Effect of protein content on low temperature inactivation of the extracellular proteinase from Pseudomonas fluorescens 22F

The formation of cholic acid and chenodeoxycholic acid through cleavage of the side chains of CoA esters of 3alpha,7alpha,12alpha-trihydroxy-5beta-choles tan-26-oic acid and 3alpha,7alpha-dihydroxy-5beta-cholestan-26-oic acid is believed to occur in peroxisomes. Recently, we found a new peroxisomal enzyme, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and suggested that this bifunctional protein is responsible for the conversion of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-2 4-en-26-oyl-CoA and 3alpha,7alpha-dihydroxy-5beta-cholest-24-en-26-oyl-CoA to their 24-oxo-forms. In the present study, the products of this bifunctional protein reaction were analyzed by gas chromatography-mass spectrometry, and the formation of 24-oxo-27-nor-cholestanes was confirmed. Previously, we found a new thiolase in Caenorhabditis elegans, P-44, and suggested that P-44 and sterol carrier protein x, a peroxisomal protein, constitute a second group of 3-oxoacyl-CoA thiolases. The production of cholic acid and chenodeoxycholic acid from the precursors on incubation with the bifunctional protein and sterol carrier protein x or P-44 was confirmed by gas chromatography.     

Substrate specificity of THCA-CoA oxidases from rat liver light mitochondrial fractions on dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid CoA thioester

The substrate specificity of rat liver peroxisomal 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) oxidases, which catalyze the dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) CoA thioester, having an asymmetric center at C-25, to form (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) CoA thioester, was studied. The stable isotope labeled substrates, [3,7,12-18O3]-(25R)- and (25S)-THCA CoA thioesters were synthesized by an exchange reaction of carbonyl oxygens on a steroid nucleus of 3,7,12-trioxo-5 beta-cholestanoic acid, followed by metal hydride reduction and condensation reaction with CoA. After incubation of a mixture of unlabeled (25R)- and 18O-labeled (25S)-THCA CoA thioester, or vice versa, with hepatic peroxisomal THCA-CoA oxidases, biotransformed delta 24-THCA was determined by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. The delta 24-THCA was derived only from (25S)-THCA CoA thioester, indicating that the 25S epimer of THCA is a preferential substrate on dehydrogenation by THCA-CoA oxidases.     

Formation of varanic acid, 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid from 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid in Bombina orientalis

Varanic acid (3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid; 24-OH-THCA) is almost the sole component of bile acids in the bile of Bombina orientalis. To examine in the mechanism of the formation of 24-OH-THCA, radiolabeled (25R)- and (25S)-3 alpha, 7 alpha, 12 alpha-trihdroxy-5 beta-cholestanoic acids [(25R)- and (25S)-THCA] and (24E)-3 alpha, 7 alpha, 12 alpha-trihdroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) were administered intraperitoneally to B. orientalis, gallbladder bile was collected after 24 h, and bile acids were subsequently extracted. Then the bile acids were analyzed by means of radio thin-layer chromatography and radio high-performance liquid chromatography after conversion to p-bromophenacyl ester derivatives. Although delta 24-THCA was not converted to 24-OH-THCA, (25R)-THCA and (25S)-THCA were transformed to (24R,25R)-24-OH-THCA and (24R,25S)-24-OH-THCA, respectively. These results strongly suggest that 24-OH-THCA was transformed via direct hydroxylation of the saturated side chain of THCA, not via hydration to an alpha, beta-unsaturated acid, delta 24-THCA, in B. orientalis.     

