Diamyl Aspartate Amidat - ACS Publications - American Chemical

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Expanding the Antiviral Spectrum of 3‑Fluoro-2(phosphonomethoxy)propyl Acyclic Nucleoside Phosphonates: Diamyl Aspartate Amidate Prodrugs Min Luo,† Elisabetta Groaz,† Graciela Andrei,‡ Robert Snoeck,‡ Raj Kalkeri,§ Roger G. Ptak,§ Tracy Hartman,∥ Robert W. Buckheit, Jr.,∥ Dominique Schols,‡ Steven De Jonghe,† and Piet Herdewijn*,† †

Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Herestraat 49, 3000 Leuven, Belgium Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, Herestraat 49 bus 1043, 3000 Leuven, Belgium § Department of Infectious Disease Research, Southern Research Institute, 431 Aviation Way, Frederick, Maryland 21701, United States ∥ Anti-Infective Research, ImQuest BioSciences, Frederick, Maryland 21704, United States ‡

S Supporting Information *

ABSTRACT: Acyclic nucleosides containing a 3-fluoro-2-(phosphonomethoxy)propyl (FPMP) side chain are known to be moderately potent antihuman immunodeficiency virus (HIV) agents, while being completely devoid of antiviral activity against a wide range of DNA viruses. The derivatization of the phosphonic acid functionality of FPMPs with a diamyl aspartate phenoxyamidate group led to a novel generation of compounds that not only demonstrate drastically improved antiretroviral potency but also are characterized by an expanded spectrum of activity that also covers hepatitis B and herpes viruses. The best compound, the (S)-FPMPA amidate prodrug, exerts anti-HIV-1 activity in TZM-bl and peripheral blood mononuclear cells at low nanomolar concentrations and displays excellent potency against hepatitis B virus (HBV) and varicella-zoster virus (VZV). This prodrug is stable in acid and human plasma media, but it is efficiently processed in human liver microsomes with a half-life of 2 min. The (R) isomeric guanine derivative emerged as a selectively active anti-HIV and anti-HBV inhibitor, while being nontoxic to human hepatoblastoma cells. Notably, the pyrimidine containing prodrug (S)-Asp-FPMPC is the only congener within this series to demonstrate micromolar antihuman cytomegalovirus (HCMV) potency.

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INTRODUCTION

thus, it is enzymatically more stable; however, it can likewise undergo enzymatic phosphorylation. Nucleoside phosphonates can be converted into their corresponding phosphonate diphosphates, which act as mimics of naturally occurring nucleoside triphosphates. Thus, ANPs only need two (instead of three for regular nucleoside analogues) phosphorylation steps to reach their biologically active stage. This is particularly advantageous as the first phosphorylation step is very often inefficient and rate-limiting in the formation of nucleoside triphosphates. The flexibility of the acyclic sugar side chain allows these compounds, once diphosphorylated, to adopt a conformation appropriate for interaction with the active sites of a DNA polymerase or reverse transcriptase.

The synthesis of modified nucleosides continues to constitute an attractive platform for the discovery of new bioactive compounds.1 The replacement of an atom or a functional group in biologically relevant molecules with isopolar and isosteric motifs is a popular approach to provide agents with improved therapeutic profiles, often through conveying greater metabolic stability and enhanced pharmacokinetic properties.2 For instance, the electronic and structural properties of the phosphonomethoxy functionality (P−C−O) in place of the phospho-oxymethyl (P−O−C) moiety in nucleoside monophosphates have found widespread application in the development of several classes of antiviral drugs,3 including the acyclic nucleoside phosphonates (ANPs).4 In contrast to the phosphate group, a phosphonate is not susceptible to phosphodiesterase and phosphatase hydrolysis; © 2017 American Chemical Society

Received: March 29, 2017 Published: July 6, 2017 6220

DOI: 10.1021/acs.jmedchem.7b00416 J. Med. Chem. 2017, 60, 6220−6238

Journal of Medicinal Chemistry

Article

Figure 1. Selected examples of acyclic nucleoside phosphonates endowed with antiviral activity.

activation to the pharmacologically active species. Therefore, various prodrug or “pronucleotide” approaches have been investigated to enable a better cellular permeability through passive diffusion by masking the phosphonate moiety with lipophilic functionalities.1b,10 However, most of the work carried out thus far has been dedicated to the synthesis of ANP (acyloxy)alkyl ester [i.e., adefovir dipivoxil, tenofovir disoproxil fumarate (TDF)] and aryloxyphosphonoamidate prodrugs, ultimately leading to the marketing approval of tenofovir alafenamide (TAF) for the treatment of HIV- and HBV-infected patients as components in different single tablet regimens.11 Despite the promising antiviral activity of FPMP ANPs, no examples of FPMP prodrugs can be found in the literature, with the only exception being the bis(amino acid) phosphonamidate of (S)-FPMPA (Figure 2), which resulted in a 13-fold improved anti-HIV activity (EC50 = 0.54 μM) compared to the parent phosphonate (EC50 = 7.04 μM).12

Extensive research on the synthesis and antiviral evaluation of ANPs has led to the marketing of three ANPs with an adenine or cytosine base (Figure 1).5 Cidofovir (HPMPC, (S)-1-(3hydroxy-2-phosphonylmethoxypropyl)cytosine) received marketing approval for the treatment of human cytomegalovirus (HCMV) retinitis in AIDS patients. Adefovir (PMEA, 9-(2phosphonylmethoxyethyl)adenine) has been licensed for the treatment of hepatitis B virus (HBV)-infected patients, while tenofovir (PMPA, (R)-9-(2-phosphonylmethoxypropyl)adenine) is being used as an antihuman immunodeficiency virus (HIV) and an anti-HBV agent. The latter two are on the market as orally bioavailable prodrug forms. Aiming at exploring further variations, ANPs with a fluorine atom in the acyclic side chain or at the base moiety have been synthesized and tested for antiviral activity.6 The best studied class of fluorinated ANPs are the 3-fluoro-2(phosphonomethoxy)propyl (FPMP) derivatives,7 obtained by replacing the hydroxyl group in HPMP-derivatives with a fluorine atom. Both enantiomers of FPMPA and FPMPG (Figure 1) are completely devoid of antiviral activity against a broad range of DNA viruses [herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), CMV, varicella-zoster virus (VZV), vaccinia virus (VV)].8 On the other hand, (S)-FPMPA showed potent and selective antiviral activity against HIV-1 and HIV-2, with EC50 (50% effective concentration) values in the 8 μM range.8b This compound has also proven effective as an inhibitor of HBV DNA synthesis with an EC50 value of 1.2 μM.9 The corresponding (R)-FPMPA completely lacks antiHIV activity.8b For the guanine containing congeners (FPMPG), both enantiomers are equally active against HIV-1 and HIV-2, with EC50 values for both isomers in the 5 μM range. Also, nucleobase modified FPMP derivatives have been synthesized and evaluated for antiviral activity. The diaminopurine congener (FPMPDAP) shows no activity against a panel of DNA viruses, whereas (R)-FPMPDAP displays an EC50 value of 4.3 and 4.6 μM, against HIV-1 and HIV-2, respectively.8b Although a weak antiviral activity of FPMP pyrimidine derivatives is mentioned throughout the literature, no detailed data are available. Since phosphonates are negatively charged at physiological pH, the main drawback linked to these derivatives lies in their poor ability to penetrate the lipid-rich cell membrane, which can limit their intracellular availability and preclude their further

Figure 2. Prodrugs of acyclic nucleoside phosphonates relevant to this study.

On the basis of the favorable pharmacokinetic profile and tissue distribution pattern demonstrated by the phosphonoamidate prodrugs of tenofovir,11 we wished to apply a similar strategy to FPMP ANPs. In addition, we recently discovered that the use of L-aspartic acid di(iso)amyl esters in place of the classical L-alanine esters in the aryloxyphosphoramidate and aryloxyphosphonoamidate prodrug approaches led to a drastic improvement in the antiviral activity of 2′-C-methyl-uridine13 and 2′-deoxy-L-threose nucleoside phosphonates,14 respectively. This improved activity prompted us to synthesize unprecedented L-aspartic acid diamyl esters containing phosphonoa6221



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Scheme 1. Synthesis of (S)/(R)-Fluorinated Acyclic Phosphonate Esters 4a/ba

Reagents and conditions: (a) KHF2, PhCl, cat. Bu4NH2F3, 135 °C, 15 h; (b) diethyl tosyloxymethylphosphonate, NaH, THF, 0 °C to rt, 5 h; (c) 80% AcOH, 90 °C, 1 h. a

midate prodrugs of FPMP ANPs and to assess their ability to inhibit the replication of retro (HIV) and DNA viruses, the details of which are reported below.

Scheme 2. Synthesis of (S)/(R)-Fluorinated Acyclic Thymine Phosphonate Acids 8a/b (FPMPTs)a

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RESULTS AND DISCUSSION Chemistry. Synthesis of Enantiomeric Fluorinated Acyclic Phosphonate Ester Precursors. Conventional methods to achieve the synthesis of FPMPs (fluorinated ANPs bearing a 3-fluoro-2-(phosphonomethoxy)propyl side chain) are based on the direct fluorination of the hydroxymethyl moiety of HPMP derivatives15 or building upon suitably functionalized chiral side-chain synthons.7,16 Initial attempts to use the former procedure in the presence of a fluorinating agent such as perfluorobutane-1-sulfonyl chloride15 or DAST resulted in the formation of significant amounts of undesired side products mainly as a result of competing elimination at the 2′-position, thus precluding the use of this route. Switching to (opting for) the second approach, we decided to prepare fluorinated acyclic phosphonate esters 4a/b (Scheme 1), following a modification of a recently reported method.17 Under solid−liquid phase transfer catalysis conditions, commercially available enantiomerically pure O-tritylated glycidols 1a/b underwent regioselective ring opening to afford fluorohydrines 2a/b upon inversion of the configuration. The replacement of diisopropyl tosyloxymethylphosphonate in the original alkylation protocol with diethyl tosyloxymethylphosphonate furnished 3a/b in 42% and 46% yield, respectively, over two steps. After acidic treatment, the resulting detritylated products 4a/b were isolated in good yields. Revisited Synthesis of (S)- and (R)-FPMP Analogues. Precursors 4a/b were directly used as substrates for the condensation with suitably protected purine and pyrimidine bases under Mitsunobu conditions (Ph3P, DIAD), providing a rapid entry to both series of enantiomerically pure FPMPs. The preparation of (S)- and (R)-FPMPT commenced from easily accessible N3-Bom-thymine 518 and 4a/b to deliver compounds 6a/b in high yields, as illustrated in Scheme 2. The removal of the N3-Bom protecting group was accomplished by catalytic hydrogenation using 10% palladium on carbon to provide compounds 7a/b, followed by standard hydrolysis of the diester groups with TMSBr in dry acetonitrile overnight to form the corresponding phosphonate acids 8a/b. This constitutes an improvement in terms of overall yield when compared to the original route.15 Although it was reported that a similar Mitsunobu reaction in the presence of 6-chloropurine proceeded in low yield (26%),17 surprisingly good yields (60−65%) of compounds 10a/b were obtained when we performed these reactions with alcohols 4a/ b (Scheme 3). The chloropurine moiety was then converted to adenine by treatment with NH3 in EtOH in a sealed tube, and finally, the phosphonate esters were cleaved with TMSBr to provide compounds 12a/b in 65 and 70% yields, respectively.

a

Reagents and conditions: (a) Ph3P, DIAD, THF, rt, 12 h; (b) Pd/C, H2, EtOH, rt, 24 h; (c) TMSBr, 2,6-lutidine, CH3CN, 0 °C to rt, 12 h.

In the cytosine series, all attempts to affect the alkylation of 4a/b using N4-benzoylcytosine as starting material were unsuccessful. Eventually, the desired products were accessible by protecting the primary amino functionality with an N4isobutyryl group. As shown in Scheme 4, compounds 14a/b were isolated in moderate yields (40%), mainly due to a competing O-2-alkylation reaction. However, the desired Nregioisomers 14a/b were separable by column chromatography and were then completely deprotected by treatment with methanolic ammonia followed by cleavage of the phosphonate esters to afford compounds 16a/b in good yields. Finally, guanine phosphonate acids 20a/b were prepared using the approach illustrated in Scheme 5. The reaction between 6-O-benzylguanine 17 and compounds 4a/b generated the requisite products 18a/b along with minor quantities of the corresponding triphenylphosphine adducts (not shown). The decomposition of these undesired adducts could be achieved by refluxing the crude reaction mixtures for 24 h in THF/H2O.19 Sequential removal of the 6-O-benzyl protecting group using 10% palladium on carbon and treatment with TMSBr provided the target compounds. Synthesis of FPMP Amidate Prodrugs. Next, we proceeded to convert the free phosphonic acids into the corresponding aryloxyphosphonamidates, according to the routes presented in Schemes 6 and 7. Previous work carried out in our group demonstrated that the use of an aspartic acid diester in place of classical L-alanine as the amino acid motif in nucleoside phosphoramidates was marked in some cases by significantly improved antiviral activity, particularly when higher alkyl groups were present as ester residues.13 In addition, such functionalized amino acids might offer an unprecedented versatility, providing bioconjugates that allow for bioavailability tuning as well as targeted drug delivery (“prodrug of a prodrug”).20 In light of these results, phosphonamidates 21a/ 6222



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Scheme 3. Synthesis of (S)/(R)-Fluorinated Acyclic Adenine Phosphonate Acids 12a/b (FPMPAs)a

a

Reagents and conditions: (a) Ph3P, DIAD, THF, rt, 12 h; (b) NH3/EtOH, 50 °C, 24 h; (c) TMSBr, 2,6-lutidine, CH3CN, 0 °C to rt, 12 h.

Anti-HIV Activity. Both (S)- and (R)-series of enantiomerically pure FPMPs and their corresponding diamyl aspartate phosphonamidate prodrugs were evaluated for antiviral activity by luciferase assay in TZM-bl cells22 infected with wild type HIV-1 strains NL4.3 (CXCR4-tropic HIV-1 strain) and BaL (CCR5-tropic HIV-1 strain). The antiviral results are summarized in Table 1, along with the assessment of their potential toxic effects on the same cell line. Tenofovir (PMPA) was included as a positive control. In agreement with previous findings, the SAR for FPMP purine derivatives was found to follow closely the one observed in MT-4 cells.8b The (S)-enantiomer of FPMPA 12a showed higher antiretroviral activity against both strains than its (R)enantiomer 12b, with EC50 values of 5.95 and 6.47 μM against NL4.3 and BaL strains, respectively. The best anti-HIV-1 activity in this series was found for analogues containing guanine as base, with (S)-FPMG 20a being comparably as effective as its (R) counterpart 20b. The corresponding fluorinated acyclic pyrimidine nucleoside phosphonate diacids 8a/b and 16a/b displayed little or no activity, most likely as a result of their poor conversion to the corresponding diphosphates or inability to inhibit polymerase activity. Notably, enhanced levels of activity, reaching in some cases the nanomolar range, were observed in the phosphonamidate series. The (S)-aspartate prodrug of FPMPA (22a) emerged as the most effective inhibitor of HIV-1 replication with EC50 values of 0.01 μM (NL4.3 strain) and 0.004 μM (BaL strain). This analogue was 600- and 1500-fold more potent than its parent phosphonate form 12a against the HIV-1 NL4.3 and BaL strains, respectively, and exhibited a 15- to 30-fold difference in potency compared to the less active (R)-AspFPMPA 22b. In an interesting reversal of SAR, the (R)enantiomer of Asp-FPMPG (25b) other than its (S) counterpart 25a was endowed with higher antiretroviral activity, displaying comparable EC50 values in the two strains (0.073 and 0.060 μM). This result suggests a dependence of the enantiospecificity of the biological effect on the type of heterocyclic base. Although (S)-Asp-FPMPA 22a was responsible for some degree of cytotoxicity, good selectivity indexes up to 7900 were nonetheless obtained. Most interestingly, (R)Asp-FPMPG 25b lacked significant cytotoxicity with a CC50

Scheme 4. Synthesis of (S)/(R)-Fluorinated Acyclic Cytosine Phosphonate Acids 16a/b (FPMPCs)a

a

Reagents and conditions: (a) Ph3P, DIAD, THF, rt, 12 h; (b) NH3/ MeOH, 45 °C, 15 h; (c) TMSBr, 2,6-lutidine, CH3CN, 0 °C to rt, 12 h.

b−23a/b were effectively prepared by treatment of 8a/b (B = T), 12a/b (B = A), and 16a/b (B = C) with a mixture of phenol and readily accessible L-aspartic acid amyl diester,13 followed by 2,2′-dithiodipyridine and triphenylphosphine activation,21 as depicted in Scheme 6. This method affords diastereomeric mixtures (ratio: 1/1−1/1.2) of FPMP amidate prodrugs due to the chirality of the phosphorus atom, and compound mixtures were biologically tested as such. However, under the same reactions conditions guanine derivatives 20a/b were transformed exclusively into the unexpected triphenylphosphine adducts 24a/b (Scheme 7). It should be noted that in the case of FPMPC derivatives, the formation of analogous adducts was also observed by mass spectroscopic analysis, although only in negligible quantities. Intermediates 24a/b could be successfully cleaved by treatment with 1 M HCl at room temperature to furnish 25a/b as diastereomeric mixtures (ratio: 1/1.1−1/1.2) albeit in low yields. 6223



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Scheme 5. Synthesis of (S)/(R)-Fluorinated Acyclic Guanine Phosphonate Acids 20a/b (FPMPGs)a

a

Reagents and conditions: (a) Ph3P, DIAD, THF, rt, 12 h; (b) Pd/C, H2, EtOAc, 10 h.; (c) TMSBr, 2,6-lutidine, CH3CN, 0 °C to rt, 12 h.

