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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Nucleosides Nucleotides Nucleic Acids. Author manuscript; available in PMC 2010 May 1.
Published in final edited form as:
Nucleosides Nucleotides Nucleic Acids. 2009 May; 28(5): 408–423.
doi:  10.1080/15257770903044465
PMCID: PMC2829736
NIHMSID: NIHMS133069

BASE-FUNCTIONALIZED CARBOCYCLIC NUCLEOSIDES: DESIGN,SYNTHESIS AND MECHANISM OF ANTIVIRAL ACTIVITY

Abstract

New carbocyclic ribonucleosides with unsaturated groups at the C-2 position of the nucleobase were designed as potential RNA antiviral compounds. The design was based on the expectation that the monophosphates of these compounds would be inhibitors of the enzyme, IMPDH. Appropriate methodologies were developed to achieve the target molecules. Results from the initial in vitro antiviral studies are mentioned. The IMPDH-associated mechanism of the antiviral activity of the most active compound is supported by enzyme inhibition studies.

Keywords: Carbocyclic nucleosides, synthesis, antiviral, IMPDH inhibitor

INTRODUCTION

Inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) catalyzes NAD (nicotinamide adenine dinucleotide)-dependent oxidation of inosine 5'-monophosphate (IMP) into xanthosine 5'-monophosphate (XMP), which is the rate-limiting reaction in the de novo biosynthesis of GTP.[1] Two distinct but nearly identical IMPDH isoforms (Type I and II) exist in mammalian cells sharing 85% sequence identity. IMPDH Type I is ubiquitous and is present in normal cells, whereas IMPDH Type II is upregulated in malignant cells, including human neoplastic cells [2-4] and human leukemic cells.[5] Inhibition of IMPDH causes the reduction of guanine nucleotide pools, impeding DNA and RNA synthesis, guanine nucleotide-coupled signaling and oncogene expression. IMPDH inhibition suppresses cell proliferation and induces cell differentiation and apoptosis.[6] As the demand for purine nucleotides needed for RNA and DNA synthesis increases significantly in virus-infected cells, inhibition of IMPDH may also lead to antiviral activity.[7] Thus, IMPDH has emerged as an important target enzyme for the development of chemotherapeutic agents and extensive efforts have been directed towards the discovery of IMPDH inhibitors.[8-11]

The mechanism of the biochemical conversion of IMP to XMP catalyzed by IMPDH is initiated by nucleophilic attack of the active-site residue, Cys-331, on IMP to form a covalent bond between the 2-position of IMP and the sulfhydryl group of Cys-331.[12,13] A hydride is then transferred to the cofactor, NAD+, to produce NADH and E-XMP*. Subsequently, the resulting intermediate, E-XMP,* is subject to hydrolysis, which liberates XMP via a tetrahedral intermediate E-XMP. Based on this mechanism, some nucleoside 5'-monophosphate derivatives containing modified purines as base moieties such as 3-deazaguanosine[14] and 2-vinylinosine[15] (Figure 1) have been identified as potent IMPDH inhibitors. 3-Deazaguanosine has been reported to possess broad spectrum antiviral activity against a variety of DNA and RNA viruses, as well as antitumor activity against the L1210 leukemia and several mammary adenocarcinomas in mice. [16,17] 2-Vinylinosine is a modified nucleoside with broad-spectrum RNA antiviral activity against a number of virus including JEV, PIC, PT, VEE and YF.[18]

Figure 1
3-Deazaguanosine and 2-vinylinosine are IMPDH inhibitors as their monophosphates

Compared to conventional nucleosides with natural sugar moieties, carbocyclic nucleosides are chemically more stable with respect to cellular degradation, particularly with respect to cleavage by nucleoside phosphorylases, because of the alteration of the N-glycosidic linkage. For example, 2-vinylinosine is a substrate for mammalian purine nucleoside phosphorylase,[19] while isonucleosides, where the base is translocated from the 1'-position to the non-glycosidic 2'-position, are not cleaved by nucleoside phosphorylases.[20] In the search for new ribonucleosides with RNA antiviral activity, we have synthesized new carbocyclic ribonucleosides functionalized at the C-2 position of the hypoxanthine nucleobase (Figure 2). This paper reports on the methodologies for the synthesis of these compounds, their antiviral activities and the IMPDH inhibition data and mechanism of antiviral activity of the most active compound.

Figure 2
Structures of target compounds

RESULTS AND DISCUSSION

There are two general approaches to this class of compounds, which can be described as linear or convergent. In terms of chemical regioselectivity, the approach that may be preferable is the linear approach rather than the convergent approach because the former avoids side products arising from alkylation at different nitrogens on the purine base. This also simplifies purifications. However, both approaches were used in this paper, depending on the target molecule. The bicyclic lactam, 2-azabicyclo[2.2.1]hept-5-en-3-one, in its racemic or chiral form, has been shown to be a versatile synthon for the preparation of carbocyclic nucleosides.[21] We used the commercially available chiral building block, (-)-2-azabicyclo[2.2.1]hept-5-en-3-one 1, as the starting material for these syntheses (Scheme 1). In several steps (cis-hydroxylation, isopropylidene and Boc protection, reduction, MOM protection and Boc removal), compound 1 was converted to the starting compound 2,[22] on which the nucleobase was constructed and elaborated at the C-2 position. We had also attempted protection of the primary hydroxyl group of 2 through formation of the corresponding tert-butyldimethylsilyl ether, but this protecting group could not be removed successfully under aqueous conditions due to the poor solubility of the compound in water.

