Photoactive monofunctional platinum(II) anticancer complexes of multidentate phenanthridine-containing ligands: photocytotoxicity and evidence for interaction with DNA.

Pt(II) complexes supported by chelating, multidentate ligands containing π-extended, planar phenanthridine (benzo[c]quinoline) donors (RLPtCl) exhibit a promising in vitro therapeutic index compared with phenanthriplatin, a leading preclinical anticancer complex containing a monodentate phenanthridine ligand. Here, we report evidence for non-specific interactions of CF3LPtCl with DNA through intercalation-mediated turn-on luminescence in O2-saturated aqueous buffer. Brief irradiation with visible light (490 nm) was also found to drastically increase the activity of CF3LPtCl, with photocytotoxicity increased up to 87% against a variety of human cancer cell lines. Mechanistic studies highlight significantly improved cellular uptake of CF3LPtCl compared with cisplatin, with localization in the nucleus and mitochondria triggering effective apoptosis. Photosensitization experiments with 1,3-diphenylisobenzofuran demonstrate that CF3LPtCl efficiently mediates the generation of singlet dioxygen (1O2), highlighting the potential of RLPtCl in photodynamic therapy.

Novel nucleocapsid protein-targeting phenanthridine inhibitors of SARS-CoV-2.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unprecedented in human history. As a major structural protein, nucleocapsid protein (NPro) is critical to the replication of SARS-CoV-2. In this work, 17 NPro-targeting phenanthridine derivatives were rationally designed and synthesized, based on the crystal structure of NPro. Most of these compounds can interact with SARS-CoV-2 NPro tightly and inhibit the replication of SARS-CoV-2 in vitro. Compounds 12 and 16 exhibited the most potent anti-viral activities with 50% effective concentration values of 3.69 and 2.18 µM, respectively. Furthermore, site-directed mutagenesis of NPro and Surface Plasmon Resonance (SPR) assays revealed that 12 and 16 target N-terminal domain (NTD) of NPro by binding to Tyr109. This work found two potent anti-SARS-CoV-2 bioactive compounds and also indicated that SARS-CoV-2 NPro-NTD can be a target for new anti-virus agents.

An ATP-Binding Cassette Transporter, LaABCB11, Contributes to Alkaloid Transport in Lycoris aurea.

As a kind of Amaryllidaceae alkaloid which is accumulated in the species of Lycoris plants, lycorine has a range of physiological effects. The biosynthesis pathway of lycorine has been partly revealed, but the transport and accumulation mechanisms of lycorine have rarely been studied. In this study, an ATP-binding cassette (ABC) transporter from Lycoris aurea (L'Hér) Herb., namely LaABCB11, was cloned and functionally characterized. Heterologous expression showed that LaABCB11 transported lycorine in an outward direction, increased the tolerance of yeast cells to lycorine, and caused a lower lycorine accumulation in transformants than control or mutant in yeast. LaABCB11 is associated with the plasma membrane, and in situ hybridization indicated that LaABCB11 was mainly expressed in the phloem of leaves and bulbs, as well as in the cortical cells of roots. These findings suggest that LaABCB11 functions as a lycorine transport and it might be related to the translocation and accumulation of lycorine from the leaves and bulbs to the roots.

Manoalide Shows Mutual Interaction between Cellular and Mitochondrial Reactive Species with Apoptosis in Oral Cancer Cells.

