Bis-Ketol Nucleoside Triesters as Prodrugs of the Antiviral Nucleoside Triphosphate Analogues of 3′-Deoxythymidine and 3′-Deoxy-2′ ,3′-didehydrothymidine

Article abstract of DOI:10.1081/NCN-120030723

Derivatives of 3′-deoxythymidine (ddT) and 3′-deoxy-2′ ,3′-didehydrothymidine (d4T) were prepared in which the 5′-hydroxyl group of the nucleoside was esterified to a bisketol phosphate. The resulting phosphate triesters are postulated to be prodrugs of t

Full text of DOI:10.1081/NCN-120030723

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, No. 3, pp. 637–646, 2004
Bis-Ketol Nucleoside Triesters as Prodrugs of the Antiviral
Nucleoside Triphosphate Analogues of 3’-Deoxythymidine
and 3’-Deoxy-2’,3’-didehydrothymidine
*
Kim C. Calvo, Xiaohong Wang, and Gerald F. Koser
Department of Chemistry, The University of Akron, Akron, Ohio, USA
ABSTRACT
Derivatives of 3’-deoxythymidine (ddT) and 3’-deoxy-2’,3’-didehydrothymidine (d4T)
were prepared in which the 5’-hydroxyl group of the nucleoside was esterified to a bis-
ketol phosphate. The resulting phosphate triesters are postulated to be prodrugs of the
corresponding 5’-mononucleotides, which are formed intracellularly by the hydrolysis
of the two ketol ester groups. The triesters were tested for anti-HIV activity with the
result that those derived from ddT showed enhanced antiviral activity when compared
to the parent nucleoside.
Key Words: Bis-ketol; Triesters; Prodrugs; Triphosphate; Didehydrothymidine.
INTRODUCTION
Analogs of deoxyribonucleosides and nucleotides find use as antiviral com-
pounds.[1–4] They constitute one of the important components of the combination
therapy currently used in the treatment of AIDS.[5] These nucleoside analogs are really
prodrugs of the biologically active form, the 5’-triphosphate,[6] which inhibits viral
replication by interaction with the viral reverse transcriptase. The nucleoside analogs
*
Correspondence: Kim C. Calvo, Ph.D., Department of Chemistry, The University of Akron,
Akron, OH 44325-3601, USA; Fax: (330) 972-7370; E-mail: kimcalvo@uakron.edu.
637
DOI: 10.1081/NCN-120030723
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

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Calvo, Wang, and Koser
are converted to the active triphosphate form by three successive phosphorylation
reactions catalyzed by various cellular kinases. Several nucleoside analogs, which are
capable of potent inhibition of reverse transcriptase when present as the triphosphate
form, do not show antiviral activity in screening assays because they are poor
substrates for the cellular kinases.[7] The mono-, di- or triphosphate forms of the
nucleosides are too polar to pass through the cell membrane and so cannot be used
as drugs.
McGuigan has examined a strategy, named the kinase bypass, which attempts to
circumvent the first phosphorylation reaction needed to activate all nucleoside analog
prodrugs.[8–12] In this strategy, the double negative charge associated with a nucleoside
analog monophosphate is masked by forming a nucleoside analog monophosphate
triester. The triester is sufficiently lipophilic that it is able to penetrate the cell
membrane. Related strategies include the use of a bis-S-acyl-2-thioethyl ester[13] and
the use of the bis-pivaloyloxymethyl ester.[14] It appears that by judicious choice of the
alcohols used in triester formation, it should be possible to direct intracellular
hydrolysis to form the nucleoside analog monophosphate. The alcohols incorporated
into the triester group should allow rapid triester hydrolysis to the diester monoanion,
which should be retained within the cell since it is too polar to pass through the
membrane, and subsequently to the monoester dianion. In addition, hydrolysis of the
triester and diester should occur in a way that leads to formation of the nucleoside
analog monophosphate, and not to the original nucleoside.
The ketol esters of phosphoric acid satisfy both of these criteria. The rate constant
for the hydrolysis of a bis-ketol alkyl phosphate triester is about 6.2 Â 10 À4 s À1 at pH
7.4 and 37°C, giving a half life for the triester of about 19 min.[15] In addition, the
mechanism for the hydrolysis as given in Scheme 1, demands that the ketol group must
always be lost during hydrolysis to the diester.[16] Pseudorotation of the intermediate
phosphorane places the alcohol oxygen of the ketol in the apical position. After the
ketol ester group is expelled, the phosphorylated carbonyl hydrate eliminates the
phosphate diester. Once the diester is formed, two hydrolysis reactions are possible.
Scheme 1. Mechanism of base hydrolysis of ketol phosphate triesters.

