Novel 7α-alkoxy-17α-(4'-halophenylethynyl)estradiols as potential SPECT/PET imaging agents for estrogen receptor expressing tumours: Synthesis and binding affinity evaluation

In order to develop potential radiolabelled probes for imaging estrogen receptor (ER) positive tumours, we have synthesized and characterized a series of novel 7α-alkoxy-17α-(4'-iodophenylethynyl)estra-1,3,5(10)-triene-3,17β-diols and 7α-alkoxy-17α-(4'-fluorophenylethynyl)estra-1,3,5(10)-triene-3,17β-diols. The fluoro-substituted compounds showed a higher ER binding affinity than the corresponding iodo-derivatives, where 7α-methoxy- and 17α-(4'-fluorophenylethynyl)estra-1,3,5(10)-triene-3,17β-diol showed the highest ER binding affinities (RBA=80.9% and 78.9%, respectively), among the halophenylethynyl compounds studied and should be further explored as potential PET biomarkers for imaging of ER expressing tumours.     

Synthesis and receptor-binding examination of 16-hydroxymethyl-3,17-estradiol stereoisomers

The four 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers were synthesized and tested in a radioligand-binding assay. The estrogen receptor recognizes these compounds, but their relative binding affinities are lower than 2.0% relative to that of the reference molecule estra-1,3,5(10)-triene-3,17beta-diol. The affinities of the tested compounds for the androgen and progesterone receptors are very low (K(i)> 100 microm and 1 microM, respectively). The prepared 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers are therefore estrogen receptor-selective molecules.     

Synthesis and receptor-binding examination of 16-hydroxymethyl-3,17-estradiol stereoisomers

The four 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers were synthesized and tested in a radioligand-binding assay. The estrogen receptor recognizes these compounds, but their relative binding affinities are lower than 2.0% relative to that of the reference molecule estra-1,3,5(10)-triene-3,17beta-diol. The affinities of the tested compounds for the androgen and progesterone receptors are very low (K(i)> 100 microm and 1 microM, respectively). The prepared 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers are therefore estrogen receptor-selective molecules.     

11β-Amidoalkyl estradiols, a new series of pure antiestrogens

In order to find new antiestrogens, devoid of any agonistic activity, a series of 11β-amidoalkyl estradiols were prepared. These compounds have been studied in comparison with tamoxifen (TAM): in vitro, for their relative binding affinities (RBA) for mouse and MCF-7 estrogen receptors (ER) and for their antiproliferative effect on MCF-7 (estradiol or EGF/PDGF stimulated) and Ly2 human breast cancer cell lines; in vivo, for their uterotrophic/antiuterotrophic activities in the mouse and for their antitumoral activities on MCF-7 tumors implanted in nude mice.

The most representative compounds are N-methyl-N-isopropyl-(3,17β-dihydroxy-estra-1,3,5(10)-trien-11β-yl)-undecanamide (RU 51625) and its 17-ethynyl derivative (RU 53637). They showed good RBAs for ER and a stronger antiproliferative effect than TAM in vitro. Unlike TAM, these compounds inhibited growth factor stimulated MCF-7 proliferation, and the growth of the TAM resistant cell line Ly2. In vivo, they were completely devoid of uterotrophic activity, when given subcutaneously in mice, but exhibited a slight agonistic effect when administered orally. They showed interesting antitumor activities in nude mice by the percutaneous route, but RU 53637 was significantly more potent than RU 51625 when given orally.     

Precursor role of 15alpha-hydroxyestradiol and 15alpha-hydroxyandrostenedione in the formation of estetrol

Studies were designed to elucidate the origin of estetrol (15alpha-hydroxyestriol (estra-1,3,5(10)triene-3,15alpha,17beta-tetrol) or E4) during late human pregnancy. 3H-Labelled 15alpha-hydroxyestradiol (3,15alpha-dihydroxyestra-1,3,5(10)-trien-17-one or 15E2) and 14C-labelled 17beta-estradiol (estra-1,3,5(10)-triene-3,17beta-diol or E2) were infused into the fetus during transfusion in utero for erythroblastosis fetalis, and in another study the same substrates were injected intravenously into the maternal circulation. In a third study, 3H-labelled 15alpha-hydroxyandrostenedion (15alpha-hydroxyandrost-4-ene-3,17-dione or 15delta4) and 14C-labelled E2 were infused into the fetus. Maternal urine was collected for 5--6 days, and after Glusulase hydrolysis, the following metabolites were isolated: estriol (estra-1,3,5(10)-triene-3,16alpha,17beta-triol or E3) containing 14C only and 15alpha-hydroxyestrone (3,15alpha-dihydroxyestra-1,3,5(10)-trien-17-one or 15E1), 15E2, and E4, all containing both labels. From the isotope content of these metabolites, it was concluded that E4 was derived from both fetal E2 and 15delta4 and only partially via 15E2. When administered to the fetus E2 and 15delta4 contributed approximately equal amounts to urinary E4. The yield of 15alpha-hydroxylated estrogens from E2 injected into the mother was very low indicating the predominantly fetal origin of the 15alpha-hydroxylase. 15delta4 was a better precursor than E2 for urinary 15E2.     

