Anti-tumor activity of a new series of benzoxazepine derivatives in breast cancer

Article abstract of DOI:10.1016/j.bmcl.2009.10.115

A series of new benzoxazepine derivatives substituted with different alkoxy and aryloxy group were synthesized comprising synthetic steps of Mitsunobu reaction, lithium aluminum hydride (LAH) reduction, followed by debenzylation and finally intramolecular Mitsunobu cyclization. The new benzoxazepines specifically inhibited growth of breast cancer cell lines, MCF-7 and MDA-MB-231, but lack cytotoxicity to normal HEK-293 cells. The cell growth inhibition induced by the active compounds was due to cell cycle arrest at G₀/G₁ phase. The active compound could cause significant reduction in tumor volume of MCF-7 xenograft tumor in nude mice model and their activity was comparable to that of tamoxifen citrate at 16 mg kg^-1 dose at 30 days of treatment. The identified most active compounds of the series have specific advantages as anti-cancer agent in breast cancer than tamoxifen.

Full text of DOI:10.1016/j.bmcl.2009.10.115

Bioorganic & Medicinal Chemistry Letters 20 (2010) 283–287
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anti-tumor activity of a new series of benzoxazepine derivatives
in breast cancer
Krishnananda Samanta a, Bandana Chakravarti b, Jitendra Kumar Mishra a,
Shailendra Kumar Dhar Dwivedi b, Lakshma Vadithe Nayak b, Preeti Choudhry b,
Hemant Kumar Bid b, Rituraj Konwar b, Naibedya Chattopadhyay b, Gautam Panda a,
*
a Endocrinology, Central Drug Research Institute, Lucknow 226 001, UP, India
b Medicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow 226 001, UP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new benzoxazepine derivatives substituted with different alkoxy and aryloxy group were syn-
thesized comprising synthetic steps of Mitsunobu reaction, lithium aluminum hydride (LAH) reduction,
followed by debenzylation and finally intramolecular Mitsunobu cyclization. The new benzoxazepines
specifically inhibited growth of breast cancer cell lines, MCF-7 and MDA-MB-231, but lack cytotoxicity
to normal HEK-293 cells. The cell growth inhibition induced by the active compounds was due to cell
cycle arrest at G0/G1 phase. The active compound could cause significant reduction in tumor volume of
MCF-7 xenograft tumor in nude mice model and their activity was comparable to that of tamoxifen cit-
rate at 16 mg kgÀ1 dose at 30 days of treatment. The identified most active compounds of the series have
specific advantages as anti-cancer agent in breast cancer than tamoxifen.
Received 12 August 2009
Revised 22 October 2009
Accepted 27 October 2009
Available online 30 October 2009
Keywords:
Amino acids
Benzoxazepines derivatives
Anti-tumor activity
Intramolecular Mitsunobu cyclization
Breast cancer
Ó 2009 Elsevier Ltd. All rights reserved.
Amino acids
Currently, tamoxifen is most commonly used adjuvant drug for
estrogen receptor (ER)-positive breast cancer.1 Tamoxifen, classi-
fied under selective estrogen receptor modulators (SERM), com-
petes with estrogen and downregulates estrogenic actions in
breast tissue. As a result, tamoxifen is less effective in ER-negative
breast cancer. Postmenopausal patients with ER-positive metastatic
breast cancer exhibit a high response rate of more than 50% with
mean duration of response of nine months,2,3 whereas the response
rate of premenopausal patients were reported in the range of 20–
45%.4–7 The safety of tamoxifen is controversial as several clinical
trial reports revealed increase risk of uterine cancer in postmeno-
pausal women.8–10 Therefore, there is a need for anti-breast cancer
agents with improved properties such as enhanced and specific
activity against cancer with reduced uterine side effects.
In our ongoing effort toward searching new pharmacophores as
anti-cancer breast agents, phenanthrene based compounds con-
taining amino alkyl chains were reported to have promising activ-
ity.11 Benzoxazepines are well-known pharmacophore in
medicinal chemistry showing promising activity against various
diseases such as antipsychotic, central nervous system activity
along with anti-cancer profile against breast cancer cells.12 One
of these groups of benzoxazepines have been identified to target
microtubule assembly as a way to induce anti-cancer activity.12
However, amino acid based benzoxazepines were not explored
much to evaluate their pharmacological activity. Syntheses of ami-
no acid based polycycles were earlier reported from our labora-
tory.13a–c Easy availability of amino acid based benzoxazepines
promoted us to evaluate them for anti-cancer breast activity. We
designed new class of amino acid-derived benzoxazepine deriva-
tives with alkyl amino ethyl chains and evaluated anti-tumor
activity in human breast cancer cells and xenograft model.
The synthesis of benzoxazepines (Scheme 1) ring followed the
methodology developed in our laboratory. S-Amino acid derived
N-tosyl amino esters and substituted benzene derivatives were
used as building blocks for the construction of benzannulated chi-
ral heterocycles. The Mitsunobu reaction of S-amino acid deriva-
tives 2a–h with 1a–c provided the esters 3a–h, 4a, 5a in 60–75%
yield. The lithium aluminum hydride (LAH) reduction of 3a–h,
4a, 5a afforded the corresponding alcohols 6a–h, 7a, 8a which on
subsequent debenzylation by H2/Pd (10% on carbon) gave 9a–h,
10a, 11a containing free alcoholic and phenolic hydroxyl groups
in 60–70% yield in two steps. Exposure of 9a–h, 10a, 11a to Mits-
unobu reaction conditions,14 that is, diethylazodicarboxylate
(DEAD), triphenylphosphine (TPP) at 0 °C resulted in the formation
of desired enantiomerically pure benzoxazepine derivatives 12a–h,
13a and 14a in 63–76% yield. The biological result of the S-amino
* Corresponding author. Tel.: +91 522 2612411x18; fax: +91 522 2623938.
E-mail addresses:
(G. Panda).
gautam.panda@gmail.com,
gautam_panda@cdri.res.in
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.115

