Methoxy-naphthyl-Linked N-Benzyl Pyridinium Styryls as Dual Cholinesterase Inhibitors: Design, Synthesis, Biological Evaluation, and Structure-Activity Relationship.

The multifaceted nature of Alzheimer's disease (AD) indicates the need for multitargeted agents as potential therapeutics. Both cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), play a vital role in disease progression. Thus, inhibiting both ChEs is more beneficial than only one for effectively managing AD. The present study provides a detailed lead optimization of the e-pharmacophore-generated pyridinium styryl scaffold to discover a dual ChE inhibitor. A structure-activity relationship analysis indicated the importance of three structural fragments, methoxy-naphthyl, vinyl-pyridinium, and substituted-benzyl, in a dual ChE inhibitor pharmacophore. The optimized 6-methoxy-naphthyl derivative, 7av (SB-1436), inhibits EeAChE and eqBChE with IC50 values of 176 and 370 nM, respectively. The kinetic study has shown that 7av inhibits AChE and BChE in a non-competitive manner with ki values of 46 and 115 nM, respectively. The docking and molecular dynamics simulation demonstrated that 7av binds with the catalytic and peripheral anionic sites of AChE and BChE. Compound 7av also significantly stops the self-aggregation of Aß. The data presented herein indicate the potential of 7av for further investigation in preclinical models of AD.

Blood-brain barrier permeable benzylpiperidin-4-yl-linked benzylamino benzamides as dual cholinesterase inhibitors.

Alzheimer's disease (AD) is a multifaceted neurodegenerative disorder involving various pathological events. The existing options for managing the disease utterly rely on cholinesterase (ChE) inhibitors. In recent years, the dual inhibition of ChEs as a potential AD therapeutics has substantially attracted the attention of medicinal chemists. Recently, we reported benzyl piperidinyl-linked methoxy-naphthamides as dual ChE inhibitors. Herein, we investigated the peripheral anionic binding site-binding methoxy-naphthamide fragment that yielded benzyl piperidinyl-linked benzyl aminobenzamide as another class of dual ChE inhibitors. The 3,5-dimethoxy benzyl aminobenzamide, 8c1, exhibits inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with half-maximal inhibitory concentration values of 0.61 and 2.04 µM, respectively. The enzyme kinetics and molecular modeling study indicated the noncompetitive and mixed-type mode of inhibition for AChE and BChE with ki values of 0.14 and 0.46 µM, respectively. The derivative 8c1 crosses the blood-brain barrier as indicated by the Pe value of 14.34 × 10-6 cm/s in the parallel artificial membrane permeability assay. Besides this, it also inhibits the self-aggregation of amyloid-ß. The results presented herein indicate the potential of benzamide 8c1 for further investigation in preclinical models of AD.

Tetramethoxystilbene Inhibits NLRP3 Inflammasome Assembly via Blocking the Oligomerization of Apoptosis-Associated Speck-like Protein Containing Caspase Recruitment Domain: In Vitro and In Vivo Evaluation.

Nucleotide-binding domain leucine-rich repeat family pyrin domain containing 3 (NLRP3) inflammasome complex regulates the caspase-1 activity and subsequent processing of interleukin-1ß (IL-1ß). Various inflammatory diseases involve the activation of inflammasome complexes; thus, the intervention in complex formation via small molecules offers a new therapeutic opportunity. The structure-guided design and synthesis of a series of methoxystilbenes and methoxy-2-phenylnaphthalenes identified new inhibitors of NLRP3 inflammasome complex. The tetramethoxystilbene 4o and trimethoxy 2-phenylnaphthalene 1t inhibit the release of a mature form of IL-1ß in J774A.1 cells with IC50 values of 1.39 and 2.07 µM, respectively. Mechanistic investigation revealed that tetramethoxystilbene 4o blocks the oligomerization of apoptosis-associated speck-like protein (ASC), which is the vital step in the formation of NLRP3 inflammasome assembly, thus preventing the activation of caspase-1 and the IL-1ß release. Treatment of LPS+ATP challenged mice with 20 mg/kg of 4o significantly suppressed the levels of IL-1ß. The data presented herein warrant further investigation of methoxystilbenes in disease-specific models of inflammatory diseases.

