Next-Generation Reduction Sensitive Lipid Conjugates of Tenofovir: Antiviral Activity and Mechanism of Release

Article abstract of DOI:10.1021/acs.jmedchem.6b01292

The pharmacokinetic properties of tenofovir (TFV) and other charged nucleoside analogues are dramatically improved upon conjugation to a lipid prodrug. We previously prepared reduction-sensitive lipid conjugates of TFV that demonstrate superior antiviral

Full text of DOI:10.1021/acs.jmedchem.6b01292

Article
pubs.acs.org/jmc
Next-Generation Reduction Sensitive Lipid Conjugates of Tenofovir:
Antiviral Activity and Mechanism of Release
Kyle E. Giesler* and Dennis C. Liotta*
Department of Chemistry, Emory University, 1521 Dickey Drive NE, Emerson 305, Atlanta, Georgia 30322, United States
S
* Supporting Information
ABSTRACT: The pharmacokinetic properties of tenofovir (TFV) and other charged nucleoside analogues are dramatically
improved upon conjugation to a lipid prodrug. We previously prepared reduction-sensitive lipid conjugates of TFV that
demonstrate superior antiviral activity compared to other lipid conjugates including the clinically approved formulation, tenofovir
disoproxil fumarate (TDF). In continuation of that work, we have synthesized next-generation conjugates with reduced
cytotoxicity that retain potent antiviral activity against HIV-1 and HBV with a therapeutic index >100000 for our most potent
conjugate. We also show that disulfide reduction is not responsible for prodrug cleavage unless 3-exo-tet intramolecular
cyclization can occur, suggesting that enzymatic hydrolysis is predominantly responsible for activity of our prodrugs in vitro.
cathepsin A to selectively release TFV within the cytosol and
demonstrates little to no nephrotoxicity.17,18 CMX-157 is a lipid
prodrug formulation of TFV that is also cleaved by intracellular
enzymes to minimize plasma exposure and reduce nephrotox-
icity. CMX-157 is endowed with interesting pharmacokinetic
properties and retains significant antiviral activity for up to a week
after a single administration.19,20 The potential for reduced
dosing frequency is highly attractive and inspired us to develop
novel reduction sensitive lipid conjugates of TFV that rival CMX-
157.21 We previously reported that our first-generation
conjugates shown in Figure 1 release TFV in vitro to potently
inhibit HIV-1 and HBV replication in PBMCs and hepatocytes,
respectively. These compounds demonstrate subnanomolar
HIV-1 activity, have a wider therapeutic index (TI) than TDF
and CMX-157 at the EC50, and are stable in human plasma for
more than 24 h. Despite these therapeutic gains, we speculated
that the β-mercaptoethyl spacer is a precursor for thiirane, which
has been implicated in the decomposition of S-acyl-2-thioethyl
(SATE) and dithioethanol (DTE) prodrugs. The mutagenic
potential of thiirane has largely precluded the clinical use of
SATE and DTE-bound nucleosides, which prompted us to
prepare next-generation reduction-sensitive TFV conjugates that
exhibit potent anti-HIV-1 and anti-HBV activity with reduced
cytotoxicity. Herein, we disclose the details of these efforts and
also probe the mechanism of prodrug release.
INTRODUCTION
■
Many drug candidates and natural products are flanked with
structural features that may limit their therapeutic potential in
vivo.1−3 These tend to include carboxylic acids,4 amines,5
dianionic phosphates,6 and polyhydroxylated aromatic rings,7,8
which often require temporary protection with a prodrug to
improve the absorption, distribution, metabolism, and excretion
(ADME) properties of a pharmacologically active compound
within the body.9,10
Tenofovir (TFV) is an acyclic antiviral nucleoside analogue
that has achieved renowned clinical success for the treatment of
hepatitis B virus (HBV)11,12 and human immunodeficiency virus
(HIV)13,14 when administered as its prodrug, tenofovir
disoproxil fumarate (TDF), which contains two isopropylox-
ymethyl carbonate prodrugs esterified to the phosphonate
moiety of TFV. These masking groups are cleaved both
extracellularly and intracellularly by nonspecific carboxyes-
terases, releasing TFV, which is subsequently phosphorylated
to its active diphosphate to arrest viral replication. In the absence
of prodrug conjugation, the considerable dianionic character of
TFV at physiological pH reduces its oral bioavailability to <10%
in various animal models.14 Although TDF largely overcomes
this limitation, continuous exposure to TDF has been linked to a
series of conditions including lactic acidosis, Fanconi syndrome,
acute renal failure, and bone loss, due in part to its cleavage in the
bloodstream.15,16
Systemic exposure to TFV may be reduced by altering the
identity of the prodrug. For instance, tenofovir alafenamide
(TAF) features a phosphoramidate prodrug that is cleaved by
Received: August 26, 2016
Published: October 19, 2016
© XXXX American Chemical Society
A
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address from our previous work was the mechanism by which
first-generation conjugate 1b liberates TFV. Presumably 1b
undergoes glutathione (GSH)-mediated reduction within the
cytosol to reveal transient intermediate 8 shown in Scheme 2.
This intermediate is thought to collapse upon itself to generate
thiirane with concomitant release of TFV. To validate this
mechanism, we initially treated 1b with GSH in PBS at 37.4 0.2
°C and monitored decomposition by LC-MS. Unfortunately, the
poor aqueous solubility of 1b precluded accurate spectral
measurements and necessitated an alternative model system.
We therefore chose to access thiolate 8 via deprotonation of 6
rather than subjecting 1b to GSH with the underlying
assumption that intramolecular cyclization governs TFV release.
Thiol 6 was incubated in various aqueous solutions buffered at
pH 2.0, 9.19, and 11.56 spiked with 0.1 M DTT at 37.4 °C. 6
demonstrated high stability in glycine/HCl buffer (pH = 2.0)
with no observed decomposition. Moderate stability was noted
in PBS (pH = 7.43) with the appearance of a new UV signal that
faintly presented itself at a significantly polar retention time (tr =
0.66 min). This species was identified as TFV when compared to
an authentic sample of the parent nucleoside. When 6 was
incubated in aqueous carbonate/bicarbonate buffer (pH = 9.19),
significant decomposition occurred and complete consumption
of starting material was noted within 8 min at pH 10.16. Figure 2
graphically reveals a pseudo-first-order rate dependence wrt 6
and clearly illustrates the link between pH and rate of reaction.
Using the method of initial rates, the differential rate law was
found to be first order wrt [OH−], resulting in the overall second-
order rate expression for the decomposition of 6.
Figure 1. Comparison of structural features previously examined with
those investigated in the current study.
RESULTS AND DISCUSSION
■
Chemistry. Conjugates 1−5b, 6, and 7 were readily prepared
using our previous protocol outlined in Scheme 1. Briefly,
hexadecanethiol or dodecanethiol was oxidized with select thiols
in the presence of iodine to furnish disulfides 1−5a as low-
melting-point solids that were purified by normal phase column
chromatography. Compounds 1−5a were then coupled to TFV
with oxalyl chloride and DMF to afford the corresponding
conjugates 1−5b after formimidine deprotection and purification
on silica gel using a DCM:MeOH:NH4OH gradient. Both 6 and
7 were purified on a C18 reverse phase column and were isolated
as free acids following lyophilization. The structures of 1−5b, 6,
and 7 are presented in Table 1.
d[6]
dt
= k[6][OH−]
(1)
The [OH−] dependence in eq 1 suggests that thiolate 8
performs the 3-exo-tet cyclization to release TFV rather than thiol
6, which is consistent with the mechanism presented in Scheme
2. These results are also in agreement with previous work22,23 and
confirm that thiolates of SATE, DTE, and 1b have the capacity to
cyclize and release the parent nucleoside. When assayed against
HIV-infected PBMCs, 6 is 58-fold less active than TFV, which
indicates that the mercaptoethyl spacer is not cleaved enzymati-
cally and reinforces the notion that a single proton governs TFV
release.
