Phenanthroindolizidine Alkaloids Research Articles

Mass spectrometry-based metabolomics for the elucidation of alkaloid biosynthesis and function in invasive Vincetoxicum rossicum populations

The genus Vincetoxicum includes a couple of highly invasive vines in North America that threaten biodiversity and challenge land management strategies. Vincetoxicum species are known to produce bioactive phenanthroindolizidine alkaloids that might play a role in the invasiveness of these plants via chemical interactions with other organisms. Untargeted, high-resolution mass spectrometry-based metabolomics approaches were used to explore specialized metabolism in Vincetoxicum plants collected from invaded sites in Ontario, Canada. All metabolites corresponding to alkaloids in lab and field samples of V. rossicum and V. nigrum were identified, which collectively contained 25 different alkaloidal features. The biosynthesis of these alkaloids was investigated by the incorporation of the stable isotope-labelled phenylalanine precursor providing a basis for an updated biosynthetic pathway accounting for the rapid generation of chemical diversity in invasive Vincetoxicum. Aqueous extracts of aerial Vincetoxicum rossicum foliage had phytotoxic activity against seedlings of several species, resulting in identification of tylophorine as a phytotoxin; tylophorine and 14 other alkaloids from Vincetoxicum accumulated in soils associated with full-sun and a high-density of V. rossicum. Using desorption-electrospray ionization mass spectrometry, 15 alkaloids were found to accumulate at wounded sites of V. rossicum leaves, a chemical cocktail that would be encountered by feeding herbivores. Understanding the specialized metabolism of V. rossicum provides insight into the roles and influences of phenanthroindolizidine alkaloids in ecological systems and enables potential, natural product-based approaches for the control of invasive Vincetoxicum and other weedy species.

Drug-like properties of serial phenanthroindolizidine alkaloid compounds: ADMET characteristic prediction and validation

Phenanthroindolizidine alkaloids (PAs) are a series of compounds that have been isolated from traditional herbal medicines and have significant therapeutic potential, such as anti-arthritic, anti-viral, anti-inflammatory, and anti-glioma effects in vitro and in vivo. This study aimed to predict the absorption, distribution, metabolism, excretion, and toxicity (ADMET) characteristics of 44 compounds in silico and to verify the ADMET characteristics. The 2-dimensional structures of these compounds were generated using ChemDraw and the characteristics were predicted using ADMET Predictor™ software. Key characteristics, such as pK a, logP/logD, solubility, permeability, absolute bioavailability in rats, and preliminary toxicity, were measured on some typical compounds to verify the accuracy of the prediction results. The results showed that ADMET predicts physicochemical and biological properties quickly and accurately for PAs. PAs are biopharmaceutics classification system (BCS) class IV compounds with low bioavailability. Moreover, these compounds have higher lipophilicity and are easily distributed into the brain after oral administration to treat brain diseases. However, some of these compounds exhibited colonic toxicity. To improve the drug-like availability of these compounds, more in-depth research should be conducted on drug delivery systems.

Isolation of GLP-1 enhancing indolizidine alkaloids from Boehmeria formosana.

Boehmeria formosana, with its related species, demonstrates anti-glycemic effect, inhibition of HBV production, anti-cancer activities, etc. Some indolizidine alkaloids from the same genus are bioactive but sensitive to light. To overcome this problem and obtain more phenanthroindolizidine alkaloids, isolation was performed in darkness, yielding 10 new indolizidine alkaloids and 17 known compounds. Among them, seven enhanced glucagon-like receptor 1 (GLP-1) activity at 50 mM, especially 14 and 6 (3.5- and 2.3-fold than the negative control). This procedure yielded bioactive indolizidine alkaloids with novel structures.

Annulation of O-silyl N,O-ketene acetals with alkynes for the synthesis of dihydropyridinones and its application in concise total synthesis of phenanthroindolizidine alkaloids.

The formation of N-heterocycles with multiple substituents is important in organic synthesis. Herein, we report a novel method for the construction of functionalized dihydropyridinone rings through the annulation of an amide α-carbon with a tethered alkyne moiety. The reaction of the amide with the alkyne was achieved via O-silyl N,O-ketene acetal formation and silver-mediated addition. Furthermore, the developed method was applied for the total synthesis of phenanthroindolizidine and phenanthroquinolizidine alkaloids. By varying the coupling partners, a concise and collective total synthesis of these alkaloids was achieved.

