N6-Substituted-5\xe2\x80\xb2-N-Methylcarbamoyl-4\xe2\x80\xb2-selenoadenosines as Potent and Selective A3 Adenosine Receptor Agonists with Unusual Sugar Puckering and Nucleobase Orientation
Potent and selective A3 adenosine receptor (AR) agonists were identified by replacement of 4\xe2\x80\xb2-oxo- or 4\xe2\x80\xb2-thionucleosides with bioisosteric selenium. Unlike previous agonists, 4\xe2\x80\xb2-seleno analogues preferred a glycosidic syn conformation and a South sugar puckering, as shown in the X-ray crystal structure of 5\xe2\x80\xb2-N-methylcarbamoyl derivative 3p. Among compounds tested, N6-3-iodobenzyl analogue 3d was found to be the most potent A3AR full agonist (Ki = 0.57 nM), which was \xe2\x89\xa5 800- and 1900-fold selective for A1 and A2AARs, respectively. In the N6-cycloalkyl series, 2-Cl analogues generally exhibited better hA3AR affinity than 2-H analogues, while 2-H > 2-Cl in the N6-3-halobenzyl series. The N7-isomers 3t and 3u were much weaker in binding than corresponding N9-isomers, but compound 3t lacked A3AR activation, appearing to be a weak antagonist. 2-Cl-N6-3-iodobenzyl analogue 3p inhibited chemoattractant-induced migration of microglia/monocytes without inducing cell death at \xe2\x89\xa4 50 \xce\xbcM. This suggests the potential for the development of 4\xe2\x80\xb2-selenonucleoside A3AR agonists as novel anti-stroke agents.
Introduction<!>Chemistry<!>Conformational analysis<!>Binding affinity<!>Molecular modeling study<!>Biological activity<!>Conclusion<!>General Methods<!>General procedure for the synthesis of 3a-s<!>(2S,3S,4R,5R)-5-(6-((3-Fluorobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydroselenophene-2-carboxamide (3a)<!>(2S,3S,4R,5R)-5-(6-((3-Chlorobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydroselenophene-2-carboxamide (3b)<!>(2S,3S,4R,5R)-5-(6-((3-Bromobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydroselenophene-2-carboxamide (3c)<!>(2S,3S,4R,5R)-3,4-Dihydroxy-5-(6-((3-iodobenzyl)amino)-9H-purin-9-yl)-N-methyltetrahydroselenophene-2-carboxamide (3d)<!>(2S,3S,4R,5R)-3,4-Dihydroxy-5-(6-((2-methoxybenzyl)amino)-9H-purin-9-yl)-N-methyltetrahydroselenophene-2-carboxamide (3e)<!>(2S,3S,4R,5R)-5-(6-((2-Chlorobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydroselenophene-2-carboxamide (3f)<!>(2S,3S,4R,5R)-3,4-Dihydroxy-N-methyl-5-(6-((naphthalen-1-ylmethyl)amino)-9H-purin-9-yl)tetrahydroselenophene-2-carboxamide (3g)<!>(2S,3S,4R,5R)-3,4-Dihydroxy-N-methyl-5-(6-(((1R,2R)-2-phenylcyclopropyl)amino)-9H-purin-9-yl)tetrahydroselenophene-2-carboxamide (3h)<!>(2S,3S,4R,5R)-5-(6-(Cyclopropylamino)-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydroselenophene-2-carboxamide (3i)<!>(2S,3S,4R,5R)-5-(6-(Cyclobutylamino)-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydroselenophene-2-carboxamide (3j)<!>(2S,3S,4R,5R)-5-(6-(Cyclopentylamino)-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydroselenophene-2-carboxamide (3k)<!>(2S,3S,4R,5R)-3,4-Dihydroxy-N-methyl-5-(6-(methylamino)-9H-purin-9-yl)tetrahydroselenophene-2-carboxamide (3l)<!>(2S,3S,4R,5R)-5-(2-Chloro-6-((3-fluorobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydroselenophene-2-carboxamide (3m)<!>(2S,3S,4R,5R)-5-(2-Chloro-6-((3-chlorobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydroselenophene-2-carboxamide (3n)<!>(2S,3S,4R,5R)-5-(6-((3-Bromobenzyl)amino)-2-chloro-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydroselenophene-2-carboxamide (3o)<!>(2S,3S,4R,5R)-5-(2-Chloro-6-((3-iodobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydroselenophene-2-carboxamide (3p)<!>(2S,3S,4R,5R)-5-(2-Chloro-6-(cyclopropylamino)-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydroselenophene-2-carboxamide (3q)<!>(2S,3S,4R,5R)-5-(2-Chloro-6-(cyclobutylamino)-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydroselenophene-2-carboxamide (3r)<!>(2S,3S,4R,5R)-5-(2-Chloro-6-(cyclopentylamino)-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydroselenophene-2-carboxamide (3s)<!>(2S,3S,4R,5R)-3,4-Dihydroxy-5-(6-((3-iodobenzyl)amino)-7H-purin-7-yl)-N-methyl tetrahydroselenophene-2-carboxamide (3t)<!>(2S,3S,4R,5R)-5-(2-Chloro-6-((3-iodobenzyl)amino)-7H-purin-7-yl)-3,4-dihydroxy-N-methyltetrahydroselenophene-2-carboxamide (3u)<!>9-[6-(tert-Butyl-diphenyl-silanyloxymethyl)-2,2-dimethyl-tetrahydro-1,3-dioxa-5-selena-pentalen-4-yl]-6-chloro-9H-purine (6a) and its N7-isomer 6b<!>Compound 6a<!>Compound 6b<!>9-[6-(tert-Butyl-diphenyl-silanyloxymethyl)-2,2-dimethyl-tetrahydro-1,3-dioxa-5-selena-pentalen-4-yl]-2,6-dichloro-9H-purine (7a) and its N7-isomer 7b<!>Compound 7a<!>Compound 7b<!>Conversion of 6b to 6a<!>Conversion of 7b to 7a<!>((3aS,4R,6R,6aR)-6-(6-Chloro-9H-purin-9-yl)-2,2-dimethyltetrahydroselenopheno[3,4-d][1,3]dioxol-4-yl)methanol (8a)<!>((3aS,4R,6R,6aR)-6-(2,6-dichloro-9H-purin-9-yl)-2,2-dimethyltetrahydroselenopheno[3,4 -d][1,3]dioxol-4-yl)methanol (8b)<!>((3aS,4R,6R,6aR)-6-(6-Chloro-9H-purin-9-yl)-2,2-dimethyltetrahydroselenopheno[3,4-d][1,3]dioxol-4-yl)methyl 4-nitrobenzoate (9a)<!>((3aS,4R,6R,6aR)-6-(2,6-Dichloro-9H-purin-9-yl)-2,2-dimethyltetrahydroselenopheno [3,4-d][1,3]dioxol-4-yl)methyl 4-nitrobenzoate (9b)<!>((2R,3S,4R,5R)-5-(6-Chloro-9H-purin-9-yl)-3,4-dihydroxytetrahydroselenophen-2-yl)methyl 4-nitrobenzoate (10a)<!>((2R,3S,4R,5R)-5-(2,6-Dichloro-9H-purin-9-yl)-3,4-dihydroxytetrahydroselenophen-2-yl)methyl 4-nitrobenzoate (10b)<!>((2R,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(6-chloro-9H-purin-9-yl) tetrahydroselenophen-2-yl)methyl 4-nitrobenzoate (11a)<!>((2R,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(2,6-dichloro-9H-purin-9-yl)tetrahydroselenophen-2-yl)methyl 4-nitrobenzoate (11b)<!>((2R,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(6-chloro-9H-purin-9-yl)tetra hydroselenophen-2-yl)methanol (12a)<!>((2R,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(2,6-dichloro-9H-purin-9-yl)tetra hydroselenophen-2-yl)methanol (12b)<!>(2S,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(6-chloro-9H-purin-9-yl)tetra hydroselenophene-2-carbaldehyde (13a)<!>(2S,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(2,6-dichloro-9H-purin-9-yl)tetra hydroselenophene-2-carbaldehyde (13b)<!>(2S,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(6-chloro-9H-purin-9-yl)-N-methyltetrahydroselenophene-2-carboxamide (14a)<!>(2S,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(2,6-dichloro-9H-purin-9-yl)-N-methyltetrahydroselenophene-2-carboxamide (14b)<!>(2S,3S,4R,5R)-5-(6-Chloro-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydroseleno phene-2-carboxamide (15a)<!>(2S,3S,4R,5R)-5-(2,6-Dichloro-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydroseleno phene-2-carboxamide (15b)<!>7-((3aR,4R,6R,6aS)-6-(((tert-Butyldiphenylsilyl)oxy)methyl)-2,2-dimethyltetrahydro selenopheno[3,4-d][1,3]dioxol-4-yl)-N-(3-iodobenzyl)-7H-purin-6-amine (16a)<!>7-((3aR,4R,6R,6aS)-6-(((tert-Butyldiphenylsilyl)oxy)methyl)-2,2-dimethyltetrahydro selenopheno[3,4-d][1,3]dioxol-4-yl)-2-chloro-N-(3-iodobenzyl)-7H-purin-6-amine (16b)<!>((3aR,4R,6R,6aS)-6-(6-((3-Iodobenzyl)amino)-7H-purin-7-yl)-2,2-dimethyltetrahydro selenopheno[3,4-d][1,3]dioxol-4-yl)methanol (17a)<!>((3aR,4R,6R,6aS)-6-(2-Chloro-6-((3-iodobenzyl)amino)-7H-purin-7-yl)-2,2-dimethyltetrahydroselenopheno[3,4-d][1,3]dioxol-4-yl)methanol (17b)<!>((3aR,4R,6R,6aS)-6-(6-((3-Iodobenzyl)amino)-7H-purin-7-yl)-2,2-dimethyltetrahydro selenopheno[3,4-d][1,3]dioxol-4-yl)methyl benzoate (18a)<!>((3aR,4R,6R,6aS)-6-(2-Chloro-6-((3-iodobenzyl)amino)-7H-purin-7-yl)-2,2-dimethyl tetrahydroselenopheno[3,4-d][1,3]dioxol-4-yl)methyl benzoate (18b)<!>((2R,3S,4R,5R)-3,4-Dihydroxy-5-(6-((3-iodobenzyl)amino)-7H-purin-7-yl)tetrahydro selenophen-2-yl)methyl benzoate (19a)<!>((2R,3S,4R,5R)-5-(2-Chloro-6-((3-iodobenzyl)amino)-7H-purin-7-yl)-3,4-dihydroxytetra hydroselenophen-2-yl)methyl benzoate (19b)<!>((2R,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(6-((3-iodobenzyl)amino)-7H-purin-7-yl)tetrahydroselenophen-2-yl)methyl benzoate (20a)<!>((2R,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(2-chloro-6-((3-iodobenzyl) amino)-7H-purin-7-yl)tetrahydroselenophen-2-yl)methyl benzoate (20b)<!>((2R,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(6-((3-iodobenzyl)amino)-7H-purin-7-yl)tetrahydroselenophen-2-yl)methanol (21a)<!>((2R,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(2-chloro-6-((3-iodobenzyl) amino)-7H-purin-7-yl)tetrahydroselenophen-2-yl)methanol (21b)\\<!>(2S,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(6-((3-iodobenzyl)amino)-7H-purin-7-yl)-N-methyltetrahydroselenophene-2-carboxamide (22a)<!>(2S,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(2-chloro-6-((3-iodobenzyl)amino)-7H-purin-7-yl)-N-methyltetrahydroselenophene-2-carboxamide (22b)<!>Cell culture and membrane preparation<!>Binding Assays at the hA1 and hA2AARs<!>Binding Assay at the hA3AR<!>Cyclic AMP accumulation assay<!>Statistical analysis<!>Homology Modeling<!>Protein Preparation<!>Induced-fit docking<!>Chemotaxis assay
