Diverse combinatorial design, synthesis and in vitro evaluation of new HEPT analogues as potential non-nucleoside HIV-1 reverse transcription inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.04.038

New analogues of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) were synthesized and evaluated for their in vitro activities against HIV-1 in MT-4 cell cultures. Chemical diversity was introduced in 4 of the six positions of the core and the influence of each substituent was studied. This library was built on the basis of a rational diversity analysis with the objective of maximizing diversity and thus, the activity range with a minimum number of synthesized compounds. Among them, 2{1,2,3,1} and 2{1,2,3,4} exhibited the most potent anti-HIV-1 activities (EC₅₀ = 0.015 μg/mL; 0.046 μM, SI >1667) and (EC₅₀ = 0.025 μg/mL; 0.086 μM, SI >1000), respectively, which were about 71-fold and 38-fold more active than the reference compound HEPT (EC₅₀ = 1.01 μg/mL; 3.27 μM, SI >25).

Full text of DOI:10.1016/j.ejmech.2012.04.038

European Journal of Medicinal Chemistry 54 (2012) 159e174
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Diverse combinatorial design, synthesis and in vitro evaluation of new HEPT
analogues as potential non-nucleoside HIV-1 reverse transcription inhibitors
Raimon Puig-de-la-Bellacasa a, Laura Giménez a, Sofia Pettersson a, Rosalia Pascual a, Encarna Gonzalo b,
,
José A. Esté b, Bonaventura Clotet b, José I. Borrell a, Jordi Teixidó a
*
a Grup d’Enginyeria Molecular, Institut Químic de Sarrià, Universitat Ramon Llull, Via Augusta 390, Barcelona E-08017, Spain
b IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona E-08916, Spain
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
< A library of HEPT analogues based on a rational diversity analysis is synthesized.
< The synthesized compounds are evaluated against HIV-1.
< The compounds showed a huge diversity of inhibitory activities against HIV-1.
< Some compounds showed high activity and selectivity index values.
< Diverse combionatorial sub-library design minimizes synthesis and maximizes activity.
a r t i c l e i n f o
a b s t r a c t
New analogues of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) were synthesized and
evaluated for their in vitro activities against HIV-1 in MT-4 cell cultures. Chemical diversity was intro-
duced in 4 of the six positions of the core and the influence of each substituent was studied. This library
was built on the basis of a rational diversity analysis with the objective of maximizing diversity and thus,
the activity range with a minimum number of synthesized compounds. Among them, 2{1,2,3,1} and 2
Article history:
Received 1 December 2011
Received in revised form
26 April 2012
Accepted 26 April 2012
Available online 4 May 2012
{1,2,3,4} exhibited the most potent anti-HIV-1 activities (EC50 ¼ 0.015
mg/mL; 0.046 mM, SI >1667) and
Abbreviations: HEPT, 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine; CC, combinatorial chemistry; HIV, human immunodeficiency virus; HIV-1, human immu-
nodeficiency virus type-1; NNRTI, non-nucleside reverse transcriptase inhibitor; PCA, principal component analysis; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; SEM, 2-trimethylsilylethoxymethyl; BTAC, benzyltriethylammonium chloride; BSA, N,O-bis(trimethylsilyl)acetamide; TBAI, tetrabutylammo-
nium iodide; g-PCC, picolinium chlorochromate; LR, Lawesson’s reagent; NVP, nevirapine; AZT, azidothymidine; EFV, efavirenz.
* Corresponding author. Tel.: þ34 932672000; fax: þ34 932056265.
E-mail addresses: raimonpc@gmail.com (R. Puig-de-la-Bellacasa), laura.gimenez@ono.com (L. Giménez), ps.sofia@gmail.com (S. Pettersson), rpascual@esteve.es
(R. Pascual), egonzalo@irsicaixa.es (E. Gonzalo), jaeste@irsicaixa.es (J.A. Esté), bclotet@irsicaixa.es (B. Clotet), j.i.borrell@iqs.url.edu (J.I. Borrell), jordi.teixido@iqs.url.edu
(J. Teixidó).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.038

160
R. Puig-de-la-Bellacasa et al. / European Journal of Medicinal Chemistry 54 (2012) 159e174
(EC50 ¼ 0.025
m
g/mL; 0.086
m
M, SI >1000), respectively, which were about 71-fold and 38-fold more
g/mL; 3.27 M, SI >25).
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
active than the reference compound HEPT (EC50 ¼ 1.01
m
m
Combinatorial design
Diversity selection HIV-1
NNRTIs HEPT
Antiviral activity
1. Introduction
analogues and their diversity space coverage assessed in terms of
broad biological activity.
Combinatorial Chemistry (CC) has been one of the major
advances introduced in medicinal chemistry during the two past
decades. The concept of diversity drove the construction of large
combinatorial libraries around almost any possible scaffold. The use
of parallel synthesis allowed the synthesis of massive collections of
compounds, even as mixtures, that were tested for relevant bio-
logical activities. However, the results obtained were worse than
expected with a large number of false positives. Consequently, the
use of computational selection methodologies prior to synthesis
has been included as a mandatory step in CC [1,2]. In this context,
our group developed Pralins (Program for Rational Analysis of
Libraries In Silico) [3], a program aimed to design smaller libraries
but covering the same chemical space than the complete ones. The
present paper deals with its application in the field of HEPT
analogues.
1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT)
(1, Fig.1) was discovered in 1989 by Tanaka et al. [4,5] being the first
non-nucleoside reverse transcriptase inhibitor (NNRTI) ever
described that was specifically targeted at the HIV-1 Reverse
Transcriptase (RT) (the enzyme responsible for catalyzing the
synthesis of double strain viral DNA and therefore for the replica-
tion of the virus) at an allosteric binding site [6]. Due to the high
rate of virus mutation, it is important to research new molecules
with improved activity and minor vulnerability to resistance [7]. A
large collection of HEPT analogues 2 has been reported [5,8e13]
where new substituents were chosen by medicinal chemistry
intuition and synthetic accessibility.
In a previous work, we explored a large combinatorial virtual
library of 125,396 HEPT analogues 2 built, following a Markush
formula [14], by combining all fragments present in the published
180-compound HEPT family. There we analyzed that randomly
picking 180 compounds from the library let to better coverage than
the 180 synthesized compounds and that a careful selection of only
24 compounds should represent the 90% of the population. We
applied Pralins to select combinatorial sub-libraries with four
diversity centers (substitution at C-2 and C-5, the side chain linked
to position N1 and the thiophenol ring linked to C6) showing that
optimal combinatorial sub-libraries selection ranging from 24 up to
2 ꢀ 7 ꢀ 5 ꢀ 8 ¼ 560 compounds should span from the 90% to the
entirely chemical space [15].
2. Results and discussion
2.1. Design and selection of the combinatorial sub-library
In order to select the sub-library to be synthesized we used our
standard protocol which include the following steps.
First, we construct the complete combinatorial library by using
all the relevant available fragments (typically coming from
commercial reagents or from previously designed molecules). As
stated before, in this case we used all fragments present in the
published 180-compound HEPT family. The library enumeration,
conversion of the models to 3D and geometry optimization is
carried out by using either Molecular Mechanics protocols (i.e. MOE
[16], Discovery Studio [17], etc.) or rule-based systems such as
Corina [18,19].
Second, the chemical space is described by calculating the
desired molecular descriptors by a convenient computational
software suite (Discovery Studio [17], MOE [16], Dragon [20],
ADRIANA.Code [21], etc.). In this case we used 9 standard combi-
natorial chemistry descriptors (area, molecular volume, molecular
weight, radius of gyration, density, principal moment of inertia,
number of rotable bonds, number of hydrogen-bond acceptors and
number of hydrogen-bond donors) and 96 autocorrelogram vectors
provided by ADRIANA.Code [21] (formerly PETRA [22]).
Third, the dimensionality is reduced by principal component
analysis (PCA) down to a number of components that account for at
least 80% of the variance. In this case 12 components were enough
to represent 90% of the variance.
Fourth, we divide the PCA space by an Optimum Binning algo-
rithm implemented in Pralins until the number of occupied cells
approaches the desired selection size (typically the square-root of
the total number of compounds present in the library). In this case
we decided to select combinatorial sub-libraries up to
2 ꢀ 7 ꢀ 5 ꢀ 8 ¼ 560 compounds.
Fifth, we run Pralins to carry out the selections to choose
combinatorial sub-libraries which represent the original library,
ideally byselecting one member of each occupied cell, in the practice
more than 80% of the total number of cells or 90% of the population.
By using this approach, we usually found more than one combina-
torial sub-library that fulfills these criteria, so a final selection can be
carried out according to complementary factors such as real avail-
ability of the reagents, cost, ADMET-Lipinski rules [23], etc.
With that objective in mind, our group proposed a single
synthetic scheme (Scheme 1) for the combinatorial synthesis of
HEPT analogues. Now we wish to report the results obtained in the
synthesis and anti-HIV-1 evaluation of a sub-library of these new
2.2. Building blocks
In this case, with the aim of synthesizing all the selected
compounds by using the synthetic pathway depicted in Scheme 1,
the initial 560 member sub-library was finally reduced to 64
analogues 2 (2X ꢀ 2R1 ꢀ4R2 ꢀ 4R3) mimicking the aforementioned
protocol by selecting the corresponding building blocks (see
Table 1) on the basis of commercial availability, cost and compati-
bility with the synthetic route.
Such synthetic route was proposed as a combination of several
literature protocols. Thus, in the first step, barbiturate structures 5
Fig. 1. HEPT and general structure of analogues 2.

R. Puig-de-la-Bellacasa et al. / European Journal of Medicinal Chemistry 54 (2012) 159e174
161
Scheme 1. Proposed synthetic route to analogues 2{w,x,y,z} (X ¼ O, S; R1 ¼ H, Me; R2 ¼ 2-trimethylsilylethoxymethyl (SEM), p-chlorophenoxymethyl, ethoxymethyl, 2,5-
dimethylbenzyl; R3 ¼ H, m-amino, 2,6-dichloro, m-chloro).
{w,x} would be synthesized, as described by Dickley and Gray [24],
by the reaction between urea 3{1} (X ¼O) or thiourea 3{2} (X ¼ S)
and the corresponding malonate 4{x} (R1 ¼ H, Me). Then, using the
methodology of Lee et al. [25], compounds 5{w,x} would be treated
with POCl3 to achieve chlorination at position C-6 followed by the
selective derivatization at N1 by using a silylating agent 7{y} (R ¼ 2-
trimethylsilylethoxymethyl
ethoxymethyl, 2,5-dimethylbenzyl) to afford intermediates
{w,x,y}. Finally, as reported by Sun et al. [26], the chlorine atom at C-
6 of compounds 8{w,x,y} would be substituted by a thiophenol
group by using disulfides 9{z} (R3 ¼ H, m-amino, 2,6-dichloro, m-
chloro) to yield the HEPT analogues 2{w,x,y,z}.
(SEM),
p-chlorophenoxymethyl,
8
Table 1
Building blocks.
Building block
Substituents
X ¼ O (3{1})
X ¼ S (3{2})
R1 ¼ H (4{1})
R1 ¼ Me (4{2})
Cl e R2 7{y}
R3 ¼ H (10{1})
R3 ¼ m-NH2 (10{2})
R3 ¼ m-Cl (10{4})
R3 ¼ 2,6-Cl2 (10{3})

162
R. Puig-de-la-Bellacasa et al. / European Journal of Medicinal Chemistry 54 (2012) 159e174
Table 2
Interestingly, these compounds exhibit tautomerism depending
on the substitution at positions C-5 and C-2, and the major
tautomer present in each case was elucidated by recording the
NMR spectrum in DMSO-d6 as shown in Table 2.
Tautomerism in barbiturate structures 5. Tautomer content determined by NMR in
DMSO-d6.
In the second step, POCl3 and benzyltriethylammonium chloride
(BTAC) were used to achieve chlorination at position C-6 of the
barbiturate structures 5{w,x} [25]. When the reaction was tested
with the 5-methylbarbituric acid (5{1,2}), the corresponding 6-
chloro-5-methylpyrimidine (6{1,2}) was obtained in 53% yield
(Scheme 2). However, treatment of barbituric acid (5{1,1}) led to
2,4,6-trichloropyrimidine (11) instead of 6-chloropyrimidine 6{1,1}.
11 was subsequently converted to the desired 6{1,1} upon treat-
ment with 1 M NaOH solution [28]. The formation of the corre-
sponding 2-thio substituted analogues 6{2,1} and 6{2,2} was not
possible, probably due to the formation of the aforementioned
trichloro substituted intermediates which cause the loosing of the
sulfur atom. Such limitation halved the total number of analogues 2
to be obtained.
keto
keto-enol
enol
5{1,1} (X ¼ O, R1 ¼ H)
5{1,2} (X ¼ O, R1 ¼ Me)
5{2,1} (X ¼ S, R1 ¼ H)
5{2,2} (X ¼ S, R1 ¼ Me)
1
0.83
e
e
e
1
e
0.17
e
e
e
1
2.3. Chemistry
The 6-chloropyrimidines 6{1,1} and 6{1,2} weretreated with N,O-
bis(trimethylsilyl)acetamide (BSA) as silylating agent and tetrabu-
tylammonium iodide (TBAI) as catalyst [25] followed by the corre-
sponding chloro derivative 7{1-4} to achieve alkylation at N1.
However, only in two of the four alkyl chlorides 7{1-4} tested the
reaction was completed satisfactorily. Thus, the SEM (7{1}) and the
ethoxymethyl (7{3}) substituents were introduced at N1 to afford
compounds 8{1,x,1} and 8{1,x,3} (Scheme 2), the yields being better
when R1 ¼ Me (x ¼ 2). An alternative methodology consisting in
treating 6-chloropyrimidines 6{1,1} and 6{1,2} with 7{2} and 7{4} in
the presence of K2CO3 in DMSO [28] was tested to achieve the N1
Barbiturate structures 5 were synthesized for the first time in
1938 by Dickley and Gray [24]. Although these authors described
the addition of urea (3{1}) to a solution of a malonate (4{1}) in
NaOEt/EtOH; in the present work the addition order was reversed
to increase the yield. The reason for this change has been, in
accordance with the reaction mechanism, to avoid secondary
reactions between malonates and the base [27]. With this modifi-
cation, the four barbiturate structures 5{w,x} were synthesized in
38e95% yields.
Scheme 2. Complete synthetic pathway for HEPT analogues 2{w,x,y,z} and 12{w,x,y,z}.

R. Puig-de-la-Bellacasa et al. / European Journal of Medicinal Chemistry 54 (2012) 159e174
163
alkylation with these chloro compounds. Although the reaction
proceeded well with 7{4}, the chloro derivative 7{2} was discarded
as alkylating agent due to the low yield (6%) caused by the decom-
position of 7{2} to p-chlorophenol under the reaction conditions.
This finally reduces the initial 2 ꢀ 2 ꢀ 4 ꢀ 4 ¼ 64 selection to the
actual 1 ꢀ2 ꢀ 3 ꢀ 4 ¼ 24 synthesizable compound library.
modifying reaction time, temperature, presence or absence of
solvent, proportion between reagents, or the use of microwave
irradiation [34]. Instead of position C-2, the thionation occurred at
position C-4, yielding two examples of this substitution (12
{1,2,3,1}; 12{1,2,3,4}). (Scheme 2)
As we planned to carry out our anti-HIV assays with MT-4 cells
and NL4-3 virus type and there was no literature reference of the
HEPT activity in those conditions, we decided to synthesize HEPT
for comparison purposes.
Thus, the N1-alkylation of 6{1,2} with the chloromethoxyethyl
acetate derivate (15) [35] was assayed by using the two method-
ologies used in this work not affording the desired product. It was
necessary to use 2-(hydroxymethoxy)ethan-1-ol diacetate (17)
[36e38] in the presence of BSA as silylating agent and SnCl4 as
catalyst to yield 18 [39]. The nucleophilic substitution of the chlo-
rine atom present in 18 by the corresponding disulfide 9{1} in the
presence of NaBH4 and the concomitant deacetylation of the N1-
alkyl chain directly yielded HEPT in good yield (Scheme 3). Simi-
larly we decided to synthesize the p-chloro substituted analogue of
HEPT 19 by treating 18 with the corresponding disulfide 9{4} for
comparison purposes.
To accomplish the last step, the substitution of the chloro atom
present in compounds 8{w,x,y} by the selected thiophenols 10{z}, it
was necessary to synthesize the corresponding disulfides 9{z}
because many of them are not commercially available. With the aim
to oxidize all the selected thiophenols 9{z} into disulfides by using
the same methodology, several oxidizing agents were tested:
picolinium chlorochromate (
g-PCC) [29,30], sodium perborate
(NaBO3) [31] and DMSO at 85 ꢁC [32]. As is depicted in Table 3,
g-
PCC and NaBO3 were discarded due to the difficulties in obtaining
pure 9{2} and the low yield of 9{3}, respectively. On the contrary,
the use of DMSO as oxidizing agent afforded disulfides 9{1-4} in
very good yields.
Finally, the treatment of 6-chloro substituted compounds 8
{w,x,y} with the disulfides 9{z} and sodium borohydride [26]
afforded the corresponding HEPT analogues 2{w,x,y,z} in
moderate to good yields.
Our convincement that in this process sodium borohydride acts
both as a reducing agent and as a base that ionizes the resulting
thiophenol, led us to test the direct substitution of the chlorine
atom present in compounds 8{w,x,y} by the thiophenolate anion
formed from thiophenols 10{z}. The reaction was assayed in three
cases and the desired analogues 2{1,1,1,1}, 2{1,2,1,1} and 2{1,2,3,1}
were afforded in good yields. By this way, it is possible to obtaining
HEPT analogues without the need to synthesize the disulfides thus
increasing the available chemical space.
The synthesis of compounds 2{w,x,y,z} has allowed us to confirm
the regiospecificty of the N-alkylation at position N1 by a NOESY
experiment which showed the coupling between the protons of the
methylene group linked to N1 and the aromatic protons of the
thiophenol ring.
2.4. Anti-HIV activity
The activity and cytotoxicity of the synthesized HEPT analogues
were evaluated with the tetrazolium-based colorimetric method
(MTT method) in MT-4 cells. Anti-HIV activity (EC50), cytotoxicity
(CC50) and selectivity index (SI) are illustrated in Table 4 with HEPT,
AZT (Azidothymidine), NVP (Nevirapine) and EFV (Efavirenz) as
references.
The results showed that the HEPT analogues with a thymine
core (2{1,2,y,z}, R1 ¼ Me) gave better results in anti-HIV activity
than series with uracil core (2{1,1,y,z}, R1 ¼ H). The analogues con-
taining an uracil core (R1 ¼ H) did not show anti-HIV activity to the
highest concentration tested (25
{1,1,1,3} and 2{1,1,1,4}) which
(EC50 ꢂ 10.58 g/mL; 25.23 M and EC50 ꢂ 11.55
m
g/mL) except in two cases (2
presented cytotoxicity
g/mL; 30.00 M,
Not forgetting that one of our objectives was the synthesis of
HEPT analogues with a sulfur atom at position C-2, we tried to
obtain these structures starting from compounds 2{w,x,y,z} by
using the Lawesso’s reagent (LR) as the thiation agent [33]. Unfor-
tunately, it was not possible to thionate the C-2 carbonyl even
m
m
m
m
respectively). This cytotoxic effect could be caused by the presence
on the molecule of the SEM group at N1 position. In fact, 5 of the 8
analogues bearing the SEM group presented a cytotoxic effect at
concentration around 9.2e11.6
m
g/mL; 21.32e30.00
mM and only
Table 3
Oxidation of thiophenols 10{z} to disulfides 9{z}.
Compound
Structure
Oxidizing reagent
g
-PCC (%)
NaBO3$H2O (%)
DMSO (%)
97
9{1}
66
0
e
9{2}
9{3}
e
92
91
68
15
9{4}
51
e
92

