Pyrogalloloestrogens: enzymic methylation of pyrogalloloestrogens and interaction of pyrogalloloestrogens with the enzymic methylation of catecholamines in rat liver in vitro.

During incubation of 2,4-dihydroxyoestrone with the 105000 X g supernatant of rat liver in the presence of S-adenosyl-[Me-14C]methionine, the formation of radioactive mono- as well as dimethyl ether derivatives was demonstrated. The products were identified as: 2,4-dihydroxyoestrone 2-methyl ether, 2,4-dihydroxyoestrone 3-methyl ether, 2,4-dihydroxyoestrone 4-methyl ether, 2,4-dihydroxyoestrone 2,3-dimethyl ether, 2,4-dihydroxyoestrone 2,4-dimethyl ether and 2,4-dihydroxyoestrone 3,4-dimethyl ether. The monomethyl ethers were the main products; within this group the 3-methyl ether of 2,4-dihydroxyoestrone was the main metabolite. Among the dimethyl ether derivatives, the 2,4-dihydroxyoestrone 2,3-dimethyl ether represented the quantitatively most important product. When 2,4-dihydroxyoestrone 2-methyl ether was incubated under the same conditions, 2,4-dihydroxyoestrone 2,3- as well as 2,4-dimethyl ether was formed. The 2,3-dimethyl ether was again the main metabolite. The incubation of 2,4-dihydroxyoestrone 4-methyl ether yielded the 2,4- and 3,4-dimethyl ethers, the first being the main product. In contrast, the 3-methyl ether of 2,4-dihydroxyoestrone was not further methylated by the catechol methyltransferase preparation. In further experiments, the effect of the pyrogalloloestrogen and its monomethyl ether derivatives on the enzymatic methylation of catecholamines was investigated. It was demonstrated that the methylation of adrenalin and dopamine was competitively inhibited by 2,4-dihydroxyoestrone and the 2,4-dihydroxyoestrone monomethyl ethers. Only a weak inhibitory effect was observed with the 3- and 4-monomenthyl ethers (Ki values 200 and 160muM). The unsubstituted pyrogalloloestrogen produced a marked inhibition (Ki value 50muM), but the strongest inhibition was found with the 2-monomethyl ether of 2,4-dihydroxyoestrone (Ki value 14muM). The extent of inhibition caused by the addition of the 2-monomethyl ether of 2,4-dihydroxyoestrone was thereby in the same range as the inhibition caused by pyrogallol and the catecholoestrogens.     

Synthesis and study of the estrogenic activity of 2,4-bis(bromomethyl)estradiol-17 beta 3-methyl ether.

Synthesis of 2,4-bis(bromomethyl)estradiol-17 beta 3-methyl ether (BBE2M) was accomplished by reducing a methanolic solution of 2,4-bis(bromomethyl)estrone methyl ether with sodium borohydride. In 0.5 M phosphate buffer, pH 7.0, 25 degrees, BBE2M readily reacts with Ellman's anion and alkylates cysteine to form a steroid-amino acid conjugate. Stoichiometry of the reaction indicates that the bromosteroid is divalent with cysteine. Tryptophan and histidine react more slowly with the bromosteroid. Estrogenic activity of BBE2M was evaluated in ovariectomized rats by uterine intraluminal administration and quantitation of glucose-6-phosphate dehydrogenase (D-glucose-6-P:NADP+ oxidoreductase, EC 1.1.1.49) activity in the uterus. BBE2M induced glucose-6-phosphate dehydrogenase activity as did estradiol-17 beta or estradiol-17 beta 3-methyl ether (E2M). BBE2M was more persistent in activity than E2M. Histological examination of uterus following BBE2M treatment shows classic estrogenic morphology. BBE2M covalently binds to the cytoplasmic estrogen receptor of calf uterus. Such binding is prevented by pretreatment of the receptor protein with estradiol-17 beta. The covalently bound steroid-receptor complex appears to stimulate RNA synthesis in isolated nuclei from calf endometrium.     

