Amino acids derived benzoxazepines: Design, synthesis and antitumor activity

Article abstract of DOI:10.1016/j.bmcl.2013.10.013

Two series of new benzoxazepines substituted with different alkyl amino ethyl chains were synthesized comprising synthetic steps of inter and intramolecular Mitsunobu reaction, lithium aluminium hydride (LAH) reduction, debenzylation, bimolecular nucleophilic substitution (S_N2) reaction. The present study investigates the effect of a tyrosine-based benzoxazepine derivative in human breast cancer cells MCF-7 and MDA-MB-231 and in breast cancer animal model. The anti-proliferative effect of 15a on MCF-7 cells was associated with G1 cell-cycle arrest. This G1 growth arrest was followed by apoptosis as 15a dose dependently increased phosphatidylserine exposure, PARP cleavage and DNA fragmentation that are hallmarks of apoptotic cell death. Interestingly, 15a activated components of both intrinsic and extrinsic pathways of apoptosis characterized by activation of caspase-8 and -9, mitochondrial membrane depolarization and increase in Bax/Bcl2 ratio. However, use of selective caspase inhibitors revealed that the caspase-8-dependent pathway is the major contributor to 15a-induced apoptosis. Compound 15a also significantly reduced the growth of MCF-7 xenograft tumors in athymic nude mice. Together, 15a could serve as a base for the development of a new group of effective breast cancer therapeutics.

Full text of DOI:10.1016/j.bmcl.2013.10.013

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6816–6821
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Amino acids derived benzoxazepines: Design, synthesis
and antitumor activity
c
b
c
Shailendra Kumar Dhar Dwivedi a, , Krishnananda Samanta , Manisha Yadav , Amit Kumar Jana ,
⇑
Abhishek Kumar Singh b, Bandana Chakravarti a, Sankalan Mondal c, Rituraj Konwar a,
Arun Kumar Trivedi b, Naibedya Chattopadhyay a, Sabyasachi Sanyal b, Gautam Panda c,
⇑
a Division of Endocrinology, CSIR-Central Drug Research Institute, 10 Janakipuram Extension, Sitapur Road, Lucknow 226031, UP, India
b Division of Drug Target Discovery and Development, CSIR-Central Drug Research Institute, 10 Janakipuram Extension, Sitapur Road, Lucknow 226031, UP, India
c Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, 10 Janakipuram Extension, Sitapur Road, Lucknow 226031, UP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of new benzoxazepines substituted with different alkyl amino ethyl chains were synthesized
comprising synthetic steps of inter and intramolecular Mitsunobu reaction, lithium aluminium hydride
(LAH) reduction, debenzylation, bimolecular nucleophilic substitution (SN2) reaction. The present study
investigates the effect of a tyrosine-based benzoxazepine derivative in human breast cancer cells MCF-
7 and MDA-MB-231 and in breast cancer animal model. The anti-proliferative effect of 15a on MCF-7 cells
was associated with G1 cell-cycle arrest. This G1 growth arrest was followed by apoptosis as 15a dose
dependently increased phosphatidylserine exposure, PARP cleavage and DNA fragmentation that are hall-
marks of apoptotic cell death. Interestingly, 15a activated components of both intrinsic and extrinsic
pathways of apoptosis characterized by activation of caspase-8 and -9, mitochondrial membrane depo-
larization and increase in Bax/Bcl2 ratio. However, use of selective caspase inhibitors revealed that the
caspase-8-dependent pathway is the major contributor to 15a-induced apoptosis. Compound 15a also
significantly reduced the growth of MCF-7 xenograft tumors in athymic nude mice. Together, 15a could
serve as a base for the development of a new group of effective breast cancer therapeutics.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 23 April 2013
Revised 23 September 2013
Accepted 5 October 2013
Available online 11 October 2013
Keywords:
Amino acids
Benzoxazepine
Mitsunobu reaction
Anticancer activity
Capasase
Breast cancer is the most frequently diagnosed life-threatening
disease in women and more than 1 million women are diagnosed
with breast cancer every year, accounting for 10% of all new can-
cers and 23% of all female cancer cases. In India the prevalence of
this cancer is estimated around 2.5 million, with over 0.8 million
new cases and 0.5 million deaths occurring each year.1 Current
available therapies suffer from several drawbacks including resis-
tance and toxic side effects.2,3 We have previously shown that
the benzoxazepine derivatives are active against breast cancer
and are non-toxic in normal cell lines tested4 the present study
is designed to investigate the effect of new compounds synthesized
as lead optimization process.
