Design, synthesis and biological evaluation of novel 2-methoxyestradiol analogs as dual selective estrogen receptor modulators (SERMs) and antiangiogenic agents

European Journal of Medicinal Chemistry 139 (2017) 390e400

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Design, synthesis and biological evaluation of novel 2-methoxyestradiol analogs as dual selective estrogen receptor modulators (SERMs) and antiangiogenic agents Kejing Lao a, c, 1, Yejun Wang b, c, 1, Mingqi Chen d, Jingjing Zhang e, Qidong You b, c, Hua Xiang b, c, * a

Institute of Basic and Translational Medicine, and School of Basic Medical Science, Xi'an Medical University, No.1 Xinwang Road, Xi'an, 710021, PR China Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, PR China Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, PR China d Laboratory of Biology, School of Higher Vocational Education, China Pharmaceutical University, 639 Longmian Avenue, Nanjing, 211198, PR China e Jiangning Hospital Affiliated to Nanjing Medical University, Gushan Road 168, Nanjing, 211100, PR China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 May 2017 Received in revised form 3 August 2017 Accepted 4 August 2017 Available online 7 August 2017

2-methoxyestradiol is a novel agent showing both anti-angiogenic and vascular disrupting properties. In this study, a series of 11a-substituted 2-methoxyestradiol analogs have been designed and synthesized targeting dual ERa and microtubulin. Biological evaluation was performed on their anti-proliferative activities against 5 different cell lines. The results indicated that most compounds exhibited good activities, in which compound 24c and 30c showed the best activity with low micromolar IC50 (2.73 mM
7.75 mM) in all cell lines. The investigation of ER affinity showed that the majority of the compounds displayed good activity at the concentration of 50 mM. In further mechanism study, it was observed that 24c and 30c could induce G2/M cell cycle arrest as well as significant anti-estrogenic activity. In CAM assay, compound 24c and 30c presented significantly anti-angiogenesis activity comparable with 2-methoxyestradiol. Overall, based on biological activities data, 24c and 30c can be identified as a potential lead molecule which might be of therapeutic importance for cancer treatment. © 2017 Elsevier Masson SAS. All rights reserved.

Keywords: 2-Methoxyestradiol Estrogen receptor Antiangiogenesis Steroids

1. Introduction 2-methoxyestradiol (2-ME2) is a novel agent targeting tumor vasculature, showing both anti-angiogenic and vascular disrupting properties. 2-ME2, a metabolite of 17b-estradiol (E2, 1), is produced by sequential hydroxylation and O-methylation at its 2-position. 17b-estradiol is firstly converted to 2-hydroxyestradiol catalyzed by CYP1A2 and CYP3A enzymes in liver. Further, the methylation of 2hydroxyestradiol occurs by the catechol-O-methyltransferase (COMT) producing 2-ME2(2) (Fig. 1) [1]. As a natural metabolite of endogenous estrogen, 2-ME2 is devoid of estrogenic activity and shows strong growth-inhibitory effect on the various cancers due to its antiproliferative,

* Corresponding author. Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, PR China. E-mail address: [email protected] (H. Xiang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ejmech.2017.08.016 0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

proapoptotic, antiangiogenic, antitubulin and antimetastatic effects [2]. It has been reported that 2-ME2 inhibits tumor growth and angiogenesis at concentrations that efficiently disrupt tumor microtubules. 2-ME2 has been shown to bind to the colchicine binding site of tubulin and to depolymerize microtubules in endothelial as well as in tumor cells, resulting in mitotic arrest and cell death [3e5]. It has been suggested that this effect is associated with the ability to induce G2/M cell cycle arrest [6]. Presently, there are several clinical trials of 2-ME2 under way in the United States [7,8]. Although 2-ME2 is found with a range of pharmacological actions that confer on it unique potential as an anti-tumour agent, its bioavailability remains one of the main problems as a good anticancer drug [9]. During this decade, several derivatives have been reported with modifications on ring A, B and D of 2-ME2. Several analogues such as ENMD-1198 and ENMD-2076 have been prepared with better bioavailability. These analogues are also under clinical trials (Fig. 2) [10,11]. Selective estrogen receptor modulators (SERMs) are structurally diverse synthetic ligands which act as antagonists in breast tissue

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Fig. 1. The biological synthesis of 2-ME2.

Fig. 2. Structures of 2-ME2 and its analogs.

but agonists in other tissues such as cardiovascular system and bone [12,13]. Tamoxifen is the first marketed SERM and is currently widely used in the treatment and prevention of breast cancer (BC). Although SERMs have shown great benefits in treating ERa positive BC, they still have disadvantages. For example, long term treatment of Tamoxifen increases the occurrence of endometrial cancer [14]. And another common deficiency that limits the use of SERMs is intrinsic and acquired drug resistance, in which breast tumors become refractory to endocrine therapies and relapse [15,16]. Therefore, the exploration of developing new SERMs with increased activity and fewer side effects still draws much attention. Recently, several experimental investigations demonstrated that the combination of 2-ME2 with other tumor-suppressing agents brought about an additive or synergistic inhibition of cell proliferation. It is reported that the combination of 2-ME2 and 4OH TAM was able to elicit a synergistic action on the antiproliferation of MCF-7 cells [2,17]. ER ligands could be assembled in a modular fashion by combining multiple components around a structurally simple and synthetically accessible core that occupies a space in the ligand binding pocket having essentially no contact with the protein [18]. Despite of the very faint ER affinity of 2-ME2, its estrogen skeleton could be taken as the core for novel SERMs. Several steroidal SERMs were developed by placing aryl group at the 11b-position of 17bestradiol (Fig. 3) [19,20]. It suggested that the flexible side chain with tertiary amine substituent in the end is critical since it leads to the formation of ER antagonist conformation when binding to ER by preventing the movement of helix 12 [21]. Thus, we designed and synthesized a series of novel antiangiogenic agents by involving an aromatic basic side chain at the C ring of 2-ME2. It was expected for these compounds with dual targets to gain more satisfactory effects for anti-breast cancer with fewer side effects. They were evaluated for their anti-proliferative and ER affinity. Besides, two selected compounds were further

investigated for their mechanism and anti-angiogenetic effect in vivo. 2. Chemistry In our previous study, an efficient and practical scheme to synthesize 2-ME2 has been developed [22]. Taking E2 (1) as starting material, 2-ME2 can be got with 61% overall yield (Scheme 1). The key step was the copper-mediated methoxylation using ethyl acetate as a co-catalyst to introduce 2-methoxyl group. By taking E2(1) or 2-ME2(2) as starting material, compound 17a~17c and 24a~24c were synthesized by 7 steps (Scheme 2). Compound 11 and 18 were afforded by the oxidation of E2(1) or 2ME2(2) under DDQ. Being protected by CH3I at 3-OH and 11a-OH, compound 13 and 20 were got by hydroboration. After connected with 4-nitrobenzoyl chloride, compound 14 and 21 were subjected to hydrogenolysis using 10% Pd/C and NH4OAc. The key intermediate 16 and 23 were given by taking chloroacetyl chloride as the

Scheme 1. The synthesis of 2-methoxyestradiol.

Fig. 3. Structures of steroidal SERMs.

