Apoptosis induction and modulation of P-glycoprotein mediated multidrug resistance by new macrocyclic lathyrane-type diterpenoids

The macrocyclic lathyrane diterpenes, latilagascenes D-F (1-3) and jolkinol B (4), were isolated from the methanol extract of Euphorbia lagascae, and evaluated for multidrug resistance reversing activity on mouse lymphoma cells. All compounds displayed very strong activity compared with that of the positive control, verapamil. The structure-activity relationship is discussed. The evaluation of compounds 1 and 4, and of latigascenes A-C (5-7), isolated from the same species, as apoptosis-inducers was also carried out. Compound 1 was the most active. Furthermore, in the model of combination chemotherapy, the interaction between the doxorubicine and latilagascene B (6) was studied in vitro, on human MDR1 gene transfected mouse lymphoma cells, showing that the type of interaction was synergistic. Latilagascenes D-F (1-3) are new compounds whose structures were established on the basis of spectroscopic methods, including 2D NMR experiments (COSY, HMQC, HMBC and NOESY).     

Jatrophane diterpenoids from Euphorbia peplus.

From the pro-inflammatory active extract of Euphorbia peplus, a new diterpene polyester (1) based on the jatrophane skeleton was isolated together with the known compounds 2-5. The irritant activities of some jatrophane diterpenes (2, 3 and 6-9) were also investigated: only compound 2 was found to exert a weak pro-inflammatory activity on mouse ear.     

Euphorbia and Momordica metabolites for overcoming multidrug resistance

Multidrug resistance (MDR) is the major obstacle for cancer chemotherapy. MDR is a multifactorial phenomenon that can result from several mechanisms, including an increased drug efflux, due to overexpression of P-glycoprotein (P-gp) that transports anticancer drugs out of the cells. Thus, the role of this transporter has made it a therapeutic target and the development of P-gp modulators considered among the most realistic approaches for overcoming P-gp-mediated MDR. Many other strategies have been proposed. One of them is the identification of compounds that selectively kill multidrug resistant cells. In our search for MDR modulators from plants, the P-gp inhibition ability of a large number of compounds on resistant cancer cells was evaluated. These compounds, presented in this review, comprise mainly diterpenes, triterpenes and phenolic derivatives. The most relevant results were obtained from two sets of compounds: macrocyclic diterpenes with the jatrophane and lathyrane scaffold, and triterpenes of the cucurbitane-type isolated from Euphorbia species and Momordica balsamina L., respectively. Additionally, some of those macrocyclic diterpenes, and ent-abietane diterpenic lactones, also isolated from Euphorbia species, were found to be selectively toxic to drug-resistant phenotypes.     

A comparative phytochemical study of the diterpenes of some species of the genera Euphorbia and Elaeophorbia (Euphorhiaceae)

Nearly 60 species from the genera Euphorbia L. and Elaeophorbia Stapf were investigated for diterpenes of the classes tiglianes, ingenanes and ortho-esters. The diterpenes were isolated as their acetates by a micro-technique from latex or fresh herb material collected from several countries around the world. Authentication of the diterpenes was by chromatographic means using spectroscopic methods as back-up data. These compounds were absent from only 9% of the species examined. The most commonly occurring diterpene was ingenol, followed closely by 12-Deoxy phorbol. The ingenane derivative 5-Deoxy-ingenol was always detected as a minor companion to ingenol from species of the section Tithymalus. Species of the section Euphorbium contained mainly the diterpene ingenol although both tigliane and ortho-ester diterpenes were also present in some species. Species from the sections Anisophylhtm and Poinsettia did not contain diterpenes of the type in question, whilst species from the genus Elaeophorbia yielded ingenol. These results provide additional chemical evidence concerning recent suggestions on the subgeneric and generic status of Anisophyllum, Poinsettia, Tithymalus and Elaeophorbia.     

