Neuroprotective and Antioxidant Activities of 4-Methylcoumarins: Development of Structure-Activity Relationships.

Coumarins are a major class of polyphenols that are abundantly present in many dietary plants and possess different biological activities. Neuroprotective effect of 28 variously substituted 4-methylcoumarins was evaluated in a cell model of oxidative stress-induced neurodegeneration, which measures viability in PC12 cells challenged with hydrogen peroxide by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The inhibitory activity of these compounds against intracellular reactive oxygen species (ROS) formation was also determined by 2',7'-dichlorofluorescein diacetate method in the same cells. Chemical redox-based assays including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) tests were employed to explore structure-antioxidant activity relationships in a cell-free environment. The results demonstrated that 4-methylcoumarins containing ortho-dihydroxy or ortho-diacetoxy substituents on the benzenoid ring possess considerable neuroprotective effects. ortho-Dihydroxy compounds inhibited cytotoxicity (44.7-62.9%) and ROS formation (41.6-71.1%) at 50 µM and showed considerable antioxidant effects. We conclude that 4-methylcoumarins are promising neuroprotective and antioxidant scaffolds potentially usefull for management of neurodegenerative diseases.

Microwave-assisted Cu(I)-catalyzed, three-component synthesis of 2-(4-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benzo[d]imidazoles.

A microwave-assisted synthesis of 2-(4-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benzo[d]imidazoles from a phenylazide, propargyloxybenzaldehyde and a 1,2-diaminobenzene is proposed.

4-Methylcoumarin derivatives with anti-inflammatory effects in activated microglial cells.

Inflammation contributes to the pathogenesis of neurodegenerative diseases and anti-inflammatory compounds may have a role in prevention or treatment of these pathologies. 4-Methylcoumarins are effective antioxidants with anti-inflammatory properties. In this study, the inhibitory effects of two 4-methylcoumarin derivatives, 7,8-dihydroxy-3-ethoxycarbonylmethyl-4-methylcoumarin (DHEMC) and 7,8-diacetoxy-3-ethoxycarbonylmethyl-4-methylcoumarin (DAEMC) were examined on the inflammatory processes induced by lipopolysaccharide (LPS) in activated primary rat microglial cultures. LPS-induced production of nitric oxide (NO, measured by Griess method) and other pro-inflammatory mediators, thromboxane (TX) B2 and prostaglandin (PG) E2 (both determined by radioimmunoassay (RIA)), as well as tumor necrosis factor (TNF)-α (determined by enzyme-linked immunosorbent assay (ELISA)) were inhibited in the presence of 100 µM DHEMC and DAEMC. DAEMC was able to significantly inhibit NO, TXB2 and TNF-α production also at 50 µM. Both compounds at 100 µM significantly lowered cyclooxygenase-2 (COX-2) protein expression in LPS-stimulated microglial cells measured by Western blot, but only DAEMC showed an inhibitory effect on inducible nitric oxide synthase (iNOS) protein expression at 100 µM. In conclusion, our findings show that 4-methylcoumarin derivatives can modulate inflammatory pathways in microglial cells, probably by acting at the protein expression level.

N-heterocyclic carbene catalyzed aroylation of 3,5-dichloro-2(1H)-pyrazinones.

The N-heterocyclic carbene catalyzed chemoselective C3-aroylation of 3,5-dichloro-2(1H)-pyrazinones with various aldehydes is reported. We herein describe results of this remarkable mild and efficient procedure.

Protein acyltransferase function of purified calreticulin. Part 1: characterization of propionylation of protein utilizing propoxycoumarin as the propionyl group donor.

We have earlier reported that an endoplasmic reticulum luminal protein calreticulin (CR) mediated the acetylation of certain receptor proteins such as glutathione S-transferase (GST) by polyphenolic acetates, leading to irreversible inhibition. This function of calreticulin was termed calreticulin transacetylase. In this communication, we have demonstrated for the first time the ability of the purified recombinant calreticulin of a parasitic nematode Haemonchus contortus to transfer propionyl group from 7,8-Dipropoxy-4-methylcoumarin (DPMC) to recombinant Schistosoma japonicum glutathione S-transferase (rGST). Calreticulin transacetylase exhibited hyperbolic kinetics and yielded K(m) (140 microM) and V(max) (105 units) when the concentration of DPMC was varied keeping the concentration of rGST constant. rGST thus propionylated was found to positively interact with anti-acetyl lysine antibody. Also, the nanoscale LC-MS/MS analysis identified the propionylation sites on three lysine residues: Lys-11, -180 and -181 of rGST. These results highlight the transacylase function of calreticulin (CRTAase).

The role of calreticulin transacetylase in the activation of human platelet nitrite reductase by polyphenolic acetates.

