Synthesis and Anticancer Activity of 4b-Triazolepodophyllotoxin Glycosides

A series of novel 4b-triazole-podophyllotoxin glycosides were synthesized by utilizing the Click reaction.Evaluation of cytotoxicity against a panel of five human cancer cell lines(HL-60,SMMC-7721,A-549,MCF-7,SW480)using MTT assay shows that most of these compounds show weak cytotoxicity.It was observed that compound 16 shows the highest activity with IC50 values ranging from 2.85 to 7.28 lM,which is more potent than the control drugs etoposide and cisplatin against four of five cancer cell lines tested.Compound 16 is characterized with an a-D-galactosyl residue directly linked to the triazole ring and a 40-OH group on the E ring of the podophyllotoxin scaffold.HPLC investigation of representative compound indicates that incorporation of a sugar moiety seems to improve the chemical stability of the podophyllotoxin scaffold.

Biological Activity of N-Hydroxyethyl-4-aza-2,3-didehydropodophyllotoxin Derivatives upon Colorectal Adenocarcinoma Cells

Etoposide is a chemotherapy drug derived from the natural lignin podophyllotoxin. Our novel generated Aza-podophyllotoxin compounds (AZP 8a & AZP 9a) are analogues of podophyllotoxin and were previously screened for anti-cancer activity through the NCI 60 cell line screening panel showing activity on various cell types including colon cancer. This study expands the toxicological screening by studying apoptosis and various hallmark events as part of the mechanism of action of these compounds on colon cancer cells. The COLO 205 cell line was selected and exposed to AZP to determine the IC50 doses at 24 hours treatment. Apoptosis hallmark events such as migration of phosphatidylserine (PS) to the cell membrane, DNA fragmentation, cell cycle effects, mitochondrial membrane permeabilization and caspase activation were included. Experiments were performed in triplicates for all tested compounds including AZP 8a, AZP 9a, camptothecin as positive control and vehicle as negative control. Our results present contrasting apoptotic activity between the experimental compounds. Compound 8a presented migration of PS (annexin V assay), DNA fragmentation and cell cycle arrest at S phase. Compound 9a presented PS migration with fragmented DNA, cell cycle arrest at S phase, mitochondrial membrane permeabilization and activation of caspase 3, 8 and 9. Compound 8a without the oxygen atoms in ring A appears to cause effects similarly to autophagy as induced by etoposide, a cancer drug analogue of our heterocyclic compounds. Compound 9a with the oxygen atoms in expanded ring A presented induction of cell death following activation of a classical apoptosis pathway. Our results suggest that minor structural differences among these AZP can account for the difference in biological response and cancer cell toxicity.

Anticancer Drugs ( Ⅳ )——New Derivatives of Podophyllotoxin

Four new derivatives of podophyllotoxin, N'-podophyllic acid-N-[3-(2, 2, 5, 5-te-tramethyl-pyrrolinenyloxy)] semicarbazide(GP-11, 6), podophyllic acid [3-(2,2,5,5-te-tramethyl-pyrrolinenyloxy)]hydrazone (GP-12, 7), podophyllic acid-[4-(2, 2, 6, 6-tetramethyl-1-hydroxy piperidine)]hydrazone(GP-1-OH, 8) and podophyllic acid[4-(2,2, 6, 6-tetramethyl piperidine)]hydrazone(GP-1-H, 9) were synthesized. The inhibition effect of the four new compounds on L-1210 cells were determined. The antitumor activity and toxicity of GP-1(2), GP-1-OH(8), GP-1-H(9) and VP-16-213(1) were discussed.

Experimental Podophyllotoxin (Bajiaolian) Poisoning:——Ⅰ. Effects on the Nervous System

Bajiaolian, one of the species in the Mayapple family ( Podophyllum pelatum ), has been widely used as a traditional Chinese herbal medicine for the remedies of snake bites, general weakness, poisons, condyloma accuminata, lymphadenopathy, and certain tumors in China. In Western medicine, Podophyllum was first used medically as a laxative in the early 19th century. Since 1940, the resin of podophyllum has also been used topically for various skin lesions, such as warts and condyloma. Human poisonings have been reported.An animal model was established to investigate the neurotoxic effects of Bajiaolian. Podophyllotoxin, the major active ingredient in Podophyllum, was injected (ip) to young adult male rats at doses of 0, 5, 10, or 15 mg-kg-1 b.w.. The animals were sacrificed 72 h after injection.Neuronal changes were readily observable in animals treated with 10 or 15 mg-kg-1 of the toxin. Edematous changes of the anterior horn motoneurons were observed in the spinal cord. No neuronal necrosis was found. The type of neuronal swelling is believed to be only a transient change and would probably subside with time if no further assaults occur. More serious and perhaps longer term of changes were found in the dorsal ganglion neurons and the nerve fibers (axons) in the central and peripheral nervous system. Severe depletion of the Nissl substance (RNA/polyribosomes) was observed in the dorsal root ganglion neurons. Alterations in these sensory neurons would give rise to and correlate with the sensory disturbances experienced by the patients. Bodian staining also revealed a dose-related increase in the coarseness (thickness) of the nerve fibers (axons) in the cerebellum, cerebral cortex, brainstem, and spinal cord.This is the first scientific study showing the neurotoxicity of Bajiaolian, a commonly used Chinese herbal medicine. Toxicities on other organ systems by this drug certainly exist. Caution should be exercised in the dispensing and usage of this medicine.

