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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Med Chem. Author manuscript; available in PMC 2012 July 31.
Published in final edited form as:
PMCID: PMC3408611
NIHMSID: NIHMS31407

2-Substitution of Adenine Nucleotide Analogues Containing a Bicyclo[3.1.0]hexane Ring System Locked in a Northern Conformation: Enhanced Potency as P2Y1 Receptor Antagonists

Abstract

Preference for the northern (N) ring conformation of the ribose moiety of adenine nucleotide 3′,5′-bisphosphate antagonists of P2Y1 receptors was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute (Nandanan et al. J. Med. Chem. 2000, 43, 829–842). We have now combined the ring-constrained (N)-methanocarba modification with other functionalities at the 2-position of the adenine moiety. A new synthetic route to this series of bisphosphate derivatives was introduced, consisting of phosphorylation of the pseudoribose moiety prior to coupling with the adenine base. The activity of the newly synthesized analogues was determined by measuring antagonism of 2-methylthio-ADP-stimulated phospholipase C (PLC) activity in 1321N1 human astrocytoma cells expressing the recombinant human P2Y1 receptor and by using the radiolabeled antagonist [3H]2-chloro-N6-methyl-(N)-methanocarba-2′-deoxyadenosine 3′,5′-bisphosphate 5 in a newly developed binding assay in Sf9 cell membranes. Within the series of 2-halo analogues, the most potent molecule at the hP2Y1 receptor was an (N)-methanocarba N6-methyl-2-iodo analogue 12, which displayed a Ki value in competition for binding of [3H]5 of 0.79 nM and a KB value of 1.74 nM for inhibition of PLC. Thus, 12 is the most potent antagonist selective for the P2Y1 receptor yet reported. The 2-iodo group was substituted with trimethyltin, thus providing a parallel synthetic route for the introduction of an iodo group in this high-affinity antagonist. The (N)-methanocarba-2-methylthio, 2-methylseleno, 2-hexyl, 2-(1-hexenyl), and 2-(1-hexynyl) analogues bound less well, exhibiting micromolar affinity at P2Y1 receptors. An enzymatic method of synthesis of the 3′,5′-bisphosphate from the corresponding 3′-monophosphate, suitable for the preparation of a radiophosphorylated analogue, was explored.

Introduction

Two families of P2 nucleotide receptors exist: G protein-coupled P2Y receptors, of which eight mammalian subtypes (P2Y1,2,4,6,11–14) are currently sequence-defined, and P2X ligand-gated cation channels, of which seven mammalian subtypes (P2X1–7) have been cloned.14 Adenine nucleotides are required for activation of the P2Y1, P2Y11, P2Y12, and P2Y13 subtypes, and uracil nucleotides activate P2Y2, P2Y4, and P2Y6, subtypes. The P2Y2 receptor is equipotently activated by UTP and ATP. The most recently identified subtypes are the P2Y13 receptor, which is present in the immune system,5 and the P2Y14 receptor, which is activated by UDP-glucose.6 There is evidence that multiple P2Y receptors may be involved in the activation of eosinophils and neutrophils.7

The P2Y1 receptor is distributed in the heart, skeletal, and various smooth muscles and the prostate, ovary, brain, and circulating blood cells.8 This receptor was first cloned from chick brain;9 its regional brain distribution and presence in glial cells have been described,1012 and abnormalities in P2Y1 receptor levels may exist in the brains of persons with Alzheimer’s disease.13 Activation of this receptor by ADP mediates relaxation in the guinea pig aorta.14 The P2Y1 receptor plays a major physiological role in platelet aggregation.15

P2Y receptor ligands are being explored for therapeutic applications in the cardiovascular, endocrine, and other systems. The P2Y1 and P2Y12 receptors are the furthest advanced in the P2Y family with respect to selective agonists and antagonists.1626 3′,5′-Bisphosphate nucleotides have been developed as selective antagonists of the P2Y1 receptor, and high receptor affinities have been achieved in this series.20 The 2′-deoxyribose moiety of widely used P2Y1 receptor antagonists such as N6-methyl-2′-deoxyadenosine 3′,5′-bisphosphate 1 may be replaced with carbocyclics, smaller and larger rings, acyclics, and conformationally constrained rings,1719 resulting in retention or enhancement of affinity for the receptor. A 2-chloro group was found to enhance potency and selectivity in the case of nucleotide 2 and the acyclic bisphosphate analogue 3.19,27 An acyclic bisphosphonate analogue 4 was also shown to be a P2Y1 receptor antagonist.19

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The most recently introduced P2Y1 receptor antagonists contain novel substitutions of the ribose moiety or of the adenine ring system. In solution, the conformation of the ribose ring of nucleosides and nucleotides exists in a rapid, dynamic two-state equilibrium between a (N) (northern, 2′-exo) or (S) (southern, 2′-endo) conformation, although their target receptors probably prefer specific conformations. We have replaced the ribose moiety for P2Y1 antagonists with a carbocyclic ring locked in a preferred conformation, a fusion of cyclopropane and cyclopentane rings known as the methanocarba modification.2830 Two structural variations, depending on the position of the cyclopropane ring, restrict the ring pucker, i.e., hold the riboselike ring (pseudosugar) in either a (N)- or (S)-envelope conformation, as defined in the pseudorotational cycle. A prototypical ring-constrained P2Y1 antagonist is the highly potent (N)-methanocarba derivative 2-chloro-N6-methyl-(N)-methanocarba-2′-deoxyadenosine 3′,5′-bisphosphate 5.18,20 This molecule was also prepared in [3H]-labeled form and provides the first broadly reliable high-affinity antagonist for quantitation of a P2Y receptor in a radioligand binding assay.31 The recently reported C-nucleoside pyrazolo[1,5-a]-1,3,5-triazine bisphosphate 6 is also a potent P2Y1 antagonist.25

In the present study, we combined the ring-constrained (N)-methanocarba modification of adenine nucleotides with other adenine 2-position functionalities to prepare novel analogues, which were evaluated for affinity at the human P2Y1 receptor. In one case, a 2-iodo substitution conferred a significant increase in antagonist potency. New synthetic routes to antagonists in this series, including methods for introduction of phosphate groups as either the terminal step or prior to the coupling of sugar and nucleobase, were explored. The 2-iodo substituent could also be introduced at various stages of the synthesis.

Experimental Section

Chemical Synthesis

Nucleosides and synthetic reagents were purchased from Sigma Chemical Co. (St. Louis, MO) and Aldrich (Milwaukee, WI). 2,6-Dichloropurine was obtained from Sigma. The protected intermediate 19 was synthesized in our laboratory as described.45 5 and 9 were prepared as described.18 37 and 38 were prepared as described.37

1H NMR spectra were obtained with a Varian Gemini-300 spectrometer (300 MHz) with D2O, CDCl3, CD3OD, and DMSOd6 as a solvent. Tetramethylsilane (TMS) was the external standard. 31P NMR spectra were recorded at room temperature with a Varian XL-300 spectrometer (121.42 MHz); orthophosphoric acid (85%) was used as an external standard.

Purity of compounds was checked with a Hewlett-Packard 1100 HPLC apparatus equipped with a Luna 5µ C18(2) analytical column (250 × 4.6 mm, Phenomenex, Torrance, CA) in two solvent systems.

System A: linear gradient solvent system, 0.1 M TEAA/CH3-CN from 95/5 to 40/60 in 20 min; the flow rate was 1 mL/min.

System B: linear gradient solvent system, 5 mM TBAP/CH3-CN from 80/20 to 40/60 in 20 min; the flow rate was 1 mL/min.

Peaks were detected by UV absorption with a diode array detector. All derivatives tested for biological activity showed ≥ 97% purity in the HPLC systems.

Low-resolution CI–NH3 (chemical ionization) mass spectra were measured with a Finnigan 4600 mass spectrometer, and high-resolution EI (electron impact) mass spectrometry was performed with a VG7070F mass spectrometer at 6 kV. High-resolution FAB (fast atom bombardment) mass spectrometry was performed with a JEOL SX102 spectrometer with 6-kV Xe atoms following desorption from a glycerol matrix.

Purification of the nucleotide analogues for biological testing was carried out on Sephadex-DEAE-A-25 resin columns with a linear gradient (0.01–0.5 M) of 0.5 M ammonium bicarbonate applied as the mobile phase.

(1′R,2′S,4′S,5′S)-4-(6-Amino-2-iodo-9H-purin-9-yl)-1-[(phosphato)methyl]-2-(phosphato)bicyclo[3.1.0]-hexane Tetraammonium Salt (7)

Compound 29 (1.48 mg, 0.0023 mmol) was dissolved in 30% NH3 in water (0.50 mL) and stirred for 6.5 h at 60 °C in a sealed tube. The solvent was removed under reduced pressure.

Purification of the obtained residue was performed on an ion-exchange column packed with Sephadex-DEAE A-25 resin. A linear gradient (0.01–0.5 M) of 0.5 M ammonium bicarbonate was applied as the mobile phase, and UV and HPLC were used to monitor the elution, which furnished 7 (0.77 mg, 54%): 1H NMR (D2O) δ 8.58 (s, 1H), 5.20–4.80 (m, 2H), 4.60–4.40 (m, 1H), 3.60–3.56 (m, 1H), 2.35–2.20 (m, 1H), 2.00–1.88 (m, 1H), 1.88–1.80 (m, 1H), 1.18–1.12 (m, 1H), 0.99–0.91 (m, 1H); 31P NMR (D2O) δ 0.17, −0.25 (2s, 3′-P, 5′-P); MS (m/e) (negative-FAB) 546 (M+ − 1); HRMS (negative−FAB) calcd for C12H15N5O8P2I 545.9441, found 545.9454; HPLC 8.3 min (99%) (system A), 14.3 min (99%) (system B).

(1′R,2′S,4′S,5′S)-4-(6-Amino-2-methylthio-9H-purin-9-yl)-1-[(phosphato)methyl]-2-(phosphato)bicyclo[3.1.0]-hexane Tetraammonium Salt (8)

To a solution of 26 (1.70 mg, 0.0020 mmol) in CH2Cl2 (0.30 mL) was added 1.0 M BCl3 in CH2Cl2 (0.50 mL) and the mixture stirred for 2 d at 5 °C. The reaction was quenched upon addition of 5.0 mL of triethylammonium bicarbonate buffer (1.0 M). The mixture was subsequently frozen and lyophilized, which furnished crude 8 (1.90 mg): MS (m/e) (negative-FAB) 485, 487 (peak height ratio = 3:1) (M+ − 1); HPLC 15.6 min (99%) (system B).

The crude sample of 8 (1.90 mg) was dissolved in 30% NH3 in water (3.0 mL) and stirred for 2 h at 70 °C in a sealed tube. The solvent was removed under reduced pressure, the obtained residue was purified on an ion-exchange column packed with Sephadex-DEAE A-25 resin, a linear gradient (0.01–0.5 M) of 0.5 M ammonium bicarbonate was applied as the mobile phase, and UV and HPLC were used to monitor the elution, which furnished homogeneous 8 (1.90 mg, 94%): 1H NMR (D2O) δ 8.44 (s, 1H), 5.30–5.20 (m, 1H), 5.09 (d, 1H, J = 6.5 Hz), 4.90–4.75 (m, 1H), 3.70–3.55 (m, 1H), 2.60 (s, 3H), 2.30–2.20 (m, 1H), 2.00–1.94 (m, 1H), 1.85–1.80 (m, 1H), 1.17–1.14 (m, 1H), 0.96–0.94 (m, 1H); 31P NMR (D2O) δ 2.03, 1.36 (2s, 3′-P, 5′-P); MS (m/e) (negative-FAB) 466 (M+ − 1); HRMS (negative−FAB) calcd for C13H18N5O8P2S 466.0374, found 466.0363; HPLC 8.7 min (99%) in solvent system A, 14.1 min (99%) in a solvent system B.

