Investigation of acyclic uridine amide and 5′-amido nucleoside analogues as potential inhibitors of the Plasmodium falciparum dUTPase

Article abstract of DOI:10.1016/j.bmc.2013.07.004

Previously we have shown that trityl and diphenyl deoxyuridine derivatives and their acyclic analogues can inhibit Plasmodium falciparum dUTPase (PfdUTPase). We report the synthesis of conformationally restrained amide derivatives as inhibitors PfdUTPase,

Full text of DOI:10.1016/j.bmc.2013.07.004

Bioorganic & Medicinal Chemistry 21 (2013) 5876–5885
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Investigation of acyclic uridine amide and 50-amido nucleoside
analogues as potential inhibitors of the Plasmodium falciparum
dUTPase q
Shahienaz E. Hampton a, Alessandro Schipani a, Cristina Bosch-Navarrete b, Eliseo Recio b,
Marcel Kaiser c,d, Pia Kahnberg e, Dolores González-Pacanowska b, Nils Gunnar Johansson e,
Ian H. Gilbert a,
⇑
a Division of Biological Chemistry and Drug Discovery, College of Life Science, University of Dundee, Sir James Black Centre, Dundee DD1 5EH, UK
b Instituto de Parasitología y Biomedicina, Consejo Superior de Investigaciones Científicas, Parque Tecnológico de Ciencias de la Salud, Avenida del Conocimiento,
18016 Armilla, Granada, Spain
c Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
d University of Basel, Petersplatz 1, CH-4051 Basel, Switzerland
e Medivir AB, P.O. Box 1086, S-14122 Huddinge, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Previously we have shown that trityl and diphenyl deoxyuridine derivatives and their acyclic analogues
can inhibit Plasmodium falciparum dUTPase (PfdUTPase). We report the synthesis of conformationally
restrained amide derivatives as inhibitors PfdUTPase, including both acyclic and cyclic examples. Activity
was dependent on the orientation and location of the amide constraining group. In the case of the acyclic
series, we were able to obtain amide-constrained analogues which showed similar or greater potency
than the unconstrained analogues. Unfortunately these compounds showed lower selectivity in cellular
assays.
Received 20 April 2013
Revised 25 June 2013
Accepted 1 July 2013
Available online 12 July 2013
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
1. Introduction
cerevisiae,6–8 it is therefore likely that dUTPases represent a novel
target that yet remains to be explored in malaria.
Malaria is a major global problem, affecting millions of people
each year. If not treated promptly; malaria can kill rapidly, espe-
cially children. In 2010, this accounted for an estimated 655,000
global malaria deaths (possibly ranging up to almost 1 million),
91% of which were in the African region.1,2 Following the
announcement of the goal of the global elimination of malaria;
there is an urgent need for novel drug targets in order to overcome
the current problems of resistance in the available antimalarial
drugs3,4 and to address new aspects of the disease.
We have previously reported the synthesis and biological eval-
uation of tritylated acyclic uracil analogues,9–11 which were shown
to be potent and selective inhibitors of the Plasmodium falciparum
dUTPase (PfdUTPase). Interestingly these analogues, which were
synthesised with varying chain lengths (Table 1), showed compa-
rable activities to their preceding cyclic counterparts,12 therefore
are of equal interest in terms of inhibitory activities. More recently
in an attempt to decrease lipophilicity and increase water solubil-
ity, we have shown when replacing the trityl group with a diphenyl
moiety, in both acyclic and cyclic molecules, that it is possible un-
der certain circumstances to retain PfdUTPase enzyme inhibition13
(Table 1 and Table 2).
A clear advantage of the acyclic analogues is that they have re-
duced molecular weight in addition to decreased clogP values,
therefore are possibly better candidates for the synthesis of an oral
compound.14 Additionally, these derivatives lack rigidity in their
structure, thus could allow access to binding pockets not accessible
with more rigid templates. The downsides of this strategy are
entropic disadvantages and the possibility of multiple binding
modes. One way in which to overcome this problem is to try and
conformationally restrain the flexible chain by the insertion of
one or more functional groups that are restricted in their rotation,
thereby introducing a certain degree of rigidity. Appropriate choice
Deoxyuridine 50-triphosphate nucleotidohydrolase (dUTPase) is
the enzyme that catalyses the hydrolysis of dUTP to dUMP. This
provides dUMP, a precursor required for the biosynthesis of dTTP,
whilst controlling the dUTP:dTTP concentration within the cell to
levels that will prevent mis-incorporation of dUTP into DNA.5
Due to the essentiality of dUTPases for cell viability in all organ-
isms studied to date, including Escherichia coli and Saccharomyces
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original author and source are credited.
⇑
Corresponding author. Tel.: +44 1382 386 240; fax: +44 1382 386 373.
E-mail address: i.h.gilbert@dundee.ac.uk (I.H. Gilbert).
0968-0896/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.004

S. E. Hampton et al. / Bioorg. Med. Chem. 21 (2013) 5876–5885
5877
Table 1
Biological results for selected acyclic PfdUTPase inhibitors as previously reported9
O
N
NH
O
n
X
R
Compound
X
n
R
MW
clogP
Enzyme assay Ki (
lM)
In vitro assays EC50 (lM)
PfdUTPase
HsdUTPase
SIa
P.f.b
L6 cellsc
SId
1a
1b
1c
1d
1e
1f
1g
1h
1i
O
O
O
O
NH
NH
NH
NH
NH
O
3
4
5
6
3
4
5
6
3
4
4
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
H
412
426
440
454
411
425
439
453
335
350
349
3.7
4.1
4.7
5.2
3.5
3.8
4.3
4.9
2.2
2.9
2.6
87
313
4
>617
>1
50
7
>1111
>233
>556
2
98
11
7.5
4.9
1.1
2.3
4.4
3.8
2.2
1.1
5.5
14
34
42
24
44
107
33
39
5
7
1.6
2.0
2.3
0.2
0.9
4.3
1.8
5.7
0.5
5.7
>1000
>1000
476
21
19
24
9
18
36
49
7
1.4
>1000
>1000
>1000
10
49
63
40
269
104
99
1j
1k
H
H
NH
2.6
38
a
b
c
Selectivity index (SI) for enzyme calculated as [Ki HsdUTPase/Ki PfdUTPase].
P. falciparum K1, a chloroquine and pyrimethamine resistant strain.
Cytotoxicity on rat L6 myoblasts.
d
Cell selectivity index (SI) was calculated as [EC50 L6/EC50 P. falciparum]. Controls: for P. falciparum, chloroquine, EC50 = 0.1 lM; for cytotoxicity, podophyllotoxin,
EC50 = 0.012 lM. The EC50 values are the means of two independent assays; the individual values vary less than a factor 2.
