Design and Synthesis of New Pyrazole-Based Heterotricycles and their Derivatization by Automated Library Synthesis.

Small-molecule heterocycles bearing orthogonal functionality have the potential to deliver diverse structural motifs that aid the drug-discovery effort. This work highlights how a readily assembled N-hydroxyethyl pyrazole trifluoroborate offers rapid access to architecturally distinct 5-6-6- and 5-7-6-fused tricyclic compounds. This chemistry is not only amenable to single compound synthesis, but also to high-throughput experimentation. It gives easy access to diverse compound arrays with various physicochemical and ADME profiles by fully automated library synthesis. The combination of the high-throughput experimentation with rapid testing of the compounds in an integrated physicochemical and ADME profiling workflow allows accelerated design of novel lead compounds in drug-discovery projects.

From identification to functional characterization of cyriotoxin-1a, an antinociceptive toxin from the spider Cyriopagopus schioedtei.

BACKGROUND AND PURPOSE: The NaV 1.7 channel is highly expressed in dorsal root ganglia of the sensory nervous system and plays a central role in the pain signalling process. We investigated a library prepared from original venoms of 117 different animals to identify new selective inhibitors of this target. EXPERIMENTAL APPROACH: We used high throughput screening of a large venom collection using automated patch-clamp experiments on human voltage-gated sodium channel subtypes and then in vitro and in vivo electrophysiological experiments to characterize the active peptides that have been purified, sequenced, and chemically synthesized. Analgesic effects were evaluated in vivo in mice models. KEY RESULTS: We identified cyriotoxin-1a (CyrTx-1a), a novel peptide isolated from Cyriopagopus schioedtei spider venom, as a candidate for further characterization. This 33 amino acids toxin belongs to the inhibitor cystine knot structural family and inhibits hNaV 1.1-1.3 and 1.6-1.7 channels in the low nanomolar range, compared to the micromolar range for hNaV 1.4-1.5 and 1.8 channels. CyrTx-1a was 920 times more efficient at inhibiting tetrodotoxin (TTX)-sensitive than TTX-resistant sodium currents recorded from adult mouse dorsal root ganglia neurons and in vivo electrophysiological experiments showed that CyrTx-1a was approximately 170 times less efficient than huwentoxin-IV at altering mouse skeletal neuromuscular excitability properties. CyrTx-1a exhibited an analgesic effect in mice by increasing reaction time in the hot-plate assay. CONCLUSIONS AND IMPLICATIONS: The pharmacological profile of CyrTx-1a paves the way for further molecular engineering aimed to optimize the potential antinociceptive properties of this peptide.

Synthetic and Mechanistic Investigation of an Oxime Ether Electrocyclization Approach to Heteroaromatic Boronic Acid Derivatives.

A range of functionalized heteroaromatic boronic acid derivatives are readily accessed by a diboration/6π-electrocyclization sequence. This study revealed the surprising observation that there is a direct relationship between oxime ether stereochemistry and reactivity towards electrocyclization. Specifically, E-oxime ethers are found to be significantly more reactive than their Z-counterparts (stereochemistry relative to azatriene scaffold). In contrast, the configuration at the azatriene alkene terminus has little impact on reaction rates. Computational analysis offers a rationale for this observation; a Nlone pair →C=C π* orbital interaction lowers the energy of the transition state in the electrocyclization of E-oxime ethers. Finally, unreactive Z-oxime ethers can be converted to the corresponding heterocyclic products by a photolytically promoted E→Z isomerization and electrocyclization sequence.

Synthesis of Bifunctional Thiophenes via Fiesselmann Condensation of Ynone Trifluoroborate Salts.

Ynone trifluoroborate salts undergo a base-promoted condensation reaction with alkylthiols to generate thiophene boronates with complete regiocontrol. The products are isolated in high yield and can be further derivatized through conventional C-B bond functionalization reactions.

Synthesis and Modular Reactivity of Pyrazole 5-Trifluoroborates: Intermediates for the Preparation of Fully Functionalized Pyrazoles.

The regioselective condensation of hydrazines and ynone trifluoroborates provides access to a range of pyrazole 5-trifluoroborates. The stability of the borate unit allows chemoselective halogenation of the heteroaromatic ring, thereby delivering pyrazole scaffolds that allow orthogonal functionalization at C5 and C4. The modular reactivity of these intermediates is exemplified by cross-coupling reactions, enabling regiocontrolled synthesis of fully functionalized pyrazole derivatives.

Discovery and Optimization of 1-Phenoxy-2-aminoindanes as Potent, Selective, and Orally Bioavailable Inhibitors of the Na+/H+ Exchanger Type 3 (NHE3).

The design, synthesis, and structure-activity relationship of 1-phenoxy-2-aminoindanes as inhibitors of the Na+/H+ exchanger type 3 (NHE3) are described based on a hit from high-throughput screening (HTS). The chemical optimization resulted in the discovery of potent, selective, and orally bioavailable NHE3 inhibitors with 13d as best compound, showing high in vitro permeability and lacking CYP2D6 inhibition as main optimization parameters. Aligning 1-phenoxy-2-aminoindanes onto the X-ray structure of 13d then provided 3D-QSAR models for NHE3 inhibition capturing guidelines for optimization. These models showed good correlation coefficients and allowed for activity estimation. In silico ADMET models for Caco-2 permeability and CYP2D6 inhibition were also successfully applied for this series. Moreover, docking into the CYP2D6 X-ray structure provided a reliable alignment for 3D-QSAR models. Finally 13d, renamed as SAR197, was characterized in vitro and by in vivo pharmacokinetic (PK) and pharmacological studies to unveil its potential for reduction of obstructive sleep apneas.

