Synthesis and biological activity of fluorinated analogues of the DAF-12 receptor antagonist 24-hydroxy-4-cholen-3-one.

The DAF-12 receptor is a ligand-activated transcription factor that in its ligand-bound form allows the expression of target genes needed to support the reproductive life cycle of the free-living nematode Caenorhabditis elegans, whereas unbound DAF-12 receptor leads to the developmentally arrested "dauer larvae", specialized for survival and dispersal. The endogenous ligands of the DAF-12 receptor are 3-keto-cholestenoic acids dubbed dafachronic acids. In a previous publication we reported that oxysterols with a shorter side chain (C24) modulate the DAF-12 receptor activity either as partial agonists or, in the case of the C24 alcohol 24-hydroxy-4-cholen-3-one, as an antagonist both in vitro and in vivo. Preliminary structure-activity relationships suggested that this activity profile could be improved with more lipophilic and less acidic functional groups at the end of the side chain. Thus, we have now synthesized two fluorine containing analogues in which the C-24 hydroxyl was replaced by a difluoromethyl group (regarded as a "lipophilic hydroxyl") or a difluoromethylidene group with similar lipophilicity but lacking the hydrogen bond donor capacity. Activity was evaluated in vitro using transactivation cell-based assays and in vivo by the effect on the development of wild-type C. elegans. The 24-difluoromethyl analogue retained the antagonist activity in vitro, being completely devoid of agonist activity and exhibited improved activity in vivo. The difluoromethylidene showed a slight antagonist tendency in vitro (statistically not significant), in the concentration range tested and was weakly active in vivo. None of the compounds were toxic, as treated worms recovered to normal development, when transferred to fresh media without added steroids.

Bufadienolide and spirostanol glycosides from the rhizomes of helleborusorientalis.

The rhizomes of Helleborus orientalis have been analyzed for the bufadienolide glycoside and spirostanol saponin constituents, resulting in the isolation of a new bufadienolide rhamnoside (1), along with two known bufadienolide glycosides (2 and 3) and five new spirostanol saponins (4-8). The structures of the new compounds were determined on the basis of extensive spectroscopic analysis, including 2D NMR, and the results of hydrolytic cleavage. The isolated compounds were evaluated for their cytotoxic activities against cultured tumor and normal cells.

The effect of three bufadienolide cardiac glycosides on contraction of isolated rat jejunum.

Three cardiac glycosides were screened for pharmacological effects on isolated rat jejunum. The contraction of rat jejunum with epoxyscillirosidin, a non-cumulative bufadienolide, and cotyledoside and tyledoside D, both cumulative neurotoxic bufadienolides were compared with methacholine. The results indicate that all three bufadienolides cause contraction of jejunal smooth muscle. When combined with atropine (1 x 10(-6) M) the response of epoxyscillirosidin and tyledoside D decreased, indicating suppression of a cholinergic response caused by the cardiac glycosides.

New insecticidal bufadienolide, bryophyllin C, from Kalanchoe pinnata.

Two insecticidal bufadienolides (1 and 2) were isolated from a methanol extract of the leaves of Kalanchoe pinnata by bioassay-guided fractionation. Compound 1 was identified as known bryophyllin A (bryotoxin C). The structure of new bufadienolide 2, named bryophyllin C, was determined by spectroscopic methods and the chemical transformation of 1. Compounds 1 and 2 showed strong insecticidal activity against third instar larvae of the silkworm (Bombyx mori), their LD50 values being evaluated as 3 and 5 microg/g of diet, respectively.

Limonoids from Azadirachta excelsa.

Activity-directed fractionation of a stem extract of Azadirachta excelsa using KB (human oral epidermoid carcinoma) cells led to the isolation of four meliacin-type limonoids. Two of these constituents were novel, namely, 2,3-dihydronimbolide and 3-deoxymethylnimbidate, and these were purified along with the known compounds, nimbolide and 28-deoxonimbolide. The structures of the new compounds were determined by spectroscopic methods. Nimbolide and 28-deoxonimbolide were broadly cytotoxic when evaluated against a panel of human cancer cell lines, while the two novel compounds were inactive in this regard. The defection of nimbolide and 28-deoxonimbolide as cytotoxic constituents was facilitated by an electrospray LC/MS dereplication procedure.

Ovine cardiac Na,K-ATPase: isolation by means of selective solubilization in Lubrol and the effect of 1 alpha,2 alpha-epoxyscillirosidin on this enzyme.

