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. 2017 May 24;8(7):1485–1497. doi: 10.1039/c7md00163k

Rational design and optimization of selenophenes with basic side chains as novel potent selective estrogen receptor modulators (SERMs) for breast cancer therapy†

Junjie Luo a,‡, Zhiye Hu a,‡, Yuan Xiao a, Tongxin Yang a, Chune Dong a,c, Jian Huang b, Hai-Bing Zhou a,✉
PMCID: PMC6072463  PMID: 30108860

graphic file with name c7md00163k-ga.jpgSelenophenes with basic side chains showed excellent antagonist activity for ERs and increased antiproliferative activity than that of 4OHT in MCF-7 cells.

Abstract

To increase the diversity of estrogen receptor (ER) ligands having novel structures and activities, series of selenophene derivatives with a basic side chain (BSC) were synthesized and their biological activity as subtype-selective antagonists for the ER was explored. Compared with the selenophenes without a BSC, most compounds showed an increase in binding affinity, and several compounds displayed enhanced antagonist potency and antiproliferative activity. Especially, compound 16c exhibited excellent transcriptional activity for ERα (IC50 = 13 nM) which made this compound the most potent antagonist for ERα of the whole series and is 66-fold better than the best selenophene compound without a BSC. Moreover, several compounds showed values of IC50 better than that of 4-hydroxytamoxifen in breast cancer MCF-7 cells. The modeling study indicated that the basic side chain might contribute to their increased antagonist potency and antiproliferative activity. These new ligands have the potential to be further developed as novel agents to improve therapeutics that target the estrogen receptor.

1. Introduction

Selenium (Se) is an important nutritional trace element involved in different physiological functions and possesses antioxidative, antitumor and chemopreventive properties. Dietary Se has been inversely associated with the risk of cancer, and substantial evidence shows that selenium has a significant influence on the incidence of many cancers.1 Epidemiological studies reveal that a low Se status may contribute to the etiology of different diseases, for example, viral infections, reproductive deficiencies, loss of immunocompetence, thyroid and cardiovascular diseases, and pancreatitis.2 Selenium is present in more than twenty five human selenoproteins, and most of them have been involved in anti-oxidant defence systems and cancer prevention, etc.3,4 The anti-carcinogenic potential of Se has also been reported in geographical studies during the past 40 years.5 A variety of selenium-containing compounds with diverse chemical structures are known to inhibit cell proliferation in vitro, such as inorganic selenium salts,6–12 selenoamino acids,5 methylselenocyanate,12,13 as well as benzyl and phenyl selenium derivatives.14 In particular, many diarylselenides possess anticancer, antitumor, antiviral, antimicrobial, and antioxidant properties.15–17 Various biologically active selenaheterocycles such as ebselen have been discovered in recent years.18 Furthermore, anticancer mechanisms of MSeA have been hypothesized to involve estrogen receptor (ER) stress signal mediators and apoptosis.19,20

As has been known, estrogens are important regulators of many physiological functions related to the reproductive and non reproductive tissues in both women and men.21,22 The effects of estrogens are mediated via two estrogen receptor subtypes, ERα and ERβ,23 which are ligand-regulated transcription factors that regulate many physiological and pathological processes. The estrogen receptors have different tissue distributions and significant differences in their ligand binding preferences.24,25 While estrogens are necessarily beneficial in some tissues, including the reproductive,26 skeletal,27 cardiovascular,28 and central nervous systems,29 inappropriate or over-expression of ER is associated with a number of endocrine disorders. For example, the estrogen receptors play a predominant role in breast cancer growth because of the pro-proliferative effect.30 Thus, developing ER subtype-selective ligands with tissue- and gene-selective biological activities is a critical clinical objective. In order to discover “ideal” selective estrogen receptor modulators (SERMs), extensive investigation has been made to increase the chemical diversity of these compounds, especially the non-steroidal ones. Apart from the preserved peripheral substituents, e.g. phenols, simple alkyl groups, and polar phenyl substituents, a wide variety of heterocyclic cores have been explored,31 from the five-membered ring heterocycles to six-membered ring heterocycles as well as fused heterocycles. Some examples of the five-membered ring heterocycles are presented below (Fig. 1), including furan (1),31 imidazole (2),32,33 propyl pyrazole triol (3),34–36 thiophene 4),37 selenophene (5),38 raloxifene (RAL), etc. Because the activity profiles of the current SERMs, e.g. tamoxifen, are not ideal and resistance to their effectiveness as antitumor agents can develop with time (Fig. 1), there has been interest in finding new SERMs that might prove more effective as hormonal or therapeutic agents.

Fig. 1. Examples of the five-membered ring heterocycles (1–5) as ER ligands and structures of known SERMs raloxifene and 4-hydroxytamoxifen.

Fig. 1

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As part of our long-term interest in the development of ligands for the ERs having novel structures and activities,39,40 we have recently described a new series of analogues based on the selenophene scaffold that showed good binding affinity to ERs. The 2,5-bis(2-fluoro-4-hydroxylphenyl) selenophene 8c showed the highest relative binding affinity (RBA, estradiol = 100) of 24.3 for ERβ. In transcription assays, most of the selenophenes exhibited partial to full agonist activity for both ER subtypes, but several compounds displayed a range of ERα or ERβ antagonistic activities.38 A few selenophenes exhibited antiproliferative activity comparable to that of 4OHT in breast cancer MCF-7 cells. Interestingly, from the modeling study of the complex of ERα-8c, it is observed that one of the phenolic groups of 8c interacts with helix 11 through H-bonds and does not further destabilize helix 12; rather, it mimics the role of the D-ring phenol of E2 and stabilizes helix 12 in the agonist conformation.

Therapeutic targeting of the estrogen receptor has traditionally involved direct disruption of the surface coactivator binding sites by a basic side chain (BSC) that is a characteristic structural feature of SERMs, such as tamoxifen and raloxifene (Fig. 1). The nature and spatial orientation of the basic side chains in SERMs can influence their tissue selectivity and affect the balance of desired and undesired activities.41 Thus, we wondered whether the introduction of basic side chains into a selenophene-based core structure (e.g., a pyrrolidine or piperidine side chain) might provide ER ligands with interesting antagonistic activities (Fig. 2). Herein, we introduced two different types of aminoethoxy moieties into the selenophene core system, placing these at different positions of the phenolic groups. We expected that these selenophene-core derivatives could act as models with improved biological activity for the development of novel estrogen receptor ligands. It was proved that several compounds (e.g.16c) showed values of IC50 better than that of 4-hydroxytamoxifen in breast cancer MCF-7 cells.

Fig. 2. Rationale for design of novel potent selenophenes with a BSC for breast cancer therapy.

Fig. 2

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2. Results and discussion

2.1. Chemical synthesis

Three series of novel selenophene derivatives, as depicted in Scheme 1, were prepared from selenophenes according to the synthetic procedures established in our laboratory (described in the Experimental section).

Scheme 1. Synthesis of substituted selenophene derivatives. Reagents and conditions: (a) [Pd] catalyst, Na2CO3, toluene/water (1 : 1), reflux, 24 h; (b) BBr3, CH2Cl2, –20 °C to rt, 4 h; (c) N-(2-chloroethyl)pyrrolidine or N-(2-chloroethyl)piperidine, KOH, K2CO3, DMF, microwave, 120 °C, 45 min; (d) pyridine hydrochloride, 190 °C, 3 h.

Scheme 1

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In the synthesis of compounds 9a–g (Scheme 1A), key intermediates 7a–g were obtained using the Suzuki cross-coupling reaction of aryl boronic acids with 2,5-disubstituted selenophene 6a.38,42 Then, compounds 7a–g were treated with boron tribromide affording the corresponding 2,5-disubstituted diphenolic selenophenes 8a–g. Finally, the target products 9a–g were obtained from corresponding phenols and chloroethyl pyrrolidine or piperidine by Williamson ether synthesis under microwave conditions. This step produced a mixture of monoalkylated products 9a–g (yield 60–70%) and was accompanied by about 30–40% of dialkylated by-products (Scheme 1A).

In the synthesis of 3,4-disubstituted selenophene derivatives 12a–i (Scheme 1B), 10a–i served as the key intermediates, which were prepared by treating 3,4-dibromoselenophene 6b38 with aryl boronic acids by using Pd(OAc)2/PPh3 as the catalyst. Subsequent ether cleavage of 10a–i by pyridine hydrochloride yielded the intermediates 11a–i. Finally, treatment of intermediates 11a–i with chloroethyl pyrrolidine or piperidine afforded the desired products 12a–i in good yields.