In vitro metabolic conjugation of catechol estrogens

In vitro metabolic conjugation of the catechol estrogens, 2-hydroxyestrone and 4-hydroxyestrone, has been investigated by means of HPLC with electrochemical detection. Sulfation of 2-hydroxyestrone and 4-hydroxyestrone with the rat liver 105 000 g supernatant fortified with 3'-phosphoadenosine-5'-phosphosulfate provided the 2- and 4-monosulfates, respectively. Glucuronidation of the two catechols with the rat and human liver 1500 g supernatant in the presence of uridine-5'- phosphoglucuronic acid gave the 2- and 4-glucuronides, respectively. In contrast, incubation with the guinea pig liver 1500 g supernatant yielded both isomeric monoglucuronides . When 2'-hydroxyestrone was incubated with rat liver 1500 g supernatant and S-adenosyl-L-methionine, the 2- and 3-monomethyl ethers were formed in an equal amount, while 4-hydroxyestrone was transformed into the 4-methyl ether in 12 times greater yield than the 3-methyl ether. The participation of sulfation and glucuronidation in the formation of guaiacol estrogens is discussed.     

Synthesis of C-2 and C-4 deuterium-labeled estradiol-17 beta

Estradiol-17 beta labeled with deuterium in the positions 2 or 4 can be prepared from 2-chloromercurio-1,3,5(10)-estratriene-3,17 beta-diol 3-methyl ether 17-acetate or 4-chloromercurio-1,3,5(10)-estratriene-3,17 beta-diol, respectively, in refluxing CH3COO(2)H/(2)H2O. The same reaction performed on 4-acetoxymercurio-1,3,5(10)-estratriene-3,17 beta-diol afforded 2,4-dideuterio-estradiol-17 beta in good yields.     

Synthesis of 16alpha-bromoacetoxyestradiol 3-methyl ether and study of the steroid binding site of human placental estradiol 17beta-dehydrogenase.

Homogeneous estradiol 17beta-dehydrogenase (EC 1.1.1.62) was prepared from human placenta by affinity chromatography and the steroid binding site was studied with affinity-labeling techniques. 16alpha-Bromoacetoxyestradiol 3-methyl ether and the tritated compound were synthesized by condensation of estriol 3-methyl ether with bromoacetic acid or [2-3H]bromoacetic acid in the presence of dicyclohexylcarbodiimide. 16alpha-Bromoacetoxyestradiol 3-methyl ether is stable in 0.01 M phosphate buffer at pH 7.0, 25 degrees, for at least 24 hours. It alkylates cysteine, histidine, methionine, lysine, and tryptophan under physiological conditions. The steroid is a substrate of estradiol 17beta-dehydrogenase, thus it must bind at the steroid binding site. The inactivation of estradiol 17beta-dehydrogenase by 150-fold molar concentrations of 16alpha-bromoacetoxyestradiol 3-methyl ether follows pseudo-first order kinetics with a half-time of 1.5 hours. Estradiol-17beta, NADH, and NADPH slow the rate of inactivation. 2-Mercaptoethanol in molar concentrations 50-fold that of 16alpha-bromoacetoxyestradiol 3-methyl ether stops the inactivation, but does not reverse it. 16alpha-Bromoacetoxyestradiol 3-methyl ether alkylates both NADH and NADPH; the presence of small amounts of enzyme markedly increases the rate of this alkylation. When the enzyme is inactivated with 16alpha-[2-3H]bromoacetoxyestradiol 3-methyl ether, amino acid analysis of acid hydrolysates reveals 3-carboxymethylhistidine and 1,3-dicarboxymethylhistidine. Comparison of 28 and 51% inactivated samples indicates that, as inactivation proceeds, the total amount of 3-carboxymethylhistidine decreases, while 1,3-dicarboxymethylhistidine increases, suggesting that the former is converted to the latter by a second alkylation step. When the enzyme is inactivated in the presence of a large excess of NADPH, only 1,3-dicarboxymethylhistidine is found. From the present study it is concluded that estradiol 17beta-dehydrogenase has a histidyl residue present in the catalytic region of the active site as does the previously studied 20beta-hydroxysteroid dehydrogenase.     