OBn
Ts
OBn
b
TsHN
CO2Me
a
R1
+
R1
N
CO2Me
OH
R
2
3, 4, 5
R
1 R1 = H, OMe
O
OH
OBn
c
d
R1
R
Ts
R1
Ts
R1
N
CH2OH
N
CH2OH
NTs
9, 10, 11
12, 13a-b, 14
6, 7, 8
R
R
Scheme 1. Reagents and conditions: (a) 1, 2, DEAD, PPh3, THF, 0 °C (2 h) to rt (10 h),
N2, 60–75%; (b) LAH, THF, 0 °C, 1 h, 59–70%; (c) H2, 10% Pd/C, MeOH, rt, 2 h, 50 psi,
61–70%; (d) DEAD, PPh3, THF, 0 °C (1 h) to rt, (14 h), N2, 63–76%.
Benzoxazepines are well-known pharmacophore in medicinal
chemistry with promising activity against various diseases such
as antipsychotic, central nervous system activity along with anti-
cancer profile against breast cancer cells.5 One of these groups of
benzoxazepines has been identified to target microtubule assem-
bly to induce anti-cancer activity.5 In our previous study, we have
synthesized and explored the amino acid based benzoxazepines6a–d
with antitumor activity. Thus, in search of new lead molecule, we
synthesized two different series of benzoxazepine (A, B) from ami-
no acids. Easy availability of amino acid-based benzoxazepines
promoted us to evaluate them for anticancer activity.
We followed the same methodology developed in our labora-
tory for the synthesis of benzoxazepines (Scheme 1). N-tosylated
S-amino acid esters and substituted benzyl alcohol were used as
building blocks for the construction of benzannulated chiral het-
erocycles. The intermolecular Mitsunobu reaction of S-amino acid
⇑
Corresponding authors.
E-mail address: gautam.panda@gmail.com (G. Panda).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.10.013

S. K. D. Dwivedi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6816–6821
6817
OR2
Table 1
OH
IC50
(lM) value of benzoxazepine series of compounds in breast cancer cell lines
O
O
a
using MTT assay after 24 h treatment
R1
R1
NTs
NTs
OR2
15a-b
O
12
14
16a-e
R1
NR
Scheme 2. Reagents and conditions: (a) anhyd K2CO3, R2Cl, dry acetone, reflux, 5 h,
Benzoxazepine
60–70%.
Compd no.
R
R1
R2
MCF-7
MDA-MB-
231
OMe
OR2
OR2
O
O
15a
Ts
H
H
6.78
9.62
7.2
R1
HCl
N
N
NTs
B
A
15b
13a
Ts
Ts
10.2
HCl
N
R1 = H, 2-OMe, 3-OMe
R2 = alkyl amino ethyl chain
3-
OMe
3-
OMe
2-
R4
R5
R3
Inactive Inactive
Inactive Inactive
Inactive Inactive
R2 = alkyl amino ethyl chain
Figure 1. Two different types of benzoxazepine.
