Synthesis and Antiviral Evaluation of Carbocyclic Nucleoside Analogs

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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 L. Fillgrove, Ming-Tain Lai, Jean-Christophe Meillon, Min Xu, James Small, Cyril Dousson, and Izzat Raheem J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00141 • Publication Date (Web): 28 Sep 2018 Downloaded from http://pubs.acs.org on September 29, 2018

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Journal of Medicinal Chemistry

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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 Alexandrea,*,‡, Rachid Rahalia, Houcine Rahalia, Sandra Guillonb, Thierry Convarda, Kerry Fillgrovec, Ming-Tain Laic, Jean-Christophe Meillonb, Min Xuc, James Smallc, Cyril B. Doussona and Izzat T. Raheemc,*,‡ a

Idenix an MSD Company, Cap Gamma, 1682 rue de la Valsière, 34189 Montpellier Cedex 4, France

b

c

Oxeltis, Cap Delta, 1682 rue de la Valsière, 34189 Montpellier Cedex 4, France

Merck & Co., Inc., PO Box 4, 770 Sumneytown Pike, West Point, PA 19486, USA

KEYWORDS Nucleoside reverse transcriptase inhibitor, antiviral, nucleoside analog, carbocycle, anabolite

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 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.

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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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 in two classes: nucleoside (or nucleotide) reverse transcriptase inhibitors (NRTIs or NtRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs).2 NNRTIs allosterically inhibit reverse transcription upon binding to a hydrophobic site near to 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 toxicity and the emergence of mutants that are resistant to the drugs emphasize the need for novel structurally and functionally differentiated nucleosides.4

O O

HO

N

N HO

NH

O

O

O

H 2N

F

H2N N

N

H N

N

N

N

N OH

S

O

O S

NH 2

ABC

3TC

N N

HO

O

O

NH

d4T

ddI

AZT

N

NH

N

O

N3

O O HO

N

OH

FTC H2 N

NH 2 HO

O

N

N O

ddC

N

N

R1

O P

N O

R2 R 1=

N

CO2H

HO2C

R2=

TDF: OCH 2 OCOOiPr TAF: R 1= OPh, R 2=NHCH(Me)CO2 iPr (S)

Chart 1: Approved NRTIs and NtRTIs.

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 translocation-defective 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


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incorporated, MK-8591-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 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 both preclinical species and in the clinic,8 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 naïve patients at oral doses as low as 0.5 mg given once weekly.9

O

HO HO

N NH2

N N

N

HO X

Base HO

F

MK-8591 PBMC IC50 = 0.21 nM MK-8591-TP RT-Pol IC50 = 263 nM

Chart 2: Chemical structure of MK-8591 (left) and representative 4’-substituted carbocyclic targeted structures

As part of a continued discovery effort driven by the further exploration of the structure activity relationship (SAR) of MK-8591 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 HIVRT, and these carbocyclic analogues 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 towards 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.

▊ 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 (±)-



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3 was then protected with a methoxymethyl (MOM) group to provide (±)-4. The ester functionalities were reduced with lithium aluminium 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-butyldimethylsilyl chloride (TBSCl), 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 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.

Si O

H H

HO

2

H

O

O O

H H

O

HO

H4

O Si

H

O H

H

H

H2

H4

O (±)-6

(±)-7

Chart 3: Primary NOESY NMR correlations (red arrows) in (±)-6 and (±)-7 used for stereochemical assignment.

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.

Scheme 1: Synthesis of intermediates (±)-6 and (±)-7a


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O

O O

OMe

a

HO

H

b O

O

O

O

TBDPSO e

HO

+ TBDPSO

O

O

O

d

OO

O

HO + TBDPSO

O

O

(±)- 4

O (±)- 6

(±)- 5

O O

c

O

HO

HO

O

O

O HO (±)- 3

2

1

HO

O O

OMe

OMe

O

O

OMe

O

+

HO O

O

O

O (±)- 7

O

TBDPSO

(±)- 9

(±)- 8

a

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.

Scheme 2: Synthesis of alcohols (±)-13 and (±)-16a

(±)-6

a

O

TBDPSO

O

TBDPSO O O

or c (X=N)

X O O

O (±)-10

OH

TBDPSO d

b (X=CH)

(±)- 11 X= CH (±)- 14 X= N

OH

TBDPSO e

X

O O (±)- 12 X= CH (±)- 15 X= N

X

O O

(±)- 13 X= CH (±)- 16 X= N

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.

