Journal of Medicinal Chemistry with triethylamine (4 mL), methylamine hydrochloride (500 mg, 7.35 mmol), and ethanol (4 mL). The mixture was heated at 60 °C for 6 h and left at room temperature for 2 days. TLC of the mixture indicated only one major spot at Rf = 0.25 (DCM−MeOH, 10:1). The solvent was removed and the crude compound was purified by flash column chromatography, eluting with DCM−MeOH, 30:1, to give a mixture of the desired compound and the methylamine hydrochloride. Pure title compound was obtained as a white solid by washing the resulting mixture with Milli-Q water (420 mg, 67%). 1H (DMSO-d6, 270 MHz) δ 8.35 (s, 1H, H-8), 8.24 (brs, 1H, H-2), 7.81 (brs, 1H, NH), 5.91 (d, 1H, J1′,2′ = 5.0 Hz, H-1′), 5.61 (m, 1H, 2′-OH), 5.42 (m, 1H, 3′-OH), 4.67 (m, 1H, H-2′), 4.31−4.18 (m, 4H, H-3′, H-4′, H-5′), 2.95 (brs, 3H, CH3N), 1.95 (s, 3H, CH3); HRMS (ES+) calcd for C13H18N5O5 324.1308 (MH)+, found 324.1302. 6-N-Methyladenosine (7). A suspension of 6-N-methyladenosine 5′-acetate (400 mg, 1.23 mmol) in a saturated methanolic ammonia (50 mL) was stirred at room temperature for 3 h, after which the solvent was removed in vacuo. The residue obtained was purified by flash column chromatography, eluting with DCM−methanol, 10:1, to produce the title compound as a white solid (300 mg, 86%); mp 178− 180 °C; 1H (DMSO-d6, 270 MHz, D2O shake) δ 8.31 (s, 1H, H-8), 8.20 (brs, 1H, H-2), 5.85 (d, 1H, J1′,2′ = 6.4 Hz, H-1′), 4.57 (dd, 1H, J2′,1′ = 6.4 Hz and J2′,3 = 5.2 Hz, H-2′), 4.12 (dd, 1H, J3′,2′ = 5.2 Hz and J3′,4′ = 3.0 Hz, H-3′), 3.97 (app.q, 1H, J4′,5′a = J4′,5′b = J4′,3′ = 3.0 Hz, H- 4′), 3.64 (dd, J5′a,5′b = 12.9 Hz and J5′a,4′ = 3.0 Hz, 1H, H-5′a), 3.55 (dd, 1H, J5′b,5′a = 12.9 Hz and J5′b,4′ = 3.0 Hz, H-5′b), and 2.94 (brs, 3H, CH3N); HRMS (ES+) calcd for [M + H]+ C11H16N5O4 282.1202 (MH+), found 282.1201. 6-N-Methyladenosine 5′-Monophosphate (8a, 6-N-Methyl AMP). A suspension of 7 (110 mg, 0.39 mmol, dried in vacuo at 100 °C for 2 h) in triethyl phosphate (1.4 mL), was heated strongly with a heat gun for 5 min. To the resulting clear solution were added POCl3 (0.3 mL, 3.20 mmol) and H2O (1 μL) at 0 °C, and the mixture was stirred for 1 h. The reaction mixture was quenched by addition of ice (15 mL). The resulting solution was extracted with cold ethyl acetate (5 × 20 mL). The aqueous layer was neutralized with NaOH (5 M) and loaded onto a reverse phase column, eluting with a gradient of 0−30% MeCN against 0.05 M TEAB. Fractions containing the desired compound were pooled, evaporated and excess TEAB was coevaporated with MeOH (3×) to give a mixture of title compound 8a (150 mg, 0.20 mmol, 51%) and the bis-phosphate 8b. 1H (D2O, 270 MHz) δ 8.17 (s, 1H, H-8), 7.71 (s, 1H, H-2), 5.83 (d, 1H, J1′,2′ = 5.7 Hz, H-1′), 4.57 (m, 1H, H-2′), 4.37 (m, 1H, H-3′), 4.25 (m, 1H, H-4′), 4.03 (m, 2H, H-5′) and 2.78 (m, 3H, CH3N); 31P (D2O, 109 MHz) δ 1.12 (s). Bisphosphate 8b: 1H (D2O, 270 MHz) δ 8.13 (s, 1H, H-8), 7.81 (s, 1H, H-2), 6.06 (d, 1H, J1′,2′ = 4.5 Hz, H-1′), 5.25 (m, 1H, H-2′), 5.05 (m, 1H, H-3′), 4.75 (m, 1H, H-4′ half overlap with HOD signal), 4.55 (m, 2H, H-5′), and 2.80 (m, 3H, CH3). 