Acyclic nucleoside analogues as novel inhibitors of human mitochondrial thymidine kinase

Article abstract of DOI:10.1021/jm011128+

A series of acyclic nucleoside analogues of 5′-O-tritylthymidine have been synthesized and evaluated as potential human mitochondrial thymidine kinase (TK-2) inhibitors. In this series, the sugar moiety of the parent 5′-O-tritylthymidine has been replaced by aliphatic chains including (E)- and (Z)-butenol, butynol, or butanol. Among them the (Z)-butenyl derivative (10) showed an IC₅₀ against TK-2 of 1.5 μM, being 1 order of magnitude more potent than the parent 5′-O-tritylthymidine. This lead compound has been further modified by replacing the thymine base by other pyrimidine bases such as 5-iodouracil, 5-ethyluracil, 5-methylcytosine, 3-N-methylthymine, or 5,6-dihydrothymine, as well as by the purine base guanine. The trityl group has also been replaced by different aliphatic and aromatic acyl moieties including tertbutylacetyl, hexanoyl, decanoyl, and diphenylacetyl moieties. The evaluation of the compounds against TK-2 and the phylogenetically close HSV-1 TK has shown that the base moiety plays a crucial role in their interaction against these pyrimidine nucleoside kinases. Also, the presence of a lipophilic substituent, preferentially an aromatic moiety such as diphenylmethyl or triphenylmethyl, is required for efficient TK-2 inhibition. Whereas some compounds showed marked specificity for either TK-2 (i.e, the 5,6-dihydrothymine derivative, 26) or HSV-1 TK (i.e., the butynyl derivative, 11), some others, including the (Z)-and (E)-butenyl derivatives 10 and 12, showed significant inhibition against both enzymes. They also proved to be inhibitory against HSV-1 TK in intact human osteosarcoma cells that were transduced with the HSV-1 TK gene.

Full text of DOI:10.1021/jm011128+

4254
J . Med. Chem. 2002, 45, 4254-4263
Acyclic Nu cleosid e An a logu es a s Novel In h ibitor s of Hu m a n Mitoch on d r ia l
Th ym id in e Kin a se
Ana-Isabel Herna´ndez,† J an Balzarini,‡ Anna Karlsson,§ Mar´ıa-J ose´ Camarasa,† and Mar´ıa-J esu´s Pe´rez-Pe´rez*,†
Instituto de Quı´mica Me´dica (CSIC), J uan de la Cierva 3, 28006 Madrid, Spain, Rega Institute for Medical Research,
Katholieke Universiteit Leuven, B-3000 Leuven, Belgium, and Karolinska Institute, S-141 86 Huddinge/ Stockholm, Sweden
Received December 18, 2001
A series of acyclic nucleoside analogues of 5′-O-tritylthymidine have been synthesized and
evaluated as potential human mitochondrial thymidine kinase (TK-2) inhibitors. In this series,
the sugar moiety of the parent 5′-O-tritylthymidine has been replaced by aliphatic chains
including (E)- and (Z)-butenol, butynol, or butanol. Among them the (Z)-butenyl derivative
(10) showed an IC50 against TK-2 of 1.5 µM, being 1 order of magnitude more potent than the
parent 5′-O-tritylthymidine. This lead compound has been further modified by replacing the
thymine base by other pyrimidine bases such as 5-iodouracil, 5-ethyluracil, 5-methylcytosine,
3-N-methylthymine, or 5,6-dihydrothymine, as well as by the purine base guanine. The trityl
group has also been replaced by different aliphatic and aromatic acyl moieties including tert-
butylacetyl, hexanoyl, decanoyl, and diphenylacetyl moieties. The evaluation of the compounds
against TK-2 and the phylogenetically close HSV-1 TK has shown that the base moiety plays
a crucial role in their interaction against these pyrimidine nucleoside kinases. Also, the presence
of a lipophilic substituent, preferentially an aromatic moiety such as diphenylmethyl or
triphenylmethyl, is required for efficient TK-2 inhibition. Whereas some compounds showed
marked specificity for either TK-2 (i.e, the 5,6-dihydrothymine derivative, 26) or HSV-1 TK
(i.e., the butynyl derivative, 11), some others, including the (Z)-and (E)-butenyl derivatives 10
and 12, showed significant inhibition against both enzymes. They also proved to be inhibitory
against HSV-1 TK in intact human osteosarcoma cells that were transduced with the HSV-1
TK gene.
In tr od u ction
particularly under debate. First, its low level of expres-
sion, its mitochondrial localization, and the concomitant
presence of the cytosolic TK-1 make it difficult to
evaluate its contribution either in mitochondrial or in
cellular events, as well as in activation of antiviral or
anticancer nucleoside analogues. Recently, mutant TK-2
was found to be the genetic basis for the mitochondrial
DNA depletion in the hereditary disorder of several
individuals who suffered from severe devastating my-
opathy.7 This points indeed to a crucial role of TK-2 in
mitochondrial DNA synthesis. Second, it has been
proposed that the mitochondrial toxicity observed under
prolonged treatments with the anti-hepatitis B virus
nucleoside analogue FIAU8 or with the anti-HIV drug
AZT9 is due to activation of these nucleoside analogues
by TK-2. Therefore, the use of TK-2 inhibitors may help
to resolve fundamental questions such as the physi-
ological role of TK-2 and may also reveal and clarify the
real contribution of TK-2-catalyzed phosphorylation of
antiviral drugs in their mitochondrial toxicity. There-
fore, there is an increasing interest and need for specific
human mitochondrial thymidine kinase inhibitors.10,11
Our approach to the identification of new TK-2
inhibitors has relied on the evaluation of nucleoside
analogues previously synthesized in our laboratories12
or easily obtained from commercial sources. From this
last approach, it was found that 5′-O-(4,4′-dimethoxy-
trityl)thymidine (DMTr-dThd, 1)13 showed slight inhibi-
tory activity against TK-2-catalyzed dThd phos-
phorylation (IC50 ) 468 ( 45 µM). This compound was
1 order of magnitude more active against the related
In mammalian cells, there are two different nucleo-
side kinases that are able to phosphorylate thymidine
to its 5′-monophosphate derivative, dTMP, namely,
cytosolic thymidine kinase (TK-1) and mitochondrial
thymidine kinase (TK-2).1 Both enzymes show impor-
tant differences in their primary amino acid sequence,
substrate specificity, and level of expression in the
different phases of the cell cycle. TK-1 reaches its peak
of expression in the early S phase and declines in the
G2 phase.2,3 TK-1 phosphorylates thymidine (dThd) and
2′-deoxyuridine (dUrd).4 TK-2 is a mitochondrial en-
zyme5 and represents only a small fraction of the
cellular TK levels in proliferating cells. However, TK-2
has been suggested to be responsible for most of the TK
activity in resting or terminally differentiated cells (i.e.,
monocytes/macrophages). TK-2 not only recognizes dThd
and dUrd but also efficiently phosphorylates 2′-deoxy-
cytidine (dCyd). It should be pointed out that TK-2
resembles more the herpes simplex virus thymidine
kinase (HSV-TK) than human TK-1, both in substrate
specificity and in primary sequence.6
As enzymes involved in the activation of pyrimidine
nucleosides and nucleoside analogues, thymidine ki-
nases are an important area of research in both the
anticancer and antiviral fields. The role of TK-2 is
* Corresponding author [telephone (+34) 915622900; fax (+34)
915644853; e-mail mjperez@iqm.csic.es].
† Instituto de Qu´ımica Me´dica (CSIC).
‡ Rega Institute for Medical Research.
§ Karolinska Institute.
10.1021/jm011128+ CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/16/2002

Acyclic Nucleoside Inhibitors of Mitochondril Thymidine Kinase J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4255
Sch em e 1a
a
(a) TrCl, DMAP, Et3N, CH2Cl2. (b) LiAlH4, THF, 0 °C. (c) 1, N3-BzT, PS-Ph3P, DIAD, THF; 2, dioxane/1 M NaOH (1:1).
Ch a r t 1
Ch em istr y
One of the very well-known strategies in nucleoside
analogue synthesis is the replacement of the sugar
moiety by conformationally constrained acyclic chains.15
Among them, residues containing butynols or (E)- or (Z)-
butenols are widely used.16,17 Alkylation of the thymine
moiety with acyclic chains through their corresponding
tosylates, mesylates, or bromides is hampered, in gen-
eral, by low yields of the coupling products. The Mit-
sunobu condensation employing N3-benzoylthymine (N3-
BzT) represents an interesting alternative.18 However,
it also suffers from some drawbacks mostly due to the
difficult elimination of the excess of reagents used (Ph3P,
DIAD) and the byproducts of the reaction, in particular
Ph3PO, that make difficult not only the isolation of the
final compounds but also the follow-up of the reaction.
The use of polystyrene-bound triphenylphosphine (PS-
triphenylphosphine), prepared by Ford19 and now com-
mercially available, can be very helpful to simplify
isolation of the target compounds, because both the
excess of Ph3P and the generated Ph3PO remained
attached to the resin and can be removed by simple
filtration.
