Design, synthesis and bioevaluation of novel 6-(4-Hydroxypiperidino)naphthalen-2-ol-based potential selective estrogen receptor modulators for breast cancer

Article abstract of DOI:10.1016/j.ejmech.2014.12.037

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

Full text of DOI:10.1016/j.ejmech.2014.12.037

European Journal of Medicinal Chemistry 92 (2015) 103e114
Contents lists available at ScienceDirect
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
b 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
a r t i c l e i n f o
a b s t r a c t
Article history:
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
Received 30 September 2014
Received in revised form
1 November 2014
Accepted 21 December 2014
Available online 23 December 2014
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 sta-
bility and were well-tolerated in experimental mice at 300 mg/kg dose.
Keywords:
Breast cancer
Anticancer
Cytotoxic
© 2014 Elsevier Masson SAS. All rights reserved.
SERM
1-(4-Dialkylaminoethoxy)phenylmethyl-6-
(4-hydroxypiperidino)naphthalen-2-ols
Pharmacokinetics
1. Introduction
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
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
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.
others [5,6]. The pharmacodynamic response to
a SERM is
The generic structure of the prototypical novel potential SERMs
designed for our present study is shown in Fig. 1, along with
* Corresponding author.
E-mail address: ajha@acadiau.ca (A. Jha).
http://dx.doi.org/10.1016/j.ejmech.2014.12.037
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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A. Jha et al. / European Journal of Medicinal Chemistry 92 (2015) 103e114
Fig. 1. Prototypical novel potential SERMs compared with 17b-estradiol, tamoxifen, afimoxifene, raloxifene and 2-napthol-based SERMs.
structures of 17
b
-estradiol (the most potent estrogen), prodrug
(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)
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
phenyl)methanols (5aeh) [15] to yield series
1 compounds.
each other: a) mono- or dihydroxy scaffold to mimic 17
b
-estradiol;
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 com-
mercial 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 C-
monoarylmethylation 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 deter-
mine 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
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 dia-
lkylaminoethoxyphenyl 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
(eNR02
¼
piperidino). Calculations on the energy-minimized
structures of these compounds indicated a distance of 11.4 Å be-
tween 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 (eNR02 ¼ piperidine). These
revealed that at least two out of these three functional groups, the
piperidinol hydroxyl group and piperidine
idinoethoxyphenyl were in the realm of interaction with respective
amino acid residues on the ligand binding domain of ER (vide
N of the piper-
a
infra) [19]. We believed that these features will render potent se-
lective 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)piperidin-
4-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

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.
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
Fig. 2. Two possible regioisomers after C-arylmethylation of compound 4 or 7.
cytotoxicity IC50 value greater than 25
mM. The poor activity of
these phenolic compounds could be attributed to their higher po-
larity leading to poor permeation through lipophilic cell
membranes.
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.
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 com-
pounds, 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].
The authenticity in our structural assignment was further sub-
stantiated 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-(4-
sulfophenyl)-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).
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 leuke-
mia, 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). Raloxi-
fene 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
Since the only difference in the structures of compounds 1a
Fig. 3. NOE enhancements on compounds 1b and 8b.

106
A. Jha et al. / European Journal of Medicinal Chemistry 92 (2015) 103e114
Fig. 4. ORTEP diagrams of 1b and 8b.
Table 3
Table 1
Evaluation of compounds 1b, 1d, 1g and 1h and reference drugs against a panel of
human tumor cell lines.
In-vitro cytotoxicity data of compounds of series 1 and 2 against MCF-7 breast cancer
cells.
