Structure-activity relationships of C6-uridine derivatives targeting Plasmodia orotidine monophosphate decarboxylase

Article abstract of DOI:10.1021/jm7010673

Malaria, caused by Plasmodia parasites, has re-emerged as a major problem, imposing its fatal effects on human health, especially due to multidrug resistance. In Plasmodia, orotidine 5′-monophosphate decarboxylase (ODCase) is an essential enzyme for the de novo synthesis of uridine 5′-monophosphate. Impairing ODCase in these pathogens is a promising strategy to develop novel classes of therapeutics. Encouraged by our recent discovery that 6-iodo uridine is a potent inhibitor of P. falciparum, we investigated the structure-activity relationships of various C6 derivatives of UMP. 6-Cyano, 6-azido, 6-amino, 6-methyl, 6-N-methylamino, and 6-N,N-dimethylamino derivatives of uridine were evaluated against P. falciparum. The mononucleotides of 6-cyano, 6-azido, 6-amino, and 6-methyl uridine derivatives were studied as inhibitors of plasmodial ODCase. 6-Azidouridine 5′-monophosphate is a potent covalent inhibitor of P. falciparum ODCase. 6-Methyluridine exhibited weak antimalarial activity against P. falciparum 3D7 isolate. 6-N-Methylamino and 6-N,N-dimethylamino uridine derivatives exhibited moderate antimalarial activities.

Full text of DOI:10.1021/jm7010673

J. Med. Chem. 2008, 51, 439–448
439
Structure–Activity Relationships of C6-Uridine Derivatives Targeting Plasmodia Orotidine
Monophosphate Decarboxylase†
Angelica M. Bello,‡ Ewa Poduch,‡ Yan Liu,§ Lianhu Wei,‡ Ian Crandall,4, Xiaoyang Wang,‡ Christopher Dyanand,‡,#
,#,O
Kevin C. Kain,4, Emil F. Pai,§, and Lakshmi P. Kotra*,‡,
Center for Molecular Design and Preformulations and DiVision of Cell and Molecular Biology, Toronto General Research Institute/UniVersity
Health Network, MaRS/TMDT, 101 College Street, Toronto, Ontario M5G 1L7, Canada, Departments of Pharmaceutical Sciences and
Chemistry, UniVersity of Toronto, Toronto, Ontario, Canada, DiVision of Cancer Genomics and Proteomics, Ontario Cancer Institute/
UniVersity Health Network, MaRS/TMDT, 101 College Street, Toronto, Ontario M5G 1L7, Canada, Departments of Biochemistry, Medical
Biophysics, and Molecular Genetics, 1 King’s College Circle, Toronto, Ontario M5S 1A8, Canada, Tropical Disease Unit, DiVision of
Infectious Diseases, Department of Medicine, UHN-Toronto General Hospital and the UniVersity of Toronto and McLaughlin-Rotman Center/
UHN, McLaughlin Center for Molecular Medicine, UniVersity of Toronto, Toronto, Ontario, Canada, and Department of Chemistry and
Biochemistry, The UniVersity of North Carolina at Greensboro, Greensboro, North Carolina 27412
ReceiVed August 28, 2007
Malaria, caused by Plasmodia parasites, has re-emerged as a major problem, imposing its fatal effects on
human health, especially due to multidrug resistance. In Plasmodia, orotidine 5′-monophosphate decarboxylase
(ODCase) is an essential enzyme for the de novo synthesis of uridine 5′-monophosphate. Impairing ODCase
in these pathogens is a promising strategy to develop novel classes of therapeutics. Encouraged by our
recent discovery that 6-iodo uridine is a potent inhibitor of P. falciparum, we investigated the structure–activity
relationships of various C6 derivatives of UMP. 6-Cyano, 6-azido, 6-amino, 6-methyl, 6-N-methylamino,
and 6-N,N-dimethylamino derivatives of uridine were evaluated against P. falciparum. The mononucleotides
of 6-cyano, 6-azido, 6-amino, and 6-methyl uridine derivatives were studied as inhibitors of plasmodial
ODCase. 6-Azidouridine 5′-monophosphate is a potent covalent inhibitor of P. falciparum ODCase.
6-Methyluridine exhibited weak antimalarial activity against P. falciparum 3D7 isolate. 6-N-Methylamino
and 6-N,N-dimethylamino uridine derivatives exhibited moderate antimalarial activities.
Introduction
new classes of drugs against new targets is an important effort
in the fight against malaria as well as against other emerging/
re-emerging infectious diseases.
In the past few years, malaria attracted considerable attention
in new drug discovery initiatives worldwide.1 This infection is
becoming a major burden on human health because drug-
resistant strains of the malaria parasite are increasingly prevalent
in most endemic areas, and current vaccine trials are not very
encouraging.2–4 While the majority of cases are now found in
sub-Saharan Africa, it is predicted that global warming and
demographic changes will lead to an increase in the distribution
of clinical malaria cases in the Western world, including Europe
and North America.5 Existing drug pipelines, most of which
are driven by public-private ventures, contain either derivatives
or combinations of existing drugs.1–3,5 Thus, the discovery of
Malaria is caused by the protozoa Plasmodia, and P.
falciparum (Pf) is the most prevalent human parasite with
approximately 70% of the human cases. It also causes the most
lethal and severe form of malaria. Other human malaria parasites
include P. ViVax (PV), P. oVale, and P. malariae. More than
95% of worldwide infections are caused by Pf and PV alone.6
Plasmodia species including Pf and PV are dependent on their
own de novo synthesis of pyrimidine nucleotides due to the
absence of the salvage pathway in these parasites.7–9 Therefore,
if enzymes in this de novo pathway are inhibited in a plasmodial
parasite, its only supply of pyrimidine nucleotides will be
impaired. In humans, however, pyrimidine nucleotides are
replenished via both the de novo and salvage pathways.10 This
makes inhibitors of de novo pyrimidine biosynthesis in Plas-
modia quite attractive as novel therapeutic agents. In addition,
the increasing prevalence of multidrug-resistant malaria parasites
underscores the need for new drugs based on new molecular
mechanisms and interacting with new targets, to complement
the current therapeutic options.5,6,11–15
†
PDB IDs for the coordinates of the complexes of ODCase with 6-iodo-
UMP, 6-amino-UMP and 6-azido-UMP are 2QAF, 2Q8Z, and 3BAR,
respectively.
* To whom correspondence should be addressed. Address: #418 New
Science Building, The University of North Carolina at Greensboro, Dept.
of Chemistry and Biochemistry, Greensboro, NC 27402. Tel.: (336) 334-
9862. Fax: (416) 581-7621. E-mail: lpkotra@uncg.edu.
‡
Toronto General Research Institute/University Health Network.
Departments of Biochemistry, Medical Biophysics, and Molecular
§
Orotidine 5′-monophosphate decarboxylase (ODCasea) plays
a central role in the de novo synthesis of UMP, which in turn
serves as the substrate for the synthesis of other pyrimidine
nucleotides. ODCase catalyzes the decarboxylation of orotidine
monophosphate (OMP, 1) to uridine 5′-monophosphate (UMP,
2, Chart 1).16–18 This enzyme seems to be ubiquitous, present
from bacteria, archaea, and parasites to higher species, including
mammals and man, with viruses being the only exception. In
the past three decades, ODCase has been investigated as a drug
target, especially against various infectious diseases and
Genetics.
