The effect of 17-N substituents on the activity of the opioid κ receptor in nalfurafine derivatives

We have previously reported the essential structure of the opioid κ receptor agonist nalfurafine hydrochloride (TRK-820) for binding to the κ receptor. In the course of this study, we focused on the effect of the substituent at 17-N in nalfurafine on the binding affinity for the κ receptor. The exchange of the 17-N substituent in nalfurafine from cyclopropylmethyl to fluoro-substituted alkyl groups, which are strong electron withdrawing substituents, almost completely diminished the binding affinities for the μ and δ opioid receptors, but the binding affinity for the κ receptor was still maintained. As a result, nalfurafine derivatives with 17-fluoro-substituted alkyl groups showed higher selectivities for the κ receptor than did nalfurafine itself. With regard to the κ agonistic activities, the conversion of the 17-N substituent in nalfurafine from cyclopropylmethyl to fluoro-substituted alkyl groups led to the gradual decrease of the agonistic activities in the order corresponding to their binding affinities for the κ receptor. In contrast, the derivative with the bulky 17-isobutyl group showed lower affinity and agonistic activity for the κ receptor than the derivatives with the smaller functional groups. This research suggested that both the electronic property and the steric characteristics of the 17-N substituent would have a great influence on the binding property for the κ receptor.     

Estrogen and androgen receptor binding affinity of 10 beta-chloro-estrenen derivatives

10 beta-Chloroestradien-3-one and its derivatives with chlorine substitution in ring A have been prepared. Efficient synthetic methods for 2-chloro- and 4-chloroestradiol are described. The binding affinity of these chlorinated estrogens to the uterine estrogen receptor was measured by a competitive binding assay using [3H]estradiol as ligand. 4-Chloroestradiol showed high binding affinity for the receptor (110% of that of estradiol). 2-Chloroestradiol, 10 beta-chloroestradien-3-one and 4,10 beta-dichloroestradien-3-one had moderate binding affinity. The structures of 10 beta-chloroestradien-3-one and androst-1,4-dien-3-one are very similar and can almost be superimposed. However, their binding affinities to the estrogen and androgen receptor were different. Androst-1,4-dien-3-one displayed no measurable affinity for the estrogen receptor and measurable affinity for the androgen receptor whereas 10 beta-chloroestradien-3-one had very low affinity for the androgen receptor.     

17 alpha-substituted analogs of estradiol for the development of fluorescent estrogen receptor ligands.

For the successful development of a high-affinity fluorophore-estradiol conjugate, the fluorophore must be attached to the estradiol molecule at a position that interferes least with its binding to the receptor. We have concentrated on 17 alpha substituents as models for fluorophore attachment, based on literature precedent and on our earlier work with small 17 alpha side chains. In this report, we describe syntheses and estrogen receptor binding affinities of 19 analogs of estradiol substituted in the 17 alpha position with larger side chains (of six to 11 carbons), some of which may be synthetically modified to link a fluorophore. These analogs were synthesized either by nucleophilic cleavage of estrone-17 beta-oxirane 3-benzyl ether and subsequent debenzylation (4 to 18), by cross-coupling of alkynes (21 to 24), by alkylation of 17 alpha-ethynylestradiol 3,17-bis(tetrahydropyranyl ether) and subsequent acidic hydrolysis (25 to 28), or by reacting estrone either with appropriate aryl/alkynyllithium reagents (29, 30, and 32) or with benzylmagnesium bromide (31). Relative binding affinities of these newly synthesized analogs were determined for estrogen receptor (rat uterus) using a standard competition assay. The results suggest that analogs with reduced mobility and/or more polarizable electron density in the side chain generally bind more strongly to the receptor. The relative affinities of several selected compounds were also determined in the presence of 4% dimethylformamide; some compounds bearing larger, nonpolar 17 alpha substituents showed dramatically improved affinities, while affinities for compounds with shorter nonpolar side chains remained largely unchanged. These binding affinity results should be useful in designing new high-affinity fluorescent ligands for the estrogen receptor.     

