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Article Cite This: J. Med. Chem. 2018, 61, 9218−9228

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Synthesis and Antiviral Evaluation of Carbocyclic Nucleoside Analogs of Nucleoside Reverse Transcriptase Translocation Inhibitor MK-8591 (4′-Ethynyl-2-fluoro-2′-deoxyadenosine) François-René Alexandre,*,†,∥ Rachid Rahali,† Houcine Rahali,† Sandra Guillon,‡ Thierry Convard,† Kerry Fillgrove,§ Ming-Tain Lai,§ Jean-Christophe Meillon,‡ Min Xu,§ James Small,§ Cyril B. Dousson,† and Izzat T. Raheem*,§,∥ J. Med. Chem. 2018.61:9218-9228. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 10/25/18. For personal use only.

†

Idenix an MSD Company, Cap Gamma, 1682 Rue de la Valsière, 34189 Montpellier Cedex 4, France Oxeltis, Cap Delta, 1682 Rue de la Valsière, 34189 Montpellier Cedex 4, France § Merck & Co., Inc., P.O. Box 4, 770 Sumneytown Pike, West Point, Pennsylvania 19486, United States ‡

S Supporting Information *

ABSTRACT: MK-8591 (4′-ethynyl-2-fluoro-2′-deoxyadenosine) is a novel nucleoside analog that displays a differentiated mechanism of action as a nucleoside reverse transcriptase translocation inhibitor (NRTTI) compared to approved NRTIs. Herein, we describe our recent efforts to explore the impact of structural changes to the properties of MK-8591 through the synthesis and antiviral evaluation of carbocyclic derivatives. Synthesized analogs were evaluated for their antiviral activity, and the corresponding triphosphates were synthesized and evaluated in a biochemical assay. 4′-Ethynyl-G derivative (±)-29 displayed a promising IC50 of 33 nM in a hPBMC cell-based antiviral assay, and its triphosphate (TP), (±)-29-TP, displayed an IC50 of 324 nM in a biochemical RT-polymerase assay. Improved TP anabolite delivery resulting in improved in vitro potency was achieved by preparing the corresponding phosphoramidate prodrug of single enantiomer 29b, with 6-ethoxy G derivative 34b displaying a significantly improved IC50 of 3.0 nM, paving the way for new directions for this novel class of nucleoside analogs.

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INTRODUCTION Reverse transcriptase (RT) is a key enzyme in the human immunodeficiency virus type 1 (HIV-1) replication cycle that converts single-stranded viral RNA into double-stranded DNA that is subsequently integrated into the host genome of infected cells by HIV integrase. RT inhibitors that prevent viral replication are critical components of highly active antiretroviral therapy (HAART).1 They are generally divided into two classes: nucleoside (or nucleotide) reverse transcriptase inhibitors (NRTIs or NtRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs).2 NNRTIs allosterically inhibit reverse transcription upon binding to a hydrophobic site near but distinct from the active site. All NRTIs are pronucleotides that are phosphorylated by host nucleoside and nucleotide kinases to their active triphosphate (TP) forms, which are subsequently incorporated into the elongating viral DNA.3 A common feature of all approved NRTIs (Chart 1) is the lack of a 3′-hydroxyl group, which prevents further elongation of the growing DNA strand during the polymerization process, an inhibition mechanism referred to as chain termination. While a number of NRTIs are critical components of highly efficacious current standard-of-care regimens, cellular © 2018 American Chemical Society

toxicity and the emergence of mutants that are resistant to the drugs emphasize the need for novel structurally and functionally differentiated nucleosides.4 MK-8591 (4′-ethynyl-2-fluoro-2′-deoxyadenosine), a novel nucleoside analog that possesses a 3′-hydroxyl group, is more potent against wild-type and most clinical drug-resistant HIV strains than any approved nucleoside reverse transcriptase inhibitor (NRTI).5 Unlike other NRTIs and due to its unique structure bearing a 4′-ethynyl substituent and a 3′-hydroxy group (Chart 2), MK-8591 has been shown to display multiple novel modes of action, acting in part as a translocationdefective RT inhibitor (NRTTI).6 Biochemical mechanistic studies have revealed that MK-8591-TP is a better substrate for RT than its natural substrate dATP. Once incorporated, MK8591-MP functions either as a de facto chain terminator by preventing RT translocation on the nucleic acid primer possessing the 3′-terminal MK-8591-MP or as a delayed chain terminator, incorporating one incoming deoxynucleoside TP (dNTP) before DNA synthesis stops.6 Another novel Received: June 7, 2018 Published: September 28, 2018 9218

DOI: 10.1021/acs.jmedchem.8b00141 J. Med. Chem. 2018, 61, 9218−9228

Journal of Medicinal Chemistry

Article

As part of a continued discovery effort driven by the further exploration of the structure activity relationship (SAR) of MK8591 and subsequent structural diversification, we turned our attention to analogs in which the ribose sugar ring was replaced by a carbocycle. The NTP anabolites of carbocyclic nucleoside analogues such as abacavir (ABC, Chart 1) are recognized by and inhibit HIV-RT, and these carbocyclic analogs are established components of antiviral regimens.10 Due to their carbocyclic nature, this unique nucleoside analog class is often more chemically robust and metabolically stable toward glycosidases compared to their ribose counterparts, characteristics that could further impart improved pharmacokinetic properties.11 Herein, we describe the synthesis and antiviral evaluation of novel carbocyclic analogs of MK-8591.

Chart 1. : Approved NRTIs and NtRTIs

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CHEMISTRY Syntheses of 4′-substituted carbocyclic nucleoside analogs available in the literature are scarce, and those reported are often long and low-yielding, particularly for our targets of interest.12 To this end, we chose to perform a racemic synthesis of carbocyclic intermediates 6−9 based on the work of Holy et al. as depicted in Scheme 1.13 Beginning with substituted malonate derivative 1, oxidation was carried out with pyridinium dichromate to afford aldehyde 2. Without purification, 2 was directly engaged in a cyclization reaction in allyl alcohol solvent to afford racemic 3 as a mixture of cis/ trans isomers. The pendant 2-hydroxy group of (±)-3 was then protected with a methoxymethyl (MOM) group to provide (±)-4. The ester functionalities were reduced with lithium aluminum hydride, and (±)-5 was obtained, still as a cis/trans mixture of isomers in a 1:4 ratio. This resulting prochiral diol was monoprotected with tert-butyldiphenylsilyl chloride (TBDPSCl), producing four isomers, of which two could be isolated by chromatography on silica gel, with trans-(±)-6, obtained in 43% yield, and trans-(±)-7 in 12% yield. The corresponding relative stereochemistry of (±)-6 and (±)-7 was assigned by COSY and NOESY NMR experiments and was in accordance with closely related structures described in the literature.13 In (±)-6 and (±)-7, as indicated by the NOESY NMR spectra (Chart 3), a strong correlation between H-2 and 1-CH2OTBDPS indicated that the 1-CH2OTBDPS and 2-OMOM were trans to one another in both compounds. A separate weaker correlation between H-2 and H-4 indicated that the 2-OMOM and 4-O-allyl groups were cis to one

Chart 2. : Chemical Structures of MK-8591 (Left) and Representative 4′-Substituted Carbocyclic Targeted Structures

structural feature of MK-8591 is the 2-fluoro substituent on the adenine nucleobase that renders it highly resistant to metabolism by adenosine deaminase, enabling more efficient uptake and phosphorylation to MK-8591-TP.7 MK-8591-TP has a very long intracellular half-life (>120 h) compared to other NRTIs, imparting the potential for a long duration of action.7 Indeed, MK-8591 has displayed an excellent pharmacokinetic profile in preclinical species and in the clinic8 and has been well-tolerated in phase 1 clinical trials. Additionally, driven by its exquisite potency, long intracellular half-life, and high oral bioavailability, MK-8591 has demonstrated robust efficacy in naive patients at oral doses as low as 0.5 mg given once weekly.9 Scheme 1. Synthesis of Intermediates (±)-6 and (±)-7a

