Journal of Medicinal Chemistry
Article
with 1 N HCl until pH 5 was reached. The mixtures were extracted
with ethyl acetate. The collected organic extracts were washed with
brine (three times), dried, and the solvent was evaporated under
reduced pressure to obtain pure derivatives 9a−g.
General Procedure for Diketo Esters 1a−r and 3a−g. The
appropriate acetyl derivative 6a−r or 9a−g (31 mmol) and diethyl
oxalate (62 mol) were dissolved in 31 mL of dry THF and treated,
under argon stream, with NaOEt obtained by the dissolution of Na
(63 mmol) in 56 mL of absolute ethanol. The mixture was stirred
at room temperature for 1 h 30 min, then was pured into n-hexane
(704 mL). The collected precipitate was vigorously stirred for 30 min
in 1 N HCl (704 mL). The solid that formed was filtered, washed with
water and light petroleum ether, and dried under IR lamp to afford the
pure diketo esters 1a−r and 3a−g.
General Procedure for Diketo Acids 2a−r and 4a−g. A
mixture of 1 N NaOH (9.5 mL) and the appropriate ester 1a−r or
3a−g (1.9 mmol) in 1:1 THF−methanol (9.3 mL) was stirred at room
temperature for 30 min and then poured into crushed ice. The mixture
was treated with 1 N HCl until pH 3 was reached and extracted with
ethyl acetate (three times). The collected organic extract was washed
with brine (three times) and dried, and the solvent was evaporated
under reduced pressure to give the pure diketo acids 2a−r and 4a−g.
Details concerning characterization data are reported in the
Supporting Information for compounds 1a−r, 2a−r, 3a−g, 4a−g,
5a−t, 6a−r, 7a−e, 8a−c, 9a−g.
Biological Assay. Enzymes and Proteins. Recombinant full
length human DNA polymerase λ was generated and purified as de-
scribed.25 After purification, the protein was >90% homogeneous, as
judged by sodium dodecyl sulfate (SDS)−polyacrylamide gel electro-
phoresis (PAGE) and Coomassie staining. Recombinant DNA poly-
merase β and TdT were from Trevigen.
DNA Polymerase Assay. Human DNA polymerase λ activity on
poly(dA)/oligo(dT)10:1 was assayed in a final volume of 25 μL
containing 50 mM Tris-HCl (pH 7.0), 0.25 mg/mL BSA, 1 mM
DTT, 0.5 mM MnCl2, 0.2 μM poly(dA)/oligo(dT)10:1 (3′-OH ends),
50 nM DNA polymerase λ, and 5 μM [3H]-2′-deoxythymidine 5′-
triphosphate (dTTP) (5 Ci/mmol), unless otherwise indicated in the
figure captions. All reactions were incubated for 15 min at 37 °C
unless otherwise stated, and the DNA precipitated with 10% tri-
chloroacetic acid. Insoluble radioactive material was determined by
scintillation counting as described. DNA polymerase β activity was
assayed as described.25
Terminal Deoxyribonucleotidyl Transferase Assay. DNA
polymerase λ and TdT terminal transferase activities were assayed
in a final volume of 25 μL containing 50 mM Tris-HCl (pH 7.0),
0.25 mg/mL BSA, 1 mM DTT, 0.5 mM MnCl2, 0.2 μM ss 27-mer
DNA oligonucleotide, unless otherwise stated. Enzymes and
[3H]dNTPs (10 Ci/mmol) were added as indicated in the figure
captions. All mixtures were incubated at 37 °C for 10 min, unless
otherwise indicated in the figures, and the DNA precipitated with 10%
trichloroacetic acid. Insoluble radioactive material was determined by
scintillation counting as described.26
colleagues.14,27 Human lymphoblastic leukemia MOLT-4 (TdT-
positive) and cervical adenocarcinoma HeLa (TdT-negative) cell
lines were grown at 37 °C in RPMI-1640 and Dulbecco’s modified
Eagle medium, respectively, both containing 10 mM glucose supple-
mented with 10% fetal calf serum and 100 units/mL each of penicillin
and streptomycin and 2 mmol/L glutamine. For each experiment,
cells were placed in fresh medium, cultured in the presence of
synthesized compounds (from 0.1 to 100 mm), and followed for
further analyses.
Cell viability was determined using the 3-[4,5-dimethylthiazol-2,5-
diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay. The test
is based on the ability of mitochondrial dehydrogenase to convert, in
viable cells, the yellow MTT reagent (Sigma Chemical Co.; St. Louis,
MO) into a soluble blue formazan dye. Cells were seeded into 96-well
plates to a density of 105 cells/100 μL well. After 24 h of growth to
allow attachment of adherent cells to the wells, compounds were
added at various concentrations (from 0.1 to 100 mM). After 24 or
48 h of growth and after removal of the culture medium, 100 μL/well
of medium containing 1 mg/mL of MTT was added. Cell cultures
were further incubated at 37 °C for 2 h in the dark. The solution was
then gently aspirated from each well, and the formazan crystals within
the cells were dissolved with 100 μL of dimethylsulfoxide (DMSO).