The inhibition of cardiac Na,K-ATPase by 1 alpha,2 alpha-epoxyscillirosidin is the principal cause of poisoning of cattle by the tulip, Homeria pallida. The ultimate goals of this study were to study the interaction between 1 alpha,2 alpha-epoxyscillirosidin and ovine Na,K-ATPase by means of inhibition and displacement binding studies. Ovine cardiac Na,K-ATPase was isolated in membrane-bound form by means of deoxycholate treatment, high-speed ultracentrifugation, NaI treatment and selective solubilization in Lubrol. The inhibition of ovine cardiac and commercial porcine cerebral cortex Na,K-ATPase by 1 alpha,2 alpha-epoxyscilirosidin and ouabain was studied using a discontinuous Na,K-ATPase assay. The binding of 1 alpha,2 alpha-epoxyscillirosidin, ouabain and digoxin to the above enzymes was compared using a displacement binding assay with [3H] oubain. The Lubrol-solubilized ovine cardiac Na,K-ATPase showed a specific activity of 0.3 U/mg with no ouabain insensitive activity. I50 values of 2.1 x 10(-8) and 2.7 x 10(-8) were obtained for the inhibition of this enzyme by 1 alpha,2 alpha-epoxyscillirosidin and ouabain, respectively. 1 alpha,2 alpha-Epoxyscillirosidin has a much higher KD value (1.5 x 10(-7) M), however, than ouabain (9.5 x 10(-9) M) and digoxin (1.7 x 10(-8) M) in displacement binding studies with [3H]ouabain. 1 alpha,2 alpha-Epoxyscillirosidin is a potent inhibitor of ovine cardiac Na,K-ATPase and is a slightly stronger inhibitor of the enzyme than ouabain. The anomalous result for the displacement of 1 alpha,2 alpha-epoxyscillirosidin from its receptor is either a result of different affinities that K+ has for the enzyme ouabain and enzyme-1 alpha,2 alpha-epoxyscillirosidin complexes or because of different complex stabilities of these complexes.

Bufadienolides and other constituents of Urginea sanguinea.

Fresh bulbs of Urginea sanguinea yielded stigmasterol, phloroglucinol, phloroglucinol 1-beta-D-glucopyranoside (phlorin), scillaren A, a novel compound 5 alpha-4,5-dihydroscillaren A (1), salicylic acid, and 3-hydroxy-4-methylbenzoic acid. The latter two showed weak antibacterial activity. The compounds were identified using spectroscopic techniques such as 1D and 2D NMR, EI-MS and FAB-MS.

Potent growth inhibitory activity of a novel Ornithogalum cholestane glycoside on human cells: induction of apoptosis in promyelocytic leukemia HL-60 cells.

Growth inhibitory activities of a novel 22-homo-23-norcholestane glycoside found in bulbs of Ornithogalum saundersiae were examined in vitro using human promyelocytic leukemia HL-60 cells, human T-lymphocytic leukemia MOLT-4 cells, and mitogen-stimulated human peripheral-blood mononuclear cells (PBMC). The growth of HL-60 cells and MOLT-4 cells was strongly suppressed in the presence of the glycoside; the IC50s of which were 21.0 and 18.0 nM, respectively. Suppressive effect of the glycoside on HL-60 cell growth appears to be mediated partially through induction of apoptosis which was demonstrated by the presence of DNA fragmentation of the leukemic cells. Flow cytometric analysis of glycoside-treated HL-60 cells also demonstrated apoptotic cells with low DNA content and showed a decrease of G0/G1 cells and a concomitant increase of S and/or G2M cells. The growth inhibiting effect of the glycoside on HL-60 cells was promoted by calcium and was inhibited in the presence of zinc, which support involvement of endonuclease activation in the glycoside-induced apoptosis. The glycoside also inhibited mitogen-stimulated blastogenesis of PBMC, the IC50 of which was 6.2 nM. These results provided the first evidence ever for the potent growth inhibitory activity of Ornithogalum glycoside on human leukemia cell lines and PBMC.

Inhibition of androgen synthesis by 22-hydroximino-23,24-bisnor-4-cholen-3-one.

The 22-hydroximino-23,24-bisnor-4-cholen-3-one (22-oxime) was synthesized and evaluated as an inhibitor of 17 alpha-hydroxylase/C17,20-lyase in rat testicular microsomes and the 5 alpha-reducatase of human prostatic microsomes from patients with benign prostatic hypertrophy. The 22-oxime demonstrated moderate inhibition for the 17 alpha-hydroxylase (Ki 74 nM vs. Km 29 nM) with progesterone as substrate and potent inhibition (Ki 18 nM vs. Km 76 nM) for the C17,20-lyase activity with 17 alpha-hydroxyprogesterone as substrate. Further investigation of this enzyme with progesterone as substrate demonstrated the inhibition occurred mainly at the 17 alpha-hydroxylation step of the progesterone substrate. The 22-oxime also demonstrated potent and competitive inhibition of 5 alpha-reductase in human prostatic microsomes (Ki 1.4 nM vs. Km 14 nM). When adult male rats were injected subcutaneously (sc) daily with 22-oxime (50 mg/kg/day) for 21 days, the concentrations of serum and testicular testosterone were significantly reduced by 65% and 59%, respectively, in comparison to vehicle-treated controls. Furthermore, both testosterone and DHT concentrations in rat prostatic tissue were significantly decreased by 60% and 44% compared to control tissue. Serum LH concentrations were unchanged in the 22-oxime-treated group compared to the control group. This indicates that the reduction in androgen concentrations in animals treated with this compound is not due to its influence on pituitary feedback mechanisms which result in reduced LH secretion. These results suggest that 22-oxime is effective in reducing androgen synthesis through the inhibition of 17 alpha-hydroxylase, C17,20-lyase, and 5 alpha-reductase both in vitro and in vivo.

