Cytotoxic leuconoxine-type diazaspiroindole alkaloids isolated from Cryptolepis dubia.

Two leuconoxine-type diazaspiroindole alkaloids, the known compound, (+)-melodinine E (1), and its new analogue, (+)-11-chloromelodinine E (2), were isolated from the stems of Cryptolepis dubia (Burm.f.) M.R. Almeida (Apocynaceae), collected in Laos. The chemical structures of these compounds were determined by analysis of their spectroscopic data and by comparison of these data with literature values, of which the molecular structure of 1 has been determined previously by analysis of its single-crystal X-ray diffraction data. The absolute configurations of 1 and 2 have been defined by their experimental and simulated electronic circular dichroism (ECD) spectroscopic data and supported by 1H and 13C NMR-based DP4+ probability analysis and specific rotation calculations. When tested against a small panel of human cancer cell lines, these two compounds exhibited selective cytotoxicity toward OVCAR3 human ovarian cancer cells.

Strategies for the discovery of potential anticancer agents from plants collected from Southeast Asian tropical rainforests as a case study.

Covering up to early 2023The present review summarizes recent accomplishments made as part of a multidisciplinary, multi-institutional anticancer drug discovery project, wherein samples comprising higher plants were collected primarily from Southeast Asia, and also from Central America, and the West Indies. In the introductory paragraphs, a short perspective is provided on the current importance of plants in the discovery of cancer therapeutic agents, and the contributions of other groups working towards this objective are mentioned. For our own investigations, following their collection, tropical plants have been subjected to solvent extraction and biological evaluation for their antitumor potential. Several examples of purified plant lead bioactive compounds were obtained and characterized, and found to exhibit diverse structures, including those of the alkaloid, cardiac glycoside, coumarin, cucurbitacin, cyclobenzofuran (rocaglate), flavonoid, lignan, and terpenoid types. In order to maximize the efficiency of work on drug discovery from tropical plant species, strategies to optimize various research components have been developed, including those for the plant collections and taxonomic identification, in accordance with the requirements of contemporary international treaties and with a focus on species conservation. A major component of this aspect of the work is the development of collaborative research agreements with representatives of the source countries of tropical rainforest plants. The phytochemical aspects have included the preparation of plant extracts for initial screening and the selection of promising extracts for activity-guided fractionation. In an attempt to facilitate this process, a TOCSY-based NMR procedure has been applied for the determination of bioactive rocaglate derivatives in samples of Aglaia species (Meliaceae) collected for the project. Preliminary in vitro and in vivo mechanistic studies carried out by the authors are described for two tropical plant-derived bioactive lead compounds, corchorusoside C and (+)-betulin, including work conducted with a zebrafish (Danio rerio) model. In the concluding remarks, a number of lessons are summarized that our group has learned as a result of working on anticancer drug discovery using tropical plants, which we hope will be of interest to future workers.

The Cytotoxic Cardiac Glycoside (-)-Cryptanoside A from the Stems of Cryptolepis dubia and Its Molecular Targets.

A cardiac glycoside epoxide, (-)-cryptanoside A (1), was isolated from the stems of Cryptolepis dubia collected in Laos, for which the complete structure was confirmed by analysis of its spectroscopic and single-crystal X-ray diffraction data, using copper radiation at a low temperature. This cardiac glycoside epoxide exhibited potent cytotoxicity against several human cancer cell lines tested, including HT-29 colon, MDA-MB-231 breast, OVCAR3 and OVCAR5 ovarian cancer, and MDA-MB-435 melanoma cells, with the IC50 values found to be in the range 0.1-0.5 µM, which is comparable with that observed for digoxin. However, it exhibited less potent activity (IC50 1.1 µM) against FT194 benign/nonmalignant human fallopian tube secretory epithelial cells when compared with digoxin (IC50 0.16 µM), indicating its more selective activity toward human cancer versus benign/nonmalignant cells. (-)-Cryptanoside A (1) also inhibited Na+/K+-ATPase activity and increased the expression of Akt and the p65 subunit of NF-κB but did not show any effects on the expression of PI3K. A molecular docking profile showed that (-)-cryptanoside A (1) binds to Na+/K+-ATPase, and thus 1 may directly target Na+/K+-ATPase to mediate its cancer cell cytotoxicity.

