Synthesis and biological evaluation of cinnamido linked pyrrolo[2,1-c][1,4]benzodiazepines as antimitotic agents

Article abstract of DOI:10.1016/j.ejmech.2010.05.041

A series of new cinnamido-pyrrolo[2,1-c][1,4]benzodiazepine conjugates (4a-d and 5a-d) and their dimers (6a-d) have been designed, synthesized and evaluated for their biological activity. The anticancer screening of compound 4a by the NCI exhibited signif

Full text of DOI:10.1016/j.ejmech.2010.05.041

European Journal of Medicinal Chemistry 45 (2010) 3870e3884
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of cinnamido linked pyrrolo[2,1-c][1,4]
benzodiazepines as antimitotic agents
*
Ahmed Kamal , G. Balakishan, G. Ramakrishna, T. Basha Shaik, K. Sreekanth, M. Balakrishna,
Rajender, D. Dastagiri, Shasi V. Kalivendi
Division of Organic Chemistry-I, Indian Institute of Chemical Technology, Hyderabad 500 607, 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 cinnamido-pyrrolo[2,1-c][1,4]benzodiazepine conjugates (4aed and 5aed) and their
dimers (6aed) have been designed, synthesized and evaluated for their biological activity. The anticancer
screening of compound 4a by the NCI exhibited significant GI50 values ranging from 68 to 732 nM
against 53 of 59 human cancer cell lines tested. Compounds 5aed and 6aed have also shown remarkable
Received 12 March 2010
Received in revised form
19 May 2010
Accepted 20 May 2010
Available online 1 June 2010
cytotoxic activity with GI50 values <0.1 mM concentrations in a large number of cell lines. Interestingly,
compounds 5b and 6b have been identified as a new class of inhibitors of tubulin polymerization and
their action has been rationalized by the cell cycle arrest in G0 and G2/M phase.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Invitro cytotoxicity
Inhibition of tubulin polymerization
Cell cycle arrest
Pyrrolobenzodiazepine
Cinnamido component
1. Introduction
as improve the safety aspects in relation to the drugs that interact
on a single target [1e3], and one such recent example is of bleo-
Cancer remains one of the leading cause of death in the World
and as a result there is a pressing need for the development of novel
and effective treatments. Cancer cells differ from their normal
counterparts in a number of biochemical processes, particularly
during the control of cell growth and division. Despite major
breakthroughs in many areas of modern medicine over the past 100
years, the successful treatment of cancer remains a significant
challenge at the start of the 21st century. Mainly because it is
difficult to discover novel agents that selectively kill tumor cells or
inhibit their proliferation without the general toxicity, therefore the
use of traditional cancer chemotherapy is still very limited.
Currently, combination chemotherapy with different mechanisms
of action is one of the methods that is being adopted to treat cancer.
Therefore, a single molecule containing more than one pharma-
cophore, each with different mode of action could be beneficial for
the treatment of cancer. In recent years there has been growing
interest in the design of ligands that could act in a specific manner
on more than one target. The development of such hybrid mole-
cules not only lowers the risk of drugedrug interaction in
comparison to cocktails but also could enhance the efficacy as well
mycin [4].
Microtubules are important in mitosis and cell division; they
have been a target for the development of a number of new anti-
cancer drugs [5]. There are two major groups of these antitumor
agents, microtubule stabilizers such as paclitaxel [6] and microtu-
bule destabilizers such as colchicine [7], combretastatin A-4 [8], and
vinca alkaloids [9]. Combretastatin A-4 (CA-4, Fig. 1), isolated from
the bark of the South African tree Combretum caffrum, is one of the
well-known natural tubulin binding molecule that affects micro-
tubule dynamics [10]. The spatial relationship between the two
aromatic rings of combretastatin A-4 or colchicine and similar other
drugs is an important structural feature that determines their ability
to bind to tubulin [11]. A number of chalcones have been reported
that are similar to CA-4, as active antimitotic agents inhibiting
tubulin polymerization [12]. Similarly, there are also several reports
on the antiproliferative properties of chalcones substituted with
basic groups [13,14]. Xia and coworkers are among the first to
demonstrate the improved antiproliferative activity of chalcones
that have amino groups [15]. Further, Liu and coworkers have
synthesized piperidinyl chalcones as promising antiproliferative
agents, many of them have interesting activities (IC50 5
mM) and
they also disrupt the cell cycle at G1 and G2/M phases at these
concentrations. In contrast, chalcones without the basic substituent
have no effect on the cell cycle when tested at their IC50 [16].
