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J A Katzenellenbogen

Publications and source records attributed to J A Katzenellenbogen.

At least 145 records · Page 8Linked to original sources
PubMed1988

N-(3-[18F]fluoropropyl)-spiperone: the preferred 18F labeled spiperone analog for positron emission tomographic studies of the dopamine receptor.

The ligands currently used for PET studies of the dopamine receptor are fluorine-18-labeled spiperone (FSp) and carbon-11 or fluorine-18-labeled N-methyl-spiperone. All three of these ligands have drawbacks in either their chemical preparation or their biological behavior. We have previously prepared a series of N-fluoroalkyl-spiperone derivatives which are simple to prepare in high radiochemical yield. N-[18F]fluoropropyl-spiperone (3-F-Pr-Sp) and N-[18F]fluoroethyl-spiperone (2-F-Et-Sp) were the most promising ligands. In vitro competitive binding studies showed affinities for the dopamine receptor of 3-F-Pr-Sp greater than FSp greater than 2-F-Et-Sp. Brain extraction studies in a primate model showed that FSp, 2-F-Et-Sp, and 3-F-Pr-Sp were not completely extracted by the brain. High bone uptake and kidney clearance was observed with 3-F-Pr-Sp, while 2-F-Et-Sp cleared through the intestine in rats. This is in contrast to FSp where clearance is through the kidney. Studies to evaluate the extraction of metabolites in the brain were carried out by administering large doses (10 mCi) of FSp, 2-F-Et-Sp and 3-F-Pr-Sp to rats and reinjecting the metabolites in blood into other rats. These experiments showed that less than 0.02% of the metabolites from FSp and 3-F-Pr-Sp entered the brain, while 0.5% of the metabolites from 2-F-Et-Sp entered the brain. The majority of the activity present in the cerebellum after the administration of 2-F-Et-Sp is metabolites; therefore 2-F-Et-Sp is unsuitable for PET imaging studies. PET imaging studies in baboons and in one normal human volunteer with 3-F-Pr-Sp showed a high striatum-to-cerebellum ratio, showing that 3-F-Pr-Sp can replace ligands currently in use to study dopamine receptors.

Animals↗
PubMed1988

Optimizing of 2,3-diarylindenes as fluorescent estrogens: variation of the acceptor group, ortho substitution of the 2-ring, and C-1 methylation.

In an attempt to elucidate steric and electronic factors that affect the fluorescence and estrogen receptor binding properties of 2,3-diarylindenes, we have prepared and examined the behavior of 11 analogues bearing substituents on the 1-position or on the 2-aryl ring. These compounds were synthesized by alkylation of a 1,2-diarylethanone with 3-methoxybenzyl chloride, followed by cyclodehydration to the indene. The electronic spectra of those compounds without tau-electron accepting groups on the 2-aryl ring display the absorbance and fluorescence of a hindered stilbene system; those with nitro and cyano substituents on the 2-aryl ring show charge-transfer character, having a more bathochromic absorption and fluorescence. One bisphenolic nitroindene, in particular, shows a strong, long-wavelength absorption and an intense emission, with a large Stokes' shift that is highly sensitive to solvent polarity. Estrogen receptor binding affinity measurements on these compounds indicate that substituents that twist the pendant aryl rings (such as a 1-methyl group, or an o-methyl or trifluoromethyl group on the 2-phenyl ring) increase binding affinity. Bulky (4-bromo) or electron-withdrawing groups (3- and 4-nitro, 4-cyano) on the 2-phenyl group, or its replacement with a 3-pyridyl group, greatly reduce binding affinity, suggesting that the complementary region of the receptor is relatively intolerant of bulk and may have specific hydrogen-bonding requirements. This investigation of the concurrent effects of substituents on the fluorescence properties and receptor binding affinity of 2,3-diarylindenes should assist in the development of effective, inherently fluorescent ligands for the estrogen receptor.

