Design, Synthesis, and Evaluation of 5′-Diphenyl Nucleoside Analogues as Inhibitors of the Plasmodium falciparum dUTPase

Article abstract of DOI:10.1002/cmdc.201100255

Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) is a potential drug target for malaria. We previously reported some 5′-tritylated deoxyuridine analogues (both cyclic and acyclic) as selective inhibitors of the Plasmodium falciparum dUTPase. Mod

Full text of DOI:10.1002/cmdc.201100255

MED
DOI: 10.1002/cmdc.201100255
Design, Synthesis, and Evaluation of 5’-Diphenyl
Nucleoside Analogues as Inhibitors of the Plasmodium
falciparum dUTPase
Shahienaz E. Hampton,[a] Beatriz BaragaÇa,[a] Alessandro Schipani,[a] Cristina Bosch-
Navarrete,[b] J. Alexander Musso-Buendꢀa,[b] Eliseo Recio,[b] Marcel Kaiser,[c, d]
Jean L. Whittingham,[e] Shirley M. Roberts,[e] Mikhail Shevtsov,[e] James A. Brannigan,[e]
Pia Kahnberg,[f] Reto Brun,[c, d] Keith S. Wilson,[e] Dolores Gonzꢁlez-Pacanowska,[b] Nils
Gunnar Johansson,[f] and Ian H. Gilbert*[a]
Deoxyuridine 5’-triphosphate nucleotidohydrolase (dUTPase) is
a potential drug target for malaria. We previously reported
some 5’-tritylated deoxyuridine analogues (both cyclic and acy-
clic) as selective inhibitors of the Plasmodium falciparum dUT-
Pase. Modelling studies indicated that it might be possible to
replace the trityl group with a diphenyl moiety, as two of the
phenyl groups are buried, whereas the third is exposed to sol-
vent. Herein we report the synthesis and evaluation of some
diphenyl analogues that have lower lipophilicity and molecular
weight than the trityl lead compound. Co-crystal structures
show that the diphenyl inhibitors bind in a similar manner to
the corresponding trityl derivatives, with the two phenyl moi-
eties occupying the predicted buried phenyl binding sites. The
diphenyl compounds prepared show similar or slightly lower
inhibition of PfdUTPase, and similar or weaker inhibition of par-
asite growth than the trityl compounds.
Introduction
Approximately half of the world’s population is at risk from
malaria, which is a health problem in more than 100 countries.
Approximately 90% of the one million deaths that occur each
year from this disease occur in sub-Saharan Africa, mainly
amongst children. The causative agent of malaria is the parasit-
ic protozoan of the genus Plasmodium. This is transmitted to
humans via the bite of the Anopheles mosquito, whereby Plas-
modium falciparum is responsible for the most severe form of
the disease.[1]
these compounds for the Plasmodium over the human enzyme
(Figure 1).
The reason for this selectivity lies in the different dUTPase
sequences between the two species. Val42 and Gly87 in the
human enzyme are respectively replaced by Phe46 and Ile117
in the Plasmodium dUTPase. This has been shown crystallo-
graphically in a previous publication reporting the co-crystalli-
[a] Dr. S. E. Hampton, Dr. B. BaragaÇa, Dr. A. Schipani, Prof. I. H. Gilbert
Division of Biological Chemistry and Drug Discovery
College of Life Science, University of Dundee
Sir James Black Centre, Dundee DD1 5EH (UK)
Fax: (+44)1382 386 373
Deoxyuridine 5’-triphosphate nucleotidohydrolase (dUTPase)
is a ubiquitous enzyme[2] that is found in prokaryotes and eu-
karyotes, in addition to a variety of viruses.[3,4] Its function is to
catalyse the hydrolysis of dUTP to dUMP,[2,3] fulfilling two main
roles within the cell. Firstly it provides dUMP, an essential pre-
cursor required for the production of dTMP, and secondly it
maintains a low dUTP/dTTP concentration, thus minimising
uracil incorporation into DNA.[3,4,5] The dUTPases have been
shown to be essential for cell viability in all organisms studied
to date, including E. coli and S. cerevisiae.[6] As a validated drug
target,[6,7,8] and owing to its essentiality in cell viability,
dUTPase is therefore a good choice with which to proceed in
the development of novel and selective inhibitors.
E-mail: i.h.gilbert@dundee.ac.uk
[b] C. Bosch-Navarrete, J. A. Musso-Buendꢀa, Dr. E. Recio,
Prof. D. Gonzꢁlez-Pacanowska
Instituto de Parasitologꢀa y Biomedicina
Consejo Superior de Investigaciones Cientꢀficas
Parque Tecnolꢂgico de Ciencias de la Salud
Avenida del Conocimiento, 18100 Armilla (Granada) (Spain)
[c] M. Kaiser, Prof. R. Brun
Swiss Tropical and Public Health Institute
Socinstrasse 57, 4002 Basel (Switzerland)
[d] M. Kaiser, Prof. R. Brun
University of Basel, Petersplatz 1, 4051 Basel (Switzerland)
We previously reported potent inhibitors of P. falciparum
dUTPase (PfdUTPase; Table 1), which show good selectivity
over the corresponding human enzyme.[4,9] These consist of
both cyclic and acyclic nucleoside analogues in which the po-
tency and selectivity arises from those that carry the trityl func-
tionality. Complete removal of the trityl group results in a sig-
nificant decrease in both biological activity and selectivity of
[e] Dr. J. L. Whittingham, S. M. Roberts, Dr. M. Shevtsov, Dr. J. A. Brannigan,
Prof. K. S. Wilson
Structural Biology Laboratory, Department of Chemistry
University of York, York YO10 5DD (UK)
[f] Dr. P. Kahnberg, Dr. N. G. Johansson
Medivir AB, PO Box 1086, 14122 Huddinge (Sweden)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100255.
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P. falciparum dUTPase Inhibitors
Table 1. Biological results for selected cyclic and acyclic PfdUTPase inhibitors.
Compd
Structure
Mr [Da]
clogP
Enzyme assays: Ki [mm]
In vitro assays: EC50 [mm]
P. fal.[a]
Human
SI[b]
P. fal.
L6 cells[c]
SI[d]
A
470
3.0
1.8
0.2
17.7
10
6.0
192
32
B
469
428
2.8
3.1
46.3
17.0
232
4.5
0.9
–
–
C
0.7
0.9
0.2
24
>1111
29
70
33
55
78
D
425
3.8
>1 mm
3.8
9
E
455
3.2
5.7
3.2
17
[a] P. falciparum K1 chloroquine- and pyrimethamine-resistant strain. [b] Enzyme selectivity index (SI) calculated as [Ki HsdUTPase/Ki PfdUTPase]. [c] Cytotox-
icity on rat L6 myoblasts; controls: for P. falciparum, chloroquine, EC50 =0.1 mm; for cytotoxicity, podophyllotoxin, EC50 =0.012 mm. The EC50 values are the
means of two independent assays; the individual values vary by less than a factor of 2. [d] Cell selectivity index (SI) calculated as [EC50 L6/EC50 P. falcipa-
rum]. Calculated logP values were determined with StarDropꢃ 2009 ver. 4.2.1 (http://www.optibrium.com).
sation of P. falciparum dUTPase with a cyclic trityl inhibitor,
compound F (Figure 2).[3] In the PfdUTPase structure there is a
hydrophobic pocket, which includes residues Phe46 and
Ile117, in which the trityl group binds. There is no correspond-
ing pocket in the human enzyme.
rings interact with Phe46. This suggests that compounds
which lack the exposed ring may still be able to interact in the
same manner and retain their activity. We have experimental
evidence to support the postulate that at least two phenyl
rings are a minimum requirement. We previously reported
some triphenylsilyl and tert-butyldiphenylsilyl (TBDPS) deriva-
tives (Figure 4).[4,9] By comparing appropriate pairs of com-
pounds, it is evident that the two-phenyl-ring derivatives
retain Ki values similar to that of the triphenyl analogues.
Herein we report a series of acyclic and cyclic nucleoside di-
phenyl derivatives to probe this further. These compounds
should have decreased lipophilicity relative to the triphenyl de-
rivatives, and so their water solubility should be increased. The
compounds also have lower molecular weights. It is anticipat-
ed that these will contribute to the creation of derivatives that
are more drug-like in accordance with the Lipinski parame-
ters.[10]
However, a major issue with the trityl analogues is their lipo-
philicity. To improve their solubility, water-solubilising groups
can be included in the molecule, but extra groups result in an
increase in molecular weight. For example, cyclic compounds
A and B (Table 1) have molecular weights near 500 Da. The
acyclic derivatives have lower molecular weights, but lipophi-
licity and water solubility remain an issue.
Our crystallographic and molecular modelling studies indi-
cate that only two of the three phenyl rings of the trityl group
have significant interactions with the enzyme; the third phenyl
ring has only limited interaction with the protein and is direct-
ed outwards to the solvent (Figure 3). The two buried phenyl
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I. H. Gilbert et al.
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Figure 3. Trityl PfdUTPase inhibitor interaction with Phe46 in the active site.
From our previous work, the optimum length for the acyclic
derivatives was shown to be a four-carbon chain, so we made
the appropriate diphenyl derivatives. We also prepared three-
carbon chain acyclic compounds for comparison (Figure 5).
With the cyclic analogues, in addition to preparation of the di-
phenyl derivatives, we prepared derivatives in which one of
the phenyl groups is replaced by a heteroaromatic ring, with
the aim of increasing solubility. Furthermore, we investigated
the effect of adding simple alkyl groups to the 5’-amino group.
Figure 1. Comparison of biological results for selected PfdUTPase inhibitors
with and without the trityl group.
Figure 2. Published trityl PfdUTPase inhibitor (F) and PfdUTPase with com-
pound F bound into the three active sites.
Figure 5. Structures of acyclic compounds prepared.
Figure 4. Comparison of biological activities for previously reported triphenylsilyl and TBDPS analogues.
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P. falciparum dUTPase Inhibitors
protection was carried out with sodium methoxide in metha-
Chemistry
nol to yield the desired compound, 1.
Acyclic derivatives
Four-C analogues
Following some initial work, we decided to develop our meth-
odology with the acyclic series and then apply this to the
cyclic series.
The preparation of the four-carbon derivatives was carried out
through an alternate synthesis, summarised in Scheme 2, with
the intention of producing compound 10 as a common inter-
mediate. Initially monobenzoyl-protected 1,4-butanediol was
used following a similar methodology to the three-carbon
route. However, following Mitsunobu coupling and benzoyl de-
protection, a low yield of alcohol intermediate 10 was ob-
tained. It was found that coupling of ethyl 4-bromobutanoate
to N3 benzoyl-protected uracil 7[9] could be achieved cleanly in
good yield after heating in N,N-dimethylformamide for 1 h. Re-
duction of the ester using a sodium borohydride/methanol
system reported by Da Costa et al.[11] led to simultaneous re-
duction and benzoyl deprotection to give 10.
Three-C analogue
The three-carbon diphenylamino derivative 1 (Scheme 1) was
synthesised by initially protecting the hydroxy functionality of
3-bromopropanol as a tert-butyldimethylsilyl ether. Attachment
of the diphenyl moiety was then carried out with the use of
benzhydrylamine and heating at 1208C in acetonitrile. Having
completed the diphenyl linker chain, attachment of the uracil
was carried out with Mitsunobu methodology. The use of poly-
mer-supported triphenylphosphine, a procedure previously
used in our laboratories,[9] ensured a cleaner reaction without
the triphenylphosphine oxide by-product, which is sometimes
difficult to remove. To ensure monoalkylation at the N1 posi-
tion of uracil, N3-protected uracil was used. Finally, benzoyl de-
This was then used to synthesise each acyclic derivative.
