Structure-activity relationships of orotidine-5′-monophosphate decarboxylase inhibitors as anticancer agents

Article abstract of DOI:10.1021/jm801224t

A series of 6-substituted and 5-fluoro-6-substituted uridine derivatives were synthesized and evaluated for their potential as anticancer agents. The designed molecules were synthesized from either fully protected uridine or the corresponding 5-fluorourid

Full text of DOI:10.1021/jm801224t

1648
J. Med. Chem. 2009, 52, 1648–1658
Structure-Activity Relationships of Orotidine-5′-Monophosphate Decarboxylase Inhibitors as
Anticancer Agents
Angelica M. Bello,†,‡ Danijela Konforte,†,§ Ewa Poduch,‡ Caren Furlonger,§ Lianhu Wei,‡ Yan Liu, Melissa Lewis,‡
,O
Emil F. Pai,§, Christopher J. Paige,§,| and Lakshmi P. Kotra*,‡,#,
Center for Molecular Design and Preformulations and DiVision of Cellular and Molecular Biology, Toronto General Research Institute,
Toronto General Hospital, Toronto, Ontario M5G 2C4, Canada, Department of Medical Biophysics, Ontario Cancer Institute, Princess
Margaret Hospital, 620 UniVersity AVenue, Toronto, Ontario M5G 2M9, Canada, Department of Immunology, UniVersity of Toronto,
Toronto, Ontario, Canada, Departments of Biochemistry and Molecular Genetics, UniVersity of Toronto, 1 King’s College Circle, Toronto,
Ontario, Canada, Departments of Pharmaceutical Sciences and Chemistry, UniVersity of Toronto, Toronto, Ontario, Canada, McLaughlin
Center for Molecular Medicine, UniVersity of Toronto, Toronto, Ontario, Canada, Department of Chemistry & Biochemistry, The UniVersity of
North Carolina at Greensboro, Greensboro, North Carolina 27412
ReceiVed September 27, 2008
A series of 6-substituted and 5-fluoro-6-substituted uridine derivatives were synthesized and evaluated for
their potential as anticancer agents. The designed molecules were synthesized from either fully protected
uridine or the corresponding 5-fluorouridine derivatives. The mononucleotide derivatives were used for
enzyme inhibition investigations against ODCase. Anticancer activities of all the synthesized derivatives
were evaluated using the nucleoside forms of the inhibitors. 5-Fluoro-UMP was a very weak inhibitor of
ODCase. 6-Azido-5-fluoro and 5-fluoro-6-iodo derivatives are covalent inhibitors of ODCase, and the active
site Lys145 residue covalently binds to the ligand after the elimination of the 6-substitution. Among the
synthesized nucleoside derivatives, 6-azido-5-fluoro, 6-amino-5-fluoro, and 6-carbaldehyde-5-fluoro derivatives
showed potent anticancer activities in cell-based assays against various leukemia cell lines. On the basis of
the overall profile, 6-azido-5-fluoro and 6-amino-5-fluoro uridine derivatives exhibited potential for further
investigations.
Introduction
synthesis of the ribonucleotide, uridine-5′-O-monophosphate
(UMP, 2), from orotidine-5′-O-monophosphate (OMP, 1). This
enzyme catalyzes the decarboxylation of OMP (1) to UMP (2)
(Figure 1). UMP, synthesized de novo from aspartic acid, is a
building block for the synthesis of pyrimidine nucleotides such
as uridine-5′-triphosphate (UTP), cytidine-5′-triphosphate (CTP),
thymidine-5′-triphosphate (TTP), and 2′-deoxycytidine-5′-triph-
osphate (dCTP). During the decarboxylation reaction, ODCase
exhibits an extraordinary level of catalytic rate enhancement
of over 17 orders of magnitude compared to the uncatalyzed
decarboxylation reaction in water at pH 7.0 at 25 °C.3-6 One
interesting difference when one looks at this enzyme across the
species is that in certain higher level organisms such as human,
rat, or mouse, ODCase is a part of the bifunctional enzyme,
UMP synthase.7
Antimetabolites have been a useful tool in the fight against
cancer over the past several decades.1 Most of these drugs such
as 5-fluorouracil and methotrexate impair the synthesis of
nucleotides in the cancer cells, thus seriously impairing the
uncontrolled proliferation of these cells.2 In humans, these
nucleosides, in particular the pyrimidine mononucleotides, are
recruited into the nucleic acid biosynthesis either via the de novo
pathway or the salvage pathway. During the uncontrolled and
fast replication of cells, de novo pathway must also contribute
toward supplying nucleotides despite the existence of salvage
pathway because of the higher demand for nucleotides in a short
period of time. Orotidine-5′-monophosphate decarboxylase
(ODCase,a EC 4.1.1.23) plays a central role in the de novo
* Corresponding Author: Phone: (416) 581-7601or (336) 334-9862. Fax:
(336) 334-5402. E-mail: lkotra@uhnres.utoronto.ca or lpkotra@uncg.edu.
Mailing address: #5-356, Toronto Medical Discoveries Tower/MaRS Center,
101 College Street, Toronto, Ontario M5G 1L7, Canada.
A handful of investigations in the past focused on developing
ODCase inhibitors against cancer, malaria, and RNA viral
infections.8 6-Aza-UMP (8), 6-hydroxy-UMP (or BMP, 9),
Pyrazofurin-5′-monophosphate (12), xanthosine-5′-monophos-
phate (XMP, 13), and 6-thiocarboxamido-UMP (10) are some
of the potent inhibitors that were studied against ODCase (Figure
2).9-11 Although, ODCase did not gain much traction in the
1980s and 1990s as a drug target, its inhibitors were explored
as potential drugs due to its essential role in the de novo
synthetic pathway. For example, Plasmodia species such as
Plasmodium falciparum and Plasmodium ViVax are dependent
on their own de novo synthesis of pyrimidine nucleotides due
to the absence of the salvage pathway in these parasites.12-19
Similarly, ODCase inhibitors were also proposed as potential
antiviral agents, although very limited studies have been carried
out.
†
Authors contributed equally.
Center for Molecular Design and Preformulations and Division of
‡
Cellular and Molecular Biology, Toronto General Research Institute, Toronto
General Hospital.
§
Department of Medical Biophysics, Ontario Cancer Institute, Princess
Margaret Hospital.
|
Department of Immunology, University of Toronto.
Departments of Biochemistry and Molecular Genetics, University of
Toronto.
#
Departments of Pharmaceutical Sciences and Chemistry, University
of Toronto.
McLaughlin Center for Molecular Medicine, University of Toronto.
Department of Chemistry & Biochemistry, The University of North
O
Carolina at Greensboro.
a Abbreviations: ODCase, orotidine 5′-monophosphate decarboxylase;
OMP, orotidine 5′-monophosphate; UMP, uridine 5′-monophosphate; Hs,
Homo sapiens; Mt, Methanobacterium thermoautotrophicum; 5-FU, 5-fluo-
rouracil; PBMC, peripheral blood mononuclear cells; PBS, phosphate buffer
saline.
Inhibitors of ODCase such as 6-aza-uridine (7) and Pyrazo-
furin (11) (Figure 2) exhibited good anticancer activities against
10.1021/jm801224t CCC: $40.75
2009 American Chemical Society
Published on Web 03/04/2009

Orotidine-5′-Monophosphate Decarboxylase Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1649
Figure 1. De novo synthesis of UMP from OMP by ODCase.
