Phenanthroindolizidine Alkaloids Research Articles

Chemical constituents from Cynanchum paniculatum (Bunge) Kitag

Twenty-nine compounds, including five acetophenone derivatives (1–5), three phenanthroindolizidine alkaloids (12–14), seven pentacyclic triterpenoids (15–21) and five C21 steroidal sapogenins (22–26), were isolated from the root of Cynanchum paniculatum (Bunge) Kitag. Their structures were determined by spectroscopic methods and comparison with reported data. Moreover, the chemotaxonomic relationships were also discussed. As a result, acetophenone derivatives, pentacyclic triterpenoids and C21 pregnane sapogenins can be recognized as chemotaxonomic markers for Cynanchum genus, and C. paniculatum has close relationships with some species of genus Cynanchum.

Design, synthesis, antiviral activity and mode of action of phenanthrene-containing N-heterocyclic compounds inspired by the phenanthroindolizidine alkaloid antofine.

The phenanthroindolizidine alkaloid antofine and its analogues have excellent antiviral activity against tobacco mosaic virus (TMV). To simplify the structure and the synthesis of the phenanthroindolizidine alkaloid, a series of phenanthrene-containing N-heterocyclic compounds (compounds 1 to 33) were designed and synthesised, based on the intermolecular interaction of antofine and TMV RNA, and systematically evaluated for their anti-TMV activity. Most of these compounds exhibited good to reasonable anti-TMV activity. The optimum compounds 5, 12 and 21 displayed higher activity than the lead compound antofine and commercial ribavirin. Compound 12 was chosen for field trials of antiviral efficacy against TMV, and was found to exhibit better activity than control plant virus inhibitors. Compounds 5 and 12 were chosen for mode of action studies. The changes in fluorescence intensity of compounds 5 and 12 on separated TMV RNA showed that these small molecules can also bind to TMV RNA, but the mode is very different from that of antofine. The compounds combining phenanthrene and an N-heterocyclic ring could maintain the anti-TMV activity of phenanthroindolizidines, but their modes of action are different from that of antofine. The present study lays a good foundation for us to find more efficient anti-plant virus reagents.

Screening of promising chemotherapeutic candidates from plants against human adult T-cell leukemia/lymphoma (IV): phenanthroindolizidine alkaloids from Tylophora tanakae leaves.

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature peripheral T lymphocytes caused by human T-cell lymphotropic virus type 1 (HTLV-1). There are an estimated 5 million to 20 million HTLV-1-infected individuals worldwide; their lifetime risk of developing ATL is 3-5%, and high HTLV-1 proviral loads have been shown to be an independent risk factor. Although conventional chemotherapeutic regimens used against other malignant lymphomas have been administered to ATL patients, the prognosis is often poor. In previous studies, we screened 459 extracts from 344 plants to isolate components exhibiting antiproliferative activity against HTLV-1-infected T-cell lines (MT-1 and MT-2). In our continuing search for potential anti-HTLV-1 natural products, 15 extracts of Asclepiadaceae plants were further tested against MT-1 and MT-2 cells. The MeOH extract of aerial parts of Tylophora tanakae showed antiproliferative activity. Activity-guided fractionation resulted in the isolation of 6 phenanthroindolizidine alkaloids (including a new compound), and we examined their antiproliferative activity against MT-1 and MT-2 cells. The EC50 value of some of the alkaloids was in the low nanomolar range, comparable to that of the clinically used antineoplastic drug doxorubicin. Structure-activity relationship analyses suggested that a 14β-hydroxy moiety is essential for activity against HTLV-1-infected T cells. In contrast, the presence of a 2-methoxy moiety, a 7-methoxy moiety, or an N-oxide moiety appears to reduce the potency of the antiproliferative activity against HTLV-1-infected T cells.

Antitumor Activity of Phenanthroindolizidine Alkaloids Is Associated with Negative Regulation of Met Endosomal Signaling in Renal Cancer Cells

Met is a receptor tyrosine kinase for hepatocyte growth factor. Met mutations have been considered as a major cause of primary resistance to Met tyrosine kinase inhibitors (TKIs). Mutated Met enhances its endosomal signaling, which includes internalization, signaling within endosomes, recycling to membrane, and sorting for degradation. These sequential events lead to a plausible mechanism for resistance. (-)-Antofine, a phenanthroindolizidine alkaloid, has exhibited potent antitumor activity but the precise underlying mechanism has been poorly understood. We found that (-)-antofine effectively inhibited the proliferation of Met-mutated Caki-1 cells, which were resistant to well-known Met TKIs. (-)-Antofine negatively regulated Met endosomal signaling and consequently inhibited the nuclear translocation of STAT3 both invitro and invivo. These findings emphasize the potential of Met endosomal signaling as a novel target for Met TKI-resistant cancers and (-)-antofine as a novel lead compound associated with the suppression of Met endosomal signaling.

