Synthesis, biological evaluation, and computational studies of N-benzyl pyridinium-curcumin derivatives as potent AChE inhibitors with antioxidant activity.

A library of N-benzylpyridinium-based compounds, 7a-j and 8a-j, was designed and synthesised as potential acetylcholinesterase) AChE (inhibitors. An in vitro assay for the synthesised compounds showed that most compounds had significant AChE inhibitory activities at the nanomolar and submicromolar levels. The benzyl (8a) and fluoro (8b) derivatives were the most active, with IC50 values ≤56 nM. Compound 7f, which had a benzyl moiety, showed the highest potency among all the target compounds, with an IC50 value of 7.5 ± 0.19 nM against AChE, which was higher than that of the activities of tacrine (IC50 = 30 ± 0.2 nM) and donepezil (IC50 = 14 ± 0.12 nM). Compounds with vanillin moieties exhibited antioxidant activity. Among the tested compounds, four derivatives (7f, 7 g, 8f, and 8 g) exhibited superior AChE inhibitory activity, with Ki values of 6-16 nM, which were potent in the same range as the approved drug, donepezil. These compounds showed moderate antioxidant activities, as indicated by the results of the ABTS assay.

Solriamfetol impurities: Synthesis, characterization, and analytical method (UPLC-UV) validation.

Given that impurities may affect the quality and safety of drug products, impurity identification and profiling is an integral part of drug quality control and is particularly important for newly developed medications such as solriamfetol, which is used to treat excessive daytime sleepiness. Although the high-performance liquid chromatography analysis of commercial solriamfetol has revealed the presence of several impurities, their synthesis, structure elucidation, and chromatographic determination have not been reported yet. To bridge this gap, we herein identified, synthesized, and isolated eight process-related solriamfetol impurities, characterized them using spectroscopic and chromatographic techniques, and proposed plausible mechanisms of their formation. Moreover, we developed and validated a prompt impurity analysis method based on ultrahigh-performance liquid chromatography with UV detection, revealing that its selectivity, linearity, accuracy, precision, and quantitation limit meet the acceptance criteria of method validation stipulated by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use. Thus, the developed method was concluded to be suitable for the routine analysis of solriamfetol substances.

Solriamfetol impurities:Synthesis,characterization,and analytical method(UPLC-UV)validation / 药物分析学报

Given that impurities may affect the quality and safety of drug products,impurity identification and profiling is an integral part of drug quality control and is particularly important for newly developed medications such as solriamfetol,which is used to treat excessive daytime sleepiness.Although the high-performance liquid chromatography analysis of commercial solriamfetol has revealed the presence of several impurities,their synthesis,structure elucidation,and chromatographic determination have not been reported yet.To bridge this gap,we herein identified,synthesized,and isolated eight process-related solriamfetol impurities,characterized them using spectroscopic and chromatographic tech-niques,and proposed plausible mechanisms of their formation.Moreover,we developed and validated a prompt impurity analysis method based on ultrahigh-performance liquid chromatography with UV detection,revealing that its selectivity,linearity,accuracy,precision,and quantitation limit meet the acceptance criteria of method validation stipulated by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use.Thus,the developed method was concluded to be suitable for the routine analysis of solriamfetol substances.

The Cytoprotective Effects of E-α-(4-Methoxyphenyl)-2',3,4,4'-Tetramethoxychalcone (E-α-p-OMe-C6H4-TMC)--A Novel and Non-Cytotoxic HO-1 Inducer.

Cell protection against different noxious stimuli like oxidative stress or chemical toxins plays a central role in the treatment of many diseases. The inducible heme oxygenase isoform, heme oxygenase-1 (HO-1), is known to protect cells against a variety of harmful conditions including apoptosis. Because a number of medium strong electrophiles from a series of α-X-substituted 2',3,4,4'-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO2-C6H4, Ph, p-OMe-C6H4, NO2, CF3, COOEt, COOH) had proven to activate Nrf2 resulting in HO-1 induction and inhibit NF-κB downstream target genes, their protective effect against staurosporine induced apoptosis and reactive oxygen species (ROS) production was investigated. RAW264.7 macrophages treated with 19 different chalcones (15 α-X-TMCs, chalcone, 2'-hydroxychalcone, calythropsin and 2'-hydroxy-3,4,4'-trimethoxychalcone) prior to staurosporine treatment were analyzed for apoptosis and ROS production, as well as HO-1 protein expression and enzyme activity. Additionally, Nrf2 and NF-κB activity was assessed. We found that amongst all tested chalcones only E-α-(4-methoxyphenyl)-2',3,4,4'-tetramethoxychalcone (E-α-p-OMe-C6H4-TMC) demonstrated a distinct, statistically significant antiapoptotic effect in a dose dependent manner, showing no toxic effects, while its double bond isomer Z-α-p-OMe-C6H4-TMC displayed no significant activity. Also, E-α-p-OMe-C6H4-TMC induced HO-1 protein expression and increased HO-1 activity, whilst inhibition of HO-1 by SnPP-IX abolished its antiapoptotic effect. The only weakly electrophilic chalcone E-α-p-OMe-C6H4-TMC reduced the staurosporine triggered formation of ROS, while inducing the translocation of Nrf2 into the nucleus. Furthermore, staurosporine induced NF-κB activity was attenuated following E-α-p-OMe-C6H4-TMC treatment. Overall, E-α-p-OMe-C6H4-TMC demonstrated its effective cytoprotective potential via a non-toxic induction of HO-1 in RAW264.7 macrophages. The observed cytoprotective effect may partly be related to both, the activation of the Nrf2- and inhibition of the NF-κB pathway.

