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J. Med. Chem.

2005, 48, 1389-1394 1389

Synthesis of Phosphonated Carbocyclic 2′-Oxa-3′-aza-nucleosides: Novel
Inhibitors of Reverse Transcriptase

Ugo Chiacchio,*,† Emanuela Balestrieri,‡ Beatrice Macchi,‡ Daniela Iannazzo, Anna Piperno, Antonio Rescifina,†
Roberto Romeo, Monica Saglimbeni, M. Teresa Sciortino,§ Vincenza Valveri,§ Antonio Mastino,*,§ and
Giovanni Romeo*
Dipartimento Farmaco-Chimico, Università di Messina, Via SS. Annunziata, Messina 98168, Italy, Dipartimento di Scienze
Chimiche, Università di Catania, Viale Andrea Doria 6, Catania 95125, Italy, Dipartimento di Neuroscienze,
Università di Roma ‘Tor Vergata’, Via Montpellier 1, and IRCCS S. Lucia, Roma 00133, Italy, and Dipartimento di Scienze
Microbiologiche, Genetiche e Molecolari, Università di Messina, Salita Sperone 31, Messina 98168, Italy

Received July 28, 2004

Phosphonated carbocyclic 2′-oxa-3′-aza-nucleosides have been synthesized in good yields by
1,3-dipolar cycloaddition methodology. The cytotoxicity and the reverse transcriptase inhibitory
activity of the obtained compounds have been investigated. Phosphonated carbocyclic 2′-oxa-
3′-aza-nucleosides, while showing low levels of cytotoxicity, exert a specific inhibitor activity
on two different reverse transcriptases, which is comparable with that of AZT, opening new
perspectives on their possible use as therapeutic agents, in anti-retroviral and anti-HBV

Introduction improve the uptake of ddNs might be to bypass the
phosphorylating step; unfortunately, nucleotides, due
The number of modified nucleosides has grown expo-
to their polar nature, are not able to cross the cell
nentially in recent years in response to the pressing
membrane efficiently. Moreover, they are readily de-
need for new treatments against virus infections. In
phosphorylated in extracellular fluids and on cell sur-
particular, modified nucleosides have been proved to
faces by nonspecific phosphohydrolases.
efficiently inhibit in vitro and in vivo virus infections
caused by HIV, HBV, and HTLV-1.1 Many of the Several strategies to overcome the problem of the
nucleoside analogues proposed against the above- initial selective and regulated phosphorylation step
mentioned viruses present similar mechanism of action could be foreseen. A good delivery system for a nucleo-
as inhibitors of viral replication: following intracellular side across membranes might be represented by phos-
phosphorylation to their 5′-triphosphate forms, they photriester molecules, which should ensure the absorp-
serve as chain terminators, thus acting as inhibitors in tion and the transport of the active molecule and should
the viral reverse transcription reaction.2 then be able to deliver intracellular monophosphate
However, several problems associated with this kind
of nucleoside analogues, due to their high toxicity and On this basis, recently, the use of nucleotide prodrugs
the appearance of cross-resistance, have led to the (pronucleotides) incorporating enzyme-labile transient
search for different structural solutions which have phosphate protecting groups has emerged, and a large
afforded new prodrugs comparable in their antiviral number of prodrugs derivatives of nucleoside mono-
activity to the clinically used nucleoside analogues, but phosphates have been prepared.
without their drawbacks. In this respect, an alternative approach to the dis-
The biological activity of nucleoside analogues (ddNs) covery of new and potent RT inhibitors involves the
showing antiviral properties is strictly linked to their design of phosphate analogues where the phosphate
conversion, through cellular enzymes, to the corre- moiety is changed to isosteric and isoelectronic phos-
sponding 5′-mono-, di, and triphosphates, which interact phonates.5 Those enzimatically and chemically stable
with viral reverse transcriptase (RT) or interfere with phosphonate analogues, which mimic the nucleoside
cell growth, slowing the cell cycle progression. In many monophosphates, are able to overcome the instability
cases, among the three successive phosphorylation of nucleotides toward phosphodiesterases and to en-
steps, the first is rate-limiting, and further conversions hance their cellular uptake by bypassing the initial
to di- and triphosphates are catalyzed by less specific enzymatic phosphorylation and could potentially be
kinases.3 effective antiviral agents.