Total Synthesis of Phenanthroindolizidines Using Strained Azacyclic Alkynes.

We report a concise approach to phenanthroindolizidine alkaloids, wherein strained azacyclic alkynes are intercepted in Pd-catalyzed annulations. Two types of strained intermediates were evaluated: a functionalized piperidyne and a new strained intermediate, an indolizidyne. We show that each can be employed, ultimately allowing access to three natural products: tylophorine, tylocrebine, and isotylocrebine. These efforts demonstrate the successful merger of strained azacyclic alkyne chemistry with transition-metal catalysis for the construction of complex heterocycles.

Phenanthroindolizidine Alkaloids Secondary Metabolites Diversity in Medicinally Viable Plants of the Genus Tylophora

Plants of the genus Tylophora have commonly been used in traditional medicine in various communities, especially in the tropical and subtropical regions of climatic zones. Of the nearly 300 species reported in the Tylophora genus, eight are primarily used in various forms to treat a variety of bodily disorders based on the symptoms. Certain plants from the genus have found use as anti-inflammatory, anti-tumor, anti-allergic, anti-microbial, hypoglycemic, hypolipidemic, anti-oxidant, smooth muscle relaxant, immunomodulatory, and anti-plasmodium agents, as well as free-radical scavengers. Pharmacologically, a few plant species from the genus have exhibited broad-spectrum anti-microbial and anti-cancer activity, which has been proven through experimental evaluations. Some of the plants in the genus have also helped in alcohol-induced anxiety amelioration and myocardial damage repair. The plants belonging to the genus have also shown diuretic, anti-asthmatic, and hepato-protective activities. Tylophora plants have afforded diverse structural bases for secondary metabolites, mainly belonging to phenanthroindolizidine alkaloids, which have been found to treat several diseases with promising pharmacological activity levels. This review encompasses information on various Tylophora species, their distribution, corresponding plant synonyms, and chemical diversity of the secondary metabolic phytochemicals as reported in the literature, together with their prominent biological activities.

Integrative cytology and proteome alterations unravel the multitarget effect of a novel alkaloid antofine againstPenicillium italicum

AbstractPenicillium italicum is the causal agent of citrus blue mold, which is a major threat to the global citrus fruit industry. Antofine, a natural phenanthroindolizidine alkaloid, is water-soluble and exhibits a broad range of biological activities. However, whether it can inhibit P. italicum growth and the potential inhibitory mechanism remains to be elucidated. This study aimed to investigate the antifungal mechanism of antofine against P. italicum using scanning electron microscopy, transmission electron microscopy (TEM), propidium iodide staining, and tandem mass tag-labeled quantitative proteomic analysis. Antofine was found to exhibit its preeminent antifungal activity against P. italicum with a minimum inhibitory concentration of 1.56 mg/L and a minimum fungicidal concentration of 6.25 mg/L. The challenge test revealed that antofine inhibited the development of citrus blue mold during a 6-d P. italicum-infected period. Antofine acted on its potential multitargets to inhibit P. italicum growth by synergistically activating oxidative stress through accumulating excess reactive oxygen species, impairing membrane integrity, inducing membrane lipid peroxidation, and disrupting mitochondrial function, thereby disrupting the membrane system and reducing cell viability. Moreover, antofine treatment downregulated most differentially expressed proteins involved in carbon metabolism, pyruvate metabolism, and the tricarboxylic acid cycle (TCA) in P. italicum mycelia, which may explain the mitochondrial decomposition observed by TEM and the declines in ATP levels as well as the activities of TCA-related enzymes. These results indicate that antofine treatment inhibited P. italicum growth by targeting the cell membrane and mitochondria.