Chemosystematic investigations of irregular diterpenes in Anisotome and related New Zealand Apiaceae

A chemosystematic HPLC-UV and HPLC-MS investigation of New Zealand members of the Apiaceae was performed. Diterpenes were identified and quantified in methanolic extracts from subaerial parts of 28 taxa and 54 samples of Aciphylla, Anisotome, Apium, Gingidia, Lignocarpa, Oreomyrrhis, and Scandia. Six diterpenes (1-2, 4-7) and four polyacetylenes (8-11) were identified. The known compounds were the diterpenes anisotomenoic acid 1, anisotomene-1-ol 2, 16-acetoxyanisotomenoic acid 4 and anisotomene-1,12-diol 5; and the polyacetylenes falcarinol 8, falcarindiol 9, (+)-9(Z),17-octadecadiene-12,14-diyne-1,11,16-triol 10, and (+)-9(Z),17-octadecadiene-12,14-diyne-1,11,16-triol 1-acetate 11. New irregular diterpenes 13,14-dihydroanisotom-12E-ene-1,14-diol 6 and 14-methoxy-13,14-dihydroanisotom-12E-ene-1-ol 7 were isolated from A. haastii. Isomers of the new semi-synthetic diterpene 16-hydroxyanisotomenoic acid 3 were detected in extracts of Anisitone flexuosa. Structure elucidation was performed by HR mass spectrometry and 1D and 2D NMR spectroscopy. In crude extracts, compounds were identified by their HPLC retention times and their on-line HPLC-UV and MS spectra. Anisotomene diterpenes occurred in eight out of 16 species of the genus Anisotome, but were not detected in any of the other genera. In contrast, polyacetylenes were present in all the genera investigated.     

Labdane diterpenes from Otostegia fruticosa

The new labdane diterpenes otostegin A (2), otostegin B (6) and 15-epi-otostegin B (7) were isolated from the aerial parts of Otostegia. fruticosa, besides the previously known labdanes preleoheterin (1), leoheterin (3), leopersin C and 15-epi-leopersin C (4, 5), ballonigrin (9) and vulgarol (11), along with the iridoid glucoside 8-O-acetylharpagide (10). The structure elucidation of all the isolated compounds was based on their spectral data and chemical derivatization.     

Cloning of casbene and neocembrene synthases from Euphorbiaceae plants and expression in Saccharomycescerevisiae

A large number of diterpenes have been isolated from Euphorbiaceae plants, many of which are of interest due to toxicity or potential therapeutic activity. Specific Euphorbiaceae diterpenes of medical interest include the latent HIV-1 activator prostratin (and related 12-deoxyphorbol esters), the analgesic resiniferatoxin, and the anticancer drug candidate ingenol 3-angelate. In spite of the large number of diterpenes isolated from these plants and the similarity of their core structures, there is little known about their biosynthetic pathways. Other than the enzymes involved in gibberellin biosynthesis, the only diterpene synthase isolated to date from the Euphorbiaceae has been casbene synthase, responsible for biosynthesis of a macrocyclic diterpene in the castor bean (Ricinus communis). Here, we have selected five Euphorbiaceae species in which to investigate terpene biosynthesis and report on the distribution of diterpene synthases within this family. We have discovered genes encoding putative casbene synthases in all of our selected Euphorbiaceae species and have demonstrated high-level casbene production through expression of four of these genes in a metabolically engineered strain of Saccharomyces cerevisiae. The only other diterpene synthase found among the five plants was a neocembrene synthase from R. communis (this being the first report of a neocembrene synthase gene). Based on the prevalence of casbene synthases, the lack of other candidates, and the structure of the casbene skeleton, we consider it likely that casbene is the precursor to a large number of Euphorbiaceae diterpenes. Casbene production levels of 31 mg/L were achieved in S. cerevisiae and we discuss strategies to further increase production by maximizing flux through the mevalonate pathway.     

Paraliane and pepluane diterpenes as anti-inflammatory agents: first insights in structure-activity relationships

Two new diterpenes, named paralianone (2) and pepluene (3), based, respectively, on rare paraliane and pepluane skeletons, have been isolated from Euphorbia paralias, together with two known analogues (4 and 5), and their stereostructure determined by spectroscopic methods. The isolated compounds were tested as anti-inflammatory agents in vitro for evaluating their ability to inhibit the nitric oxide production in LPS-stimulated J774 murine macrophages. Compound 4 showed the highest anti-inflammatory activity comparable to those recently discovered for pepluanone (1). The data obtained provided first insights towards the structure-activity relationship of this class of compounds highlighting the key role of D-ring structure.     

