Directed Evolution and Immobilization of Lactobacillus brevis Alcohol Dehydrogenase for Chemo-Enzymatic Synthesis of Rivastigmine.

Rivastigmine is one of the several pharmaceuticals widely prescribed for the treatment of Alzheimer's disease. However, its practical synthesis still faces many issues, such as the involvement of toxic metals and harsh reaction conditions. Herein, we report a chemo-enzymatic synthesis of Rivastigmine. The key chiral intermediate was synthesized by an engineered alcohol dehydrogenase from Lactobacillus brevis (LbADH). A semi-rational approach was employed to improve its catalytic activity and thermal stability. Several LbADH variants were obtained with a remarkable increase in activity and melting temperature. Exploration of the substrate scope of these variants demonstrated improved activities toward various ketones, especially acetophenone analogs. To further recycle and reuse the biocatalyst, one LbADH variant and glucose dehydrogenase were co-immobilized on nanoparticles. By integrating enzymatic and chemical steps, Rivastigmine was successfully synthesized with an overall yield of 66 %. This study offers an efficient chemo-enzymatic route for Rivastigmine and provides several efficient LbADH variants with a broad range of potential applications.

Discovery of acetophenone/piperazin-2-one hybrids as selective anti-TNBC cancer agents by causing DNA damage.

Twenty-five acetophenone/piperazin-2-one (APPA) hybrids were designed and synthesized based on key pharmacophores found in anti-breast cancer drugs Neratinib, Palbociclib, and Olaparib. Compound 1j exhibited good in vitro antiproliferative activity (IC50 = 6.50 µM) and high selectivity (SI = 9.2 vs HER2-positive breast cancer cells SKBr3; SI = 7.3 vs normal breast cells MCF-10A) against triple negative breast cancer (TNBC) cells MDA-MB-468. In addition, 1j could selectively cause DNA damage, inducing the accumulation of γH2AX and P53 in MDA-MB-468 cells. It also reduced the phosphorylation level of P38 and the expression of HSP70, which further prevented the repair of DNA damage and caused cells S/G2-arrest leading to MDA-MB-468 cells death.

Synthesis of Cytotoxic Benzofurans and Ethers Derivatives of Paeonol.

Paeonol is a broadly studied natural product due to its many biological activities. Using a methodology previously employed by our research group, 11 derivatives of paeonol were synthesized (seven of them are unpublished compounds), including four ethers and seven benzofurans. Additionally, we determined the crystal structure of one of these ether derivatives (1 a) and of five benzofuran derivatives (2 a, 2 b, 2 c, 2 f and 2 g) by single crystal X-ray diffraction. To continue studying the cytotoxicity of this natural product and its derivatives, all compounds were tested against two cancer cell lines, HCT116 and MCF-7. Compounds 2 b, 2 e, and 2 g were considered active against the colorectal adenocarcinoma cells HCT116 (Growth inhibition >60 %). Compound 2 e showed an IC50 of 0.2 µM and was selected for further analysis, results reinforce its anticancer potential.

Inhibitory Activity Against Rhizoctonia Solani and Chemical Composition of Extract from Moutan Cortex.

Rice sheath blight (RSB), caused by Rhizoctonia solani, is a significant disease of rice. The negative effects of chemical fungicides have created an urgent need for low-toxicity botanical fungicides. Our previous research revealed that the ethanol crude extract of Moutan Cortex (MC) exhibited superior antifungal activity against R. solani at 1000 µg/mL, resulting in a 100 % inhibition rate. The antifungal properties were mainly found in the petroleum ether extract. However, the active ingredients of the extract are still unclear. In this study, gas chromatography-mass spectrometry (GC-MS) was utilised for the analysis of its chemical components. The mycelium growth rate method was utilized to detect the antifungal activity. The findings indicated that paeonol constituted the primary active component, with a content of more than 96 %. Meanwhile, paeonol was the most significant antifungal active ingredient, the antifungal activity of paeonol (EC50=44.83 µg/mL) was much higher than that of ß-sitosterol and ethyl propionate against R. solani. Observation under an optical microscope revealed that paeonol resulted in abnormal mycelial morphology. This study provided theoretical support for identifying monomer antifungal compounds and developing biological fungicides for R. solani.

