2894 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12
Roma et al.
From each series of experiments, in which the inhibitors were tested
in at least five concentrations, a percentage inhibition-concentration
curve was derived. From this curve, the IC50 value was calculated
as the concentration of inhibitor causing a 50% inhibition of the
aggregation.
Statistical Analysis. The IC50 values reported in Table 2 are
averages ((standard deviation) of those obtained from at least four
independent determinations carried out on different batches of
platelets (usually 5-8 batches). Statistical analysis was performed
using the Student’s t-test, considering significant the activity
difference between two compounds when corresponding to P <
0.05.
PDE3 Activity Assay. Platelet soluble cyclic nucleotide PDE3
was purified as in Weishaar et al.21 The enzyme activity was tested
on PDE3 isolated from human platelets according to Thompson
and Strada22 with minor modifications. Briefly, phosphodiesterase
activity was assayed after 5 min of preincubation at 30 °C of suitable
aliquots of the purified enzyme both in the solvent and in the
solutions containing the agents. The reaction started by the addition
of the substrate (0.1 µCi/µL [3H]cAMP and 0.1 µM cAMP). The
incubation, prolonged for 30 min at 30 °C, was stopped by boiling
samples for 90 s. The cooled mixtures were added to 25 µg/mL
5′-nucleotidase (5′-ribonucleotide phosphohydrolase from Crotalus
atrox venom: EC 3.1.3.5) and incubated for additional 15 min at
30 °C. The [3H]adenosine formed was eluted with 50% methanol
from 2 mL AG1 × 8 resin (200-400 mesh, BIO-RAD pre-packed
column). The eluates were collected in scintillation vials, and the
radioactivity was counted in a Beckman LS6500 liquid scintillation
counter. The values of IC50 (concentration that inhibits substrate
hydrolysis by 50%) were determined from dose-response curves
in which the concentrations of agents ranged from 10-9 M to 10-5
M for the more potent inhibitors and from 10-8 M to 10-4 M for
the less potent ones.
Molecular Modeling. Molecular structures of ligands 5f, 5u,
milrinone, and cilostazol were built and energy minimized within
MacroModel (MacroModel 7.0, Schro¨dinger LLC).23 Conforma-
tional analysis was carried out using the AMBER* force field, as
included in MacroModel. For all compounds, the resulting geom-
etries of the lowest energy conformers were re-optimized with
semiempirical quantum mechanic calculations, using the Hamilto-
nian AM1, as implemented in Spartan (Spartan ‘02, Wavefunction
Inc., Irvine, CA.).
The three-dimensional model of PDE3A, already published by
some of us,18 was used for these docking studies. Each inhibitor
was docked into the active site of the enzyme by means of the
FlexX module, as implemented in Sybyl 7.1 (Sybyl 7.1, Tripos
Inc., 1699 South Hanley Road, St. Louis, Missouri, 63144, U.S.A.),
which keeps the macromolecule and the ligands flexible. Because
the preparation of the protein for FlexX requires definition of the
binding pocket in terms of “interaction points”, the active site was
defined as all atoms located within a distance of 10 Å from residues
His752, Ile951, Ile968, Phe972, Leu1000, and Phe1004, which,
according to the results of our previous study,18 interact with cAMP,
the natural substrate of the enzyme. This specific distance was
determined to ensure that a significant portion of the catalytic site
was available for the docking experiments. The results were
evaluated in terms of total estimated binding energy, internal strain
energy of the ligand, and van der Waals and electrostatic interaction
energies. All calculations were carried out on a SGI O2 workstation
and on a standard personal computer running under Linux.
analogue 5x, 4-chlorocoumarin derivative 20b, and quinoline
derivatives 22a,b. This material is available free of charge via the
References
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Acknowledgment. The financial support from MIUR (Min-
istero dell’Istruzione, dell’Universita` e della Ricerca, Rome,
Italy) is gratefully acknowledged.
Supporting Information Available: Elemental analysis data
of all new compounds described; experimental details of synthetic
1
procedures for compounds 18a-o and 19a-o; IR and H NMR
data of substituted acetophenone 18m (chosen as an example) and
1
of substituted 4-hydroxycoumarins 19a-o; IR and H NMR data
of substituted 4-(1-piperazinyl)coumarins 5i-l,n-t,v,w, and their