Sensitive analysis of serum 3alpha, 7alpha, 12alpha,24-tetrahydroxy- 5beta-cholestan-26-oic acid diastereomers using gas chromatography-mass spectrometry and its application in peroxisomal D-bifunctional protein deficiency

The final steps in bile acid biosynthesis take place in peroxisomes and involve oxidative cleavage of the side chain of C27-5beta-cholestanoic acids leading to the formation of the primary bile acids cholic acid and chenodeoxycholic acid. The enoyl-CoA hydratase and beta-hydroxy acyl-CoA dehydrogenase reactions involved in the chain shortening of C27-5beta-cholestanoic acids are catalyzed by the recently identified peroxisomal d-bifunctional protein. Deficiencies of d-bifunctional protein lead, among others, to an accumulation of 3alpha,7alpha,12alpha, 24-tetrahydroxy-5beta-cholest-26-oic acid (varanic acid). The ability to resolve the four C24, C25 diastereomers of varanic acid has, so far, only been carried out on biliary bile acids using p -bromophenacyl derivatives. Here, we describe a sensitive gas chromatography-mass spectrometry (GC/MS) method that enables good separation of the four varanic acid diastereomers by use of 2R-butylester-trimethylsilylether derivatives. This method showed the specific accumulation of (24R,25R)-varanic acid in the serum of a patient with isolated deficiency of the d-3-hydroxy acyl-CoA dehydrogenase part of peroxisomal d-bifunctional protein, whereas this diastereomer was absent in a serum sample from a patient suffering from complete d-bifunctional protein deficiency. In samples from both patients an accumulation of (24S,25S)-varanic acid was observed, most likely due to the action of l-bifunctional protein on Delta24E-THCA-CoA. This GC/MS method is applicable to serum samples, obviating the use of bile fluid, and is a helpful tool in the subclassification of patients with peroxisomal d-bifunctional protein deficiency.     

Comparison of side chain oxidation of potential C27-bile acid intermediates between mitochondria and peroxisomes of the rat liver: presence of beta-oxidation activity for bile acid biosynthesis in mitochondria

The oxidation of the side chains of two potential bile acid intermediates, 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) and 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA), were investigated in rat liver mitochondria and peroxisomes. Both THCA and DHCA were efficiently oxidized to yield cholic acid and chenodeoxycholic acid, along with 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid and 3 alpha,7 alpha-dihydroxy-5 beta-cholest-24-enoic acid, respectively, in both the mitochondria and peroxisomes. However, the spectrum of the metabolites in the mitochondria differed greatly from those in the peroxisomes. The major products from THCA and DHCA in the mitochondria were 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-chol-22-enoic acid and 3 alpha,7 alpha-trihydroxy-5 beta-chol-22-enoic acid, respectively, which were tentatively identified from the mass spectral data. However, the formation of these C24-unsaturated bile acids was not observed in the peroxisomes. These results strongly suggest that the cleavage of the side chain of the C27-intermediates for bile acid biosynthesis also occurs independently in the mitochondria, not due to the contamination of peroxisomes.     

Metabolism and choleretic activity of homochenodeoxycholic acid in the hamster

The hepatic metabolism and the choleretic effect of homochenodeoxycholic acid, the C25 homologue of chenodeoxycholic acid, were investigated in the hamster. After intravenous administration of 3H-labeled homochenodeoxycholic acid into biliary fistula hamsters, more than 80% of the radioactivity was recovered in bile in 4 h. A relatively small proportion of homochenodeoxycholic acid was present in bile as the taurine (22%) or glycine (4%) conjugate. However, more than 70% of the administered compound was biotransformed into C23 bile acids. The major C23 metabolites in bile were norchenodeoxycholic acid (17%), tauronorchenodeoxycholic acid (33%), and a trihydroxy norbile acid (identified as 3 alpha, 5 beta, 7 alpha-trihydroxy-24-nor-5 beta-cholan-23-oic acid, 19%). Small amounts (< 5%) of sulfate(s) and glucuronide(s) were also detected. Homochenodeoxycholic acid, when infused intravenously into the hamster, produced a striking choleresis. The increase in bile flow after infusion of this compound was 6- to 7-times that induced by chenodeoxycholic acid. The apparent choleretic activity of homochenodeoxycholic acid, 181 microliters/mumol, was much greater than that of chenodeoxycholic acid, 11 microliters/mumol. In conclusion, homochenodeoxycholic acid induced a hypercholeresis of the same order of magnitude as norchenodeoxycholic acid, presumably because considerable proportions of this compound were degraded to the hypercholeretic norchenodeoxycholic acid via beta-oxidation in the liver.     