We previously found that marine sponge-derived manoalide induced antiproliferation and apoptosis of oral cancer cells as well as reactive species generations probed by dichloro-dihydrofluorescein diacetate (DCFH-DA) and MitoSOX Red. However, the sources of cellular and mitochondrial redox stresses and the mutual interacting effects between these redox stresses and apoptosis remain unclear. To address this issue, we examined a panel of reactive species and used the inhibitors of cellular reactive species (N-acetylcysteine (NAC)), mitochondrial reactive species (MitoTEMPO), and apoptosis (Z-VAD-FMK; ZVAD) to explore their interactions in manoalide-treated oral cancer Ca9-22 and CAL 27 cells. Hydroxyl (˙OH), nitrogen dioxide (NO2˙), nitric oxide (˙NO), carbonate radical-anion (CO3 ˙-), peroxynitrite (ONOO-), and superoxide (O2 ˙-) were increased in oral cancer cells following manoalide treatments in terms of fluorescence staining and flow cytometry. Cellular reactive species (˙OH, NO2 ·, ˙NO, CO3 ˙-, and ONOO-) as well as cellular and mitochondrial reactive species (O2 ˙-) were induced in oral cancer cells following manoalide treatment for 6 h. NAC, MitoTEMPO, and ZVAD inhibit manoalide-induced apoptosis in terms of annexin V and pancaspase activity assays. Moreover, NAC inhibits mitochondrial reactive species and MitoTEMPO inhibits cellular reactive species, suggesting that cellular and mitochondrial reactive species can crosstalk to regulate each other. ZVAD shows suppressing effects on the generation of both cellular and mitochondrial reactive species. In conclusion, manoalide induces reciprocally activation between cellular and mitochondrial reactive species and apoptosis in oral cancer cells.

Protective effects of dapagliflozin against oxidative stress-induced cell injury in human proximal tubular cells.

Elevated reactive oxygen species (ROS) in type 2 diabetes cause cellular damage in many organs. Recently, the new class of glucose-lowering agents, SGLT-2 inhibitors, have been shown to reduce the risk of developing diabetic complications; however, the mechanisms of such beneficial effect are largely unknown. Here we aimed to investigate the effects of dapagliflozin on cell proliferation and cell death under oxidative stress conditions and explore its underlying mechanisms. Human proximal tubular cells (HK-2) were used. Cell growth and death were monitored by cell counting, water-soluble tetrazolium-1 (WST-1) and lactate dehydrogenase (LDH) assays, and flow cytometry. The cytosolic and mitochondrial (ROS) production was measured using fluorescent probes (H2DCFDA and MitoSOX) under normal and oxidative stress conditions mimicked by addition of H2O2. Intracellular Ca2+ dynamics was monitored by FlexStation 3 using cell-permeable Ca2+ dye Fura-PE3/AM. Dapagliflozin (0.1-10 µM) had no effect on HK-2 cell proliferation under normal conditions, but an inhibitory effect was seen at an extreme high concentration (100 µM). However, dapagliflozin at 0.1 to 5 µM showed remarkable protective effects against H2O2-induced cell injury via increasing the viable cell number at phase G0/G1. The elevated cytosolic and mitochondrial ROS under oxidative stress was significantly decreased by dapagliflozin. Dapagliflozin increased the basal intracellular [Ca2+]i in proximal tubular cells, but did not affect calcium release from endoplasmic reticulum and store-operated Ca2+ entry. The H2O2-sensitive TRPM2 channel seemed to be involved in the Ca2+ dynamics regulated by dapagliflozin. However, dapagliflozin had no direct effects on ORAI1, ORAI3, TRPC4 and TRPC5 channels. Our results suggest that dapagliflozin shows anti-oxidative properties by reducing cytosolic and mitochondrial ROS production and altering Ca2+ dynamics, and thus exerts its protective effects against cell damage under oxidative stress environment.

Two new monofunctional platinum(II) dithiocarbamate complexes: phenanthriplatin-type axial protection, equatorial-axial conformational isomerism, and anticancer and DNA binding studies.

The syntheses of two platinum(ii) dithiocarbamate complexes (1 and 2) that show quinoplatin- and phenanthriplatin-type axial protection of the Pt-plane are described. The Pt-plane of complex 2 is axially more protected than that of complex 1. Furthermore, both complexes adopt two different stereochemical conformations in the solid state (based on single-crystal X-ray structures) owing to the structurally flexible piperazine backbone; i.e., C-e,e-Anti (1) and C-e,a-Syn (2), where "C" stands for the chair configuration, "e" and "a" stand for the equatorial and axial positions and "Anti" (opposite side) and "Syn" (same side) represent the relative orientations in space of the terminal substituents on the piperazine ring. In complex 2, the C-e,a-Syn conformation may provide additional steric hindrance to the Pt-plane. Despite the lower lipophilicity of 2 as compared to that of 1, the in vitro anticancer action against selected cancer cell lines is better for the former revealing the superior role of the axial protection over lipophilicity in modulating anticancer activity. The activity against the cancer promoting protein NF-κB signifies that the mode of cancer cell death may be the result of hindering the activity of NF-κB in the initiation of apoptosis. The apoptotic mode of cell death has been established earlier in a study using Annexin V-FITC. Finally, DNA binding studies revealed that the complex-DNA adduct formation is spontaneous and the mode of interaction is non-intercalative (electrostatic/covalent).

