Design, Synthesis, and Biological Evaluation of Pyrimidine Dihydroquinoxalinone Derivatives as Tubulin Colchicine Site-Binding Agents That Displayed Potent Anticancer Activity Both In Vitro and In Vivo

Polymerization of tubulin dimers to form microtubules
is one of
the key events in cell proliferation. The inhibition of this event
has long been recognized as a potential treatment option for various
types of cancer. Compound 1e was previously developed
by our team as a potent inhibitor of tubulin polymerization that binds
to the colchicine site. To further improve the potency and therapeutic
properties of compound 1e, we hypothesized based on the
X-ray crystal structure that modification of the pyrimidine dihydroquinoxalinone
scaffold with additional hetero-atom (N, O, and S) substituents could
allow the resulting new compounds to bind more tightly to the colchicine
site and display greater efficacy in cancer therapy. We therefore
synthesized a series of new pyrimidine dihydroquinoxalinone derivatives,
compounds 10, 12b–c, 12e, 12h, and 12j–l, and evaluated
their cytotoxicity and relative ability to inhibit proliferation,
resulting in the discovery of new tubulin-polymerization inhibitors.
Among these, the most potent new inhibitor was compound 12k, which exhibited high cytotoxic activity in vitro, a longer half-life
than the parental compound in liver microsomes (IC50 =
0.2 nM, t1/2 = >300 min), and significant
potency against a wide range of cancer cell lines including those
from melanoma and breast, pancreatic, and prostate cancers. High-resolution
X-ray crystal structures of the best compounds in this scaffold series, 12e, 12j, and 12k, confirmed their
direct binding to the colchicine site of tubulin and revealed their
detailed molecular interactions. Further evaluation of 12k in vivo using a highly taxane-resistant prostate cancer xenograft
model, PC-3/TxR, demonstrated the strong tumor growth inhibition at
the low dose of 2.5 mg/kg (i.v., twice per week). Collectively, these
results strongly support further preclinical evaluations of 12k as a potential candidate for development.