<p>Adenosine receptors (ARs), consisting of four subtypes (A1, A2A, A2B, and A3), have continued to be promising targets for the developments of new therapeutic agents.1,2 The pharmacological actions of ARs are initiated upon binding of extracellular adenosine to each receptor subtype, to modulate the levels of second messengers such as adenosine 3′,5′-cyclic phosphate (cAMP), diacylglycerol (DAG), and inositol trisphosphate (IP3), in turn affecting cellular signal transduction.3 Thus, altering an imbalance in activation of ARs can correct abnormal cell signaling that °Ccurs in certain pathological states.4 Therefore, agonists or antagonists that can modulate the AR activity can be developed as novel chemotherapeutic agents.</p><p>A3AR is the most recently identified receptor among the ARs, yet numerous A3AR agonists and antagonists have already been developed as potential therapeutic agents for the treatments of many diseases such as cancer, cardiac ischemia, cerebral ischemia, renal ischemia, glaucoma, and rheumatoid arthritis.5 Among these, 2-chloro-N6-(3-iodobenzyl)-5′-N-methylcarbamoyladenosine (1, Cl-IB-MECA, X = O, R1 = 3-iodobenzyl, R2 = Cl, Ki = 1.4 nM for human (h) A3AR)6 which belong to the 4′-oxonucleosides, is regarded as a first generation A3AR agonist (Figure 1). Compound 1 is in a phase II clinical trial for hepatocellular carcinoma and is projected to enter a clinical trial for nonalcoholic steatohepatitis.7 On the basis of the structure of 1, its bioisosteric 4′-thio analogue 2 (thio-Cl-IB-MECA, X = S, R1 = 3-iodobenzyl, R2 = Cl, Ki = 0.38 nM for A3AR)8 was discovered as the second generation A3AR agonist. Compound 2 exhibited potent biological activities in models of cancer9 and cerebral ischemia10 with novel mechanisms of action.</p><p>On the other hand, it has long been known that the sugar conformation of nucleoside derivatives exists in dynamic equilibrium between North and South conformations, whose energy barrier in the 4′-oxo series is less than roughly 4 Kcal/mol.11 The nucleobase of nucleoside derivatives also exists in either an anti or syn orientation, which is affected by the substitution of the nucleobase. It has been reported that compound 1 prefers the North (3T2) sugar puckering to the South (2T3) sugar puckering in binding to the A3AR, which was confirmed by high affinity A3AR agonist (N)-methanocarba derivative (Ki = 1.5 nM for A3AR) that was fixed in the North (3T2) sugar conformation,12 while South (2T3)-fixed analogues exhibited much lower binding affinity at the A3AR than corresponding North analogues.13 Compound 2 was also reported to show the same sugar puckering as 1.8</p><p>Another structural requirement that is essential for binding to A3AR is the orientation of purine base. Nucleoside analogues with a syn orientation exhibited much lower binding affinity to adenosine receptors, indicating that A3AR agonists should adopt the North (3T2) sugar puckering with an anti nucleobase orientation.13,14</p><p>We have recently reported the syntheses and unusual sugar conformations of 4′-selenonucleosides based on the bioisosteric concept of substituting native oxygen with selenium.15 From these studies, 4′-selenonucleosides showed an opposite South conformation in comparison to the 4′-oxonucleosides adopting a North conformation, probably due to steric effects induced by a bulky selenium atom.16 Thus, it was interesting to design the 4′-selenonucleoside derivatives 3 and to compare their conformation with that of 1 with respect to A3AR agonist activity. It was also interesting to compare the orientation of the purine base of 3 with that of 1, with the previous assumption that an A3AR agonist attained an anti orientation when bound to the receptor. In addition to the desired N9-derivatives 3, we also synthesized the corresponding N7-analogues for comparing binding affinities at the A3AR. Herein, we report the synthesis, binding affinity, and conformational study of 4′-selenonucleoside derivatives 3 as potent and selective A3AR agonists with unusual sugar puckering and nucleobase orientation.</p><!><p>For the synthesis of the final compound 3, the key precursors 6a and 6b were first synthesized from d-ribose as shown in Scheme 1.</p><p>Using a reported procedure17 developed by our laboratory, d-ribose was converted to the 4-selenosugar 4, which was converted to the glycosyl donor 517 by treatment with mCPBA followed by reaction with acetic anhydride at 100 °C. Condensation of 5 with silylated 6-chloropurine and 2,6-dichloropurine in the presence of TMSOTf as a Lewis acid catalyst afforded the β-N9-anomers 6a (49%) and 7a (20%) along with the β-N7-anomer 6b (15%) and 7b (29%), respectively.17 The β-N7-anomers 6b and 7b were smoothly converted to the β-N9-anomers 6a and 7a by heating with TMSOTf in toluene, respectively.17 The anomeric β-configurations of 6a and 7a were readily confirmed by NOE experiments between 1′-H and 4′-H, while the N9- and N7-regioisomers were confirmed by the X-ray crystal structures.17</p><p>The N9-precursors 6a and 7a were converted to the key intermediates 12a and 12b, respectively, as depicted in Scheme 2. The TBDPS protecting groups in 6a and 7a were first changed to the benzoyl (Bz) group for the selective conversion to the 5′-N-methyluronamide, but its removal with base such as NaOMe, methanolic ammonia, NaBH4, or MeMgCl resulted in N6-substitution along with debenzoylation. Thus, a p-nitrobenzoyl (PNB) group was finally selected because it can be removed under milder basic conditions than the Bz group. Compounds 6a and 7a were treated with HF-Et3N in THF to give the 5′-hydroxy compounds 8a and 8b, which were protected with a PNB group to afford 9a and 9b, respectively. The 2′,3′-acetonide protecting groups of 9a and 9b were also changed to the TBS groups because the 2′,3′-acetonide was problematic on removal under acidic conditions at the final stage. Thus, treatment of 9a and 9b with 50% aqueous TFA yielded diols 10a and 10b, which were again protected with TBS groups to afford 11a and 11b, respectively. The PNB groups of 11a and 11b were smoothly removed by treating with 1 N NaOH in 1,4-dioxane to give the 5′-hydroxyl derivatives 12a and 12b, respectively, which are ready for the transformation to the 5′-N-methyluronamide.</p><p>For the conversion of the primary alcohol into the carboxylic acid, compounds 12a and 12b were treated with PDC in DMF, used in the preparations of the corresponding 4′-oxo- and 4′-thionucleosides, but this treatment resulted in extensive decomposition and very low yields. Thus, we decided to synthesize the acid in two steps via the aldehyde. Albright-Goldman oxidation18 of 12a and 12b with DMSO/Ac2O at 100 ° for 1 h produced the aldehydes 13a and 13b, respectively in good yields. Further oxidation of 13a and 13b using Tollens' reagents (Ag(NH3)2(OH)),19 prepared by treating AgNO3, NaOH, and NH4OH, afforded the corresponding acids, which without purification, were treated with HATU and methylamine hydrochloride in the presence of N,N-diisopropylethylamine (DIPEA) to afford the 5′-uronamides 14a and 14b, respectively in good yields. Removals of the TBS groups of 14a and 14b with TBAF or HF-Et3N yielded 15a and 15b, respectively. Compounds 15a and 15b were treated with various amines such as alkylamines and arylalkylamines to afford the final 4′-selenonucleoside derivatives 3a-s.</p><p>The corresponding N7-isomers 3t and 3u were also synthesized as shown in Scheme 4 and binding affinities at the ARs compared. Compounds 6b and 7b were treated with 3-iodobenzylamine in EtOH to give 16a and 16b, respectively. Removal of the TBDPS groups in 16a and 16b with TBAF yielded 17a and 17b, which were benzoylated and successively treated with 88% HCO2H to afford diols 19a and 19b, respectively. Protection of 19a and 19b with TBS groups yielded 20a and 20b, which were treated with NaOMe to give the primary alcohols 21a and 21b, respectively. Oxidation of 21a and 21b with PDC in DMF, followed by the treatment of the crude acid with CH3NH2-HCl in the presence of HOBt and EDC•HCl yielded the 5′-uronamides 22a and 22b, respectively in reasonable yields. It is interesting to note that the N7-isomer was better tolerated under the PDC oxidation conditions than the N9-isomer. Removal of the TBS groups of 22a and 22b with TBAF afforded the final nucleosides 3t and 3u, respectively.</p><!><p>The conformation of a representative synthesized 4′-seleno compound was analyzed using 1H NMR and X-ray crystallography. The X-ray crystal structure of 3p was obtained as illustrated in Figure 2.20</p><p>Surprisingly, compound 3p exhibited a 2′-exo/3′-endo (South) conformation, which is opposite to that of the corresponding 4′-oxo- or 4′-thionucleoside, probably due to the steric effects induced by bulky selenium atom instead of gauche effects.16 Furthermore, 3p exhibited a syn orientation of the purine base, which is not common to the corresponding to 4′-oxo- or 4′-thionucleoside. The three parameters that are essential for the description of the nucleoside conformation were measured from the X-ray structure of 3p. The torsion angle χ (C5-N9-C1′-Se4′), which describes the geometry of the glycosyl bond between the purine base and the sugar ring was 61.92°, indicating a syn conformation. Another torsion angle γ (C3′-C4′-C5′-N6′) describing the rotation about the exocyclic C4′-C5′ bond was -83.77°, indicating that it adopts a –sc orientation. The last parameter is the pseudorotational phase angle P, indicating which atoms are displaced out of the plane and the direction of the displacement. The P value was 0.030, which is the characteristic of C3′-endo South conformation (0 < P < 18°).</p><p>The reason why compound 3p assumes a syn conformation despite the possible steric repulsion of the purine base with the sugar ring might be explained by the following factors: (1) Intramolecular hydrogen bonding between N3 of purine base and the amide NH of N6′.21 (2) The intermolecular π-π stacking interaction between purine ring and other aromatic rings on crystal stacking (Figure 1S). (3) The 'South' sugar puckering.</p><p>1H NMR experiments (Figure 2S) were also performed to confirm the sugar puckering and base orientation of 3p in the solution state. The population (64.5%) of South conformation was preferred over that of North conformation at 25 °C, showing the P and υmax (maximum torsion angle) values were 190.93° and 47.45, respectively. 1H-1H NOESY spectrum of 3p confirmed the syn conformation of the purine base. In summary, compound 3p preferred the South conformation and the syn orientation in the solid as well as in the solution state.</p><p>However, correlation of biological activity with a crystal structure or 1H NMR data must be carefully interpreted because conformational preferences of a molecule are greatly influenced by its environments such as the solid state (X-ray crystal structure), solution (NMR experiments), gas state (theoretical calculations), or binding to a protein.22 The syn crystal structure conformation of a nucleoside obtained in the solid state is just one of the many possible ones, due to the flexibility of the glycosidic bond. It has been also reported that the potent nonselective AR agonist adenosine-5′-N-ethyluronamide (NECA, 23) prefers a syn conformation as determined in NMR experiments, demonstrating that a syn conformation in solution was permissive to potent biological activity.23 However, when bound to the receptor, adenosine adopted an anti conformation, which was confirmed by conformationally restricted analogues12,13 and X-ray crystallography24 of agonist-bound A2AAR complexes. The conformational state of a nucleoside can be also indicated by the chemical shift of the 2′-H in 1H NMR.25 The chemical shifts of 2′-H of the syn conformation nucleosides are approximately 5.2 ppm, while those of the anti conformation are around 4.2 ppm. Adenosine shows an intermediate value of 4.62, suggesting that it rapidly changes its conformation with a slight preference for the anti conformation. For 3p, the chemical shift of 2′-H was found to be close to that of adenosine (4.56 and 4.62, respectively), suggesting that it adopts both conformations in solution, similar to adenosine, although it showed the syn conformation in the solid state.