Metabolism of exogenous 4- and 2-hydroxyestradiol in the human male

The metabolic fate of the isomeric catecholestrogens 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol (2-OHE2) was studied to elucidate possible differences in their metabolism as an explanation for their different bioactivities. Healthy young men (n = 3 each) were infused (90 min) with 4-OHE2 (60 micrograms/h) or 2-OHE2 (100 micrograms/h). The main metabolites were determined in plasma and urine before, during and after infusion. Unconjugated and conjugated steroids, the latter after hot acid hydrolysis, were subjected to chromatography on LH-20 columns and measured by specific RIAs. During the infusion 4-OHE2 reached significant plasma concentrations whereas 2-OHE2 was so rapidly metabolised that its plasma levels remained virtually undetectable in spite of a higher infusion rate. The metabolism of 4-OHE2 was dominated by direct conjugation, that of 2-OHE2 by methyl ether formation. These findings were corroborated by the urinary excretion rates: during the infusion and the first hours afterwards, 4-OHE2 was mainly excreted as 4-OHE2 and 4-hydroxyestrone, while 2-OHE2 was predominantly excreted as 2-hydroxyestradiol 2-methyl ether and 2-hydroxyestrone 2-methyl ether.     

Role of porcine endometrial estrogen sulfotransferase in progesterone mediated downregulation of estrogen receptor

Estrogen sulfotransferase (EST) is a progesterone (Pg) induced secretory endometrial enzyme which may effect estrogen receptor levels by esterifying estradiol-17 beta (E2) to an inactive, sulfate form. The effects of this enzyme were studied using specific inhibitors of EST that do not bind to estrogen receptor (ER): 4-nitroestrone 3-methyl ether and 4-fluoroestrone 3-methyl ether. A 1 h pulse with 4 nM E2 caused ERn (i.e. E2-bound, chromatin-bound receptor) to increase 40% in incubations of proliferative gilt endometrium (no EST activity), while the same E2 treatment of secretory endometrium (high EST activity) caused no increase in ERn. ERn accumulation was completely restored in these experiments by preincubating secretory endometrium with 4 microM 4-fluoroestrone 3-methyl ether. Gilt endometrial explants cultured 7 days with 1 nM E2 plus 1 microM Pg (which induced EST activity) possessed half the ERn as explants devoid of EST activity which were cultured in E2 alone. The addition of 10 microM 4-nitroestrone 3-methyl ester to the cultures of secretory endometrium restored ERn to the levels seen in minces cultured with E2 alone. Furthermore, ovariectomized gilts injected daily with 250 micrograms E2 plus 25 mg Pg had much lower ERn (0.06 fmol/micrograms DNA) than gilts injected with E2 only (0.21 fmol/microgram DNA). ERn was restored completely by supplementing the E2 plus Pg injections with 0.5 g 4-nitroestrone 3-methyl ether administered by vaginal suppositories.(ABSTRACT TRUNCATED AT 250 WORDS)     

The transformation and resolution of 8-aza steroids by fungi.

1. When (+/-)-8-aza-d-homo-oestrone 3-methyl ether was added to a culture of Aspergillus ochraceus, the laevo-rotatory enantiomer is reduced to a 17A alcohol. Thus (+/-)-8-aza-d-homo-oestrone 3-methyl ether has been resolved into both its components, the laevo-rotatory enantiomer being obtained by chromic acid oxidation of the alcohol, and the dextro-rotatory enantiomer as residual precursor from the fermentation. 2. Fungi have been found to demethylate the 3-methyl ethers of 8-aza steroids to the corresponding phenols. In particular, 8-aza-d-homo-oestrone 3-methyl ether was demethylated by Aspergillus flavus and 8-aza-d-homo-oestradiol 3-methyl ether by Cunninghamella blakesleeana.     

Metabolism of radioactive 17 beta-estradiol 3-methyl ether by humans.

A mixture of 2-3H and 4-14C-17beta-estradiol 3-methyl ether was administered orally to a man and to a woman. 34 and 35 percent of the 3H was liberated into the body water of the man and of the woman, respectively, reflecting reactions involving position 2. The metabolism of estradiol methyl ether was qualitatively similar to that observed previously for radioactive estradiol administered intravenously to the same subjects, as judged by the measurement of various urinary metabolites by reverse isotope dilution. Evidence was obtained for hydroxylation at position 2 without demethylation by the isolation of urinary 2-hydroxyestrone 3-methyl ether which retained 33% of the original 3H. This 3H was presumably at position 1, resulted from an NIH shift which does not occur during hydroxylation of estrone or estradiol. This was confirmed by subsequent administration of a mixture of 4-14C and 3H-(methoxyl)-estradiol 3-methyl ether to the man. There was no evidence (by reverse isotope dilution) for 1-hydroxyestrone, 1-hydroxyestrone 3-methyl ether, 4-hydroxyestrone 3-methyl ether or 4-hydroxyestradiol 3-methyl ether as urinary metabolites of estradiol 3-methyl ether.     