Holding compound 10 at reflux with diphenyl carbinol in the
presence of para-toluenesulfonic acid (PTSA) gave 2a. Fluori-
nated compound 2b, however, could not be prepared in the
same manner. As an alternate procedure, bis(4-fluorophenyl)-
Scheme 1. Synthetic route for the three-carbon derivative. Reagents and conditions: a) TBDMSCl, imidazole, DMF, 08C!RT, 87%; b) (C6H5)2CHNH2, Et3N, CH3CN,
1208C, 39%; c) TBAF, THF, RT, 84%; d) 1. PhCOCl, pyridine, CH3CN, RT, 2. K2CO3, dioxane, 59%; e) PS–PPh3, DIAD, THF, RT, 35%; f) NaOCH3, CH3OH, RT, 28%.
Scheme 2. Synthetic route for the four-carbon derivatives. Reagents and conditions: a) 7, Cs2CO3, DMF, 608C, 1 h, 88%; b) NaBH4, CH3OH, THF, reflux, 1 h, 39%;
c) (p-FC6H5)2CHOH, MsCl, pyridine, RT; d) (C6H5)2CHOH, PTSA, toluene, 4 ꢄ MS, reflux, 20%; e) 11, Et3N, CH3CN, 1208C, 12% (two steps from 4,4’-difluorobenzhy-
drol); f) MsCl, pyridine, RT, 49%; g) (C6H5)2CHNH2, Et3N, CH3CN, 1208C, 7%.
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methanol was initially mesylated (compound 11) and subse-
quently displaced by the acyclic alcohol, 10. Finally the four-
carbon amino diphenyl analogue 2c was synthesised in a simi-
lar manner by mesylating alcohol 10 followed by reaction with
benzhydrylamine.
Scheme 5. Synthetic route for 16d–16 f. Reagents and conditions: a) TiCl4,
RNH2 08C!RT, 3 h; b) NaBH3CN, RT.
with titanium(IV) chloride to yield the desired products. For
purposes of the initial in vitro screening, these were used as a
racemic mixture as shown in Scheme 3. Upon reaction with 5’-
mesyl-2’-deoxyuridine, the isopropyl (16e) and cyclopropyl
(16 f) derivatives failed to react; only the ethyl derivative 16d
reacted successfully to give 15h.
Cyclic derivatives
Following on from the methodology used for the acyclouridine
diphenyl derivatives, mesylation of the 2’-deoxyuridine fol-
lowed by displacement with the relevant diphenyl derivative
was chosen as the route to synthesise the cyclic diphenyl ana-
logues (Scheme 3). Displacement of the mesylate with the cor-
responding diphenyl methanamines was successful, although a
similar displacement with the diphenyl carbinols was not.
Results and Discussion
Compounds were evaluated for
biological activity by testing
against the recombinant PfdUT-
Pase and recombinant human
dUTPase in order to determine
inhibition and selectivity be-
tween the two enzymes. Com-
pounds were also tested in vitro
against the chloroquine-resistant
K1 strain of P. falciparum and ad-
ditionally against mammalian L6
cells as a measure of cytotoxicity
(Table 1, Table 2, and Table 3).
Scheme 3. Synthesis of cyclic derivatives. Reagents and conditions: a) MsCl, pyridine, RT, 52%; b) amine, Et3N,
CH3CN, 1208C.
Acyclic analogues
The diphenyl methanamines used to prepare compounds
15a–c and 15g were obtained commercially. The diaryl meth-
anamines for compounds 15d–f and 15h were prepared in
house. The diaryl methanamines 16a–c were prepared by
using the two-step procedure of Torregrosa et al.[12]
(Scheme 4), which consists of formation of the N-silylated
imine prepared from benzaldehyde and lithium hexamethyldi-
silazanide. The corresponding lithiated heterocycle was then
added to this, which upon workup generated the primary
amino compounds 16a–c. These were isolated and used as a
racemic mixture in the coupling reaction with the base.
For the three-carbon linked compound 1, loss of one of the
phenyl groups led to a significant (30-fold) loss in PfdUTPase
inhibition relative to the trityl derivative 21 (Ki =5.7 versus
0.2 mm). However, for the four-carbon linked molecules, the
effect on inhibition of PfdUTPase was less marked. Thus the
ether-linked compounds 2a and 22 had reasonably similar ac-
tivity (Ki =0.5 versus 1.6 mm, respectively), although the amino-
linked compounds 2c and 23 (Ki =5.7 versus 0.9 mm, respec-
tively) showed a sixfold decrease in activity. The activity against
parasites for the trityl and diphenyls are all similar, with EC50
values in the range 2.6–14 mm.
Diphenyl methanamines 16d–f were prepared as shown in
Scheme 5 from benzophenone.[13] Reductive aminations were
carried out with sodium cyanoborohydride in combination
Cyclic analogues
Removal of one phenyl ring from the trityl derivative
24 gave compound 15a, which retained the same
inhibition of PfdUTPase (Ki =0.2 mm), whereas the
presence of an extra methylene unit (in 15b) led to
a significant decrease (Ki =14 mm). Replacing one of
the phenyl rings with a heteroaromatic ring (com-
pounds 15d–f) resulted in decreased activity (~70-
fold) relative to the trityl derivative 24, with Ki values
in the range 1.7–3.8 mm. Finally, addition of an alkyl
group to the 5’-amino position (for 15g and 15h)
Scheme 4. Synthetic route for phenylmethanamines. Reagents and conditions:
a) (Me3Si)2NLi, THF, 08C; b) RLi, THF, 08C.
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P. falciparum dUTPase Inhibitors
Table 2. Activity data for the acyclic derivatives.
Compd
Structure
Enzyme assays: Ki [mm]
In vitro assays: EC50 [mm]
P. fal.[a]
Human
SI[b]
P. fal.
L6 cells[c]
SI[d]
1
5.7
10
49
2
5.5
269
104
49
2a
0.5
98
14
7
2b
3.6
46
13
6.8
87
13
2c
5.7
63
11
2.6
99
38
21[e]
0.2
1.4
7
4.4
107
24
22[e]
1.6
>1000
>625
4.9
42
9
23[e]
0.9
>1000
>1111
3.8
33
9
[a] P. falciparum K1 chloroquine- and pyrimethamine-resistant strain. [b] Enzyme selectivity index (SI) calculated as [Ki HsdUTPase/Ki PfdUTPase]. [c] Cytotox-
icity on rat L6 myoblasts; controls: for P. falciparum, chloroquine, EC50 =0.1 mm; for cytotoxicity, podophyllotoxin, EC50 =0.012 mm. The EC50 values are the
means of two independent assays; the individual values vary by less than a factor of 2. [d] Cell selectivity index (SI) calculated as [EC50 L6/EC50 P. falcipa-
rum]. [e] Compounds 21–23 have been published,[9] but are included here for comparison.
gave compounds with only slightly lower activity (Ki =0.5 and
0.9 mm) than compound 24. Selectivity of PfdUTPase over the
human enzyme was retained in compound 15a versus the
trityl derivative 24 (>500 versus 230). Introduction of the addi-
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Table 3. Biological results for cyclic derivatives.
Compd
Structure
Enzyme assays: Ki [mm]
In vitro assays: EC50 [mm]
L6 cells[c]
P. fal.[a]
Human
SI[b]
P. fal.
SI[d]
24[e]
0.2
46
230
4.5
–
–
15a
0.2
>100
>500
12
>229
>20
15b
14
33
2
>12
219
>18
15c
3.8
>100
>26
10
145
15
15d
15e
15 f
15g
1.7
2.3
3.0
0.5
>100
>100
>100
26
>58
>43
>33
52
>12
>13
>12.6
5.8
>219
>18
215
17
>226
18
>222
>38
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P. falciparum dUTPase Inhibitors
Table 3. (Continued)
Compd
Structure
Enzyme assays: Ki [mm]
In vitro assays: EC50 [mm]
P. fal.[a]
Human
SI[b]
P. fal.
L6 cells[c]
SI[d]
15h
0.9
33
37
9.1
117
13
[a] P. falciparum K1 chloroquine- and pyrimethamine-resistant strain. [b] Enzyme selectivity index (SI) calculated as [Ki HsdUTPase/Ki PfdUTPase]. [c] Cytotox-
icity on rat L6 myoblasts; controls: for P. falciparum, chloroquine, EC50 =0.1 mm; for cytotoxicity, podophyllotoxin, EC50 =0.012 mm. The EC50 values are the
means of two independent assays; the individual values vary by less than a factor of 2. [d] Cell selectivity index (SI) calculated as [EC50 L6/EC50 P. falcipa-
rum]. [e] Compound 24 has been published,[9] but is included here for comparison.
tional methylene group led to a significant decrease in selec-
tivity, with only twofold preference for PfdUTPase observed
over the human enzyme. In the cellular assay, all the com-
pounds showed similar or reduced activity compared to the
starting trityl derivative 24.
RMSD drops to ~0.25 ꢄ if the present four ligands in the same
space group are superposed, showing only a minor effect of
crystal packing. The N terminus is well ordered, from residue
Met1. There is a disordered loop with no visible density from
residue 58 to 81 (the latter number varies slightly over the in-
dividual subunits of the four complexes), corresponding to the
low complexity region in the sequence, well removed from the
active site. As often observed in dUTPase structures, there is
no electron density for the C terminus of each subunit: chains
A and C are ordered up to residue 165, chain B only to 155.
The ordering of residues 156–165 in chains A and C is a result
of crystal packing against a symmetry-related trimer in a similar
way, and these point away from the body of the trimer. There
are no such contacts in subunit B, so this is likely to represent
the conformation in solution, and our description of the struc-
tures focuses on this active site.
Water solubility
A comparison of water solubility between the original tritylat-
ed compound 24 and the methylated diphenyl compound
15g is shown in Figure 6. A decrease in the molecular weight
Compound 24 (Figure 7A): Compound 24 binds in an es-
sentially identical manner to that of the previously published
trityl compound F, Figure 2. The ligand is well ordered and in
the same orientation in all three active sites. The uracil and
sugar rings occupy the same positions, making the same hy-
drogen bonds to the protein. Two trityl rings stack against
Phe46, with one ring (R1) pointing down into a hydrophobic
pocket, a second (R2) stacking against the hydrophobic protein
surface, while the third (R3) points out towards the solvent
and is more exposed. The position of Phe46 is rotated some-
what from its position in the original compound F complex.
Tyr112 stacks against the hydrophobic side of the sugar. The
substitution of the linking O atom by an NH group has little
impact on the position of the three aromatic rings of the trityl
moiety. There is a water (W103) in the compound 24 complex,
hydrogen bonded to the NH group of the ligand, which is not
present in the complex of compound F.
Figure 6. Measured water solubility [mm] comparison of trityl and diphenyl
analogues 24 and 15g.
and lipophilicity of these compounds by removal of just one
phenyl ring is reflected in the significant improvement in aque-
ous solubility. The solubility of the trityl derivative 24, which is
<0.5 mm, is increased to >180 mm for the diphenyl 15g. This
is additionally reflected in the clogP value, which decreases
from 3.7 to 2.5.