Hydroxylated 2,3-diarylindenes: synthesis, estrogen receptor binding affinity, and binding orientation considerations

In order to develop high affinity, fluorescent ligands for the estrogen receptor based on 2-arylindenes, it is important to understand how this non-steroidal estrogen is oriented within the binding site and to know how hydroxyl substituents affect binding. To investigate these issues a series of dihydroxyl-substituted 2,3-diphenylindenes were prepared by the cyclization of appropriately substituted alpha-benzyldesoxybenzoins, and their binding affinities for the estrogen receptor measured by a competitive radiometric binding assay. Introduction of a p-hydroxyl group in the 2-phenyl ring of two 2,3-diphenyl-6-hydroxyindene systems causes a 3-fold increase in binding affinity, whereas, p-hydroxylation in the 3-phenyl ring of these systems causes a 2-fold reduction in binding affinity. The parallel change in binding affinity in these two systems suggests a consistent binding orientation of the 2,3-diarylindene systems, which, on the basis of earlier studies, has the indene system corresponding to the A/B-ring system of estradiol. This orientation model and the enhanced affinity of the p-hydroxy 2-ring derivatives are suggestive of a new hydrogen bonding site below the D-ring binding site. Changes in receptor binding affinity upon hydroxylation in triphenylacrylonitrile ligands for the estrogen receptor, reported by others, do not show such parallelism, suggesting that different derivatives may not be bound in congruent orientations. A m-hydroxyl substituent in ring-3 of the 2,3-diarylindene has very little effect on receptor binding. In designing fluorescent 2,3-diarylindene ligands for the estrogen receptor, 3-ring hydroxylation may be useful in reducing non-specific binding and in modifying electron donation to the fluorophore with only modest or no reduction in binding affinity. p-Hydroxylation of the 2-ring, although increasing receptor binding, is not consistent with the electron accepting nature required of this ring.     

Synthesis and receptor binding of polynuclear organometallic estradiol derivatives

Twelve novel organometallic derivatives of estradiol were synthesized with the aim of utilizing organometallic cold bioprobes as radioisotopic labels substitutes for steroid hormone receptor assays. For this purpose, we envisaged the attachment of several stable cobalt, molybdenum, osmium carbonyl clusters (tetra- and pentanuclear species) at estradiol 17 alpha-, 16 alpha-, 2- or 4-positions. The binding affinity of these new complexes for uterine estradiol receptor has been measured by the competitive binding method. The results show that the 17 alpha-position can tolerate substitution by bulky organometallic groups (especially in the case of cobalt and molybdenum carbonyl clusters). Estradiol derivatives which are functionalized at C-4 and C-16 alpha bind estradiol receptor with reasonable affinity and the RBA values are the same for the complexed and uncomplexed hormones. The 2- position is more sensitive to organometallic substitution and the complexation at the 2- alkyne results in a dramatic decrease of the RBA values. These results show that the attachment of polynuclear moieties in estradiol 17 alpha-, 4- and 16 alpha-, positions gives rise to compounds which are of potential utility in a new non-radioisotopic receptor assay since the metal-carbonyl markers are readily detected by high-sensitivity Fourier-transform infra-red spectroscopy.     

The size and/or configuration of the cycloalkane D' ring in pentacyclic progesterone derivatives are crucial for their high-affinity binding to a protein in addition to progesterone receptor in rat uterine cytosol

[(3)H]labeled progesterone and a number of its 16alpha, 17alpha-cycloalkano derivatives with an additional three to six-membered D' ring were investigated for mutual competition and equilibrium binding to proteins from rat uterine cytosol. The interaction of all studied [(3)H]ligands with proteins was characterized by comparable affinity (K(d) in nM region) and apparent homogeneity in terms of affinity. At the same time, the concentrations of binding sites for ligands bearing 16alpha,17alpha cyclopentano, cyclohexano, or cyclohexeno substituents were several-fold higher than those for progesterone or 16alpha, 17alpha-cyclopropanoprogesterone. In mutual competition experiments, when [(3)H]progesterone or [(3)H]16alpha, 17alpha-cyclopropanoprogesterone were used, the curves of 'bound radioactivity-log of competitor concentration' for all compounds studied were parallel and corresponded to a model of 'one protein-two ligands.' However, when [(3)H]ligands with bulky 16alpha, 17alpha-substituents (with the possible exception of cyclohexene derivative) were used, competitive curves for various ligands had different appearances and fell into two groups. Parallel curves for derivatives with 5 or 6 carbons in D' ring described by a model of 'one protein-two ligands' formed the 1st group. The 2nd group comprised curves for progesterone or 16alpha, 17alpha-cyclopropanoprogesterone that had lower slopes and could be described by a model of 'two proteins-two ligands.' Taken together, the results suggest the presence in rat uterine cytosol, of a protein in addition to progesterone receptor capable of discriminating between ligands with no or small 16alpha, 17alpha-cycloalkano substituents and ligands with more bulky substituents.     

