Dual-action compounds targeting adenosine A2A receptor and adenosine transporter for prevention and treatment of neurodegenerative diseases

Abstract

The present invention provides therapeutic agents for preventing and treating neurodegenerative diseases. These agents synergistically target both the adenosine A.sub.2A receptor (A.sub.2AR) and the equilibrative nucleoside transporter 1 (ENT1).

Claims

1. A method for treating neurodegenerative disease, comprising administering to a subject in need thereof an effective amount of a compound having the formula: ##STR00011##

2. The method of claim 1, wherein the neurodegenerative disease is a protein-misfolding disease.

3. The method of claim 1, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Prion disease, Huntington's disease, and spinal cerebellar ataxias.

4. The method of claim 1, wherein the neurodegenerative disease is Huntington's disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows structures of CGS, NBTI and some designed adenosine derivatives with modification at the N.sup.6- and C.sup.5-positions.

(2) FIGS. 2A-F show the 3D pharmacophore model of the adenosine A.sub.2A receptor agonists. (A) The geometric features of the pharmacophore model. Cyan: hydrophobic (HP), gold: ring aromatic (RA), magenta: hydrogen bond donor (HBD), green: hydrogen bond acceptor (HBA) (B) Fitting of CGS into the pharmacophore model. (C) Fitting of compound 1. (D) Fitting of NBTI. (E) Fitting of compound 6. (F) Fitting of compound 11.

(3) FIGS. 3A-F show the 3D pharmacophore model of the human equilibrative nucleoside transporter (hENT1) inhibitors. (A) The geometric features of the pharmacophore model. Gold: ring aromatic (RA), green: hydrogen bond acceptor (HBA1: 3.431 distance from RA; HBA2: 10.388 distance from RA). (B) Fitting of NBTI into the pharmacophore model. (C) Fitting of compound 1. (D) Fitting of CGS. (E) Fitting of compound 6. (F) Fitting of compound 11.

(4) FIG. 4 shows the scatter plot of the predicted pK.sub.i values of A.sub.2A-PR agonists versus the measured pK.sub.i values. The filled circles represent the training compounds, and the open circles the synthesized compounds.

(5) FIG. 5 shows the scatter plot of the predicted pK.sub.i values of ENT1 inhibitors versus the measured pK.sub.i values. The filled circles represent the training compounds, and the open circles the synthesized compounds.

DESCRIPTION OF THE EMBODIMENTS

(6) Pharmacophore Model of the Human Adenosine A.sub.2A Receptor Agonists

(7) As part of the dual-pharmacophore drug design approach, a 3D-pharmacophore model of the human A.sub.2AR (hA.sub.2AR) agonists was first constructed to design the compounds that could function as hA.sub.2AR agonists. The training set includes 25 compounds having large range of structural diversity and hA.sub.2AR activity (K.sub.i from 1.2 nM to 187 M) selected from the literature. A potent hA.sub.2AR agonist CGS,.sup.33 is also included in this training set. The HypoGen module of Catalyst of Accelrys.sup.34 was used to construct the pharmacophore model of these ligands. The constructed pharmacophore is illustrated in FIG. 2A, which shows four geometric features including hydrophobic (HP, in cyan), ring aromatic (RA, in gold), hydrogen bond donor (HBD, in magenta) and hydrogen bond acceptor (HBA, in green). For CGS all the four features of the constructed pharmacophore can be fitted nicely (FIG. 2B). In contrast, S-(4-nitrobenzyl)-6-thioinosine (NBTI).sup.35 lacks a ring-aromatic fitting (FIG. 2D), in agreement with its weak affinity as hA.sub.2AR ligand, though it exhibits a strong binding with adenosine transporter. The designed dual-action ligands 1, 6 and 11 fit at least three features in this pharmacophore model for A.sub.2AR agonists (FIGS. 2C, 2E and 2F).

(8) Pharmacophore Model of the Equilibrative Nucleoside Transporter Inhibitors.

(9) To design dual-function compounds that act cooperatively as the hA.sub.2AR agonists and the hENT1 inhibitors, the pharmacophore of hENT1 inhibitors was also constructed (FIG. 3A). The training set includes 25 compounds possessing hENT1 inhibitory activity ranging from IC.sub.50 of 0.29 nM to 32 M, which were selected from the literature (see Supporting information). The constructed pharmacophore model of the hENT1 inhibitors consists of only three features, including two hydrogen bond acceptors and one ring aromatic. All the five compounds (NBTI, 1, CGS, 6, and 11) can fit into all these three features (FIGS. 3B-F). The different number of features between the pharmacophores of hA.sub.2AR agonists and hENT1 inhibitors could be attributed to the nature of the training set compounds.

(10) By carefully scrutinizing the structures in the hA.sub.2AR agonists training set (see Supporting Information), we found that many of them possess a hydrophobic group in the 5 end of nucleoside, especially those compounds with higher potency, including CGS. Therefore, the constructed pharmacophore must include this important feature. On the contrary, in the hENT1 inhibitors training set, almost all the 5 end of the nucleoside possesses a polar hydroxyl group.

(11) The pharmacophore differences among the two investigating targets also shed some light on the design of dual-function compounds. For example, compound 6 does not fit well to the hydrophobic feature in hA.sub.2AR receptor pharmacophore model, and it is indeed less potent than CGS, which fits well to all features. However, compound 6 could fit well in all three features in hENT1 model.

(12) As for compound 11, it fits well to all features including hydrophobic of A.sub.2AR pharmacophore mode. Nevertheless, it still fits well to hENT1 pharmacophore model, indicating the higher tolerance of ENT1 pharmacophore model. In summary, by comparing the features and compound-fitting qualities of these two pharmacophore models, we may hypothesize that the hA.sub.2AR binding pocket has an important hydrophobic site, and the hENT1 binding pocket may be more flexible to accommodate the nucleosides with hydrophobic moieties at the 5 end in this series of compounds. Pharmacophore analyses provide us with an insight into the design and understanding of dual-function compound in the absence of the structural information of hENT1.

(13) Synthesis of Adenosine Derivatives.

(14) A representative library of adenosine analogues (FIG. 1) was developed based on the pharmacophore models. Compound 1, originally isolated from Gastrodia elata,.sup.3 was synthesized in a high yield by the substitution reaction of 6-chloropurine ribofuranoside (17) with 4-hydroxybenzylamine (as the hydrochloric salt) in the presence of a base of diisopropylethylamine. As 4-hydroxybenzylamine was not commercially available, it was prepared in two steps from 4-hydroxybenzaldehyde via the condensation reaction with hydroxylamine to give an intermediate oxime, which was subsequently hydrogenated by the catalysis of Pd/C and HCl. By the procedures similar to that for compound 1, a series of N.sup.6-substituted adenosine derivatives 2-6 were prepared by the substitution reactions of 6-chloropurine ribofuranoside with appropriate substituted benzylamines. In lieu of the conventional heating method, microwave irradiation was also applied to shorten the reaction time in the preparation of N.sup.6-(3-indolylethyl)adenosine (6).

