Synthesis and anticancer activity of chalcone-pyrrolobenzodiazepine conjugates linked via 1,2,3-triazole ring side-armed with alkane spacers

Article abstract of DOI:10.1016/j.ejmech.2011.05.050

Aiming to develop multitarget drugs for the anticancer treatment, a new class of chalcone-pyrrolo[2,1-c] [1,4]benzodiazepine (PBD) conjugates linked through a 1,2,3-triazole moiety containing alkane spacers has been designed and synthesized. Combining the

Full text of DOI:10.1016/j.ejmech.2011.05.050

European Journal of Medicinal Chemistry 46 (2011) 3820e3831
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticancer activity of chalcone-pyrrolobenzodiazepine conjugates
linked via 1,2,3-triazole ring side-armed with alkane spacers
,
a
b
a
a
Ahmed Kamal a , S. Prabhakar , M. Janaki Ramaiah , P. Venkat Reddy , Ch. Ratna Reddy ,
A. Mallareddy a, Nagula Shankaraiah c, T. Lakshmi Narayan Reddy b, S.N.C.V.L. Pushpavalli b,
Manika Pal-Bhadra b
*
a Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 607, India
b Chemical Biology Laboratory, Indian Institute of Chemical Technology, Hyderabad 500 607, India
c National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Aiming to develop multitarget drugs for the anticancer treatment, a new class of chalcone-pyrrolo[2,1-c]
[1,4]benzodiazepine (PBD) conjugates linked through a 1,2,3-triazole moiety containing alkane spacers
has been designed and synthesized. Combining these two core pharmacophore structures with modi-
fications at A-C8/C-C2-position of PBD ring system yielded analogs with improved efficacy and have
Received 11 April 2011
Received in revised form
18 May 2011
Accepted 22 May 2011
Available online 30 May 2011
shown promising in vitro anticancer activity ranging from <0.1e2.92 mM. These PBD-conjugates caused
G1 cell cycle arrest with effect on G1 cell cycle regulatory proteins such as Cyclin D1 and Cdk4. These
conjugates also exhibited inhibitory effect on NF-kB, Bcl-XL proteins that play a vital role in breast cancer
cell proliferation. These findings suggest that one of the compound 4d among this series is most effective
and has potential for detailed investigations.
Keywords:
Pyrrolo[2,1-c][1,4]benzodiazepines
1,2,3-Triazoles
Chalcones
Ó 2011 Elsevier Masson SAS. All rights reserved.
Anticancer activity
Apoptosis
Cyclin D1
Cdk4
NF-kB
1. Introduction
Chalcones represent an important group of natural products [9,10],
that received significant attention for their antitumor properties,
Cancer is a multifactor disease with superfluous and robust
biological networks. It may require treatment with compounds that
could target multiple intracellular components. Currently, cancer
therapy interfering with a single biological molecule or pathway
has been successfully utilized for the treatment in clinics [1e4].
There is general belief that agents modulating more than one target
could have superior efficacy compared to single target drugs [5,6].
Therefore, modulating multiple targets simultaneously can be
achieved by the combination of multiple drugs with different
mechanisms or by single chemical entity that could modulate
several targets of a multifactorial disease. As a result, there is
increasing interest in the discovery of agents that concomitantly
address more than one biological target for cancer treatment [7,8].
particularly in view of their similar mode of action to the struc-
turally related natural combretastatin (3, Fig. 1) [11]. In addition,
chalcones have been reported for a wide range of pharmacological
activities including cytotoxicity [12] and anticancer activity [13,14].
Moreover, recent studies have shown that these chalcones induce
apoptosis in a variety of cell types, including breast cancers [15,16].
The development of anticancer agents by performing structural
modifications in chalcone scaffold led to the improvement of their
bioavailability [17] and some synthetic chalcone derivatives are
showing selective cytotoxicity against MCF-7 cells [18].
Over the past few years, 1,2,3-triazole molecules have been
synthesized as useful chemotherapeutic agents for various diseases
[19]. This class of heterocycles is known to exhibit interesting
biological activities, such as antibiotic, antifungal, antehelmintic
[20e23], and anticancer activity [24]. Whereas, the imine
or carbinolamine-containing pyrrolo[2,1-c][1,4]benzodiazepines
(PBDs) are a family of low molecular weight antitumor antibiotics
originally isolated from various Streptomyces species [25] and
* Corresponding author. Tel.: þ91 40 27193157; fax: þ91 40 27193189.
E-mail addresses: ahmedkamal@iict.res.in (A. Kamal), manika@iict.res.in (M.
Pal-Bhadra).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.050

A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3820e3831
3821
O
10
N
9
A
6
11
MeO
MeO
HO
OH
H
8
B11a
1
C
7
N
4
H3CO
2
5
HO
OMe
OMe
3
O
2
OH
3
OMe
1
R2
N
N
N
R3
R4
O
N
R1
R1
R2
H
N
H3CO
O
H
O
n
N
N
N
O
H3CO
OCH3
HO
N
N
O
O
O
R1 = OH, R2, R3, R4 = H; 4a–c
R1, R2 = OCH3; 5a
R
R1, R2, R4 = H, R3 = OH; 4d–f
n = 3–5
1 = OBn, R2 = OCH3; 5b
R1 = OH, R2, R3 = H, R4 = Cl; 4g–i
Fig. 1. Structures of DC-81 (1), skeleton of chalcone (2), combretastatin (3), 1,2,3- triazoloechalcone-PBD hybrids (4aei and 5a,b).
R2
R1
O
R3
R4
R2
H3C
R3
R4
R1
O
O
N3
HO
N3
O
i
8a–c ii
n
n
OMe
MeO
CHO
MeO
CHO
6
–
7a c; n = 3 5
–
R1 = OH, R2, R3, R4 = H; 9a–c
R1, R2, R4 = H, R3 = OH; 9d f
–
R
1 = OH, R2, R3 = H, R4 = Cl; 9g–i
NO
NO
2 CH(SEt)2
HO
MeO
O
2 CH(SEt)2
iii
11 + 9a–i
iv 'click' reaction
N
N
MeO
O
O
11
10
R2
N
O
N
N
R3
R1
NO
2 CH(SEt)2
O
n
R4
N
H3CO
OCH3
O
O
–
12a i; n = 1 3
–
v, vi
–
4a–i; n = 1 3
Scheme 1. Reagents and conditions: (i) a) K2CO3, dibromo alkane spacers, dry DMF, rt, 12 h; b) NaN3, (0.5 M in DMSO), 80 ꢀC, 6 h; (ii) KOH/MeOH, rt, 12 h; (iii) K2CO3, propargyl
bromide, dry DMF, rt, 12 h; (iv) CuSO4$5H2O (1 mol%), sodium ascorbate (5 mol%), t-BuOH/H2O (1:1), rt, 12 h; (v) SnCl2$2H2O, MeOH, reflux, 2 h; (vi) HgCl2eCaCO3, CH3CN-H2O
(4:1), rt, 12 h.
examples of which include, DC-81 (1), anthramycin, tomaymycin
and sibiromycin. These compounds bind selectively in the minor
groove of DNA while a covalent aminal bond formation between
the electrophilic C11-position of the PBD and the nucleophilic N2-
amino group of a guanine base [26,27] results for their biological
activity. Some representative structures of chalcone, DC-81 and
their conjugates are illustrated in Fig. 1.
In the past few years, several conjugates in which known anti-
tumor agents linked to PBD moiety have been designed, synthe-
sized and evaluated for their biological activity [28e31]. Recently,
Wang and co-workers have reported DC-81-indole and DC-81-
enediyne conjugates as potential anticancer agents and a correla-
tion between antitumor activity and apoptosis has been well
explained [32,33]. In the last few years, we have not only been
involved in the preparation of the PBD ring system by developing
new synthetic strategies [34,35] but also in the design of struc-
turally modified PBDs and their conjugates [36e40]. In continua-
tion of these efforts, we herein describe the synthesis and biological
evaluation of chalcone-PBD conjugates linked through the 1,2,3-
triazole ring side-armed with alkane spacers. In view of the
promising anticancer activity observed by these new PBD conju-
gates, it was considered of interest to examine their role by cell
proliferation and apoptosis by using the breast carcinoma cell line
MCF-7. Detailed biological assays revealed that these conjugates
Scheme 2. Reagents and conditions: (i) K2CO3, propargyl bromide, dry DMF, rt, 12 h;
(ii) KOH/MeOH, rt, 12 h; (iii) CuSO4$5H2O (1 mol%), sodium ascorbate (5 mol%),
t-BuOH/H2O (1:1), rt, 12 h; (iv) SnCl2$2H2O, MeOH, reflux, 2 h; (v) HgCl2eCaCO3,
CH3CNeH2O (4:1), rt, 12 h.

3822
A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3820e3831
Table 1
GI50 valuesa (in
mM) for compounds 4aei and 5a in selected human cancer cell lines.
Compound
Zr-75-1b
MCF
7b
HOP
62c
Guravc
A549c
A2780d
DWDe
KBe
Colo-205f
Pc3g
SiHah
4a
4b
4c
4d
4e
4h
4i
5a
2.20
0.15
0.17
0.17
0.16
0.19
2.02
NA
2.40
0.14
2.10
0.16
0.16
0.16
0.18
2.35
0.17
0.17
2.92
0.16
2.56
0.17
0.19
2.50
2.28
NA
2.31
0.12
0.16
0.13
0.13
1.66
0.19
2.51
0.17
0.16
2.33
<0.16
0.17
0.15
<0.15
0.19
2.09
2.78
7.25
0.16
0.17
0.10
0.14
0.12
0.11
0.13
0.15
0.15
0.16
0.14
NA
2.19
0.17
0.18
2.03
0.16
2.66
NA
NA
0.17
0.17
0.15
NA
0.14
NA
0.11
<0.1
0.10
0.11
0.14
0.11
2.48
0.13
2.04
0.16
0.15
0.16
0.19
2.23
1.81
0.20
NA
<0.13
2.30
0.12
<0.16
<0.17
2.30
2.70
0.10
1.49
0.14
2.71
0.18
2.52
NA
NA
NA
0.17
0.17
ADRi
DC-81
1.79
2.37
0.14
0.15
a
50% growth inhibition and the values are mean of three determinations and are reported as mean ꢁ standard error of the mean.
b
c
d
e
f
breast cancer.
lung cancer.
ovarian cancer.
oral cancer.
colon cancer.
prostate cancer.
cervix cancer.
ADR (adriamycin).
g
h
i
show G1 cell cycle arrest and effect cell cycle regulatory proteins
a CuSO4$5H2O catalyst (1 mol%) with sodium ascorbate (5 mol%) to
afford nitro intermediates 12aei. Later, the nitro functionality was
reduced with SnCl2$2H2O followed by deprotective-cyclizationwith
HgCl2-CaCO3 to afford the target compounds 4aei as depicted in
Scheme 1.
such as Cyclin D1 and Cdk4. This study also revealed that the
inhibitory activity of these conjugates on NF-kB (p65) protein there
by controlling active cell proliferation in MCF-7 cells.
Similarly, in order to explore their diversity at C-C2-position of
the PBD ring system, the chalcone linked PBD (5a,b) conjugates are
attached through a 1,2,3-triazole obtained from compounds 15a,b,
which were prepared from the previously reported method [40].
Etherification of the vanillin (6) with propargyl bromide followed
by aldol condensation using hydroxy acetopheonone (8a) affords
the alkyne precursor 14. The two key intermediates 14 and 15a,b
were subjected to ‘click’ chemistry to produce 16a,b. Finally, the
nitro reduction followed by diethanethiol deprotective-cyclization
provide the target compounds (5a,b) as shown in Scheme 2.
2. Chemistry
The synthesis of azido-chalcone intermediates 9aei was carried
out by employing commercially available substituted-aceto-
phenones (8aec) and vanillin (6) [41] as starting materials. Ether-
ification of vanillin (6) with dibromoalkanes followed by azidation
[42] of bromine using NaN3 produces the corresponding azido
precursors 7aec. Then, the aldol condensation reaction was per-
formed by reacting 7aec with a variety of acetophenones (8aec)
using KOH as the base to afford the azido-chalcone key intermedi-
ates 9aei. The synthesis of A-C8 chalcone linked PBD conjugates
(4aei and 5a,b) that are attached through a triazole containing
alkane spacer was carried out from 4-hydroxy nitrothioacetal 10,
which was synthesized by the methods reported in the previous
studies [40,42,43]. The etherification of compound 10 with prop-
argyl bromide in presence of K2CO3 provides one of the key alkyne
intermediate 11 [42]. At this stage the ‘click’ chemistry protocol is
introduced between alkyne 11 and azides 9aei by employing
3. Results and discussion
3.1. Evaluation of biological activity
3.1.1. Anticancer activity
These PBD conjugates 4aei and 5a,b were evaluated for their
anticancer activity in selected human cancer cell lines of lung, breast,
Fig. 2. Cell viability observed after the treatment with 1,2,3-triazolo chalcone-PBD compounds at 4
mM concentration by MTT assay. MCF-7 breast carcinoma cells were treated with
various compounds 1, 2, 4a, 4b, 4c, 4d, 4h, 4i, 5a and 5b at 4 M concentration as indicated for 24 h in 96 well plates at 10,000 cells per well. Compound 1 and 2 were used as
m
starting materials in this experiment. Control indicates the untreated cells. Each experiment was repeated three times and Standard Deviations were derived from three inde-
pendent experiments.