</p><!><p>The binding affinities of all final compounds at the ARs were measured using standard radioligands and membrane preparations.26 The hA1AR or hA3AR was expressed in Chinese hamster ovary (CHO) cells, and the hA2AAR was expressed in human embryonic kidney (HEK)-293 cells. [125I]N6-(3-Iodo-4-aminobenzyl)-5′-N-methylcarboxamidoadenosine (I-AB-MECA) was used to measure the binding affinity at the hA3AR, while [3H](-)-N6-2-phenylisopropyl adenosine (R-PIA) and [3H]2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine) (CGS21680) were used for the hA1AR and hA2AAR, respectively. The percent inhibition of radioligand binding to the hA1AR and hA2AAR at 10 μM was used in cases of weak binding. Nonspecific binding was defined using 10 μM of 23.</p><p>As shown in Table 1, all final N9-4′-selenonucleosides 3a-s produced nanomolar binding affinity with Ki values in the range of 0.5 ∼ 7 nM at the hA3AR subtype with high selectivity to other subtypes hA1 and hA2AARs, among which 3d exhibited the best binding affinity (Ki = 0.57 nM) at the hA3AR. Compound 3d (Ki = 0.57 nM) showed about 3 times higher binding affinity at the hA3AR subtype than the corresponding 4′-oxonucleoside 1a (Ki = 1.8 nM), while exhibiting about 2 times lower binding affinity than the corresponding 4′-thionucleoside 2a (Ki = 0.25 nM). This result is unusual in that these nucleosides 3a-s with a possible South conformation and syn orientation could also act as potent and selective A3AR agonists, because the North conformation and anti orientation have been regarded as the key structural requirements for A3AR agonists. In the N6-cycloalkyl series, 2-Cl analogues 3q-s exhibited better binding affinity at the hA3AR than 2-H analogues 3i-k and the binding affinity at the hA3AR decreased in the following order: cyclobutyl > cyclopropyl > cyclopentyl. In the N6-3-halobenzyl series, 2-H analogues 3a-d generally exhibited higher binding affinity at the hA3AR than 2-Cl analogues 3m-p. The binding affinity at the hA3AR in the 2-H series decreased in the following order: 3-I derivative 3d > 3-Br derivative 3c > 3-F derivative 3a > 3-Cl derivative 3b, while in the 2-Cl series, 3-Cl derivative 3n ≈ 3-Br derivative 3o > 3-F derivative 3m > 3-I derivative 3p. As expected, the corresponding N7-isomers 3t and 3u showed much weaker binding affinity at all three subtypes maybe due to poor hydrophobic interaction of 3-iodobenzyl group in the binding site of hA3AR. From the SAR analysis, it was found that the 4′-selenonucleosides had similar requirements for the A3AR affinity to those of the corresponding 4′-oxo- or 4′-thionucleosides.8,12 For example, a bulky hydrophobic substituent at the N6 position was compatible with high affinity, but with better selectivity to other subtypes, hA1 and hA2AARs. The wide range of steric bulk tolerated at the N6 substituent was indicated by the N6-methyl 3l (Ki = 1.49 nM) and naphthyl 3g (Ki = 1.76 nM) analogues producing similarly high A3AR affinity. It should also be mentioned that despite the high binding affinity at the hA3AR of the 4′-selenonucleosides they adopted the unusual inactive conformations such as South sugar puckering as well as a syn base orientation because of a steric effect induced by the bulky selenium atom. This result demonstrates that they may be converted to the active North and anti conformation in the binding site of the receptor, overwhelming the steric effect caused by the selenium atom.</p><p>At the hA1 and hA2AARs, moderate affinity was observed for many Se analogues, with the most potent at the hA1 being N6-cycloalkyl derivative 3j (Ki = 38 nM) and 3k (Ki = 25 nM). In the N6-cycloalkyl series, the presence of a 2-Cl reduced the hA1AR affinity by ∼2-fold, while the hA2AAR affinity nearly identical. The most potent affinities at the hA2AAR (∼200 nM) were observed for 3g and 3n, which do not share C2 and N6 substituent.</p><p>In a cAMP functional assay26 at the hA3AR expressed in CHO cells, the most potent compound 3d behaved as a potent full agonist, similar to 1a,b and 2a,b. In a full concentration-response curve compound 3d displayed an EC50 value of 2.7 ± 2.2 nM in stimulation of cAMP formation via the hA3AR (Figure 3).</p><p>Compound 3d displayed a maximal hA3AR-mediated inhibition of cAMP formation of 91 ± 16% (n = 3, 10 μM), relative to the full agonist 23 (= 100%) and similarly other compounds proved to be full agonists of the hA3AR. Interestingly, the N7-isomer 3t induced 0.2% of activation seen with full agonist 23, indicating that it was an A3AR antagonist of μM affinity.</p><!><p>2-Chloro-N6-(3-iodobenzyl) analogue 3p exhibited high hA3AR binding affinity (Ki = 4.2 nM), and its X-ray crystal structure showed extraordinary 2′-endo/3′-exo (South) puckering and syn conformation via intra-molecular H-bonding between the side-chain amide and the adenine ring (Figure 4C). To investigate how 3p binds with hA3AR, we first built the homology model of hA3AR using an agonist-bound hA2AAR X-ray crystal structure (PDB id: 3QAK)24 as the template, which was used to perform the induced-fit docking study. As shown in Figures 4A and 4B, 3p bound very well at the orthosteric agonist binding site of hA3AR. The Se-containing sugar ring maintained its 2′-endo/3′-exo (South) puckering and displayed H-bonds with Ser271 and His272. The side chain amide, involved in syn conformation of the glycosidic bond in the X-ray crystal structure of 3p, exhibited H-bonding interaction with Thr94 which was suggested as one of the key residues for agonist activity.29 The adenine ring exhibited π-π interactions with Phe168 and H-bonding with Asn250. In addition, the C2-chlorine showed hydrophobic interactions with Val72, Phe168, and Ile268. The iodobenzene ring displayed hydrophobic interactions with Val169, Ile253, Leu264, Tyr265, and Ile268.</p><p>The docked conformation was superimposed on the X-ray crystal structure of 3p as shown in Figure 4D. Compound 3p preserved its ring puckering as a South form. However, it transformed into an anti conformation by losing the original intra-molecular H-bonding. It appeared that the side chain amide and the adenine ring dramatically moved away to obtain more favorable interactions with binding site residues in hA3AR.</p><p>Thus, the syn conformation in solution or in the pure crystal state of the potent 4′-selenonucleoside 3p appears to be modified when bound to the A3AR in order to maintain conserved H-bonding and hydrophobic contacts of the ribose and adenine moieties with the receptor</p><!><p>We have recently reported that A3AR agonist 2b reduced brain ischemic injury and inhibited inflammatory cell migration in rats with higher potency than 1b.10 In order to compare the inhibition by 2b and 3d of inflammatory cell migration, we examined the effects of these agonists on chemoattractant-induced migration of microglia/monocytes in a chemotaxis chamber (Figure 5). The chemotaxis assay revealed that compound 3d inhibited monocyte chemotactic protein 1 (MCP-1, 100 ng/mL)-induced migration of microglial cells with higher potency than 2b. Furthermore, 3d at concentrations up to 50 μM exhibited much less cytotoxicity than 2b in PrestoBlue cell viability test. These results demonstrate that 4′-selenonucleosides may serve as better candidates than the corresponding 4′-thionucleosides as protective agents in cerebral ischemia.</p><p>We have also examined the cytotoxic effects of the representative A3AR agonists, 3a-d in MRC-9 (lung normal cells).30 All compounds did not show any cytotoxicity up to 20 μM.</p><!><p>We have discovered novel 4′-selenonucleosides 3a-s as potent and selective hA3AR agonists, whose binding affinity at the hA3AR was almost comparable to that of 1 or 2. They were found to adopt an unusual South sugar puckering and syn nucleobase orientation, which were confirmed by X-ray crystallography and 1H NMR analyses. These findings are surprising in that known hA3AR agonists prefer the North conformation and anti nucleobase orientation. Compound 3d exhibited more potent inhibition of MCP-1 induced microglial chemotaxis than 1b or 2b, indicating that 4′-selenonucleosides have a great potential to be developed as novel anti-stroke agents. It is believed that this study opens up a new field in designing new A3AR ligands.</p><!><p>1H and 13C NMR spectra (CDCl3, CD3OD or DMSO-d6) were recorded on Bruker AV 400 (400/100 MHz), Bruker AMX 500 (500/125 MHz), or Jeol JNM-ECA600 (600/150 MHz) instrument. Chemical shifts are reported as parts per million (δ) relative to the solvent peak. Coupling constants (J) are reported in hertz (Hz). Mass spectra were recored on a Thermo LCQ XP instrument. Optical rotations were determined on Jasco III in appropriate solvent. UV spectra were recorded on U-3000 made by Hitachi in methanol or water. Infrared spectra were recorded on FT-IR (FTS-135) made by Bio-Rad. Melting points were determined on a Buchan B-540 instrument and are uncorrected. Elemental analyses (C, H, and N) were used to determine the purity of all synthesized compounds, and the results were within ± 0.4% of the calculated values, confirming ≥ 95% purity. Reactions were checked with TLC (Merck precoated 60F254 plates). Flash column chromatography was performed on silica gel 60 (230-400 mesh, Merck). Unless otherwise noted, materials were obtained from commercial suppliers and were used without purification. All solvents were purified and dried by standard techniques just before use. THF and Et2O were freshly distilled from sodium and benzophenone. Methylene chloride, toluene, and benzene were purified by refluxing with CaH2. Hexanes and ethylacetate were purified by simple distillation.</p><!><p>To a stirred solution of 15a-b (1 equiv) in ethanol were added amine (3 equiv) and triethylamine (6 equiv), and the reaction mixture was stirred at 95 °C for 15 - 30 h. All volatiles was evaporated, and the residue was purified by silica gel column chromatography (dichloromethane : methanol = 100 : 1 - 10 : 1) to give 3a-s as a white solid.</p><!