Maternal and developmental toxicity of halogenated 4'-nitrodiphenyl ethers in mice

In an ongoing effort to delineate structure-activity relationships in the developmental toxicity of diphenyl ethers, we evaluated the maternal and developmental toxicity of 10 diphenyl ethers related to the herbicide nitrofen. All possible trichlorophenyl 4'-nitrophenyl ethers were evaluated, as were the 2,4-difluorophenyl and 2,4-dibromophenyl 4'-nitrophenyl ethers. We also evaluated bifenox and chlomethoxyfen, which are 2,4-dichlorophenyl congeners with meta-substituents on the 4'-nitrophenyl ring. Nitrofen (2,4-dichlorophenyl 4'-nitrophenyl ether) was included for comparison. Identity of the halogen affected the postnatal (but not prenatal) mortality induced by 2,4-dihalogenated 4'-nitrophenyl ethers. The presence of 3'-substituents on the 4'-nitrophenyl ring reduced both pre- and postnatal toxicity of 2,4-dichlorinated congeners. Among chlorinated 4'-nitrophenyl congeners without meta-substituents on the nitrophenyl ring, the position of chlorine substituents strongly affected the congener's potential for inducing prenatal vs. postnatal syndromes. All congeners increased liver to body weight ratios in unmated females, but such increases were not well-correlated with either prenatal or postnatal embryotoxicity.     

Biodegradation of diphenyl ether and transformation of selected brominated congeners by <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. PH-07

Polybrominated diphenyl ethers (PBDEs) are common flame-retardant chemicals that are used in diverse commercial products such as textiles, circuit boards, and plastics. Because of the widespread production and improper disposal of materials that contain PBDEs, there has been an increasing accumulation of these compounds in the environment. The toxicity and bioavailability of PBDEs are variable for different congeners, with some congeners showing dioxin-like activities and estrogenicity. The diphenyl ether-utilizing bacterium Sphingomonas sp. PH-07 was enriched from activated sludge of a wastewater treatment plant. In liquid cultures, this strain mineralized 1 g of diphenyl ether per liter completely within 6 days. The metabolites detected and identified by gas chromatography/mass spectrometry (MS) and electrospray ionization/MS analysis corresponded with a feasible degradative pathway. However, the strain PH-07 even catabolized several brominated congeners such as mono-, di-, and tribrominated diphenyl ethers thereby producing the corresponding metabolites.     

Diphenyl ethers as androgen receptor antagonists for the topical suppression of sebum production

A series of diphenyl ethers was prepared and evaluated for androgen receptor antagonist activity in human androgen receptor binding and cellular functional assays. Analogs with potent in vitro activities were evaluated for topical in vivo efficacy in the Golden Syrian Hamster ear model. Several compounds showed reduction in wax esters in this validated animal model.     

Nitrofen induces apoptosis independently of retinaldehyde dehydrogenase (RALDH) inhibition

BACKGROUND: Nitrofen is a diphenyl ether that induces congenital diaphragmatic hernia (CDH) in rodents. Its mechanism of action has been hypothesized as inhibition of the retinaldehyde dehydrogenase (RALDH) enzymes with consequent reduced retinoic acid signaling. METHODS: To determine if nitrofen inhibits RALDH enzymes, a reporter gene construct containing a retinoic acid response‐element (RARE) was transfected into HEK‐293 cells and treated with varying concentrations of nitrofen in the presence of retinaldehyde (retinal). Cell death was characterized by caspace‐cleavage microplate assays and terminal deoxynucleotidyl transferase dUTP nick end‐labeling (TUNEL) assays. Ex vivo analyses of cell viability were characterized in fetal rat lung explants using Live/Dead staining. Cell proliferation and apoptosis were assessed using fluorescent immunohistochemistry with phosphorylated histone and activated caspase antibodies on explant tissues. Nile red staining was used to identify intracellular lipid droplets. RESULTS: Nitrofen‐induced dose‐dependent declines in RARE‐reporter gene expression. However, similar reductions were observed in control‐reporter constructs suggesting that nitrofen compromised cell viability. These observed declines in cell viability resulted from increased cell death and were confirmed using two independent assays. Ex vivo analyses showed that mesenchymal cells were particularly susceptible to nitrofen‐induced apoptosis while epithelial cell proliferation was dramatically reduced in fetal rat lung explants. Nitrofen treatment of these explants also showed profound lipid redistribution, primarily to phagocytes. CONCLUSIONS: The observed declines in nitrofen‐associated retinoic acid signaling appear to be independent of RALDH inhibition and likely result from nitrofen induced cell death/apoptosis. These results support a cellular apoptotic mechanism of CDH development, independent of RALDH inhibition. Birth Defects Res (Part B) 89:223–232, 2010. © 2010 Wiley‐Liss, Inc.     