Figure 2. Various nucleosides and nucleotides with anticancer and ODCase inhibition activities.
a number of clinical tumor models.20,21 6-Aza-uridine (7) is
transformed into its mononucleotide form, 6-aza-uridine-5′-O-
monophosphate (8), in vivo and the resulting mononucleotide
inhibits ODCase, impairing the de novo production of pyrimi-
dine nucleotides.22 Compound 8 inhibits yeast and Mt ODCases
with the inhibition constants (Ki) of 64 nM and 11 µM,
respectively.23,34 Another potent inhibitor of ODCase, Pyrazo-
furin (11), has been investigated as an anticancer agent including
in clinical trials. Pyrazofurin-5′-O-monophosphate (12), which
is a C-nucleotide, inhibits ODCase with a Ki of 5 nM and its
nucleoside analogue is readily taken up by the cells and
transformed into its monophosphate form.24 In a separate study,
measurement of levels of pyrimidine and purine intermediates
in cultured mouse L1210 leukemia cells showed that 25 µM
Pyrazofurin induced an 8-fold increase in the accumulation of
OMP and an abrupt decrease in the pyrimidine ribosyl mono-
nucleotides.25 Pyrazofurin, however, was not clinically devel-
oped further due to its toxicity to patients in phase I studies.
With the above studies available on ODCase that illustrate the
role and relevance of this target in anticancer drug development,
we became interested in exploring novel ODCase inhibitors
carrying C6 substitutions on the pyrimidine nucleosides as
anticancer agents.
As alluded above, ODCase carries out the decarboxylation
of OMP very efficientlysits only but essential function in the
cell. A number of mechanisms for the decarboxylation of OMP
by ODCase were proposed prior to and after the availability of
its X-ray crystal structures.26-32 Our groups were interested in
a series of C6-substituted pyrimidine nucleotides to investigate
the mechanism of ODCase. These probe molecules present small
substitutions at the C6 position of the uridine moiety, such that
they can sterically and/or electronically mimic the carboxyl
group present on the substrate, OMP.33,34 For example, 6-cyano-
UMP (34) was designed as a potential bioisosteric inhibitor of
ODCase and its interactions with the enzyme were investigated
using X-ray crystallography and enzymology.33,34 ODCase
catalyzed the surprising conversion of the chemically stable
6-cyano-UMP (34) into 6-hydroxy-UMP (BMP, 9), albeit slowly
with a half-life of 5 h, in what can be categorized as a
“pseudohydrolysis” process.33 The conversion of compound 34
into 9 by ODCase established the catalytic promiscuity for this
fascinating decarboxylase.33,34 Compound 34 also exhibited
noncovalent, competitive inhibition of ODCase activity with a
Ki of 29 ( 2 µM.34,35 We also revealed two covalent inhibitors
to ODCase and the potential of these compounds in antimalarial
drug development.35,36 As part of an ongoing program exploring
ODCase inhibitors, we synthesized a series of 6-substituted and
5-fluoro-6-substituted uridine derivatives and evaluated their
potential as anticancer agents. Here, we reveal the medicinal
chemistry of these novel compounds, their anticancer activities,
their ODCase inhibition activities, and the structural determi-
nants for their binding to ODCase in the context of the potential
of ODCase inhibitors for targeting various types of cancer.
Results and Discussion
Nucleosides have played a key role in cancer chemotherapy
during the past four decades and continue to provide therapeutic
options for hard-to-treat neoplastic diseases.37 Most of these
nucleoside analogues mimic various metabolites or inhibit an
essential enzyme, ultimately affecting the synthesis of nucleic
acids in the fast replicating cancer cells. Nucleotides are essential
molecules for the synthesis of ribo and deoxyribo nucleic acids
during replication, and thus this is a very attractive pathway
for anticancer drug design. The recent discovery of novel
mechanism-based inhibitors targeting ODCase inspired us to
evaluate the potential of these compounds as anticancer agents.
The designed compounds are various 6-substituted pyrimidine
nucleosides with substitutions such as 6-cyano, 6-azido, 6-amino,
6-iodo, 6-N-methylamino, 6-N,N-dimethylamino, 6-aldehyde,
6-ethyl ester, and 6-hydroxyl amino moieties (Figure 3).

1650 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Bello et al.
Scheme 1. Synthesis of Compounds 26-28 and 40-42a
Figure 3. Various C6-substituted pyrimidine derivatives.
Additionally, we designed derivatives carrying 5-fluoro moiety
along with the above 6-substitutions to study its effect on
binding to human ODCase and potentially their anticancer
activities (Figure 3). An introduction of the fluorine moiety at
C5 position would have stabilizing effects on the C6 substitu-
tions due to the electronegativity of the fluorine moiety. We
also envisioned that the C6 center could be more electrophilic
due to the 5-fluoro substitution. The designed molecules were
synthesized from either fully protected uridine or the corre-
sponding 5-fluorouridine derivatives. Synthesis of uridine
derivatives 14-18, 21, 22, and 34-38 was already published
by our group earlier.36 5-Fluorouridine derivatives were syn-
thesized according to Schemes 1 and 2. 5-Fluorouridine (25)
was fully protected and treated with LDA followed by iodine
to obtain 6-iodo derivative 45. This compound 45 served as a
key intermediate to synthesize other C6 derivatives. Thus
6-azido derivative 46 was synthesized by the treatment of 45
with sodium azide. Deprotection of the silyl and isopropylidene
moieties on 45 and 46 led to the corresponding nucleoside
derivatives 26 and 27, respectively. Monophosphorylation of
the nucleosides was accomplished using the standard conditions
of phosphorus chloride/pyridine. 6-Amino derivatives 28 and
42 were synthesized by the reduction of corresponding azido
derivatives 27 and 41 (Scheme 1). When compound 44 was
treated with LDA and methyl iodide, 6-ethyl derivative was
obtained due to the additional methylation of the 6-monom-
ethylated 5-fluorouridine derivative. We did not observe the
formation of any monomethylation at C6 position, and 6-ethyl
derivative 47 was isolated as the only product. Treatment of 44
with methyl formate led to the synthesis of 6-aldehyde derivative
48. Compounds 47 and 48 were fully deprotected under acidic
conditions, leading to the nucleoside derivatives 29 and 30,
respectively (Scheme 2). Monophosphorylation of compound
29 generated the mononucleotide derivative of 6-ethyl-5-
fluorouridine derivative (Scheme 2). The mononucleotide
derivatives were all transformed into the corresponding am-
monium salt forms for enzyme inhibition investigations against
human ODCase. Anticancer activities of all the synthesized
derivatives were evaluated using the nucleoside forms of the
inhibitors.
a Reagents: (a) acetone, H+; (b) TBDMSiCl, imidazole, CH2Cl2; (c) LDA,
I2, THF, -78 °C; (d) NaN3, DMF; (e) 50% TFA/H2O; (f) POCl3, Py,
CH3CN, 0 °C; (g) NH4OH, 0 °C; (h) Pd/C, H2.
Compound 39, which is a 5-fluorouridine derivative with no
substitution at C6 position, inhibited Hs ODCase weakly, with
a Ki of 98 µM, and this inhibition was much worse against that
of Mt, with a Ki of 645 µM. This perhaps is anticipated because
5-fluorouracil derivative 39 does not carry any substitution at
C6 position, and although 5-fluoro moiety fits well within the
binding pocket, it may not enhance the binding affinity
significantly. The 5-fluoro-6-amino derivative 42 is a moderate
inhibitor of both Hs and Mt ODCases, with Kis of 16.6 and
11.4 µM, respectively. The most potent inhibitor of Hs ODCase
is the 5-fluoro-6-ethyl derivative 43 and inhibited Hs ODCase
with a Ki of 0.35 µM. However, compound 43 is approximately
2 orders of magnitude weaker against Mt ODCase. In light of
the differences in inhibition patterns that exist between ODCases
from different species for the same inhibitor,39 the differences
of up to 2 orders of magnitude in the inhibition constants for
compounds 39 and 43 is not surprising in this case.
