Synthesis and evaluation of α-thymidine analogues as novel antimalarials

Article abstract of DOI:10.1021/jm301328h

Plasmodium falciparum thymidylate kinase (PfTMPK) is a key enzyme in pyrimidine nucleotide biosynthesis. 3-Trifluoromethyl-4-chloro-phenyl-urea- α-thymidine has been reported as an inhibitor of Mycobacterium tuberculosis TMPK (MtTMPK). Starting from this

Full text of DOI:10.1021/jm301328h

Article
pubs.acs.org/jmc
Synthesis and Evaluation of α‑Thymidine Analogues as Novel
Antimalarials
Huaqing Cui,†,‡ Juana Carrero-Lerida,‡ Ana P. G. Silva,§ Jean L. Whittingham,§ James A. Brannigan,§
́
Luis M. Ruiz-Perez,‡ Kevin D. Read,† Keith S. Wilson,*,§ Dolores Gonzalez-Pacanowska,*,‡
́
́
and Ian H. Gilbert*,†
†Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Sir James Black Centre,
Dundee, DD1 5EH, U.K.
‡Instituto de Parasitología y Biomedicina “Lop
Spain
́
ez-Neyra”, Consejo Superior de Investigaciones Científicas, 18100-Armilla, Granada,
§Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD, U.K.
S
* Supporting Information
ABSTRACT: Plasmodium falciparum thymidylate kinase
(Pf TMPK) is a key enzyme in pyrimidine nucleotide
biosynthesis. 3-Trifluoromethyl-4-chloro-phenyl-urea-α-thymi-
dine has been reported as an inhibitor of Mycobacterium
tuberculosis TMPK (MtTMPK). Starting from this point, we
designed, synthesized and evaluated a number of thymidine
analogues as antimalarials. Both 5′-urea-α- and β-thymidine
derivatives were moderate inhibitors of Pf TMPK and
furthermore showed moderate inhibition of parasite growth.
The structure of several enzyme−inhibitor complexes provides
a basis for improved inhibitor design. However, we found that
certain 5′-urea-α-thymidine analogues had antimalarial activity where inhibition of PfTMPK is not the major mode of action.
Optimization of this series resulted in a compound with potent antimalarial activity (EC50 = 28 nM; CC50 = 29 μM).
essential in most organisms. We have recently reported structural
and kinetic studies of Pf TMPK, which indicate significant
differences from the human homologue.12
TMPK is a homodimer with a subunit fold consisting of a five-
stranded parallel β-sheet surrounded by 7−11 α-helices.12 Two
conserved sequence motifs (Figure 1) are found in the
nucleotide substrate binding domain: the P-loop containing
structural elements required for substrate recognition and
catalysis and a LID domain that partially encloses the phosphate
donor and is important for catalysis.12
Superposition of the structure of Pf TMPK-TMP-ADP with
the equivalent complex of human TMPK (hTMPK) shows a
highly conserved topology and mode of nucleotide binding, the
TMP binding sites being essentially identical. However, several
significant differences were observed in the P-loop and the LID
domain.12 Therefore, reported TMPK inhibitors containing the
thymidine base, but an optimized 5′-OH motif, will potentially
target the P-loop and LID domain, and could lead to selective
inhibition of Pf TMPK compared to hTMPK.
INTRODUCTION
■
There are approximately 300−500 million clinical cases of
malaria each year.1−5 While there is significant progress in
development of vaccines, they are still on trial and not available,3
so chemotherapy remains the mainstay for dealing with this
enormous problem. Furthermore, owing to problems with
resistance, there is a need for new drugs to treat the disease.
Analysis of the Plasmodium falciparum genome6 indicates that
these parasites lack the enzymes required for pyrimidine salvage7
and are totally dependent on de novo pyrimidine nucleoside
synthesis for DNA replication. In contrast, erythrocyte
maturation is accompanied by the loss of the capacity to carry
out de novo synthesis of pyrimidines8 reinforcing the importance
of pyrimidine biosynthesis as a potential antimalarial drug target.
