Amidate Prodrugs of Deoxythreosyl Nucleoside ... - ACS Publications

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Amidate Prodrugs of Deoxythreosyl Nucleoside Phosphonates as Dual Inhibitors of HIV and HBV Replication Chao Liu,† Shrinivas G. Dumbre,† Christophe Pannecouque,‡ Chunsheng Huang,§ Roger G. Ptak,§ Michael G. Murray,§ Steven De Jonghe,† and Piet Herdewijn*,† †

Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium § Infectious Disease Research, Southern Research, 431 Aviation Way, Frederick, Maryland 21701, United States ‡

S Supporting Information *

ABSTRACT: The synthesis of four L-2′-deoxy-threose nucleoside phosphonates with the natural nucleobases adenine, thymine, cytosine, and guanosine has been performed. Especially the adenine containing analogue (PMDTA) was endowed with potent antiviral activity displaying an EC50 of 4.69 μM against HIV-1 and an EC50 value of 0.5 μM against HBV, whereas completely lacking cytotoxicity. The synthesis of a number of phosphonomonoamidate and phosphonobisamidate prodrugs of PMDTA led to a boost in antiviral potency. The most potent congeners were a L-aspartic acid diisoamyl ester phenoxy prodrug and a Lphenylalanine propyl ester phosphonobisamidate prodrug that both display anti-HIV and anti-HBV activities in the low nanomolar range and selectivity indexes of more than 300.

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treatment of hepatitis B (HBV) infections.4 The substrate binding site of the reverse transcriptase enzyme of the human immunodeficiency virus (HIV) has proved to be an attractive target as seven nucleoside analogues have been licensed as antiHIV drugs.5 Several of these nucleoside analogues (lamivudine and emtricitabine) received also marketing approval for the treatment of HBV infections.4 Ribavirin and sofosbuvir are the only marketed nucleoside analogues for RNA viral infections. Ribavirin is being used for the treatment of respiratory syncytial virus (RSV) infections and in combination with interferon-α for the treatment of hepatitis C (HCV) infections. It is also licensed for the use in treatment of Lassa virus infections.6 Sofosbuvir is an uridine nucleotide analogue inhibitor (developed as a phosphoramidate prodrug) of the HCV NS5B RNA polymerase.7 The antiviral activity of these “classical” nucleosides depends upon their intracellular metabolism within virus-infected cells to form sequentially the mono-, di-, and triphosphates. It is these

INTRODUCTION The currently licensed antiviral medications are based on a wide variety of chemical structures (e.g., heterocycles, peptidomimetics). However, nucleosides are especially attractive as antiviral drugs for a number of reasons.1 Nucleoside analogues target the viral polymerase active sites, which are validated as targets for antiviral drug discovery. Moreover, the active sites of these polymerases are often conserved and nucleoside analogues have a high barrier of resistance emergence compared to other classes of inhibitors. In addition, nucleoside based inhibitors usually display equivalent potency against different serotypes/genotypes of a particular virus and even related viruses and are therefore excellent candidates for the development of broad-spectrum antivirals. At present, all antiviral agents available for the treatment of herpesvirus infections are nucleoside analogues: either acyclic guanosine analogues (acyclovir, penciclovir, ganciclovir)2 or thymidine analogues, such as (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU).3 All of these compounds target the viral DNA polymerase. DNA polymerase inhibitors based on a nucleoside scaffold (telbivudine, entecavir) have been licensed for the © 2016 American Chemical Society

Received: August 19, 2016 Published: October 17, 2016 9513

DOI: 10.1021/acs.jmedchem.6b01260 J. Med. Chem. 2016, 59, 9513−9531

Journal of Medicinal Chemistry

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Figure 1. Examples of biologically active nucleoside phosphonates.

Figure 2. Prodrugs of nucleoside phosphonates.

nucleoside phosphonate analogues, from which two congeners showed excellent activity against HIV. Both phosphonomethoxydeoxythreosyl adenine (PMDTA) and phosphonomethoxydeoxythreosyl thymine (PMDTT) displayed low μM EC50 values against HIV-1 and HIV-2 and lacked cellular toxicity.11 Despite the favorable characteristics of the nucleoside phosphonates (metabolic stability and bypassing of the first phosphorylation step), the fact that they are acidic implies that they are negatively charged at physiological pH and hence are not able to easily penetrate the lipid-rich cell membrane, which hampers their antiviral activity. Therefore, various prodrug or pronucleotide approaches have been investigated to promote the passive diffusion through the lipophilic cell membranes and to liberate the parent nucleotide intracellularly where it can be further phosphorylated to the pharmacologically active species.12 The (acyloxy)alkyl esters have been successfully used as prodrug moieties. Adefovir dipivoxil is a pivaloyloxymethylester prodrug, and tenofovir disoproxil fumarate is the fumarate salt of a isopropyloxycarbonyloxymethyl prodrug. More recently, the aryloxyphosphonoamidate prodrug approach13 has been applied to nucleoside phosphonates. A well-known example is tenofovir alafenamide (TAF), which recently received marketing approval as a component in different single tablet regimens for the treatment of HIV infected patients (Figure 2).14 Although the aforementioned PMDTA and PMDT are endowed with promising anti-HIV activity, prodrugs of these phosphonates have never been described in literature. In this paper, the design and synthesis of phosphonomonoamidate and phosphonodiamidate prodrugs of L-α-2′-deoxythreosyl phosphonate nucleosides are presented. In addition, to complete the structure−activity relationship (SAR) study of deoxythreosyl phosphonate nucleosides as antiviral agents, the synthesis of a previously unknown guanosine containing L-2′-deoxythreose nucleoside phosphonate and a 2′-fluoro-2′-deoxy modified PMDTA analogue was effected. Moreover, the compounds were subjected to a broader antiviral profiling, including HIV and HBV.

nucleotides, and especially the triphosphates that are the pharmacologically active species, as they are incorporated into a growing DNA or RNA strand by a DNA or RNA polymerase, resulting in chain termination or fraudulent DNA/RNA. The first phosphorylation step leading to the formation of the nucleoside 5′-monophosphate is commonly catalyzed by a nucleoside kinase encoded by the host cell or the virus infecting the host cell. Conversion of the nucleoside monophosphate to the corresponding 5′-diphosphate and triphosphate is carried out by nucleoside, nucleotide, and nucleoside diphosphate kinases, respectively. Hence, cellular kinases, as well as virally encoded kinases, play a vital role in the activation of nucleoside drugs. The first phosphorylation step in the generation of the biologically active nucleoside triphosphate analogue is very often inefficient. To bypass this rate-limiting step in the conversion to the nucleoside-5′-triphosphate, nucleoside phosphonates have been synthesized, allowing the first phosphorylation step required for nucleoside activation to be skipped. Nucleoside phosphonates are essentially nucleoside monophosphate analogues, having the advantage of being metabolically stable. Several phosphate isosteres have been adopted to prepare nucleoside phosphonates. The phosphonomethoxy functionality (P−C−O) emerged as the most promising isostere of the naturally occurring phospho-oxymethyl (P−O−C) moiety in nucleoside monophosphate. This has successfully been used in the field of HIV, with the discovery of cyclic phosphonates [5-(6-amino-purin-9-yl)-2,5dihydro-furan-2-yloxymethyl]-phosphonic acid (d4AP)8 and its fluorinated counterpart 2′Fd4AP (also known as GS-9148)9 (Figure 1). The same phosphonate motif is also present in the acyclic nucleoside phosphonates cidofovir, adefovir, and tenofovir.10 The success of this motif is due to the fact that it is isopolar and isosteric with the phosphate group. Hence, they can undergo enzymatic phosphorylation that converts them into the corresponding phosphonate diphosphates, which act as analogues of the natural nucleoside triphosphates. Our group previously described before a series of L-2′-deoxythreose 9514



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Scheme 1. Synthesis of the 1′,2′-Diacylated L-Threose Derivatives 3 and 6a

Reagents and conditions: (a) TBSCl, cat. DMAP, imidazole, 0 °C to rt, overnight, 97%; (b) DIBAL-H, dry THF, −70 to −60 °C, 3 h; (c) acetic anhydride, Et3N, dry CH2Cl2, 0 °C to rt, overnight, 73% for 2 steps; (d) acetic chloride, MeOH, rt, 2 h, 83% for 2 steps; (e) (iPrO)2POCH2OTf, NaH, THF, −5 to 0 °C, 15 min, 87%; (f) acetic anhydride, H2SO4, CH2Cl2, rt, overnight, 83%.

a

Scheme 2. Synthesis of PMDTA and PMDTTa

Reagents and conditions: (a) BSA, TMSOTf, CH3CN, 65 °C, 16 h for 7a (56%) and 2 h for 7b (83%); (b) Et3N·3HF, THF, rt, 24 h, 97% for 8a and 98% for 8b; (c) (iPrO)2POCH2OTf, NaH, THF, −5 to 0 °C, 80% for 9a and 85% for 9b; (d) NaOH, THF/MeOH/H2O, 0 °C, 20 min, 80% for 10a, LiOH, CH3CN/MeOH/H2O, 0 °C, 30 min, 85% for 10b; (e) (1) TCDI, DMAP, CH2Cl2, 40 °C, overnight, (2) Bu3SnH, AIBN, toluene, reflux, 20 min, 98% for 11a and 97% for 11b; (f) satd NH3 in MeOH, rt, overnight, 92%; (g) TMSBr, 2,6-lutidine, CH3CN, rt, overnight, 60% for 12a, TMSI, 2,6-lutidine, CH3CN, rt, overnight, 65% for 12b. a

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RESULTS AND DISCUSSION

phosphonomethyl triflate and NaH in THF to afford 3′-Ophosphonomethylated 5 as a mixture of diastereoisomers in 87% yield. This methyl threonoside was converted into the 1′,2′-diacyl glycosyl donor 6 in 83% yield. Synthesis of PMDTA and PMDTT. The synthesis of PMDTA and PMDTT has been described earlier starting from 2′-OTBDMS-3′-O-benzoyl-L-threose lactone, using a lengthy procedure involving multiple protection and deprotection steps.11 The synthetic approach shown in Scheme 2 allows to reduce the number of steps. The 1′,2′-diacylated α/β mixture 3 is a suitable glycosyl donor for the preparation of threose nucleoside phosphonates. The Vorbrüggen glycosylation with silylated benzoyl adenine and thymine using trimethylsilyl trifluoromethanesulfonate (TMSOTf) at 65 °C in acetonitrile gave compounds 7a and 7b in 56% and 83% yield, respectively. Deprotection of the TBS group by treatment with Et3N·3HF in

Chemistry. Synthesis of the 1′,2′-Diacylated L-Threose Building Blocks. 2′-O-Benzoyl-L-threonolactone 1 was synthesized from L-ascorbic acid following a literature procedure (Scheme 1).15,16 Although silylation of the 3′-hydroxyl group of 1 has been performed before using the bulky and lipophilic tertbutyldiphenylsilyl chloride, we opted to use a tert-butyldimethylsilyl protecting group. DIBAL-H mediated reduction of the lactone 2 to the corresponding lactol was followed by acetylation, affording key intermediate 3 as a mixture of α/β anomers in 73% yield.17 To introduce a phosphonate group early on in the synthetic sequence, lactone 2, after reduction, was treated with methanol in the presence of acetic chloride, furnishing methyl threonoside 4 in 83% yield. The phosphonate function was then introduced using diisopropyl9515



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Scheme 3. Synthesis of PMDTCa

Reagents and conditions: (a) BSA, TMSOTf, CH3CN, 70 °C, 1.5 h, 84%; (b) Et3N·3HF, THF, rt, 24 h, 97% for 14 and 79% for 20; (c) (iPrO)2POCH2OTf, NaH, THF, −5 to 0 °C, 45 min, 72%; (d) BSA, TMSOTf, CH3CN, 0 °C, overnight, 40%; (e) NaOH, THF/MeOH/H2O, 0 °C, 20 min, 80% for 16 and 71% for 18; (f) (1) TCDI, DMAP, CH2Cl2, 40 °C, overnight, (2) Bu3SnH, AIBN, toluene, reflux, 20 min, 70% for 17 and 91% for 19; (g) satd NH3 in MeOH, rt, overnight, 88%; (h) TMSI, 2,6-lutidine, CH3CN, rt, overnight, 48%. a

the N-alkylated product as the major compound. Therefore, an alternative strategy, starting from phosphonomethylated Lthreose sugar 6, was envisioned. Vorbrüggen glycosylation of presilylated N4-benzoylcytosine using trimethylsilyl trifluoromethanesulfonate (TMSOTf) as the Lewis acid at 0 °C in anhydrous acetonitrile gave cytosine threonucleoside 15 in 40% yield. Deprotection of the benzoyl group, followed by a Barton deoxygenation reaction, yielded nucleoside derivative 17. Alternatively, alkaline deprotection of the 2′-benzoyl group of 13, followed by 2′-deoxygenation, yielded the 3′-O-TBSprotected threose nucleoside derivative 19. Fluoride-mediated cleavage of the silyl protecting group and 3′-O-phosphonomethylation afforded the protected 2′-deoxy-threose cytidine analogue 17. Finally, alkaline deprotection of the benzoyl group and Lewis acid mediated cleavage of the phosphonate esters afforded PMDTC (compound 22). Synthesis of PMDTG. In an initial attempt of the synthesis of PMDTG, the N2-acetyl-O6-diphenyl-carbamoyl protected guanine derivative (GDPCAc) was selected as a coupling partner in the Vorbrü ggen glycosylation condensation, yielding compound 22a in 66% yield (Scheme 4). Deprotection of the 3′-hydroxyl group furnished intermediate 23a.18 Extensive efforts to phosphonomethylate nucleoside 23a under various reaction conditions (e.g., sodium hydride, silver oxide mediated alkylation) remained unsuccessful. Similarly, Vorbrüggen

THF afforded nucleosides 8a and 8b. Introduction of the phosphonate functionality using diisopropylphosphonomethyl triflate and NaH in THF afforded nucleosides 9a and 9b in 80% and 85% yield, respectively. Removal of the 2′-benzoyl group under basic conditions afforded nucleosides 10a and 10b. 2′Deoxygenation under standard Barton deoxygenation conditions gave nucleosides 11a and 11b. The benzoyl group, protecting the exocyclic amino group of the adenine moiety of compound 11a, was removed with ammonia in methanol, and hydrolysis of the phosphonate ester groups of 11b and 11c was carried out with TMSBr or TMSI in the presence of 2,6lutidine at room temperature to furnish target compounds PMDTA 12a and PMDTT 12b. Synthesis of PMDTC. PMDTC has been synthesized before in literature starting from the corresponding uridine analogue.11 We opted for the direct introduction of the cytosine nucleobase on the threose sugar moiety, as shown in Scheme 3. Coupling of N4-benzoylcytosine (CBz) with the protected L-threose sugar derivative 3 under Vorbrüggen reaction conditions afforded the desired L-threose nucleoside derivative 13. Fluoride-mediated deprotection of the 3′-hydroxyl group afforded 14. Phosphonomethylation of the nucleosides 14 afforded only minor amounts of 15, as evidenced from TLC analysis, due to the low solubility of 14 in tetrahydrofuran. However, switching the reaction solvent to N,N-dimethylformamide led to formation of 9516



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Scheme 4. Synthesis of PMDTGa

Reagents and conditions: (a) BSA, TMSOTf, toluene, 80 °C, 2 h, 66% for 22a, DBU, TMSOTf, CH3CN, 70 °C, 1.5 h, 67% for 22b; (b) Et3N·3HF, THF, rt, 24 h, 96% for 23a and 97% for 23b; (c) (iPrO)2POCH2OTf, NaH, THF, −5 to 0 °C, 67%; (d) 2 N NH3 in MeOH, rt, 2 h, 75%; (e) (1) TCDI, DMAP, CH2Cl2, 40 °C, overnight, (2) Bu3SnH, AIBN, toluene, reflux, 20 min, 68%; (f) (1) TMSBr, 2,6-lutidine, CH3CN, rt, overnight, (2) 2-mercaptoethanol, NaOMe, MeOH, reflux, 19 h, 49%. a

Scheme 5. Synthesis of 2′-Fluoro-PMDTAa

Reagents and conditions: (a) DMP oxidation (15% in CH2Cl2), 0 °C to rt, 1 h; (b) NaBH4, MeOH, 0 °C to rt, 1 h, 86% for two steps; (c) DAST, pyridine, CH2Cl2, −10 °C to rt, overnight, 63%; (d) TMSBr, 2,6-lutidine, CH3CN, rt, 24 h, 62%.

a

phosphonate function was introduced using diisopropylphosphonomethyl triflate and NaH in THF to afford nucleoside 24 in 67% yield. The benzoyl group at the 2′ position was hydrolyzed with 2 N NH3 in methanol, which upon Barton’s reductive 2′-deoxygenation gave the 2′-deoxy nucleoside 26 in 68% yield. After the hydrolysis of the phosphonate ester function in 26, the 6-chloro group was transformed to hydroxyl group by refluxing with 2-mercaptoethanol and NaOMe in methanol to afford PMDTG (compound 27) in 49% yield. Synthesis of 2′-fluoro-PMDTA. The synthesis of 2′-deoxy2′-fluoro modified PMDTA analogue 31 is shown in Scheme 5. Prior to the nucleophilic fluorination of the 2′ hydroxyl group,

nucleosidation of the diacylated L-threose devivative 6 with silylated N 2 -acetyl-O 6 -diphenyl-carbamoylguanine using TMSOTf or SnCl4 as the Lewis catalyst, which was successful for the synthesis of PMDTC, failed for the guanine-containing congener. Therefore, 2-amino-6-chloropurine was selected as coupling partner in the glycosylation reaction. The Vorbrü ggen glycosylation of the threose derivative 3 with 2-amino-6chloropurine using trimethylsilyl trifluoromethanesulfonate (TMSOTf) as the Lewis acid at 70 °C in anhydrous acetonitrile gave compound 22b in 67% yield. The TBS group was removed by treatment with Et3N·3HF in THF, and the 9517



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Scheme 6. Synthesis of Prodrugs of PMDTA, PMDTT, PMDTC, and PMDTGa

a

Reagents and conditions: (a) amino acid ester, PhOH, Et3N, 2,2′-dithiodipyridine, PPh3, pyridine, 13−66% for 32a−b, 33a−b, 34a, and 35a, amino acid ester, Et3N, 2,2′-dithiodipyridine, PPh3, pyridine, 23−73% for 32c, 33c, 34d, 35c, and 35d.