A Novel Asymmetric Synthesis of (+)-Deoxytylophorinine

A novel asymmetric synthesis of a phenanthroindolizidine alkaloid, (+)-deoxytylophorinine, is reported, which uses a chiral nickel(II) complex of a glycine Schiff base to synthesize a substituted ( R )-3-phenanthrylalanine as a key intermediate and Meldrum’s acid to construct the E ring.

Development and validation of a liquid chromatography–tandem mass spectroscopy method for simultaneous determination of (+)-(13aS)-deoxytylophorinine and its pharmacologically active 3-O-desmethyl metabolite in rat plasma

CAT ((+)-(13aS)-deoxytylophorinine) is a novel anticancer drug belonging to phenanthroindolizidine alkaloids. A sensitive and reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous quantification of CAT and its pharmacologically active 3-O-desmethyl metabolite (S-4) was developed and validated in rat plasma using rotundine as the internal standard (IS). CAT, S-4 and IS were extracted by acetonitrile protein precipitation and separated on an Eclipse XDB-C18 column (1.8μm, 4.6mm×50mm) with acetonitrile–water (27:73, v/v) mobile phase containing 0.1% formic acid at a 0.4mL/min flow rate. Positive ion electrospray ionization in multiple reaction monitoring mode was employed to measure CAT, S-4 and IS by monitoring the transitions m/z 364.2→70.1 for CAT, 350.1→70.1 for S-4 and 356.2→192.2 for IS. Good linear correlation (r2>0.991) was achieved for CAT and S-4 over the range of 0.214–128.16 and 0.044–11.00ng/mL, respectively. The lower limit of quantification was 0.214ng/mL for CAT and 0.044ng/mL for S-4, using 50μL rat plasma samples. The intra- and inter-day precisions were not exceed 15% and the accuracy ranged between 94.80% and 108.22%. The average extraction recoveries of both analytes were greater than 94.62%. The method was successfully applied to the pharmacokinetic study of CAT and S-4 in rats after oral administration.

Hispidacine, an unusual 8,4′-oxyneolignan-alkaloid with vasorelaxant activity, and hispiloscine, an antiproliferative phenanthroindolizidine alkaloid, from Ficus hispida Linn.

Hispidacine, an 8,4′-oxyneolignan featuring incorporation of an unusual 2-hydroxyethylamine moiety at C-7, and hispiloscine, a phenanthroindolizidine alkaloid, were isolated from the stem-bark and leaves of the Malaysian Ficus hispida Linn. Their structures were established by spectroscopic analysis. Hispidacine induced a moderate vasorelaxant activity in rat isolated aorta, while hispiloscine showed appreciable antiproliferative activities against MDA-MB-231, MCF-7, A549, HCT-116 and MRC-5 cell lines.

ChemInform Abstract: An Enantioselective Strategy for the Total Synthesis of (S)‐Tylophorine via Catalytic Asymmetric Allylation and a One‐Pot DMAP‐Promoted Isocyanate Formation/Lewis Acid Catalyzed Cyclization Sequence.

A new asymmetric total synthesis of a phenanthroindolizidine alkaloid (S)-tylophorine is reported, which features a catalytic asymmetric allylation of aldehydes and an unexpected one-pot DMAP promoted isocyanate formation and Lewis acid catalyzed intramolecular cyclization reaction. In addition, White's direct C–H oxidation catalyst system converting monosubstituted olefins to linear allylic acetates was also employed for late-stage transformation.