Phenanthroindolizidine Alkaloids Isolated from Tylophora ovata as Potent Inhibitors of Inflammation, Spheroid Growth, and Invasion of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC), representing the most aggressive form of breast cancer with currently no targeted therapy available, is characterized by an inflammatory and hypoxic tumor microenvironment. To date, a broad spectrum of anti-tumor activities has been reported for phenanthroindolizidine alkaloids (PAs), however, their mode of action in TNBC remains elusive. Thus, we investigated six naturally occurring PAs extracted from the plant Tylophora ovata: O-methyltylophorinidine (1) and its five derivatives tylophorinidine (2), tylophoridicine E (3), 2-demethoxytylophorine (4), tylophoridicine D (5), and anhydrodehydrotylophorinidine (6). In comparison to natural (1) and for more-in depth studies, we also utilized a sample of synthetic O-methyltylophorinidine (1s). Our results indicate a remarkably effective blockade of nuclear factor kappa B (NFκB) within 2 h for compounds (1) and (1s) (IC50 = 17.1 ± 2.0 nM and 3.3 ± 0.2 nM) that is different from its effect on cell viability within 24 h (IC50 = 13.6 ± 0.4 nM and 4.2 ± 1 nM). Furthermore, NFκB inhibition data for the additional five analogues indicate a structure–activity relationship (SAR). Mechanistically, NFκB is significantly blocked through the stabilization of its inhibitor protein kappa B alpha (IκBα) under normoxic as well as hypoxic conditions. To better mimic the TNBC microenvironment in vitro, we established a 3D co-culture by combining the human TNBC cell line MDA-MB-231 with primary murine cancer-associated fibroblasts (CAF) and type I collagen. Compound (1) demonstrates superiority against the therapeutic gold standard paclitaxel by diminishing spheroid growth by 40% at 100 nM. The anti-proliferative effect of (1s) is distinct from paclitaxel in that it arrests the cell cycle at the G0/G1 state, thereby mediating a time-dependent delay in cell cycle progression. Furthermore, (1s) inhibited invasion of TNBC monoculture spheroids into a matrigel®-based environment at 10 nM. In conclusion, PAs serve as promising agents with presumably multiple target sites to combat inflammatory and hypoxia-driven cancer, such as TNBC, with a different mode of action than the currently applied chemotherapeutic drugs.

(R)-13aα-Densiindolizidine, A New Phenanthroindolizidine Alkaloid From Cryptocarya densiflora Blume (Lauraceae) and Molecular Docking Against SARS-CoV-2

Cryptocarya densiflora Blume (Lauraceae) is an evergreen tree widely distributed throughout the hills and mountain forests up to 1500 m in Malaysia and Indonesia. The plant has been reported to contain phenanthroindolizidine-type of alkaloids. In the present work, a new phenanthroindolizidine alkaloid named ( R)-13a α-densiindolizidine, was isolated from the dichloromethane (DCM) extract of the leaves. The structure of the alkaloid was established based on 1D and 2D nuclear magnetic resonance (NMR) and liquid chromatography mass spectrometry-ion trap-time of flight (LCMS-IT-TOF) analysis. ( R)-13a α-densiindolizidine displayed binding interactions with crucial amino acid residues in the active sites ofsevere acute respiratory syndrome coronavirus 2 Mpro (SARS-CoV-2 Mpro) and RNA-dependent protease (RdRp) in silico, whilst fulfilling theabsorption, distribution, metabolism, excretion, and toxicity (ADMET) criteria and Lipinsky's rule, thus revealing its potential as a lead compound.

Lignin Biosynthesis Pathway and Redox Balance Act Synergistically in Conferring Resistance against Penicillium italicum Infection in 7-Demethoxytylophorine-Treated Navel Orange.