epimerization of the 2′-hydroxyl of compound 10a from a threose to an erythrose is needed. This was successfully accomplished via Dess−Martin periodinane (DMP) oxidation of 10a, followed by sodium borohydride reduction to afford selectively compound 29. Fluorination of 29 was carried out with diethylaminosulfur trifluoride (DAST) from −10 °C to rt to furnish 2′-fluorinated nucleoside 30 in 63% yield, with inversion of configuration. Under these reaction condition, concomitantly, benzoyl deprotection took place. Finally, hydrolysis of the phosphonate ester function of 30 with TMSBr in the presence of 2,6-lutidine afforded the target nucleoside 31. Synthesis of Prodrugs of PMDTA, PMDTT, PMDTC, and PMDTG. The phosphonomonoamidate prodrugs (32a−b, 33a−b, 34a, and 35a) were prepared from the parent nucleoside phosphonate using 2,2′-dithiodipyridine and triphenylphosphine as activating agents and utilizing a mixture of phenol and the appropriate amino acid ester (Scheme 6).9 This method affords diasteromeric mixtures of compounds due to the chirality of the phosphorus atom. The phosphonobisamidate prodrugs (32c, 33c, 34d, 35c, and 35d) were prepared in a similar way, using the desired amino acid ester.9 Anti-HIV Activity. The α-L-2′-deoxythreose nucleoside phosphonates 12a, 12b, 22, 27, and 31 were assessed for antiviral activity using the MTT method in MT-4 cells infected with a wild-type HIV-1 strain IIIB and a wild-type HIV-2 strain (ROD). In parallel, cytotoxicity of the compounds in the MT-4 cell line was assessed (Table 1).19,20 Lamivudine (2′,3′-dideoxy3-thiacytidine, commonly called 3TC) was included as positive control as it has dual anti-HIV (Table 1) and anti-HBV (Table 2) activity. PMDTA (compound 12a) is endowed with an EC50 value of 4.69 μM against HIV-1 and 5.23 μM against HIV-2. The

Table 1. Anti-HIV Activity and Cytotoxicity of Deoxythreosyl Nucleoside Phosphonates and Their Prodrugs EC50a (μM) compd 12a (PMDTA) 12b (PMDTT) 22 (PMDTC) 27 (PMDTG) 31 (2′-F-PMDTA) 32a 32b 32c 33a 33b 33c 34a 34d 35a 35c 35d 3TC

SI b

HIV-1 (IIIB)

HIV-2 (ROD)

CC50 (μM)

HIV-1

HIV-2

4.69 27.07 >343 >302 ≥222

5.23 23.55 >343 >302 ≥214.0

≥315 297 >343 >302 262

≥67 11

≥60 13

0.103 0.0216 0.0033 0.565 0.0173 0.0292 >160 >193 >112 >62 >97.6 2.22

0.0535 0.0036 0.0020 0.424 0.0110 0.0204 >160 >193 >112 >62 >97.6 8.81

61.8 16.9 1.12 127.3 42.2 56.6 >160 ≥193 ≥112 >62 ≥97.6 >87.2

597 792 347 223 2353 1971

1148 4812 570 295 3862 2781

>39

>10

a

EC50: compound concentration required to achieve 50% protection of MT-4 cells against HIV-induced cytopathicity. bCC50: compound concentration required to reduce the viability of mock-infected cells by 50%.

corresponding thymidine analogue PMDTT (compound 12b) is slightly less active and displayed an EC50 value of 27.07 μM against HIV-1 and 23.55 μM against HIV-2. The cytosine 9518



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or they are not recognized by the HIV reverse transcriptase. This is in sharp contrast with PMDTA for which it has been demonstrated that the diphosphate metabolite is an efficient substrate for HIV-1 reverse transcriptase (functioning as a chain terminator), whereas it is only a very poor substrate for human DNA polymerase α.11 Anti-HBV Activity. Acyclic nucleoside phosphonates adefovir and tenofovir are endowed with potent anti-HIV as well as anti-HBV activity. Therefore, the ability of the α-L-2′deoxythreose nucleoside phosphonates to inhibit the replication of HBV was assessed in the HepG2 2.2.15 cell line, which 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.22 Real-time qPCR (TaqMan) was used to measure 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.23,24 The biological data obtained in the HBV assay are quite similar to the anti-HIV data (Table 2). Among the parent

(PMDTC, compound 22) and guanosine (PMDTG, compound 27) containing congeners completely lack anti-HIV activity. The 2′-F analogue of PMDTA (compound 31) is also completely devoid of anti-HIV activity. Despite the only mediocre anti-HIV activity of PMDTT and especially PMDTA, their anti-HIV activity still warrants further investigation, as it is known that the application of a suitable prodrug strategy can boost antiviral activity. A noteworthy example is tenofovir alafenamide (also known as TAF), which is 1000-fold more active against HIV-1 (EC50 = 5 nM) when compared to the parent nucleoside tenofovir (EC50 = 5 μM).14 Different prodrugs of the α-L-2′-deoxythreose nucleoside phosphonates were designed. As L-alanine is often the preferred amino acid motif in ProTides, an alanine containing aryloxyphosphonomonoamidate prodrug of each nucleoside phosphonate was therefore prepared (series a in Scheme 6). Recently, we discovered that appropriately substituted Laspartic acid diester phosphoramidate prodrugs confer high anti-HCV activity to 2′-C-Me-uridine.21 In analogy, L-aspartic acid di-isoamylesters of α-L-2′-deoxythreose nucleoside phosphonates were also synthesized (series b in Scheme 6) and studied for antiviral activity. The main drawback of these phosphonomonoamidate prodrugs is that diastereomeric mixtures are generated due to the chirality of the phosphorus atom. In the early phase of drug discovery, this usually is not an issue as these compounds are biologically evaluated as mixtures. However, upon further development, separation of both diastereomers is necessary imposing an additional hurdle in the development of these prodrugs. Therefore, we also envisioned the synthesis of a phosphono-diamidate prodrug, avoiding the problem of diastereomeric mixtures (series c and d in Scheme 6). The alanine containing prodrug of PMDTA (compound 32a) shows a 50-fold increased activity against HIV-1 and a 100-fold improvement of activity against HIV-2. The phosphono-amidate prodrug of PMDTA carrying a diisoamylester L-aspartic acid as amino acid motif (compound 32b) is endowed with potent antiviral activity displaying EC50 values of 0.022 and 0.0036 μM against HIV-1 and HIV-2, respectively. The phosphonodiamidate analogue 32c is the most potent compound with EC50 values of 0.0033 μM (HIV1) and 0.002 μM (HIV-2). The same prodrug strategy was applied to PMDTT (compound 12b), and similar trends as in the PMDTA series were observed. The L-alanine phosphonoamidate prodrug 33a is endowed with a 50-fold increased antiHIV activity as compared to the parent PMDTT (compound 12b). The L-aspartic acid containing phenoxyphosphonomonoamidate prodrug (compound 33b) and the phosphonodiamidate prodrug (compound 33c) are endowed with comparable anti-HIV activities, with EC50 values in the range of 11−30 nM. In general, the PMDTA (compounds 32a−c) and PMDT prodrugs (compounds 33a−c) do show an increased cytotoxicity in the MT-4 cell line when compared to the free phosphonates. However, because of their excellent antiviral activity, selectivity indexes of 200 or more are still obtained. Anti-HIV evaluation of the different prodrugs of PMDTC (compounds 34a and 34d) and PMDTG (compounds 35a, 35c, and 35d) did not reveal an improvement in antiviral activity. This suggests that the limited cellular permeability of the parent phosphonates is not the reason for the lack of antiviral activity. Either PMDTC and PMDTG are not phosphorylated to their corresponding diphosphophosphonates

Table 2. Anti-HBV Activity and Cytotoxicity of Deoxythreosyl Nucleoside Phosphonates and Their Prodrugs compd

EC50a (μM)

EC90b (μM)

CC50c (μM)

SI50

12a (PMDTA) 12b (PMDTT) 22 (PMDTC) 27 (PMDTG) 31 (2′-F-PMDTA) 32a 32b 32c 33a 33b 33c 34a 34d 35a 35c 35d 3TC

0.50 40.2 >100 >100 79.58 0.03 0.01 0.01 0.26 0.25 0.28 47.89 >100 >100 >100 >100 0.01

>100 >100 >100 >100 >100 >10 0.98 4.20 >10 >100 >10 >100 >100 >100 >100 >100 >2.0

>100 >100 >100 >100 >100 >10 >10 >10 >100 34.03 >10 >100 >100 >100 >100 >100 >2.0

>200 >2.49

>333 >1250 >1000 >38 136 >36 >2

>200

a

EC50: compound concentration that reduces viral replication by 50%. b EC90: compound concentration that reduces viral replication by 90%. c CC50: compound concentration that reduces cell viability by 50%.

phosphonates, PMDTA (compound 12a) is the most potent anti-HBV compound, with an EC50 value of 0.5 μM. PMDTT (compound 12b) is 10-fold less potent (EC50 value 40.2 μM), whereas PMDTC (compound 22) and PMDTG (compound 27) are completely devoid of activity against HBV (EC50 values greater than 100 μM). The fluorinated counterpart of PMDTA (compound 31) shows very weak activity against HBV (EC50: 79 μM). To see whether, analogously to the anti-HIV activity, the prodrug had an improved inhibitory activity against HBV, the prodrugs were also evaluated. The different prodrugs of PMDTA (compounds 32a−c) are approximately 50 times more potent than PMDTA. Although the EC50 values of the different prodrugs are in the same range (EC50 values of 0.01− 0.03 μM), a close inspection of the EC90 values reveals that the phosphonodiamidate prodrug 32c (EC90 = 4.20 μM) and 9519



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vitro chemical and enzymatic hydrolysis of representative PMDTA prodrugs was investigated. Chemical Stability at pH 1 and pH 8. The chemical stability of prodrug 32b was evaluated in phosphate buffer at pH values of biological relevance (i.e., pH 1 and 8). The disappearance of the two peaks in the phosphorus NMR spectrum (due to the existence of diastereomers) was monitored for 14 h by 31P NMR at 37 °C. Under acidic conditions (pH 1, see Supporting Information, Figure S1), the prodrug 32b started to degrade after 1 h and new peaks arose in the 31P NMR spectrum (with chemical shift values of 24.60, 23.84, and 25.05, 24.31 ppm). Although these degradation products have not been unambiguously identified, these might arise from epimerization at the anomeric carbon or opening of the sugar ring under the acidic conditions. After 2 h, almost no original prodrug 32b was left and a clear signal at 15.31 ppm emerged, which gradually increased over time. The signals at the chemical shift 15.31 ppm may correspond to a product with cleavage of the phenol moiety. On the other hand, prodrug 32b is reasonably stable under mild basic conditions (pH 8, see Supporting Information, Figure S2), as after 14 h, 43% of the prodrug 32b was still present. A single peak at 15.48 ppm became apparent after 30 min, and it gradually increased over time. At the end of the experiment, HPLC and mass spectral analysis of the NMR sample (see Supporting Information, Figure S3) revealed that this peak in 31P NMR can be assigned to intermediate A (Figure 3), which was formed by cleavage of the phenol moiety of 32b. Incubations with Carboxypeptidase Y. To exert their antiviral activity, the prodrugs need to be metabolized intracellularly to their corresponding nucleoside-phosphonates. On the basis of the proposed intracellular activation route reported for other ProTide families,13 a hypothetical metabolic route is shown in Figure 4. It starts with the hydrolysis of the αcarboxylic acid ester group, mediated by a carboxyesterase type of enzyme. Spontaneous cyclization and concomitant release of the phenoxy moiety yields intermediate C. The cyclic mixed anhydride intermediate is then hydrolyzed, affording intermediate D. The final step involves a phosphoramidase type of enzyme, which cleaves off the amino acid, generating the nucleoside phosphonate. To investigate whether enzymatic cleavage of the ester group is sufficient to trigger the following steps, an enzymatic experiment incubating the prodrug 32b with carboxypeptidase Y enzyme in acetone-d6 and Trizma buffer (pH = 7.6) at 25 °C was performed, whereby 31P NMR spectra were recorded at specific time intervals. To improve the visualization of results, the spectra were processed using the Lorentz−Gauss deconvolution method (Figure 5). A difference in the rate of

especially the L-aspartic acid di-isoamylester prodrug 32b (EC90 = 0.98 μM) are much more potent than the classical L-alanine containing PMDTA prodrug 32a (EC90 > 10 μM). The CC50 values of the different prodrugs in the hepatoma cell line exceeds 10 μM, resulting in SI values of more than 300 for the alanine prodrug 32a, whereas the more potent prodrugs 32b and 32c are endowed with SI over 1000. The prodrugs of PMDTT (compounds 33a−c) all have very similar potencies in the HBV assay, as they all are endowed with an EC50 value of 0.25 μM, making them about 150 times more potent than PMDTT itself. To determine whether reductions in extracellular HBV DNA copy number that were observed in the primary assay (Table 2) correlate to a concomitant reduction in intracellular HBV DNA copy number, a secondary HBV assay was performed. This assay was carried out in a manner similar to the primary assay, but at the end of the assay, the cells were processed to isolate total intracellular DNA. Real-time TaqMan qPCR assay was then performed using the isolated DNA to measure the reductions in intracellular HBV DNA copy number. The two most potent prodrugs from the primary anti-HBV assay (32b and 32c) were tested in the secondary assay (Table 3). The Table 3. Anti-HBV Activity of Compounds 32b and 32c: Secondary Assay compd

EC50a (μM)

EC90b (μM)

CC50c (μM)

SI

32b 32c 3TC

100 >2.0

8810 >3333 >254

a

EC50: compound concentration that reduces viral replication by 50%. EC90: compound concentration that reduces viral replication by 90%. c CC50: compound concentration that reduces cell viability by 50%. b

results from the primary assay were confirmed. Both prodrugs display pronounced anti-HBV activity, and especially the Laspartic acid based prodrug 32b is highly potent based on the calculated EC50 and EC90 values. The anti-HBV activity of both compounds is in the same range as the activity of 3TC and with a selectivity index better than that of the control drug. Stability Studies. Although we observe very potent in vitro antiviral activity against HIV and HBV with the PMDTA prodrugs, in vivo, the prodrugs can only reach the target cells (the hepatocytes in case of HBV infection and lymphoid cell for HIV infected patients), on the condition that they are resistant to hydrolysis by extracellular and intracellular carboxylesterases during the absorption and distribution process. Partial or full conversion of these prodrugs to intermediates or the free nucleoside monophosphate will result in a lower cell penetration and a reduced antiviral potency. Therefore, the in

Figure 3. Degradation of compound 32b at pH 8. 9520



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Figure 4. Proposed activation pathway for prodrug 32b.

Figure 5. Deconvoluted 31P NMR spectra of prodrug 32b over time after carboxypeptidase Y digestion.

be metabolized intracellularly to release the L-2′-deoxythreose nucleoside phosphonate, which will then be further phosphorylated, yielding the corresponding diphosphophosphonate analogue that acts as an inhibitor of the HIV reverse transcriptase or HBV DNA polymerase. The stability of prodrugs 32a−c in the human liver S9 fraction and human liver microsomes was investigated (Table 4). The L-alanine containing monoamidate prodrug 32a is stable against degradation in the human liver S9 fraction and human liver microsomes. It suggests that this type of prodrug will survive first-pass metabolism, and they are suitable to be developed as

hydrolysis of the two diastereomers was observed. After incubation for 16 h, diastereomer (δ = 23.63 pm) was completely metabolized, whereas still substantial amounts of the other diastereomer (δ = 24.71 ppm) were still present. After 3 h, a new single peak (δ = 17.13 ppm) appeared in the 31 P NMR spectrum, which was assigned to metabolite D (Figure 4), as confirmed by HPLC and mass spectral analysis of the NMR sample at the end of the experiment (see Supporting Information, Figure S4). Mass spectroscopy does not allow discrimination whether the α- or β-carboxyl ester moiety is cleaved. However, recent research demonstrated that in the case of L-aspartate acid diester based nucleoside phosphoramidates, the β-carboxyl ester of the L-aspartate moiety was the first to be hydrolyzed. On the other hand, considering the fact that the α-carboxyl group is responsible for the nucleophilic attack at the phosphorus center to displace the aryl group, the α-carboxyl ester of the aspartate moiety should then be hydrolyzed first. Stability in Human Liver S9 Fraction and Human Liver Microsomes. To exert their antiviral activity, the ProTides must

Table 4. Stability of Prodrugs 32a−c in Human Liver S9 Fraction and Human Liver Microsomes

9521

compd

human liver S9 t1/2 (min)

human liver microsomes t1/2 (min)

32a 32b 32c

>60 12.1 60 8 7 DOI: 10.1021/acs.jmedchem.6b01260 J. Med. Chem. 2016, 59, 9513−9531