(15) ##STR00008##

(16) Treatment of N.sup.6-(4-methoxybenzyl)adenosine 3 with 2,2-dimethoxypropane in anhydrous acetone afforded the corresponding 3-acetonide, which reacted with p-toluenesulfonyl chloride in the presence of pyridine to give a mixture of tosylate and chloride derivatives (Scheme 1). The tosylate derivative was unstable, whereas the chloride compound (7-acetonide) could be isolated by chromatography and subsequently hydrolyzed to give 7. Without separation, this mixture was treated with sodium azide, followed by acid-catalyzed hydrolysis, to give an azido compound 8. Alternatively, Staudinger reduction of 8-acetonide gave an intermediate amine, which was converted to the corresponding acetamide 9 and sulfonamide 10 after removal of the 2,3-isopropylidene group in acid.

(17) ##STR00009##

(18) In another approach (Scheme 2), the Cu.sup.+-catalyzed 1,3-dipolar cycloaddition (click reaction).sup.38 of azido compound 8 with 1-hexyne, 1-octyne, and 3-phenyl-1-propyne afforded the triazole derivatives 12, 13 and 14, respectively. Likewise, the click reaction of 8-acetonide with propargyl alcohol gave a triazole compound, of which hydroxyl group was activated as a mesylate and then reduced by NaBH.sub.4 to give a methyl group. Compound 11 was obtained after removal of the 2,3-isopropylidene group.

(19) ##STR00010##

(20) The acetonide of 6-chloropurine ribofuranoside was oxidized by (diacetoxyiodo)benzene with catalysis of 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO).sup.39 to give a carboxylic acid 18. The coupling reactions of acid 18 with ethylamine and ammonia gave amides 19 and 20, respectively. The chlorine atom in 19 was replaced by 4-hydroxybenzylamine, and the subsequent hydrolysis of the acetonide furnished amide 15. On the other hand, amide 20 was converted to nitrile 21 on treatment with Me.sub.2SO, oxalyl chloride and i-Pr.sub.2NEt..sup.40 After the chlorine atom was substituted by 4-methoxybenzylamine, a 1,3-dipolar cycloaddition of the cyano group with NaN.sub.3 introduced the desired tetrazole moiety at the C-5 position,.sup.41 giving 16 after removal of the 2,3-isopropylidene group under acidic conditions.

(21) Biological Evaluation of N.sup.6- and C.sup.5-Modified Adenosine Derivatives.

(22) The pharmacological properties of the prepared adenosine analogues were characterized by MDS Pharma Services using radioligand binding assays. The binding constants (K.sub.i) of some representative compounds are shown in Table 1. The potent A.sub.2AR agonist CGS appears to lack the activity against ENT1, whereas the ENT1 inhibitor NBTI shows no binding ability with A.sub.2AR. Neither CGS nor NBTI is dual-functional drug. Some prepared adenosine analogues exhibit the dual actions on A.sub.2AR and ENT1, in particular, compounds 1, 4 and 6 showing the K.sub.i values in low micromolar and sub-micromolar range with A.sub.2AR and ENT1, respectively. Except for 11 and 15, the adenosine derivatives having modification at the C-5 position appeared to deteriorate their binding with A.sub.2AR, though they still maintained high affinity with ENT1.

(23) TABLE-US-00001 TABLE 1 Binding activity of the N.sup.6- and C.sup.5-modified adenosine derivatives with adenosine receptor and transporter..sup.a K.sub.i (M) Compound A.sub.2A-R.sup.b ENT1.sup.c CGS 7.77 10.sup.2 NBTI >10 2.9 10.sup.4 1 2.62 5.38 10.sup.1 1-acetonide >100 >100 2 14.4 1.44 10.sup.2 3 30.1 3.18 10.sup.1 4 3.21 3.72 5 27.7 6.51 10.sup.3 6 4.39 3.47 7 >100 2.98 8 5.81 10.sup.1 9 >100 1.43 10 >100 2 10.sup.1 11 41.8 9.60 10.sup.1 12 >100 5.11 10.sup.1 13 >100 5.2 10.sup.2 14 >100 1.16 10.sup.1 15 20.3 >10 16 >100 1.17 .sup.aThe radioligand binding assays were performed by MDS Pharma Services Taiwan (Taipei, Taiwan) using standard binding protocols. .sup.bHuman adenosine A.sub.2A receptor. .sup.cGuinea pig equilibrium transporter 1.

(24) We have previously reported that compound 1 isolated from an aqueous methanolic extract of Gastrodia elata prevented apoptosis of serum-deprived PC12 cells by suppressing JNK activity..sup.15 In this study, serum-deprived PC12 cells were treated with the compound at the indicated dose for 24 h. Cell viability was monitored by the MTT assay, and is expressed as a percentage of the MTT activity measured in the serum-containing group. At a concentration of 0.01 M, compounds 4 and 6 also rescued PC12 cells from the apoptosis evoked by serum withdrawal equally as well as 1 according to the cell viability of MTT assays. Collectively, the dual function of these compounds in activation of adenosine receptor and in inhibition of adenosine transporter might synergistically increase the effective concentration of adenosine, especially when these two proteins are in proximity.

(25) Statistical Assessment of Pharmacophore Models.

(26) FIG. 4 shows the scatter plot of the experimental pK.sub.i versus predicted pK.sub.i values from the pharmacophore model of A.sub.2AR agonists. The r.sup.2 value of the predicted K.sub.i values versus the experimental K.sub.i values is 0.962, and the root-mean-square of error (rmse) is 0.658 kcal/mol. This pharmacophore model was further evaluated using the Fisher's randomization test for statistical significance, as implemented in the CatScramble module. The CatScrambler module scrambled the pK.sub.i values randomly for 19 times to generate new hypotheses (i.e., pharmacophore models). None of the 19 hypotheses from the scrambled data had a cost lower than the reported hypothesis. Table 2 summarizes the fitted features of the compounds in FIG. 4, along with the distance deviation of the fitted location of the feature on the compound from the center of the feature in the pharmacophore model. To reiterate, Table 2 is a quantitative representation of FIG. 4. When a ligand is fitted into a pharmacophore, the quality of fitting (or mapping) is indicated by the fit value. A higher fit value represents a better fit, and the computer fit values depends on two factors: the weights assigned to the pharmacophore features and how close the features in the molecules are to the exact locations of the features in the pharmacophore model.

