Novel cytidine-based orotidine-5′-monophosphate decarboxylase inhibitors with an unusual twist

Article abstract of DOI:10.1021/jm301176r

Orotidine-5′-monophosphate decarboxylase (ODCase) is an interesting enzyme with an unusual catalytic activity and a potential drug target in Plasmodium falciparum, which causes malaria. ODCase has been shown to exhibit unusual and interesting interactions with a variety of nucleotide ligands. Cytidine-5′-monophosphate (CMP) is a poor ligand of ODCase, and CMP binds to the active site of ODCase with an unusual orientation and conformation. We designed N3- and N4-modified CMP derivatives as novel ligands to ODCase. These novel CMP derivatives and their corresponding nucleosides were evaluated against Plasmodium falciparum ODCase and parasitic cultures, respectively. These derivatives exhibited improved inhibition of the enzyme catalytic activity, displayed interesting binding conformations and unusual molecular rearrangements of the ligands. These findings with the modified CMP nucleotides underscored the potential of transformation of poor ligands to ODCase into novel inhibitors of this drug target.

Full text of DOI:10.1021/jm301176r

Article
pubs.acs.org/jmc
Novel Cytidine-Based Orotidine-5′-Monophosphate Decarboxylase
Inhibitors with an Unusual Twist
†
,‡
†
†
§
⊥
Meena K. Purohit, Ewa Poduch, Lianhu William Wei, Ian Edward Crandall, Terrence To,
Kevin C. Kain,#,▽ Emil F. Pai,§,⊥,∥ and Lakshmi P. Kotra*,†,‡,§,▽
†Center for Molecular Design and Preformulations, Toronto General Research Institute, University Health Network, Toronto,
Ontario M5G 1L7, Canada
‡
Department of Pharmacy, Birla Institute of Technology and Science, Pilani, Rajasthan, India
§
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto,
Ontario M5S 3M2, Canada
⊥
Campbell Family Cancer Research Institute, Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada
∥
Departments of Medical Biophysics, Biochemistry, and Molecular Genetics, University of Toronto, 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, University of Toronto, Toronto, Ontario, Canada
▽McLaughlin Center for Molecular Medicine, University of Toronto, Toronto, Ontario, Canada
*
S Supporting Information
ABSTRACT: Orotidine-5′-monophosphate decarboxylase
ODCase) is an interesting enzyme with an unusual catalytic
(
activity and a potential drug target in Plasmodium falciparum,
which causes malaria. ODCase has been shown to exhibit
unusual and interesting interactions with a variety of
nucleotide ligands. Cytidine-5′-monophosphate (CMP) is a
poor ligand of ODCase, and CMP binds to the active site of
ODCase with an unusual orientation and conformation. We designed N3- and N4-modified CMP derivatives as novel ligands to
ODCase. These novel CMP derivatives and their corresponding nucleosides were evaluated against Plasmodium falciparum
ODCase and parasitic cultures, respectively. These derivatives exhibited improved inhibition of the enzyme catalytic activity,
displayed interesting binding conformations and unusual molecular rearrangements of the ligands. These findings with the
modified CMP nucleotides underscored the potential of transformation of poor ligands to ODCase into novel inhibitors of this
drug target.
INTRODUCTION
Chart 1. Structures of ODCase Substrate 2 and Various
Known Inhibitors
■
4
Orotidine-5′-monophosphate decarboxylase (ODCase, EC
.1.1.23) catalyzes the decarboxylation of orotidine-5′-mono-
phosphate (OMP, 2) to uridine-5′-monophosphate (UMP, 1)
during de novo pyrimidine biosynthesis. Pyrimidine nucleotides
are necessary for the biological construction of DNA and RNA.
The catalytic activity of ODCase does not involve any
cofactors, metal ions, or intermittent covalent bond formation.
The half-time (t1/2) for the uncatalyzed decarboxylation
reaction of 2 is about 78 million years, but when catalyzed by
1,2
ODCase, it is 18 ms. ODCase is present in most living
organisms including humans, bacteria, and various parasites.
Due to the significant physiological role of ODCase in living
organisms, this proficient enzyme could be a valuable drug
target if compounds can be designed to selectively inhibit
5
,6
also demonstrate its potential for inhibitor development.
These investigations provided insights into the biochemical
nature of this enzyme, binding conformations of various ligands
3
pathogen enzymes. Several known C-6 substituted nucleoside
inhibitors of ODCase exhibit potent antimalarial activities
4
(
Chart 1). Recent discoveries of fluorinated nucleosides as
Received: August 9, 2012
ODCase inhibitors that exhibit anticancer and antiviral activities
Published: September 19, 2012
©
2012 American Chemical Society
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Journal of Medicinal Chemistry
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in its active site, and binding of inhibitors to this therapeutic
target.
