Isolation of Ingenol-Type Diterpenoids from Euphorbia kansui by Offline Coupling of HPLC-ESI-MSn and HSCCC

A method based on an offline coupling of high-performance liquid chromatography/electrospray ionization multi-stage mass spectrometry (HPLC-ESI-MSⁿ) and high-speed counter-current chromatography (HSCCC), was established for targeted separation and purification of skin-irritative and structurally similar ingenol-type diterpenoids in Euphorbia kansui. The crude dichloromethane extract of Euphorbia kansui after an initial clean-up step on silica gel and targeted fractionation according to HPLC-ESI-MSⁿ, was separated by HSCCC. As a result, 3-O-(2′E,4′Z-decadienoyl)-20-O-acetylingenol and 3-O-(2′E,4′E-decadienoyl)-20-O-acetylingenol as well as a pair of geometrical isomers, 5-O-(2′E,4′Z-decadienoyl)-20-O-acetylingenol and 5-O-(2′E,4′E-decadienoyl)-20-O-acetylingenol, were successfully separated and purified. The established method is rapid and efficient without irritation to operators, providing an extensible and feasible way for the separation and purification of other toxic and skin-irritative compounds.     

Phenolic Compounds from Halimodendron halodendron (Pall.) Voss and Their Antimicrobial and Antioxidant Activities

Halimodendron halodendron has been used as forage in northwestern China for a long time. Its young leaves and flowers are edible and favored by indigenous people. In this study, eleven phenolic compounds were bioassay-guided and isolated from the aerial parts of H. halodendron for the first time. They were identified by means of physicochemical and spectrometric analysis as quercetin (1), 3,5,7,8,4′-pentahydroxy-3′-methoxy flavone (2), 3-O-methylquercetin (3), 3,3′-di-O-methylquercetin (4), 3,3′-di-O-methylquercetin-7-O-β-d-glucopyranoside (5), isorhamentin-3-O-β-d-rutinoside (6), 8-O-methylretusin (7), 8-O-methylretusin-7-O-β-d-glucopyranoside (8), salicylic acid (9), p-hydroxybenzoic acid (ferulic acid) (10), and 4-hydroxy-3-methoxy cinnamic acid (11). They were sorted as flavonols (1–6), soflavones (7 and 8), and phenolic acids (9–11). Among the compounds, flanools 1–4 revealed a strong antibacterial activity with minimum inhibitory concentration (MIC) values of 50–150 μg/mL, and median inhibitory concentration (IC₅₀) values of 26.8–125.1 μg/mL. The two isoflavones (7 and 8) showed moderate inhibitory activity on the test bacteria. Three phenolic acids (9, 10 and 11) showed strong antibacterial activity with IC₅₀ values of 28.1–149.7 μg/mL. Antifungal activities of the compounds were similar to their antibacterial activities. All these phenolic compounds showed significant antimicrobial activity with a broad spectrum as well as antioxidant activity based on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and β-carotene-linoleic acid bleaching assays. In general, the flavonol aglycones with relatively low polarity exhibited stronger activities than the glycosides. The results suggest the potential of this plant as a source of functional food ingredients and provide support data for its utilization as forage as well.     

The Effect of the Aerial Part of Lindera akoensis on Lipopolysaccharides (LPS)-Induced Nitric Oxide Production in RAW264.7 Cells

Four new secondary metabolites, 3α-((E)-Dodec-1-enyl)-4β-hydroxy-5β-methyldihydrofuran-2-one (1), linderinol (6), 4′-O-methylkaempferol 3-O-α-l-(4″-E-p-coumaroyl)rhamnoside (11) and kaempferol 3-O-α-l-(4″-Z-p-coumaroyl) rhamnoside (12) with eleven known compounds—3-epilistenolide D₁ (2), 3-epilistenolide D₂ (3), (3Z,4α,5β)-3-(dodec-11-ynylidene)-4-hydroxy-5-methylbutanolide (4), (3E,4β,5β)-3-(dodec-11-ynylidene)-4-hydroxy-5-methylbutanolide (5), matairesinol (7), syringaresinol (8), (+)-pinoresinol (9), salicifoliol (10), 4″-p-coumaroylafzelin (13), catechin (14) and epicatechin (15)—were first isolated from the aerial part of Lindera akoensis. Their structures were determined by detailed analysis of 1D- and 2D-NMR spectroscopic data. All of the compounds isolated from Lindera akoensis showed that in vitro anti-inflammatory activity decreases the LPS-stimulated production of nitric oxide (NO) in RAW 264.7 cell, with IC₅₀ values of 4.1–413.8 μM.     