A pentamethoxylated flavone from Glycosmis ovoidea promotes apoptosis through the intrinsic pathway and inhibits migration of MCF-7 breast cancer cells.

The rare flavone 5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone (PMF) has been isolated from several plant species, and its cytotoxic activity has been reported against many types of cancer cells. In this study, PMF was purified from Glycomis ovoidea collected in Vietnam, and its antiproliferative effects and underlying mechanism of action were investigated against MCF-7 cells. PMF inhibited growth in MCF-7 > MCF-10A > MDA-MB-231 cells after 72 hr treatment, with IC50 values of 1.5, 1.9, and 8.6 µg/ml, respectively. Further experiments conducted with this compound in MCF-7 cells, showed the loss of mitochondrial membrane potential, reactive oxygen species overproduction, upregulation of BAX, cytochrome c, caspase-3 and PARP-1 and down-regulation of BCL-2 proteins as well as an increase in caspase-3/-7 activity, suggesting induction of the apoptotic intrinsic pathway. Furthermore, PMF increased cell cycle arrest in the G1 phase, which correlated with increments in the p53 and p21 levels. Additionally, MCF-7 cell migration was inhibited, which could be related to NF-κB p65 downregulation. Finally, PMF did not show toxicity in vivo in a zebrafish (Danio rerio) model. In conclusion, PMF induces cell death in MCF-7 cells through regulation of the BCL-2 protein family and may be proposed as a lead as a potential alternative for breast cancer therapy.

Cytotoxic and non-cytotoxic cardiac glycosides isolated from the combined flowers, leaves, and twigs of Streblus asper.

A new non-cytotoxic [(+)-17ß-hydroxystrebloside (1)] and two known cytotoxic [(+)-3'-de-O-methylkamaloside (2) and (+)-strebloside (3)] cardiac glycosides were isolated and identified from the combined flowers, leaves, and twigs of Streblus asper collected in Vietnam, with the absolute configuration of 1 established from analysis of its ECD and NMR spectroscopic data and confirmed by computational ECD calculations. A new 14,21-epoxycardanolide (3a) was synthesized from 3 that was treated with base. A preliminary structure-activity relationship study indicated that the C-14 hydroxy group and the C-17 lactone unit and the established conformation are important for the mediation of the cytotoxicity of 3. Molecular docking profiles showed that the cytotoxic 3 and its non-cytotoxic analogue 1 bind differentially to Na+/K+-ATPase. Compound 3 docks deeply in the Na+/K+-ATPase pocket with a sole pose, and its C-10 formyl and C-5, C-14, and C-4' hydroxy groups may form hydrogen bonds with the side-chains of Glu111, Glu117, Thr797, and Arg880 of Na+/K+-ATPase, respectively. However, 1 fits the cation binding sites with at least three different poses, which all depotentiate the binding between 1 and Na+/K+-ATPase. Thus, 3 was found to inhibit Na+/K+-ATPase, but 1 did not. In addition, the cytotoxic and Na+/K+-ATPase inhibitory 3 did not affect glucose uptake in human lung cancer cells, against which it showed potent activity, indicating that this cardiac glycoside mediates its cytotoxicity by targeting Na+/K+-ATPase but not by interacting with glucose transporters.

Potential Anticancer Agents Characterized from Selected Tropical Plants.

Higher plants are well known for their value in affording clinically useful anticancer agents, with such compounds acting against cancer cells by a range of mechanisms of action. There remains a strong interest in the discovery and development of plant secondary metabolites as additional cancer chemotherapeutic lead compounds. In the present review, progress on the discovery of plant-derived compounds of the biflavonoid, lignan, sesquiterpene, steroid, and xanthone structural types is presented. Several potential anticancer leads of these types have been characterized from tropical plants collected in three countries as part of our ongoing collaborative multi-institutional project. Preliminary structure-activity relationships and work on in vivo testing and cellular mechanisms of action are also discussed. In addition, the relevant work reported by other groups on the same compound classes is included herein.

