Improved Synthesis of the Selected Serine Protease uPA Inhibitor UAMC-00050, a Lead Compound for the Treatment of Dry Eye Disease.

The α-aminophosphonate UAMC-00050, a newly developed trypsin-like serine protease inhibitor, is a lead compound for the treatment of dry eye syndrome and ocular inflammation. The medicinal chemistry route developed at the University of Antwerp possessed several problems hampering the scale-up such as poor yields for some of the steps, hazardous reagents, and environmental footprint. Herein, we report an optimized route for the UAMC-00050, in which environmental unfriendly solvents were excluded, hazardous reagents were replaced with safer alternatives, and are more efficient in terms of atom economy. Every reaction step was optimized to reach a higher yield, and design of experiment was used to find the optimum conditions in the last step. Furthermore, all the flash chromatography purifications of intermediates were replaced with plug filtration, slurry purifications, or crystallization. The overall yield was increased from 3% in the medicinal chemistry route to 22% in the process development route.

Chemically diverse activity-based probes with unexpected inhibitory mechanisms targeting trypsin-like serine proteases.

Activity-based probes (ABP) are molecules that bind covalently to the active form of an enzyme family, making them an attractive tool for target and biomarker identification and drug discovery. The present study describes the synthesis and biochemical characterization of novel activity-based probes targeting trypsin-like serine proteases. We developed an extensive library of activity-based probes with "clickable" affinity tags and a diaryl phosphonate warhead. A wide diversity was achieved by including natural amino acid analogs as well as basic polar residues as side chains. A detailed enzymatic characterization was performed in a panel of trypsin-like serine proteases. Their inhibitory potencies and kinetic profile were examined, and their IC50 values, mechanism of inhibition, and kinetic constants were determined. The activity-based probes with a benzyl guanidine side chain showed the highest inhibitory effects in the panel. Surprisingly, some of the high-affinity probes presented a reversible inhibitory mechanism. On the other hand, probes with different side chains exhibited the expected irreversible mechanism. For the first time, we demonstrate that not only irreversible probes but also reversible probes can tightly label recombinant proteases and proteases released from human mast cells. Even under denaturing SDS-PAGE conditions, reversible slow-tight-binding probes can label proteases due to the formation of high-affinity complexes and slow dissociation rates. This unexpected finding will transform the view on the required irreversible nature of activity-based probes. The diversity of this library of activity-based probes combined with a detailed enzyme kinetic characterization will advance their applications in proteomic studies and drug discovery.

Amphiphilic peptide-based MMP3 inhibitors for intra-articular treatment of knee OA.

Since 2007, Metalloproteases (MMPs) have been considered potential targets for treating osteoarthritis (OA), for which the primary pathogenic event is the extensive degeneration of articular cartilage. MMP3 is an enzyme critical for these degenerative changes. However, problems of selectivity, low bioavailability and poor metabolic profile during clinical trials of MMPs inhibitors (MMPIs) led to limited beneficial effect and thus did not justify further pursuit of the clinical studies. In a previous work, a new alkyl derivative of hyaluronic acid (HA), HYADD4®, previously approved as intra-articular treatment for knee OA, was studied in vitro and in vivo as MMP3I. Molecular simulation studies confirmed the interaction between the alkyl side chain of this HA derivative and the additional S1' pocket of MMP3. However, the high MW and the polar HA backbone of HYADD4® imply a high desolvation energy cost, which can potentially decrease its inhibitory potency. In this study, a new class of MMP3Is based on a small peptide backbone (CGV) chemically derivatized with an alkyl chain was developed through interactive cycles of design, synthesis and screening, accompanied by computational evaluation and optimization. Two MMP3Is, e(I) and l(II), were selected because of their effective inhibitory activity (3.2 and 10.2 µM, respectively) and water solubility. Both MMPIs showed a broad range of inhibitory effects against almost all the MMPs tested. In an in vitro model of inflammatory OA, e(I) was the most effective MMPI: at the concentration of 93 µM, it reversed inflammatory outcomes. Moreover, because of its amphiphilic structure, the e(I) MMPI promoted stable micellar formulation at concentrations higher than 0.2 mg/mL in water. The findings were confirmed by TEM and Nile red staining analysis. Based on these results, the e(I) MMPI can be considered a good candidate for the intra-articular treatment of OA, and the micellar formulation of this peptide in an aqueous buffer can potentially increase the bioavailability and, thus, the efficacy of the MMPIs.

One-pot synthesis of α-aminophosphonates by yttrium-catalyzed Birum-Oleksyszyn reaction.

For the first time, yttrium triflate was used as an efficient green catalyst for the synthesis of α-aminophosphonates through a one-pot three-component Birum-Oleksyszyn reaction. Under the action of this Lewis acid, enhancement of the yield and reaction chemoselectivity was provided by the achievement of an appropriate balance in the complex network of reactions.