Article

acid (0.05 mmol, 1 equiv), appropriate amino acid ester hydrochloride (0.10 mmol, 2 equiv), and phenol (0.25 mmol, 5 equiv) in anhydrous pyridine (1.0 mL). The resultant mixture was stirred at 60 °C under argon atmosphere. In a separate flask, 2,2′-dithiodipyridine (0.42 mmol, 8.3 equiv) and PPh3 (0.30 mmol, 6 equiv) were dissolved in anhydrous pyridine (1.0 mL) and the resultant yellow solution was stirred at rt for 15 min. The solution was then added to the solution of phosphonic acid in one portion. The combined mixture was stirred at 60 °C for overnight. The mixture was then concentrated under reduced pressure. The resultant residue was first purified by silica gel chromatography (60:1 to 10:1, DCM/MeOH) and then purified by RP-HPLC (linear gradient, 5−95% CH3CN in water) to give the pure product as a mixture of P(R) and P(S) isomers. General Procedure for Preparation of Bisamidate Prodrugs (32c, 33c, 34d, 35c, and 35d). Anhydrous trimethylamine (0.70 mmol, 12 equiv) was added to a solution of phosphonic acid (0.05 mmol, 1 equiv) and appropriate amino acid ester hydrochloride (0.30 mmol, 6 equiv) in anhydrous pyridine (1.0 mL). The resultant mixture was stirred at 60 °C under argon atmosphere. In a separate flask, 2,2′dithiodipyridine (0.35 mmol, 7 equiv) and PPh3 (0.35 mmol, 7 equiv) were dissolved in anhydrous pyridine (1.0 mL) and the resultant yellow solution was stirred at rt for 15 min. The solution was then added to the solution of phosphonic acid in one portion. The combined mixture was stirred at 60 °C for overnight. The mixture was then concentrated under reduced pressure. The resultant residue was first purified by silica gel chromatography (60:1 to 10:1, DCM/ MeOH) and then purified by RP-HPLC (linear gradient, 5−95% CH3CN in water) to give the pure product. 2-O-Benzoyl-3-O-tert-butyldimethylsilyl-L-threonolactone (2). To a solution of lactone 1 (29.8 g, 134 mmol), cat. DMAP (0.1 g), and imidazole (15.8 g, 231 mmol) in dry acetonitrile at 0 °C, a solution of tert-butyldimethylchlorosilane (22.2 g, 148 mmol) in anhydrous acetonitrile was added dropwise. The reaction mixture was stirred at room temperature for overnight. The acetonitrile was evaporated under reduced pressure, the residue was taken up into 500 mL of EtOAc, and 300 mL of water was added. The resulting mixture was sequentially washed with ice-cold 1 M HCl solution, water, satd aq NaHCO3, and brine. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by column chromatography (4:1 hexane/EtOAc) to afford 2 (44.0 g, 97% yield) as a colorless oil. 1 H NMR (300 MHz, CDCl3): δ 8.06−8.09 (m, 2H, Ph), 7.58−7.63 (m, 1H, Ph), 7.44−7.49 (m, 2H, Ph), 5.59 (d, J = 7.3 Hz, 1H, H-2′), 4.80 (q, J = 7.2 Hz, 1H, H-3′), 4.53 (dd, J = 9.1, 7.0 Hz, 1H, H-4a′), 4.09 (dd, J = 9.1, 7.3 Hz, 1H, H-4b′), 0.86 (s, 9H, CH3), 0.07 (s, 3H, SiCH3), 0.06 (s, 3H, SiCH3). 13C NMR (75 MHz, CDCl3): δ 170.4 (C-1′), 165.2 (PhCO), 133.9, 130.1, 128.7 (Ph), 75.1 (C-2′), 71.7 (C3′), 70.6 (C-4′), 25.6 [C(CH3)3], 18.0 [C(CH3)3], −4.7, −4.9 (SiCH3). HRMS: [M + H]+ calcd for C17H25O5Si, 337.1466; found, 337.1460. 1-O-Acetyl-2-O-benzoyl-3-O-tert-butyldimethylsilyl-L-threofuranose (3).17 Prepared according to the literature17 and obtained as a colorless oil. Yield 73%. α-Anomer: 1H NMR (300 MHz, CDCl3): δ 8.01−8.04 (m, 2H, Ph), 7.57−7.62 (m, 1H, Ph), 7.43−7.48 (m, 2H, Ph), 6.23 (s, 1H, H-1′), 5.29 (d, J = 1.7 Hz, 1H, H-2′), 4.44−4.49 (m, 1H, H-3′), 4.28 (dd, J = 9.3, 5.7 Hz, 1H, H-4a′), 4.01 (dd, J = 9.3, 4.2 Hz, 1H, H-4b′), 2.12 (s, 3H, CH3CO), 0.91 (s, 9H, CH3), 0.15 (s, 3H, SiCH3), 0.10 (s, 3H, SiCH3). 13C NMR (75 MHz, CDCl3): δ 169.8 (CH3CO), 165.3 (PhCO), 133.5, 129.8, 129.1, 128.5 (Ph), 99.9 (C1′), 83.2 (C-2′), 75.4 (C-4′), 74.9 (C-3′), 25.6 [C(CH3)3], 21.1 (CH3CO), 17.9 [C(CH3)3], −4.8, −5.1 (SiCH3). β-Anomer: 1H NMR (300 MHz, CDCl3): δ 8.02−8.05 (m, 2H, Ph), 7.54−7.60 (m, 1H, Ph), 7.41−7.46 (m, 2H, Ph), 6.49 (d, J = 4.4 Hz, 1H, H-1′), 5.26 (t, J = 4.7 Hz, 1H, H-2′), 4.68−4.73 (m, 1H, H-3′), 4.27 (dd, J = 9.1, 6.5 Hz, 1H, H-4a′), 3.78 (dd, J = 9.1, 4.7 Hz, 1H, H-4b′), 1.94 (s, 3H, CH3CO), 0.87 (s, 9H, CH3), 0.09 (s, 3H, SiCH3), 0.08 (s, 3H, SiCH3). 13 C NMR (75 MHz, CDCl3): δ 169.4 (CH3CO), 165.4 (PhCO), 133.4, 129.7, 129.2, 128.5 (Ph), 94.65 (C-1′), 80.0 (C-2′), 73.4 (C-3′), 72.5 (C-4′), 25.6 [C(CH3)3], 20.9 (CH3CO), 17.9 [C(CH3)3], −4.7, −5.0 (SiCH3). HRMS: [M + Na]+ calcd for C19H28O6SiNa, 403.1548; found, 403.1548.

anti-HIV drugs, as the target cells for anti-HIV medication are lymphocytes, which are present in the systemic circulation. On the other hand, the L-aspartic acid based phosphonomonoamidate prodrug 32b and the bisamidate prodrug 32c are both metabolically labile in liver S9 fraction and human liver microsomes with half-lives of less than 10 min. This type of prodrug is well suited for further development as an anti-HBV drug, as fast metabolism in the liver will quickly generate high levels of the nucleoside phosphonate (the pharmacologically active metabolite) in hepatocytes, which are the target cells for HBV infection.

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CONCLUSION The synthesis of four L-2′-deoxy-threose nucleoside phosphonates containing the natural nucleobases adenine, thymine, cytosine, and guanosine has been performed, using an improved synthetic method. In addition, a fluorinated congener of PMDTA has been synthesized. PMDTT, and especially PMDTA, are both endowed with antiviral activity against HIV and HBV, whereas other congeners within this series completely lack antiviral activity. Different phosphonomonoamidate and phosphonobisamidate prodrugs of these nucleoside phosphonates were also prepared. The application of these prodrug technologies to PMDTA and PMDTT led to a boost in antiviral potency. The anti-HIV potency increased by a factor of 100 to 1000, depending on the prodrug moiety, whereas 50− 150-fold enhancements for anti-HBV activity were observed. The prodrugs of PMDTA are reasonably stable in pH 8 buffer but can be activated by carboxypeptidase Y type enzyme and human liver enzymes. The promising antiviral data of the phosphono-amidate prodrugs of PMDTA and PMDTT suggest that they deserve further investigation as dual anti-HIV and anti-HBV drugs.

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EXPERIMENTAL SECTION

Chemistry. NMR spectra were recorded on a Bruker Advance 300 MHz (1H NMR, 300 MHz; 13C NMR, 75 MHz; 31P NMR, 121 MHz) or 500 MHz (1H NMR, 500 MHz; 13C NMR, 125 MHz; 31P NMR, 202 MHz) or 600 MHz (1H NMR, 600 MHz; 13C NMR, 150 MHz) spectrometer with tetramethylsilane as internal standard or referenced to the residual solvent signal and 85% H3PO4 for 31P NMR experiments. Two-dimensional NMRs (H-COSY, NOESY, HSQC, and HMBC) were used for the assignment of the intermediates and final compounds. High resolution mass spectra were measured on a quadrupole 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 negative ionization mode with a resolution of 15000 (fwhm) using leucine enkephalin as lock mass. Precoated aluminum sheets (Fluka silica gel/TLC-cards, 254 nm) were used for TLC. Purities of the prodrugs were verified to be >95% by HPLC analysis. HPLC conditions to assess purity were as follows: Shimadzu HPLC equipped with a LC-20AT pump, a DGU20A5 degasser, and a SPD-20A UV detector; Inertsil ODS-3 (4 μm, 4.6 mm × 100 mm) or Symmetry C18 column (5 μm, 4.6 mm × 150 mm); gradient elution of H2O/CH3CN or H2O/MeOH from 95/5 to 5/95 in 15 min; flow rate, 1 mL/min; wavelength, UV 254 nm. Preparative HPLC purifications were performed on a Phenomenex Gemini 110A column (C18, 10 μm, 21.2 mm × 250 mm). Column chromatography was performed on silica gel 60 Å, 0.035−0.070 mm (Acros Organics). All reagents and solvents were obtained from commercial sources and were used as received. Moisture sensitive reactions were performed under an argon atmosphere using ovendried glassware. General Procedure for Preparation of Monoamidate Podrugs (32a−b, 33a−b, 34a, and 35a). Anhydrous trimethylamine (0.70 mmol, 12 equiv) was added to a solution of phosphonic 9522



Article

1′α-(N6-Benzoyladenin-9-yl)-2′-O-benzoyl-3′-O-tert-butyldimethylsilyl-L-threose (7a). A suspension of 3 (0.51g, 1.35 mmol) and N6-benzoyladenine (0.36 g, 1.49 mmol) in anhydrous CH3CN (15 mL) was treated with BSA (0.73 mL, 3.0 mmol) and heated to 65 °C. Stirring and heating was continued until a clear solution was formed (ca. 1 h), and TMSOTf (0.49 mL, 2.70 mmol) was added. After heating at 65 °C for 16 h, the mixture was cooled to rt and poured into an ice-cold 100 mL of satd aq NaHCO3/EtOAc 1:1. The organic layer was separated, the water layer was extracted with EtOAc. The combined organic layers were washed with brine and dried over Na2SO4. Evaporation of the solvent and column chromatography (5:2 CH2Cl2/EtOAc) gave 7a (0.42 g, 56% yield) as a white foam. 1H NMR (300 MHz, CDCl3): δ 9.60 (s, 1H, NH), 8.67 (s, 1H, H-2), 8.37 (s, 1H, H-8), 7.94−8.00 (m, 4H, Ph), 7.47−7.55 (m, 2H, Ph), 7.36− 7.40 (m, 4H, Ph), 6.45 (s, 1H, H-1′), 5.56 (s, 1H, H-2′), 4.48 (d, J = 3.2 Hz, 1H, H-3′), 4.20−4.31 (m, 2H, H-4′), 0.77 [s, 9H, C(CH3)3], 0.07 (s, 3H, SiCH3), −0.02 (s, 3H, SiCH3). 13C NMR (75 MHz, CDCl3): δ 164.7 (PhCO), 152.3 (C-2), 151.2 (C-4), 149.2 (C-6), 141.6 (C-8), 133.5, 133.5, 132.2, 129.5, 128.3, 128.3, 127.6 (Ph), 122.9 (C-5), 87.8 (C-1′), 82.3 (C-2′), 76.3 (C-4′), 74.8 (C-3′), 25.3 [C(CH3)3], 17.6 [C(CH3)3], −5.2, −5.6 (SiCH3). HRMS: [M + H]+ calcd for C29H34N5O5Si, 560.2324; found, 560.2330. 1′α-(Thymin-1-yl)-2′-O-benzoyl-3′-O-tert-butyldimethylsilylL-threose (7b). This compound was prepared using a similar procedure as described for 7a. Obtained from 3 (10.4 g, 27.3 mmol), thymine (3.44 g, 27.3 mmol), BSA (13.3 mL, 54.5 mmol), and TMSOTf (14.8 mL, 81.7 mmol) as a white foam, yield 83% (10.2 g). 1 H NMR (300 MHz, CDCl3): δ 9.50 (s, 1H, NH), 8.02−8.05 (m, 2H, Ph), 7.56−7.62 (m, 1H, Ph), 7.52 (d, J = 1.2 Hz, 1H, H-6), 7.42−7.47 (m, 2H, Ph), 6.19 (d, J = 0.9 Hz, 1H, H-1′), 5.23 (s, 1H, H-2′), 4.36 (d, J = 2.6 Hz, 1H, H-3′), 4.15−4.23 (m, 2H, H-4′), 1.92 (d, J = 1.2 Hz, 3H, T CH3), 0.90 [s, 9H, C(CH3)3], 0.16 (s, 3H, SiCH3), 0.15 (s, 3H, SiCH3). 13C NMR (75 MHz, CDCl3): δ 165.0 (PhCO), 164.3 (C4), 150.4 (C-2), 136.5 (C-6), 133.7, 129.9, 129.8, 128.9, 128.5 (Ph), 110.2 (C-5), 89.6 (C-1′), 82.7 (C-2′), 76.5 (C-3′), 74.7 (C-4′), 25.6 [C(CH3)3], 17.9 [C(CH3)3], 12.6 (T CH3), −4.8, −5.3 (SiCH3). HRMS: [M + H]+ calcd for C22H31N2O6Si, 447.1946; found, 447.1941. 1′α-(N6-Benzoyladenin-9-yl)-2′-O-benzoyl-L-threose (8a).16 To a solution of compound 7a (11.6 g, 20.7 mmol) in 200 mL of anhydrous THF at rt was added triethylamine trihydrofluoride. The reaction mixture was stirred at rt for 24 h and found to be complete. The solvent was removed under reduced pressure. The residue was taken up into 300 mL of EtOAc, and 200 mL of water was added. The organic layer was separated and washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (50:1 CH2Cl2/MeOH) to obtain 8a (9.0 g, 97% yield) as a white solid. 1H NMR (300 MHz, CDCl3): δ 9.45 (s, 1H, NH), 8.74 (s, 1H, H-2), 8.32 (s, 1H, H-8), 7.99−8.02 (m, 4H, Ph), 7.43−7.63 (m, 6H, Ph), 6.13 (d, J = 1.3 Hz, 1H, H-1′), 5.63 (s, 1H, H-2′), 4.64 (d, J = 3.0 Hz, 1H, H-3′), 4.36 (d, J = 10.1 Hz, 1H, H4a′), 4.27 (dd, J = 10.1, 3.8 Hz, 1H, H-4b′). 13C NMR (75 MHz, CDCl3): δ 165.6, 164.8 (PhCO), 152.0 (C-2), 150.4 (C-4), 150.0 (C6), 143.1 (C-8), 133.9, 133.4, 132.8, 129.8, 128.7, 128.6, 128.5, 127.9 (Ph), 123.5 (C-5), 90.3 (C-1′), 84.1 (C-2′), 76.1 (C-4′), 74.6 (C-3′). HRMS: [M + H]+ calcd for C23H20N5O5, 446.1459; found, 446.1451. 1′α-(Thymin-1-yl)-2′-O-benzoyl-L-threose (8b).15 This compound was prepared as described for 8a. Obtained from 7b (11.6 g, 26.0 mmol) and triethylamine trihydrofluoride (8.5 mL, 52.0 mmol) as a white solid. Yield 98% (8.5 g). 1H NMR (300 MHz, CDCl3): δ 9.93 (s,1H, NH), 7.96−7.98 (m, 2H, Ph), 7.52−7.57 (m, 1H, Ph), 7.47 (d, J = 0.9 Hz, 1H, H-6), 7.37−7.42 (m, 2H, Ph), 5.99 (d, J = 1.2 Hz, 1H, H-1′), 5.48 (s, 1H, H-2′), 4.77 (brs, 1H, OH), 4.46 (brs, 1H, H-3′), 4.31 (d, J = 10.0 Hz, H-4a′), 4.16 (dd, J = 10.0, 3.6 Hz, H-4b′), 1.79 (s, 3H, T CH3). 13C NMR (75 MHz, CDCl3): δ 165.5 (PhCO), 164.4 (C-4), 150.5 (C-2), 137.2 (C-6), 133.6, 129.7, 128.6, 128.4 (Ph), 109.9 (C-5), 90.7 (C-1′), 82.6 (C-2′), 75.51 (C-4′), 73.9 (C-3′), 12.2 (T CH3). HRMS: [M + H]+ calcd for C16H17N2O6, 333.1081; found, 333.1082.

1-O-Methyl-2-O-benzoyl-L-threose (4). To a solution of lactol (8.04 g, 23.75 mmol) in anhydrous methanol (59 mL) was added acetyl chloride (1.69 mL, 23.75 mmol). After stirring at rt for 2 h, Et3N (4 mL) was added and the mixture was concentrated under reduced pressure. The residue was partitioned between water and EtOAc, and the organic layer was washed with satd aq NaHCO3 and brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (3:1 to 2:1, hexane/EtOAc) to afford 4 (4.7 g, 83% yield) as a colorless oil. 1H NMR (300 MHz, CDCl3): δ 8.00−8.06 (m, 2H, Ph), 7.56−7.62 (m, 1H, Ph), 7.42−7.48 (m, 2H, Ph), 5.14 (brs, 1H, H-1′), 5.11 (s, 1H, H-2′), 4.30−4.38 (m, 2H, H-3′ and H-4a′), 3.96−4.02 (m, 1H, H-4b′), 3.45 (s, 3H, OMe). 13 C NMR (75 MHz, CDCl3): δ 166.1 (PhCO), 133.5, 129.8, 129.1, 128.5 (Ph), 106.5 (C-1′), 83.6 (C-2′), 75.2 (C-3′), 73.8 (C-4′), 55.0 (OMe). HRMS: [M + Na]+ calcd for C12H14O6Na, 261.0734; found, 261.0738. 1-O-Methyl-2-O-benzoyl-3-O-diisopropylphosphonomethyl-L-threose (5). To a solution of 4 (2.23 g, 9.36 mmol) and triflate diisopropylphosphonomethanol (4.61 g, 14.04 mmol) in anhydrous THF (50 mL) was added NaH (60% in mineral oil, 0.45 g, 11.23 mmol) at −5 °C. The reaction mixture was warmed to 0 °C and stirred for 15 min. The reaction was quenched with satd aq NH4Cl and was subsequently concentrated. The residue was partitioned between water and EtOAc. The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (2:1 to 1:1, hexane/EtOAc) to afford 5 (3.43 g, 87% yield) as a colorless oil containing about 10% of β-anomer. α-Anomer: 1H NMR (600 MHz, CDCl3): δ 8.02−8.05 (m, 2H, Ph), 7.57−7.61 (m, 1H, Ph), 7.44−7.47 (m, 2H, Ph), 5.29 (s, 1H, H-2′), 5.04 (s, 1H, H-1′), 4.71−4.81 [m, 2H, CH(CH3)2], 4.38 (dd, J = 9.8, 7.1 Hz, 1H, H-4a′), 4.28−4.30 (m, 1H, H-3′), 3.95 (dd, J = 9.6, 5.1 Hz, 1H, H-4b′), 3.86−4.07 (m, 2H, PCH2), 3.40 (s, 3H, OMe), 1.32−1.34 [m, 12H, CH(CH3)2]. 13C NMR (150 MHz, CDCl3): δ 165.5 (PhCO), 133.5, 129.8, 129.3, 128.5 (Ph), 107.0 (C-1′), 84.8 (d, 3 JP,C = 10.4 Hz, C-3′), 80.9 (C-2′), 71.4, 71.1 [CH(CH3)2], 71.1 (C4′), 65.0 (d, 1JP,C = 168.4 Hz, PCH2), 54.7 (OMe), 24.0 [CH(CH3)2]. 31 P NMR (121 MHz, CDCl3): δ 18.5. β-anomer: 1H NMR (600 MHz, CDCl3): δ 8.06−8.08 (m, 2H, Ph), 7.58−7.61 (m, 2H, Ph), 7.45−7.48 (m, 2H, Ph), 5.27 (d, J = 4.5 Hz, 1H, H-1′), 5.10 (t, J = 4.6 Hz, 1H, H2′), 4.71−4.78 [m, 2H, CH(CH3)2], 4.56−4.58 (m, 1H, H-3′), 4.22 (dd, J = 9.9, 6.7 Hz, 1H, H-4a′), 3.89 (dd, J = 9.9, 3.6 Hz, 1H, H-4b′), 3.76−3.89 (m, 2H, PCH2), 3.33 (s, 3H, OMe), 1.29−1.34 [m, 2H, CH(CH3)2]. 13C NMR (150 MHz, CDCl3): δ 165.8 (PhCO), 133.4, 129.8, 129.3, 128.5 (Ph), 101.8 (C-1′), 83.2 (d, 3JP,C = 12.5 Hz, C-3′), 79.5 (C-2′), 71.4, 71.3 [CH(CH3)2], 68.5 (C-4′), 64.6 (d, 1JP,C = 169.9 Hz, PCH2), 55.4 (OMe), 24.0 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 18.1. HRMS: [M + H]+ calcd for C19H30O8P, 417.1673; found, 417.1673. 1-O-Acetyl-2-O-benzoyl-3-O-diisopropylphosphonomethylL-threose (6). To a solution of 5 (0.20 g, 0.48 mmol) in anhydrous CH2Cl2 (5 mL) was added acetic anhydride (0.18 mL, 1.92 mmol) and a catalytic amount of sulfuric acid. After stirring at room temperature for overnight, the reaction mixture was neutralized by Et3N (3 mL) at 0 °C and concentrated under reduced pressure. The crude product was diluted by EtOAc (20 mL), washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (1:1, hexane/EtOAc) to afford 6 (0.18 g, 83% yield) as a colorless oil containing about 10% of β-anomer, and the major α-anomer was characterized. 1H NMR (600 MHz, CDCl3): δ 8.02−8.04 (m, 2H, Ph), 7.59−7.62 (m, 1H, Ph), 7.45−7.48 (m, 2H, Ph), 6.31 (s,1H, H-1′), 5.42 (d, J = 0.9 Hz, 1H, H-2′), 4.74−4.80 [m, 2H, CH(CH3)2], 4.44 (dd, J = 10.1, 6.7 Hz, H-4a′), 4.30−4.32 (m, 1H, H-3′), 4.12 (dd, J = 10.1, 4.3 Hz, 1H, H4b′), 3.86−4.10 (m, 2H, PCH2), 2.12 (s, 3H, OCH3), 1.33−1.35 [m, 12H, CH(CH3)2]. 13C NMR (125 MHz, CDCl3): δ 169.7 (CH3CO), 165.3 (PhCO), 133.7, 129.8, 128.9, 128.6 (Ph), 99.8 (C-1′), 84.3 (d, 3 JP,C = 11.6 Hz, C-3′), 80.3 (C-2′), 73.1 (C-4′), 71.3, 71.2 [CH(CH3)2], 65.2 (d, 1JP,C = 168.6 Hz, PCH2), 24.0 [CH(CH3)2], 21.1 (CH3CO). 31P NMR (121 MHz, CDCl3): δ 18.2. HRMS: [M + Na]+ calcd for C20H29O9PNa, 467.1442; found, 467.1444. 9523



