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Sickle cell anemia disease has been a great challenge to the world in the present situation. It occurs only due to the polymerization of sickle hemoglobin (HbS) having Pro-Val-Glu typed mutation, while the polymerization does not occur in normal hemoglobin (HbA) having Pro-Glu-Glu peptides. It is also well confirmed that the oxygenated HbS (OHbS) does not participate in the polymerization, while the deoxygenated HbS (dHbS) does, which causes the shape of red blood cells sickled. After polymerization, the blood has a low oxygen affinity. Keeping this fact into consideration, only those drugs are being synthesized that stabilize the OHbS structure so that the polymerization of HbS can be stopped. The literature data showed no systematic description of the changes occurring during the OHbS conversion to dHbS before polymerization. Hence, an innovative reasonable study between HbA and HbS, when they convert into their deoxygenated forms, was done computationally. In this evaluation, physiochemical parameters in HbA/HbS before and after deoxygenation were studied and compared deeply. The computationally collected data was used to understand the abnormal behaviour of dHbS arising due to the replacement of Glu6 with Val6. Consequently, during the presented computational study, the changes occurring in HbS were found opposite/abnormal as compared to HbA after the deoxygenation of both. The mechanism of Voxelotor (GBT-440) action to stop the HbS polymerization was also explained with the help of computationally collected data. Besides, a comparative study between GBT-440 and another suggested drug was also done to know their antisickling strength. Additionally, the effect of pH, CO, CO 2 , and 2,3-diphosphoglycerate (2,3-DPG) on HbS structure was also studied computationally., (© 2024. The Author(s).)

A thin, active shell layer on core-shell columns provides high efficiency in HPLC at moderately high pressures. We revisited three models of mobile phase effects on retention for core-shell columns in mixed aqueous-organic mobile phases: linear solvent strength and Snyder-Soczewiński two-parameter models and a three-parameter model. For some compounds, two-parameter models show minor deviations from linearity due to neglect of possible minor retention in pure weak solvent, which is compensated for in the three-parameter model, which does not explicitly assume either the adsorption or the partition retention mechanism in normal- or reversed-phase systems. The model retention equation can be formulated as a function of solute retention factors of nonionic compounds in pure organic solvent and in pure water (or aqueous buffer) and of the volume fraction of an either aqueous or organic solvent component in a two-component mobile phase. With core-shell columns, the impervious solid core does not participate in the retention process. Hence, the thermodynamic retention factors, defined as the ratio of the mass of the analyte mass contained in the stationary phase to its mass in the mobile phase in the column, should not include the particle core volume. The values of the thermodynamic factors are lower than the retention factors determined using a convention including the inert core in the stationary phase. However, both conventions produce correct results if consistently used to predict the effects of changing mobile phase composition on retention. We compared three types of core-shell columns with C18-, phenyl-hexyl-, and biphenyl-bonded phases. The core-shell columns with phenyl-hexyl- and biphenyl-bonded ligands provided lower errors in two-parameter model predictions for alkylbenzenes, phenolic acids, and flavonoid compounds in comparison with C18-bonded ligands.

Cationic amphiphilic drugs and aminoglycoside antibiotics can induce phospholipidosis (PLD), an abnormal accumulation of phospholipids in lysosome-derived vesicles, in preclinical studies. The incidence of PLD in patients and its clinical relevance are difficult to assess without noninvasive biomarkers. Di-docosahexaenoyl bis(monoacylglycerol)phosphate (di-22:6-BMP) is a phospholipid that is enriched in lysosomal membranes and a proposed urinary biomarker of drug-induced PLD. The specificity of di-22:6-BMP for PLD was compared to other phospholipid species that can increase in urine with nephrotoxicity. Using liquid chromatography coupled to mass spectrometry, 12 phospholipids were assayed in the urine of rats treated with drugs that induced PLD or caused renal or skeletal muscle injury. In receiver operating curve analyses, urinary di-22:6-BMP was a significantly better predictor of PLD and the least predictive of tissue injury of the phospholipids assayed. The data provide evidence supporting the use of di-22:6-BMP as a urinary biomarker of PLD in rats.

The research was performed on 24 male sexually mature domestic rabbits, divided in two equal batches. The rabbits from the first batch were administered 0.5 mg/kg of body weight hexestrol diacetate intramuscularly twice a week for four consecutive weeks. Batch 2 was used for control. The testicular tissue samples obtained after orchidectomy were processed in order to obtain histological samples, stained using the Goldner's trichrome method. Examination of histological sections from the control batch showed that a natural aspect of seminal tubule epithelium, without reported injuries, even discrete. In the experimental batch were recorded a number of changes to all categories of the seminal cells. The severity and extent of damages varied greatly from one seminal tube to another and even from one part to another of the same seminal tube. These changes were the appearance of apoptotic cells and apoptotic bodies, vacuolar degenerescence of spermatocytes and spermatides, syncitialisation of spermatides, areas of necrosis accompanied by severe disruption of the seminiferous epithelium. In some areas, the lesions were so severe that the affected area of the cells forms "basal area". If lesions in the "adluminal area" affects only temporarily gametogenetic function the lesions in the "basal area" are irreversible as there are named "reserve cells" which is the starting point of spermatogenesis. Highlighted issue raised on the opportunity use of hexestrol diacetate in therapy or animal production stimulation as it gametogenetic function in males while their risk transfer to humans through consumption of foods of animal origin.