Establishing Composition of Solid Solution Based on Single Crystal and Powder X-ray Measurement: The Case of Halogenated Bismuth(III) Complexes with Acetophenone-4-methyl-3-thiosemicarbazone.

New bismuth (III) complexes with acetophenone-4-methyl-3-thiosemicarbazone (L) and halogens (Cl and Br) in both bridging and terminal positions have been synthesized and structurally characterized using single-crystal X-ray diffraction. The pure complexes (Cl or Br) were found to be highly isostructural, which motivated our attempts to create solid solutions of these complexes. A series of such compounds was prepared using various procedures and stoichiometries. A method for determining the mutual concentrations of different halogens, based on the positions of selected peaks in powder diffraction patterns, was tested and compared with other methods.

Traveling across Life Sciences with Acetophenone-A Simple Ketone That Has Special Multipurpose Missions.

Each metabolite, regardless of its molecular simplicity or complexity, has a mission or function in the organism biosynthesizing it. In this review, the biological, allelochemical, and chemical properties of acetophenone, as a metabolite involved in multiple interactions with various (mi-cro)organisms, are discussed. Further, the details of its biogenesis and chemical synthesis are provided, and the possibility of its application in different areas of life sciences, i.e., the status quo of acetophenone and its simple substituted analogs, is examined. In particular, natural and synthetic simple acetophenone derivatives are analyzed as promising agrochemicals and useful scaffolds for drug research and development.

Discovery of novel paeonol-based derivatives against skin inflammation in vitro and in vivo.

T-LAK-cell-originated protein kinase (TOPK), a novel member of the mitogen-activated protein kinase family, is considered an effective therapeutic target for skin inflammation. In this study, a series (A - D) of paeonol derivatives was designed and synthesised using a fragment growing approach, and their anti-inflammatory activities against lipopolysaccharide (LPS)-induced nitric oxide production in RAW264.7 cells were tested. Among them, compound B12 yielded the best results (IC50 = 2.14 µM) with low toxicity (IC50 > 50 µM). Preliminary mechanistic studies indicated that this compound could inhibit the TOPK-p38/JNK signalling pathway and phosphorylate downstream related proteins. A murine psoriasis-like skin inflammation model was used to determine its therapeutic effect.

Paeonol attenuates inflammation by confining HMGB1 to the nucleus.

Inflammation is a biological process that exists in a large number of diseases. If the magnitude or duration of inflammation becomes uncontrolled, inflammation may cause pathological damage to the host. HMGB1 and NF-κB have been shown to play pivotal roles in inflammation-related diseases. New drugs aimed at inhibiting HMGB1 expression have become a key research focus. In the present study, we showed that paeonol (Pae), the main active component of Paeonia suffruticosa, decreases the expression of inflammatory cytokines and inhibits the translocation of HMGB1 induced by lipopolysaccharide (LPS). By constructing HMGB1-overexpressing (HMGB1+ ) and HMGB1-mutant (HMGB1m ) RAW264.7 cells, we found that the nuclear HMGB1 could induce an LPS-tolerant state in RAW264.7 cells and that paeonol had no influence on the expression of inflammatory cytokines in HMGB1m RAW264.7 cells. In addition, the anti-inflammatory property of paeonol was lost in HMGB1 conditional knockout mice, indicating that HMGB1 is a target of paeonol and a mediator through which paeonol exerts its anti-inflammatory function. Additionally, we also found that HMGB1 and P50 competitively bound with P65, thus inactivating the NF-κB pathway. Our research confirmed the anti-inflammation property of paeonol and suggests that inhibiting the translocation of HMGB1 could be a new strategy for treating inflammation.

An improved and highly selective fluorescence assay for measuring phosphatidylserine decarboxylase activity.