4
UHN-Toronto General Hospital and the University of Toronto and
McLaughlin-Rotman Center/UHN.
McLaughlin Center for Molecular Medicine.
Departments of Pharmaceutical Sciences and Chemistry, University
#
of Toronto.
Ontario Cancer Institute/University Health Network.
O
The University of North Carolina at Greensboro.
aAbbreviations: ODCase, orotidine 5′-monophosphate decarboxylase;
UMP, uridine 5′-monophosphate; OMP, orotidine 5′-monophosphate; BMP,
barbiturate ribonucleoside 5′-monophosphate; TCEP HCl, tris(2-carboxy-
ethyl)phosphine hydrochloride.
10.1021/jm7010673 CCC: $40.75
2008 American Chemical Society
Published on Web 01/12/2008

440 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Chart 1. Structures of ODCase Substrate and Inhibitors
Bello et al.
elimination of the 6-iodo moiety.26 Prompted by these findings,
we studied the structure–activity relationships of various C6-
substituted uridine nucleotides as inhibitors of two Plasmodia
ODCases and the potential of their nucleoside analogs as
antimalarial agents. Here we present the design and the synthesis
of several C6 derivatives of uridine and uridine 5′-monophos-
phate, inhibition profiles against Plasmodia ODCases, structural
interactions of the novel inhibitors with ODCases and their
antiplasmodial activities.
Experimental Section
General. All anhydrous reactions were performed under a
nitrogen atmosphere. All solvents and reagents were obtained from
commercial sources; anhydrous solvents were dried following
standard procedures. Column chromatography was performed using
silica gel (60 Å, 70–230 mesh). NMR spectra were recorded on a
1
Varian spectrometer (300 and 400 MHz for H, 75 and 100 MHz
for 13C, and 121.46 MHz for 31P). The chemical shifts are reported
in δ ppm using tetramethylsilane (TMS) as the reference for the
1H NMR spectra and phosphoric acid as an external standard for
the 31P spectrum. All ODCase enzyme assays were performed at
55 or 37 °C using a VP-ITC microcalorimeter (MicroCal, Northamp-
ton, MA) using previously published procedures.24 Mass spectra
for the enzymes and the complexes were obtained in a Q-TOF mass
spectrometer with MassLynx software for data analysis (Waters
Micromass, Manchester, U.K.) at the Mass Spectrometry Facility,
Advanced Protein Technology Centre, The Hospital for Sick
Children, Toronto, Canada, and an AB/Sciex QStar mass spec-
trometer with an ESI source and on a an Agilent 1100 capillary
LC attached (MDS Sciex, U.S.) at the Mass Spectrometry-AIMS
Laboratory, Department of Chemistry, University of Toronto.
Chart 2. Structures of C6-Uridine Derivatives
Synthesis. 6-Cyano-uridine (8) and 6-Cyano-uridine 5′-O-
Monophosphate Ammonium Salt (6). These compounds were
synthesized as reported earlier.23
6-Azido-uridine (9), 6-Azido-uridine 5′-O-Monophosphate
Ammonium Salt (14) and 6-Amino-uridine 5′-O-Monophos-
phate Ammonium Salt (15). These compounds were synthesized
as reported earlier.24
cancer.19–21 The mononucleotide derivatives of 6-aza-uridine
(3), barbiturate ribonucleoside (4), and pyrazofurin (5) are
some of the inhibitors tested against ODCase
(Chart 1).20,22 The development of historic ODCase inhibitors
as therapeutics did not gain much momentum due to their
toxicities, lack of specificity, and the limited understanding of
their mode of action.20
6-Amino-uridine (10). 6-Azido-uridine (9; 0.2 g, 0.7 mmol) was
dissolved in 5 mL of 50% aqueous methanol and 10% Pd/C (5
mg) was added. The reaction mixture was stirred in the dark for
2 h under a hydrogen atmosphere at room temperature and
atmospheric pressure. Then the reaction was filtered through a Celite
pad and the solvent was evaporated to dryness to yield compound
10 as a light yellow solid in 94% yield (185 mg). UV (H2O) λmax
Our group recently reported novel inhibitors of ODCase that
also interact via novel mechanisms. In this context, 6-cyano-
uridine 5′-monophosphate (6, Chart 1) was investigated as a
potential bioisosteric inhibitor of Methanobacterium thermoau-
totrophicum (Mt) ODCase using X-ray crystallography and
enzymology.23–25 ODCase catalyzed the unusual conversion of
the chemically stable 6-cyano-uridine 5′-monophosphate (6) into
barbiturate ribonucleoside 5′-monophosphate (BMP), with a
half-life of 5 h, via what can be categorized as a “pseudo-
hydrolysis” process.23 In this unusual enzymatic conversion, no
covalent interaction between the residues in the catalytic site
of ODCase and the substrate was observed.23,24 6-Cyano-uridine
5′-monophosphate (6) also exhibited noncovalent, competitive
inhibition of Mt ODCase activity with a Ki of 29 ( 2 µM.24
We also recently discovered the first covalent inhibitor of
ODCase, 6-iodo-uridine 5′-monophosphate (6-iodo-UMP, 7),
and reported that its nucleoside analog exhibits potent antima-
larial activities.26 This compound 7 inhibited Mt ODCase
irreversibly in a time-dependent fashion and 6-iodo-uridine, the
nucleoside form of 7, inhibited Pf in cell-based assays, with an
IC50 of 4.4 ( 1.3 and 6.2 ( 0.7 µM (ItG and 3D7 isolates,
respectively).26 A cocrystal structure of the product of the suicide
inhibitor 6-iodo-UMP (7), bound in the active site of Mt
ODCase, clearly identified a covalent bond between Lys72 and
the C6 atom on the pyrimidine ring, accompanied by the
1
) 271 nm; ꢀ271 18301; H NMR (D2O) δ 6.39 (d, J ) 7.5 Hz,
1H), 4.83 (s, 1H), 4.61 (t, 6.9 Hz, 1H), 4.23 (dd, J ) 3.3 and 6 Hz,
1H), 3.98 (m, 1H), 3.78 (m, 2H); 13C NMR (D2O) δ 166.2, 158.7,
152.9, 90.0, 86.9, 71.5, 71.2, 61.8, 30.7; HRMS (ESI, +ve) calcd
for C9H13N3O6Na+ (MNa+), 282.0696; found, 282.0690.
6-Methyl-uridine (11). A stirred solution of LDA (0.6 mL, 1.2
mmol, 2.0 M solution in THF) in anhyd THF (2 mL) was treated
with compound 19 (0.25 g, 0.63 mmol) dissolved in anhyd THF
(1.5 mL) at -78 °C. After stirring for 1 h, methyl iodide (0.63
mmol) in dry THF (2 mL) was added and the mixture was stirred
for an additional 5 h at -78 °C. The reaction was quenched with
glacial AcOH (0.3 mL), then brought to room temperature and
dissolved in ethyl acetate (25 mL). The organic layer was washed
with saturated NaHCO3 solution (10 mL) and brine (10 mL) and
dried (Na2SO4). Evaporation of the solvent and purification of the
crude by column chromatography (hexanes-ethyl acetate, 70:30)
yielded the fully protected 20 as a foamy white solid.
A stirred aqueous suspension of compound 20 (300 mg, 0.73
mmol) was then treated with 50% aqueous TFA (3 mL) at 0 °C
then brought to room temperature and stirred for an additional 2 h.
