Synthesis of targeted dibenzo[b,f]thiepines and dibenzo[b,f]oxepines as potential lead molecules with promising anti-breast cancer activity

Article abstract of DOI:10.1016/j.ejmech.2015.05.035

A targeted library of substituted dibenzo[b,f]thiepines and dibenzo[b,f]oxepines (prototypes I, II and III), and structurally analogous to tamoxifen have been synthesized as a new class of anti-breast cancer agents. All the prototype molecules exhibited potential antiproliferative activity against ER + ve and ER-ve breast cancer cell lines. Dibenzo[b,f]thiepine prototypes were found to be more active. Of all the compound tested, 14b exhibited potent in-vitro antiproliferative activity at 1.33 μM and 5 μM concentration in MCF-7 and MDA-MB-231 cell lines and was devoid of any cytotoxicity in normal HEK cells even at 50 μM. Cell cycle analysis showed that the compound 14b inhibited cell proliferation due to G0/G1 arrest in MCF-7 cells. Annexin-V FITC and PI staining experiments confirmed that the cell inhibition was primarily due to apoptosis and not by necrosis, which was also supported by LDH release assay experiment. Molecular docking studies showed better binding interaction of the new dibenzo[b,f]thiepine analogue 14b with the estrogen receptor (ER) as compared to 4-hydroxy-tamoxifen and this enhanced binding might be responsible for its estrogen antagonistic activity that induces cell cycle arrest, apoptosis and inhibition of breast cancer cells.

Full text of DOI:10.1016/j.ejmech.2015.05.035

Accepted Manuscript
Synthesis of Targeted Dibenzo[b,f]thiepines and Dibenzo[b,f]oxepines as Potential
Lead Molecules with Promising Anti-breast Cancer Activity
Mohd. Imran Ansari, Mohd. Kamil Hussain, Ashutosh Arun, Bandana Chakravarti,
Rituraj Konwar, K. Hajela
PII:
S0223-5234(15)30062-3
10.1016/j.ejmech.2015.05.035
EJMECH 7914
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 December 2014
Revised Date: 19 May 2015
Accepted Date: 22 May 2015
Please cite this article as: M.I. Ansari,, M.K. Hussain, A. Arun, B. Chakravarti, R. Konwar, K. Hajela,
Synthesis of Targeted Dibenzo[b,f]thiepines and Dibenzo[b,f]oxepines as Potential Lead Molecules with
Promising Anti-breast Cancer Activity, European Journal of Medicinal Chemistry (2015), doi: 10.1016/
j.ejmech.2015.05.035.
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ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT
Conformationally constrained tricyclic compounds namely, substituted dibenzo[b,f]thiepines and
dibenzo[b,f]oxepines ( prototypes I, II and III), have been synthesized as new lead molecules exhibiting
potential antiproliferative activity against ER+ve & ER-ve breast cancer cell lines.

ACCEPTED MANUSCRIPT
Synthesis of Targeted Dibenzo[b,f]thiepines and
Dibenzo[b,f]oxepines as Potential Lead Molecules with Promising
Anti-breast Cancer Activity
a#
a
b
b
Mohd. Imran Ansari, Mohd. Kamil Hussain, Ashutosh Arun, Bandana Chakravarti,
b
a
Rituraj Konwar, *and K. Hajela *
a
Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Sector 10,
Jankipuram Extension, Sitapur Road, Uttar Pradesh, Lucknow-226031, India.
b
Endocrinology Division, CSIR-Central Drug Research Institute, Sector10, Jankipuram Extension,
Sitapur Road, Uttar Pradesh, Lucknow-226031, India.
#
Present address:Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy,
Baltimore, Maryland 21201, United States.
*
To whom correspondence should be addressed
Chemistry related queries: kanchan_hajela@cdri.res.in
Biology related queries: r_konwar@cdri.res.in
1

ACCEPTED MANUSCRIPT
Abstract
A targeted library of substituted dibenzo[b,f]thiepines and dibenzo[b,f]oxepines (prototypes I, II
and III), and structurally analogous to tamoxifen have been synthesized as a new class of anti-
breast cancer agents. All the prototype molecules exhibited potential antiproliferative activity
against ER+ve and ER-ve breast cancer cell lines. Dibenzo[b,f]thiepine prototypes were found
to be more active. Of all the compound tested, 14b exhibited potent in-vitro antiproliferative
activity at 1.33 µM and 5 µM concentration in MCF-7 and MDA-MB-231 cell lines and was
devoid of any cytotoxicity in normal HEK cells even at 50 µM. Cell cycle analysis showed that
the compound 14b inhibited cell proliferation due to G0/G1 arrest in MCF-7 cells. Annexin-V
FITC and PI staining experiments confirmed that the cell inhibition was primarily due to
apoptosis and not by necrosis, which was also supported by LDH release assay experiment.
Molecular docking studies showed better binding interaction of the new dibenzo[b,f]thiepine
analogue 14b with the estrogen receptor (ER) as compared to 4-hydroxy-tamoxifen and this
enhanced binding might be responsible for its estrogen antagonistic activity that induces cell
cycle arrest, apoptosis and inhibition of breast cancer cells.
Keywords: Anti-breast cancer, estrogen receptors, antiestrogen, dibenzo[b,f]thiepines,
dibenzo[b,f]oxepines, synthesis
2

ACCEPTED MANUSCRIPT
1
. Introduction
Breast cancer is a major health concern worldwide and the leading cause of death among
women in economically developed as well as in developing countries. An estimated 1.7 million
women will be diagnosed with breast cancer in 2020 - a 26% increase from current levels [1]. In
India, almost 100,000 women are diagnosed with breast cancer every year, and a rise to 131,000
cases is predicted by 2020 [2, 3]. Estrogens are a group of steroid hormones and key regulators of
growth, differentiation and function of the female reproductive organs including the breast,
uterus and ovaries. In addition, they also help in the regulation of cholesterol, lipid levels and the
functioning of skeletal, central nervous and cardiovascular systems [4]. The biological actions of
estrogens are mediated through intracellular transcription factors called estrogen receptors (ER)
which belong to the nuclear receptor superfamily. In spite of these beneficial effects, estradiol
stimulation also influences the pathological processes of hormone-dependent cancers of breast,
endometrial, prostrate, ovary and many other type of malignant tumors [5,6]. The important
finding of the hormone responsiveness of ER+ve breast cancers, triggered the development of
molecules which can compete with estradiol at its receptor and counterbalance its proliferative
action [7]. The discovery of non-steroidal antiestrogen, tamoxifen as the therapy of choice for
patients with ER+ve breast cancer [8] revolutionized the approach to regulate the endocrine
system by disrupting the signalling cascade. However, it was later discovered that tamoxifen
exists as two geometrical stereoisomers which had opposite actions [9, 10]. This pharmaceutical
limitation steered the medicinal chemists towards development of nonisomerizable
antiestrogenic compounds such as, nafoxidine, containing a cyclic hexene ring with a fixed
double bond configuration [11,12] and some ring fused analogues like benzocycloheptene
[13,14], benzoxepine [15] and benzothiepines [16] with stereochemical and endocrinological
features similar to that of tamoxifen (Fig.1).
<
Figure 1>
The discovery of these compounds solved the issue of isomerisation, but were devoid of
any potential antiproliferative or antiestrogenic activity. In our continuous endeavour towards the
search and development of estrogen antagonists as potential anti-breast cancer agents [17], we
3

ACCEPTED MANUSCRIPT
report in this communication, synthesis of some new hetrocyclic compounds having a planar
tricyclic core to which is attached a pendant phenyl ring with basic tert.aminoalkoxy group (side
chain) perpendicularly oriented in the desired antiestrogenic conformation, an essential requisite
for the antiproliferative activity. Following prototypes of molecules I, II and III were
successfully synthesized for the structure - activity relationship studies and biologically screened
for anti-proliferative activity against breast cancer cell lines (Fig.2).
<
Figure 2>
2
. Results and discussion
2
.1 Chemistry
The target prototype molecules were synthesized as shown in Schemes 1 to 6. The key
precursors, dibenzo[b,f]thiepinone 3 and dibenzo[b,f]oxepinone 9 required for the synthesis of
prototype I molecules were prepared by some modification of the reported methods [18, 19].
Hetero-Ullmann coupling of thiophenol with methyl 2-(2-iodophenyl)acetate formed the
thioether 1, which on basic hydrolysis followed by intramolecular cyclization gave the desired
cyclic ketone 3 in 78% yields (Scheme 1).
<
Scheme 1>
The dibenzo[b,f]oxepinone 9 was synthesised following the procedure shown in Scheme
2
. Nucleophilic substitution of 2-fluoro-benzaldehyde with phenol [20] formed the 2-
phenoxybenzaldehyde 4, which on reduction with sodium borohydride gave the corresponding
alcohol 5. Reaction with thionyl chloride yielded the 2-phenoxy benzyl chloride 6 followed by
its subsequent conversion to 2-phenoxy benzyl cyanide 7. Alkaline hydrolysis of cyanide 7
furnished the desired 2-phenoxy phenyl acetic acid 8 which on intramolecular Friedel-Crafts
acylation formed the dibenzo [b,f]oxepin-10(11H)-one scaffold 9 in good yields.
<
Scheme 2 >
Grignard reaction of the cyclic ketones 3 and 9 with 4-methoxyphenylmagnesium
bromide formed the 7-arylated products 10 and 11 in low yields. Alternatively, reacting the
4