Phosphatidylserine decarboxylases (PSDs) catalyze the conversion of phosphatidylserine (PS) to phosphatidylethanolamine (PE), a critical step in membrane biogenesis and a potential target for development of antimicrobial and anti-cancer drugs. PSD activity has typically been quantified using radioactive substrates and products. Recently, we described a fluorescence-based assay that measures the PSD reaction using distyrylbenzene-bis-aldehyde (DSB-3), whose reaction with PE produces a fluorescence signal. However, DSB-3 is not widely available and also reacts with PSD's substrate, PS, producing an adduct with lower fluorescence yield than that of PE. Here, we report a new fluorescence-based assay that is specific for PSD and in which the presence of PS causes only negligible background. This new assay uses 1,2-diacetyl benzene/ß-mercaptoethanol, which forms a fluorescent iso-indole-mercaptide conjugate with PE. PE detection with this method is very sensitive and comparable with detection by radiochemical methods. Model reactions examining adduct formation with ethanolamine produced stable products of exact masses (m/z) of 342.119 and 264.105. The assay is robust, with a signal/background ratio of 24, and can readily detect formation of 100 pmol of PE produced from Escherichia coli membranes, Candida albicans mitochondria, or HeLa cell mitochondria. PSD activity can easily be quantified by sequential reagent additions in 96- or 384-well plates, making it readily adaptable to high-throughput screening for PSD inhibitors. This new assay now enables straightforward large-scale screening for PSD inhibitors against pathogenic fungi, antibiotic-resistant bacteria, and neoplastic mammalian cells.

2,2,2-trifluoro-1-(1,4,5,6-tetrahydropyridin-3-yl)ethanone derivative as efflux pump inhibitor in Mycobacterium tuberculosis.

Although the administration of combined therapy is efficient to tuberculosis (TB) treatment caused by susceptible Mycobacterium tuberculosis strains, to overcome the multidrug resistance is still a challenge. Some studies have reported evidence about tetrahydropyridines as a putative efflux pump inhibitor, including in mycobacteria, being a promising strategy against M. tuberculosis. Thus, we investigated the biological potential of 2,2,2-trifluoro-1-(1,4,5,6-tetrahydropyridin-3-yl)ethanone derivative (NUNL02) against two strains of M. tuberculosis. NUNL02 was able to increase the susceptibility of the multidrug resistant strain to the anti-TB drugs, resulting in synergism with rifampicin. Still, we assume that this compound plays a role in the efflux mechanism in M. tuberculosis, besides, to be able to kill the bacillus under the deprivation of essential nutrients. Thus, our findings highlight NUNL02 as a promising prototype to develop a new adjuvant for TB treatment, mainly as EPI.

Computational Structural Revision of a 4-Hydroxy-3-(1'-angeloyloxy-2',3'-epoxy-3'-methyl)butylacetophenone Compound from Ageratina grandifolia.

The structure of the reported compound 4-hydroxy-3-((S)-1'-angeloyloxy-(R)-2',3'-epoxy-3'-methyl)butylacetophenone (1), isolated from Ageratina grandifolia, has been revised through the use of DFT computational predictions. Re-examination of the reported experimental and DFT computed chemical shifts has led to the proposal of a chromane skeleton rather than the original epoxide derivative. Empirical predictions of the 13C and 1H NMR shifts showed a much better fit for the chromane structure than for the epoxide. The relative configuration of the molecule was established using CASE-3D methodology on the basis of new DFT chemical shielding and J-coupling predictions, allowing the proposal of a new rel-2,2-dimethyl-3R-hydroxy-4S-(1-angeloyloxy)chromane structure (2) for the isolated compound. However, DFT prediction of the optical rotation for the CASE-3D selected configuration/conformations did not provide a conclusive answer for the absolute configuration.

Design, synthesis of Cinnamyl-paeonol derivatives with 1, 3-Dioxypropyl as link arm and screening of tyrosinase inhibition activity in vitro.

