Improvement of Tool Steel Powder Cold Sprayability Via Softening and Agglomeration Heat Treatments.

Cold spray can produce deposits from a broad range of materials but reports on cold spray of steels are still limited to the few steel families demonstrating high ductility and medium strength. Softening and agglomeration of steel powders via heat treatment in a rotary tube furnace were investigated as promising ways to improve H13 tool steel powder cold sprayability. By adjusting starting powder size, as well as heat treatment conditions (maximum temperature, cooling rate and heat treatment atmosphere), cold spray of H13 powder improved from virtually no deposition to the production of dense, sound and thick deposits with a powder deposition efficiency of 70%. Powder agglomeration, surface state, microstructure evolution and softening are identified as key factors determining the powder deposition efficiency and resulting deposit microstructure. The developed powder modification method has the potential to facilitate the cold spray of all steels subjected to martensitic transformation.

The effect of organism density on bioaccumulation of contaminants from sediment in three aquatic test species: a case for standardizing to sediment organic carbon.

Laboratory methods for measuring bioaccumulation of organic contaminants from sediment into aquatic organisms continue to improve, but some aspects are still in need of standardization. From a review of published methods, we noted that the loading density of organisms was determined inconsistently and was primarily based on either sediment volume or total organic carbon (TOC). The rationale mainly expressed for standardizing to TOC was to minimize the depletion of sediment contaminants. However, even when density was standardized to TOC, the relative amount of TOC provided (i.e., ratio of TOC to organism dry weight [dw]) was highly variable. In this study, we examined the effect of organism density (standardized to sediment TOC or volume) on bioaccumulation in three freshwater organisms. The oligochaete Lumbriculus variegatus, mayfly nymph Hexagenia spp., and fathead minnow Pimephales promelas were exposed for 28 days to two field-contaminated sediments that varied in concentration of PCBs and TOC. Densities tested were 50:1 and 27:1 ratios of TOC to organism dw and 140 ml sediment/g wet weight (ww) biomass, yielding low to high organism densities. Bioaccumulation in Hexagenia spp. was significantly higher at the lowest organism density compared with the highest organism density when exposed to site 2 sediment (1.1% TOC) but only with tissue concentrations expressed on a ww basis. Otherwise, there was no significant effect of density on bioaccumulation in organisms exposed to sediments from site 1 (12% TOC) or site 2. Survival of Hexagenia spp. was adversely affected at the highest organism density when the relative amount of TOC was low. The results of this study support the recommendation of standardizing organism density relative to a particular amount of TOC for invertebrate species. A 27:1 ratio of TOC:organism dw was selected as a standard organism density for a new bioaccumulation method because survival, growth, and bioaccumulation were not impacted relative to a 50:1 ratio, and less sediment was required. This density is recommended as an appropriate ratio for sediment bioaccumulation assessments in general.

Evaluation of hydrogen peroxide and ozone residue levels on N95 masks following chemical decontamination.

BACKGROUND: Hydrogen peroxide and ozone have been used as chemical decontamination agents for N95 masks during supply shortages. If left behind on the masks, the residues of both chemicals represent a potential health hazard by skin contact and respiratory exposure. AIM: Characterization of hydrogen peroxide and ozone residues on mask surfaces after chemical decontamination. METHODS: Various N95 masks were decontaminated using two commercial systems employing either aerosol spray or vaporization of hydrogen peroxide in the presence of ozone. Following the decontamination, the masks were aired out to eliminate moisture and potential chemical residues. The residual hydrogen peroxide and ozone were monitored in the gas phase above the mask surface, and hydrogen peroxide residue directly on mask surfaces using a colorimetric assay. FINDINGS: After decontamination, hydrogen peroxide and ozone were detectable in the gas phase in the vicinity of masks even after 5 h of aeration. Hydrogen peroxide was also detected on all studied masks, and levels up to 56 mg per mask were observed after 0.5 h of aeration. All residues gradually decreased with aeration, likely due to decomposition and vaporization. CONCLUSION: Hydrogen peroxide and ozone were present on N95 masks after decontamination. With appropriate aeration, the gaseous residue levels in the vicinity of the masks decreased to permissible levels as defined by the US Occupational Safety and Health Administration. Reliable assays to monitor these residues are necessary to ensure the safety of the mask users.

Formation of fullerides and fullerene-based heterostructures.