Scheme 3: Synthesis of 2’-deoxy-carbocyclic nucleosidesa



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(±)-13 X= CH (±)-16 X= N

b,d

a,d

c,d

O N

HO X

N

N

Bz

O

X

O

O

O (±)-17 X= CH (±)-18 X= N

X

N

HO

NH O

HO (±)-19 X= CH (±)-20 X= N

X

NH 2

N

HO

O

HN

O (±)-27 X= CH (±)-28 X= N e,f

O N

N

N O

e,g

e,f

X

N

R2 (±)-21 X= CH, R 2= F (±)-22 X= CH, R 2= Cl (±)-23 X= N, R2= F

ODPC

N

HO N

O

N

Cl

N

HO

N HO

N N

HO

N X R2

(±)-24 X= CH, R 2 = F (±)-25 X= CH, R 2 = Cl (±)-26 X= N, R2= F

N HO (±)-29 X= CH (±)-30 X= N

O NH NH2

a

Reagents and conditions: (a) N3-Bz-thymine, PPh3, DIAD, THF, −41 °C to room temperature, 16 h; (b) 2-fluoro-6-chloropurine or 2,6-

dichloropurine, 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 h to 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) 7N 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.

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 de-silylation using TBAF in the presence of AcOH in THF to afford 5’OH carbocyclic nucleoside analogues, 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.

▊ 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


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series, thymine (T) derivative (±)-19 showed no activity (Table 1, entry 3), adenosine (A) derivatives (±)-24 and (±)-25 were modestly active in the mid-nanomolar range (Table 1, entries 4−5), 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 13). Based on 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 which was consistent with a modeling study of their corresponding TPs described latter in this manuscript.

Scheme 4: Chiral separation of (±)-29a N (±)- 29

a

N

HO

N HO 29a

N

O NH

+

O

N

HO

NH

N HO

NH2

29b

NH 2

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.

Table 1. Structure and in vitro activity of 2’-deoxy− −carbocyclic nucleosides against wild-type HIV-1-infected hPBMCs. HO X

Entry

Cpd

Base HO

X

Base

IC50a

CC50b

(µM)

(µM)

1

MK-8591

0.00021

>4.2

2

3TC

0.171

>42.0

3

(±)-19

CH

T

>2.10

>42.0

4

(±)-24

CH

2FA

0.201

>42.0



a

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5

(±)-25

CH

2ClA

0.232

>42.0

6

(±)-29

CH

G

0.033

>42.0

7

(±)-20

N

T

>2.10

>42.0

8

(±)-26

N

2FA

>2.10

>42.0

9

(±)-30

N

G

1.15

>42.0

10

29a

CH

G

>2.10

>42.0

11

29b

CH

G

0.024

>42.0

12

34a

CH

GOEt

0.382

>4.2

13

34b

CH

GOEt

0.0030

>42.0

IC50 values were determined in 10% NHS in a single-cycle HIV replication assay in hPBMCs.

b

CC50 values were determined in 10%

NHS condition in an MT4 cell line (See Supporting Information for details).

Table 2. Structure and in vitro activity of 2’-deoxy-carbocyclic nucleoside triphosphates against wild-type HIV-1 RT. O O O HO P P O HO P O HO HO O X

Entry

Cpd

Base HO

X

Base

IC50a (µM)

1

(±)-19-TP

CH

T

0.114

2

(±)-24-TP

CH

2FA

3.46

3

(±)-25-TP

CH

2ClA

20.2

4

(±)-29-TP

CH

G

0.324

5

(±)-20-TP

N

T

0.523

6

(±)-26-TP

N

2FA

ND

7

(±)-30-TP

N

G

0.444

8

MK-8591-TP


0.263

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9 a

3TC-TP

0.015

HIV reverse transcriptase polymerase assay (RT-Pol); IC50 values were determined by non-linear 4-parameter curve fitting (See Sup-

porting Information for details).