Indeed, several enzymes in the pyrimidine metabolism pathway
are validated antimalarial targets, such as dihydrofolate
reductase9 and dihydroorotate dehydrogenase.10
Another enzyme involved in pyrimidine biosynthesis, P.
falciparum thymidylate kinase (Pf TMPK), which catalyzes
phosphorylation of thymidine monophosphate (TMP) to
thymidine diphosphate (TDP), represents a potentially
attractive drug target for malaria.11,12 TMPK has been
characterized in several organisms including human,13,14
Escherichia coli,13,15 Saccharomyces cerevisiae,13,15 Mycobacterium
tuberculosis,16 Vaccinia virus,17 and P. falciparum,5,12 and is
TMPK has been investigated as a target against M.
tuberculosis.16 Various series of inhibitors have been reported
for MtTMPK.16,19,20 An interesting class is a series of 5′-
Received: September 15, 2012
© XXXX American Chemical Society
A
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concerning chemical solubility and cell permeability. Super-
imposition of the structures of hTMPK and MtTMPK revealed
significant amino acid differences in the active site close to the 5′-
position of the deoxyribose.21 Moreover, 5′-thiourea α-
thymidine derivatives were completely inactive against human
thymidine kinase 1 and only showed weak inhibition of human
mitochondrial nucleoside kinase TK-2.22,23
As a starting point we were interested to investigate if α-
thymidine derivatives could act against PfTMPK, as these have
been shown to inhibit MtTMPK, but not hTMPK.
RESULTS AND DISCUSSION
■
Synthesis of α-Thymidine. The synthesis of α-thymidine
was based on literature methods.24,25 The key step was
epimerization of the base. Several methods are reported for
epimerization of β-thymidine including the use of TMSOTf
reported by Sato25 and use of acetic anhydride/sulphuric acid as
reported by Ward.24 Initially the method by Sato was followed.
First, β-thymidine was selectively protected with diphenylacetyl
chloride on the primary position in 50% yield (Scheme 1). The
3′-position was then protected with p-toluoyl chloride to give
compound 3, as this substituent was reported to be selective for
generation of the α-analogue on epimerization of the anomeric
center.25 However, epimerization using TMSOTf was un-
successful. It was decided to retain the 5′ and 3′ protecting
groups suggested by Sato, since the compounds crystallized well,
but epimerize the base using the acetic anhydride/sulphuric acid
method of Ward.24 The ratio of α/β analogues after
epimerization was around 3/1. The 3′,5′-O-diacylated α-
thymidine derivative was readily separated by crystallization,
giving the required product 4 in around 70% yield. Identification
of the α/β-thymidine derivatives (3 and 4) was carried out by 1H
NMR (Supporting Information, Figure S4), in particular, by
looking at the chemical shifts of H1′ and H2′, which were
affected by the stereochemistry of C1′. The two acyl groups were
Figure 1. The crystal structure of the Pf TMPK TMP-ADP complex
showing the P-loop (red) and LID domain (blue), PDB code 2wwf,
Whittingham et al.12 Figures 1 and 3 were made with CCP4mg.18
substituted α-thymidine derivatives (Figure 2), some of which
showed sub-micromolar activity against MtTMPK, and also good
Figure 2. Example of a 5′- substituted α-thymidine inhibitor of
MtTMPK.16
selectivity compared to hTMPK (Figure 2).16 A number of
MtTMPK inhibitors were active against M. tuberculosis in cell
culture, although others were inactive possibly due to issues
a
Scheme 1. Synthesis of α-Thymidine
a
(a) Diphenylacetyl chloride, pyridine, 0 °C, 50%; (b) p-toluoyl chloride, pyridine, RT, 56%; (c) acetic anhydride, H2SO4, CH3CN, 51%; (d),
NaOMe, MeOH, 90%.
B
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a
Scheme 2. Synthesis of 5′-Substituted α- and β-Thymidine Derivatives
a
(a) Methanesulfonyl chloride, pyridine, −38 °C, [α: 43%; β: 50%]; (b) sodium azide, DMF, 60°C, [α: 43%; β 64%]; (c) 10% Pd/C, MeOH, [α:
95%; β 95%]; (d) sulfonyl chloride, DMF, RT; (e) carbonyl chloride, DMF, RT; (f) aryl chloride, DMF, RT; (g) (thio)isocyanate, DMF, RT.
removed from compound 4 by sodium methoxide to give α-
thymidine (5).
derivatives. 5′-(Thio)urea α-thymidine derivatives have been
previously reported to be inhibitors of MtTMPK but are less
active against hTMPK,16 human thymidine kinase23 and human
mitochondrial thymidine kinase.23 They have also been shown to
inhibit growth of M. tuberculosis and are selective compared to
human cells.16
The 5′-urea β-thymidine derivatives (39−55) (Table 1)
exhibited moderate inhibitory activity against PfTMPK in a
similar fashion to the α-derivatives. Most of them inhibited in a
similar range (50−100 μM), with again the 3-trifluoromethyl-4-
chlorophenyl derivatives 53 (Ki = 25 μM) and 54 (Ki = 27 μM)
and the 4-nitrophenyl derivative 55 (Ki = 11 μM) showing
slightly higher potency.