Scheme 6. Synthesis of (S)/(R)-Fluorinated Acyclic Nucleoside Phosphonamidates 21a/b−23a/ba

a

Reagents and conditions: (a) L-aspartic acid amyl diester HCl salt, PhOH, 2,2′-dithiodipyridine, PPh3, Et3N, Pyr, 60 °C, 12 h.

greater than 100 μM. The corresponding amidate prodrugs of thymine and cytosine containing FPMPs (compounds 21a/b and 23a/b) are completely devoid of anti-HIV activity. The higher antiviral activity of 22a and 25b, when compared to the parent phosphonates 12a and 20b, can be explained by their improved cellular permeability due to increased lipophilicity. This is supported by the higher cLogP values of compounds 22a and 25b (5.29 and 4.30, respectively) versus those of 12a and 20b (−1.69 and −2.68, respectively), as predicted using ChemBioDraw Ultra version 14.0 from CambridgeSoft. Besides testing the antiviral activity and cytotoxicity in TZMbl cells, the most promising amidate prodrugs (compounds 22a/b and 25a/b), as well as their parent phosphonates (compounds 12a/b and 20a/b) were also evaluated by clinically relevant antiviral assays in primary peripheral blood mononuclear cells (PBMCs). The antiviral activity of the compounds was evaluated in phytohemagglutinin (PHA)stimulated PBMCs via measurements of viral replication through HIV-1 p-24 Ag production by using a specific HIV-1 p-24 Ag ELISA. Tenofovir (PMPA), tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide (TAF) were used as reference drugs, and the results are illustrated in Table 2. The antiviral inhibitory activities of the parent phosphonates

(compounds 12a/b and 20a/b) were slightly increased in PBMC cultures (Table 2), as compared to the ones observed in the TZM-bl cells (Table 1). This might be due to a more efficient metabolism and/or an improved cellular permeability in PBMCs, compared to the TZM-bl cancer cell line. Similarly, the cytotoxicity of these parent phosphonates was more pronounced in PBMCs than in the TZM-bl cells. The most potent phosphonate analogue in PBMCs was the (S) guanine containing analogue 20a, whose profile (antiviral activity and cytotoxicity) was very similar to that of tenofovir. Similar to the trend observed in TZM-bl cells, all phosphonoamidate prodrugs showed an increased anti-HIV activity compared to that of the free phosphonates when tested in PBMCs. In particular, the prodrug of (S)-FPMPA (22a) displayed very potent anti-HIV-1 activity against both CXCR4using (X4) NL4.3 and CCR5-using (R5) BaL strains with IC50 values of 0.003 and 0.022 μM, respectively, whereas the corresponding diastereomer 22b showed a 10-fold reduced anti-HIV activity (0.026 and 0.33 μM for the NL4.3WT and BaL strain, respectively). Prodrugs of FPMPG (compounds 25a/b) were less active than the corresponding adenine containing analogue 22a against either the NL4.3WT or the BaL strain. All prodrugs, including the positive controls (TDF and TAF), were quite cytotoxic in PBMCs. However, the very 6224



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Scheme 7. Synthesis of (S)/(R)-Fluorinated Acyclic Guanine Phosphonamidates 25a/ba

Reagents and conditions: (a) L-aspartic acid amyl diester HCl salt, PhOH, 2,2′-dithiodipyridine, PPh3, Et3N, Pyr, 60 °C, 12 h; (b) 1 M HCl, 0 °C to rt, 1 h. a

Table 1. Anti-HIV-1 Activity and Cellular Cytotoxicity of FPMPs and Their Prodrugs Evaluated in TZM-bl Cells EC50a (μM)

Table 2. Anti-HIV Activity and Cellular Cytotoxicity of Compounds 12a/b, 20a/b, 22a/b, and 25a/b in (PHA)Stimulated PBMCs

SIc b

compd

HIV-1 X4 (NL4.3)

HIV-1 R5 (BaL)

CC50 (μM)

HIV-1 (NL4.3)

HIV-1 (BaL)

8a 8b 12a 12b 16a 16b 20a 20b 21a 21b 22a 22b 23a 23b 25a 25b PMPA

56.77 54.80 5.95 29.24 41.86 54.11 4.81 4.57 >43.44 >100 0.01 0.14 >68.98 >54.19 0.16 0.073 4.00

>100 >100 6.47 30.73 68.46 >100 3.79 6.65 >43.44 >100 0.004 0.11 >68.98 >54.19 0.22 0.060 6.91

>100 >100 >100 >100 >100 >100 >100 >100 43.44 >100 33.21 37.61 68.98 54.19 >100 >100 >100

>2 >2 >17 >3 >2 >2 >21 >22 25

1 1 >15 >3 >1 1 >26 >15 14

IC50a (μM)

SIc

compd

HIV-1 (NL4.3)

HIV-1 (BaL)

CC50b (μM)

HIV-1 (NL4.3)

HIV-1 (BaL)

12a 12b 20a 20b 22a 22b 25a 25b PMPA TDF TAF

1.19 4.04 0.28 1.42 0.003 0.026 0.03 0.02 0.38 0.08 0.001

3.73 11.93 1.14 6.41 0.022 0.33 0.13 0.17 1.40 0.22 0.01

>100 93.88 36.40 93.14 2.48 36.09 11.22 9.31 >25 14.56 1.32

84 23 130 66 >827 >1388 374 466 >66 182 1320

27 8 32 14.5 >113 109 86 55 >18 66 132

a Effective concentration required to inhibit HIV-1 replication by 50% in phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells as measured by HIV-1 p-24 Ag ELISA. bCytotoxic concentration required to reduce cell growth by 50%. cSelectivity index (SI): CC50/EC50.

a

Effective concentration required to reduce HIV-induced cytopathicity by 50%. bCytotoxic concentration required to reduce cell viability by 50%. cSelectivity index (SI): CC50/EC50.

been demonstrated by in vitro experiments, as well as in clinical settings. Prompted by the prior SAR results in wild-type HIV-1 strains, the two most promising analogues [(S)-Asp-FPMPA 22a and (R)-Asp-FPMPG 25b] were selected and evaluated in parallel with azidothymidine (AZT) and tenofovir for efficacy and toxicity in a standard anti-HIV-1 virus cytoprotection assay in MT-4 cells. AZT was included as a control compound because K65R mutants remain susceptible to AZT.24 The wild type HIV-1 NL4-3 strain was evaluated in parallel with tenofovir-resistant NL4-3K65R HIV-1. The cell viability was measured by a standard tetrazolium dye assay. The results given in Table 3 indicate that both phosphonoamidates 22a and 25b possess a more attractive resistance profile compared to that of TDF in terms of fold-change from the wild type assay data.

potent antiviral activity of the amidate prodrugs (especially compounds 22a/b) still gave rise to favorable selectivity indexes. HIV Resistance Profiling. The efficacy of new antiretroviral drugs against drug-resistant viruses is an important consideration in the selection of new anti-HIV drug candidates. Currently, tenofovir is the only approved nucleotide analogue for the treatment of HIV infections. Tenofovir is known to select for the K65R mutation in HIV-1 reverse transcriptase, which results in a 2−4-fold reduced susceptibility to this drug.23 The role of this mutation in the development of resistance has 6225



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pyrimidine derivatives FPMPT 8a/b and FPMPC 16a/b were largely inactive when tested up to 100 μM. The conversion of the thymidine (compounds 8a/b) and cytosine (compounds 16a/b) containing phosphonates to their corresponding phenoxy L-aspartic acid amyl ester phosphonoamidate prodrugs afforded analogues 21a/b and 23a/b, respectively, with an improved but still very weak anti-HBV activity, displaying EC50 values in the 28 to 47 μM range. The application of the amidate prodrug technology was more successful for the purine containing FPMP ANPs. For instance, the (S)-isomeric adenine analogue 22a had an antiviral EC50 = 0.01 μM and was >100-fold more potent than (R)-Asp-FPMPA 22b. The (R)-Asp-FPMPG prodrug 25b was also extremely active with an EC50 value of 0.02 μM. Further inspection of the EC90 values revealed that compound 25b (EC90 = 0.62 μM) surpassed the antiviral potency of the controls (EC90 > 2 μM for lamivudine; EC90 > 1 μM for entecavir). Not only did (R)-Asp-FPMPG exhibit potent anti-HBV activity but also the CC50 in the hepatoma cell line exceeded 100 μM, suggesting that 25b did not possess significant cytotoxicity. Although the cytotoxicity of 22a was greater (CC50 = 22.56 μM), this compound nonetheless possessed a selective mode of action. The high selectivity indexes of both guanine and adenine containing Asp-prodrugs 25b and 22a are particularly noteworthy in view of future studies. Since it is important to establish whether the reductions in extracellular HBV DNA copy number observed in the primary assay (Table 4) correlate to a concomitant reduction in intracellular HBV DNA copy number, a secondary HBV assay was performed using compound 22a as a model compound (Table 5). This assay resembles the primary assay, except for

Table 3. Comparison between the Anti-HIV-1 Activity and Cytotoxicity of Compounds 22a and 25b against Wild-Type (NL4.3) and Resistant (K65R Mutant) HIV-1 Strains in MT4 Cells HIV-1 (NL4.3)

HIV-1 NL4.3 (K65R)

compd

EC50a (μM)

EC50a (μM)

EC50 fold changeb

CC50c (μM)

22a 25b TDF PMPA AZT

0.03 0.01 16.7 2.3 1.3

>1 >1 >20 >20 >0.5

a Effective concentration required to reduce HIV-induced cytopathicity by 50%. bEC50 fold changes calculated from the EC50 across HIV1wild-type (NL4.3) and resistant (K65R Mutant) strains. cCytotoxic concentration required to reduce cell viability by 50%.

Anti-HBV Activity. All synthesized compounds were also tested in the human hepatoblastoma cell line HepG2.2.15 for inhibition of HBV replication and cytotoxicity (Table 4). Table 4. Anti-HBV Activity and Cytotoxicity of FPMPs and Their Prodrugs in HepG2.2.15 Cells (Primary Assay) compd

EC50a (μM)

EC90b (μM)

CC50c (μM)

SId

8a 8b 12a 12b 16a 16b 20a 20b 21a 21b 22a 22b 23a 23b 25a 25b lamivudine entecavir

>100 >100 >1.65 11.2 >100 >100 0.59 0.49 42.37 47.04 0.01 1.06 41.89 28.01 0.13 0.02 0.02 100 >100 >100 >100 >100 >100 42.74 82.49 >100 >100 1.97 16.89 >100 91.55 2.86 0.62 >2.00 >1.00

>100 >100 >100 >100 >100 >100 >100 >100 33.55 34.25 22.56 33.03 46.31 33.50 >100 >100 >2.00 >1.00

1 1 >61 >9 1 1 >169 >204 5000 >105 >2381

Table 5. Anti-HBV Activity and Cytotoxicity of Compound 22a in HepG2.2.15 Cells (Secondary Assay) compd

EC50a (μM)

EC90b (μM)

CC50c (μM)

SId

22a lamivudine entecavir

0.02 0.00887 0.00035

6.08 >2.00 >1.00

20.34 >2.00 >1.00

1017 >225 >2857

a

Effective concentration required to reduce HBV replication by 50%. Effective concentration required to reduce HBV replication by 90%. c Cytotoxic concentration required to reduce cell viability by 50%. d Selectivity index (SI): CC50/EC50. b

the final stage where the cells are processed to isolate the total intracellular DNA. The real-time TaqMan qPCR assay was then carried out using the isolated DNA to measure the reductions in intracellular HBV DNA copy number. From the obtained results, compound 22a displayed a pronounced anti-HBV activity based on the calculated EC50, EC90, and selectivity index values (SI50 = 1017), confirming the primary assay outcome. Anti-HCMV and VZV Activity. As outlined above, HPMPs have long been recognized as effective inhibitors of a wide range of DNA viruses. However, the replacement of the hydroxyl group in HPMPs with a fluorine atom resulted in a complete loss of activity against HSV-1, HSV-2, CMV, VZV, and VV.8b Since higher levels of active metabolites in the cell can be achieved by nucleoside prodrugs, it was anticipated that the newly prepared analogues might be able to take advantage of this property leading to an expanded activity spectrum. Thus, the antiherpes virus activity of all nucleoside phosphonamidates was evaluated in parallel with the parent FPMPs against

a

Effective concentration required to reduce HBV replication by 50%. Effective concentration required to reduce HBV replication by 90%. c Cytotoxic concentration required to reduce cell viability by 50%. d Selectivity index (SI): CC50/EC50. b

HepG2.2.15 is a widely used cell line in antiviral research that contains two copies of the HBV wild-type strain ayw1 genome and constitutively produces high levels of HBV.25 Real-time qPCR (TaqMan) was used to measure the extracellular HBV DNA copy number associated with virions released from HepG2 2.2.15 cells. A tetrazolium dye uptake assay was employed to measure cell viability and to calculate CC50 values. Among the parent compounds, both enantiomers of FPMPG exhibited the highest potency against HBV, with similar EC50 values of 0.59 and 0.49 μM for 20a and 20b, respectively. (S)FPMPA 12a proved 7-fold more inhibitory than the corresponding (R)-enantiomer 12b, with an EC50 value of 1.65 μM that was consistent with a previous report.9 The 6226



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Table 6. Antiviral Activity and Cytotoxicity of FPMPs and Their Prodrugs against HCMV and VZV in HEL Cells antiviral activity EC50a (μM) HCMV

cytotoxicity (μM) cell morphology (MCC)b

VZV

compound

AD-169 strain

Davis strain

TK+ VZV strain

TK− VZV strain

HEL

8a 8b 12a 12b 16a 16b 20a 20b 21a 21b 22a 22b 23a 23b 25a 25b ganciclovir cidofovir acyclovir brivudin

>338 >338 >328 >328 143.3 ± 43.3 >356 127 ± 18 139 >31.9 >31.9 3.97 >31.4 0.76 ± 0.10 >32.6 5.94 ± 2.92 4.43 ± 2.39 8.70 ± 0.66 2.49 ± 2.91 NDd NDd

>338 >338 >328 >328 27.1 ± 2.9 >356 57 ± 29 62.3 >31.9 >31.9 6.28 >31.4 2.02 ± 1.78 >32.6 3.05 ± 0.43 1.98 ± 1.07 16.5 ± 22.5 3.74 ± 4.48 NDd NDd

>338 >338 98.0 172 71.1 >356 15.5 ± 6.1 62.3 19.7 31.9 0.050 3.81 ± 1.41 3.15 ± 2.61 >32.6 0.59 ± 0.31 0.89 ± 0.48 NDd NDd 3.02 ± 3.26 0.055 ± 0.019

338 >338 131 >328 71.1 >356 12.5 62.3 23.1 12.7 0.057 ± 0.0089 8.24 ± 5.72 1.32 ± 0.46 >32.6 0.079 ± 0.008 0.11 ± 0.042 NDd NDd 20.7 ± 8.25 5.21 ± 3.97

>338 >338 >328 >328 >356 >356 >311 >311 159 31.9 31.4 157 163 32.6 153 153 >391 >358 >444 >300

cell growth (CC50)c HEL NDd NDd NDd NDd >356 NDd 65.4 NDd NDd NDd 1.96 ± 7.47 ± 74.7 ± NDd 27.8 ± 10.2 ± >391 >358 >444 >300

0.15 0.34 9.17 1.11 1.21

a

Effective concentration required to reduce virus-induced cytopathicity (HCMV) or plaque formation (VZV) by 50%. bMinimum concentration required to cause a microscopically detectable alteration of cell morphology. cCytotoxic concentration required to reduce cell viability by 50%. dNot determined.