Kinetic Studies and Antiviral Activity. Conjugates 1b, 2b,
and 5b feature the disulfide moiety at various positions along the
lipid tail with increasing distance from the phosphonic acid
whereas 3b and 4b are endowed with a benzylic ring to restrict
the conformational flexibility of the spacer. The influence of
these modifications on anti-HIV and anti-HBV activity is shown
in Table 1 and is discussed herein. The first question we sought to
Scheme 1. Synthesis of Conjugates 1−5b, 6, and 7
B
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a
Table 1. HIV-1 and HBV Activity of Conjugates 1−5b, 6, and 7 Compared to TFV and TDF
a
b
All data represent an average of triplicate experiments. R = tenofovir. EC50, effective concentration (in μM) required to inhibit HIV-1 or HBV by
c
50%. CC50, effective concentration (in μM) required to reduce the viability of uninfected cells by 50%.
Scheme 2. Posited Decomposition Mechanism of Relevant Prodrug Strategies in Vitro
In light of the fact that 1b has the potential to generate thiirane,
we prepared conjugate 2b whose predicated mechanism of
cleavage involves a 5-exo-tet cyclization to yield nonelectrophilic
tetrahydrothiophene and TFV (Scheme 2). To our delight, 2b
exhibited subnanomolar anti-HIV activity comparable to 1b with
reduced toxicity and a TI50 > 100000. Unfortunately, the poor
C
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Figure 2. Decomposition profile of 6 and 7 in various buffer solutions containing 0.1 M DTT at 37.4 °C.
aqueous solubility of 2b prevented accurate CC50 measurements
beyond 50 μM. To determine if 2b passes through intermediate 9
to release TFV, thiol 7 was synthesized and subjected to various
aqueous buffers in a similar manner done for 6. Surprisingly, 7
was stable at all tested pH values with no detectable
decomposition after several hours at 37 °C (Figure 2). Clearly
9 does not cyclize to liberate TFV as shown in Scheme 2.
Furthermore, 7 demonstrates dismal anti-HIV (EC50 = 5.13 μM)
and anti-HBV (EC50 > 100 μM) activity, which suggests poor
enzymatic hydrolysis of the bare thiobutyl spacer. A search of the
literature revealed that Gosselin and collaborators reported
similar results for their S-pivaloyl-4-thiobutyl (SATB) prodrug
whose structure is shown in Scheme 2.24 In that study, the
authors conjugated SATB to azidothymidine (AZT) and
dideoxyadenosine (ddA) and noted a significant reduction in
HIV-1 activity relative to the parent nucleoside. The
compromised activity of SATB conjugates can be traced to
either poor enzymatic cleavage of the thiopivaloyl ester or the
robust nature of the intermediate thiol/ate (7/9). The authors
comment that their bis(SATB) nucleoside conjugates are
suitable substrates for esterases and speculate that the source
of inactivity is due to the persistence of the resultant 4-thiobutyl
linker in vitro. Our results confirm this to be the case. However,
in contrast to SATB prodrug conjugates, 2b demonstrates potent
antiviral activity that appears to rely on the structural integrity of
the entire lipid. This leads us to believe that 2b itself is a substrate
for cellular enzymes, specifically phospholipase C and/or
sphingomyelinase, which have been implicated in the cleavage
of CMX-157 and CMX-001.19,25 The kinetic data and antiviral
activity collected for 7 supports this hypothesis and suggest that
the P−O bond is severed before disulfide reduction. Additional
evidence for enzymatic hydrolysis is provided by the observed
anti-HIV activity for 5b (7.0 nM), which features the disulfide
moiety eight atoms away from the phosphonate headgroup to
abolish the potential for kinetic and thermodynamic cyclization
following reduction. Translocation of the disulfide away from the
phosphonate reduces the antiviral activity of 5b when compared
to 1b (note that 5b and 1b are isomers). This effect may be
countered when translocation is accompanied by a concomitant
increase in lipid chain length, such as is the case for 2b, which
bears a C16 lipid and features the S−S moiety five atoms away
from the phosphonate. Even though 5b is less active than
congeners 1b and 2b, all three compounds are more potent than
CMX-157 against HIV-1 (EC50 = 20 nM),26 thereby
demonstrating an advantage of our disulfide prodrugs over
hexadecyloxypropyl lipids. Although the mechanistic rationale
for this advantage is unclear at this time, it is possible that the
“kinked” S−S bond (i.e., its 90° dihedral angle) increases
membrane fluidity to accelerate translocation of the conjugate
from the outer leaflet of the plasma membrane to the inner leaflet
where it is then enzymatically cleaved to release TFV within the
cytosol.
In light of these findings, the following question arises: how
significant is enzymatic hydrolysis of 1b over the reductive
mechanism shown in Scheme 2? This question remains largely
unanswered. It is likely that 1−5b produce micelles in solution
that hinder GSH and other hydrophilic reductants from
approaching the S−S bond. This would imply that enzymatic
hydrolysis is exclusive mechanism of release, however, future
experiments will characterize the aggregation properties of 1−5b
and tease out these mechanistic details.
The final goal of this study was to probe the rigidity of the
spacer which resulted in the preparation of compounds 3b and
4b. Conjugate 3b demonstrated unremarkable antiviral activity
against HIV and HBV, whereas 4b exhibited potency comparable
to 2b at the expense of increased cytotoxicity. 3b was tailored to
cleave via an o-thioquinone methide elimination following
disulfide reduction, whereas 4b was designed to accelerate the
rate of 5-exo-tet cyclization post reduction. However, in the wake
of our previous findings, these cleavage mechanisms may be
insignificant. Nonetheless, it is clear that other spacers such as (2-
(mercaptomethyl)phenyl)methanol can be used in place of
undecorated aliphatic thioalcohols to furnish potent disulfide
lipid conjugates of TFV.
CONCLUSIONS
■
We have successfully synthesized a second-generation disulfide
lipid conjugate of TFV (2b) that demonstrates potent antiviral
activity against HIV-1 and HBV with reduced cytotoxicity. The
data suggest that 2b does not cleave by chemical means, and the
structural integrity of the disulfide linkage is required to release
TFV in vitro. When taken together, we conclude that enzymatic
hydrolysis is central to the antiviral activity of 2b and speculate
that phospholipase C and/or sphingomyelinase may be
responsible for prodrug cleavage, which have also been
implicated in the decomposition of other lipid nucleoside
conjugates.19,20,27 We further demonstrate that 1b has the
potential to undergo intramolecular prodrug cleavage following
disulfide reduction, however, the significance of this mechanism
remains to be determined in vitro. Future studies will probe the
aggregation of 1−5b and elucidate the enzyme(s) responsible for
D
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NMR (400 MHz, CDCl3) δ 3.90 (q, J = 5.6 Hz, 2H), 2.95−2.80 (m,
2H), 2.77−2.63 (m, 2H), 2.01 (t, J = 6.0 Hz, 1H), 1.69 (dt, J = 14.9, 7.3
Hz, 2H), 1.46−1.17 (m, 26H), 0.89 (t, J = 6.9 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ 60.29, 41.16, 39.05, 31.92, 29.69 (4), 29.66, 29.63,
29.58, 29.49, 29.36, 29.21, 29.13, 28.51, 22.69, 14.13. HRMS (ESI) m/z
calculated for C18H38OS2Na [M + Na]+, 357.22563; found, 357.22533.
Melting point: 50−51 °C.
prodrug cleavage. Additional efforts will focus on the design of
novel disulfide prodrugs bound to antiviral cargo which may
exhibit compounded activity when conjugated to TFV or other
phosphonate nucleoside analogues.