Compounds 40 and 41 exhibited potent inhibition of Hs
ODCase as covalent inhibitors (Table 2). The 5-fluoro-6-azido
derivative 41 exhibited interesting results, where it did not show
competitive inhibition with Hs ODCase, but with Mt ODCase,
we were able to derive a competitive inhibition constant by
measuring the initial rate constants. This again is further
confirmation of the species-related differences for the inhibition
of ODCases by the same inhibitor. Compound 40 inhibits human
ODCase with an equilibrium inhibition constant (KI) of 1.1 µM,
where as compound 41 inhibited with a KI of 18.2 µM (Figure
6). The second-order rate constant (kobs/[I]) for the inactivation
of Hs ODCase by compound 40 is estimated to be 1.9 M-1 s-1
(Table 2). On the basis of the enzymatic studies, compounds
40, 41, and 43 are good inhibitors of Hs ODCase among
5-fluoro-6-substituted uridine mononucleotide derivatives. Mass
First, mononucleotide derivatives 34-38 and 39-43 were
evaluated as potential inhibitors of Hs ODCase and/or Mt
ODCase (Table 1). Compounds 39, 42, and 43 exhibited an
interesting pattern of inhibition of Hs ODCase (Figure 5).

Orotidine-5′-Monophosphate Decarboxylase Inhibitors
Scheme 2. Synthesis of Compounds 29-30 and 43a
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1651
a Reagents: (a) LDA, methyl iodide, THF, -78 °C; (b) 50% TFA/H2O; (c) POCl3, Py, CH3CN, 0 °C; (d) NH4OH, 0 °C; (e) LDA, methyl formate, THF,
-78 °C.
Figure 4. X-ray crystal structure of the complex of 41 and Hs ODCase. (A) Active site region with the ligand 41 bound covalently to Lys125
residue. The enzyme is represented by the secondary structural elements. The critical residues Asp128, Lys125, Asp123, and Lys92 and the ligands
41 are shown as a capped stick model, and the atoms are color-coded (C, gray; N, blue; O, red; P, orange; F, turquoise). (B) Hydrogen bonding
network between the ligand 41 and various residues in the active site of Hs ODCase.
Table 1. Enzyme Inhibition Studies on ODCases Using Various
Inhibitors
To understand the molecular interactions of compounds 40
and 41 with ODCase, this compound was cocrystallized with
Hs ODCase (Table 3). Compounds 40 and 41 generated identical
complex structures with Hs ODCase, and here the discussion
is thus facilitated with the structure of compound 41 bound in
the active site of Hs ODCase (Figure 4). Compound 41 (similar
to compound 40) is a covalent inhibitor of Hs ODCase, and
this is clearly seen in the cocrystal structure, supporting the
enzyme kinetics (Table 2). The ligand is bound in the catalytic
pocket formed by the TIM barrel similar to other ligands. The
Nε of Lys125 in Hs ODCase is clearly seen bound to C6 atom
of the inhibitor 41 (Figure 4A). The 5′-monophosphate group
engaged in a tight hydrogen bonding network with residues
Asn241, Tyr243, Arg262, Gln261, and three water molecules
providing the most binding energy for the inhibitor, as expected
(Figure 4B). The 2′- and 3′-hydroxyl moieties as well as the
urea portion of the nucleic base are interacting with the enzyme
with residues Ser68, Asp70, Lys92, Thr132, Asp128, Ser183,
and Asn241, as was seen in previous cocrystal structures with
Ki (µM)
compd
Hs
Mt
3434,35
3634,35
3734,35
3834,35
39
41
42
43
ND
ND
ND
ND
29 ( 2
0.2 ( 0.1
0.84 ( 0.02
134 ( 5
98 ( 6
645 ( 15
0.36 ( 0.03
11.4 ( 0.6
29.0 ( 0.3
NDa
16.6 ( 0.7
0.35 ( 0.01
a Note: Compound 41 did not exhibit reversible inhibition in a competi-
tive assay with Hs ODCase.
spectral analyses of the samples of Hs ODCase treated with
compound 40 and 41 revealed that these compounds bind
covalently to the enzyme after the elimination of the C6-
substitutions (iodo and azido moieties, respectively), further
supporting the enzyme kinetics analyses (see Supporting
Information).

1652 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Bello et al.
( 0.02 µM, respectively. Compound 36 and 38 inhibited Mt
ODCase (considering initial rates of inhibition for the former
compound) with Ki of 0.19 ( 0.1 and 134 ( 5 µM, respectively.
Compounds 36 and 37 have exhibited submicromolar inhibition
against Mt ODCase, and are the most potent among the
nonfluorinated compounds, next to compound 35. Compound
35 inhibited Mt ODCase with a second-order rate constant (kobs
/
[I]) of 260000 M-1 s-1. Compound 36, on the other hand, is a
relatively poor inactivator, with kinact of 61.2 h-1 and inactivation
constant of 0.6 ( 0.1 µM.36 The general trend of the covalent
inhibition with 6-iodo and 6-azido moieties at C6 position was
observed in all cases. On the basis of the in vitro cell-based
assays (vide infra), we did not further evaluate compounds
34-38 against human ODCase because their nucleoside forms
did not exhibit any significant anticancer activities.
The nucleoside derivatives of compounds 8, 9, and 14-33
were screened against various cancer cell lines in order to test
their potential to inhibit cancer cell growth in vitro (Table 4).
The evaluation of all these compounds in WST.1 assays revealed
that 27, 28, 29, and 30 strongly inhibited cell lines of
hematopoietic origin, including leukemias (acute myelogenous
leukemia lines OCI-AML-1 and OCI-AML-2, and T-cell
leukemia line SKW3), lymphomas (non-Hodgkin’s B cell
lymphoma-OCI-Ly-7), and multiple myeloma (OCI-My-2). The
inhibitory concentrations (IC50) for these compounds were in
the range of 0.3-2.1 µM (Table 4). Expansion of adherent
cancer cell lines including breast cancer lines (MCF7 and
MDA468) were generally less sensitive to the above compounds,
with IC50 values ranging between 1-31 µM. None of the other
compounds showed inhibitory activities against the adherent
cancer lines up to 50 µM. Melanoma cell line Lox was highly
sensitive to compound 30, with an IC50 of 300 nM. In further
evaluations, compounds 27 and 28 revealed the inhibitory
concentrations (IC50) against healthy human PBMCs stimulated
with concanavalin A were greater than 10 µM.
Following this encouraging outcome, compounds 27, 28, and
30 were further evaluated for their ability to induce apoptosis
in different leukemia and lymphoma cell lines. To determine
the relationships between the cell survival and the reduced
expansion of different cell lines in the presence of 27, 28, and
30, the cell lines were treated with Annexin V and PI. Four
cell lines (OCI-AML1, OCI-Ly7, OCI-My2, and SKW3) were
observed for 3 days in the presence and absence of the above
inhibitors. The results clearly showed an increase in the
percentage of apoptotic cells in the presence of the inhibitors,
27, 28, and 30 compared to the control cells (Figure 7). Increased
cell death was first observed on day 2 after exposing the cancer
cells to the compounds. While all three compounds induced
apoptosis in these cell lines, compound 27 was relatively more
effective than 28 and 30 (Figure 7).