Figure 3. Stability assay of compound 22a upon incubation with a pH 1 buffer, monitored by 31P NMR.

varicella-zoster virus [VZV, strains (TK+) Oka and thymidine kinase deficient (TK−) 07−1] and human cytomegalovirus (HCMV, strains AD-169 and Davis) in human embryonic lung

(HEL) cells (Table 6). Ganciclovir and cidofovir were included as positive controls for the HCMV assay, while acyclovir and brivudin were used as reference drugs for VZV testing. Each 6227



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Figure 4. Stability assay of compound 22a upon incubation with a pH 8 buffer, monitored by 31P NMR.

2.02 μM when tested against the AD-169 and Davis HCMV strains, respectively. Stability Studies. Significant antiviral activity against HIV, HBV, and VZV was observed in vitro for FPMP purine prodrugs. However, good chemical and enzymatic stabilities of these phosphonoamidate prodrugs are required to achieve antiviral efficacy in vivo since the partial or full hydrolysis of the prodrugs to form the corresponding free nucleoside phosphonate during absorption or distribution may hamper their antiviral effect. Therefore, the in vitro chemical and enzymatic stability of these compounds was further investigated, choosing the (S)-adenine containing analogue 22a as model compound. Chemical Stability at pH 1 and pH 8. The chemical stability of prodrug 22a was evaluated at different biologically relevant pH values using 31P NMR spectroscopy, by monitoring the changes of the set of two peaks representing the two phosphorus diastereomers. Under acidic conditions (pH 1), prodrug 22a was highly stable over 5 h (Figure 3), and only after 6 h the formation of a minor degradation product at 13.3 ppm was observed. On the other hand, in basic medium (pH 8), prodrug 22a was stable only for 1 h. After 2 h, the formation of a new single peak in the 31P NMR spectrum was observed, which gradually increased over time. Despite this instability, 58% of prodrug 22a was still present in the phosphate buffer after 14 h (Figure 4). Stability Study in Human Plasma and Human Liver Microsomes. In order to exert their antiviral activity, the diamyl aspartate phosphonoamidate prodrugs should be stable in plasma and metabolized in the target cells to release intracellularly their parent phosphonates. Further phosphorylation, catalyzed by cellular kinases, would then afford the corresponding FPMP diphosphates that may act as inhibitors of

FPMP (compounds 8a/b, 12a/b, 16a/b, and 20a/b) was found to be inactive against HCMV and VZV at subtoxic concentrations in agreement with the reported data.8b With respect to the Asp-prodrugs containing a purine nucleobase (compounds 22a/b and 25a/b), an overall improvement of their antiviral activity was evident when compared to that of the parent phosphonates. Amidate prodrug (S)-Asp-FPMPA 22a emerged as one of the most active compounds with 2000-fold increased activity compared to that of 12a against both VZV strain mutants either expressing a functional thymidine kinase (TK+) or enzyme deficient (TK−) with EC50 values in the 0.05 μM range. Compound 22a proved also active against HCMV, but it was not selective since it inhibited HEL growth with a CC50 of 1.96 μM, which was lower than the EC50 values obtained for both HCMV strains. Despite a low CC50 value, 22a selectively inhibited VZV replication with selectivity indices of 34 (TK− strain) and 39 (TK+ strain) for VZV. While 22b was unable to decrease HCMV replication at the highest concentration tested, it inhibited VZV plaque formation with EC50 values in the same range as the CC50 value. With regard to the guanine containing amidate prodrugs (compounds 25a/b), selectivity indices lower than 10 were calculated for HCMV, while these compounds proved more active and selective against the different strains of VZV (EC50 values in the 0.079− 0.89 μM range). Although compound 25b is endowed with more pronounced HIV and HBV activity when compared to its diasteromer 25a, for VZV and HCMV, the antiviral activity of both diastereomers is very similar. Among the pyrimidine analogues, only the cytosine containing (S)-Asp-prodrug 23a was found to be moderately active against both VZV TK+ and TK− viruses, while being endowed with EC50 values of 0.76 and 6228



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the HIV reverse transcriptase or viral DNA polymerases. The in vitro stability of prodrugs 22a and 25b in human plasma and human liver microsomes was evaluated, and their half-life values were calculated (Table 7). Both prodrugs were found to be

investigation as anti-HBV drugs. On the other hand, for the development as anti-HIV drugs, further fine-tuning of the prodrug moiety will be necessary to improve microsomal stability and hence decrease the first-pass metabolism. Incubations with Carboxypeptidase Y. The intracellular activation pathway for various classes of ProTides has been extensively studied.20 Recently, our group investigated and proposed a metabolic process for deoxythreosyl aryloxyphosphonamidate prodrugs containing a L-aspartate acid diester as the amino acid moiety.14 Putative activation routes for prodrug 22a are shown in Figure 5. In order to verify whether prodrug 22a is indeed activated via one of these routes, an enzymatic experiment was carried out. Prodrug 22a and carboxypeptidase Y were incubated in acetone-d6 and Trizma buffer (pH 7.6) at 25 °C. 31P NMR spectra were recorded at regular time intervals (Figure 6). A fast hydrolysis of the two diastereomers of compound 22a (represented as two signals at δP = 24.2 and 23.2 ppm) to the proposed intermediate (E or F at δP = 16.3 ppm) was observed. Within 30 min of incubation, the

Table 7. Metabolic Stability of Prodrugs 22a and 25b in Human Plasma and Human Liver Microsomes compd

human plasma t1/2 (min)a

human liver microsomes t1/2 (min)a

22a 25b

89 73

2 2

a

Results are the mean of two independent experiments.

stable in human plasma with t1/2 values exceeding 1 h. On the other hand, very short half-lives of prodrugs 22a and 25b (t1/2 = 2 min) were observed in human liver microsomes. This suggests that prodrugs 22a and 25b are quickly metabolized in liver cells, releasing the parent phosphonates 12a and 22b. Therefore, prodrugs 22a and 25b are well suited for further

Figure 5. Proposed activation pathway for prodrug 22a. 6229



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Figure 6. Carboxypeptidase Y assay applied on prodrug 22a and monitored by 31P NMR, 25 °C.

FPMPG amidate prodrugs selectively inhibited VZV plaque formation at submicromolar concentrations, while the (S)aspartate prodrug of FPMPC was endowed with micromolar anti-HCMV potency. The in vitro chemical and enzymatic stability of the prodrug of (S)-FPMPA was investigated. This compound was stable at acidic pH, whereas it was only stable for 1 h under alkaline conditions. The prodrug was stable in human plasma but was prone to extensive metabolism in human liver microsomes. Enzymatic experiments revealed that carboxypeptidase Y treatment triggered further metabolism of the prodrug.

appearance of a small downfield new peak was observed at 16.3 ppm in the 31P NMR spectrum. After 2 h, more than 70% of the parent prodrug 22a was converted to this intermediate. One of the diastereomers was metabolized at a slightly faster rate compared to that of the other one. The diastereomer (δP = 23.2 ppm) was completely metabolized to the intermediate after 5 h, whereas the other diastereomer (δP = 24.2 ppm) was processed within 6 h. Mass spectral analysis of the isolated peak at δP = 16.3 ppm confirmed the structure of the metabolite to be intermediate E or F (see Supporting Information). Whether the α- or β-carboxyl ester is cleaved first is not known at this moment and needs to be further explored. Overall, the carboxypeptidase assay demonstrates that the enzymatic cleavage of the ester group is sufficient to trigger further activation of prodrug 22a to generate metabolite E or F.

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EXPERIMENTAL SECTION

Chemistry. For reactions, all reagents and solvents were purchased from commercial sources and were used as obtained. Moisture sensitive reactions were carried out in oven-dried glassware under a nitrogen or argon atmosphere. 1H, 13C, and 31P NMR spectra were recorded on Bruker Avance 300, 500, or 600 MHz spectrometers with tetramethylsilane as internal standard or referenced to the residual solvent signal and 85% H3PO4 for 31P NMR experiments. The intermediates and final compounds were characterized by using 2D NMR (H−COSY, HSQC, and HMBC) spectroscopic techniques. High-resolution mass spectra (HRMS) were obtained on a quadruple orthogonal acceleration time-of-flight mass spectrometer (Synapt G2 HDMS, Waters, Milford, MA). Samples were infused at 3 μL/min, and spectra were obtained in positive (or in negative) ionization mode with a resolution of 15 000 (fwhm) using leucine enkephalin as the lock mass. Precoated aluminum sheets (254 nm) were used for TLC. Products were purified by column chromatography on silica gel (60 Å, 0.035−0.070 mm, Acros Organics). Preparative RP-HPLC purifications were performed on a Phenomenex Gemini 110A column (C18, 10 μm, 21.2 mm × 250 mm) using CH3CN/0.05 M TEAB buffer or H2O/CH3CN as eluent gradient. Purities of all of the tested compounds were above 95% by HPLC analysis.

■

CONCLUSIONS In summary, an expedient method for the synthesis of both enantiomeric series of 3-fluoro-2-(phosphonomethoxy)propyl acyclic nucleosides bearing the four natural nucleobases was developed. The purine containing analogues FPMPA and FPMPG displayed moderate anti-HIV and -HBV activity, whereas their pyrimidine counterparts were largely inactive. The introduction of a diamyl aspartate phenoxyamidate group as the phosphonate prodrug motif led to a significant enhancement of antiviral potency. The anti-HIV activity increased by a factor up to 1500, depending on the nucleobase moiety, whereas improvements up to 160-fold for the anti-HBV activity were observed. Importantly, some of the synthesized compounds exhibited a broader spectrum activity than the parent phosphonate analogues since they also suppressed DNA virus replication. For instance, (S)-Asp-FPMPA and (R)-Asp6230



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(S)-1-{3-Fluoro-2-[(diethylphosphoryl)methoxy]propyl}-N 3 (benzyloxymethyl)thymine (6a). A solution of DIAD (0.32 mL, 1.64 mmol) in anhydrous THF (1 mL) was added dropwise to a mixture of compound 4a (200 mg, 0.82 mmol), compound 518 (240 mg, 0.98 mmol), and Ph3P (430 mg, 1.64 mmol) in anhydrous THF (5 mL) at room temperature. The reaction mixture was stirred for 12 h, and it was then concentrated under reduced pressure. The crude residue was purified by column chromatography on silica gel (gradient DCM/ MeOH, 80:1, v/v; DCM/MeOH, 50:1, v/v) to give 6a (310 mg, 80%) as a colorless oil. 1H NMR (300 MHz, CDCl3): δ 7.39−7.25 (m, 5H, ArH), 7.14−7.12 (m, 1 H, H-6), 5.50 (s, 2H, OCH2N), 4.73−4.33 (m, 4H, CH2-Ar, H-3′), 4.17−3.92 (m, 7H, H-1′, H-2′, 2 × CH2CH3), 3.84−3.66 (m, 2H, PCH2), 1.93 (d, J = 1.2 Hz, 3H, CH3-5), 1.34−1.28 (m, 6H, 2 × CH2CH3). 13C NMR (75 MHz, CDCl3): δ 163.8 (C-4), 151.8 (C-2), 140.5 (C-6), 138.1 (Ar-C), 128.3 (Ar-C), 127.7 (Ar-C), 109.7 (C-5), 82.3 (d, 1JC,F = 173.4 Hz, C-3′), 78.6 (dd, 2JC,F = 18.5 Hz, 3 JC,P = 9.8 Hz, C-2′), 72.3 (OCH2N), 70.8 (CH2−Ar), 64.6 (d, 1JC,P = 167.4 Hz, CH2P), 62.6, 62.5 (2 × d, 2JC,P = 3.8 Hz, CH2CH3), 62.5 (d, 2 JC,P = 3.7 Hz, CH2CH3), 49.3 (d, 3JC,F = 8.6 Hz, C-1′), 16.6, 16.5 (CH2CH3), 13.0 (CH3-5). 31P NMR (121 MHz, CDCl3): δ 20.8. HRMS for C21H30FN2O7P [M + H]+ calcd.: 473.1847; found, 473.1848. (R)-1-{3-Fluoro-2-[(diethylphosphoryl)methoxy]propyl}-N 3 (denzyloxymethyl)thymine (6b). Compound 6b was obtained as a colorless oil (460 mg, 70%) according to the procedure used for the preparation of 6a, starting from compound 4b (300 mg, 1.23 mmol), compound 518 (360 mg, 1.47 mmol), Ph3P (640 mg, 2.46 mmol), and DIAD (0.48 mL, 2.46 mmol) in anhydrous THF (8 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/MeOH, 80:1, v/v; DCM/MeOH, 50:1, v/v). 1H NMR (300 MHz, CDCl3): δ 7.39−7.25 (m, 5H, ArH), 7.13 (t, J = 1.3 Hz, 1 H, H6), 5.50 (s, 2H, OCH2N), 4.73−4.33 (m, 4H, CH2-Ar, H-3′), 4.17− 3.91 (m, 7H, H-1′, H-2′, 2 × CH2CH3), 3.84−3.66 (m, 2H, PCH2), 1.93 (d, J = 1.1 Hz, 3H, CH3-5), 1.34−1.28 (m, 6H, 2 × CH2CH3). 13 C NMR (75 MHz, CDCl3): δ 163.8 (C-4), 151.8 (C-2), 140.5 (C6), 138.0 (Ar-C), 128.3 (Ar-C), 127.7 (Ar-C), 109.7 (C-5), 82.3 (d, 1 JC,F = 173.5 Hz, C-3′), 78.6 (dd, 2JC,F = 18.5 Hz, 3JC,P = 9.7 Hz, C-2′), 72.3 (OCH2N), 70.7 (CH2−Ar), 64.6 (d, 1JC,P = 167.3 Hz, CH2P), 62.6, 62.5 (2 × d, 2JC,P = 3.5 Hz, CH2CH3), 49.3 (d, 3JC,F = 8.8 Hz, C1′), 16.6, 16.5 (CH2CH3), 13.0 (CH3-5). 31P NMR (121 MHz, CDCl3): δ 20.8. HRMS for C21H30FN2O7P [M + H]+ calcd.: 473.1847; found, 473.1841. (S)-1-{3-Fluoro-2-[(diethylphosphoryl)methoxy]propyl}thymine (7a). A suspension of compound 6a (240 mg, 0.51 mmol) and Pd/C on charcoal (10%w/w, 120 mg) in EtOH (10 mL) was purged with nitrogen, followed by hydrogen, and then allowed to stir under a hydrogen atmosphere. After 24 h, the mixture was filtered through a pad of Celite, and the filtrate was evaporated under reduced pressure to give a crude product. The residue was then redissolved in MeOH (10 mL) and Et3N (1 mL), and the solution was stirred for 3 h at room temperature. The reaction mixture was concentrated under reduced pressure, and the resulting crude residue was purified by column chromatography on silica gel (gradient DCM/MeOH, 30:1, v/ v; DCM/MeOH, 20:1, v/v) to give 7a (160 mg, 90%) as a colorless oil. 1H NMR (300 MHz, CDCl3): δ 9.63 (s, 1H, NHCO), 7.16 (t, J = 1.3 Hz, 1H, H-6), 4.72−4.33 (m, 2H, H-3′), 4.17−3.90 (m, 7H, H-1′, H-2′, 2 × CH2CH3), 3.83−3.63 (m, 2H, PCH2), 1.89 (d, J = 1.2 Hz, 3H, CH3-5), 1.33−1.28 (m, 6H, 2 × CH2CH3). 13C NMR (75 MHz, CDCl3): δ 164.5 (C-4), 151.3 (C-2), 141.9 (C-6), 110.4 (C-5), 82.3 (d, 1JC,F = 173.4 Hz, C-3′), 78.7 (dd, 2JC,F = 18.5 Hz, 3JC,P = 9.5 Hz, C2′), 64.6 (d, 1JC,P = 167.3 Hz, CH2P), 62.7, 62.6 (CH2CH3), 48.6 (d, 3 JC,F = 8.8 Hz, C-1′), 16.6, 16.5 (CH2CH3), 12.3 (CH3-5). 31P NMR (121 MHz, CDCl3): δ 21.0. HRMS for C9H14FN2O6P [M + H]+ calcd.: 353.1272; found, 353.1262. (R)-1-{3-Fluoro-2-[(diethylphosphoryl)methoxy]propyl}thymine (7b). Compound 7b was obtained as a colorless oil (270 mg, 90%) according to the procedure used for the preparation of 7a, starting from compound 6b (400 mg, 0.85 mmol), Pd/C on charcoal (10%w/ w, 200 mg) in EtOH (20 mL), and Et3N (1 mL) in MeOH (20 mL). The crude residue was purified by column chromatography on silica

