Tamoxifen Analogues Research Articles

Increasing the number of tandem estrogen response elements increases the estrogenic activity of a tamoxifen analogue.

There have been several reports of women who have tumor relapse while on tamoxifen therapy, followed by tumor regression after tamoxifen withdrawal. In such apparently tamoxifen-stimulated tumors, there is likely a genetic change which increases the estrogenicity of tamoxifen. In this study, we determine if increasing the number of estrogen response elements (EREs) in the promoter region of a reporter gene can alter the agonistic activity of fixed-ring 4-hydroxytamoxifen. We show that increasing the number of EREs in the promotor region increases the transcriptional response of the reporter plasmid to estradiol. We also find that while fixed-ring 4-hydroxytamoxifen is unable to stimulate transcription when one ERE is present, transcriptional activation can occur with multiple EREs. These results demonstrate that ERE amplification could explain the agonistic properties of tamoxifen, and suggests a novel mechanism to explain tamoxifen-stimulated breast cancer growth.

The biological action of cDNAs from mutated estrogen receptors transfected into breast cancer cells.

While tamoxifen may inhibit breast cancer proliferation, mutations in the estrogen receptor could potentially result in breast cancer cells which can circumvent the tamoxifen blockade. Previously, we identified a mutation at codon 351 in the estrogen receptor from a tamoxifen-stimulated human breast cancer. This receptor was stably transfected into the estrogen receptor-negative human breast cancer cell line MDA-MB-231 (clone 10A). Clones were compared to stably transfected cell lines containing either the wild type or codon 400 mutant estrogen receptor to study the effect of either estradiol or the tamoxifen analogue, fixed-ring 4-hydroxytamoxifen ((fr)4-OH TAM), on cell growth and reporter gene activation. (fr)4-OH TAM reduced the growth rate in cell lines containing mutant estrogen receptors, while the cell line containing the wild type estrogen receptor is minimally influenced by (fr)4-OH TAM. We then needed to show that the ligand-estrogen receptor interaction resulted in estrogen receptor activation. As a ligand-dependent transcription factor, estrogen receptor activation is measured by its ability to stimulate reporter gene (luciferase) transcription when bound to an estrogenic ligand. We found that the wild type estrogen receptor is activated by estradiol but not by the tamoxifen analogue, while the codon 351 estrogen receptor is activated by both (fr)4-OH TAM and estradiol.

The synthesis of three indolizine derivatives of interest as non‐isomerizable analogues of tamoxifen

AbstractThree novel derivatives of indolizine 21, 23, and 25 have been prepared as analogues of the anti‐cancer agent tamoxifen 1. The compounds showed low relative binding affinity at the estrogen receptor.

Alternate antiestrogens and approaches to the prevention of breast cancer.

The biological rationale and extensive clinical experience with the breast cancer drug tamoxifen make it the agent of choice for testing as a breast cancer preventive. However, concerns (Jordan and Morrow, Eur J Cancer, in press) about development of endometrial cancer in patients and liver tumors in rats with tamoxifen has encouraged the investigation of other antiestrogens. At present no compounds are available to replace tamoxifen, but two triphenylethylenes, toremifene and droloxifene, have been tested in postmenopausal women to treat advanced breast cancer. The response rates are similar to those observed with tamoxifen (i.e., approximately 35% [CR+PR] in unselected patients), although dosage regimens of the new antiestrogens are higher than the 20 mg tamoxifen required daily. Doses of up to 200 mg toremifene daily are being tested and studies use up to 100 mg droloxifene daily. Side effects appear comparable, but neither droloxifene nor toremifene produce liver tumors in rats. Tamoxifen produces DNA adducts, whereas toremifene and droloxifene appear to be only weakly active. A new tamoxifen analogue, idoxifene, is entering clinical trial. The drug is designed to be metabolically stable so that there will be low carcinogenic potential. In contrast, a novel strategy may be considered to be of value to protect women from developing breast cancer. It is known from laboratory and clinical studies that antiestrogens protect bone and prevent rat mammary cancer. One compound, raloxifene, is being tested as an agent to treat osteoporosis. If the drug becomes generally available to prevent osteoporosis in postmenopausal women, a beneficial side effect may be a reduction in breast cancer risk.(ABSTRACT TRUNCATED AT 250 WORDS)

Rationally designed analogues of tamoxifen with improved calmodulin antagonism.

