Design, synthesis and bioevaluation of novel 6-(4-Hydroxypiperidino)naphthalen-2-ol-based potential Selective Estrogen Receptor Modulators for breast cancer

European Journal of Medicinal Chemistry 92 (2015) 103e114

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

Original article

Design, synthesis and bioevaluation of novel 6-(4-Hydroxypiperidino) naphthalen-2-ol-based potential Selective Estrogen Receptor Modulators for breast cancer Amitabh Jha a, *, Yogesh Yadav a, b, Ajay B. Naidu a, V. Kameswara Rao a, c, Anil Kumar c, Virinder S. Parmar b, William J. MacDonald d, Catherine K.L. Too d, Jan Balzarini e, Christopher J. Barden f, T. Stanley Cameron g a

Department of Chemistry, Acadia University, Wolfville, NS B4P 2R6, Canada Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India c Department of Chemistry, Birla Institute of Technology and Science, Pilani, Rajasthan, India d Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Canada e Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium f Toronto Western Research Institute, Toronto M5T 2S8, Canada g Department of Chemistry, Dalhousie University, Halifax B3H 4R2, Canada b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 September 2014 Received in revised form 1 November 2014 Accepted 21 December 2014 Available online 23 December 2014

In a study directed towards development of novel Selective Estrogen Receptor Modulators (SERMs), 1-(4(2-(dialkylamino)ethoxy)benzyl)-6-(4-hydroxypiperidin-1-yl)-2-naphthol and corresponding aryl methyl ethers were synthesized and bioevaluated against the estrogen-responsive human MCF-7 breast cancer cell line. The phenolic analogs displayed little or no activity, but aryl methyl ether analogs showed significant cytotoxic potency. Also, representative compounds from the aryl methyl ether series showed significant binding and antagonistic activity against ERa. Two representative compounds were also evaluated for in vitro membrane permeability, plasma stability as well as in-vivo toxicity in mice. The compounds displayed well-acceptable drug-like in vitro membrane permeability as well as plasma stability and were well-tolerated in experimental mice at 300 mg/kg dose. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Breast cancer Anticancer Cytotoxic SERM 1-(4-Dialkylaminoethoxy)phenylmethyl-6(4-hydroxypiperidino)naphthalen-2-ols Pharmacokinetics

1. Introduction Breast cancer is the most frequently encountered cancer in women and the second leading cause of cancer related deaths [1]. There are evidences to show that estrogens play an important role in the proliferation of breast cancer [2]. Selective estrogen receptor modulators (SERMs) are compounds used to treat breast cancer by blocking the effect of estrogens in breast tissues [3,4]. These are chemical entities which bind to the estrogen receptors (ERa or ERb subtypes) in much the same way as estrogens do, however, their effects range from anti-estrogenic in some cases to estrogenic in others [5,6]. The pharmacodynamic response to a SERM is

* Corresponding author. E-mail address: [email protected] (A. Jha). http://dx.doi.org/10.1016/j.ejmech.2014.12.037 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

determined by both the location of the specific ER to which it binds, as well as the ER subtype [7]. Although most SERMs decrease the risk of breast cancer, the side effects of many of these drugs are deleterious [8]. The most widely used therapy for the treatment of estrogen-responsive breast cancer is using considerably effective SERMs, such as tamoxifen and raloxifene [9,10]. The administration of tamoxifen is sometimes accompanied by negative side effects such as uterine cancer [11]. Raloxifene is as effective as tamoxifen in the treatment of estrogen-responsive breast cancer and, while it shows lower incidence of uterine cancer, it causes thrombosis and fatal stroke in postmenopausal women [12,13]. Thus, there is still a need to develop new and more potent SERMs which are devoid of deleterious side effects. The generic structure of the prototypical novel potential SERMs designed for our present study is shown in Fig. 1, along with

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Fig. 1. Prototypical novel potential SERMs compared with 17b-estradiol, tamoxifen, afimoxifene, raloxifene and 2-napthol-based SERMs.

structures of 17b-estradiol (the most potent estrogen), prodrug tamoxifen and its active metabolite afimoxifene [14], raloxifene and potential SERMs based on 2-naphthol Mannich bases previously studied in our laboratory [15]. Generally speaking, most SERMs possess two important structural features aptly placed in relation to each other: a) mono- or dihydroxy scaffold to mimic 17b-estradiol; in case of dihydroxy SERMs, the two hydroxyl groups are placed ~11 Å apart [16], and b) a dialkylaminoethoxyphenyl activity modulating side chain found on most clinical SERMs such as tamoxifen, afimoxifene, raloxifene (Fig. 1), etc. [17]. It has been discerned by X-ray crystallographic studies that the two hydroxyl groups on the SERMs H-bond with Glu 353-H2O-Arg 394 triad and His 524 whereas the basic amino group of the dialkylaminoethoxyphenyl side chain creates electrostatic interaction with Asp 351 of the ligand binding domain of the ERa [17]. Based on this information, we have designed two series of compounds, series 1 and 2 (Fig. 1). In silico molecular modeling experiments were performed on one representative compound from each series (eNR0 2 ¼ piperidino). Calculations on the energy-minimized structures of these compounds indicated a distance of 11.4 Å between the oxygen atoms of piperidinol eOH and eOCH3 or phenolic eOH in series 1 and 2 compounds, respectively [18]. To decipher the expected interaction of the three critical functional groups with the appropriate amino acid residues in the ligand binding domain of human ERa, docking studies were undertaken on a representative series 1 compound (eNR0 2 ¼ piperidine). These revealed that at least two out of these three functional groups, the piperidinol hydroxyl group and piperidine N of the piperidinoethoxyphenyl were in the realm of interaction with respective amino acid residues on the ligand binding domain of ERa (vide infra) [19]. We believed that these features will render potent selective ER modulatory activity to the designed molecules. Thus, towards our continuous effort to develop novel anticancer agents [20e23] including SERM candidates [15,24], herein we report the design, synthesis and relevant bioevaluation of several 1-(5-(4-(2(dialkylamino)ethoxy)benzyl)-6-alkoxynaphthalen-2-yl)piperidin4-ols and related compounds as potential SERMs.

2. Results and discussion 2.1. Chemistry The synthesis of compounds in series 1 and 2 was envisioned as follows. Reaction of commercial 2-bromo-6-methoxynaphthalene

(3) with 4-piperidinol under Ullmann coupling conditions was expected to produce 1-(6-methoxynaphthalen-2-yl)piperidin-4-ol (4). Compound 4 was anticipated to undergo Lewis acid-catalyzed electrophilic C-monoalkylation with (4-(2-(dialkylamino)ethoxy) phenyl)methanols (5aeh) [15] to yield series 1 compounds. Demethylation of series 1 compounds was expected to produce series 2 compounds. We succeeded in synthesizing compounds 1aeh following this protocol (Scheme 1). However, the subsequent demethylation reaction to compounds 2aeh failed under numerous conditions (Aq HBr; BBr3 in dichloromethane; LiCl in dimethylformamide; NaSEt in dimethylformamide, etc). To circumvent this impasse, we initiated the synthesis from commercial 2-benzyloxy-6-bromonaphthalene (6) and followed the same synthetic strategy to produce compounds 8aeh in two steps. Catalytic hydrogenolysis of compounds 8aeh led to the formation series 2 compounds (Scheme 1). All compounds belonging to series 1, 2 and 8 reported in this paper are new to chemical literature. All compounds were characterized by usual spectroscopic means, however the absolute confirmation of their chemical structures was not as simple as it initially seemed. Electrophilic Cmonoarylmethylation of 4 (or 7) with (4-(2-(dialkylamino)ethoxy) phenyl)methanols 5 in the presence of BF3. Et2O is expected to occur either at C-1 or at C-5 position resulting in the formation of the regioisomer A or B owing to the highly activating nature of the alkoxy and amino functionalities, respectively (Fig. 2). However, we believe that regioisomer A should be the most likely (or exclusive) product even if amines are known to be more activating than ethers [25]. This is because BF3 etherate-mediated arylmethylation at C-5 (ortho to piperidine group) will be retarded due to formation of deactivating Lewis adduct on the amine in the reaction mixture [26,27], as well as the steric hindrance by the larger piperidine group. As can be expected, the common spectroscopic data of the products of these reactions (1aeh and 8aeh) were inadequate to distinguish which regioisomer was formed. In order to conclusively ascertain correct structural assignment, we resorted to nuclear Overhauser effect (NOE) experiment on two products, viz. 1b and 8b. For the compounds to have structure corresponding to regioisomer B (Fig. 2), irradiation on ArCH2- group should lead to strong NOE enhancement on piperidine NeCH2 protons. Since there are five NeCH2 groups in the molecule, it was important to determine which resonance belonged to piperidine NeCH2 protons. This was achieved by performing 1H and COSY experiments on the compounds. 1D NOESY experiments on test compounds did not

A. Jha et al. / European Journal of Medicinal Chemistry 92 (2015) 103e114

105

Scheme 1. Synthesis of novel 6-(4-hydroxypiperidin-1-yl)naphthalen-2-ol-derived SERMs.

Fig. 2. Two possible regioisomers after C-arylmethylation of compound 4 or 7.

show any NOE enhancements on piperidine protons. Furthermore, small NOE enhancement was observed on the eOCH3 of 1b and eOCH2 of 8b. This led to the conclusion that the products obtained corresponded to regioisomers A (Fig. 3). Also, as expected, strong NOE enhancements were observed on aromatic protons indicated by blue dots (in the web version) in Fig. 3. The authenticity in our structural assignment was further substantiated by performing single X-ray crystallography on the same pair of molecules, 1b and 8b. As depicted in Fig. 4, these structures, 1b (CCDC No. 1025756) and 8b (CCDC No. 1025757), unambiguously establish that the structures of these compounds as correspond to regioisomer A.

