. The effects of a series of 2-substituted thioadenosine nucleoside and nucleotide analogues were evaluated on the adenosine receptors using radioligand binding in competition assays, and the results are given in Table 1. Affinities for A2 receptors were OH
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Table 1. Ki Values for A1 and A2 Receptor Binding Affinities for 2-Substituted Thioadenine Nucleoside and Nucleotide Analogues. Ki (PW, Ki (PM), selectivity compd A2 bindingb A1 binding AilAz 0.0013 f 0.00042 0.0032 @)-PIA 0.4f 0.06 1.4 f 0.92 127 CGS 21680 0.011f 0.0009 6.5 2.4 f 0.16 15.7 f 1.3 3 9.2f 1.2 4.6 5 2.0 f 0.10 4.1 3.67 f 0.30 15.0 f 1.4 7 17.2f 2.5 4.6 9 3.67f 0.25 12 2.04 f 0.08 16.0f 1.0 7.8 13 1.2 f 0.09 10.0 f 0.9 8.3 The assays were performed three times in triplicate and values represent mean f SE. (R)-Phenylisopropyladenosine(@)-PIA)and 2-[[4-(2-Carboxyethyl)phenethyl]amino]adenosine 5’-N-ethyluronamide (CGS 21680)were used at 10-11-104M while compounds were used at lO-9-lV M for the competitionexperiments. * [SHICGS 21680. [3H]8-Cyclopentyl-l,3-dipropylxanthine ( [3HlDPCPX). (I
determined in radioligand assays for the receptors of rat striatum using f3H1CGS 21680 as the radioligand. Affinities for A1 receptors were determined in radioligand competition assays for the receptors of rat cortex using [3HlDPCPX as radioligand. CGS 21680and (23)-PIAwere used as reference compounds, for A2 and AI, respectively. The Ki values of @)-PIA and CGS 21680 for A1 and A2 receptors, respectively, were found in accordance with the earlier reports (44) suggesting the validity of the receptor binding assays. The nucleotides 4, 6, 8, and 10 exhibited poor affinities for both A1 and A2 receptors (10-20% inhibition of PHI DPCPX binding and approximately 4050% inhibition of PHICGS 21680 binding) at 100 pM concentrations. It could be expected since the nucleotide, AMP, had been reported earlier to be a less potent on adenosine receptors (45). Compounds 3, 5, 7, 9, 12, and 13 have Ki 1.2-3.6 pM for the A2 receptor and 10-17 pM for the A1 receptor suggesting low affinities but with 4-8fold higher selectivity for A2 as compared to AI. The A2 selectivity of these compounds was apparently based on the accepted notion (46) that adenosine with N6 substituents is more selective for A1 receptor while those with the C-2 substituents are more selective for A2 receptor. However, the difference in A2 selectivity of compounds 12 and 13 (8-fold) and compound 3 (6-fold) may be explained on the basis of the carbon chain length and the nature of substituents on the phenyl ring. The presence of a phenyl group increases the hydrophobicity of these compounds resulting in higher selectivity. The decrease in hydrophobicity of compounds 5,7, and 9 compared to compounds 3, 12, and 13 led to a slight attenuation in A2 selectivity (4-fold). The presence of a boronic acid moiety in compound 7 or iodine in compound 9 apparently made no significant difference in either the affinity or selectivity of these compounds. Also, it appears that alkylvinyl and alkyl phenyl moieties seriously perturb the adenosine molecule and impair its ability for A1/A2 receptor selectivity. The agonist activity of these compounds was tested in isolated rat aortic rings (for A2 response in terms of vasorelaxation) and left atria (for A1 response in terms of inotropic effect) using the organ bath technique (47). Compound 3 produced vasorelaxation with an IC50 of 7.5 pM. The force of contraction (inotropic effect) was inhibited by 20 % at 100 pM concentration of compound 3. These data suggest that the compound exhibits the agonist activity as well as the A2 selectivity. A similar pattern of agonist activity and selectivity was observed with other compounds. These preliminary data did not support pursuing radiolabeling of these compounds with
Hasan et al.
1231for receptor imaging application by single photon emission computed tomography. In summary, the binding affinities observed for 2-substituted thioadenine nucleoside and nucleotide analogues indicate that introduction of the sulfur atom at the C-2 position of adenine does not contribute toward the affinity or selectivity of these compounds. However, the presence of a hydrophobic group a t the C-2 position appears to increase the A2 receptor selectivity. ACKNOWLEDGMENT
Research supported by the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc. LITERATURE CITED (1) Presented in part (as an abstract) at the 204th American
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