5 beta-hydroxylation by the liver. Identification of 3,5,7-trihydroxy nor-bile acids as new major biotransformation products of 3,7-dihydroxy nor-bile acids in rodents

24-Norursodeoxycholic acid (nor-UDCA), when administered into the anesthetized biliary fistula hamster or injected into the perfusate of an isolated liver, was hydroxylated at C-5 to give 5 beta-hydroxynorursodeoxycholic acid 2 (3 alpha,5,7 beta-trihydroxy-24-nor-5 beta-cholan-23-oic acid), which was secreted into bile mainly as such. Similarly, 24-norchenodeoxycholic acid (nor-CDCA) was 5 beta-hydroxylated to give 5 beta-hydroxynor-chenodeoxycholic acid 4 (3 alpha,5,7 alpha-trihydroxy-24-nor-5 beta-cholan-23-oic acid), which was also secreted into bile without appreciable further biotransformation. The site of hydroxylation was assigned by 13C and 1H NMR and mass spectrometry. 5-Hydroxylation was a major biotransformation pathway at physiological bile acid loads. 5-Hydroxylation of UDCA also occurred in the perfused rat liver but to a lesser extent. 5-Hydroxylation of nor-UDCA was not observed in rabbit, dog, or man, indicating that its formation is species-specific. 5-Hydroxylation of nor-CDCA and nor-UDCA is the first reported example of hydroxylation of a tertiary carbon atom of bile acids. Nor-dihydroxy bile acids appear to be useful for the detection of minor hydroxylation pathways, because their prolonged hepatobiliary retention exposes them repeatedly to hydroxylases present in the hepatobiliary system.     

Uptake of 3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate into isolated rat hepatocytes by three transport systems

Uptake of norcholansulfonate (3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate), an isogeometric analogue of cholate into isolated rat liver hepatocytes occurs only by saturable transport. In order to identify the transport systems involved, uptake of norcholansulfonate was studied using 7 beta-NBD-NCT ({N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-7 beta-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl})-2'-aminoethanesulfonate) as a competing substrate. For transport of both bile salt derivatives, which mutually inhibit their mediated transport competitively, the existence of at least three transport systems must be assumed. Uptake studies using the cloned hepatic Na+/cholyltaurine cotransporting polypeptide stably expressed in CHO cells (Chinese hamster ovary cells) showed that both bile salt derivatives were transported and furnished the definite KT values of this single transport system and the ratio of the maximal uptake velocities. On the basis of these data, uptake of both bile salt derivatives into rat hepatocytes and their mutual competitive inhibition could be analyzed for three transport systems. The maximal flux rates J2 and the half-saturation constants KT2 in the presence of Na+ (143 mM) are for norcholansulfonate: J1(Na+ 143) = 1.0 +/- 0.2 nmol/(min . mg protein), KT1(Na+ 143) = 15 +/- 4 microM, J2(Na+ 143) = 0.5 +/- 0.2 nmol/(min.mg protein), KT2(Na+ 143) = 15 +/- 2 microM, J3(Na+ 143) = 0.5 +/- 0.2 nmol/(min.mg protein), KT3(Na+ 143) = 60 +/- 15 microM, and for 7 beta-NBD-NCT J1(Na+ 143) = 0.14 +/- 0.04 nmol/(min.mg protein), KT1(Na+ 143) = 3.1 +/- 0.5 microM, J2(Na+ 143) = 0.014 +/- 0.005 nmol/(min.mg protein), KT2(Na+ 143) = 21 +/- 2 microM, J3(Na+ 143) = 1.0 +/- 0.1 nmol/(min.mg protein), KT3(Na+ 143) = 190 +/- 25 microM. The kinetic parameters are in accordance with the assumptions that the cloned Na+/cholyltaurine cotransporting polypeptide represents transport system 2 and that the kinetically identified additional transport system 1 is either strictly or partially Na(+)-dependent.     