ACCEPTED MANUSCRIPT
ketones with 4-bromoanisole and n-butyllithium at -78ºC, formed the carbinols which on acidic
dehydration furnished the desired compounds 10 and 11 in good yields [21]. Subsequent
demethylation with boron tribromide yielded the phenolic compounds 12 and 13 which were O-
alkylated with different tert-aminoalkyl halides to form the prototype I molecules, 10-(4-
ethoxyaminophenyl)
dibenzo[b,f]thiepines,
14a-d
and
10-(4-ethoxyamino
phenyl)dibenzo[b,f]oxepines 15 a-d respectively (Scheme 3).
<
Scheme 3 >
To mimic the biological effect of the ethyl group present in tamoxifen on the anti-breast
cancer activity, prototype II molecules with an ethyl substituent at 6-position were synthesized
as shown in Schemes 4 and 5. Alkylation at the α-position of 2-(2-(phenylthio)phenyl)butanoic
acid 2 with ethyl iodide using freshly prepared lithium diisopropyl amide under strict anhydrous
conditions furnished the product 16 in good yields. Subsequent cyclisation of 16 to ketones 17
&
18 using either PPA, AlCl -(COCl) or CH SO H was sluggish giving very low yields of the
3 2, 3 3
ketones. However, successful use of Eaton’s reagent (PPA and CH SO H in 1:1 ratio) resulted
3
3
in the formation of cyclized product 11-ethyl dibenzo[b,f]thiepin-10(11H)-one 17 in more than
0% yields. With dibenzo[b,f]oxepin-10(11H)-one 9, α-ethylation was successfully achieved
6
using PTC conditions at room temperature to accomplish the ethyl substituted
dibenzo[b,f]oxepinone 18, [22] (Scheme 4).
<
Scheme 4 >
The target molecules of prototype II, 10-ethyl-11-(4-ethoxyaminophenyl)dibenzo[b,f]
thiepines 23a-d and 10-ethyl-11-(4-ethoxyaminophenyl) dibenzo [b,f]oxepines 24a-e
respectively (Scheme 5) were synthesized following the same protocol of arylation at 7-position,
demethylation and O-alkylation of compounds 17 and 18 as described for compounds 14a-d and
1
5a-d.
<
Scheme 5 >
Further advancement in the modification of the pharmcophore by replacing the ethyl
substituent with an aryl ring and its effect on the biological activity led to the synthesis of tetra-
aryl derivatives of prototypes III, which were synthesized as summarized in Scheme 6.
5

ACCEPTED MANUSCRIPT
Bromination of the double bond in compounds 10 and 11 with pyridinium hydrobromide
perbromide readily accomplished the bromo compounds 25 and 26 which on Suzuki coupling
with phenyl boronic acid afforded the tetra-arylated compounds 27 and 28. Subsequent
,
3
demethylation with BBr led to the phenolic derivatives 29 and 30 which were finally O-
alkylated with different aminoalkyl halides furnishing the target prototype III molecules, 11-(4-
aminoethoxyphenyl)-10-phenyldibenzo[b,f]thiepines 31a-e and 11-(4-aminoethoxy-phenyl)-10-
phenyldibenzo[b,f] oxepines 32a-d in good yields.
<
Scheme 6 >
2
.2 Biological evaluation
Anti-proliferative activity against breast cancer
As the anti-estrogenic compounds act antagonistically on the breast cancer cells
inhibiting their proliferation, all the targeted compounds were screened for their antiproliferative
activity on breast cancer cell lines namely, MCF-7 (ER+ve) and MDA-MB-231 (ER-ve) cells
using MTT assay. The results are shown in Table 1. Twelve compounds of the series, 14a, 14b
and 14d, 15d, 24d, 29, 31a, 31d and 31e, 32b, 32c and 32d were found to inhibit growth of
MCF-7 cells with IC less than 20µM. In MDA-MB-231 cell lines, eighteen compounds 14b,
5
0
1
3
4c and 14d, 13, 15a and 15d, 23a, 23b, 23d and 23e, 29, 31b, 31c and 31e, 32a, 32b, 32c and
2d inhibited cell proliferation with IC less than 20µM. Among the active compounds, nine
5
0
compounds, viz. 13, 14b, 14d, 15d, 24d, 29, 32b, c and d showed activity against both ER+ve
and ER-ve cell lines. Of all the active compounds, 14a, 14b and 31e showed maximum inhibition
of cell growth in MCF-7 cells with an IC50 of 6.6 µM, 1.33 µM and 9.25 µM respectively,
whereas, four compounds 14b, 24d, 31e and 32d showed maximum inhibitory effect on cell
growth against MDA-MB-231 cells with an IC50 of 5 µM, 5.74 µm, 10.8 µM, and 7.02 µM
respectively (Table 1). A close observation of the data showed that sulphur containing prototypes
were more active than their oxygen counterpart. Further, of the three prototype molecules,
compound 14b (prototype I with piperidino amino alkyl group) was the most active followed by
compound 14a. It was important to note that prototype II compounds with a α-ethyl substituent
were devoid of any significant activity, whereas alpha arylated compounds exhibited better
activity and compound 31e (prototype III, with dimethyl amino group) was equiactive to
tamoxifen in both the cell lines. Similarly compound 32b also showed good activity but was
6

ACCEPTED MANUSCRIPT
found to be toxic. Though compound 14a is more selective towards MCF-7 cells than MDA-
MB-231 cells (6.6 µM versus 22.9 µM respectively), compound 14b showed potent activity
against both the breast cancer cell lines (1.33 µM and 5.0 µM respectively and was therefore
chosen as lead molecule for further investigations.
<
Table 1>
Cytotoxicity towards normal cells
Specific killing of the cancer cells without affecting the normal cell growth is a key
safety feature of cancer chemotherapy, therefore, all the compounds were evaluated for possible
cytotoxicity in normal cells, HEK-293. Of the active molecules, two compounds, 24d and 32d
induced significant cell death in HEK-293 cells. Significantly, compound 14b showed no effect
on the growth of HEK-293, suggesting that the molecule is specifically active against breast
cancer cells, whereas compound 14a, which is active against ER+ve MCF-7 cells, was also
found to be relatively safe against HEK-293 cells (Table 1).
Morphological study of compound 14b in bright field microscopy
The ER+ve MCF-7 cells were treated with IC50 (1.33 µM), sub IC50 (0.87 µM) and
higher than IC50 concentration(10.5 µM) of compound 14b for 24 hours along with untreated
control and 5 µM of standard drug, tamoxifen in 24-well culture plates. Random fields under
bright light inverted microscope were acquired after 24 hours. On viewing the plates, visible
rounding and shrinking of cell size was observed in the treated groups substantiating the
inhibitory effect of compound 14b. In addition, cell numbers were also found to be visibly
reduced in higher concentration treatment possibly due to excessive cell death (Fig.3).
<
Figure 3 >
Analysis of the effect of compound 14b on cell cycle distribution of MCF-7 cells
For cell cycle analysis, MCF-7 cells grown in T25 cell culture flask were treated with
IC (1.33 µM), sub IC (0.87 µM) and higher than IC (10.5 µM) concentration of compound
5
0
50
50
1
1
4b for 24 hours along with untreated control and 5 µM of standard drug, tamoxifen. Compound
4b induced arrest of cells in G1 phase and reduction in S-phase cells in a dose dependent
7