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639
Scheme 2. Proposed routes for intracellular hydrolysis of mono-ketol phosphate diesters.
The monoketol diester can either hydrolyze non-enzymatically and regioselectively via
phosphate elimination from the ketol[17] to give the nucleoside monophosphate or
enzymatically to produce either the nucleoside monophosphate and the ketol, or the
ketol phosphate and the nucleoside analog (Scheme 2). Hydrolysis to give the ketol
phosphate does not accomplish the kinase bypass.
In this paper we describe the synthesis of various bis-ketol ddT and d4T triesters
and the results of antiviral screening of the compounds.
EXPERIMENTAL
General. NMR spectra were recorded on a Varian 300 MHZ NMR. The
chemical shifts for 1H spectra are referenced to the residual protio form of the
deuterated NMR solvent. The chemical shifts for 13C spectra are referenced to
the solvent carbons, while the 31P NMR shifts (proton-decoupled) are referenced to
external 85% phosphoric acid capillary in the NMR solvent. Reagents were obtained
from Aldrich, Lancaster or Acros. Solvents used in reactions were purified by standard
methods by distillation from appropriate drying agents. The ddT was prepared by
literature procedures.[18]
Tris-Ketol Phosphoric Acid Esters (1a–c). These compounds were prepared by
treatment of crystalline phosphoric acid with 3 mole equivalents of PhI(OAc)2 and 6 to
6.5 mole equivalents of the appropriate silyl enol ethers in t-butyl alcohol as reported
in[19] for the analogous procedure with p-(difluoroiodo)toluene . Beginning with H3PO4
(10.0 mmol for 1a, 15.3 mmol for 1b, and 25.5 mmol for 1c), the product yields were
1a (22%, mp 107–9°C), 1b (44%, mp 43–44°C), and 1c (56%, mp 142–4°C). Selected
NMR data (1H, 13C, and 31P) for 1a and 1c are reported in.[19]
Lithium Bis-Ketol Phosphate Diesters (2a–c). These compounds were prepared
as described in[19] Solutions of the tris-ketol phosphates [1a (5.89 mmol), 1b
(3.90 mmol), and 1c (3.49 mmol)] and LiBr (1 to 1.3 equivalents) in dry acetone (120
or 130 mL) were stirred and heated under reflux (3 to 5 hr). During this time, the

