Phosphoroamidate derivatives of N , O -nucleosides as inhibitors of reverse transcriptase

Phosphoroamidate derivatives of adenine and 5-fluorouracil N , O -nucleoside analogues have been synthesized as potential antiviral prodrugs. In particular, dimethoxyphenyl phosphates, linked via nitrogen to L -leucine methyl ester were studied. The synthesized compounds were also subjected to in vitro evaluation for their RT inhibition. Results show that phosphoroamidate derivatives, in comparison with their corresponding N,O-nucleosides, present a promising antiviral activity, though of micro-molar order.


Introduction
The nucleoside structure has proven to be an effective template for the development of therapeutically useful agents with antiviral and antitumor activity.In particular nucleoside analogues were approved for the treatment of pathologies induced by infections from HCMV, HSV, HIV, and HBV. 1 Dideoxynucleoside analogues exert their antiviral activity by the competitive reversible inhibition of Reverse Transcriptase (RT) and/or viral DNA chain termination.These antiviral agents, acting via a nucleoside analogue mode, need to be phosphorylated into their corresponding 5'-triphosphates by cellular and/or viral enzymes. 2 As a part of our continuing efforts to the search of new templates for the development of new antiviral agents, in the last years, our research group has been interested in the synthesis and evaluation of the antiviral activity of nucleoside analogues, where the ribose moiety is replaced by an isoxazolidine ring. 3ome N,O-nucleosides have shown relevant biological properties as antiviral agents or antimetabolites. 4However, the analysis of the biological profile for some other derivatives revealed a moderate or no antiviral activity.The conversion of these latter nucleosides into the corresponding phosphonated structures 5 have furnished often a higher potency in activity, suggesting that the inefficient conversion of N,O-nucleosides into the 5'-monophosphate forms by the kinase, is responsible of the lack of biological activity.
Several strategies directed to overcome the problem of the initial selective and regulated phosphorylation step could be foreseen.The use of the nucleotide monophosphate might bypass the first phosphorylation step.However, as unmodified agents, nucleoside monophosphates are unstable in biological media and they also show poor membrane permeation owing to the associated negative charges at physiological pH.
On the basis of these considerations, we decided to improve the pharmacological characteristics of these N,O-nucleosides designing the corresponding phosphoramidates as derivatives which can mask the negative charge of phosphate group conferring better lipophifilic features.These nucleotide prodrugs, after their cellular uptake, may release the monophosphorylated nucleoside analogue which will be converted into di-and triphosphates by unspecific kinases.This technology greatly increases the lipophilicity of the nucleoside monophosphate analogue with a consequent increase of membrane permeation and intracellular availability.
Herein, we describe the preparation of phosphoramidates of adenine and 5-fluorouracil N,Onucleosides, 15 and 17 respectively, and their antiviral evaluation against two commercial RT [avian myeloblastosis virus RT (AMV-RT), and moloney murine leukemia virus RT (MLV-RT)] in comparison with the corresponding N,O-nucleosides.In particular, phosphoramidate derivatives with dimethoxyphenyl phosphates linked via nitrogen to methyl-esterified L-leucine were examined.Besides the strong effect activating of dimethoxyphenyl in enhancing the lipophilicity of molecules 6 it would appear that a more potent antiviral action of the phosphoramidate derivatives requires an aminoacid which has a methylene unit adjacent to its chiral center, such as L-leucine in spite of its steric encumbrance. 7

Results and Discussion
The synthesis of phosphoramidate 15, containing the adenine as nucleobase, was accomplished through two simple steps: i) the preparation of isoxazolidinyladenine 6, and ii) the phosphoramidation of this modified nucleoside by means of methyl N-[chloro- (2,6dimethoxyphenoxy)phosphoryl]-L-leucinate 14.
Cis-β-nucleoside 6 was prepared by two different synthetic approaches of which the first is based on the nitrone 1,3-dipolar cycloaddition, and the other one on the Hilbert Jones nucleosidation performed on 5-O-acetylated isoxazolidine 8. 3a The cycloaddition reaction of N-glycosyl nitrone 2, 8 easily prepared in situ as previously described by us 3c and vinyl-N 6 -benzoyladenine 3 9 in chloroform solution were allowed to react in a sealed tube for 18 h.After evaporation and purification, three adducts 4a,b and 5 (Scheme 1), in a 1:6:6 ratio, and 65% global yield, were obtained and separated.The diastereomeric ratio of the products was determined by 1 H-NMR spectroscopy on crude mixture, while the relative configuration was assigned by NOEDS and T-Roesy spectra.