Crystal structures of the ligand complexes
PfdUTPase was co-crystallised with each of the four ligands 24,
15a, 15 f, and 15g, and the structures of the complexes were
refined. All are in the same space group, P41, with similar cell
dimensions, each with a trimer in the asymmetric unit. The
overall fold is closely similar to that of the complex with the
trityl ligand, compound F (Figure 2).[3] Superposition of the
trimers of the four ligand complexes on that of compound F
gives an RMSD of 0.5–0.6 ꢄ on ~400 equivalent Ca atoms. The
Compound 15a (Figure 7B): This structure has the highest
resolution (1.65 ꢄ) of the PfdUTPase complexes to date, with
the ligand again well defined in all three active sites. The uracil
and the sugar rings are bound in a very similar manner to
those in the compound F and compound 24 complexes,
Figure 7. The diphenyl moiety is oriented so that the two
phenyl groups lie in the “buried” sites (R1 and R2). R1 super-
poses almost exactly on the compound F/compound 24 ring,
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I. H. Gilbert et al.
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there is once more good density in all three active sites, with
the two aromatic rings of the ligand occupying sites R1 and
R2. The ligand binds in a very similar position to that observed
for 15a, with rotation of Phe46 from its position in the com-
plex with compound F, and almost identical positions of the
uracil and sugar rings. The R2 aromatic ring is rotated slightly
from its position in 15a.
In summary, all four ligands bind in a very similar manner,
with the trityl or diphenyl and analogue moieties in the hydro-
phobic site identified in the original complex with compound
F. The trityl amino compound 24 binds in a very similar
manner to the original ligand F, showing that the substitution
of oxygen by nitrogen in the linkage to the trityl group is
easily accommodated. The three other complexes with 15a,
15 f, and 15g confirm that two aromatic rings are sufficient to
provide binding in this site. As expected, if one ring is deleted
from the trityl group, it is the exposed R3 position which is un-
filled in the resulting complex. Most of the protein conforma-
tion is unchanged, but the side chain of Phe46 can rotate to
accommodate the various ligands. Modified rings in the di-
phenyl complexes are bound at the half-exposed R2 position.
Conclusions
In an attempt to decrease lipophilicity and molecular weight
of the trityl derivatives, we decided to remove one of the
phenyl rings. A selection of both acyclic and cyclic derivatives
were synthesised, both for comparison and in order to serve as
a validation for the diphenyl-type analogues. The simple di-
phenyl derivatives 15a and 2a showed only slightly lower ac-
tivity against both dUTPase and parasite than the correspond-
ing trityl derivatives. The more complex heteroaromatic re-
placements generally showed lower activity against the
enzyme and the parasite. Alkylation of the 5’-amino group ap-
peared to be tolerated (15g and 15h). Furthermore, com-
pound 15g showed improved solubility, a key goal of the proj-
ect.
Figure 7. Views of the compounds bound to PfDUTPase in the chain B
active site. The compounds A) 24, B) 15a, C) 15 f, and D) 15g are coloured
by atom type, each superimposed on compound F with its carbon atoms
shown in coral. The electrostatic surface of the protein and the hydrogen
bonds from the compound to the protein are shown, together with key pro-
tein residues in contact with the compound. The protein residues around
the ligand site are shown, colour coded as for the compounds. Stereoviews
of these structures are given in the Supporting Information.
while the orientation of the R2 ring is slightly rotated, probably
reflecting the greater mobility of the diphenyl over the trityl
unit. As expected, the R3 site is empty in this complex. The po-
sition of Tyr112 is the same as that observed in the compound
F and compound 24 complexes. The plane of Phe46 has rotat-
ed even further in this complex relative to compound F and
compound 24, reflecting the small change in position and ori-
entation of R2. This structure confirms that a diphenyl group is
sufficient to bind as a substrate mimic, occupying the hydro-
phobic site present in the Plasmodium enzyme but absent in
other species.
Crystallographic data show that the compounds bound in a
very similar manner to the trityl derivatives. For the diphenyl
derivatives, the two phenyl rings were bound in the “buried”
sites occupied by phenyls from the trityl group. The position
occupied by the solvent-exposed phenyl in the trityl group is
not occupied in the diphenyl derivatives, as predicted. There-
fore, it may be feasible in certain circumstances to replace the
trityl group with a diphenyl group with relatively small loss in
activity against the enzyme and parasite. Further optimisation
is required in order to exploit these compounds as potential
anti-malarial agents.
Compound 15 f (Figure 7C): There is well-defined density
for all the methylimidazole diaryl derivative ligands in all three
active sites. The phenyl ring is buried in site R1, while the
methylimidazole ring is accommodated in R2. The exposed R3
site is empty. There is a hydrogen bond from the ligand O41
to a water molecule. As in the complex with 15a, Phe46 is ro-
tated by ~908 from its position in the compound F complex,
presumably reflecting the change in the nature of the ring at
the R2 position, and lies in roughly the same position as in the
complex with compound 15a.
Experimental Section
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 expression vectors, respec-
tively (kindly provided by P. O. Nyman, Lund University, Sweden).
For dUTPase purification, the same procedure was used for both
the human and Plasmodium enzymes. Cell pellets from a 2.8 L
Compound 15g (Figure 7D): For the 15g ligand—the di-
phenyl compound with the methylated 5’-N substituent—
1824
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ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1816 – 1831

P. falciparum dUTPase Inhibitors
IPTG-induced culture were resuspended in 70 mL buffer A (20 mm
MES, 50 mm NaCl, 1 mm DTT, pH 5.5) containing a protease inhibi-
tor cocktail. The cells were lysed by sonication, and the cell extract
was cleared by centrifugation at 14000 rpm (23700 g) for 30 min.
The supernatant was loaded onto a 40 mL phosphocellulose
(Whatman P-11) column at 48C and eluted with a gradient of NaCl
(50 mm!1m) in buffer A. Protein was further purified by gel filtra-
tion chromatography on a Superdex 200 HA 10/30 column at 48C.
Pooled fractions were concentrated by centrifugation at 48C and
desalted using a PD-10 column. The enzyme was stored in 10 mm
bicine and 5 mm MgCl2, pH 8 at ꢀ808C. Purified fractions con-
tained dUTPase of ꢁ96% purity.
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.[6] Protons, re-
leased through the hydrolysis of nucleotides, were neutralised by a
pH indicator in weak buffered medium with similar pKa and moni-
tored spectrophotometrically at the absorbance peak of the basic
form of the indicator. The molar ratio between indicator and buffer
concentration was 5:200, 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 mgmLꢀ1 BSA, and 100 mm KCl. Vmax
and KMapp were calculated by fitting the resulting data to the inte-
grated Michaelis–Menten equation. The apparent KM values were
plotted against inhibitor concentration, and Ki values (Table 1) were
obtained according to Equation (1).
er MicroTof mass spectrometer using an Agilent HPLC 1100 with a
diode array detector in series, or using an Agilent 6130 quadrupole
mass spectrometer in series with an Agilent 1200 binary pump,
using one of the following methods:
Method 1. Phenomenex Gemini 5.0ꢅ50 mm column, acidic gradi-
ent running 5!95% CH3CN/H2O + 0.1% formic acid (FA), run time
6.1 min;
Method 2. Waters Xbridge C18, 3.5 mm particle size, 2.1ꢅ50 mm
column, acidic gradient running 5!95% CH3CN/H2O + 0.1% FA,
run time 6.1 min;
Method 3. Waters Xbridge 5.0ꢅ50 mm column, basic gradient run-
ning 5!95% CH3CN/H2O + 0.1% NH3;
Method 4. Waters Xbridge C18, 3.5 mm particle size, 2.1ꢅ50 mm
column, basic gradient running 20!95% CH3CN/H2O + 0.1% NH3,
run time 5.1 min;
Method 5. Waters Xbridge C18, 3.5 mm particle size, 2.1ꢅ50 mm
column, acidic gradient running 20!95% + 0.1% FA in CH3CN,
run time 6.5 min.
(3-Bromopropyloxy)tert-butyldimethylsilane (4): 3-Bromopropan-
1-ol (1.31 mL, 15.0 mmol) was added to a cooled solution of
TBDMSCl (2.49 g, 16.5 mmol) and imidazole (1.23 g, 18.0 mmol) in
DMF (10 mL). The mixture was left to warn to room temperature
overnight. The mixture was then diluted with Et2O (20 mL) and ex-
tracted with NaHCO3 (2ꢅ20 mL) and 3m HCl (2ꢅ20 mL). The or-
ganic layer was dried (MgSO4) and concentrated in vacuo to yield a
colourless oil (3.3 g, 87%) that did not require further purification.
1
Rf =0.22 (hexane/EtOAc 90:10); H NMR (500 MHz, CDCl3): d=0.00
KMapp ¼ KM=Ki½Iꢂ þ KM
ð1Þ
(s, 6H, CH3), 0.83 (s, 9H, CH3), 1.96 (m, 2H, OCH2CH2), 3.45 (t, J=
6.4 Hz, 2H, CH2Br), 3.67 (t, J=5.8 Hz, 2H, OCH2); 13C NMR
(125 MHz, CDCl3): d=ꢀ6.5 (CH3), 17.2 (C), 24.8 (CH3), 29.5 (CH2),
34.4 (CH2), 59.2 (CH2).
Activity against the P. falciparum K1 strain and cytotoxicity assess-
ment against L6 (rat skeletal myoblast) cells was determined as
previously reported.[9]
N-Benzhydryl-3-(tert-butyldimethylsilyloxy)propan-1-amine (5):
Et3N (3.51 mL, 25.3 mmol) and compound 4 (1.28 g, 5.06 mmol) in
CH3CN (8 mL) were added to a solution of benzhydrylamine
(3.93 g, 4.55 mmol) in CH3CN (10 mL). The mixture was heated at
reflux until reaction completion. After cooling, the solvent was re-
moved to leave the crude as an oil, which was purified by flash
chromatography (EtOAc/hexane 0!4%) to give the product as a
Chemistry
Solvents and reagents were purchased from commercial suppliers
and used without further purification. Dry solvents were purchased
in sure-sealed bottles stored over molecular sieves. Reactions were
performed in a pre-dried apparatus under an atmosphere of argon
unless otherwise stated. Normal-phase TLC was carried out on pre-
coated silica plates (Kieselgel 60 F254, BDH) with visualisation by
either ninhydrin, PMA, or 254 nm UV light. Flash chromatography
was performed using Combiflash Companion or Combiflash Rf and
pre-packed columns (silica gel) purchased from Redisep (Presearch)
or Sylicycle (Anachem). HPLC was performed using a Gilson instru-
ment (321-Pump, 153-UV/Vis Detector) equipped with a Gilson
liquid handler for injection and fraction collection, and an XBridge
Prep C18 column (5 mm, ODB, 19ꢅ100 mm; Waters) with 0.1% NH3
in H2O (solvent A) and CH3CN (solvent B) as mobile phase. Melting
points (mp) were measured on a Gallenkamp melting point appa-
1
yellow oil (0.627 g, 39%); Rf =0.33 (hexane/EtOAc 90:10); H NMR
(500 MHz, CDCl3): d=0.00 (s, 6H, CH3), 0.83 (s, 9H, CH3), 1.70 (m,
2H, OCH2CH2), 2.64 (m, 2H, CH2NH), 3.66 (m, 2H, OCH2), 4.75 (s,
1H, NHCH), 7.15 (m, 2H, H-Ar), 7.22 (m, 4H, H-Ar), 7.35 (m, 4H, H-
Ar); 13C NMR (125 MHz, CDCl3): d=ꢀ5.2 (CH3), 18.4 (C), 26.1 (CH3),
33.1 (CH2), 45.8 (CH2), 32.1 (CH2), 67.8 (CH), 127.0–128.5 (CH), 144.4
(C); LRMS (ES+): m/z 167.0 [Ph2C]+, 356.2 [M+H]+; HRMS (ES+):
found 356.2416 [M+H]+ C22H34NOSi+ requires 356.2404.