Table 2
Biological results for selected cyclic PfdUTPase inhibitors as previously reported12,13
O
O
N
R
X
NH
O
HO
X=O, NH
Compd
X
R
MW
clogP
Enzyme assay Ki (
PfdUTPase
l
M)
In vitro assays EC50 (lM)
HsdUTPase
SIa
P.f.b
L6 cellsc
SId
2a
2b
2c
O
NH
NH
Ph
Ph
H
470
469
393
3.0
2.8
1.6
1.8
0.2
0.2
18
46
>100
10
232
>500
6.0
4.5
12
192
—
>229
32
—
>20
a
b
c
Selectivity index (SI) for enzyme calculated as [Ki HsdUTPase/Ki PfdUTPase].
P. falciparum K1 chloroquine and pyrimethamine resistant strain.
Cytotoxicity on rat L6 myoblasts.
d
Cell selectivity index (SI) was calculated as [EC50 L6/EC50 P. falciparum]. Controls: for P. falciparum, chloroquine, EC50 = 0.1
M. The EC50 values are the means of two independent assays; the individual values vary less than a factor 2.
lM; for cytotoxicity, podophyllotoxin,
EC50 = 0.012
l
of functional group may also give additional interactions with the
active site, which may lead to an increase in potency and possibly
also selectively. Additionally there is also potential for alteration
and improvement of the pharmacokinetic properties of these com-
pounds as anti-parasitic agents.
We have previously synthesised within our laboratories mono
alkyl chain uracil acetamides with the amide bond insertion into
the alkyl linker chain at the C-2,3 position (Fig. 1).10 These were
shown to exhibit weak inhibition of the PfdUTPase; therefore it
did not seem that an amide linkage at this position was favourable.
O
O
NH
NH
Structural modification
N
O
N
O
H
N
n
X
X
n
O
n=2,3,4
X=N, O
n=1,2,3
X=N, O
Lead compounds
Target compounds
Figure 1. Derivatives synthesised by McCarthy et al.10 with the C-2,3 amide functionality.

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S. E. Hampton et al. / Bioorg. Med. Chem. 21 (2013) 5876–5885
O
O
N
NH
NH
Amide bond insertion
N
O
O
H
N
X
R
R
O
X = O, NH
R = H, Ph
Figure 2. Proposed structural modification to linker chain at C-4 position.
R
R
NH
NH
O
O
O
O
N
N
NH
NH
O
O
O
HO
HO
Figure 3. Proposed insertion of amide bond into cyclic nucleoside.
O
N
O
NH
NBz
O
O
O
O
N
O
N
N
H
OH
N
H
R
H
R
COOH
R
R
(i)
(ii)
(iii)
R = H,Ph
3
5,
7,
, R=H, 69%
R=H, 37%
R=H, 84%
4, R=Ph, 86%
6, R=Ph, 35%
8, R=Ph, 40%
Scheme 1. Synthesis of acyclic uridine amide analogues: (i) NH2(CH2)3OH, HOBt, TBTU, DIPEA, DMF, rt,16 h; (ii) 3N-benzoyl uracil, PS-PPh3, DIAD, THF, rt, 16 h; (iii) 0.2 M
NaOMe, CH3OH, rt, 16 h.
O
N
O
N
O
N
NH
NBz
O
NH
O
Br
O
O
EtO
H
N
(i)
(ii)
(iii)
EtO
HO
9
10
R
O
O
O
11, R=H, 14%
12,
R=Ph, 18%
Scheme 2. Synthetic route to acyclic derivatives with amide bond reversed: (i) 3N-benzoyl uracil, Cs2CO3, DMF, 60 °C, 1 h, 88%; (ii) 1 M NaOH, THF, rt, 16 h 65%; (iii) TrtNH2 or
(C6H5)2(CH)NH2, HOBt, TBTU, DIPEA, DMF, rt,16 h.
Here we describe insertion of the amide bond at the C-4 posi-
tion into the alkyl linker chain in order to probe the effects of a
movement in the amide linkage in the tritylated derivatives. Di-
phenyl analogues were also included in this study, and only the
4C chain was synthesised as they were shown to have optimal
activity and selectivity in the straight chain tritylated derivatives
(Table 1). Additionally reversal of the amide linkage was also
investigated (Fig. 2).
1.1. Chemistry
1.1.1. Acyclic analogues
The overall synthetic strategy (Scheme 1) in order to synthesise
the first set of amides involved coupling of the relevant carboxylic
acids to the amino alcohol linker chain, followed by attachment to
the uracil base.
The initial amidation step was achieved starting from either
diphenylacetic acid or triphenylacetic acid. HOBt and TBTU were
used as coupling reagents with 3-aminopropanol to give interme-
diate compounds 3 and 4. Mitsunobu coupling using polymer
Finally insertion of the amide bond into the cyclic compounds
gave an overall comparison of the effect of this increased rigidity
in the restrained nucleoside upon enzyme inhibition (Fig. 3).

S. E. Hampton et al. / Bioorg. Med. Chem. 21 (2013) 5876–5885
5879
O
O
O
O
O
O
N
N
N
NH
NH
NH
HO
TBDMSO
TBDMSO
HO
(i)
(ii)
O
O
O
HO
TBDMSO
13
14
(iii)
O
O
O
O
O
O
O
O
O
N
N
N
NH
NH
NH
HO
N
H
N
H
(iv)
(v)
O
O
O
TBDMSO
HO
TBDMSO
16
17
15
Scheme 3. Synthetic route to the cyclic amide derivatives: (i) TBDMSCl, imidazole, THF/DMF, rt, 3 h, 72%-quantitative; (ii) PPTS, CH3OH, rt, 16 h, 30%; (iii) BAIB, TEMPO,
CH3CN/H2O, rt, 14 h, 91%; (iv) (C6H5)2(CH)NH2, HOBt, TBTU, DIPEA, DMF, rt,16 h, 69%; (v) TBAF–Si, THF, rt, 24 h, 92%.
supported triphenylphosphine was carried out in order to attach
the nucleobase, prior to deprotection of the benzoyl group yielding
final compounds 7 and 8.
The synthesis was modified slightly in order to produce the cor-
responding compounds with the amide bond reversed (Scheme 2).
Following ester hydrolysis of 9,13 amidations of the resultant acid
gave the desired compounds, 11 and 12.
ꢀ Compounds 3 and 4 have been included and show the require-
ment of the uracil in inhibition of PfdUTPases (Ki = >100 and
1000
ꢀ The N-benzoyl derivative 6 (Ki >100
removal of the benzoyl group from this trityl forward amide
improves this value to 6.3 M (compound 8).
lM).
lM) is essentially inactive;
l
ꢀ The acid intermediate, 10, which lacks the trityl or diphenyl
moiety clearly shows low affinity for PfdUTPase together with
1.1.2. Cyclic analogues
weak activity (25 lM) against P. falciparum.