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K. Lao et al. / European Journal of Medicinal Chemistry 139 (2017) 390e400

Scheme 2. The synthesis of compound 17a~17c and 24a~24c.

acylation reagent. The targeting compounds 17a~17c and 24a~24c were eventually obtained by substituted with different amide respectively. Compound 30a~30c were synthesized under similar condition with the only difference of taking MOM as the protection group (Scheme 3). Compound 31a~31c were then obtained by deprotection under HCl/MeOH.

3. Results and discussion To investigate their anti-cancer effects, all the synthesized compounds were tested for their anti-proliferative activities in different cell lines. The anti-proliferative activities were firstly evaluated in human breast cancer cell MCF-7 by taking tamoxifen and 2-ME2 as positive control. As shown in Table 1, most compounds displayed good activity with the IC50 less than 50 mM. Among them, compounds with 3, 17-dihydroxy substitution (compounds 31a, 31b and 31c) were obviously inferior to the others, which proved that hydrophobic groups at 3-OH was beneficial for the anti-proliferative activity. The compounds with 3-OH protected by MOM (compounds 30a, 30b and 30c) displayed better anti-proliferative activities than those protected by methyl (compounds 17a, 17b and 17c). It is suggested that -OMOM could slightly enhance the cytotoxic potential. Moreover, it is obviously that increasing of the steric hindrance of R3 led to better inhibitory activity. The compounds with 4methylpiperazine substitution showed better inhibitory activities

than others, in which, the most potential compounds 24c and 30c, with low micromolar IC50 (4.33 mM and 4.06 mM), were better than the positive control 2-ME2. The anti-proliferative activity of these compounds was then evaluated in human endometrial adenocarcinoma cell Ishikawa considering the fact that the occurrence of endometrial cancer is one of the side effects of tamoxifen. As shown in Table 1, most of the compounds displayed anti-proliferative activity comparable with 2-ME2. At low concentration of 10 mM, all of the synthesized compounds inhibited the growth of Ishikawa while tamoxifen exhibited a proliferative effect of 60.08% (data not shown). To investigate if there were non-estrogen signaling pathway attributed to the anti-proliferative effects, the growth inhibitory activity was evaluated in 3 other cell lines including human umbilical vein endothelial cells HUVEC, human liver carcinoma cells HepG2 and human prostate adenocarcinoma cells PC-3 (Table 1). Most of the synthesized compounds exhibited comparative activities with positive control 2-ME2. Consistent with the trend observed in MCF-7 and Ishikawa, 3, 17-dihydroxy analogs (compounds 31a, 31b and 31c) still presented the growth inhibitory activity inferior to the others. The ERa binding affinities of synthesized compounds were assessed by following a fluorescence polarization procedure. Tamoxifen was used as positive control. As shown in Fig. 4, at the concentration of 10 mM, the majority of compounds presented the affinity rate higher than 50% only except compound 17c. Several compounds, 24b, 24c and 30c exhibited good binding affinities

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Scheme 3. Synthesis of compound 30a~30c and 31a~31c.

comparable with the positive control. The high affinity observed in compound 24c and 30c was consisted with their good antiproliferative activities toward the ER positive cell MCF-7. Thus, compounds 24c and 30c were chosen for the further mechanism study. It has been reported that 2ME-2 is able to inducing G2/M cell cycle arrest, which is associated with the ability to interact with microtubules disrupting normal microtubule functions. Thus, the effects of 24c and 30c on cell cycle changes were evaluated in MCF7 cell lines by flow cytometric analysis. As shown in Fig. 5, 24c and 30c significantly increased the combined G2/M cell populations in a dose-response manner at 1 mM and 2 mM. Time-course experiments showed that the induced G2/M arrest peaked around 6 h after treatment. Although slightly effective than 24c at 6 h, the G2/M inducement effect of 30c could hardly be observed at 24 h, which might be associated with the fact that 3-OMOM substitution was more susceptible to hydrolysis. To further assess the mechanism of compounds 24c and 30c associated with ER, we used real-time polymerase chain reaction (RT-PCR) in the ER positive MCF-7 cells to evaluate the modulation of progesterone receptor (PgR). The progesterone receptor expression is commonly used to assess estrogenic or antiestrogenic activity. As shown in Fig. 6, presence of 10 nM E2 was able to remarkably elevate the mRNA expression of PgR gene compared to the vehicle control. The mRNA expression level of PgR with 10 nM E2 was regard as 100%. Tamoxifen was used as the positive control at the concentration of 2 mM in combination with 10 nM E2 and could reduce the expression of PgR mRNA by about 80%. Compounds 24c and 30c exhibited strong antagonism against the expression of PgR mRNA in a dose-response manner at the concentration of 0.5 mM, 1 mM and 2 mM in the presence of 10 nM E2

comparable to tamoxifen, indicating that 24c and 30c presented significantly anti-estrogenic bioactivity. In order to exclude unspecific effects, the formation of AIM2 mRNA, which is independent from ER pathway was also measured as a reference. As shown in Fig. 6, No effect on AIM2 mRNA expression in MCF-7 cells was observed by treated with neither our synthesized compounds nor tamoxifen. Based on the preliminary results, compounds 24c and 30c were selected to perform chicken chorioallantoic membrane (CAM) assay to investigate their inhibition of angiogenesis in vivo. Test compounds and the positive control Sunitinib dissolved in DMSO were placed on sterile methyl cellulose filter papers at 1 mM, 10 mM and 40 mM with phosphate buffered saline (PBS) as the blank control. Results are shown in Fig. 7. Compared with blank control group, compounds 24c and 30c could significantly inhibit angiogenesis. And the inhibitory ability was proportional to the concentration. At 40 mM, 24c and 30c presented comparable inhibitory activity with 2-ME2 group. Overall, compounds 24c and 30c showed potential anti-angiogenesis activities in vivo.

4. Conclusion In this study, a series of 11a-substituted 2-ME2 analogs have been designed and synthesized. Biological evaluation was performed on their anti-proliferative activities against 5 different cell lines. The results indicated that most compounds exhibited good activities. Among them, compound 24c and 30c showed the best activity with low micromolar IC50 (2.73 mM
7.75 mM) toward all the cell lines. The investigation of ER affinity showed that the majority of the compounds displayed good activity at the concentration of 50 mM. In further mechanism study, the result of flow

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Table 1 The anti-proliferative activities of synthesized compounds toward 5 cell lines.

