Design, synthesis and evaluation of novel uracil acetamide derivatives as potential inhibitors of Plasmodium falciparum dUTP nucleotidohydrolase

Article abstract of DOI:10.1016/j.ejmech.2008.05.018

The ubiquitous enzyme dUTP nucleotidohydrolase (dUTPase) catalyses the hydrolysis of dUTP to dUMP and can be considered as the first line of defence against incorporation of uracil into DNA. Inhibition of this enzyme results in over-incorporation of uraci

Full text of DOI:10.1016/j.ejmech.2008.05.018

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 678e688
http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and evaluation of novel uracil acetamide derivatives as
potential inhibitors of Plasmodium falciparum dUTP nucleotidohydrolase
Orla McCarthy a,b, Alex Musso-Buendia c, Marcel Kaiser d, Reto Brun d, Luis M. Ruiz-Perez c,
Nils Gunnar Johansson e, Dolores Gonzalez Pacanowska c, Ian H. Gilbert a,b,
*
a Welsh School of Pharmacy, University of Wales Cardiff, King Edward VII Avenue, Cardiff CF10 3XF, UK
b College of Life Sciences, University of Dundee, Sir James Black Centre, Dundee DD1 5EH, UK
c Instituto de Parasitologia y Biomedicina ‘‘Lopez-Neyra’’, Consejo Superior de Investigaciones Cient´ıficas,
Parque Tecnolo´gico de Ciencias de la Salud, Avenida del Conocimiento, s/n, 18100-Armilla, Granada, Spain
d Swiss Tropical Institute, Socinstrasse 57, Basel, Switzerland
e Medivir AB, Lunastigen 7, S-141 44 Huddinge, Sweden
Received 10 October 2007; received in revised form 12 May 2008; accepted 13 May 2008
Available online 10 July 2008
Abstract
The ubiquitous enzyme dUTP nucleotidohydrolase (dUTPase) catalyses the hydrolysis of dUTP to dUMP and can be considered as the first
line of defence against incorporation of uracil into DNA. Inhibition of this enzyme results in over-incorporation of uracil into DNA, leading to
DNA fragmentation and cell death and is therefore lethal. By taking advantage of structural differences between the human and Plasmodium
dUTPase, selective inhibitors of the enzyme can be designed and synthesised with the aim of being developed into novel anti-parasitic drugs.
Analogue based design was used to target the Plasmodium falciparum dUTPase (PfdUTPase). The structures of previously discovered selective
inhibitors of the PfdUTPase were modified by insertion of an amide bond. A series of tritylated uracil acetamide derivatives were synthesised
and assessed for inhibition of the enzyme and parasite growth in vitro. These compounds were weak inhibitors of the PfdUTPase.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: dUTP nucleotidohydrolase; Uracil acetamide; Anti-plasmodial; Anti-malarial
1. Introduction
Annually, there are over 300 million cases of malaria and
more than 1 million deaths, mainly amongst children [1]. The
parasite Plasmodium falciparum, transmitted to the human
host through the female Anopheles mosquito, is the main cause
of severe clinical malaria. Resistance of the pathogenic parasite
to commonly used chemotherapeutic agents is a major issue and
the constant need for new drugs to treat this disease is urgent.
dUTP nucleotidohydrolase, also called dUTP pyrophosphatase
or dUTPase is an enzyme which catalyses the hydrolysis of dUTP
to dUMP in the presence of magnesium ions [2]. The enzyme has
two major roles in maintaining the correct free nucleotide balance
in the cell. It provides dUMP, a major cellular source for dTMP
which is in turn needed for dTTP formation. It also maintains
the concentration of dUTP 10ꢀ5 times lower than that of dTTP
thereby minimizing mistaken incorporation of dUTP into DNA
Malaria is a health problem in more than 90 countries, plac-
ing about 40% of the world’s population at risk of contracting
the disease. It is endemic to tropical and subtropical regions.
Abbreviations: PfdUTPase, Plasmodium falciparum dUTPase; dUTPase,
dUTP nucleotidohydrolase; TBDPS, tert-butyl diphenyl silyl; EDC, N-ethyl-
N0-(3-dimethylaminopropyl)carbodiimide; CbZ, carbobenzoxy; DCC, dicyclo-
hexylcarbodiimide;
benzotriazine.
DhbtOH,
3-hydroxy-3,4-dihydro-4-oxo-1,2,3-
* Corresponding author. College of Life Sciences, University of Dundee, Sir
James Black Centre, Dundee DD1 5EH, UK. Tel.: þ44 0 1382 386 240; fax:
þ44 0 1382 386 373.
E-mail address: i.h.gilbert@dundee.ac.uk (I.H. Gilbert).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.05.018

O. McCarthy et al. / European Journal of Medicinal Chemistry 44 (2009) 678e688
679
[3]. dUTPase is an ubiquitous enzyme and it has been shown to be
essential for cell viability in many organisms [4e6].
shown in Scheme 3. The first step was selective protection
of the terminal amino functions of the starting linear triamines
which was carried out using carbobenzyloxy imidazole (CbZ-
imidazole) [11]. CbZ-imidazole was synthesised from the
reaction of CbZeCl with imidazole at 0 ꢁC. In the case of
compound 9a, the uracil acetamide was synthesised from the
reaction of compound 1 with the protected triamine 7a using
dicyclohexylcarbodiimide (DCC) as a coupling reagent and
3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (DhbtOH)
as an additive [12]. Compound 9b, however, was synthesised
from the reaction of the activated succinimidyl ester, 8 of com-
pound 1 with the protected triamine 7b [12]. Subsequent
deprotection of the terminal amino groups was carried out
by hydrogenation over 5% Pd/C catalyst. NMR analyses of
compounds 10a and 10b, however, revealed that acyl migra-
tion to the terminal amino groups had occurred to some extent
(see Scheme 4). Tritylation was then carried out. However, the
final product was not the expected product. During the trityla-
tion process migration of the uracil acetamide to the terminal
amino group was observed. Migration from the secondary
amine to the terminal amine is presumably an equilibrium pro-
cess, and addition of the bulky trityl group to one of the termi-
nal amines pushes the equilibrium in favour of migration of
the uracil acetamide from the secondary amine to the other ter-
minal amine (Scheme 4). The migration was detected by
studying the NMR spectra. If no migration had occurred in
11a or 11b, then there would be 4 protons in the spectra cor-
responding to C(O)NCH2e and 2 protons corresponding to e
CH2NH-trityl and 2 protons corresponding to eCH2NH2. The
C(O)NCH2e (d w 3.4 ppm) protons are distinctive (down-
field) compared to the eCH2NH-trityl (d w 2.2e2.4 ppm)
and eCH2NH2 (d w 2.8e2.9 ppm). However, in the spectra
Initially, we have shown tritylated deoxyuridine analogues
and then subsequently tritylated acyclic uridine analogues
were able to inhibit the P. falciparum dUTPase (PfdUTPase)
enzyme selectively in the low micromolar range (Fig. 1).
These compounds also inhibited parasite growth in the low
micromolar range [7e9]. This enzyme is a good target for de-
velopment of novel anti-malarials.
In this paper we report an analogue based design, in which an
amide bond was inserted into the scaffold of these previously
discovered selective acyclic inhibitors. It was thought that
through amide coupling methodology, a wide range offunctional
variability could be introduced to these molecules and therefore
this modification would be advantageous for diversification
studies and ultimately to increase the potency of our inhibitors.
Furthermore, the presence of an amide bond would introduce
rigidity into the conformationally flexible acyclic inhibitors.
2. Chemistry
The synthesis of the tert-butyl diphenyl silyl (TBDPS) ura-
cil acetamides (3a, 3b) was carried out by coupling the
TBDPS protected amino alcohol (2a, 2b) to 1-carboxymethyl
uracil (1) [10] using EDC as a coupling reagent (Scheme 1).
The tritylamino analogues were synthesised by mono-trity-
lation of the relevant diamines (4aec) followed by coupling to
1-methylcarboxyuracil (1) again using EDC as a coupling re-
agent (Scheme 2).
Subsequent to the synthesis of monoalkyl chain acetamide
uracil derivatives, dialkyl chain derivatives were synthesised.