8-Alkylcoumarins from the Fruits of Cnidium monnieri Protect against Hydrogen Peroxide Induced Oxidative Stress Damage

Three new 8-alkylcoumarins, 7-O-methylphellodenol-B (1), 7-methoxy-8-(3-methyl-2,3-epoxy-1-oxobutyl)chromen-2-one (2), and 3′-O-methylvaginol (3), together with seven known compounds (4–10) were isolated from the fruits of Cnidium monnieri. Their structures were determined by detailed analysis of spectroscopic data and comparison with the data of known analogues. All the isolates were evaluated the cytoprotective activity by MTS cell proliferation assay and the results showed that all the three new 8-alkylcoumarins exhibited cytoprotective effect on Neuro-2a neuroblastoma cells injured by hydrogen peroxide.     

Isolation and Structural Characterization of Lignin from Cotton Stalk Treated in an Ammonia Hydrothermal System

To investigate the potential for the utilization of cotton stalk, ammonia hydrothermal treatment was applied to fractionate the samples into aqueous ammonia-soluble and ammonia-insoluble portions. The ammonia-soluble portion was purified to yield lignin fractions. The lignin fractions obtained were characterized by wet chemistry (carbohydrate analysis) and spectroscopy methods (FT-IR, ¹³C and ¹H-¹³C HSQC NMR spectroscopy) as well as gel permeation chromatography (GPC). The results showed that the cotton stalk lignin fractions were almost absent of neutral sugars (0.43%–1.29%) and had relatively low average molecular weights (1255–1746 g/mol). The lignin fractions belonged to typical G-S lignin, which was composed predominately of G-type units (59%) and noticeable amounts of S-type units (40%) together with a small amount of H-type units (~1%). Furthermore, the ammonia-extractable lignin fractions were mainly composed of β-O-4′ inter-unit linkages (75.6%), and small quantities of β-β′ (12.2%), together with lower amounts of β-5′ carbon-carbon linkages (7.4%) and p-hydroxycinnamyl alcohol end groups.     

Coumarins from the Herb Cnidium monnieri and Chemically Modified Derivatives as Antifoulants against Balanus albicostatus and Bugula neritina Larvae

In the search for new environmental friendly antifouling (AF) agents, four coumarins were isolated from the herbal plant Cnidium monnieri, known as osthole (1), imperatorin (2), isopimpinellin (3) and auraptenol (4). Furthermore, five coumarin derivatives, namely 8-epoxypentylcoumarin (5), meranzin hydrate (6), 2′-deoxymetranzin hydrate (7), 8-methylbutenalcoumarin (8), and micromarin-F (9) were synthesized from osthole. Compounds 1, 2, 4, 7 showed high inhibitory activities against larval settlement of Balanus albicostatus with EC₅₀ values of 4.64, 3.39, 3.38, 4.67 μg mL⁻¹. Compound 8 could significantly inhibit larval settlement of Bugula neritina with an EC₅₀ value of 3.87 μg mL⁻¹. The impact of functional groups on anti-larval settlement activities suggested that the groups on C-5′ and C-2′/C-3′ of isoamylene chian could affect the AF activities.     

