Rh(I)-Catalyzed Allenic Pauson-Khand Reaction to Access the Thapsigargin Core: Influence of Furan and Allenyl Chloroacetate Groups on Enantioselectivity.

Thapsigargin (Tg) is a potent SERCA pump inhibitor with the potential to treat cancer and COVID-19. We have extended the scope of the asymmetric allenic Pauson-Khand reaction to furan-tethered allene-ynes, a stereoconvergent transformation affording the 5,7,5-ring system of Tg in good yields and high enantioselectivity. Computational studies of the oxidative cyclization step show that the furan and chloroacetate groups contribute to this high selectivity.

Thapsigargin-From Traditional Medicine to Anticancer Drug.

A sesquiterpene lactone, thapsigargin, is a phytochemical found in the roots and fruits of Mediterranean plants from Thapsia L. species that have been used for centuries in folk medicine to treat rheumatic pain, lung diseases, and female infertility. More recently thapsigargin was found to be a potent cytotoxin that induces apoptosis by inhibiting the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pump, which is necessary for cellular viability. This biological activity encouraged studies on the use of thapsigargin as a novel antineoplastic agent, which were, however, hampered due to high toxicity of this compound to normal cells. In this review, we summarized the recent knowledge on the biological activity and molecular mechanisms of thapsigargin action and advances in the synthesis of less-toxic thapsigargin derivatives that are being developed as novel anticancer drugs.

Thapsigargin induces apoptosis in adrenocortical carcinoma by activating endoplasmic reticulum stress and the JNK signaling pathway: an in vitro and in vivo study.

OBJECTIVE: Thapsigargin (TG) is a natural product that exists in most parts of the plant Thapsia garganica L. and possesses potential anticancer activities against variety tumor cell lines. TG induces endoplasmic reticulum (ER) stress and apoptosis by inhibiting cancer growth. However, the antineoplastic effect of TG in human adrenocortical carcinoma (ACC) cells is still unknown. METHODS: In this study, two human ACC cell lines including SW-13 and NCI-H295R were employed to explore the potential role of TG in ACC. A mouse xenograft model of SW-13 cells was established to verify the role of TG in vivo. The cell viability was tested using Cell Counting Kit-8 and Transwell assays. Flow cytometry and Hoechst 33,258 staining were employed to analyze cell apoptosis. RT-qPCR and Western blot (WB) were performed to explore the underlying mechanism of TG-induced apoptosis in ACC cells. RESULTS: The results indicated that TG dose-dependently inhibited proliferation, migration and invasion in human ACC cells. TG significantly increased the mitochondrial rate of apoptosis and ER stress activity in ACC cells and suppressed ACC xenograft growth in vivo. In addition, the expression of Jun N-terminal kinase (JNK) signaling-related genes and proteins was upregulated by the treatment with TG. CONCLUSION: Our findings suggest that TG inhibits the viability of ACC cells by inducing apoptosis through the activation of JNK signaling. Thus, TG is expected to be a potential candidate for the treatment of ACC.

Seeds, Leaves and Roots of Thapsia garganica as a Source of New Potent Lipases Inhibitors: In vitro and In silico Studies.

BACKGROUND AND OBJECTIVE: Lipase inhibitors have gained great interest because they could help in the therapy of many diseases, however, unfortunately, only a few drugs are currently available on the market. Therefore, the aim of this work was to evaluate for the first time the lipase inhibition effect of Thapsia garganica extracts from seeds, leaves and roots. METHODS: Polyphenols and flavonoids contents were determined using spectrophotometric method. Inhibitory activity of ethyl acetate extracts from seeds, leaves and roots of T. garganica against Candida rugosa lipase was determined. To uncover the active constituents responsible for this anti-lipase activity, further investigations were performed by employing theoretical docking simulations, using AutoDock Vina program to discuss the nature of interactions and the inhibition mechanism by major bioactive compounds synthesized by this plant. RESULTS: Seeds, leaves and roots extracts of T. garganica showed appreciable contents of polyphenols and flavonoids which is most in seeds extract with 2.90±0.02mg GAE/gdw and 1.53±0.05mg QE/gdw, respectively. Hence, their inhibitory activities against Candida rugosa lipase were determined as IC50 of 1.19mg/ml, 1.96mg/ml and 1.87mg/ml, respectively. Docking simulations have shown that nortribolid and tribolid are best inhibitors for both lipases (Candida rugosa and human pancreatic lipases). CONCLUSION: Testing the anti-lipase activity of the ethyl acetate extracts of T. garganica revealed a potent lipase inhibition activity, which suggests the use of these molecules as anti-obesity drugs.

Antifungal Activity of Thapsia villosa Essential Oil against Candida, Cryptococcus, Malassezia, Aspergillus and Dermatophyte Species.