Diamino uracil acetamides 11a and 11b were synthesised as
O
O
O
NH
NH
NH
H
O
N
Si
O
N
N
O
O
O
N
O
O
O
OH
OH
OH
K PfdUTPase = 0.2 µM
K
PfdUTPase = 1.8 µM
HsdUTPase = 18 µM
K
PfdUTPase = 4.2 µM
HsdUTPase = 806 µM
Ki
i
HsdUTPase = 46 µM
Ki
i
Ki
i
ED50 Pf = 4.5 µM
ED50 Pf = 6 µM
ED50 Pf = 6.6 µM
O
N
O
O
N
NH
NH
NH
N
O
O
O
H
N
O
Si
N
H
16
17
18
K
PfdUTPase = 4.3 µM
HsdUTPase = >1000 µM
K
PfdUTPase = 1.8 µM
HsdUTPase = >1000 µM
K
PfdUTPase = 8.5 µM
HsdUTPase = >1000 µM
Ki
i
Ki
i
Ki
i
ED50 Pf = 2.2 µM
ED50 Pf = 1.1 µM
ED50 Pf = 3.9 µM
Fig. 1. Previously reported PfdUTPase inhibitors [8,9].

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O. McCarthy et al. / European Journal of Medicinal Chemistry 44 (2009) 678e688
O
O
a
NH
O
NH
+
Cl
O
N
O
HO
N
H
O
N
HO
1
NH
O
O
c
+
Si
O
NH
n
O
n
NH2
n
NH2
3a, n=3, 61%
3b, n=4, 68%
b
Si
O
HO
Si Cl
+
n=3,4
2a, n=3, 7%
2b, n=4, 14%
Scheme 1. Synthesis of TBDPS uracil acetamide derivatives (a) H2O, KOH, reflux 1 h, HCl, 4 ꢁC, 16 h; (b) DMF, imidazole, RT, 18 h; (c) EDC, DMF, 18 h.
for 11a or 11b, there are 2 protons in the spectra correspond-
ing to C(O)NCH2e and 2 protons corresponding to eCH2NH-
trityl and 4 protons corresponding to eCH2NH2.
[8]. A continuous spectrophotometric method was used for the
enzyme assays using indicator, based on the fact that the hydro-
lysis of dUTP to dUMP is coupled to release of protons.
The compounds were also tested for in vitro activity against
the erythocytic stages of the chloroquine resistant K1 strain of
P. falciparum and against rat L6 cells for toxicity.
Various attempts were made to prevent this migration. Tri-
tylation of 10b was attempted at 0 ꢁC in pyridine to prevent
further acyl migration from occurring, but there was no reac-
tion at this temperature; heating was required (70 ꢁC for 8 h)
and migration was again observed. It was found that carrying
out the reaction at RT in DCM using Et3N as base reduced re-
action time to 2 h and this procedure was used for the synthe-
sis of compound 11a. However, it did not prove possible to
isolate the compound where migration of the uracil acetamide
did not occur.
Hydroxy functionalised branched chain derivatives were
synthesised as shown in Scheme 5. The starting diamino alco-
hol was protected at the hydroxy function using the TBDPS
group. Selective tritylation of the terminal amine was then car-
ried out by slow addition of a dilute solution of trityl chloride
to diamines 13 at 0 ꢁC followed by the addition of Et3N. Cou-
pling to 1-carboxymethyl uracil to yield the uracil acetamide
14 was again carried out using the DCC, DhbtOH methodol-
ogy. Finally, removal of the TBDPS group was carried out us-
ing TBAF to yield the desired uracil acetamide 15.
4. Results and discussion
The results of the biological assays for the uracil acetamide
derivatives synthesised are shown in Table 1. Compounds 16e
18 are some of the lead inhibitors previously synthesised and
reported [9] in which the amide bond is not present, the struc-
tures of which are shown in Fig. 1.
Two of the uracil acetamide compounds 5a and 5b ex-
hibited weak but selective inhibition of the PfdUTPase en-
zyme (Ki ¼ 37.3, 23.1 mM, respectively). However, the
activity of these compounds against the enzyme was about
10-fold lower than their acyclic analogues 17 and 18
(Ki ¼ 4.3, 1.84 mM, respectively). It would therefore seem
that introduction of an amide bond is unfavourable for the in-
hibitory action of these nucleoside derivatives against the
PfdUTPase.
Despite their lack of activity as inhibitors of the dUTPase
enzyme, some of these compounds show quite potent activity
against P. falciparum cultured in red blood cells. These com-
pounds may therefore be exerting their effect on a different
target which has not yet been identified.
3. Biological activity
The compounds were assayed against the recombinant form
of the PfdUTPase and the human dUTPase as reported previously
O
NH
O
n
NH2
a
N
b
N
H
NH2
H
N
H
N
H2N
n
n
n=2,3,4
O
4a, n=2, 51%
4b, n=3, 20%
4c, n=4, 31%
5a, n=2, 33%
5b, n=3, 42%
5c, n=4, 26%
Scheme 2. Synthesis of tritylamino uracil acetamide derivatives (a) TrtCl, DCM, RT, 18 h; (b) EDC, DMF, RT, 18 h.

O. McCarthy et al. / European Journal of Medicinal Chemistry 44 (2009) 678e688
681
n
n
N
n
n
n
N
n
a
c or d
O
CbZHN
NHCbZ
NHCbZ
CbZHN
H2N
NH2
N
H
7a, n=2,2, 9%
H
O
O
O
7b, n=3,3, 35%
N
O
NH
O
N
HN
O
O
N
N
6, 80%
N
O
9a, n=2,2, 69%
9b, n=3,3, 89%
b
O
8, 100%
O
O
N
O
Cl
e
+
O
N
N
O
NH
O
N OH
+
HO
O
O
O
N
O
NH
NH
N
f
g
O
O
O
O
N
H
N
HN
NHTrt
n
H2N
NH2
n
n
n
10a, n=2,2, 41%
10b, n=3,3, 50%
11a, n=2,2, 6%
11b, n=3,3, 17%
Scheme 3. Synthesis of branched chain trityldiamino uracil acetamide derivatives (a) DCM, 2 d, RT; (b) DCM, 0 ꢁC, RT, 10 min; (c) DMF, DMAP, 3 d, 60 ꢁC; (d)
1-carboxymethyl uracil, DMF, DCC, DhbtOH, 0 ꢁC, 1 h, RT 2d; (e) DMF, DCC, 0 ꢁC, 3 h, RT, 24 h; (f) MeOH/EtOH, H2, 5% Pd/C; (g) TrtCl, Et3N, DCM, RT,
2 h.
4.1. Molecular modelling
orientations found with a tritylated deoxyuridine analogue
[7]. This was in comparison to other acyclic derivatives, which
were docked in conformations consistent with the crystallo-
graphic data [9]. Therefore 5b was manually placed into the ac-
tive site in a conformation consistent with the crystallographic
In order to try and understand the lack of activity, some mo-
lecular modelling studies were undertaken. Attempts to dock
compound 5b in the active site failed to reproduce the
O
O
NH
NH
N,N acyl migration,
O
N
isomers in equilibrium
O
N
O
O
H
N
n
n
N
H2N
NH
H2N
NH2
n
n
O
N
O
N
NH
Bulky terminal group
attached and equilibrium
shifted to the right
NH
Ph
Ph
Ph
O
O
Ph
Ph
Ph
O
n
NH
O
H
N
n
NH
n
NH
N
n
NH2
Scheme 4. Equilibrium of N,N acyl migration.

682
O. McCarthy et al. / European Journal of Medicinal Chemistry 44 (2009) 678e688
a
b
H
N
TBDPSO
NH2
N
H
HO
NH2
12, 32%
O
N
O
NH
NH
H
N
d
c
O
O
N
NHTrt
TBDPSO
O
O
13, 63%
N
N
HO
NHTrt
TBDPSO
NHTrt
14, 52%
15, 78%
Scheme 5. Synthesis of branched chain hydroxyl tritylamino uracil acetamide derivatives (a) TBDPSCl, pyridine, 24 h, RT; (b) TrtCl, DCM, Et3N, 0 ꢁC, RT, 24 h;
(c) 1-carboxymethyl uracil, DCC, DhbtOH, 0 ꢁC, 1 h, RT, 24 h; (d) THF, TBAF, RT, 30 min.
structure of the tritylated deoxyuridine analogue. Compound 5b
was then minimised in the active site using the MAB force field
in Moloc [13] in order to identify any large conformational
change due to interaction between the protein and ligand. These
studies suggest the reason for the loss of activity of the uracil
acetamides may be due to an unfavourable conformation the
molecules must adopt to bind in the active site of the enzyme.