15 beta-hydroxysteroids (Part IV). Steroids of the human perinatal period: the synthesis of 3 alpha,15 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one and its A/B-ring configurational isomers

In recent years several 15 beta-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3 alpha,15 beta,17 alpha-trihydroxy-5 beta-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3 xi,5 xi-isomers, namely 3 alpha,15 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one (3), 3 beta,15 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one (7) and 3 beta,15 beta,17 alpha-trihydroxy-5 beta-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3 beta,15 beta-Diacetoxy-17 alpha-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15 beta,17 alpha-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15 beta-acetoxy-3 beta,17 alpha-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15 beta-acetoxy-3 beta,17 alpha-dihydroxy-5 alpha-pregnan-20-one (13) a common intermediate for the synthesis of the 3 beta(and alpha),5 alpha-isomers. Hydrolysis of the 15 beta-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15 beta-acetoxy-17 alpha-hydroxy-5 alpha-pregnan-3,20-dione (14) which on reduction with L-Selectride and hydrolysis of the 15 beta-acetate gave 3. Finally, hydrogenation of 4 gave 15 beta, 17 alpha-dihydroxy-5 beta-pregnan-3,20-dione (10) which on reduction with L-Selectride gave 8.     

Transformation of cholanic acid derivatives into pharmacologically active esters of phenolic acids by heterogeneous Wittig reaction

Steroid esters of cynnamic acid derivatives have been synthesized by a heterogeneous Wittig reaction under sonochemical conditions from the corresponding triphenylphosphonium bromides and unprotected phenolic aldehyds using K2CO3 as a base. 5 beta-Cholan-3 alpha, 7 alpha, 12 alpha, 24-E-ferulate (11') exhibited a marked inhibitory effect on influenza virus A. The synthetic 3 alpha, 24-E-diferulates of 5 beta-cholan-3 alpha, 24- diol, 5 beta-cholan-3 alpha, 12 alpha, 24-triol and 5 beta-cholan-3 alpha, 7 alpha, 12 alpha, 24-tetrol (8, 9 and 12) showed antitumor activity on leukemia P-388 in mice.     

Synthesis and biological evaluation of (23R)- and (23S)-24,24-difluoro-1 alpha,23,25-trihydroxyvitamin D3

The syntheses and biological evaluations of (23R)- and (23S)-24,24-difluoro-1 alpha,23,25-trithydroxyvitamin D3 (3a and 3b), new C-24 fluorinated analogs of 1 alpha,25-dihydroxyvitamin D3, are described. The syntheses of these compounds were achieved in 3 steps from (5Z,7E,20R)-1 alpha,3 beta-bis-[(tert-butyldimethylsilyl)oxy]-20-formylmethyl-9,10-seco- 5,7,10(19) pregnatriene (5) which is derived from vitamin D2. The absolute configuration at the C-23 position of 3a and 3b was determined by the modified Mosher method. The relative affinities of 3a and 3b to the vitamin D receptor were both 10 and 14 times lower than that of 1 alpha,25-dihydroxyvitamin D3 (1), and to vitamin D binding protein were also both 130 and 40 times lower. The HL-60 cell differentiating activity of 3a was 6 times more potent than that of 1, while there was no remarkable difference in activity between 3b and 1.     

Synthesis, stereochemistry, and biological activity of 1alpha,23,25-trihydroxy-24-oxovitamin D3, a major natural metabolite of 1alpha,25-dihydroxyvitamin D3

