Synthesis of Fused Indolines by Interrupted Fischer Indolization in a Microfluidic Reactor.

This study describes our development of a microfluidic reaction scheme for the synthesis of fused indoline ring systems found in several bioactive compounds. We have utilized a continuous-flow microfluidic reactor for the reaction of hydrazines with latent aldehydes through the interrupted Fischer indolization reaction to form fused indoline and azaindoline products. We have identified optimal conditions and evaluated the scope of this microfluidic reaction using various hydrazine and latent aldehyde surrogates. This green chemistry approach can be of general utility to rapidly produce indoline scaffolds and intermediates in a continuous manner.

Suppression of tau propagation using an inhibitor that targets the DK-switch of nSMase2.

Targeting of molecular pathways involved in the cell-to-cell propagation of pathological tau species is a novel approach for development of disease-modifying therapies that could block tau pathology and attenuate cognitive decline in patients with Alzheimer's disease and other tauopathies. We discovered cambinol through a screening effort and show that it is an inhibitor of cell-to-cell tau propagation. Our in vitro data demonstrate that cambinol inhibits neutral sphingomyelinase 2 (nSMase2) enzyme activity in dose response fashion, and suppresses extracellular vesicle (EV) production while reducing tau seed propagation. Our in vivo testing with cambinol shows that it can reduce the nSMase2 activity in the brain after oral administration. Our molecular docking and simulation analysis reveals that cambinol can target the DK-switch in the nSMase2 active site.

Drug-induced modulation of gp130 signalling prevents articular cartilage degeneration and promotes repair.

OBJECTIVE: Human adult articular cartilage (AC) has little capacity for repair, and joint surface injuries often result in osteoarthritis (OA), characterised by loss of matrix, hypertrophy and chondrocyte apoptosis. Inflammation mediated by interleukin (IL)-6 family cytokines has been identified as a critical driver of proarthritic changes in mouse and human joints, resulting in a feed-forward process driving expression of matrix degrading enzymes and IL-6 itself. Here we show that signalling through glycoprotein 130 (gp130), the common receptor for IL-6 family cytokines, can have both context-specific and cytokine-specific effects on articular chondrocytes and that a small molecule gp130 modulator can bias signalling towards anti-inflammatory and antidegenerative outputs. METHODS: High throughput screening of 170 000 compounds identified a small molecule gp130 modulator termed regulator of cartilage growth and differentiation (RCGD 423) that promotes atypical homodimeric signalling in the absence of cytokine ligands, driving transient increases in MYC and pSTAT3 while suppressing oncostatin M- and IL-6-mediated activation of ERK and NF-κB via direct competition for gp130 occupancy. RESULTS: This small molecule increased proliferation while reducing apoptosis and hypertrophic responses in adult chondrocytes in vitro. In a rat partial meniscectomy model, RCGD 423 greatly reduced chondrocyte hypertrophy, loss and degeneration while increasing chondrocyte proliferation beyond that observed in response to injury. Moreover, RCGD 423 improved cartilage healing in a rat full-thickness osteochondral defect model, increasing proliferation of mesenchymal cells in the defect and also inhibiting breakdown of cartilage matrix in de novo generated cartilage. CONCLUSION: These results identify a novel strategy for AC remediation via small molecule-mediated modulation of gp130 signalling.

Optimization of pyrimidinol antioxidants as mitochondrial protective agents: ATP production and metabolic stability.

Previously we described a novel series of pyrimidinol antioxidants and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Our initial lead compound was a potent antioxidant in vitro, but was subsequently found to exhibit poor stability to oxidative metabolism. The current study focused on balancing potency with metabolic stability through structural modification, and involved modifications at positions 2 and 4 of the pyrimidinol redox core, likely sites of oxidative metabolism. Eight new analogues have been prepared and their ability to suppress lipid peroxidation and reactive oxygen species (ROS), and to preserve mitochondrial membrane potential (Δψm) and support ATP production, has been investigated. The metabolic stability of the prepared compounds was also assessed in vitro using bovine liver microsomes to obtain preliminary insight on this class of compounds. This study revealed the complexity of balancing reasonable metabolic stability with efficient antioxidant properties. While a few analogues appear promising, especially in terms of metabolic stability, a 4-isopropoxy derivative conserved the favorable biological activity and exhibited good metabolic stability. The favorable metabolic stability conferred by the combination of the azetidine and isopropoxy moieties in analogue 6 makes this compound an excellent candidate for further evaluation.

C-O bond Formation in a Microfluidic Reactor: High Yield SNAr Substitution of Heteroaryl Chlorides.

This study describes our development of a novel and efficient procedure for C-O bond formation under mild conditions, for coupling heteroaryl chlorides with phenols or primary aliphatic alcohols. We utilized a continuous-flow microfluidic reactor for C-O bond formation in electron-deficient pyrimidines and pyridines in a much more facile manner with a cleaner reaction profile, high yield, quick scalability and without the need for the transition metal catalyst. This approach can be of general utility to make C-O bond containing intermediates of industrial importance in a continuous and safe manner.

Cytoprotective pyridinol antioxidants as potential therapeutic agents for neurodegenerative and mitochondrial diseases.