Mallory-Denk bodies (MDB) are hepatocyte inclusions containing cytokeratin 8 (CK8) which can develop, along with other steatohepatitis lesions, in patients treated with amiodarone, perhexiline maleate or 4,4'-diethylaminoethoxyhexestrol. These drugs accumulate lipids, whose subsequent peroxidation liberates reactive by-products, like malondialdehyde (MDA). The formation of MDB has been previously reproduced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine or griseofulvin administration which cross-link CK8 by tissue transglutaminase, thus forming an entangled network, from which MDB progressively arise. The present study depicts the mechanisms initiating MDB formation by steatohepatitis-inducing drugs. Short incubation of hepatocytes with amiodarone (50 microM), 4,4'-diethylaminoethoxyhexestrol (50 microM) or perhexiline maleate (25 microM) increased the pool of CK8 monomers and increased cell calcium to activate Ca(++)-dependent transglutaminases which cross-linked the CK8 monomers into CK8-containing oligomers. The present study also provides the first evidence that MDA might directly participate in MDB formation, as this reactive agent cross-linked purified CK8 or albumin in vitro, disrupted the cytokeratin network of isolated hepatocytes, and bridged CK8 molecules. In conclusion, steatohepatitis-inducing drugs increase cell calcium and activate tissue transglutaminase, which cross-links CK8 to form a molecular scaffold, from which MDB might secondarily arise. Malondialdehyde also cross-links CK8, albeit through a different mechanism, and might also contribute to MDB formation.

Fenestranes are regarded as a particularly challenging synthetic targets, and only few syntheses have been reported in the recent past. These rare compounds of synthetic and theoretical interest are a class of tetracyclic skeletons, defined as doubly a,a'-bridged spiroalkanes. The reported results are focused on the synthesis of new and original [4.6.4.6]fenestradienes 3a-f and [4.6.4.6]fenestrenes 4a-e. Our approach implies the formation of this tetracyclic structure by a reaction cascade, based on consecutive transformations starting from the trienyne 1a-f: an initial soft hydrogenation using a P-2 Nickel catalyst at room temperature, followed by a conrotatory 8p electrocyclization and a disrotatory 6p electrocyclization and a final oxidation. Several examples of this type of new compounds are described in this Communication.

A series of thermal pericyclic reactions of beta-allenylfuranones have been studied. It was observed that beta-allenylfuranones would undergo 1,5-hydrogen shift to afford a new type of trienes upon heating. Due to their high reactivity, these trienes would undergo subsequent pericyclic reactions based on the nature of the substituent group R: When R is an alkyl group, the intermediate 4a or 4b would undergo a further 1,7-hydrogen shift to afford a more stable conjugated triene 3; with R being phenyl or cyclopropyl group, the 1,7-hydrogen shift was inhibited and the 4-type conjugated triene would form a six-membered ring 5 via 6 pi-electrocyclization. Interestingly, introducing another C=C double bond into the triene intermediate (R = CH=CH2, the 18-type intermediate would undergo 8 pi-electrocyclization reaction to form an eight-membered ring. Such a transformation was also observed with 2-allyl-3-allenylcyclohex-2-enones. The deuterium-labeling mechanistic studies show that the alkyl groups at the allenyl moiety of 1 participated in the isomerization process via 1,7-hydrogen shifts between 18 A, 20 A, and 29 A.

The nonsteroidal synthetic estrogen hexestrol (HES), which is diethylstilbestrol hydrogenated at the C-3-C-4 double bond, is carcinogenic. Its major metabolite is the catechol, 3'-OH-HES, which can be metabolically converted to the catechol quinone, HES-3',4'-Q. Study of HES was undertaken with the scope to substantiate evidence that natural catechol estrogen-3,4-quinones are endogenous carcinogenic metabolites. HES-3',4'-Q was previously shown to react with deoxyguanosine to form the depurinating adduct 3'-OH-HES-6'-N7Gua by 1,4-Michael addition [Jan S-T, Devanesan PD, Stack DE, Ramanathan R, Byun J, Gross ML, et al. Metabolic activation and formation of DNAadducts of hexestrol,a synthetic nonsteroidal carcinogenic estrogen. Chem Res Toxicol 1998;11:412-9.]. We report here formation of the depurinating adduct 3'-OH-HES-6'-N3Ade by reaction of HES-3',4'-Q with Ade by 1,4-Michael addition. The structure of the N3Ade adduct was established by NMR and MS. We also report here formation of the depurinating 3'-OH-HES-6'-N7Gua and 3'-OH-HES-6'-N3Ade adducts by reaction of HES-3',4'-Q with DNA or by activation of 3'-OH-HES by tyrosinase, lactoperoxidase, prostaglandin H synthase or 3-methylcholanthrene-induced rat liver microsomes in the presence of DNA. The N3Ade adduct was released instantaneously from DNA, whereas the N7Gua adduct was released with a half-life of approximately 3 h. Much lower (<1%) levels of unidentified stable adducts were detected in the DNA from these reactions. These results are similar to those obtained by reaction of endogenous catechol estrogen-3,4-quinones with DNA. The similarities extend to the instantaneously-depurinating N3Ade adducts and relatively slowly-depurinating N7Gua adducts. The endogenous estrogens, estrone and estradiol, their 4-catechol estrogens and HES are carcinogenic in the kidney of Syrian golden hamsters. These results suggest that estrone (estradiol)-3,4-quinones and HES-3',4'-Q are the ultimate carcinogenic metabolites of the natural and synthetic estrogens, respectively. Reaction of the electrophilic quinones by 1,4-Michael addition with DNA at the nucleophilic N-3 of Ade and N-7 of Gua is suggested to be the major critical step in tumor initiation by these compounds.