Novel diphenyl ethers: design, docking studies, synthesis and inhibition of enoyl ACP reductase of Plasmodium falciparum and Escherichia coli

We designed some novel diphenyl ethers and determined their binding energies for Enoyl-Acyl Carrier Protein Reductase (ENR) of Plasmodium falciparum using Autodock. Out of these, we synthesized the promising compounds and tested them for their inhibitory activity against ENRs of P. falciparum as well as Escherichia coli. Some of these compounds show nanomolar inhibition of PfENR and low micromolar inhibition of EcENR. They also exhibit low micromolar potency against in vitro cultures of P. falciparum and E. coli. The study of structure-activity relationship of these compounds paves the way for further improvements in the design of novel diphenyl ethers with improved activity against purified enzyme and the pathogens.     

Effects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes

Several diphenyl ether herbicides, such as acifluorfen methyl, have been previously shown to cause large accumulations of the heme and chlorophyll precursor, protoporphyrin, in plants. Lightinduced herbicidal damage is mediated by the photoactive porphyrin. Here we investigate whether diphenyl ether herbicides can affect porphyrin synthesis in rat and chick hepatocytes. In rat hepatocyte cultures, protoporphyrin, as well as coproporphyrin, accumulated after treatment with acifluorfen or acifluorfen methyl. Combination of acifluorfen methyl with an esterase inhibitor to prevent the conversion of acifluorfen methyl to acifluorfen resulted in a greater accumulation of porphyrins than caused by acifluorfen methyl or acifluorfen alone. In vitro enzyme studies of hepatic mitochondria isolated from rat and chick embryos demonstrated that protopor-phyrinogen oxidase, the penultimate enzyme of heme biosynthesis, was inhibited by low concentrations of acifluorfen, nitrofen, or acifluorfen methyl with the latter being the most potent inhibitor. These findings indicate that diphenyl ether treatment can cause protoporphyrin accumulation in rat hepatocyte cultures and suggest that this accumulation was associated with the inhibition of protoporphyrinogen oxidase. In cultured chick embryo hepatocytes, treatment with acifluorfen methyl plus an esterase inhibitor caused massive accumulation of uroporphyrin rather than protoporphyrin or coproporphyrin. Specific isozymes of cytochrome P450 were also induced in chick embryo hepatocytes. These effects were not observed in the absence of an esterase inhibitor. These results suggest that diphenyl ether herbicides can cause uroporphyrin accumulation similar to that induced by other cytochrome P450-inducing chemicals such as polyhalogenated aromatic hydrocarbons in the chick hepatocyte system.     