Synthesis and cytotoxicity of novel podophyllotoxin derivatives

In order to find novel synthetic antitumor agents with superior cytotoxicity and overcoming multidrug resistance, a novel series of 4β-N-substituted podophyllotoxin derivatives were synthesized and evaluated as potential antitumor agents. Seven novel podophyllotoxin derivatives were synthesized by linking 4β-amino-4-deoxypodophyllotoxin with N-substituted 5-methylindol-3- yl-glyoxyl chlorides and tested against K562 and K562/A02 using SRB methods in vitro, KB and KBVusing MTT methods in vitro.

Synthesis and Antitumor Activity of4β-(1,2,3-Triazol-1-yl) podophyllotoxins

Three 4β-(1,2,3-triazol-1-yl) podophyllotoxins 2～4 have been synthesized and testedtar their antitumor activity in vitro. The compound 3 was found to be much more cytotoxic than theclinically used etoposide (VP-16) against L 1210 cells.

Design and synthesis of novel cytotoxic podophyllotoxin derivatives

In order to investigate the effect of different C4 linkage moieties on the cytotoxicity of podophyllotoxin derivatives, novel 4-N- and 4-C-substituted 4＇-O-demethylepipodophyllotoxin derivatives were designed and synthesized. All the compounds were tested against A549 and MCF-7 tumor cells in vitro, and six compounds showed significant cytotoxicity. The most active compound 9f was superior to GL-331, and exhibited potent cytotoxicity with IC50 value at 10^-7 mol/L level.

Isolation, purification and characterization of carboxymethyl cellulase (CMCase) from endophytic Fusarium oxysporum producing podophyllotoxin

Endophytic fungus Fusarium oxysporum is a rich source of cellulases. In the present study, the highest activity was reported at 28 ° C, pH 5.6 with 2% Carboxymethyl cellulose (CMC) as carbon source. CMC was purified using Sephadex G and DEAE cellulose chromatography to 15.9 folds and the molecular weight was determined to be 84 kDa by SDS-PAGE analysis and was subsequently characterized. The purified enzyme was stable over the pH range from 4.0 to 8.0 and at temperatures below 50 ° C. The enzyme was highly active on CMC and reduced or no activity on Avicel, cellobiose and it was suggested to be CMCase/endoglucanase. The activity of endoglucanase was enhanced in the presence of MgCl2, CoCl2, FeCl3, CaCl2, FeCl2 and intensive to HgCl2. The purified enzyme showed its optimum activity at pH 5.0 - 6.0 and was quite stable at 50 ° C for 30 min and retained 45% of original activity.

Experimental Podophyllotoxin (Bajiaolian) Poisoning:——Ⅱ. Effects on the Liver, Intestine, Kidney, Pancreas and Testis

Young male rats were orally intubated with podophyllotoxin: Group I, control animals, orally fed with vehicle only; Group Ⅱ, fed with an initial dose of 5 mg.kg-1 b.w., followed by a daily dose of 1.67 mg-kg-1 b.w. for 7d. Group III, fed with an initial dose of 15 mg.kg-1 b.w., followed by a daily dose of 5 mg.kg-1 b.w. for 7d. All animals were sacrificed 72 h after the last dosing.Histopathological examination revealed dose-related fatty change of the liver, atrophy andi degenerative changes of the intestinal epithelial linings and testicular seminiferous tubules. Depletion of the pancreatic acinar cell granules was also apparent in the Group III animals. No pathology, however, was observed in the kidneys. The present study demonstrated for the first time degenerative changes in the liver, intestine, testis, and pancreas of animals ingested podophyllotoxin. These pathological changes correlate well with the clinical signs/symptoms of abnormal liver function, abdominal pain and diarrhea, and reduced serum amylase in humans poisonded by podophyllum. Inhibition of protein synthesis and mitosis (disruption of microtubules) are believed to be the underlying mechanisms of these changes observed in the animals intoxicated by. podophyllotoxin.