Background & Aims: 4,4'-Diethylaminoethoxyhexestrol (DEAEH), amiodarone, and perhexiline cause steatohepatitis in humans. The mechanisms of these effects are unknown for DEAEH and have not been completely elucidated for amiodarone and perhexiline. The aim of this study was to determine these mechanisms., Methods: Rat liver mitochondria, cultured rat hepatocytes, or rats were treated with these drugs, and the effects on mitochondrial respiration, beta-oxidation, reactive oxygen species formation, and lipid peroxidation were determined., Results: DEAEH accumulated in mitochondria and inhibited carnitine palmitoyl transferase I and acyl-coenzyme A dehydrogenases; it decreased beta-oxidation and caused lipid deposits in hepatocytes. DEAEH also inhibited mitochondrial respiration and decreased adenosine triphosphate (ATP) levels in hepatocytes. DEAEH, amiodarone, and perhexiline augmented the mitochondrial formation of reactive oxygen species and caused lipid peroxidation in rats., Conclusions: Like amiodarone and perhexiline, DEAEH accumulates in mitochondria, where it inhibits both beta-oxidation (causing steatosis) and respiration. Inhibition of respiration decreases ATP and also increases the mitochondrial formation of reactive oxygen species. The latter oxidize fat deposits, causing lipid peroxidation. We suggest that ATP depletion and lipid peroxidation may cause cell death and that lipid peroxidation products may account, in part, for other steatohepatitis lesions.

The immunomodulatory drug tilorone (2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one) and several congeners are known to disturb the lysosomal degradation of sulphated glycosaminoglycans and thereby induce lysosomal storage of glycosaminoglycans in cultured cells and intact organisms. The molecules of tilorone and congeners consist of a planar aromatic ring system symmetrically substituted with two aliphatic side chains each carrying a protonizable nitrogen. In a previous study it was proposed that non-degradable glycosaminoglycan-drug complexes are formed by electrostatic interactions and that additionally intermolecular interactions between the drug molecules due to electronic coupling of their central planar ring system are important for formation and stabilization of the glycosaminoglycan-drug complexes and thus for the drug side effect in question. The significance of the central planar ring system was tested in the present study by comparing tilorone and the compound bis(beta-diethylamino-ethylether)hexestrol (DH) with respect to their potencies to cause lysosomal glycosaminoglycan storage in cultured bovine corneal fibroblasts. DH has the same side chains as tilorone, but its central apolar moiety lacks planarity. At a concentration (1.75 muM) which did not cause enhanced secretion of the lysosomal enzyme beta-hexosaminidase (E.C. 3.2.1.52), DH was significantly less potent than tilorone in causing storage of [35S]glycosaminoglycans. This is taken as support of the hypothesis that the planar tricyclic ring system is essential for the high potency of tilorone and its congeners to exert this adverse action.

Aminoglycoside antibiotics cause aggregation but not fusion of negatively-charged liposomes at an extent proportional to their capacity to interact with acidic phospholipids (Van Bambeke et al., 1995, Eur. J. Pharmacol., 289, 321-333). To understand why aggregation is not followed by fusion, we have examined here the influence of two aminoglycosides with markedly different toxic potential (gentamicin > isepamicin) on lipid phase transition in negatively-charged liposomes using 31P-NMR spectroscopy, in comparison with spermine (an aggregating agent) and bis(beta-diethylaminoethylether)hexestrol or DEH (a fusogenic cationic amphiphile). Gentamicin, spermine, and, to a lesser extent, isepamicin inhibit the appearance of the isotropic signal seen upon warming of control liposomes and denoting the presence of mobile structures. This non-bilayer signal appeared most prominently when liposomes were incubated with DEH, a strong fusogenic agent. We conclude that aminoglycosides, like spermine, have the potential to prevent membrane fusion, by inhibiting the development of a critical change in membrane organization, which is associated with fusion. We suggest that this capacity could be a determinant in aminoglycoside toxicity.