Biosynthesis, synthetic studies, and biological activities of the jadomycin alkaloids and related analogues.

The jadomycins are an expanding class of compounds produced from Streptomyces venezuelae, by diverting the normal biosynthesis which provides the antibiotic chloramphenicol. In the presence of amino acids, and either by heat shock, supplementation with ethanol, or when phage SV1 is added to the culture, the formation of substituted jadomycins and benzo[b]phenanthridines can be achieved. The first part of this review provides details of intermediates involved in the biosynthesis of the jadomycins and the related benzo[b]phenanthridines. Both the jadomycins and the benzo[b]phenanthridines share biosynthetic pathways with a large class of naturally occurring compounds known as the angucyclines. The biosynthetic pathways diverge when it is postulated that an intermediate quinone, such as 3-(2-formyl-6-hydroxy-4-methylphenyl)-8-hydroxy-1,4-naphthoquinone-2-carboxylic acid is formed. The quinone then undergoes reactions with amino acids and derivatives in the culture medium to ultimately afford a library of jadomycins and a few benzo[b]phenanthridines. The second part of the review initially details synthetic efforts toward the synthesis of the naturally occurring benzo[b]phenanthridine, phenanthroviridin, and then outlines methods that have been used to assemble a selection of jadomycins. Total syntheses of jadomycin A and B, derived from l-isoleucine, are described. In addition, the synthesis of the aglycon of jadomycins M, W, S, and T is outlined. These four jadomycins were derived from l-methionine, l-tryptophan, l-serine and l-threonine respectively. As a result of these synthetic efforts, the structures of jadomycin S and T have been revised. The third part of the review describes the reported antibacterial and anticancer activities of both the jadomycins and some naturally occurring benzo[b]phenanthridines.

Functionalized aromatic esters of the Amaryllidaceae alkaloid haemanthamine and their in vitro and in silico biological activity connected to Alzheimer's disease.

A novel series of aromatic esters (1a-1m) related to the Amaryllidaceae alkaloid (AA) haemanthamine were designed, synthesized and tested in vitro with particular emphasis on the treatment of neurodegenerative diseases. Some of the synthesized compounds revealed promising acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory profile. Significant human AChE (hAChE) inhibition was demonstrated by 11-O-(3-nitrobenzoyl)haemanthamine (1j) with IC50value of 4.0 ± 0.3 µM. The strongest human BuChE (hBuChE) inhibition generated 1-O-(2-methoxybenzoyl)haemanthamine (1g) with IC50 value 3.3 ± 0.4 µM. Moreover, 11-O-(2-chlorbenzoyl)haemanthamine (1m) was able to inhibit both enzymes in dose-dependent manner. The mode of hAChE and hBuChE inhibition was minutely inspected using enzyme kinetic analysis in tandem with in silico experiments, the latter elucidating crucial interaction in 1j-, 1m-hAChE and 1g-, 1m-hBuChE complexes. The blood-brain barrier (BBB) permeability was investigated applying the parallel artificial membrane permeation assay (PAMPA) to predict the CNS availability of the compounds.

Factors for the emission enhancement of dimidium in specific media such as in DNA and on a clay surface.