Cell lines
1b
5.93
11.0
3.94
10.20 10.77
3.48
8.38
3.00
12.54 12.75
6.84 9.28
12.16 11.75
3.34 4.47
1d
6.34
11.0
3.02
1g
3.11
16.2
1.97
3.48
2.03
3.60
1.92
7.01
3.61
4.95
2.86
1h
TEMc RALd
Entry
Compound
IC50
(
m
M)
Entry
Compound
IC50 (m
M)a
All cell lines
GI50
SIb
(
m
M)a
2.44 3.94 8.29
20.0 7.9
1
2
3
4
5
6
7
8
9
1a
1b
1c
1d
1e
1f
1g
1h
8.49
4.73
3.44
5.20
4.32
>25
3.41
2.46
10
11
12
13
14
15
16
17
18
2a
2b
2c
2d
2e
2f
2g
2h
>50
>50
>25, <50
>25, <50
>25, <50
>50
>25, <50
>25, <50
16.3
251.2
Leukemia
GI50
GI50
GI50
GI50
GI50
(m
(m
(m
(m
(m
(m
(m
(m
(m
M)
M)
M)
M)
M)
M)
M)
M)
M)
1.69 2.24
3.04 5.55
1.70 3.06
2.28 4.30
1.76 3.41
4.96 6.11
2.71 4.47
3.35 4.47
2.04 3.29
3.16
13.44
7.94
10.00
6.99
15.84
11.22
14.12
3.55
Lung cancer
Colon cancer
CNS cancer
Melanoma
Ovarian cancer GI50
Renal cancer GI50
Prostate cancer GI50
3.09
8.35
3.60
Tamoxifen
10.3
Raloxifene
a
The IC50 represents the concentration required to inhibit tumor cell prolifera-
Breast cancer
GI50
tion by 50%.
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
Table 2
SI refers to the selectivity index. The SI figures for all cell lines were obtained by
Cytostatic potency of compounds 1aeh against murine L1210, human CEM T-
lymphocyte and human HeLa cell lines.
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).
Compound
GI50
(
m
M)a
d
GI50 values for raloxifene (RAL) were obtained from online NCI database
L1210
CEM
HeLa
(COMPARE data vector search, compound ID NSC 747974).
1a
1b
1c
1d
1e
1f
1g
1h
5.9 1.3
1.7 0.7
2.1 0.8
5.2 1.5
2.4 1.1
7.2 0.9
4.9 2.4
6.7 0.3
9.8 4.0
15
16
19
21
9.8 6.5
18
13
3.8 0.3
17
15
2
1
0
1
human ER
kit (Carlsbad, CA) at 25 uM and 2.5
pound 1h was used at concentrations of 25
agonist 17 -estradiol (E2) and antagonist tamoxifen (TEM) were
used as controls at 25 M concentration. The results of the colori-
metric 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 M concentrations
a
using ActiveMotif's NR peptide ER
M concentration [32]. Com-
M and 2.5 M. The
a colorimetric ELISA
m
11
18
6
1
11
2
1
6
m
m
2.5 1.7
1.9 0.3
6.7 3.0
5.0 1.7
8.0 3.7
3.5 1.2
6.3 2.1
5.6 1.9
b
m
Tamoxifen
Raloxifene
4
1
m
a
GI50 refers to the compound concentrations required to inhibit the growth of the
used, and is a more potent ER binder and antagonist than
tamoxifen.
cells by 50%.
2.2.5. In vitro pharmacokinetics studies
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.
As described so far, compounds 1g and 1h displayed impressive
in vitro pharmacodynamics as candidate SERMs. We next deter-
mined their preliminary pharmacokinetic profiles to see whether
they warranted further studies. Therefore, in vitro metabolic sta-
bility 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).
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

A. Jha et al. / European Journal of Medicinal Chemistry 92 (2015) 103e114
107
90
80
70
60
50
40
30
20
10
0.45
0.4
0.35
0.3
0.25
0.2
Mouse Liver Microsome
Human Liver Microsome
0.15
0.1
0.05
0
E2
(25μM)
TEM
(25μM)
1h
(25μM)
1h
(2.5μM)
0
Propranolol
1g
1h
Compound (conc.)
Compound
Fig. 5. ER
a
binding affinity assay using commercial ER
a
colorimetric ELISA kit.
Fig. 7. Percent remaining values of control and test compounds 1g and 1h after 60 min
incubation with mouse and human liver microsomes.