Computerized molecular modeling studies on the interactions of the antiestrogen tamoxifen (1) and its analogues bound to the calcium-binding protein calmodulin have guided the rational design of more potent antagonists. Compounds with either three or four methylene units in the basic side chain or slim lipophilic 4-substituents were expected to be more potent. All compounds were tested for antagonism of the calmodulin-dependent activity of cAMP phosphodiesterase and for binding affinity to the estrogen receptor from rat uteri. Some compounds were assayed for cytotoxicity against MCF-7 breast tumor cells in vitro. Introduction of lipophilic 4-substituents was accomplished by using palladium(0)-catalyzed coupling reactions with a 4-iodinated precursor. Both the 4-ethynyl (16 and 17) and 4-butyl (18 and 19) compounds were more potent calmodulin antagonists than tamoxifen. Extension of the basic aminoethoxy side chain of 4-iodotamoxifen (3) and idoxifene (2) ((E)-1-[4-[2-(N-pyrrolidino)ethoxy]phenyl]-1-(4-iodophenyl)-2-phen yl-1- butene) by one or two methylene units resulted in modest gains in calmodulin antagonism (10-13). All the compounds assayed retained estrogen receptor binding characteristics. The compound possessing the optimal combination of calmodulin antagonism and estrogen receptor binding was 12 ((E)-1-[4-[3-(N-pyrrolidino)propoxy]phenyl]-1-(4-iodophenyl)-2-phe nyl-1 - butene) (IC50 = 1.1 microM, RBA = 23). Correlation between calmodulin antagonism and cytotoxicity was demonstrated for selected compounds.

Evidence that tamoxifen binds to calmodulin in a conformation different to that when binding to estrogen receptors, through structure-activity study on ring-fused analogues

A ring-fused analogue of tamoxifen, which had previously been shown to have practically identical estrogen receptor (ER) affinity and antitumour potency against estrogen responsive cells as tamoxifen, failed to inhibit calmodulin-dependent cyclic AMP phosphodiesterase. The substitution of an extra methyl group into the ring-fusef analogue, at a position which the ethyl group of tamoxifen can occupy in one of its conformations, restored the calmodulin inhibition. Also, the replacement of the tamoxifen ethyl group by methyl diminishes calmodulin inhibition. Direct interaction of these tamoxifen analogues with calmodulin was demonstrated through the use of the fluorescent probe, 2- p-toluidinyl-napthalene-6-sulfonic acid (TNS). These findings lead to the conclusion that tamoxifen binds to calmodulin in a conformation not accessible to the fused analogue and therefore likely to be different to that which binds to the ER. Also, the results on the ring-fused analogues indicate that the calmodulin binding cannot be essential for antitumour activity.

Synthesis of 4‐stannylated tamoxifen analogues; useful precursors to radiolabelled idoxifene and aziridinyl 4‐iodotamoxifen

AbstractThe 4‐stannylated tamoxifen analogues (E)‐1‐[4‐[2‐(N‐pyrrolidino)ethoxy]phenyl]‐1‐[4‐(tributylstannyl)phenyl]‐2‐phenyl‐1‐butene and (E)1‐[4‐[2‐(aziridin‐1‐yl)ethoxy]phenyI]‐1‐[4‐(tributylstannyl)phenyl]‐2‐phenyl‐1‐butene have been synthesised as precursors to radiolabelled idoxifene, an important new drug for the treatment of breast cancer, and to aziridinyl 4‐iodotamoxifen, a potentially useful biological research tool for studying the action of tamoxifen, respectively.

