The reaction of guanidines 14 with substituted acetoacetic esters (Scheme 3)20,21 afforded the dihydropyrimidones 19aCt. including the family of signal transducers and activators of transcription (STATs).4-6 STAT3 is a tumor promoting transcription factor that has been shown to be constitutively activated in numerous cancers, and suppression of STAT3 leads to inhibition of tumor growth in both in vitro and in vivo experiments. In contrast, the related transcription factor, STAT1, activates genes that promote tumor suppression. Therefore, molecules that selectively inhibit STAT3-mediated pathways with no effect on STAT1 pathways, have the potential to be highly effective anti-tumor agents. Several small organic molecules that inhibit the STAT3 pathway have been reported in the literature.7 One strategy has been to design molecules that directly target the Src homology 2 (SH2) domain in STAT3 (1C4, Fig. 1).8 Other approaches include focusing on inhibiting kinases operative in the CHR2797 (Tosedostat) STAT3 pathway, such as Janus activated kinases (JAKs), and identified quinolones, pyridones, and the pyridine carboxamide, sorafenib (5, 6 and 7, respectively, Fig. 1).9 Additionally, natural products, including STA-21 (8), curcumin (9), and cucurbitacin Q (10), inhibit the STAT3 pathway; however, specific inhibitory mechanisms are still being elucidated (Fig. 2).5b Finally, anti-sense oligonucleotides (AZD9150) and decoy nucleotides directed at STAT3 also exhibit promising anti-proliferative activities in cellular assays.5,10 Open in a separate window Figure 1 SH2 targeted phosphopeptide mimetics and JAK inhibitors of the STAT3 pathway. Open in a separate window Figure 2 Natural product STAT3 inhibitors. By using a high content phenotypic screen (HCS) to identify selective inhibitors of IL-6 induced activation of the STAT3 pathway,11 we identified the quinazoline 11a (Fig. 3). In Cal33 head and neck tumor cells, 11a inhibited IL-6-induced STAT3 tyrosine phosphorylation and nuclear translocation (IC50 = 15.7 HVH3 M), but had no effect on IFN-induced activation of the STAT1 pathway at 50 M (Fig. 3). Western blot analysis indicated a 69% decrease in phospho-STAT3 (pSTAT3) levels upon treatment of 11a at 39.6 M concentration (Fig. 4, A and B). Unlike the JAK inhibitor 6 that displayed nanomolar potencies against both STAT3 and STAT1 (data not shown),11 compound 11a selectively inhibited STAT3 compared to STAT1 and displayed no effects on JAK1/JAK2 as determined by Western blot analysis (Figs. 3 and ?and4,4, panels C and D). In addition, 11a exhibited anti-proliferative activities (IC50s = 17-37 lM in four HNSCC cell lines (CAL33, FADU, 686 LN, OSC19, Fig. 3). Examination of the literature and PubChem revealed limited examples of biological effects for this chemotype, and Lipinski and Veber parameters fell into the generally desirable ranges (Fig. 3).12-15 While the specific mode of action of 11a was not determined, its apparent lack of activity in the STAT1 assay likely rules out direct binding to SH2 domains. Furthermore, this hit compound did not exhibit any significant activity against a panel of 80 kinases (data not shown). The promising selectivity for STAT3, the notable anti-proliferative activity and desirable physical properties made this compound CHR2797 (Tosedostat) an attractive lead structure for further medicinal chemistry optimization, and herein we report the results of these efforts. Open in a separate window Figure 3 Guanidinoquinazoline hit 11a. Open in a separate window Figure 4 Inhibition of STAT3 phosphorylation using Western blot analysis of compound 11a versus vehicle in interleukin 6 (IL6, 50 ng/mL)-stimulated CAL33 cells (A & B). Compound 11a did not show any effects on pJAK1/JAK1 (C) or pJAK2/JAK2 (D). Our initial strategy was to incorporate modest structural modifications onto the 2-guanidinoquinazoline core in order to establish preliminary structure-activity relationships. Using established synthetic procedures,16 the dihydroquinolines 13 were generated through the treatment of the substituted anilines 12 with acetone under modified Skraup conditions (Scheme 1).17 Conversion to the guanidines 14 occurred by reaction with cyano-guanidine under aqueous acidic conditions.18 The final products, dihydropyrimidinyl-aminoquinazolines 11a-d, were formed via thermal cyclodehydrations using mesityl oxide in DMSO. The structure of 11b was confirmed by X-ray analysis (Scheme 1).16 In this subset of analogs (Table 1), it was apparent that structural modification was tolerated and modulated the biological profile; the C6-methyl (11b) and C6-,C8-dimethyl (11c) analogs exhibited improved potency (4- and 30-fold, respectively) while maintaining selectivity versus STAT1 compared to the original hit 11a. Unlike 11a, 11b and 11d, which failed to achieve 50% inhibition of IFN-induced STAT1 activation at 50 CHR2797 (Tosedostat) M, 11c exhibited an IC50 of 5.9 M for STAT1 but still maintained a good selectivity index (Table 1). Open in a separate window Scheme 1 Preparation of 2-guanidinoquinazolines 11aCd and X-ray structure of 11b (CCDC 1020633). Table 1 STAT3 and.