There was no or very low inhibitory activity with the other 5′
substituents in either the α- or the β- series: this includes the
sulphonamides (9, 10, 34, 35), the amides (11, 12, 36, 37), and
the amines (13, 38). Interestingly, some 5′-substituted β-
thymidine derivatives, including 5′-sulfonamides, 5′-(thio) ureas
and 5′-amides, have been reported as moderate inhibitors of
Bacillus anthracis TMPK but do not inhibit bacterial growth.26
Since the 5′-urea α-thymidine derivatives and the 5′-urea β-
thymidine derivatives showed moderate inhibitory activity
against Pf TMPK, they were further investigated by cocrystall-
izing them with Pf TMPK to assist in possible optimization.
Preparation of 5′ Substituted α- and β-Thymidine.
Synthesis of 5′-amino substituted α-thymidine was carried out as
shown in Scheme 2, by sulfonation, displacement with sodium
azide and then hydrogenation to give the amine 8. Amine 8 was
then coupled with thioisocyanate to give thioureas. The ureas,
sulphonamides, amides and amines were also prepared to explore
the SAR.
For comparison, some 5′-substituted β-thymidine analogues
were prepared (Scheme 2) using similar conditions as for the
preparation of 5′-substituted α-thymidine. The β-thymidine
amine 33 was coupled to give the urea, sulfonamide, amide and
amine.
PfTMPK Inhibition Assay. Compounds were screened
against recombinant Pf TMPK using a coupled assay with
pyruvate kinase and lactate dehydrogenase (Figure S1).12
Activity was measured spectrophotometrically by following the
change in absorption at 340 nm due to the oxidation of NADH.
The assay was carried out using TMP as substrate (Km = 11 μM).
Control spectrophotometric assays were performed to verify that
the compounds were inhibitors of PfTMPK and not of the
coupling enzymes, by using ADP as substrate and the two
coupling enzymes pyruvate kinase and lactate dehydrogenase,
but no PfTMPK or TMP.12
The 5′-urea α-thymidine derivatives (14−30) showed
moderate inhibition of Pf TMPK, with most compounds having
Ki values between 80 and 200 μM (Table 1). The most active
were the 3-trifluoromethyl-4-chlorophenyl derivative 28 (Ki = 31
μM) and the 4-nitrophenyl derivative 30 (Ki = 11 μM). There
were no significant differences between urea and thio-urea
CRYSTAL STRUCTURE OF LIGAND COMPLEXES
■
Co-crystallization with Pf TMPK was successful with three of our
active Pf TMPK inhibitors (28, 30 and 53) (Figure 3).
Crystallization conditions of the three inhibitors are listed in
Supplementary Table S1, Supporting Information. Statistics for
the X-ray data and refinement are summarized in Supplementary
C
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c
Table 1. Evaluation of 5′-α and β-Thymidine Derivatives against Pf TMPK, P. falciparum and MRC5 Cells
a
b
c
hTMPK Ki > 1000 μM.16 hTMPK Ki = 362 μM.16 Reference compound: chloroquine EC50 = 0.007 μM.
Table S2, and data for the three complexes have been deposited
in the PDB with codes, 2YOG (28), 2YOH (30) and 2YOF (53).