164
R. Puig-de-la-Bellacasa et al. / European Journal of Medicinal Chemistry 54 (2012) 159e174
Scheme 3. Synthetic pathway for HEPT (1) and 19.
one compound showed measurable anti-HIV activity (2{1,2,1,2},
g/mL; 1.15 M).
On the other hand, in the case of the thymine core (R1 ¼ Me) the
inhibitory activity is better by one or two orders of magnitude
when R2 ¼ ethoxymethyl (2{1,2,3,z}), with respect to R2 ¼ 3,5-
dimethylbenzyl (2{1,2,4,z}).
With regard to the substitution pattern of the thiophenol ring,
the ortho dichloro disubstitution led to a low inhibitory effect of the
compound. The effect of a substituent in the meta position depends
on its nature. Thus, it is better the presence of a chlorine atom than
an amine group by comparing the EC50 of compounds 2{1,2,3,4}
The analogues with better anti-HIV activity were 2{1,2,3,4}
(EC50 ¼ 0.015 g/mL; 0.046 M) and 2{1,2,3,1} (EC50 ¼ 0.025 g/mL;
0.086 M) which showed a m-chlorophenylthio and a phenylthio
EC50 ¼ 0.44
m
m
m
m
m
m
substituent at position C6, respectively.
The introduction of a thiocarbonyl group at position C-4 (12
{1,2,3,4}
(EC50 ꢂ 1.70 g/mL;
m
4.96
m
M)
and
12{1,2,3,1}
(EC50 ¼ 0.6
m
g/mL; 1.95 mM)) caused a noticeable increase of the
cytotoxicity and a remarkable decrease of the inhibitory activity.
The improvement of the inhibitory activity observed for 2
{1,2,3,4} (EC50 ¼ 0.015
a chlorine atom in meta position of the thiophenol ring was
confirmed by the anti-HIV evaluation of 19 (EC50 ¼ 0.42 g/mL;
1.23 g/mL; 3.28 M) were
mg/mL; 0.046 mM) due to the presence of
(EC50 ¼ 0.015
m
g/mL; 0.046
m
M) and 2{1,2,4,4} (EC50 ¼ 0.336
m
g/mL;
m
0.87
m
M) in front of 2{1,2,3,2} (EC50 ¼ 0.4
m
g/mL; 1.30
m
M) and 2
m
M) compared with HEPT (EC50 ¼ 1.01
m
m
{1,2,4,2} (EC50 ¼ 1.9
mg/mL; 5.17
mM) respectively.
the activity was increased one order of magnitude. On the other
hand, a comparison of inhibitory activities of 2{1,2,3,1} and 2
{1,2,3,4} with HEPT and 19 respectively, seem to indicate that the
presence of the hydroxyl group in the alkyl chain is not good for the
inhibitory activity.
Table 4
EC50, CC50 and selectivity index (SI) of HEPT analogues.
Finally, in comparison with the references (HEPT, AZT, NVP,
RTV), the most active HEPT analogues exhibited activity values two
orders of magnitude better than HEPT, in the case of 2{1,2,3,4}
about 67-fold more active, similar values as NVP and only one order
of magnitude worse respect to AZT and EFV. The diversity of the
1 ꢀ 2 ꢀ 3 ꢀ 4 ¼ 24 compound combinatorial library has been
confirmed by the broad range of biological activities obtained. This
confirms our computational findings that a careful selection of
a minimal combinatorial sub-library can actually span the biolog-
ical activity space.
Compound
EC50
(
m
g/mL)
EC50
(
m
M)
CC50
(
m
g/mL)
SI
AZT
HEPT
NVP
EFV
0.001
1.01
0.03
0.001
>25
0.004
3.28
0.11
0.003
>71
>68
>25.23
>30.00
>90
>85
>72
>1
>25
>5
>1
>25
>25
10.6
11.6
>25
>25
>25
>25
>25
>25
>25
>25
9.9
>25
9.2
9.9
>25
>25
>25
>25
>25
>25
>25
>1000
>25
>167
>1000
e
2{1,1,1,1}
2{1,1,1,2}
2{1,1,1,3}
2{1,1,1,4}
2{1,1,3,1}
2{1,1,3,2}
2{1,1,3,3}
2{1,1,3,4}
2{1,1,4,1}
2{1,1,4,2}
2{1,1,4,3}
2{1,1,4,4}
2{1,2,1,1}
2{1,2,1,2}
2{1,2,1,3}
2{1,2,1,4}
2{1,2,3,1}
2{1,2,3,2}
2{1,2,3,3}
2{1,2,3,4}
2{1,2,4,1}
2{1,2,4,2}
2{1,2,4,3}
2{1,2,4,4}
19
>25
e
>10.58
>11.55
>25
>25
>25
>25
>25
>25
>25
e
e
e
e
e
>80
>74
>71
>61
e
3. Conclusion
e
e
In summary, we have described the synthesis and anti-HIV-1
activity of 25 new HEPT analogues of general structures 2{w,x,y,z}
and 12{w,x,y,z}. Unfortunately, the thionated analogues at position
C-2 were not accessible. The bioassay results revealed a wide range
of activity values but, in general, we can conclude that the thymine
core (R1 ¼ Me) is better than the uracil core (R1 ¼ H) and that the
presence of an hydroxyl group at the N1 alkyl group seems to
unfavour the inhibitory activity. It is also remarkable that the thi-
ophenol ring should be unsubstituted or, even better, with a chlo-
rine atom at the meta position. Furthermore, the thionation of the
carbonyl at position C-4 led to a fall of the activity values. Some of
the compounds exhibited potent HIV-1 inhibitory activity, in
e
>25
>67
e
>9.94
0.436
>9.24
>9.94
0.025
0.4
1.7
0.015
1.26
1.9
>25
0.336
0.42
0.6
>27.3
1.15
>21.3
>24.9
0.086
1.3
e
>57
e
e
>1000
>63
>15
>1667
>20
>13
e
4.7
0.046
3.57
5.2
>59
0.868
1.2
13.6
>25
2.38
1.7
>40
>60
>4
e
particular 2{1,2,3,4} and 2{1,2,3,1} with EC50 ¼ 0.015
0.046 g/mL; 0.086 M, being about 67-fold
M and EC50 ¼ 0.025
and 40-fold more active than HEPT, respectively. In addition, the
mg/mL;
12{1,2,3,1}
12{1,2,3,4}
1.9
>4.96
m
m
m
>1.70

R. Puig-de-la-Bellacasa et al. / European Journal of Medicinal Chemistry 54 (2012) 159e174
165
two best compounds have a good SI, >1667 and >1000, respec-
tively. Moreover, we have demonstrated that it is not necessary to
synthesize a huge combinatorial library to explore the chemical
space, obtaining a large diversity of anti-HIV-1 activity values from
a small sub-library with a rational selection of substituents.
16.0 g (0.1 mol) of diethyl malonate (4{1}). Spectroscopic data was
identical with those reported in the literature [24].
m.p. ¼ 252e253 ꢁC. 1H NMR (300 MHz, DMSO-d6)
d (ppm): 11.12
(br s, 2H, NH), 3.46 (s, 2H, CH2). IR (KBr, d
cmꢄ1): 3192, 3098 (NeH),
1766, 1744, 1718, 1696 (C]O), 1418 (NeH), 1366 (CeN), 805 (NeH).
4. Experimental
4.2.2. Thiobarbituric acid (5{2,1})
This compound was obtained as a pale yellow solid in a 38%
yield (5.44 g, 0.38 mol) but by using 7.61 g (0.1 mol) of thiourea (3
{2}) and 16.0 g (0.1 mol) of diethyl malonate (4{1}). The product is
described in the literature, but not the tautomeric form obtained.
4.1. Chemistry
Melting points were determined on a Büchi-Tottoli 530 capillary
apparatus and are uncorrected. Infrared (IR) spectra were recorded
on a Nicolet Magna 560 FTIR spectrophotometer. 1H and 13C NMR
spectra were recorded on either a Varian Gemini 300HC apparatus
or a Varian 400-MR spectrometer that were operating at a field
strength of 300 and 75.5 MHz; 400 and 100.6 MHz, respectively.
1H NMR (300 MHz, DMSO-d6)
d
(ppm): 12.19 (br s, 2H, NH), 4.91 (br
s, 1H, CH or OH), 3.55 (br s, CH or OH). 13C NMR (75.5 MHz, DMSO-
d6) (ppm): 174.9 (C]S), 165.9 (C]O), 161.9 (CeOH), 81.9 (CH). IR
(KBr,
cmꢄ1): 3380, 3305 (OeH), 3197, 3096 (NeH), 2941, 2871
d
n
(CeH), 1742, 1720, 1702 (C]O), 1652 (C]C), 1435 (OeH), 1163 (C]
S), 812 (NeH). MS (EI): m/z (%) ¼ 144.1 (85) [M]þ, 116.2 (44)
[M ꢄ CO]þ, 43.2 (100) [CONH]þ.
Chemical shifts were reported in parts per million (d) and coupling
constants (J) were in Hz by using, in the case of 1H NMR spectros-
copy, TMS as an internal standard, and in the case of 13C NMR
spectroscopy the solvent at 77.0 pm (CDCl3), 39.5 ppm DMSO-d6,
49.0 ppm (methanol-d4), 29.8 (acetone-d6) as an internal reference.
Standard and peak multiplicities are designed as follows: s, singlet;
d, doublet; t, triplet; q, quartet; br s, broad signal. MS data were
obtained by using HewlettePackard HP5988A spectrometer or on
Agilent Technologies 5975 spectrometer operating in Electron
Ionisation (EI) at 70 eV, or on a VG AutoSpec (Micromass Instru-
ments) Triosector EBE spectrometer operating in Fast Atom
Bombardment (FAB) mode or on a Biotoff II (Bruker) in Electrospray
ionization (ESI) mode with a Time Of Flight (TOF) detector. HRMS
data was conducted on a VG AutoSpec (Micromass Instruments)
Trisector EBE of high resolution spectrometer operating in FAB or EI
mode and on Biotoff II (Bruker) apparatus in ESI-TOF mode.
Elemental microanalyses were performed on a Carlo-Erba CHNS-O/
EA 1180 analyzer and the results of elemental analyses for C, H, N
and S were within ꢃ0.4% of the theoretical values. A Biotage Initi-
ator microwave oven was used for reactions requiring microwave
irradiation. All air-sensitive reactions were run under nitrogen
atmosphere. Thin-layer chromatography (TLC) was performed on
precoated sheets of silica 60 polygram SIL N-HR/UV254 (Macherey-
Nagel, art. 804023). Flash chromatography was performed with
4.2.3. 5-Methylbarbituric acid (5{1,2})
This compound was obtained as a white solid in a 95% yield
(13.49 g, 0.95 mol) but by using 6.0 g (0.1 mol) of urea (3{1}) and
17.42 g (0.1 mol) of diethyl methylmalonate (4{2}). It was obtained
as a mixture of tautomers. m.p. ¼198e200 ꢁC. 1H NMR (300 MHz,
DMSO-d6)
d (ppm): Keto form: 11.07 (br s, 2H, NH), 3.63 (q,
3J ¼ 7.2 Hz,1H, CH),1.30 (d, 3J ¼ 7.2 Hz, 3H, CH3). Enol form: 11.19 (br
s, 2H, OH), 6.16 (br s, 1H, OH), 1.45 (s, 3H, CH3). IR (KBr,
n
cmꢄ1):
3394 (OeH), 3128 (NeH), 3004, 2936, 2854 (Csp3eH), 1701, 1630
(C]O), 1403, 1366 (CH3), 859 (Csp3eH), 764 (NeH).
4.2.4. 5-Methylthiobarbituric acid (5{2,2})
This compound was obtained as a white solid in a 80% yield
(12.66 g, 0.80 mol) but by using 7.61 g (0.1 mol) of thiourea (3{2})
and 17.42 g (0.1 mol) of diethyl methylmalonate (4{2}). It was
obtained as an enol tautomer. m.p. ¼ 253e254 ꢁC. 1H NMR
(300 MHz, DMSO-d6)
d (ppm): 12.06 (br s, 2H, OH), 3.78 (br s, 1H,
SH), 1.70 (s, 3H, CH3). 13C NMR (75.5 MHz, DMSO-d6)
(CeOH), 160.0 (C-SH), 90.1 (C5), 7.5 (CH3). IR (KBr,
d
(ppm): 172.5
n
cmꢄ1): 3166,
3105 (OeH), 2728, 2655 (SeH), 1603, 1551 (C]C), 1211, 1130 (CeO).
MS (ESI-TOF): m/z (%) ¼ 159.0 (100) [M þ H]þ. HRMS (ESI-TOF):
calcd. for C5H7N2O2S: 159.0223, [M þ H]þ, found: 159.0223.
silica gel 35e70
solvents as eluent.
mm (SDS, art. 2000027) using suitable mixture of
The synthesized compounds have been checked for their
melting points, physical nature, IR, 1H NMR, 13C NMR, Mass spec-
troscopy and Elemental analysis for individual compounds and the
data are summarized as under.
4.3. Preparation of 6-chlorouracil (6{1,1})
The synthesis consists of two steps.
4.3.1. 2,4,6-Trichloropyrimidine (11)
4.2. General preparation of barbituric acids (5{w,x})
A stirred suspension of barbituric acid (5{1,1}) (12.81 g, 0.1 mol)
and benzyltrielthylammonium chloride (BTAC) (45.56 g, 0.2 mol) in
phosphorus oxychloride (POCl3) (230 mL, 2.5 mol) was heated at
50 ꢁC for 7 h under nitrogen atmosphere. The reaction mixture was
cooled to room temperature and evaporated to dryness in vacuo.
The residue was carefully quenched with 285 g of ice chips at 0 ꢁC
and the slurry was kept in a refrigerator overnight. The precipitate
was collected by filtration, washed with cold water and dried in
vacuo over phosphorus pentoxide. 10.21 g (56.2 mmol, 56%) of
2,4,6-trichloropyrimidine (11) were obtained as a white solid
slightly brown. Spectroscopic data was identical with those repor-
ted in the literature [40]. m.p. ¼ 21e23 ꢁC. 1H NMR (300 MHz,
In a 250 mL round-bottomed flask fitted with a reflux condenser
protected by a calcium chloride tube, 2.3 g (0.1 mol) of finely cut
sodium in 35 mL of anhydrous methanol. To this solution is added
urea or thiourea (3{w}) (0.1 mol) in 35 mL of anhydrous methanol
at 60 ꢁC, followed by a dropwise addition of the corresponding
malonate (4{x}) (0.1 mol). After being well shaken mixture is
refluxed for 16 h. A white solid separates rapidly. After the reaction
is completed, 100 mL of hot (50 ꢁC) water is added and then enough
hydrochloric acid to make the solution acidic and solve the solid.
The resulting clear solution is filtered and cooled in a refrigerator
overnight. The white product is collected by filtration, washed with
cold water and then dried in an oven at 110 ꢁC for 4 h to give 5{w,x}.
CDCl3)
d (ppm): 7.40 (s, 1H, CH). IR (film, n
cmꢄ1): 3110 (Csp2eH),
1522, 1369, 1279 (skeletal vibration), 1105 (CeCl).
4.2.1. Barbituric acid (5{1,1})
4.3.2. 6-Chlorouracil (6{1,1})
This compound was obtained as a white solid in a 64% yield
(8.11 g, 0.64 mol) but by using 6.0 g (0.1 mol) of urea (3{1}) and
A solution of 2,4,6-trichloropyrimidine (11) (10.21 g, 0.56 mol)
in water (100 mL) containing NaOH (8.9 g, 0.22 mol) was stirred