* Corresponding author. Tel.: þ91 40 27193157; fax: þ91 40 27193189.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.041

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3870e3884
3871
Fig. 1. Chemical structures of tubulin polymerization inhibitors, PBDs, CinnamidoePBD Conjugates (4aed, 5aed and 6aed).
Whereas, pyrrolo[2,1-c][1,4]benzodiazepines [17] (PBDs), CC-
1065 and distamycin exhibit cytotoxic activity that is derived from
their ability to bind and monoalkylate DNA in the minor groove
[18,19]. Moreover, PBDs are a family of naturally occurring anti-
tumour agents that are known to interact with DNA in a sequences
elective manner. PBD monomers such as DC-81 (Fig. 1), anthra-
mycin and tomaymycin are known to monoalkylate DNA by cova-
lently binding to the N2 of a guanine base in the DNA minor groove
through their electrophilic N10eC11 imine (or equivalent carbi-
nolamine) moiety, a process which can block transcription and lead
to cytotoxicity [20]. Recently a large number of structurally modi-
fied PBDs have been synthesized that have exhibited improved
anticancer activity [21e27]. More recently, Hu and coworkers
reported that a PBD hybrid (IN6CPBD) can activate the apoptotic
pathway mediated by mitochondria and its continuation to the
possess not only high potency but also different binding sites, both
the aspects are likely to be useful for the treatment of tumors.
While piperazine is an attractive pharmacological scaffold that
is present in many important drugs [31]. It is well known that this
heterocyclic backbone could act on various pharmacological targets
and that display anticancer [32e39], calcium channel blocking
[40e43] and histamine antagonist properties [44,45].
We have been interested in the structural modifications of the
PBD ring system [46e49] apart from the development of new
synthetic strategies [50e52] for its preparation. Furthermore,
modifications of the ethylene bridge in the combretastatin based
compounds and changes in chalcones have led to interesting bio-
logical properties, particularly cytotoxicity. As mentioned, in view
of the importance of the piperazine moiety, it has been considered
of interest to incorporate this moiety in such new hybrids of PBD
which is also flanked by carbonyl functionalities. Moreover, in
another set of series, the aromatic ring of the chalcones has been
replaced by a morpholine moiety. This modified cinnamido
component has been linked to the PBD ring system through flexible
spacers with a view to explore their anticancer activity and to
examine their effect on the tubulin polymerization. Further this
report investigates the effect of these compounds on cell cycle
progression.
transcription factors like nuclear factor-kB (NF-kB) and activator
protein-1 (AP-1) [28,29]. The naturally occurring and structurally
modified PBDs are known for their potent anticancer activity,
however they have been precluded from clinical application due to
problems relating to solubility and cardiovascular side-effects [30].
Hence, the development of conjugates or hybrid molecules that
contain two types of cytotoxic moieties represent a potential
approach in the discovery of new antitumor agents, as they could

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A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3870e3884
O
O
O
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
Cl
b
N
N
a
NH
NBoc
OCH3
7
OCH3
OCH3
9
8
O
c
H3CO
H3CO
N
N
O
OCH3
10
OCH3
OH
Scheme 1. Preparation of Cinnamido promoiety 10. Reagents and conditions: a) TEA, N-Boc piperazine, dry THF, 0 ꢁC to RT, 2 h; b) CF3COOH, CHCl3, RT, 8 h; c) trans-3-methoxy-4-
hydroxy cinnamoyl chloride, TEA, dry THF, 0 ꢁC to RT, 2 h.
2. Results and discussion
15a. Further coupling of this with the cinnamido promoieties 10, 11
and 12 respectively in the presence of K2CO3 in acetonitrile produce
corresponding nitro compounds 16, 17 and 20. Further these upon
reduction with SnCl2$2H2O provide the aminothioacetal interme-
diates (18, 19 and 21). Finally deprotection of these amino-
thioacetals by employing HgCl2/CaCO3 affords the target imine
containing PBD conjugates (4, 5 and 6) as shown in Schemes 3 and 4.
