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.

Bioactivity-Guided Isolation of Totarane-Derived Diterpenes from Podocarpus neriifolius and Structure Revision of 3-Deoxy-2α-hydroxynagilactone E.

Bioactivity-guided phytochemical investigation of Podocarpus neriifolius D. Don. (Podocarpaceae) has led to the isolation of one new (2) and three known (1, 3, and 4) B-type podolactones, along with three totarane-type diterpenes (5-7). Their structures were determined by interpretation of High Resolution ElectroSpray Ionization Mass Spectrometry (HRESIMS) and 1D and 2D NMR data, and comparison with the values reported in the literature. The structure of compound 1, previously identified as 3-deoxy-2α-hydroxynagilactone E (8), was revised as its 2ß-epimer, which has been reported recently as a new compound. All of the isolates were evaluated for their antiproliferative activity against a panel of four human cancer cell lines, namely, ovarian (OVCAR3), breast (MDA-MB-231), colon (HT-29), and melanoma (MDA-MB-435), and compounds 1 and 3 were found to be cytotoxic with IC50 values in the low micromolar range for most of the cell lines used. The major compound, inumakilactone A (3), was further tested in vivo using the HT-29, MDA-MB-435, and OVCAR3 cells in a murine hollow fiber model, for the first time.

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.

Synthesis and antiproliferative activity of derivatives of the phyllanthusmin class of arylnaphthalene lignan lactones.

A series of arylnaphthalene lignan lactones based on the structure of the phyllanthusmins, a class of potent natural products possessing diphyllin as the aglycone, has been synthesized and screened for activity against multiple cancer cell lines. SAR exploration was performed on both the carbohydrate and lactone moieties of this structural class. These studies have revealed the importance of functionalization of the carbohydrate hydroxy groups with both acetylated and methylated analogues showing increased potency relative to those with unsubstituted sugar moieties. In addition, the requirement for the presence and position of the C-ring lactone has been demonstrated through reduction and selective re-oxidation of the lactone ring. The most potent compound in this study displayed an IC50 value of 18 nM in an HT-29 assay with several others ranging from 50 to 200 nM. In an effort to elucidate their potential mechanism(s) of action, the DNA topoisomerase IIa inhibitory activity of the most potent compounds was examined based on previous reports of structurally similar compounds, but does not appear to contribute significantly to their antiproliferative effects.

Halogenated Compounds from Directed Fermentation of Penicillium concentricum, an Endophytic Fungus of the Liverwort Trichocolea tomentella.

One new chlorinated xanthone, 6-chloro-3,8-dihydroxy-1-methylxanthone (1), a new 2-bromo-gentisyl alcohol (2), and a mixture of 6-epimers of 6-dehydroxy-6-bromogabosine C (3a and 3b), together with 19 previously identified compounds, epoxydon (4), norlichexanthone (5), 2-chlorogentisyl alcohol (6), hydroxychlorogentisyl quinone (7), 6-dehydroxy-6α-chlorogabosine C (8a), 6-dehydroxy-6ß-chlorogabosine C (8b), gentisyl alcohol (9), gentisyl quinone (10), (R,S)-1-phenyl-1,2-ethanediol (11), dehydrodechlorogriseofulvin (12), dechlorogriseofulvin (13), dehydrogriseofulvin (14), griseofulvin (15), ethylene glycol benzoate (16), alternariol (17), griseoxanthone C (18), drimiopsin H (19), griseophenone C (20), and griseophenone B (21), were isolated from cultures of Penicillium concentricum, a fungal endophyte of the liverwort Trichocolea tomentella. The structures of the new compounds (1, 2, 3a, and 3b) were elucidated by interpretation of spectroscopic data including one- and two-dimensional NMR techniques. Among these, compounds 2-4 displayed modest cytotoxicity to the MCF-7 hormone-dependent breast cancer cell line with IC50 values of 8.4, 9.7, and 5.7 µM, respectively, whereas compound 9 exhibited selective cytotoxicity against the HT-29 colon cancer cell line with an IC50 value of 6.4 µM. During this study we confirmed that the brominated gentisyl alcohol (2) was formed by chemical conversion of 4 during bromide salt addition to culture media.

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.

Cyclopenta[b]benzofuran and Secodammarane Derivatives from the Stems of Aglaia stellatopilosa.

Four new 2,3-secodammarane triterpenoids, stellatonins A-D (3-6), together with a new 3,4-secodammarane triterpenoid, stellatonin E (7), and the known silvestrol (1), 5‴-episilvestrol (2), and ß-sitosterol, were isolated from a methanol extract of the stems of Aglaia stellatopilosa through bioassay-guided fractionation. The structures of the new compounds were elucidated using spectroscopic and chemical methods. The compounds were evaluated for their cytotoxic activity against three human cancer cell lines and for their antimicrobial activity using a microtiter plate assay against a panel of Gram-positive and Gram-negative bacteria and fungi.

Cytotoxic Barrigenol-like Triterpenoids from an Extract of Cyrilla racemiflora Housed in a Repository.