EXPERIMENTAL SECTION
■
2-(Hexadecyldisulfanyl)ethyl Hydrogen ((((R)-1-(6-Amino-9H-
purin-9-yl)propan-2-yl)oxy)methyl)phosphonate (1b). Following
general procedure A, a solution of 2-(hexadecyldisulfanyl)ethanol
(0.070 g, 0.209 mmol) and pyridine (0.084 mL, 1.045 mmol) in
anhydrous DCM was slowly added dropwise to the mixture. After
stirring for 10 min at this temperature, the solution was naturally
warmed to room temperature and stirred for an hour. The reaction was
then quenched with excess water and stirred for 30 min at room
temperature followed by HCl and methanol. Complete hydrolysis of
formimidine was confirmed by LC-MS (isocratic 95% MeOH, 5% H2O,
7 min) after several hours. The mixture was washed with brine and the
crude extracted into DCM. The organic layer was collected,
concentrated, and purified on a silica column using a DCM/
DCM:MeOH:NH4OH (90:10:0.1) gradient (0−65%) to afford the
title compound 2-(hexadecyldisulfanyl)ethyl hydrogen ((((R)-1-(6-
amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphonate (29.8 mg,
0.049 mmol, 28.3% yield) as a white solid. 1H NMR (400 MHz,
CD3OD) δ 8.32 (s, 1H), 8.20 (s, 1H), 4.43−4.33 (m, 1H), 4.23 (dd, J =J
= 14.4, 6.7 Hz, 1H), 4.00 (qd, J = 7.0, 1.7 Hz, 2H), 3.90 (pd, J = 6.3, 3.1
Hz, 1H), 3.73 (dd, J = 12.8, 9.4 Hz, 1H), 3.49 (dd, J = 12.7, 10.1 Hz, 1H),
2.86−2.73 (m, 2H), 2.64 (dd, J = 7.7, 6.8 Hz, 2H), 1.68−1.55 (m, 2H),
1.39−1.25 (m, 26H), 1.17 (d, J = 6.2 Hz, 3H), 0.94−0.85 (m, 3H). 13C
NMR (101 MHz, CD3OD) δ 155.36, 151.48, 149.41, 142.91, 139.91,
118.10, 75.52 (d, J = 13.1 Hz), 64.05 (d, J = 160.2 Hz), 62.96 (d, J = 5.7
Hz), 38.87 (d, J = 6.2 Hz), 38.46, 31.65, 29.38, 29.35, 29.34, 29.28, 29.23,
29.06, 28.93, 28.71, 28.06, 22.32, 15.41, 13.04. 31P NMR (162 MHz,
CD3OD) δ 16.87. HRMS (ESI) m/z calculated for C27H51O4N5PS2 [M
+ H]+, 604.31146; found, 604.31149. Anal. Calculated for
C27H53O5N6PS2 (as an ammonium salt monohydrate): C, 50.76; H,
8.68; N, 13.15. Found: C, 51.45; H, 8.42; N, 12.79. Melting point: 138−
142 °C
4-(Hexadecyldisulfanyl)butan-1-ol (2a). To a stirring solution of
hexadecane-1-thiol (5.94 mL, 19.30 mmol) and 4-mercaptobutan-1-ol
(1.990 mL, 19.30 mmol) in MeOH/DCM (1:2, 150 mL) was added
pyridine (3.36 mL, 38.6 mmol) followed by the gradual addition of
diiodine (4.90 g, 19.30 mmol) at room temperature. The solution stirred
for 3 h at room temperature, and reaction progress was monitored by
TLC (hexanes/EtOAc 4:1, PMA stain). Then the solvents were
evaporated under reduced pressure to afford a white residue, which was
redissolved in DCM and partitioned with water. The organic layer was
collected, dried over anhydrous sodium sulfate, filtered, and purified on a
silica column using a hexanes/EtOAc gradient (0−16% EtOAc) to
afford the title compound 4-(hexadecyldisulfanyl)butan-1-ol (2.7 g, 7.44
mmol, 38.6% yield) as a pearlescent white powder. 1H NMR (400 MHz,
CDCl3) δ 3.68 (t, J = 6.3 Hz, 2H), 2.78−2.65 (m, 4H), 1.85−1.73 (m,
2H), 1.73−1.62 (m, 4H), 1.43−1.22 (m, 26H), 0.88 (t, J = 6.9 Hz, 3H).
13C NMR (101 MHz, CDCl3) δ 62.42, 39.10, 38.65, 31.90, 31.42, 29.67
Chemical Synthesis. All reagents were obtained from commercial
suppliers and used without further purification. Reaction progress was
monitored by either thin layer chromatography (TLC) using precoated
aluminum-backed silica gel plates (60 F254 Merck, article 5554) or liquid
chromatography−mass spectrometry (LC-MS) on an Agilent Tech-
nologies 6100 quadrupole instrument equipped with UV detection at
254 and 210 nm and a Varian C8 analytical column. Hanes reagent and
UV detection at 254 nm were the preferred visualization agents for TLC.
LC-MS analysis was performed using a stepwise H2O/MeOH gradient
with the % MeOH increasing from 75% to 95% over the course of 3 min
unless otherwise specified. Flash column chromatography was
conducted using CombiFlash Rf 200 (Telendyne-Isco) automated
flash chromatography system with hand-packed RediSep columns.
Evaporation of solvents was carried out on a rotary evaporator under
reduced pressure and under ultrahigh vacuum (UHV) where
appropriate. 1H NMR and 13C NMR spectra were recorded at ambient
temperature on a Varian 400 spectrometer. 31P spectra were recorded at
ambient temperature on either a Mercury 300 or Varian 400
spectrometer. Unless otherwise specified, all NMR spectra were
obtained in deuterated chloroform (CDCl3) and referenced to the
residual solvent peak. Chemical shifts are given in δ values and coupling
constants are reported in hertz (Hz). Coupling constants are not
reported in the 13C spectrum of diastereomeric mixtures. Melting points
were determined on a MelTemp melting apparatus and are uncorrected.
High resolution mass-spectra (HRMS) were acquired on a VG 70-S
Nier Johnson or JEOL mass spectrometer. Elemental analyses were
performed by Atlantic Microlabs (Norcross, GA) for C, H, N analysis
and are in agreement with the proposed structures with purity ≥95%.
General Procedure A. To a stirring solution of dry tenofovir (0.05 g,
0.174 mmol) in anhydrous DCM (6 mL) and N,N-dimethylformamide
(0.016 mL, 0.209 mmol) was gradually added excess oxalyl chloride
(0.075 mL, 0.870 mmol). The mixture stirred exposed to atmosphere for
1 h at room temperature until a colorless, transparent solution was
observed and no starting material coated the walls of the vessel. The
solvent and excess oxalyl chloride was evaporated under reduced
pressure to produce a pale-yellow foam, which was redissolved in
anhydrous DCM (5 mL) and placed under argon. The vessel was
equipped with a magnetic stir bar and chilled to 0 °C. Then, a solution of
2-(hexadecyldisulfanyl)ethanol (0.070 g, 0.209 mmol) and pyridine
(0.084 mL, 1.045 mmol) in anhydrous DCM was slowly added
dropwise. After stirring for 10 min at this temperature, the mixture was
naturally warmed to room temperature and stirred for an hour.
General Procedure B. To a stirring solution of dry tenofovir (0.1 g,
0.348 mmol) in anhydrous DCM (6 mL) and N,N-dimethylformamide
(0.032 mL, 0.418 mmol) was gradually added excess oxalyl chloride
(0.149 mL, 1.741 mmol). The mixture stirred at rt exposed to air for 15
min or until complete dissolution of starting material was observed. The
solvent and excess oxalyl chloride was evaporated under reduced
pressure, and the resulting residue was placed under argon and
redissolved in anhydrous DCM (5 mL) to afford a clear colorless
solution. The mixture was then chilled to 0 °C.
