Semisynthesis, Characterization, and Biological Evaluation of Fluorinated Analogues of the Spirobisnaphthalene, Diepoxin-η.

Diepoxin-η (1) is a cytotoxic fungal metabolite belonging to the spirobisnaphthalene structural class. In this study, four mono fluorinated analogues (2-5) of diepoxin-η (1) were semisynthesized in a single-step by selectively fluorinating the naphthalene moiety with Selectfluor. The structures of 2-5 were elucidated using a set of spectroscopic and spectrometric techniques and were further confirmed by means of TDDFT-ECD and isotropic shielding tensors calculations. Compounds 2-5 showed equipotent cytotoxic activity to 1 when tested against OVCAR3 (ovarian) and MDA-MB-435 (melanoma) cancer cell lines with IC50 values that range from 5.7-8.2 µM.

Acetogenins from the Stem of Uvaria rufa and Their Cytotoxic Activity.

Four new adjacent bis-tetrahydrofuran acetogenins, bullacin C (7), uvarirufin (9), and uvariasolins III (12) and IV (13), along with 11 known acetogenins, were isolated from the stem of Uvaria rufa. Their structures were elucidated based on spectroscopic data analysis, including 1D and 2D NMR, HRESIMS, and MALDI-MS/MS of the lithium adducts. Absolute configurations were assigned using Mosher ester analysis and ECD measurements. Uvarirufin (9) possesses a unique C-39 skeleton among acetogenins. Most tested acetogenins exhibited cytotoxicity against human cancer cell lines (HCT 116, 22Rv1, MDA-MB-435, OVCAR3). Squamocin (8) and uvarirufin (9) were found to be the most potent, with an IC50 value of 1.2 µM for both in HCT 116 colon cancer cells. Additionally, a new application of Dragendorff's reagent is proposed herein for the TLC detection of acetogenins.

Probing the Cytotoxic Signaling Induced by Eupenifeldin in Ovarian Cancer Models.

High-grade serous ovarian cancer (HGSOC) is the most common and lethal ovarian cancer histotype. Lack of early detection methods, limited therapeutic agents, and low 5-year survival rate reflect the urgent need to develop new therapies. Eupenifeldin, a bistropolone, originally isolated from Eupenicillium brefeldianum, is a cytotoxic fungal metabolite. In three HSGOC cell lines (OVCAR3, OVCAR5, OVCAR8), eupenifeldin was found to have an IC50 value less than 10 nM, while 10 times higher concentrations were required for cytotoxicity in nontumorigenic fallopian tube secretory epithelial cell lines (FTSEC). An in vivo hollow fiber assay showed significant cytotoxicity in OVCAR3. Eupenifeldin significantly increased Annexin V staining in OVCAR3 and -8, but not OVCAR5. Eupenifeldin activated caspases 3/7 in OVCAR3, OVCAR5, and OVCAR8; however, cleaved PARP was only detected in OVCAR3. Quantitative proteomics performed on OVCAR3 implicated ferroptosis as the most enriched cell death pathway. However, validation experiments did not support ferroptosis as part of the cytotoxic mechanism of eupenifeldin. Autophagic flux and LC3B puncta assays found that eupenifeldin displayed weak autophagic induction in OVCAR3. Inhibition of autophagy by cotreatment with bafilomycin reduced the toxicity of eupenifeldin, supporting the idea that induction of autophagy contributes to the cytotoxic mechanism of eupenifeldin.

Semisynthesis of Hypothemycin Analogues Targeting the C8-C9 Diol.

Hypothemycin, an epoxide derivative of (5Z)-7-oxozeaenol, was used in the semisynthesis of a series of C8-C9 diol derivatives, with many inhibiting TAK1 at submicromolar concentrations. A step-economical approach was chosen, whereby nonselective reactions functionalized the diol to generate multiple analogues in a single reaction. Using this approach, 35 analogues were synthesized using 12 reactions, providing a wealth of information about the role that the C8-C9 diol plays in TAK1 inhibition and cytotoxicity in ovarian and breast cancer cell lines. Monofunctionalized analogues exhibited strong inhibition of TAK1, showing potential for modification of this section of the molecule to assist with solubility, formulation, and other desirable properties. Most analogues were cytotoxic, and three compounds had similar or slightly increased potency with >100-fold improvement in solubility profiles.

