Regioselective addition of Grignard reagents to N-acylpyrazinium salts: synthesis of substituted 1,2-dihydropyrazines and Δ5-2-oxopiperazines.

The regioselective addition of Grignard reagents to mono- and disubstituted N-acylpyrazinium salts affording substituted 1,2-dihydropyrazines in modest to excellent yields (45-100%) is described. Under acidic conditions, these 1,2-dihydropyrazines can be converted to substituted Δ5-2-oxopiperazines providing a simple and efficient approach towards their preparation.

Discovery and characterization of an iminocoumarin scaffold as an inhibitor of MEKK2 (MAP3K2).

The kinase MEKK2 (MAP3K2) activates the MEK5/ERK5 cell signaling pathway and may play an important role in tumor growth and metastasis. Thus, MEKK2 may represent a novel kinase target for cancer. In order to identify inhibitors of MEKK2, we screened a library of compounds using a high throughput MEKK2 intrinsic ATPase enzyme assay. We identified two hits with validated structures and confirmed activity in the primary assay (IC50 values = 322 nM and 7.7 µM) and two orthogonal MEKK2 biochemical assays. Compound 1, the more potent hit, was the subject of further investigation. Limited structure-activity relationship (SAR) studies were performed on this iminocoumarin hit which resulted in ≥20-fold more potent analogs (e.g. 8 and 16 nM IC50). Two analogs had improved selectivity in a 50-member kinase profiling panel compared to the hit. These studies suggested that substitutions around the phenoxy ring of this scaffold can impart improved potency and selectivity for MEKK2. Analog Compound 1s (16 nM IC50) was further verified by external testing to inhibit MEKK2 and MEKK3 with similar potencies. Compound 1s displayed activity in cell-based assays in which it inhibited ERK5 pathway activation in cells and inhibited cell migration in a scratch assay. Thus, we have identified a scaffold that has promising potential to be developed into a highly selective and potent inhibitor of MEKK2. Information from these SAR studies provides specific guidance for the future design of MEKK2 inhibitor probes.

Neuroprotective effects of an in vitro BBB permeable phenoxythiophene sulfonamide small molecule in glutamate-induced oxidative injury.

Reactive oxygen species (ROS) play a central role in oxidative stress-associated neuronal cell death during ischemia. Further investigation into the inhibition of excessive ROS generation post-stroke is urgently required for the treatment of ischemic stroke. In the present study, the neuroprotective properties of the blood-brain barrier (BBB) penetrant B355227 were investigated. B355227 is a chemical analogue of B355252, and the role of the phenoxythiophene sulfonamide compound B355227 was further investigated in a glutamate-induced oxidative injury model. An in vitro model of the BBB was established in the immortalized mouse brain capillary endothelial cell line, bEnd.3. Formation of barrier in Transwell inserts was confirmed using EVOM resistance meter and Caffeine, Imatinib and Axitinib were used to validate the efficacy of the model. The validated BBB assay in combination with high performance liquid chromatography were used to analyse and verify the permeability of B355227 through the barrier. The integrity of the cell junctions after the BBB assays were confirmed using immunofluorescence to visualize the expression of the barrier junction protein zonula occludens-1. Cell survival was measured with Resazurin, a redox indicator dye, in HT22, a hippocampal neuronal cell treated with 5 mM glutamate or co-treated with the B355227 recovered from the BBB permeability experiment. Changes in glutathione levels were detected using a glutathione detection kit, while analyses of ROS, calcium (Ca2+), and mitochondrial membrane potential (MMP) were accomplished with the fluorescent dyes 2',7'-dichlorofluorescein diacetate, Fura-2 AM and MitoTracker Red dyes, respectively. Immunoblotting was also performed to detect the expression and activation of Erk1/2, p-38, JNK, Bax and Bcl-2. The results of the present study demonstrated that B355227 crossed the BBB in vitro and protected HT22 from oxidative injury induced by glutamate exposure. Treatment of cells with B355227 blocked the glutamate-dependent depletion of intracellular glutathione and significantly reduced ROS production. Increased Ca2+ influx and subsequent collapse of the MMP was attenuated by B355227. Furthermore, the results of the present study demonstrated that B355227 protected against oxidative stress via the MAPK pathway, by increasing the activation of Erk1/2, JNK and P38, and restoring anti-apoptotic Bcl-2. Collectively, the results of the present study indicate that B355227 has potent antioxidant and neuroprotective attributes in glutamate-induced neuronal cell death. Further investigation into the role of B355227 in the modulation of glutamate-dependent oxidative stress is required.

A trifluoromethyl analog of verbenachalcone promotes neurite outgrowth and cell proliferation of NeuroScreen-1 cells.