166
R. Puig-de-la-Bellacasa et al. / European Journal of Medicinal Chemistry 54 (2012) 159e174
under reflux for 1 h. The reaction mixture was adjusted to pH 2e3
with conc. aqueous HCl and was kept in a refrigerator overnight.
The precipitate was collected by filtration, washed with cold water
and dried in vacuo over phosphorus pentoxide. 6.43 g (0.54 mol,
97%) of 6-clorouracil (6{1,1}) were obtained as a white crystalline
solid. Spectroscopic data was identical with those reported in the
literature [28,40]. m.p. >280 ꢁC. 1H NMR (300 MHz, DMSO-d6)
of 6-chloro-1-(2-trimethylsilyl)ethoxymethyluracil (8{1,1,1}) were
obtained as a white solid. m.p. ¼104e106 ꢁC. 1H NMR (300 MHz,
CDCl3)
d
(ppm): 8.64 (br s, 1H, NH), 5.92 (d, 4J ¼ 2.1 Hz, 1H, CH),
5.45 (s, 2H, NeCH2eO), 3.72e3.67 (m, 2H, CH2eCH2eSi(Me)3),
0.99e0.93 (m, 2H, CH2eCH2eSi(Me)3), 0.02 (s, 9H, Si(Me)3). 13C
NMR (75.5 MHz, CDCl3)
(C6), 103.4 (CH), 74.2 (CH2), 67.6 (CH2eCH2eSi(Me)3), 18.1
cmꢄ1): 3127 (NeH),
d (ppm): 161.0 (C4), 150.3 (C2), 147.5
d
(ppm): 12.00 (br s, 1H, NH), 11.09 (br s, 1H, NH), 5.66 (s, 1H, CH). IR
(CH2eCH2eSi(Me)3), ꢄ1.3 (Si(Me)3). IR (film,
n
(KBr,
n
cmꢄ1): 3095 (NeH), 1729, 1709, 1652 (C]O), 1616 (C]C),
3028 (Csp2eH), 2952, 2921, 2870 (Csp3eH), 1706, 1664 (C]O),
1555 (C]C), 1454 (NeH), 1244 (SieCH3), 1095 (CeOeC), 845, 756
(SieCH3). Anal. calcd for C10H17N2O3ClSi: C, 43.39; H, 6.19; Cl, 12.81;
N, 10.12; O, 17.35; Si, 10.15. Found: C, 43.30; H, 6.34; N, 10.00%. MS
(EI): m/z (%) ¼ 261.2 (1) [M ꢄ CH3]þ, 241.3 (1) [M ꢄ 35Cl]þ, 203.2 (33)
[M ꢄ Si(Me)3]þ, 159.2 (2) [M-O(CH2)2Si(Me)3]þ, 116.2 (21)
[O(CH2)2Si(Me)3]þ, 73.2 (100) [Si(Me)3]þ.
1380 (CeN), 841 (NeH), 790 (CeCl).
The overall yield of the reaction for obtaining 6-chlorouracil (6
{1,1}) is 54%.
4.4. Preparation of 6-chlorothymine (6{1,2})
A stirred suspension of 5-methylbarbituric acid (5{1,2}) (9.40 g,
0.66 mol) and benzyltrielthylammonium chloride (BTAC) (30.14 g,
0.13 mol) in phosphorus oxychloride (POCl3) (154 mL, 1.65 mol)
was heated at 50 ꢁC for 7 h under nitrogen atmosphere. The reac-
tion mixture was cooled to room temperature and evaporated to
dryness in vacuo. The residue was carefully quenched with 285 g of
ice chips at 0 ꢁC and the slurry was kept in a refrigerator overnight.
The precipitate was collected by filtration, washed with cold water
and dried in vacuo over phosphorus pentoxide. 5.60 g (0.35 mol,
53%) of 6-chloro-5-thymine (6{1,2}) were obtained as a white solid.
4.5.2.2. 6-chloro-1-(ethoxymethyl)uracil (8{1,1,3}). The procedure
used was the methodology A, but carried out by using 4.51 g
(30.7 mmol) of 6-clorouracil (6{1,1}) in 90 mL of anhydrous CH2Cl2,
17.6 mL (67.6 mmol) of BSA, 112 mg (0.37 mmol) of TBAI and
5.93 mL (61.4 mmol) of chloromethyl ethyl ether (7{3}). The residue
was purified by flash column chromatography on silica gel with
a mixture of ethyl acetate and hexane (2:3, v/v) as eluent. 2.31 g
(11.3 mmol, 37%) of 6-chloro-1-(2-ethoxymethyl)uracil (8{1,1,3})
were obtained as a pale yellow solid. m.p. ¼142e143 ꢁC. 1H NMR
m.p. 270e272 ꢁC. 1H NMR (300 MHz, DMSO-d6)
d
(ppm): 11.81 (br s,
(300 MHz, CDCl3) d (ppm): 8.75 (br s, 1H, NH), 5.93 (s, 1H, CH), 5.47
1H, NH), 11.32 (br s, 1H, NH), 1.82 (s, 3H, CH3). IR (KBr,
n
cmꢄ1): 3158
(s, 2H, NeCH2eO), 3.67 (q, 3J ¼ 6.9 Hz, 2H, CH2eCH3), 1.24
(NeH), 3013, 2814 (Csp2eH), 1711, 1647 (C]O), 1625 (C]C), 1491
(NeH), 1424 (CeN), 891 (NeH), 753 (CeCl).
(t, 3J ¼ 6.9 Hz, 3H, Me). 13C NMR (75.5 MHz, CDCl3)
d (ppm): 161.1
(C4), 150.4 (C2), 147.38 (C6), 103.5 (CH), 74.5 (CH2), 65.6 CH2eCH3),
15.1 (CH3). IR (film,
n
cmꢄ1): 3159 (NeH), 3093, 3036 (Csp2eH),
4.5. Preparation of N1-derivatized compounds (8{w,x,y})
2974, 2875, 2789 (Csp3eH), 1705 (C]O), 1603 (C]C), 1442 (NeH),
1098 (CeOeC), 828 (NeH), 752 (CeCl). Anal. calcd for C7H9N2O3Cl:
C, 41.09; H, 4.43; Cl, 17.33; N, 13.69; O, 23.46. Found: C, 41.11; H,
4.62; N, 13.40%. MS (EI): m/z (%) ¼ 204.2 (2) [M]þ, 175.2 (5)
[M ꢄ Et]þ, 169.2 (6) [M ꢄ 35Cl]þ, 59.2 (100) [CH2OEt]þ.
4.5.1. Methodology A
A suspension of 6{w,x} and N,O-bis-(trimethylsilyl)-acetamide
(BSA) in anhydrous dichloromethane was stirred at room temper-
ature for 2 h under nitrogen atmosphere. To the resulting solution,
tetrabutylammonium iodide (TBAI) in one portion at room
temperature, and the mixture was immediately cooled to 0 ꢁC. 7{y}
was slowly added to the reaction mixture at 0 ꢁC, and the mixture
was stirred for an additional 2 h in an ice bath. The reaction mixture
was poured into saturated sodium bicarbonate solution and ice,
and stirred for 30 min. The organic phase was separated and the
aqueous phase was extracted with dichloromethane. The combined
organic phase was washed with brine, dried over anhydrous
magnesium sulfate and evaporated to dryness in vacuo. The residue
was purified by flash column chromatography on silica gel with the
corresponding eluent.
4.5.2.3. 6-Chloro-1-(2,5-dimethylbenzyl)uracil (8{1,1,4}). The proce-
dure used was the methodology B, but carried out by using 4.50 g
(31.0 mmol) of 6-chlorouracil (6{1,1}), 6.83 mL (46.0 mmol) of 2,5-
dimethylbenzyl chloride (7{4}) and 2.13 g (15.0 mmol), K2CO3 in
30 mL of DMSO and 30 mL of aqueous NaOH solution (4%, w/v) to
adjust the pH. The solid obtained was purified by recrystallization from
MeOH to give 3.78 g (14.3 mmol, 47%) of 6-chloro-1-(2,5-
dimethylbenzyl)uracil (8{1,1,4}) as a white solid. m.p. ¼ 249e251 ꢁC.
1H NMR (300 MHz, DMSO-d6)
d (ppm): 11.75 (br s, 1H, NH), 7.09 (d,
3J ¼ 7.8 Hz, 1H, ArC3eH), 6.99 (d, 3J ¼ 7.8 Hz, 1H, ArC4eH), 6.67 (s, 1H,
ArC6eH), 6.05 (d, 4J ¼ 2.1 Hz, 1H, C5-H), 5.07 (s, 2H, CH2), 2.25 (s, 3H,
Me), 2.23 (s, 3H, Me). 13C NMR (75.5 MHz, DMSO-d6)
d (ppm): 160.9
4.5.2. Methodology B
(C4), 150.2 (C2), 146.8 (C6), 135.1 (ArC1)**, 133.8 (ArC5)**, 131.1 (ArC2)
**, 130.0 (ArC3)*, 127.4 (ArC6)*, 124.1 (ArC4)*, 102.4 (C5H), 46.2 (CH2),
A mixture of 6{w,x}, 7{y} and K2CO3 in DMSO was stirred at
60e70 ꢁC for 1 h. An aqueous NaOH solution (4%, w/v) was added to
the hot reaction mixture with stirring. The reaction was washed
with toluene, and the aqueous phase was adjusted to pH 2e3 with
conc. aqueous HCl. The resulting aqueous mixture was kept in
a refrigerator overnight and the precipitate was collected by
filtration, washed with cold water, and dried. The solid was purified
by recrystallization from MeOH.
20.8 (CH3), 18.1 (CH3). IR (film,
n
cmꢄ1): 3145 (NeH), 3090, 3015
(Csp2eH), 2921, 2856, 2795 (Csp3eH), 1727, 1715, 1703, 1656 (C]O),
1585 (C]C), 1456 (NeH), 753 (CeCl). MS (ESI-TOF): m/z (%) ¼ 265.1
(14) [M þ H]þ, 119.1 (100) [C9H11]þ. HRMS (ESI-TOF): calcd. for
C13H1435ClN2O2: 265.0738, [M þ H]þ, found: 265.0740.
4.5.2.4. 6-Chloro-1-(2-trimethylsilyl)ethoxymethylthymine (8{1,2,1}).
The procedure used was the methodology A, but carried out by using
2.41 g (15.0 mmol) of 6-clorothymine (6{1,2}) in 45 mL of anhy-
drous CH2Cl2, 8.6 mL (33.0 mmol) of BSA, 55 mg (0.15 mmol) of
TBAI and 5.28 mL (30.0 mmol) of SEM (7{1}). The residue was
purified by flash column chromatography on silica gel with
a mixture of ethyl acetate and hexane (1:1, v/v) as eluent. 3.91 g
(13.4 mmol, 90%) of 6-chloro-1-(2-trimethylsilyl)ethoxymethylth-
ymine (8{1,2,1}) were obtained as a white solid. m.p. ¼ 96e97 ꢁC.
4.5.2.1. 6-chloro-1-(2-trimethylsilyl)ethoxymethyluracil (8{1,1,1}). The
procedure used was the methodology A, but carried out by using
4.51 g (30.7 mmol) of 6-clorouracil (6{1,1}) in 110 mL of anhydrous
CH2Cl2, 21.8 mL (83.5 mmol) of BSA, 139 mg (0.38 mmol) of TBAI and
13.40 mL (75.9 mmol) of SEM (7{1}). The residue was purified by
flash column chromatography on silica gel with a mixture of ethyl-
acetate and hexane (1:9, v/v) as eluent. 5.73 g (21.0 mmol, 68%)