The required conformation forces the O-3 of the uracil ring to
be in close proximity to the carbonyl oxygen of the amide
bond causing unfavourable electrostatic repulsion. There also
may be unfavourable interactions between the carbonyl oxygen
of the amide bond and the carbonyl oxygen of Glu115 and phe-
nyl OH of Tyr112 (Fig. 2).
N-1 of the uracil ring resulted in a loss of activity against
the PfdUTPase enzyme. Molecular modelling and conforma-
tional studies showed that this may have been due to the
fact that the low energy conformation of the uracil acetamide
compounds must overcome an energy barrier to adopt the re-
quired binding conformation. Additionally, there may be an
unfavourable interaction between the O-2 of the uracil ring
and the carbonyl oxygen of the amide bond in the proposed
bound conformation leading to a decrease in potency of these
compounds.
The uracil acetamide compounds do retain good activity
in vitro, however. Their mode of action has not yet been
ascertained.
In fact, as calculated using the Tripos force field, the energy
difference between the lowest energy conformation of com-
pound 5b in vacuo and the conformation it would have to
adopt in the active site of the PfdUTPase is 7.5 kcal/mol.
6. Experimental
Low resolution mass spectra were recorded at a Platform II
mass spectrometer (Micromass) from Fisons where ionisation
was achieved in the positive and negative electrospray modes
using a 1:1 mixture of acetonitrile and water plus 0.2% formic
acid or a 95:5 mixture of methanol and water plus 0.2% formic
acid as a mobile phase. High resolution mass spectra were re-
corded by the National Mass Spectrometry Service Centre in
Swansea at a MAT 900 XLT high resolution double focussing
mass spectrometer from Finnigan or by University of Birming-
ham at a Micromass LCT spectrometer. High resolution mass
spectra were also recorded at a MicrOTOF mass spectrometer
from Bruker Daltonics. Accurate mass measurement was
performed by peak matching. NMR spectra were recorded at
a Bruker 300 MHz NMR spectrometer or at a Bruker
500 MHz NMR spectrometer. Purification by column chro-
matography was performed on Sorbosil C60A silica gel
40e60 mm from Merck. Flash chromatography was performed
using the Flashmaster II from Jones Chromatography and Iso-
luteÒ propylene columns which were prepacked with silica.
Qualitative thin layer chromatography (TLC) was done on pre-
coated aluminium sheets Silica gel 60 F254 from Merck.
Compounds were detected either with ninhydrin or 254 nm
UV light. Elemental analysis was carried out by the analytical
and chemical consultancy services MEDAC Ltd. Found per-
centage values are reported together with calculated values
5. Conclusion
The introduction of an amide bond into the scaffold of pre-
viously discovered selective inhibitors of the P. falciparum
dUTPase led to the synthesis of a range of uracil acetamide
derivatives. Introduction of an amide bond at the b-C to the
Table 1
Biological results of uracil acetamides against the trimeric dUTPase and in vi-
tro results against malaria parasites
Compound Enzyme assay Ki (mM)
P. falciparum Human
In vitro assay IC50 (mM)
P. falciparum Toxicityb
SIa
>1
3a
3b
5a
>1 mM
>1 mM
37.3
>1 mM
>1 mM
>1 mM
>1 mM
>1 mM
>1 mM
>1 mM
>1 mM
4.6
3.7
4.2
2.5
2.9
4.5
3.9
88.9
33.4
53.0
102.2
42.7
52.7
76.7
>1
>26
>43
>1
5b
5c
23.1
>1 mM
>1 mM
>1 mM
>1 mM
4.3
11a
11b
15
>1
>1
>1 >83.0
>373
39
16
17
>1 mM >232
>1 mM >555
>1 mM >117
2.2
1.1
3.9
1.8
8.5
40
18.0
18
Data for 16-18 has been previously reported [9].
a
SI ¼ selectivity index [Ki(human dUTPase)/Ki(PfdUTPase)].
b
Toxicity tests were carried out on L6 cells.

O. McCarthy et al. / European Journal of Medicinal Chemistry 44 (2009) 678e688
683
Fig. 2. Possible repulsive interactions to the carbonyl oxygen of the amide bond in uracil acetamides compounds in the PfdUTPase active site.
for different salt compositions as indicated by the preceding
formula. Melting points were determined with a Gallenkamp
melting point apparatus and are not corrected. Solvents and re-
agents were purchased from SigmaeAldrich, Fluka or Lancas-
ter and were used without further purification. Dry solvents
were purchased in sure sealed bottles stored over molecular
sieves. Deuterated solvents were purchased from Goss. All
molecular modelling was carried out on a silicon graphics
work station or a Mac OS using SYBYL 6.9 or 7.2.
(1 eq.) dissolved in DMF (0.1 mL/mmol) under N2 or Ar
atmosphere. The mixture was left stirring at RT until the dis-
appearance of the starting material was observed by TLC.
6.1.2.1. Synthesis of 3-(tert-butyl diphenyl silanyloxy)-propyl-
amine (2a). Yield: 7%. Rf: 0.3 in CH2Cl2/MeOH/NH3,
90:8:2. 1H NMR (300 MHz, MeOD) d 0.95 (s, 9H,
CH3 ꢂ 3), 1.71 (qn, J ¼ 6.50, 2H, CH2CH2CH2), 2.78 (t,
J ¼ 7.1, 2H, CH2NH2), 3.73 (t, J ¼ 6.01, 2H, CH2O), 7.36
(m, 6H, meta,para-PhH), 7.71 (m, 4H, ortho-PhH). 13C
NMR (75 MHz, MeOD) d 20.5 (tCeSi), 27.9 (CH3 ꢂ 3),
36.5 (CH2CH2CH2), 40.3 (CNH2), 63.6 (CO), 129.3 (meta-
PhC ), 131.4 ( para-PhC ), 135.2 (PhCeSi), 136.7e137.4 (or-
tho-PhC ). LRMS (ESþ): m/z ¼ 314.2 [M þ Hþ], 636.9 2
[M þ Hþ]. HRMS (ESþ): found: 314.1935 [M þ Hþ]
C19H28ONSi requires 314.1935.
6.1. Chemistry
6.1.1. Synthesis of 1-carboxymethyl uracil (1) [10]
Uracil (2.98 g, 26.64 mmol), chloroacetic acid (4.41 g,
47.67 mmol) and KOH (6.57 g, 117.12 mmol) were dissolved
in 100 mL water and refluxed for 1 h. The reaction mixture
was cooled to room temperature and acidified to pH 2 with
HCl (32% w/w in water). The mixture was left at 4 ꢁC over-
night. The white precipitate formed was collected by filtration
and washed with water (50 mL), ethanol (50 mL) and EtOAc
(50 mL) to afford 3.16 g of the product as a white powder.
Yield: 71%. Melting point: 287e290 ꢁC (Lit. 287 ꢁC [10]).
6.1.2.2. Synthesis of 4-(tert-butyl diphenyl silanyloxy)-butyl-
amine (2b). Yield: 14%. Rf: 0.2 in CH2Cl2/MeOH/NH3,
90:8:2. 1H NMR (300 MHz, MeOD) d 1.05 (s, 9H,
CH3 ꢂ 3), 1.01 (m, 4H, CH2CH2CH2CH2), 2.63 (t, J ¼ 6.65,
2H, CH2NH2), 3.69 (m, 2H, CH2O), 7.36 (m, 6H, meta,-
para-PhH), 7.60 (m, 4H, ortho-PhH). 13C NMR (75 MHz,
MeOD) d 20.5 (CeSi), 27.9 (CH3 ꢂ 3), 30.5 (CH2CH2CH2),
31.5 (CH2CH2CH2), 42.8 (CNH2), 65.0 (CO), 129.3 (meta-
PhC ), 131.3 ( para-PhC ), 135.3 (PhCeSi), 137.1 (ortho-
PhC ). LRMS (ESþ): m/z ¼ 328.2 [M þ Hþ]. HRMS (ESþ):
found: 328.2098 [M þ H]þ C20H30ONSi requires 328.2091.