2.2. Biological results 2.2.1. Cytotoxicity against estrogen-responsive human MCF-7 breast cancer cells After successful synthesis of a total of sixteen novel compounds belonging to series 1 and 2, their cytotoxicity was evaluated against estrogen-responsive human MCF-7 breast cancer cells using the 3(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium (MTS) cell viability assay (Promega Corp., Madison, USA). The cytotoxicity results are summarized in Table 1. Compounds 1aeh except 1f, showed significant in vitro cytotoxicity against MCF-7 cells that surpassed those of clinical SERMs tamoxifen and raloxifene. However, to our surprise and disappointment, their dihydroxy analogs 2aeh displayed poor cytotoxicity in the same assay system (Table 1). Since the only difference in the structures of compounds 1a

Fig. 3. NOE enhancements on compounds 1b and 8b.

through 1h is in the dialkylaminoethoxy group, it appeared reasonable to assume that the difference in their cytotoxicities in MCF-7 cells was due to the structures of the respective secondary amino groups. The most potent compound was found to be 1h with homopiperdino group as the dialkylamino substituent. Also, among the tested series 1 compounds, 1f bearing morpholino group was found to possess the lowest cytotoxic potency. Similar observations were made in our previous study on potential SERMs bearing morpholino group [15]. Most compounds in series 2 had the MCF-7 cytotoxicity IC50 value greater than 25 mM. The poor activity of these phenolic compounds could be attributed to their higher polarity leading to poor permeation through lipophilic cell membranes. 2.2.2. Cytostatic activity against human CEM and HeLa cells, and murine L1210 cells To assess the selectivity of compounds 1aeh, their cytostatic potency was tested against murine L1210 lymphocytic leukemia, human HeLa cervical cancer and human CEM T-lymphoblast cell lines. These results are summarized in Table 2. Among these compounds, 1b, 1c, 1e, 1g and 1h exhibited higher cytostatic activity than either clinical SERM tamoxifen or raloxifene towards murine L1210 cells, while other compounds exhibited moderate to good cytostatic potency. Here again, compound 1h turned out to be most potent against all three cell lines. In general, the murine L1210 cells were found to be most sensitive, followed by CEM cells and then HeLa cells (Table 2). It should be noted that nuclear receptors, such as ER play a role in proliferation of L1210 [28] and CEM [29] cells but not in wild-type HeLa cells [30]. 2.2.3. Cytostatic activity against NCI's 60 tumor cell line panel Encouraged by these results, compounds 1b, 1d, 1g and 1h were also evaluated against NCI's panel of 60 human tumor cell lines representing nine different neoplastic conditions, namely leukemia, melanoma, non-small cell lung, colon, central nervous system, ovarian, renal, prostate, and breast cancers [31]. The results are summarized in Table 3. As shown in Table 3, when all cell lines were considered, both compounds 1g and 1h were significantly more cytostatic than tamoxifen and raloxifene, compound 1h being the most potent. An important requirement of a candidate anticancer drug is selectively toxic towards certain tumor cell types rather than indiscriminate toxicity to all cells. The selectivity index (SI) figures for all tumor cell lines indicated that the test compounds from series 1 were moderately selective in their cytostatic action (SI range: 11e20), but were considerably more selective than tamoxifen (SI 7.9). Raloxifene boasted an impressive selectivity index (SI 251.2) in favor of inhibiting the growth of leukemia and breast cancer cell lines. A comparative review of the GI50 values with respect to various cell line classes in Table 3 revealed that the test compounds from series

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Fig. 4. ORTEP diagrams of 1b and 8b.

Table 1 In-vitro cytotoxicity data of compounds of series 1 and 2 against MCF-7 breast cancer cells. Entry 1 2 3 4 5 6 7 8 9

Compound 1a 1b 1c 1d 1e 1f 1g 1h Tamoxifen

IC50 (mM) 8.49 4.73 3.44 5.20 4.32 >25 3.41 2.46 10.3

Entry

Compound

10 11 12 13 14 15 16 17 18

2a 2b 2c 2d 2e 2f 2g 2h Raloxifene

IC50 (mM)a >50 >50 >25, >25, >25, >50 >25, >25, 16.3

<50 <50 <50 <50 <50

Table 2 Cytostatic potency of compounds 1aeh against murine L1210, human CEM Tlymphocyte and human HeLa cell lines. GI50 (mM)a L1210 1a 1b 1c 1d 1e 1f 1g 1h Tamoxifen Raloxifene

5.9 1.7 2.1 5.2 2.4 11 2.5 1.9 6.7 5.0

± ± ± ± ± ± ± ± ± ±

1.3 0.7 0.8 1.5 1.1 2 1.7 0.3 3.0 1.7

CEM 7.2 4.9 6.7 9.8 11 18 8.0 3.5 6.3 5.6

± ± ± ± ± ± ± ± ± ±

HeLa 0.9 2.4 0.3 4.0 6 1 3.7 1.2 2.1 1.9

15 16 19 21 9.8 18 13 3.8 17 15

Cell lines All cell lines

a The IC50 represents the concentration required to inhibit tumor cell proliferation by 50%.

Compound

Table 3 Evaluation of compounds 1b, 1d, 1g and 1h and reference drugs against a panel of human tumor cell lines.

± ± ± ± ± ± ± ± ± ±

2 1 0 1 6.5 1 6 0.3 4 1

a GI50 refers to the compound concentrations required to inhibit the growth of the cells by 50%.

1 exerted greater toxicity against leukemic, colon, melanoma and breast cancer cell lines than against those cell lines representing other neoplastic diseases. These findings are going to be of value in designing better SERMs. 2.2.4. ERa binding affinity assay Compound 1h, with most potent cytotoxicity against MCF-7 cells, was next evaluated for its ability to bind and antagonize

Leukemia Lung cancer Colon cancer CNS cancer Melanoma Ovarian cancer Renal cancer Prostate cancer Breast cancer

GI50 SIb GI50 GI50 GI50 GI50 GI50 GI50 GI50 GI50 GI50

(mM) (mM) (mM) (mM) (mM) (mM) (mM) (mM) (mM) (mM)

a

1b

1d

1g

1h

TEMc

RALd

5.93 11.0 3.94 10.20 3.48 8.38 3.00 12.54 6.84 12.16 3.34

6.34 11.0 3.02 10.77 3.09 8.35 3.60 12.75 9.28 11.75 4.47

3.11 16.2 1.97 3.48 2.03 3.60 1.92 7.01 3.61 4.95 2.86

2.44 20.0 1.69 3.04 1.70 2.28 1.76 4.96 2.71 3.35 2.04

3.94 7.9 2.24 5.55 3.06 4.30 3.41 6.11 4.47 4.47 3.29

8.29 251.2 3.16 13.44 7.94 10.00 6.99 15.84 11.22 14.12 3.55

a GI50 refers to the compound concentrations required to inhibit the growth of the cells by 50%. The value presented here are the average of individual GI50 against each cell line in the panel. b SI refers to the selectivity index. The SI figures for all cell lines were obtained by dividing the GI50 values of the least and most sensitive cells. c GI50 values for tamoxifen (TEM) were obtained from online NCI database (COMPARE data vector search, compound ID NSC 180973). d GI50 values for raloxifene (RAL) were obtained from online NCI database (COMPARE data vector search, compound ID NSC 747974).

human ERa using ActiveMotif's NR peptide ERa colorimetric ELISA kit (Carlsbad, CA) at 25 uM and 2.5 mM concentration [32]. Compound 1h was used at concentrations of 25 mM and 2.5 mM. The agonist 17b-estradiol (E2) and antagonist tamoxifen (TEM) were used as controls at 25 mM concentration. The results of the colorimetric ELISA assay are shown in Fig. 5. It is clear from these assay results that 1h is an antagonist at the 25 and 2.5 mM concentrations used, and is a more potent ER binder and antagonist than tamoxifen. 2.2.5. In vitro pharmacokinetics studies As described so far, compounds 1g and 1h displayed impressive in vitro pharmacodynamics as candidate SERMs. We next determined their preliminary pharmacokinetic profiles to see whether they warranted further studies. Therefore, in vitro metabolic stability studies using mouse and human liver microsomes, as well as membrane permeability studies on these two compounds were contracted out to Drumetix Laboratories (Greensboro, NC, USA; www.drumetix.com).

107

90

0.45

80

0.4

% Remaining aŌer 1 hr incubaƟon

ERα Activation (OD 450 nm)

A. Jha et al. / European Journal of Medicinal Chemistry 92 (2015) 103e114

0.35 0.3

0.25 0.2 0.15 0.1 0.05 0

E2 (25μM)

TEM (25μM)

1h (25μM)

1h (2.5μM)

Compound (conc.)

70 60 50 Mouse Liver Microsome

40

Human Liver Microsome

30 20 10 0 Propranolol

1g

1h

Compound Fig. 5. ERa binding affinity assay using commercial ERa colorimetric ELISA kit.

2.2.5.1. In vitro membrane permeability studies. Parallel Artificial Membrane Permeability Assay (PAMPA) was performed on compounds 1g and 1h, along with reference b-blocker drug propranolol. Their average permeability is presented in Fig. 6. Propranolol showed an impressive average permeability of 94 and 88 nm/s respectively at the two test concentrations of 25 mM and 50 mM, respectively. Both test compounds, 1g and 1h showed a comparable average permeability range of 39e57 nm/s at the test concentrations. These results are respectable when taking account the average permeability values of clinical drugs raloxifene (27 nm/s at 20 mM) [33] and tamoxifen (57 nm/s at 50 mM) [34]. 2.2.5.2. In vitro metabolic stability studies. The average in vitro metabolic stability data for reference drug propranolol and compounds 1g and 1h, when incubated with mouse and human liver microsomes are presented in Fig. 7. The metabolism rate appeared to be considerably faster in mouse liver microsomes, as compared to human liver microsomes. Compared to the reference propranolol, compound 1g was found to be significantly more stable in both microsome systems. However, compound 1h was far less stable in mouse liver microsomes (only ~7% compound remaining after 1h incubation) than in human liver microsome (~58% compound remaining after 1 h incubation as opposed to ~52% propranolol remaining under identical conditions). These results suggest that both 1g and 1h may survive the first pass metabolism in humans. It is not certain yet whether these compounds were active in their own right or if one or more of their metabolites were responsible for the activity shown in Fig. 7, as is the case in tamoxifen where the

100 90

Permeability (nm/s)

80 70 60 50

20 μM Incuba on

40

25 μM Incuba on

30

50 μM Incuba on

20 10 0 Propranolol

1g

1h

Raloxifene

Tamoxifen

Compound

Fig. 6. Permeability of test and reference compounds at pH 7.4. The values for raloxifene (at pH 6.4) and tamoxifen were obtained from literature sources [33,34].

Fig. 7. Percent remaining values of control and test compounds 1g and 1h after 60 min incubation with mouse and human liver microsomes.

active metabolite is 4-hydroxytamoxifen (afimoxifene; Fig. 1) [35].

2.2.6. In vivo neurotoxicity studies The two potent cytotoxic compounds, 1g and 1h were evaluated for murine toxicity in vivo as previously described [36]. Briefly, the compounds were administered intraperitoneally into mice at doses of 30, 100 and 300 mg/kg. The animals were then monitored for 0.5 and 4 h after the administration of the compounds. The compounds were found to be well-tolerated, with 1g and 1h showing no mortality even up to 300 mg/kg animal body weight in 24 experimental animals for each compound. Among animals receiving compound 1g, one test animal administered with 300 mg/kg dose displayed signs of mild tremors. In the case of 1h, one test animal receiving 100 mg/kg suffered diarrhea during the 0.5 h observation. These results indicate that the compounds 1g and 1h are welltolerated and are not general biocidal agents.