To prepare the cyclic amide analogues (trityl and diphenyl
derivatives), 20-deoxyuridine was protected at both the 50 and
the 30-positions as t-butyldimethylsilyl (TBDMS) ethers (Scheme 3).
This was followed by selective monodeprotection of the 50 hydro-
xyl using pyridinium para-toluene sulphonate (PPTS). Oxidation
of the alcohol was carried out in the presence of [bis(acetoxy)-
iodo]benzene (BAIB) and 2,2,6,6-tetramethylpiperidine-1-oxyl
(TEMPO) in water to give compound 15 in excellent yield.15,16
The final amidations were once again carried out with tritylamine
and diphenylmethylamine. The diphenyl product (16) was ob-
tained in 69% yield; however no triphenyl amide was obtained. It
is likely that the trityl group was too bulky to be able to react at
the crowded centre of the activated ester intermediate. It is antic-
ipated that had the trityl amide been formed, then it is likely that
this would have been more potent than that of the diphenyl ana-
logue as has been seen previously, although larger and more lipo-
philic. The diphenyl derivative on its own however is sufficient to
serve as an indicator as to whether these compounds would show
any affinity for the PfdUTPase. The final step was the removal of the
protecting group from 16 in the presence of TBAF on silica.
ꢀ Comparison of ‘forward’ amides 7 and 8 with ‘reverse’ amides 11
and 12 shows that the ‘reverse’ amides are more potent against
the PfdUTPase (Ki = 11 and 6.3
ꢀ The trityl ‘reverse amide’ (12) is also very comparable with the
straight chain (unconstrained) trityl derivative, 1f (Ki = 0.9 M),
whilst the diphenyl ‘reverse amide’ (11) is 8-fold more potent
than the straight chain diphenyl compound, 1k (Ki = 5.7 M).
lM vs Ki = 0.7 and 0.2 lM).
l
l
These results are noteworthy as a small change, such as revers-
ing an amide bond has had a significant impact on the observed
activities and illustrates the importance of correct orientation of
any functionality. Additionally this is encouraging as it means that
this extra rigidity in the chain is tolerated and the insertion of the
amide group is not detrimental to activity if the correct orientation
is attained.
The antiparasitic data remains constant for all the compounds
ranging between 7 and 14 lM for 7, 8, 11 and 12, although the
selectivity decreases compared to mammalian cells. There was
no correlation between inhibition of the enzyme and inhibition
of the parasite growth.
2.2. Cyclic analogues
2. Results and discussion
The results of cyclic amide 17 alongside the original trityl (2b)
and diphenyl (2c) derivatives are shown in Table 4. Compound 17
Biological activity was evaluated by testing all compounds
against the recombinant PfdUTPase and human dUTPase (HsdUT-
Pase) in order to determine inhibition constants and selectivity.
Additionally compounds were screened in vitro against the
chloroquine and pyrimethamine resistant, K1 strain of Plasmodium
falciparum cultured in erythrocytes to evaluate antiplasmodial
activity and the mammalian L6 cell line as a measure for cytotox-
icity (Tables 3 and 4).
showed some inhibition of the PfdUTPase (Ki = 8.3
lM), and selec-
tivity compared to the human dUTPase was retained (Ki = >100
lM).
This may suggest that the diphenyl group is constrained in a sub-
optimal conformation within the Plasmodium enzyme active site.
Activity against the parasite is also poor (EC50 = >5 lM). In contrast
to the acyclic series, this cyclic ‘reverse amide’ 17 was a weaker
inhibitor of PfdUTPase than the corresponding amine 2c.
2.1. Acyclic analogues
3. Conclusions
The results for all the compounds tested are shown in Table 3
including the data for the straight chain acyclic analogues, 1f and
1k, for comparison.9 A number of conclusions can be drawn from
this data:
We have successfully prepared some conformationally re-
strained amide derivatives of the tritylated acyclic uridine deriva-
tives. The aim of this work was to see if we could increase the

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S. E. Hampton et al. / Bioorg. Med. Chem. 21 (2013) 5876–5885
Table 3
Activity data for the acyclic derivatives
Compound No.
Structure
Enzyme assay, Ki (
lM)
In vitro assays, EC50
L6 cellsc
(lM)
P.fal
H. sap.
SIa
P.fal.b
SId
O
O
OH
OH
N
H
3
>100
1000
1000
13
19
18
19
1.0
4
6
1000
>100
1
9.8
6.7
1.9
1.4
N
H
O
N
O
N
O
O
1000
56
9
N
H
O
N
NH
O
O
7
11
286
26
14
18
1.3
N
H
O
N
NH
O
O
8
6.3
1000
159
11
18
1.6
N
H
O
NH
10
>100
1000
400
25
18
0.7
N
O
HO
O
O
NH
N
O
11
0.7
1000
1429
13
16
1.2
H
N
O
O
N
NH
O
12
0.2
1000
4545
7.2
4
0.6
H
N
O

S. E. Hampton et al. / Bioorg. Med. Chem. 21 (2013) 5876–5885
5881
Table 3 (continued)
Compound No.
Structure
Enzyme assay, Ki (
lM)
In vitro assays, EC50
L6 cellsc
(lM)
P.fal
H. sap.
SIa
P.fal.b
SId
O
N
NH
O
1fe
0.9
>1000
>1111
3.8
33
9
H
N
O
N
NH
O
1ke
5.7
63
11
2.6
99
38
H
N
a
Selectivity index (SI) for enzyme calculated as [Ki HsdUTPase/Ki PfdUTPase].
P. falciparum K1 chloroquine and pyrimethamine resistant strain.
Cytotoxicity on rat L6 myoblasts.
b
c
d
e
Cell selectivity index (SI) was calculated as [EC50 L6/EC50 P. falciparum]
Compounds 1f and 1k have previously been published,9 however have been included for comparative purposes. Controls: for P. falciparum, chloroquine, EC50 = 0.1
lM; for
cytotoxicity, podophyllotoxin, EC50 = 0.012 lM. The EC50 values are the means of two independent assays, the individual values vary less than a factor 2.
Table 4
Biological results for selected cyclic and acyclic PfdUTPase inhibitors
Compound No.
Structure
clogP
Enzyme assay, Ki (
lM)
In vitro assays, EC50 (lM)
P.fal.
H. sap.
SIa
P.fal.b
Toxc
SId
—
O
O
N
2be
2.8
0.2
46
230
4.5
—
NH
N
H
O
HO
O
2ce
1.6
1.4
0.2
8.3
>100
>100
>500
12
>5
>229
>20
NH
O
N
NH
O
HO
O
O
O
N
17
>12
>90
—
NH
N
H
O
HO
a
b
c
Selectivity index (SI) for enzyme calculated as [Ki HsdUTPase/Ki PfdUTPase].
P. falciparum K1 chloroquine and pyrimethamine resistant strain.
Cytotoxicity on rat L6 myoblasts.
d
e
Cell selectivity index (SI) was calculated as [EC50 L6/EC50 P. falciparum].