R. Puig-de-la-Bellacasa et al. / European Journal of Medicinal Chemistry 54 (2012) 159e174
167
1H NMR (300 MHz, CDCl3)
d
(ppm): 8.59 (br s, 1H, NH), 5.47 (s,
(ArC1), 149.3 (C2), 142.3 (C6), 129.6 (ArC3), 127.9 (ArC4), 117.7 (ArC2),
111.2 (C5), 72.7 (CH2), 12.3 (CH3). IR (film,
cmꢄ1): 3164, 3121, 3080
2H, NeCH2eO), 3.71e3.66 (m, 2H, CH2eCH2eSi(Me)3), 2.07 (s, 3H,
Me), 0.99e0.93 (m, 2H, CH2eCH2eSi(Me)3), 0.01 (s, 9H,
n
(NeH), 3033, 2925, 2859, 2818 (Csp3eH), 1724, 1671 (C]O), 1615,
1597, 1584, 1494 (C]C), 1459 (NeH), 1239, 1216 (CeOeAr), 1013
(CeCl), 873 (NeH), 743 (CeH), 666 (C]C). MS (ESI-TOF): m/z (%) ¼
301.0 (13) [M þ H]þ, 173.0 (7) [C6H6ClN2O2]þ, 141.0 (14) [C7H6ClO]þ.
HRMS (ESI-TOF): calcd. for C12H1135Cl2N2O3: 301.0138, [M þ H]þ,
found: 301.0141; calcd. for C12H1135Cl37ClN2O3: 303.0118, [M þ H]þ,
found: 303.0114.
Si(Me)3). 13C NMR (75.5 MHz, CDCl3)
d
(ppm): 161.7 (C4), 150.0
(C2), 143.3 (C6), 110.4 (C5), 74.5 (CH2), 67.4 (CH2eCH2eSi(Me)3),
18.1 (CH2eCH2eSi(Me)3), 12.2 (CH3), ꢄ1.3 (Si(Me)3). IR (film,
n
cmꢄ1): 3199, 3053 (NeH), 2953, 2927, 2896 (Csp3eH), 1723,
1694 (C]O), 1617 (C]C), 1447 (NeH), 1249 (SieCH3), 1091
(CeOeC), 837, 756 (SieCH3). Anal. calcd for C11H19N2O3ClSi: C,
45.43; H, 6.59; Cl, 12.19; N, 9.63; O, 16.50; Si, 9.66. Found: C, 45.42;
H, 6.83; N, 9.46%. MS (EI): m/z (%) ¼ 217.2 (50) [M ꢄ Si(Me)3]þ, 73.2
(100) [Si(Me)3]þ.
4.6. General preparation of disulfides (9{z})
An equimolar mixture of thiophenol (10{z}) and DMSO was
heated at 80e90 ꢁC for 4 h. The reaction was cooled to room
temperature, diethyl ether was added and washed with water (ꢀ5).
The organic phase was dried over anhydrous magnesium sulfate
and evaporated to dryness in vacuo to give (9{z}).
4.5.2.5. 6-chloro-1-(ethoxymethyl)thymine (8{1,2,3}). The proce-
dure used was the methodology A, but carried out by using 2.0 g
(12.5 mmol) of 6-clorothymine (6{1,2}) in 35 mL of anhydrous
CH2Cl2, 7.14 mL (24.9 mmol) of BSA, 46 mg (0.13 mmol) of TBAI and
2.40 mL (24.9 mmol) of chloromethyl ethyl ether (7{3}). 2.00 g
(9.15 mmol, 73%) of 6-chloro-1-(ethoxymethyl)thymine (8{1,2,3})
were obtained, without further purification, as a pale yellow sol-
4.6.1. Diphenyl disulfide (9{1})
This compound was obtained as a white crystalline solid in
a 97% yield (1.626 g, 14.9 mmol) but by using 1.57 mL (15.32 mmol)
of thiophenol (10{1}) and 0.90 mL (15.32 mmol) of DMSO. m.
id. m.p. ¼132e133 ꢁC. 1H NMR (300 MHz, CDCl3)
d (ppm): 8.67 (br
s, 1H, NH), 5.49 (s, 2H, NeCH2eO), 3.66 (q, 3J ¼ 6.9 Hz, 2H,
CH2eCH3), 2.07 (s, 3H, Me), 1.23 (t, 3J ¼ 6.9 Hz, 3H, CH2eCH3). 13C
p. ¼ 61e62 ꢁC. 1H NMR (300 MHz, CDCl3)
d (ppm): 7.52e7.48 (m,
4H, ArC2eH), 7.33e7.19 (m, 6H, ArC3eH, ArC4eH). IR (film, n
NMR (75.5 MHz, CDCl3)
d
(ppm): 161.5 (C4), 149.9 (C2), 142.1 (C6),
108.9 (C5), 74.1 (CH2), 64.0 (CH2eCH3), 15.0 (CH2eCH3), 12.0 (CH3).
cmꢄ1): 3071, 3056 (Csp2eH), 1575, 1475, 1438 (C]C), 739 (CeH),
684 (C]C).
IR (film,
n
cmꢄ1): 3158, 3119 (NeH), 3023, 2978, 2824 (Csp3eH),
1723, 1683, 1671 (C]O), 1606 (C]C), 1456 (NeH), 1098 (CeOeC),
896 (NeH), 754 (CeCl). Anal. calcd for C8H11N2O3Cl: C, 43.95; H,
5.07; Cl, 16.22; N, 12.81; O, 21.95. Found: C, 43.96; H, 5.53; N,
12.45%. MS (EI): m/z (%) ¼ 218.2 (5) [M]þ, 189.2 (4) [M ꢄ Et]þ, 183.3
(2) [M ꢄ 35Cl]þ, 173.2 (4) [M-OEt]þ, 59.2 (100) [CH2OEt]þ.
4.6.2. Bis(3-aminophenyl) disulfide (9{2})
This compound was obtained as a yellow oil which crystallizes
slowly in a 92% yield (5.36 g, 43.2 mmol) but by using 5.0 mL
(47.0 mmol) of 3-aminothiophenol (10{2}) and 3.35 mL
(47.0 mmol) of DMSO. m.p. ¼ 55e58 ꢁC. 1H NMR (300 MHz, CDCl3)
4.5.2.6. 6-Chloro-1-(2,5-dimethylbenzyl)thymine (8{1,2,4}). The pro-
cedure used was the methodology B, but carried out by using 1.60 g
(10.0 mmol) of 6-chlorothymine (6{1,2}), 2.20 mL (15.0 mmol) of 2,5-
dimethylbenzyl chloride (7{4}) and 691 mg (5.0 mmol), K2CO3
in 15 mL of DMSO and 10 mL of aqueous NaOH solution (4%, w/v)
to adjust the pH. The solid obtained was purified by
recrystallization from MeOH to give 883 mg (3.18 mmol, 32%) of 6-
chloro-1-(2,5-dimethylbenzyl)thymine (8{1,2,4}) as a white solid.
d
(ppm): 7.07 (t, 3J ¼ 7.8 Hz, 2H, ArC5eH), 6.87 (ddd, 4J ¼ 0.9 Hz,
4J ¼ 2.1 Hz, 3J ¼ 7.8 Hz, 2H, ArC6eH), 6.82 (t, 4J ¼ 1.8 Hz, 2H,
ArC2eH), 6.52 (ddd, 4J ¼ 0.9 Hz, 4J ¼ 2.1 Hz, 3J ¼ 7.8 Hz, 2H,
ArC4eH), 3.69 (br s, 4H, NH2). IR (film, n
cmꢄ1): 3432, 3353 (NeH),
3214 (Fermi resonance NH), 3051 (Csp2eH), 1618 (NeH), 1589, 1479
(C]C), 883, 853 (NeH), 773 (CeH), 684 (C]C).
4.6.3. Bis(2,6-dichlorophenyl) disulfide (9{3})
m.p. ¼ 249e251 ꢁC.1H NMR (300 MHz, CDCl3)
d
(ppm): 8.69 (br s,1H,
This compound was obtained as a white solid in a 91% yield
(4.55 g, 25.41 mmol) but by using 5.0 g (27.92 mmol) of 2,6-
dichlorothiophenol (10{3}) and 2.0 mL (27.92 mmol) of DMSO.
NH), 7.07 (d, 3J ¼ 7.8 Hz, 1H, ArC3eH), 7.00 (d, 3J ¼ 7.8 Hz, 1H,
ArC4eH), 6.64 (s,1H, ArC6eH), 5.22 (s, 2H, CH2), 2.32 (s, 3H, AreMe),
2.28 (s, 3H, AreMe), 2.09 (s, 3H, Me). 13C NMR (75.5 MHz, CDCl3)
m.p. ¼ 217e218 ꢁC. 1H NMR (300 MHz, CDCl3)
d
(ppm): 7.35e7.33
d
(ppm): 161.6 (C4),149.8 (C2),142.8 (C6),135.0 (ArC1)**,134.0 (ArC5)
**, 131.0 (ArC2)**, 130.0 (ArC3)*, 127.3 (ArC6)*, 124.2 (ArC4)*, 108.5
(C5), 46.6 (CH2), 20.8, (AreMe), 18.1 (AreMe), 12.3 (CH3). IR (film,
(m, 4H, ArC3eH), 7.23e7.18 (m, 2H, ArC4eH). IR (KBr, n
cmꢄ1):
3077, 3068 (Csp2eH),1551,1424,1398 (C]C),1187,1146 (CeCl), 776
(CeH), 704 (C]C).
n
cmꢄ1): 3158 (NeH), 3027 (Csp2eH), 2967, 2921, 2856, 2804
(Csp3eH), 1714, 1690, 1667 (C]O), 1612 (C]C), 1442 (NeH), 807
(NeH), 740 (CeCl). Anal. calcd for C14H15ClN2O2: C, 60.33; H, 5.42; Cl,
12.72; N, 10.05; O, 11.48. Found: C, 60.43; H, 5.66; N, 9.82%. MS (EI):
m/z (%) ¼ 278.9 (29) [M]þ, 119.1 (100) [C9H11]þ, 91.0 (28) [C7H7]þ.
4.6.4. Bis(3-chlorophenyl) disulfide (9{4})
This compound was obtained as a yellow oil in a 92% yield
(9.54 g, 66 mmol) but by using 8.4 mL (72.6 mmol) of 3-
chlorothiophenol (10{4}) and 5.17 mL (72.6 mmol) of DMSO. 1H
NMR (300 MHz, CDCl3)
7.37e7.34 (m, 2H, ArC6eH), 7.27e7.19 (m, 4H, ArC4eH, ArC5eH).
IR (film,
cmꢄ1): 3054 (Csp2eH), 1574, 1495, 1406 (C]C), 1115,
d (ppm): 7.48e7.47 (m, 2H, ArC2eH),
4.5.2.7. 6-Chloro-1-((4-chlorophenoxy)methyl)thymine (8{1,2,2}). The
procedure used was the methodology B, but carried out by using
160.5 mg (1.0 mmol) of 6-chlorothymine (6{1,2}), 266 mg (1.5 mmol)
n
1072 (CeCl), 773 (CeH), 676 (C]C).
of a,4-dichloroanisole (7{2}) and 69.1 mg (0.5 mmol), K2CO3 in 1.5 mL
of DMSO and 1.0 mL of aqueous NaOH solution (4%, w/v) to adjust the
pH. The residue was purified by flash column chromatography on
silica gel with a mixture of ethyl acetate and hexane (1:1, v/v) as
eluent. 17.3 mg (0.057 mmol, 6%) of 6-chloro-1-((4-chlorophenoxy)
methyl)thymine (8{1,2,2}) were obtained as a white solid. 1H NMR
4.7. Preparation of HEPT analogues (2{w,x,y,z})
4.7.1. Methodology C
To an ice-cooled stirring solution of N1-derivatized compound
(8{w,x,y}), disulfide (9{z}) in MeOH and NaBH4 were added in
portions under nitrogen atmosphere, and the mixture was warmed
to room temperature and stirred for 16 h. The evaporation of
solvent gave a solid which was dissolved in water, and the solution
(300 MHz, CDCl3)
d
(ppm): 8.57 (br s, 1H, NH), 7.27 (d, 3J ¼ 9.0 Hz, 2H,
ArC3eH), 6.97 (d, 3J ¼ 9.0 Hz, 2H, ArC2eH), 5.93 (s, 2H, CH2), 2.09 (s,
3H, CH3). 13C NMR (75.5 MHz, CDCl3)
d (ppm): 161.3 (C4), 154.3

168
R. Puig-de-la-Bellacasa et al. / European Journal of Medicinal Chemistry 54 (2012) 159e174
was adjusted to pH 6e7 with 1M HCl. The solution was then
extracted with dichloromethane. The combined organic phase was
washed with brine and dried over anhydrous magnesium sulfate,
filtered, and evaporated to dryness in vacuo. The residue was
washed with hexane to give the corresponding HEPT analogue (2
{w,x,y,z}).
4.7.2.3. 1-(2-Trimethylsilyl)ethoxymethyl-6-(2,6-dichlorophenylthio)
uracil (2{1,1,1,3}). The procedure used was the methodology C, but
carried out by using 100 mg (0.36 mmol) of 6-chloro-1-(2-
trimethylsilyl)ethoxymethyluracil (8{1,1,1}), 642 mg (1.81 mmol)
of bis(2,6-dichlorophenyl) disulfide (9{3}) and 130 mg (3.44 mmol)
of NaBH4 in MeOH (5 mL). The residue was purified by flash column
chromatography on silica gel with a mixture of ethyl acetate and
chloroform (1:9, v/v) as eluent. 106 mg (0.25 mmol, 70%) of 1-(2-
trimethylsilyl)ethoxymethyl-6-(2,6-dichlorophenylthio)uracil (2
{1,1,1,3}) were obtained as a white solid. m.p. ¼171e173 ꢁC. 1H NMR
4.7.2. Methodology D
The procedure was similar to the methodology C, but carried out
by using thiophenol (10{z}) instead of disulfide (9{z}).
(300 MHz, CDCl3)
d (ppm): 9.38 (br s, 1H, NH), 7.53e7.49 (m, 2H,
4.7.2.1. 1-(2-Trimethylsilyl)ethoxymethyl-6-(phenylthio)uracil
(2
ArC3eH), 7.43e7.37 (m, 1H, ArC4eH), 5.60 (s, 2H, NeCH2eO), 4.99
(d, 4J ¼ 2.1 Hz, 1H, C5-H), 3.76e3.71 (m, 2H, CH2eCH2eSi(Me)3),
1.04e0.99 (m, 2H, CH2eCH2eSi(Me)3), 0.04 (s, 9H, Me). 13C NMR
{1,1,1,1}). The procedure used was the methodology C, but carried
out by using 100 mg (0.36 mmol) of 6-chloro-1-(2-trimethylsilyl)
ethoxymethyluracil (8{1,1,1}), 786 mg (3.61 mmol) of bisphenyl
disulfide (9{1}) and 260 mg (6.86 mmol) of NaBH4 in MeOH (5 mL).
The residue was washed with hexane to give 106 mg (0.30 mmol,
84%) of 1-(2-trimethylsilyl)ethoxymethyl-6-(phenylthio)uracil (2
{1,1,1,1}) as a white solid.
(75.5 MHz, CDCl3)
d (ppm): 161.2 (C4), 156.4 (C6), 150.8
(C2), 141.7 (ArC2), 132.7 (ArC4), 129.4 (ArC3), 126.8 (ArC1),
98.9 (C5), 73.8 (NeCH2eO), 67.1 (CH2eCH2eSi(Me)3), 18.1
(CH2eCH2eSi(Me)3), ꢄ1.2 (Me). IR (film,
n
cmꢄ1): 3186 (NeH), 3038
(Csp2eH), 2953, 2895, 2803 (Csp3eH), 1717, 1690 (C]O),1669,1569
(C]C), 1429 (NeH), 1248 (SieCH3), 1086 (CeOeC), 837 (NeH), 738
(CeH), 695 (C]C). Anal. calcd for C16H20Cl2N2O3SSi: C, 45.82; H,
4.81; Cl, 16.91; N, 6.68; O, 11.44; S, 7.65; Si, 6.70. Found: C, 45.92; H,
4.86; N, 6.67; S, 7.60%. MS (EI): m/z (%) ¼ 418.0 (0.1) [M]þ, 345.9 (19)
[M ꢄ Si(Me)3]þ, 248.9 (11) [M ꢄ O(CH2)2Si(Me)3]þ, 142.0 (7)
[SC6H3Cl]þ, 73.0 (9) [Si(Me)3]þ.
1-(2-trimethylsilyl)ethoxymethyl-6-(phenylthio)uracil
(2
{1,1,1,1}) was also synthesized through the methodology D, but
carried out by using 0.368 mL (3.61 mmol) of thiophenol 10{1}. The
residue was washed with pentane to give 106 mg (0.30 mmol, 84%)
of 2{1,1,1,1}. m.p. ¼157e158 ꢁC. 1H NMR (300 MHz, CDCl3)
d (ppm):
8.41 (br s, 1H, NH), 7.59e7.45 (m, 5H, AreH), 5.53 (s, 2H,
NeCH2eO), 5.00 (d, 4J ¼ 2.1 Hz, 1H, C5-H), 3.76e3.71 (m, 2H,
CH2eCH2eSi(Me)3), 1.04e0.99 (m, 2H, CH2eCH2eSi(Me)3), 0.05 (s,
4.7.2.4. 1-(2-Trimethylsilyl)ethoxymethyl-6-(3-chlorophenylthio)
uracil (2{1,1,1,4}). The procedure used was the methodology C, but
carried out by using 100 mg (0.36 mmol) of 6-chloro-1-(2-
trimethylsilyl)ethoxymethyluracil (8{1,1,1}), 518 mg (1.81 mmol)
of bis(3-chlorophenyl) disulfide (9{4}) and 130 mg (3.44 mmol) of
NaBH4 in MeOH (5 mL). The residue was washed with hexane to
give 107 mg (0.28 mmol, 77%) of 1-(2-trimethylsilyl)ethoxymethyl-
9H, Me). 13C-RMN (75.5 MHz, CDCl3)
d (ppm): 161.3 (C4), 160.5
(C6), 151.0 (C2), 136.0 (ArC3), 131.1 (ArC4), 130.3 (ArC2), 126.4
(ArC1), 98.9 (C5), 73.3 (NeCH2eO), 67.3 (CH2eCH2eSi(Me)3),
18.1 (CH2eCH2eSi(Me)3), ꢄ1.3 (CH3). IR (film,
n
cmꢄ1): 3139
(NeH), 3015 (Csp2eH), 2950, 2895, 2842 (Csp3eH), 1719, 1666 (C]
O), 1560 (C]C), 1459 (NH), 1248 (SieCH3), 1080 (CeOeC), 750
(CeH), 691 (C]C). Anal. calcd for C16H22N2O3SSi: C, 54.83; H, 6.33;
N, 7.99; O, 13.69; S, 9.15; Si, 8.01. Found: C, 54.84; H, 6.52; N, 7.85; S,
9.41%. MS (EI): m/z (%) ¼ 350.0 (3) [M]þ, 277.0 (33) [M ꢄ Si(Me)3]þ,
233.0 (7) [M ꢄ O(CH2)2Si(Me)3]þ, 109.0 (29) [SC6H5]þ, 73.0 (100)
[Si(Me)3]þ.
6-(3-chlorophenylthio)uracil (2{1,1,1,4}) as
a
white solid.
m.p. ¼118e120 ꢁC. 1H NMR (300 MHz, CDCl3)
d
(ppm): 8.40 (br s,
1H, NH), 7.58e7.43 (m, 4H, AreH), 5.52 (s, 2H, NeCH2eO), 5.03 (d,
4J ¼ 2.1 Hz, 1H, C5-H), 3.74e3.69 (m, 2H, CH2eCH2eSi(Me)3),
1.03e0.98 (m, 2H, CH2eCH2eSi(Me)3), 0.05 (s, 9H, Me). 13C NMR
(75.5 MHz, CDCl3)
d (ppm): 161.1 (C4), 159.4 (C6), 150.8 (C2), 135.8
4.7.2.2. 1-(2-Trimethylsilyl)ethoxymethyl-6-(3-aminophenylthio)-
uracil (2{1,1,1,2}). The procedure used was the methodology C, but
carried out by using 100 mg (0.36 mmol) of 6-chloro-1-(2-
trimethylsilyl)ethoxymethyluracil (8{1,1,1}), 448 mg (1.80 mmol)
of bis(3-aminophenyl) disulfide (9{2}) and 130 mg (3.44 mmol) of
NaBH4 in MeOH (5 mL). The residue was purified by flash column
chromatography on silica gel with a mixture of acetonitrile and
dichloromethane (1:2, v/v) as eluent. 118 mg (0.33 mmol, 90%) of
1-(2-trimethylsilyl)ethoxymethyl-6-(3-aminophenylthio)uracil (2
{1,1,1,2}) were obtained as a pale yellow solid. m.p. ¼169e171 ꢁC.
(ArC3), 135.6 (ArC2)*, 134.0 (ArC5)*, 131.4 (ArC6)*, 131.3 (ArC4)*,
128.3 (ArC1), 99.5 (C5), 73.4 (NeCH2eO), 67.3 (CH2eCH2eSi(Me)3),
18.1 (CH2eCH2eSi(Me)3), ꢄ1.3 (Me). IR (film,
n
cmꢄ1): 3184 (NeH),
3036 (Csp2eH), 2953, 2894 (Csp3eH),1717,1691 (C]O),1668, 1566,
1402 (C]C), 1448 (NeH), 1248 (SieCH3), 1085 (CeOeC), 859
(NeH), 836, 782 (Si-CH3), 683 (C]C). Anal. calcd for
C16H21ClN2O3SSi: C, 49.92; H, 5.50; Cl, 9.21; N, 7.28; O, 12.47; S,
8.33; Si, 7.30. Found: C, 50.03; H, 5.48; N, 7.18; S, 8.47%. MS (EI): m/z
(%) ¼ 384.0 (1) [M]þ, 311.0 (66) [M ꢄ Si(Me)3]þ, 267.0 (9)
[M ꢄ O(CH2)2Si(Me)3]þ, 143.0 (8) [SC6H4Cl]þ, 73.0 (21) [Si(Me)3]þ.
1H NMR (300 MHz, CDCl3)
d (ppm): 9.32 (br s, 1H, NeH), 7.23 (t,
3J ¼ 7.8 Hz, 1H, ArC5eH), 6.91 (d, 3J ¼ 7.8 Hz, 1H, ArC6eH), 6.83 (t,
4J ¼ 2.1 Hz, 1H, ArC2eH), 6.78 (dd, 3J ¼ 7.8 Hz, 4J ¼ 2.1 Hz, 1H,
ArC4eH), 5.50 (s, 2H, NeCH2eO), 5.15 (s, 1H, C5-H), 3.86 (br s, 2H,
NH2), 3.75e3.69 (m, 2H, CH2eCH2eSi(Me)3), 1.03e0.98 (m, 2H,
CH2eCH2eSi(Me)3), 0.04 (s, 9H, Me). 13C NMR (75.5 MHz,
4.7.2.5. 1-Ethoxymethyl-6-(phenylthio)uracil (2{1,1,3,1}). The proce-
dure used was the methodology C, but carried out by using 100 mg
(0.49 mmol) of 6-chloro-1-ethoxymethyluracil (8{1,1,3}), 532 mg
(2.44 mmol) of bisphenyl disulfide (9{1}) and 176 mg (4.65 mmol) of
NaBH4 in MeOH (5 mL). The residue was washed with hexane to give
114 mg (0.41 mmol, 84%) of 1-ethoxymethyl-6-(phenylthio)uracil (2
{1,1,3,1}) as a white solid. m.p. ¼161e163 ꢁC. 1H NMR (300 MHz,
CDCl3)
d (ppm): 161.3 (C4), 160.7 (C6), 151.0 (C2), 148.0 (ArC3),
131.0 (ArC5), 126.8 (ArC1), 125.5 (ArC6), 121.5 (ArC2), 117.5 (ArC4),
98.7 (C5), 73.2 (NeCH2eO), 67.2 (CH2eCH2eSi(Me)3), 18.1
CDCl3) d (ppm): 8.34 (br s, 1H, NH), 7.59e7.48 (m, 5H, AreH), 5.55 (s,
(CH2eCH2eSi(Me)3), ꢄ1.3 (Me). IR (film,
n
cmꢄ1): 3415, 3338
2H, NeCH2eO), 5.01 (d, 4J ¼ 1.8 Hz, 1H, C5-H), 3.71 (q, 3J ¼ 7.2 Hz, 2H,
(NH2), 3126 (NeH), 3057 (Csp2eH), 2997, 2952, 2848, 2799
(Csp3eH), 1713, 1663 (C]O), 1593, 1558 (C]C), 1462, 1413 (NeH),
1249 (SieCH3), 1078 (CeOeC) 859, 836 (NeH), 757 (CeH), 687
(C]C). MS (EI): m/z (%) ¼ 365.1 (5) [M]þ, 292.0 (18)
[M ꢄ Si(Me)3]þ, 248.0 (11) [M-O(CH2)2Si(Me)3]þ, 124.0 (13)
[SC6H5NH]þ, 73.0 (100) [Si(Me)3]þ.
CH2eCH3), 1.28 (t, 3J ¼ 7.2 Hz, 3H, CH2eCH3). 13C NMR (75.5 MHz,
CDCl3): d (ppm): 161.1 (C4), 160.4 (C6), 151.0 (C2), 136.0 (ArC3), 131.1
(ArC4), 130.3 (ArC2), 126.4 (ArC1), 99.1 (C5), 73.6 (NeCH2eO), 65.3
(CH2eCH3), 15.1 (CH2eCH3). IR (KBr,
n
cmꢄ1): 3143 (NeH), 3070,
3023 (Csp2eH), 2975, 2916, 2882, 2843, 2799 (Csp3eH), 1718, 1675
(C]O), 1660, 1558, 1407 (C]C), 1455 (NeH), 1096 (CeOeC), 886