EXPERIMENTAL SECTION
■
General. All solvents and reagents were obtained from commercial
sources. Column chromatography purifications were performed on
Biotage flash chromatography systems using normal silica gel (60 Å,
70−230 mesh) and reverse-phase (C18) cartridges. Reactions were
monitored by thin layer chromatography (Merck). NMR spectra were
Thus, ODCase has captivated biochemists for the last three
decades. To date, 152 crystal structures of ODCase and its
complexes from both prokaryotic and eukaryotic organisms
7
have been solved and appear in the Protein Data Bank. Still
1
recorded on Bruker spectrometers (400 MHz for H and 162.03 MHz
after a significant number of mechanistic studies and three-
dimensional structure elucidations, the underlying chemical
mechanism for its catalytic biochemical activity remains unclear.
However, the active site of the enzyme has been very well
mapped.
for 31P). Chemical shifts are reported in δ ppm using the residual
1
solvent peak as the reference for the H NMR spectra, and phosphoric
3
1
acid as external standard for the P spectrum. Purity of the
synthesized compounds was determined by a Waters HPLC system
Delta 600) or LC-MS system (Waters 2545 binary gradient module).
(
The active site of ODCase is fairly small and rigid, with four
conserved catalytic amino acid residues, Lys42, Asp70, Lys72,
Mass spectra (ESI) were recorded on a Waters LC/MS system
equipped with a Waters 3100 mass detector. HPLC methodology was
used to determine purity of the final compounds, and their purity is
≥95%. All enzyme assays were performed at either 37 or 55 °C using a
VP-ITC microcalorimeter (MicroCal, Northampton, MA) according
B
8
and Asp75 , in its center. Despite the restricted environment,
both pyrimidine- and purine-based nucleotides fit in the
9
,10
ODCase active site exposing its plasticity.
ODCase is
1
3
to previously published procedures. The pH of the buffers was
naturally evolved to bind to nucleotides carrying uracil-type
nucleobases, considering its dedicated function in the cell for
the decarboxylation of 2 to yield 1. Additionally, the binding
conformation of 1 in the enzymatic pocket of ODCase gives
insight into the types of preferable interactions and binding
positions within the active site. It has been reported that the
nucleobase of cytidine-5′-monophosphate (CMP) binds to the
active site of ODCase in a mode different from that of 1. In this
binding mode, the phosphate and ribosyl moieties sit in a
similar fashion as found for 1, but the pyrimidine assumes a
completely different orientation as well as conformation, almost
measured with a Corning 430 pH meter.
Synthesis. 4-Amino-1-(3,4-dihydroxy-5-hydroxymethyl-tetrahy-
drofuran-2-yl)-1H-pyrimidin-2-one-3-oxide (Cyd-N -oxide, 13). Cy-
3
tidine (100 mg, 0.41 mmol) was suspended in anhydrous methanol (4
mL). m-Chloroperbenzoic acid (213 mg, 1.23 mmol) was added in
fractions to the reaction suspension at 0 °C. The reaction mixture was
allowed to stir at 0 °C for 15 min. After 24 h of stirring at room
temperature, the reaction solvent was evaporated to dryness. Water (4
mL) was added to precipitate m-chlorobenzoic acid. The byproduct
was filtered off, and the filtrate was purified by reverse phase
chromatography on HPLC (Supporting Information). Fractions
containing the compound of interest were mixed together and
1
1,12
reaching outside of the binding pocket.
Due to its unusual
1
lyophilized to give compound 13 as a white solid (62 mg, 58%). H
binding conformation in the active site of ODCase, CMP (11)
fails to undergo optimum interactions and is thus unable to
effectively inhibit the catalytic site. This is further demonstrated
NMR (D O) δ 3.85 (dd, J = 12, 44 Hz, 2H, 5′ and 5″), 4.13−4.19 (m,
2
2
H, 4′ and 3′), 4.35 (dd, J = 3.36, 1.16 Hz, 1H, 2′), 5.87 (d, J = 3.24
Hz, 1H, 1′), 6.31 (d, J = 8.00 Hz, 1H, 5), 8.04 (d, J = 8.04 Hz, 1H, 6).
+
by the difference in K values between 1 and CMP for the
ESI (+) m/z calcd for C H N O [M + H ] 260.09, found 260.11 Da.
i
9
13
3
6
inhibition of HsODCase (220 ± 10 and 1200 ± 700 μM,
(5-(4-Amino-3-oxido-2-oxopyrimidin-1-yl)-3,4-dihydroxyoxolan-
2-yl)-methyl Dihydrogen Phosphate (CMP-N -oxide, 14). Com-
pound 14 was synthesized similar to compound 13 from CMP (100
mg, 0.27 mmol). The product was purified by HPLC using a reverse
phase semipreparatory column (Supporting Information). Fractions
containing the desired compound were collected and lyophilized. The
3
9
respectively).
N-Modified CMP derivatives, especially at the N3 and N4
positions of CMP, were conceived as novel ligands to ODCase
(Chart 2), with the anticipation to create novel interactions for
free acid form of compound 14 was neutralized by NH OH to yield
4
3
1
Chart 2. Design of Unusual CMP Analogs
the ammonium salt of CMP-N -oxide (48 mg, 42%). H NMR (D O)
2
δ 4.04 (dd, J = 12.24, 37.16 Hz, 2H, 5′ and 5″), 4.26 (m, 1H, 4′),
4
7
.30−4.36 (m, 2H, 3′and 2′), 5.98 (d, J = 2.6 Hz, 1H, 1′), 6.36 (d, J =
31
.84 Hz, 5), 8.10 (d, J = 7.6 Hz, 6). P (D O) δ −3.647. ESI (−) m/z
2
+
calcd for C H N O P[M − H ] 339.05, found 338.21 Da.
