Inhibitor of Orotidine 5′-Monophosphate Decarboxylase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 917
stirred for an additional 1 h. The solvent was evaporated under
vacuum, and the crude material was dissolved in ethyl acetate
(30 mL), washed with water (15 mL) and brine (15 mL), and
dried (Na2SO4). Evaporation of the solvent and purification of
the crude by column chromatography (5% MeOH in CHCl3)
yielded compound 5 (0.27 mg, 96% yield) as a foam: 1H NMR
(CDCl3) δ 0.10 (s, 6H, CH3), 0.90 (s, 9H, CH3), 1.36 (s, 3H,
CH3) 1.59 (s, 3H, CH3), 3.79 (dd, 1H, H-5′), 3.92 (dd, 1H,
H-5′′), 4.30-4.33 (m, 1H, H-4′), 4.67 (dd, 1H, H-3′), 4.75 (dd,
1H, H-2′), 5.66 (d, 1H, H-5), 5.96 (d, 1H, H-1′), 7.68 (d, 1H,
H-6), 8.47 (brs, 1H, -NH).
modified for infusion cloning and addition of an N-terminal
hexahistidine affinity tag. The proteins were expressed in E.
coli BL21 (DE3) CodonPlus-RIL in Terrific Broth (TB) in the
presence of kanamycin/ chloramphenicol (50 µg/mL and 25 µg/
mL, respectively). The cultures were incubated at 37 °C in a
water bath with aeration and mixing provided by means of
bubbling air at a rate between 4 and 6 L/min. The cultures were
cooled to 15 °C when the OD600 reached 5. After addition of
isopropyl 1-thio-D-galactopyranoside (IPTG) 0.4 mM, they were
incubated at 15 °C overnight. The cultures were harvested by
centrifugation, and the cell pellets were suspended in 160 mL
of binding buffer (50 mM HEPES, pH 7.5, 0.5 M NaCl, 5%
glycerol, and 15 mM imidazole) with protease inhibitors (1 mM
benzamidine-HCl and 1 mM phenylmethylsulfonyl fluoride,
PMSF) added. The suspensions were kept in 50 mL Falcon tubes
at -80 °C.
Before purification, the cell suspensions were thawed over-
night at 4 °C. Prior to mechanical lysis, each tube of cell
suspension was pretreated with 0.5% CHAPS and 500 units of
benzonase (per 40 mL of resuspended cell pellet) for 40 min at
room temperature. Subsequently, the cells were mechanically
lysed in a microfluidizer (Microfluidizer Processor, M-110EH)
at 18 000 psi. The lysates were centrifuged at 24 000 rpm for
20 min at 10 °C. Each cleared cell lysate was loaded onto a
column containing 10 g DE-52 resin (Whatman) and then
directly onto a 3 mL Ni-NTA (Qiagen) column. When all the
lysate was loaded, the two-column system was washed with 20
mL of binding buffer. The Ni-NTA column was then washed
with 200 mL of wash buffer (50 mM HEPES pH 7.5, 500 mM
NaCl, 30 mM imidazole, and 5% glycerol). After washing, the
protein was eluted from the Ni-NTA column with 15-20 mL
of elution buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 250
mM imidazole, and 5% glycerol). EDTA was added im-
mediately to 1 mM, with DTT added to 1 mM 15 min later.
The mixture was dialyzed overnight in 10 mM HEPES and 500
mM NaCl. The following day, it was concentrated using a 15
mL Amicon Ultra centrifugal filter device (Millipore). Protein
5′-O-(tert-Butyldimethylsilyl)-6-iodo-2′,3′-O-isopropylide-
neuridine (6). A stirred solution of LDA (0.62 mL, 1.3 mmol,
2.0 M solution in THF) in anhydrous THF (2 mL) was treated
with compound 5 (0.25 g, 0.6 mmol) dissolved in 1.5 mL of
anhydrous THF, at -78 °C. After stirring for 1 h, iodine (0.16
g, 0.6 mmol) in anhydrous THF (2 mL) was added and the
mixture was stirred for an additional 5 h at the same temperature.