Optical densities were read at 550 nm using a Multiskan Spectrum
Thermo Electron Corporation reader. Results were expressed as per-
centage relative to vehicle-treated control (0.5% DMSO was added to
untreated cells). IC50 (concentration eliciting 50% inhibition) values
were determined by linear and polynomial regression. Experiments
were performed in triplicate.
Molt-4 and HeLa cells (2.5 × 105 cells/mL) in 12-well tissue culture
plates were incubated for 24 h in the presence or absence (vehicle-
treated cells) of 3e, 2b, and 4g compounds. Cells were then washed
with phosphate buffered saline (PBS) 1× and suspended by tryp-
sinization. Cells were centrifuged at 2000 rpm for 5 min, then washed
with PBS 1× and resuspended in fresh medium. Finally, cells were
incubated in the dark with a staining solution containing 0.1% sodium
citrate, 0.1% Triton X-100, and 50 mg/mL propidium iodide at 4 °C
for 30 min. Samples were analyzed by Necton Dickinson FACScan
flow cytometer. Cell cycle distribution, expressed as percentage of
cells in the G0/G1, S, and G2/M phases, was calculated using ModFit
LT 3.0 software. Apoptotic cells are expressed as percentage of
hypodiploid nuclei.
Cells were plated in flasks (1 × 106 cells) in normal culture con-
ditions and incubated with or without 3e, 2b, and 4g compounds. At
the indicated times, cells were lysed using an ice cold lysis buffer
(50 mM Tris, 150 mM NaCl, 10 mM ethylenediaminetetraacetic
acid (EDTA), 1% Triton) supplemented with a mixture of protease
inhibitors containing antipain, bestatin, chymostatin, leupeptin, pep-
statin, phosphoramidon, Pefabloc, EDTA, and aprotinin (Boehringer,
Mannheim, Germany). Equivalent amounts of protein were loaded
on 8−12% sodium dodecyl sulfate (SDS)−polyacrylamide gels and
electrophoresed followed by blotting onto nitrocellulose membranes
(Bio-Rad, Germany). After blotting with 5% (w/v) fat-free milk
powder and 0.1% Tween 20 in TBS, the membrane was incubated
overnight at 4 °C with specific antibodies against cyclin E2, cyclin A,
p53, and caspase 3 at the concentrations indicated by the manufacter’s
protocol (Santa Cruz Biotechnology). The antibody was diluted in
Tris-buffered saline/Tween 20 5% milk powder. Following incubation
with horseradish peroxidase-conjugated secondary antibodies, bands
were detected by enhanced chemiluminescence (ECL kit, Amersham,
Germany). Each filter was then probed with rabbit polyclonal anti-
actin (Santa Cruz Biotechnology). Level of expression of detected
bands was quantified by NIH ImageJ 1.40 after normalization with
actin.
Inhibition Assays. Reactions were performed under the
conditions described for the terminal deoxyribonucleotidyl transferase
activity assay. Incorporation of radioactive dTTP into the ss 27-mer
oligodeoxynucleotide at different concentrations of DNA or dNTP
was monitored in the presence of increasing amounts of inhibitor as
indicated in the figure captions. Dose−response curves were generated
by computer fitting of the data to the relationship E(%) = Emax/(1 + I/
ID50) where E(%) is the fraction of enzyme’s activity measured in the
presence of the inhibitor, Emax is the activity in the absence of the
inhibitor, I is the inhibitor concentration, and ID50 is the inhibitor
concentration at which E(%) = 0.5Emax. The ID50 values at different
substrate concentrations where used to determine the Ki, according to
the Cheng−Prusoff relationship for a fully competitive model, in the
form ID50 = Ki(1 + S/Km) where S is the concentration and Km is the
apparent affinity of the competing substrate.
Details concerning the biological results are reported in the
Supporting Information for 1a−r, 2a−r, 3a−g, and 4a−g.
Structural Study. Crystallization and Data Collection. TdT
(10 mg/mL) was incubated with inhibitors (1 mM final) for 30 min at
4 °C prior to setting up crystallization drops. All crystals were grown
overnight at room temperature. Complexes with 4c were grown in
Greiner plates in a solution containing 22% polyethylene glycol (PEG)
Cell-Based Assay. The TDT+ cell line MOLT-4 (a human early T
cell leukemia cell line) and the TDT-cell line HeLa were used. The
TdT status of MOLT-4 cells has been described by McCaffrey and
7439
dx.doi.org/10.1021/jm4010187 | J. Med. Chem. 2013, 56, 7431−7441
Costi, Roberta
