Modified 5'-Trityl Nucleosides as Inhibitors of Plasmodium falciparum dUTPase

Article abstract of DOI:10.1002/cmdc.201000445

2'-Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) is a potential drug target for the treatment of malaria. We previously reported the discovery of 5'-tritylated analogues of deoxyuridine as selective inhibitors of this Plasmodium falciparum enzyme. Herein we report further structure-activity studies; in particular, variations of the 5'-trityl group, the introduction of various substituents at the 3'-position of deoxyuridine, and modifications of the base. Compounds were tested against both the enzyme and the parasite. Variations of the 5'-trityl group and of the 3'-substituent were well tolerated and yielded active compounds. However, there is a clear requirement for the uracil base for activity, because modifications of the uracil ring result in loss of enzyme inhibition and significant decreases in antiplasmodial action. Fewer trips to the dUMP: dUTPase is a potential drug target for the treatment of malaria. We previously reported the discovery of 5'-tritylated analogues of deoxyuridine as selective inhibitors of this P.falciparum enzyme. Herein we report further structure-activity studies of the 5'-trityl group, the introduction of various substituents at the 3'-position of deoxyuridine, and modifications of the base.

Full text of DOI:10.1002/cmdc.201000445

DOI: 10.1002/cmdc.201000445
Modified 5’-Trityl Nucleosides as Inhibitors of Plasmodium
falciparum dUTPase
Gian Filippo Ruda,[b] Corinne Nguyen,[b] Przemysław Ziemkowski,[c] Krzysztof Felczak,[c, d]
Ganasan Kasinathan,[b] Alexander Musso-Buendia,[e] Christian Sund,[f] Xiao Xiong Zhou,[f]
Marcel Kaiser,[g] Luis M. Ruiz-Pꢀrez,[e] Reto Brun,[g] Tadeusz Kulikowski,[c]
Nils Gunnar Johansson,[f] Dolores Gonzꢁlez-Pacanowska,[e] and Ian H. Gilbert*[a, b]
2’-Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) is
a potential drug target for the treatment of malaria. We previ-
ously reported the discovery of 5’-tritylated analogues of deox-
yuridine as selective inhibitors of this Plasmodium falciparum
enzyme. Herein we report further structure–activity studies; in
particular, variations of the 5’-trityl group, the introduction of
various substituents at the 3’-position of deoxyuridine, and
modifications of the base. Compounds were tested against
both the enzyme and the parasite. Variations of the 5’-trityl
group and of the 3’-substituent were well tolerated and yield-
ed active compounds. However, there is a clear requirement
for the uracil base for activity, because modifications of the
uracil ring result in loss of enzyme inhibition and significant
decreases in antiplasmodial action.
Introduction
Malaria is a widespread, life-threatening tropical disease, with
over 500 million clinical cases per year. It causes over a million
deaths each year, mainly amongst children in sub-Saharan
Africa.[1] Due to drug resistance developed by Plasmodium fal-
ciparum (the causative agent of the most severe form of malar-
ia) against current treatments, there is an urgent need for new
drugs that act on new molecular targets in order to circumvent
the problem of increasing resistance.
ure 1A), 3’-modified 5’-trityl nucleosides, and some base-modi-
fied trityl nucleosides (Figure 1B,C).
Such structures would further probe the P. falciparum dUT-
Pase active site, giving new insight into the development of
more potent and selective inhibitors. Herein we present the
synthesis of the listed structures and their biological evaluation
against the P. falciparum dUTPase and the parasite cultured in
vitro.
A potential target of interest is the enzyme 2’-deoxyuridine
triphosphate nucleotidohydrolase (dUTPase), which is an es-
sential enzyme in nucleotide metabolism. The role of dUTPase
is to hydrolyse dUTP to dUMP. This has two major effects on
nucleotide metabolism: first, it decreases the level of dUTP;
second, the enzyme provides a source of dUMP for biosynthe-
sis of dTMP, a precursor of dTTP. The combination of these ef-
fects decreases the dUTP/dTTP ratio, and hence reduces the er-
roneous incorporation of uracil into DNA by DNA polymerases,
as these enzymes can substitute dUTP for dTTP.
[a] Prof. I. H. Gilbert
Current address: Division of Biological Chemistry and Drug Discovery
College of Life Sciences, University of Dundee
Sir James Black Centre, Dundee, DD1 5EH (UK)
Fax: (+44)1382-386-373
E-mail: i.h.gilbert@dundee.ac.uk
[b] Dr. G. F. Ruda,+ Dr. C. Nguyen,+ Dr. G. Kasinathan, Prof. I. H. Gilbert
Welsh School of Pharmacy, Cardiff University
King Edward VII Avenue, Cardiff, CF10 3XF (UK)
[c] Dr. P. Ziemkowski, Dr. K. Felczak, Prof. T. Kulikowski
Institute of Biochemistry and Biophysics, Polish Academy of Sciences
There is good evidence that dUTPase is an essential enzyme.
Gene inactivation has been reported to be lethal in several or-
ganisms such as S. cerevisiae[2] and E. coli,[3] and therefore inhib-
ition of the enzyme could represent a new avenue for chemo-
therapeutic development.
´
Pawinskiego 5A, 02-106 Warszawa (Poland)
[d] Dr. K. Felczak
Current address: University of Minnesota, Center for Drug Design
7-159 Phillips-Wangensteen Building
516 Delaware Street SE, Minneapolis, MN 55455 (USA)
[e] A. Musso-Buendia, Prof. L. M. Ruiz-Pꢀrez, Prof. D. Gonzꢁlez-Pacanowska
Instituto de Parasitologꢂa y Biomedicina, Consejo Superior de Investiga-
ciones Cientꢂficas, Parque Tecnolꢃgico de Ciencias de la Salud
Avenida del Conocimiento, 18100 Armilla (Granada) (Spain)
We previously reported a series of 5’-tritylated nucleosides
as inhibitors of dUTPase that are leads for the development of
potential medicines for the treatment of malaria.[4,5] These
compounds are highly selective for the Plasmodium enzyme
over the human enzyme. Further SAR studies were performed
that show it is possible to prepare “acyclic” analogues in which
the deoxyribose ring is replaced by an aliphatic linker, whilst
retaining activity.[6] Herein we report additional studies to
extend our SAR analysis to 5’-substituted trityl-2’-deoxyuridine
analogues, 5’-heterotrityl-2’-deoxyuridine analogues (Fig-
[f] C. Sund, X. X. Zhou, Dr. N. G. Johansson
Medivir AB, P.O. Box 1086, SE-14122 Huddinge (Sweden)
[g] M. Kaiser, Dr. R. Brun
Swiss Tropical and Public Health Institute
Socinstrasse 57, 4002 Basel (Switzerland)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000445.
ChemMedChem 2011, 6, 309 – 320
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
309

I. H. Gilbert et al.
MED
Figure 1. The modified nucleosides synthesised in this work (compounds 1a, 1b, 14a, 14e, and 15 were reported previously;[4,5] data are reported here for
comparative purposes).
Chemistry
5’-O-tosyl-,[10] or 5’-deoxy-5’-halonucleoside.[11] To our knowl-
edge there is no reported application of the Mitsunobu proto-
col for the introduction of S-trityl groups into nucleosides. We
successfully applied this method for the synthesis of 5’-S-tri-
tylthio-5’,2’-dideoxyuridine 1c (Scheme 1).
Substituted trityl analogues
Several modified 5’-trityl nucleosides were prepared from com-
mercially available trityl chlorides (e.g., 4-methoxytrityl, 4,4’-di-
methoxytrityl) by starting from either 2’-deoxyuridine or 5’-
amino-2’-deoxyuridine. The latter was synthesised in a three-
step procedure[7] (Scheme 1).
Heterocyclic trityl derivatives
We investigated the replacement
of one of the phenyl rings with
an aromatic heterocycle, in par-
ticular, the 4-pyridyl and 5-pyri-
midyl derivatives. These hetero-
cycles are similar in size to the
phenyl group; they may favour
additional interactions with the
enzyme active site through their
nitrogen heteroatoms, should
improve water solubility, and
should also provide increased
stability in acidic medium.
Scheme 1. a) TsCl, Py, 08C, 5 h; b) NaN3, DMF, 908C, 16 h; c) H2, 5% Pd/C, EtOH/H2O; d) Ar3C-Cl, Py, DMAP, 40–
508C, 12–92 h, or Bn3SiCl, Py, 08C, 2.5–3 h; e) PPh3, Tr-SH, DEAD, 1,4-dioxane; f) Ar3C-Cl, Py, 40–508C, 12–48 h.
Diphenyl(4-pyridyl)methyl
chloride itself was prepared from
the
corresponding
carbinol
The modified trityl groups were introduced by using the
general conditions reported for the synthesis of 5’-tritylated
nucleosides[8] (heating in pyridine with, if necessary, catalytic 4-
dimethylaminopyridine (DMAP)),[9] Scheme 1. The tribenzylsilyl
derivatives 8a,b were synthesised by the same procedure from
the commercially available chloride (Scheme 1).
(Scheme 2) by following the method of Tzerpos et al.[12] First
diphenyl(4-pyridyl)methanol was converted into its hydrochlo-
ride, which was then treated with a mixture of thionyl chloride
and acetyl chloride at room temperature to produce the de-
sired chloride 20a in quantitative yield. However, diphenyl(4-
pyridyl)methyl chloride decomposed rapidly in contact with
moisture, which limited storage even under vacuum. 5’-Amino-
2’-deoxyuridine was successfully reacted with 20a in the pres-
ence of triethylamine to form the diphenyl(4-pyridyl)methyl-
A 5’-S-trityl group is usually introduced into the nucleoside
by direct tritylation of 5’-deoxy-5’-thionucleoside in the pres-
ence of base[10] or by reaction of trityl thiol with 5’-O-mesyl-,[10]
310
www.chemmedchem.org
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 309 – 320