2.1. Chemistry
The synthetic pathway (Scheme 1) involves the use of
commercially available 3,4,5-trimethoxy benzoic acid as the start-
ing material, which was treated with thionyl chloride and then
coupled to N-Boc piperazine in the presence of a base to give the
corresponding compound 8. This upon deprotection of boc by
employing TFA in CH2Cl2 gives the compound 9, which was
coupled with trans 4-hydroxy-3-methoxy cinnamic acid chloride
provides the required cinnamido promoiety 10.
Addition of trans 4-hydroxy-3-methoxy cinnamic acid chloride
to morpholine in the presence of triethylamine provides compound
11. Whereas, addition of trans 4-hydroxy-3-methoxy cinnamic acid
chloride to piperazine in the presence of base gives a dimer cin-
namido promoiety 12 as shown in Scheme 2.
The synthesis of cinnamido-pyrrolo[2,1-c][1,4]benzodiazepine
(PBD) conjugates (4, 5) and their dimers (6) was carried out starting
from the (2S)-N-(4-benzyloxy-5-methoxy-2-nitrobenzoyl)pyrroli-
dine-2-carboxaldehyde diethylthioacetal (13), which was prepared
by literature method [53] and this upon debenzylation provides
(2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-2-car-
boxaldehyde diethylthioacetal precursor (14). Etherification of this
hydroxyl compound 14 with dibromopropane in the presence of
K2CO3 in acetone affords (2S)-N-[4-(3-bromopropoxy-5-methoxy-
2.2. Evoluation of biological activity
2.2.1. Anticancer activity
Compound 4a has been evaluated for the anticancer activity
against a panel of 60 human cancer cell lines of the National Cancer
Institute (NCI), Bathesda and the results are shown in Table 1.
Compound 4a is remarkably potent with GI50 values ranging from
68 to 732 nM towards 53 of the 59 human cancer cell lines tested.
This compound is active between 73 and 695 nM in all leukemia,
CNS, prostrate and melanoma cancer cell lines. Interestingly it is
highly active against some specific cell lines like CCRF-CEM
(leukemia), NCI-H522 (non-small cell lung cancer), and MCF7,
MDA-MB-468 (breast cancer). The mean graph midpoint values of
compound 4a are log10 GI50 (ꢀ6.48), log10 TGI (ꢀ5.4) and log10
LC50 (ꢀ4.43) and these results indicate that 4a possesses potent
broad-spectrum anticancer activity.
The promising activity of compound 4a prompted us to evaluate
the anticancer activity of other related compounds 4bed, 5aed and
6aed in selected human cancer cell lines like A-549, Hop62 (lung);
2-nitrobenzoyl)]pyrrolidine-2-carboxaldehyde
diethylthioacetal
O
H3CO
N
a
O
11
HO
O
O
H3CO
HO
Cl
H3CO
HO
N
b
N
O
12
OCH3
OH
Scheme 2. Preparation of Cinnamido promoieties11 and 12. Reagents and conditions: a) dry THF, morpholine, TEA, 0 ꢁC to RT, 2 h; b) dry THF, piperazine, TEA, 0 ꢁC to RT, 2 h.

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3870e3884
3873
polymerization. One of the possible explanations of compounds
showing anticancer activity and cell cycle arrest is the inhibition of
tubulin polymerization to functional microtubules as it is observed
with antimitotic agents such as nocodazole and cholchicine. As
tubulin subunits heterodimerize and self-assemble to form
microtubules in a time dependent manner, we have investigated
the progression of tubulin polymerization by monitoring the
absorbance at 340 nm in 384 well plate for 1 h at 37 ꢁC with and
without the compounds at 2.5 mM (final concentration) concen-
tration solubilized in DMSO. Amongst the ten molecules examined,
5b and 6b inhibited tubulin polymerization to nearly 40% compared
to controls and a similar pattern of inhibition has been observed
with the positive control, nocodozole (Fig. 2). The compounds 5b
and 6b also demonstrated a dose dependent inhibition with IC50
values of w16.4 mM and w27.9 mM respectively.
2.2.3. Immunohistochemistry of tubulin
In order to substantiate the observed in vitro effects of the
compoundson theinhibitionof tubulinpolymerization tofunctional
microtubules, immunohistochemistry studies have been carried out
to examine the in situ effects of compounds 5b and 6b on cellular
microtubules and their special arrangement. In this study, untreated
human breast cancer cells (MCF-7) displayed the normal distribu-
tion of microtubules (Fig. 3a). However, cells treated with 2.5 mM
concentration of compounds 5b, 6b and nocodazole demonstrated
disrupted microtubule organization as seen in Fig. 3bed, thus
exemplifying the inhibition of tubulin polymerization.