A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3820e3831
3823
Table 2
respectively in the cell lines employed. Interestingly, all the PBD
conjugates showed promising anticancer activity, particularly
against A2780, Gurav, MCF-7, Colo-205 and A549 cell lines, and some
of the compounds like 4a, 4b, 4d, 4h and 4i have shown significant
anticancer activity as illustrated in Table 1
FACS analysis of cell cycle distribution of MCF-7 cells after treatment with
compounds (1, 2, 4a, 4b, 4c, 4d, 4h, 4i, 5a and 5b) at 10
Compound 1 and 2 were used as starting materials in this experiment.
m
Ma concentration for 24 h.
Compound
G0
1.27 ꢁ 0.08
G1
S
G2/M
Control
1
2
4a
4b
4c
4d
4h
4i
47.64 ꢁ 1.49
61.81 ꢁ 1.71
51.61 ꢁ 2.21
64.08 ꢁ 1.04
77.47 ꢁ 0.76
46.39 ꢁ 0.87
72.51 ꢁ 1.32
50.63 ꢁ 1.18
60.56 ꢁ 1.32
50.9 ꢁ 1.82
54.61 ꢁ 0.94
12.92 ꢁ 1
38.15 ꢁ 1.068
20.89 ꢁ 1.05
31.5 ꢁ 2.14
14.32 ꢁ 0.5
7.07 ꢁ 0.88
35.88 ꢁ 1.37
7.33 ꢁ 0.75
31.07 ꢁ 1
MTT assay was also carried out to identify the cytotoxic effect of
some of these chalcone linked PBD conjugates (4aed, 4h, 4i, 5a,
7.62 ꢁ 0.32
10.15 ꢁ 0.29
12.06 ꢁ 0.35
10.79 ꢁ 0.42
7.81 ꢁ 0.94
13.67 ꢁ 0.63
10.51 ꢁ 0.83
10.75 ꢁ 0.3
5.19 ꢁ 0.3
9.67 ꢁ 0.62
6.55 ꢁ 0.5
9.52 ꢁ 1.04
4.99 ꢁ 0.21
9.89 ꢁ 0.43
6.48 ꢁ 0.66
7.74 ꢁ 0.38
7.28 ꢁ 0.26
8.71 ꢁ 0.66
6.65 ꢁ 0.35
and 5b) in MCF-7 cells at 4
mM concentration. The response of cells
at 2 M was not that effective in producing cytotoxic activity.
m
Moreover, the anticancer activity of DC-81 (1) and compound 2,
which are the starting materials of this series was also examined to
substantiate the activity of these new chalcone-PBD conjugates. It is
interesting to observe that some of the compounds (4a, 4b and 4d)
possess improved cytotoxic effect compared to the naturally
occurring 1 and 2 as shown in Fig. 2.
22.06 ꢁ 0.07
35.19 ꢁ 1.05
32.06 ꢁ 2.53
5a
5b
5.21 ꢁ 0.37
a
Values represent the mean ꢁ SEM of three determinations.
3.1.2. Effect of conjugates on cell cycle in MCF-7 cell line
oral, colon, prostate, ovarian and cervix by using sulforhodamine B
(SRB) method. The compounds that exhibit GI50 ꢂ 10ꢃ5 M have been
considered to be active on the respective cell lines and the results are
illustrated in Table 1. All the compounds (4aei and 5a,b) showed
significant anticancer activity with GI50 values ranging from <0.1 to
To investigate the mechanism underlying the anti-proliferative
nature, the cell cycle distribution of MCF-7 cells was analyzed by
flow cytometry. MCF-7 cells were treated with compounds 1, 2,
4aed, 4h, 4i, 5a and 5b at 4 mM concentration. The majority of
control cells treated with DMSO showed 50% of cells in G0/G1
phase. The starting materials such as 1 and 2 have shown 71.10%
and 64.11% cells in G0/G1 phase. Compounds 4aed, 4h, 4i, 5a and
2.92
mM, while the positive controls, adriamycin and DC-81 (1)
demonstrated the GI50 in the range of 0.1e7.25
m
M and 0.1e2.37 M,
m
Fig. 3. a. Histogram of cell cycle distribution of MCF-7 cells after treatment with compounds (1, 2, 4a, 4b, 4c, 4d, 4h, 4i, 5a and 5b) at 4
were used as starting materials in this experiment. Each experiment was repeated three times and Standard Deviations were derived from three independent experiments.
b. Histogram depicting the percentage of apoptotic cells of MCF-7 cells after treatment with compounds (1, 2, 4a, 4b, 4c, 4d, 4h, 4i, 5a and 5b) at 4 M concentration for 24 h.
mM concentration for 24 h. Compound 1 and 2
m

3824
A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3820e3831
Fig. 4. a. Effect compounds on the expression of Cell cycle regulated (Cyclin D1 and
Cdk4) proteins. MCF-7 Cells were treated with 4 M concentration of compounds
(1, 4a, 4b, 4d, and 4i) for 24 h. The cell lysates were collected and observed for
expression levels of proteins such as Cyclin D1 and Cdk4 using specific antibodies.
Fig. 6. a. Effect of compounds on the expression Bcl2 protein. MCF-7 cells treated with
M concentration of compounds 1, 4a, 4b, 4d, and 4i for 24 h. The cell lysates were
collected and observed for expression level of Bcl2 protein using Bcl2 ELISA kit (Bender
Med Systems, ALEXIS Biochemicals Company). b. Effect of compounds on the expres-
m
4
m
b-actin was used as loading control. C: control (untreated). b. Effect compounds on the
sion Bcl-xL protein. MCF-7 cells were treated with 4
mM concentration of compounds
Cyclin D1 promoter (ꢃ1745 Luc) activity. MCF-7 Cells were transfected with Cyclin D1
(1, 4a, 4b, 4d and 4i) for 24 h. The cell lysates were collected and observed for
expression level of Bcl-xL protein using Bcl-xL specific antibody, b-actin was used as
a loading control. C: control (untreated).
promoter (Cy) for 24 h followed by compound treatments (1, 4a, 4b, 4d, and 4i) at final
concentration of 4
mM for further 24 h. Here Cy represents Cyclin D1 promoter
transfection. C: represents the value of promoter activity without any transfection and
can be considered as negative control. The C þ Cy, represents the control untreated
cells transfected with Cyclin D1 promoter (without any treatment) and can be
considered as positive control. X þ Cy: represents Cyclin D1 promoter transfection
followed by compound treatments. X may be 1, 4a, 4b, 4d, and 4i.
analysis about apoptotic cell death (G0 phase) is comparable to the
in vitro cytotoxicity data (Fig. 3b). It is observed from the data of cell
cycle analysis that PBD conjugates show G1 arrest and whereas
chalcone based compounds are known to cause G2/M cell cycle
arrest. However, there are evidences that chalcones (HTMC-chal-
cone) cause G1 arrest in A549 adenocarcinoma cells [44] and
accumulation of cells in sub G1 phase of cell cycle in MDA-MB-231
breast cancer cells [45]. Whereas in the present study G1 cell cycle
arrest is clearly seen in case of PBD-chalcone conjugates.
5b showed 76.87%, 89.10%, 56.23%, 87.70%, 61.38%, 60.18%, 55.24%,
and 59.79% of cells in G0/G1 phase respectively. It is observed from
this data that 4b and 4d was highly effective in causing G0/G1
arrest (Table 2 and Fig. 3a). The data obtained from the FACS
3.1.3. Effect of compounds on cell cycle regulator proteins at G1
phase of cell cycle
Cell cycle regulatory proteins such as Cyclin D1 and its Cyclin
dependent kinase component (Cdk4) [46] are known to play a vital
role in G1 to S-phase transition [47,48]. Therefore, we examined the
effect of some of the promising compounds (results obtained from
FACS analysis) on these proteins. Thus MCF-7 cells were treated
with compounds 1, 4a, 4b, 4d, and 4i at 4 mM concentration. We
observed a significant down regulation of Cyclin D1 for these
conjugates revealing the involvement of G1 cell cycle proteins in
this event. Similarly, Cdk4, an important protein associated with
Cyclin D1 was down-regulated as illustrated in Fig. 4a. Further, we
focused our efforts on the effect of these compounds on the Cyclin
D1 promoter (ꢃ1745 Luc) activity. Interestingly we have observed
down regulation of promoter activity (Fig. 4b) thus revealing the
possible action of these compounds on Cyclin D1, the major regu-
lator of cell proliferation in breast cancer [49].
3.1.4. Effect of compounds on NF-kB protein
Fig. 5. Effect of compounds on NF-kB proteins. MCF-7 Cells were transfected with NF-
kB SEAP Reporter by lipofectamine 2000. After 24 h the cells were treated with (1, 4a,
4b, 4d, and 4i) 4 mM concentration of compounds for 24 h. The cell lysates have been
collected and studied the expression levels of protein NF-kB. C þ NF: the Control cells
transfected with NF-kB SEAP Reporter construct without any compound treatment.
Y þ NF: are the compound treatments after transfection with NF-kB SEAP Reporter. Y
may be 1, 4a, 4b, 4d, 4i.
It is well known that NF-kB protein regulates Cyclin D1 [50]. As
a reduction in the level of cyclin D1 protein and its promoter
activity is observed in the compound treated cells, the possibility of
involvement of NF-kB protein in this event was anticipated. Inter-
estingly upon ectopic expression (transfection) of NF-kB SEAP