Diterpenoids from the fruits of Vitex trifolia

An abietane-type diterpene, named vitetrifolin A, and two labdane-type diterpenes, named vitetrifolins B and C, were isolated from the acetone extract of the fruits of Vitex trifolia L. (Viticis Fructus; Verbenaceae) along with three known diterpenes, rotundifuran, dihydrosolidagenone and abietatriene 3beta-ol. The structures of these compounds were elucidated on the basis of spectroscopic analysis, X-ray crystallographic analysis and chemical evidence.     

Macrocyclic diterpene esters of the cytotoxic fraction from Euphorbia kamerunica

The ether-soluble resin of the latex of Euphorbia kamerunica was separated into a non-polar and a polar fraction by gradient elution column chromatography. From the non-polar fraction three macrocyclic diterpene esters were isolated by adsorption and partition preparative TLC methods. These compounds were identified as 3,7,8-triacetyl-ingol-12-tigliate,3,7-diacetyl-ingol-12-tigliate and ingol-tetra-acetate by means of spectroscopic data, together with hydrolysis and partial synthesis.     

Minor labdane diterpenes from Marrubium thessalum

Five new labdane diterpenes, thessalines A, B, and D, 14β-hydroxythessaline A, and 14β-hydroxythessaline B (1-5, resp.) were isolated from the aerial parts of Marrubium thessalum, along with the known labdane diterpene deacetylvitexilactone (6) and the methoxylated flavones 4',7-dimethylapigenin and salvigenin. (3S,5R)-Loliolide was also found in the same source. Their structures were established by 1D- and 2D-NMR (COSY, HSQC, HMBC, NOESY, and ROESY) and MS analyses. The plant produces a great variety of labdane-type diterpenes, with variations in functionalities, particularly in the side chain. Their structures could be of chemotaxonomic significance for the genus Marrubium.     

Terpenes from Euphorbia antiquorum and Their in Vitro Anti‐HIV Activity

Three new compounds ( 1 – 3 ), including two euphane type triterpenes, 24,24‐dimethoxy‐25,26,27‐trinoreuphan‐3β‐ol ( 1 ) and (24S)‐24‐hydroperoxyeupha‐8,25‐dien‐3β‐ol ( 2 ), and an ent‐atisine diterpene, ent‐atisane‐3α,16α,17‐triol ( 3 ), were isolated from an acetone extract of the stems of Euphorbia antiquorum, together with eight known diterpenes ( 4 – 11 ). The structures of compounds ( 1 – 11 ) were elucidated using NMR and MS spectroscopic methods. Compound 7 showed moderate activity against HIV‐1 replication in vitro (EC₅₀ = 1.38 μm ).     

Antinematodal activities of ingenane diterpenes from Euphorbia kansui and their derivatives against the pine wood nematode (Bursaphelenchus xylophilus)

Under the bioassay-guided method, two diterpenes, 3-O-(2",3"-dimethylbutanoyl)-13-O-dodecanoylingenol (1) and 3-O-(2",3"-dimethylbutanoyl)-13-O-decanoylingenol (2) isolated from Euphorbia kansui, showed a pronounced antinematodal activity against the nematode Bursaphelenchus xylophilus at the same minimum effective dose (MED) of 5 microg per cotton ball and still displayed antinematodal activity at a dose of 2.5 microg per cotton ball. Compounds 3-6 were obtained, and the structure of the new compound 6 was elucidated based on 1D- and 2D-NMR analyses and physicochemical data. Preliminary structure-biological activity relationships of ingenane-type compounds were deduced.     

