Development of thieno- and benzopyrimidinone inhibitors of the Hedgehog signaling pathway reveals PDE4-dependent and PDE4-independent mechanisms of action

Abstract

From a high content in vivo screen for modulators of developmental patterning in embryonic zebrafish, we previously identified eggmanone (EGM1, 3) as a Hedgehog (Hh) signaling inhibitor functioning downstream of Smoothened. Phenotypic optimization studies for in vitro probe development utilizing a Gli transcription-linked stable luciferase reporter cell line identified EGM1 analogs with improved potency and aqueous solubility. Mechanistic profiling of optimized analogs indicated two distinct scaffold clusters: PDE4 inhibitors able to inhibit downstream of Sufu, and PDE4-independent Hh inhibitors functioning between Smo and Sufu. Each class represents valuable in vitro probes for elucidating the complex mechanisms of Hh regulation.

Keywords: Eggmanone; Hedgehog signaling pathway; Phenotypic screening; Probe development; Suppressor of Fused.

Figure 1

Structures of FDA-approved Smo antagonists (1, 2), eggmanone (EGM1, 3), and the Gli antagonist GANT-61 (4).

Figure 2

SAR of the linker functionality of 19c.

Figure 3

EGM1 analogs lacking activity downstream of Sufu do not displace the binding of BODIPY-cyclopamine (5 nM) from its Smo binding site at the indicated concentrations, in contrast to KAAD-cyclopamine (200 nM). Green = BODIPY-cyclopamine; blue = DAPI. Scale bar = 20 μm.

Scheme 1

Reagents and conditions: (a) S₈, NCCH₂CO₂Me, Et₃N, EtOH, rt, 16 h, 49–80%; (b) CS₂, NaOH, DMSO, H₂O, rt, 1 h then Me₂SO₄, rt, 3 h, 72–93%; (c) R¹NH₂, Et₃N, CH₃CN, 90 °C, 16 h; KOH, EtOH, H₂O, 70 °C, 4 h, 17–72%, two steps; (d) R¹NCS, PhMe, 115 °C, 72 h; KOH, EtOH, H₂O, 70 °C, 4 h, 16–80%, two steps; (e) XCH₂C(O)R², Cs₂CO₃, CH₃CN, rt, 3 h, 9–88%; (f) TFA, CH₂Cl₂, 0–25 °C, 3 h, 27%. X = Cl, Br.

Scheme 2

Reagents and conditions: (a) AcCl, DMAP, THF, rt, 2 h, 82%; (b) DDQ, PhH, 85 °C, 5 d, 34%; (c) pyrrolidine, PhMe, 100 °C, 18 h, 87%.

Scheme 3

Reagents and conditions: (a) NCCH₂CO₂Me, Et₃N, MeOH, DMF, 50 °C via μW, 3 min, 83%; (b) CS₂, NaOH, DMSO, H₂O, rt, 1 h then Me₂SO₄, rt, 3 h, 74%; (c) R¹NH₂, Et₃N, CH₃CN, 90 °C, 16 h; KOH, EtOH, H₂O, 70 °C, 4 h, 11–84%, two steps; (d) XCH₂C(O)R², Cs₂CO₃, CH₃CN, rt, 3 h, 21–71%; (e) NaBH₄, THF, EtOH, 0–25 °C, 2 h, 66%; (f) MeMgCl, THF, 0 °C, 1 h, 19%; (g) POCl₃, DMF, 0–55 °C, 72 h, 30%; (h) PhC(O)CH₂OH, NaH, THF, rt, 0.5 h, then 21, 65 °C, 18 h, 1%; (i) PhC(O)CH₂NH₂·HCl, DIPEA, i-PrOH, rt, 0.5 h, then 21, 100 °C, 18 h, 8%. X = Cl, Br.