The reaction was quenched with AcOH (0.3 mL) and then
brought to room temperature and dissolved in ethyl acetate (25
mL). The organic layer was washed with saturated NaHCO3
solution (10 mL), 5% Na2S2O3 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) gave 6 (0.224 g, 68%) as a yellow foam:
1H NMR (CDCl3) δ 0.06 (s, 6H, CH3), 0.89 (s, 9H, 3CH3),
1.35 (s, 3H, CH3) 1.56 (s, 3H, CH3), 3.76-3.86 (m, 2H, H5′,
H-5′′), 4.15-4.20 (m, 1H, H-4′), 4.81 (dd, 1H, J ) 4.2, 6.3
Hz, H-3′), 5.18 (dd, 1H, J ) 2.0, 6.3 Hz, H-2′), 6.09 (d, 1H, J
) 2.0 Hz, H-1′), 6.45 (s, 1H, H-5), 8.78 (brs, 1H, NH).
6-Iodouridine (7). A stirred suspension of compound 6 (0.300
g, 0.572 mmol) in water (2 mL) was treated with 50% aqueous
TFA (3 mL) at 0 °C, brought to room temperature, and stirred
for 2 h in the dark. Evaporation of the solvent and purification
of the crude by column chromatography (10-15% EtOH in
CHCl3) afforded compound 7 (0.182 g, 0.49 mmol, 86%) as a
1
light brown solid. UV (H2O): λmax ) 268 nm (ꢀ ) 8975); H
concentration was measured by taking absorbance at OD280
.
NMR (D2O) δ 3.77 (dd, 1H, H-5′), 3.91 (dd, 1H, H-5′′), 3.978-
4.032 (m, 1H, H-4′), 4.43 (t, 1H, H-3′), 4.84 (dd, 1H, H-2′),
6.06 (d, 1H, H-1′), 6.67 (s, 1H, H-5). HRMS (ESI) calculated
for C9H11N2O6NaI (M + Na+) 392.9554, found 392.9565.
Finally, aliquots of the purified protein were labeled and stored
at -80 °C.
P. falciparum Cultures and CHO Cell Assays. Assays
comparing the antiplasmodial activities of the inhibitors were
performed using the SYBR-Green method.24 Briefly, the inhibi-
tors were dissolved in DMSO to achieve a concentration of 10
mg/mL. Fifty microliters of RPMI-A were added to each well
in a 96-well plate before 40 µL of RPMI-A and 10 µL of
compound 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. Fifty microliters of parasite
culture (2% hematocrit, 2% parasitemia) were added to each
well, and the plates were then incubated at 37 °C in 95% N2,
3% CO2, and 2% O2 for 72 h.
6-Iodouridine 5′-Monophosphate (3). A stirred solution of
H2O (0.034 g, 1.89 mmol) and POCl3 (0.28 mL, 2.97 mmol) in
anhydrous acetonitrile (3 mL) was treated with pyridine (0.261
mL, 3.24 mmol) at 0 °C and stirred for 10 min. Compound 7
(0.250 g, 0.67 mmol) was added, and the mixture was stirred
for an additional 5 h at 0 °C. The reaction mixture was then
quenched with 25 mL of cold water and continued stirring for
an additional 1 h. 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 3 (0.207 g,
68%) as a syrup. The monophosphate compound was trans-
formed into the ammonium salt by neutralization with 0.5 M
NH4OH solution at 0 °C and freeze-dried to obtain the
ammonium salt as a pale yellow powder. UV (H2O): λmax
)
1
267 nm (ꢀ ) 2890); H NMR (D2O) δ 3.78 (dd, 1H, H-5′),
3.91 (dd, 1H, H-5′′), 3.98-4.03 (m, 1H, H-4′), 4.43 (t, H-3′),
4.84 (dd, 1H, H-2′), 6.05 (d, 1H, H-1′), 6.67 (s, 1H, H-5). 31P
NMR (D2O) δ ppm 2.214. HRMS (ESI, negative) calculated
for C9H11N2O9PI (M-) 448.9252, found 448.9263.
CHO cells (ATCC, Manassas, VA) were grown in RPMI-
1640 supplemented with 10% fetal calf serum (Sigma, St. Louis,
MO), 25 mM HEPES, and gentimicin (RPMI-10). Cells were
seeded in 96-well plates and 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. Compound gradients were prepared by adding
90 µL of RPMI-10 mixed with 10µL of compound solution to
the first well in the series, mixing, transferring 100 µL to the
ODCase Cloning, Expression, and Purification. ODCase
from Methanobacterium thermoautotrophicum was produced as
described earlier (ref 14). The gene coding for ODCase from
Plasmodium falciparum was cloned into a pET28 vector