HSV-1 TK enzyme (IC50 ) 13.6 ( 1.0 µM). Replacement
of the strongly acid labile 4,4′-dimethoxytrityl group by
the more stable trityl analogue (5′-O-tritylthymidine,
Tr-dThd, 2) markedly improved the inhibitory potency
against both enzymes, in particular against TK-2. The
presence of the trityl or dimethoxytrityl substituent at
position 5′ of thymidine converts the thymidine sub-
strate into an inhibitor because both compounds (1 and
2, Chart 1) lack a 5′-OH group that allows phosphory-
lation. This observation led us to hypothesize that the
role of the sugar moiety in compounds 1 and 2 may serve
only to position the base and the trityl substituents in
the right way to interact with the enzyme and, thus,
the sugar moiety has been replaced by acyclic spacers
that may fulfill the same spatial function. Moreover,
these acyclic nucleoside structures may reduce the
possibility of other toxicities from accidental interference
with other nucleoside-processing enzymes (i.e., TK-1).
Therefore, our approach to the synthesis of the [Thy]-
spacer-[trityl] series, shown in Scheme 1, has been the
introduction of the thymine base on tritylated spacers
under Mitsunobu conditions. The intermediate alcohols
6-9 were obtained by monotritylation of commercially
available diols 3-5, with the exception of 8, the corre-
sponding diol of which is not commercially available.
This (E)-buten-2-yl derivative 820 was prepared by
reduction of the butynyl alcohol 7 with LiAlH4. Con-
densation of the alcohols 6-9 with N3-BzT21 in the
presence of PS-triphenylphosphine and DIAD in dry
THF, followed by debenzoylation by treatment with 1
M NaOH in a dioxane/H2O (1:1) solution, afforded the
target compounds 10-13 in good yields (61-89%).
Under these reaction conditions, the excess of thymine
is retained in the aqueous phase and the target com-
pounds 10-13 remain in the organic phase and are
easily isolated by chromatography. In every case, the
only condensation product detected was the alkyl com-
pound at position 1 of the thymine ring.
In the present study, we describe the synthesis and
enzymatic evaluation of a series of acyclic nucleoside
analogues of the general formula [Thy]-spacer-[trityl]
against TK-2 and the phylogenetically closely related
HSV-1 TK. The lead compound has been further ex-
plored and optimized by replacing or modifying the
thymine base and the trityl substituent. Due to the
difficulties encountered in evaluating TK-2 inhibitors
in cell culture,14 and based on the similarities in
substrate specificity between TK-2 and HSV-1 TK, the
most potent inhibitors in the enzymatic assays (i.e., 10
and 12) have been studied in a human osteosarcoma
cell line (OST-TK-/HSV-1 TK+) that is transduced by
and expresses the HSV-1 TK gene.

4256 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Herna´ndez et al.
Sch em e 2a
Sch em e 3
a
(a) Acetone, pTSA, rt. (b) 1, N3-BzT, PS-Ph3P, DIAD, THF; 2,
dioxane/1 M NaOH (1:1). (c) AcOH 80%, ∆. (d) TrCl, DMAP, Py, ∆.
To increase the conformational flexibility on the
spacer and/or to include hydroxyl groups that could
mimic the OH of the (2-deoxy)ribose moiety of the
parent nucleosides (1 and 2), a second series of modi-
fications were carried out. The synthesis of the 2-de-
oxyribose mimetic (18) is shown in Scheme 2. On the
basis of the good yields obtained under Mitsunobu
conditions, this was also the key step in this reaction
sequence. Thus, treatment of 1,2,4-butanetriol (14) with
p-toluenesulfonic acid in acetone, under described con-
ditions, afforded the five-membered ring acetonide 15.22
Reaction of this alcohol (15) with N3-BzT21 under
Mitsonubu conditions (PS-triphenylphosphine, DIAD)
followed by debenzoylation gave the N-1 alkyl compound
16. Removal of the isopropylidene moiety by treatment
with 80% AcOH afforded the diol 17 (40% yield starting
from 14). Previous synthesis of this compound by Holy,23
following a two-step procedure, gave 17 in 23% yield.
Tritylation of the primary alcohol in 17 afforded the
target compound 18.
The (Z)-butenyl derivative 10 was used to obtain the
saturated and bis-hydroxylated derivatives in the spacer
(Scheme 3). Thus, treatment of 10 with OsO4 in the
presence of N-methylmorpholine-N-oxide afforded the
enantiomeric mixture of cis-diols 19 (67% yield). On the
other hand, reduction of 10 by catalytic hydrogenation
(H2, 10% Pd/C) yielded the butyl derivative 20 in 47%
yield. When compounds 10-13 and 18-20 were evalu-
ated against TK-2 and HSV-1 TK (see Biological Results
and Discussion), it was found that the (Z)-butenyl
derivative 10 showed the most potent inhibition against
TK-2, being 1 order of magnitude more potent than the
parent nucleoside (2). Therefore, a new series of modi-
fications were performed now using compound 10 as the
lead, keeping the (Z)-butenyl as the spacer and replacing
or modifying the thymine base and the trityl susbtitu-
ent.
purine, under the same reaction conditions, yielded the
coupling product at position 9 of the purine ring (23,
87% yield). Treatment of 23 with 1 N NaOH in dioxane25
afforded the guanine derivative 24 in 84% yield.
Derivative 10 was used to perform some modifications
on the thymine ring (Scheme 3). Thus, reaction of 10
with POCl3 and 1,2,4-triazole in Et3N and acetonitrile,26
followed by treatment with NH4OH in dioxane, gave the
5-methylcytosine derivative 25 (21% yield from 10). On
the other hand, the 5,6-dihydrothymine derivative 26
was obtained in 54% yield by a novel procedure that
involved reduction of the pyrimidine ring with DIBAL-H
in THF, as an interesting alternative to described
procedures that perform such reduction by catalytic
hydrogenation in the presence of the expensive Rh
catalysts.27 By avoiding the catalytic hydrogenation, the
double bond of the spacer remains unaltered and the
trityl substituent is not removed. Treatment of 10 with
CH3I in the presence of K2CO3 leads to the N3-meth-
ylthymine derivative 27 in 93% yield.
Finally, replacement of the trityl group in the lead
compound 10 by other lipophilic moieties, both aromatic
and aliphatic, was assayed. Because the inhibitory
capacity of the synthesized compounds would be tested
in a cell-free assay using purified enzyme, it was
considered that ester derivatives, which are easily
synthesized, would provide interesting information
about the structural requirements for inhibitory activ-
ity. In a very recent paper, a one-step method for the
deprotection and esterification of trityl ethers has been
described by treatment of the trityl ether with a large
excess (200-1000 equiv) of the corresponding acyl
chloride.28 When these reaction conditions were assayed
on the trityl ether 10 using pivaloyl chloride (100 equiv)
as the acylating agent, only extensive decomposition
was observed. In the above-mentioned paper, it is
proposed that the large excess of the acyl chloride could
be required to ensure the presence of enough HCl in
the reaction medium to perform the deprotection of the
trityl ethers.28 As an alternative to this acid medium,
addition of an acid resin (Dowex 50WX4, H+ form) was
Reaction of the synthon 6 (Scheme 3) with N3-benzoyl-
5-ethyluracil21,24 or N3-benzoyl-5-iodouracil21,24 in the
presence of PS-triphenylphosphine and DIAD, followed
by treatment with 1M NaOH in dioxane/water (1:1) gave
the N-1 alkyl derivatives of 5-ethyl and 5-iodouracil, 21
and 22, in 50 and 30% yields, respectively. On the other
hand, reaction of the alcohol 6 with 6-amino-2-chloro-

Acyclic Nucleoside Inhibitors of Mitochondril Thymidine Kinase J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4257
Ta ble 1. Inhibitory Effect of the Synthesized Compounds on
inhibitory capacity of several derivatives but further
increased the inhibitory potency against the target
enzymes. Thus, derivative 12 containing an (E)-butenyl
the Phosphorylation of [methyl-3H]dThd by TK-2 and HSV-1
TK
a,b
a,b
IC50 (µM)
TK-2 HSV-1 TK
NEAc
33 ( 20
IC50 (µM)
TK-2 HSV-1 TK
NEAc
spacer showed a behavior similar to that of compound
2, being slightly more active than 2 against HSV-1 TK.
Replacement of the furanosyl moiety by a (Z)-butenyl
spacer (10) increased the inhibitory potency 20-fold
against TK-2, but at the same time reduced the inhibi-
tory potency 6-fold against HSV-1 TK. The butynyl
derivative 11 showed a very weak activity at the level
of TK-2 while being as potent as compound 12 against
HSV-1 TK. Thus, in this case, there is a marked
specificity of compound 11 for HSV-1 TK. The benzylic
derivative 13 was completely inactive against both
enzymes. By increasing the conformational freedom in
the spacer, as shown for the butyl derivatives 18-20,
potent inhibitors were obtained against both enzymes
and the enzyme selectivity was reduced. From the
above-mentioned data, it was concluded that the (Z)-
butenyl derivative 10 was the most potent and selective
compound against TK-2 in this series.
compd
compd
1
2
14 ( 1.0
7.8 ( 0.3
25
26
27
28
29
30
31
32
33
34
36
NEAc
NEAc
NEAc
NEAc
NEAc
NEAc
NEAc
NEAc
NEAc
NEAc
NEAc
9.8 ( 1.9
10
11
12
13
18
19
20
21
22
24
1.5 ( 0.16 45 ( 1
NEAc
NEAc
3.1 ( 0.2
40 ( 11
28 ( 5
11 ( 7
19 ( 12
4.6 ( 0.5
NEAc
25 ( 13
3.0 ( 0.0
NEAc
NEAc
3.6 ( 0.4
17 ( 0.3
3.3 ( 1.2
20 ( 7
1.2 ( 0.7
13 ( 1.5
10 ( 1
NEAc
30 ( 4
22 ( 4
4.6 ( 0.4
48 ( 14
NEAc
NEAc
a
50% inhibitory concentration or compound concentration (ex-
presed in µM) required to inhibit dThd phosphorylation by 50%.