The known antiviral nucleoside analogues have often In our laboratory, we have developed the synthesis
shown inefficiency in the first intracellular phosphory- of a series of ddNs in which the furanose ring has been
lation step by nucleoside kinases. A possibility to replaced by a N,O-heterocyclic ring.6 Carbocyclic 2′-oxa-
3′-aza-nucleosides 1 are emerging as a new important
* To whom correspondence should be addressed. Phone: +39 090
class of compounds that are interesting to synthesize
356230. Fax: +39 090 6766562. E-mail: in order to investigate their pharmacological activities.7
† Università di Catania.
‡ Università di Roma ‘Tor Vergata’.
In connection with our synthetic studies on the utility
§ Dipartimento di Scienze Microbiologiche, Genetiche e Molecolari, of nitrones for the synthesis of interesting biologically
Università di Messina. nitrogenated compounds,8 we have devised a synthetic
10.1021/jm049399i CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/08/2005

The crude mixture was formed in acetonitrile at 55 °C in the presence of 0. and H6′a signals these protons.28. The stereochemical outcome of the cycloaddition Furthermore. Carbocyclic 2′-oxa-3′-aza-nucleosides. 48.6:1.167 0. shows the H5 proton as doublet of doublets at δ were further treated with 5. the same protons give resonances at δ Pure anomers were isolated by flash chromatography. indicating a cis relationship between irradiated. Their structure was yields: a mixture of β. Scheme 1. Synthesis of Isoxazolidines 6 and 7 Figure 2. methodology. and the relative configuration was assigned on the basis Stereochemical assignments have been performed by of 1H NMR and NOE experiments.57 (E)-endo (cis adduct 6) -228.31 (as a doublet) and 3. These data unam- of the upfield resonance of the methylene protons at C4 biguously indicate a β-configuration where H4′ and the (Figure 2). the R-configuration for anomers 12 was process can be explained by considering that nitrone 5 supported by the strong NOE effect observed for H6 exists as a mixture of E/Z isomers: NOE data indicated when irradiating H4′. The compounds 11d and 12d isomer.43 (Z)-exo (cis adduct 6) -226. Thus. irradiation of H1′ increased the reso- of the H5 resonance induces a positive NOE effect on nance of H6.12 The condensation with silylated thymine ture of epimeric isoxazolidines 6 and 7 (90% overall (8).1390 Journal of Medicinal Chemistry.45 kcal/mol more stable than the (E)-exo one leading sponding carbonyl derivative 4 by treatment with to trans stereoisomer 7. pyrimidine base are in a trans relationship. in agreement with the results reported The strategy of the synthetic approach is based on for similar R-alkoxynitrones. 6.10 AM1 calculations11 give the construction of the phosphonated nitrone 5. In compound 7. Vol. Synthesis of Phosphonated Carbocyclic 2′-Oxa-3′-aza-nucleosides 11 and 12 route to phoshonated carbocyclic 2′-oxa-3′-aza-nucleo- sides of type 2 via 1. ate to give the deacetylated derivatives 11b and 12b.3-dipolar cycloaddition of phospho- nated nitrones (Figure 1). irradiation of the same were observed. 0. N-acetylcytosine (9). 5 Chiacchio et al. the major product 6 could be formed by the (Z)-nitrone Results and Discussion reacting in an exo mode or by the (E)-nitrone reacting in an endo mode. Thus.5:1 cis/trans ratio (Table 1). . in compound 6 irradiation β-compounds 11. AM1 Calculations Results on Transition State Structures for Isoxazolidines 6 and 7 ∆Hf percent transition state (kcal/mol) calcd obsd (E)-exo (trans adduct 7) -228.752 66.07 (Z)-endo (trans adduct 7) -226. H4′.35 (as a multiplet). and 5-fluorouracil (10). according to the Vorbrüeggen The cycloaddition with vinyl acetate leads to a mix. 5: the (E)-endo transition state leading to 6 is about pound 3 has been efficiently hydrolyzed to the corre.87 71. These values agree satisfacto- Amberlist-159 in acetone at room temperature: the rily with