Absorption,Translocation and Metabolism of Chlomethoxynil (X-52) in Plants

Rapid absorption and degradation of chlomethoxynil were observed in seedlings of rice and barnyard millet. The amounts of absorption by rice and barnyard millet, at the 2-leaf and 4-leaf stages of plants, were determined in water and soil cultures.

The major metabolic products of chlomethoxynil were 2,4-dichlorophenyl 3′-hydroxy-4′-nitrophenyl ether, 2,4-dichlorophenyl 3′-methoxy-4′-aminophenyl ether and their conjugates, and 2,4-dichlorophenyl 3′-methoxy-4′-acetylaminophenyl ether. Several minor unknown substances were also detected. A major metabolic pathway of chlomethoxynil in plants was postulated.     

Characterisation of bacterial cultures enriched on the chlorophenoxyalkanoic acid herbicides 4-(2,4-dichlorophenoxy) butyric acid and 4-(4-chloro-2-methylphenoxy) butyric acid

The aim of this study was to enrich and characterise bacterial consortia from soils around a herbicide production plant through their capability to degrade the herbicides 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB) and 4-(4-chloro-2-methylphenoxy) butyric acid (MCPB). Partial 16S rRNA gene sequencing revealed members of the genera Stenotrophomonas, Brevundimonas, Pseudomonas, and Ochrobactrum in the 2,4-DB- and MCPB-degrading communities. The degradation of 2,4-DB and MCPB was facilitated by the combined activities of the community members. Some of the members were able to utilise other herbicides from the family of chlorophenoxyalkanoic acids. During degradation of 2,4-DB and MCPB, phenol intermediates were detected, indicating ether cleavage of the side chain as the initial step responsible for the breakdown. This was also verified using an indicator medium. Repeated attempts to amplify putatively conserved tfd genes by PCR indicated the absence of tfd genes among the consortia members. First step cleavage of the chlorophenoxybutyric acid herbicides is by ether cleavage in bacteria and is encoded by divergent or different tfd gene types. The isolation of mixed cultures capable of degrading 2,4-DB and MCPB will aid future investigations to determine both the metabolic route for dissimilation and the fate of these herbicides in natural environments.     

Lumi-mestranol and epi-lumi-mestranol.

Treatment of lumi-estrone 3-methyl ether (I) with acetylene gave the C-17-epimeric compounds lumi-mestranol (3-methoxy-17 alpha-ethynyl-13 alpha-estra-1,3,5(10)-trien-17 beta-ol, III ) and epi-lumi-mestranol (3-methoxy-17 beta-ethynyl-13 alpha-estra-1,3,5(10)-trien-17 alpha-ol, IV). The structures of the two isomers were assigned on the basis of their molecular rotations and shift-reagent experiments in the NMR. The irradiation of estrone 3-methyl ether (II) to provide compound I was investigated in two solvent systems. Minor products of these reactions were the seco-steroids VII, VIII and X.     

Degradation of the multiple branched alkane 2,6,10,14-tetramethyl-pentadecane (pristane) in Rhodococcus ruber and Mycobacterium neoaurum