(1′R,2′S,4′S,5′S)-4-(2-Fluoro-6-methylamino-9H-purin-9-yl)-1-[(phosphato)methyl]-2-(phosphato)bicyclo[3.1.0]-hexane Tetraammonium Salt (10)

The mixture of 44 (5 mg, 0.0074 mmol) in 5% TFA/CH2Cl2 (5 mL) was stirred at room temperature for 3 h. After solvent was removed, the residue was treated with triethylammonium bicarbonate buffer (1 M, 0.5 mL) and stirred for 30 min. The aqueous layer was washed twice with CH2Cl2 and evaporated to dryness under reduced pressure. The residue was purified with ion-exchange column chromatography with the use of Sephadex-DEAE-A-25 resin with a linear gradient (0.01–0.5 M) of 0.5 M NH4-HCO3 as the mobile phase. After lyophilization, 10 (1.38 mg, 36%) was obtained as a white solid: 1H NMR (D2O) δ 8.25 (s, 1H), 5.25–5.1 (m, 2H), 3.8–3.65 (m, 2H), 3.05 (s, 3H), 2.4–2.1 (1H), 2.1–1.9 (m, 2H), 1.1–0.9 (1H); 31P NMR (D2O) 0.878 (s), 1.2 (s); HRMS (m/e) (negative-FAB) calcd forC13H18FN5O8P2 452.0536, found 452.0548; HPLC 8.2 (99%) min in solvent system A, 16.1 min (99%) in solvent system B.

(1′R,2′S,4′S,5′S)-[4-(2-Bromo-6-methylamino-9H-purin-9-yl)-1-[(phosphato)methyl]-2-(phosphato)bicyclo[3.1.0]-hexane Tetraammonium Salt (11)

The mixture of 45 (3.0 mg, 0.0041 mmol) in 5% TFA/CH2Cl2 (5 mL) was stirred at room temperature for 3 h. After removal of the solvent, the residue was treated with triethylammonium bicarbonate buffer (1 M, 0.5 mL) and stirred for 30 min. The aqueous layer was washed twice with CH2Cl2 and evaporated to dryness. The residue was purified with ion-exchange column chromatography with the use of Sephadex-DEAE-A-25 resin with a linear gradient (0.01–0.5 M) of 0.5 M NH4HCO3 as the mobile phase. After lyophilization, 11 (1.36 mg, 58%) was obtained as a white solid: 1H NMR (D2O) δ 8.42 (s, 1H), 5.4–5.1 (m, 2H), 3.8–3.6 (m, 2H), 3.1 (s, 3H), 2.4–2.1 (m, 2H), 2.1–1.8 (m, 2H), 1.1–0.9 (1H); 31P NMR (D2O) δ 0.4 (s), 0.9 (s); HRMS (m/e) (negative-FAB) calcd for C13H18BrN5O8P2 511.9736, found 511.9763; HPLC 9.3 min (99%) in solvent system A, 14.5 min (99%) in solvent system B.

(1′R,2′S,4′S,5′S)-4-(2-Iodo-6-methylaminopurin-9-yl)-1-[(phosphato)methyl]-2-(phosphato)bicyclo[3.1.0]-hexane (12)

Method A

Compound 29 (3.61 mg, 0.0057 mmol) was dissolved in 40% MeNH2 in water (2.0 mL) and stirred for 26 h at room temperature. The solvent was removed under reduced pressure. Purification of the obtained residue was performed on an ion-exchange column packed with Sephadex-DEAE A-25 resin, a linear gradient (0.01–0.5 M) of 0.5 M ammonium bicarbonate was applied as the mobile phase, and UV and HPLC were used to monitor the elution, which furnished 12 (0.73 mg, 20%).

Method B

A solution of the 2-iodo bis(di-tert-butyl phosphate) derivative 46 (7.1 mg, 9.03 µmol) in 5% trifluoroacetic acid in methylene chloride was stirred for 2 h at room temperature. All volatile material was removed in vacuo. The residue was purified by using ion-exchange column chromatography on Sephadex-DEAE-25 resin and a linear gradient (0–0.5 M) of 0.5 M ammonium bicarbonate as the mobile phase to give the 2-iodo bisphosphate derivative 12 (2.9 mg, 55%) as an ammonium salt: 1H NMR (D2O) δ 8.54 (bs, 1H), 5.19 (m, 1H), 5.01 (d, 1H, J = 6.9 Hz), 4.58 (dd, 1H, J = 4.7, 11.3 Hz), 3.73 (dd, 1H, J = 4.4, 11.0 Hz), 3.07 (bs, 3H), 2.28 (dd, 1H, J = 7.7, 14.6 Hz), 1.92–2.09 (m, 2H), 1.26 (dd, 1H, J = 4.1, 6.1 Hz), 1.06 (dd, 1H, J = 9.7, 16.3 Hz); 31P NMR (D2O) 0.651 (s); HRMS (negative-ion FAB) calcd for C13H17N5O8P2I 559.9597, found 559.9604; HPLC 9.8 min (97%) in solvent system A, 15.4 min (97%) in system B.

(1′R,2′S,4′S,5′S)-4-(6-Methylamino-2-methylthio-9H-purin-9-yl)-1-[(phosphato)methyl]-2-(phosphato)bicyclo-[3.1.0]hexane Tetraammonium Salt (13)

To a solution of 28 (4.2 mg, 0.0050 mmol) in CH2Cl2 (0.30 mL) was added 1.0 M BCl3 in CH2Cl2 (0.60 mL) and the mixture stirred for 2 d at 5 °C. The reaction was quenched upon the addition of 5.0 mL of triethylammonium bicarbonate buffer (1.0 M). The mixture was subsequently frozen and lyophilized.

Purification of the obtained residue was performed on an ion-exchange column packed with Sephadex-DEAE A-25 resin, a linear gradient (0.01–0.5 M) of 0.5 M ammonium bicarbonate was applied as the mobile phase, and UV and HPLC were used to monitor the elution, which furnished 13 (1.69 mg, 81%): 1H NMR (D2O) δ 8.45 (s, 1H), 5.25–5.15 (m, 1H), 5.08–5.02 (m, 1H), 4.65–4.60 (m, 1H), 3.65–3.55 (m, 1H), 3.13 (bs, 3H), 2.62 (s, 3H), 2.30–2.20 (m, 1H), 2.00–1.85 (m, 1H), 1.85–1.75 (m, 1H), 1.15–1.10 (m, 1H), 1.00–0.95 (m, 1H); 31P NMR (D2O) δ 2.21, 2.90 (2s, 3′-P, 5′-P); MS (m/e) (negative-FAB) 480 (M+ − 1); HRMS (negative–FAB) calcd for C14H20N5O8P2S 480.0488, found 480.0498; HPLC 10.3 min (99%) in solvent system A, 16.4 min (99%) in solvent system B.

(1′R,2′S,4′S,5′S)-4-(6-Methylamino-2-methylselenopurin-9-yl)-1-[(phosphato)methyl]-2-(phosphato)bicyclo-[3.1.0]hexane Tetraammonium Salt (14)

Compound 30 (0.38 mg, 0.63 µmol) was dissolved in 40% MeNH2 in water (3.0 mL) and stirred for 8 h at 80 °C. The solvent was removed under reduced pressure. Purification of the obtained residue was performed on an ion-exchange column packed with Sephadex-DEAE A-25 resin, a linear gradient (0.01–0.5 M) of 0.5 M ammonium bicarbonate was applied as the mobile phase, and UV and HPLC were used to monitor the elution, which furnished 14 (0.30 mg, 79%): 1H NMR (D2O) δ 8.51 (s, 1H), 5.10–5.00 (m, 1H), 5.00–4.90 (m, 1H), 4.74–4.72 (m, 1H), 3.65–3.50 (m, 1H), 3.14 (bs, 3H), 2.53 (s, 3H), 2.40–2.18 (m, 1H), 2.05–1.85 (m, 1H), 1.85–1.75 (m, 1H), 1.25–1.14 (m, 1H), 1.00–0.95 (m, 1H); 31P NMR (D2O) δ 3.00, 3.53 (2s, 3′-P, 5′-P); MS (m/e) (negative-FAB) 528 (M+ − 1), HRMS (negative–FAB) calcd for C14H20N5O8P2Se 527.9948, found 527.9950; HPLC 10.8 min (99%) in solvent system A, 16.8 min (99%) in solvent system B.

(1′R,2′S,4′S,5′S)-Phosphoric Acid [4-(2-Methyl-6-methylaminopurin-9-yl)-1-phosphonooxymethylbicyclo[3.1.0]-hex-2-yl] Monoester (15)

A solution of 47 (8.0 mg, 0.012 mmol) in 5% TFA/CH2Cl2 (2.0 mL) was stirred for 3 h at 25 °C. The solvent was removed under reduced pressure and the residue was quenched by addition of 5.0 mL of triethylammonium bicarbonate buffer (1.0 M). The mixture was subsequently frozen and lyophilized. Purification of the obtained residue was performed on an ion-exchange column packed with Sephadex-DEAE A-25 resin, a linear gradient (0.01–0.5 M) of 0.5 M ammonium bicarbonate was applied as the mobile phase, and UV and HPLC were used to monitor the elution, which furnished 15 (4.5 mg, 73%): 1H NMR (D2O) δ 8.45 (s, 1H), 5.18 (m, 1H), 5.00 (d, 1H, J = 5.4 Hz), 4.57–4.50 (m, 1H), 3.69–3.62 (m, 1H), 3.13 (s, 3H), 2.53 (s, 3H), 2.30–2.20 (m, 1H), 2.24–1.92 (m, 1H), 1.88 (m, 1H), 1.19 (m, 1H), 0.98 (m, 1H); 31P NMR (D2O) δ 1.05, 0.65 (2s, 3′-P, 5′-P); MS (m/e) (negative-FAB) 448 (M+ − 1); HRMS (negative-FAB) calcd for C14H20N5O8P2 448.0787, found 448.0807; HPLC 7.3 min (99%) (system A), 14.2 min (99%) (system B).

(1′R,2′S,4′S,5′S)-4-(2-Hexyl-6-methylaminopurin-9-yl)-1-[(phosphato)methyl]-2-(phosphato)bicyclo[3.1.0]-hexane (16)

The 2-hexanyl bisphosphate derivative 16 (5.6 mg, 69%) was prepared by the same method as above for 11, except the 2-hexanyl bis(di-tert-butyl phosphate) derivative 48 (11.2 mg, 0.015 mmol) was used as the starting material: 1H NMR (D2O) δ 8.54 (bs, 1H), 5.23 (m, 1H), 5.10 (d, 1H, J = 6.6 Hz), 4.57 (dd, 1H, J = 5.2, 11.3 Hz), 3.70 (m, 1H), 3.25 (bs, 3H), 2.85 (dd, 2H, J = 7.4, 7.7 Hz), 2.29 (dd, 1H, J = 7.7, 15.1 Hz), 2.03 (dt, 1H, J = 6.6, 16.8 Hz), 1.92 (dd, 1H, J = 3.8, 9.1 Hz), 1.773 (m, 2H), 1.13–1.43 (m, 9H), 1.03 (dd, 1H, J = 6.6, 7.7 Hz), 0.85 (m, 3H); 31P NMR (D2O) 0.827 (s), 0.375 (s); HRMS (negative-ion FAB) calcd for C19H30N5O8P2 518.1570, found 518.1544; HPLC 13.2 min (98%) in solvent system A, 16.6 min (97%) in system B.