Reagents and conditions: (a) PDC, DCM, 0 °C to room temperature, 16 h; (b) NaH, allyl alcohol, 0 °C to room temperature, 16 h, 22% (over 2 steps); (c) dimethoxymethane, Tf2O, room temperature, 1 h, 57%; (d) LiAlH4, THF, 0 °C, then reflux, 90 min, 92%; (e) TBDPSCl, imidazole, DMAP, DCM, 0 °C to room temperature, 16 h; (±)-6 (43%), (±)-7 (12%); (±)-8 and (±)-9 not isolated. a

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Chart 3. : Primary NOESY NMR Correlations (Red Arrows) in (±)-6 and (±)-7 Used for Stereochemical Assignment

Article

RESULTS AND DISCUSSION

The antiviral activity of our novel nucleoside analogs against wild-type HIV-1 was evaluated in a cell-based assay in HIV-1 infected human peripheral blood mononuclear cells (hPBMCs),15 and the results are summarized in Table 1. Among the 4′-ethynyl series, thymine (T) derivative (±)-19 showed no activity (Table 1, entry 3), adenosine (A) derivatives (±)-24 and (±)-25 were modestly active in the midnanomolar range (Table 1, entries 4 and 5), while guanine (G) analogs were most potent, with (±)-29 displaying an IC50 of 33 nM. Analogs in the 4′-cyano series were significantly less potent than the 4′-ethynyl series (Table 1, entries 7−9), with the most potent compound in this series being G analog (±)-30 with an IC50 of 1.15 μM, significantly less potent than (±)-29. Importantly, all compounds displayed no measurable cellular toxicity. The most potent G derivative (±)-29 was resolved into its constituent enantiomers via chiral supercritical fluid chromatography (SFC) (Scheme 4) to afford 29a (faster eluting isomer) and 29b (slower eluting isomer). The two enantiomers were then evaluated in the cell-based antiviral assay, and only 29b retained antiviral activity (Table 1, entry 11). On the basis of these results, we attributed the potent 29b to be the D-series (1S,3R,4R) and 29a to be the L-series (1R,3S,4S), a hypothesis that was consistent with a modeling study of their corresponding TPs described later in this manuscript. In addition to the in vitro evaluation of the parent nucleoside analogs, the corresponding TPs were synthesized using known methods16 and were evaluated in a biochemical HIV-1 RT polymerase (RT-Pol) assay (Table 2). The results were in accordance with the trend observed in the cell-based assay, with G analogs (±)-29-TP and (±)-30-TP being potent inhibitors of HIV RT, superior to the corresponding A derivatives. The T derivatives (±)-19-TP and (±)-20-TP displayed IC50’s of 114 nM and 523 nM, respectively (Table 2, entries 1 and 5), while their corresponding nucleosides were inactive in the cell-based assay (Table 1). Interestingly, compared to MK-8591-TP, carbocyclic analog 24-TP, which also bears a 2-fluoroadenine base (2FA), displayed an 13-fold higher IC50 (Table 2, entry 2). In order to rationalize the observed potency differences between MK-8591-TP and 24-TP, a computational modeling study of these TPs bound in the RT polymerase active site was undertaken (see Supporting Information for details). In this process, lowest energy 3D conformations of the TP ligands, within a 45 kcal/mol range between the lowest and the highest energy conformation, were first generated using the Discovery

another in (±)-7, while the absence of such a correlation in (±)-6 indicated that these groups were trans. The cis isomers (±)-8 and (±)-9 could not be separated at this stage and were not used further in the synthesis. Alcohol (±)-6 was functionalized at its 4′ position as shown in Scheme 2. First, oxidation of the free hydroxyl group to the corresponding aldehyde with Dess−Martin periodinane provided (±)-10 in good yield. The aldehyde (±)-10 was next transformed into the corresponding ethynyl derivative (±)-11 through Seyferth−Gilbert homologation or converted to the 4′-cyano derivative (±)-14 through a two-step sequence involving hydroxylamine formation followed by dehydration. Next, allyl deprotection was carried out with Pd(PPh3)4 in the presence of barbituric acid in methanol14 to afford (±)-12 and (±)-15. Finally, (±)-12 was converted to (±)-13 and (±)-15 was converted to (±)-16 by inverting the stereochemistry of the hydroxyl using a Mitsunobu reaction followed by hydrolysis of the pendant phenyl ester. Alternatively, the same reaction sequence could be carried out beginning with (±)-7 obtained previously, to produce additional (±)-13 and (±)-16 and eliminating the final Mitsunobu inversion. Starting from alcohol intermediates (±)-13 and (±)-16, final carbocyclic nucleosides derivatives bearing various bases were synthesized as depicted in Scheme 3. Protected bases were coupled to (±)-13 and (±)-16 using a standard Mitsunobu process. The resulting compounds were subjected to desilylation using TBAF in the presence of AcOH in THF to afford 5′-OH carbocyclic nucleoside analogs, which were purified and characterized at this stage. The final racemic nucleoside analogs were obtained in good yields by a subsequent deprotection of the MOM protecting group using TFA, followed by either deprotection of the nucleobase or amination of the nucleobase at the purine 6-position using ammonia in methanol or 1,4-dioxane. Scheme 2. Synthesis of Alcohols (±)-13 and (±)-16a

a

Reagents and conditions: (a) Dess−Martin periodinane, DCM, room temperature, 16 h, 76%; (b) Seyferth−Gilbert reagent, K2CO3, MeOH, room temperature, 16 h, 78%; (c) (1) hydroxylamine, pyridine, EtOH, 2 h; (2) Burgess reagent, toluene, 1 h, 78% (over 2 steps); (d) Pd(PPh3)4, 1,3-dimethylbarbituric acid, MeOH, room temperature, 16 h, 89%; (e) (1) benzoic acid, PPh3, DIAD, THF (quant), 16 h; (2) NaOH, MeOH, 2 h, 80% (over 2 steps) for (±)-13, 66% (over 2 steps) for (±)-16. 9220



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Scheme 3. Synthesis of 2′-Deoxycarbocyclic Nucleosidesa

a Reagents and conditions: (a) N3-Bz-thymine, PPh3, DIAD, THF, −41 °C to room temperature, 16 h; (b) 2-fluoro-6-chloropurine or 2,6dichloropurine, DIAD, THF, −41 °C to room temperature, 16 h; (c) N2-Ac-6-ODPC-guanine, DIAD, THF, −41 °C to room temperature, 16 h; (d) TBAF, AcOH, THF, room temperature, 24 −48 h, 45% (over 2 steps) for (±-17, 33% (over 2 steps) for (±)-18, 56% (over 2 steps) for (±)-21, 78% (over 2 steps) for (±)-22, 42% (over 2 steps) for (±)-23, 74% (over 2 steps) for (±)-27, 54% (over 2 steps) for (±)-28; (e) TFA, DCM, rt, 16 h; (f) 7 N NH3 in MeOH, sealed tube, room temperature, 16 h, 56% (over 2 steps) for (±)-19, 27% (over 2 steps) for (±)-20, 73% (over 2 steps) for (±)-29, 26% (over 2 steps) for (±)-30; (g) NH3 in 1,4-dioxane, sealed tube, 50 °C, 16 h, 19% (over 2 steps) for (±)-24, 27% (over 2 steps) for (±)-25, 10% (over 2 steps) for (±)-26.