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CDCl3): δ 164.6 (C-4), 151.0 (C-2), 136.4 (C-6), 110.0 (C-5), 93.0 (C-1′), 85.2 (d, 3JP,C = 10.7 Hz, C-3′), 78.6 (C-2′), 73.6 (C-4′), 71.3, 71.2 [CH(CH3)2], 64.3 (d, 1JP,C = 168.4 Hz, PCH2), 24.0, 23.9 [CH(CH3)2], 12.4 (T CH3). 31P NMR (121 MHz, CDCl3): δ 18.3. HRMS: [M + H]+ calcd for C16H28N2O8P, 407.1578; found, 407.1582. 1′α-(N 6 -Benzoyladenin-9-yl)-2′-deoxy-3′-O-diisopropylphosphonomethyl-L-threose (11a). To a solution of 10a (137 mg, 0.26 mmol) and DMAP (10 mg, 0.08 mmol) in andydrous CH2Cl2 (2.70 mL) was added 1,1′-thiocarbonyldiimidazole (TCDI) (94 mg, 0.53 mmol) at rt. The reaction mixture was stirred at 40 °C for overnight. The mixture was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. To the residue in toluene (5 mL) was added azobis(isobutyronitrile) (AIBN) (17 mg, 0.11 mmol) and tributytin hydride (0.28 mL, 1.05 mmol). The reaction mixture was refluxed for 20 min. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (30:1 CH2Cl2/MeOH) to afford 11a (130 mg, 98% yield) as a white solid. 1H NMR (500 MHz, CDCl3): δ 9.15 (s, 1H, NH), 8.79 (s, 1H, H-2), 8.49 (s, 1H, H-8), 8.00−8.02 (m, 2H, Ph), 7.57−7.59 (m, 1H, Ph), 7.50−7.53 (m, 2H, Ph), 6.55 (dd, J = 7.7, 2.0 Hz, 1H, H-1′), 4.69−4.75 [m, 2H, CH(CH3)2], 4.51−4.53 (m, 1H, H-3′), 4.38 (d, J = 10.5 Hz, 1H, H-4a′), 4.08 (dd, J = 10.5, 4.1 Hz, 1H, H-4b′), 3.67−3.75 (m, 2H, PCH2), 2.62−2.74 (m, 2H, H-2′). 1.28−1.33 [m, 12H, CH(CH3)2]. 13C NMR (125 MHz, CDCl3): δ 164.5 (PhCO), 152.5 (C-2), 151.5 (C-4), 149.3 (C-6), 142.1 (C-8), 133.8, 132.6, 128.8, 127.8 (Ph), 122.8 (C-5), 83.7 (C-1′), 80.2 (d, 3JP,C = 9.5 Hz, C-3′), 73.9 (C-4′), 71.3, 71.3 [CH(CH3)2], 64.0 (d, 1JP,C = 169.2 Hz, PCH2), 38.6 (C-2′), 24.0 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 18.2. HRMS: [M + H]+ calcd for C23H31N5O6P, 504.2006; found, 504.2023. 1′α-(Thymin-1-yl)-2′-deoxy-3′-O-diisopropylphosphonomethyl-L-threose (11b).11 This compound was prepared as described for 11a. Obtained from 10b (1.53 g, 3.8 mmol) as a white solid. Yield 97% (1.43 g). 1H NMR (300 MHz, CDCl3): δ 8.97 (brs, 1H, NH), 7.55 (d, J = 1.2 Hz, 1H, H-6), 6.24 (dd, J = 8.1, 2.6 Hz, 1H, H-1′), 4.69−4.80 [m, 2H, CH(CH3)2], 4.30−4.36 (m, 2H, H-4a′ and H-3′), 3.84 (dd, J = 10.8, 3.6 Hz, 1H, H-4b′), 3.71 (d, J = 9.2 Hz, 2H, PCH2), 2.50−2.60 (m, 1H, H-2a′), 2.16 (d, J = 15.3 Hz, 1H, H2b′), 1.97 (d, J = 1.1 Hz, 3H, T CH3), 1.31−1.35 [m, 12H, CH(CH3)2]. 13C NMR (75 MHz, CDCl3): δ 163.8 (C-4), 150.6 (C2), 136.4 (C-6), 110.7 (C-5), 84.8 (C-1′), 80.2 (d, 3JP,C = 11.4 Hz, C3′), 73.4 (C-4′), 71.2 [CH(CH3)2], 64.0 (d, 1JP,C = 169.8 Hz, PCH2), 38.3 (C-2′), 24.0 [CH(CH3)2], 12.5 (T CH3). 31P NMR (121 MHz, CDCl3): δ 18.2. HRMS: [M + H]+ calcd for C16H28N2O7P, 391.1629; found, 391.1628. 1′α-(Adenin-9-yl)-2′-deoxy-3′-O-diisopropylphosphonomethyl-L-threose (11c).11 A solution of 11a (2.17 g, 4.32 mmol) in satd NH3 in MeOH (300 mL) was stirred at rt for overnight. After removing the volatiles, the crude residue was purified by column chromatography (20:1 CH2Cl2/MeOH) to afford 11c (1.58 g, 92% yield) as a white solid. 1H NMR (300 MHz, CDCl3): δ 8.35 (s, 1H, H8), 8.31 (s, 1H, H-2), 6.48 (dd, J = 7.5, 2.4 Hz, 1H, H-1′), 6.10 (brs, 2H, NH2), 4.71−4.78 [m, 2H, CH(CH3)2], 4.45−4.48 (m, 1H, H-3′), 4.36 (d, J = 10.5 Hz, 1H, H-4a′), 4.06 (dd, J = 10.5, 4.3 Hz, 1H, H4b′), 3.71−3.75 (m, 2H, PCH2), 2.57−2.73 (m, 2H, H-2′), 1.28−1.35 [m, 12H, CH(CH3)2]. 13C NMR (75 MHz, CDCl3): δ 155.5 (C-6), 153.0 (C-2), 149.7 (C-4), 139.5 (C-8), 119.5 (C-5), 83.4 (C-1′), 80.5 (d, 3JP,C = 11.2 Hz, C-3′), 73.7 (C-4′), 71.4, 71.3 [CH(CH3)2], 64.1 (d, 1JP,C = 169.6 Hz, 2H, PCH2), 38.5 (C-2′), 24.0, 23.9 [CH(CH3)2]. 31 P NMR (121 MHz, CDCl3): δ 18.2. HRMS: [M + H]+ calcd for C16H27N5O5P, 400.1744; found, 400.1746. 1′α-(Adenin-9-yl)-2′-deoxy-3′-O-phosphonomethyl- L threose Triethylammonium Salt (PMDTA, 12a).11 To a solution of 11c (30 mg, 0.075 mmol) and 2,6-lutidine (0.07 mL, 0.60 mmol) in dry CH3CN (3 mL) was added bromotrimethylsilane (0.08 mL, 0.6 mmol) at 0 °C. The reaction mixture was stirred at rt overnight and quenched with 1.0 M TEAB solution (1 mL). The solvent was removed under reduced pressure. The residue was partitioned between water and EtOAc/ether (1:1), the water layer was lyophilized, and the residue was first purified by chromatography on a silica gel column (10:1:0 to 10:5:1, CH2Cl2/MeOH/1 M TEAB) to give the crude

1′α-(N6-Benzoyladenin-9-yl)-2′-O-benzoyl-3′-O-diisopropylphosphonomethyl-L-threose (9a).11 This compound was prepared as described for 5. Obtained from 8a (0.345 g, 0.77 mmol), triflate diisopropylphosphonomethanol (0.254 g, 0.77 mmol), and NaH (60% in mineral oil, 0.102 g, 2.56 mmol) as a colorless oil. Yield 80% (0.385 g). 1H NMR (300 MHz, CDCl3): δ 9.32 (brs, 1H, NH), 8.80 (s, 1H, H-2), 8.51 (s, 1H, H-8), 8.03−8.08 (m, 1H, Ph), 7.47−7.66 (m, 6H, Ph), 6.56 (s, 1H, H-1′), 5.80 (s, 1H, H-2′), 4.71−4.82 [m, 2H, CH(CH3)2], 4.49−4.51 (m, 2H, H-3′ and H-4a′), 4.35−4.40 (m, 1H, H-4b′), 3.95−4.01 (m, 2H, PCH2), 1.31−1.36 [m, 12H, CH(CH3)2]. 13 C NMR (75 MHz, CDCl3): δ 165.1, 164.6 (PhCO), 152.7 (C-2), 151.5 (C-4), 149.5 (C-6), 141.9 (C-8), 133.9, 133.7, 132.6, 129.9, 128.7, 128.6, 128.4, 127.9 (Ph), 122.7 (C-5), 87.8 (C-1′), 83.7 (d, 3JP,C = 9.5 Hz, C-3′), 80.2 (C-2′), 73.5 (C-4′), 71.5, 71.4 [CH(CH3)2], 64.6 (d, 1JP,C = 169.4 Hz, PCH2), 23.9 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 17.8. HRMS: [M + H]+ calcd for C30H35N5O8P, 624.2218; found, 624.2217. 1′α-(Thymin-1-yl)-2′-O-benzoyl-3′-O-diisopropylphosphonomethyl-L-threose (9b).11 This compound was prepared as described for 5. Obtained from 8b (2.0 g, 6.02 mmol), triflate diisopropylphosphonomethanol (1.1 g, 6.62 mmol), and NaH (60% in mineral oil, 0.794 g, 19.86 mmol) as a white solid. Yield 85% (2.6 g). 1 H NMR (300 MHz, CDCl3): δ 9.25 (brs, 1H, NH), 8.01−8.04 (m, 2H, Ph), 7.58−7.60 (m, 1H, Ph), 7.43−7.48 (m, 3H, Ph and H-6), 6.26 (d, J = 1.5 Hz, 1H, H-1′), 5.39 (s, 1H, H-2′), 4.74−4.81 [m, 2H, CH(CH3)2], 4.39 (d, J = 10.7 Hz, 1H, H-4a′), 4.26 (d, J = 3.6 Hz, H3′), 4.13 (dd, J = 10.7, 3.7 Hz, H-4b′), 3.88−4.06 (m, 2H, PCH2), 1.97 (d, J = 0.9 Hz, 3H, T CH3), 1.32−1.35 [m, 12H, CH(CH3)2]. 13C NMR (75 MHz, CDCl3): δ 165.2 (PhCO), 163.8 (C-4), 150.4 (C-2), 136.0 (C-6), 133.8, 129.8, 128.5 (Ph), 111.3 (C-5), 89.0 (C-1′), 83.7 (d, 3JP,C = 10.9 Hz, C-3′), 80.2 (C-2′), 72.7 (C-4′), 71.4, 71.3 [CH(CH3)2], 64.5 (d, 1JP,C = 168.6 Hz, PCH2), 23.9 [CH(CH3)2], 12.5 (T CH3). 31P NMR (121 MHz, CDCl3): δ 17.9. HRMS: [M + H]+ calcd for C23H32N2O9P, 511.1840; found, 511.1853. 1′α-(N6-Benzoyladenin-9-yl)-3′-O-diisopropylphosphonomethyl-L-threose (10a). To a suspension of 9a (8.65 g, 13.87 mmol) in 280 mL of THF/MeOH/H2O 5:4:1 at 0 °C was added 6.94 mL (13.87 mmol) of 2 N aq NaOH. After 20 min, the solution was neutralized with acetic acid. The mixture was concentrated under reduced pressure. The residue was partitioned between water and EtOAc. The organic layer was washed with water and brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (30:1 CH2Cl2/MeOH) to afford 10a (5.7 g, 80% yield) as a white solid. 1H NMR (300 MHz, CDCl3): δ 9.53 (brs, 1H, NH), 8.66 (s, 1H, H-2), 8.36 (1H, H-8), 7.98−8.00 (m, 2H, Ph), 7.44−7.58 (m, 3H, Ph), 6.21 (d, J = 1.7 Hz, 1H, H-1′), 4.80 (s, 1H, H-2′), 4.63−4.73 [m, 2H, CH(CH3)2], 4.28−4.34 (m, 3H, H-4′ and H-3′), 3.82 (d, J = 8.8 Hz, 2H, PCH2), 1.24−1.31 [m, 12H, CH(CH3)2]. 13C NMR (75 MHz, CDCl3): δ 164.8 (PhCO), 152.1 (C-2), 151.2 (C-4), 149.1 (C-6), 142.1 (C-8), 133.4, 132.5, 128.5, 127.8 (Ph), 122.7 (C-5), 90.5 (C-1′), 85.9 (d, 3JP,C = 9.7 Hz, C3′), 78.9 (C-2′), 72.5 (C-4′), 71.4 [CH(CH3)2], 64.4 (d, 1JP,C = 169.5 Hz, PCH2), 23.8, 23.7 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 18.4. HRMS: [M + H]+ calcd for C23H31N5O7P, 520.1955; found, 520.1961. 1′α-(Thymin-1-yl)-3′-O-diisopropylphosphonomethyl- L threose (10b).11 A solution of 9b (0.186 g, 0.364 mmol) in 0.4 mL of acetonitrile was treated with LiOH (8.7 mg, 0.364 mmol) in 0.2 mL of water and 0.4 mL of MeOH. The mixture was stirred at rt for 0.5 h, and the reaction mixture was neutralized with acetic acid. The solvent was removed under reduced pressure, and the residue was partitioned between water and EtOAc. The organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (20:1 CH2Cl2/ MeOH) to afford 10b (126 mg, 85% yield) as a white foam. 1H NMR (300 MHz, CDCl3): δ 10.59 (brs, 1H, NH), 7.41 (d, J = 1.2 Hz, H-6), 5.84 (s, 1H, H-1′), 5.67 (brs, 1H, OH), 4.64−4.77 [m, 2H, CH(CH3)2], 4.39 (s, 1H, H-2′), 4.26−4.36 (m, 2H, H-4′), 4.14 (d, J = 2.4 Hz, H-3′), 3.75 (d, J = 9.1 Hz, 2H, PCH2), 1.93 (d, J = 1.0 Hz, 3H, T CH3), 1.28−1.33 [m, 12H, CH(CH3)2]. 13C NMR (75 MHz, 9524