Pristane, a highly branched hydrocarbon that also contains iso-branched termini, was used as a substrate for several alkane-metabolizing bacteria. Rhodococcus ruber and Mycobacterium neoaurum were able to utilize pristane for growth effectively. The intermediates produced by these bacteria during incubation with pristane were analyzed by gas chromatography (GC) and gas chromatography/mass spectra (GC/MS). The products revealed as products of 4-methyl pentanoic acid; methyl butanedioic acid; 2-methyl pentadioic acid; methyl propanedioic acid; 4-methyl heptanedioic acid; and 2,6,10,14-tetramethyl-pentadecan-3-one were detected in M. neoaurum cultures. In R. ruber, methyl butanedioic acid; 2-methyl pentadioic acid; 4,8-dimethylnonanoic acid, 4-methyl heptanedioic acid; 2,6,10-trimethylundecanoic acid; 3,7-dimethyl decanedioic acid; and 2,6,10,14-tetramethyl-pentadecan-3-one were detected. The occurrence of these intermediates showed that pristane could be catabolized not only via mono- but also by a di-terminal oxidation pathway. Furthermore, the presence of 2,6,10,14-tetramethyl-pentadecan-3-one; 3,7-dimethyldecandioate; and 2-methylbutandioate established a third pathway initiated by sub-terminal oxidation at the third carbon atom of pristane. Novel intermediates detected suggest simultaneous sub-terminal and di-terminal oxidation pathways.     

The relative amounts and identification of some 2,4-dichlorophenoxyacetic Acid metabolites isolated from soybean cotyledon callus cultures

Soybean (Glycine max L.) cotyledon callus grown on radioactive 2,4-dichlorophenoxyacetic acid (2,4-D-1-¹⁴C) as an auxin produced 2,4-D metabolites, which qualitatively and quantitatively changed with time. Water soluble fractions from the tissue exhibited a steady increase in radioactivity during the course of 24 days. Following β-glucosidase treatment, at least eight aglycones were obtained from the water soluble fraction of the tissue after 8 days. The metabolite, 4-hydroxy-2,5-dichlorophenoxyacetic acid was the most abundant aglycone during the entire 32 day growth period while 4-hydroxy-2,3-dichlorophenoxyacetic acid was detected as a minor metabolite. Radioactivity in the ether soluble acidic fractions reached a maximum of 82% of the total in the tissue after 2 days. The level then decreased to 44% by the end of 24 days. A total of seven ether soluble components were detected. In addition to 2,4-D glutamic acid, which was detected in high amounts after 24 hours, 2,4-D aspartic acid was found to be the most abundant ether soluble metabolite after longer time periods. Mass spectral data and a fragmentation pattern are presented for 2,4-D aspartic acid.     

Radioimmunoassay of contraceptive steroids. I. Synthesis of 6-oxomestranol 6-(O-carboxymethyl) oxime

A synthesis of 6-(O-carboxymethyl) oxime of 6-oxo-mestranol (3-methoxy-17-ethinyl-17β -hydroxy-1,3,5 (10)-estratrien-6-one) which was required for coupling with bovine serum albumin in order to produce a specific anti-sera for mestranol (3-methoxy-17-ethinyl-1,3,5 (10)-estratrien-17β-ol) has been described. 6-Oxoestradiol-17β 3-methyl ether was prepared from estradiol-17β 3,17-diacetate by chromic acid oxidation, followed by hydrolysis and methylation. It was converted to its O-carboxymethyloxime derivative which was smoothly oxidized by Jones reagent to the corresponding es estrone derivative. This was easily ethinylated with lithium acetylide-ethylenediamine complex to the desired compound. In an alternate approach to the desired compound, it was found that 6-oxoestradiol-17β 3- methyl ether could not be converted to its ketal under any of a variety of conditions. Ethinylation of 6-oxoestrone 3-methyl ether with limited amount of lithium acetylide reagent probably gave the 17α -ethinyl derivative as was indicated from IR and UV spectra, but its identity could not be further confirmed due to its extremely poor yield.     

Synthesis of the 3-methyl ether and 4-deoxy derivatives of 4-cyanophenyl 1,5-dithio-beta-D-xylopyranoside (Beciparcil).

Treatment of the 2,3-isopropylidene acetal of the title dithioxyloside with 2,4,5-triiodoimidazole-PPh3 caused replacement of the 4-hydroxyl group by iodine to afford 82% of the 4-axial iodide 6, converted by base into 4-cyanophenyl 2,3-O-isopropylidene-1,5-dithio-beta-D-glycero-pent-3-enopyranoside++ + (8). Acid treatment of 8 gave 87% of the deacetonated glycos-3-ulose, borohydride reduction of which afforded 63% of 4-cyanophenyl 4-deoxy-1,5-dithio-alpha-L-threo-pentopyranoside (3), together with 27% of the 3-axial epimer. The 3-methyl ether of the title dithioxyloside was satisfactorily prepared via 2,4-protection as the cyclic phenylboronate, methylation, and deprotection; alternative strategy via the 2,4-bis(triisopropylsilyl) ether was complicated because of silyl-group migration under methylation conditions.     