As part of our ongoing efforts to identify compounds having potential utility in treating neurodegenerative and mitochondrial disorders, a series of pyridinol analogues have been prepared. The synthetic route employed for the preparation of the new analogues is different, and considerably more efficient, than that used in previously reported studies. The new route yields a pair of pyridinol regioisomers that can be readily separated and evaluated. Their ability to quench lipid peroxidation and reactive oxygen species (ROS), and to preserve mitochondrial membrane potential (Δψm) and support ATP synthesis is reported. The optimal side chain length was found to be 16 carbon atoms. The metabolic stability of those compounds having optimal biological activities was evaluated in vitro using bovine liver microsomes. The omission of any side chain hydroxyl group and introduction of an azetidine moiety at position 6 of the pyridinol redox core (8 and 9) increased their microsomal stability as compared to the exocyclic dimethylamino group. The favorable metabolic stability conferred by the azetidine moiety in compounds 8 and 9 makes these compounds excellent candidates for further evaluation.

A Small Molecule Mimetic of the Humanin Peptide as a Candidate for Modulating NMDA-Induced Neurotoxicity.

Humanin (HN), a 24-amino acid bioactive peptide, has been shown to increase cell survival of neurons after exposure to Aß and NMDA-induced toxicity and thus could be beneficial in the treatment of Alzheimer's disease (AD). The neuroprotection by HN is reported to be primarily through its agonist binding properties to the gp130 receptor. However, the peptidic nature of HN presents challenges in its development as a therapeutic for AD. We report here for the first time the elucidation of the binding site of Humanin (HN) peptide to the gp130 receptor extracellular domain through modeling and the synthesis of small molecule mimetics that interact with the HN binding site on the gp130 receptor and provide protection against NMDA-induced neurotoxicity in primary hippocampal neurons. A brain permeable small molecule mimetic was identified through exploratory medicinal chemistry using microfluidic flow chemistry to facilitate the synthesis of new analogues for screening and SAR optimization.

Deformable Nanovesicles Synthesized through an Adaptable Microfluidic Platform for Enhanced Localized Transdermal Drug Delivery.

Phospholipid-based deformable nanovesicles (DNVs) that have flexibility in shape offer an adaptable and facile method to encapsulate diverse classes of therapeutics and facilitate localized transdermal delivery while minimizing systemic exposure. Here we report the use of a microfluidic reactor for the synthesis of DNVs and show that alteration of input parameters such as flow speeds as well as molar and flow rate ratios increases entrapment efficiency of drugs and allows fine-tuning of DNV size, elasticity, and surface charge. To determine the ability of DNV-encapsulated drug to be delivered transdermally to a local site, we synthesized, characterized, and tested DNVs carrying the fluorescently labeled hydrophilic bisphosphonate drug AF-647 zoledronate (AF647-Zol). AF647-Zol DNVs were lyophilized, resuspended, and applied topically as a paste to the calvarial skin of mice. High-resolution fluorescent imaging and confocal microscopy revealed significant increase of encapsulated payload delivery to the target tissue-cranial bone-by DNVs as compared to nondeformable nanovesicles (NVs) or aqueous drug solutions. Interestingly, NV delivery was not superior to aqueous drug solution. Our studies show that microfluidic reactor-synthesized DNVs can be produced in good yield, with high encapsulation efficiency, reproducibility, and stability after storage, and represent a useful vehicle for localized transdermal drug delivery.

Thermodynamic, diffusional, and structural anomalies in rigid-body water models.

Structural, density, entropy, and diffusivity anomalies of the TIP4P/2005 model of water are mapped out over a wide range of densities and temperatures. The locus of temperatures of maximum density (TMD) for this model is very close to the experimental TMD locus for temperatures between 250 and 275 K. Four different water models (mTIP3P, TIP4P, TIP5P, and SPC/E) are compared with the TIP4P/2005 model in terms of their anomalous behavior. For all the water models, the density regimes for anomalous behavior are bounded by a low-density limit at around 0.85-0.90 g cm(-3) and a high-density limit at about 1.10-1.15 g cm(-3). The onset temperatures of the density anomaly in the various models show a much greater variation, ranging from 202 K for mTIP3P to 289 K for TIP5P. The order maps for the various water models are qualitatively very similar with the structurally anomalous regions almost superimposable in the q(tet)-τ plane. Comparison of the phase diagrams of water models with the region of liquid-state anomalies shows that the crystalline phases are much more sensitive to the choice of water models than the liquid state anomalies; for example, SPC/E and TIP4P/2005 show qualitatively similar liquid state anomalies but very different phase diagrams. The anomalies in the liquid in all the models occur at much lower pressures than those at which the melting line changes from negative to positive slope. The results in this study demonstrate several aspects of structure-entropy-diffusivity relationships of water models that can be compared with experiment and used to develop better atomistic and coarse-grained models for water.

Relationship between structure, entropy, and diffusivity in water and water-like liquids.

Anomalous behavior of the excess entropy (S(e)) and the associated scaling relationship with diffusivity are compared in liquids with very different underlying interactions but similar water-like anomalies: water (SPC/E and TIP3P models), tetrahedral ionic melts (SiO(2) and BeF(2)), and a fluid with core-softened, two-scale ramp (2SRP) interactions. We demonstrate the presence of an excess entropy anomaly in the two water models. Using length and energy scales appropriate for onset of anomalous behavior, we show the density range of the excess entropy anomaly to be much narrower in water than in ionic melts or the 2SRP fluid. While the reduced diffusivities (D*) conform to the excess-entropy-scaling relation, D* = A exp(alphaS(e)) for all the systems (Rosenfeld, Y. Phys. Rev. A 1977, 15, 2545), the exponential scaling parameter, alpha, shows a small isochore dependence in the case of water. Replacing S(e) by pair correlation-based approximants accentuates the isochore dependence of the diffusivity scaling. Isochores with similar diffusivity-scaling parameters are shown to have the temperature dependence of the corresponding entropic contribution. The relationship between diffusivity, excess entropy, and pair correlation approximants to the excess entropy are very similar in all the tetrahedral liquids.