Caspase-Dependent Apoptosis in Prostate Cancer Cells and Zebrafish by Corchorusoside C from Streptocaulon juventas.

Corchorusoside C (1), isolated from Streptocaulon juventas collected in Vietnam, was found to be nontoxic in a zebrafish ( Danio rerio) model and to induce cytotoxicity in several cancer cell lines with notable selective activity against prostate DU-145 cancer cells (IC50 0.08 µM). Moreover, corchorusoside C induced DU-145 cell shrinkage and cell detachment. In CCD-112CoN colon normal cells, 1 showed significantly reduced cytotoxic activity (IC50 2.3 µM). A preliminary mechanistic study indicated that 1 inhibits activity and protein expression of NF-κB (p50 and p65), IKK (α and ß), and ICAM-1 in DU-145 cells. ROS concentrations increased at 5 h post-treatment, and MTP decreased in a dose-dependent manner. Moreover, decreased protein expression of Bcl-2 and increased expression of PARP-1 was observed. Furthermore, corchorusoside C increased both the activity and protein levels of caspases 3 and 7. Additionally, 1 induced sub-G1 population increase of DU-145 cells and modulated caspases in zebrafish with nondifferential morphological effects. Therefore, corchorusoside C (1) induces apoptosis in DU-145 cells and targets the same pathways both in vitro and in vivo in zebrafish. Thus, the use of zebrafish assays seems worthy of wider application than is currently employed for the evaluation of potential anticancer agents of natural origin.

Structurally Modified Cyclopenta[b]benzofuran Analogues Isolated from Aglaia perviridis.

Four new cyclopenta[b]benzofuran derivatives based on an unprecedented carbon skeleton (1-4), with a dihydrofuran ring fused to dioxanyl and aryl rings, along with a new structural analogue (5) of 5‴-episilvestrol (episilvestrol, 7), were isolated from an aqueous extract of a large-scale re-collection of the roots of Aglaia perviridis collected in Vietnam. Compound 5 demonstrated mutarotation in solution due to the presence of a hydroxy group at C-2‴, leading to the isolation of a racemic mixture, despite being purified on a chiral-phase HPLC column. Silvestrol (6) and episilvestrol (7) were isolated from the most potently cytotoxic chloroform subfraction of the roots. All new structures were elucidated using 1D and 2D NMR, HRESIMS, IR, UV, and ECD spectroscopic data. Of the five newly isolated compounds, only compound 5 exhibited cytotoxic activity against a human colon cancer (HT-29) and human prostate cancer cell line (PC-3), with IC50 values of 2.3 µM in both cases. The isolated compounds (1-5) double the number of dioxanyl ring-containing rocaglate analogues reported to date from Aglaia species and present additional information on the structural requirements for cancer cell line cytotoxicity within this compound class.

Cytotoxic and NF-κB and mitochondrial transmembrane potential inhibitory pentacyclic triterpenoids from Syzygium corticosum and their semi-synthetic derivatives.

Syzygium is a large genus of flowering plants, with several species, including the clove tree, used as important resources in the food and pharmaceutical industries. In our continuing search for anticancer agents from higher plants, a chloroform extract of the leaves and twigs of Syzygium corticosum collected in Vietnam was found to be active toward the HT-29 human colon cancer cell line. Separation of this extract guided by HT-29 cells and nuclear factor-kappa B (NF-κB) inhibition yielded 19 known natural products, including seven triterpenoids, three ellagic acid derivatives, two methylated flavonoids, a cyclohexanone, four megastigmanes, a small lactone, and an aromatic aldehyde. The full stereochemistry of (+)-fouquierol (2) was defined for the first time. Biological investigations showed that (+)-ursolic acid (1) is the major cytotoxic component of S. corticosum, which exhibited also potent activities in the NF-κB and mitochondrial transmembrane potential (MTP) inhibition assays conducted, with IC50 values of 31 nM and 3.5 µM, respectively. Several analogues of (+)-ursolic acid (1) were synthesized, and a preliminary structure-activity relationship (SAR) study indicated that the C-3 hydroxy and C-28 carboxylic acid groups and 19,20-dimethyl substitution are all essential in the mediation of the bioactivities observed for this triterpenoid.