2.2.5.1. In vitro membrane permeability studies. Parallel Artificial
Membrane Permeability Assay (PAMPA) was performed on com-
pounds 1g and 1h, along with reference b-blocker drug propran-
active metabolite is 4-hydroxytamoxifen (afimoxifene; Fig. 1) [35].
olol. 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,
2.2.6. In vivo neurotoxicity studies
respectively. Both test compounds, 1g and 1h showed a comparable
average permeability range of 39e57 nm/s at the test concentra-
tions. These results are respectable when taking account the
average permeability values of clinical drugs raloxifene (27 nm/s at
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 experi-
mental 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 well-
tolerated and are not general biocidal agents.
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 com-
pounds 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 proprano-
lol, 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
2.3. Molecular modeling
Based on the results of cytotoxicity of series 1 compounds
against estrogen-responsive MCF-7 cells (Table 1) and ER
a binding
affinity assay, it became increasingly evident that ER , the prog-
a
nostic 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 com-
pounds (vide supra) support this hypothesis even further [19]. The
screenshots of the docking studies are included in Fig. 8. Fig. 8A
100
90
shows the ligand 1g bound in the ligand binding domain of ER
a
80
with important amino acid residues on the protein surface. Fig. 8B
is a proximity contour of ligand 1g indicating three major in-
teractions, viz., piperidinol eOH forming H-bond with Glu 353,
hydrogen bonding between methoxy and His 524, and the piperi-
dine 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]:
70
60
50
40
30
20
10
0
20 μM Incubaꢀon
25 μM Incubaꢀon
50 μM Incubaꢀon
Propranolol
1g
1h
Raloxifene Tamoxifen
Compound
Fig. 6. Permeability of test and reference compounds at pH 7.4. The values for ralox-
ifene (at pH 6.4) and tamoxifen were obtained from literature sources [33,34].

108
A. Jha et al. / European Journal of Medicinal Chemistry 92 (2015) 103e114
Fig. 8. Compound 1g docked in the ER
a
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.
300 and 75 MHz, respectively, in CDCl3 where residual CHCl3
¼ 7.27) served as the internal standard for 1H NMR, and CDCl3
DDGbinding ¼ DGligand 1 ꢀ DGligand 2 ¼ RT lnr
(
d
was used as the internal standard (
d
¼ 77.0) for 13C NMR. Melting
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].
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, Ger-
many) and silica gel 60 (SiliaFlash P60, Silicycle, Quebec City, Can-
ada) were used for thin layer chromatography (TLC) and flash
chromatography, respectively.
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 ꢁ10ꢀ3 kcal/K.mol) and
temperature T (298 K or 25 ꢂC) 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).
4.1.2. General procedure for the synthesis of 1-(6-
alkoxynaphthalen-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 ꢂC for 24 h. After completion of the re-
action, 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 pres-
sure 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.
3. Conclusion
In conclusion, the present study has led to the identification of
1-(5-(4-(2-(dialkylamino)ethoxy)benzyl)-6-methoxynaphthalen-
2-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 ac-
tivity 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
4.1.2.1. 1-(6-Methoxynaphthalen-2-yl)piperidin-4-ol
(4).
1.57 (brs,
Colorless solid, yield 72%. 1H NMR (300 MHz, CDCl3):
d
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,
cytotoxic efficacy and antagonistic activity against ER
a-dependent
1H), 7.60e7.69 (m, 2H). 13C NMR (75.5 MHz, CDCl3):
d 34.1, 47.5,
breast cancer cells in vitro.
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. Experimental section
4.1. Chemistry
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
4.1.1. General information
(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,
All chemicals and reagents were purchased from Aldrich
Chemical Co. and were used without further purification. 1H and
13C NMR were recorded on a Bruker AV300 spectrophotometer at
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
109
119.4,120.8,127.6,127.8,128.0,128.4,128.7,129.7,130.6,137.7,148.2,
155.7.
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. General procedure for the synthesis of 1-(5-(4-(2-
(dialkylamino)ethoxy)benzyl)-6-alkoxynaphthalen-2-yl)piperidin-
4-ols (1aeh & 8aeh)
4.1. 3. 5. 1-(5-(4-(2-(Piperidin-1-yl)ethoxy)benzyl)-6-
methoxynaphthalen-2-yl)piperidin-4-ol (1e). Colorless solid, yield
To an ice-cold solution of appropriate 1-(6-(alkoxyoxy)naph-
thalen-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 drop-
wise addition of borontrifluoride etherate (4 equiv.). The reaction
mixture turned brown. The stirring was continued at room tem-
parature 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 pres-
sure. 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
71%, mp 193e195 ꢂC. 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).