Compounds 28 and 53 are pairs of α- and β-thymidine
derivatives (3-trifluoromethyl-4-chlorophenyl) that allow for
comparison. The structures of all three complexes show that the
enzyme is well ordered, with an essentially identical fold to that
reported for the complexes with nucleotides and nucleotide
derivatives.12 There was clear electron density for most of the
ligands in all three complexes. A summary of some key features of
the structural data on the complexes is given in Supporting
Information Table S3. One substantial change in the protein is
that the extended loop, residues 141−152, is disordered with no
visible density in all three ligand structures, with the exception of
Chain C in the complex with 53 where it packs against one of the
two alternate conformations of the ligand.
to take up optimum interactions with the enzyme. The thymidine
and deoxyribose are buried deep within the pocket, which also
accommodates two water molecules, W27 and W303, that form
bridges between ligand and protein (Figure 3C). Two additional
waters form bridging H-bonds between ligand and protein. The
H-bonds between the ligand and the protein and surrounding
ordered water molecules (red spheres) are shown. The aromatic
ring with its fluoro substituents extends out of the pocket and
packs against the protein surface.
Chain C of the 53 complex displays the least well-ordered
ligand (Figure 3D), and we have modeled this in two
conformations. There is reasonable density for the thymidine
and deoxyribose, though the deoxyribose is already showing
disorder over two alternative puckers. There is essentially no
density for the aromatic ring, and its positions are solely based on
density for the fluorine groups. This ligand is well ordered in
Chain B. A summary of the experimental electron density for the
ligands is given in Supporting Table S3.
The thymidine ring of the synthetic ligands mimics that of the
natural TMP substrate (Figure 3B). The deoxyribose ring in
contrast shows its typical variation in pucker and orientation
relative to the thymidine allowing the end of the synthetic ligands
D
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Figure 3. (A) Superposition of three ligand complexes 53 (blue), 30 (coral) and 28 (tan) on the TMP−ADP complex (colored by atom type)
determined previously (pdb: 1wwf). The chain was selected in which the ligand was best ordered. The two sodium atoms in the TMP−ADP complex are
shown as spheres. The peptide backbone is shown for the 53 complex (pale blue). (B) The binding pocket for 28, showing interactions with the protein.
(C) The binding pocket for 28. The surface of the protein is colored by electrostatic potential. The four waters that form H-bond bridges to the protein
are numbered. (D) The two conformations for 53 in Chain C, the least well-ordered ligand site.
cells. For instance, compound 26 has an EC50 of 2.5 μM, but a
CC50 > 50 μM. Additional experiments were performed to probe
the antimalarial activity of these derivatives.
ANTI-MALARIAL ACTIVITY
■
All of the compounds were evaluated for growth inhibition
activity against P. falciparum using a SYBR green assay as
reported in the literature.27 Most compounds from both series
with Ki values for PfTMPK below 50 μM inhibited parasite
growth. A number of compounds gave low micromolar inhibition
of growth, the actives belonging almost exclusively to the α-
thymidine analogue series (Table 1). The most potent
compounds were 20, 26, 27 and 28, which had EC50 values of
approximately 2 μM. They were all 4′-substituted phenyl
compounds, which was the most active subset of this series of
inhibitors in producing inhibition of parasite growth. All of the
tested inhibitors showed good selectivity between malaria
parasites and human MRC5 cells (Table 1). The β-thymidine
derivatives had very low or no activity against the parasite (Table
1), and we therefore focused our attention on the α-derivatives.
There was only a low correlation between inhibition of
PfTMPK and inhibition of the growth of P. falciparum. Most
compounds were more potent inhibitors of parasite growth than
inhibitors of the enzyme. For example, compound 17 (Figure 4)
has a Ki of 166 μM against Pf TMPK, but an EC50 of 23 μM
against the parasite suggesting that the principal mode of action is
not through inhibition of PfTMPK. These compounds showed
good selectivity between the malaria parasite and human MRC5
Compound 17 is a representative of the 5′-phenyl urea α-
thymidine derivatives (Figure 4). Compound 17 can be divided
into three components: the α-anomeric thymidine base, the
deoxyribose ring and the substituted phenyl urea. The initial SAR
showed that a substitution of the 5′-phenyl urea gave a significant
increase in the activity against the parasite. For example, a para-
methoxy group on the phenyl increased potency against the
parasite 10-fold (compound 26, Figure 4). Work was carried out
to optimize the phenyl group in the 5′-position, and the
corresponding β-thymidine analogues were also evaluated for
comparison purposes. Compounds were prepared as shown in
Scheme 2 and tested against the parasite (Table 2).