><p>White solid; Yield: 85%; mp 123-130 °C; [α]D20 -19.35 (c 0.34, CH2Cl2+CH3OH); UV (CH2Cl2) λmax 269.0 nm; 1H NMR (400 MHz, CD3OD) δ 8.54 (s, 1 H), 8.29 (s, 1 H), 7.35-7.30 (m, 1 H), 7.20 (d, 1 H, J = 7.6 Hz), 7.11 (d, 1 H, J = 10.0 Hz), 6.97 (td, 1 H, J = 2.6, 8.2 Hz), 6.21 (d, 1 H, J = 6.0 Hz), 4.87 (dd, 1 H, J = 5.4, 7.8 Hz), 4.58 (dd, 1 H, J = 3.4, 4.2 Hz), 4.10 (d, 1 H, J = 4.4 Hz), 2.82 (s, 3 H); 13C NMR (100 MHz, CD3OD) δ 174.4, 153.9, 142.1, 131.3, 131.2, 124.24, 124.21, 115.2, 115.0, 114.9, 114.7, 81.6, 77.8, 56.9, 47.2, 26.9; HRMS (FAB) found 467.0754 [calcd for C18H19FN6O3Se (M+H)+ 467.0746]; Anal. (C18H19FN6O3Se) C, H, N.</p><!><p>White solid; Yield: 99%; mp 219-227 °C; [α]D20 -18.24 (c 0.27, CH2Cl2+CH3OH); UV (CH2Cl2) λmax 269.0 nm; 1H NMR (400 MHz, CD3OD+ CDCl3) δ 8.54 (s, 1 H), 8.29 (s, 1 H), 7.43-7.23 (m, 4 H), 6.21 (d, 1 H, J = 6.0 Hz), 4.89-4.88 (m, 1 H), 4.85 (bs, 1 H), 4.58 (q, 1 H, J = 3.4, 4.2 Hz), 4.10 (d, 1 H, J = 4.0 Hz), 4.06 (s, 1 H), 2.82 (s, 3 H); 13C NMR (100 MHz, CD3OD+ CDCl3) δ 173.1, 155.3, 153.3, 141.5, 135.5, 134.9, 130.4, 129.5, 128.0, 127.9, 126.2, 120.4, 80.8, 78.2, 77.2, 56.4, 47.2, 26.9; HRMS (FAB) found 483.0456 [calcd for C18H19ClN6O3Se (M+H)+ 483.0451]; Anal. (C18H19ClN6O3Se) C, H, N.</p><!><p>White solid; Yield: 79%; mp 252 ° (decomposed); [α]D20 -15.12 (c 0.17, CH2Cl2+CH3OH); UV (CH2Cl2) λmax 269.0 nm; 1H NMR (400 MHz, CD3OD) δ 8.54 (s, 1 H), 8.29 (s, 1 H), 7.56 (s, 1 H), 7.41-7.35 (m, 2 H), 7.24 (t, 1 H, J = 7.6 Hz), 6.21 (d, 1 H, J = 5.6 Hz), 4.89-4.85 (m, 1 H), 4.58 (t, 1 H, J = 3.8 Hz), 4.10 (d, 1 H, J = 4.0 Hz), 2.82 (s, 3 H); 13C NMR (100 MHz, CD3OD+CDCl3) δ 173.0, 155.1, 153.0, 141.6, 132.9, 132.4, 131.3, 130.9, 130.7, 128.0, 126.6, 123.0, 80.8, 78.2, 77.1, 56.4, 47.2, 26.9; HRMS (FAB) found 526.9947 [calcd for C18H19BrN6O3Se (M+H)+ 526.9945]; Anal. (C18H19BrN6O3Se) C, H, N.</p><!><p>White solid; Yield: 80%; mp 180-185 °C; [α]D20 157.50 (c 0.10, CH2Cl2+CH3OH); UV (CH2Cl2) λmax 269.0 nm; 1H NMR (400 MHz, DMSO-d6) δ 8.51 (bs, 1 H), 8.45 (s, 1 H), 8.24 (s, 1 H), 7.72 (s, 1 H), 7.58 (d, 1 H, J = 8.0 Hz), 7.36 (d, 1 H, J = 8.0 Hz), 7.11 (t, 1 H, J = 7.8 Hz), 6.02 (d, 1 H, J = 6.0 Hz), 5.72 (d, 1 H, J = 6.0 Hz), 5.50 (d, 1 H, J = 5.2 Hz), 4.78 (td, 1 H, J = 3.0, 6.0 Hz), 4.66 (bs, 1 H), 4.44 (q, 1 H, J = 4.6, 7.8 Hz), 3.95 (d, 1 H, J = 4.0 Hz), 2.64 (d, 3 H, J = 4.4 Hz); 13C NMR (100 MHz, DMSO-d6) δ 171.6, 154.3, 152.6, 142.9, 140.0, 135.8, 135.5, 130.6, 126.7, 94.8, 79.5, 75.7, 55.2, 45.3, 26.1; HRMS (FAB) found 574.9800 [calcd for C18H19IN6O3Se (M+H)+ 574.9807]; Anal. (C18H19IN6O3Se) C, H, N.</p><!><p>White solid; Yield: 74%; mp 103-108 °C; [α]D20 14.67 (c 0.20, CH3OH); UV (CH2Cl2) λmax 272.0 nm; 1H NMR (400 MHz, CD3OD) δ 8.53 (s, 1 H), 8.28 (s, 1 H), 7.30-7.23 (m, 2 H), 6.99 (d, 1 H, J = 7.6 Hz), 6.88 (dd, 1 H, J = 6.8, 7.2 Hz), 6.20 (d, 1 H, J = 6.0 Hz), 4.58 (dd, 1 H, J = 3.4, 4.2 Hz), 4.10 (d, 1 H, J = 4.4 Hz), 3.89 (s, 3 H), 2.81 (s, 3 H); 13C NMR (300 MHz, CD3OD) δ 175.0, 159.7, 156.8, 154.6, 142.6, 132.8, 132.4, 130.5, 130.4, 122.6, 122.1, 112.6, 112.1, 82.3, 78.6, 57.7, 56.7, 47.9, 27.7; HRMS (FAB) found 479.0945 [calcd for C19H22N6O4Se (M+H)+ 479.0946]; Anal. (C19H22FN6O4Se) C, H, N.</p><!><p>White solid; Yield: 84%; mp 154-160 °C; [α]D20 -35.26 (c 0.58, CH3OH); UV (CH2Cl2) λmax 268.5 nm; 1H NMR (400 MHz, CD3OD) δ 8.55 (s, 1 H), 8.29 (s, 1 H), 7.44-7.41 (m, 2 H), 7.28-7.23 (m, 2 H), 6.21 (d, 1 H, J = 6.0 Hz), 4.92 (bs, 1 H), 4.88 (dd, 2 H, J = 3.2, 6.0 Hz), 4.58 (dd, 1 H, J = 3.4, 4.2 Hz), 4.10 (d, 1 H, J = 4.4 Hz); 13C NMR (100 MHz, CD3OD) δ 174.4, 153.9, 142.1, 134.4, 130.5, 130.1, 129.8, 128.1, 81.6, 77.8, 56.9, 47.2, 26.9; HRMS (FAB) found 483.0458 [calcd for C18H19ClN6O3Se (M+H)+ 483.0451]; Anal. (C18H19ClN6O3Se) C, H, N.</p><!><p>White solid; Yield: 79%; mp 233 ° (decomposed); [α]D20 -22.18 (c 0.28, CH2Cl2+CH3OH); UV (CH2Cl2) λmax 273.0 nm; 1H NMR (400 MHz, CD3OD) δ 8.51 (s, 1 H), 8.35 (s, 1 H), 7.92-7.89 (m, 1 H), 7.83 (d, 1 H, J = 8.4 Hz), 7.56-7.48 (m, 2 H), 7.44 (t, 1 H, J = 7.8 Hz), 6.22 (d, 1 H, J = 5.6 Hz), 5.29 (bs, 2 H), 4.58 (dd, 1 H, J = 3.2, 4.0 Hz), 4.10 (d, 1 H, J = 4.0 Hz); 13C NMR (100 MHz, CD3OD) δ 173.9, 153.8, 141.8, 135.1, 132.6, 130.0, 129.6, 129.2, 128.2, 127.4, 127.3, 126.3, 124.2, 123.2, 81.4, 77.6, 56.7, 47.1, 27.0; HRMS (FAB) found 499.0992 [calcd for C22H22N6O3Se (M+H)+ 499.0997]; Anal. (C21H20N6O3Se) C, H, N.</p><!><p>White solid; Yield: 78%; mp 125-129 °C; [α]D20 34.53 (c 0.35, CH3OH); UV (CH2Cl2) λmax 274.0 nm; 1H NMR (400 MHz, CD3OD) δ 8.53 (s, 1 H), 8.32 (s, 1 H), 7.30-7.15 (m, 6 H), 6.21 (d, 1 H, J = 6.0 Hz), 4.59-4.57 (m, 2 H), 4.10 (d, 1 H, J = 4.4 Hz), 2.82 (s, 3 H), 2.23-2.16 (m, 1 H), 1.40-1.29 (m, 2 H); 13C NMR (100 MHz, CD3OD) δ 173.6, 156.4, 153.4, 141.8, 141.6, 129.4, 129.1, 127.0, 126.8, 81.2, 78.7, 77.4, 56.6, 47.1, 34.0, 27.0, 25.7; HRMS (FAB) found 475.0998 [calcd for C20H22N6O3Se (M+H)+ 475.0997]; Anal. (C20H22N6O3Se) C, H, N.</p><!><p>White solid; Yield: 98%; mp 165-170 °C; [α]D20 -29.83 (c 0.58, CH3OH); UV (CH2Cl2) λmax 270.5 nm; 1H NMR (400 MHz, CD3OD) δ 8.53 (s, 1 H), 8.32 (s, 1 H), 6.21 (d, 1 H, J = 6.0 Hz), 4.58 (dd, 1 H, J = 3.4, 4.2 Hz), 4.10 (d, 1 H, J = 4.4 Hz), 2.81 (s, 3 H), 0.92-0.87 (m, 2 H), 0.67-0.63 (m, 2 H); 13C NMR (100 MHz, CD3OD) δ 174.4, 157.2, 153.7, 142.1, 81.7, 77.8, 56.9, 47.1, 26.9, 7.6; HRMS (FAB) found 399.0685 [calcd for C14H18N6O3Se (M+H)+ 399.0684]; Anal. (C14H18N6O3Se) C, H, N.</p><!><p>White solid; Yield: 79%; mp 153-158 °C; [α]D20 -38.92 (c 0.65, CH3OH); UV (CH2Cl2) λmax 271.0 nm; 1H NMR (400 MHz, CD3OD) δ 8.53 (s, 1 H), 8.25 (s, 1 H), 6.19 (d, 1 H, J = 6.0 Hz), 4.57 (dd, 1 H, J = 3.4, 4.2 Hz), 4.10 (d, 1 H, J = 4.4 Hz), 2.81 (s, 3 H), 2.49-2.41 (m, 2 H), 2.12-2.04 (m, 2 H), 1.86-1.79 (m, 2 H); 13C NMR (100 MHz, CD3OD) δ 174.4, 153.9, 141.9, 81.6, 77.8, 56.9, 47.1, 32.0, 26.9, 15.9; HRMS (FAB) found 413.0841 [calcd for C15H20N6O3Se (M+H)+ 413.0840]; Anal. (C15H20N6O3Se) C, H, N.</p><!><p>White solid; Yield: 94%; mp 135 ° (decomposed); [α]D20 -110.77 (c 0.13, CH3OH); UV (CH2Cl2) λmax 271.0 nm; 1H NMR (400 MHz, CD3OD+CDCl3) δ 8.50 (s, 1 H), 8.26 (s, 1 H), 6.17 (d, 1 H, J = 5.6 Hz), 4.83 (dd, 1 H, J = 3.2, 6.0 Hz), 4.58 (dd, 1 H, J = 3.2, 4.4 Hz), 4.10 (d, 1 H, J = 4.8 Hz), 2.82 (s, 3 H), 2.16-2.05 (m, 2 H), 1.83-1.79 (m, 2 H), 1.73-1.59 (m, 4 H); 13C NMR (100 MHz, CD3OD+CDCl3) δ 174.0, 155.5, 153.7, 149.7, 141.5, 120.6, 81.5, 81.4, 77.6, 56.8, 56.7, 53.5, 47.1, 33.9, 27.0, 26.9, 24.6; HRMS (FAB) found 427.0993 [calcd for C16H22N6O3Se (M+H)+ 427.0997]; Anal. (C16H22N6O3Se) C, H, N.</p><!><p>White solid; Yield: 92%; mp 173-178 °C; [α]D20 -29.57 (c 0.47, CH3OH); UV (CH2Cl2) λmax 267.5 nm; 1H NMR (400 MHz, CD3OD) δ 8.51 (s, 1 H), 8.28 (s, 1 H), 6.20 (d, 1 H, J = 6.0 Hz), 4.58 (dd, 1 H, J = 3.6, 4.0 Hz), 4.10 (d, 1 H, J = 4.4 Hz), 3.12 (bs, 3 H), 2.82 (s, 3 H); 13C NMR (100 MHz, CD3OD) δ 174.4, 153.9, 141.8, 81.6, 77.8, 56.9, 47.1, 26.9; HRMS (FAB) found 373.0527 [calcd for C12H16N6O3Se (M+H)+ 373.0527]; Anal. (C12H16N6O3Se) C, H, N.</p><!><p>White solid; Yield: 85%; mp 139-144 °C; [α]20D -15.0 (c 0.34, CH3OH); UV (CH2Cl2) λmax 273.0 nm; 1H NMR (500 MHz, CD3OD + CDCl3) δ 8.41 (s, 1 H), 7.28 (dd, J = 7.6, 13.9 Hz, 1 H), 7.15 (d, J = 7.5 Hz, 1 H), 7.07 (d, J = 9.6 Hz, 1 H), 6.93 (t, J = 7.6 Hz, 1 H), 6.06 (d, J = 5.8 Hz, 1 H), 4.75 (s, 3 H), 4.54 (t, J = 3.5 Hz, 1 H), 4.07 (d, J = 4.4 Hz, 1 H), 2.84 (s, 3 H); 13C NMR (75 MHz, CD3OD+CDCl3) δ 174.3, 166.3, 163.0, 156.8, 155.9, 151.7, 142.9, 131.7 (d, J = 8.3 Hz), 125.0, 120.3, 116.0 (d, J = 22.6 Hz), 115.5 (d, J = 21.3 Hz), 81.9, 78.2, 57.5, 48.0, 45.2, 27.9; HRMS (ESI) found 501.0349 [calcd for C18H19ClFN6O3Se (M+H)+ 501.0356]; Anal. (C18H18ClFN6O3Se) C, H, N.</p><!><p>White solid; Yield: 76%; mp 166-170 °C; [α]20D -14.6 (c 1.00, CH3OH); UV (CH2Cl2) λmax 275.0 nm; 1H NMR (500 MHz, CDCl3) δ 8.14 (s, 1 H), 7.32 (s, 1 H), 7.21 (s, 3 H), 5.92 (d, J = 6.0 Hz, 1 H), 4.71 (s, 2 H), 4.68-4.67 (m, 1 H), 4.46 (t, J = 3.8 Hz, 1 H), 4.05-4.04 (m, 1 H), 3.32 (s, 1 H), 2.88 (s, 3 H); 13C NMR (75 MHz, CD3OD+CDCl3) δ 174.4, 143.0, 142.5, 135.9, 131.5, 129.4, 129.2, 129.0, 127.7, 82.0, 79.7, 78.3, 57.5, 48.0, 45.3, 41.4, 31.3, 27.9; HRMS (ESI) found 517.0049 [calcd for C18H19Cl2N6O3Se (M+H)+ 517.0061]; Anal. (C18H18Cl2N6O3Se) C, H, N.</p><!><p>White solid; Yield: 81%; mp 165-170 °C; [α]20D -36.5 (c 0.33, CH3OH); UV (CH2Cl2) λmax 276.0 nm; 1H NMR (400 MHz, CDCl3 + CD3OD) δ 8.16 (s, 1 H), 7.45 (s, 1 H), 7.32 (d, J = 7.8 Hz, 1 H), 7.22 (d, J = 6.9 Hz, 1 H), 7.12 (t, J = 7.8 Hz, 1 H), 5.91 (d, J = 5.8 Hz, 1 H), 4.67 (s, 2 H), 4.65-4.63 (m, 1 H), 4.43 (t, J = 4.7 Hz, 1 H), 2.84 (s, 3 H); 13C NMR (150 MHz, CD3OD) δ 175.0, 145.7, 144.3, 143.3, 132.5, 132.17, 132.15, 132.8, 129.6, 128.4, 124.2, 101.9, 62.1, 58.3, 57.7, 48.0, 45.3, 27.9; HRMS (ESI) found 560.9463 [calcd for C18H19BrClN6O3Se (M+H)+ 560.9556]; Anal. (C18H18BrN6O3Se) C, H, N.</p><!><p>White solid; Yield: 87%; mp 154-158 °C; [α]20D -19.5 (c 0.48, CH3OH); UV (CH2Cl2) λmax 273.0 nm; 1H NMR (500 MHz, CD3OD) δ 8.48 (s, 1 H), 7.77 (s, 1 H), 7.60 (d, J = 7.8 Hz, 1 H), 7.39 (d, J = 7.7 Hz, 1 H), 7.09 (t, J = 7.8 Hz, 1 H), 6.10 (d, J = 7.5 Hz, 1 H), 4.79 (dd, J = 3.3, 5.8 Hz, 1 H), 4.75 (broad s, 1 H), 4.56 (t, J = 3.9 Hz, 1 H), 4.09 (d, J = 4.5 Hz, 1 H), 2.82 (s, 3 H); 13C NMR (125 MHz, CD3OD) δ 175.0, 157.2, 156.3, 152.4, 143.4, 143.3, 138.7, 138.2, 132.2, 129.0, 120.7, 95.7, 82.3, 78.6, 57.8, 48.0, 45.2, 27.8; HRMS (ESI) found 608.9481 [calcd for C18H19ClIN6O3Se (M+H)+ 608.9417]; Anal. (C18H18ClIN6O3Se) C, H, N.</p><!><p>White solid; Yield: 86%; mp 130-135 °C; [α]20D -27.3 (c 1.98, CH3OH); UV (CH2Cl2) λmax 275.0 nm; 1H NMR (400 MHz, CD3OD) δ 8.47 (s, 1 H), 6.11 (d, J = 5.8 Hz, 1 H), 4.79 (dd, J = 3.3, 5.8 Hz, 1 H, partially merged with H2O), 4.56 (t, J = 3.8 Hz, 1 H), 4.09 (d, J = 4.5 Hz, 1 H), 2.82 (s, 3 H), 0.89-0.85 (m, 2 H), 0.65-0.62 (m, 2 H); 13C NMR (75 MHz, CD3OD) δ 175.0, 158.7, 156.2, 143.1, 142.8, 120.7, 82.3, 80.6, 78.6, 76.3, 66.9, 57.7, 48.0, 27.8, 27.3; HRMS (ESI) found 433.0268 [calcd for C14H18ClN6O3Se (M+H)+ 433.0294]; Anal. (C14H17ClN6O3Se) C, H, N.</p><!><p>White solid; Yield: 82%; mp 152-157 °C; [α]20D -20.5 (c 1.66, CH3OH); UV (CH2Cl2) λmax 277.0 nm; 1H NMR (500 MHz, DMSO-d6) δ 8.68 (d, J = 7.6 Hz, 1 H), 8.44 (s, 1 H), 8.16 (d, J = 4.0 Hz, 1 H), 5.92 (d, J = 6.1 Hz, 1 H), 5.74 (d, J = 5.5 Hz, 1 H), 5.51 (d, J = 4.9 Hz, 1 H), 4.71 (s, 1 H), 4.62-4.58 (m, 1 H), 4.41-4.40 (m, 1 H), 3.95 (d, J = 4.0 Hz, 1 H), 2.64 (d, J = 4.5 Hz, 3 H), 2.30-2.22 (m, 2 H), 2.13-2.05 (m, 2 H), 1.70-1.63 (m, 2 H); 13C NMR (100 MHz, DMSO-d6) δ 181.5, 163.9, 163.2, 160.2, 150.1, 128.0, 89.7, 85.5, 65.0, 55.1, 40.8, 39.98, 39.93, 36.0, 24.6; HRMS (ESI) found 447.0443 [calcd for C15H20ClN6O3Se (M+H)+ 447.0451]; Anal. (C15H19ClN6O3Se) C, H, N.