The ligand binding domain of the human estrogen receptor (hER-LBD), encompassing the sequence MDPS282AG...V595, has been expressed at high levels in Escherichia coli from a pET-23d vector, and a purified preparation has been characterized both by mass spectrometry and biochemical methods. Inclusion bodies from the bacterial expression were solubilized by sonication and the hER-LBD was purified to near homogeneity by affinity chromatography over an estradiol-Sepharose column in urea-containing buffer. This material ran as a single peak on reversed-phase HPLC, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band with apparent molecular mass of 31-32 kilodaltons (kDa), somewhat smaller than that expected from the construct (35.6 kDa). Edman degradation revealed a single sequence of MDPSAGDMRA, consistent with an intact N terminus. Further characterization of this material using low resolution matrix-assisted laser desorption ionization mass spectrometry indicated an apparent single protein species of average mass 33,143 daltons (Da). Detailed molecular analysis by electrospray ionization mass spectrometry, however, revealed that this purified hER-LBD preparation was actually composed of a number of both modified and unmodified LBD protein fragments between 32,900-33,400 Da. The bulk of this 33-kDa protein mixture consisted of three LBD protein fragments with C termini at Ala571 (70%), Ala569 (23%), and Ser575 (4%), with only a trace amount (3%) of the full length expressed LBD (MDPS282...V595; 35, 602 Da). These four protein species also appear to be partially chemically modified by carbamylation. The functional integrity of this hER-LBD preparation was investigated by a ligand-exchange assay, which showed that the hER-LBD retained full estradiol-binding capacity; specific, covalent labeling was also observed using either the electrophilic affinity-labeling ligand tamoxifen aziridine (TAZ) or the photoaffinity-labeling ligand hexestrol diazirine. Thus, this expressed hER-LBD preparation, while appearing nominally pure by conventional biochemical techniques and having the expected ligand-binding capacity, was shown by sensitive high resolution electrospray ionization mass spectrometry techniques to be largely truncated 20-26 amino acids from the expected C terminus and to have a substantial level of covalent modification arising from the urea.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Azibutyl (2R*,3S*)-2,3-bis(4-hydroxyphenyl)pentyl sulfide (1), a photoaffinity labeling reagent for the estrogen receptor (ER), has been prepared in unlabeled and in high specific activity tritium-labeled form (32 Ci/mmol) and has been shown to undergo selective and efficient photocovalent attachment to rat uterine ER. Diazirine 1 demonstrates high binding affinity for ER, as determined by both a competitive binding assay and a direct binding assay (relative binding: estradiol = 100; (1) = 17. Kd: estradiol = 0.19 nM; (1) = 0.98 nM, respectively). It is efficient in site-specific photoinactivation of ER, reaching the level of 31% after 5 min of irradiation at > 315 nm. The tritium-labeled diazirine [3H]-1 undergoes specific photocovalent attachment to ER with an attachment efficiency of 29% and a selectivity of 90%. Both of these values are quite high for a photoaffinity reagent. SDS-polyacrylamide gel electrophoretic analysis of the photolabeled proteins shows specific labeling of a major species at M(r) 65,000, the same species that is labeled by [3H]tamoxifen aziridine, a well-characterized affinity label for ER. Hexestrol diazirine 1 is the first carbene-generating photoaffinity label that covalently labels ER with high efficiency and selectivity, and it should be useful in further studies on the hormone-binding domain of ER.

We have prepared two non-steroidal estrogens in the 2-oxohexestrol series labeled with the positron-emitting radionuclide fluorine-18, 1-fluoro-5-oxohexestrol (4) and 1-fluoro-2-oxohexesterol (5). We anticipated that the polar ketone function at the interior of these ligands would reduce their level of non-specific binding, which might increase the selectivity of their uptake in vivo. The two compounds were prepared by total synthesis: compound 4 was prepared in fluorine-18 labeled form by [18F]fluorine ion displacement on a suitably protected methanesulfonate precursor followed by deprotection under acidic hydrogenolytic conditions; the isomer 5 was prepared from a protected alpha-keto trifluoromethanesulfonate precursor with deprotection under basic conditions as the final step. The binding affinity of these hexestrol derivatives for the estrogen receptor was determined by competitive radiometric binding assays at 0 and 25 degrees C, and their lipophilicity (as octanol-water partition coefficients, log P values) and non-specific binding were estimated. The log P values determined by a reversed phase HPLC method were higher, relative to estradiol, than those calculated by the fragment method of Rekker. In tissue distribution studies in immature (50 g) rats, both of these compounds showed selective uptake in estrogen target tissues. At 1 h, activity in the uterus reached the level of 2.5-3.0% of the injected dose per gram tissue, with uterus-to-blood and uterus-to-muscle ratios of 14-20 and 8-14, respectively. The uptake efficiency and selectivity of these fluoro-oxohexestrols in principal estrogen target tissues is less than that of fluorine-18 labeled steroidal estrogens we have prepared previously, but their receptor-mediated uptake in certain secondary target tissues is substantial. The specific and non-specific components of target tissue uptake of these two compounds appear to be directly related to their non-specific binding and their binding selectivity.