ACCEPTED MANUSCRIPT
manner (Fig. 4). However, only higher concentration of 10.5 µM of compound 14b could induce
significant G1 arrest in comparison to untreated control and comparable to G1 arrest induced by
5
µM of tamoxifen. This indicates that cell growth inhibition by compounds 14b is due to G0/G1
arrest in MCF-7 cells.
<
Figure 4 >
Effect of Compound 14b on apoptosis of MCF-7cells
In order to differentiate whether the resultant cell inhibition is due to necrosis or
apoptosis, we carried out Annexin-V FITC and PI staining technique. Compound 14b caused a
significant decrease in live cells counts and increased early and late apoptotic cells counts at 10.5
ꢀ
M concentration. Interestingly, compound 14b did not increase necrotic cells in comparison to
untreated control group (Fig. 5).
<
Figure 5 >
LDH release assay
When assessed for the percent cytotoxicity in terms of LDH release, 14b showed less
cytotoxity towards MCF-7 and HEK-293 cells. This suggested that at tested concentration and
time point, 14b did not disrupt cell membrane integrity for release of cytoplasmic LDH which
supported the assumption that cell inhibition is due to apoptosis and not by necrosis (Fig.6).
<
Figure 6 >
Molecular docking of three active compounds with ER alpha
The series of compounds were designed as potential estrogen antagonists for anti-breast
cancer activity by their interaction with the estrogen receptor in the ER-α cavity of ER positive
breast cancer cells. Therefore, to get an insight of the binding interactions and to validate the
antagonistic activity profile of the three active compounds 14a, 14b and 31e, a comparative
docking study of computationally energy minimized conformation of the three molecules was
performed in comparision with the antiestrogenic drug, 4-hydroxy tamoxifen (OHT) as shown in
Fig.7. As can be clearly observed in the figure, the tricyclic core of all the three molecules
occupies the same binding space in the ER pocket and is similarly oriented in the plane of the
biphenyl stilbene residue of tamoxifen, (the superimposed structure of OH-TAM with
8

ACCEPTED MANUSCRIPT
compounds 14a, 14b and 31e shown Fig.7). Both the active compounds 14a,14b and OH-TAM
share significant homology ( the binding energy and interacting residues of the compounds and
OH-TAM are shown in Table 2) in interacting with critical amino acid residues (GLU353,
GLY390, ILE326, ILE386, LEU387, LEU391, LEU403, LYS449), however, the
tert.aminoalkoxy group (basic side chain) is placed in the opposite orientation to that of
tamoxifen.
It has been demonstrated that molecular basis of antagonism is a complicated
phenomenon and is a combination of several factors such as, spatial conformation within the ER
cavity, specific interactions of the amino acid residues, hydrogen bonding, vander waal’s forces
that come into interplay with the ligand and the position of the basic side chain that may occupy
either 7α-position or 11β-position to displace the helix 12 in forming the lid of the binding
cavity. In compounds 14a and 14b, this chain probably orients itself in the 7α-position in
contrast to tamoxifen (oriented in the 11-β position) thereby imparting the antagonistic activity to
the molecules.
<
Figure 7 >
However, it is most surprising to note that compound 31e having very similar
conformation with OH-TAM and showing high docking energy is active at a higher
concentration as compared to compounds 32d or 14a and 14b. This difference in the activity of
the compound 31e gives credence to interplay of other factors critical for inhibitory activity.
<
Table 2 >
3
. Conclusion
A targeted series of constrained tricyclic compounds (three prototypes I, II, and III ) of
substituted dibenzo[b,f]thiepine and dibenzo[b,f]oxepines and structurally analogous to
tamoxifen were prepared and evaluated for their antiproliferative activity on breast cancer cell
lines. Among the evaluated compounds, sulphur analogues showed better anti-breast cancer
activity than their oxygen analogues. Three compounds 14a, 14b and 31e showed good activity,
particularly compound 14b which effectively inhibited both the breast cancer cell lines, MCF-7
with IC of 1.33 ꢀM and MDA-MB-231 with IC of 5 ꢀM. It was devoid of any cytotoxic
5
0
50
effect on the normal HEK-293 cells at 50 ꢀM concentration, which was more than 35 fold of
9

ACCEPTED MANUSCRIPT
their IC50 value and much better than the anti-breast cancer drug, tamoxifen. Further, molecular
docking studies with estrogen receptor indicated ER-α as the plausible target of compound 14b
for exerting its anti-cancer action.
4
. Experimental
4
.1 Materials and methods
All the glass apparatus were oven dried prior to use. All chemicals and reagents were purchased
from Sigma- Aldrich or ACROS ORGANICS and were used without further purification.
Solvents THF, DCM, DMF, DMSO, diethyl ether, acetone were purified using standard
methods. 100-200 mesh silica gel was used for column chromatography, and TLC was
performed on Merck-precoated silica gel 60-F254 and aluminum oxide 60-F254 plates. Melting
points were recorded with COMPLAB melting point apparatus and are uncorrected. The
chromatographic solvents are mentioned as v/v ratios. All the synthesized compounds were fully
1
13
characterized by H, C NMR, IR, and further confirmed through ESI-MS, ESI-HRMS analysis.
IR spectra were recorded on a Perkin–Elmer FT-IR RXI spectrophotometer and values reported
-1
1
13
1
in cm . H NMR and C NMR spectra were recorded on Bruker DRX-300 (300 MHz for H and
1
3
13
at 75 MHz for C or DPX-200 (at 50 MHz for C) spectrometers using CDCl , DMSO-
3
d ,CD OD or acetone-d as solvents using tetramethylsilane as internal standard. Chemical shifts
6
3
6
are reported in parts per million. ESI-MS spectra were obtained on a LCQ Advantage Ion trap
mass spectrometer (Finnigan thermo fischer scientific) and High-resolution mass spectra (ESI-
HRMS) were recorded on Agilent 6520 ESI-QTOP mass spectrometer. All products reported
1
13
showed H NMR and C NMR spectra in agreement with the assigned structures. The purity of
compounds was determined by HRMS, and all tested compounds yielded data consistent with a
purity of at least 95% compared with the theoretical values.
4
.2. Representative procedure for the preparation of thiepine derivatives (14a-d and 15 a-d)
A mixture of 4-(dbenzo[b,f]thiepin-10-yl)phenol 10 (302 mg, 1 mmol), 1-(2-chloro
ethyl)pyrrolidine monohydrochloride (255 mg, 1.5 mmol), dry acetone (10 mL) and anhydrous
K CO (345 mg, 2.5 mmol) were refluxed overnight. On completion, the reaction mixture was
2
3
1
0