2.2.4. Cell cycle analysis and apoptosis (flow cytometry)
Since the compounds 5b and 6b mediate their cytotoxic effects by
inhibiting the polymerization of tubulin, we have examined whether
the same could be reflected in the pattern of cell cycle arrest.
Towards this, the cell cycle dependent DNA content was determined
by flow cytometry using propidium iodide staining according to the
standard protocol. The effect of compounds 5b and 6b (2
with positive controls i.e., nocodazole and podophyllotoxin (1
m
M) along
Scheme 3. Preparation of CinnamidoePBD conjugates 4aed and 5aed. Reagents and
conditions: a) EtSH, BF3$OEt2, CH2Cl2, 12 h; b) dibromoalkanes, K2CO3, acetone, reflux,
48 h; (c) K2CO3, acetonitrile, 18 h, reflux, 65e73%; (d) SnCl2$2H2O, MeOH, reflux, 4 h; e)
HgCl2, CaCO3, CH3CN$H2O (4:1), RT, 12 h 60e65%.
mM)
were examined in MCF-7 breast cancer cells treated individually
with these compounds for 48 h. The results demonstrate that there is
an increase in G0 and G2/M phase in cells treated with compounds
5b and 6b compared to the controls. Although the effect of 5b and 6b
at G0 phase is comparable tothatof nocodazole and podophyllotoxin
(Figs. 4 and 5), they have demonstrated a less pronounced effect at
G2/M phase possibly due to the presence of two distinctively
different functional moieties, ie., PBD and chalcones. Nevertheless,
these results indicate that the induction of cellular apoptosis by 5b
and 6b could be by virtue of their inhibitory effects on tubulin
polymerization as there is an increase in cell cycle arrest at meta-
phase with parallel increase in apoptotic cells at G0 phase.
KB, GURAV, DWD (oral); SiHa (cervix); MCF7 and ZR75-1 (breast);
Colon205 (colon); PC3 (prostate) and A2780 (ovarian) by employ-
ing the sulforhodamine B (SRB) assay. The results described in
Table 2 show that all the compounds are significantly cytotoxic
with the concentration of the drug that produced 50% inhibition of
cell growth (GI50) ranging from <0.1 to 2.5
compounds synthesized, compounds 5c, 5d, 6b and 6d displayed
significant cytotoxicity with a GI50 value <0.1 M against six cell
mM. Among all the
m
lines of the eleven cell lines examined. Similarly, compounds 5a
and 6a (in five cell lines), 6c (in four cell lines), 4b and 4c (in two cell
lines) and 5b (in one cell line) have shown promising anticancer
2.2.5. Effect of 5b and 6b on nuclear condensation
activity with a GI50 value <0.1
m
M.
Since several of the microtubule disrupting agents are also
known to induce apoptosis in cells. We have analyzed the cellular
effects of compounds 5b and 6b on nuclear condensation by
Hoechst staining. The results indicate that compared to controls,
cells treated with compounds 5b and 6b that show a dose depen-
The anticancer activity of these new PBD conjugates has been
compared to DC-81 (PBD monomer) and it is interesting to observe
that all these conjugates possess betteractivity profile than DC-81 as
shown inTable 2. The cinnamido moieties (10 and 11) have also been
evaluated for their anticancer potential and it is observed that they
do not exhibit any significant activity (GI50 > 10ꢀ4). These results
clearly indicate that the hybrid compounds are more effective than
their individual moieties towards their anticancer potential and also
emphasized that linking/combination approach certainly has
benefits in designing in superior anticancer compounds.
dent effect on nuclear condensation between 1 and 3
However, significant nuclear condensation is observed with noco-
dazole and podophyllotoxin even at 2 M concentration (Fig. 6).
mM (Fig. 6).
m
These results clearly demonstrate that compounds 5b and 6b are
effective in inducing cellular apoptosis.