Two potassium fulleride phases, metallic K(3)C(60) and nonmetallic K(6)C(60), are formed when potassium is incorporated into thin C(60) films under ultrahigh vacuum conditions. Phase separation is observed for intermediate stoichiometries. Results obtained for the C(60)-K(3)C(60) heterostructure demonstrate that it is stable against potassium migration from the K(3)C(60) phase. In contrast, the C(60)-K(6)C(60) interface is not stable and K(3)C(60) is formed.

Design and synthesis of bisubstrate inhibitors of type 1 17beta-hydroxysteroid dehydrogenase: overview and perspectives.

Type 1 17beta-hydroxysteroid dehydrogenase (17beta-HSD1) is a key steroidogenic enzyme that catalyses the reduction of steroid estrone into the most potent endogenous estrogen estradiol using the cofactor NAD(P)H. Bisubstrate inhibition is a good way to enhance the potency of inhibitors of cofactor-assisted enzymes. The design of a bisubstrate inhibitor of 17beta-HSD1, the estradiol/adenosine hybrid EM-1745, is reviewed and strategies for future designs of inhibitors are proposed.

Validated, stability-indicated quantitative purity test for triethylenetetramine tetrachlorhydrate by automated multiple development.

There is a monography of Triethylenetetramine dichlorhydrate (Trientine) in the United States Pharmacopeia. But neither the base nor the salts di- or tetra-chlorhydrate are in the European Pharmacopeia. Triethylène tetramine tetrachlorhydrate, used by AGEPS now as matural, is more soluble then triethylene tetramine dichlorhydrate. It is administred to patients with Wilson's disease, which results from a congenital lack of the copper metabolism. A quantitative purity test of this drug by automated multiple development high-performance thin-layer chromatography is developed and validated. The validation parameters tested are specifically characterized by retention factor, linearity, limits of detection and quantitation of several nanograms, reliability, and accuracy. To determine impurities, the monography of triethylenetetramine dichlorhydrate in the American Pharmacopeia is tested. This method in classic developing tank requires two mobile phases and is not quantitative. Assays in high-performance liquid chromatography with a different column and mobile phase did not give good results for the separation of impurities. Thus, it is not possible to perform comparative validation of the separation of the impurities. Only the assay of triethylenetetramine with potentiometer detection has been validated.

Blockade of the stimulatory effect of estrogens, OH-tamoxifen, OH-toremifene, droloxifene, and raloxifene on alkaline phosphatase activity by the antiestrogen EM-800 in human endometrial adenocarcinoma Ishikawa cells.

Although temporary benefits of tamoxifen therapy are observed in up to 40% of women with breast cancer, this compound, which is known to possess mixed estrogenic and antiestrogenic activities, has been associated with increased risk of endometrial carcinoma. This study compares the effects of the novel nonsteroidal pure antiestrogen EM-800 and related compounds with those of a series of antiestrogens on the estrogen-sensitive alkaline phosphatase (AP) activity in human endometrial adenocarcinoma Ishikawa cells. Exposure to increasing concentrations of up to 1000 nM EM-800 or its active metabolite EM-652 alone failed to affect basal AP activity. In contrast, incubation with 10 nM (Z)-4-OH-tamoxifen, (Z)-4-OH-toremifene, droloxifene, or raloxifene increased the value of this estrogen-sensitive parameter by 3.3-, 3.5-, 2.2-, and 1.6-fold, respectively, a stimulatory effect that was completely reversed by simultaneous exposure to 30 nM EM-800. Moreover, the stimulation of AP activity induced by 1 nM 17beta-estradiol was completely reversed by EM-800, EM-652, or ICI-182780, at the IC50 value of 1.98 +/- 0.23, 1.01 +/- 0.16, and 5.64 +/- 0.59 nM, respectively, whereas the partial blockade exerted by (Z)-4-OH-tamoxifen, (Z)-4-OH-toremifene, or raloxifene was observed at IC50 values of 13.5 +/- 3.80, 41.0 +/- 7.2, and 3.74 +/- 0.43 nM, respectively. Thus, as assessed by their activity in the human Ishikawa endometrial carcinoma cells, EM-800 and EM-652 are the most potent known antiestrogens in Ishikawa cells, and, most importantly, they are devoid of the estrogenic activity observed in these human endometrial cancer cells with (Z)-4-OH-tamoxifen, (Z)-4-OH-toremifene, droloxifene, and raloxifene.