2-(Hexadecyldisulfanyl)ethanol (1a). To a stirring solution of
hexadecane-1-thiol (8.74 mL, 28.4 mmol) and 2-mercaptoethanol (2
mL, 28.4 mmol) in MeOH/DCM (50:50, 200 mL) was added pyridine
(4.94 mL, 56.8 mmol) followed by the gradual added diiodine (7.21 g,
28.4 mmol) until the color of the solution remained brown. The solution
was stirred for 2 h at room temperature and then the resulting
suspension was filtered and the supernatant collected. The solvents were
evaporated under reduced pressure, and the resulting solid was washed
with water and extracted into DCM. The organic layer was concentrated
and the residue purified on a silica column using hexanes/EtOAc (0−
8%) gradient to afford the title compound 2-(hexadecyldisulfanyl)-
ethanol (3.98 g, 11.89 mmol, 41.9% yield) as a fluffy white powder. 1H
(2), 29.66 (2), 29.64 (2), 29.58, 29.50, 29.35, 29.23, 29.20, 28.51, 25.42,
22.68, 14.12. HRMS (ESI) m/z calculated for C20H42OS2 [M + Na]+,
385.25693; found, 385.25621. Melting point: 51−52 °C.
(R)-4-(Hexadecyldisulfanyl)butyl (((1-(6-Amino-9H-purin-9-yl)-
propan-2-yl)oxy)methyl)phosphonate (2b). Following general proce-
dure B, a mixture of 4-(hexadecyldisulfanyl)butan-1-ol (0.152 g, 0.418
mmol) and pyridine (0.168 mL, 2.089 mmol) in anhydrous DCM was
slowly added dropwise to the solution. The mixture stirred at 0 °C for 15
min and then naturally warmed to room temperature and stirred for 1 h.
Water (0.094 mL, 5.22 mmol) was added and the mixture continued
stirring for an additional 30 min. The solvent was evaporated under
reduced pressure, and the resulting residue was dried under UHV and
redissolved in EtOH (5 mL) and stirred at 40 °C overnight. Reaction
progress could be monitored by TLC (80:20:1 DCM/MeOH/
NH4OH). The solvent was evaporated under reduced pressure, and
the crude was dissolved in minimal DCM and purified on a silica column
E
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using a DCM/DCM:MeOH:NH4OH (80:20:1) gradient (0−45%) to
afford the title compound (R)-4-(hexadecyldisulfanyl)butyl (((1-(6-
amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphonate (140 mg,
0.222 mmol, 63.7% yield) as a waxy solid. 1H and 13C spectra referenced
Hz, 3H), 0.86 (t, J = 6.9 Hz, 3H). 13C NMR (101 MHz, CD3OD/
CDCl3, referenced to CD3OD) δ 156.16, 152.29, 150.44, 143.92, 138.79
(d, J = 6.9 Hz), 135.94, 129.88, 128.86, 128.77, 128.13, 119.21, 76.79 (d,
J = 13.2 Hz), 65.46 (d, J = 159.8 Hz), 65.23 (d, J = 5.0 Hz), 48.88, 39.57,
32.85, 30.60, 30.59, 30.59, 30.56, 30.54, 30.48, 30.39, 30.28, 30.08, 29.57,
29.23, 23.55, 16.77, 14.53. 31P NMR (121 MHz, CDCl3/CD3OD) δ
16.49. HRMS (ESI) m/z calculated for C32H51O4N5PS2 [M − H]−,
664.31146; found, 664.31306. Anal. Calculated for C32H57N6O5PS2
(NH4+ monohydrate): C,54.83; H, 8.20; N, 11.99. Found: C, 54.23; H,
8.16; N, 12.38. Melting point: 177−180 °C.
Methyl 2-((Acetylthio)methyl)benzoate. To a stirring solution of
methyl 2-(bromomethyl)benzoate (7 g, 30.6 mmol) in THF (100 mL)
and DMF (5 mL) at 0 °C was added potassium ethanethioate (3.84 g,
33.6 mmol), followed by catalytic tetrabutylammonium iodide (2.257 g,
6.11 mmol). The mixture stirred at this temperature for 15 min and then
naturally warmed to room temperature and stirred for 12 h. Progress was
monitored by TLC (hexanes/EtOAc, 4:1). Upon completion, the
organic solvent was evaporated under reduced pressure and the resulting
oil was partitioned between EtOAc and brine (3×). The organic layer
was collected, dried over anhydrous sodium sulfate, and purified on a
silica column using a hexanes/EtOAc gradient (0−6% EtOAc) to afford
the title compound methyl 2-((acetylthio)methyl)benzoate (6.53 g,
29.1 mmol, 95% yield) as a foul-smelling yellow oil. 1H NMR (400 MHz,
CDCl3) δ 7.95 (d, J = 7.8 Hz, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.47−7.39
(m, 1H), 7.35−7.26 (m, 1H), 4.47 (s, 2H), 3.90 (s, 3H), 2.29 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 195.64, 167.30, 140.04, 132.54, 131.64,
131.00, 128.48, 127.43, 52.11, 32.24, 30.15. HRMS (ESI) m/z calculated
for C11H13O3S [M + H]+, 225.05799; found, 225.05809.
1
to CD3OD (3.31 δ and 49.15 δ, respectively). H NMR (400 MHz,
CDCl3/CD3OD) δ 8.30 (s, 1H), 8.20 (s, 1H), 4.37 (dd, J = 14.4, 3.1 Hz,
1H), 4.22 (dd, J = 14.4, 6.8 Hz, 1H), 3.94−3.82 (m, 1H), 3.82−3.65 (m,
3H), 3.45 (dd, J = 12.7, 10.2 Hz, 1H), 2.68−2.58 (m, 4H), 1.74−1.54
(m, 6H), 1.42−1.20 (m, 25H), 1.18 (d, J = 6.2 Hz, 3H), 0.88 (t, J = 6.9
Hz, 3H). 13C NMR (101 MHz, CDCl3/CD3OD) δ 156.92, 153.23,
150.78, 144.12, 119.53, 76.96 (d, J = 13.2 Hz), 65.40 (d, J = 160.3 Hz),
65.37 (d, J = 5.9 Hz), 66.20, 65.40, 65.34, 64.61, 39.71, 39.29, 33.08,
30.81, 30.80, 30.79, 30.78, 30.74, 30.69 (d, J = 5.9 Hz), 30.49, 30.37,
30.22, 29.53, 26.57, 23.76, 16.95, 14.64. 31P NMR (162 MHz, CDCl3/
CD3OD) δ 16.54. HRMS (ESI) m/z calculated for C29H55N5O4PS2 [M
+ H]+, 632.34276; found, 632.34515. Anal. Calculated for
C29H58N6O5PS2 (NH4+ monohydrate): C, 52.31; H, 8.78; N, 12.62.
Found: C, 52.49; H, 8.78; N, 12.64. Melting point: 140−150 °C. Solid is
amorphous.
(2-(Hexadecyldisulfanyl)phenyl)methanol (3a). To a stirring
solution of hexadecane-1-thiol (5.43 mL, 17.65 mmol) and (2-
mercaptophenyl)methanol (2.474 g, 17.65 mmol) in MeOH/DCM
(1:2, 150 mL) was added pyridine (3.07 mL, 35.3 mmol), followed by
the gradual addition of diiodine (4.48 g, 17.65 mmol) at room
temperature. The solution stirred for 4 h at room temperature, and
reaction progress was monitored by TLC (hexanes/EtOAc 4:1, UV).
Then, the solvents were evaporated under reduced pressure to afford a
white residue which was redissolved in DCM and partitioned with water.