Dimidium (3,8-diamino-5-methyl-6-phenylphenanthridinium: NH2PhP) is a well-known fluorophore as a DNA probe, although its fluorescence enhancement mechanism is not clear. In this study, we investigated the fluorescence enhancement mechanism of NH2PhP on a clay surface by observing the fluorescence behavior. Four systematically selected phenanthridinium derivatives (PDs): NH2PhP, 3,8-bisdimethylamino-5-methyl-6-phenylphenanthridinium (NMe2PhP), 5-methyl-6-phenylphenanthridinium (PhP) and 5-methylphenanthridinium (P) and synthetic clay were used as guest and host materials, respectively. It was revealed that the suppression of hydrogen bonding with water (N-HOH or NH-OH2) is the dominant factor for the fluorescence enhancement on the clay surface for NH2PhP and NMe2PhP. In addition, judging from the fluorescence enhancement for NH2PhP, NMe2PhP and PhP and no fluorescence enhancement for P on the clay surface, the suppression of rotation of the phenyl ring was indicated to make a partial contribution to the fluorescence enhancement mechanism. Because the fluorescence enhancement behavior was quite similar on the clay surface and in DNA, the obtained results afford an important clue to discuss the fluorescence enhancement mechanism of NH2PhP in DNA.

HPLC-Based Monitoring of Oxidation of Hydroethidine for the Detection of NADPH Oxidase-Derived Superoxide Radical Anion.

Hydroethidine is a fluorogenic probe that in the presence of the superoxide radical anion is oxidized to a red fluorescent product, 2-hydroxyethidium. In cells, hydroethidine is also oxidized to other products, including red fluorescent ethidium. Thus, selective monitoring of 2-hydroxyethidium is required for specific detection of the superoxide radical anion. Here, we provide protocols for HPLC- and LC-MS-based quantitation of 2-hydroxyethidium, among other oxidation products. Also, a protocol for continuous sampling for real-time monitoring of superoxide production using rapid HPLC measurements of 2-hydroxyethidium is described.

High-Throughput Screening of NOX Inhibitors.

Development of new, selective inhibitors of nicotinamide adenine dinucleotide phosphate oxidase (NOX) isoforms is important both for basic studies on the role of these enzymes in cellular redox signaling, cell physiology, and proliferation and for development of new drugs for diseases carrying a component of increased NOX activity, such as several types of cancer and cardiovascular and neurodegenerative diseases. High-throughput screening (HTS) of large libraries of compounds remains the major approach for development of new NOX inhibitors. Here, we describe the protocol for the HTS campaign for NOX inhibitors using rigorous assays for superoxide radical anion and hydrogen peroxide, based on oxidation of hydropropidine, coumarin boronic acid, and Amplex Red. We propose using these three probes to screen for and identify new inhibitors, by selecting positive hits that show inhibitory effects in all three assays. Protocols for the synthesis of hydropropidine and for confirmatory assays, including oxygen consumption measurements, electron paramagnetic resonance spin trapping of superoxide, and simultaneous monitoring of superoxide and hydrogen peroxide, are also provided.

Derivatives of the ß-Crinane Amaryllidaceae Alkaloid Haemanthamine as Multi-Target Directed Ligands for Alzheimer's Disease.

Twelve derivatives 1a-1m of the ß-crinane-type alkaloid haemanthamine were developed. All the semisynthetic derivatives were studied for their inhibitory potential against both acetylcholinesterase and butyrylcholinesterase. In addition, glycogen synthase kinase 3ß (GSK-3ß) inhibition potency was evaluated in the active derivatives. In order to reveal the availability of the drugs to the CNS, we elucidated the potential of selected derivatives to penetrate through the blood-brain barrier (BBB). Two compounds, namely 11-O-(2-methylbenzoyl)-haemanthamine (1j) and 11-O-(4-nitrobenzoyl)-haemanthamine (1m), revealed the most intriguing profile, both being acetylcholinesterase (hAChE) inhibitors on a micromolar scale, with GSK-3ß inhibition properties, and predicted permeation through the BBB. In vitro data were further corroborated by detailed inspection of the compounds' plausible binding modes in the active sites of hAChE and hBuChE, which led us to provide the structural determinants responsible for the activity towards these enzymes.

DNA Intercalation Facilitates Efficient DNA-Targeted Covalent Binding of Phenanthriplatin.