Compounds 2b and 2c have previously been published,12,13 however have been included for comparative purposes. Controls: for P. falciparum, chloroquine, EC50 = 0.1
lM;
for cytotoxicity, podophyllotoxin, EC50 = 0.012 lM. The EC50 values are the means of two independent assays; the individual values vary less than a factor 2.
potency of the compounds. Our ‘reverse amides’, 11 and 12,
showed similar or greater potency to the alkyl analogues against
PfdUTPase; whereas the ‘normal amides’ 7 and 8, showed reduced
activity. This suggests that the normal amides constrain the trityl/
diphenyl group in a sub-optimal orientation and gives rise to unfa-
vourable interactions in the active site.
The ‘reverse amides’ retain activity against the enzyme, but
show reduced selectivity in the cellular assay, indicating off-target
effects of these compounds.
4. Experimental section
4.1. Enzyme purification and inhibition assays
Both recombinant P. falciparum and human dUTPases were ex-
pressed in E. coli BL21 (DE3) cells which had been transformed
with the pET11Pfdut and pET3Hudut (kindly provided by P.O. Ny-
man, Lund University, Sweden) expression vectors, respectively.
For dUTPase purification, the same procedure was used for both

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S. E. Hampton et al. / Bioorg. Med. Chem. 21 (2013) 5876–5885
the human and the Plasmodium enzymes. Cell pellets from a 2.8 L
IPTG-induced culture were resuspended in 70 mL of buffer A
(20 mM MES, 50 mM NaCl, 1 mM DTT, pH 5.5) containing a prote-
ase inhibitor cocktail. The cells were lysed by sonication, and the
cell extract was cleared by centrifugation at 14,000 rpm for
30 min. The supernatant was loaded onto a 40 mL phosphocellu-
lose (Whatman P-11) column at 4 °C and eluted with a 50 mM to
1 M NaCl gradient in buffer A. Protein was further purified by gel
filtration chromatography on a Superdex 200 HA 10/30 column
at 4 °C. Pooled fractions were concentrated by centrifugation at
4 °C and desalted using a PD-10 column. The enzyme was stored
in 10 mM bicine and 5 mM MgCl2, pH 8 at ꢁ80 °C. Purified fractions
contained dUTPase of P96% purity.
Nucleotide hydrolysis was monitored by mixing enzyme and
substrate with a rapid kinetic accessory (Hi-Tech Scientific) at-
tached to a spectrophotometer (Cary 50) and connected to a com-
puter for data acquisition and storage as described previously.17
Protons, released through the hydrolysis of nucleotides, were neu-
tralized by a pH indicator in weak 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 (M), and the absorbance
changes were kept within 0.1 units. The indicator/buffer pair used
was red cresol/bicine (pH 8, 573 nm). Assay mixes contained
and the compounds were eluted with a gradient of 5–95% Acetoni-
trile/H2O + 0.1% ammonia.
4.3. General procedure A for the synthesis of amides 3–4, 11–12
The relevant carboxylic acid (1 equiv), HOBt (1.4 equiv) and
TBTU (1.4 equiv) were dissolved in DMF. To this was added DIPEA
(3 equiv) and stirred at room temperature for 1 h. To the mixture,
the amine (3.2 equiv) was added and the reaction left to stir at
room temperature overnight under an atmosphere of Ar. The mix-
ture was diluted with CHCl3 (25 mL) and extracted with H2O (5
ꢃ 30 mL). The organic layer was dried with MgSO4 and the solvent
concentrated under reduced pressure. The product was purified by
flash chromatography.
4.3.1. General procedure B: Mitsunobu coupling of alcohols with
3-N-benzoyluracil for the synthesis of 5–6
Polymer supported triphenylphosphine (2.5 equiv; 3 mmol/g)
was swelled in THF for 15 min. To this was added the correspond-
ing alcohol (1 equiv) and the N-3 benzoyluracil (2 equiv) which
were shaken at room temperature for a further 15 min before DIAD
(2 equiv) in THF was added to the mixture. The reaction was sha-
ken until consumption of the alcohol was seen by TLC. The resin
was then filtered off and washed with THF and the solvent re-
moved under reduced pressure. The crude product was then puri-
fied by flash chromatography (Hexane/EtOAc 40:60 and/or CHCl3/
CH3OH 95:5).
30 nM PfdUTPase, 50 lM dUTP, 5 mM MgCl2, 1 mg/mL BSA, and
100 mM KCl. Vmax and KMapp were calculated by fitting the result-
ing data to the integrated Michaelis–Menten equation. The appar-
ent KM values were plotted against inhibitor concentration, and Ki
values (Table 1) were obtained according to Eq. 1.
4.3.2. General procedure C: hydrolysis of benzoyl esters for the
synthesis of compounds 7–8
KM
Ki
KMapp
¼
½Iꢂ þ KM
ð1Þ
The benzoyl ester intermediate was dissolved in a solution of
0.2 M sodium methoxide in CH3OH and the reaction stirred at
room temperature overnight until the disappearance of the start-
ing esters was observed (TLC). The solution was neutralised with
Dowex H+ ion exchange resin, filtered and washed with methanol.
The solution was concentrated in vacuo and the crude residue was
purified by chromatography.
Activity against P. falciparum K1 strain and cytotoxicity assess-
ment against L6 cells (rat skeletal myoblast cells) was determined
as previously reported.9
4.2. Chemistry
Solvents and reagents were purchased from commercial suppli-
ers and used without further purification. Dry solvents were
purchased in sure sealed bottles stored over molecular sieves.