Enhancing the anti-inflammatory activity of chalcones by tuning the Michael acceptor site.

Inflammatory signaling pathways orchestrate the cellular response to infection and injury. These pathways are known to be modulated by compounds that alkylate cysteinyl thiols. One class of phytochemicals with strong thiol alkylating activity is the chalcones. In this study we tested fourteen chalcone derivatives, α-X-substituted 2',3,4,4'-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO2-C6H4, Ph, p-OMe-C6H4, NO2, CF3, COOEt, COOH), for their ability to modulate inflammatory responses, as monitored by their influence on heme oxygenase-1 (HO-1) activity, inducible nitric oxide synthase (iNOS) activity, and cytokine expression levels. We confirmed that the transcriptional activity of Nrf2 was activated by α-X-TMCs while for NF-κB it was inhibited. For most α-X-TMCs, anti-inflammatory activity was positively correlated with thiol alkylating activity, i.e. stronger electrophiles (X = CF3, Br and Cl) being more potent. Notably, this correlation did not hold true for the strongest electrophiles (X = CN and NO2) which were found to be ineffective as anti-inflammatory compounds. These results emphasize the idea that chemical fine-tuning of electrophilicity is needed to achieve and optimize desired therapeutic effects.

The synthetic α-bromo-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) inhibits the JAK/STAT signaling pathway.

Signal transducer and activator of transcription STAT5 and its upstream activating kinase JAK2 are essential mediators of cytokine signaling. Their activity is normally tightly regulated and transient. However, constitutive activation of STAT5 is found in numerous cancers and a driving force for malignant transformation. We describe here the identification of the synthetic chalcone α-Br-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) as a novel JAK/STAT inhibitor. Using the non-transformed IL-3-dependent B cell line Ba/F3 and its oncogenic derivative Ba/F3-1*6 expressing constitutively activated STAT5, we show that α-Br-TMC targets the JAK/STAT pathway at multiple levels, inhibiting both JAK2 and STAT5 phosphorylation. Moreover, α-Br-TMC alters the mobility of STAT5A/B proteins in SDS-PAGE, indicating a change in their post-translational modification state. These alterations correlate with a decreased association of STAT5 and RNA polymerase II with STAT5 target genes in chromatin immunoprecipitation assays. Interestingly, expression of STAT5 target genes such as Cis and c-Myc was differentially regulated by α-Br-TMC in normal and cancer cells. While both genes were inhibited in IL-3-stimulated Ba/F3 cells, expression of the oncogene c-Myc was down-regulated and that of the tumor suppressor gene Cis was up-regulated in transformed Ba/F3-1*6 cells. The synthetic chalcone α-Br-TMC might therefore represent a promising novel anticancer agent for therapeutic intervention in STAT5-associated malignancies.

Opening or closing the lock? When reactivity is the key to biological activity.

Thiol-mediated processes play a key role to induce or inhibit inflammation proteins. Tailoring the reactivity of electrophiles can enhance the selectivity to address only certain surface cysteines. Fourteen 2',3,4,4'-tetramethoxychalcones with different α-X substituents (X=H, F, Cl, Br, I, CN, Me, p-NO2-C6H4, Ph, p-OMe-C6H4, NO2, CF3, COOEt, COOH) were synthesized, containing the potentially electrophilic α,ß-unsaturated carbonyl unit. The assessment of their reactivity as electrophiles in thia-Michael additions with cysteamine shows a change in the reactivity of more than six orders of magnitude. Moreover, a clear correlation between their reactivity and an influence on the inflammation proteins heme oxygenase-1 (HO-1) and the inducible NO synthase (iNOS) is demonstrated. As the biologically most active compound, the α-CF3 -chalcone is shown to inhibit the NO production in RAW264.7 mouse macrophages in the nanomolar range.

Reactivity assessment of chalcones by a kinetic thiol assay.

The electrophilic nature of chalcones (1,3-diphenylprop-2-en-1-ones) and many other α,ß-unsaturated carbonyl compounds is crucial for their biological activity, which is often based on thiol-mediated regulation processes. To better predict their biological activity a simple screening assay for the assessment of the second-order rate constants (k(2)) in thia-Michael additions was developed. Hence, a clear structure-activity relationship of 16 differentially decorated hydroxy-alkoxychalcones upon addition of cysteamine could be established. Moreover, amongst other naturally occurring α,ß-unsaturated carbonyl compounds k(2) values for curcumin and cinnamaldehyde were gained while cinnamic acids or esters gave no or very slow reactions.