R3

IC50(mM)

Compound

R1

R2

MCF-7

Ishikawa

HUVEC

HEPG2

PC-3

17a

-H

-Me

34.30

15.59

6.97

3.42

6.49

17b

-H

-Me

13.71

4.79

3.69

8.64

10.59

17c

-H

-Me

12.92

7.52

5.88

8.12

18.11

24a

-OMe

-Me

28.19

6.32

5.88

1.78

8.94

24b

-OMe

-Me

15.02

5.51

2.63

3.05

3.74

24c

-OMe

-Me

4.33

5.55

4.85

5.22

4.29

30a

-H

-MOM

28.01

28.48

17.67

23.49

17.62

30b

-H

-MOM

14.31

7.30

9.28

25.40

17.07

30c

-H

-MOM

4.06

7.75

5.23

2.73

2.85

31a

-H

-H

47.22

37.52

32.00

8.92

23.45

31b

-H

-H

34.67

17.34

36.05

21.05

39.06

31c

-H

-H

29.6

9.66

36.95

15.07

19.36

2-ME2

-OMe

-H

6.01

8.96

14.17

11.03

12.31

e

24c and 30c in MCF-7 cells, which suggested that 24c and 30c presented significant anti-estrogenic activity. Based on the preliminary results, compounds 24c and 30c were selected to perform CAM assay to investigate their inhibition of angiogenesis in vivo. Compared with blank control group, compounds 24c and 30c could significantly inhibit angiogenesis. At 40 mM, 24c and 30c presented comparable inhibitory activity with 2-ME2 group. Based on biological activities data, 24c and 30c can be identified as potential lead molecules which might be of therapeutic importance for cancer treatment. 5. Experiment Fig. 4. The relative binding affinities of synthesized compounds towards ERa.

cytometric analysis indicated that 24c and 30c could significantly increase the combined G2/M cell population. Moreover, the increased mRNA expression of PR induced by E2 was reversed by

5.1. Chemistry 5.1.1. General procedure Melting points of compounds were measured on a RY-1 melting point apparatus and were uncorrected. Nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker AV-300

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Fig. 5. (A) Cell cycle analysis of MCF-7 cells treatment of compound 24c and 30c (1 mM and 2 mM) and no treatment (DMSO) as reference control at 6 h. (B) and (C) Percentage of cells in G2/M phases of the cycle after treatment of 24c and 30c versus time. Results are expressed as mean ± SEM of three independent experiments.

(300 MHz) spectrometer as deuterochloroform (CDCl3) solutions using tetramethylsilane (TMS) as an internal standard (d ¼ 0) unless noted otherwise. Electron impact mass spectral (EI-MS) data were obtained on a SHIMADZU GCMS-QP2010 system. All chemicals were purchased from commercial sources and were used without further purification unless otherwise noted. The solvents (such as MeOH, EtOAc, EtOH, CH2Cl2 and others) were C.P. grade purchased from Nanjing Chemical Co., Ltd. And used without further purification. Column chromatography (CC) was carried out on silica gel (200e300 mesh, Qingdao Ocean Chemical Company, China). Thinlayer chromatography (TLC) analyses were carried out on silica gel GF254 (Qingdao Ocean Chemical Company, China) glass plates (2.5 cm  10 cm with 250 mm layer). Concentration and evaporation of the solvent after reaction or extraction was carried out on a rotary evaporator operated at reduced pressure. Analytical HPLC for assessing purity was performed on Agilent 1260 Infinity System equipped with a UV detector at 254 nm. The column employed is an Agilent Zorbax AB-C18 column (5 mm, 4.6  250 mm). The purities of allfinal compounds were at least

>97%. 5.1.2. Synthesis of 3,17b-dihydroxyestra-1,3,5(10), 9(11)-tetraen (11) Estradiol (20.0 g, 73.53 mmol) and DDQ (25 g, 110.13 mmol) was dissolved in 300 ml methanol, refluxed under N2 for 4 h. Upon completion, the reaction mixture was poured into saturated aqueous NaHCO3 (300 ml), and extracted with ethyl acetate (300ml  3). The combined organic layers were dried and the solvent was evaporated in vacuo to provide crude product. The compound was purified by column chromatography (PE/EA, 9:1) to give white solid 7.8 g, yield 39.29%. MS (ESI, m/z):269[M
H]þ 5.1.3. Synthesis of 3,17b-dimethyoxyestra-1,3,5(10), 9(11)-tetraen (12) Compound 11 (3 g, 11.11 mmol) was dissolved in 30 ml dry THF and slowly added in NaH (1.33 g, 55.56 mmol). The CH3I (3.46 ml, 55.56 mmol) was added after 1 h. The reaction mixture was stirred for another 4 h. Upon completion, the reaction mixture was poured

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Fig. 6. 24c and 30c dose-dependently inhibit the mRNA expression of PR and have no effect on AIM2 mRNA expression in MCF-7 cells. (A) The increased mRNA expression of PR induced by E2 was reversed by 24c and 30c in MCF-7 cells. (B) No effect on AIM2 mRNA expression in MCF-7 cells was observed. The mRNA expression of PR and AIM2 were examined by real-time PCR. Values are mean ± SD (n ¼ 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. E2 group. #P < 0.05, ##P < 0.01,###P < 0.001.

[MþH]þ

5.1.4. Synthesis of 11a-hydroxy-3,17b-dimethyoxyestra-1,3,5(10) -triene (13) Compound 12 (2.9 g, 9.73 mmol) was added in 47.5 ml BH3$THF at 0  C and stirred for 6 h. 30% aqueous NaOH (19.73 ml) and 30% H2O2 was then added dropwise and stirred at room temperature for another 3 h. Upon completion, the reaction mixture was poured into 50 ml water and extracted with ethyl acetate (100ml  3). The combined organic layers were dried and the solvent was evaporated in vacuo to provide white solid. The compound was purified by column chromatography (PE/EA, 10:1) to give white solid 2.03 g, yield 66.1%. MS (ESI, m/z): 339[MþNa]þ

Fig. 7. The result of CAM assay.

into water and extracted with ethyl acetate (60ml  3). The combined organic layers were dried and the solvent was evaporated in vacuo to provide yellow solid 2.9 g, yield 87.58%. MS (ESI, m/z):299

5.1.5. Synthesis of 11a-(4-nitrobenzoyl)oxy-3,17b-dimethyoxyestra1,3,5(10) -triene (14) Compound 13 (2.03 g, 6.42 mmol) was dissolved in 40 ml dry THF, with 4-DMAP (1.57 g, 12.85 mmol) and 4-nitrobenzoyl chloride (1.79 g, 9.63 mmol) added at 0  C. The reaction mixture was stirred at 0  C for 5 h. Upon completion, the reaction mixture was poured into 80 ml water and extracted with ethyl acetate (100ml  3). The combined organic layers were dried and the solvent was evaporated in vacuo to provide white solid 2.97 g, yield 93.3%. MS (ESI, m/z): 488[MþNa]þ