Scheme 4. : Chiral Separation of (±)-29a

Table 1. Structure and in Vitro Activity of 2′Deoxycarbocyclic Nucleosides against Wild-Type HIV-1Infected hPBMCs

entry

compd

1 2 3 4 5 6 7 8 9 10 11 12 13

MK-8591 3TC (±)-19 (±)-24 (±)-25 (±)-29 (±)-20 (±)-26 (±)-30 29a 29b 34a 34b

X

CH CH CH CH N N N CH CH CH CH

base

IC50a (μM)

CC50b (μM)

T 2FA 2ClA G T 2FA G G G GOEt GOEt

0.00021 0.171 >2.10 0.201 0.232 0.033 >2.10 >2.10 1.15 >2.10 0.024 0.382 0.0030

>4.2 >42.0 >42.0 >42.0 >42.0 >42.0 >42.0 >42.0 >42.0 >42.0 >42.0 >4.2 >42.0

a

Reagents and conditions: (a) chiral SFC separation, CHIRALCEL OD-H column, mobile phase CO2/MeOH; faster eluting isomer 29a and slower eluting isomer 29b.

allowed to be unconstrained, and the calculation was repeated. A maximum of 10 conformations, based on the best calculated fit, were kept. These were then docked into the available complex of HIV RT polymerase protein, primer, template, and 2 Mg2+ ions (PDB code 1rtd17), and their geometries were optimized using the CHARMm force field while keeping most components of the complex fixed except for specific residues. Next, simulated annealing of the ligand/enzyme complexes was performed using CDOCKER. The resulting complexes were relaxed by energy minimization using the CHARMm force field while keeping other parts of the complex fixed. Finally, ligand geometries from the minimized complexes were remapped to the pharmacophore model, keeping the 3D coordinates of the ligand fixed, and the fit values were calculated. These scores were used to evaluate the quality of the modeled structures. At this final stage only the highest fit value conformation was kept, with these solutions depicted in Figure 1 for several TPs. The primary interactions of MK-8591-TP and (1S,3R,4R)24-TP are shown in Figure 1A and Figure 1B, respectively. We assumed that the active enantiomer of (±)-24-TP would be

a IC50 values were determined in 10% NHS in a single-cycle HIV replication assay in hPBMCs. bCC50 values were determined in 10% NHS condition in an MT4 cell line (see Supporting Information for details).

Studio Catalyst (Biovia) software package. Next, the minimized TP ligand geometries were flexibly mapped to a previously generated and validated pharmacophore model using the same software package. If no solution was found, two fixed points of the pharmacophore model were sequentially 9221



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Having gained a better understanding of the structure and active site binding interactions of our novel carbocyclic nucleoside analogs, we chose to evaluate the in vivo pharmacokinetics of 29b as a leading representative of this new class. Pharmacokinetic parameters obtained in male Sprague-Dawley rats after intravenous (iv, 1 mg/kg) and oral (po, 2 mg/kg) administration are summarized in Table 3, compared to MK-8591. After oral administration, 29b exhibited an excellent oral bioavailability of 91%, good oral exposure, and a moderate plasma clearance (32.4 mL min−1 kg−1) and half-life (9.8 h). These data provided early confidence in the in vivo performance of this class. In order to improve the in vitro cellular potency of the 4′ethynyl-G carbocyclic series, a prodrug approach was investigated. Prodrugs have historically been employed in the nucleoside space to directly deliver a masked monophosphate into target cells rather than the nucleoside itself, expediting the route to the triphosphate form by bypassing the commonly rate-determining first phosphorylation step.19 As a benchmark prodrug, we chose to synthesize the L-Ala phosphoramidate, 6ethoxyguanine derivative. In this prodrug class, it is well established that the isopropyl ester is cleaved intracellularly by cathepsin A, and the resulting carboxylic acid is subsequently degraded to the monophosphate over a sequence of three steps.20 Upon liberation of the free monophosphate, the 2amino-6-ethoxypurine is transformed to a G by intracellular adenosine deaminase.21 The 2-amino-6-ethoxypurine intermediate (±)-32 was synthesized in a manner similar to carbocyclic nucleoside (±)-29 via a Mitsunobu coupling followed by subsequent deprotections (Scheme 5). The pendant phosphoramidate was installed using commercially available isopropyl ((S)(perfluorophenoxy)(phenoxy)phosphoryl)-L-alaninate in the presence of tert-butylmagnesium chloride in THF.22 The diastereomeric mixture obtained was deprotected at the 3′-OH and then separated by chiral SFC to afford 34a and 34b. In the hPBMC cell-based antiviral assay, 34b was identified as the active enantiomer (3.0 nM), 127-fold more active than 34a (382 nM). Gratifyingly, compared to our lead parent nucleoside analog 29b, the corresponding phosphoramidate prodrug 34b provided an 8-fold improvement in activity (Table 1, entries 11, 12, and 13). Upon the basis of these exciting preliminary results, we anticipate this improvement in the in vitro potency should result in improved TP delivery in vivo, and efforts are focused toward optimizing our NMP delivery to this end.

Table 2. Structure and in Vitro Activity of 2′Deoxycarbocyclic Nucleoside Triphosphates against WildType HIV-1 RT

entry

compd

X

base

IC50a (μM)

1 2 3 4 5 6 7 8 9

(±)-19-TP (±)-24-TP (±)-25-TP (±)-29-TP (±)-20-TP (±)-26-TP (±)-30-TP MK-8591-TP 3TC-TP

CH CH CH CH N N N

T 2FA 2ClA G T 2FA G

0.114 3.46 20.2 0.324 0.523 ND 0.444 0.263 0.015

a HIV reverse transcriptase polymerase assay (RT-Pol). IC50 values were determined by nonlinear four-parameter curve fitting (see Supporting Information for details).

the (1S,3R,4R)-24-TP, corresponding to the configuration of a D-nucleoside as no solution for the other enantiomer (1R,3S,4S)-24-TP could be found in the previously described pharmacophore model computational mapping study. In this precatalytic complex of RT bound to the dideoxyguanosine (ddG) terminated double-stranded DNA, MK-8591-TP adopts a C-3′ endo ring conformation (Figure 1A), displays Watson− Crick base pairing interactions, and shows strong hydrophobic interactions of the 4′-ethynyl with Phe160 (π−σ), Tyr115 (πalkyl), Met184 (alkyl), and Ala114 (alkyl). In the case of (1S,3R,4R)-24-TP (Figure 1B), which has a flat ring conformation, base pairing is of the Hoogsteen type with the 2-fluoroadenine ring system adopting a syn conformation. The interactions with the carbocyclic ring are less numerous and less efficient than those for MK-8591-TP, with the 4′-ethynyl making a weaker interaction with Phe160 (d = 4.41 Å as compared to d = 2.79 Å for MK-8591-TP) and no interaction with Ala114. These observations suggest that (1S,3R,4R)-24TP is less favorably bound to the RT compared to MK-8591TP. Unlike (±)-24-TP, (±)-29-TP is a potent inhibitor of the HIV RT polymerase and adopts a C-3′ endo ring conformation. The corresponding modeling study (Figure 1C) performed on (1S,3R,4R)-29-TP shows a binding mode similar to MK-8591-TP, with Watson−Crick base pairing interactions (losing only interaction with Gly-152), strong hydrophobic interactions with 4′-ethynyl, and presence of the metal (Figure 1, in green) in an optimal distance to the ddG 3′-OH of the primer. To validate our assumption that the active nucleoside analog enantiomer 29b does indeed bear the (1S,3R,4R) configuration, we also modeled the TP derivative of the inactive enantiomer 29a in RT (Figure 1D). Interestingly, and in support of our assignment, this triphosphate displayed fewer interactions with the protein with longer distances between the 4′-ethynyl and Tyr115 and Phe160, as well as no interactions with Ala114 and Met184. Furthermore, the distance from the metal chelating the α-phosphate to the 3′-OH of the primer was 3.78 Å, which renders the likelihood of the further polymerization less favorable.18