Our earlier investigations demonstrated the remarkable activation of cytochrome P-450 reductase and nitric oxide synthase by 7,8-diacetoxy-4-methylcoumarin, a model polyphenolic acetate by way of acetylation, catalyzed by the Calreticulin. Protein acetyltransferase action of Calreticulin was hence termed Calreticulin transacetylase (CRTAase). Nitric oxide synthase and nitrite reductase are now considered as parts of nitric oxide cycle. The activation of platelets nitric oxide synthase by 7,8-diacetoxy-4-methylcoumarin has already been demonstrated by us. Also, there are reports that certain proteins such as cytochrome P-450 reductase and cytochrome P-450 are endowed with the nitrite reductase activity in mammalian cells. Keeping these facts in view, we turned our attention to probe whether 7,8-diacetoxy-4-methylcoumarin could alter the levels of nitric oxide independent of the action of nitric oxide synthase in the human platelets model. The incubation of 7,8-diacetoxy-4-methylcoumarin and nitrite with platelets caused significant elevation of nitric oxide and cyclic guanosine monophosphate levels possibly due to the activation of nitrite reductase. Several polyphenolic acetates were similarly found to activate the nitrite reductase in tune with their affinities as substrate to CRTAase. N-omega-Nitro-L-arginine methyl ester, the inhibitor of nitric oxide synthase, failed to reverse such an effect of 7,8-diacetoxy-4-methylcoumarin. Clotrimazole which is known to be an inhibitor of nitrite reductase, effectively abolished the 7,8-diacetoxy-4-methylcoumarin mediated enhancement of nitric oxide levels in platelets as well as the nitric oxide mediated effects; such as cyclic guanosine monophosphate levels as well as adenosine diphospate induced platelets aggregation due to nitrite.

Establishment of glutamine synthetase of Mycobacterium smegmatis as a protein acetyltransferase utilizing polyphenolic acetates as the acetyl group donors.

Acetoxy Drug: Protein Transacetylase (TAase) mediating the transfer of acetyl group(s) from polyphenolic acetates (PA) to certain functional proteins in mammalian cells was identified by our earlier investigations. TAase activity was characterized in the cell lysates of Mycobacterium smegmatis and the purified protein was found to have M(r) 58,000. TAase catalysed protein acetylation by a model acetoxy drug 7,8-diacetoxy-4-methylcoumarin (DAMC) was established by the demonstration of immunoreactivity of the acetylated target protein with an anti-acetyllysine antibody. The specificity of the TAase of M. smegmatis (MTAase) to various acetoxycoumarins was found to be in the order DAMC > 7-AMC > 6-AMC > 4-AC > 3-AC > ABP. Also, the N-terminal sequence of purified MTAase was found to perfectly match with glutamine synthetase (GS) of M. smegmatis. The identity of MTAase with GS was confirmed by the observation that the purified MTAase as well as the purified recombinant GS exhibited all the properties of GS. The finding that purified Escherichia coli GS was found to have substantial TAase activity highlighted the TAase function of GS in other bacteria. These results conclusively established for the first time the protein acetyltransferase function of GS of M. smegmatis.

Calreticulin transacetylase catalyzed activation of rat tracheal smooth muscle cell nitric oxide synthase by acetoxycoumarins.

The Transacetylase function of Calreticulin (CR) catalyzing the transfer of acetyl groups from acetoxycoumarins (AC) to certain proteins was identified for the first time in our laboratory. Protein acetyltransferase action of CR was termed Calreticulin Transacetylase (CRTAase). In the present work, CRTAase of rat tracheal smooth muscle cells (TSMC) was characterized with respect to the specificity for various AC and its role in the activation of nitric oxide synthase (NOS). 7,8-Diacetoxy-4-methylcoumarin (DAMC), a model AC, when incubated with TSMC along with L-arginine caused profound activation of NOS as compared to that with L-arginine alone. Further, the inclusion of N-omega-nitro-L-arginine methyl ester (L-NAME) along with DAMC resulted in the reduction of NO levels of TSMC to that of control, there by confirming the activation of TSMC NOS. Also, several AC were found to activate TSMC NOS in tune with their specificities to CRTAase. The results presented in this paper bear evidence for the activation of TSMC NOS by AC and their effectiveness to enhance NO of airway cells may be expected to find useful applications in respiratory diseases.

Palladium-catalyzed copper(I)-mediated cross-coupling of arylboronic acids and 2(1H)-pyrazinones facilitated by microwave irradiation with simultaneous cooling.

The application of a palladium-catalyzed Cu(I)-mediated Liebeskind-Srogl protocol for the decoration of the 2(1H)-pyrazinone scaffold resulted in significantly improved yields and rates when performed under microwave irradiation with simultaneous cooling.

Copper(II)-mediated cross-coupling of arylboronic acids and 2(1H)-pyrazinones facilitated by microwave irradiation with simultaneous cooling.

[reaction: see text] Optimized conditions for the decoration of the 2(1H)-pyrazinone scaffold were developed by applying the Chan-Lam protocol. It was demonstrated that this Cu(II)-mediated cross-coupling reaction resulted in significantly improved yields and rates when performed under microwave irradiation with simultaneous cooling at 0 degrees C, applying a mixture of bases Et(3)N/pyridine.