Synthesis, anticancer activity and apoptosis inducing ability of anthranilamide-PBD conjugates

Article abstract of DOI:10.1016/j.bmcl.2010.04.037

A series of novel anthranilamide linked pyrrolo[2,1-c][1,4]benzodiazepine conjugates were prepared and evaluated for their anticancer activity. The effects of three promising PBD conjugates on cell cycle of cancerous cell line A375 were investigated. Thes

Full text of DOI:10.1016/j.bmcl.2010.04.037

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3310–3313
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, anticancer activity and apoptosis inducing ability
of anthranilamide-PBD conjugates
a
a
a
a
Ahmed Kamal a, , E. Vijaya Bharathi , M. Janaki Ramaiah , J. Surendranadha Reddy , D. Dastagiri ,
*
A. Viswanath a, Farheen Sultana a, S. N. C. V. L. Pushpavalli a, Manika Pal-Bhadra a, , Aarti Juvekar ,
b
*
Subrata Sen b, Surekha Zingde b
a Chemical Biology Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 607, India
b Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 December 2009
Revised 3 April 2010
Accepted 12 April 2010
Available online 14 April 2010
A series of novel anthranilamide linked pyrrolo[2,1-c][1,4]benzodiazepine conjugates were prepared and
evaluated for their anticancer activity. The effects of three promising PBD conjugates on cell cycle of can-
cerous cell line A375 were investigated. These promising compounds showed the characteristic features
of apoptosis like enhancement in the levels of p53 and activation of caspase-3.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Pyrrolobenzodiazepine
Anthranilamide
Anticancer activity
Apoptosis
p53
Active caspase-3
Pyrrolo[2,1-c][1,4]benzodiazepines (PBD) are naturally occur-
ring DNA interactive antitumour antibiotics isolated from various
Streptomyces species.1 The PBD cytotoxins exert a powerful antitu-
moural activity by binding to the minor groove of double stranded
DNA by forming a covalent bond to the exocyclic amino group of a
central guanine within a three base pair recognition site.2,3 The
natural occurring PBDs have potent anticancer activity, however
they have been precluded from clinical studies due to problems
relating to side effects.4 Hence, there has been a considerable inter-
est in the design and synthesis of conjugate agents with active
moieties of known antitumour agents to enhance the sequence
selectivity as well as antitumour activity. An anthranilamide moi-
ety present in the structures of agents like AAL-993 (2) and PD
184352 (CI-1040, 3)5,6 is considered responsible for the potent
antitumour properties.7,8 Similarly, it is interesting to observe that
in the last decade, a number of piperazine derivatives have been
synthesized to exploit their chemotherapeutic potential.9–11
Michejda and co-workers12 reported symmetrical bifunctional
agents as promising antitumour class of compounds with remark-
able selectivity against colon cancers that has a piperazine moiety
in its linker spacer. Recently, transdiamine dichloroplatinum (II)
complexes with piperazine ligands have also exhibited significant
cytotoxicity.13 Some representative members of the anthranila-
mides, DC-81 and its conjugates are illustrated in Figure 1.
In the past few years, several hybrid compounds, in which known
antitumour agents tethered to PBD moiety, have been designed, syn-
thesized and evaluated for their biological activity.14–18 Recently, we
have been not only involved in the development of new synthetic
strategies19,20 for the preparation of PBD ring system but also in
the design of structurally modified PBDs and their hybrids.21–23 In
continuation of these efforts, it is considered of interest to design
and synthesize hybrid molecules in which anthranilamide moiety
is linked through 4-piperazinyl alkane spacer to the C8-position of
the A ring of PBD ring system to explore their potential as a new class
of anticancer agents.
The synthesis of the target conjugates was performed in a con-
venient manner as outlined in Schemes 1 and 2. Isatoic anhydride
(5a) or 6-chloroisatoic anhydride (5b) was treated with N-boc
piperazine in 1:1.2 M ratio, and refluxed in 1,4-dioxane to give
the 4-(2-aminobenzoyl)N-boc piperazine (6a) and 4-(2-amino-5-
chlorobenzoyl) N-boc piperazine (6b), respectively, in almost
quantitative yield. These compounds 6a and 6b upon reductive
amination with different aromatic aldehydes by using sodium cy-
ano borohydride (NaCNBH3) in methanol with catalytic amount
of acetic acid afforded compounds 7a–f. These on deprotection
* Corresponding authors. Tel.: +91 40 27193157; fax: +91 40 27193189 (A.K.);
tel.: +91 40 27193236 (M.P.-B.).
E-mail addresses: ahmedkamal@iict.res.in (A. Kamal), manika@iict.res.in (M. Pal-
Bhadra).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.037