Potent Inhibitor of Drug-Resistant HIV-1 Strains Identified from the Medicinal Plant Justicia gendarussa.

Justicia gendarussa, a medicinal plant collected in Vietnam, was identified as a potent anti-HIV-1 active lead from the evaluation of over 4500 plant extracts. Bioassay-guided separation of the extracts of the stems and roots of this plant led to the isolation of an anti-HIV arylnaphthalene lignan (ANL) glycoside, patentiflorin A (1). Evaluation of the compound against both the M- and T-tropic HIV-1 isolates showed it to possess a significantly higher inhibition effect than the clinically used anti-HIV drug AZT. Patentiflorin A and two congeners were synthesized, de novo, as an efficient strategy for resupply as well as for further structural modification of the anti-HIV ANL glycosides in the search for drug leads. Subsequently, it was determined that the presence of a quinovopyranosyloxy group in the structure is likely essential to retain the high degree of anti-HIV activity of this type of compounds. Patentiflorin A was further investigated against the HIV-1 gene expression of the R/U5 and U5/gag transcripts, and the data showed that the compound acts as a potential inhibitor of HIV-1 reverse transcription. Importantly, the compound displayed potent inhibitory activity against drug-resistant HIV-1 isolates of both the nucleotide analogue (AZT) and non-nucleotide analogue (nevaripine). Thus, the ANL glycosides have the potential to be developed as novel anti-HIV drugs.

Cardiac Glycoside Constituents of Streblus asper with Potential Antineoplastic Activity.

Three new (1-3) and two known (4 and 5) cytotoxic cardiac glycosides were isolated and characterized from a medicinal plant, Streblus asper Lour. (Moraceae), collected in Vietnam, with six new analogues and one known derivative (5a-g) synthesized from (+)-strebloside (5). A preliminary structure-activity relationship study indicated that the C-10 formyl and C-5 and C-14 hydroxy groups and C-3 sugar unit play important roles in the mediation of the cytotoxicity of (+)-strebloside (5) against HT-29 human colon cancer cells. When evaluated in NCr nu/nu mice implanted intraperitoneally with hollow fibers facilitated with either MDA-MB-231 human breast or OVCAR3 human ovarian cancer cells, (+)-strebloside (5) showed significant cell growth inhibitory activity in both cases, in the dose range 5-30 mg/kg.

Isolation of Bioactive Rotenoids and Isoflavonoids from the Fruits of Millettia caerulea.

Three new rotenoids (1-3), two new isoflavonoids (4 and 5), and six known analogues (6-11) were isolated from an n-hexane partition of a methanol extract of the fruits of Millettia caerulea, with the structures of the new compounds elucidated by analysis of their spectroscopic data. The relative configurations of the rotenoids were determined by interpretation of their NMR spectroscopic data, and their absolute configurations were established using electronic circular dichroism spectra and specific rotation values. All compounds isolated were evaluated for their cell growth inhibitory activity against the HT-29 human colon cancer cell line, and the known compounds, (-)-3-hydroxyrotenone (6) and (-)-rotenone (7), were found to be potently active. When tested in an NF-κB inhibition assay, compound 6 showed activity. This compound, along with the new compound, (-)-caeruleanone D (1), and the known compound, ichthynone (8), exhibited K-Ras inhibitory potency. Further bioactivity studies showed that the new compounds, (-)-3-deoxycaeruleanone D (2) and (-)-3-hydroxycaeruleanone A (3), and the known compounds 8 and 11 induced quinone reductase in murine Hepa 1c1c7 cells.

New Bioactive Lupane Triterpene Coumaroyl Esters Isolated from Buxus cochinchinensis.