This afforded a third layer between the organic and aqueous interface
and was identified as the homodimer of (2-mercaptophenyl)methanol
by LC-MS. The organic layer was collected and dried over sodium
sulfate. The mixture was filtered and the solvents evaporated under
reduced pressure. The pale-yellow residue was redissolved in minimal
DCM. A substantial quantity of the solid resisted dissolution and was
subsequently filtered over a fine glass frit. The supernatant was collected,
concentrated, and purified on a silica column via flash chromatography
using a hexanes/EtOAc gradient (0−6% EtOAc) to afford the title
compound (2-(hexadecyldisulfanyl)phenyl)methanol (3.45 g, 8.70
mmol, 49.3% yield) as an off-white solid. 1H NMR (400 MHz,
CDCl3) δ 7.78−7.72 (m, 1H), 7.46−7.40 (m, 1H), 7.33−7.25 (m, 2H),
4.84 (s, 2H), 2.75−2.69 (m, 2H), 1.65 (dt, J = 14.9, 7.3 Hz, 2H), 1.39−
1.17 (m, 26H), 0.88 (t, J = 6.9 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ
140.23, 135.65, 129.78, 128.51, 128.24, 127.67, 63.26, 38.73, 31.92,
29.69 (3), 29.67 (2), 29.63, 29.57, 29.46, 29.37, 29.16, 28.68, 28.44,
22.69, 14.14. HRMS (ESI) m/z calculated for C23H40S2O [M + Na]+,
419.24128; found, 419.24099. Melting point: 40−42 °C.
(R)-2-(Hexadecyldisulfanyl)benzyl (((1-(6-Amino-9H-purin-9-yl)-
propan-2-yl)oxy)methyl)phosphonate (3b). Following general proce-
dure B, a mixture of (2-(hexadecyldisulfanyl)phenyl)methanol (0.166 g,
0.418 mmol) (KEG-4-164) and pyridine (0.168 mL, 2.089 mmol) in
anhydrous DCM was slowly added dropwise to the solution. The
mixture stirred at this temperature for 15 min and then naturally warmed
to room temperature and stirred for 3 h. Then water (0.094 mL, 5.22
mmol) was added and the mixture continued stirring for an additional
30 min. The solvent was evaporated under reduced pressure, and the
resulting residue was dried under UHV. Then, the residue was
redissolved in EtOH (5 mL) and stirred at 40 °C overnight. The
product precipitated from the reaction mixture and was filtered with
additional EtOH and dried under UHV. The solid was dissolved in
chloroform and further purified on a silica column using a DCM/
DCM:MeOH:NH4OH (80:20:1) gradient (0−66%) to afford the title
compound (R)-2-(hexadecyldisulfanyl)benzyl (((1-(6-amino-9H-
purin-9-yl)propan-2-yl)oxy)methyl)phosphonate (94.5 mg, 0.142
mmol, 40.8% yield) as an off-white solid. 1H NMR (400 MHz,
CD3OD/CDCl3, referenced to CD3OD) δ 8.26 (s, 1H), 8.18 (s, 1H),
7.67 (dd, J = 7.7, 1.3 Hz, 1H), 7.46 (dd, J = 7.5, 1.3 Hz, 1H), 7.22 (dtd, J
= 21.2, 7.4, 1.5 Hz, 2H), 5.13−5.03 (m, 2H), 4.32 (dd, J = 14.4, 3.1 Hz,
1H), 4.17 (dd, J = 14.4, 6.5 Hz, 1H), 3.88−3.81 (m, 1H), 3.77 (dd, J =
12.7, 9.4 Hz, 1H), 3.53 (dd, J = 12.7, 10.0 Hz, 1H), 2.65 (t, J = 7.2 Hz,
2H), 1.57 (dt, J = 14.8, 7.2 Hz, 2H), 1.34−1.14 (m, 26H), 1.11 (d, J = 6.3
(2-(Mercaptomethyl)phenyl)methanol. To a stirring solution of
lithium aluminum hydride (82 mL, 163 mmol) in THF under inert
atmosphere at 0 °C was added methyl 2-((acetylthio)methyl)benzoate
(9.15 g, 40.8 mmol), dropwise. The solution stirred at this temperature
for 15 min and then naturally warmed to room temperature. Progress
was monitored by LC-MS (H2O/MeOH gradient, 75−95% MeOH, 3
min). The reaction reached completion after stirring for 1 h and was
subsequently chilled to 0 °C. Then acetone (8.99 mL, 122 mmol) was
added slowly dropwise, followed by the slow addition of 15% aqueous
NaOH (37 mL) with vigorous stirring under inert atmosphere. The
reaction mixture was then diluted with a saturated solution of sodium
potassium tartrate and vigorously stirred for 2 h. After stirring, the pH
was adjusted to 9 with solid ammonium chloride and the reaction
mixture was allowed to settle. The supernatant was collected and
concentrated under reduced pressure, and the resulting aqueous mixture
was partitioned with DCM. The solids were also partitioned with DCM
and brine. The organic layers were collected, dried over anhydrous
sodium sulfate, and purified via flash chromatography on a silica column
using a hexanes/EtOAc gradient (0−41% EtOAc) to afford the title
compound (2-(mercaptomethyl)phenyl)methanol (3.76 g, 24.38 mmol,
59.8% yield) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.41−7.33
(m, 1H), 7.30−7.23 (m, 3H), 4.76 (s, 2H), 3.83 (d, J = 7.2 Hz, 2H), 1.87
(t, J = 7.2 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 139.37, 138.15,
129.37, 129.33, 128.51, 127.75, 63.12, 26.10. HRMS (ESI) m/z
calculated for C8H10SOCl [M + Cl]−, 189.01464; found, 189.01465.
(2-((Hexadecyldisulfanyl)methyl)phenyl)methanol (4a). To a
stirring solution of hexadecane-1-thiol (2.99 mL, 9.73 mmol) and (2-
(mercaptomethyl)phenyl)methanol (1.5 g, 9.73 mmol) in MeOH/
DCM (1:3, 150 mL) was added pyridine (1.691 mL, 19.45 mmol),
followed by the gradual addition of diiodine (2.468 g, 9.73 mmol) at
room temperature. The solution stirred for 3 h at room temperature and
reaction progress was monitored by TLC (DCM:MeOH:NH4OH,
95:5:0.1, PMA stain). The reaction mixture was diluted with methanol
(25 mL), and the resulting precipitate was filtered and discarded. The
supernatant was collected and the solvent evaporated under reduced
pressure to afford a pale-orange residue that was redissolved in DCM
and partitioned with water. The organic layer was collected, dried over
anhydrous sodium sulfate, filtered, and purified on a silica column using
a hexanes/EtOAc gradient (0−7% EtOAc) to afford the title compound
(2-((hexadecyldisulfanyl)methyl)phenyl)methanol (1.79 g, 4.36 mmol,
1
44.8% yield) as a light-orange solid. H NMR (400 MHz, CDCl3) δ
7.44−7.37 (m, 1H), 7.34−7.21 (m, 3H), 4.79 (d, J = 5.6 Hz, 2H), 4.01
F
DOI: 10.1021/acs.jmedchem.6b01292
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(s, 2H), 2.42−2.33 (m, 2H), 2.08 (t, J = 5.8 Hz, 1H), 1.53 (dt, J = 14.8,
7.3 Hz, 2H), 1.43−1.02 (m, 26H), 0.88 (t, J = 6.9 Hz, 3H). 13C NMR
(101 MHz, CDCl3) δ 139.03, 135.22, 131.02, 129.11, 128.06, 127.97,
63.03, 40.45, 38.69, 31.92, 29.69 (2), 29.68 (2), 29.66, 29.65, 29.59,
29.48, 29.36, 29.16, 28.98, 28.46, 22.70, 14.14. HRMS (ESI) m/z
calculated for C24H42OS2 [M + Na]+, 433.25693; found, 433.25619.