A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3820e3831
3825
reporter construct in MCF-7 cells followed by compound treat-
ments resulted in significant reduction in NF-kB activity and was
highly down-regulated in cells treated by 4d (Fig. 5). Infact similar
results were obtained in the literature for the studies against
3-hydroxy-4,30,40,50-tetramethoxy chalcones [51].
voltage 3.98 kV and ESI mode positive ion trap detector. High-
resolution mass spectra (HRMS) were recorded on QSTAR XL
Hybrid MS/MS mass spectrometer. Melting points were determined
with an Electro thermal melting point apparatus, and are
uncorrected.
3.1.5. Effect of chalcone-1,2,3-triazoleepyrrolobenzodiazepine
conjugates on anti- apoptotic proteins
5.1.1. General procedures for synthesis of 4-(azidoalkyloxy)-3-
methoxybenzaldehyde (7aec)
Bcl-xL and Bcl2 are the proteins that play an important role for
causing rapid cell proliferation in breast cancer [52e54]. Thus,
MCF-7 breast cancer cells were treated with compounds 1, 4a, 4b,
To a stirred solution of compound 6aec (5 mmol) in DMF
(20 mL), anhydrous K2CO3 (15 mmol) and dibromo alkane spacers
(7.5 mmol) were added and the mixture was refluxed for 12 h.
After completion of the reaction, K2CO3 was removed by filtration
and solvent was evaporated under reduced pressure to give the
crude products. This was further purified by column chromatog-
raphy using ethyl acetateehexane (10%) to afford the bromo
compounds as white solids. Later, these compounds (2.5 mmol)
dissolved in DMF (30 mL), NaN3 (3 mmol) was added and the
mixture was heated to 80 ꢀC for 6 h. The residue was extracted
with ethyl acetate in ice-water and organic layer was dried over
anhydrous Na2SO4 and the solvent was removed under vacuum to
afford the crude products. These were further purified by column
chromatography using (15% EtOAcehexane) as eluant to afford the
pure compounds 7aec.
4d and 4i at 4 mM concentration and Western blot analysis against
Bcl-XL and ELISA against Bcl2 was carried out. As expected a down
regulation of these proteins resulting in apoptotic event, and this
was more pronounced in case of 4d. The possible action of these
compounds on cell proliferation is illustrated in Fig. 6a and b.
4. Conclusion
In conclusion, a series of new A-C8/C-C2-chalconeelinked pyr-
rolo[2,1-c] [1,4]benzodiazepine conjugates connected through
a 1,2,3-triazole side-armed with alkane spacers have been designed
and synthesized by employing ‘click’ chemistry protocol. These
conjugates have exhibited promising anticancer activity against
selected human cancer cell lines. The effect of these chalcone-PBD
conjugates on MCF-7 cells has been studied extensively. It is
observed from the MTT proliferation assay that the conjugate 4d is
more effective as an anti-proliferative agent than the naturally
5.1.1.1. 4-(3-Azidopropyloxy)-3-methoxybenzaldehyde
88%; Yellow solid; mp:137e140 ꢀC; 1H NMR (CDCl3, 300 MHz):
9.87 (s, 1H, aldehydeeH), 7.40e7.38 (m, 1H, eArH), 7.33 (s, 1H,
(7a). Yield
d
eArH), 6.78 (d, J ¼ 8.30 Hz, 1H, eArH), 4.11 (t, 2H, eOCH2e), 3.92 (s,
3H, eOCH3), 3.50 (t, 2H, eNCH2e), 2.16e2.04 (m, 2H, eCH2e): MS
(ESI); m/z 236 (M þ H)þ.
occurring (DC-81) at 4 mM concentration. The FACS analysis showed
more population in G0/G1 phase indicating that all these PBD
conjugates have cell cycle regulatory properties. Involvement of G1
cell cycle proteins such as cyclin D1 and Cdk 4 has also been
investigated. Detailed studies have revealed that these conjugates
inhibit NF-kB protein as well as transcriptional activation of the NF-
kB dependent genes such as (Cyclin D1 and Bcl-XL) that help in
active cell proliferation [55]. Interestingly, these conjugates func-
tion as inhibitors of Cyclin D1 as well as NF-kB protein and such
inhibitors are known to play a crucial role in breast cancer. The
results obtained in this study has shown the functional importance
of NF-kB and its dependent genes such as (Cyclin D1 and Bcl-XL)
that help in cell cycle and control of cell proliferation [56,57]. The
effect of these new conjugates on cell cycle proteins such as inhi-
bition on Cyclin D1, Cdk4, NF-kB, Bcl-XL and Bcl2 protein observed
and could be considered as promising leads against breast cancer.
Hence from this data it can be concluded that linking of a chalcone
moiety with PBD scaffold through a 1,2,3-triazole ring side-armed
with alkane spacers has not only enhanced the anticancer activity
but has provided an insight in the design of such conjugates.
5.1.1.2. 4-(4-Azidobutyloxy)-3-methoxybenzaldehyde
90%; Yellow solid; mp 144e148 ꢀC; 1H NMR (CDCl3, 300 MHz):
9.81 (s, 1H, aldehydeeH), 7.39e7.36 (m, 2H, eArH), 6.88 (d,
(7b). Yield
d
J ¼ 8.30 Hz, 1H, eArH), 4.10 (t, 2H, eOCH2e), 3.91 (s, 3H, eOCH3),
3.40 (t, 2H, eNCH2e), 2.02e1.77 (m, 4H, eCH2e); MS (ESI): m/z
250 (M þ H)þ.
5.1.1.3. 4-(5-Azidopentyloxy)-3-methoxybenzaldehyde
83.2%; Yellow solid; mp 142e146 ꢀC; 1H NMR (CDCl3, 300 MHz):
9.85 (s, 1H, aldehydeeH), 7.39e7.37 (m, 1H, eArH), 7.36 (s, 1H,
(7c). Yield
d
eArH), 6.87 (d, J ¼ 8.30 Hz,1H, eArH), 4.04 (t, 2H, eOCH2e), 3.90 (s,
3H, eOCH3), 3.39 (t, 2H, eNCH2e), 1.88e1.27 (m, 6H, eCH2e); MS
(ESI): m/z 264 (M þ H)þ.
5.1.2. General procedures for synthesis of (E)-3-(4-(azidoalkyloxy)-
3-methoxyphenyl)-1-(substitute phenyl)prop-2-en-1-one(9aei)
Potassium hydroxide (1 mL of a 50% w/v aqueous solution) was
added to a stirred solution of azido-benzaldehyde (7aec, 2 mmol)
and substituted acetophenone (8aec, 2 mmol) in methanol (30 mL).
The mixture was stirred overnight at room temperature. After
completion of reaction, the mixture was acidified to pH 1 with 1 N
aq. solution of HCl and extracted with dichloromethane (2 ꢄ 50 mL).
The combined organic layers were dried over anhydrous Na2SO4,
evaporated in vacuo and purified by column chromatography (15%
ethyl acetateehexane) to afford the pure chalcones.
5. Experimental section
5.1. General
All chemicals and reagents were obtained from Aldrich (Sigmae
`Aldrich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey
Company, Ward Hill, MA, USA) or Spectrochem Pvt. Ltd (Mumbai,
India) and were used without further purification. Reactions were
monitored by TLC, performed on silica gel glass plates containing
60 GF-254, and visualization on TLC was achieved by UV light or
iodine indicator. Column chromatography was performed with
Merck 60e120 mesh silica gel. 1H spectra were recorded on Gemini
Varian-VXR-unity (200 MHz) or Bruker UXNMR/XWIN-NMR
5.1.2.1. (E)-3-(4-(3-Azidopropyloxy)-3-methoxyphenyl)-1-(2-
hydroxyphenyl)prop-2-en-1-one(9a). Yield 90%; Yellow solid; mp
180e182 ꢀC; 1H NMR (CDCl3, 300 MHz):
d 12.92 (s, 1H, eArOH),
7.96e7.92 (m, 1H, eArH), 7.91 (d, 1H, J ¼ 15.62 Hz, dbeH), 7.54e7.45
(m, 1H, eArH), 7.50 (d, 1H, J ¼ 15.62 Hz, dbeH), 7.28e7.17 (m, 2H,
eArH), 7.05e6.90 (m, 3H, eArH), 4.16 (t, 2H, eOCH2e), 3.95 (s, 3H,
eOCH3), 3.57 (t, 2H, eNCH2e), 2.20e2.04 (m, 2H, eCH2e); MS
(ESI): m/z 354 (M þ H)þ.
(300 MHz) instruments. Chemical shifts (d) are reported in ppm
downfield from internal TMS standard. ESI spectra were recorded
on Micro mass, Quattro LC using ESI þ software with capillary

