Cyclic ADP-Ribose Analogues Containing the

Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455. Received July 2, 2004. Analogues of cyclic ADP-ribos...
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J. Med. Chem. 2005, 48, 4177-4181

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Cyclic ADP-Ribose Analogues Containing the Methylenebisphosphonate Linkage: Effect of Pyrophosphate Modifications on Ca2+ Release Activity Libo Xu,§,† Timothy F. Walseth,‡ and James T. Slama*,§ Department of Medicinal and Biological Chemistry, College of Pharmacy, The University of Toledo, Toledo, Ohio 43606, and Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455 Received July 2, 2004

Analogues of cyclic ADP-ribose (cADPR) incorporating a methylenebisphosphonate linkage in the place of the pyrophosphate have been synthesized from nicotinamide adenine dinucleotide analogues enzymatically using Aplysia californica ADP-ribosyl cyclase. Methylenebisphosphonate cyclic ADP-ribose (cADPR[CH2]) and methylenebisphosphonate cyclic 3-deaza-ADP-ribose (3-deaza-cADPR[CH2]) showed full agonist activity for release of Ca2+ ions from sea urchin egg homogenates. The EC50 for cADPR[CH2] was 856 nM and that for 3-deaza-cADPR[CH2] was 300 nM, about 15- and 5-fold less potent than cADPR, respectively. Introduction Cyclic ADP-ribose (1, cADPR) is a natural metabolite of nicotinamide adenine dinucleotide (NAD) and a potent calcium-releasing second-messenger.1,2 Calcium release induced by cADPR is regulated independently of the IP33,4 by the ryanodine receptor.5 Ryanodinesensitive calcium channels are known to be present within a variety of organisms and in many human tissues and to regulate a physiologically important intracellular calcium store. Many cADPR analogues have been synthesized through chemoenzymatic or chemical synthesis,6-9 and structural modifications have been made to the purine ring and to both ribotides. Replacement of a ribofuranosyl oxygen with a methylene group (-CH2-) in cyclic aristeromycin diphosphate ribose10 and in cyclic ADP-carbocyclic-ribose11 was reported to result in the production of hydrolysisresistant cADPR analogues with significant Ca2+ mobilizing activity. There are as yet few reports of modification to the pyrophosphate of cADPR. The only such compounds reported in the literature are cyclic ATPribose (3, cATPR) in which the pyrophosphate is replaced with a triphosphate12 and caged cADPR in which a photoactivatable 2-(nitrophenyl)ethyl caging group modifies one of the two phosphate moieties.13 In this study, we describe the synthesis and Ca2+ mobilizing activity of two novel analogues of cADPR in which the pyrophosphate is replaced with a methylenebisphosphonate (4, cADPR[CH2] and 5, 3-deaza-cADPR[CH2]). Results and Discussion Synthesis of Phosphate Modified cADPR Analogues. A chemoenzymatic approach was employed for the synthesis of the two novel cADPR analogues, i.e., via cyclization of the corresponding linear NAD analogues catalyzed by Aplysia californica ADP-ribosyl cyclase. The precursor for cADPR[CH2] (4), NAD meth* To whom correspondence should be addressed. Telephone: 419.530.1925. Fax: 419.530.7946. E-mail: [email protected]. § The University of Toledo. † Current Address: Merck & Co., P.O. Box 2000, Rahway, NJ 070650900. ‡ University of Minnesota Medical School.

ylenebisphosphonate (12, Scheme 1; NAD[CH2]), was potentially available by coupling of adenosine 5′-methylenebisphosphonate derivatives with protected nicotinamide-β-D-riboside,14,15 but in our hands these methods failed to produce the coupled product in usable yields. Nucleophilic displacement of 5′-O-tosyl (p-methylbenzenesulfonyl-) nucleosides with phosphate salts has been used to synthesize nucleoside 5′-diphosphates and triphosphates,16 and the method had been adapted to the synthesis of a nicotinamide aristeromycin dinucleotide (a carbocyclic NAD analogue) containing a methylenebisphosphonate linkage.17 We attempted to make 5′tosylnicotinamide riboside from nicotinamide riboside using p-methylbenzenesulfonyl chloride (tosyl chloride) and p-dimethyaminopyridine (DMAP) catalyst;16 however, no product was formed even under forcing conditions. We next investigated coupling 5′-tosyladenosine and nicotinamide ribose 5′-methylenebisphosphonate but needed to devise an effective preparation of the later compound. We found that protected nicotinamide ribose 5′-methylenebisphosphonate (9) can be readily synthesized by treatment of nicotinamide-β-D-riboside 2′,3′isopropylidene acetal with a 3-fold excess of methylenebisphosphoryl tetrachloride in trimethyl phosphate.