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K. Samanta et al. / Bioorg. Med. Chem. Lett. 20 (2010) 283–287
TsHN
CO CH
2
3
OH
Ts
OBn
OBn
Ts
1
2a-h
a
+
OBn
c
b
R
Ts
R
R
R
N
CH OH
2
N
CH OH
2
N
CO CH
2
3
R
1
1
1
9a-h
10a
11a
OH
R
6a-h R
7a
R
3a-h
4a
5a
d
1a, R = H
1b, R = 3-OMe
1c, R = 4-OMe
8a
O
1
12a-h
13a
R
R
NTs
14a
Scheme 1. Reagents and conditions: (a) 1a–c, 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–
75%; (d) DEAD, PPh3, THF, 0 °C (1 h) to rt, (14 h), N2, 63–76%.
Table 1
The in vitro cell growth inhibitory effect of benzoxazepine series of compounds in variOous cell lines with MTT assay 12a–h, 13a, 14a
1
R
R
N
Ts
12a-h
13a
14a
Compound no.
R
R1 a
Cell growth inhibition in terms of IC50 (lM)
MCF-7
NAb
MDA-MB 231
Ishikawa
DU-145
NA
12a
H
NA
NA
HN
12b
12c
H
H
NA
NA
NA
NA
NA
NA
NA
NA
12d
12e
H
H
NA
NA
NA
NA
NA
NA
NA
NA
12f
H
H
NA
NA
NA
NA
NA
NA
NA
NA
12g
12h
13a
H
29.1
NDc
>50
ND
34
OH
OH
OH
3-OMe
4-OMe
ND
ND
14a
ND
ND
ND
ND
Tamoxifen citrate
10.2
12.3
12.5
15.25
Synthesized benzoxazepines, 12a–h, 13a, 14a.
a
R1 means alkyl derivatives of different amino acids.
b
NA means not active and having IC50 >50
ND means not determined.
lM.
c
acid derived benzoxazepines showed the compounds having ben-
zene ring in side chain exhibiting better activity and the compound
derived from tyrosine was most potent, Table 1. This observation
influenced us to further derivatize the tyrosine-based benzoxaze-
pine compound. In this context we made various amino alkyl chain
derivatives 15a–d, 16a–f, 17a–d, Scheme 2 (Table 2).
did not increase the activity in vitro. Among compounds without
methoxy substituent, the chains containing ethylamino group
exhibited better activity. Compound 15b had the highest cell
growth inhibitory effect in MCF-7 cells and more active than
tamoxifen citrate appeared from the IC50 value (6.53
lM vs
10.2 M). Overall, within this series, 15b and 15c appear to be
l
Structure–activity relationships. Introduction of methoxy substi-
tuent in the benzene ring of benzoxazepine series of compounds
more effective in inhibiting cell growth of breast cancer cells than
other type of cancer cells (endometrial cancer-Ishikawa and pros-