(27) TABLE-US-00002 TABLE 2 Comparison of activities of compounds with the fitted number of features of the A.sub.2A-R agonist pharmacophore model. The numbers are in the unit of . Fit Compound ID HBD HBA RA HP Value CGS21680 /0.166 /0.125 /0.201 /0.436 10.6482 1 (T1-11) /0.121 /0.135 /0.248 x 8.68049 NBTI /0.422 /0.304 x /0.901 8.62848 6 /0.377 /0.359 /0.403 x 8.52907 11 /1.181 /0.445 /0.434 /0.544 9.59155

(28) The potent A.sub.2AR agonist CGS fits all the four features of the constructed pharmacophore, and the deviations of the fitted feature locations from the exact locations of the features of the pharmacophore model are also very small. Compared to CGS, compound 1 lacks a hydrophobic moiety to fit into the feature of the pharmacophore model, and therefore exhibited a reduced affinity. In contrast, NBTI lacks a ring-aromatic moiety, which completely abolishes the activity to agonize the adenosine A.sub.2A receptor. This indicates that the ring-aromatic feature may be more important than the hydrophobic moiety in this pharmacophore model. Compound 6 does not fit the hydrophobic feature of the pharmacophore model, while compound 11 does show higher fit value. In contrast, the binding assays indicate that compound 6 (K.sub.i=4.39 M) exhibits 10-fold stronger binding affinity than compound 11 (K.sub.i=41.8 M). This can be rationalized by the fact that all the fitted locations of the features of the compound 6 have less deviations from the exact locations of the features.

(29) The constructed pharmacophore model of the hENT1 inhibitors consists of only three features, namely, a ring aromatic feature and two hydrogen bond acceptors. The r.sup.2 value of the predicted K.sub.i values versus the experimental K.sub.i values is 0.927, and the rmse is 0.85 kcal/mol (FIG. 5). This pharmacophore model was further evaluated by the CatScrambler module. All the five compounds (NBTI, 1, CGS, 6, and 11) can fit into all these three features (FIGS. 3B-F), and therefore the deviations from the exact locations of the features need to be compared (Table 3). Apparently, the most potent inhibitor, NBTI, has the highest fit value and smallest deviation of all three features. The fit values of compounds 1, 11 and 6 are 6.61, 6.4 and 5.86, respectively, which are consistent with the ranking of their measured activity. CGS (with a high fit value 7.1) is obviously an outlier of this model, since this compound has no inhibitory activity toward hENT1. However, CGS is the only compound with a ring aromatic feature fitted on to the nucleoside moiety (FIG. 3).

(30) It is thus important to carefully examine whether the fitted functional group is indeed the same as the functional groups of the training set compounds that define the consensus feature in the pharmacophore analysis. The fit value alone can not be considered the measure of fitness.

(31) TABLE-US-00003 TABLE 3 Comparison of activities of compounds with the fitted number of features of the ENT1 inhibitor pharmacophore model. The numbers are in the unit of . Fit Compound ID HBD HBA RA Value NBTI /0.421 /0.647 /0.793 5.92748 1 /0.544 /0.619 /0.586 4.94133 CGS21680 /0.567 /1.276 /0.358 5.37611 6 /0.421 /0.647 /0.493 5.40138 11 /0.74 /0.588 /0.942 4.35311

CONCLUSION

(32) We have adopted a dual-pharmacophore modeling approach to design dual-action compounds targeting the A.sub.2AR signaling system. Based on the structural scaffold of 1, we designed and synthesized a series of adenosine derivatives and carried out chemical modifications of adenosine if the pharmacophore fitting of the modified compound predicts acceptable activity. The competitive ligand binding assays verified that the designed compounds indeed bind to both A.sub.2AR and ENT1 with moderate affinity. The effective amount of the designed compounds for therapeutic treatment of neurodegenerative diseases, including Huntington's disease, is 1.5-2.5 mg/kg, based on oral dosage of the representative T1-11 in mice. The preferred route of administration of the designed compounds is oral administration, either in immediate release or slow release forms. Finally, these compounds were shown to prevent apoptosis of the serum-deprived PC12 cells, which is a crucial indication for their potential for treating neurodegenerative diseases.

Experimental Section

(33) Materials and Methods.

(34) All reagents and solvents were of reagents grade and were used without further purification unless otherwise specified. Tetrahydrofuran and diethyl ether were distilled from Na/benzophenone and CH.sub.2Cl.sub.2 was distilled from CaH.sub.2. All air or moisture sensitive experiments were performed under argon. All glasses were dried in oven for more than 2 hours and used after cooling to room temperature in desiccators. Microwave reactions were conducted using a focused single mode microwave unit (CEM Discover). The machine consists of a continuous focused microwave power delivery system with operator selectable power output.

(35) Melting points were recorded on a Yanaco micro apparatus. Optical rotations were measured on digital polarimeter of Japan JASCO Co. DIP-1000. [].sub.D values are given in units of 10.sup.1 deg cm.sup.2 g.sup.1. Infrared (IR) spectra were recorded on Nicolet Magna 550-II. NMR spectra were obtained on Varian Unity Plus-400 (400 MHz) and chemical shifts () were recorded in parts per million (ppm) relative to .sub.H 7.24/.sub.C 77.0 (central line of t) for CHCl.sub.3/CDCl.sub.3, .sub.H 2.05/.sub.C 29.92 for CH.sub.3).sub.2CO/(CD).sub.2CO, .sub.H 3.31/.sub.C 49.0 for CH.sub.3OH/CD.sub.3OD, and .sub.H 2.49 (m)/.sub.C 39.5 (m) for (CH.sub.3).sub.2SO/(CD.sub.3).sub.2SO. The splitting patterns are reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad). Coupling constants (J) are given in Hz. The ESI-MS experiments were conducted on a Bruker Daltonics BioTOF III high-resolution mass spectrometer. Analytical thin-layer chromatography (TLC) was performed on E. Merck silica gel 60 F.sub.254 plates (0.25 mm). Compounds were visualized by UV, anisaldehyde or ninhydrin spray. Column chromatography was carried out on columns packed with 70-230 mesh silica gel. Purity of compounds tested on A.sub.2A-R and ENT1 was assessed to be 295% by HPLC (Agilent HP-1100) with detection at 280 nm wavelength.

(36) Construction of Pharmacophore Models.

(37) The HypoGen module of Catalyst of Accelrys was used to construct the pharmacophore model of human A.sub.2AR agonists and human ENT 1 inhibitors. The chemical structures of the training set compounds and their binding affinities to the human A.sub.2AR (or human ENT 1) were collected from the literature (see Supporting information)..sup.41-44 It is important to ensure that the activity of the training compounds cover at least four orders of magnitude, with at least three compounds in each log scale..sup.23,45 It is also recommended to select compounds with larger chemical diversity as the training set..sup.45 Each compound was sketched with ChemDraw, and then imported into Catalyst 4.11. The Best option was used for conformation generation. In Catalyst, the Poling algorithm was used to generate 250 conformations, whose energies are less than 20.0 kcal/mol from the lowest energy of all the conformations. Five molecular features were selected, namely, hydrophobic (HPh), hydrogen bond acceptor (HBA), hydrogen bond donor (HBD), positively ionizable atom (PI), and negatively ionizable atom (NI). All these compounds were loaded into the Catalyst's spreadsheet and the default uncertainty of 3 were assigned. All other parameters are as default.