9
14
3
9
1
-(3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-
4
(
hydroxyamino)pyrimidin-2[1H]-one (N -OH-Cyd, 15). Hydroxyl
14
ammonium acetate was synthesized by adjusting the pH of 5 N
NH OH with acetic acid to 5.98. Cytidine (50 mg, 0.20 mmol) was
2
dissolved in 2.5 mL of hydroxyl ammonium acetate at room
temperature. The reaction mixture was then warmed to 37 °C and
stirred for 15 h. The reaction solvent was evaporated, and the reaction
crude was dissolved in 2 mL of water. It was then purified by reverse
phase chromatography on HPLC (Supporting Information). Aqueous
fractions containing the target compound were collected and
1
lyophilized to yield compound 15 (32 mg, 60%). H NMR (D O) δ
2
3
.85 (dd, J = 12, 44 Hz, 2H, 5′and 5″), 4.07 (br m, 1H, 4′), 4.18 (br
dd, J = 4.68, 5.40 Hz, 1H, 3′), 4.29 (br dd, J = 5.28, 5.00 Hz, 1H, 2′),
.84−5.86 (m, 2H, 1′ and 5), 7.34 (d, J = 8.28 Hz, 1H, 6). ESI (+) m/
5
+
z calcd for C H N O [M + H ] 260.09, found 260.11 Da.
9
13
3
6
4
-Amino-1-(6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-
d][1,3]dioxol-4-yl)pyrimidin-2[1H]-one (24). In a flame-dried flask, a
suspension of cytidine (500 mg, 2.05 mmol) in anhydrous acetone (40
mL) was cooled to 0 °C and treated with conc. H SO (0.25 mL)
the CMP ligands in the ODCase binding site, as well as to
improve on its affinity. Thus, N3 and N4 oxygen-substituted
cytidine-based derivatives were synthesized and screened for
their inhibition of ODCases. Here, we disclose the chemistry
and biochemistry of N3- and N4-substituted CMP derivatives
in the context of the therapeutic target, ODCase.
2
4
dropwise. After stirring at room temperature for 12 h, the reaction
mixture was neutralized by NH OH and purified over silica gel (10%
4
MeOH in DCM). Organic fractions containing the compound of
interest were evaporated to obtain compound 24 as a white solid (547
9
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1
mg, 93%). H NMR (CDCl ) δ 1.34 (s, 3H, iso CH ), 1.55 (s, 3H, iso
Data Analysis. The raw kinetic data obtained from isothermal
titration calorimetry (ITC) experiments were analyzed using the
Origin 7.0 program. Raw and analyzed kinetic data sets of inhibitor 14
with MtODCase are provided in Figure 1. The reaction progress
3
3
CH ) 3.83 (dd, J = 35.42, 11.16 Hz, 2H, 5′ and 5″), 4.30 (br m, 1H,
′), 4.96 (br m, 1H, 3′), 5.02 (br m,1H, 2′), 5.57 (br s, 1H, 1′), 6.00
d, J = 7.36 Hz, 1H, 5), 7.57 (d, J = 7.44 Hz, 1H, 6).
6 - ( 4 - A m i n o - 2 - o x o p y r i m i d i n - 1 ( 2 H ) - y l ) - 2 , 2 -
3
4
(
(
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl Diethyl Phos-
phate (25). Compound 24 (547 mg, 1.93 mmol) was dissolved in 2
mL of anhydrous pyridine. The reaction mixture was cooled to 0 °C
and diethyl chlorophosphate (333 mg, 1.93 mmol) was added
dropwise. The reaction was stirred at 0 °C for 30 min and then for
an additional 15 min at room temperature. The reaction was quenched
with 2 mL of MeOH and purified over silica gel (10% MeOH in
1
DCM) to give compound 25 as a white solid (479 mg, 67%). H NMR
(
3
CDCl ) δ 1.25−1.27 (br, m, 9H, 3 CH ), 1.48 (s, 3H, iso CH ),
3
3
3
.65−4.27 (m, 7H, 5′, 5″, 4′ and diethyl 2CH ), 4.80−4.89 (m, 2H, 3′
2
and 2′), 5.75 (br s, 1H, 1′), 6.28 (br s, 1H,5), 7.63 (br s, 1H, 6), 8.59
(
br s, 1H, NH), 9.16 (br s, 1H, NH).