Binding, Competitive↗
PubMed1988

Bis(4-hydroxyphenyl)[2-(phenoxysulfonyl)phenyl]methane: isolation and structure elucidation of a novel estrogen from commercial preparations of phenol red (phenolsulfonphthalein)

Commercial preparations of phenolsulfonphthalein (Phenol Red), a pH indicator dye widely added to cell culture media, have weak estrogenic activity that can be accounted for by a minor lipophilic impurity (ca. 0.002%). We have isolated this impurity, determined its structure to be bis(4-hydroxyphenyl)[2-(phenoxysulfonyl)phenyl]methane, and synthesized it from phenolsulfonphthalein. This compound binds to the estrogen receptor with an affinity 50% that of estradiol; it stimulates the proliferation and increases the progesterone receptor content of estrogen-responsive breast cancer cells in vitro, and it stimulates uterine weight gain in rats in vivo, but shows a potency in these assays only 0.1-0.2% that of estradiol. We suggest how this novel estrogen may be generated during the preparation of phenolsulfonphthalein.

Animals↗
PubMed1988

2,3-Diarylindenes and 2,3-diarylindenones: synthesis, molecular structure, photochemistry, estrogen receptor binding affinity, and comparisons with related triarylethylenes.

Two 2,3-diphenylindene and -indenone systems, with potential fluorescent and photofluorogenic properties, were prepared and studied as ligands for the estrogen receptor. The indene systems were prepared by Friedel-Crafts cyclization of appropriate alpha-benzyl desoxybenzoin systems, and the indenones either by oxidation of the indenes, by cyclization of alpha-benzoyl desoxybenzoins, or by acylium ion attack on tolan. Crystallographic analysis of the 2,3-diphenylindene and -indenone systems shows the phenyl substituents twisted out of the plane of the indene/indenone systems, with both torsional angles greater in the indenone than indene system; the phenyl attachment to the five-membered ring allows these systems to be considerably more planar than the related 1,2-diphenyl-3,4-dihydronaphthalene and the triarylethylene nonsteroidal estrogens. In contrast to the diphenyldihydronaphthalenes, the diarylindene and -indenone systems undergo photocyclization to phenanthrenes inefficiently. The estrogen receptor binding affinity of these systems is reasonably high (9-59% relative to estradiol), with the indenone systems having higher affinity than the indenes; additional hydroxyl substitution raises the affinity of the indenes but lowers that of the indenones. These trends can be rationalized by considering differences in molecular volumes or surface areas (related to torsional angles) and specific polar interactions.

Binding, Competitive↗
PubMed1988

21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone: synthesis and target tissue selective uptake of a progestin receptor based radiotracer for positron emission tomography.

We have synthesized 21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone (FENP), a high affinity ligand for the progesterone receptor, labeled with the positron-emitting radionuclide fluorine-18 (t1/2 = 110 min). The synthesis proceeds in two steps from 21-hydroxy-16 alpha-ethyl-19-norprogesterone and involves [18F]fluoride ion displacement of the 21-trifluoromethanesulfonate (21-triflate). This material is purified by HPLC and is obtained in 4-30% overall yield (decay corrected) within 40 min after the end of bombardment to produce [18F]fluoride ion. The effective specific activity, determined by competitive radioreceptor binding assays, is 700-1400 Ci/mmol. In vivo, [18F]FENP demonstrates highly selective, receptor-mediated uptake by the uterus of estrogen-primed rats; the uterus to blood and uterus to muscle ratios were respectively 26 and 16 at 1 h and 71 and 41 at 3 h after injection. The high target tissue selectivity of this uptake suggests that this compound may be useful for the in vivo imaging of progestin target tissues and receptor-rich tumors (such as human breast tumors) by positron emission tomography.

Animals↗
PubMed1988

Breast cancer: PET imaging of estrogen receptors.

Thirteen patients with primary breast masses were studied with positron emission tomography (PET) and 16 alpha-[fluorine-18]-fluoroestradiol-17 beta. PET images demonstrated uptake of the labeled estrogen analog at sites of primary carcinomas and in several foci of axillary lymph node metastases, as well as in one distant metastatic site. There was excellent correlation between uptake within the primary tumor measured on the PET images and the tumor estrogen-receptor concentration measured in vitro after excision (r = .96). This technique may provide an in vivo method of assessing estrogen receptors in primary and metastatic breast cancers and thus guide management of this disease with antiestrogen chemotherapy.