Imaging, biodistribution and therapy potential of Halogenated tamoxifen analogues

Tamoxifen binds to estrogen receptors (ERs) and prevents breast cancer cell proliferation. This study is aimed at developing a ligand for imaging ER (+) breast tumors by positron emission tomography (PET) or single photon emission computed tomography (SPECT). [ 18F]-Labeled tamoxifen analogue ([ 18F]FTX) was prepared in 30–40% yield and [ 131I]-labeled tamoxifen analogue ([ 131I]ITX) was prepared in 20–25% yield. In mammary tumor-bearing rats, the biodistribution of [ 18F]FTX at 2 h showed a tumor uptake value (% injected dose/gram tissue) of 0.41 ± 0.07; when rats were pretreated with diethylstilbestrol (DES), the value changed to 0.24 ± 0.017. [ 131]ITX at 6 h showed a tumor uptake value of 0.26 ± 0.166; when rats were pretreated with DES, the value changed to 0.22 ± 0.044. Priming tumor-bearing rats with estradiol, a tumor uptake value for [ 131]ITX was increased to 0.48±0.107 at 6 h. In the [ 3H]estradiol receptor assay, tumors had a mean estrogen receptor density of 7.5 fmol/mg of protein. In gamma scintigraphic imaging studies with [ 131I]ITX, the rabbit uterus uptake can be blocked by pretreatment with DES. Both iodotamoxifen and tamoxifen reduced ER(+) breast tumor growth at the dose of 50 μg in tumor-bearing mice. The findings indicate that tamoxifen analogue uptake in tumors occurs via an ER-mediated process. Both analogues should have potential for diagnosing functioning ER(+) breast cancer.

A mechanistic hypothesis for DNA adduct formation by tamoxifen following hepatic oxidative metabolism.

A mechanism for the generation of electrophilic alkylating agents from tamoxifen by hepatic alpha-oxidation of its ethyl group is presented which accounts for the formation of covalent DNA adducts associated with its hepatic carcinogenicity. The key process leading to the generation of electrophilic alkylating agents from tamoxifen is postulated to be alpha-hydroxylation in conjunction with 4-hydroxylation or secondary metabolic conjugation. The mechanism explains the lack of DNA adducts observed for the tamoxifen analogues toremifene, droloxifene and idoxifene and accounts for the species variation in hepatic carcinogenicity of tamoxifen.

Reaction of Pyrylium Salts with Nucleophiles. 23: Triarylethene Derivatives Containing an Oxyalkyleneamino or Oxyalkylene-N-pyridinium Side Chain

A synthetic design was devised for preparing primary amines related to anticancer drugs clomiphene and tamoxifen on the basis of key intermediates with a phenolic group, to which a side chain (ω-aminoethoxy or ω-aminopropoxy) was attached. These compounds were then reacted with 2,4,6-trimethyl- or 2,4,6-triphenylpyrylium salts.1 This afforded pyridinium analogues of clomiphene and tamoxifen as potential therapeutic agents for treatment against hormone-dependent tumors.

Synthesis of [18F]Fluoroalanine and [18F]Fluorotamoxifen for Imaging Breast Tumors

To develop ligands for imaging breast tumors, [18F]fluoro analogue of tamoxifen and [18F]fluoroalanine were radiosynthesized. In vivo biodistribution studies were performed in mammary tumor-bearing rats. In studies on the biodistribution of an [18F]fluoro analogue of tamoxifen, tumor uptake decreased when rats were pretreated with diethylstilbestrol (DES), suggesting that tracer uptake in tumors was receptor-mediated. An estrogen receptor assay indicated that tumors have a receptor density of 7.5 fmol/mg protein. Studies of the distribution of [18F]fluoroalanine in tissue showed that the tumor-to-tissue ratio increases as a function of time. Positron emission tomography (PET) images of tumor-bearing rats demonstrated that tumors can be visualized 1 h after rats are injected with an [18F]fluoro analogue of tamoxifen. PET imaging of pigs after injection of 10 mCi of [18F]fluoro analogue of tamoxifen showed uterine uptake that could be blocked by DES (50 mg). The findings suggest that both radiotracers are useful for imaging breast tumors.