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(d, J = 10.0 Hz, H-4a′), 4.30−4.33 (m, 2H, H-4b′ and H-3′), 3.85− 3.95 (m, 2H, PCH2), 1.31−1.34 [m, 12H, CH(CH3)2]. 13C NMR (125 MHz, CDCl3): δ 165.1 (PhCO), 162.4 (C-4), 155.0 (C-2), 145.1 (C-6), 133.7, 133.2, 129.9, 129.0, 128.8, 128.5, 127.5 (Ph), 96.2 (C-5), 90.9 (C-1′), 83.3 (3JP,C = 9.6 Hz, C-3′), 79.6 (C-2′), 74.5 (C-4′), 73.9 (C-3′), 71.3 [CH(CH3)2], 64.6 (1JP,C = 168.5 Hz, PCH2), 24.0 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 17.9. HRMS: [M + H]+ calcd for C29H35N3O9P, 600.2105; found, 600.2130. 1′α-(N4-Benzoylcytosin-1-yl)-3′-O-diisopropylphosphonomethyl-L-threose (16). This compound was prepared as described for 10a. Obtained from 15 (260 mg, 0.43 mmol) as a white solid. Yield 80% (72 mg). 1H NMR (500 MHz, CDCl3): δ 9.31 (brs, 1H, NH), 8.02 (d, J = 7.5 Hz, 1H, H-6), 7.93−7.95 (m, 2H, Ph), 7.57−7.60 (m, 3H, H-5 and Ph), 7.49 (t, J = 8.0 Hz, 2H, Ph), 5.87 (s, 1H, H-1′), 5.52 (brs, 1H, OH), 4.64−4.72 [m, 2H, CH(CH3)2], 4.52 (s, 1H, H-2′), 4.34−4.39 (m, 2H, H-4′), 4.18 (s, 1H, H-3′), 3.73−3.87 (m, 2H, PCH2), 1.26−1.30 [m, 12H, CH(CH3)2]. 13C NMR (125 MHz, CDCl3): δ 166.7 (PhCO), 162.7 (C-4), 155.8 (C-2), 144.8 (C-6), 133.1, 132.9, 128.8, 127.8 (Ph), 96.3 (C-5), 94.6 (C-1′), 85.5 (3JP,C = 10.4 Hz, C-3′), 78.7 (C-2′), 74.0 (C-4′), 71.2 [CH(CH3)2], 64.2 (d, 1 JP,C = 168.8 Hz, PCH2), 23.9 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 18.5. HRMS: [M + H]+ calcd for C22H31N3O8P, 496.1843; found, 496.1856. 1′α-(N 4 -Benzoylcytosin-1-yl)-2′-deoxy-3′-O-diisopropylphosphonomethyl-L-threose (17). This compound was prepared as described for 11a or 5. Obtained from 16 (60 mg, 0.12 mmol) or 20 (137 mg, 0.46 mmol) as a white foam, Yield 70% (41 mg) and 72% (156 mg), respectively. 1H NMR (300 MHz, CDCl3): δ 8.63 (brs, 1H, NH), 8.09 (d, J = 7.2 Hz, 1H, H-6), 7.91 (d, J = 7.6 Hz, 2H, Ph), 7.60−7.66 (m, 1H, Ph), 7.51−7.56 (m, 3H, H-5 and Ph), 6.21 (dd, J = 7.1 Hz, 1.8 Hz, 1H, H-1′), 4.66−4.79 [m, 2H, CH(CH3)2], 4.49 (dd, J = 10.5, 1.2 Hz, 1H, H-4a′), 4.39 (t, J = 4.0 Hz, 1H, H-3′), 4.06 (dd, J = 10.5, 3.5 Hz, 1H, H-4b′), 3.57−3.70 (m, 2H, PCH2), 2.52−2.61 (m, 1H, H-2a′), 2.46 (d, J = 15.2 Hz, 1H, H-2b′), 1.28−1.36 [m, 12H, CH(CH3)2]. 13C NMR (75 MHz, CDCl3): δ 166.1 (PhCO), 162.1 (C-4), 155.4 (C-2), 145.3 (C-6), 133.1, 129.1, 127.4 (Ph), 95.6 (C-5), 87.8 (C-1′), 80.2 (3JP,C = 9.7 Hz, C-3′), 75.1 (C-4′), 71.2 [CH(CH3)2], 63.8 (d, 1JP,C = 169.2 Hz, PCH2), 38.7 (C-2′), 24.0 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 18.3. HRMS: [M + H]+ calcd for C22H31N3O7P, 480.1894; found, 480.1889. 1′α-(N4-Benzoylcytosin-1-yl)-3′-O-tert-butyldimethylsilyl-Lthreose (18). This compound was prepared as described for 10a. Obtained from 13 (2.65 g, 4.95 mmol) as a white foam. Yield 71% (1.52 g). 1H NMR (300 MHz, CDCl3): δ 9.25 (brs, 1H, NH), 8.05 (d, J = 7.5 Hz, 1H, H-6), 7.92 (d, J = 7.6 Hz, 2H, Ph), 7.44−7.58 (m, 4H, Ph and H-5), 5.88 (s, 2H, H-1′ and OH), 4.40 (dd, J = 9.4, 3.0 Hz, 1H, H-4a′), 4.30 (s, 1H, H-2′), 4.25 (s, 1H, H-3′), 4.16 (d, J = 9.4 Hz, 1H, H-4b′), 0.75 [s, 9H, C(CH3)3], 0.03 (s, 3H, SiCH3), −0.07 (s, 3H, SiCH3). 13C NMR (75 MHz, CDCl3): δ 166.5 (PhCO), 162.4 (C-2), 155.7 (C-4), 145.3 (C-6), 13 2.9, 132.8, 128.7, 127.6 (Ph), 95.8 (C-5), 94.5 (C-1′), 80.8 (C-2′), 77.6 (C-4′), 76.3 (C-3′), 25.4 [C(CH3)3], 17.7 [C(CH3)3], −5.2, −5.3 (SiCH3). HRMS: [M + H]+ calcd for C21H30N3O5Si, 432.1949; found, 432.1949. 1′α-(N4-Benzoylcytosin-1-yl)-2′-deoxy-3′-O-tert-butyldimethylsilyl-L-threose (19). This compound was prepared as described for 11a. Obtained from 18 (1.24 g, 2.87 mmol) as a white foam. Yield 91% (1.1 g). 1H NMR (300 MHz, CDCl3): δ 8.98 (brs, 1H, NH), 8.10 (d, J = 7.5 Hz, 1H, H-6), 7.91 (d, J = 7.2 Hz, 2H, Ph), 7.56−7.61 (m, 1H, Ph), 7.46−7.51 (m, 3H, Ph and H-5), 6.15 (d, J = 6.6 Hz, 1H, H1′), 4.47 (t, J = 3.7 Hz, 1H, H-3′), 4.20 (d, J = 9.6 Hz, 1H, H-4a′), 4.16 (dd, J = 9.6, 3.3 Hz, 1H, H-4b′), 2.23−2.53 (m, 2H, H-2′), 0.79 [s, 9H, C(CH3)3], 0.04 (s, 3H, SiCH3), −0.05 (s, 3H, SiCH3). 13C NMR (75 MHz, CDCl3): δ 166.6 (PhCO), 162.1 (C-4), 155.0 (C-2), 145.5 (C6), 132.9, 128.8, 128.5, 127.5 (Ph), 95.4 (C-5), 88.1 (C-1′), 78.8 (C4′), 71.0 (C-3′), 41.9 (C-2′), 25.5 [C(CH3)3], 17.7 [C(CH3)3], −5.1, −5.2 (SiCH3). HRMS: [M + H]+ calcd for C21H30N3O4Si, 416.2000; found, 416.2003. 1′α-(N4-Benzoylcytosin-1-yl)-2′-deoxy-L-threose (20). This compound was prepared as described for 8a. Obtained from 19 (1.14 g, 2.74 mmol) as a white foam. Yield 79% (0.65 g). 1H NMR

product. Further purification using preparative reverse phase HPLC with gradient CH3CN in 0.05 M TEAB solution from 2% to 30% gave 12a (19 mg, 60%) as a white foam. 1H NMR (500 MHz, D2O): δ 8.55 (s, 1H, H-8), 8.15 (s, 1H, H-2), 6.35 (dd, J = 8.2, 2.2 Hz, 1H, H-1′), 4.51−4.53 (m, 1H, H-3′), 4.31 (d, J = 10.4 Hz, 1H, H-4a′), 4.06 (dd, J = 10.3, 4.1 Hz, 1H, H-4b′), 3.45−3.53 (m, 2H, PCH2), 2.75−2.81 (m, 1H, H-2a′), 2.64 (d, J = 15.1 Hz, 1H, H-2b′). 13C NMR (125 MHz, D2O): δ 155.0 (C-6), 152.0 (C-2), 148.2 (C-4), 140.9 (C-8), 117.9 (C-5), 82.9 (C-1′), 79.0 (d, 3JP,C = 10.3 Hz, C-3′), 73.3 (C-4′), 66.6 (d, 1JP,C = 151.3 Hz, PCH2), 36.5 (C-2′). 31P NMR (121 MHz, D2O): δ 13.1. HRMS: [M − H]− calcd for C10H13N5O5P, 314.0660; found, 314.0657. 1′α-(Thymin-1-yl)-2′-deoxy-3′-O-phosphonomethyl- L threose Triethylammonium Salt (PMDTT, 12b).11 This compound was prepared as described for 12a. Obtained from 11b (205 mg, 0.53 mmol), 2,6-lutidine (0.49 mL, 4.20 mmol), and iodotrimethylsilane (0.60 mL, 4.20 mmol) as a white foam. Yield 65% (138 mg). 1H NMR (300 MHz, D2O): δ 7.70 (s, 1H, H-6), 6.11 (d, J = 6.3 Hz, 1H, H-1′), 4.30 (brs, 2H, H-4a′ and H-3′), 3.86 (d, J = 7.5 Hz, H-4b′), 3.51 (d, J = 9.1 Hz, PCH2), 2.50−2.55 (m, 1H, H-2a′), 2.20 (d, J = 15.1 Hz, 1H, H-2b′), 1.82 (s, 3H, T CH3). 13C NMR (75 MHz, D2O): δ 166.3 (C-4), 151.4 (C-2), 138.2 (C-6), 110.7 (C-5), 85.1 (C-1′), 79.4 (d, 3JP,C = 11.5 Hz, C-3′), 73.4 (C-4′), 64.6 (d, 1JP,C = 157.6 Hz, PCH2), 36.4 (C-2′), 11.4 (T CH3). 31P NMR (121 MHz, D2O): δ 15.3. HRMS: [M − H]− calcd for C10H14N2O7P, 305.0544; found, 305.0555. 1′α-(N4-Benzoylcytosin-1-yl)-2′-O-benzoyl-3′-O-tert-butyldimethylsilyl-L-threose (13). This compound was prepared using a similar procedure as described for 7a. Obtained from 3 (3.17 g, 8.3 mmol), N4-benzoylcytosine (2.15 g, 10.0 mmol), BSA (5.09 mL, 20.8 mmol), and TMSOTf (4.52 mL, 25.0 mmol) as a white solid. Yield 84% (3.76 g). 1H NMR (500 MHz, CDCl3): δ 8.67 (s, 1H, NH), 8.11 (d, J = 7.4 Hz, H-6), 8.06−8.08 (m, 2H, Ph), 7.90−7.92 (m, 2H, Ph and H-5), 7.45−7.62 (m, 7H, Ph), 6.26 (s, 1H, H-1′), 5.37 (s, 1H, H2′), 4.41 (d, J = 2.8 Hz, 1H, H-3′), 4.30−4.35 (m, 2H, H-4′), 0.85 [s, 9H, C(CH3)3], 0.16 (s, 3H, SiCH3), 0.09 (s, 3H, SiCH3). 13C NMR (125 MHz, CDCl3): δ 165.0 (PhCO), 162.3 (PhCO), 154.9 (C-2), 145.5 (C-6), 133.6, 133.2, 129.9, 129.0, 128.5 (Ph), 127.5 (C-5), 91.1 (C-1′), 82.0 (C-2′), 77.6 (C-4′), 74.4 (C-3′), 25.6 [C(CH3)3], 17.9 [C(CH3)3]. HRMS: [M + H]+ calcd for C28H34N3O6Si, 536.2211; found, 536.2225. 1′α-(N4-Benzoylcytosin-1-yl)-2′-O-benzoyl-L-threose (14).18 This compound was prepared as described for 8a. Obtained from 13 (3.8 g, 7.0 mmol) and triethylamine trihydrofluoride (2.3 mL, 14 mmol) as a white solid. Yield 97% (2.9 g). 1H NMR (600 MHz, DMSO-d6): δ 11.28 (s, 1H, NH), 8.22 (d, J = 7.5 Hz, 1H, H-6), 8.01− 8.05 (m, 4H, Ph), 7.70−7.73 (m, 1H, Ph), 7.63 (t, J = 7.4 Hz, 1H, Ph), 7.56−7.60 (m, 2H, Ph), 7.51−7.54 (m, 2H, Ph), 7.42 (d, J = 7.7 Hz, 1H, H-5), 6.00 (d, J = 0.5 Hz, 1H, H-1′), 5.83 (d, J = 2.8 Hz, 1H, OH), 5.40 (s, 1H, H-2′), 4.33−4.36 (m, H-3′ and H-4a′), 4.29 (dd, J = 9.9 Hz, 3.9 Hz, H-4b′). 13C NMR (150 MHz, DMSO-d6): δ 167.4 (PhCO), 164.6 (PhCO), 163.5 (C-4), 154.6 (C-2), 145.8 (C-6), 134.0, 133.2, 132.8, 129.6, 129.0, 128.5 (Ph), 95.7 (C-5), 91.0 (C-1′), 81.6 (C-2′), 76.7 (C-4′), 72.5 (C-3′). HRMS: [M + H]+ calcd for C22H20N3O6, 422.1347; found, 422.1346. 1′α-(N4-Benzoylcytosin-1-yl)-2′-O-benzoyl-3′-O-diisopropylphosphonomethyl-L-threose (15). A suspension of N6-benzoylcytosine (97 mg, 0.45 mmol) in anhydrous CH3CN (2 mL) was treated with BSA (0.25 mL, 1.01 mmol) and heated to 65 °C. After stirring for 1 h, a solution of 6 (100 mg, 0.23 mmol) in anhydrous CH3CN (1 mL) was added followed by TMSOTf (0.12 mL, 0.68 mmol) at 0 °C. The mixture was stirred at 0 °C for overnight. The reaction mixture was poured into an ice-cold 50 mL of satd aq NaHCO3 soln/EtOAc 1:1. The organic layer was washed with brine, dried over Na2SO4, and concentrated. The residue was purified by column chromatography (50:1, DCM/MeOH) gave 15 (54 mg, 40% yield) as a white foam. 1H NMR (500 MHz, CDCl3): δ 8.72 (brs, 1H, NH), 8.04−8.06 (m, 3H, H-6 and Ph), 7.90 (d, J = 7.4 Hz, Ph), 7.59−7.63 (m, 3H, H-5 and Ph), 7.52 (t, J = 7.9 Hz, 2H, Ph), 7.47 (t, J = 8.1 Hz, 2H, Ph), 6.32 (s, 1H, H-1′), 5.51 (s, 1H, H-2′), 4.70−4.76 [m, 2H, CH(CH3)2], 4.52 9525



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(300 MHz, CDCl3): δ 9.09 (brs, 1H, NH), 8.09 (d, J = 7.5 Hz, 1H, H6), 7.88 (d, J = 7.2 Hz, 2H, Ph), 7.55−7.60 (m, 1H, Ph), 7.40−7.50 (m, 3H, Ph and H-5), 6.00 (d, J = 6.6 Hz, 1H, H-1′), 4.61 (t, J = 3.7 Hz, 1H, H-3′), 4.33 (d, J = 9.6 Hz, 1H, H-4a′), 4.09 (dd, J = 9.6, 3.3 Hz, 1H, H-4b′), 2.71 (d, J = 14.9 Hz, 1H, H-2a′), 2.42−2.50 (m, 1H, H-2b′). 13C NMR (75 MHz, CDCl3): δ 166.4 (PhCO), 162.2 (C-4), 155.2 (C-2), 145.6 (C-6), 132.9, 128.8, 127.7 (Ph), 95.6 (C-5), 89.1 (C-1′), 78.5 (C-4′), 70.0 (C-3′), 41.3 (C-2′). HRMS: [M + H]+ calcd for C15H16N3O4, 302.1135; found, 302.1140. 1′α-(Cytosin-1-yl)-2′-deoxy-3′-O-diisopropylphosphonomethyl-L-threose (21).11 This compound was prepared as described for 11c. Obtained from 17 (177 mg, 0.37 mmol) as a white foam. Yield 88% (122 mg). 1H NMR (300 MHz, CDCl3): δ 7.68 (d, J = 7.4 Hz, 1H, H-6), 6.19 (dd, J = 7.4, 1.9 Hz, 1H, H-1′), 5.78 (d, J = 7.5 Hz, 1H, H-5), 4.61−4.76 [m, 2H, CH(CH3)2], 4.35 (dd, J = 10.4, 1.3 Hz, 1H, H-4a′), 4.27−4.30 (m, 1H, H-3′), 3.93 (dd, J = 10.4, 3.6 Hz, 1H, H-4b′), 3.63 (d, J = 9.2 Hz, 2H, PCH2), 2.44−2.54 (m, 1H, H-2a′), 2.89 (d, J = 15.1 Hz, 1H, H-2b′), 1.26−1.33 [m, 12H, CH(CH3)2]. 13 C NMR (75 MHz, CDCl3): δ 165.8 (C-4), 156.1 (C-2), 141.7 (C6), 93.6 (C-5), 86.6 (C-1′), 80.3 (d, 3JP,C = 11.6 Hz, C-3′), 74.1 (C4′), 71.3, 71.1 [CH(CH3)2], 63.7 (d, 1JP,C = 170.2 Hz, PCH2), 38.6 (C2′), 24.0 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 18.4. HRMS: [M + H]+ calcd for C15H27N3O6P, 376.1632; found, 376.1629. 1′α-(Cytosin-1-yl)-2′-deoxy-3′-O-phosphonomethyl- L threose Triethylammonium Salt (PMDTC, 22).11 This compound was prepared as described for 12a. Obtained from 21 (120 mg, 0.32 mmol), 2,6-lutidine (0.30 mL, 2.56 mmol), and iodotrimethylsilane (0.36 mL, 2.56 mmol) as a white foam. Yield 48% (60 mg). 1H NMR (600 MHz, D2O): δ 7.95 (d, J = 7.5 Hz, 1H, H-6), 6.19 (dd, J = 8.0, 2.0 Hz, 1H, H-1′), 6.04 (d, J = 7.6 Hz, 1H, H-5), 4.40 (d, J = 10.2 Hz, 1H, H-4a′), 4.36−4.38 (m, 1H, H-3′), 3.95 (dd, J = 10.4, 3.6 Hz, 1H, H-4b′), 3.45−3.52 (m, 2H, PCH2), 2.52−2.58 (m, 1H, H-2a′), 2.26 (d, J = 15.2 Hz, 1H, H-2b′). 13C NMR (150 MHz, D2O): δ 165.8 (C4), 157.3 (C-2), 142.4 (C-6), 95.6 (C-5), 85.9 (C-1′), 79.2 (d, 3JP,C = 11.6 Hz, C-3′), 73.6 (C-4′), 64.9 (d, 1JP,C = 155.3 Hz, PCH2), 36.6 (C2′). 31P NMR (121 MHz, D2O): δ 14.6. HRMS: [M − H]− calcd for C9H13N3O6P, 290.0547; found, 290.0545. 1′α-(N2-Acetyl-O6-diphenylcarbamoyl-9-yl)-2′-O-benzoyl-3′O-tert-butyldimethylsilyl-L-threose (22a). A suspension of N2acetyl-O6-(diphenylcarbamoyl)guanine (9.94 g, 25.6 mmol) in 60 mL of (CH2Cl)2 was treated with BSA (13.77 mL, 56.3 mmol) and heated to 80 °C for 1 h. The solvent was evaporated and replaced with 30 mL of dry toluene and TMSOTf (5.09 mL, 28.2 mmol), and a solution of 3 (4.87 g, 12.8 mmol) in 30 mL of dry toluene was added. The mixture was heated to 80 °C for 2 h, cooled to rt, and poured into an ice-cold mixture of EtOAc and aq satd NaHCO3 with stirring. The organic layer was separated and washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (2:1 to 3:2, hexane/EtOAc) gave 22a (6.0 g, 66% yield) as a white foam. 1H NMR (500 MHz, CDCl3): δ 8.34 (s, 1H, H-8), 8.05−8.07 (m, 2H, Ph), 7.95 (brs, 1H, NH), 7.65−7.24 (m, 13H, Ph), 6.31 (s, 1H, H-1′), 5.55 (s, 1H, H-2′), 4.54 (brs, 1H, H-3′), 4.34 (dd, J = 9.9, 3.7 Hz, 1H, H-4a′), 4.30 (d, J = 9.8 Hz, 1H, H-4b′), 2.53 (s, 3H, CH3CO), 0.88 [s, 9H, C(CH3)3], 0.16 (s, 3H, SiCH3), 0.09 (s, 3H, SiCH3). 13C NMR (125 MHz, CDCl3): δ 171.4 (CH3CO), 165.0 (PhCO), 156.2, 154.4(C-4), 152.2, 150.2, 142.6 (C8), 134.0, 129.8, 129.2, 128.7, 127.2 (Ph), 120.6 (C-5), 88.4 (C-1′), 82.7 (C-2′), 76.6 (C-4′), 75.1 (C-3′), 25.6 [C(CH3)3], 21.0 (CH3CO), 14.2 [C(CH3)3]. HRMS: [M + H]+ calcd for C37H41N6O7Si, 709.2800; found, 709.2806. 1′α-(2-Amino-6-chloropurin-9-yl)-2′-O-benzoyl-3′-O-tert-butyldimethylsilyl-L-threose (22b). To a solution of 3 (0.35g, 0.92 mmol), 2-amino-6-chloro-9H-purine (0.17 g, 1.01 mmol), and DBU (0.41 mL, 2.76 mmol) in dry MeCN (9 mL) was added dropwise TMSOTf (0.66 mL, 3.68 mmol) at 0 °C. The resulting clear-brown solution was stirred for 1.5 h at 70 °C, after which it was cooled to room temperature and aq satd NaHCO3 (20 mL) was added. The aqueous phase was extracted with EtOAc, dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by column chromatography (10:1 to 5:1, hexane/EtOAc) to afford 22b