IIIM-941, a Stilbene Derivative Inhibits NLRP3 Inflammasome Activation by Inducing Autophagy.

Aberrant activation of NLRP3 inflammasome has been implicated in several inflammatory diseases. Autophagy is one of the primary mechanisms that regulate NLRP3 inflammasome activity. In this study, we attempted to target NLRP3 inflammasome activity by a synthetic compound IIIM-941. We found that IIIM-941 inhibits ATP induced NLRP3 inflammasome by induction of autophagy through AMPK pathway in bone marrow derived macrophages (BMDMs) and J774A.1 cells. It was interesting to observe that IIIM-941 did not show any inhibitory activity against LPS induced pro-inflammatory cytokines TNF-α and IL-6. The anti-NLRP3 activity of IIIM-941 was significantly reversed when we attempted to block autophagy by using either pharmacological inhibitor bafilomycin A1or by using siRNA against AMPK. Further, we found that IIIM-941 downregulated the expression of NLRP3 and prevented the oligomerization of ASC to exert its anti-NLRP3 inflammasome effect in J774A.1 cells. We validated inhibitory activity of IIIM-941 against NLRP3 in three different mice models. The anti-inflammatory effect of IIIM-941 was highly significant in ATP induced peritoneal inflammation model. IIIM-941 was similarly effective in suppressing MSU induced IL-1ß in the air pouch model of inflammation without affecting the levels of TNF-α and IL-6. Finally, oral efficacy of IIIM-941 was also proved in MSU indued foot paw edema model of inflammation in mice at 10 and 20 mg/kg (b.w.). The compounds like IIIM-941 can be explored further for the development of therapies against diseases such as Alzheimer's disease and Parkinson's disease, where hampered autophagy and NLRP3 activation play a crucial role in the pathological development.

Design, synthesis and comparative analysis of triphenyl-1,2,3-triazoles as anti-proliferative agents.

Herein, a series of triaryl-1,2,3-triazoles, in order to check cytotoxicity on breast cancer cell lines have been synthesized with pendent benzyl ring to mimic the phenolic A ring of Tamoxifene. The biological results indicated that most of the compounds possessed comparative anti-proliferative activities in both ER + ve (MCF-7) and ER-ve (MDA-MB-231) breast cancer cell lines. Among synthesized derivatives, five compounds 8f, 8i, 8j, 8n and 8p showed anti-proliferative activities at <5 µM against MCF-7 cell line and three compounds 8e, 8f and 8j show IC50 value greater than 30 µM in FR-2 cells (normal cell). Moreover, to understand the mechanistic behavior of the selective compound 8f, various studies performed viz. surface morphological changes by bright field microscopic examination, nuclear morphological alteration by DAPI staining, measurement of intracellular ROS level and determination of change in mitochondrial membrane potential. It was observed that, the selective compound 8f associated with higher ROS generation along with decrease in mitochondrial membrane potential in addition to surface and nuclear morphological alterations such as reduction in number and shrinkage of cells coupled with nuclear blabbing indicating sign of apoptosis. Further, molecular docking study in comparison to tamoxifen was also carried out to investigate the interaction of 8f with ER-α which favors its possible mode of anticancer action.

Discovery of methoxy-naphthyl linked N-(1-benzylpiperidine) benzamide as a blood-brain permeable dual inhibitor of acetylcholinesterase and butyrylcholinesterase.