Jatrophane diterpenes from Euphorbia spp. as modulators of multidrug resistance in cancer therapy

A phytochemical investigation of the aerial parts of Euphorbia spp., considered one of the most common elements of Mediterranean landascape, led to the isolation of a large number of bioactive plant metabolites, belonging to the diterpenes family. Above all, over seventy jatrophane, modified jatrophane, segetane, pepluane, and paraliane diterpenoids, fifty of them reported for the first time, were extracted, purified and characterized from Euphorbia dendroides, Euphorbia characias, Euphorbia peplus, Euphorbia paralias and Euphorbia helioscopia. These compounds showed interesting pharmacological activities. In particular, jatrophanes, modified jatrophanes and lathyranes exhibited a potent inhibitory activity against P-glycoprotein (Pgp), a membrane protein that confers cellular ability to resist lethal doses of certain cytotoxic drugs by pumping them to the outside, thus resulting in a reduced cytotoxicity. Among the others, our chemical survey led to isolation of the most powerful inhibitors of daunomycin-efflux activity isolated to date for this class of inhibitors, named euphodendroidin D and pepluanin A. Their efficiency was found to be at least two-fold higher than the conventional modulator cyclosporin A, taken as a reference. In addition, the isolation of a high number of natural structurally-related analogues allowed us to perform Structure Activity Relationship (SAR) studies, without any chemical modification, which gave information on the key pharmacophoric elements of these new class of promising drugs. A further set of diterpene analogues, very recently isolated from sun spurge, E. helioscopia, individually investigated for their Pgp- and BCRP-inhibiting properties, appeared to be specific inhibitors of Pgp since they showed no significant activity against BCRP, thus resembling to the third-generation class of specific MDR inhibitors.     

Gut microbiota of the pine weevil degrades conifer diterpenes and increases insect fitness

The pine weevil (Hylobius abietis), a major pest of conifer forests throughout Europe, feeds on the bark and cambium, tissues rich in terpenoid resins that are toxic to many insect herbivores. Here, we report the ability of the pine weevil gut microbiota to degrade the diterpene acids of Norway spruce. The diterpene acid levels present in ingested bark were substantially reduced on passage through the pine weevil gut. This reduction was significantly less upon antibiotic treatment, and supplementing the diet with gut suspensions from untreated insects restored the ability to degrade diterpenes. In addition, cultured bacteria isolated from pine weevil guts were shown to degrade a Norway spruce diterpene acid. In a metagenomic survey of the insect's bacterial community, we were able to annotate several genes of a previously described diterpene degradation (dit) gene cluster. Antibiotic treatment disrupted the core bacterial community of H. abietis guts and eliminated nearly all dit genes concordant with its reduction in diterpene degradation. Pine weevils reared on an artificial diet spiked with diterpenes, but without antibiotics, were found to lay more eggs with a higher hatching rate than weevils raised on diets with antibiotics or without diterpenes. These results suggest that gut symbionts contribute towards host fitness, but not by detoxification of diterpenes, as these compounds do not show toxic effects with or without antibiotics. Rather the ability to thrive in a terpene‐rich environment appears to allow gut microbes to benefit the weevil in other ways, such as increasing the nutritional properties of their diet.     

ent-Rosane and abietane diterpenoids as cancer chemopreventive agents

Two ent-rosane- (cuzcol, 1 and 6-dehydroxycuzcol, 2) and a abietatriene- (salvadoriol, 3) type diterpenoids have been isolated from Maytenus cuzcoina and Crossopetalum uragoga, respectively, along with five known diterpene compounds (4-8). Their stereostructures have been elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques, and computational data. The absolute configuration of cuzcol was determined by application of Riguera ester procedure. This is the first instance of isolation of ent-rosane diterpenoids from species of the Celastraceae. The isolated diterpenes were found to be potent anti-tumour-promoter agents, and carnosol (7) also showed a remarkable chemopreventive effect in an in vivo two-stage carcinogenesis model.     