3. Conclusion
2.2.2. Inhibition of tubulin polymerization
Since these ten new conjugates possess a chalcone subunit, it
has been considered of interest to investigate their effect on tubulin
In conclusion, we have designed and synthesized new cinna-
mido- pyrrolobenzodiazepine conjugates and their dimers. These

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A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3870e3884
Scheme 4. Preparation of CinnamidoePBD dimers 6aed. Reagents and conditions: a) K2CO3, acetonitrile, reflux, 18 h; 63e75% b) SnCl2$2H2O, MeOH, reflux, 4 h; c) HgCl2, CaCO3,
CH3CN$H2O (4:1), RT, 12 h 60e63%.
compounds exhibit significant anticancer activity with GI50 values
ranging from nanomolar to micromolar concentrations. Among
these compounds 5b and 6b have shown potent antitubulin activity
in vitro as well as disruption of microtubule organization within
cells. Further, cell cycle analysis indicated that these compounds
induced cell cycle arrest at metaphase with a concomitant increase
in apoptotic cells. The compounds 5b and 6b have also shown
a significant increase in apoptotic cells as observed by DNA
condensation and fragmented nuclei. Cytotoxic assays employing
individual moieties present in 5b and 6b along with DC-81 clearly
indicate that the hybrid molecules (5b and 6b) are more potent
than their individual moieties. These results provide an insight for
future direction in the development of such conjugates.
4.1. Synthesis of cinnamido-PBD conjugates
4.1.1. tert-Butyl-4-(3,4,5-trimethoxybenzoyl)-1-
piperazinecarboxylate (8)
To a stirred solution of N-boc piperazine (372 mg, 1.0 mmol) in
dry THF was added triethylamine (1.1 mL, 4.0 mmol) followed by
3,4,5-trimethoxy benzoylchloride (458 mg, 1.0 mmol) at 0 ꢁC. The
reaction mixture was stirred for 2 h and the reaction was monitored
by TLC. After completion of the reaction, THF was evaporated under
vacuum to get the crude product. This was further purified by
column chromatography (30% EtOAceHexane) to afford the pure
compound 8 (Yield 646 mg, 85%). Rf ¼ 0.58 (EtOAceHexane 2:8); 1H
NMR (300 MHz, CDCl3): d 6.59 (s, 2H, ArH), 3.88 (s, 6H, OCH3), 3.84
(s, 3H, OCH3), 3.44e3.66 (m, 8H, NeCH2e), 1.47 ppm (s, 9H, 3CH3);
4. Experimental protocols
ESI-MS: m/z 382 [M þ 1]þ
Reaction progress was monitored by thin-layer chromatography
(TLC) using GF254 silica gel with fluorescent indicator on glass
plates. Visualization was achieved with UV light and iodine vapor
unless otherwise stated. Chromatography was performed using
Acme silica gel (100e200 and 60e120 mesh). The majority of
reaction solvents were purified by distillation under nitrogen from
the indicated drying agent and used fresh: dichloromethane
(calcium hydride), tetrahydrofuran (sodium benzophenone ketyl),
methanol (magnesium methoxide), and acetonitrile (calcium
hydride). 1H NMR spectra were recorded on Varian Gemini
200 MHz and Avance 300 MHz spectrometer using tetramethyl
silane (TMS) as an internal standard. Chemical shifts are reported in
parts per million (ppm) down field from tetramethyl silane. Spin
multiplicities are described as s (singlet), d (doublet), t (triplet),
q (quartet), m (multiplet). Coupling constants are reported in Hertz
(Hz). Optical rotations are measured on Horiba, high sensitive
polarimeter, SEPA-300.
4.1.2. Piperazino(3,4,5-trimethoxyphenyl)methanone (9)
To a solution of boc-protected compound 8 (660 mg, 1 mmol) in
dry dichloromethane was added trifluoroacetic acid (1.73 mL) at
0
ꢁC and stirred under nitrogen for 12 h, the reaction mixture was
concentrated under vacuum to afford compound 9 and then it was
used directly in the next step.
4.1.3. (E)-3-(4-Hydroxy-3-methoxyphenyl)-1-[4-(3,4,5-
trimethoxybenzoyl)piperazino]-2-propen-1-one (10)
To a stirred solution of piperazino(3,4,5-trimethoxyphenyl)
methanone (840 mg,1.0 mmol) in dry THF was added triethylamine
(1.5 mL) followed by 4-hydroxy-3-methoxy cinnamic acid chloride
in dry THF (636 mg, 1.0 mmol) at 0 ꢁC. The reaction mixture was
stirred for 2 h and the reaction was monitored by TLC. After
completion of the reaction, THF was evaporated under vacuum to
get the crude product. This was further purified by column chro-
matography (98:2 CHCl3eMeOH) to afford the pure compound 10.