Past research has shown that natural products of plant and marine origins and their congeners enhance the actions of neuritogenic factors of the central nervous system (CNS) such as nerve growth factor (NGF). However, the role of fluorine substitutions in their structure-activity relationship (SAR) has not been explored. We have synthesized a trifluoromethyl analog of verbenachalcone (VC), a pharmacologically active natural compound previously shown to potentiate NGF activity. This analog, designated C278, enhances neurite outgrowth and proliferation of NeuroScreen-1™ (NS-1) cells, a subclone of PC12 pheochromocytoma cells. C278 increases the percentage of neurite bearing cells in the presence of suboptimal doses of NGF in comparison with controls treated with NGF alone. In addition, C278 stimulates cell growth in reduced serum and serum-free cell culture conditions based on our observation of increases in cell number and metabolic assessment with MTT reduction and resazurin assays. The addition of C278 partially restored inhibition of NGF-induced neurite outgrowth by the mitogen-activated protein kinase kinase (MEK) inhibitors PD98059 and U0126. Short-term sequential exposure of cells to U0126, C278, and NGF enhanced phosphorylation of extracellular signal-regulated kinase (ERK) in comparison with cells treated with only the MEK inhibitor and NGF. C278 also attenuated cell growth arrest caused by exposure to PD98059, U0126 and the phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002 but did not alter phosphorylation of Akt, a classic downstream target of PI3K during cell survival. These data suggest that C278 promotes NGF-dependent neurite outgrowth in NS-1 cells through a MEK signaling pathway by a mechanism that alters short-term activation of ERK. In contrast, C278 promotes PI3K-mediated survival independently of Akt phosphorylation.

High molecular weight polyglycerol-based multivalent mannose conjugates.

We report the synthesis and characterization of multivalent mannose conjugates based on high molecular weight hyperbranched polyglycerols (HPG). A range of glycoconjugates were synthesized from high molecular weight HPGs (up to 493 kDa) and varying mannose units (22-303 per HPG). Hemagglutination assays using fresh human red blood cells and concanavalin A (Con A) showed that HPG-mannose conjugates exhibited a large enhancement in the relative potency of conjugates (as high as 40000) along with a significant increment in relative activity per sugar (up to 255). The size of the HPG scaffold and the number of mannose residues per HPG were all shown to influence the enhancement of binding interactions with Con A. Isothermal titration calorimetry (ITC) experiments confirmed the enhanced binding affinity and showed that both molecular size and ligand density play important roles. The enhancement in Con A binding to the high molecular weight HPG-mannose conjugates is due to a combination of inter- and intramolecular mannose binding. A few fold increments in the binding constant were obtained over mannose upon covalent attachment to HPG. The binding enhancement is due to the highly favorable entropic contribution to the multiple interactions of Con A to mannose residues on HPG. The high molecular weight HPG-mannose conjugates showed positive cooperativity in binding to Con A. Although carbohydrate density has less of an effect on functional valency of the conjugate compared to the molecular size, it determines the binding affinity.

Microwave-assisted synthesis of 4-chloro-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide (B-355252): a new potentiator of Nerve Growth Factor (NGF)-induced neurite outgrowth.

The synthesis of 4-chloro-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide (B-355252) using a MW-assisted nucleophilic aromatic substitution (S(N)Ar) reaction will be discussed. Utilization of this method allowed for the rapid generation of B-355252 heteroaryl ether core structure in the presence of cesium carbonate in dimethylformamide or tripotassium phosphate in N-methyl-2-pyrrolidone in 94% yield. Evaluation of B-355252 enhancement of nerve growth factor's ability to stimulate neurite outgrowths was determined using NS-1 cells.

A p-methoxybenzyl (PMB) protection/deprotection approach toward the synthesis of 5-phenoxy-4-chloro-N-(aryl/alkyl) thiophene-2-sulfonamides.

The synthesis of 17 phenoxy substituted 4-chloro-N-(aryl/alkyl)thiophene-2-sulfonamides using a PMB protection/deprotection strategy is described. Nucleophilic displacement of p-methoxybenzyl (PMB) protected 4,5-dichloro-N-(aryl/alkyl)-thiophene-2-sulfonamides was carried out with different phenols under mild basic conditions. Reaction times of 3-6 h and overall yields of 78-98% were achieved with the PMB group in place compared to no reaction without this protecting group. The PMB group was easily and selectively removed in 68-98% yield using TFA in DCM.

A synthetic analog of verbenachalcone potentiates NGF-induced neurite outgrowth and enhances cell survival in neuronal cell models.

This study uses NeuroScreen-1 (NS-1) cells, a derivative of pheochromocytoma (PC12) cells, to examine neurite outgrowth induced by a novel synthetic verbenachalcone derivative, DSRB20-022 (C22). We treated NS-1 cells with varying concentrations of C22 in the presence of 2ng/mL nerve growth factor (NGF). A dose-dependent effect of C22 was observed at concentrations of 2microM and above, resulting in significant enhancement of NGF-dependent neurite outgrowth in NS-1 cells. C22 did not exhibit neuritogenic activity in the absence of NGF, but promoted a concentration-dependent increase in neurite-bearing cells without inducing cytotoxicity. Cell viability assays showed that C22 and the parent compound verbenachalcone (VC) are neuroprotective and enhanced survival of NS-1, PC12, and the murine neuro-2A (N2a) cell lines under conditions of serum deprivation. The results show that augmentation of NGF-induced neurite outgrowth by C22 in NS-1 was dependent on MAP kinase. Furthermore, the neuroprotective function of C22 and VC was accompanied by suppression of caspase-3/7 activation. However, C22 and VC exerted their antagonistic effects on caspase-3/7 activation through potentially different mechanisms of action.