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. Author manuscript; available in PMC: 2013 Aug 29.
Published in final edited form as: Mini Rev Med Chem. 2013 Jul;13(9):1381–1388. doi: 10.2174/1389557511313090012

Synthesis and Biological Activity of 3-N-Substituted Estrogen Derivatives as Breast Cancer Agents

Zhongliang Wan 1, Musiliyu A Musa 2, Patrick Joseph 1, John S Cooperwood 1,*
PMCID: PMC3756546  NIHMSID: NIHMS506490  PMID: 22876946

Abstract

3-N-substituted-estrogen derivatives were synthesized and characterized. Their antiproliferative activities against human ER (+) MCF-7 (Breast), ER (−) MDA-MB-231 (breast) and Ishikawa (endometrial) cancer cell lines were determined after 72 hours drug exposure employing CellTiter-Glo assay at concentrations ranging from (0.01-100,000 nM). The antiproliferative activities of these compounds were compared to tamoxifen (TAM), 4-hydroxytamoxifen (4-OHT, active metabolite of tamoxifen) and raloxifene (RAL). In vitro results indicated that compound 5 (IC50 = 12μM) displayed comparable antiproliferative activity against MDA-MB 231 cell line; while compounds 6, 7 and 13 (IC50 = 12μM) displayed higher activity against MCF-7 and Ishikawa cell lines, in comparison to TAM activity (19-33μM).

Keywords: 3-N-substituted estrogen derivatives, antiproliferative activity, breast cancer

INTRODUCTION

Estrogens are known to affect growth, development and maintenance of many tissues and organs [1-3]. The physiological effects of these steroids are mediated by two estrogen receptor (ER) subtypes; estrogen receptor alpha (ER-α) and estrogen receptor beta (ER-β) with differences in tissue distribution and transcriptional activity [4-7]. Estrogens stimulate the proliferation of normal and malignant cells through the ER via the induction of nucleic acid synthesis and activation of growth regulatory genes. Estrogen functions primarily by binding to the ER that dimerizes; then binds to estrogen responsive elements (EREs) in DNA, followed by the regulation of estrogen responsive transcription genes [7].

There is strong evidence that estrogen plays a role in the development and progression of breast cancer, the most frequently diagnosed cancers among women [8]. Currently one of way of blocking estrogen action on tumor cells is to prevent the binding of estrogen to ER by using an antiestrogen compound capable of blocking the effects of 17β-estradiol (E2) without displaying any estrogenic activity on their own [9-11]. Antiestrogenic compounds display an antagonist action at the ER, exhibit antitumor effect and are widely used in the treatment of hormone-dependent ER (+) breast cancer [12, 13]. Example of antiestrogenic compounds are Selective estrogen receptor modulators (SERMs) drugs such as tamoxifen (TAM, 1) and Raloxifen (RAL, 2) [14, 15]. TAM (Fig. 1), a triphenylethylene (TPE) nonsteroidal antiestrogen, is the first chemotherapeutic drug used in the treatment of estrogen receptor positive (ER +) breast cancer [16]. It reduces the risk of contralateral breast cancer, behaves as ER antagonist in the breast tissue and as ER agonist in bone and lipids [17, 18]. TPE antiestrogen compounds contain a dialkylaminoethoxy side chain group, which is essential for their physiological activity. Studies have shown that these compounds lose their antiestrogenic activity and become potent estrogenic compounds in the absence of such groups [19, 20]. For example, replacement of dimethylaminoethoxy group of TAM with a methoxy group decreases TAM inhibitory growth activity in MCF-7 cells, while the addition of dimethylaminoethoxy group to gem-diphenyldichloroethylene, a weak estrogenic compound, resulted in an enhanced inhibitory growth activity in MCF-7 cells [21, 22].

Fig. (1).

Fig. (1)

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Structures of tamoxifen (TAM, 1), 4-hydroxytamoxifen (4-OHT, 2), raloxifene (RAL, 3), 17β-estradiol (E2), ICI-182,780 (fulvestrant, 4a), and ICI-164,384 (4b).

E2 (the most biological active estrogen) and derivative are used by millions of women in Hormone Replacement Therapy (HRT) for the treatment of peri- and post-menopausal related symptoms [23-25]. E2 also served as a framework for the attachment of various substituents for ER therapeutic applications in the treatment of hormone-dependent breast cancer [26, 27]. Studies have shown that E2 derivatives ICI-182,780 (Fulvestrant, 4a) and ICI-164,384 (4b) are as effective as tamoxifen in the treatment breast cancer [28, 29]. This finding and others led to Food & Drugs Administration (FDA) approving Fulvestrant for the treatment of post-menopausal ER (+) breast cancers [30].