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3870e3884
3875
Table 1
In vitro cytotoxicity of the compound 4a on selected human cancer cell lines.a
Cancer panel/cell line GI50 (
m
M) 4a Cancer panel/cell line GI50 (mM) 4a
Leukemia
Renal
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
SR
0.08
0.18
0.69
0.23
0.28
786-0
A498
0.27
0.19
2.54
2.11
0.54
0.27
0.29
2.59
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Non-small cell lung
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Prostate
PC-3
DU-145
Fig. 2. Effect of compounds on tubulin polymerization: tubulin polymerization assay
was carried out in a reaction mixture that contained PEM buffer, GTP (1 mM) and in
the presence or absence of test compounds at 2.5 mM concentration. The reaction was
initiated by the addition of GTP to all the wells. Tubulin polymerization was monitored
by the increase in absorbance at 340 nm using Dynex multimode plate reader at 37 ꢁC.
Absorbance was recorded at every 2 min intervals for up to 1 h. Nocodazole was used
as positive control. Data shown are the representative of three separate experiments.
1.89
0.72
0.23
0.49
0.31
0.26
0.34
0.24
0.06
0.73
0.33
Ovarian
IGROV1
0.24
0.21
1.07
0.29
0.45
0.61
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
(Yield 1.14 g, 84%): Rf ¼ 0.62 (EtOAceHexane 8:2); 1H NMR
(300 MHz, CDCl3)
d
: 7.54 (d, 1H, J ¼ 15.10 Hz, olefinic-H), 7.02 (d, 1H,
Colon
Breast
MCF7
J ¼ 7.55 Hz, ArH), 6.90 (s, 1H, ArH), 6.81 (d, 1H, J ¼ 8.30 Hz, ArH),
6.53e6.64 (m, 3H ArH and Olefinic-H), 5.86e5.99 (s, H, OH), 3.87
(s, 3H, OCH3); 3.81 (s, 6H, OCH3), 3.79 (s, 3H, OCH3),
3.53e3.74 ppm (m, 8H, NeCH2e). ESI-MS: m/z 457 [M þ 1]þ
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
0.19
0.24
0.17
2.98
0.26
0.21
0.17
0.08
NCI/ADR-RES
MDA-MB-
231/ATCC
HS 578T
MDA-MB-435
BT-549
8.91
0.23
0.23
0.18
0.15
0.23
0.07
KM12
SW-620
4.1.4. (E)-3-(4-Hydroxy-3-methoxyphenyl)-1-morpholino-2-
propen-1-one (11)
T-47D
MDA-MB-468
To a stirred solution of morpholine (372 mg, 1.0 mmol) in dry
THF was added triethylamine (1.1 mL, 4.0 mmol) followed by 4-
hydroxy-3-methoxy cinnamic acid chloride in dry THF (458 mg,
1.0 mmol) at 0 ꢁC. The reaction mixture was stirred for 2 h and the
reaction was monitored by TLC. After completion of the reaction,
THF was evaporated under vacuum to get the crude product. This
was further purified by column chromatography (70%
EtOAceHexane) to afford the pure compound 11 (Yield 646 mg,
CNS
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
0.24
0.31
0.24
0.38
0.21
0.18
0.24
0.17
0.32
0.26
0.20
0.19
0.35
0.16
85%): Rf ¼ 0.59 (EtOAceHexane 7:3); 1H NMR (300 MHz, CDCl3)
d:
7.64 (d, 1H, J ¼ 15.42 Hz, olefinic-H); 7.08 (d, 1H, J ¼ 8.08 Hz, ArH);
6.99 (s, 1H, ArH); 6.89 (d, 1H, J ¼ 8.08 Hz, ArH); 6.68 (d, 1H,
J ¼ 14.69 Hz, olefinic-H); 6.03 (s, 1H, OH); 3.93 (s, 3H, OCH3);
3.62e3.72 ppm (m, 8H, morpholine-H). ESI-MS: m/z 264 [Mþ]
a
Data obtained from NCI’s in vitro anticancer activity cells screen.