Aryl cyclopentadienyl tricarbonyl rhenium complexes: novel ligands for the estrogen receptor with potential use as estrogen radiopharmaceuticals

The need for imaging agents for estrogen receptor positive (ER+) tumors that are both cost effective and widely available, as well as the need for novel radiotherapeutic agents for the treatment of breast cancer, has prompted us to investigate cyclopentadienyl tricarbonyl metal [CpMet(CO)(3), Met=Re, Tc-99m] complexes that bind well to the ER. Thus, we have prepared a series of p-hydroxyphenyl-substituted CpRe(CO)(3) complexes and evaluated them (and, in some cases, their cyclopentadiene precursors) for binding to ER. These compounds constitute a new class of structurally integrated organometallic ligands for ER in which the CpMet(CO)(3 )organometallic unit forms the very structural core of these molecules and thus is necessarily intimately involved in their interaction with the receptor. The CpRe(CO)(3) compounds were prepared by reaction of the lithium salt of the arene-substituted cyclopentadiene with a suitable Re(CO)(3)(+) precursor, followed by deprotection of the methyl ether. The X-ray crystal structure of one of these analogues shows that it has the classical 'piano stool'-like geometry, with the alkyl groups directed upward, away from the tripodyl metal carbonyl base. The aryl-substituted CpRe(CO)(3) complexes that we have prepared all bind to the ER, some with affinity as great as 20% that of the native ligand, estradiol. In general, at least two p-hydroxyphenyl substituents and one to two alkyl groups attached to the organometallic cyclopentadienyl core are needed for high ER affinity. Where we have been able to make comparisons, the metal complexes bind to ER with an affinity greater than their cyclopentadiene precursors. The high affinity of some of these complexes indicates that the bulky Re(CO)(3) unit is able to exploit the considerable volume in the center of the ER ligand binding pocket that is not occupied by most ligands, a consideration that is supported by molecular modeling. The preparation of the best of these agents in technetium-99m labeled form is currently being investigated.     

The use of the biotinyl estradiol-avidin system for the purification of "nontransformed" estrogen receptor by biohormonal affinity chromatography

Several biotinyl estradiol derivatives have been prepared by coupling estradiol 7 alpha-carboxylic acid to biotin via different linear linkers. All these compounds exhibit a high affinity for the estrogen receptor as determined by competitive binding assays against [3H]estradiol. These compounds also displaced the dye 4-hydroxyazobenzene-2'-carboxylic acid from the biotin-binding sites of avidin free or immobilized on agarose. It was demonstrated that only the derivatives bearing a long spacer chain (greater than 42 A greater than) between estradiol and biotin were able to bind receptor and avidin simultaneously, suggesting some steric hindrance. The biotin-avidin system has been investigated for the purification of the cytosoluble "nontransformed" estrogen receptor stabilized by sodium molybdate. The method relies on: 1) high biohormonal affinity of receptor for biotinyl estradiol derivative; 2) the specific selection by avidin-agarose column of biotinyl estradiol-receptor complexes; and 3) the biohormonal elution step by an excess of radioactive estradiol. Starting from unfractionated cytosol containing molybdate-stabilized nontransformed 8S estrogen receptor with estradiol 7 alpha-(CH2)10-CO-NH-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)3-NH-biotin, preliminary experiments using avidin-agarose chromatography and then a specific elution step by exchange with free [3H]estradiol, allowed a 500-1,500-fold purification. Further purification of estrogen receptor was obtained by ion exchange chromatography through a DEAE-Sephacel column and led to a congruent to 20% pure protein, assuming one binding site/65,000-Da unit. The hydrodynamic parameters of the purified receptor were essentially identical to those of molybdate-stabilized nontransformed receptor present in crude cytosol. The advantages of this double biotinyl steroid derivative-avidin chromatographic technique over more conventional affinity procedures are discussed and make it applicable to the purification of minute amounts of steroid receptors in a wide variety of tissues.     