7-Demethoxytylophorine (DEM), a natural water-soluble phenanthroindolizidine alkaloid, has a great potential for in vitro suppression of Penicillium italicum growth. In the present study, we investigated the ability of DEM to confer resistance against P. italicum in harvested "Newhall" navel orange and the underlying mechanism. Results from the in vivo experiment showed that DEM treatment delayed blue mold development. The water-soaked lesion diameter in 40 mg L-1 DEM-treated fruit was 35.2% lower than that in the control after 96 h. Moreover, the decrease in peel firmness loss and increase in electrolyte leakage, superoxide anion (O2•-) production, and malondialdehyde (MDA) content were significantly inhibited by DEM treatment. Hydrogen peroxide (H2O2) burst in DEM-treated fruit at the early stage of P. italicum infection contributed to the conferred resistance by increasing the activities of lignin biosynthesis-related enzymes, along with the expressions of their encoding genes, resulting in lignin accumulation. The DEM-treated fruit maintained an elevated antioxidant capacity, as evidenced by high levels of ascorbic acid and glutathione content, and enhanced or upregulated the activities and gene expression levels of APX, GR, MDHAR, DHAR, GPX, and GST, thereby maintaining ROS homeostasis and reducing postharvest blue mold. Collectively, the results in the present study revealed a control mechanism in which DEM treatment conferred the resistance against P. italicum infection in harvested "Newhall" navel orange fruit by activating lignin biosynthesis and maintaining the redox balance.

In Vitro Anti-Proliferative, and Kinase Inhibitory Activity of Phenanthroindolizidine Alkaloids Isolated from Tylophora indica.

The phenanthroindolizidine alkaloid (-)-tylophorine has been reported for its significant anticancer activity working through different biomechanistic pathways. The current study aimed to evaluate the anticancer activity of phenanthroindolizidine alkaloids isolated from Tylophora indica. Six phenanthroindolizidine alkaloid (compounds 1–6) in addition to septicine (7), chlorogenic acid (8), and chlorogenic acid methyl ester (9) were isolated from Tylophora indica using different chromatographic techniques including vacuum liquid chromatography (VLC) and preparative high performance liquid chromatography (HPLC). The isolated compounds structures’ were determined using various spectro-analytical techniques, i.e., 1H-NMR, 13C-NMR, and mass spectrometry. The isolates’ structural stereochemistry and structural geometries were determined with the help of chiroptical techniques together with comparisons with the available standard samples. The in vitro anti-proliferative activity on three different cell lines, MCF-7, HepG2, and HCT-116 were evaluated. Among all the isolated compounds, tylophorinidine (5) was the most active cytotoxic agent with the lowest IC50 values at 6.45, 4.77, and 20.08 μM against MCF-7, HepG2, and HCT-116 cell lines, respectively. The bioactivities were also validated by the in vitro kinase receptors inhibition assay. Compound (5) also exhibited the highest activity with lowest IC50 values (0.6 and 1.3 μM against the Aurora-A and Aurora-B enzymes, respectively), as compared with all the isolated alkaloidal products. The structure activity relationship on the molecular properties, molecular attributes, and bioactivity levels were analyzed, interrelated, and the molecular docking studies on two different receptors, Aurora-A and Aurora-B, were determined, which provided the confirmations of the bioactivity with receptor-ligand geometric disposition, energy requirements, lipophilicity, and detailed the binding pharmacophore involvements responsible for bioactivity elicitations.

Total Synthesis of the Proposed Structure of Tyloindane and Its Diastereoisomer

AbstractIt is intriguing that tyloindane, which was isolated together with phenanthroindolizidine alkaloids, contains no nitrogen atom. Attracted by its unique structure and uncovered biological activity, we synthesized the proposed structure of tyloindane and its diastereoisomer. To achieve this goal, several strategies that include an aryl/alkene oxidative coupling, radical cyclization, and intramolecular Parham alkylation, were explored. The 1H NMR data of the synthesized compounds do not match those of the isolated sample (tyloindane), indicating that the structure of tyloindane should be reassigned.

Target-Directed Design, Synthesis, Antiviral Activity, and SARs of 9-Substituted Phenanthroindolizidine Alkaloid Derivatives.

On the basis of our previous studies on the antiviral mechanism against tobacco mosaic virus (TMV) and structure-activity relationship of phenanthroindolizidine alkaloids, a series of 9-substituted tylophorine derivatives targeting TMV RNA were designed, synthesized, and assessed for their anti-TMV activities. The bioassay results indicated that most of these compounds showed good in vivo anti-TMV activities, and some of them displayed higher activity than that of commercial ribavirin. Especially, the anti-TMV activities of compound 3b, 4, and 6 are 2-3 times higher than that of commercial ribavirin, according to EC50 values. In this work, we have demonstrated an effective way to design new inhibitors against plant virus and developed 9-ethoxy methyl tylophorine (4) with excellent anti-TMV activity (in vitro activity, 70.2%/500 μg/mL and 27.1%/100 μg/mL; inactivation activity, 67.7%/500 μg/mL and 30.5%/100 μg/mL; curative activity, 65.3%/500 μg/mL and 30.8%/100 μg/mL; and protection activity, 65.9%/500 μg/mL and 36.0%/100 μg/mL) as a potential plant viral inhibitor.