1
Rf: 0.1 in CHCl3/MeOH, 9:1. H NMR (300 MHz, DMSO)
d 4.43 (s, 2H, CH2), 5.60 (d, J ¼ 7.8, 1H, ]CH ), 7.62 (d,
J ¼ 7.8, 1H, ]CH ). 13C NMR (75 MHz, DMSO) d 49.4
(CH2), 101.7 (CO2H), 146.9 (C]C), 151.9 (C]C ),
164.7(NHCO), 170.4 (HNCO). LRMS (ESꢀ): m/z ¼ 168.8
[M ꢀ Hþ].
6.1.2. General procedure for tert-butyl diphenyl silylation of
hydroxyl group using DMF and imidazole
6.1.3. General procedure for the mono-tritylation
of diamines
TBDPSCl (1.1 eq.) dissolved in DMF (0.2 mL/mmol) was
added to a mixture of imidazole (2.2 eq.) and the alcohol
TrtCl (0.1 eq.) dissolved in DCM (2.5 mL/mmol) was
added to the diamines in DCM (0.1 mL/mmol) and left stirring

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O. McCarthy et al. / European Journal of Medicinal Chemistry 44 (2009) 678e688
under N2 at RT overnight. The mixture was poured into 50 mL
ice water and extracted with ether (1 ꢂ 100 mL, 2 ꢂ 50 mL).
The organic layer was washed with water (2 ꢂ 50 mL), brine
(1 ꢂ 50 mL) and dried with MgSO4. The solvent was evapo-
rated off. The product was purified by flash chromatography.
DMSO) d 18.8 (3 ꢂ CH3), 26.6 (CeSi), 32.0 (CH2CH2CH2),
45.1 (CH2NH), 49.4 (COCH2uracil), 61.2 (CH2O), 100.4
(C]C), 127.9 ( para-PhC ), 129.8 (meta-PhC ), 133.2 (ortho-
PhC ), 135.0 (SiePhC ), 146.6 (C]C ), 151.0, (NCONH),
163.9 (CCONH), 166.5 (NHCOCH2). LRMS (ESꢀ): m/
z ¼ 464.0 [M ꢀ Hþ]. HRMS (ESþ): found: 466.2159
[M þ Hþ] C25H32O4N3Si1þ requires 466.2157. Analysis: found:
C, 64.35; H, 6.75; N, 8.86%; calc. for C25H31N3O4Si$0.1 mol
H2O: C, 64.24; H, 6.73, N, 8.99%.
6.1.3.1. Synthesis of 2-(triphenylmethylamino) ethylamine (4a).
1
Yield: 51%. Rf: 0.6 in CH2Cl2/MeOH/NH3, 90:7:3. H NMR
(300 MHz, MeOD) d 2.14 (t, J ¼ 6.19, 2H, CH2NHTrt), 2.64
(t, J ¼ 6.26, 2H, CH2NH2), 7.19 (m, 3H, para-PhH ), 7.29
(m, 6H, meta-PhH ), 7.5 (m, 6H, ortho-PhH ). 13C NMR
(75 MHz, MeOD) d 43.4 (CH2NH2), 47.6 (CH2NHTrt), 83.3
(Trt quat. C), 127.7 ( para-PhC ), 129.1 (ortho-PhC ), 129.7
(meta-PhC ), 147.9 (Ph quat. C ). LRMS (ESþ): m/z ¼ 303
[M þ Hþ]. HRMS (ESþ): found: 303.1853 [MHþ]
C21H23Nþ2 requires 303.1856.
6.1.4.2. Synthesis of N-[4-(tert-butyl diphenyl silanyloxy)-bu-
tyl]-2-uracil acetamide (3b). Yield: 68%. Melting point:
1
174 ꢁC. Rf: 0.5 in CH2Cl2/MeOH, 9:1. H NMR (300 MHz,
MeOD)
d
1.05 (s, 9H, CH3 ꢂ 3), 1.63 (m, 4H,
CH2CH2CH2CH2), 3.23 (t, J ¼ 6.53, 2H, CH2NH2), 3.71 (t,
J ¼ 5.77, 2H, CH2O), 4.41 (s, 2H, COCH2uracil), 5.65 (d,
J ¼ 7.65, 1H, ]CH ), 7.40 (m, 6H, meta,para-PhH), 7.51 (d,
J ¼ 7.87, 1H, ]CH ), 7.66 (m, 4H, ortho-PhH). 13C NMR
(75 MHz, MeOD) d 20.4 (Me3CeSi), 27.8 (CH3 ꢂ 3), 30.3
(CH2CH2CH2), 31.3 (CH2CH2CH2), 40.8 (CH2NH2), 51.6
(CH2uracil), 65.0 (CH2O), 102.6 (C]C), 129.2 (meta-PhC ),
131.2 ( para-PhC ), 135.4 (PhCeSi), 137.0 (ortho-PhC ),
148.3 (C]C), 153.1 (NCONH), 156.7 (CCONH), 169.5
(NHCOCH2). LRMS (ESꢀ): m/z ¼ 478 [M ꢀ Hþ]. HRMS
(ESþ): found: 480.2306 [M þ Hþ] C26H34O4N3Siþ1 requires:
480.2313 [MH]. Analysis: found: C, 64.43; H, 6.92; N,
8.69%; calc. for C26H33O4N3Si$0.2 mol H2O: C, 64.62; H,
6.97; N, 8.70%.
6.1.3.2. Synthesis of 3-tritylamino propylamine (4b). Yield:
20%. Rf: 0.4 in CH2Cl2/MeOH/NH3, 92:6.4:1.6. 1H NMR
(300 MHz, MeOD) d 1.63 (qn, J ¼ 7.05, 2H, CH2CH2CH2),
2.15 (t, J ¼ 6.96, 2H, CH2NHTrt), 2.67 (t, J ¼ 7.27, 2H,
CH2NH2), 7.15 (m, 3H, para-PhH), 7.24 (m, 6H, meta-
PhH), 7.42 (m, 66ortho6H, ortho-PhH). 13C NMR (75 MHz,
MeOD)
d 35.1 (CH2CH2CH2), 41.4 (CH2NH2), 43.3
(CH2NHTrt), 72.6 (Trt quat. C), 127.7 ( para-PhC ), 129.1 (or-
tho-PhC ), 130.3 (meta-PhC ), 147.9 (Ph quat. C). LRMS
(ESþ): m/z ¼ 317 [M þ Hþ]. HRMS (ESþ): found: 317.2015
[MHþ] C22H25Nþ2 requires 317.2012.
6.1.3.3. Synthesis of 4-(triphenylamino) butylamine (4c).
6.1.4.3. Synthesis of 1-[N-(2-triphenylmethylaminoethyl)-acet-
1
Yield: 31%. Rf: 0.3 in CH2Cl2/MeOH/NH3, 90:7:3. H NMR
amide] uracil (5a). Yield: 33%. Melting point: 219 ꢁC. Rf: 0.4
1
in CH2Cl2/MeOH, 95:5. H NMR (300 MHz, MeOD) d 2.28
(300 MHz, MeOD) d 1.51 (m, 4H, CH2CH2CH2CH2), 2.15
(d, J ¼ 6.76, 2H, CH2NHTrt), 2.60 (d, J ¼ 6.79, 2H,
CH2NH2), 7.17 (m, 3H, para-PhH), 7.26 (m, 6H, meta-
PhH), 7.47 (m, 6H, ortho-PhH). 13C NMR (75 MHz, MeOD)
d 29.3 (CH2), 31.7 (CH2), 42.8 (CH2NH2), 45.2 (CH2NHTrt),
72.6 (Trt quat. C), 127.7 ( para-PhC ), 129.1 (ortho-PhC ),
130.3 (meta-PhC ), 147.9 (Ph quat. C). LRMS (ESþ): m/
z ¼ 331 [M þ Hþ]. HRMS (ESþ): found: 331.2005
[M þ H]þ C23H27Nþ2 requires 331.2169.