the observed 2. Table 1.0% aqueous sodium carbon- 6.289 1.4 purified by radial chromatography. for NOE measurements. The compounds obtained have been proved to be potential antiviral agents: data on the biological activity show that 2 are low toxic and exert a relevant activity as RT inhibitors. Relevant NOE effects on compounds 6 and 7. On the contrary. Two cycloadducts were independently coupled with forded nitrone 5 in good yield (95%) (Scheme 1). Com. equiv of TMSOTf as catalyst. Figure 1. No. enhancements of the H1′. the cis been obtained (Scheme 2). In particular. which theoretical support to the experimental data. 6. that the Z form is predominant (Z/E ratio ) 5:1).297 31.18 28. subsequent reaction with N-methylhydroxylamine af. and two cycload.85. proceeded with good ducts were obtained in pure form. when H6′b was protons at C4. while irradiation of H6′a gives rise to a resonance in compound 7 resulted in the enhancement positive NOE effect for H6 and H6′b. and H6′b (the downfield resonance of H3 proton and on the downfield resonance of methylene methylene protons at C6′). thus. per- yield) in a relative ratio 2. while H3 proton resonates as a multiplet at δ 2.and R-nucleosides 11 and 12 has confirmed by 1H NMR experiments. indicating has been prepared from the commercially available that the (E)-isomer as the more reactive form of nitrone diethylphosphonoacetaldehyde diethyl acetal (3). H4′. 2005. silylated nucleobases.88 Scheme 2. Conversely.

11a). we assayed the ability of the new compounds inhibitory activity in preliminary tests. and AdF-phosphonate (AdF-P. the concen. detected by microscopy analysis following staining with acridine orange. The ratio between R. showing that all the phosphonate com- would not be expected to have any significant facial bias. the concentration that causes 50% toxicity by trypan crude extract from human peripheral blood mononuclear blue) and AC50 (apoptotic concentration 50. Results. and triphosphate or phosphonate trypan blue test and the apoptosis assay. 2005. originally developed β-anomers clearly predominate as nearly exclusive by us for a preliminary screening of potential inhibitors products. Table 3. Evaluation of Apoptosis Induced by Phosphonates in Lymphoid and Monocytoid Cell Linesa 24 h 48 h 72 h cell line AdT-P AdC-P AdF-P AdT-P AdC-P AdF-P AdT-P AdC-P AdF-P Molt-3 >1000 >1000 457 ( 31 >1000 >1000 277b >1000 >1000 170 ( 30 U937 >1000 >1000 NDc >1000 >1000 NDc >1000 >1000 NDc HL-60 >1000 >1000 250 ( 32 >1000 >1000 151 ( 22 >1000 >1000 NDc a Cells were exposed under optimal culture conditions to concentrations of AdT-phosphonate (AdT-P. These two RTs were chosen on the basis of major limitations in the use of antiviral drugs is their their well-known characteristic of being extensively toxicity to uninfected cells.Novel Inhibitors of Reverse Transcriptase Journal of Medicinal Chemistry. for the controlled 5-yl-1H-pyrimidine-2. anomeric center. was detected forms for effective inhibition of RT activity and cDNA in 3 days of treatment in the lymphoid (Molt-3) and elongation. CC50 and AC50 calculated for AdF-P were the formation of an intermediate oxonium ion. 11b) and AdF-phosphonate (AdF-P. 11b). considering the pivotal role of specific primers. respectively. a significant amount of the R-nucleo. which relatively high. ring. pounds tested were of low toxicity or nontoxic. The RT activity was revealed by directly using a conventional viability assay. copy. a well-known nu- apoptosis as a mechanism. respectively. 48. since the substituents at C3 and C4 are not in close Inhibition of Reverse Transcriptase Activity in proximity to induce any steric effect upon the incoming Vitro. Before being assayed. Cytotoxicity Assays. 11c). Conversely. detected by trypan blue exclusion test. described here for the first time. For this reason. 