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CONCLUSIONS In summary, we have synthesized and evaluated a series of novel carbocyclic analogs of MK-8591. Prepared racemates were resolved, and the stereochemistry of the active analogs was postulated to be from the (1S,3R,4R) series. This hypothesis was further supported by a computational modeling study and was in line with corresponding in vitro potency data. NTP anabolites from this class exhibit potent inhibitory activity against HIV-1 RT similar to MK-8591-TP. In addition, lead carbocyclic derivatives display potent inhibitory activity in a hPBMC cell-based antiviral assay, although in a higher range compared to the exceptional potency of MK-8591. These results demonstrate that the highly conserved ribose moiety can be replaced by a carbocyclic backbone to open up new structural space. Compared to MK-8591, lead compound 29b displayed a good overall PK profile in rat with improved 9222



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Figure 1. Computational models illustrating the primary interactions of MK-8591-TP (A), (1S,3R,4R)-24-TP (B), (1S,3R,4R)-29-TP (C), and (1R,3S,4S)-29-TP (D) in the polymerase active site of HIV-1 RT/DNA. NTPs are colored by atom type. Template is displayed in yellow. Primer is displayed in pink. RT amino acids are displayed in in cyan, and metal ions are displayed in green.

Table 3. Rat Pharmacokinetic Profile of MK-8591 and 29b compd

routea

dose (mg/kg)

AUC0−∞ (μM·h)

MK-8591

iv po iv po

1 5 1 2

0.78 5.0 1.82 3.30

29b a

Cl (mL min−1 kg−1)

t1/2 (h)

72

0.7

32.4

9.8

F (%) >100 91

Intravenous (iv) arm formulated in 50:50 DMSO/saline; oral (po) arm formulated in 0.5% methylcellulose (aq).

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plasma clearance and half-life. A prodrug strategy was interrogated in order to further improve in vitro potency, and an 8-fold improvement in cellular IC50 was obtained by employing a phosphoramidate prodrug. This early result validated the prodrug strategy for this promising new class of nucleosides, and additional studies exploring this approach will be reported in due course.

EXPERIMENTAL SECTION

Synthesis of Representative Carbocyclic Nucleosides. The syntheses of 29a, 29b, 34a, and 34b are described in this section, while the syntheses of all other compounds presented in this manuscript are described in the Supporting Information. Chemistry. General. All reactions were performed with reagentgrade materials under an atmosphere of nitrogen. Solvents were 9223



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Scheme 5. Synthesis and Chiral Separation of Phosphoramidate Prodrugs 34a and 34ba

Reagents and conditions: (a) N2-COiPr-6-ODPC-guanine, DIAD, THF, −41 °C to room temperature, 16 h, 50%; (b) TBAF, AcOH, THF, room temperature, 16 h, 98%; (c) KOH, MeOH, room temperature, 0 °C, 16 h, 66%; (d) isopropyl ((S)-(perfluorophenoxy)(phenoxy)phosphoryl)-Lalaninate, tBuMgCl 1 M in THF, 0 °C to rt, 16 h, 82%; (e) TFA, DCM, room temperature, then 0 °C, 2.5 h, 57%; (f) chiral SFC separation, CHIRALPAK AD-H column, mobile phase CO2/MeOH; faster eluting isomer 34a (28%) and slower eluting isomer 34b (30%). a

chiral analytical chromatography under supercritical fluid (SFC) conditions. Procedures. (±)-cis- and (±)-trans-(4-(Allyloxy)-2(methoxymethoxy)cyclopentane-1,1-diyl)dimethanol (±)-5. Step a: To a solution of dimethyl (Z)-2-(4-hydroxybut-2-en-1yl)malonate13 (40 g, 0.198 mol) in DCM (800 mL) cooled at 0 °C was added portionwise pyridinium dichromate (126.5 g, 0.336 mol). The reaction mixture was stirred at room temperature for 16 h, filtered on silica gel eluting with EtOAc, and then concentrated in vacuo. The crude dimethyl (Z)-2-(4-oxobut-2-en-1-yl)malonate (±)-2 was used in the subsequent step without further purification. Step b: To allyl alcohol (700 mL) was slowly added sodium hydride (60% in oil, 5 g, 0.124 mol) at 0 °C. The reaction mixture was stirred at room temperature for 1 h. To this reaction mixture was added dropwise a solution of crude (±)-2 (35.4 g, 0.177 mol) in allyl alcohol (190 mL). The reaction mixture was stirred at room temperature for 16 h. The reaction was quenched with addition of acetic acid until pH = 7, and then the reaction mixture was concentrated in vacuo. The crude residue was dissolved in EtOAc and water, the organic layer was separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with a saturated NaHCO3 solution and brine, dried over Na2SO4, and concentrated in vacuo. The crude residue was purified by gradient elution on SiO2 (PE/ EtOAc: 0−70%) to afford (±)-cis- and (±)-trans-diallyl-4-(allyloxy)2-hydroxycyclopentane-1,1-dicarboxylate (±)-3 (13.7 g, 22%). Step c: To a solution of (±)-3 (13.7 g, 44 mmol) in dimethoxymethane (560 mL) was added dropwise triflic acid (3.1 mL, 35 mmol). The reaction mixture was stirred at room temperature for 1 h and then poured into a saturated NaHCO3 solution and extracted twice with DCM. The organic layer was dried over Na2SO4 and concentrated in vacuo. The crude residue was purified by gradient elution on SiO2 (PE/EtOAc: 0−100%) to afford (±)-cis- and (±)-trans-diallyl-4-(allyloxy)-2-(methoxymethoxy)cyclopentane-1,1dicarboxylate (±)-4 (8.94 g, 57%). Step d: To a solution of (±)-4 (8.94 g, 25 mmol) in THF (70 mL), cooled at 0 °C was added dropwise LiAlH4 at 0 °C. The reaction mixture was stirred at reflux for 90 min and then cooled to 0 °C, quenched with dropwise addition of water, and diluted with EtOAc. The resulting reaction mixture was filtered on Celite and washed with water and EtOAc. The organic layer was dried, filtered, and concentrated in vacuo. The crude residue was purified by gradient elution on SiO2 (DCM/MeOH: 0−10%) to afford the expected compound (±)-5 (5.66 g, 92%) as a mixture of isomer. 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 5.91−5.81 (m, 1H), 5.24−5.19 (m, 1H), 5.12−5.08 (m, 1H), 4.58−4.51 (m, 3H), 4.17−4.12 (m, 1H), 3.99−3.93 (m, 1.5H), 3.88−3.79 (m, 2.5H), 3.45−3.35 (m, 2H), 3.33−3.24 (m, 2H), 3.23 (s, 3H), 2.30−2.23 (m, 0.2H), 1.95−1.84