Evaporation of solvent and purification of the crude by column
chromatography (10–15% EtOH/CHCl3) provided compound 11
as a white solid (169 mg, 90% yield). UV (H2O) λmax ) 261 nm;
1H NMR (D2O) δ 5.78 (s, 1H), 5.68 (d, J ) 3.2 Hz, 1H), 4.84 (dd,

Structure-ActiVity Relationships of C6-Uridine DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 441
J ) 4 and 6 Hz, 1H), 4.41 (t, J ) 6 Hz, 1H), 4.01–3.96 (m, 1H),
3.91 (dd, J ) 3.2 and 12 Hz, 1H), 3.77 (dd, J ) 6 and 12 Hz),
2.41 (s, 3H); HRMS (ESI, +ve) calcd for C10H15N3O6Na+ (MNa+),
296.0853; found, 296.0842.
(14) was determined after the coinjection of the substrate and the
inhibitor. The concentrations of 6-azido-UMP (14) in the assay
samples were 0.25, 0.50, and 1.0 µM.
Irreversible Inhibition Studies. The Mt ODCase was incubated
in the absence and presence of an inhibitor (for example, 6-azido-
UMP, 14) for up to 48 h at room temperature. The control reaction
contained 20 µM ODCase in 50 mM Tris (pH 7.5), 20 mM DTT,
and 40 mM NaCl. The inhibition reactions contained 20, 40, or 60
mM of the inhibitor. The remaining enzyme activity was measured
after 0.3, 1, 2, 4, 8, 12, 24, and 48 h after the initiation of the
enzyme reaction. A single injection of 5 mM substrate (11.4 µL)
provided the initial concentration of the substrate OMP in the
reaction cell at 40 µM. The inactivation of ODCase by 6-azido-
UMP without incubation was also determined following a coin-
jection of the substrate and the inhibitor into the enzyme solution.
Enzyme samples were prepared in degassed 50 mM Tris buffer
containing 1 mM DTT. The final enzyme concentration was 20
nM. Inhibitor was added to the sample cell in a single 22.8 µL
injection together with the substrate, and the enzyme activity was
recorded. The final substrate concentration in the 1.3 mL cell was
40 µM. The inhibitor concentration after a single injection was 0,
0.25, 0.50, 1, 3, or 6 µM.
Pf ODCase. Pf ODCase stock was prepared in 50 mM Tris (pH
7.5), 10 mM DTT, and 20 mM NaCl and was incubated overnight
at room temperature. The reversible inhibition of ODCase was
assayed after the dilution of a 2.5 µL aliquot of the concentrated
enzyme and an appropriate volume of concentrated inhibitor in 50
mM Tris, containing 1 mM DTT to a final volume of 2.5 mL. The
enzyme activity of Pf ODCase was measured after a single injection
of the substrate.
Reversible Inhibition Studies. The inhibition of Pf ODCase by
6-cyano-UMP (6), 6-aza-UMP (18), and 6-methyl-UMP (16) was
evaluated using competitive inhibition assays. The mixture of the
enzyme and the inhibitor was prepared from the stock solutions
and the samples were immediately transferred into the calorimeter
cell. The concentration of the enzyme was 60 nM for the inhibition
assays using 6-cyano-UMP (6) and 6-aza-UMP (18), and was 100
nM with 6-methyl-UMP (16). The final concentrations of the
inhibitor in the reaction cell were 25, 50, and 75 µM for 6-cyano-
UMP (6) and 1, 2, 3, 5, 7.5, and 10 µM for 6-aza-UMP (18). In
the case of 6-methyl-UMP (16), the concentrations of the inhibitor
in the sample cell were 20, 150, and 250 µM. The substrate
concentration was 12 µM after a single injection of OMP. The
reversible inhibition assays with 6-amino-UMP (15) and 6-azido-
UMP (14) were conducted with 30 nM Pf ODCase. The final
substrate concentration was 10 µM. The enzyme was exposed to
the final concentrations of 1, 2.5, 3.5, and 5 µM of 6-amino-UMP
(15) or 2, 5, 10, and 15 µM of 6-azido-UMP (14).
Time-Dependent Inhibition Studies. Pf ODCase (50 µM) was
incubated in the presence of 12.5, 25.0, and 50.0 mM of 6-cyano-
UMP (6). All samples were prepared in 50 mM Tris (pH 7.5), 20
mM DTT, and 40 mM NaCl and incubated at room temperature.
The control reaction contained no inhibitor. The samples were
incubated for 0, 0.3, 1, 2, 4, 8, 12, 24, and 48 h. The remaining
enzyme activity at each time point was measured by using 2.5 µL
aliquots of the reaction mixture, which were diluted 1000-fold with
50 mM Tris containing 1 mM DTT. The final substrate concentra-
tion was 12 µM.
6-N-Methylamino Uridine (12). 6-Iodo uridine26 (21; 200 mg,
0.54 mmol) was dissolved in 20 mL of anhyd ethanol and then
methylamine (0.60 mL, 2.16 mmol, soln 33 wt % in absolute EtOH)
and triethylamine (1 mL) were added dropwise. The reaction
mixture was stirred for 3 h at room temperature until compound
21 was consumed. The solvent was evaporated under vacuum and
the product was purified by column chromatography (CHCl3/MeOH,
9:1) to yield compound 12 as a pale yellow solid (73 mg, 50%
1
yield). UV (H2O) λmax ) 272 nm; H NMR (CD3OD) δ 7.89 (s,
1H), 6.45 (d, J ) 7.6 Hz, 1H), 4.69 (s, 1H), 4.48 (dd, J ) 6.4 and
7.6 Hz, 1H), 3.99 (m, 1H), 3.78 (m, 2H), 2.74 (s, 3H); HRMS
(ESI, +ve) calcd for C10H15N3O6Na+ (MNa+), 296.0853; found,
296.0842.
6-N,N-Dimethylamino Uridine (13). Compound 13 was syn-
thesized using the method described for compound 12, starting from
6-iodo uridine 21 (100 mg, 0.27 mmol) and 0.55 mL of 2 M
dimethylamine solution in THF (1.1 mmol) to yield compound 13
(70 mg, 90% yield). UV (H2O) λmax ) 285 nm; 1H NMR (CD3OD)
δ 5.70 (d, J ) 4 Hz, 1H), 5.09 (s, 1H), 6.67 (dd, J ) 4 and 6 Hz,
1H), 4.29 (t, J ) 6 Hz, 1H), 3.86 (m, 1H), 3.79 (dd, J ) 12 and
2.8 Hz, 1H), 3.67 (dd, 12 and 5.6 Hz, 1H), 2.85 (s, 6H); HRMS
(ESI, +ve) calcd for C11H17N3O6Na+ (MNa+), 310.1009; found,
310.1012.