3-(Benzhydrylamino)propan-1-ol (6): TBAF (600 mL, 1.94 mmol)
was added to a solution of 5 (0.625 g, 1.76 mmol) in THF (5 mL).
This was left to stir at room temperature until reaction completion.
Upon removal of the solvent, the crude was purified by flash chro-
matography (EtOAc/hexane 30!100%) to give the product as an
off-white solid (0.358 g, 84%); Rf =0.39 (hexane/EtOAc 40:60);
1H NMR (500 MHz, CDCl3): d=1.63 (m, 2H, NHCH2CH2), 2.72 (m,
2H, NHCH2), 3.69 (m, 2H, CH2OH), 4.69 (d, J=4.8 Hz, 1H, CHNH),
7.11–7.28 (m, 10H, H-Ar); 13C NMR (125 MHz, CDCl3): d=32.7 (CH2),
48.3 (CH2), 64.1 (CH2), 68.0 (CH), 127.7–128.7 (CH), 143.3 (C); LRMS
(ES+): m/z 242.3 [M+H]+.
ratus and are uncorrected. H NMR and 13C NMR spectra were re-
1
corded on a Bruker Avance DPX500 spectrometer or on a Bruker
Avance DPX300 using the applied solvent simultaneously as inter-
nal standard. Deuterated solvents were purchased from Goss.
Chemical shifts (d) are given in ppm together with the multiplicity,
relative frequency, coupling constants (J, in Hz), and assignment.
Low-resolution electrospray (ES) mass spectra were recorded on a
Bruker MicroTof mass spectrometer, run in positive or negative ion
mode. High-resolution mass spectra were performed on a Bruker
MicroTof mass spectrometer at University of Dundee. LC–MS analy-
sis and chromatographic separation were conducted with a Bruck-
3-N-Benzoyluracil (7): Benzoyl chloride (5.2 mL, 44.8 mmol) was
added to a suspension of uracil (2.24 g, 20 mmol) in CH3CN (20 mL)
ChemMedChem 2011, 6, 1816 – 1831
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1825

I. H. Gilbert et al.
MED
and pyridine (8 mL) under argon. After 5 min the suspension
turned clear green, which was left to stir overnight at room tem-
perature. After 24 h the solution, which contained a precipitate,
was concentrated under reduced pressure. The resultant residue
was partitioned between H2O (100 mL) and CH2Cl2 (100 mL), and
the organic layer was concentrated. The residue was taken into a
mixture of dioxane (40 mL) and K2CO3 (0.5m, 20 mL) and stirred for
30 min. The solution was lowered to pH 5 with glacial acetic acid
to leave a residue that was then dissolved in saturated NaHCO3
(100 mL). This was stirred at room temperature for 1 h, and the
precipitate was filtered off and washed with cold H2O. This was re-
crystallised from aqueous acetone (70% v/v) to yield an off-white
solid (2.54 g, 59%); Rf =0.11 (CHCl3/CH3OH 95:5); 1H NMR
(500 MHz, (CD3)2SO): d=5.75 (d, J=7.7 Hz, 1H, CHCO), 7.61 (t, J=
7.4 Hz, 2H, H-Ar), 7.68 (d, J=7.8 Hz, 1H, CHN), 7.79 (t, J=7.6 Hz,
1H, H-Ar), 7.97 (d, J=7.2 Hz, 2H, H-Ar), 11.63 (bs, 1H, NH); 13C NMR
(125 MHz, (CD3)2SO): d=100.5 (CH), 129.9 (CH), 130.7 (CH), 131.7
(C), 135.9 (CH), 143.8 (CH), 150.5 (C), 163.4 (C), 170.5 (C).
(10 mL). This was left to stir at room temperature overnight, or al-
ternatively was heated at 608C for 1 h. The mixture was then dilut-
ed with EtOAc (50 mL) and extracted with H2O (5ꢅ30 mL). The
aqueous layers were then combined and back extracted with
EtOAc (50 mL). The organic layers were then combined, dried
(MgSO4), and the solvent removed. Purification was carried out by
flash chromatography (EtOAc/hexane 0!100%) to give the title
compound as a viscous yellow oil (1.336 g, 87%). Purity by LCMS
(UV chromatogram, l 190–450 nm): >99%, tR =3.3 min (Method
1); Rf =0.63 (CHCl3/CH3OH 90:10); 1H NMR (500 MHz, CDCl3): d=
1.18 (t, J=7.7 Hz, 3H, CH3CH2O), 1.95 (quint, J=7.1 Hz, 2H,
CH2CH2N), 2.31 (t, J=7.0 Hz, 2H, COCH2), 3.73 (t, J=7.1 Hz, 2H,
CH2N), 4.07 (q, J=7.1 Hz, 2H, CH2O), 5.69 (d, J=8.0 Hz, 1H, CHCO),
7.29 (d, J=8.0 Hz, 1H, CHN), 7.45 (t, J=7.5 Hz, 2H, H-Ar), 7.61 (t,
J=7.5 Hz, 1H, H-Ar), 7.90 (d, J=8.4 Hz, 2H, H-Ar); 13C NMR
(125 MHz, CDCl3): d=14.1 (CH3), 23.9 (CH2), 30.7 (CH2), 48.2 (CH2),
60.6 (CH2), 101.8 (CH), 129.2 (CH), 130.4 (CH), 131.4 (C), 135.2 (CH),
144.7 (CH), 149.8 (C), 162.5 (C), 169.1 (C), 172.4 (C); LRMS (ES+):
m/z 331.1 [M+H]+.
1-(3-(Benzhydrylamino)propyl)-3-benzoyluracil (8): Polymer-sup-
ported PPh3 (2.5 equiv, 3 mmolgꢀ1) was swelled in THF for 15 min.
Compound 6 (0.356 g, 1.48 mmol) and the 3-N-benzoyluracil 7
(0.640 g, 2.96 mmol) were added, and the mixture was shaken at
room temperature for a further 15 min before addition of DIAD
(2 equiv) in THF. The reaction was shaken until consumption of the
alcohol was observed by TLC. The resin was then filtered off and
washed with THF, and the solvent was removed under reduced
pressure. The crude product was then purified by flash chromatog-
raphy (hexane/EtOAc 40:60) to give the product as a white foam
3-Benzoyl-1-(4-hydroxybutyl)uracil
(10):
NaBH4
(3.65 g,
96.39 mmol) was added to a solution of 9 (3.53 g, 10.71 mmol) in
THF (20 mL). This was heated at reflux for 15 min. CH3OH (21.7 mL,
0.536 mmol) was then added very slowly over 25 min. After 1 h,
the reaction was complete as evidenced by TLC. After cooling, H2O
(30 mL) was added to quench the reaction and stirred for a further
15 min. This was washed with CH2Cl2 (3ꢅ50 mL), and the aqueous
layer was concentrated to an oil. This was further purified by flash
chromatography (CH3OH/CH2Cl2 0!10%) to give 10 (0.604 g,
31%) as a white wax. Rf =0.13 (CHCl3/CH3OH 90:10); 1H NMR
(500 MHz, CDCl3): d=1.55 (m, 2H, HOCH2CH2), 1.75 (m, 2H,
CH2CH2N), 3.65 (q, J1 =11.1 Hz, J2 =6.1 Hz, 2H, HOCH2), 3.72 (m, 2H,
CH2N), 5.63 (dd, J1 =7.9 Hz, J2 =1.8 Hz, 1H, CHCO), 7.12 (d, J=
7.9 Hz, 1H, CHN), 8.19 (bs, 1H, NH); 13C NMR (125 MHz, CDCl3): d=
25.8 (CH2), 29.0 (CH2), 48.7 (CH2), 62.2 (CH2), 102.2 (CH), 144.4 (CH),
150.6 (C), 163.1 (C); LRMS (ES+): m/z 185.1 [M+H]+, 369.2 [2M+
H]+.
1
(0.230 g, 35%); Rf =0.11 (EtOAc/hexane 60:40); H NMR (500 MHz,
CDCl3): d=1.89 (m, 2H, CH2CH2N), 2.65 (t, J=6.3 Hz, 2H, NHCH2),
3.90 (t, J=6.7 Hz, 2H, CH2N), 4.80 (s, 1H, CHNH), 5.68 (d, J=7.9 Hz,
1H, CHCO), 7.19 (d, J=8.0 Hz, 1H, CHN), 7.25 (t, J=7.3 Hz, 2H, H-
Ar), 7.33 (t, J=7.6 Hz, 4H, H-Ar), 7.41 (d, J=7.4 Hz, 4H, H-Ar), 7.51
(t, J=7.8 Hz, 2H, H-Ar), 7.67 (d, J=7.5 Hz, 1H, H-Ar), 7.93 (d, J=
7.2 Hz, 2H, H-Ar); 13C NMR (125 MHz, CDCl3): d=29.2 (CH2), 44.2
(CH2), 47.1 (CH2), 67.6 (CH), 101.7 (CH), 127.3–143.7 (C-Ar), 144.9
(CH), 149.8 (C), 162.6 (C), 169.0 (C); LRMS (ES+): m/z 440.2 [M+H]+
; HRMS (ES+): found 440.1954 [M+H]+ C27H26N3O3 requires
+
Bis(4-fluorophenyl)methyl
methanesulfonate
(11):
MsCl
(0.527 mL, 6.81 mmol) was added to a solution of 4,4’-difluoro-
benzhydrol (1 g, 4.54 mmol) in pyridine (5 mL). This was left to stir
at room temperature overnight. Upon reaction completion, the
crude product was used in the following reaction as such. Rf =0.78
(CHCl3/CH3OH 90:10); 1H NMR (500 MHz, CDCl3): d=2.78 (s, 3H,
CH3), 4.68 (s, 1H, CHO), 7.09 (m, 4H, H-Ar), 7.35 (m, 4H, H-Ar).
440.1969.