Bioaccumulation of perfluorinated carboxylates and sulfonates and polychlorinated biphenyls in laboratory-cultured Hexagenia spp., Lumbriculus variegatus and Pimephales promelas from field-collected sediments.

Polychlorinated biphenyls (PCBs) and perfluorinated carboxylates and sulfonates (PFASs) are persistent pollutants in sediment that can potentially bioaccumulate in aquatic organisms. The current study investigates variation in the accumulation of PCBs and PFASs in laboratory-cultured Hexagenia spp., Lumbriculus variegatus and Pimephales promelas from contaminated field-collected sediment using 28-day tests. BSAF(lipid) (lipid-normalized biota-sediment accumulation factor) values for total concentration of PCBs were greater in Hexagenia spp. relative to L. variegatus and P. promelas. The distribution of congeners contributing to the total concentration of PCBs in tissue varied among the three species. Trichlorobiphenyl congeners composed the greatest proportion of the total concentration of PCBs in L. variegatus while tetra- and pentabiphenyl congeners dominated in Hexagenia spp. and P. promelas. Perfluorooctane sulfonate (PFOS) was present in all three species at concentrations greater than all other PFASs analyzed. Hexagenia spp. also produced the greatest BSAF(lipid) and BSAF(ww) (non-lipid-normalized biota-sediment accumulation factor) values for PFOS relative to the other two species. However, this was not the case for all PFASs. The trend of BSAF values and number of carbon atoms in the perfluoroalkyl chain of perfluorinated carboxylates varied among the three species but was similar for perfluorinated sulfonates. Differences in the dominant pathways of exposure (e.g., water, sediment ingestion) likely explain a large proportion of the variation in accumulation observed across the three species.

The role of poly(ADP-ribose) metabolism in response to active oxygen cytotoxicity.

These experiments are a continuation of our work describing the effect of H2O2 and O2- on DNA strand breaks, NAD pools and poly(ADP-ribose) synthesis in C3H10T1/2 cells (Lautier et al. (1990) Biochem. Cell Biol. 68, 602-608). The current experiments were carried out firstly to evaluate the polymer synthesis in C3H10T1/2 cells exposed to benzamide, oxygen radicals and hyperthermia. Secondly, using four different protocols for the time of addition and removal of benzamide, the lowest benzamide levels shown to inhibit polymer synthesis were used to study the effect on plating efficiency and colony-forming ability of cells exposed to H2O2 and O2(-). Plating efficiency and colony-forming ability were affected by the active oxygen-species-generating system xanthine-xanthine oxidase and 100 microM benzamide. With higher levels of benzamide, this effect disappeared, and 0.5 to 1 mM benzamide were actually protective against the effects of xanthine-xanthine oxidase, suggesting the involvement of other processes in addition to poly(ADP-ribosyl)ation in response to oxygen radical damage.

Complete inhibition of poly(ADP-ribose) polymerase activity prevents the recovery of C3H10T1/2 cells from oxidative stress.

Activation of the poly(ADP-ribose) polymerase after oxidative damage is implicated in different responses of the cells, for example, cell recovery after sublethal damage or cell death after lethal damage. However, the extent and mechanism of involvement of the enzyme in these two processes appear to be different. Inhibitors of this polymerase, such as benzamides, which do not completely inhibit PARP have been shown to protect the cells from killing by massive oxidant damage, could neither reduce the cellular recovery after mild oxidant damage nor completely inhibit DNA repair in vitro. We report here that 1,5-dihydroxyisoquinoline, which was earlier shown to be a strong inhibitor of this polymerase in vitro, is also its potent inhibitor in vivo. Using sensitive techniques for measuring low levels of cellular poly(ADP-ribose) polymer, we show that this inhibitor can completely abolish oxidant-induced activation of the polymerase in C3H10T1/2 cells. We show that only a minor fraction of the poly(ADP-ribose) polymerase activity is sufficient in cellular recovery after sublethal oxidant damage. We also demonstrate that cells are unable to recover from oxidant damage in the complete absence of polymerase activity.

Synthesis and antiestrogenic activity of diaryl thioether derivatives.