Kratom (Mitragyna speciosa) Validation: Quantitative Analysis of Indole and Oxindole Alkaloids Reveals Chemotypes of Plants and Products.

Many consumers are turning to kratom (Mitragyna speciosa) to self-manage pain and opioid addiction. In the United States, an array of capsules, powders, and loose-leaf kratom products are readily available. Additionally, several online sites supply live kratom plants. A prerequisite to establishing quality control and quality assurance standards for the kratom industry, or understanding how alkaloid levels effect clinical outcomes, is the identification and quantitation of major and minor alkaloid constituents within available products and preparations. To this end, an ultra-high performance liquid chromatography-high resolution mass spectrometry method was developed for the analysis of 8 indole alkaloids (7-hydroxymitragynine, ajmalicine, paynantheine, mitragynine, speciogynine, isopaynantheine, speciociliatine, and mitraciliatine) and 6 oxindole alkaloids (isomitraphylline, isospeciofoleine, speciofoline, corynoxine A, corynoxeine, and rhynchophylline) in US-grown kratom plants and commercial products. These commercial products shared a qualitatively similar alkaloid profile, with 12 - 13 detected alkaloids and high levels of the indole alkaloid mitragynine (13.9 ± 1.1 - 270 ± 24 mg/g). The levels of the other major alkaloids (paynantheine, speciociliatine, speciogynine, mitraciliatine, and isopaynantheine) and the minor alkaloids varied in concentration from product to product. The alkaloid profile of US-grown M. speciosa "Rifat" showed high levels of the indole alkaloid speciogynine (7.94 ± 0.83 - 11.55 ± 0.18 mg/g) and quantifiable levels of isomitraphylline (0.943 ± 0.033 - 1.47 ± 0.18 mg/g). Notably, the alkaloid profile of a US-grown M. speciosa seedling was comparable to the commercial products with a high level of mitragynine (15.01 ± 0.20 mg/g). This work suggests that there are several M. speciosa chemotypes.

Aulosirazoles B and C from the Cyanobacterium Nostoc sp. UIC 10771: Analogues of an Isothiazolonaphthoquinone Scaffold that Activate Nuclear Transcription Factor FOXO3a in Ovarian Cancer Cells.

The known solid-tumor-selective cytotoxin aulosirazole (1) was identified from bioactive extracts from the culture medium of the cyanobacterium Nostoc sp. UIC 10771. Here, we demonstrate that 1 induces the nuclear accumulation of FOXO3a in OVCAR3 using both Western blot analysis and immunofluorescence confocal microscopy. We also report the discovery of two additional analogues, aulosirazoles B (2) and C (3). Structures for compounds 2 and 3 were determined using HR-ESI-LC-MS/MS and 1D and 2D NMR experiments. Aulosirazoles B (2) and C (3) represent the first natural analogues of the FOXO-activating compound aulosirazole (1) and are the second and third isothiazole-containing metabolites reported from this phylum.

Capturing the antimicrobial profile of Rosmarinus officinalis against methicillin-resistant Staphylococcus aureus (MRSA) with bioassay-guided fractionation and bioinformatics.