Inhibitors of sterol synthesis. Synthesis and spectral properties of 3 beta-hydroxy-24-dimethylamino-5 alpha-chol-8(14)-en-15-one and its effects on HMG-CoA reductase activity in CHO-K1 cells.

A simple, three-step synthesis of the 25-aza analog of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I) is described. Treatment of 3 beta-acetoxy-24-hydroxy-5 alpha-chol-8(14)-en-15-one (III) with 1.75 equivalents of tosyl chloride in pyridine for 24 h at 5 degrees C gave 3 beta-acetoxy-24-tosyloxy-5 alpha-chol-8(14)-en-15-one (IV). In contrast, treatment of III with 3.95 equivalents of tosyl chloride in pyridine for 12 h at 48 degrees C gave 3 beta-acetoxy-24-chloro-5 alpha-chol-8(14)-en-15-one (V). Treatment of IV with dimethylamine in dioxane yielded 3 beta-acetoxy-24-dimethylamino-5 alpha-chol-8(14)-en-15-one (VI). Hydrolysis of VI with LiOH.H2O in methanol gave 3 beta-hydroxy-24-dimethylamino-5 alpha-chol-8(14)-en-15-one (VII). 1H- and 13C-NMR assignments are presented for compounds IV-VII. The 25-aza analog (VII) of the 15-ketosterol (I), at a concentration of 1.0 microM, caused a 47% lowering of the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in Chinese hamster ovary cells.

Spectroscopic and biological investigation of nimbolide and 28-deoxonimbolide from Azadirachta indica.

The reisolation of nimbolide [1] from Azadirachta indica of Tanzanian origin and the isolation and structure elucidation of a new limonoid, 28-deoxonimbolide [2], from the same plant source are reported. For the first time, unambiguous 1H- and 13C-nmr assignments of compounds 1 and 2 are presented, as well as their in vitro cytotoxic activity against human tumor cell lines.

Monohydroxy bile salt sulfates: tauro-3 beta-hydroxy-5-cholenoate-3-sulfate induces intrahepatic cholestasis in rats.

The biologic effects of sulfation of tauro-3 beta-hydroxy-5-cholenoate and of taurolithocholate were compared. Equimolar amounts (100 nmol/min X 100 g body wt) of the following were administered intravenously to male Sprague-Dawley rats over a 180-min period: taurolithocholate, [14C]taurolithocholate-3-sulfate, tauro-3 beta-hydroxy-5-cholenoate, [14C]tauro-3 beta-hydroxy-5-cholenoate-3-sulfate, its combination with taurocholate, and a saline-albumin solution (control). Sulfation of taurolithocholate and of tauro-3 beta-hydroxy-5-cholenoate only prevented the cholestatic effect of the former. Bile flow during infusion of [14C]tauro-3 beta-hydroxy-5-cholenoate-3-sulfate was reduced by 80% at the end of the experiment. A dose-dependent bile flow reduction was demonstrated. Recovery of the administered bile acid was 3% in urine, 13% in serum, 23% in the liver tissue, and 52% in bile, respectively. Excretion of biliary cholesterol and phospholipids was significantly reduced during the first hour of infusion. Coadministration of taurocholate abolished the cholestatic effect and enhanced the renal excretion of the sulfated bile salt. These data suggest that (a) the cholestatic effect of tauro-3 beta-hydroxy-5-cholenoate-3-sulfate is comparable with or may even exceed the effect of taurolithocholate and (b) although sulfation renders some bile salts more water soluble, it does not prevent the cholestatic effect of all monohydroxy bile salts.

Oxidation of furans. 3. Estrogenic properties of lactones and anhydrides derived from the oxidation of 17alpha-[3-furyl]estradiol derivatives.

The oxidation of 17alpha-[3-furyl]estradiol derivatives and the estrogenic properties of the resulting isomeric 17,21-and 17,23-dihydroxy-19,24-dinor-17alpha-chola-1,3,5(10)20(22)-tetraenoic acid(20leads to 23) gamma-lactones as well as those of the related 17-hydroxy-19-nor-17alpha-pregna-1,3,5(10)-triene-20,21-dicarboxylic acid anhydrides and gamma-lactones are described. Of these, only lactones 3c,e,h and 5b retained the same degree and profile of estrogenic activity as the starting 17alpha-[3-furyl]estradiols.