2.3. Molecular modeling Based on the results of cytotoxicity of series 1 compounds against estrogen-responsive MCF-7 cells (Table 1) and ERa binding affinity assay, it became increasingly evident that ERa, the prognostic maker for estrogen-responsive breast cancer [37], is the molecular target for the candidate SERMs under investigation. The docking studies performed during the designing of these compounds (vide supra) support this hypothesis even further [19]. The screenshots of the docking studies are included in Fig. 8. Fig. 8A shows the ligand 1g bound in the ligand binding domain of ERa with important amino acid residues on the protein surface. Fig. 8B is a proximity contour of ligand 1g indicating three major interactions, viz., piperidinol eOH forming H-bond with Glu 353, hydrogen bonding between methoxy and His 524, and the piperidine group of the piperidinoethoxyphenyl side chain engaging in electrostatic interaction with Asp 351. Through the docking studies, the calculated binding affinities of 1g, 1h and tamoxifen were found to be 10.52, 10.78 and 9.79 kcal/mol, respectively. Clearly, the binding of tamoxifen is weaker than that of 1g as well as 1h. These binding affinity values can also be used to predict the relative potency of the compounds. The difference in binding free energies of two ligands is given by Ref. [38]:

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Fig. 8. Compound 1g docked in the ERa Ligand binding domain. A. Important amino acid residues on the protein surface in the ligand binding domain shown with 1g. B. Proximity contour of 1g indicating important interactions with amino acid residues in the ERa ligand binding domain.

DDGbinding ¼ DGligand 1  DGligand 2 ¼ RT lnr where r is the ratio of the respective IC50 values of the two ligands, assuming competitive antagonism and that the fixed substrate concentration and Km are held constant for each screening assay, as per the ChengePrusoff equation [39]. Thus, for tamoxifen and compound 1h, DDGbinding is 0.99 kcal/ mol (difference in respective calculated binding energies). Placing the values for the gas constant R (1.9872041  103 kcal/K.mol) and temperature T (298 K or 25  C) in the equation above, the value of r (ratio between IC50 values of tamoxifan and 1h) is returned as 5.3. Using the experimental IC50 values from Table 1, r turns out to be 4.2. Thus, we notice a reasonable correlation between experimental and theoretical calculations, assuming that cytotoxicity is driven by the SERM modulation (based on Fig. 5, this is a reasonable assumption). 3. Conclusion In conclusion, the present study has led to the identification of 1-(5-(4-(2-(dialkylamino)ethoxy)benzyl)-6-methoxynaphthalen2-yl)piperidin-4-ols as potential SERMs. Two of the newly designed compounds 1g and 1h displayed far superior cytotoxicity towards estrogen-responsive human MCF-7 breast cancer cells than tamoxifen. Compound 1h also showed significant binding and antagonistic effects against human ERa in an in-vitro ELISA assay. In cytostatic potency assays, compounds 1aeh exhibited better activity than either clinical SERM tamoxifen or raloxifene, when evaluated against murine L1210, human HeLa and CEM cell lines. We are actively pursuing modifications on this model to improve cytotoxic efficacy and antagonistic activity against ERa-dependent breast cancer cells in vitro. 4. Experimental section 4.1. Chemistry 4.1.1. General information All chemicals and reagents were purchased from Aldrich Chemical Co. and were used without further purification. 1H and 13 C NMR were recorded on a Bruker AV300 spectrophotometer at

300 and 75 MHz, respectively, in CDCl3 where residual CHCl3 (d ¼ 7.27) served as the internal standard for 1H NMR, and CDCl3 was used as the internal standard (d ¼ 77.0) for 13C NMR. Melting points were recorded on a MEL-TEMP II apparatus and are reported as uncorrected values. ESI-HRMS spectra were recorded on a microTOF (Bruker Daltonics) spectrometer at Dalhousie University. Silica 60 F254 aluminum-backed sheets (Merck, Darmstadt, Germany) and silica gel 60 (SiliaFlash P60, Silicycle, Quebec City, Canada) were used for thin layer chromatography (TLC) and flash chromatography, respectively. 4.1.2. General procedure for the synthesis of 1-(6alkoxynaphthalen-2-yl)piperidin-4-ol (4 or 7) In a dry single-neck round bottom flask equipped with septum, appropriate 2-alkyloxy-6-bromonaphthalene (3 or 6; 9.6 mmol, 1 equiv.), copper iodide (3.84 mmol, 0.40 equiv.), L-proline (1.92 mmol, 0.2 equiv.), potassium carbonate (19.2 mmol, 2 equiv.) were taken and the contents were dried under vacuum followed by flushing with nitrogen. 4-Piperidinol (9.6 mmol, 1 equiv.) dissolved in dimethyl sulfoxide (DMSO, 30 mL) was added and the reaction mixture was stirred at 110  C for 24 h. After completion of the reaction, DMSO was removed by extraction with water and ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate. The organic solvent was evaporated under reduced pressure to give the product as a light brown solid. The crude product was purified on silica gel by using 8e15% of ethyl acetate in hexanes as eluant to get the products. 4.1.2.1. 1-(6-Methoxynaphthalen-2-yl)piperidin-4-ol (4). Colorless solid, yield 72%. 1H NMR (300 MHz, CDCl3): d 1.57 (brs, 1H), 1.65e1.84 (m, 2H), 2.03e2.17 (m, 2H), 2.91e3.09 (m, 2H), 3.58e3.71 (m, 2H), 3.83e3.95 (m, 4H), 7.08e7.18 (m, 3H), 7.32 (brs, 1H), 7.60e7.69 (m, 2H). 13C NMR (75.5 MHz, CDCl3): d 34.1, 47.5, 48.4, 55.3, 105.9, 111.6, 118.9, 120.6, 127.6, 128.3, 129.7, 129.8, 146.2, 156.3. 4.1.2.2. 1-(6-Benzyloxynaphthalen-2-yl)piperidin-4-ol (7). Colorless solid, yield 69%. 1H NMR (300 MHz, CDCl3): d 1.70e1.83 (m, 3H), 2.04e2.11 (m, 2H), 2.93e3.02 (m, 2H), 3.59e3.67 (m, 2H), 3.80e3.94 (m, 1H), 5.17 (s, 2H), 6.77 (d, J ¼ 8.4 Hz, 2H), 7.07e7.18 (m, 2H), 7.22e7.42 (m, 5H), 7.61 (d, J ¼ 9.0 Hz, 1H), 7.83 (d, J ¼ 9.6 Hz, 1H). 13C NMR (75.5 MHz, CDCl3): d 34.7, 48.1, 68.1, 70.7, 108.4, 111.5,

A. Jha et al. / European Journal of Medicinal Chemistry 92 (2015) 103e114

119.4, 120.8, 127.6, 127.8, 128.0, 128.4, 128.7, 129.7, 130.6, 137.7, 148.2, 155.7. 4.1.3. General procedure for the synthesis of 1-(5-(4-(2(dialkylamino)ethoxy)benzyl)-6-alkoxynaphthalen-2-yl)piperidin4-ols (1aeh & 8aeh) To an ice-cold solution of appropriate 1-(6-(alkoxyoxy)naphthalen-2-yl)piperidin-4-ol (4 or 7) (12.7 mmol) in 1,4-dioxane (60 mL), 4-(dialkylaminoethoxy)benzyl alcohol (5aeh [15]; 12.7 mmol) in 1,4-dioxane (60 mL) was added, followed by dropwise addition of borontrifluoride etherate (4 equiv.). The reaction mixture turned brown. The stirring was continued at room temparature until 4-(dialkylaminoethoxy)benzyl alcohol was completely consumed as indicated by TLC. Generally, the reaction took two days to complete. After completion of the reaction, excess of borontrifluoride was quenched with aq. NaHCO3 solution (3  100 mL) and 1,4-dioxane was removed under reduced pressure. The reaction mixture was extracted with dichlromethane (2  40 mL). The combined organic layer was dried over anhydrous sodium sulfate and concentrated to give crude products which were purified on silica gel using 6e10 % of methanol in dichlorometahne. 4 .1. 3 .1. 1 - ( 5 - ( 4 - ( 2 - ( D i m e t h y l a m i n o ) e t h o x y ) b e n z y l ) - 6 methoxynaphthalen-2-yl)piperidin-4-ol (1a). Colorless solid, yield 42%, mp 220e222  C. 1H NMR (300 MHz, CDCl3): d 1.69e1.81 (m, 2H), 2.04e2.08 (m, 2H), 2.47e2.61 (m, 6H), 2.93e3.00 (m, 4H), 3.60e3.64 (m, 2H), 3.81e3.91 (m, 4H), 4.11 (brs, 2H), 4.38 (s, 2H), 6.77 (d, J ¼ 8.29 Hz), 7.08e7.13 (m, 3H), 7.22e7.28 (m, 2H), 7.64 (d, J ¼ 9.0 Hz, 1H) 7.80 (d, J ¼ 9.4 Hz, 1H). 13C NMR (75.5 MHz): d 29.7, 34.3, 45.3, 47.8, 56.9, 57.8, 65.1, 67.9, 111.3, 114.3, 120.8, 122.2, 124.7, 126.8, 128.2, 129.1, 130.4, 133.9, 147.2, 153.2, 156.3. ESI-HRMS (m/z): calcd. for C27H34N2O3: 435.2648 [MþH]þ; found: 435.2658. 4 .1. 3 . 2 . 1 - ( 5 - ( 4 - ( 2 - ( D i e t h y l a m i n o ) e t h o x y ) b e n z y l ) - 6 methoxynaphthalen-2-yl)piperidin-4-ol (1b). Colorless solid, yield 67%, mp 124e127  C. 1H NMR (300 MHz, CDCl3): d 1.11 (t, J ¼ 7.16 Hz, 6H), 1.69e1.76 (m, 3H), 2.03e2.07 (m, 2H), 2.66e2.72 (m, 4H), 2.89e2.99 (m, 4H), 3.58e3.91 (m, 2H), 3.82e3.94 (m, 4H), 4.04 (t, J ¼ 6.0 Hz, 2H), 4.38 (s, 2H), 6.76 (d, J ¼ 8.6 Hz, 2H), 7.08e7.13 (m, 3H), 7.22e7.29 (m, 2H), 7.65 (d, J ¼ 8.7 Hz, 1H), 7.81 (d, J ¼ 9.3 Hz, 1H). 13C NMR (75.5 MHz): d 11.4, 29.7, 34.3, 47.7, 47.8, 51.6, 56.9, 65.9, 67.9, 111.3, 114.3, 120.8, 122.3, 124.7, 126.8, 128.2, 129.1, 130.4, 133.6, 147.2, 153.3, 156.6. ESI-HRMS (m/z): calcd. for C29H38N2O3: 463.2961 [MþH]þ; found: 463.2953. 4 .1. 3 . 3 . 1 - ( 5 - ( 4 - ( 2 - ( D i i s o p ro p yl a m i n o ) e t h o x y ) b e n z yl ) - 6 methoxynaphthalen-2-yl)piperidin-4-ol (1c). Colorless solid, yield 49%, mp 224e225  C. 1H NMR (300 MHz, CDCl3): d 1.06 (d, J ¼ 6.0 Hz, 12H), 1.71e1.81 (m, 2H), 2.05e2.08 (m, 2H), 2.76e2.84 (m, 2H), 2.93e3.00 (m, 2H), 3.07 (brs, 2H), 3.60e3.64 (m, 2H), 3.81e3.91 (m, 6H), 4.38 (s, 2H), 6.76 (d, J ¼ 8.1 Hz, 2H), 7.10e7.13 (m, 3H), 7.22e7.29 (m, 2H), 7.64 (d, J ¼ 9.0 Hz, 1H), 7.81 (d, J ¼ 9.3 Hz, 1H). 13C NMR (75.5 MHz): d 20.6, 29.7, 34.3, 44.5, 47.8, 49.9, 56.9, 67.9, 111.3, 114.2, 114.3, 120.8, 122.3, 124.7, 126.8, 128.2, 129.1, 130.4, 133.4, 147.2, 153.3, 156.8. ESI-HRMS (m/z): calcd. for C31H42N2O3: 491.3274 [MþH]þ; found: 491.3277. 4 .1. 3 . 4 . 1 - ( 5 - ( 4 - ( 2 - ( P y r r o l i d i n - 1 - y l ) e t h o x y ) b e n z y l ) - 6 methoxynaphthalen-2-yl)piperidin-4-ol (1d). Colorless solid, yield 65%, mp 220e222  C. 1H NMR (300 MHz, CDCl3): d 1.73e1.76 (m, 3H), 1.85e1.97 (m, 4H), 1.03e2.07 (m, 2H), 2.73 (brs, 4H), 2.93e2.99 (m, 4H), 3.79e3.94 (m, 4H), 4.11 (t, J ¼ 5.6 Hz, 2H), 4.38 (s, 2H), 6.77 (d, J ¼ 8.4 Hz, 2H), 7.07e7.13 (m, 3H), 7.22e7.29 (m, 2H), 7.64 (d, J ¼ 8.7 Hz, 1H) 7.80 (d, J ¼ 9.3 Hz, 1H). 13C NMR (75.5 MHz): d 23.4,