Scheme 1
In particular, the irradiation of the H 5' proton in compound 5, chosen as a reference compound, induced a positive NOE effect on the H 4'b proton (3.11 ppm); in turn, the irradiation of this latter H 4'b proton gave rise to an enhancement of the H 3' resonance.Moreover, the irradiation of the H 1" proton produced a strong positive effect on the H 4" proton, thus indicating that all these protons are in a topological cis arrangement.As a confirmation, T-Roesy experiments reveal a NOE effect between H 5' -H 3" protons, and H 1" -H 4" protons.
Besides a complete diastereofacial selectivity, the cycloaddition reaction, therefore, shows a low cis/trans diastereoselectivity, which can be rationalized by assuming that the E-endo attack of the dipolarophile on the re-face of the nitrone is the preferred reaction pathway, because of secondary orbital interactions exerted by the purine ring of nucleobases.PM3 semi-empirical calculations confirm this result insofar as, an energy difference of 2.3 kcal/mol is observed in favour of the re-face attack with respect to the si-face one.This behaviour is also in agreement with DFT calculations on C-methoxycarbonyl-N-methyl nitrone with electron-rich dipolarophiles. 10On these basis, the absolute configuration of the cycloadducts 4b and 4a,5 can be tentatively assigned as (3R,5R) and (3R,5S) respectively.
A valuable improvement of the synthetic strategy leading to 6 has been exploited through the reaction of 6-chloropurine 7 with an epimeric mixture of 5-O-acetyl-N,O-isoxazolidinyl derivatives 8 (cis/trans), 3a via the intermediate 9, followed by NaBH 4 reduction to give 10 and finally by methanol/ammonia treatment (Scheme 2).

Scheme 2
In particular, 6-chloropurine 7 was refluxed with HMDS for 2 h; after removal of HMDS, the residue was treated with a dichloroethane solution of epimeric mixture of isoxazolidines 8 and TMSOTf in the presence of molecular sieves at 60 °C for 2 h to afford 9 in 75% yield.
According to similar nucleosidations performed at high temperature, 11 nucleoside 9 show to be the β-anomer as a result of its major thermodynamic stability in comparison with α-anomer.
The synthetic approach to phosphoramidate 15, was accomplished by means of the three simple steps illustrated in Scheme 3. 2,6-Dimethoxyphenyl phosphorodichloridate 12 was prepared according to the procedure of Orloff et al. 12 by the reaction of 11 with POCl 3 in the presence of AlCl 3 , and immediately used to prepare a diastereomeric mixture of 14 in 80 % yield by reaction with L-leucine methyl ester hydrochloride 13.Diastereomeric mixture of 14 was then converted into 15, as single diastereomer, in 57% yield by reaction with 6 in THF solution in the presence of imidazole at room temperature.Structures of phosphorochloridates 14 and phosphoroamidate 15 were assigned on the basis of 1 H-NMR data.In particular, as expected, the 1 H-NMR spectrum of 14 reveals the presence of duplicate peaks due to the presence of two diastereoisomers, in a 1:1 ratio, produced by nucleophilic reaction displacement of chlorine in 12 by 13.On the contrary, the 1 H-NMR spectrum of 15 shows the presence of a single stereoisomer, whose formation is, probably, attributable to the bulky effect promoted by nucleoside moiety during the reaction of 14 with 6 at the phosphorus centre.
In order to valuate the antiviral activity of one pyridine and one purine phosphoroamidate derivatives, compound 17 as single stereoisomer was similarly prepared, in 60% yield, by reaction of 14 with 16. 3a