ACCEPTED MANUSCRIPT
cooled, filtered and washed with acetone (5 mL × 3). The filtrate was concentrated and the
residue chromatographed over a column of basic alumina to furnish compounds 14a-d and 15a-d
(prototype I).
1
-(2-(4-(Dibenzo[b,f]thiepin-10-yl)phenoxy)ethyl)pyrrolidine (compound 14a)::Yellow solid,
-1 1
Yield 71%; Mp (76-78°C); IR (KBr): 3427, 2925, 1602, 1458, 1248, 1038, 746 cm ; H NMR
300 MHz, CDCl ): δ 7.60-7.53 (m, 2H), 7.38-7.27 (m, 7H), 7.20-7.17 (m, 1H), 7.03 (d, J = 1.32
(
3
Hz, 1H), 6.94 (d, J = 8.7 Hz, 2H), 4.18 (t, J = 5.96 Hz, 2H), 2.96 (t, J = 5.96 Hz, 2H), 2.69 (m,
1
3
4
1
3
H), 1.87-1.83 (m, 4H); C NMR (75 MHz, CDCl ): δ 158.8, 145.5, 142.1, 136.8, 136.2, 132.9,
32.1, 130.9, 130.7, 130.1, 129.1, 128.3, 127.9, 127.7, 114.3, 67.0, 55.0, 54.7, 29.7, 23.5; MS
+
(
ESI) m/z 400 (M+H); HRMS (ESI) exact mass calcd for [C H NOS+H] 400.1735, found
2
6
25
4
00.1769.
1
-(2-(4-(Dibenzo[b,f]thiepin-10-yl)phenoxy)ethyl)piperidine (compound 14b):Off white solid,
-1 1
Yield 68%; Mp (100-102°C); IR (KBr) 3010, 2920, 1602, 1456, 1243, 1038, 746 cm ; H NMR
300 MHz, CDCl ): δ 7.64-7.54 (m, 2H), 7.38-7.29 (m, 7H), 7.20-7.15 (m, 1H), 7.06 (d, J = 7.68
Hz, 1H), 6.94 (d, J = 8.58 Hz, 2H), 4.17 (t, J = 6.03 Hz, 2H), 2.82 (t, J = 6.03 Hz, 2H), 2.57-2.54
(
3
13
(
m, 4H), 1.67-1.61 (m, 4H), 1.52-1.48 (m, 2H); C NMR (75 MHz, CDCl ): δ 158.6, 145.7,
3
1
1
42.1, 140.3, 136.9, 136.7, 136.1, 132.8, 132.1, 130.9, 130.9, 130.7, 130.1, 129.1, 128.3, 127.9,
27.7, 114.3, 66.0, 57.9, 55.1, 25.9, 24.2; MS (ESI) m/z 414 (M+H); HRMS (ESI) exact mass
+
calcd for [C H NOS+H] 414.1892, found 414.1892.
2
7
27
4
-(2-(4-(Dibenzo[b,f]thiepin-10-yl)phenoxy)ethyl)morpholine (compound 14c): Solid, Yield
-1 1
6
4%; Mp (84°C); IR (KBr) 3020, 2923, 1600, 1450, 1248, 1038, 746 cm ; H NMR (300 MHz,
CDCl ): δ 7.62 (d, J = 6.93 Hz, 1H), 7.56-7.53 (m, 1H), 7.38-7.15 (m, 8H), 7.04 (d, J = 7.59
3
Hz, 1H), 6.93 (d, J = 8.61 Hz, 2H), 4.17 (t, J = 5.73 Hz, 2H), 3.77 (t, J = 4.56 Hz, 4H), 2.85 (t, J
1
3
=
3
5.7 Hz, 2H), 2.62 (t, J = 4.47 Hz, 4H); C NMR (75 MHz, CDCl ): δ 158.4, 145.6, 142.1,
1
5
4
40.3, 137.0, 136.2, 132.9, 132.2, 130.9, 130.8, 130.2, 129.2, 128.4, 127.8, 114.4, 66.8, 65.7,
+
7.6, 54.1; MS (ESI) m/z 416 (M+H); HRMS (ESI) exact mass calcd for [C H NO S+H]
2
6
25
2
16.1684, found 416.1695.
1
1

ACCEPTED MANUSCRIPT
1
-(2-(4-(Dibenzo[b,f]thiepin-10-yl)phenoxy)ethyl)azepane (compound 14d): Viscous oil, Yield
-1 1
7
1%; IR (KBr): 3012, 2920, 1602, 1452, 1240, 1036, 743 cm ; H NMR (300 MHz, CDCl ):
3
δ 7.63-7.53 (m, 2H), 7.37-7.28 (m, 7H), 7.20-7.15 (m, 1H), 7.05 (d, J = 7.53 Hz, 1H), 6.93 (d, J
8.55 Hz, 2H), 4.15 (t, J = 6.10 Hz, 2H), 3.02 (t, J = 6.09 Hz, 2H), 2.86-2.83 (m, 4H), 1.71-1.65
=
1
3
(
m, 8H); C NMR (50 MHz, CDCl
3
): δ 158.5, 145.6, 142.1, 140.3, 136.9, 136.1, 132.8, 132.1,
1
30.9, 130.1, 129.1, 128.3, 127.9, 114.3, 66.2, 56.3, 55.8, 27.4, 27.0; MS (ESI) m/z 428 (M+H).
1
-(2-(4-(Dibenzo[b,f]oxepin-10-yl)phenoxy)ethyl)pyrrolidine (compound 15a): Yellow solid,
-1 1
Yield 76%; Mp (80-82°C); IR (KBr): 2930, 2770, 1604, 1453, 1234, 1036, 757 cm ; H NMR
(
300 MHz, CDCl ): δ 7.40 (d, J = 8.52 Hz, 2H), 7.34-7.23 (m, 5H), 7.18-7.06 (m, 1H), 7.03-
3
1
3
6
.93 (m, 5H), 4.18 (t, J = 5.97 Hz, 2H), 2.96 (t, J = 6.02 Hz, 2H), 2.67 (s, 4H), 1.84 (m, 4H); C
NMR (75 MHz, CDCl ): δ 158.6, 158.5,157.9, 141.7, 134.9, 132.2, 130.8, 130.5, 130.1, 130.0,
3
1
29.8, 129.2, 127.2, 124.8, 124.5, 121.4, 120.8, 114.5, 114.4, 67.1, 55.1, 54.7, 23.5; MS (ESI)
+
m/z 384 (M+H); HRMS (ESI) exact mass calcd for [C H NO +H] 384.1964, found 384.1965.
2
6
25
2
1
-(2-(4-(Dibenzo[b,f]oxepin-10-yl)phenoxy)ethyl)piperidine (compound 15b): Yellow solid,
-1 1
Yield 50%; Mp (94-96°C); IR (KBr): 2934, 2775, 1606, 1458, 1236, 1038, 757 cm ; H NMR
300 MHz, CDCl ): δ 7.40 ( d, J = 6.72 Hz, 2H), 7.38-7.23 (m, 5H), 7.18-7.13 (m, 1H), 7.12-
(
3
7
.00 ( m, 2H), 6.97-6.93 (m, 3H), 4.18 (t, J = 6.01 Hz, 2H), 2.83 (t, J = 6.06 Hz, 2H), 2.56-2.54
13
(
m, 4H), 1.67-1.63 (m, 4H), 1.50-1.48 (m, 2H); C NMR (75 MHz, CDCl ): δ 158.6, 157.9,
3
1
1
41.7, 134.9, 132.1, 130.8, 130.1, 130.0, 129.8, 129.4, 129.2, 127.2, 124.8, 124.5, 121.4, 120.8,
14.5, 65.9, 57.9, 55.0, 25.8, 24.1; MS (ESI) m/z 398 (M+H); HRMS (ESI) exact mass calcd for
+
[C
26
H27NO
2
+H] 398.2120, found 398.2115.
4
-(2-(4-(Dibenzo[b,f]oxepin-10-yl)phenoxy)ethyl)morpholine (compound 15c): Yellow solid,
-1 1
Yield 86%; Mp (116°C); IR (KBr): 2932, 2770, 1600, 1453, 1236, 1038, 757 cm ; H NMR
(
300 MHz, CDCl ): δ 7.34-7.31 (m, 2H), 7.29-7.15 (m, 6H), 7.09-7.04 (m, 1H), 7.00-6.84 (m,
3
1
3
4
H), 4.16 (t, J = 5.4 Hz, 2H), 3.74 (t, J = 4.5Hz, 4H), 2.85 (t, J = 5.38 Hz, 2H), 2.64 (m, 4H); C
NMR (75 MHz, CDCl ): δ 157.4, 156.8, 140.6, 134.0, 131.1, 129.8, 129.4, 129.1, 128.8, 128.2,
3
1
27.3, 126.2, 124.1, 122.7, 122.0, 121.2, 119.7, 116.1, 115.8, 113.3, 65.9, 64.8, 56.6, 53.0; MS
+
(ESI) m/z 400 (M+H); HRMS (ESI) exact mass calcd for [C26
H25NO
3
+H] 400.1913, found
4
00.1900.
1
2