Influence of chlorine substituents on rates of oxidation of chlorinated biphenyls by the biphenyl dioxygenase of Burkholderia sp. strain LB400

Biphenyl dioxygenase from Burkholderia (Pseudomonas) sp. strain LB400 catalyzes the first reaction of a pathway for the degradation of biphenyl and a broad range of chlorinated biphenyls (CBs). The effect of chlorine substituents on catalysis was determined by measuring the specific activity of the enzyme with biphenyl and 18 congeners. The catalytic oxygenase component was purified and incubated with individual CBs in the presence of electron transport proteins and cofactors that were required for enzyme activity. The rate of depletion of biphenyl from the assay mixture and the rate of formation of cis-biphenyl 2,3-dihydrodiol, the oxidation product, were almost equal, indicating that the assay accurately measured enzyme-specific activity. Four classes of CBs were defined based on their oxidation rates. Class I contained 3-CB and 2,5-CB, which gave rates that were approximately twice that of biphenyl. Class II contained 2,5,3',4'-CB, 2,3,2',5'-CB, 2,3,4,5-CB, 2,3,2',3'-CB, 2,4, 5,2',5'-CB, 2,5,3'-CB, 2,5,4'-CB, 2-CB, and 3,4,5-CB, which gave rates that ranged from 97 to 35% of the biphenyl rate. Class III contained only 2,3,4,2',5'-CB, which gave a rate that was 4% of the biphenyl rate. Class IV contained 2,4,4'-CB, 2,4,2',4'-CB, 3,4,5, 2'-CB, 3,4,5,3'-CB, 3,5,3',5'-CB, and 3,4,5,2',5'-CB, which showed no detectable depletion. Rates were not significantly correlated with the aqueous solubilities of the CBs or the number of chlorine substituents on the rings. Oxidation products were detected for all class I, II, and III congeners and were identified as chlorinated cis-dihydrodiols for classes I and II. The specificity of biphenyl dioxygenase for the CBs examined in this study was determined by the relative positions of the chlorine substituents on the aromatic rings rather than the number of chlorine substituents on the rings.     

The influence of tetraphenylborates (hydrophobic anions) on yeast cell electro-rotation

The action of a series of tetraphenylborate ion (TPB) derivatives on yeast cells was studied by electro-rotation of the pre-treated cells. TPB derivatives in which all four phenyl groups were substituted with fluorine, chlorine or trifluoromethyl were much more toxic than the unsubstituted compound, the effect increasing dramatically with increasing size of substituents. These observations suggest that the toxicity of these hydrophobic ions is determined mainly by their size and possibly also by the chemical inductivity of their substituent groups. The order of the toxicities of these ions was in fair agreement with literature values for their translocation rates across artificial bilayers. Incubation times of 3 h were used as standard, longer incubations (up to 48 h) showed that the number of cells affected by low doses of TPB increased with the logarithm of time after the first hour of incubation. Although measurements of the percentage of cells showing co-field rotation showed that controls were not adversely affected by incubations as long as 9 h, rotation spectra showed that some cells suffer loss of internal conductivity during extended incubations. Decrease of the pH of the incubation medium, or inclusion of high concentrations of NaCl or KCl, potentiated the effects of these hydrophobic ions. The toxicity developed slowly, and the sensitivity of the assay was only very weakly dependent on the cell suspension density.     

The in vivo and in vitro genotoxicity of aromatic amines in relationship to the genotoxicity of benzidine.

Benzidine and 12 related aromatic amines have been studied for the effects of substituent groups and pi orbital conjugation on their genotoxicity as measured by their mutagenicity in vitro with Salmonella and by chromosomal aberrations (CA) in vivo in the bone-marrow cells of mice. The in vitro studies indicated increases in mutagenicity with increases in the electron withdrawing ability of para' substituents. Mutagenicity also increases with increased conjugation as shown by the degree of planarity of the biphenyl compounds and by comparing the mutagenicities of biphenyl amines to stilbenes as well as to ethylene bridged diphenyl compounds. The relative in vitro mutagenicity results were not predictive of relative in vivo CA results. The 3 most genotoxic compounds in vivo were the conjugated amines without substituents in the para' position. The CA values for 4-aminostilbene were exceptionally high. These in vivo results indicate increased genotoxicity for benzidine analogs without substitution in the para' position.     

