Concise Synthesis of (+)-ß- and γ-Apopicropodophyllins, and Dehydrodesoxypodophyllotoxin.

Herein, we present an expeditous synthesis of bioactive aryldihydronaphthalene lignans (+)-ß- and γ-apopicropodophyllins, and arylnaphthalene lignan dehydrodesoxypodophyllotoxin. The key reaction is regiocontrolled oxidations of stereodivergent aryltetralin lactones, which were easily accessed from a nickel-catalyzed reductive cascade approach developed in our group.

A novel anti-cancer role of ß-apopicropodophyllin against non-small cell lung cancer cells.

We previously reported that podophyllotoxin acetate (PA) inhibits the growth and proliferation of non-small cell lung cancer (NSCLC) cells and also makes them more sensitive to radiation and chemotherapeutic agents. In an attempt to enhance PA activity, we synthesized 34 derivatives based on podophyllotoxin (PPT). Screening of the derivative compounds for anti-cancer activity against NSCLC led to the identification of ß-apopicropodophyllin (APP) as a strong anti-cancer agent. In addition to its role as an immunosuppressive regulator of the T-cell mediated immune response, the compound additionally showed anti-cancer activity against A549, NCI-H1299 and NCI-460 cell lines with IC50 values of 16.9, 13.1 and 17.1 nM, respectively. The intracellular mechanisms underlying the effects of APP were additionally examined. APP treatment caused disruption of microtubule polymerization and DNA damage, which led to cell cycle arrest, as evident from accumulation of phospho-CHK2, p21, and phospho-Cdc2. Moreover, APP stimulated the pro-apoptotic ER stress signaling pathway, indicated by elevated levels of BiP, phospho-PERK, phospho-eIF2α, CHOP and ATF4. We further observed activation of caspase-3, -8 and -9, providing evidence that both intrinsic and extrinsic apoptotic pathways were triggered. In vivo, APP inhibited tumor growth of NSCLC xenografts in nude mice by promoting apoptosis. Our results collectively support a novel role of APP as an anticancer agent that evokes apoptosis by inducing microtubule disruption, DNA damage, cell cycle arrest and ER stress.

Antileishmanial activity and tubulin polymerization inhibition of podophyllotoxin derivatives on Leishmania infantum.

Leishmania microtubules play an important role not only in cell division, but also in keeping the shape of the parasite and motility of its free-living stages. Microtubules result from the self-assembly of alpha and beta tubulins, two phylogenetically conserved and very abundant eukaryotic proteins in kinetoplastids. The colchicine binding domain has inspired the discovery and development of several drugs currently in clinical use against parasites. However, this domain is less conserved in kinetoplastids and may be selectively targeted by new compounds. This report shows the antileishmanial effect of several series of compounds (53), derived from podophyllotoxin (a natural cyclolignan isolated from rhizomes of Podophyllum spp.) and podophyllic aldehyde, on a transgenic, fluorescence-emitting strain of Leishmania infantum. These compounds were tested on both promastigotes and amastigote-infected mouse splenocytes, and in mammalian - mouse non-infected splenocytes and liver HepG2 cells - in order to determine selective indexes of the drugs. Results obtained with podophyllotoxin derivatives showed that the hydroxyl group at position C-7α was a structural requisite to kill the parasites. On regards podophyllic aldehyde, derivatives with C9-aldehyde group integrated into a bicyclic heterostructure displayed more potent antileishmanial effects and were relatively safe for host cells. Docking studies of podophyllotoxin and podophyllic aldehyde derivatives showed that these compounds share a similar pattern of interaction at the colchicine site of Leishmania tubulin, thus pointing to a common mechanism of action. However, the results obtained suggested that despite tubulin is a remarkable target against leishmaniasis, there is a poor correlation between inhibition of tubulin polymerization and antileishmanial effect of many of the compounds tested, fact that points to alternative pathways to kill the parasites.

Total Synthesis of the Claimed Structure of (±)-Hyptinin and Structural Revision of Natural Hyptinin.

A total synthesis of (±)-hyptinin was achieved via a convergent route using the key phosphonate, cyclic ketone, and aryl Grignard components. The 1H and 13C NMR spectra of natural hyptinin did not agree with those of the synthesized compound. In particular, there were considerable differences between the signals assigned to the protons and carbons surrounding the lactone carbonyl group for the natural and synthesized compounds. The NMR data strongly suggested that the naturally occurring compound, hyptinin, was a structural isomer of the synthesized compound. The structure of the natural compound was eventually established as (+)-ß-apopicropodophyllin, based on the synthesis results.