Recently, our group has demonstrated that 3-N-alkylaminoethoxy derivatives of E2 showed sufficient potent activity in inhibiting the E2-stimulated proliferation of MCF-7 cancer cells [31]. Therefore, based on this and other previous investigations involving the attachment of side chain group to biologically active E2, we herein report the synthesis of new series of estrogen derivatives (Scheme 1) with various alkylaminopropoxy groups at the Carbon-3 position. These compounds were also evaluated for in vitro antiproliferative activities against MCF-7, MDA-MB-231 human breast cancer cell lines, and Ishikawa human endometrial cell line. These cell lines are widely accepted models for assessing potent anti-proliferative and antiestrogenic compounds. TAM, 4-OHT and RAL were used as standards for comparison purposes in all these studies.

Scheme (1).

Scheme (1)

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Reagents and Conditions: (i) Appropriate alkyl halide (R-Cl), K2CO3, NaI, acetone, reflux (method A).

MATERIALS AND METHODS

Experimental Section

General

Commercial grade solvents and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Alfa Aeser (Ward Hill, MA, USA) and used without further purification. The NMR spectra were recorded on Varian 300 MHz spectrometer (1H: 300 MHz, 13C: 75MHz,). The appropriate deuterated solvents are indicated in the procedure, and line positions recorded in ppm from the reference signal. ESI-TOF Mass Spectrometer was recorded on Finnigan LCQ - Quadrupole Ion Trap (Thermo Finnigan, San Jose, CA), and the HPLC pump was an Agilent (HP) 1100 series pump or Applied Biosystems model 400 pump. The purification was performed using Flash column chromatography with silica gel 60 (160–200 mesh) obtained from Sigma-Aldrich (St. Louis, MO, USA)

General Procedures for the Synthesis of 3-N-alkylaminopropoxyl Derivatives of Estrogen

Method A

To a solution of estradiol/estrone (1 eq) in anhydrous acetone were added 1-(3-chloropropyl)-alkyl amino hydrochloride (1.1 eq), anhydrous K2CO3 (2.5 eq) and NaI (catalytic amount). The mixture was heated under reflux overnight. After TLC indicated that the reaction was completed, the solvent was removed in vacuum and extracted with ethyl acetate (3 times). The combined organic layer was washed with brine water, dried over anhydrous magnesium sulfate and the solvent removed to afford the crude product that was purified using silica gel flash column chromatography [eluting with mixture of CH2Cl2 - MeOH (9:1, V/V)] to provide the title product.

3-(3-piperidinylpropoxy)estra-1,3,5(10)-trien-17β-ol (5) as white solid

1H-NMR (300MHz, CDCl3) δ 7.18 (d, J=8.4 Hz, 1H, 1-CH), 6.69 (dd, J=8.4, 2.7 Hz, 1H, 2-CH), 6.62 (d, J=2.7 Hz, 1H, 4-CH), 3.97 (t, J=6.3Hz, 2H, 3-OCH2-), 3.73 (td, J=8.6, 5.1Hz, 1H, 17α-CH-), 2.87-2.81 (m, 2H, 6-CH2), 2.46 (t, J=7.5Hz, 2H, -NCH2CH2O-), 2.40 (t, J=5.4 Hz, 4H, -NCH2CH2- of piperidinyl), 2.35-2.26 (m, 1H), 2.25-2.06 (m, 2H), 1.99-1.83 (m, 4H), 1.74-1.16 (m, 15H), 0.78 (s, 3H, 13-CH3); 13C-NMR (75MHz, CDCl3) δ 157.1, 138.1, 132.9, 126.5, 114.8, 112.3, 82.0, 66.7, 56.3, 54.8 (two carbon), 50.3, 44.2, 43.5, 39.1, 37.0, 30.8, 30.0, 27.5, 27.1, 26.6, 26.1 (two carbon), 24.6, 23.4, 11.3; HRMS (ESI-MS) calculated for C26H40NO2 [(M+H)+] 398.3054, found 398.3029.