The composition of the essential oil (EO) of Thapsia villosa (Apiaceae), isolated by hydrodistillation from the plant's aerial parts, was analysed by GC and GC-MS. Antifungal activity of the EO and its main components, limonene (57.5%) and methyleugenol (35.9%), were evaluated against clinically relevant yeasts (Candida spp., Cryptococcus neoformans and Malassezia furfur) and moulds (Aspergillus spp. and dermatophytes). Minimum inhibitory concentrations (MICs) were measured according to the broth macrodilution protocols by Clinical and Laboratory Standards Institute (CLSI). The EO, limonene and methyleugenol displayed low MIC and MFC (minimum fungicidal concentration) values against Candida spp., Cryptococcus neoformans, dermatophytes, and Aspergillus spp. Regarding Candida species, an inhibition of yeast-mycelium transition was demonstrated at sub-inhibitory concentrations of the EO (MIC/128; 0.01 µL/mL) and their major compounds in Candida albicans. Fluconazole does not show this activity, and the combination with low concentrations of EO could associate a supplementary target for the antifungal activity. The association of fluconazole with T. villosa oil does not show antagonism, but the combination limonene/fluconazole displays synergism. The fungistatic and fungicidal activities revealed by T. villosa EO and its main compounds, associated with their low haemolytic activity, confirm their potential antimicrobial interest against fungal species often associated with human mycoses.

Phytochemical composition and antioxidant activity of medicinal plants collected from the Tunisian flora.

Polyphenols, flavonoids and condensed tannins contents, as well as the antioxidant activity of the ethanolic and aqueous extracts obtained from aerial parts of 10 wild Tunisian plants, have been determined. Extracts showed appreciable levels of polyphenols and flavonoids, which reached 215.16 mg GAE g-1 DW in Lavandula stoechas ethanolic extract, and 49.12 mg RE g-1 DW in Thapsia garganica aqueous extract. The majority of tested extracts exhibited low total condensed tannins content, except for Rhus tripartitum and Periploca laevigata. The antioxidant activity tests showed great activity, especially for R. tripartitum and Lavandula multifida (IC50 = 5.16 and 5.1 µg mL-1, respectively). Canonical Correspondence Analysis revealed clear groupings of species according to the solvent used.

Chemical composition of the essential oil from Thapsia garganica L. (Apiaceae) grown wild in Sicily and its antimicrobial activity.

In this study, the chemical composition of the essential oil from flowers and leaves of Thapsia garganica L. collected in Sicily was evaluated by GC and GC-MS. The main components of T. garganica flower oil (T.f.) were chamazulene (58.3%), humulene oxide II (9.0%), tricosane (8.2%) and pentacosane (8.2%). Also the oil from leaves (T.l.) was characterised by high content of chamazulene (49.2%). Other abundant metabolites were 1,4-dimethylazulene (18.5%), (E)-phytol (6.3%) and neophytadiene (5.1%). The comparison with other studied oils of genus Thapsia is discussed. Antimicrobial activity against several micro-organisms, including some ones infesting historical art craft, was also determined.

Chemo- and Regioselective Functionalization of Nortrilobolide: Application for Semisynthesis of the Natural Product 2-Acetoxytrilobolide.

The difference in reactivity of the hexaoxygenated natural product thapsigargin (1) and the pentaoxygenated nortrilobolide (3) was compared in order to develop a chemo- and regioselective method for the conversion of nortrilobolide (3) into the natural product 2-acetoxytrilobolide (4). For the first time, a stereoselective synthesis of 2-acetoxytrilobolide (4) is described, which involves two key reactions: the first chemical step was a one-pot substitution-oxidation reaction of an allylic ester into its corresponding α,ß-unsaturated ketone. The second process consisted of a stereoselective α'-acyloxylation of the key intermediate α,ß-unsaturated ketone to afford its corresponding acetoxyketone, which was converted into 2-acetoxytrilobolide (4) in a few steps. This innovative approach would allow the synthesis of a broad library of novel and valuable penta- and hexaoxygenated guaianolides as potential anticancer agents.

Chemodiversity of volatile oils in Thapsia garganica L. (Apiaceae).

The chemical composition of the volatile oils obtained from the roots, leaves, flowers, and stems of Thapsia garganica of Tunisian origin was investigated by GC-FID and GC/MS analyses. Sesquiterpene hydrocarbons and oxygenated monoterpenes were predominant in the oils of all plant parts. Bicyclogermacrene (21.59-35.09%) was the main component in the former compound class, whereas geranial (3.31-14.84%) and linalool (0.81-10.9%) were the most prominent ones in the latter compound class. Principal-component (PCA) and hierarchical-cluster (HCA) analyses revealed some common constituents, but also significant variability amongst the oils of the different plant parts. This organ-specific oil composition was discussed in relation to their biological and ecological functions. For the evaluation of the intraspecific chemical variability in T. garganica, the composition of the flower volatile oils from four wild populations was investigated. Bicyclogermacrene, linalool, and geranial were predominant in the oils of three populations, whereas epicubenol, ß-sesquiphellandrene, and cadina-1,4-diene were the most prominent components of the oil of one population. PCA and HCA allowed the separation of the flower oils into three distinct groups, however, no relationship was found between the volatile-oil composition and the geographical distribution and pedoclimatic conditions of the studied populations.