6-Methyl-uridine 5′-O-Monophosphate (16). A stirred solution
of water (34 mg, 1.89 mmol) and POCl3 (0.277 mL, 2.973 mmol)
in anhyd acetonitrile (3 mL) was treated with pyridine (0.261 mL,
3.24 mmol) at 0 °C and stirred for 10 min. Compound 11 was added
(185 mg, 0.675 mmol) and the mixture was stirred for an additional
5 h at the same temperature. The reaction mixture was quenched
with 25 mL of cold water and the stirring was continued for an
additional hour. The evaporation of the solvent and the purification
of the crude by column chromatography (Dowex ion-exchange basic
resin, 0.1 M formic acid) yielded compound 16 as a syrup. The
mononucleotide derivative was then converted into the correspond-
ing ammonium salt by neutralization with 0.5 M NH4OH solution
at 0 °C and lyophilized to yield the corresponding ammonium salt
as a white powder (140 mg, 56% yield). UV (H2O) λmax ) 260
nm; ꢀ260 (H2O) ) 2271; 1H NMR (D2O) δ ppm 5.75 (s, 1H), 5.67
(d, J ) 6 Hz, 1H), 4.63 (dd, J ) 6, 7 Hz, 1H), 4.44 (t, J ) 7 Hz,
1H), 4.14–4.02 (m, 2H), 4.00–3.92 (m, 1H), 2.39 (s, 3H); HRMS
(ESI, positive) calcd for C10H15N2O9PH+ (MH+), 339.0587; found,
339.0587.
Enzymology. Cloning, Expression, and Purification of
ODCases. The ODCase enzymes from Mt and Pf were produced
as described earlier.26,27
General Methods for Enzyme Assays. All enzymatic assays
were performed on a VP-ITC calorimeter (MicroCal, MA). Tris
buffer (50 mM, pH 7.5) was prepared using Millipore water and
filtered through a membrane (0.45 µm) to remove any fine particles.
The substrate stock solution and the inhibitor solutions were
prepared using 50 mM Tris and diluted as required. All solutions
were degassed for 5 min using a Thermovac vacuum degasser
(MicroCal, MA). The control reaction contained no inhibitor. The
enzyme activity was determined either at 37 or 55 °C for Pf and
Mt ODCases, respectively. The assays were performed using the
single injection method, where the ligand is injected into the reaction
cell containing the enzyme solution. The inhibitor was either
injected together with the substrate into the enzyme solution or
mixed with the enzyme. In the second scenario, the reaction was
initiated by the addition of substrate only.
Mt ODCase. Reversible Inhibition Studies. The final concen-
tration of the enzyme in all assays was 20 nM and that of the
substrate was 40 µM. The inhibition of Mt ODCase by 6-methyl-
UMP (16) was recorded in the presence of 50, 150, 350, and 500
µM of the inhibitor. The enzyme was mixed with the inhibitor in
50 mM Tris and 1 mM DTT, and the reaction was initiated by the
addition of the substrate. The reversible inhibition by 6-azido-UMP
The inhibition of ODCase due to 6-azido-UMP (14) was
monitored in a time-dependent assay. The control reaction contained
30 µM of the enzyme in 50 mM Tris (pH 7.5), 20 mM DTT, and
83 mM NaCl. The incubation samples contained 30 µM Pf ODCase
and 0.4, 1.0, 5.0, or 10.0 mM of 14 in 50 mM Tris (pH 7.5), 20
mM DTT, and 83 mM NaCl. The samples were incubated at room
temperature for 0.5, 1, 2, 4, 8, 12, 24, and 48 h. At each incubation
time point, 2.5 µL aliquots of the control and reaction samples were
removed from the incubation mixtures and diluted to 2.5 mL with
50 mM Tris buffer containing 1 mM DTT. The remaining activity
of Pf ODCase in the presence of the inhibitor was measured after
a single 5 µL injection of 2.85 mM OMP into the sample cell. The
final substrate concentration was 10 µM.

442 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Bello et al.
PW ODCase. Competitive Inhibition Assay. The enzyme was
mixed with various concentrations of the inhibitor in 50 mM Tris
containing 1 mM DTT to a final volume of 2.5 mL. The activity
of PV ODCase (50 nM) was monitored in the presence of 40, 75,
and 150 µM of 6-cyano-UMP (6). The inhibition assay for 6-methyl-
UMP (16) was performed using 50, 150, and 250 µM inhibitor
and 70 nM ODCase enzyme. The final concentration of the substrate
was 12 µM in the reaction cell after a single injection of OMP into
the calorimetric sample cell.
Time-Dependent Assay. The time-dependent inhibition of PV
ODCase was studied at 25 °C. The reaction and the control samples
contained PV ODCase (50 µM) in 50 mM Tris (pH 8.0), 20 mM
DTT, and 10 mM NaCl. The initial concentrations of the inhibitor
(added at t ) 0) were 37.5, 50, and 75 mM, corresponding to 750-,
1000-, and 1500-fold molar excess of the inhibitor (6-cyano-UMP,
6). Small aliquots (2.5 µL) were withdrawn from the incubation
samples and diluted to 2.5 mL with the reaction buffer (50 mM
Tris, 1 mM DTT, pH 8). The enzyme activity was measured in the
inhibition reaction sample and the control after 1, 2, 4, 8, 12, 24,
36, and 48 h of incubation. The enzyme activity was recorded after
a single injection of the substrate into the sample solution. A 6 µL
injection of OMP (2.85 mM) gave a final substrate concentration
of 12 µM.
Mass Spectral Analyses. The control reaction with Pf ODCase
(300 µM) was prepared in 20 mM Tris buffer (pH 8) containing
10 mM DTT and 20 mM NaCl. Samples contained 300 µM of the
enzyme, 6 mM 6-azido-UMP (14), 20 mM Tris (pH 8), 10 mM
DTT, and 20 mM NaCl. Mass spectral analyses for samples at t )
0 s were conducted immediately before mixing the inhibitor with
the enzyme. The control sample and the mixture of Pf ODCase
with 6-azido-UMP (14) were incubated at room temperature in the
dark for 72 h, and the mass spectral analyses were repeated.
Data Analyses. The data were analyzed using the “Enzyme
Assay” module available from the Origin 7.0 data analysis package.
First the raw data were adjusted for the instrument response time.
The baselines were set to zero using the “Baseline” function, and
the data were truncated to remove the initial baseline, dilution heat,
and the final baseline. The remaining data representing the
enzymatic decarboxylation of OMP in the absence or in the presence
of a reversible inhibitor were transformed into Michaelis–Menten
plots by fitting them to the equation
Vi
kobs
[P] )
[1 - exp(-kobst)]
(3)
where [P] represents the product concentration, Vi is the initial
reaction rate, and t is the time. The calculated kobs and the inhibitor
concentrations [I] were used to calculate the inactivation constant
KI and the rate of inactivation kinact from the equation
kinact[I]
kobs
)
(4)
KI + [I]
The kinetic parameters for the inactivation of Pf ODCase were
derived using the data points for up to 4 h of incubation with the
inhibitor. The remaining enzyme activity was calculated for each
incubation time point as a percentage of the control sample. The
natural logarithm of % enzyme activity was plotted against time to
obtain the observed rate of enzyme inactivation (kobs) at each
inhibitor concentration. Then kobs and inhibitor concentrations were
fitted to the eq 4 to calculate KI and kinact
.
P. falciparum and CHO Cell Inhibition Assays. P. falciparum
cultures were grown in O+ blood obtained by venipuncture of
volunteers. The cultures of the laboratory line ItG were maintained
by the method of Trager and Jensen29 using RPMI 1640 supple-
mented with 10% human serum (a kind gift obtained under ethical
consent from the Chemo Day Care Department, Princess Margaret
Hospital, Toronto, Canada) and 50 µM hypoxanthine (RPMI-A).