3-(3-N,N-dimethylaminopropoxy)estra-1,3,5(10)-trien-17β-ol (6) as white solid

1H-NMR (300MHz, CDCl3) δ 7.18 (d, J=8.4 Hz, 1H, 1-CH), 6.70 (dd, J=8.4, 2.7 Hz, 1H, 2-CH), 6.63 (d, J=2.7 Hz, 1H, 4-CH), 3.98 (t, J=6.3 Hz, 2H, 3-OCH2-), 3.72 (t, J=8.4 Hz, 1H, 17α-CH-), 2.87-2.81 (m, 2H, 6-CH2), 2.49 (t, J=7.4 Hz, 2H, -NCH2CH2O-), 2.29 (s, 6H, -NCH3), 2.22-2.06 (m, 3H), 2.00-1.83 (m, 4H), 1.74-1.64 (m, 1H), 1.56-1.13 (m, 8H), 0.78 (s, 3H, 13-CH3); 13C-NMR (75MHz, CDCl3) δ 157.0, 138.2, 132.9, 126.6, 114.6, 112.2, 82.0, 66.2, 56.7, 50.2, 45.6 (two carbon), 44.2, 43.5, 39.1, 37.0, 30.8, 30.0, 27.6, 27.5, 26.6, 23.4, 11.3; HRMS (ESI-MS) calculated for C23H35NO2 [(M+H)+] 358.2741, found 358.2755.

3-(β-N,N-dimethylaminopropoxy)estra-1,3,5(10)-trien-17δ-ol (7) as white solid

1H-NMR (300MHz, CDCl3) δ 7.19 (d, J=8.4 Hz, 1H, 1-CH), 6.72 (dd, J=8.4, 2.7 Hz, 1H, 2-CH), 6.64 (d, J=2.7 Hz, 1H, 4-CH), 4.02 (ddd, J=9.6, 5.7, 2.1 Hz, 1H, 3-OCH2-), 3.82 (ddd, J=9.6, 6.0, 2.4 Hz, 1H, 3-OCH2-), 3.73 (t, J=8.4 Hz, 1H, 17α-CH-), 3.00 (m, 1H, CH3CHNCH3 of isopropyl), 2.87-2.78 (m, 2H, 6-CH2), 2.37 (s, 6H, -CHNCH3), 2.23-2.06 (m, 2H), 1.98-1.84 (m, 3H), 1.74-1.65 (m, 2H), 1.57-1.20 (m, 6H), 1.24 (d, J=6.6Hz, 3H, -NCHCH3), 0.78 (s, 3H, 13-CH3); 13C-NMR (75MHz, CDCl3) δ 156.9, 138.2, 133.0, 126.6, 114.7, 112.3, 82.1, 69.8, 58.7, 50.2, 46.4, 44.2, 43.5, 41.7 (two carbon), 39.0, 37.0, 30.8, 30.0, 27.5, 26.6, 23.4, 11.4; HRMS (ESI-MS) calculated for C23H35NO2 [(M+H)+] 358.2741, found 358.2754.

3-(3-N,N-dimethylaminopropoxy)estra-1,3,5(10)-trien-17-one (8) as white solid

1H-NMR (300MHz, CDCl3) δ 7.18 (d, J=8.4 Hz, 1H, 1-CH), 6.71 (dd, J=8.4, 2.7 Hz, 1H, 2-CH), 6.65 (d, J=2.7 Hz, 1H, 4-CH), 3.98 (t, J=6.3 Hz, 2H, 3-OCH2-), 2.91-2.86 (m, 2H, 6-CH2), 2.43 (t, J=7.4 Hz, 2H, -NCH2CH2O-), 2.49 (dd, 1H, J=8.7, 18.5 Hz), 2.43 (t, J=7.4 Hz, 2H, -NCH2CH2CH2O-), 2.41-2.36 (m, 1H), 2.24 (s, 6H, -NCH3), 2.21-2.19 (m, 1H), 2.16-2.08 (dd, 1H, J=8.7, 18.5 Hz), 2.05-1.88 (m, 5H), 1.66-1.39 (m, 6H), 0.91 (s, 3H, 13-CH3); 13C-NMR (75MHz, CDCl3) δ 221.2, 157.3, 138.0, 132.1, 126.5, 114.7, 112.4, 66.4, 56.7, 50.6, 48.2, 45.8 (two carbon), 44.2, 38.6, 36.1, 31.8, 29.9, 27.9, 27.0, 26.2, 21.8, 14.1; HRMS (ESI-MS) calculated for C23H33NO2 [(M+H)+] 356.2584, found 356.2598.

