Switching on a signaling pathway with an organoruthenium complex.

Altering cellular function with small synthetic molecules is a general approach for the design of drugs (medicinal chemistry) and molecular probes (chemical genetics). Medicinal chemistry and chemical genetics are focused predominately on the design of organic molecules, whereas inorganic compounds find applications mainly for their reactivity (e.g., cisplatin as DNA-reactive therapeutic) or imaging properties (e.g., gadolinium complexes as MRI diagnostics). 2] We are exploring a new direction that aims at utilizing the unique structural opportunities of metal complexes for the design of “organiclike” small-molecule probes and drugs. 4] We recently described an organoruthenium compound to be a low-nanomolar inhibitor for the protein kinase glycogen synthase kinase 3 (GSK-3). Here we disclose our success in developing this compound into a molecular probe for cellular signal transduction pathways involving GSK-3. Our strategy for the design of metal complexes as protein kinase inhibitors uses the class of indolocarbazole alkaloids (e.g., staurosporine) as a lead structure. Accordingly, these metal complexes bear a bidentate ligand which retains the structural features of the indolocarbazole heterocycle. This targets the metal complexes to the ATP-binding site by enabling two hydrogen bonds to the backbone of the hinge between the N-terminal and C-terminal kinase domains, analogous to ATP and conventional organic indolocarbazole inhibitors (Scheme 1). The remaining ligand sphere of the ruthenium atom gives the opportunity to create interactions with other parts of the ATP-binding site. Following this strategy, we recently described ruthenium compound 1-H as a selective low-nanomolar and ATP-competitive inhibitor of GSK-3. The key component of the design was the novel pyridocarbazole ligand 2-H, derived from arcyriaflavin A by just replacing one indole moiety with a pyridine residue. Here we introduce the ruthenium complex 1-OH (Scheme 1), derived from 1-H by adding a single hydroxy group to the indole moiety. Molecular modeling suggests that this OH group can occupy a small hydrophilic cavity within the ATP-binding site of GSK-3. Indeed, this modification not only increases the affinity for GSK-3 by an order of magnitude, but at the same time increases water solubility considerably, and thus makes it a promising molecular probe for GSK-3. GSK-3 is a component and negative regulator of the wnt signaling pathway, and we demonstrate in the following that 1-OH can switch on the wnt signaling pathway by inhibiting GSK-3 inside living cells and in Xenopus embryos. We devised a short and economic synthetic route to this class of metalated pyridocarbazoles (Scheme 2). Starting with aryl hydrazine hydrochloride 3, Fischer indole synthesis and protecting-group exchange lead to pyridoindole 4, which is deprotonated and treated with the TBS-protected dibromomaleimide 5 to yield monosubstitution product 6. The key step of the synthetic scheme is the following smooth anaerobic photocyclization to the TBS-protected pyridocarbazole 7 in 78% yield. Compound 7 can be deprotected with TBAF to give 2-OH or treated with [Ru(Cp)(CO)(CH3CN)2]PF6 in the presence of K2CO3 to yield cyclometalated ruthenium complex 8. Subsequent TBS deprotection with TBAF gives 1-OH. Complex 1-OH is stable in aqueous solution, in cell culture medium, under air, and can even withstand a 5 mm solution of 2-mercaptoethanol for 12 h without any sign of decomposition, as determined by 1H NMR spectroscopy. Furthermore, 1-OH also has favorable solubility in water. For example, it can be dissolved in 3% DMSO/water at a concentration of 1 mm. We next examined the potency of 1-OH by determining the concentration required for 50% inhibition (IC50) of GSK3a (the a isoform) and GSK-3b (the b isoform). The IC50 of 1OH is 300 pm and 500 pm for GSK-3a and GSK-3b, respectively, approximately an order of magnitude lower than that [*] D. S. Williams, G. E. Atilla, H. Bregman, Prof. Dr. E. Meggers Department of Chemistry University of Pennsylvania 231 South 34th Street, Philadelphia, PA 19104 (USA) Fax: (+1)215-746-0348 E-mail: meggers@sas.upenn.edu