13C 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
C
30H38N2O3: 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 ꢂC. 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,
were purified on silica gel using 6e10
dichlorometahne.
% of methanol in
4.1. 3.1. 1-(5-(4-(2-(Dimethylamino)ethoxy)benzyl)-6-
1H). 13C NMR (75.5 MHz):
d 29.7, 34.3, 47.8, 53.9, 56.9, 57.6, 65.5,
methoxynaphthalen-2-yl)piperidin-4-ol (1a). Colorless solid, yield
42%, mp 220e222 ꢂC. 1H NMR (300 MHz, CDCl3):
d 1.69e1.81 (m,
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
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,
C
29H36N2O4: 477.2753 [MþH]þ; found: 477.2772.
J ¼ 9.0 Hz, 1H) 7.80 (d, J ¼ 9.4 Hz, 1H). 13C NMR (75.5 MHz):
d 29.7,
4.1.3.7. 1-(5-(4-(2-(4-Methylpiperidin-1-yl)ethoxy)benzyl)-6-
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.
methoxynaphthalen-2-yl)piperidin-4-ol (1g). Colorless solid, yield
66%, mp 198e201 ꢂC. 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,
4 .1. 3 . 2 . 1 - ( 5 - ( 4- ( 2 - ( Di e t hyl a mi no ) e t h o xy ) b e nz yl ) - 6 -
methoxynaphthalen-2-yl)piperidin-4-ol (1b). Colorless solid, yield
67%, mp 124e127 ꢂC. 1H NMR (300 MHz, CDCl3):
d 1.11 (t,
J ¼ 9.0 Hz, 1H), 7.80 (d, J ¼ 9.3 Hz, 1H). 13C NMR (75.5 MHz):
d 21.7,
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,
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.
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,
4 .1. 3 . 8 . 1 - ( 5 - ( 4 - ( 2 - ( A z e p a n - 1 - yl ) e t h o x y ) b e n z yl ) - 6 -
129.1, 130.4, 133.6, 147.2, 153.3, 156.6. ESI-HRMS (m/z): calcd. for
methoxynaphthalen-2-yl)piperidin-4-ol (1h). Colorless solid, yield
C
29H38N2O3: 463.2961 [MþH]þ; found: 463.2953.
67%, mp 146e148 ꢂC. 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):
4.1.3.3. 1-(5-(4-(2-(Diisopropylamino)ethoxy)benzyl)-6-
methoxynaphthalen-2-yl)piperidin-4-ol (1c). Colorless solid, yield
49%, mp 224e225 ꢂC. 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,
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. ESI-
HRMS (m/z): calcd. for C31H40N2O3: 489.3117 [MþH]þ; found:
489.3120.
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.
4.1.3.9. 1-(6-(Benzyloxy)-5-(4-(2-(dimethylamino)ethoxy)benzyl)
naphthalen-2-yl)piperidin-4-ol (8a). Brown colored foamy solid,
yield 40%, mp 145e147 ꢂC. 1H NMR (300 MHz, CDCl3):
d 1.75e1.81
4.1.3.4. 1-(5-(4-(2-(Pyrrolidin-1-yl)ethoxy)benzyl)-6-
(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),
methoxynaphthalen-2-yl)piperidin-4-ol (1d). Colorless solid, yield
65%, mp 220e222 ꢂC. 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,
7.21e7.40 (m, 6H), 7.60 (d, J ¼ 9.0 Hz, 1H), 7.80 (d, J ¼ 9.6 Hz, 1H). 13
C
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.
J ¼ 8.7 Hz, 1H) 7.80 (d, J ¼ 9.3 Hz, 1H). 13C NMR (75.5 MHz):
d 23.4,

110
A. Jha et al. / European Journal of Medicinal Chemistry 92 (2015) 103e114
4.1.3.10. 1-(6-(Benzyloxy)-5-(4-(2-(diethylamino)ethoxy)benzyl)
114.9, 116.3, 120.7, 123.7, 125.2, 127.0, 127.6, 128, 128.6, 129.5, 131.2,
naphthalen-2-yl)piperidin-4-ol (8b). Colorless foamy solid, yield
134, 138, 147.7, 152.8, 157.2.