Halogenated Analogues of Tamoxifen: Synthesis, Receptor Assay, and Inhibition of MCF7 Cells

AbstractThis study was conducted to develop a ligand for imaging estrogen-receptor-positive breast tumors by positron emission tomography or single photon emission computed tomography. We synthesized fluoro and iodo analogues of tamoxifen, and these halogenated analogues produced greater affinity for binding to the receptor than tamoxifen. Values of the inhibition affinity constants were as follows: tamoxifen, 15 000 nM; fluoromethyl-N,N-diethyltamoxifen, 2500nM for the cis isomer and 500nM for the trans isomer; and iodomethyl-N,N-diethyltamoxifen, 1500nM for the cis isomer and 1000nM for the transisomer. In studies of human MCF7 breast tumor cell growth, concentrations that inhibited tumor growth in 50% of the cases were as follows: tamoxifen, 11 μM; fluoromethyl-N,N-diethyltamoxifen, 4.5 and 11.8 μM for the cis and trans isomers, respectively; and iodomethyl-N,N-diethyltamoxifen, 2.4 and 6.3 μM for the cis and trans isomers, respectively. These studies suggest that both fluoro and iodo analogues of tamoxifen may be useful diagnostic compounds for predicting the response of estrogen-receptor-positive breast tumors to tamoxifen analogues used in chemotherapy.

Effects of nonsteroidal antiestrogens in the in vitro rat uterus.

We have studied the effect of nonsteroidal antiestrogens on rat uterine contractions induced by oxytocin (8 nmol/l), methacholine (10 mumol/l), prostaglandin F2 alpha (1 mumol/l), KCl (60 mmol/l) and CaCl2 (6 mmol/l). In a concentration-dependent way, the antiestrogens tamoxifen, clomiphene, nafoxidine and ethamoxytriphetol inhibited the amplitude and frequency of the oxytocin-induced contractions and the contraction produced by CaCl2. At a concentration of 30 mumol/l the four drugs inhibited the contractions induced by methacholine and prostaglandin F2 alpha. They also relaxed the tonic contraction to KCl in a concentration-dependent way. This action was partially counteracted by CaCl2 (0.1-10 mmol/l). Bay k 8644 (0.3 nmol/l to 3 mumol/l) only partially reversed the inhibition by ethamoxytriphetol (0.1 mmol/l) of CaCl2 (6 mmol/l)-induced contractions. The steroidal antiestrogen, ICI 164,384, which lacks agonist activity, had an inhibitory effect (44 +/- 4%, n = 7) on KCl-induced contractions only at a concentration of 0.1 mmol/l. However, the quaternary analogue of tamoxifen (tamoxifen ethyl bromide) produced 86 +/- 3% relaxation of the KCl-induced contracture (IC50 1.52 +/- 0.1 mumol/l, n = 10) and this effect was counteracted by addition of CaCl2. Taken together the results indicate that the inhibitory effects of nonsteroidal antiestrogens on rat uterine contractions could be mediated by an action to block Ca2+ entry through an agonist action on extracellular estrogen receptors.

Coupling of low-order organocopper complexes with organoiron cations; synthesis of tamandron, a novel potentially antiandrogenic analogue of tamoxifen

Low-order alkyl, aryl and vinyl organocopper complexes react efficiently with tricarbonyl(cyclohexadienylium)iron cations; the low-order vinylcopper complex derived from an (E)-1-bromo-1-(4-alkoxyphenyl)-2-phenylbut-1-ene couples the bulky vinyl group regioselectively, and with retention of the double-bond stereochemistry, to the encumbered C-5 methylsubstituted position of a tricarbonyl(2-alkoxy-5-methylcyclohexadienylium)iron cation, thereby forming a chiral quaternary carbon centre, and providing access to tamandron, a novel potentially antiandrogenic analogue of tamoxifen.

