Synthesis and biological studies of steroidal pyran based derivatives.

Steroid based cancer chemotherapeutic agents of the type 2'-amino-3'-cyanocholest-6-eno[5,7-de]4H-pyrans (1c-3c) have been synthesized and characterized by the various spectroscopic and analytical techniques. The DNA binding studies of compounds (1c-3c) with CT DNA were carried out by UV-vis and fluorescence spectroscopy and gel electrophoresis. The compounds (1c-3c) bind to DNA preferentially through electrostatic and hydrophobic interactions with Kb values found to be 5.4 × 10(3), 2.3 × 10(3)M(-1) and 1.97 × 10(3)M(-1), respectively indicating the higher binding affinity of compound (1c) towards DNA. The molecular docking study suggested that the electrostatic interaction of compounds (1c-3c) in between the nucleotide base pairs is due to the presence of pyran moiety in steroid molecule. All the compounds (1c-3c) cleave supercoiled pBR322 DNA via hydrolytic pathway, as validated by T4 DNA ligase assay. The compounds (1c-3c) were screened for in vitro cytotoxicity against the cancer and non-cancer cells SW480, A549, HepG2, HeLa, MCF-7, HL-60, DU-145, NL-20, HPC and HPLF by MTT assay. The compounds (1c-3c) were tested for genotoxicity (comet assay) involving apoptotic degradation of DNA and was analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. The results revealed that compound (1c) has better prospectus to act as cancer chemotherapeutic candidate which warrants further in vivo anticancer investigations.

First synthesis of 3,16,20-polyoxygenated cholestanes, new cytotoxic steroids from the gorgonian Leptogorgia sarmentosa.

Using tigogenin as starting material, (20S)-20-hydroxycholestane-3,6-dione (1), (16S, 20S)-16,20-dihydroxycholestan-3-one (2), (20S)-20-hydroxycholest-1-ene-3,16-dione (3) and (20S)-20-hydroxycholest-4-ene-3,16-dione (4), natural polyoxygenated steroids from the gorgonian, Leptogorgia sarmentosa, were synthesized in four steps. Antitumor activity against three tumor cell lines (breast cancer, MCF7, lung cancer NCI and oral cancer KB) was evaluated. Two compounds (3 and 4) showed strong activity against NCI (IC(50) 6.16 and 10.51 microM) and moderate activity against MCF7 and KB, the IC(50) being in the range 30.65-47.22 microM. Compound 2 showed moderate activity against NCI (IC(50) 42.68 microM) but was inactive against MCF7 and KB whereas compound 1 showed no activity against all tested cells.

Cytotoxic cholestane and pregnane glycosides from Tribulus macropterus.

The methanol extract of the whole parts of Tribulus macropterus Boiss. (family Zygophyllaceae) showed cytotoxic activity against a human tumour cell line (hepatocyte generation 2, HepG2) (IC50 = 2.9 microg/ml). The n-butanolic fraction obtained from successive fractionation of the methanolic extract exhibited activity against HepG2 (IC50 = 2.6 microg/ml). Therefore, this fraction was subjected to separation using different chromatographic techniques. Five compounds, 1-5, were isolated and identified as: (22S,25S)-16beta,22,26-trihydroxy-cholest-4-en-3-one-16-O-beta-D-glucopyranosyl-(1-->3)-beta-D-xylopyranoside (1), (22S,25S)-16beta,22,26-trihydroxy-cholest-4-en-3-one-16-O-beta-D-glucopyranosyl-(1-->3)-beta-D-glucopyranoside (2), sucrose (3), D-pinitol (4) and 3beta-hydroxy-5a-pregn-16(17)en-20-one-3-O-beta-D-xylopyranosyl-(1-->2)-[beta-D-xylopyranosyl-(1-->3)]-beta-D-glucopyranosyl-(1-->4)-[alpha-L-rhamnopyranosyl-(1-->2)]-beta-D-ga-lactopyranoside (5) on the basis of spectroscopic and chemical data. The three steroidal compounds 1, 2 and 5 were also tested against the same cell line HepG2 and their IC50 values were 2.4, 2.2 and 1.1 microg/ml, respectively.