3826
A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3820e3831
5.1.2.2. (E)-3-(4-(4-Azidobutyloxy)-3-methoxyphenyl)-1-(2-hydroxy-
3.94 (s, 3H, eOCH3), 3.40 (t, 2H, eNCH2e), 2.00e1.79 (m, 4H,
eCH2e); MS (ESI): m/z 402 (M þ H)þ.
phenyl)prop-2-en-1-one(9b). Yield 95%; Yellow solid; mp
174e176 ꢀC; 1H NMR (CDCl3, 300 MHz):
d 12.81 (s, 1H, eArOH),
7.95e7.90 (m, 1H, eArH), 7.89 (d, 1H, J ¼ 15.62 Hz, dbeH), 7.55e7.50
(m, 1H, eArH), 7.48 (d, 1H, J ¼ 15.62 Hz, dbeH), 7.21e7.16 (m, 1H,
eArH), 7.12 (s, 1H, eArH), 6.98 (d, J ¼ 8.49 Hz, 1H, eArH), 6.92e6.88
(m, 1H, eArH), 6.85 (d, J ¼ 8.49 Hz,1H, eArH), 4.07 (t, 2H, eOCH2e),
3.92 (s, 3H, eOCH3), 3.40 (t, 2H, eNCH2e), 2.00e1.78 (m, 4H,
eCH2e); MS (ESI): m/z 368 (M þ H)þ.
5.1.2.9. (E)-3-(4-(5-Azidopentyloxy)-3-methoxyphenyl)-1-(5-chloro-
2-hydroxyphenyl) prop-2-en-1-one (9i). Yield 95%; Yellow solid; mp
148e150 ꢀC; 1H NMR (CDCl3, 400 MHz):
d 12.84 (s,1H, eArOH), 7.87
(d, J ¼ 15.67 Hz, 1H, dbeH), 7.86 (s, 1H, eArH), 7.45e7.41 (m, 1H,
eArH), 7.41 (d, J ¼ 15.29 Hz, 1H, dbeH), 7.28e7.24 (m, 1H, eArH),
7.17 (s, 1H, eArH), 6.99 (d, J ¼ 8.87 Hz, 1H, eArH), 6.90 (d,
J ¼ 8.30 Hz, 1H, eArH), 4.09 (t, 2H, eOCH2e), 3.96 (s, 3H, eOCH3),
3.32 (t, 2H, eNCH2e), 1.97e1.87 (m, 2H, eCH2e), 1.77e1.56 (m, 4H,
eCH2e); MS (ESI): m/z 416 (M þ H)þ.
5.1.2.3. (E)-3-(4-(5-Azidopentyloxy)-3-methoxyphenyl)-1-(2-hydroxy-
phenyl)prop-2-en-1-one (9c). Yield 95%; Yellow solid; mp
182e184 ꢀC; 1H NMR (CDCl3, 200 MHz):
d 12.81 (s, 1H, eArOH),
7.91e7.87 (m, 1H, eArH), 7.84 (d, 1H, J ¼ 15.89 Hz, dbeH), 7.49e7.40
(m, 1H, eArH), 7.44 (d, 1H, J ¼ 15.89 Hz, dbeH), 7.23e7.17 (m, 1H,
eArH), 7.12 (s, 1H, eArH), 6.98 (d, J ¼ 8.32 Hz, 1H, eArH), 6.92e6.88
(m, 1H, eArH), 6.81 (m, 1H, eArH), 4.04 (t, 2H, eOCH2e), 3.93 (s, 3H,
eOCH3), 3.31 (t, 2H, eNCH2e), 2.04e1.49 (m, 6H, eCH2e); MS (ESI):
m/z 382 (M þ H)þ.
5.1.3. 3-Methoxy-4-(prop-2-ynyloxy)benzaldehyde (13)
To a solution of compound 6 (760 mg, 5 mmol) in DMF (20 mL),
anhydrous K2CO3 (2.07 g, 15 mmol) and propargyl bromide
(0.54 mL, 6 mmol) were added and the mixture at room temper-
ature for 12 h. After completion of the reaction, potassium
carbonate was removed by filtration and solvent was evaporated
under reduced pressure to get the crude product. This was further
purified by column chromatography using ethyl acetateehexane
(10%) to afford the compound 13 as a yellow solid; Yield 93%; mp
5.1.2.4. (E)-3-(4-(3-Azidopropoxy)-3-methoxyphenyl)-1-(4-hydroxy-
phenyl)prop-2-en-1-one (9d). Yield 95%; Yellow solid; mp
174e176 ꢀC; 1H NMR (CDCl3, 300 MHz):
d
9.04 (s, 1H, eArH), 7.84
105e108 ꢀC; 1H NMR (CDCl3, 300 MHz):
d 9.84 (s, 1H, eArH),
(d, J ¼ 8.30 Hz, 2H, eArH), 7.68 (d, J ¼ 15.10 Hz, 1H, dbeH), 7.39
(d, J ¼ 15.10 Hz, 1H, dbeH), 7.15e7.09 (m, 2H, eArH), 6.95 (d,
J ¼ 8.30 Hz, 2H, eArH), 6.85e6.90 (m, 1H, eArH), 4.14e4.07 (m,
2H, eOCH2e), 3.86 (s, 3H, eOCH2e), 3.59 (m, 2H, eNCH2e),
2.13e2.00 (m, 2H, eCH2e); MS (ESI): m/z 354 (M þ H)þ.
7.42e7.39 (m, 2H, eArH), 7.09 (d, J ¼ 7.55 Hz,1H, eArH), 4.82 (d, 2H,
J ¼ 2.26 Hz, eOCH2e), 3.94 (s, 3H, eOCH3), 2.50 (t, 1H, J ¼ 2.26 Hz,
alkyneeH); MS (ESI): m/z 191 (M þ H)þ.
5.1.3.1. (E)-1-(2-Hydroxyphenyl)-3-(3-methoxy-4-(prop-2-ynyloxy)
phenyl)prop-2-en-1-one (14). The compound 14 was prepared
following the method described for the preparation of the
5.1.2.5. (E)-3-(4-(4-Azidobutyloxy)-3-methoxyphenyl)-1-(4-hydroxy-
phenyl) prop-2-en-1-one (9e). Yield 92.5%; Yellow solid; mp
compound 9a, employing 13 (956 mg, 5 mmol) and 2-
170e172 ꢀC; 1H NMR (CDCl3, 300 MHz):
d
8.51 (s, 1H, eArOH), 7.97
hydroxyacetophenone 8a (0.52 ml, 5 mmol), and the crude
product was purified by column chromatography (30% ethyl ace-
tateehexane) to afford the compound 14 as a yellow solid; Yield
(d, J ¼ 8.30 Hz, 2H, eArH), 7.70 (d, J ¼ 15.10 Hz, 1H, dbeH), 7.49 (d,
J ¼ 15.10 Hz,1H, dbeH), 7.39e7.34 (m,1H, eArH), 7.16 (d, J ¼ 8.30 Hz,
1H, eArH), 7.11 (s,1H, eArH), 6.89 (d, J ¼ 8.30 Hz,1H, eArH), 6.84 (d,
J ¼ 8.30 Hz, 1H, eArH), 4.12e4.05 (m, 2H, eOCH2e), 3.91 (s, 3H,
eOCH3), 3.49 (t, 2H, eNCH2e), 2.14e1.99 (m, 4H, eCH2e); MS (ESI):
m/z 368 (M þ H)þ.
88%; mp 145e147 ꢀC; 1H NMR (CDCl3, 300 MHz):
d 12.77 (s, 1H,
eArOH), 7.92e7.82 (m, 1H, eArH), 7.56e7.42 (m, 1H, eArH), 7.50 (d,
J ¼ 15.10 Hz, 1H, edbH), 7.29e7.14 (m, 1H, eArH), 7.12 (s, 1H, eArH),
7.04e6.86 (m, 3H, eArH), 6.89 (d, J ¼ 15.10 Hz, 1H, edbH), 4.80 (s,
2H, eOCH2e), 3.94 (s, 3H, eOCH3), 2.48 (s, 1H, alkyneeH); MS (ESI):
m/z 309 (M þ H)þ.
5.1.2.6. (E)-3-(4-(5-Azidopentyloxy)-3-methoxyphenyl)-1-(4-hydroxy-
phenyl)prop-2-en-1-one (9f). Yield 94%; Yellow solid; mp
176e178 ꢀC; 1H NMR (CDCl3, 300 MHz):
d
8.93 (s, 1H, eArOH), 7.91
5.1.4. (2S)-N-[4-(Prop-2-ynyloxy)-5-methoxy-2-nitrobenzoyl]
pyrrolidine-2-carbox-aldehyde diethylthioacetal (11)
(d, J ¼ 8.30 Hz, 2H, eArH), 7.69 (d, J ¼ 15.48 Hz, 1H, dbeH), 7.32
(d, J ¼ 15.48 Hz, 1H, dbeH), 7.12 (d, J ¼ 8.49 Hz, 1H, eArH), 7.10 (s,
1H, eArH), 6.85 (d, J ¼ 8.30 Hz, 2H, eArH), 6.80 (d, J ¼ 8.49 Hz, 1H,
eArH), 4.00 (t, 2H, eOCH2e), 3.86 (s, 3H, eOCH3), 3.24 (t, 2H,
eNCH2e), 1.89e1.77 (m, 2H, eCH2e), 1.67e1.45 (m, 4H, eCH2e); MS
(ESI): m/z 382 (M þ H)þ.
To a solution of compound 10 (400 mg, 1 mmol) in DMF (15 mL)
was added, anhydrous K2CO3 (553 mg, 4 mmol), propargyl bromide
(106 mL,1.2 mmol) and the mixturewas stirred at room temperature
for 12 h. The reaction was monitored by TLC using EtOAcehexane
(1:1). After completion of the reaction as indicated by TLC, diluted in
water and extracted with ethyl acetate. The combined organic
phases were dried over Na2SO4 and evaporated under vacuum. The
residue, thus obtained was purified by column chromatography
using ethyl acetate and hexane (2:3) to afford compound 11 as
5.1.2.7. (E)-3-(4-(3-Azidopropoxy)-3-methoxyphenyl)-1-(5-chloro-2-
hydroxyphenyl) prop-2-en-1-one (9g). Yield 92.5%; yellow solid; mp
146e148 ꢀC;1H NMR (CDCl3, 300 MHz):
d 12.74 (s,1H, eArOH), 7.88(d,
J ¼ 15.10 Hz, 1H, dbeH), 7.84e7.83 (m, 1H, eArH), 7.44e7.39 (m, 1H,
eArH), 7.39 (d, J¼ 15.10Hz,1H, dbeH), 7.27e7.18 (m, 1H, eArH), 7.13 (s,
1H, eArH), 6.95 (d, J ¼ 9.06 Hz, 1H, eArH), 6.90 (d, J ¼ 8.30 Hz, 1H,
eArH), 4.14 (t, 2H, eOCH2e), 3.94 (s, 3H, eOCH3), 3.58 (t, 2H,
eNCH2e), 2.16e2.08 (m, 2H, eCH2e); MS (ESI): m/z 388 (M þ H)þ.
yellow liquid; Yield 93%; 1H NMR (CDCl3, 300 Mhz):
d 7.84 (s, 1H,
eArH), 6.81 (s, 1H, eArH), 4.82 (s, 2H, eOCH2e), 4.74e4.62 (m, 1H,
eSCHe), 4.17e4.04 (m, 1H, eNCHe), 3.96 (s, 3H, eOCH3), 3.33e3.16
(m, 2H, eNCH2e), 2.88e2.64 (m, 4H, eSCH2e), 2.42e2.02 (m, 2H,
eCH2e), 2.01 (s, 1H, alkyneeH), 2.00e1.69 (m 2H, eCH2e),
1.40e1.29 (m, 6H, eCH3); MS(ESI): m/z 439 (M þ H)þ.
5.1.2.8. (E)-3-(4-(4-Azidobutyloxy)-3-methoxyphenyl)-1-(5-chloro-2-
hydroxyphenyl) prop-2-en-1-one (9h). Yield 93%; Yellow solid; mp
5.1.5. General procedure of synthesis (2S)-N-4-{(1-(3-(4-((E)-3-
(phenyl)-3-oxoprop-1-enyl)-2-methoxyphenoxy)alkyl)-1H-1,2,
3-triazol-4-yl}methoxy)-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-
carboxaldehyde diethylthioacetal (12aei)
To a stirred solution of compound 11 (438 mg, 1 mmol) and (E)-
3-(4-(azido alkyloxy)-3-methoxyphenyl)-1-(substituted phenyl)
144e146 ꢀC; 1H NMR (CDCl3, 400 MHz):
d 12.75 (s, 1H, eArOH),
7.89e7.85 (m, 1H, eArH), 7.83 (d, J ¼ 15.10 Hz, 1H, dbeH),
7.42e7.36 (m, 1H, eArH), 7.36 (d, J ¼ 15.10 Hz, 1H, dbeH),
7.25e7.23 (m, 1H, eArH), 7.13 (s, 1H, eArH), 6.94 (d, J ¼ 8.79 Hz,
1H, eArH), 6.84 (d, J ¼ 7.91 Hz, 1H, eArH), 4.08 (t, 2H, eOCH2e),