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5.1.6. Synthesis of 11a-(4-aminobenzoyl)oxy-3,17bdimethyoxyestra-1,3,5(10) -triene (15) Compound 14 (2.79 g, 6 mmol), 0.28 g Pd/C and 0.756 g ammonium formate was added in 40 ml ethyl acetate, stirred for 3 h at 50  C with H2. Upon completion, the reaction mixture was filtered to remove Pd/C. The filtrate was dried and the solvent was evaporated in vacuo to provide white solid 2.35 g, yield 90.45%. MS (ESI, m/z): 458[MþNa]þ 5.1.7. Synthesis of 11a-(4-(2-chloroacetamido)benzoyl)oxy-3,17bdimethyoxyestra-1,3,5 (10) -triene (16) Compound 15(2.35 g, 5.40 mmol), triethylamine (1.12 ml, 8.10 mmol) was dissolved in 35 ml dry THF, with 0.61 ml chloroacetyl chloride slowly added at 0  C. The reaction mixture was stirred at room temperature for 3 h. Upon completion, the reaction mixture was poured into 50 ml water and extracted with ethyl acetate (100ml  3). The combined organic layers were dried and the solvent was evaporated in vacuo to provide yellow solid 2 g, yield 71.45%. MS (ESI, m/z): 534[MþNa]þ 5.1.8. General procedure for the preparation of compound (17a~17c) Compound 16 (0.3 g, 0.59 mmol), amine (0.65 mmol), triethylamine (0.24 ml, 1.77 mmol) and KI (0.03 g) was dissolved in 6 ml acetonitrile. The reaction mixture was refluxed for 3 h. Upon completion, the reaction mixture was poured into 6 ml water and extracted with ethyl acetate (10ml  3). The combined organic layers were dried and the solvent was evaporated in vacuo to provide brown solid. The compound was purified by column chromatography (PE/EA, 10:1). 5.1.9. 11a-(4-(2-dimethylaminoacetamido)benzoyl)oxy-3,17bdimethoxyestra-1,3,5(10)-triene (17a) White solid, yield 54.3%. m.p.: 83e85  C; 1H NMR (CDCl3, 300 MHz) d: 9.33 (s, 1H, CONH), 8.04 (d, J ¼ 8.64, 2H, 20 ,60 -H), 7.68 (d, J ¼ 8.7, 2H, 30 ,50 -H), 7.10 (d, J ¼ 8.67, 1H, 1-H), 6.71 (d, J¼8.61, 1H, 2-H), 6.76 (s, 1H, 4-H), 5.57e5.66 (td, J¼10.40, 5.19, 1H, 11-H), 3.73 (s, 3H, 3-OCH3), 3.37 (s, 3H, 17-OCH3), 3.37 (s, 1H, 17-H), 3.10 (s, 2H, -COCH2N-), 2.89 (t, J¼6.33, 2H, 6-H), 2.69 (m, 1H, 9-H), 2.39 (s, 6H, N(CH3)2), 1.00e2.30 (m, 10H), 0.91 (s, 3H, 18-CH3)ppm; 13C NMR (CDCl3, 75 Hz) d: 168.70, 165.06, 157.23, 141.37, 138.56, 131.23, 130.47, 125.46, 125.26, 118.10, 113.50, 110.56, 89.45, 74.02, 63.16, 57.58, 54.64, 49.52, 45.96, 45.55, 43.41, 43.12, 36.75, 28.05, 27.40, 26.46, 22.47, 11.61; HRMS (ESI) m/z: calcd for C31H41N2O5 [MþH]þ 521.2937 Found5 21.3020. 5.1.10. 11a-(4-(2-(pyrrolidin-1-yl)acetamido)benzoyl)oxy-3,17bdimethoxyestra-1,3,5(10)-triene (17b) White solid, yield 65.52%. m.p.: 196e198  C; 1H NMR (CDCl3, 300 MHz) d: 9.35 (s, 1H, CONH), 8.05 (d, J ¼ 8.64, 2H, 20 ,60 -H), 7.66 (d, J ¼ 8.7, 2H, 30 ,50 -H), 7.10 (d, J ¼ 8.67, 1H, 1-H), 6.68 (s, 1H, 4-H), 6.59 (d, J¼8.61, 1H, 2-H), 5.57e5.66 (td, J¼10.40, 5.19, 1H, 11-H), 3.73 (s, 3H, 3-OCH3), 3.37 (s, 3H, 17-OCH3), 3.37 (s, 1H, 17-H), 3.10 (s, 2H, -COCH2N-), 2.87 (t, 2H, 6-H), 2.72 (m, 5H, 9-H, N(CH2CH2)2), 1.00e2.30 (m, 10H), 0.89 (s, 3H, 18-CH3)ppm; 13C NMR (CDCl3, 75 Hz) d: 170.07, 165.07,157.22, 141.32, 138.55, 131.23, 130.50, 125.40, 125.28, 117.97, 113.50, 110.55, 89.56, 74.03, 57.59, 54.64, 49.52, 48.41, 45.96, 43.41, 43.13, 36.75, 28.06, 27.40, 26.46, 22.47, 11.93, 11.60; HRMS (ESI) m/z: calcd for C33H45N2O5 [MþH]þ 547.3094 Found 547.3164. 5.1.11. 11a-(4-(4-methylpiperazin-1-yl)acetamido)benzoyl)oxy3,17b-dimethoxyestra-1,3,5(10)-triene (17c) White solid, yield 45.62%. m.p.: 92e93  C; 1H NMR (CDCl3, 300 MHz) d: 9.31 (s, 1H, CONH), 8.05 (d, J ¼ 8.64, 2H, 20 ,60 -H), 7.66