N6-(4-Hydroxybenzyl)adenosine (1).9

(38) Hydroxylamine hydrochloride (1.29 g, 18.6 mmol) and sodium acetate (1.67 g, 20.4 mmol) were added to a solution of 4-hydroxybenzaldehyde (1.25 g, 10.2 mmol) in ethanol (20 mL). The reaction mixture was stirred at zoom temperature for 6 h. Ethanol was removed under reduced pressure. The residue was added water, and then extracted with Et.sub.2O (3). The combined organic layer was dried over MgSO.sub.4. After the volatiles were removed by rotary evaporation under reduced pressure, the residue was recrystallized from CH.sub.2Cl.sub.2 to give the oxime of 4-hydroxybenzaldehyde (1.3 g, 93%). C.sub.2H.sub.7NO.sub.2; light yellow solid, mp 92.0-93.6 C.

(39) A solution of the above-prepared oxime (342 mg, 2.5 mmol) and concentrated hydrochloric acid (1 mL) in ethanol. (20 mL) was subjected to hydrogenation at atmospheric pressure in the presence of 10% Pd/C (80 mg) for 4 h. The reaction mixture was filtered through Celite. The filtrate was concentrated to yield the hydrochloric salt of 4-hydroxybenzylamine as light yellow solids, which were used for the next step without further purification.

(40) A mixture of 4-hydroxybenzylamine (395 mg, as the hydrochloric salt), 6-chloropurine riboside (143 mg, 0.5 mmol), and diisopropylethylamine (DIEA, 2 mL, 12 mmol) in 1-propanol (25 mL) was heated to 70 C. for 6 h. The mixture was concentrated under reduced pressure, and triturated with water to give white precipitates, which were filtered to yield the title compound 1 (151 mg, 81%). The purity of product was >99% as shown by HPLC on an Inertsil ODS-3 column (4.6250 mm, 5 m) with elution of 0.1% TFA/MeOH (6:4). C.sub.7H.sub.19N.sub.5O.sub.9; white powder, mp 208.7-209.2 C. (lit..sup.9 mp 216-219 C.); [].sup.20.sub.D=64.5 (DMSO, c=1) (lit..sup.9 [].sup.25.sub.D=87 (MeOH, c=0.1)); TLC (MeOH/EtOAc (1:9)) R.sub.f=0.3; .sup.1H NMR (DMSO-d.sub.6, 400 MHz) 9.22 (1H, s), 8.34 (1H, s), 8.30 (1H, br s), 8.18 (1H, s), 7.12 (2H, d, J=8.0 Hz), 6.65 (2H, d, J=8.0 Hz), 5.86 (1H, d, J=5.6 Hz), 5.41 (2H, m), 5.18 (1H, d, J=5.6 Hz), 4.60 (2H, m), 4.13 (1H, q, J=4.6, 7.4 Hz), 3.95 (1H, q, J=3.4, 6.2 Hz), 3.66 (1H, m), 3.53 (1H, m); .sup.13C NMR (DMSO-d.sub.6, 400 MHz) 155.3, 153.6, 151.6, 147.6, 139.1, 129.5, 127.9 (2), 119.2, 114.4 (2), 87.6, 85.6, 73.3, 70.5, 61.5, 42.4; ESI-HRMS calcd for C.sub.17H.sub.20N.sub.5O.sub.5: 374.1459. found: m/z 374.1412 [M+H].sup.+.

N6-(3-Indolylethyl)adenosine (6)

(41) In a round-bottomed flask (10 mL) were placed a solution of 6-chloropurine ribonucleoside (71 mg, 0.25 mmol), trypamine (101 mg, 0.63 mmol) and diisopropylethylamine (0.24 mL, 2.88 mmol) in EtOH (3 mL). The flask was placed into the cavity of a focused monomode microwave reactor, and irradiated at 150 W for 10 min in refluxing EtOH. The mixture was concentrated by rotary evaporation, and the residue was purified by flash chromatography (silica gel; MeOH/EtOAc (1:9)) to give the title compound 6 (85 mg, 83%). The purity of product was >99% as shown by HPLC on an HC-C18 column (Agilent, 4.6250 mm, 5 m) with elution of gradients of 30-60% aqueous CH.sub.3CN. C.sub.20H.sub.22N.sub.6O.sub.4; white powder; mp 187.0-187.2 C.; [].sup.20.sub.D=55.7 (CH.sub.3OH, c=1.0); TLC (MeOH/EtOAc (1:9)) R.sub.f=0.41; .sup.1H NMR (DMSO-d.sub.6, 400 MHz) 10.78 (1H, s), 8.33 (1H, s), 8.25 (1H, s), 7.96 (1H, br s), 7.61 (1H, d, J=7.2 Hz), 7.32 (1H, d, J=9.2 Hz), 7.18 (1H, s), 7.05 (1H, t, J=8.0 Hz), 5.80 (1H, d, J=6.0 Hz), 5.47-5.44 (2H, m), 5.20 (1H, d, J=4.4 Hz), 4.61 (1H, d, J=5.6 Hz), 4.14 (1H, d, J=2.8 Hz), 3.96 (1H, d, J=3.2 Hz), 3.77 (1H, br s), 3.69-3.52 (2H, m), 3.01 (2H, t, J=7.2 Hz); .sup.13C NMR (DMSO-d.sub.6, 100 MHz) 154.3, 152.2, 148.0, 139.5, 136.0, 127.1, 122.4, 120.8, 119.6, 118.3, 118.1, 111.7, 111.2, 87.9, 85.8, 73.4, 70.6, 61.6, 40.5, 25.1; ESI-HRMS calcd for C.sub.20H.sub.23N.sub.5O.sub.4: 411.1775. found: m/z 411.1750 [M+H].sup.+.

5-Azido-5-deoxy-2,3-O-isopropylidene-N6-(4-methoxybenzyl)adenosine (8-actonide)

(42) To the acetonide of N.sup.6-(4-methoxybenzyl) adenosine (3-acetonide, 2.96 g, 6.9 mmol) in anhydrous pyridine (36 mL) was added a solution of p-toluenesulfonyl chloride (6.3 g, 34.6 mmol) in anhydrous pyridine (6.0 mL) dropwise via syringe to a solution of. The mixture was stirred at room temperature for 6 h. Pyridine was removed under reduced pressure, and the residue was extracted with CH.sub.2Cl.sub.2 and H.sub.2O. The organic layer was dried over MgSO.sub.4, filtered, and concentrated to give a mixture of sulfonate and the chloride derivatives (5:1) as shown by the .sup.1H NMR spectrum.