ACCEPTED MANUSCRIPT
1
-(2-(4-(Dibenzo[b,f]oxepin-10-yl)phenoxy)ethyl)azepane (compound 15d): Viscous oil, Yield
-1 1
7
6.6%; IR (KBr): 2934, 2770, 1602, 1460, 1234, 1038, 753 cm ; H NMR (300 MHz, CDCl ):
3
δ 7.40 ( d, J = 6.78 Hz, 2H), 7.37-7.26 (m, 5H), 7.23-713 (m, 1H), 7.06-7.02 (m, 2H), 7.00-6.93
(m, 3H), 4.21 (t, J = 5.98 Hz, 2H), 3.08 (t, J = 5.98 Hz, 2H), 2.92-2.98 (m, 4H), 1.75-1.64 (m,
1
3
8
1
4
3
H); C NMR (75 MHz, CDCl ): δ 157.6, 156.8, 140.7, 133.9, 131.1, 129.8, 129.0, 128.3,
26.1, 123.5, 123.5, 120.4, 119.7, 113.5, 113.3, 65.3, 55.3, 54.8, 52.3, 26.7, 26.0; MS (ESI) m/z
12 (M+H).
4
.3 Representative procedure for the synthesis of compounds 23a-d and 24a-e as described for
compounds 14a-d and 15a-d.
1
-(2-(4-(11-Ethyldibenzo[b,f]thiepin-10-yl)phenoxy)ethyl) pyrrolidine (compound 23a): Yellow
solid, Yield 54%; Mp (109-111ºC); IR (KBr): 3010, 2920, 2810, 1602, 1454, 1243, 1038, 747
-1 1
cm ; H NMR (300 MHz, CDCl ): δ 7.58-7.47 (m. 2H), 7.45-7.30 (m, 1H), 7.26-7.10 (m, 5H),
3
7
2
.09-7.02 (m, 2H), 6.96-6.92 (m, 2H), 4.15 (t, J = 6.06 Hz, 2H), 2.94 (t, J = 6.0 Hz, 2H), 2.66-
1
3
.61 (m, 4H), 1.85-1.83 (m, 4H), 1.56 (q, J = 7.32 Hz, 2H), 0.65 (t, J = 6.85 Hz, 3H); C NMR
(
75MHz,CDCl ):δ 157.7, 144.2, 142.6, 140.0, 139.5, 138.5, 136.2, 132.0,131.9,131.3,128.2,127.
3
1
31.9, 131.3, 128.2, 127.6,127.2,114.1, 66.9, 55.1, 54.7, 23.5, 13.9; MS (ESI) m/z 428 (M+H);
+
HRMS (ESI) exact mass calcd for [C H NOS+H] 428.2048, found 428.2040.
2
8
30
1
-(2-(4-(11-Ethyldibenzo[b,f]thiepin-10-yl)phenoxy)ethyl) piperidine (compound 23b): Yellow
-1
solid, Yield 62%; Mp (92-94ºC); IR (KBr): 3020, 2928, 2810, 1600, 1456, 1248, 1038, 747 cm ;
1
H NMR (300 MHz, CDCl ): δ 7.15−7.12 (m, 2Η), 7.11 (d, J = 6.06 Hz, 1H), 7.07- 6.92 (m,
3
6
1
H), 6.91-6.79 (m, 3H), 4.08 (t, J = 5.91 Hz, 2H), 2.76 (t, J = 5.88 Hz, 2H), 2.51-2.46 (m, 4H),
13
.59-1.56 (m, 4H), 1.43 (q, J = 6.98 Hz, 2H), 0.68 (t, J = 7.40 Hz, 3H); C NMR (75 MHz,
CDCl ): δ 157.6, 144.3, 143.5, 142.5, 140.0, 139.8, 138.5, 136.5, 136.3, 131.9, 131.7, 13
3
1
.2, 128.2, 127.8, 127.6, 127.4, 114.1, 65.8, 58.0, 55.0, 29.6, 25.9, 24.1, 13.8; MS (ESI) m/z 442
+
(
M+H); HRMS (ESI) exact mass calcd for [C H NOS+H] 442.2204, found 442.2204.
2
9
32
N,N-Diethyl-2-(4-(11-ethyldibenzo[b,f]thiepin-10-yl)phenoxy)ethanamine(compound
23c):
Yellow solid, Yield 57%; Mp (89°C); IR (KBr): 3010, 2920, 2810, 1602, 1454, 1243, 1038, 747
-1 1
cm ; H NMR (300 MHz, CDCl ): δ 7.65 (d, J = 7.74 Hz, 1H), 7.42-7.23 (m, 4H), 7.13-7.09 (m,
3
1
3

ACCEPTED MANUSCRIPT
1
2
H), 6.91-6.86 (m, 1H), 6.70-6.54 (m, 5H), 3.98 (t, J = 6.07 Hz, 2H), 2.85 (t, J = 6.36 Hz, 2H),
1
3
.68-2.61 (m, 4H), 1.92-1.21 (m, 2H), 1.08 (t, J = 7.11 Hz, 3H), 0.95-0.82 (m, 6H); C NMR
(
75 MHz, CDCl ): δ 160.8, 158.4, 152.3, 140.5, 137.3, 136.6, 135.7, 134.7, 133.2, 132.2, 131.8,
3
1
29.8, 129.4, 128.8, 126.3, 125.5, 125.0, 121.5, 120.9, 115.0, 66.7, 59.1, 46.7, 31.1, 14.0,
+
1
3.2;MS (ESI) m/z 430 (M+H); HRMS (ESI) exact mass calcd for [C H NOS+H] 430.2205,
2
8
31
found 430.2108.
2
-(4-(11-Ethyldibenzo[b,f]thiepin-10-yl)phenoxy)-N,N-dimethylethanamine(compound
23d):
Yellow solid, Yield 59%; Mp (74-76ºC); IR (KBr): 3010, 2920, 2810, 1602, 1454, 1243, 1038,
-
1
1
7
7
7
1
1
47 cm ; H NMR (300 MHz, CDCl ): δ 7.24-7.14 (m, 2Η), 7.10 (d, J = 6.0 Hz, 1H), 7.18-
3
.02 (m, 6H), 6.98-6.82 (m, 3H), 4.03 (t, J = 5.34 Hz, 2H), 2.54 (t, J = 5.32 Hz, 2H), 2.43 (q, J =
13
3
.29, Hz, 2H), 2.18 (s, 6H), 0.76 (t, J = 7.36 Hz, 3H); C NMR (75 MHz, CDCl ): δ 159.9,
57.6, 151.5, 139.7, 136.5, 135.8, 134.9, 133.9, 132.4, 131.4, 131.0, 129.0, 128.6, 128.0, 125.5,
24.7, 124.2, 120.7, 120.1, 114.2, 65.9, 58.3, 45.9, 30.3, 14.0; MS (ESI) m/z 402 (M+H); HRMS
+
(ESI) exact mass calcd for [C28
H
28NOS+H] 402.1869, found 402.1891.
1
-(2-(4-(11-Ethyldibenzo[b,f]oxepin-10-yl)phenoxy)ethyl)pyrrolidine(compound 24a): Brown
-1 1
solid, Yield 65%; Mp (67°C); IR (KBr): 3010, 2920, 2810, 1602, 1454, 1243, 1038, 747 cm ; H
NMR (300 MHz, CDCl ): δ 7.36 (dd, J = 1.35, 7.92 Hz, 1H), 7.21-7.19 (m, 2H), 7.12-7.09 (m,
H), 6.89-6.84 (m, 3H), 6.82-6.66 (m, 1H), 4.12 (t, J = 5.91 Hz, 2H), 2.91 (t, J = 5.85 Hz, 2H),
3
2
2
1
3
.65-2.58 (m, 4H), 1.98 (m, 2H), 1.80-1.76 (m, 4H), 0.78 (t, J = 7.39 Hz, 3H); C NMR (50
): δ 159.9, 158.8, 157.6, 139.7, 136.5, 133.9, 132.4, 131.4, 129.0, 128.6, 128.0,
24.7, 120.2, 120.1, 114.2, 66.5, 55.0, 33.8, 30.3, 29.7, 26.9, 23.4, 22.7, 14.0; MS (ESI) m/z 412
MHz, CDCl
3
1
+
(
M+H); HRMS (ESI) exact mass calcd for [C H NO +H] 412.2277, found 412.2268.
28 29 2
1
-(2-(4-(11-Ethyldibenzo[b,f]oxepin-10-yl)phenoxy)ethyl) piperidine (compound 24b): Brown
-1 1
solid, Yield 87%; Mp (90-92°C); IR (KBr): 2934, 2775, 1606, 1458, 1236, 1038, 757 cm ; H
NMR (300 MHz, CDCl ): δ 7.40-7.49 (m, 1H), 7.12-7.32 (m, 7H), 6.88-7.01 (m, 3H), 6.72-
3
6
2
.80 (m, 1H), 4.18 (t, J = 6.00 Hz, 2H), 2.84 (t, J = 6.02 Hz, 2H), 2.68 (q, J = 7.35 Hz, 2H), 2.62-
1
3
.52 (m, 4H), 1.59-1.71 (m, 4H), 1.42-1.54 (m, 2H), 0.87 (t, J = 7.41 Hz, 3H); C NMR (50
MHz, CDCl ): δ 159.9, 158.8, 157.7, 139.7, 136.6, 134.9, 133.9, 132.4, 131.4, 131.0, 129.0,
3
1
28.6, 128.0, 124.7, 124.2, 120.7, 120.1, 114.4, 114.3, 65.8, 58.0, 55.0, 29.7, 26.9, 25.8, 24.1,
1
4