Since the 4-benzyloxy derivatives appeared to give very potent
activity (compound 60), this series was expanded by preparing 4-
benzyloxy phenyl isocyanates using the conditions reported by
Knaggs et al.28 with triphosgene. The isocyanates were rapidly
passed through a column for purification and then reacted with
α- or β-thymidine amine to give the final compounds 84−90
(Scheme 3 and Table 3).
ANTI-MALARIAL STRUCTURE−ACTIVITY
RELATIONSHIP
■
Data for the antiparasite assays are given in Tables 1−3.
Compounds 57, 60, 63, 66, 84, 85, 86, 87 and 89 showed sub-
micromolar activity. Most notably, compound 84 had an EC50 of
28 nM, which represents an increase in inhibition of
approximately 1000-fold compared to the starting compound
17 (EC50 = 23 μM). We can summarize the key trends as follows:
• α-Anomers are much more potent than β-anomers. Five of
Figure 4. Two 5′-phenyl urea α-thymidine derivatives.
the most active pairs of derivatives are compared in Table
E
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a
Table 2. Evaluation of 5′-Phenyl (thio)urea α- and β-Thymidine Derivatives against P. falciparum and MRC5 Cells
a
Reference compound: chloroquine EC50 = 0.007 μM.
F
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a
Scheme 3. Preparation of 4-Benzyloxy-phenyl Urea α- and β-Thymidine Derivatives
a
(a) NaH, N-Boc protected 4-aminophenol, DMF; (b) TFA, DCM; (c) Et3N, triphosgene, EtOAC; (d) DMF, RT.
DMPK Studies. To assess the further developability of the
compounds, we carried out in vitro DMPK studies on five key
compounds (Table 6).29 All showed reasonable microsomal
stability (CLi < 5 mL/min/g) except for compound 84, which
exhibited rapid turnover. Plasma protein binding was less than
95% in most cases, with compound 63 showing a particularly
large unbound fraction. Unfortunately the most potent
compound 84 was the most unstable when incubated with
hepatic microsomes. However, related compounds showed good
in vitro DMPK properties (57, 60, 63, 66), suggesting that there
is nothing inherently problematic associated with the scaffold in
terms of microsomal stability and protein binding.
4 which shows that α- had at least 10 times more activity
than β-derivatives, presumably due to the α-anomer
constraining the base in a much more favorable
orientation. Interestingly compound 84, which has an
ortho substitution in the benzyl group, gave the best
antimalarial activity with an EC50 of 28 nM, and the related
β-derivative 89 also gave the best inhibition activity of the
β-derivatives, albeit with a 20-fold drop in activity.
• A para-substitution gives enhanced activity. The phenyl
urea is optimally substituted in the para-position rather
than the ortho or meta positions for both the α- and β-
anomers. For example, 56 (2-phenyl, EC50 = 96 μM) is
much less active than 57 (4-phenyl, EC50 = 0.29 μM)
(Table 2). Most of the active compounds in this study are
para-substituted derivatives, for both α- and β- derivatives
of the same pairs.
• Ureas are more potent than thiourea. In the α-derivatives,
thiourea and urea derivatives were investigated for their
contribution to the inhibitory activity. Four pairs of
inhibitors were chosen (Table 5). Urea derivatives are
more active than thioureas except for the pair containing
the 3-trifluoromethyl-4-chloro substituent, which is
slightly more active for the thio-urea (28 and 29).
• The para-substituent of the phenyl urea should be
preferably a hydrophobic group. For the α-derivatives,
improved activity is observed when the para-subsituents
are hydrophobic groups. This is evident in comparisons of
66 (4-phenyloxy, EC50 = 0.24 μM) versus 62 (4-
tetrahydropyran-oxy, EC50 = 1.3 μM); 63 (4-piperidine,
EC50 = 0.34 μM) versus 67 (4-morpholino, EC50 = 35
μM) or 64 (4-(4-methyl-piperazine), EC50 = 29 μM).
CONCLUSION
■
In this study, we optimized a series of 5′-para substituted phenyl
urea α-thymidine derivatives to produce compounds with
improved antimalarial activity. Initially different series of
compounds were designed as inhibitors of PfTMPK. While
inhibition of the enzyme and parasite growth was moderate, the
elucidation here of the crystal structure of several enzyme−
inhibitor complexes provides the basis for a future structure-
based drug discovery program.