R. Puig-de-la-Bellacasa et al. / European Journal of Medicinal Chemistry 54 (2012) 159e174
169
(NeH), 756 (CeH), 693 (C]C). Anal. calcd for C13H14N2O3S: C, 56.10;
H, 5.07; N, 10.06; O, 17.25; S, 11.52. Found: C, 56.14; H, 4.99; N, 10.05;
S, 11.46%. MS (EI): m/z (%) ¼ 278.0 (100) [M]þ, 249.0 (18) [M ꢄ Et]þ,
233.0 (30) [M ꢄ OEt]þ, 219.0 (35) [M ꢄ CH2OEt]þ, 109.0 (10)
[SC6H5]þ.
solid. m.p. ¼132e134 ꢁC. 1H NMR (300 MHz, CDCl3)
d
(ppm): 8.41
(br s, 1H, NH), 7.59e7.58 (m, 1H, ArC2eH), 7.51e7.40 (m, 3H,
ArC4eH, ArC5eH, ArC6eH), 5.53 (s, 2H, NeCH2eO), 5.05 (d,
4J ¼ 1.5 Hz, 1H, C5-H), 3.69 (q, 3J ¼ 7.2 Hz, 2H, CH2eCH3), 1.27 (t,
3J ¼ 7.2 Hz, 3H, CH2eCH3).13C NMR (75.5 MHz, CDCl3)
d (ppm): 160.9
(C4),159.3 (C6),150.9 (C2),135.9 (ArC3),135.5 (ArC2)*,133.9 (ArC5)*,
131.4 (ArC6)*, 131.4 (ArC4)*, 128.3 (ArC1), 99.7 (C5), 73.7
4.7.2.6. 1-Ethoxymethyl-6-(3-aminophenylthio)uracil (2{1,1,3,2}). The
procedure used was the methodology C, but carried out by using
100 mg (0.49 mmol) of 6-chloro-1-ethoxymethyluracil (8{1,1,3}),
607 mg (2.44 mmol) of bis(3-aminophenyl) disulfide (9{2}) and
176 mg (4.65 mmol) of NaBH4 in MeOH (5 mL). The residue was
purified by flash column chromatography on silica gel with
(NeCH2eO), 65.3 (CH2eCH3), 15.1 (CH2eCH3). IR (KBr,
n
cmꢄ1):
3125 (NeH), 3062 (Csp2eH), 2981, 2925, 2892, 2839, 2794 (Csp3eH),
1723, 1658 (C]O), 1562, 1551, 1404 (C]C), 1454 (NeH), 1096
(CeOeC), 906 (NeH), 781 (CeH), 684 (C]C). Anal. calcd for
C13H13ClN2O3S: C, 49.92; H, 4.19; Cl, 11.34; N, 8.96; O, 15.35; S, 10.25.
Found: C, 49.94; H, 4.04; N, 8.66; S, 10.17%. MS (EI): m/z (%) ¼ 312.0
(100) [M]þ, 267.0 (36) [M-OEt]þ, 253.0 (38) [M ꢄ CH2OEt]þ, 143.0
(28) [SC6H4Cl]þ, 108.0 (48) [SC6H4]þ.
a
mixture of acetonitrile and dichloromethane (1:1, v/v) as
eluent. 121 mg (0.41 mmol, 84%) of 1-ethoxymethyl-6-(3-
aminophenylthio)uracil (2{1,1,3,2}) were obtained as a pale yellow
solid. m.p. ¼193e195 ꢁC. 1H NMR (300 MHz, CDCl3)
d (ppm): 8.38
(br s, 1H, NeH), 7.24 (t, 3J ¼ 7.8 Hz, 1H, ArC5eH), 6.92 (ddd,
3J ¼ 7.8 Hz, 4J ¼ 1.5 Hz, 4J ¼ 0.9 Hz, 1H, ArC6eH), 6.85 (t, 4J ¼ 2.1 Hz,
1H, ArC2eH), 6.79 (ddd, 3J ¼ 7.8 Hz, 4J ¼ 2.1 Hz, 4J ¼ 0.9 Hz, 1H,
ArC4eH), 5.52 (s, 2H, NeCH2eO), 5.16 (d, 4J ¼ 2.1 Hz, 1H, C5-H), 3.83
(br s, 2H, NH2), 3.75 (q, 3J ¼ 7.2 Hz, 2H, CH2eCH3), 1.28 (t, 3J ¼ 7.2 Hz,
4.7.2.9. 1-(2,5-Dimethylbenzyl)-6-(phenylthio)uracil (2{1,1,4,1}). The
procedure used was the methodology C, but carried out by using
100 mg (0.38 mmol) of 6-chloro-1-(2,5-dimethylbenzyl)uracil (8
{1,1,4}), 410 mg (1.88 mmol) of bisphenyl disulfide (9{1}) and 136 mg
(3.58 mmol) of NaBH4 in MeOH (5 mL). The residue was washed
with hexane to give 116v (0.34 mmol, 91%) of 1-(2,5-
dimethylbenzyl)-6-(phenylthio)uracil (2{1,1,4,1}) as a white solid.
3H, CH2eCH3). 13C NMR (75.5 MHz, CDCl3)
d (ppm): 161.0 (C4), 160.6
(C6), 151.0 (C2), 148.0 (ArC3), 131.0 (ArC5),126.8 (ArC1), 125.5 (ArC6),
121.5 (ArC2), 117.5 (ArC4), 98.8 (C5), 73.6 (NeCH2eO), 65.3
m.p. ¼ 241e243 ꢁC. 1H NMR (300 MHz, CDCl3)
d (ppm): 8.34 (br s,
(CH2eCH3),15.1 (CH2eCH3). IR (KBr,
n
cmꢄ1): 3416, 3335 (NH2), 3125
1H, NH), 7.49e7.45 (m, 5H, AreH), 7.10 (d, 3J ¼ 7.8 Hz, 1H, BzC3-H),
7.03 (d, 3J ¼ 7.8 Hz, 1H, BzC4eH), 6.74 (s, 1H, BzC6eH), 5.24 (s, 2H,
CH2), 5.03 (s, 1H, C5eH), 2.36 (s, 3H, Me), 2.33 (s, 3H, Me). 13C NMR
(NeH), 2979, 2901, 2841, 2800 (Csp3eH), 1713, 1669 (C]O), 1594,
1558 (C]C), 1461, 1409 (NeH), 1096 (CeOeC), 888 (NeH), 757
(CeH), 686 (C]C). Anal. calcd for C13H15N3O3S: C, 53.23; H, 5.15; N,
14.32; O, 16.36; S, 10.93. Found: C, 53.71; H, 5.24; N, 14.19; S, 10.70%.
MS (EI): m/z (%) ¼ 293.0 (30) [M]þ, 292.0 (35) [M ꢄ H]þ, 234.0 (98)
[M ꢄ CH2OEt]þ, 124.3 (8) [SC6H4NH2]þ. HRMS (EI): calcd. for
C13H15N3O3S: 293.0834, [M]þ, found: 293.0827.
(75.5 MHz, CDCl3)
d (ppm): 161.3 (C4), 160.9 (C6), 150.9 (C2), 136.0
(ArC3), 135.9 (BzC1)**, 132.6 (BzC5)**, 131.5 (BzC2)**, 131.2 (ArC4),
130.4 (BzC3)*, 130.3 (ArC2), 128.1 (BzC6)*, 126.1 (ArC1), 124.6 (BzC4)
*, 98.4 (C5), 45.8 (CH2), 21.4 (Me), 18.7 (Me). IR (KBr,
n
cmꢄ1): 3124
(NeH), 3064, 3000 (Csp2eH), 2905, 2841, 2799 (Csp3eH), 1717, 1650
(C]O), 1557, 1412 (C]C), 1458 (NeH), 813 (NeH), 749 (CeH), 688
(C]C). Anal. calcd for C19H18N2O2S: C, 67.43; H, 5.36; N, 8.28; O,
9.46; S, 9.48. Found: C, 67.68; H, 5.22; N, 8.17; S, 9.61%. MS (EI): m/z
(%) ¼ 338.1 (45) [M]þ, 229.1 (12) [M ꢄ SC6H5]þ, 119.0 (100) [C9H11]þ.
4.7.2.7. 1-Ethoxymethyl-6-(2,6-dichlorophenylthio)uracil (2{1,1,3,3}).
The procedure used was the methodology C, but carried out by using
100 mg (0.49 mmol) of 6-chloro-1-ethoxymethyluracil (8{1,1,3}),
868 mg (2.44 mmol) of bis(2,6-dichlorophenyl) disulfide (9{3}) and
176 mg (4.65 mmol) of NaBH4 in MeOH (5 mL). The residue was
purified by flash column chromatography on silica gel with a mixture
of ethyl acetate and hexane (1:2, v/v) as eluent. 87 mg (0.25 mmol,
51%) of 1-ethoxymethyl-6-(2,6-dichlorophenylthio)uracil (2{1,1,3,3})
were obtained as a white solid. m.p. ¼165e167 ꢁC. 1H NMR (300 MHz,
4.7.2.10. 1-(2,5-Dimethylbenzyl)-6-(3-aminophenylthio)uracil
(2
{1,1,4,2}). The procedure used was the methodology C, but carried
out by using 100 mg (0.38 mmol) of 6-chloro-1-(2,5-
dimethylbenzyl)uracil (8{1,1,4}), 469 mg (1.89 mmol) of bis(3-
aminophenyl) disulfide (9{2}) and 136 mg (3.60 mmol) of NaBH4
in MeOH (5 mL). The residue was washed with methanol to give
49 mg (0.14 mmol, 37%) of 1-(2,5-dimethylbenzyl)-6-(3-
aminophenylthio)uracil (2{1,1,4,2}) as a white solid. m.p. >288 ꢁC.
CDCl3)
d (ppm): 9.47 (br s, 1H, NH), 7.53e7.50 (m, 2H, ArC3eH),
7.43e7.38 (m, 1H, ArC4eH), 5.62 (s, 2H, NeCH2eO), 5.00 (s, 1H, C5-
H), 3.71 (q, 3J ¼ 7.2 Hz, 2H, CH2eCH3), 1.28 (t, 3J ¼ 7.2 Hz, 3H,
CH2eCH3). 13C NMR (75.5 MHz, CDCl3)
d
(ppm): 161.1 (C4), 156.4 (C6),
1H NMR (300 MHz, DMSO-d6)
d (ppm): 11.43 (br s, 1H, NH), 7.18 (t,
150.9 (C2), 141.7 (ArC2), 132.7 (ArC4), 129.4 (ArC3), 126.7 (ArC1), 99.0
3J ¼ 7.5 Hz, 1H, ArC5eH), 7.11 (d, 3J ¼ 7.9 Hz, 1H, BzC3-H), 7.00 (d,
3J ¼ 7.9 Hz, 1H, BzC4eH), 6.75e6.62 (m, 4H, ArC2eH, ArC4eH,
ArC6eH, BzC6eH), 5.52 (br s, 2H, NH2), 5.07 (s, 2H, CH2), 4.84 (d,
4J ¼ 2.1 Hz, 1H, C5-H), 2.28 (s, 3H, Me), 2.26 (s, 3H, Me). 13C NMR
(C5), 74.1 (NeCH2eO), 65.0 (CH2eCH3), 15.2 (CH2eCH3). IR (KBr,
n
cmꢄ1): 3165 (NeH), 3039 (Csp2eH), 2969, 2930, 2888, 2841
(Csp3eH), 1692 (C]O), 1579, 1566, 1427, 1411 (C]C), 1443 (NeH),
1190, 1164 (CeCl), 1097 (CeOeC), 877 (NeH), 790 (CeH), 660 (C]
C). Anal. calcd for C13H12Cl2N2O3S: C, 44.97; H, 3.48; Cl, 20.42; N, 8.07;
O,13.82; S, 9.23. Found: C, 45.39; H, 3.55; N, 8.22; S, 8.99%. MS (EI): m/z
(%) ¼ 346.0 (14) [M]þ, 311.0 (100) [M ꢄ 35Cl]þ, 142.0 (25) [SC6H3Cl]þ.
HRMS (EI): calcd. for C13H1235Cl2N2O3S: 345.9946, [M]þ, found:
345.9953; calcd. for C13H1235Cl37ClN2O3S: 345.9916, [M]þ, found:
345.9925.
(75.5 MHz, DMSO-d6)
d (ppm): 160.7 (C4), 159.3 (C6), 150.7 (C2),
150.4 (ArC3), 135.0 (BzC1)**, 133.6 (BzC5)**, 131.3 (BzC2)**, 130.7
(ArC5)*, 130.1 (BzC3)*, 127.4 (BzC6)*, 126.1 (ArC1), 124.0 (BzC4)*,
121.7 (ArC6)*, 119.4 (ArC2)*, 116.1 (ArC4)*, 97.5 (C5), 45.2 (CH2), 21.0
(Me), 18.2 (Me). IR (KBr,
n
cmꢄ1): 3425, 3346 (NH2), 3126 (NeH),
2990 (Csp2eH), 2922, 2847, 2800 (Csp3eH), 1710, 1660 (C]O),
1592, 1558 (C]C), 1468, 1419 (NeH), 814, 784 (NeH), 755 (CeH),
688 (C]C). MS (EI): m/z (%) ¼ 353.1 (46) [M]þ, 234.0 (32)
[M ꢄ C9H11]þ, 119.2 (100) [C9H11]þ. HRMS (EI): calcd. for
C19H19N3O2S: 353.1198, [M]þ, found: 353.1190.
4.7.2.8. 1-Ethoxymethyl-6-(3-chlorophenylthio)uracil (2{1,1,3,4}). The
procedure used was the methodology C, but carried out by using
100 mg (0.49 mmol) of 6-chloro-1-ethoxymethyluracil (8{1,1,3}),
700 mg (2.44 mmol) of bis(3-chlorophenyl) disulfide (9{4}) and
176 mg (4.65 mmol) of NaBH4 in MeOH (5 mL). The residue was
washed with hexane to give 126 mg (0.40 mmol, 82%) of 1-
ethoxymethyl-6-(3-chlorophenylthio)uracil (2{1,1,3,4}) as a white
4.7.2.11. 1-(2,5-Dimethylbenzyl)-6-(2,6-dichlorophenylthio)uracil (2
{1,1,4,3}). The procedure used was the methodology C, but carried
out by using 100 mg (0.38 mmol) of 6-chloro-1-(2,5-
dimethylbenzyl)uracil (8{1,1,4}), 673 mg (1.89 mmol) of bis(2,6-