Reactions were performed in pre-dried apparatus under an
atmosphere of argon unless otherwise stated. Normal phase TLC
4.3.3. N-(3-Hydroxypropyl)-2,2-diphenylacetamide (3)
Compound 3 was synthesised following general procedure A,
using diphenyl acetic acid (1.00 g, 4.71 mmol), 3-amino-1-propa-
nol (1.15 mL, 15.04 mmol), HOBt (1.07 g, 6.59 mmol), TBTU
(2.12 g, 6.59 mmol) and DIPEA (2.46 mL, 14.13 mmol) dissolved
in DMF (20 mL). The product was purified by flash chromatography
(MeOH/CHCl3 0 ? 2%) to give the product as a white crystalline so-
lid (508 mg, 40%). Purity by LCMS (UV chromatogram, 190–
450 nm): >98%, Rt = 4.7 min; Rf = 0.10 (CHCl3/CH3OH 95:5); 1H
NMR (500 MHz; CDCl3): d 1.66 (m, 2H, HOCH2CH2), 3.10 (t,
J = 6.3 Hz, 1H, OH), 3.46 (q, 6.2 Hz, 2H, NHCH2), 3.61 (q, J = 6.2 Hz,
2H, HOCH2), 4.96 (s, 1H, COCH), 6.06 (bs, 1H, NH), 7.27–7.38 (m,
10H, H–Ar); 13C NMR (125 MHz; CDCl3): d 32.2 (CH2), 36.6 (CH2),
59.2 (CH2), 59.3 (CH), 127.4 (2 ꢃ CH–Ar), 128.9 (2 ꢃ CH–Ar),
139.2 (CH–Ar), 173.4 (CO); LRMS (ES+): m/z 270.1 [M+H]+, 539.3
was carried out on pre-coated silica plates (Kieselgel 60 F254
,
BDH) with visualisation via either ninhydrin, PMA, or 254 nm UV
light. Flash chromatography was performed using Combiflash
Companion or Combiflash Rf and prepacked columns (silica gel)
purchased from Redisep (Presearch), Silicycle (Anachem) or Grace
Resolve. Preparative HPLC was performed using
a Gilson
(321-Pump, 153-UV–vis Detector) equipped with a Gilson liquid
handler for injection and fraction collection and XBridge Prep
C18, 5
lm, ODB, 19 ꢃ 100 mm column (Waters) with 0.1% ammo-
nia in water (solvent A) and acetonitrile (solvent B) as mobile
phase. Melting points (mp) were measured on a Gallenkamp melt-
ing point apparatus and are uncorrected. 1H NMR and 13C NMR
spectra were recorded on a Bruker Avance DPX500 spectrometer
or on a Bruker Avance DPX300 using the applied solvent simulta-
neously as internal standard. Deuterated solvents were purchased
from Goss. Chemical shifts (d) are given in ppm together with the
multiplicity, relative frequency, coupling constants (J, Hz) and
assignment. High resolution mass spectra were performed on a
Bruker MicroTof mass spectrometer at University of Dundee.
LC–MS analysis and chromatographic separation were conducted
with a Bruker MicroTof mass spectrometer using an Agilent HPLC
1100 with a diode array detector in series. The column used was
[2M+H]+; HRMS (ES+): found 270.1483 [M+H]+ C17H20NO2 re-
quires 270.1489.
+
4.3.4. N-(3-Hydroxypropyl)-2,2,2-triphenylacetamide (4)
Compound 4 was following using general procedure A, using
triphenyl acetic acid (1.00 g, 3.47 mmol), 3-amino-1-propanol
(845 lL, 11.1 mmol), HOBt (656.68 mg, 4.86 mmol), TBTU (1.56 g,
4.86 mmol) and DIPEA (1.82 mL, 10.41 mmol) dissolved in DMF
(15 mL). Following extraction the product was sufficiently pure
and did not require chromatography. 4 was obtained as an off
white solid (1.03 g, 86%). Purity by LCMS (UV chromatogram,
190–450 nm): >98%, Rt = 5.1 min; Rf = 0.60 (CHCl3/CH3OH 90:10);
1H NMR (500 MHz; CDCl3): d 1.66 (m, 2H, HOCH2CH2), 3.03 (t,
a Waters Xbridge C18, 3.5
lm particle size, 2.1 ꢃ 50 mm column

S. E. Hampton et al. / Bioorg. Med. Chem. 21 (2013) 5876–5885
5883
J = 5.9 Hz, 1H, OH), 3.51 (q, J = 6.2 Hz, 2H, NHCH2), 3.56 (q,
J = 5.6 Hz, 2H, HOCH2), 6.26 (bs, 1H, NH), 7.28–7.35 (m, 15H, H–
Ar); 13C NMR (125 MHz; CDCl3): d 32.3(CH2), 36.9 (CH2), 59.3
(CH2), 67.9 (CH), 127.1 (CH–Ar), 128.1–130.4 (2 ꢃ CH–Ar), 143.2
(CH–Ar), 174.7 (CO); LRMS (ES+): m/z 346.1 [M+H]+, 691.3
Ar), 7.50 (d, J = 7.8 Hz, 1H, NCH), 11.24 (s, 1H,CONHCO); 13C NMR
(125 MHz; (CD3)2SO): d 29.0 (CH2), 37.1 (CH2), 45.9 (CH2), 67.8
(CH), 101.3 (CH), 126.9 (C–Ar), 128.2 (C–Ar), 130.6 (C–Ar), 144.2
(C–Ar), 146.1 (CH), 151.4 (C), 164.1 (C), 172.7 (C); LRMS (ES+): m/
z
440.2 [M+Na]+, m/z 901.4 [2M+Na]+; HRMS (ES+): found
[2M+H]+; HRMS (ES+): found 346.1808 [M+H]+ C23H24NO2 re-
440.1950 [M+H]+ C27H26N3O3 requires 440.1969.
+
+
quires 346.1802.
4.3.9. 1-(Carboxypropyl)uracil (10)18
4.3.5. 3-Benzoyl-(1-(3-diphenylacetylamino)propyl)uracil) (5)
To a mixture of compound 9 (2.92 g, 8.84 mmol) suspended in
THF (25 mL) was added NaOH (90 mL. 88.40 mmol, 1 M) and the
mixture stirred vigorously at rt overnight. The solvents were re-
moved under reduced pressure and the residue taken up in H2O
(50 mL) and acidified with HCl (1 M), followed by extraction with
CHCl3 (3 ꢃ 100 mL). The aqueous layer was concentrated and the
residue purified by flash chromatography (0 ? 20% CH3OH/
CHCl3 + 0.3% TFA) to give a white solid (1.13 g, 5.71 mmol, 65%).
Purity by LCMS (UV chromatogram, 190–450 nm): >98%,
Rt = 0.6 min; Rf = 0.15 (CHCl3/ CH3OH 80:20); Mp: 174–176 °C;
1H NMR (500 MHz; CDCl3): d 1.80 (m, 2H, NCH2CH2), 2.24 (t,
J = 7.4 Hz, 2H, NCH2), 3.68 (t, J = 7.0 Hz, 1H, COCH2), 5.54 (dd,
J = 2.3, 7.8 Hz, 1H, NCHCH), 7.62 (d, J = 7.8 Hz, 1H, NCH), 11.23
(bs, 1H, NH), 12.17 (bs, OH); 13C NMR (125 MHz; CDCl3): d 24.3
(CH2), 31.0 (CH2), 47.4 (CH2), 101.4 (CH), 146.1 (CH), 151.4 (C),
164.2 (C), 174.2 (C); LRMS (ES+): m/z 199.0 [M+H]+, 397.1
Compound 5 was prepared following general procedure B from
the alcohol
3 (499 mg; 1.85 mmol) and 3-N-benzoyl uracil
(800 mg; 3.70 mmol) to give a white powder (0.317 g, 37%).