This study aimed to obtain tyrosinase inhibitors for treating hyperpigmentation. A series of cinnamyl ester analogues were designed and synthesized with cinnamic acid (CA) and peaonol compounds. The safety, melanin content and inhibitory effects of all target compounds were evaluated. In the enzymatic activity test, the inhibitory rate of compounds 8, 13 and 14 had stronger inhibitory activity with the IC50 values of 20.7 µM, 13.98 µM and 15.16 µM, respectively than the positive drug kojic acid (IC50 with 30.83 µM). The cytotoxicity evaluation showed that compounds 13 and 14 have higher safety than the other compounds to the proliferation of B16F10 cells. The result of the melanocyte test supported that compound13 has stronger cellular tyrosinase inhibitory activity than kojic acid and arbutin at 100 µM and 200 µM. The enzyme kinetics mechanism revealed that compound 13 was a non-competitive inhibitor while compounds 8 and 14 were mixed inhibitors. For the experiments of melanin content and tyrosinase activity in the B16F10 melanona cells, the inhibition rates of compounds 8, 14 and 13 were with 19.62%, 20.59% and 23.83%, respectively. In addition, compound 13 revealed the highest inhibitory activity to tyrosinase in the melanocyte with inhibition rates of 23.83%, which was better than kojic acid and arbutin (19.21% and 20.45%) at the same concentration. In the anti-melanogenesis experiment, compounds 8 and 13 had better anti-melanin effects than kojic acid from 25 µM to 100 µM. In summary, the results indicated that compounds 8, 13 and 14 had better tyrosinase inhibitory activity and anti-melanogenesis activity. Especially, the compound 13 has potentiality to develop novel tyrosinase inhibitors and whitening agents. The docking studies results revealed that the functional group of compound 13 mostly depends on the phenolic hydroxyl moiety, and its hydroxyl group did not insert into the active site of tyrosinase, which was in agreement with the results of the kinetics study.

Synthesis of chalcones derived from 1-naphthylacetophenone and evaluation of their cytotoxic and apoptotic effects in acute leukemia cell lines.

Chalcones and their derivatives have been described as promising compounds with antiproliferative activity against leukemic cells. This study aimed to investigate the cytotoxic effect of three synthetic chalcones derived from 1-naphthylacetophenone (F07, F09, and F10) in acute leukemia cell lines (K562 and Jurkat) and examine the mechanisms of cell death induced by these compounds. The three compounds were cytotoxic to K562 and Jurkat cells, with IC50 values ranging from 1.03 to 31.66 µM. Chalcones induced intrinsic and extrinsic apoptosis, resulting in activation of caspase-3 and DNA fragmentation. F07, F09, and F10 were not cytotoxic to human peripheral blood mononuclear cells, did not produce any significant hemolytic activity, and did not affect platelet aggregation after ADP stimulation. These results, combined with calculations of molecular properties, suggest that chalcones F07, F09, and F10 are promising molecules for the development of novel antileukemic drugs.

Identification of a newly isolated Rhodotorula mucilaginosa NQ1 and its development for the synthesis of bulky carbonyl compounds by whole-cell bioreduction.

A strain NQ1, which showed efficient asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to enantiopure (S)-[3,5-bis(trifluoromethyl)phenyl]ethanol ((S)-BTPE), which is the key intermediate for the synthesis of a receptor antagonist and antidepressant, was isolated from a soil sample. Based on its morphological and internal transcribed spacer sequence, the strain NQ1 was identified to be Rhodotorula mucilaginosa NQ1. Some key reaction parameters involved in the bioreduction catalyzed by whole cells of R. mucilaginosa NQ1 were subsequently optimized, and the optimized conditions for the synthesis of (S)-BTPE were determined to be as follows: 5·0 ml phosphate buffer (200 mmol l-1 , pH 7·0), 80 mmol l-1 of BTAP, 250 g (wet weight) l-1 of resting cell, 35 g l-1 of glucose and a reaction for 18 h at 30°C and 180 rev min-1 . The strain NQ1 exhibited a best yield of 99% and an excellent enantiomeric excess of 99% for the preparation of (S)-BTPE under the above optimal conditions, and could also asymmetrically reduce a variety of bulky prochiral carbonyl compounds to their corresponding optical hydroxyl compound with excellent enantioselectivity. These results indicated that R. mucilaginosa NQ1 had a good capacity to reduce BTAP to its corresponding (S)-BTPE, and might be a new potential biocatalyst for the production of valuable chiral hydroxyl compounds in industry.

Natural Benzo/Acetophenones as Leads for New Synthetic Acetophenone Hybrids Containing a 1,2,3-Triazole Ring as Potential Antifouling Agents.