Interestingly, while poorly active against the enzyme, the α-
thymidine derivatives showed promising antimalarial activity and
optimization was performed in order to increase potency. Several
features were found to contribute to antiplasmodial action. The
α-thymidine derivatives with para substituted phenyl groups
(preferably hydrophobic substitutents) and ureas (better than
thiourea) exhibited increased growth inhibition. Testing of the
inhibitors gave activities in the nanomolar range and compounds
showed a good selectivity between P. falciparum and human
G
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Table 3. Biological Evaluation of 4-Benzyloxy-phenyl Urea α-
and β-Thymidine Derivatives
Table 4. Comparison in Parallel of the α- and β-Urea
Derivatives
a
a
a
Reference compound: chloroquine EC50 = 0.007 μM.
Table 5. Comparison in Parallel of the Thiourea and Urea α-
a
Derivatives
a
Reference compound: chloroquine EC50 = 0.007 μM.
MRC5 cells. The most potent inhibitor from this series is
compound 84 with an EC50 of 28 nM and CC50 of 29 μM, an
increase in potency of nearly 1000 times compared to the starting
compound 17 (EC50 = 23 μM). Furthermore some of the most
active compounds have reasonable microsomal stability and free
fractions. The resulting SAR information obtained for this series
of inhibitors is shown in Figure 5.
EXPERIMENTAL SECTION
Chemistry. General. Chemicals and solvents were purchased from
■
the Sigma-Aldrich Chemical Co., Fluka, VWR, Acros, Fisher Chemicals
1
and Alfa Aesar. H NMR and 13C NMR were recorded on a Bruker
Avance DPX 500 spectrometer (1H at 500.1 MHz and 13C at 125.8
MHz). Chemical shifts (δ) are expressed in ppm. Signal splitting patters
are described as singlet (s), double (d), double doublet (dd), triplet (t),
quarter (qt), multiplet (m). Low resolution electrospray (ES) mass
spectra were recorded either on an Agilent Technologies 1200 series
HPLC connected to an Agilent Technologies 6130 quadrupole
spectrometer and to an Agilent diode array detector or on a Bruker
MicroTof mass spectrometer, run in a positive ion mode, using either
methanol, methanol/water (95:5), or water/acetonitrile (1:1) + 0.2%
formic acid as the mobile phase. High resolution electrospray
measurements were performed on a Bruker Daltonics MicrOTOF
mass spectrometer. Column chromatography was carried out using silica
gel 60 from Fluka. Thin layer chromatography (TLC) was carried out on
Merck silica gel 60 F254 plates using UV light or PMA for visualization.
Purity was determined using both LCMS and NMR spectroscopy.
Compounds had a purity of >95%.
a
Reference compound: chloroquine EC50 = 0.007 μM.
mixture was allowed to stir at room temperature for 3 h. After the
completion of the reaction, the reaction mixture was evaporated to dry
(ethanol and toluene were used to coevaporate), and the residue was
purified by column chromatography to yield the compounds 84−90 as a
solid with the yields ranging from 67% to 83%.
General Procedure for Compounds 84−90. For the synthesis of
compounds 84−90, amine 8 or 33 (1 equiv) was dissolved in DMF at 0
°C. The coupling reagents (1.1 equiv) were added, and the reaction
N-(5′-Deoxy-α-thymidin-5′-yl)-N′-(4-(2-chlorobenzyloxy)phenyl)-
urea 84. 4-(2-Chlorobenzyloxy)phenyl isocyanate reacted with amine 8
to yield compound 84 as a solid; 1H NMR (500 MHz, DMSO): δ 11.28
H
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(m, 2H, H1′ and NH), 5.45 (d, J = 3.25 Hz, 1H, OH), 5.01 (s, 2H, CH2),
4.16−4.18 (m, 2H, H3′ and H4′), 3.75 (s, 3H, CH3), 3.22−3.32 (m, 1H,
H5′), 3.15−3.27 (m, 1H, H5′), 2.57 (m, 1H, H2′), 1.94 (m, 1H, H2′),
1.78 (s, 3H, CH3); 13C NMR (125 MHz, DMSO): δ 163.8 (C4), 155.4
(CO), 150.5 (C2), 136.9 (C6), 159.3, 152.9, 138.9, 129.5, 119.2, 114.9,
113.1, 113.0 (C-Ph), 108.8 (C5), 87.0 (C1′), 84.6 (C4′), 70.8 (C3′),
69.2 (CH2), 55.0 (CH3), 41.0 (C5′), 39.0 (C2′), 12.3 (CH3); LCMS
(ES+): m/z (%) 497 (100) [M + H]+; HRMS (ES+): calcd for
C25H29N4O7 [M + H]+ 497.2031 m/z, found 497.2031 m/z (−0.12
ppm).