Data are the mean value (( SD) of at least two to three
b
independent experiments
IC50 values were obtained in the
presence of 10% DMSO at each inhibitor concentration, including
the control assays (in the absence of inhibitor). c NEA, not
estimated accurately. No inhibition of enzyme activity was noted
at any drug concentration used (up to 500 µM). However, because
full solubility of the drug could not be assured at 500 and 50 µM
(even in the presence of 1% DMSO), accurate values at which
inactivity has been shown cannot be given.
Further modifications performed at the base level
revealed that this fragment is crucial for the interaction
of this family of nucleoside kinase inhibitors. Replace-
ment of the thymine base in compound 10 by guanine
(24) or by 5-methylcytosine (25) abolished the inhibitory
capacity. Methylation of position 3 of the thymine ring,
as shown in compound 27, resulted in complete inactiv-
ity. Substitution of the thymine base by the close
analogue 5-iodouracil (22) kept activity against both
enzymes, and the inhibitory behavior was quite similar
to that of the lead compound 10. The 5-ethyluracil
derivative (21) was 1 order of magnitude less potent
against TK-2 and lost inhibitory activity against HSV-1
TK. It should also be pointed out that the 5,6-dihy-
drothymine derivative (26) exclusively showed activity
against TK-2 with an IC50 value of 9.8 µM, therefore
being a highly selective TK-2 inhibitor.
assayed, and under these conditions the number of
equivalents of acyl chloride required was reduced. Thus,
treatment of 10 (Scheme 3) with pivaloyl chloride (50
equiv) in CH2Cl2 and in the presence of Dowex 50WX4
(H+ form) (100 equiv) led to the acyl derivative 28 in
66% yield. This procedure allows the easy isolation of
the reaction product 28 by filtration of the resin,
hydrolysis of the excess of acyl chloride, washing with
aqueous NaHCO3, and a short chromatography. Fol-
lowing this procedure, compound 10 was easily trans-
formed into the acyl derivatives 29-32 carrying tert-
butylacetyl, hexanoyl, decanoyl, and diphenylacetyl
moieties, respectively (Scheme 3). It is interesting to
mention that reaction of 10 with benzoyl chloride, under
the same reaction conditions, gave only traces of the
benzoyl derivative (34). This is in agreement with the
published results28 which indicate that aromatic acyl
chlorides afford only low yields of the corresponding acyl
products. Alternatively, the O-benzoyl compound29 was
prepared by treatment of 10 with Dowex 50WX4 (H+
form) in MeOH to yield the free OH17 (33) (98% yield),
which by treatment with benzoyl chloride in pyridine
afforded the O-benzoyl derivative 34 in 53% yield.
Finally, the O-benzyl derivative 36 was prepared in
59% yield by condensation of the benzyl alcohol 3530
with N3-BzT under Mitsunobu conditions.
In the last series of modifications, the trityl moiety
was replaced by aromatic and aliphatic acyl residues.
All compounds in the acyl series were inactive or almost
inactive against the herpes TK enzyme while moder-
ately active against TK-2. Among them, the diphenyl
acetyl derivative 32 showed the most potent inhibition
of TK-2 (IC50 ) 4.6 µM). It should also be pointed out
that compound 33 shows an IC50 of ∼200 µM against
TK-2, stressing the importance of the presence of a
highly lipophilic entity, preferentially a diphenylmethyl
or triphenylmethyl moiety, for enzyme inhibition. Fi-
nally, the O-benzoyl and O-benzyl derivatives (34 and
36) were equally inhibitory to TK-2 (IC50 ) 22-30 µM),
being inactive against HSV-1 TK.
Biologica l Resu lts a n d Discu ssion
The most potent TK-2 inhibitors, including com-
pounds 10, 12, 18, and 26, were also tested for their
inhibitory activity against cytosolic TK-1-catalyzed phos-
phorylation of the natural substrate dThd. All of them
showed no inhibitory activity against TK-1 at 100 µM.
In h ibition of HSV-1 TK-Catalyzed [3H]GCV P h os-
p h or yla tion by Com p ou n d 10. From the above-
discussed data, it became clear that the base part of this
new family of nucleoside kinase inhibitors played a
crucial role in the interaction with their target enzymes.
Because no crystallographic X-ray structure has been
available for TK2, it is difficult to speculate how these
In h ibition of [m eth yl-3H]d Th d P h osp h or yla tion
by TK-2 a n d HSV-1 TK. Compounds 10-13, 18-22,
and 24-36 have been tested for their inhibitory effect
against phosphorylation of 2 µM dThd by recombinant
TK-2 and HSV-1 TK (see Experimental Section), and
the results are shown in Table 1. Compounds 1 and 2
are also included in the table for comparative reasons.
Evaluation of the first series of compounds (10-13 and
18-20) indicated that replacement of the furanosyl
moiety in compounds 1 and 2 by aliphatic chain spacers,
according to our working hypothesis, not only kept the

4258 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Herna´ndez et al.
Ta ble 2. Inhibition of HSV-1 TK-Catalyzed [3H]GCV
Phosphorylation by dThd, dGuo, and Compounds 10 and 12
against the enzyme (13) in OST-TK-/HSV-1 TK+ cell
lines in combination with HSV-1 TK substrates, such
as ganciclovir (GCV) and (E)-5-(2-bromovinyl)-1-â-D-
(arabinofuranosyl)uracil (BVaraU) (see Table 3). The
OST-TK-/HSV-1 TK+ cells represent human osteosar-
coma cells that are deficient for cytosolic TK but express
HSV-1 TK in the cytosol.32 When the substrates GCV
and BVaraU were exposed to OST-TK-/HSV-1 TK+ cell
cultures, a marked cytotoxicity was observed with IC50
values in the low nanomolar range (0.004-0.008 µM).
Addition of HSV-1 TK inhibitors, such as compound 10
or 12 at a concentration of 20 µM (see Experimental
Section), to these cell cultures allowed a marked reduc-
tion of 3 orders of magnitude in the cytotoxic effect of
both GCV and BVaraU. This “detoxifying” effect is more
pronounced and efficient by the addition of these inhibi-
tors than by the addition of thymidine as the natural
substrate. In contrast, compound 13 that showed no
inhibitory effect against HSV-1 TK did not change the
inhibitory values of GCV and BvaraU (Table 3). Thus,
potential alterations of the cell membrane properties by
the lipophilic compounds affecting uptake and/or efflux
of the drugs cannot be the reason for the reversing
activity of 10 and 12. Although compound 12 is a more
potent inhibitor of HSV-1 TK than compound 10, it
reversed the cytostatic activity of GCV and BVaraU to
a similar extent as compound 10. Although puzzling, it
is not known whether the administered drug concentra-
tions (20 µM for 10 and 12) are at saturating concentra-
tions to allow full inhibition of the enzyme in the
cytoplasma because no dose-response experiment has
been performed. However, the main purpose of this
experiment was to prove whether compounds 10 and
12 may enter the cells and inhibit cytosolic HSV-1 TK
at subtoxic concentrations, and this turned out to be the
case. These results support the potential interest of the
synthesized inhibitors such as 10 or 12 to be combined
in cell culture with TK-dependent nucleoside analogues
to avoid or prevent potential toxic side effects.
50% inhibitory concentrationa (µM)
compd
HSV-1 TK (WT)
HSV-1 TK (A167Y)
dThd
dGuo
10
1.8 ( 1.0
364 ( 90
g500
115 ( 11
8.7 ( 2.5
>50
12
0.39 ( 0.07
48 ( 3.2
a
50% inhibitory concentration or compound concentration,
expressed in µM, required to inhibit [3H]GCV phosphorylation by
50%.
inhibitors fit into the enzyme. However, the crystal
structures of HSV-1 TK complexed with either the
natural substrate dThd or antiherpetic drugs such as
the guanine nucleoside analogue ganciclovir (GCV) have
been resolved and revealed that both pyrimidine and
purine nucleoside analogues bind to the same substrate
binding site of the enzyme. On the basis of these
crystallographic structures, we have recently designed
a mutant HSV-1 TK (A167Y) that discriminates be-
tween purine and pyrimidine nucleoside substrates.31
Indeed, as shown in Table 2, dThd and, to a lesser
extent, dGuo inhibit [3H]GCV phosphorylation by the
wild-type HSV-1 TK, presumably by competitive inhibi-
tion. However, pyrimidine nucleosides, such as dThd,
are not recognized by the mutant enzyme (A167Y) either
as substrates or as inhibitors (the mutant enzyme does
not have a decreased GCV phosphorylation), thus not
affecting GCV phosphorylation by the mutant enzyme.