1-(3-(Benzhydrylamino)propyl)uracil (1): Compound 8 (0.200 g,
0.455 mmol) was dissolved in a solution of 0.2m NaOCH3 in
CH3OH, and the reaction was stirred at room temperature over-
night until the disappearance of the starting esters was observed
(TLC). The solution was neutralised with Dowex H+ ion-exchange
resin, filtered, and washed with CH3OH. The solution was concen-
trated in vacuo, and the crude residue was purified by column
chromatography (0!2% CH3OH/CHCl3). The title compound was
obtained as an off-white crystalline foam (0.043 g, 28%). Purity by
LCMS (UV chromatogram, l 190–450 nm): 93%, tR =0.7 min
(Method 4); Rf =0.36 (CHCl3/CH3OH 90:10); 1H NMR (500 MHz,
(CD3)2SO): d=1.6 (m, 2H, CH2CH2N), 2.40 (t, J=6.5 Hz, 2H, NHCH2),
3.73 (t, J=7.0 Hz, 2H, CH2N), 4.76 (s, 1H, CHNH), 5.50 (d, J=7.8 Hz,
1H, CHCO), 7.18 (t, J=7.3 Hz, 2H, H-Ar), 7.28 (t, J=7.6 Hz, 4H, H-
Ar), 7.41 (d, J=7.3 Hz, 4H, H-Ar), 7.61 (d, J=7.8 Hz, 1H, CHN), 11.22
(bs, 1H, NHCO); 13C NMR (125 MHz, (CD3)2SO): d=28.5 (CH2), 44.1
(CH2), 45.6 (CH2), 66.4 (CH), 100.6 (CH), 126.6 (CH), 127.0 (CH), 128.2
(CH), 144.7 (C), 145.8 (CH), 150.9 (C), 163.7 (C); LRMS (ES+): m/z
167.0 [Ph2C]+, 336.1 [M+H]+; HRMS (ES+): found 336.1715 [M+
1-(4-Mesyloxybutyl)uracil (12): MsCl (1.25 mL, 16.08 mmol) was
added to a solution of 10 (0.305 g, 1.66 mmol) in pyridine (10 mL)
cooled to 08C. This was left to warm to room temperature over-
night. The solvent was concentrated in vacuo and purified by flash
chromatography (EtOAc/hexane 0!100%) to give 12 (0.215 g,
49%). Purity by LCMS (UV chromatogram, l 190–450 nm): 91%,
tR =0.6 min (Method 2); Rf =0.57 (CHCl3/CH3OH 90:10); 1H NMR
(500 MHz, (CD3)2SO): d=1.65 (m, 4H, CH2CH2N), 3.18 (s, 3H, CH3),
3.69 (t, J=6.4 Hz, 2H, CH2N), 4.22 (t, J=5.8 Hz, 2H, OCH2CH2), 5.56
(dd, J=7.8, 2.3 Hz 1H, CHCO), 7.67 (d, J=7.8 Hz, 1H, CHN), 11.27
(bs, 1H, NH); 13C NMR (125 MHz, (CD3)2SO): d=24.6 (CH2), 25.4
(CH2), 36.4 (CH3), 46.8 (CH2), 69.9 (CH2), 100.9 (CH), 145.6 (CH), 150.9
(C), 163.7 (C); LRMS (ES+): m/z 263.0 [M+H]+, 525.1 [2M+H]+;
HRMS (ES+): found 263.0686 [M+H]+ C9H15N2O5S+ requires
263.0696.
H]+ C20H22N3O2 requires 336.1707.
+
3-Benzoyl-1-(3-(ethoxycarbonyl)propyl)uracil (9): 3-N-Benzoylura-
cil 7 (1.00 g, 4.63 mmol) was combined with ethyl 4-bromobuta-
noate (1.35 g, 6.95 mmol) and Cs2CO3 (1.66 g, 5.09 mmol) in DMF
1-(4-(Benzhydryloxy)butyl)uracil (2a): 1-(4-Hydroxybutyl)uracil 10
(0.068 g, 0.372 mmol), benzhydrol (0.068 g, 0.370 mmol), and PTSA
(0.070 g, 0.372 mmol) were combined in toluene (10 mL). Molecular
1826
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ChemMedChem 2011, 6, 1816 – 1831

P. falciparum dUTPase Inhibitors
1
sieves (4 ꢄ) were then added, and this was heated at reflux. After
30 min a further portion of benzhydrol (0.013 g, 0.074 mmol) was
added, and heating at reflux continued for 1 h. The crude mixture
was filtered, and the filtrate crude was purified by flash chromatog-
raphy (EtOAc/hexane 60:40) to give 2a as a colourless oil (0.026 g,
20%). Purity by LCMS (UV chromatogram, l 190–450 nm): 92%,
tR =3.5 min (Method 4); Rf =0.18 (hexane/EtOAc 40:60); 1H NMR
(500 MHz, CDCl3): d=1.60 (m, 2H, OCH2CH2), 1.75 (m, 2H,
CH2CH2N), 3.42 (t, J=6.0 Hz, 2H, OCH2), 3.68 (t, J=7.3 Hz, 2H,
CH2N), 5.25 (s, 1H, CHO), 5.58 (dd, J=7.9, 1.8 Hz, 1H, CHCO), 7.02
(d, J=7.9 Hz, 1H, CHN), 7.15–7.26 (m, 10H, H-Ar), 9.09 (bs, 1H,
NH); 13C NMR (125 MHz, CDCl3): d=26.2 (CH2), 26.6 (CH2), 48.7
(CH2), 68.4 (CH2), 83.9 (CH), 102.0 (CH), 126.4–128.4 (C-Ar), 142.1
(CH), 144.6 (CH), 150.8 (C), 163.7 (C); LRMS (ES+): m/z 167.0
[Ph2C]+, 351.1 [M+H]+, 701.3 [2M+H]+; HRMS (ES+): found
(CHCl3/CH3OH 90:10); mp: 172–1748C; H NMR (500 MHz, DMSO):
d=2.18 (m, 2H, H-2’), 3.23 (s, 3H, CH3), 3.98 (m, 1H, H-4’), 4.24 (m,
1H, H-3’), 4.36 (m, 2H, H-5’), 5.53 (bs, 1H, 3’-OH), 5.66 (dd, J=8.1,
2.2 Hz, 1H, H-5), 6.20 (t, J=6.9 Hz, 1H, H-1’), 7.65 (d, J=8.1 Hz, 1H,
H-6), 11.38 (bs, 1H, 3-NH); 13C NMR (125 MHz, DMSO): d=36.6
(CH3), 38.3 (CH2), 69.4 (CH2), 70.0 (CH), 83.4 (CH), 84.3 (CH), 102.0
(CH), 140.5 (CH), 150.3 (C), 162.9 (C); LRMS (ES+): m/z 307.0 [M+
H]+, 613.1 [2M+H]+; HRMS (ES+): found 329.0413 [M+H]+
C10H15N2O7S+ requires 329.0414.
5’-(Benzhydrylamino)-2’,5’-dideoxyuridine (15a): Benzhydryla-
mine (0.178 mL, 1.03 mmol) and Et3N (0.045 mL, 0.326 mmol) were
added to
a solution of 5’-mesyl-2’-deoxyuridine 14 (0.100 g,
0.326 mmol) in CH3CN (2 mL), and the mixture was heated at
1208C overnight. After cooling, this was diluted with CH2Cl2
(10 mL) and extracted with H2O (3ꢅ10 mL). The organic layer was
dried (MgSO4), the solvent was removed, and purification by flash
chromatography (CH3OH/CHCl3 0!5%) gave 15a as a pale-orange
powder (0.061 g, 48%). Purity by LCMS (UV chromatogram, l 190–
450 nm): 91%, tR =4.0 min (Method 2); Rf =0.40 (CHCl3/CH3OH
90:10); mp: 188–1908C; 1H NMR (500 MHz, DMSO): d=2.10 (m,
2H, CH-2’), 2.63 (m, 3H, CH-5’+NH), 3.86 (m, 1H, CH-4’), 4.21 (m,
1H, CH-3’), 4.83 (d, J=4.1 Hz, 1H, CHNHCH2), 5.24 (d, J=4.3 Hz,
1H, OH), 5.48 (d, J=8.1 Hz, 1H, H-5), 6.12 (t, J=6.7 Hz, 1H, H-1’),
7.20 (t, J=7.3 Hz, 2H, H-Ar), 7.30 (t, J=7.6 Hz, 4H, H-Ar), 7.42 (d,
J=7.2 Hz, 4H, H-Ar), 7.76 (d, J=8.1 Hz, 1H, H-6), 11.28 (s, 1H, NH-
3); 13C NMR (125 MHz, DMSO): d=38.9 (CH2), 49.5 (CH2), 66.4 (CH),
71.2 (CH), 84.0 (CH), 85.7 (CH), 101.6 (CH), 126.6 (CH), 126.9 (CH),
128.3 (CH), 140.8 (CH), 144.5 (C), 150.3 (C), 163.0 (C); LRMS (ES+):
m/z 167.0 [Ph2C]+, 394.1 [M+H]+; HRMS (ES+): found 394.1747
351.1716 [M+H]+ C21H23N2O3 requires 351.1703.
+
1-(4-(4,4’-Difluorobenzhydryloxy)butyl)uracil (2b): Compounds
10 (0.194 g, 1.05 mmol), 11 (0.480 g, 2.18 mmol), and Et3N
(0.152 mL, 1.09 mmol) were combined in CH3CN (5 mL). This was
heated at reflux overnight. The solvent was then concentrated
under reduced pressure, and the crude residue was purified by
semi-preparative HPLC (tR =5.7 min, 1 min hold 95% A, 8 min ramp
to 95% B, 2 min hold 95% B) to give 2b (0.071 g, 17%). Purity by
LCMS (UV chromatogram, l 190–450 nm): 94%, tR =4.9 min
(Method 2); Rf =0.72 (CHCl3/CH3OH 90:10); 1H NMR (500 MHz,
CH3OD): d=1.65–1.68 (m, 2H, CH2CH2N), 1.79–1.82 (m, 2H,
OCH2CH2), 3.47–3.49 (t, J=6.1 Hz, 2H, CH2N), 3.77–3.79 (t, J=
7.3 Hz, 2H, OCH2), 5.40 (s, 1H, CHO), 5.64 (d, J=7.8 Hz, 1H, CHCO),
7.05–7.07 (m, 4H, H-Ar), 7.35–7.38 (m, 4H, H-Ar), 7.56 (d, J=7.8 Hz,
1H, CHN); 13C NMR (125 MHz, CH3OD): d=27.0 (CH2), 27.7 (CH2),
49.5 (CH2), 69.4 (CH2), 83.5 (CH), 102.2 (CH), 116.1 (CH), 129.8 (CH),
140.0 (C), 147.38 (CH), 163.5 (d, J=242.6 Hz, C), 166.8 (C); 19F NMR
(470 MHz, CH3OD): d=ꢀ117.2 (F); LRMS (ES+): m/z 203.0 [FPh2C]+,
387.0 [M+H]+, 773.3 [2M+H]+; HRMS (ES+): found 387.1519 [M+
[M+H]+ C22H24N3O4 requires 394.1761.
+
5’-(2,2-Diphenylethylamino)-2’,5’-dideoxyuridine (15b): Follow-
ing the procedure for 15a, compound 14 (0.075 g, 0.244 mmol),
2,2’-diphenylethylamine (0.073 g, 0.366 mmol), and Et3N (0.052 mL,
0.366 mmol) were suspended in CH3CN (2 mL) and heated at
1208C overnight. The solvent was removed, and purification by
semi-preparative HPLC (tR =5.1 min, 1 min hold 95% A, 8 min ramp
to 95% B, 2 min hold 95% B) gave 15b (0.007 g, 7%). Purity by
LCMS (UV chromatogram, l 190–450 nm): 95%, tR =4.2 min
(Method 2); Rf =0.36 (CHCl3/CH3OH 90:10); 1H NMR (500 MHz,
CDCl3): d=1.92 (m, 1H, CHH-2’), 2.16 (m, 1H, CHH-2’), 2.70 (dd, J=
12.5, 6.1 Hz 1H, CHH-5’), 2.79 (dd, J=12.5, 5.1 Hz 1H, CHH-5’), 3.11
(m, 2H, CHCH2NH), 3.64 (m, 1H, H-4’), 3.97 (t, J=7.7 Hz, 1H,
CHCH2NH), 4.03 (m, 1H, H-3’), 5.43 (d, J=8.1 Hz, 1H, H-5), 5.97 (t,
J=6.3 Hz, 1H, H-1’), 7.02–7.13 (m, 11H, H-Ar+H-6); 13C NMR
(125 MHz, CDCl3): d=40.1 (CH2), 51.1 (CH2), 51.2 (CH2), 54.7 (CH),
72.9 (CH), 84.5 (CH), 84.7 (CH), 102.6 (CH), 126.8–128.7 (C-Ar), 139.5
(CH), 142.3 (C), 142.5 (C); LRMS (ES+): m/z 408.2 [M+H]+, 815.3
H]+ C21H21F2N2O3 requires 387.1515.