Four new sacculatane-type diterpenoids from Trichocoleopsis sacculata and Pellia endiviaefolia

The characteristic pungency of the liverwort, Trichocoleopsis sacculata is due to the mixture of three sacculatane-type diterpene dialdehydes. Two have new structures, 18-hydroxysacculatal and 19-hydroxysacculatal, and the third is the previously known sacculatal. The absolute configurations of the new diterpene dialdehydes were tentatively shown by the co-occurrence of sacculatal and isosacculatal. Two new non-pungent sacculatanes, 3-hydroxyisosacculatal and 3-hydroxysacculatanolide have been isolated from the liverwort Pellia endiviaefolia together with sacculatal and isosacculatal. The position of the hydroxyl group at C-3 in the new diterpenes was also assumed on biogenetic considerations and a structural analogy with perrottetianal B, a previously known sacculatane-type diterpene dialdehyde isolated from the liverwort.     

Terpenes from Otostegia integrifolia

The essential oil and chloroform extract of air-dried leaves of Otostegia integrifolia Benth. were investigated for the first time using analytical and preparative gas chromatography (GC), GC-mass spectrometry (MS) and NMR techniques. A total of 40 constituents including monoterpenes, sesquiterpenes, diterpenes and their derivatives were identified. A prenylbisabolane type diterpene, 1-methyl-4-(5,9-dimethyl-1-methylene-deca-4,8-dienyl)cyclohexene was identified as a major component. The chloroform extract of the leaves yielded two labdane type diterpenoids, 15,16-epoxy-3alpha,9alpha-dihydroxy-labda-13(16),14-diene and 9(13),15(16)-diepoxy-3alpha-hydroxy-16-dihydrolabda-14-ene, a saturated hydrocarbon, pentatriacontane, and stigmasterol. The structures of the isolated compounds were established by spectroscopic methods.     

A New Harziane Diterpene,Harziaketal A,and a New Sterol,Trichosterol A,from the Marine-Alga-Epiphytic Trichoderma sp. Z43

One new diterpene, harziaketal A ( 1 ), and one new highly degraded sterol, trichosterol A ( 2 ), along with three known compounds, including one diterpene, harzianone ( 3 ), and two steroids, (22E,24R)-5α,6α-epoxy-ergosta-8(14),22-dien-3β,7α-diol ( 4 ) and isoergokonin B ( 5 ), were isolated from the culture of the marine-alga-epiphytic fungus Trichoderma sp. Z43 by silica gel column chromatography (CC), Sephadex LH-20 CC, and preparative thin-layer chromatography (TLC). Their structures and relative configurations were assigned by nuclear magnetic resonance (NMR) and high resolution electrospray ionisation mass spectrometry (HR-ESI-MS) data, and the absolute configuration of 1 was established by X-ray diffraction. Compound 1 features a hemiketal unit situated at the four-membered ring of harziane-type diterpenes for the first time, while 2 represents the rare occurrence of sterols with rings A and B being degraded. Compounds 1 and 2 displayed weak inhibition against the tested phytoplankton (Amphidinium carterae, Heterocapsa circularisquama, Heterosigma akashiwo, and Prorocentrum donghaiense) with half maximal inhibitory concentration (IC₅₀) ranging from 14 to 53 μg/mL.     

Phytotoxic Allelochemicals From Roots and Root Exudates of Leafy Spurge (Euphorbia esula L.)

Invasive plants are a widespread problem but the mechanisms used by these plants to become invasive are often unknown. The production of phytotoxic natural products by invasive weeds is one mechanism by which these species may become successful competitors. Here we conducted a bioactivity-driven fractionation of root extracts and exudates from the invasive plant leafy spurge (Euphorbia esula L.), and structurally characterized jatrophane diterpenes and ellagic acid derivatives. Ellagic acid derivatives and one of the jatrophane diterpenes, esulone A, have been previously reported from leafy spurge, but another of the jatrophane diterpenes, kasuinine B, has not. We show that these compounds are phytotoxic but affect plants in different ways, either inducing overall plant necrosis or reducing root branching and elongation.Key Words: phytotoxicity, allelochemicals, roots, root exudates, jatrophane diterpenes, kansuinine B, ellagic acid derivatives, leafy spurge, Euphorbia esula, Arabidopsis thaliana