Method B

To a solution of estradiol (1 eq) in anhydrous acetone was added 1-bromo-3-chloroprane (1.1 eq), anhydrous K2CO3 (2.5 eq) and NaI (catalytic amount). The mixture was heated under reflux overnight. After TLC indicated that the reaction was completed, the solvent was removed in vacuum and extracted with ethyl acetate (3 times). The combined organic layer was washed with brine water, dried over anhydrous magnesium sulfate and the solvent removed to afford the crude product that was purified by recrystallization from hexane to provide 3-chloropropoxyl-estradiol.

To a solution of 3-chloropropoxyl-estradiol (1 eq) in anhydrous acetone was added alkyl amino (1.5 eq), anhydrous K2CO3 (2.5 eq) and NaI (catalytic amount). The mixture was heated under reflux overnight. After TLC indicated that the reaction was completed, the solvent was removed in vacuum and extracted with ethyl acetate (3 times). The combined organic layer was washed with brine water, dried over anhydrous magnesium sulfate and the solvent removed to afford the crude product that was purified using flush column chromatography [eluting with mixture of CH2Cl2 - MeOH (9:1, V/V)] to provide the title product.

3-(3-(4-methylpiperazinyl)-propoxy)estra-1,3,5(10)-trien-17β-ol (9) as white solid

1H-NMR (300MHz, CDCl3) δ 7.18 (d, J=8.4 Hz, 1H, 1-CH), 6.69 (dd, J=8.4, 2.7 Hz, 1H, 2-CH), 6.62 (d, J=2.7 Hz, 1H, 4-CH), 3.98 (t, J=6.6Hz, 2H, 3-OCH2-), 3.73 (t, J=8.7 Hz, 1H, 17α-CH-), 2.87-2.81 (m, 2H, 6-CH2), 2.54-2.47 (m, 10H, -NCH2-), 2.35-2.30(m, 1H), 2.28 (s, 3H, CH3N- of piperazinyl), 2.21-2.04 (m, 3H), 1.98-1.83 (m, 4H), 1.74-0.88 (m, 8H), 0.78 (s, 3H, 13-CH3); 13C-NMR (75MHz, CDCl3) δ 157.1, 138.1, 132.9, 126.5, 114.8, 112.3, 82.0, 66.4, 55.4, 55.3 (two carbon), 53.4 (two carbon), 50.3, 46.2, 44.2, 43.5, 39.1, 37.0, 30.8, 30.0, 27.5, 27.1, 26.6, 23.4, 11.3; HRMS (ESI-MS) calculated for C26H41N2O2 [(M+H)+] 413.3163, found 413.3191.

3-(3-morpholinylpropoxy)estra-1,3,5(10)-trien-17β-ol (10) as white solid

1H-NMR (300MHz, CDCl3) δ 7.19 (d, J=8.4 Hz, 1H, 1-CH), 6.70 (dd, J=8.4, 2.7 Hz, 1H, 2-CH), 6.62 (d, J=2.7 Hz, 1H, 4-CH), 3.99 (t, J=6.3Hz, 2H, 3-OCH2-), 3.71 (t, J=4.8Hz, 4H, -OCH2CH2N- in morpholinyl; m, 1H, 17α-CH-), 2.87-2.82 (m, 2H, 6-CH2), 2.50(t, J=6.9Hz, 2H, -NCH2CH2O-), 2.46 (t, J=4.5Hz, 4H, -NCH2CH2 of morpholinyl), 2.35-2.25 (m, 1H), 2.21-2.05 (m, 2H), 1.99-1.84 (m, 4H), 1.64-1.74 (m, 1H), 1.51-1.41 (m, 8H), 0.78 (s, 3H, 13-CH3); 13C-NMR (75MHz, CDCl3) δ 157.1, 138.1, 132.8, 126.5, 114.7, 112.2, 81.9, 67.1 (two carbon), 66.2, 55.9, 53.9 (two carbon), 50.3, 44.2, 43.5, 39.1, 37.0, 30.7, 30.0, 27.5, 26.7, 26.5, 23.3, 11.3; HRMS (ESI-MS) calculated for C25H38NO3 [(M+H)+] 400.2846, found 400.2805.