Figure 5. Reversible inhibition of Hs ODCase by the compounds 39
(A), 42 (B), and 43 (C) shown in double reciprocal plots. All
experiments are conducted in triplicate.
Table 2. Kinetic Characterization of ODCase in the Presence of
Irreversible Inhibitors
coinjection assay
KI (µM)
kinact (h-1
time-dependent assay
kobs/[I] (M-1 s-1
)
compd
)
Mt ODCase
35
36
ND
0.6 ( 0.1
ND
61.2
260000
ND
Hs ODCase
It occurred to us that 5-fluoro-uridine derivatives such as
25-30 might inhibit thymidylate synthase, similar to 5-fluoro-
2′-deoxyuridine. However, thymidylate synthase accepts 2′-
deoxynucleosides as substrates, and the nucleosides evaluated
in this report are ribonucleosides. In fact, we evaluated 5-fluoro-
UMP (39) against Hs and Mt ODCases for its inhibitory
potential. This compound was not a potent inhibitor of ODCases
and did not indicate that the 5-fluoro-UMP has favorable effect
without a C6-substitution. The other possibility could be that
the above nucleosides, including compound 25, are degraded
into their nucleic bases in the cells, which then are further
transformed into the corresponding 2′-deoxynucleosides and
exhibit anticancer activities through the inhibition of thymidylate
synthase. Thus, there may potentially be multiple mechanisms,
40
41
1.1 ( 0.4
158 ( 18
41 ( 3
1.9
NA
18.2 ( 3.6
ligands.34-36 The 5-fluoro moiety due to its small size fits into
the enzyme pocket well and does not specifically engage in any
direct interactions with the enzyme, as seen from this structure
(Figure 4B).
Among other compounds, 34-38 showed a range of inhibi-
tion potencies against Mt ODCase and were revealed in
previously published reports from our group.34-36 Compounds
34, 37, and 38 are competitive inhibitors of ODCase, while
compounds 35 and 36 inhibited ODCase covalently. Compounds
34 and 37 inhibited Mt ODCase with a Ki of 29.2 ( 2 and 0.84

Orotidine-5′-Monophosphate Decarboxylase Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1653
Figure 6. Inactivation of Hs ODCase by 40 and 41 (A and B, respectively) following the simultaneous addition of substrate mixed with the
inhibitor. Nonlinear least-squares fit of kobs at different inhibitor concentrations (eq 3) was used to determine the inactivation rate (kinact) and inactivation
constant (KI). All experiments are conducted in triplicate.
MHz for 13C). The chemical shifts are reported in δ ppm using
Table 3. Crystallographic Parameters for the Structure of the Complex
tetramethylsilane as the reference for the 1H and 13C NMR spectra.
of Human ODCase with Compounds 40 and 41
Mass spectra were obtained on a Q-Star mass spectrometer using
either ESI or EI techniques. The monophosphate compounds (free
acid forms) were transformed into the corresponding ammonium
salts by neutralization with 0.5 M NH4OH solution at 0 °C, followed
by lyophilization to obtain the corresponding ammonium salts.
ODCase enzyme assays were performed at 55 or 37 °C using a
VP-ITC microcalorimeter (MicroCal, Northampton, MA) using
previously published procedures.34 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, UK) at the Mass Spectrometry Facility, Advanced
Protein Technology Centre, The Hospital for Sick Children,
Toronto, Canada, and an AB/Sciex QStar mass spectrometer with
an ESI source and on a an Agilent 1100 capillary LC attached (MDS
Sciex, USA) at the Mass Spectrometry-AIMS Laboratory, Depart-
ment of Chemistry, University of Toronto.
Hs ODCase + 40 Hs ODCase + 41
Diffraction Data
1.40 (1.45-1.40) 1.70 (1.73-1.70)
resolution (Å)a
measured reflections (n)
321875
52359
205710
58035
unique reflections (n)
completeness (%)a
93.1 (60.7)
5.7 (21.3)
C2221
94.9 (61.3)
7.4 (49.3)
P21
Rsym (%)a
apace group
unit cell axes (Å)
a
b
78.3
116.6
62.1
69.7
61.9
70.3
c
unit cell angle (deg)
ꢀ
112.7
2
molecules in asymmetric unit (n)
1
Synthesis of uridine derivatives 14-18, 21, 22, and 34-38 was
already published by our group earlier.36
Refinement Statistics
resolution (Å)a
64.96-1.40
28.72-1.70
(1.70-1.74)
4286
5′-O-(t-Butyldimethylsilyl)-2′,3′-O-isopropylidene-5-fluorou-
ridine (44). A stirred suspension of 5-fluorouridine 25 (1 g, 3.8
mmol) in anhydrous acetone (70 mL) was treated with H2SO4 (0.5
mL) dropwise at 0 °C. The reaction mixture was then stirred at rt
for an hour. The reaction was then neutralized with triethylamine
and was concentrated. The crude mixture was purified by column
chromatography (5-8% MeOH:CHCl3) to afford 2′,3′-O-isopro-
pylidene-5-fluorouridine (1.15 g, quantitative) as a white solid. A
solution of this isopropylidene-protected derivative (0.5 g, 1.7
mmol) in anhydrous CH2Cl2 (20 mL) was treated with imidazole
(225 mg, 3.3 mmol) and TBDMSCl (250 mg, 1.7 mmol) at 0 °C.
The reaction mixture was brought to rt and was stirred for an hour.
The solvent was evaporated under vacuum, and the crude was
dissolved in ethyl acetate (30 mL), washed with water (15 mL),
brine (15 mL), and dried (Na2SO4). Evaporation of the solvent and
purification of the crude by column chromatography (30% EtOAc:
(1.436-1.400)
protein atoms (n)
2261
water molecules (n)
reflections used for Rfree (n)a
Rwork (%)a
273
333
2643 (122)
16.5 (27.6)
18.6 (29.2)
0.010
2927 (159)
17.5 (30.1)
21.0 (37.8)
0.010
Rfree (%)a
root mean square deviation
bond length (Å)
root mean square deviation
bond angle (deg)
1.55
1.54
average B factor (Å2)
15.0
23.9
a Numbers in brackets are for the highest resolution shells. Rsym ) ∑|I -
I |/∑I, where I is the observed intensity and is the average intensity
from multiple observations of symmetry-related reflections.
I
however, based on the enzymatic data ODCase inhibition,
certainly is one of the predominant means of activities for these
compounds such as 27 and 28.
Here, we reveal novel ODCase inhibitors with potent
anticancer activities. Although at the enzymatic level, several
compounds inhibited ODCase, at the cellular level, only three
compounds 27, 28, and 30 showed potential as anticancer agents
against various leukemias. Among these compounds, compound
27 is the best compound for further evaluation based on the in
vitro data.
1
Hx) yielded compound 44 (674 mg, 98%) as a foam. H NMR
(CDCl3) 9.54 (s, 1H), 7.78 (d, J ) 6 Hz, 1H), 5.91 (d, J ) 1.6 Hz,
1H), 4.66 (dd, 2.4, 5.6 Hz, 1H), 5.56 (dd, J ) 2.8, 5.6 Hz), 4.27
(m, 1H), 3.86 (dd, J ) 1.6, 11.6 Hz), 3.72 (dd, J ) 2, 11.6 Hz,
1H), 1.50 (s, 3H), 1.27 (s, 3H), 0.82 (s, 9 H), 0.02 (s, 6H).