Thapsigargin--from Thapsia L. to mipsagargin.

The sesquiterpene lactone thapsigargin is found in the plant Thapsia garganica L., and is one of the major constituents of the roots and fruits of this Mediterranean species. In 1978, the first pharmacological effects of thapsigargin were established and the full structure was elucidated in 1985. Shortly after, the overall mechanism of the Sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibition that leads to apoptosis was discovered. Thapsigargin has a potent antagonistic effect on the SERCA and is widely used to study Ca2+-signaling. The effect on SERCA has also been utilized in the treatment of solid tumors. A prodrug has been designed to target the blood vessels of cancer cells; the death of these blood vessels then leads to tumor necrosis. The first clinical trials of this drug were initiated in 2008, and the potent drug is expected to enter the market in the near future under the generic name Mipsagargin (G-202). This review will describe the discovery of the new drug, the on-going elucidation of the biosynthesis of thapsigargin in the plant and attempts to supply the global market with a novel potent anti-cancer drug.

Large scale purification of the SERCA inhibitor thapsigargin from Thapsia garganica L. roots using centrifugal partition chromatography.

Thapsigargin (Tg) is a selective and irreversible inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)-dependent pump at subnanomolecular concentrations. As such, it has become a powerful tool in the study of Ca(2+) signaling pathway. Purification of Tg from Thapsia species requires repeated chromatographic steps with normal-phase alumina or silica and reverse phase chromatography. We thus developed an innovative procedure coupling high pressure automatized extraction with centrifugal partition chromatography allowing a fast and safe large-scale isolation of highly pure Tg, in two steps from Thapsia garganica L. roots. Comparison of influence of extraction procedures, storage conditions and harvesting areas on Tg content in different Algerian specimens of Thapsia garganica L. roots has been precised by mean of HPLC quantification procedure. Highest Tg content were found in the fresh material of the sample from Setif area.

Inhibition of thrombin-induced Ca²âº influx in platelets by R59949, an inhibitor of diacylglycerol kinase.

OBJECTIVES: The aim of this study was to determine whether diacylglycerol kinase (DGK) is involved in transplasmalemmal Ca²âº influx of platelets. METHODS: Effects of R59949, an inhibitor of diacylglycerol kinase, on intracellular Ca²âº concentration ([Ca²âº](i) ) and mRNA expression of DGK isozymes were investigated using washed human platelet suspensions. KEY FINDINGS: Thrombin-induced increase in [Ca²âº](i) was significantly inhibited by pretreatment of platelets with R59949, while thapsigargin-induced increase in [Ca²âº](i) was comparable in platelets with and without R59949 pretreatment. Thapsigargin-induced increase in [Ca²âº](i) was markedly attenuated in the presence of SKF-96365. In the presence of SKF-96365, thrombin-induced increase in [Ca²âº](i) was significantly attenuated, and additional treatment with R59949 caused a further decrease in [Ca²âº](i) . Pretreatment of platelets with 1-butanol significantly attenuated thrombin-induced increase in [Ca²âº](i) , while thrombin-induced increase in [Ca²âº](i) was augmented in the presence of propranolol. mRNA expression of DGK-α and DGK-γ, which are known to be inhibited by R59949, in platelets was confirmed by RT-PCR analysis. CONCLUSIONS: R59949 inhibited a store-depletion-insensitive component of transplasmalemmal Ca²âº entry induced by thrombin, while store-operated Ca²âº entry was not affected by R59949. The results of this study suggest that phosphatidic acid is involved in thrombin-induced Ca²âº influx of platelets.

A comparison of headspace solid-phase microextraction and classic hydrodistillation for the identification of volatile constituents from Thapsia spp. provides insights into guaianolide biosynthesis in Apiaceae.