reagent-grade or better. Evaporation of the solvents was carried out in a rotary evaporator in vacuo. Thin layer chromatography (TLC) was performed on precoated aluminum sheets of silica gel 60 F254 (Merck, 60F-254), visualization of products being accomplished by UV absorbance at 254 nm; column chromatography was performed on silica gel (15−45 μm, 40−63 μm, or spheric silica prepacked cartridges) using a Biotage flash chromatography apparatus (Isolera). 1 H (400 MHz) and 31P (162 MHz) NMR spectra were acquired on a Bruker AVANCE II 400 MHz spectrometer using DMSO-d6 or CDCl3 as solvents. Chemical shifts (δ) are quoted in parts per million (ppm) referenced to the residual solvent peak (DMSO-d6 set at 2.49 ppm or CDCl3 set at 7.26 ppm). 13C (176 MHz) NMR data were acquired on a Bruker AVANCE III HD 700 MHz NMR spectrometer equipped with a 1.7 mm HCN TCI MicroCryoProbe using CD3OD as solvent. Chemical shifts (δ) are quoted in parts per million (ppm) referenced to the residual solvent peak (CD3OD set at 49.15 ppm). The accepted abbreviations are as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Low-resolution LC mass spectra (LR UPLCMS) were recorded on a WATERS unit [Acquity UPLC, SQD2 (ESCI source)] using a reverse phase analytical column, CORTECS UPLC C18 1.6 μm, 2.1 mm × 30 mm for standard methods and ACQUITY UPLC HSST3 1.8 μm, 2.1 mm × 30 mm for polar methods. The compound to be analyzed was eluted using a linear gradient of 5−95% acetonitrile in water with 0.05% formic acid programmed over a 1.7 min period with a flow rate of 0.7 mL/min. Purity of all compounds was determined to be >95% by analytical LC−MS system consisting of an Agilent 6140 quadrupole LC/MS platform with PDA detector. The column for standard methods was Purospher STAR RP-18 end-capped 2 μm, Hibar HR 50-2.1, the column temperature was 60 °C, the flow rate was 0.8 mL/min, and injection volume was 0.5−5 μL. UV detection was in the range 210− 400 nm. The mobile phase consisted of solvent A (water plus 0.05% formic acid) and solvent B (acetonitrile plus 0.05% formic acid) with different gradients for two different methods: (1) starting with 98% solvent A changing to 100% solvent B over 1.8 min, maintained for 0.8 min; (2) starting with 98% solvent A changing to 100% solvent B over 5.8 min, maintained for 0.3 min. Preparative HPLC was performed on a Gilson system 233XL with 735 (Unipoint). The column was a Waters XBridge Prep C18 5 μm OBD, dimension 30 mm × 250 mm. The mobile phase consisted of mixture of acetonitrile/ammonium carbonate 0.02 N (3−15% over 70 min or 3−30% over 50 min). Flow rates were maintained at 50 mL/min, the injection volume was 1000 μL, and the UV detection range was 260 nm. Chiral preparative chromatography was conducted on CHIRALCEL OD-H or CHIRALPAK AD-H columns (Daicel Chemical Industries, Ltd.) with desired isocratic solvent systems identified on 9224



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temperature. The reaction mixture was stirred at room temperature for 16 h and then concentrated in vacuo and directly purified by gradient elution on SiO2 (PE/Et2O: 0−100%) to afford the expected compound (±)-12 (1.98 g, 87%). 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 7.67−7.62 (m, 4H), 7.50−7.42 (m, 6H), 4.75−4.74 (m, 1H), 4.60 (d, J = 6.69 Hz, 1H), 4.57 (d, J = 6.69 Hz, 1H), 4.27−4.25 (m, 1H), 4.19 (t, J = 7.40 Hz, 1H), 3.66−3.60 (m, 2H), 3.22 (s, 3H), 2.97 (s, 1H), 2.13−2.08 (m, 1H), 2.01−1.93 (m, 1H), 1.87−1.81 (m, 1H), 1.79−1.75 (m, 1H), 1.02 (s, 9H); MS (ESI) m/z = 461.4 (MNa+). (1S,3S,4S)- and (1R,3R,4R)-3-(((tert-Butyldiphenylsilyl)oxy)methyl)-3-ethynyl-4-(methoxymethoxy)cyclopentan-1-ol (±)-13. To a suspension of PPh3 (2.368 g, 9.03 mmol) in Et2O (60 mL) under nitrogen at 0 °C was added DIAD (1.778 mL, 9.03 mmol). This reaction mixture was stirred at 0 °C for 30 min. The resulting mixture was added to a solution of compound (±)-12 (1.98 g, 4.51 mmol) and benzoic acid (1.103 g, 9.03 mmol) in Et2O (60 mL) at 0 °C. The reaction mixture was stirred at room temperature for 16 h and then concentrated in vacuo. A solution of 1% NaOH in MeOH (23 mL) was added, and the reaction mixture was stirred for 2 h. EtOAc (250 mL) was added followed by a 1 M HCl solution (250 mL). The aqueous layer was extracted twice with EtOAc, and the combined organic layer was dried over Na2SO4, filtered, and concentrated in vacuo to afford the title compound (±)-13 (1.581 g, 80%). 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 7.64−7.60 (m, 4H), 7.51−7.42 (m, 6H), 4.80−4.74 (m, 1H), 4.60−4.56 (m, 2H), 4.05−3.98 (m, 1H), 3.95−3.92 (m, 1H), 3.57 (d, J = 9.92 Hz, 1H), 3.46 (d, J = 9.92 Hz, 1H), 3.22 (s, 3H), 2.99 (s, 1H), 2.29−2.18 (m, 2H), 1.77−1.65 (m, 2H), 1.01 (s, 9H); MS (ESI) m/z = 461.4 (MNa+). 2-Acetamido-9-((1R,3S,4S)- and (1S,3R,4R)-3-Ethynyl-3-(hydroxymethyl)-4-(methoxymethoxy)cyclopentyl)-9H-purin-6yl Diphenylcarbamate (±)-27. Step a: PPh3 (0.718 g, 3 equiv) was suspended in anhydrous THF (15 mL) under nitrogen and cooled to 0 °C using an ice bath. Then DIAD (0.503 mL, 2.8 equiv) was added dropwise, and the reaction mixture was stirred 30 min. The resulting mixture was added to a solution of (±)-13 (400 mg, 0.912 mmol) and 2-acetamido-9H-purin-6-yl-diphenylcarbamate (0.531 g, 1.5 equiv) in anhydrous THF (15 mL) at −41 °C (N2/acetonitrile bath). The reaction mixture was stirred at −41 °C for 2 h, then at room temperature for 16 h. The reaction mixture was concentrated in vacuo, then the crude residue was purified by gradient elution on SiO2 (PE/Et2O: 0−10%) to afford the intermediate 2-acetamido-9((1R,3S,4S)- and (1S,3R,4R)-3-(((tert-butyldiphenylsilyl)oxy)methyl)-3-ethynyl-4-(methoxymethoxy)cyclopentyl)-9H-purin-6-yl diphenylcarbamate (0.545 g) contaminated with DIAD-H2, which was used in the subsequent step without further purification. 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 10.68 (s, 1H), 8.50 (s, 1H), 7.64− 7.30 (m, 20H), 5.27−5.18 (m, 1H), 4.71−4.66 (m, 3H), 3.83 (d, J = 9.72 Hz, 1H), 3.77 (d, J = 9.72 Hz, 1H), 3.22 (s, 3H), 3.19 (s, 1H), 2.58−2.54 (m, 2H), 2.42−2.32 (m, 2H), 2.13 (s, 3H), 0.98 (s, 9H); MS (ESI) m/z = 807.5 (MH−). Step b: To a solution of product from step a in anhydrous THF (11 mL) and acetic acid (0.190 mL, 3.37 mmol) was added TBAF (1.35 mL, 1.35 mmol). The reaction mixture was stirred 16 h. Solvents were removed in vacuo, and the crude residue was purified by gradient elution on SiO2 (DCM/MeOH: 0−5%) to afford (±)-27 (384 mg, 74%). 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 10.70 (s, 1H), 8.54 (s, 1H), 7.50−7.31 (m, 10H), 5.25−5.12 (m, 2H), 4.69 (d, J = 6.63 Hz, 1H), 4.64 (d, J = 6.63 Hz, 1H), 4.45 (t, J = 7.22 Hz, 1H), 3.61− 3.54 (m, 2H), 3.28 (s, 3H), 3.11 (s, 1H), 2.46−2.41 (m, 1H), 2.36− 2.29 (m, 3H), 2.22 (s, 3H); MS (ESI) m/z = 571.7 (MH+). 2-Amino-9-((1R,3S,4S)- and (1S,3R,4R)-3-ethynyl-4-hydroxy3-(hydroxymethyl)cyclopentyl)-1,9-dihydro-6H-purin-6-one (±)-29. Step a: (±)-27 (0.235 g, 0.412 mmol) was dissolved in DCM (8.8 mL), and TFA (1.2 mL, 40 equiv) was added. The reaction mixture was stirred at room temperature for 16 h, then solvents were removed in vacuo. The product N-(9-((1R,3S,4S)- and (1S,3R,4R)-3ethynyl-4-hydroxy-3-(hydroxymethyl)cyclopentyl)-6-oxo-6,9-dihydro1H-purin-2-yl)acetamide was used in the subsequent step without further purification. 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 12.01