Inactivation of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase by 16-methylene estrone, an affinity alkylator enzymatically generated from 16-methylene estradiol-17 beta

The substrate 16-methylene estra-1,3,5(10)-triene-3,17 beta-diol (16-methylene estradiol-17 beta) and its enzyme-generated alkylating product, 3-hydroxy-16-methylene estra-1,3,5(10)-triene-17-one (16-methylene estrone), were synthesized to study the 17 beta- and 20 alpha-hydroxysteroid dehydrogenase activities which coexist in homogeneous enzyme purified from human placental cytosol. 16-Methylene estradiol, an excellent substrate (Km = 8.0 microM; Vmax = 2.8 mumol/mg/min) when enzymatically oxidized to 16-methylene estrone in the presence of NAD+ (256 microM), inactivates simultaneously the 17 beta- and 20 alpha-activities in a time-dependent and irreversible manner following pseudo-first order kinetics (t1/2 = 1.0 h, 100 microM, pH 9.2). 16-Methylene estradiol does not inactivate the enzyme in the absence of NAD+. 16-Methylene estrone (Km = 2.7 microM; Vmax = 2.9 mumol/mg/min) is an affinity alkylator (biomolecular rate constant k'3 = 63.3 liters/mol-s, pH 9.2; KI = 261 microM; k3 = 8.0 X 10(-4) S-1, pH 7.0) which also simultaneously inhibits both activities in an irreversible time-dependent manner (at 25 microM; t1/2 = 7.2 min, pH 9.2; t1/2 = 2.7 h, pH 7.0). Substrates (estradiol-17 beta, estrone, and progesterone) protect against inhibition of enzyme activity by 16-methylene estrone and 16-methylene estradiol. Affinity radioalkylation studies using 16-methylene [6,7-3H]estrone demonstrate that 1 mol of alkylator binds per mol of inactivated enzyme dimer. Thus, 16-methylene estradiol functions as a unique substrate for the enzymatic generation of a powerful affinity alkylator of 17 beta,20 alpha-hydroxysteroid dehydrogenase and should be a useful pharmacological tool.     

Iodoestrogens, syntheses, and interaction with uterine receptors.

The rationale for undertaking the present study was to evaluate the utility of iodoestradiol analogs made highly radioactive with iodine isotopes in (a) the non-invasive differentiation of estrogen-dependent from estrogen-independent breast tumors, (b) spread of metastases containing estrogen receptors, and (c) potential application in therapeutic irradiation of target tissues. In the present paper, the model syntheses of a number of nonradioactive 127I-estrogen analogs are described. The analogs were tested for their ability to displace (compete with) [3H]estradiol from receptor sites. The most active compounds, 16beta-iodoestra-1,3,5(10)-triene-3,17 beta-diol (17) and 6-iodoestra-1,3,5(10),6-tetraene-3,17 beta-diol (10b), showed a relative binding affinity of 0.57 and 0.49, respectively.     

Biological activities of 4-fluoro estrogen analogues

As part of a study on the biological activity of gamma-emitting estradiol analogues, a series of fluorinated and/or brominated analogues of estradiol were synthesized. 4-Fluoro-1,3,5(10-estratrien-3,17 beta-diol (4-fluoro-estradiol) and 16 alpha-bromo-1,3,5(10)-estratrien-3,17 beta-diol (16 alpha-bromo-estradiol) had relative binding affinities (RBA) for the rabbit uterine cytosol receptor which were comparable to those of estradiol. However, while 4-fluoro-estradiol stimulated the increase in uterine weight of immature mice to the same amount as estradiol, 16 alpha-bromo-estradiol was less effective in this assay indicating that its metabolism is probably more rapid than that of estradiol. 16 beta-Bromo-1,3,5(10)-estratrien-3,17 beta-diol had a low RBA and was ineffective in stimulating uterine weight at the doses used. 4-Fluoro-16 alpha-bromo-estradiol had a lower RBA than either the 4-fluoro- or 16 alpha-bromo-estradiol and all the 4-fluoro-seleno-estrogens tested possessed low RBA's.     