13b
14
Ts
Ts
OMe
OBn
2-
OMe
b
ClH.H2N
O
16a
16b
Ts
Ts
Inactive Inactive
Inactive Inactive
OBn
Br
H
OBn
N
N
a
N
CO2Me
CO2Me
OH
2-
OMe
1a
17
18
OMe
2-
OMe
OMe
OBn
16c
Ts
Inactive Inactive
N
OBn
Boc
2-
OMe
2-
OMe
H
H
H
16d
16e
Ts
Ts
Inactive Inactive
Inactive Inactive
c
Boc
N
N
e
d
N
CO2Me
N
OH
20
19
22
23
25
Boc
H
Me
Me
Me
Inactive Inactive
Inactive Inactive
OMe
f
OMe
R3
10.34
13.21
OH
OMe
Boc
R3
R3
N
N
26a
26b
H
H
Me
Me
Inactive Inactive
N
O
OH
g
14.5
Inactive Inactive
12.45 15.67
Inactive Inactive
7.2 10.4
18.6
21
NBoc
22
OMe
OMe
26c
26d
H
H
Me
Me
OMe
R3
R3
N
N
O
h
O
N
OTBDMS
NH
OH
Br
25
26e
H
Me
24
23
R3
N
4OH-T
OMe
O
26a, R2 = CH2CH2N(CH3)2
i
Results were calculated from three independent experiments with triplicate of each
observation.
(i) Compound with IC50 more than 20
26b, R2 = CH2CH2N(CH2CH3)2
26c, R2 = CH2CH2-piperidine
N
26d, R2 = CH2CH2-pyrolidine
OR2
l
M were considered inactive
2
H
26e
, R = CH2CH2-azepane
N
4
26
R3
=
(iii) R
=
(iv) R5 = CH2CHMe2.
OH
Scheme 3. Reagents and conditions: (a) PPh3, CBr4, DCM; (b) K2CO3, DMF, 2 h, 70%
in two steps; (c) NaHCO3, Boc2O, EtOH, 74%; (d) LiBH4, THF, 85%; (e) H2, 10% Pd–C,
50 psi, 75%; (f) PPh3, DEAD, 2 h, 75%; (g) 6 N HCl, MeOH, 70 °C, 2 h, 70%; (h) K2CO3,
DMF, 74%; (i) R2Cl, K2CO3, acetone, 70–80%.
was most potent.6d This observation influenced us to further deriv-
atize the tyrosine-based benzoxazepine compound. In this context
we made various amino alkyl chain derivatives 15a–b, 16a–e (see
Scheme 2).
derivatives 2 with 1 provided the esters 3, 4, 5 in 60–75% yield. The
Lithium aluminium hydride (LAH) reduction of 3, 4, 5 afforded the
corresponding alcohols 6, 7, 8 which on subsequent debenzylation
by H2/Pd (10% on carbon) gave 9, 10, 11 containing free alcoholic
and phenolic hydroxyl groups in 60–70% yield in two steps. Expo-
sure of 9, 10, 11 to Mitsunobu reaction conditions7, that is, dieth-
ylazodicarboxylate (DEAD), triphenylphosphine (TPP) at 0 °C in
tetrahydrofuran as solvent resulted in the formation of desired
enantiomerically pure benzoxazepine derivatives 12, 13 and 14
in 63–76% yield.
Table 2
IC50 value (lM) of compounds in MCF-7 and MDA-MB-231 cells
Treatment
Time (h)
MCF-7
MDA-MB-231
15a
24
48
72
24
48
72
6.78
3.55
1.83
9.62
4.31
3.23
7.2
5.45
4.6
10.2
5.12
4.2
15b
The biological results of the S-amino acid derived benzoxaze-
pines showed the compounds having benzene ring in side chain
exhibiting better activity and the compound derived from tyrosine

6818
S. K. D. Dwivedi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6816–6821
Figure 2. Effect of novel Benzoxazepines on breast cancer and ‘non-cancer’ cells. (A–C) MDA-MB-231 and MCF-7 cells were treated with vehicle (V), 15a or 4OHT (positive
control) as indicated in the figure and cell death was determined by MTT assay. % cell death compared to vehicle is plotted. (D) Vero, Hek 293 and primary rat calvarial
osteoblast cells were treated with vehicle or different concentrations of 15a for 24 h and % cell death compared to V is plotted. (E) MCF cells were treated with different
concentrations of 15a and LDH release in the media was measured. Data represent mean SEM from three independent experiments.