(m, 2.4H), 1.79−1.74 (m, 0.2H), 1.65−1.53 (m, 0.4H), 1.40−1.35 (m, 0.8H). ((1S,2S,4R)- and ((1R,2R,4S)-4-(Allyloxy)-1-(((tertbutyldiphenylsilyl)oxy)methyl)-2-(methoxymethoxy)cyclopentyl)methanol (±)-6 and ((1R,2R,4R)- and (1S,2S,4S)-4(Allyloxy)-1-(((tert-butyldiphenylsilyl)oxy)methyl)-2(methoxymethoxy)cyclopentyl)methanol (±)-7. To a solution of compound (±)-5 (5.75 g, 23.35 mmol) in DCM (280 mL) at 0 °C were added imidazole (1.91 g, 28.0 mmol), DMAP (0.28 g, 2.33 mmol), and tert-butyldiphenylchlorosilane (7.28 mL, 28.0 mmol). The reaction mixture was stirred from 0 °C to room temperature for 3 h and at room temperature for 16 h. The reaction mixture was then diluted with EtOAc (500 mL) and washed with a 1 M HCl solution (500 mL), water (500 mL), and brine (500 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by gradient elution on SiO2 (PE/ Et2O: 0−70%) to afford the expected compounds (±)-6 (4.86 g, 43%), (±)-7 (1.416 g, 12%) and a mixture of isomers (±)-8 and (±)-9 (5.0 g, 44%). Compound (±)-6: 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 7.66−7.61 (m, 4H), 7.48−7.40 (m, 6H), 5.87−5.77 (m, 1H), 5.20−5.15 (m, 1H), 5.09−5.05 (m, 1H), 4.55 (d, J = 6.62 Hz, 1H), 4.53 (d, J = 6.62 Hz, 1H), 4.29 (t, J = 4.97 Hz, 1H), 4.14 (t, J = 6.55 Hz, 1H), 3.99−3.93 (m, 1H), 3.84−3.82 (m, 2H), 3.62−3.52 (m, 2H), 3.53−3.43 (m, 2H), 3.21 (s, 3H), 1.99−1.85 (m, 3H), 1.53−1.49 (m, 1H), 1.01 (s, 9H); MS (ESI) m/z = 507.8 (MNa+). Compound (±)-7: 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 7.68− 7.60 (m, 4H), 7.49−7.41 (m, 6H), 5.91−5.82 (m, 1H), 5.25−5.20 (m, 1H), 5.13−5.09 (m, 1H), 4.55 (d, J = 6.62 Hz, 1H), 4.50 (d, J = 6.62 Hz, 1H), 4.26 (t, J = 4.95 Hz, 1H), 4.04−3.99 (m, 1H), 3.88− 3.78 (m, 3H), 3.49−3.48 (m, 4H), 3.21 (s, 3H), 2.31−2.24 (m, 1H), 1.95−1.86 (m, 1H), 1.68−1.60 (m, 2H), 1.01 (s, 9H); MS (ESI) m/z = 507.6 (MNa+). ( 1S,2 S,4R)- an d ( 1R,2 R,4 S)-4-( Allyloxy)-1-((( tertbutyldiphenylsilyl)oxy)methyl)-2-(methoxymethoxy)cyclopentane-1-carbaldehyde (±)-10. To a solution of compound (±)-6 (2.68 g, 5.53 mmol) in DCM (40 mL) under nitrogen was added Dess−Martin periodinane (4.69 g, 11.06 mmol) dissolved in DCM (40 mL). The reaction mixture was stirred at room temperature for 16 h. The resulting reaction mixture was partially concentrated in vacuo (2/3), and the compound was purified by gradient elution on SiO2 (PE/Et2O: 0−70%) to afford the expected compound (±)-10 (2.67 g, 76%). 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 9.66 (s, 1H), 7.61−7.59 (m, 4H), 7.50−7.41 (m, 6H), 5.86− 5.76 (m, 1H), 5.20−5.15 (m, 1H), 5.11−5.07 (m, 1H), 4.53 (d, J = 6.79 Hz, 1H), 4.50 (d, J = 6.79 Hz, 1H), 4.22 (t, J = 6.79 Hz, 1H), 4.12 (d, J = 9.78 Hz, 1H), 4.06−4.01 (m, 1H), 3.85−3.84 (m, 2H), 3.72 (d, J = 9.78 Hz, 1H), 3.17 (s, 3H), 2.44−2.39 (m, 1H), 2.02− 1.96 (m, 1H), 1.85−1.78 (m, 1H), 1.70−1.66 (m, 1H), 0.98 (s, 9H); MS (ESI) m/z = 505.5 (MNa+). (((1S,2S,4R)- and (1R,2R,4S)-4-(Allyloxy)-1-ethynyl-2(methoxymethoxy)cyclopentyl)methoxy)(tert-butyl)diphenylsilane (±)-11. To a solution of compound (±)-10 (3.24 g, 6.71 mmol) in MeOH (192 mL) under nitrogen was added potassium carbonate (2.78 g, 20.14 mmol). The reaction mixture was stirred at 0 °C, and then dimethyl(1-diazo-2-oxopropyl)phosphonate (2.015 mL, 13.42 mmol) was added dropwise under nitrogen. The reaction mixture was stirred at room temperature for 16 h and then concentrated in vacuo. The crude residue was purified by gradient elution on SiO2 (PE/Et2O: 0−70%) to afford the expected compound (±)-11 (3.21 g, 78%). 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 7.66−7.61 (m, 4H), 7.50−7.42 (m, 6H), 5.88−5.79 (m, 1H), 5.22− 5.17 (m, 1H), 5.11−5.08 (m, 1H), 4.59 (s, 2H), 4.16 (t, J = 7.49 Hz, 1H), 4.08−4.03 (m, 1H), 3.86−3.84 (m, 2H), 3.66 (d, J = 9.76 Hz, 1H), 3.60 (d, J = 9.76 Hz, 1H), 3.22 (s, 3H), 3.02 (s, 1H), 2.15−2.10 (m, 1H), 1.99−1.96 (m, 2H), 1.93−1.89 (m, 1H), 1.02 (s, 9H); MS (ESI) m/z = 501.5 (MNa+). (1R,3S,4S)- and (1S,3R,4R)-3-(((tert-Butyldiphenylsilyl)oxy)methyl)-3-ethynyl-4-(methoxymethoxy)cyclopentan-1-ol (±)-12. To a solution of compound (±)-11 (2.50 g, 5.22 mmol) in MeOH (11.5 mL) were added 1,3-dimethylbarbituric acid (1.631 g, 10.45 mmol) and Pd(PPh3)4 (302 mg, 0.261 mmol) at room 9225