(43) The above-prepared mixture was dissolved in anhydrous DMF (70 mL), and sodium azide (1.34 g, 20.6 mmol) was added. The mixture was stirred at 80 C. for 6 h, and then concentrated under reduced pressure. The residue was extracted with CH.sub.2Cl.sub.2 and H.sub.2O. The organic layer was dried over MgSO.sub.4, filtered, and concentrated to give a pale yellow oil, which was purified by flash chromatography (silica gel; CH.sub.2Cl.sub.2/MeOH (100:1)) to give 8-acetonide (653 mg, 21% overall yield). C.sub.21H.sub.24N.sub.8O.sub.4; colorless oil; TLC (EtOAc/Hexane (6:4)) R.sub.f=0.39; [].sup.23.sub.D=+5.0 (EtOAc, c=1.0); IR .sub.max (neat) 3280, 2987, 2931, 2101, 1618, 1512, 1478, 1375, 1330, 1296, 1218, 1211, 1154, 1091 cm.sup.1; .sup.1H NMR (CDCl.sub.3, 400 MHz) 8.38 (1H, br s), 7.72 (1H, br s), 7.26 (2H, d, J=8.8 Hz), 6.84 (2H, d, J=8.8 Hz), 6.37 (1H, br s), 6.06 (1H, d, J=2.0 Hz), 5.46-5.44 (1H, m), 5.07-5.05 (1H, m), 4.77 (2H, br s), 4.38-4.35 (1H, m), 3.77 (3H, s), 3.51-3.62 (2H, m), 1.61 (3H, s), 1.39 (3H, s); .sup.13C NMR (CDCl.sub.3, 100 MHz) 158.7, 154.5, 153.1, 147.9, 139.0, 130.2, 128.8 (2), 120.1, 114.4, 113.8 (2), 90.4, 85.6, 83.9, 82.0, 55.2, 52.2, 43.7, 29.7, 27.1, 25.3; ESI-HRMS calcd for C.sub.21H.sub.24N.sub.8O.sub.4: 453.1999. found: m/z 453.1999 [M+H].sup.+.

5-Acetamido-5-deoxy-N6-(4-methoxybenzyl)adenosine (9)

(44) The azido compound 8-acetonide (95 mg, 0.21 mmol) was stirred with triphenylphosphine (66 mg, 0.24 mmol) in THF/H.sub.2O (10:1, 2 mL) at room temperature for 4.5 h. The mixture was concentrated under reduced pressure. The residue was taken up with CH.sub.2Cl.sub.2 and H.sub.2O, and acidified with HCl solution (1 M) until pH=2. The aqueous phase was separated, neutralized with saturated NaHCO.sub.3 aqueous solution, and extracted with CH.sub.2Cl.sub.2. The organic extract was dried over MgSO.sub.4, filtered, and concentrated to yield a crude amine product.

(45) The crude amine was treated with acetic anhydride (98.6 L, 1.05 mmol) in anhydrous pyridine (0.2 mL). The mixture was stirred at room temperature for 1.5 h, and then concentrated under reduced pressure. The residue was extracted with CH.sub.2Cl.sub.2 and H.sub.2O. The organic layer was dried over MgSO.sub.4, filtered, concentrated, and purified by flash chromatography (silica gel; CH.sub.2Cl.sub.2/MeOH (98:2)) to give the acetonide of compound 9 (56 mg, 57% yield for 2 steps). The purity of product was >99% as shown by HPLC on an HC-C18 column (Agilent, 4.6250 mm, 5 in) with elution of gradients of 30-60% aqueous CH.sub.3CN. C.sub.23H.sub.28N.sub.6O.sub.5; colorless oil; TLC (CH.sub.2Cl.sub.2/MeOH (98:2)) R.sub.f=0.2; [].sup.28.sub.D=146.6 (CHCl.sub.3, c=1.0); IR .sub.max (neat) 3280, 3062, 2989, 2930, 2835, 2358, 1667, 1620, 1513, 1376, 1336, 1296, 1246, 1215, 1096, 1034 cm.sup.1; .sup.1H NMR (CDCl.sub.3, 400 MHz) 8.36-8.38 (2H, m), 7.73 (1H, s), 7.28 (2H, d, J=8.8 Hz), 6.86 (2H, d, J=8.8 Hz), 6.15 (1H, br s), 5.77 (1H, d, J=4.8 Hz), 5.26 (1H, t, J=4.8 Hz), 4.81 (1H, dd, J=4.0, 2.4 Hz), 4.76 (2H, br s), 4.47-4.48 (1H, m), 4.11-4.17 (1H, m), 3.79 (3H, s), 3.24 (1H, d, J=14.4 Hz), 2.15 (3H, s), 1.61 (3H, s), 1.34 (3H, s); .sup.13C NMR (CDCl.sub.3, 100 MHz) 170.5, 158.8, 154.8, 152.7, 147.7, 139.7, 130.0, 128.9 (2), 121.1, 114.6, 113.9 (2), 92.5, 83.3, 82.2, 81.3, 55.3, 43.9, 41.1, 27.6, 25.4, 23.2; ESI-HRMS calcd. for C.sub.23H.sub.28N.sub.6O.sub.5: 469.2190. found m/z 469.2193 [M+H].sup.+.

(46) The acetonide of 9 (17.2 mg, 0.037 mmol) was stirred in 3 M HCl/THF (1:1, 0.1 mL) at room temperature for 14 h, and then neutralized with saturated NaHCO.sub.3 aqueous solution. The mixture was concentrated under reduced pressure, and the residue was extracted with CH.sub.2Cl.sub.2 and H.sub.2O. The organic layer was dried over MgSO.sub.4, filtered, and concentrated to give the title compound 9 (11 mg, 70%). The purity of product 9 was 99% as shown by HPLC on an HC-C18 column (Agilent, 4.6250 mm, 5 m) with elution of gradients of 30-60% aqueous CH.sub.3CN in 20 min. C.sub.20H.sub.24N.sub.6O.sub.5; white powder; mp 121.1-121.6 C.; TLC (CH.sub.2Cl.sub.2/MeOH (9:1)) R.sub.f=0.5; [].sup.25.sub.D==108.7 (THF, c=0.89); IR .sub.max (neat) 3275, 3071, 2923, 2852, 2360, 1621, 1512, 1375, 1339, 1297, 1245, 1175, 1126, 1076 cm.sup.1; .sup.1H NMR (CDCl.sub.3, 400 MHz) 8.76 (1H, s), 8.27 (1H, s), 7.24 (2H, d, J=8.4 Hz), 6.81 (2H, d, J=8.4 Hz), 6.54 (1H, s), 5.70 (1H, d, J=5.6 Hz), 4.72 (3H, d, J=5.6 Hz), 4.23 (1H, s), 4.18 (1H, s), 3.98-4.03 (1H, m), 3.75 (3H, s), 3.13 (1H, d, J=14.0 Hz), 2.02 (3H, s); .sup.13C NMR (DMSO, 100 MHz) 169.4, 157.9, 154.3, 152.3, 148.3, 140.2, 131.8, 128.4 (2), 119.8, 113.5 (2), 87.9, 83.6, 72.6, 71.3, 55.1, 42.4, 41.1, 22.7; ESI-HRMS calcd for C.sub.26H.sub.24N.sub.6O.sub.5: 427.1730. found: m/z 427.1727 [M+H].sup.+.