5’-Trityl Nucleoside dUTPase Inhibitors
Scheme 2. a) HCl/H2O, reflux, 2 h; b) SOCl2, AcCl, RT, 72 h; c) 5’-amino-2’,5’-di-
deoxyuridine, TEA, Py, 40–708C; d) SOCl2/AcCl, RT, 72 h; e) 5’-amino-2’,5’-di-
deoxyuridine, Py, TEA, microwave (1 min at 808C, 1 min at 1008C); f) 2’-deoxy-
uridine, Py, DMAP, microwave (2ꢃ1 min at 1008C).
amino target 7a. The diphenyl(5-pyrimidyl)methoxy analogue
7b and the diphenyl(5-pyrimidyl)methylamino derivative 7c
were similarly prepared from diphenyl(5-pyrimidyl)methyl chlo-
ride hydrochloride 20b (prepared by reacting the correspond-
ing carbinol[13] 19b with thionyl chloride and acetyl chloride at
room temperature).
Scheme 3. a) Benzoyl chloride (R: C6H5), tolyl chloride (R: 4-CH3-C6H4), para-
chlorobenzoyl chloride (4-Cl-C6H4), para-methoxybenzoyl chloride (R: 4-
(OCH3)-C6H4), meta-trifluoromethylbenzoyl chloride (R: 3-(CF3)-C6H4), ortho-
fluorobenzoyl chloride (R: 2-F-C6H4), 2-furoyl chloride (R: 2-furoyl), Py, 80–
908C, 1.5 h; b) same as in a), N,N-dimethylaniline instead of Py; c) AcBr,
PhMe, reflux, 1.5 h; d) 2’-deoxyuridine, Py, 808C, 3 h, or 2’-deoxyuridine, Py,
microwave (2ꢃ30 s at 1008C).
Polysubstituted trityl groups
We decided to extend the range of trityl groups ex-
amined, with some polysubstituted trityl groups. To
extend application of trityl analogues, Sekine et al. in-
troduced tris[4-(acyloxy)trityl] groups, which are base
labile.[14–16] Only reports of acetoxy,[17] benzoyloxy,[15]
and 4-methoxybenzoyl[16] derivatives have been pub-
lished so far. We extended the reported methodology
for the synthesis of a new series of aroyloxy ana-
logues by starting from the commercially available
aurine 21 (rosolic acid), as outlined in Scheme 3.
Starting from the corresponding aroyl chlorides, a
series of tris[4-(substituted)]trityl methanols were pre-
pared (22a–g). Treatment of compounds 22a–g with
acetyl bromide in toluene converted them into to
the corresponding bromides 23a[14] and 23b–g.
These were then coupled with the 2’-deoxyuridine in
pyridine by conventional heating for compound
Scheme 4. a) TrCl, DMAP, Py, 808C, 3 h; b) CH2N2/Et2O, CH3OH; c) Py, NCS (X=Cl), or NBS
(X=Br).
6a,b, or by microwave irradiation with two cycles of
30 s at 1008C, with the addition of extra tritylating
agent after the first cycle, to give compounds 6c–g
(Scheme 3).
1a to 5-bromo-5’-O-trityl-2’-deoxyuridine 10a with elemental
bromine was unsuccessful and led only to decomposition. N-
Halo-substituted succinimides in pyridine have been reported
for the synthesis of 5-halogenated N-1-propargyluracils.[21]
Modified base analogues
Bases modified at the 5-position, 5-fluoro-5’-O-trityl-2’-deoxyur-
idine[18] 10b and 5-ethyl-5’-O-trityl-2’-deoxyuridine[19] 10d,
were synthesised in good yield by starting from their respec-
tive parent nucleosides: 5-ethyl-2’deoxyuridine[20] 24a and 5-
fluoro-2’-deoxyuridine 24b (Scheme 4). Direct bromination of
Chlorination of 1a with N-chlorosuccinimide (NCS) by this
method led to 5-chloro-5’-O-trityl-2’-deoxyuridine 10c in good
yield (Scheme 4). The 5-bromo analogue[22] 10a was prepared
in the same way with N-bromosuccinimide (NBS) instead of
NCS. Direct treatment of 1a with diazomethane resulted in
ChemMedChem 2011, 6, 309 – 320
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
311

I. H. Gilbert et al.
MED
quantitative synthesis of 3-methyl-5’-O-trityl-2’-deoxyuridine
11 a, which was then used as the starting material for the syn-
thesis of 3-methyl-5-bromo-5’-O-trityl-2’-deoxyuridine 11 b and
3-methyl-5-chloro-5’-O-trityl-2’-deoxyuridine 11 c.
The 5’-trityl-2’-deoxyuridine 1a was converted into the 3’-di-
phenyl phosphate ester 14a by reaction with diphenylchloro-
phosphate.[5] The azido derivative 14b was prepared by mesy-
lation, and then formation of the 5’-trityl anhydro nucleoside
29. This was opened with lithium azide (generated in situ from
lithium fluoride and azidotrimethylsilane in the presence of tet-
ramethylethylenediamine (TMEDA), as suggested by Kirschen-
heuter and colleagues[28]) to form 14b with retention of 2’-de-
oxyribose stereochemistry at the 3’-position. In the next stage,
azide 14b was converted into the amine 14c by mild hydroge-
nation using Lindlar’s catalyst. Previous attempts with 10%
and 5% Pd/C were unsuccessful due to the lability of the trityl
ether under the hydrogenation conditions. The 3’-amino nu-
cleoside 14c was then reacted with acetic anhydride to pro-
duce the acetamide derivative 14d.
Our efforts in the direct thiation of 1a with Lawesson’s re-
agent[23] were unsuccessful. Therefore, we decided to tritylate
4-thio-2’-deoxyuridine 27 to 4-thio-5’-O-trityl-2’-deoxyuridine[24]
9. Lawesson’s-reagent-promoted thiation of 5’,3’-di-O-p-tolyl-2’-
deoxyuridine[25] 25 resulted in the formation of 4-thio-5’,3’-di-
O-p-tolyl-2’-deoxyuridine 26 in high yield. This intermediate
was deprotected with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
in methanol into 27, which was used directly for tritylation.
Methylation of 9 into its 4-methylthio derivative 12 was carried
out in very good yield with methyl iodide (Scheme 5).
Results and Discussion
Biological results
All the compounds were evaluated against recombinant P. fal-
ciparum and human dUTPases in order to estimate inhibition
constants and selectivity. They were also screened against cul-
tures of the parasite to evaluate in vitro IC50 values.
Enzyme inhibition
The lead compounds for the inhibition of P. falciparum dUT-
Pase are 5’-O-trityl-2’-deoxyuridine 1a and 5’-tritylamino-2’-de-
oxyuridine 1b, the latter of which is the most potent inhibitor
reported so far (Ki =0.2 mm).[4–6] The thio-linked analogue 1c
exhibited activity against the enzyme similar to that of the
oxygen-linked analogue 1a, but with slightly better selectivity
(Table 1).
Scheme 5. a) Lawesson’s reagent, dioxane; b) DBU, CH3OH; c) TrCl, DMAP,
Py; d) MeI, NaOH, TDA-1, dioxane.
Modified 3’-nucleosides
Modifications of the 3’-position of the sugar ring were based
on the synthetic approaches developed by Horwitz and co-
workers[26] for the synthesis of AZT and by Kumar et al.[27] for
the synthesis of threothymidine, as illustrated in Scheme 6.
Methoxy, nitrile, and chloro substituents on the trityl group
(compounds 1–5) were generally well tolerated, with the 4-cya-
notrityl compounds 3a and 3b and the 4-methoxytrityl 2b
presenting the best Ki values in the sub-micromolar range. Al-
though the inhibition constants are similar, but not better than
that of the lead 1b, all these substituted trityl derivatives were
selective for the Plasmodium dUTPase over the human
enzyme.
None of the polysubstituted trityl derivatives 6a–g showed
any activity, with Ki values all greater than 1000 mm. This may
be due to steric hindrance or hydrolysis with concomitant loss
of the trityl group.
The “heterotrityl” derivatives 7a–c had similar activity to the
lead compounds. The pyridyldiphenyl derivative 7a was the
most active, with a Ki value of 0.23 mm comparable with the
lead 1b. Remarkably, all heterotrityl nucleosides 7a–c were
more selective for the parasitic enzyme, with inhibition con-
stants >1000 mm for the human dUTPase, and consequently
high selectivity indexes (Kihuman/KiP.fal.).
Scheme 6. a) TrCl, Py, 508C, 12 h; b) CH3SO2Cl, Py, 08C then RT, 4 h;
c) (PhO)2P(O)Cl, Py, RT, 12 h; d) DBU, CH2Cl2, RT, 30 h; e) LiF, TMEDA, Me3SiN3,
DMF, 1108C, 20 h; f) H2, Lindlar’s catalyst, EtOH, 5 h; g) Ac2O, TEA, CH2Cl2, RT,
3 h.
Replacement of the oxygen atom in the 5’-position with an
amino group improved the activity in some cases, as shown by
the pairs 1a/1b and 2a/2b, whilst in other cases the effect
312
www.chemmedchem.org
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 309 – 320

5’-Trityl Nucleoside dUTPase Inhibitors
Table 1. Biological results for the modified 5’-trityl nucleosides.
Table 2. Biological results (enzymatic and cellular) for the 3’-modified nu-
cleosides.
Ki [mm]
IC50 [mm]
L6 cells[b]
Compd
P. fal.
Human
P. fal.[a]
Ki [mm]
IC50 [mm]
dUTPase
dUTPase
Compd
P. fal.
dUTPase
Human
dUTPase
P. fal.[a]
L6 cells[b]
14a
14b
14c
14d
14e[4]
15[4]
30[4]
31[4]
32[4]
33[4]
112
47
1.3
1.3
5.0
nd
>1000
>1000
>1000
457.0
>1000
>1000
>1000
>1000
>1000
0.8
1.6
0.99
1.92
2.0
>11
1.0
5.3
18
25
3.1
40
nd
nd
85
30
8
1a
1b
1c
2a
2b
3a
3b
4
5a
5b
6a
6b
6c
6d
6e
6 f
6g
7a
7b
7c
8a
8b
1.8[4]
0.2[4]
4.2
4.7
0.68
0.94
0.37
1.9
17.7[4]
46.3[4]
>1000
>1000
351
696
213
>1000
>1000
238
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
6[4]
4.5[4]
1.1
192[4]
nd
21
nd
nd
44
28
24
1.9
6.2
1.3
3.0
2.8
2.0
2.0
3.7
2.7
1.3
1.6
1.0
4.3
1.2
0.70
1.3
12.4
313
>1000
1.8
1.0
85
2.3
3.4
35
30
[a] P. falciparum K1. [b] Cytotoxicity toward rat L6 myoblasts; nd: not de-
termined. Controls: for P. falciparum, chloroquine, IC50 =0.1 mm; for cyto-
toxicity, podophyllotoxin, IC50 =0.012 mm. IC50 values are the means of
four values of two independent assays performed in duplicate. Com-
pounds 14a, 14e, 15, and 30–33 were published previously, and are
listed here for comparative purposes only.
>1000
>1000
>1000
>1000
>1000
>1000
>1000
0.23
1.8
0.98
>1000
103
108
>96
>102
>103
>98
>87
10
>10
7.7
nd
ent does not favour enzyme inhibition. Interestingly, the 5’-tri-
phenylsilyl-3’-fluoro-2’,3’-dideoxyuridine 33 was the least active
of this series of compounds, with a Ki value greater than the
highest concentration tested in the biological assay. This com-
pares with 14e and 30, which have activity at ~1–5 mm.
>190
>190
14
7.2
2.1
1.2
29
[a] P. falciparum K1. [b] Cytotoxicity toward rat L6 myoblasts; nd: not de-
termined. Controls: for P. falciparum, chloroquine, IC50 =0.1 mm; for cyto-
toxicity, podophyllotoxin, IC50 =0.012 mm. IC50 values are the means of
four values of two independent assays performed in duplicate.
Base analogues
Modification of the base of the nucleosides was clearly not
well tolerated, as all the resulting compounds were essentially
inactive against the enzyme. This probably reflects the tight
hydrogen-bonding network that binds the uracil in the dUT-
Pase active site. The enzyme is very specific in its binding, only
binding dUTP and not dTTP, dCTP, dATP, or dGTP. The only ex-
ception to this lack of inhibitory potency among the base-
modified compounds is the 5-fluorouracil derivative 10b,
which has a Ki value of 22 mm. This is perhaps not surprising,
as the fluorine has relatively small steric requirements (Table 3).
The acyclic analogues of thymidine 13a,b and cytidine 13c
are completely devoid of activity, confirming the high selectivi-
ty of the enzyme for the uracil base. In fact, the latter three
compounds are analogues of acyclic uracil derivatives, which
were previously reported as very potent inhibitors of P. falci-
parum dUTPase.[6]
was marginal (3a/3b and 7b/7c). With the sulfur, instead, a
loss of activity was observed (Ki =4.5 mm for 1c).
3’ Variants
Substitution of the OH group at the 3’-position of the ribose
with NH2 rendered Ki values (Ki =1.3 mm for 14c) similar to that
of the lead compound 1a, and the N-acetylated derivative
14d was equally active (Ki =1.3 mm). Replacement of the 3’-OH
group with fluorine led to a slight loss of inhibition as shown
by compounds 14e and 31. These compounds had similar ac-
tivity to the tritylated d4U 15, with no substituent at the 3’-po-
sition (Table 2).[5]
Compounds with bulky lipophilic groups such as 14a and
32 showed high Ki values, indicating that this kind of substitu-
ChemMedChem 2011, 6, 309 – 320
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
313