3-(3-pyrrolidinylpropoxy)estra-1,3,5(10)-trien-17β-ol (11) as white solid

1H-NMR (300MHz, CDCl3) δ 7.18 (d, J=8.4 Hz, 1H, 1-CH), 6.70 (dd, J=8.4, 2.7 Hz, 1H, 2-CH), 6.63 (d, J=2.7 Hz, 1H, 4-CH), 3.99 (t, J=6.3Hz, 2H, 3-OCH2-), 3.73 (t, J=8.4, 1H, 17α-CH-), 2.87-2.81 (m, 2H, 6-CH2), 2.61 (t, J=7.5 Hz, 2H, -NCH2CH2O-), 2.55-2.47 (m, 4H, -NCH2CH2 of pyrrolidinyl), 2.33-2.28 (m, 1H), 2.22-2.07 (m, 2H), 2.03-1.83 (m, 4H), 1.80-1.72 (m, 4H, -NCH2CH2- in pyrrolidinyl), 1.71-1.16 (m, 8H), 0.78 (s, 3H, 13-CH3); 13C-NMR (75MHz, CDCl3) δ 157.1, 138.1, 132.8, 126.5, 114.8, 112.3, 82.1, 66.6, 54.5 (two carbon), 53.4, 50.3, 44.2, 43.5, 39.1, 37.0, 30.9, 30.0, 29.1, 27.5, 26.6, 23.7 (two carbon), 23.4, 11.3; HRMS (ESI-MS) calculated for C25H39NO2 [(M+H)+] 384.2897, found 384.2867.

3-(3-N,N-diisopropylaminopropoxy)estra-1,3,5(10)-trien-17β-ol (12) as white solid

1H-NMR (300MHz, CDCl3) δ 7.18 (d, J=8.4 Hz, 1H, 1-CH), 6.70 (dd, J=8.4, 2.7 Hz, 1H, 2-CH), 6.63 (d, J=2.7 Hz, 1H, 4-CH), 3.97 (t, J=7.2Hz, 2H, 3-OCH2-), 3.72 (t, J=8.4Hz, 1H, 17α-CH-), 3.02 (m, J=6.9Hz, 2H, -NCHCH3 of isopropyl), 2.85-2.80 (m, 2H, 6-CH2), 2.60 (t, J=6.9Hz, 2H, -NCH2CH2CH2O- ), 2.35-2.25 (m, 1H), 2.20-2.06 (m, 3H), 1.98-1.79 (m, 3H), 1.74-1.65 (m, 1H), 1.40-1.07 (m, 8H), 1.01 (d, J=6.9Hz, 12H, -NCHCH3), 0.78 (s, 3H, 13-CH3); 13C-NMR (75MHz, CDCl3) δ 157.0, 138.2, 133.0, 126.5, 114.8, 112.4, 82.0, 66.3, 64.8, 50.4, 48.7, 44.2, 43.5, 41.9, 39.1, 37.0, 30.8, 30.0, 27.6, 27.5, 26.6, 23.4, 21.0 (four carbon), 11.4; HRMS (ESI-MS) calculated for C27H44NO2 [(M+H)+] 414.3367, found 414.3367.

3-(3-N,N-diethylaminopropoxy)estra-1,3,5(10)-trien-17β-ol (13) as white solid

1H-NMR (300MHz, CDCl3) δ 7.18 (d, J=8.4 Hz, 1H, 1-CH), 6.69 (dd, J=8.4, 2.7 Hz, 1H, 2-CH), 6.63 (d, J=2.7 Hz, 1H, 4-CH), 3.97 (t, J=6.6Hz, 2H, 3-OCH2-), 3.72 (t, J=8.4Hz, 1H, 17α-CH-), 2.87-2.82 (m, 2H, 6-CH2), 2.60 (t, J=7.2Hz, 2H, -NCH2CH2O-), 2.54 (q, J=7.2 Hz, 4H, -NCH2CH3), 2.34-2.22 (m, 1H), 2.17-2.05 (m, 2H), 1.98-1.83 (m, 4H), 1.74-1.64 (m, 1H), 1.56-1.10 (m, 8H), 1.02 (t, J=7.2Hz, 6H, -NCH2CH3, 0.78 (s, 3H, 13-CH3); 13C-NMR (75MHz, CDCl3) δ 157.2, 138.1, 132.8, 126.4, 114.8, 112.3, 81.9, 66.6, 52.1, 50.3, 47.2 (two carbon), 44.2, 43.5, 39.1, 37.0, 30.8, 30.0, 27.5, 27.2, 26.6, 23.4, 12.0 (two carbon), 11.3; HRMS (ESI-MS) calculated for C25H40NO2 [(M+H)+] 386.3054, found 386.3058.