10.1021/jm049469l CCC: $30.25 © 2005 American Chemical Society Published on Web 05/19/2005

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Scheme 1

Although this method has been used by others to incorporate methylene bisphosphonate into simple alcohols or purine nucleosides in generally low yield,18,19 we are the first ones to demonstrate that this can be a useful method to make the zwitterionic nicotinamide ribosyl methylenebisphosphonate. When 2′,3′-isopropylidene-5′-tosyladenosine (6)20 was treated with the bis(tetrabutylammonium) salt of 9 in acetonitrile,16 protected NAD[CH2] (10) was formed in 25% yield (Scheme 1). Most of the 2′,3′-isopropylidene5′-tosyladenosine underwent intramolecular cyclization to form N3,5′-cyclonucleoside. For the synthesis of bis(2′,3′-O-isopropylidene)nicotinamide 3-deazaadenine dinucleotide methylenebisphosphonate (11, bis-(2′,3′-Oisopropylidene)-3-deaza-NAD[CH2]) we were unable to prepare 5′-tosyl 3-deazaadenosine (8) (Scheme 1) under standard conditions; however, reaction with methanesulfonyl chloride proceeded quickly to afford the 5′methanesulfonyl nucleoside (7) in 60% yield (Scheme 1). Reaction of 7 with 916 gave the desired dinucleotide 11 in 43% yield, with starting material 9 recovered in 44% yield. Deprotection of 10 and 11 with 1 M HCl afforded NAD methylenebisphosphonate (12, NAD[CH2]) and nicotinamide 3-deazaadenine dinucleotide methylenebisphosphonate (13, 3-deaza-NAD[CH2]), respectively. Commercial Aplysia ADP-ribosyl cyclase was used to make cADPR[CH2] (4) and 3-deaza-cADPR[CH2] (5) from their linear precursors, NAD[CH2] (12) and 3-deazaNAD[CH2] (13). This enzyme has been used to prepare many cADPR analogues.12,21 We found that NAD[CH2] (12) and 3-deaza-NAD[CH2] (13) were well-recognized by the enzyme and formed the cyclic product when incubated with excess enzyme. We expected that cADPR[CH2] (4) and deaza-cADPR[CH2] (5) were generated by formation of a new glycosyl linkage at N1 of adenine by analogy to the transformation of NAD to cADPR22 and 3-deaza-NAD to 3-deazacADPR (2).21 This expectation was confirmed by the spectroscopic properties and biological activity of the new cyclic nucleotides. First, the UV chromaphore and 1H NMR of the PCP containing cyclic nucleotides correspond closely to that of the parent cyclic nucleotides