The C(23) epimers of 1alpha,23,25(OH)3-24-oxovitamin D3, a major natural metabolite of the secosteroid hormone, 1alpha,25(OH)2D3, were chemically synthesized for the first time. The metabolite was synthesized by palladium coupling of the appropriate CD ring analog with an A ring enyne. Various approaches from quinic acid to the A ring precursors were explored, and a new route to the A ring enyne from quinic acid was developed. The C(23) stereochemistry of the natural 1alpha,23,25(OH)3-24-oxovitamin D3 produced in neonatal human keratinocytes was determined to be S on the basis of the 1H NMR and the HPLC data. The biological activity of 1alpha,23(S), 25(OH)3-24-oxovitamin D3 in primary cultures of bovine parathyroid cells was determined by comparing the potency of this metabolite to that of 1alpha,25(OH)2D3 in suppression of parathyroid hormone (PTH) secretion. The results indicate that 1alpha,23(S), 25(OH)3-24-oxovitamin D3 potently suppressed PTH secretion even at concentrations as low as 10(-)12 M and is equipotent with 1alpha, 25(OH)2D3. The high activity of 1alpha,23(S),25(OH)3-24-oxovitamin D3 cannot be explained on the basis of its affinity for the vitamin D receptor as this metabolite was found to be 10 times less effective than radioinert 1alpha,25(OH)2D3 in blocking the uptake and receptor binding of [3H]-1alpha,25(OH)2D3 in intact parathyroid cells. Further studies are required to explain the molecular basis for the activity of 1alpha,23(S),25(OH)3-24-oxovitamin D3 in its ability to suppress PTH secretion. In summary, our present study indicates that the C(23) stereochemistry of the natural 1alpha,23, 25(OH)3-24-oxovitamin D3 is S and this metabolite is equipotent to 1alpha,25(OH)2D3 in suppressing PTH secretion.     

A GC-MS study of three major acidic metabolites of delta 1-tetrahydrocannabinol

The major urinary metabolite of delta 1-tetrahydrocannabinol (delta 1-THC) (1), delta 1-THC-7-oic acid (2), has been extensively studied for several purposes, including testing in the workplace for drug abuse. Immunoassays in combination with more specific methods such as gas chromatography-mass spectrometry (GC-MS), are commonly used for verification of positive results in the screening. Two additional and recently synthesized acidic metabolites of 1, 4",5"-bisnor-delta 1-THC-7,3"-dioic acid (3) and 4"-hydroxy-delta 1-THC-7-oic acid (4), were studied to widen the scientific basis in the analysis. Five different derivatives were examined using GC-MS. In addition, a new deuterated internal standard for 2, [2H10]-2, was evaluated. According to our results, suitable derivatives of 2, 3, and 4, according to chromatographic properties, are the methyl ester/silyl ether (procedure a), the methyl ester/trifluoroacetate (procedure b), or the silyl ester/silyl ether (procedure c). The estimated recoveries of [2H5]-3 and [2H6]-4 using liquid-liquid extraction were 24% and 50%, respectively. The properties of [2H10]-2 as internal standard were equivalent to those of [2H9]-2 and, under the conditions used, did not appear to give rise to a significantly higher chromatographic resolution from that of 2. However, [2H10]-2 produces ions at different mass numbers, which makes it useful as a complement to the existing deuterated internal standards of 2.     

The 16,17-double bond is needed for irreversible inhibition of human cytochrome p45017alpha by abiraterone (17-(3-pyridyl)androsta-5, 16-dien-3beta-ol) and related steroidal inhibitors

Abiraterone (17-(3-pyridyl)androsta-5,16-dien-3beta-ol, 1) is a potent inhibitor (IC50 4 nM for hydroxylase) of human cytochrome P45017alpha. To assist in studies of the role of the 16,17-double bond in its mechanism of action, the novel 17alpha-(4-pyridyl)androst-5-en-3beta-ol (5) and 17beta-(3-pyridyl)-16,17alpha-epoxy-5alpha-androst-3beta-ol (6) were synthesized. 3beta-Acetoxyetienic acid was converted in three steps into 5 via photolysis of the thiohydroxamic ester 8. Oxidation of an appropriate 16,17-unsaturated precursor (21) with CrO3-pyridine afforded the acetate (23) of 6. Inhibition of the enzyme by 1, the similarly potent 5,6-reduced analogue 19 (IC50 5 nM), and the 4, 16-dien-3-one 26 (IC50 3 nM) and by the less potent (IC50 13 nM) 3,5, 16-triene 25 is slow to occur but is enhanced by preincubation of the inhibitor with the enzyme. Inhibition following preincubation with these compounds is not lessened by dialysis for 24 h, implying irreversible binding to the enzyme. In contrast under these conditions the still potent (IC50 27 nM) 17alpha-(4-pyridyl)androst-5-en-3beta-ol (5) showed partial reversal after 5 h of dialysis and complete reversal of inhibition after 24 h. This behavior was also shown by the less potent 16,17-reduced 3-pyridyl compounds 3 and 24. Further, in contrast to the compounds (1, 19, 25, 26) with the 16,17-double bond, the inhibition of the enzymic reaction was not enhanced by preincubation either with 5 or with the 17beta-pyridyl analogues 3, 4, and 24 which also lack this structural feature. The results show that the 16,17-double bond is necessary for irreversible binding of these pyridyl steroids to cytochrome P45017alpha. However oxidation to an epoxide is probably not involved since epoxide 6 was only a moderately potent inhibitor (IC50 260 nM).     