</p><!><p>White solid; Yield: 79%; mp 148-152 °C; [α]20D -21.7 (c 0.18, CH3OH); UV (CH2Cl2) λmax 277.0 nm; 1H NMR (500 MHz, CDCl3) δ 8.24 (s, 1 H), 8.10 (s, 1 H), 6.29 (s, 1 H), 6.11 (d, J = 4.3 Hz, 1 H), 5.88 (s, 1 H), 5.02 (s, 1 H), 4.76 (s, 1 H), 4.42 (d, J = 4.7 Hz, 1 H), 4.17 (s, 1 H), 2.88 (d, J = 3.7 Hz, 3 H), 2.37 (s, 1 H), 2.02 (s, 2 H), 1.66 (s, 2 H), 1.59 (s, 2 H), 1.43 (s, 2 H); 13C NMR (75 MHz, CD3OD) δ 190.4, 175.0, 157.0, 142.8, 82.3, 78.5, 57.8, 54.4, 47.9, 34.5, 27.8, 25.5; HRMS (ESI) found 461.0607 [calcd for C16H22ClN6O3Se (M+H)+ 461.0607]; Anal. (C16H21ClN6O3Se) C, H, N.</p><!><p>To a solution of 22a (480 mg, 0.6 mmol) in THF (5 mL) was added tetra-n-butylammonuim fluoride (0.39 mL, 1.32 mmol) and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was evaporated and the residue was purified by silica gel chromatography (dichloromethane : methanol = 10 : 1) to give 3t (300 mg, 87%) as a white solid: [α]20D 22.00 (c 0.10, CH3OH); UV (CH3OH) λmax 283.5 nm; 1H NMR (400 MHz, CD3OD) δ 8.46 (s, 1 H), 8.31 (s, 1 H), 7.82 (s, 1 H), 7.55 (d, J = 7.2 Hz, 1 H), 7.44 (d, J =7.6 Hz, 1 H), 7.06 (t, J = 7.6 Hz, 1 H), 6.39 (d, J = 9.2 Hz, 1 H), 5.12 (d, J =15.6 Hz, 1 H), 5.03 (dd, J = 3.6, 9.2 Hz, 1 H), 4.73 (d, J = 15.2 Hz, 1 H), 4.36 (dd, J = 1.0, 3.4 Hz, 1 H), 4.06 (d, J = 1.2 Hz, 1 H), 2.68 (s, 3 H); 13C NMR (75 MHz, CDCl3+CD3OD) δ 174.2, 160.9, 154.0, 152.1, 148.0, 143.4, 137.7, 137.2, 131.4, 128.0, 113.1, 95.3, 82.9, 76.4, 60.8, 47.7, 45.3, 36.1, 27.5; HRMS (ESI) found 574.9803 [calcd for C18H19IN6O3Se (M+H)+ 574.9807]; Anal. (C18H19IN6O3Se) C, H, N.</p><!><p>Compound 22b (254 mg, 0.30 mmol) was converted to 3u (160 mg, 87%) as a white solid, using similar procedure used in the preparation of 3t: [α]20D 40.00 (c 0.10,CH3OH); UV (MeOH) λmax 280.5 nm; 1H NMR (400 MHz, CD3OD) δ 8.40 (s, 1 H), 7.88 (t, J = 1.4 Hz, 1 H), 7.57 (d, J = 7.6 Hz, 1 H), 7.49 (d, 1 H, J = 8.0 Hz), 7.07 (t, 1 H, J = 7.6 Hz), 6.34 (d, J = 9.2 Hz, 1 H), 5.02 (d, 1 H, J = 14.8 Hz), 4.97 (dd, 1 H, J = 3.6, 9.6 Hz), 4.69 (d, 1 H, J = 15.2 Hz), 4.34 (dd, J = 1.2, 3.6 Hz, 1 H), 4.05 (d, J = 1.2 Hz, 1 H), 2.70 (s, 3 H); 13C NMR (100 MHz, CD3OD) δ 174.6, 163.1, 155.5, 152.3, 149.2, 143.1, 138.3, 137.2, 131.2, 128.5, 106.2, 94.8, 82.7, 76.3, 60.7, 47.8, 45.3, 26.8; HRMS (ESI) found 608.9404 [calcd for C18H18ClIN6O3Se (M+H)+ 608.9417]; Anal. (C18H18ClIN6O3Se) C, H, N.</p><!><p>A suspension of 6-chloropurine (0.89 g, 5.76 mmol) and BSA (1.95 g, 2.35 mL, 9.60 mmol) in dry toluene (100 mL) was stirred at 95 °C for 1 h under nitrogen. To this clear solution were added a solution of 5 (2.56 g, 4.80 mmol) in dry toluene (50 ml) and TMSOTf (0.54 g, 0.45 mL, 2.40 mmol) at 95 °C, and the reaction mixture was stirred at same temperature for 15 h, cooled to room temperature, diluted with saturated aqueous NaHCO3 solution (10 mL), and stirred for 15 min. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (70 mL × 2). The combined organic layers were washed with brine, dried (MgSO4), filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (hexane : ethyl acetate = 4:1) to give N9-isomer 6a (1.47 g, 49%) as a pale yellow foam and N7-isomer 6b (0.44 g, 15%) as a pale yellow foam.</p><!><p>[α]20D -6.78 (c 5.4, CH2Cl2); UV (CH2Cl2) λmax 265.0 nm; 1H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), 8.31 (s, 1H), 7.66-7.61 (m, 4H), 7.43-7.32 (m, 6H), 6.27 (d, 1H, J = 3.2 Hz), 5.04 (dd, 1H, J = 3.2, 5.6 Hz), 4.97 (dd, 1H, J = 2.8, 5.6 Hz), 4.13 (td, 1H, J = 2.9, 7.3Hz), 4.10-3.96 (m, 1H) 1.61 (s, 3H), 1.30 (s, 3H), 1.06 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 152.2, 151.6, 151.4, 144.7, 135.75, 135.71, 133.0, 132.9, 132.6, 130.17, 130.15, 128.00, 127.98, 112.4, 90.4, 85.7, 65.0, 58.2, 51.6, 27.8, 27.0, 25.3, 19.4; Anal. (C29H33ClN4O3SeSi) C, H, N.</p><!><p>[α]20D 0.69 (c 5.5, CH2Cl2); UV (CH2Cl2) λmax 253.5 nm; 1H NMR (400 MHz, CDCl3) δ 8.89 (s, 1 H), 8.65 (s, 1 H), 7.68-7.65 (m, 4 H), 7.45-7.37 (m, 6 H), 6.74 (d, 1 H, J = 3.6 Hz), 4.85-4.78 (m, 2 H), 4.10 (td, 1 H, J = 3.1, 6.8 Hz), 4.00-3.90 (m, 2 H), 1.60 (s, 3 H), 1.29 (s, 3 H), 1.09 (s, 9 H); 13C NMR (100 MHz, CDCl3) δ 162.5, 152.9, 148.3, 143.3, 135.81, 135.77, 132.9, 132.8, 130.4, 130.3, 128.2, 122.6, 112.8, 91.5, 85.3, 66.0, 59.4, 51.2, 27.8, 27.1, 25.3, 19.5; Anal. (C29H33ClN4O3SeSi) C, H, N.</p><!><p>A suspension of 2,6-dichloropurine (87 mg, 0.46 mmol) and BSA (0.19 mL, 0.76 mmol) in dry acetonitrile (10 mL) was stirred at 60 ° for 1 h under nitrogen. To this clear solution were added a solution of 5 (205 mg, 0.38 mmol) in dry acetonitrile (5 mL) and TMSOTf (34 μL, 0.19 mmol) at 60 °C, and the reaction mixture was stirred at same temperature for 15 h, cooled to room temperature, diluted with saturated aqueous NaHCO3 solution (3 mL), and stirred for 15 min. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (7 mL × 2). The combined organic layers were washed with brine, dried (MgSO4), filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (hexane : ethyl acetate = 4:1) to give 6b (50 mg, 20%) as a pale yellow foam and N7-isomer 7b (75 mg, 29%) as a pale-yellow foam.</p><!><p>[α]20D 13.55 (c 8.6, CH2Cl2); UV (CH2Cl2) λmax 273.0 nm; 1H NMR (500 MHz, CDCl3) δ 8.22 (s, 1 H), 7.65-7.61 (m, 4 H), 7.42-7.31 (m, 6 H), 6.24 (d, J = 3.7 Hz, 1 H), 5.01-5.00 (m, 1 H), 4.94-4.92 (m, 1 H), 4.13-4.09 (m, 2 H), 3.97 (dd, J = 10.5, 12.8 Hz, 1 H), 1.59 (s, 3 H), 1.28 (s, 3 H), 1.05 (s, 9 H); 13C NMR (75 MHz, CDCl3) δ 153.0, 152.3, 152.0, 144.9, 135.5, 135.5, 132.8, 132.6, 131.3, 129.9, 127.8, 127.7, 112.4, 90.1, 85.3, 77.2, 65.7, 57.1, 51.5, 27.7, 26.8, 25.2, 19.2; Anal. (C29H32Cl2N4O3SeSi) C, H, N.</p><!><p>[α]20D -15.94; (c 0.32, CH2Cl2); UV (CH2Cl2) λmax 258.0 nm; 1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1 H), 7.64-7.67 (m, 4 H), 7.37-7.48 (m, 6 H), 6.64 (d, 1 H, J = 3.6 Hz), 4.81-4.83 (dd, J = 3.2, 5.6 Hz, 1 H), 4.73-4.75 (dd, J = 3.6, 5.6 Hz, 1 H), 4.08-4.15 (m, 1 H), 3.89-3.98 (m, 2 H), 1.59 (s, 3 H), 1.28 (s, 3 H), 1.09 (s, 9 H); 13C NMR (100 MHz, CDCl3) δ 164.1, 153.7, 149.8, 144.1, 135.79, 135.75, 132.8, 132.7, 130.4, 130.3, 128.2, 128.0, 121.8, 112.8, 91.5, 85.3, 65.9, 59.8, 51.4, 27.7, 27.1, 25.2, 19.5; Anal. (C29H32Cl2N4O3SeSi) C, H, N.</p><!><p>A solution of 6b (3.93 g, 6.26 mmol) in toluene (50 mL) was added TMSOTf (0.71 g, 0.59 mL, 3.15 mmol) at 95 °C, and the reaction mixture was stirred at the same temperature for 30 min, cooled to room temperature, and diluted with saturated aqueous solution of NaHCO3 (10 mL). The aqueous layer was extracted with ethylene acetate (10 mL × 2) and the organic layer was dried (MgSO4), filtered and evaporated under reduced pressure. The crude residue was purified on flash silica gel column chromatography (hexane : ethyl acetate = 4 : 1) to give 6a (1.95 g, 50%) as a pale yellow foam.</p><!><p>A solution of 7b (56 mg, 0.085 mmol) in toluene (5 mL) was added TMSOTf (8 μL, 0.042 mmol) at 70 °C, and the reaction mixture was stirred at the same temperature for 30 min, cooled to room temperature, and diluted with saturated aqueous solution of NaHCO3 (5 mL). The aqueous layer was extracted with ethylene acetate (10 mL × 2) and the organic layer was dried (MgSO4), filtered and evaporated under reduced pressure. The crude residue was purified on flash silica gel column chromatography (hexane : ethyl acetate = 4 : 1) to give 7a (26 mg, 46%) as a pale yellow foam.</p><!><p>To a stirred solution of 7a (745 mg, 1.19 mmol) in THF (5 mL) was added HF·Et3N (0.29 mL, 1.78 mmol) and the reaction mixture stirred at room temperature for 15 h and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane : ethyl acetate = 1 : 1) to give 8a (372 mg, 80%) as a yellow form: [α]D20 -43.57 (c 5.10, CH2Cl2); UV (CH2Cl2) λmax 265.5 nm; 1H NMR (400 MHz, CDCl3) δ 9.10 (s, 1 H), 8.76 (s, 1 H), 6.35 (d, 1 H, J = 2.8 Hz), 5.27 (bs, 1 H), 5.12 (dd, 1 H, J = 2.8, 4.8 Hz), 5.03 (dd, 1 H, J = 1.2, 5.2 Hz), 4.25 (dd, 1 H, J = 3.4, 11 Hz), 4.18 (bs, 1 H), 4.07 (dd, 1 H, J = 2.6, 11 Hz), 1.62 (s, 3 H), 1.31 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 152.1, 151.3, 151.0, 146.5, 131.9, 111.6, 92.9, 89.0, 65.3, 61.8, 51.8, 27.9, 25.2; Anal. (C13H15ClN4O3Se) C, H, N.</p><!><p>Compound 7b (7.28 g, 10.99 mmol) was converted to 8b (3.77 g, 81%) using similar procedure used in the preparation of 8a: mp 181-186 °C; [α]20D -53.7 (c 1.8, CH2Cl2+CH3OH); UV (CH2Cl2) λmax 274.0 nm; 1H NMR (500 MHz, CDCl3) δ 8.79 (s, 1 H), 6.28 (d, J = 3.2 Hz, 1 H), 5.15 (t, J = 8.1 Hz, 1 H), 5.03 (d, J = 4.9 Hz, 1 H), 4.22-4.19 (m, 2 H), 4.13-4.12 (m, 1 H), 4.07-4.04 (m, 1 H), 1.63 (s, 3 H), 1.33 (s, 3 H); 13C NMR (75 MHz, CDCl3) δ 152.9, 152.2, 151.8, 146.3, 131.1, 111.7, 92.0, 88.3, 77.2, 65.2, 61.2, 51.2, 27.9, 25.1; Anal. (C13H14Cl2N4O3Se) C, H, N.</p><!><p>To a stirred solution of 8a (372 mg, 0.96 mmol) in dry dichloromethane (5 mL) were added p-nitrobenzoyl chloride (266 mg, 1.43 mmol) and triethylamine (0.27 mL, 1.91 mmol) at room temperature, and the reaction mixture was stirred at room temperature for 1 h and diluted with water (5 mL × 2). The aqueous layer was extracted with ethyl acetate (10 mL × 2), and the combined organic layers were washed with brine (5 mL), dried (MgSO4), filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane : ethyl acetate = 3 : 1) to give 9a (500 mg, 97%) as a yellow form: [α]D20 -160.3 (c 2.10, CH2Cl2+ CH3OH); UV (CH2Cl2) λmax 262.0 nm; 1H NMR (400 MHz, CDCl3) δ 8.80 (s, 1 H), 8.29 (s, 1 H), 8.28 (d, J = 8.5 Hz, 2 H), 8.17 (d, J = 9.0 Hz, 2 H), 6.35 (d, J = 2.7 Hz, 1 H), 5.41 (dd, J = 2.8, 5.5 Hz, 1 H), 5.22 (d, J = 3.5, 5.5 Hz, 1 H), 5.00 (dd, J = 8.1, 11.5 Hz, 1 H), 4.70 (dd, J = 7.0, 11.5 Hz, 1 H), 4.29 (td, J = 1.7, 7.5 Hz, 1 H), 1.63 (s, 3 H), 1.34 (s, 3 H); 13C NMR (75 MHz, CDCl3) δ 167.3, 151.8, 151.4, 150.8, 150.5, 144.6, 134.6, 132.3, 130.8, 123.6, 123.5, 112.5, 90.2, 86.8, 66.7, 58.8, 47.9, 29.5, 27.5, 25.0; Anal. (C20H18ClN5O6Se) C, H, N.</p><!><p>Compound 8b (1.50 g, 3.54 mmol) was converted to 9b (1.98 g, 98%) as a white solid, using similar procedure used in the preparation of 9a: mp 177-182 °C; [α]20D -3.86 (c 12.6, CH2Cl2); UV (CH2Cl2) λmax 268.0 nm; 1H NMR (500 MHz, CDCl3) δ 8.26 (d, J = 8.6 Hz, 2 H), 8.24 (s, 1H), 8.18 (d, J = 8.5 Hz, 2 H), 6.31 (d, J = 8.5 Hz, 1 H), 5.33-5.31 (m, 1 H), 5.27-5.25 (m, 1 H), 4.98 (dd, J = 8.3, 11.4 Hz, 1 H), 4.74 (dd, J = 7.2, 11.4 Hz 1 H), 1.60 (s, 3 H), 1.33 (s, 3 H); 13C NMR (75 MHz, CDCl3) δ 164.1, 152.9, 152.4, 152.0, 150.6, 144.9, 134.7, 131.6, 130.8, 123.6, 112.8, 90.3, 87.0, 66.7, 58.3, 48.2, 27.7, 25.2; Anal. (C20H17Cl2N5O6Se) C, H, N.