11a). following change when the nucleosidation reaction was performed treatment with AdC-P (11b) or AdT-P (11a). the attack on the stable transfectants expressing constitutively the gly- intermediate oxonium ion from either the R. Evidently. or control medium for the times indicated. the means of a novel. triggered by cellular signals cleoside RT inhibitor. negative controls. AdC-phosphonate (AdC-P. in cell lines of lymphoid and mono. using micros. No.13 to inhibit retroviral RNA-dependent DNA polymerase The anomeric distribution obtained depends on the (reverse transcriptase) activity was determined by attacking nucleobase. expressed as CC50 (cytotoxic concentration the new compounds and AZT were incubated with a 50. RNA isolated from side has been obtained. which is acetylcytosine (9). with the cytotoxicity of the newly synthesized compounds high activity. such as the trypan detecting specific HSV-1-gD DNA following a successive blue exclusion test. are reported in Tables 2 and 3. the mono- the coupling reaction of isoxazolidines with silylated cytoid HL-60 cell line was more sensitive to apoptosis bases occurs without selectivity with respect to the induced by AdF-P rather than the lymphoid cells. c Not done. ranging from 16 to 1000 µM. As previously reported. b Mean of two values. Results obtained using AMV-RT are shown . 11c).14 and hence the product distribution is the RT activity of commercial avian myeloblastosis virus sensitive to structural changes of the reactants. In particular. as expected by to specifically induce apoptosis in the same cell lines.or β-side coprotein D (gD) of HSV-1 was used as a template for is possible. One of the (MLV-RT). Moreover. di-. HL-60) cell lines assayed. Each value is determined by three or more experiments. AdC-phosphonate (AdC-P. Each value is determined by three or more experiments. when using both the classical esters to mono-. ranging from 16 to 1000 µM. this is due to Nevertheless. In this assay. polymerase chain reaction (PCR) amplification using cytoid origin. particularly of the immune pound showing biological activity6 but lacking RT- system. calculated as the concentrations of the drug required to cause 50% toxicity. Evaluation of Cytotoxicity of Phosphonates in Lymphoid and Monocytoid Cell Linesa 24 h 48 h 72 h cell line AdT-P AdC-P AdF-P AdT-P AdC-P AdF-P AdT-P AdC-P AdF-P Molt-3 >1000 >1000 247 ( 37 >1000 >1000 197b >1000 >1000 141 ( 25 U937 >1000 >1000 NDc >1000 >1000 NDc >1000 >1000 NDc HL-60 >1000 >1000 >1000 >1000 >1000 457 ( 53 >1000 >1000 408b a Cells were exposed under optimal culture conditions to concentrations of AdT-phosphonate (AdT-P. Preincu- tration required to cause apoptosis in 50% of treated bation with the PBMC crude extract served to supply cells).and β-nucleosides does not monocytoid (U937. or control medium for the times indicated. the absence of a hydroxymethyl group in the furanose Apoptosis was detected by morphological analysis. while. Zidovudine (AZT). calculated as the concentrations of the drug required to cause 50% apoptosis. RT (AMV-RT) and moloney murine leukemia virus RT Biological Tests. Values indicate the CC50. 5 1391 Table 2. These results show that toxicity using both the assays. cells (PBMCs) stimulated with PHA for 72 h. fol. starting from the crude mixture of isoxazolidines with. of reverse transcriptase (RT). The ability of the newly synthesized compounds group at C5. Values indicate the AC50. c Not done. we tested utilized commercial sources for routine RT-PCR. cell-free assay. were utilized in the assay as internal positive and lowing staining with a DNA-binding dye. b Mean of two values. and (5′S)-5-fluoro-1-isoxazolidin- as well as by a variety of drugs.4-dione (ADF). a nucleoside com- removal of dead cells. in the case of thymine (8) and N. With 5-fluorouracil (10). the necessary enzymes to transform the prodrugs from No significant toxicity. Vol. AdF-P (11c) was shown to cause detectable levels of out any preventive separation.