In our previous work on furan or thiophene derived ER ligands,37 triphenol furans and thiophenes were proved to be more effective than the corresponding bisphenol analogues with higher binding affinity and subtype selectivity. Particularly, a few triaryl selenophenes exhibited substantial antiproliferative activity in breast cancer MCF-7 cells.38 Thus, we wanted to introduce a basic side chain into the triphenol selenophene scaffold, as such selenophenes 16a–c were obtained by demethylation of 15a–c, which were prepared through Suzuki cross-coupling reactions followed by installation of a BSC (Scheme 1C). In the first step, 1 equiv. of 2,3,5-tribromoselenophene 6c38 was reacted with 4 equiv. of aryl boronic acid under standard conditions, and the resulting 2,5-bis-substituted selenophenes 13 were subsequently submitted to a cross-coupling reaction with 2 equiv. of phenyl boronic acid to yield the intermediates 14a–c. Installation of the two types of basic side chains in the free phenolic positions in 14a–c was effected by a Williamson reaction, and the remaining methyl group was then selectively cleaved with boron tribromide, leaving the basic side chain unaffected. By this approach, we prepared the analogues containing the two types of basic side chains at the para position of the C-3 phenyl group, 16a–16c.

2.2. Relative binding affinities

The binding affinities of the selenophene compounds for both ERα and ERβ were determined by a competitive fluorometric receptor-binding assay and are summarized in Table 1. These affinities are presented as relative binding affinity (RBA) values, where estradiol (E2) has an affinity of 100%.

Table 1. Relative binding affinity (RBA) of compounds 9a–g, 12a–i, 16a–c for ERα and ERβ a .

Inline graphic
Entry Compound Core R n ERα b ERβ b α/β
1 9a graphic file with name c7md00163k-u2.jpg H 1 12.4 ± 0.42 1.02 ± 0.08 12.2
2 9b 2-Me 1 1.96 ± 0.11 0.58 ± 0.07 3.37
3 9c 2-Me 2 <0.1 0.62 ± 0.08 <0.1
4 9d 2-F 1 0.76 ± 0.01 <0.1 >10
5 9e 2-F 2 6.89 ± 1.02 0.63 ± 0.09 10.9
6 9f 2-Cl 1 4.59 ± 0.35 23.6 ± 0.49 0.19
7 9g 2-Cl 2 <0.1 <0.1 —
8 12a graphic file with name c7md00163k-u3.jpg H 1 2.06 ± 0.09 5.92 ± 0.28 0.35
9 12b H 2 7.58 ± 0.11 2.35 ± 0.04 3.3
10 12c 2-Me 1 1.65 ± 0.03 0.46 ± 0.08 3.6
11 12d 2-Me 2 0.56 ± 0.06 10.2 ± 0.17 0.05
12 12e 3-Me 1 2.03 ± 0.23 5.92 ± 0.14 0.34
13 12f 3-Me 2 <0.1 <0.1 —
14 12g 3-F 2 4.82 ± 0.32 15.3 ± 0.29 0.32
15 12h 2-Cl 1 <0.1 23.3 ± 0.06 <0.1
16 12i 2-Cl 2 5.38 ± 0.08 18.4 ± 0.31 0.29
17 16a graphic file with name c7md00163k-u4.jpg H 1 3.14 ± 0.31 16.7 ± 0.10 0.18
18 16d H 2 4.59 ± 0.35 <0.1 >10
19 16c 2-Me 1 13.8 ± 0.27 3.4 ± 0.29 4.06
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aTo simplify comparisons of the compounds in related series, we designated locant positions of the substituents on the phenyl groups with respect to the selenophene core; locant positions on the selenophene core itself are given by numbers in italics.

bRelative Binding Affinity (RBA) values are determined by competitive fluorometric binding assays and are expressed as IC50estradiol/IC50compound × 100 ± the range (RBA, estradiol = 100%). In these assays, the Kd value of estradiol is 3.1 nM for ERα and 3.4 nM for ERβ, respectively. For details, see the Experimental section.

As a global observation, it is noteworthy that the position of basic side chains in the phenyl ring of selenophene derivatives has very significant effects on the binding affinity of conjugates. In general, most of the compounds in series I (except 9c and 9f) that possess the basic side chain in the 2-phenol moiety exhibit better binding affinity for ERα. In contrast, the vast majority of the members of series II (apart from 12b and 12c) with the basic side chain located in the 3-phenol moiety display moderate levels of ERβ selectivity. The compound that shows the highest binding affinity for ERβ and moderate ER subtype selectivity of all of the ligands is 9f, which possesses a basic side chain on the 2-phenol unit. The RBA values of this compound are 4.59 and 23.6 for ERα and ERβ, respectively; and it has an ERα/ERβ selectivity as low as 0.19 (Table 1, entry 6). Compared to the parent compound 2,5-bis(2-chloro-4-hydroxylphenyl)-selenophene38 (RBA values were 6.11 for ERα and 12.7 for ERβ; α/β was 0.48), 9f still retained high binding affinity for ERβ and displayed higher selectivity. The compound that has the highest ERα/ERβ selectivity is 12h (ERα/ERβ ratio < 0.1), which also shows a high RBA value for ERβ (23.3). As we expected, the halogens Cl and F bind better than the other ligands, with Cl being better than F in most cases (9e, 9f, and 12g–i).

Compared to the diphenolic selenophene compounds previously reported by our group, the introduction of the basic side chains can alter the ER subtype selectivity through changing the substitution position. Moreover, introduction of a substituent in the phenol rings has significant effects on the binding affinity and selectivity. Among the compounds of series I, the nonsubstituted compound 9a showed a good binding affinity (12.4 and 1.02 for ERα and ERβ, respectively). When the substitution occurred at the meta position of the phenol ring (9b–9g), moderate affinity, ranging from 0.1 to 23.6, was observed. Interestingly, most of the compounds in series I show better selectivity for ERα, and when basic side chains were introduced at the C-3 position of the selenophene ring (series II), most of the compounds show good selectivity for ERβ (Table 1, entries 8–16). For the disubstituted selenophene derivatives, when the substitution occurred at 3- and 4-positions (12a–12i), lower binding affinities were observed. A similar trend was also observed for furan and thiophene derivatives.38 Nevertheless, it should be pointed out that these compounds also show a moderate affinity for ERβ. Moreover, the binding affinities and selectivities for ERβ dramatically changed when the substitution was moved from the C-2 position to the C-3 position (12c–12f, entries 10–13). For comparison, trisubstituted selenophene analogues with a basic side chain were also prepared (series III). With the exception of 16b with undetectable ERβ binding affinity of less than 0.1, all showed good binding affinity for both ERα and ERβ (entries 17–19). The desired compound showing the highest binding affinity for ERα was 16c (13.8 for ERα), which also displayed a moderate ER subtype selectivity.

Overall, we found that the compounds synthesized with N-(2-chloroethyl)pyrrolidine might display a better binding affinity for ER than those obtained with N-(2-chloroethyl)piperidine.

Compared with our previous work, it was interesting to find that most of the biaryl substituted selenophene compounds with basic side chains exhibited an equivalent or better binding affinity for ER than the corresponding parent selenophenes except 9b (Table 2).

Table 2. A direct comparison of RBA values to ERα and ERβ for the various biaryl selenophene compounds with a BSC in this work and the corresponding ones without a BSC studied previously38.

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This work
 Biaryl selenophenes (without BSC)
Entry R Compound RBA
 RBA
ERα ERβ ERα ERβ
1 H 9a 12.4 ± 0.42 1.02 ± 0.08 0.61 ± 0.034 2.87 ± 0.20
2 2-Me 9b 1.96 ± 0.11 0.58 ± 0.07 5.60 ± 0.43 11.1 ± 0.73
3 2-F 9e 6.89 ± 1.02 0.63 ± 0.09 5.90 ± 0.90 24.3 ± 0.52
4 2-Cl 9f 4.59 ± 0.35 23.6 ± 0.49 6.11 ± 0.05 12.7 ± 3.66
5 H 12a 2.06 ± 0.09 5.92 ± 0.28 0.32 ± 0.04 2.01 ± 0.11
6 2-Me 12d 0.56 ± 0.06 10.02 ± 0.17 0.27 ± 0.06 6.70 ± 0.49
7 3-Me 12e 2.03 ± 0.23 5.92 ± 0.14 0.71 ± 0.09 0.62 ± 0.12
8 3-F 12g 4.32 ± 0.32 15.3 ± 0.29 0.58 ± 0.14 1.56 ± 0.23
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2.3. Transcription activation assays

The effects of these selenophene derivatives on ER transcriptional activity were determined using an ER responsive luciferase reporter gene. HEK 293 cells were seeded in 24-cell plates at a concentration of 2 × 106 cells per plates and allowed to settle for 24 hours. Then the cells were transfected with a widely used 3 × ERE-luciferase reporter and an ERα or ERβ expression plasmid for agonist activity (% efficacy) and potency (EC50) determinations. These cells were stimulated with increasing concentrations of 17β-estradiol (E2) or these compounds. For antagonist mode assays (% efficacy and IC50), cells were stimulated with a combination of estradiol (10 nM) and an increasing concentration of the various selenophene compounds. The luciferase activity was measured the next day. These results are summarized in Table 3, and dose–response curves for representative samples are shown in Fig. 3.