Five new lupane triterpene coumaroyl esters (1-5), together with betulin (6) and a known Buxus alkaloid, N-3-benzoyldihydrocyclomicrophylline F (7), were isolated from a CHCl3-soluble partition of a methanol extract of Buxus cochinchinensis Pierre ex Gagnep. (Buxaceae) collected in Vietnam. Isolation work was monitored using human colon cancer cells (HT-29). The structures of the new compounds (1-5) were determined on the basis of spectroscopic data interpretation. In addition to their cytotoxicity against HT-29 cells and nuclear factor-kappa B (p65) inhibitory activity in an enzyme-linked immunosorbent assay, all isolates as well as two semisynthetic compounds derived from betulin and 5, respectively, were also evaluated for their in vitro antiplasmodial activities against the drug-resistant Dd2 strain of Plasmodium falciparum and antifungal effects on the growth of the pathogenic yeast Candida albicans. The new lupane triterpene coumaroyl esters (1-5), along with a betulin derivative and the known Buxus alkaloid, were found to show significant in vitro antimalarial activities, with IC50 values ranging from 0.26 to 2.07 µM.

Investigation of Vietnamese plants for potential anticancer agents.

Higher plants continue to afford humankind with many new drugs, for a variety of disease types. In this review, recent phytochemical and biological progress is presented for part of a collaborative multi-institutional project directed towards the discovery of new antitumor agents. The specific focus is on bioactive natural products isolated and characterized structurally from tropical plants collected in Vietnam. The plant collection, identification, and processing steps are described, and the natural products isolated from these species are summarized with their biological activities.

Potent cytotoxic arylnaphthalene lignan lactones from Phyllanthus poilanei.

Two new (1 and 2) and four known arylnaphthalene lignan lactones (3-6) were isolated from different plant parts of Phyllanthus poilanei collected in Vietnam, with two further known analogues (7 and 8) being prepared from phyllanthusmin C (4). The structures of the new compounds were determined by interpretation of their spectroscopic data and by chemical methods, and the structure of phyllanthusmin D (1) was confirmed by single-crystal X-ray diffraction analysis. Several of these arylnaphthalene lignan lactones were cytotoxic toward HT-29 human colon cancer cells, with compounds 1 and 7-O-[(2,3,4-tri-O-acetyl)-α-L-arabinopyranosyl)]diphyllin (7) found to be the most potent, exhibiting IC50 values of 170 and 110 nM, respectively. Compound 1 showed activity when tested in an in vivo hollow fiber assay using HT-29 cells implanted in immunodeficient NCr nu/nu mice. Mechanistic studies showed that this compound mediated its cytotoxic effects by inducing tumor cell apoptosis through activation of caspase-3, but it did not inhibit DNA topoisomerase IIα activity.

Species-specific Standardisation of Licorice by Metabolomic Profiling of Flavanones and Chalcones.

INTRODUCTION: Major phenolics from licorice roots (Glycyrrhiza sp.) are glycosides of the flavanone liquiritigenin (F) and its 2'-hydroxychalcone isomer, isoliquiritigenin (C). As the F and C contents fluctuate between batches of licorice, both quality control and standardisation of its preparations become complex tasks. OBJECTIVE: To characterise the F and C metabolome in extracts from Glycyrrhiza glabra L. and Glycyrrhiza uralensis Fisch. ex DC. by addressing their composition in major F­C pairs and defining the total F:C proportion. MATERIAL AND METHODS: Three types of extracts from DNA-authenticated samples were analysed by a validated UHPLC/UV method to quantify major F and C glycosides. Each extract was characterised by the identity of major F­C pairs and the proportion of Fs among all quantified Fs:Cs. RESULTS: The F and C compositions and proportions were found to be constant for all extracts from a Glycyrrhiza species. All G. uralensis extracts contained up to 2.5 more Fs than G. glabra extracts. Major F­C pairs were B-ring glycosidated in G. uralensis, and A-/B-ring apiosyl-glucosidated in the G. glabra extracts. The F:C proportion was found to be linked to the glycosidation site: the more B-ring F-C glycosides were present, the higher was the final F:C proportion in the extract. These results enable the chemical differentiation of extracts from G. uralensis and G. glabra, which are characterised by total F:C proportions of 8.37:1.63 and 7.18:2.82, respectively. CONCLUSION: Extracts from G. glabra and G. uralensis can be differentiated by their respective F and C compositions and proportions, which are both useful for further standardisation of licorice botanicals.

Sphenostylisins A-K: bioactive modified isoflavonoid constituents of the root bark of Sphenostylis marginata ssp. erecta.