Diethyl (S)-({[1-Fluoro-3-(trityloxy)propan-2-yl]oxy}methyl)phosphonate (3a). A solution of 2a17 (800 mg, 2.38 mmol) and diethyl tosyloxymethylphosphonate (1.53 g, 4.76 mmol) in anhydrous tetrahydrofuran (30 mL) was cooled to −20 °C. Sodium hydride (182 mg, 4.76 mmol, 60% in mineral oil) was then added, and the mixture was slowly warmed to room temperature and stirred for further 6−10 h. The reaction mixture was filtered through a pad of Celite, and the filtrate was concentrated in vacuo to give a crude product. The residue was then purified by column chromatography on silica gel (petroleum ether/EtOAc, 1:1, v/v) to give 3a (600 mg, 52%) as a colorless oil. 1H NMR (300 MHz, CDCl3): δ 7.49−7.45 (m, 6H, ArH), 7.35−7.22 (m, 9H, ArH), 4.65 (dd, J = 4.7, 1.6 Hz, 1H, H-3′a), 4.49 (dd, J = 4.7, 2.0 Hz, 1H, H-3′b), 4.24−4.13 (m, 4H, 2 × CH2CH3), 3.97 (dd, J = 8.9, 1.5 Hz, 2H, PCH2), 3.88−3.77 (m, 1H, H-2′), 3.34−3.28 (m, 2H, H1′), 1.36−1.30 (m, 6H, 2 × CH2CH3). 13C NMR (75 MHz, CDCl3): δ 143.3 (Ar-C), 128.3 (Ar-C), 127.6 (Ar-C), 126.9 (Ar-C), 86.7 (CTr), 83.2 (d, 1JC,F = 171.4 Hz, C-3′), 79.8 (dd, 2JC,F = 18.6 Hz, 3JC,P = 11.4 Hz, C-2′), 64.4 (d, 1JC,P = 166.3 Hz, CH2P), 62.2, 62.1 (CH2CH3), 61.8 (d, 3JC,F = 8.2 Hz, C-1′), 16.2, 16.1 (CH2CH3). 31P NMR (121 MHz, CDCl3): δ 20.7. HRMS for C27H32FO5P [M + Na]+ calcd.: 509.1864; found, 509.1860. Diethyl (R)-({[1-Fluoro-3-(trityloxy)propan-2-yl]oxy}methyl)phosphonate (3b). Compound 3b was obtained as a colorless oil (7.00 g, 54%) according to the procedure used for the preparation of 3a, starting from compound 2b17 (9.00 g, 26.8 mmol), diethyl tosyloxymethylphosphonate (17.24 g, 53.50 mmol), and sodium hydride (2.14 g, 53.51 mmol, 60% in mineral oil) in anhydrous THF (300 mL). The crude residue was purified by column chromatography on silica gel (petroleum ether/EtOAc, 1:1, v/v). 1H NMR (300 MHz, CDCl3): δ 7.45−7.42 (m, 6H, ArH), 7.31−7.22 (m, 9H, ArH), 4.62− 4.60 (m, 1H, H-3′a), 4.46 (dd, J = 4.6, 2.5 Hz, 1H, H-3′b), 4.20−4.09 (m, 4H, 2 × CH2CH3), 3.93 (dd, J = 8.9, 1.5 Hz, 2H, PCH2), 3.83− 3.74 (m, 1H, H-2′), 3.29−3.23 (m, 2H, H-1′), 1.33−1.26 (m, 6H, 2 × CH2CH3). 13C NMR (75 MHz, CDCl3): δ 143.6 (Ar-C), 128.6 (ArC), 127.9 (Ar-C), 127.1 (Ar-C), 86.9 (CTr), 83.5 (d, 1JC,F = 171.4 Hz, C-3′), 80.0 (dd, 2JC,F = 18.7 Hz, 3JC,P = 11.2 Hz, C-2′), 64.7 (d, 1JC,P = 166.3 Hz, CH2P), 62.5, 62.4 (CH2CH3), 62.1 (d, 3JC,F = 8.1 Hz, C-1′), 16.5, 16.4 (CH2CH3). 31P NMR (121 MHz, CDCl3): δ 21.3. HRMS for C27H32FO5P [M + Na]+ calcd.: 509.1864; found, 509.1857. Diethyl (S)-{[(1-Fluoro-3-hydroxypropan-2-yl)oxy]methyl}phosphonate (4a). A solution of 3a (570 mg, 1.17 mmol) in 80% aqueous acetic acid (10 mL) was stirred at 90 °C for 1 h. After removal of all the volatiles, the residue was purified by column chromatography on silica gel (EtOAc/MeOH, 50:1, v/v; 30:1, v/v) to give 4a (170 mg, 60%) as a colorless oil. 1H NMR (500 MHz, CDCl3): δ 4.58−4.52 (m, 1H, H-3′a), 4.48−4.42 (m, 1H, H-3′b), 4.25−4.09 (m, 5H, 2 × CH2CH3, PCH2a), 3.89 (dd, J = 14.2, 8.5 Hz, 1H, PCH2b), 3.82−3.74 (m, 2H, H-2′, H-1′a), 3.65 (dd, J = 12.3, 6.0 Hz, 1H, H-1′b), 1.38− 1.34 (m, 6H, 2 × CH2CH3). 13C NMR (125 MHz, CDCl3): δ 82.7 (d, 1 JC,F = 170.8 Hz, C-3′), 81.9 (dd, 2JC,F = 18.6 Hz, 3JC,P = 8.5 Hz, C-2′), 64.5 (d, 1JC,P = 167.5 Hz, CH2P), 62.7, 62.2 (CH2CH3), 60.7 (d, 3JC,F = 8.1 Hz, C-1′), 16.1, 16.0 (CH2CH3). 31P NMR (121 MHz, CDCl3): δ 21.8. HRMS for C8H18FO5P [M + H]+ calcd.: 245.0949; found, 245.0952. Diethyl (R)-{[(1-Fluoro-3-hydroxypropan-2- yl)oxy]methyl}phosphonate (4b). Compound 4b was obtained as a colorless oil (1.80 g, 62%) according to the procedure used for the preparation of 4a, starting from compound 3b (6.00 g, 12.3 mmol) in 80% aqueous acetic acid (150 mL). The crude residue was purified by column chromatography on silica gel (petroleum ether/EtOAc, 1:1, v/v). 1H NMR (300 MHz, CDCl3): δ 4.63−4.52 (m, 1H, H-3′a), 4.47−4.36 (m, 1H, H-3′b), 4.23−3.99 (m, 5H, 2 × CH2CH3, PCH2a), 3.91 (dd, J = 13.9, 8.7 Hz, 1H, PCH2b), 3.79−3.60 (m, 3H, H-2′, H-1′), 1.36− 1.30 (m, 6H, 2 × CH2CH3). 13C NMR (75 MHz, CDCl3): δ 82.9 (d, 1 JC,F = 170.4 Hz, C-3′), 81.6 (dd, 2JC,F = 18.4 Hz, 3JC,P = 10.0 Hz, C2′), 64.3 (d, 1JC,P = 167.3 Hz, CH2P), 62.7, 62.4 (CH2CH3), 60.3 (d, 3 JC,F = 8.2 Hz, C-1′), 16.2, 16.1 (CH2CH3). 31P NMR (121 MHz, CDCl3): δ 22.0. HRMS for C8H18FO5P [M + H]+ calcd.: 245.0949; found, 245.0953. 6231



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gel (gradient DCM/MeOH, 30:1, v/v; DCM/MeOH, 20:1, v/v). 1H NMR (300 MHz, CDCl3): δ 9.67 (s, 1H, NHCO), 7.18 (d, J = 1.4 Hz, 1H, H-6), 4.75−4.35 (m, 2H, H-3′), 4.19−3.93 (m, 7H, H-1′, H-2′, 2 × CH2CH3), 3.86−3.66 (m, 2H, PCH2), 1.92 (d, J = 1.2 Hz, 3H, CH35), 1.35−1.31 (m, 6H, 2 × CH2CH3). 13C NMR (75 MHz, CDCl3): δ 164.5 (C-4), 151.3 (C-2), 141.9 (C-6), 110.4 (C-5), 82.3 (d, 1JC,F = 173.4 Hz, C-3′), 78.7 (dd, 2JC,F = 18.5 Hz, 3JC,P = 9.5 Hz, C-2′), 64.6 (d, 1JC,P = 167.4 Hz, CH2P), 62.7, 62.6 (2 × d, 2JC,P = 5.5 Hz, CH2CH3), 48.5 (d, 3JC,F = 8.8 Hz, C-1′), 16.6, 16.5 (CH2CH3), 12.3 (CH3-5). 31P NMR (121 MHz, CDCl3): δ 21.0. HRMS for C9H14FN2O6P [M + H]+ calcd.: 353.1272; found, 353.1278. (S)-1-[3-Fluoro-2-(phosphonomethoxy)propyl]thymine triethylammonium Salt (8a). Bromotrimethylsilane (0.15 mL, 1.14 mmol) was added dropwise to a solution of diethyl phosphonate ester 7a (100 mg, 0.28 mmol) and 2,6-lutidine (0.26 mL, 2.27 mmol) in anhydrous acetonitrile (5 mL) at 0 °C. After the addition was completed, the mixture was slowly warmed to room temperature and set aside in the dark for 12 h. The reaction was quenched with 0.1 M TEAB and was then concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (gradient DCM/MeOH/Et3N, 10:5:1, v/v/v; 7.5:5:1, v/v/v), followed by RP-HPLC (linear gradient, 2−98% CH3CN in 0.05 M TEAB solution) to give the desired phosphonate acid triethylammonium salt 8a (59 mg, 70%) as a white foam. 1H NMR (300 MHz, D2O): δ 7.52 (s, 1H, H-6), 4.77−4.48 (m, 2H, H-3′, overlapped with H2O), 3.93−3.79 (m, 3H, H-1′, H-2′), 3.58−3.46 (m, 2H, PCH2), 1.80 (s, 3H, CH3-5). 13C NMR (75 MHz, D2O): δ 166.8 (C-4), 152.2 (C-2), 143.5 (C-6), 110.3 (C-5), 82.1 (d, 1 JC,F = 167.3 Hz, C-3′), 77.1 (dd, 2JC,F = 18.1 Hz, 3JC,P = 10.4 Hz, C2′), 67.7 (d, 1JC,P = 150.9 Hz, CH2P), 47.3 (d, 3JC,F = 6.9 Hz, C-1′), 11.0 (CH3-5). 31P NMR (121 MHz, D2O): δ 12.9. HRMS for C9H14FN2O6P [M − H]− calcd.: 295.0501; found, 295.0499. (R)-1-[3-Fluoro-2-(phosphonomethoxy)propyl]thymine triethylammonium Salt (8b). Compound 8b was obtained as a white foam (176 mg, 70%) according to the procedure used for the preparation of 8a, starting from compound 7b (300 mg, 0.85 mmol), bromotrimethylsilane (0.45 mL, 3.41 mmol), and 2,6-lutidine (0.79 mL, 6.81 mmol) in anhydrous acetonitrile (10 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/ MeOH/Et3N, 10:5:1, v/v/v; 7.5:5:1, v/v/v). 1H NMR (300 MHz, D2O): δ 7.55−7.54 (m, 1H, H-6), 4.80−4.43 (m, 2H, H-3′, overlapped with H2O), 4.09−3.88 (m, 3H, H-1′, H-2′), 3.79−3.60 (m, 2H, PCH2), 1.87 (d, J = 1.1 Hz, 3H, CH3). 13C NMR (75 MHz, D2O): δ 166.7 (C-4), 152.0 (C-2), 143.5 (C-6), 110.2 (C-5), 82.0 (d, 1 JC,F = 168.0 Hz, C-3′), 77.5 (dd, 2JC,F = 18.2 Hz, 3JC,P = 11.5 Hz, C2′), 66.4 (d, 1JC,P = 156.3 Hz, CH2P), 47.6 (d, 3JC,F = 7.6 Hz, C-1′), 11.0 (CH3). 31P NMR (121 MHz, D2O): δ 14.7. HRMS for C9H14FN2O6P [M − H]− calcd.: 295.0501; found, 295.0498. (S)-6-Chloro-9-{3-fluoro-2-[(diethylphosphoryl)methoxy]propyl}purine (10a). Compound 10a was obtained as a colorless oil (190 mg, 60%) according to the procedure used for the preparation of 6a, starting from compound 4a (200 mg, 0.82 mmol), compound 9 (150 mg, 0.98 mmol), Ph3P (430 mg, 1.64 mmol), and DIAD (0.32 mL, 1.64 mmol) in anhydrous THF (6 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/ MeOH, 50:1, v/v; DCM/MeOH, 30:1, v/v). 1H NMR (300 MHz, CDCl3): δ 8.75 (s, 1H, H-2), 8.32 (s, 1H, H-8), 4.74−4.40 (m, 4H, H3′, H-1′), 4.22−3.94 (m, 6H, H-2′, 2 × CH2CH3, PCH2a), 3.78 (dd, J = 14.0, 8.5 Hz, 1H, PCH2b), 1.34−1.24 (m, 6H, 2 × CH2CH3). 13C NMR (75 MHz, CDCl3): δ 152.0 (C-2, C-4), 151.1 (C-6), 146.5 (C8), 131.4 (C-5), 81.6 (d, 1JC,F = 174.3 Hz, C-3′), 78.0 (dd, 2JC,F = 19.6 Hz, 3JC,P = 9.0 Hz, C-2′), 64.4 (d, 1JC,P = 167.1 Hz, CH2P), 62.7, 62.5 (2 × d, 2JC,P = 6.7 Hz, CH2CH3), 44.2 (d, 3JC,F = 8.0 Hz, C-1′), 16.5, 16.4 (2 × d, 3JC,P = 3.8 Hz, CH2CH3). 31P NMR (121 MHz, CDCl3): δ 20.3. HRMS for C13H19ClFN4O4P [M + H]+ calcd.: 381.0889; found, 381.0884. (R)-6-Chloro-9-{3-fluoro-2-[(diethylphosphoryl)methoxy]propyl}purine (10b). Compound 10b was obtained as a colorless oil (300 mg, 65%) according to the procedure used for the preparation of 6a, starting from compound 4b (300 mg, 1.23 mmol), compound 9 (230 mg, 1.50 mmol), Ph3P (640 mg, 2.46 mmol), and DIAD (0.48 mL,