397

(d, J ¼ 8.7, 2H, 30 ,50 -H), 7.10 (d, J ¼ 8.67, 1H, 1-H), 6.67 (s, 1H, 4-H), 6.59 (d, J¼8.61, 1H, 2-H), 5.57e5.66 (td, J¼10.40, 5.19, 1H, 11-H), 3.73 (s, 3H, 3-OCH3), 3.37 (s, 3H, 17-OCH3), 3.37 (s, 1H, 17-H), 3.10 (s, 2H, -COCH2N-), 2.86 (t, J¼6.33, 2H, 6-H), 2.58e2.72 (m, 9H, 9-H, N(CH2CH2)2N), 1.00e2.30 (m, 10H), 0.89 (s, 3H, 18-CH3)ppm; 13C NMR (CDCl3, 75 Hz) d: 168.11, 165.01, 157.21, 141.19, 138.58, 131.21, 130.50, 125.56, 125.23, 118.10, 113.45, 110.56, 89.42, 74.07, 61.33, 57.58, 54.64, 54.54, 52.67, 49.49, 45.93, 45.27, 43.40, 43.10, 36.73, 28.05, 27.38, 26.44, 22.47, 11.61; HRMS (ESI) m/z: calcd for C34H46N3O5 [MþH]þ 576.3359 Found 576.3429. 5.1.12. Synthesis of 2-methyoxy-3,17b-dihydroxyestra-1,3,5(10), 9(11)-tetraen (18) Using the synthetic method for 11, compound 18 was given as white solid 7.8 g, yield 39.29%. MS (ESI, m/z): 299[M
H]þ 5.1.13. Synthesis of 2,3,17b-trihydroxyestra-1,3,5(10), 9(11)-tetraen (19) Using the synthetic method for 12, compound 19 was given as yellow solid 4.03 g, yield 85.64%. MS (ESI, m/z): 351[MþNa]þ 5.1.14. Synthesis of 11a-hydroxy-2,3,17b-trihydroxyestra-1,3,5(10), 9(11)-tetraen (20) Using the synthetic method for 13, compound 20 was given as white solid 3.04 g, yield 71.44%. MS (ESI, m/z): 369[MþNa]þ 5.1.15. Synthesis of 11a-(4-nitrobenzoyl)oxy-2,3,17bdimethoxyestra-1,3,5(10) -triene (21) Using the synthetic method for 14, compound 21 was given as 4.04 g, yield 92.09%. MS (ESI, m/z): 518[MþNa]þ 5.1.16. Synthesis of 11a-(4-aminobenzoyl)oxy-2,3,17bdimethoxyestra
1,3,5(10)-triene (22) Using the synthetic method for 15, compound 22 was given as white solid 3.43 g, yield 90.45%. MS (ESI, m/z):488[MþNa]þ 5.1.17. Synthesis of 11a-(4-(2-chloroacetamido)benzoyl)oxy-2,3,17b -dimethyoxyestra-1,3,5 (10) -triene (23) Using the synthetic method for 16, compound 23 was given as yellow solid 2.9 g, yield 70.34%. MS (ESI, m/z): 564[MþNa]þ 5.1.18. 11a-(4-(2-dimethylaminoacetamido)benzoyl)oxy-2,3,17bdimethyoxyestra-1,3,5(10) -triene (24a) White solid, yield 54.10%. m.p.: 90e92  C; 1H NMR (CDCl3, 300 MHz) d: 9.62 (s, 1H, CONH), 8.07 (d, J ¼ 8.64, 2H, 20 ,60 -H), 7.66 (d, J ¼ 8.7, 2H, 30 ,50 -H), 6.84 (s, 1H, 4-H), 6.60 (s, 1H, 1-H), 5.57e5.67 (td, J¼10.35, 5.13, 1H, 11-H), 3.81 (d, 6H,, -OCH3), 3.38 (s, 3H, -OCH3), 3.34 (m, 4H, -OCH3, 17-H), 3.09 (s, 2H, 200 -H), 2.86 (t, J¼7.74, 2H, 6H), 2.61e2.74 (m, 1H, 9-H), 2.39 (s, 6H, N(CH3), 0.89 (s, 3H, 18-CH3) ppm; 13C NMR (CDCl3, 75 Hz) d: 168.73, 164.99, 146.41, 141.52, 130.43, 130.36, 128.79, 125.22, 118.08, 111.07, 108.18, 89.47, 74.43, 63.14, 57.56, 55.28, 54.74, 49.19, 46.45, 45.5743.51, 43.38, 36.77, 27.88, 27.39, 26.88, 22.45, 11.66; HRMS (ESI) m/z: calcd for C32H43N2O6 [MþH]þ 551.3043, Found 551.3111. 5.1.19. 11a-(2-(pyrrolidin-1-yl)acetamido)benzoyl)oxy-2,3,17bdimethyoxyestra-1,3,5(10) -triene (24b) White solid, yield 72.32%. m.p.: 95e97  C; 1H NMR (CDCl3, 300 MHz) d: 9.37 (s, 1H, CONH), 8.07 (d, J ¼ 8.64, 2H, 20 ,60 -H), 7.69 (d, J ¼ 8.7, 2H, 30 ,50 -H), 6.84 (s, 1H, 4-H), 6.60 (s, 1H, 1-H), 5.57e5.67 (td, J¼10.35, 5.13, 1H, 11-H), 3.81 (d, 6H,, -OCH3), 3.38 (s, 3H, -OCH3), 3.34 (m, 4H, -OCH3, 17-H), 3.30 (s, 2H, 200 -H), 2.86 (t, J¼7.74, 2H, 6H), 2.61e2.75 (m, 5H, 9-H, N(CH2CH2)2), 0.89 (s, 3H, 18-CH3)ppm; 13 C NMR (CDCl3, 75 Hz) d: 169.00, 164.99, 146.52, 146.18, 141.56, 130.43, 130.35, 128.78, 125.20, 118.13, 111.06, 108.19, 89.47, 74.44,

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59.25, 57.57, 55.29, 54.76, 54.15, 49.19, 46.45, 43.51, 43.29, 36.77, 27.89, 27.39, 26.88, 23.59, 22.45, 11.66; HRMS (ESI) m/z: calcd for C34H45N2O6 [MþH]þ 577.3199, Found 577.3266. 5.1.20. 11a-(4-(4-methylpiperazin-1-yl)acetamido)benzoyl)oxy2,3,17b-dimethyoxyestra-1,3,5(10) -triene (24c) White solid, yield 44.26%. m.p.: 95e97  C; 1H NMR (CDCl3, 300 MHz) d: 9.35 (s, 1H, CONH), 8.07 (d, J ¼ 8.64, 2H, 20 ,60 -H), 7.66 (d, J ¼ 8.7, 2H, 30 ,50 -H), 6.84 (s, 1H, 4-H), 6.60 (s, 1H, 1-H), 5.57e5.67 (td, J¼10.35, 5.13, 1H, 11-H), 3.81 (d, 6H,, -OCH3), 3.38 (s, 3H, -OCH3), 3.35 (m, 4H, -OCH3, 17-H), 3.16 (s, 2H, 200 -H), 2.86 (t, J¼7.74, 2H, 6H), 2.53e2.67 (m, 9H, 9-H, N(CH2CH2)2), 2.34 (s, 3H, NCH3), 0.89 (s, 3H, 18-CH3)ppm; 13C NMR (CDCl3, 75 Hz) d: 168.22, 164.96, 146.42, 146.12, 141.36, 130.40, 128.81, 125.34, 118.05, 117.97, 111.06, 108.22, 108.17, 89.46, 74.47, 61.37, 57.55, 55.28, 54.76, 54.70, 52.92, 49.26, 49.19, 46.44, 45.46, 4329, 43.45, 43.39, 36.76, 27.86, 27.38, 26.87, 22.45, 11.66; HRMS (ESI) m/z: calcd for C35H48N3O6 [MþH]þ 606.3465, Found 606.3532. 5.1.21. Synthesis of 3, 17b-dimethoxymethoxyestra-1, 3, 5(10) -triene (25) Compound 11(4.8 g, 17.78 mmol) was dissolved in 100 ml dry CH2Cl2 with DIEA (20.53 ml, 124.46 mmol) slowly added at 0  C. 0.83 ml chloromethyl methyl ether was added after 1 h. The reaction mixture was stirred at room temperature for 7 h. Upon completion, the reaction mixture was poured into 50 ml water and extracted with ethyl acetate (100ml  3). The combined organic layers were dried and the solvent was evaporated in vacuo to provide yellow solid 5.7 g, yield 89.1%. MS (ESI, m/z): 357[M
H]þ 5.1.22. Synthesis of 11a-hydroxy-3, 17b-dimethoxymethoxyestra-1, 3, 5(10) -triene (26) Using the synthetic method for 13, compound 26 was given as white solide, yield 89.10%. MS (ESI, m/z): 399[MþNa]þ 5.1.23. Synthesis of 11a-(4-nitrobenzoyl)oxy-3,17bdimethyoxymethyoxyestra-1,3,5(10)-triene (27) Using the synthetic method for 14, compound 27 was given as yellow solid, yield 92.90%. MS (ESI. m/z): 548[MþNa]þ 5.1.24. Synthesis of 11a-(4-aminobenzoyl)oxy-3,17bdimethyoxymethyoxyestra-1,3,5(10) -triene (28) Using the synthetic method for 15, compound 28 was given as yellow solid, yield 89.02%. MS (ESI, m/z): 518[MþNa]þ 5.1.25. Synthesis of 11a-(4-(2-chloroacetamido)benzoyl)oxy-3,17bdimethyoxymethyoxyestra
1,3,5(10)-triene (29) Using the synthetic method for 16, compound 29 was given as yellow solid, yield 70.62%. MS (ESI, m/z): 594[MþNa]þ 5.1.26. 11a-(4-(2-dimethylaminoacetamido)benzoyl)oxy-3,17bbis(methoxymethoxy)estra-1,3, 5(10) -triene (30a) White solid, yield 36.38%. m.p.: 73e76  C; 1H NMR (CDCl3, 300 MHz) d: 9.33 (s, 1H, CONH), 8.07 (d, J ¼ 8.64, 2H, 20 ,60 -H), 7.66 (d, J ¼ 8.7, 2H, 30 ,50 -H), 7.10(d, J¼8.61, 1H, 1-H), 6.84 (s, 1H, 4-H), 6.60 (d, J¼8.61, 1H, 2-H), 5.57e5.67 (td, J¼10.35, 5.13, 1H, 11-H), 5.10 (s, 2H, OCH2OCH3), 4.61 (s, 2H, OCH2OCH3), 3.68 (t, J¼8.25, 1H, 17-H), 3.43 (s, 3H, OCH2OCH3), 3.34 (s, 3H, OCH2OCH3), 3.10 (s, 2H, 200 -H), 2.86 (t, J¼6.33, 2H, 6-H), 2.61e2.74 (m, 1H, 9-H), 2.39 (s, 6H, N(CH3), 0.89 (s, 3H, 18-CH3)ppm; 13C NMR (CDCl3, 75 Hz) d: 168.62, 165.07, 154.85, 141.38, 138.67, 132.57, 130.69, 125.42.125.28, 118.09, 115.37, 113.20, 95.49, 93.88.85.10, 73.88, 63.14, 55.41, 54.77, 49.26, 46.07, 45.53, 43.21, 42.25, 36.85, 27.98.27.68, 26.39, 22.53, 11.82; HRMS (ESI) m/z: calcd for C33H45N2O7 [MþH]þ 581.3149, Found 581.3220.