+
1-(4-(Benzhydrylamino)butyl)uracil (2c): 1-(4-Mesyloxybutyl)uracil
12 (0.050 g, 0.191 mmol), benzhydrylamine (0.039 mg, 0.229 mmol)
and Et3N (0.039 mL, 0.287 mmol) were combined in CH3CN (2 mL).
This was heated at reflux overnight. The mixture was diluted with
CH2Cl2 (10 mL) and extracted with H2O (3ꢅ10 mL). The organic
layer was dried (MgSO4) and concentrated. This was purified by
flash chromatography (EtOAc/hexane 0!100%), followed by semi-
preparative HPLC [tR =5.2 min, 1 min hold 95% A, 8 min ramp to
95% B, 2 min hold 95% B to give a white wax (0.009 g, 14%)].
Purity by LCMS (UV chromatogram, l 190–450 nm): 93%, tR =
4.3 min (Method 2); Rf =0.68 (CHCl3/CH3OH 90:10); 1H NMR
(500 MHz, CH3OD): d=1.46 (m, 2H, CH2CH2N), 1.58 (m, 2H,
NHCH2CH2), 2.45 (m, 2H, CH2N), 3.62 (t, J=7.2 Hz, 2H, NHCH2), 4.71
(s, 1H, CHNH), 5.52 (d, J=7.8 Hz, 1H, CHCO), 7.10 (m, 2H, H-Ar),
7.19 (m, 4H, H-Ar), 7.28 (d, J=7.2 Hz, 4H, H-Ar), 7.43 (d, J=7.8 Hz,
1H, CHN); 13C NMR (125 MHz, CH3OD): d=27.3 (CH2), 27.7 (CH2),
48.5 (CH2), 48.6 (CH2), 68.5 (C), 102.1 (CH), 128.0 (C), 128.4 (CH),
128.7 (C), 129.4 (CH), 145.0 (CH), 147.3 (C), 166.8 (C); LRMS (ES+):
m/z 167.0 [Ph2C]+, 350.2 [M+H]+; HRMS (ES+): found 350.1864
[2M+H]+; HRMS (ES+): found 408.1901 [M+H]+ C23H26N3O4 re-
+
quires 408.1918.
5’-(Bis(4-fluorophenyl)methylamino)-2’,5’-dideoxyuridine (15c):
5’-Amino-2’-deoxyuridine (0.200 g, 0.881 mmol), 11 (0.194 g,
0.881 mmol), and Et3N (0.124 mL, 0.891 mmol) were combined in
CH3CN (5 mL). This was heated at 1208C overnight. The solvent
was then concentrated under reduced pressure, and the crude resi-
due was purified by semi-preparative HPLC (tR =4.9 min, 1 min
hold 95% A, 8 min ramp to 95% B, 2 min hold 95% B) to give 15c
(0.006 g, 2%). Purity by LCMS (UV chromatogram, l 190–450 nm):
91%, tR =4.4 min (Method 2); Rf =0.52 (CHCl3/CH3OH 90:10);
1H NMR (500 MHz, CH3OD): d=2.28 (m, 2H, CH-2’), 2.79 (m, 2H,
CH-5’), 3.99 (dt, J=4.2, 6.9 Hz,1H, CH-4’), 4.29 (m, 1H, CH-3’), 4.90
(s, 1H, CHNHCH2), 5.62 (d, J=8.1 Hz, 1H, H-5), 6.22 (t, J=6.5 Hz,
1H, H-1’), 7.05 (m, 4H, H-Ar), 7.43 (m, 4H, H-Ar), 7.73 (d, J=8.1 Hz,
[M+H]+ C21H24N3O2 requires 350.1863.
+
5’-O-Mesyl-2’-deoxyuridine (14): MsCl (0.169 mL, 2.19 mmol) was
added to a cooled solution of 2’-deoxyuridine (0.500 g, 2.19 mmol)
in pyridine (10 mL), and this was left at room temperature over-
night. The reaction mixture was concentrated and purified by flash
chromatography (CH3OH/CHCl3 0!5%) to give the title compound
as a white crystalline solid (0.347 g, 52%). Purity by LCMS (UV chro-
matogram, l 190–450 nm): 99%, tR =0.5 min (Method 2); Rf =0.27
ChemMedChem 2011, 6, 1816 – 1831
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1827

I. H. Gilbert et al.
MED
1H, H-6); 13C NMR (125 MHz, CH3OD): d=40.5 (CH2), 50.6 (CH2),
67.0 (CH), 73.1 (CH), 86.7 (CH), 87.2 (CH), 102.7 (CH), 116.1 (CH),
130.1 (CH), 142.6 (CH), 152.0 (C), 162.2 (C); 19F NMR (470 MHz,
CH3OD): d=ꢀ117.9 (F); LRMS (ES+): m/z 203.0 [(FPh)2C]+, 430.1
4.0 min, 1 min hold 95% A, 8 min ramp to 95% B, 2 min hold 95%
B) gave 15 f as a white wax (0.008 g, 3%), isolated as a mixture of
two diastereomers. Purity by LCMS (UV chromatogram, l 190–
450 nm): 100%, tR =0.6 min (Method 2); Rf =0.15 (CHCl3/CH3OH
90:10); 1H NMR (500 MHz, CH3OD): d=2.24–2.32 (m, 4H, H-2’),
2.77–2.93 (m, 4H, H-5’), 5.50 (s, 3H, CH3), 3.53 (s, 3H, CH3), 3.99–
4.02 (m, 2H, H-4’), 4.28–4.31 (m, 2H, H-3’), 5.12 (s, 2H, CHNH), 5.58
(d, J=8.0 Hz, 1H, H-5), 5.64 (d, J=8.0 Hz, 1H, H-5), 6.23 (t, J=
6.4 Hz, 2H, H-1’), 7.00–7.01 (m, 4H, (N)CHCH(N)), 7.30–7.38 (m,
10H, H-Ar), 7.61 (d, J=8.0 Hz, 1H, H-6), 7.84 (d, J=8.0 Hz, 1H, H-6);
13C NMR (125 MHz, CH3OD): d=33.2 (CH3), 33.3 (CH3), 40.4 (CH2),
40.5 (CH2), 50.2 (CH2), 50.6 (CH2), 60.2 (CH), 60.5 (CH), 73.0 (CH),
73.2 (CH), 86.6 (CH), 86.7 (CH), 87.2 (CH), 87.4 (CH), 102.9 (CH),
123.0 (CH), 123.2 (CH), 127.3–129.9 (C-Ar), 141.0 (CH), 141.1 (CH),
142.4 (C), 142.7 (C), 149.5 (C), 149.6 (C), 152.7 (C), 167.0 (C); LRMS
(ES+): m/z 398.1 [M+H]+, 795.3 [2M+H]+; HRMS (ES+): found
[M+H]+; HRMS (ES+): found 430.1580 [M+H]+ C22H22F2N3O4 re-
+
quires 430.1573.
5’-(2-(1-Methyl-1H-imidazole-2-yl)-2-phenylethylamino]-2’,5’-di-
deoxyuridine (15d): Following the procedure for 15a, compounds
14 (0.100 g, 0.326 mmol), 16a (0.085 g, 0.424 mmol), and Et3N
(0.045 mL, 0.326 mmol) were suspended in CH3CN (2 mL) and
heated at 1208C overnight. After cooling, the mixture was concen-
trated and purification by semi-preparative HPLC (tR =1.6 min,
1 min hold 95% A, 8 min ramp to 95% B, 2 min hold 95% B) gave
15d (0.005 g, 4%), isolated as a mixture of two diastereomers.
Purity by LCMS (UV chromatogram, l 190–450 nm): 100%, tR =
0.6 min (Method 2); Rf =0.26 (CHCl3/CH3OH 90:10); 1H NMR
(500 MHz, CH3OD): d=2.17–2.28 (m, 4H, H-2’), 2.59–2.80 (m, 4H,
H-5’), 2.29–3.01 (m, 2H, CH2CHNH), 3.12–3.18 (m, 2H, CH2CHNH),
3.23 (s, 6H, CH3), 3.92–3.96 (m, 2H, H-4’), 4.07–4.13 (m, 2H,
CH2CHNH), 4.18–4.23 (m, 2H, H-3’), 5.65 (d, J=8.1 Hz, 1H, H-5),
5.68 (d, J=8.1 Hz, 1H, H-5), 6.19 (d, J=6.7 Hz, 1H, H-1’), 6.20 (d,
J=6.7 Hz, 1H, H-1’), 6.84–6.87 (m, 4H, (N)CHCH(N)CH3), 7.23–7.34
(m, 10H, H-Ar), 7.62 (d, J=8.1 Hz, 1H, H-6), 7.72 (d, J=8.1 Hz, 1H,
H-6); 13C NMR (125 MHz, CH3OD): d=31.3 (CH3), 31.4 (CH3), 34.3
(CH2), 34.4 (CH2), 39.1 (CH2), 39.2 (CH2), 49.0 (CH2), 49.2 (CH2), 62.4
(CH), 62.7 (CH), 71.9 (CH), 85.2 (CH), 85.3 (CH), 85.6 (CH), 86.3 (CH),
101.4 (CH), 120.7 (CH), 125.7 (CH), 125.8 (CH), 126.8 (CH), 127.2
(CH), 127.3 (CH), 128.3 (CH), 140.9 (C), 141.1 (CH), 142.3 (CH), 145.6
398.1807 [M+H]+ C20H24N5O4 requires 398.1823.
+
5’-(N-Methylbenzhydrylamino)-2’,5’-dideoxyuridine (15g): 5’-Me-
syldeoxyuridine 14 (0.050 g, 0.163 mmol) was combined with N-
methylbenzhydrylamine (0.094 g, 0.489 mmol) and Et3N (0.050 g,
0.489 mmol) in CH3CN (2 mL) and heated at 1208C for five days
until reaction completion. Upon cooling, the crude was diluted
with CH2Cl2 (15 mL) and washed with H2O (3ꢅ15 mL). The organic
layer was dried (MgSO4) and concentrated. Purification was carried
out by flash chromatography (CH3OH/CH3Cl 0!5%) to give the
product as a pale-brown crystalline powder (0.019 g, 29%). Purity
by LCMS (UV chromatogram, l 190–450 nm): 96%, tR =4.3 min
1
(Method 5); Rf =0.16 (CH3OH/CHCl3 10:90); mp: 99–1018C; H NMR
(C), 150.7 (C),164.8 (C), 164.9 (C); LRMS (ES+): m/z 412.2 [M+H]+;
(500 MHz, CDCl3): d=1.97 (ddd, J=13.9, 7.1, 5.0 Hz, 1H, CHH-2’),
2.24 (s, 3H, H-12), 2.33 (m, 1H, CHH-2’), 2.57 (m, 2H, H-5’), 3.94 (q,
J=6.4 Hz, 1H, H-4’), 4.09 (dd, J=12.6, 6.3 Hz, 1H, H-3’), 4.36 (s, 1H,
CHNCH3), 5.54 (d, J=8.1 Hz, 1H, H-5), 6.11 (dd, J=6.8, 5.1 Hz, 1H,
H-1’), 7.08 (d, J=8.1 Hz, 1H, H-6), 7.14–7.33 (m, 10H, H-Ar);
13C NMR (125 MHz, CDCl3): d=39.4 (CH2), 41.8 (CH3), 57.3 (CH2),
73.0 (CH), 76.3 (CH), 83.0 (CH), 84.9 (CH), 102.4 (CH), 127.3–128.7
(CH), 139.4 (CH), 142.2 (C), 150.0 (C), 163.0 (C); LRMS (ES+): m/z
HRMS (ES+): found 412.1985 [M+H]+ C21H26N5O4
requires
+
412.1979.