ACCEPTED MANUSCRIPT
+
1
2
4.0; MS (ESI) m/z 426 (M+H); HRMS (ESI) exact mass calcd for [C29H31NO +H] 426.2433,
found 426.2427.
4
-(2-(4-(11-Ethyldibenzo[b,f]oxepin-10-yl)phenoxy)ethyl) morpholine(compound 24c): Solid,
-1 1
Yield: 72%; Mp (86-88ºC); IR (KBr): 3018, 2926, 2810, 1609, 1458, 1246, 1036, 747 cm ; H
NMR (300 MHz, CDCl ): δ 7.48-7.41 (m, 1H), 7.31-7.14 (m, 7H), 7.00-6.89 (m, 3H), 6.79-
3
6
.73 (m, 1H), 4.18 (t, J = 5.64 Hz, 2H), 3.82-3.73 (m, 4H), 2.87 (t, J = 5.64 Hz, 2H), 2.71-2.60
1
3
(
m, 6H), 0.87 (t, J = 7.39 Hz, 3H); C NMR (75 MHz, CDCl ): δ 159.9, 158.9, 157.6, 139.8,
3
1
6
36.5, 135.1, 133.9, 132.4, 131.4, 130.9, 129.0, 128.7, 128.0, 124.7, 124.2, 120.7, 120.2, 114.3,
6.8, 65.7, 57.7, 54.1, 29.7, 26.9, 14.0; MS (ESI) m/z 428 (M+H); HRMS (ESI) exact mass calcd
+
for [C H NO +H] 428.2226, found 428.2224.
2
8
29
3
1
-(2-(4-(11-Ethyldibenzo[b,f]oxepin-10-yl)phenoxy)ethyl) azepane (compound 24d): Yellow oil,
-1
1
Yield: 64%; IR (KBr): 3016, 2905, 2800, 1608, 1460, 1250, 1038, 747 cm ; H NMR (300
MHz, CDCl ): δ 7.47-7.43 (m, 1H), 7.30-7.20 (m, 7H), 6.97-6.95 (m, 3H), 6.76-6.73 (m, 1H),
3
4
1
1
6
.37 (t, J = 4.95 Hz, 2H), 3.27 (t, J = 4.89 Hz, 2H), 3.15-3.12 (m, 4H), 2.78 (t, J = 4.93 Hz, 2H),
13
.72 (m, 6H), 0.87 (t, J = 7.24 Hz, 3H); C NMR (75 MHz, CDCl ): δ 160.9, 159.1, 157.7,
3
40.2, 137.3, 135.1, 133.5, 132.0, 131.9, 130.3, 129.0, 129.2, 127.8, 124.7, 124.0, 120.6, 114.3,
6.8, 56.7, 55.1, 29.7, 26.9, 14.2; MS (ESI) m/z 440 (M+H); HRMS (ESI) exact mass calcd for
+
[
C H NO +H] 440.2590, found 440.2584.
3
0
33
2
2
-(4-(11-Ethyldibenzo[b,f]oxepin-10-yl)phenoxy)-N,N-dimeth-ylethanamine(compound24e):
-1
1
Brown viscous oil, Yield 64%; IR (KBr): 3010, 2928, 1612, 1458, 1245, 1036, 747 cm ; H
NMR (300 MHz, CDCl ): δ (dd, J = 7.08 Hz, 1H), 7.17-7.01 (m, 7H), 6.87-6.77 (m, 4H), 6.65-
3
6
2
1
1
.62 (m, 1H), 4.00 (t, J = 4.00 Hz, 2H), 2.65 (t, J = 5.76 Hz, 2H), 2.55 (q, J = 7.24, 14.67 Hz,
13
H), 2.25 (s, 6H), 0.74 (t, J = 7.39 Hz, 3H); C NMR (75 MHz, CDCl ): δ 159.9, 157.6, 151.5,
3
39.7, 136.5, 135.8, 134.9, 133.9, 132.4, 131.4, 131.0, 129.0, 128.6, 128.0, 125.5, 124.7, 124.2,
20.7, 120.1, 114.2, 65.9, 58.3, 45.9, 30.3, 14.0; MS (ESI) m/z 386 (M+H); HRMS (ESI) exact
+
mass calcd for [C H NO +H] 386.2120, found 428.2127.
2
6
27
2
4
.4. Representative procedure for the synthesis of target molecules 31a-e and 32a-d as described
for compounds 23a-d and 24a-e.
1
5

ACCEPTED MANUSCRIPT
1
-(2-(4-(11-Phenyldibenzo[b,f]thiepin-10-yl)phenoxy)ethyl) pyrrolidine (compound 31a):Yellow
-1
solid, Yield 67.3%; Mp (111-113°C); IR (KBr): 3010, 2920, 1602, 1456, 1243, 1038, 746 cm ;
1
H NMR (300 MHz, CDCl ): δ 7.61-7.48 (m, 1H), 7.46-7.44 (m, 1H), 7.29-7.05 (m, 12H), 6.96-
3
6
4
1
.84 (m, 1H), 6.75-6.71 (m, 2H), 4.10 (t, J = 2.89 Hz, 2H), 2.97 (t, J = 3.27 Hz, 2H), 2.75 (m,
1
3
3
H), 1.88-1.85 (m, 4H); C NMR (75 MHz, CDCl ): δ 157.1, 155.6, 140.1, 138.0, 137.7, 132.6,
31.8, 130.1, 129.2, 129.0, 127.8, 126.0, 124.8, 122.9, 119.0, 112.0, 65.2, 53.5, 53.1, 21.9; MS
+
(
ESI) m/z 476 (M+H); HRMS (ESI) exact mass calcd for [C H NOS+H] 476.2048, found
3
2
29
4
76.2006.
1
-(2-(4-(11-Phenyldibenzo[b,f]thiepin-10-yl)phenoxy)ethyl) piperidine (compound 31b):Yellow
-1 1
viscous oil, Yield 69.28%; IR (KBr): 3030, 2928, 1609, 1458, 1246, 1030, 757 cm ; H NMR
300 MHz, CDCl ): δ 7.63-7.56 (m, 1H), 7.49-7.45 (m, 1H), 7.29 (m, 12H), 6.99-6.90 (m, 1H),
.75-6.67 (m, 2H), 2.73 (t, J = 6.1 Hz, 2H), 2.50 (M, 4H), 1.63-1.60 (m, 4H), 1.47-1.45 (m, 2H);
(
3
6
1
3
C NMR (75 MHz, CDCl ): δ 158.6, 157.1, 142.0, 139.7, 134.2, 133.4, 132.9, 131.6, 130.8,
3
1
4
30.6, 130.5, 129.3, 127.6, 126.3, 124.5, 120.6, 113.5, 65.7, 57.9, 55.0, 25.9, 24.2; MS (ESI) m/z
+
90 (M+H); HRMS (ESI) exact mass calcd for [C H NOS+H] 490.2205, found 490.2241.
3
3
31
4
-(2-(4-(11-Phenyldibenzo[b,f]thiepin-10-yl)phenoxy)ethyl) morpholine (compound 31c): White
-1 1
solid, Yield: 59%; Mp (97-99°C); IR (KBr): 3020, 2910, 1609, 1458, 1243, 1038, 746 cm ; H
NMR (300 MHz, CDCl ): δ 7.31-7.23 (m, 4H), 7.19-7.14 (m, 4H), 7.11-6.94 (m, 7H), 6.47-
.35 (m, 2H), 3.96 (t, J = 5.71 Hz, 2H), 3.68 (t, J = 4.59 Hz, 4H), 2.69 (t, J = 5.71 Hz, 2H), 2.47
3
6
1
3
(
t, J = 4.64 Hz, 4H); C NMR (75 MHz, CDCl ): δ 158.1, 156.9, 142.1, 141.2, 139.2, 136.3,
3
1
5
4
34.8, 134.1, 133.2, 131.4, 130.6, 129.5, 127.6, 126.8, 125.1, 124.0, 120.8, 112.9, 66.1, 64.9,
+
8.2, 53.7; MS (ESI) m/z 492 (M+H); HRMS (ESI) exact mass calcd for [C H NO S+H]
3
2
29
2
92.1997, found 492.2002.
N,N-Dimethyl-2-(4-(11-phenyldibenzo[b,f]thiepin-10-yl)
1d):Yellow oil, Yield 52%; IR (KBr): 3032, 2926, 1606, 1450, 1242, 1030, 757 cm ; H NMR
300 MHz, CDCl ): δ 7.29-7.24 (m, 4H), 7.21-7.16 (m, 4H), 7.14-6.99 (m, 7H), 6.72 (d, J = 6.03
phenoxy)ethanamine
(compound
-1 1
3
(
3
Hz, 2H), 4.03 (t, J = 5.98 Hz, 2H), 2.86 (t, J = 6.03 Hz, 2H),2.88-2.72 (m, 4H), 1.08-0.89 (m,
1
3
6
3
H); C NMR (50 MHz, CDCl ): δ 158.2, 141.5, 139.3, 139.0, 132.9, 132.6, 131.9, 130.8,
1
6