A. Kamal et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3310–3313
3311
NO2
HO
NO2
Br
O
( )
CH(SEt)2
CH(SEt)2
(i)
n
HN
O
O
N
H3CO
N
H3CO
10a−c
O
N
HO
H
O
O
N
CF3
9
F
NH
H
n = 1, 3, 4
N
H3CO
NH
F
Cl
8a−f (ii)
O
O
N
X
I
N
DC-81(1)
AAL993 (2)
CI-1040 (3)
N
NO2
O
( )
CH(SEt)2
NH
n
O
R
N
H3CO
X
N
11a−l
O
N
N
O
( )
H
NH
n
(iii), (iv)
O
N
R
H3CO
X = H, Cl
X
O
N
4a−l
n = 1, 3, 4
N
N
( )
O
H
NH
O
O
n
R
N
H3CO
4a−l
n = 1,3,4
R =
4a−c: R = 3, 4, 5-trimethoxy phenyl; X = H
O
4d−f: R = 4 -pyridinyl; X = H
H3CO
OCH3
OCH3
N
4g−i: R = 2- napthyl; X = H
4j: R = 3-chromenyl; X = H; n = 3
4k: R = 2-napthyl;
X = Cl;n = 3
4l: R = 3-chromenyl; X = Cl;n = 3
Figure 1. Chemical structures of DC-81 (1), anthranilamide AAL993 (2), CI-1040 (3),
anthranilamide-PBD conjugates (4al).
Scheme 2. Reagents and conditions: (i) dibromoalkane, K2CO3, acetone, reflux,
24 h; (ii) K2CO3, acetone, reflux, 24 h; (iii) SnCl2ꢀ2H2O, MeOH, reflux, 6 h; (iv) HgCl2,
CaCO3, CH3CN/H2O (4:1), rt, o/n.
with triflouroacetic acid gave precursors 8a–f as shown in
Scheme 1. The synthesis of C8-linked anthranilamide-PBD conju-
gates (4a–l) was carried out by coupling (2S)-N-{4-[n-bromoalk-
oxy-5-methoxy-2-nitrobenzoyl} pyrrolidine-2-carboxaldehyde
diethyl thioacetal (10a–c) (prepared by the methods reported in
our previous studies21–23) with anthranilamide precursors (8a–f)
using K2CO3 in acetone to provide the nitro thioacetal intermedi-
ates 11a–l. Finally, these upon reduction with SnCl2ꢀ2H2O in meth-
anol followed by deprotection using HgCl2/CaCO3 provided the
desired PBD conjugates (4a–l)24 as shown in Scheme 2.
for DC-81 the GI50 ranged from 0.10 to 2.37 lM. As shown in
Table 1, most of these conjugates exhibited higher anticancer
activity than 1 (DC-81), particularly against Zr-75-1, MCF7, KB,
and DWD cell lines. The significant anticancer activity showed by
some of the compounds like 4g, 4h and 4j prompted us to evaluate
the cell viability of human melanoma cell line A375 cells, as this
cell line is rapidly proliferating and malignant cell line resistant
to radio and chemotherapy.26 Further, to confirm the effectiveness
of these conjugates, they were compared to both the precursors 1
(DC-81), 8c and 8d (anthranilamide precursors used to obtain com-
pounds 4g, 4h and 4j, respectively). As shown in Supplementary
Figure 1, all these three conjugates (4g, 4h and 4j) exhibited higher
anticancer activity relative to 1 (DC-81), 8c and 8d in A375 cells.
In order to study the effect of these conjugates on cell cycle pro-
gression, human melanoma cell lines (A375) were treated with
compounds 1, 8c, 8d, 4g, 4h and 4j and analyzed using fluores-