5-Deoxy-5-(4-methyl-1,2,3-triazol-1-yl)-N6-(4-methoxybenzyl)adenosine (11)

(47) A mixture of azido compound 8-acetonide (313 mg, 0.69 mmol), CuSO.sub.4.5H.sub.2O (24.9 mg), sodium ascorbate (61.4 mg) and propargyl alcohol in H.sub.2O/t-BuOH (1:1, 7 mL) was stirred at room temperature for 12 h, and then concentrated under reduced pressure. The residue was extracted with CH.sub.2Cl.sub.2 and H.sub.2O. The organic layer was dried over MgSO.sub.4, filtered, and concentrated to yield a triazole acetonide (220 mg) as colorless oil. TLC (CH.sub.2Cl.sub.2/MeOH (9:1)) R.sub.f=0.5; ESI-HRMS calcd for C.sub.24H.sub.26N.sub.8O.sub.5: 509.2261. found: m/z 509.2267 [M+H].sup.+.

(48) The above-prepared triazole compound was stirred with triethylamine (0.15 mL, 1.08 mmol) and methylsulfonyl chloride (0.08 mL, 1.08 mmol) in anhydrous CH.sub.2Cl.sub.2 (4.3 mL) at room temperature for 2 h. The mixture was concentrated under reduced pressure, and the residue was extracted with CH.sub.2Cl.sub.2 and H.sub.2O. The organic layer was dried over MgSO.sub.4, filtered, and concentrated to yield a mesylate compound as colorless oil. TLC (EtOAc/Hex (4:1)) R.sub.f=0.45; ESI-HRMS calcd for C.sub.25H.sub.30N.sub.6O.sub.7SNa: 609.1856. found: m/z 609.1876 [M+Na].sup.+.

(49) The mesylate was treated with NaBH.sub.4 (24.5 mg, 0.65 mmol) at 0 C. in DMF, and then heated to 60 C. for 6 h. The mixture was concentrated under reduced pressure, and the residue was extracted with CH.sub.2Cl.sub.2 and H.sub.2O. The organic layer was dried over MgSO.sub.4, filtered, and concentrated to give 11-acetonide as colorless oil. TLC (EtOAc/Hex (4:1)) R.sub.f=0.25; ESI-HRMS calcd for C.sub.24H.sub.28N.sub.8O.sub.4: 493.2312. found: m/z 493.2312 [M+H].sup.+.

(50) The acetonide of 11 was stirred in 3 M HCl/THF (1:1, 0.33 mL) at room temperature for 14 h, and then neutralized with saturated NaHCO.sub.3 aqueous solution. The mixture was concentrated under reduced pressure, and the residue was dissolved in THF, filtered, and concentrated to give the title compound 11 (48.1 mg, 25% overall yield). The purity of product was 98% as shown by HPLC on an HC-C18 column (Agilent, 4.6250 mm, 5 m) with elution of gradients of 30-60% aqueous CH.sub.3CN. C.sub.21H.sub.24N.sub.6O.sub.4; white powder; mp 183.0-183.2 C.; TLC (CH.sub.2Cl.sub.2/MeOH (9:1)) R.sub.f=0.12; [].sup.27.sub.D+20.3 (CH.sub.3OH, c=0.45); IR .sub.max (neat) 3217, 2921, 2850, 2685, 1620, 1513, 1470, 1337, 1297, 1244, 1176, 1111, 1058 cm.sup.1; .sup.1H NMR (CD.sub.3OD, 400 MHz) 8.22 (1H, s), 7.99 (1H, s), 7.45 (1H, s), 7.31 (2H, d, J=8.8 Hz), 6.87 (2H, d, J=8.8 Hz), 5.96 (1H, d, J=4.0 Hz), 4.82-4.68 (5H, m), 4.46 (1H, t, J=4.0 Hz), 4.34 (1H, q, J=4.0 Hz), 3.77 (3H, s), 2.15 (3H, s); .sup.13C NMR (CDCl.sub.3, 100 MHz) 158.7, 154.2, 152.9, 148.0, 142.8, 138.8, 130.1, 128.8 (2), 123.1, 119.6, 113.8 (2), 89.3, 82.2, 73.4, 70.8, 55.2, 50.9, 43.9, 10.5; ESI-HRMS (negative mode) calcd for C.sub.21H.sub.24N.sub.8O.sub.4: 451.1842. found: m/z 451.1843 [MH].sup..

3,4-Dihydroxy-5-[6-(4-hydroxybenzylamino)-purin-9-yl]-tetrahydrofuran-2-carboxylic Acid Ethylamide (15)

(51) The acetonide derived from 6-chloropurine ribofuranoside (17-acetonide, 158 mg, 0.48 mmol) was stirred with PhI(OAc).sub.2 (509 mg, 1.56 mmol) and 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO, 15.4 mg, 0.1 mmol) in a degassed CH.sub.3CN/H.sub.2O solution (1:1, 2.6 mL) at 40 C. for 4 h. The mixture was concentrated under reduced pressure to yield a crude acid product 18.

(52) The crude acid was treated with ethylamine (117 mg, as the hydrochloric salt), O-(benzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (HBTU, 375 mg, 0.72 mmol) and diisopropylethylamine (0.5 ml, 2.89 mmol) in anhydrous DMF (11.6 mL) at room temperature for 14 h. The mixture was concentrated under reduced pressure. The residue was extracted with CH.sub.2Cl.sub.2 and H.sub.2O. The organic layer was dried over MgSO.sub.4, filtered, concentrated, and purified by flash chromatography (silica gel; EtOAc/hexane (1:1)) to yield an amide product 19 as colorless oil. The amide product was treated with 4-hydroxybenzylamine (385 mg, 2.4 mmol as the hydrochloric salt) and diisopropylethylamine (2.8 mL, 16.9 mmol) in 1-propanol (28 mL) at 70 C. for 2 h. The mixture was concentrated under reduced pressure, and the residue was extracted with CH.sub.2Cl.sub.2 and H.sub.2O. The organic layer was dried over MgSO.sub.4, filtered, concentrated, and purified by flash chromatography (silica gel; CH.sub.2Cl.sub.2/MeOH (97:3)) to give 15-acetonide (179 mg, 82%). C.sub.23H.sub.23N.sub.6O.sub.5; colorless oil; TLC (EtOAc/hexane (4:1)) R.sub.f=0.13; [].sup.22.sub.D=32.0 (EtOAc, c=1.0); IR .sub.max (neat) 3347, 3103, 2982, 2933, 1732, 1667, 1615, 1516, 1479, 1461, 1376, 1332, 1295, 1245, 1212, 1154, 1088 cm.sup.1; .sup.1H NMR (CDCl.sub.3, 400 MHz) 9.01 (1H, br s), 8.35 (1H, br s), 7.81 (1H, s), 7.14 (1H, br s), 7.04 (2H, d, J=8.0 Hz), 6.68 (2H, d, J=8.0 Hz), 6.49 (1H, t, 4.8 Hz), 6.03 (1H, d, J=2.4 Hz), 5.33-5.38 (2H, m), 4.70 (3H, s), 3.09-3.16 (2H, m), 1.62 (3H, s), 1.37 (3H, s), 0.90 (3H, t, J=7.2 Hz); .sup.13C NMR (CDCl.sub.3, 100 MHz) 168.7, 155.8, 154.2, 153.1, 147.7, 139.1, 128.8 (3), 119.6, 115.4 (2), 114.3, 91.6, 85.6, 83.3, 82.4, 43.9, 34.0, 27.0, 25.1, 14.2; ESI-HRMS calcd for C.sub.22H.sub.26N.sub.6O.sub.5: 455.2043. found: m/z 455.2037 [M+H].sup.+.