In an attempt to further explore the biological properties of
benzoxazepines as active pharmacophore, we further modified
the scheme to prepare new kind of tyrosine based benzoxazepine
derivatives with alkyl amino ethyl chains (Fig. 1B). The synthesis
of the new kind of core is outlined in Scheme 3. First, benzyl alco-
hol 1a was converted to its bromo derivative 17 and proceeds next
step without purification. It was reacted with tyrosine derivative in
presence of K2CO3/DMF to obtain 18 in good overall yield. Second-
ary amine of 18 was protected with Boc-group, followed by lithium
borohydride reduction to afford alcohol 20. Next, debenzylation of
20, followed by Mitsunobu reaction afforded benzoxazepane deriv-
ative 22. Boc-group of 22 was deprotected under acidic condition
(6 N HCl in MeOH) to obtain 23. 23 was treated with 24 furnishing
25. Different kinds of amino alkyl chain were introduced in 25 to
obtain 26a–e.
IC50 of less than 20.0 lM., Tables 1 and 2. Out of these compounds,
15a exhibited highest cytotoxicity in breast cancer cells MCF-7 and
MDA-MB-231 (Fig. 2A–C) Comparison of 15a with the previously
published THBP4 is given in Table 3.
Cytotoxicity towards normal cells: Specific cytotoxicity against
cancer cells without affecting normal cell growth is a key safety
feature of cancer chemotherapy. Five active compounds of the
series were evaluated for their cytotoxicity in normal cells, (Vero,
HEK-293 and primary rat calvarial osteoblasts) and 15a mediated
cytotoxicity in these cells were visible at a much higher dose than
in MCF-7 and MFA-MB-231 cells, signifying that the molecules
have selectivity for inducing growth inhibition of cancer cells
(Table 3 and Fig. 2D). Next, effect of 15a on membrane integrity
in MCF-7 cell line was assessed by LDH release. At IC50 value,
15a did not significantly affect membrane integrity of MCF-7 cells
(Fig. 2E), while it did cause LDH release at higher doses, indicating
that although at high concentration 15a affected membrane integ-
rity, the 15a-induced cytotoxicity was probably independent of its
effect on membrane integrity.
The newly synthesized compounds were evaluated for anti-pro-
liferative activity in both MCF-7 (ER+ve) and MDA-MB-231 (ER-ve)
cells using MTT assay. Five compounds (15a, 15b, 25, 26b and 26d)
were found to inhibit growth of MCF-7 and MDA-MB-231 cells at
Compound 15a induces apoptosis in MCF-7 cells: To determine
whether cell death induced by 15a was apoptotic in nature, cellular
processes associated with apoptosis rather than necrosis such as:
phosphatidylserine exposure (Annexin V-PI; Flow cytometry),
DNA fragmentation and PARP Cleavage were assessed. As evident
from Figure 3, 15a concentration dependently increased phospha-
tidylserine exposure (Fig. 3A). Compound 15a also induced PARP
cleavage (Fig. 3B) and DNA fragmentation (Fig. 3C).
Table 3
Safety index and therapeutic potential of compounds at 72 h
Activity/
treatments
MCF-7
(EC50
HEK-293
Safety index
(IC50/EC50)
Therapeutic
potential
)
(IC50
)
versus THBP4
15a
THBP
1.83
10
30
30
16.39
3
5.46
1
Effect of 15a on breast cancer cell cycle progression: Effect of 15a
on breast cancer cell line (MDA-MB-231and MCF-7) cell cycle

S. K. D. Dwivedi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6816–6821
6819
Figure 3. Compound 15a induces Apoptosis in MCF-7 cells. (A) Compound 15a increases phosphatidylserine exposure- MCF-7 cells were treated either with vehicle (V) or
indicated concentrations of 15a for 24 h and phosphatidylserine exposure was measured by flow cytometry using FITC labelled Annexin V & PI staining. Representative dot
plots and relative apoptotic cells are shown. (B) Effect of 15a treatment on PARP Cleavage—15a treated MCF-7 cell lysates were immunoblotted against PARP, Densitometry
data of cleaved PARP is plotted. (C) Effect of 15a treatment on DNA Fragmentation—cells were treated with 15a and 4 OHT as indicated and DNA fragmentation after 24 h of
treatment was quantified. Graph represents relative DNA fragmentation taking +ve control (PC) as 100% (#). Data represent mean SEM from three independent
experiments; ⁄P <0.05, ⁄⁄P <0.01, and ⁄⁄⁄P <0.001 compared to V.