Phenanthriplatin, a monofunctional anticancer agent derived from cisplatin, shows significantly more rapid DNA covalent-binding activity compared to its parent complex. To understand the underlying molecular mechanism, we used single-molecule studies with optical tweezers to probe the kinetics of DNA-phenanthriplatin binding as well as DNA binding to several control complexes. The time-dependent extensions of single λ-DNA molecules were monitored at constant applied forces and compound concentrations, followed by rinsing with a compound-free solution. DNA-phenanthriplatin association consisted of fast and reversible DNA lengthening with time constant τ ≈ 10 s, followed by slow and irreversible DNA elongation that reached equilibrium in ∼30 min. In contrast, only reversible fast DNA elongation occured for its stereoisomer  trans-phenanthriplatin, suggesting that the distinct two-rate kinetics of phenanthriplatin is sensitive to the geometric conformation of the complex. Furthermore, no DNA unwinding was observed for pyriplatin, in which the phenanthridine ligand of phenanthriplatin is replaced by the smaller pyridine molecule, indicating that the size of the aromatic group is responsible for the rapid DNA elongation. These findings suggest that the mechanism of binding of phenanthriplatin to DNA involves rapid, partial intercalation of the phenanthridine ring followed by slower substitution of the adjacent chloride ligand by, most likely, the N7 atom of a purine base. The cis isomer affords the proper stereochemistry at the metal center to facilitate essentially irreversible DNA covalent binding, a geometric advantage not afforded by trans-phenanthriplatin. This study demonstrates that reversible DNA intercalation provides a robust transition state that is efficiently converted to an irreversible DNA-Pt bound state.

Are Hydroethidine-Based Probes Reliable for Reactive Oxygen Species Detection?

Detection and quantification of the highly reactive and short-lived superoxide (O•2-) can be challenging. Here, we present a new mass spectrometry (MS)-based method to detect and quantify O•2- using three fluorogenic hydroethidine probes: hydroethidine (HE), mito-hydroethidine (mito-HE), and hydropropidine (HPr+), which measure cytosolic, mitochondrial, and extracellular O•2-, respectively. The probes and their oxidation products were simultaneously quantified by applying multiple reaction monitoring (MRM) with MS that allowed the specific measurement of reactive oxygen species (ROS) distribution within the cell. The advantage of this liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is that coeluting compounds can be precisely distinguished using specific precursor and fragment masses. This method overcomes limitations from spectral overlap of O•2--specific and nonspecific products in fluorescence spectra or the low specificity associated with chromatography-based approaches. However, our experiments showed that these HE probes can be prone to autoxidation during incubation at 37°C in Hank's solution. Cell treatments with strong oxidants did not significantly increase levels of the O•2- radical. Thus, subtle changes in ROS levels in cell culture experiments might not be quantifiable. Our findings raise the question of whether HE-based probes can be used for the reliable detection of O•2- radicals in cell culture. Antioxid. Redox Signal. 00, 000-000.

Design, synthesis and structure-activity relationship optimization of phenanthridine derivatives as new Wnt/ß-catenin signalling pathway agonists.

Phenanthridine derivativeHLY78 has previously been identified as the first Wnt/ß-catenin signalling pathway agonist that targets the DAX domain of axin. However, due to the relatively weak activation on the Wnt/ß-catenin signalling pathway, HLY78 is insufficient for further pharmacological study. Herein, the structural optimization of HLY78 and analyses of the structure-activity relationships (SARs) of HLY78-derived phenanthridine derivatives as agonists of the Wnt/ß-catenin signalling pathway are presented. In this work, 36 derivatives were designed and synthesized with some derivatives exhibiting stronger Wnt activity than the activity of HLY78. In particular, one of them, 8-((1,3-dimethy-pyrazol-5-yl)methoxy)-5-ethyl-4-methyl-5,6-dihydro-phenanthridin-9-ol, exhibited strong Wnt active activity and is 10 times more potent than HLY78. The following SAR analysis suggests that a pyrazole group, especially at the C-8 position, is important for Wnt activation; a methyl group at the C-4position seems to be more beneficial for Wnt activation than ethyl; and oxidation of the C-6 position reduces the Wnt activation.