5 mL/min. in comparison with ADF (negative control) performed with a Perkin-Elmer elemental analyzer. (b) Experimental runs in which RT of the multiplet. it is a steady-state NOE in which a single activity was assayed in the presence of AdC-P or AdT-P at resonance is saturated at low power for approximately 5 × the concentrations of 100. following exposure of the nucleosides to the stirred. Preparative centrifugally acceler- in which RT activity was assayed in the presence of AZT at ated radial thin-layer chromatography (PCAR-TLC) was per- the concentrations of 100. for 3 h. acetal (3). which does alternate scan subtraction of two FIDs absence of the nucleoside compounds. Experimental Section General. the obtained phospho- nate nucleosides seem to act as RT inhibitors. chemical shifts are given in ppm in the presence of the compounds. which was completely inactive. No of 0. in the ylphosphonate (5). NMR and AZT (reference control). AdT-P. Conclusions Phosphonated N. Melting points were determined with a Kofler Figure 3. No. which is comparable with that of AZT. The reaction mixture crude extract from (PBMCs). 10.3-dipolar cycloaddition methodology. opening new perspectives in future investigations on their possible use as therapeutic agents. 5 Chiacchio et al. or 1 nM. and the residue was purified by silica gel flash chroma- . (c) Experimental runs chromatographic separations were performed on Merck silica in which RT activity was assayed in the presence of AdF-P or gel 60-F254 precoated aluminum plates. search. as well as by detection of cell death by apoptosis. Diethylphosphonoacetaldehyde diethyl amplification product was detected when isolated RNA. Elemental analyses were compounds 11a-c. No Starting Materials. Power may be reduced from ordinary NOE single subtraction. evaluated by a cell-free assay spectra were recorded on a Varian instrument at 500 MHz based on a RT-PCR reaction. with the exception of ADF. and cytosine. and triethylamine (20 mmol) in toluene (70 mL) was versely. The quality and the specificity of the assay with binder and fluorescence indicator (Aldrich 34. in Figure 3. where RT activity was the eluting solvents were delivered by the pump at a flow rate assayed in the absence of the nucleoside compounds. difference was observed with or without the addition of The identification of samples from different experiments was the crude extract to the complete reaction mixture in secured by mixed melting point and superimposable NMR spectra. All solvents were dried reaction mixture. or 1 nM. as powerfully as AZT is. No RT-inhibitory activity was observed for all the compounds at 1 nM concentration.O-nucleosides. The same results were obtained when a concentration of 100 nM was used (not shown). when tested in a simple cell-free assay. containing thymine. thymine (8). with or without formed with a Chromatotron Model 7924 T (Harrison Re- preincubation with the crude extract. AdC-P. 48. the activity of AZT overlapped with that of phos- phonates. such as the trypan blue exclusion test. These results demonstrate that the phos- phonate compounds exert a specific inhibitory action on RT from at least two different retroviruses. N-acetylcytosine (9). Vol. fluorouracil. mmol). As shown in Figure 3d. implemented in (a) Control runs in which RT activity was assayed. 