The assays comparing the antiplasmodial activities of the inhibitors
were performed using the SYBR-Green method.30 Briefly, the
inhibitors were dissolved in DMSO to achieve a concentration of
10 mg/mL. RPMI-A (50 µL) was added to each well in a 96-well
plate before 40 µL of RPMI-A and 10 µL of the inhibitor solution
were added to the first well, the contents of the well were mixed,
50 µL were removed and added to the next well in the series, and
the process was repeated until the next-to-last well was reached.
This produced a plate with a series of 2-fold dilutions across it,
except for the last well in the series, which contained RPMI-A
alone. The parasite culture (50 µL, 2% hematocrit, 2% parasitemia)
was added to each well and the plates were incubated at 37 °C in
95% N2, 3% CO2, and 2% O2 for 48 h. Relative fluorescence was
determined using a Fluostar Optima plate reader (BMG Labtech,
Offenburg, Germany). The IC50 values of individual compounds
were determined using a nonlinear regression analysis of the data
using the computer program SigmaPlot (Jandel Scientific). The IC50
values represent the mean ( SD, n ) 4.
kcat[E][S]
V )
(1)
KM + [S]
The CHO cells (ATCC, Manassas, VA) were grown in RPMI-
1640 supplemented with 10% fetal calf serum (Sigma, St. Louis,
MO), 25 mM HEPES, and gentamicin (RPMI-10). The cells were
seeded in 96-well plates and were grown to 50% confluency in
100 µL of RPMI-10 per well prior to the addition of either DMSO
alone or a test compound dissolved in DMSO to a concentration
of 10 mg/mL. The inhibitor solutions were prepared by adding 90
µL of RPMI-10 mixed with 10 µL of inhibitor solution to the first
well in the series, mixing, transferring 100 µL to the next well,
and repeating until the next-to-last well was reached. After 48 h,
the media in the wells were discarded, 100 µL of 10 mg/mL of
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT,
Sigma, St. Louis, MO) in RPMI-10 was added, and the plates were
incubated for an additional hour. The viability of the cells was
determined by removing the media followed by the addition of 100
µL of DMSO and reading the absorbance at 650 nm.31 The IC50
values of individual compounds were determined, applying a
nonlinear regression analysis of the dose–response curve using the
computer program SigmaPlot (Jandel Scientific).
Crystallographic Analyses. All Pf ODCase concentrations were
determined using a BioRAD protein assay kit and BSA as a
standard. ODCase protein was dissolved in 20 mM Tris (pH 8.5),
10 mM NaCl, and 1 mM TCEP-HCl. Pf ODCase complex crystals
were grown in hanging drops using ∼30% PEG1000, pH 7.0–9.0,
as the main precipitant. To obtain larger single crystals of the
enzyme complexes, microseeding was performed the following day.
All crystals grew in space group P21212, with unit cell dimensions
The curves were averaged for each set of samples. Several data
points representing the reaction rate at specific substrate concentra-
tions were extracted from the Michaelis–Menten curves and were
used to generate a Dixon plot.24,28 The inhibition constant Ki was
established for each inhibitor as the intersection point on the x-axis
from the plots of [I] vs rate-1. For the time-dependent inhibition
of ODCase, the remaining enzyme activity in the incubation sample
was determined as a percentage of the control at each time point.
The half-time of the enzyme inhibition for the assay with
6-cyano-UMP was calculated using the equation for a single
exponential decay.
A ) A0e-kt
(2)
where A represents 50% of enzyme activity, A0 is the initial activity
at time 0 (extrapolated from the nonlinear least-squares fitting of
the data to eq 2, k is the rate constant, and t represents the time
required to inhibit 50% of the enzyme activity.
Time-Dependent Inhibition. The remaining enzyme activity was
determined for both Mt and Pf ODCase enzymes using 6-azido-
UMP (14) as a percentage of the activity of the control. To
determine the inactivation of Mt ODCase after the coinjection of
substrate and inhibitor, kobs was first computed from each progress
curve. The value (Power, µcal/sec) at each inflection point was
normalized by setting the inflection point value to zero. The data
representing the change in power over time were fitted to the
following equation to calculate kobs

Structure-ActiVity Relationships of C6-Uridine DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 443
Table 1. Inhibition Kinetics of C6-Substituted UMP Derivatives against
ODCases from P. falciparum (Pf), P. ViVax (PV), and M.
thermoautotrophicum (Mt)
forded the mononucleotide 16. Compound 16 was transformed
into its ammonium salt by neutralization with 0.5 M NH4OH
solution.
Mt
Ki (µM)
Pf
Ki (µM)
PV
Ki (µM)
These C6-substituted UMP derivatives were investigated as
antiplasmodial agents following the discovery that C6 substitu-
tions on UMP exhibit potent ODCase inhibition and confer
potent antiplasmodial activities.24,26 Previously, compound 6
exhibited interesting activities against Mt ODCase and was
converted into BMP by this enzyme. Additionally, 6-cyano-
UMP (6) inhibited this enzyme competitively with moderate
potency.24 Thus, compound 6 was of interest for evaluation
against Pf and PV ODCases. Compound 14 carries an azido
moiety at the C6 position, a substitution with one atom “longer”
than the nitrile moiety and is linear in shape. It is also a
reasonably good leaving group similar to the iodo moiety. The
6-methyl derivative 16 addressed the question what effect a
small hydrophobic moiety at this position has on ODCase
inhibition and antiplasmodial activity.
cmpd
6-CN-UMP (6)
6-N3-UMP (14)
6-NH2-UMP (15)
6-Me-UMP (16)
6-Aza-UMP (18)
29 ( 2
26 ( 2
2.0 ( 0.1
2.1
34.1 ( 0.4
1.1 ( 0.03
62 ( 5
ND
ND
22.4 ( 0.7
ND
0.19 ( 0.01
0.84 ( 0.025
134 ( 5
12.4 ( 0.7
Time-Dependent Inactivation of Pf ODCase
6-N3-UMP (14)
(KI and kinact
0.63 ( 0.11 µM
7.3 ( 0.4 µM
ND
)
61.2 h-1
0.35 h-1
deviating less than 1% from a ) 80.8 Å, b ) 83.1 Å, and c ) 90.0
Å. For data collection, the crystals were cryo-protected by Paro-
tone-N oil before flash-freezing in a stream of boiling nitrogen.
Diffraction data for the crystals of Pf ODCase cocrystallized with
both 6-amino-UMP (15) and 6-iodo-UMP (7) were collected at 100
K and λ ) 0.9002 Å on beamline 14BM-C BioCARS, APS, and
those for the 6-azido-UMP (14) exposed crystals at 100 K and λ )
0.97934 Å on beamline 08ID-1 at the Canadian Macromolecular
Crystallography Facility, Canadian Light Source, Inc. Data were
reduced and scaled using HKL2000.32 Data collection statistics are
given in Table 2. The structures of all complexes were determined
using molecular replacement techniques with the help of the
program package MOLPREP;33 subsequent refinements were done
with Refmac-5.234 and model building used COOT.35 The refine-
ment statistics are also given in Table 2. Atomic coordinates and
structure factors have been deposited into the Protein Data Bank
(PDB IDs: 2QAF, 2Q8Z, and 3BAR).