1 and 2 (see Supporting Information). Second, the Ca2+ mobilizing activity of the PCP cyclic nucleotides suggests that the glycosidic linkage to the N1 of adenine is formed, since cyclic nucleotides linked via the N7 usually lack such activity. Cyclic ADPR-ribose[CH2] (4), like cADPR, is hydrolyzed at the N1-C1′ glycoside bond in neutral aqueous solution and catalytically by NAD glycohydrolase.6 We found that cADPR[CH2] is less stable than cADPR, having a half-life of 16.5 h at 37 °C at pH 7, compared to t1/2 of 53 h for cADPR. However, cADPR[CH 2] (4) was hydrolyzed more slowly than cADPR (1) by NADase. 3-Deaza-cADPR[CH2] (5), like 3-deaza-cADPR (2),21 was stable toward chemical and enzymatic hydrolysis. Calcium Release Properties of 4 and 5. When tested for calcium-ion releasing activity in vitro, cADPR[CH2] (4) and 3-deaza-cADPR[CH2] (5) released calcium effectively from sea urchin egg homogenates. Linear ADPR[CH2] (ADP-ribose containing the methylenebisphosphonate linkage) and linear 3-deaza-ADPR[CH2] (3deaza-ADP-ribose containing the methylenebisphosphonate linkage) were purified from hydrolyzed samples of the cyclic nucleotides, and both failed to elicit Ca2+ release under comparable conditions. The effects of cADPR[CH2] (4) and 3-deaza-cADPR[CH2] (5) on Ca2+ release are shown in Figure 1. cADPR[CH2] (4) induces Ca2+ release at concentrations as low as 300 nM, and at saturating concentration (10 µM), it releases Ca2+ equivalent to that released by saturating concentrations of the full-agonist cADPR (1). 3-Deaza-cADPR[CH2] (5) induces Ca2+ release at concentrations as low as 60 nM, and the Ca2+ release is inhibited by 8-Br-cADPR, a known selective antagonist of the cADPR system,23 suggesting that 3-deaza-cADPR[CH2] releases Ca2+ through the same mechanism as does cADPR. Ca2+ release by cADPR[CH2] was similarly inhibited in the presence of low concentrations of 8-Br-cADPR (data not shown). Figure 2 shows the concentration-response curves for cADPR[CH2], 3-deaza-cADPR[CH2], cADPR, and 3-deaza-cADPR. The half-maximal concentrations for Ca2+ release are 856 ( 111 nM for cADPR[CH2], 302 ( 38 nM for 3-deaza-cADPR[CH2], 59 ( 7 nM for cADPR, and 0.76 ( 0.02 nM for 3-deaza-cADPR. Thus,

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starting point for the development of membrane permeant cADPR prodrugs in which the phosphoryl oxygens are protected as bioreversible phosphonate esters. Previously Zhang et al.12 had found that cATPR (3), the triphosphate analogue of cADPR, induced Ca2+ release from the same Ca2+ stores as that of cADPR, and it was 20 times more potent than cADPR in releasing Ca2+ from rat brain microsomes. cATPR was up to now the only cADPR analogue with modification to the pyrophosphate region. Our results indicate that the agonist activity of cADPR is very sensitive to modifications in the pyrophosphate moiety. The change in activity could be the result of changed bond angles, the lowering of pKa of the phosphonate group compared to that of the pyrophosphate, or the absence of a crucial hydrogen bond acceptor. Although the methylene substitution could cause some steric hindrance because CH2 is bigger than oxygen, it is less likely that steric hindrance is the major mechanism because the receptor apparently recognizes the much larger cATPR.12 However, care should be taken when comparing the activities of cADPR[CH2] and cATPR because they were evaluated using different organisms, i.e., sea urchin egg homogenates versus rat brain microsomes, with demonstrated differences in agonist recognition.24 Experimental Section Figure 1. Calcium release properties of the analogues. The effect of increasing concentrations of cADPR (1) (A), cADPR[CH2] (4) (B), 3-deaza-cADPR (2) (C), and 3-deaza-cADPR[CH2] (5) (D) are shown. (E) 8-Br-cADPR (48 µM) blocks the calcium release stimulated by 300 nM 3-deaza-cADPR[CH2] (5).

Figure 2. Concentration-response curves for calcium release by cADPR[CH2] (4) (9), 3-deaza-cADPR[CH2] (5) (b), cADPR (1) (0), and 3-deaza-cADPR (2) (O). The data represent the mean ( SD (n ) 3). The EC50 values are 0.76 ( 0.02, 59 ( 7, 302 ( 38, and 856 ( 111 nM for 2, 1, 5, and 4, respectively.

cADPR[CH2] is 15 times less potent than cADPR and 3-deaza-cADPR[CH2] is 5 times less potent than cADPR and about 375 times less potent than 3-deaza-cADPR. The EC50 values determined for cADPR and 3-deazacADPR in this study agree closely with those determined previously.21 The results of this study suggest that the pyrophosphate moiety of cADPR contributes to cADPR receptor binding. Substitution of the bridging pyrophosphate oxygen with methylene resulted in compounds that are full agonists but with decreased agonist potency. Even with some loss of potency, 4 and 5 will be useful as a