The reaction of 1,2-diarylethanol and mercapto side chain catalyzed by ZnI2 was used as a key step in the short (three to five steps) and efficient synthesis of 17 diaryl thioether derivatives. Several of these compounds contain a methyl butyl amide chain and an hydroxyaryl moiety, respectively, for antiestrogenic activity and binding affinity on estrogen receptor. No binding affinity for crude cytosolic preparation of the estrogen receptor was observed for compounds without phenolic group, while a low affinity (0.01-0.05%) was measured for mono- or diphenol derivatives. Like the pure steroidal antiestrogen EM-139, these novel nonsteroidal compounds did not exert any stimulatory effect on cell proliferation of (ER+) ZR-75-1 human breast cancer cells and partially reversed the amplitude of the stimulatory effect induced by estradiol on this (ER+) cell line. No proliferative or antiproliferative effect on (ER-) MDA-MB-231 human breast cancer cells was also observed for three of these compounds (39-41). Among the newly synthesized nonsteroidal compounds, the thioether derivative 41 (N-butyl-N-methyl-13,14-bis(4'-hydroxyphenyl)-12-thiatetradecanamide+ ++), with a long methylbutylalkanamide side chain and a diphenolic nucleus, was selected as the best antiestrogenic compound. However, this compound was 100-fold less antiestrogenic in (ER+) ZR-75-1 cells than the steroidal antiestrogen EM-139.

Steroidal spiro-gamma-lactones that inhibit 17 beta-hydroxysteroid dehydrogenase activity in human placental microsomes.

The important enzyme 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) is known to regulate intracellular levels of biologically active steroids, namely, androgens and estrogens. In an effort to develop potent inhibitors of 17 beta-HSD for reducing the levels of active steroids, we found that steroidal spiro-gamma-lactones inhibit 17 beta-HSD activity. In this report, we describe the synthesis of 11 spiro-gamma-lactone analogs containing a steroidal C-18 or C-19 nucleus and compare their relative inhibitory effects on 17 beta-HSD activity in the human placenta microsomes that catalyze the interconversion of androgens and estrogens. To void the interaction of the cytosolic 17 beta-HSD activity that is specific for the interconversion of estrone and estradiol, we used 4-androstenedione as substrate. Analysis of the inhibitory effect exerted by these analogs on microsomal 17 beta-HSD activity indicates that spiro-gamma-lactones containing the C-18 nucleus are more potent inhibitors than C-19 nucleus analogs. The best inhibition was obtained with the phenolic spiro-gamma-lactone 7 (3-hydroxy-19-nor-17 alpha-pregna-1,3,5(10)-triene 21,17-carbolactone), which has an IC50 value of 0.27 microM, and was much lower than the competitive effect of the unlabeled substrate 4-androstenedione, which has an IC50 value of 1.40 microM. Preincubation with lactone 7 did not inactivate 17 beta-HSD activity. The results thus suggest that lactone 7 is a reversible in inhibitor. Lactone 7 is selective for microsomal 17 beta-HSD activity, as no inhibition was observed for cytosolic 17 beta-HSD activity.

Structure-activity relationships of 17alpha-derivatives of estradiol as inhibitors of steroid sulfatase.

The steroid sulfatase or steryl sulfatase is a microsomal enzyme widely distributed in human tissues that catalyzes the hydrolysis of sulfated 3-hydroxy steroids to the corresponding free active 3-hydroxy steroids. Since androgens and estrogens may be synthesized inside the cancerous cells starting from dehydroepiandrosterone sulfate (DHEAS) and estrone sulfate (E(1)S) available in blood circulation, the use of therapeutic agents that inhibit steroid sulfatase activity may be a rewarding approach to the treatment of androgeno-sensitive and estrogeno-sensitive diseases. In the present study, we report the chemical synthesis and biological evaluation of a new family of steroid sulfatase inhibitors. The inhibitors were designed by adding an alkyl, a phenyl, a benzyl, or a benzyl substituted at position 17alpha of estradiol (E(2)), a C18-steroid, and enzymatic assays were performed using the steroid sulfatase of homogenized JEG-3 cells or transfected in HEK-293 cells. We observed that a hydrophobic substituent induces powerful inhibition of steroid sulfatase while a hydrophilic one was weak. Although a hydrophobic group at the 17alpha-position increased the inhibitory activity, the steric factors contribute to the opposite effect. As exemplified by 17alpha-decyl-E(2) and 17alpha-dodecyl-E(2), a long flexible side chain prevents adequate fitting into the enzyme catalytic site, thus decreasing capacity to inhibit the steroid sulfatase activity. In the alkyl series, the best compromise between hydrophobicity and steric hindrance was obtained with the octyl group (IC(50) = 440 nM), but judicious branching of side chain could improve this further. Benzyl substituted derivatives of estradiol were better inhibitors than alkyl analogues. Among the series of 17alpha-(benzyl substituted)-E(2) derivatives studied, the 3'-bromobenzyl, 4'-tert-butylbenzyl, 4'-butylbenzyl, and 4'-benzyloxybenzyl groups provided the most potent inhibition of steroid sulfatase transformation of E(1)S into E(1) (IC(50) = 24, 28, 25, and 22 nM, respectively). As an example, the tert-butylbenzyl group increases the ability of the E(2) nucleus to inhibit the steroid sulfatase by 3000-fold, and it also inhibits similarly the steroid sulfatase transformations of both natural substrates, E(1)S and DHEAS. Interestingly, the newly reported family of steroid sulfatase inhibitors acts by a reversible mechanism of action that is different from the irreversible mechanism of the known inhibitor estrone sulfamate (EMATE).