Adenocarcinoma↗
PubMed1987

Desmethylnafoxidine aziridine: an electrophilic affinity label for the estrogen receptor with high efficiency and selectivity.

Desmethylnafoxidine aziridine (Naf-Az), an affinity label for the estrogen receptor based structurally on the antiestrogen nafoxidine, has been prepared in unlabeled and in high specific activity, tritium-labeled form and has been evaluated for its apparent competitive binding, and time-dependent irreversible, covalent attachment to the estrogen receptor. Naf-Az was synthesized through a key 1,2-diaryl-3,4-dihydronaphthalene intermediate that was prepared from 6-methoxy-1-tetralone by two routes involving alternate strategies for arylation. Conversion of the diaryldihydronaphthalene to Naf-Az through a series of deprotection-activation reactions culminated in ethyleneimine displacement of a methanesulfonate. The tritium-labeled material was prepared by tritium-iodine exchange on an iodinated methanesulfonate precursor, followed by ethyleneimine displacement. Compared to our previously-prepared reagent tamoxifen aziridine (Tam-Az), Naf-Az has a higher apparent competitive binding affinity, and it reacts with the estrogen receptor in cytosol preparations and in intact MCF-7 breast cancer cells rapidly and with at least comparable efficiency and selectivity. SDS-polyacrylamide gel electrophoretic analysis confirms its selective labeling of the Mr 66,000 estrogen receptor. Naf-Az should prove to be useful in studies aimed at characterizing the properties and structure of estrogen receptors.

Affinity Labels↗
PubMed1987

11 beta-chloromethyl-[3H]estradiol-17 beta: a very high affinity, reversible ligand for the estrogen receptor.

It has been suggested that binding of 11 beta-chloromethyl estradiol (11 beta-CME2) to the estrogen receptor is irreversible, since its complex with receptor fails to undergo exchange with estradiol (E2). To investigate this behavior directly, 11 beta-CME2 was prepared in high specific activity, tritium-labeled form: The binding of [3H]11 beta-CME2 to the estrogen receptor from lamb and rat uterus and MCF-7 human breast cancer cells was shown to be fully reversible; the 11 beta-CME2 complex with receptor, as well as that of a structural analog 11 beta-ethyl estradiol, however, do not dissociate or exchange with [3H]E2 over a 22 h period at 25 degrees C. By competitive or direct binding assays, the affinity of 11 beta-CME2 for the estrogen receptor can be estimated to be as much as 10- to 30-fold higher than that of E2. The complexes of estrogen receptor from MCF-7 cells with [3H]11 beta-CME2 and [3H]E2 show identical velocity sedimentation profiles on sucrose gradients, under conditions when the receptor is either a monomer of a dimer. Because of its very high affinity and unusual dissociation kinetics, [3H]11 beta-CME2 should be a very useful ligand for studies of estrogen receptor dynamics and in the assay of estrogen receptor concentrations in tumors and tissues.

Animals↗
PubMed1987

Characterization of the uptake of 16 alpha-([18F]fluoro)-17 beta-estradiol in DMBA-induced mammary tumors.

In order to investigate possible correlations between the uptake of 16 alpha-([18F]fluoro)-17 beta-estradiol (18F-ES) by 7,12-dimethylbenz(a) anthracene (DMBA)-induced tumors in rats and the estrogen receptor (ER) content of these tumors, a comprehensive study was performed in which the tissue distribution of 18F-ES was measured in tumor-bearing rats, together with simultaneous measurements of blood volume (by technetium-labeled red blood cells) and blood flow (by iodoantipyrine infusion). In addition, the time course of 18F-ES metabolism and the tissue distribution of the metabolites was studied. Metabolism of 18F-ES is very rapid, and after 2 h, most of the activity in blood and nontarget tissues is due to metabolites; target tissue activity, however, is due mainly to unmetabolized compound. Most of the circulating activity, both 18F-ES and its metabolites, is strongly associated with macromolecules or cells, and while the metabolites are not taken up selectively by target tissues, they do enter nontarget tissues. Tumor blood volume and blood flow vary widely, but not in a way that appears related to tumor necrosis. The uptake of 18F-ES by the uterus and DMBA-induced mammary tumors of adult rats reaches maximum levels (ca 0.35 and 0.10% I.D./g X kg, respectively) at early times (0-1 h), and drops slowly thereafter. The uterus to nontarget or tumour to nontarget tissue ratios, however, start low and continue to increase, reaching maximum levels (ca 20 and 15, respectively) at 2-3 h. There does not, however, appear to be a simple relationship between tumor uptake (either as % I.D./g X kg or tumor to nontarget ratio) measured at a single 3 h time point and tumor ER content, even considering differences in tumor blood flow. This suggests that an estimation of tumor ER content will require the application of more complex pharmacodynamic models that involve the measurement of the complete profile of receptor lignad uptake, retention, and washout from target to nontarget areas. The application of such models will be assisted by the development of estrogen receptor binding ligands that are not converted to circulating metabolites.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1987