Therefore, recognition of dThd by the mutant (A167Y)
HSV-1 TK enzyme is seriously compromised. In con-
trast, GCV phosphorylation by the mutant enzyme is
equally affected by dGuo as the wild-type enzyme.
Interestingly, when compound 10, a thymine-derived
TK-2/HSV-1 TK inhibitor, was investigated against the
wild-type and the mutant HSV-1 TK enzymes, its
inhibitory capacity against GCV phosphorylation by the
wild-type enzyme was very pronounced, whereas GCV
phosphorylation by the mutant enzyme was unaffected
by the compound. This could indicate that the thymine-
based TK inhibitors such as 10 interact with HSV-1 TK
in the substrate binding site and may bind to the
enzyme in a fashion similar to that of the natural
substrate dThd.
Con clu sion s
A series of acyclic nucleoside analogues of 5′-O-
tritylthymidine have been synthesized and tested against
human mitochondrial thymidine kinase (TK-2) and
herpes simplex virus TK (HSV-1 TK).
Replacement of the sugar moiety by acyclic chains
resulted in inhibitors that were 1 order of magnitude
more potent than the parent 5′-O-tritylthymidine. Among
them, the (Z)-butenyl derivative (10) showed an IC50
against TK-2 of 1.5 µM.
To the best of our knowledge, the here-described
inhibitors represent the first example of nucleoside
analogues lacking a (deoxy)ribose moiety that show
pronounced inhibition of human mitochondrial thymi-
dine kinase 2. Finally, the results obtained in the cell
culture model (OST-TK-/HSV-1 TK+ cells) combining
Eva lu a tion of 10, 12, a n d 13 in th e OST-TK-/
HSV-1 TK+ Cell Lin es. Direct evaluation of TK-2
inhibitors in cell culture has proven to be difficult, and
alternative ways to express TK-2 in tumor cells and to
evaluate the inhibitors in an intact cell system have
been proposed.14 However, a tumor cell line expressing
TK-2 in the cytosol is not available yet. On the basis of
the existing similarities between TK-2 and HSV-1 TK,
and because some of our most potent enzyme inhibitors
showed inhibitory activity against both enzymes, we
have investigated two of these inhibitors (10 and 12)
and one derivative that show no inhibitory activity
Ta ble 3. Effect of dThd and Inhibitors of HSV-1 TK on the Cytostatic Activity of GCV and BVAraU in OST-TK-/HSV-1 TK+ Cell
Lines
a
IC50 (µM)
as such
+ 10 (20 µM)
+ 12 (20 µM)
+ 13 (20 µM)
+ dThd (50 µM)
GCV
BVAraU
0.004 ( 0.002
0.008 ( 0.006
0.18 ( 0.17
2.2 ( 1.6
0.23 ( 0.19
3.1 ( 0.85
0.002 ( 0.0003
0.008 ( 0.001
0.026( 0.002
0.016( 0.009
a
50% inhibitory concentration or compound concentration, expressed in µM, required to inhibit cell proliferation by 50%.

Acyclic Nucleoside Inhibitors of Mitochondril Thymidine Kinase J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4259
for an additional hour. After cooling to 0 °C, Na2SO4 (85 mg,
0.60 mmol) and water (0.11 mL, 6 mmol) were added and
stirring was continued overnight. Then CH2Cl2 (40 mL) and
brine (20 mL) were added. The organic phase was dried on
anhydrous Na2SO4, filtered, and evaporated. The residue was
purified by CCTLC in the Chromatotron (hexane/EtOAc, 1:1)
to yield 146 mg (96%) of 8 as a solid: mp 62-63 °C (lit.20 61-
HSV-1 TK substrates and the here-reported inhibitors
demonstrate that TK inhibitors are efficiently taken up
by intact cells to reach the cytosolic compartment and
are sufficiently stable and potent to exert their inhibi-
tory activity in intact cells. These data also support the
potential of this new family of compounds to avoid or
prevent the toxic effects observed with TK-dependent
nucleoside analogues.
1
62 °C); H NMR was identical to that described.20
(2-Tr ip h en ylm eth oxyp h en yl)m eth a n ol (9) was prepared
from 1,2-bis(hydroxymethyl)benzene (5) according to the gen-
eral procedure for monotritylation of diols. The residue was
purified by column chromatography (hexane/EtOAc, 1:1):
yield, 83%; mp 102-105 °C; MS (ES, positive mode), m/z 403
Exp er im en ta l Section
Ch em ica l P r oced u r es. Melting points were obtained on
a Reichert-J ung Kofler apparatus and are uncorrected. Mi-
croanalyses were obtained with a Heraeus CHN-O-RAPID
instrument. Electrospray mass spectra were measured on a
quadrupole mass spectrometer equipped with an electrospray
1
(M + Na)+; H NMR (CDCl3) δ 2.89 (t, J ) 6.6 Hz, 1H, OH),
4.13 (s, 2H, CH2OTr), 4.37 (d, J ) 6.4 Hz, 2H, CH2OH), 7.25-
7.41 (m, 19H, Ph).
Gen er a l P r oced u r e for th e Mitsu n obu Con d en sa tion
of Alcoh ols w ith N3-Ben zoylth ym in e. To a suspension
containing the corresponding alcohol (6-9) (0.5 mmol), PS-
Ph3P (3 mmol/g, 416 mg, 1.25 mmol), and N3-BzT (230 mg,
1.0 mmol) in dry THF (5 mL) was slowly added a solution of
DIAD (0.19 mL, 1.0 mmol) in dry THF (2 mL). The mixture
was stirred at room temperature overnight. The reaction was
filtered, the residue was washed with THF (2 × 5 mL), and
the combined filtrates were evaporated to dryness. The residue
was dissolved in a dioxane/NaOH 1 M (1:1) mixture (9 mL)
and stirred overnight. Then, EtOAc (10 mL) and brine (5 mL)
were added. The aqueous phase was further extracted with
EtOAc (3 × 10 mL). The combined organic extracts were dried
on anhydrous Na2SO4, filtered, and evaporated. The residue
was purified by CCTLC in the Chromatotron (hexane/EtOAc,
1:1) to yield the target compounds.
1
source (Hewlett-Packard, LC/MS HP 1100). H and 13C NMR
spectra were recorded on a Varian Gemini operating at 200
MHz (1H) and 50 MHz (13C), respectively, on a Varian INNOVA
300 operating at 299 MHz (1H) and 75 MHz (13C), respectively,
and on a Varian INNOVA 400 operating at 399 MHz (1H) and
99 MHz (13C), respectively. Monodimensional 1H and 13C
spectra were obtained using standard conditions. Two-
dimensional inverse proton detected heteronuclear one-bond
shift correlation spectra were obtained using the pulsed field
gradient HSQC pulse sequence.
Analytical TLC was performed on silica gel 60 F254 (Merck)
precoated plates (0.2 mm). Spots were detected under UV light
(254 nm) and/or by charring with phosphomolybdic acid.
Separations on silica gel were performed by preparative
centrifugal circular thin-layer chromatography (CCTLC) on a
Chromatotron [Kiesegel 60 PF254 gipshaltig (Merck)], layer
thickness ) 1 or 2 mm, flow rate ) 4 or 8 mL/min, respectively.
Flash column chromatography was performed with silica gel
60 (230-400 mesh) (Merck).
1-[(Z)-4-(Tr iph en ylm eth oxy)-2-bu ten yl]th ym in e (10) was
prepared from alcohol 6 (165 mg, 0.50 mmol) according to the
general procedure: yield, 82%; mp 148-150 °C; MS (ES,
1
positive mode), m/z 461 (M + Na)+; H NMR (CDCl3) δ 1.91
(s, 3H, 5-CH3), 3.72 (d, J ) 6.9 Hz, 2H, CH2O), 4.18 (d, J )
7.1 Hz, 2H, CH2N), 5.56, 5.95 (m, 2H, CHdCH), 6.86 (s, 1H,
H-6), 7.23-7.46 (m, 15H, Ph), 7.94 (br s, 1H, 3-NH); 13C NMR
(CDCl3) δ 12.23 (5-CH3), 44.28 (CH2N), 59.55 (CH2O), 87.29
(CPh3), 110.86 (C-5), 125.86, 131.69 (CHdCH), 127.21, 127.96,
128.53, 143.69 (Ph), 139.53 (C-6), 150.53 (C-2), 164.00 (C-4).
Anal. for C28H26N2O3: C, H, N.