Computer Modeling. Computer modeling was performed using
the SYBYL suite of software on an SGI workstation at the Center
for Molecular Design and Preformulations at University Health
Network, Toronto.36 The structure of the complex of 6-methyl-
UMP (16) and Pf ODCase was modeled by mutating the 6-amino
group to a 6-methyl group in the X-ray crystal structure of the
complex of 6-amino-UMP (15) bound to Pf ODCase. Similarly,
Pf ODCase complexes with 6-methylamino-UMP or 6-dimethyl-
amino-UMP structures were modeled by mutating one or two
hydrogen atoms on the 6-amino group of compound 15 with one
or two methyl groups, respectively. Conformations of the N-
methylamino and N,N-dimethylamino moieties were positioned in
the best possible orientation within the binding site with minimal
steric crowding.
First, all nucleotide derivatives 6, 14-16, and 18 were first
evaluated for their inhibitory potential against Pf ODCase,
followed by the PV and Mt enzymes. 6-Aza-UMP (18), which
is a classic inhibitor of ODCases, provided excellent comparative
values for Pf ODCase. While compound 18 was a moderate
inhibitor of Mt ODCase, with a Ki of 12.4 ( 0.7 µM, when
challenged with Pf ODCase, it exhibited stronger inhibition
potential (Ki ) 1.1 ( 0.03 µM). However, this is still
approximately 15-fold less potent than its effect on the yeast
enzyme.24 6-Cyano-UMP (6) showed a typical competitive
inhibition pattern, as was observed with Mt ODCase in earlier
studies.24 However, when 6-cyano-UMP (6) was incubated with
Pf ODCase for several hours, it was transformed into BMP (4),
as had been observed with Mt ODCase (Figure 3B).23 The time
required for the enzyme to lose 50% of its activity was 7, 5,
and 3 h in the presence of 12.5, 25, and 50 mM of 6-cyano-
UMP, and such loss of activity is due to the conversion of
6-cyano-UMP into BMP. The inhibition constant (Ki) against
Pf ODCase for 6 in the competitive assay was 29 ( 2 µM,
which is very close to that with Mt ODCase (26 ( 2 µM; Figure
3A and Table 1). However, compound 6 exhibited a slightly
weaker inhibition against PV ODCase, with a Ki of 62 ( 5 µM
(Table 1). 6-Methyl-UMP (17) is a competitive inhibitor with
moderate potency against Pf and PV ODCases (Ki ) 34.1 (
0.4 and 22.4 ( 0.7 µM, respectively; Figure 3C).
6-Amino-UMP (15) and 6-azido-UMP (14) exhibited quite
interesting inhibition profiles (Figure 3D, and E, respectively).
Compound 15 inhibited Mt ODCase competitively, with a Ki
of 840 ( 25 nM. This compound, however, showed slightly
less potency against Pf ODCase, with a Ki of approximately
2.1 ( 0.0 µM (Table 1). Potent inhibition due to 6-amino-UMP
(15) is perhaps expected based on the well-characterized
inhibition pattern of ODCases by BMP, which carries a hydroxyl
moiety at the C6 position.24
Results and Discussion
All nucleoside and mononucleotide derivatives (6, 8–13, and
14-16) were synthesized from the fully protected uridine
derivative 19. The 6-cyano derivatives 6 and 8 were synthesized
according to a previous report.23 6-Amino-uridine (10) was
synthesized by catalytic hydrogenation of 6-azido-uridine (9)
at room temperature for 3 h (Scheme 1A).24 6-Amino-UMP (15)
was similarly obtained by the reduction of 6-azido-UMP (14).24
Compounds 12 and 13 were synthesized by the displacement
of the iodo moiety from 6-iodo-uridine (23) with methylamine
and dimethylamine, respectively, in ethanol at room temperature
(Scheme 1B). Our attempts to phosphorylate 6-N-methylamino-
uridine (12) and 6-N,N-dimethylamino-uridine (13) were not
successful and products of decomposition were observed.
6-Methyl-UMP (16) was synthesized according to Scheme 1C.
The methyl group at the C6 position was introduced onto the
fully protected uridine 19 first by treating with lithium diiso-
propylamide followed by methyl iodide. The deprotection of
20 with trifluoroacetic acid yielded 6-methyl-uridine (11),
followed by phosporylation with phosphorus oxychloride af-
We earlier reported 6-iodo-UMP (7) as the first covalent
inhibitor of ODCase and showed that an irreversible enzyme
complex with the inhibitor was observed using mass spectro-
metry (Pf ODCase), enzymology (Pf ODCase), and X-ray
crystallography (Mt ODCase).26 In the present studies, 6-azido-
UMP (14) was identified as another covalent inhibitor for
ODCase, exhibiting a potent, covalent inhibition of the enzyme
(Figures 1C,D, 3E,F, and 4B). When initial rates were moni-
tored, compound 14 inhibited Mt and Pf ODCases, with Ki )
0.19 ( 0.01 and 2.0 ( 0.1 µM, respectively. In a time-dependent
assay, 6-azido-UMP (14) inactivated Mt and Pf ODCases, with
an equilibrium inhibition constant (KI) of 628 ( 110 nM and

444 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Bello et al.
Table 2. Data Collection and Refinement Statistics for the X-ray Crystal Structures of the Complexes of ODCase with the Inhibitors 7, 14, and 15a
Pf ODCase +
6-I-UMP (7)
Pf ODCase +
6-N3-UMP (14)
Pf ODCase +
6-NH2-UMP (15)
Diffraction Data
resolution (Å)
1.95 (1.98–1.95)
203475
43303
95.6 (98.0)
10.2 (49.2)
P21212
1.90 (1.97–1.90)
241881
46532
95.5 (97.9)
8.5 (45.9)
P21212
1.80 (1.83–1.80)
266845
54102
94.6 (97.0)
9.7 (43.5)
P21212
measured reflections (n)
unique reflections (n)
completeness (%)
Rsym (%)
space group
molecules in asymmetric unit (n)
2
2
2
Refinement Statistics
resolution (Å)
protein atoms (n)
28.30–1.95 (2.00–1.95)
5461
26.0–1.90 (1.95–1.90)
5670
24.57–1.80 (1.84–1.80)
5478
water molecules (n)
252
257
247
reflections used for Rfree (n)
Rwork (%)
Rfree (%)
root mean square deviation bond length (Å)
root mean square deviation bond angle (°)
avg B-factor (Å2)
2191 (152)
17.13 (18.7)
22.51 (24.7)
0.010
1.665
19.84
2357 (167)
17.2 (19.6)
21.9 (25.4)
0.010
1.487
22.1
2752 (366)
16.7 (19.1)
21.2 (27.6)
0.010
1.500
22.27
a Numbers in brackets are for the highest resolution shells. Rsym ) S|I - <I>|/SI, where I is the observed intensity and <I> is the average intensity from
multiple observations of symmetry-related reflections.
Scheme 1. Synthesis of ODCase Inhibitorsa
(7) showed that the iodo moiety was lost and a covalent bond
was formed between Nꢀ of Lys138 and C6 of the inhibitor, as
was previously observed with Mt ODCase. The same result was
obtained when 6-azido-UMP (14) was used instead; again,
the X-ray crystal structure (1.90 Å resolution) confirmed the
covalent bond formation (Figure 1C,D). In both cases, the
pyrimidine moiety engages in three strong hydrogen bonding
interactions with the ODCase binding site, specifically between
Nꢀ of Gln269 and O2 of the ligand, the hydroxyl moiety of
Thr195 and N3 of the ligand, and from the backbone NH of
Thr195 to O4 of the ligand (bond lengths of 2.83, 2.75, and
2.92 Å, respectively). This pattern is similar to that found with
other noncovalently bound ligands.23,27 The 5′-monophosphate
portion of the ligand, which is responsible for most of the
binding energy, interacts via five tightly bound water molecules
and the critical Arg294 residue in the Pf ODCase binding site.