Melting point: 38−40 °C.
directly on silica gel using a DCM/DCM:MeOH:NH4OH (80:20:3)
gradient (0−50%) to afford the title compound 6-(dodecyldisulfanyl)-
hexyl hydrogen ((((R)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)-
methyl)phosphonate (40 mg, 0.066 mmol, 19.03% yield) as a waxy
solid. 1H NMR (400 MHz, CD3OD/CDCl3 referenced to TMS) δ 8.31
(s, 1H), 8.24 (s, 1H), 4.39 (dd, J = 14.4, 3.0 Hz, 1H), 4.20 (dd, J = 14.4,
6.9 Hz, 1H), 3.85 (ddd, J = 9.9, 6.4, 3.1 Hz, 1H), 3.83−3.71 (m, 3H),
3.45 (dd, J = 12.6, 10.2 Hz, 1H), 2.67 (dt, J = 10.8, 3.8 Hz, 4H), 1.72−
1.61 (m, 4H), 1.56 (dt, J = 13.5, 6.8 Hz, 2H), 1.44−1.23 (m, 22H), 1.20
(d, J = 6.2 Hz, 3H), 0.89 (t, J = 6.9 Hz, 3H). 13C NMR (101 MHz,
CDCl3/CD3OD referenced to CD3OD) δ 155.82, 152.46, 150.01,
143.31, 118.77, 76.26 (d, J = 13.2 Hz), 65.23 (d, J = 6.0 Hz), 64.63 (d, J =
159.7 Hz), 48.47, 39.34, 39.17, 34.90, 32.33, 31.27 (d, J = 6.3 Hz), 30.05,
30.03, 29.99, 29.92, 29.75, 29.65, 29.58, 29.47, 28.89, 28.58, 25.80, 23.07,
16.61, 14.30. 31P NMR (162 MHz, CDCl3/CD3OD) δ 16.36. HRMS
(ESI) m/z calculated for C27H49O4N5PS2 [M − H]−, 602.29691; found,
602.29703. Elemental Analysis Calculated for C27H53N6O4PS2 (as an
ammonium salt): C, 52.23; H, 8.60; N, 13.54. Found: C, 52.21; H, 8.65;
N, 12.42.
2-Mercaptoethyl Hydrogen ((((R)-1-(6-Amino-9H-purin-9-yl)-
propan-2yl)oxy)methyl)phosphonate (6). To a stirring solution of
dry tenofovir (1 g, 3.48 mmol) in anhydrous DCM (100 mL) and DMF
(0.537 mL, 6.96 mmol) was gradually added excess oxalyl chloride (1.19
mL, 13.93 mmol) at room temperature. After stirring for 1 h, the mixture
was then cooled to 0 °C and quenched with excess 2-mercaptoethanol
(2.451 mL, 34.8 mmol). Progress was monitored by LC-MS (H2O/
MeOH gradient, 50−95% MeOH, 3 min). The mixture stirred for an
additional hour at this temperature. Aqueous HCl was then added (3
mL), followed by 15 mL of methanol (pH = 1). The mixture stirred at
room temperature overnight, and then the pH was gradually raised to 5
using saturated aqueous sodium bicarbonate. The organic solvents were
evaporated under reduced pressure at 30 °C, and the resulting residue
was purified on a C18 reverse phase column using a H2O/MeOH
gradient (isocratic 10% MeOH) to afford the title compound 2-
mercaptoethyl hydrogen ((((R)-1-(6-amino-9H-purin-9-yl)propan-2-
yl)oxy)methyl)phosphonate (0.6043 g, 1.740 mmol, 50.0% yield) as a
white solid. 1H NMR (400 MHz, D2O) δ 8.38 (s, 1H), 8.37 (s, 1H), 4.44
(dd, J = 14.7, 3.1 Hz, 1H), 4.26 (dd, J = 14.7, 7.7 Hz, 1H), 4.04−3.92 (m,
1H), 3.77−3.66 (m, 3H), 3.48 (dd, J = 13.4, 9.6 Hz, 1H), 2.53 (td, J =
6.4, 4.2 Hz, 2H), 1.19 (d, J = 6.3 Hz, 3H). 13C NMR (101 MHz, D2O) δ
149.73, 148.53, 145.45, 144.35, 117.52, 75.55 (d, J = 12.5 Hz), 66.10 (d, J
= 5.6 Hz), 62.92 (d, J = 159.5 Hz), 48.37, 24.40 (d, J = 6.5 Hz), 15.58. 31P
NMR (162 MHz, D2O) δ 20.77. HRMS (ESI) m/z calculated for
C11H19O4N5PS [M + H]+, 348.08899; found, 348.08929. Anal.
Calculated for C11H20N5O5PS (as a monohydrate): C, 36.16; H, 5.52;
N, 19.17. Found: C, 35.73; H, 5.53; N, 19.07. Melting point:
decomposes at 100 °C.
4-Mercaptobutyl Hydrogen ((((R)-1-(6-Amino-9H-purin-9-yl)-
propan-2-yl)oxy)methyl)phosphonate (7). To a stirring solution of
tenofovir (1 g, 3.48 mmol) in anhydrous DCM (34.8 mL) and N,N-
dimethylformamide (0.322 mL, 4.18 mmol) was added excess oxalyl
chloride (1.493 mL, 17.41 mmol) at room temperature. The mixture
stirred for 15 min with progress monitored with LC-MS (H2O/MeOH
gradient, 35−75% MeOH, 3 min) by quenching an aliquot of the
reaction mixture with MeOH. Upon completion, the solvents and excess
oxalyl chloride were evaporated under reduced pressure and the
resulting yellow foam was further dried under UHV. The foam was
redissolved in anhydrous DCM (34 mL), and the mixture was chilled to
0 °C. Then a solution of 4-mercaptobutan-1-ol (0.395 mL, 3.83 mmol)
and pyridine (1.683 mL, 20.89 mmol) in DCM (2 mL) was added
dropwise and the reaction stirred at this temperature for 15 min and then
naturally warmed to room temperature with progress monitored by LC-
MS. After stirring for 2 h, the mixture was quenched with water (0.25
mL) and stirring continued at room temperature for 30 min. The
solution was then acidified with HCl (1.2 M) and homogenized with
MeOH until the aqueous/organic interface disappeared and stirred
overnight to facilitate the cleavage of formimidine. The solvents were
then evaporated under reduced pressure, and the resulting yellow oil was
purified on a C18 reverse phase column using a H2O/MeOH gradient
with 0.1% formic acid (gradient 0−25% MeOH) to afford the title
(R)-2-((Hexadecyldisulfanyl)methyl)benzyl (((1-(6-Amino-9H-
purin-9-yl)propan-2-yl)oxy)methyl)phosphonate (4b). Following
general procedure B, a mixture of (2-((hexadecyldisulfanyl)methyl)-
phenyl)methanol (0.172 g, 0.418 mmol) and pyridine (0.168 mL, 2.089
mmol) in anhydrous DCM (2 mL) was slowly added dropwise to the
reacting solution. The mixture stirred at this temperature for 15 min and
then naturally warmed to room temperature and stirred for 3 h. Water
(0.094 mL, 5.22 mmol) was added, and the mixture continued stirring
for an additional 30 min. The solvent was evaporated under reduced
pressure, and the resulting residue was dried under UHV. The residue
was redissolved in 190 proof EtOH (5 mL) and stirred at 40 °C
overnight. The mixture was diluted with water (5 mL), and the
precipitate was filtered over a glass frit. The resulting solid was dried
under UHV and purified via flash chromatography on a silica column
using a DCM/DCM/MeOH/NH4OH (80:20:1) gradient (0−62%) to
afford the title compound (R)-2-((hexadecyldisulfanyl)methyl)benzyl
(((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphonate
(114 mg, 0.168 mmol, 48.2% yield) as a white solid. Proton spectrum
referenced to CD3OD (3.31 ppm). 1H NMR (400 MHz, CDCl3/
CD3OD) δ 8.23 (s, 1H), 8.19−8.15 (m, 1H), 7.40−7.33 (m, 1H), 7.26−
7.15 (m, 3H), 5.07 (d, J = 6.9 Hz, 2H), 4.29 (dd, J = 14.4, 3.0 Hz, 1H),
4.11 (dd, J = 14.4, 6.7 Hz, 1H), 3.92 (s, 2H), 3.74 (ddd, J = 17.0, 10.3, 6.2
Hz, 2H), 3.42 (dd, J = 12.7, 10.3 Hz, 1H), 2.31−2.22 (m, 2H), 1.44 (dd,
J = 14.2, 7.1 Hz, 2H), 1.31−1.13 (m, 26H), 1.09 (d, J = 6.3 Hz, 3H), 0.84
(t, J = 6.9 Hz, 3H). Carbon referenced to CD3OD (49.00 ppm). 13C
NMR (101 MHz, CD3OD/CDCl3) δ 155.54, 151.93, 149.95, 143.42,
136.93 (d, J = 6.5 Hz), 135.78, 131.22, 129.17, 128.22, 128.07, 118.74,
76.34 (d, J = 13.3 Hz), 64.96 (d, J = 5.3 Hz), 64.90 (d, J = 159.4 Hz),
48.44, 40.54, 39.02, 32.37, 30.13 (3), 30.09 (3), 30.03, 29.94, 29.80,
29.61, 29.36, 28.88, 23.11, 16.62, 14.36. 31P NMR (162 MHz, CDCl3/
CD3OD) δ 16.30. HRMS (ESI) m/z calculated for C33H53O4N5PS2 [M
− H]−, 678.32821; found, 678.32746. Anal. Calculated for
C33H59N6O5PS2 (NH4+ monohydrate): C, 55.44; H, 8.32; N, 11.75.