The cholinesterase enzymes play a vital role in maintaining balanced levels of the neurotransmitter acetylcholine in the central nervous system. However, the overexpression of these enzymes results in hampered neurotransmission. Both the major forms of cholinesterase enzymes viz. acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) play a crucial role in blocking neurotransmission; therefore, in recent years, a strategy of dual cholinesterase inhibition is being explored. Herein, we developed an energy-optimized e-pharmacophore hypothesis AHHPRR from AChE-donepezil complex and screened a set of 15 scaffolds that were designed imaginarily. The ligand with N-(1-benzylpyridinium) benzamide framework has shown the highest fitness and volume score, which was chosen for synthesis and validation. A series of pyridinium benzamides were synthesized and screened for cholinesterase inhibition that led to the identification of 7b, a naphthalene containing N-(1-benzylpiperidine) benzamide as a potent dual AChE and BChE inhibitor with IC50 values of 0.176, and 0.47 µM, respectively. The kinetic study indicated that 7b inhibits AChE in a non-competitive manner with Ki value of 0.21 µM, and BChE in a mixed-fashion with Ki of 0.15 µM. The observed mode of inhibition was corroborated with molecular docking studies. The MD simulation studies pointed out that both AChE and BChE undergo low conformational changes in complex with 7b. The benzamide 7b displayed high BBB permeability in PAMPA assay, which indicates its potential for further exploration in preclinical studies for Alzheimer's disease.

Discovery of Helminthosporin, an Anthraquinone Isolated from Rumex abyssinicus Jacq as a Dual Cholinesterase Inhibitor.

Natural products have extensively contributed toward the discovery of new leads for Alzheimer's disease. During our search for new inhibitors of cholinesterase enzymes from natural sources, the ethyl acetate (EtOAc) extract of Rumex abyssinicus Jacq was identified as a dual cholinesterase inhibitor with IC50 values of 2.7 and 11.4 µg/mL against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), respectively. The phytochemical investigation of the EtOAc extract has resulted in isolation of four anthraquinones, namely, helminthosporin, emodin, chrysophanol, and physcion, amongst which the helminthosporin has been isolated for the first time from Rumex sp. All isolated secondary metabolites have displayed significant inhibition of EeAChE with IC50 values of 2.63, 15.21, 33.7, and 12.16 µM, respectively. In addition, the helminthosporin was also found to inhibit BChE with an IC50 value of 2.99 µM. The enzyme kinetic study has indicated that helminthosporin inhibits AChE and BChE in a noncompetitive manner with k i values of 10.3 and 12.3 µM, respectively. The results of molecular modeling and propidium iodide displacement assay have revealed that helminthosporin occupies the peripheral anionic site of the active site gorge of AChE. In the PAMPA-BBB permeability assay, helminthosporin was found to possess high BBB permeability (P e = 6.16 × 10-6 cm/s). In a nutshell, helminthosporin has been identified as a brain permeable dual cholinesterase inhibitor, and thus its further synthetic exploration is warranted for optimization of its potency.

Discovery of benzo[cd]indol-2-one and benzylidene-thiazolidine-2,4-dione as new classes of NLRP3 inflammasome inhibitors via ER-ß structure based virtual screening.

The structure-guided virtual screening (VS) has proved to be successful strategy in identification of new scaffolds for biological targets. The overactivity of NLRP3 inflammasome has been implicated in variety of inflammatory diseases including Alzheimer's disease. The up-regulation of estrogen-receptor ß (ER-ß) activity has been directly linked with inhibition of NLRP3 inflammasome activity. In the present study, we report discovery of new NLRP3 inflammasome inhibitors via ER-ß crystal structure (PDB: 5TOA) guided virtual screening of 20,000 compound library. For experimental validation, top 10 ligands were selected based on structure novelty, docking score, prime MMGB/SA binding affinity and interaction pattern analysis. Amongst the tested compounds, three thiazolidin-4-ones IIIM-1268, IIIM-1269 and IIIM-1270 and benzo[cd]indol-2-one IIIM-1266 have shown 73, 69, 75 and 77% suppression of IL-1ß release in mouse macrophages (J774A.1 cells) at 10 µM. Benzylidene-thiazolidine-2,4-diones IIIM-1268 and IIIM-1270 inhibited IL-1ß release with IC50 of 2.3 and 3.5 µM and also significantly decreased the protein expression level of mature form of IL-1ß in western-blot analysis. IIIM-1266 and IIIM-1270 displayed bidentate H-bonding with Arg 346 and Glu 305 residues in the active site of ER-ß; and they also strongly occupied the ADP-binding site of NLRP3 protein. The results presented herein, indicate that ER-ß guided VS can be successfully used to identify new NLRP3 inflammasome inhibitors, which may have potential in the development of novel anti-Alzheimer agents.