[Trofosides A and B and other cytostatic steroid-derived compounds from the Far East starfish Trofodiscus über].

Three new polar steroids identified as trofoside A, (20R,24S)-24-O-(3-O-methyl-beta-D-xylopyranosyl)-3beta,6alpha,8,15beta,24-pentahydroxy-5alpha-cholestane, its 22(23)-dehydro derivative (trofoside B), and 15-sulfoxy-(20R,24S)-5alpha-cholestane-3beta,6beta,8,15alpha,24-pentaol sodium salt, were isolated from Trofodiscus uber starfish extracts collected in the Sea of Okhotsk. Two known compounds, trofoside A aglycone, (20R,24S)-3beta,6alpha,8,15beta,24-pentahydroxy-5alpha-cholestane, and triseramide, (20R,24R,25S,22E)-24-methyl-3beta,6alpha,8,15beta-tetrahydroxy-5alpha-cholest-22-en-27-oic acid (2-sulfoethyl)amide sodium salt, were also found. The structures of the isolated polyoxysteroids were established from their spectra. Minimal concentrations causing degradation of unfertilized egg-cells of the sea-urchin Strongylocentrotus intermedius (C(min)) and terminating the cell division at the stage of the first division (C(min) embr.), as well as the concentrations causing 50% immobilization of sperm cells (ImC50) and inhibiting their ability to fertilize egg-cells by 50% (IC50) were determined for the isolated compounds. Of three compounds highly toxic in embryos and sea-urchin sperm cells, the polyol with a sulfo group in the steroid core was the most active; two glycosides with monosaccharide chains located at C3 and C24 atoms were less toxic. Note that all the compounds with the spermiotoxic activities differently affected the embryo development. The positions of monosaccharide residues in the core considerably influence the compound activity. For example, both mono- and double chained glycosides with the monosaccharide fragment at C3 and C24 atoms are active against sea-urchin sperm cells and embryos, whereas the C24 glycosylated trofoside A does not affect embryos and displays a poor spermiotoxicity.

Ozonation of cholesterol in the presence of ethanol: identification of a cytotoxic ethoxyhydroperoxide molecule.

Cholesterol ozonation was carried out in ethanol-containing aqueous or nonaqueous solvent, and the ozonized products were analyzed by chemiluminescence detection-HPLC with on-line electrospray MS (HPLC-CL-MS) and characterized on the basis of NMR and FABMS. After the ozonolysis of cholesterol in water/ethanol (aqueous system) as well as in chloroform/ethanol (nonaqueous system), a unique ethoxyhydroperoxide molecule (7alpha-ethoxy-3beta-hydroxy-5alpha-B-homo-6-oxacholestane-5-hydroperoxide, termed "7alpha-ethoxy-5-OOH") appeared as main ozonation product. In addition to structural analysis, we confirmed the remarkable cytotoxicity of 7alpha-ethoxy-5-OOH toward human lung adenocarcinoma A549 cells and found that its cytotoxicity is superior to that of the commonly known autoxidized cholesterol (3beta-hydroxycholest-5-ene-7-one). Hence, 7alpha-ethoxy-5-OOH is a toxic molecule of primary importance, arising during cholesterol ozonation in the presence of ethanol.

[Oxysterol (3,5-cholestadien-7-one, 5 beta-cholestan-3-one, 5,24-cholestadien-3 beta-OL) induced cytotoxicity and apoptosis in gallbladder epithelial cells].