Table 6. The Stability and Plasma Protein Binding Data of
Five Selected Compounds
a
microsomal stability
(mL/min/g)
plasma protein binding
(% bound)
EC50
(μM)
CC50
(μM)
cmpd
84
57
60
63
66
6
94.1
92.0
81.2
52.3
90.5
0.028
0.29
0.24
0.34
0.24
29
1.9
3
>50
>50
>50
43.6
0.5
1.8
N-(5′-Deoxy-α-thymidin-5′-yl)-N′-(4-(4-tertbutylbenzyloxy)-
a
Reference compound: chloroquine EC50 = 0.007 μM.
phenyl)urea 88. 4-(4-tert-Butylbenzyloxy)phenyl isocyanate reacted
1
with amine 8 to yield compound 88 as a solid; H NMR (500 MHz,
DMSO): δ 11.23 (s, 1H, NH), 8.37 (s, 1H, NH), 7.77 (d, J = 1.20 Hz,
1H, H6), 7.67−7.76, 7.26−7.41, 6.77−6.89 (m, 8H, H-Ph), 6.16−6.19
(m, 2H, H1′ and NH), 5.45 (d, J = 3.20 Hz, 1H, OH), 4.99 (s, 2H, CH2),
4.18−4.21 (m, 2H, H3′ and H4′), 3.21−3.28, 3.09−3.13 (m, 2H, H5′),
2.52−2.59, 1.91−1.98 (m, 2H, H2′), 1.78 (s, 3H, CH3), 1.28 (s, 9H,
CH3); LCMS (ES+): m/z (%) 523 (100) [M + H]+; HRMS (ES+): calcd
for C28H35N4O6 [M + H]+ 523.2551 m/z, found 523.2554 m/z (−0.57
ppm).
ASSOCIATED CONTENT
* Supporting Information
■
Figure 5. Summary of the SAR data for the thymidine-derived
S
inhibitors.
Methodology for enzyme assays, parasite assays, crystallography,
DMPK and chemistry. Stereo views of several protein−ligand
complexes. This material is available free of charge via the
Internet at http://pubs.acs.org.
(s, 1H, NH), 8.40 (s, 1H, NH), 7.78 (d, J = 1.20 Hz, 1H, H6), 7.57−7.60,
7.50−7.53, 7.37−7.40, 7.29−7.31, 6.90−6.94 (m, 8H, H-Ph), 6.16−6.19
(m, 2H, H1′ and NH), 5.45 (d, J = 3.20 Hz, 1H, OH), 5.09 (s, 2H, CH2),
4.18−4.21 (m, 2H, H3′ and H4′), 3.21−3.28, 3.09−3.13 (m, 2H, H5′),
2.53−2.60, 1.91−1.97 (m, 2H, H2′), 1.78 (s, 3H, CH3); 13C NMR (125
MHz, DMSO): δ 163.8 (C4), 155.4 (CO), 150.5 (C2), 136.9 (C6),
152.8, 134.6, 132.5, 130.0, 129.7, 129.3, 127.3, 119.3, 114.9 (C-Ph),
108.8 (C5), 86.9 (C1′), 84.7 (C4′), 70.8 (C3′), 67.0 (CH2), 55.0 (C5′),
41.0 (C5′), 39.0 (C2′), 12.3 (CH3); LCMS (ES+): m/z (%) 501 (100)
[M + H]+; HRMS (ES+): calcd for C24H26Cl1N4O6 [M + H]+ 501.1535
m/z, found 501.1533 m/z (0.54 ppm).