Two new [(+)-cyrillins A (1) and B (2)] and four known barrigenol-like triterpenoids (3-6), along with betulinic acid and (+)-3ß-O-trans-feruloylbetulinic acid, were isolated from a sample-restricted CH2Cl2-soluble extract of the bark of Cyrilla racemiflora, collected in Dominica. The structures of the new compounds were elucidated by interpretation of their spectroscopic data, and the absolute configuration of the cyclic 1,2-diol unit of (+)-cyrillin A (1) was ascertained by analysis of the electronic circular dichroism (ECD) spectrum induced with [Mo2(OAc)4]. In the case of (+)-cyrillin B (2), which was found to contain a diangeloylated glucose residue, the structure proposed was supported by analysis of its MS(2) and MS(3) spectra. All compounds isolated were evaluated for their cytotoxicity against HT-29 human colon cancer cells, and the known compound, (+)-barringtogenol B (3), was found to be the most potent, exhibiting an IC50 value of 1.7 µM. This compound also showed inhibitory activity toward the CCD-112CoN human normal colon cell line, with an IC50 value of 5.9 µM, indicating a lack of cytotoxic selectivity.

Antiproliferative Constituents of the Roots of Ethiopian Podocarpus falcatus and Structure Revision of 2α-Hydroxynagilactone F and Nagilactone I.

Bioassay-guided fractionation using the human colorectal adenocarcinoma (HT-29) cell line of the methanol extract of dried roots of Podocarpus falcatus led to the isolation of two new type C nagilactones, 16-hydroxynagilactone F (1) and 2ß,16-dihydroxynagilactone F (2), and the new totarane-type bisditerpenoid 7ß-hydroxymacrophyllic acid (4), along with the seven known compounds 2ß-hydroxynagilactone F (3), macrophyllic acid (5), nagilactone D (6), 15-hydroxynagilactone D (7), nagilactone I (8), inumakiol D (9), and ponasterone A (10). The structures of the new compounds were determined by 1D and 2D NMR, HRESIMS, UV, and IR and by comparison with the reported spectroscopic data of their congeners. The orientation of the C-2 hydroxy group of 3 and 8 was revised to be ß based on evidence from detailed analysis of 1D and 2D NMR data and single-crystal X-ray diffraction studies. Among the isolated compounds, the nagilactones, including the new dilactones 16-hydroxynagilactone F (1) and 2ß,16-dihydroxynagilactone F (2), were the most active (IC50 0.3-5.1 µM range) against the HT-29 cell line, whereas the bisditerpenoids (4 and 5) and the other known compounds 9 and 10 were inactive. The presence of the bioactive nagilactones in P. falcatus supports its traditional use.

Cytotoxic and natural killer cell stimulatory constituents of Phyllanthus songboiensis.

A dichapetalin-type triterpenoid and a dibenzylbutyrolactone-type lignan, together with five known lignans, a known aromatic diterpenoid, and a known acylated phytosterol, were isolated from the aerial parts of Phyllanthus songboiensis, collected in Vietnam. Their structures were determined by interpretation of the spectroscopic data, and the inhibitory activity toward HT-29 human colon cancer cells of all isolates was evaluated by a cytotoxicity assay. The known arylnaphthalene lignan, (+)-acutissimalignan A, was highly cytotoxic toward HT-29 cells, with an IC50 value of 19 nM, but this compound was inactive as a DNA topoisomerase IIα (topo IIα) poison. The known phytosterol, (-)-ß-sitosterol-3-O-ß-D-(6-O-palmitoyl)glucopyranoside, was found to stimulate natural killer (NK) cells at a concentration of 10µM in the presence of interleukin 12 (IL-12).

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.

Caeruleanone A, a rotenoid with a new arrangement of the D-ring from the fruits of Millettia caerulea.

Caeruleanone A (1), a novel rotenoid with an unprecedented arrangement of the D-ring, was isolated with another two new analogues, caeruleanones B (2) and C (3), together with 11 known rotenoids from the fruits of Millettia caerulea. The structures of the new compounds were determined by spectroscopic data analysis, with that of 1 being confirmed by single-crystal X-ray diffraction. Compounds 2 and 3 displayed potent mitochondrial transmembrane potential inhibitory and quinone reductase induction activities.

Constituents of an extract of Cryptocarya rubra housed in a repository with cytotoxic and glucose transport inhibitory effects.

A new alkylated chalcone (1), a new 1,16-hexadecanediol diester (2), and eight known compounds were isolated from a dichloromethane-soluble repository extract of the leaves and twigs of Cryptocarya rubra collected in Hawaii. The structures of the new compounds were determined by interpretation of their spectroscopic data, and the absolute configurations of the two known cryptocaryanone-type flavonoid dimers, (+)-bicaryanone A (3) and (+)-chalcocaryanone C (4), were ascertained by analysis of their electronic circular dichroism and NOESY NMR spectra. All compounds isolated were evaluated against HT-29 human colon cancer cells, and, of these, (+)-cryptocaryone (5) was found to be potently cytotoxic toward this cancer cell line, with an IC50 value of 0.32 µM. This compound also exhibited glucose transport inhibitory activity when tested in a glucose uptake assay.