5′-O-(t-Butyldimethylsilyl)-2′,3′-O-isopropylidene-5-fluoro-6-
iodouridine (45).38 Compound 44 (250 mg, 0.6 mmol) was
dissolved in 3 mL anhyd THF at -78 °C and was treated with
LDA (0.9 mL, 1.8 mmol, 2.0 M solution in THF). After stirring
for an hour, iodine (152 mg, 0.6 mmol) dissolved in anhyd THF
(2 mL) was added and the mixture was stirred for an additional
5 h in dark. The reaction was quenched with water (0.5 mL) and
then brought to rt and dissolved in ethyl acetate (25 mL). The
organic layer was washed with water (10 mL), brine (10 mL), and
dried (Na2SO4). Evaporation of the solvent and purification of the
crude by column chromatography (30% EtOAc:Hx) provided
compound 45 (310 mg, 95%) as an yellow foam. 1H NMR (CDCl3)
9.47 (s, 1H), 6.09 (d, J ) 1.6 Hz, 1H), 5.20 (dd, J ) 1.6, 6.8 Hz,
Experimental Section
General. All anhydrous reactions were performed under the
nitrogen atmosphere. All solvents and reagents were obtained from
commercial sources. Column chromatography was performed using
silica gel (60 Å, 70-230 mesh). The NMR spectra were recorded
1
on a Varian spectrometer (300 and 400 MHz for H, 75 and 100

1654 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Bello et al.
Table 4. Anticancer Activities (IC50) for Compounds 8, 9, and 14-30 in Various Cell Linesa
IC50 (µM)
compd
OCI-AML-1
OCI-AML-2
OCI-Ly-7
OCI-My-2
SKW3
MCF7
LOX
MDA 468
OVCR 4
COLO 205
8
9
6.1
16
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.7
18.7
>50
>50
27.8
>50
>50
>50
42.3
>50
>50
>50
>50
>50
>50
0.6
2.8
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
1.0
6.7
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
1.8
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
NA
NA
>50
1.5
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
NA
NA
>50
3.8
>50
>50
15.3
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.6
0.7
2.1
0.3
>50
15.2
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.7
0.9
ND
0.7
>50
11.7
>50
>50
>50
18.9
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.4
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
2.3
14
15
16
17
18
19
20
21
22
23
24
26
27
28
29
30
31
32
33
1.3
1.9
0.6
>50
36.2
>50
0.9
1.4
0.4
>50
1.3
0.6
1.2
0.3
>50
1.6
1.4
3.4
4.5
0.4
>50
1.1
2.6
5.3
1.9
>50
>50
>50
2.0
3.3
31.4
23.7
>50
4.4
ND
ND
>50
>50
>50
ND
ND
>50
>50
>50
>50
>50
>50
>50
a The details of the cell lines used in this study are detailed in Supporting Information Table S-1. There is no activity detected in CCD 967, HS58T,
T47D, PC3, and SNB19 cell lines (for compounds 27, 28 up to 4 µM, and for all other compounds up to 50 µM).
quenched with 25 mL of cold water and stirring was continued for
an additional hour. The evaporation of the solvent and purification
of the crude by column chromatography (Dowex ion-exchange basic
resin, 0.1 M formic acid) afforded compound 40 as syrup. Free
acid form was transformed into the corresponding ammonium salt
by neutralization with 0.5 M NH4OH solution at 0 °C to yield
compound 40 as powder (202 mg, 60%). UV (H2O) λmax ) 271
nm; ε271 ) 6400. 1H NMR (D2O) 5.89 (d, J ) 3.1 Hz, 1H), 4.7 (m,
1H), 4.31 (t, J ) 6.9 Hz, 1H), 4.01-3.42 (m, 3H). HRMS (ESI,
+ve): calculated (M + H+) C9H10N2O9FPI, 466.9158; found,
466.9145.
5′-O-(t-Butyldimethylsilyl)-2′,3′-O-isopropylidene-5-fluoro-6-
azido Uridine (46). Compound 45 (260 mg, 0.5 mmol) was
dissolved in anhydrous DMF (3 mL) and NaN3 (34 mg, 0.5 mmol)
was added. The reaction mixture was stirred at room temperature
for two hours in dark. The reaction mixture was concentrated under
vacuum at room temperature, and the crude was dissolved in ethyl
acetate (15 mL), washed with brine, and dried (Na2SO4). The
combined organic layers were evaporated, and the residue was
Figure 7. Apoptosis as a result of treatment with compounds 27, 28,
purified by silica gel column chromatography (1% MeOH:CHCl3)
and 30 in various cancer cell lines. The increase in apoptotic cells as
1
seen through the annexin V+/PI- stain in comparison to control cells
when treated with each compound on days 1, 2, and 3.
to yield compound 46 (201 mg, 92%) as a light-yellow solid. H
NMR (CDCl3) 9. 47 (s, 1H), 6.07 (d, J ) 1.6 Hz, 1H), 5.13 (dd,
J ) 1.2, 6.8 Hz, 1H), 4.78 (dd, J ) 84.8, 6.4 Hz, 1H), 4.12 (m,
1H), 3.79 (m, 2H), 1.55 (s, 3H), 1.34 (s, 3H), 0.89 (s, 9H), 0.06 (s,
6H). 9. 47 (s, 1H), 6.07 (d, J ) 1.6 Hz, 1H), 5.13 (dd, J ) 1.2, 6.8
Hz, 1H), 4.78 (dd, J ) 84.8, 6.4 Hz, 1H), 4.12 (m, 1H), 3.79 (m,
2H), 1.55 (s, 3H), 1.34 (s, 3H), 0.89 (s, 9H), 0.06 (s, 6H).
1H), 4.81 (dd, J ) 4.4, 6.4 Hz, 1H), 4.18 (m, 1H), 3.79 (dd, J )
2.4, 6 Hz, 2H), 1.56 (s, 3H), 1.35 (s, 3H), 0.89 (s, 9H), 0.05 (s,
6H).
5-Fluoro-6-iodouridine (26).38 A stirred suspension of com-
pound 45 (300 mg, 0.55 mmol) in water (2 mL) was treated with
50% aqueous TFA (2 mL) at 0 °C, brought to room temperature,
and stirred for an additional 2 h, in dark. The reaction mixture was
concentrated under vacuum and was purified by column chroma-
tography (10-15% EtOH:CHCl3) to afford compound 26 (193 mg,
90%) as a light-yellow solid. UV (H2O) λmax ) 272 nm; ε272 7440.
1H NMR (CD3OD) 5.95 (d, J ) 3.2 Hz, 1H), 4.73 (dd, 3.2, 6.4 Hz,
1H), 4.33 (t, 6.4 Hz, 1H), 3.89 (m, 1H), 3.80 (dd, J ) 2.8, 12 Hz,
1H), 3.67 (dd, 6, 12 Hz). 13C NMR (CD3OD) δ 154.8 (2JCF ) 30.3
Hz), 147.5, 144.0 (1JCF ) 228.4 Hz), 103.9 (2JCF ) 39.0 Hz), 102.0,
85.1, 72.1, 70.1, 62.5. HRMS (ESI, +ve): calculated (M + Na+)
C9H10N2O6FNaI, 410.9459; found, 410.9448.
5-Fluoro-6-azido Uridine (27). A stirred solution of compound
46 (200 mg, 0.4 mmol) was treated with 50% aqueous trifluoroacetic
acid (4 mL) at 0 °C. The reaction mixture was then brought to
room temperature and was stirred for an additional two hours in
the dark. Evaporation of the solvent and purification of the crude
by silica gel column chromatography (10-15% MeOH: CHCl3)
afforded the product 27 (121 mg, 89%) as a light-yellow solid.