Abstract 4236: Antitumor activity of a novel (-)-antofine analog in human lung cancer cells and paclitaxel-resistant lung cancer cells

Abstract Our previous study revealed that (-)-antofine, a phenanthroindolizidine alkaloid from Cynanchum paniculatum Kitagawa (Asclepiadaceae), exhibited a potent growth inhibition activity against A549 human lung cancer cells. Based on the information several analogs were designed and synthesized to procure new entities. A novel (-)-antofine C-13a analog ((R)-4b) was found to be a potential growth inhibition and antitumor activities in A549 cells. Treatment with (R)-4b for 24 hours did not result in the induction of apoptotic cell death but moderately induced cell cycle arrest in the G0/G1 phase and inhibited the expression of cyclin D1, cyclin E, CDK4 and pRb. In addition, (R)-4b suppressed the phosphorylation of mTOR and its downstream effectors such as 4E-BP-1 and eIF4E. The paclitaxel-resistance was also overcome in human lung cancer cells (A549-Pa) via the suppression of p-glycoprotein expression. Furthermore, (R)-4b significantly inhibited the tumor growth of A549 and A549-Pa xenografts in a nude mouse model. Taken together, these findings suggest that (R)-4b might be a potential candidate for the development of cancer chemotherapeutic agents derived from natural products. Citation Format: Jayoung Song, Song Yi Bae, Yoonho Shin, Won Kyung Kim, Jedo Oh, Tae Joon Choi, Eun Ju Jeong, So Hyun Park, Eun Jeong Jang, Ji In Kang, Hyen Joo Park, Ji-Young Hong, Hwa-Jin Chung, Yongseok Kwon, Sanghee Kim, Sang Kook Lee. Antitumor activity of a novel (-)-antofine analog in human lung cancer cells and paclitaxel-resistant lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4236. doi:10.1158/1538-7445.AM2014-4236

Anti-adipogenic activity of the naturally occurring phenanthroindolizidine alkaloid antofine via direct suppression of PPARγ expression.

Antofine (ANTF) is a phenanthroindolizidine alkaloid isolated from the root of Cynanchum paniculatum Kitagawa (Asclepiadaceae), which is used as an herbal remedy for pain and inflammation. ANTF also possesses antiviral and antitumorigenic activities. In this study, we investigated the role of ANTF in adipogenesis. Chronic ABTF administration suppressed adipocyte differentiation and marker expression in a dose-dependent manner. Furthermore, acute administration of ANTF at early stages of differentiation process inhibited lipid droplet formation and adipogenic gene expression. ANTF Treatment decreased expression of PPARγ protein, a master transcription factor in the regulation of adipocyte differentiation, leading to a suppression of aP2 promoter activity. These results suggest that ANTF exerts potent anti-adipogenic effects via direct suppression of PPARγ protein expression, with consequent downregulation of adipogenic gene expression.

Design, synthesis, antiviral activity, and SARs of 13a-substituted phenanthroindolizidine alkaloid derivatives

On the basis of our previous structure–activity relationship (SAR) and antiviral mechanism studies, a series of 13a-substituted phenanthroindolizidine alkaloid analogues (3a–16a, 3b, 4b, 6b, 7b, 10b, and 14b) were designed targeting tobacco mosaic virus (TMV) RNA, synthesized, and evaluated for their antiviral activity against TMV for the first time. The bioassay results showed that most of the synthesized compounds (such as 4a, 6a, 7a, 11a, 14a, 6b, and 14b) exhibited good to excellent antiviral activity against TMV both in vitro and in vivo. Especially, for inactivation effect and curative effect, compounds 4a, 6a, 7a, 11a, 14a, and 14b showed higher activity at both concentrations (500μgmL−1 and 100μgmL−1) than commercial Ningnanmycin. Preliminary SARs showed that the substituted groups with hydrogen donor at 13a position were found to be favorable for keeping high antiviral activity. The present work demonstrates that 13a-substituted phenanthroindolizidines can be used as possible lead compounds for developing anti-TMV agents.

Total synthesis of phenanthroindolizidine alkaloids via asymmetric deprotonation of N-Boc-pyrrolidine

A concise synthesis of two typical phenanthroindolizidine alkaloids via an asymmetric deprotonation/diastereoselective carbonyl addition strategy is described.

Synthesis of Alkaloids: Recent Advances in the Synthesis of Phenanthroindolizidine Alkaloids

Phenanthroindolizidine alkaloids are a well-known class of compounds due to their interesting biological activities, especially anticancer ones. Represented by more than 60 substances, they are mainly isolated from plants of the Moraceae and Asclepiadaceae families. In the last 30 years, a great effort has been made aiming the synthesis of these compounds and analogues to be applied in medicinal chemistry.