Bioactive constituents of Indigofera spicata.

Four new flavanones, designated as (+)-5″-deacetylpurpurin (1), (+)-5-methoxypurpurin (2), (2S)-2,3-dihydrotephroglabrin (3), and (2S)-2,3-dihydrotephroapollin C (4), together with two known flavanones (5 and 6), three known rotenoids (7-9), and one known chalcone (10) were isolated from a chloroform-soluble partition of a methanol extract from the combined flowers, fruits, leaves, and twigs of Indigofera spicata, collected in Vietnam. The compounds were obtained by bioactivity-guided isolation using the HT-29 human colon cancer, 697 human acute lymphoblastic leukemia, and Raji human Burkitt's lymphoma cell lines. The structures of 1-4 were established by extensive 1D- and 2D-NMR experiments, and the absolute configurations were determined by the measurement of specific rotations and CD spectra. The cytotoxic activities of the isolated compounds were tested against the HT-29, 697, Raji, and CCD-112CoN human normal colon cells. Also, the quinone reductase induction activities of the isolates were determined using the Hepa 1c1c7 murine hepatoma cell line. In addition, cis-(6aß,12aß)-hydroxyrotenone (7) was evaluated in an in vivo hollow fiber bioassay using HT-29, MCF-7 human breast cancer, and MDA-MB-435 human melanoma cells.

Alkaloids from Microcos paniculata with cytotoxic and nicotinic receptor antagonistic activities.

Microcos paniculata is a large shrub or small tree that grows in several countries in South and Southeast Asia. In the present study, three new piperidine alkaloids, microgrewiapines A-C (1-3), as well as three known compounds, inclusive of microcosamine A (4), 7'-(3',4'-dihydroxyphenyl)-N-[4-methoxyphenyl)ethyl]propenamide (5), and liriodenine (6), were isolated from cytotoxic fractions of the separate chloroform-soluble extracts of the stem bark, branches, and leaves of M. paniculata. Compounds 1-6 and 1a (microgrewiapine A 3-acetate) showed a range of cytotoxicity values against the HT-29 human colon cancer cell line. When evaluated for their effects on human α3ß4 or α4ß2 nicotinic acetylcholine receptors (nAChRs), several of these compounds were shown to be active as nAChR antagonists. As a result of this study, microgrewiapine A (1) was found to be a selective cytotoxic agent for colon cancer cells over normal colon cells and to exhibit nicotinic receptor antagonistic activity for both the hα3ß4 and hα4ß2 receptor subtypes.

Discovery of new anticancer agents from higher plants.

Small organic molecules derived from higher plants have been one of the mainstays of cancer chemotherapy for approximately the past half a century. In the present review, selected single chemical entity natural products of plant origin and their semi-synthetic derivatives currently in clinical trials are featured as examples of new cancer chemotherapeutic drug candidates. Several more recently isolated compounds obtained from plants showing promising in vivo biological activity are also discussed in terms of their potential as anticancer agents, with many of these obtained from species that grow in tropical regions. Since extracts of only a relatively small proportion of the ca. 300,000 higher plants on earth have been screened biologically to date, bioactive compounds from plants should play an important role in future anticancer drug discovery efforts.

Phytochemical and Bioactivity Studies on Constituents of the Leaves of Vitex Quinata.

A phytochemical investigation of the leaves of Vitex quinata (Lour.) F.N. Williams (Verbenaceae), guided by the MCF-7 human breast cancer cell line, led to the isolation of a new δ-truxinate derivative (1) and a new phytonoic acid derivative (2), together with 12 known compounds. The structures of the new compounds were determined by spectroscopic methods as dimethyl 3,4,3',4'-tetrahydroxy-δ-truxinate (1) and methyl 10R-methoxy-12-oxo-9(13),16E-phytodienoate (2), respectively. In a cytotoxicity assay, (S)-5-hydroxy-7,4'-dimethoxyflavanone (3) was found to be the sole active principle, with ED(50) values of 1.1-6.7 µM, respectively, when tested against a panel of three human cancer cells. Methyl 3,4,5-O-tricaffeoyl quinate (4) showed activity in an enzyme-based ELISA NF-κB p65 assay, with an ED(50) value of 10.3 µM.

The classical drug discovery approach to defining bioactive constituents of botanicals.

In this review, several recently identified biologically active principles of selected botanical dietary supplement ingredients are described, and were isolated using classical phytochemical chromatographic methods, with various spectroscopic procedures used for their isolation and structure elucidation. A central component of such an approach is "activity-guided fractionation" to monitor the compound purification process. In vitro assays germane to cancer chemoprevention were used to facilitate the work performed. Bioactive compounds, including several new substances, were characterized from açai (Euterpe oleracea), baobab (Adansonia digitata), licorice (Glycyrrhiza glabra), mangosteen (Garcinia mangostana), and noni (Morinda citrifolia). Many of these compounds exhibited quite potent biological activity, but tended to be present in their plant of origin only at low concentration levels.