6-N-Methyladenosine 5′-Monophosphate Morpholidate (9). A suspension of 6-N-methyl-AMP and the bisphosphate as above 8a/8b (0.20 mmol, calculated by 1H NMR integration) in dry DMSO (0.9 mL) was evaporated with dry DMF (3 × 2 mL). To the residue were added in sequence triphenylphosphine (280 mg, 1.07 mmol), morpholine (0.15 mL, 1.72 mmol), and dipyridyl disulfide (235 mg, 1.07 mmol). The resulting yellow solution was stirred at room tem- perature for 4 h, after which a solution of sodium iodide in acetone (0.2 M, 15 mL) was added and the resulting precipitate was filtered and washed with acetone. The crude product was further purified by ion-exchange chromatography, eluting with a gradient of 0−50% 1 M TEAB against Milli-Q water. The solvent was evaporated in vacuo and excess TEAB was coevaporated with MeOH (3 times) to give the title compound as its triethylammonium salt (93 mg, 78%). HPLC, tR = 2.8 min at 254 nm; UV (H2O) λmax 266.4 nm; 1H (D2O, 270 MHz) δ 8.23 (s, 1H, H-8), 7.98 (s, 1H, H-2), 5.93 (d, 1H, J1′,2′ = 4.9 Hz, H-1′), 4.68 (m, 1H, H-2′), 4.42 (m, 1H, H-3′), 4.26 (m, 1H, H-4′), 3.94 (m, 2H, H-5′), 3.44 (m, 4H, 2 × CH2O), 2.91 (m, 3H, CH3N), and 2.80 (m, 4H, 2 × CH2N); 31P (D2O, 109 MHz) δ 8.10 (s); HRMS (ES+) C15H24N6O7P 431.1444 calcd for MH+, found 431.1441. 6-N-Methyl nicotinamide Adenine Dinucleotide (6-N-Methyl NAD+, 10). To a mixture of 9 (56 mg, 95 μmol), β-NMN+ (55 mg, Article 165 μmol), and MgSO4 (38 mg, 317 μmol) was added a solution of MnCl2 in formamide (0.2 M, 1.18 mL). The resulting suspension was stirred at room temperature for 48 h under a nitrogen atmosphere, and the reaction mixture was quenched by dropwise addition of MeCN (2 mL). The yellow precipitate was filtered, washed with acetone, and dissolved in small amount of Milli-Q water. The aqueous solution was treated with Chelex 100 (sodium form) to remove any residual manganese and purified by reverse-phase chromatography, eluting with a gradient of 0.05 M TEAB against MeCN. The title compound was isolated as a glassy solid in the triethylammonium form (51 mg, 64%). HPLC, tR = 3.4 min at 254 nm; UV (H2O) λmax 262.5 nm (ε/dm3 mol−1 cm−1 14 560); 1H (D2O, 400 MHz) δ 9.25 (s, 1H, HN-2), 9.07 (d, 1H, J6,5 = 5.9 Hz, HN-6), 8.71 (d, 1H, J4,5 = 8.2 Hz, HN-4), 8.26 (s, 1H, H-8), 8.09 (dd, 1H, J5,4 = 8.2 Hz and J5,6 = 5.9 Hz, HN-5), 7.97 (s, 1H, H-2), 5.99 (d, 1H, J1″,2″ = 5.5 Hz, H-1″), 5.91 (d, 1H, J1′,2′ = 5.9 Hz, H-1′), 4.70 (dd, 1H, J2′,1′ = 5.9 Hz and J2′,3′ = 5.5 Hz, H-2′), 4.48 (m, 1H, H-4″), 4.44 (m, 2H, H-3″ and H-3′), 4.37 (m, 1H, H-2″), 4.32−4.16 (m, 5H, H-4′, H-5′, and H-5″), and 2.93 (CH3N); 31P (D2O, 109 MHz) δ −10.73 (brs); HRMS (ES−) calcd for C22H28- N7O14P2 676.1169 [M − H]−, found 676.1158. 