170
R. Puig-de-la-Bellacasa et al. / European Journal of Medicinal Chemistry 54 (2012) 159e174
dichlorophenyl) disulfide (9{3}) and 136 mg (3.60 mmol) of NaBH4
in MeOH (5 mL). The residue was purified by flash column chro-
matography on silica gel with a mixture of ethyl acetate and chlo-
roform (1:9, v/v) as eluent. 105 mg (0.26 mmol, 68%) of 1-(2,5-
dimethylbenzyl)-6-(2,6-dichlorophenylthio)uracil (2{1,1,4,3}) as
a white solid. d.p. ¼ 265e267 ꢁC. 1H NMR (300 MHz, DMSO-d6)
2922, 2895, 2827 (Csp3eH), 1710, 1685 (C]O), 1583 (C]C), 1441
(NeH), 1249 (SieCH3), 1085 (CeOeC), 837 (NeH), 741 (CeH), 689
(C]C). Anal. calcd for C17H24N2O3SSi: C, 56.01; H, 6.64; N, 7.68; O,
13.17; S, 8.80; Si, 7.70. Found: C, 56.27; H, 6.75; N, 7.63; S, 8.74%. MS
(EI): v (%) ¼ 291.0 (21) [M ꢄ Si(Me)3]þ, 109.0 (22) [SC6H5]þ, 73.0
(100) [Si(Me)3]þ.
d
(ppm): 8.51 (br s, 1H, NH), 7.51e7.48 (m, 2H, ArC3eH), 7.43e7.37
(m, 1H, ArC4eH), 7.10 (d, 3J ¼ 7.5 Hz, 1H, BzC3-H), 7.02 (d,
3J ¼ 7.5 Hz, 1H, BzC4eH), 6.84 (s, 1H, BzC6eH), 5.27 (s, 2H, CH2),
4.92 (s, 1H, C5-H), 2.37 (s, 3H, Me), 2.30 (s, 3H, Me). 13C NMR
4.7.2.14. 1-(2-Trimethylsilyl)ethoxymethyl-6-(3-aminophenylthio)
thymine (2{1,2,1,2}). The procedure used was the methodology C,
but carried out by using 100 mg (0.34 mmol) of 6-chloro-1-(2-
(75.5 MHz, DMSO-d6)
d
(ppm): 160.9 (C4), 156.6 (C6), 150.5 (C2),
trimethylsilyl)ethoxymethylthymine
(8{1,2,1}),
427 mg
142.1 (ArC2), 135.8 (BzC1)**, 133.1 (ArC4), 132.3 (BzC5)**, 131.5
(BzC2)**, 130.4 (BzC3)*, 129.5 (ArC3), 128.2 (BzC6)*, 125.4 (ArC1),
(1.72 mmol) of bis(3-aminophenyl) disulfide (9{2}) and 124 mg
(3.27 mmol) of NaBH4 in MeOH (5 mL). The residue was purified by
flash column chromatography on silica gel with a mixture of
acetonitrile and dichloromethane (1:3, v/v) as eluent. 105 mg
(0.28 mmol, 81%) of 1-(2-trimethylsilyl)ethoxymethyl-6-(3-
aminophenylthio)thymine (2{1,2,1,2}) were obtained as a white
124.8 (BzC4)*, 97.1 (C5), 46.2 (CH2), 21.3 (Me), 18.8 (Me). IR (KBr,
n
cmꢄ1): 3121 (NeH), 3067, 3017 (Csp2eH), 2958, 2923, 2898, 2845,
2804 (Csp3eH), 1711, 1662 (C]O), 1559, 1422, 1409 (C]C), 1452
(NeH), 858 (NeH), 797 (CeH), 706 (C]C). MS (EI): m/z (%) ¼ 406.1
(2) [M]þ, 371.1 (5) [M ꢄ 35Cl]þ, 229.1 (1) [M ꢄ SC6H3Cl2]þ, 119.2
(100) [C9H11]þ. HRMS (EI): calcd. for C19H1635Cl2N2O2S: 406.0298,
[M]þ, found: 406.0310; calcd. for C19H1635Cl 37ClN2O2S: 408.0271,
[M]þ, found: 408.0280; calcd. for C19H1637Cl2N2O2S: 410.0248,
[M]þ, found: 408.0251.
solid. m.p. ¼169e171 ꢁC. 1H NMR (300 MHz, CDCl3)
d (ppm): 10.34
(br s, 1H, NH), 7.27 (t, 3J ¼ 7.8 Hz, 1H, ArC5eH), 6.78e6.71 (m, 3H,
ArC2eH, ArC4eH, ArC6eH), 5.70 (s, 2H, NeCH2eO), 4.12 (br s, 2H,
NH2), 3.85e3.79 (m, 2H, CH2eCH2eSi(Me)3), 2.25 (s, 3H, Me),
1.13e1.08 (m, 2H, CH2eCH2eSi(Me)3), 0.20 (s, 9H, Si(Me)3). 13C NMR
(75.5 MHz, CDCl3)
d (ppm): 162.7 (C4), 151.1 (C2), 147.5 (C6), 146.2
4.7.2.12. 1-(2,5-Dimethylbenzyl)-6-(3-chlorophenylthio)uracil
(2
(ArC3), 133.4 (ArC1), 130.3 (ArC5), 119.7 (C5), 117.4 (ArC6), 113.9
{1,1,4,4}). The procedure used was the methodology C, but carried
out by using 100 mg (0.38 mmol) of 6-chloro-1-(2,5-
dimethylbenzyl)uracil (8{1,1,4}), 540 mg (1.89 mmol) of bis(3-
chlorophenyl) disulfide (9{4}) and 136 mg (3.60 mmol) of NaBH4
in MeOH (5 mL). The residue was purified by flash column chro-
matography on silica gel with a mixture of ethyl acetate and hexane
(1:1, v/v) as eluent. 118 mg (0.32 mmol, 84%) of 1-(2,5-
(ArC2), 113.4 (ArC4), 74.3 (NeCH2eO), 66.6 (CH2eCH2eSi(Me)3),
18.1 (CH2eCH2eSi(Me)3), 13.9 (Me), ꢄ1.3 (Si(Me)3). IR (film,
n
cmꢄ1): 3464, 3367 (NH2), 3182 (NeH), 3040 (Csp2eH), 2953, 2924,
2895, 2830 (Csp3eH), 1688, 1627 (C]O), 1592 (C]C), 1482, 1450
(NeH), 1249 (SieCH3), 1083 (CeOeC) 858, 837 (NeH), 765 (CeH),
689 (C]C). MS (EI): m/z (%) ¼ 379.3 (12) [M]þ, 306.2 (39)
[M ꢄ Si(Me)3]þ, 124.0 (12) [SC6H5NH2]þ, 73.0 (100) [Si(Me)3]þ.
HRMS (EI): calcd. for C17H25N3O3SSi: 379.1386, [M]þ, found:
379.1375.
dimethylbenzyl)-6-(3-chlorophenylthio)uracil
(2{1,1,4,4})
as
a white solid. m.p. ¼ 240e241 ꢁC. 1H NMR (300 MHz, CDCl3)
d
(ppm): 9.42 (br s, 1H, NH), 7.49e7.46 (m, 1H, ArC2eH), 7.42e7.37
(m, 3H, ArC4eH, ArC5eH, ArC6eH), 7.09 (d, 3J ¼ 8.1 Hz, 1H,
BzC3eH), 7.02 (d, 3J ¼ 8.1 Hz, 1H, BzC4eH), 6.71 (s, 1H, BzC6eH),
5.23 (s, 2H, CH2), 5.08 (d, 4J ¼ 2.1 Hz, 1H, C5eH), 2.34 (s, 3H, Me),
4.7.2.15. 1-(2-Trimethylsilyl)ethoxymethyl-6-(2,6-dichlorophenylthio)
thymine (2{1,2,1,3}). The procedure used was the methodology C,
but carried out by using 100 mg (0.34 mmol) of 6-chloro-1-(2-
trimethylsilyl)ethoxymethylthymine (8{1,2,1}), 612 mg (1.72 mmol)
of bis(2,6-dichlorophenyl) disulfide (9{3}) and 124 mg (3.27 mmol)
of NaBH4 in MeOH (5 mL). The residue was purified by flash column
chromatography on silica gel with a mixture of ethyl acetate and
hexane (1:4, v/v) as eluent. 59 mg (0.14 mmol, 40%) of 1-(2-
trimethylsilyl)ethoxymethyl-6-(2,6-dichlorophenylthio)thymine (2
{1,2,1,3}) were obtained as a white solid. m.p. ¼176e177 ꢁC. 1H NMR
2.32 (s, 3H, Me). 13C NMR (75.5 MHz, CDCl3)
d (ppm): 161.1 (C4),
159.4 (C6), 150.8 (C2), 136.0 (ArC3)**, 135.9 (BzC1)**, 135.5 (ArC2)*,
133.9 (ArC5)*, 132.5 (BzC5)**, 131.5 (ArC6)*, 131.4 (BzC2)*, 131.3
(ArC4)*, 130.5 (BzC3)*, 128.2 (BzC6)*, 127.9 (ArC1), 124.6 (BzC4)*,
99.1 (C5), 45.9 (CH2), 21.4 (Me), 18.7 (Me). IR (KBr,
n
cmꢄ1): 3190,
3117 (NeH), 3062, 3001 (Csp2eH), 2950, 2913, 2847, 2805
(Csp3eH), 1718, 1655 (C]O), 1557, 1501, 1413 (C]C), 1460 (NeH),
900 (NeH), 781 (CeH), 680 (C]C). Anal. calcd for C19H17N2O2ClS:
C, 61.20; H, 4.60; Cl, 9.51; N, 7.51; O, 8.58; S, 8.60; Found: C, 6.21; H,
4.83; N, 7.44; S, 8.20%. MS (EI): m/z (%) ¼ 372.1 (2) [M]þ, 229.0 (74)
[M ꢄ SC6H3Cl]þ, 119.1 (100) [C9H11]þ.
(300 MHz, CDCl3)
d (ppm): 8.98 (br s, 1H, NH), 7.41e7.38 (m, 2H,
ArC3eH), 7.27e7.21 (m, 1H, ArC4eH), 5.76 (s, 2H, NeCH2eO),
3.67e3.61 (m, 2H, CH2eCH2eSi(Me)3), 1.62 (s, 3H, Me), 1.00e0.95
(m, 2H, CH2eCH2eSi(Me)3), 0.03 (s, 9H, Si(Me)3). 13C NMR
(75.5 MHz, CDCl3)
d (ppm):161.9 (C4), 150.4 (C2), 148.3 (C6), 138.5
4.7.2.13. 1-(2-Trimethylsilyl)ethoxymethyl-6-(phenylthio)thymine (2
{1,2,1,1}). The procedure used was the methodology D, but carried
out by using 100 mg (0.38 mmol) of 6-chloro-1-(2-trimethylsilyl)
ethoxymethylthymine (8{1,2,1}), 378 mg (3.43 mmol) of thio-
phenol (13{1}) and 248 mg (6.53 mmol) of NaBH4 in MeOH (5 mL).
The residue was washed with pentane to give 105 mg (0.30 mmol,
79%) of 1-(2-trimethylsilyl)ethoxymethyl-6-(phenylthio)thymine
(2{1,2,1,1}) as a white solid. m.p. ¼ 84e85 ꢁC. 1H NMR (300 MHz,
(ArC2), 130.3 (ArC1), 130.2 (ArC4), 129.1 (ArC3), 115.6 (C5), 74.3
(NeCH2eO), 66.6 (CH2eCH2eSi(Me)3), 18.1 (CH2eCH2eSi(Me)3),
12.9 (Me), ꢄ1.3 (Si(Me)3). IR (KBr,
n
cmꢄ1): 3160 (NeH), 3021
(Csp2eH), 2960, 2927, 2899, 2858, 2831 (Csp3eH),1714,1663 (C]O),
1586, 1555, 1424 (C]C), 1460 (NeH), 1249 (SieCH3), 1081 (CeOeC),
863, 835 (SieCH3), 776 (CeH). Anal. calcd for C17H22Cl2N2O3SSi: C,
47.11; H, 5.12; Cl, 16.36; N, 6.46; O, 11.07; S, 7.40; Si, 6.48. Found: C,
47.53; H, 5.43; N, 6.39; S: 7.62%. MS (EI): m/z (%) ¼ 358.9 (6)
[M ꢄ Si(Me)3]þ, 141.9 (23) [SC6H3Cl]þ, 73.0 (100) [Si(Me)3]þ. HRMS
(FAB): calcd. for C17H2335Cl2N2O3SSi: 433.0576, [M þ H]þ, found:
433.0588.
CDCl3)
d (ppm): 8.59 (br s, 1H, NH), 7.35e7.20 (m, 5H, AreH), 5.53
(s, 2H, NeCH2eO), 5.00 (d, 4J ¼ 2.1 Hz, 1H, C5-H), 3.63e3.58 (m, 2H,
CH2eCH2eSi(Me)3), 2.03 (s, 3H, Me), 0.90e0.85 (m, 2H,
CH2eCH2eSi(Me)3), 0.01 (s, 9H, Si(Me)3). 13C NMR (75.5 MHz,
CDCl3)
d
(ppm): 162.3 (C4), 150.8 (C2), 146.4 (C6), 132.8 (ArC1),
4.7.2.16. 1-(2-Trimethylsilyl)ethoxymethyl-6-(3-chlorophenylthio)
thymine (2{1,2,1,4}). The procedure used was the methodology
C, but carried out by using 100 mg (0.34 mmol) of 6-chloro-
129.6 (ArC3),127.9 (ArC2),127.2 (ArC4),119.6 (C5), 74.4 (NeCH2eO),
66.7 (CH2eCH2eSi(Me)3), 18.1 (CH2eCH2eSi(Me)3), 14.0 (Me), ꢄ1.3
(Si(Me)3). IR (film,
n
cmꢄ1): 3182 (NeH), 3053 (Csp2eH), 2953,
1-(2-trimethylsilyl)ethoxymethylthymine
(8{1,2,1}),
494 mg