2.46 mmol) in anhydrous THF (10 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/ MeOH, 50:1, v/v; DCM/MeOH, 30:1, v/v). 1H NMR (300 MHz, CDCl3): δ 8.71 (s, 1H, H-2), 8.26 (s, 1H, H-8), 4.69−4.35 (m, 4H, H3′, H-1′), 4.17−3.89 (m, 6H, H-2′, 2 × CH2CH3, PCH2a), 3.73 (dd, J = 14.0, 8.4 Hz, 1H, PCH2b), 1.30−1.19 (m, 6H, 2 × CH2CH3). 13C NMR (75 MHz, CDCl3): δ 152.0 (C-2, C-4), 151.1 (C-6), 146.5 (C8), 131.5 (C-5), 81.6 (d, 1JC,F = 174.3 Hz, C-3′), 78.1 (dd, 2JC,F = 19.6 Hz, 3JC,P = 8.9 Hz, C-2′), 64.5 (d, 1JC,P = 167.0 Hz, CH2P), 62.7, 62.6 (2 × d, 2JC,P = 6.6 Hz, CH2CH3), 44.2 (d, 3JC,F = 8.2 Hz, C-1′), 16.5, 16.4 (2 × d, 3JC,P = 4.5 Hz, CH2CH3). 31P NMR (121 MHz, CDCl3): δ 20.6. HRMS for C13H19ClFN4O4P [M + H]+ calcd.: 381.0889; found, 381.0883. (S)-9-{3-Fluoro-2-[(diethylphosphoryl)methoxy]propyl}adenine (11a). A solution of 10a (150 mg, 0.40 mmol) in 10% ethanolic ammonia (20 mL) was stirred at 50 °C for 24 h. After the removal of all the volatiles, the residue was purified by column chromatography on silica gel (gradient DCM/MeOH, 20:1, v/v; 15:1, v/v) to give 11a (110 mg, 80%) as a colorless oil. 1H NMR (300 MHz, CDCl3): δ 8.30 (s, 1H, H-2), 8.00 (s, 1H, H-8), 6.75 (s, 2H, NH2), 4.71−4.27 (m, 4H, H-3′, H-1′), 4.14−3.90 (m, 6H, H-2′, 2 × CH2CH3, PCH2a), 3.76 (dd, J = 13.9, 8.9 Hz, 1H, PCH2b), 1.30−1.22 (m, 6H, 2 × CH2CH3). 13C NMR (75 MHz, CDCl3): δ 155.0 (C-6), 151.6 (C-2), 159.9 (C-4), 142.1 (C-8), 119.2 (C-5), 82.0 (d, 1JC,F = 173.8 Hz, C-3′), 78.5 (dd, 2 JC,F = 19.3 Hz, 3JC,P = 10.0 Hz, C-2′), 64.6 (d, 1JC,P = 167.3 Hz, CH2P), 62.7, 62.6 (CH2CH3), 43.6 (d, 3JC,F = 8.3 Hz, C-1′), 16.5, 16.4 (CH2CH3). 31P NMR (121 MHz, CDCl3): δ 20.5. HRMS for C13H21FN5O4P [M + H]+ calcd.: 362.1389; found, 362.1386. (R)-9-{3-Fluoro-2-[(diethylphosphoryl)methoxy]propyl}adenine (11b). Compound 11b was obtained as a colorless oil (260 mg, 90%) according to the procedure used for the preparation of 11a, starting from compound 10b (300 mg, 0.80 mmol) in 10% ethanolic ammonia (30 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/MeOH, 20:1, v/v; 15:1, v/v). 1H NMR (300 MHz, CDCl3): δ 8.35 (s, 1H, H-2), 7.95 (s, 1H, H-8), 5.86 (s, 2H, NH2), 4.74−4.28 (m, 4H, H-3′, H-1′), 4.18−3.91 (m, 6H, H-2′, 2 × CH2CH3, PCH2a), 3.78 (dd, J = 13.9, 8.9 Hz, 1H, PCH2b), 1.34− 1.25 (m, 6H, 2 × CH2CH3). 13C NMR (150 MHz, CDCl3): δ 155.4 (C-6), 153.1 (C-2), 150.2 (C-4), 141.6 (C-8), 119.4 (C-5), 82.0 (d, 1 JC,F = 173.8 Hz, C-3′), 78.5 (dd, 2JC,F = 19.4 Hz, 3JC,P = 9.4 Hz, C-2′), 64.6 (d, 1JC,P = 167.3 Hz, CH2P), 62.5, 62.4 (d, 2JC,P = 6.5 Hz, CH2CH3), 43.6 (d, 3JC,F = 8.1 Hz, C-1′), 16.4, 16.3 (CH2CH3). 31P NMR (121 MHz, CDCl3): δ 20.6. HRMS for C13H21FN5O4P [M + H]+ calcd.: 362.1389; found, 362.1390. (S)-9-[3-Fluoro-2-(phosphonomethoxy)propyl]adenine triethylammonium Salt (12a). Compound 12a was obtained as a white foam (38 mg, 65%) according to the procedure used for the preparation of 8a, starting from compound 11a (70 mg, 0.19 mmol), bromotrimethylsilane (0.10 mL, 0.77 mmol), and 2,6-lutidine (0.18 mL, 1.59 mmol) in anhydrous acetonitrile (5 mL). The crude residue was purified by column chromatography on silica gel (gradient acetone/H2O/Et3N, 6:1:1, v/v/v). 1H NMR (300 MHz, D2O): δ 8.23 (s, 1H, H-2), 8.14 (s, 1H, H-8), 4.97−4.27 (m, 4H, H-3′, H-1′), 4.05− 3.95 (m, 1H, H-2′), 3.55−3.40 (m, 2H, PCH2). 13C NMR (75 MHz, D2O): δ 154.2 (C-6), 150.7 (C-2), 148.7 (C-4), 143.4 (C-8), 117.8 (C-5), 81.7 (d, 1JC,F = 168.0 Hz, C-3′), 77.6 (dd, 2JC,F = 18.9 Hz, 3JC,P = 11.4 Hz, C-2′), 66.1 (d, 1JC,P = 157.0 Hz, CH2P), 43.3 (d, 3JC,F = 7.5 Hz, C-1′). 31P NMR (121 MHz, D2O): δ 12.7. HRMS for C9H13FN5O4P [M − H]− calcd.: 304.0616; found, 304.0603. (R)-9-[3-Fluoro-2-(phosphonomethoxy)propyl]adenine triethylammonium Salt (12b). Compound 12b was obtained as a white foam (148 mg, 70%) according to the procedure used for the preparation of 8a, starting from compound 11b (250 mg, 0.67 mmol), bromotrimethylsilane (0.37 mL, 2.77 mmol), and 2,6-lutidine (0.64 mL, 5.54 mmol) in anhydrous acetonitrile (10 mL). The crude residue was purified by column chromatography on silica gel (gradient acetone/H2O/Et3N, 6:1:1, v/v/v). 1H NMR (300 MHz, D2O): δ 8.01 (d, J = 1.0 Hz, 1H, H-2), 7.89 (d, J = 1.0 Hz, 1H, H-8), 4.71−4.18 (m, 4H, H-3′, H-1′), 3.96−3.85 (m, 1H, H-2′), 3.62−3.40 (m, 2H, PCH2). 13 C NMR (75 MHz, D2O): δ 154.6 (C-6), 151.6 (C-2), 148.2 (C-4), 6232



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2H, PCH2), 1.36−1.30 (m, 6H, 2 × CH2CH3). 13C NMR (75 MHz, CDCl3): δ 166.5 (C-4), 156.8 (C-2), 146.8 (C-6), 94.5 (C-5), 82.7 (d, 1 JC,F = 172.9 Hz, C-3′), 78.8 (dd, 2JC,F = 18.6 Hz, 3JC,P = 10.6 Hz, C2′), 64.7 (d, 1JC,P = 167.1 Hz, CH2P), 62.7, 62.6 (2 × d, 2JC,P = 6.4 Hz, CH2CH3), 50.1 (d, 3JC,F = 8.4 Hz, C-1′), 16.6, 16.5 (CH2CH3). 31P NMR (121 MHz, CDCl3): δ 20.9. HRMS for C12H21FN3O5P [M + H]+ calcd.: 338.1275; found, 338.1278. (S)-1-[3-Fluoro-2-(phosphonomethoxy)propyl]cytosine triethylammonium Salt (16a). Compound 16a was obtained as a white foam (58 mg, 70%) according to the procedure used for the preparation of 8a, starting from compound 15a (100 mg, 0.30 mmol), bromotrimethylsilane (0.16 mL, 1.19 mmol), and 2,6-lutidine (0.27 mL, 2.37 mmol) in anhydrous acetonitrile (5 mL). The crude residue was purified by column chromatography on silica gel (gradient acetone/H2O/Et3N, 6:1:1, v/v/v; acetone/H2O/Et3N, 5:1:1, v/v/v). 1 H NMR (600 MHz, D2O): δ 7.75−7.74 (m, 1H, H-6), 6.07−6.06 (m, 1H, H-5), 4.80−4.48 (m, 1H, H-3′, overlapped with H2O), 4.14 (dd, J = 14.1, 3.4 Hz, 1H, H-1′a), 3.98−3.88 (m, 2H, H-2′, H-1′b), 3.77 (dd, J = 13.1, 9.1 Hz, 1H, PCH2a), 3.59 (dd, J = 13.1, 9.6 Hz, 1H, PCH2b). 13 C NMR (75 MHz, D2O): δ 162.4 (C-4), 153.0 (C-2), 149.0 (C-6), 94.5 (C-5), 82.0 (d, 1JC,F = 167.9 Hz, C-3′), 77.4 (dd, 2JC,F = 18.4 Hz, 3 JC,P = 11.5 Hz, C-2′), 66.1 (d, 1JC,P = 157.2 Hz, CH2P), 49.1 (d, 3JC,F = 7.9 Hz, C-1′). 31P NMR (121 MHz, D2O): δ 14.9. HRMS for C8H13FN3O5P [M − H]− calcd.: 280.0504; found, 280.0501. (R)-1-[3-Fluoro-2-(phosphonomethoxy)propyl]cytosine triethylammonium Salt (16b). Compound 16b was obtained as a white foam (82 mg, 70%) according to the procedure used for the preparation of 8a, starting from compound 15b (140 mg, 0.42 mmol), bromotrimethylsilane (0.22 mL, 1.66 mmol), and 2,6-lutidine (0.38 mL, 3.32 mmol) in anhydrous acetonitrile (5 mL). The crude residue was purified by column chromatography on silica gel (gradient acetone/H2O/Et3N, 6:1:1, v/v/v; acetone/H2O/Et3N, 5:1:1, v/v/v). 1 H NMR (300 MHz, D2O): δ 7.72 (d, J = 7.6 Hz, 1H, H-6), 6.05 (d, J = 7.5 Hz, 1H, H-5), 4.82−4.45 (m, 1H, H-3′, overlapped with H2O), 4.18−4.12 (m, 1H, H-1′a), 4.02−3.75 (m, 3H, H-2′, H-1′b, PCH2a), 3.64−3.57 (m, 1H, PCH2b). 13C NMR (75 MHz, D2O): δ 163.9 (C4), 155.0 (C-2), 148.4 (C-6), 94.7 (C-5), 82.1 (d, 1JC,F = 167.7 Hz, C3′), 77.6 (dd, 2JC,F = 18.3 Hz, 3JC,P = 11.6 Hz, C-2′), 66.2 (d, 1JC,P = 157.1 Hz, CH2P), 49.2 (d, 3JC,F = 7.9 Hz, C-1′). 31P NMR (121 MHz, D2O): δ 14.9. HRMS for C8H13FN3O5P [M − H]− calcd.: 280.0504; found, 280.0507. (S)-9-{3-Fluoro-2-[(diethylphosphoryl)methoxy]propyl}-O6-benzylguanine (18a). Compound 18a was obtained as a colorless oil (290 mg, 50%) according to the procedure used for the preparation of 6a, starting from compound 4a (300 mg, 1.23 mmol), compound 17 (360 mg, 1.47 mmol), Ph3P (650 mg, 2.46 mmol), and DIAD (0.48 mL, 2.46 mmol) in anhydrous THF (8 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/ MeOH, 40:1, v/v; DCM/MeOH, 30:1, v/v). 1H NMR (300 MHz, CDCl3): δ 7.70 (s, 1H, H-8), 7.51−7.29 (m, 5H, ArH), 5.56 (s, 2H, CH2-Ar), 5.01 (s, 2H, NH2), 4.69−4.28 (m, 3H, H-3′, H-1′a), 4.20− 4.01 (m, 6H, H-1′b, H-2′, 2 × CH2CH3), 3.95−3.77 (m, 2H, PCH2), 1.34−1.23 (m, 6H, 2 × CH2CH3). 13C NMR (75 MHz, CDCl3): δ 161.1 (C-6), 159.4 (C-2), 154.2 (C-4), 140.3 (C-8), 136.5 (Ar-C), 128.4 (Ar-C), 128.3 (Ar-C), 128.0 (Ar-C), 115.4 (C-5), 82.1 (d, 1JC,F = 173.5 Hz, C-3′), 78.6 (dd, 2JC,F = 19.3 Hz, 3JC,P = 10.3 Hz, C-2′), 68.1 (CH2−Ar), 64.6 (d, 1JC,P = 166.9 Hz, CH2P), 62.7, 62.6 (2 × d, 2JC,P = 7.0 Hz, CH2CH3), 44.3 (d, 3JC,F = 7.9 Hz, C-1′), 16.5, 16.4 (CH2CH3). 31 P NMR (121 MHz, CDCl3): δ 19.9. HRMS for C20H27FN5O5P [M + H]+ calcd.: 468.1806; found, 468.1801. (R)-9-{3-Fluoro-2-[(diethylphosphoryl)methoxy]propyl}-O6-benzylguanine (18b). Compound 18b was obtained as a colorless oil (250 mg, 44%) according to the procedure used for the preparation of 6a, starting from compound 4b (300 mg, 1.23 mmol), compound 17 (360 mg, 1.47 mmol), Ph3P (650 mg, 2.46 mmol), and DIAD (0.48 mL, 2.46 mmol) in anhydrous THF (8 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/ MeOH, 40:1, v/v; DCM/MeOH, 30:1, v/v). 1H NMR (300 MHz, CDCl3): δ 7.70 (s, 1H, H-8), 7.52−7.28 (m, 5H, ArH), 5.57 (s, 2H, CH2-Ar), 4.94 (s, 2H, NH2), 4.64−4.28 (m, 3H, H-3′, H-1′a), 4.21−

142.6 (C-8), 117.3 (C-5), 81.7 (d, 1JC,F = 168.1 Hz, C-3′), 77.3 (dd, 2 JC,F = 18.9 Hz, 3JC,P = 11.4 Hz, C-2′), 66.4 (d, 1JC,P = 155.5 Hz, CH2P), 43.0 (d, 3JC,F = 7.4 Hz, C-1′). 31P NMR (121 MHz, D2O): δ 14.2. HRMS for C9H13FN5O4P [M − H]− calcd.: 304.0616; found, 304.0609. (S)-1-{3-Fluoro-2-[(diethylphosphoryl)methoxy]propyl}-N4-isobutyrylcytosine (14a). Compound 14a was obtained as a colorless oil (130 mg, 40%) according to the procedure used for the preparation of 6a, starting from compound 4a (200 mg, 0.82 mmol), compound 1326 (180 mg, 0.98 mmol), Ph3P (430 mg, 1.63 mmol), and DIAD (0.33 mL, 1.63 mmol) in anhydrous THF (6 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/ MeOH, 30:1, v/v; DCM/MeOH, 25:1, v/v). 1H NMR (300 MHz, CDCl3): δ 9.14 (s, 1H, NHCO), 7.71 (d, J = 7.3 Hz, 1H, H-6), 7.41 (d, J = 7.3 Hz, 1H, H-5), 4.82−4.31 (m, 3H, H-3′, H-1′a), 4.22−3.95 (m, 7H, H-1′b, H-2′, 2 × CH2CH3, PCH2a), 3.70 (dd, J = 13.6, 8.3 Hz, 1H, PCH2b), 2.72−2.63 (m, 1H, CH(CH3)2), 1.36−1.20 (m, 12H, 2 × CH2CH3, CH(CH3)2). 13C NMR (75 MHz, CDCl3): δ 177.4 (CONH), 163.0 (C-4), 156.1 (C-2), 150.4 (C-6), 96.3 (C-5), 82.3 (d, 1JC,F = 173.4 Hz, C-3′), 78.3 (dd, 2JC,F = 18.3 Hz, 3JC,P = 11.9 Hz, C-2′), 64.4 (d, 1JC,P = 166.9 Hz, CH2P), 62.7, 62.5 (2 × d, 2JC,P = 6.6 Hz, CH2CH3), 51.0 (d, 3JC,F = 8.7 Hz, C-1′), 36.7 (CH(CH3)2), 19.3, 19.1 (CH(CH3)2), 16.6, 16.5 (CH2CH3). 31P NMR (121 MHz, CDCl3): δ 20.7. HRMS for C16H27FN3O6P [M + H]+ calcd.: 408.1694; found, 408.1690. (R)-1-{3-Fluoro-2-[(diethylphosphoryl)methoxy]propyl}-N4-isobutyrylcytosine (14b). Compound 14b was obtained as a colorless oil (260 mg, 40%) according to the procedure used for the preparation of 6a, starting from compound 4a (400 mg, 1.64 mmol), compound 1326 (360 mg, 1.96 mmol), Ph3P (860 mg, 3.26 mmol), and DIAD (0.66 mL, 3.26 mmol) in anhydrous THF (10 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/ MeOH, 30:1, v/v; DCM/MeOH, 25:1, v/v). 1H NMR (300 MHz, CDCl3): δ 8.89 (s, 1H, NHCO), 7.71 (d, J = 7.3 Hz, 1H, H-6), 7.40 (d, J = 7.4 Hz, 1H, H-5), 4.82−4.31 (m, 3H, H-3′, H-1′a), 4.24−3.95 (m, 7H, H-1′b, H-2′, 2 × CH2CH3, PCH2a), 3.70 (dd, J = 13.4, 8.2 Hz, 1H, PCH2b), 2.69−2.60 (m, 1H, CH(CH3)2), 1.35−1.20 (m, 12H, 2 × CH2CH3, CH(CH3)2). 13C NMR (75 MHz, CDCl3): δ 177.2 (CONH), 162.9 (C-4), 156.1 (C-2), 150.4 (C-6), 96.3 (C-5), 82.3 (d, 1JC,F = 173.3 Hz, C-3′), 78.3 (dd, 2JC,F = 18.4 Hz, 3JC,P = 11.8 Hz, C-2′), 64.4 (d, 1JC,P = 166.9 Hz, CH2P), 62.7, 62.5 (2 × d, 2JC,P = 6.6 Hz, CH2CH3), 51.0 (d, 3JC,F = 9.1 Hz, C-1′), 36.7 (CH(CH3)2), 19.3, 19.1 (CH(CH3)2), 16.6, 16.5 (CH2CH3). 31P NMR (121 MHz, CDCl3): δ 21.4. HRMS for C16H27FN3O6P [M + H]+ calcd.: 408.1694; found, 408.1691. (S)-1-{3-Fluoro-2-[(diethylphosphoryl)methoxy]propyl}cytosine (15a). A solution of 14a (120 mg, 0.31 mmol) in 30% methanolic ammonia (20 mL) was stirred at 45 °C for 15 h. After the removal of all of the volatiles, the residue was purified by column chromatography on silica gel (gradient DCM/MeOH, 10:1, v/v; DCM/MeOH, 8:1, v/ v) to give 15a (90 mg, 90%) as a colorless foam. 1H NMR (300 MHz, CDCl3): δ 7.40 (d, J = 7.2 Hz, 1H, H-6), 5.92 (d, J = 7.2 Hz, 1H, H-5), 4.77−4.35 (m, 2H, H-3′), 4.18−3.95 (m, 7H, H-1′, H-2′, 2 × CH2CH3), 3.83 (dd, J = 13.8, 9.0 Hz, 1H, PCH2a), 3.70 (dd, J = 13.4, 7.2 Hz, 1H, PCH2b), 1.36−1.30 (m, 6H, 2 × CH2CH3). 13C NMR (75 MHz, CDCl3): δ 165.8 (C-4), 156.3 (C-2), 147.1 (C-6), 94.7 (C-5), 82.6 (d, 1JC,F = 172.9 Hz, C-3′), 78.7 (dd, 2JC,F = 18.4 Hz, 3JC,P = 10.8 Hz, C-2′), 64.6 (d, 1JC,P = 167.6 Hz, CH2P), 62.8, 62.6 (2 × d, 2JC,P = 6.6 Hz, CH2CH3), 50.1 (d, 3JC,F = 8.7 Hz, C-1′), 16.6, 16.5 (CH2CH3). 31 P NMR (121 MHz, CDCl3): δ 20.4. HRMS for C12H21FN3O5P [M − H]+ calcd.: 338.1275; found, 338.1280. (R)-1-{3-Fluoro-2-[(diethylphosphoryl)methoxy]propyl}cytosine (15b). Compound 15b was obtained as a white foam (140 mg, 82%) according to the procedure used for the preparation of 15a, starting from compound 14b (200 mg, 0.49 mmol) in 30% methanolic ammonia (20 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/MeOH, 10:1, v/v; DCM/MeOH, 8:1, v/v). 1H NMR (300 MHz, CDCl3): δ 7.35 (d, J = 7.2 Hz, 1H, H-6), 5.81 (m, d, J = 7.2 Hz, 1H, H-5), 4.76−4.34 (m, 2H, H-3′), 4.18−3.93 (m, 7H, H-1′, H-2′, 2 × CH2CH3), 3.87−3.66 (m, 6233