Congener Selectivity During Polychlorinated Biphenyls Degradation by Enterobacter sp. LY402

The relationship between the selectivity of a particular polychlorinated biphenyls (PCBs) congener and its biodegradability under the same concentration, especially by Enterobacter sp. LY402, is less well studied. To measure congener selectivity of Enterobacter sp. LY402, several influencing factors were studied. The results showed LY402 effectively degraded coplanar 3,4,3',4'-chlorobiphenyl (CB) at a concentration of 0.05 μM, but not 0.5 μM. The degradation rates of 2,4,5,2',3'-CB and 2,4,5,2',4',5'-CB were increased significantly when the sample constituents were changed from 12 to 5 congeners or to one congener. This indicated that bioremediation of individual congener was affected by other congeners present in the mixture. Moreover, for PCBs containing one chlorine on each phenyl ring, the reactivity preference of LY402 was 2,2'-CB ≥ 3,3'-CB ? 4,4'-CB. For two ortho chlorines congeners of PCBs, 2,2'-CB was degraded faster than 2,6-CB. Although 2,6-CB and 4,4'-CB were poorly degraded, the addition of one (i.e., 2,4,4'-CB and 2,6,3'-CB) or two more chlorines (i.e., 2,4,2',4'-CB) on the phenyl ring significantly increased their biodegradability. In addition, comparing the two congeners of ortho-meta-chlorinated biphenyl, 2,3,2',3'-CB with neighbor meta chlorines was degraded slower than 2,5,2',5'-CB with interval meta chlorines. All these indicated that the transformation rates of PCBs were not consistent with the number of chlorines, and PCBs containing the same numbers of chlorines but at different positions also resulted in different conversions. In principle, the extents of effect caused by the position of chlorine substituents on the degradation of PCBs by LY402 were ortho- > meta- > para-CB. In conclusion, the congener selectivity of LY402 was determined by many factors, including the composition of the congeners, their concentrations in the mixture and location and number of chlorine substituents on the phenyl rings.     

Beta-keto-ester chemistry and ketolides. Synthesis and antibacterial activity of 2-halogeno, 2-methyl and 2,3 enol-ether ketolides

The effect of 2,3 modifications on the antibacterial activity of ketolides was evaluated by introducing substituents in position 2 and converting the C-1, C-2, C-3 beta-keto-ester into stable 2,3 enol-ether or 2,3 anhydro derivatives. Introduction of a fluorine in C-2 is beneficial with regard to the overall antibacterial spectrum whereas the enol-ether and 2,3 unsaturated compounds, as well as the bulky gem dimethyl or 2-chloro derivatives, are less active particularly against erythromycin resistant strains. A 2-fluoro ketolide derivative demonstrates good antibacterial activity and in vivo efficacy against multi-resistant Streptococcus pneumoniae. Compared to azithromycin against Haemophilus influenzae, this compound is equivalent in vitro and slightly more active in vivo. These results demonstrate that within the ketolide class, to retain good antibacterial activity, position 2 needs to remain tetrahedral and tolerates only very small substituents such as fluorine.     

Biodegradation of diphenyl ether and its monohalogenated derivatives by Sphingomonas sp. strain SS3.

The bacterium Sphingomonas sp. strain SS3, which utilizes diphenyl ether and its 4-fluoro, 4-chloro, and (to a considerably lesser extent) 4-bromo derivatives as sole sources of carbon and energy, was enriched from soil samples of an industrial waste deposit. The bacterium showed cometabolic activities toward all other isomeric monohalogenated diphenyl ethers. During diphenyl ether degradation in batch culture experiments, phenol and catechol were produced as intermediates which were then channeled into the 3-oxoadipate pathway. The initial step in the degradation follows the recently discovered mechanism of 1,2-dioxygenation, which yields unstable phenolic hemiacetals from diphenyl ether structures. Oxidation of the structure-related dibenzo-p-dioxin yielded 2-(2-hydroxyphenoxy)-muconate upon ortho cleavage of the intermediate 2,2',3-trihydroxydiphenyl ether. Formation of phenol, catechol, halophenol, and halocatechol from the conversion of monohalogenated diphenyl ethers gives evidence for a nonspecific attack of the dioxygenating enzyme system.     