Potentiometric study of the interaction of Cr and O ions in fused NaCl-2CsCl eutectic

The physical and chemical properties of chromium chlorides have been studied in fused NaCl-2CsCl eutectic at the temperature range 843-1008 K using an electrochemical cell containing a platinum-oxygen electrode with solid electrolyte membrane, which was used as pO²⁻ indicator electrode. The concentration of oxygen ions in the solution was modifying by dropping the calculated amount of BaO. Titration of Cr³⁺ chlorocomplex by O²⁻ ions demonstrated the precipitation of CrOCl and Cr₂O₃. The solubility constants of these compounds were calculated. E-pO²⁻ and three-dimensional E-pO²⁻-T type diagrams, which summarized the properties of chromium species in the melt, were determined.     

A Comparison of B16 Melanoma Cells and 3T3 Fibroblasts Concerning Cell Viability and ROS Production in the Presence of Melatonin,Tested Over a Wide Range of Concentrations

Melatonin is a pleiotropic molecule with many cellular and systemic actions, including chronobiotic effects. Beneficial effects are widely documented concerning the treatment of neoplastic diseases in vivo as well as reductions in viability of cultured cells from melanoma, one of the most aggressive cancers in humans. However, studies of its effects on non-tumor cells in vitro have not focused on viability, except for experiments aiming to protect against oxidotoxicity or other toxicological insults. Furthermore, there is no agreement on the range of effective melatonin concentrations in vitro, and the mechanisms that reduce cell viability have remained unclear. Tumor cell-specific increases in the production of reactive oxygen and nitrogen species (ROS/RNS) may provide a possible explanation. Our aim was to analyze the potential inhibition of tumor (B16 melanoma 4A5) and non-tumor cell (3T3 Swiss albino) viability using a wide range of melatonin concentrations (10⁻¹¹–10⁻² M), and to determine whether intracellular ROS enhancement was involved in this process. In the absence of fetal bovine serum (FBS), low melatonin concentrations (10⁻⁹–10⁻⁵ M) reduced the proliferation of melanoma cells with no effect in fibroblasts, whereas, in the presence of FBS, they had no effect or even increased the proliferation of both fibroblast and melanoma cells. Melatonin concentrations in the upper millimolar range increased ROS levels and reduced the viability of both cell types, but more markedly so in non-tumor cells. Thus, low melatonin concentrations reduce proliferation in this specific melanoma cell line, whereas high concentrations affect the viability of both tumor (B16 4A5 melanoma) and non-tumor (3T3 fibroblasts) cells. Increased ROS levels in both lines indicate a role for ROS production in the reduction of cell viability at high—but not low—melatonin concentrations, although the mechanism of action still remains to be elucidated.     

New Glycosylated Dihydrochalcones Obtained by Biotransformation of 2′-Hydroxy-2-methylchalcone in Cultures of Entomopathogenic Filamentous Fungi

Flavonoids, including chalcones, are more stable and bioavailable in the form of glycosylated and methylated derivatives. The combined chemical and biotechnological methods can be applied to obtain such compounds. In the present study, 2′-hydroxy-2-methylchalcone was synthesized and biotransformed in the cultures of entomopathogenic filamentous fungi Beauveria bassiana KCH J1.5, Isaria fumosorosea KCH J2 and Isaria farinosa KCH J2.6, which have been known for their extensive enzymatic system and ability to perform glycosylation of flavonoids. As a result, five new glycosylated dihydrochalcones were obtained. Biotransformation of 2′-hydroxy-2-methylchalcone by B. bassiana KCH J1.5 resulted in four glycosylated dihydrochalcones: 2′-hydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, 2′,3-dihydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, 2′-hydroxy-2-hydroxymethyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, and 2′,4-dihydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside. In the culture of I. fumosorosea KCH J2 only one product was formed—3-hydroxy-2-methyldihydrochalcone 2′-O-β-d-(4″-O-methyl)-glucopyranoside. Biotransformation performed by I. farinosa KCH J2.6 resulted in the formation of two products: 2′-hydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside and 2′,3-dihydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside. The structures of all obtained products were established based on the NMR spectroscopy. All products mentioned above may be used in further studies as potentially bioactive compounds with improved stability and bioavailability. These compounds can be considered as flavor enhancers and potential sweeteners.     