Cyanidin Protects SH-SY5Y Human Neuroblastoma Cells from 1-Methyl-4-Phenylpyridinium-Induced Neurotoxicity.

Cyanidin is an anthocyanidin extracted from a variety of fruits and vegetables. Cyanidin showed benefits against diabetes, cancer, and atherosclerosis. However, the potential neuroprotective effects of cyanidin against Parkinson's disease (PD) have not been examined. Indicated concentrations of cyanidin (1, 3, 10, and 30 µmol/L) were incubated together with 0.5 mmol/L 1-methyl-4-phenylpyridinium (MPP+) to human neuroblastoma SH-SY5Y cells. We found cyanidin prevented MPP+-induced cell demise in a concentration-dependent manner. Cyanidin significantly reduced MPP+-induced apoptosis, this is reflected by decreased TdT-mediated dUTP nick-end labeling staining and caspase-3 expressions. Further, MPP+ increased the Bax/Bcl-2 ratio, which was partly reversed by cyanidin. We also found cyanidin attenuated the MPP+-induced mitochondrial oxidative stress as revealed by decreased MitoSOX staining. Taken together, these data for the first time indicated the -neuroprotective effects of cyanidin against MPP+-induced -SH-SY5Y cell death. These findings shed light on the potential implications of cyanidin for PD treatment.

Speciation of Phenanthriplatin and Its Analogs in the Core of Tobacco Mosaic Virus.

Efficient loading of drugs in novel delivery agents has the potential to substantially improve therapy by targeting the diseased tissue while avoiding unwanted side effects. Here we report the first systematic study of the loading mechanism of phenanthriplatin and its analogs into tobacco mosaic virus (TMV), previously used by our group as an efficient carrier for anticancer drug delivery. A detailed investigation of the preferential uptake of phenanthriplatin in its aquated form (∼2000 molecules per TMV particle versus ∼1000 for the chlorido form) is provided. Whereas the net charge of phenanthriplatin analogs and their ionic mobilities have no effect on loading, the reactivity of aqua phenanthriplatin with the glutamates, lining the interior walls of the channel of TMV, has a pronounced effect on its loading. MALDI-MS analysis along with NMR spectroscopic studies of a model reaction of hydroxy-phenanthriplatin with acetate establish the formation of stable covalent adducts. The increased number of heteroaromatic rings on the platinum ligand appears to enhance loading, possibly by stabilizing hydrophobic stacking interactions with TMV core components, specifically Pro102 and Thr103 residues neighboring Glu97 and Glu106 in the channel. Electron transfer dissociation MS/MS fragmentation, a technique that can prevent mass-condition-vulnerable modification of proteins, reveals that Glu97 preferentially participates over Glu106 in covalent bond formation to the platinum center.

Anti-cancer characteristics and ototoxicity of platinum(II) amine complexes with only one leaving ligand.

Unlike cisplatin, which forms bifunctional DNA adducts, monofunctional platinum(II) complexes bind only one strand of DNA and might target cancer without causing auditory side-effects associated with cisplatin treatment. We synthesized the monofunctional triamine-ligated platinum(II) complexes, Pt(diethylenetriamine)Cl, [Pt(dien)Cl]+, and Pt(N,N-diethyldiethylenetriamine)Cl, [Pt(Et2dien)Cl]+, and the monofunctional heterocyclic-ligated platinum(II) complexes, pyriplatin and phenanthriplatin, and compared their 5'-GMP binding rates, cellular compartmental distribution and cellular viability effects. A zebrafish inner ear model was used to determine if the monofunctional complexes and cisplatin caused hearing threshold shifts and reduced auditory hair cell density. The four monofunctional complexes had varied relative GMP binding rates, but similar cytosolic and nuclear compartmental uptake in three cancer cell lines (A549, Caco2, HTB16) and a control cell line (IMR90). Phenanthriplatin had the strongest effect against cellular viability, comparable to cisplatin, followed by [Pt(Et2dien)Cl]+, pyriplatin and [Pt(dien)Cl]+. Phenanthriplatin also produced the highest hearing threshold shifts followed by [Pt(dien)Cl]+, [Pt(Et2dien)Cl]+, cisplatin and pyriplatin. Hair cell counts taken from four regions of the zebrafish saccule showed that cisplatin significantly reduced hair cell density in three regions and phenanthriplatin in only one region, with the other complexes having no significant effect. Utricular hair cell density was not reduced by any of the compounds. Our results suggest that placing greater steric hindrance cis to one side of the platinum coordinating center in monofunctional complexes promotes efficient targeting of the nuclear compartment and guanosine residues, and may be responsible for reducing cancer cell viability. Also, the monofunctional compounds caused hearing threshold shifts with minimal effect on hair cell density, which suggests that they may affect different pathways than cisplatin.

