Modulation of Mitochondrial Metabolic Parameters and Antioxidant Enzymes in Healthy and Glaucomatous Trabecular Meshwork Cells with Hybrid Small Molecule SA-2.

Oxidative stress (OS)-induced mitochondrial damage is a risk factor for primary open-angle glaucoma (POAG). Mitochondria-targeted novel antioxidant therapies could unearth promising drug candidates for the management of POAG. Previously, our dual-acting hybrid molecule SA-2 with nitric oxide-donating and antioxidant activity reduced intraocular pressure and improved aqueous humor outflow in rodent eyes. Here, we examined the mechanistic role of SA-2 in trabecular meshwork (TM) cells in vitro and measured the activity of intracellular antioxidant enzymes during OS. Primary human TM cells isolated from normal (hNTM) or glaucomatous (hGTM) post-mortem donors and transformed glaucomatous TM cells (GTM-3) were used for in vitro assays. We examined the effect of SA-2 on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro using Seahorse Analyzer with or without the oxidant, tert-butyl hydroperoxide (TBHP) treatment. Concentrations of total antioxidant enzymes, catalase (CAT), malondialdehyde (MDA), and glutathione peroxidase (GPx) were measured. We observed significant protection of both hNTM and hGTM cells from TBHP-induced cell death by SA-2. Antioxidant enzymes were elevated in SA-2-treated cells compared to TBHP-treated cells. In addition, SA-2 demonstrated an increase in mitochondrial metabolic parameters. Altogether, SA-2 protected both normal and glaucomatous TM cells from OS via increasing mitochondrial energy parameters and the activity of antioxidant enzymes.

Acid-Catalyzed Synthesis of Isatoic Anhydride-8-Secondary Amides Enables IASA Transformations for Medicinal Chemistry.

Quinazolin-dione-N-3-alklyl derivatives are the core scaffolds for several categories of bioactive small molecules, but current synthetic methods are costly, involve environmental hazards, and are not uniformly scalable. Here, we report an inexpensive, flexible, and scalable method for the one-pot synthesis of substituted quinazolin-dione-N-3-alkyls (isomers of isatoic-8-secondary amides (IASAs)) from isatin that take advantage of in situ capture of imidic acid under acidic conditions. We further show that this method can be used for the synthesis of a wide variety of derivatives with medicinal uses.

Novel Thiol Containing Hybrid Antioxidant-Nitric Oxide Donor Small Molecules for Treatment of Glaucoma.

Oxidative stress induced death and dysregulation of trabecular meshwork (TM) cells contribute to the increased intraocular pressure (IOP) in primary open angle (POAG) glaucoma patients. POAG is one of the major causes of irreversible vision loss worldwide. Nitric oxide (NO), a small gas molecule, has demonstrated IOP lowering activity in glaucoma by increasing aqueous humor outflow and relaxing TM. Glaucomatous pathology is associated with decreased antioxidant enzyme levels in ocular tissues causing increased reactive oxygen species (ROS) production that reduce the bioavailability of NO. Here, we designed, synthesized, and conducted in vitro studies of novel second-generation sulfur containing hybrid NO donor-antioxidants SA-9 and its active metabolite SA-10 to scavenge broad-spectrum ROS as well as provide efficient protection from t-butyl hydrogen peroxide (TBHP) induced oxidative stress while maintaining NO bioavailability in TM cells. To allow a better drug delivery, a slow release nanosuspension SA-9 nanoparticles (SA-9 NPs) was prepared, characterized, and tested in dexamethasone induced ocular hypertensive (OHT) mice model for IOP lowering activity. A single topical eye drop of SA-9 NPs significantly lowered IOP (61%) at 3 h post-dose, with the effect lasting up to 72 h. This class of molecule has high potential to be useful for treatment of glaucoma.

Green Synthesis of Substituted Anilines and Quinazolines from Isatoic Anhydride-8-amide.