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29.7, 34.3, 47.8, 54.6, 54.9, 56.9, 66.5, 67.8, 111.3, 114.3, 120.8, 122.2, 124.7, 126.8, 128.2, 129.1, 130.4, 133.6, 147.2, 153.3, 156.6. ESI-HRMS (m/z): calcd. for C29H36N2O3: 461.2804 [MþH]þ; found: 461.2797. 4 .1. 3 . 5 . 1 - ( 5 - ( 4 - ( 2 - ( P i p e r i d i n - 1 - y l ) e t h o x y ) b e n z y l ) - 6 methoxynaphthalen-2-yl)piperidin-4-ol (1e). Colorless solid, yield 71%, mp 193e195  C. 1H NMR (300 MHz, CDCl3): d 1.49 (brs, 2H), 1.69e1.81 (m, 6H), 2.04e2.08 (m, 2H), 2.62 (brs, 4H), 2.85 (brs, 2H), 2.93e3.00 (m, 2H), 3.60e3.64 (m, 2H), 3.81e3.91 (m, 4H), 4.13 (brs, 2H), 4.38 (s, 2H), 6.75 (d, J ¼ 8.1 Hz, 2H), 7.07e7.13 (m, 3H), 7.22e7.28 (m, 2H), 7.64 (d, J ¼ 9.0 Hz, 2H), 7.80 (d, J ¼ 9.3 Hz, 2H). 13 C NMR (75.5 MHz): d 23.8, 25.3, 29.7, 30.9, 34.3, 47.8, 54.8, 56.9, 57.6, 65.3, 67.9, 111.3, 114.3, 120.8, 122.2, 124.7, 126.8, 128.2, 129.1, 130.4, 133.7, 147.2, 153.3, 156.5. ESI-HRMS (m/z): calcd. for C30H38N2O3: 475.2961 [MþH]þ; found: 475.2982. 4 .1. 3 . 6 . 1 - ( 5 - ( 4 - ( 2 - M o r p h o l i n o e t h o x y ) b e n z y l ) - 6 methoxynaphthalen-2-yl)piperidin-4-ol (1f). Colorless solid, yield 64%, mp 225e227  C. 1H NMR (300 MHz, CDCl3): d 1.70e1.80 (m, 3H), 2.04e2.08 (m, 2H), 2.60 (brs, 4H), 2.80 (brs, 2H), 2.97 (t, J ¼ 10.2 Hz, 2H), 3.60e3.64 (m, 2H), 3.76 (brs, 4H), 3.83e3.92 (m, 4H), 4.08 (brs, 2H), 4.38 (s, 2H), 6.76 (d, J ¼ 7.5 Hz, 2H), 7.08e7.13 (m, 3H), 7.22e7.29 (m, 2H), 7.65 (d, J ¼ 8.7 Hz, 1H), 7.80 (d, J ¼ 9.0 Hz, 1H). 13C NMR (75.5 MHz): d 29.7, 34.3, 47.8, 53.9, 56.9, 57.6, 65.5, 66.7, 67.8, 111.4, 114.4, 120.8, 122.2, 124.7, 126.8, 128.2, 129.1, 129.4, 130.4, 133.8, 147.2, 153.3, 156.5. ESI-HRMS (m/z): calcd. for C29H36N2O4: 477.2753 [MþH]þ; found: 477.2772. 4.1.3.7. 1-(5-(4-(2-(4-Methylpiperidin-1-yl)ethoxy)benzyl)-6methoxynaphthalen-2-yl)piperidin-4-ol (1g). Colorless solid, yield 66%, mp 198e201  C. 1H NMR (300 MHz, CDCl3): d 0.90 (s, 3H), 1.39 (brs, 3H), 1.64e1.81 (m, 5H), 2.04e2.07 (m, 2H), 2.17e2.19 (m, 2H), 2.83e2.85 (m, 2H), 2.92e3.04 (m, 4H), 3.60e3.64 (m, 2H), 3.81e3.91 (m, 4H), 4.10 (t, J ¼ 5.7 Hz, 2H), 4.38 (s, 2H), 6.76 (d, J ¼ 8.4 Hz, 2H), 7.07e7.13 (m, 3H), 7.22e7.28 (m, 2H), 7.64 (d, J ¼ 9.0 Hz, 1H), 7.80 (d, J ¼ 9.3 Hz, 1H). 13C NMR (75.5 MHz): d 21.7, 29.7, 30.3, 33.7, 34.3, 47.8, 54.2, 56.9, 57.3, 65.5, 67.1, 67.9, 111.3, 114.3, 120.8, 122.2, 124.7, 126.8, 128.2, 129.1, 130.4, 133.6, 147.2, 153.3, 156.6. ESI-HRMS (m/z): calcd. for C31H40N2O3: 489.3117 [MþH]þ; found: 489.3101. 4 . 1. 3 . 8 . 1 - ( 5 - ( 4 - ( 2 - ( A z e p a n - 1 - y l ) e t h o x y ) b e n z y l ) - 6 methoxynaphthalen-2-yl)piperidin-4-ol (1h). Colorless solid, yield 67%, mp 146e148  C. 1H NMR (300 MHz, CDCl3): d 1.62e1.77 (m, 12H), 2.04e2.19 (m, 2H), 2.83 (brs, 4H), 2.93e3.00 (m, 4H), 3.60e3.64 (m, 2H), 3.83e3.91 (m, 4H), 4.06e4.08 (m, 2H), 4.38 (s, 2H), 6.76 (d, J ¼ 8.1 Hz, 2H), 7.08e7.13 (m, 3H), 7.22e7.28 (m, 2H), 7.64 (d, J ¼ 9.0 Hz, 1H), 7.80 (d, J ¼ 9.3 Hz, 1H). 13C NMR (75.5 MHz): d 27.0, 27.3, 29.7, 34.3, 47.8, 55.7, 56.3, 56.9, 65.9, 67.9, 111.3, 114.3, 120.8, 122.3, 124.7, 126.8, 128.2, 130.4, 133.6, 147.2, 153.3, 156.6. ESIHRMS (m/z): calcd. for C31H40N2O3: 489.3117 [MþH]þ; found: 489.3120. 4.1.3.9. 1-(6-(Benzyloxy)-5-(4-(2-(dimethylamino)ethoxy)benzyl) naphthalen-2-yl)piperidin-4-ol (8a). Brown colored foamy solid, yield 40%, mp 145e147  C. 1H NMR (300 MHz, CDCl3): d 1.75e1.81 (m, 2H), 2.01e2.07 (m, 2H), 2.34 (s, 6H), 2.73 (t, J ¼ 5.7 Hz, 2H), 3.40e3.70 (m, 2H), 3.75e3.90 (m, 1H), 4.02 (t, J ¼ 5.7 Hz, 2H), 4.42 (s, 2H), 5.2 (s, 2H), 6.7 (d, J ¼ 8.4 Hz, 2H), 7.05e7.13 (m, 3H), 7.21e7.40 (m, 6H), 7.60 (d, J ¼ 9.0 Hz, 1H), 7.80 (d, J ¼ 9.6 Hz, 1H). 13C NMR (75 MHz, CDCl3): d 30.2, 34.6, 45.8, 47.7, 58.5, 66.6, 68.0, 72.2, 111.6, 114.8, 116.3, 120.6, 123.7, 125.1, 126.9, 127.5, 127.8, 128.5 (2C), 129.4, 133.0, 137.2, 147.5, 134.0, 152.0, 157.0.