(t, J ¼ 6.30, 2H, CH2NHTrt), 3.37 (t, J ¼ 6.33, 2H, CH2NHCO),
4.43 (s, 2H, CH2CO), 5.66 (d, J ¼ 7.86, 1H, ]CH ), 7.18 (m, 1H,
]CH ), 7.18 (m, 3H, para-PhH), 7.29 (m, 6H, meta-PhH), 7.45,
(m, 6H, ortho-PhH). 13C NMR (75 MHz, MeOD) d 41.4
(CH2NHTrt), 45.5 (CH2NHCO), 50.3 (CH2uracil), 70.5 (Trt
quat. C), 100.8 (C]C), 126.4 (ortho-PhC ), 128.1 ( para-
PhC ), 128.7 (meta-PhC ), 145.4 (C]C ), 146.4 (Ph quat. C),
164.3 (CCONH), 167.2 (NHCOCH2). LRMS (ESꢀ): m/
z ¼ 453 [M ꢀ Hþ]. HRMS (ESꢀ): found: 453.1940 [M þ Hþ]
C27H27N4O3 requires 453.1927.
6.1.4. General procedure for EDC mediated coupling to
form amide bonds
1-Carboxyuracil 1 (1.3 eq.) and EDC (1.4 eq.) dissolved in
DMF (2 mL/mmol) was added to the amine (1 eq.) under N2
or Ar atmosphere. The reaction was left at RT, usually over-
night. The solvent was evaporated off and the crude purified
by column chromatography.
6.1.4.4. Synthesis of 1-[N-(3-triphenylmethylaminopropyl)-
acetamide] uracil (5b). Yield: 42%. Melting point: 189 ꢁC.
Rf: 0.6 in CH2Cl2/MeOH, 90:10. 1H NMR (300 MHz,
CDCl3) d 1.62 (m, 2H, CH2CH2CH2), 2.13 (m, 2H,
CH2NHTrt), 3.31 (m, 2H, CH2NHCO), 4.18 (s, 2H,
CH2CO), 5.61 (d, J ¼ 7.76, 1H, ]CH ), 7.19 (m, 1H,
]CH), 7.14 (m, 3H, para-PhH), 7.22 (m, 6H, meta-PhH),
7.40, (m, 6H, ortho-PhH). 13C NMR (75 MHz, CDCl3)
d 30.6 (CH2CH2CH2), 38.8 (CH2NHTrt), 41.6 (CH2NHCO),
51.1 (CH2uracil), 71.4 (Trt quat. C), 102.9 (C]C), 126.8
( para-PhC ), 128.3 (ortho-PhC ), 129.1 (meta-PhC ), 145.7
(C]C ), 146.4 (Ph quat. C), 151.7 (HNCONH), 164.5
(CCONH), 166.6 (HNCOCH2). LRMS (ESþ): m/z ¼ 469
[M þ Hþ]. HRMS (ESþ): found: 469.2239 [M þ Hþ]
6.1.4.1. Synthesis of N-[3-(tert-butyl diphenyl silanyloxy)-pro-
pyl]-2-uracil acetamide (3a). Yield: 61%. Melting point:
186 ꢁC. Rf: 0.5 in CH2Cl2/MeOH, 90:10. 1H NMR (300 MHz,
MeOD) d 1.05 (s, 9H, CH3 ꢂ 3), 1.79 (qn, J ¼ 6.63, 2H,
CH2CH2CH2), 3.37 (m, 2H, CH2NH2), 3.75 (t, J ¼ 6.11, 2H,
CH2O), 4.35 (s, 2H, COCH2uracil), 5.67 (d, J ¼ 7.9, 1H,
]CH ), 7.40 (m, 6H, meta,para-PhH ), 7.42 (d, J ¼ 7.44, 1H,
]CH ), 7.68 (m, 4H, ortho-PhH ). 13C NMR (75 MHz,

O. McCarthy et al. / European Journal of Medicinal Chemistry 44 (2009) 678e688
685
C28H29N4Oþ3 requires 469.2240. Analysis: found: C, 69.16; H,
5.91; N, 11.43%; calc. for C28H28N4O3$1.0H2O: C, 69.12; H,
6.21; N, 11.51%.
138.3 (quat. PheC), 158.9 (NHCOO). LRMS (ESþ): m/
z ¼ 372.2 [M þ H]þ.
0
6.1.6.2. Synthesis of N1N1 -(dicarbobenzoxy) di (n-propyl) tria-
6.1.4.5. Synthesis of 1-[N-(3-triphenylmethylaminobutyl)-acet-
mine (7b). Yield: 38%. Melting point: 208 ꢁC. Rf: 0.4in
1
CH2Cl2/MeOH, 75:25. H NMR (500 MHz, MeOD) d 1.85
amide] uracil (5c). Yield: 26%. Melting point: 180 ꢁC. Rf:
1
0.3 in CH2Cl2/MeOH, 90:10. H NMR: (300 MHz, CDCl3)
(qn, J ¼ 6.9, 4H, 2 ꢂ CH2CH2CH2), 2.94 (t, J ¼ 7.5, 4H,
2 ꢂ CH2NH), 3.25 (t, J ¼ 6.5, 4H, 2 ꢂ CH2NHCbZ), 5.10 (s,
4H, 2 ꢂ CH2CbZ), 7.36 (m, 10H, PhCH). 13C NMR
(125 MHz, MeOD) d 26.4 (CH2CH2CH2), 38.8 (CH2NH),
46.2 (CH2NHCbZ), 65.9 (CH2O), 126.6e128.8 (PhCH),
137.0 (quat. PheC), 157.5 (NHCOO). LRMS (ESþ): m/
z ¼ 399.4 [M þ H]þ.
d 1.57 (m, 4H, CH2CH2CH2CH2), 2.19 (m, 2H, CH2NHTrt),
3.27 (m, 2H, CH2NHCO), 4.29 (s, 2H, CH2CO), 5.75 (d,
J ¼ 7.90, 1H, ]CH ), 7.25 (m, 1H, ]CH ), 7.23 (m, 3H,
para-PhH ), 7.32 (m, 6H, meta-PhH ), 7.50 (m, 6H, ortho-
PhH ). 13C NMR: (75 MHz, CDCl3)
d
27.6, 28.6
(CH2CH2CH2CH2), 40.4 (CH2NHTrt), 43.6 (CH2NHCO),
51.3 (CH2CO), 71.3 (Trt quat. C), 102.9 (C]C), 126.7
( para-PhC ), 128.2 (ortho-PhC ), 129.0 (meta-PhC ), 145.6
(C]C ), 146.5 (quat. PheC ), 151.5 (HNCONH), 164.1
(CCONH), 166.4 (HNCOCH2). LRMS (ESþ): m/z ¼ 483
[M þ Hþ]. HRMS (ESþ): found: 483.2396 [M þ Hþ]
C29H31N4Oþ3 requires 483.2391. Analysis: found: C, 71.35;
H, 6.23; N, 11.37%; calc. for C29H30N4O3$0.3H2O: C,
71.38; H, 6.32; N, 11.48%.
6.1.7. Synthesis of 1-carboxymethyluracil succinimidyl
ester (8) [12]
1-Carboxymethyl uracil 1 (2.95 g, 17.35 mmol) and N-hy-
droxysuccinimide (2.21 g, 19.09 mmol) were dissolved in
DMF (20 mL) and cooled to 0 ꢁC in an ice bath. A solution
of DCC (3.94 g, 19.09 mmol) in DMF (5 mL) was added to
the mixture under N2 atmosphere. The solution was left stir-
ring at 0 ꢁC for 3 h and then at RT for a further 24 h. The
pure product was precipitated from MeOH to yield a white
solid (4.70 g). The product could not be detected by ES MS
as it was too unstable to ionisation.
6.1.5. Synthesis of benzyl 1H-imidazole-1-
carboxylate (6) [11]
Benzyl chloroformate (19.92 g, 117.23 mmol) was cooled
to 0 ꢁC in an ice bath. Imidazole (15.96 g, 234.47 mmol) in
DCM (70 mL) was added slowly. The reaction was left at
RT for 15 min. The mixture was washed with citric acid
(3 ꢂ 150 mL) and the organic layer was dried to give the
pure product as a colourless oil (20.60 g). The product could
not be detected by ES MS as it is more than likely too unstable
to the ionisation conditions.