3-(3-N-methylaminopropoxy)estra-1,3,5(10)-trien-17β-ol (14) as white solid

1H-NMR (300MHz, CDCl3) δ 7.18 (d, J=8.4 Hz, 1H, 1-CH), 6.70 (dd, J=8.4, 2.7 Hz, 1H, 2-CH), 6.63 (d, J=2.7 Hz, 1H, 4-CH), 4.01 (t, J=6.3Hz, 2H, 3-OCH2-), 3.72 (t, J=8.6 Hz, 1H, 17α-CH-), 2.87-2.81 (m, 2H, 6-CH2), 2.77 (t, J=6.9 Hz, 2H, -NCH2CH2O-), 2.46 (s, 3H, CH3NH-), 2.35-2.25 (m, 1H), 2.20-2.05 (m, 2H), 2.00-1.83 (m, 4H), 1.74-1.65 (m, 1H), 1.56 (br, s, 1H, -NHCH3), 1.52-1.14 (m, 8H), 0.78 (s, 3H, 13-CH3); 13C-NMR (75MHz, CDCl3) δ 157.0, 138.2, 132.9, 126.5, 114.7, 112.3, 82.1, 66.5, 50.3, 49.3, 44.2, 43.5, 39.1, 37.0, 36.6, 30.8, 30.0, 29.7, 27.5, 26.6, 23.4, 11.3; HRMS (ESI-MS) calculated for C22H33NO2 [(M+H)+] 344.2584, found 344.2598.

3-(3-aminopropoxy)estra-1,3,5(10)-trien-17β-ol (15) as white solid

1H-NMR (300MHz, CDCl3) δ 7.18 (d, J=8.4 Hz, 1H, 1-CH), 6.70 (dd, J=8.4, 2.7 Hz, 1H, 2-CH), 6.63 (d, J=2.7 Hz, 1H, 4-CH), 4.01 (t, J=6.0Hz, 2H, 3-OCH2-), 3.72 (t, J=8.7 Hz, 1H, 17α-CH-), 3.38 (t, J=6.8 Hz, 1H, H2NCH2-), 2.89 (t, J=6.8 Hz, 1H, H2NCH2-), 2.85-2.81 (m, 2H, 6-CH2), 2.34-2.26 (m, 1H), 2.18-1.87 (m, 6H), 1.73-1.64 (m, 3H), 1.56-1.13 (m, 8H), 0.78 (s, 3H, 13-CH3); 13C-NMR (75MHz, CDCl3) δ 157.0, 138.2, 132.9, 126.6, 114.6, 112.2, 82.0, 66.0, 50.2, 44.2, 43.5, 39.6, 39.1, 37.0, 33.2, 30.8, 30.0, 27.5, 26.6, 23.4, 11.3; HRMS (ESI-MS) calculated for C21H31NO2 [(M+H)+] 330.2428, found 330.2437.

Antiproliferative Activity Studies

The antiproliferative activity of compounds 5-15 was evaluated at the Southern Research Institute (SRI, Birmingham, Alabama, USA) according to procedures [32]. The compounds were screened against human ER (+) MCF-7 (breast), ER (−) MDA-MB-231 (breast) and Ishikawa (endometrial) cancer cell lines in comparison to TAM, 4-OHT and RAL.

Material

Human MCF-7 and MDA-MB-231 breast cancer cell lines were purchased from the NCI. The human Ishikawa endometrial cancer cell line was purchased from Sigma Aldrich company. All three-cell lines were cultured in phenol red-free RPMI-1640 (Hyclone) (500 mL) supplemented with L-glutamine-dipeptide (Hyclone) (5 mL), and 10% fetal bovine serum (Atlanta Biologicals) (50 mL).

Method

All three cell lines were cultured and treated with compounds 5-15, TAM, 4-OHT and RAL ranging from 0.01-100,000 nM concentration in the presence of 10 nM estradiol using the previous reported method [33]. The results expressed as IC50 (Inhibitory concentration of 50%) values were the averages of three data points for each concentration and were calculated using GraphPad Prism 4.0.

RESULTS

Chemistry

The 3-N-aminopropoxy derivatives of estrogen 5-8 were synthesized according to the general procedure outlined scheme 1. In this investigation, the alkylation of the 3-position was achieved by reacting 17β-estradiol/estrone with chloropropyl-alkylamino group in the presence of potassium carbonate (K2CO3) and NaI (catalytic amount) in acetone (method A) to afford the desired compounds in moderate to good yields. This method has also been widely using in the alkylation of phenolic group to afford the desired product in up to 80% yield and is said to involve a Finkelstein reaction, followed by an SN2 reaction to afford the desired product [34-36]. Compounds 5-8 were purified using silica gel flash column chromatography eluting with mixture of CH2Cl2-MeOH (9: 1 V/V) to afford the pure analogs between 54-77%, which were characterized using proton (1H) NMR, carbon (13C) NMR and high resolution mass spectra (ESI-MS, Electrospray Ionization) analysis as indicated in the experimental section. Mass spectroscopic (ESI, Electrospray Ionization) analysis revealed molecular ion at m/z 398.3029 [M+H]+ corresponding to compound 5, m/z 358.2755 [M+H]+ corresponding to compound 6, m/z 358.2754 [M+H]+ corresponding to compound 7 and m/z 356.2598 [M+H]+ corresponding to compound 8.