Scheme 1
Synthesis of a carbocyclic analog of 2-vinylinosine

With intermediate 2 in hand as the building intermediate for the carbocyclic moiety, the strategy of first constructing carbocyclic guanosine and then modifying it was adopted for synthesis of target compounds. For carbocyclic 2-vinylinosine, the amine 2 and 2-amino-4,6-dichloro-5-formamidopyrimidine (prepared according to a literature method[23]) in ethanol in the presence of triethylamine was heated under reflux to afford compound 3. The chloropurine 4 was obtained by treating 3 with 4 equivalents of concentrated HCl in triethylorthoformate.[24] However, under these acidic conditions, the cyclization reaction was accompanied by removal of the isopropylidene protecting group. Thus, continuation of the synthesis began with the diacetate 5, which underwent deamination-halogenation[25] on treatment with isoamyl nitrite in the presence of diiodomethane at 75 °C to afford the 2-iodo derivative 6. Cross coupling of 6 with tributylvinylstannane catalyzed by Pd(CH3CN)2Cl2 was carried out in CH3CN under reflux conditions[26] to introduce the vinyl group at the C-2 position of the purine ring. The product, 7, was subjected first to acidic hydrolysis conditions (removal of MOM protecting group) and the resulting product was treated with ammonium hydroxide to disconnect the acetate protecting groups to afford target compound 8.

While compound 8 and its precursor, 7, are relatively stable with respect to the C-2 vinyl substituent under the harsh acidic conditions used for the hydrolysis of compound 7, target compounds and their precursors with reactive Michael acceptors at C-2 (e.g., α,β-unsaturated carbonyls) were found to be less stable under these conditions. So a new synthetic route based on radical deamination-iodination of partially protected carbocyclic guanosine and palladium-catalyzed cross-coupling between unprotected carbocyclic 2-iodoinosine and appropriate tin reagents was designed in which those substituents susceptible to nucleophilic attack were introduced into the purine ring in the final step. In this way, deprotection under acidic conditions was avoided and the sequence of protection and deprotection were necessary to achieve the target compounds described below.

Protected carbocyclic guanosine precursor 9 was synthesized from 2 in one step[22,23,27] and then converted in three further steps[24] to provide 10 (Scheme 2). Compound 10 was treated with acetic anhydride and triethylamine and the resulting triacetate, 11, subjected to aprotic diazotization-halogenation[28] with isoamyl nitrite in the presence of iodoform in refluxing CH3CN to afford the 2-iodopurine 12. After formation of the 2-iodo compound, the protective groups of compound 12 were removed to provide the deprotected coupling precursor 13. The 3-oxocyclohex-1-enyl functionality was introduced at C-2 through cross-coupling of 13 with 3-(tributylstannyl)cyclohex-2-enone under catalysis with Pd(PPh3)2Cl2 in DMF to directly afford target compound 14. Synthesis of the 2-ethynyl compound 15 required two steps[29] from precursor intermediate 12.

Scheme 2
Methodology to purine carbocyclic nucleosides with Michael acceptors at C-2.

A synthetic strategy similar to that used for 14 was adopted for the synthesis of compounds with other Michael acceptors at C-2: 16 (3-oxocyclopent-1-enyl), 17 [(E)-3-oxobut-1-enyl] and 18 [(E)-buta-1,3-dienyl] (Scheme 3).

Scheme 3
Carbocyclic hypoxanthine nucleosides with other Michael acceptors.

The compounds synthesized in this paper were submitted for initial antiviral assays against a number of RNA viruses. The most active compound in this series was compound 8, which is moderately active against Flu A (H5N1), [EC50 = 3μg/ml, CC50 74 μg/ml, TI = 23 (MDCK, NR Assay)]. It also exhibits low activity against the Punta Toro virus [EC50 = 7μg/ml, CC50 28 μg/ml TI = 4 (LLC-MK2), NR Assay]. The mechanism of the antiviral activity of compound 8 may be associated with the inherent ability of its monophosphate to inhibit IMPDH through blocking the formation of the ternary complex normally formed between the enzyme, the substrate IMP and the cofactor, NAD+.7 Support for this comes from our observation of the inhibition of E. coli IMPDH by the monophosphate of 8 (graphical data shown below in Figures 3 and and4).4). We found that 8-MP was a strong inactivator of IMPDH with a kon of 2.12 × 104 M-1s-1 (the second-order rate constant, kon = kinact/Ki, is a measure of the potency of the inhibition). In comparison, 2-vinylinosine 5'-monophosphate exhibited a kon of 0.73 × 104 M-1s-1 and 6-chloropurine ribonucleoside 5'-monophosphate had a kon of 1.55 × 102 M-1s-1 (E. coli IMPDH).12,15

Figure 3
Progress curves for the inhibition of IMPDH by inhibitor 8-MP monitored by the UV absorption of cofactor product, NADH, at 340 nM (see reference 15 for detailed procedure for inhibition studies).
Figure 4
Plot of Kobs versus inhibitor concentration derived from progress curves of Figure 3. The relevant equations are: A - Ao = Vo/Kobs[1-exp(-Kobst) and Kobs = kon[I]/(1 +[IMP]/Km.