Biodegradation of diphenyl ether and its monohalogenated derivatives by Sphingomonas sp. strain SS3.

The bacterium Sphingomonas sp. strain SS3, which utilizes diphenyl ether and its 4-fluoro, 4-chloro, and (to a considerably lesser extent) 4-bromo derivatives as sole sources of carbon and energy, was enriched from soil samples of an industrial waste deposit. The bacterium showed cometabolic activities toward all other isomeric monohalogenated diphenyl ethers. During diphenyl ether degradation in batch culture experiments, phenol and catechol were produced as intermediates which were then channeled into the 3-oxoadipate pathway. The initial step in the degradation follows the recently discovered mechanism of 1,2-dioxygenation, which yields unstable phenolic hemiacetals from diphenyl ether structures. Oxidation of the structure-related dibenzo-p-dioxin yielded 2-(2-hydroxyphenoxy)-muconate upon ortho cleavage of the intermediate 2,2',3-trihydroxydiphenyl ether. Formation of phenol, catechol, halophenol, and halocatechol from the conversion of monohalogenated diphenyl ethers gives evidence for a nonspecific attack of the dioxygenating enzyme system.     

Design, synthesis and evaluation of novel molecules with a diphenyl ether nucleus as potential antitubercular agents

A series of compounds with a diphenyl ether nucleus were synthesized by incorporating various amines into the diphenyl ether scaffold with an amide bond. Their antitubercular activities were evaluated against Mycobacterium tuberculosis H(37)Rv by a microdilution method, with MIC values ranging from 4 to 64μg/mL. Through structure-activity relationship studies, the two chlorine atoms at 3 and 4 positions in the phenyl ring of R(2) group were found to play a significant role in the antitubercular activity. The most potent compound 6c showed an MIC value of 4μg/mL and a good safety profile in HepG2 cell line by the MTT assay. Compound 6c was further found to be effective in a murine model of BCG infection, providing a good lead for subsequent optimization.     

Aerobic degradation of polychlorinated biphenyls by Alcaligenes sp. JB1: metabolites and enzymes

In contrast to the degradation of penta-and hexachlorobiphenyls in chemostat cultures, the metabolism of PCBs by Alcaligenes sp. JB1 was shown to be restricted to PCBs with up to four chlorine substituents in resting-cell assays. Among these, the PCB congeners containing ortho chlorine substituents on both phenyl rings were found to be least degraded. Monochloro-benzoates and dichlorobenzoates were detected as metabolites. Resting cell assays with chlorobenzoates showed that JB1 could metabolize all three monochlorobenzoates and dichlorobenzoates containing only meta and para chlorine substituents, but not dichlorobenzoates possessing an ortho chlorine substituent. In enzyme activity assays, meta cleaving 2,3-dihydroxybiphenyl 1,2-dioxygenase and catechol 2,3-dioxygenase activities were constitutive, whereas benzoate dioxygenase and ortho cleaving catechol 1,2-dioxygenase activities were induced by their substrates. No activity was found for pyrocatechase II, the enzyme that is specific for chlorocatechols. The data suggest that complete mineralization of PCBs with three or more chlorine substituents by Alcaligenes sp. JB1 is unlikely.Abbreviations PCB polychlorinated biphenyls - CBA chlorobenzoate - D di - Tr tri - Te tetra - Pe penta- - H hexa     

Degradation of Delor 103, a technical mixture of polychlorinated biphenyls,by selected bacteria

Ability to utilize a technical mixture of polychlorinated biphenyls (PCB), Delor 103, as the sole carbon source, has been tested in 14 bacterial strains. For the five best growing strains (Alcaligenes latus, Alcalgenes eutrophus, Comamonas testosteroni, Micrococcus varians and Pseudomonas putida), the dependence of the degradation of individual PCB congeners on the number of chlorine substituents is discussed.     