Potent inhibition of steroid sulfatase activity by 3-O-sulfamate 17alpha-benzyl(or 4'-tert-butylbenzyl)estra-1,3,5(10)-trienes: combination of two substituents at positions C3 and c17alpha of estradiol.

Steroid sulfates are precursors of hormones that stimulate androgen- and estrogen-dependent cancers. Thus, steroid sulfatase, the enzyme that catalyzes conversion of DHEAS and E1S to the corresponding unconjugated steroids DHEA and E1, appears to be one of the key enzymes regulating the level of active androgenic and estrogenic steroids. Since 17alpha-substituted benzylestradiols and 3-O-sulfamate estrone (EMATE) represent two families of steroid sulfatase inhibitors that probably act through different mechanisms, we synthesized compounds 3-O-sulfamate 17alpha-benzylestradiol (4) and 3-O-sulfamate 17alpha-(tert-butylbenzyl)estradiol (5) that contain two kinds of substituents on the same molecule. In our enzymatic assay using a homogenate of human embryonal (293) cells transfected with steroid sulfatase, compounds 4 and 5 were found to be more potent inhibitors than already known steroid sulfatase inhibitors that have only a C17alpha-substituent or only a C3-sulfamate group (EMATE). The IC50 values of 4 and 5 were, respectively, 0.39 and 0.15 nM for the transformation of E1S to E1 and 4.1 and 1.4 nM for the transformation of DHEAS to DHEA. Compound 5 inhibited the steroid sulfatase activity in intact transfected (293) cell culture assays by inactivating the enzyme activity. Compound 5 also inactivates the steroid sulfatase activity at lower concentration than EMATE in microsomes of transfected (293) cells. In this assay, an excess of natural substrate E1S protects enzyme against inactivation by 5 or EMATE. Furthermore, the unsulfamoylated analogue of 5, compound 3, did not inactivate the steroid sulfatase.

Intracellular regulation of 17 beta-hydroxysteroid dehydrogenase type 2 catalytic activity in A431 cells.

There is growing evidence that various isoforms of 17 beta-hydroxysteroid dehydrogenase (17-HSD) are regulated at the level of catalysis in intact cells. A number of investigators have proposed that the NAD(P)/NAD(P)H ratio may control the direction of reaction. In a previous study, we obtained evidence that A431 cells, derived from an epidermoid carcinoma of the vulva, are enriched in 17-HSD type 2, a membrane-bound isoform reactive with C18 and C19 17 beta-hydroxysteroids and 17-ketosteroids. The present investigation was undertaken to confirm the presence of 17-HSD type 2 in A431 cells and to assess intracellular regulation of 17-HSD at the level of catalysis by comparing the activity of homogenates and microsomes with that of cell monolayers. Northern blot analysis confirmed the presence of 17-HSD type 2 mRNA. Exposure of cells to epidermal growth factor resulted in an increase in type 2 mRNA and, for microsomes, increases in maximum velocity (Vmax) with no change in Michaelis constant (Km) for testosterone and androstenedione, resulting in equivalent increases in the Vmax/Km ratio consistent with the presence of a single enzyme. Initial velocity data and inhibition patterns were consistent with a highly ordered reaction sequence in vitro in which testosterone and androstenedione bind only to either an enzyme-NAD or an enzyme-NADH complex respectively. Microsomal dehydrogenase activity with testosterone was 2- to 3-fold higher than reductase activity with androstenedione. In contrast, although cell monolayers rapidly converted testosterone to androstenedione, reductase activity with androstenedione or dehydroepiandrosterone (DHEA) was barely detectable. lactate but not glucose, pyruvate or isocitrate stimulated the conversion of androstenedione to testosterone by monolayers, suggesting that cytoplasmic NADH may be the cofactor for 17-HSD type 2 reductase activity with androstenedione. However, exposure to lactate did not result in a significant change in the NAD/NADH ratio of cell monolayers. It appears that within A431 cells 17-HSD type 2 is regulated at the level of catalysis to function almost exclusively as a dehydrogenase. These findings give further support to the concept that 17-HSD type 2 functions in vivo principally as a dehydrogenase and that its role as a reductase in testosterone formation by either the delta 4 or delta 5 pathway is limited.