INTRODUCTION: Thapsia spp. (Apiaceae) are the major natural source of polyoxygenated guaianolide sesquiterpene lactones known as thapsigargins, which induce apoptosis in mammalian cells via a high affinity inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase. The mechanism of biosynthesis of thapsigargins has not been elucidated, and probable biochemical precursors such as hydrocarbon or oxygenated sesquiterpenes have not been identified in previous phytochemical analyses of essential oils from this genus. OBJECTIVE: To investigate the utility of solid phase micro-extraction (SPME), when compared with classical essential oil distillates, for identifying potential precursors of guaianolide sesquiterpene lactones from Thapsia garganica L. and Thapsia villosa L. type II. METHODOLOGY: A systematic description of the volatile components of roots, flowers, stems and fruits of T. villosa and of root, flower and fruits of T. garganica was constructed via GC-MS analyses of SPME-adsorbed compounds and of essential oils obtained through hydrodistillation of the same tissues. RESULTS: The sesquiterpenoids δ-cadinene, α- and δ-guaiene, elemol and guaiols were found to be major volatile constituents of the roots of T. garganica and T. villosa trapped using SPME. In contrast, these sesquiterpenoids were not detected or were at negligible levels in essential oils, where sesquiterpenoids are potentially converted to azulenes during hydrodistillation. CONCLUSION: The new data reported in this study demonstrates that SPME is a valuable tool for the identification of volatile sesquiterpenes when compared with analysis of essential oils, and we postulate that guaiene is the likely precursor of guaianolide sesquiterpenes from Thapsia.

A phenylpropanoid, a slovenolide, two sulphur-containing germacranes and Ca2+-ATPase inhibitors from Thapsia villosa.

A phenylpropanoid 1, a slovenolide 2, and two germacranes bearing a methylthiopropenoate moiety, 3 and 4, along with twenty known metabolites have been isolated from the roots of Thapsia villosa var. villosa L. The structures of two known phenylpropanoids 5 and 6 have been corrected. Compounds 7 and 8 showed activity as potential inhibitors of the sarco- and endoplasmic Ca(2+)-dependent ATPases (SERCA) pump. Compounds 9, 10 and 11 increased significantly the cytoplasmic free calcium concentration ([Ca(2+)](c)) in human platelets in a concentration-dependent manner.

Base-catalyzed Diels-Alder reactions of 2H-pyran-2,5-diones: a mild approach to basiliolide B.

A new class of base-catalyzed Diels-Alder reactions of 2H-pyran-2,5-diones has been developed using catalytic amount of dicyclohexylmethylamine in tert-butyl alcohol. This method has been successfully employed for construction of the tricyclic core of basiliolide B at room temperature with good yields and exclusive endo selectivity.

Progress toward the synthesis of the basiliolides and transtaganolides: an intramolecular pyrone Diels-Alder entry into a novel class of natural products.

Efforts directed toward the synthesis of a basiliolide/transtaganolide model system are disclosed. A highly endo-selective intramolecular pyrone Diels-Alder (IMPDA) cycloaddition rapidly constructs the tricyclic core of the basiliolides and transtaganolides.

A pyran-2-one and four meroterpenoids from Thapsia transtagana and their implication in the biosynthesis of transtaganolides.

Four meroterpenoids (3a, 3b, 4 and 5), a prenylated pyran-2-one (2) along with the known compounds 7-O-geranylscopoletin (1), and thapsitranstagin (6) have been isolated from the roots of Thapsia transtagana. The presence of 1 and 2 supports the biogenetic hypothesis that transtaganolides, a group of bioactive metabolites, are meroterpenoids which come from an O-prenylated coumarin via successive pericyclic reactions.

Identification of natural products using HPLC-SPE combined with CapNMR.

Two major development areas in HPLC-NMR hyphenation are postcolumn solid-phase extraction (HPLC-SPE-NMR) and capillary separations with NMR detection by means of solenoidal microcoils (CapNMR). These two techniques were combined off-line into HPLC-SPE-CapNMR, which combines the advantage of high loadability of normal-bore HPLC columns with high mass sensitivity of capillary NMR probes with an active volume of 1.5 microL. The technique was used for rapid identification of complex sesquiterpene lactones and esterified phenylpropanoids present in an essentially crude plant extract (toluene fraction of an ethanolic extract of Thapsia garganica fruits). Elution profiles of 10 x 1 mm i.d. SPE cartridges filled with poly(divinylbenzene) resin were found to be only marginally broader than those observed upon direct injection of 6-microL samples into the probe. Thus, the technique focuses analytes emerging in the HPLC elution bands of 0.5-1 mL into volumes of approximately 10 microL, compatible with the CapNMR probe. Using this technique, nine natural products (1-9) present in the plant extract in amounts varying from 0.1 to 20% were identified by means of 1D and 2D NMR spectra, supported by parallel HPLC-ESIMS measurements. Therefore, HPLC-SPE-CapNMR should be regarded as an attractive alternative to other applications of CapNMR for mixture analysis.

Cytotoxic phenylpropanoids and an additional thapsigargin analogue isolated from Thapsia garganica.

Four phenylpropanoids and a thapsigargin analogue have been isolated from the fruits of Thapsia garganica. A spectroscopic method for elucidating the relative stereochemistry at the two pairs of stereogenic centers in the phenylpropanoids has been developed. The phenylpropanoids were found to be potent cytotoxins.