Table 3. Effects of selenophene-core compounds on the transcriptional activities of estrogen receptors α and β and antiproliferative activity in MCF-7 cells a .

Inline graphic
Agonist mode b
Antagonist mode c tab3fnd tab3fne
MCF-7
R n ERα
 ERβ
 ERα
 ERβ
 IC50 e (μM)
Entry Cmpd EC50 (μM) Eff (% E2) EC50 (μM) Eff (% E2) IC50 (μM) Eff c (% E2) IC50 (μM) Eff (% E2)
1 9a H 1 0.219 15.3 ± 4.8 — — 1.213 71.5 ± 16.7 1.266 83.9 ± 7.7 5.77 ± 0.06
2 9b 2-Me 1 — — 0.24 72.3 ± 12.5 0.874 78.3 ± 27.8 0.146 4.6 ± 15 53.8 ± 4.24
3 9c 2-Me 2 2.369 11.2 ± 17.2 — — 0.041 52.3 ± 7.7 1.235 78.3 ± 29.4 25.3 ± 0.55
4 9d 2-F 1 0.136 86.9 ± 26.6 — — — — 1.120 92.1 ± 10.9 >100
5 9e 2-F 2 0.204 68.4 ± 13.5 — — — — 1.945 75.4 ± 4.2 >100
6 9f 2-Cl 1 — — — — 0.415 69.7 ± 21.9 0.299 49.4 ± 11.1 7.81 ± 0.13
7 9g 2-Cl 2 0.911 19.9 ± 5.0 — — — — 2.96 24.9 ± 14.4 >100
8 12a H 1 1.003 45.9 ± 6.3 — — — — 0.102 46.7 ± 12.3 >100
9 12b H 2 — — 1.147 53.5 ± 11 1.589 89.2 ± 8.3 0.451 22.8 ± 8.9 8.13 ± 0.46
10 12c 2-Me 1 — — — — 0.179 21.4 ± 1.7 0.043 66.7 ± 3.6 16.6 ± 1.34
11 12d 2-Me 2 0.158 23.6 ± 19.7 — — 0.237 84.6 ± 21.3 1.335 43.4 ± 10.2 15.8 ± 0.98
12 12e 3-Me 1 0.116 36.5 ± 4.9 — 20.8 ± 3 0.836 87.4 ± 5.6 0.005 34.1 ± 5.8 12.8 ± 2.40
13 12f 3-Me 2 0.721 30.3 ± 12.2 — — — — 0.415 30.3 ± 12.2 >100
14 12g 3-F 2 — — — — 0.069 15.8 ± 2.6 0.54 45.0 ± 1.2 15.6 ± 2.41
15 12h 2-Cl 1 — — — — 0.769 38.5 ± 16.4 1.159 68.1 ± 19.6 10.9 ± 1.12
16 12i 2-Cl 2 — — — — 0.413 77.8 ± 25.7 0.331 37.7 ± 11.0 14.1 ± 2.27
17 16a H 1 — — — — 0.974 56.2 ± 9.4 0.475 49.2 ± 9.9 4.95 ± 0.26
18 16b H 2 1.831 9.2 ± 3.3 — — 0.779 91.2 ± 10.4 5.391 33.8 ± 14.2 7.04 ± 0.49
19 16c 2-Me 1 — — — — 0.013 24.8 ± 4.5 0.016 170.0 ± 1.0 7.05 ± 0.82
20 4OHT 0.008 35 ± 3 — –1 ± 1 0.003 35 ± 3 0.001 –20 ± 2 15.6 ± 1.77
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aLuciferase activity was measured in HEK293T cells transfected with a 3 × ERE-driven luciferase reporter and expression vectors encoding ERα or ERβ and treated in triplicate with increasing doses (up to 10–5 M) of the compounds.

bEC50 and standard deviation (mean ± SD), shown as a percentage of 10–8 M 17β-estradiol (E2), were determined.

cIC50 and standard deviation (mean ± SD) were determined in the percentage of 10–8 M 17β-estradiol (E2) on ERα or ERβ.

dERs have considerable basal activity in HEK293T cells; compounds with inverse agonist activity are given negative efficacy values. Omitted EC50 or IC50 values were too high to be determined accurately; omitted Eff (% E2) values were too low to be accurately determined.

eIC50 values are an average of at least three independent experiments ± standard deviation (mean ± SD).

Fig. 3. Illustrative dose–response curves for the ERα antagonist effects of 4TOH, and two selenophene-core compounds 12g and 16c. Efficacy values are the mean ± SD from three experiments. For details, see the Experimental section.

Fig. 3

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The interesting activities are seen in compounds of series I, 2,5-disubstituted selenophenes 9a–g. These ligands displayed a wide range of activities at both ERα and ERβ, most of which are potent and highly efficacious ERα agonists and ERβ antagonists, respectively. Compound 9a with a pyrrolidinyl side chain was a weak agonist of ERα and antagonist of ERβ with a nanomolar to micromolar IC50 range; however, addition of a methyl group to 9a converted it from being an agonist to an antagonist of ERα and a strong antagonist of ERβ (9b, IC50 = 0.24 and 0.146 μM for ERα and ERβ, respectively). Compared with 9b, compound 9c prepared with N-(2-chloroethyl) piperidine didn't have obvious influence on the activity (Table 3, entries 2 and 3). Moreover, replacing the methyl group (9b) with a halogen group also had significant effects on the transcriptional activity of the ER subtypes. The fluorine-substituted compounds 9d and 9e profiled as ERα agonists, being about 4 to 6-fold more potent than 9a, whereas the chloro analogues 9f and 9g had little effect on ERα (Table 3, entries 4, 5 and 6, 7). However, when the halogen group was introduced into these compounds, the antagonistic activity for ERβ significantly increased (Table 3, entries 4 and 6).

Comparisons of the position of basic side chains in the selenophene core indicate that 3,4-disubstituted compounds (series II) had decreased ERα binding affinity (Table 1, 12a–i), which also demonstrate decreased efficacy as ERα agonists in general (Table 3). Interestingly, these 3,4-disubstituted ligands show improved ERβ binding affinity and also display increased potency as ERβ antagonists, whereas 12b profiled as a moderate ERβ agonist with a μM range IC50 but had low efficacy. When the methyl group was changed from the C2- to C3-position (12e, 12f), these compounds profiled as antagonists for ERβ, and were more efficacious than 12c and 12d (Table 3, entries 12 vs. 10; 13 vs. 11). It was remarkable that 12e could act as a potent ERβ antagonist with a nanomolar IC50 value (5 nM).

Addition of a third substituent onto the selenophene core also has drastic effects on transcriptional activity. We prepared three compounds 16a–c and these were potent and highly efficacious ERα and ERβ antagonists, especially compound 16c, which gave low IC50 values of 0.013 and 0.016 μM for ERα and ERβ, respectively. This makes 16c the most potent antagonist for ERα of the whole series, and the transcription activity of 16c is 66-fold better than the best compound in selenophenes without a BSC.38 The trend is consistent with the RBA of these compounds. Again, there was also a trend that most compounds with pyrrolidine side chains displayed better activities than those with piperidine side chains.

2.4. Antiproliferative activity on breast cancer cells

To evaluate the antiproliferative activity of this type of compounds, all selenophene-core compounds were screened against MCF-7, and the results are summarized in Table 3.

Overall, most of the selenophene derivatives were effective in inhibiting the MCF-7 breast cancer cells. Among them, six compounds, 9a, 12h–i and 16a–c, exhibited potent antiproliferative activity with values of IC50 better than that of 4-hydroxytamoxifen in breast cancer MCF-7 cells. Generally, the compounds that displayed potent and highly efficacious antiproliferative activity on MCF-7 breast cancer cells also showed moderate to good binding affinities for ERα and ERβ; meanwhile, two compounds, 9g and 12f, had low antiproliferative activity due to their unobvious RBA values (less than 1%). However, the compounds 9d, 9e, and 12a possessing high binding affinities showed weak inhibition for MCF-7 cells, which suggested that the binding affinity and antiproliferative potency should be independent. Furthermore, introduction of the basic side chains in compounds demonstrated more promising antiproliferative activity.

