Compromised Porcn activity commonly results in developmental disorders including focal dermal hypoplasia (Goltz syndrome) whereas hyperactivity of Porcn is associated with cancerous cell growth.5 We envision that inhibition of Porcn will be an effective strategy for broadly suppressing Wnt signaling and thus hold potential in regenerative medicine and anti-cancer applications. responses. Compromised Porcn activity commonly results in developmental disorders including focal dermal hypoplasia (Goltz syndrome) whereas hyperactivity of Porcn is associated with cancerous cell growth.5 We envision that inhibition of Porcn will be an effective strategy for broadly suppressing Wnt signaling and thus hold potential in regenerative medicine and anti-cancer applications. Although genetically based targeting of Wnt signaling components suggests that chemical inhibitors of Wnt signaling may give rise to toxic effects, Porcn inhibitors have proven to be remarkably non-toxic in rodents.6 Indeed, we surmise that these favorable results in preclinical tests were a pre-requisite to the Phase I trials underway for LGK974, a novel Porcn inhibitor.2 The four IWP molecules (1C4) identified in the initial screen of 200,000 compounds7 bear similar molecular skeletons (Figure 1). They all suppress cell-autonomous Wnt signaling in mouse fibroblasts at nanomolar concentrations.3 We consider the phthalazinone moiety of IWP-1 (1) and pyrimidinone moiety of IWP-2C4 (2C4) exchangeable scaffolding motifs. The benzothiazole moiety appears to be a conserved motif and the phenyl group tolerates both electronic and steric perturbations. Based on this information, we prepared an IWP-biotin conjugate and an IWP-Cy3 conjugate (5), and used them to demonstrate that IWP-2 (2) directly binds to Porcn.3 We report herein the subsequent structure-activity relationship (SAR) studies yielding new Porcn inhibitors that suppress Wnt signaling at sub-nanomolar concentrations. Open in a separate window Figure 1 The structures and activities of IWPs identified from a high-throughput screen in cells exhibiting cell-autonomous Wnt signaling. RESULTS AND DISCUSSION We recently identified 13 additional Porcn inhibitors from the same screen that netted IWP-1C4 (1C4).8 Five of them (6C10) possess similar molecular skeletons as IWP-1C4 (1C4) and provided further SAR information. The discovery of 6C10 as active Porcn inhibitors confirmed that the phthalazinone and pyrimidinone moieties are scaffolding motifs. Most importantly, the phenyl and benzothiazole groups of IWP-1C4 (1C4) can be replaced by an alkyl group and a simple aromatic group, respectively. We therefore hypothesized that IWPs bind to Porcn by fitting the phthalazinone/pyrimidinone and the benzothiazole regions into the binding pocket (Figure 2). Consistent with this model, we prepared 11 and 12 and found that they both failed to suppress the Wnt signaling at up to 25 M Cxcr7 in L-Wnt-STF cells, potentially due to reduced hydrophobic interactions. In addition, effective biotin and Cyclothiazide Cy3 conjugates (5) were obtained from derivatizing the phenyl group of IWP-2 (2), a region that is believed to be exposed to the solvent. Open in a separate window Figure 2 The phthalazinone/pyrimidinone and the benzothiazole moieties of IWPs are important for their binding to Porcn. We started our investigation by examining effects of substituent groups on the benzothiazole and phenyl moieties (Table 1). As expected, there is no significant difference in potency for either the IWP-1 (A) or the IWP-2 (B) scaffolds harboring adducts to these moieties. Exchanging the substituent patterns observed in IWP-1C4 (1C4) (R1=OMe or Me; R2=H, Cyclothiazide = 8.0 Hz, 2H), 3.03 (t, = 8.0 Hz, 2H), 3.68 (s, 3H).18 A solution of 3-amino-2-(methoxycarbonyl)4,5-dihydrothiophene (1.00 g, 5.78 mmol) and phenyl isothiocyanate (937 mg, 6.94 mmol) in pyridine (18 mL) was stirred at 100 C overnight. The solvent was then evaporated and the residue was purified by silica gel column chromatography (30% ethyl acetate/hexanes then acetone) and then washed three times with ethyl acetate to give pure 3-phenyl-6,7-dihydrothieno[3,2-= 2.1 Hz, 1H), 8.25 (d, = 8.6 Hz, 1H), 7.95 (dd, = 8.6, 2.4 Hz, 1H), 7.56C7.63 (m, 5H), 7.48C7.56 (m, 2H), 7.40C7.47 (m, 1H), 7.29C7.35 (m, 2H), 3.85 (s, 2H), 3.54C3.62 (m, 2H), 3.44C3.52 (m, 2H); 13C NMR (100 MHz, CDCl3) 166.6, 160.2, 159.2, 157.4, 150.5, 146.3, 137.4, 136.7, 135.0, 133.0, 130.6, 130.0, 129.1, 128.6, 127.9, 126.8, 122.3, 113.7, 37.6, 37.0, 29.3. MS(ES)+ calcd for C25H21N4O2S2 (M+H)+ 473.1, found 473.1. Supplementary Material 1_si_001Click here to view.(336K, pdf) Acknowledgments Financial support was provided by the Cancer Prevention and Research Institute of Texas (RP100119 to L.L. and C.C.), National Institute of Health (5R21HD061303 to J.F.A., C.C., and L.L., R01CA135731 to J.F.A., and R01DK080004 & P30DK079328 to T.C.), the Welch Foundation (I-1596 to C.C., and I-1665 to L.L.), and UT Southwestern. L.L. is a Virginia Murchison Linthicum Scholar in Medical Research, and C.C. is a Southwestern Cyclothiazide Medical Foundation Scholar in Biomedical Research. X.P. is supported.