Estrogen receptor-binding affinity of tamoxifen analogs with various side chains and their biologic profile in immature rat uterus

Estrogen receptor-binding affinity and estrogenic and antie strogenic activity have been evaluated for tamoxifen analogs substituted with various side chains. Antagonist activity of the compounds of this series appears to be dependent on the presence of the β-tert- aminoethoxy moiety. The results also indicate that the dissociation of these compounds from the estrogen receptor-binding site at 25 C is very slow.

Variation of the inhibition of calmodulin dependent cyclic AMP phosphodiesterase amongst analogues of tamoxifen; Correlations with cytotoxicity

The ability of a variety of analogues of tamoxifen to inhibit calmodulin dependent cyclic AMP phosphodiesterase has been determined. Effective inhibition requires that the aminoethoxy side chain bears a positive charge at physiological pH and is not too bulky. Amongst 4-substituents, inhibitory potency increases with lipophilicity. The stereochemistry about the olefinic linkage is not important. The most potent agent found ( ic 50 1.4 μM, compare tamoxifen = 6.75 μM) has a 4-iodine substituent and pyrrolidino in place of dimethylamino. This analogue is also more cytotoxic than tamoxifen against MCF-7 human breast cancer cells as determined in a 24-hr assay, but there was no correlation found between calmodulin inhibition and cytotoxicity against the L1210 murine leukaemia or Walker rat carcinosarcoma cells in culture. The results are consistent with the possibility that calmodulin is important to the functioning of oestrogen receptor mediated growth in MCF-7 cells.

Comparison of the effects of tamoxifen and of a tamoxifen analogue that does not bind the estrogen receptor on serum lipid profiles in the cockerel

Administration of the nonsteroidal antiestrogen tamoxifen to cockerels results in dose- and time-dependent decreases in the levels of free and esterified cholesterol, phospholipids, and triglycerides in serum and in very low density and low density lipoprotein fractions. Similar changes can be elicited using a tamoxifen analogue, N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine.HCl (DPPE). Like tamoxifen, this compound is capable of binding antiestrogen binding sites and exhibits a relative binding affinity of 90% compared with tamoxifen (Ki approximately 4-5 nM). Unlike tamoxifen, DPPE shows no measureable affinity for the cockerel liver nuclear estrogen receptor. Further, DPPE exhibits no estrogen agonist or antagonist activity as measured at the level of synthesis of apolipoprotein II of very low density lipoprotein by liver, synthesis of ovalbumin by oviduct, or growth of the oviduct. Although it is possible that the lipid-lowering effects of tamoxifen result from the opposition of endogenous estrogen action in the cockerel, the similarity of the effects of tamoxifen and DPPE on the lipid profiles suggests common mechanisms that do not involve the estrogen receptor.

An analogue of the antioestrogen tamoxifen of sufficient rigidity to exist as distinct enantiomers: synthesis and conformational dynamics studies

The conformationally constrained tamoxifen analogue 1-methyl-8-phenyl-4-[4-[2-(dimethylamino)ethoxy]phenyl-6,7-dihydro-5- H -benzocycloheptane has been synthesised. A key synthetic step is a novel method for introduction of a methyl group into the aromatic ring of bnenzosuberone by enolate oxygen directed lithiation. Conformational studies were carried out on the methoxy of the above compound. Dynamic 1H NMR revealed a free energy barrier to racemisation of enantiomeric atropisomers, ΔG ‡ - 20 (±1) kcal mol −1. It could be separated by chromatography at −5°C on (+)-poly(triphenylmethylmethacryate) into enantiomers which racemised with t 1 2 1.8 h at −5.2°C corresponding to ΔG ‡-20.9 20.9 kcal mol −1. The difficulties in designing analogues having a greater degree of conformational constraint are discussed.