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 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 allowed to be unconstrained, and the calculation was repeated. A maximum of ten 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: 1rtd17), and their geometries 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 were 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 re-mapped 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 on Figure 1A and 1B, respectively. We assumed that the active enantiomer of (±)-24-TP would be 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 pre-catalytic complex of RT bound to the dideoxyguanosine (ddG) terminated double-stranded DNA, MK-8591-TP adopts a C-3’endo ring conformation (Figure 1, A), displays Watson–Crick base pairing interactions, and shows strong hydrophobic interactions of the 4’-ethynyl with Phe160 (π-σ), Tyr115 (π-alkyl), Met184 (alkyl) and Ala114 (alkyl). In the



Page 10 of 24

case of (1S,3R,4R)-24-TP (Figure 1, B), 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 as d= 2.79 Å for MK-8591-TP) and no interaction with Ala114. These observations suggest that (1S,3R,4R)-24-TP is less favorably bound to the RT compared to MK-8591-TP. 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 bases 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

A

B


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C

D

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; metal ions are displayed in green.

Scheme 5: Synthesis and chiral separation of phosphoramidate prodrugs 34a and 34b

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)-L-alaninate, tBuMgCl 1M 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, Chiralcel OD-H column, mobile phase CO2/MeOH; faster eluting isomer 34a (28%) and slower eluting isomer 34b (30%).



Page 12 of 24

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 (p.o., 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/kg) and half-life (9.8 h). These data provided early confidence in the in vivo performance of this class.

Table 3. Rat pharmacokinetic profile of MK-8591 and 29b. Dose

AUC0-∞

Cl

t1/2

F

(mg/kg)

(µM•h)

(mL/min/kg)

(h)

(%)

iv

1

0.78

72

0.7

p.o.

5

5.0

iv

1

1.82

p.o.

2

3.30

Routea

Cpd

MK8591

29b

a

>100 32.4

9.8 91

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

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, 6-ethoxy guanine 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 3 steps.20 Upon liberation of the free monophosphate, the 2-amino-6-ethoxy-purine is transformed to a G by intracellular adenosine deaminase.21 The 2-amino-6-ethoxy-purine 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


Page 13 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 13, 14 and 15). Based upon 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.

▊ 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 analogues was postulated to be from the (1S,3R,4S) 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 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 reagent-grade materials under an atmosphere of nitrogen. Solvents were

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 (15−45µm, 40−63 µm, or spheric silica pre-packed cartridges) using a Biotage® Flash Chromatography apparatus (Isolera). 1H (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). 13

C (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, quar-



Page 14 of 24

tet; 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, 2.1x30 mm for standard methods and ACQUITY UPLC HSST3 1.8 µm, 2.1x30 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 consisted of an Agilent 6140 quadrupole LC/MS platform with PDA detector. The column for standard methods was Purospher® STAR RP-18 endcapped 2µm, 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 2 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 were performed on a Gilson system 233XL WITH 735 (Unipoint). The column was a Waters XBridge Prep C18 5µm OBD, dimension 30x250 mm. The mobile phase consisted of mixture of acetonitrile/ammonium carbonate 0.02N (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, 20x250 mm) (Daicel Chemical Industries, Ltd.) with desired isocratic solvent systems identified on 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-1-yl)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 16h, 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 hour. 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 to 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 hour, 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 to 100%) to


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afford (±)-cis and (±)-trans diallyl-4-(allyloxy)-2-(methoxymethoxy)cyclopentane-1,1-dicarboxylate (±)-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 to 10%) to afford the expected compound (±)-5 (5.66 g, 92%) as a mixture of isomer.1H NMR (DMSO-d6, 400 MHz) δ (ppm) 5.915.81 (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), 3.333.24 (m, 2H), 3.23 (s, 3H), 2.30-2.23 (m, 0.2H), 1.95-1.84 (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-(((tert-butyldiphenylsilyl)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 ethyl acetate (500 mL), and washed with a 1M 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 to 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), 3.993.93 (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,2S,4R) and (1R,2R,4S)-4-(allyloxy)-1-(((tert-butyldiphenylsilyl)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 to 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


Page 16 of 24

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 to 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 so-

lution 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 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 to 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 = 6.69Hz, 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 sus-

pension of triphenylphosphine (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 hours. EtOAc (250 mL) was added followed by a 1M HCl solution (250 mL). The aqueous layer was extracted twice with EtOAc, and the combined organic layer were 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-6-yl diphenylcarbamate (±)-27. Step a: Triphenylphosphine (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