5.1.27. 11a-(4-(4-(pyrrolidin-1-yl)acetamido)benzoyl)oxy-3,17bbis(methoxymethoxy)estra-1,3, 5(10) -triene (30b) White solid, yield 45.96%. m.p.: 67e68  C; 1H NMR (CDCl3, 300 MHz) d: 9.50 (s, 1H, CONH), 8.07 (d, J ¼ 8.64, 2H, 20 ,60 -H), 7.66 (d, J ¼ 8.7, 2H, 30 ,50 -H), 7.10(d, J ¼ 8.61, 1H, 1-H), 6.81 (s, 1H, 4-H), 6.69 (d, J¼8.61, 1H, 2-H), 5.57e5.67 (td, J¼10.35, 5.13, 1H, 11-H), 5.12 (s, 2H, OCH2OCH3), 4.63 (s, 2H, OCH2OCH3), 3.68 (t, J¼8.25, 1H, 17H), 3.45 (s, 3H, OCH2OCH3), 3.35 (s, 3H, OCH2OCH3), 3.31 (s, 2H, 200 H), 2.85 (t, 2H, 6-H), 2.86 (m, 9H, 9-H, N(CH2CH2)2), 0.89 (s, 3H, 18CH3)ppm; 13C NMR (CDCl3, 75 Hz) d: 169.04, 165.09, 154.84, 141.42, 138.66, 132.56, 130.48, 125.37, 125.30, 118.13, 115.56, 113.20, 95.49, 93.87, 85.09, 76.98, 76.56, 76.14, 73.89, 59.30, 55.41, 54.77, 54.15, 49.25, 46.07, 43.21, 42.25, 36.80, 27.99, 27.68, 26.39, 23.59, 22.53, 11.82; HRMS (ESI) m/z: calcd for C35H47N2O7 [MþH]þ 607.3305, Found 607.3387. 5.1.28. 11a-(4-(4-(2-(4-methylpiperazin-1-yl)acetamido)benzoyl) oxy-3,17b-bis(methoxymethoxy)estra-1,3, 5(10) -triene (30c) White solid, yield 41.34%. m.p.: 70e72  C; 1H NMR (CDCl3, 300 MHz) d: 9.33 (s, 1H, CONH), 8.07 (d, J ¼ 8.64, 2H, 20 ,6-H), 7.66 (d, J ¼ 8.7, 2H, 30 ,50 -H), 7.10(d, J ¼ 8.61, 1H, 1-H), 6.84 (s, 1H, 4-H), 6.60 (d, J ¼ 8.61, 1H, 2-H), 5.57e5.67 (td, J ¼ 10.35, 5.13, 1H, 11-H), 5.10 (s, 2H, OCH2OCH3), 4.61 (s, 2H, OCH2OCH3), 3.68 (t, J ¼ 8.25, 1H, 17-H), 3.43 (s, 3H, OCH2OCH3), 3.34 (s, 3H, OCH2OCH3), 3.10 (s, 2H, 200 -H), 2.86 (t, J ¼ 6.33, 2H, 6-H), 2.61e2.74 (m, 1H, 9-H), 2.56 (m, 9H, 9-H, N(CH2CH2)2N), 2.36 (s, 3H, -NCH3),0.89 (s, 3H, 18-CH3)ppm; 13C NMR (CDCl3, 75 Hz) d: 168.13, 165.04, 154.84, 141.21, 138.68, 132.54, 130.52, 125.21, 125.27, 118.07, 115.58, 113.16, 95.49, 93.86, 85.10, 73.95, 61.36, 55.41, 54.77, 54.66, 52.81, 49.51, 46.06, 45.60, 43.21, 42.25, 36.79, 27.99, 27.68, 26.38, 22.53, 11.82; HRMS (ESI) m/z: calcd for C36H50N3O7 [MþH]þ 636.3571, Found 636.3640. 5.1.29. General procedure for the preparation of compound (31a~31c) Compound 30(0.22 mmol) was dissolved in 3 ml methanol, with hydrochloric acid (0.15 ml, 4.93 mmol) added. The reaction mixture was stirred at 60  C for 2 h. Upon completion, the solvent was evaporated in vacuo to provide yellow oily product. The compound was purified by column chromatography (DCM/MeOH, 20:1). 5.1.30. 11a-(4-(2-dimethylaminoacetamido)benzoyl)oxy-3,17bdihydroxyestra-1,3,5(10)-triene (31a) White solid, yield 63.48%. m.p.: 218e220  C; 1H NMR (DMSO-d6, 300 MHz) d: 10.65 (s, 1H, 3-OH), 9.08 (s, 1H, CONH), 8.07 (d, J ¼ 8.64, 2H, 20 ,60 -H), 7.75 (d, J ¼ 8.7, 2H, 30 ,50 -H), 6.81(d, J¼8.52, 1H, 1-H), 6.51 (s, 1H, 4-H), 6.34 (d, J¼8.61, 1H, 2-H), 5.36e5.45 (td, J¼9.72, 5.01, 1H, 11-H), 4.61 (s, 1H, 17-OH), 4.14 (s, 2H, 200 -H), 3.58 (t, 1H, 17H), 2.85 (t, J¼6.9, 2H, 6-H), 2.61 (s, 6H, N(CH3)), 2.74 (m, 1H, 9-H), 0.89 (s, 3H, 18-CH3)ppm; 13C NMR (DMSO-d6, 75 Hz) d: 168.55, 164.85, 142.70, 138.70, 130.30, 129.75, 124.86, 124.64, 118.93, 118.85, 112.39, 79.20, 74.35, 60.22, 48.94, 45.71, 44.20, 44.13, 43.53, 42.16, 37.37, 29.68, 27.73, 27.69, 26.31, 22.56, 11.74; HRMS (ESI) m/z: calcd for C29H37N2O5 [MþH]þ 493.2624, Found 493.2696. 5.1.31. 11a-(4-(2-(pyrrolidin-1-yl)acetamido)benzoyl)oxy-3,17bdihydroxyestra-1,3,5(10)-triene (31b) White solid, yield 70.19%. m.p.: 207e209  C; 1H NMR (DMSO-d6, 300 MHz) d: 10.29 (s, 1H, 3-OH), 9.08 (s, 1H, CONH), 7.94 (d, J ¼ 8.64, 2H, 20 ,60 -H), 7.75 (d, J ¼ 8.7, 2H, 30 ,50 -H), 6.81(d, J¼8.52, 1H, 1-H), 6.51 (s, 1H, 4-H), 6.34 (d, J¼8.61, 1H, 2-H), 5.36e5.45 (td, J¼9.72, 5.01, 1H, 11-H), 4.61 (s, 1H, 17-OH), 3.58 (t, 1H, 17-H), 3.16 (s, 2H, 200 H), 2.74 (t, J¼6.9, 2H, 6-H), 2.34 (m, 1H, 9-H), 2.60 (m, 4H, N(CH2CH3)2), 0.89 (s, 3H, 18-CH3)ppm; 13C NMR (DMSO-d6, 75 Hz) d: 169.03, 164.82, 155.28, 142.27, 138.71, 130.42, 129.75, 125.07, 124.62, 118.89, 114.95, 112.38, 74.39, 56.23, 54.12, 48.96, 45.73,