In order to determine that the antiproliferative activity of these compounds arises from their antiestrogenicity on ER, the cells were incubated with 50 μM of 9a, 12b and 16a in the presence and absence of 10 nM E2. As shown in Fig. 4, after incubation with different doses of ligands, relative cell activities decreased substantially. Exogenous estradiol could also restore a portion of the MCF-7 cells, which demonstrated that these selenophene derivatives inhibited the MCF-7 cells by interacting with ER. As such, those potent compounds in suppressing the proliferation of MCF-7 cell showed moderate to excellent antiproliferative activity, which might be attributed to their antagonistic potency on ER.

Fig. 4. Effect of E2, 9a, 12b and 16a on the proliferation of hormone-dependent breast cancer MCF-7 cells after 5 days of culture. Nontreated MCF-7 cells are used as the control set at 100%. Mean of three separate experiments ± range.

Fig. 4

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2.5. Structural analysis of the origin of the enhanced antagonist character of these ligands

Estradiol supports transcriptional activation of ERα and ERβ by stabilizing helix 12 in a position where it forms a hydrophobic groove for binding transcriptional coactivators. The traditional SERMs or full antagonists have typically been developed by adding a bulky side group that directly obstructs the agonist position of helix 12, relocating it out of this position and thereby blocking the recruitment of transcriptional coactivators.43

Consistent with this model, we find that 11c (Fig. 5A) can similarly form a hydrogen bond with residue Glu353 on helix 3 and residue Arg394 on helix 6, respectively.44 In contrast, the SERM with a close structure, raloxifene (Fig. 5C), has a bulky side chain that projects between helices 3 and 11, directly displacing helix 12 from its active conformation and destroying the transcriptional coactivator binding site; moreover, compound 12e (Fig. 5B) mimics the binding orientation of raloxifene, with the BSC on one of the phenol groups directly interacting with any helix 12 residues, which is consistent with raloxifene, thus giving 12e potent ERα antagonist activity. For triaryl substituted selenophene 16c, the ligand is consistent with raloxifene: there are similar hydrogen bonds and direct displacement of helix 12 with a BSC to those of raloxifene and 12e. The second phenolic hydroxy of 16c also forms a hydrogen bond with the residue His524 on helix 11 (Fig. 5D), which makes 16c the most potent antagonist for ERα of the whole series.

Fig. 5. Model of selenophene ligands 11c, 12e and 16c bound to ERα and comparisons with raloxifene. (A) Computer-developed model of 11c bound to the ERα (PDB: ; 1ERR). The phenolic hydroxyl group of 11c forms hydrogen bonding interactions with residue Glu353 on helix 3 and residue Arg394 on helix 6, respectively. (B) Computer-developed model of 12e bound to the ERα with the conserved H-bonds to Glu353 and Arg394 residues. The basic side chain of the 12e is oriented toward helix 12. (C) Crystal structure of the ERα LBD in complex with raloxifene. Raloxifene forms H-bonds to the conserved Glu353, Arg394, Asp351, and His524 residues. The basic side chain displaces helix 12. (D) Computer-developed model of the ERα LBD in complex with 16c. Consistent with the complex of raloxifene, the second phenol forms a hydrogen bond with the residue His524 on helix 11, and the basic side chain displaces helix 12.

Fig. 5

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3. Conclusions

Therapeutic targeting of the estrogen receptor has traditionally involved direct disruption of the surface coactivator binding sites by a basic side chain that is a characteristic structural feature of SERMs, such as tamoxifen and raloxifene. In this study, we sequentially introduced different basic side chains into the selenophene scaffold to form new subtype-selective antagonists for the estrogen receptor. Interestingly, most of the compounds displayed good binding affinity and increased antagonistic activity for both ERs in comparison with the parent selenophenes without a BSC. Several compounds showed values of IC50 better than that of 4-hydroxytamoxifen in breast cancer MCF-7 cells. The preliminary mechanistic study indicated the antiproliferative activity arises from their antiestrogenicity; the modeling study showed that the introduction of a basic side chain had a significant effect on the antiproliferative activity of these compounds via directly interacting with helix 12. These new ligands could act as models for the development of novel agents to improve therapeutics that target the estrogen receptor.

4. Experimental section

4.1. Chemistry

4.1.1. General

Starting materials were purchased from Aldrich, Acros, Aladdin-Reagent, and Alfa-Aesar and were used without purification. Toluene was freshly distilled with sodium, and dichloromethane was distilled with anhydrous CaH2. Glassware was oven-dried, assembled while hot, and cooled under an inert atmosphere. Unless otherwise noted, all reactions were conducted in an inert atmosphere. All reactions were performed under an Ar atmosphere unless otherwise specified. Reaction progress was monitored by analytical thin-layer chromatography (TLC). Visualization was achieved by UV light (254 nm).

1H NMR and 13C NMR spectra were measured on a Bruker AVANCE III 400 (400 MHz, 1H NMR; 101 MHz, 13C NMR) instrument. Chemical shifts are reported in ppm (parts per million) and are referenced to either tetramethylsilane or the solvent. Melting points were determined on a X-4 Beijing Tech melting point apparatus, and the data were uncorrected.

General procedure for Suzuki coupling

Under an Ar atmosphere, a mixture of bromoselenophene (1 equiv.), arylboronic acid (3 equiv. for disubstituted, 4 equiv. for trisubstituted selenophenes), the Pd catalyst, sodium carbonate (2 equiv.) in an oxygen-free toluene/water (1:1) solution was stirred at 120 °C for 24 h, after which, the reaction mixture was cooled to room temperature. The aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous Na2SO4 and then filtered and concentrated under vacuum. The product was purified by column chromatography (CC).

General procedure for ether cleavage

Method A: under an Ar atmosphere, to a solution of the methoxyphenyl selenophene derivative (1 equiv.) in dry dichloromethane, boron tribromide (3 equiv. per methoxy function) was added dropwise at –20 °C. The mixture was allowed to stir for 4 h, and quenched with MeOH. The reaction mixture was poured into water, and extracted with ethyl acetate. The extracts were dried (Na2SO4) and evaporated. The residue was purified by silica gel column chromatography (CC).

Method B: a mixture of the methoxyphenyl selenophene derivative (1 equiv.) and pyridine hydrochloride (10 equiv.) was stirred at 190 °C for 3 h, after which, the reaction mixture was cooled to room temperature. The aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with dilute hydrochloric acid, dried over anhydrous Na2SO4 and then filtered and concentrated under vacuum. The product was purified by column chromatography (CC).

General procedure for the installation of basic side chains by Williamson ether synthesis

To a mixture of the phenolic selenophene derivative (1 equiv.) and N-(2-chloroethyl)pyrrolidine or N-(2-chloroethyl)piperidine (1.1 equiv.) in DMF was added K2CO3 (4 equiv.) and KOH (4 equiv.). The reaction mixture was stirred for 45 min under microwave conditions. The reaction mixture was poured into water, and extracted with ethyl acetate. The extracts were dried (Na2SO4) and evaporated. The residue was purified by silica gel column chromatography (CC).

2-(4-(2-(Pyrrolidin-1-yl)ethoxy)phenyl)-5-(4-hydroxylphenyl)-selenophene 9a

Compound 9a was prepared with 2,5-bis(4-hydroxylphenyl)selenophene (8a) and N-(2-chloroethyl)pyrrolidine according to the general procedure for Williamson ether synthesis. Purification by CC (petroleum ether : ethyl acetate = 1 : 1) gave the title compound as a yellow solid (65% yield; mp 89–92 °C); 1H NMR (400 MHz, acetone-d6) δ 7.97 (dd, J = 10.2, 2.8 Hz, 2H), 7.07 (d, J = 8.8 Hz, 2H), 7.01–6.95 (m, 2H), 6.82 (d, J = 8.8 Hz, 2H), 6.72 (t, J = 10.1 Hz, 2H), 4.15–4.04 (m, 2H), 2.90 (t, J = 5.9 Hz, 2H), 2.65 (t, J = 6.0 Hz, 4H), 1.82–1.71 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 157.69, 156.76, 143.96, 143.58, 131.09, 130.26, 129.37, 129.29, 129.01, 127.71, 115.40, 114.48, 66.62, 54.65, 54.47, 23.54. HRMS (ESI) calcd. for C22H23NO2Se [M + H]+, 414.0972; found 414.0976.