(1′R,2′S,4′S,5′S)-4-(2-Hex-1-ynyl-6-methylaminopurin-9-yl)-1-[(phosphato)methyl]-2-(phosphato)bicyclo[3.1.0]-hexane (17)

The 2-hexenyl bisphosphate derivative 17 (3.2 mg, 68%) was prepared by the same method as above for 11, except the 2-hexenyl bis(di-tert-butyl phosphate) derivative 49 (6.0 mg, 8.08 µmol) was used as the starting material: 1H NMR (D2O) δ 8.54 (s, 1H), 7.18 (dt, 1H, J = 7.1, 15.4 Hz), 6.45 (m, 1H), 5.22 (m, 1H), 5.08 (d, 1H, J = 6.3 Hz), 4.57 (dd, 1H, J = 4.7, 11.0 Hz), 3.71 (dd, 1H, J = 5.0, 10.7 Hz), 3.22 (bs, 3H), 2.23–2.44 (m, 3H), 2.03 (m, 1H), 1.95 (dd, 1H, J = 3.7, 8.8 Hz), 1.32–1.61 (m, 4H), 1.24 (dd, 1H, J = 4.1, 5.5 Hz), 1.05 (dd, 1H, J = 6.3, 8.2 Hz), 0.94 (m, 3H); 31P NMR (D2O) 0.601 (s), 0.299 (s); HRMS (negative-ion FAB) calcd for C19H28N5O8P2 516.1413, found 516.1403; HPLC 13.5 min (99%) in solvent system A, 16.9 min (99%) in solvent system B.

(1′R,2′S,4′S,5′S)-4-(2-Hex-1-enyl-6-methylaminopurin-9-yl)-1-[(phosphato)methyl]-2-(phosphato)bicyclo[3.1.0]-hexane (18)

The 2-hexynyl bisphosphate derivative 18 (4.0 mg, 73%) was prepared by the same method as above for 11, except the 2-hexynyl bis(di-tert-butyl phosphate) derivative 50 (7.0 mg, 9.46 µmol) was used as the starting material: 1H NMR (D2O) δ 8.46 (s, 1H), 5.16 (m, 1H), 4.97 (d, 1H, J = 6.3 Hz), 4.56 (dd, 1H, J = 4.7, 11.3 Hz), 3.70 (dd, 1H, J = 6.3, 11.3 Hz), 3.07 (bs, 3H), 2.50 (t, 2H, J = 7.1 Hz), 2.24 (dd, 1H, J = 7.4, 14.6 Hz), 1.91–2.05 (m, 2H), 1.42–1.71 (m, 4H), 1.23 (dd, 1H, J = 4.1, 5.8 Hz), 1.03 (dd, 1H, J = 6.1, 8.2 Hz), 0.95 (t, 3H, J = 7.2 Hz); 31P NMR (D2O) 0.777 (s), 0.375 (s); HRMS (negative-ion FAB) calcd for C19H26N5O8P2 514.1257, found 514.1234; HPLC 12.4 min (98%) in solvent system A, 12.3 min (99%) in solvent system B.

General (Phosphorylation) Procedure: Synthesis of (N)-Methanocarbaadenosine Derivatives (21–57). (1′R,2′S,-4′S,5′S)-4-(2-Amino-6-chloro-9H-purin-9-yl)-1-(benzyloxymethyl)-2-benzyloxybicyclo[3.1.0]hexane (21)

To a solution of triphenylphosphine (0.20 g, 0.76 mmol) in anhydrous tetrehydrofuran (THF, 1.0 mL) was added dropwise diisopropyl azodicarboxylate (DIAD, 0.15 mL, 0.76 mmol) at −20 °C with stirring for 0.5 h. Compound 19 (76 mg, 0.23 mmol) and 2-amino-6-chloropurine 20 (92 mg, 0.54 mmol) in THF (5.0 mL) were added to the reaction mixture and it stirred for 18.5 h at room temperature. The solvent was removed under vacuum, and the residue obtained was purified by using flash chromatography with 1/1 petroleum ether/ethyl acetate to furnish 21 (103 mg, 92%): 1H NMR (CDCl3) δ 8.58 (s, 1H), 7.40–7.21 (m, 10H), 5.01 (bs, 1H), 4.97 (d, 1H, J = 6.6 Hz), 4.66 (t, 1H, J = 8.25 Hz), 4.54 (s, 2H), 4.45 (d, 1H, J = 11.7 Hz), 4.38 (d, 1H, J = 11.7 Hz), 4.09 (d, 1H, J = 9.8 Hz), 3.14 (d, 1H, J = 9.8 Hz), 2.09–2.00 (m, 1H), 1.85–1.74 (m, 1H), 1.59–1.54 (m, 1H), 1.11–1.06 (m, 1H), 0.81–0.75 (m, 1H); MS (m/e) (positive-FAB) 476, 478 (peak height ratio = 3:1) (M+ + 1).

(1′R,2′S,4′S,5′S)-4-(2-Amino-6-chloro-9H-purin-9-yl)-1-(hydroxymethyl)-2-hydroxybicyclo[3.1.0]hexane (22)

Compound 21 (169 mg, 0.353 mmol) was dissolved in CH2Cl2 (5.0 mL) and treated with BCl3 (1 M in CH2Cl2, 1.50 mL) for 1 h at −40 °C under argon. i-PrOH (0.40 mL) was added to the reaction mixture and it was warmed to room temperature. The solvent was evaporated, and the crude product was purified by flash column chromatography with AcOEt/i-PrOH (1/1) to yield 22 as a white solid (72 mg, 69%): 1H NMR (CD3OD) δ 8.54 (s, 1H), 4.94 (d, 1H, J = 6.6 Hz), 4.26 (d, 1H, J = 11.7 Hz), 4.03 (s, 1H), 3.33 (d, 1H, J = 11.7 Hz), 2.09–1.98 (m, 1H), 1.84–1.73 (m, 1H), 1.67–1.62 (m, 1H), 1.04–0.99 (m, 1H), 0.79–0.74 (m, 1H); MS (m/e) (negative-FAB) 294, 296 (peak height ratio = 3:1) (M+ − 1).

(1′R,2′S,4′S,5′S)-4-(2-Amino-6-chloro-9H-purin-9-yl)-1-[(dibenzylphosphato)methyl]-2-(dibenzylphosphato)-bicyclo[3.1.0]hexane (24)

To a stirred solution of 22 (56 mg, 0.190 mmol) and 1H-tetrazole (130 mg, 1.86 mmol) in 3.0 mL of anhydrous THF was added dibenzyl diisopropylphosphoramidite (23, 0.30 mL, 0.89 mmol), and the mixture was stirred for 4.5 h at room temperature. The reaction mixture was cooled to −78 °C and treated with a solution of m-chloroperbenzoic acid (m-CPBA, 70% max, 350 mg) in CH2Cl2 (5.0 mL). The resulting mixture was warmed to room temperature, and the solvent was removed under reduced pressure. The residue obtained was purified by flash column chromatography (1/1 = AcOEt/CH2Cl2), which furnished 24 (52 mg, 35%): 1H NMR (CDCl3) δ 7.82 (s, 1H), 7.33–7.25 (m, 20H), 5.41 (dd, 1H, J = 7.8, 14.1 Hz), 5.27 (bs, 2H), 5.05–4.91 (m, 8H), 4.74 (d, 1H, J = 6.9 Hz), 4.54 (dd, 1H, J = 5.7, 11.1 Hz), 3.94 (dd, 1H, J = 6.0, 11.1 Hz), 2.14–2.04 (m, 1H), 1.98–1.87 (m, 1H), 1.62–1.52 (m, 1H), 1.00–0.93 (m, 1H), 0.87–0.80 (m, 1H); MS (m/e) (positive-FAB) 816, 818 (peak height ratio = 3:1) (M+ + 1).

(1′R,2′S,4′S,5′S)-4-(6-Chloro-2-iodo-9H-purin-9-yl)-1-[(dibenzylphosphato)methyl]-2-(dibenzylphosphato)-bicyclo[3.1.0]hexane (25)

To a solution of 24 (62.1 mg, 0.0761 mmol) in acetonitrile (0.50 mL) were added diiodomethane (2.0 mL) and tert-butyl nitrite (0.10 mL, 1.11 mmol) were added, and oxygen was purged by N2 bubbling. The tube was sealed and stirred at 85 °C for 3.0 h. Diiodomethane was removed upon passage though a short column (silica gel, eluting with CHCl3, then AcOEt). The residue obtained was purified by preparative thin-layer chromatography (AcOEt), which furnished 25 (25.8 mg, 37%): 1H NMR (CDCl3) δ 8.19 (s, 1H), 7.36–7.25 (m, 20H), 5.29 (q, 1H, J = 6.9 Hz), 5.10–4.92 (m, 9H), 4.61 (dd, 1H, J = 6.3, 12.0 Hz), 3.84 (dd, 1H, J = 6.6, 12.0 Hz), 2.05–1.88 (m, 2H), 1.62–1.52 (m, 1H), 1.02 (m, 1H), 0.95–0.84 (m, 1H); MS (m/e) (positive-FAB) 928, 930 (peak height ratio = 3:1) (M+ + 1).

(1′R,2′S,4′S,5′S)-4-(6-Chloro-2-methylthio-9H-purin-9-yl)-1-[(dibenzylphosphato)methyl]-2-(dibenzylphosphato)-bicyclo[3.1.0]hexane (26)

To a solution of 24 (4.5 mg, 0.0056 mmol) in MeCN (0.25 mL) were added dimethyl disulfide (0.15 mL, 1.26 mmol) and tert-butylnitrite (0.050 mL, 0.56 mmol), and oxygen was purged by nitrogen bubbling. The tube was sealed and stirred at 70 °C for 5.5 h. The solvent was removed under reduced pressure. The residue obtained was purified by preparative thin-layer chromatography (2/1=AcOEt/CH2Cl2), which furnished 26 (3.0 mg, 64%): 1H NMR (CDCl3) δ 8.15 (s, 1H), 7.33–7.25 (m, 20H), 5.30 (dd, 1H, J = 7.8, 14.7 Hz), 5.07–4.91 (m, 9H), 4.62 (dd, 1H, J = 6.0, 11.5 Hz), 3.74 (dd, 1H, J = 6.6, 11.5 Hz), 2.61 (s, 3H), 2.14–2.04 (m, 1H), 1.98–1.87 (m, 1H), 1.63–1.56 (m, 1H), 1.07–1.03 (m, 1H), 0.88–0.81 (m, 1H); MS (m/e) (positive-FAB) 847, 849 (peak height ratio = 3:1) (M+ + 1).

(1′R,2′S,4′S,5′S)-4-(6-Chloro-2-methylseleno-9H-purin-9-yl)-1-[(dibenzylphosphato)methyl]-2-(dibenzylphosphato)bicyclo[3.1.0]hexane (27)

To a solution of 24 (16.0 mg, 0.0196 mmol) in MeCN (0.30 mL) were added dimethyldiselenide (0.10 mL, 1.06 mmol) and tert-butyl nitrite (0.100 mL, 1.11 mmol), and oxygen was purged by nitrogen bubbling. The tube was sealed and stirred at 65 °C for 20 h. The solvent was removed under reduced pressure. The residue obtained was purified by preparative thin-layer chromatography (1/1 = AcOEt/CH2Cl2), which furnished 27 (6.7 mg, 38%): 1H NMR (CDCl3) δ 8.16 (s, 1H), 7.40–7.25 (m, 20H), 5.28 (dd, 1H, J = 8.4, 14.4 Hz), 5.10–4.90 (m, 9H), 4.62 (dd, 1H, J = 6.0, 11.4 Hz), 3.74 (dd, 1H, J = 6.6, 14.4 Hz), 2.53 (s, 3H), 2.15–2.00 (m, 1H), 2.00–1.85 (m, 1H), 1.75–1.65 (m, 1H), 1.45–1.35 (m, 1H), 1.10–1.00 (m, 1H); MS (m/e) (positive-FAB) 895, 897 (peak height ratio = 3:1) (M+ + 1).

