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Biomedical subjects

J A Katzenellenbogen

Publications and source records attributed to J A Katzenellenbogen.

At least 163 records · Page 9Linked to original sources
PubMed1986

Synthesis of no-carrier-added N-([18F]fluoroalkyl)spiperone derivatives.

3-N-([18F]fluoroalkyl)spiperone derivatives (2,3) can be prepared by N-alkylation of spiperone (1) with fluoroalkyl halides. The fluoroalkylating species 2-[18F]fluoroethyl bromide (7), 3-[18F]fluoropropyl bromide (8) and 4-[18F]fluorobutyl bromide (9) were prepared by [18F]fluoride ion displacement of the corresponding trifluoromethanesulfonates (triflates 4,5,6). By this method, the 2-[18F]fluoroethyl-, 3-[18F]fluoropropyl-, and 4-[18F]fluorobutyl spiperone derivatives (2a-c) can be prepared and purified rapidly and conveniently, within 40 min, in yields of 30-40% (end of synthesis, EOS). An alternative approach, suitable for the preparation of 2-[18F]fluoroalkyl (ethyl, propyl, butyl, pentyl and hexyl) spiperone derivatives (3a-d), involves iodo[18F]fluorination of terminal olefins, followed by N-alkylation of spiperone. This sequence is less convenient and proceeds in lower overall yields (less than 5%).

Chromatography, High Pressure Liquid↗
PubMed1986

NCA 16 alpha-[18F]fluoroestradiol-17 beta: the effect of reaction vessel on fluorine-18 resolubilization, product yield, and effective specific activity.

Although the reported synthesis of the title compound resulted in a high radiochemical yield (43% based on resolubilized 18F), the effective specific activity at EOS was low (166 Ci/mmol). Reduction in the amount of carrier fluoride in the target water improved the effective specific activity of the product, but with a concommitant decrease in the resolubilized yield of the fluoroestradiol (12.7%). A re-examination of the labeling parameters was performed to determine the conditions that would increase the yield of the fluoroestrogen and maintain a high effective activity for the product. Since the amount of resolubilized 18F in THF is important in obtaining high specific activity compounds in this type of synthesis, several types of vessels were investigated to determine their effect on the evaporation of the [18O]H2O target water and subsequent resolubilization of 18F into THF. Of the vessels (Pt crucible, borosilicate glass, siliconized borosilicate glass, Vacutainer), the Vacutainer afforded the highest resolubilization of 18F into THF (90%), resulting in an improved resolubilized yield for the fluoroestradiol (28%) and an increased effective specific activity at EOS for the product (1600-3939 Ci/mmol).

Estradiol↗
PubMed1986

Biodistribution of N-alkyl and N-fluoroalkyl derivatives of spiroperidol; radiopharmaceuticals for PET studies of dopamine receptors.

There is great interest in the application of positron labeled ligands to map the dopamine receptor in vivo. A series of fluorine-18-labeled N-alkyl and N-fluoroalkyl spiroperidol (SP) derivatives N-methyl[18F]SP; N-ethyl[18F]SP; N-(2-[18F]fluoroethyl)SP; N-propyl[18F]fluoropropyl) SP; N-(3-fluoropropyl) [18F]SP; N-(2-[18F]fluoropropyl)SP; N-(2-[18F]fluorobutyl)SP; N-(2-[18F]fluoropentyl)SP; and N-(2-[18F]fluorohexyl)SP were synthesized. The lipophilicity of these ligands (log octanol/water partition coefficient) varies from 2.67 to 5.56 and the initial brain uptake in rats, measured at 2 min, was greatest with the methyl, ethyl, propyl, fluoroethyl, and fluoropropyl derivatives. The highest striatum/cerebellum values 1 h after administration were obtained with the N-methyl, N-propyl, and N-3-fluoropropyl derivatives, while that of N-2-fluoroethyl showed the greatest uptake of total activity in the brain at this time. The uptake of all these ligands in the striatum could be blocked by cold SP showing the striatal uptake to be by the dopamine receptors.

Alkylation↗
PubMed1986

Enol lactone inhibitors of serine proteases. The effect of regiochemistry on the inactivation behavior of phenyl-substituted (halomethylene)tetra- and -dihydrofuranones and (halomethylene)tetrahydropyranones toward alpha-chymotrypsin: stable acyl enzyme intermediate.