Found: C, 56.09; H, 8.10; N, 11.42. Melting point: decomposes at 150
°C.
6-(Dodecyldisulfanyl)hexan-1-ol (5a). To a stirring solution of
dodecane-1-thiol (3.50 mL, 14.62 mmol) and 6-mercaptohexan-1-ol (2
mL, 14.62 mmol) in MeOH/DCM (25:75, 70 mL) was added pyridine
(2.54 mL, 29.2 mmol) followed by the gradual addition of iodine (4.08 g,
16.08 mmol). The solution stirred for 5 h at room temperature under
nitrogen. Then the solvents were evaporated under reduced pressure
and the residue was partitioned between brine and DCM. The organic
layer was concentrated and purified on a silica column using a hexanes/
EtOAc (0−10% EtOAc) gradient to afford the title compound 6-
(dodecyldisulfanyl)hexan-1-ol (2.27 g, 6.78 mmol, 46.4% yield) as a
1
white solid. H NMR (400 MHz, CDCl3) δ 3.65 (t, J = 6.6 Hz, 2H),
2.71−2.66 (m, 4H), 1.75−1.63 (m, 4H), 1.63−1.54 (m, 2H), 1.48−1.35
(m, 6H), 1.34−1.21 (m, 16H), 0.88 (t, J = 6.9 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ 62.87, 39.14, 38.95, 32.58, 31.90, 29.64, 29.61, 29.58,
29.50, 29.33, 29.23, 29.20, 29.10, 28.51, 28.24, 25.36, 22.68, 14.12.
HRMS (ESI) m/z calculated for C18H38OS2 [M + H]+, 335.24368;
found, 335.24391. Melting point: 38−39 °C.
6-(Dodecyldisulfanyl)hexyl Hydrogen ((((R)-1-(6-Amino-9H-purin-
9-yl)propan-2-yl)oxy)methyl)phosphonate (5b). Following general
procedure B, a solution of 6-(dodecyldisulfanyl)hexan-1-ol (0.117 g,
0.348 mmol) and pyridine (0.337 mL, 4.18 mmol) in DCM (2 mL) was
slowly added dropwise to the solution stirring at 0 °C. The mixture
stirred at this temperature for 15 min and then naturally warmed to
room temperature and stirred for 3 h. Then water (0.094 mL, 5.22
mmol) was added and the mixture was continued stirring for an
additional 30 min and a 30% solution of NH4OH was added. Stirring
continued for 1 h and then the solvents were evaporated under reduced
pressure and the resulting residue was dried under UHV and purified
G
DOI: 10.1021/acs.jmedchem.6b01292
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
compound 4-mercaptobutyl hydrogen ((((R)-1-(6-amino-9H-purin-9-
yl)propan-2-yl)oxy)methyl)phosphonate (0.7075 g, 1.885 mmol, 54.1%
yield) as a white foam. 1H NMR (400 MHz, CD3OD) δ 8.40 (s, 1H),
8.27 (s, 1H), 4.46 (dd, J = 14.4, 3.0 Hz, 1H), 4.27 (dd, J = 14.5, 7.1 Hz,
1H), 4.08−3.98 (m, 1H), 3.83 (ddd, J = 22.4, 12.6, 7.7 Hz, 3H), 3.63
(dd, J = 13.0, 9.5 Hz, 1H), 2.99 (t, J = 6.9 Hz, 1H), 2.49 (t, J = 6.7 Hz,
2H), 1.72−1.58 (m, 4H), 1.19 (d, J = 6.3 Hz, 3H). 13C NMR (101 MHz,
CD3OD) δ 152.23, 150.40, 146.30, 146.15, 119.17, 76.58 (d, J = 12.2
Hz), 65.56 (d, J = 6.0 Hz), 64.72 (d, J = 162.9 Hz), 31.47, 30.63 (d, J =
6.1 Hz), 27.40, 24.67, 16.96. 31P NMR (162 MHz, CD3OD) δ 17.70.
HRMS (ESI) m/z calculated for C13H23N5O4PS [M + H]+, 376.12029;
found, 376.11987. Anal. Calculated for C13H24O5N5PS (as a
monohydrate): C, 39.69; H, 6.15; N, 17.80. Found: C, 39.88; H, 5.73;
N, 17.76. Melting Point: 151−152 °C.
(8) Manach, C.; Williamson, G.; Morand, C.; Scalbert, A.; Remesy, C.
Bioavailability and Bioefficacy of Polyphenols in Humans. I. Review of
97 Bioavailability Studies. Am. J. Clin. Nutr. 2005, 81, 230S−242S.
(9) Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.;
Jarvinen, T.; Savolainen, J. Prodrugs: Design and Clinical Applications.
Nat. Rev. Drug Discovery 2008, 7, 255−270.
(10) Kang, M. J.; Cho, J. Y.; Shim, B. H.; Kim, D. K.; Lee, J.
Bioavailability Enhancing Activities of Natural Compounds from
Medicinal Plants. J. Med. Plant Res. 2009, 3, 1204−1211.
(11) Kuo, A.; Dienstag, J. L.; Chung, R. T. Tenofovir Disoproxil
Fumarate for the Treatment of Lamivudine-Resistant Hepatitis B. Clin.
Gastroenterol. Hepatol. 2004, 2, 266−272.
(12) Heathcote, E. J.; Marcellin, P.; Buti, M.; Gane, E.; De Man, R. A.;
Krastev, Z.; Germanidis, G.; Lee, S. S.; Flisiak, R.; Kaita, K.; Manns, M.;
Kotzev, I.; Tchernev, K.; Buggisch, P.; Weilert, F.; Kurdas, O. O.;
Shiffman, M. L.; Trinh, H.; Gurel, S.; Snow-Lampart, A.; Borroto-Esoda,
K.; Mondou, E.; Anderson, J.; Sorbel, J.; Rousseau, F. Three-Year
Efficacy and Safety of Tenofovir Disoproxil Fumarate Treatment for
Chronic Hepatitis B. Gastroenterology 2011, 140, 132−143.