5’-(Phenyl(thiazole-2-yl)methylamino)-2’-deoxyuridine (15e): Fol-
lowing the procedure for 15a, compounds 14 (0.200 g,
0.652 mmol), 16b (0.124 g, 0.848 mmol), and Et3N (0.165 mL,
0.652 mmol) were suspended in CH3CN (2 mL) and heated at
1208C overnight. After cooling, the mixture was diluted with
CH2Cl2 (2 mL), and washed with H2O (2 mL). The organic layer was
concentrated and purification by semi-preparative HPLC (tR =
4.1 min, 1 min hold 95% A, 8 min ramp to 95% B, 2 min hold 95%
B) gave 15e (6.70 mg, 3%), isolated as a mixture of two diastereo-
mers. Purity by LCMS (UV chromatogram, l 190–450 nm): 94%, tR =
0.7 min (Method 2); Rf =0.47 (CHCl3/CH3OH 90:10); 1H NMR
(500 MHz, CH3OD): d=2.25–2.31 (m, 2H, H-2’), 2.84–2.89 (m, 1H,
H-5’), 2.93–2.97 (m, 1H, H-5’), 3.99–4.02 (m, 1H, H-4’), 4.33–4.36 (m,
1H, H-3’), 5.26 (d, J=6.2 Hz, 1H, CHNHCH2), 5.64 (d, J=8.1 Hz, 1H,
H-5), 5.65 (d, J=8.1 Hz, 1H, H-5), 6.22–6.25 (m, 1H, H-1’), 7.32 (m,
1H, H-Ar), 7.38 (m, 2H, H-Ar), 7.48 (d, J=7.5 Hz, 2H, H-Ar), 7.54(d,
J=3.2 Hz, 1H, (N)CHCH(S)), 7.72 (m, 1H, (N)CHCH(S)), 7.75 (d, J=
8.1 Hz, 1H, H-6), 7.81 (d, J=8.1 Hz, 1H, H-6); 13C NMR (125 MHz,
CH3OD): d=40.5 (CH2), 50.5 (CH2), 50.6 (CH2), 66.2 (CH), 66.3 (CH),
73.0 (CH), 73.1 (CH), 86.7 (CH), 86.8 (CH), 87.2 (CH), 87.3 (CH), 102.8
(CH), 121.0 (CH), 128.7 (CH), 129.1 (CH), 129.8 (CH), 129.9 (CH),
142.5 (CH), 143.0 (CH), 152.1 (C), 166.2 (C), 177.2 (C), 177.3(C);
LRMS (ES+): m/z 401.1 [M+H]+; HRMS (ES+): found 401.1264 [M+
H]+ C19H21N4O4S+ requires 401.1278.
408.2 [M+H]+; HRMS (ES+): found 408.1922 [M+H]+ C23H26N3O4
+
requires 408.1918.
5’-(N-Ethylbenzhydrylamino)-2’,5’-dideoxyuridine (15h): To a so-
lution of 5’-mesyl-2’-deoxyuridine (83 mg, 0.270 mmol) in anhy-
drous DMF (4 mL), benzhydryl ethylamine (171 mg, 0.810 mmol)
and Et3N (0.141 mL, 0.810 mmol) were added. The reaction was
stirred at 1208C for four days in a sealed tube. After four days, fur-
ther 5’-mesyl-2’-deoxyuridine (83 mg, 0.270 mmol) was added, and
the mixture was stirred at 1408C overnight. The reaction was then
allowed to reach room temperature, and the solvents were re-
moved under reduced pressure. The product was purified by semi-
preparative HPLC (tR =6.7 min, 1 min hold 95% A, 9 min ramp to
95% B, 3 min hold 95% B) gave the title compound as a white
solid (31 mg, 27%). Purity by LC–MS (UV chromatogram, l 190–
1
450 nm) 97% (Method 3); Rf =0.27 (CH3OH/CH2Cl2 10:90); H NMR
(500 MHz, CDCl3) d=1.07 (t, J=7.1 Hz, 3H, CH3), 2.06 (ddd, J=
13.9, 7.2, 5.0 Hz, 1H, H-2’), 2.39 (td, J=13.7, 6.8 Hz, 1H, H-2’), 2.72–
2.80 (m, 3H, H-5’ and CH2CH3), 2.91 (dd, J=13.6, 5.7 Hz, 1H, H-5’),
3.52 (s, 1H, OH), 3.90 (dd, J=12.8, 5.8 Hz, 1H, H-4’), 4.14 (dd, J=
12.9, 6.5 Hz, 1H, H-3’), 5.60 (d, J=8.1 Hz, 1H, H-5), 6.17 (dd, J=6.9,
5.0 Hz, 1H, H-1’), 7.09 (d, J=8.2 Hz, 1H, H-6), 7.25–7.41 (m, 10H, H-
Ar); 13C NMR (125 MHz, CDCl3) d=10.8 (CH3), 39.7 (CH2), 45.3 (CH2),
51.9 (CH2), 71.3 (CH), 72.8 (CH), 83.2 (CH), 84.6 (CH), 102.3 (CH),
127.4 (CH), 128.5 (CH), 128.6 (CH), 139.4 (CH), 141.6 (C), 149.8 (C),
162.7 (C); LRMS (ES+) m/z 422.2 [(M+H)+, 100%]; MS (ES+) found
5’-[(Phenyl)(1-methyl-1H-imidazol-2-yl)methylamino)]-2’,5’-di-
deoxyuridine (15 f): Following the procedure for 15a, compounds
14 (0.200 g, 0.652 mmol), 16c (0.124 g, 0.848 mmol), and Et3N
(0.165 mL, 0.652 mmol) were suspended in CH3CN (2 mL) and
heated at reflux overnight. After cooling the mixture was diluted
with CH2Cl2 (2 mL) and washed with H2O (2 mL). The organic layer
was concentrated, and purification by semi-preparative HPLC (tR =
+
422.2066, C24H28N3O4 requires 422.2074.
1828
www.chemmedchem.org
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1816 – 1831

P. falciparum dUTPase Inhibitors
2-(1-Methyl-1H-imidazol-2-yl)-1-phenylethanamine (16a): A solu-
tion of lithium hexamethyldisilazanide (LiHMDS; 1m in THF,
20.3 mL, 20.3 mmol), was added dropwise to a solution of benzal-
dehyde (1.5 mL, 14.87 mmol) in THF at 08C under argon. The mix-
ture was stirred for 30 min at the same temperature. In a separate
round-bottom flask, 1,2-dimethylimidazole (1.3 g, 13.52 mmol) was
dissolved in THF, and n-butyllithium (1.6m in hexane, 9.3 mL,
14.87 mmol) was added dropwise at 08C under argon. The result-
ing mixture was stirred for 30 min at the same temperature. After
this time, this solution was added dropwise to the benzylimine so-
lution 08C. Upon completion of the addition, the reaction mixture
was allowed to reach room temperature, hydrolysed with HCl (3m,
90 mL), and washed with EtOAc (3ꢅ120 mL). The aqueous layer
was then made alkaline with NaOH (2.5m, 150 mL), extracted with
EtOAc (3ꢅ150 mL), and the combined organic layers were dried
over MgSO4, and the solvents removed under reduced pressure.
The product was purified by flash chromatography using the fol-
lowing gradient: 1 min hold at 100% CH2Cl2, 20 min ramp to 10%
CH3OH/NH3 in CH2Cl2, 5 min hold to 10% CH3OH/NH3 in CH2Cl2.
After removing the solvents, the title compound was obtained as
pale-yellow oil (488 mg, 18%). Purity by LC–MS (UV chromatogram,
the same temperature. After this time, this solution was added
dropwise to the benzylimine solution 08C. Upon completion of the
addition, the reaction mixture was allowed to reach room tempera-
ture, hydrolysed with HCl (3m, 90 mL), and washed with EtOAc (3ꢅ
120 mL). The aqueous layer was then made alkaline with NaOH
(2.5m, 150 mL), extracted with EtOAc (3ꢅ150 mL), and the com-
bined organic layers were dried over MgSO4, and the solvents
were removed under reduced pressure. The product was purified
by flash chromatography using the following gradient: 5 min hold
at 100% CH2Cl2, 15 min ramp to 20% CH3OH/NH3 in CH2Cl2, 5 min
hold to 20% CH3OH/NH3 in CH2Cl2. After removing solvents, the
title compound was obtained as a yellow oil (532 mg, 23%). Purity
by LC–MS (UV chromatogram, l 190–450 nm) 93% (Method 3);
1H NMR (500 MHz, CDCl3) d=2.20 (bs, 2H, NH2), 3.41 (s, 3H, CH3),
5.20 (s, 1H, CH), 6.84 (d, J=1.1 Hz, 1H, CHN), 7.04 (d, J=1.2 Hz,
1H, CHNCH3), 7.27–7.37 (m, 5H, H-Ar); 13C NMR (125 MHz, CDCl3)
d=32.7 (CH3), 53.5 (CH), 121.5 (CH), 126.9 (CH), 127.0 (CH), 127.6
(CH), 128.9 (CH), 142.7 (C), 149.7 (C); LRMS (ES+) m/z 171.1
[(MꢀNH2)+, 100%], 188.1 [(M+H)+, 22%].
Benzhydryl ethylamine (16d): Benzophenone (1 g, 5.5 mmol) was
dissolved in dry CH2Cl2 (30 mL), and a 1m solution of TiCl4 in
CH2Cl2 (6 mL, 6 mmol) was added. The mixture was placed in an
ice bath, and 2m ethylamine solution in THF (6 mL, 12 mmol) was
added. The resulting yellow suspension was stirred at room tem-
perature for 3 h under argon. NaCNBH3 (1m solution in THF,
6.5 mL, 6.5 mmol) was then added to the reaction followed by
10 mL anhydrous CH3OH, and the mixture was stirred for 1 h at
room temperature. The mixture was made basic (pHꢃ10) with a
1.5m solution of NaOH and filtered. The filtrate was partitioned be-
tween EtOAc (100 mL) and H2O (100 mL). The organic layer was
separated, washed with H2O (100 mL) and brine (100 mL), dried
over MgSO4, and concentrated under reduced pressure. The resi-
due was dissolved in Et2O (100 mL), acidified with concentrated
HCl, and extracted with H2O (100 mL). The aqueous extract was ad-
justed to pHꢃ10 with aqueous NH3 and extracted with Et2O (2ꢅ
100 mL). The organic layer was dried over MgSO4, and solvents
were removed under reduced pressure to give the title compound
as a yellow oil (518 mg, 45%). Purity by LC–MS (UV chromatogram,
1
l 190–450 nm) 99% (Method 3); H NMR (500 MHz, CDCl3) d=1.95
(bs, 2H, NH2), 2.94–2.96 (m, 2H, CH2), 3.31 (s, 3H, CH3), 4.59 (dd,
J=7.7, 5.4 Hz, 1H, CH), 6.77 (d, J=1.2 Hz, 1H, CHN), 7.00 (d, J=
1.2 Hz, 1H, CHNCH3), 7.26–7.37 (m, 5H, H-Ar); 13C NMR (125 MHz,
CDCl3) d=32.4 (CH3), 37.0 (CH2), 53.8 (CH), 120.5 (CH), 126.3 (CH),
127.30 (CH), 127.35 (CH), 128.5 (CH), 145.3 (C), 146.0 (C); LRMS (ES+
) m/z 202.1 [(M+H)+, 100%].