I. H. Gilbert et al.
MED
An interesting result was observed with the heteroaryldi-
phenyl derivatives 7a–c. In these cases one of the phenyl rings
could be substituted with a heteroaryl group without compro-
mising enzyme inhibition, although there was a loss of in vitro
activity for compound 7a (IC50 >10 mm), whilst 7b and 7c re-
tained activity. The introduction of a heteroaryl group such as
pyridine or pyrimidine should increase water solubility and de-
crease the logP value.
Table 3. Biological results for the base-modified nucleosides.
The general lack of variation of enzyme inhibition as the
substituent on the trityl group is modified, is consistent with
the hydrophobic character of the trityl-binding pocket of P. fal-
ciparum dUTPase. Moreover, one of the phenyl rings is ex-
posed to the solvent, and this is likely to be where the substi-
tuted phenyl or heteroaromatic ring of the inhibitors would sit
upon binding the enzyme.
Ki [mm]
IC50 [mm]
Compd
P. fal.
dUTPase
Human
dUTPase
P. fal.[a]
L6 cells[b]
Increased enzyme inhibition is observed if the oxygen atom
at the 5’-position is replaced by an amino group in compound
pairs 1a/1b and 2a/2b. However, a similar trend was not ob-
tained for growth inhibition in vitro, although all pairs gave
IC50 values within the 1–6 mm range. This is presumably due to
other factors such as cell permeability, which undoubtedly
plays a major role in activity against the parasite. With the
other pairs 3a/3b, 5a/5b, and 7b/7c, there was no significant
change observed in enzyme inhibition on replacing the 5’-
oxygen with a 5’-nitrogen. The 5’-thiotrityl derivative 1c
showed moderate enzyme inhibition (Ki =4.2 mm), yet an IC50
value of 1.1 mm against the parasite. However, this result could
be influenced by the slightly increased toxicity of 1c, as shown
by the L6 cell assay: IC50 =21 mm (Table 1).
9
201
>1000
22
>1000
480
>1000
800
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
4.0
9.1
10.2
9.9
5.6
2.6
3.3
4.8
0.4
6.6
6.2
nd
35
35
23
29
6
14
14
12
18
nd
nd
nd
10a
10b
10c
10d
11 a
11 b
11 c
12
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
13a
13b
13c
[a] P. falciparum K1. [b] Cytotoxicity toward rat L6 myoblasts; nd: not de-
termined. Controls: for P. falciparum, chloroquine, IC50 =0.1 mm; for cyto-
toxicity, podophyllotoxin, IC50 =0.012 mm. IC50 values are the means of
four values of two independent assays performed in duplicate.
3’ Variants
In vitro assay
In analysis of the 3’-position of the sugar we observed that the
replacement of the OH group with NH2 retained enzyme inhib-
ition, as seen for the 3’-amino derivative 14c, but this also in-
creased toxicity. Acylation of the amino moiety (compound
14d) did not affect enzyme inhibition but decreased the toxici-
ty against mammalian cells and retained antiplasmodial activity
(IC50 =2.1 mm). The 3’-diphenyl phosphate ester 14a, the 3’-
azide 14b, and the 3’-tert-butyldimethylsilyl ether 32 all have
Ki values >40 mm; however, for two of these compounds the
presence of a bulky lipophilic group could limit solubility and
affect activity against the enzyme in vitro. Nonetheless, it is
possible to replace the 3’-hydroxy group with a fluorine, as ob-
served for 14e (Table 2).
All compounds were assayed against intact P. falciparum para-
site cultured in human erythrocytes and against mammalian L6
cells as a counterscreen for toxicity. A number of compounds
showed activity against P. falciparum in the low or sub-micro-
molar levels, with good selectivity over the mammalian cells.
However, others showed poor selectivity (<10-fold) and are
probably generally toxic to the cells (notably compounds 3b,
4, 6g, 8a, 9, 10a–d, 11 a–c, 14c, 31, and 32).
Discussion
No significant conclusions on the requirements for antipara-
sitic activity can be obtained for the 3’-substituted compounds,
as 14a, 14b, 14d, 30, and 33 are all active, with IC50 values
~1–2 mm. However, comparing these results with the previous-
ly reported 3’-fluoro 14e and didehydro derivatives 15,[5] we
can deduce that the substitution of the 3’-hydroxy group with
small polar groups does not affect inhibition of either the
enzyme or parasite growth in vitro. This is in agreement with
the structural data, which suggest that the 3’-substitutent can
point toward the solvent and away from the active site, thus
allowing substitutions at this position.[4]
5’-Trityl analogues
SAR analysis of the modified 5’-trityl nucleosides shows that
substituents in the triphenyl moiety can be introduced without
compromising enzyme inhibition, with the exception of the
bulky aryl substituents in the series of compounds 6a–g. The
cyano derivative 3, the chloro 5, the methoxy 2, and dime-
thoxy 4 all exhibit similar Ki values. The lack of activity of the
large aryl groups (compounds 6a–g) is probably due either to
the group being too large to fit into the active site, or to a
lack of chemical stability.
314
www.chemmedchem.org
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 309 – 320

5’-Trityl Nucleoside dUTPase Inhibitors
Base analogues
IC50 value of 0.3 mm. However, there is clearly a base require-
ment for selective activity, because analogues (compounds 9–
13) in which the uracil is modified or replaced by another base
are generally less active and toxic to both P. falciparum and L6
cells in culture.
The high selectivity of dUTPase for uridine analogues strongly
limits the modifications that can be introduced on the nucleo-
base. As shown in Table 3, the 5-bromo 10a, 5-chloro 10c, and
5-ethyl 10d nucleosides were all poor inhibitors, and none of
the N3-methylated derivatives 11 a, 11 b, or 11 c gave Ki values
less than 1000 mm. The best-tolerated substitution was the in-
troduction of a fluorine atom at position 5 for compound 10b.
This compound showed a moderate inhibition of P. falciparum
dUTPase (Ki =22 mm). The base analogues (compounds 9–12),
in addition to showing poor inhibition of dUTPase, appear to
be toxic in cell culture and lack selectivity.
Conclusions
Some new 5’-trityl-2’,3’-dideoxyuridine analogues were syn-
thesised. The modifications were introduced on the oxygen
atom at the 5’-position of the sugar moiety, on the phenyl
rings of the trityl group, at the 3’-position of the sugar, and on
the nucleobase. There is almost no tolerance for modifications
on the base. However, there is room for variation of the trityl
group and the 3’-position. Some of the compounds prepared
failed to inhibit dUTPase, yet intracellular metabolism and ad-
ditional modes of action could explain this. New compounds
with improved activity and selectivity against P. falciparum
dUTPase may give rise to nontoxic entities that show increased
activity against the parasite.
We had previously reported some acyclic uracil derivatives
as potent inhibitors of the parasitic enzyme, and it was there-
fore of interest to test whether the corresponding thymidine
derivatives 13a, 13b, and cytosine derivative 13c are active.
As reported in Table 3, none of the three acyclic analogues
showed Ki values lower than 1 mm, again indicating the strong
requirement of the uracil ring.
Enzyme and cell activity
Compounds that showed inhibition of P. falciparum dUTPase
also inhibited the growth of the intact parasite, with the ex-
ception of compounds 7a and 15. The reason for the lack of in
vitro activity for these compounds is still unclear. This could
possibly be due to instability in the cell culture medium or
issues related to uptake/efflux or protein binding. The majority
of compounds that are active and selective in the enzyme in-
hibition assays are also selective in vitro for P. falciparum over
mammalian L6 cells, with the exception of compounds 3b, 4,
14c, and 31. This result is consistent with the selective inhibi-
tion of the P. falciparum dUTPase. Most of the compounds that
are active against P. falciparum dUTPase show inhibition in the
fairly narrow Ki value range of 0.2–5 mm. These compounds in-
hibit growth of the parasite in the range of 1–7 mm. Conversely,
compounds that are not selective for P. falciparum dUTPase
over the human enzyme are nonselective (toxic) at the cellular
level.
Experimental Section
General methods
1H NMR, 13C NMR, 19F NMR, and 2D NMR spectra were recorded
either on a Bruker Avance DPX 300 spectrometer, on a Bruker
Avance DPX 500 spectrometer, or on a Varian 400 MHz instrument.
Chemical shifts (d) are expressed in ppm. Signal splitting patterns
are described as singlet (s), broad singlet (bs), doublet (d), triplet
(t), quartet (q), multiplet (m), or a combination thereof; 2D NMR
spectra were performed to aid in assignments.
Low-resolution electrospray (ES) mass spectra were recorded either
on a Fisons VG Platform mass spectrometer, a Bruker MicroTof
mass spectrometer, or on a Waters Micromass ESI Q-ToF spectrom-
eter, run in either positive or negative ion modes, using either
CH3OH, CH3OH/H2O (95:5) or H2O/CH3CN (1:1) + 0.2% HCOOH as
the mobile phase. For each spectrum, the most abundant and rele-
vant peaks are listed with their relative intensities (%) given in
brackets. High-resolution electrospray MS measurements were per-
formed by the EPSRC Mass Spectrometry Centre, Swansea (UK).
The exceptions to this are compounds 6a–g. These are inac-
tive against P. falciparum dUTPase, yet are active and selective
against the parasite. These compounds almost certainly act
through a mechanism of action other than inhibition of dUT-
Pase. A supposed mechanism of action could involve intracel-
lular esterase-mediated hydrolysis of the ester groups at posi-
tion 4 of the phenyl rings. This would lead to a 4,4’,4’’-tris-
(hydroxy)trityl group that could undergo cleavage under basic
conditions, as reported by Sekine and co-workers,[14] releasing
the unprotected nucleoside and rosolic acid. The latter is
known to have toxic effects in mammals.[29] This cleavage pre-
sumably occurs more rapidly in the parasite than in L6 cells,
accounting for the selectivity.
Infrared (IR) spectra were recorded on a PerkinElmer 1600 FTIR
spectrometer using NaCl plates for liquids (neat) and with a diffuse
reflectance accessory using a KBr matrix for solids. IR data are
given as wavenumbers expressed in cmꢀ1
.
Melting points (mp) were measured on a Griffin analogue melting-
point apparatus and are uncorrected. Thin-layer chromatography
(TLC) was carried out on Merck silica gel 60 F254 plates using UV
light and/or PMA or ninhydrin for visualisation. TLC data are given
as the Rf value with the corresponding eluent system specified in
brackets. Column chromatography was performed either with pre-
packed silica columns (ISOLUTE SI columns) on a Jones Chroma-
tography Flashmaster apparatus or with Fluka silica gel 60.
As we previously reported,[5] the trityl moiety itself has cer-
tain activity against P. falciparum, although not against the
enzyme. Thus the trityl alcohol has an IC50 value of 12.3 mm
against P. falciparum, whereas the triphenylsilyl alcohol has an
All reactions were carried out under dry and inert conditions (N2 at-
mosphere) unless otherwise stated. Reactions using microwave ir-
radiation were carried in a Biotage InitiatorTM microwave.
ChemMedChem 2011, 6, 309 – 320
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
315