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4.00 (m, 6H, H-1′b, H-2′, 2 × CH2CH3), 3.95−3.77 (m, 2H, PCH2), 1.34−1.24 (m, 6H, 2 × CH2CH3). 13C NMR (75 MHz, CDCl3): δ 161.2 (C-6), 159.4 (C-2), 154.3 (C-4), 140.4 (C-8), 136.5 (Ar-C), 128.4 (d, J = 8.2 Hz, Ar-C), 128.1 (Ar-C), 115.4 (C-5), 82.2 (d, 1JC,F = 173.5 Hz, C-3′), 78.6 (dd, 2JC,F = 19.3 Hz, 3JC,P = 10.1 Hz, C-2′), 68.2 (CH2−Ar), 64.7 (d, 1JC,P = 167.0 Hz, CH2P), 62.7, 62.6 (2 × d, 2JC,P = 7.0 Hz, CH2CH3), 44.3 (d, 3JC,F = 8.1 Hz, C-1′), 16.5, 16.4 (2 × d, 3 JC,P = 2.9 Hz, CH2CH3). 31P NMR (121 MHz, CDCl3): δ 20.5. HRMS for C20H27FN5O5P [M + H]+ calcd.: 468.1806; found, 468.1811. (S)-9-{3-Fluoro-2-[(diethylphosphoryl)methoxy]propyl}guanine (19a). Compound 18a (300 mg, 0.64 mmol) and Pd/C on charcoal (10% w/w, 150 mg) were suspended in EtOAc (20 mL), and the solution was purged first with nitrogen, then hydrogen, and allowed to stir under a hydrogen atmosphere. After 10 h, the mixture was filtered through a pad of Celite, and the filtrate was evaporated under reduced pressure to give a crude product. The residue was then purified by column chromatography on silica gel (gradient DCM/MeOH, 10:1, v/ v; 7:1, v/v) to give 19a (170 mg, 70%) as a colorless foam. 1H NMR (300 MHz, MeOD): δ 7.74 (s, 1H, H-8), 4.77−4.37 (m, 2H, H-3′), 4.33−3.85 (m, 9H, H-1′, H-2′, 2 × CH2CH3, PCH2), 1.31−1.23 (m, 6H, 2 × CH2CH3). 13C NMR (75 MHz, MeOD): δ 159.5 (C-6), 155.3 (C-2), 153.4 (C-4), 140.4 (C-8), 117.3 (C-5), 83.2 (d, 1JC,F = 171.7 Hz, C-3′), 79.9 (dd, 2JC,F = 19.1 Hz, 3JC,P = 11.9 Hz, C-2′), 64.5 (d, 1JC,P = 165.0 Hz, CH2P), 64.3, 64.1 (2 × d, 2JC,P = 6.6 Hz, CH2CH3), 44.3 (d, 3JC,F = 8.6 Hz, C-1′), 16.7, 16.6 (CH2CH3). 31P NMR (121 MHz, MeOD): δ 21.1. HRMS for C13H21FN5O5P [M + H]+ calcd.: 378.1337; found, 378.1329. (R)-9-{3-Fluoro-2-[(diethylphosphoryl)methoxy]propyl}guanine (19b). Compound 19b was obtained as a colorless foam (160 mg, 80%) according to the general procedure used for the preparation of 19a, starting from compound 18b (250 mg, 0.54 mmol) and Pd/C on charcoal (10% w/w, 120 mg) in EtOAc (30 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/ MeOH, 10:1, v/v; 7:1, v/v). 1H NMR (300 MHz, MeOD): δ 7.74 (s, 1H, H-8), 4.76−4.37 (m, 2H, H-3′), 4.33−3.85 (m, 9H, H-1′, H-2′, 2 × CH2CH3, PCH2), 1.31−1.23 (m, 6H, 2 × CH2CH3). 13C NMR (75 MHz, MeOD): δ 159.5 (C-6), 155.4 (C-2), 153.4 (C-4), 140.4 (C-8), 117.3 (C-5), 83.2 (d, 1JC,F = 171.6 Hz, C-3′), 79.9 (dd, 2JC,F = 19.0 Hz, 3 JC,P = 11.9 Hz, C-2′), 64.5 (d, 1JC,P = 165.0 Hz, CH2P), 64.3, 64.1 (2 × d, 2JC,P = 6.6 Hz, CH2CH3), 44.3 (d, 3JC,F = 8.6 Hz, C-1′), 16.7, 16.6 (CH2CH3). 31P NMR (121 MHz, MeOD): δ 21.1. HRMS for C13H21FN5O5P [M + H]+ calcd.: 378.1337; found, 378.1329. (S)-9-[3-Fluoro-2-(phosphonomethoxy)propyl]guanine triethylammonium Salt (20a). Compound 20a was obtained as a white foam (87 mg, 60%) according to the procedure used for the preparation of 8a, starting from compound 19a (170 mg, 0.45 mmol), bromotrimethylsilane (0.24 mL, 1.80 mmol), and 2,6-lutidine (0.42 mL, 3.60 mmol) in anhydrous acetonitrile (5 mL). The crude residue was purified by column chromatography on silica gel (gradient acetone/H2O/Et3N, 5:1:1, v/v/v; acetone/H2O/Et3N, 4:1:1, v/v/v). 1 H NMR (300 MHz, D2O): δ 7.74 (s, 1H, H-8), 4.79−4.59 (m, 4H, H-3′, H-1′), 3.97−3.88 (m, 1H, H-2′), 3.51−3.39 (m, 2H, PCH2). 13C NMR (75 MHz, D2O): δ 166.8 (C-6), 160.0 (C-2), 151.2 (C-4), 138.8 (C-8), 116.7 (C-5), 81.9 (d, 1JC,F = 167.4 Hz, C-3′), 77.2 (dd, 2JC,F = 18.7 Hz, 3JC,P = 10.6 Hz, C-2′), 68.0 (d, 1JC,P = 150.0 Hz, CH2P), 42.2 (d, 3JC,F = 7.1 Hz, C-1′). 31P NMR (121 MHz, D2O): δ 13.6. HRMS for C9H13FN5O5P [M − H]− calcd.: 320.0565; found, 320.0565. (R)-9-[3-Fluoro-2-(phosphonomethoxy)propyl]guanine triethylammonium Salt (20b). Compound 20b was obtained as a white foam (87 mg, 60%) according to the procedure used for the preparation of 8a, starting from compound 19b (160 mg, 0.42 mmol), bromotrimethylsilane (0.22 mL, 1.70 mmol), and 2,6-lutidine (0.39 mL, 3.40 mmol) in anhydrous acetonitrile (5 mL). The crude residue was purified by column chromatography on silica gel (gradient acetone/H2O/Et3N, 5:1:1, v/v/v; acetone/H2O/Et3N, 4:1:1, v/v/v). 1 H NMR (300 MHz, D2O): δ 7.76 (s, 1H, H-8), 4.70−4.29 (m, 2H, H-3′), 4.25−4.09 (m, 2H, H-1′), 4.02−3.88 (m, 1H, H-2′), 3.65−3.47 (m, 2H, PCH2). 13C NMR (75 MHz, D2O): δ 158.4 (C-6), 153.3 (C2), 151.2 (C-4), 140.3 (C-8), 115.2 (C-5), 81.9 (d, 1JC,F = 167.9 Hz,

C-3′), 77.5 (dd, 2JC,F = 18.7 Hz, 3JC,P = 11.3 Hz, C-2′), 66.4 (d, 1JC,P = 156.0 Hz, CH2P), 42.7 (d, 1JC,F = 7.6 Hz, C-1′). 31P NMR (121 MHz, D2O): δ 14.5. HRMS for C9H13FN5O5P [M − H]− calcd.: 320.0565; found, 320.0571. General Procedure for the Synthesis of Phosphonamidates. The relevant phosphonic acid (1 equiv) was mixed with L-aspartic acid amyl diester HCl salt13 (1.7 equiv) and phenol (4.4 equiv) in anhydrous pyridine. Then Et3N (10 equiv) was added, and the mixture was stirred at 60 °C under a nitrogen atmosphere for 15−20 min. 2,2′Dithiodipyridine (7 equiv) was mixed in a separate flask with PPh3 (7 equiv) in anhydrous pyridine, and the resultant mixture was stirred for 10−15 min to give a clear light yellow solution. This solution was then added to the above solution, and the combined mixture was stirred at 60 °C for 12 h. The mixture was then concentrated under reduced pressure to give a residue that was redissolved in EtOAc. This solution was washed with saturated aq. NaHCO3 and brine, the organic layer was separated, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography, followed by RP-HPLC (linear gradient, 40−95% CH3CN in water) to give the desired phosphonamidate in pure form as a mixture of P (S) and P (R) isomers. (S)-1-{3-Fluoro-2-[phenyloxy-bis(amyl-L-aspartyl)phosphoryl]methoxy]propyl}thymine (21a). Compound 21a was obtained as a colorless oil (45 mg, 45%) according to the general procedure, starting from compound 8a (50 mg, 0.17 mmol), L-aspartic acid amyl diester HCl salt (90 mg, 0.29 mmol), PhOH (70 mg, 0.75 mmol), Et3N (0.24 mL, 1.7 mmol), 2,2′-dithiodipyridine (260 mg, 1.20 mmol), and PPh3 (310 mg, 1.20 mmol) in anhydrous pyridine (5 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/MeOH, 60:1, v/v; 50:1, v/v). 1H NMR (300 MHz, CD3CN): δ 9.35 (s, 1H, NHCO), 7.36−7.13 (m, 6H, H-6, ArH), 4.72−3.65 (m, 13H, H-3′, H-2′, H-1′, 2 × OCH2(CH2)3CH3, PCH2, H-α-Asp, NHPO), 2.77−2.49 (m, 2H, H-β-Asp), 1.74 (d, J = 1.2 Hz, 3H, CH35), 1.71 (d, J = 1.2 Hz, 3H, CH3-5), 1.60−1.50 (m, 4H, 2 × OCH2CH2(CH2)2CH3), 1.35−1.26 (m, 8H, 2 × O(CH2)2(CH2)2CH3), 0.91−0.85 (m, 6H, 2 × O(CH2)4CH3). 13C NMR (75 MHz, CD3CN): δ 172.9 (d, 3JC,P = 4.7 Hz, CO-α), 172.6 (d, 3 JC,P = 4.6 Hz, CO-α), 171.5 (CO-β), 165.3 (C-4), 152.3, 152.2 (C-2), 151.3 (Ar-C), 143.0 (C-6), 130.7 (Ar-C), 125.9, 125.8 (Ar-C), 121.9 (d, 3JC,P = 4.3 Hz, Ar-C), 121.7 (d, 3JC,P = 4.5 Hz, Ar-C), 110.4, 110.3 (C-5), 83.5 (d, 1JC,F = 172.5 Hz, C-3′), 83.2 (d, 1JC,F = 165.0 Hz, C3′), 79.9, 79.8, 79.7, 79.6, 79.5, 79.4, 79.2 (C-2′), 66.9 (d, 1JC,P = 157.5 Hz, CH2P), 66.5, 66.4, 65.8, 65.7 (OCH2(CH2)3CH3), 51.5, 51.4 (C1′), 49.0, 48.8 (C-α-Asp), 39.8 (d, 3JC,P = 4.0 Hz, C-β-Asp), 39.6 (d, 3 JC,P = 4.1 Hz, C-β-Asp), 29.0, 28.9, 28.7, 28.6 (OCH2(CH2)2CH2CH3), 23.0, 22.9 (O(CH2)3CH2CH3), 14.3 (O(CH2)4CH3), 12.3 (CH3-5). 31P NMR (121 MHz, CD3CN): δ 22.3, 21.3. HRMS for C29H43FN3O9P [M + Na]+ calcd.: 650.2613; found, 650.2629. (R)-1-{3-Fluoro-2-[phenyloxy-bis(amyl-L-aspartyl)phosphoryl]methoxy]propyl}thymine (21b). Compound 21b was obtained as a colorless oil (80 mg, 50%) according to the general procedure, starting from compound 8b (80 mg, 0.27 mmol), L-aspartic acid amyl diester HCl salt (142 mg, 0.46 mmol), PhOH (112 mg, 1.20 mmol), Et3N (0.38 mL, 2.70 mmol), 2,2′-dithiodipyridine (420 mg, 1.90 mmol), and PPh3 (500 mg, 1.90 mmol) in anhydrous pyridine (5 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/MeOH, 60:1, v/v; 50:1, v/v). 1H NMR (300 MHz, CD3CN): δ 9.59 (s, 1H, NHCO), 7.39−7.13 (m, 6H, H-6, ArH), 4.77−3.71 (m, 13H, H-3′, H-2′, H-1′, 2 × OCH2(CH2)3CH3, PCH2, H-α-Asp, NHPO), 2.82−2.53 (m, 2H, H-β-Asp), 1.79 (s, 3H, CH3-5), 1.73 (s, 3H, CH3-5), 1.64−1.55 (m, 4H, 2 × OCH2CH2(CH2)2CH3), 1.41−1.28 (m, 8H, 2 × O(CH2)2(CH2)2CH3), 0.97−0.88 (m, 6H, 2 × O(CH2)4CH3). 13C NMR (75 MHz, CD3CN): δ 172.8 (d, 3JC,P = 5.0 Hz, CO-α), 172.6 (d, 3JC,P = 4.5 Hz, CO-α), 171.6, 171.5 (CO-β), 165.4 (C-4), 152.3 (C-2), 151.4, 151.3 (Ar-C), 143.1, 143.0 (C-6), 130.7, 130.6 (Ar-C), 125.8 (Ar-C), 121.8 (d, 3JC,P = 4.0 Hz, Ar-C), 121.7 (d, 3JC,P = 4.4 Hz, Ar-C), 110.4, 110.3 (C-5), 83.4 (d, J = 172.5 Hz, C-3′), 82.2 (d, 1JC,F = 165.0 Hz, C-3′), 79.8, 79.6, 79.5, 79.4, 79.2 (C-2′), 66.9 (d, 1JC,P = 157.5 Hz, CH2P), 66.5, 66.4, 65.7 6234