Metabolite profiling and isolation of biologically active compounds from Scadoxus puniceus, a highly traded South African medicinal plant.

Scadoxus puniceus (Amaryllidaceae), a medicinal plant of high value in South Africa, is used as a component of a traditional herbal tonic prescribed to treat several ailments. Ultra-high performance liquid chromatography-tandem mass spectrometry quantified the phenolic compounds in different organs of S. puniceus. Gravity column chromatography was used to separate fractions and active compounds. The structure of these compounds was determined using 1D and 2D nuclear magnetic resonance and mass spectroscopic techniques. A microplate technique was used to determine the acetylcholinesterase inhibitory activity of the pure compounds. Metabolite profiling revealed a greater profusion of hydroxycinnamic acids (69.5%), as opposed to hydroxybenzoic acids (30.5%). Chlorogenic acid was the most abundant (49.6% of hydroxycinnamic acids) compound. In addition to chlorogenic acid, the study is the first to report the presence of sinapic, gallic, and m-hydroxybenzoic acids in the Amaryllidaceae. Chromatographic separation of S. puniceus led to the isolation of haemanthamine (1), haemanthidine (2), and a rare chlorinated amide, metolachlor (3), the natural occurrence of which is described for the first time. Haemanthamine, haemanthidine, and metolachlor displayed strong acetylcholinesterase inhibitory activity (IC50 ; 23.1, 23.7, and 11.5 µM, respectively). These results substantiate the frequent use of S. puniceus as a medicinal plant and hold much promise for further pharmaceutical development.

Evaluation of a novel radiotracer for positron emission tomography imaging of reactive oxygen species in the central nervous system.

INTRODUCTION: Few, if any, radiotracers are available for the in vivo imaging of reactive oxygen species (ROS) in the central nervous system. ROS play a critical role in normal cell processes such as signaling and homeostasis but overproduction of ROS is implicated in several disorders. We describe here the radiosynthesis and initial ex vivo and in vivo evaluation of [11C]hydromethidine ([11C]HM) as a radiotracer to image ROS using positron emission tomography (PET). METHODS: [11C]HM and its deuterated isotopologue [11C](4) were produced using [11C]methyl triflate in a one-pot, two-step reaction and purified by high performance liquid chromatography. Ex vivo biodistribution studies were performed after tail vein injections of both radiotracers. To demonstrate sensitivity of uptake to ROS, [11C]HM was administered to rats treated systemically with lipopolysaccharide (LPS). In addition, ex vivo autoradiography and in vivo PET imaging were performed using [11C]HM on rats which had been microinjected with sodium nitroprusside (SNP) to induce ROS. RESULTS: [11C]HM and [11C](4) radiosyntheses were reliable and produced the radiotracers at high specific activities and radiochemical purities. Both radiotracers demonstrated good brain uptake and fast washout of radioactivity, but [11C](4) washout was faster. Pretreatment with LPS resulted in a significant increase in brain retention of radioactivity. Ex vivo autoradiography and PET imaging of rats unilaterally treated with microinjections of SNP demonstrated increased retention of radioactivity in the treated side of the brain. CONCLUSIONS: [11C]HM has the attributes of a radiotracer for PET imaging of ROS in the brain including good brain penetration and increased retention of radioactivity in animal models of oxidative stress.