I. H. Gilbert et al.
MED
pressure. The residue was co-evaporated with toluene several
times and purified by chromatography. ISOLUTE SI column eluted
with 0!3% CH3OH in CHCl3.
Enzyme purification and inhibition assays
Both recombinant P. falciparum and human dUTPases were ex-
pressed in E. coli BL21 (DE3) cells that had been transformed with
the pET11Pfdut and pET3Hudut (kindly provided by P. O. Nyman,
Lund University, Sweden) expression vectors, respectively. For dUT-
Pase purification, the same procedure was used for both the
human and Plasmodium enzymes. Cell pellets from a 2-L IPTG-in-
duced culture were resuspended in 40 mL buffer A (20 mm 2-(N-
morpholino)ethanesulfonic acid (MES) pH 5.5, 50 mm NaCl, 5 mm
MgCl2, 1 mm DTT) and 20 mm PMSF. The cells were lysed by soni-
cation, and the cell extract was cleared by centrifugation at
16000 g for 45 min. The supernatant was loaded onto a 50 mL
phosphocellulose (Whatman P-11) column at 48C and was eluted
with a gradient of NaCl (50 mm!2m) in buffer A. The enzyme was
then dialyzed against buffer A prior to gel filtration chromatogra-
phy on a Superdex 200 HA 10/30 column at 48C. Purified fractions
contained dUTPase of ~96% purity.
General procedure F for the synthesis of 5-chloro- and 5-bromo-
2’-deoxynucleosides: To
a
stirred solution of nucleoside
(0.2 mmol) in 3 mL dry pyridine, protected from light, was added
the appropriate N-halosuccinimide (NCS or NBS; 0.4 mmol), and
the resulting mixture was kept at 1008C for 30 min. The mixture
was cooled, excess volatiles were removed by evaporation, and the
crude residue was purified by PTLC in 10% CH3OH in CHCl3 and
precipitated with hexane from iPrOH.
General
procedure G
for
the
synthesis
of
tris[4-(aroyloxy)phenyl]methanols in the presence of pyridine:
To a solution of rosolic acid (10 g, 34.5 mmol) in dry pyridine
(25 mL) was added the appropriate aroyl chloride (183.2 mmol).
The mixture was heated at 908C for 1.5 h, and progress of the re-
action was monitored by TLC in 3% (CH3)2CO in CHCl3. The mixture
was cooled to room temperature, quenched with ice-H2O and final-
ly extracted with CH2Cl2. The organic phase was washed several
times with 5% NaHCO3, dried over Na2SO4, filtered, and evaporated
under reduced pressure to a gum. The gummy residue was co-
evaporated several times with toluene to remove the last traces of
pyridine to give the crude product, which was recrystallised from
toluene/cyclohexane or CH3OH.
Nucleotide hydrolysis was monitored by mixing enzyme and sub-
strate with a rapid kinetic accessory (Hi-Tech Scientific) attached to
a spectrophotometer (Cary 50) and connected to a computer for
data acquisition and storage as described previously.[30] Protons, re-
leased through the hydrolysis of nucleotides, were neutralised by a
pH indicator in weakly buffered medium with similar pKa and
monitored spectrophotometrically at the absorbance peak of the
basic form of the indicator. The ratio between the indicator and
the buffer concentration was 50:2000 (mm), and the absorbance
changes were kept within 0.1 units. The indicator/buffer pair used
was red cresol/bicine (pH 8, l=573 nm). Assay mixes contained
30 nm PfdUTPase, 50 mm dUTP, 5 mm MgCl2, 1.25 mgmLꢀ1 BSA,
and 100 mm KCl. Vmax and KMapp were calculated by fitting the re-
sulting data to the integrated Michaelis–Menten equation. The ap-
parent KM values were plotted against inhibitor concentration, and
Ki values (Table 2) were obtained according to Equation (1).
General
procedure H
for
the
synthesis
of
tris[4-(aroyloxy)phenyl]methanols in the presence of 4-(N,N-di-
methyl)aniline: This procedure is similar to that mentioned above
with the use of dry 4-(N,N-dimethyl)aniline (25 mL) instead of dry
pyridine. The crude gummy residue was purified by column chro-
matography on silica gel in a stepwise gradient of toluene/CHCl3
1:1, CHCl3, CHCl3/acetone 98:2 (v/v), and finally recrystallised from
toluene/cyclohexane or CH3OH.
General procedure I for the synthesis of trityl bromides: To a
suspension of appropriate trityl analogue (5.66 mmol) in dry tolu-
ene (10 mL) was added acetyl bromide (1 mL, 13.5 mmol). The mix-
ture was heated at 858C for 1.5 h, and the hot solution was filtered
to remove a small amount of insoluble material. Dry hexane was
added portionwise to the hot filtrate. After cooling, the resulting
precipitate was collected by filtration and washed with dry
hexane/toluene (2:1 v/v) and then with dry hexane.
KMapp ¼ KM=Ki ½Iꢁ þ KM
ð1Þ
General procedure A for the synthesis of 5’-tritylated and modi-
fied 5’-trityl nucleosides 1a, 1b, 2a, 2b, 3a, 3b, 4, 5a, 5b, 10b,
10d, 13a, 13b, and 13c: To a stirred solution of nucleoside
(1 equiv) in pyridine was added the trityl precursor (1.1–1.3 equiv)
and DMAP (0.1 equiv) when stated. The reaction was stirred at 40–
808C for 3–92 h (as reported for each compound). The progress of
the reaction was monitored by TLC. The mixture was cooled to
room temperature, quenched with H2O or CH3OH, and extracted
with CH2Cl2 or EtOAc. The organic solution was washed with H2O,
saturated NaHCO3, and diluted HCl or AcOH, or otherwise stated.
The solution was dried over MgSO4, concentrated, and purified by
chromatography.
5’-S-Tritylthio-5’,2’-dideoxyuridine (1c): To a stirred solution of 2’-
deoxyuridine (50 mg, 0.22 mmol) in 3 mL dry 1,4-dioxane were
subsequently added in four portions PPh3 (281.6 mg, 1.05 mmol;
70.4 mmol, 0.263 mmol in each portion) and TrSH (362.0 mg,
1.28 mmol; 90.5 mg, 0.32 mmol in each portion) followed by DEAD
also in four portions (0.18 mL, 1.09 mmol; 45 mL, 0.272 mmol in
each portion). The resulting mixture was stirred at room tempera-
ture. The mixture was concentrated to dryness. The residue was
purified by PTLC eluting with 10% CH3OH in CHCl3 and crystallised
from toluene to give 1c (50.3 mg, 47%); mp: 91–938C; Rf =0.43
(5% CH3OH/CHCl3); 1HNMR (CDCl3): d=1.98, 2.31 (1H, ddd, J=
6.8 Hz, J=13.8 Hz, J=6.5 Hz, and 1H, ddd, J=4.3 Hz, J=6.4 Hz, J=
14.0 Hz, 2’-H an 2’’-H), 2.48, 2.60 (1H, dd, J=5.4 Hz, J=12.9 Hz and
1H, dd, J=6.5 Hz, J=12.9 Hz, 5’-H and 5’’-H), 3.68 (1H, m, 4’-H),
4.00 (1H, m, 3’-H), 5.70 (1H, d, J=7.6 Hz, 5-H), 6.10 (dd, 1H, J=
6.5 Hz, J=6.5 Hz, 1’-H), 7.22–7.45 (16H, m, ArH, 6-H), 8.29 (1H, bs,
NH); MS (EI) m/z 509 [M+Na]+; Anal. (%) calcd for (C28H26N2O5S,
1CH3OH): C 67.16, H 5.83, N 5.40, found: C 66.97, H 5.88, N 5.71.
General procedure B for the synthesis of modified trityl nucleo-
sides 6b–g: A microwave vial was charged with 2’-deoxyuridine
(1 equiv), tris(substituted)trityl bromide (0.5 equiv) and pyridine.
The reaction was heated at 1008C under microwave irradiation for
30 s. After the first cycle more trityl precursor (0.5 equiv) was
added to the solution, and the reaction was again irradiated at
1008C for 30 s. The solvent was co-evaporated with toluene, and
the crude residue was purified by chromatography. ISOLUTE SI
column eluted with 0!5% CH3OH in CHCl3.
General procedure C for the synthesis of 5’-tribenzylsilyl nucleo-
sides 8a and 8b: Tribenzylsilyl chloride (1.3 equiv) was added to a
solution of 2’-deoxyuridine (1 equiv) in pyridine. The reaction was
stirred at 08C for 2.5–3 h. The solvent was removed under reduced
5’-O-(4-Methoxytrityl)-2’-deoxyuridine (2a): This compound was
prepared by following general procedure A, starting from 4-me-
316
www.chemmedchem.org
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 309 – 320