Diethyl(6-(4-(hydroxyamino)-2-oxopyrimidin-1(2H)-yl)-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl Phosphate
(26). Compound 25 (450 mg, 1.07 mmol) was dissolved in 1 mL of
ethanol. Hydroxyl ammonium acetate (pH 6.5, 5N, 2 mL) was added
to it, and the mixture was stirred at 37 °C for 17 h. The reaction crude
was then purified over silica gel (9% MeOH in DCM). Organic
fractions containing the desired compound were mixed together and
1
concentrated to yield compound 26 (45 mg, 9%). H NMR (CDCl )
3
δ 1.30−1.31 (m, 9H, iso CH and diethyl 2CH ), 1.53 (s, 3H, iso CH ),
3
2
3
4
4
6
.08−4.24 (m, 7H, 5′, 5″, 4′, and diethyl 2CH ), 4.80 (br m, 1H, 3′),
2
.88 (br m, 1H, 2′), 5.68 (br s, 1H, 1′), 6.70 (d, J = 7.36 Hz, 1H, 5),
.92 (br s, 1H, NH), 7.48 (d, J = 6.52 Hz, 1H, 6).
(
3,4-Dihydroxy-5-(4-(hydroxyamino)-2-oxopyrimidin-1(2H)-yl)-
tetrahydrofuran-2-yl)methyl Phosphate (27). Compound 26 (37
mg, 0.08 mmol) was dissolved in 1 mL of anhydrous DCM and cooled
to 0 °C. TMSBr (130 mg, 0.84 mmol) was added to the reaction
mixture and stirred for 10 min at 0 °C. After additional stirring at
room temperature for 30 min, the reaction mixture was concentrated,
and 1 mL of water was added to it. The aqueous reaction mixture was
then purified over HPLC by reverse phase chromatography
(Supporting Information). Aqueous fractions containing compound
2
7 were lyophilized and neutralized by NH OH to yield the
4
1
ammonium salt of the target compound 27 (10 mg, 33%). H NMR
(
D O) δ 3.93 (br, m, 2H, 5′ and 5″), 4.18 (m, 1H, 4′), 4.29 (dd, J =
2
2
8
1
.84, 2.30 Hz, 1H, 3′), 4.36 (dd, J = 5.60, 6.12 Hz, 1H, 2′), 5.80 (d, J =
.24 Hz, 1H, 5), 5.92 (d, J = 6.44 Hz, 1H, 1′), 7.26 (d, J = 8.08 Hz,
31
H, 6). P (D O) δ 3.39. ESI (−) m/z calcd for C H N O P[M −
2
9
14
3
9
+
H ] 339.05, found 338.21 Da.
Enzymology. ODCases from Methanobacterium thermoautotrophi-
cum (Mt), Plasmodium falciparum (Pf), and Homo sapiens (Hs) were
4,12
cloned, expressed, and purified, as described earlier.
Enzyme
activity measurements were performed at 55 °C (MtODCase) and 37
°
C (Pf- and HsODCases) on the isothermal titration calorimeter (VP-
13
ITC, MicroCal, Northampton, MA) as described previously.
Enzyme Inhibition. The inhibition of ODCase from different
species by compounds 14 and 27 was evaluated by the competitive
1
3
Figure 1. Double-reciprocal plots for compound 14 against Mt- (A),
Hs- (B), and Pf ODCases (C).
inhibition method. Enzyme stock samples were prepared in assay
buffer (50 mM Tris, 40 mM NaCl, and 20 mM DTT) and incubated
overnight at room temperature. Assay samples were prepared in 50
mM Tris, 20 mM DTT buffer, pH 7.5. Small aliquots of enzyme stock
were diluted into assay buffer to give a final enzyme concentration of
curves obtained from the ITC experiment at various concentrations of
inhibitor (raw data) are represented in Figure 1A. The inhibition
20 nM (Mt), 60 nM (Hs), and 60 nM (Pf). Control reactions, without
constant (K ) was derived by converting the raw data from inhibited
i
inhibitor, were initiated by a single injection of 2. The final substrate
concentration was 40 μM (Mt), 20 μM (Hs), and 12 μM (Pf). The
final assay concentrations of compound 14 were as follows: for
MtODCase, 0, 15, 30, 50, and 100 μM; for HsODCase, 0, 25, 50, 100,
and 150 μM; for Pf ODCase, 0, 25, 50, and 100 μM. The
concentrations of compound 27 in the assay sample were 0, 25, 50,
reactions to Michaelis−Menten curves (Figure 1B) by use of the
Substrate Only mode in the Origin software to fit the raw data. From
this typical rate versus substrate concentration, [S], plot, reaction rate
at each substrate concentration in the absence and presence of the
inhibitor could be computed. The data from inhibition assays were
plotted using the double reciprocal plots (1/rate vs 1/[S]) at various
concentrations of inhibitor to confirm that the inhibition is
competitive in nature (Figure 1C). For a detailed description of the
data analysis, please refer to ref 13.
100, and 150 μM for the HsODCase. In the competitive inhibition
assay of compounds 14 and 27, the reaction was initiated by addition
of the substrate 2 to the mixture of enzyme and inhibitor. Reactions
were performed in triplicate.
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Crystallographic Analyses. All HsODCase concentrations were
determined using a BioRAD protein assay kit and BSA as a standard.
ODCase protein was dissolved in 20 mM Tris (pH 8.0), 10 mM NaCl.