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min. The resulting mixture was added to a solution of (±)-13 (400 mg, 0.912 mmol) and 2-acetamido-9H-purin-6-yldiphenylcarbamate (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 to 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 to 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-hydroxy-3-(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)-3-ethynyl4-hydroxy-3-(hydroxymethyl)cyclopentyl)-6-oxo-6,9-dihydro-1H-purin-2-yl)acetamide was used in the subsequent step without further purification. 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 12.01 (s, 1H), 11.68 (s, 1H), 8.15 (s, 1H), 5.10-5.03 (m, 3H), 4.314.27 (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 CH3CN (10 mL) was added a 7N 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/CH3CN (1:1), filtered and washed with water/CH3CN (1:1), CH3CN 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 = 5.82Hz, 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 condition - Column: OD-H, 2x25 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; to afford: Isomer 29a (Faster eluting: Rt= 1.76 min; MS (ESI) m/z = 290.2 (MH+)) and Isomer 29b (Slower eluting: Rt= 2.22 min; MS (ESI) m/z = 290.2 (MH+)).


N-{6-ethoxy-9-[(1R,3S,4S)

and

Page 18 of 24

(1S,3R,4R)-3-ethynyl-3-(hydroxymethyl)-4-(methoxymethoxy)cyclopentyl]-9H-purin-2-yl}-2-

methylpropanamide (±)-31. Step a: Triphenylphosphine (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 to 10%) to afford 9-[(2R,4S,5R) and (2S,4R,5S)-5-[[tert-butyl(diphenyl)silyl]oxymethyl]-5ethynyl-4-(methoxymethoxy)tetrahydrofuran-2-yl]-6-ethoxy-purin-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 1M 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 to 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 = 6.62Hz, 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-purin-9-yl)-1-ethynyl-2-(methoxymethoxy)cyclopentyl]methanol (±)-32. A so-

lution 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 hours 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 to 10%) to afford (±)-32 (212 mg, 66%).1H NMR (DMSO-d6, 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-9H-purin-9-yl)-1-ethynyl-2-(methoxymethoxy)cyclopentyl]

(phenoxy)phosphoryl]-L-alaninate

33a

and

Propan-2-yl

methoxy}-

N-[(S)-{[(1S,2S,4R)-4-(2-amino-6-ethoxy-9H-purin-9-yl)-1-ethynyl-2-

(methoxymethoxy)cyclopentyl]methoxy}-(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 1M 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 to 10%) to afford the product a mixture of diastere-


Page 19 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


omers 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), 7.237.21 (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), 2.402.11 (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 (phenoxy)phosphoryl]-L-alaninate

N-[(S)-{[(1R,2R,4S)-4-(2-amino-6-ethoxy-9H-purin-9-yl)-1-ethynyl-2-hydroxycyclopentyl]methoxy}34a

and

Propan-2-yl

N-[(S)-{[(1S,2S,4R)-4-(2-amino-6-ethoxy-9H-purin-9-yl)-1-ethynyl-2-

hydroxycyclopentyl]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 °C 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 to 4%) to afford the title compound as a mixture of diastereomers 34a and 34b (145 mg, 57%). 31P NMR (DMSOd6, 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: AD-H, 2×25 cm, 5 µm; Mobile Phase A: CO2; Mobile Phase B: iPrOH; Gradient: 30% B in 8 min; Flow rate: 60 mL/min; Detector: UV 220 nm; to afford: Diastereomer 34a (40mg, 28%): Faster eluting: Rt= 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), 7.177.14 (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+) and diastereomer 34b (42 mg, 30%) : Slower eluting: Rt= 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), 3.773.71 (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+).

▊ AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected] (I. T. Raheem)



Page 20 of 24

* E-mail: [email protected] (F.-R. Alexandre)

Author Contributions ‡ F.-R.A. and I.T.R. contributed equally.

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

▊ 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; NNRTI non-nucleoside reverse transcriptase inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; NRTTI, nucleoside reverse transcriptase translocation inhibitor; PDC, pyridinium dichromate; PK, pharmacokinetic; p.o., oral; RT, reverse transcriptase; TBAF, tetrabutyl ammonium fluoride; TBS, tert-butyldimethylsilyl; TBDMS tert-butyldiphenylsilyl; THF, tetrahydrofuran; TP, triphosphate; SAR, structure-activity relationship; SFC, supercritical fluid chromatography.