1
UV (H2O) λmax ) 291 nm; ε291 ) 8905. H NMR (CD3OD) 5.88
(d, J ) 2.8 Hz, 1H), 4.58 (dd, J ) 4, 6 Hz, 1H), 4.28 (t, J ) 6.8
Hz, 1H), 4.23 (dd, J ) 3.2, 6.4 Hz, 1H), 4.00 (m, 1H), 3.79 (m,
2H). 13C NMR (CD3OD) δ 156.4 (2JCF ) 23.4 Hz), 148.0, 137.1
(1JCF ) 212.7 Hz), 132.8, 91.3, 84.5, 71.9, 69.3, 61.7. HRMS (ESI,
+ve): calculated (M + Na+) C9H10N5O6FNa, 326.0507; found,
326.0519.
5-Fluoro-6-iodouridine-5′-O-monophosphate (40). A stirred
solution of H2O (34 mg, 1.9 mmol) and POCl3 (0.3 mL, 3 mmol)
in anhydrous acetonitrile (3 mL) was treated with pyridine (0.3
mL, 3 mmol) at 0 °C and stirred for 10 min. Compound 26 (260
mg, 0.7 mmol) was then added and the mixture was stirred for an
additional 5 h at 0 °C in dark. The reaction mixture was then
5-Fluoro-6-azidouridine-5′-O-monophosphate (41). A stirred
solution of water (30 mg, 1.9 mmol) and POCl3 (0.3 mL, 3 mmol)
in anhydrous acetonitrile (3 mL) was treated with pyridine (0.3
mL, 3.2 mmol) at 0 °C and was stirred for 10 min. Compound 27

Orotidine-5′-Monophosphate Decarboxylase Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1655
was then added (206 mg, 0.7 mmol) to the reaction mixture and
was stirred for an additional 5 h at 0 °C in dark. The reaction
mixture was quenched with 2.5 mL of cold water. Evaporation of
the solvent and the purification of the crude by column chroma-
tography (Dowex ion-exchange basic resin, 0.1 M formic acid) gave
the mononucleotide as a syrup. This was transformed into the
corresponding ammonium salt by neutralization with 0.5 M NH4OH
solution at 0 °C and freeze-dried to yield compound 41 as a white
powder (0.199 mg, 70%). UV (H2O) λmax ) 286 nm; ε286 ) 1585.
1H NMR (D2O) 5.01 (bs, 1H), 4.42 (m, 1H), 4.08 (m, 2H), 3.95
(m, 1H), 3.51 (m, 1H). HRMS (ESI, +ve): calculated (M + H+)
C9H10N5O9FP, 382.0205; found, 382.0196.
5-Fluoro-6-aminouridine (28). Compound (27) (200 mg, 0.66
mmol) was dissolved in methanol and 10% Pd/C (10 mg) was
added. The reaction mixture was stirred for 3 h in dark under a
hydrogen atmosphere at rt. The mixture was filtered through celite,
and the solvent was evaporated to dryness to give compound 28 as
a white powder (222 mg, 82%). UV (H2O) λmax ) 265 nm; ε265 )
3018. 1H NMR (D2O) 5.39 (bs, 1H), 4.73 (m, 1H), 4.30 (t, J ) 6.0
Hz, 1H), 3.89 (m, 2H), 3.82 (m, 1H), 2.77 (m, 2H), 1.30 (t, J )
7.1 Hz, 3H). HRMS (ESI, +ve): calculated (M
C11H15N2O9FP, 369.0504; found, 369.0505.
+
H+)
5′-O-(t-Butyldimethylsilyl)-2′,3′-O-isopropylidene-5-fluoro-6-
carbaldehydeuridine (48). Compound 44 (250 mg, 0.6 mmol)
dissolved in 3 mL of anhyd THF at -78 °C and was treated with
a solution of LDA (1.5 mL, 3.0 mmol, 2.0 M solution in THF).
After stirring for an hour, methyl formate (54 mg, 0.9 mmol) in
anhyd THF (1 mL) was added and the mixture was stirred for an
additional 5 h at the same temperature. The reaction was quenched
with water (0.5 mL), brought to room temperature, and dissolved
in ethyl acetate (25 mL). The organic layer was washed with water
(10 mL), brine (10 mL), and dried (Na2SO4). Evaporation of the
solvent and purification of the crude by column chromatography
(MeOH:CHCl3, 5:95) gave 48 (0.187 g, 70%) as a brown foam.
1H NMR (CDCl3) δ 10.1 (d, J ) 0.8 Hz, 1H), 6.25 (d, J ) 1.6 Hz,
1H), 5.10 (dd, J ) 4, 8 Hz, 1H), 4.74 (dd, J ) 4, 8 Hz, 1H), 4.09
(m, 1H), 3.82 (dd, J ) 4, 12 Hz, 1H), 3.75 (dd, J ) 8, 12 Hz, 1H),
1.56 (s, 3H), 1.36 (s, 3H), 0.90 (s, 9H), 0.07 (s, 6H).
a white solid (165 mg, 90%). UV (H2O) λmax ) 281 nm; ε281
)
17806. 1H NMR (CD3OD) 6.36 (dd, J ) 1.2, 7.6 Hz, 1H), 4.61 (t,
J ) 4, 6 Hz, 1H), 4.28 (t, 6 Hz, 1H), 3.77 (m, 2H), 3.62 (dd, J )
6, 12.8 Hz, 1H). 13C NMR (CD3OD) δ 156.2, 149.6, 125.8, 123.1,
89.2, 85.5, 70.1, 69.8, 60.5. HRMS (ESI, +ve): calculated (M +
Na+) C9H12N3O6FNa, 300.0602; found, 300.0608.
5-Fluoro-6-carbaldehyde Uridine (30). A stirred suspension of
compound 48 (200 mg, 0.5 mmol) in water (2 mL) was treated
with 50% aqueous TFA (2 mL) at 0 °C and was stirred at room
temperature for 2 h. Evaporation of the solvent and the purification
of the crude by silica gel column chromatography (10-15% MeOH:
CHCl3) afforded compound 30 (115 mg, 88%) as a light-brown
5-Fluoro-6-aminouridine-5′-O-monophosphate (42). The mono-
nucleotide 41 (R1 ) -PO42-) (120 mg, 0.29 mmol) was dissolved
in 50% aqueous methanol and 10% Pd/C (10 mg) was added. A
procedure similar to that for 41 was used to obtain compound 42
1
as a white solid (101 mg, 90%). H NMR (D2O) δ 5.92 (bs, 1H),
1
solid. UV (H2O) λmax ) 269 nm; ε269 ) 5734. H NMR (D2O)
4.53 (m, 1H), 4.12 (m, 2H), 3.86 (m, 2H). HRMS (ESI, +ve):
6.26 (d, 1H), 6.92 (dd, 1H), 6.58 (m, 1H), 6.38 (t, 1H), 3.79 (m,
2H), 3.69 (m, 1H). 13C NMR (D2O) δ 159.1, 149.8, 138.7, 137.4
(1JCF ) 225.0 Hz), 93.5, 89.0, 83.3, 72.1, 69.2, 61.5. HRMS (ESI,
+ve): calculated (M + H+) C10H12N2O7F, 291.0623; found,
291.0600.
calculated (M + H+) C9H12N3O9FP, 356.0300; found, 56.0301.