2005. N-methylhydroxylamine hydrochloride (20 absence of crude extract activation (Figure 3b.5-1. and 5-fluorouracil primers. as Diethyl (2E). was filtered. have been synthesized in good yields by 1.035-0. the appearance of a clear band of amplified DNA.1392 Journal of Medicinal Chemistry. TLC grade. The results indicate that the synthesized phosphonate nucleosides presented low levels of cytotoxicity assessed by a conventional assay to detect viability. the solvent was evaporated under reduced pres- pletely inhibited the formation of amplified products at sure. the rotors (1 or 2 mm layer thickness) were coated with silica gel Merck grade type 7749. with or without tions were carried out by flash chromatography using Merck preincubation with the crude extract. in the Darmstadt. Thin-layer preincubation with the crude extract. DNA was then amplified from TMS as internal standard. or AdF-P at 100 nM concentration. A solution of diethyl 2-oxoethylphospho- nate9 (4) (20 mmol).644-6) and are illustrated in Figure 3a. Inhibition of reverse transcriptase (RT) activity of apparatus and are uncorrected.and (2Z)-2-[Methyl(oxido)imino]eth- well as at lower concentrations (not shown). (d) Experimental runs silica gel 0. all phosphonates com. 10. RNA template from glycoprotein (1H) and at 125 MHz (13C) using deuteriochloroform or deu- D of HSV-1 was reversely transcribed by AMV-RT into cDNA terated methanol as solvent. of isolated RNA. The cytotoxicity and the RT-inhibitory activity of the obtained compounds have been investigated. at room temperature. or 1 nM. experiments because the irradiation is cycled through the lines primers. or AMV-RT was singly subtracted from the (10) were purchased from Aldrich Co.c). CA). Preparative separa- ADF at the concentrations of 100. from the reaction mixture. or AMV-RT. with or without T1 (5 s in our case) before acquiring the FID.070 mm. Similar results were obtained when MLV-RT was utilized. 10. No inhibitory effect was exerted by according to literature methods. particularly in antiretroviral and anti-HBV chemotherapy. 10 nM concentration. Palo Alto. performed by a modified cyclenoe sequence. with or without the in which the saturation frequency is moved on-resonance and addition of a crude extract from stimulated PBMCs and with off-resonance. Con. NOE experiments were and visualized on ethidium bromide containing agarose gel. More importantly.

4. The crude careful addition of aqueous 5% sodium bicarbonate and then extract was then inactivated for 5 min at 95 °C. 20 mM Na3F.54 mmol) and refluxed for 15 min under Triton-X. separated by density gradient from peripheral isoxazolidin-5-yl acetate (6): yield 65%.4′RS)-1′-[4-(acetylamino)-2-oxopyrimi. (Italy) is also acknowledged for a chromatography (CHCl3/CH3OH 95:5) to give the deacetylated fellowship to M. Annu.. A suspension of bases 8-10 (0. Yarchoan. spot evidenced with AdFU6 or AZT (Sigma-Aldrich Co.4-dioxo-3.d and (0. The residue was purified by flash chroma.05% acetamide (BSA) (2. (progetto P. R. Apoptosis was evaluated by morphological 5-7. No. 2002. (d) Turner. 30.5 µM primers (US6 reverse 5′- methylphosphonate (11d): yield 56%. The choice of a nonretro.R. For the General Procedure for the Preparation of Nucleo. washed with water Trizol (Gibco-Invitrogen Co. PBMCs were rinsed three sides 11 and 12. 285.62 mmol) in times in cold PBS and then solubilized in lysis buffer (50 mM dry acetonitrile (3 mL) was treated with bis(trimethylsilyl). 5 1393 tography (CHCl3/CH3OH 95:5) to give nitrone 5 as colorless template was motivated to avoid problems with retroviral or oil. 5 mM MgCl2. G.. To the clear solution obtained were added a solution 40. 7. Summers. and. 249. 