An Alkyne Diboration/6π-Electrocyclization Strategy for the Synthesis of Pyridine Boronic Acid Derivatives.

A new and efficient synthesis of pyridine-based heteroaromatic boronic acid derivatives is reported through a novel diboration/6π-electrocyclization strategy. This method delivers a range of functionalized heterocycles from readily available starting materials.

A 10,000 member PNA-encoded peptide library for profiling tyrosine kinases.

A 10,000 member peptide nucleic acid (PNA) encoded peptide library was prepared, treated with the Abelson tyrosine kinase (Abl), and decoded using a DNA microarray and a fluorescently labeled secondary antiphosphotyrosine antibody. A dual-color approach ensured internal referencing for each and every member of the library and the generation of robust data sets. Analysis identified 155 peptides (out of 10,000) that were strongly phosphorylated by Abl in full agreement with known Abl specificities. BLAST analysis identified known cellular Abl substrates such as c-Jun amino-terminal kinase as well as new potential target proteins such as the G-protein coupled receptor kinase 6 and diacylglycerol kinase gamma. To illustrate the generalization of this approach, two other tyrosine kinases, human epidermal growth factor 2 (Her2) and vascular endothelial growth factor receptor 2/kinase insert domain protein receptor (VEGFR2/KDR), were profiled allowing characterization of specific peptide sequences known to interact with these kinases; under these conditions Her2 was demonstrated to have a marked preference for D-proline perhaps offering a unique means of targeting and inhibiting this kinase.

Dual colour, microarray-based, analysis of 10,000 protease substrates.

A 10,000 member PNA-encoded library of FRET based peptides was synthesised for global analysis of protease cleavage specificity; analysis was achieved using a DNA microarray and consumed minimal quantities of enzyme (60 pmole) and library (3.5 nmole).

Synthesis and cellular uptake of cell delivering PNA-peptide conjugates.

The synthesis and cellular uptake of fluorescently labelled PNA-peptide conjugates is described; Dde/Mmt protected PNA monomers, fully orthogonal to Fmoc chemistry, were used to develop a flexible strategy to give Peptide Nucleic Acids conjugated to tri- and hepta-arginine and the short basic Tat(48-57) peptide as examples of cellular penetrating peptides, thereby allowing efficient cellular delivery of PNA into cells.

Inhibitors of protein tyrosine phosphatases: next-generation drugs?

The protein tyrosine phosphatases (PTPs) constitute a family of closely related key regulatory enzymes that dephosphorylate phosphotyrosine residues in their protein substrates. Malfunctions in PTP activity are linked to various diseases, ranging from cancer to neurological disorders and diabetes. Consequently, PTPs have emerged as promising targets for therapeutic intervention in recent years. In this review, general aspects of PTPs and the development of small-molecule inhibitors of PTPs by both academic research groups and pharmaceutical companies are discussed. Different strategies have been successfully applied to identify potent and selective inhibitors. These studies constitute the basis for the future development of PTP inhibitors as drugs.

Combinatorial libraries - from solution to 2D microarrays.

Enzymatic modifications of split and mix libraries were followed by "pulling down" onto a 2-dimensional DNA microarray, via PNA tagging; this allowed complete library interrogation of all members of the split and mix library.

Total synthesis and biological evaluation of the protein phosphatase 2A inhibitor cytostatin and analogues.

The total synthesis of the natural product cytostatin is described which inhibits protein phosphatase 2A. Cytostatin has anti-metastatic properties and induces apoptosis. On the basis of this synthesis the relative and absolute configuration of cytostatin could be assigned. Key structural elements of cytostatin are an alpha,beta-unsaturated lactone group and a side chain embodying a phosphate and a rather unstable (Z,Z,E)-triene subunit. In addition, the natural product carries six stereocenters. For the construction of the stereocenters reagent-controlled transformations were used in order to ensure maximum stereochemical flexibility. The Evans syn-aldol reaction was chosen to establish the stereochemistry at C-4, C-5, C-9 and C-10; C-6 was introduced by means of the Evans asymmetric alkylation. In all cases the same chiral auxiliary was employed as stereodirecting group. The stereocenter at C-11 was established by an asymmetric reduction using CBS-oxazaborolidine. Temporary protection of the phosphate group was achieved best by using the base-labile 9-fluorenylmethyl group, which could be cleanly cleaved by an excess of triethylamine; this reaction yielded analytically pure phosphates after a simple aqueous work-up. The (Z,Z,E)-triene embodied in cytostatin was synthesized by means of a Stille coupling as key transformation. The synthesis sequence established in this way readily gave access to a series of analogues with simplified structure. Initial biological testing of these analogues proved that the alpha,beta-unsaturated lactone, the C-11-hydroxy group and a fully deprotected phosphate moiety at C-9 are essential for the PP2A-inhibitory activity of cytostatin. The rather unstable triene moiety in the side chain can be replaced by other lipophilic residues with only moderate decrease of biological activity. Other phosphatases, that is, PP1, VHR, PTP1B, CD45, were not inhibited by cytostatin or any of the analogues, demonstrating the high selectivity of this compound. These findings will be useful for the design and synthesis of cytostatin-derived chemical tools for the study of biological processes influenced by PP2A.

Full orthogonality between Dde and Fmoc: the direct synthesis of PNA--peptide conjugates.

New deprotection conditions for the Dde amine protecting group that are fully orthogonal to Fmoc are described and successfully applied to the dual synthesis of PNA-peptide conjugates.

Synthesis and biological evaluation of cytostatin analogues.

Analogues of the serine/threonine phosphatase 2A inhibitor cytostatin were synthesized and evaluated as inhibitors of various phosphatases in order to establish a basic structure-activity relationship (SAR) of the natural product.