Click-Addressable Cassette for Photoaffinity Labeling

A small molecule <b>1</b> was designed to contain an alkyne, a trifluoromethyl phenyldiazirine, and a free piperidine-<i>NH</i> for facile conjugation to protein binding ligands. This “cassette” <b>1</b> was synthesized via a relatively direct route involving only routine steps. In this proof-of-concept study, putative ligands for carbonic anhydrase IX and for TrkC were conjugated to <b>1</b>. Photoaffinity labeling was performed using purified extracellular regions of both these protein-receptors, and using cells that express these receptors (isolation via a pull-down procedure), labeling of the protein was observed in all four experiments

Double-Targeting Using a TrkC Ligand Conjugated to Dipyrrometheneboron Difluoride (BODIPY) Based Photodynamic Therapy (PDT) Agent

A molecule <b>1</b> (IY-IY-PDT) was designed to contain a fragment (IY-IY) that targets the TrkC receptor and a photosensitizer that acts as an agent for photodynamic therapy (PDT). Molecule <b>1</b> had submicromolar photocytotoxicities to cells that were engineered to stably express TrkC (NIH3T3-TrkC) or that naturally express high levels of TrkC (SY5Y neuroblastoma lines). Control experiments showed that <b>1</b> is not cytotoxic in the dark and has significantly less photocytotoxicity toward cells that do not express TrkC (NIH3T3-WT). Other controls featuring a similar agent <b>2</b> (YI-YI-PDT), which is identical and isomeric with <b>1</b> except that the targeting region is scrambled (a YI-YI motif, see text), showed that <b>1</b> is considerably more photocytotoxic than <b>2</b> on TrkC⁺ cells. Imaging live TrkC⁺ cells after treatment with a fluorescent agent <b>1</b> (IY-IY-PDT) proved that <b>1</b> permeates into TrkC⁺ cells and is localized in the lysosomes. This observation indirectly indicates that agent <b>1</b> enters the cells via the TrkC receptor. Consistent with this, the dose-dependent PDT effects of <b>1</b> can be competitively reduced by the natural TrkC ligand, neurotrophin NT3

A Chemoselective Route to β‑Enamino Esters and Thioesters

Conditions were developed for syntheses of β-enamino esters, thioesters, and amides. These reactions involve hydroxy­benzo­triazole derivatives in buffered media. Illustrative syntheses of some heterocyclic systems are given, including some related to protein–protein interface mimics

Small Molecule Ligands for Active Targeting of TrkC-Expressing Tumor Cells

A small molecule motif was used in “active targeting” to deliver cytotoxic substances into tumor cells that express the TrkC receptor. Underlying this study was the hypothesis that internalization of targeted conjugates into cells would be facile if mediated by receptor binding and receptor–ligand internalization. Initial experiments using 6-mercaptopurine gave encouraging data but demonstrated the importance of maintaining solubility and high cytotoxicity. Conjugates of the targeting agent with a cytotoxic rosamine (similar to a rhodamine) were more successful. Targeting of TrkC was observed, validated in a series of competition experiments featuring other TrkC ligands, and accumulation into lysosomes was observed, as expected for receptor-mediated internalization

Homo-Roche Ester Derivatives by Asymmetric Hydrogenation and Organocatalysis

Asymmetric hydrogenation routes to homologues of The Roche ester tend to be restricted to hydrogenations of itaconic acid derivatives, that is, substrates that contain a relatively unhindered, 1,1-disubstituted alkene. This is because in hydrogenations mediated by RhP₂ complexes, the typical catalysts, it is difficult to obtain high conversions using the alternative substrate for the same product, the isomeric trisubstituted alkenes (<b>D</b> in the text). However, chemoselective modification of the identical functional groups in itaconic acid derivatives are difficult; hence, it would be favorable to use the trisubstituted alkene. Trisubstituted alkene substrates can be hydrogenated with high conversions using chiral analogs of Crabtree’s catalyst of the type IrN­(carbene). This paper demonstrates that such reactions are scalable (tens of grams) and can be manipulated to give optically pure homo-Roche ester chirons. Organocatalytic fluorination, chlorination, and amination of the homo-Roche building blocks was performed to demonstrate that they could easily be transformed into functionalized materials with two chiral centers and α,ω-groups that provide extensive scope for modifications. A synthesis of (<i>S,S</i>)- and (<i>R,S</i>)-γ-hydroxyvaline was performed to illustrate one application of the amination product

Interplay Of Stereochemistry, Conformational Rigidity, And Ease Of Synthesis For 13-Membered Cyclic Peptidomimetics Containing APC Residues