</p><!><p>A solution of 9a (2.15 g, 4.35 mmol) in aqueous trifluoroacetic acid (60 mL) and THF (10 mL) was stirred at room temperature for 2 h and evaporated under reduced pressure. The residue was dissolved in ethyl acetate (50 mL) and the organic layer was washed with water (30 mL × 2). The aqueous layer was again extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (5 mL), dried (MgSO4), filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane : ethyl acetate = 1 : 1 to 1 : 5) to give 10a (1.50 g, 76%) as a white solid: [α]D20 -108.9 (c 2.53, CH2Cl2+CH3OH); UV (CH2Cl2) λmax 262.0 nm; 1H NMR (500 MHz, CD3OD) δ 8.83 (s, 1 H), 8.71 (s, 1 H), 8.30 (d, J = 8.8 Hz, 2 H), 8.21 (d, J = 8.7 Hz, 2 H), 6.31 (d, J = 6.9 Hz, 1 H), 5.05 (dd, J = 3.2, 6.7 Hz, 1 H), 4.99 (d, J = 8.2, 11.5 Hz, 1 H), 4.73 (dd, J = 6.7, 11.5 Hz, 1 H), 4.56 (t, J = 3.3 Hz, 1 H), 4.00-3.97 (m, 1 H); 13C NMR (75 MHz, CD3OD) δ 166.4, 154.0, 153.6, 152.8, 152.1, 148.7, 137.2, 133.7, 132.6, 125.5, 80.5, 77.2, 68.8, 57.0, 46.3; Anal. (C17H14ClN5O6Se) C, H, N.</p><!><p>Compound 9b (1.98 g, 3.45 mmol) was converted to 10b (1.60 g, 87%) as a yellow foam, using similar procedure used in the preparation of 10a: [α]20D -11.59 (c 12.7, CH2Cl2+CH3OH); UV (CH2Cl2) λmax 267.0 nm; 1H NMR (500 MHz, CD3OD+CDCl3) δ 8.78 (s, 1 H), 8.26 (d, J = 8.7 Hz, 2 H), 8.17 (d, J = 8.7 Hz, 2, H), 6.23 (d, J = 6.6 Hz, 1 H), 4.98-4.93 (m, 2 H), 4.76-4.71 (m, 1 H), 4.54-4.53 (t, J = 3.5 Hz, 1 H), 4.01-3.98 (m, 1 H), 3.63 (d, J = 6.2 Hz, 1 H), 3.08 (d, J = 4.8 Hz, 1 H); 13C NMR (75 MHz, CD3OD +CDCl3) δ 166.1, 154.9, 154.2, 152.7, 152.4, 149.0, 136.7, 132.8, 132.4, 125.2, 80.5, 79.8, 77.0, 68.7, 56.9, 46.2; Anal. (C17H13Cl2N5O6Se) C, H, N.</p><!><p>To a stirred solution of 10a (1.07 g, 2.36 mmol) in dry dichloromethane (30 mL) were added TMSOTf (1.87 g, 1.62 mL, 7.07 mmol), triethylamine (1.43 g, 1.97 ml, 14.15 mmol) and 4-dimethylaminopyridine (29 mg, 0.24 mmol), the reaction mixture was stirred at room temperature for 1 h and diluted with NaHCO3 (60 mL). The aqueous layer was extracted with dichloromethane (30 mL × 2). The organic layer was washed with birne (5 mL), dried (MgSO4), filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane : ethyl acetate = 9 : 1) to give 11a (1.54 g, 96%) as a white foam: [α]D20 -85.6 (c 4.00, CH2Cl2); UV (CH2Cl2) λmax 263.0 nm; 1H NMR (400 MHz, CDCl3) δ 8.77 (s, 1 H), 8.34 (s, 1 H), 8.31 (d, J = 8.8 Hz, 1 H), 8.20 (d, J = 8.8 Hz, 2 H), 6.08 (d, J = 6.3 Hz, 1 H), 5.09 (dd, J = 8.3, 11.7 Hz, 1 H), 4.99 (broad s, 1 H), 4.66 (dd, J = 6.5, 11.7 Hz, 1 H), 4.33 (t, J = 3.2 Hz, 1 H), 3.88 (broad s, 1 H), 0.91 (s, 9 H), 0.73 (s, 9 H), 0.066 (s, 3 H), 0.042 (s, 3 H), -0.077 (s, 3 H), -0.34 (s, 3 H); 13C NMR (75 MHz, CDCl3) δ 164.3, 151.8, 151.6, 151.5, 150.8, 145.5, 134.7, 132.6, 130.7, 123.8, 78.8, 75.9, 66.5, 55.5, 44.7, 25.7, 25.5, 18.0, 17.7, -4.4, -4.7, -4.8, -5.3; Anal. (C29H42ClN5O6SeSi2) C, H, N.</p><!><p>Compound 10b (1.47 g, 2.76 mmol) was converted to 11b (1.76 g, 84%) as a yellow foam, using similar procedure used in the preparation of 11a: [α]20D -8.07 (c 7.4, CH2Cl2); UV (CH2Cl2) λmax 266.0 nm; 1H NMR (500 MHz, CDCl3) δ 8.35 (s, 1 H), 8.27 (d, J = 8.8 Hz, 2 H), 8.18 (d, J = 8.9 Hz, 2 H), 5.98 (s, 1 H), 5.06 (dd, J = 8.2, 11.7 Hz, 1 H), 4.88 (broad s, 1 H), 4.62 (dd, J = 6.5, 11.5 Hz, 1 H), 4.30-4.29 (m, 1 H), 3.88 (broad s, 1 H), 0.87 (s, 9 H), 0.73 (s, 9 H), 0.015 (s, 3 H), -0.018 (s, 3 H), -0.061 (s, 3 H), -0.30 (s, 3 H); 13C NMR (75 MHz, CDCl3) δ 64.1, 152.8, 152.7, 152.1, 150.7, 146.1, 134.6, 131.6, 130.6, 123.7, 78.9, 75.9, 66.2, 55.5, 44.8, 25.6, 25.5, 17.9,17.7, -4.42, -4.72, -4.95, -5.34; Anal. (C29H41Cl2N5O6SeSi2) C, H, N.</p><!><p>To a stirred solution of 11a (400 mg, 0.55 mmol) in dioxane (10 mL) was added 1N NaOH solution in water (1 mL) at room temperature, and the mixture was stirred at same temperature for 3 h. The reaction mixture was neutralized with glacial acetic acid and diluted with saturated NaHCO3 solution (5 mL × 2). The aqueous layer was extracted with ethyl acetate (10 mL × 2), and the organic layer was washed with brine, dried (MgSO4), filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane : ethyl acetate = 5 : 1) to give 12a (277 mg, 87%) as a white foam: [α]D20 -25.52 (c 4.62, CH2Cl2); UV (CH2Cl2) λmax 265.0 nm; 1H NMR (400 MHz, CDCl3) δ 8.79 (s, 1 H), 8.27 (s, 1 H), 5.97 (d, 1 H, J = 7.2 Hz), 5.18 (bs, 1 H), 4.29 (t, 1 H, J = 2.8 Hz), 4.06-3.95 (m, 2 H), 3.67 (d, 1 H, J = 0.8 Hz), 0.95 (s, 9 H), 0.73 (s, 9 H), 0.12 (s, 3 H), 0.1 (s, 3 H), -0.13 (s, 3 H), -0.61 (d, 3 H, J = 1.2 Hz); 13C NMR (100 MHz, CDCl3) δ 152.1, 151.5, 151.4, 146.3, 133.4, 78.9, 78.6, 64.2, 57.5, 51.9, 26.0, 25.94, 25.89, 25.85, 25.82, 18.2, 17.9, -4.19, -4.51, -5.53; Anal. (C22H39ClN4O3SeSi2) C, H, N.</p><!><p>Compound 11b (1.62 g, 2.13 mmol) was converted to 12b (1.07 g, 83%) as a yellow foam, using similar procedure used in the preparation of 12a: [α]20D -9.11 (c 15.8, CH2Cl2); UV (CH2Cl2) λmax 276.0 nm; 1H NMR (400 MHz, CDCl3) δ 8.33 (s, 1 H), 5.89 (d, J = 6.9 Hz, 1 H), 5.00 (broad s, 1 H), 4.25 (t, J = 2.9 Hz, 1 H), 4.02-3.94 (m, 2 H), 3.66 (s, 1 H), 0.90 (s, 9 H), 0.72 (s, 9 H), 0.080 (s, 3 H), 0.046 (s, 3 H), -0.11 (s, 3 H), -0.50 (s, 3 H); 13C NMR (75 MHz, CDCl3) δ 152.5, 152.2, 146.7, 131.9, 79.7, 77.8, 77.2, 63.5, 56.9, 51.3, 25.7, 25.6, 18.0, 17.7, -4.40, -4.68, -4.78, -5.58; Anal. (C22H38Cl2N4O3SeSi2) C, H, N.</p><!><p>To a solution of 12a (558 mg, 0.97 mmol) in dimethyl sufoxide (5 mL) was added acetic anhydride (0.18 mL, 1.93 mmol) and the reaction mixture was stirred at 100 ° for 1 h, cooled to room temperature, and diluted with dichloromethane (20 mL). The mixture was washed with water (10 mL × 2), and the aqueous layer was further extracted with dichloromethane (20 mL × 2). The combined organic layers were washed with brine (5 mL), dried (MgSO4), filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane : ethyl acetate = 4 : 1) to give 13a (483 mg, 87%) as a white foam: [α]D20 -74.88 (c 1.23, CH2Cl2); UV (CH2Cl2) λmax 265.0 nm; 1H NMR (400 MHz, CDCl3) δ 9.76 (d, 1 H, J = 2.4 Hz), 8.78 (s, 1 H), 8.32 (s, 1 H), 6.24 (d, 1 H, J = 7.2 Hz), 4.86 (dd, 1 H, J = 2.6, 7.0 Hz), 4.68 (t, 1 H, J = 3.0 Hz), 4.12 (dd, 1 H, J = 2.4, 2.8 Hz), 0.95 (s, 9 H), 0.71 (s, 9 H), 0.12 (s, 6 H), -0.05 (s, 3 H), -0.4 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 194.6, 152.3, 152.1, 151.7, 145.3, 132.5, 81.0, 77.4, 74.6, 56.9, 56.8, 53.0, 25.9, 25.7, 18.3, 17.9, 0.21, -4.2, -4.45, -4.55, -5.3; Anal. (C22H37ClN4O3SeSi2) C, H, N.</p><!><p>Compound 12b (3.99 g, 5.50 mmol) was converted to 13b (2.98 g, 75%) as a yellow foam, using similar procedure used in the preparation of 13a: [α]20D -33.2 (c 10.8, CH2Cl2); UV (CH2Cl2) λmax 276.0 nm; 1H NMR (500 MHz, CDCl3) δ 9.73 (d, J = 2.1 Hz, 1 H), 8.40 (s, 1 H), 6.13 (d, J = 6.8 Hz, 1 H), 4.74-4.73 (m, 1 H), 4.63 (t, J = 2.9 Hz, 1 H), 4.11 (s, 1 H), 0.91 (s, 9 H), 0.71 (s, 9 H), 0.090 (s, 6 H), -0.043 (s, 3 H), -0.38 (s, 3 H); 13C NMR (75 MHz, CDCl3) δ 194.3, 153.2, 153.0, 152.2, 145.7, 131.3, 81.0, 74.4, 56.7, 52.9, 25.69, 25.66, 25.6, 18.0, 17.6, -4.42, -4.70, -4.81, -4.86, -5.55; Anal. (C22H36Cl2N4O3SeSi2) C, H, N.</p><!><p>To a solution of AgNO3 (51 mg, 0.30 mmol) was added 1 N NaOH (0.30 mL, 0.30 mmol) at 0 °C. After Ag2O was precipitated, 24% ammonia water (0.09 mL) was added to reaction mixture until the mixture was clear. The solution of 13a (87 mg, 0.15 mmol) in THF (2 mL) was added to above Tollens' reagent at 0 °C, and the reaction mixture was stirred at same temperature for 30 min and diluted with water (6 mL). The aqueous layer was extracted with ethyl acetate (10 mL × 2), and the aqueous layer was acidified with 1 N HCl solution (3 mL) and extracted with ethyl acetate (10 mL × 2). The combined organic layers were washed with brine, dried (MgSO4), filtered and evaporated under reduced pressure to give the crude acid. To this dried the residue were added a solution of crude acid in THF (5 mL), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluoro phosphate (HATU) (50 mg, 0.13 mmol), methylamine hydrochloride (20 mg, 0.30 mmol), and diisopropylamine (0.06 ml, 0.34 mmol) at room temperature. The reaction mixture was stirred at same temperature for 1 h, and diluted with ethyl acetate (5 mL). The organic layer was washed with water (3 mL × 2) and the aqueous layer was further extracted with ethyl acetate (10 mL × 2). The combined organic layers were washed with brine (2 mL), dried (MgSO4), filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane : ethyl acetate = 3 : 1) to give 14a (50 mg, 54%) as a white foam: [α]D20 -36.04 (c 1.92, CH2Cl2); UV (CH2Cl2) λmax 265.5 nm; 1H NMR (400 MHz, CDCl3) δ 8.77 (s, 1 H), 8.40 (bs, 1 H), 6.06 (d, 1 H, J = 7.6 Hz), 4.1 (bs, 1 H), 4.55 (t, 1 H, J = 2.4 Hz), 3.96 (d, 1 H, J = 2.8 Hz), 2.98 (d, 3 H, J = 4.8 Hz), 0.94 (s, 9 H), 0.7 (s, 9 H), 0.16 (s, 3 H), -0.17 (s, 3 H), -0.53 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 170.2, 152.1, 151.5, 146.6, 133.2, 79.5, 78.3, 56.6, 49.9, 27.2, 25.9, 25.7, 18.2, 17.9, -4.3, -4.6, -5.4; Anal. (C23H40ClN5O3SeSi2) C, H, N.</p><!><p>Compound 13b (86 mg, 0.14 mmol) was converted to 14b (56 mg, 62%) as a yellow foam, using similar procedure used in the preparation of 14a: 1H NMR (400 MHz, CDCl3) δ 8.35 (s, 1 H), 5.97 (d, J = 7.6 Hz, 1 H), 4.83 (broad s, 1 H), 4.52 (s, 1 H), 3.95 (d, J = 2.7 Hz, 1 H), 2.99 (d, J = 4.7 Hz, 3 H), 0.91 (s, 9 H), 0.69 (s, 9 H), 0.15 (s, 3 H), 0.096 (s, 3 H), -0.16 (s, 3 H), -0.50 (s, 3 H); Anal. (C23H39Cl2N5O3SeSi2) C, H, N.</p><!><p>To a stirred solution of 14a (78 mg, 0.13 mmol) in THF (3 mL) was added tetra-n-butylammonium fluoride hydrate (34 mg, 0.13 mmol) and the reaction mixture was stirred at room temperature for 1 h. The solvent was evaporated and the resulting residue was purified by silica gel column chromatography (dichloromethane : methanol = 100 : 1 - 10 : 1) to give 15a (34 mg, 70%) as a white solid: mp 212 °C (decomposed); [α]D20 -19.05 (c 0.20, CH2Cl2); UV (CH2Cl2) λmax 265.0 nm; 1H NMR (400 MHz, CD3OD) δ 9.03 (s, 1 H), 8.77 (s, 1 H), 6.35 (d, 1 H, J = 6.4 Hz), 4.95 (dd, 1 H, J = 3.6, 6.0 Hz), 4.60 (t, 1 H, J = 3.6 Hz), 4.12 (d, 1 H, J = 4.4 Hz), 2.81 (s, 3 H); 13C NMR (100 MHz, CD3OD) δ 173.4, 152.8, 152.5, 151.4, 147.4, 132.5, 81.5, 78.6, 77.2, 56.5, 47.1, 26.9; Anal. (C11H12ClN5O3Se) C, H, N.</p><!