Design, synthesis, and anti-tobacco mosaic virus (TMV) activity of glycoconjugates of phenanthroindolizidines alkaloids

Glycoconjugates of phenanthroindolizidine alkaloids targeting tobacco mosaic virus (TMV) RNA were designed, synthesized, and evaluated for their antiviral activity against TMV for the first time. The glycoconjugation of (S)-6-O-desmethylantofine (2) and 14-hydroxyltylophorines (3-6) was accomplished in three ways (O-glycosylation manner, using carbamoyloxy as linker arm, and using 1,2,3-triazole as linker arm) with three different sugar units (glucose, galactose, and mannose). The glycoconjugates showed improved water solubility and molecule polarity compared with phenanthroindolizidine alkaloids. The bioassay results showed that C6 was a suitable position for glycoconjugation and O-glycosylation can increase the antiviral activity of phenanthroindolizidine alkaloids indicating that the introduction of sugar units can improve the antiviral activity profile of glycoconjugates. Two O-glycosides of (S)-6-O-desmethylantofine, (13aS)-6-O-β-D-galactopyranosyl-2,3-dimethoxyphenanthro [9,10-b]-11-indolizidinone (10) and (13aS)-6-O-β-D-mannopyranosyl-2,3-dimethoxyphenanthro [9,10-b]-11-indolizidinone (11) displayed significant higher activity than commercial ningnanmycin, and thus could be considered for novel therapy against plant virus infection.

Total Synthesis of Phenanthroindolizidine Alkaloids through an Amidyl Radical Cascade/Rearrangement Reaction

A short and general synthesis of the phenanthroindolizidine alkaloids is reported, featuring an unusual amidyl radical 5-exo/5-exo/rearrangement cascade of a xanthate precursor. Second, using an amidyl radical 5-exo/6-endo cascade to synthesize a phenanthroindolizidine alkaloid exclusively has been developed through a small structural modification.

Mechanisms in endovascular differentiation of the trophoblast and its regulation by decorin

Although antofine, a natural phenanthroindolizidine alkaloid, exerts potential biological activities, including anticancer effect and anti-angiogenic activity, the underlying mechanisms have not yet been investigated. In the present study, the inhibitory effect of antofine on angiogenesis was determined in cultured mouse embryonic stem (mES)/embryoid body (EB)-derived endothelial cells and vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVECs). Antofine effectively inhibited VEGF-induced cell migration and tube formation of HUVECs. Antofine also significantly decreased ex vivo microvessel sprouting in cultured mouse aortic rings, and inhibited the vascular formation and platelet/endothelial cell adhesion molecule (PECAM) expression of mES/EB-derived cells in 3-D collagen gel. The underlying mechanism of anti-angiogenic activity of antofine was, in part, associated with the modulation of AKT/mTOR and AMP-activated protein kinase (AMPK) signaling in VEGF-stimulated HUVECs.

Simplification of antitumoral phenanthroindolizidine alkaloids: Short synthesis of cytotoxic indolizidinone and pyrrolidine analogs

Hydroxylated seco-analogs of cytotoxic phenanthroindolizidine alkaloids were prepared in good yields from inexpensive 4-hydroxyproline derivatives, in just two steps. Thus, a sequential oxidative radical scission–oxidation was used for the direct conversion of the proline derivative into a 2-(2-aryl-oxoethyl)pyrrolidine with a variety of aryl and heteroaryl groups. The 4R-stereogenic center allowed ready isomer separation, and stereocontrol in the introduction of new chains (interestingly, the 2,4-cis isomers predominated). In the second step, a cyclization reaction afforded alkaloid analogs with an indolizidinone core; a partial isomerization took place but the isomers were readily purified. Then the cytotoxic activity of the bicyclic indolizidinones and the simpler pyrrolidine derivatives was compared against tumorogenic human neuronal SHSY-5Y and breast cancer MCF7 cells. All the biphenyl derivatives displayed a potent activity (one derivative caused >80% cell death in both tumor lines at micromolar dosis), being comparable in the pyrrolidine and indolizidinone series.