Yield: quantitative. Melting point: 218e222 ꢁC. Rf: 0.2 in
1
CH2Cl2/MeOH/NH3, 80:18:2. H NMR (500 MHz, DMSO)
d 2.83 (s, 4H, CH2succ), 5.02 (s, 2H, CH2uracil), 5.85 (d,
J ¼ 7.9, 1H, ]CH ), 7.74 (d, J ¼ 7.9, 1H, ]CH ). 13C NMR
(125 MHz, DMSO) d 25.8 (2 ꢂ CH2succ), 46.9 (CH2uracil),
102.1 (]C ), 145.6 (]C ), 151.0 (NCONH), 164.0 (CCONH),
165.3 (CH2CON), 170.1 (2 ꢂ COsucc).
Yield: 87%. Melting point: 41 ꢁC. Rf: 0.7 in CH2Cl2/MeOH
1
90:10. H NMR (500 MHz, CDCl3) d 5.39 (s, 2H, CH2), 7.02
110
6.1.8. Synthesis of 1-[N11N -(dicarbobenzoxy) diamino
(m, 1H, ]CH ), 7.34e7.43 (m, 5H, PhCH ), 7.49 (m, 1H,
]CH ), 8.20 (m, 1H, CH ). 13C NMR (125 MHz, CDCl3)
d 70.3 (CH2), 117.6 (NCH]), 128.8e129.6 (PhC ), 131.1
(NCH]), 134.4 (quat. PheC), 137.6 (NCH]), 149.1
(OCON).
diethylacetamide] uracil (9a)
Compound 7a (1.53 g, 4.12 mmol) was dissolved in DMF
(5 mL). 1-Carboxymethyluracil (700 mg, 4.12 mmol) in
DMF (5 mL) was added under N2 atmosphere, followed by
DhbtOH (770 mg, 4.74 mmol) in DMF (5 mL). The solution
was cooled to 0 ꢁC and DCC (1.02 g, 4.95 mmol) in DMF
(5 mL) was added under N2 atmosphere. The mixture was
left stirring at 0 ꢁC for 1 h and then at RT for 48 h after which
time a precipitate had formed. The solvent was evaporated off
under reduced pressure and DCU was precipitated out using
EtOAc. On addition of diethyl ether to the EtOAc solution,
a further gel precipitate formed. This was collected by decan-
tation. On evaporation of the remaining solvent, a foamy white
solid formed and identified as the pure product (783 mg).
Yield: 36%. Rf: 0.4 in CH2Cl2/MeOH, 90:10. 1H NMR
(500 MHz, MeOD) d 3.25e3.30 (m, 4H, 2 ꢂ CH2NHCbZ),
3.40e3.45 (m, 4H, 2 ꢂ CH2NHuracil), 4.57 (s, 2H, CH2ura-
cil), 5.10 (m, 4H, 2 ꢂ CH2CbZ), 5.65 (d, J ¼ 7.9, 1H,
]CH ), 7.25e7.39 (m, 11H, PhCH þ ]CH). 13C NMR
(125 MHz, MeOD) d 35.0, 39.4, 40.0 (CH2NH), 49.9
(CH2uracil), 67.6 (CH2CbZ), 102.2 (]C), 129.0e130.0
(PhCH), 147.9 (]C), 154.7, 159.0 (quat. CO). LRMS
6.1.6. General procedure for carbobenzoxy protection
of terminal NH groups of triamines
The relevant triamine (1 eq.) and DMAP (0.2 eq.) were dis-
solved in DCM (0.7 mL/mmol). Compound 6 (2 eq.) in DCM
(1 mL/mol) was added under N2 atmosphere. The reaction was
left stirring overnight at 50 ꢁC. The solvent was evaporated off
under reduced pressure and the product purified by flash
chromatography.
0
6.1.6.1. Synthesis of N1,N1 -(dicarbobenzoxy) diethylene tria-
mine (7a). Yield: 35%. Melting point: 53e55 ꢁC. Rf: 0.9 in
CH2Cl2/MeOH/NH3, 80:18:2. 1H NMR (500 MHz, MeOD)
d 2.64 (t, J ¼ 6.2, 4H, 2 ꢂ CH2NH), 3.19 (t, J ¼ 6.2, 4H,
2 ꢂ CH2NHCbZ), 5.01 (s, 4H, 2 ꢂ CH2CbZ), 7.28 (m, 10H,
PhCH ). 13C NMR (125 MHz, MeOD) d 41.1, (CH2NH),
49.5 (CH2NHCbZ), 67.5 (CH2CbZ), 128.5e129.5 (PhCH),

686
O. McCarthy et al. / European Journal of Medicinal Chemistry 44 (2009) 678e688
(ESþ): m/z ¼ 524 [M þ H]þ. HRMS (ESþ): found: 546.1953
[M þ Na]þ C26H29N5O7Naþ requires 546.1965.
(NCOCH2). LRMS (ESþ): m/z ¼ 284 [M þ H]þ. HRMS
(ESþ): found: 284.1715 [M þ H]þ C12H22N5Oþ3 requires
284.1717.
0
6.1.9. Synthesis of 1-[N12N12 -(dicarbobenzoxy) diaminodi
(n-propyl) acetamide] uracil (9b)
6.1.11. Synthesis of 1-[N-(triphenylmethylaminoethyl)-N-
(aminoethyl) acetamide] uracil (11a)
Compound 7b (1.67 g, 6.26 mmol) and DMAP (10% w/w)
were dissolved in DMF (10 mL). Compound 8 (2.50 g,
6.26 mmol) was added under N2 atmosphere. The reaction
was heated to 60 ꢁC and left stirring for 4 d. The solvent
was removed in vacuo. White crystals were recrystallised
from EtOAc and were washed with H2O (10 mL) to yield
the pure product (2.37 g).
A suspension of 10a (100 mg, 0.39 mmol) in DCM
(30 mL) was placed under N2 atmosphere and Et3N (80 mg,
0.78 mmol) in DCM (2 mL) was added followed by TrtCl
(110 mg, 0.39 mmol) in DCM (10 mL). The solution which
became cloudy was left stirring at RT for 2 h. The solvent
was removed in vacuo and both mono- and ditritylated prod-
ucts were observed by mass spectrometry. The crude was
washed with diethyl ether and water and purified by flash
Yield: 69%. Melting point: 141e144 ꢁC. Rf: 0.4 in CH2Cl2/
1
MeOH, 90:10. H NMR (500 MHz, MeOD) d 1.73, 1.88 (qn,
J ¼ 6.7, J ¼ 6.9, 4H, 2 ꢂ CH2CH2CH2), 3.11, 3.20 (2 ꢂ t,
J ¼ 6.49, 4H, CH2NH), 3.38 (m, 4H, 2 ꢂ CH2NHCbZ), 4.58
(s, 2H, CH2uracil), 5.08 (m, 4H, 2 ꢂ CH2CbZ), 5.67 (d,
J ¼ 7.8, 1H, ]CH ), 7.28e7.35 (m, 10H, PhCH ), 7.43 (d,
J ¼ 7.8, ]CH ). 13C NMR (125 MHz, MeOD) d 27.2, 28.2
(2 ꢂ CH2CH2CH2), 37.6 (CH2NHCbZ), 43.3, 44.3 (CH2NHur-
acil), 48.5 (CH2uracil), 65.9 (CH2CbZ), 100.6 (]C), 127.3e
128.0 (PhCH), 137.0 (quat. PheC), 146.6 (]C), 151.4
(NHCONH), 157.4 (NHCOO), 165.4 (HCCONH), 167.2
(H2CCON). LRMS (ESþ): m/z ¼ 552.4 [M þ H]þ.
chromatography
80:18.6:0.4). The product was collected as a white solid
(11 mg).