3,3'-Dihydroxy-alpha,beta-diethyldiphenylethane (I), 3,3'-dihydroxy-alpha,beta-diethylstilbene (II), hexestrol (III) and diethylstilbestrol (IV) have already been reported to show hypotensive effects on rats and exhibit phytogrowth-inhibitory activities. We have proposed that two phenolic hydroxyl groups in these compounds are necessary for the biological activities, and a structure-activity relationship for I-related compounds was accomplished using molecular-mechanics (MM) calculations. As a result, the following three findings were obtained; 1) the minimized conformational energy obtained from MM calculations, which is a parameter expressing the molecular stability, showed a relatively high correlation with the biological activities, 2) as results of quantitative structure-activity relationship (QSAR) analyses, the combination of the distance between two phenolic hydroxyl oxygens led to the regression equations with high correlation values, and 3) the idealized molecular model of the most active compound (I) showed the highest stability and had a particular conformation which differed from the other compounds (II-IV).

Carbon-11-labeled estradiol and hexestrol derivatives were prepared via the reaction of [11C]ethylchloroformate with the 2- and 4-amino derivatives of estradiol, the 3'-amino derivatives of hexestrol, and the 1-aminophenoxy derivatives of hexestrol and 1-norhexestrol. The corresponding nonradioactive carbamates were prepared for chemical characterization and in vitro receptor binding assays. The positions of the substituents on the parent molecules were selected with a view to minimize interference with the receptor binding process. In spite of this, affinity for the estrogen receptor was strongly impaired for all carbamate derivatives. Likewise, in vivo, the [11C]carbamate analogs failed to localize in receptor rich tissue via an estrogen receptor mediated process.

Selective Birch-reduction of one of the phenolic rings of meso- or d,l-HES led to compounds 6a and 6b which exert a higher binding affinity to the androgen receptor as the respective parent HES. In vivo testing of 6a and 6b shows that 6a has the same potency in reducing accessory sex organ weights and testosterone levels in the intact mouse as has meso-HES, but strongly decreased estrogenic activity. Syntheses and testing of 7 having no ethyl side chains revealed the necessity of these groups for biological activity.

The diethylstilbestrol-related compounds 3,3'-dihydroxy-alpha, beta-diethyldiphenylethane (I), diethylstilbestrol (II) and hexestrol (III) showed hypotensive effects on spontaneously hypertensive rats (SHR) and antifungal activities against all Fusarium oxysporum sp. tested. As previously reported, I had strong hypotensive action on normotensive rats at the dose of 10 mg/kg, while II and III showed weak hypotensive effects on these rats at the same dose. In this work, all three compounds also had hypotensive actions on SHR at the same dose. I showed the strongest hypotensive effect (-80.0 +/- 5.0 mmHg, 10 mg/kg, i.v.) on both SHR and normotensive rats. The three compounds also had antifungal activities against five kinds of Fusarium oxysporum sp. tested. Especially, II strongly inhibited the growth of Fusarium oxysporum f. sp. raphani IFO-9972 (minimum inhibitory concentration (MIC): 1.0 micrograms/ml).

The synthesis of the bis-acrylate 12, the bis-beta-chloropropionate 13 and the bis-beta-bromopropionate 14 of the "partial" antiestrogen 2,3-bis(2-fluoro-4-hydroxyphenyl)-2,3-dimethylbutane (7) is described. In the case of 13 and 14 the introduction of the beta-haloester-functions moderately reduces the estrogen receptor affinity of 7. However, it was quite strongly diminished in 12. The hydrolytic stability under in vitro-receptor-assay conditions decreases in the order 12 greater than 14 greater than 13. Compared with 7 the estrogenic potency of 12-14 is increased to a great extent. The title compounds cause a strong inhibition of the hormone-dependent MXT-M3.2 mouse mammary tumor.

Treatment of rats, monkeys and man with diethylaminoethoxyhexestrol causes phospholipid storage in liver and other tissues. However, this drug has not been reported to alter plasma lipoprotein levels. When guinea pigs were treated with diethylaminoethoxyhexestrol, the fasting plasma triacylglycerol levels increased dramatically, from 43 to 1281 mg/dl, after only five doses of 12.5 mg/kg. Diethylaminoethoxyhexestrol-treated guinea pigs had reduced postheparin lipase activity. In addition, in vitro assays showed that this agent inhibited guinea pig postheparin lipoprotein lipase. It is hypothesized that diethylaminoethoxyhexestrol causes hypertriglyceridemia in guinea pigs because these animals are known to have low levels of serum activator for lipoprotein lipase and may be unusually susceptible to agents that inhibit lipoprotein lipase activity. The ability to produce hypertriglyceridemia in guinea pigs provides an animal model in which the metabolic consequences of hypertriglyceridemia can be studied.