Nicotinamide Riboside 5′-Methylenebis(phosphonate) 2′,3′-O-Isopropylidene Acetal Mono(triethylammonium) Salt (9). Nicotinamide-β-D-riboside 2′,3′-O-isopropylidene acetal25 (220 mg, 0.59 mmol) was added to a mixture of methylenebis(phosphonyl) tetrachloride (735 mg, 2.94 mmol) in TMP (5 mL) at 0 °C. The mixture was kept at 0 °C for 5 h, and the reaction was quenched by adding the mixture to aqueous triethylamine (18.2 mmol) and stirring it for 2 h. After lyophilization, the residue was purified by anion exchange (DEAE-cellulose) followed by reversed-phase HPLC to give the monotriethylammonium salt of 9 as an amorphous glass (190 mg, 59% yield): 1H NMR (600 MHz, D2O) δ 9.36 (s, 1H), 9.21 (d, J ) 6.3 Hz, 1H), 8.92 (d, J ) 7.9 Hz, 1H), 8.27 (t, J ) 7 Hz, 1H), 6.39 (s, 1H), 5.38 (m, 1H), 5.18 (d, J ) 5.6 Hz, 1H), 5.00 (s, 1H), 4.27 (m, 1H), 3.16 (q, J ) 7.3 Hz, 6H), 1.90 (m, 2H, PCH2P), 1.64 (s, 3H), 1.43 (s, 3H), 1.23 (t, J ) 7.3 Hz, 9H); LC-MS (ESI) m/z 453 (M + 1). Bis(2′,3′-isopropylidene)-β-NAD[CH2] (10). The bis(tetrabutylammonium) salt of 9 (177 mg, 0.19 mmol) and 2′,3′-Oisopropylidene-5′-tosyladenosine (6)20 (105 mg, 0.23 mmol) were mixed in anhydrous acetonitrile (0.3 mL) and stirred at room temperature for 48 h. The mixture was diluted, and the product was purified by anion-exchange (DEAE-cellulose; linear gradient, 0-100 mM NH4HCO3). Compound 10 was isolated as a white solid (30 mg, 21% yield) along with a significant amount of starting material 9 (54 mg, 63% yield). 1 H NMR (500 MHz, D2O) δ 9.33 (s, 1H), 9.17 (d, 1H, J ) 6.0 Hz), 8.88 (d, 1H, J ) 7.5 Hz), 8.48 (s, 1H), 8.31 (s, 1H), 8.25 (t, 1H, J ) 7.0 Hz), 6.31 (s, 1H), 6.26 (d, 1H, J ) 2.5 Hz), 5.41 (m, 1H), 5.31 (m, 1H), 5.16 (m, 1H), 5.12 (d, 1H, J ) 5.5 Hz), 4.87 (m, 1H), 4.63 (m, 1H), 4.04-4.23 (m, 3H), 1.88 (t, 2H, J ) 19.0 Hz), 1.64 (s, 6H), 1.42 (s, 3H), 1.40 (s, 3H); LC-MS (ESI) m/z 742 (M + 1). NAD[CH2] (12). Compound 10 was dissolved in 1 N HCl and stirred for 4 h at room temperature. The solution was diluted and lyophilized twice to give NAD[CH2], which was primarily β-isomer but contained 10% R-NAD[CH2]. An NMR sample was purified by reversed-phase HPLC (Zorbax C-18; 2.1 cm × 25 cm) to give β-NAD[CH2] as the monotriethylammonium salt. 1H NMR (600 MHz, D2O) δ 9.34 (s, 1H), 9.20 (d, 1H, J ) 6 Hz), 8.79 (d, 1H, J ) 8.4 Hz), 8.44 (s, 1H), 8.17 (m, 2H), 6.02 (d, 1H, J ) 5.4 Hz), 5.99 (d, 1H, J ) 5.4 Hz), 4.094.51 (10 ribosyl Hs), 2.15 (t, 2H, J ) 17.4 Hz); LC-MS (ESI) m/z 662 (M + 1).