HsODCase complex crystals were grown in hanging drops using 1.6−
Table 1. Inhibition Parameters (K ) for Nucleotide Ligands
against ODCases from Mt, Hs, and Pf
i
ODCase inhibition, K
(μM)
i
1.7 M ammonium sulfate, pH 7.0−9.0, as the precipitant. All crystals
Ligand
Mt
Hs
Pf
grew in space group P2 , with unit cell dimensions deviating less than
1
1
330 ± 10
1200 ± 700
11.1 ± 0.4
220 ± 100
1400 ± 100
28.3 ± 1.5
210 ± 10
>10000
1% from a = 69.8 Å, b = 61.6 Å, c = 71.4 Å, and β = 112.5°. For data
CMP
14
collection, the crystals were cryoprotected by 25% glycerol or
Parotone-N oil before flash-freezing in a stream of boiling nitrogen.
Diffraction data for the crystals of HsODCase cocrystallized with
compound 27 were collected at 100 K and λ = 0.979 490 Å on
beamline 08ID-1 at the Canadian Macromolecular Crystallography
22.1 ± 3.2
>1000
27
the active site. The ribose ring adopts the 3′-endo conformation
15
Facility, Canadian Light Source, Inc. All data were reduced and
11,12
and the base is oriented anti to the ribose ring.
16
scaled using XDS. The diffraction data for HsODCase cocrystallized
with compound 14 were collected at 100 K and λ = 1.541 79 Å in-
house on a Rigaku 007 rotating anode equipped with multilayer optics
and a MAR345 IP area detector. Data were reduced and scaled using
In the case of uracil-based nucleotide ligands, the backbone
-
NH- of Ser127 in the binding pocket of ODCase interacts with
the O4 atom of the uracil ring while the Ser127 side chain
OH serves as a hydrogen bond acceptor to the N3 proton.
17
−
HKL2000. Data collection and refinement statistics for all data sets
are given in Table S3 (Supporting Information). The structures of all
complexes were determined using molecular replacement techniques
Earlier structural studies by Wu et al. with the S127A ODCase
mutant showed that the hydrogen bonds with the side chain of
Ser127 residue are important for the correct placement of the
18
with the help of the program package MOLPREP; subsequent
19
23
refinements were done with Refmac-5.5 and model building using
uracil ring in the active site. In the case of CMP, the
phosphate and ribosyl moieties are held in place by similar
hydrogen bonding patterns to the one observed with 1. The
cytosine base, however, adopts an anti instead of a syn
conformation. In this conformation the cytosine moiety is
pointing away from the Ser127 residue. Even if the cytosine
position were more favorable, the tertiary N3 could not act as
hydrogen bond donor to Ser127 nor is there any other suitable
atom to form a hydrogen bond interaction with backbone
atoms, as was seen in the case of 1 (Figure 2 and 3C).
2
0
COOT. Atomic coordinates and structure factors have been
deposited into the Protein Data Bank (PDB IDs: 4HIB and 4HKP).
In Vitro Parasitology. Human malaria parasites were grown in O
blood obtained by venipuncture of volunteers. Cultures of the
laboratory line ItG were maintained by the method of Trager and
21
Jensen using RPMI 1640 supplemented with 10% human serum and
0 μM hypoxanthine. In vitro viability assays were performed using the
5
22
SYBR Green method. Briefly, the compound was dissolved in RPMI
1640 to a final concentration of 10 mg/mL and was then sterilized by
passing it through a 0.22 μM filter. The compound was then serially
diluted across a 96 well plate (concentrations up to 500 μg/mL)
containing a constant number of parasites per well, with the final wells
in the series receiving no compound. After 48 h in an atmosphere of
3
% CO , 2% O , and 95% N , the growth of the parasites in individual
2 2 2
wells was determined by adding 100 μL of a solution containing 2 μL
of SYBR Green stock solution in 11 mL of a solution containing buffer
and Triton X-100. The relative fluorescence was determined using a
GMG fluorescent plate reader and was compared with the results
obtained with a chloroquine control. Assays were done in
quadruplicate. The concentration of compound that inhibited the
growth of 50% of the cultures (IC50 values) was determined using a
nonlinear regression analysis of the dose−response curve using the
following equation:
Abs = Nmax(1 − ([I]/([I] + IC ))) + Bkg
50
where the experimentally observed absorption (Abs) is fit to the
inhibitor concentrations, [I]. Sigma Plot 2000 (Jandel Scientific) was
then used to best-fit and determine the observed values for completely
dead parasites (Bkg), the difference between Bkg and completely alive
parasites (Nmax, i.e., when no inhibitor is present), and the 50% value
Figure 2. Hydrogen bonding interactions between 1 and the various
active site residues of HsODCase.
(
IC ) between Bkg and Nmax.
50
Therefore, we envisioned that modification of the N3 and N4
positions of CMP with oxygen may improve its potential to
interact within the active site and increase its affinity for
ODCase. Thus, the synthesis of compounds 14 and 27 was
attempted.