Design, Synthesis, Antivirus Activity, and SARs of Phenanthroquinolizidine Alkaloid Derivatives

Our previous investigation revealed that phenanthroindolizidine alkaloid derivatives have a good inhibitory effect against tabacco mosaic virus (TMV) and that the conformation of these alkaloid analogues has a big influence on the anti-TMV activity. Compared with the well-studied phenanthroindolizidine alkaloids, phenanthroquinolizidine alkaloids have gained less attention. To uncover their anti-TMV activity and structure and activity relationships, a series of D-ring opened phenanthroquinolizidine derivatives and 14a-substituted pentacyclic alkaloid analogues were designed, synthesized, and evaluated against TMV for the first time. Bioassay results revealed that most of these synthesized compounds exhibited a good to excellent inhibitory effect, and three of them displayed better activity than Ningnanmycin—one of the most successful plant virucides—thus providing a new anti-TMV lead for further development. Preliminary SARs showed that the pentacyclic structure, 14a-substituent, and hydrogen-bond donors in these alkaloids are crucial for keeping high potent activities.

Abstract 6383: HTBPI inhibits liver tumorigenesis by combined inhibition of Akt

Abstract Akt, a crucial protein involved in a variety of signaling pathways in cancer, acts as an important regulator of cell survival in hepatocellular carcinoma (HCC), and provides curative option for the related drug development. We have found an active phenanthroindolizidine alkaloid, (13aR,14R)-9,11,12,13,13a,14-hexahydro-3,6,7- trimethoxydibenzo[f,h]pyrrolo[1,2-b]isoquinolin-14-ol (HTBPI), which is a promising Akt inhibitor effective in the suppression of HCC cells proliferation through stimulating apoptotic and autophagic capability in vivo and in vitro. Treatment of HTBPI combined with a classical autophagy-lysosomal inhibitor (bafilomycin A1), could enhance stimulation effects of apoptosis on HCC cell lines. In addition, we confirmed that HTBPI, targeting Akt, occupied the kinase binding domain (Thr 308) of Akt to inactivate its function by CETSA and DARTS assay. Moreover, HTBPI showed a potential therapeutic effect in patient-derived xenograft (PDX) tumors without observed toxicity. Furthermore, high p-Akt (Thr 308) expression was collated with liver tumor formation and poor survival in HCC patients. In conclusions, HTBPI impeded HCC progress through regulation of apoptosis and autophagy machinery via interaction with p-Akt (Thr 308). This may provide potential molecular candidate by inhibiting Akt for the therapy of HCC patients. Citation Format: Haiyang Yu, Hongwei Liu, Qian Chen, Xu Pang, Shuangshuang Yin, Kailong Wang, Rui Wang, Tao Wang, Yuling Qiu. HTBPI inhibits liver tumorigenesis by combined inhibition of Akt [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6383.

HTBPI, an active phenanthroindolizidine alkaloid, inhibits liver tumorigenesis by targeting Akt.

Akt, a crucial protein involved in a variety of signaling pathways in cancer, acts as an important regulator of survival in hepatocellular carcinoma (HCC), and provides curative option for the related drugs development. We have found an active phenanthroindolizidine alkaloid, (13aR,14R)-9,11,12,13,13a,14-hexahydro-3,6,7-trimethoxydibenzo[f,h]pyrrolo[1,2-b]isoquinolin-14-ol (HTBPI), is a promising Akt inhibitor effective in the suppression of HCC cells proliferation through stimulating apoptotic and autophagic capability in vivo and in vitro. Treatment of HTBPI combined with a classical autophagy-lysosomal inhibitor (bafilomycin A1), could enhance stimulation effects of apoptosis on HCC cell lines. In addition, we confirmed HTBPI targeting Akt, occupied the kinase binding domain (Thr 308) of Akt to inactivate its function by CETSA and DARTS assay. In contrast, ectopic Akt-induced overexpression significantly abrogated inhibitory effects of HTBPI on cell viability and proliferation. Furthermore, high p-Akt (Thr 308) expression is collated with liver tumor formation and poor survival in HCC patients. In conclusions, HTBPI impeded HCC progress through regulation of apoptosis and autophagy machinery via interaction with p-Akt (Thr 308). This may provide potential molecular candidate by targeting Akt for the therapy of HCC patients.