Inhibitors of type II 17beta-hydroxysteroid dehydrogenase.

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are involved in the last step of the biosynthesis of sex steroids from cholesterol. This family of steroidogenic enzymes constitutes an interesting target in the control of the concentration of estrogens and androgens. Among the isoforms of 17beta-HSD, type II preferentially catalyzes the oxidation of estradiol (E(2)), testosterone (T), dihydrotestosterone (DHT), and 20alpha-dihydroprogesterone (20alpha-DHP). Based on structure-activity relationship studies, we have developed steroidal spirolactones as inhibitors of type II 17beta-HSD using different steroid nuclei: a C18-steroid (lactones 1 and 10), an antiestrogenic nucleus (lactone 2), and a C19-steroid (lactone 28). We know these inhibitors are selective for type II 17beta-HSD as no or only weak inhibition was observed for types I and III. They also have no proliferative (androgenic) activity on androgen sensitive (AR(+)) Shionogi cells whereas their proliferative (estrogenic) activity on estrogen sensitive (ER(+)) ZR-75-1 cells depends on the nature of the steroid nucleus. Lactones 1 and 10 are weak estrogens, while lactones 2 and 28 do not exert estrogenic activity, in fact lactone 2 is an antiestrogen. Lactones 1, 2, 10 and 28 were also tested in an identical assay with a series of enzyme substrates, C19-steroid diols, and known inhibitors, for the oxidation of testosterone and estradiol into androstenedione and estrone, respectively. From this comparative study, the best inhibitors of type II 17beta-HSD (oxidase activity) were identified, but none of them were clearly more potent than the hydroxylated (reduced) forms of enzyme substrates, E2, T, and DHT. Such inhibitors remain, however, useful tools to, (1) further elucidate the role of type II 17beta-HSD, and (2) regulate the level of active estrogens, androgens and progesterone.

Overview of a rational approach to design type I 17beta-hydroxysteroid dehydrogenase inhibitors without estrogenic activity: chemical synthesis and biological evaluation.

Hormone-sensitive diseases such as breast cancer are health problems of major importance in North America and Europe. Endocrine therapies using antiestrogens for the treatment and the prevention of breast cancer are presently under clinical trials. Antiestrogens are drugs that compete with estrogens for the estrogen receptor without activating the transcription of estrogen-sensitive genes. However, an optimal blockade of estrogen action could ideally be achieved by a dual-action compound that would antagonize the estrogen receptor and inhibit the biosynthesis of estradiol. Type I 17beta-hydroxysteroid dehydrogenase (17beta-HSD) was chosen as a key steroidogenic target enzyme to inhibit the formation of estradiol, which is the most potent estrogen. This article describes a rational approach that could lead to the development of compounds that exhibit both actions. The chemical syntheses of estradiol derivatives bearing a bromoalkyl and a bromoalkylamide side chain at the 16alpha-position are summarized. Two parameters were studied for biological evaluation of our synthetic inhibitors: (1) the inhibition of estrone reduction into estradiol by type I 17beta-HSD, and (2) the proliferative/antiproliferative cell assays performed on the estrogen-sensitive ZR-75-1 breast tumor cell line. First, the substitution of the 16alpha-position of estradiol by bromoalkyl side chain led to potent inhibitors of type I 17beta-HSD, but the estrogenic activity remained. Secondly, an alkylamide functionality at the 16alpha- or 7alpha-position of estradiol cannot abolish the estrogenic activity without affecting considerably the inhibitory potency on type I 17beta-HSD. In conclusion, the best dual-action inhibitor synthesized showed an IC50 of 13 +/- 1 microM for type I 17beta-HSD, while displaying antiestrogenic activity at 1.0 microM. Despite the fact that we did not obtain an ideal dual-action blocker, we have optimized several structural parameters providing important structure-activity relationship.