The ribosyl moiety is held via a number of interactions,
including residues from the second subunit of the dimer. The
2′-hydroxyl moiety shares three hydrogen bonds with the side
chains of Asp141B (Oγ), Thr145B (Oγ), and Asn104 (Nꢀ; bond
lengths of 2.44, 2.83, and 2.80 Å, respectively); the 3′-hydroxyl
moiety interacts with the side chains of Lys102 (Nꢀ), Asp23
(Oγ), and Asn104 (Oγ; bond lengths of 3.45, 2.76 and 2.94,
a Reagents and conditions: (a) LDA/THF, CH3I, -78 °C; (b) 50% TFA/
Å, respectively) via hydrogen bonds (Figure 1A,C).
H2O, 0–25 °C; (c) POCl3, Py, H2O, CH3CN, 0 °C; (d) H2, Pd/C, MeOH,
Mass spectral analyses for the reactions of 6-iodo-UMP (7)
and 6-azido-UMP (14) with Pf ODCase revealed that identical
rt; (e) CH3NH2, EtOH, rt; (f) NH(CH3)2, EtOH, rt.
covalent adducts are formed as expected (Figure 4). Thus, the
7.3 ( 0.4 µM, respectively. The first-order rates of inactivation
(kinact) were 61.2 h-1 and 0.35 h-1, respectively. 6-Iodo-UMP
reaction between 7 and Pf ODCase showed a peak at 40042
daltons (Figure 4A, peak 1), which is equivalent to the sum of
(7) inhibited Pf ODCase with a second-order rate constant (kobs
/
[I]) of 680000 M-1sec-1. On the other hand, when Pf ODCase
was challenged with 6-azido-UMP (14), the second-order rate
constant (kinact/KI) for the inactivation of the enzyme was 13.2
M-1sec-1, indicating that fewer molecules of 6-azido-UMP were
forming a covalent bond at the active site Lys138 than had been
observed with 7.
the average mass of Pf ODCase (39720 dalton, inset in Figure
4A, peak 2) and 7 without the iodo moiety (323 dalton). This
resulting mass additionally reflects the loss of two protons from
the Lys138 residue, which is engaged in a covalent bond with
C6 of the ligand. These results parallel those obtained with Mt
ODCase.26 A separate study using 6-azido-UMP 14 and Pf
ODCase generated an identical adduct with an average mass of
40042 dalton (Figure 4B, Peak 4), which is equivalent to the
major adduct observed after the reaction between 6-iodo-UMP
and Pf ODCase.
High-resolution X-ray crystallography experiments with the
inhibitors 7 and 14 revealed in detail the interactions of these
inhibitors with the active site of ODCase. Crystal structures of
Pf ODCase incubated with 6-iodo-UMP (7) and 6-azido-UMP
(14) were almost identical, both revealing covalent enzyme–in-
hibitor complexes (Figure 1 and Table 2). The crystal structure
(1.95 Å resolution) of Pf ODCase incubated with 6-iodo-UMP
The three-dimensional (3D) structure (1.8 Å resolution) of
the complex between 6-amino-UMP (15) and Pf ODCase
confirmed that the complementary interactions at the C6 position

Structure-ActiVity Relationships of C6-Uridine DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 445
Figure 1. X-ray crystal structures of the complexes generated by incubating Pf ODCase with 6-iodo-UMP (A and B), 6-azido-UMP (C and D),
and 6-amino-UMP (E and F). Panels A and C depict the covalent ligand resulting from 6-iodo-UMP and 6-azido-UMP (7 and 14, respectively),
panel E shows 6-amino UMP (15) bound to the active site of ODCase; inhibitor molecules and the covalently bound residue Lys138 (in A and C)
are shown in capped-stick representation and the other protein residues are shown in line representation. Covalent bonds between the enzyme and
the inhibitors are highlighted by blue arrows. Panels B, D, and F depict the electron densities (2Fo - Fc) corresponding to the inhibitor molecules,
displayed at 1σ level. Enzyme backbones are rendered according to their secondary structures and the ligands are shown in capped-stick representation.
on the pyrimidine moiety are necessary for potent inhibition
(Figure 1E,F). The hydrogen bonding interactions with the
pyrimidine moiety at positions O2, N3, and O4 were similar to
those observed with 6-iodo-UMP (Figure 1E). The charged 5′-
monophosphate moiety exhibited the well-characterized interac-
tions with five water molecules and the Arg294 residue. The
ribosyl moiety was held in a conformation similar to that with
7 as a result of (i) the interactions of the 2′-hydroxyl moiety
with the side chains of Asp141B, Thr145B, and Asn104 and
(ii) the hydrogen bonding interactions of the 3′-hydroxyl moiety
with the side chains of Lys102, Asp23, and Asn104. Interest-
ingly, the 6-amino moiety in 15 is in close proximity to an active
site water molecule (N7-HOH265 ) 2.58 Å) and the Nꢀ moiety
of Lys138 (N7-Nꢀ of Lys138 ) 2.37 Å). It also interacts with
the side chain of Asp136 (Figure 1E). These strong interactions
of the 6-amino moiety (N7) in compound 15 are contributing
to the potent inhibition of Pf ODCase.
Structures of the complexes of 6-methylamino-UMP, 6-di-
methylamino-UMP, and 6-methyl-UMP (16) with Pf ODCase
were modeled based on the structure of the complex of Pf
ODCase and 6-amino-UMP to shed light on the potential
interactions between these compounds and the enzyme active
site. Although 6-N-methylamino-UMP and 6-N,N-dimeth-
ylamino-UMP could not be synthesized, we were nevertheless
interested in modeling these compounds in the binding site of
ODCase because the corresponding nucleoside derivatives (12
and 13) exhibited weak antimalarial activities (Table 3). Most
likely, compounds 12 and 13 were phosphorylated by kinases
in vivo, transforming them into the inhibitors of Pf ODCase,

446 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Bello et al.
Figure 2. Computational models of the 3D structures of the complexes of Pf ODCase with 6-methyl-UMP, 16 (Panel A), and 6-N-methylamino-
UMP (Panel B).
Figure 3. Kinetics profiles of Pf ODCase when challenged with 6-CN-UMP, 6-methyl-UMP, 6-amino-UMP, and 6-azido-UMP (6, 16, 15, and 14,
respectively). (A) Dixon plot for the competitive inhibition of Pf ODCase by 6-cyano-UMP (6). (B) Time-dependent inhibition of Pf ODCase by
6-cyano-UMP (6). (C) Competitive inhibition of Pf ODCase by 6-methyl-UMP (16). (D) Nonlinear least-squares fit of the raw data for the competitive
inhibition of Pf ODCase by 6-amino-UMP (15). (E) Competitive inhibition of Pf ODCase by 6-azido-UMP (14). (F) Nonlinear least-squares fit of
the kobs vs [I] for the inhibition of Pf ODCase by 6-azido-UMP (14).
although we could not chemically synthesize the mononucle-
otides of 12 and 13.