Four halogenated estrogen analogs--o-fluorohexestrol, and 1-fluoro-, 1-bromo-, and 1-iodohexestrol--have been prepared and tritium-labeled in high specific activity, to investigate their potential as estrogen-receptor-based agents for imaging breast tumors. These compounds bind with high affinity in vitro to the cytoplasmic uterine estrogen receptor from rat and lamb and sediment as 8S receptor complexes on sucrose gradients. After 1 hr in immature rats, these compounds show high uptake into the uterus, but low uptakes (10--25% of the uterine levels) into most nontarget tissues. The uterine uptake is estrogen specific since it is depressed by excess nonradioactive estradiol. Uptake selectivity is greatest for the fluorohexestrols and decreases for the bromo and iodo compounds. In mature rats bearing DMBA-induced mammary tumors, selective uptake by the uterus and tumors is seen with 1-fluoro[3H4]hexestrol and o-fluoro[3H3]hexestrol. The studies indicate that these four halogenated hexestrols are promising candidates as estrogen-receptor-based agents for the imaging of human breast tumors.

The activity of two isoenzymes of alkaline phosphatase was studied in the uteri of ovariectomized golden hamsters. Animals belonging to different groups were daily injected with 10 micrograms of estrogen (octoestrol) once, for 4 and 16 days. The estrogen did not affect the overall activity lf alkaline phosphatase in the uterine luminal epithelium, but decreased the enzyme activity in stroma. Moreover, it was found that prolonged estrogen treatment increases significantly the proportion of alkaline phosphatase isoenzyme of the intestinal type in the uterine luminal epithelium.

After ovariectomy DMBA tumor-bearing SD rats were treated with the estrogen DES, a combination of DES plus mammary tumor-inhibiting antiestrogen (tetramethylHES, 3,3'HES and tamoxifen respectively) or with tetramethylHES alone. Small doses of DES led to a stimulation of tumor growth showing a maximum effect at 1 microgram/kg/day, whereas higher doses inhibited tumor growth. In the combination experiment only tetramethylHES reduced, i.e. antagonized, the DES effect. Tamoxifen and 3,3'HES, however, led to an increase of the tumor growth-stimulating and inhibiting effects of DES. Only the dose of 10 mg/kg/day of tetramethylHES led to a slight stimulation of the tumor growth in ovariectomized DMBA tumor-bearing rats. These results indicate that 3,3'HES and tamoxifen possibly unfold their mammary tumor-inhibiting activity by means of their estrogenic potency, whereas tetramethylHES might act as an antiestrogen, though the latter compound does not lack estrogenic activity completely.

The syntheses of the meso-1,2-dialkyl-1,2-bis(3'-hydroxyphenyl)ethanes [alkyl substituent: CH3 (19), C2H5 (20), C3H7 (22), C4H9 (23), i-C4H9 (24), and C5H11 (25)] and of d,l-3,4-bis(3'-hydroxyphenyl)hexane (21) are described. In vitro these compounds inhibited the [3H]estradiol receptor interaction competitively, exhibiting Ka values between 0.20 x 10(9) (20) and 0.11 x 10(6) M-1 (24). In vivo the meso compounds reduced the estrone-stimulated mouse uterine growth; the most effective compounds were 20, 22, and 23 (53, 50, and 45% inhibition, respectively). Compounds 20 and 22-24 showed weak estrogenic activity in the mouse uterine weight test and in the vaginal cornification test. Compounds 19 (NSC-297169), 20 (NSC-297170), and 22 (NSC-297171) exhibited a dose-dependent growth inhibition on the MCF-7 human breast tumor cell line (10(-6) to 10(-9) M). These compounds also showed a marked dose-dependent inhibition on the DMBA-induced, hormone-dependent mammary carcinoma of the Sprague-Dawley rat corresponding to their association constants.

The antimetastatic effect of thaliblastine alkaloid isolated from Thalictrum min. ssp. elatum was evaluated using Wexler's technique for accurate identification of experimental pulmonary metastases. Thaliblastine was found to inhibit the growth of lung metastases as compared with the control.

The effect of 4,4'-diethylamino-ethoxyhexestrol (DH) on acid esterase in rat liver was studied in vivo and in vitro. The acid esterase activity in the livers of rats treated with 0.125% DH for 1 week was found to decrease more than 60% as compared with that in untreated rats. The addition of DH to the incubation medium caused considerable inhibition of the acid esterase activity in lysosome from untreated rat liver, and the inhibition type appears to be noncompetitive. The acid lipase activity in rat liver lysosome was also inhibited by DH. Some antihistamic agents and chloroquine also inhibited the acid esterase activity in rat liver lysosome.

The synthesis of monohydroxy- and dihydroxy-bifluranol, and of glucuronide and sulphate conjugates of bifluranol are described. Bifluranol administered orally to rats, ferrets and dogs at a dosage of 50 to 200 microgram kg-1 is mostly excreted in the faeces as unchanged bifluranol and bifluranol monosulphate, disulphate and monoglucuronide. The bifluranol is well absorbed and is mostly excreted in the bile, as six different conjugates, including a glucuronide sulphate found in all 3 species, and a glucuronide phosphate found only in ferret and dog bile. Hydroxylation of the aromatic rings occurs in the rat, to an extent of about 8% of the dose, but was not detected in ferret or dog.

