■ Journal of Medicinal Chemistry CONCLUSION In summary, we present a synthetic focus upon enzymatic C-6 structural modifications with a view to their enzymatic incor- poration into cADPR. The substituted N6-NAD+ analogue 6- NMe-NAD+, upon incubation with Aplysia cyclase, hydrolyzes to the linear product 6-NMe-ADPR, presumably because of steric hindrance and/or reduction of hydrogen bonding interaction with the enzyme. A high yielding route also generated another C-6 + modified analogue, 6-thio NHD . Surprisingly, this analogue cyclizes at N1 in a similar manner to NAD+, to give the novel fluorescent cADPR analogue 6-thio N1-cIDPR, although the parent oxo-congener NHD+ cyclizes at N7. A mechanistic study implies a more complex explanation than simply a hydrogen bonding interaction, as the enzyme recognizes this thio deriva- tive as a normal substrate, although it is structurally closer to NHD+ than it is to NAD+. Biological evaluation in permeabi- lized Jurkat T cells reveals that this compound is decreased in its agonist activity relative to cIDPR but possesses new anta- gonist activity against both cADPR- and cIDPR-induced Ca2+ release, showing that substitution of the oxygen of the hypo- xanthine ring for sulfur interferes with the functional con- sequences of ligand binding to its receptor. Like two known classical antagonists, 6-thio cIDPR shows agonist activity at high concentrations. Antagonist activity in cADPR analogues has thus now been demonstrated in compounds substituted at the 8-position of the purine, the 3′-hydroxyl group of the “southern” ribose, and now for the first time in a compound with a C-6 structural modification. Both the fundamental causes of antagonism and the switch to agonism at higher concen- tration in some cases thus seem highly complex and seem to defy a simple explanation. The present study demonstrates, however, that future synthetic efforts to facilitate further C-6- structural modification should be a fruitful enterprise in eluci- dating cADPR SAR and defining useful new tools for dissection of its signaling pathway. ■ EXPERIMENTAL SECTION General. All reagents and solvents were of commercial quality and were used without further purification unless described otherwise. Triethylamine and morpholine were dried over potassium hydroxide, distilled, and then stored over potassium hydroxide pellets. ADP- ribosyl cyclase was purified from the ovotestis of Aplysia californica.67 H2O was of Milli-Q quality. All 1H, 13C, and 31P NMR spectra of final compounds were collected in D2O on a JEOL Delta machine at 270 MHz (1H) or 109 MHz (31P) or on a Varian Mercury-vx system at 400 MHz (1H) or 100 MHz (13C). All 1H and 13C NMR assignments are based on gCOSY, gHMBC, gHMQC, and DEPT experiments. Abbreviations for splitting patterns are as follows: br, broad; s, singlet; d, doublet; t, triplet; m, multiplet. UV spectra were collected in aqueous solution on a Perkin-Elmer Lambda EZ 201 or Lambda 3B spectrophotometer. High resolution time-of-flight mass spectra were obtained on a Bruker Daltonics micrOTOF mass spectrometer using electrospray ionization (ESI). HPLC analyses were carried out on a Waters 2695 Alliance module equipped with a Waters 2996 photo- diode array detector (210−350 nm). The chromatographic system consisted of a Hichrom guard column for HPLC and a Phenomenex Synergi 4 μm MAX-RP 80A column (150 × 4.60 mm), with elution at 1 mL/min with the following ion-pair buffer: 0.17% (m/v) cetrimide and 45% (v/v) phosphate buffer (pH 6.4) in MeOH. The purities of all phosphate containing compounds were determined by HPLC, and they were in all cases over 95%. All other compounds were analyzed using a Phenomenex Gemini column 5 μm C18 (150 mm × 4.6 mm), with elution at 1 mL/min with a MeCN/H2O gradient (5−65% over 20 min). Preparative chromatography was performed on a Pharmacia Biotech Gradifrac system equipped with a peristaltic P-1 pump and a Article fixed wavelength UV-1 optical unit (280 nm). The following puri- fication methods were employed: LiChroprep RP-18 equilibrated with 0.05 M TEAB buffer (pH 6.0−6.4), gradient of 0.05 M TEAB buffer against MeCN at 5 mL/min, Q-Sepharose washed with H2O, gradient of 1 M TEAB buffer (pH 7.1−7.6) against H2O at 5 mL/min, and AG MP-1 washed with H2O, gradient 150 mM TFA against H2O at 3 mL/min. Synthetic phosphates were assayed by an adaptation of the Briggs phosphate test.68 Computational Details. cADPR, N1-cIDPR, and 6-thio N1-cIDPR were built using Sybyl-X 1.1.1, and the volumes were calculated. The pKa values were calculated using the Sparc online calculator http:// sparc.chem.uga.edu/sparc/. Pharmacology. Materials. Fluo-3 was purchased from Molecular Probes. Saponin and KH2PO4 were obtained from Fluka. ATP, crea- tine phosphate, EGTA, Tris, and NaCl were provided from Sigma Aldrich. MgCI2, CaCl2, and KCl were procured from Merck Chemicals. HEPES was purchased from Biomol. Creatine kinase was obtained from Roche. Culture medium reagents were supplied by Invitrogen or Biochrom. Cell Culture. Jurkat T-lymphocytes (subclone JMP) were cultured as described previously69 at 37 °C in the presence of 5% CO2 in RPMI 1640 medium containing Glutamax I and HEPES (25 mM) and supplemented with 7.5% (v/v) NCS (newborn calf serum), 100 units/ mL penicillin, and 100 μg/mL streptomycin. Ca2+ Release Experiments in Permeabilized Cells. Permeabi- lized cells were prepared as described,70 and the Ca2+ concentration was measured by the use of Fluo-3. In brief, cells were permeabilized in the presence of saponin (55 μg/mL) for 20 min in an intracellular buffer (20 mM HEPES, 110 mM KCI, 2 mM MgCI2, 5 mM KH2PO4, 10 mM NaCI, pH 7.2) at 37 °C. An aliquot containing 1 × 108 cells was transferred to a cuvette, and fluorescence was measured in a Hitachi F-2000 spectrofluorometer (excitation 504 nm, emission 524 nm) at 37 °C in the presence of Fluo-3 (1 μM) with continuous stirring. Reuptake of Ca2+ into stores was achieved by addition of ATP (1 mM), creatine phosphate (20 mM), and creatine kinase (20 U/mL). At the end of each experiment, the free Ca2+ concentration was calibrated by addition of CaCl2 and subsequently by addition of EGTA/Tris and calculated by using the following equation: [Ca2+] = Kd(F − Fmin)/ (Fmax − F) where Fmin is the fluorescence intensity in the absence of Ca2+, Fmax is the fluorescence intensity of the Ca2+-saturated indicator, F is the fluorescence during the measurement, and Kd is the dissociation constant of Fluo-3. Kd of Fluo-3 (503 nM) was determined on the basis of the calcium calibration buffer kit (Molecular Probes) for intracellular buffer containing 1 mM ATP, 20 mM creatine phosphate, and 20 U/mL creatine kinase at 37 °C, pH 7.06. 6-Chloroadenosine 2′,3′,5′-Triacetate (6). Compound 6 was synthesized according to a modified Vorbrüggen condensation. To a vigorously stirred solution of 6-chloropurine (500 mg, 3.23 mmol), β- D-ribofuranose 1,2,3,5-tetraacetate (926 mg, 2.91 mmol), and DBU (1.3 mL, 8.70 mmol) in dry MeCN (22 mL) was added TMSOTf (2.12 mL) at 0 °C under an argon atmosphere. The reaction mixture was heated at 60 °C for 1 h and carefully quenched by addition of a saturated solution of NaHCO3 (100 mL). The crude compound was extracted with DCM (3 × 100 mL), and the organic layers were combined, dried over MgSO4, and evaporated under reduced pressure. The yellow residue obtained was further purified by flash column chromatography, eluting with DCM−MeOH, 20:1, to give the desired compound as a yellow oil (960 mg, 77%). 1H (CDCl3, 270 MHz) δ 8.76 (s, 1H, H-8), 8.28 (s, 1H, H-2), 6.21 (d, 1H, J1′,2′ = 5.2 Hz, H-1′), 5.92 (dd, 1H, J2′,3′ = 5.4 Hz and J2′,1′ = 5.2 Hz, H-2′), 5.62 (dd, 1H, J3′,2′ = 5.4 Hz and J3′,4′ = 4.9 Hz, H-3′), 4.37 (m, 3H, H-4′ and H-5′), 2.14 (s, 3H, CH3), 2.10 (s, 3H, CH3), and 2.07 (s, 3H, CH3). 6-N-Methyladenosine 5′-Acetate. The title compound was synthesized by adaptation of a literature protocol.71 To a suspension of 6-chloroadenosine triacetate 6 (800 mg, 1.94 mmol) and methylamine hydrochloride (573 mg, 8.42 mmol) in a mixture of DCM (20 mL) and ethanol (4.2 mL) was added triethylamine (3.8 mL). The resulting mixture was stirred at 60 °C overnight. TLC analysis indicated that the starting material was completely consumed and three more polar spots were given. The reaction mixture was transferred into a pressure tube 1485 dx.doi.org/10.1021/jm201127y | J. Med. Chem. 2012, 55, 1478−1489