R. Puig-de-la-Bellacasa et al. / European Journal of Medicinal Chemistry 54 (2012) 159e174
171
(1.72 mmol) of bis(3-chlorophenyl) disulfide (9{4}) and 124 mg
(3.27 mmol) of NaBH4 in MeOH (5 mL). The residue was purified by
flash column chromatography on silica gel with a mixture of ethyl
acetate and hexane (1:3, v/v) as eluent. 136 mg (0.34 mmol, 99%) of
1-(2-trimethylsilyl)ethoxymethyl-6-(3-chlorophenylthio)uracil (2
{1,2,1,4}) were obtained as a white solid. m.p. ¼ 98e100 ꢁC. 1H NMR
146.1 (ArC3), 133.4 (ArC1), 130.4 (ArC5), 119.9 (C5), 117.6(ArC6), 114.0
(ArC2), 113.5 (ArC4), 74.6 (NeCH2eO), 64.8 (CH2eCH3), 15.1
(CH2eCH3), 14.0 (Me). IR (film,
n
cmꢄ1): 3463, 3372 (NH2), 3151
(NeH), 3014 (Csp2eH) 2977, 2923, 2878, 2820 (Csp3eH), 1703, 1646
(C]O), 1591 (C]C), 1482, 1450, 1420 (NeH), 1093 (CeOeC), 893, 854
(NeH), 764 (CeH), 682 (C]C). MS (EI): m/z (%) ¼ 307.1 (26) [M]þ,
248.0 (59) [M ꢄ CH2OEt]þ. HRMS (EI): calcd. for C14H17N3O3S:
307.0990, [M]þ, found: 307.0988.
(300 MHz, CDCl3)
d (ppm): 9.29 (br s, 1H, NH), 7.29e7.19 (m, 3H,
AreH), 7.11e7.08 (m, 1H, AreH), 5.53 (s, 2H, NeCH2eO), 3.63e3.57
(m, 2H, CH2eCH2eSi(Me)3), 2.05 (s, 3H, Me), 0.88e0.83 (m, 2H,
CH2eCH2eSi(Me)3), 0.01 (s, 9H, (Me)3). 13C NMR (75.5 MHz, CDCl3)
4.7.2.19. 1-Ethoxymethyl-6-(2,6-dichlorophenylthio)thymine (2{1,2,3,
3}). The procedure used was the methodology C, but carried out by
using 100 mg (0.46 mmol) of 6-chloro-1-ethoxymethylthymine (8
{1,2,3}), 815 mg (2.29 mmol) of bis(2,6-dichlorophenyl) disulfide (9
{3}) and 165 mg (4.36 mmol) of NaBH4 in MeOH (5 mL). The residue
was purified by flash column chromatography on silica gel with
a mixture of ethyl acetate and chloroform (1:9, v/v) as eluent. 15 mg
(0.32 mmol, 70%) of 1-ethoxymethyl-6-(2,6-dichlorophenylthio)
d
(ppm): 162.2 (C4), 150.8 (C2), 145.2 (C6), 135.4 (ArC3)**, 134.7
(ArC1)**, 130.6 (ArC2)*, 127.4 (ArC5)*, 127.3 (ArC6)*, 125.7 (ArC4)*,
120.3 (C5), 74.5 (NeCH2eO), 66.8 (CH2eCH2eSi(Me)3), 18.1
(CH2eCH2eSi(Me)3), 14.0 (Me), ꢄ1.3 (Si(Me)3). IR (KBr,
n
cmꢄ1):
3155 (NeH), 3018 (Csp2eH), 2953, 2923, 2894, 2862, 2837
(Csp3eH), 1715, 1693 (C]O), 1591, 1575, 1426 (C]C), 1461 (NeH),
1250 (SieCH3), 1086 (CeOeC), 836, 755 (SieCH3), 772 (CeH), 677
(C]C). Anal. calcd for C17H23ClN2O3SSi: C, 51.18; H, 5.81; Cl, 8.89; N,
7.02; O, 12.03; S, 8.04; Si, 7.04. Found: C, 51.43; H, 5.78; N, 7.21; S,
8.26%. MS (EI): m/z (%) ¼ 325.0 (44) [M ꢄ Si(Me)3]þ, 281.0 (10)
[M ꢄ O(CH2)2Si(Me)3]þ, 108.0 (3) [SC6H4]þ, 73.0 (13) [Si(Me)3]þ.
thymine (2{1,2,3,3}) were obtained as
a
white solid.
m.p. ¼ 206e207 ꢁC.1H NMR (300 MHz, CDCl3)
d (ppm): 9.41 (br s,1H,
NH), 7.41e7.38 (m, 2H, ArC3eH), 7.27e7.21 (m,1H, ArC4eH), 5.78 (s,
2H, NeCH2eO), 3.62 (q, 3J ¼ 7.2 Hz, 2H, CH2eCH3), 1.64 (s, 3H, Me),
1.23 (t, 3J ¼ 7.2 Hz, 3H, CH2eCH3). 13C NMR (75.5 MHz, CDCl3)
4.7.2.17. 1-Ethoxymethyl-6-(phenylthio)thymine
(2{1,2,3,1}). The
d (ppm): 162.0 (C4),150.6 (C2),148.1 (C6),138.5 (ArC2),130.19 (ArC1),
procedure used was the methodology C, but carried out by using
100 mg (0.47 mmol) of 6-chloro-1-ethoxymethylthymine (8
{1,2,3}), 500 mg (2.29 mmol) of bisphenyl disulfide (9{1}) and
165 mg (4.36 mmol) of NaBH4 in MeOH (5 mL). The residue was
washed with hexane to give 102 mg (0.35 mmol, 76%) of 1-
ethoxymethyl-6-(phenylthio)thymine (2{1,2,3,1}) as a white solid.
This compound was also synthesized through the methodology
D, but carried out by using 600 mg (2.74 mmol) of 6-chloro-1-
ethoxymethylthymine (8{1,2,3}), 2.80 mL (27.4 mmol) of thio-
phenol (10{1}) and 1.98 g (52.1 mmol) of NaBH4 in MeOH (30 mL).
The residue was washed with hexane to give 708 mg (2.41 mmol,
88%) of 2{1,2,3,1}. m.p. ¼132e133 ꢁC. 1H NMR (300 MHz, CDCl3)
130.17 (ArC4), 129.1 (ArC3), 115.7 (C5), 74.7 (NeCH2eO), 64.6
(CH2eCH3), 15.1 (CH2eCH3), 12.9 (Me). IR (KBr,
n
cmꢄ1): 3158,
3089 (NeH), 3016 (Csp2eH), 2969, 2916, 2885, 2835 (Csp3eH),1705,
1665 (C]O), 1585, 1555, 1425 (C]C), 1463 (NeH), 1096 (CeOeC),
885 (NeH), 777 (CeH). Anal. calcd for C14H14Cl2N2O3S: C, 46.55; H,
3.91; Cl, 19.63; N, 7.75; O, 13.29; S, 8.88. Found: C, 46.61; H, 3.83; N,
7.61; S, 8.50%. MS (EI): m/z (%) ¼ 360.0 (17) [M]þ, 325.0 (35)
[M ꢄ 35Cl]þ, 141.9 (98) [SC6H3Cl]þ.
4.7.2.20. 1-Ethoxymethyl-6-(3-chlorophenylthio)thymine (2{1,2,3,4}).
The procedure used was the methodology C, but carried out by using
100 mg (0.46 mmol) of 6-chloro-1-ethoxymethylthymine (8{1,2,3}),
657 mg (2.29 mmol) of bis(3-chlorophenyl) disulfide (9{4}) and
165 mg (4.36 mmol) of NaBH4 in MeOH (5 mL). The residue was
washed with pentane to give 128 mg (0.39 mmol, 86%) of 1-
ethoxymethyl-6-(3-chlorophenylthio)thymine (2{1,2,3,4}) as a white
d
(ppm): 8.56 (br s, 1H, NH), 7.37e7.23 (m, 5H, AreH), 5.57 (s, 2H,
NeCH2eO), 3.61 (q, 3J ¼ 7.2 Hz, 2H, CH2eCH3), 2.06 (s, 3H, Me), 1.17
(t, 3J ¼ 7.2 Hz, 3H, CH2eCH3). 13C NMR (75.5 MHz, CDCl3):
d (ppm):
162.3 (C4), 151.0 (C2), 146.2 (C6), 132.7 (ArC1), 129.6 (ArC3), 127.9
(ArC2), 127.2 (ArC4), 119.8 (C5), 74.7 (NeCH2eO), 64.8 (CH2eCH3),
solid. m. p. 126e127 ꢁC. 1H NMR (300 MHz, CDCl3)
d (ppm): 8.57 (br
15.0 (CH2eCH3), 14.0 (Me). NOESY (300 MHz, CDCl3)
(AreH, NeCH2eO), (AreH, Me), (NeCH2eO, CH2eCH3). IR (film,
d
(ppm):
s, 1H, NH), 7.24e7.20 (m, 3H, AreH), 7.11e7.08 (m, 1H, AreH), 5.53
(s, 2H, NeCH2eO), 3.58 (q, 3J ¼ 6.9 Hz, 2H, CH2eCH3), 2.06 (s, 3H, Me),
1.13 (t, 3J ¼ 6.9 Hz, 3H, CH2eCH3). 13C NMR (75.5 MHz, CDCl3)
n
cmꢄ1): 3178 (NeH), 3049 (Csp2eH), 2977, 2928, 2881, 2824
(Csp3eH), 1708, 1680 (C]O), 1582 (C]C), 1441 (NeH), 1094
(CeOeC), 853 (NeH), 742 (CeH), 689 (C]C). Anal. calcd for
C14H16N2O3S: C, 57.52; H, 5.52; N, 9.58; O, 16.42; S, 10.97. Found: C,
57.82; H, 5.59; N, 9.49; S, 10.89%. MS (EI): m/z (%) ¼ 291.9 (64) [M]þ,
246.8 (26) [M ꢄ OEt]þ, 232.8 (100) [M ꢄ CH2OEt]þ, 109.0 (74)
[SC6H5]þ. HRMS (ESI-TOF): calcd. for C14H16N2NaO3S: 315.0774,
[M þ Na]þ, found: 315.0785.
d
(ppm): 162.3 (C4), 151.0 (C2), 145.1 (C6), 135.4 (ArC3)**, 134.6 (ArC1)
**,130.5 (ArC2)*,127.4 (ArC5)*,127.3 (ArC6)*,125.7 (ArC4)*,120.5 (C5),
74.8 (NeCH2eO), 64.9 (CH2eCH3), 15.0 (CH2eCH3), 14.0 (Me). IR (KBr,
n
cmꢄ1): 3146, 3117, 3081 (NeH), 3020 (Csp2eH), 2975, 2931,
2890, 2865, 2820 (Csp3eH), 1713, 1686 (C]O), 1586, 1574, 1565, 1418
(C]C), 1453 (NeH), 1094 (CeOeC), 884 (NeH), 774 (CeH), 682 (C]
C). Anal. calcd for C14H15ClN2O3S: C, 51.45; H, 4.63; Cl, 10.85; N, 8.57;
O, 14.69; S, 9.81. Found: C, 51.38; H, 4.49; N, 8.44; S, 9.65%. MS (EI): m/
z (%) ¼ 326.0 (100) [M]þ, 267.0 (33) [M ꢄ CH2OEt]þ, 143.0 (4)
[SC6H4Cl]þ, 108.0 (48) [SC6H4]þ.
4.7.2.18. 1-Ethoxymethyl-6-(3-aminophenylthio)thymine (2{1,2,3,2}).
The procedure used was the methodology C, but carried out by using
300 mg (1.37 mmol) of 6-chloro-1-ethoxymethylthymine (8{1,2,3}),
1.70 g (6.87 mmol) of bis(3-aminophenyl) disulfide (9{2}) and 495 mg
(13.1 mmol) of NaBH4 in MeOH (15 mL). The residue was purified by
flash column chromatography on silica gel with a mixture of ethyl
acetate and hexane (2:1, v/v) as eluent. 159 mg (0.52 mmol, 38%) of 1-
ethoxymethyl-6-(3-aminophenylthio)thymine (2{1,2,3,2}) were
obtained as a white solid. m.p. ¼143e144 ꢁC. 1H NMR (300 MHz,
4.7.2.21. 1-(2,5-Dimethylbenzyl)-6-(phenylthio)thymine (2{1,2,4,1}).
The procedure used was the methodology C, but carried out by using
100 mg (0.36 mmol) of 6-chloro-1-(2,5-dimethylbenzyl)thymine (8
{1,2,4}), 390 mg (1.79 mmol) of bisphenyl disulfide (9{1}) and 129 mg
(3.40 mmol) of NaBH4 in MeOH (5 mL). The residue was washed with
hexane to give 101 mg (0.29 mmol, 80%) of 1-(2,5-dimethylbenzyl)-
CDCl3)
d
(ppm): 8.49 (br s, 1H, NH), 7.08 (t, 3J ¼ 7.8 Hz, 1H, ArC5eH),
6-(phenylthio)thymine
(2{1,2,4,1})
as
a
white
solid.
6.59e6.48 (m, 3H, ArC2eH, ArC4eH, ArC6eH), 5.52 (s, 2H,
NeCH2eO), 3.73 (br s, 2H, NH2), 3.60 (q, 3J ¼ 7.2 Hz, 2H, CH2eCH3),
2.07 (s, 3H, Me), 1.17 (t, 3J ¼ 7.2 Hz, 3H, CH2eCH3). 13C
m.p. ¼ 216e217 ꢁC.1H NMR (300 MHz, CDCl3)
d (ppm): 8.69 (br s,1H,
NH), 7.30e7.19 (m, 3H, ArC4eH, ArC2eH or ArC3eH), 7.11e7.08 (m,
2H, ArC2eH or ArC3eH), 7.01 (d, 3J ¼ 7.5 Hz, 1H, BzC3eH), 6.93 (d,
3J ¼ 7.5 Hz, 1H, BzC4eH), 6.52 (s, 1H, BzC6eH), 5.24 (s, 2H, CH2), 2.23
NMR (75.5 MHz, CDCl3)
d (ppm): 162.4 (C4), 151.0 (C2), 147.5 (C6),