cence activated cell sorter (FACS shown in Supplementary Figs. 2
These conjugates 4a–l were evaluated for their anticancer activ-
ity in selected human cancer cell lines of lung, breast, oral, colon,
cervix, ovary and prostate by using sulforhodamine B (SRB) protein
assay. This assay is used for cell density determination, based on
the measurement of cellular protein content. The experimental
protocol is given in Supplementary data. The compounds exhibit-
ing GI50 6 10ꢁ5 M (10
respective cell lines. All these compounds 4a–l exhibited potent
anticancer activity with GI50 values ranging from 0.13 to 29 M.
The positive control compound adriamycin25 demonstrated signif-
lM) are considered to be active on the
l
and 3). Treatment of cells with these compounds at 1 lM concen-
icant activity with the GI50 in the range from 0.10 to 7.25
lM and
tration for 24 h induced apoptotic effects up to 2.63%, 1.23%, 1.15%,
5.48%, 7.34% and 3.48%, respectively, of sub G1 DNA peak (G0
phase) of A375 cells compared to 0.74% for the control cells. These
results show that all these conjugates (4g, 4h and 4j) were more
efficient in inducing apoptosis than 1 (DC-81) in A375 cells as
shown in Figure 2.
O
O
O
X
X
X
(ii)
(i)
N
N
O
NBoc
NBoc
NH
NH2
N
H
O
Activation of tumour suppressor genes such as p53 is found to
be important in the regulation of apoptotic pathway induced by
various stimuli.27 The p53 gene, is a tumour suppressor gene, that
is, its activity stops the formation of tumours. To investigate
whether these PBD conjugates induce apoptosis through p53-
dependent pathway, the expression levels of p53 protein was
checked after treating the A375 cells with compounds and western
blot analysis was carried out using p53-specific antibody. As
shown in Figure 3, it was observed that p53 protein expression lev-
els were up-regulated when treated with compound 4h indicating
a clear-cut evidence of p53-dependent pathway. Further, all these
conjugates increased the levels of p53 when compared to DC-81.
As we observed the increase in levels of p53, we tried to
investigate the down stream signalling events in the process of
apoptosis that is mainly mediated by caspases. Caspase-3 is
5a
R
6a, b
7a−f
5a: X = H
5b: X = Cl
(iii)
N
8a: R = 3, 4, 5-trimethoxyphenyl; X = H
8b: R = 4 -pyridinyl; X = H
O
8c: R = 2- napthyl; X = H
X
8d: R = 3-chromenyl; X = H
8e: R = 2-napthyl;
X = Cl
8f: R = 3-chromenyl; X = Cl
NH
NH
O
O
R
R =
H3CO
8a−f
OCH3
N
OCH3
Scheme 1. Reagents and conditions: (i) N-boc piperazine, 1,4-dioxane, reflux, 4 h;
(ii) R-CHO, NaCNBH3, MeOH, CH3COOH, rt, 22 h; (iii) TFA, CH2Cl2, rt, 5 h.