(0.30 g, 67%) as a white foam. 1H NMR (300 MHz, CDCl3): δ 8.22 (s, 1H, H-8), 8.03 (d, J = 7.2 Hz, 2H, Ph), 7.60 (t, J = 7.3 Hz, 1H, Ph), 7.45 (t, J = 7.5 Hz, 2H, Ph), 6.29 (s, 1H, H-1′), 5.54 (s, 1H, H-2′), 5.50 (brs, 2H, NH2), 4.52 (s, 1H, H-3′), 4.33 (dd, J = 9.8, 3.7 Hz, 1H, H-4a′), 4.25 (d, J = 9.8 Hz, 1H, H-4b′). 0.88 [s, 9H, C(CH3)3], 0.15 (s, 3H, SiCH3), 0.11 (s, 3H, SiCH3). 13C NMR (75 MHz, CDCl3): δ 164.9 (PhCO), 159.1 (C-2), 153.2 (C-4), 151.0 (C-6), 141.0 (C-8), 133.7, 129.7, 128.6, 128.5 (Ph), 125.0 (C-5), 87.7 (C-1′), 82.5 (C-2′), 76.5 (C-4′), 75.1 (C-3′), 25.5 [C(CH3)3], 17.8 [C(CH3)3], −5.0, −5.3 (SiCH3). HRMS: [M + H]+ calcd for C22H29ClN5O4Si, 490.1672; found, 490.1681. 1′α-(N2-Acetyl-O6-diphenylcarbamoyl-9-yl)-2′-O-benzoyl-Lthreose (23a).18 This compound was prepared as described for 8a. Obtained from 22a (6.0 g, 8.5 mmol) and triethylamine trihydrofluoride (2.76 mL, 16.9 mmol) as a white solid. Yield 96% (4.8 g). 1H NMR (500 MHz, CDCl3): δ 8.45 (s, 1H, NH), 8.06 (s, 1H, H-8), 8.02−8.04 (m, 2H, Ph), 7.58−7.62 (m, 1H, Ph), 7.34−7.48 (m, 10H, Ph), 7.36 (t, J = 7.8 Hz, 1H, Ph), 7.25 (brs, 1H, Ph), 5.93 (d, J = 1.7 Hz, 1H, H-1′), 5.86 (s, 1H, H-2′), 5.58 (brs, 1H, OH), 4.71−4.73 (m, 1H, H-3′), 4.44 (dd, J = 9.7, 4.3 Hz, 1H, H-4a′), 4.33 (dd, J = 9.7, 5.6 Hz, 1H, H-4b′), 2.24 (s, 3H, CH3CO). 13C NMR (125 MHz, CDCl3): δ 168.6 (CH3CO), 166.2 (PhCO), 156.3, 154.0 (C-4), 151.4, 150.2, 144.5 (C-8), 141.6, 133.9, 129.9, 129.2, 128.7, 128.6, 121.9 (C-5), 91.0 (C-1′), 84.5 (C-2′), 75.5 (C-4′), 75.4 (C-3′), 24.88 (CH3CO). HRMS: [M + H]+ calcd for C31H27N6O7, 595.1936; found, 595.1943. 1′α-(2-Amino-6-chloropurin-9-yl)-2′-O-benzoyl-L-threose (23b). This compound was prepared as described for 8a. Obtained from 22b (220 mg, 0.45 mmol) and triethylamine trihydrofluoride (0.15 mL, 0.90 mmol) as a white foam. Yield 97% (165 mg). 1H NMR (300 MHz, CDCl3): δ 7.98 (s, 1H, H-8), 8.01 (d, J = 7.2 Hz, 2H, Ph), 7.63 (t, J = 7.3 Hz, 1H, Ph), 7.47 (t, J = 7.5 Hz, 2H, Ph), 5.92 (d, J = 1.7 Hz, 1H, H-1′), 5.58 (s, 1H, H-2′), 5.42 (brs, 2H, NH2), 4.57 (d, J = 3.2 Hz, 1H, H-3′), 4.33 (d, J = 9.8 Hz, 1H, H-4a′), 4.22 (dd, J = 9.8, 3.7 Hz, 1H, H-4b′). 13C NMR (75 MHz, CDCl3): δ 165.7 (PhCO), 158.4 (C-2), 152.5 (C-4), 151.9 (C-6), 142.1 (C-8), 134.0, 129.8, 128.6, 128.4 (Ph), 126.0 (C-5), 90.4 (C-1′), 83.8 (C-2′), 75.8 (C-4′), 74.6 (C-3′). HRMS: [M + H]+ calcd for C16H15ClN5O4, 376.0807; found, 376.0808. 1′α-(2-Amino-6-chloropurin-9-yl)-2′-O-benzoyl-3′-O-diisopropylphosphonomethyl-L-threose (24). This compound was prepared using a similar procedure as described for 5. Obtained from 23b (85 mg, 0.23 mmol), triflate diisopropylphosphonomethanol (148 mg, 0.45 mmol), and NaH (60% in mineral oil, 18 mg, 0.45 mmol) as a white foam. Yield 67% (84 mg). 1H NMR (300 MHz, CDCl3): δ 8.12 (s, 1H, H-8), 8.05 (d, J = 7.2 Hz, 2H, Ph), 7.64 (t, J = 7.4 Hz, 1H, Ph), 7.47 (t, J = 7.9 Hz, 2H, Ph), 6.22 (d, J = 1.3 Hz, 1H, H-1′), 5.85 (s, 1H, H-2′), 5.33 (brs, 2H, NH2), 4.71−4.82 [m, 2H, CH(CH3)2], 4.42−4.45 (m, 2H, H-3′ and H-4a′), 4.33 (dd, J = 10.7, 4.7 Hz, 1H, H4b′), 3.86−4.06 (m, 2H, PCH2), 1.30−1.37 [m, 12H, CH(CH3)2]. 13C NMR (75 MHz, CDCl3): δ 165.1 (PhCO), 159.2 (C-2), 153.4 (C-4), 151.4 (C-6), 141.1 (C-8), 134.0, 129.9, 128.7, 128.6 (Ph), 125.3 (C5), 88.2 (C-1′), 84.1 (d, 3JP,C = 10.6 Hz, C-3′), 79.8 (C-2′), 73.3 (C4′), 71.5 [CH(CH3)2], 64.7 (d, 1JP,C = 168.5 Hz, PCH2). 23.9 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 17.8. HRMS: [M + H]+ calcd for C23H30ClN5O7P, 554.1566; found, 554.1568. 1′α-(2-Amino-6-chloropurin-9-yl)-3′-O-diisopropylphosphonomethyl-L-threose (25). A solution of 24 (5.0 g, 9.03 mmol) in 2 N NH3 in MeOH (150 mL) was stirred at rt for 2 h. The mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (20:1 CH2Cl2/MeOH) to give compound 25 (3.0 g, 75% yield) as a white foam. 1H NMR (300 MHz, CDCl3): δ 8.02 (s, 1H, H-8), 5.91 (d, J = 2.7 Hz, 1H, H-1′), 5.72 (brs, 3H, NH2 and OH), 4.76 (s, 1H, H-2′), 4.62−4.73 [m, 2H, CH(CH3)2], 4.25− 4.29 (m, 3H, H-3′ and H-4′), 3.76−3.91 (m, 2H, PCH2), 1.26−1.33 [m, 12H, CH(CH3)2]. 13C NMR (75 MHz, CDCl3): δ 159.2 (C-2), 153.2 (C-4), 151.1 (C-6), 141.1 (C-8), 125.0 (C-5), 90.4 (C-1′), 86.4 (3JP,C = 8.9 Hz, C-3′), 78.9 (C-2′), 72.3 (C-4′), 71.8 [CH(CH3)2], 64.8 (1JP,C = 167.8 Hz, PCH2). 23.9 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 18.6. HRMS: [M + H]+ calcd for C16H26ClN5O6P, 450.1304; found, 450.1301. 9526



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mmol) in 4.5 mL of anhydrous CH2Cl2 at −78 °C was treated with diethylaminosulfur trifluoride (DAST) (0.12 mL, 0.908 mmol). The reaction mixture was allowed to warm to rt and was stirred overnight. The resulting solution was then carefully poured into an ice-cold satd aq sodium bicarbonate (10 mL) and extracted with CH2Cl2 (3 × 10 mL). The combined organic extracts were washed with brine, dried over NaSO4, filtered, and concentrated. The residue was purified by column chromatography (30:1 to 15:1, DCM/MeOH) to obtain 28 (60 mg, 63% yield) as a white foam. 1H NMR (500 MHz, CDCl3): δ 8.38 (s, 1H, H-2), 8.11 (s, 1H, H-8), 6.38 (d, 2JF,H = 19.5 Hz, 1H, H1′), 5.77 (brs, 2H, NH2), 5.48 (d, 1JF,H = 49.0 Hz, 1H, H-2′), 4.71− 4.75 [m, 2H, CH(CH3)2], 4.43−4.48 (m, 2H, H-3′ and H-4a′), 4.31− 4.34 (m, 1H, H-4b′), 3.76−3.82 (m, 2H, PCH2), 1.29−1.34 [m, 12H, CH(CH3)2]. 13C NMR (125 MHz, CDCl3): δ 155.4 (C-6), 153.3 (C2), 149.6 (C-4), 139.2 (C-8), 119.6 (C-5), 96.2 (d, 1JF,C = 186.4 Hz, C-2′), 87.9 (d, 2JF,C = 36.7 Hz, C-1′), 83.1 (C-3′, 3JP,C = 9.8 Hz, 2JF,C = 27.3 Hz), 72.8 (C-4′), 71.6 [CH(CH3)2, 2JP,C = 6.0 Hz] 64.9 (d, 1JP,C = 169.0 Hz, PCH2), 24.0 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 17.4. HRMS: [M + H]+ calcd for C16H26FN5O5P, 418.1650; found, 418.1647. (1′α,2′R)-1′-(Adenin-9-yl)-2′-deoxy-2′-fluoro-3′-O-phosphonomethyl-L-threose Triethylammonium Salt (2′-F-PMDTA, 31). This compound was prepared as described for 12a. Obtained from 30 (60 mg, 0.14 mmol), 2,6-lutidine (0.13 mL, 1.2 mmol), and bromotrimethylsilane (0.15 mL, 1.2 mmol) as a white foam. Yield 62% (48 mg). 1H NMR (600 MHz, D2O): δ 8.38 (s, 1H, H-8), 8.16 (s, 1H, H-2), 6.33 (d, 2JF,H = 19.5 Hz, 1H, H-1′), 5.57 (d, 1JF,H = 49.0 Hz, 1H, H-2′), 4.54−4.56 (m, 1H, H-3′), 4.46 (d, J = 12.0 Hz, H-4a′), 4.33−4.35 (m, 1H, H-4b′), 3.54−3.61 (m, 2H, PCH2). 13C NMR (150 MHz, D2O): δ 155.44 (C-6), 152.68 (C-2), 148.41 (C-4), 140.81 (C8), 118.19 (C-5), 96.67 (d, 1JF,C = 184.0 Hz, C-2′), 87.36 (d, 2JF,C = 37.7 Hz, C-1′), 82.12 (C-3′, 3JP,C = 10.6 Hz, 2JF,C = 27.2 Hz), 73.08 (C-4′), 67.32 (d, 1JP,C = 150.9 Hz, PCH2). 31P NMR (121 MHz, D2O): δ 12.7. HRMS: [M-H]− calcd for C10H12FN5O5P, 332.0565; found, 332.0558. 1′α-(Adenin-9-yl)-2′-deoxy-3′-O-{[N-(isopropyl-L-alaninate)](phenoxy)methylphosphonoamidate]}-L-threose (32a). Obtained from PMDTA (28 mg, 0.067 mmol, Et3N salt), L-alanine isopropyl ester hydrochloride (23 mg, 0.134 mmol), and PhOH (32 mg, 0.336 mmol) as a white solid. Yield 47% (16 mg). 1H NMR (500 MHz, acetone-d6): δ 8.44 (s, 1H, H-8), 8.20 (s, 1H, H-2), 7.16−7.36 (m, 5H, Ph), 6.45−6.47 (m, 1H, H-1′), 4.89−4.95 [m, 1H, CH(CH3)2], 4.66−4.85 (m, 1H, Ala-NH), 4.52−4.56 (m, 1H, H-3′), 4.37−4.42 (m, 1H, H-4a′), 4.02−4.08 (m, 4H, H-4b′, PCH2 and AlaCH), 2.76−2.84 (m, 1H, H-2a′), 2.56−2.63 (m, 1H, H-2b′), 1.25− 1.28 (m, 3H, Ala-CH3), 1.15−1.19 [m, 6H, CH(CH3)2]. 13C NMR (125 MHz, acetone-d6): δ 174.1, 173.7 (Ala-CO), 157.0 (C-6), 153.7, 153.6 (C-2), 151.7, 151.6 (Ph), 150.62, 150.59 (C-4), 140.6, 140.5 (C8), 130.4, 130.31, 125.32, 125.3, 121.8, 121.73, 121.67, 121.6 (Ph), 120.02, 119.98 (C-5), 84.2, 83.9 (C-1′), 81.88, 81.85 (3JP,C = 13.4, 13.0 Hz, C-3′), 74.36, 73.90 (C-4′), 69.14, 69.08 [CH(CH3)2], 65.99, 65.91 (1JP,C = 156.5, 155.7 Hz, PCH2), 50.5, 50.4 (Ala-CH), 39.10, 39.07 (C2′), 21.84, 21.81 [CH(CH3)2], 21.3, 21.0 (Ala-CH3). 31P NMR (121 MHz, acetone-d6): δ 21.6, 20.6. HRMS: [M + H]+ calcd for C22H30N6O6P, 505.1959; found, 505.1965. 1 ′ α - ( A d e n i n - 9 - y l) - 2 ′ - d e o x y- 3 ′ -O -{ [N -( di is oam yl - L aspartate)](phenoxy)methylphosphonoamidate}-L -threose (32b). Obtained from PMDTA (26 mg, 0.062 mmol, Et3N salt), Laspartic acid isoamyl ester hydrochloride (39 mg, 0.125 mmol), and PhOH (29 mg, 0.312 mmol) as a white solid. Yield 66% (27 mg). 1H NMR (500 MHz, acetone-d6): δ 8.40 (s, 1H, H-8), 8.19 (s, 1H, H-2), 7.14−7.37 (m, 5H, Ph), 6.70 (brs, 2H, NH2), 6.44−6.47 (m, 1H, H1′), 4.73−4.87 (m, 1H, Asp-NH), 4.51−4.57 (m, 1H, H-3′), 4.36− 4.42 (m, 2H, H-4a′ and Asp-CH), 3.97−4.12 [m, 7H, H-4b′, PCH2 and OCH2CH2CH(CH3)2], 2.53−2.84 (m, 4H, Asp-CH2, H-2′), 1.58−1.68 [m, 2H, OCH2CH2CH(CH3)2], 1.42−1.50 [m, 4H, OCH2CH2CH(CH3)2], 0.86−0.89 [m, 12H, OCH2CH2CH(CH3)2]. 13 C NMR (125 MHz, acetone-d6): δ 172.7, 172.4, 171.1 (Asp-CO), 157.0, 156.9 (C-6), 153.7, 153.6 (C-2), 151.68, 151.66 (Ph), 150.63 (C-4), 140.6, 140.5 (C-8), 130.4, 130.3, 125.40, 125.38, 121.82,