Some characteristics of cytosol binding protein for 1alpha,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol in rat intestinal mucosa

Cytosol binding protein for 1alpha,25-(OH)2D3 and 25-OHD3 was demonstrated in rat intestinal mucosa using sucrose density gradient ultracentrifugation analysis, Sephadex G-200 column chromatography and polyacrylamide disc gel electrophoresis. The binding protein has a sedimentation constant of 5-6S, a molecular weight of 100,000-120,000 daltons and a mobility in electrophoresis of Rf 0.42. Further analysis of binding behavior by DEAE-cellulose filter assays revealed that the bindig protein had a higher affinity for 25-OHD3 than for 1alpha,25-(OH)2D3, and that 1alpha,25-(OH)2D3 competed with 25-OHD3 for the same binding site on the protein. The apparent dissociation constant for 1alpha,25-(OH)2D3 and 25-OHD3 was 9.2 X 10(-9) M and 1.5 X 10(-9) M, respectively. The binding capacity was 3.1 pmoles/mg protein for both 1alpha,25-(OH)2D3 and 25-OHD3. The order of binding affinity was 25-OHD3 greater than 1alpha,25-(OH)2D3 greater than D3 = 1alpha-OHD3.     

Steroidal saponins from the rhizomes of Hosta sieboldii and their cytostatic activity on HL-60 cells

A total of eighteen steroidal saponins were isolated from the rhizomes of Hosta sieboldii, one of which appeared to be the first isolation from a plant source and six to be new compounds. The structures of the new saponins were determined by spectral data and a few chemical transformations to be (25R)-2 alpha, 3 beta-dihydroxy-5 alpha-spirostan-12-one (manogenin) 3-O-?O-beta-D-glucopyranosyl-(1-->2)-O-beta-D-glucopyranosyl -(1-->4)-beta-D-galactopyranoside?, (25R)-2 alpha,3 beta-dihydroxy-5 alpha-spirost-9-en-12-one (9,11-dehydromanogenin) 3-O-?O-beta-D-glucopyranosyl-(1-->2)-O-beta-D- glucopyranosyl-(1-->4)-beta-D-galactopyranoside?, 9,11-dehydromanogenin 3-O-?O-beta-D-glucopyranosyl-(1-->2)-O-[O-alpha-L- rhamnopyranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->3)]-O-beta-D- glucopyranosyl-(1-->4)-beta-D-galactopyranoside?, (25R)-2 alpha,3 beta-dihydroxy-26-beta-D-glucopyranosyloxy-22-methoxy-5 alpha-furostan-12-one 3-O-?O-beta-D-glucopyranosyl-(1-->2)-O-[beta-D-xylopyranosyl-(1-->3)]-O- beta-D-glucopyranosyl-(1-->4)-beta-D-galactopyranoside?, (25R)-2 alpha, 3 beta-dihydroxy-26-beta-D-glucopyranosyloxy-22-methoxy-5 alpha-furost-9-en-12-one 3-O-?O-beta-D-glucopyranosyl-(1-->2)-O-[beta-D-xylopyranosyl-(1-->3)]-O- beta-D-glucopyranosyl-(1-->4)-beta-D-galactopyranoside? and (25R)-5 alpha-spirostan-2 alpha,3 beta,12 beta-triol 3-O-?O-alpha-L- rhamnopyranosyl-(1-->2)-beta-D-galactopyranoside?, respectively. Cytostatic activity of the isolated saponins on leukaemia HL-60 cells was examined.     