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2′,3′-O-Isopropylidene-5′-methanesulfonyl-3-deazaadenosine (7). 3-Deazaadenosine was prepared and converted to its acetonide as described previously.26 Methanesulfonyl chloride (0.21 mL, 2.68 mmol) was added to a solution of 2′,3′O-isopropylidene-3-deazaadenosine (0.4 g, 1.4 mmol) in pyridine (5 mL) at 0 °C. After 2 h, methanol (0.5 mL) was added to the mixture to destroy excess methanesulfonyl chloride, and after 30 min volatiles were removed in vacuo and the residue was partitioned between ethyl acetate (20 mL) and saturated sodium bicarbonate (10 mL). Phases were separated, and the organic phase was washed with brine, dried over Na2SO4, and evaporated. Purification by silica gel chromatography (4-10% MeOH-CH2Cl2) yielded 7 as a white solid (0.23 g, 43% yield). 1 H NMR (600 MHz, CDCl3) δ 8.00 (s, 1H), 7.86 (d, 1H, J ) 6.0 Hz), 6.88 (d, 1H, J ) 6.0 Hz), 5.97 (d, 1H, J ) 3.6 Hz), 5.02 (dd, 1H, J ) 3.6 and 6.6 Hz), 4.98 (dd, 1H, J ) 3.6 and 6.6 Hz), 4.55 (m, 1H), 4.49 (dd, 1H, J ) 3.0 and 11.4 Hz), 4.44 (dd, 1H, J ) 3.0 and 11.4 Hz), 2.95 (s, 3H), 1.66 (s, 3H), 1.41 (s, 3H). Bis(2′,3′-isopropylidene)-3-deaza-NAD[CH2] (11). The bis(tetrabutylammonium) salt of 9 (200 mg, 0.44 mmol) and 7 (115 mg, 0.3 mmol) were mixed in acetonitrile (0.4 mL) and stirred for 48 h. LC-MS analysis indicated that the reaction did not progress much after 48 h. Product was purified similarly as described for 10, resulting in the isolation of 11 as a white solid (95 mg, 43% yield) along with 90 mg (42%) of recovered 9. 1H NMR (600 MHz, D2O) δ 9.33 (s, 1H), 9.18 (d, 1H, J ) 6.5 Hz), 8.86 (d, 1H, J ) 8.0 Hz), 8.49 (s, 1H), 8.23 (t, 1H, J ) 7.3 Hz), 7.64 (d, 1H, J ) 7.0 Hz), 7.17 (d, 1H, J ) 7.0 Hz), 6.33 (d, 1H, J ) 2.5 Hz), 6.19 (d, 1H, J ) 3.5 Hz), 5.32 (m, 1H), 5.23 (m, 1H), 5.15 (dd, 1H, J ) 3.0 and 6.6 Hz), 5.12 (d, 1H, J ) 5.5 Hz), 4.89 (s, 1H), 4.63 (m, 1H), 4.22 (m, 1H), 4.02-4.13 (m, 3H), 1.86 (t, 2H, J ) 19.8 Hz), 1.63 (s, 3H), 1.62 (s, 3H), 1.40 (s, 3H), 1.37 (s, 3H); LC-MS (ESI) m/z 741 (M + 1). 3-Deaza-NAD[CH2] (13). Compound 11 (85 mg, 0.11 mmol) was mixed with 1 N HCl (3 mL) and stirred at room temperature for 4 h. The solution was diluted and lyophilized twice to give 3-deaza-NAD[CH2] as a white solid (73 mg, 100% yield). 1 H NMR (600 MHz, D2O) δ 9.42 (s, 1H), 7.27 (d, 1H, J ) 6.6 Hz), 8.87 (d, 1H, J ) 7.8 Hz), 8.56 (s, 1H), 8.23 (t, 1H, J ) 6.9 Hz), 7.63 (d, 1H, J ) 6.6 Hz), 7.27 (d, 1H, J ) 6.6 Hz), 6.13 (d, 1H, J ) 5.4 Hz), 6.00 (d, 1H, J ) 6.6 Hz), 4.55-4.61 (m, 3H), 4.43 (m, 2H), 4.36 (m, 1H), 4.32 (m, 1H), 4.19 (m, 3H), 2.23 (t, 2H, J ) 19.8 Hz); LC-MS (ESI) m/z 661 (M + 1). cADPR[CH2] (4). NAD[CH2] (12) (10 mg, 0.015 mmol) was dissolved in 10 mL of 0.1 M NaHCO3 (pH 8.5) and treated with 20 units of Aplysia ADP-ribosyl cyclase (Sigma, product no. A8950) for 4 h. Product was purified by chromatography on DEAE-cellulose with a linear gradient of 0-200 mM NH4HCO3 followed by rechromatography on AG 1-X2 (trifluoroacetate form) using a linear gradient of 0-25 mM TFA. cADPR[CH2] was isolated as a white solid (5 mg, 62% yield). HPLC: Waters S5 SAX column, 4.6 mm × 250 mm, 0.05 M KH2PO4, pH 3, 1 mL/min, cADPR[CH2] retention time of 8.44 min. Additional HPLC data confirming purity are reported in Supporting Information. 1H NMR (500 MHz, D2O) δ 9.10 (s, 1H), 8.42 (s, 1H), 6.17 (d, 1H, J ) 3 Hz), 6.06 (d, 1H, J ) 6.5 Hz), 5.40 (t, 1H, J ) 5.5 Hz), 4.65 (m, 1H), 4.48 (m, 1H), 4.35 (m, 2H), 4.23 (m, 1H), 4.12 (m, 1H), 3.99 (m, 1H), 2.05 (m, 2H); 31P NMR (162 MHz, D2O) δ 17.41. HRMS-ESI (m/z): [M + 1]+ calcd for C16H23N5O12P2, 540.089; found, 540.088. UV (H2O, pH 7) λmax ) 259 nm. 3-Deaza-cADPR[CH2] (5). 3-Deaza-NAD[CH2] (13) (15 mg, 0.02 mmol) was mixed with 0.1 M sodium bicarbonate (pH 8.5, 10 mL) and treated with 10 units of Aplysia ADP-ribosyl cyclase for 4 h. Product was purified as described above for 12. 3-Deaza-cADPR[CH2] (5) was isolated as a white solid (5 mg, 46% yield). HPLC: Waters S5 SAX column, 4.6 mm × 250 mm, 0.05 M KH2PO4, pH 3, 1 mL/min, 3-deaza-cADPR[CH2] retention time of 8.35 min. Additional HPLC data confirming purity is reported in Supporting Information. 1H NMR (600 MHz, D2O): δ 8.37 (s, 1H), 8.20 (d, 1H, J ) 7.8 Hz), 7.61 (d, 1H, J ) 7.8 Hz), 6.04 (s, 1H), 6.02 (d, 1H, J ) 5.4