ACCEPTED MANUSCRIPT
1
4
30.4, 130.0, 128.6, 127.9, 127.0, 124.1, 120.2, 119.8, 114.5, 68.7, 56.2, 13.8; MS (ESI) m/z
+
78 (M+H); HRMS (ESI) exact mass calcd for [C H NOS+H] 478.2205, found 478.2225.
3
2
32
N,N-Dimethyl-2-(4-(11-phenyldibenzo[b,f]thiepin-10-yl) phenoxy)ethanamine (compound 31e):
Off white solid, Yield 59%; Mp (98-100ºC); IR (KBr): 3010, 2920, 1602, 1456, 1243, 1038, 746
-1 1
cm ; H NMR (300 MHz, CDCl ): δ 7.31-7.28 (m, 4H), 7.26-7.17 (m, 4H), 7.14-6.98 (m, 7H),
3
1
3
6
.71 (d, J = 8.7 Hz, 2H), 4.07 (t, J = 5.88 Hz, 2H), 3.0 (t, J = 5.92 Hz, 2H), 2.88-2.85(m, 4H); C
NMR (75 MHz, CDCl ): δ 158.6, 141.7, 139.6, 139.2, 133.3, 132.9, 131.6, 130.6, 130.6, 130.5,
3
1
29.3, 127.5, 126.3, 124.5, 120.6, 120.5, 113.5, 68.1, 56.2, 55.7, 27.0; MS (ESI) m/z 450
+
(
M+H); HRMS (ESI) exact mass calcd for [C H NOS+H] 450.1892, found 450.2002.
3
0
28
1
-(2-(4-(11-Phenyldibenzo[b,f]oxepin-10-yl)phenoxy)ethyl) pyrrolidine (compound 32a): White
-1
solid, Yield 81.69%; Mp (93-95°C); IR (KBr): 3010, 2920, 1602, 1456, 1243, 1038, 746 cm ;
1
H NMR (300 MHz, CDCl ): δ 7.29-7.24 (m, 4H), 7.21-7.16 (m, 4H), 7.14-6.99 (m, 7H), 6.72
3
(d, J = 6.03 Hz, 2H), 4.03 (t, J = 5.98 Hz, 2H), 2.86 (t, J = 6.03 Hz, 2H), 2.61 (m, 2H), 1.82 (m,
1
3
4
1
3
H); C NMR (100 MHz, CDCl ): δ 157.6, 156.1, 140.7, 138.5, 138.2, 133.1, 132.3, 130.6,
29.7, 129.5, 128.3, 126.5, 125.3, 123.4, 119.5, 112.5, 65.7, 54.0, 53.6, 22.4; MS (ESI) m/z 460
+
(
M+H); HRMS (ESI) exact mass calcd for [C H NO +H] 460.2277, found 460.2285.
3
2
29
2
1
-(2-(4-(11-Phenyldibenzo[b,f]oxepin-10-yl)phenoxy)ethyl) piperidine (compound 32b): Off
white solid, Yield 76%; Mp (112-114°C); IR (KBr): 3030, 2928, 1610, 1460, 1248, 1038, 746
-1 1
cm ; H NMR (300 MHz, CDCl ): δ 7.29-7.26 (m, 4H), 7.21-7.19 (m, 4H), 7.15-6.99 (m, 7H),
3
6
1
1
5
.70 (d, J = 8.94 Hz, 2H), 4.02 (t, J = 6.09 Hz, 2H), 2.72 (t, J = 6.09 Hz, 2H,), 2.49 (m, 4H),
1
3
3
.65-1.57 (m, 4H), 1.48-143 (m, 2H); C NMR (75 MHz, CDCl ): δ 158.6, 157.1, 142.0, 139.7,
34.2, 133.4, 132.9, 131.6, 130.8, 130.6, 130.5, 129.3, 127.6, 126.3, 124.5, 120.6, 113.5, 65.7,
7.9, 55.0, 25.9, 24.2; MS (ESI) m/z 474 (M+H); HRMS (ESI) exact mass calcd for
+
[C
33
H31NO
2
+H] 474.2433, found 474.2426.
4
-(2-(4-(11-Phenyldibenzo[b,f]oxepin-10-yl)phenoxy)ethyl) morpholine (compound 32c): Yellow
-1 1
solid, Yield 71%; Mp (122-123°C); IR (KBr): 3018, 2926, 1602, 1458, 1245, 1036, 746 cm ; H
NMR (300 MHz, CDCl ): δ 7.19-7.17 (m, 4H), 7.15-7.10 (m, 4H), 7.05-6.89 (m, 7H), 6.39-
.31 (m, 2H), 3.93 (t, J = 5.76 Hz, 2H), 3.64 (t, J = 4.63 Hz, 4H), 2.66 (t, J = 5.76 Hz, 2H), 2.47
3
6
1
7

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1
3
(
t, J = 4.60 Hz, 4H); C NMR (75 MHz, CDCl ): δ 158.7, 157.0, 141.8, 139.6, 139.2, 135.8,
3
1
5
4
34.3, 133.4, 132.9, 131.7, 130.6, 129.5, 127.6, 126.3, 125.5, 124.5, 120.6, 113.6, 66.9, 65.5,
+
7.7, 54.1; MS (ESI) m/z 476 (M+H); HRMS (ESI) exact mass calcd for [C H NO +H]
3
2
29
3
76.2226, found 476.2222.
1
-(2-(4-(11-Phenyldibenzo[b,f]oxepin-10-yl)phenoxy)ethyl) azepane (compound 32d): Brown
-1 1
oil, Yield 65%; IR (KBr): 3010, 2920, 1602, 1456, 1243, 1038, 746 cm ; H NMR (300 MHz,
CDCl ): δ 7.31-7.28 (m, 4H), 7.26-7.17 (m, 4H), 7.14-6.98 (m, 7H), 6.71 (d, J = 8.7 Hz, 2H),
3
1
3
4
.07 (t, J = 5.88 Hz, 2H), 3.0 (t, J = 5.92 Hz, 2H), 2.88-2.85(m, 4H), 1.72-1.63 (m, 8H); C
NMR (75 MHz, CDCl ): δ 158.6, 141.7, 139.6, 139.2, 133.3, 132.9, 131.6, 130.6, 130.6, 130.5,
3
1
29.3, 127.5, 126.3, 124.5, 120.6, 120.5, 113.5, 68.1, 56.2, 55.7, 27.0; MS (ESI) m/z 488 (M+H).
4
.5 Materials and Methods
Cells and cell culture condition
MCF-7 (estrogen receptor positive breast cancer cell line), MDA-MB- 231 (estrogen
receptor negative breast cancer cell lines) cells were originally obtained from ATCC, USA and
maintained in laboratory. HEK-293 (human embryonic kidney epithelial cell line) cells were
obtained from Institutional repository of CSIR-CDRI. Cells were routinely sub-cultured in
DMEM medium with 10% fetal bovine serum (FBS), 100U/ml penicillin and 100 U/ml
o
streptomycin incubated at 37 C with 5% CO . FBS supplement was reduced to 2% during MTT
2
assays.
MTT assay
The antiproliferative activities of the series of compounds were determined using MTT
4
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction assay 1 x 10
cells/well (MCF-7, MDA-MB-231 and HEK-293) were seeded in 200 ꢀl DMEM (Dulbecco’s
Modified Eagle’s Medium) supplemented with 10% FBS in each well of 96-well microculture
o
plates and incubated for 24 h at 37 C in CO
2
incubator. Compound stock solutions were prepared
in DMSO and were diluted to the desired concentrations in culture medium. Cells cultured in 96-
well plate were treated with various concentrations of test compounds for 24 h. After the period
of incubation, media were removed and to each well 20 µl of MTT (5 mg/mL) was added and the
1
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plates were further incubated for 4 h. Supernatant from each well was carefully removed,
formazon crystals were dissolved in 200 µl of DMSO and absorbance at 540 nM wavelength was
recorded. Percentage cell inhibition was calculated by comparing test compound absorbance
versus untreated control group absorbance. Percent cell inhibition was plotted against various
concentrations of each compound and IC50 concentration of test compounds were calculated
using Graphpad Prism software [17b].
Morphological analysis of lead compound treated cells
MTT assay quantitatively measures live cells based upon treatment of test compounds
based on activity of mitochondrial enzyme. In order to see how the lead compounds are affecting
breast cancer cells in a concentration dependent manner, we observed the compound treated cells
under bright field microscope. Briefly, cells were seeded at a density of 80,000 cells in 6-well
plate and incubated for 24 h. Then, the medium was replaced, and cells were treated with desired
concentrations of lead compounds for 24 h. For untreated control group in the experiment, cells
were treated with vehicle (0.001% DMSO in culture medium). After the period of treatment,
random fields of cells from each group were captured under bright field inverted microscope
(
Leica, Germany) [23].
Cell cycle analysis assays
The lead compounds based on activity against breast cancer cells and cytotoxicity
towards normal HEK-293 cells were identified and subjected to cell cycle analysis. Briefly,
MCF-7 cells were treated with compounds, at the end of the incubation adherent cells were
trypsinized and combined with any floating cells present and then washed with cold PBS. Cells
were fixed in 70% ethanol and incubated with RNase A and stained with 50g/ml propidium
iodide (PI) for 30 min before acquiring the flow cytometry reading (FACScan, BD Biosciences,
USA) [24].
Morphological analysis for apoptosis using Hoechst staining
Hoechst is fluorescent dye and binds specifically to DNA and therefore used to visually
asses the status of nuclear DNA in treated cancer cells. Briefly, cells were seeded at a density of
1
0,000 cells over 18-mm coverslips placed at the bottom of 6-well cell culture plates and
incubated for 24 h. Then, the medium was replaced, and cells were treated with desired
concentrations of lead compounds for 24 h. Cells treated with vehicle (0.001% DMSO in culture
1
9

