Development of novel, highly potent inhibitors of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF): increasing cellular potency through optimization of a distal heteroaromatic group.

We describe the design, synthesis, and optimization of a series of new inhibitors of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF), a kinase whose mutant form (V600E) is implicated in several types of cancer, with a particularly high frequency in melanoma. Our previously described inhibitors with a tripartite A-B-C system (where A is a hinge binding pyrido[4,5-b]imidazolone system, B is an aryl spacer group, and C is a heteroaromatic group) were potent against purified (V600E)BRAF in vitro but were less potent in accompanying cellular assays. Substitution of different aromatic heterocycles for the phenyl based C-ring is evaluated herein as a potential means of improving the cellular potencies of these inhibitors. Substituted pyrazoles, particularly 3-tert-butyl-1-aryl-1H-pyrazoles, increase the cellular potencies without detrimental effects on the potency on isolated (V600E)BRAF. Thus, compounds have been synthesized that inhibit, with low nanomolar concentrations, (V600E)BRAF, its downstream signaling in cells [as measured by the reduction of the phosphorylation of extracellular regulated kinase (ERK)], and the proliferation of mutant BRAF-dependent cells. Concomitant benefits are good oral bioavailability and high plasma concentrations in vivo.

BRAF inhibitors based on an imidazo[4,5]pyridin-2-one scaffold and a meta substituted middle ring.

We recently reported on the development of a novel series of BRAF inhibitors based on a tripartite A-B-C system characterized by a para-substituted central aromatic core connected to an imidazo[4,5]pyridin-2-one scaffold and a substituted urea linker. Here, we present a new series of BRAF inhibitors in which the central phenyl ring connects to the hinge binder and substrate pocket of BRAF with a meta-substitution pattern. The optimization of this new scaffold led to the development of low-nanomolar inhibitors that permits the use of a wider range of linkers and terminal C rings while enhancing the selectivity for the BRAF enzyme in comparison to the para series.

Sulfato-bridged ECE-pincer palladium(II) complexes: structures in the solid-state and in solution, and catalytic properties.

ECE-pincer sulfato palladium complexes (pincer = [C(6)H(3)(CH(2)E)(2)-2,6](-); E = SPh (), SMe (), S(t)Bu (), NMe(2) ()) were synthesized and characterized. In the solid-state (X-ray determinations) and exist as neutral ECE-pincer palladium sulfato complexes with a mu(2)-O,O' bridging sulfato ligand. IR and Raman spectroscopic studies revealed that in the solid-state the complexes can be present as either solely neutral or as a mixture of neutral and ionic species, depending on the preparation and morphology of the solids. In water, ionic complexes with non-coordinating sulfate ions prevail. Preliminary studies of the catalytic activity of in the Suzuki-Miyaura C-C cross-coupling reaction of 3-iodobenzoic acid and sodium tetraphenylborate in water reveal that the C-C cross-coupling product is efficiently formed in good yields at room temperature.

Novel potent BRAF inhibitors: toward 1 nM compounds through optimization of the central phenyl ring.

BRAF, a serine/threonine specific protein kinase that is part of the MAPK pathway and acts as a downstream effector of RAS, is a potential therapeutic target in melanoma. We have developed a series of small-molecule BRAF inhibitors based on a 1H-imidazo[4,5-b]pyridine-2(3H)-one scaffold (ring A) as the hinge binding moiety and a number of substituted phenyl rings C that interact with the allosteric binding site. The introduction of various groups on the central phenyl ring B combined with appropriate A- and C-ring modifications afford very potent compounds that inhibit (V600E)BRAF kinase activity in vitro and oncogenic BRAF signaling in melanoma cells. Substitution on the central phenyl ring of a 3-fluoro, a naphthyl, or a 3-thiomethyl group improves activity to yield compounds with an IC(50) of 1 nM for purified (V600E)BRAF and nanomolar activity in cells.

Pyridoimidazolones as novel potent inhibitors of v-Raf murine sarcoma viral oncogene homologue B1 (BRAF).

BRAF is a serine/threonine kinase that is mutated in a range of cancers, including 50-70% of melanomas, and has been validated as a therapeutic target. We have designed and synthesized mutant BRAF inhibitors containing pyridoimidazolone as a new hinge-binding scaffold. Compounds have been obtained which have low nanomolar potency for mutant BRAF (12 nM for compound 5i) and low micromolar cellular potency against a mutant BRAF melanoma cell line, WM266.4. The series benefits from very low metabolism, and pharmacokinetics (PK) that can be modulated by methylation of the NH groups of the imidazolone, resulting in compounds with fewer H-donors and a better PK profile. These compounds have great potential in the treatment of mutant BRAF melanomas.

Selective and diagnostic labelling of serine hydrolases with reactive phosphonate inhibitors.

Reactive phosphonates are important probes to target the active site of serine hydrolases, one of the largest and most diverse family of enzymes. Developing such inhibitory probes is of special importance in activity based protein profiling, a strategy that is increasingly used to gain information about a certain class of enzymes in complex proteosomes. Therefore, gaining detailed information about these inhibition events on the individual protein level is important since it affords information that can be used to fine-tune the probe for a specific task. Here, we report a novel and versatile synthesis protocol to access a variety of functionalised p-nitrophenyl phosphonate (PNPP) inhibitors from a common azide functionalised precursor using click chemistry. The obtained PNPPs were successfully used to covalently label serine hydrolases in their active sites with molecular tags. Furthermore, a model study is described in which we developed straightforward protocols that can be used to study protein inhibition events. Kinetic studies using UV-Vis and fluorescence spectroscopy techniques revealed that these PNPPs possess different inhibition rates for various proteins and were shown to be suitable probes to discriminate between various lipases. Additionally, we demonstrate that PNPPs are highly selective for serine hydrolases, making these probes very interesting as diagnostic or affinity probes for studying proteins in complex proteosomes.

Transmission of binding information across lipid bilayers.

A synthetic transmembrane receptor that is capable of transmitting binding information across a lipid bilayer membrane is reported. The binding event is based on aggregation of the receptor triggered by copper(II) complexation to ethylenediamine functionalities. By labelling the receptor with fluorescent dansyl groups, the copper(II) binding event could be monitored by measuring the extent of fluorescence quenching. Comparing the receptor with a control receptor lacking the transmembrane linkage revealed that the transmembrane receptor binds copper(II) ions more tightly than the non-spanning control receptor at low copper(II) concentrations. Since the intrinsic binding to copper(II) is the same for both receptors, this effect was attributed to synergy between the connected interior and exterior binding sides of the transmembrane receptor. Thus, this is the first reported artificial signalling event in which binding of a messenger on one side of the membrane leads to a cooperative binding event on the opposite side of the membrane, resembling biological signalling systems and helping us to get a better understanding of the requirements for more effective artificial signalling systems.