(CH2Cl2/MeOH/NH3,
100:0:0 /
Yield: 6%. Rf: 0.5 in CH2Cl2/MeOH/NH3, 85:14:1. 1H
NMR (500 MHz, MeOD) d 2.39 (m, 2H, CH2NHTrt), 2.88
(m, 4H, 2 ꢂ CH2NHCH2), 3.42 (m, 2H, CH2NHuracil), 4.45
(s, 2H, CH2uracil), 5.67 (d, J ¼ 7.87, 1H, ]CH ), 7.18e7.50
(m, 16H, PhCH þ ]CH ). 13C NMR (175 MHz, MeOD)
d
30.7 (CH2NHTrt), 39.1, 43.2 (CH2NHCH2), 51.4
(CH2NHuracil), 54.8 (CH2uracil), 72.1 (2 ꢂ quat. Trt C),
102.4 (]C), 127.5e129.9 (PheCH), 147.8 (]C6), 147.2
(quat. Ph C), 147.2 (NCONH), 170.2 (NHCOCH2). LRMS
(ESþ): m/z ¼ 498 [M þ H]þ. HRMS (ESþ): found: 498.2498
[M þ H]þ C29H32N5Oþ3 requires 498.2500.
6.1.10. General procedure for the removal of the CbZ
protecting group by hydrogenation
5% Pd/C (0.1 eq. w/w) was pre-activated with hydrogen
in a round-bottomed flask using a hydrogen balloon. The
relevant protected amine dissolved in either MeOH or
a 1:1 mixture of MeOH:EtOH was added. The mixture
was flushed with hydrogen three times using a hydrogen bal-
loon and finally left stirring under hydrogen until the disap-
pearance of the starting material was observed by TLC. The
catalyst was removed by filtration after flushing the system
with N2.
6.1.12. Synthesis of 1-[N-(triphenylmethyl) diaminodi
(n-propyl) acetamide] uracil (11b)
Compound 10b (140 mg, 0.49 mmol) and Et3N (0.03 mg,
0.25 mmol) were dissolved in pyridine (50 mL). TrtCl
(138 mg, 0.49 mmol) in pyridine (10 mL) was added over
3 h. After 1 h the solution had begun to turn cloudy. After ad-
dition had ceased the mixture was heated to 70 ꢁC, left stirring
for 2 d and monitored periodically by TLC. The solvent was
evaporated off under reduced pressure. The remaining starting
material was removed by precipitation firstly from EtOH
(10 mL) and subsequently EtOAc (10 mL). The remaining fil-
trate was purified by flash chromatography (CH2Cl2/MeOH/
NH3, 100:0:0 / 97:2.4:0.6) to yield 11b (45 mg) as a yellow
crystalline solid.
6.1.10.1. Synthesis of 1-[diaminodiethylacetamide] uracil
(10a). Yield: 41%. Rf: 0.2 in CH2Cl2/MeOH/NH3, 80:16:4.
1H NMR (500 MHz, MeOD)
d 2.70e2.97 (m, 4H,
CH2NH2), 3.30e3.59 (m, 4H, CH2Nuracil), 4.46, 4.75
(2 ꢂ s, 2H, CH2uracil), 5.70 (m, 1H, ]CH), 7.53 (m, 1H,
]CH). 13C NMR (175 MHz, MeOD)
d
39.8e41.2
(CH2NH2), 49.1e51.3 (CH2Nuracil þ CH2uracil), 102.3
(]C), 147.9 (]C). LRMS (ESþ): m/z ¼ 256 [M þ H]þ.
HRMS (ESþ): found: 256.1400 [M þ H]þ C10H18N5Oþ3 re-
quires 256.1404.
Yield: 17%. Melting point: 103e105 ꢁC. Rf: 0.1 in CH2Cl2/
1
MeOH/NH3, 85:14:1. H NMR (500 MHz, MeOD) d 1.75e
1.80 (m, 4H, 2 ꢂ CH2CH2CH2), 2.23e2.27 (m, 2H,
CH2NHTrt), 2.75e2.81 (m, 4H, 2 ꢂ CH2NH), 3.37e3.51 (m,
2H, CH2NHuracil), 4.41 (s, 2H, CH2uracil), 5.67 (d, J ¼ 7.9,
1H, ]CH), 7.19e7.52 (m, 16H, PheCH þ ]CH). 13C
NMR (175 MHz, MeOD) d 29.2, 30.0 (2 ꢂ CH2CH2CH2),
38.0 (CH2NHTrt), 43.1 (CH2NHCH2), 58.6 (CH2NHuracil),
51.5 (CH2uracil), 72.3 (quat. Trt-C), 102.3 (]C), 127.3e
129.9 (PheCH), 147.9 (]C), 147.4 (quat. PheC), 169.8
(NHCOCH2). LRMS (ESþ): m/z ¼ 526 [M þ H]þ. HRMS
(ESþ): found: 548.2639 [M þ Na]þ C31H35N5O3Naþ requires
548.2632.
6.1.10.2. Synthesis of 1-[diaminodi (n-propyl) acetamide] ura-
cil (10b). Yield: 35%. Rf: 0.2 in CH2Cl2/MeOH/NH3, 80:16:4.
1H NMR (500 MHz, MeOD)
d 1.78e1.94 (m, 4H,
CH2CH2CH2), 2.70e2.83 (m, 4H, CH2NH2), 3.40e3.51 (m,
4H, CH2Nuracil), 4.34, 4.72 (2 ꢂ s, 2H, CH2uracil), 5.69 (m,
1H, ]CH), 7.51 (m, 1H, ]CH). 13C NMR (175 MHz,
MeOD) d 25.1e26.0 (CH2CH2CH2), 38.1e38.8 (CH2NH2),
45.1e45.7 (CH2Nuracil), 48.5 (CH2uracil), 101.2 (]C),
147.6 (]C), 150.4 (NCONH), 165.4 (NHCOCH), 167.2

O. McCarthy et al. / European Journal of Medicinal Chemistry 44 (2009) 678e688
687
6.1.13. Synthesis of N-(tert-butyl diphenyl silyloxy)
ethyl ethylenediamine (12)
1.08 (2 ꢂ s, 9H, CH3t-Bu), 2.15, 2.22 (2 ꢂ m, 2H, CH2NHTrt),
3.41, 3.47 (2 ꢂ m, 4H, CH2NHuracil), 3.71, 3.85 (2 ꢂ m, 2H,
CH2O), 4.58, 4.75 (2 ꢂ s, 2H, CH2uracil), 5.56, 5.66 (2 ꢂ d,
J ¼ 7.8, ]CH), 7.20e7.69 (m, 26H, PhCH þ ]CH). 13C
NMR (75 MHz, MeOD) d 19.8, 20.3 (t-Bu quat. C), 26.5,
27.0 (t-Bu CH3), 43.2, 43.6, (CH2NHTrt), 47.3, 48.2
(2 ꢂ CH2Nuracil), 50.5, 51.6 (CH2uracil), 63.7, 64.3
(CH2O), 72.3 (Trt quat. C), 127.4e135.0 (PhCH), 147.5
(TrtPh quat. C), 154.2 (NCONH), 166.5 (NHCOC]), 168.8
(NCOCH2). LRMS (ESþ): m/z ¼ 738 [M þ H]þ.
The amino alcohol, N-(2-hydroxyethyl) ethylenediamine
(2.13 mL, 21.07 mmol) was dissolved in pyridine (20 mL),
and tert-butyl diphenyl silyl chloride (5.50 mL, 21.15 mmol)
in pyridine (30 mL) was added. The reaction was left stirring
at RT overnight. The solvent was removed in vacuo and the
crude product was purified by column chromatography
(CH2Cl2/MeOH/NH3, 100:0:0 / 90:8:2) to yield the pure
product as a yellow oil (2.27 g). Yield: 32%. Rf: 0.7 in
CH2Cl2/MeOH/NH3, 80:18:2. 1H NMR (250 MHz, MeOD)
d 1.08 (s, 9H, CH3t-Bu), 2.76, 2.80, 2.84 (3 ꢂ t, J ¼ 6.2,
J ¼ 5.7, J ¼ 6.00, 3 ꢂ CH2NH), 3.81 (t, J ¼ 5.6, CH2O),
7.42, 7.71 (m, 10H, PhCH). 13C NMR (125 MHz, MeOD)
d 20.1 (t-Bu CH3), 27.4 (t-Bu quat. C), 41.1 (CH2NH2),
50.5, 52.0 (2 ꢂ CH2NH), 64.2 (CH2O), 128.9, 131.1, 136.7
(PheCH), 134.6 (quat. PheC). LRMS (ESþ): m/z ¼ 343.4
[M þ H]þ.