2-(2-Methyl-4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-5-(2-methyl-4-hydroxylphenyl)selenophene 9b

Compound 9b was prepared with 2,5-bis(2-methyl-4-hydroxylphenyl)selenophene (8b) and N-(2-chloroethyl)pyrrolidine according to the general procedure for Williamson ether synthesis. Purification by CC (petroleum ether : ethyl acetate = 1 : 1) gave the title compound as a yellow solid (67% yield; mp 82–86 °C); 1H NMR (400 MHz, acetone-d6) δ 7.30 (d, J = 8.5 Hz, 1H), 7.22 (d, J = 8.3 Hz, 1H), 7.10 (dd, J = 7.9, 3.8 Hz, 2H), 6.85 (d, J = 2.4 Hz, 1H), 6.79–6.75 (m, 2H), 6.71 (dd, J = 8.3, 2.4 Hz, 1H), 4.14 (dd, J = 7.7, 4.2 Hz, 2H), 2.92–2.88 (m, 2H), 2.67–2.62 (m, 4H), 2.41 (s, 3H), 2.36 (s, 3H), 1.75 (dd, J = 6.8, 3.2 Hz, 4H). 13C NMR (101 MHz, acetone-d6) δ 159.31, 158.18, 150.15, 149.24, 137.57, 137.50, 132.38, 132.28, 129.58, 129.27, 129.02, 128.09, 118.43, 117.69, 114.01, 112.81, 67.68, 55.49, 55.15, 24.20, 21.80, 21.70. HRMS (ESI) calcd. for C24H27NO2Se [M + H]+, 442.1285; found 442.1286.

2-(2-Methyl-4-(2-(piperidin-1-yl)ethoxy)phenyl)-5-(2-methyl-4-hydroxylphenyl)selenophene 9c

Compound 9c was prepared with 2,5-bis(2-methyl-4-hydroxylphenyl)selenophene (8b) and N-(2-chloroethyl)piperidine according to the general procedure for Williamson ether synthesis. Purification by CC (petroleum ether : ethyl acetate = 1 : 1) gave the title compound as a yellow solid (65% yield; mp 156–158 °C); 1H NMR (400 MHz, acetone-d6) δ 7.30 (t, J = 6.8 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 7.12 (q, J = 3.8 Hz, 2H), 6.86 (d, J = 2.5 Hz, 1H), 6.79 (dt, J = 5.5, 2.8 Hz, 2H), 6.72 (dd, J = 8.3, 2.6 Hz, 1H), 4.12 (s, 2H), 2.72 (t, J = 6.0 Hz, 2H), 2.50 (s, 4H), 2.42 (s, 3H), 2.38 (s, 3H), 1.56 (dt, J = 10.9, 5.6 Hz, 4H), 1.42 (dd, J = 11.1, 6.0 Hz, 2H). 13C NMR (101 MHz, acetone-d6) δ 159.41, 158.01, 150.07, 149.31, 137.56, 136.73, 132.39, 132.26, 129.50, 129.26, 129.06, 128.23, 118.36, 117.70, 113.94, 112.86, 66.85, 58.69, 55.74, 26.76, 25.05, 21.77, 21.66. HRMS (ESI) calcd. for C25H29NO2Se [M + H]+, 454.1450; found 454.1452.

2-(2-Fluoro-4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-5-(2-fluoro-4-hydroxylphenyl)selenophene 9d

Compound 9d was prepared with 2,5-bis(2-fluoro-4-hydroxylphenyl)selenophene (8c) and N-(2-chloroethyl)pyrrolidine according to the general procedure for Williamson ether synthesis. Purification by CC (petroleum ether : ethyl acetate = 1 : 1) gave the title compound as a yellow solid (70% yield; mp 162–165 °C); 1H NMR (400 MHz, CD3OD) δ 7.65 (t, J = 8.7 Hz, 1H), 7.56 (dd, J = 5.5, 3.2 Hz, 2H), 7.52 (dd, J = 4.3, 1.6 Hz, 1H), 6.87 (dd, J = 8.0, 6.0 Hz, 2H), 6.65 (ddd, J = 15.5, 10.8, 2.5 Hz, 2H), 4.21 (t, J = 5.5 Hz, 2H), 3.05 (t, J = 5.4 Hz, 2H), 2.80 (t, J = 6.7 Hz, 4H), 1.95–1.85 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 159.33 (d, J = 247.1 Hz), 159.06 (d, J = 247.3 Hz), 141.32 (d, J = 29.0 Hz), 140.02 (d, J = 29.7 Hz), 129.17 (d, J = 12.1 Hz), 129.01 (d, J = 11.4 Hz), 127.35, 126.71, 116.05, 114.37, 113.07, 112.33, 103.67, 103.42, 103.04, 102.78, 67.84, 54.56, 54.42, 23.60. HRMS (ESI) calcd. for C22H21F2NO2Se [M + H]+, 450.0784; found 450.0781.

2-(2-Fluoro-4-(2-(piperidin-1-yl)ethoxy)phenyl)-5-(2-fluoro-4-hydroxylphenyl)selenophene 9e

Compound 9e was prepared with 2,5-bis(2-fluoro-4-hydroxylphenyl)selenophene (8c) and N-(2-chloroethyl)piperidine according to the general procedure for Williamson ether synthesis. Purification by CC (petroleum ether : ethyl acetate = 1 : 1) gave the title compound as a yellow solid (65% yield; mp 158–162 °C); 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 7.76–7.68 (m, 1H), 7.68–7.58 (m, 2H), 6.99 (dd, J = 13.6, 2.5 Hz, 1H), 6.86 (dd, J = 8.8, 2.4 Hz, 2H), 6.75–6.64 (m, 2H), 4.11 (t, J = 5.9 Hz, 2H), 2.65 (t, J = 5.8 Hz, 2H), 2.43 (s, 4H), 1.50 (dt, J = 10.8, 5.5 Hz, 4H), 1.38 (d, J = 5.1 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 159.28 (d, J = 247.2 Hz), 159.05 (d, J = 249.1 Hz), 141.30 (d, J = 31.4 Hz), 140.06 (d, J = 32.3 Hz), 129.16 (d, J = 11.3 Hz), 128.96 (d, J = 12.1 Hz), 127.32, 126.72, 116.04, 114.44, 113.06, 112.34, 103.67, 103.42, 103.06, 102.80, 66.65, 57.60, 54.79, 25.96, 24.33. HRMS (ESI) calcd. for C23H23F2NO2Se [M + H]+, 464.0940; found 464.0948.

2-(2-Chloro-4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-5-(2-chloro-4-hydroxylphenyl)selenophene 9f

Compound 9f was prepared with 2,5-bis(2-chloro-4-hydroxylphenyl)selenophene (8d) and N-(2-chloroethyl)pyrrolidine according to the general procedure for Williamson ether synthesis. Purification by CC (petroleum ether : ethyl acetate = 1 : 1) gave the title compound as a yellow solid (55% yield; mp 137–140 °C); 1H NMR (400 MHz, DMSO-d6) δ 7.56 (t, J = 7.0 Hz, 1H), 7.51–7.42 (m, 3H), 7.13 (d, J = 2.6 Hz, 1H), 6.98–6.93 (m, 2H), 6.82 (dt, J = 4.6, 2.3 Hz, 1H), 4.11 (t, J = 5.7 Hz, 2H), 2.81 (t, J = 5.8 Hz, 2H), 2.58–2.52 (m, 4H), 1.70–1.63 (m, 4H).13C NMR (101 MHz, DMSO-d6) δ 158.27, 157.95, 145.89, 144.80, 131.55, 131.36, 131.11, 130.92, 128.95, 128.53, 126.51, 124.72, 116.71, 115.84, 115.18, 114.37, 66.96, 53.90, 53.83, 23.05. HRMS (ESI) calcd. for C22H21Cl2NO2Se [M + H]+, 482.2830; found 482.2830.

2-(2-Chloro-4-(2-(piperidin-1-yl)ethoxy)phenyl)-5-(2-chloro-4-hydroxylphenyl)selenophene 9g

Compound 9g was prepared with 2,5-bis(2-fluoro-4-hydroxylphenyl)selenophene (8d) and N-(2-chloroethyl)piperidine according to the general procedure for Williamson ether synthesis. Purification by CC (petroleum ether : ethyl acetate = 1 : 1) gave the title compound as a yellow solid (65% yield; mp 92–95 °C); 1H NMR (400 MHz, DMSO-d6) δ 7.59 (d, J = 8.7 Hz, 1H), 7.54–7.45 (m, 3H), 7.16 (d, J = 2.5 Hz, 1H), 7.01–6.94 (m, 2H), 6.83 (dt, J = 11.9, 5.9 Hz, 1H), 4.15–4.04 (m, 2H), 2.65 (t, J = 5.8 Hz, 2H), 2.42 (s, 4H), 1.48 (dd, J = 10.8, 5.4 Hz, 4H), 1.37 (d, J = 4.8 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 158.40, 157.91, 145.84, 144.83, 131.60, 131.36, 131.09, 130.91, 128.97, 128.58, 126.43, 124.74, 116.68, 115.87, 115.18, 114.50, 66.10, 57.13, 54.28, 25.48, 23.84. HRMS (ESI) calcd. for C23H23Cl2NO2Se [M + H]+, 496.0349; found 496.0341.