Diethylstilbestrol metabolites and analogs. Biochemical probes for differential stimulation of uterine estrogen responses

Diethylstilbestrol (DES) and certain chemically structural derivatives and analogs, indenestrol A (IA), indenestrol B (IB), indanestrol (IN), and pseudo-DES (PD), have been used as probes to examine various estrogenic responses previously considered interrelated and obligatory to the stimulation of uterine growth. All the analogs had poor uterotropic activity in vivo which ranged from 10-200 times less than that of estradiol or DES. The poor uterotropic activity was not due to poor binding affinity for the receptor. All compounds except IN interacted with the mouse uterine estrogen receptor with high affinity (approximately Ka 1.5-2.2 X 10(10) M-1). In addition, the compounds were able to translocate similar levels of receptor to the nucleus in vivo. Nuclear retention and occupancy of the estrogen receptor by the compounds was comparable to the patterns produced by DES or estradiol. The activity of uterine tissue responses was investigated during treatment with the compounds. Only IA stimulated uterine glucose-6-phosphate dehydrogenase to significant levels similar to DES or estradiol. Uterine progesterone receptor was induced to varying degrees by all compounds; the indenestrol isomers (IA and IB) were the most active. Uterine DNA synthesis was marginally stimulated by the derivatives and analogs except for IB which showed a response increase comparable to DES or estradiol. Because of the differential stimulation, these data suggest that in uterine tissue estrogen receptor stimulates certain biochemical responses independently and not in concert. The ability of a particular response to be increased may depend on the chemical nature of the ligand receptor complex and its interaction at genomic sites.     

Synthesis and evaluation of novel 1,5-Benzodiazepines as potent and selective CCK-B ligands. Effect of the substitution of the N-5 phenyl with alkyl groups

The synthesis and biological evaluation of both 3-ureido and 3-carbamate derivatives of 1,5-benzodiazepines bearing bulky alkyl substituents at N-1 and N-5 positions is reported. Their activity as CCK-B receptor antagonists is discussed and compared with the related N-5-phenyl derivatives.     

Cytosol and nuclear estrogen receptor binding in the preoptic area and hypothalamus of female rats during pregnancy and ovariectomized, nulliparous rats after steroid priming: correlation with maternal behavior

In a previous study, high nuclear estrogen receptor concentrations in the preoptic area (POA) were found on Day 16 of pregnancy to prime females to respond to a subsequent low dose of estradiol benzoate (EB) after hysterectomy-ovariectomy by exhibiting maternal behavior in 48 hr. Receptor concentrations in the POA were found to be higher than those in the hypothalamus (HYP). The present study investigated when nuclear estrogen receptors increase during pregnancy in POA and when the difference in receptor concentrations between POA and HYP occurs. An attempt was made to reproduce these pregnancy changes with a 16-day treatment of estrogen and progesterone in ovariectomized (OVX), nulliparous rats. In Experiment 1, we measured cytosol and nuclear estrogen receptor concentrations in the POA and HYP of female rats during pregnancy. Nuclear receptor concentrations in the POA increased beginning on Day 10, increased again on Day 16, and continued at this high level for the remainder of pregnancy. Nuclear estrogen receptor concentrations in the HYP remained at a lower level throughout most of pregnancy until Day 22 when they increased significantly. In Experiment 2, we tested the maternal behavior and measured estrogen receptor concentrations in OVX, steroid-primed, nulliparous rats after hysterectomy (H) and EB treatment. While 90% of estradiol (E) + progesterone (P)-primed females displayed short-latency maternal behavior 48 hr after H and EB treatment, 46% of E + vehicle (V)-treated controls were maternal. At 0 hr (prior to H and EB treatment), there was a significantly larger nuclear receptor accumulation in the POA but significantly attenuated receptor binding in the HYP. P treatment significantly affected cytosol and nuclear estrogen receptor dynamics. Differences in nuclear estrogen receptor concentrations were shown to be based on the number of available binding sites and not to changes in receptor affinity for estradiol.     