1-[4-(Tr ip h en ylm eth oxy)-2-bu tyn yl]th ym in e (11) was
prepared from alcohol 7 (179 mg, 0.50 mmol) according to the
general procedure: yield, 61%; mp 80-82 °C; MS (ES, positive
mode), m/z 459 (M + Na)+; 1H NMR (CDCl3) δ 1.92 (s, 3H,
5-CH3), 3.83 (t, J ) 1.8 Hz, 2H, CH2O), 4.49 (t, J ) 1.8 Hz,
2H, CH2N), 7.17 (s, 1H, H-6), 7.23-7.34 (m, 15H, Ph), 8.43
(br s, 1H, 3-NH); 13C NMR (CDCl3) δ 12.43 (5-CH3), 36.97
(CH2N), 52.98 (CH2O), 77.68, 83.80 (HCtCH), 87.70 (CPh3),
111.18 (C-5), 127.29, 127.97, 128.53, 143.21 (Ph), 138.55 (C-
6), 150.22 (C-2), 163.75 (C-4). Anal. for C28H24N2O3: C, H, N.
Polystyrene-triphenylphosphine (PS-Ph3P) was purchased
from Fluka. Diisopropyl azodicarboxylate (DIAD) was pur-
chased from Aldrich.
Triethylamine and acetonitrile were dried by refluxing over
calcium hydride. Tetrahydrofuran was dried by refluxing over
sodium/benzophenone. Anhydrous N,N′-dimethylformamide
was purchased from Aldrich.
The radiolabeled substrates [CH3-3H]dThd (70 Ci/mmol) and
[2,8-3H]ganciclovir (12.4 Ci/mmol) were obtained from Moravek
Biochemicals (Brea, CA).
Gen er a l P r oced u r e for Mon otr ityla tion of Diols. Syn -
th esis of 6, 7, a n d 9. To a stirred solution of the corresponding
diol (3-5) (10 mmol) in dry CH2Cl2 (4 mL) at 0 °C were added
Et3N (0.15 mL, 1.1 mmol), DMAP (5 mg, 0.04 mmol), and trityl
chloride (278 mg, 1.0 mmol). The mixture was stirred at room
temperature for 24 h. Then it was diluted with CH2Cl2 (20
mL) and water (10 mL). The organic phase was washed with
water (10 mL) and brine (10 mL). The organic layer was dried
on anhydrous Na2SO4, filtered, and evaporated. The residue
was purified by flash column chromatography or in the
Chromatotron as indicated.
1-[(E)-4-(Tr ip h en ylm et h oxy)-2-bu t en yl]t h ym in e (12)
was prepared from alcohol 8 (100 mg, 0.30 mmol) according
to the general procedure: yield, 85%; amorphous solid; MS (ES,
1
positive mode), m/z 461 (M + Na)+; H NMR (CDCl3) δ 1.90
(s, 3H, 5-CH3), 3.64 (t, J ) 2.8 Hz, 2H, CH2O), 4.31 (t, J ) 5.3
Hz, 2H, CH2N), 5.73-5.82 (m, 2H, CHdCH), 6.93 (s, 1H, H-6),
7.21-7.40 (m, 15H, Ph), 8.19 (br s, 1H, 3-NH); 13C NMR
(CDCl3) δ 12.32 (5-CH3), 48.97 (CH2N), 63.71 (CH2O), 87.14
(CPh3), 110.86 (C-5), 124.09, 132.54 (CHdCH), 127.13, 127.88,
128.59, 143.93 (Ph), 139.65 (C-6), 150.51 (C-2), 163.71 (C-4).
Anal. for C28H26N2O3: C, H, N.
(Z)-1-Hyd r oxy-4-(tr ip h en ylm eth oxy)-2-bu ten e (6) was
p repared from (Z)-2-butene-1,4-diol (3) and purified by CCTLC
in the Chromatotron (hexane/EtOAc, 1:1): yield, 80%; 1H NMR
was identical to that described.33
1-Hyd r oxy-4-(tr ip h en ylm eth oxy)-2-bu tyn e (7) was pre-
pared from 2-butyne-1,4-diol (4) and purified by CCTLC in the
Chromatotron (hexane/EtOAc, 3:1): yield, 50% of a white solid;
mp 74-76 °C; MS (ES, positive mode), m/z 451 (M + Na)+; 1H
NMR (CDCl3) δ 1.56 (t, J ) 5.7 Hz, 1H, OH), 3.83 (s, 2H, CH2-
OTr), 4.26 (d, J ) 5.7 Hz, 2H, CH2OH), 7.25-7.49 (m, 19H,
Ph); 13C NMR (CDCl3) δ 51.20, 53.05 (CH2OH, CH2OTr), 82.50,
83.57 (CtC), 87.46 (CPh3), 127.15, 127.92, 128.59, 134.38 (Ph).
(E)-1-Hyd r oxy-4-(tr ip h en ylm eth oxy)-2-bu ten e (8). A
suspension of LiAlH4 (48 mg, 1.2 mmol) in dry THF (1.2 mL)
was cooled to 0 °C in an ice bath. A solution of 7 (150 mg, 0.46
mmol) in dry THF (7.6 mL) was added, and the resulting
suspension was stirred at 4 °C for 2 h and at room temperature
1-[2-((Tr ip h en ylm eth oxy)m eth yl)ben zyl]th ym in e (13)
was prepared from alcohol 9 (190 mg, 0.50 mmol) according
to the general procedure: yield, 67%; mp 103-105 °C; MS (ES,
1
positive mode), m/z 511 (M + Na)+; H NMR (CDCl3) δ 1.73
(s, 3H, 5-CH3), 4.14 (s, 2H, CH2O), 4.71 (s, 2H, CH2N), 6.76 (s,
1H, H-6), 7.13-7.50 (m, 19H, Ph), 8.67 (br s, 1H, 3-NH); 13C
NMR (CDCl3) δ 12.25 (5-CH3), 47.16 (CH2N), 63.82 (CH2O),
87.60 (CPh3), 110.89 (C-5), 127.27, 128.00, 128.42, 128.54,
129.46, 133.96, 136.58, 143.57 (Ph), 139.69 (C-6), 151.09 (C-
2), 163.97 (C-4). Anal. for C32H28N2O3: C, H, N.

4260 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Herna´ndez et al.
(RS)-1-[2-(2,2-Dim et h yl-[1,3]d ioxola n -4-yl)et h yl]t h y-
m in e (16) was prepared from alcohol 1522 (100 mg, 0.68 mmol)
according to the general procedure: yield, 67%; mp 108-110
°C; MS (ES, positive mode), m/z 255 (M + 1)+, 277 (M + Na)+;
1H NMR (CDCl3) δ 1.31, 1.38 [2s, 6H, (CH3)2C], 1.89 (s, 3H,
5-CH3), 1.81, 1.99 (m, 2H, CH2), 3.53, 4.07 (m, 2H, CH2O), 3.71,
3.93 (m, 2H, CH2N), 4.05 (m, 1H, CH), 7.05 (s, 1H, H-6), 9.53
(br s, 1H, 3-NH); 13C NMR (CDCl3) δ 12.23 (5-CH3), 25.47,
26.92 [(CH3)2C], 32.42 (CH2), 45.92 (CH2N), 69.03 (CH2O),
72.65 (CH), 109.23 [(CH3)2C], 110.42 (C-5), 140.94 (C-6), 150.98
(C-2), 164.49 (C-4). Anal. for C12H18N2O4: C, H, N.
(R S )-1-[3-H yd r oxy-4-(t r ip h e n ylm e t h oxy)b u t yl]t h y-
m in e (18). A solution of 16 (100 mg, 0.40 mmol) in 80% acetic
acid (6 mL) was heated at 80 °C for 1 h. Volatiles were
removed, and the residue was coevaporated with EtOH (4 ×
10 mL) and pyridine (2 × 5 mL). The residue, containing (RS)-
1-(3,4-dihydroxybutyl)thymine23 (17) was dissolved in pyridine
(3 mL), and DMAP (catalytic amount) and trityl chloride (181
mg, 0.64 mmol) were added. The mixture was heated at 80 °C
for 24 h. Volatiles were removed, amd the residue was treated
with CH2Cl2 (20 mL) and water (10 mL). The aqueous phase
was further extracted with CH2Cl2 (2 × 20 mL). The combined
organic extracts were dried on anhydrous Na2SO4, filtered, and
evaporated. The residue was purified in a silica SPE cartridge
eluting with CH2Cl2/MeOH (200:1); 73 mg (40%) of 18 were
obtained: mp 183-185 °C; MS (ES, positive mode), m/z 479
(M + Na)+; 1H NMR (CDCl3) δ 1.31 (m, 2H, CH2), 1.89 (s, 3H,
5-CH3), 3.12 (m, 2H, CH2O), 3.63 (m, 1H, CHOH), 3.80 (m,
2H, CH2N), 7.00 (s, 1H, H-6), 7.17-7.42 (m, 15H, Ph), 8.29
(br s, 1H, 3-NH). Anal. for C28H28N2O4: C, H, N.
