The majority of cancer therapeutics induces DNA damage to kill cells. Normal proliferating cells undergo cell cycle arrest in response to DNA damage, thus allowing DNA repair to protect the genome. DNA damage induced cell cycle arrest depends on an evolutionarily conserved signal transduction network in which the Chk1 kinase plays a critical role. In mammalian cells, the p53 and RB pathways further augment the cell cycle arrest response to prevent catastrophic cell death. Given the fact that most tumor cells suffer defects in the p53 and RB pathways, it is likely that tumor cells would depend more on the Chk1 kinase to maintain cell cycle arrest than would normal cells. Therefore Chk1 inhibition could be used to specifically sensitize tumor cells to DNA-damaging agents. We have previously shown that siRNA-mediated Chk1 knockdown abrogates DNA damage-induced checkpoints and potentiates the cytotoxicity of several DNA-damaging agents in p53-deficient cell lines. In this study, we have developed 2 potent and selective Chk1 inhibitors, A-690002 and A-641397, and shown that these compounds abrogate cell cycle checkpoints and potentiate the cytotoxicity of topoisomerase inhibitors and c-radiation in p53-deficient but not in p53-proficient cells of different tissue origins. These results indicate that it is feasible to achieve a therapeutic window with 1 or more Chk1 inhibitors in potentiation of cancer therapy based on the status of the p53 pathway in a wide spectrum of tumor types. ' 2006 Wiley-Liss, Inc.Key words: Chk1; Cdc25A; small molecule inhibitor; cell cycle checkpoint; topoisomerase inhibitors; camptothecin; doxorubicin DNA damaging agents are mainstays of cancer therapy. In response to DNA damage, proliferating cells arrest at distinct transition points along the cell cycle to allow time for DNA repair. The successful repair of DNA lesions is critical for clonogenic survival and the restoration of genome integrity. Excessive and persistent DNA damage, on the other hand, leads to cell death by causing premature senescence, apoptosis, necrosis or mitotic catastrophe.

One and two electron oxidation of N,N',N'',N'''-tetramethyl-1,5,12,16-tetraaza[5,5]paracyclophane (Me3C), a bis-trimethylene bridged bis-p-phenylene diamine (PD), and its ethyl and isopropyl analogues are discussed. The monocation and dication are both stable, as demonstrated by optical studies that show they are in equilibrium in solution, with an especially small difference in first and second oxidation potentials for Me3C in MeCN (+23 to -20 mV, measured by simulation of the optical spectrum and of the cyclic voltammogram, respectively). The monocations have charge localized in one PD unit and show a Hush-type mixed valence transition between their PD0 and PD.+ groups. The dications Me3C2+ and Et3C2+ have optical spectra that appear to show large splittings between their PD.+ groups and have a weak ESR spectrum, and 1H NMR data show that the former is a ground-state singlet. iPr3C2+ has a very different optical spectrum and exhibits a triplet ESR spectrum at 120 K. X-ray crystal structures show that for Me3C0 the N(CH2)3N units on each side are in doubly anti (aa) conformations that put the aryl rings as far apart as possible, but Me3C2+ has doubly gg N(CH2)3N trimethylene bridges and both N,N and C,C distances between the PD.+ groups that are significantly below van der Walls contact. In contrast, iPr3C0 is in a doubly ag conformation, and its diradical dication is suggested to be a triplet because it does not attain the doubly gg conformation.

X-ray crystallographic structures are reported for 1(Me)(2+)(SbCl(6)(-))(2) x 2 CH(3)CN, 2(Et)(2+)(SbF(6)(-))(2) x 2 CH(3)CN x 2 CH(2)Cl(2), and 1(iPr)(2+)(SbF(6)(-))(2), which also contained unresolved solvent and is in a completely different conformation than the methyl- and ethyl-substituted compounds. A quite different structure of 1(Me)(2+)(SbF(6)(-))(2) than that previously published was obtained upon crystallizing it from a mixture rich in monocation. It does not contain close intramolecular PD(+),PD(+) contacts but has close intermolecular ones. Low temperature NMR spectra of 1(Me)(2+) and 1(Et)(2+) in 2:1 CD(3)OD/CD(3)CN showed that both contain three conformations of all-gauche NCCC unit material with close intramolecular PD(+),PD(+) contacts. In addition to the both PD(+) ring syn and anti material that had been seen in the crystal structure of 1(Me)(2+)(SbF(6)(-))(2) x 2 CH(3)CN published previously, an unsymmetrical conformation having one PD(+) ring syn and the other anti (abbreviated uns) was seen, and the relative amounts of these conformations were significantly different for 1(Me)(2+) and 1(Et)(2+). Calculations that correctly obtain the relative amounts of both the methyl- and ethyl-substituted material as well as changes in the optical spectra between 1(Me)(2+) and 1(Et)(2+), which contains much less of the uns conformation, are reported.

The discovery of 1-(5-chloro-2-alkoxyphenyl)-3-(5-cyanopyrazin-2-yl)ureas as a new class of potent (IC(50) values of 3-10 nM) and selective inhibitors of Chk1 kinase was described. One of these compounds (2e) potentiates HeLa cells by over 22-fold against doxorubicin in an antiproliferation assay, and SW620 cells against camptothecin by 20-fold in an antiproliferation assay and 14-fold in a soft agar assay. Flow cytometry (FACS) analysis confirmed that 2e abrogated G2 checkpoint arrest of H1299 cells caused by doxorubicin and S checkpoint arrest caused by camptothecin.