Synthesis and Biological Evaluation of Phthalazinone Inhibitors of ...

oom temperature prior to addition of substrates. The following conditions were
used: 50 mM Tris-HCl, pH 8.0, 100 mM KCl, 3 mM EDTA, 1 mM dithiothreitol (DTT)
(assay buffer) at 25 °C, 10 nM CpIMPDH, 300 μM NAD and 150 μM IMP. To
characterize the non-specific binding of inhibitors, assays were also carried out in
the presence of 0.05% BSA (fatty acid free). IC50 values were calculated for each
inhibitor according to Equation 1 using the SigmaPlot program (SPSS, Inc.):
υi = υ0/(1+[I]/IC50) (Eq. 1)
where υi is initial velocity in the presence of inhibitor (I) and υo is the initial velocity
in the absence of inhibitor. Inhibition at each inhibitor concentration was measured
in quadruplicate and averaged; this value was used as υi. The IC50 values were
determined three times except those marked by a *; the averages are reported in
Tables 1, 2, and 3.
Experimental
Unless otherwise noted, all reagents and solvents were purchased from
commercial sources and used without further purification. All reactions were
performed under nitrogen atmosphere unless otherwise noted. The NMR spectra
were obtained using a 400 or 500 MHz spectrometer. All 1 H NMR spectra are
reported in δ units ppm and are referenced to tetramethylsilane (TMS) if conducted
in CDCl3 or to the central line of the quintet at 2.49 ppm for samples in d6-DMSO. All
20