(1′R,2′S,4′S,5′S)-4-(6-Methylamino-2-methylthio-9H-purin-9-yl)-1-[(dibenzylphosphato)-methyl]-2-(dibenzylphosphato)bicyclo[3.1.0]hexane (28)

Compound 26 (3.0 mg, 0.0035 mmol) was dissolved in 2.0 M MeNH2 in THF (0.50 mL) and stirred for 2.5 h at room temperature. The solvent was removed under reduced pressure. The residue obtained was purified by preparative thin-layer chromatography (AcOEt), which furnished 28 (2.7 mg, 91%): 1H NMR (CDCl3) δ 7.77 (s, 1H), 7.30–7.25 (m, 20H), 5.72 (bs, 1H), 5.38 (dd, 1H, J = 7.5, 14.1 Hz), 5.03–4.91 (m, 9H), 4.59 (dd, 1H, J = 6.3, 11.4 Hz), 3.78 (dd, 1H, J = 5.1, 11.4 Hz), 3.19 (bs, 3H), 2.57 (s, 3H), 2.19–2.10 (m, 1H), 1.96–1.82 (m, 1H), 1.60–1.55 (m, 1H), 1.04–1.00 (m, 1H), 0.88–0.78 (m, 1H); MS (m/e) (positive-FAB) 842 (M+ + 1).

(1′R,2′S,4′S,5′S)-4-(6-Chloro-2-iodo-9H-purin-9-yl)-1-[(phosphato)methyl]-2-(phosphato)bicyclo[3.1.0]-hexane Tetraammonium Salt (29)

To a solution of 25 (13.6 mg, 0.015 mmol) in CH2Cl2 (0.80 mL) was added 1.0 M BCl3 in CH2Cl2 (0.80 mL) and the mixture stirred for 24 h at 5 °C. The reaction was quenched upon addition of 3.0 mL of triethylammonium bicarbonate buffer (1.0 M). The mixture was subsequently frozen and lyophilized. Purification of the residue obtained was performed on an ion-exchange column packed with Sephadex-DEAE A-25 resin. A linear gradient (0.01–0.5 M) of 0.5 M ammonium bicarbonate was applied as the mobile phase, and UV and HPLC were used to monitor the elution, which furnished 29 (5.56 mg, 60%): 1H NMR (D2O) δ 8.83 (s, 1H), 5.30–5.20 (m, 1H), 5.16 (d, 1H, J = 6.3 Hz), 4.60–4.50 (m, 1H), 3.75–3.65 (m, 1H), 2.40–2.20 (m, 1H), 2.10–1.95 (m, 1H), 1.95–1.90 (m, 1H), 1.25–1.20 (m, 1H), 1.05–1.00 (m, 1H); 31P NMR (D2O) δ 2.02, 1.40 (2s, 3′-P, 5′-P); MS (m/e) (negative-FAB) 565, 567 (peak height ratio = 3:1) (M+ − 1); HPLC 9.8 min (98%) in solvent system A, 16.0 min (98%) in solvent system B.

(1′R,2′S,4′S,5′S)-4-(6-Chloro-2-methylseleno-9H-purin-9-yl)-1-[(phosphato)methyl]-2-(phosphato)bicyclo[3.1.0]-hexane Tetraammonium Salt (30)

To a solution of 27 (6.7 mg, 0.0075 mmol) in CH2Cl2 (0.50 mL) was added 1.0 M BCl3 in CH2Cl2 (0.80 mL) and the mixture stirred for 2 d at 5 °C. The reaction was quenched upon addition of 5.0 mL of triethylammonium bicarbonate buffer (1.0 M). The mixture was subsequently frozen and lyophilized. Purification of the obtained residue was performed on an ion-exchange column packed with Sephadex-DEAE A-25 resin, a linear gradient (0.01–0.5 M) of 0.5 M ammonium bicarbonate was applied as the mobile phase, and UV and HPLC were used to monitor the elution, which furnished 30 (1.28 mg, 28%): 1H NMR (D2O) δ 8.78 (s, 1H), 5.35–5.22 (m, 2H), 4.60–4.50 (m, 1H), 3.70–3.65 (m, 1H), 2.61 (s, 3H), 2.45–2.30 (m, 1H), 2.15–1.95 (m, 1H), 1.95–1.85 (m, 1H), 1.25–1.18 (m, 1H), 1.05–0.95 (m, 1H); 31P NMR (D2O) δ 1.67, 1.09 (2s, 3′-P, 5′-P); MS (m/e) (negative-FAB) 533, 535 (peak height ratio = 3:1) (M+ − 1), HRMS (negative–FAB) calcd for C13H16N4O8P2Se 532.9293, found 532.9295; HPLC 11.0 min (99%) in solvent system A, 15.5 min (99%) in solvent system B.

(1′R,2′S,4′R,5′S)-Acetic Acid 4-Hydroxy-5-hydroxymethylbicyclo[3.1.0]hex-2-yl Ester (32)

A mixture of dibenzyl alcohol 19 (137 mg, 0.422 mmol), acetic anhydride (140.4 mg, 1.38 mmol), triethylamine (436 mg, 4.3 mmol), 4-(dimethylamino)pyridine (10 mg, 0.2 mmol), and dichloromethane (5 mL) was stirred at room temperature for 4 h. The resulting mixture was partitioned between ethyl acetate (50 mL) and water/brine (1/1, 20 mL). The organic layer was washed with saturated aqueous sodium bicarbonate (10 mL) and brine (10 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was used in the next reaction without further purification. The dibenzyl acetate 31 (crude 164 mg) was dissolved in methanol (5 mL). Five percent Pd/C (150 mg) was added. The reaction flask was applied to a vacuum and filled with hydrogen several times. The reaction mixture was stirred for 4 h at room temperature under a hydrogen balloon. The resulting mixture was filtrated through a pad of Celite and washed with methanol (10 mL × 3). The residue was purified by flash column chromatography (chloroform/methanol = 10/1) to give a diol 32 (76 mg, 97% for two steps yield): 1H NMR (CDCl3) δ 5.13 (m, 1H), 4.45 (t, 1H, J = 8.2 Hz), 3.96 (d, 1H, J = 11.8 Hz), 3.24 (d, 1H, J = 11.8 Hz), 2.33 (dt, 1H, J = 8.0, 13.2 Hz), 2.01 (s, 3H), 1.66 (m, 1H), 1.27 (dt, 1H, J = 8.8, 13.5 Hz), 1.09 (dd, 1H, J = 3.8, 5.5 Hz), 0.55 (dd, 1H, J = 5.5, 8.0 Hz).

(1′R,2′S,4′R,5′S)-Acetic Acid 4-(Di-tert-butoxyphosphoryloxy)-5-(di-tert-butoxyphosphoryloxymethyl)-bicyclo[3.1.0]hex-2-yl Ester (33)

To a stirred suspension of diol 32 (25 mg, 0.134 mmol) and diethyl di-tert-butylphosphoramidite (100.5 mg, 0.402 mmol) in anhydrous tetrahydrofuran (4 mL) at room temperature was added solid tetrazole (94 mg, 1.34 mmol) in one pot. The reaction mixture was stirred at room temperature for 1 h and then cooled to −78 °C. To this cooled reaction mixture was added solid m-CPBA (57–85%, calculated by 57%, 240 mg), and the resulting mixture was stirred at −78 °C for 20 min and at room temperature for 10 min. After removal of tetrahydrofuran in vacuo, the residue was directly purified by flash column chromatography (CHCl3/MeOH = 10/1) to give 33 (71 mg, 93%): 1H NMR (CDCl3) δ 4.87 (dd, 1H, J = 8.0, 14.6 Hz), 4.41 (m, 1H), 4.40 (dd, 1H, J = 7.7, 11.0 Hz), 3.69 (dd, 1H, J = 6.9, 11.0 Hz), 2.46 (dt, 1H, J = 7.7, 13.5 Hz), 1.78 (m, 1H), 1.485 (s, 9H), 1.478 (s, 9H), 1.469 (s, 9H), 1.457 (s, 9H), 1.39 (m, 1H), 1.25 (dd, 1H, J = 5.0, 5.2 Hz), 0.67 (dd, 1H, J = 6.3, 7.4 Hz).

(1′R,2′S,4′R,5′S)-Phosphoric Acid Di-tert-butyl Ester 1-(Di-tert-butoxyphosphoryloxymethyl)-4-hydroxybicyclo[3.1.0]hex-2-yl Ester (34)

Acetate 33 (70 mg, 0.122 mmol) was dissolved in methanol (3 mL), and then solid potassium carbonate (20 mg, 0.145 mmol) was added. After the reaction mixture was stirred for 2 h, methanol was removed in vacuo and the residue was purified by flash column chromatography (CHCl3/MeOH = 30/1) to produce the bisphosphate alcohol 34 (51 mg, 79%): 1H NMR (CDCl3) δ 5.13 (m, 1H), 4.45 (t, 1H, J = 8.2 Hz), 3.96 (d, 1H, J = 11.8 Hz), 3.24 (d, 1H, J = 11.8 Hz), 2.33 (dt, 1H, J = 8.0, 13.2 Hz), 2.01 (s, 3H), 1.66 (m, 1H), 1.27 (dt, 1H, J = 8.8, 13.5 Hz), 1.09 (dd, 1H, J = 3.8, 5.5 Hz), 0.55 (dd, 1H, J = 5.5, 8.0 Hz); MS (m/e) (positive-FAB) 529.35 (M+ + 1), 551.31 (M+ + Na + 1).

2-Bromo-6-chloro-9H-purine (35)

To a stirred solution of 20 (0.35 g, 2 mmol) in CH2Br2 (5 mL) was added a solution of SbBr3 in CH2Br2 (5 mL) at −10 °C. This solution was stirred at this temperature for 10 min, and tert-butyl nitrite (4.5 mL) was added. The reaction mixture was stirred for 2.5 h at −10 °C and then was poured into a stirred mixture of 2.4 g of NaHCO3 in 100 mL of water and crushed ice. This mixture was filtered, and the filtrate was extracted with CHCl3 and dried over Na2SO4. After removal of the solvent, the residue was purified by flash chromatography (MeOH/CHCl3 = 1/10) to provide 35 (0.404 g, 87%): 1H NMR (DMSO-d6) δ 8.72 (s, 1H); MS (m/e) (positive-FAB) 233 (M+ + H).

6-Chloro-2-fluoro-9H-purine (36)

Compound 20 (100 mg, 0.572 mmol) was added to 60% HF–pyridine (which was prepared from 1.5 g of 70% HF–pyridine diluted with 0.25 g of pyridine) at −50 °C with stirring. The temperature was allowed to rise from −30 °C, and 0.1 mL of tert-butyl nitrite (0.1 mL) was added dropwise. Stirring was continued for 10 min, and the reaction mixture was poured into ice water. The aqueous layer was extracted with CHCl3 and ethyl acetate. The combined organic layer was washed with H2O, dried over Na2SO4, and filtered, and the filtrate was evaporated. The given residue was purified on a flash column chromatography (MeOH/CHCl3 = 1/10) to give 36 (90 mg, 91%) as a white solid: 1H NMR (CD3OD) δ 8.5 (s, 1H); MS (m/e) (positive-FAB) 173 (M+ + H).