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DCM/MeOH, 20:1, v/v; 10:1, v/v). 1H NMR (300 MHz, CD3CN): δ 7.45−7.16 (m, 6H, ArH, H-6), 5.95 (s, 2H, NH2), 5.70, 5.68 (s, 1H, H-5), 4.75−3.66 (m, 13H, H-3′, H-2′, H-1′, 2 × OCH2(CH2)3CH3, PCH2, H-α-Asp, NHPO), 2.82−2.56 (m, 2H, H-β-Asp), 1.63−1.55 (m, 4H, 2 × OCH2CH2(CH2)2CH3), 1.35−1.27 (m, 8H, 2 × O(CH2)2(CH2)2CH3), 0.94−0.88 (m, 6H, O(CH2)4CH3). 13C NMR (75 MHz, CD3CN): δ 172.9 (d, 3JC,P = 4.7 Hz, CO-α), 172.7 (d, 3JC,P = 4.5 Hz, CO-α), 171.5 (CO-β), 167.5 (C-4), 157.5, 157.4 (C-2), 151.5, 151.4, 151.3, 151.2 (Ar−C), 148.2 (C-6), 130.7, 130.6 (Ar-C), 125.8, 125.7 (Ar-C), 122.0 (d, 3JC,P = 4.4 Hz, Ar-C), 121.8 (d, 3JC,P = 4.4 Hz, Ar-C), 94.2, 94.1 (C-5), 83.8 (d, 1JC,F = 172.5 Hz, C-3′), 83.4 (d, 1JC,F = 165.0 Hz, C-3′), 80.0, 79.8, 79.7, 79.6, 79.5, 79.4, 79.3 (C2′), 66.9 (d, J = 150.0 Hz, CH2P), 66.8 (d, 1JC,P = 150.0 Hz, CH2P), 66.5, 66.4, 65.8, 65.7 (OCH2(CH2)3CH3), 51.6, 51.5 (C-α-Asp), 50.6, 50.5, 50.4 (C-1′), 39.8 (d, 3JC,P = 4.0 Hz, C-β-Asp), 39.5 (d, 3JC,P = 4.2 Hz, C-β-Asp), 29.0, 28.9, 28.8, 28.7 (OCH2(CH2)2CH2CH3), 23.0, 22.9 (O(CH2)3CH2CH3), 14.3 (O(CH2)4CH3). 31P NMR (121 MHz, CD3CN): δ 22.4, 21.4. HRMS for C28H42FN4O8P [M + H]+ calcd.: 613.2797; found, 613.2802. (R)-1-{3-Fluoro-2-[phenyloxy-bis(amyl-L-aspartyl)phosphoryl]methoxy]propyl}cytosine (23b). Compound 23b was obtained as a colorless oil (20 mg, 20%) according to the general procedure, starting from compound 16b (40 mg, 0.14 mmol), L-aspartic acid amyl diester HCl salt (75 mg, 0.24 mmol), PhOH (59 mg, 0.63 mmol), Et3N (0.20 mL, 1.40 mmol), 2,2′-dithiodipyridine (220 mg, 1.00 mmol), and PPh3 (260 mg, 1.00 mmol) in anhydrous pyridine (3 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/MeOH, 20:1, v/v; 10:1, v/v). 1H NMR (300 MHz, CD3CN): δ 7.45−7.13 (m, 6H, ArH, H-6), 5.74−5.69 (m, 1H, H-5), 4.78−3.70 (m, 13H, H-3′, H-2′, H-1′, 2 × OCH2(CH2)3CH3, PCH2, H-α-Asp, NHPO), 2.82−2.56 (m, 2H, H-β-Asp), 1.64−1.52 (m, 4H, 2 × OCH2CH2(CH2)2CH3), 1.40−1.24 (m, 8H, 2 × O(CH2)2(CH2)2CH3), 0.93−0.87 (m, 6H, 2 × O(CH2)4CH3). 13C NMR (75 MHz, CD3CN): δ 172.8 (d, 3JC,P = 5.1 Hz, CO-α), 172.7 (d, 3 JC,P = 4.4 Hz, CO-α), 171.6, 171.5 (CO-β), 167.5 (C-4), 157.5, 157.4 (C-2), 151.5, 151.3, 151.2 (Ar−C), 148.2, 148.1 (C-6), 130.6 (Ar-C), 125.8, 125.7 (Ar-C), 121.9 (d, 3JC,P = 4.4 Hz, Ar-C), 121.8 (d, 3JC,P = 4.4 Hz, Ar-C), 94.3, 94.2 (C-5), 88.7 (d, 1JC,F = 172.5 Hz, C-3′), 83.5 (d, 1JC,F = 165 Hz, C-3′), 79.8, 79.7, 79.6, 79.4, 79.3 (C-2′), 66.9 (d, 1 JC,P = 157.5 Hz, CH2P), 66.7 (d, 1JC,P = 150.0 Hz, CH2P), 66.5, 66.4, 65.7 (OCH2(CH2)3CH3), 51.5 (C-α-Asp), 50.5 (d, 2JC,P = 8.3 Hz, C1′), 50.4 (d, 2JC,P = 8.3 Hz, C-1′), 39.8 (d, 3JC,P = 4.2 Hz, C-β-Asp), 39.6 (d, 3 J C,P = 3.8 Hz, C-β-Asp), 29.0, 28.9, 28.8, 28.7 (OCH2(CH2)2CH2CH3), 23.0, 22.9 (O(CH2)3CH2CH3), 14.3 (O(CH2)4CH3). 31P NMR (121 MHz, CD3CN): δ 22.4, 21.7. HRMS for C28H42FN4O8P [M − H]− calcd.: 611.2651; found, 611.2655. (S)-9-{3-Fluoro-2-[phenyloxy-bis(amyl-L-aspartyl)phosphoryl]methoxy]propyl}guanine (25a). According to the general procedure, starting from compound 20a (30 mg, 0.09 mmol), L-aspartic acid amyl diester HCl salt (50 mg, 0.16 mmol), PhOH (40 mg, 0.41 mmol), Et3N (0.13 mL, 0.90 mmol), 2,2′-dithiodipyridine (145 mg, 0.65 mmol), and PPh3 (170 mg, 0.65 mmol) in anhydrous pyridine (3 mL), compound 25a was obtained. The crude residue was purified by column chromatography on silica gel (gradient DCM/MeOH, 40:1, v/ v; 30:1, v/v) to give the triphosphine adduct 24a. The collected eluate was evaporated and redissolved in CH3CN/H2O (1:1, v/v, 10 mL). One drop of a 1 M HCl solution was added at 0 °C, and then, the mixture was stirred for another 1 h at room temperature. The solution was neutralized with 2 M TEAB. After removal of all the volatiles, the resulting residue was purified by RP-HPLC (CH3CN, H2O) to give compound 25a (3 mg, 5%) as a colorless oil. 1H NMR (300 MHz, CD3CN): δ 7.64, 7.61 (s, 1H, H-8), 7.38−7.13 (m, 5H, ArH), 4.74− 3.92 (m, 13H, H-3′, H-2′, H-1′, 2 × OCH2(CH2)3CH3, PCH2, H-αAsp, NHPO), 2.82−2.54 (m, 2H, H-β-Asp), 1.61−1.52 (m, 4H, 2 × OCH2CH2(CH2)2CH3), 1.33−1.26 (m, 8H, 2 × O(CH2)2(CH2)2CH3), 0.92−0.85 (m, 6H, O(CH2)4CH3). 13C NMR (75 MHz, CD3CN): δ 172.8, 172.7 (CO-α), 171.6, 171.5 (CO-β), 154.7, 154.6 (C-2), 152.8 (C-4), 151.2 (Ar−C), 139.3 (C-8), 130.6 (Ar-C), 125.9, 125.8 (Ar-C), 121.9 (d, 3JC,P = 4.3 Hz, Ar-C), 121.7 (d, 3 JC,P = 4.4 Hz, Ar-C), 118.3 (C-5, overlapped with CD3CN), 83.5 (d,

(OCH2(CH2)3CH3), 51.5 (C-1′), 49.0 (d, 2JC,P = 8.3 Hz, C-α-Asp), 48.8 (d, 2JC,P = 8.0 Hz, C-α-Asp), 39.7 (d, 3JC,P = 4.0 Hz, C-β-Asp), 39.6 (d, 3 J C,P = 4.6 Hz, C-β-Asp), 29.0, 28.9, 28.7, 28.6 (OCH2(CH2)2CH2CH3), 23.0, 22.9 (O(CH2)3CH2CH3), 14.3 (O(CH2)4CH3), 12.3 (CH3-5). 31P NMR (121 MHz, CD3CN): δ 22.2, 21.5. HRMS for C29H43FN3O9P [M − H]− calcd.: 626.2648; found, 626.2648. (S)-9-{3-Fluoro-2-[phenyloxy-bis(amyl-L-aspartyl)phosphoryl]methoxy]propyl}adenine (22a). Compound 22a was obtained as a colorless oil (26 mg, 40%) according to the general procedure, starting from compound 12a (30 mg, 0.10 mmol), L-aspartic acid amyl diester HCl salt (52 mg, 0.17 mmol), PhOH (40 mg, 0.43 mmol), Et3N (0.14 mL, 1.00 mmol), 2,2′-dithiodipyridine (150 mg, 0.69 mmol), and PPh3 (180 mg, 0.69 mmol) in anhydrous pyridine (3 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/MeOH, 25:1, v/v; 20:1, v/v). 1H NMR (300 MHz, CD3CN): δ 8.25, 8.22 (s, 1H, H-2), 7.99, 7.97 (s, 1H, H-8), 7.37−7.05 (m, 6H, ArH), 6.02 (s, 2H, NH2), 4.75−3.83 (m, 13H, H-3′, H-2′, H-1′, 2 × OCH2(CH2)3CH3, PCH2, H-α-Asp, NHPO), 2.79−2.53 (m, 2H, H-βAsp), 1.61−1.53 (m, 4H, 2 × OCH2CH2(CH2)2CH3), 1.36−1.28 (m, 8H, 2 × O(CH2)2(CH2)2CH3), 0.93−0.87 (m, 6H, O(CH2)4CH3). 13 C NMR (75 MHz, CD3CN): δ 172.9 (d, 3JC,P = 4.4 Hz, CO-α), 172.6 (d, 3JC,P = 4.7 Hz, CO-α), 171.5, 171.4 (CO-β), 156.9 (C-6), 153.8, 153.7 (C-2), 151.4, 151.2, 151.1 (Ar−C, C-4), 142.9, 142.8 (C8), 130.6 (Ar-C), 125.8, 125.7 (Ar-C), 121.9 (d, 3JC,P = 4.4 Hz, Ar-C), 121.7 (d, 3JC,P = 4.4 Hz, Ar-C), 120.1, 120.0 (C-5), 83.3 (d, 1JC,F = 165.0 Hz, C-3′), 83.1 (d, 1JC,F = 172.5 Hz, C-3′), 79.9, 79.8, 79.7, 79.5, 79.4, 79.3 (C-2′), 67.0 (d, 1JC,P = 157.5 Hz, CH2P), 66.7 (d, 1JC,P = 157.5 Hz, CH2P), 66.5, 66.4, 65.8, 65.7 (OCH2(CH2)3CH3), 51.6, 51.5 (C-α-Asp), 44.3 (d, 2JC,P = 2.4 Hz, C-1′), 44.2 (d, 2JC,P = 2.4 Hz, C-1′), 39.8 (d, 3JC,P = 3.9 Hz, C-β-Asp), 39.6 (d, 3JC,P = 4.2 Hz, C-β-Asp), 29.0, 28.9, 28.7, 28.6 (OCH2(CH2)2CH2CH3), 23.0, 22.9 (O(CH2)3CH2CH3), 14.3 (O(CH2)4CH3). 31P NMR (121 MHz, CD3CN): δ 22.2, 21.2. HRMS for C29H42FN6O7P [M + H]+ calcd.: 637.2909; found, 637.2913. (R)-9-{3-Fluoro-2-[phenyloxy-bis(amyl-L-aspartyl)phosphoryl]methoxy]propyl}adenine (22b). Compound 22b was obtained as a colorless oil (110 mg, 55%) according to the general procedure, starting from compound 12b (80 mg, 0.26 mmol), L-aspartic acid amyl diester HCl salt (140 mg, 0.45 mmol), PhOH (110 mg, 1.15 mmol), Et3N (0.36 mL, 2.60 mmol), 2,2′-dithiodipyridine (400 mg, 1.83 mmol), and PPh3 (480 mg, 1.83 mmol) in anhydrous pyridine (5 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/MeOH, 25:1, v/v; 20:1, v/v). 1H NMR (300 MHz, CD3CN): δ 8.26 (s, 1H, H-2), 8.05, 8.03 (s, 1H, H-8), 7.35− 7.01 (m, 5H, ArH), 6.02 (s, 2H, NH2), 4.87−3.90 (m, 13H, H-3′, H2′, H-1′, 2 × OCH2(CH2)3CH3, PCH2, H-α-Asp, NHPO), 2.82−2.56 (m, 2H, H-β-Asp),1.57−1.48 (m, 4H, OCH2CH2(CH2)2CH3), 1.32− 1.24 (m, 8H, 2 × O(CH2)2(CH2)2CH3), 0.90−0.81 (m, 6H, 2 × O(CH2)4CH3). 13C NMR (75 MHz, CD3CN): δ 172.8 (d, 3JC,P = 5.0 Hz, CO-α), 172.7 (d, 3JC,P = 4.4 Hz, CO-α), 171.5, 171.4 (CO-β), 157.0 (C-6), 153.8 (C-2), 151.3, 151.2, 151.1, 151.0 (Ar−C, C-4), 142.8 (C-8), 130.6, 130.5 (Ar-C), 125.7 (Ar-C), 121.7 (d, 3JC,P = 4.6 Hz, Ar-C), 119.9 (C-5), 83.3 (d, 1JC,F = 172.5 Hz, C-3′), 83.1 (d, 1JC,F = 172.5 Hz, C-3′), 79.7, 79.6, 79.5, 79.4, 79.3 (C-2′), 66.8 (d, 1JC,P = 150.0 Hz, CH2P), 66.6 (d, 1JC,P = 157.5 Hz, CH2P), 66.4, 65.7 (OCH2(CH2)3CH3), 51.5 (C-α-Asp), 44.2 (d, 2JC,P = 8.2 Hz, C-1′), 44.0 (d, 2JC,P = 7.8 Hz, C-1′), 39.7 (d, 3JC,P = 3.9 Hz, C-β-Asp), 39.6 (d, 3 J C , P = 3.8 Hz, C-β-Asp), 28.9, 28.8, 28.7, 28.6 (OCH2(CH2)2CH2CH3), 23.0, 22.9 (O(CH2)3CH2CH3), 14.2 (O(CH2)4CH3). 31P NMR (121 MHz, CD3CN): δ 22.1, 21.5. HRMS for C29H42FN6O7P [M − H]− calcd.: 635.2764; found, 635.2775. (S)-1-{3-Fluoro-2-[phenyloxy-bis(amyl-L-aspartyl)phosphoryl]methoxy]propyl}cytosine (23a). Compound 23a was obtained as a colorless oil (23 mg, 26%) according to the general procedure, starting from compound 16a (40 mg, 0.14 mmol), L-aspartic acid amyl diester HCl salt (75 mg, 0.24 mmol), PhOH (59 mg, 0.63 mmol), Et3N (0.20 mL, 1.40 mmol), 2,2′-dithiodipyridine (220 mg, 1.00 mmol), and PPh3 (260 mg, 1.00 mmol) in anhydrous pyridine (3 mL). The crude residue was purified by column chromatography on silica gel (gradient 6235