K. Lao et al. / European Journal of Medicinal Chemistry 139 (2017) 390e400

43.54, 37.38, 27.72, 22.79, 22.57, 11.74; HRMS (ESI) m/z: calcd for C31H39N2O5 [MþH]þ 519.2781, Found 519.2860. 5.1.32. 11a-(4-(4-(4-methylpiperazin-1-yl)acetamido)benzoyl)oxy3,17b-dihydroxyestra-1,3,5(10)-triene (31c) White solid, yield 75.63%. m.p.: 198e203  C; 1H NMR (DMSO-d6, 300 MHz) d: 10.31 (s, 1H, 3-OH), 9.14 (s, 1H, CONH), 7.94 (d, J ¼ 8.64, 2H, 20 ,60 -H), 7.75 (d, J ¼ 8.7, 2H, 30 ,50 -H), 6.81(d, J¼8.52, 1H, 1-H), 6.51 (s, 1H, 4-H), 6.34 (d, J¼8.61, 1H, 2-H), 5.36e5.45 (td, 1H, 11-H), 4.63 (s, 1H, 17-OH), 3.58 (t, 1H, 17-H), 3.16 (s, 2H, 200 -H), 2.74 (t, J¼6.9, 2H, 6-H), 2.58e3.00 (m, 8H, N(CH2CH2)2), 2.58 (s, 3H, -NCH3), 2.34 (m, 1H, 9-H), 0.89 (s, 3H, 18-CH3)ppm; 13C NMR (DMSO-d6, 75 Hz) d: 168.22, 164.89, 155.31, 142.94, 138.64, 130.23, 129.71, 124.68, 118.95, 118.89, 114.99, 112.42, 79.17, 74.36, 48.89, 48.56, 45.72, 43.52, 42.17, 41.99, 41.91, 29.66, 27.76, 26.31, 11.75; HRMS (ESI) m/z: calcd for C32H42N3O5 [MþH]þ 548.3046, Found 548.3149. 5.2. Biological evaluation 5.2.1. ERa binding affinity assay The recombinant ERa (Thermo Fisher Scientific Inc., Invitrogen, USA) and the fluorescent estrogen ligands (Thermo Fisher Scientific Inc., Invitrogen, USA) were removed from the
80  C freezer and thawed on ice for 1 h prior to use. The fluorescent estrogen ligand was added to the ERa and screening buffer (ES2 Screening Buffer, Invitrogen, USA) was added to make the final concentration 9 nM for fluorescent estrogen and 30 nM for ERa. Test compounds were accurately weighed and dissolved in DMSO, screening buffer was added to dilute to required concentration. Test compound (1 mL) was added to 9 mL screening buffer in each well (384-well microplate, Corning, USA). To this 10 mL of the fluorescent estrogen/ER complex was added to make up a final volume of 20 mL. A positive control contained 10 mL estradiol buffer and 10 mL fluorescent estrogen/ER complex. A negative control contained 10 mL screening buffer and 10 mL fluorescent estrogen/ER complex. The negative control was used to determine the polarization value when no competitor was present (theoretical maximum polarization). The microplate was incubated in the dark at room temperature for 2 h and shaken on a plate shaker. The polarization values were read on a Safire microplate reader and used to calculate the IC50 values. 5.2.2. MTT assay for anti-proliferative activities Cells were cultured in RPMI1640 medium (containing 10% (v/v) FBS, 100 U/mL Penicillin and 100 mg/mL Streptomycin) in a 5% CO2humidified atmosphere at 37  C. Cells were trypsinized and seeded at a density of 1  105/mL into a 96-well plate (100 mL/well) and incubated at 37  C, 5% CO2 atmosphere for 24 h. After this time they were treated with 100 mL/well medium containing test compounds which had been pre-prepared to provide the concentration range of 1  10
4 mol/L, 1  10
5 mol/L, 1  10
6 mol/L and 1  10
7 mol/L, and re-incubated for a further 48 h. Control wells were added the equivalent volume of medium containing 1% (v/v) DMSO. 20 mL MTT (5 mg/mL) was added and cells continued to incubate in darkness at 37  C for 4 h. The culture medium was then removed carefully and 150 mL DMSO was added. The cells were maintained at room temperature in darkness for 20 min to ensure thorough color diffusion before reading the absorbance. The absorbance values were read at 490 nm for determination of IC50 values. 5.2.3. Cell cycle analysis The cell cycle status and nuclear DNA contents were determined using propidium iodide (PI) staining and flow cytometry. Human breast cancer cell line MCF-7 was plated in 12-well plates at a density of 3  105 per well in 2 mL medium. After incubation overnight, cells were treated with compound 30c (1 and 2 mM) and