New Triterpenoids with Cytotoxic Activity from Actinidia Valvata

Two new triterpenoids, 30-O-β-d-glucopyranosyloxy-2α,3α,24-trihydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester (1) and 2α,3β,3,30-tetrahydroxyurs-12, 18-diene-28-oic acid O-β-d-glucopyranosyl ester (2) were isolated from roots of Actinidia valvata Dunn. Their structures were elucidated by means of extensive spectroscopic studies. Both these two new compounds showed moderate cytotoxic activity in vitro against BEL-7402 and SMMC-7721 tumor cell line.     

Polyoxygenated Cembrane Diterpenoids from the Soft Coral Sarcophyton ehrenbergi

Five new polyoxygenated cembranoids, named (+)-1,15-epoxy-2-methoxy-12-methoxycarbonyl-11E-sarcophytoxide (1), (+)-2-epi-12-methoxycarbonyl-11E-sarcophine (2), 3,4-epoxyehrenberoxide A (3), ehrenbergol D (4) and ehrenbergol E (5), were obtained from the soft coral Sarcophyton ehrenbergi. The structures of 1–5 were established on the basis of comprehensive NMR and HR-ESI-MS analyses and by comparison with reported data in the literature. Compounds 4 and 5 showed moderate cytotoxicity against P-388 (mouse lymphocytic leukemia) cancer cell line with EC₅₀ values of 2.0 and 3.0 μM, respectively. Compound 2 exhibited slight antiviral activity against HCMV (human cytomegalovirus) with IC₅₀ values of 25.0 μg/mL.     

Mitochondrial Protection and Anti-aging Activity of Astragalus Polysaccharides and Their Potential Mechanism

The current study was performed to investigate mitochondrial protection and anti-aging activity of Astragalus polysaccharides (APS) and the potential underlying mechanism. Lipid peroxidation of liver and brain mitochondria was induced by Fe²⁺–Vit C in vitro. Thiobarbituric acid (TBA) colorimetry was used to measure the content of thiobarbituric acid reactive substances (TBARS). Mouse liver mitochondrial permeability transition (PT) was induced by calcium overload in vitro and spectrophotometry was used to measure it. The scavenging activities of APS on superoxide anion (O₂^•−) and hydroxyl radical (•OH), which were produced by reduced nicotinamide adenine dinucleotide (NADH)—N-Methylphenazonium methyl sulfate (PMS) and hydrogen peroxide (H₂O₂)–Fe²⁺ system respectively, were measured by 4-nitrobluetetrazolium chloride (NBT) reduction and Fenton reaction colorimetry respectively. The Na₂S₂O₃ titration method was used to measure the scavenging activities of APS on H₂O₂. APS could inhibit TBARS production, protect mitochondria from PT, and scavenge O₂^•−, •OH and H₂O₂ significantly in a concentration-dependent manner respectively. The back of the neck of mice was injected subcutaneously with D-galactose to induce aging at a dose of 100 mg/kg/d for seven weeks. Moreover, the activities of catalase (CAT), surperoxide dismutase (SOD) and glutathione peroxidase (GPx) and anti-hydroxyl radical which were assayed by using commercial monitoring kits were increased significantly in vivo by APS. According to this research, APS protects mitochondria by scavenging reactive oxygen species (ROS), inhibiting mitochondrial PT and increasing the activities of antioxidases. Therefore, APS has the effect of promoting health.     

Fungal Biotransformation of 2′-Methylflavanone and 2′-Methylflavone as a Method to Obtain Glycosylated Derivatives