2-Fluoro-6-methylamino-9H-purine (37)

The mixture of 36 (56 mg, 0.324 mmol) in CH3NH2 (2.0 M in THF, 3 mL) was stirred at room temperature overnight. After removal of the solvent, the residue was purified by preparative thin-layer chromatography (MeOH/CHCl3 = 1/10) to yield 37 (43 mg, 80%): 1H NMR (DMSO-d6) δ 8.12 (br, 1H), 8.06 (s, 1H), 3.30 (s, 1H), 2.91 (s, 3H); MS (m/e) (positive-FAB) 168 (M+ + H).

2-Bromo-6-methylamino-9H-purine (38)

The mixture of 35 (130 mg, 0.557 mmol) in CH3NH2 (40% in H2O, 2 mL) was heated to 70 °C and stirred at this temperature for 6 h. The reaction mixture was cooled to room temperature, and the formed white precipitate was filtered and washed with a small amount of water/MeOH. The precipitate was dried in a vacuum to give 38 (118 mg, 93%): 1H NMR (DMSO-d6) δ 8.08 (s, 1H), 8.06 (bs, 1H), 3.30 (s, 1H), 2.91 (s, 3H); MS (m/e) (positive-FAB) 228 (M+ + H).

(1′R,2′S,4′S,5′S)-4-(2-Fluoro-6-methylamino-9H-purin-9-yl)-1-(di-tert-butoxyphosphoryloxymethyl)bicyclo[3.1.0]-hex-2-yl-phosphoric Acid Di-tert-butyl Ester (44)

To an ice-cold solution of triphenylphosphine (23.6 mg, 0.09 mmol) in dry THF (0.5 mL) was added DIAD (18.7 µL, 0.09 mmol) dropwise under a nitrogen atmosphere, and the mixture was stirred for 20 min until the solution turned red. This mixture was added dropwise to a cold, stirred mixture of 34 (14.4 mg, 0.027 mmol) and 37 (15.03 mg, 0.09 mmol) under a nitrogen atmosphere. The reaction mixture was stirred in an ice bath for 30 min and then allowed to warm to room temperature, and stirring continued for 12 h. Solvent was removed by nitrogen purge, and the residue was purified by preparative thin-layer chromatography (MeOH/CHCl3 = 1/20) to produce 44 (8.61 mg, 47%) as a thick liquid: 1H NMR (CDCl3) δ 8.08 (s, 1H), 6.18 (br, 1H), 5.45–5.25 (m, 1H), 5.10–4.9 (m, 1H), 4.75–4.55 (m, 1H), 4.0–3.8 (m, 1H), 3.17 (s, 3H), 2.45–2.2 (m, 1H), 2.14–1.95 (m, 1H), 1.86–1.75 (m, 1H), 1.49 (s, 18H), 1.47 (s, 18H), 1.2–1.05 (m, 1H), 1.05–0.9 (m, 1H); MS (m/e) (positive-FAB) 678 (M+ + H).

(1′R,2′S,4′S,5′S)-4-(2-Bromo-6-methylamino-9H-purin-9-yl)-1-(di-tert-butoxyphosphoryloxymethyl)bicyclo[3.1.0]-hex-2-yl-phosphoric Acid Di-tert-butyl Ester (45)

To a solution of triphenylphosphine (15 mg, 0.057 mmol) in anhydrous THF (0.5 mL) was added DIAD (12 µL, 0.057 mmol) dropwise at 0 °C under a nitrogen atmosphere, and the mixture was stirred at this temperature for 20 min until the solution turned red. This reaction mixture was added slowly to a cold stirred solution of alcohol 34 (9.2 mg, 0.0174 mmol) and 38 (13.1 mg, 0.057 mmol) under a nitrogen atmosphere. The reaction mixture was stirred in an ice bath for 30 min and then allowed to warm to room temperature while stirring continued for 12 h. Solvent was removed by nitrogen purge, and the residue was purified by preparative thin-layer chromatography (MeOH/CHCl3 = 1/20) to produce 45 (3.1 mg, 23%) as a thick liquid: 1H NMR (CDCl3) δ 8.05 (s, 1H), 5.92 (br, 1H), 5.45–5.25 (m, 1H), 5.15–5.0 (m, 1H), 4.75–4.55 (m, 1H), 3.95–3.80 (m, 1H), 3.18 (s, 3H), 2.4–2.2 (m, 1H), 2.20–2.0 (m, 1H), 1.85–1.7 (m, 1H), 1.58 (s, 18H), 1.49 (s, 18H), 1.2–1.05 (m, 1H), 1.05–0.9 (m, 1H); MS (m/e) (positive-FAB) 738 (M+ + H).

(1′R,2′S,4′S,5′S)-Phosphoric Acid Di-tert-butyl Ester 1-(Di-tert-butoxyphosphoryloxymethyl)-4-(2-iodo-6-methylaminopurin-9-yl)bicyclo[3.1.0]hex-2-yl Ester (46)

The alcohol 34 (10 mg, 0.0189 mmol), triphenylphosphine (20 mg, 0.0756 mmol), and 2-iodo-6-methylaminopurine 39 (10.4 mg, 0.0378 mmol) were mixed and dried under high vacuum for ~30 min. The residue was dissolved in anhydrous tetrahydrofuran (1 mL) and cooled to 0 °C. Diethyl azodicarboxylate (DEAD, 9.9 mg, 0.0567 mmol) was added dropwise via syringe to this solution. After the solution was stirred for 2 h at room temperature (the progress of the reaction was monitored by thin-layer chromatography), tetrahydrofuran was removed in vacuo. The residue was purified by preparative thin-layer chromatography (CHCl3/MeOH = 20/1) to give the desired 2-iodo bis(di-tert-butyl phosphate) derivative 46 (7.1 mg, 48%): 1H NMR (CDCl3) δ 7.98 (s, 1H), 5.85 (bs, 1H, –NH), 5.35 (dd, 1H, J = 7.7, 14.3 Hz), 5.07 (d, 1H, J = 6.9 Hz), 4.66 (dd, 1H, J = 4.7, 11.0 Hz), 3.91 (dd, 1H, J = 6.6, 11.3 Hz), 3.17 (bs, 3H), 2.31 (dd, 1H, J = 8.0, 15.4 Hz), 2.09 (dt, 1H, J = 8.2, 14.8 Hz), 1.76 (dd, 1H, J = 4.4, 8.5 Hz), 1.32 (s, 18H), 1.29 (s, 9H), 1.26 (s, 9H), 1.10 (dd, 1H, J = 4.1, 6.0 Hz), 0.99 (m, 1H); MS (m/e) (positive-FAB) 786.3 (M+ + 1).

2-Amino-6-chloropurin-9-ylmethyl 2,2-Dimethylpropionate (54)

2-Amino-6-chloropurine 20 (1.00 g, 5.90 mmol) was dissolved in DMSO (5.0 mL) under heating. To this solution were added DMF (20.0 mL), chloromethyl pivalate 53 (1.0 mL, 6.94 mmol), and K2CO3 (990 mg, 7.16 mmol), and the mixture was stirred at room temperature for 3 days. The reaction mixture was filtered and the filtrate was evaporated. The obtained residue was purified by flash chromatography (AcOEt/petroleum ether = 2/1) and recrystallized from AcOEt/petroleum ether, which furnished 54 (1.33 g, 79%): 1H NMR (CDCl3) δ 8.01 (s, 1H), 6.00 (s, 2H), 5.20 (bs, 2H), 1.18 (s, 9H); MS (m/e) (positive-FAB) 284, 286 (peak height ratio 3:1) (M+ + 1).

6-Chloro-2-iodopurin-9-ylmethyl 2,2-Dimethylpropionate (55)

To a solution of 2-amino-6-chloropurin-9-yl-methyl 2,2-dimethylpropionate 54 (704 mg, 2.48 mmol) in MeCN (2.0 mL) were added diiodomethane (8.0 mL) and tert-butylnitrite (0.90 mL, 9.99 mmol), and oxygen was purged by N2 bubbling. The tube was sealed and the contents stirred at 80 °C for 2.5 h. The solvent was removed under vacuum and the obtained residue was purified by flash chromatography (AcOEt/petroleum ether = 1/2), which furnished 55 (561 mg, 57%): 1H NMR (CDCl3) 8.29 (s, 1H), 6.14 (s, 2H), 1.19 (s, 9H); MS (m/e) (positive-FAB) 395, 397 (peak height ratio 3:1) (M+ + 1).

6-Chloro-2-iodopurine (43)

To a solution of 6-chloro-2-iodopurin-9-ylmethyl 2,2-dimethylpropionate 55 (125 mg, 0.317 mmol) in i-PrOH (5.0 mL) and H2O (0.500 mL) was added 5 N aq NaOH (0.200 mL) and the mixture stirred at room temperature for 3 days. The solvent was removed under reduced pressure, the residue was poured into saturated aq NaHCO3 and extracted with THF–AcOEt. The organic layer was washed with brine, dried over Na2SO4, and evaporated. The residue obtained was purified by flash chromatography (AcOEt), which furnished 43 (42 mg, 48%): 1H NMR (DMSO) δ 8.63 (s, 1H); MS (m/e) (positive-FAB) 281, 283 (peak height ratio 3:1) (M++1).

(1′R,2′S,4′S,5′S)-Phosphoric Acid Di-tert-butyl Ester 1-(Di-tert-butoxyphosphoryloxymethyl)-4-(6-chloro-2-iodopurin-9-yl)bicyclo[3.1.0]hex-2-yl Ester (51)

To a solution of triphenylphosphine (70 mg, 0.26 mmol) in anhydrous THF (0.50 mL) was added DEAD (0.035 mL, 0.22 mmol) dropwise at −20 °C with stirring for 1.0 h. Compound 34 (18 mg, 0.035 mmol) and 6-chloro-2-iodopurine 43 (65 mg, 0.23 mmol) in THF (1.50 mL) were added to the reaction mixture and it stirred at room temperature for 3 days. The solvent was removed under vacuum and the residue obtained was purified by preparative thin-layer chromatography (AcOEt), which furnished 51 (15.5 mg, 57%): 1H NMR (CDCl3) δ 8.44 (s, 1H), 5.34 (dd, 1H, J = 8.1, 15.0 Hz), 5.16 (d, 1H, J = 6.9 Hz), 4.69 (dd, 1H, J = 5.1, 11.4 Hz), 3.94 (dd, 1H, J = 6.6, 11.4 Hz), 2.40–2.30 (m, 1H), 2.22–2.10 (m, 1H), 1.85–1.80 (m, 1H), 1.50 (s, 9H), 1.49 (s × 2, 18H), 1.48 (s, 9H), 1.18–1.14 (m, 1H), 1.09–1.03 (m, 1H); MS (m/e) (positive-FAB) 791, 793 (peak height ratio 3:1) (M+ + 1).