Synthesis of A Novel Fulleroaziridine Carrying 4-Deoxy-4'-demethylepipodophyllotoxin

A novel fulleroaziridine 2 with a closed 6,6-ring junction was synthesized from [60]fullerene and 4-azido-4-deoxy- 4?demethylepipodophyllotoxin 1 in 1, 2-dichlorobenzene at 110℃ for 45 h, in a yield of 39.3 % based on consumed C60. The structure of the product was characterized by NMR and MS.

X-Ray Diffraction and NMR Study of Inclusion Complex of Podophyllotoxin with β-Cyclodextrin

The inclusion complex of podophyllotoxin (P) with β-cyclodextrin (β-CD) has been studied by X-ray diffractometry and 1HNMR、13CNMR spectroscopy. The complex structure is deduced.

The Synthesis of Podophyllotoxin Derivatives with Macrocyclic Dilactone

Starting from podophyllotoxin, five new derivatives with macrocyclic dilactone 5 similar to 9 have been synthesized,and their structures were confirmed by IR, MS,H-1 NMR as well as HRMS. The key step is cyclization by high dilution method.

Reactions of Podophyllotoxin with DDQ

Podophyllotoxin I reacts with DDQ in acetic acid to give podophyllotoxone 2 at 75 degreesC and the naphthol 3 at 90 degreesC, and in TFA to give the naphthalene 4. 4'-O-Benzyl-epipodophyllotoxin 5 reacts with DDQ in acetic acid at 70 degreesC to give the acetate 8. The structural elucidation of these products is described.

THEORETICAL STUDY OF PODOPHYLLOTOXIN AND QUINOLONE ANALOGUES AS ANTITUMOR DRUGS

Objective: To study the active sites of podophyllotoxin derivatives. Methods: Some podophyllotoxin derivatives were analyzed by quantum and mechanics method. Results: Some information was given according to the calculation results about HOMO and LUMO electron density. The C-4 position is the position for effective modification. The B ring and E ring are important active centers. Conclusion: The hole of positive charge in B ring easily combines with an acceptor within the molecular. Some quinolones with similar electronic construction to podophyllotoxin may have antitumor activity.

Synthesis and Characterization of a Novel Podophyllotoxin Derivative Containing a Cyclohexadienone Group

4β-Azido-4-demethyl-4＇-O-demethyl-epipedophyllotoxin reacted with phenyliodonium diacetate in MeOH at room temperature to synthesize a novel pedophyllotoxin derivative containing a cyclohexadienone group with the original configurations of C2 and C4, which are required for the antitumor activities. The component and structure of the pro-duct were confirmed using elemental analysis, high-resolution mass spectrum, ^1H and ^13C NMR spectra, and infrared spectrum. Furthermore, the fragmentation routes of the product were fully assigned with electron-impaet time-of-flight mass spectrometry（ EI-TOF MS）, which can also be used for the structural elucidation based on the molecular ion at m/z 455（16% ） and three novel characteristic fragment ions at m/z 183（37% ）, 425（7% ）, and 199（5% ）. The cyclohexadienone product is sensitive to acidic media. When it is treated with a trace of acid, another novel derivative containing an orthoquinone moiety is formed, which links with the metabolism of the cancer inhihitors.

Stereoselective Synthesis and Anticancer Activities of New Podophyllotoxin Derivatives:4-β-Cyano-4-Deoxy-4'-Demethylepipodophyllotoxin and 4-β-Carboxyl-4-Deoxy-4'-Demethylepipodophyllotoxin

4-β-Cyano-4-deoxy-4'-demethylepipodophyllotoxin 4 was synthesized from 4'-de -methylepipodophyllotoxin and Me3SiCN in the presence of BF3· Et2O. 4-β-Carboxyl-4-deoxy-4'-demethylepipodophyllotoxin 5 was obtained by hydrolyzing 4 in HOAc. Both of them show very high anticancer activities against L1210 and KB cell lines in vitro.

Experimental Podophyllotoxin (Bajiaolian) Poisoning:Ⅲ. Biochemical Bases for Toxic Effects

The biochemical mechanism underlying the toxicity of podophyllotoxin is investigated.Previous studies from our laboratories suggested that hepatocytes were extremely sensitive to the toxicity of podophyllotoxin and a disruption of protein synthesis was suspected.Dose-response and time-course studies on the effects of podophyllotoxin on protein, RNA,and DNA syniheses on hepatocellular cultures were made. Inhibitions of protein, RNA,and DNA syntheses were demonstrated, and a direct correlated dose-response relationshipon such effects was also evident. Inhibition of protein synthesis appeared to be a direet toxic effect of podophyllotoxin and occurred independently from that of RNA. The reduction of DNA synthesis was reflected by a reduction of H3-thymidine incorporation,which is believed to be the consequence of inhibition on the mitotic activity by podophyllotoxin