Synthesis of Quaternary Carbon-Centered Benzoindolizidinones via Novel Photoredox-Catalyzed Alkene Aminoarylation: Facile Access to Tylophorine and Analogues

Photoredox-catalyzed aminoarylation and thioamination of unactivated alkenes have been developed, providing novel synthetic routes to access synthetically challenging quaternary carbon-centered ben...

Design, synthesis, and biological activity evaluation of (-)-6-O-desmethylantofine analogues as potent anti-cancer agents.

Phenanthroindolizidine alkaloids that possess profound anti-proliferative activity and unique mode of action have recently attracted much attention as potential anti-cancer drug candidates. To intensively study the structure-activity-relationship, we designed, synthesized, and evaluated a series of derivatives of 6-desmethylantofine at C-6 position. Most of the derivatives exhibited potent anti-proliferative activity in BEL-7402 and HL60cells. Compound R-12, the cyanomethyl ether of 6-desmethylantofine, exhibited significant anti-cancer activity and inhibited the proliferation of a panel of 30 cancer cell lines including 2 multi-drug-resistant cell lines with an average IC50 value of 18.7 nM, which suggests that R-12 is a promising new anti-cancer agent. Our studies suggest that R-12 displayed potent inhibitory effect on cell growth and colony formation, which is associated with delaying S phase progression by inhibiting DNA synthesis in human hepatoma cancer BEL-7402, SMMC-7721 and ZIP-177 cells.

A phenanthroindolizidine glycoside with HIF-1 inhibitory activity from Tylophora atrofolliculata

A phenanthroindolizidine glycoside 6-O-β-D-glucopyranosyl-tylophorinidine (1), the first glycoside of phenanthroindolizidine alkaloid in nature, was isolated from whole plants of Tylophora atrofolliculata (Asclepiadaceae). Its structure was elucidated by means of spectral and chemical evidences. Compound 1 showed potent inhibitory effect (IC50: 69 ± 13 nM) on hypoxia induced factor-1 (HIF-1) activation. It was discovered that glucosylation at C-6 of 1 could lead to reduction in cytotoxicity against normal cells and enhance its selectivity in tumor cells inhibition.

Inhibitory Effect of 7-Demethoxytylophorine on Penicillium italicum and its Possible Mechanism.

7-demethoxytylophorine (DEM) is a phenanthroindolizidine alkaloid, which is reported to be effective in inhibiting leucocytes and regulation of human immunity. However, few studies reported the inhibitory effect of DEM against plant-pathogenic fungi, particularly postharvest pathogen Penicillium italicum (P. italicum). Current studies have investigated the antifungal activity of DEM through membrane damage and energy deficit in P. italicum. The results showed that the DEM potentially inhibits the growth of P. italicum in a dose-dependent manner. In vitro (mycelial growth and spore germination) tests showed great minimal inhibitory concentration (MIC) (1.56 µg mL−1) and minimum fugicide concentration (MFC) (6.25 µg mL−1). Microscopic analyses showed that mycelial morphology of P. italicum was severely damaged following DEM treatment. Moreover, relative electrical conductivity and lysis ability assays showed that DEM treatment aids in destroying the integrity of plasma membranes that deplete reducing sugars and soluble proteins. The activity of malate dehydrogenase (MDH) and succinate dehydrogenase (SDH) demonstrated that DEM led to the disruption of TCA cycle in P. italicum mycelia. The results of this study led us to conclude that, DEM could be used as a natural antifungal agent for controlling postharvest blue mold disease of citrus fruits caused by P. italicum.