We have synthesized as potential imaging agents for human breast tumors a series of hexestrol analogues bearing the halogens fluorine, chlorine, bromine, and iodine at the terminus of the hexane chain. The binding affinity of these compounds for the estrogen receptor from uterine tissues forms a monotonically decreasing series, starting at 129% of that of estradiol for the fluoro analogue and decreasing to 60% for the iodo analogue. Such a modest decrease in binding affinity is thought to reflect the preference of the receptor for lipophilic groups and for substituents of moderate steric size at this site, parameters which change in opposite directions in the halogen sequence going from fluorine to iodine. Three estrogenic bis(trifluoromethyl)diphenylethylenes, prepared by DuPont, also showed substantial binding affinities for the estrogen receptor. In terms of ease of radiolabeling and high receptor binding selectivity, the compound that appears to be the most promising candidate for a breast tumor imaging agent in these series is the chain terminal fluorohexestrol.

The influence of amphiphilic drugs and phospholipids on the activities of beta-galactosidase and beta-glucosidase from liver lysosomal fractions of untreated rats, isolated by affinity chromatography using castor bean lectins, was studied in vitro. Chloroquine (93 microM) inhibited beta-galactosidase activity by about 30%, while O,O'-bis(diethylaminoethyl)hexestrol showed no inhibitory effect. Neutral phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelin) inhibited the enzyme slightly, while the enzyme activity was drastically reduced in the presence of acidic phospholipids (phosphatidylinositol, phosphatidylserine, bis-(monoacylglycero)phosphate). Lysosomal beta-glucosidase was strongly inhibited by chloroquine and O,O'-bis(diethylaminoethyl)-hexestrol. The neutral phospholipids showed only a moderate inhibitory effect, whereas the acidic phospholipids were stimulators. Bis(monoacylglycero)phosphate was by far the best stimulating compound.

1 Endocrine and anti-fertility studies were carried out on a fluorinated bibenzyl, bifluranol, in rats and mice. 2 A potent anti-prostatic activity of bifluranol was shown to be comparable to diethylstilboestrol (DES). In contrast, its oestrogenic potency by the oral route was about eight times less than that of DES. 3 In comparative short and long-term anti-androgenic and fertility studies in rats and in studies on sexual potency and reproductive performance in male mice, bifluranol given orally was shown to produce fully reversible suppression of accessory sexual structures without impairment of spermatogenesis and fertility. In contrast, DES administered in the same dose reduced spermatogenesis as well as accessory sexual glands. 4 Bifluranol lowered serum luteinising hormone (LH) levels without affecting follicle stimulating hormone (FSH). Under similar conditions DES reduces both LH and FSH levels. Since bifluranol does not antagonize androgen-induced stimulation of the prostate in castrated rats, its anti-prostatic effect is interpreted as a negative, hormonostatic feedback activity, mediated through a selective inhibition of LH secretion.

Two gamma-emitting estrogen analogues, (2R*,3S*)-1-[125I]iodo-2,3-bis(4-hydroxyphenyl)pentane ([125I]iodonorhexestrol) and (2R*,3S*)-1-[77Br]bromo-2,3-bis(4-hydroxyphenyl]pentane ([77Br]bromonorhexestrol), have been prepared by halide ion displacement on a labile trifluoromethanesulfonate derivative of a suitably protected precursor, followed by mild acid deprotection. Although halide displacement on a more stable tristrifluoromethanesulfonate derivative was successful, the basic conditions required for deprotection of this precursor resulted in destruction of the products by a base-induced spiroelimination reaction. In immature female rats, both of these halonorhexestrols demonstrated preferential uptake by the uterus that could be blocked selectively by coadministration of a large dose of unlabeled estradiol. In a double label comparison with 16 alpha-[125I]iodo-17 beta-estradiol the uterine uptake of [77Br]bromonorhexestrol was notably less selective. Stability studies in vitro and in vitro have indicated that both iodo- and bromonorhexestrol are quite labile, and this lability compromises the selectivity of their uptake by estrogen target tissues in vivo. p-Hydroxyphenethyl halides are known to be unusually prone to a base-catalyzed solvolysis, via cyclization of the phenolate to a spirocyclohexadienone intermediate. This unusual solvolytic mechanism may contribute to the lability of these halonorhexestrols in vivo.

In the framework of a study on nonsteroidal androgens, the authors previously observed that in perhydrohexestrol series, the (+/-)-[(cis-4 hydroxycyclohexyl)-4(trans-4 hydroxycyclohexyl)-hexane] or cis-trans perhydrohexestrol and especially the (+/-)-(3,4 bis (trans-4-oxocyclohexyl)-hexane) or trans-trans perhydrodiketone, there is no affinity for AR (androgen receptor of the prostate) but they bind with high and specific affinity to the testosterone binding sites on ABP (epididymal androgen-binding protein). In this work, we describe the preparation, the stereochemistry and biological activities of trans-trans and cis-trans perhydrohexestrols and of trans-trans perhydrodiketone as well as their corresponding enantiomers. Biologically the tests AR and ABP are negative for the trans-trans perhydrohexestrol and of trans-trans perhydrodiketone and for its enantiomers. However for the enantiomers of cis-trans perhydrohexestrol and of trans-trans perhydrodiketone, the affinity of the (+) enantiomer for ABP is superior to that of the racemic and that of the (-) enantiomer, whereas the affinity for AR are zero. Chemically, the stereochemistry of the three racemics has been established by X-ray crystallographic analysis or by 1H n.m.r. The n.m.r. spectra have been analyzed in terms of chemical shifts and coupling constants.