Rf = 0.04 (EtOAc/Hexane 60:40); 1H NMR (500 MHz; CDCl3): d
1.89 (m, 2H, NCH2CH2), 3.33 (q, J = 6.29 Hz, 2H, NHCH2), 3.73 (t,
J = 6.32 Hz, 2H, NCH2), 4.90 (s, 1H, COCH), 5.80 (d, J = 7.96 Hz, 1H,
NCHCH), 6.18 (t, J = 6.17 Hz, 1H, NH), 7.25–7.34 (m,10H, H–Ar),
7.36 (d, J = 7.97 Hz, 1H, NCH), 7.53 (t, J = 7.61 Hz, 2H, H–Ar), 7.69
(t, J = 7.46 Hz, 1H, H–Ar), 7.94 (d, J = 7.30 Hz, 2H, H–Ar); 13C NMR
(125 MHz; CDCl3): d 29.3 (CH2), 36.2 (CH2), 46.5 (CH2), 59.0 (CH),
102.4 (CH), 127.3–131.4 (C–Ar), 135.2 (C–Ar), 139.2 (C–Ar), 144.5
(CH), 150.2 (C), 162.3 (C), 168.8 (C), 172.6 (C);
LRMS (ES+): m/z 490.2 [M+Na]+, 957.4 [2M+Na]+; HRMS (ES+):
found 468.1920 [M+H]+ C28H26N3O4 requires 468.1918.
+
[2M+H]+; HRMS (ES+): found 199.0718 [M+H]+ C8H11N2O4 re-
+
4.3.6. 3-Benzoyl-(1-(3-(triphenylacetylamino)propyl)uracil) (6)
Compound 6 was prepared following general procedure A from
the alcohol 3 (200 mg; 0.579 mmol) and 3-N-benzoyl uracil 3
(253 mg; 1.16 mmol). This was obtained as a white powder
(0.111 g, 35%). Rf = 0.16 (CHCl3/CH3OH 95:5); 1H NMR (500 MHz;
(CD3)2SO): d 1.75 (m, 2H, NCH2CH2), 3.17 (m, 2H, NHCH2), 3.60
(t, J = 6.9 Hz, 2H, NCH2), 5.84 (d, J = 8.0 Hz, 1H, NCHCH), 7.19–
7.30 (m, 15H, H–Ar), 7.36 (t, J = 5. 9 Hz, 1H, NH), 7.58 (t, J = 7.9,
2H, H–Ar), 7.78 (m, 2H, NCH+H–Ar), 7.96 (d, J = 8.4 Hz, 2H, H–
Ar); 13C NMR (125 MHz; (CD3)2SO): d 28.9 (CH2), 37.2 (CH2), 46.7
(CH2), 67.7 (C), 101.1 (CH), 126.9–130.7 (C–Ar), 131.6 (C–Ar),
135.9 (CH), 144.2 (C–Ar), 147.1 (C–Ar), 150.0 (C), 162.7 (C), 170.1
(C), 172.7 (C); LRMS (ES+): m/z 544.2 [M+H]+; HRMS (ES+): found
quires 199.0713.
4.3.10. 1-[3-(Benzhydrylaminocarbonyl)propyl]uracil (11)
Compound 11 was synthesised using following procedure A,
using acid 10 (400 mg, 2.02 mmol), benzhydryl amine (1.20 mL,
6.46 mmol), HOBt (384 mg, 2.83 mmol), TBTU (9.09 mg,
2.83 mmol) and DIPEA (1.06 mL, 6.06 mmol) dissolved in DMF
(20 mL). The product was purified by flash chromatography
(0 ? 5% CH3OH/CH2Cl2 + 3% Et3N) as an off-white solid (102 mg;
0.281 mmol, 14%). Purity by LCMS (UV chromatogram, 190–
450 nm): 97%, Rt = 4.2 min; Rf = 0.23 (CHCl3/ CH3OH 95:5); 1H
NMR (500 MHz; CDCl3): d 2.06 (m, Hz, 2H, NCH2CH2), 2.35 (t,
J = 6.8 Hz, 2H, NCH2), 3.82 (t, J = 6.8 Hz, 2H, COCH2), 5.66 (d,
J = 7.9 Hz, 1H, NCHCH), 6.26 (d, J = 8.1 Hz, 1H, NHCH), 6.57 (d, J =
8.1 Hz, 1H, CHNHCO); 7.21 (d, J = 7.9 Hz, 1H, NCH), 7.25–7.37 (m,
10H, H–Ar), 8.65 (bs, 1H, CONHCO); 13C NMR (125 MHz; CDCl3):
d 24.8 (CH2), 32.4 (CH2), 47.8 (CH2), 57.1 (CH), 102.4 (CH), 127.3–
128.7 (C–Ar ꢃ 3), 141.4 (C–Ar), 144.7 (CH), 151.1 (CO), 163.3
(CO), 170.6 (CO); LRMS (ES+): m/z 364.1 [M+H]+, 749.3 [2M+Na]+;
544.2217 [M+H]+ C34H30N3O4 requires 544.2231.
+
4.3.7. 1-(3-(Diphenylacetylamino)propyl)uracil (7)
Compound 7 was prepared following general procedure B from
the 3-N-benzoyl protected intermediate 6 (298 mg; 0.638 mmol).
The crude was purified by flash chromatography (CH3OH/CHCl3
0 ? 10%) to yield the product as a white solid (0.195 g, 84%). Purity
by LCMS (UV chromatogram, 190–450 nm): >98%, Rt=3.1 min;
Rf = 0.12 (CHCl3/CH3OH 95:5); Mp = 145–147 °C; 1H NMR
(500 MHz; CDCl3): d 1.87 (m, 2H, NCH2CH2), 3.31 (m, 2H, NCH2),
3.70 (t, J = 6.47 Hz, 2H, NCH2), 4.95 (s, 1H, COCH), 5.71 (dd,
J = 7.9, 2.2 Hz, 1H, NCHCH), 6.29 (t, J = 6.11 Hz, 1H, NH), 7.24 (d,
J = 7.9 Hz, NCH), 7.27–7.37 (m, 10H, H–Ar), 9.15 (s, 1H, CONHCO);
13C NMR (125 MHz; CDCl3): d 29.3 (CH2), 36.1 (CH2), 46.0 (CH2),
59.1 (CH), 102.3 (CH), 127.3 (C–Ar), 128.8 (C–Ar), 139.3 (C–Ar),
144.6 (CH), 151.2 (C), 163.5 (C), 172.6 (C); LRMS (ES+): m/z 364.1
[M+H]+, 727.3 [2M+H]+; HRMS (ES+): found 364.1657 [M+H]+
HRMS (ES+): found 364.1638 [M+H]+ C21H22N3O3 requires
+
364.1656.