52%, mp 108e109 ꢂC. 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),
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 ꢂC. 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,
7.25e7.40 (m, 6H), 6.61 (d, J ¼ 9 Hz, 1H), 7.83 (d, J ¼ 9.3 Hz, 1H). 13
C
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.
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.
4.1.3.11. 1-(6-(Benzyloxy)-5-(4-(2-(diisopropylamino)ethoxy)benzyl)
naphthalen-2-yl)piperidin-4-ol (8c). Colorless foamysolid, yield 62%,
mp 79e81 ꢂC. 1H NMR (300 MHz, CDCl3):
d
1.13e1.15 (m, 12H),
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)
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,
A
mixture of 1-(1-(4-(2-(dialkylamino)ethoxy)benzyl)-6-
J¼8.7Hz,1H),7.82(d, J¼ 9.0Hz,1H).13CNMR(75MHz,CDCl3):
d
20.6,
hydroxynaphthalen-2-yl)piperidin-4-ol (1.5 g) and 10% palla-
diumecarbon (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)piper-
idin-4-ols (2aeh) as white solids. The compounds were of high
purity and further purification was not required.
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 ꢂC. 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),
4.1.4.1. 1-(1-(4-(2-(Dimethylamino)ethoxy)benzyl)-6-
hydroxynaphthalen-2-yl)piperidin-4-ol (2a). Pale pink colored solid,
yield 91%, mp 93e95 ꢂC. FTIR (Nujol): 740, 1034, 1377, 1462, 1643,
7.20e7.40 (m, 7H), 7.60 (d, J ¼ 9.0 Hz,1H), 7.84 (d, J ¼ 9.3 Hz,1H). 13
C
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.
2800, 3419 cmꢀ1. 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,
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 ꢂC. 1H NMR (300 MHz, CDCl3):
d
1.42e1.48 (m,
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,
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),
135.3, 136.0, 137.6, 155.8, 157.3. ESI-HRMS (m/z): calcd. for
C
26H33N2O3: 421.2491 [MþH]þ; found: 421.2484.
7.22e7.40 (m, 7H), 7.61 (d, J ¼ 9 Hz, 1H), 7.83 (d, J ¼ 9.3 Hz, 1H). 13
C
4.1. 4. 2 . 1 - ( 1- ( 4 - ( 2 - ( Di e t hyla min o ) etho x y)ben z yl)- 6 -
hydroxynaphthalen-2-yl)piperidin-4-ol (2b). Off white solid, yield
88%, mp 75e78 ꢂC. FTIR (Nujol): 732, 805, 990, 1060, 1174, 1242,
1383, 1465, 1510, 1607, 2947, 3336 cmꢀ1. 1H NMR (300 MHz, CDCl3):
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.
d
1.07 (t, J ¼ 6.9 Hz, 6H), 1.65e1.82 (m, 2H), 1.98e2.10 (m, 2H), 2.68
4.1.3.14. 1-(6-(Benzyloxy)-5-(4-(2-morpholinoethoxy)benzyl)naph-
(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),
thalen-2-yl)piperidin-4-ol (8f). Colorless foamy solid, yield 45%, mp
54e55 ꢂC.1H NMR (300 MHz, CDCl3):
d
1.65e1.82 (m, 4H), 2.01e2.07
7.0e7.30 (m, 5H), 7.50 (d, J ¼ 8.7 Hz, 1H), 7.77 (d, J ¼ 9.3 Hz; 1H). 13
C
(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,
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.
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.
4.1.4.3. 1-(1-(4-(2-(Diisopropylamino)ethoxy)benzyl)-6-
hydroxynaphthalen-2-yl)piperidin-4-ol (2c). Pink colored solid,
yield 90%, mp 94e96 ꢂC. FTIR (Nujol): 743, 808, 1061, 1173, 1263,
4.1.3.15. 1-(6-(Benzyloxy)-5-(4-(2-(4-methylpiperidin-1-yl)ethoxy)
1464, 1609, 2800, 3200 cmꢀ1
.