Hz), 4.85 (t, 1H, J ) 5.4 Hz), 4.67 (d, 1H, J ) 4.8 Hz), 4.52 (t, 1H, J ) 4.8 Hz), 4.44 (m, 1H), 4.36 (t, 1H, J ) 5.5 Hz), 4.30 (m, 2H), 4.14 (dd, 1H, J ) 6.6 and 12 Hz), 4.02 (m, 2H), 2.00 (m, 1H), 1.77 (m, 1H); 13C NMR (150 MHz, D2O) δ 148.33, 144.96, 138.30, 128.31, 128.20, 101.68, 93.91, 90.93, 84.90 (d, J ) 8 Hz), 84.42 (d, J ) 8.9 Hz), 74.84, 73.24, 69.91, 69.19, 63.60 (d, J ) 4.6 Hz), 61.14 (d, J ) 5.2 Hz), 25.95 (t, J ) 131 Hz). HRMS-ESI (m/z): [M + 1]+ calcd for C17H24N4O12P2, 539.094; found, 539.091. UV (H2O, pH 7) λmax ) 263 nm. Calcium Release from Sea Urchin Egg Homogenates. Homogenates (1.25%) of Strongylocentrotus purpuratus eggs were prepared, and the fluorescent Ca2+ assay was conducted as described previously.27

Acknowledgment. We thank Merck & Co., Inc. for financial support through an MRL Doctoral Program Studies Award to Libo Xu. This work was supported by NIH Grant DA11806 to T.F.W. Supporting Information Available: Detailed information on synthetic methods, analytical and spectroscopic data, and Ca2+ releasing activity. This material is available free of charge via the Internet at http://pubs.acs.org.

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