RESULTS AND DISCUSSION
■
In our earlier study, we reported the plasticity of the ODCase
catalytic site toward purine and pyrimidine nucleotide ligands
and discussed their unusual mode of binding.
pyrimidine nucleotide, is a weak inhibitor of ODCase with a Ki
of 1200 ± 700 and 1400 ± 100 μM against Mt- and
HsODCases, respectively (Table 1). K values were determined
9,12
CMP, a
These compounds could be synthesized from either cytidine
24
or CMP. The basic tertiary nitrogen of cytidine (pKa 4.2) and
24
CMP (pKa 5.6) was oxidized with meta-chloroperbenzoic
i
3
3
in ITC-based enzymatic assays as reported in earlier work
acid (m-CPBA) to yield cytidine-N -oxide (Cyd-N -oxide, 13)
25
1
3
3
4
(
Figure 1). Structurally, the main difference between CMP
and CMP-N -oxide (14), respectively (Scheme 1). N -
hydroxy-cytidine (N -OH-Cyd, 15) was obtained by reacting
4
and 1 is an amino moiety replacing the ketone moiety at the C4
position of CMP. Regardless of its close similarity to 1, CMP
binds to ODCase in an unusual position, retaining its low-
energy conformation, with its nucleobase projecting away from
cytidine with hydroxyl ammonium acetate (pH 6.5). Synthesis
4
of N -OH-CMP (27) required the protection of the 2′- and 3′-
hydroxyl moieties of cytidine with isopropylidene and the 5′-
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Journal of Medicinal Chemistry
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a
Scheme 1. Synthesis of Cytidine Derivatives
a
Reagents and conditions: (i) m-CPBA, methanol, rt; (ii) 5 N
hydroxyl ammonium acetate, pH 6.5, 35 °C.
a
Scheme 2. Synthesis of Compound 27
a
+
Reagents and conditions: (i) acetone/H , rt; (ii) diethylchlorophos-
phate, py, 0 °C to rt; (iii) 5 N hydroxyl ammonium acetate, 35 °C, 13
h; (iv) TMSBr, DCM, 0 °C to rt.
26−28
on CMP.
For the compounds oxidized at the N3 position
Figure 3. Comparison of the bound conformations of 1, CMP, and
compound 27 in ODCase active site. (A) HsODCase bound by 1
(13 and 14), an orange-red color was observed, whereas for the
N4-oxidized cytidine derivatives 15 and 27, a blue color was
observed (Figure 5). The interactions between the mono-
nucleotide derivatives 14 and 27, and ODCase were
investigated, and the nucleoside derivatives 13 and 15 were
evaluated in cell-based assays.
(
yellow) superimposed onto MtODCase complexed with CMP
(
cyan); (B) HsODCase bound by 1 (yellow) superimposed onto
4
HsODCase bound to N -OH-CMP (27) (green); (C) MtODCase
with CMP (cyan) superimposed onto HsODCase bound to 27
(
green).
3
CMP-N -oxide (14) inhibited Mt-, Hs-, and Pf ODCases
reversibly with K values of 11.1 ± 0.4, 28.3 ± 1.5, and 22.1 ±
i
hydroxyl moiety with diethyl chlorophosphate followed by
treatment with hydroxyl ammonium acetate at pH 6.5. All
protecting groups were removed by trimethylsilyl bromide
3.2 μM, respectively (Table 1). Compared with CMP,
compound 14 showed a 100-fold increase in ODCase
inhibition. Compound 27, which is a derivative of CMP at
the N4 position, did not display significant inhibition of
Pf ODCase up to a concentration of 1 mM of the inhibitor.
Antiplasmodial activities against P. falciparum of nucleosides
13 and 15 (nucleoside forms of 14 and 27, respectively) were
determined by a fluorescence-based SYBR-green assay.
Inhibitory concentrations (IC ) for compounds 13 and 15
(TMSBr) to yield the desired compound 27 (Scheme 2).
Compounds 13, 14, 15, and 27 were characterized confirming
the structures unambiguously as the desired cytidine and CMP
derivatives by infrared spectroscopy (Figures S1−S4, Support-
1
ing Information), and H NMR spectroscopy (Figure 4, and
Figures S5−S8, Supporting Information). Additionally, chem-
ical analyses using the classical ferric chloride test were
employed to characterize the oxidation at N3 vs N4 position
50
were 411 ± 22 and 610 ± 38 μg/mL against P. falciparum ItG,
respectively. Compound 13 showed better potency than the
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orientations of these two rearranged products in the active
site of ODCase and the corresponding interactions with the
active site residues are similar. In fact, the orientation and the
binding site interactions of the rearranged products are also
similar to the ones observed in the cocrystal structure of 1 with
human ODCase. This is indeed a surprising outcome since
CMP itself binds in atypical fashion in the binding site of
ODCase in comparison to other pyrimidine nucleotides
(
Figures 2, 3, and 6E,F).
The rearranged ligand N -OH-CMP from compound 14
4
engaged in three hydrogen bond interactions with Ser183, one
with the backbone −NH−, one with the side chain hydroxyl
moiety, and a third one with a tightly bound water molecule
(
H O637) in the ODCase active site (Figure 6E). The second
2
rearranged ligand, 5-OH-CMP from compound 14, formed two
hydrogen bonds: one between N3 of the ligand and the side
chain of Ser183 and the other between the 4-amino moiety and
the same water molecule H O637 (Figure 6F).