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4.1.3.10. 1-(6-(Benzyloxy)-5-(4-(2-(diethylamino)ethoxy)benzyl) naphthalen-2-yl)piperidin-4-ol (8b). Colorless foamy solid, yield 52%, mp 108e109  C. 1H NMR (300 MHz, CDCl3): d 1.08 (t, J ¼ 7.2 Hz, 6H), 1.55e1.82 (m, 2H), 2.01e2.09 (m, 2H), 2.65 (q, J ¼ 7.2 Hz, 4H), 2.90 (t, J ¼ 6.3 Hz, 2H), 2.92e2.3.01 (m, 2H), 3.40 (3.65 (m, 2H), 3.50e3.70 (m, 2H), 3.82e3.92 (m, 1H), 4.01 (t, J ¼ 6.3 Hz, 2H), 4.40 (s, 2H), 5.20 (s, 2H), 6.80 (d, J ¼ 8.4 Hz, 2H), 7.05e7.14 (m, 2H), 7.25e7.40 (m, 6H), 6.61 (d, J ¼ 9 Hz, 1H), 7.83 (d, J ¼ 9.3 Hz, 1H). 13C NMR (75 MHz, CDCl3): d 11.9, 30.2, 34.4, 47.7, 47.9, 52.1, 66.8, 67.8, 72.0, 76.7, 77.2, 77.6, 111.4, 114.6, 116.1, 120.6, 123.5, 125.0, 126.9, 127.5, 127.8, 128.5, 129.3, 131.0, 133.4, 137.8, 147.5, 152.6, 157.1. 4.1.3.11. 1-(6-(Benzyloxy)-5-(4-(2-(diisopropylamino)ethoxy)benzyl) naphthalen-2-yl)piperidin-4-ol (8c). Colorless foamy solid, yield 62%, mp 79e81  C. 1H NMR (300 MHz, CDCl3): d 1.13e1.15 (m, 12H), 1.71e1.80 (2H), 2.02e2.10 (m, 3H), 3.0 (t, J ¼ 9.9 Hz, 4 Hz), 3.15e3.20 (m, 2H), 3.54e3.65 (m, 2H), 3.80e4.01 (m, 3H), 4.42 (s, 2H), 5.2 (s, 2H), 6.75 (d, J ¼ 8.4 Hz, 2H), 7.05e7.13 (m, 3H), 7.21e7.40 (m, 7H), 7.60 (d, J ¼ 8.7 Hz,1H), 7.82 (d, J ¼ 9.0 Hz,1H). 13C NMR (75 MHz, CDCl3): d 20.6, 30.4, 34.6, 45.0, 47.8, 50.9, 68.1, 72.4, 111.7, 114.9, 116.4, 120.7, 123.8, 125.2, 127.0, 127.6, 127.9, 128.6, 129.5, 131.3, 134.1, 138.1, 147.7, 152.8. 4.1.3.12. 1-(6-(Benzyloxy)-5-(4-(2-(pyrrolidin-1-yl)ethoxy)benzyl) naphthalen-2-yl)piperidin-4-ol (8d). Colorless foamy solid, yield 25%, mp 123e125  C. 1H NMR (300 MHz, CDCl3): d 1.06e1.85 (m, 6H), 1.97e2.05 (m, 2H), 2.65e2.75 (m, 4H), 2.85e3.0 (m, 4H), 3.45e3.64 (m, 2H), 3.65e3.86 (m, 1H), 4.10 (t, J ¼ 6.0 Hz, 2H), 4.44 (s, 2H), 5.20 (s, 2H), 6.80 (d, J ¼ 8.4 Hz, 2H), 6.95e7.14 (m, 3H), 7.20e7.40 (m, 7H), 7.60 (d, J ¼ 9.0 Hz, 1H), 7.84 (d, J ¼ 9.3 Hz, 1H). 13C NMR (75 MHz, CDCl3): NMR d 23.8, 30.3, 34.5, 47.7, 54.7, 55.2, 67.2, 72.2, 111.5, 114.9, 116.3, 120.6, 123.7, 125.1, 126.9, 127.6, 127.8, 128.6, 129.4, 131.2, 134.1, 138.0, 147.7, 152.7, 157.1. 4.1.3.13. 1-(6-(Benzyloxy)-5-(4-(2-(piperidin-1-yl)ethoxy)benzyl) naphthalen-2-yl)piperidin-4-ol (8e). Colorless foamy solid, yield 40%, mp 156e158  C. 1H NMR (300 MHz, CDCl3): d 1.42e1.48 (m, 2H), 1.50e1.68 (m, 4H), 1.70e1.82 (m, 2H), 2.01e2.08 (m, 2H), 2.45e2.55 (m, 4H), 2.80 (t, J ¼ 5.7 Hz, 2H), 2.70 (t, J ¼ 10.2 Hz, 2H), 3.61 (t, J ¼ 8.1 Hz, 2H), 3.80e3.93 (m, 1H), 4.07 (t, J ¼ 6 Hz, 2H), 4.42 (s, 2H), 5.20 (s, 2H), 6.80 (d, J ¼ 8.4 Hz, 2H), 7.06e7.13 (m, 3H), 7.22e7.40 (m, 7H), 7.61 (d, J ¼ 9 Hz, 1H), 7.83 (d, J ¼ 9.3 Hz, 1H). 13C NMR (75 MHz, CDCl3): d 23.8, 25.2, 29.9, 33.9, 47.7, 54.6, 57.6, 65.5, 71.8, 111.4, 114.4, 115.9, 120.4, 123.2, 124.7, 126.7, 127.2, 127.5, 128.2, 129.1, 130.8, 133.8, 137.6, 147.3, 152.4, 156.7. 4.1.3.14. 1-(6-(Benzyloxy)-5-(4-(2-morpholinoethoxy)benzyl)naphthalen-2-yl)piperidin-4-ol (8f). Colorless foamy solid, yield 45%, mp 54e55  C. 1H NMR (300 MHz, CDCl3): d 1.65e1.82 (m, 4H), 2.01e2.07 (m, 2H), 2.60 (d, J ¼ 4.5 Hz, 4H), 2.80 (t, J ¼ 5.7 Hz, 2H), 2.85e3.10 (m, 2H), 3.45e3.70 (m, 2H), 3.74 (t, J ¼ 4.5 Hz, 2H), 3.80e3.90 (m, 1H), 4.10 (t, J ¼ 5.7 Hz, 2H), 4.43 (s, 2H), 5.20 (s, 2H), 6.80 (d, J ¼ 8.7 Hz, 2H), 7.08e7.14 (m, 3H), 7.21e7.40 (m, 7H), 7.61 (d, J ¼ 9 Hz, 1H), 7.83 (d, J ¼ 9.3 Hz,1H). 13C NMR (75 MHz, CDCl3): d 30.3, 34.6, 47.8, 54.3, 58.0, 67.2, 68.1, 72.2, 111.6, 114.9, 116.3, 120.7, 123.6, 125.1 (2C), 127.0, 127.6, 128.6, 129.4 (3C), 138.0, 147.7, 152.5, 157.2. 4.1.3.15. 1-(6-(Benzyloxy)-5-(4-(2-(4-methylpiperidin-1-yl)ethoxy) benzyl)naphthalen-2-yl)piperidin-4-ol (8g). Colorless foamy solid, yield 45%, mp 58e60  C. 1H NMR (300 MHz, CDCl3): d 0.93 (d, J ¼ 5.4 Hz, 3H), 1.25e1.37 (m, 3H), 1.60e1.81 (m, 5H), 2.02e2.20 (m, 4H), 2.80 (t, J ¼ 6 Hz, 2H), 2.93e3.10 (m, 4H), 3.45e3.70 (m, 2H), 3.80e3.90 (m, 1H), 4.07 (t, J ¼ 5.7 Hz, 2H), 4.42 (s, 2H), 5.2 (s, 2H), 6.75 (d, J ¼ 8.1 Hz, 2H), 7.05e7.13 (m, 3H), 7.20e7.40 (m, 7H), 7.63 (d, J ¼ 9 Hz, 1H), 7.83 (d, J ¼ 9.3 Hz, 1H). 13C NMR (75 MHz, CDCl3): d 21.8, 30.3, 30.7, 34.3, 34.6, 47.8, 54.6, 57.8, 66.4, 68.1, 72.2, 111.6,

114.9, 116.3, 120.7, 123.7, 125.2, 127.0, 127.6, 128, 128.6, 129.5, 131.2, 134, 138, 147.7, 152.8, 157.2. 4.1.3.16. 1-(5-(4-(2-(Azepan-1-yl)ethoxy)benzyl)-6-(benzyloxy) naphthalen-2-yl)piperidin-4-ol (8h). Colorless foamy solid, yield 55%, mp 60e62  C. 1H NMR (300 MHz, CDCl3): d 1.60e1.80 (m, 10H), 1.95e2.02 (m, 3H), 2.75e2.85 (m, 4H), 2.90e3.0 (m, 4H), 3.40e3.70 (m, 2H), 3.80e3.90 (m, 1H), 4.1 (t, J ¼ 6 Hz, 2H), 4.45 9s, 2H), 5.2 (s, 2H), 6.80 (d, J ¼ 8.7 Hz, 2H), 6.96e7.15 (m, 3H), 7.20e7.40 (m, 7H), 7.62 (d, J ¼ 9 Hz, 1H), 7.9 (d, J ¼ 9.3 Hz, 1H). 13C NMR (75 MHz, CDCl3): d 27.3, 28.0, 30.3, 34.5, 47.7, 56.0, 56.9, 66.9, 67.9, 72.2, 111.6, 114.9, 116.3, 120.6, 123.7, 125.1, 127.0, 127.6, 127.8, 128.6, 129.5, 131.2, 134.0, 138.0, 147.7, 152.7, 157.2. 4.1.4. General procedure for the debenzylation of 1-(6-(benzyloxy)1-(4-(2-(dimethylamino)ethoxy)benzyl)-naphthalen-2-yl) piperidin-4-ol (8aeh) A mixture of 1-(1-(4-(2-(dialkylamino)ethoxy)benzyl)-6hydroxynaphthalen-2-yl)piperidin-4-ol (1.5 g) and 10% palladiumecarbon (1.0 g) in methanol (25 mL) was stirred for 4 h at room temperature under a hydrogen atmosphere. After completion of the reaction, palladiumecarbon was removed by filtration and the filtrate was concentrated on a rotavapor to afford 1-(1-(4-(2(dialkylamino)ethoxy)benzyl)-6-hydroxynaphthalen-2-yl)piperidin-4-ols (2aeh) as white solids. The compounds were of high purity and further purification was not required. 4 .1. 4 .1. 1 - ( 1 - ( 4 - ( 2 - ( D i m e t h y l a m i n o ) e t h o x y ) b e n z y l ) - 6 hydroxynaphthalen-2-yl)piperidin-4-ol (2a). Pale pink colored solid, yield 91%, mp 93e95  C. FTIR (Nujol): 740, 1034, 1377, 1462, 1643, 2800, 3419 cm1. 1H NMR (300 MHz, CDCl3): d 1.74 (q, J ¼ 9.0 Hz, 2H), 1.98e2.10 (m, 2H), 2.36 (s, 6H), 2.75 (t, J ¼ 5.7 Hz, 2H), 2.93 (t, J ¼ 10.8 Hz, 2H), 3.56e3.62 (m, 2H), 3.80e3.86 (m, 1H), 4.00 (t, J ¼ 5.7 Hz, 2H), 4.32 (s, 2H), 6.68 (d, J ¼ 7.8 Hz, 2H), 6.99e7.13 (m, 4H), 7.20 (d, J ¼ 9.3 Hz, 1H), 7.50 (d, J ¼ 8.7 Hz, 1H), 7.75 (d, J ¼ 9.0 Hz, 1H). 13C NMR (75 MHz, MeOD): d 18.3, 30.5, 32.3, 58.1, 58.4, 63.7, 116.0, 119.0, 120.6, 121.2, 121.8, 126.8, 127.4, 129.7, 130.5, 135.3, 136.0, 137.6, 155.8, 157.3. ESI-HRMS (m/z): calcd. for C26H33N2O3: 421.2491 [MþH]þ; found: 421.2484. 4 .1. 4 . 2 . 1 - ( 1 - ( 4 - ( 2 - ( D i e t h y l a m i n o ) e t h o x y ) b e n z y l ) - 6 hydroxynaphthalen-2-yl)piperidin-4-ol (2b). Off white solid, yield 88%, mp 75e78  C. FTIR (Nujol): 732, 805, 990, 1060, 1174, 1242, 1383, 1465, 1510, 1607, 2947, 3336 cm1. 1H NMR (300 MHz, CDCl3): d 1.07 (t, J ¼ 6.9 Hz, 6H), 1.65e1.82 (m, 2H), 1.98e2.10 (m, 2H), 2.68 (q, J ¼ 6.9 Hz, 4H), 2.82e3.0 (m, 4H), 3.52e3.68 (m, 2H), 3.80e3.92 (m, 1H), 3.98 (t, J ¼ 6.0 Hz, 2H), 4.32 (s, 2H), 6.69 (d, J ¼ 4.0 Hz, 2H), 7.0e7.30 (m, 5H), 7.50 (d, J ¼ 8.7 Hz, 1H), 7.77 (d, J ¼ 9.3 Hz; 1H). 13C NMR (75 MHz, CDCl3): d 11.9, 30.3, 34.6, 48.1, 52.3, 53.5, 67.0, 68.0, 112.1, 115.1, 118.8, 120.6, 124.5, 125.1, 127.0, 127.6, 128.6, 129.4, 135.0, 136.5, 157.5, 159.0. ESI-HRMS (m/z): calcd. for C28H37N2O3: 449.2804 [MþH]þ; found: 449.2809. 4 .1. 4 . 3 . 1 - ( 1 - ( 4 - ( 2 - ( D i i s o p r o p yl a m i n o ) e t h o x y ) b e n z yl ) - 6 hydroxynaphthalen-2-yl)piperidin-4-ol (2c). Pink colored solid, yield 90%, mp 94e96  C. FTIR (Nujol): 743, 808, 1061, 1173, 1263, 1464, 1609, 2800, 3200 cm1. 1H NMR (300 MHz, CDCl3þMeOD): d 0.91 (d, J ¼ 6.6 Hz, 12H), 1.43e1.60 (m, 2H), 1.75e1.87 (m, 2H), 2.60e2.70 (m, 4H), 2.93e3.04 (m, 2H), 3.30e3.40 (m, 2H), 3.53e3.57 (m, 1H), 3.72 (t, J ¼ 6.6 Hz, 2H), 4.13 (s, 2H), 6.53 (d, J ¼ 8.4 Hz, 2H), 6.89e7.01 (m, 5H), 7.30 (d, J ¼ 8.7 Hz, 1H), 7.53 (d, J ¼ 9.3 Hz, 1H). 13C NMR (75 MHz, CDCl3þMeOD): d 19.4, 29.5, 33.7, 44.9, 48.1, 51.0, 67.1, 67.5, 112.1, 114.2, 118.1, 118.7, 120.2, 124.1, 126.5, 128.7, 129.0, 133.8, 146.5, 150.4, 156.3. ESI-HRMS (m/z): calcd. for C30H41N2O3: 477.3117 [MþH]þ; found: 477.3104.