Preparation, receptor binding, and fluorescence properties of hexestrol-fluorophore conjugates: evaluation of site of attachment, fluorophore structure, and fluorophore-ligand spacing.

We have undertaken a staged development of certain estrogen-fluorophore conjugates, in order to prepare a fluorescent estrogen suitable for determination of the estrogen receptor content of individual cells. Since non-steroidal estrogens with bulky substituents are often more readily bound by receptor than their steroidal counterparts, we have investigated fluorophore conjugates with derivatives of the non-steroidal estrogen hexestrol [3R*, 4S*)-3,4-bis(4-hydroxyphenyl)hexane). On the basis of the receptor-binding affinity of model compounds, we prepared a prototypical set of three ring- and side-chain-substituted fluorescent hexestrol derivatives, whose binding and fluorescence properties ultimately led to the preparation of a series of side-chain-substituted nitrobenzoxadiazole derivatives. The compounds prepared have binding affinities for the estrogen receptors that range from ca. 1% to 5% that of estradiol, and they have very favorable fluorescence characteristics, similar to those of fluorescein.

Animals↗
PubMed1987

Estrogenic affinity labels: synthesis, irreversible receptor binding, and bioactivity of aziridine-substituted hexestrol derivatives.

To develop an affinity label for the estrogen receptor that would be an estrogen agonist, rather than antagonist, we prepared several aziridine derivatives of the potent nonsteroidal estrogen hexestrol [3R,4S)-3,4-bis(4-hydroxyphenyl)hexane) bearing an aziridine function on the side chain. Three functional groups link the hexestrol ligand and the aziridine: a carbonyl group (ketone or ester), a thioether, or a methylene chain. The apparent competitive binding affinity of these derivatives for the estrogen receptor ranges from 1.8% to 25% that of estradiol, and most of them bind in a time-dependent, irreversible manner with the receptor, although the rate and efficiency of this binding vary widely, often with relatively small changes in structure. This is consistent with the irreversible attachment requiring a precise alignment of activating and reacting residues in the binding site of the receptor. The estrogenic and antiestrogenic activity of these aziridine derivatives was investigated in MCF-7 human breast cancer cells. Most of the compounds are agonists, with one being an antagonist. The derivative (6R,7S)-1-N-aziridinyl-6,7-bis(4-hydroxyphenyl)-5-nonanone (keto-nonestrol aziridine 3) appears to have the most ideal behavior of the estrogenic affinity labeling agents prepared: It is an agonist, and it binds to receptor irreversibly, efficiently, and quite rapidly.

Affinity Labels↗
PubMed1987

Ketononestrol aziridine, an agonistic estrogen receptor affinity label: study of its bioactivity and estrogen receptor covalent labeling.