Antitumor Activity of Natural Phenanthroindolizines and Daphnane Diterpenes

Natural products have played major roles in drug discovery programs including development of anticancer agents. In our continuing efforts to identify and characterize antitumor agents from natural products, we found the extract of the root of Cynanchum paniculatum (Asclepiadaceae) exhibited potent anti-proliferative effects in cultured human cancer cells. Bioassay-guided fractionation further led to the isolation of the bioactive constituent and was elucidated as antofine, a phenanthroindolizidine alkaloid. Antofine markedly inhibited the growth of several human cancer cells with the IC50 values in the nanomolar range. In view of the potential anti-proliferative effects against cancer cells we developed the procedure of total synthesis of antofine and further investigated the anti-proliferative mechanisms and antitumor effects. In HCT 116 human colon cancer cells, the plausible anti-proliferative mechanisms of antofine might be associated with the down-regulation of cyclin D1, cyclin E, and CDK4 expression, the inhibition of DNA synthesis, the modulation of Wnt signaling activation, and the induction of differentiation. Antofine also exhibited the potential growth inhibition of paclitaxel-resistant human lung cancer cells (A549-PA) along with the down-regulation of P-glycoprotein expression. Antofine also effectively suppressed tumor growth in the tumor xenograft model. In addition, daphnane-type diterpenoids were isolated from the flower of Daphne genkwa (Thymelaceae) with a potential anti-proliferative activity against human lung cancer cells. Yuanhuadine, one of the most potent isolates from D. genkwa, showed potent growth inhibition and a relatively strong selectivity against human lung cancer cells compared to a panel of several cancer cell lines. In A549 human lung cancer cells, yuanhuadine induced cell cycle arrest at either G0/G1 or G2/M phase depending on incubation time. The cell cycle arrest was well correlated with the expression of checkpoint proteins including the upregulation of cyclin-dependent kinase inhibitor p21, and down-regulation of cyclin B1, cyclin dependent kinases and c-myc. Yuanhuadine also inhibited the phosphorylation of Akt, the mammalian target of rapamycin (mTOR) and its downstream effector molecules, p70 S6 kinase 1 and eukaryotic initiation factor 4E binding protein 1, indicating that the induction of cell cycle arrest by yuanhuadine might be in part mediated by the suppression of Akt/mTOR signaling pathway. Taken together, these findings suggest that antofine and yuanhuadine might provide potential lead candidates for the development of cancer chemotherapeutic agents derived from natural products.

Antofine-induced connexin43 gap junction disassembly in rat astrocytes involves protein kinase Cβ

Antofine, a phenanthroindolizidine alkaloid derived from Cryptocaryachinensis and Ficusseptica in the Asclepiadaceae milkweed family, is cytotoxic for various cancer cell lines. In this study, we demonstrated that treatment of rat primary astrocytes with antofine induced dose-dependent inhibition of gap junction intercellular communication (GJIC), as assessed by scrape-loading 6-carboxyfluorescein dye transfer. Levels of Cx43 protein were also decreased in a dose- and time-dependent manner following antofine treatment. Double-labeling immunofluorescence microscopy showed that antofine (10ng/ml) induced endocytosis of surface gap junctions into the cytoplasm, where Cx43 was co-localized with the early endosome marker EEA1. Inhibition of lysosomes or proteasomes by co-treatment with antofine and their respective specific inhibitors, NH4Cl or MG132, partially inhibited the antofine-induced decrease in Cx43 protein levels, but did not inhibit the antofine-induced inhibition of GJIC. After 30min of treatment, antofine induced a rapid increase in the intracellular Ca2+ concentration and activation of protein kinase C (PKC)α/βII, which was maintained for at least 6h. Co-treatment of astrocytes with antofine and the intracellular Ca2+ chelator BAPTA-AM prevented downregulation of Cx43 and inhibition of GJIC. Moreover, co-treatment with antofine and a specific PKCβ inhibitor prevented endocytosis of gap junctions, downregulation of Cx43, and inhibition of GJIC. Taken together, these findings indicate that antofine induces Cx43 gap junction disassembly by the PKCβ signaling pathway. Inhibition of GJIC by antofine may undermine the neuroprotective effect of astrocytes in CNS.

Concise synthesis of tylophorine

The phenanthroindolizidine alkaloid tylophorine has been synthesized in the (R)- and racemic forms. One of the routes involves three steps from a known compound employing a Stevens rearrangement as the pivotal reaction. The phenanthrene moiety was constructed by either a base-catalyzed cyclization of 2-alkynylbiphenyls or a double Suzuki coupling of a 2,2′-dibromobiphenyl with vic-bis(pinacolatoboryl)alkene in other routes.