(1′R,2′S,4′S,5′S)-Phosphoric Acid Di-tert-butyl Ester 1-(Di-tert-butoxyphosphoryloxymethyl)-4-(2-iodo-6-methylaminopurin-9-yl)bicyclo[3.1.0]hex-2-yl Ester (46)

To a solution of 6-chloro-2-methylpurin-9-ylmethyl 2,2-dimethylpropionate 51 (13.0 mg, 0.016 mmol) in THF (0.10 mL) was added 2.0 N MeNH2 in THF (1.0 mL) and the mixture stirred for 3 h. The solvent was removed under reduced pressure and the obtained residue was purified by flash chromatography (CHCl3/MeOH = 5/1), which furnished 46 (6.1 mg, 47%).

6-Chloro-2-trimethylstannylpurin-9-ylmethyl 2,2-Dimethylpropionate (56)

To a solution of 6-chloro-2-iodopurin-9-ylmethyl 2,2-dimethylpropionate 55 (323 mg, 0.819 mmol) and PdCl2(PPh3)2 (30 mg, 0.043 mmol) in dioxane (0.75 mL) was added Me3SnSnMe3 (820 mg, 2.50 mmol) in dioxane (2.0 mL) and the mixture stirred at 100 °C for 5 h. The solvent was removed under vacuum and the residue obtained was purified by flash chromatography (AcOEt/petroleum ether = 1/2), which furnished 56 (310 mg, 88%): 1H NMR (CDCl3) δ 8.26 (s, 1H), 6.21 (s, 2H), 1.18 (s, 9H), 0.45 (s, 9H); MS (m/e) (positive-FAB) 431(M+ + 1).

6-Chloro-2-methylpurin-9-ylmethyl 2,2-Dimethylpropionate (57)

A mixture of tris(dibenzylideneacetone)dipalladium(0) chloroform adduct (Pd2(dba)3, 16 mg, 0.031 mmol), tri o-tolylphosphine (P(o-Tol)3, 31 mg, 0.102 mmol), CuBr (19 mg, 0.135 mmol), and K2CO3 (26 mg, 0.188 mmol) in a flask was dried under reduced pressure, and the vacuum was replaced by dry nitrogen. A solution of 6-chloro-2-trimethylstannylpurin-9-ylmethyl 2,2-dimethylpropionate 56 (109 mg, 0.253 mmol) in DMF (2.0 mL) and MeI (0.15 mL, 2.41 mmol) was added, and the mixture was stirred at 60 °C for 3 h. The resulting reaction mixture was directly pass through a short column (SiO2, 2.0 mL, eluent AcOEt) and evaporated. The residue obtained was purified by flash chromatography (AcOEt/petroleum ether = 1/2), which furnished 57 (45 mg, 62%): 1H NMR (CDCl3) δ 8.29 (s, 1H), 6.16 (s, 2H), 2.83 (s, 3H), 1.18 (s, 9H); MS (m/e) (positive-FAB) 283, 285 (peak height ratio 3:1) (M+ + 1).

6-Methylamino-2-methylpurine (40)

To a solution of 6-chloro-2-methylpurin-9-yl-methyl 2,2-dimethylpropionate 57 (34 mg, 0.120 mmol) in THF (1.0 mL) was added 2.0 N MeNH2 in THF (2.0 mL) and the mixture stirred for 22 h. The solvent was removed under reduced pressure and the obtained residue was purified by preparative thin-layer chromatography (first, AcOEt; second, CHCl3/MeOH = 5/1), which also furnished 6-methylamino-2-methylpurine 40 (27 mg, 81%) and 6-methylamino-2-methylpurin-9-ylmethyl 2,2-dimethylpropionate (4 mg, 12%): 1H NMR (DMSO) δ 7.96 (s, 1H), 7.40 (bs, 1H), 2.94 (s, 3H), 2.40 (s, 3H); MS (m/e) (positive-FAB) 164 (M+ + 1).

6-Methylamino-2-methylpurin-9-yl-methyl 2,2-dimethylpropionate

1H NMR (DMSO) δ 8.11 (s, 1H), 7.65 (bs, 1H), 6.10 (s, 2H), 2.94 (s, 3H), 2.44 (s, 3H), 1.10 (s, 9H); MS (m/e) (positive-FAB) 278 (M++1).

(1′R,2′S,4′S,5′S)-Phosphoric Acid Di-tert-butyl Ester 1-(Di-tert-butoxyphosphoryloxymethyl)-4-(2-methyl-6-methylaminopurin-9-yl)bicyclo[3.1.0]hex-2-yl Ester (47)

To a solution of triphenylphosphine (52 mg, 0.198 mmol) in anhydrous THF (0.40 mL) was added DEAD (0.030 mL, 0.191 mmol) dropwise at −20 °C with stirring for 1.0 h. Compound 34 (16.0 mg, 0.030 mmol) and 6-methylamino-2-methylpurine 40 (27 mg, 0.0975 mmol) in THF (1.50 mL) were added to the reaction mixture, which stirred at room temperature for 3 d. The solvent was removed under vacuum and the residue obtained was purified by preparative thin-layer chromatography (AcOEt), which furnished 47 (10.0 mg, 49%): 1H NMR (CDCl3) δ 7.99 (s, 1H), 5.62 (br, 1H), 5.38 (m, 1H), 5.09 (d, 1H, J = 6.9 Hz), 4.65 (dd, 1H, J = 4.5, 11.4 Hz), 3.92 (dd, 1H, J = 6.3, 11.4 Hz), 3.22 (s, 3H), 2.60 (s, 3H), 2.40–2.29 (m, 1H), 2.18–2.05 (m, 1H), 1.80 (m, 1H), 1.49 (s, 18H), 1.47 (s, 9H), 1.45 (s, 9H), 1.13–1.08 (m, 1H), 1.01–0.95 (m, 1H).

(1′R,2′S,4′S,5′S)-Phosphoric Acid Di-tert-butyl Ester 1-(Di-tert-butoxyphosphoryloxymethyl)-4-(2-hexyl-6-methylaminopurin-9-yl)bicyclo[3.1.0]hex-2-yl Ester (48)

2-Hexynyl bis(di-tert-butyl phosphate) derivative 50 (13 mg, 0.0176 mmol) was dissolved in methanol (5 mL), and 5% Pd/C (150 mg) was added. The reaction flask was applied to a vacuum and filled with hydrogen several times. The reaction mixture was stirred for 2 h at room temperature under a hydrogen balloon. The resulting mixture was filtrated through a pad of Celite and washed with methanol (5 mL × 3). The residue was purified by preparative thin-layer chromatography (CHCl3/MeOH = 20/1) to give the desired 2-hexanyl bis(di-tert-butyl phosphate) derivative 48 (11.5 mg, 88%): 1H NMR (CDCl3) δ 8.02 (s, 1H), 5.62 (bs, 1H, –NH), 5.35 (dd, 1H, J = 7.7, 14.6 Hz), 5.12 (d, 1H, J = 6.9 Hz), 4.64 (dd, 1H, J = 4.4, 11.0 Hz), 3.90 (dd, 1H, J = 6.3, 11.0 Hz), 3.23 (d, 3H, J = 4.9 Hz), 2.81 (dd, 2H, J = 7.7, 8.0 Hz), 2.34 (dd, 1H, J = 7.7, 15.1 Hz), 2.09 (dt, 1H, J = 8.0, 15.7 Hz), 1.23–1.88 (m, 9H), 1.497 (s, 9H), 1.489 (s, 9H), 1.479 (s, 9H), 1.455 (s, 9H), 1.13 (dd, 1H, J = 4.4, 5.8 Hz), 0.98 (dd, 1H, J = 6.3, 8.5 Hz), 0.89 (m, 3H); MS (m/e) (positive-FAB) 744.5 (M+ + 1).

(1′R,2′S,4′S,5′S)-Phosphoric Acid Di-tert-butyl Ester 1-(Di-tert-butoxy-phosphoryloxymethyl)-4-(2-hex-1-enyl-6-methylaminopurin-9-yl)bicyclo[3.1.0]hex-2-yl Ester (49)

2-Hexenyl bis(di-tert-butyl phosphate) derivative 49 (6.1 mg, 44%) was prepared by a method similar to that for 46, except 2-hexenyl-6-methylaminopurine 41 (10 mg, 0.0189 mmol) was used as the starting material: 1H NMR (CDCl3) δ 8.01 (s, 1H), 7.11 (dt, 1H, J = 6.9, 15.4 Hz), 6.48 (dt, 1H, J = 1.4, 15.6 Hz), 5.62 (bq, 1H, –NH), 5.38 (dd, 1H, J = 7.7, 14.3 Hz), 5.14 (d, 1H, J = 6.9 Hz), 4.65 (dd, 1H, J = 4.7, 11.3 Hz), 3.91 (dd, 1H, J = 6.3, 11.3 Hz), 3.24 (d, 3H, J = 5.0 Hz), 2.35 (dd, 1H, J = 8.5, 15.4 Hz), 2.29 (m, 2H), 2.11 (dt, 1H, J = 7.7, 15.4 Hz), 1.80 (dd, 1H, J = 4.1, 8.5 Hz), 1.33–1.61 (m, 4H), 1.496 (s, 9H), 1.490 (s, 9H), 1.482 (s, 9H), 1.446 (s, 9H), 1.13 (dd, 1H, J = 4.4, 5.8 Hz), 0.98 (dd, 1H, J = 6.3, 8.5 Hz), 0.94 (m, 3H); MS (m/e) (positive-FAB) 742.5 (M+ + 1).

(1′R,2′S,4′S,5′S)-Phosphoric Acid Di-tert-butyl Ester 1-(Di-tert-butoxyphosphoryloxymethyl)-4-(2-hex-1-ynyl-6-methylaminopurin-9-yl)bicyclo[3.1.0]hex-2-yl Ester (50)

2-Hexynyl bis(di-tert-butyl phosphate) derivative 50 (20.1 mg, 72%) was prepared by a method similar to that for 44, except 2-hexynyl-6-methylaminopurine 42 (20 mg, 0.0378 mmol) was used as the starting material: 1H NMR (CDCl3) δ 8.14 (s, 1H), 5.77 (bs, 1H), 5.33 (dd, 1H, J = 8.2, 14.8 Hz), 5.21 (d, 1H, J = 6.9 Hz), 4.67 (dd, 1H, J = 4.4, 11.3 Hz), 3.83 (dd, 1H, J = 6.6, 11.3 Hz), 3.25 (bs, 3H), 2.46 (t, 2H, J = 7.1 Hz), 2.29 (dd, 1H, J = 8.0, 15.4 Hz), 2.08 (m, 1H), 1.60–1.82 (m, 5H), 1.499 (s, 9H), 1.488 (s, 9H), 1.472 (s, 9H), 1.462 (s, 9H), 1.12 (dd, 1H, J = 4.4, 5.8 Hz), 0.9 (m, 1H), 0.94 (t, 3H, J = 7.1 Hz); MS (m/e) (positive-FAB) 740.5 (M+ + 1).

(1′R,2′S,4′S,5′S)-Phosphoric Acid Di-tert-butyl Ester 1-(Di-tert-butoxy-phosphoryloxymethyl)-4-(6-chloro-2-trimethylstannylpurin-9-yl)bicyclo[3.1.0]hex-2-yl Ester (52)

To a solution of 51 (2.7 mg, 0.0034 mmol) and PdCl2-(PPh3)2 (3.5 mg, 0.0050 mmol) in dioxane (0.30 mL) was added Me3SnSnMe3 (20 mg, 0.061 mmol) and the mixture stirred at 60 °C for 3.5 h. The solvent was removed under vacuum and the residue obtained was purified by preparative TLC (AcOEt), which furnished 52 (0.7 mg, 25%): 1H NMR (CDCl3) δ 8.42 (s, 1H), 5.40–5.20 (m, 2H), 4.65 (m, 1H), 3.90 (m, 1H), 2.40–2.25 (m, 1H), 2.25–2.10 (m, 1H), 1.90–1.80 (m, 1H), 1.50 (s, 9H), 1.48 (s, 18H), 1.44 (s, 9H), 1.25–1.20 (m, 1H), 1.05–1.00 (m, 1H), 0.43 (s, 9H); MS (m/e) (positive-FAB) 827 (M++1), 849 (M++Na).