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31c (1 and 2 mM) in serum-free medium for 24 h. Then the cells were collected and fixed with 70% ice-cold ethanol. After they were washed with PBS, the cells were stained with PI at 4  C. The percentage of cells in G1, S and G2/M phase of the cell cycle was determined by using a FACSort flow-cytometer provided by the Cell-Quest software (Becton-Dickinson, USA). 5.2.4. Real-time polymerase chain reaction (RT-PCR) RNA samples were reverse transcribed to cDNA and the PCR reactions were performed using TaKaRa SYBR Green Master Mix (Code. no. 638320) carried out in StepOnePlus™Real-Time PCR instrument (4376600, Life Technologies). The program for amplification was 1 cycle of 95  C for 2 min followed by 40 cycles of 95  C for 10s, 60  C for 30s, and 95  C for 10s. The PCR results were normalized to GAPDH expression and were quantified by the DDCT method. 5.2.5. Chicken chorioallantoic membrane (CAM) assay Fertilized eggs were incubated for 7 days in a humidified environment at 37  C with 5% CO2 in air and saturated humidity. Then, a window of approximately 1 cm2 was opened on the egg shell to expose the CAM. Test compounds and positive control Sunitinib dissolved in DMSO were placed on sterile methyl cellulose filter papers at 1 mM, 10 mM and 40 mM with phosphate buffered saline as the blank control. The papers were then placed on the CAM. The window was sealed with sterile cellophane tape. The eggs were further incubated at 37  C under a constant relative humidity of 60% for 72 h. After fixed with acetone and ethanol for 10min, the CAM was cut and papers removed to observe angiogenesis. Images of the control and sample-treated areas were captured. Acknowledgments Research reported in this publication was supported by the NSFC (81373279), the Twelfth Five-Year Plan Major Project of Candidate Drugs (Ministry of National Science and Technology, 2012ZX09103101048), the Jiangsu Province Science and Technology Support Program of Social Development Projects (BE2012745) and the Innovation Project of Jiangsu Province (201610316107). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2017.08.016. References [1] T. Aoyama, K. Korzekwa, K. Nagata, J. Gillette, H.V. Gelboin, F.J. Gonzalez, Estradiol metabolism by complementary deoxyribonucleic acid-expressed human cytochrome p450s, Endocrinology 126 (1990) 3101e3106. [2] A.O. Mueck, H. Seeger, 2-methoxyestradiolebiology and mechanism of action, Steroids 75 (2010) 625e631. [3] R.J. D'Amato, C.M. Lin, E. Flynn, J. Folkman, E. Hamel, 2-methoxyestradiol, an endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site, P. Natl. Acad. Sci. U. S. A. 91 (1994) 3964e3968. [4] T. Fotsis, Y. Zhang, M.S. Pepper, H. Adlercreutz, R. Montesano, P.P. Nawroth, L. Schweigerer, The endogenous oestrogen metabolite 2-methoxyoestradiol inhibits angiogenesis and suppresses tumour growth, Nature 368 (1994) 237e239. [5] E. Aizuyokota, A. Susaki, Y. Sato, Natural estrogens induce modulation of microtubules in Chinese hamster v79 cells in culture, Cancer Res. 55 (1995) 1863e1868. [6] L.R. Qadan, C.M. Perez-Stable, C. Anderson, G. D'Ippolito, A. Herron, G.A. Howard, B.A. Roos, 2-methoxyestradiol induces g2/m arrest and apoptosis in prostate cancer, Biochem. Biophys. Res. Commun. 285 (2001) 1259e2126. [7] J. James, D.J. Murry, A.M. Treston, A.M. Storniolo, G.W. Sledge, C. Sidor, K.D. Miller, Phase I safety, pharmacokinetic and pharmacodynamic studies of 2-methoxyestradiol alone or in combination with docetaxel in patients with

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[8] [9]

[10]

[11]

[12]

[13]

[14]

K. Lao et al. / European Journal of Medicinal Chemistry 139 (2017) 390e400 locally recurrent or metastatic breast cancer, Invest. New Drugs 25 (2007) 41e48. N.J. Lakhani, M.A. Sarkar, J. Venitz, W.D. Figg, 2-methoxyestradiol, a promising anticancer agent, Pharmacotherapy 23 (2003) 165e172. D.P. Squillace, J.M. Reid, M.J. Kuffel, M.M. Ames, Bioavailability andin vivo metabolism of 2-methoxyestradiol in mice, Proc. Am. Assoc. Cancer Res. 39 (1998) 523. B.S. Kumar, D.S. Raghuvanshi, M. Hasanain, S. Alam, J. Sarkar, K. Mitra, F. Khan, A.S. Negi, Recent advances in chemistry and pharmacology of 2methoxyestradiol: an anticancer investigational drug, Steroids 110 (2016) 9e34. T.M. Lavallee, P.A. Burke, G.M. Swartz, E. Hamel, G.E. Agoston, J. Shah, L. Suwandi, A.D. Hanson, W.E. Fogler, C.F. Sidor, A.M. Treston, Significant antitumor activity in vivo following treatment with the microtubule agent ENMD-1198, Mol. Cancer. Ther. 7 (2008) 1472e1482. V.C. Jordan, Antiestrogens and selective estrogen receptor modulators as multifunctional medicines, part 1. Recept. Interact. Cheminform 46 (2003) 883e908. V.C. Jordan, Antiestrogens and selective estrogen receptor modulators as multifunctional medicines. 2. clinical considerations and new agents, J. Med. Chem. 46 (2003) 1081e1111. re, F.E. van Leeuwen, J. Benraadt, J.W. Coebergh, L.A. Kiemeney, C.H. Gimbre R. Otter, L.J. Schouten, R.A. Damhuis, M. Bontenbal, F.W. Diepenhorst, Risk of endometrial cancer after tamoxifen treatment of breast cancer, Lancet 343 (1994) 448e452.

[15] R. Clarke, M.C. Liu, K.B. Bouker, Z. Gu, R.Y. Lee, Y. Zhu, T.C. Skaar, B. Gomez, K. O'Brien, Y. Wang, L.A. Hilakivi-Clarke, Antiestrogen resistance in breast cancer and the role of estrogen receptor signaling, Oncogene 22 (2003) 7316e7339. [16] A. Ring, M. Dowsett, Mechanisms of tamoxifen resistance, Endocr. Relat. Cancer 11 (2004) 643e658. [17] C. Fotopoulou, D. Baumunk, S.C. Schmidt, G. Schumacher, Additive growth inhibition after combined treatment of 2-methoxyestradiol and conventional chemotherapeutic agents in human pancreatic cancer cells, Anticancer Res. 30 (2010) 4619e4624. [18] J.A. Katzenellenbogen, The 2010 Philip S. Portoghese Medicinal Chemistry Lectureship: addressing the “core issue” in the design of estrogen receptor ligands, J. Med. Chem. 15 (2011) 5271e5282. [19] A. Segaloff, R.B. Gabbard, Structure-activity relationships of estrogens: effects of esterification of the 11 beta-hydroxyl group, Steroids 43 (1984) 111e123. [20] T.E. Vogelvang, M.J. van der Mooren, V. Mijatovic, P. Kenemans, Emerging selective estrogen receptor modulators: special focus on effects on coronary heart disease in postmenopausal women, Drugs 66 (2006) 191e221. [21] R.N. Hanson, E. Hua, J.A. Hendricks, D. Labaree, R.B. Hochberg, Synthesis and evaluation of 11b-(4-substituted phenyl) estradiol analogs: transition from estrogen receptor agonists to antagonists, Bioorg. Med. Chem. 20 (2012) 3768e3780. [22] M. Xin, Q. You, H. Xiang, An efficient, practical synthesis of 2-methoxyestradiol, Steroids 75 (2010) 53e56.