The activity/toxicity of two other compounds of the series synthesized need to be mentioned. Compound 15 shows some toxicity and it was difficult to determine whether any observed activity (e.g., against the West Nile virus) was due to activity or was a manifestation of the toxicity of this compound. Target compound 18 exhibited low activity against SARS (Vero cells). Further antiviral studies are in progress.

SUMMARY AND CONCLUSIONS

In summary, six new carbocyclic hypoxanthine ribonucleosides, 8 and 14-18, with different unsaturated groups at the C-2 position of the nucleobase were designed as inhibitors of IMPDH as their monophosphates. Because of the reactivity of the exocyclic groups at C-2, the multi-step syntheses of these compounds were challenging but appropriate methodologies were developed to achieve the target molecules. Evaluation of the compounds for in vitro RNA antiviral activity revealed that one of the compounds (8) had good activity against the FluA (H5N1) virus. The mechanism of the antiviral activity of compound 8 may be associated with the inherent ability of its monophosphate to inhibit IMPDH and experimental support for this came from inhibition studies, which showed that the monophosphate of this compound was a strong inactivator of IMPDH.

EXPERIMENTAL SECTION

(1R,2S,3R,4R)-1-[(2-Amino-6-chloro-5-formamido-4-pyrimidinyl) amino] -2,3-isopropyl-idenedioxy-4-(methoxymethoxymethyl)cyclopentane (3)

A mixture of compound 2 (1.32g, 5.70mmol), [22] 2-amino-4,6-dichloro-5-formamidopyrimidine (1.18g, 5.70mmol) and triethylamine (2mL) in ethanol (15mL) was heated under reflux for 7h. After cooling to 5 °C, the mixture was filtered and washed with ethanol (50mL). The combined filtrate was concentrated and the residue was purified on a silica gel column using chloroform/methanol (97:3) as eluent to give compound 3 (1.18g, 51.5%): 1H NMR (500MHz, DMSO-d6) δ 9.05 and 8.66 (s and d, J=11Hz, total 1H), 8.17 and 7.81 (s and d, J=11Hz, total 1H), 7.11 and 6.82 (two d, J=8, 8Hz, total 1H), 6.64 and 6.53 (two br.s, total 2H), 4.60 (s, 2H), 4.54-4.51 (m, 1H), 4.42-4.39 (m, 2H), 3.51-3.48 (m, 2H), 3.27 (s, 3H), 2.23-2.16 (m, 2H), 1.47-1.43 (m, 1H), 1.42 (s, 3H), 1.23 (s, 3H). 13C NMR (125MHz, CD3OD) δ 167.4, 162.5, 161.3, 159.9, 155.4, 111.3, 96.3, 86.0, 83.0, 69.0,.56.7, 54.4, 45.0, 33.0, 26.1,23.6; UV λmax (MeOH) 212 nm (ε 33500), 241 nm (ε 16000), 289 nm (ε 10100); HRMS (ESI) calcd for C16H25Cl N5O5 [M+H]+ for 402.1544, found 402.1531.

(1R,2S,3R,5R)-3-(2-Amino-6-chloro-9H-purin-9-yl)-5-[(methoxymethoxy)methyl] cyclopentane-1,2-diol (4)

A solution of compound 3 (100mg, 0.25mmol) and triethylorthoformate (60mL) was stirred while conc. HCl (37%, 1.8mL) was added in one portion. A clear, light yellow solution resulted within one minute. After 4 h, the solution was neutralized with saturated NaHCO3, extracted with EtOAc, washed with water and dried over anhydrous Na2SO4. The solvent was removed under reduced pressure, and the residue was dissolved in 0.6 N HCl. After stirring for 30 min at room temperature, the pH of the reaction mixture was adjusted to 10 with ammonium hydroxide. The solution was concentrated to dryness and the residue was dissolved in acetone and filtered. The filtrate was evaporated to give the crude product, which was purified by silica gel chromatography to afford pure compound 4 (70 mg, 81.4%): 1H NMR (500MHz, DMSO-d6) δ 8.28 (s, 1H), 6.89 (br.s, 2H), 5.05 (d, J=6Hz, 1H), 4.78 (d, J=4Hz, 1H), 4.68-4.61 (m,3H), 4.35-4.31 (m, 1H), 3.83-3.81 (m, 1H), 3.60-3.57(m, 1H), 3.52-3.48 (m, 1H), 3.29 (s, 3H), 2.28-2.26 (m, 1H), 2.20-2.16 (m, 1H), 1.64-1.61 (m, 1H); 13C NMR (125MHz, CD3OD) δ 159.9, 154.1, 150.1, 142.5, 124.0, 96.2, 74.5, 72.5, 69.0, 60.2, 54.2, 43.2, 28.6; UV λmax (MeOH) 225 nm (ε 34300), 248 nm (ε 6800), 310 nm (ε 9700); HRMS (ESI) calcd for C13H19ClN5O4 [M+H]+ for 344.1126, found 344.1107.