Polychlorinated biphenyls and their different level metabolites as inhibitors of glutathione S-transferase isoenzymes

Research on the effects of polychlorinated biphenyl (PCB) toxicity tends to focus on commercial PCB congeners and parent PCBs themselves. However, studies have suggested that PCB metabolites may be more interesting than the parent compounds because of their high reactivity. As a key metabolic enzyme, glutathione S-transferases (GSTs) are responsible for detoxification by catalyzing the conjugation reaction of glutathione (GSH) to xenobiotics. Inhibition of GST activity indicates reduced detoxification ability. We investigated the inhibition of chicken liver GSTs by parent PCBs and their metabolites and observed dose-dependent inhibition in vitro; inhibitory efficiency declined in the order GSH-conjugate > mono-hydroxyl ≈ quinone ≈ hydroquinone > parent PCB. Structure-inhibitory activity relationship studies indicated that with the inhibitory activity greatly increases with the number of GSH moieties or chlorine substituents on the quinone ring. However, no significant linear relationship was observed for chlorine pattern changes on the phenyl ring. The reversibility of PCB metabolite inhibition of GSTs is discussed. PCB mono-hydroxyl, hydroquinone and quinone forms showed irreversible inhibition of GSTs, which suggests a mechanism involving covalent binding to cysteine residues in the GST active site. PCB glutathionyl conjugates showed reversible GST inhibition, implying non-covalent binding. Furthermore, reactive oxygen species did not significantly affect GST activity.     

Transformation of diphenyl ethers by Trametes versicolor and characterization of ring cleavage products

The white-rot fungi Trametes versicolor SBUG 1050, DSM 11269 and DSM 11309 are able to oxidize diphenyl ether and its halogenated derivatives 4-bromo- and 4-chlorodiphenyl ether. The products formed from diphenyl ether were 2- and 4-hydroxydiphenyl ether. Both 4-bromo- and 4-chlorodiphenyl ether were transformed to the corresponding products hydroxylated at the non-halogenated ring. Additionally, ring-cleavage products were detected by high perfomance liquid chromatography and characterized by gas chromatography/mass spectrometry and proton nuclear magnetic resonance spectroscopy. Unhalogenated diphenyl ether was degraded to 2-hydroxy-4-phenoxymuconic acid and 6-carboxy-4-phenoxy-2-pyrone. Brominated derivatives of both these compounds were formed from 4-bromodiphenyl ether, and 4-chlorodiphenyl ether was transformed in the same way to the analogous chlorinated ring cleavage products. Additionally, 4-bromo- and 4-chlorophenol were detected as intermediates from 4-bromo- and 4-chlorodiphenyl ether, respectively. In the presence of the cytochrome-P450 inhibitor 1-aminobenzotriazole, no metabolites were formed by cells of Trametes versicolor from the diphenyl ethers investigated. Cell-free supernatants of whole cultures with high laccase and manganese peroxidase activities were not able to transform the unhydroxylated diphenyl ethers used.     

Contraceptive progestins. Various 11-substituents combined with four 17-substituents: 17alpha-ethynyl, five- and six-membered spiromethylene ethers or six-membered spiromethylene lactones

