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Single-agent CHK1 inhibition in CLL
P<0.05. Detailed information is available in the Online Supplementary Appendix.
Results
Enantioselective synthesis of MU380
In order to prepare sufficient quantities of MU380 for in vivo studies, we developed its enantioselective synthesis from commercially available N-Boc-(R)-nipecotic acid 1 (Figure 1). Briefly, acid 1 was converted into the Weinreb amide 2, whose treatment with deprotonated acetonitrile at low temperature afforded the required β-ketonitrile 3 with high optical purity [99% ee, determined by high per- formance liquid chromatography (HPLC) on chiral sta- tionary phase]. Subsequent cyclization of 3 with 3- aminopyrazole proved to be quite challenging when, under a variety of reaction conditions including alcoholic solvents used in analogous synthesis of the CHK1 inhibitor SCH900776,31 we observed significant loss of stereochemical integrity. Finally, we found neat acetic acid to be the optimal solvent: cyclization was rapid and pro- vided the desired pyrazolo[1,5-a]pyrimidine intermediate
4 in high yield (95%) and optical purity (96% ee). Using part of the sequence we had previously reported,26 com- pound 4 was converted into advanced intermediate 11, utilizing the in-house prepared boronate 9. Optical purity of 11 (96% ee) was again determined by HPLC on chiral stationary phase, confirming that no loss of stereochemi- cal integrity was associated with the post-cyclization steps of the sequence. Subsequent Boc-protection fol- lowed by regioselective bromination and deprotection provided the target compound MU380 (overall yield 33% over 10 steps). Final recrystallization from acetonitrile afforded optically pure MU380 (> 99% ee) on gram scale. For detailed procedure see the Online Supplementary Appendix.
MU380 effectively inhibits CHK1 kinase and sensitizes lymphoid tumor cells to gemcitabine
Our novel inhibitor showed satisfactory target-specific effects in a pilot study using cell lines established from solid tumors.26 Herein, we first explored whether MU380 also efficiently inhibits CHK1 in lymphoid tumor cells. We treated NALM-6 and MEC-1 cell lines with gemc- itabine, a potent inductor of RS, and analyzed impact of
Figure 1. Scheme of enantioselective synthesis of MU380. A brief description is pro- vided in the Results section; detailed procedures and structural characterizations are provided in the Online Supplementary Appendix.
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