Bile acids. LI. Formation of 12alpha-hydroxyl derivatives and companions from 5 alpha-sterols by rabbit liver microsomes

A comparison of the activity of rabbit liver microsomes fortified with 0.1 mM NADPH to promote 12alpha-hydroxylation over 60 minutes with appropriate sterols provided the following relative order of activities: kalpha-cholestane-3alpha, 7alpha-diol, 100; 7alpha-hydroxy-5alpha-cholestane-3one, 76; 7alpha-hydroxycholest-4-en-3-one, 64; 5alpha-cholestane-3-3beta, 7alpha-diol, 26; allochendoexycholate, 22. If the more polar products formation. Investigation by gas-liquid chromatography-mass spectrometry of the nature of these more polar products derived form 5alpha-cholestane-3alpha, 7alpha-diol showed that a series of tetrols was dormed; i.e., 5alpha-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol as the major product and lesser amounts of 5alpha-cholestane-3alpha, 7alpha, 12alpha, 24- and 3alpha, 7alpha, 12alpha, 23tetrols. No significant amount of 26-hydroxylated product was formed.     

Studies on quinazolines and 1,2,4-benzothiadiazine 1,1-dioxides. 8.1, 2 synthesis and pharmacological evaluation of tricyclic fused quinazolines and 1,2,4-benzothiadiazine 1,1-dioxides as potential alpha1-adrenoceptor antagonists

A series of 2-substituted methyl 2,3-dihydroimidazo[1, 2-c]quinazolin-5(6H)-ones (4), 3-substituted methyl 2, 3-dihydroimidazo[1,2-c]quinazolin-5(6H)-ones (5), 3-substituted methyl 2,3-dihydro-5H-thiazolo[2,3-b]quinazolin-5-ones (15a,b), 3-substituted methyl 2,3-dihydroimidazo[2,1-b]quinazolin-5(1H)-ones (16a,b), 3-substituted methyl 2,3-dihydro-1H-imidazo[1,2-b][1,2, 4]benzothiadiazine 5,5-dioxides (33a,b), 2-substituted methyl imidazo[1,2-c]quinazolin-5(6H)-ones (42-45a,b), 3-substituted methyl imidazo[1,2-c]quinazolin-5(6H)-ones (50-53a,b), 3-substituted methyl 5H-thiazolo[2,3-b]quinazolin-5-ones (55-56a,b), and 3-substituted methyl 5-(methylthio)-2,3-dihydroimidazo[1,2-c]quinazoline (57) were synthesized as compound 1conformational rigid congeners for pharmacological evaluation as potential alpha1-adrenoceptor antagonists. Compounds 4, 5, 33a,b, 44a,b, 45a,b, 52a,b, 53a,b, and 57 were found to possess high affinity for the alpha1-adrenoceptor. Compounds 5 and 57 were the most highly selective and potent alpha1 antagonists with Ki = 0.21 +/- 0.02 and 0.90 +/- 0.08 nM, respectively. The S-enantiomers of these two compounds (Ki = 0.13 +/- 0.01 nM for (S)-(-)-5; Ki = 1.0 +/- 0.2 nM for (S)-(+)-57) were 144-200-fold more potent than the R-enantiomers (Ki = 26 +/- 8 nM for (R)-(+)-5; Ki = 144 +/- 23 nM for (R)-(-)-57). Compound 4 showed 8-fold higher affinity to alpha1A-AR better than alpha1B-AR. These compounds possessed weak to no activity against the 5-HT1A receptor.     