5’-Trityl Nucleoside dUTPase Inhibitors
thoxytrityl chloride (0.61 g, 1.98 mmol), 2’-deoxyuridine (0.41 g,
1.80 mmol), and pyridine (10 mL), stirred at 508C for 40 h. Purified
by flash column chromatography, eluting the column (ISOLUTE SI)
with a gradient of 0!5% CH3OH in CHCl3. The title compound
was obtained as a white crystalline solid (0.625 g, 69%). Rf =0.28
(CN), 1685, 1463, 1273, 1088 cmꢀ1; Anal. (%) calcd for (C29H25N3O5,
2.35HCl) C 59.93, H 4.74, N 7.23, found: C 59.89, H 4.45, N 7.02.
5’-(4-Cyanotrityl)amino-2’,5’-dideoxyuridine (3b): The compound
was prepared by following general procedure A, starting from 4-cy-
anotrityl chloride (0.41 g, 1.34 mmol), 5’-amino-2’,5’-dideoxyuridine
(23) (0.24 g, 1.05 mmol) and pyridine (5 mL), stirred at 408C for
48 h. Purified by flash column chromatography eluting the column
(ISOLUTE SI) with a gradient of 0!5% CH3OH in CHCl3. White crys-
talline solid (0.386 g, 37%). Rf =0.31 (10% CH3OH/CHCl3); mp: 160–
1
(10% CH3OH/CHCl3); mp: 96–978C; H NMR (300 MHz, CDCl3): d=
2.27 (1H, m, 2’-H), 2.42 (1H, m, 2’-H), 2.57 (1H, bs, 3’-OH), 3.42 (2H,
m, 5’-H), 3.77 (3H, s, OCH3), 4.00 (1H, m, 4’-H), 4.54 (1H, m, 3’-H),
5.37 (1H, d, J=8.1 Hz, 5-H), 6.29 (1H, t, J=6.3 Hz, 1’-H), 6.82 (2H,
d, J=8.9 Hz, Ar-H), 7.18–7.38 (12H, m, Ph-H and Ar-H), 7.74 (1H, d,
J=8.1 Hz, 6-H), 9.20 (1H, bs, 3-NH); 13C NMR (75 MHz, CDCl3): d=
41.6 (2’-CH2), 55.7 (5’-CH2), 63.5 (OCH3), 71.9 (3’-CH2), 85.5 (4’-CH),
86.5 (1’-CH), 87.8 (ArPh2-C), 102.7 (5-CH), 113.8 (Ar-CH), 127.8 (Ph-
CH), 128.5 (Ph-CH), 128.8 (Ph-CH), 130.8 (Ar-CH), 135.1 (Ar-C), 140.6
(6-CH), 144.1 (Ph-C), 144.3 (Ar-CH), 150.8 (2-C), 159.3 (Ar-C), 163.8
(4-C); LRMS (ES+) m/z 523 ([M+Na]+, 100%); HRMS (ES+) calcd for
C29H28N2O6Na+ [M+Na]+: 523.1840, found: 523.1837; IR (KBr): n˜ =
3208, 3054, 1714, 1694, 1682, 1507, 147, 1250, 1092, 1034, 759,
702 cmꢀ1; Anal. (%) calcd for (C29H28N2O6, 1.42HCl, 0.40H2O): C
62.25, H 5.44, N 5.01, Cl 9.00, found: C 62.17, H 5.05, N 4.85, Cl
8.86.
1
1638C; H NMR (300 MHz, CDCl3): d=2.03 (2H, m, 2’-CHH and 5’-
NH), 2.21 (1H, m, 5’-CHH), 2.37 (1H, m, 2’-CHH), 2.58 (1H, m, 5’-
CHH), 3.31 (1H, bs, 3’-OH), 4.10 (1H, m, 4’-H), 4.24 (1H, m, 3’-H),
5.63 (1H, d, J=8.1 Hz, 5-H), 6.24 (1H, t, J=6.3 Hz, 1’-H), 7.00 (1H,
dd, J=2.1, 8.1 Hz, 6-H), 7.18–7.48 (10H, m, Ph-H), 7.54 (2H, d, J=
8.1 Hz, Ar-H), 7.66 (2H, d, J=8.1 Hz, Ar-H), 9.74 (1H, bs, 3-NH);
13C NMR (75 MHz, CDCl3): d=40.7 (2’-CH2), 46.5 (5’-CH2), 71.3
(ArPh2-C), 72.8 (3’-CH), 85.6 (1’-CH), 86.5 (4’-CH), 103.3 (5-CH), 110.7
(Ar-CH), 119.2 (CN), 127.6 (Ph-CH), 128.7 (Ph-CH), 128.9 (Ph-CH),
129.1 (Ph-CH), 129.5 (Ar-CH), 132.4 (Ar-CH), 139.9 (6-CH), 144.3 (Ph-
C), 145.0 (Ar-C), 150.8 (2-C), 151.6 (Ar-C), 163.8 (4-C); LRMS (ES+)
m/z 517 ([M+Na]+, 19%), 495 ([M+H]+, 9%), 517 ([CNPh3C+],
100%); HRMS (ES+) calcd for C29H27N4O4 [M+H]+ 495.2027,
+
5’-(4-Methoxytrityl)amino-2’,5’-dideoxyuridine (2b): The com-
pound was prepared following the general procedure A, starting
from 5’-amino-2’,5’-dideoxyuridine 23 (0.20 g, 0.90 mmol), 4-me-
thoxytrityl chloride (0.29 g, 0.99 mmol) and pyridine (5 mL), stirred
at 508C for 40 h. Purified by flash column chromatography eluting
the column (ISOLUTE SI) with a gradient of 0!5% CH3OH in
CHCl3. Compound 2b was obtained as a white solid (0.115 g, 26%).
found: 495.2023; IR (KBr): n˜ =3387, 3177, 3027, 2230 (CN), 1699,
1661, 1466, 1267, 1097, 1039 cmꢀ1; Anal. (%) calcd for (C29H26N4O4,
1.0HCl, 0.20H2O) C 64.80, H 5.21, N 10.42, Cl 6.46, found: C 64.69,
H 4.90, N 10.28, Cl 6.84.
5’-O-(4,4’-Dimethoxytrityl)-2’,5’-dideoxyuridine (4): The com-
pound was prepared by following general procedure A, starting
from 2’-deoxyuridine (0.51 g, 2.23 mmol), 4,4’-dimethoxytrityl chlo-
ride (0.98 g, 2.89 mmol), DMAP (32 mg, 0.26 mmol) and pyridine
(10 mL), stirred at 408C for 48 h. Purified by flash column chroma-
tography eluting the column (ISOLUTE SI) with a gradient of 0!
5% CH3OH in CHCl3. Compound 4 was obtained as a pale-yellow
solid (0.678 g, 57%). Rf =0.33 (10% CH3OH/CHCl3); mp: 85–868C;
1H NMR (300 MHz, CDCl3): d=2.31 (1H, m, 2’-H), 2.51 (1H, m, 2’-H),
2.80–2.90 (1H, b signal, 3’-OH), 3.84 (6H, s, 2ꢃOCH3), 3.50 (2H, m,
5’-H), 4.10 (1H, m, 4’-H), 4.62 (1H, m, 3’-H), 5.46 (1H, d, J=8.1 Hz,
5-H), 6.38 (1H, t, J=6.3 Hz, 1’-H), 6.89 (4H, d, J=8.7 Hz, Ar-H),
7.25–7.45 (9H, m, Ph-H and Ar-H), 7.85 (1H, m, 6-H), 9.50 (1H, bs,
3-NH); 13C NMR (75 MHz, CDCl3): d=41.6 (2’-CH2), 55.7 (OCH3), 64.4
(5’-CH2), 72.0 (3’-CH), 85.5 (4’-CH or 1’-CH), 86.5 (4’-CH or 1’-CH),
87.5 (Ar2Ph-C), 102.7 (5-CH), 113.7 (Ar-CH), 127.6 (Ph-CH), 128.4 (Ph-
CH), 128.5 (Ph-CH), 130.5 (Ar-CH), 135.6 (Ar-C), 135.8 (Ar-C), 140.7
(6-CH), 144.7 (Ph-C), 150.7 (2-C), 159.1 (Ar-C), 163.7 (4-C). (ES+) m/z
553 ([M+Na]+, 49%); HRMS (ES+) calcd for C30H30N2O7Na+
[M+Na]+ 553.1945, found: 553.1948; Anal. (%) calcd for
(C30H30N2O7, 2.20 HCl) C 58.99, H 5.31, N 4.59, Cl 12.77, found: C
58.72, H 5.13, N 4.33, Cl 12.68.
1
Rf =0.29 (10% CH3OH/CHCl3); mp: 140–1438C; H NMR (300 MHz,
CDCl3): d=2.04 (2H, m, 2’-H), 2.29–2.48 (2H, m, 5’-H), 2.69 (1H, dd,
J=3.7, 12.1 Hz, 5’-NH), 3.83 (3H, s, OCH3), 4.15 (1H, m, 4’-H), 4.30
(1H, m, 3’-H), 5.69 (1H, d, J=8.1 Hz, 5-H), 6.32 (1H, t, J=6.4 Hz, 1’-
H), 6.88 (2H, m, Ar-H), 7.17 (1H, d, J=8.1 Hz, 6-H), 7.21–7.61 (14H,
m, Ph-H and Ar-H); 13C NMR (75 MHz, CDCl3): d=40.8 (2’-CH2), 46.6
(5’-CH2), 55.7 (OCH3), 70.7 (ArPh2-C), 73.0 (3’-CH), 85.4 (1’-CH), 86.8
(4’-CH), 103.2 (5-CH), 113.7 (Ar-CH), 126.9 (Ph-CH), 128.4 (Ph-CH),
128.9 (Ph-CH), 130.2 (Ar-CH), 138.0 (Ar-C), 139.9 (6-CH), 146.2 (Ph-
C), 150.8 (2-C), 158.4 (Ar-C), 163.8 (4-C); LRMS (ES+) m/z 522
([M+Na]+, 27%), 87 (100); HRMS (ES+) calcd for C29H30N3O5
+
[M+H]+: 500.2180, found: 500.2174; IR (KBr): n˜ =(3408), 3052,
1713, 1694, 1682, 1666, 1650, 1501, 1250, 1034, 760 cmꢀ1; Anal. (%)
calcd for (C29H29N3O5, 0.88HCl): C 65.52, H 5.66, N 7.90, Cl 5.87,
found: C 65.69, H 5.52, N 7.86, Cl 6.05.
5’-O-(4-Cyanotrityl)-2’-deoxyuridine (3a): The compound was pre-
pared following the general procedure A, starting from 4-cyanotri-
tyl chloride (0.40 g, 1.31 mmol), 2’-deoxyuridine (0.23 g,
1.00 mmol), pyridine (5 mL) and DMAP (11 mg, 0.09 mmol) stirred
at 508C for 92 h. Purified by flash column chromatography with a
gradient of 0!6% CH3OH in CHCl3. The title compound was a
white solid (0.215 g, 43%). Rf =0.29 (10% CH3OH/CHCl3); mp: 92–
958C; 1H NMR (300 MHz, CDCl3): d=2.19 (1H, m, 2’-CHH), 2.45
(1H, m, 2’- CHH), 2.94 (1H, bs, 3’-OH), 3.38 (2H, m, 5’-H), 4.06 (1H,
m, 4’-H), 4.50 (1H, m, 3’-H), 5.45 (1H, d, J=8.1 Hz, 5-H), 6.27 (1H, t,
J=6.2 Hz, 1’-H), 7.24–7.34 (10H, m, Ph-H), 7.53–7.60 (5H, m, 6-H
and Ar-H), 9.50 (1H, bs, 3-NH); 13C NMR (75 MHz, CDCl3): d=41.4
(2’-CH2), 63.9 (5’-CH2), 71.7 (3’-CH), 85.6 (1’-CH), 86.1 (4’-CH), 87.6
(ArPh2-C), 102.8 (5-CH), 111.4 (Ar-C), 119.0 (CꢂN), 128.6 (Ph-CH),
128.8 (Ph-CH), 129.0 (Ar-CH), 132.4 (Ar-CH), 140.3 (6-CH), 142.0 (Ph-
C), 142.1 (Ph-C),150.1 (Ar-C), 150.8 (2-C), 163.8 (4-C); LRMS (ES+) m/
z 518 ([M+Na]+, 23%), 268 (Ph3C+, 100%); LRMS (ESꢀ) m/z 494
5’-O-(2-Chlorotrityl)-2’-deoxyuridine (5a): The compound was
prepared by following general procedure A, starting from 2’-deoxy-
uridine (0.24 g, 1.04 mmol), 2-chlorotrityl chloride (0.64 g,
2.04 mmol), DMAP (18 mg, 0.15 mmol) and pyridine (5 mL), stirred
at 408C for 50 h. Purified by flash column chromatography (ISO-
LUTE SI column) eluting with 0!5% CH3OH in CHCl3. The title
compound was as a white solid (0.176 g, 34%). Rf =0.14 (10%
CH3OH/CHCl3); mp: 95–988C; 1H NMR (300 MHz, CDCl3): d=2.31
(1H, m, 2’-H), 2.49 (1H, m, 2’-H), 2.66 (1H, bs, 3’-OH), 3.48 (2H, m,
5’-H), 4.09 (1H, m, 4’-H), 4.71 (1H, m, 3’-H), 5.53 (1H, d, J=8.1 Hz,
5-H), 6.37 (1H, t, J=6.2 Hz, 1’-H), 7.25–7.50 (13H, m, Ph-H and Ar-
H), 7.70–7.85 (2H, m, 6-H and Ar-H), 9.18 (1H, bs, 3-NH); 13C NMR
(75 MHz, CDCl3): d=41.4 (2’-CH2), 64.0 (5’-CH2), 71.6 (3’-CH), 85.3
(4’-CH or 1’-CH), 86.1 (4’-CH or 1’-CH), 87.6 (ArPh2-C), 102.7 (5-CH),
([MꢀH]+, 100%); HRMS (ES+) calcd for C29H29N4O5 [M+NH4]+
+
513.2132, found: 513.2132; IR (KBr): n˜ =3401, 3180, 3060, 2230
ChemMedChem 2011, 6, 309 – 320
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
317