2004. Balestrieri. including those showing typical apoptotic characteristics.N. After this period. and evapo. De Clercq. and 45 s at 72 °C) on a Cetus Diethyl [(1′SR. Med. 0. . M. filtered. were previously stimulated with PHA (2 µg/mL) isoxazolidin-5-yl acetate (7): yield 25%. blood of healthy donors. H. 0. (c) De Clercq.S. and 12d. 133- 173. Antimicrob. in a reaction mix containing RT buffer (1×). based on routinely adopted RT-PCR procedures. HIV Resistance to Reverse Transcriptase Inhibitors. This material is available free of analysis of the cells. Scotland. 115-133.4-dihydro. Norwalk. containing 10 µg/mL ethidium bromide in 1× TAE buffer.5 µM) specific for a sequence of the HSV-1 US6 gene that R-nucleosides 12a. DTT (10 mM). and 1 nM. for 72 h in RPMI plus 20% FBS. CT). 1999. ]methylphosphonate (11b). 129-154. Diethyl {(1′SR. 5′-AGA CTT GTT GTA GGA GCA TTC G-3′). After the addition of dichloromethane from 5 × 106 gD-expressing transfectants was performed using (8 mL). Molecular Targets for RNA from stable transfectants expressing gD of HSV-1.4-dihy. the organic phase was separated.4-dioxo-3. E. A. 51. D. using the trypan blue (1H and 13C NMR) and elemental analyses data of compounds exclusion test. 11a-d. sticky oil. 15 µM DTT.I. Vol.16 Transfectants were grown in D-MEM plus Pharmacol. H. 20. moloney murine leukemia virus RT (MLV-RT) (all from Promega Co. Mastino. using the trypan blue dye exclusion under reduced pressure and the residue purified by radial test. the solution was neutralized by extract from stimulated PBMCs. MO). (e) Jonckheere. Paisley. Med. (Sigma-Aldrich Co. E. M. and IL-2 (20 U/mL). 995- apoptotic cells was based on the presence of uniformly stained 1001.4′RS)-1′-[4-(acetylamino)-2-oxopyrimi- was used for DNA PCR in a reaction mix containing 1× Taq din-1(2H)-yl]-3′-methyl-2′-oxa-3′-azacyclopent-4′-yl}- Gold buffer (Promega Co. at dropyrimidin-1(2H)-yl)]-3′-methyl-2′-oxa-3′-azacyclopent- the concentration of 100. Rev. E. sticky oil. 10. Mol. U of avian myeloblastosis virus RT (AMV-RT) or 40 U of 4′-yl]}methylphosphonate (11a): yield 68%.5. Skalka. Structural Biology of Invitrogen Co.. compound 11b: yield 90%.3 mM dNTP.25% sodium deoxycholate. RNA isolation concentrated in vacuo.. Virol. 12a. DNA thermal cycler 2400 (Perkin-Elmer. 1327-1330. (c) Katz. A solution of 11d (100 mg) in a mixture of aqueous K2CO3 (5%. This included (2) (a) Mitsuya. a reverse primer (5′-TGT CGT CAT AGT GGG CCT CCA T-3′) tography (CHCl3/MeOH 95:5) to give β-nucleosides 11a. the reaction mixture was evaporated evaluating living cells. Biochim. 0. R. for 1 h at 37 °C. Res. scribed elsewhere. After being cooled at 0 °C. samples were incubated at 72 °C for 20 yl]methylphosphonate (11c): yield 75%. din-1(2H)-yl]-3′-methyl-2′-oxa-3′-azacyclopent-4′-yl}. Biophys. as a Aids Therapy. orange as previously described. Fol- pyrimidin-1(2H)-yl)-3′-methyl-2′-oxa-3′-azacyclopent-4′. The compounds were incubated with the crude h. (e) De Clercq. 1-32. 531-565. tation.P. Clin. for 30 cycles methylphosphonate (12d): yield 14%. over 600 cells. A.I. S. HTLV-1/2.c. Rev. 1 mM Na3- trile (3 mL) and trimethylsilyltriflate (TMSOTf) (0.. Biochem. (d) Macchi. Amplified DNA (350 bp) was visualized on 1% agarose gel rimidin-1(2H)-yl)-3′-methyl-2′-oxa-3′-azacyclopent-4′-yl. The new synthesized compounds and the nucleosides chromatography (CHCl3/CH3OH 99. Tris-HCl pH 7. J. Toxicity was Supporting Information Available: Spectroscopic data evaluated by a standard viability assay.I.4-dioxo-3. dNTP (4 mM). Cells underwent passage every 5 (5. 258-275. Evaluation of Toxicity and Apoptosis. 47. 