In order to synthesize the 3-N-alkylaminopropoxyl E2 derivatives 9-15, we first synthesized the key intermediate, 3-chloropropoxy substituted E2, by reacting a mixture of 1-bromo-3-chloro propane and E2 in the presence of potassium carbonate (K2CO3) in acetone (Scheme 2). The intermediate, 3-chloropropoxy substituted E2, was then reacted with appropriate tertiary amino groups to afford 3-N-aminopropoxy E2 9-15 in good to moderate yields (65-78%). The advantage of this method is that it does provide access to other derivatives with increase –CH2 group at the tertiary aminoalkoxy side chain group.

Scheme (2).

Scheme (2)

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Reagents and Conditions: (i) 1-bromo-3-chloro propane, K2CO3, acetone, reflux, (ii) Appropriate tertiary amino groups, K2CO3, NaI, acetone, reflux (method B).

Antiproliferative Activity

In vitro antiproliferative activity of compounds 5-15 were evaluated against human ER (+) MCF-7 (breast), ER (−) MDA-MB-231 (breast) and Ishikawa (endometrial) cancer cell lines at concentration ranging from (0.01 – 100,000 nM) in the presence of 10 nM E2 using CellTiter-Glo assay (E2 was used for competitive growth inhibitory studies). As shown in Table 1, compounds 5-9 and 11- 15 (IC50 = 10-25 μM) demonstrated significant antiproliferative activity against human ER (+) MCF-7 breast cancer cell line in comparison to TAM (IC50 = 26 μM), but were less potent than 4-OHT and RAL (IC50 = 2 and 3 μM, respectively) (Note: IC50 is the concentration of test drug where a 50% reduction is observed in cell growth compared to the untreated control after a 72 h period of exposure to test drug). Compounds bearing dimethylaminopropoxy (6) and diethylaminopropoxy (13) groups, the most active of the series in this cell line, exhibit higher antiproliferative activity against MCF-7 cell line based on the IC50 value (Fig. 2a).

Table 1. IC50 Values (μM) for Compounds 1-15 Tested against Human (ER+) MCF-7 (Breast), (ER−) MDA-MB231 (Breast) and Ishikawa (Endometrial) Cancer Cells.
Compounds No. Ishikawa (μM) MDA-MB-231 (μM) MCF-7 (μM)
1 (TAM) 33 19 26
2 (4-OHT) 20 22 2
3 (RAL) 23 28 3
5 39 12 20
6 25 25 16
7 16 28 25
8 19 26 21
9 25 26 22
10 >100 82 >100
11 21 25 25
12 28 29 18
13 19 36 16
14 24 29 28
15 18 20 20
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1

The data represent the average of triplicate determinations at various concentrations.

2

The IC50 values were determined from the graphs (GraphPad Prism) using mean values of data points at various concentrations.

Fig. (2).

Fig. (2)

Open in a new tab

In vitro antiproliferative activity of (a) compound 6 against (ER+) MCF-7 cell line, (b) compound 5 against (ER−) MDA-MB-231 cell line and (c) compound 7 against Ishikawa cell line; in comparison to TAM, 4-OHT and RAL.

The antiproliferative activity of compounds 5-15 against human ER (−) MDA-MB-231 breast cancer cell lines were also investigated to shed some light on their mechanism of action. It was observed in this cell line that (i) compounds 6-9, 11, 12, 14 and 15 (IC50 = 20-29 μM) showed comparable cytotoxicity to the standard drugs, TAM (IC50 = 19 μM), 4-OHT (IC50 = 22 μM), and RAL (IC50 = 28 μM), respectively (Table 1); and (ii) compound 5 (IC50 = 12 μM) possessing piperidinylpropoxyl group, the most active of the series in this cell line, exhibits similar antiproliferative activity based on the IC50 value in comparison to TAM, 4-OHT and RAL (Fig. 2b). This result suggests that compound 5 may also inhibit cell proliferation via ER-independent mechanism in comparison to TAM.