1H NMR (300 MHz, CDCl3þMeOD):
benzyl)naphthalen-2-yl)piperidin-4-ol (8g). Colorless foamy solid,
d
0.91 (d, J ¼ 6.6 Hz, 12H), 1.43e1.60 (m, 2H), 1.75e1.87 (m, 2H),
yield 45%, mp 58e60 ꢂC. 1H NMR (300 MHz, CDCl3):
d
0.93 (d,
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 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):
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
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,
C
30H41N2O3: 477.3117 [MþH]þ; found: 477.3104.

A. Jha et al. / European Journal of Medicinal Chemistry 92 (2015) 103e114
111
4.1.4.4. 1-(1-(4-(2-(Pyrrolidin-1-yl)ethoxy)benzyl)-6-
4.2. Biological assay methods
4.2.1. Cell cytotoxicity assays
hydroxynaphthalen-2-yl)piperidin-4-ol (2d). Brown colored solid,
yield 94%, mp 92e94 ꢂC. FTIR (Nujol): 723, 881, 1051, 1154, 1376,
1457, 2724, 3331 cmꢀ1
.
1H NMR (300 MHz, CDCl3þMeOD):
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 per-
formed following the manufacturer's instructions. The results are
presented in Table 1.
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.
The cytostatic activities of the synthesized compounds for mu-
rine 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 Insti-
tute (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.
4.1.4.5. 1-(6-Hydroxy-1-(4-(2-(piperidin-1-yl)ethoxy)benzyl)naph-
thalen-2-yl)piperidin-4-ol (2e). Off white solid, yield 93%, mp
118e120 ꢂC. FTIR (Nujol): 759, 819, 990, 1060, 1176, 1263, 1382, 1470,
1510, 1609, 2943, 3404 cmꢀ1 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
C
29H37N2O3: 461.2804 [MþH]þ; found: 461.2804.
4.1.4.6. 1-(1-(4-(2-Morpholinoethoxy)benzyl)-6-hydroxynaphthalen-
2-yl)piperidin-4-ol (2f). Light brown colored solid, yield 91%, mp
79e82 ꢂC. FTIR (Nujol): 777, 808, 988, 1062, 1173, 1243, 1382, 1456,
4.2.2. NR peptide ER
In this assay, 96-well plates were coated with a peptide con-
taining an ER co-activation binding motif that bound only to the
a ELISA assay
1510, 1609, 2926, 3345 cmꢀ1
.
1H NMR (300 MHz, CDCl3þMeOD):
a
d
1.66e1.81 (m, 2H), 1.98e2.10 (m, 2H), 2.52e2.62 (m, 4H), 2.80 (t,
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 so-
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.
lutions of tamoxifen (TEM, antagonist control) and 17b-estradiol
(E2, agonist control) were diluted to 25 mM. Compound 1h (as
4.1.4.7. 1-(1-(4-(2-(4-Methylpiperidin-1-yl)ethoxy)benzyl)-6-
hydroxynaphthalen-2-yl)piperidin-4-ol (2g). Pale yellow colored
solid, yield 89%, mp 91e93 ꢂC. FTIR (Nujol): 730, 877, 1049, 1124,
hydrochloride salt) was dissolved in water to form a 1 mM stock
solution. This stock solution was diluted to 25
the ELISA assay. Nuclear extracts of 50 g per well were used,
except for the agonist and antagonist controls, which had 15 g of
mM and 2.5 mM for
m
1377, 1460, 2725, 3334 cmꢀ1
.
1H NMR (300 MHz, CDCl3þMeOD):
m
d
0.70 (d, J ¼ 4.0 Hz, 3H), 1.0e1.24 (m, 3H), 1.30e1.60 (m, 4H),
nuclear extract per well as suggested by the manufacturer. The
developing and stop solutions were added at the end of the pro-
cedure, allowing the amount of active co-activator-ERa complex to
be quantified. The absorbance at 450 nm was measured on a Bio-
Rad microplate reader. This assay was performed in duplicate at
both the concentrations.