A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3820e3831
3827
prop-2-en-1-one 9aei (1.2 mmol) in t-BuOH:H2O (1:1) (50 mL),
CuSO4$5H2O catalyst (1 mol%) (1.36 g, 3 mmol) and sodium
ascorbate (5 mol%) were added and this mixture was stirred for
12 h at room temperature. After completion of the reaction,
quenched with NH3 solution and n-butanol was removed under
reduced pressure. Chloroform and water added to the above
residue and stirred for another 30 min followed by celite filtration,
extracted with chloroform, and organic solvent was evaporated
under reduced pressure to afford the crude products. These were
further purified by column chromatography using ethyl aceta-
teehexane (90%) provides the pure compounds 12aei.
6.84 (s, 1H, ArH), 5.35 (s, 2H, eOCH2e), 4.89 (d, J ¼ 3.77 Hz, 1H,
eSCHe), 4.75e4.67 (m, 1H, eNCHe), 4.55 (t, 2H, eOCH2e),
4.17e4.10 (m, 2H, eNCH2e), 3.94 (s, 3H, eOCH3), 3.93 (s, 3H,
eOCH3), 3.34e3.18 (m, 2H, eNCH2e), 2.87e2.66 (m, 4H, eSCH2e),
2.38e1.74 (m, 6H, eCH2e), 1.39e1.29 (m, 6H, eCH3); MS (ESI): m/z
792 (M þ H)þ.
5.1.5.5. (2S)-N-{4-{1-(4-(4-((E)-3-(4-Hydroxyphenyl)-3-oxoprop-
1-enyl)-2-methoxy phenoxy)butyl)-1H-1,2,3-triazol-4-yl)methoxy)-
5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylth-
ioacetal (12e). Yield 80%; yellow solid; mp 123e125 ꢀC; 1H NMR
(CDCl3, 300 MHz):
d
9.81 (s, 1H, eArOH), 7.94 (d, J ¼ 8.30 Hz, 2H,
5.1.5.1. (2S)-N-4-{(1-(3-(4-((E)-3-(2-hydroxyphenyl)-3-oxoprop-1-
enyl)-2-methoxy phenoxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy}-5-
methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal
(12a). Yield 80%; Yellow solid; mp 125e127 ꢀC; 1H NMR (CDCl3,
eArH), 7.91 (s, 1H, eArH), 7.85 (s, 1H, triazoleeH), 7.62 (d, 1H,
J ¼ 15.10 Hz, dbeH), 7.47 (d, J ¼ 15.10 Hz, 1H, dbeH), 7.15e7.07 (m,
1H, eArH), 6.95 (d, J ¼ 8.30 Hz, 2H, eArH), 6.93e6.85 (m, 3H,
eArH), 5.34 (s, 2H, eOCH2e), 4.87 (d, J ¼ 3.77 Hz, 1H, eSCHe),
4.73e4.67 (m, 1H, eNCHe), 4.54 (t, 2H, eOCH2e), 4.15e4.10 (m, 2H,
eNCH2e), 3.93 (s, 3H, eOCH3), 3.92 (s, 3H, eOCH3), 3.34e3.18 (m,
2H, eNCH2e), 2.87e2.66 (m, 4H, eSCH2e), 2.39e1.75 (m, 8H,
eCH2e), 1.38e1.29 (m, 6H, eCH3); MS (ESI): m/z 806 (M þ H)þ.
300 MHz):
d 12.90 (s, 1H, eArOH), 7.95e7.85 (m, 2H, eArH), 7.87
(d, J ¼ 15.86 Hz, 1H, dbeH), 7.75 (m, 1H, triazoleeH), 7.54
(d, J ¼ 15.10 Hz, 1H, dbeH), 7.53e7.42 (m, 1H, eArH), 7.24e7.18
(m, 1H, eArH), 7.06e6.83 (m, 5H, eArH), 5.32 (s, 2H, eOCH2e), 4.86
(d, J ¼ 3.77 Hz, 1H, eSCHe), 4.73e4.70 (m, 1H, eNCHe), 4.70e4.64
(m, 2H, eOCH2e), 4.15e4.03 (m, 2H, eNCH2e), 3.96 (s, 3H, eOCH3),
3.92 (s, 3H, eOCH3), 3.32e3.15 (m, 2H, eNCH2e), 2.86e2.65 (m, 4H,
eSCH2e), 2.56e2.44 (m, 2H, eCH2e), 2.35e1.73 (m, 4H, eCH2e),
1.38e1.29 (m, 6H, eCH3); MS (ESI): m/z 792 (M þ H)þ.
5.1.5.6. (2S)-N-{4-{1-(5-(4-((E)-3-(4-Hydroxyphenyl)-3-oxoprop-1-
enyl)-2-methoxy phenoxy)pentyl)-1H-1,2,3-triazol-4-yl)methoxy)–
5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylth-
ioacetal (12f). Yield 78%; Yellow solid; mp 122e124 ꢀC; 1H NMR
(CDCl3, 300 MHz): 9.86 (s, 1H, eArOH), 8.14 (d, J ¼ 8.30 Hz, 2H,
eArH), 7.89 (s, 1H, eArH), 7.83 (s, 1H, triazoleeH), 7.75 (d,
J ¼ 15.62 Hz, 1H, dbeH), 7.45 (d, J ¼ 15.62 Hz, 1H, dbeH), 7.25e7.15
(m, 1H, eArH), 6.94 (d, J ¼ 8.30 Hz, 2H, eArH), 6.93e6.86 (m, 3H,
eArH), 5.33 (s, 2H, eOCH2e), 4.86 (d, J ¼ 3.77 Hz, 1H, eSCHe),
4.71e4.67 (m, 1H, eNCHe), 4.53 (t, 2H, eOCH2e), 4.14e4.10 (m, 2H,
eNCH2e), 3.94 (s, 3H, eOCH3), 3.93 (s, 3H, eOCH3), 3.33e3.17 (m,
2H, eNCH2e), 2.88e2.65 (m, 4H, eSCH2e), 2.36e2.17 (m, 2H,
eCH2e), 2.16e1.74 (m, 4H, eCH2e), 1.63e1.41 (m, 4H, eCH2e),
1.38e1.28 (m, 6H, eCH3); MS (ESI): m/z 820 (M þ H)þ.
5.1.5.2. (2S)-N-4-{(1-(4-(4-((E)-3-(2-hydroxyphenyl)-3-oxoprop-1-
enyl)-2-methoxy phenoxy)butyl)-1H-1,2,3-triazol-4-yl)methoxy}-5-
methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde
ioacetal (12b). Yield 85%; Yellow solid; mp 122e124 ꢀC; 1H NMR
(CDCl3, 300 MHz): 12.90 (s, 1H, eArOH), 7.94e7.87 (m, 3H, eArH,
diethylth-
d
triazoleeH), 7.89 (d, J ¼ 15.67 Hz, 1H, dbeH), 7.51 (d, J ¼ 15.98 Hz,
1H, dbeH), 7.51e7.46 (m, 1H, eArH), 7.26e7.23 (m, 1H, eArH),
7.17 (s, 1H, eArH), 7.02 (d, J ¼ 8.30 Hz, 1H, eArH), 6.96e6.91
(m, 1H, eArH), 6.86 (d, J ¼ 8.30 Hz, 1H, eArH), 6.82 (s, 1H, eArH),
5.33 (s, 2H, eOCH2e), 4.85 (d, J ¼ 3.77 Hz, 1H, eOCH2e), 4.72e4.66
(m, 1H, eOCH2e), 4.54 (t, 2H, eOCH2e), 4.11 (t, 2H, eNCH2e), 3.94
(s, 3H, eOCH3), 3.93 (s, 3H, eOCH3), 3.33e3.18 (m, 2H, eNCH2e),
2.81e2.67 (m, 4H, eSCH2e), 2.40e1.74(m, 8H, eCH2e),
1.38e1.30(m, 6H, eCH3); MS (ESI): m/z 806 (M þ H)þ.
5.1.5.7. (2S)-N-{4(1-(3-(4-((E)-3-(5-Chloro-2-hydroxyphenyl)-3-
oxoprop-1-enyl)-2-methoxyphenoxy)propyl)-1H-1,2,3-triazol-4-yl)
methoxy}-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde
diethylthioacetal (12g). Yield 84%; Yellow solid; mp 120e122 ꢀC; 1H
NMR (CDCl3, 300 MHz): 1H NMR (CDCl3, 300 MHz):
d 12.84 (s, 1H,
5.1.5.3. (2S)-4-{(1-(5-(4-((E)-3-(2-Hydroxyphenyl)-3-oxoprop-1-
enyl)-2-methoxy phenoxy)pentyl)-1H-1,2,3-triazol-4-yl)methoxy}-5-
methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal
(12c). Yield 86%; Yellow solid; mp 118e120 ꢀC; 1H NMR (CDCl3,
eArOH), 7.95e7.85 (m, 3H, eArH), 7.90 (d, J ¼ 15.10 Hz, 1H, dbeH),
7.46e7.43 (m, 1H, ArH), 7.43 (s, 1H, d, J ¼ 15.10 Hz, 1H, dbeH),
7.29e7.25 (m, 1H, eArH), 7.18 (s, 1H, eArH), 6.98 (d, J ¼ 9.06 Hz, 1H,
eArH), 6.95e6.90 (m, 1H, dbeH), 6.86 (d, J ¼ 8.30 Hz, 1H, eArH),
6.84 (s, 1H, eArH), 5.35 (s, 2H, eOCH2e), 4.86 (d, J ¼ 3.77 Hz, 1H,
eSCHe), 4.73e4.67 (m, 1H, eNCHe), 4.53 (t, 2H, eOCH2e), 4.10 (t,
2H, eNCH2e), 3.97 (s, 3H, eOCH3), 3.94 (s, 3H, eOCH3), 3.32e3.19
(m, 2H, eNCH2e), 2.85e2.67 (m, 4H, eSCH2e), 2.40e1.74 (m, 6H,
eCH2e), 1.37e1.29 (m, 6H, eCH3); MS (ESI): m/z 827 (M þ H)þ.
300 MHz): d 12.80 (s, 1H, eArOH), 7.92e7.80 (m, 2H, eArH), 7.82 (d,
J ¼ 15.86 Hz, 1H, dbeH), 7.64 (s, 1H, eArH), 7.47 (d, J ¼ 15.10 Hz, 1H,
dbeH), 7.47e7.35 (m, 1H, eArH), 7.20 (d, J ¼ 8.30 Hz, 1H, eArH),
7.12 (s, 1H, eArH), 6.99 (d, J ¼ 9.06 Hz, 1H, eArH), 6.91e6.84
(m, 1H, eArH), 6.81 (d, J ¼ 8.30 Hz,1H, eArH), 6.78 (s, 1H, ArH), 5.30
(s, 2H, eOCH2e), 4.81 (d, J ¼ 3.77 Hz, 1H, eSCH2e), 4.70e4.61 (m,
1H, eNCH2e), 4.41 (t, 2H, eOCH2e), 4.07e4.05 (m, 2H, eNCH2e),
3.92 (s, 3H, eOCH3), 3.91 (s, 3H, eOCH3), 3.29e3.16 (m,
2H, eNCH2e), 2.83e2.65 (m, 4H, eSCH2e), 2.34e1.74 (m,
8H, eCH2e), 1.63e1.52 (m, 2H, eCH2e), 1.38e1.30 (m, 6H, eCH3);
MS (ESI): m/z 820 (M þ H)þ.
5.1.5.8. (2S)-N-{4(1-(4-(4-((E)-3-(5-Chloro-2-hydroxyphenyl)-3-
oxoprop-1-enyl)-2-methoxyphenoxy)butyl)-1H-1,2,3-triazol-4-yl)
methoxy}-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde
diethylthioacetal (12h). Yield 85%; Yellow solid; mp 118e120 ꢀC; 1H
NMR (CDCl3, 300 MHz):
d 12.83 (s, 1H, eArOH), 7.94e7.84 (m, 3H,
eArH), 7.89 (d, J ¼ 15.10 Hz, 1H, dbeH), 7.44e7.40 (m, 1H, ArH), 7.42
(d, J ¼ 15.86 Hz, 1H, dbeH), 7.29e7.25 (m, 1H, eArH), 7.19 (s, 1H,
eArH), 6.99 (d, J ¼ 9.06 Hz, 1H, eArH), 6.89 (d, J ¼ 8.30 Hz, 1H,
eArH), 6.84 (s,1H, eArH), 5.34 (s, 2H, eOCH2e), 4.86 (d, J ¼ 3.77 Hz,
1H, eSCHe), 4.73e4.67 (m, 1H, eNCHe), 4.54 (t, 2H, eOCH2e), 4.12
(t, 2H, eNCH2e), 3.96 (s, 3H, eOCH3), 3.93 (s, 3H, eOCH3),
3.32e3.19 (m, 2H, eNCH2e), 2.85e2.67 (m, 4H, eSCH2e),
2.40e2.21 (m, 2H, eCH2e), 2.14e1.74 (m, 6H, eCH2e), 1.37e1.30
(m, 6H, eOCH3); MS (ESI): m/z 841 (M þ H)þ.
5.1.5.4. (2S)-N-{4-{1-(3-(4-((E)-3-(4-Hydroxyphenyl)-3-oxoprop-1-
enyl)-2-methoxy phenoxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy)}-
5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylth-
ioacetal (12d). Yield 82%; Yellow solid; mp 117e119 ꢀC; 1H NMR
(CDCl3, 300 MHz):
d
9.87 (s, 1H, eArOH), 8.01 (d, J ¼ 8.30 Hz, 2H,
eArH), 7.89 (s, 1H, eArH), 7.83 (s, 1H, triazoleeH), 7.75 (d, 1H,
J ¼ 15.10 Hz, dbeH), 7.56 (d, 1H, J ¼ 15.10 Hz, dbeH), 7.46e7.41 (m,
2H, eArH), 6.95 (d, J ¼ 8.30 Hz, 2H, eArH), 6.91e6.85 (m,1H, eArH),