(1R,2S,3R,5R)-3-(2-Amino-6-chloro-9H-purin-9-yl)-5-[(methoxymethoxy)methyl] cyclopentane-1,2-diol diacetate (5)

A solution of compound 4 (100mg, 0.29mmol) in acetonitrile (5mL) was treated with acetic anhydride (0.15mL, 1.60mmol) in the presence of pyridine (5mL) and stirred at room temperature overnight. The solvent was evaporated under reduced pressure and the residue was purified by column chromatography on silica gel to give the title compound 5 (98mg, 0.23mmol, 79.3%):1H NMR (500MHz, DMSO-d6) δ 8.34 (s, 1H), 6.99 (br. s, 2H), 5.65-5.62 (m, 1H), 5.25-5.23 (m, 1H), 4.99-4.93 (m, 1H), 4.65 (s, 2H), 3.66-3.62 (m, 2H), 3.30 (s, 3H), 2.47-2.38 (m, 2H), 2.10 (s, 3H), 2.01-1.93 (m, 1H), 1.91 (s, 3H). 13C NMR (125MHz, CDCl3) δ 170.0, 169.9, 159.0, 153.7, 151.4, 141.2, 125.6, 96.6, 74.4, 73.3, 67.9, 57.3, 55.6, 41.2, 28.7, 20.9, 20.5; UV λmax (MeOH) 221 nm (ε 23200), 247 nm (ε 5800), 308 nm (ε 6030); HRMS (ESI) calcd for C17H23Cl N5O6 [M+H]+ for 428.1337, found 428.1346.

(1R,2S,3R,5R)-3-(2-Iodo-6-chloro-9H-purin-9-yl)-5-[(methoxymethoxy) methyl]cyclopentane-1,2-diol diacetate (6)

A dry, one-necked round bottom flask equipped with a magnetic stirring bar and an argon bubble was charged with compound 5 (235mg, 0.55mmol) and acetonitrile (25mL). The solution was stirred under the argon atmosphere and cooled to about 5°C in an ice/water bath. To the solution was added diiodomethane (735mg, 2.75mmol) and isoamyl nitrite (332mg, 2.75mmol). The solution was purged with argon for 30 min and then heated at 90°C under an argon atmosphere for 5 h. After removal of solvent under reduced pressure, the residue was purified on a silica gel column to give compound 6 (135mg, 45.6%): 1H NMR (500MHz, CDCl3) δ 8.22 (s, 1H), 5.75-5.72 (m, 1H), 5.42-5.40 (m, 1H), 5.22-5.16 (m, 1H), 4.78-4.74 (m, 2H), 3.85-3.82 (m,1H), 3.69-3.66 (m, 1H), 3.45 (s, 3H), 2.74-2.69 (m, 1H), 2.57-2.54 (m, 1H), 2.18 (s, 3H), 2.17-2.13 (m, 1H), 1.99 (s, 3H). 13C NMR (CDCl3, 125MHz) δ 170.0, 169.9, 152.7, 150.7, 143.6, 132.0, 116.6, 96.8, 74.8, 73.7, 67.8, 57.8, 55.7, 41.3, 29.3, 20.9, 20.5; UV λmax (MeOH) 277nm (ε 11300); HRMS (ESI) calcd for C17H21 Cl IN4O6 [M+H]+ for 539.0194, found 539.0199.

(1R,2S,3R,5R)-3-(2-Vinyl-6-chloro-9H-purin-9-yl)-5-[(methoxymethoxy)methyl]cyclopentane-1,2-diol diacetate (7)

Compound 6 (260mg, 0.48mmol), Pd(CH3CN)2Cl2 (8mg, 0.031mmol) and tributylvinylstannane (780mg, 2.46mmol) in anhydrous CH3CN (18mL) was heated under reflux for 2 h. The solution was evaporated to dryness under reduced pressure and the residue was purified by silica gel column chromatography to afford compound 7 (140mg, 66.5%): 1H NMR (500MHz, CDCl3) δ 8.16 (s, 1H), 6.93-6.88 (m, 1H), 6.71-6.68 (m, 1H), 5.81-5.76 (m, 2H), 5.47-5.45 (m, 1H), 5.12-5.08 (m,1H), 4.71 (s, 2H), 3.80-3.77 (m 1H), 3.70-3.67 (m, 1H), 3.41 (s, 3H), 2.64-2.60 (m,1H), 2.56-2.54 (m,1H), 2.29 (s, 3H), 2.15-2.11 (m, 1H), 1.95 (s, 3H). 13C NMR (125MHz, CDCl3) δ169.9, 169.8, 158.9, 152.2, 150.9, 144.0, 135.4, 130.6, 124.4, 96.7, 74.6, 73.3, 67.8, 58.0, 55.6, 41.3, 29.0, 20.9, 20.5; UV λmax (MeOH) 229 nm (ε 21500), 276 nm (ε 12400); HRMS (ESI) calcd for C19H24 Cl N4O6 [M+H]+ for 439.1384, found 439.1383.