4.3.11. 1-[3-(Tritylaminocarbonyl)propyl]uracil (12)
Compound 12 was synthesised following general procedure A,
using acid 10 (482 mg, 2.43 mmol), tritylamine (2.02 g,
7.78 mmol), HOBt (459 mg, 3.40 mmol), TBTU (1.09 g, 3.40 mmol)
and DIPEA (1.30 mL, 7.29 mmol) dissolved in DMF (20 mL). The
product was purified by flash chromatography (0 ? 10% CH3OH/
CH2Cl2 + 3% Et3N, then 0–100% EtOAc/Hexane) as a white solid
(179 mg, 0.408 mmol, 18%). Purity by LCMS (UV chromatogram,
190–450 nm): >98%, Rt = 3.5 min; Rf = 0.36 (CHCl3/CH3OH 95:5);
1H NMR (500 MHz; CDCl3): d 2.00 (m, 2H, NCH2CH2); 2.37 (t,
J = 6.6 Hz, 2H, COCH2), 3.72 (t, J = 6.9 Hz, 2H, NCH2), 5.64 (d,
J = 7.9 Hz, 1H, NCHCH), 6.75 (s, 1H, CHNH), 7.12 (d, J = 7.9 Hz, 1H,
NCH), 7.21–7.34 (m, 15H, H–Ar), 8.49 (bs, 1H, CONHCO); 13C
NMR (125 MHz; CDCl3): d 13.9 (CH2), 24.6 (CH2), 33.1 (CH2), 64.8
(CH), 102.1 (CH), 127.2–130.0 (C–Ar ꢃ 3), 144.4 (CH), 145.0 (CO),
170.6 (CO), 176.2 (CO); LRMS (ES+): m/z 243.0 [Ph3C]+, 440.1
[M+H]+, 896.4 [M+NH4]+; HRMS (ES+): found 440.1977 [M+H]+
+
C21H22N3O3 requires 364.1656.
4.3.8. 1-(3-(Triphenylacetylamino)propyl)uracil (8)
Deprotection was carried out following general procedure C
from the benzoyl intermediate 6 (73 mg; 0.166 mmol). The residue
was purified by flash chromatography (CH3OH/CH2Cl2 0 ? 2%) to
yield a white powder (0.029 g, 0.066 mmol, 40%). Purity by LCMS
(UV chromatogram, 190–450 nm): 99%, Rt=3.5 min; Rf = 0.16
(CHCl3/CH3OH 95:5); mp: 154–156 °C; 1H NMR (500 MHz; (CD3)2-
SO): d 1.67 (m, 2H, NCH2CH2), 3.13 (m, 2H, NCH2), 3.49 (t, J = 7.0 Hz,
2H, NCH2), 5.53 (d, J = 7.8 Hz, 1H, NCHCH), 7.21–7.33 (m, 15H, H–
+
C27H26N3O3 requires 440.1969.

5884
S. E. Hampton et al. / Bioorg. Med. Chem. 21 (2013) 5876–5885
4.3.12. 30,50-O-Di(tert-butyldimethylsilyl)-20-deoxyuridine (13)19
To a solution of 20-deoxyuridine (1.00 g, 4.39 mmol) in THF/
DMF (1:1) (10 mL) was added imidazole (1.20 g, 17.56 mmol) fol-
lowed by TBDMSCl (1.46 g, 9.66 mmol), and stirred at rt for 3 h.
The mixture was diluted with EtOAc (25 mL) and washed with
H2O (3 ꢃ 25 mL). The organic layer was dried (MgSO4), concen-
trated and purified by flash chromatography (CH3OH/CH3Cl
0 ? 20%) to yield the product as a white crystalline foam (1.44 g,
3.16 mmol, 72%). Purity by LCMS (UV chromatogram, 190–
450 nm): >98%, Rt = 5.8 min; Rf = 0.51 (CH3OH/CHCl3 10:90); Mp:
45–47 °C; 1H NMR (500 MHz, CDCl3): d 0.00 (d, J = 2.8 Hz, 6H,
Si(CH3)2), 0.03 (s, 6H, Si(CH3)2), 0.79 (s, 9H, (CH3)3), 0.81 (s, 9H,
(CH3)3), 1.96 (m, 1H, CHH-20), 2.22 (m, 1H, CHH-20), 3.66 (dd,
J = 11.2, 1.8 Hz 1H, CHH-50), 3.81 (dd, J = 8.1, 2.2 Hz, 1H, CHH-
50 + H-40), 4.31 (m, 1H, H-30), 5.58 (d, J = 8.2 Hz, 1H, H-5), 6.19 (t,
J = 6.2 Hz, 1H, H-10), 7.80 (d, J = 8.2 Hz, 1H, H-6), 8.31 (bs, 1H,
NH-3); 13C NMR (125 MHz, CDCl3): d [ꢁ5.5]–[ꢁ4.6] (Si((CH3)2) -
ꢃ 2), 18.0–18.3 ((CH3)3 ꢃ 2), 25.7–25.8 (SiC ꢃ 2), 41.8(CH2-20),
62.4 (CH-30), 71.1 (CH2-50), 85.1 (CH-40), 87.7 (CH-10), 102.1 (CH-
5), 140.2 (CH-6), 150.0 (C-2), 163.0 (C-4); LRMS (ES+): m/z 457.2
[M+H]+, 479.3 [M+Na]+, 913.5 [2M+H]+; HRMS (ES+): found
0.397 mmol), HOBt (0.026 g, 0.149 mmol), TBTU (0.048 g,
0.149 mmol) and DIPEA (0.048 g, 0.372 mmol) dissolved in DMF
(10 mL). Upon completion, the mixture was diluted with CH2Cl2
(15 mL) and washed with H2O (3 ꢃ 15 mL). The organic portion
was dried (MgSO4) and concentrated. Purification by HPLC
(Rt = 10.9 min, 1 min hold 95% A, 3.5 min ramp to 95% B, 3.5 min
hold 95% B) gave 16 (0.045 g, 0.086 mmol, 69%) as a white powder.
Rf = 0.32 (CH3OH/CHCl3 10:90); Mp: 94–96 °C; 1H NMR (500 MHz,
CDCl3): d 0.00 (d, J = 5.0 Hz, 6H, Si((CH3)2), 0.78 (s, 9H, SiC(CH3)3),
2.02 (dd, J = 12.1, 4.9 Hz, 1H, CHH-20), 2.22 (m, 1H, CHH-20), 4.27
(s, 1H, H-40), 4.58 (d, J = 4.7 Hz, 1H, H-30), 5.42 (d, J = 8.1 Hz, 1H,
H-5), 6.14–6.19 (m, 2H, H-10 + CHNH), 7.13–7.25 (m, 12H, H–
Ar + H-6 + NH-3); 13C NMR (125 MHz, CDCl3): d ꢁ4.8 ((CH3)2),
18.0 (SiC), 25.7 ((CH3)3), 38.5(C-20), 56.8 (CH3)2), 75.1 (CH-30),
87.1 (CH-40), 88.6 (CH-10), 103.2 (CH-5), 127.1–128.9 (CH–
Ar ꢃ 3), 140.6–141.3 (CH-6 + C-Ar), 150.4 (C-2), 163.1 (C-4),
168.8 (C-50); LRMS (ES+): m/z 167.0 [Ph2C]+, 522.2 [M+H]+; HRMS
(ES+): found 522.2405 [M+H]+ C28H36N3O5Si+ requires 522.2405.