Figure 4. Compound 15a inhibits growth of MCF-7 cancer cell xenograft in athymic nude mice. (A) Increase in relative tumor volume and (B) average body weight over time
in control and 15a treatment groups indicated. N = 10 mice/group; data represent mean SEM. ⁄P <0.05 and ⁄⁄P <0.01 compared to control.
progression was evaluated by flow cytometry using propidium io-
dide (PI) staining and a significant increase in G1 sub-populations
and decrease in S sub-populations were observed following 15a
treatment (Fig. 5A). To further confirm G1 growth arrest, expres-
sion of cell cycle regulatory proteins including p21, p27 and cyclin
D1 were measured by quantitative real time PCR (QRT-PCR) and
western blotting. Compound 15a significantly increased mRNA
levels of cyclin dependent kinase inhibitors p21 and p27, and sup-
pressed cyclin D1 and E2F in MCF-7 cells (Fig. 5B). Concomitant to
the mRNA expression, 15a caused robust increase in the protein
levels of p21 and p27 while it suppressed cyclin-D1 protein expres-
sion (Fig. 5C).
Compound 15a induces both extrinsic and intrinsic pathways of
apoptosis: To determine if 15a-induced apoptosis was mediated
by extrinsic or intrinsic apoptotic pathways, cleaved caspase levels
were assessed. Compound 15a induced cleavage of both caspase-8
and -9, the initiator caspases that are usually activated by extrinsic
and intrinsic pathways, respectively; within 3 h (Fig. 6A). It also in-
duced the cleavage of caspase-3, the effector caspase that is acti-
vated by both caspase-8 and 9 (Fig. 6A) at 6 h onwards. In line
with caspase-9 activation, 15a significantly decreased mitochon-
drial membrane potential (Fig. 6B) and caused a robust increase
in Bax/Bcl2 ratio as determined by immunoblotting (Fig. 6C).
To further evaluate the relative importance of the caspase-8 and
-9-dependent pathways in 15a-induced apoptosis, 15a-induced
cell death was assessed in presence of various selective caspase
inhibitors. Figure 6D showed that z—VAD- FMK (an inhibitor of
caspases 1, 3, 4 and 7) and z—IETD-FMK (a caspase-8 inhibitor)
protected against 15a-induced loss of cell viability but LEHD-
FMK (a caspase-9 inhibitor) failed to do so, indicating that although
15a may induce both caspase-8 and -9-dependent apoptotic path-
ways, the primary pathway related to 15a-induced apoptosis ap-
peared to be caspase-8-dependent.
Effect of 15a on MCF-7 xenograft tumor model: Injection of MCF-7
cells subcutaneously into dorsal flank region of nude mice resulted
in the appearance of palpable tumors that rapidly grew from 9 to
21 days. Treatment of 15a (16 mg kgÀ1 dayÀ1) to these mice for
21 days resulted in nearly 50% reduction in relative tumor volume
(Fig. 4A). However, no significant differences in the initial and final
body weights were observed when compared with vehicle-treated
controls (Fig. 4B).
The present study sought to investigate the effects of a tyrosine-
based benzoxazepine, 15a in human breast cancer cells and animal
model. Compound 15a was synthesized in lead optimization
process using THBP4 as a template. The anti-proliferative effect
of 15a on MCF-7 cells was associated with G1 cell-cycle arrest.