172
R. Puig-de-la-Bellacasa et al. / European Journal of Medicinal Chemistry 54 (2012) 159e174
(s, 6H, 2 BzeMe), 2.11 (s, 3H, Me). 13C NMR (75.5 MHz, CDCl3)
(ppm): 162.5 (C4), 150.9 (C2), 146.7 (C6), 135.6 (BzC1)**, 134.1
chlorophenyl) disulfide (9{4}) and 129 mg (3.40 mmol) of NaBH4
in MeOH (5 mL). The residue was washed with hexane to give
122 mg (0.32 mmol, 88%) of 1-(2,5-dimethylbenzyl)-6-(3-
chlorophenylthio)thymine (2{1,2,4,4}) as a white solid. m. p.
d
(ArC1), 132.3 (BzC5)**, 131.3 (BzC2)**, 130.2 (BzC3)*, 129.6 (ArC3),
127.7 (BzC6)*, 127.6 (ArC2), 127.3 (ArC4)*, 124.8 (BzC4)*, 119.7 (C5),
47.3 (CH2), 21.3 (BzeMe), 18.7 (BzeMe), 14.4 (Me). NOESY (300 MHz,
211e213 ꢁC. 1H NMR (300 MHz, CDCl3)
d (ppm): 8.61 (br s, 1H, NH),
CDCl3):
d
(ppm): (CH2, ArC3eH), (CH2, ArC2eH). IR (film,
n
cmꢄ1):
7.17e7.15 (m, 2H, ArC2eH, ArC6eH), 7.00e6.90 (m, 4H, BzC3eH,
BzC4eH, ArC4eH, ArC5eH), 6.50 (s, 1H, BzC6eH), 5.24 (s, 2H,
3166 (NeH), 3021 (Csp2eH), 2921, 2858, 2825 (Csp3eH), 1683 (C]
O), 1582, (C]C), 1452 (NeH), 855 (NeH), 740 (CeH). Anal. calcd for
C20H20N2O2S: C, 68.16; H, 5.72; N, 7.95; O, 9.08; S, 9.10. Found: C,
67.85; H, 5.69; N, 7.94; S, 8.90%. MS (EI): m/z (%) ¼ 352.2 (9) [M]þ,
243.2 (2) [M ꢄ SC6H5]þ, 233.2 (2) [M ꢄ C9H11]þ, 119.0 (100) [C9H11]þ.
CH2), 2.24 (s, 3H, BzeMe), 2.22 (s, 3H, BzeMe), 2.12 (s, 3H, Me). 13
C
NMR (75.5 MHz, CDCl3)
d (ppm): 162.4 (C4), 150.9 (C2), 145.6 (C6),
135.7 (ArC3)**, 135.4 (BzC1)**, 134.0 (ArC1)**, 133.8 (BzC5)**, 131.3
(BzC2)**, 130.4 (ArC2)*, 130.3 (BzC3)*, 127.9 (ArC5)*, 127.5 (BzC6)*,
127.1 (ArC6)*, 125.4 (ArC4)*, 124.9 (BzC4)*, 120.3 (C5), 47.4 (CH2),
4.7.2.22. 1-(2,5-Dimethylbenzyl)-6-(3-aminophenylthio)thymine (2
{1,2,4,2}). The procedure used was the methodology C, but carried
out by using 100 mg (0.36 mmol) of 6-chloro-1-(2,5-
dimethylbenzyl)thymine (8{1,2,4}), 445 mg (1.79 mmol) of bis(3-
aminophenyl) disulfide (9{2}) and 129 mg (3.40 mmol) of NaBH4
in MeOH (5 mL). The residue was washed with methanol to give
102 mg (0.28 mmol, 78%) of 1-(2,5-dimethylbenzyl)-6-(3-
aminophenylthio)thymine (2{1,2,4,2}) as a white solid. 1H NMR
21.3 (BzeMe), 18.7 (BzeMe), 14.4 (C22). IR (KBr, n
cmꢄ1): Two
crystalline structures were obtained: 3189 (NeH), 3056 (Csp2eH),
2922, 2857 (Csp3eH), 1699, 1678 (C]O), 1582, 1572, 1563, 1502,
1436, 1401 (C]C), 1458 (NeH), 775 (CeH), 679 (C]C). 3144 (NeH),
3011 (Csp2eH), 2918, 2861, 2820 (Csp3eH), 1714, 1653 (C]O), 1576,
1500, 1420, (C]C), 1460 (NeH), 775 (CeH), 675 (C]C).Anal. calcd
for C20H19N2O2ClS: C, 62.09; H, 4.95; Cl, 9.16; N, 7.24; O, 8.27; S,
8.29; Found: C, 61.83; H, 4.90; N, 7.12; S, 8.51%. MS (EI): m/z (%) ¼
386.0 (9) [M]þ, 119.0 (100) [C9H11]þ.
(300 MHz, CDCl3)
d (ppm): 8.47 (br s, 1H, NH), 7.07e7.02 (m, 2H,
ArC5eH, BzC3eH), 6.95 (d, 3J ¼ 7.8 Hz, 1H, BzC4eH), 6.54e6.48 (m,
3H, ArC2eH, ArC6eH, BzC6eH), 6.34e6.32 (m, 1H, ArC4eH), 5.22
(s, 2H, CH2), 3.70 (br s, 2H, NH2), 2.25 (s, 3H, BzeMe), 2.24 (s, 3H,
4.8. Preparation of HEPT analogues (12{w,x,y,z})
BzeMe), 2.13 (s, 3H, Me). 13C NMR (75.5 MHz, CDCl3)
d
(ppm): 162.0
4.8.1. Methodology E
(C4), 150.5 (C2), 147.5 (C6), 146.8 (ArC3), 135.6 (BzC1)**, 134.2
(ArC1), 133.0 (BzC5)**, 131.4 (BzC2)**, 130.4 (ArC5)*, 130.3 (BzC3)*,
127.7 (BzC6)*, 124.8 (BzC4)*, 119.6 (C5), 117.6 (ArC6), 114.2 (ArC2),
113.3 (ArC4), 47.3 (CH2), 21.4 (BzeMe), 18.8 (BzeMe), 14.4 (Me). IR
To a solution of 2{w,x,y,z} in toluene was added Lawesson’s
reagent (LR) and the mixture was refluxed for 2 h. The solution was
allowed to cool to room temperature and the mixture was filtered.
The filtrate was evaporated in vacuo obtaining a yellow solid, which
was dissolved in chloroform and washed with saturated NaHCO3
solution. The organic layer was dried over anhydrous magnesium
sulfate, filtered and concentrated to dryness. The residue was
purified by flash column chromatography on silica gel with the
corresponding eluent.
(KBr, n
cmꢄ1): 3449, 3368 (NH2), 3146 (NeH), 3016 (Csp2eH), 2914,
2828 (Csp3eH), 1683 (C]O), 1630, 1591 (C]C), 1456, 1417 (NeH),
881 (NeH), 742 (CeH), 688 (C]C). MS (EI): v (%) ¼ 367.2 (30)
[M]þ, 248.0 (28) [M ꢄ C9H11]þ, 119.1 (100) [C9H11]þ.
HRMS (EI): calcd. for C20H21N3O2S: 367.1354, [M]þ, found:
367.1355.
4.8.1.1. 1-Ethoxymethyl-3,4-dihydro-5-methyl-6-(phenylthio)-4-
thioxopyrimidin-2(1H)-one (12{1,2,3,1}). The procedure used was
the methodology E, but carried out by using 30 mg (0.1 mmol) of 1-
4.7.2.23. 1-(2,5-Dimethylbenzyl)-6-(2,6-dichlorophenylthio)thymine
(2{1,2,4,3}). The procedure used was the methodology C, but carried
out by using 100 mg (0.36 mmol) of 6-chloro-1-(2,5-
dimethylbenzyl)thymine (8{1,2,4}), 637 mg (1.79 mmol) of bis(2,6-
dichlorophenyl) disulfide (9{3}) and 129 mg (3.40 mmol) of NaBH4
in MeOH (5 mL). The residue was purified by flash column chro-
matography on silica gel with a mixture of ethyl acetate and chlo-
roform (1:9, v/v) as eluent. A 140 mg (0.33 mmol, 93%) of 1-(2,5-
dimethylbenzyl)-6-(2,6-dichlorophenylthio)thymine (2{1,2,4,3}) as
ethoxymethyl-6-(phenylthio)thymine
(2{1,2,3,1}),
122 mg
(0.3 mmol) of LR in toluene (1 mL) and heating with microwaves
during 3 min at 110 ꢁC. The residue was purified by flash column
chromatography on silica gel with a mixture of ethyl acetate and
hexane (1:2, v/v) as eluent. 22.0 mg (0.071 mmol, 71%) of 1-
ethoxymethyl-3,4-dihydro-5-methyl-6-(phenylthio)-4-
thioxopyrimidin-2(1H)-one (12{1,2,3,4}) were obtained as a yellow
a
d
white solid. m.p. ¼ 275e276 ꢁC. 1H NMR (300 MHz, CDCl3)
(ppm): 8.37 (br s, 1H, NH), 7.32e7.31 (m, 2H, ArC3eH), 7.16e7.11
solid. 1H NMR (300 MHz, CDCl3)
d (ppm): 10.28 (br s, 1H, NH), 7.28
(m, 5H, AreH), 5.59 (s, 2H, NeCH2eO), 3.62 (q, 3J ¼ 7.2 Hz, 2H,
(m, 1H, ArC4eH), 7.02 (d, 3J ¼ 7.5 Hz, 1H, BzC3eH), 6.94 (d,
3J ¼ 7.5 Hz, 1H, BzC4eH), 6.55 (s, 1H, BzC6eH), 5.46 (s, 2H, CH2), 2.30
(s, 3H, BzeMe), 2.27 (s, 3H, BzeMe), 1.80 (s, 3H, Me). 13C NMR
CH2eCH3), 2.27 (s, 3H, Me), 1.16 (t, 3J ¼ 7.2 Hz, 3H, CH2eCH3). 13C
NMR (75.5 MHz, CDCl3)
d (ppm): 188.8 (C4), 148.5 (C6), 144.2
(C2), 132.5 (ArC1), 129.7(ArC2), 128.2 (ArC3), 127.5 (ArC4), 126.6
(75.5 MHz, CDCl3)
d
(ppm): 162.1 (C4), 150.6 (C2), 148.3 (C6), 138.5
(C5), 75.4 (NeCH2eO), 65.1 (CH2eCH3), 19.3 (CH2eCH3), 15.1 (Me).
(ArC2), 135.7 (BzC1)**, 133.7 (BzC5)**, 131.4 (BzC2)**, 130.33 (ArC4)*,
130.27 (BzC3)*,129.4 (ArC1),129.1 (ArC3),127.9 (BzC6)*,124.7 (BzC4)
*, 116.1 (C5), 47.9 (CH2), 21.3 (BzeMe), 18.7 (BzeMe), 13.5 (Me). IR
IR (KBr, n
cmꢄ1): 3188 (NeH), 3084 (Csp2eH), 2971, 2925,
2884 (Csp3eH), 1673 (C]O), 1568, 1420 (C]C), 1466 (NeH), 1114
(C]S), 1068 (CeOeC), 871 (NeH), 739 (CeH), 688 (C]C). MS (EI): v
(%) ¼ 308.0 (51) [M]þ, 264.0 (100) [M ꢄ C2H4O]þ, 249.0 (73)
[M ꢄ CH2OEt]þ, 231.1 (19) [M ꢄ C6H5]þ. HRMS (EI): calcd for
C14H16N2O2S2: 308.0653, [M]þ, found: 308.0660.
(KBr,
n
cmꢄ1): 3154 (NeH), 3028 (Csp2eH), 2958, 2919, 2854,
2816 (Csp3eH), 1694, 1669 (C]O), 1589, 1556, 1429, 1419 (C]C),
1456 (NeH), 870 (NeH), 779 (CeH), 745 (C]C). Anal. calcd for
C20H18Cl2N2O2S: C, 57.01; H, 4.31; Cl, 16.83; N, 6.65; O, 7.59; S, 7.61.
Found: C, 57.27; H, 4.70; N, 6.43; S, 7.25%. MS (EI): m/z (%) ¼ 176.9
(10) [SC6H3Cl2]þ, 119.1 (100) [C9H11]þ, 91.0 (15) [C7H7]þ.
4.8.1.2. 6-(3-Chlorophenylthio)-1-(ethoxymethyl)-3,4-dihydro-5-
methyl-4-thioxopyrimidin-2(1H)-one (12{1,2,3,4}). The proce-
dure used was the methodology E, but carried out by using 33 mg
(0.1 mmol) of 1-ethoxymethyl-6-(3-chlorophenylthio)thymine
(2{1,2,3,4}), 121 mg (0.3 mmol) of LR in toluene (1 mL). The
residue was purified by flash column chromatography on silica
gel with a mixture of ethyl acetate and hexane (1:2, v/v) as eluent.
4.7.2.24. 1-(2,5-Dimethylbenzyl)-6-(3-chlorophenylthio)thymine (2
{1,2,4,4}). The procedure used was the methodology C, but carried
out by using 100 mg (0.36 mmol) of 6-chloro-1-(2,5-
dimethylbenzyl)thymine (8{1,2,4}), 514 mg (1.79 mmol) of bis(3-

R. Puig-de-la-Bellacasa et al. / European Journal of Medicinal Chemistry 54 (2012) 159e174
173
18.8 mg (0.055 mmol, 55%) of 6-(3-chlorophenylthio)-1-(ethox-
ymethyl)-3,4-dihydro-5-methyl-4-thioxopyrimidin-2(1H)-one
(12{1,2,3,4}) were obtained as a yellow solid. m.p. ¼101e103 ꢁC.
2842 (Csp3eH), 1716, 1686 (C]O), 1595, 1581, 1426 (C]C), 1480
(NeH), 1082 (CeOeC), 873 (NeH), 743 (CeH). Anal. calcd for
C14H16N2O4S: C, 54.53; H, 5.23; N, 9.08; O, 20.75; S, 10.40. Found: C,
54.84; H, 5.29; N, 9.21; S, 10.86%. MS (EI): m/z (%) ¼ 308.1 (2) [M]þ,
247.1 (7) [M ꢄ O(CH2)2OH]þ.
1H NMR (300 MHz, CDCl3)
d (ppm): 10.23 (br s, 1H, NH), 7.30e7.21
(m, 3H, AreH), 7.11e7.07 (m, 1H, AreH), 5.57 (s, 2H, NeCH2eO),
3.61 (q, 3J ¼ 7.2 Hz, 2H, CH2eCH3), 2.30 (s, 3H, Me), 1.15
(t, 3J ¼ 7.2 Hz, 3H, CH2eCH3). 13C NMR (75.5 MHz, CDCl3)
4.9.4. 1-[(2-Hydroxyethoxy)methyl]-6-(3-chlorophenylthio)
thymine (19)
d
(ppm): 188.7 (C4), 148.5 (C6), 142.7 (C2), 135.5 (ArC3)**, 134.4
(ArC1)**, 130.7 (ArC2)*, 127.7 (ArC5, ArC6)*, 127.1 (C5), 126.0
The procedure used was the methodology C, but carried out by
using 126 mg (0.46 mmol) of 1-[(2-acetoxyethoxy)methyl]-6-
chlorothymine (18), 658 mg (2.29 mmol) of bisphenyl disulfide (9
{4}) and 330 mg (8.75 mmol) of NaBH4 in MeOH (5 mL). Purifica-
tion of the residue by flash chromatography (silica, hexaneeethyl
acetate; 100:0 to 0:100 in 25 min) to afford 140 mg (0.41 mmol,
89%) of 1-[(2-hydroxyethoxy)methyl]-6-(3-chlorophenylthio)
thymine (19) as a white solid. m.p. ¼ 80e82 ꢁC. 1H NMR (400 MHz,
(ArC4)*, 75.5 (NeCH2eO), 65.2 (CH2eCH3), 19.3 (CH2eCH3), 15.0
(Me). IR (film,
n
cmꢄ1): 3177 (NeH), 3075 (Csp2eH), 2977,
2919 (Csp3eH), 1696 (C]O), 1563, 1441, 1414, (C]C), 1460 (NeH),
1117 (C]S), 1062 (CeOeC), 887 (NeH), 774 (CeH), 677 (C]C). MS
(EI): m/z (%) ¼ 342.0 (57) [M]þ, 298.0 (100) [M ꢄ OEt]þ, 283.0 (61)
[M-CH2OEt]þ. HRMS (EI): calcd. for C14H1535ClN2O2S2: 342.0263,
[M]þ, found: 342.0267; calcd. for C14H1537ClN2O2S2: 344.0234,
[M]þ, found: 344.0239.
CDCl3)
2H, NeCH2eO), 3.67 (s, 4H, CH2eCH2eOH), 2.08 (s, 3H, Me). 13C
NMR (100.6 MHz, CDCl3) (ppm): 162.6 (C4), 151.5 (C2), 144.6 (C6),
d (ppm): 9.30 (s, 1H, NH), 7.27e7.09 (m, 4H, AreH), 5.59 (s,
4.9. Preparation of HEPT and HEPT analogue 19
d
135.5 (ArC3)**, 134.5 (ArC1)**, 130.7 (ArC2)*, 127.5 (ArC5)*, 127.3
4.9.1. Diacetate of 2eoxa-1,4-butanediol (17)
The compound was synthesized according to the literature
procedure [36e38].
(ArC6)*, 125.6 (ArC4)*, 120.9 (C5), 75.2 (NeCH2eO), 70.6
(CH2eCH2eOH), 61.4 (CH2eCH2eOH), 13.9 (Me). IR (KBr,
n
cmꢄ1):
3420 (OeH), 3164 (NeH), 3039 (Csp2eH), 2925 (Csp3eH), 1704,
1643 (C]O), 1574 (C]C), 1456 (NeH), 1082 (CeOeC), 887 (NeH),
775 (CeH). Anal. calcd for C14H15ClN2O4S: C, 49.05; H, 4.41; Cl,
10.34; N, 8.17; O, 18.67; S, 9.35. Found: C, 49.20; H, 4.75; N, 7.90; S,
9.26%. MS (EI): m/z (%) ¼ 342.0 (1) [M]þ.
4.9.2. 1-[(2-Acetoxyethoxy)methyl]-6-chlorothymine (18)
To a stirred mixture of 6-chlorothymine (6{1,2}) (642 mg,
4.0 mmol) and diacetate of 2-oxa-1,4-butanediol (17) (1.057 g,
6.0 mmol) in anhydrous CH2Cl2 (10 mL), BSA (2.4 mL, 9.6 mmol)
was added dropwise. After stirring at room temperature for
4.10. Anti-HIV assays
30 min., the resulting solution was cooled to 0 ꢁC, SnCl4 (80
mL,
0.8 mmol) was added, and the mixture left to stir at room
temperature overnight, then poured into cold saturated NaHCO3
solution and extracted with CH2Cl2. The combined organic phases
were washed with brine (100 mL), dried over MgSO4, and evapo-
rated in vacuo to dryness. Purification of the residue by flash
chromatography (silica, hexaneeethyl acetate; 100:0 to 25:75 in
27 min) to afford 422 mg (1.52 mmol, 38%) of 1-[(2-acetoxyethoxy)
methyl]-6-chlorothymine (18) as a white solid. m.p. ¼ 98e100 ꢁC.
HIV-1 NL4-3 strain was titered in MT-4 cells after acute infection
and infectivity was measured by evaluation the cytopathic effect
that induced after 5-day cultures as described [41]. Anti-HIV activity
(EC50) and cytotoxicity (CC50) measurements in MT-4 cells were
based on the viability of cells that had been infected or not infected
with HIV-1, all were exposed to various concentrations of the test
compound. After the MT-4 cells were allowed to proliferate for 5
days, the numberof viable cells was quantified bytetrazolium-based
colorimetric method (MTT method) as described.
1H NMR (400 MHz, CDCl3)
d (ppm): 9.16 (br s, 1H, NH), 5.54 (s, 2H,
NeCH2eO), 4.25e4.20 (m, 2H, CH2eCH2eOAc), 3.87e3.81 (m, 2H,
CH2eCH2eOAc), 2.075 (s, 3H, Me)*, 2.072 (s, 3H, Me)*. 13C NMR
Acknowledgements
(100.6 MHz, CDCl3)
(C2), 143.1 (C6), 110.7 (C5), 75.0 (NeCH2eO), 67.7 (CH2eCH2eOAc),
63.1 (CH2eCH2eOAc), 20.8 (OeCOeCH3),12.1 (Me). IR (KBr,
cmꢄ1):
d (ppm): 170.8 (OeCOeCH3), 161.8 (C4), 150.3
This work was supported in part by the Fundació Marató de TV3
project 020930 and Spanish Ministerio de Ciencia e Innovación
project SAF2010-C21617-C02 (J.I.B. and B.C.). R. Puig de la Bellacasa
holds an FI scholarship from Generalitat de Catalunya and S. Pet-
tersson from IQS.
n
3215 (NeH), 3017 (Csp2eH), 2825 (Csp3eH), 1736, 1724, 1674 (C]
O), 1605, 1426, (C]C), 1455 (NeH), 1099 (CeOeC). Anal. calcd for
C10H13N2O5Cl: C, 43.41; H, 4.74; Cl, 12.81; N, 10.13; O, 28.91. Found:
C, 43.85; H, 4.78; N, 9.71%. MS (EI): m/z (%) ¼ 276.1 (1) [M]þ, 173.0
(9) [M ꢄ O(CH2)2OAc]þ, 117.1 (50) [CH2O(CH2)2OAc]þ, 87.1 (100)
[(CH2)2OAc]þ.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2012.04.038. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
4.9.3. 1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)thymine (1)
The procedure used was the methodology C, but carried out by
using 126 mg (0.46 mmol) of 1-[(2-acetoxyethoxy)methyl]-6-
chlorothymine (18), 500 mg (2.29 mmol) of bisphenyl disulfide (9
{1}) and 330 mg (8.75 mmol) of NaBH4 in MeOH (5 mL). The residue
was washed with hexane to give 107 mg (0.35 mmol, 76%) of 1-[(2-
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