9-[(1'R,2'S,3'R,4'R)-2',3'-Dihydroxy-4'-(hydroxymethyl)cyclopentyl]-2-vinyl-1H-purin-6(9H)-one (8)

Compound 7 (140mg, 0.31mmol) was heated under reflux in 1N HCl (10mL) for 1 h. After cooling to room temperature, the reaction mixture was treated with ammonium hydroxide (10mL, pH 10-11). Then the solvent was removed under reduced pressure and the residue was purified initially by silica gel column chromatography and then by HPLC to provide compound 8 (43mg, 47.5%): 1H NMR (500MHz, CD3OD) δ 8.16 (s, 1H), 6.69-6.60 (m, 2H), 5.85 (dd, J=9, 2Hz, 1H), 4.91-4.87 (m, 1H), 4.58-4.55 (m,1H), 4.10-4.09 (m,1H), 3.74-3.72 (m, 2H), 2.49-2.45 (m, 1H), 2.27 (m, 1H), 2.04-1.99 (m, 1H); 13C NMR (125MHz, CD3OD) δ 157.9, 151.7, 149.4, 140.4, 129.2, 124.5, 123.2, 75.2, 72.3, 63.2, 60.6, 45.4, 29.0; UV (H2O) λmax 295 nm (ε 7100), 262 nm (ε 7700); HRMS (ESI) calcd for C13H17N4O4 [M+H]+ for 293.1250, found 293.1222.

2-Amino-6-[(1'R,2'S,3'R,4'R)-2',3'-isopropylidenedioxy-4'-(methoxymethoxymethyl)-cyclopentylamino]-5-nitropyrimidin-4(3H)-o n e (9)

2-Amino-6-chloro-5-nitropyrimidin-4(3H)-one (2.65g, 13.9mmol) and the amine 2 (1.34g, 5.79mmol) were suspended in dry EtOH (100mL) and stirred for 1h over 4A molecular sieves. The reaction mixture was subsequently heated under reflux for 2h. After cooling to room temperature, the mixture was filtered and the residue was washed with (3 × 10 mL). The combined filtrate was evaporated to dryness and the residue was purified by silica gel column chromatography to give compound 9 (1.53g, 65.7%): 1H NMR (500MHz, DMSO-d6) δ 10.66 (s, 1H), 9.74 (d, J=8Hz, 1H), 7.95 (br.s, 1H), 6.61 (br.s, 1H), 4.68-4.66 (m, 1H), 4.64-4.61 (m, 2H), 4.56-4.54 (m, 1H), 4.48-4.47 (m,1H), 3.55-3.48 (m, 2H), 3.26 (s, 3H), 2.41-2.37 (m, 1H), 2.28-2.26 (m,1H), 1.59-1.54 (m,1H), 1.42 (s, 3H), 1.24 (s, 3H); 13C NMR (125MHz, DMSO-d6) δ 159.0, 156.7, 154.5, 111.3, 110.9, 96.1, 86.2, 83.1, 69.2, 57.3, 55.1, 44.9, 34.3, 27.6, 25.1; UV (MeOH) λmax 215 nm (ε 31500), 238 nm (ε 21900), 283 nm (ε 7600), 330 nm (ε 16300); HRMS (ESI) calcd for C15H24N5O7 [M+H]+ for 386.1676, found 386.1675.

2-Amino-9-[(1'R,2'S,3'R,4'R)-2',3'-dihydroxy-4'-(hydroxymethyl)cyclopentyl]-1H-purin -6(9H)-one (10)

The nitro product from the previous step (1000 mg, 2.60 mmol) was dissolved in dry methanol (50mL). Under an argon atmosphere, 10% Pt/C (1000 mg) was added and the reaction mixture was stirred under a H2 atmosphere (14 psi). After 30 min, diisopropylethylamine (1mL) was added and the hydrogenation was continued for 3h. The solvent was removed under reduced pressure, and the crude product was dissolved in formamide (10 mL) under argon, and the solution was heated under reflux for 2h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate and filtered through Celite. The filtrate was condensed to dryness under reduced pressure and the residue was purified by silica gel column chromatography to give the cyclized protected nucleoside (670mg, 70.5% for two steps): 1H NMR (500MHz, DMSO-d6) δ10.61 (s, 1H), 7.89 (s, 1H), 6.64 (br.s, 2H), 4.65-4.62 (m, 1H), 4.61(s, 2H), 4.54-4.52 (m, 1H), 4.46-4.44 (m,1H), 3.56-3.54 (m, 2H), 3.28 (s, 3H), 2.31-2.28 (m, 2H), 2.03-2.00 (m,1H), 1.50 (s, 3H), 1.25 (s, 3H); 13C NMR (125MHz, DMSO-d6) δ 161.9, 157.3, 153.9, 151.5, (137.5), 117.3, 113.1, 96.2, 83.8, 81.6, 68.8, 59.5, 55.1, 43.8, 35.1, 27.9, 25.5; UV λ (MeOH) 254 nm (ε 12600); HRMS (ESI) calcd for C16H24N5O5 [M+H]+ for 366.1777, found 366.1761.