Discovery of Quinazolin-4(3 H)-ones as NLRP3 Inflammasome Inhibitors: Computational Design, Metal-Free Synthesis, and in Vitro Biological Evaluation.

NLRP3 inflammasome is an important therapeutic target for a number of human diseases. Herein, computationally designed series of quinazolin-4(3 H)-ones were synthesized using iodine-catalyzed coupling of arylalkynes (or styrenes) with O-aminobenzamides. The key event in this transformation involves the oxidative cleavage of the C-C triple/double bond and the release of formaldehyde. The reaction relies on the C-N bond formation along with the C-C bond cleavage under metal-free conditions. The nitro-substituted quinazolin-4(3 H)-one 2k inhibited NLRP3 inflammasome (IC50 5 µM) via the suppression of IL-1ß release from ATP-stimulated J774A.1 cells.

Nonantioxidant Tetramethoxystilbene Abrogates α-Synuclein-Induced Yeast Cell Death but Not That Triggered by the Bax or ßA4 Peptide.

The overexpression of α-synuclein (α-syn) and its aggregation is the hallmark of Parkinson's disease. The α-syn aggregation results in the formation of Lewy bodies that causes neuronal cell death. Therefore, the small molecules that can protect neuronal cells from α-syn toxicity or inhibit the aggregation of α-syn could emerge as anti-Parkinson agents. Herein, a library of methoxy-stilbenes was screened for their ability to restore the cell growth from α-syn toxicity, using a yeast strain that stably expresses two copies of a chromosomally integrated human α-syn gene. Tetramethoxy-stilbene 4s, a nonantioxidant, was the most capable of restoring cell growth. It also rescues the more toxic cells that bear three copies of wild-type or A53T-mutant α-syn, from cell growth block. Its EC50 values for growth restoration of the 2-copy wild-type and the 3-copy mutant α-syn strains are 0.95 and 0.35 µM, respectively. Stilbene 4s mitigates mitochondrial membrane potential loss, negates ROS production, and prevents nuclear DNA-fragmentation, all hallmarks of apoptosis. However, 4s does not rescue cells from the death-inducing effects of Bax and ßA4, which suggest that 4s specifically inhibits α-syn-mediated toxicity in the yeast. Our results signify that simultaneous use of multiple yeast-cell-based screens can facilitate revelation of compounds that may have the potential for further investigation as anti-Parkinson's agents.

Oxidant-Controlled C-sp2/sp3-H Cross-Dehydrogenative Coupling of N-Heterocycles with Benzylamines.

Oxidant controlled ionic liquid mediated cross-dehydrogenative coupling (CDC) of benzylamines with N-heterocycles having sp2 or sp3 carbon resulted in the formation of C-benzoylated or alkenylated products. Benzoylation of N-heterocycles occurs via (NH4)2S2O8 catalyzed benzoyl radical formation. An oxidative alkenylation of N-heterocycles having C-sp3 carbon (2-methylaza-arenes) occurs via deamination of benzylamine followed by C-sp3-H bond activation in high stereoselectivity. Both benzoylation and alkenylation protocols are metal-free, green, simple, efficient, and tolerate a wide variety of functional groups.