Sphenostylisins A-C (1-3), three complex dimeric compounds representing two novel carbon skeletons, along with an additional eight new compounds, sphenostylisins D-K (4-11), were isolated from the active chloroform-soluble extract of the root bark of Sphenostylis marginata ssp. erecta using a bioactivity-guided isolation approach. The structures were elucidated by means of detailed spectroscopic analysis, including NMR and HRESIMS analysis, and tandem MS fragmentation was utilized to further support the structures of 1-3. The absolute configuration of sphenostylisin C (3) was established by electronic circular dichroism analysis. Plausible biogenetic relationships between the modified isoflavonoids 1-11 are proposed, and a cyclization reaction of 9 was conducted to support one of the biogenetic proposals made. All of these pure isolates were evaluated against a panel of in vitro bioassays, and among the results obtained, sphenostylisin A (1) was found to be a very potent NF-κB inhibitor (IC50 = 6 nM).

Bioactive flavaglines and other constituents isolated from Aglaia perviridis.

Eight new compounds, including two cyclopenta[b]benzopyran derivatives (1, 2), two cyclopenta[b]benzofuran derivatives (3, 4), three cycloartane triterpenoids (5-7), and an apocarotenoid (8), together with 16 known compounds, were isolated from the chloroform-soluble partitions of separate methanol extracts of a combination of the fruits, leaves, and twigs and of the roots of Aglaia perviridis collected in Vietnam. Isolation work was monitored using human colon cancer cells (HT-29) and facilitated with an LC/MS dereplication procedure. The structures of the new compounds (1-8) were determined on the basis of spectroscopic data interpretation. The Mosher ester method was employed to determine the absolute configurations of 5-7, and the absolute configuration of the 9,10-diol unit of compound 8 was established by a dimolybdenum tetraacetate [Mo2(AcO)4] induced circular dichroism procedure. Seven known rocaglate derivatives (9-15) exhibited significant cytotoxicity against the HT-29 cell line, with rocaglaol (9) being the most potent (ED50 0.0007 µM). The new compounds 2-4 were also active against this cell line, with ED50 values ranging from 0.46 to 4.7 µM. The cytotoxic compounds were evaluated against a normal colon cell line, CCD-112CoN. In addition, the new compound perviridicin B (2), three known rocaglate derivatives (9, 11, 12), and a known sesquiterpene, 2-oxaisodauc-5-en-12-al (17), showed significant NF-κB (p65) inhibitory activity in an ELISA assay.

A cytotoxic dimeric furanoheliangolide from Piptocoma rufescens.

A new sesquiterpene lactone, rufescenolide C (1), the first furanoheliangolide dimer, was isolated from the leaves of Piptocoma rufescens, collected in the Dominican Republic. Its structure was determined by analysis of its spectroscopic data, with the absolute configuration being established by analysis of the CD spectrum. A plausible biogenesis of this dimer is proposed. This compound showed potent cytotoxicity with an IC50 value of 150 nM, when tested against HT-29 human colon cancer cells.

Diarylheptanoids from Dioscorea villosa (Wild Yam).

A fractionation methodology aimed at the metabolomic mining of new phytoconstituents for the widely used botanical, wild yam (Dioscorea villosa), makes use of 1D qHNMR and 2D NMR profiles along the preparative fractionation pathway. This quantifiable and structural guidance led to the isolation of 14 diarylheptanoids (1-14), including five new compounds (1-5) with a tetrahydropyrano core skeleton. The structures, including the absolute configurations of both new and previously known diarylheptanoids, were assigned by a combination of HRESIMS, 1D and 2D NMR, (1)H iterative full spin analysis (HiFSA), and Mosher's ester method. The isolation yields were consistent with yields predicted by qHNMR, which confirms the (semi)quantifiable capabilities of NMR-based preparative metabolomic mining. The qHNMR-aided approach enabled the identification of new and potentially significant chemical entities from a small fraction of the plant extract and, thereby, facilitated the characterization of the residual complexity of the D. villosa secondary metabolome. LC-MS profiling of different D. villosa accessions further confirmed that the diarylheptanoids represent genuine secondary metabolites, which can serve as a new class of markers for botanical integrity analysis of D. villosa.