K. Samanta et al. / Bioorg. Med. Chem. Lett. 20 (2010) 283–287
285
OR2
OH
O
O
a
R
R
N
N
Ts
Ts
15a-d
16a-f
17a-d
12h
13a
14a
Scheme 2. Synthesis of amino ethyl alkyl chain derivatives with reagents and conditions: (a) anhyd K2CO3, R2Cl, dry acetone, reflux, 5 h, 60–70%.
tate cancer DU-145, Table 1). Furthermore, 12h, 15b and 15c
showed time dependent inhibition of cell growth of MCF-7 and
MDA-MB-231 cells at IC50 concentration (Fig. 1a and b).
Since 15b and 15c showed better cell growth inhibition of
breast cancer cell line than tamoxifen citrate, we tested cytotoxic-
ity in normal cells (HEK-293). Data show that 15b had no cell
Table 2
The in vitro cell growth inhibitory effect of benzoxazepine series of compounds in various cell lines with MTT assay 15a–e, 16a–f, 17a–d
OR2
O
R
N
Ts
15a-d
16a-f
17a-d
Compound no.
R
R2
Cell growth inhibition in terms of IC50 (lM)
MCF-7
NDa
MDA-MB 231
Ishikawa
DU-145
ND
CH2CH2N
CH2CH2N
CH2CH2N
O
15a
15b
15c
H
H
H
ND
ND
6.53
9.34
17.45
13.01
22.6
23.8
23
30
NAb
NA
NA
NA
CH2CH2N
15d
H
CH2CH2N
16a
16b
16c
3-OMe
3-OMe
3-OMe
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
O
CH2CH2N
CH2CH2N
CH2CH2N
16d
16e
3-OMe
3-OMe
NA
NA
NA
NA
NA
NA
NA
NA
CH2CH2N
O
16f
17a
17b
17c
3-OMe
4-OMe
4-OMe
4-OMe
4-OMe
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
17d
NA
NA
NA
NA
Tamoxifen citrate
10.2
12.3
12.5
15.25
Synthesized alkyl amine ethyl chain derivatives of benzoxazepines 15a–d, 16a–f, 17a–d.
a
ND means not determined.
NA means not active and having IC50 >50 lM.
b

286
K. Samanta et al. / Bioorg. Med. Chem. Lett. 20 (2010) 283–287
(a)
(b)
120
100
80
60
40
20
0
15b(6.53µM)
15c(9.34µM)
12h(23µM)
15b(17.45µM)
15c(13.01µM)
12h(29.1µM)
100
80
60
40
20
0
12hrs
24hrs
48hrs
Time
96hrs
12hrs
24hrs
48hrs
Time
96hrs
(c) 90
80
70
60
50
40
30
20
10
0
-10
-20
5um
10um
20um
15b
5um
10um
20um
5um
12h
10um
20um
15c
Figure 1. Percent cell growth inhibition of (a) MCF-7 cells, (b) MDA-MB-231 cells and (c) HEK-293 cells by various compounds.
Necrotic cells
growth inhibitory effect in HEK-293 cells whereas, 15c signifi-
100
90
80
70
60
50
40
30
20
10
0
Live cells
cantly inhibited cell growth (Fig. 1c). Because of its highest cyto-
static effect in MCF-7 cells with no effect in normal cells, we
took 15b for subsequent experiments.
Flow cytometric evaluation of induction of apoptosis. Compound
15b significantly (p >0.05) increased Annexin-V positive and PI
negative cell population at 5 and 10 lM indicating induction of
apoptosis compared with untreated control (Fig. 2).
Cell growth inhibition by benzoxazepine is due to G0/G1 arrest. Cell
cycle analysis by PI staining showed that 15b at 5 and 10 lM
Early apoptotic cells
Late apoptotic cells
induced cell cycle arrest at G0/G1 phase suggesting that cell cycle
arrest is responsible for cell growth inhibitory activity in MCF-7
cells (Fig. 3).
Control
5µM
10µM
15b
Figure 2. Negative cell population at 5
l
M and 10 lM of 15b; after 24 h, the cells
were harvested, stained with Annexin-V and propidium iodide, and analyzed by
flow cytometry. Data are expressed as% of total cell Count.
6000
control
15b(8mg/kg)
15b(16mg/kg)
5000
100
**
Tamoxifen(20mg/kg)
90
80
70
60
50
40
30
20
10
0
*
4000
Dip.G1
DipG2
DipS
3000
2000
1000
0
Apoptosis
Control
5µM
10µM
1
5
9
12
16
19
23
26
30
Days
15b
Figure 3. Apoptosis of MCF-7 by 15b occurs due to G0/G1 arrest. MCF-7 cells were
incubated in DMEM supplemented with 10% FBS and treated with 5 M and 10
of 15b. After 24 h, the cells were harvested, stained with PI, and analyzed by flow
Figure 4. Regression in mean relative tumor volume in human breast cancer cell
(MCF-7) xenograft model. Animals were treated with 15b after tumor induction for
30 days. The data presented are average of relative tumor volume (n = 10 animals/
group).
l
lM
cytometry. Data are expressed as% of total cell count.