BACKGROUND/AIMS: Biliary epithelial cells are exposed to highly concentrated oxysterols. Therefore, oxysterols may play a role in pathogenesis of biliary tract diseases. We investigated the cytotoxic effect and apoptosis inducing effect of oxysterol on gallbladder epithelial cells. METHODS: We studied the cytotoxic effect of 3,5- cholestadien-7-one, 5 beta-cholestan-3-one and 5,24-cholestadien-3 beta-OL which are identified in human bile and pigment gallstones on dog gallbladder epithelial cells (DGBE) and mouse gallbladder epithelial cells (MGBE). We used model bile to dissolve oxysterols as in vitro experiment and also used MTT, cell count, Diff-Quick stain, and flow cytometry to investigate cytotoxicity and apoptosis. RESULTS: Oxysterols dissolved in model bile have cytotoxic effects in a dose dependent fashion. In oxysterol containing model bile, viable cells are 51% in 500 microM 5 beta-cholestan-3-one (cholesterol:oxysterol 50:50) and 47% in 5 mM 3,5-cholestadien-7-one (90:10) on MGBE, and are 129% and 38% in 500 microM (50:50) 3,5-cholestadien-7-one and 5 beta-cholestan-3-one on DGBE, and are 74% and 71.5% in 5 mM (90:10) 3,5-cholestadien-7-one and 5 beta-cholestan-3-one on DGBE, respectively. 500 microM (50:50) 3,5- cholestadien-7-one, 5 beta-cholestan-3-one, and 5,24-cholestadien-3 beta-OL treated on DGBE increase the apoptotic cell number as 22.0+/-8.8, 30.2+/-12.6, and 45.5+/-13.2%, respectively, compared with control (14.6+/-10.0%). 500 microM (50:50) 3,5-cholestadien-7-one, 5 beta-cholestan-3-one, and 5,24-cholestadien-3 beta-OL also affect the changes in cell cycles compared with the control. CONCLUSIONS: We concluded that oxysterol containing model bile is useful as an in vitro experiment as model to analyze the effects of oxysterols on biliary epithelial cells and that adequate concentration of oxysterols can induce the cytotoxic effect and the apoptosis on gallbladder epithelial cells.

Oxysterol (3,5-cholestadien-7-one, 5beta-cholestan-3-one, 5,24-cholestadien-3beta-OL) Induced Cytotoxicity and Apoptosis in Gallbladder Epithelial Cells / 대한소화기학회지

BACKGROUND/AIMS: Biliary epithelial cells are exposed to highly concentrated oxysterols. Therefore, oxysterols may play a role in pathogenesis of biliary tract diseases. We investigated the cytotoxic effect and apoptosis inducing effect of oxysterol on gallbladder epithelial cells. METHODS: We studied the cytotoxic effect of 3,5- cholestadien-7-one, 5beta-cholestan-3-one and 5,24-cholestadien-3beta-OL which are identified in human bile and pigment gallstones on dog gallbladder epithelial cells (DGBE) and mouse gallbladder epithelial cells (MGBE). We used model bile to dissolve oxysterols as in vitro experiment and also used MTT, cell count, Diff-Quick stain, and flow cytometry to investigate cytotoxicity and apoptosis. RESULTS: Oxysterols dissolved in model bile have cytotoxic effects in a dose dependent fashion. In oxysterol containing model bile, viable cells are 51% in 500 microM 5beta-cholestan-3-one (cholesterol : oxysterol 50:50) and 47% in 5 mM 3,5-cholestadien-7-one (90:10) on MGBE, and are 129% and 38% in 500 microM (50:50) 3,5-cholestadien-7-one and 5beta-cholestan-3-one on DGBE, and are 74% and 71.5% in 5 mM (90:10) 3,5-cholestadien-7-one and 5beta-cholestan-3-one on DGBE, respectively. 500 microM (50:50) 3,5- cholestadien-7-one, 5beta-cholestan-3-one, and 5,24-cholestadien-3beta-OL treated on DGBE increase the apoptotic cell number as 22.0+/-8.8, 30.2+/-12.6, and 45.5+/-13.2%, respectively, compared with control (14.6+/-10.0%). 500 microM (50:50) 3,5-cholestadien-7-one, 5beta-cholestan-3-one, and 5,24-cholestadien-3beta-OL also affect the changes in cell cycles compared with the control. CONCLUSIONS: We concluded that oxysterol containing model bile is useful as an in vitro experiment as model to analyze the effects of oxysterols on biliary epithelial cells and that adequate concentration of oxysterols can induce the cytotoxic effect and the apoptosis on gallbladder epithelial cells.

Cholestane glycosides from the bulbs of Galtonia candicans and their cytotoxicity.