3828
A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3820e3831
5.1.5.9. (2S)-N-{4(1-(5-(4-((E)-3-(5-Chloro-2-hydroxyphenyl)-3-
oxoprop-1-enyl)-2-methoxyphenoxy)pentyl)-1H-1,2,3-triazol-4-yl)
methoxy}-5-methoxy-2-nitrobenzoyl pyrrolidine-2-carboxaldehyde
7.95e7.90 (m, 1H, eArH), 7.88 (d, J ¼ 15.10 Hz, 1H, dbeH), 7.81 (s,
1H, triazoleeH), 7.65 (d, J ¼ 4.53 Hz, 1H, imineeH), 7.53 (d,
J ¼ 15.86 Hz,1H, dbeH), 7.53 (s,1H, eArH), 7.51e7.47 (m,1H, eArH),
7.16 (s, 1H, eArH), 7.09e6.85 (m, 5H, eArH), 5.31 (s, 2H, eOCH2e),
4.53e48 (m, 2H, eOCH2e), 4.14e4.04 (m, 2H, eNCH2e), 3.92 (s, 6H,
eOCH3), 3.85e3.48 (m, 3H, eNCH2e, eNCHe), 2.41e2.28 (m, 2H,
diethylthioacetal
(12i). Yield
84.5%;
Yellow
12.86 (s, 1H, eArOH),
solid;
mp
121e123 ꢀC; 1H NMR (CDCl3, 400 MHz):
d
7.89 (s, 2H, eArH), 7.89 (d, J ¼ 14.88 Hz, 1H, dbeH), 7.69 (s, 1H,
triazoleeH), 7.45e7.42 (m, 1H, eArH), 7.43 (d, J ¼ 14.88 Hz, 1H,
dbeH), 7.28e7.24 (m, 1H, eArH), 7.18 (s, 1H, eArH), 6.97 (d,
J ¼ 8.62 Hz, 1H, eArH), 6.91 (d, J ¼ 8.62 Hz, 1H, eArH), 6.84 (s,
1H, eArH), 5.34 (s, 2H, eOCH2e), 4.86 (d, J ¼ 3.13 Hz 1H, eSCHe),
4.73e4.68 (m, 1H, eNCHe), 4.42 (t, 2H, eOCH2e), 4.08 (t,
2H, eNCH2e), 3.96 (s, 3H, eOCH3), 3.94 (s, 3H, eOCH3), 3.41e3.18
(m, 2H, eNCH2e), 2.88e2.66 (m, 4H, eSCH2e), 2.41e1.75 (m,
8H, eCH2e), 1.61e1.51 (m, 2H, eCH2e), 1.37e1.31 (m, 6H, eOCH3);
MS (ESI): m/z 855 (M þ H)þ.
eCH2e), 2.22e2.02 (m, 4H, eCH2e), 1.92e1.81 (m, 2H, eCH2e); 13
C
NMR (CDCl3, 75 MHz): 191.6, 164.4, 163.5, 162.6, 150.7, 149.9, 149.4,
147.7, 145.4, 143.1, 136.1, 131.5, 129.4, 127.7, 127.0, 123.5, 121.5, 118.7,
117.8, 112.7, 112.3, 111.6, 111.2, 110.0, 109.6, 68.3, 62.8, 56.0, 53.6,
49.9, 46.6, 44.5, 29.4, 27.3, 25.7, 24.0; IR (KBr): 3354 (br), 2933,
1682, 1602, 1497, 1462, 1420, 1370, 1313, 1259, 1140, 1020, 818, 760,
655 cmꢃ1; MS (ESI): m/z 652 (M þ H)þ; Anal. Calcd for: C36H37N5O7
C, 66.35; H, 5.72; N, 10.75; found: C, 66.30; H, 5.68; N, 10.71.
5.1.6.3. 7-Methoxy-8-{(1-(5-(4-((E)-3-(2-hydroxyphenyl)-3-oxoprop-
1-enyl)-2-methox yphenoxy)pentyl)-1H-1,2,3-triazol-4-yl)methoxy}-
(11aS)-1,2,3,11atetrahydro-5H-pyrrolo [2,1-c] [1,4]benzodiazepine-5-one
5.1.6. General procedure for synthesis of 7-methoxy-8-{(1-(n-(4-
((E)-3-(substitute phenyl)-3-oxoprop-1-enyl)-2-methoxyphenoxy)
alkyl)-1H-1,2,3-triazol-4-yl)methoxy}-(11aS)-1,2,3,11atetrahydro-
5H-pyrrolo [2,1-c] [1,4]benzodiazepine-5-one (4a)
(4c). Yield 55%; Yellow solid; mp 100e102 ꢀC; [
a]
25 þ147.5 (c ¼ 0.1,
D
CHCl3); 1H NMR (CDCl3, 200 MHz):
d
12.92 (s, 1H, ArOH), 7.98e7.91
To a stirred solution of compounds 12aei (1.0 mmol) in meth-
anol (20 mL), SnCl2$2H2O (5 mmol) was added and refluxed for 2 h.
The methanol was evaporated in vacuum and the aqueous layer
was then carefully adjusted to pH 8 with 10% NaHCO3 solution and
then extracted with ethyl acetate (20e30 mL). The combined
organic phase was dried over anhydrous Na2SO4 and evaporated
under vacuum to afford the amino diethylthioacetals, which due to
potential stability problems proceeded for the next step. A solution
of amino diethylthioacetal (1 mmol), HgCl2 (2.26 mmol) and CaCO3
(2.26 mmol) in CH3CNewater (4:1) was stirred slowly at room
temperature until TLC indicated complete loss of starting material
(12 h). The reaction mixture was diluted with ethyl acetate (30 mL)
and filtered through a Celite bed. The clear yellow organic super-
natant was washed with saturated 5% NaHCO3 (20 mL), brine wash
(20 mL), and the combined organic phase was dried over anhydrous
Na2SO4. The organic layer was evaporated in vacuum and purified
by column chromatography (MeOH/CHCl3, 5%) to give the final
products 4aei.
(m, 1H, eArH), 7.87 (d, J ¼ 14.69 Hz, 1H, dbeH), 7.67 (s, 1H, tri-
azoleeH), 7.64 (d, J ¼ 3.37 Hz, 1H, imineeH), 7.53e7.40 (m, 2H,
eArH), 7.53 (d, J ¼ 14.69 Hz,1H, dbeH), 7.16 (s,1H, eArH), 7.10e6.81
(m, 5H, eArH), 5.30 (s, 2H, eOCH2e), 4.39 (t, 2H, eOCH2e),
4.13e3.99 (m, 2H, eNCH2e), 3.93 (s, 3H, eOCH3), 3.91 (s, 3H,
eOCH3), 3.88e3.48 (m, 3H, eNCH2e, eNCHe), 2.37e2.23 (m, 2H,
eCH2e), 2.13e1.82 (m, 6H, eCH2e), 1.64e1.44 (m, 2H, eCH2e); 13
C
NMR (CDCl3, 75 MHz): 191.5, 164.3, 163.2, 162.5, 151.0, 149.8, 149.5,
147.5,145.5,143.2,136.2,131.4,129.4,127.8,126.9,123.3,121.4,118.4,
117.6,112.5,112.4,111.5,111.4,110.1,109.5, 68.2, 62.6, 55.8, 53.4, 49.7,
46.4, 44.2, 31.4, 28.3, 27.3, 25.7, 22.2; IR (KBr): 3364 (br), 2933,1600,
1497, 1463, 1422, 1338, 1313, 1245, 1179, 1127, 1009, 865, 737,
653 cmꢃ1; MS (ESI): m/z 666 (M þ H)þ; Anal. Calcd for: C37H39N5O7
C, 66.75; H, 5.90; N, 10.52; found: C, 66.70; H, 5.85; N, 10.46.
5.1.6.4. 7-Methoxy-8(1-(3-(4-((E)-3-(4-hydroxyphenyl)-3-oxoprop-
1-enyl)-2-methoxy phenoxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy}-
(11aS)-1,2,3,11atetrahydro-5H-pyr rolo [2,1-c] [1,4]benzodiazepine-5-
25
one (4d). Yield 55%; Yellow solid; mp 95e97 ꢀC; [
a
]
þ 142.5
D
5.1.6.1. 7-Methoxy-8-{(1-(3-(4-((E)-3-(2-hydroxyphenyl)-3-oxoprop-
1-enyl)-2-methoxyphenoxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy}-
(11aS)-1,2,3,11atetrahydro-5H-pyrrolo [2,1-c] [1,4]benzodiazepine-
5-one (4a). Yield 56%; Yellow solid; mp 100e102 ꢀC;
(c ¼ 0.1, CHCl3); 1H NMR (CDCl3, 200 MHz):
d 9.95 (s, 1H, eArOH),
7.92 (d, J ¼ 8.81 Hz, 2H, eArH), 7.75 (s, 1H, triazoleeH), 7.71 (d,
J ¼ 16.89 Hz, 1H, dbeH), 7.65 (d, 1H, J ¼ 3.90 Hz, imineeH), 7.50 (s,
1H, eArH), 7.40 (m, J ¼ 16.16 Hz,1H, dbeH), 7.13 (m,1H, eArH), 6.97
(s, 1H, eArH), 6.96 (d, J ¼ 8.81 Hz, 2H, eArH), 6.79 (d, J ¼ 8.08 Hz,
2H, ArH), 5.29 (s, 2H, eOCH2e), 4.62 (t, 2H, eOCH2e), 4.08e3.94
(m, 2H, eNCH2e), 3.92 (s, 3H, eOCH3), 3.88 (s, 3H, eOCH3),
3.77e3.44 (m, 3H, eNCH2e, eNCHe), 2.52e2.23 (m, 4H, eCH2e),
2.16e1.94 (m, 2H, eCH2e); 13C NMR (CDCl3, 75 MHz): 193.2, 163.3,
162.4, 156.4, 150.8, 149.4, 147.6, 145.4, 143.1, 140.2, 136.1, 129.4,
127.6, 127.0, 123.2, 121.4, 118.6, 117.7, 112.6, 112.2, 111.4, 111.1, 110.4,
109.4, 68.1, 62.6, 56.2, 53.4, 50.9, 46.4, 44.3, 30.0, 25.4, 24.2; IR
(KBr): 3360 (br), 2930, 1678, 1632, 1497, 1463, 1422, 1338, 1313,
1245, 1179, 1127, 1009, 865, 737, 653 cmꢃ1; MS (ESI): m/z 638
[M þ H]þ; Anal. Calcd for: C35H35N5O7 C, 65.92; H, 5.53; N, 10.98;
found: C, 65.88; H, 5.48; N, 10.92.
[
a
]
25 þ 118.5 (c ¼ 0.1, CHCl3); 1H NMR (CDCl3, 200 MHz):
d 12.90
D
(s, 1H, eArOH), 7.96e7.91 (m, 1H, eArH), 7.88 (d, J ¼ 14.84 Hz, 1H,
dbeH), 7.74 (s, 1H, triazoleeH), 7.70 (d, J ¼ 4.68 Hz, 1H, imineeH),
7.62 (s, 1H, eArH), 7.50 (d, J ¼ 14.84 Hz, 1H, dbeH), 7.47e7.40 (m,
1H, eArH), 7.18 (s, 1H, eArH), 7.10e6.86 (m, 5H, eArH), 5.30 (s,
2H, eOCH2e), 4.63 (t, 2H, eOCH2e), 4.08e4.00 (m, 2H, eNCH2e),
3.95 (s, 3H, eOCH3), 3.91 (s, 3H, eOCH3), 3.86e3.48 (m,
3H, eNCH2e, eNCHe), 2.53e2.27 (m, 4H, eCH2e), 2.16e1.97 (m,
2H, eCH2e); 13C NMR (CDCl3, 75 MHz): 195.0, 163.6, 162.4, 152.3,
149.7, 149.1, 147.3, 145.5, 143.5, 136.2, 132.4, 129.6, 128.2, 127.0,
123.6, 123.4, 118.6, 118.0, 114.8, 113.8, 111.6, 111.3, 111.0, 109.9,
106.7, 65.4, 56.1, 55.8, 53.6, 46.9, 46.6, 44.5, 34.6, 29.6, 24.1; IR
(KBr): 3348 (br), 2931, 1684, 1632, 1602, 1510, 1463, 1422, 1374,
1309, 1261, 1137, 1025, 816, 762, 659 cmꢃ1; MS (ESI): m/z 638
(M þ H)þ; Anal. Calcd for: C35H35N5O7 C, 65.92; H, 5.53; N, 10.98;
found: C, 65.85; H, 5.48; N, 9.71.
5.1.6.5. 7-Methoxy-8(1-(4-(4-((E)-3-(4-hydroxyphenyl)-3-oxoprop-
1-enyl)-2-methoxy phenoxy)butyl)-1H-1,2,3-triazol-4-yl)methoxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrr olo [2,1-c] [1,4]benzodiazepine-5-one
25
(4e). Yield 56%; Yellow solid; mp 94e96 ꢀC; [
a
]
D
þ 112.0
5.1.6.2. 7-Methoxy-8-{(1-(4-(4-((E)-3-(2-hydroxyphenyl)-3-oxoprop-
1-enyl)-2-me thoxyphenoxy)butyl)-1H-1,2,3-triazol-4-yl)methoxy}-
(11aS)-1,2,3,11atetrahydro-5H-pyrrolo [2,1-c] [1,4]benzodiazepine-5-one
(c ¼ 0.1, CHCl3); 1H NMR (CDCl3, 200 MHz):
d 9.95 (s, 1H, eArOH),
7.92 (d, J ¼ 8.81 Hz, 2H, eArH), 7.74 (m, 1H, triazoleeH), 7.73 (d,
J ¼ 15.60 Hz, 1H, dbeH), 7.64 (d, J ¼ 4.10 Hz, 1H, imineeH), 7.50 (s,
1H, eArH), 7.42 (d, J ¼ 15.60 Hz, 1H, dbeH), 7.13 (s, 1H, eArH), 7.09
(s, 1H, eArH), 6.97e6.91 (m, 4H, eArH), 5.28 (s, 2H, eOCH2e), 4.61
(4b). Yield 58%; Yellow solid; mp 95e97 ꢀC; [
CHCl3); 1H NMR (CDCl3, 300 MHz):
a
]
25 þ 159.5 (c ¼ 0.1,
D
d
12.92 (s, 1H, eArOH),