I. H. Gilbert et al.
MED
127.0 (Ar-CH), 127.8 (Ph-CH), 127.9 (Ph-CH), 128.4 (Ph-CH), 128.5
(Ph-CH), 128.7 (Ph-CH), 128.8 (Ph-CH), 129.9 (Ar-CH), 131.1 (Ar-CH),
132.6 (Ar-CH), 134.7 (Ar-C), 140.2 (Ar-C), 140.7 (6-CH), 141.7 (Ar-C),
142.3 (Ph-C), 150.7 (2-C), 163.7 (4-C); LRMS (ES+) m/z 529 ([M+Na]+
, 12%), 527 ([M+Na]+, 29%), 87 (100%); HRMS (ES+) calcd for
C28H25N2O5NaCl+ [M+Na]+ 527.1344, found: 527.1350; Anal. (%)
calcd for (C28H25N2O5Cl, 1.65 HCl, 0.30 H2O) C 58.95, H 4.81, N 4.91,
Cl 16.47, found: C 58.93, H 4.39, N 4.76, Cl 16.15.
5’-[Diphenyl(4-pyridyl)methyl]amino-2’,5’-dideoxyuridine
(7a):
5’-Amino-2’,5’-dideoxyuridine (18) (0.11 g, 0.47 mmol) was added
to a solution of diphenyl(4-pyridyl)methyl chloride (20a) (0.15 g,
0.47 mmol), pyridine (3 mL) and Et3N (0.12 mL, 0.86 mmol). The re-
action mixture was heated at 408C for 4 h then the temperature
was increased to 708C for 10 h. The crude solution was partitioned
between H2O (5 mL) and EtOAc (3ꢃ7 mL). The organic extracts
were combined, dried over Na2SO4 and concentrated in vacuo. The
brown solid obtained was taken in CH3OH and the remaining in-
soluble material was filtered off. The filtrate was concentrated
under reduced pressure and further purified using flash column
chromatography eluting the column (ISOLUTE SI) with a gradient
of 0!8% CH3OH in CHCl3. The title compound was a pale-yellow
solid (61 mg, 27%). Rf =0.24 (10% CH3OH/CHCl3); mp: 131–1338C;
1H NMR (300 MHz, CD3OD): d=2.15–2.60 (4H, m, 2-H and 5’-H),
4.03 (1H, m, 3’-H or 4’-H), 4.18 (1H, m, 3’-H or 4’-H), 5.63 (1H, d,
J=8.0 Hz, 5-H), 6.21 (1H, t, J=6.4 Hz, 1’-H), 7.20–7.48 (11H, m, 6-H
and Ph-H), 7.59 (2H, d, J=5.9 Hz, Ar-H), 8.42 (2H, d, J=5.9 Hz, Ar-
H); 13C NMR (75 MHz, CD3OD): d=40.9 (2’-CH2), 47.6 (5’-CH2), 72.2
(ArPh2-C), 73.3 (3’-CH), 87.1 (1’-CH or 4’-CH), 87.9 (1’-CH or 4’-CH),
103.4 (5-CH), 125.7 (Ar-CH), 128.6 (Ph-CH), 129.7 (Ph-CH), 130.2 (Ph-
CH), 130.3 (Ph-CH), 142.7 (6-CH), 145.8 (Ph-C), 146.1 (Ph-C), 150.2
(Ar-CH), 152.4 (Ar-C), 158.1 (2-C), 166.4 (4-C); LRMS (ES+) m/z 963
5’-(2-Chlorotrityl)amino-2’,5’-dideoxyuridine (5b): The compound
was prepared by following general procedure A, starting from 5’-
amino-2’,5’-dideoxyuridine (23) (0.24 g, 1.04 mmol), 2-chlorotrityl
chloride (0.41 g, 1.33 mmol) and pyridine (5 mL), stirred at 408C for
24 h. Purified by flash column chromatography eluting the column
(ISOLUTE SI) with a gradient of 0!5% CH3OH in CHCl3. Compound
5b was obtained as a white solid (85 mg, 16%). Rf =0.17 (10%
1
CH3OH/CHCl3); mp: 129–1318C; H NMR (300 MHz, CDCl3): d=2.02
(1H, m, 2’-H), 2.20 (1H, m, 5’-H), 2.37 (1H, m, 2’-H), 2.53 (1H, m, 5’-
H), 4.16 (1H, m, 3’-H), 4.24 (1H, m, 4’-H), 5.62 (1H, d, J=8.1 Hz, 5-
H), 6.26 (1H, t, J=6.4 Hz, 1’-H), 7.05–7.45 (15H, m, 6-H and Ph-H
and Ar-H); 13C NMR (75 MHz, CDCl3): d=41.1 (2’-CH2), 47.3 (5’-CH2),
71.5 (ArPh2-C), 72.9 (3’-CH), 85.5 (1’-CH), 86.8 (4’-CH), 103.0 (5-CH),
126.8 (Ar-CH), 126.9 (Ar-CH), 127.6 (Ar-CH), 128.4 (Ar-CH), 128.5 (Ar-
CH), 128.6 (Ar-CH), 129.2 (Ar-CH), 132.3 (Ar-CH), 132.9 (Ar-CH),
134.7 (A-C), 140.0 (6-CH), 140.9 (Ar-C), 144.8 (Ar-C), 146.0 (Ar-C),
150.7 (2-C), 163.7 (4-C); LRMS (ES+) m/z 526 ([M+Na]+, 11%), 504
([M+H]+, 14%), 277 ([Ph2 ArC]+, 100%); HRMS (ES+) calcd for
C28H27N3O4Cl+ [M+H]+ 504.1685, found: 504.1689.
([2M+Na]+, 13%), 493 ([M+Na]+, 84%), 471 ([M+H]+, 13%); HRMS
+
(ES+) calcd for C27H27N4O4 [M+H]+ 471.2027, found: 471.2033.
,
5’-[Diphenyl(5-pyrimidyl)methyl]-2’-deoxyuridine (7b): 2’-Deoxy-
uridine (0.15 g, 0.47 mmol) was added to a solution of 5-(chlorodi-
phenylmethyl)pyrimidine hydrochloride (20b) (0.26 g, 0.83 mmol)
and DMAP (10 mg, 0.08 mmol) in dry pyridine (1 mL). The reaction
mixture was heated at 1008C in a microwave twice for 1 min. The
solvent was removed in vacuo and the residue co-evaporated with
toluene. The crude product was then purified by flash column
chromatography (ISOLUTE SI column) eluting with 0!8% CH3OH
in CHCl3. The title compound was obtained a pale-yellow solid
(26 mg, 8%). Rf =0.40 (10% CH3OH/CHCl3); mp: 103–1088C;
1H NMR (500 MHz, CDCl3): d=2.23 (1H, m, 2’-CHH), 2.48 (1H, m,
2’-CHH), 3.43 (2H, m, 5’-CH2), 3.78 (1H, bs, 3’-OH), 4.10 (1H, m, 4’-
H), 4.58 (1H, m, 3’-H), 5.51 (1H, d, J=8.1 Hz, 5-H), 6.26 (1H, t, J=
6.2 Hz, 1’-H), 7.37 (10H, m, Ph-H), 7.57 (1H, d, J=8.1 Hz, 6-H), 8.85
(2H, s, Ar-H), 9.12 (1H, s, Ar-H), 9.94 (1H, bs, 3-NH); 13C NMR
(500 MHz, CDCl3): d=40.6 (2’-CH2), 63.0 (5’-CH2), 70.4 (3’-CH), 76.9
(ArPh2-C), 84.9 (1’-CH), 85.1 (4’-CH), 102.1 (5-CH), 128.09 (Ph-CH),
128.14 (Ph-CH), 128.3 (Ph-CH), 128.4 (Ph-CH), 137.7 (Ar-C), 139.4 (6-
CH), 140.2 (Ph-C), 140.9 (Ph-C), 150.1 (2-C), 156.0 (Ar-CH), 156.7 (Ar-
CH), 163.2 (4-C); LRMS (ES+) m/z 967 ([2M+Na]+, 7%), 945
([2M+H]+, 8%), 495 ([M+Na]+, 58%), 473 ([M+H]+, 100%); HRMS
4-(Chlorodiphenylmethyl)pyridine hydrochloride (20a): Diphen-
yl(4-pyridyl)methanol (2.12 g, 8.11 mmol) was taken in H2O (15 mL).
Concentrated HCl (1 mL) was added and the mixture was held at
reflux for 2 h. The crude solution obtained was concentrated in va-
cuo to yield diphenyl(4-pyridyl)methanol hydrochloride as a white/
cream solid (2.173 g, 90%); mp: 204–2058C; 1H NMR (300 MHz,
CDCl3): d=7.37 (10H, m, Ph-H), 8.14 (2H, d, J=6.7 Hz, Ar-H), 8.81
(2H, d, J=6.7 Hz, Ar-H); 13C NMR (75 MHz, CDCl3): d=82.6
(ArPh2C), 127.7 (Ar-CH), 129.6 (Ph-H), 129.7 (Ph-H), 129.9 (Ph-H),
142.7 (Ar-H), 146.2 (Ph-C), 170.3 (Ar-C); LRMS (ES+) m/z 262
([PyPh2COH+H]+, 100%).
Diphenyl(4-pyridyl)methanol hydrochloride (1.01 g, 3.41 mmol),
SOCl2 (9 mL), and CH3COCl (6 mL) were stirred at room tempera-
ture for 72 h. Excess SOCl2 and CH3COCl was removed under re-
duced pressure, and the crude product was co-evaporated several
times with toluene to yield 4-(chlorodiphenylmethyl)pyridine hy-
drochloride (1.073 g, 100%); mp: 154–1558C; 1H NMR (300 MHz,
CDCl3): d=7.19–7.44 (10H, m, Ph-H), 7.86 (2H, d, J=5.2 Hz, Ar-H),
8.95 (2H, d, J=5.2 Hz, Ar-H); 13C NMR (75 MHz, CDCl3): d=78.5
(ArPh2C), 127.8 (Ar-CH), 129.2 (Ph-H), 129.5 (Ph -H), 129.7 (Ph -H),
141.5 (Ar-H), 141.9 (Ph -C), 165.0 (Ar-C); LRMS (ES+) m/z 303
([M+Na]+, 11%), 262 ([PyPh2COH+H]+, 100%).
(ES+) calcd for C26H25N4O5 [M+H]+ 473.1819, found: 473.1821.
+
5’-[Diphenyl-(5-pyrimidyl)methyl]amino-2’,5’-dideoxyuridine
(7c): 5-(Chlorodiphenylmethyl)pyrimidine hydrochloride 20b
(0.25 g, 0.79 mmol) was taken up in dry pyridine (1 mL) in the pres-
ence of TEA (0.15 mL) under N2. 5’-Amino-2’,5’-dideoxyuridine 18
(0.15 g, 0.66 mmol) was added and the reaction mixture was
heated using microwave irradiation, first at 808C for 1 min and
then at 1008C for 1 min. The solvent was removed in vacuo and
the residue co-evaporated with toluene. The crude product was
then purified by flash column chromatography (ISOLUTE SI
column) eluting with 0!12% CH3OH in CHCl3 yielding the title
compound as a pale-yellow solid (44 mg, 14%). Rf =0.30 (10%
CH3OH/CHCl3); mp: 121–1268C; 1H NMR (500 MHz, CDCl3): d=
2.10–2.25 (2H, m, 2’-CHH and ArPH2C-NH), 2.30–2.45 (2H, m, 2’-
CHH and 5’-CHH), 2.64 (1H, m, 5’-CHH), 3.34 (1H, bs, 3’-OH), 4.10
(1H, m, 3’-H), 4.40 (1H, m, 4’-H), 5.68 (1H, d, J=8.1 Hz, 5-H), 6.20
5-(Chlorodiphenylmethyl)pyrimidine hydrochloride (20b): Di-
phenyl(5-pyrimidyl)methanol (1.06 g, 4.05 mmol), SOCl2 (9 mL) and
CH3COCl (6 mL) were stirred at room temperature for 72 h. The
excess of SOCl2 and CH3COCl was removed under reduced pressure
and the residue was co-evaporated several times with toluene to
yield 5-(chlorodiphenylmethyl)pyrimidine hydrochloride (25b) as a
1
thick yellow oil (1.28 g, 99%); H NMR (500 MHz, CDCl3): d=7.20–
7.27 (4H, m, Ph-H), 7.37–7.45 (6H, m, Ph-H), 7.84 (2H, s, Ar-H), 9.20
(1H, s, Ar-H); 13C NMR (125 MHz, CDCl3): d=75.7 (Ph2 Ar-C), 128.8
(Ph-CH), 129.1 (Ph-CH), 129.2 (Ph-CH), 141.3 (Ar-C), 141.8 (Ph-C), 1
(Ar-CH), 156.8 (Ar-CH); LRMS (ES+) m/z 263 ([MꢀCl+OH+H]+,
100%).
318
www.chemmedchem.org
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 309 – 320