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Calvo, Wang, and Koser
lithium bis-ketol phosphates precipitated and were isolated by filtration: yields; 2a
(89%, mp 221–2°C), 2b (93%, mp 214–6°C), and 2c (100 %, mp 189–93°C). Selected
NMR data (1H, 13C, and 31P) for 2a and 2c are reported in.[19] Combustion analysis of
samples of 2a–2c, previously prepared in our laboratory, indicate that these compounds
may be hemi-hydrates.[19]
Bis-Ketol Hydrogen Phosphoric Acid Diesters (3a-c). Mixtures of the lithium
bis-ketol phosphates [2a (2.57 mmol), 2b (2.00 mmol), and 2c (1.35 mmol)] and
Dowex (50w  8, H+ form) ion exchange resin [14 g for 2a, 7 g for 2b and 2c] in
acetonitrile (50 mL) were stirred and heated under reflux for 3 h. The salts dissolved as
they were converted to 3. The resin was then removed by filtration and washed with
methylene chloride (2 Â 20 mL). Concentration of the filtrate and washings gave white
solids, identified as 3a (89%, mp 105–7°C), 3b (90%, mp 72–4°C), and 3c (92%).
4b-3’-Deoxythymidine-5’-bis(heptanoylmethyl)phosphate. Dicyclohexylcarbo-
diimide (DCC, 0.660 g, 3.20 mmol) was added at rt to a stirred solution of the bis-
ketol hydrogen phosphate 3b (0.561 g, 1.60 mmol) in dry CH2Cl2 (10 mL); a white
solid (dicyclohexyl urea) separated immediately. After 4 h, the mixture was passed
through a pipet packed with a glass wool plug. Triethylamine (25 drops) and ddT
(91 mg, 0.40 mmol) were added to the filtrate, and the reaction mixture was
allowed to stir for 20 h at rt. The solvent was then removed (rotary evaporator), and
the residual yellow oil was subjected to flash column chromatography on silica gel
(20 g) with CHCl3 (300 mL), CHCl3/CH3OH (200:1 (v/v), 100 mL), CHCl3/CH3OH
(100:1 (v/v), 100 mL), and CHCl3/CH3OH (75:1 (v/v), 150 mL) to give 4b as a
pale yellow oil which gradually (1 week) solidified under mechanical vacuum; white
1
solid; yield 202 mg (90%); mp 51.5–53°C. H NMR (CDCl3): 0.88 (t, J = 6.9 Hz,
6H), 1.28 (m, 12H), 1.61 (m, 4H), 1.93 (d, J = 1.1 Hz, 3H), 2.01–2.18 (m, 3H),
2.38–2.46 (m, 5H), 4.28–4.54 (m, 3H), 4.63–4.82 (m, 4H), 6.11–6.15 (m, 1H),
7.54 (d, J = 1.1 Hz, 1H), 8.50 (br s, 1H). 13C NMR (CDCl3): 12.60, 14.22, 22.65,
23.39, 25.56, 28.99, 31.69, 32.21, 38.60, 68.98 (d, JCP = 5.4 Hz), 71.16 (t,
JCP = 5.1 Hz), 78.79 (d, JCP = 8.5 Hz), 86.14, 110.96, 135.86, 150.42, 163.78,
204.05 (t, JCP = 3.8 Hz). 31P NMR (CDCl3): 0.009 (s). IR (neat): 1694 cmÀ1
(C = O), 1270 cmÀ1 (P = O). Anal. Calcd. for C26H43N2O9P; C, 55.90; H, 7.76; N,
5.01. Found: C, 56.19; H, 7.99; N, 5.10.
4a-3’-Deoxythymidine-5’-bis(2,2-dimethylpropanoylmethyl)phosphate. Yield
221 mg (88%). H NMR (CDCl3): 1.19 (d, J = 2.1 Hz, 18H), 1.93 (d, J = 1.0 Hz, 3H),
1
2.02–2.15 (m, 3H), 2.35–2.44 (m, 1H), 4.29–4.59 (m, 3H), 4.89–5.11 (m, 4H), 6.13–
6.16 (m, 1H), 7.58 (d, J = 1.1 Hz, 1H), 8.15 (br s, 1H). 13C NMR (CDCl3): 12.58,
25.53, 26.35, 32.26, 42.87, 68.03 (dd, JCP = 5.3 Hz), 68.83 (d, JCP = 5.5 Hz), 78.85 (d,
JCP = 7.3 Hz), 86.05, 110.92, 135.96, 150.50, 163.90, 208.16 (t, JCP = 3 Hz). 31P NMR
(CDCl3): 0.46 (s). mp: 114–115°C. IR (neat): 1695 cmÀ1 (C = O), 1270 cmÀ1 (P = O).
Anal. Calcd. for C22H35N2O9P; C, 52.59; H, 7.02; N, 5.57. Found: C, 52.54; H, 7.04;
N, 5.59.
4c-3’-Deoxythymidine-5’-bis(phenacyl)phosphate. Yield 153 mg (88%). 1H
NMR (CDCl3): 1.90 (d, J = 0.9 Hz, 3H), 2.03–2.19 (m, 3H), 2.36–2.46 (m, 1H), 4.32–
4.42 (m, 1H), 4.46–4.68 (m, 2H), 5.41–5.61 (m, 4H), 6.12–6.16 (m, 1H), 7.48–7.55 (m,