G1 to S phase transition is known to be dependent on the
action of cyclin D1 and p27 protein levels,8 and that cyclin D1 is

6820
S. K. D. Dwivedi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6816–6821
Figure 5. Effect of 15a on cancer cell cycle progression. (A) MDA-MB-231 and MCF-7 cells were treated either with vehicle or 15a for 24 h and were then stained with PI and
analyzed by flow cytometry. Quantification of flow cytometry data has been shown as percent of total cells (right panel). (B) MCF-7 cells were treated with different
concentrations of 15a as indicated for 12 h, mRNA from these samples were then isolated, reverse transcribed, and the expressions of P21, P27 and cyclin D1 were measured
by QRT-PCR. (C) Total Protein isolated from MCF-7 cells treated with 15a as indicated was used for immunoblotting and relative density of p21, p27 & cyclin D1 were
measured (right panel). Data represent mean SEM from three independent experiments; ⁄P <0.05, ⁄⁄P <0.01, and ⁄⁄⁄P <0.001 compared to control.
Figure 6. Compound 15a activates both extrinsic and intrinsic apoptotic pathways. (A) Caspase activation by 15a was assessed by treating MCF-7 cells with 5
indicated time periods and then determination of cleaved caspase levels by immunoblotting with cleaved caspase-3, -8 and -9 antibodies. b-Actin was used as a loading
control (B). Loss of mitochondrial membrane potential by 15a—changes in mitochondrial membrane potential ( ) was assessed by JC-1 staining of MCF-7 cells treated with
15a for 12 h followed by flow cytometry analysis. Graph represents relative membrane potential. (C) Cell lysates from MCF-7 cells treated with 15a were blotted against Bcl2
and Bax, b-actin was used as a loading control. Graph represents Bax/BCL2 ratio. (D) MCF-7 cells were treated with 5 M 15a in the presence of different caspase inhibitors
lM 15a for
D
W
l
zVAD-FMK (pan caspase inhibitor), IETD-FMK (caspase-8 inhibitor) and LEHD-FMK (caspase-9 inhibitor). After 24 h, cell viability was determined using MTT assay. Data
represent mean SEM from three independent experiments; ⁄P <0.05, ⁄⁄P <0.01, and ⁄⁄⁄P <0.001 compared to control, $P <0.001 compared to 15a treatment.
over-expressed in mammary epithelial cell carcinoma that favors
tumor growth and up-regulation of p27 protein levels.9,10 At IC50
cleavage and DNA fragmentation which are considered as hallmarks
of apoptotic cell death. Compound 15a activated both extrinsic and
intrinsic apoptosis pathways as illustrated by activation of caspase
-8 and -9, loss of mitochondrial membrane potential (indicating
mitochondrial damage) and increased Bax-to-Bcl-2 ratio in
MCF-7 cells. However, use of caspase-selective inhibitors revealed
that the apoptosis induced by 15a could be reversed by a selective
inhibitor of caspase-8 but not that of caspase-9. Prior studies
value (ꢀ5
lM); 15a resulted in G1 arrest which was associated
with a decline in expression levels of E2F and cyclin D1 and
enhancement in expression of cyclin dependent kinase inhibitors
p21 and p27.
G1 arrest of MCF-7 cell was followed by apoptosis as 15a
dose dependently increased phosphatidylserine exposure, PARP

S. K. D. Dwivedi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6816–6821
6821
indicate that caspase-8-dependent truncation of BCL-2 homology 3
References and notes
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induced mitochondrial membrane depolarization and caspase-9
cleavage may result from the caspase-8-dependent BID-activation
pathway.
In conclusion, 15a could serve as a base for the development of
a new group of effective breast cancer therapeutics, although
detailed mechanistic studies, including identifications of possible
targets will be essential for future rational drug design.
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Acknowledgments
S.K.D.D., A.K.J., S.M. thank CSIR for providing fellowships. S.S.,
G.P. thank BSC0201 and DST, New Delhi, respectively for funding.
We would like to thank the referees for their valuable comments
which helped us a great deal to improve the quality of the paper.
This has CDRI communication no 8558.
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Supplementary data (NMR spectra) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2013.10.013.