Synthesis and in vitro activity of some epimeric 20 alpha-hydroxy, 20-oxime and aziridine pregnene derivatives as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase and 5 alpha-reductase

Some epimeric 20-hydroxy, 20-oxime, 16 alpha, 17 alpha-, 17,20- and 20,21-aziridine derivatives of progesterone were synthesized and evaluated as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase (P450(17) alpha) and 5 alpha-reductase (5 alpha-R). The reduction of 16-dehydropregenolone acetate (3a) was reinvestigated. NaBH4 in the presence of CeCl3 gave better stereo-selectivity for 20 beta-ol [20 alpha/20 beta-OH (4 alpha/4 beta) = 1/2.7] than LTBAH or the Meerwein-Pondroff method reported; reduction with Zn in HOAc formed exclusively 20 alpha-ol (4 alpha b). The 20 alpha- and 20 beta-hydroxy-4,16-pregnadien-3-one (9 alpha) and (9 beta) were synthesized from the alcohols 4 alpha b and 4 beta b. Several 20-oxime pregnadienes and 16 alpha, 17 alpha-, 17,20- and 20,21-aziridinyl-5-pregnene derivatives were also synthesized. LiAlH4 reduction of the 16-en-20-oxime (12b) yielded 20 (R)-(13a) and 20(S)-17 alpha,20-aziridine (13b) and 20(R)-17 beta,20-aziridine (14a). Several compounds inhibited the human P450(17) alpha with greater potency than ketoconzole. The 5 alpha-R enzyme assay showed that while (9 alpha) did not have any activity, (9 beta) and (3b) were potent 5 alpha-reductase (IC50 = 21 and 31 nM) inhibitors with activities similar to finasteride. The 20-oximes (17a) and (17b) were potent dual inhibitors for both 5 alpha-R (IC50 = 63 and 115 nM, compared to 33 nM for finasteride) and P450(17) alpha (IC50 = 43 and 25 nM, compared to 78 nM for ketoconazole).     

Duration of treatment with progesterone and regression of persistent ovarian follicles in cattle

The objective of the present study was to determine the duration of elevated concentrations of progesterone necessary to induce atresia of persistent ovarian follicles. Heifers were administered 25 mg of PGF2alpha on d 6 and 7 (d 0 = d of synchronized estrus) and a norgestomet implant from d 6 to 14. Ovaries were monitored by ultrasonography, and blood samples were collected on d 3, 5, 7, 9, 11, and 12 and daily from d 14 until ovulation. On d 12, heifers received either two progesterone-releasing intravaginal devices (PRID) for 6 h (6-h; n = 5), two PRID for 24 h (24-h; n = 5), or no treatment (CON; n = 5). Blood samples were collected at 15-min intervals from h -6 to 30 (PRID insertion = h 0) and analyzed for concentrations of LH. Characteristics of LH secretion were determined for consecutive 6-h periods (Period 0 to 5). Hourly blood samples, collected from h 0 to 29, were analyzed for concentrations of 17beta-estradiol (estradiol) and progesterone. The dominant ovarian follicles present on d 7 increased in size to 15.4+/-.3 mm on d 12 ("persistent follicle"). Following removal of the PRID and norgestomet implants, atresia of persistent follicles and ovulation of new follicles were induced in one of five and in four of five heifers in the 6-h and 24-h treatments, respectively. Persistent follicles ovulated after withdrawal of norgestomet in all other heifers. Concentrations of progesterone were increased from h 1 to 7 in the 6-h and h 1 to 26 in the 24-h treatment. Frequency of LH pulses was reduced (P < .05) during Periods 1 to 2 in the 6-h and Periods 1 to 5 in the 24-h treatment relative to the CON treatment. By h 10, concentrations of estradiol in the 6-h and 24-h treatments were lower (P < . 05) than in the CON treatment. This suppression continued through h 29 in the 24-h treatment (P < .05), whereas concentrations in the 6-h treatment were intermediate to those of the CON and 24-h treatments after h 14. Suppression of pulsatile LH release and estradiol secretion was evident with 6 and 24 h of treatment with progesterone, but only the 24-h treatment induced atresia of persistent follicles in a majority of the heifers.