Small-Scale Conversion of Compound 52 into Compound 12

A 20 µL (0.35 µmol) portion of an iodine solution (4.5 mg of I2 in 1.00 mL of AcOEt) was added to a solution of 52 (0.15 mg, 0.18 µmol) in AcOEt (0.100 mL) and the mixture stirred at room temperature for 10 min. Then 0.01 mL of 5% aqueous NaHSO3 was added to stop the reaction, and the solvent was removed under vacuum. The residue (crude 29) was dissolved in 5% TFA/CH2Cl2 (0.50 mL) and stirred for 3 h at 25 °C. The solvent was removed under reduced pressure, and the residue was quenched by addition of 5.0 mL of triethylammonium bicarbonate buffer (1.0 M). The mixture was subsequently frozen and lyophilized. The residue (crude 29) was dissolved in 40% MeNH2 in water (0.50 mL) and stirred for 3 h at room temperature. The solvent was removed under reduced pressure. The residue (crude 12) was purified by HPLC (system A or system B).

Enzymatic Synthesis of 5

ATP (20 nmol, 2 µL of a 10-mM solution in water) and polynucleotide kinase (Sigma, 10 IU) were added to 20 µL of a buffer consisting of 50 mM Tris HCl, 10 mM MgCl2, 5 mM dithiothreitol, 0.1 mM spermidine, and 0.1 mM EDTA. The nucleotide substrate 5841 (1 µL of a 10-mM solution in water) was then added, and the mixture was mixed with vortexing and incubated at 37 °C for the time indicated (18 h incubation was found to be optimal). Aliquots (10 µL) were injected for HPLC analysis. A C18 Zorbax column (Agilent) was used, with the flow rate 1 mL/min and the mobile phase consisting of a gradient of acetonitrile/0.1 M triethylammonium acetate (aq) of 10/90 to 15/85 in 10 min, then to 30/70 in 10 min. The product of the enzymatic transformation (5) was detected in ~40% yield, as judged with HPLC. The retention time of the product was 6.2 min, compared with the starting material, which had a retention time of 8.1 min. These three individual peaks were collected and lyophilized. Mass spectrometry (FAB) confirmed the identity of the starting material (m/z 387, M − 1) and product 5 (m/z 468, M − 1). The side product at retention time 11.6 min displayed an m/z of 310, consistent with the corresponding nucleoside (M + 1). An unidentified impurity present in the starting material was also detected at 7.7 min.

Compound 58 was found to be chemically stable. The HPLC profile of a solution in H2O/acetonitrile/buffer was unchanged following 60 h incubation at 37 °C. However, when enzyme was added in the absence of ATP, ~20% of the compound was dephosphorylated during an incubation of 17 h to the nucleoside 59 (HPLC retention time = 11.6 min). Under the same incubation conditions in the presence of the enzyme, 5 was stable.

Pharmacological Analyses

The affinities of 2-substituted (N)-methanocarbo bisphosphate analogues for the human P2Y1 receptor were directly determined by using [3H]5 in a radioligand binding assay, as we recently described in detail.31 Briefly, the human P2Y1 receptor was expressed to high levels in Sf9 insect cells with a recombinant baculovirus. Membranes prepared from these cells were incubated for 30 min at 4 °C in the presence of ~20 nM [3H]5 and a wide range of concentrations of the newly synthesized analogues. Binding reactions were terminated by the addition of ice-cold tris-(hydroxymethyl)aminomethane (Tris) wash buffer (10 mM Tris, pH 7.5, and 145 mM NaCl) to the samples followed by rapid filtration over GF/A glass-fiber filters. Each filter was washed with an additional 4 mL of wash buffer, and radioactivity retained by the filters was quantified by liquid scintillation spectrometry. All assays were carried out in triplicate, and competition curves for all molecules were generated in three separate experiments. IC50 values were determined from each competition curve, and a Ki value was calculated for each analogue according to the relationship Ki = IC50/1 + [[3H]5]/Kd of [3H]5, where the Kd of [3H]5 determined in separate experiments was 8 nM.

P2Y receptor-promoted stimulation of inositol phosphate formation was measured at human P2Y1 receptors stably expressed in 1321N1 human astrocytoma cells as previously described.19,42,43 The K0.5 values were averaged from three to eight independently determined concentration-effect curves for each compound. Briefly, cells plated in 24-well dishes were labeled in inositol-free medium (DMEM; Gibco, Gaithersburg, MD) containing 1.0 µCi of 2-[3H]myo-inositol (20 Ci/mmol; American Radiolabeled Chemicals, Inc., St. Louis, MO) for 18–24 h in a humidified atmosphere of 95% air/5% CO2 at 37 °C. Phospholipase C activity was measured the following day by quantitating [3H]inositol phosphate accumulation after a 10-min incubation at 37 °C in the presence of 10 mM LiCl. Total [3H]inositol phosphates were quantified by anion-exchange chromatography as previously described.42,43

Data Analysis

IC50 values obtained in radioligand binding assays and in assays of inhibition of 2MeSADP-stimulated inositol phosphate accumulation were calculated by a four-parameter logistic equation and the GraphPad software package (GraphPad, San Diego, CA). All concentration–effect curves were repeated in at least three separate experiments, carried out in duplicate or triplicate with different membrane preparations or different 1321N1 cell cultures.

Results

Chemical Synthesis

We prepared methanocarbocyclic analogues of various bisphosphate derivatives (Table 1) in which fused cyclopropane and cyclopentane rings were fixed the pseudoribose moiety in a rigid (N)-envelope conformation. Synthetic intermediates included O-benzyl-protected phosphoesters, carbocyclic derivatives, adenine derivatives, and O-tert-butyl-protected phosphoesters (Schemes 15). The nucleotide analogues 7–18 were prepared in the ammonium salt form according to the methods shown in Schemes 1 and and44 and tested biologically as antagonists at the P2Y1 receptor (Table 1). Identity was confirmed by NMR (1H and 31P) and high-resolution mass spectrometry (HRMS), and purity was demonstrated with high-performance liquid chromatography (HPLC) in two different solvent systems.

Scheme 1a
a Reagents: (i) DIAD, Ph3P, rt, 2-amino-6-chloropurine; (ii) BCl3, CH2Cl2, −40 °C; (iii) 1H-tetrazole then m-CPBA; (iv) CH2I2, or MeSSMe, or MeSeSeMe, t-BuONO, MeCN; (v) MeNH2/THF; (vi) BCl3, CH2Cl2, 5 °C; (vii) NH3/H2O, 70 °C, ...
Scheme 4a
a Reagents: (i) 2-substituted (iodo, methyl, 2-hexenyl) 6-methylaminopurine or 2-iodo-6-chloropurine, Ph3P, DEAD, THF, rt, 2 h; (ii) 2-substituted (fluoro, bromo, or 2-hexynyl) 6-methylaminopurine, Ph3P, DIAD, THF; (iii) 5% Pd/C, H2, MeOH, rt, 2 h; (iv) ...
Scheme 5a
a Reagents: (i) K2CO3, DMSO–DMF, rt, 3 d; (ii) CH2I2, t-BuONO, acetonitrile, 60 °C, 2.5 h; (iii) PdCl2(PPh3)2, Me3SnSnMe3, dioxane, 100 °C, 5 h; (iv) Pd2(dba)3, P(o-tol)3, CuBr, K2CO3, MeI, DMF, 60 °C, 3 h; (v) CH3NH2/THF, ...
Table 1
In Vitro Pharmacological Data at Human P2Y1 Receptors for Inhibition of PLC Elicited by 30 nM 2-MeSADP (receptor expressed in 1321N astrocytoma cells) and in Binding (receptor expressed in Sf9 cells)31 d

To construct the pseudoribose ring leading to the 2′-deoxy-(N)-methanocarba-2′-deoxyadenosine precursors, a bicyclo[3.1.0]hexane intermediate 19 was prepared by the new, optimized approach of Yoshimura et al.,32 which used simple starting materials and enzymatic transformations. Scheme 1 illustrates the usual synthetic route to the bisphosphate derivatives, in which the phosphorylation was carried out on the preformed nucleoside precursor. To prepare that nucleoside, a commercially available 2-substituted adenine intermediate, 6-chloro-2-aminopurine 20, was condensed with the protected bicyclohexane derivative 19 via a Mitsunobu reaction to give 21, which was then deprotected to give diol 22. The 3′- and 5′-hydroxyl groups then were phosphorylated by the phosphoramidite method33 using dibenzyl diisopropyl phosphoramidite 23 to give the benzyl-protected derivative 24. The 2-amino group of 24 was replaced with an iodo, methylthio, or methylseleno group (protected intermediates 25–27) with the use of a neutral purinyl radical, which was generated transiently from the thermal homolysis of the 2-diazonium intermediate. This was accomplished by using the approach introduced by Nair and Fasbender34 for the preparation of 2-methylthio and other 2-derivatives of adenosine. Selenium was selected for inclusion because of its similarity to sulfur and because of its known use in X-ray crystallography of proteins, when incorporated into ligands as a heavy atom.35 To introduce an amino or methylamino group at the 6-position, it was necessary to substitute 6-Cl with the corresponding amine, which could be accomplished either before (to give 13) or following (to give 7, 8, 12, or 14) deprotection of the phosphate groups. First we synthesized the N6-methyl-2-methylthio derivative 13, following the procedure in Scheme 1. However, we were unable to apply this route for the corresponding N6-amino derivative 8 because treatment of 26 with ammonia to replace the 6-chloro group caused side reactions. Therefore, we carried out an ammonia replacement reaction following deprotection of the benzyl groups to obtain the N6-amino derivative 8. We used similar procedures of phosphoester deprotection followed by replacement of the 6-chloro group for the preparation of the 2-methylseleno derivative 14.

As an alternate synthetic route to the bisphosphate derivatives, phosphate groups were introduced into a versatile phosporylated bicyclo[3.1.0]hexane precursor 34, protected with tert-butyl ester groups, prior to the Mitsunobu coupling (Scheme 2).

Scheme 2a
a Reagents: (i) Ac2O, cat. DMAP, TEA, CH2Cl2, rt, 4 h; (ii) wet 5% Pd/C, H2, MeOH, rt, 4 h; (iii) (t-BuO)2PNEt, tetrazole, THF, rt, 1 h then m-CPBA, −78 to 0 °C, 20 min; (iv) K2CO3, MeOH, rt, 2 h.

We first attempted to synthesize 2-fluoro 10 and 2-bromo 11 derivatives by the approach shown in Scheme 1, which used the phosphoramidite route to phosphorylate the corresponding nucleoside diol derivative.18 However, only traces of the products were obtained due to side reactions, which seemed to result from the high nucleophilicity of the 6-amino group. Thus, an alternate route was needed. For substitution of the 2-position of the purine ring with bromo or fluoro, the corresponding adenine precursors 37 and 38 were prepared (Scheme 3). We used 20, a commercially available material, to prepare the dihaloadenines 35 and 36 by a method from the literature.36 Subsequent methylamination of the 6-position gave 37 and 38, which were coupled with the methanocarba moiety 34 by a Mitsunobu reaction. 2-Iodo-N6-methyladenine 39 was prepared with the method of Nair and Fasbender.34 The two 2-alkene/alkyne-substitituted adenines 41 and 42 were prepared as previously reported.37 These 2-haloadenine derivatives and several 2-C-substituted analogues were incorporated into the nucleoside structure as shown in Scheme 4, via the protected bicyclo[3.1.0]-hexane precursor 34. Following the Mitsunobu reaction, the hexynyl intermediate 50 was reduced to the corresponding alkane to give 48. Deprotection of the tert-butyl groups was carried out with TFA to give the bisphosphate derivatives 10–12 and 15–18.