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641
4H), 7.58–7.68 (m, 2H), 7.87–7.93 (m, 4H), 8.65 (br s, 1H). 13C NMR (CDCl3): 12.57,
25.61, 32.18, 69.14 (d, JCP = 6.0 Hz), 69.73 (dd, JCP = 6.4 Hz), 78.79 (d, JCP = 8.5 Hz),
86.06, 111.02, 127.90, 129.21, 133.93, 134.41, 135.89, 150.45, 163.77, 192.32 (t,
JCP = 3.7 Hz). 31P NMR (CDCl3): 0.52 (s). mp: 58–59°C. IR (neat): 1690 cmÀ1 (C = O),
1266 cmÀ1 (P = O). Anal. Calcd. for C26H27N2O9P.1/2H2O; C, 56.62; H, 5.12; N, 5.08.
Found: C, 56.63; H, 5.12; N, 5.08.
5b-3’-Deoxy-2’,3’-didehydrothymidine-5’-bis(heptanoylmethyl)phosphate. DCC
(1.41 g, 6.83 mmol) was added at rt to a stirred solution of 3b (1.20 g, 3.42 mmol)
in dry CH2Cl2 (15 mL); a white solid separated immediately. After 5h, the mixture was
passed through a pipet packed with a glass wool plug. Triethylamine (25 drops) and
d4T (0.192 g, 0.86 mmol) were added to the filtrate and the reaction mixture was
allowed to stir for 2–3 days at rt. The solvent was then removed and the residual
yellow oil was subjected to flash column chromatography on silica gel (26 g) with
CHCl3 (300 mL), CHCl3/CH3OH (200:1 (v/v), 100 mL), CHCl3/CH3OH (100:1 (v/v),
100 mL), and CHCl3/CH3OH (75:1 (v/v), 150 mL) to give 5b as a pale yellow oil
which gradually solidified (1 week) under mechanical vacuum. yield 320 mg (67%).
Further purification of this material by a second chromatography on silica gel (6 g)
with CHCl3 (150 mL) and CHCl3/CH3OH (200:1 (v/v), 150 mL) returned 0.280 g of
1
5b. mp: 90–92°C. H NMR (CDCl3): 0.88 (t, J = 7.0 Hz, 6H), 1.21–1.28 (m, 12H),
1.54–1.64 (m, 4H), 1.89 (d, J = 1.2 Hz, 3H), 2.38–2.42 (m, 4H), 4.35–4.52 (m, 2H),
4.60–4.83 (m, 4H), 4.99–5.06 (m, 1H), 5.86–5.92 (m, 1H), 6.39–6.42 (m, 1H), 7.01–
7.04 (m, 1H), 7.36 (d, J = 1.2 Hz, 1H), 8.82 (br s, 1H). 13C NMR (CDCl3): 12.39,