High increment of triglycerols with ether linkages in the retina during anoxia

In the bovine retina incubated during 90 min in a medium lacking glucose without preoxygenation and gassed with O₂-free N₂ a striking rise in triglycerides with ether bonds was found. NaF and 2,4-DNP decrease the production of these molecules while cycloheximide (1.78mM) completely abolishes the phenomenon. The free fatty acid pool increases about six-fold and this rise is not affected by the inhibitors. Bovine serum albumin stimulates the free fatty acid and triglycerols production. The amount of these lipids decrease by further incubation under normoxia in the presence of glucose.     

Alumina-supported synthesis of antibacterial quinolines using microwaves

7-(5'-Alkyl-1',3',4'-thiadiazol/oxadiazol-2'-ylthio)-6 -fluoro-2,4-dimethylquinolines and 3-formyl-2-(2'-hydroxy- 1',4'-naphthoquinon-3'-yl)-4-methyl/6-methyl/7-quinolines have been synthesised by the reaction of 5-alkyl-1,3,4-thiadiazol/oxadiazol-2-thiols with 7-chloro-6-fluoro-2,4-dimethylquinoline and by the reaction of 2-hydroxy-1,4-naphthoquinone with 2-chloro-3-formyl-4-methyl/6-methyl/7-methyl/8-methylquinolines respectively on basic alumina using microwaves, the reaction time has been brought down from hours to seconds with improved yield as compared to conventional heating. The compounds were tested for their in vitro antibacterial activity. All compounds showed promising antibacterial activity. The best activity was observed by compounds 3a and 3f.     

Synthesis of ( + )-Methyl Phaseate and Its Isomer from ( — )-β-Pinene

The total synthesis of ( + )-methyI phaseate (2b) and its epimer (25) is described. The known β- ketoester (8), which was prepared from ( — )-/f-pinene (4), was converted to a key intermediate (5) via a 1, 4-dioxoester (7). The reaction of 5 with a lithium reagent of the acetylene TBDMS ether (6) in THF-HMPA at — 70°C afforded the desired acetylene alcohol (17) and its epimer (18) in high yields. 17 was transformed into ( + )-methyl phaseate (2b). From this synthetic work, the absolute configuration of natural ( — )-phaseic acid (2a) was confirmed.     

Structure-effect relationship in the induction of mitotic phase-specific abnormality of centrosome integrity and multipolar spindles by steroidal estrogens and their derivatives in cultured mammalian cells

In order to determine the structure-effect relationship in the induction of centrosome disintegrity (abnormality of gamma-tubulin signals) and multipolar spindles in a cultured fibroblast cell line V79 by steroidal estrogens, the activities of various estrogens and their derivatives were investigated. Induction of centrosome disintegrity by estrogens was specific in cells in the mitotic phase and was not observed in interphase cells. The centrosome disintegrity induced 24 h after exposure to estrogens was accompanied by the appearance of multinucleated cells, but the microtubule network was organized. The rank order of potency of estrogens in inducing mitotic phase-specific centrosome disintegrity and multipolar spindles was as follows: 2-methoxyestradiol>dihydroequilin 3-methyl ether=equilin 3-methyl ether>17alpha-estradiol>17beta-estradiol 3-methyl ether=17beta-estradiol>dihydroequilin>estrone 3-methyl ether. Equilin and estrone were not effective in causing centrosome disintegrity. These results suggest that the 17-hydroxyl group, irrespective of whether it is the sterically alpha or beta form, is necessary for estradiol and dihydroequilin to cause centrosome disintegrity and that O-methylation at the C-3 position was effective for equilin and dihydroequilin in enhancing the centrosome abnormality. 2-Methoxyestradiol was the most potent inducer of the centrosome disintegrity among the tested compounds and caused the induction of multiple signals of gamma-tubulin, including more than five signals.