Marine biofouling is a natural process that represents major economic, environmental, and health concerns. Some booster biocides have been used in biofouling control, however, they were found to accumulate in environmental compartments, showing negative effects on marine organisms. Therefore, it is urgent to develop new eco-friendly alternatives. Phenyl ketones, such as benzophenones and acetophenones, have been described as modulators of several biological activities, including antifouling activity (AF). In this work, acetophenones were combined with other chemical substrates through a 1,2,3-triazole ring, a strategy commonly used in Medicinal Chemistry. In our approach, a library of 14 new acetophenone-triazole hybrids was obtained through the copper(I)-catalyzed alkyne-azide cycloaddition "click" reaction. All of the synthesized compounds were evaluated against the settlement of a representative macrofouling species, Mytilus galloprovincialis, as well as on biofilm-forming marine microorganisms, including bacteria and fungi. The growth of the microalgae Navicula sp. was also evaluated after exposure to the most promising compounds. While compounds 6a, 7a, and 9a caused significant inhibition of the settlement of mussel larvae, compounds 3b, 4b, and 7b were able to inhibit Roseobacter litoralis bacterial biofilm growth. Interestingly, acetophenone 7a displayed activity against both mussel larvae and the microalgae Navicula sp., suggesting a complementary action of this compound against macro- and microfouling species. The most potent compounds (6a, 7a, and 9a) also showed to be less toxic to the non-target species Artemia salina than the biocide Econea®. Regarding both AF potency and ecotoxicity activity evaluation, acetophenones 7a and 9a were put forward in this work as promising eco-friendly AF agents.

Dibenzofuran, 4-Chromanone, Acetophenone, and Dithiecine Derivatives: Cytotoxic Constituents from Eupatorium fortunei.

Five new compounds, eupatodibenzofuran A (1), eupatodibenzofuran B (2), 6-acetyl-8-methoxy-2,2-dimethylchroman-4-one (3), eupatofortunone (4), and eupatodithiecine (5), have been isolated from the aerial part of Eupatorium fortunei, together with 11 known compounds (6‒16). Compounds 1 and 2 featured a new carbon skeleton with an unprecedented 1-(9-(4-methylphenyl)-6-methyldibe nzo[b,d]furan-2-yl)ethenone. Among the isolates, compound 1 exhibited potent inhibitory activity with IC50 values of 5.95 ± 0.89 and 5.55 ± 0.23 µM, respectively, against A549 and MCF-7 cells. The colony-formation assay demonstrated that compound 1 (5 µM) obviously decreased A549 and MCF-7 cell proliferation, and Western blot test confirmed that compound 1 markedly induced apoptosis of A549 and MCF-7 cells through mitochondrial- and caspase-3-dependent pathways.

Lignin-Derived Syringol and Acetosyringone from Palm Bunch Using Heterogeneous Oxidative Depolymerization over Mixed Metal Oxide Catalysts under Microwave Heating.

Biomass valorization to building block chemicals in food and pharmaceutical industries has tremendously gained attention. To produce monophenolic compounds from palm empty fruit bunch (EFB), EFB was subjected to alkaline hydrothermal extraction using NaOH or K2CO3 as a promotor. Subsequently, EFB-derived lignin was subjected to an oxidative depolymerization using Cu(II) and Fe(III) mixed metal oxides catalyst supported on γ-Al2O3 or SiO2 as the catalyst in the presence of hydrogen peroxide. The highest percentage of total phenolic compounds of 63.87 wt% was obtained from microwave-induced oxidative degradation of K2CO3 extracted lignin catalyzed by Cu-Fe/SiO2 catalyst. Main products from the aforementioned condition included 27.29 wt% of 2,4-di-tert-butylphenol, 19.21 wt% of syringol, 9.36 wt% of acetosyringone, 3.69 wt% of acetovanillone, 2.16 wt% of syringaldehyde, and 2.16 wt% of vanillin. Although the total phenolic compound from Cu-Fe/Al2O3 catalyst was lower (49.52 wt%) compared with that from Cu-Fe/SiO2 catalyst (63.87 wt%), Cu-Fe/Al2O3 catalyst provided the greater selectivity of main two value-added products, syringol and acetosyrigone, at 54.64% and 23.65%, respectively (78.29% total selectivity of two products) from the NaOH extracted lignin. The findings suggested a promising method for syringol and acetosyringone production from the oxidative heterogeneous lignin depolymerization under low power intensity microwave heating within a short reaction time of 30 min.