5’-Trityl Nucleoside dUTPase Inhibitors
(1H, t, J=6.5 Hz, 1’-H), 7.06 (1H, d, J=8.1 Hz, 6-H), 7.25–7.50 (10H,
m, Ph-H), 8.94 (2H, s, Ar-H), 9.09 (1H, s, Ar-H), 9.51 (1H, bs, 3-NH);
13C NMR (500 MHz, CDCl3): d=40.1 (2’-CH2), 45.6 (5’-CH2), 68.4
(ArPH2-C), 72.0 (3’-CH), 85.6 (1’-CH), 85.8 (4’-CH), 102.9 (5-CH), 127.5
(Ph-CH), 128.1 (Ph-CH), 128.3 (Ph-CH), 128.62 (Ph-CH), 128.64 (Ph-
CH), 138.8 (Ar-C), 139.7 (6-CH), 143.5 (Ph-C), 143.9 (Ph-C), 150.2 (2-
C), 156.5 (Ar-CH), 157.0 (Ar-CH), 163.1 (4-C); LRMS (ES+) m/z 943
([2M+H]+, 8%), 494 ([M+Na]+, 53%), 472 ([M+H]+, 100%); HRMS
(0.103 g, 95%). Rf =0.45 (10% CH3OH/CH2Cl2); mp: 135–1408C;
1H NMR (300 MHz; CDCl3): d=2.04 (3H, s, COCH3), 2.32–2.50 (2H,
m, 2’-H-), 3.47–3.59 (2H, m, 5’-H), 4.07 (1H, s, 4’-H), 4.72–4.79 (1H,
m, 3’-H), 5.39 (1H, d, J=8.1 Hz, 5-H), 6.34 (1H, t, J=6.3 Hz, 1’-H),
6.94 (1H, s, CONH), 7.28–7.46 (15H, m, Ph-H), 7.83 (1H, d, J=
8.2 Hz, 6-H), 9.82 (1H, s, N-H); 13C NMR (75 MHz; CDCl3): d=38.8
(2’-CH2), 50.9 (3’-CH), 62.1 (5’-CH2), 85.2 (4’-CH), 87.3 (1’-CH), 88.1
(Ph3-C), 103.1 (5-CH), 127.9 (Ph-CH), 128.5 (Ph-CH), 129.1 (Ph-CH),
140.5 (6-CH), 143.6 (Ph-C), 154.9 (2-C), 163.6 (4-C); LRMS (ES+) m/z
533.8 ([M+Na]+, 20%); HRMS (ES+) calcd for C30H29N3O5Na
[M+Na]+ 534.2005, found: 534.2009; Anal. (%) calcd for
(C30H29N3O5, 1.0 HCl, 1.0 H2O) C 63.66, H 5.70, N 7.42, found: C
63.20, H 5.15, N 7.11.
(ES+) calcd for C26H26N5O4 [M+H]+ 472.1979, found: 472.1983.
+
3’-Azido-5’-O-trityl-2’,3’-dideoxyuridine
(14b):
LiF
(0.14 g,
5.61 mmol) was suspended in DMF (3 mL) and heated at 1058C
with stirring. To the stirred suspension was added TMEDA (5 mL)
followed by azidotrimethylsilane (0.64 g, 5.61 mmol). After stirring
for 1 h, 29 (1.41 g, 3.11 mmol) dissolved in DMF (2 mL) was added,
and the reaction was allowed to proceed for 20 h at 1108C. The
mixture was cooled, poured into CHCl3 (110 mL) and filtered
through Celite. The solvent was removed under reduced pressure,
and the residue (brown oil) was taken in EtOAc (100 mL). The or-
ganic phase was washed with H2O (4ꢃ180 mL), dried (MgSO4), fil-
tered and concentrated. The concentrated mixture was purified by
column chromatography eluting with 3% CH3OH in CHCl3. The title
compound was isolated as an orange solid (0.996 g, 65%). Rf =0.78
(5% CH3OH/CHCl3); mp: 67–708C; 1H NMR (300 MHz; CDCl3): d=
2.41–2.61 (2H, m, 2’-H), 3.49 (1H, dd, J=2.8, 11.1 Hz, 5’-CHH), 3.61
(1H, dd, J=1.9, 11.1 Hz, 5’-CHH), 3.98–4.02 (1H, m, 3’-H-), 4.40 (1H,
q, J=6.3 Hz, 4’-H), 5.44 (1H, dd, J=1.9, 8.1 Hz, 5-H), 6.25 (1H, t, J=
5.3 Hz, 1’-H), 7.32–7.46 (15H, m, Ph-H), 7.90 (1H, d, J=8.2 Hz, 6-H),
8.41 (1H, s, N-H); 13C NMR (75 MHz; CDCl3)d 36.8 (2’-CH2), 59.8 (3’-
CH), 64.0 (5’-CH2), 84.1 (1’-CH), 85.2 (4’-CH), 88.2 (Ph3-C), 102.8 (5-
CH), 128.0 (Ph-CH), 128.6 (Ph-CH), 129.0 (Ph-CH), 140.2 (6-CH),
143.4 (Ph-C), 151.2 (2-C), 161.4 (2-C); LRMS (ES+) m/z 518.0
([M+Na]+, 60%); HRMS (ES+) calcd for C28H25N5O4Na [M+Na]+
Acknowledgements
We thank the European Union (FP5 Cell Factory QLRT-2001-
00305), the FIS Network RICET/C03, and the State Secretariat for
Education and Research of Switzerland (R.B. and M.K.) for fund-
ing. The EPSRC National Mass Spectrometry Service Centre in
Swansea (UK) is acknowledged for carrying out accurate mass
spectrometry. Yvette Veerbeck is acknowledged for technical as-
sistance.
Keywords: dUTPase
· malaria · nucleoside chemistry ·
nucleotide metabolism · Plasmodium falciparum
[1] World Health Organisation (WHO), Health topics:
http://www.who.int/topics/malaria/en/ (accessed December 14, 2010).
[2] M. H. Gadsden, E. M. McIntosh, J. C. Game, P. J. Wilson, R. H. Haynes,
EMBO J. 1993, 12, 4425–4431.
518.1804, found: 518.1824; IR (KBr): n˜ =3038, 2108, 1691 cmꢀ1
;
[3] H. H. Elhajj, H. Zhang, B. Weiss, J. Bacteriol. 1988, 170, 1069–1075.
[4] J. L. Whittingham, I. Leal, C. Nguyen, G. Kasinathan, E. Bell, A. F. Jone, C.
Berry, A. Benito, J. P. Turkenburg, E. J. Dodson, L. M. Ruiz-Pꢀrez, A. J. Wil-
kinson, N. G. Johansson, R. Brun, I. H. Gilbert, D. Gonzꢁlez-Pacanowska,
K. S. Wilson, Structure 2005, 13, 329–338.
[5] C. Nguyen, G. Kanisathan, I. Leal-Cortijo, A. Musso-Buendia, M. Kaiser, R.
Brun, M. Ruiz-Pꢀrez, N.-G. Johansson, D. Gonzꢁlez-Pacanowska, I. H. Gil-
bert, J. Med. Chem. 2005, 48, 5942–5954.
[6] C. Nguyen, G. F. Ruda, A. Schipani, G. Kasinathan, I. Leal, A. Musso-Buen-
dia, M. Kaiser, R. Brun, L. M. Ruiz-Pꢀrez, B.-L. Sahlberg, N. G. Johansson,
D. Gonzꢁlez-Pacanowska, I. H. Gilbert, J. Med. Chem. 2006, 49, 4183–
4195.
[7] T. F. G. Henn, M. C. Garnett, S. R. Chhabra, B. W. Bycroft, J. Med. Chem.
1993, 36, 1570–1579.
Anal. (%) calcd for (C28H25N4.5O4, 0.6 H2O) C 66.87, H 5.33, N 12.53,
found: C 66.59, H 4.68, N 12.70.
3’-Amino-5’-O-trityl-2’,3’-dideoxyuridine (14c): Compound 14b
(0.10 g, 0.20 mmol) was hydrogenated in presence of Lindlar’s cata-
lyst (20 mg) in EtOH (5 mL) for 5 h. The suspension was filtered
through Celite, and fresh Lindlar’s catalyst (20 mg) was added to
the filtrate. The black suspension was then hydrogenated for fur-
ther 3 h. The solvent was evaporated and the concentrated solu-
tion was purified by column chromatography eluting with a gradi-
ent of 2!10% CH3OH in CH2Cl2. The title compound was obtained
as white solid (0.065 g, 70%). Rf =0.3 (10% CH3OH/CH2Cl2); mp:
1
110–1148C; H NMR (300 MHz; CDCl3): d=2.18–2.44 (2H, m, 2’-H),
[8] T. W. Greene, Protective Groups in Organic Synthesis, 2nd ed. (Ed.:
P. G. M. Wutts), John Wiley & Sons, Chichester, 1991.
3.38–3.73 (5H, m, 3’-H, 4’-H-, 5’-H), 5.41 (1H, d, J=8.1 Hz, 5-H), 6.20
(1H, q, J=3.3 Hz, 1’-H), 7.27–7.47 (15H, m, Ph-H), 7.97 (1H, d, J=
8.2 Hz, 6-H); 13C NMR (75 MHz, CDCl3): d=42.7 (2’-CH2), 50.7 (3’-
CH), 62.2 (5’-CH2), 85.2 (1’-CH), 87.2 (4’-CH), 87.9 (Ph3-C), 102.2 (5-
CH), 127.8 (Ph-CH), 128.5 (Ph-CH), 129.1 (Ph-CH), 140.7 (6-CH),
143.7 (Ph-C), 150.5 (2-C), 163.5 (4-C); LRMS (ES+) m/z 491.7
([M+Na]+, 45%); 243.2 ([Ph3C]+, 100%); HRMS (ES+) calcd for
C28H27N3O4Na [M+Na]+ 492.1899, found: 492.1902; Anal. (%) calcd
for (C28H27N3O4, 0.5 H2O) C 70.28, H 5.90, N 8.78, found: C 70.64, H
5.92, N 8.41.
[9] S. K. Chaudhary, O. Hernandez, Tetrahedron Lett. 1979, 20, 95–98.
[10] K. Jahn-Hofmann, J. W. Engels, Helv. Chim. Acta 2004, 87, 2812–2828.
[11] P. Kumar, K. C. Gupta, Chem. Lett. 1996, 635–636.
[12] N. I. Tzerpos, A. K. Zarkadis, R. P. Kreher, L. Repas, M. Lehnig, J. Chem.
Soc. Perkin Trans. 2 1995, 755–761.
[13] J.-M. Wang, T. Asami, S. Yoshida, N. Murofushi, Biosci. Biotechnol. Bio-
chem. 2001, 65, 817–822.
[14] M. Sekine, T. Hata, J. Org. Chem. 1983, 48, 3011–3014.
[15] M. Sekine, N. Masuda, T. Hata, Tetrahedron 1985, 41, 5445–5454.
[16] M. Sekine, R. Iwase, N. Masuda, T. Hata, Bull. Chem. Soc. Jpn. 1988, 61,
1669–1678.
[17] M. Gomberg, H. R. Snow, J. Am. Chem. Soc. 1925, 47, 189–211.
[18] A. Uemura, Y. Tada, S. Takeda, J. Uchida, J. Yamashita, Chem. Pharm. Bull.
1996, 44, 150–155.
[19] E. Strum, H. Reinhard, K. J. Kreutz, Neue Nucleosid-Verbindungen and Ver-
fahren zu ihrer Herstellung, Robugen GmbH, DE2658672, 1978; [Chem.
Abstr. 89, 163903].
3’-Aacetylamino-5’-O-trityl-2’,3’-dideoxyuridine
(14d):
Ac2O
(0.05 g, 0.044 mL, 0.469 mmol) and TEA (0.065 mL, 0.469 mmol)
were added to suspension of 14c (0.10 g, 0.213 mmol) in CH2Cl2
(5 mL). The mixture was stirred at room temperature for 3 h. The
solvent was evaporated to give a white solid, which was purified
by column chromatography eluting with a gradient of 2!6%
CH3OH in CH2Cl2. Compound 14d was obtained as white solid
[20] T. Kulikowski, D. Shugar, J. Med. Chem. 1974, 17, 269–273.
ChemMedChem 2011, 6, 309 – 320
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
319