Ketononestrol aziridine [(6R,TS)1-(N-aziridinyl)6,7-bis-(4-hydroxyphenyl)5-nonamone (KNA)], an aziridine derivative of hexestrol, is an estrogenic affinity label for the estrogen receptor (ER). It has an apparent relative binding affinity 8% that of estradiol and shows time-dependent irreversible binding to the ER in uterine cytosol preparations and intact human breast cancer cells (MCF-7). The agonistic activity of KNA is evident in MCF-7 cells in culture, where it increases the cell growth rate and elevates the level of progesterone receptor. KNA was prepared in high specific activity tritium-labeled form by iodination of a methanesulfonate precursor, followed by catalytic tritium-iodine exchange and aziridinylation; the material prepared has high radiochemical purity and a specific activity of 67 Ci/mmol. The covalent attachment of [3H]KNA to the ER can be followed directly by a solvent precipitation assay. In cytosol preparations of uterine ER, labeling with [3H]KNA proceeds in a time-, concentration-, and temperature-dependent manner; labeling is efficient and selective and, by competition studies, was shown to be estrogen specific. ER in intact MCF-7 cells can also be covalently labeled by treatment with [3H]KNA. Receptor covalently labeled with [3H]KNA sediments as a 4S species on high salt sucrose gradients, and its sedimentation position is shifted by treatment with monoclonal antireceptor antibodies. On sodium dodecyl sulfate-polyacrylamide gels, the principal labeled species migrates with a mol wt of 66,000. KNA should prove to be a useful probe for studies on receptor structure, function, and chromatin interactions, particularly when the behavior of a receptor-agonist complex is being investigated.

Affinity Labels↗
PubMed1987

Fluorinated androgens and progestins: molecular probes for androgen and progesterone receptors with potential use in positron emission tomography.

In order to develop imaging agents for receptor-positive tumors of the breast and prostate, we have investigated the binding affinity of several fluorine-substituted steroids in the testosterone and nortestosterone series for the androgen receptor and the progesterone receptor. The 6 alpha- and 11 beta-fluoro-, and 16 alpha-fluoroalkyl-substituted steroids were prepared by an olefin bromofluorination reaction followed by dehydrobromination or reductive debromination. The 17 alpha-fluoromethyl derivatives were prepared by fluoride ion attack on the 17-spiroepoxide or 17-spiro sulfate and the 17 alpha-fluoropropynyl derivative, by reaction of a propargyl alcohol precursor with diethylaminosulfur trifluoride. Of the compounds synthesized, 17 alpha-(3-fluoro-I-propynyl)nortestosterone was found to possess the highest binding affinity and selectivity for the progesterone receptor, and 11 beta-fluoronordihydrotestosterone had the greatest affinity for the androgen receptor. Both receptor systems seem to tolerate reasonably well the substitution of fluorine for hydrogen.

Androgens↗
PubMed1986

Compounds that inhibit chymotrypsin and cell replication.

Several compounds have been tested for their ability to inhibit bovine pancreatic alpha-chymotrypsin (Ki) and their ability to inhibit cell replication (IC50). There is good agreement over three orders of magnitude between the Ki and the IC50 values of these compounds. The data support the hypothesis that a cellular, chymotrypsin-like activity is necessary for cell replication.

Animals↗
PubMed1986

Halo enol lactones: studies on the mechanism of inactivation of alpha-chymotrypsin.

In a previous investigation [Daniels, S. B., Cooney, E., Sofia, M. J., Chakravarty, P. K., & Katzenellenbogen, J. A. (1983) J. Biol. Chem. 258, 15046-15053], we demonstrated that alpha-aryl-substituted five- and six-membered ring halo enol lactones were effective inhibitors of chymotrypsin, and we proposed that they reacted by an enzyme-activated mechanism: acyl transfer to the active site serine generates a halomethyl ketone that remains tethered in the catalytic site until it alkylates an accessible nucleophilic residue. In this study, we have investigated in greater detail the process of chymotrypsin inactivation by an alpha-naphthyl-substituted five- and six-membered bromo enol lactone. Inactivation by both compounds appears to be active site directed, since the time-dependent inactivation is retarded by competing substrate. The possible involvement of a paracatalytic mechanism for inactivation (generation of a free, rather than active site bound, inactivating species) was investigated by comparing the inactivation efficiencies of the lactones with that of the bromomethyl keto acid hydrolysis products. The bromomethyl ketone derived from the five-membered lactone is ineffective, whereas that derived from the six-membered lactone is highly efficient. However, the possible involvement of the free keto acid in chymotrypsin inactivation by the six-membered lactone is ruled out by experiments involving selective scavenging. The long-term inactivation of chymotrypsin requires the presence of the bromine substituent and appears to involve an alkylation rather than an acylation reaction (hydrazine resistant). Furthermore, a 1:1 lactone:enzyme stoichiometry is demonstrated with the 14C-labeled six-membered lactone. These results are consistent with the mechanism-based inactivation process previously presented.