14.19, 22.61, 23.37, 28.96, 31.66, 38.55, 68.37 (d, JCP = 6.1 Hz), 71.07 (dd, JCP = 6.1
Hz), 84.78 (d, JCP = 8.6 Hz), 89.77, 111.46, 127.58, 133.40, 136.28, 151.03, 163.96,
203.92 (t, JCP = 4.8 Hz). 31P NMR (CDCl3): À 0.15 (s). . IR (neat): 1699 cmÀ1
(C = O), 1265 cmÀ1 (P = O). Anal. Calcd. for C26H41N2O9P; C, 56.11; H, 7.42; N,
5.03. Found: C, 56.32; H, 7.08; N, 5.31.
5a-3’-Deoxy-2’,3’-didehydrothymidine-5’-bis(2,2-dimethylpropanoylmethyl)-
phosphate. Yield 300 mg (66%). H NMR (CDCl3): 1.18 (d, J = 3.5 Hz, 18H), 1.90
1
(d, J = 1.2 Hz, 3H), 4.37–4.57 (m, 2H), 4.83–5.12 (m, 5H), 5.87–5.91 (m, 1H), 6.42–
6.46 (m, 1H), 7.04–7.07 (m, 1H), 7.40 (d, J = 1.2 Hz, 1H), 8.55 (br s, 1H). 13C NMR
(CDCl3): 12.36, 26.35, 42.87, 67.93 (dd, JCP = 4.8 Hz), 68.26 (d, JCP = 6.1 Hz), 84.90
(d, JCP = 8.6 Hz), 89.74, 111.43, 127.40, 133.64, 136.47, 150.97, 163.88, 208.15 (t,
JCP = 3 Hz). 31P NMR (CDCl3): 0.22 (s). mp: 121–122°C. IR (neat): 1700 cmÀ1
(C = O), 1262 cmÀ1 (P = O). Anal. Calcd. for C22H33N2O9P; C, 52.80; H, 6.65; N,
5.60. Found: C, 53.06; H, 6.84; N, 5.85.
5c-3’-Deoxy-2’,3’-didehydrothymidine-5’-bis(phenacyl)phosphate. Yield 310 mg
(87%). H NMR (CDCl3): 1.89 (d, J = 1.1 Hz, 3H), 4.48–4.68 (m, 2H), 5.05–5.15 (m,
1
1H), 5.37–5.61 (m, 4H), 5.89–5.92 (m, 1H), 6.47–6.51 (m, 1H), 7.04–7.08 (m, 1H), 7.44
(d, J = 1.3 Hz, 1H), 7.48–7.54 (m, 4H), 7.60–7.67 (m, 2H), 7.86–7.92 (m, 4H), 8.56 (br s,
1H). 13C NMR (CDCl3): 12.39, 68.48 (d, JCP = 6.1 Hz), 69.64 (dd, JCP = 5.3 Hz), 84.89
(d, JCP = 8.5 Hz), 89.74, 111.48, 127.49, 127.86, 129.21, 133.58, 133.84, 134.42, 136.39,
151.00, 163.91, 192.24 (t, JCP = 4.2 Hz). 31P NMR (CDCl3): 0.29 (s). mp: 72–74°C. IR
(neat): 1703 cmÀ1 (C = O), 1102 cmÀ1 (P = O). Anal. Calcd. for C26H25N2O9P.1/2H2O; C,
56.83; H, 4.77; N, 5.10. Found: C, 56.94; H, 4.68; N, 5.15.