Table 2
GI50 values (in m
M) for compounds 4bed, 5aed & 6aed in selected human cancer cell lines.a
Compd
ZR-75b
A-549c
A2870d
Hop62c
KBe
SiHaf
Guravc
MCF7b
Colo205g
DWDe
PC3h
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
DC-81
ADR
0.15
0.14
0.21
0.12
0.15
<0.1
0.14
<0.1
0.15
<0.1
0.21
2.37
0.12
0.12
0.11
0.16
<0.1
0.11
<0.1
<0.1
<0.1
<0.1
0.11
0.15
0.16
<0.1
0.15
0.11
0.18
<0.1
0.12
0.14
<0.1
0.12
<0.1
0.16
<0.1
0.14
<0.1
0.17
0.15
2.6
0.15
0.12
1.9
<0.1
0.13
0.15
<0.1
0.14
<0.1
2.0
<0.1
0.17
0.14
2.5
0.12
0.14
0.17
<0.1
0.16
<0.1
2.0
0.11
0.13
1.9
0.10
0.13
0.16
<0.1
0.14
<0.1
2.3
0.14
0.13
0.18
<0.1
0.14
<0.1
0.15
<0.1
0.15
1.0
<0.1
<0.1
2.1
0.11
<0.1
0.17
<0.1
<0.1
<0.1
0.15
<0.1
0.12
<0.1
<0.1
1.49
0.12
<0.1
0.14
1.9
0.13
0.14
0.14
0.16
0.17
<0.1
0.15
<0.1
0.18
<0.1
0.15
0.12
0.10
0.12
<0.1
0.15
<0.1
0.15
<0.1
<0.1
0.11
0.13
<0.1
0.17
0.15
0.16
<0.1
<0.1
0.17
0.15
<0.1
0.16
0.14
1.9
0.17
<0.1
0.15
0.20
0.16
0.17
0.13
a
50% Growth inhibition and the values are mean of three determinations.
Breast cancer.
Lung cancer.
Ovarian cancer.
Oral cancer.
Cervix cancer.
Colon cancer.
Prostate cancer.
b
c
d
e
f
g
h
ADR is adriamycin.

3876
A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3870e3884
Fig. 3. Immunohistochemistry analysis of microtubules: MCF-7 cells were grown on glass cover slips; incubated for 48 h with Nocodazole (b) 5b (c) and 6b (d) at 2.5
mM
concentration, untreated cells were considered as controls (a). Following the termination of incubation, cells were fixed and immunohistochemistry was performed as described in
the “Experimental Section” employing anti tubulin (mouse monoclonal) antibody followed by FITC conjugated secondary antibody. Photographs were taken using Olympus confocal
microscope. Data is the representative of pictures.
4.1.5. (2E,20E)-1,10-(Piperazine-1,4-diyl)bis(4-hydroxy-3-
methoxyphenyl)prop-2-en-1-one (12)
acetate (3 ꢂ 25 mL). The combined organic phases were washed
with saturated brine (1 ꢂ 25 mL), dried over Na2SO4 and the
solvent removed under vacuum to afford the crude product. This
was further purified by column chromatography using
EtOAcehexane (7:3) as eluent to afford compound 14 (yield
300 mg, 75%): Rf ¼ 0.59 (EtOAcehexane 7:3); 1H NMR (300 MHz,
To a stirred solution of piperazine (200 mg, 1.0 mmol) in dry THF
was added triethylamine (1.1 mL, 4.0 mmol) followed by
4-hydroxy-3-methoxy cinnamic acid chloride in dry THF (1.06 g,
2.0 mmol) at 0 ꢁC. The reaction mixture was stirred for 2 h and the
reaction was monitored by TLC. After completion of the reaction,
THF was evaporated under vacuum to get the crude product. This
was further purified by column chromatography (100% EtOAc) to
afford the pure compound (Yield 1.64 g, 75%): Rf ¼ 0.59 (EtOAc); mp
CDCl3)
d
: 7.6 (s, 1H, ArH), 6.75 (s, 1H, ArH), 4.85 (d, 1H, J ¼ 7.0 Hz,
eCHe), 4.60e4.70 (m, 1H, eCHe), 3.95 (s, 3H, OCH3), 3.20e3.32
(m, 2H, NCH2e), 2.70e2.88 (m, 4H, eCH2e), 1.75e2.35 (m, 4H,
eCH2e), 1.20e1.40 (m, 6H eCH3). EI-MS: m/z 400 [Mþ]
152e154 ꢁC; 1H NMR (300 MHz, CDCl3)
d
: 7.59 (d, 2H, J ¼ 14.69 Hz,
olefinic-H), 7.08 (d, 2H, J ¼ 10.28 Hz, ArH), 6.92 (d, 2H, J ¼ 12.48 Hz,
ArH), 6.85 (s, 2H, ArH), 6.64 (d, 2H, J ¼ 15.42 Hz, olefinic-H), 5.76
(brs, 2H, OH), 3.95 (s, 6H, OCH3), 3.42e3.70, ppm (m, 8H, NCH2).