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4 .1. 4 . 4 . 1 - ( 1 - ( 4 - ( 2 - ( P y r r o l i d i n - 1 - y l ) e t h o x y ) b e n z y l ) - 6 hydroxynaphthalen-2-yl)piperidin-4-ol (2d). Brown colored solid, yield 94%, mp 92e94  C. FTIR (Nujol): 723, 881, 1051, 1154, 1376, 1457, 2724, 3331 cm1. 1H NMR (300 MHz, CDCl3þMeOD): d 1.68e1.81 (6H, m), 1.90e2.20 (2H, m), 2.57e2.67 (4H, m), 2.78e2.90 (4H, m), 3.42e3.60 (2H, m), 3.70e3.81 (1H, m), 4.00 (2H, t, J ¼ 5.7 Hz), 4.30 (2H, s), 6.60 (2H, d, J ¼ 7.2 Hz), 7.04 (3H, d, J ¼ 8.4 Hz), 7.13(2H, d, J ¼ 9.0 Hz), 7.43 (1H, d, J ¼ 8.7 Hz), 7.7 (1H, d, J ¼ 9.0 Hz). 13C NMR (75 MHz, CDCl3þMeOD): 23.5, 30.0, 34.3, 48.1, 54.6, 55.1, 66.8, 67.7, 112.2, 114.8, 118.5, 119.1, 120.6, 124.4, 126.9, 128.9, 129.3, 130.4, 133.5, 147.0, 150.4, 157.0. ESI-HRMS (m/z): calcd. for C28H35N2O3: 447.2648 [MþH]þ; found: 447.2640. 4.1.4.5. 1-(6-Hydroxy-1-(4-(2-(piperidin-1-yl)ethoxy)benzyl)naphthalen-2-yl)piperidin-4-ol (2e). Off white solid, yield 93%, mp 118e120  C. FTIR (Nujol): 759, 819, 990, 1060, 1176, 1263, 1382, 1470, 1510, 1609, 2943, 3404 cm1. 1H NMR (300 MHz, d6-DMSO): d 1.22e1.68 (m,10H),1.80e1.95 (m, 2H), 2.57e2.67 (m, 3H), 2.70e2.82 (m, 2H), 3.35e3.54 (m, 3H), 3.58e3.75 (m, 1H), 3.90 (m, 2H), 4.20 (m, 2H), 6.60 (d, J ¼ 7.5 Hz, 2H), 6.53e7.10 (m, 5H), 7.33 (d, J ¼ 8.1 Hz, 1H), 7.60 (d, J ¼ 9.0 Hz,1H). 13C NMR (75 MHz, d6-DMSO): d 24.4, 26.0, 29.7, 34.6, 47.7, 54.8, 57.9, 66.6, 111.5, 115.0, 119.0, 120.2, 124.3, 126.7, 128.3, 129.6,130.2,134.2,134.5,147.0,150.9,157.1. ESI-HRMS (m/z): calcd. for C29H37N2O3: 461.2804 [MþH]þ; found: 461.2804. 4.1.4.6. 1-(1-(4-(2-Morpholinoethoxy)benzyl)-6-hydroxynaphthalen2-yl)piperidin-4-ol (2f). Light brown colored solid, yield 91%, mp 79e82  C. FTIR (Nujol): 777, 808, 988, 1062, 1173, 1243, 1382, 1456, 1510, 1609, 2926, 3345 cm1. 1H NMR (300 MHz, CDCl3þMeOD): d 1.66e1.81 (m, 2H), 1.98e2.10 (m, 2H), 2.52e2.62 (m, 4H), 2.80 (t, J ¼ 5.4 Hz, 2H), 2.93 (t, J ¼ 9.9 Hz, 2H), 3.56e3.62 (m, 2H), 3.73 (t, J ¼ 4.5 Hz, 4H), 3.80e3.91 (m, 1H), 4.03 (t, J ¼ 5.4 Hz, 2H), 4.33 (s, 2H), 6.72 (d, J ¼ 8.4 Hz, 2H), 6.96e7.17 (m, 3H), 7.21 (d, J ¼ 8.7 Hz, 2H), 7.5 (d, J ¼ 8.7 Hz, 1H), 7.70 (d, J ¼ 9.0 Hz, 1H). 13C NMR (75 MHz, CDCl3þMeOD): d 29.9, 34.4, 48.0, 54.1, 57.8, 66.0, 66.9, 67.7, 112.2, 114.8, 118.4, 119.1, 120.5, 124.4, 126.9, 128.8, 129.3, 130.5, 133.6, 147.1, 150.2, 157.0. ESI-HRMS (m/z): calcd. for C28H35N2O4: 463.2597 [MþH]þ; found: 463.2587. 4.1.4.7. 1-(1-(4-(2-(4-Methylpiperidin-1-yl)ethoxy)benzyl)-6hydroxynaphthalen-2-yl)piperidin-4-ol (2g). Pale yellow colored solid, yield 89%, mp 91e93  C. FTIR (Nujol): 730, 877, 1049, 1124, 1377, 1460, 2725, 3334 cm1. 1H NMR (300 MHz, CDCl3þMeOD): d 0.70 (d, J ¼ 4.0 Hz, 3H), 1.0e1.24 (m, 3H), 1.30e1.60 (m, 4H), 1.71e2.0 (m, 4H), 2.58 (t, J ¼ 5.7 Hz, 2H), 2.63e2.98 (m, 4H), 3.30e3.41 (m, 2H), 3.57e3.70 (m, 1H), 3.81 (t, J ¼ 5.7 Hz, 2H), 4.1 (s, 2H), 6.50 (d, J ¼ 8.1 Hz, 2H), 6.74e6.91 (m, 3H), 6.99 (d, J ¼ 9.0 Hz, 2H), 7.30 (d, J ¼ 8.7 Hz, 1H), 7.53 (d, J ¼ 9.0 Hz, 1H). 13C NMR (75 MHz, CDCl3þMeOD): d 20.5, 24.8, 27.4, 28.4, 29.4, 31.0, 45.9, 53.8, 63.3, 66.9, 111.0, 114.6, 117.8, 120.2, 120.7, 124.8, 125.6, 125.9, 128.4, 129.2, 133.8, 134.3, 154.2, 155.5. ESI-HRMS (m/z): calcd. for C30H39N2O3: 475.2961 [MþH]þ; found: 475.2944. 4 . 1. 4 . 8 . 1 - ( 1 - ( 4 - ( 2 - ( A z e p a n - 1 - y l ) e t h o x y ) b e n z y l ) - 6 hydroxynaphthalen-2-yl)piperidin-4-ol (2h). Pale pink colored solid, yield 95%, mp 88e90  C. FTIR (Nujol): 744, 1154, 1263, 1376, 1460, 2724, 3300 cm1. 1H NMR (300 MHz, CDCl3þMeOD): d 1.50e1.80 (m, 10H), 1.90e2.10 (m, 2H), 2.72e3.0 (m, 8H), 3.17 (bs, 1H), 3.50e3.60 (m, 2H), 3.70e3.84 (m, 1H), 4.03 (s, 2H), 4.3 (s, 2H), 6.70 (d, J ¼ 8.4 Hz, 2H), 7.0e7.12 (m, 3H), 7.20 (d, J ¼ 9.0 Hz, 2H), 7.50 (d, J ¼ 8.7 Hz, 1H), 7.72 (d, J ¼ 9.3 Hz, 1H). 13C NMR (75 MHz, CDCl3þMeOD): d 26.1, 27.0, 30.0, 34.0, 48.1, 56.0, 56.1, 65.3, 67.2, 112.1, 114.3, 118.2, 118.8, 120.2, 124.1, 126.6, 128.7, 129.1, 129.9, 133.9, 146.6, 150.4, 156.4. ESI-HRMS (m/z): calcd. for C30H39N2O3: 475.2961 [MþH]þ; found: 475.2958.