Mechanism of inactivation of 3-oxosteroid delta 5-isomerase by 17 beta-oxiranes

The affinity label (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran]-3-ol (5 beta) inactivates 3-oxosteroid delta 5-isomerase from Pseudomonas testosteroni by formation of a covalent bond between Asp-38 of the enzyme and the steroid. High-performance liquid chromatography (HPLC) analysis of tryptic digests of inactivated enzyme shows that two isomeric steroid-containing peptides are formed in a ratio of 9:1 at pH 7 (TPS1 and TPS2). Hydrolysis of each of these peptides produces a different steroid: TPS1 releases 17 alpha-(hydroxymethyl)estra-1,3,5(10),6,8-pentaene-3,17 beta-diol (S1) whereas TPS2 yields 17 beta-(hydroxymethyl)estra-1,3,5(10),6,8-pentaene-3,17 alpha-diol (S2). Inactivation of the enzyme by (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran-18O]-3-ol, followed by mass spectral analysis of the diacetate of the steroid released upon hydrolysis of the enzyme-inhibitor bond, reveals that TPS1 is formed by attack of Asp-38 at the methylene carbon of the oxirane. In contrast, TPS2 is produced by Asp-38 attack at the tertiary carbon. These results imply that inactivation occurs through concurrent SN1 and SN2 reactions of Asp-38 with the protonated inhibitor and that Asp-38 is located on the alpha face of the steroid when it is bound to the active site in the correct manner to react for both the SN1 and SN2 processes.     

Expression of cytochrome P-450d by Saccharomyces cerevisiae

Rat liver microsomal cytochrome P-450d was abundantly expressed in the yeast Saccharomyces cerevisiae by using a yeast-Escherichia coli shuttle vector consisting of rat liver P-450d cDNA and yeast acid phosphatase promoter. The expressed cytochrome P-450d was immunologically crossed with rat liver P-450d. The hydroxylase activity of estra-1,3,5(10)-triene-3, 17 beta-diol was 11 nmol/min per nmol P-450d, which is comparable to that reported previously for rat liver P-450d. The expressed P-450d content was nearlyt 1% of total yeast protein as estimated from immunoblotting, hydroxylase activity and optical absorpton of the reduced CO form.     

Binding, X-ray and NMR studies of the three A-ring isomers of natural estradiol

The effect of the position of the phenolic hydroxyl on the conformations of the three A-ring isomers of estradiol, namely, estra-1,3,5(10)-trien-1,17 beta-diol (10), estra-1,3,5(10)-trien-2,17 beta-diol (3), and estra-1,3,5(10)-trien-4,17 beta-diol (6), has been analyzed by X-ray crystallography. The results of these analyses were correlated with the absorptions of the angular methyl groups in the [1H]NMR spectra of these isomers and natural estradiol (E2). It was observed that the changes in chemical shift of protons at C18 corresponded to skeletal modifications in the steroid structure which changed the anisotropic effect of the hydroxyl group at C17. Examination of the affinity of these A-ring isomers of E2 for the estrogen receptor has shown the 2-hydroxylated isomer 3 to retain 1/5th the affinity of E2 for its binding protein. The 1- and 4-hydroxylated derivatives (10 and 6, respectively) bound to a much lesser extent. The receptor affinities of these estrogen analogues may be related to the angle between the 18-methyl and the 17 beta-hydroxyl groups (or the dihedral angle between the planar A-ring and the angular C18 methyl) as well as the position of the A-ring hydroxyl group.     

Biology and mode of action of pure antioestrogens

The properties of a series of 7 alpha-alkyl analogues of oestradiol are described. Studies of chemical structure and activity in the immature rat uterotrophic/antiuterotrophic assay revealed that molecules containing a terminal functional group (acid, alcohol, amine, amide) linked to the steroid by a decamethylene bridge possess both oestradiol agonist and antagonist activity. However, certain amides, exemplified by the compound ICI 164,384 [N-n-butyl-11-(3,17 beta-dihydroxyoestra-1,3,5(10)-trien-7 alpha-yl)-N-methylundecanamide], were devoid of oestrogenic activity but possessed potent antioestrogenic activity. Comparison of receptor binding and biological potency of steroid 7 alpha- and 7 beta-isomers showed that activity is confined largely to the 7 alpha-isomer. Comparison of the effects of tamoxifen and ICI 164,384 on progesterone receptor (PR) concentration in the rat uterus showed that, unlike tamoxifen, ICI 164,384 did not induce PR and blocked induction of PR by oestradiol. Chronic treatment of mature female rats with ICI 164,384 led to an ovariectomy-like regression of the uterus without affecting LH secretion or the rate of growth. ICI 164,384 was also an effective antitumour agent in rats bearing carcinogen-induced mammary tumours.     