K. Samanta et al. / Bioorg. Med. Chem. Lett. 20 (2010) 283–287
287
Anti-tumor effect in MCF-7 xenograft model. As shown in Figure 4,
Supplementary data
treatment of mice by 15b (16 mg kgÀ1 dose) resulted in signifi-
cantly higher relative tumor volume regression than the control
group. At 30 days, 15b (16 mg kgÀ1 dose) exhibited comparable tu-
mor regression with that of tamoxifen citrate. Our data showed
that out of 24 molecules, three (12h, 15b, and 15c) exhibited sig-
nificant cell growth inhibition when subjected to the MTT assay
on a panel of three human cancer cell lines (IC50 ranging from
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.10.115.
References and notes
1. Jaiyesimi, I. A.; Buzdar, A. U.; Decker, D. A.; Hortobagyi, G. N. J. Clin. Oncol. 1995,
13, 513.
2. Litherland, S.; Jackson, I. M. Cancer Treat. Rep. 1988, 15, 183.
3. Rose, C.; Mourisden, H. T. Cancer Res. 1984, 91, 230.
6.5 to 29.1 lM). However, only one compound 15b showed signif-
icantly better IC50 values than tamoxifen citrate (p >0.05) in inhib-
iting cell growth of MCF-7 cells. Compound 15b had no effect on
the growth of HEK-293 cells, suggesting that this compound may
be safe in normal cells. In flow cytometry analyses, 15b was found
to induce cell cycle arrest at G0/G1 phase. The in vitro ability to in-
hibit cell growth inhibition of MCF-7 cells by 15b was confirmed
in vivo in MCF-7 xenograft in nude mice model. Compound 15b
significantly regressed tumor volume that was comparable to that
of tamoxifen citrate. Given its cytostatic effect in MCF-7 cells,
in vivo anti-tumor activity in MCF-7 xenograft model, 15b is a po-
tential lead suitable for further improvement for developing as
anti-cancer breast therapy.
4. Henson, J. C. Cancer Treat. Rep. 1976, 60, 1463.
5. Manni, A.; Pearson, O. H. Cancer Treat. Rep. 1980, 64, 779.
6. Planting, A. S. T.; Alexieva-Figusch, J.; Blonk vd, W. J. Cancer Treat. Rep. 1985, 69,
363.
7. Sawka, C. A.; Pritchard, K. I.; Paterson, A. H. G.; Sutherland, D. J. A.; Thomson, D.
B.; Shelley, W. E.; Myers, R. E.; Mobbs, B. G.; Malkin, A.; Meakin, J. W. Cancer
Res. 1986, 46, 3152.
8. Magriples, U.; Naftolin, F.; Schwartz, P. E. J. Clin. Oncol. 1993, 11, 485.
9. Mourits, M. J.; De Vries, E. G.; Willemse, P. H.; Ten Hoor, K. A.; Hollema, H.; Van
der Zee, A. G. Obstet. Gynecol. 2001, 97, 855.
10. Chalas, E.; Costantino, J. P.; Wickerham, D. L. Am. J. Obstet. Gynecol. 2005, 192,
1230.
11. Shagufta; Srivastava, A. K.; Sharma, R.; Mishra, R.; Balapure, A. K.; Murthy, P. S.
R.; Panda, G. Bioorg. Med. Chem. 2006, 14, 1497.
12. (a) Liao, Y.; Venhuis, B. J.; Rodenhuis, N.; Timmerman, W.; Wikstrom, H.; Meire,
E.; Bartoszyk, G. D.; Bottcher, H.; Seyfried, C. A.; Sundell, S. J. Med. Chem. 1999,
42, 2235; (b) James, G. L.; Goldstein, J. L.; Brown, M. S.; Rawson, T. E.; Somers, T.
C.; McDowell, R. S.; Crowley, C. W.; Lucas, B. K.; Levinson, A. D.; Marsters, J. C.,
Jr. Science 1993, 260, 1937; (c) Mulligan, J. M.; Greene, L. M.; Cloonan, S.; Mc
Gee, M. M.; Onnis, V.; Campiani, G.; Fattorusso, C.; Lawler, M.; Williams, D. C.;
Zisterer, D. Mol. Pharmacol. 2006, 70, 60.
13. (a) Mishra, J. K.; Garg, P.; Dohare, P.; Kumar, A.; Siddiqi, M. I.; Ray, M.; Panda, G.
Bioorg. Med. Chem. Lett. 2007, 17, 1326; (b) Mishra, J. K.; Panda, G. J. Comb.
Chem. 2007, 9, 321; (a) Mishra, J. K.; Panda, G. Synthesis 2005, 11, 1881.
14. Mitsunobu, O. Synthesis 1981, 1.
Acknowledgments
Financial assistance from Indian Council of Medical Research
(ICMR 58/1/2003-BMS) and Ministry of Health and Family Welfare
are gratefully acknowledged. Jitendra, Krishnananda, Shailendra
and Bandana thank CSIR for fellowships. This has CDRI communi-
cation number 7720.