(53) The acetonide of 15 (26 mg, 0.057 mmol) was stirred in 1 M HCl/THE (1:1, 0.3 mL) at room temperature for 16 h, and then neutralized with saturated NaHCO.sub.3 aqueous solution. After concentration, the residue was triturated with H.sub.2O to give the title compound 15, which was then recrystallized from MeOH (14.65 mg, 62%). C.sub.19H.sub.22N.sub.6O.sub.5; white powder, mp 179.7-180.5 C.; TLC (EtOAc) R.sub.f=0.04; [].sup.23.sub.D=27.7 (MeOH, c=1.0); IR .sub.max, (neat) 3256, 2688, 2360, 1618, 1515, 1335, 1294, 1232, 1128, 1052 cm.sup.1; .sup.1H NMR (CD.sub.3OD, 400 MHz) 8.29 (1H, s), 8.22 (1H, s), 7.20 (2H, d, J=8.4 Hz), 6.73 (2H, d, J=8.4 Hz), 6.00 (1H, d, J=7.6 Hz), 4.76-4.73 (1H, m), 4.70 (2H, br s), 4.46 (1H, s), 4.30-4.31 (1H, m), 3.36 (2H, q, 7.2 Hz), 1.21 (3H, t, 7.2 Hz); .sup.13C NMR (CD.sub.3OD, 100 MHz) 171.8, 157.5, 155.8, 153.6, 149.1, 141.9, 130.5, 129.9 (2), 121.3, 116.2 (2), 90.5, 86.3, 74.9, 73.4, 44.9, 35.2, 15.3; ESI-HRMS calcd for CF.sub.19H.sub.21N.sub.6O.sub.5: 413.1573. found: m/z 413.1573 [MH].sup.+. Anal. Calcd for C.sub.19H.sub.22N.sub.6O.sub.5.H.sub.2O: C, 52.77; H, 5.59; N, 19.43. found: C, 52.88; H, 5.40; N, 19.44.

2-[6-(4-Methoxybenzylamino)-purin-9-yl]-5-(1H-tetrazol-5-yl)-tetrahydrofuran-3,4-diol (16)

(54) The crude acid 18 obtained from oxidation of 17-acetonide (ca. 3.98 mmol) with PhI(OAc).sub.2/TEMPO was treated with ammonium chloride (426 mg, 7.96 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, 3.07 g, 5.97 mmol), hydroxybenzotriazole (HOBt, 807 mg, 5.97 mmol) and diisopropylethylamine (2.5 mL, 15.9 mmol) in anhydrous DMF (40 mL) at 50 C. for 14 h. The mixture was concentrated under reduced pressure. The residue was extracted with CH.sub.2Cl.sub.2 and H.sub.2O. The organic layer was dried over MgSO.sub.4, filtered, concentrated, and purified by flash chromatography (silica gel; EtOAc/hexane (1:1 to 4:1)) to yield an amide product 20 as colorless oil.

(55) A solution of dimethyl sulfoxide (0.85 mL, 11.9 mmol) in CH.sub.2Cl.sub.2 (10 mL) was added to a solution of oxalyl chloride (0.7 mL, 7.96 mmol) in CH.sub.2Cl.sub.2 (10 mL) at 78 C. The mixture was stirred for 30 min, and a solution of amide 20 (ca. 3.98 mmol) in CH.sub.2Cl.sub.2 (20 mL) was added. The mixture was stirred at 78 C. for another 30 min, and diisopropylethylamine (2.6 mL, 15.9 mmol) was added. After 1 h stirring, formation of nitrile 21 was monitored by TLC analysis. The mixture was extracted with CH.sub.2Cl.sub.2 and H.sub.2O. The organic phase was dried over MgSO.sub.4, filtered, concentrated, and purified by flash chromatography (silica gel; EtOAc/hexane (2:3)) to yield nitrile 21 as colorless oil (863 mg) contaminated with a small amount of HOBt.

(56) The above-prepared nitrile product (863 mg, 2.68 mmol) was treated with 4-methoxybenzylamine (1.84 g, 13.4 mmol) and diisopropylethylamine (15.5 mL) in 1-propanol (26 mL) at 70 C. for 4 h. The mixture was concentrated under reduced pressure, and the residue was extracted with CH.sub.2Cl.sub.2 and H.sub.2O. The organic layer was dried over MgSO.sub.4, filtered, concentrated, and purified by flash chromatography (silica gel; CH.sub.2Cl.sub.2/MeOH (300:1 to 150:1)) to give compound 22 (905 mg, 54% overall yield). C.sub.21H.sub.22NO.sub.4; colorless oil; TLC (EtOAc/hexane (4:1)) R.sub.f=0.55; [].sup.26.sub.D=+25.8 (EtOAc, c=1.0); IR .sub.max (neat) 3373, 3282, 2990, 2925, 2853, 1679, 1618, 1.512, 1465, 1376, 1331, 1295, 1249, 1212, 1135, 1086 cm.sup.1; .sup.1H NMR (CDCl.sub.3, 400 MHz) 8.39 (1H, br s), 7.64 (1H, br s), 7.26 (2H, d, J=10.4 Hz), 6.83 (2H, d, J=10.4 Hz), 6.54 (1H, t, J=5.6 Hz), 6.13 (1H, s), 5.77 (1H, d, J=4.0 Hz), 5.68 (1H, dd, J=1.6, 4.0 Hz), 4.95 (1H, d, J=1.6 Hz), 4.75 (2H, br s), 3.79 (3H, s), 1.57 (3H, s), 1.42 (3H, s); .sup.13C NMR (CDCl.sub.3, 100 MHz) 158.8, 154.5, 153.2, 148.2, 138.9, 130.2, 128.9 (2), 119.7, 115.9, 114.5, 113.9 (2), 91.6, 84.6, 83.9, 75.1, 55.3, 44.0, 26.6, 25.1; ESI-HRMS (negative mode) calcd for C.sub.21H.sub.22N.sub.6O.sub.4: 421.1624. found: m/z 421.1612 [MH].sup..