111

4.2. Biological assay methods 4.2.1. Cell cytotoxicity assays The cytostatic activity and cytotoxicity assays were performed in triplicate on the HCl salt of test compounds. MCF-7 cell cytotoxicity was measured using the MTS assay [40]. MCF-7 cells in DMEM containing 10% heat-inactivated fetal bovine serum (FBS) were seeded into 24-well plates (30,000 cells/well). After treatment, the MTS assay (CellTiter 96 Aqueous Non-radioactive Proliferation Assay; Fisher Scientific Ltd., Nepean, Ontario, Canada) was performed following the manufacturer's instructions. The results are presented in Table 1. The cytostatic activities of the synthesized compounds for murine L1210 lymphocytic leukemia, human HeLa cervical cancer and CEM T-lymphocyte cell lines were evaluated as previously described [41]. Briefly, the tumor cells were seeded in 96-well microtiter plates and exposed to different concentrations of the test compounds. After 2 days (L1210) and 3 days (Hela and CEM), cell numbers were determined using a Particle counter (Coulter Z-1, Analis, Ghent, Belgium). Table 2 contained the GI50 values of test compounds against these three cell lines. The human tumor cell line screen at the National Cancer Institute (NCI) was undertaken as previously reported [31] using the selected compounds on a panel of 60 tumor cell lines. The data are summarized in Table 3. 4.2.2. NR peptide ERa ELISA assay In this assay, 96-well plates were coated with a peptide containing an ERa co-activation binding motif that bound only to the ER that was in the ligand-activated conformation. The ER captured through this interaction was then quantified using an anti-ER antibody and followed by colorimetric detection, according to the manufacturer's instructions. This assay was performed to evaluate the antagonistic action of the most potent compound 1h. All of the reagents were included in the kit. The vehicle control solution contained the MCF-7 nuclear extract as well as diluent buffers. Blank solution contained only diluent buffer. The 1 mM stock solutions of tamoxifen (TEM, antagonist control) and 17b-estradiol (E2, agonist control) were diluted to 25 mM. Compound 1h (as hydrochloride salt) was dissolved in water to form a 1 mM stock solution. This stock solution was diluted to 25 mM and 2.5 mM for the ELISA assay. Nuclear extracts of 50 mg per well were used, except for the agonist and antagonist controls, which had 15 mg of nuclear extract per well as suggested by the manufacturer. The developing and stop solutions were added at the end of the procedure, allowing the amount of active co-activator-ERa complex to be quantified. The absorbance at 450 nm was measured on a BioRad microplate reader. This assay was performed in duplicate at both the concentrations. 4.2.3. In vitro pharmacokinetics studies 4.2.3.1. Membrane permeability assay. Parallel Artificial Membrane Permeability Assay (PAMPA) was performed in a 96-well BD Gentest Pre-coated PAMPA Plate System (BD Biosciences, Woburn, MA). Prior to use, the pre-coated PAMPA plate system was warmed to room temperature for 30 min 325 mL of 50 or 25 mM test compound solutions in 5% DMSO in phosphate-buffered saline (PBS) was added into the wells in the receiver (donor) plate. 200 mL of 5% DMSO in PBS was added into the wells in the filter (acceptor) plate. The filter plate was placed on the receiver plate by slowly lowering the pre-coated PAMPA plate until it sits on the receiver plate. The assembly was incubated in water bath at 25  C for 5 h. After incubation, buffer samples collected from the acceptor plate and the donor plate, together with calibration standard samples, were prepared in 96-well plates as shown in the table below.

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A. Jha et al. / European Journal of Medicinal Chemistry 92 (2015) 103e114

Blank 5% DMSO in PBS (mL) Donor Sample (mL) Acceptor Sample (mL) Standard working solutiona (mL) MeOH (mL) a b

Cal. std.

Donor sampleb

Acceptor sample

0 0 0 400 400

360 40 0 0 400

0 0 150 0 150

Standard working solution: 5, 50, 500, and 5000 nM in 5% DMSO in PBS. The donor samples were analyzed with 10 times dilution.

The plates were then capped, vortexed, and centrifuged at 3000 rpm for 10 min. The supernatant was injected into LC-MS/MS. Spiked and back-calculated concentrations of propranolol and test compound calibration standards in buffer as well as their concentrations in buffer samples from donor and acceptor wells were used for calculation of permeability (Pe) in nm/s as shown below:

Pe ¼ 107 *

i h . ln 1  CA ðtÞ Cequilibrium A*ð1=VD þ 1=VA Þ*t

Where: CD(t) ¼ compound concentration in donor well at time t (nM); CA(t) ¼ compound concentration in acceptor well at time t (nM); VD ¼ donor well volume, 0.325 mL; VA ¼ acceptor well volume, 0.2 mL; Cequilibrium ¼ [CD(t)*VD þ CA(t)*VA]/(VD þ VA); A ¼ filter area ¼ 0.3 cm2; t ¼ incubation time ¼ 18,000 s (¼ 5 h) The calculated average PAMPA permeability values are presented in Fig. 5. 4.2.3.2. Metabolic stability assay. Compounds 1g, 1h and control (propranolol) were incubated at a concentration of 1 mM with mouse and human liver microsomes, in the presence of an excess of NADPH. The duplicate incubations, conducted in 0.5-mL 96-well plates in a shaking water bath maintained at 37  C, were performed for 0 and 60 min and quenched by addition of one volume of acetonitrile. Ingredients for different incubations were added as shown below.

Add (mL)

Component

0h 1h Incubation Incubation 0.1 M K2HPO4eKH2PO4 Buffer (pH 7.4) 33 mM MgCl2 5 mg/mL Microsomal Protein

145 20 20

145 20 20

40 mM Test Compounds/Control in 0.1 M Phosphate Buffer: Acetonitrile 60:40 Acetonitrile Preincubated at 37  C for 5 min. 26 mM NADPH Incubated at 37  C for 60 min. Acetonitrile

5

5

200 No 10 No 0

0 Yes 10 Yes 200

Final conc.

90 mM 3.3 mM 0.5 mg/ mL 1 mM

1.3 mM

After quenching by acetonitrile, the plates were capped, vortexed, and centrifuged at 3000 rpm for 10 min. The supernatant (10 mL) was injected into LC-MS/MS. Peak area of propranolol and test compounds at 0 and 1h incubation were used to calculate the % remaining values as follows:

% Remaining ¼ 100*

PeakArea1hr PeakArea0hReplicate1 þ PeakArea0hReplicate2

The average % remaining values for control and test compounds after 1 h incubation in mouse and human liver microsomes are presented in Fig. 6.

4.2.4. In vivo toxicity assay Compounds 1g and 1h were examined for short-term survival and neurotoxicity by the National Institute of Neurological Disorders and Stroke according to their protocols [36]. In brief, mice were injected intraperitoneally with doses of 30, 100 and 300 mg/kg for each compound and the animals were observed at the end of 0.5 h and 4 h. Both compounds showed mild toxicity in just a few of the 24 assays performed for them by the rotarod method [42]. The data was presented as follows: (number of animals demonstrating toxicity/total number of animals tested, time of test in h, dose of compound in mg/kg): For 1g (0/4, 0.5, 30; 0/8, 0.5, 100; 3/4 tremors, 0.5, 300; 0/2., 4, 30; 0/4, 4, 100; 0/2, 4, 300); for 1h (0/4, 0.5, 30; 1/8 diarrhea, 0.5, 100; 0/4, 0.5, 300; 0/2., 4, 30; 0/4, 4, 100; 0/2, 4, 300). There were no mortalities. Mice were fed, handled and housed in accordance with the procedures outlined in the National Research Council publication “Guide for the Care and Use of Laboratory Animals”. The animals were euthanized using the guidelines of the Institute of Laboratory Resources.

4.3. Molecular modeling Molecular mechanics and docking calculations were performed using the Molecular Operating Environment (Chemical Computing Group, Montreal, Canada). The ligand-binding domain of the human estrogen receptor as deduced by protein X-ray crystallography (PDB ID: 1ERR) was retrieved from the Protein Data Bank (www. pdb.org) and prepared using the MMFF94 force field and charge groups. The model was reduced to include only one of the two active sites, residues were physiologically protonated/deprotonated to pH 7.4, and minimization with heavy atom tethers was used to remove potential bad contacts. The bound raloxifene was used as the primary template for docking of the molecules described in literature [43]. Docking was performed using a two-step methodology. In the first step, up to 30 conformations were placed by aligning triplets of atoms to triplets of alpha spheres generated by the receptor site points. These placed conformations were then scored using the London dG function, which estimates free energy of binding for each pose by considering rotational and translational entropy, flexibility upon binding, hydrogen bonding, and desolvation. In the second stop, docking refinement was performed using the above force field, allowing for movement within 6 Å of the pocket residues (backbones tethered slightly by applying constraints with a force constant of 10). Solvation effects were included in this step using a Generalized Born (GB/VI) implicit solvation model. Finally, re-scoring was performed using the GBVI/WSA dG function, which considers rotational and translational entropy, solvent-weighted electrostatic energy, van der Waals contributions, and any energy penalties associated with exposed surface area [44]. Binding modes presented graphically are representative of the highest-scored conformations.

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Acknowledgments The authors thank the following agencies for financial support: the Canadian Breast Cancer Foundation (Atlantic chapter) (to AJ and CKLT), Natural Sciences and Engineering Research Council of Canada (AJ and TSC), Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (to JB) and University of Delhi e Department of Science and Technology PURSE Grant (to VSP). ABN thanks Beatrice Hunter Cancer Research Institute for the Trainee Award for which funds were provided by the Bruce and Dorothy Rossetti Scholarship in Cancer Research as part of The Terry Fox Foundation Strategic Health Research Training Program in Cancer Research at CIHR. YY and VKR acknowledge Center for Scientific and Industrial Research (CSIR), New Delhi, for fellowship support. Technical support from Dr Michael Lumsden of Nuclear Magnetic Resonance Research Resource (NMR-3) at Dalhousie University is gratefully acknowledged for providing NOE results on compounds 1b and 8b. Appreciation is also extended to Mrs. Lizette van Berckelaer for conducting the HeLa, CEM and L1210 assays, the National Cancer Institute, USA, which provided the data using the panel of human tumor cell lines and the National Institute of Neurological Disorders and Stroke, USA for undertaking the short-term toxicity studies in mice. Dr Zenbiao Li of Drumetix Laboratories, Greensboro, NC (USA) is thanked for undertaking PAMPA permeability and metabolic stability assays. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.12.037. References [1] J. Sariego, Breast cancer in the young patient, Am. Surg. 76 (2010) 1397e1400. [2] I. Russo, J. Russo, Role of hormones in mammary cancer initiation and progression, J. Mammary Gland Biol. Neoplasia 3 (1998) 49e61.
langer, J. Simard, S. Gauthier, V. Luu-The, Y. Me
rand, [3] F. Labrie, C. Labrie, A. Be V. Giguere, B. Candas, S. Luo, C. Martel, S.M. Singh, M. Fournier, A. Coquet, V. Richard, R. Charbonneau, G. Charpenet, A. Tremblay, G. Tremblay, L. Cusan, R. Veilleux, EM-652 (SCH 57068), a third generation SERM acting as pure antiestrogen in the mammary gland and endometrium, J. Steroid Biochem. Mol. Biol. 69 (1999) 51e84. [4] V.C. Jordan, W.J. Gradishar, Molecular mechanisms and future uses of antiestrogens, Mol. Asp. Med. 18 (1997) 167e247. [5] K. Maruyama, M. Nakamura, S. Tomoshige, K. Sugita, M. Makishima, Y. Hashimoto, M. Ishikawa, Structureeactivity relationships of bisphenol A analogs at estrogen receptors (ERs): discovery of an ERa-selective antagonist, Bioorg. Med. Chem. Lett. 23 (2013) 4031e4036. [6] C. Yang, G. Xu, J. Li, X. Wu, B. Liu, X. Yan, M. Wang, Y. Xie, Benzothiophenes containing a piperazine side chain as selective ligands for the estrogen receptor a and their bioactivities in vivo, Bioorg. Med. Chem. Lett. 15 (2005) 1505e1507. [7] S.J. Howell, S.R.D. Johnston, A. Howell, The use of selective estrogen receptor modulators and selective estrogen receptor down-regulators in breast cancer, Best Pract. Res. Clin. Endocrinol. Metab. 18 (2004) 47e66. [8] B.S. Katzenellenbogen, I. Choi, R. Delage-Mourroux, T.R. Ediger, P.G.V. Martini, M. Montano, J. Sun, K. Weis, J.A. Katzenellenbogen, Molecular mechanisms of estrogen action: selective ligands and receptor pharmacology, J. Steroid Biochem. Mol. Biol. 74 (2000) 279e285. [9] 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. 54 (2011) 5271e5282. [10] V.C. Jordan, Antiestrogens and selective estrogen receptor modulators as multifunctional medicines. 2. Clinical considerations and new agents, J. Med. Chem. 46 (2003) 1081e1111. [11] V.C. Jordan, Selective estrogen receptor modulation: concept and consequences in cancer, Cancer Cell 5 (2004) 207e213. [12] L. Mosca, D. Grady, E. Barrett-Connor, P. Collins, N. Wenger, B.L. Abramson, A. Paganini-Hill, M.J. Geiger, S.A. Dowsett, M. Amewou-Atisso, M. Kornitzer, Effect of raloxifene on stroke and venous thromboembolism according to subgroups in postmenopausal women at increased risk of coronary heart disease, Stroke 40 (2009) 147e155. [13] D. Grady, J.A. Cauley, J.L. Stock, D.A. Cox, B.H. Mitlak, J. Song, S.R. Cummings, Effect of raloxifene on all-cause mortality, Am. J. Med. 123 (2010), 469e469. [14] Z. Desta, B.A. Ward, N.V. Soukhova, D.A. Flockhart, Comprehensive evaluation