6.1.16. Synthesis of 1-[N-hydroxyethyl-N-(triphenylmethyl
amino) ethyl-acetamide] uracil (15)
Compound 14 (365 mg, 0.495 mmol) was dissolved in THF
(5 mL) and TBAF in THF (1.0 mL, 1.0 mmol). After 30 min,
no starting material could be seen by TLC. The solvent was
removed in vacuo and purified by flash chromatography
(CH2Cl2/MeOH, 100:0 / 95:5) and the pure product was iso-
lated as a white solid (200 mg). NMR again showed peak split-
ting. It was proven, however, by variable temperature NMR
that this was due to restricted rotation around the nitrogen
bond as J C(7)H2 reduced from 0.4 to 0.1 ppm on heating to
40 ꢁC. Yield: 79%. Rf: 0.25 in CH2Cl2/MeOH, 95:5. 1H
NMR (500 MHz, MeOD) d 2.38, 2.41 (2 ꢂ t, J ¼ 6.0, 2H,
CH2NHTrt), 3.45, 3.55 (2 ꢂ m, 4H, CH2Nuracil), 3.63, 3.73
(2 ꢂ t, J ¼ 6.0, 2H, CH2O), 4.78, 4.79 (2 ꢂ s, 2H, CH2uracil),
5.57 (d, J ¼ 7.9, 1H, ]CH), 7.17e7.48 (m, 16H,
PhCH þ ]CH). 13C NMR (125 MHz, MeOD) d 43.1, 43.7
(CH2NHTrt), 48.7 (CH2uracil), 50.4, 51.2, (CH2NHuracil),
60.4 (CH2O), 72.6 (Trt quat. C), 102.1 (]C), 127.4, 128.9,
129.9 (PhCH), 147.4 (Ph quat. C), 147.9 (]C), 152.9
(NCONH), 166.9 (HNCOC]), 169.7 (NCOCH2). LRMS
(ESþ): m/z ¼ 499 [M þ H]þ. HRMS (ESþ): found: 499.2361
[M þ H]þ C29H31N4Oþ4 requires 499.2340.
6.1.14. Synthesis of N-(tert-butyl diphenyl silyloxy) ethyl-N-
(triphenylmethylamino) ethyl amine (13)
A solution of 12 (500 mg, 1.46 mmol) in DCM (50 mL)
was cooled to 0 ꢁC under N2 atmosphere. TrtCl (407 mg,
1.46 mmol) in DCM (80 mL) was added slowly. The reaction
was brought to RT and Et3N was added (0.20 mL, 1.46 mmol).
The reaction was left stirring at RT overnight over which time
the solution turned cloudy. The solvent was removed in vacuo.
The crude was redissolved in DCM (30 mL), washed with
H2O and dried with MgSO4 to yield a sticky yellow oil
(613 mg). This crude product was used for the subsequent syn-
thetic step without further purification. Yield: 72%. Rf: 0.6 in
1
CH2Cl2/MeOH/NH3, 95:4.5:0.5. H NMR (500 MHz, MeOD)
d 0.97 (s, 9H, CH3t-Bu), 2.20 (t, J ¼ 5.65, 2H, CH2NHTrt),
2.57, 2.61 (2 ꢂ t, J ¼ 5.6, J ¼ 6.0, 2H, 2 ꢂ CH2NH), 3.70 (t,
J ¼ 5.6, 2H, CH2O), 7.05e7.37 (m, 25H, PhCH). 13C NMR
(125 MHz, MeOD) d 20.1 (t-Bu quat. C), 27.4 (t-Bu CH3),
44.2 (CH2NHTrt), 50.7, 52.1 (2 ꢂ CH2NH), 63.9 (CH2O),
72.1 (Trt quat. C), 127.4, 130.5 (TrtPhCH), 128.9, 130.5,
136.7 (TBDPS PhCH), 134.6 (TBDPS quat. PheC), 147.5
(TrtPh quat. C). LRMS (ESþ): m/z ¼ 585 [M þ H]þ.
6.2. Biological assays
Methods used for the enzyme assays and parasite assays
have been described previously [7e9].
Acknowledgements
6.1.15. Synthesis of 1-[N-(tert-butyl diphenyl silyloxy) ethyl-
N-(triphenylmethylamino) ethyl-acetamide] uracil (14)
The authors would like to thank the European Union
(QLRT-2001-00305) and the Welsh School of Pharmacy for
funding and the EPSRC National Mass Spectrometry Service
Centre for accurate mass spectrometry.
Compound 13 (614 mg, 1.05 mmol) was dissolved in DMF
(3 mL) and cooled to 0 ꢁC under N2 atmosphere. A solution of
1 (180 mg, 1.05 mmol) and DhbtOH (200 mg, 1.21 mmol) in
DMF (3 mL) was added while stirring under N2 atmosphere
followed by a solution of DCC (260 mg, 1.25 mmol) in
DMF (6 mL). The mixture was left stirring at 0 ꢁC for 1 h
and then at RT overnight. The solvent was evaporated off un-
der reduced pressure. The crude was redissolved in DCM
(30 mL) and washed with NaHCO3 (3 ꢂ 30 mL), NaCl
(2 ꢂ 30 mL) and dried with MgSO4. The solvent was removed
in vacuo and the pure product was precipitated from MeOH as
a white solid (365 mg). NMR showed splitting of peaks due to
restricted rotation about the amide bond. Yield: 52%. Rf: 0.8 in
References
[1] http://www.who.int/tdr.
[2] P.O. Nyman, Curr. Protein Pept. Sci. 2 (2001) 277e285.
[3] A.W. Studebaker, W.P. Lafuse, R. Kloesel, M.V. Williams, Biochem. Bi-
ophys. Res. Commun. 327 (2005) 306e310.
[4] H.H. Elhajj, H. Zhang, B. Weiss, J. Bacteriol. 170 (1988) 1069e1075.
[5] M.H. Gadsden, E.M. McIntosh, J.C. Game, P.J. Wilson, R.H. Haynes,
EMBO J. 12 (1993) 4425e4431.
1
CH2Cl2/MeOH, 95:5. H NMR (500 MHz, MeOD) d 1.02,
[6] E.M. McIntosh, R.H. Haynes, Acta Biochim. Pol. 44 (1997) 159e172.

688
O. McCarthy et al. / European Journal of Medicinal Chemistry 44 (2009) 678e688
N.G. Johansson, D. Gonzalez-Pacanowska, I.H. Gilbert, J. Med. Chem.
[7] J.L. Whittingham, I. Leal, C. Nguyen, G. Kasinathan, E. Bell, A.F. Jones,
C. Berry, A. Benito, J.P. Turkenburg, E.J. Dodson, L.M.R. Perez,
A.J. Wilkinson, N.G. Johansson, R. Brun, I.H. Gilbert,
D.G. Pacanowska, K.S. Wilson, Structure 13 (2005) 329e338.
49 (2006) 4183e4195.
[10] J.R. Jacobsen, A.G. Cochran, J.C. Stephans, D.S. King, P.G. Schultz, J.
Am. Chem. Soc. 117 (1995) 5453e5461.
[8] C. Nguyen, G. Kasinathan, I. Leal-Cortijo, A. Musso-Buendia,
M. Kaiser, R. Brun, L.M. Ruiz-Perez, N.G. Johansson, D. Gonzalez Pa-
canowska, I.H. Gilbert, J. Med. Chem. 48 (2005) 5942e5954.
[11] S.K. Sharma, M.J. Miller, S.M. Payne, J. Med. Chem. 32 (1989) 357e
367.
[12] D. Wenninger, H. Seliger, Nucleosides Nucleotides 16 (1997) 977e
980.
[13] P.R. Gerber, K. Muller, J. Comput.-Aided Mol. Des. 9 (1995) 251e268.
[9] C. Nguyen, G.F. Ruda, A. Schipani, G. Kasinathan, I. Leal, A. Musso-
Buendia, M. Kaiser, R. Brun, L.M. Ruiz-Perez, B.L. Sahlberg,