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
C
4.2.3. In vitro pharmacokinetics studies
30H39N2O3: 475.2961 [MþH]þ; found: 475.2944.
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
4 .1. 4 . 8 . 1 - ( 1 - ( 4 - ( 2 - ( A z e p a n - 1 - yl ) e t h o x y ) b e n z yl ) - 6 -
hydroxynaphthalen-2-yl)piperidin-4-ol (2h). Pale pink colored
solid, yield 95%, mp 88e90 ꢂC. FTIR (Nujol): 744, 1154, 1263, 1376,
to room temperature for 30 min 325
pound solutions in 5% DMSO in phosphate-buffered saline (PBS)
was added into the wells in the receiver (donor) plate. 200 L of 5%
mL of 50 or 25 mM test com-
1460, 2724, 3300 cmꢀ1
.
1H NMR (300 MHz, CDCl3þMeOD):
d
1.50e1.80 (m, 10H), 1.90e2.10 (m, 2H), 2.72e3.0 (m, 8H), 3.17 (bs,
m
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,
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 ꢂC for 5 h. After in-
cubation, 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.
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.

112
A. Jha et al. / European Journal of Medicinal Chemistry 92 (2015) 103e114
Cal. std. Donor sampleb Acceptor sample
The average % remaining values for control and test compounds
after 1 h incubation in mouse and human liver microsomes are
presented in Fig. 6.
Blank 5% DMSO in PBS (
Donor Sample ( L)
Acceptor Sample (
Standard working solutiona
m
L)
0
0
0
360
40
0
0
400
0
0
150
0
m
mL)
(
m
L) 400
400
MeOH (
mL)
150
4.2.4. In vivo toxicity assay
a
Standard working solution: 5, 50, 500, and 5000 nM in 5% DMSO in PBS.
The donor samples were analyzed with 10 times dilution.
Compounds 1g and 1h were examined for short-term survival
and neurotoxicity by the National Institute of Neurological Disor-
ders 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 An-
imals”. The animals were euthanized using the guidelines of the
Institute of Laboratory Resources.
b
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 concen-
trations in buffer samples from donor and acceptor wells were used
for calculation of permeability (Pe) in nm/s as shown below:
h
.
i
ꢀln 1 ꢀ CAðtÞ Cequilibrium
A*ð1=VD þ 1=VAÞ*t
Pe ¼ 107*
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 vol-
ume, 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 pre-
sented in Fig. 5.
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 hu-
man 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/deproto-
nated 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].
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 ꢂC, were per-
formed for 0 and 60 min and quenched by addition of one volume
of acetonitrile. Ingredients for different incubations were added as
shown below.
Component
Add (
0 h
m
L)
Final
conc.
1 h
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 resi-
dues (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.
Incubation Incubation
0.1 M K2HPO4eKH2PO4 Buffer (pH 7.4)
33 mM MgCl2
5 mg/mL Microsomal Protein
145
20
145
20
90 mM
3.3 mM
0.5 mg/
mL
20
20
40
m
M Test Compounds/Control in 0.1 M
5
5
1 mM
Phosphate Buffer: Acetonitrile 60:40
Acetonitrile
200
No
10
No
0
0
Yes
10
Yes
200
Preincubated at 37 ꢂC for 5 min.
26 mM NADPH
1.3 mM
Incubated at 37 ꢂC for 60 min.
Acetonitrile
After quenching by acetonitrile, the plates were capped, vor-
texed, 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:
PeakArea1hr
ꢀ
ꢁ.
% Remaining ¼ 100*
PeakArea0hꢀReplicate1 þ PeakArea0hꢀReplicate2
2

A. Jha et al. / European Journal of Medicinal Chemistry 92 (2015) 103e114
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.
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 Can-
ada (AJ and TSC), Fonds voor Wetenschappelijk Onderzoek-Vlaan-
deren (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 acknowl-
edged 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.
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(version 8.0) after energy minimization of the molecules using MOPAC.
Chem3D® is obtainable from Perkin Elmer, MA, USA.
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Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A
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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.
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