Species differences in biotransformation of 17α-cyanomethylestradiol 3-methyl ether

Following oral administration of 9,11- ³H-17α-cyano-methylestra-1,3,5(10)-triene-3,17-diol 3-methyl ether, urinary metabolites were studied in man, baboon, beagle dog, minipig and rat. The metabolite pattern revealed remarkable species differences, especially in quantitative respects. 17α-Cyanomethylestra-1,3,5(10)-triene-3,17-diol, 17α-cyanomethylestra-1,3,5(10)-triene-2,3,17-triol 2-methyl ether, 17α-hydroxymethylestra-1,3,5(10)-triene-3,17-diol and 17α-cyanomethylestra-1,3,5(10)-triene-3,16$\text{6}\text{5}$,17-triol were isolated as principal metabolites. In rat bile, a metabolite was tentatively identified as aγ-lactone of a 17α-carbozymethyl-16α-hydroxy compound.     

Conjugates of steroids and anti-cancer agents. III. The synthesis of estrynamine and certain derivatives

Propargyl amine was protected by condensing it with 2,5-hexane-dione to give 2,5-dimethyl-N-(2'-propyn-1'-yl)pyrrole (2). The latter was converted to the corresponding Grignard reagent with ethylmagnesium bromide, and then condensed with estrone tetrahydropyranyl ether to give 17 alpha-[3'-(2',5'-dimethyl-1'-pyrryl)-1'-propyn-1'-yl)-1,3 ,5( 10)- estratriene-3,17 beta-diol 3-tetrahydropyranyl ether (3), in 85% yield. Acetic acid and methanol cleaved the tetrahydropyranyl ether group, and hydroxylamine and sodium bicarbonate cleaved the pyrrole ring to give 17 alpha-(3'-amino-1'-propyn-1'-yl)-1,3,5(10)-estratriene-3,17 beta-diol (1), estrynamine. Several derivatives and analogs of 1 were also synthesized. Estrynamine binds to estrogen receptor with an RBA of 0.0045 (estradiol = 1.0). Several of the compounds, including estrynamine, are weak estrogens (stimulation of prolactin synthesis).     

The preparation of 2,4-dibromoestra-1,3,5(10),6-tetraene-3,17 beta-diol diacetate.

[2,4,6,7-3H4]Estradiol is used for the quantitative determination of estradiol receptors. The synthesis of 2,4-dibromoestra-1,3,5(10),6-tetraene-3,17 beta-diol 3,17-diacetate, the convenient precursor of [2,4,6,7-3H4]estradiol, is described. 6-Alkoxy compounds were also prepared and investigated.     

Novel steroidal pure antiestrogens

A series of steroidal estrogen antagonists with no intrinsic estrogenicity in rat uterotrophic-antiuterotrophic tests has been discovered. The compounds are derivatives of estradiol containing amidoalkyl side chains at the 7 alpha-position. The most potent compounds are N-n-butyl-N-methyl-11-(3,17 beta-dihydroxyestra- 1,3,5(10)-trien-7 alpha-yl) undecanamide and N-2,2,3,3,4,4,4-heptafluorobutyl-N-methyl-11-(3,17 beta-dihydroxyestra- 1,3,5(10)-trien-7 alpha-yl) undecanamide. Structure activity relationships are discussed.     

A comparison of three methods of hydrolysis for estrogen conjugates

The efficiencies for estrogen conjugate hydrolysis were compared between enzyme hydrolysis, acid solvolysis and a new method, ammonolysis. Samples included: 1) crystalline 1,3,5(10)-estratriene-3, 17 beta-diol disulfate (estradiol 3,17-disulfate), 2) squirrel monkey urine collected following an intravenous injection of [2,4,6,7-H] 1,3,5(10)-estratriene-3,17 beta-diol (estradiol) and 3) a pool of human pregnancy urine. Ammonolysis demonstrated a significant increase over the other techniques in "free" estrogen yields, specifically, from estradiol 3,17-disulfate.     

Water-soluble metabolites of the estrogens. Quantitation of C-18 tetrols in rat feces.

Several radioactive estrogens possessing one, two and three hydroxyl groups were injected orally (and in the case of estrone sulfate also intraperitoneally) into adult male rats. The rats were either intact or had ligated or cannulated bile ducts. Two unconjugated estrogen tetrols together represented 21 - 87% of the total metabolites in the intact rat. One of the tetrols was 2-hydroxyestriol (estra-1,3,5(10)-triene-2,3,16alpha,17beta-tetrol); the other may be estra-1,3,5(10)-triene-2,3,6xi,17beta-tetrol but this was not confirmed. It is concluded that poly-hydroxylated estrogens represent a very large proportion of the previously unidentified water-soluble metabolites of the estrogens in the adult male rat.     

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

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