The protected nucleoside from the previous step (300mg, 0.82mmol) was heated to 80°C in 1N HCl for 20 min. After cooling to room temperature, the solution was neutralized by saturated NaHCO3. The solvent was then removed and the residue was dissolved in methanol and filtered. The filtrate was concentrated and purified by HPLC to give compound 10 (166mg, 71.3%):1H NMR (500MHz, CD3OD) δ 7.84 (s,1H), 4.71-4.66 (m, 1H), 4.47-4.44 (m, 1H), 4.05-4.03 (m, 1H), 3.72-3.69 (m, 2H), 2.42-2.39 (m, 1H), 2.24-2.22 (m, 1H), 1.91-1.88 (m, 1H); 13C NMR (125MHz, CD3OD) δ 158.0, 153.5, 152.0, 137.4, 116.7, 75.1, 72.3, 63.2, 59.9, 45.4, 28.8; UV λmax (MeOH) 254nm (ε 9600); HRMS (ESI) calcd for C11H16N5O4 [M+H]+ for 282.1202, found 282. 1190.

2-Amino-9-[(1'R,2'S,3'R,4'R)-2',3'-diacetoxy-4'-(acetoxymethyl)cyclopentyl]-1H-purin-6(9H)-one (11)

Compound 10 (127 mg, 0.45 mmol), triethylamine (1010 mg, 10 mmol) and DMAP (10 mg, 0.08 mmol) were suspended in dry acetonitrile (10 mL). To the solution was added acetic anhydride (163mg, 1.6mmol) and the reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography to give compound 11 (135 mg, 73.1%): 1H NMR (500MHz, CD3OD) δ 7.77 (s, 1H), 5.72-5.68 (m,1H), 5.37-5.34 (m, 1H), 4.94-4.91 (m, 1H), 4.29-4.26 (m, 2H), 2.66-2.63 (m,1H), 2.45-2.42 (m, 1H), 2.11-2.07 (m,1H), 2.09 (s, 3H), 2.07 (s, 3H), 1.96 (s, 3H); 13C NMR (125MHz, CD3OD) δ171.4, 170.3, 170.2, 158.0, 153.8, 151.7, 137.6, 116.8, 74.4, 72.4, 64.4, 57.8, 40.6, 28.2, 19.5, 19.4, 19.1; UV λmax (MeOH) 254nm (ε 10000); HRMS (ESI) calcd for C17H22N5O7 [M+H]+ for 408.1519, found 408.1509.

9-[(1'R,2'S,3'R,4'R)-2',3'-Diacetoxy-4'-(acetoxymethyl)cyclopentyl]-2-iodo-1H-purin-6(9H)-one (12)

To a solution of compound 11 (135mg, 0.33 mmol) and iodoform (1299 mg, 3.3 mmol) in dry acetonitrile (15 mL) under argon was added isoamyl nitrite (387 mg, 3.3 mmol) at 5°C. The solution was purged with argon for 30 min and then heated under reflux under argon for 2h. After removal of solvent under reduced pressure, the residue was purified by silica gel column chromatography to give compound 12 (25 mg, 14.6%): 1H NMR (500MHz, CD3OD) δ 8.06 (s, 1H), 5.70-5.66 (m, 1H), 5.40-5.38 (m, 1H), 5.09-5.07 (m, 1H), 4.32-4.30 (m, 2H), 2.64-2.61 (m,1H), 2.55-2.52 (m, 1H), 2.15-2.13 (m,1H), 2.14 (s, 3H), 2.13 (s, 3H), 2.02 (s, 3H); 13C NMR (125MHz, CDCl3) δ 171.0, 169.8, 169.7, 158.7, 148.9, 139.0, 124.9, 105.0, 74.3, 72.2, 64.1, 58.3, 40.5, 29.0, 21.0, 20.9, 20.5; UV λmax (MeOH) 254nm (ε 12900); HRMS (ESI) calcd for C17H20IN4O7 [M+H]+ for 519.0377, found 519.0376.