Synthetic methods used to generate substituted anilines and quinazolines, both privileged pharmacological structures, are cumbersome, hazardous or, in some cases, unavailable. We developed a straightforward method for making isatoic anhydride-8-amide from isatin-7-carboxylic acid as a tool to easily produce a range of quinazoline and substituted aniline derivatives using adaptable pH-sensitive cyclization chemistry. The approaches are inexpensive, simple, fast, efficient at room temperature and scalable, enabling the synthesis of both established and new quinazolines and also highly substituted anilines including cyano derivatives.

Publisher Correction: Green Synthesis of Substituted Anilines and Quinazolines from Isatoic Anhydride-8-amide.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Development of biodegradable crosslinked urethane-doped polyester elastomers.

Traditional crosslinked polyester elastomers are inherently weak, and the strategy of increasing crosslink density to improve their mechanical properties makes them brittle materials. Biodegradable polyurethanes, although strong and elastic, do not fare well in dynamic environments due to the onset of permanent deformation. The design and development of a soft, strong and completely elastic (100% recovery from deformation) material for tissue engineering still remains a challenge. Herein, we report the synthesis and evaluation of a new class of biodegradable elastomers, crosslinked urethane-doped polyesters (CUPEs), which is able to satisfy the need for soft, strong, and elastic biomaterials. Tensile strength of CUPE was as high as 41.07+/-6.85 MPa with corresponding elongation at break of 222.66+/-27.84%. The initial modulus ranged from 4.14+/-1.71 MPa to 38.35+/-4.5 MPa. Mechanical properties and degradation rates of CUPE could be controlled by varying the choice of diol used for synthesis, the polymerization conditions, as well as the concentration of urethane bonds in the polymer. The polymers demonstrated good in vitro and in vivo biocompatibilities. Preliminary hemocompatibility evaluation indicated that CUPE adhered and activated lesser number of platelets compared to PLLA. Good mechanical properties and easy processability make these materials well suited for soft tissue engineering applications. The introduction of CUPEs provides new avenues to meet the versatile requirements of tissue engineering and other biomedical applications.

Temperature-regulated activity of responsive polymer-protein conjugates prepared by grafting-from via RAFT polymerization.

A facile route to well-defined "smart" polymer-protein conjugates with tunable bioactivity is reported. Protein modification with a reversible addition-fragmentation chain transfer (RAFT) agent and subsequent room temperature polymerization in aqueous media led to conjugates of poly(N-isopropylacrylamide) and a model protein. Representing the first example of polymer-protein conjugation with RAFT agent immobilization via the "R-group" approach, high molecular weight and reductively stable conjugates were accessible without extensive purification or adverse effects on the protein structure. An increase in molecular weight with conversion was observed for the chains grafted from the protein surface, confirming the controlled nature of the polymerization. The responsive behavior of the immobilized polymer facilitated conjugate isolation and also allowed environmental modulation of bioactivity.

Folate-conjugated thermoresponsive block copolymers: highly efficient conjugation and solution self-assembly.

A combination of controlled radical polymerization and azide-alkyne click chemistry was employed to prepare temperature-responsive block copolymer micelles conjugated with biological ligands with potential for active targeting of cancer tissues. Block copolymers of N-isopropylacrylamide (NIPAM) and N,N-dimethylacrylamide (DMA) were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization with an azido chain transfer agent (CTA). Pseudo-first-order kinetics and linear molecular weight dependence on conversion were observed for the RAFT polymerizations. CuI-catalyzed coupling with propargyl folate resulted in folic acid residues being efficiently conjugated to the alpha-azido chain ends of the homo and block copolymers. Temperature-induced self-assembly resulted in aggregates capable of controlled release of a model hydrophobic drug. CuI-catalyzed azide-alkyne cycloaddition has proven superior to conventional methods for conjugation of biological ligands to macromolecules, and the general strategy presented herein can potentially be extended to the preparation of folate-functionalized assemblies with other stimuli susceptibility (e.g., pH) for therapeutic and imaging applications.