We have found that alpha-aryl-substituted halo enol lactones (I and II) are effective mechanism-based inactivators for chymotrypsin. In this study, we have investigated, for comparative purposes, halo enol lactones with aryl functions situated beta and gamma to the lactone carbonyl group. We synthesized 4-phenyl-5(E)-(iodomethylidene)tetrahydro-2-furanone (1), 4-phenyl-5(E)-(iodomethylidene)dihydro-2-furanone (2), 4-phenyl-6(E)-(iodomethylidene)tetrahydro-2-pyranone (3), and 5-phenyl-6(E)-(iodomethylidene)tetrahydro-2-pyranone (4), using a halolactonization reaction to convert the appropriate phenyl-substituted acetylenic acid precursor into the corresponding 5(E)-(halomethylidene)furanone and 6(E)-(halomethylidene)pyranone system. The 4-phenylfuranone (1 and 2) and the 5-phenylpyranone (4) proved to be only reversible, competitive inhibitors. By contrast, the 4-phenyltetrahydropyranone (3) inactivated alpha-chymotrypsin in a time-dependent manner. This inactivation was very rapid but reversible, with regeneration of enzyme activity being spontaneous and hydrazine-accelerated, suggestive of the intermediacy of a stable acyl enzyme. Kinetic comparison of the iodomethylene lactone 3 with the corresponding protio lactone 25 indicates that the iodine accelerates the rate of chymotrypsin acylation but produces an acyl enzyme that is more hydrolytically labile than that formed from lactone 25. From the results of this study, we conclude that a phenyl group situated at C-3 (alpha to the lactone carbonyl group) in both the 5(E)-(iodomethylidene)tetrahydro-2-furanone (I) and 6(E)-(iodomethylidene)tetrahydro-2-pyranone (II) series is essential for their activity as mechanism-based irreversible inactivators of chymotrypsin. The corresponding beta-aryl-substituted lactones, by contrast, are potent acylating agents that lead to acyl enzymes of high stability.

Acylation↗
PubMed1986

Structure-activity study of 6-substituted 2-pyranones as inactivators of alpha-chymotrypsin.

A series of 2-pyranones, bearing halogens or electron-withdrawing groups at the 6-position and alkyl, aryl, or aralkyl groups at positions 3, 4, and 5, were synthesized to investigate their binding to and inactivation of chymotrypsin. Both binding and inactivation by 2-pyranones are sensitive to substitutions on positions 3, 4, 5, and 6. Binding was poorest with alkyl substituents on position 3 and best with phenyl substitution, with benzyl or benzyl-like substitution falling in between. The sequence of binding of 6-substituted pyrones is Cl greater than Br greater than H greater than CF3. 6-Chloro-2-pyranones bearing 4-phenyl or 3-(2-naphthylmethyl) substituents effected rapid inactivation of chymotrypsin, while those having 3-benzyl or 3-(1-naphthylmethyl) substituents gave slow inactivation and those with 3-phenyl or 3-alkyl substituents gave no inactivation. Only the 6-halopyrones demonstrated inactivation, with chloro-substituted ones acting faster than bromo-substituted ones.

Chymotrypsin↗
PubMed1986

5-(Halomethyl)-2-pyranones as irreversible inhibitors of alpha-chymotrypsin.

A series of 5-(halomethyl)-2-pyranones were prepared and assayed as potential mechanism-based inhibitors of chymotrypsin, in order to test whether substitution of a suitable electron-withdrawing group at position 5 in a 2-pyranone can activate the ring for catalytic hydrolysis, as well as form a mechanism-based inhibitor. 3-Benzyl-5-(chloromethyl)-2-pyranone (3) and 3-(1-naphthylmethyl)-5-(chloromethyl)-2-pyranone (4) rapidly and irreversibly inactivate chymotrypsin with high efficiency (I/E = 2.0 for 3 and 1.38 for 4). However, evidence for their formation of an acyl enzyme is lacking, and thus they may simply be active-site alkylating agents. 6-Methyl substitution on 4, or replacement of the chloromethyl of 3 with CF3, prevents inactivation. Since 6-bromo substitution is capable of ring activation, but 5-bromo substitution (3-benzyl-5-bromo-2-pyranone (11)) is not, the 5-position of the pyrone does not appear to affect catalytic hydrolysis.

Alkylation↗
PubMed1986

Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture.