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medium) were included as controls for all experiments. After the period of treatment, Hoechst
3
3342 (Sigma-Aldrich) stain were added to medium at a concentration of 0.5 g/ml containing 4%
o
paraformaldehyde under low light condition. After incubation for 30 min at 37 C in dark
condition, the coverslips from each well were mounted on slides and random fields of cells from
each group were captured under fluorescent microscope (Leica, Germany) using blue filter [25].
LDH release assay
Release of lactate dehydrogenase (LDH) is an indicator of membrane integrity and hence
can be used to determine cellular membrane injury. LDH assay was performed to assess the LDH
release to the media following compounds treatment on HEK-293 cells for 24h as per the
manufactures’ protocols (Roche, Germany) [26].
Molecular docking
The X-ray crystallographic structure of human estrogen receptor with ligand binding
domain (LBD) in complex with the endogenous estrogen, 4-hydroxytamoxifen and reference
protein coordinates used for structural alignment was downloaded from Protein Data Bank (PDB
Id: 3ERT) [1]. The energy minimized, 3D structure of the all the three compounds 5, 10, 11 and
OH-tamoxifen were generated from PRODRG2 server. MGL Tools 1.5.4 was used to perform
flexible docking. A grid spacing of 0.374 Ǻ with 60x60x60 grid points were prepared. The
docking was performed for 150 population size for 100 runs applying Lamarckian genetic
algorithm (LGA). Rest all the parameters were set default for the analysis. The top ranked
complex with minimum scoring energy was selected as best docking complex [27].
Evaluation of apoptosis with flow cytometry
6
MCF-7 cells (1x 10 ) were plated in six well plate and allowed to grow overnight. The
medium was then replaced and cells were treated with desired concentrations of lead compounds
for 24 h. Cells treated with vehicle (0.001% DMSO in culture medium) were included as
controls for all experiments. After 24 hours of treatment, cells from supernatant and adherent
monolayer cells were harvested by trypsinization, washed with PBS at 300 g. Then the cells
5
(
1x10 ) were stained with Annexin V-FITC and propidium iodide using the Annexin-V-PI
apoptosis detection kit (Sigma). Flow cytometry was performed using a FACScan (Becton
Dickinson) equipped with a single 488-nm argon laser. Annexin V-FITC were analyzed using
excitation and emission settings of 488 nm and 535 nm (FL-1channel) whereas PI was recorded
2
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with 488 nm and 610 nm (FL-2 channel). Debris and clumps were gated out using forward and
orthogonal light scatter [28].
Acknowledgments
The authors (M.I.A., M.K.H., A.A.,) are thankful for the financial assistance as SRF by
CSIR, New Delhi, India. All the authors are also grateful to the Sophisticated Analytical
Instrument Facility (SAIF) CDRI, Lucknow, India, for providing spectral analysis/data of pure
compounds. Financial assistance from the Ministry of Health and Family Welfare for the
procurement of chemicals/solvents is highly acknowledged. The CDRI Communication No. for
this paper is 8967.
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Figures and Tables:
N
O
O
N
N
O
H CO
3
Nafoxidine
8,9-diphenylbenzocycloheptene
O
Tamoxifen
N
O
N
O
S
4,5-diphenylbenzoxepine
4,5-diphenylbenzothiepine
Fig.1. Fused analogues of tamoxifen
N
O
A
RO
RO
RO
B
C
X
X
X
Prototype I
PrototypeII
Prototype III
Tamoxifen
Target molecules
X = O, S, R = OH, OCH , amino alkyl chain
3
Fig.2. Synthesized prototype molecules I, II and III.
2
4

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Fig.3. White light image after treatment with compound 14b
2
5

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Fig. 4. Effect of Compound 14b in cell cycle of breast cancer cells.
MCF-7 cells were incubated in DMEM supplemented with 10% FBS and treated with IC (1.33 µM), sub
50
IC (0.87 µM) and higher than IC (10.5 µM) concentration of compound 14b for 24 hours. After 24 h,
50
50
the cells were harvested, stained with PI, and analyzed by flow cytometry. Data are expressed as % of
total cell count.
Fig. 5. Effect of compound 14b treatment on apoptosis in terms of Annexin-V and PI staining of MCF-7
cells.
Fig. 6. % cytoxicity induced by compound 14b in MCF-7 and HEK-293 cells in terms of LDH release.
2
6

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Fig. 7. Molecular docking showing orientation and interaction of active molecules within the ERα cavity
in comparison to 4-hydroxy tamoxifen (OH-TAM).
Schemes 1 - 6
O
O
HO
O
O
O
O
SH
c
a
b
S
S
S
I
2
3
1
Scheme1. Reagents and conditions: (a) CuI, Neocuproine, dry toluene, 6 h; (b) KOH/EtOH (80% v/v),
reflux 4 h; (c) (i) (COCl) , DMF, CH Cl , 30 min; (ii) AlCl , CH Cl , rt, 60 min.
2
2
2
3
2
2
2
7

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CHO
CN
OH
Cl
OH
CHO
F
d
O
a
O
b
O
c
O
4
5
6
7
COOH
O
O
e
f
O
9
8
Scheme 2. Reagents and conditions: (a) Cs CO , DMA, 150ºC, 2 h; (b) NaBH , methanol, 0ºC to rt; (c)
2
3
4
SOCl , pyridine, benzene, reflux, 24 h; (d) NaCN, DMSO, rt, 24 h; (e) KOH, EtOH, H O, reflux, 4 h; (f)
2
2
(
i) (COCl)
2
, DMF, CH
2
Cl
2
, 30 min.; (ii) AlCl
3
, CH
2
Cl
2
, rt, 60 min.
H CO
HO
3
O
a
b
X
X
X
X = S, 3
X = O, 9
X = S, 10
X = O, 11
X = S,
X = O, 13
12
RO
1
1
1
4 a. R = ethylpyrrolidine
4 b. R = ethylpiperidine
4 c. R = ethylmorpholine
c
14 d. R = ethylazepane
X
15 a. R = ethylpyrrolidine
X = S, 14 a-d
X = O, 15 a-d
15 b. R = ethylpiperidine
15 c. R = ethylmorpholine
15 d. R = ethylazepane
PROTOTYPE I
Scheme 3. Reagents and conditions: (a) (i) 4-bromoanisole, n-BuLi, THF, 0ºC-rt; (ii) 2-3 drops H SO ,
2
4
MeOH, 60ºC, 30 min.; (b) BBr , CH Cl , -30ºC to rt, overnight; (c) aminoalkyl monohydrochlorides,
3
2
2
2 3
anhyd. K CO , acetone, reflux, overnight.
2
8