5′-O-(t-Butyldimethylsilyl)-2′,3′-O-isopropylidene-5-fluoro-6-
ethyluridine (47). Compound 44 (250 mg, 0.6 mmol) was dissolved
in 3 mL of anhyd THF at -78 °C and was treated with a solution
of LDA (1.5 mL, 3.0 mmol, 2.0 M solution in THF). After stirring
for 1 h, CH3I (225 mg, 1.8 mmol) in anhyd THF (1 mL) was added,
and the mixture was stirred for an additional 5 h at the same
temperature. The reaction was quenched with water (0.5 mL) and
then brought to room temperature and dissolved in ethyl acetate
(25 mL). The organic layer was washed with water (10 mL), brine
(10 mL), and dried (Na2SO4). Evaporation of the solvent and
purification of the crude by column chromatography (hexanes:
EtOAc, 70:30) yielded compound 47 (187 mg, 70%) as a white
Enzymology. ODCase from human (Hs) or from Methanobac-
terium thermoautotrophicum (Mt) was used in enzyme activity
studies. Hs ODCase is a C-terminal portion of the bifunctional
enzyme UMP synthase.
Reversible Inhibition. The inhibition of Mt ODCase by various
inhibitors was evaluated in a competitive inhibition assay as
described previously.34 The activity of 20 nM ODCase was
monitored at 55 °C. The substrate concentration was 40 µM. The
concentrations of 42 were 0, 10, 20, 35, and 50 µM and for 41
were 0, 0.25, 0.50, 0.75, and 1.0 µM. The concentration of 43 in
the assay samples was 0, 20, 40, 100, and 200 µM. The
concentration of 39 was 0, 0.5, 1.0, 2.5, and 4.0 mM and was
evaluated as a competitive inhibitor of Mt ODCase.
The reversible inhibition of human ODCase was studied at 37
°C, as described previously.39 The enzyme stock was diluted with
50 mM Tris buffer containing 1 mM DTT to prepare 60 nM enzyme
assay samples. The final substrate concentration was either 15 or
20 µM. Concentrated inhibitors stock solutions were prepared in
50 mM Tris (pH 7.5). All four compounds were tested in a
competitive inhibition assay where enzyme was mixed with the
inhibitor and the reaction was initiated by the substrate addition.
The final assay concentrations of 41 were 0, 0.25, 0.50, 0.75, and
1.0 µM, for 42 were 0, 10, 25, 50, and 120 µM, for 43, they were
0, 0.2, 0.4, 1.0, and 2.0 µM and for 39, 0, 50, 100, 250, and 500
µM.
Time-Dependent Inhibition. The inactivation of human ODCase
by 40 was monitored at 37 °C following the coinjection of the
substrate and inhibitor into the calorimetric cell containing the
enzyme. Concentrated enzyme sample (60 µM) was prepared in
50 mM Tris (pH 7.5), 20 mM DTT, and 40 mM NaCl and incubated
overnight at 4 °C. The assay samples were prepared in 50 mM
Tris, 1 mM DTT to obtain 60 nM enzyme concentration. Substrate
and the inhibitor were mixed and loaded into the automatic injection
syringe. The substrate concentration was kept constant while the
final concentration of the inhibitor was varied. A single, 5.7 µL
injection of the ligands’ mixture resulted in 0.5, 1.0, 1.5, 1.8, or
2.5 µM concentration of 40, and 20 µM OMP.
1
foam. H NMR (CDCl3) 9.60 (s, 1H), 5.60 (d, J ) 1.3 Hz, 1H),
5.20 (dd, J ) 1.4, 6.5 Hz, 1H), 4.81 (dd, J ) 4.5, 6.4 Hz, 1H),
4.16 (m, 1H), 3.81 (m, 2H), 2.86-2.67 (m, 2H), 1.55 (s, 3H), 1.345
(s, 3H), 1.31 (t, J ) 8.3 Hz, 3H), 0.88 (s, 9H), 0.04 (s, 6H).
5-Fluoro-6-ethyluridine (29). A stirred suspension of compound
47 (0.200 g, 0.45 mmol) in water (2 mL) was treated with 50%
aqueous TFA (2 mL) at 0 °C and the reaction mixture was stirred
for 2 h at rt. Evaporation of the solvent and purification of the
crude by column chromatography (10-15% MeOH:CHCl3) af-
forded compound 29 (118 mg, 94%) as a light-yellow solid. UV
(H2O) λmax ) 269 nm; ε269 ) 8205. 1H NMR (CD3OD) δ 5.45 (d,
J ) 3.6 Hz, 1H), 4.77 (dd, J ) 4.1, 6.3 Hz, 1H), 4.31 (t, J ) 6.1
Hz, 1H), 3. 90 (m, 1H), 3.80 (dd, J ) 3, 11.9, 1H), 3.66 (dd, J )
5.4, 11.9 1H), 2.78 (m, 2H), 1.29 (t, J ) 7.3 Hz, 3H). 13C NMR
(CD3OD) δ 157.8, 149.7, 143.8 (2JCF ) 24.3 Hz), 137.8 (1JCF
)
226.8 Hz), 92.9, 85.2, 71.6, 70.2, 62.5, 18.5, 11.4. HRMS (ESI,
+ve): calculated (M + H+) C11H16N2O6F, 291.0986; found,
291.0986.
5-Fluoro-6-ethyluridine-5′-O-monophosphate (43). A stirred
solution of H2O (34 mg, 1.9 mmol) and POCl3 (0.3 mL, 3 mmol)
in anhyd acetonitrile (3 mL) was treated with pyridine (0.3 mL,
3.3 mmol) at 0 °C and stirred for 10 min. Compound 29 (195 mg,
0.7 mmol) was added, and the mixture was stirred for an additional
5 h at 0 °C. The reaction mixture was then quenched with cold
water (25 mL) and stirring was continued for an additional hour.
Evaporation of the solvent and purification of the crude by column
chromatography (Dowex ion-exchange basic resin, 0.1 M formic
acid) afforded the free acid form of 43 as a syrup. The product
was transformed into the corresponding ammonium salt to afford

1656 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Bello et al.
Second-order rate of inactivation of Hs ODCase by 40 was
studied in a time-dependent assay. The incubation samples of the
enzyme (control) or the enzyme with inhibitor were set up at room
temperature, and the remaining enzymatic activity of ODCase was
monitored for up to 48 h. All samples were prepared in the buffer
(pH 7.5) consisted of 50 mM Tris, 20 mM DTT, and 40 mM NaCl.
The control reaction contained 60 µM Hs ODCase. The initial
concentration of the enzyme in the inactivation samples was also
60 µM, and of 40 was varied from 60, 120, 150, or 300 µM. All
samples were incubated for 0.25, 0.5, 1, 2, 4, 6, 24, and 48 h. At
each incubation time point, 2.5 µL aliquots were removed from
the concentrated samples and diluted to 2.5 mL in 50 mM Tris, 1
mM DTT. The remaining activity of ODCase was measured after
5.7 µL injection of 5 mM OMP that resulted in 20 µM final
substrate concentration.
Inactivation of Hs ODCase was investigated in the presence of
41 by coinjecting the substrate OMP and the inhibitor. Final
substrate concentration was 20 µM in all assays. The concentrations
of the inhibitor 41 after the 5.7 µL injection (containing the substrate
and inhibitor) in the reaction cell were (control), 25, 35, 50, and
75 µM.