I. H. Gilbert et al.
MED
[21] H. B. Lazrek, M. Taourirte, T. Oulih, J. L. Barascut, J. L. Imbach, C. Panne-
couque, M. Witrouw, E. De Clercq, Nucleosides Nucleotides Nucleic Acids
2001, 20, 1949–1960.
[22] W. S. Saari, J. E. Schwering, P. A. Lyle, S. J. Smith, E. L. Engelhardt, J. Med.
Chem. 1990, 33, 2590–2595.
[27] P. Kumar, K. Ohkura, J. Balzarini, E. De Clercq, S. Koh-ichi, I. L. Wiebe, Nu-
cleosides Nucleotides Nucleic Acids 2004, 23, 7–29.
[28] G. P. Kirschenheuter, Y. Zhai, W. A. Pieken, Tetrahedron Lett. 1994, 35,
8517–8520.
[29] K. Maeda, H. Oya, K. Yamada, J. Yamamoto, M. Nakashima, Nagoya J.
Med. Sci. 1970, 32, 453–458.
[30] F. Hidalgo-Zarco, A. G. Camacho, V. Bernier-Villamor, J. Nord, L. M. Ruiz-
Pꢀrez, D. Gonzꢁlez-Pacanowska, Protein Sci. 2008, 10, 1426–1433.
[23] K. A. Jørgensen, A. B. A. G. Ghattas, S.-O. Lawesson, Tetrahedron 1982,
38, 1163–1168.
[24] S. Czernecki, T. Le Diguarher, J. M. Valery, Nucleosides & Nucleotides
1993, 12, 369–380; Nucleotides 1993, 12, 369–380.
ˇ
ˇ
ˇ
[25] M. Prystas, J. Farkas, F. Sorm, Collect. Czech. Chem. Commun. 1963, 28,
Received: October 12, 2010
3140–3143.
Revised: December 3, 2010
[26] J. P. Horwitz, J. Chua, M. Noel, J. Org. Chem. 1964, 29, 2076–2078.
Published online on January 18, 2011
320
www.chemmedchem.org
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 309 – 320