Scheme 3a
a Reagents: (i) SbBr3/CH2Br2, t-Bu nitrite; (ii) HF/pyridine, t-Bu nitrite; (iii) CH3NH2/THF or H2O.

The preparation of additional 2-substituted adenine intermediates, including 6-chloro-2-iodopurine 43 used in Scheme 4, required a separate synthetic strategy (Scheme 5). The adenine precursor 20 dissolved in polar organic solvents, e.g. pyridine or DMSO, only to a limited extent, which made the selection of transformation conditions difficult. To proceed further, we applied a protecting group at the N-9 position, expecting an improvement in solubility. The pivaloyloxymethyl group was chosen as the protecting group, and 54 was prepared with the method of Rasmussen and Leonard.41 Compound 54 dissolved well in the usual organic solvents, allowing the 2-iodination of 54 to be performed by the method of Nair and Fasbender.34

With the success of [3H]5 as a selective radioligand for P2Y1 receptor characterization,31 we sought to design new antagonists in this series as high-affinity receptor probes. As a route to a novel iodinated analogue that could be potentially radiolabeled, we explored the substitution of the iodo group of 12 (Scheme 4) and its adenine precursors (Scheme 5) with trimethyltin.38,39 This provides a means of rapidly obtaining the iodinated ligand 12. Similar methodology based on trialkyl tin precursors has been used to prepare radioiodinated ligands for SPECT (single photon emission computed tomography).39,40 The 2-iodo product 55 was converted to the 2-trimethylstannyl derivative 56 by the method of Baranowska-Kortylewicz et al.42 Compound 56 was a useful intermediate because it could serve as a suitable substrate for Stille coupling; thus, we decided to prepare the 2-methyl derivative. Compound 56 reacted with methyl iodide under the conditions of Suzuki et al.43 The product 57 was treated with methylamine to make 40, which was coupled with the methanocarba moiety by a Mitsunobu reaction. Deprotection was carried out with TFA to give the bisphosphate derivative 15.

As an alternate route to introduce iodine in the antagonist 12, we also attempted stannylation of 46 with the method of Baranowska-Kortylewicz et al., to be followed by reiodination.42 However, we obtained only the 2-H derivative, which formed upon replacement of the trimethylstannyl group by hydrogen, which was readily available from an acidic proton such as the 6-NH(Me). As an alternative (Scheme 4), we tried stannylation using a 6-chloro derivative 51. This intermediate was synthesized from 43, which was obtained by hydrolysis of 55, and was coupled with the methanocarba moiety by a Mitsunobu reaction. Intermediate 51 was successfully stannylated with the method of Baranowska-Kortylewicz et al.42 The trimethyl stannyl derivative 52 reacted with iodine (which could be generated rapidly from iodide) and re-formed 51. By mass spectral analysis of the iodination reaction at various time points (1–40 min), an optimal reaction time was determined to be 10 min. Deprotection of 51 on a small scale with TFA was accomplished to give the bisphosphate derivative 29, which was then converted to the antagonist 12 (Scheme 1).

We have explored a synthetic methodology that would allow a phosphate group to be incorporated in a potent P2Y1 receptor antagonist in the final synthetic step. Although chemical phosphorylation methods were used until now to prepare P2Y1 receptor antagonists in this series, we devised an enzymatic method for the phosphorylation at the 5′-position, leading to compound 5. This method (Scheme 6) is based on phosphorylation with polynucleotide kinase (EC 2.7.1.78).44 The 3′-monophosphorylated intermediate 5845 was treated with the enzyme and ATP as the phosphate source. Following incubation for 60 min, the product 5 was detected in ~40% yield, as judged by using HPLC. The intermediate 58 was found to be chemically stable under the incubation conditions. A side reaction, removal of the 3′-phosphate group of 58 to yield the nucleoside analogue 59, occurred to a significant degree only in the absence of ATP.

Scheme 6a
a Reagents: (i) polynucleotide kinase, ATP; (ii) polynucleotide kinase, alone.

Pharmacological Activity

We recently developed [3H]5 as a high-affinity and selective radioligand for quantification of the P2Y1 receptor.31 Therefore, the human P2Y1 receptor can be expressed from a baculovirus to high levels in Sf9 insect cells, and membranes prepared from these cells can be labeled specifically with [3H]5 to directly assess the affinity of newly synthesized molecules at the P2Y1 receptor. Therefore, human P2Y1 receptor-expressing membranes were incubated with 10 nM [3H]5 and a wide range of concentrations of 2-substituted (N)-methanocarba bisphosphate analogues. As illustrated in Figure 1, all of the bisphosphate derivatives tested interacted with the P2Y1 receptor, as shown by their capacity to inhibit [3H]5 binding, and this inhibition occurred with kinetics consistent with interaction by the law of mass action kinetics at a single site. IC50 values were determined (Table 1) from each competition curve, and a Ki value was calculated for each analogue according to the relationship Ki = IC50/1 + [[3H]5]/Kd of [3H]5.

Figure 1
Inhibition of [3H]5 binding to human P2Y1 receptors by 2-substituted analogues. [3H]5 binding assays were carried out as described in the Experimental Section using Sf9 membranes expressing recombinant human P2Y1 receptor. The data are presented as percent ...

We also tested the functional activities of (N)-methanocarba bisphosphate analogues as agonists and antagonists at the human P2Y1 receptor stably expressed in 1321N1 human astrocytoma cells (Table 1). Agonist activity was tested by measuring the capacity of molecules to increase inositol phosphate accumulation by activating the phospholipase C-coupled P2Y1 receptor, and antagonist activity at the P2Y1 receptor was assessed by measuring the capacity of these molecules to inhibit 2-methylthio-adenosine 5′-diphosphate (2Me-SADP)-stimulated accumulation of inositol phosphates.

None of these 2-substituted molecules exhibited agonist activity in the 1321N1 cell-test system, but all displayed a capacity to inhibit 2MeSADP-stimulated inositol phosphate accumulation, with an IC50 value consistent with their Ki values determined in binding experiments. Within the series of 2-halo analogues, the most potent antagonist in both functional and binding models was a (N)-methanocarba-2-iodo analogue 12, and all other 2-halo derivatives were clearly less potent. This derivative displayed a Ki value in competition for binding of [3H]5 at the hP2Y1 receptor of 0.79 nM. The N6-methyl derivative 12 was 16-fold more potent in binding than the corresponding 6-amino analogue 7. The corresponding N6-methyl 2-bromo and 2-chloro derivatives 11 and 5 exhibited similar Ki values and affinities that were 5–10-fold less than that for 12, and the fluoro derivative 10 exhibited a much lower affinity (Figure 1). The (N)-methanocarba-2-methylthio-, 2-methylseleno, 2-hexyl, 2-(1-hexenyl), and 2-(1-hexynyl) analogues exhibited lower affinities than did most of the 2-halo derivatives, with Ki values in the micromolar range observed for these compounds at P2Y1 receptors. No significant difference was observed between the alkene and alkyne derivatives 17 and 18, whereas the corresponding alkane 16 exhibited a 4–5-fold-lower affinity, with a Ki value in binding calculated to be 80 nM. However, the 2-methyl derivative 15 was nearly as potent as the corresponding 2-chloro analogue 5.

Schild analysis of antagonism by 12 of inositol phosphate accumulation in 1321N1 human astrocytoma cells stably expressing the human P2Y1 receptor, stimulated by 2-MeS-ADP, was carried out (Figure 2). A KB value of 1.74 nM was determined, in close agreement with binding data for this compound.

Figure 2
Competitive antagonism of 2MeSADP-promoted activation of the human P2Y1 receptor. Phospholipase C activity was measured as described in the Experimental Section in 1321N1 human astrocytoma cells stably expressing the human P2Y1 receptor. Top panel: assays ...

A correlation of binding affinities (expressed as Ki values) and functional antagonistic potencies (expressed as IC50 values) at the human P2Y1 receptor was examined for nine compounds (Figure 3). The degree of correlation was high, with an r2 value of 0.98. Thus, as we showed recently in radioligand binding assays with [3H]5 and previously studied bisphosphate analogues,31 the radioligand binding assay is a readily applicable predictor of the functional effects of the P2Y1 receptor antagonists.

Figure 3
Correlation of binding affinities at the human P2Y1 receptor expressed in Sf9 cells and functional potencies as P2Y1 receptor antagonists (measuring inhibition of inositol phosphate accumulation in 1321N1 human astrocytoma cells stably expressing the ...

Discussion

In previous studies,18,20 we concluded that substitution of the ribose moiety with the (N)-methanocarba ring system provides an enhancement of affinity at the P2Y1 receptor. We strengthened this conclusion in the present study. For example, the compounds homologous to 8, 12, and 13 were reported in the ribose series,17 and the IC50 values in the same phospholipase C assay in turkey erythrocytes expressing an endogenous P2Y1 receptor were 1890, 891, and 362 µM, respectively. This is to be compared to the values for 8, 12, and 13 of 367, 8.4, and 221 nM, respectively. Thus, the enhancement achieved upon conformationally constraining the ribose-like ring in the preferred (N)-conformation was present in all three cases. However, the degree of enhancement ranged from slight, in the case of the N6-methyl-2-methylthio analogue 13, to dramatic, i.e., 106-fold, in the case of the N6-methyl-2-iodo analogue 12.

In the present study, we focused on optimization of the 2-position substitution. Halo substitution demonstrated a potency order of I > Br, Cl > F, which did not correspond to the relative effects of these substitutions in the ribose series.17 The 2-alkylthio substitution, which was found to greatly enhance potency in the agonist series, i.e., 5′-mono-, di-, and triphosphates,2123 did not enhance antagonist potency in the present series. This observation is similar to previous findings with other nucleotide antagonists of the P2Y1 receptor.17 In contrast with C-substitution at the 2-position of adenine reported previously24 (i.e., 2-methyl in the ribose series), we did not find an enhancement of affinity in the alkene and alkyne analogues synthesized here.

The methods explored in this study for incorporation of iodo and phosphate groups from simple chemical precursors might be suitable for the preparation of radioligands (containing 125I, 33P, etc.) of high affinity for the P2Y1 receptor.

In conclusion, we have enhanced the potency of ring-constrained P2Y1 receptor antagonists by 2-position substitution. The structure–activity relationship (SAR) patterns were not entirely in parallel to the SAR observed in the ribose series. Thus, subtle differences in the mode of receptor binding probably occur between ribose-containing and (N)-methanocarba nucleotides. A key finding in this study is that compound 12 is the most potent antagonist selective for the P2Y1 receptor yet reported. These findings promise to be useful in defining the microscopic determinants of the binding sites on these receptors and in designing novel pharmacological probes, including radioligands, and/or therapeutic agents.

Acknowledgment

This work was supported by USPHS grants GM38213 and HL54889 (to T.K.H.). M.O. is on sabbatical from Toray Industries (Kamakura, Japan) and thanks them for financial support. Mass spectral measurements were carried out by Victor Livengood and NMR by Wesley White (NIDDK). We thank Ariel Gross (NIDDK) for proofreading the manuscript.

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