(2S,3S)- a n d (2R,3R)-1-[2,3-Dih yd r oxy-4-(t r ip h en yl-
m eth oxy)bu tyl]th ym in e (19). To a solution of 10 (150 mg,
0.34 mmol) in acetone/H2O (90:10) (9 mL) were added N-
methylmorpholine-N-oxide (0.071 mL of a 60% aqueous solu-
tion, 0.41 mmol) and OsO4 (0.085 mL of a 2.5% solution in
tert-butyl alcohol, 0.007 mmol). The mixture was stirred at
room temperature for 3 days. A small portion of Na2S2O5 was
added, and then the mixture was filtered through Celite. The
filtrate was evaporated and purified by CCTLC in the Chro-
matotron (CH2Cl2/MeOH, 15:1) to yield 103 mg (67%) of 19:
mp 189-191 °C; MS (ES, positive mode), m/z 495 (M + Na)+;
1H NMR (CDCl3) δ 1.93 (s, 3H, 5-CH3), 3.37, 3.50 (m, 2H, OH),
3.46 (m, 2H, CH2O), 3.64, 3.82 (m, 2H, CHOH), 3.76, 4.05 (dd,
J ) 3.3, 14.3 Hz, 2H, CH2N), 7.28 (s, 1H, H-6), 7.31-7.42 (m,
15H, Ph), 8.66 (br s, 1H, 3-NH); 13C NMR (CDCl3) δ 12.22 (5-
CH3), 50.72 (CH2N), 66.90 (CH2O), 68.91, 73.52, (CHOH), 87.84
(CPh3), 110.82 (C-5), 127.39, 128.07, 128.54, 142.41 (Ph),
143.35 (C-6), 152.58 (C-2), 163.52 (C-4). Anal. for C28H28N2O5‚
2H2O: C, H, N.
1-[4-(Tr ip h en ylm eth oxy)bu tyl]th ym in e (20). A solution
of 10 (150 mg, 0.34 mmol) in EtOH (20 mL) was hydrogenated
at room temperature in the presence of 10% Pd/C (40 mg) at
30 psi for 2 h. The mixture was filtered and the filtrate purified
by CCTLC in the Chromatotron (hexane/EtOAc 1:1) to give
70 mg (47%) of 20: mp 163-166 °C; MS (ES, positive mode),
m/z 463 (M + Na)+; 1H NMR (CDCl3) δ 1.63, 1.77 (m, 4H, CH2),
1.90 (s, 3H, 5-CH3), 3.12 (t, J ) 6.2 Hz, 2H, CH2O), 3.66 (t, J
) 6.1 Hz, 2H, CH2N), 6.89 (s, 1H, H-6), 7.20-7.45 (m, 15H,
Ph), 9.29 (br s, 1H, 3-NH); 13C NMR (CDCl3) δ 12.24 (5-CH3),
25.99, 26.79 (CH2), 48.28 (CH2N), 62.71 (CH2O), 86.52 (CPh3),
110.49 (C-5), 126.91, 127.73, 128.56, 140.31 (Ph), 144.12 (C-
6), 150.89 (C-2), 164.31 (C-4). Anal. for C28H28N2O3: C, H, N.
CH), 127.18, 127.93, 128.51, 143.66 (Ph), 138.79 (C-6), 150.72
(C-2), 163.67 (C-4). Anal. for C29H28N2O3: C, H, N.
5-Iodo-1-[(Z)-4-(tr iph en ylm eth oxy)-2-bu ten yl]u r acil (22).
Following a procedure analogous to that described for the
Mitsunobu condensation of alcohols with N3-BzT, alcohol 6
(330 mg, 1.0 mmol) reacted with N3-benzoyl 5-iodouracil (684
mg, 2.0 mmol) to afford 22 (136 mg, 30%): mp 150-155 °C;
MS (ES, positive mode), m/z 573 (M + Na)+; 1H NMR (CDCl3)
δ 3.74 (d, J ) 6.4 Hz, 2H, CH2O), 4.23 (d, J ) 7.0 Hz, 2H,
CH2N), 5.50, 6.00 (m, 2H, CHdCH), 7.26-7.44 (m, 16H, Ph,
H-6), 8.20 (br s, 1H, 3-NH); 13C NMR (CDCl3) δ 45.68 (CH2N),
48.86 (C-5), 58.48 (CH2O), 82.01 (CPh3), 124.03, 128.83 (CHd
CH), 127.25, 127.92, 128.52, 142.28 (Ph), 146.82 (C-6), 148.51
(C-2), 159.97 (C-4). Anal. for C27H23IN2O3: C, H, N.
2-Am in o-6-ch lor o-9-[(Z)-4-(t r ip h en ylm et h oxy)-2-b u t -
en yl]p u r in e (23). To a suspension containing alcohol 10 (150
mg, 0.34 mmol), PS-Ph3P (3 mmol/g, 283 mg, 0.85 mmol), and
2-amino-6-chloropurine (115 mg, 0.68 mmol) in dry THF (5
mL) was slowly added a solution of DIAD (0.13 mL, 0.68 mmol)
in THF (2 mL). The mixture was stirred at room temperature
overnight. The reaction was filtered, the residue was washed
with THF (2 × 5 mL), and the combined filtrates were
evaporated to dryness. The residue was purified by CCTLC
in the Chromatotron (hexane/EtOAc, 1:1) to yield 143 mg (87%)
of 23: mp 187-190 °C; MS (ES, positive mode), m/z 482 (M)+;
1H NMR (CDCl3) δ 3.81 (d, J ) 7.4 Hz, 2H, CH2O), 4.53 (d, J
) 5.9 Hz, 2H, CH2N), 4.91 (br s, 2H, NH2), 5.68, 5.94 (m, 2H,
CHdCH), 7.26-7.43 (m, 15H, Ph), 7.62 (s, 1H, H-8); 13C NMR
(CDCl3) δ 48.50 (CH2N), 60.02 (CH2O), 87.22 (CPh3), 124.46,
132.32 (CHdCH), 125.05 (C-5), 127.15, 127.90, 128.51, 143.67
(Ph), 141.83 (br s, C-8), 151.10 (C-4), 153.48 (C-2), 158.97 (C-
6). Anal. for C28H24ClN5O: C, H, N.
9-[(Z)-4-(Tr ip h en ylm eth oxy)-2-bu ten yl]gu a n in e (24).
To a solution of 23 (150 mg, 0.31 mmol) in 1,4-dioxane (23
mL) was added 1 N sodium hydroxide (14.3 mL). The mixture
was stirred at 95 °C for 27 h, neutralized with acetic acid, and
concentrated to dryness. The residue was purified by flash
column chromatography (CH2Cl2/MeOH, 10:1) to yield 117 mg
(81%) of 24 as a white solid: mp 252-255 °C; MS (ES, positive
1
mode), m/z 464 (M)+; H NMR (DMSO-d6) δ 3.69 (d, J ) 6.0
Hz, 2H, CH2O), 4.40 (d, J ) 6.0 Hz, 2H, CH2N), 5.58-5.77 (m,
2H, CHdCH), 6.33 (br s, 2H, NH2), 7.23-7.39 (m, 15H, Ph),
7.48 (s, 1H, H-8), 10.51 (br s, 1H, NH); 13C NMR (DMSO-d6) δ
39.50 (CH2N), 59.83 (CH2O), 86.51 (CPh3), 116.49 (C-5), 126.30,
130.29 (CHdCH), 127.11, 127.99, 128.20, 143.70 (Ph), 150.91
(C-4), 153.48 (C-2), 156.77 (C-6). Anal. for C28H25N5O2: C, H,
N.
5-Me t h yl-1-[(Z)-4-(t r ip h e n ylm e t h oxy)-2-b u t e n yl]cy-
tosin e (25). A suspension of 1,2,4-triazole (189 mg, 2.74 mmol)
and POCl3 (0.15 mL, 1.80 mmol) in dry CH3CN (2 mL) was
stirred at 0 °C for 5 min. Then Et3N (0.78 mL, 5.60 mmol)
was slowly added. The resulting mixture was stirred at 0 °C
for 1 h, and then a solution of 10 (150 mg, 0.34 mmol) in dry
CH3CN (1 mL) was added. The mixture was stirred at room
temperature for 18 h and then filtered. The filtrate was diluted
with EtOAc (20 mL) and washed with aqueuos NaHCO3 (10
mL). The aqueous phase was further extracted with EtOAc (2
× 15 mL). The combined organic extracts were dried on
anhydrous Na2SO4, filtered, evaporated, and coevaporated
with dioxane. The residual oil was dissolved in dioxane (2 mL)
and treated with NH4OH (2 mL) for 1 h. Volatiles were
removed, and the residue was purified by flash column
chromatography (hexane/EtOAc, 2:1). The fractions containing
25 were further purified by CCTLC in the Chromatotron
(EtOAc/MeOH, 10:1) to yield 32 mg (22%) of 25: mp 167-171
°C; MS (ES, positive mode), m/z 460 (M + Na)+; 1H NMR
(CDCl3) δ 1.80 (s, 3H, 5-CH3), 3.71 (d, J ) 6.6 Hz, 2H, CH2O),
4.24 (d, J ) 7.1 Hz, 2H, CH2N), 5.60, 5.90 (m, 2H, CHdCH),
6.92 (s, 1H, H-6), 7.17-7.46 (m, 15H, Ph), 8.13 (br s, 2H, NH2);
13C NMR (CDCl3) δ 12.93 (5-CH3), 45.48 (CH2N), 59.58 (CH2O),
87.14 (CPh3), 101.63 (C-5), 127.09, 130.69 (CHdCH), 127.13,
127.92, 128.52, 142.18 (Ph), 143.76 (C-6), 156.64 (C-2), 165.47
(C-4). Anal. for C28H27N3O2: C, H, N.