A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3820e3831
3829
(t, 2H, eOCH2e), 4.02 (t, 2H, eNCH2e), 3.90 (s, 3H, eOCH3), 3.86 (s,
3H, eOCH3), 3.77e3.46 (m, 3H, eNCH2e, eNCHe), 2.52e1.80 (m,
8H, eCH2e); 13C NMR (CDCl3, 75 MHz): 193.4, 163.4, 162.5, 156.5,
150.9, 149.5, 147.8, 145.5, 143.0, 136.0, 129.6, 127.8, 127.1, 123.4,
121.5, 118.8, 117.8, 112.7, 112.3, 111.6, 111.3, 110.5, 109.6, 68.3, 62.8,
56.0, 53.6, 50.1, 46.6, 44.4, 29.6, 27.4, 25.7, 24.0; IR (KBr): 3352 (br),
2934, 1684, 1632, 1599, 1509, 1462, 1422, 1375, 1311, 1260, 1138,
1024, 815, 756, 659, 565 cmꢃ1; MS (ESI): m/z 652 [M þ H]þ; Anal.
Calcd for: C36H37N5O7 C, 66.35; H, 5.72; N, 10.75; found: C, 66.28; H,
5.66; N, 10.69.
2.23e2.01 (m, 2H, eCH2e), 1.94e1.83 (m, 2H, eCH2e); 13C NMR
(CDCl3, 75 MHz): 192.5, 164.6, 162.6, 161.8, 149.8, 149.4, 147.6,
146.6, 143.1, 140.3, 135.8, 128.6, 127.3, 126.2, 123.8, 123.3, 120.0,
118.7, 117.2, 112.7, 112.4, 111.5, 111.1, 110.5, 109.6, 68.1, 62.9, 56.0,
53.6, 50.1, 46.6, 44.0, 29.4, 27.3, 25.6, 24.0; IR (KBr): 3357 (br),
2932, 1691, 1632, 1600, 1567, 1510, 1466, 1428, 1428, 1336, 1313,
1264, 1181, 1136, 1024, 820, 753, 645 cmꢃ1; MS (ESI): m/z 686
(M þ H)þ; Anal. Calcd for: C36H36ClN5O7 C, 63.02; H, 5.29; N,
10.21; found: C, 63.06; H, 5.25; N, 10.15.
5.1.6.9. 7-Methoxy-8(1-(5-(4-((E)-3-(5-chloro-2-hydroxyphenyl)-3-
oxoprop-1-enyl)-2-methoxyphenoxy)pentyl)-1H-1,2,3-triazol-4-yl)
methoxy}-(11aS)-1,2,3,11atetrahydro-5H-pyrrolo [2,1-c] [1,4]benzo-
diazepine-5-one (4i). Yield 52%; Yellow solid; mp 93e95 ꢀC;
5.1.6.6. 7-Methoxy-8(1-(5-(4-((E)-3-(4-hydroxyphenyl)-3-oxoprop-
1-enyl)-2-methoxy phenoxy)pentyl)-1H-1,2,3-triazol-4-yl)methoxy}-
(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo [2,1-c] [1,4]benzodiazepine-
5-one (4f). Yield 56%; Yellow solid; mp 100e102 ꢀC; [
a
]
25 þ 159.5
[a
]
25 þ159.5 (c ¼ 0.1, CHCl3); 1H NMR (CDCl3, 400 MHz):
d 12.86 (s,
D
D
(c ¼ 0.1, CHCl3); 1H NMR (CDCl3, 200 MHz):
d
9.82 (s, 1H, eArOH),
1H, eArOH), 7.92 (s, 1H, eArH), 7.89 (d, J ¼ 15.38 Hz, 1H, dbeH),
7.70 (s, 1H, TriazoleeH), 7.64 (d, 1H, J ¼ 4.35 Hz, imineeH), 7.53 (s,
1H, ArH),7.46e7.40 (m, 1H, eArH), 7.42 (d, J ¼ 15.38 Hz, 1H, dbeH),
7.32e7.24 (m, 1H, eArH), 7.18 (s, 1H, eArH), 7.03e6.86 (m, 3H,
eArH), 5.31 (s, 2H, eOCH2e), 4.42e4.37 (m, 2H, eOCH2e), 4.07 (t,
2H, eNCH2e), 3.95 (s, 3H, eOCH3), 3.93 (s, 3H, eOCH3), 3.86e3.53
(m, 3H, eNCH2e, eNCHe), 2.41e2.30 (m, 4H, eCH2e), 2.12e1.50
(m, 6H, eCH2e); 13C NMR (CDCl3, 75 MHz): 192.7,167.0, 162.1,161.9,
150.0, 149.6, 147.6, 146.7, 143.1, 140.8, 135.9, 128.6, 127.5, 126.3,
123.9, 123.4, 121.6, 120.6, 118.8, 116.6, 112.3, 111.3, 111.0, 110.9, 109.7,
68.6, 65.2, 56.1, 52.4, 50.7, 44.6, 42.7, 29.8, 28.2, 24.5, 23.3, 22.6; IR
(KBr): 3357 (br), 2932,1690,1633,1567,1511,1467,1428,1338,1254,
1179, 1127, 1020, 820, 753, 648 cmꢃ1; MS (ESI): m/z 700 [M þ H]þ;
Anal. Calcd for: C37H38ClN5O7 C, 63.47; H, 5.47; N, 10.00; found: C,
63.40; H, 5.40; N, 10.08.
7.92 (m, 2H, eArH), 7.71 (d, J ¼ 15.60 Hz, 1H, dbeH), 7.78e7.64 (m,
1H, triazoleeH), 7.64 (m, 1H, imineeH), 7.51 (s, 1H, eArH),
7.44e7.36 (m, 1H, eArH), 7.40 (d, J ¼ 15.60 Hz, 1H, dbeH), 7.19e7.11
(m, 1H, eArH), 7.07 (s, 1H, eArH), 7.02e6.86 (m, 3H, eArH), 5.21
(s, 2H, eOCH2e), 4.47e4.28 (m, 2H, eOCH2e), 4.18e4.00 (m,
2H, eNCH2e), 3.97 (s, 3H, eOCH3), 3.90 (s, 3H, eOCH3), 3.84e3.53
(m, 3H, eNCH2e, eNCHe), 2.53e2.23 (m, 2H, eCH2e), 2.11e1.38
(m, 8H, eCH2e); 13C NMR (CDCl3, 75 MHz): 192.5, 163.2, 162.3,
156.8, 149.9,147.7, 145.4, 143.1, 142.6, 136.1, 129.5, 127.8, 127.0, 123.3,
121.4, 118.7, 118.0, 112.6, 112.2, 111.5, 111.1, 109.1, 105.6, 68.1, 62.7,
55.9, 53.5, 49.8, 46.5, 44.3, 29.4, 27.3, 26.7, 25.6, 23.2; IR (KBr): 3355
(br), 2934,1684, 1632,1597, 1509, 1462, 1422, 1375, 1312,1260, 1155,
1025, 815, 758, 659, 565 cmꢃ1; MS (ESI): m/z 666 [M þ H]þ; Anal.
Calcd for: C37H39N5O7 C, 66.75; H, 5.90; N, 10.52; found: C, 66.68; H,
5.82; N, 10.44.
5.1.7. General procedure for synthesis of substituted-2(4-(4-((E)-3-
(2-hydroxyphenyl)-3-oxoprop-1-enyl)-2-thoxyphenoxy)methyl)-
1H-1,2,3-triazol-1-yl)-2-nitrobenzoyl}pyrrolidine-5-carboxaldehyde
diethylthioacetal (16a,b)
5.1.6.7. 7-Methoxy-8(1-(3-(4-((E)-3-(5-chloro-2-hydroxyphenyl)-3-
oxo prop-1-enyl)-2-methoxyphenoxy)propyl)-1H-1,2,3-triazol-4-yl)
methoxy}-(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo [2,1-c] [1,4]benzo-
diaepine-5-one (4g). Yield 56%; Yellow solid; mp 104e106 ꢀC;
To a stirred solution of alkyne 14 (1.0 mmol) and PBD-C2-azide
15a,b (1.2 mmol) in t-Butanol-water (1:1) (50 mL), CuSO4$5H2O
catalyst (1 mol%) (3 mmol) and sodium ascorbate (5 mol%) were
added and the mixture was refluxed for 12 h. After completion of
the reaction, quenched with NH3 solution and butanol was
removed under reduced pressure. Chloroform, water added to the
above residue and stirred for another 30 min followed by celite
filtration, extraction with chloroform, and organic solvent was
evaporated under reduced pressure to get the crude product. These
were further purified by column chromatography using ethyl ace-
tateehexane (90%) to afford the compounds 16a,b.
[
a]
25 þ130.5 (c ¼ 0.1, CHCl3); 1H NMR (CDCl3, 200 MHz):
d 12.87 (s,
D
1H, eArOH), 7.95e7.82 (m, 2H, triazoleeH, eArH), 7.88 (d,
J ¼ 15.10 Hz, 1H, olifiniceH), 7.64 (d, J ¼ 4.15 Hz, 1H, imineeH), 7.57
(s, 1H, eArH), 7.56e7.37 (m, 2H, eArH), 7.54 (d, J ¼ 15.10 Hz,
1H, olifiniceH), 7.18 (s, 1H, eArH), 7.10e6.84 (m, 3H, eArH), 5.31
(s, 2H, eOCH2e), 4.62 (t, 2H, eOCH2e), 4.09e4.00 (m,
2H, eNCH2e), 3.94 (s, 3H, eOCH3), 3.92 (s, 3H, eOCH3), 3.86e3.48
(m, 3H, eNCH2e, eNCHe), 2.53e2.27 (m, 4H, eCH2e), 2.16e1.97
(m, 2H, eCH2e); 13C NMR (CDCl3, 75 MHz): 192.5, 164.5, 162.7,
161.8, 151.0, 149.4, 147.5, 146.5, 143.3, 140.4, 135.6, 128.4, 127.3,
126.0, 123.6, 123.1, 120.0, 118.5, 116.7, 112.6, 112.0, 111.3, 111.0, 110.3,
109.4, 68.2, 62.5, 55.8, 53.4, 49.9, 46.4, 43.8, 29.2, 27.6, 23.8; IR
(KBr): 3357 (br), 2933, 1685, 1632, 1600, 1567, 1510, 1466, 1428,
1338, 1313, 1245, 1179, 1136, 1024, 820, 753, 645 cmꢃ1; MS (ESI):
m/z 672 (M þ H)þ; Anal. Calcd for: C35H34ClN5O7 C, 62.54; H, 5.10;
N, 10.42; found: C, 66.50; H, 5.06; N, 10.38.
5.1.7.1. 7,8-Dimethoxy-2(4-(4-((E)-3-(2-hydroxyphenyl)-3-oxoprop-
1-enyl)-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)}-2-nitro-
benzoyl}pyrrolidine-5-carboxaldehyde diethylthioacetal (16a). Yield
85%; Yellow solid; 120e122 ꢀC; 1H NMR (CDCl3, 300 MHz):
d 12.75
(s, 1H, eArOH), 7.98e7.94 (d, 3H, eArH), 7.84 (d, J ¼ 15.38 Hz, 1H,
dbeH), 7.75 (s, 1H, eArH), 7.43 (d, J ¼ 15.38 Hz, 1H, dbeH), 7.45 (s,
1H, eArH), 7.29e7.24 (m, 1H, eArH), 7.15 (s, 1H, eArH), 6.96e6.86
(m, 3H, eArH), 5.01 (s, 2H, eOCH2e), 4.96e4.92 (m, 1H, eNCHe),
4.86 (d, 1H, eSCHe), 4.80e4.68 (m, 1H, eNCHe), 3.99 (s, 3H,
eOCH3), 3.97 (s, 3H, eOCH3), 3.90 (s, 3H, eOCH3), 3.20e3.15 (t, 2H,
eNCH2e), 2.87e2.65 (m, 4H, eSCH2e), 2.11e2.04 (m, 2H, eCH2e),
1.38e1.33 (t, 6H, eCH3); MS (ESI): m/z 764(M þ H)þ.
5.1.6.8. 7-Methoxy-8(1-(4-(4-((E)-3-(5-chloro-2-hydroxyphenyl)-3-
oxo
prop-1-enyl)-2-methoxyphenoxy)butyl)-1H-1,2,3-triazol-4-yl)
methoxy}-(11aS)-,2,3,11atetrahydro-5H-pyrrolo [2,1-c] [1,4]benzodi-
azepine-5-one (4h). Yield 54%; Yellow solid; mp 106e108 ꢀC;
[
a
]
25 þ125.5 (c ¼ 0.1, CHCl3); 1H NMR (CDCl3, 300 MHz):
d 12.85
D
(s, 1H, eArOH), 7.90 (d, J ¼ 15.10 Hz, 1H, dbeH), 7.88 (s, 1H, ArH),
7.82 (s, 1H, triazoleeH), 7.65 (d, J ¼ 4.15 Hz, 1H, imineeH), 7.52 (s,
1H, ArH), 7.47e7.38 (m, 2H, eArH), 7.43 (s, J ¼ 15.10 Hz, 1H,
dbeH), 7.18 (s, 1H, eArH), 7.04e6.84 (m, 3H, eArH), 5.30 (s,
2H, eOCH2e), 4.56e4.47 (m, 2H, eOCH2e), 4.16e4.03 (m,
2H, eNCH2e), 3.94 (s, 3H, eOCH3), 3.92 (s, 3H, eOCH3),
3.87e3.50 (m, 3H, eNCH2e, eNCHe), 2.38e2.28 (m, 4H, eCH2e),
5.1.7.2. 8-(Benzyloxy)6-methoxy-2(4-(4-((E)-3-(2-hydroxyphenyl)-
3-oxoprop-1-enyl)-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-
yl)}- 2-nitrobenzoyl}pyrrolidine-5-carboxaldehyde diethylthioacetal
(16b). Yield 86%; Yellow solid; 118e120 ꢀC; 1H NMR (CDCl3,
300 MHz):
d
12.75 (s, 1H, eArOH), 7.98 (d, J ¼ 9.06 Hz, 1H, eArH),