Methylated flavonoids are promising pharmaceutical agents due to their improved metabolic stability and increased activity compared to unmethylated forms. The biotransformation in cultures of entomopathogenic filamentous fungi is a valuable method to obtain glycosylated flavones and flavanones with increased aqueous solubility and bioavailability. In the present study, we combined chemical synthesis and biotransformation to obtain methylated and glycosylated flavonoid derivatives. In the first step, we synthesized 2′-methylflavanone and 2′-methylflavone. Afterwards, both compounds were biotransformed in the cultures of two strains of entomopathogenic filamentous fungi Beauveria bassiana KCH J1.5 and Isaria fumosorosea KCH J2. We determined the structures of biotransformation products based on NMR spectroscopy. Biotransformations of 2′-methyflavanone in the culture of B. bassiana KCH J1.5 resulted in three glycosylated flavanones: 2′-methylflavanone 6-O-β-d-(4″-O-methyl)-glucopyranoside, 3′-hydroxy-2′-methylflavanone 6-O-β-d-(4″-O-methyl)-glucopyranoside, and 2-(2′-methylphenyl)-chromane 4-O-β-d-(4″-O-methyl)-glucopyranoside, whereas in the culture of I. fumosorosea KCH J2, two other products were obtained: 2′-methylflavanone 3′-O-β-d-(4″-O-methyl)-glucopyranoside and 2-methylbenzoic acid 4-O-β-d-(4′-O-methyl)-glucopyranoside. 2′-Methylflavone was effectively biotransformed only by I. fumosorosea KCH J2 into three derivatives: 2′-methylflavone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, 2′-methylflavone 4′-O-β-d-(4″-O-methyl)-glucopyranoside, and 2′-methylflavone 5′-O-β-d-(4″-O-methyl)-glucopyranoside. All obtained glycosylated flavonoids have not been described in the literature until now and need further research on their biological activity and pharmacological efficacy as potential drugs.     

Yeast α-Glucosidase Inhibitory Phenolic Compounds Isolated from Gynura medica Leaf

Gynura medica leaf extract contains significant amounts of flavonols and phenolic acids and exhibits powerful hypoglycemic activity against diabetic rats in vivo. However, the hypoglycemic active constituents that exist in the plant have not been fully elaborated. The purpose of this study is to isolate and elaborate the hypoglycemic activity compounds against inhibition the yeast α-glucosidase in vitro. Seven phenolic compounds including five flavonols and two phenolic acids were isolated from the leaf of G. medica. Their structures were identified by the extensive NMR and mass spectral analyses as: kaempferol (1), quercetin (2), kaempferol-3-O-β-D-glucopyranoside (3), kaempferol-3-O-rutinoside (4), rutin (5), chlorogenic acid (6) and 3,5-dicaffeoylquinic acid methyl ester (7). All of the compounds except 1 and 3 were isolated for the first time from G. medica. Compounds 1–7 were also assayed for their hypoglycemic activity against yeast α-glucosidase in vitro. All of the compounds except 1 and 6 showed good yeast α-glucosidase inhibitory activity with the IC₅₀ values of 1.67 mg/mL, 1.46 mg/mL, 0.38 mg/mL, 0.10 mg/mL and 0.53 mg/mL, respectively.     

Synthesis and Characterization of Some New Bis-Pyrazolyl-Thiazoles Incorporating the Thiophene Moiety as Potent Anti-Tumor Agents

A new series of 1,4-bis(1-(5-(aryldiazenyl)thiazol-2-yl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)benzenes 3a–i were synthesized via reaction of 5,5′-(1,4-phenylene)bis(3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide) (1) with hydrazonoyl halides 2a–i. In addition, reaction of 1 with ethyl chloroacetate afforded bis-thiazolone derivative 8 as the end product. Reaction of compound 8 with methyl glyoxalate gave bis-thiazolone derivative 10. The structures of the newly synthesized compounds were established on the basis of spectroscopic evidences and their alternative syntheses. All the synthesized compounds were evaluated for their anti-tumor activities against hepatocellular carcinoma (HepG2) cell lines, and the results revealed promising activities of compounds 3g, 5e, 3e, 10, 5f, 3i, and 3f with IC₅₀ equal 1.37 ± 0.15, 1.41 ± 0.17, 1.62 ± 0.20, 1.86 ± 0.20, 1.93 ± 0.08, 2.03 ± 0.25, and 2.09 ± 0.19 μM, respectively.     