(57) A solution of nitrile 22 (905 mg, 2.14 mmol) and NH.sub.4Cl (429 mg, 8.04 mmol) in DMF (20 mL) was cooled to 0 C., and added NaN.sub.3 (523 mg, 8.04 mmol). The ice bath was removed; the mixture was heated to 40 C. for 1 h, slowly to 90 C., and kept stirring at 90 C. for 9 h. The mixture was cooled, concentrated under reduced pressure, dissolved in EtOAc, and extracted with NaHCO.sub.3 aqueous solution (pH=8). The combined aqueous phase was acidified by addition of HCl solution (1 M) until pH=2, and extracted with CH.sub.2Cl.sub.2. The organic layer was dried over MgSO.sub.4, filtered, and concentrated to give a practically pure tetrazole product 16-acetonide as colorless oil (460 mg, 46% yield). C.sub.23H.sub.23N.sub.3O.sub.4; TLC (CH.sub.2Cl.sub.2/MeOH (9:1)) R.sub.f=0.25; [].sup.27.sub.D=13.2 (EtOAc, c=1.0); IR .sub.max (neat) 3361, 2926, 2852, 1613, 1513, 1481, 1375, 1333, 1293, 1249, 1210, 1176, 1154, 1101, 1034 cm.sup.1, .sup.1H NMR (CDCl.sub.3, 400 MHz) 7.90 (1H, br s), 7.68 (1H, br s), 7.35 (2H, d, J=8.4 Hz), 6.86 (2H, d, J=8.4 Hz), 6.83 (1H, br s), 6.18 (1H, s), 5.85 (1H, s), 5.73 (1H, d, J=6.0 Hz), 5.49 (1H, d, J=6.0 Hz), 4.92 (1H, dd, J=6.8, 7.6 Hz), 4.39 (1H, dd, J=4.0, 10.4 Hz), 3.77 (3H, s), 1.69 (3H, s), 1.43 (3H, s); .sup.13C NMR (CDCl.sub.3, 100 MHz) 158.4, 154.9, 152.9, 152.6, 146.2, 138.5, 129.5, 129.2 (2), 118.4, 114.2, 113.7 (2), 93.4, 85.9, 83.7, 82.3, 55.4, 44.1, 27.1, 25.2; ESI-HRMS (negative mode) calcd for C.sub.21H.sub.23N.sub.9O.sub.4: 464.1795. found: m/z 1786 [MH].sup..

(58) Compound 16-acetonide (460 mg, 0.99 mmol) was stirred in 3 M HCl/THF (1:1, 0.1 mL) at room temperature for 14 h, and then neutralized with saturated NaHCO.sub.3 aqueous solution. The mixture was concentrated under reduced pressure; the residue was taken up with THF, filtered, and concentrated to give the title compound 16 (320 mg, 76%). The purity of product was 99% as shown by HPLC on an HC-C18 column (Agilent, 4.6250 mm, 5 m) with elution of gradients of 30-60% aqueous CH.sub.3CN in 20 min. C.sub.18H.sub.19N.sub.9O.sub.4; white powder; mp 210.0-210.6 C.; TLC (CH.sub.2Cl.sub.2/MeOH (9:1)) R.sub.f=0.05; [].sup.26.sub.D=25.8 (THF, c=1.0); IR .sub.max (neat) 3397, 2841, 2692, 1623, 1511, 1475, 1419, 1339, 1302, 1236, 1180, 1124, 1045 cm.sup.1; .sup.1H NMR (DMSO-d.sub.6, 400 MHz) 8.82 (1H, s), 8.30 (1H, br s), 8.20 (1H, s), 7.26 (2H, d, J=8.0 Hz), 6.83 (2H, d, J=8.0 Hz), 6.08 (1H, d, J=5.6 Hz), 5.53 (1H, d, J=6.0 Hz), 5.46 (1H, d, J=2.8 Hz), 5.18 (1H, s), 4.91 (1H, d, J=5.2 Hz), 4.62 (2H, br s), 4.20 (1H, s), 3.69 (3H, d, J=2.0 Hz); .sup.13C NMR (DMSO-d.sub.6, 100 MHz) 159.9, 157.4, 153.7, 151.9, 138.8, 131.5, 127.9 (2), 113.1 (2), 85.9, 79.1, 75.4, 74.3, 54.6, 41.9; ESI-HRMS (negative mode) calcd for C.sub.18H.sub.19N.sub.9O.sub.4: 427.1730. found: m/z 427.1727 [MH].sup..

(59) Radioligand Binding Assays.

(60) Radioligand binding assays were performed by MDS Pharma Services Taiwan (Taipei, Taiwan) using standard binding protocols. For the binding assay of the A.sub.2A receptor,.sup.46 membrane proteins collected from HEK293 cells overexpressing the human A.sub.2A receptor were incubated in reaction buffer [50 mM Tris-HCl (pH 7.4), 10 mM MgCl.sub.2, 1 mM EDTA, and 2 U/mL adenosine deaminase] containing .sup.3H-CGS21680 (50 nM) for 90 min at 25 C. Nonspecific binding was assessed in the presence of 50 M adenosine-5-N-ethylcarboxamide (NECA).

(61) Binding assays for adenosine transporters were conducted as described earlier..sup.47 Membrane fractions collected from the cerebral cortex of Duncan Hartley derived guinea pigs were incubated with .sup.3H-labeled NBTI (0.5 nM) for 30 min at 25 C. in an incubation buffer containing 50 mM Tris-HCl (pH 7.4). Nonspecific binding was assessed in the presence of 5 M NBTI, a high-affinity inhibitor of equilibrative nucleoside transporter 1 (ENT1), which inhibits only ENT1 at 0.5 nM..sup.48 Reactions were terminated by filtration over GF/B glass fibers and washing with the corresponding reaction buffer.

(62) MTT Metabolism Assay.

(63) PC12 cells purchased from ATCC (Manassas, Va., USA) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% horse serum and 5% fetal bovine serum and incubated in a CO.sub.2 incubator (5%) at 37 C. Survival was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) metabolism assay as described elsewhere..sup.49,50 In brief, cells grown on 150-mm plates were washed three times with PBS and resuspended in DMEM. Suspended cells (110.sup.4 cells) were plated on 96-well plates and treated with or without the indicated reagent. After incubation for 24 h, MTT (0.5 mg/mL) was added to the medium and incubated for 3 h. After discarding the medium, DMSO (100 L) was then applied to the well to dissolve the formazan crystals derived from the mitochondrial cleavage of the tetrazolium ring by live cells. The absorbance at 570/630 nm in each well was measured on a micro-enzyme-linked immunosorbent assay (ELISA) reader.

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