When the 6-methyl group was present on the pyrimidine
of UMP, a hydrophobic pocket formed by the residues
Tyr149, Thr163, Val164, Ile166, and Leu191 and adjacent
to the critical catalytic residues was occupied by this small
hydrophobic group (Figure 2A). This provided a reasonable
explanation for the moderate inhibition of Pf ODCase by
6-methyl-UMP (16). Similarly, 6-methylamino and 6-di-
methylamino moieties could easily be accommodated into

Structure-ActiVity Relationships of C6-Uridine DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 447
( 10 µM, respectively. Compound 11 carrying the 6-methyl
moiety exhibited a very weak activity against the P. falciparum
3D7 isolate, with an IC50 of 530.5 ( 0.1 µM. None of the other
compounds exhibited significant activity, including 6-azido-
uridine (9). 6-Iodo-uridine 5′-monophosphate, which is the most
potent covalent inhibitor of Pf ODCase, exhibited inhibition of
several strains of P. falciparum (L. Kotra and K. Kain,
unpublished results).26 This is in distinct contrast to what was
found with 6-azido-uridine, which, despite the fast covalent
inhibition of ODCase by its mononucleotide derivative, did not
show any activity against Plasmodia cultures. The most probable
explanation for this somewhat surprising result is that compound
9, unlike 6-iodo-uridine, is not effectively phosphorylated by
cellular kinases. All nucleoside derivatives 9–13 were also
evaluated for their cellular toxicities. Compound 11 was well
tolerated by the CHO cells in the toxicity assays, and the IC50
was more than 1162 µM. Compounds 12 and 13 inhibited CHO
cells with IC50s in the range of 68–72 µM, a value only about
3-fold higher than the concentrations required for the inhibition
of P. falciparum cultures. The monophosphate derivatives 6 and
14–16 were not evaluated for their antimalarial activities in cell-
based assays because these compounds are highly polar
mononucleotides.
In summary, 6-azido, 6-amino, 6-methyl, 6-N-methylamino,
and 6-N,N-dimethylamino derivatives of uridine were synthe-
sized and evaluated for their activity against the P. falciparum
pathogen. In addition, the inhibitory potential of several of the
corresponding mononucleotides against their enzymic target,
ODCase, was measured. UMP derivatives exhibited moderate
to potent inhibitory activities against Pf ODCase, including the
covalent inhibition of ODCase by 6-azido-UMP (14). Specif-
ically, 6-N-methylamino and 6-N,N-dimethylamino uridine
derivatives (12 and 13, respectively) exhibited moderate activi-
ties against the Pf 3D7 pathogen. For these latter compounds,
however, moderate toxicities were also observed in a CHO cell
assay, but compound 11 exhibited low toxicity in the same
system. Interactions of various C6-substituted UMP derivatives
with Pf ODCase indicated that both polar and hydrophobic
groups can be accommodated at the C6 position of the aromatic
ring of mononucleotide inhibitor molecules tightly bound in the
active site of ODCase. However, monophosphorylation of the
nucleoside derivatives in cell-based assays was unpredictable,
thus, the cellular activities of these nucleoside derivatives may
not necessarily reflect the inhibition patterns of ODCase by the
corresponding mononucleotides. This particular observation is
not unique to ODCase nucleoside inhibitors. Various anticancer
and antiviral nucleoside inhibitors were subjects of similar
limitations.37 Thus, the investigation of new ODCase inhibitors
as antiplasmodial compounds using structure-based design
should be complemented with cell-based assays for optimal drug
design.
Figure 4. Mass spectral analyses of the complexes of Pf ODCase with
6-iodo-UMP, 7 (panel A), and with 6-azido-UMP 14 (panel B) after
72 h incubation at room temperature. A shift in the peak of Pf ODCase
from 39720.0 daltons to 40042.0 daltons (peaks 2 and 1, panel A) is
due to the formation of the Pf ODCase covalent complex with inhibitor
7 but without the iodo moiety and the loss of a proton (322.0 daltons).
Inset in panel A shows the mass spectrum for Pf ODCase alone, as a
control experiment (Peak 2). Panel B shows peak 3 (39720.0 daltons)
that corresponds to uncomplexed Pf ODCase and peak 4 (40042.0
daltons) representing the covalent complex between Pf ODCase and
14 but without the azido moiety and an additional proton.
Table 3. Antimalarial Activities of C6-Substituted Uridine Derivatives
against P. falciparum (3D7) and their Toxicity in CHO Cells
Pf (3D7)
(µM)
CHO cells
(µM)
cmpd
6-iodo-uridine (ref 26)
6-CN-uridine (8)
6-N3-uridine (9)
6-NH2-uridine (10)
6-Me-uridine (11)
6-NHMe-uridine (12)
6-N(Me)2-uridine (13)
6.2 ( 0.7
446 ( 35
1226 ( 161
>1500
530.5 ( 0.1
28.5 ( 8.9
31.7 ( 10.0
366 ( 45
>1500
>1500
>1500
>1162
71.9 ( 5.3
68.3 ( 11.4
this region (Figure 2B). The moderate binding potencies of
these compounds may be the result of balancing moderate
entropic gains generated by accommodating hydrophobic C6-
ligands in a hydrophobic pocket and the loss of binding
interactions with the polar/ionic catalytic residues. However,
these entropic gains may be important for efficient inhibition
of the enzyme activity when a sufficiently high concentration
of the inhibitor is present (vide infra).
The nucleoside derivatives 9–13 were evaluated for their
antimalarial activities against the cultures of P. falciparum 3D7,
as well as for their cellular toxicities using the Chinese Hamster
Ovary (CHO) cells (Table 3). 6-N-Methylamino uridine and
6-N,N-dimethylamino uridine (12 and 13, respectively) exhibited
moderate antiplasmodial effects with an IC50 of 28 ( 9 and 32
Acknowledgment. The authors thank Ms. Wing Lau for help
with the expression and purification of ODCase enzymes and
Professor David Clarke for generously allowing the use of the
ITC instrument. The authors thank Dr. Junquan Wang for the
synthesis of 6-methyl-uridine. This work was supported by
grants from the Canadian Institutes of Health Research (E.F.P.
and L.P.K.), a CIHR Team Grant in Malaria (K.C.K.), an
operating grant MT-13721 (K.C.K.), Genome Canada (K.C.K.),
and the Canadian Genetics Disease Network (CGDN; K.C.K.),
PSI (K.C.K.). L.P.K. is a recipient of an Rx and D HRF-CIHR
research career award. An infrastructure grant from the Ontario
Innovation Trust provides support for the Molecular Design and
Information Technology Centre (MDIT). E.F.P. and K.C.K.

448 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Bello et al.
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acknowledge support through the Canada Research Chairs
program. We thank the staff at BioCARS, sector 14 beamlines
at the Advanced Photon Source, Argonne National Laboratories,
for their generous time commitments and support. Use of the
Advanced Photon Source was supported by the Basic Energy
Sciences, Office of Science, United States Department of
Energy, under Contract W-31-109-Eng-38. Use of the BioCARS
sector 14 was supported by the National Center for Research
Resources, National Institutes of Health, under Grant RR07707.
We also gratefully acknowledge the help we received from the
staff of the Canadian Macromolecular Crystallography Facility,
at the Canadian Light Source, which is supported by NSERC,
NRC, CIHR, and the University of Saskatchewan.
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Supporting Information Available: Details of HPLC purity data
on compounds 11–14. This material is available free of charge via
the Internet at http://pubs.acs.org.
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