><p>Compound 14b (290 mg, 0.450 mmol) was converted to 15b (155 mg, 83%) as a white solid, using similar procedure used in the preparation of 15a: mp 141-144 °C; [α]20D -7.58 (c 2.3, CH3OH); UV (CH2Cl2) λmax 276.0 nm; 1H NMR (500 MHz, CD3OD) δ 9.03 (s, 1 H), 6.23 (d, J = 5.9 Hz, 1 H), 4.88 (dd, J = 3.2, 5.9 Hz, 1 H), 4.58 (t, J = 3.7 Hz, 1 H), 4.11 (d, J = 4.4 Hz, 1 H), 2.81 (s, 3 H); 13C NMR (75 MHz, CD3OD+CDCl3) δ 173.9, 154.7, 154.1, 148.7, 132.4, 82.1, 77.8, 57.3, 48.0, 41.1, 27.8; Anal. (C11H11Cl2N5O3Se) C, H, N.</p><!><p>A solution of 7a (1.49 g, 2.39 mmol), 3-iodobenzylamine hydrochloride (840 mg, 3.10 mmol), and triethylamine (1.05 ml, 7.16 mmol) in ethanol (50 mL) was stirred at room temperature for 15 h. The reaction mixture was evaporated under reduced pressure, and the resulting residue was purified by silica gel chromatography (hexane : ethyl acetate = 3 : 1) to give 16a (1.70 g, 87%) as a white foam: [α]20D 8.00 (c 0.20, CH2Cl2); UV (CH2Cl2) λmax 280.5 nm; 1H NMR (400 MHz, CDCl3) δ 8.57 (s, 1 H), 8.37 (s, 1 H), 7.73 (t, J = 3.2 Hz, 1 H), 7.64-7.61 (m, 1 H), 7.57-7.52 (m, 4 H), 7.37-7.21 (m, 7 H), 7.06 (t, J = 7.8 Hz, 1 H), 6.60 (t, J = 10.0 Hz, 1 H), 5.93 (d, J = 3.2 Hz, 1 H), 4.79 (m, 2 H), 4.64 (dd, J = 5.2, 14.4 Hz, 1 H), 4.20 (dd, J = 3.2, 5.6 Hz, 1 H), 4.02-3.93 (m, 2 H), 3.76 (dd, J = 8.0, 10.0 Hz, 1 H), 1.31 (s, 3 H), 1.13 (s, 3 H), 1.05 (s, 9 H); 13C NMR (100 MHz, CDCl3) δ 161.5, 153.6, 150.5, 144.4, 141.0, 137.5, 136.9, 135.71, 135.66, 132.7, 132.6, 130.6, 130.3, 130.2, 128.11, 128.08, 128.0, 113.2, 112.3, 94.7, 91.3, 86.1, 65.9, 63.0, 51.4, 44.8, 27.1, 24.9, 19.4; Anal. (C36H40IN5O3SeSi) C, H, N.</p><!><p>Compound 7b (1.80 g, 2.72 mmol) was converted to 16b (1.39 g, 60%) as a white foam, using similar procedure used in the preparation of 16a: [α]20D 12.17 (c 0.23, CH2Cl2); UV (CH2Cl2) λmax 285.5 nm; 1H NMR (400 MHz, CDCl3) δ 8.35 (s, 1 H), 7.74 (t, J = 1.6 Hz, 1 H), 7.66-7.64 (m, 1 H), 7.57-7.51 (m, 4 H), 7.40-7.21 (m, 7 H), 7.09 (t, J = 7.6 Hz, 1 H), 6.79 (t, J = 5.0 Hz, 1 H), 5.84 (d, J = 2.8 Hz, 1 H), 4.81-4.73 (m, 2 H), 4.59 (dd, J = 5.2, 14.4 Hz, 1 H), 4.13-4.01 (m, 1 H), 4.01-3.94 (m, 2 H), 3.74-3.69 (m, 1 H), 1.29 (s, 3 H), 1.12 (s, 3 H), 1.04 (s, 9 H); 13C NMR (100 MHz, CDCl3) δ 163.1, 154.8, 151.0, 145.2, 140.3, 137.1, 135.74, 135.68, 132.7, 132.6, 130.7, 130.2, 128.4, 128.2, 128.1, 113.2, 111.1, 94.8, 91.3, 86.4, 65.9, 63.6, 51.4, 45.2, 27.1, 27.0, 24.9, 19.4; Anal. (C36H39ClIN5O3SeSi) C, H, N.</p><!><p>To a solution of 16a (1.71 g, 2.06 mmol) in THF (20 mL) was added tetra-n-butylammonium fluoride (0.9 ml, 3.10 mmol) and the reaction mixture stirred at room temperature for 1 h. The solvent was evaporated and the residue was purified by silica gel chromatography (dichloromethane : methanol = 20 : 1) to give to give 17a (1.11 g, 90%) as a white solid: [α]20D 73.66 (c 0.30, CH2Cl2); UV (CH2Cl2) λmax 279.5 nm; 1H NMR (400 MHz, CDCl3) δ 9.04 (s, 1 H), 8.47 (s, 1 H), 7.76 (t, J = 1.6 Hz, 1 H), 7.66-7.63 (m, 1 H), 7.37 (d, J = 7.6 Hz, 1 H), 7.09 (t, J = 7.8 Hz, 1 H), 6.82 (t, J = 5.0 Hz, 1 H), 6.07 (d, J = 2.4 Hz, 1 H), 5.05 (d, J = 5.2 Hz, 1 H), 4.95 (d, J = 5.2 Hz, 1 H), 4.23-4.09 (m, 3 H), 1.28 (s, 3 H), 1.22 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 160.3, 153.4, 150.7, 146.1, 140.8, 137.7, 136.9, 130.6, 128.1, 111.9, 94.7, 93.6, 90.1, 66.4, 64.5, 52.8, 45.1, 27.2, 25.1; Anal. (C20H22IN5O3Se) C, H, N.</p><!><p>Compound 16b (1.39 g, 1.62 mmol) was converted to 17b (0.86 g, 86%) as a white solid, using similar procedure used in the preparation of 17a: [α]20D 83.33 (c 0.10, CH2Cl2); UV (CH2Cl2) λmax 283.5 nm; 1H NMR (400 MHz, CDCl3) δ 8.93 (s, 1 H), 7.76 (t, 1 H, J = 1.2 Hz), 7.68-7.65 (m, 1 H), 7.40-7.38 (m, 1 H), 7.11 (t, 1 H, J = 15.6 Hz), 6.98 (t, 1 H, J = 5.2 Hz), 5.97 (d, 1 H, J = 2 Hz), 5.06 (d, 1 H, J = 4.8 Hz), 4.98 (dd, 1 H, J = 2.0, 4.8 Hz), 4.68-4.67 (m, 2 H), 4.18 (dd, J = 3.2, 10.8 Hz, 1 H), 4.12 (t, J = 3.0 Hz, 1 H), 4.04 (dd, J = 3.4, 10.6 Hz, 1 H), 1.26 (s, 3 H).23 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 161.8, 154.5, 151.1, 146.9, 140.1, 137.1, 130.7, 128.5, 112.1, 94.7, 93.5, 66.8, 64.6, 53.0, 45.5, 27.1, 25.1; Anal. (C20H21ClIN5O3Se) C, H, N.</p><!><p>To a solution of 17a (1.10 g, 1.88 mmol) in pyridine (20 mL) was added benzoyl chloride (0.26 mL, 2.25 mmol) and the reaction mixture stirred at room temperature for 3 h. The reaction mixture was evaporated under reduced pressure, and the resulting residue was purified by silica gel chromatography (hexane : ethyl acetate = 3 : 1) to give 18a (1.20 g, 96%) as a white foam: [α]20D 76.57 (c 0.10, CH2Cl2); UV (CH2Cl2) λmax 275.0 nm; 1H NMR (400 MHz, CDCl3) δ 8.54 (s, 1 H), 8.45 (s, 1 H), 7.87 (d, J = 7.52 Hz, 2 H), 7.74 (s, 1 H), 7.62 (d, J = 7.9 Hz, 1 H), 7.54 (t, J = 7.4 Hz, 1 H), 7.39-7.35 (m, 3 H), 7.08 (t, J =7.7 Hz, 1 H), 6.66 (s, 1 H), 6.13 (d, J = 3.2 Hz, 1 H), 5.02-5.00 (m, 1 H), 4.94-4.91 (m, 1H), 4.70 (d, J = 4.96 Hz, 2 H), 4.57 (d, J = 6.4 Hz, 2 H), 4.24-4.20 (m, 1 H), 1.33 (s, 3 H),1.20 (s, 3 H); 13C NMR (75 MHz, CDCl3) δ 165.9, 161.3, 153.5, 150.4, 144.4, 140.6,137.3, 136.8, 133.7, 130.5, 129.6, 128.9, 128.6, 127.8, 113.8, 112.0, 94.6, 91.5, 86.4, 77.2,65.6, 62.7, 47.3, 44.9, 27.0, 24.9; Anal. (C20H22IN5O3Se) C, H, N.</p><!><p>Compound 17b (0.86 g, 1.39 mmol) was converted to 18b (0.91 g, 99 %) as a white foam, using similar procedure used in the preparation of 18a: [α]20D 96.59 (c 0.10, CH2Cl2); UV (CH2Cl2) λmax 283.5 nm; 1H NMR (400 MHz, CDCl3) δ 8.56 (s, 1 H), 7.86 (d, J = 7.4 Hz, 2 H), 7.77 (s, 1 H), 7.67 (d, J = 8.0 Hz, 1 H), 7.56 (t, J = 8.0 Hz, 1 H), 7.38 (dd, J = 8.8, 20.0 Hz, 3 H), 7.12 (t, 1 H, J = 8.0 Hz), 6.93 (t, J = 4.4 Hz, 1 H), 6.08 (s, 1 H), 5.09-5.01 (m, 2 H), 4.69 (d, J = 4.8 Hz, 2 H), 4.67-4.58 (m, 2 H), 4.23-4.27 (m, 1 H), 1.32 (s, 3 H),1.22 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 166.2, 162.2, 155.0, 151.1, 145.3, 137.9,137.3, 133.9, 130.8, 129.8, 129.1, 128.5, 114.0, 111.0, 94.8, 91.9, 87.3, 66.0, 64.0, 48.0, 45.6, 27.1, 25.1; Anal. (C20H21ClIN5O3Se) C, H, N.</p><!><p>A solution of 18a (1.25g, 1.81 mmol) in 88 % formic acid (10 mL) was stirred at room temperature for 12 h and the reaction mixture was evaporated. The residue was purified by silica gel chromatography (dichloromethane : methanol = 20 : 1) to give 19a (860 mg, 73%) as a white foam: [α]20D 29.60 (c 0.10, CH3OH); UV (CH3OH) λmax 274.5 nm; 1H NMR (400 MHz, CDCl3) δ 8.22 (s, 1 H), 8.02 (s, 1 H), 7.99 (d, J = 2.8 Hz, 2 H), 7.78 (s, 1 H), 7.58-7.52 (m, 2 H), 7.44-7.35 (m, 3 H), 7.02 (t, J = 7.6 Hz, 1 H), 6.24 (d, J = 8.4 Hz, 1 H), 5.04 (d, J = 7.2 Hz, 1 H), 4.71-4.59 (m, 5 H), 4.01 (t, J = 8.0 Hz, 1 H); 13C NMR (100 MHz, CDCl3) δ 166.2, 158.1, 152.2, 151.1, 145.5, 140.8, 137.0, 136.8, 133.7, 130.6, 129.9, 129.5, 128.7, 127.3, 112.3, 94.8, 80.8, 75.4, 66.0, 59.1, 44.6; Anal. (C24H22IN5O4Se) C, H, N.</p><!><p>Compound 18b (0.91 g, 1.25 mmol) was converted to 19b (0.77 mg, 89%) as a white foam, using similar procedure used in the preparation of 19a: [α]20D 20.53 (c 0.20, CH3OH); UV (CH3OH) λmax 280.0 nm; 1H NMR (400 MHz, CD3OD) δ 8.68 (s, 1 H), 8.04-8.02 (m, 2 H), 7.84 (t, J = 1.6 Hz, 1 H), 7.65-7.61 (m, 1 H), 7.59-7.56 (m, 1 H), 7.51-7.48 (m, 2 H), 7.45-7.43 (m, 1 H), 7.07 (t, J = 7.6 Hz, 1 H), 6.32 (d, J = 8.0 Hz, 1 H), 4.80-4.70 (m, 3 H), 4.61-4.56 (m, 2 H), 4.44-4.30 (m, 1 H), 3.96-3.92 (m, 1 H); 13C NMR (100 MHz, CDCl3) δ 166.1, 160.2, 153.8, 151.4, 140.1, 137.4, 136.9, 133.7, 130.6, 130.0, 129.4, 128.7, 127.8, 110.8, 94.8, 80.6, 75.2, 65.8, 59.5, 45.2; Anal. (C24H21ClIN5O4Se) C, H, N.</p><!><p>To a solution of 19a (860 mg, 1.32 mmol) in dichloromethane (30 mL) were added TMSOTf (1.5 mL, 6.61 mmol) and 2,6-lutidine (1.5 mL, 13.2 mmol) at 0 °C, and the reaction mixture was stirred at room temperature for 15 h. The reaction mixture was diluted with water (20 mL) and extracted with dichloromethane (20 mL × 2). The organic layer was washed with brine, dried (MgSO4), filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane : ethyl acetate = 3 : 1) to give 20a (1.14 g, 98%) as a white foam: [α]20D 2.67 (c 0.10, CH2Cl2); UV (CH2Cl2) λmax 280.0 nm; 1H NMR (400 MHz, CDCl3) δ 8.60 (s, 1 H), 8.02 (d, J = 7.2 Hz, 2 H), 7.77 (s, 1 H), 7.64-7.60 (m, 1 H), 7.56 (d, J = 7.6 Hz, 1 H), 7.50-7.46 (m, 2 H), 7.39 (d, J = 7.6 Hz, 1 H), 7.03 (t, J = 7.8 Hz, 1 H), 6.20 (broad s, 1 H), 4.92 (broad s, 2 H), 4.62-4.53 (m, 2 H), 4.42 (dd, J = 2.6, 9.4 Hz, 1 H), 4.31 (broad s, 1 H), 3.73-3.71 (m, 1 H); 13C NMR (100 MHz, CDCl3) δ 166.0, 153.5, 151.0, 141.2, 137.4, 136.8, 133.9, 130.6, 129.8, 129.3, 128.9, 127.7, 94.8, 44.6, 25.8, 125.7, 18.1, 14.4, -4.3, -4.5, -4.8, -5.7; Anal. (C36H50IN5O4SeSi2) C, H, N.</p><!><p>Compound 19b (0.77 g, 1.12 mmol) was converted 20b (0.94 g, 92 %) as a white foam, using similar procedure used in the preparation of 20a: [α]20D 44.44 (c 0.10, CHCl3); UV (CH2Cl2) λmax 282.0 nm; 1H NMR (400 MHz, CDCl3) δ 8.52 (broad s, 1 H), 8.02 (d, J = 7.8 Hz, 1 H), 7.78 (s, 1 H), 7.65-7.61 (m, 1 H), 7.58 (d, J = 8.0 Hz, 1 H), 7.50-7.47 (m, 2 H), 7.41 (d, J = 8.0 Hz, 1 H), 7.05 (t, 1 H, J = 4.0 Hz), 6.13 (broad s, 1 H), 4.88 (broad s, 1 H), 4.58 (broad s, 2 H), 4.36 (dd, J = 2.8, 6 Hz, 1 H), 4.21 (broad s, 1 H), 3.96 (s, 1 H) 3.70 (s, 1 H), 0.89 (s, 9 H), 0.65 (s, 9 H), 0.05 (s, 3 H), 0.02 (s, 3 H), -0.16 (s, 3 H), -0.46 (broad s, 3 H); 13C NMR (100 MHz, CDCl3) δ 166.0, 154.7, 154.7, 151.6, 140.4, 137.8, 137.1, 133.9, 130.7, 129.9, 129.2, 128.9, 128.2, 112.2, 96.3, 94.8, 80.6, 74.6, 64.7, 58.3, 45.0, 25.9, 25.85, 25.77, 18.1, 0.2, -4.3, -4.5, -4.8, -5.6; Anal. (C36H49ClIN5O4SeSi2) C, H, N.</p><!><p>To a solution of 20a (1.14 g, 1.30 mmol) in methanol (25 mL) was added sodium methoxide (105 mg, 1.95 mmol) and the mixture was stirred at room temperature for 1 h. The reaction mixture was evaporated under reduced pressure and the residue was purified by silica gel column chromatography (hexane : ethyl acetate = 1 : 1) to give 21a (1.00 g, 99%) as a white foam: [α]20D 26.00 (c 0.10, CH2Cl2); UV (CH2Cl2) λmax 278.5 nm; 1H NMR (400 MHz, CDCl3) δ 8.48 (s, 1 H), 7.77 (s, 1 H), 7.55 (d, J = 8.0 Hz, 1 H), 7.39 (d, J = 8.4 Hz, 1 H), 7.02 (t, J = 7.6 Hz, 1 H), 6.08 (broad s, 1 H), 4.87 (broad s, 2 H), 4.61 (broad s, 1 H), 4.30 (broad s, 1 H), 4.01 (dd, J = 4.2, 11.4 Hz, 1 H), 3.77 (broad s, 1 H), 3.62 (t, J = 4.4 Hz, 1 H), 0.94 (s, 9 H), 0.64 (s, 9 H), 0.15 (s, 3 H), 0.12 (s, 3 H), -0.20 (s, 3 H), -0.62 (s, 3 H); 13C NMR (75 MHz, CDCl3) δ 152.6, 151.0, 141.8, 137.1, 136.5, 130.4, 127.5, 94.6, 50.5, 44.6, 26.0, 25.9, 18.2, -4.17, -4.21, -4.50, -5.67; Anal. (C29H46IN5O3SeSi2) C, H, N.