Although much attention has been paid to the removal of hormones from sera and to the development of serum-free media for studies on hormone-responsive cells in culture, little consideration has been given to the possibility that the media components themselves may have hormonal activity. We have found that phenol red, which bears a structural resemblance to some nonsteroidal estrogens and which is used ubiquitously as a pH indicator in tissue culture media, has significant estrogenic activity at the concentrations (15-45 microM) at which it is found in tissue culture media. Phenol red binds to the estrogen receptor of MCF-7 human breast cancer cells with an affinity 0.001% that of estradiol (Kd = 2 X 10(-5) M). It stimulates the proliferation of estrogen receptor-positive MCF-7 breast cancer cells in a dose-dependent manner but has no effect on the growth of estrogen receptor-negative MDA-MB-231 breast cancer cells. At the concentrations present in tissue culture media, phenol red causes partial estrogenic stimulation, increasing cell number to 200% and progesterone receptor content to 300% of that found for cells grown in phenol red-free media, thereby reducing the degree to which exogenous estrogen is able to stimulate responses. The antiestrogens tamoxifen and hydroxytamoxifen inhibit cell proliferation below the control level only when cells are grown in the presence of phenol red; in the absence of phenol red, the antiestrogens do not suppress growth. The estrogenic activity of phenol red should be considered in any studies that utilize estrogen-responsive cells in culture.

Binding, Competitive↗
PubMed1986

Application of robotics to radiopharmaceutical preparation: controlled synthesis of fluorine-18 16 alpha-fluoroestradiol-17 beta.

A commercially available robot system, the Zymark Zymate Laboratory Automation System, has been utilized for the preparation of a positron-emitting radiopharmaceutical, 16 alpha-[18F] fluoroestradiol-17 beta. This radiopharmaceutical is prepared in a three-step synthesis (preparation of [18F]-tetrabutylammonium fluoride, SN2 displacement of a triflate, and ketone reduction) and is purified by high performance liquid chromatography (HPLC). All steps in the synthesis and HPLC purification are controlled by the robot system with no manual intervention. This represents a new approach to the complete automation of radiopharmaceutical production.

Automation↗
PubMed1985

1,2-Diaryl-3,4-dihydronaphthalenes: photofluorogenic ligands for the estrogen receptor.

1,2-Diaryl-3,4-dihydronaphthalene systems related to the Upjohn antiestrogen nafoxidine (U11, 110A) (1) have high affinity for the estrogen receptor, but are essentially non-fluorescent. Upon u.v. irradiation, however, the cis-stilbene system within their structure undergo a photocyclization (2) followed by a spontaneous oxidation, forming a dihydrobenzochrysene system (3) that is highly fluorescent, but is only weakly bound by the estrogen receptor. This system can be oxidized further to the benzochrysene system, which exhibits similar fluorescence. The 1,2-diarylnaphthalene systems, obtained by direct oxidation of the dihydronaphthalenes, however, do not undergo photocyclization. The binding affinity of these derivatives for the estrogen receptor appears to be related to their degree of non-planarity and can be correlated with their molecular surface area. This facile photochemical conversion of a high-affinity ligand to a highly fluorescent species offers a new approach to the design of fluorescent estrogens.

Animals↗
PubMed1985

Facile geometric isomerization of phenolic non-steroidal estrogens and antiestrogens: limitations to the interpretation of experiments characterizing the activity of individual isomers.

In many estrogen responsive systems the isomers of tamoxifen are known to have different biological character-the trans isomer is generally an antagonist and the cis isomer an agonist. Attempts to similarly characterize the isomers of hydroxytamoxifen (which differ greatly in their affinity for the estrogen receptor) are shown to be complicated by their facile isomerization. This isomerization was studied in cultures of estrogen receptor positive MCF-7 human breast cancer cells and monitored by HPLC under reversed phase conditions. Hydroxytamoxifen isomers that are initially 99% pure, undergo a time and temperature dependent isomerization, so that after 2 days in tissue culture medium at 37 degrees C they have isomerized to the extent of 20%. This isomerization occurs in the cell-free medium alone and cannot be attributed to a metabolic conversion by the cells. The isomerization occurs much more slowly at 4 than at 37 degrees C and can be reduced considerably by various antioxidants (butylated hydroxytoluene, ascorbate, alpha-tocopherol, retinoic acid and retinal); however, at concentrations that block isomerization, these antioxidants are toxic to the cells. Although the medium contains both the cis and trans isomers of hydroxytamoxifen, the MCF-7 cells preferentially accumulate the trans isomer and the material associated with the nuclear estrogen receptor is, in all cases, mainly the higher affinity trans isomer. A similar preference of the estrogen receptor for the trans isomer is seen with diethylstilbestrol, resulting again in almost exclusive accumulation of the trans isomer in the receptor binding site. These experiments indicate the importance of verifying the isomer compositions of easily isomerizable non-steroidal estrogens and antiestrogens, such as diethylstilbestrol and hydroxytamoxifen, both in stock solutions and in experimental samples (especially those derived from receptor-associated material), so as to ascertain that the activity of the individual isomers is being correctly assigned.

Animals↗
PubMed1985

Acylamino boronic acids and difluoroborane analogues of amino acids: potent inhibitors of chymotrypsin and elastase.