Synthesis of 4��,7-Diacetoxyapigenin and Its Apoptotic Induction in Human Hep G2 Cells

In this study, 4′,7-diacetoxyapigenin [4-(7-acetoxy-5-hydroxy-4-oxo-4H-chromen-2-yl) phenyl acetate] was synthesized for the first time. Its chemical structure was identified by UV, ESI-MS, ¹H and ¹³C-NMR. It could inhibit the proliferation of Hep G2 cells in a dose-dependent manner and induce the significant increase of the G0/G1 cell population. After treatment by 4′,7-diacetoxyapigenin, phosphatidylserine of Hep G2 cells could significantly translocate to the surface of the membrane. The increase of an early apoptotic population was observed by both annexin-FITC and PI staining. It was concluded that 4′,7-diacetoxyapigenin not only induced cells to enter into apoptosis, but also affected the progress of the cell cycle.     

Altered DNA Binding and Amplification of Human Breast Cancer Suppressor Gene BRCA1 Induced by a Novel Antitumor Compound, [Ru(��6-p-phenylethacrynate)Cl2(pta)]

The ruthenium-based complex [Ru(η⁶-p-phenylethacrynate)Cl₂(pta)] (pta = 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane), termed ethaRAPTA, is an interesting antitumor compound. The elucidation of the molecular mechanism of drug activity is central to the drug development program. To this end, we have characterized the ethaRAPTA interaction with DNA, including probing the sequence specific modified DNA structural stability and DNA amplification using the breast cancer suppressor gene 1 (BRCA1) of human breast and colon adenocarcinoma cell lines as models. The preference of ethaRAPTA base binding is in the order A > G > T > C. Once modified, the ethaRAPTA-induced BRCA1 structure has higher thermal stability than the modified equivalents of its related compound, RAPTA-C. EthaRAPTA exhibits a higher efficiency than RAPTA-C in inhibiting BRCA1 amplification. With respect to both compounds, the inhibition of BRCA1 amplification is more effective in an isolated system than in cell lines. These data provide evidence that will help to understand the process of elucidating the pathways involved in the response induced by ethaRAPTA.     

Copper(II) Complexes Containing Natural Flavonoid Pomiferin Show Considerable In Vitro Cytotoxicity and Anti-inflammatory Effects

A series of new heteroleptic copper(II) complexes of the composition [Cu(L)(bpy)]NO₃·2MeOH (1), [Cu(L)(dimebpy)]NO₃·2H₂O (2), [Cu(L)(phen)]NO₃·2MeOH (3), [Cu(L)(bphen)]NO₃·MeOH (4), [Cu(L)(dppz)]NO₃·MeOH (5) was prepared, where HL = 3-(3,4-dihydroxyphenyl)-5-hydroxy-8,8-dimethyl-6-(3-methylbut-2-ene-1-yl)-4H,8H-benzo[1,2-b:3,4-b′]dipyran-4-one, (pomiferin) and bpy = 2,2′-bipyridine, dimebpy = 4,4′-dimethyl-2,2′-bipyridine, phen = 1,10-phenanthroline, bphen = 4,7-diphenyl-1,10-phenanthroline, and dppz = dipyrido[3,2-a:2′,3′-c]phenazine. The complexes were characterized using elemental analysis, infrared and UV/Vis spectroscopies, mass spectrometry, thermal analysis and conductivity measurements. The in vitro cytotoxicity, screened against eight human cancer cell lines (breast adenocarcinoma (MCF-7), osteosarcoma (HOS), lung adenocarcinoma (A549), prostate adenocarcinoma (PC-3), ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780R), colorectal adenocarcinoma (Caco-2) and monocytic leukemia (THP-1), revealed the complexes as effective antiproliferative agents, with the IC₅₀ values of 2.2–13.0 μM for the best performing complexes 3 and 5. All the complexes 1–5 showed the best activity against the A2780R cells (IC₅₀ = 2.2–6.6 μM), and moreover, the complexes demonstrated relatively low toxicity on healthy human hepatocytes, with IC₅₀ > 100 μM. The complexes were evaluated by the Annexin V/propidium iodide apoptosis assay, induction of cell cycle modifications in A2780 cells, production of reactive oxygen species (ROS), perturbation of mitochondrial membrane potential, inhibition of apoptosis and inflammation-related signaling pathways (NF-κB/AP-1 activity, NF-κB translocation, TNF-α secretion), and tested for nuclease mimicking activity. The obtained results revealed the corresponding complexes to be effective antiproliferative and anti-inflammatory agents.     