Natural products have been a primary source of medicines throughout the history of human existence. It is estimated that close to 70 % of small molecule pharmaceuticals on the market are derived from natural products. With increasing antibiotic resistance, natural products remain an important source for the discovery of novel antimicrobial compounds. The plant rosemary (Rosmarinus officinalis), has been widely and commonly used as a food preservative due to its antimicrobial potential. To evaluate the antimicrobial profile of this plant, we used bioassay-guided fractionation and bioinformatics approaches. Through bioassay-guided fractionation, we tested in vitro activities of a R. officinalis extract and fractions thereof, as well as pure compounds micromeric acid (1), oleanolic acid (2), and ursolic acid (3) against methicillin-resistant Staphylococcus aureus (MRSA). Compounds 1 and 3 showed complete inhibition of MRSA (with MIC values of 32 µg/mL and 8 µg/mL, respectively) while compound 2 displayed only partial inhibition (MIC > 64 µg/mL). In addition, we utilized orthogonal partial least square-discriminant analysis (OPLS-DA) and selectivity ratio (SR) analysis to correlate the isolated compounds 1-3 with the observed antimicrobial activity, as well as to identify antimicrobials present in trace quantities. For mass spectrometry (MS) data collected in the negative ionization mode, compound 1 was the most positively correlated with activity, while for MS data collected in the positive ion mode, compounds 2-3 had the highest positive correlation. Using the bioinformatics approaches, we highlighted additional antimicrobials associated with the antimicrobial activity of R. officinalis, including genkwanin (4), rosmadial (5a) and/or 16-hydroxyrosmadial (5b), rosmanol (6), and hesperetin (7). Compounds 1-3 resulting from the bioassay-guided fractionation were identified by MS-MS fragmentation patterns and 1H NMR spectra. Among the compounds highlighted by the biochemical analysis, compound 6 was identified by comparison with its commercial standard by employed ultra-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS), while 4, 5a-b and 7 were putatively identified based on MS data and in comparison with the literature. This is the first reported antimicrobial activity of micromeric acid (1) against MRSA.

Chemical Evaluation of the Effects of Storage Conditions on the Botanical Goldenseal using Marker-based and Metabolomics Approaches.

Hydrastis canadensis, commonly known as goldenseal, is a botanical native to the southeastern United States that has been used for the treatment of infection. The activity of goldenseal is often attributed to the presence of alkaloids (cyclic, nitrogen-containing compounds) present within its roots. Chemical components of botanical supplements like goldenseal may face degradation if not stored properly. The purpose of the research was to analyze the stability of known and unknown metabolites of H. canadensis during exposure to different storage conditions using mass spectrometry. Three abundant metabolites of H. canadensis, berberine, canadine, and hydrastine, were chosen for targeted analysis, and the stability of unknown metabolites was evaluated using untargeted metabolomics. The analysis and evaluation of H. canadensis samples were performed utilizing LC-MS and Principal Component Analysis (PCA). The research project focused on identifying the chemical changes in the metabolite content of H. canadensis under different temperature conditions (40°C ± 5°C, 20°C ± 5°C , and 4°C ± 5°C), different light:dark (hr:hr) cycles (16:8, 12:12, and 0:24), and different sample conditions (powdered roots versus whole roots) over a six month period. The results of this 6-month study revealed that the storage conditions evaluated had no significant effects on the chemical composition of H. canadensis roots. Hence, as long as H. canadensis roots are stored within the storage conditions tested in the study, no significant changes in chemical compositions of metabolites are expected.

Phytochemical Analysis and Antimicrobial Efficacy of Macleaya cordata against Extensively Drug-Resistant Staphylococcus aureus.

The antibiotic resistant threat is continuing to grow, due in part to the overuse of antibiotics in livestock feed. Many nations in Europe have banned the use of antibiotics in feed, leading to higher rates of infection in livestock animals and reduced productivity for the food market. Increasingly, researchers are looking into the efficacy of phytopreparations to replace antibiotics in feed, allowing for increased animal health without the development of resistance. Macleaya cordata, or Chinese plume poppy, shows promise as a food additive. To evaluate the antimicrobial efficacy of this plant, we tested in vitro activity of M. cordata extract, as well as pure compounds sanguinarine and chelerythrine against wild-type, methicillin-resistant, and multiply-resistant strains of Staphylococcus aureus (SA1199, AH1263, and IA116, respectively). Combination tests to evaluate synergy, additivity, and antagonism within the extract were also completed for the first time. Sanguinarine and chelerythrine showed complete growth inhibition of all strains of S. aureus at concentrations ranging from 3-10 µg/mL, and were equal in activity or were more potent than the reference compound chloramphenicol. Combination studies of pure sanguinarine and chelerythrine with M. cordata extract revealed additivity or indifference of mixture components with these compounds. Because sanguinarine and chelerythrine represent the major active constituents of M. cordata, the pooled amounts of these two compounds may be useful for establishing potency for quality control purposes. This is the first report of activity of chelerythrine and sanguinarine against methicillin-resistant S. aureus AH1263 and multiply-resistant S. aureus IA116, and illustrates the promise of M. cordata extract as an alternative to antibiotics in feed additives.