A series of 1-acylamino boronic acids (IA-VA), analogues of the amino acids phenylalanine, phenylglycine, alanine, valine, and isoleucine, were prepared as potential transition-state inhibitors of the serine proteases alpha-chymotrypsin and elastase, by a boronate homologation reaction. The corresponding difluoroboranes (IB-VB), produced from the boronic acids by treatment with HF, were more easily purified than the boronic acids. Since the difluoroboranes readily hydrolyze in water, they proved to be convenient precursors for the boronic acids. The phenylalanine and phenylglycine analogues I and II were good competitive inhibitors of alpha-chymotrypsin (Ki = 0.3-8 microM), and the alanine, valine, and isoleucine analogues (III-IV) proved to be good inhibitors of elastase (Ki = 0.1-35 microM). On the basis of their high affinity and the tendency of boronic acids to form borate complexes, these acylamino boronic acids may be behaving as transition-state inhibitors.

Acylation↗
PubMed1984

Characterization of the estrogen receptor and its dynamics in MCF-7 human breast cancer cells using a covalently attaching antiestrogen.

We have used a covalently attaching antiestrogen, tamoxifen aziridine [TA; (Z)-(1-[4-(2-[N-aziridinyl] ethoxy)phenyl])1,2-diphenyl-1-butene], to analyze the structure and dynamics of the estrogen receptor in MCF-7 human breast cancer cells. The labeling of receptor with [3H]TA is specific, being blocked only by estrogens and antiestrogens, and the labeling is very efficient in that TA labels covalently the same number of receptors that are labeled reversibly by estradiol. In cells exposed to [3H]TA for 1 h, most of the covalently associated radioactivity is found in the 0.6 M KCl extract of the nuclear fraction; this receptor has an apparent mol wt of 63,000 +/- 2000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.7 by gel isoelectric focusing in the presence of 8 M urea. The mol wt and pI of cytosol receptor labeled with [3H] TA are identical. In cells labeled with [3H]TA (20 nM) for 1 h and then exposed to a chase of 10(-6) M estradiol, [3H]TA-labeled nuclear receptor disappears with a half-life of 4 h. Analysis of nuclear receptor by sodium dodecyl sulfate-gels during the chase period reveals that this loss reflects a decrease in the 63,000 mol wt species; no significant quantities of lower mol wt TA-labeled fragments are observed in the nuclear, cytosol, or membrane fractions. Affinity labeled receptor interacts with several monoclonal antibodies to MCF-7 estrogen receptor, and it can be purified extensively by immunoadsorbent chromatography. TA has a low affinity (8% that of tamoxifen) for microsomal antiestrogen-binding sites that are distinct from the estrogen receptor, but TA reacts reversibly, rather than covalently, with these sites. The findings of similar mol wt and isoelectric points for soluble cytosol and nuclear extracted receptors under strongly denaturing and disaggregating conditions reveal that nuclear localization of receptor after ligand binding is not associated with major structural alterations in the receptor component labeled by TA. In addition, the receptor, even when occupied by a covalently attached ligand, is rapidly turned over in these cells.

Affinity Labels↗
PubMed1984

Preparation of four fluorine- 18-labeled estrogens and their selective uptakes in target tissues of immature rats.

Four fluorine- 18-labeled estrogens--16 alpha-[18F]fluoro-estradiol-17 beta (1), 16 beta-[18F]fluoro-estradiol-17 beta (2), (2R, 3S)-1-[18F]fluoro-2,3-bis(4-hydroxyphenyl)-pentane (1-[18F]fluoropentestrol) (3), and (3R, 4S)-1-[18F]fluoro-3,4-bis(4-hydroxyphenyl)hexane (1-[18F]fluorohexestrol) (4)--have been prepared by simple displacement reactions utilizing reactive trifluoromethane sulfonate (triflate) precursors and F-18 fluoride ion. All four fluoroestrogens have high affinity for the estrogen receptor. In immature female rats, they are taken up by target tissues, such as the uterus, with very high selectivity: uterus-to-blood ratios at 1 hr are: Compound 1, 39; Compound 2, 12; Compound 3, 13; and Compound 4, 19. Average uterus-to-blood ratios exceed 80 for Compound 1 at 2 hr. That the uptake process involves an estrogen-specific binder of limited capacity is demonstrated by the suppressive effect of coadministered unlabeled estradiol on target tissue uptake.

Animals↗
PubMed1984

Biological activity and receptor binding of a strongly interacting estrogen in human breast cancer cells.