Norethisterone (NET) is a 19-nortestosterone derivative with progestagenic and some androgenic activity, which was used in the first generation of contraceptives. NET was succeeded by levonorgestrel (LNG) and later on by desogestrel (DSG) and gestodene (GSD). Although these latter two progestins had increased potency, there was still androgenicity with gestodene and to a lesser extent with desogestrel. New progestins were synthesized in order to further enhance progestagenic and to reduce androgenic activity. Four different chemical moieties were introduced in position 17 of 19-nortestosterone, viz. 17alpha-ethynyl, five- and six-membered spiromethylene ethers, and a six-membered-spiromethylene lactone. In combination with these structures seven different substituents were added at position 11, i.e. methylene, methyl, ethyl, ethenyl, ethynyl, 2-propenyl and 1-propynyl. All substituents except for methylene occupied the 11beta-position. All these 32 compounds were synthesized and analysed in vitro and in vivo against etonogestrel (ETG, 3-keto-desogestrel), the biologically active metabolite of desogestrel. Their relative binding potency to progesterone (PR), androgen (AR) and estrogen (ER) receptors were determined in cell lysates of human breast tumor MCF-7 cells and to glucocorticoid (GR) receptors in that of human leukemic IM-9 cells. Moreover, their relative agonistic activities were assessed in Chinese hamster ovary cell-based transactivation assays. All in vivo activities were determined in McPhail (progestagenic), ovulation inhibition (progestagenic and estrogenic), Hershberger (androgenic), hormone screening (glucocorticoid and estrogen) and Allen-Doisy (estrogenic) tests after oral and for the McPhail test also after subcutaneous administration. The progestagenic binding and transactivation potencies of all compounds in the three 17-spiro series were higher than those of the corresponding analogues in the 17alpha-ethynyl series. None of the compounds showed estrogenic or clear androgenic binding and transactivation potential except for a six-membered-spiromethylene lactone with a propynyl group. This compound showed strong androgenic binding. The glucocorticoid binding and transactivation were very low for the compounds with the 17alpha-ethynyl and the five-membered-spiromethylene ether groups, whereas both six-membered-spiro series showed, clearly with methyl and ethynyl substituents, and less pronounced with methylene and ethenyl, higher binding and transactivation values. For the 17alpha-ethynyl series, the McPhail test showed high potencies with methylene, methyl and ethenyl substituents after oral treatment or with propenyl after subcutaneous administration. The introduction of the spiro substituents in position 17 led to high potencies for other 11-substituents as well. Besides methyl, also ethyl, ethynyl and propynyl were potent substituents. With ovulation inhibition tests, the ethyl, ethenyl and ethynyl substituents were the more potent compounds in all four series. However, compounds with methyl or ethynyl additions appeared to be glucocorticoidal in the hormone screening test irrespective of the 17-substituent, while with the three spiro series even methylene and ethenyl groups became active. Androgenicity was only observed at dose levels at or above 5 mg/kg, which is 2.5-fold weaker than ETG. Moreover, estrogenicity appeared negligible with the three spiro series, while with the 17alpha-ethynyl series methyl, ethyl, ethenyl and ethynyl substituents, a very high estrogenic potential was assessed. Based on the high efficacy and low side-effects, the following compounds show a high selectivity: 17alpha-ethynyl with ethyl, ethenyl and 2-propenyl substituents, six-membered spiromethylene ether with ethyl and six-membered-spiromethylene lactone with ethyl, 2-propenyl or 1-propynyl substituents. (ABSTRACT TRUNCATED)     

Interaction between thyroid hormones and erythrocyte membranes: competitive inhibition of binding 131 I-L-triiodothyronine and 131 I-L-thyroxine by their analogs.

Molecular structural characteristics of thyroid hormones which influence binding to the erythrocyte membranes were investigated by competitive binding experiments. The ability of thyroid hormone analogs to displace 131 I-L-thyroxine and 131 I-L-triiodothyronine from the membranes was considered evidence of their competitive binding. The diphenyl ether linkage (thyronine) was essential as compounds with a single aromatic ring were weakly competitive. The presence of three iodine atoms at 3, 5 and 3' positions on thyronine was optimal for maximal competitive binding. There was weak competitive binding of analogs if chlorine or bromine was substituted for iodine. The alanine side chain was required for optimal binding as N-acetyl-l-thyroxine and various deaminated analogs were poor competitors compared to T4 and T3. L-isomers of T4 and T3 showed greater competitive binding to erythrocyte membranes than the corresponding d-isomers.