(13) Fung, H. B.; Stone, E. A.; Piacenti, F. J. Tenofovir Disoproxil
Fumarate: A Nucleotide Reverse Transcriptase Inhibitor for the
Treatment of Hiv Infection. Clin. Ther. 2002, 24, 1515−1548.
(14) Kearney, B. P.; Flaherty, J. F.; Shah, J. Tenofovir Disoproxil
Fumarate - Clinical Pharmacology and Pharmacokinetics. Clin.
Pharmacokinet. 2004, 43, 595−612.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b01292.
Assay details, kinetic studies, and associated spectra for
synthesized compounds (PDF)
Molecular formula strings (CSV)
(15) Lewis, W.; Day, B. J.; Copeland, W. C. Mitochondrial Toxicity of
Nrti Antiviral Drugs: An Integrated Cellular Perspective. Nat. Rev. Drug
Discovery 2003, 2, 812−822.
AUTHOR INFORMATION
Corresponding Authors
*For D.C.L.: phone, 404-727-8130; fax, 404-712-8679; E-mail,
dliotta@emory.edu.
*For K.E.G.: E-mail, kgiesle@emory.edu.
Notes
The authors declare no competing financial interest.
■
(16) Herlitz, L. C.; Mohan, S.; Stokes, M. B.; Radhakrishnan, J.;
D’Agati, V. D.; Markowitz, G. S. Tenofovir Nephrotoxicity: Acute
Tubular Necrosis with Distinctive Clinical, Pathological, and
Mitochondrial Abnormalities. Kidney Int. 2010, 78, 1171−1177.
(17) Ruane, P. J.; DeJesus, E.; Berger, D.; Markowitz, M.; Bredeek, U.
F.; Callebaut, C.; Zhong, L. J.; Ramanathan, S.; Rhee, M. S.; Fordyce, M.
W.; Yale, K. Antiviral Activity, Safety, and Pharmacokinetics/
Pharmacodynamics of Tenofovir Alafenamide as 10-Day Monotherapy
in Hiv-1-Positive Adults. JAIDS, J. Acquired Immune Defic. Syndr. 2013,
63, 449−455.
ACKNOWLEDGMENTS
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The authors would like to acknowledge Manohar Saindane for
transporting numerous samples from Emory to the EIDD for
analysis. Work presented herein was funded internally.
(18) Markowitz, M.; Zolopa, A.; Squires, K.; Ruane, P.; Coakley, D.;
Kearney, B.; Zhong, L. J.; Wulfsohn, M.; Miller, M. D.; Lee, W. A. Phase
I/Ii Study of the Pharmacokinetics, Safety and Antiretroviral Activity of
Tenofovir Alafenamide, a New Prodrug of the Hiv Reverse Tran-
scriptase Inhibitor Tenofovir, in Hiv-Infected Adults. J. Antimicrob.
Chemother. 2014, 69, 1362−1369.
ABBREVIATIONS USED
DCM, dichloromethane; DMF, dimethylformamide; TFV,
tenofovir; TDF, tenofovir disoproxil fumarate
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(19) Hostetler, K. Y. Alkoxyalkyl Prodrugs of Acyclic Nucleoside
Phosphonates Enhance Oral Antiviral Activity and Reduce Toxicity:
Current State of the Art. Antiviral Res. 2009, 82, A84−98.
(20) Painter, G. R.; Almond, M. R.; Trost, L. C.; Lampert, B. M.; Neyts,
J.; De Clercq, E.; Korba, B. E.; Aldern, K. A.; Beadle, J. R.; Hostetler, K.
Y. Evaluation of Hexadecyloxypropyl-9-R-[2-(Phosphonomethoxy)-
Propyl]-Adenine, Cmx157, as a Potential Treatment for Human
Immunodeficiency Virus Type 1 and Hepatitis B Virus Infections.
Antimicrob. Agents Chemother. 2007, 51, 4538−4538.
(21) Giesler, K. E.; Marengo, J.; Liotta, D. C. Reduction Sensitive Lipid
Conjugates of Tenofovir: Synthesis, Stability, and Antiviral Activity. J.
Med. Chem. 2016, 59, 7097−7110.
(22) Iyer, S.; Hengge, A. C. The Effects of Sulfur Substitution for the
Nucleophile and Bridging Oxygen Atoms in Reactions of Hydroxyalkyl
Phosphate Esters. J. Org. Chem. 2008, 73, 4819−4829.
REFERENCES
■
(1) Walle, T.; Hsieh, F.; DeLegge, M. H.; Oatis, J. E.; Walle, U. K. High
Absorption but Very Low Bioavailability of Oral Resveratrol in Humans.
Drug Metab. Dispos. 2004, 32, 1377−1382.
(2) Anand, P.; Kunnumakkara, A. B.; Newman, R. A.; Aggarwal, B. B.
Bioavailability of Curcumin: Problems and Promises. Mol. Pharmaceutics
2007, 4, 807−818.
(3) Veber, D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B. R.; Ward, K.
W.; Kopple, K. D. Molecular Properties That Influence the Oral
Bioavailability of Drug Candidates. J. Med. Chem. 2002, 45, 2615−2623.
(4) Li, W. X.; Escarpe, P. A.; Eisenberg, E. J.; Cundy, K. C.; Sweet, C.;
Jakeman, K. J.; Merson, J.; Lew, W.; Williams, M.; Zhang, L. J.; Kim, C.
U.; Bischofberger, N.; Chen, M. S.; Mendel, D. B. Identification of Gs
4104 as an Orally Bioavailable Prodrug of the Influenza Virus
Neuraminidase Inhibitor Gs 4071. Antimicrob. Agents Chemother.
1998, 42, 647−653.
(23) Xia, F.; Zhu, H. Density Functional Calculations on the Effect of
Sulfur Substitution for 2′-Hydroxypropyl-P-Nitrophenyl Phosphate: Co
Vs. Po Bond Cleavage. Bioorg. Chem. 2012, 40, 99−107.
(5) Simplicio, A. L.; Clancy, J. M.; Gilmer, J. F. Prodrugs for Amines.
Molecules 2008, 13, 519−547.
(24) Egron, D.; Perigaud, C.; Aubertin, A. M.; Imbach, J. L.; Gosselin,
C. Effect of the Thioalkyl Chain Variation in the Efficiency of Sate
Pronucleotides. Nucleosides Nucleotides 1998, 17, 1725−1729.
(25) Pertusat, F.; Serpi, M.; McGuigan, C. Medicinal Chemistry of
Nucleoside Phosphonate Prodrugs for Antiviral Therapy. Antivir. Chem.
Chemother. 2012, 22, 181−203.
(6) Pradere, U.; Garnier-Amblard, E. C.; Coats, S. J.; Amblard, F.;
Schinazi, R. F. Synthesis of Nucleoside Phosphate and Phosphonate
Prodrugs. Chem. Rev. 2014, 114, 9154−9218.
(7) Thilakarathna, S. H.; Rupasinghe, H. P. Flavonoid Bioavailability
and Attempts for Bioavailability Enhancement. Nutrients 2013, 5, 3367−
3387.
H
DOI: 10.1021/acs.jmedchem.6b01292
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(26) Giesler, K. E.; Marengo, J.; Liotta, D. C. Reduction Sensitive Lipid
Conjugates of Tenofovir: Synthesis, Stability, and Antiviral Activity. J.
Med. Chem. 2016, 59, 7097−7110.
(27) Ray, A. S.; Hostetler, K. Y. Application of Kinase Bypass Strategies
to Nucleoside Antivirals. Antiviral Res. 2011, 92, 277−291.
I
DOI: 10.1021/acs.jmedchem.6b01292
J. Med. Chem. XXXX, XXX, XXX−XXX