5-E t h yl-1-[(Z)-4-(t r ip h en ylm et h oxy)-2-b u t en yl]u r a cil
(21). Following a procedure analogous to that described for
the Mitsunobu condensation of alcohols with N3-BzT, alcohol
6 (165 mg, 0.5 mmol) reacted with N3-benzoyl-5-ethyluracil
(244 mg, 1.0 mmol) to afford 21 (226 mg, 50%): mp 118-120
°C; MS (ES, positive mode), m/z 475 (M + Na)+; 1H NMR
(CDCl3) δ 1.05 (t, J ) 7.5 Hz, 3H, 5-CH2CH3), 2.26 (q, J ) 7.5
Hz, 2H, 5-CH2CH3), 3.74 (d, J ) 6.4 Hz, 2H, CH2O), 4.21 (d, J
) 7.1 Hz, 2H, CH2N), 5.55, 5.95 (m, 2H, CHdCH), 6.80 (s, 1H,
H-6), 7.21-7.47 (m, 15H, Ph), 8.94 (br s, 1H, 3-NH); 13C NMR
(CDCl3) δ 12.99 (5-CH2CH3), 20.00 (5-CH2CH3), 44.36 (CH2N),
59.64 (CH2O), 87.24 (CPh3), 116.74 (C-5), 125.83, 131.65 (CHd
1-[(Z)-4-(Tr ip h en ylm et h oxy)-2-b u t en yl]-5,6-d ih yd r o-

Acyclic Nucleoside Inhibitors of Mitochondril Thymidine Kinase J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4261
th ym in e (26). To a solution of 10 (150 mg, 0.34 mmol) in dry
THF (2 mL) at 0 °C and under Ar atmosphere was added a
1.2 M solution of DibalH in hexane (0.7 mL, 3.40 mmol). The
mixture was allowed to reach room temperature and stirred
overnight. The reaction was quenched by the addition of MeOH
(2 mL) and evaporated. The residue was extracted several
times with hot MeOH and filtered. The filtrate was evaporated
and then purified by CCTLC in the Chromatotron (hexane/
EtOAc, 1:1) to yield 81 mg (54%) of 26 as an amorphous solid:
MS (ES, positive mode), m/z 463 (M + Na)+; 1H NMR (CDCl3)
δ 1.58 (d, J ) 7.0 Hz, 3H, 5-CH3), 2.57 (m, 1H, H-5), 2.94 (pt,
1H, H-6), 3.17 (dd, J ) 12.4, 6.2 Hz, 1H, H-6), 3.67 (d, J ) 6.4
Hz, 2H, CH2O), 3.87 (d, J ) 5.7 Hz, 2H, CH2N), 5.46, 5.94 (m,
2H, CHdCH), 7.18-7.45 (m, 15H, Ph), 8.34 (br s, 1H, 3-NH);
13C NMR (CDCl3) δ 12.67 (5-CH3), 35.35 (C-5), 43.47 (CH2N),
47.74 (C-6), 59.51 (CH2O), 87.04 (CPh3), 126.31, 131.26 (CHd
CH), 127.14, 127.89, 128.56, 143.82 (Ph), 152.35 (C-2), 172.42
(C-4). Anal. for C28H28N2O3: C, H, N.
3-Met h yl-1-[(Z)-4-(t r ip h en ylm et h oxy)-2-b u t en yl]t h y-
m in e (27). A suspension containing compound 10 (150 mg,
0.34 mmol) and K2CO3 (24 mg, 0.17 mmol) in acetone (3 mL)
was stirred at room temperature for 10 min. Then CH3I (0.032
mL, 0.51 mmol) was added, and the mixture was stirred at
room temperature for 48 h. Then it was diluted with CH2Cl2
(20 mL) and aqueous NaHCO3 (10 mL). The aqueous phase
was further extracted with CH2Cl2 (2 × 10 mL). The combined
organic extracts were dried on anhydrous Na2SO4, filtered, and
evaporated. The residue was purified by CCTLC in the
Chromatotron (hexane/EtOAc, 1:1) to yield 143 mg (93%) of
27: mp 133-135 °C; MS (ES, positive mode), m/z 475 (M +
Na)+; 1H NMR (CDCl3) δ 1.84 (s, 3H, 5-CH3), 3.32 (s, 3H,
3-NCH3), 3.75 (d, J ) 5.3 Hz, 2H, CH2O), 4.22 (d, J ) 5.9 Hz,
2H, CH2N), 5.55, 5.92 (m, 2H, CHdCH), 6.84 (s, 1H, H-6),
7.20-7.34 (m, 15H, Ph); 13C NMR (CDCl3) δ 12.99 (5-CH3),
27.98 (3-NCH3), 45.42 (CH2N), 59.69 (CH2O), 87.32 (CPh3),
109.89 (C-5), 126.11, 131.51 (CHdCH), 127.20, 127.96, 128.59,
143.78 (Ph), 137.39 (C-6), 151.61 (C-2), 163.88 (C-4). Anal. for
hexanoyl chloride: yield, 65%; mp 93-95 °C; MS (ES, positive
1
mode), m/z 295 (M + 1)+, 317 (M + Na)+; H NMR (CDCl3) δ
0.86 (t, J ) 6.6 Hz, 3H, CH3), 1.22-1.39 (m, 4H, CH2), 1.61 (q,
4H, CH2), 1.88 (s, 3H, 5-CH3), 2.30 (t, J ) 6.3 Hz, 2H, CH2-
CO), 4.44 (d, J ) 6.6 Hz, 2H, CH2N), 4.68 (d, J ) 6.8 Hz, 2H,
CH2O), 5.60, 5.76 (m, 2H, CHdCH), 7.09 (s, 1H, H-6), 9.08 (br
s, 1H, 3-NH); 13C NMR (CDCl3) δ 12.28, 13.85 (5-CH3, CH3),
22.26, 24.55, 31.22, 34.12 (CH2), 44.26 (CH2N), 59.16 (CH2O),
111.09 (C-5), 127.97, 128.60 (CHdCH), 139.87 (C-6), 150.89
(C-2), 164.15 (C-4), 173.67 (CO). Anal. for C15H22N2O4: C, H,
N.
4-(Th ym in -1-yl)bu t-2-en yl la u r ea te (31) was prepared
according to the general procedure by reaction of 10 with
lauroyl chloride and purified by CCTLC in the Chromatotron
(hexane/EtOAc, 1:1): yield, 49%; mp 62-65 °C; MS (ES,
positive mode), m/z 379 (M + 1)+, 401 (M + Na)+; 1H NMR
(CDCl3) δ 0.85 (t, 3H, J ) 6.8 Hz, CH3), 1.22-1.37 (m, 16H,
CH2), 1.59 (q, J ) 7.1 Hz, 2H, CH2CH2CO), 1.88 (s, 3H, 5-CH3),
2.30 (t, J ) 7.3 Hz, 2H, CH2CO), 4.45 (d, J ) 6.8 Hz, 2H,
CH2N), 4.68 (d, J ) 6.6 Hz, 2H, CH2O), 5.58-5.79 (m, 2H, CHd
CH), 7.10 (s, 1H, H-6), 8.87 (br s, 1H, 3-NH). Anal. for
C
21H34N2O4: C, H, N.
4-(Th ym in -1-yl)bu t-2-en yl-diph en ylacetate (32) was pre-
pared according to the general procedure by reaction of 10 with
diphenylacetyl chloride: yield, 81%; mp 105-110 °C; MS (ES,
positive mode), m/z 391 (M + 1)+, 413 (M + Na)+; 1H NMR
(CDCl3) δ 1.85 (s, 3H, 5-CH3), 4.43 (d, J ) 6.6 Hz, 2H, CH2N),
4.80 (d, J ) 6.8 Hz, 2H, CH2O), 5.07 [s, 1H, CH(Ph)2], 5.60-
5.75 (m, 2H, CHdCH), 7.05 (s, 1H, H-6), 7.28-7.40 (m, 10H,
Ph), 9.32 (br s, 1H, 3-NH); 13C NMR (CDCl3) δ 12.20 (5-CH3),
44.28 (CH2N), 56.89 (CPh2), 60.06 (CH2O), 111.02 (C-5), 127.46,
127.83 (CHdCH), 128.50, 128.59, 128.69, 138.26 (Ph), 139.98
(C-6), 150.70 (C-2), 164.02 (C-4), 172.49 (CO). Anal. for
C
23H22N2O4: C, H, N.
1-[(Z)-4-Hyd r oxy-2-bu ten yl]th ym in e (33). A solution of
10 (150 mg, 0.34 mmol) in MeOH (12 mL) was treated with a
Dowex resin 50WX4 (H+ form) (34 mmol) at room temperature
overnight. The mixture was diluted by the addition of MeOH
(50 mL) and filtered. The filtrate was evaporated and purified
in the Chromatotron CCTLC (CH2Cl2/MeOH, 15:1) to yield 65
mg (98% yield) of 33 as a white solid: mp 166-169 °C (lit.17
mp 163-166 °C); Anal. for C9H12N2O3: C, H, N.