Further search for cytotoxic compounds contained in the bulbs of Galtonia candicans (Liliaceae) led to the isolation of four potent cytotoxic cholestane glycosides (1-4) based upon 3beta,16beta,17alpha-trihydroxycholest-5-en-22-one, three of which (2-4) have not been reported previously. A new cholestane bisdesmoside (5) and a new rearranged cholestane glycoside (6) were also isolated. The structural assignment of the new constituents was carried out by spectroscopic analysis and a few chemical transformations.

Damage and mutagenesis of E. coli and bacteriophage lambda induced by oxathiolane and aziridinyl steroids.

lambda-Escherichia coli complexes exhibited remarkable sensitivity to the treatment with test steroidal derivatives in the presence of Cu(II). The decline in plaque-forming units after steroid treatment was more pronounced in complexes with some of the radiation repair-defective mutants of E. coli K-12, i.e., recA, lexA and polA, as compared to uvrA and wild-type strains. The red gene of lambda phage and recA gene of E. coli seem to have a complementary effect on the steroid-induced lesions. An enhanced level of mutagenesis was observed when steroid-treated E. coli cells were transformed with steroid-treated pBR322 plasmid DNA. A remarkable degree of c mutation was also observed when steroid I-treated phage particles were allowed to adsorb on steroid-treated wild-type bacteria. Moreover, the oxathiolane steroid treatment of lambda cI857-E. coli lysogen resulted in prophage induction in nutrient broth even at 32 degrees C. Thus on the basis of these results, the role of SOS repair system in steroid-induced mutagenesis and repair of DNA lesions in E. coli and bacteriophage lambda has been suggested.

Steroid induced single strand breaks in DNA mediated by active oxygen species and its biological consequences.

A mutagenic steroidal derivative (3 beta-Acetoxy-5 alpha-Cholestano[6 alpha,5-d']1'-3'oxathiolane-2' thione) structurally related to cholesterol caused strand scission and induced nicks in calf thymus, supercoiled pBR322 and single stranded M13 mp8 phage DNAs. S1 nuclease hydrolysis, reaction with pBR322 and M13 phage DNA as well as treatment of E. coil mutant strains and phage was used to evaluate the effect of test steroid on the DNA molecule. The strand scission/nicking of DNA by the test steroid was enhanced by some metal ions, especially the Cu(II). Scavengers of active oxygen radical species significantly inhibited the S1 nuclease hydrolysis by the test steroid indicating the major role of active oxygen species in DNA strand scission and nicking. The steroid brought about the DNA degradation even in the absence of S1 nuclease. There was an appreciable reduction in the survival of steroid treated polA and lig mutants of E. coli K12 compared to the wild type strain. Phage on steroid treatment also lost its plaque forming units (P.F.U.) which was more pronounced in the polA and rec A background.

Mutagenic activity of oxathiolane steroids: structural requirement for the genotoxic activity in Salmonella and E. coli.

Oxathiolanes and disulfonyl derivatives of steroids were tested for mutagenic activity in the Ames tester strains. The test compounds exhibited mutagenic activity without metabolic activation although metabolic activation markedly enhanced their activity. A significant decrease in the survival of the radiation-sensitive mutants recA, lexA and rer of E. coli was observed as compared to their wild-type counterpart in the presence of the test steroid. Structural features which appear to be crucial for the mutagenic activity in these steroidal drugs are: (i) an electron-donating group at position 3, and (ii) a bulky group anchored at the 5th and 6th positions. The test steroids appear to damage DNA which in turn initiates the SOS repair with the concomitant induction of mutation.

Genotoxicity of two fecal steroids in murine colonic epithelium assessed by the sister chromatid exchange technique.

Two components of human feces are known to induce nuclear anomalies in mice when applied intrarectally, but to be nonmutagenic in Salmonella. We have tested these two compounds for their ability to induce sister chromatid exchanges in the colonic epithelium of mice, the same tissue in which they induce nuclear anomalies when administered by the same route. One, 4-cholesten-3-one, induced sister chromatid exchanges whereas the other, 5-alpha-cholestan-3-one did not, even at the maximum feasible dose. The results suggest that 4-cholesten-3-one is more likely to be a significant factor in human colon cancer than the 5-alpha analog.