[15]

[16]

[17] [18]

[19]

[20]

[21]

[22]

[23]

[24] [25]

[26]

[27]

[28] [29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

113

of tamoxifen sequential biotransformation by the human cytochrome P450 system in vitro: prominent roles for CYP3A and CYP2D6, J. Pharmacol. Exp. Ther. 310 (2004) 1062e1075. Y. Yadav, E.D. MacLean, A. Bhattacharyya, V.S. Parmar, J. Balzarini, C.J. Barden, C.K.L. Too, A. Jha, Design, synthesis and bioevaluation of novel candidate selective estrogen receptor modulators, Eur. J. Med. Chem. 46 (2011) 3858e3866. B.F. Rasulev, A.I. Saidkhodzhaev, S.S. Nazrullaev, K.S. Akhmedkhodzhaeva, Z.A. Khushbaktova, J. Leszczynski, Molecular modelling and QSAR analysis of the estrogenic activity of terpenoids isolated from Ferula plants, SAR QSAR Environ. Res. 18 (2007) 663e673. Z. Bai, R. Gust, Breast cancer, estrogen receptor and ligands, Arch. Pharm. 342 (2009) 133e149. The interatomic distances were calculated by Chem3D® computer program (version 8.0) after energy minimization of the molecules using MOPAC. Chem3D® is obtainable from Perkin Elmer, MA, USA. Molecular Operating Environment (MOE), 2013.08; Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2014. A. Jha, C. Mukherjee, A.K. Prasad, V.S. Parmar, E.D. Clercq, J. Balzarini, J.P. Stables, E.K. Manavathu, A. Shrivastav, R.K. Sharma, K.H. Nienaber, G.A. Zello, J.R. Dimmock, E, E,E-1-(4-Arylamino-4-oxo-2-butenoyl)-3,5bis(arylidene)-4-piperidones: a topographical study of some novel potent cytotoxins, Bioorg. Med. Chem. 15 (2007) 5854e5865. A. Jha, J. Zhao, T.S. Cameron, E. De Clercq, J. Balzarini, E.K. Manavathu, J.P. Stables, Design, synthesis and biological evaluation of novel curcumin analogues as anti-neoplastic agents, Lett. Drug Des. Discov. 3 (2006) 304e310. H.G. Raj, S. Gupta, G. Biswas, S. Singh, A. Singh, A. Jha, K.S. Bisht, S.K. Sharma, S.C. Jain, V.S. Parmar, Chemoprevention of carcinogen-DNA binding: the relative role of different oxygenated substituents on 4-methylcoumarins in the inhibition of aflatoxin B1-DNA binding in vitro, Bioorg. Med. Chem. 4 (1996) 2225e2228. D. Youssef, C.E. Nichols, T.S. Cameron, J. Balzarini, E. De Clercq, A. Jha, Design, synthesis, and cytostatic activity of novel cyclic curcumin analogues, Biorog. Med. Chem. Lett. 17 (2007) 5624e5629. A. Jha, Selective estrogen receptor modulators, PCT Patent, WO2012079154 (2012).
rez, L.R. Domingo, M. Duque-Noren ~ a, E. Chamorro, A condensed-to-atom P. Pe nucleophilicity index. An application to the director effects on the electrophilic aromatic substitutions, J. Mol. Struct. THEOCHEM 895 (2009) 86e91. M. Beller, O.R. Thiel, H. Trauthwein, Catalytic alkylation of aromatic amines with styrene in the presence of cationic rhodium complexes and acid, Synlett 1999 (1999) 243e245. T. Sugasawa, T. Toyoda, M. Adachi, K. Sasakura, Aminohaloborane in organic synthesis. 1. Specific ortho substitution reaction of anilines, J. Am. Chem. Soc. 100 (1978) 4842e4852. D.S. Salomon, D.T. Vistica, Steroid receptors and steroid response in cultured L1210 murine leukemia cells, Mol. Cell. Endocrinol. 13 (1979) 55e71. M. Castro-Caldas, C.B. Duarte, A. Carvalho, M.C. Lopes, 17b-Estradiol promotes the synthesis and the secretion of annexin I in the CCRF-CEM human cell line, Mediat. Inflamm. 10 (2001) 245e251. M.L. Maminta, A. Molteni, S.T. Rosen, Stable expression of the human estrogen receptor in HeLa cells by infection: effect of estrogen on cell proliferation and c-myc expression, Mol. Cell Endocrinol. 78 (1991) 61e69. M.R. Boyd, K.D. Paull, Some practical considerations and applications of the National Cancer Institute in vitro anticancer drug discovery screen, Drug Dev. Res. 34 (1995) 91e109. €nen, M.K. Karvonen, M. Unkila, T. Paimela, J.M.T. Hyttinen, J. Viiri, T. Ryha H. Uusitalo, A. Salminen, K. Kaarniranta, Influence of selective estrogen receptor modulators on interleukin-6 expression in human retinal pigment epithelial cells (ARPE-19), Eur. J. Pharmacol. 640 (2010) 219e225. T. Trdan Lusin, A. Mrhar, B. Stieger, G.A. Kullak-Ublick, J. Marc, B. Ostanek, A. Zavratnik, A. Kristl, K. Berginc, K. Deli
c, J. Trontelj, Influence of hepatic and intestinal efflux transporters and their genetic variants on the pharmacokinetics and pharmacodynamics of raloxifene in osteoporosis treatment, Transl. Res. 160 (2012) 298e308. J.A. Ruell, O. Tsinman, A. Avdeef, Acid-base cosolvent method for determining aqueous permeability of amiodarone, itraconazole, tamoxifen, terfenadine and other very insoluble molecules, Chem. Pharm. Bull. 52 (2004) 561e565. J. Kim, C.C. Coss, C.M. Barrett, M.L. Mohler, C.E. Bohl, C.-M. Li, Y. He, K.A. Veverka, J.T. Dalton, Role and pharmacologic significance of cytochrome P-450 2D6 in oxidative metabolism of toremifene and tamoxifen, Int. J. Cancer 132 (2013) 1475e1485. J.P. Stables, H.J. Kupferberg, The NIH Anticonvulsant Drug Development (ADD) program: preclinical anticonvulsant, in: Molecular and Cellular Targets for Anti-epileptic Drugs, 1997, p. 191. S.J. Han, Q.Q. Guo, T. Wang, Y.X. Wang, Y.X. Zhang, F. Liu, Y.X. Luo, J. Zhang, Y.L. Wang, Y.X. Yan, X.X. Peng, Y.X. Yan, R. Ling, Y. He, Prognostic significance of interactions between ER alpha and ER beta and lymph node status in breast cancer cases, Asian Pac. J. Cancer Prev. 14 (2013) 6081e6084. E. Marco, W. Laine, C. Tardy, A. Lansiaux, M. Iwao, F. Ishibashi, C. Bailly, F. Gago, Molecular determinants of topoisomerase I poisoning by lamellarins: comparison with camptothecin and structureeactivity relationships, J. Med. Chem. 48 (2005) 3796e3807. Y.-C. Cheng, W.H. Prusoff, Relationship between the inhibition constant (KI)

114

A. Jha et al. / European Journal of Medicinal Chemistry 92 (2015) 103e114

and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction, Biochem. Pharmacol. 22 (1973) 3099e3108. [40] S.A. Abdelmagid, C.K.L. Too, Prolactin and estrogen up-regulate carboxypeptidase-D to promote nitric oxide production and survival of MCF-7 breast cancer cells, Endocrinology 149 (2008) 4821e4828. [41] J. Balzarini, E. De Clercq, M.P. Mertes, D. Shugar, P.F. Torrence, 5-Substituted 20 -deoxyuridines: correlation between inhibition of tumor cell growth and inhibition of thymidine kinase and thymidylate synthetase, Biochem. Pharmacol. 31 (1982) 3673e3682.

[42] N.W. Dunham, T.S. Miya, A note on a simple apparatus for detecting neurological deficit in rats and mice, J. Am. Pharm. Assoc. 46 (1957) 208e209. [43] A.M. Brzozowski, A.C.W. Pike, Z. Dauter, R.E. Hubbard, T. Bonn, O. Engstrom, L. Ohman, G.L. Greene, J.-A. Gustafsson, M. Carlquist, Molecular basis of agonism and antagonism in the oestrogen receptor, Nature 389 (1997) 753e758. [44] M. Naïm, S. Bhat, K.N. Rankin, S. Dennis, S.F. Chowdhury, I. Siddiqi, P. Drabik, T. Sulea, C.I. Bayly, A. Jakalian, E.O. Purisima, Solvated interaction energy (SIE) for scoring proteineligand binding affinities. 1. Exploring the parameter space, J. Chem. Inf. Model. 47 (2007) 122e133.