2
It was indeed intriguing to observe these unusual products
4
3
N -OH-CMP and 5-OH-CMP, when CMP-N -oxide (14) was
incubated with HsODCase. However, our laboratories have
observed such unusual and unexpected behavior for various
ligands in the presence of ODCase, for example, with 6-cyano-
1
Figure 4. H NMR spectra for compounds 14 (A) and 27 (B).
29−31
4
UMP (4), 6-iodo-UMP (6), 6-azido-UMP (5), and others.
N -hydroxy derivative 15, a trend that correlates with the
Interestingly, pyrimidine-N-oxides are known to undergo
weaker enzymatic inhibition by the nucleotide derivative 27. In
vitro cellular activities of these cytidine nucleosides are also a
reflection of the fact that these compounds have entered the
cell and were phosphorylated by a kinase in order to inhibit the
target enzyme, ODCase.
Dimroth rearrangements, a classical mechanism where
2
8,32
endocyclic and exocyclic nitrogens switch places.
In fact,
the Dimroth rearrangement, also known as amidine rearrange-
3
ment, was reported on cytidine-N -oxide (13) under alkaline
33
3
4
conditions. Presumably, if one were to accept the idea that a
Dimroth rearrangement occurred in the binding site of
ODCase after the binding of compound 14, Figure 6G outlines
a plausible mechanism. According to this, the electron-
withdrawing nature of the N3-oxide of 14 weakens the C2−
N3 bond (Figure 6G, species II), and the labile C2−N3 bond
yields to the acyclic reactive isocyanate species III with the
participation of the lone pair of electrons (Figure 6G).
Subsequently, a 180° rotation around the C4−C5 bond
CMP-N -oxide (14) and N -OH-CMP (27) were cocrystal-
3
lized with HsODCase to investigate the influence of N -oxide
and N -hydroxy substitutions, respectively, on the ligand
4
orientations and their interactions in the ODCase active site.
These cocrystal structures revealed very interesting outcomes.
In the X-ray crystal structure of compound 14 bound to
HsODCase, migration of oxygen was observed from N3 to N4,
as well as to the C5 position (Figure 6). Compound 27 was
bound to HsODCase in both cis and trans conformations
exhibiting an orientation and conformation similar to that with
4
followed by the ring closure via species IV leads to the N -
OH-CMP product, observed as one of the two products bound
in the active site of ODCase. Alternately, species III could
undergo tautomerization yielding species VI, which then sets
the stage for a totally different outcome. Species VI, after a 180°
rotation of the N3−C4 and C4−C5 bonds, yields species VII.
This species could lead to the oxazete-like species VIII.
Oxazetes are relatively stable entities that could even be isolated
1
in the ODCase active site (Figure 7).
Crystals of compound 14 with ODCase proved to be difficult
to grow in comparison to cocrystals with other pyrimidine
derivatives in our laboratory. In the electron density map from
the cocrystal with 14, the pyrimidine portion exhibited changes
in the atomic arrangement. The oxygen atom migrated from
N3 to N4 or the C5 position of the cytosine ring (Figure
34,35
6
A,C,D). These rearranged or modified nucleotides, presum-
with appropriate stabilizing groups.
Further, N3−C2 ring
ably during the crystallization process, were each bound to
closure and deprotonation at C5 position followed by
ODCase in a 2′-endo,syn conformation. The binding
rearrangement of the electrons leads to species IX. Collapse
Figure 5. Ferric chloride color test for (A) deionized water alone, (B) CMP, (C) compound 14, and (D) compound 27. Deionized water and CMP
3
were used as control experiments (panels A and B). N -Oxidized compounds 13 and 14 produced the characteristic red-orange color as shown in
4
panel C. N -Oxidized compounds 15 and 27 produced deep blue color, as shown in panel D.
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Figure 6. X-ray crystal structure of the complex generated by incubating ODCase with compound 14. Panel A shows the rearranged products, N4-
OH-CMP and 5-OH-CMP, generated from 14 during cocrystallization with HsODCase. Panel B shows the electron density map and the resulting
4
products fit into the observed electron density for the rearranged products N -OH-CMP and 5-OH-CMP when 14 and HsODCase were
4
cocrystallized. Panels C and D show the products N -OH-CMP and 5-OH-CMP bound in the active site of HsODCase separately as stick models to
the experimental electron density map (2F − F ) displayed at a 1σ value. HsODCase is presented as a cartoon model and colored with respect to its
o
c
secondary structure (α-helix, red; β-sheet, yellow; loop, green). Catalytic residues of K92, D123, K125, and D128 from HsODCase are also shown as
capped-stick models. Panels E and F illustrate the hydrogen bonding networks between the various active site residues of HsODCase and the
4
4
rearranged products N -OH-CMP and 5-OH-CMP, respectively. Panel G shows a plausible rearrangement mechanism for ligand 14 to yield N -OH-
CMP and 5-OH-CMP: (a) ring-opening at C2−N3, (b) 180° rotation about C4−C5 bond leads to formation of the Z-isomer, species IV, (c) ring
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Article
Figure 6. continued
closing due to the intramolecular proton extraction by N-oxide from the amino moiety leading to species V, one of the two products identified in the
cocrystal structure, (d) amine−imine (N3 and N4) tautomerization, (e) first, rotation around C4−C5, followed by torsional rotation about C4−N3
leads to the E-isomer leading to species VII, (f) nucleophilic attachment of hydroxylamine onto C5 to yield species VIII, (g) deprotonation at C5
and followed by rearrangement of electrons to generate ene−amine species IX, then ring closure at C2−N3, followed by regeneration of the C5−C6
double bond and the migration of the oxygen from N4 to C5 leading to iminohydroxy species X, or its tautomer XI, the second product observed in
3
the binding site of ODCase cocrystallized with CMP-N -oxide (14).