4-(Th ym in -1-yl)bu t-2-en yl ben zoa te (34). To a solution
of 33 (50 mg, 0.25 mmol) in dry CH2Cl2 (3 mL) and pyridine
(0.5 mL) was added benzoyl chloride (0.035 mL, 0.30 mmol).
The mixture was stirred at room temperature for 6 h. Then it
was treated with CH2Cl2 (20 mL) and 1 N HCl solution (10
mL). The organic phase was washed with H2O, dried on
anhydrous Na2SO4, filtered, and evaporated. The residue was
purified in an SPE cartridge (silica) eluting with CH2Cl2/EtOAc
to yield 40 mg (53%) of 34 as a white solid: MS (ES, positive
mode), m/z 287 (M + 1)+, 309 (M + Na)+; 1H NMR was
identical to that reported.29 Anal. for C16H16N2O4: C, H, N.
C
29H28N2O3: C, H, N.
Gen er a l P r oced u r e for th e Ester ifica tion of th e Tr ityl
Eth er 10. Syn th esis of 28)32. To a solution of 10 (150 mg,
0.34 mmol) in CH2Cl2 (14 mL) at 0 °C was added a Dowex
resin 50WX4 (H+ form) (34 mmol) and the corresponding acyl
chloride (17 mmol). The mixture was allowed to reach room
temperature and stirred for 24 h. The reaction mixture was
diluted with CH2Cl2 (50 mL) and filtered. The filtrate was
washed with a solution of aqueous NaHCO3 (15 mL). The
organic phase was dried on anhydrous Na2SO4, filtered, and
evaporated. The residue was purified by CCTLC in the
Chromatotron (hexane/EtOAc, 1:1).
4-(Th ym in -1-yl)bu t-2-en yl-2,2-dim eth yl pr opion ate (28)
was prepared according to the general procedure by reaction
of 10 with pivaloyl chloride: yield, 66%; mp 168-171 °C; MS
1
(ES, positive mode), m/z 303 (M + Na)+; H NMR (CDCl3) δ
1.19 [s, 9H, (CH3)3C)], 1.89 (s, 3H, 5-CH3), 4.46 (d, J ) 6.6 Hz,
2H, CH2N), 4.67 (d, J ) 6.6 Hz, 2H, CH2O), 5.64, 5.72 (m, 2H,
CHdCH), 7.14 (s, 1H, H-6), 8.60 (br s, 1H, 3-NH); 13C NMR
(CDCl3) δ 12.28 (5-CH3), 27.09 [(CH3)3C)], 38.71 [(CH3)3C)],
44.18 (CH2N), 59.26 (CH2O), 111.07 (C-5), 128.01, 128.61
(CHdCH), 139.96 (C-6), 150.08 (C-2), 164.20 (C-4), 178.45
(CO). Anal. for C14H20N2O4: C, H, N.
4-(Th ym in -1-yl)bu t-2-en yl-3,3-d im eth yl bu tyr a te (29)
was prepared according to the general procedure by reaction
of 10 with tert-butylacetyl chloride: yield, 56%; mp 128-131
°C; MS (ES, positive mode), m/z 295 (M + 1)+, 317 (M + Na)+;
1H NMR (CDCl3) δ 1.04 [s, 9H, (CH3)3C], 1.90 (s, 3H, 5-CH3),
2.23 (s, 2H, CH2CO), 4.50 (d, J ) 6.8 Hz, 2H, CH2N), 4.70 (d,
J ) 7.0 Hz, 2H, CH2O), 5.64, 5.79 (m, 2H, CHdCH), 7.17 (s,
1H, H-6), 8.94 (br s, 1H, 3-NH); 13C NMR (CDCl3) δ 12.32 (5-
CH3), 29.69 [(CH3)3C], 30.94 [(CH3)3C], 44.29 (CH2CO), 47.84
(CH2N), 58.94 (CH2O), 111.21 (C-5), 128.16, 128.65 (CHdCH),
140.06 (C-6), 150.92 (C-2), 164.11 (C-4), 172.26 (CO). Anal. for
1-[(Z)-4-(Ben zyloxy)-2-bu ten yl]th ym in e (36). Following
a procedure analogous to that described for the Mitsunobu
condensation of alcohols with N3-BzT, alcohol 3530 (100 mg,
0.56 mmol) reacted with N3-BzT (258 mg, 1.1 mmol) to afford
36 (94 mg, 59%) as a syrup: 1H NMR (CDCl3) δ 1.80 (s, 3H,
5-CH3), 3.14 (d, J ) 5.5 Hz, 2H, CH2O), 4.37 (d, J ) 7.1 Hz,
2H, CH2N), 4.53 (s, 2H, CH2Ph), 5.59, 5.80 (m, 2H, CHdCH),
6.96 (s, 1H, H-6), 7.30 (m, 5H, Ph), 9.29 (br s, 1H, 3-NH); 13C
NMR (CDCl3) δ 12.18 (5-CH3), 44.48 (CH2N), 65.61 (CH2O),
72.88 (CH2Ph), 110.89 (C-5), 126.57, 131.26 (CHdCH), 127.86,
127.92, 128.50, 137.67 (Ph), 139.70 (C-6), 150.91 (C-2), 164.17
(C-4). Anal. for C16H18N2O3: C, H, N.
Biologica l P r oced u r es. A. TK Assa y Usin g [m eth yl-3H]-
d Th d a s th e Su bstr a te. The preparation and purification of
recombinant HSV-1 TK, HSV-1 (A167Y) TK, and TK-2 has
been described previously.6,10,12,31,32 The activity of purified
recombinant HSV-1 TK and mitochondrial TK-2 was assayed
in a 50-µL reaction mixture containing 50 mM Tris-HCl (pH
8.0), 2.5 mM MgCl2, 10 mM dithiothreitol, 0.5 mM CHAPS, 3
mg/mL BSA, 2.5 mM ATP, 2 µM [methyl-3H]dTd or 1.6 µM
[2,8-3H]GCV (1 µCi), and enzyme (CHAPS was replaced by 10
C
15H22N2O4: C, H, N.
4-(Th ym in -1-yl)bu t-2-en yl h exa n oa te (30) was prepared
according to the general procedure by reaction of 10 with

4262 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Herna´ndez et al.
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Karlsson, A. Cloning and characterization of the multisubstrate
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mM NaF in the reaction mixtures for HSV-1 TK). The samples
were incubated at 37 °C for 30 min in the presence or absence
of different concentrations of the test compounds. Due to the
poor solubility of the compounds in water, stock solutions were
made in pure DMSO. Appropriate dilutions of the test com-
pounds were performed in 100% DMSO. When added to the
enzyme reaction mixture, constant volumes of each of the
compound dilutions were used to achieve a DMSO concentra-
tion of 10% in all samples, including the enzyme controls. At
this DMSO concentration the enzyme catalytic activity was
not significantly affected. Precipitation of the (lipophilic) test
compounds is avoided for most of the compounds at 50 µM in
the presence of 10% DMSO. Therefore, compound concentra-
tions >50 µM may not be accurate due to insolubility, even in
the presence of 10% DMSO. Aliquots of 45 µL of the reaction
mixtures were spotted on Whatman DE-81 filter paper disks.
The filters were washed three times for 5 min each in 1 mM
ammonium formate, once for 1 min in water, and once for 5
min in ethanol. The radioactivity was determined by scintil-
lation counting. The IC50 was defined as the drug concentration
required to inhibit the enzyme activity by 50%. The tested
compound dilutions were 5-fold. The IC50 values were derived
from an extrapolation of the data obtained in the presence of
an immediately higher and immediately lower concentration
than the IC50 value.
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The following formula for the calculation of IC50 was used:
50% - % cell growth at A
% cell growth at B - % cell growth at A
IC50 ) A -
×
[A - B] (1)
In eq 1 A is the higher drug concentration and B is the lower
drug concentration
Effect of d Th d a n d HSV-1 TK In h ibitor s on th e Cyto-
sta tic Activity of GCV a n d BVa r a U in Hu m a n Osteosa -
r com a Cells Tr a n sd u ced w ith th e HSV-1 TK Gen e. The
cytostatic activity of GCV and BVaraU against HSV-1 TK
gene-transduced human osteosarcoma cells was evaluated as
follows: 104 OST TK-/HSV-1 TK+ cells/well were plated in 96-
well microtiter plates and subsequently incubated in the
presence of 5-fold dilutions of the test compounds GCV and
BVaraU in the absence or presence of 20 µM of compounds 10
or 12 (in the presence of 1% DMSO, which is not toxic to the
cell cultures) or 50 µM dThd (in the absence of DMSO). After
3 days, the number of cells was counted in a Coulter counter
(Coulter Electronics Ltd., Harpenden, U.K.). The IC50 was
defined as the drug concentration required to inhibit tumor
cell proliferation by 50%.
Ack n ow led gm en t. We thank Lizette van Berck-
elaer for excellent technical assistance. We also thank
FAES, S.A., for an award to A.I.H. This research was
supported by grants from the European Commission
(Project QLRT-2001-01004) and from the Spanish CICYT
(Project SAF 2000-0153-C02-01).
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J M011128+