3,4-Bis(2-chloro-4-methoxylphenyl)selenophene 10e

Compound 10e was prepared with 3,4-dibromoselenophene (6b) and 2-chloro-4-methoxylphenylboronic acid according to the general procedure for Suzuki coupling. Purification by CC (petroleum ether : ethyl acetate = 98 : 2) gave the title compound as a yellow solid; 1H NMR (400 MHz, CDCl3) δ 7.71 (s, 2H), 7.09 (t, J = 14.5 Hz, 2H), 6.74 (dt, J = 17.4, 14.8 Hz, 2H), 6.67–6.56 (m, 2H), 3.77–3.63 (m, 6H).

3,4-Bis(2-chloro-4-hydroxylphenyl)selenophene 11e

Compound 11e was prepared with 3,4-bis(2-chloro-4-methoxylphenyl)selenophene (10e) using boron tribromide according to the general procedure for ether cleavage. Purification by CC (petroleum ether : ethyl acetate = 4 : 1) gave the title compound as a yellow solid; 1H NMR (400 MHz, acetone-d6) δ 8.00 (d, J = 4.9 Hz, 2H), 7.01–6.93 (m, 2H), 6.82 (d, J = 2.5 Hz, 2H), 6.71–6.63 (m, 2H).

3-(4-(2-(Pyrrolidin-1-yl)ethoxy)phenyl)-4-(4-hydroxylphenyl)-selenophene 12a

Compound 12a was prepared with 3,4-bis(4-hydroxylphenyl)selenophene (11a) and N-(2-chloroethyl)pyrrolidine according to the general procedure for Williamson ether synthesis. Purification by CC (petroleum ether : ethyl acetate = 1 : 2) gave the title compound as a yellow solid (65% yield; mp 89–92 °C); 1H NMR (400 MHz, acetone-d6) δ 7.96 (dd, J = 10.2, 2.8 Hz, 2H), 7.06 (d, J = 8.8 Hz, 2H), 7.00–6.94 (m, 2H), 6.81 (d, J = 8.8 Hz, 2H), 6.71 (t, J = 10.1 Hz, 2H), 4.14–4.03 (m, 2H), 2.89 (t, J = 5.9 Hz, 2H), 2.64 (t, J = 6.0 Hz, 4H), 1.81–1.70 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 157.62, 156.69, 143.89, 143.51, 131.02, 130.19, 129.30, 129.22, 128.94, 127.44, 115.33, 114.41, 66.55, 54.58, 54.40, 23.47. HRMS (ESI) calcd. for C22H23NO2Se [M + H]+, 414.0972; found 414.0970.

3-(4-(2-(Piperidin-1-yl)ethoxy)phenyl)-4-(4-hydroxylphenyl)-selenophene 12b

Compound 12b was prepared with 3,4-bis(4-hydroxylphenyl)selenophene (11a) and N-(2-chloroethyl)piperidine according to the general procedure for Williamson ether synthesis. Purification by CC (petroleum ether : ethyl acetate = 1 : 2) gave the title compound as a yellow solid (72% yield; mp 159–163 °C); 1H NMR (400 MHz, acetone-d6) δ 8.01–7.90 (m, 1H), 7.98–7.93 (m, 1H), 7.08–7.04 (m, 1H), 7.09–6.92 (m, 2H), 6.99–6.94 (m, 1H), 6.83–6.78 (m, 1H), 6.75 (ddd, J = 11.3, 6.7, 2.3 Hz, 2H), 6.74–6.70 (m, 1H), 4.06 (t, J = 6.0 Hz, 2H), 2.72–2.66 (m, 2H), 2.48 (s, 4H), 1.54 (dt, J = 10.9, 5.5 Hz, 4H), 1.41 (d, J = 2.5 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 157.70, 156.63, 143.83, 143.47, 130.84, 130.13, 130.10, 129.18, 129.16, 128.85, 115.27, 114.36, 65.84, 57.80, 54.79, 25.94, 24.31. HRMS (ESI) calcd. for C23H25NO2Se [M + H]+, 428.1129; found 428.1120.

3-(2-Methyl-4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-4-(2-methyl-4-hydroxylphenyl)selenophene 12c

Compound 12c was prepared with 3,4-bis(2-methyl-4-hydroxylphenyl)selenophene (11b) and N-(2-chloroethyl)pyrrolidine according to the general procedure for Williamson ether synthesis. Purification by CC (petroleum ether : ethyl acetate = 1 : 2) gave the title compound as a yellow solid (67% yield; mp 97–101 °C); 1H NMR (400 MHz, acetone-d6) δ 7.90 (dd, J = 7.9, 2.8 Hz, 2H), 6.98 (dd, J = 11.8, 1.2 Hz, 2H), 6.86 (dd, J = 8.4, 1.8 Hz, 1H), 6.70 (dt, J = 18.3, 8.3 Hz, 3H), 4.09 (t, J = 5.8 Hz, 2H), 2.93 (t, J = 5.7 Hz, 2H), 2.67 (s, 4H), 2.11 (d, J = 4.7 Hz, 6H), 1.78–1.73 (m, 4H). 13C NMR (101 MHz, acetone-d6) δ 159.22, 158.08, 150.06, 149.15, 137.47, 137.41, 132.29, 132.19, 129.49, 129.17, 128.92, 128.00, 118.33, 117.60, 113.92, 112.71, 55.40, 55.06, 29.84, 24.11, 21.71, 21.60. HRMS (ESI) calcd. for C24H27NO2Se [M + H]+, 442.1285; found 442.1283.

3-(2-Methyl-4-(2-(piperidin-1-yl)ethoxy)phenyl)-4-(2-methyl-4-hydroxylphenyl)selenophene 12d

Compound 12d was prepared with 3,4-bis(2-methyl-4-hydroxylphenyl)selenophene (11b) and N-(2-chloroethyl)piperidine according to the general procedure for Williamson ether synthesis. Purification by CC (petroleum ether : ethyl acetate = 1 : 2) gave the title compound as a yellow solid (55% yield; mp 91–95 °C); 1H NMR (400 MHz, acetone-d6) δ 7.93 (d, J = 2.8 Hz, 1H), 7.91 (d, J = 2.8 Hz, 1H), 7.00 (d, J = 1.6 Hz, 1H), 6.97 (s, 1H), 6.86 (dd, J = 8.4, 2.0 Hz, 1H), 6.75 (d, J = 8.4 Hz, 1H), 6.70 (d, J = 2.0 Hz, 2H), 4.15 (t, J = 5.7 Hz, 2H), 2.94–2.87 (m, 2H), 2.70 (s, 4H), 2.16–2.08 (m, 6H), 1.65–1.60 (m, 4H), 1.49–1.42 (m, 2H). 13C NMR (101 MHz, acetone-d6) δ 159.38, 158.17, 150.11, 149.25, 137.56, 137.52, 132.38, 132.26, 129.51, 129.27, 129.04, 128.11, 118.39, 117.67, 113.98, 112.86, 66.74, 58.58, 55.65, 26.66, 24.96, 21.74, 21.64. HRMS (ESI) calcd. for C25H29NO2Se [M + H]+, 454.1450; found 454.1455.

3-(3-Methyl-4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-4-(3-methyl-4-hydroxylphenyl)selenophene 12e

Compound 12e was prepared with 3,4-bis(3-methyl-4-hydroxylphenyl)selenophene (11c) and N-(2-chloroethyl)pyrrolidine according to the general procedure for Williamson ether synthesis. Purification by CC (petroleum ether : ethyl acetate = 1 : 2) gave the title compound as a yellow solid (72% yield; mp 84–88 °C); 1H NMR (400 MHz, CDCl3) δ 7.73 (d, J = 4.8 Hz, 2H), 7.73 (d, J = 4.8 Hz, 1H), 6.89–6.76 (m, 1H), 6.52 (dd, J = 8.1, 5.4 Hz, 2H), 6.48–6.42 (m, 2H), 4.01 (t, J = 5.6 Hz, 2H), 2.96 (t, J = 5.6 Hz, 2H), 2.76 (d, J = 20.0 Hz, 4H), 2.01 (s, 3H), 1.96 (s, 3H), 1.84 (s, 4H). 13C NMR (101 MHz, acetone-d6) δ 156.86, 155.31, 145.22, 144.92, 132.27, 132.02, 131.61, 130.62, 128.48, 128.32, 128.27, 128.25, 126.49, 124.54, 114.88, 111.19, 68.03, 55.53, 55.26, 24.22, 16.55, 16.31. HRMS (ESI) calcd. for C24H27NO2Se [M + H]+, 442.1285; found 442.1283.