9-[(1'R,2'S,3'R,4'R)-2',3'-Dihydroxy-4'-(hydroxymethyl)-cyclopentyl]-2-iodo-1H-purin-6(9H)-one (13)

A solution of compound 12 (20 mg, 0.038 mmol) in methanolic ammonia (10 mL) was stirred under room temperature for 12 h. After the reaction was complete, the solvent was removed under reduced pressure. The crude residue was purified by silica gel column chromatography to give compound 13 (11mg, 71.1%): 1H NMR (500MHz, CD3OD) δ 8.05 (s, 1H), 4.86-4.81 (m, 1H), 4.50-4.47 (m, 1H), 4.08-4.06 (m, 1H), 3.74-3.69 (m, 2H), 2.50-2.46 (m, 1H), 2.28-2.25 (m, 1H), 1.91-1.86 (m, 1H); 13C NMR (125MHz, CD3OD) δ160.3, 149.4, 138.9, 124.0, 109.7, 75.5, 72.4, 63.2, 60.2, 45.4, 29.2; UV λmax (MeOH) 254nm (ε 12900); HRMS (ESI) calcd for C11H14IN4O4 [M+H]+ for 393.0060, found 393.0008.

9-[(1'R,2'S,3'R,4'R)-2',3'-Dihydroxy-4'-(hydroxymethyl)cyclopentyl]-2-(3-oxocyclohex-1-enyl)-1H-purin-6(9H)-one (14)

To a solution of compound 13 (50 mg, 0.13 mmol) and Pd(PPh3)2Cl2 (14 mg, 0.01mmol) in anhydrous DMF (5mL) was added 3-(tributylstannyl)cyclohex-2-enone (120mg, 0.30mmol). The reaction mixture was stirred at 95 °C under argon for 24 h. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography to give the title compound 14 (20mg, 42.2%): 1H NMR (500MHz,DMSO-d6) δ 12.28 (s, 1H), 8.17 (s, 1H), 6.75 (s, 1H), 4.92 (m, 1H), 4.68-4.64 (m, 3H), 4.27-4.25 (m, 1H), 3.78 (m, 1H), 3.42-3.36 (m, 2H), 2.77-2.74 (m, 2H), 2.37-2.35 (m, 2H), 2.20-2.15 (m, 1H), 1.98-1.93 (m, 3H), 1.68-1.65 (m, 1H); 13C NMR (125MHz, DMSO-d6) δ 199.6, 157.5, 151.9, 151.7, 148.5, 141.5, 129.5, 125.0, 75.3, 72.2, 63.6, 60.2, 45.7, 37.6, 29.9, 25.7, 22.4; UV λmax (MeOH) 243nm (ε 15500), 328 (ε 7400); HRMS (ESI) calcd for C17H21N4O5 [M+H]+ for 361.1512, found 361.1519.

Preparation of 9-[(1'R,2'S,3'R,4'R)-2',3'-dihydroxy-4'-(hydroxymethyl)cyclo pentyl]-2-ethynyl-1H-purin-6(9H)-one (15)

To a solution of compound 12 (130mg, 0.25mmol) and Pd(PPh3)2Cl2 (18mg, 0.025mmol) in anhydrous DMF (5mL) was added 2-(tributylstannyl)ethynyl)trimethylsilane (387mg, 1.00mmol). The reaction mixture was stirred at 95 °C under argon for 24 h. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography to give the title compound 15 (86mg, 70.4%): 1H NMR (500MHz, CDCl 3) δ 11.54 (br.s, 1H), 7.85 (s, 1H), 5.68-5.65 (m, 1H), 5.35-5.33 (m, 1H), 5.00-4.98 (m,1H), 4.27-4.20 (m,2H), 2.55-2.52 (m, 2H), 2.13 (s, 3H), 2.10 (s, 3H), 2.09-2.07 (m,1H), 1.96 (s, 3H), 0.11 (s, 9H); 13C NMR (125MHz, CDCl3) 171.6, 170.5, 170.4, 158.3, 149.3, 140.2, 138.4, 126.6, 101.7, 96.7, 74.7, 73.0, 65.0, 58.5, 41.3, 29.7, 21.7, 21.6, 21.2; UV λmax (MeOH) 254nm (ε 6400), 261nm (ε 6300), 301nm (ε 9300) HRMS (ESI) calcd for C22H29N4O7Si [M+H]+ for 489.1806, found 489.1801.

The aforementioned coupled product (86mg, 0.17mmol) was treated with NH3/MeOH (saturated at 0 °C, 20mL) at 0 °C for 6 h. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography and HPLC to afford the title compound 33 (24mg, 48.6%): 1H NMR (500MHz, DMSO-d6) δ 12.84 (br.s, 1H), 8.19 (s, 1H), 4.92 (m, 1H), 4.69-4.58 (m, 4H), 4.17-4.15 (m,1H), 3.75 (m,1H), 3.52-3.36 (m, 2H), 2.19-2.17 (m, 1H), 1.97-1.95 (m,1H), 1.54-1.50 (m, 1H); 13C NMR (125MHz, DMSO-d6) 157.4, 148.6, 140.4, 137.6, 125.5, 83.3, 77.5, 75.6, 72.1, 63.4, 59.5, 45.8, 30.3; UV λmax (MeOH) 252nm (ε 2900), 260nm (ε 2800), 297nm (ε 3800); HRMS (ESI) calcd for C13H15N4O4 [M+H]+ for 291.1093, found 291.1060.