Catecholestrogens are MCF-7 cell estrogen receptor agonists

Catecholestrogens are important metabolites of estradiol and estrone in the human. Considerable interest has focused on the catecholestrogens 2-hydroxy- and 4-hydroxyestradiol since they bind to the estrogen receptor with an affinity in the range of estradiol. Using the MCF-7 cell line, we analysed the capacity of purified catecholestrogens to transform the estrogen receptor into its high affinity nuclear binding form and to effect receptor-dependent processes such as proliferation and expression of the progesterone receptor (PR). Incubations with 2-hydroxy- and 4-hydroxyestradiol at 10⁻⁸ M for 1 h resulted in tight nuclear binding of the estrogen receptor. During treatment of the cells with catecholestrogens we obtained a marked increase in proliferation rate of 36 and 76% for 2-hydroxy- and 4-hydroxyestradiol, respectively, relative to the inductive effect of estradiol (100%). The PR level, was slightly increased by treatment with 2-hydroxyestradiol (10%), whereas treatment with 4-hydroxyestradiol increased the PR level at 28%, compared to estradiol (100%). Form these results we conclude that the 2- and 4-hydroxylated derivatives of estradiol are active hormones and are able to initiate estrogen receptor mediated processes in MCF-7 cells.     

Synthesis and biological properties of 7alpha-cyano derivatives of the (17alpha,20E/Z)-[125I]iodovinyl- and 16alpha-[125I]iodo-estradiols

The synthesis, receptor binding affinity, estrogenic potency and tissue distribution of the 7alpha-cyano derivatives of the (17alpha,20E/Z)-[125I]iodovinyl-(CIVE) and 16alpha-[125I]iodo-estradiols (CIE) are reported. The iodovinyl derivatives were prepared via the (17alpha,20E/Z)-tri-n-butylstannyl intermediates, derived from the addition of tri-n-butyl tin hydride to the 17alpha-ethynyl group of the 7alpha-cyano-17alpha-ethynylestradiol, using triethylborane as a catalyst. The no-carrier-added [125I]-CIVE isomers were prepared via the same stereospecific reaction. [125I]-CIE was prepared from 7alpha-cyano-16beta-bromoestradiol via halogen exchange with Na125I. Addition of the 7alpha-cyano group to 16alpha-iodoestradiol did not affect estrogen receptor binding affinity (RBA of CIE is 115). However the estrogenic potential of CIE, as measured by the capacity to stimulate the expression of the pS2 gene, was reduced to 1% as compared to that of estradiol. Addition of a 7alpha-cyano group to the (17alpha,20E/Z)-IVE isomers reduced the RBA to 21 and 36, respectively, while the estrogenic potential was reduced to 2-3% of that of estradiol. Uterus uptake in immature rats of the 125I-labeled CIVE 20E-isomer and the 16alpha-iodo CIE peaked at 0.5h post injection while the (17alpha,20Z)-CIVE isomer showed a maximum only past 5h post injection. Uptake of all three 125I-labeled 7alpha-cyanoestrogens was suppressed by the co-injection of non-radioactive estradiol confirming the role of estrogen receptors in the localization process. Uterus retention pattern differ substantially from those of the analogues 7alpha-methylestrogens, which were previously shown to give high maximum 125I-uptake values at 2h post injection. Overall our data indicate that addition of a 7alpha-cyano group to 123I-labeled estrogens does not improve their potential to serve as SPECT agents for the imaging of estrogen receptor densities in breast cancer.     

Amide derivatives of 9,11-seco-estra-1,3,5(10)-trien-11-oic acid as modified orally active estrogen agonists with moderate antagonistic activity

Synthesis of amide derivatives of 9,11-seco-estra-1,3,5(10)-trien-11-oic acid containing alkyl and aromatic amine residues has been carried out with an aim to prepare orally active estrogen antagonists. Modification of the estradiol molecule in the form of C-seco-amide derivatives has led to their high oral absorption. Compounds 7 an n-propyl amide, 8 an n-butyl amide, and 16 a p-anisidyl amide of C-seco-estrane showed significant estrogen antagonistic activity (>20%), while, the majority of compounds possessed high estrogen agonistic activity on oral administration at 10mg/kg dose in rats.     

Analogies and differences in the modulation of progesterone receptor induction and cell proliferation by estrogens and antiestrogens in MCF-7 human breast cancer cells: study with 24 triphenylacrylonitrile derivatives

Structure-activity relationships in a homogeneous series of 24 triphenylacrylonitrile derivatives were examined with respect to the stimulation of progesterone receptor induction and cell proliferation in MCF-7 cells. In general, triphenylacrylonitrile derivatives were found to be full or partial agonists for both responses; the partial agonists were also able to antagonize the stimulatory action of estradiol. The agonistic activities of these molecules decreased as the size of the lateral side chain increased, but the side-chains correlated with partial agonism of progesterone receptor induction were bulkier than those correlated with partial agonism of cell proliferation. Agonistic and antagonistic effects on both responses were correlated with affinity for the estrogen receptor. Half maximal effects on the two responses occurred at different concentrations (4-fold) of the compounds. It can be concluded that in MCF-7 cells, triphenylacrylonitrile modulation of progesterone receptor induction and cell proliferation are mediated by the estrogen receptor; the two effects, which occur at different concentrations and with slightly different substituents of the compounds, are differentially modulated.     