1′α-(2-Amino-6-chloropurin-9-yl)-2′-deoxy-3′-O-diisopropylphosphonomethyl-L-threose (26). This compound was prepared as described for 11a. Obtained from 25 (0.61 g, 1.35 mmol) as a white foam. Yield 68% (0.40 g). 1H NMR (300 MHz, CDCl3): δ 8.20 (s, 1H, H-8), 6.30 (dd, J = 6.8, 3.1 Hz, 1H, H-1′), 5.30 (brs, 2H, NH2), 4.65−4.83 [m, 2H, CH(CH3)2], 4.50 (brs, 1H, H-3′), 4.33 (d, J = 10.5 Hz, 1H, H-4a′), 4.04 (dd, J = 10.4, 4.2 Hz, 1H, H-4b′). 3.67−3.80 (m, 2H, PCH2), 2.56−2.70 (m, 2H, H-2′), 1.29−1.36 [m, 12H, CH(CH3)2]. 13C NMR (75 MHz, CDCl3): δ 158.7 (C-2), 153.2 (C-4), 151.1 (C-6), 141.0 (C-8), 125.1 (C-5), 83.1 (C-1′), 80.0 (d, 3 JP,C = 10.0 Hz, C-3′), 73.4 (C-4′), 71.1 [CH(CH3)2], 63.8 (d, 1JP,C = 167.8 Hz, PCH2), 37.8 (C-2′), 23.7 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 18.1. HRMS: [M + H]+ calcd for C16H26ClN5O5P, 434.1354; found, 434.1354. 1′α-(Guanin-9-yl)-2′-deoxy-3′-O-phosphonomethyl- L threose Triethylammonium Salt (PMDTG, 27). To a solution of 26 (184 mg, 0.42 mmol) in anhydrous CH3CN (9 mL) was added 2,6lutidine (0.40 mL, 3.39 mmol) and bromotrimethylsilane (0.45 mL, 3.39 mmol) at 0 °C. After stirring at room temperature for overnight, the solvent was removed under reduced pressure and coevaporated three times with anhydrous methanol (3 × 3 mL). The residue was dissolved in anhydrous MeOH (4 mL), and 2-mercaptoethanol (0.15 mL, 2.12 mmol) and NaOMe (5.4 M in MeOH, 0.39 mL, 2.12 mmol) were added. The mixture was refluxed for 19 h, cooled, quenched with 2 mL of 1 M TEAB buffer, and evaporated. The residue was partitioned between water and EtOAc, and the aqueous phase was lyophilized and the residue was purified by RP-HPLC running a gradient of CH3CN in 0.1 M TEAB buffer solution from 2% to 30% to afford 27 (90 mg, 49%) as a yellowish foam. 1H NMR (300 MHz, D2O): δ 8.05 (s, 1H, H-8), 6.06 (dd, J = 7.9, 2.0 Hz, 1H, H-1′), 4.39 (brs, 1H, H-3′), 4.23 (d, J = 10.4 Hz, 1H, H-4a′), 3.94 (dd, J = 10.4, 4.0 Hz, 1H, H-4b′). 3.55 (d, J = 9.3 Hz, 2H, PCH2), 2.60−2.70 (m, 1H, H-2a′), 2.49 (d, J = 15.4 Hz, H-2b′). 13C NMR (75 MHz, D2O): δ 158.3 (C-6), 153.4 (C-2), 150.7 (C-4), 138.1 (C-8), 115.4 (C-5), 82.8 (C-1′), 79.6 (3JP,C = 12.0 Hz, C-3′), 73.2 (C-4′), 65.2 (1JP,C = 155.2 Hz, PCH2), 36.6 (C-2′). 31P NMR (121 MHz, D2O): δ 15.1. HRMS: [M − H]− calcd for C10H13N5O6P, 330.0609; found, 330.0604. 1′α-(N6-Benzoyladenin-9-yl)-3′-O-diisophosphonomethyl-Lerythrose (29). To a solution of 10a (137 mg, 0.264 mmol) in 6 mL of dichloromethane was added a solution of Dess−Martin periodinane (15% in CH2Cl2, 0.82 mL, 0.396 mmol) at 0 °C. The reaction mixture was stirred at rt for 1 h (a white solid formed). To the reaction mixture was added a solution of satd aq sodium thiosulfate and satd aq NaHCO3 (1:1, 10 mL), and the mixture was stirred at rt for 20 min. After separation, the organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated to afford the crude ketone 28 (134 mg, 98%) as a white foam. The crude ketone was used for the next step without further purification. Crude ketone was taken up in 7.5 mL of dry methanol and treated with sodium borohydride (20 mg, 0.527 mmol) at 0 °C. The reaction mixture was stirred at rt for 1 h and quenched by the dropwise addition of satd aq NaHCO3 (2 mL). The reaction mixture was extracted with EtOAc, and the organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (20:1 DCM/MeOH) to obtain 29 (118 mg, 86% yield) as a white foam. 1H NMR (600 MHz, CDCl3): δ 9.18 (brs, 1H, NH), 8.77 (s, 1H, H-2), 8.46 (s, 1H, H-8), 8.04−8.05 (m, 2H, Ph), 7.60−7.61 (m, 1H, Ph), 7.52−7.54 (m, 2H, Ph), 6.48 (d, J = 4.8 Hz, 1H, H-1′), 5.05 (brs, 1H, OH), 4.71−4.81 [m, 2H, CH(CH3)2)], 4.38 (brs, 1H, H-2′), 4.35−4.38 (m, 1H, H-3′), 4.28−4.31 (m, 1H, H-4a′), 4.11−4.19 (m, 1H, H-4b′), 3.77−4.07 (m, 2H, PCH2), 1.26−1.36 [m, 12H, CH(CH3)2]. 13C NMR (150 MHz, CDCl3): δ 164.6 (PhCO), 152.6 (C-2), 151.8 (C-4), 149.0 (C-6), 143.5 (C-8), 134.0, 132.6, 128.8, 127.9 (Ph), 122.1 (C-5), 83.9 (C-1′), 81.6 (d, 3JP,C = 6.5 Hz, C-3′), 72.1, 71.9 [CH(CH3)2], 71.1 (C-2′), 70.1 (C-4′), 66.0 (d, 1JP,C = 168.6 Hz, PCH2), 24.0 [CH(CH3)2]. 31P NMR (121 MHz, CDCl3): δ 19.2. HRMS: [M + H]+ calcd for C23H31N5O7P, 520.1955; found, 520.1959. (1′α,2′R)-1′-(Adenin-9-yl)-2′-deoxy-2′-fluoro-3′-O-diisophosphonomethyl-L-threose (30). A solution of 29 (118 mg, 0.227 9527



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acetone-d6): δ 22.2, 21.2. HRMS: [M + H]+ calcd for C30H45N3O10P, 638.2837; found, 638.2820. 1′α-(Thymin-1-yl)-2′-deoxy-3′-O-[N, N′-bis(n-propyl- L phenylalaninate)methylphosphonobisamidate]- L -threose (33c). Obtained from PMDTT (16 mg, 0.038 mmol, Et3N salt) and Lphenylalanine-O-n-Pr ester hydrochloride (56 mg, 0.23 mmol) as a white solid. Yield 34% (9 mg). 1H NMR (500 MHz, acetone-d6): δ 9.92 (s, 1H, NH), 7.61−7.62 (m, 1H, H-6), 7.20−7.29 (m, 10H, Ph), 6.16, 6.15 (d, J = 2.8 Hz, 1H, H-1′), 4.09−4.28 (m, 4H, H-3′, H-4a′ and Phe-CH), 3.98−4.05 (m, 4H, OCH2CH2CH3), 3.74−3.80 (m, 2H, NH and H-4b′), 3.44−3.49 (m, 1H, NH), 3.38 (dd, J = 8.7, 1.1 Hz, 2H, PCH2), 2.82−3.08 (m, 4H, Phe-CH2), 2.52−2.57 (m, 1H, H2a′), 2.00−2.06 (m, 1H, H-2b′), 1.84 (d, J = 1.2 Hz, 3H, T CH3), 1.57−1.66 (m, 4H, OCH 2 CH 2 CH 3 ), 0.86−0.93 (m, 6H, OCH2CH2CH3). 13C NMR (125 MHz, acetone-d6): δ 173.6, 173.3 (Phe-CO), 164.2 (C-4), 151.3 (C-2), 138.0, 137.8 (Ph), 137.2 (C-6), 130.4, 130. 3, 128.90, 128.86, 127.30, 127.27 (Ph), 110.5 (C-5), 85.1 (C-1′), 80.9 (3JP,C = 12.1 Hz, C-3′), 73.4 (C-4′), 66.94, 66.92 (OCH2CH2CH3), 66.6 (1JP,C = 136.9 Hz, PCH2), 54.9, 54.6 (PheCH), 41.2, 41.0 (Phe-CH2), 38.3 (C-2′), 22.4, 22.3 (OCH2CH2CH3), 12.4 (T CH3), 10.5, 10.4 (OCH2CH2CH3). 31P NMR (121 MHz, acetone-d6): δ 19.6. HRMS: [M + H]+ calcd for C34H46N4O9P, 685.2997; found, 685.3006. 1′ α-(C ytosi n-1-yl)- 2 ′-deoxy-3′ -O -{[N-(isopropyl - L alaninate)](phenoxy)methylphosphonoamidate}- L -threose (34a). Obtained from PMDTC (17 mg, 0.043 mmol, Et3N salt), Lalanine isopropyl ester hydrochloride (14 mg, 0.087 mmol), and PhOH (20 mg, 0.22 mmol) as a white solid. Yield 15% (3 mg). 1H NMR (300 MHz, CD3OD): δ 7.83, 7.79 (d, J = 7.5 Hz, 1H, H-6), 7.32−7.38 (m, 2H, Ph), 7.14−7.21 (m, 3H, Ph), 6.07−6.12 (m, 1H, H-1′), (5.80, 5.79) (d, J = 7.5 Hz, 1H, H-5), 4.85−4.99 [m, 1H, CH(CH3)2], 4.38−4.44 (m, 1H, H-4a′), 4.27−4.35 (m, 1H, H-3′), 3.80−3.99 (m, 4H, H-4b′, PCH2 and Ala-CH), 2.48−2.58 (m, 1H, H2a′), 2.19−2.24 (m, 1H, H-2b′), 1.18−1.29 [m, 9H, Ala-CH3 and CH(CH3)2]. 13C NMR (150 MHz, CD3OD): δ 174.9, 174.5 (AlaCO), 167.7 (C-4), 158.41, 158.38 (C-2), 151.6, 151.4 (Ph), 143.2, 143.0 (C-6), 130.8, 126.2, 126.1, 122.0, 121.8 (Ph), 95.50, 95.48 (C5), 88.1 (C-1′), 82.3 (3JP,C = 13.1 Hz, C-3′), 75.2, 75.0 (C-4′), 70.2 [CH(CH3)2], 65.8, 65.7 (1JP,C = 158.3, 156.8 Hz, PCH2), 51.0 (AlaCH), 39.7 (C-2′), 21.9 [CH(CH3)2], 21.1, 20.7 (Ala-CH3). 31P NMR (121 MHz, CD3OD): δ 23.9, 22.7. HRMS: [M + H]+ calcd for C21H30N4O7P, 481.1846; found, 481.1848. 1′α-(Cytosin-1-yl)-2′-deoxy-3′-O-{[N,N′-bis(n-propyl- L alaninate)]methylphosphonobisamidate}-L-threose (34d). Obtained from PMDTC (18 mg, 0.046 mmol, Et3N salt) and L-alanine isopropyl ester hydrochloride (46 mg, 0.28 mmol) as a white solid. Yield 26% (6 mg). 1H NMR (600 MHz, CD3OD): δ 7.84 (d, J = 7.4 Hz, 1H, H-6), 6.09 (d, J = 6.6 Hz, 1H, H-1′), 5.90 (d, J = 7.4 Hz, 1H, H-5), 4.96−5.02 [m, 2H, CH(CH3)2], 4.41 (d, J = 10.3 Hz, 1H, H4a′), 4.25−4.28 (m, 1H, H-3′), 3.84−3.95 (m, 3H, H-4b′ and AlaCH), 3.61−3.75 (m, 2H, PCH2), 2.48−2.55 (m, 1H, H-2a′), 2.19 (d, J = 15.0 Hz, 1H, H-2b′), 1.33−1.36 (m, 6H, Ala-CH3). 1.23−1.26 [m, 12H, CH(CH3)2]. 13C NMR (150 MHz, CD3OD): δ 175.3, 175.2 (Ala-CO), 167.7 (C-4), 158.4 (C-2), 143.3 (C-6), 95.6 (C-5), 88.1 (C1′), 82.3 (3JP,C = 13.4 Hz, C-3′), 75.0 (C-4′), 70.2 [CH(CH3)2], 66.8 (1JP,C = 137.0 Hz, PCH2), 50.2, 49.9 (Ala-CH), 39.8 (C-2′), 22.0 [CH(CH3)2], 21.4, 21.2 (Ala-CH3). 31P NMR (121 MHz, CD3OD): δ 23.1. HRMS: [M + H]+ calcd for C21H37N5O8P, 518.2374; found, 518.2385. 1′α-(Guanin-9-yl)-2′-deoxy-3′-O-{[N-(isopropyl-L-alaninate)](phenoxy)methylphosphonoamidate}-L-threose (35a). Obtained from PMDTG (26 mg, 0.060 mmol, Et3N salt), L-alanine isopropyl ester hydrochloride (20 mg, 0.12 mmol), and PhOH (28 mg, 0.30 mmol) as a white solid. Yield 13% (4 mg). 1H NMR (300 MHz, DMSO-d6): δ 7.90, 7.88 (s, 1H, H-8), 7.15−7.40 (m, 5H, Ph), 6.57 (brs, 2H, NH2), 6.07−6.10 (m, 1H, H-1′), 5.62−5.79 (m, 1H, NH), 4.79−4.88 [m, 1H, CH(CH3)2], 4.36−4.45 (m, 1H, H-3′), 4.16−4.22 (m, 1H, H-4a′), 3.83−4.00 (m, 4H, H-4b′, PCH2 and Ala-CH), 2.63− 2.73 (m, 1H, H-2a′), 2.30−2.42 (m, 1H, H-2b′), 1.12−1.19 [m, 9H, CH(CH3)2 and Ala-CH3]. 13C NMR (75 MHz, DMSO-d6): 173.1

121.78, 121.72, 121.68 (Ph), 120.1, 120.0 (C-5), 84.2, 83.9 (C-1′), 81.9 (3JP,C = 13.5 Hz, C-3′), 74.4, 73.9 (C-4′), 65.94, 65.90 (1JP,C = 156.6, 155.9 Hz, PCH2), 64.6, 64.5, 63.8 [OCH2CH2CH(CH3)2], 51.54, 51.46 (Asp-CH), 39.6, 39.1 (Asp-CH2), 37.93, 37.90, 37.87 [OCH2CH2CH(CH3)2], 25.64, 25.62, 25.55, 25.53 [OCH2CH2CH(CH3)2], 22.7, 22.6 [OCH2CH2CH(CH3)2]. 31P NMR (121 MHz, acetone-d6): δ 22.2, 21.2. HRMS: [M + H]+ calcd for C30H44N6O8P, 647.2953; found, 647.2953. 1′α-(Adenin-9-yl)-2′-deoxy-3′-O-{[N,N-bis(n-propyl-L-phenylalaninate)] methylphosphonobisamidate}-L-threose (32c). Obtained from PMDTA (23 mg, 0.055 mmol, Et3N salt) and Lphenylalanine-O-n-Pr ester hydrochloride (81 mg, 0.331 mmol) as a white solid. Yield 73% (28 mg). 1H NMR (500 MHz, acetone-d6): δ 8.47 (s, 1H, H-8), 8.20 (s, 1H, H-2), 7.18−7.29 (m, 10H, Ph), 6.42− 6.44 (m, 1H, H-1′), 4.2−4.35 (m, 3H, Phe-CH, H-3′ and H-4a′), 4.04−4.15 (m, 1H, Phe-CH), 3.95−4.03 (m, 5H, OCH2CH2CH3 and H-4b′), 3.85−3.95 (m, 1H, Phe-NH), 3.47−3.49 (m, 2H, PCH2), 3.35−3.45 (m, 1H, Phe-NH), 2.80−3.15 (m, 4H, Ph-CH2), 2.68−2.75 (m, 1H, H-2a′), 2.40−2.45 (m, 1H, H-2b′), 1.55−1.64 (m, 4H, OCH2CH2CH3), 0.90−0.91 (m, 6H, OCH2CH2CH3). 13C NMR (125 MHz, acetone-d6): δ 173.9, 173.4 (Phe-CO), 157.1 (C-6), 153.7 (C2), 150.6 (C-4), 140.7 (C-8), 138.3, 137.9, 130.6, 130.6, 129.1, 129.0, 127.4, 127.3 (Ph), 119.9 (C-5), 83.9 (C-1′), 81.4 (d, 3JP,C = 13.8 Hz, C-3′), 74.1 (C-4′), 67.1, 67.1 (OCH2CH2CH3), 66.9 (d, 1JP,C = 136.3 Hz, PCH2), 55.3, 54.7 (Phe-CH), 41.3, 41.2 (Ph-CH2), 38.9 (C-2′), 22.6 (OCH2CH2CH3), 13.8 (OCH2CH2CH3). 31P NMR (121 MHz, acetone-d6): δ 19.7. HRMS: [M + H]+ calcd for C34H45N7O7P, 694.3112; found, 694.3120. 1 ′α - ( T h ym in -1- yl ) -2 ′- d e o xy -3′ - O -{[ N-( isop rop yl - L alaninate)](phenoxy)methylphosphonoamidate}- L-threose (33a). Obtained from PMDTT (18 mg, 0.044 mmol, Et3N salt), Lalanine isopropyl ester hydrochloride (15 mg, 0.088 mmol), and PhOH (21 mg, 0.22 mmol) as a white solid. Yield 41% (9 mg). 1H NMR (500 MHz, acetone-d6): δ 9.91 (s, 1H, NH), 7.68−7.69 (m, 1H, H-6), 7.16−7.35 (m, 5H, Ph), 6.20−6.23 (m, 1H, H-1′), 4.92−4.94 [m, 1H, CH(CH3)2], 4.59−4.71 (m, 1H, NH), 4.39−4.44 (m, 1H, H3′), 4.34−4.38 (m, 1H, H-4a′), 3.96−4.05 (m, 3H, Ala-CH and PCH2), 3.82−3.87 (m, 1H, H-4b′), 2.60−2.66 (m, 1H, H-2a′), 2.14− 2.20 (m, 1H, H-2b′), 1.80−1.81 (m, 3H, T CH3), 1.25−1.30 (m, 3H, Ala-CH3), 1.18−1.20 [m, 6H, CH(CH3)2]. 13C NMR (125 MHz, acetone-d6): δ 174.0, 173.7 (Ala-CO), 164.4 (C-4), 151.6, 151.5 (C-2), 137.5, 137.4 (C-6), 130.34, 130.30, 125.36, 125.33, 121.78, 121.75, 121.64, 121.60 (Ph), 110.64, 110.60 (C-5), 85.4, 85.3 (C-1′), 81.6 (d, 3 JP,C = 12.6 Hz, C-3′), 73.9, 73.6 (C-4′), 69.2, 69.1 [OCH(CH3)2], 66.8, 65.8 (PCH2, 1JP,C = 155.8, 156.4 Hz), 50.5, 50.4 (Ala-CH), 38.5 (C-2′), 21.87, 21.85, 21.80 [OCH(CH3)2], 21.3, 21.2 (Ala-CH3), 12.6 (T CH3). 31P NMR (121 MHz, acetone-d6): δ 21.6, 20.7. HRMS: [M + H]+ calcd for C22H31N3O8P, 496.1843; found, 496.1850. 1′α-(Thymin-1-yl)-2′-deoxy-3′-{[N-(diisoamyl-L-aspartate)](phenoxy)methylphosphonoamidate}-L-threose (33b). Obtained from PMDTT (27 mg, 0.066 mmol, Et3N salt), L-aspartic acid isoamyl ester hydrochloride (41 mg, 0.133 mmol), and PhOH (31 mg, 0.331 mmol) as a white solid. Yield 60% (25 mg). 1H NMR (500 MHz, acetone-d6): δ 10.00 (s, 1H, NH), 7.68−7.69 (m, 1H, H-6), 7.17−7.35 (m, 5H, Ph), 6.21−6.23 (m, 1H, H-1′), 4.73−4.86 (m, 1H, NH), 4.34−4.46 (m, 3H, H-3′, H-4a′ and Asp-CH), 4.02−4.17 [m, 6H, PCH2 and OCH2CH2CH(CH3)2], 3.81−3.87 (m, 1H, H-4b′), 2.78−2.82 (m, 2H,Asp-CH2), 2.60−2.65 (m, 1H, H-2a′), 2.12−2.20 (m, 1H, H-2b′), 1.80−1.81 (m, 3H, T CH3), 1.62−1.69 [m, 2H, OCH2CH2CH(CH3)2], 1.44−1.51 [m, 4H, OCH2CH2CH(CH3)2], 0.88−0.90 [m, 12H, OCH2CH2CH(CH3)2]. 13C NMR (125 MHz, acetone-d6): δ 172.5, 172.4, 171.1, 171.0 (Asp-CO), 164.4 (C-4), 151.5 (C-2), 137.5, 137.4 (C-6), 130.33, 130.31, 125.44, 125.42, 121.80, 121.77, 121.67, 121.64 (Ph), 110.74, 110.67 (C-5), 85.3, 85.2 (C-1′), 81.7, 81.6 (C-3′), 73.9, 73.5 (C-4′), 65.7, 64.8 (PCH2, 1JP,C = 155.5, 157.0 Hz), 64.51, 64.47, 63.8 [OCH2CH2CH(CH3)2], 51.4 (Asp-CH), 39.7, 39.5 (Asp-CH2), 38.5 (C-2′), 37.97, 37.94, 37.90, 37.88 [OCH2CH2CH(CH3)2], 25.6, 25.5 [OCH2CH2CH(CH3)2], 22.7 [OCH2CH2CH(CH3)2], 12.6 (T CH3). 31P NMR (121 MHz, 9528