2005. sticky oil. light yellow oil. 0. F. human DNA.7 mmol) in vinyl acetate (30 mL) was stirred at 60 °C for 2 days and at each passage cell growth was monitored by 24 h. ectopic cellular system of expression as a source of RNA as Target for RT Inhibitors. Science 1990. E. 0. This work was partially sup- was left to stir for 4 h. 12% fetal bovine serum (FBS). The HIV-1 Reverse Transcription (RT) Process viral.I. for 1 h on ice.. 1994.M. freshly added.).. E. reduced pressure. The gD-expressing transfectants have been de. incubation at 95 °C for 5 min.4′RS)-1′-[[(5-methyl-2. colorless sticky oil. 1 mM EGTA pH 7. 5 µg/mL aprotinin. solvent was then evaporated under ported by M. Synthesis of Diethyl [(1′SR. sticky oil. E. therapy.4′RS)-1′-(4-amino-2-oxopy. New Anti-HIV Agents and Targets.5RS)-3-[(Diethoxyphosphoryl)methyl]-2-methyl. preparation of the crude extract. TGT CGT CAT AGT GGG CCT CCA T-3′ and US6 forward Diethyl {(1′RS. light yellow oil.).5SR)-3-[(Diethoxyphosphoryl)methyl]-2-methyl. A. and 1. 1 mM PMSF. The capacity Effects of Nucleoside-Based Anti-Retroviral Chemotherapy on of the described compounds to inhibit RT activity was inves. 5 µL of the RT reaction product Diethyl {(1′SR. The Retroviral Enzymes. New Developments in Anti-HIV Chemo- nuclei showing chromatin condensation and nuclear fragmen. 400 µg/mL G418 (Gibco.4-dihy- the presence or in the absence of the activated compounds. 5 mL) and methanol (5 mL) Acknowledgment.R. A solution of nitrone execution of the entire assay. and the reaction mixture was stirred at 55 °C for 6 10 000 rpm. St.. J.). codes for gD. Acta 2002. all from Sigma) on ice and centrifuged at dropwise. B. 2003.) were activated in vitro cerium molibdic reagent) to give the pure isoxazolidines 6 and through incubation with a crude extract from 1 × 106 PBMCs. RNase inhibitor (100 U).N.acs. 63. 410. were References counted using a fluorescence microscope.. Anne. and (3RS. template.B. 1 mM EDTA. Broder. Madison WI). 5 µg/mL of the epimeric isoxazolidines 6/7 (0. and 30 dropyrimidin-1(2H)-yl)]-3′-methyl-2′-oxa-3′-azacyclopent. D. Louis.5:0. 155-169.25 U Taq Gold (Promega Co. The identification of (1) (a) Richman. 1587. B/C hepatitis.Novel Inhibitors of Reverse Transcriptase Journal of Medicinal Chemistry. PBMCs. instructions. negative for HIV-1/2. sticky oil. J. Total RNA (1 µg) was reverse transcribed using rated to dryness. NaCl 150 mM. Res.4′RS)-1′-(5-fluoro-2. 48. (30 s at 95 °C.U.4. dried over sodium sulfate. Rev. 22.R. UK). Biol.15 Briefly. possibly present in reagents utilized during the Synthesis of Isoxazolidines 6 and 7.. performed following staining with acridine charge via the Internet at http://pubs. and 30 µg/mL BrdU HIV.d. Nature 2001.1% NP- stirring. Human T-Cell Leukaemia/Lymphotropic Virus Type-1 (HTLV- tigated by evaluating their activity toward cDNA generation 1). HIV Chemoterapy.. min to ensure the completion of the final extension step. Yield 95% (Z/E ) 5/1).S. 2000.). lowing the final cycle. 5 µg/mL pepstatin. which served as enzyme supplier for phosphorylation pro- (3RS. Biochem. leupeptin. 1994. (b) De Clercq. from an RNA template using a cell-free RT reaction assay. (b) De Clercq. 2002 and F. cesses. Reverse Transcriptase Inhibition Assay. according to the manufacturer’s (2 × 10 mL). After 4′-yl]}methylphosphonate (12a): yield 12%.4′RS)-1′-[[(5-methyl-2.4 mmol) VO4. Antiviral Drugs in Current Clinical Use. and the residue was purified by flash The C. J. The reactions were performed in Diethyl {(1′RS. Chemother.52 mmol) in dry acetoni. B. 30 s at 60 °C.

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