Podophyllin, but not the constituents quercetin or kaempferol, induced genotoxicity in vitro and in vivo through ROS production.

The genotoxic potential of podophyllin (PD) was investigated in this study. PD increased bacterial revertants and abnormal chromosomal structures in a concentration-dependent manner, both with and without metabolic activating enzymes, and increased the incidence of micronuclei in imprinted control region mouse reticulocytes. Results from three studied constituents of PD, such as podophyllotoxin, kampferol, and quercetin, suggested that the mutagenic effect of PD was not due to the presence of podophyllotoxin, kampferol, and quercetin and might be related to other components and the formation of reactive oxygen species. The detailed mutagenic mechanisms need further investigation, and the medicinal use of PD needs to be cautioned against.

[Studies on the mechanism of the interaction between hydrazide-podophyllic Ni(II), Co(II), Zn(II) metal complexes and DNA].

The mechanism of the interaction between hydrazide-podophyllic (HDPP) metal (Me) complexes and calf thymus (ct) DNA in Tris buffer (pH 7.08) was studied by viscosity measurements, electronic absorption, gel electrophoresis, and ethidium bromide (EB) fluorescence spectroscopy. The results from varied experiments show that the intensity of the maximal absorption peaks from absorption spectra is weakened in the presence of DNA compared with that in the absence of DNA. Meanwhile, DNA can remarkably quench the emission intensity of the complex Me-HDPP system. The Me-HDPP complexes can increase the viscosity of ct DNA slightly and catalyze the cleavage of super coiled pBR322 DNA to the nicked form. The complexes of Ni-HDPP and Co-HDPP can be bound to ct DNA mainly by interaction, while the partial interaction of Zn-HDPP and ct DNA is the major modes. The binding constant of Me-HDPP complexes with ct DNA was determined.

Structure-activity relationship of chemical defenses from the freshwater plant Micranthemum umbrosum.

Vascular plants produce a variety of molecules of phenylpropanoid biosynthetic origin, including lignoids. Recent investigations indicated that in freshwater plants, some of these natural products function as chemical defenses against generalist consumers such as crayfish. Certain structural features are shared among several of these anti-herbivore compounds, including phenolic, methoxy, methylenedioxy, and lactone functional groups. To test the relative importance of various functional groups in contributing to the feeding deterrence of phenylpropanoid-based natural products, we compared the feeding behavior of crayfish offered artificial diets containing analogs of elemicin (1) and beta-apopicropodophyllin (2), chemical defenses of the freshwater macrophyte Micranthemum umbrosum. Both allyl and methoxy moieties of 1 contributed to feeding deterrence. Disruption of the lactone moiety of 2 reduced its deterrence. Finally, feeding assays testing effects of 1 and 2 at multiple concentrations established that these two natural products interact additively in deterring crayfish feeding.

[Poisoning with a podophyllin-containing wart-treating tincture]. / Vergiftung mit Podophyllin-haltiger Warzentinktur.

HISTORY AND ADMISSION FINDINGS: A 57-year-old depressive and alcohol-dependent man was admitted because of frequent nausea and vomiting and abdominal complaints after he had ingested 40 ml of a tincture for treating warts. He was under the influence of alcohol, but normally oriented and without contributory findings other than his gastrointestinal complaints and tachypnea. INVESTIGATIONS: Transaminases were raised (GOT 1197 U/l, GPT 170 U/l, gammaGT 150 U/l, LDH 2047 U/l), as were creatine phosphokinase (426 U/l) and ferritin (12 200 ng/ml). Platelet count was 36000 mm3, Leucocytes count 11 700/mm (3). Gastroscopy showed marked mucosal necrosis along the entire esophagus and the pulled-up small intestine (state after gastrectomy). DIAGNOSIS, TREATMENT AND COURSE: The patient became comatose within 5 hours, acidotic, oliguric, required ventilation and went into severe shock. The symptoms and the fact that podophyllin (pod.) was the main agent in the wart preparation confirmed the suspicion of pod. poisoning. Symptomatic and intensive care measures stabilized his critically grave condition. He was extubated on the 7th day after ingestion and on the 10th day was discharged at his own request in a relatively good general state. When he was re-admitted after 4 weeks he was without psychiatric symptoms but deeply depressed, and he had signs of a polyneuropathy in all limbs, typical of pod. toxin poisoning. CONCLUSION: Pod. toxin, a spindle poison, is the toxic agent of pod., the resin from the roots and rhizomes of various Berberis plants. While the potential toxicity of the resin, taken either orally or applied externally, has been long known, the poorly definied raw product is still being added to anti-wart tinctures.