</p><!><p>Compound 20b (0.86 g, 0.94 mmol) was converted to 21b (0.65 mg, 90%) as a white foam, using similar procedure used in the preparation of 21a: [α]20D 8.42 (c 0.10, CDCl3); UV (CH2Cl2) λmax 281.5 nm; 1H NMR (400 MHz, CDCl3) δ 8.49 (broad s, 1 H), 7.78 (s, 1 H), 7.74 (broad s, 1 H), 7.53 (d, J = 8.0 Hz, 1 H), 7.41 (d, J = 8.0 Hz, 1 H), 7.00 (t, J = 8.0 Hz, 1 H), 6.00 (broad s, 1 H), 4.89 (broad s, 1 H), 4.75 (broad s, 1 H), 4.64 (broad s, 1 H), 4.29 (broad s, 1 H), 4.14 (d, J = 8.8 Hz, 1 H), 3.79 (broad s, 1 H), 3.64-3.63 (m, 1 H), 0.95 (s, 9 H), 0.64 (s, 9 H), 0.15 (s, 3 H), 0.12 (s, 3 H), -0.20 (s, 3 H), -0.63 (broad s, 3 H); 13C NMR (100 MHz, CDCl3) δ 154.6, 151.3, 147.7, 141.5, 137.5, 136.4, 130.3, 127.9, 94.5, 81.3, 77.1, 63.0, 59.5, 50.6, 44.7, 26.0, 25.9, 18.2, 17.9, -4.2, -4.2, -4.5, -5.7; Anal. (C29H45ClIN5O3SeSi2) C, H, N.</p><!><p>To a solution of 21a (997 mg, 1.28mmol) in dry DMF (10 mL) was added PDC (4.7 g, 12.8 mmol) and the reaction mixture was stirred at room temperature for 12 h and diluted with water (100 mL), and stirred for 1 h until obtaining the precipitate. The mixture was filtered, washed with water, and dissolved in dichloromethane. The aqueous layer was extracted with dichloromethane. The combining organic layers were washed with brine, dried, filtered, and concentrated under reduced pressure to give the crude carboxylic acid, which was used for the next reaction without further purification. To a stirred solution of crude acid (900 mg, 1.14 mmol) in dry dichloromethane (50 mL) were added 1-hydroxybenzotriazole hydrate (HOBt) (309 mg, 2.28 mmol), N-(3-dimethylaminopropyl) N′-ethylcarbodiimide (EDC) hydrochloride (438 mg, 2.28 mmol), methylamine hydrochloride (156 mg, 2.28 mmol), N,N-diisopropylethylamine (0.6 mL, 3.42 mmol) and the reaction mixture was stirred at room temperature for 15 h. The mixture was diluted with ethyl acetate and the organic layer was washed with water, dried, filtered, and evaporated. The residue was purified by silica gel chromatography (hexane : ethyl acetate = 3 : 1) to give 22a (480 mg, 37%) as a white foam: [α]20D 55.87 (c 0.20, CH2Cl2); UV (CH2Cl2) λmax 277.5 nm; 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 1 H), 7.84 (s, 2 H), 7.67 (broad s, 1 H), 7.53 (d, J = 8.4 Hz, 1 H), 7.45 (d, J = 7.6 Hz, 1 H), 7.00 (t, J = 7.8 Hz, 1 H), 6.40 (d, J = 3.6 Hz, 1 H), 6.10 (s, 1 H), 5.19 (s, 2 H), 4.75 (s, 1 H), 4.30 (s, 1 H), 3.80 (d, J = 0.8 Hz, 1 H), 2.74 (s, 3 H), 0.95 (s, 9 H), 0.67 (s, 9 H), 0.15 (s, 6 H), -0.13 (s, 3 H), -0.58 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 174.0, 172.0, 160.8, 153.2, 150.5, 146.8, 142.5, 136.7, 135.8, 134.8, 130.0, 129.6, 127.7, 127.2, 109.5, 94.3, 81.5, 76.1, 75.7, 75.6, 72.0, 70.2, 67.4, 63.9, 61.0, 59.7, 46.8, 44.1, 29.7, 26.8, 26.7, 26.6, 25.7, 25.7, 25.3, 24.8, 18.0, 17.7, -0.0, -4.4, -4.6, -4.7, -6.0; Anal. (C30H47IN6O3SeSi2) C, H, N.</p><!><p>Compound 21b (0.69 g, 0.741 mmol) was converted to 22b (254 mg, 41%) as a white foam, using similar procedure used in the preparation of 21a: [α]20D -7.33 (c 0.10, CDCl3); UV (CH2Cl2) λmax 282.5 nm; 1H NMR (400 MHz, CDCl3) δ 8.06 (broad s, 1 H), 7.90 (broad s, 1 H), 7.79 (broad s, 1 H), 7.32-7.54 (m, 2 H), 7.10 (broad s, 1 H), 7.01 (t, J = 8.0 Hz, 1 H), 6.00 (d, J = 8.0 Hz, 1 H), 5.15 (d, J = 9.2 Hz, 1 H), 5.07-5.05 (m, 1 H), 4.72 (d, J = 12.0 Hz, 1 H), 4.27 (s, 1 H), 3.94 (s, 1 H), 2.78 (d, J = 3.6 Hz, 3 H), 0.91 (s, 9 H), 0.67 (s, 9 H), 0.14 (s, 3 H), 0.12 (s, 3 H), -0.13 (s, 3 H)-0.60 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 172.5, 162.8, 154.7, 151.1, 147.6, 141.8, 137.7, 136.1, 130.2, 128.1, 110.9, 94.3, 81.5, 76.0, 59.7, 47.7, 44.8, 26.9, 25.92, 25.88, 18.2, 17.9, -4.3, -4.4, -5.8; Anal. (C30H46ClIN6O3SeSi2) C, H, N.</p><!><p>CHO or HEK293 cells expressing the recombinant hA1, hA2A or A3AR were cultured in Dulbecco's modified Eagle's medium (DMEM) and F12 (1:1) supplemented with 10% fetal bovine serum, 100 Units/mL penicillin, 100 μg/mL streptomycin, and 2 μmol/mL glutamine. Cells were harvested after reaching confluency. After homogenization and suspension, cells were centrifuged at 1000 g for 10 min, and the pellet was discarded. The suspension was then recentrifuged at 20,000 g for 60 min at 4 °C. The resultant pellets were resuspended in buffer containing 3 Units/mL adenosine deaminase (from bovine spleen, Worthington Biochemical Corp., Lakewood, NJ) and incubated at 37 °C for 30 min. The aliquots of membrane preparation were stored at -80 °C until the binding experiments. The protein concentration was measured using the Bradford assay.31</p><!><p>For binding to the hA1AR, 50 μL of increasing concentrations of a test ligand and 50 μL of [3H]R-PIA (1 nM, PerkinElmer, Boston, MA) were incubated with membrane preparations (40 μg/tube) at 25 °C for 60 min in 50 mM Tris·HCl buffer (pH 7.4; MgCl2, 10 mM) in a total assay volume of 200 μL. Nonspecific binding was determined using 10 μM of 23. For hA2AAR binding, membrane preparations (20 μg/tube) were incubated at 25 °C for 60 min with a final concentration of 10 nM [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino-5′-N-ethylcarboxamidoadenosine (American Radiolabeled Chemicals, Inc., St. Louis, MO) in a mixture containing 50 μL of increasing concentrations of a test ligand and 200 μL of 50 mM Tris·HCl, pH 7.4, containing 10 mM MgCl2. Compound 23 (10 μM) was used to define nonspecific binding. The reaction was terminated by filtration with GF/B filters. Filters were placed in scintillation vials containing 5 mL of Hydrofluor scintillation buffer and counted using a PerkinElmer Tricarb 2810TR Liquid Scintillation Analyzer.</p><!><p>Each tube in the binding assay contained 100 μL membrane suspension (20 μg protein), 50 μL [125I]N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyl-uronamide (0.2 nM, PerkinElmer, Boston, MA), and 50 μL of increasing concentrations of the test ligands in TrisHCl buffer (50 mM, pH 8.0) containing 10 mM MgCl2, 1 mM EDTA. Nonspecific binding was determined using 10 μM of 23 in the buffer. The mixtures were incubated at 25 °C for 60 min. Binding reaction was terminated by filtration through Whatman GF/B filters under reduced pressure using a MT-24 cell harvester (Brandell, Gaithersburgh, MD, USA). Filters were washed three times with 9 mL ice-cold buffer. Filters were counted using a PerkinElmer Cobra II γ-counter.</p><!><p>Cells were first treated with assay buffer containing rolipram (10 μM) and adenosine deaminase (3 units/mL) for 30 min. Agonists and/or test compounds were then added for another 10 min followed by the addition of foskolin (10 μM) and incubate for another 15 min. The reaction was terminated upon removal of the supernatant, and addition of 100 μL of Tween-20 (0.03%). Intramolecular cAMP levels were measured with an ALPHAScreen cAMP assay kit as instructed by manual from manufacturer.</p><!><p>Binding and functional parameters were calculated using Prism 5.0 software (GraphPAD, San Diego, CA, USA). IC50 values obtained from competition curves were converted to Ki values using the Cheng-Prusoff equation.32 Data were expressed as mean ± standard error of the mean.</p><!><p>To predict the 3D structure of hA3AR, homology modeling study was performed. The primary sequence of hA3AR (accession: P0DMS8) was used as the query sequence and the X-ray crystal structure of the agonist-bound hA2AAR (PDB ID: 3QAK) was utilized as the template for homology modeling. The homology model was built and validated by SWISS-MODEL server.33</p><!><p>The hA3AR model went through using the Protein Preparation Wizard in Maestro v10.2 (Schrodinger, LLC, NY, USA). During the preparation process, the bond orders were assigned, hydrogen atoms were added, and the appropriate protonation states of the residues were generated at pH 7.4. All the hydrogen atoms were energy minimized with the optimized potential for liquid simulation (OPLS) 2005 force field until the average root mean square deviation for hydrogen atoms reached 0.30 Å.</p><!><p>The X-ray crystal structure of 3p was utilized and the charges were calculated with Merck molecular force field (MMFF94) in SYBYL-X 2.0 (Tripos Int., St. Louis, MO, USA). Then, it was docked into the homology model of hA3AR using Induced Fit Docking (IFD) module in Maestro v10.2 (Schrodinger, LLC, NY, USA) with the following steps: (i) The grid for the binding site was defined as a 26 Å3 box from the centroid of selected residues (Thr94, Phe168, Asn250, Ser271, His272).34 (ii) For the initial docking stage, Glide SP (standard precision) docking was performed retaining a maximum number of 20 poses per ligand. (iii) Residues within 5 Å from the ligand were allowed to move during the Prime refinement stage and further energy minimization was applied to optimize the side chain orientations. (iv) Then, re-docking of the ligands using Glide XP (extra precision) was performed within 30 kcal/mol of the best structure. The protein-ligand complexes with the best IFD score were selected and analyzed. All the molecular graphic figures were generated by PyMOL software (http://www.pymol.org). All the computational studies were undertaken on an Intel Xeon 16-Core 3.10 GHz workstation with Linux CentOS release 5.9.</p><!><p>The chemotaxis assay was performed as described in previous publication.10 Microphotographs of migrating microglial cells in the live cell chamber (Live Cell Instrument, Seoul, Korea) were monitored with inverted confocal microscopy system (Leica DMIRE2, TLC_SP2) and differential interference contrast transmission image programs (Leica, Ver. 2.5). Fifteen min after 1b or 3d was applied by micromanipulator (Narishige, Japan), the MCP-1 gradient (100 ng/mL; BD Pharmingen, San Jose, CA) was generated using a Harvard apparatus microinjection device (0.1 μL/min; PHD 2000 rogrammable). MRS1523 (1 μmol/L) or SCH58261 (100 mol/L) was applied to the cells for 20 min before treatment with MCP-1. For quantitative analysis, MCP-1 was laced in the bottom chamber of a chemotaxis chamber Neuro Probe, Cabin John, MD) and microglia (5 × 104 cells/mL in serum-free MEM) in the upper chamber were allowed to migrate to the bottom part for 2 hours, through membrane pore (8 mm2 filter area; Neuro Probe). Migrated cells on the bottom-side filter were stained for nuclei with Harris's hematoxylin and then counted. The movement or morphological change of microglia was recorded for 20 min starting from the application of MCP-1.</p>