3-(3-Methyl-4-(2-(piperidin-1-yl)ethoxy)phenyl)-4-(3-methyl-4-hydroxylphenyl)selenophene 12f

Compound 12f was prepared with 3,4-bis(3-methyl-4-hydroxylphenyl)selenophene (11c) and N-(2-chloroethyl)piperidine according to the general procedure for Williamson ether synthesis. Purification by CC (petroleum ether : ethyl acetate = 1 : 2) gave the title compound as a yellow solid (65% yield; mp 85–89 °C); 1H NMR (400 MHz, acetone-d6) δ 7.92 (dd, J = 9.9, 2.8 Hz, 2H), 7.02–6.95 (m, 2H), 6.86 (dd, J = 8.4, 2.0 Hz, 1H), 6.75 (d, J = 8.4 Hz, 1H), 6.70 (d, J = 2.0 Hz, 2H), 4.15 (t, J = 5.7 Hz, 2H), 2.92 (dd, J = 10.9, 5.3 Hz, 2H), 2.70 (s, 4H), 2.15–2.08 (m, 6H), 1.62 (dt, J = 11.0, 5.6 Hz, 4H), 1.49–1.39 (m, 2H). 13C NMR (101 MHz, acetone-d6) δ 159.39, 158.19, 150.12, 149.26, 137.58, 137.53, 132.39, 132.27, 129.52, 129.28, 129.06, 128.12, 118.41, 117.68, 113.99, 112.87, 66.75, 58.59, 55.66, 26.67, 24.97, 21.75, 21.65. HRMS (ESI) calcd. for C25H29NO2Se [M + H]+, 454.1450; found 454.1448.

3-(3-Fluoro-4-(2-(piperidin-1-yl)ethoxy)phenyl)-4-(3-fluoro-4-hydroxylphenyl)selenophene 12g

Compound 12g was prepared with 3,4-bis(3-fluoro-4-hydroxylphenyl)selenophene (11d) and N-(2-chloroethyl)piperidine according to the general procedure for Williamson ether synthesis. Purification by CC (petroleum ether : ethyl acetate = 1 : 2) gave the title compound as a yellow solid (74% yield; mp 89–93 °C); 1H NMR (400 MHz, DMSO-d6) δ 7.63 (t, J = 9.1 Hz, 1H), 7.59–7.55 (m, 2H), 7.54–7.51 (m, 1H), 6.90 (dd, J = 13.6, 2.5 Hz, 1H), 6.78 (dd, J = 8.8, 2.4 Hz, 1H), 6.65–6.58 (m, 2H), 4.03 (t, J = 5.9 Hz, 2H), 2.57 (t, J = 5.8 Hz, 2H), 2.34 (s, 4H), 1.41 (dt, J = 10.8, 5.5 Hz, 4H), 1.30 (d, J = 5.1 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 159.22 (d, J = 241.9 Hz), 158.99 (d, J = 233.1 Hz), 141.24 (d, J = 25.6 Hz), 140.00 (d, J = 27.4 Hz), 129.10 (d, J = 14.0 Hz), 128.90 (d, J = 15.9 Hz), 127.26, 126.66, 115.98, 114.38, 113.00, 112.28, 103.61, 103.36, 103.00, 102.74, 66.59, 57.54, 54.73, 25.90, 24.27. HRMS (ESI) calcd. for C23H23F2NO2Se [M + H]+, 462.0948; found 462.0942.

3-(2-Chloro-4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-4-(2-chloro-4-hydroxylphenyl)selenophene 12h

Compound 12h was prepared with 3,4-bis(2-chloro-4-hydroxylphenyl)selenophene (11e) and N-(2-chloroethyl)pyrrolidine according to the general procedure for Williamson ether synthesis. Purification by CC (petroleum ether : ethyl acetate = 1 : 2) gave the title compound as a yellow solid (73% yield; mp 79–83 °C); 1H NMR (400 MHz, CD3OD) δ 7.82 (dt, J = 8.3, 4.1 Hz, 2H), 6.93 (dd, J = 8.4, 3.3 Hz, 1H), 6.83 (ddd, J = 6.4, 5.5, 1.7 Hz, 1H), 6.66 (dd, J = 8.6, 2.6 Hz, 2H), 6.59 (t, J = 2.3 Hz, 1H), 6.44 (dd, J = 8.4, 2.5 Hz, 1H), 4.13–4.08 (m, 2H), 3.31–3.27 (m, 2H), 3.12 (dd, J = 12.4, 5.9 Hz, 4H), 1.90 (td, J = 6.8, 2.9 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) δ 158.00, 157.36, 141.15, 140.89, 132.98, 132.60, 132.36, 132.30, 130.16, 130.03, 128.76, 126.85, 115.66, 114.69, 113.84, 113.02, 66.83, 59.73, 53.89, 23.06. HRMS (ESI) calcd. for C22H21Cl2NO2Se [M + H]+, 482.0193; found 482.0190.

3-(2-Chloro-4-(2-(piperidin-1-yl)ethoxy)phenyl)-4-(2-chloro-4-hydroxylphenyl)selenophene 12i

Compound 12i was prepared with 3,4-bis(2-chloro-4-hydroxylphenyl)selenophene (11e) and N-(2-chloroethyl)piperidine according to the general procedure for Williamson ether synthesis. Purification by CC (petroleum ether : ethyl acetate = 1 : 2) gave the title compound as a yellow solid (70% yield; mp 109–113 °C); 1H NMR (400 MHz, CD3OD) δ 7.93–7.89 (m, 2H), 7.03–6.99 (m, 1H), 6.91 (dt, J = 6.6, 3.3 Hz, 2H), 6.71 (dd, J = 6.3, 4.2 Hz, 2H), 6.54 (dd, J = 8.4, 2.3 Hz, 1H), 4.12 (t, J = 5.3 Hz, 2H), 2.97 (t, J = 5.2 Hz, 2H), 2.76 (s, 4H), 1.68 (dd, J = 10.9, 5.4 Hz, 4H), 1.53 (d, J = 4.7 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 158.06, 157.30, 141.14, 140.90, 132.97, 132.60, 132.36, 132.26, 130.16, 130.03, 128.72, 126.90, 115.65, 114.75, 113.83, 113.08, 65.81, 57.22, 54.30, 25.47, 23.85. HRMS (ESI) calcd. for C23H23Cl2NO2Se [M + H]+, 496.0349; found 496.0346.

3-(4-Hydroxylphenyl)-2,5-bis(4-methoxylphenyl)selenophene 14a

Compound 14a was prepared with 3-bromo-2,5-bis(4-methoxylphenyl)selenophene (13a) and 4-hydroxylphenylboronic acid according to the general procedure for Suzuki coupling. Purification by CC (petroleum ether : ethyl acetate = 30 : 1) gave the title compound as a yellow solid; 1H NMR (400 MHz, CDCl3) δ 7.51–7.48 (m, 1H), 7.51–7.48 (m, 1H), 7.42–7.40 (m, 1H), 7.22–7.16 (m, 4H), 6.93–6.89 (m, 2H), 6.81–6.72 (m, 4H), 3.84 (s, 3H), 3.79 (s, 3H).

3-(4-Hydroxylphenyl)-2,5-bis(2-methyl-4-methoxylphenyl)-selenophene 14b

Compound 14b was prepared with 3-bromo-2,5-bis(2-methyl-4-methoxylphenyl)selenophene (13b) and 4-hydroxylphenylboronic acid according to the general procedure for Suzuki coupling. Purification by CC (petroleum ether : ethyl acetate = 30 : 1) gave the title compound as a yellow solid; 1H NMR (400 MHz, CDCl3) δ 7.48–7.33 (m, 3H), 7.21–7.16 (m, 2H), 6.88 (d, J = 2.6 Hz, 1H), 6.86–6.73 (m, 5H), 3.86–3.84 (t, J = 8.9 Hz, 6H), 2.53 (d, J = 8.1 Hz, 3H), 2.08 (d, J = 13.6 Hz, 3H).