4.3.16. 20,50-Dideoxyuridine-50-N-benzhydryl carboxamide (17)
Silyl protected compound, 16 (0.351 g, 0.674 mmol) was dis-
solved in THF (20 mL). To this was added TBAF on silica (1.13 g,
1.70 mmol, 1.5 mmol Fꢁ/g) and stirred at rt for 24 h. A further por-
tion of TBAF on silica was added and stirred for a further 7 h. The
mixture was filtered and washed 5% CH3OH/CHCl3 and the filtrate
dried (MgSO4), concentrated and purified by flash chromatography
(CH3OH/CHCl3 0 ? 5%) to give 17 (0.253 g, 92%) as a white powder.
Purity by LCMS (UV chromatogram, 190–450 nm): 96%,
Rt = 4.3 min; Rf = 0.29 (CH3OH/CHCl3 10:90); 1H NMR (500 MHz,
(CD3)2SO): d 2.15–2.17 (m, 2H, H-20), 4.32 (s, 1H, H-30) 4.49 (s,
1H, H-40), 4.63–5.65 (dd, J = 8.1, 1.8 Hz, 1H, H-5), 5.66–5.67 (d,
J = 4.4 Hz, 1H, OH-30), 6.14–6.16 (d, J = 8.5 Hz, 1H, CHNH), 6.32–
6.35 (t, J = 7.1 Hz, 1H, H-10), 7.26–7.37 (m, 10H, H–Ar), 8.34–8.35
(d, J = 8.1 Hz, 1H, H-6), 9.27–9.29 (d, J = 8.5 Hz, 1H, CHNH), 11.31
457.2538 [M+H]+ C21H41N2O5Si2 requires 457.2549.
+
4.3.13. 30-O-(tert-butyldimethylsilyl)-20-deoxyuridine (14)
13 (1.43 g, 3.14 mmol) and PPTS (4.21 g, 16.75 mmol) were
combined in CH3OH (30 mL) and stirred at rt until reaction com-
pletion. This was concentrated, the residue suspended in EtOAc
(30 mL) and washed with H2O (7 ꢃ 30 mL). The organic layer was
dried (MgSO4), concentrated to give the product (0.322 g,
0.942 mmol, 30%) which did not require further purification.
Rf = 0.44 (CH3OH/CHCl3 10:90); 1H NMR (500 MHz, CDCl3): d 0.00
(s, 6H, Si(CH3)2), 0.80 (s, 9H, SiC(CH3)3), 2.20 (m, 2H, H-20), 3.68
(m, 1H, CHH-50), 3.85 (m, 2H, CHH-50 + H-40), 4.40 (m, 1H, H-30),
5.65 (dd, J = 8.1, 1.8 Hz 1H, H-5), 6.08 (t, J = 6.6 Hz, 1H, H-10), 7.55
(d, J = 8.1 Hz, 1H, H-6), 8.60 (bs, 1H, NH-3); 13C NMR (125 MHz,
CDCl3): d ꢁ4.8 (Si(CH3)2)), 17.9 (SiC), 25.7 ((CH3)3), 40.8(CH2-20),
61.9 (CH2-50), 71.4 (CH-30), 86.8 (CH-10), 87.5 (CH-40), 102.5 (CH-
5), 141.1 (CH-6), 150.1 (C-2), 163.0 (C-4); LRMS (ES+): m/z 343.1
[M+H]+, 685.2 [2M+H]+.
13
(s, 1H, NH-3); C NMR (125 MHz, (CD3)2SO): d 38.9 (CH2-20),
56.0(CHNH), 73.7 (30-CH), 85.1 (40-CH), 85.6 (CH-10), 101.9 (CH-
5), 127.1 (CH–Ar), 127.2 (CH–Ar), 128.4 (CH–Ar), 141.1 (CH-6),
128.4 (C–Ar), 150.5 (C-2), 163.0 (C-4), 169.8 (C-50); LRMS (ES+):
m/z 167.0 [Ph2C]+, 408.2 [M+H]+; 815.3 [2M+H]+; HRMS (ES+):
found 408.1538 [M+H]+ C22H22N3O5 requires 408.1554.
+
4.3.14. 30-O-(tert-butyldimethylsilyl)-20-deoxyuridine-50-
carboxylic acid (15)
Acknowledgments
TEMPO (0.031 g, 0.198 mmol) and BAIB (0.728 g, 2.26 mmol)
were added to a solution of 14 (0.322 g, 0.942 mmol) in CH3CN/
H2O (1:1) (20 mL). This was left to stir overnight at rt. The mixture
was diluted with EtOAc (15 mL) and washed with H2O (3 ꢃ 10 mL).
The organic layer was dried (MgSO4), concentrated and purified by
flash chromatography (EtOAc/Hexane 0 ? 100%) to give 15
(0.082 g, 0.230 mmol, 24%) as a white powder. Purity by LCMS
(UV chromatogram, 190–450 nm): >98%, Rt = 0.5 min; Rf = 0.13
(CH3OH/CHCl3 10:90); 1H NMR (500 MHz, CDCl3): d 0.01 (d,
J = 6.5 Hz, 6H, Si(CH3)2), 0.77 (s, 9H, SiC(CH3)3), 1.97 (m, 1H, CHH-
20), 2.20 (m, 1H, CHH-20), 4.37 (s, 1H, H-30), 4.51 (d, J = 4.3 Hz, 1H,
H-40), 5.72 (d, J = 8.0 Hz, 1H, H-5), 6.20 (dd, J = 9.1, 5.1 Hz, 1H, H-
10), 7.98 (d, J = 8.1 Hz, 1H, H-6), 8.69 (bs, 1H, NH-3); 13C NMR
(125 MHz, CDCl3): d ꢁ4.9 Si(CH3)2), 18.0 (SiC), 25.6 ((CH3)3),
39.5(CH2-20), 75.8 (CH-30), 85.4 (CH-10), 88.9 (CH-40), 102.7 (CH-
5), 141.8 (CH-6), 150.2 (C-2), 163.5 (C-4), 172.9 (C-50); LRMS
(ES+): m/z 357.1 [M+H]+, 713.3 [2M+H]+; LRMS (ESꢁ): m/z 355.1
[MꢁH]+; HRMS (ES+): found 357.1482 [M+H]+ C15H25N2O6Si+ re-
quires 357.1476.
We would like to acknowledge the BBSRC and Medivir for a
CASE award to S.E.H. The European Union (contract 037587), the
RICET FIS Network to CSIC (RD06/0021/0018), the Junta de And-
alucía to CSIC (BIO-199) and the Welcome Trust Strategic Grant
to the University of Dundee, WT083481) are acknowledged for
funding.
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