Figure 7. X-ray crystal structure of the complex generated by incubating HsODCase with compound 27. (A) Active site region of the HsODCase
4
with ligand 27 bound in different conformations. (B) Amino−imino tautomers of 27 and the cis−trans imino isomers of the exocyclic N -OH moiety
with respect to the N3 of the pyrimidine. Panels C and D present compound 27 bound in the cis- and trans-conformation, respectively, in the active
site of HsODCase. The enzyme is presented as a cartoon model, colored with respect to the secondary structure (α-helix, red; β-sheet, yellow; loop,
green). Compound 27 is shown in capped stick representation and the electron density map (2F − F ) is rendered at a value of 1σ. Catalytic
o
c
residues K92, D123, K125, and D128 of HsODCase are shown as stick models.
of the C6 anion to generate C5−C6 double bond and
subsequent rearrangement leads to the formation of X with a
C5-OH moiety. Compounds X and XI are the amine−imine
tautomeric forms for 5-OH-CMP, and the latter species XI was
observed in the catalytic site of ODCase as the second product
spectrophotometric studies on compound 14. Thus, compound
14 was incubated with crystallization buffer, devoid of ODCase.
UV spectra were recorded for up to 24 h, the average time it
takes to generate crystals from the protein solution. There was
no change observed in the UV profiles of 14 even after this
prolonged exposure (Table S2 in Supporting Information).
This suggests that the rearrangement of 14 probably took place
during the cocrystallization process and presumably in the
active site of the enzyme itself.
3
when incubated with CMP-N -oxide (14). While there is no
proof that these complex rearrangements could happen in the
binding site of ODCase, the mechanism described above
provides a plausible explanation for what is observed when
3
4
ODCase is incubated with CMP-N -oxide (14). However,
In the complex of compound 27 (N -OH-CMP) bound to
ODCase was known for inducing unusual and energetically
demanding ligand transformations that would not happen
without the enzyme.
In order to rule out the possibility that the crystallization
buffer (Tris or HEPES) played any role in initiating or
accelerating the rearrangement of 14, we conducted UV
the active site of HsODCase, the nucleobase assumed a syn- and
the ribosyl moiety a 2′-endo-conformation. The 5′-O-phosphate
moiety of compound 27 maintained strong hydrogen bond
interactions with Gly261 and Arg262 and with three crystallo-
2
9,30
graphic water molecules H O2, -3, and -33. In fact, the
2
conformation of 27 in the active site of ODCase is similar to
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3
6
that observed with 1 (Figure 3B). Compound 27 could exis2t
Engineering Research Council, the National Research Council
Canada, the Canadian Institutes of Health Research, the
Province of Saskatchewan, Western Economic Diversification
Canada, and the University of Saskatchewan. This research was
funded in part by the Ontario Ministry of Health and Long
Term Care. The views expressed do not necessarily reflect
those of the OMOHLTC.
in two tautomeric forms, where the N4 group could assume sp
or sp hybridization, resulting in an imino or amino moiety,
respectively (Figure 7B). In an aqueous environment, it was
reported that the imino tautomer is more predominant, and the
dipole moment of the imino tautomer of N -OH cytosine is
Thus, it is conceivable that 27 binds
to the ODCase active site with a conformation similar to that of
3
4
37,38
close to that of uracil.
1
.
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(
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3
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In summary, CMP and 1, pyrimidine mononucleotides that
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(
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(
ASSOCIATED CONTENT
Supporting Information
■
2
(
*
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Purity and spectrometric data. This material is available free of
charge via the Internet at http://pubs.acs.org.
complexes reveal an alternate binding mode in orotidine-5′-mono-
phosphate decarboxaylase. J. Biol. Chem. 2002, 277 (31), 28080−
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AUTHOR INFORMATION
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(13) Poduch, E.; Bello, A. M.; Tang, S.; Fujihashi, M.; Pai, E. F.;
Corresponding Author
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Mailing address: Center for Molecular Design and Preformu-
lations, University Health Network, #5-356 MaRS/TMDT, 101
(
College Street, Toronto, ON M5G 1L7, Canada. Tel. (416)
581-7601. E-mail: lkotra@uhnres.utoronto.ca.
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Notes
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The authors declare no competing financial interest.
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