▊ ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX. All experimental procedures, characterization data for new compounds, in vitro experimental protocols, and details on computational modeling (PDF). Molecular Formula Stings for compounds 19, 20, 24, 25, 26, 29b, 30, 34a, and 34b (CSV).

▊ REFERENCES (1) (a) De Clercq, E. Chapter Nine - The Nucleoside Reverse Transcriptase Inhibitors, Nonnucleoside Reverse Transcriptase Inhibitors, and Protease Inhibitors in the Treatment of HIV Infections (AIDS). In Advances in Pharmacology; De Clercq, E., Ed.; Antiviral Agents; Academic Press, Oxford, 2013; Vol. 67, pp 317–358. (b) De Clercq, E. A 40-Year Journey in Search of Selective Antiviral Chemotherapy*. Annual Review of Pharmacology and Toxicology 2011, 51 (1), 1–24. (c) Anusha, N. P. Antiretroviral Strategies for Treatment of HIV. J. Antivir. Antiretrovir. 2011, 3, 55-59. (d) Tressler, R.; Godfrey, C. NRTI Backbone in HIV Treatment. Drugs 2012, 72 (16), 2051–2062. (e) Tan, X.; Chu, C. K.; Boudinot, F. D. Development and Optimization of Anti-HIV Nucleoside Analogs and Prodrugs: A Review of Their Cellular Pharmacology, Structure-Activity Relationships and Pharmacokinetics. Advanced Drug Delivery Reviews 1999, 39 (1), 117–151. (2) Kukhanova, M. K. Anti-HIV Nucleoside Drugs: A Retrospective View into the Future. Mol. Biol. 2012, 46 (6), 768–779.


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(11) Wang, J.; Rawal, R.K.; Chu, C.K. Chapter One – Recent Advances in Carbocyclic Nucleosides: Synthesis and Biological Activity. In Zhang, L-H.; Xi, Z.; Chattopadhyaya, J., Ed.; Medicinal Chemistry of Nucleic Acids; John Wiley & Sons, Inc., Hoboken, 2011, pp 1–100. (12) (a) Liu, P.; Sharon, A.; Chu, C. K. Enantiomeric synthesis of carbocyclic D-4′-C-methylribonucleosides as Potential Antiviral Agents. Tetrahedron: Asym. 2006, 17, 3304–3314. (b) Ko, O. H.; Hong, J. H. Efficient Synthesis of Novel Carbocyclic Nucleosides via Sequential Claisen Rearrangement and Ring-Closing Metathesis. Tetrahedron Lett. 2002, 43, 6399–6402. (c) Kim, A.; Hong, J. H. Synthesis and Antiviral Activity of Novel 2',4'-Doubly Branched Carbocyclic Nucleosides. Nucleosides Nucleotides Nucleic Acids 2004, 23, 813–822. (d) Baik, G. H.; Chung, B. Y.; Oh, C.-H.; Cho, J.-H.; Ko, O. H.; Hong, J. H. Novel Synthesis of 4′C-Aryl-Branched Acyclic Nucleoside Using [3,3]-Sigmatropic Rearrangement. Nucleosides Nucleotides Nucleic Acids 2003, 22, 1781–1788. (e) Kumamoto, H.; Haraguchi, K.; Tanaka, H.; Nitanda, T.; Baba, M.; Dutschman, G. E.; Cheng, Y.-C.; Kato, K. Synthesis of (±)-4′-Ethynyl and 4′-Cyano Carbocyclic Analogues of Stavudine (d4T). Nucleosides Nucleotides Nucleic Acids 2005, 24, 73–83. (f) Kato, K.; Suzuki, H.; Tanaka, H.; Miyasaka, T. Enantioand Diastereoselective Synthesis of 4′-α-Substituted Carbocyclic Nucleosides. Tetrahedron: Asymmetry 1998, 9, 911–914. (g) Kim, H. S.; Jacobson, K. A. Synthesis of a Novel Conformationally Locked Carbocyclic Nucleoside Ring System. Org. Lett. 2003, 5, 1665–1668. (h) Yin, K.-Q.; Schneller, S. W. A Practical Synthetic Route to 4′-Alkylaristeromycin Derivatives: 4′-Methylaristeromycin. Tetrahedron Lett. 2006, 47, 4057–4059. (13) Hřebabecký, H.; Masojídková, M.; Holý, A. Synthesis of Carba Analogues of Deoxy-4-C-(Hydroxymethyl)-Pentofuranoses, Intermediates in the Synthesis of Carbocyclic Nucleosides. Collect. Czech. Chem. Commun., CCCC 1998, 63, 2044–2064. (14) Tsukamoto, H.; Suzuki, T.; Kondo, Y. Remarkable Solvent Effect on Pd(0)-Catalyzed Deprotection of Allyl Ethers Using Barbituric Acid Derivatives: Application to Selective and Successive Removal of Allyl, Methallyl, and Prenyl Ethers. Synlett 2007, 20, 3131–3132. (15) See Supporting Information. A full manuscript reviewing the details of this assay is forthcoming (Lai, M-T., et al. manuscript in preparation). (16) Caton-Williams, J.; Lin, L.; Smith, M.; Huang, Z. Convenient Synthesis of Nucleoside 5′-Triphosphates for RNA Transcription. Chem. Commun. 2011, 47, 8142–8144. (17) Pdb: 1rtd was used as a starting point for modeling efforts. This complex contains HXB2 RT, which was also used for the program’s RT-Pol assay and therefore provided an optimal starting point. Prior to docking ligands of interest, NTP was removed and primer was modified to align with the ligand being modeled. See Supporting Information for details. (18) Sarafianos, S. G.; Marchand, B.; Das, K.; Himmel, D.; Parniak, M. A.; Hughes, S. H.; Arnold, E. Structure and Function of HIV-1 Reverse Transcriptase: Molecular Mechanisms of Polymerization and Inhibition. J. Mol. Biol. 2009, 385, 693–713. (19) Roy, B.; Depaix, A.; Périgaud, C.; Peyrottes, S. Recent Trends in Nucleotide Synthesis. Chem. Rev. 2016, 116, 7854–7897.