Chromatography, High Pressure Liquid↗
PubMed1986

Nitrosourea and nitrosocarbamate derivatives of the antiestrogen tamoxifen as potential estrogen receptor-mediated cytotoxic agents in human breast cancer cells.

We have prepared two analogs of the antiestrogen tamoxifen that incorporate known DNA-crosslinking functions, a chloroethyl nitrosourea and a nitrosocarbamate moiety, and we have tested their bioactivities in cultures of human breast cancer cells. Both compounds bind to the estrogen receptor from MCF-7 cells, with relative binding affinities of 0.18% for the nitrosocarbamate derivative and 0.35% for the nitrosourea derivative, while the affinity of tamoxifen is 1.8%, and that of estradiol is set at 100%. The tamoxifen-nitrosocarbamate compound demonstrated a dose-related cytotoxicity by the colony formation and cell proliferation assays that was not blocked by estradiol in either estrogen receptor-positive MCF-7 cells or estrogen receptor-negative MDA-MB-231 cells, and thus, was not studied further. Tamoxifen-nitrosourea (TAM-NU) showed dose-related cytotoxicity in MCF-7 cells that was blocked by estradiol, whereas its activity in MDA-MB-231 cells was unaffected by estradiol. N-2-(4-t-butylphenoxy)ethyl-N'-chloroethyl-N'-nitrosourea (BPE-NU), a control compound which contains the nitrosourea moiety but does not bind to the estrogen receptor, had no effect on cell proliferation or colony formation in MCF-7 cells, but was very inhibitory in the receptor-negative MDA-MB-231 cells. In contrast, TAM-NU was more active in the receptor-positive MCF-7 cells than in the MDA-MB-231 line. Thus, because TAM-NU appears to be active selectively against the receptor-positive cell line, and because this activity is suppressible by estradiol, its cytotoxic effect seems to be mediated via the estrogen receptor. However, since TAM-NU is active only in prolonged treatment protocols, it appears likely that its cytotoxic activity results from the hormone antagonistic effect of the hydrolysis product of TAM-NU (bis-desmethyltamoxifen), rather than from a direct receptor-mediated, DNA-directed cytotoxic action of TAM-NU itself. This study stresses the need for the use of appropriate control compounds and cell systems in order to assess whether the toxic activity displayed by hormone-cytotoxic conjugates is mediated by receptor interactions and whether it operates through the intended toxic mechanism.

Breast Neoplasms↗
PubMed1986

Fluorescence-based assay of estrogen receptor using 12-oxo-9(11)-dehydroestradiol-17 beta.

12-Oxo-9(11)-dehydroestradiol-17 beta (12-oxo-E2) was used to assay estrogen receptor binding in uterine cytosol preparations by an indirect fluorescence assay. In alkaline solution, 12-oxo-E2 has a fluorescence excitation maximum at 402 nm (epsilon = 24,000) and an emission maximum at 480 nm (phi f = 0.57), and its fluorescence can be observed down to 5 X 10(-11) M. The minimum detection limit of 12-oxo-E2 is 25 fmol by spectrofluorometry and 5 fmol by HPLC-fluorometry. Although this compound is not appreciably fluorescent at neutral pH (i.e., at conditions under which it binds to the estrogen receptor), receptor binding by fluorometry can be measured indirectly: After equilibration of 12-oxo-E2 with the receptor preparation and removal of excess free ligand, the receptor-12-oxo-E2 complex is disrupted, and fluorescence measurements are made on the dissociated 12-oxo-E2 in alkaline medium. This fluorometric assay was validated quantitatively by performing simultaneously, on the same receptor preparation, radiometric and fluorometric assays with [3H]E2 and [3H]-12-oxo-E2. The radiometric determinations with both compounds gave nearly equivalent estimates of receptor site concentrations, but the fluorometric estimate of binding site concentration was somewhat less (70-85%) than that expected on the basis of the [3H]E2 radiometric assay. The use of 12-oxo-E2 in an indirect spectro- or HPLC-fluorometric assay provides a means for assaying estrogen receptor concentrations by fluorescence with a sensitivity approaching that of radiometric techniques.

Animals↗