Biological evaluations
The two N,O-nucleosides 6 and 16 and the corresponding phosphoroamidate derivatives 15 and 17 were evaluated for their antiviral activity by measuring their ability to inhibit the reverse transcriptase of avian myeloblastosis virus and moloney murine leukaemia virus on a cell free assay along with respect to a reference antiviral compound such as AZT.4b Moreover, the same compounds were tested in vitro for their cytotoxicity on Molt-3 cells.Compounds 15 and 17 completely inhibited the RT activity at 10 and 1 µM concentration, respectively, while compounds 6 and 16 are completely inactive at the higher tested concentration.Although compounds 15 and 17 inhibit the RT at concentrations 1000-and 100-fold, respectively, higher than that showed by AZT, these data show that the conversion of N,O-nucleosides into the corresponding phosphoramidates determines the switching from not active to active compounds (Table 1).No significant toxicity was observed at the higher tested concentration

Conclusions
Two N,O-nucleosides containing 5-fluorouracil or adenine moiety and their corresponding derivatives with a phosphoramidate group at the C-5' position were synthesized and evaluated for their antiviral activity.The obtained results indicate that the introduction of the dimethoxyphenyl phosphate group, linked via nitrogen to L-leucine methyl ester, determine a switching in the activity of compounds allowing the possible speculation that the weak antiviral activity, sometimes showed by N,O-nucleosides, is linked to their inefficient conversion into the 5'-monophosphate forms, rather than to the lower affinity to viral polymerases.Therefore, the development of the phosphoramidate chemistry applied to N,O-nucleosides could improve the intracellular delivery with a concomitant overcome of the rate limiting first phosphorylation.

Experimental Section
General Procedures.Melting points were determined with a Kofler apparatus and are uncorrected.Elemental analyses were performed with a Perkin-Elmer elemental analyzer.NMR spectra were recorded at 200 or 500 MHz ( 1 H), at 50 or 125 MHz ( 13 C) and at 205 MHz ( 31 P) using CDCl 3 as solvent; chemical shifts are given in ppm from TMS as internal standard.All 31 P chemical shifts are quoted in ppm using H 3 PO 4 as external reference.Thin-layer chromatography was done on Merck silica gel 60-F 254 precoated aluminium plates.Preparative separations were made by flash column chromatography using Merck silica gel 0.063-0.200mm and 0.035-0.070mm.Chemical reagents were purchased from Aldrich except where noted and used without further purification.Solvent for chromatography were distilled at atmospheric pressure prior to use and dried using standard procedures.Isoxazolidines 4a,b and 5 3a and N 6 -benzoyl vinyladenine 13 9 were prepared according to literature methods.

Biological assays
Evaluation of toxicity.Toxicity was evaluated by a commercial viability assay (CellTiter 96 R AQueous One Solution Assay, Promega Co., Madison WI), according to manufacturer's instructions.This assay is based on the principle that cells, at death, rapidly lose the ability to reduce MTS tetrazolium.Briefly, Molt-3 and Vero cells were cultured in optimal culture conditions for 20 h in 96-well plates, in the absence of the compounds or in their presence, at concentrations ranging from 1 to 1000 µM.At the end of the incubation time, the MTStetrazolium-based reagent was added to each well.After a further incubation of one hour at 37 °C in a humidified, 5% CO 2 atmosphere, the absorbance of the samples was recorded at 490 nm using a 96-well spectrophotometer.The assays were performed in triplicate.The cytotoxic concentrations (CC 50 ) were calculated as the concentrations of the compounds required to cause 50% reduction of absorbance values.
Reverse-transcriptase inhibition assay.The capacity of the described compounds to inhibit avian myeloblastosis virus RT (Promega Co.) activity was investigated by evaluating their activity towards cDNA.Generation from an RNA template using a cell-free RT reaction assay, was recently described by us.4b This assay is based on routinely adopted RT-PCR procedures.

Table 1 .
Inhibitory activity and Cytotoxicity for Compounds 6

, 15-17
a MRTCI towards Reverse Transcriptase, determined as the minimal concentration required to completely inhibit reverse transcription evaluated in a cell free assay through an amplification program cycle.b Concentration of compound required to cause a 50% toxicity detected by a commercial viability assay (CellTiter 96 ® AQueous One Solution assay, Promega Co., Madison WI).