Phenyl(thiazol-2-yl)methanamine (16b): A solution of LiHMDS
(1m in THF, 17.6 mL, 17.6 mmol), was added dropwise to a solution
of benzaldehyde (1.39 mL, 12.92 mmol) in THF at 08C under argon.
The mixture was stirred for 30 min at the same temperature. In a
separate round-bottom flask, thiazole (1 g, 11.75 mmol) was dis-
solved in THF, and n-butyllithium (1.6m in hexane, 8.61 mL,
13.79 mmol) was added dropwise at 08C under argon. The result-
ing mixture was stirred for 30 min at the same temperature. After
this time, this solution was added dropwise to the benzylimine so-
lution 08C. Upon completion of the addition, the reaction mixture
was allowed to reach room temperature, hydrolysed with HCl (3m,
90 mL) and washed with EtOAc (3ꢅ120 mL). The aqueous layer
was then made alkaline with NaOH (2.5m, 150 mL), extracted with
EtOAc (3ꢅ150 mL), and the combined organic layers were dried
over MgSO4, and the solvents were removed under reduced pres-
sure. The product was purified by flash chromatography using the
following gradient: 5 min hold at 100% CH2Cl2, 15 min ramp to
5% CH3OH/NH3 in CH2Cl2, 5 min hold to 5% CH3OH/NH3 in CH2Cl2.
After removing solvents, the title compound was obtained as a
yellow oil (217 mg, 10%). Purity by LC–MS (UV chromatogram,
1
l 190–450 nm) 93% (Method 3); H NMR (500 MHz, CDCl3) d=1.16
(t, J=7.1 Hz, 3H, CH3), 1.58 (bs, 1H, NH), 2.64 (q, J=7.1 Hz, 2H,
CH2), 4.86 (s, 1H, CH), 7.21–7.43 (m, 10H, H-Ar); 13C NMR (125 MHz,
CDCl3) d=15.4 (CH3), 42.6 (CH2), 67.5 (CH), 126.9 (CH), 127.3 (CH),
128.5 (CH), 144.2 (C); LRMS (ES+) m/z 167.1 [(Ph2C+H)+, 100%],
212.1 [(M+H)+, 26%].
Benzhydryl isopropylamine (16e): Prepared as described above
for 16d from benzophenone (1 g, 5.5 mmol) and isopropylamine
(1.0 mL, 12 mmol) to give the title compound as a yellow oil
(636 mg, 52%); 1H NMR (500 MHz, CDCl3) d=1.11 (d, 6H, J=
6.3 Hz, 2ꢅCH3), 2.77 (sept, 1H, J=6.3 Hz, CH), 4.99 (s, 1H, CH),
7.21–7.42 (m, 10H, H-Ar); 13C NMR (125 MHz, CDCl3) d=23.2 (2ꢅ
CH3), 46.1 (CH), 64.2 (CH), 126.8 (CH), 127.4 (CH), 128.4 (CH), 144.5
(C).
1
l 190–450 nm) 84% (Method 3); H NMR (500 MHz, CDCl3) d=2.20
(bs, 2H, NH2), 5.51 (s, 1H, CH), 7.25 (d, J=3.3 Hz, 1H, CHS), 7.29–
7.48 (m, 5H, H-Ar), 7.73(d, J=3.3 Hz, 1H, CHN); 13C NMR (125 MHz,
CDCl3) d=58.3 (CH), 119.0 (CH), 126.9 (CH), 127.9 (CH), 128.8 (CH),
142.7 (CH), 143.1 (C), 176.3 (C); LRMS (ES+) m/z 174 [(MꢀNH2)+,
100%], 191.1 [(M+H)+, 13%].
Benzhydryl cyclopropylamine (16 f): Prepared as described above
for 16d from benzophenone (1 g, 5.5 mmol) and isopropylamine
(0.8 mL, 12 mmol) to give the title compound as a yellow oil
(755 mg, 61%). Purity by LC–MS (UV chromatogram, l 190–
(1-Methyl-1H-imidazol-2-yl)(phenyl)methanamine (16c): A solu-
tion of LiHMDS (1m in THF, 18.8 mL, 18.8 mmol) was added drop-
wise to a solution of benzaldehyde (1.39 mL, 12.92 mmol) in THF
at 08C under argon. The mixture was stirred for 30 min at the
same temperature. In a separate round-bottom flask, N-methylimi-
dazole (1 g, 12.18 mmol) was dissolved in THF and n-butyllithium
(1.6m in hexane, 8.6 mL, 13.79 mmol) was added dropwise at
ꢀ788C under argon. The resulting mixture was stirred for 1 h at
1
450 nm) 99%; H NMR (500 MHz, CDCl3) d=0.18–0.19 (m, 4H, 2ꢅ
CH2), 1.16 (t, 3H, J=7.1 Hz, CH3), 1.79 (bs, 1H, NH), 1.83–1.88 (m,
1H, CH2CHCH2), 4.70 (s, 1H, CH), 6.98–7.15 (m, 10H, H-Ar); 13C NMR
(125 MHz, CDCl3) d=6.7 (CH2), 22.6 (CH), 67.2 (CH), 126.9 (CH),
127.4 (CH), 128.4 (CH), 144.2 (C); LRMS (ES+) m/z 167.1 [(Ph2C+H)+
, 100%], 224 [(M+H)+, 14%].
ChemMedChem 2011, 6, 1816 – 1831
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1829

I. H. Gilbert et al.
MED
were vitrified at 110 K. For 24 the crystal was cryoprotected using
40% glycerol/60% reservoir; for 15a, 19% glycerol/78% well/3%
ligand. The others were taken straight from the drop without cryo-
protectant.
Crystallography
Protein constructs: For co-crystallisation of the complex with 24, a
construct corresponding to the full-length enzyme was overex-
pressed, and the protein was purified as described for the first
complex of a trityl derivative.[3] In brief, this involved a tag-free pro-
tein overexpressed in E. coli and then purified by using a phospho-
cellulose matrix followed by gel filtration. For the other three com-
plexes, a second construct was used in which the six C-terminal
residues, which were disordered in the first crystal structures, were
replaced by a non-cleavable His6 tag. This allowed a simplified pu-
rification protocol with no tag cleavage required. Crystals grew
more rapidly and reproducibly with this construct, and were iso-
morphous to those for 24. The construct is referred to as PfdUTPa-
seDC.
X-ray data collection and structure refinement: X-ray data were col-
lected from single crystals at 110 K on station ID23eh1 at the ESRF,
Grenoble (France) using an ADSC CCD detector for 24 and 15a.
For 15g and 15 f, data were collected at the Diamond Light
Source, on beam lines I02 and I24, respectively. The images were
integrated using MOSFLM, and all crystals were isomorphous in
space group is P41 with cell dimensions a=bꢃ77 ꢄ and cꢃ106 ꢄ,
with a trimer of the complex in the asymmetric unit. Calculations
were performed using the CCP4 suite.[14] The compound 24 struc-
ture was solved by molecular replacement using the known struc-
ture of the enzyme reported earlier[3] (PDB code: 1VYQ) as a search
model. The other three started from the refined structure of 24.
The models were refined using REFMAC[15] alternating with rebuild-
ing using COOT.[16] The coordinates and refinement dictionary for
the ligands were generated using SKETCHER. The data and refine-
ment statistics are listed in Table 4. X-ray crystallographic data and
coordinates have been deposited with the RCSB Protein Data Bank
(PDB codes: compounds 15a, 3T64; 15 f, 3T6Y; 15g, 3T70; 24,
3T60).
Crystallisation: To obtain the 24 complex, the ligand was first dis-
solved in ethanol. This was then added in threefold molar excess
to a ꢃ1 mgmLꢀ1 solution of the protein in 20 mm Tris-HCl pH 7.5,
10 mm NaCl, 5 mm MgCl2. The other inhibitors were less soluble,
and a method was devised for producing a crystallisation solution
by first dissolving the ligand to a stock concentration of 10–20 mm
in DMSO. An aliquot of stock ligand solution was slowly added to
the protein with continuous mixing to give a crystallisation solu-
tion of 15 mgmLꢀ1 protein, 1.5 mm ligand in 50 mm Tris-HCl
pH 8.5, 50 mm NaCl, and 5 mm TCEP. The protein/ligand molar
ratio was 1:2. A centrifugation step was performed after 20 min in-
cubation to remove any precipitate (14000 g). Crystallisation of
complexes was performed by sitting-drop vapour diffusion with
300 nL drops (1:1 or 3:2 protein/inhibitor solution: Ammonium Sul-
fate screen) using a Mosquito robot (TTP Labtech) in 96-well trays
(SwissCi). Crystals of each complex grew to a maximum dimension
of 100–150 mm. Crystals were obtained under the following condi-
tions: 24: 2.0m ammonium sulfate, 0.1m sodium acetate, pH 4.6;
15a and 15 f: 2.2m ammonium sulfate, 0.2m disodium phosphate;
15g: 2.2m ammonium sulfate alone. Crystals of each complex
Acknowledgements
We acknowledge the BBSRC and Medivir for a CASE award to
S.E.H. The European Union (contract 037587), the RICET FIS Net-
work (RD06/0021/0018), and the Junta de Andalucꢀa (BIO-199)
are acknowledged for funding. We acknowledge the European
Synchrotron Radiation Facility for provision of synchrotron radia-
tion facilities, and we thank the staff for assistance in using
Table 4. Data processing and refinement statistics for the complexes 15a, 15 f, 15g, and 24.
Data collection
15a
15 f
15g
24
Beamline/wavelength [ꢄ]
P41 cell dimensions: a, c [ꢄ]
Resolution limits [ꢄ]
No. unique reflections
Multiplicity
ID23eh1/1.072
77.01, 106.28
36.20–1.65
74358
I24/0.9778
76,83, 106.13
37.96–2.60
19028
IO2/0.976
76.75, 105.83
43.56–1.80
56711
ID23eh1/0.976
76.97, 106.92
40.00–2.40
24538
5.5
5.6
7.4
3.9
Completeness [%]
100.0
100.0
100.0
99.3
R-merge [%]
8.20
11.80
4.80
9.00
I/s(I)
11.5
10.7
12.5
13.6
Refinement statistics[a]
R-cryst/R-free [%]
19.14/21.56
3602
15.90/22.65
3459
20.09/23.23
3556
19.91/25.36
3226
No. protein atoms
No. water molecules
No. ligand atoms
322
87
122
87
395
90
96
105
No. sulfate atoms
20
25
30
n/a
No. glycerol atoms
n/a
n/a
n/a
18
RMS D bond lengths [ꢄ]
RMS D bond angles [8]
RMS D chiral volume [ꢄ3]
B-average on protein atoms [ꢄ2]
B-average on water molecules [ꢄ2]
B-average on ligand atoms [ꢄ2]
B-average on sulfate atoms [ꢄ2]
B-average on glycerol atoms [ꢄ2]
Ramachandran outliers [%]
0.011 (0.022)
1.335 (2.010)
0.093 (0.200)
23.12
35.13
23.73
45.54
n/a
0.8
0.025 (0.022)
2.563 (2.021)
0.166 (0.200)
37.48
36.85
40.32
69.72
n/a
0.8
0.008 (0.022)
1.133 (2.023)
0.072 (0.200)
26.60
38.64
25.93
66.44
n/a
0.8
0.024 (0.022)
2.508 (2.023)
0.430 (0.200)
36.36
36.87
36.57
n/a
51.82
0.8
[a] For the refinement statistics, average ideal values are given in parentheses.
1830
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ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1816 – 1831

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Received: May 23, 2011
Revised: June 27, 2011
Published online on August 16, 2011
ChemMedChem 2011, 6, 1816 – 1831
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1831