Toronto, Canada. Peripheral blood mononuclear cells (PBMCs)
were isolated from peripheral blood by differential density cen-
trifugation over Ficoll-Hypaque (Amersham Biosciences) according
to the manufacturer’s instructions. The blood was collected from
two healthy donors at the Ontario Cancer Institute, University Health
Network. Briefly, collected blood was diluted in phosphate buffered
saline (PBS) (1:1). A layer of Ficoll-Hypaque was carefully overlaid
with diluted blood to a final concentration of 1:1. Cells were spun
at 2000 rpm at room temperature for 30 min. Buffy coat, containing
PBMCs, was removed and washed twice in PBS. PBMCs were
then stimulated with 3 µg/mL of Concanavalin A and seeded to be
used in WST.1 assay as described below.
In Vitro Cell Proliferation WST.1 Assay. Cell lines were
seeded in triplicate at 104 cells/100 µL in 96-well plates. Peripheral
blood mononuclear cells were seeded in triplicate at 105 cells/100
µL in 96-well plates. Inhibitors were added immediately, at final
concentrations ranging between 0 to 100 µM. All compounds were
dissolved in sterile deionized water. On day 3, WST.1 assay kit
(Roche Biosciences) was used to measure the effect of inhibitors
on the cell growth. WST.1 assay relies on the cleavage of WST.1
(water soluble tetrazolium salt: 4-[3-(4-iodophenyl)-2-(4-nitrophe-
nyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) to formazan by cel-
lular enzymes. The amount of formazan dye formed correlates to
the number of metabolically active cells in the culture and was
read in an ELISA microplate reader at 440 nm. These OD values
were plotted against concentrations of different compounds and the
inhibition constants (IC50) were determined for each compound.
Apoptosis Assay. Cells were seeded in duplicate at 105 cells/
mL in 24-well plates. Immediately after seeding, the inhibitors (27,
28, and 30) were added to obtain the final concentration of 4 µM.
Water was added to the control wells. Cells were harvested every
24 h, washed in PBS, and stained with Annexin V/propidium iodide
(PI) (1:200) using Annexin-V-FLUOS staining kit (Roche). Annexin
V is a Ca2+-dependent phospholipid-binding protein with high
affinity for phosphatidylserine (PS). It is used as a probe to detect
PS exposure on the outer leaflet of the cell membrane and is
therefore widely used for detection of early apoptotic cells. Unlike
apoptotic cells (Annexin V+/PI-), necrotic cells are Annexin V+/
PI+. FACS was used to analyze the staining due to Annexin V and
PI.
Data Analysis. The initial data analyses for the reversible
inhibition were performed using Origin 7.0 software. The raw data
representing the heat changes over time were converted into the
reaction rate at each recorded time point. The raw data representing
the rate of decarboxylation at several substrate concentrations in
the absence or presence of the inhibitors were directly fitted to eq
1 to calculate the reversible inhibition constant, Ki.
Vmax[S]
V )
(1)
[I]
Ki
KM 1 +
+ [S]
(
)
To determine the inactivation of Hs ODCase following the
coinjection of the substrate and the inhibitor, kobs was first computed
from each progress curve. The value (Power, µcal/s) at each
inflection point was normalized by setting the inflection point value
to zero. The data representing the change in thermal power over
time were fitted to the eq 2 to calculate kobs
.
X-ray Crystallography. All Hs ODCase concentrations were
determined using a BioRAD protein assay kit and BSA as a
standard. ODCase protein was dissolved in 20 mM Tris (pH 8.4),
10 mM NaCl, and 1 mM TCEP-HCl. Hs ODCase complex crystals
were grown in hanging drops using 2.4 M ammonium sulfate, pH
8.4, as the main precipitant. To obtain larger single crystals of the
enzyme complexes, microseeding was performed the following day.
Crystals of the complexes with compound 41 grew in space group
P21 with unit cell dimensions of close to a ) 69.6 Å, b ) 61.7 Å,
c ) 70.7 Å, ꢀ ) 112.2°. The crystals of the complex of compound
40 with the enzyme adopted space group C2221. For data collection,
the crystals were cryo-protected by Parotone-N oil before being
flash-frozen in a stream of boiling nitrogen. Diffraction data for
the crystals of HsODCase cocrystallized with compound 41 were
collected at 100 K and λ ) 1.10 Å on beamline X08C, Brookhaven
National Laboratory; those for the other two complexes were from
beamline 08ID-1 at the Canadian Macromolecular Crystallography
Facility, Canadian Light Source, Inc., collected also at 100 K but
at λ ) 0.97034 Å. All data were reduced and scaled using
HKL2000.40 The structures of all complexes were determined using
molecular replacement techniques with the help of the program
package MOLPREP;41 subsequent refinements were done with
Refmac-5.2,42 and model building used COOT.43 Data collection
and refinement statistics are given in Table 3. Atomic coordinates
and structure factors have been deposited into the Protein Data Bank
(PDB IDs: 3G3M, and 3G3D).
Vi
kobs
[P] )
(1 - exp(-kobst))
(2)
where [P] represents the product concentration, Vi is the initial
reaction rate, and t is 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
)
(3)
KI + [I]
To analyze the time-dependent inactivation of Hs ODCase, kobs
was first determined at each inhibitor concentration. The rate
constant for loss of enzyme activity, kobs, was determined from the
slope of ln(% remaining enzyme activity) versus time. The kobs
values were then plotted against the inhibitor concentration in the
incubation samples ([I]). The potency of each inhibitor in terms of
the second-order rate constant, kobs/[I] (M-1 s-1), was calculated
from the slope of the linear fit of data.
Anticancer Activity Evaluation. Cell Lines and Cell
Culture Conditions. All cell lines were grown in Iscove’s MDM
(GIBCO no. 12440) supplemented with 5% fetal calf serum (FCS),
50 µM 2-mercaptoethanol, 100 µg/mL penicillin, and 100 µg/mL
streptomycin. Cells lines growing in suspension were maintained
at 0.5-1.5 × 106 cell/mL, while adherent cell lines were maintained
subconfluent. Cells were kept in culture for ca. 10 weeks.
Isolation and Culture of Human Peripheral Blood Mono-
nuclear Cells. The protocol for informed consent and the use of
human blood was approved by the Ethics Review Committees at
both the University Health Network and St. Michael’s Hospital in
Mass Spectral Analyses. The concentrations of 40 and 41 for
incubation with Hs ODCase for mass spectral analyses were 22.6
and 80 mM, respectively. The controls and the samples containing
the inhibitors were analyzed 2 h after the onset of incubation at
room temperature and then again after 24 h incubation. The samples

Orotidine-5′-Monophosphate Decarboxylase Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1657
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Acknowledgment. This work was supported by grant no.
200611PPP from the Canadian Institutes of Health Research,
Proof-of-Principle program (L.P.K., C.P.J., and E.F.P.). L.P.K.
is a recipient of an Rx&D HRF-CIHR research career award.
E.F.P. thanks the Canada Research Chair Program for support.
We thank staff at the NSLS beamline X8C and the Canadian
Macromolecular Crystallography Facility for their time com-
mitments and expert help. A joint grant from the Canadian
Institutes of Health Research and the National Sciences and
Engineering Research Council of Canada enabled the use of
beamline X8C at the National Synchrotron Light Source,
Brookhaven National Laboratory. Part of the research described
in this paper was performed at the Canadian Light Source, which
is supported by NSERC, NRC, CIHR, and the University of
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Supporting Information Available: Purity data, details of the
cancer cell lines, thermograms/enzyme inhibition kinetics profiles,
and mass spectral analyses of the enzyme-inhibitor covalent
complexes. This material is available free of charge via the Internet
at http://pubs.acs.org.
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