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(20) (a) Murakami, E.; Tolstykh, T.; Bao, H.; Niu, C.; Micolochick Steuer, H. M.; Bao, D.; Chang, W.; Espiritu, C.; Bansal, S.; Lam, A. M.; Otto, M. J.; Sofia, M. J.; Furman, P. A. Mechanism of Activation of PSI-7851 and its Diastereoisomer PSI7977. J. Biol. Chem. 2010, 285, 34337–34347. (b) Derudas, M.; Carta, D.; Brancale, A.; Vanpouille, C.; Lisco, A.; Margolis, L.; Balzarini, J.; McGuigan, C. The Application of Phosphoramidate Protide Technology to Acyclovir Confers Anti-HIV Inhibition. J. Med. Chem. 2009, 52, 5520–5530. (21) Niu, C.; Tolstykh, T.; Bao, H.; Park, Y.; Babusis, D.; Lam, A. M.; Bansal, S.; Du, J.; Chang, W.; Reddy, P. G.; Zhang, H.-R.; Woolley, J.; Wang, L.-Q.; Chao, P. B.; Ray, A. S.; Otto, M. J.; Sofia, M. J.; Furman, P. A.; Murakami, E. Metabolic Activation of the Anti-Hepatitis C Virus Nucleotide Prodrug PSI-352938. Antimicrob. Agents Chemother. 2012, 56, 3767–3775. (22) Ross, B. S.; Ganapati Reddy, P.; Zhang, H.-R.; Rachakonda, S.; Sofia, M. J. Synthesis of Diastereomerically Pure Nucleotide Phosphoramidates. J. Org. Chem. 2011, 76, 8311–8319.



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Table of Contents Graphic

O O HO HO

N

N NH2

N N

N MK-8591

F

HO X

Base

HO

HO

HO

Base= T, 2-FA,2-ClA, G X= CH, CN

O

N N

NH NH2

29b PBMC IC50= 24 nM

O

H N

O P O O

N N

OEt

N N HO NH 34b 2 PBMC IC50= 3.0 nM

x


24

Synthesis and Antiviral Evaluation of Carbocyclic Nucleoside Analogs

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