In vitro inhibition of hepatic steroid hydroxylation by tamoxifen, a series of tamoxifen analogues and related compounds

The in vitro inhibition of the cytochrome P-450 (P-450) isozyme specific positional hydroxylation of androst-4-ene-3,17-dione (androstenedione) by the alkylamino containing compounds trans-and cis-tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen, SKF 525-A and the non-alkylamino containing compounds tamoxifen metabolite E, and tamoxifen analogue U-23469 was assessed in pooled hepatic microsomes isolated from untreated male rats. P-450 IIA 1-mediated androstenedione 7α-hydroxylation appeared refractory to inhibition, with the lowest I 50s being approximately 200 μM ( cis-and trans-tamoxifen, 4-hydroxytamoxifen). (According to the recently recommended nomenclature for cytochromes P-450 (Nebert DW and González FJ, Ann Reu Biochem 56: 945–993, 1987), rat hepatic cytochromes P-450 UT-A, PB-B, PCN-E and UT-F are encoded by genes IIC 11, IIB 1, IIIA 1 2 and IIA 1, respectively. I 50s toward the P-450 IIC 11-, IIB 1-, and IIIA 1 2 -catalysed reactions, androstenedione 16α-, 16β-and 6β-hydroxylations, respectively, were generally in the range 70–190 μM. However, metabolite E exhibited a rather specific and potent capacity to inhibit androstenedione 16α-hydroxylase activity (I 50 = 18 μM). Since a number of alkylamine compounds have been shown to sequester microsomal P-450 as an inactive metabolite intermediate (MI), the tamoxifen analogues were investigated for their in vitro MI complexation capacity. However, spectral binding studies revealed that the incubation of these compounds with NADPH-fortified microsomal fractions did not result in MI complex formation. In binding experiments conducted with oxidised microsomal fractions it was apparent that most of the tamoxifen analogues are type I ligands of quite high affinity for ferric P-450 ( K s range 10–60 μM). It seems unlikely that MI formation is involved in the observed inhibition of androstenedione hydroxylation by tamoxifen and congeners. Instead, and in contrast to the situation observed with SKF 525-A, it would appear that the inhibitory capacity of the tamoxifen analogues is more closely related to type I binding capacity with ferric P-450. A finding of particular interest is that metabolite E, in which the alkylamino side-chain is absent, elicited a type I interaction of high capacity. The maximal absorbance change of the type I interaction of this compound with microsomal P-450 was about three-fold greater than the other compounds. It therefore appears likely that removal of the tamoxifen side-chain results in quite potent inhibition of P-450 IIC 11-mediated monooxygenase activity and an enhanced binding interaction with the cytochrome.

Microsomal binding sites for antioestrogens in rat liver. Properties and detergent solubilization.

The properties of an antioestrogen binding site (AEBS), which has high affinity and specificity for nonsteroidal antioestrogens and structurally related compounds, have been studied in rat liver microsomes. When subcellular organelles were separated on Percoll density gradients the distribution of the AEBS paralleled that of NADPH-cytochrome c reductase, indicating that the AEBS is associated with the endoplasmic reticulum. Saturation analysis showed that [3H]tamoxifen was bound to a single class of saturable binding sites in liver microsomes with a KD of 0.9 +/- 0.1 nM at 0 degrees C. The equilibrium KD was not significantly different at 22 degrees C. The KD calculated from the association and dissociation rate constants for [3H]tamoxifen binding at 0 degrees C and 22 degrees C was compatible with the KD measured at equilibrium. Ligand specificity studies using tamoxifen analogues showed qualitatively similar structure-affinity relationships for the AEBS from both rat liver and the MCF 7 breast cancer cell line. In general structural modifications caused correspondingly greater changes in affinity for rat liver AEBS than for MCF 7 AEBS. The AEBS was solubilized from microsomal membranes with sodium cholate. This was the only detergent of nine tested that solubilized the site in high yield without loss of activity. Solubilization using cholate was more effective in the presence of 1 M-NaCl. In the solubilized state there was an apparent loss of [3H]tamoxifen binding activity which could be restored by dilution of the detergent. Gel filtration indicated an Mr of 440,000-490,000 for the AEBS-cholate complex. These studies demonstrate that rat liver contains high concentrations of a microsomal AEBS which has similar properties and specificity to the AEBS previously described in human breast cancer cells. This site can be solubilized by sodium cholate to supply material suitable for further purification.