6-N-Methyladenosine 5′-Diphosphate Ribose (6-N-Methyl ADPR, 11). To a solution of 10 (20 mg, 24 μmol) in HEPES buffer (25 mM, pH 7.4, 60 mL) was added Aplysia ADP-ribosyl cyclase (80 μL). The resulting solution was stirred at room temperature until RP-HPLC indicated that all the starting material had reacted. The solution was diluted, and then product was purified by ion-exchange chromatography, eluting with a gradient of 0−50% 1 M TEAB buffer against Milli-Q water. The appropriate fractions were collected, evapo- rated and excess TEAB was removed by coevaporating with MeOH (3 times) to give the compound as a glassy solid in its triethylammo- nium form (10 mg, 59%). HPLC, tR = 11.8 min at 254 nm; UV (H2O) λmax 265.1 nm (ε/dm3 mol−1 cm−1 13 780); 1H (D2O, 270 MHz) δ 8.37 (s, 1H, H-8), 8.10 (s, 1H, H-2), 6.02 (d, 1H, J1′,2′ = 5.9 Hz, H-1′), 5.24 (d, 0.3H, J1″,2″ = 4.0 Hz, H-1″β), 5.14 (m, 0.7H, H-1″α), 4.70−3.94 (m, 10H, H-ribose), and 2.87 (m, 3H, CH3N); 31P (D2O, 109 MHz) δ −10.6 (brs); HRMS (ES−) calcd for C16H24N5O14P2 572.0795 [M − H]−, found 572.0792. 2′,3′-O-Isopropylidene-6-(2,4-dinitrophenyl)thioinosine, 15. To a suspension of 13 (80 mg, 0.236 mmol) in dry MeCN (5 mL) were added triethylamine (197 μL, 1.416 mmol) and dinitrofluoro- benzene (36 μL, 0.283 mmol). The resulting solution was stirred at room temperature for 1 h after the solvent was removed under reduced pressure and the residue obtained was purified by flash chromatography on silica gel (EtOAc/hexane, 1:1) to yield the desired product as a yellow foam (96 mg, 83%). HPLC, tR = 14.6 min at 260 nm; 1H (270 MHz, CDCl3) δ 8.98 (d, 1H, J = 2.5 Hz, Ar-H), 8.65 (s, 1H, H-2), 8.38 (dd, 1H, J = 8.8 and 2.5 Hz, Ar-H), 8.16 (s, 1H, H-8), 7.97 (d, 1H, J = 8.8 Hz, Ar-H), 5.94 (d, 1H, J1′,2′ = 4.7 Hz, H-1′), 5.21−5.17 (m, 1H, H-2′), 5.12−5.08 (m, 1H, H-3′), 4.55−4.54 (m, 1H, H-4′), 3.98−3.76 (m, 2H, H-5′), 1.64 (s, 3H, CH3), and 1.37 (s, 3H, CH3); HRMS (ES+) calcd for C19H19N6O8S 4910.0980 (MH+), found 491.0957; Rf = 0.3 (EtOAc/hexane, 6:4). 2′,3′-O-Isopropylidene-5′-O-di-tert-butylphosphoramidite- 6-(2,4-dinitrophenyl)thioinosine, 16. To a solution of 15 (70 mg, 0.138 mmol) in dry DCM (3 mL) were added tetrazole (20 mg, 0.276 mmol) and N,N-diisopropyl-di-tert-butylphosphoramidite (66 μL, 0.208 mmol). The reaction mixture was stirred at room temperature for 1 h, after which time TLC analysis (hexane/EtOAc, 6:4) indicated conversion of starting material to a single phosphite. The mixture was cooled to −78 °C, and mCPBA (47 mg, 0.276 mmol) was added. After 20 min, 10% aqueous Na2SO3 (10 mL) was added and the mixture was warmed to room temperature. The organic layer was separated and washed with a saturated solution of NaHCO3 (15 mL) and brine (15 mL), dried (Na2SO4), filtered, and evaporated under reduced pressure to leave an oil which was purified by column chromatography on silica gel (hexane/EtOAc, 1:1) to give the title compound 16 as a yellow oil (85 mg, 90%). HPLC, tR = 12.1 min at 260 nm; 1H (270 MHz, CDCl3) δ 8.98 (d, 1H, J = 2.5 Hz, Ar-H), 8.74 (s, 1H, H-2), 8.35 (d, 1H, J = 2.5 Hz, Ar-H), 8.33 (s, 1H, H-8), 7.92 (d, 1H, J = 8.8 Hz, Ar-H), 6.23 (d, 1H, J1′,2′ = 2.7 Hz, H-1′), 5.33 (dd, 1H, 1486 dx.doi.org/10.1021/jm201127y | J. Med. Chem. 2012, 55, 1478−1489