Article

dye. Cells (1 × 104 cells per well) were seeded in 96-well U-bottomed tissue culture plates. Diluted test compounds and virus diluted to a predetermined titer to yield 85 to 95% cell killing at 6 days postinfection were added to the plate. Following incubation at 37 °C, 5% CO2 for 6 days, cell viability was measured by XTT staining. The optical density of the cell culture plate was determined spectrophotometrically at 450 and 650 nm using Softmax Pro 4.6 software. Percent CPE reduction of the virus-infected wells and the percent cell viability of uninfected drug control wells were calculated to determine the EC50 and TC50 values using four parameter curve fit analysis. HBV Antiviral Assay. Primary Assay. The primary anti-HBV assay was performed as previously described28 with modifications to use real-time qPCR (TaqMan) to measure extracellular HBV DNA copy number associated with virions released from HepG2.2.15 cells. The HepG2.2.15 cell line is a stable human hepatoblastoma cell line that contains two copies of the HBV wild-type strain ayw1 genome and constitutively produces high levels of HBV.25 Briefly, HepG2.2.15 cells were plated in 96-well microtiter plates at 1.5 × 104 cells/well in Dulbecco’s modified Eagle’s medium supplemented with 2% FBS, 380 μg/mL G418, 2.0 mM L-glutamine, 100 units/mL penicillin, 100 μg/ mL streptomycin, and 0.1 mM nonessential amino acids. Only the interior wells are utilized to reduce the “edge effects” observed during cell culture; the exterior wells are filled with complete medium to help minimize sample evaporation. After 16−24 h, the confluent monolayer of HepG2.2.15 cells was washed, and the medium was replaced with complete medium containing various concentrations of a test compound in triplicate. Lamivudine (3TC) was used as the positive control, while media alone was added to cells as a negative control (virus control, VC). Three days later, the culture medium was replaced with fresh medium containing the appropriately diluted test compounds. Six days following the initial administration of the test compound, the cell culture supernatant was collected, treated with Pronase, and then used in a real-time quantitative TaqMan qPCR assay. The qPCR assay targets the HBV precore/core region of the genome using the primers HBV-F (5′-CCAAATGCCCCTATCCTATCA-3) and HBV-R (5′-GAGGCGAGGGAGTTCTTCTTCTA-3′). The PCR-amplified HBV DNA was detected in real-time by monitoring increases in fluorescent signal that result from the exonucleolytic degradation of a quenched fluorescent probe molecule ([6-FAM]-CGGAAACTACTGTTGTTAGACGACGAGGCAG-[TAMRA]) that hybridized to the amplified HBV DNA. For each PCR amplification, a standard curve was simultaneously generated using dilutions of purified HBV DNA. Antiviral activity was calculated from the reduction in HBV DNA levels (EC50 and EC90 values determined). A tetrazolium dye (MTS; 3(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; CellTiter96 Reagent, Promega) uptake assay was then employed to measure cell viability, which was used to calculate toxicity (CC50). Secondary Assay. The secondary anti-HBV assay was performed in a manner similar to that for the primary assay described above. However, at the end of the assay, the cells were processed to isolate total intracellular DNA using the Qiagen DNeasy Blood and Tissue Kit following the manufacturer’s protocol. The real-time TaqMan qPCR assay was then performed using the isolated DNA to measure reductions in intracellular HBV DNA copy number. HCMV and VZV Antiviral Assays. The compounds were evaluated against varicella-zoster virus (VZV) strain Oka, and thymidine kinase deficient (TK−) VZV strain 07−1 and human cytomegalovirus (HCMV) strains AD-169 and Davis. The antiviral assays were based on inhibition of virus-induced cytopathicity or plaque formation in human embryonic lung (HEL) fibroblasts. Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID50 of HCMV (1 CCID50 being the virus dose to infect 50% of the cell cultures) or with 20 plaque forming units (PFU) (VZV). After a 1−2 h adsorption period, the residual virus was removed, and the cell cultures were incubated in the presence of varying concentrations of the test compounds. Viral cytopathicity (HCMV) or plaque formation (VZV) was recorded as soon as it reached completion in the control virus-infected cell cultures that were

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JC,F = 172.5 Hz, C-3′), 83.2 (d, 1JC,F = 165.0 Hz, C-3′), 79.7, 79.6, 79.5 (C-2′), 66.7 (d, 1JC,P = 157.5 Hz, CH2P), 66.6, 66.5, 65.8, 65.7 (OCH2(CH2)3CH3), 51.6, 51.5 (C-α-Asp), 43.8 (C-1′), 39.8, 39.6 (Cβ-Asp), 29.0, 28.9, 28.8, 28.7 (OCH2(CH2)2CH2CH3), 23.0, 22.9 (O(CH2)3CH2CH3), 14.3 (O(CH2)4CH3). 31P NMR (121 MHz, CD3CN): δ 22.3, 21.5. HRMS for C29H42FN6O8P [M − H]− calcd.: 651.2713; found, 651.2733. (R)-9-{3-Fluoro-2-[phenyloxy-bis(amyl-L-aspartyl)phosphoryl]methoxy]propyl}guanine (25b). Compound 25b was obatined according to the general procedure, starting from compound 20b (60 mg, 0.18 mmol), L-aspartic acid amyl diester HCl salt (100 mg, 0.32 mmol), PhOH (80 mg, 0.82 mmol), Et3N (0.26 mL, 1.80 mmol), 2,2′-dithiodipyridine (290 mg, 1.30 mmol), and PPh3 (340 mg, 1.30 mmol) in anhydrous pyridine (5 mL). The crude residue was purified by column chromatography on silica gel (gradient DCM/MeOH, 40:1, v/v; 30:1, v/v) to give the triphosphine adduct 24b. The collected eluate was evaporated and redissolved in CH3CN/H2O (1:1, v/v, 10 mL). One drop of 1 M HCl solution was added at 0 °C, and then the mixture was stirred for another 1 h at room temperature. The solution was neutralized with 2 M TEAB. After removal of all the volatiles, the resulting residue was purified by RP-HPLC (CH3CN, H2O) to give compound 25b (5 mg, 4%) as a colorless oil. 1H NMR (300 MHz, CD3CN): δ 7.67, 7.65 (s, 1H, H-8), 7.38−7.09 (m, 5H, ArH), 4.76− 3.94 (m, 13H, H-3′, H-2′, H-1′, 2 × OCH2(CH2)3CH3, PCH2, H-αAsp, NHPO), 2.81−2.56 (m, 2H, H-β-Asp), 1.59−1.52 (m, 4H, 2 × OCH2CH2(CH2)2CH3), 1.32−1.28 (m, 8H, 2 × O(CH2)2(CH2)2CH3), 0.92−0.86 (m, 6H, 2 × O(CH2)4CH3). 13C NMR (75 MHz, CD3CN): δ 172.8 (CO-α), 171.6 (CO-β), 158.6 (C6), 154.6 (C-2), 152.7 (C-4), 151.4 (Ar−C), 139.3 (C-8), 130.7, 130.6 (Ar-C), 125.9 (Ar-C), 121.8 (t, 3JC,P = 4.7 Hz, Ar-C), 118.3 (C-5, overlapped with CD3CN), 83.4 (d, 1JC,F = 172.5 Hz, C-3′), 83.2 (d, 1 JC,F = 165.0 Hz, C-3′), 79.8, 79.6, 79.5, 79.4 (C-2′), 66.9 (d, 1JC,P = 150.0 Hz, CH2P), 66.6 (d, 1JC,P = 157.5 Hz, CH2P), 66.5, 65.8 (OCH2(CH2)3CH3), 51.5 (C-α-Asp), 43.8 (C-1′), 39.8, 39.6 (C-βAsp), 29.0, 28.9, 28.8, 28.7 (OCH2(CH2)2CH2CH3), 23.0, 22.9 (O(CH2)3CH2CH3), 14.3 (O(CH2)4CH3). 31P NMR (121 MHz, CD3CN): δ 22.3, 21.7. HRMS for C29H42FN6O8P [M − H]− calcd.: 651.2726; found, 651.2733. HIV Antiviral Assay. Anti-HIV-1 Activity Tested in TZM-bl Cells. TZM-bl cells22 were seeded in transparent 96-well plates at 1 × 104 cells per well in DMEM (Dulbecco’s modified Eagle’s medium; Life Technologies, Waltham, MA, USA) with 10% fetal bovine serum (FBS) and 10 mM HEPES. Subsequently, compounds were added, and the cell/compound mixture was incubated at 37 °C. After 30 min, the virus (HIV-1 X4 NL4.3 and HIV-1 R5 BaL) was added at 100 pg p-24 HIV-1 Ag per well. After 48 h of incubation, the assay plates were analyzed. For the analysis, steadylite plus substrate solution (PerkinElmer, Waltham, MA, USA) was added to the assay plates. The luminescent signal of the lysed cell suspension was analyzed in white 96-well plates on a SpectraMax L luminescence microplate reader (Molecular Devices, Sunnyvale, CA, USA) after a 10 min incubation period in the dark. Luciferase activity induced by HIV-1 Tat protein expression was measured as an assessment of the amount of HIV replication. Anti-HIV-1 Activity Tested in PMBC Cells. Freshly isolated PBMCs27 were stimulated with 2 μg/mL PHA for 3 days at 37 °C before further use in anti-HIV replication assays. These PHAstimulated PBMCs (200 μL; 5 × 105 cells/ml) were seeded in a 48well plate (Costar, Elscolab NV, Kruibeke, Belgium) and preincubated for 30 min at 37 °C with various concentrations of compound (250 μL) in the presence of 2 ng/mL interleukin (IL)-2 (Roche Applied Science, Vilvoorde, Belgium). Next, (500 μL) 1500 pg/well of HIV-1 BaL and 500 pg/well of HIV-1 clinical isolates were given, and at days 3 and 6 of infection, 2 ng/mL of IL-2 was added again. Finally, 10 days postinfection, the supernatant of the cells was collected and tested for HIV-1 p24 Ag ELISA (PerkinElmer) according to the manufacturer’s guidelines. Activity Profiling against the HIV-1 K65R Variant. Inhibition of virus-induced cytopathic effects (CPE) and cell viability following HIV-1 replication in MT-4 cells were measured by XTT tetrazolium 6236



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experiments (at 25 °C), and the spectra were recorded every 30 min over 12 h. 31P NMR recorded data were processed and analyzed with the Bruker Topspin 2.1 program.

not treated with the test compounds. Antiviral activity was expressed as the EC50 or concentration (expressed in micromolar) required for reducing virus-induced cytopathicity or viral plaque formation by 50%. Cytostatic measurements were based on the inhibition of cell growth. HEL cells were seeded at a rate of 5 × 103 cells/well into 96-well microtiter plates and allowed to proliferate for 24 h. Then, medium containing different concentrations of the test compounds was added. After 3 days of incubation at 37 °C, the cell number was determined with a Coulter counter. The cytostatic concentration (expressed in micromolar) was calculated as the CC50 or the compound concentration required for reducing cell proliferation by 50% relative to the number of cells in the untreated controls. CC50 values were estimated from graphic plots of the number of cells (percentage of control) as a function of the concentration of the test compounds. Selectivity indices were calculated as the ratio CC 50 /EC 50 . Alternatively, the cytotoxicity of the test compounds was expressed as the minimum cytotoxic concentration (MCC) or the compound concentration that caused a microscopically detectable alteration of cell morphology. 31 P NMR Stability Experiments at Acidic and Basic pH. Buffer pH 1. The stability assay toward hydrolysis by an aqueous buffer at pH 1 was conducted using in situ 31P NMR (202 MHz) spectroscopy. First, prodrug 22a (2.0 mg) was dissolved in acetone-d6 (0.2 mL), followed by the addition of a pH 1 buffer (0.3 mL), which was previously prepared from equal parts of 0.2 M HCl and 0.2 M KCl. Next, the sample was subjected to 31P NMR experiments at 37 °C, and the spectra were recorded every 30 min over a period of 14 h. Buffer pH 8: The stability assay toward hydrolysis by an aqueous buffer at pH 8 was conducted using in situ 31P NMR (202 MHz) spectroscopy. First, prodrug 22a (1 mg) was dissolved in acetone-d6 (0.2 mL), followed by the addition of a pH 8 buffer (0.3 mL), which was previously prepared from a 0.1 M Na2HPO4 solution and adjusted to the appropriate pH using 0.1 M HCl. Next, the sample was subjected to 31P NMR experiments at 37 °C, and the spectra were recorded every 30 min over a period of 14 h. Metabolic Stability Assay in Human Plasma. This stability assay was contracted and carried out by Eurofins (Eurofins ref 1127). Human plasma was preincubated with a phosphate buffer (pH 7.4) in a 2 mL-block 96-well plate for 10 min in a 37 °C shaking water bath. The reaction was initiated by adding the test compound (final concentration 1 μM) and incubated to a final volume of 700 μL in the 37 °C shaking water bath. At 0, 0.5, 1, 1.5, and 2 h after the reaction initiation, 100 μL of the incubation mixture was added to 100 μL of acetonitrile and transferred to a 0.8 mL V-bottomed 96-well plate. Samples were then mixed on a plate shaker for 5 min and centrifuged at 2550g for 15 min at room temperature. Each supernatant was transferred to a clean cluster tube, followed by HPLC MS/MS analysis on a Thermo Electron triple quadrupole system. Metabolic Stability Assay in Human Liver Microsomes. This stability assay was contracted and carried out by Eurofins (Eurofins ref 0607). Pooled liver microsomes were preincubated with an NADPHregenerating system (1 mM NADP, 5 mM G6P, and 1 U mL/1 G6PDHase) in a phosphate buffer (pH 7.4) containing 3 mM MgCl2 and 1 mM EDTA in a 2 mL-block 96-well plate for 10 min in a 37 °C shaking water bath. The reaction was initiated by adding the test compound (final concentration 0.1 μM) and incubated to a final volume of 700 μL in the 37 °C shaking water bath. At 0, 15, 30, 45, and 60 min after the reaction initiation, 100 μL of the incubation mixture was added to 100 μL of acetonitrile/methanol (50/50, v/v) and transferred to a 0.8 mL V-bottomed 96-well plate. Samples were then mixed on a plate shaker for 5 min and centrifuged at 2550g for 15 min at room temperature. Each supernatant was transferred to a clean cluster tube, followed by HPLC MS/MS analysis on a Thermo Electron triple quadrupole system. Carboxypeptidase Y (EC 3.4.16.1) Assay. The experiment was carried out by dissolving 22a (4.4 mg) in acetone-d6 (0.15 mL) and by adding 0.30 mL of Trizma buffer (pH 7.6). After the 31P NMR spectrum was recorded at 25 °C as a control, a previously defrosted carboxypeptidase Y (0.1 mg dissolved in 0.15 mL of Trizma) was added to the sample. Next, the sample was subjected to 31P NMR

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00416. NMR spectra of compounds 3a/b−25a/b; analytical HPLC traces of compounds 8a/b, 12a/b, 16a/b, 20a/b, 21a/b, 22a/b, 23a/b, and 25a/b; mass spectrum of the proposed intermediate E or F in Figure 6 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +32 16 32 26 57. E-mail: Piet.Herdewijn@kuleuven. be. ORCID

Piet Herdewijn: 0000-0003-3589-8503 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.L. acknowledges the China Scholarship Council (CSC) for funding. We express our gratitude to Mrs. Ellen De Waegenaere, Mr. Seppe Kelchtermans, Mrs. Sandra Claes, Mrs. Evelyne Van Kerckhove, Mrs. Daisy Ceusters, and Mr. Chunsheng Huang for excellent technical assistance. We thank the KU Leuven Program Financing (ref. 10/018) for financial support. Testing against HBV was conducted by Southern Research Institute using federal funds from the Division of Microbiology and Infectious Diseases (DMID), NIAID/NIH under contract HHSN272201100013I entitled “In Vitro Assessment for Antimicrobial Activity”.

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ABBREVIATIONS USED ANPs, acyclic nucleoside phosphonates; FPMP, 3-fluoro-2(phosphonomethoxy)propyl nucleosides; HPMPC, (S)-1-(3hydroxy-2-phosphonylmethoxypropyl)cytosine; PMEA, 9-(2phosphonylmethoxyethyl)adenine; TDF, tenofovir disoproxil fumarate; TAF, tenofovir alafenamide; AZT, azidothymidine; 3TC, lamivudine; HBV, hepatitis B virus; HIV, human immunodeficiency virus; HCMV, human cytomegalovirus; HSV, herpes simplex virus; VZV, varicella zoster virus; VV, vaccinia virus; PBMCs, peripheral blood mononuclear cells; HEL, human embryonic lung; TK, thymidine kinase

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REFERENCES

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Diamyl Aspartate Amidat - ACS Publications - American Chemical

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