3312
A. Kamal et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3310–3313
Table 1
GI50 valuesa (in
lM) for compounds 4al in selected human cancer cell lines
Compd
A549b
Hop62b
Zr-75-1c
MCF7c
DWDd
KBd
Guravd
PC-3e
A2780f
Colo205g
SiHah
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
2.0
1.9
0.18
2.0
2.0
2.1
1.9
1.9
2.2
2.3
25
1.7
1.8
1.9
1.8
2.0
1.8
0.14
0.15
0.17
0.16
0.18
0.18
2.0
0.14
0.14
0.14
0.13
0.16
0.15
0.14
0.13
0.14
0.14
0.14
0.15
0.17
0.17
0.15
0.14
0.15
0.17
0.15
0.17
0.14
0.13
0.16
0.16
0.17
0.17
0.10
1.49
0.15
0.14
0.15
0.15
0.15
0.15
0.15
0.13
0.15
0.15
0.14
0.16
0.17
0.17
0.17
0.16
0.17
0.18
0.17
0.18
0.17
0.14
0.17
0.17
0.17
0.17
0.17
0.16
0.16
0.14
0.16
1.8
0.17
1.8
0.14
0.14
0.15
0.14
0.17
0.16
1.81
0.20
0.18
0.16
0.17
0.19
0.17
0.19
0.17
0.14
0.19
0.18
0.18
0.18
0.16
0.14
0.17
2.2
0.16
0.19
25
0.17
0.17
1.8
1.8
1.8
2.2
2.0
2.6
1.9
0.17
0.17
0.17
0.16
2.0
0.17
7.25
0.16
0.15
0.15
0.16
0.16
0.18
0.16
1.79
2.37
0.18
0.15
0.18
2.4
29
2.3
0.16
0.15
0.15
0.13
1.8
0.16
0.17
0.17
ADRi
DC-81 (1)
0.14
0.11
a
50% growth inhibition and the values are mean of three determination.
b
c
d
e
f
Lung cancer.
Breast cancer.
Oral cancer.
Prostate cancer.
Ovary cancer.
Colon cancer.
Cervix cancer.
Adriamycin.
g
h
i
Figure 2. FACS analysis of cell cycle distribution of A375 cells after treatment with
anthranilamide-PBD conjugates 1, 4g, 4h, 4j, 8c and 8d at 1 M concentration for
Figure 4. Effect of anthranilamide-PBD conjugates on caspase-3 activity in A375
l
cells determined by flourimetry.
24 h. The cells which fall in G0 phase of cell cycle indicates the percentage of
apoptotic cells.
Further, PBD compounds are effective as DNA binding agents
particularly at G-rich sequences.30 Therefore, in order to check
the superior activity of these PBD conjugates over the naturally
occurring PBD compounds such as DC-81, the restriction enzyme
digestion assay (RED 100 assay) was carried out for the most active
compound 4h along with the naturally occurring PBD compound
DC-81 (1). In this assay pBR 322 vector having Bam HI restriction
site (50-G0GATCC-30) with the G-rich regions around the Bam HI site
was used. The experiment is based on the principle that the drug
binding at Bam HI site should inhibit the restriction digestion
mediated by Bam HI enzyme. The experimental protocol is de-
scribed in the previous study.31 In this study, we used various con-
Figure 3. Effect on p53 protein expression. A375 cells were treated with 1 lM
concentration of anthranilamide-PBD conjugates for 24 h. The cell lysates were
used for western blot analysis using p53-specific antibody. C: cells without
treatment. b-Actin was used as internal or loading control.
centration gradients ranging from 2 to 32 lM. It was observed
from Figure 5 that, compound 4h, the effective compound in our
other studies was found to inhibit the restriction digestion even
considered to be the effector caspase and is considered as the ther-
apeutic target for the treatment of cancer.28 Hence, we examined
the involvement of caspases and their role in the process of apopto-
sis. From the results obtained in caspase-3 assay, an up regulation of
caspase-3 (2.5-fold) was observed when treated with compound 4h
compared to untreated cells, while a slight increase in caspase-3 le-
vel was observed when treated with compounds 4g and 4j compared
to DC-81 as shown in Figure 4. Similar type of observations were re-
ported for the indole-PBD (IN6CPBD) conjugate.29
at 4
lM concentration, whereas DC-81 showed similar effects at
much higher concentrations (i.e., 16
lM). This showed the im-
proved selectivity of the compound 4h than the naturally occurring
DC-81(1).
In conclusion, in the present study, a series of new anthranila-
mide-PBD conjugates were synthesized. All these PBD conjugates

A. Kamal et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3310–3313
3313
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Figure 5. RED 100 assay: The figure in the top panel depicts the Red assay using
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Acknowledegments
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Supplementary data.
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The authors E.V.B., J.S.N.R., D.D.G., A.V. and F.S. are thankful to
CSIR, New Delhi, for the award of research fellowships. We are also
thankful to the Department of Biotechnology(BT/PR/7037/Med/14/
933/2006), New Delhi; for financial assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.04.037.
31. Kamal, A.; Srinivas, O.; Ramulu, P. G.; Kumar, P. P.; Kumar, M. S. Bioorg. Med.
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