A substantial proportion of human breast cancers contain estrogen receptors, and it is believed that the growth of some of these tumors and their synthesis of specific proteins are stimulated by estrogens. Since natural estrogens, such as estradiol, react reversibly with estrogen receptors, it was of interest to determine the biological consequences that would result from very strong, possibly irreversible interaction of an estradiol-based ligand with the estrogen receptor of breast cancer cells. For these studies, we have examined the receptor interactions and biological character of 11 beta- chloromethylestradiol (CME) and 11 beta- bromomethylestradiol (BME) as potential estradiol-based affinity labeling ligands in MCF-7 human breast cancer cells which contain high levels of estrogen receptors. The apparent relative binding affinities of CME and BME for MCF-7 estrogen receptor measured by competitive binding assay are 230 and 15%, respectively, whereas the affinity of estradiol is considered 100%. Incubation of receptor preparations from MCF-7 cells or rat uteri with CME at 21 degrees results in a concentration- and time-dependent decrease in receptor content measured by exchange assays with [3H]estradiol. This may be due to covalent attachment of CME to receptor and is termed "inactivation." Inactivation of 80 to 85% of the receptors occurs within 30 min at 21 degrees by exposure to 5 or 20 nM CME, with 2 nM giving 20 to 40% inactivation. This receptor inactivation is prevented by preincubation with 2000 nM estradiol, indicating that the interaction of CME is occurring at the estradiol binding site on the receptor. MCF-7 cells incubated with 20 nM CME show a rapid loss of cytosol receptor sites and no accumulation of receptors detectable by exchange assay in the nucleus, while 20 nM estradiol shows nuclear localization of receptor. BME, in contrast, inactivates only a portion (approximately 40%) of estrogen receptors. CME and BME both behave as estrogen agonists. They stimulate the proliferation of MCF-7 cells and increase cellular progesterone receptor content and plasminogen activator activity. CME is at least as potent as estradiol on a molar basis in increasing all of these activities, while BME shows a biopotency of only 1% of that of estradiol or CME. Hence, although CME reacts very strongly and apparently irreversibly with estrogen receptors in MCF-7 cells, it still behaves as a potent estrogen agonist.

Animals↗
PubMed1983

Haloenol lactones. Potent enzyme-activated irreversible inhibitors for alpha-chymotrypsin.

Haloenol lactones can act as enzyme-activated irreversible inhibitors for alpha-chymotrypsin: acyl transfer to the active site serine releases a halomethyl ketone that remains tethered in the active site during the lifetime of the acyl enzyme, poised to alkylate an accessible nucleophilic residue. To investigate the structural determinants for chymotrypsin suicide inactivation with haloenol lactones, we prepared a series of nine lactones, differing in ring size (6-membered valerolactones and 5-membered butyrolactones) and in the nature of the aromatic substituent (phenyl and alpha-naphthyl), and the halogen (bromine and iodine). The inactivating behavior of these lactones is characterized by a binding constant (Ki) and three rate constants, for inactivation (k2), catalytic hydrolysis (kc), and spontaneous hydrolysis (kh). The six-membered valerolactones were much more potent inactivators than the butyrolactones, having both higher affinity and more rapid inactivation; the alpha-naphthyl-substituted lactones were also more effective, but the nature of the halogen had relatively little effect. The spontaneous rate of hydrolysis of all of these lactones is low. The turnovers per inactivation of these lactones vary from 91-1.7, with some of the alpha-naphthyl-substituted lactones approaching ideal behavior (stoichiometric inactivation). These studies indicate that several haloenol lactones are effective enzyme-activated irreversible inhibitors of chymotrypsin, and that their potency and efficiency depends markedly upon certain structural features of the lactone system.

Chromatography, High Pressure Liquid↗
PubMed1983

(2R*, 3S*)-1-[18F]fluoro-2,3-bis(4-hydroxyphenyl)pentane [( 18F]fluoronor-hexestrol), a positron-emitting estrogen that shows highly-selective, receptor-mediated uptake by target tissues in vivo.

The positron-emitting, non-steroidal estrogen (2R*, 3S*)-1-[18F]fluoro-2,3-bis(4-hydroxyphenyl)pentane [( 18F]-fluoronor-hexestrol), has been prepared by fluoride ion displacement on a labile trifluoromethanesulfonate (triflate) derivative of a suitably protected precursor, followed by removal of the aryl triflate groups with lithium aluminum hydride and purification by HPLC. In immature female rats, this compound is taken up selectively by the uterus and is retained for prolonged periods, due to its binding to the estrogen receptor. This compound and related 18F-labeled estrogens thus appear to be promising agents for imaging estrogen receptor-positive breast tumors in humans.

Animals↗