As part of a program to design small molecules that bind proteins, we require cyclic peptides (or peptidomimetics) that are severely constrained such that they adopt one predominant conformation in solution. This paper describes syntheses of the 13-membered cyclic tetrapeptides <b>1</b> containing aminopyrrolidine carboxyl (APC) residues. A linear precursor was prepared and used to determine optimal conditions for cyclization of that substrate. A special linker was prepared to enable cyclization of similar linear peptidomimetics on a solid phase, and the solution-phase cyclization conditions were shown to be appropriate for this too. Stereochemical variations were then used to determine the ideal APC configuration for cyclization of the linear precursors (on a solid phase, using the conditions identified previously). Consequently, a series of compounds were prepared that are representative of compounds <b>1</b>. Conformational studies of representative compounds in DMSO solution were performed primarily using (i) NOE studies, (ii) quenched molecular dynamics simulations using no constraints from experiment, and (iii) MacroModel calculations with NMR constraints. All three strategies converged to the same conclusion: the backbone of molecules based on <b>1</b> tends to adopt one preferential conformation in solution and that conformation can be predicted from the stereochemistries of the α-amino acids involved

Novel Small Molecule Probes for Metastatic Melanoma

Actively targeting probe <b>1b</b>, an unsymmetrical bivalent dipeptide mimic, selectively bound melanoma over healthy skin tissue in histological samples from patients and Sinclair swine. Modifications to <b>1b</b> gave agents <b>2</b>–<b>4</b> that contain a near-IR aza-BODIPY fluor. Contrary to our expectations, symmetrical probe <b>3</b> gave the highest melanoma-to-healthy skin selectivity in histochemistry and experiments with live cells; this was surprising because <b>2</b>, not <b>3</b>, is unsymmetrical like the original lead <b>1</b>. Optical imaging of <b>3</b> in a mouse melanoma model failed to show tumor accumulation <i>in vivo</i>, but the probe did selectively accumulate in the tumor (some in lung and less in the liver) as proven by analysis of the organs post mortem

Protein–Protein Interface Mimicry by an Oxazoline Piperidine-2,4-dione

Representative minimalist mimics <b>1</b> were prepared from amino acids. Scaffold <b>1</b> was not designed to mimic any particular secondary structure, but simulated accessible conformations of this material were compared with common ideal secondary structures and with >125000 different protein–protein interaction (PPI) interfaces. This data mining exercise indicates that scaffolds <b>1</b> can mimic features of sheet-turn-sheets, somewhat fewer helical motifs, and numerous PPI interface regions that do not resemble any particular secondary structure

Small Molecule Inhibitors of the PCSK9·LDLR Interaction

The protein–protein interaction between proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR) is a relatively new, and extremely important, validated therapeutic target for treatment and prevention of heart disease. Experts in the area agree that the first small molecules to disrupt PCSK9·LDLR would represent a milestone in this field, yet few credible leads have been reported. This paper describes how side-chain orientations in preferred conformations of carefully designed chemotypes were compared with LDLR side chains at the PCSK9·LDLR interface to find molecules that would mimic interface regions of LDLR. This approach is an example of the procedure called EKO (Exploring Key Orientations). The guiding hypothesis on which EKO is based is that good matches indicate the chemotypes bearing the same side chains as the protein at the sites of overlay have the potential to disrupt the parent protein–protein interaction. In the event, the EKO procedure and one round of combinatorial fragment-based virtual docking led to the discovery of seven compounds that bound PCSK9 (SPR and ELISA) and had a favorable outcome in a cellular assay (hepatocyte uptake of fluorescently labeled low-density lipoprotein particles) and increased the expression LDLR on hepatocytes in culture. Three promising hit compounds in this series had dissociation constants for PCSK9 binding in the 20–40 μM range, and one of these was modified with a photoaffinity label and shown to form a covalent conjugate with PCSK9 on photolysis

Heterogeneous Phase Transfer Catalysis in Solid Phase Syntheses of <i>Anth</i>-Cyclic Tetrapeptides

This study features solid phase syntheses of cyclic tetrapeptides containing anthranilic acid (<i>Anth</i>) on relatively inexpensive resins derived from polystyrene. It proved to be difficult to hydrolyze a supported <i>Anth</i>-methyl ester <i>unless</i> a phase transfer catalyst was added to facilitate transport of hydroxide into the swollen hydrophobic gel state of the resin. We suggest this may be an under-appreciated strategy for improving syntheses on polystyrene supports