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(phenoxy)phosphoryl]-L-alaninate 33b. To a solution of compound (±)-32 (240 mg, 0.66 mmol) in THF (10 mL) at 0 °C under nitrogen was added a 1 M solution of tert-butylmagnesium chloride in THF (1.66 mL, 1.66 mmol). The reaction mixture was stirred at 0 °C for 5 min, and then a solution of isopropyl ((S)(perfluorophenoxy)(phenoxy)phosphoryl)-L -alaninate (331 mg, 0.731 mmol) in THF (10 mL) was added dropwise at 0 °C. The reaction mixture was allowed to warm slowly to room temperature and stirred at room temperature for 16 h. The resulting reaction mixture was diluted with EtOAc and washed with a saturated NH4Cl solution and brine. The organic layer was dried, filtered and concentrated in vacuo. The crude residue was purified by gradient elution on SiO2 (DCM/MeOH: 0−10%) to afford the product a mixture of diastereomers 33a and 33b (344 mg, 82%). 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 7.94 (s, 0.5H), 7.93 (0.5H), 7.37− 7.32 (m, 2H), 7.23−7.21 (m, 2H), 7.18−7.13 (m, 1H), 6.41−6.40 (m, 2H), 6.09−6.01 (m, 1H), 5.12−5.03 (m, 1H), 4.89−4.79 (m, 1H), 4.67−4.62 (m, 2H), 4.45 (q, J = 6.98 Hz, 2H), 4.34−4.07 (m, 3H), 3.86−3.78 (m, 1H), 3.28 (s, 1.5H), 3.275 (s, 1.5H), 3.24 (s, 0.5H), 3.23 (s, 0.5H), 2.40−2.11 (m, 4H), 1.35 (t, J = 6.98 Hz, 3H), 1.24−1.22 (m, 3H), 1.16−1.10 (m, 6H); 31P NMR (DMSO-d6, 162 MHz) δ (ppm) 3.26 (s, 0.5P), 3.15 (0.5P); MS (ESI) m/z = 631.4 (MH+). Propan-2-yl N-[(S)-{[(1R,2R,4S)-4-(2-Amino-6-ethoxy-9Hpurin-9-yl)-1-ethynyl-2-hydroxycyclopentyl]methoxy}(phenoxy)phosphoryl]-L-alaninate 34a and Propan-2-yl N-[(S){[(1S,2S,4R)-4-(2-Amino-6-ethoxy-9H-purin-9-yl)-1-ethynyl-2hydroxycyclopentyl]methoxy}(phenoxy)phosphoryl]-L-alaninate 34b. To a solution of a mixture of diastereomers 33a and 33b (270 mg, 0.43 mmol) in DCM (5 mL) at 10 °C was added TFA (0.99 mL, 12.84 mmol). The reaction mixture was stirred between 10 and 25 °C for 2 h and at 0 °C for 2.5 h and then concentrated. The crude residue was purified by gradient elution on SiO2 (DCM/MeOH: 0− 4%) to afford the title compound as a mixture of diastereomers 34a and 34b (145 mg, 57%). 31P NMR (DMSO-d6, 162 MHz) δ (ppm) 3.34 (s, 0.4P), 3.28 (0.6P); MS (ESI) m/z = 587.8 (MH+). The two diastereomers 34a and 34b (139 mg) were separated by preparative chiral SFC with the following conditions. Column: CHIRALPAK ADH, 2 cm × 25 cm, 5 μm. Mobile phase A: CO2. Mobile phase B: i PrOH. Gradient: 30% B in 8 min. Flow rate: 60 mL/min. Detector: UV 220 nm. The process afford 34a and 34b. Diastereomer 34a (40 mg, 28%): faster eluting, tR = 3.22 min; 1H NMR (CDCl3, 400 MHz) δ (ppm) 7.44 (s, 1H), 7.30−7.27 (m, 2H), 7.23−7.22 (m, 2H), 7.17− 7.14 (m, 1H), 5.18 (brs, 2H), 5.08−5.02 (m, 2H), 4.53 (q, J = 7.31 Hz, 2H), 4.48−4.46 (m, 1H), 4.44−4.42 (m, 1H), 4.38−4.35 (m, 1H), 4.08−4.00 (m, 3H), 2.51−2.46 (m, 1H), 2.41 (s, 1H), 2.40− 2.39 (m, 2H), 2.37−2.32 (m, 1H), 1.45 (t, J = 7.31 Hz, 3H), 1.405 (d, J = 6.83 Hz, 3H), 1.25 (d, J = 5.85 Hz, 3H), 1.24 (d, J = 5.85 Hz, 3H); 13C NMR (CD3OD, 176 MHz) δ (ppm) 174.2, 162.0, 161.1, 154.3, 151.9, 138.3, 129.4, 124.8, 120.1, 115.3, 83.2, 74.6, 72.2, 68.8, 68.4, 61.9, 52.2, 51.0, 50.2, 38.8, 37.4, 20.5, 19.1, 13.4; 31P NMR (CDCl3, 243 MHz) δ (ppm) 3.21 (s, 1P); MS (ESI) m/z = 587.6 (MH+). Diastereomer 34b (42 mg, 30%): slower eluting, tR = 5.37 min; 1H NMR (CDCl3, 400 MHz) δ (ppm) 7.52 (s, 1H), 7.33−7.30 (m, 2H), 7.25−7.21 (m, 2H), 7.18−7.15 (m, 1H), 5.24 (brs, 2H), 5.12−4.99 (m, 2H), 4.66−4.63 (m, 1H), 4.55−4.52 (m, 3H), 4.47− 4.45 (m, 1H), 4.38−4.35 (m, 1H), 4.03−3.99 (m, 1H), 3.77−3.71 (m, 1H), 2.62−2.58 (m, 1H), 2.46−2.43 (m, 1H), 2.42 (s, 1H), 2.40−2.36 (m, 1H), 2.33−2.29 (m, 1H), 1.45 (t, J = 6.83 Hz, 3H), 1.39 (d, J = 6.83 Hz, 3H), 1.225 (d, J = 5.85 Hz, 3H), 1.22 (d, J = 5.85 Hz, 3H); 13C NMR (CD3OD, 176 MHz) δ (ppm) 174.3, 162.3, 161.4, 154.4, 151.8, 139.7, 130.6, 126.1, 121.4, 115.6, 82.7, 75.9, 73.5, 73.4, 70.1, 69.8, 65.8, 52.4, 51.5, 49.9, 40.2, 21.9, 21.8, 20.3, 14.7; 31P NMR (CDCl3, 243 MHz) δ (ppm) 2.88 (s, 1P); MS (ESI) m/z = 587.6 (MH+).

