The c-Myc proto-oncogene is involved in many human tumors, and needs to bind to its activation partner Max for all of its known biological activities.[1–4] Inhibition of the protein–protein interactions between c-Myc and Max by cell-permeable molecules is therefore an attractive goal.[5–8] Dimerization between c-Myc and Max occurs via α-helical domains comprising leucine zipper motifs, which display no obvious binding sites for inhibitory ligands.[9] We recently identified two pyrazoloACHTUNGTRENNUNG [1, 5-a] pyrimidines which inhibit c-Myc/Max dimerization from a diverse collection of chemicals (Figure 1a).[10] These compounds, dubbed Mycro1 and Mycro2, inhibited c-Myc/Max dimerization and DNA binding with preference over other structurally related transcription factors in vitro, and exhibited c-Myc dependent effects in cellular assays. To explore the chemical space around the pyrazoloACHTUNGTRENNUNG [1, 5-a] pyrimidine core structure for substitution patterns which are associated with activity against c-Myc/Max dimerization, and to possibly identify a Myc/Max dimerization inhibitor with improved properties, we screened a 1438-membered pyrazoloACHTUNGTRENNUNG [1, 5-a] pyrimidine library based on the structures of Mycro1 and Mycro2 (Figure 1b and Figure S1 in the Supporting Information).

As c-Myc can bind DNA only as dimer with Max, we tested the compounds for their abilities to inhibit DNA binding of c-Myc in a fluorescence polarization assay.[10] Five test compounds (1–5) inhibited DNA binding of c-Myc/Max with preference over Max/Max DNA binding by more than 50% at a concentration of 100 μM (Table1). DNA binding of Max/Max dimers is the most stringent specificity control possible, as c-Myc and Max are 59% similar at the protein level in the dimerization domains, and the overall structure of the DNA-bound dimers are very similar to each other.[9, 11] As an additional specificity control, we analyzed the effect of compounds 1–5 on the function of the Src-homology 2 (SH2) domain of the structurally unrelated transcription factor STAT3. None of the compounds affected the interactions between STAT3 and a phosphotyrosine-containing peptide comprising the STAT3 binding motif to a major extent (Table 1).[12] Confirmation of compound activities in well-controlled cellular systems would provide a strong argument against the notion that any in vitro data could be influenced by factors which are irrelevant under cellular conditions. In addition, cellular assays allow the analysis of compound specificities against all relevant proteins; therefore, their scope is incomparably larger than any in vitro analysis. We chose a cell proliferation assay to further analyze the effects of inhibitor candidates 1–5. Cell cycle progression and proliferation of almost all cell types, including U-2OS osteosarcoma cells, requires c-Myc function, and hence c-Myc’s ability to bind to its activation partner Max.[3, 13] However, for reasons not yet fully understood, PC-12 pheochromocytoma cells proliferate independent of c-Myc/Max dimerization, as they express a truncated Max protein which is unable to interact with c-Myc.[14] Therefore, a selective inhibitor of c-Myc/Max dimerization can be expected to inhibit proliferation of the c-Myc/Max-dependent U-2OS cells, without inhibiting the growth of the c-Myc/Max-independent PC-12 cells, provided it is cell permeable and stable in the cellular environment. In contrast, compounds which act unspecifically in the cellular context will either cause a reduction in the proliferative rate of both U-2OS and PC-12 cells, or display toxic effects. Whereas compound 5 appeared to be toxic in both cell lines, compound 4 inhibited …