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Calvo, Wang, and Koser
Hydrolysis rate constant for 5c: The hydrolysis of 5c was determined as
indicated in reference 8. At 25°C, pH 7.68, the hydrolysis rate constant is 0.0260 minÀ1
.
Using an activation energy of 18.40 kcal/mole,[15] we estimate that at pH 7.4 and 37°C,
the half life of 5c is about 42 min.
Assay of anti-HIV activity: The bis-ketol ddT and d4T derivatives were tested
for anti-HIV activity at the National Cancer Institute by their standard protocol.[20] In
this protocol, the compounds are dissolved in DMSO and then serially diluted into the
cell culture medium. CEM cells are used as the T4 lymphocytes. These cells are added
to the culture medium followed by HIV-1. An uninfected control in the presence of the
compound being tested is used as a toxicity control, and infected and uninfected cells
without the compound serve as basic controls. Cell viability is assessed by the
formazan color development following addition of the tetrazolium salt, XTT. The IC50
values represent the molar concentration of compound that results in 50% inhibition of
Scheme 3. Synthesis of bis-ketol phosphate derivatives of deoxy and dideoxythymidine.

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643
uninfected cell growth. The EC50 values represent the molar concentration of
compound that results in survival of 50% of infected cells. The TI50 is the ratio
of IC50/EC50.
RESULTS AND DISCUSSION
The synthesis of the bis-ketol ddT and d4T monophosphate triesters was accom-
plished by phosphorylation of the nucleoside with the tetra-kis ketol pyrophosphates
generated in situ from bis-ketol hydrogen phosphates and DCC, Scheme 3. Formation
of the bis-ketol hydrogen phosphates was made possible by using an ion exchange resin
to exchange lithium for proton. The formation of the lithium bis-ketol phosphates
proceeded almost quantitatively from the tris-ketol phosphates using LiBr in acetone.
Overall, the synthesis proceeded in good yield from the nucleoside derivative to give
the desired bis-ketol phosphate triesters.
Table 1 summarizes the in vitro anti-HIV test results for the bis-ketol phosphate
triesters of ddT and d4T given in Scheme 4. In the assay conducted by the NCI, ddT
is classified as inactive in inhibiting the growth of HIV in CEM T4 lymphocytes. This
category is reserved for compounds showing an EC50 greater than about 200 mM.
When the bis-ketol phosphate is added to the 5-hydroxyl of ddT, all of the derivatives
become moderately active in inhibiting the growth of HIV. The EC50 values for the
bis-ketol phosphate ddT triesters ranged from 19 to 25 mM which represents a
decrease in EC50 of at least 10 fold (the NCI assay did not always determine the
higher limit of activity) when compared to the parent ddT. This increase in anti-viral
activity is attributed to the formation of increased levels of the monophosphate of ddT
in the cells.
Table 1. Anti-HIV activity of compounds 4a–c and 5a–c.a
Compound
IC50b mM
EC50c mM
TI50d
ddT
4a
> 200
> 200
> 200
> 200
> 200
> 200
> 180
110
INACTIVEe
23.
19.
> 8.7
> 10
> 8
4b
4c
25.
d4T
5a
0.60
1.3
333
> 154
> 353
124
5b
5c
0.51
0.89
aCompounds were tested against HIV-1 in CEM cells according to the NCI protocol.[20]
bConcentration that gives a cell growth of 50% relative to a culture not exposed to the compound or
virus.
cConcentration that leads to 50% protection of virus infected cells.
dThe ratio IC50/EC50.
eAccording to the National Cancer Institute, the EC50 of this compound is greater than 200 mM and
the compound is labeled as inactive. Compounds 4a–c are labeled moderately active. Compounds
5a–c are all labeled as active by the NCI.

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Scheme 4. Structural formulas of nucleotides tested for anti-HIV activity.
Both ddT and d4T have been assayed for anti-HIV activity in the past. When ddT
and d4T were examined in the same assay system and the data reported in the same
paper,[21,22] ddT was found to have an EC50 about 20 times larger than d4T. When the
triphosphates of ddT and d4T were analyzed as inhibitors of HIV reverse transcriptase
using the same enzyme and the same polymeric substrate,[23] the inhibition constant for
ddT was about 3 times that for d4T. It is clear that both ddT and d4T are active against
HIV in an in vitro assay or reverse transcriptase assay. The fact that d4T is only three
times better at inhibiting reverse transcriptase but 20 fold better at protecting cells from
HIV indicates that the two compounds lead to significantly different levels of
triphosphate inside the cells when administered as the nucleosides. The data on the
EC50 values of the bis-ketol phosphate triesters of ddT and d4T in this study indicate
that the bis-ketol derivatives of d4T inhibit HIV at concentrations about 25 fold lower
than the bis-ketol derivatives of ddT. However, in the assay used to assess anti-HIV
activity, the EC50 of d4T is about 300 fold lower than the EC50 of ddT. The addition
of the bis-ketol phosphate group to ddT has decreased the EC50 value by at least 10
fold. Although the addition of a bis-ketol triester to d4T did not significantly improve
the EC50, it did not render the nucleoside analog inactive. This is in contrast to a series
of bis-ketol phosphate AZT derivatives that were tested previously[24] where the bis-
ketol derivative was 1.5 to 77 times more active than the parent nucleoside analog. In
the case of ddT, the addition of a bis-ketol triester significantly improved the EC50
value. The bis-ketol phosphate triesters of ddT behave as though they were pro-drugs
of ddT monophosphate esters.
ACKNOWLEDGMENT
This research was supported in part by NIH grant AI043975 awarded to G.F. Koser
and K.C. Calvo.
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Received July 1, 2003
Accepted December 31, 2003

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