Furthermore, the antiproliferative activity of these compounds against human Ishikawa endometrial cell line was also evaluated and results (Table 1) revealed that (i) compounds 7, 8, 13 and 15 (IC50 = 16, 19, and 18 M; respectively) were more active than TAM (IC50 = 33 μM), 4-OHT (IC50 = 20 μM), and RAL (IC50 = 23 μM) in inhibiting the E2 stimulated proliferation of the endometrial cells and (ii) compound 7 possessing β-dimethylaminoisopropoxyl group, the most active of the series in this cell line, exhibits higher antiproliferative activity (Fig. 2c). This result indicates that compound 7 may lower the risk of developing uterine cancer based upon the IC50 value in comparison with TAM.

DISCUSSION

One of the chemical features of antiestrogenic compounds known as ER ligands is the basic amino side chain (tertiary aminoalkoxy group) [37-39]. The nature of the side chain and its orientation relative to the ligand backbone play an important role in the determination of their tissue-selective activity [40, 41]. In view of the above facts and in order to further develop an understanding of the structural requirement for E2 derivative containing amino the side chain group, we proposed the incorporation of the tertiary aminopropoxy group at the C-3 position of E2 to afford 3-N-alkylaminopropoxyl estradiol derivatives (Scheme 1-2). The antiproliferative activities of these compounds 5-15 against MCF-7, MDA-MB-231 and Ishikawa cell lines as a test models were evaluated using CellTiter-Glo assay [32]. Antiproliferative activities of these compounds in MCF-7 cell line indicates that compounds 6 and 13 were more active than TAM and may act as ER antagonists based on the antiproliferative mechanism (ER-dependent mechanisms) of TAM against ER (+) MCF-7 breast cancer cells; inhibition of E2 binding to the ER [42]. However, further studies are needed to ascertain this claim.

Recent studies have also shown that TAM may inhibit cell proliferation via ER-independent mechanisms against ER (−) MDA-MB-231 breast cancer cell line [43-46]. This cell line constitutes an original model for identifying the ER-independent mechanisms of TAM antiproliferative effects [47, 48]. The results obtained against MDA-MB-231 breast cancer cell line indicate that the antiproliferative activity of compound 5 were comparable to the standard drugs, thus suggesting that they may also inhibit cell proliferation via ER-independent mechanism. Also, in the Ishikawa cell line, compound 5 showed a higher IC50 value that indicates possible risk for uterine cancer development.

In the development of antiestrogenic SERM drugs, it is critical that the candidate drug minimize undesirable uterine effect (agonist activity), which could lead to both increase in uterine bleeding and an increased risk of developing uterine cancer. The Ishikawa cell line is an endometrial adenocarcinoma cell line, which has been used as test model in evaluating this effect [48]. Finally, this present results further confirm the importance of the basic amino side chain groups with respect to the antiproliferative activity of the compounds.

CONCLUSION

3-N-substituted-estrogen derivatives were synthesized, and characterized using proton (1H) NMR, carbon (13C) NMR and high-resolution mass spectra (ESI-MS, Electrospray Ionization). Their antiproliferative activities against human ER (+) MCF-7 (breast), ER (−) MDA-MB-231 (breast) and Ishikawa (endometrial) cell lines were determined and compared to that of the standard antiestrogen drugs TAM, 4-OHT, and RAL. In vitro results indicated that the synthesized E2 derivatives containing piperidinylethoxy 5, displayed slightly higher antiproliferative activity against human MDA-MB-231 (hormone-independent), MCF-7 (hormone-dependent); as evident by the lowest IC50 value in comparison with TAM. Nevertheless, compound 5 has a higher liability in causing uterine cancer based upon the higher IC50 value against Ishikawa cell line in comparison with TAM. Furthermore, it is worth noting that compound 5 displayed lower antiproliferative activities against MCF-7 (hormone-dependent) in comparison to RAL and 4-OHT. Compound 15 displayed moderate activities against Ishikawa (endometrial), MCF-7 and MDA-MB-231 (breast) cell lines, indicating that it is the overall best candidate to be considered for future studies These studies will involve ER binding studies, uterotropic and antiuterotropic activities in immature rat in order to further understand how these compounds interact with the ER.

ACKNOWLEDGEMENTS

This research was supported by the National Center for Research Resources and the National Institute of Minority Health and Health Disparities of the National Institutes of Health through Grant Number 8 G12 MD007582-28 and 2 G12 RR003020.

Footnotes

CONFLICT OF INTEREST

There is no conflict of interest associated with this publication.

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