Synthesis, characterization, and estrogen receptor binding affinity of flavone-, indole-, and furan-estradiol conjugates

Different flavone-, indole-, and furan-17beta-estradiol conjugates, linked via alkyl spacer chains extending from the 17alpha-position of the estradiol moiety, were synthesized by Pd-catalyzed cross-coupling reactions. Structures were assigned based on spectroscopic data. In vitro competitive binding assays for the estrogen receptor (alpha-ER), using [(3)H]estradiol (RBA=100) as a competitor, revealed that a two-carbon alkyl linker combined with a flavone conjugate provided the highest binding affinity (RBA approximately 9), warranting further studies on their potential use as selective estrogen-receptor modulators (SERMs) for hormone-replacement therapies.     

Synthesis and binding affinities of fluoroalkylated raloxifenes

Three fluoroalkylated derivatives (1-3) of the selective estrogen receptor modulator (SERM), raloxifene, have been synthesized. The key step in the synthesis is the C-C bond formation of benzo[b]thiophene and a substituted phenyl group (ring C) using a Stille reaction. The in vitro binding affinities of the substituted raloxifenes 1-3 are 45, 60, 89%, respectively, relative to the affinity of estradiol, which is higher than the affinity of raloxifene itself (25%). When labeled with the positron-emitting radionuclide, these compounds might be useful as PET imaging agents for estrogen receptor-positive breast tumors.     

Novel urushiol derivatives as HDAC8 inhibitors: rational design,virtual screening,molecular docking and molecular dynamics studies

Three series of novel urushiol derivatives were designed by introducing a hydroxamic acid moiety into the tail of an alkyl side chain and substituents with differing electronic properties or steric bulk onto the benzene ring and alkyl side chain. The compounds’ binding affinity toward HDAC8 was screened by Glide docking. The highest-scoring compounds were processed further with molecular docking, MD simulations, and binding free energy studies to analyze the binding modes and mechanisms. Ten compounds had Glide scores of ?8.2 to ?10.2, which revealed that introducing hydroxy, carbonyl, amino, or methyl ether groups into the alkyl side chain or addition of –F, –Cl, sulfonamide, benzamido, amino, or hydroxy substituents on the benzene ring could significantly increase binding affinity. Molecular docking studies revealed that zinc ion coordination, hydrogen bonding, and hydrophobic interactions contributed to the high calculated binding affinities of these compounds toward HDAC8. MD simulations and binding free energy studies showed that all complexes possessed good stability, as characterized by low RMSDs, low RMSFs of residues, moderate hydrogen bonding and zinc ion coordination and low values of binding free energies. Hie147, Tyr121, Phe175, Hip110, Phe119, Tyr273, Lys21, Gly118, Gln230, Leu122, Gly269, and Gly107 contributed favorably to the binding; and Van der Waals and electrostatic interactions provided major contributions to the stability of these complexes. These results show the potential of urushiol derivatives as HDAC8 binding lead compounds, which have great therapeutic potential in the treatment of various malignancies, neurological disorders, and human parasitic diseases.     

Symmetric 4,4′-(piperidin-4-ylidenemethylene)bisphenol derivatives as novel tunable estrogen receptor (ER) modulators

We designed and synthesized 4,4′-(piperidin-4-ylidenemethylene)bisphenol derivatives as novel tunable estrogen receptor (ER) modulators. The introduction of hydrophobic substituents on the nitrogen atom of the piperidine ring enhanced ERα binding affinity. In addition, the introduction of four methyl groups adjacent to the piperidine ring nitrogen atom remarkably enhanced ERα binding affinity. N-Acetyl-2,2,6,6-tetramethylpiperidine derivative 3b showed high ERα binding affinity, high MCF-7 cell proliferation inducing activity, and high metabolic stability in rat liver S9 fractions.