Zn finger and nuclear localization of transcription factor Sp1.

Transcription factor Sp1 is located in the nucleus of a mammalian cell and importantly related to expression of many cellular genes. In order to elucidate the nuclear localization mechanism of Sp1, various truncated fragments of Sp1 were fused to green fluorescent protein (GFP) and expressed in HeLa cell. The results show significance of the DNA binding region, especially, zinc finger (Zn finger) domain for nuclear localization of Sp1 in HeLa cell.

Regulation of ZiRF1 and basal SP1 transcription factor MRE-binding activity by transition metals.

The metal-dependent activation of metallothionein (MT) genes requires the interaction of positive trans-activators (MRFs) with metal-regulatory (MRE) regions of MT promoters. In this report, we examined the role of transition metals in modulating the MRE-binding activities of two different MRE-binding proteins: the metal-regulated factor ZiRF1 and the basal factor SP1. We showed the ability of both proteins to interact with a similar sequence specificity with the cognate target site (MRE-S) of another known MRE-binding protein, mMTF1. We next evaluated the role of metal ions in modulating the MRE-binding activity of recombinant ZiRF1 and basal SP1 proteins by measuring the effect of different metal chelators on DNA interaction. We observed a dose-dependent inhibition of the GST-ZiRF1/MRE-binding activity using three different metal chelators: EDTA, 1,10 PHE and TPEN. Interestingly, EDTA treatment failed to inhibit the recombinant SP1 MRE-binding activity while the effect of 1,10 PHE was comparable to that obtained analyzing 1,10 PHE-treated GST-ZiRF1. The MRE-binding complexes detected in cell extracts showed a response to metal chelator treatment very similar to that displayed by the recombinant ZiRF1 and SP1 proteins. The hypothesis of mutual interactions of both basal and metal-regulated transcription factors with the same metal-regulatory regions is discussed.

Quercetin and kaempherol: an argument against the use of podophyllin?

INTRODUCTION: Topical application of podophyllin is a routine procedure in patients with ano-genital warts. Podophyllin is a crude plant extract and is therefore not a well-defined product. It may contain variable amounts of the active lignan podophyllotoxin and the majority of the dry weight of podophyllin is made up of substances never identified. OBJECTIVE: The purpose of the study was to estimate in podophyllin 20% the amounts of two mutagenic substances, quercetin and kaempherol. METHODS: Using high-pressure liquid chromatography the amounts of quercetin and kaempherol were determined in 3 batches of podophyllin 20%. RESULTS: Quercetin and kaempherol constitutes 2.5-3.8% and 6.0-6.4% of podophyllin dry substance, respectively. Podophyllotoxin constitutes in comparison 12.7-13.8% of podophyllin dry substance. CONCLUSION: As approximately 10% of the amount of dry substance in podophyllin 20% is composed of two mutagenic flavonoids, quercetin and kaempherol, efforts should be focused on the production of a well-defined purified podophyllotoxin preparation that may replace podophyllin for clinic use in patients with genital warts. Self-medication with purified podophyllotoxin 0.5% may be considered as first-line treatment in well-instructed patients with external genital warts.

Antitumor activity of a new low immunosuppressive derivative of podophyllotoxin (GP-11) and its mechanisms.

The spin-labeled derivative of podophyllotoxin, N'-podophyllic acid-N-[3-(2,2,5,5-tetramethyl pyrrolinenyloxy)] semicarbazide (GP-11), was synthesized and tested for its antitumor activity against mouse transplantable tumors, Sarcoma-180, Hepatoma-A, P388 leukemia and Ehrlich ascites carcinoma. At an equitoxic dose, the antitumor activity of GP-11 was similar to that of etoposide (VP-16). However, the immunosuppressive effects of GP-11 were weaker than that of VP-16. In vitro, GP-11 and VP-16 inhibited the proliferation of human lymphoid leukemia Molt 4B cells and suppressed DNA and protein syntheses, but the effect of GP-11 and VP-16 on cell cycle progression was different. The mitotic index was increased by GP-11 and reduced by VP-16. On the basis of flow cytometric bromodeoxyuridine (BrdU)/DNA analysis, GP-11 and VP-16 resulted in the accumulation of cells in the S and G2/M phases. G2/M arrest by GP-11 on cell cycle progression was stronger than that of VP-16, while S arrest was weaker than that of VP-16. After the removal of drugs, the arrest by GP-11 and VP-16 still existed and was irreversible. These results may provide insights into the structure-activity relationships and the design of novel derivatives of podophyllotoxin useful in cancer chemotherapy.