Article

Metabolic Stability Assay in Human Liver S9 Fraction. The reaction mixture was prepared in a total volume of 1 mL containing 5 mM of MgCl2, 50 mM of K2HPO4 (pH 7.4), and 100 μM prodrug. The reaction was initiated by adding 4 mg/mL human liver S9 fraction (obtained from Gibco) to the reaction mixture and incubated at 37 °C. At the desired times (0, 10, 20, 30, and 60 min), 100 μL aliquots were taken and the reaction was stopped by mixing the reaction mixture sample with 300 μL of acetonitrile. The samples were centrifuged at 13000 rpm for 30 min at 4 °C. Then 10 μL of the supernatant was analyzed by HPLC on a Shimadzu HPLC system equipped with a Symmetry C18 column (4.6 mm × 150 mm, 5 μm) and UV detection at 260 nm. The mobile phase consisted of solvent A (5% acetonitrile in water, 10 mM ammonium acetate) and solvent B (95% acetonitrile in water, 10 mM ammonium acetate). Elution was performed using a linear gradient of solvent B from 0% to 100% for 20 min. The flow rate was set at 1.0 mL/min. The amount of parent compound was determined on the basis of the peak area for each time point, and the percentage remaining was calculated on the basis of the initial amount measured at 0 h. The half-life of each compound was calculated by using the GraphPad Prism software. Metabolic Stability Assay in Human Liver Microsomes. The stability assay was contracted and carried out by Eurofins (Eurofins ref 0607). Pooled liver microsomes were preincubated with NADPHregenerating system (1 mM NADP, 5 mM G6P, and 1 U/mL G6PDHase) in 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 in a final volume of 700 μL in the 37 °C shaking water bath. At 0, 15, 30, 45, and 60 min after reaction initiation, 100 μL of the incubation mixture is transferred to 100 μL of acetonitrile/methanol (50/50, v/v) in a 0.8 mL V-bottom 96-well plate. Samples are then mixed on a plate shaker for 5 min and centrifuged at 2550g for 15 min at room temperature. Each supernatant is transferred to a clean cluster tube, followed by HPLC MS/MS analysis on a Thermo Electron triplequadrupole system. HIV Antiviral Assay. Evaluation of the antiviral activity of the compounds against HIV-1 strain IIIB and HIV-2 strain ROD in MT-4 cells was performed using the MTT assay as previously described.19,20 Stock solutions (10× final concentration) of test compounds were added in 25 μL volumes to two series of triplicate wells so as to allow simultaneous evaluation of their effects on mock- and HIV-infected cells at the beginning of each experiment. Serial 5-fold dilutions of test compounds were made directly in flat-bottomed 96-well microtiter trays using a Biomek 3000 robot (Beckman instruments, Fullerton, CA). Untreated HIV- and mock-infected cell samples were included as controls. HIV-1(IIIB) stock (50 μL) at 100−300 CCID50 (50% cell culture infectious doses) or culture medium was added to either the infected or mock-infected wells of the microtiter tray. Mock-infected cells were used to evaluate the effects of test compound on uninfected cells in order to assess the cytotoxicity of the test compounds. Exponentially growing MT-4 cells were centrifuged for 5 min at 220 g, and the supernatant was discarded. The MT-4 cells were resuspended at 6 × 105 cells/mL, and 50 μL volumes were transferred to the microtiter tray wells. Five days after infection, the viability of mock-and HIV-infected cells was examined spectrophotometrically using the MTT assay. The MTT assay is based on the reduction of yellowcolored 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Acros Organics) by mitochondrial dehydrogenase activity in metabolically active cells to a blue−purple formazan that can be measured spectrophotometrically. The absorbances were read in an eight-channel computer-controlled photometer (Infinite M1000, Tecan) at two wavelengths (540 and 690 nm). All data were calculated using the median absorbance value of three wells. The 50% cytotoxic concentration (CC50) was defined as the concentration of the test compound that reduced the absorbance (OD540) of the mockinfected control sample by 50%. The concentration achieving 50% protection against the cytopathic effect of the virus in infected cells was defined as the 50% effective concentration (EC50).

(Ala-CO), 157.2 (C-6), 154.1 (C-2), 151.1 (C-4), 150.5 (Ph), 135.6 (C-8), 129.69, 129.66, 124.5, 120.9, 120.8, 120.75, 120.70 (Ph), 116.4 (C-5), 82.2, 82.1 (C-1′), 80.6, 80.4 (C-3′), 72.9, 72.5 (C-4′), 68.10, 68.05 [CH(CH3)2], 65.1 (1JP,C = 154.8 Hz, PCH2), 49.2 (Ala-CH), 37.8, 37.6 (C-2′), 21.5 [CH(CH3)2], 20.4, 20.1 (Ala-CH3). 31P NMR (121 MHz, DMSO-d6): δ 22.6, 21.9. HRMS: [M + H]+ calcd for C22H30N6O7P, 521.1908; found, 521.1914. 1′α-(Guanin-9-yl)-2′-deoxy-3′-O-[N,N′-bis(n-propyl- L phenylalaninate)]methylphosphonobisamidate]- L -threose (35c). Obtained from PMDTG (20 mg, 0.045 mmol, Et3N salt) and Lphenylalanine-O-n-Pr ester hydrochloride (65 mg, 0.27 mmol) as a white solid. Yield 24% (8 mg). 1H NMR (600 MHz, DMSO-d6): δ 10.56 (brs, 1H, NH), 7.86 (s, 1H, H-8), 7.12−7.27 (m, 10H, Ph), 6.45 (brs, 2H, NH2), 6.04 (dd, J = 8.1, 2.5 Hz, 1H, H-1′), 4.54 (t, J = 11.3 Hz, 1H, NH), 4.20 (t, J = 10.8 Hz, 1H, NH), 4.13−4.16 (m, 1H, H3′), 4.07 (d, J = 10.2 Hz, 1H, H-4a′), 3.92−4.04 (m, 4H, CH2CH2CH3), 3.85−3.91 (m, 2H, PCH2), 3.79 (dd, J = 10.1, 4.3 Hz, 1H, H-4b′), 3.15−3.16 (m, 2H, Phe-CH), 2.76−2.93 (m, 4H, PheCH2), 2.57−2.62 (m, 1H, H-2a′), 2.20 (d, J = 14.6 Hz, H-2b′), 1.45− 1.53 (m, 4H, CH2CH2CH3), 0.75−0.82 (m, 6H, CH2CH2CH3). 13C NMR (150 MHz, DMSO-d6): 173.0, 172.9 (Phe-CO), 156.9 (C-6), 153.7 (C-2), 150.9 (C-4), 137.3, 137.2 (Ph), 135.8 (C-8), 129.48, 129.47, 128.20, 128.17, 126.55, 126.49 (Ph), 116.4 (C-5), 82.2 (C-1′), 79.8 (3JP,C = 11.2 Hz, C-3′), 72.5 (C-4′), 65.94, 65.87 (CH2CH2CH3), 65.7 (1JP,C = 135.7 Hz, PCH2), 54.2, 53.0 (Phe-CH), 40.1 (Phe-CH2), 37.6 (C-2′), 21.5, 21.4 (CH2CH2CH3), 10.3, 10.2 (CH2CH2CH3). 31P NMR (121 MHz, DMSO-d6): δ 20.3. HRMS: [M + H]+ calcd for C34H45N7O8P, 710.3062; found, 710.3063. 1′α-(Guanin-9-yl)-2′-deoxy-3′-O-[N,N′-bis(n-propyl- L alaninate)]methylphosphonobisamidate]-L-threose (35d). Obtained from PMDTG (17 mg, 0.039 mmol, Et3N salt) and L-alanine isopropyl ester hydrochloride (40 mg, 0.23 mmol) as a white solid. Yield 23% (5 mg). 1H NMR (300 MHz, DMSO-d6): δ 7.86 (s, 1H, H8), 6.59 (brs, 2H, NH2), 6.05 (dd, J = 8.1, 2.5 Hz, 1H, H-1′), 4.84− 4.92 [m, 2H, CH(CH3)2], 4.42−4.59 (m, 2H, NH), 4.35 (brs, 1H, H3′), 4.18 (d, J = 9.8 Hz, H-4a′), 3.75−3.90 (m, 3H, H-4b′ and AlaCH), 3.56−3.72 (m, 2H, PCH2), 2.59−2.69 (m, 1H, H-2a′), 2.30 (d, J = 14.7 Hz, H-2b′), 1.27 (t, J = 7.5 Hz, 6H, Ala-CH3), 1.17−1.19 [m, 12H, CH(CH3)2]. 13C NMR (150 MHz, CD3OD): 175.4, 175.2 (AlaCO), 159.5 (C-6), 155.3 (C-2), 152.7 (C-4), 138.6 (C-8), 117.3 (C5), 85.3 (C-1′), 82.2 (3JP,C = 13.2 Hz, C-3′), 74.7 (C-4′), 70.1 [CH(CH3)2], 66.8 (1JP,C = 137.5 Hz, PCH2), 50.3, 50.0 (Ala-CH), 39.4 (C-2′), 22.0 [CH(CH3)2], 21.21, 21.18 (Ala-CH3). 31P NMR (121 MHz, DMSO-d6): δ 20.3. HRMS: [M + H]+ calcd for C22H37N7O8P, 558.2436; found, 558.2448. 31 P NMR Stability Experiments in Acidic and Basic pH. Buffer pH 1: The stability assay toward hydrolysis by aqueous buffer at pH 1 was conducted using in situ 31P NMR (202 MHz). The experiment was carried out by dissolving prodrug 32b (2.0 mg) in 0.2 mL of acetone-d6 and then adding buffer (0.3 mL), pH 1 (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 14 h. Buffer pH 8: The stability assay toward hydrolysis by aqueous buffer at pH 8 was conducted using in situ 31P NMR (202 MHz). The experiment was carried out by dissolving prodrug 32b (2 mg) in 0.2 mL of acetone-d6 and then adding buffer (0.3 mL), pH 8 (prepared from solution of 0.1 M Na2HPO4 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 14 h. Carboxypeptidase Y (EC 3.4.16.1) Assay. The experiment was carried out by dissolving prodrug 32b (1.0 mg) in 0.10 mL of acetoned6 followed by addition of 0.20 mL of Trizma buffer (pH 7.6). After recording the control 31P NMR (121 MHz) at 25 °C, 0.10 mL of carboxypeptidase Y (obtained from Sigma-Aldrich, 0.1 mg dissolved in 0.15 mL Trizma) was added to the sample, which was then subjected to 31P NMR experiments. The spectra were recorded every 1 h over a period of 16 h at 25 °C. The 31P NMR spectra was processed and analyzed by the Bruker Topspin 2.1 program. 9529



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HBV Antiviral Assay. Primary Assay. The primary anti-HBV assay was performed as previously described23,24 with modifications to use real-time qPCR (TaqMan) to measure extracellular HBV DNA copy number associated with virions released from HepG2 2.2.15 cells. The HepG2 2.2.15 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.22 Briefly, HepG2 2.2.15 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 “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 2.2.15 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 is 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 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 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 the primary assay described above in. 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.

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Rozenski (KU Leuven) for providing HRMS data. Mass spectrometry was made possible by the support of the Hercules Foundation of the Flemish Government (grant 20100225-7). We thank Kristien Erven and Kris Uyttersprot for dedicated technical assistance. We thank Luc Baudemprez for technical assistance. 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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DEDICATION In memory of A. Holy on the occasion of his 80th birthday ABBREVIATIONS USED PMEA, phosphonomethoxyethyladenine; PMPA, phosphonylmethoxypropyladenine; PMT, phosphonomethoxythreosyl; PMDT, phosphonomethoxydeoxythreosyl; HIV, human immunodeficiency virus; HBV, hepatitis B virus; TBSCl, tertbutylchlorodimethylsilane; BSA, N,O-bis(trimethylsilyl)acetamide; TMSOTf, trimethylsilyl trifluoromethanesulfonate; TCDI, 1,1′-thiocarbonyldiimidazole; DMAP, 4-dimethylaminopyridine; AIBN, azobis(isobutyronitrile); DMP, Dess−Martin periodinane; DAST, diethylaminosulfur trifluoride; THF, tetrahydrofuran; DMF, N,N-dimethylformamide; TEAB, triethylammonium bicarbonate; TLC, thin layer chromatography; rt, room temperature

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(1) Jordheim, L. P.; Durantel, D.; Zoulim, F.; Dumontet, C. Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases. Nat. Rev. Drug Discovery 2013, 12, 447− 464. (2) Krečmerová, M. Nucleoside and Nucleotide Analogues for the Treatment of Herpesvirus Infections: Current Stage and New Prospects in the Field of Acyclic Nucleoside Phosphonates, HerpesviridaeA Look Into This Unique Family of Viruses; Magel, G. D., Ed.; InTech: Rijeka, Croatia, 2012. (3) De Clercq, E. (E)-5-(2-Bromovinyl)-2′-deoxyuridine (BVDU). Med. Res. Rev. 2005, 25, 1−20. (4) Menéndez-Arias, L.; Á lvarez, M.; Pacheco, B. Nucleoside/ nucleotide analog inhibitors of hepatitis B virus polymerase: mechanism of action and resistance. Curr. Opin. Virol. 2014, 8, 1−9. (5) Cihlar, T.; Ray, A. S. Nucleoside and nucleotide HIV reverse transcriptase inhibitors: 25 years after zidovudine. Antiviral Res. 2010, 85, 39−58. (6) Debing, Y.; Jochmans, D.; Neyts, J. Intervention strategies for emerging viruses: use of antivirals. Curr. Opin. Virol. 2013, 3, 217−224. (7) Lawitz, E.; Jacobson, I. M.; Nelson, D. R.; Zeuzem, S.; Sulkowski, M. S.; Esteban, R.; Brainard, D.; McNally, J.; Symonds, W. T.; McHutchison, J. G.; Dieterich, D.; Gane, E. Development of sofosbuvir for the treatment of hepatitis C virus infection. Ann. N. Y. Acad. Sci. 2015, 1358, 56−67. (8) Kim, C. U.; Luh, B. Y.; Martin, J. C. Regiospecific and highly stereoselective electrophilic addition to furanoid glycals: synthesis of phosphonate nucleotide analogs with potent activity against HIV. J. Org. Chem. 1991, 56, 2642−2647. (9) Mackman, R. L.; Ray, A. S.; Hui, H. C.; Zhang, L.; Birkus, G.; Boojamra, C. G.; Desai, M. C.; Douglas, J. L.; Gao, Y.; Grant, D.; Laflamme, G.; Lin, K. Y.; Markevitch, D. Y.; Mishra, R.; McDermott, M.; Pakdaman, R.; Petrakovsky, O. V.; Vela, J. E.; Cihlar, T. Discovery of GS-9131: Design, synthesis and optimization of amidate prodrugs of the novel nucleoside phosphonate HIV reverse transcriptase (RT) inhibitor GS-9148. Bioorg. Med. Chem. 2010, 18, 3606−3617. (10) De Clercq, E.; Holý, A. Acyclic nucleoside phosphonates: a key class of antiviral drugs. Nat. Rev. Drug Discovery 2005, 4, 928−940.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01260. 31 P NMR spectra of prodrug 32b in pH 1 and pH 8 buffer; 31P NMR spectra of prodrug 32b over time after incubation with pH 8 buffer; RP-18-HPLC chromatogram of prodrug 32b after incubation with pH 8 buffer; RP-18-HPLC chromatogram of prodrug 32b after carboxypeptidase Y digestion; NMR spectra for compounds 2−35d (PDF) Molecular formula strings (CSV)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +32 16 337387. Fax: +32 16 337340. E-mail: Piet. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Chao Liu acknowledges the China Scholarship Council (CSC) for funding (grant 201306220065). We thank Prof. Jef 9530



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(11) Wu, T.; Froeyen, M.; Kempeneers, V.; Pannecouque, C.; Wang, J.; Busson, R.; De Clercq, E.; Herdewijn, P. Deoxythreosyl phosphonate nucleosides as selective anti-HIV agents. J. Am. Chem. Soc. 2005, 127, 5056−5065. (12) Pradere, U.; Garnier-Amblard, E. C.; Coats, S. J.; Amblard, F.; Schinazi, R. F. Synthesis of Nucleoside Phosphate and Phosphonate Prodrugs. Chem. Rev. 2014, 114, 9154−9218. (13) Mehellou, Y.; Balzarini, J.; McGuigan, C. Aryloxy phosphoramidate triesters: a technology for delivering monophosphorylated nucleosides and sugars into cells. ChemMedChem 2009, 4, 1779−1791. (14) Ray, A. S.; Fordyce, M. W.; Hitchcock, M. J. Tenofovir alafenamide: A novel prodrug of tenofovir for the treatment of Human Immunodeficiency Virus. Antiviral Res. 2016, 125, 63−70. (15) Schoning, K. U.; Scholz, P.; Wu, X. L.; Guntha, S.; Delgado, G.; Krishnamurthy, R.; Eschenmoser, A. The α-L-Threofuranosyl-(3′→ 2′)-oligonucleotide system (‘TNA’): synthesis and pairing properties. Helv. Chim. Acta 2002, 85, 4111−4153. (16) Dumbre, S. G.; Jang, M. Y.; Herdewijn, P. Synthesis of α-Lthreose nucleoside phosphonates via regioselective sugar protection. J. Org. Chem. 2013, 78, 7137−7144. (17) Vina, D.; Wu, T.; Renders, M.; Laflamme, G.; Herdewijn, P. Synthesis of 3′-O-phosphonomethyl nucleosides with an adenine base moiety. Tetrahedron 2007, 63, 2634−2646. (18) Sau, S. P.; Fahmi, N. E.; Liao, J. Y.; Bala, S.; Chaput, J. C. A scalable Synthesis of α-L-threose nucleic acid monomers. J. Org. Chem. 2016, 81, 2302−2307. (19) Pannecouque, C.; Daelemans, D.; De Clercq, E. Tetrazoliumbased colorimetric assay for the detection of HIV replication inhibitors: revisited 20 years later. Nat. Protoc. 2008, 3, 427−434. (20) Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.; Herdewijn, P.; Desmyter, J.; De Clercq, E. Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds. J. Virol. Methods 1988, 20, 309−321. (21) Maiti, M.; Maiti, M.; Rozenski, J.; De Jonghe, S.; Herdewijn, P. Aspartic acid based nucleoside phosphoramidate prodrugs as potent inhibitors of hepatitis C virus replication. Org. Biomol. Chem. 2015, 13, 5158−5174. (22) Sells, M. A.; Chen, M. L.; Acs, G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc. Natl. Acad. Sci. U. S. A. 1987, 84, 1005−1009. (23) Korba, B. E.; Milman, G. A cell culture assay for compounds which inhibit hepatitis B virus replication. Antiviral Res. 1991, 15, 217−228. (24) Korba, B. E.; Gerin, J. L. Use of a standardized cell culture assay to assess activities of nucleoside analogs against hepatitis B virus replication. Antiviral Res. 1992, 19, 55−70.

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