9-[(1'R,2'S,3'R,4'R)-2',3'-Dihydroxy-4'-(hydroxymethyl)cyclopentyl]-2-(3-oxocyclo- pent-1-enyl)-1H-purin-6(9H)-one (16)

Compound 13 (60mg, 0.15mmol) and Pd(PPh3)2Cl2 (8mg, 0.01mmol) in anhydrous DMF (5mL) was treated with 3-(tributylstannyl)cyclopent-2-enone (174mg, 0.45mmol) and the reaction mixture was stirred at 95 °C under argon for 24 h. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography to give compound 16 (22mg, 43.1%): 1H NMR (500MHz, CD3OD) δ 8.23 (s, 1H), 7.04 (s, 1H), 4.93-4.89 (m, 1H), 4.62-4.60 (m, 1H), 4.12-4.10 (m, 1H), 3.75-3.72 (m, 2H), 3.23-3.20 (m, 2H), 2.64-2.62 (m, 2H), 2.49-2.46 (m, 1H), 2.28-2.26 (m, 1H), 2.11-2.09 (m, 1H); 13C NMR (125MHz, DMSO-d6) δ 209.4, 166.7, 157.8, 149.0, 148.5, 142.1, 133.6, 125.7, 75.2, 72.2, 63.6, 60.7, 45.7, 35.4, 29.7, 28.2; UV λmax (MeOH) 240nm (ε 22800), 332nm (ε 14500); HRMS (ESI) calcd for C16H19N4O5 [M+H]+ for 347.1355, found 347.1351.

9-[(1'R,2'S,3'R,4'R)-2',3'-Dihydroxy-4'-(hydroxymethyl)cyclo- pentyl]-2-[(E)-3-oxobut-1-enyl]-1H-purin-6(9H)-one (17)

To a solution of compound 13 (30mg, 0.08mmol) and Pd(PPh3)2Cl2 (6mg, 0.008mmol) in anhydrous DMF (5mL) was added (E)-4-(tributylstannyl)but-3-en-2-one (41.2mg, 0.12mmol). The reaction mixture was stirred at 95 °C under argon for 24 h. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography to give compound 17 (11mg, 41.2%): 1H NMR (500MHz, CD3OD) δ 8.24 (s, 1H), 7.39 (d, J =16Hz, 1H), 7.34 (d, J=16Hz, 1H), 4.92-4.90 (m, 1H), 4.57-4.54 (m, 1H), 4.11-4.09 (m, 1H), 3.75-3.74 (m, 2H), 2.49-2.46 (m,1H), 2.45 (s, 3H), 2.29-2.27 (m, 1H), 2.05-2.02 (m, 1H); 13C NMR (125MHz, CD3OD) δ 198.1, 157.8, 150.1, 149.1, 141.0, 134.6, 133.4, 124.2, 75.3, 72.3, 63.1, 60.6, 45.4, 29.0, 26.8; UV λmax (MeOH) 233nm (ε 26100), 337nm (ε 9900); HRMS (ESI) calcd for C15H19N4O5 [M+H]+ for 335.1355, found 335.1345.

2-[(E)-Buta-1,3-dienyl]-9-[(1'R,2'S,3'R,4'R)-2',3'-dihydroxy-4'-(hydroxymethyl)cyclopentyl] -1H-purin-6(9H)-one (18)

Compound 13 (60mg, 0.15mmol) and Pd(PPh3)2Cl2 (8mg, 0.01mmol) in anhydrous DMF (5mL) was treated with (E)-buta-1,3-dienyltributylstannane (172mg, 0.50mmol). This reaction mixture was then stirred at 95 °C under argon for 24 h. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give compound 18 (21mg, 44.3%):1H NMR (500MHz, CD3OD) δ 8.15 (s, 1H), 7.64-7.59 (m, 1H), 6.67-6.63 (m, 1H), 6.48-6.45 (m, 1H), 5.73-5.70 (m, 1H), 5.55-5.53 (m, 1H), 4.92-4.88 (m, 1H), 4.58-4.55 (m, 1H), 4.12-4.10 (m, 1H), 3.76-3.75 (m, 2H), 2.49-2.44 (m, 1H), 2.30-2.27 (m, 1H), 2.08-2.05 (m, 1H); 13C NMR (125MHz, CD3OD) δ 158.0, 152.1, 149.6, 140.3, 140.0, 135.4, 123.5, 123.1, 122.9, 75.3, 72.4, 63.1, 60.5, 45.4, 28.9; UV λmax (MeOH) 236 nm (ε 34000), 260 nm (ε 29400), 321 nm (ε 22900); HRMS (ESI) calcd for C15H19N4O4 [M+H]+ for 319.1406, found 319.1404.

Acknowledgments

This project was supported by NIAID (NIH) through research award number U19 AI 056540.

Dedicated to Professor Morris Robins on the occasion of his seventieth birthday

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