3830
A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3820e3831
7.84 (d, J ¼ 16.10 Hz, 1H, dbeH), 7.82 (s, 1H, eArH), 7.76e7.52 (m,
3H, eArH), 7.42 (d, J ¼ 15.86 Hz, 1H, dbeH), 7.47 (s, 1H, eArH),
7.45e7.32 (m, 6H, eArH), 7.15 (s, 1H, eArH), 6.96e6.84 (m,
2H, eArH), 5.19 (s, 2H, eOCH2e), 5.05 (s, 2H, eOCH2e), 4.99e3.92
(m, 1H, eNCHe), 4.86 (d, 1H, eSCHe), 4.78e4.67 (m, 1H, eNCHe),
3.97 (s, 3H, eOCH2e), 3.90 (s, 3H, eOCH2e),3.88e3.80 (m,
2H, eNCH2e), 2.86e2.65 (m, 4H, eSCH2e), 2.15e2.04 (m,
2H, eCH2e), 1.40e1.33 (t, 6H, eCH3); MS (ESI): m/z 840 (M þ H)þ.
5.3. In vitro evaluation of cytotoxicity
Cell viability was assessed by the MTT assay, a mitochondrial
function assay. It is based on the ability of viable cells to reduce the
MTT to insoluble formazan crystals by mitochondrial dehydroge-
nase. In this assay MCF-7 cells were seeded in a 96-well plate at
a density of 10,000 cells/well. After overnight incubation cells were
treated with compounds 1, 2, 4a, 4b, 4c, 4d, 4h, 4i, 5a and 5b at
4
m
M concentration and incubated for 24 h. Then the medium was
discarded and replaced with 10 L MTT dye. Plates were incubated
at 37 ꢀC for 2 h. The resulting formazan crystals were solubilized in
100 L extraction buffer. The optical density (O.D) was read at
5.1.8. General procedure for synthesis of substituted-2(4-(4-((E)-3-
(2-hydroxyphenyl)-3-oxoprop-1-enyl)-2-thoxyphenoxy)methyl)-1H-
1,2,3-triazol-1-yl)}-(11aS)-1,2,3,11atetrahydro-5H-pyrrolo [2,1-c]
[1,4]benzodiazepine-5-one (5a,b)
m
m
570 nm with micro plate reader.
The compounds 5a,b ware prepared according to the method
described for the compounds 4aei, employing the compounds
16a,b (1.0 mmol). The crude product was purified by column chro-
matography (MeOH/CHCl3, 5%) to afford the pure compounds 5a,b.
5.4. Cell cycle analysis
5 ꢄ 105 MCF-7 cells were seeded in 60 mm dish and were
5.1.8.1. 7,8-Dimethoxy-2(4-(4-((E)-3-(2-hydroxyphenyl)-3-oxoprop-
1-enyl)-2-methoxy phenoxy)methyl)-1H-1,2,3-triazol-1-yl)}-(11aS)-
allowed to grow for 24 h, 4 mM concentration of 1, 2, 4a, 4b, 4c,
4d, 4h, 4i, 5a and 5b compounds were added to the culture
media, and the cells were incubated for an additional 24 h. Cells
were harvested with TrypsineEDTA, fixed with ice-cold 70%
ethanol at 4 ꢀC for 30 min, washed with PBS and incubated with
1 mg/ml RNAase solution (Sigma) at 37 ꢀC for 30 min. Cells were
collected by centrifugation at 2000 rpm for 5 min and further
1,2,3,11atetrahydro-5H-pyrrolo [2,1-c] [1,4]benzodiazepine-5-one
25
(5a). Yield 58%; Yellow solid; mp 114e116 ꢀC; [
a
]
þ 145.0
D
(c ¼ 0.1, CHCl3); 1H NMR (CDCl3, 300 MHz): 1H NMR (CDCl3,
200 MHz):
d 12.87 (s, 1H, eArOH), 7.96e7.78 (m, 2H, eArH), 7.76
(d, J ¼ 15.40 Hz, 1H, dbeH), 7.65 (d, J ¼ 4.15 Hz, 1H, imineeH),
7.65e7.50 (m, 2H, eArH), 7.42 (d, J ¼ 16.10 Hz, 1H, dbeH),
7.30e7.05 (m, 3H, eArH), 7.03e6.84 (m, 3H, eArH), 5.33 (s,
2H, eOCH2e), 5.31e5.28 (m, 1H, eNCHe), 4.56e4.47 (m,
3H, eNCH2e, eNCHe), 3.94 (s, 3H, eOCH3), 3.92 (s, 6H, eOCH3),
2.36e2.24 (m, 2H, eCH2e); 13C NMR (CDCl3, 75 MHz): 192.5,
163.4, 162.3, 151.7, 149.7, 149.1, 147.4, 145.3, 143.5, 136.6, 132.4,
129.6, 128.1, 127.0, 123.8, 123.4, 118.6, 118.0, 114.8, 113.8, 111.6,
111.3, 111.0, 109.9, 105.2, 69.9, 59.7, 56.5, 56.1, 50.4, 40.1, 32.4, 28.0;
IR (KBr): 3385 (br), 2927, 1685, 1634, 1577, 1510, 1458, 1428, 1377,
1342, 1311, 1258, 1145, 1027, 815, 763, 661, 567 cmꢃ1; MS (ESI): m/
z 610 [M þ H]þ; Anal. Calcd for: C33H31N5O7 C, 65.02; H, 5.13; N,
11.49; found: C, 64.98; H, 5.08; N, 11.45.
stained with 250 mL of DNA staining solution [10 mg of Propidium
Iodide (PI), 0.1 mg of trisodium citrate, and 0.03 mL of Triton X-
100 were dissolved in 100 mL of sterile MilliQ water at room
temperature for 30 min in the dark]. The DNA contents of 20,000
events were measured by flow cytometer (DAKO CYTOMATION,
Beckman Coulter, Brea, CA). Histograms were analyzed using
Summit Software.
5.5. Protein extraction and western blot analysis
MCF-7 cells were seeded in 60 mm dish and were allowed to
5.1.8.2. 8-(Benzyloxy)7-methoxy-2(4-(4-((E)-3-(2-hydroxyphenyl)-
3-oxoprop-1-enyl)-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-
yl)}-(11aS)-1,2,3,11atetrahydro-5H-pyrrolo [2,1-c] [1,4]benzodiaze-
pine-5-one (5b). Yield 56%; Yellow solid; mp 120e122 ꢀC;
grow to attain 80% confluency for 24 h, 4 mM concentration of 1, 4a,
4b, 4d, 4i compounds were added to the culture media, and the
cells were incubated with compounds for 24 h. After 24h total cell
lysates from cultured MCF-7 cells were obtained by lysing the cells
in ice-cold RIPA buffer (1XPBS, 1% NP-40, 0.5% sodium deoxycholate
[
a]
25 þ129.5 (c ¼ 0.1, CHCl3); 1H NMR (CDCl3, 200 MHz):
d 12.88 (s,
D
1H, eArOH), 7.94e7.80 (m, 3H, eArH), 7.83 (d, J ¼ 15.60 Hz, 1H,
dbeH), 7.66 (d, J ¼ 4.15 Hz, 1H, eArH), 7.58e7.50 (m, 2H, eArH),
7.42 (d, J ¼ 15.80 Hz, 1H, dbeH), 7.26e7.16 (m, 6H, eArH), 7.11e6.81
(m, 4H, eArH), 5.35 (s, 4H, eOCH2e), 5.31e5.18 (m, 1H, eOCH2e),
4.35e4.19 (m, 3H, eNCH2e, eNCHe), 3.93 (s, 6H, eOCH3),
3.27e2.81 (m, 2H, eCH2e); 13C NMR (CDCl3, 75 MHz): 192.8, 168.4,
164.9, 162.4, 150.7, 149.5, 148.8, 146.9, 145.4, 144.6, 142.3, 135.4,
135.0, 129.9, 129.1, 127.6, 127.2, 126.7, 122.8, 121.9, 119.1, 117.9, 117.3,
116.6, 115.4, 112.8, 111.5, 110.2, 105.2, 69.9, 61.7, 56.1, 55.1, 50.4, 39.6,
31.4, 28.6; IR (KBr): 3385 (br), 2923, 1695, 1635, 1607, 1574, 1510,
1467, 1426, 1374, 1258, 1202, 1143, 1023, 848, 812, 757, 697, 659,
562 cmꢃ1; MS (ESI): m/z 686 [M þ H]þ; Anal. Calcd for: C39H35N5O7
C, 68.31; H, 5.14; N, 10.21; found: C, 68.25; H, 5.08; N, 10.15.
and 0.1% SDS) and containing 100 g/mL Aprotinin,
mg/mL PMSF, 5
m
5
m
g/mL leupeptin, 5 g/mL pepstatin and 100 mg/mL NaF. After
m
centrifugation at 12,000 rpm for 10 min, the protein in supernatant
was quantified by Bradford method (BIO-RAD) using Multimode
varioskan instrument (Thermo-Fischer Scientifics). Fifty micro-
grams of protein per lane was applied in 12% SDS-polyacrylamide
gel. After electrophoresis, the protein was transferred to poly-
vinylidine difluoride (PVDF) membrane (Amersham Biosciences).
The membrane was blocked at room temperature for 2 h in
TBS þ 0.1% Tween20 (TBST) containing 5% blocking powder (San-
tacruz). The membrane was washed with TBST for 5 min, and
primary antibody was added and incubated at 4 ꢀC overnight (O/N).
Rabbit polyclonal antibodies Cyclin D1 (37 kDa) was purchased
from Santacruz biotech company. Rabbit polyclonal Cdk4 (34 KDa)
antibody was purchased from Prosci company. NF-kB SEAP reporter
was purchased from Clone tech Company. Rabbit polyclonal anti-
5.2. Cell culture
bodies Bcl-xL (29 kDa),
b-actin (38 kDa) was purchased from
The MCF-7 (breast carcinoma cells) were incubated by using
DMEM media, supplemented with 10% fetal calf serum, 100 mg/mL
Imgenex company. Membranes were washed with TBST three times
for 15 min and the blots were visualized with chemiluminescence
reagent (Thermo-Fischer Scientifics Ltd.). The X-ray films were
developed with developer and fixed with fixer solution (from
Kodak Company).
pencillin-G and 100 mg/mL streptomycin sulfate. The cell line was
maintained at 37 ꢀC in a humidified atmosphere containing 5% CO2
in the incubator.

A. Kamal et al. / European Journal of Medicinal Chemistry 46 (2011) 3820e3831
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The NF-kB SEAPorter assay was conducted by transiently
transfecting MCF-7 cells with NF-kB SEAP plasmid (6767 bp)
obtained from Imgenex Company. SEAP is a secreted alkaline
phosphatase tagged downstream to NF-kB promoter. After trans-
fecting NF-kB SEAP reporter construct the cells were treated with
compounds(1, 4a, 4b, 4d, 4i) at a concentration of 4 mM for 24 h, the
cell lysates were subjected to Chemiluminiscent based reagent
obtained from Clone Tech (Takara). The amount of the expression
was measured using luminometry and as well as exposing the X-ray
film kept on the top surface of the plate for 30 min.
5.8. Bcl2-ELISA
Bcl2 ELISA was conducted with Alexis Biochemicals BCl2 ELISA
kit. Here MCF-7 cells were treated with compounds (1, 4a, 4b, 4d,
4i) for 24 h at 4
added to the micro wells which contains Bcl2 antibody. After the
addition of lysates 100 L of biotin conjugated anti-human Bcl2
monoclonal antibody was added. After the incubation period and
washing steps the bound Bcl2 was detected by using 150
mM concentration. The cell lysates were isolated and
m
mL
Streptavidin-HRP secondary antibodies. The colored product
obtained was detected by O.D at 450 nm. O.D is directly propor-
tional to the amount of Bcl2 protein in the sample.
Acknowledgments
The authors S. P. R., P. V. R. Ch. R. R. and A. M. R 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. Thanks to Richard G.
Pestell, Kimmel Cancer Centre, USA for providing the Cyclin D1
promoter constructs.
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