New Acetophenones and Chromenes from the Leaves of Melicope barbigera A. Gray.

The dichloromethane extract from leaves of Melicope barbigera (Rutaceae), endemic to the Hawaiian island of Kaua'i, yielded four new and three previously known acetophenones and 2H-chromenes, all found for the first time in M. barbigera. The structures of the new compounds obtained from the dichloromethane extract after purification by chromatographic methods were unambiguously elucidated by spectroscopic analyses including 1D/2D NMR spectroscopy and HRESIMS. The absolute configuration was determined by modified Mosher's method. Compounds 2, 4 and the mixture of 6 and 7 exhibited moderate cytotoxic activities against the human ovarian cancer cell line A2780 with IC50 values of 30.0 and 75.7 µM for 2 and 4, respectively, in a nuclear shrinkage cytotoxicity assay.

Synthesis and Antinociceptive Effect of Some Thiazole-Piperazine Derivatives: Involvement of Opioidergic System in the Activity.

In this study, we aimed to design and synthesize novel molecules carrying both the thiazole and piperazine rings in their structures and to investigate their antinociceptive activity. Targeted compounds were obtained by reacting thiosemicarbazide derivative and appropriate 2-bromoacetophenone in ethanol. The structures of the obtained compounds were determined using data from various spectroscopic methods (IR, 1H-NMR, 13C-NMR, and LCMSMS). Experimental data from in vivo tests showed that test compounds 3a-3c, 3f, and 3g (50 mg/kg) significantly prolonged reaction times of animals in tail-clip and hot-plate tests compared to the controls, indicating that these compounds possess centrally mediated antinociceptive activities. Furthermore, these compounds reduced the number of writhing behaviors in the acetic acid-induced writhing tests, showing that the compounds also possess peripheral antinociceptive activity. In the mechanistic studies, naloxone pre-treatments abolished the antinociceptive activities of compounds 3a-3c, 3f, and 3g, indicating that opioidergic mechanisms were involved in their antinociceptive effects. Molecular docking studies demonstrating significant interactions between the active compounds and µ- and δ-opioid receptor proteins supported the pharmacological findings. This study is the first showing that molecules designed to bear thiazole and piperazine moieties together on their structure exert centrally and peripherally mediated antinociceptive effects by activating the opioid system.

Neuroprotective Ability of Apocynin Loaded Nanoparticles (APO-NPs) as NADPH Oxidase (NOX)-Mediated ROS Modulator for Hydrogen Peroxide-Induced Oxidative Neuronal Injuries.

Apocynin (APO) is a known multi-enzymatic complexed compound, employed as a viable NADPH oxidase (NOX) inhibitor, extensively used in both traditional and modern-day therapeutic strategies to combat neuronal disorders. However, its therapeutic efficacy is limited by lower solubility and lesser bioavailability; thus, a suitable nanocarrier system to overcome such limitations is needed. The present study is designed to fabricate APO-loaded polymeric nanoparticles (APO-NPs) to enhance its therapeutic efficacy and sustainability in the biological system. The optimized APO NPs in the study exhibited 103.6 ± 6.8 nm and -13.7 ± 0.43 mV of particle size and zeta potential, respectively, along with further confirmation by TEM. In addition, the antioxidant (AO) abilities quantified by DPPH and nitric oxide scavenging assays exhibited comparatively higher AO potential of APO-NPs than APO alone. An in-vitro release profile displayed a linear diffusion pattern of zero order kinetics for APO from the NPs, followed by its cytotoxicity evaluation on the PC12 cell line, which revealed minimal toxicity with higher cell viability, even after treatment with a stress inducer (H2O2). The stability of APO-NPs after six months showed minimal AO decline in comparison to APO only, indicating that the designed nano-formulation enhanced therapeutic efficacy for modulating NOX-mediated ROS generation.