The syntheses of the hexestrol derivatives 3,4-bis-(3'-hydroxyphenyl)hexane (4a), 3,4-bis(4'-fluoro-3'-hydroxyphenyl)hexane (4b), 3,4-bis(3',4'dihydroxyphenyl)hexane (4c), and 3,4-bis(3',4'-diacetoxyphenyl)hexane (4d) are described. All compounds showed a marked, competitive inhibition of the estradiol receptor interaction (Ka4c greater than Ka4a greater than Ka4d greater than Ka4b). Evaluated in the mouse uterine weight test compounds 4c and 4d almost reached the estrone effect, whereas 4a and 4b did not produce full uterotrophic response. Compounds 4a--d antagonized the estrone stimulated uterine growth of the immature mouse. Compound 4a (NSC-297170) exhibited a specific, dose-related growth inhibition of the estrogen responsive MCF-7 human breast tumor cell line. Tested on the 9,10-dimethyl-1,2-benzanthracene-induced hormone-dependent mammary adenocarcinoma of the Sprague-Dawley rat all compounds showed marked inhibition of tumor growth. As in all experiments compounds 4a and 4b, which is resistant to hydroxylation in 4'-position exhibited an identical pattern of action, which is different from that shown by compound 4c, the effect of compound 4a cannot be explained by its possible catechol metabolite 4c.

The syntheses of symmetrically 4,4'-, 5,5'-, and 6,6'-disubstituted derivatives of the mammary tumor inhibiting antiestrogen metahexestrol [meso-3,4-bis(3-hydroxyphenyl)hexane] (1) are described [4,4'-substituents: F, (2), Cl (3), Br (4), I (5), CH2N (CH3)2 (6), CH3 (7), CH2OCH3 (8), CH2OC2H5 (9), CH2OH (10), NO2 (11), NH2 (12), N(CH3)2 (13), COCH3 (14), and C2H5 (15); 5,5'-substituents: OH (16) and Cl (17); 6,6'-substituents: OH (18), F (19), Cl (20), and CH3 (21)]. The synthesis of 1-3, 16, and 19 was accomplished by reductive coupling of the propiophenones with TiCl4 /Zn and subsequent hydrogenation of the cis-3,4- diphenylhex -3- enes . Compounds 17, 18, 20, and 21 were synthesized by coupling the 1-phenyl-1-propanols with TiCl3 /LiAlH4 and separation of the meso diastereomers, while 4-15 were obtained by substitution of metahexestrol . The binding affinity of these compounds to the calf uterine estrogen receptor was measured relative to that of [3H]estradiol by a competitive binding assay. The test compounds showed relative binding affinity (RBA) values between 15 and less than 0.01% that of estradiol. Only compound 21 showed an estrogen receptor binding affinity exceeding that of metahexestrol (15 and 10%, respectively). Compounds exhibiting RBA values of greater than 0.5% were evaluated in the mouse uterine weight test. They showed a similar (2 and 12), slightly increased (19 and 21), or strongly enhanced (7 and 20) estrogenicity compared to that of metahexestrol . Compounds 1, 2, 7, 12, and 21 exhibited antiestrogenic activity inhibiting the estrone-stimulated uterine growth (24 to 60% inhibition).

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.

Four young (23 kg body weight) and two mature wethers (52 and 92 kg body weight) were subcutaneously injected with hexestrol (HX) or HX dicaprylate (HX-D) and killed 41 d later. The HX residues, comprising free, glucuronide and KOH hydrolyzable forms plus metabolites were determined by gas chromatography after liquid-liquid extraction and silica gel chromatographic purification of tissue sample. The HX residues were observed to be at concentrations of .1 to 1.0 ppb in most of the tissues examined. Maturity of the animals and the two hormonal formulations resulted in little difference in residues. The KOH hydrolyzable fraction was hardly detected in the tissues examined. Free HX was a major residue in muscle, representing about 70% of HX residues. Glucuronide HX represented 70 to 80% of HX residues in liver and kidney. In lung, glucuronide and free HX were present in similar amounts. This study showed that the gross metabolic patterns of HX and its esters are similar to other estrogens and steroids.

The synthesis of bifluranol, a new fluorinated bibenzyl anti-androgen, and of 3H-labelled bifluranol is described. The absorption, distribution and excretion of bifluranol has been studied in mouse, rat, ferret and dog; it is readily absorbed following oral administration, but blood concentrations of the drug are low due to hepatic uptake and biliary excretion. Enterohepatic re-circulation occurs, but the drug is excreted primarily in the faeces and only small amounts appear in urine. This pattern of disposition and excretion is similar to that reported elsewhere for the bibenzyl, hexoestrol, and for the stilbene, diethylstilboestrol.