N-(5′-Deoxy-α-thymidin-5′-yl)-N′-(4-(3-chlorobenzyloxy)phenyl)-
urea 85. 4-(3-Chlorobenzyloxy)phenyl isocyanate reacted with amine 8
to yield compound 85 as a solid; 1H NMR (500 MHz, DMSO): δ 11.28
(s, 1H, NH), 8.38 (s, 1H, NH), 7.78 (d, J = 1.20 Hz, 1H, H6), 7.37−7.42,
7.28−7.30, 6.89−6.91 (m, 8H, H-Ph), 6.16−6.19 (m, 2H, H1′ and NH),
5.45 (d, J = 3.25 Hz, 1H, OH), 5.06 (s, 2H, CH2), 4.16−4.20 (m, 2H,
H3′ and H4′), 3.12−3.22 (m, 1H, H5′), 2.57 (m, 1H, H2′), 1.92−1.95
(m, 1H, H2′), 1.78 (s, 3H, CH3); 13C NMR (125 MHz, DMSO): δ
163.8 (C4), 155.3 (CO), 150.5 (C2), 136.9 (C6), 152.7, 136.9, 133.9,
133.0, 130.0, 127.6, 127.2, 126.1, 119.3, 115.0 (C-Ph), 108.8 (C5), 86.9
(C1′), 84.7 (C4′), 70.8 (C3′), 68.4 (CH2), 41.0 (C5′), 39.0 (C2′), 12.3
(CH3); LCMS (ES+): m/z (%) 501 (100) [M + H]+; HRMS (ES+):
calcd for C24H26Cl1N4O6 [M + H]+ 501.1535 m/z, found 501.1533 m/z
(0.54 ppm).
N-(5′-Deoxy-α-thymidin-5′-yl)-N′-(4-(4-chlorobenzyloxy)phenyl)-
urea 86. 4-(4-Chlorobenzyloxy)phenyl isocyanate reacted with amine 8
to yield compound 86 as a solid; 1H NMR (500 MHz, DMSO): δ 11.28
(s, 1H, NH), 8.37 (s, 1H, NH), 7.77 (d, J = 1.20 Hz, 1H, H6), 7.44−7.45,
7.27−7.29, 6.88−6.90 (m, 8H, H-Ph), 6.16−6.19 (m, 2H, H1′ and NH),
5.45 (d, J = 3.30 Hz, 1H, OH), 5.03 (s, 2H, CH2), 4.16−4.18 (m, 2H,
H3′ and H4′), 3.75 (s, 3H, CH3), 3.22−3.32 (m, 1H, H5′), 3.15−3.27
(m, 1H, H5′), 2.57 (m, 1H, H2′), 1.92−1.94 (m, 1H, H2′), 1.78 (s, 3H,
CH3); 13C NMR (125 MHz, DMSO): δ 163.8 (C4), 155.3 (CO), 150.5
(C2), 136.9 (C6), 152.7, 136.4, 133.8, 132.2, 129.4, 128.4, 119.2, 115.0
(C-Ph), 108.8 (C5), 86.9 (C1′), 84.6 (C4′), 70.8 (C3′), 68.9 (CH2), 41.0
(C5′), 39.0 (C2′), 12.3 (CH3); LCMS (ES+): m/z (%) 501 (100) [M +
H]+; HRMS (ES+): calcd for C24H26Cl1N4O6 [M + H]+ 501.1535 m/z,
found 501.1519 m/z (3.33 ppm).
AUTHOR INFORMATION
Corresponding Author
■
*Tel: +44 1382 386240. For correspondence on medicinal
chemistry: (I.G.) i.h.gilbert@dundee.ac.uk. For correspondence
on enzyme inhibition: (D.G.-P.) dgonzalez@ipb.csic.es. For
correspondence on crystallography: (K.W.) keith.wilson@york.
ac.uk.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The research leading to these results has received funding from
AntiMal, an FP6-funded integrated project under contract
number LSHP-CT-2005-0188, the RICET FIS Network
́
(RD06/0021) and the Junta de Andalucia (BIO-199, P09-CVI-
5367). We would also like to acknowledge the support of the
Wellcome Trust (Grant 083481). We would like to thank
Suzanne Norval for the DMPK study. The authors thank the
ESRF for access to beamlines and support by staff during visits.
ABBREVIATIONS USED
■
CC50, concentration required to reduce growth of MRC5 cells by
50%; CLi, intrinsic clearance; TDP, thymidine diphosphate;
TMP, thymidine monophosphate; hTMPK, human thymidylate
kinase; Pf , Plasmodium falciparum; Mt, Mycobacterium tuber-
culosis; TMPK, thymidylate kinase; TMSOTf, trimethylsilyl
trifluoromethanesulfonate
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