A 6beta-(thiaheptanamide) derivative of estradiol as inhibitor of 17beta-hydroxysteroid dehydrogenase type 1.

In an effort to develop potent agents for reducing the levels of the active estrogen, estradiol, we developed a new category of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 1 inhibitors. The compounds described possess a butyl methyl alkylamide side chain linked to the C6 position of estradiol by a thioether. With a series of epimeric mixtures, an optimal side-chain length of five methylene groups (between the amide group and steroid part) was first determined. Thereafter, both C6 epimers of optimized mixture were obtained after high-pressure liquid chromatography separation. 1H and 13C NMR experiments were performed to confirm the stereochemistry of each epimer. The 6beta-orientation of the side-chain was found to be crucial for enzymatic inhibition. Indeed, for the optimized side-chain length, the compound with a beta-orientation (5: N-butyl,N-methyl 7-(3',17'beta-dihydroxy-1',3',5'( 10')-estratriene-6'beta-yl)-7-thiaheptanamide) was 70-fold more potent than the 6alpha-analog. Compound 5 did not inactivate 17beta-HSD type 1, suggesting a reversible inhibitor. In addition, it was found to be a more potent inhibitor than the substrate estrone itself or a panel of three known inhibitors.

Synthesis and biological activity of 17 alpha-alkynylamide derivatives of estradiol.

Seven estradiol (E2) derivatives with an alkynylamide side chain at the 17 alpha position were synthesized starting from ethynylestradiol (EE2). The main chemical step was the coupling reaction of the acetylide ion of EE2 with carbon dioxide, glutaric anhydride or bromoalkyl ortho ester. The synthesis of these compounds is fast (3-6 steps according to the compound) and is easily achieved with good yield. Five compounds with different side chain lengths were evaluated for uterotrophic and antiuterotrophic activity in the CD-1 mouse. None of the tested compounds shows estrogenic activity in this sensitive in vivo system. At low doses (1 and 3 micrograms), a 14-57% inhibition of E2-induced uterine growth was observed while no additional inhibition was observed at the 10, 20 and 30 micrograms doses. In human breast carcinoma cells in culture, all compounds show estrogenic activity at high concentrations while only compound 39 (N-butyl,N-methyl-8-[3',17' beta-dihydroxy estra-1',3',5'(10')-trien-17' alpha-yl]-7-octynamide) possesses antiproliferative or antiestrogenic effects. No significant correlation could be demonstrated between alkynylamide side chain length and estrogenic or antiestrogenic activity. Among the compounds tested, the derivative of EE2 possessing a five-methylene (CH2) side chain (compound 39) possesses the best antiestrogenic activity (44 +/- 7% in the CD-1 mouse uterus assay at the 3 micrograms dose and 57 +/- 4% at 0.1 nM in human ZR-75-1 cancer cells in culture.

Synthesis and biological activities of thioether derivatives related to the antiestrogens tamoxifen and ICI 164384.

The catalyzed coupling reaction of activated alcohol and mercaptan was used for the short and efficient synthesis of 14 thioether compounds. Two types of side chains, the methyl butyl alkylamide related to the pure steroidal antiestrogen ICI 164384 and the dimethylamino ethyloxy phenyl related to the clinically used nonsteroidal antiestrogen tamoxifen, were introduced by a thioether link on two types of nuclei (triphenylethane or estradiol). The new thioether derivatives were tested to assess their relative binding affinity for the estrogen receptor and their estrogenic or antiestrogenic activity in the ZR-75-1 (ER+) cell line. The results indicate that of the three types of compounds studied, only the nonsteroidal derivatives with an alkylamide side chain possess antiestrogenic activity. In the steroidal series, displacement of the alkylamide side chain from the 7 to the 6 position produced compounds with chemical characteristics similar to ICI 164384 or EM-139 but without antiestrogenic activity. In the nonsteroidal series of compounds with an aryl side chain, compounds with estrogenic activity were obtained. One compound, a nonsteroidal derivative with a methyl butyl alkylamide side chain 20, possesses a relative binding affinity for the estrogen receptor identical to EM-139 (1.1 and 1.2%, respectively) and a relatively good antiestrogenic activity that is 10-fold lower than EM-139 (IC50 values of 250 and 25 nM, respectively). This nonsteroidal thioether with an alkylamide side chain is free of estrogenic activity.