(s, 1H), 11.68 (s, 1H), 8.15 (s, 1H), 5.10−5.03 (m, 3H), 4.31−4.27 (m, 1H), 3.54 (brs, 2H), 3.08 (s, 1H), 2.29−2.21 (m, 2H), 2.11−2.06 (m, 2H), 2.22 (s, 3H); MS (ESI) m/z = 332.3 (MH+). Step b: To a solution of the product of step a in MeCN (10 mL) was added a 7 N ammonia solution in MeOH (2.9 mL, 20.6 mmol). The reaction mixture was stirred at 50 °C for 16 h. The reaction mixture was then concentrated in vacuo, and the crude residue was triturated in a mixture water/MeCN (1:1), filtered, and washed with water/MeCN (1:1), MeCN, and pentane to afford the title compound (±)-29 (119 mg, 73%). 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 10.55 (s, 1H), 7.81 (s, 1H), 6.44 (brs, 2H), 5.01 (t, J = 5.82 Hz, 1H), 4.98−4.91 (m, 2H), 4.29−4.24 (m, 1H), 3.56−3.48 (m, 2H), 3.04 (s, 1H), 2.22−2.11 (m, 3H), 2.08−2.02 (m, 1H); 13C NMR (CD3OD, 176 MHz) δ (ppm) 159.45, 154.93, 152.89, 138.34, 118.08, 84.98, 74.93, 73.55, 66.03, 52.51, 51.32, 40.54, 39.40; MS (ESI) m/z = 290.4 (MH+). The two enantiomers of compound (±)-29 were separated by preparative chiral SFC with the following conditions. Column: CHIRALCEL OD-H, 2 cm × 25 cm, 5 μm. Mobile phase A: CO2. Mobile phase B: MeOH. Gradient: 30% B in 6 min. Flow rate: 60 mL/min. Detector: UV 254 nm. The process afforded isomer 29a (faster eluting, tR = 1.76 min; MS (ESI) m/z = 290.2 (MH+)) and isomer 29b (slower eluting, tR = 2.22 min; MS (ESI) m/z = 290.2 (MH+)). N-{6-Ethoxy-9-[(1R,3S,4S)- and (1S,3R,4R)-3-Ethynyl-3-(hydroxymethyl)-4-(methoxymethoxy)cyclopentyl]-9H-purin-2yl}-2-methylpropanamide (±)-31. Step a: PPh3 (1.658 g, 3 equiv) was suspended in anhydrous THF (60 mL) under N2 and then cooled to 0 °C using an ice bath. DIAD (1.193 g, 2.8 equiv) was added dropwise, and the reaction mixture was stirred for 30 min. The resulting mixture was added to a solution of (±)-13 (0.924 g, 2.107 mmol) and N-(6-ethoxy-9H-purin-2-yl)isobutyramide (0.788 g, 1.5 equiv) in anhydrous THF (60 mL) at −41 °C (N2/acetonitrile bath). The reaction mixture was stirred at −41 °C for 2 h and then warmed to room temperature for 16 h. Solvent was removed in vacuo, and the crude residue was purified by gradient elution on SiO2 (DCM/ MeOH: 0−10%) to afford 9-[(2R,4S,5R) and (2S,4R,5S)-5-[[tertbutyl(diphenyl)silyl]oxymethyl]-5-ethynyl-4-(methoxymethoxy)tetrahydrofuran-2-yl]-6-ethoxypurin-2-amine (0.706 g) contaminated with DIAD-H2. It was used in the subsequent step without further purification. MS (ESI) m/z = 670.4 (MH+). Step b: To the product from step a dissolved in anhydrous THF (15 mL) and acetic acid (0.3 mL, 5.22 mmol) was added a solution of TBAF 1 M in THF (2.09 mL, 2.09 mmol). The reaction mixture was stirred at room temperature for 16 h. Solvents were removed in vacuo, and the crude residue was purified by gradient elution on SiO2 (DCM/MeOH: 0−10%) to afford (±)-31 (440 mg, 49% 2 steps). 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 10.31 (s, 1H), 8.32 (s, 1H), 5.21−5.13 (m, 1H), 5.10 (t, J = 5.96 Hz, 1H), 4.69 (d, J = 6.62 Hz, 1H), 4.65 (d, J = 6.62 Hz, 1H), 4.57 (q, J = 7.06 Hz, 2H), 4.45 (t, J = 7.06 Hz, 1H), 3.62−3.54 (m, 2H), 3.28 (s, 3H), 3.10 (s, 1H), 2.98− 2.91 (m, 1H), 2.41−2.22 (m, 4H), 1.40 (t, J = 7.06 Hz, 3H), 1.09 (d, J = 6.83 Hz, 6H); MS (ESI) m/z = 432.8 (MH+). [(1S,2S,4R)- and (1R,2R,4S)-4-(2-Amino-6-ethoxy-9H-purin9-yl)-1-ethynyl-2-(methoxymethoxy)cyclopentyl]methanol (±)-32. A solution of potassium hydroxide (629 mg, 11.22 mmol) in MeOH (12 mL) was added to compound (±)-31 (440 mg, 1.02 mmol). The reaction mixture was stirred at room temperature for 6 h and at 0 °C for 16 h and then quenched with acetic acid (0.701 mL, 12.24 mmol). The reaction mixture was concentrated in vacuo, and the crude residue purified by gradient elution on SiO2 (DCM/ MeOH: 0−10%) to afford (±)-32 (212 mg, 66%). 1H NMR (DMSOd6, 400 MHz) δ (ppm) 7.99 (s, 1H), 6.39 (brs, 2H), 5.16 (t, J = 5.77 Hz, 1H), 5.10−5.01 (m, 1H), 4.67 (d, J = 6.69 Hz, 1H), 4.63 (d, J = 6.69 Hz, 1H), 4.44 (q, J = 7.01 Hz, 2H), 4.31−4.28 (m, 1H), 3.59− 3.51 (m, 2H), 3.28 (s, 3H), 3.09 (s, 1H), 2.32−2.14 (m, 4H), 1.35 (t, J = 7.01 Hz, 3H); MS (ESI) m/z = 362.6 (MH+). Propan-2-yl N-[(S)-{[(1R,2R,4S)-4-(2-Amino-6-ethoxy-9Hpurin-9-yl)-1-ethynyl-2-(methoxymethoxy)cyclopentyl]methoxy}(phenoxy)phosphoryl]-L-alaninate 33a and Propan2-yl N-[(S)-{[(1S,2S,4R)-4-(2-Amino-6-ethoxy-9H-purin-9-yl)-1ethynyl-2-(methoxymethoxy)cyclopentyl]methoxy}9226



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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00141. All experimental procedures, characterization data for new compounds, in vitro experimental protocols, and details on computational modeling (PDF) Molecular formula strings for compounds 19, 20, 24− 26, 29b, 30, 34a, and 34b and some data (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*F.-R.A.: e-mail, [email protected]. *I.T.R.: e-mail, [email protected]. ORCID

François-René Alexandre: 0000-0003-1396-3757 Izzat T. Raheem: 0000-0002-6769-8816 Author Contributions ∥

F.-R.A. and I.T.R. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank colleagues at Idenix and Merck & Co, Inc., Kenilworth, NJ, U.S., for their help and assistance during this project. Dr. John Sanders and Dr. Jay Grobler are gratefully acknowledged for support preparing this manuscript.

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ABBREVIATIONS USED AUC,area under the plasma concentration−time curve; Cl,clearance; DCM,dichoromethane; DIAD,diisopropyl azodicarboxylate; DMAP,4-dimethylaminopyridine; DMF,dimethylformamide; DPC,diphenylcarbamoyl; F,bioavailability; HAART,highly active antiretroviral therapy; HIV-1,human immunodeficiency virus type 1; iv,intravenous; Me,methyl; MeCN,acetonitrile; MeOH,methanol; MOM,methoxymethyl

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REFERENCES

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