Phenylalanine-Derived β-Lactam TRPM8 Modulators. Configuration Effect on the Antagonist Activity

Transient receptor potential cation channel subfamily M member 8 (TRPM8) is a Ca²⁺ non-selective ion channel implicated in a variety of pathological conditions, including cancer, inflammatory and neuropathic pain. In previous works we identified a family of chiral, highly hydrophobic β–lactam derivatives, and began to intuit a possible effect of the stereogenic centers on the antagonist activity. To investigate the influence of configuration on the TRPM8 antagonist properties, here we prepare and characterize four possible diastereoisomeric derivatives of 4-benzyl-1-[(3′-phenyl-2′-dibenzylamino)prop-1′-yl]-4-benzyloxycarbonyl-3-methyl-2-oxoazetidine. In microfluorography assays, all isomers were able to reduce the menthol-induced cell Ca²⁺ entry to larger or lesser extent. Potency follows the order 3R,4R,2′R > 3S,4S,2′R ≅ 3R,4R,2′S > 3S,4S,2′S, with the most potent diastereoisomer showing a half inhibitory concentration (IC₅₀) in the low nanomolar range, confirmed by Patch-Clamp electrophysiology experiments. All four compounds display high receptor selectivity against other members of the TRP family. Furthermore, in primary cultures of rat dorsal root ganglion (DRG) neurons, the most potent diastereoisomers do not produce any alteration in neuronal excitability, indicating their high specificity for TRPM8 channels. Docking studies positioned these β-lactams at different subsites by the pore zone, suggesting a different mechanism than the known N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide (AMTB) antagonist.     

Design,Synthesis and Evaluation of Novel Derivatives of Curcuminoids with Cytotoxicity

Curcumin and curcuminoids have been discussed frequently due to their promising functional groups (such as scaffolds of α,β-unsaturated β-diketone, α,β-unsaturated ketone and β′-hydroxy-α,β-unsaturated ketone connected with aromatic rings on both sides) that play an important role in various bioactivities, including antioxidant, anti-inflammatory, anti-proliferation and anticancer activity. A series of novel curcuminoid derivatives (a total of 55 new compounds) and three reference compounds were synthesized with good yields using three-step organic synthesis. The anti-proliferative activities of curcumin derivatives were examined for six human cancer cell lines: HeLaS3, KBvin, MCF-7, HepG2, NCI-H460 and NCI-H460/MX20. Compared to the IC₅₀ values of all the synthesized derivatives, most α,β-unsaturated ketones displayed potent anti-proliferative effects against all six human cancer cell lines, whereas β′-hydroxy-α,β-unsaturated ketones and α,β-unsaturated β-diketones presented moderate anti-proliferative effects. Two potent curcuminoid derivatives were found among all the novel derivatives and reference compounds: (E)-5-hydroxy-7-phenyl-1-(3,4,5-trimethoxyphenyl)hept-1-en-3-one (compound 3) and (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a). These were selected for further analysis after the evaluation of their anti-proliferative effects against all human cancer cell lines. The results of apoptosis assays revealed that the number of dead cells was increased in early apoptosis and late apoptosis, while cell proliferation was also decreased after applying various concentrations of (E)-5-hydroxy-7-phenyl-1-(3,4,5-trimethoxyphenyl)hept-1-en-3-one (compound 3) and (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a) to MCF-7 and HpeG2 cancer cells. Analysis of the gene expression arrays showed that three genes (GADD45B, SESN2 and BBC3) were correlated with the p53 pathway. From the quantitative PCR analysis, it was seen that (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a) effectively induced the up-regulated expression of GADD45B, leading to the suppression of MCF-7 cancer cell formation and cell death. Molecular docking analysis was used to predict and sketch the interactions of the GADD45B-α,β-unsaturated ketone complex for help in drug design.