3-(4-(2-(Pyrrolidin-1-yl)ethoxy)phenyl)-2,5-bis(4-hydroxylphenyl)selenophene 16a

Compound 16a was obtained from the demethylation of compound 15a using boron tribromide; the latter was prepared with 3-(4-hydroxylphenyl)-2,5-bis(4-methoxylphenyl)selenophene (14a) and N-(2-chloroethyl)pyrrolidine according to the general procedure for Williamson ether synthesis. Purification by CC (dichloromethane : ethyl acetate = 2 : 3) gave the title compound 16a as a yellow solid (56% yield; mp 105–109 °C); 1H NMR (400 MHz, DMSO-d6) δ 9.79 (d, J = 23.2 Hz, 2H), 7.57–7.47 (m, 3H), 7.31–7.27 (m, 2H), 7.10–7.07 (m, 4H), 6.99 (d, J = 8.8 Hz, 1H), 6.85 (t, J = 8.0 Hz, 1H), 6.78–6.71 (m, 2H), 4.36 (t, J = 4.9 Hz, 2H), 3.53 (d, J = 19.2 Hz, 2H), 3.33 (d, J = 3.5 Hz, 4H), 1.97 (d, J = 3.5 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) δ 157.93, 157.40, 156.97, 147.39, 141.56, 139.30, 130.91, 130.48, 130.43, 127.99, 127.42, 127.14, 126.96, 116.32, 116.01, 114.98, 63.93, 54.19, 53.30, 23.09. HRMS (ESI) calcd. for C28H27NO3Se [M + H]+, 506.1234; found 506.1231.

3-(4-(2-(Piperidin-1-yl)ethoxy)phenyl)-2,5-bis(4-hydroxylphenyl)selenophene 16b

Compound 16b was obtained from the demethylation of compound 15b using boron tribromide; the latter was prepared with 3-(4-hydroxylphenyl)-2,5-bis(4-methoxylphenyl)selenophene (14a) and N-(2-chloroethyl)pyrrolidine according to the general procedure for Williamson ether synthesis. Purification by CC (dichloromethane : ethyl acetate = 2 : 3) gave the title compound 16b as a yellow solid (62% yield; mp 142–146 °C); 1H NMR (400 MHz, DMSO-d6) δ 7.52–7.42 (m, 3H), 7.21 (d, J = 8.7 Hz, 2H), 7.04 (d, J = 8.6 Hz, 2H), 6.84 (dd, J = 19.9, 8.7 Hz, 2H), 6.70 (d, J = 8.6 Hz, 2H), 4.09 (t, J = 5.6 Hz, 2H), 2.80 (t, J = 5.3 Hz, 2H), 2.55 (d, J = 19.8 Hz, 4H), 1.54 (dt, J = 10.8, 5.5 Hz, 4H), 1.40 (d, J = 4.4 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 157.92, 157.62, 157.36, 147.33, 141.43, 139.39, 130.41, 130.34, 128.15, 128.03, 127.40, 127.21, 127.06, 116.32, 115.97, 114.80, 65.28, 57.39, 54.54, 25.41, 23.86. HRMS (ESI) calcd. for C29H29NO3Se [M + H]+, 520.1391; found 520.1398.

3-(4-(2-(Pyrrolidin-1-yl)ethoxy)phenyl)-2,5-bis(2-methyl-4-hydroxylphenyl)selenophene 16c

Compound 16c was obtained from the demethylation of compound 15c using boron tribromide; the latter was prepared with 3-(4-hydroxylphenyl)-2,5-bis(4-methoxylphenyl)selenophene (14b) and N-(2-chloroethyl)pyrrolidine according to the general procedure for Williamson ether synthesis. Purification by CC (dichloromethane : ethyl acetate = 2 : 3) gave the title compound 16c as a yellow solid (52% yield; mp 89–93 °C); 1H NMR (400 MHz, DMSO-d6) δ 9.78 (s, 1H), 9.63 (s, 1H), 7.47 (d, J = 2.0 Hz, 1H), 7.33 (d, J = 1.9 Hz, 1H), 7.28 (dd, J = 8.2, 2.3 Hz, 1H), 7.24–7.12 (m, 2H), 6.96 (d, J = 1.8 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.73 (dd, J = 8.3, 2.2 Hz, 1H), 6.63 (d, J = 8.4 Hz, 1H), 4.45–4.33 (m, 2H), 3.50 (d, J = 38.9 Hz, 2H), 3.38 (s, 4H), 2.17 (s, 3H), 2.01 (d, J = 11.8 Hz, 3H), 1.93 (d, J = 4.9 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) δ 156.40, 155.93, 153.82, 145.42, 141.29, 137.38, 135.30, 132.37, 131.94, 131.86, 131.66, 128.17, 127.54, 125.84, 125.46, 124.78, 124.65, 115.17, 115.03, 113.62, 110.61, 110.42, 65.65, 54.98, 53.45, 23.12, 16.46, 16.43. HRMS (ESI) calcd. for C30H31NO3Se [M + H]+, 534.1537; found 534.1539.

4.2. Gene clone and protein purification

Human ERα or ERβ ligand binding domain (LBD) genes were amplified by PCR from plasmid pVP-16-ERα and pVP-16-ERβ. The PCR product was cloned into a plasmid, and PGEx-KG E. coli BL21 (DE3) was used for the overexpression of ER-LBD. The cells were induced by IPTG (10 μM) for 2 h, then the cells were harvested, frozen, and thawed in phosphate-buffered saline (PBS), containing 1 mM EDTA and 1 mM DTT. After being ultrasonicated in an icy bath, the supernatant was loaded into a column of GSH-resin. The collection was dialyzed in ice buffer for 4 h. After being checked by a combination of sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting, the protein was prepared as a 10 mM stock in potassium phosphate and stored at –80 °C.45

4.3. Estrogen receptor binding affinity

Relative binding affinities were determined by a competitive fluorometric binding assay as previously described. Briefly, 40 nM of a fluorescence tracer (coumestrol, Sigma-Aldrich, MO) and 0.8 μM purified human ERα or ERβ ligand binding domain (LBD) were diluted in 100 mM potassium phosphate buffer (pH 7.4), containing 100 μg mL–1 bovine gamma globulin (Sigma-Aldrich, MO). Incubations were for 2 h at room temperature (25 °C). Fluorescence polarization values were then measured. The binding affinities are expressed as relative binding affinity (RBA) values with the RBA of 17β-estradiol set to 100%. The values given are the average ± range of two independent determinations. IC50 values were calculated according to equations described previously.46

4.4. Gene transcriptional activity

The human embryonic kidney cell line HEK 293T was maintained in Dulbecco's Minimum Essential Medium (DMEM) (Gibco by Invitrogen Corp., CA) with 10% fetal bovine serum (FBS) (Hylcone by Thermo Scientific, UT). Cells were plated in phenol red-free DMEM with 10% FBS. HEK 293T cells were transfected with 25 μL of the mixture per well, containing 300 ng of the 3 × ERE-luciferase reporter, 100 ng of the ERα or ERβ expression vector, 125 mM calcium chloride (GuoYao, China) and 12.5 μL 2 × HBS. On the next day, the cells were treated with increasing doses of ER ligands diluted in phenol red-free DMEM with 10% FBS. After 24 h, luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega, MI) according to the manufacturer's protocol.

4.5. Cell culture and cell viability assay

The human breast cancer cell line MCF-7 was obtained from ATCC. Cells were maintained in DMEM with 10% FBS. For all experiments, cells were grown in 96-well microtiter plates (Nest Biotech Co., China) with appropriate ligand triplicates for 72 h. MTT colormetric tests (Biosharp, China) were employed to determine cell viability per manufacturer instructions. IC50 values were calculated according to the following equation using Origin software: Y = 100% inhibition + (0% inhibition – 100% inhibition)/(1 + 10[(logIC50–X)×Hillslope]), where Y = fluorescence value, X = log[inhibitor].46

4.6. Molecular modeling

Crystal structures of ER LBD in complex with raloxifene were downloaded from the Protein Data Bank (PDB ID: 3ERR). Compounds 11c, 12e and 16c were docked into the three-dimensional structure of ERα LBD with AutoDock software (version 4.2).44,45 Crystallographic coordinates of 11c, 12e and 16c were created by Biochemoffice. The crystal structure of ERα LBD (PDB ID: ; 3ERD) was obtained from the PDB, and all water molecules were removed. Preparations of all ligands and the protein were performed with AutoDockTools (ADT). A docking cube with edges of 60 Å, 60 Å, and 58 Å in the X, Y, and Z dimensions, respectively (a grid spacing of 0.375 Å), which encompassed the whole active site, was used throughout the docking. On the basis of the Lamarckian genetic algorithm (LGA), 80 runs were performed for each ligand with 500 individuals in the population.47,48 The figures were prepared using PyMOL.

Conflict of interest

The authors declare no competing interests.

Supplementary Material

Click here for additional data file. (1.7MB, pdf)

Acknowledgments

We are grateful to the NSFC (81573279, 81373255), the Major Project of Technology Innovation Program of Hubei Province (2016ACA126), Hubei Province's Outstanding Medical Academic Leader Program, the Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, and the Fundamental Research Funds for the Central Universities of China (2015306020201) for support of this research. We thank Dr. Chu Tang for help with the molecular modeling.

Footnotes

†Electronic supplementary information (ESI) available: 1H NMR and 13C NMR spectra of final compounds. See DOI: 10.1039/c7md00163k

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