ESI-MS: m/z 439 [Mþ]
4.1.7. (2S)-N-[4-(3-Bromopropoxy-5-methoxy-2-nitrobenzoyl)]
pyrrolidine-2-carboxaldehyde diethylthioacetal (15a)
To a solution of compound 14 (400 mg, 1 mmol) in dry acetone
(15 mL) was added, anhydrous K2CO3 (553 mg, 4 mmol), 1,3-
dibromopropane (256 mg,1.2 mmol) and the mixture was stirred at
reflux temperature for 48 h. The reaction was monitored by TLC
using EtOAcehexane (2:8). After completion of the reaction as
indicated by TLC, K2CO3 was removed by filtration and the solvent
was evaporated under reduced pressure, diluted with 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
EtOAcehexane (2:8) to afford compound 15a as yellow liquid (Yield
440 mg, 96%): Rf ¼ 0.61 (EtOAcehexane 2:8); 1H NMR (300 MHz,
4.1.6. (2S)-N-(4-Hydroxy-5-methoxy-2-nitrobenzoyl)pyrrolidine-
2-carboxaldehyde diethylthioacetal (14)
To a stirred solution of EtSH (1.91 g, 19.0 mmol) and BF3.OEt2
(1.41 g, 10 mmol) in dichloromethane was added drop wise to the
solution of the compound 13 (0.490 g, 1 mmol) in dichloromethane
(10 mL) at room temperature. Stirring was continued until TLC
indicated completion of the reaction. The solvent was evaporated
under vacuum. The residue, thus obtained was quenched with
bicarbonate solution (2 ꢂ 25 mL) and then extracted with ethyl

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3870e3884
3877
Fig. 4. Effect on Cell cycle and apoptosis: (A) MCF-7 breast cancer cells. In 6 well plates were treated with compounds 5b (2
(1
m
M), 6b (2
m
M), nocodozole (1
m
M) and podophyllotoxin
m
M) for 48 h. Cells were trypsinized, washed with PBS and were fixed in 70% ethanol. Before analysis, cells were finely dispersed and stained with propidium iodide as described
in the “Experimental Section”. Stained cells were analyzed by flow cytometry. (B) The percentage of cells treated with compounds 5b, 6b, nocodozole and podophyllotoxin at
different phases of cell cycle.
CDCl3):
d 7.65 (s, 1H, ArH), 6.80 (s, 1H, ArH), 4.82e4.87 (d, 1H,
J ¼ 4.3 Hz, eCHe), 4.60e4.71 (m, 1H, eCHe), 3.98e4.10 (t, 2H,
J ¼ 6.0 Hz, OCH2), 3.95 (s, 3H, OCH3), 3.52e3.59 (t, 2H, J ¼ 6.2 Hz,
eCH2), 3.15e3.30 (m, 2H, eCH2e), 2.60e2.90 (m, 4H, eCH2e),
1.70e2.40 (m, 6H, eCH2e), 1.21e1.45 ppm (m, 6H, eCH3). ESI-MS:
m/z 543 [M þ Na]
8
7
Control
5b
6b
4.1.8. (2S)-N-[4-(4-Bromobutoxy-5-methoxy-2-nitrobenzoyl)]
pyrrolidine-2-carboxaldehyde diethylthioacetal (15b)
6
5
4
3
Nocodazole
Podophyllotoxin
The compound 15b was prepared following the method
described for the compound 15a, employing 1,4-dibromobutane
(226 mg, 1.2 mmol) and anhydrous K2CO3 (483 mg, 4 mmol) and
the crude product was purified by column chromatography to
afford the compound 15b as yellow liquid (Yield 421 mg, 90%):
Rf ¼ 0.55 (EtOAcehexane 2:8); 1H NMR (300 MHz, CDCl3):
d 7.71 (s,
1H, ArH), 6.92 (s, 1H, ArH), 4.79e4.80 (d, 1H, J ¼ 4.3 Hz, eCHe),
4.61e4.70 (m, 1H, eCHe), 3.98e4.10 (t, 2H, J ¼ 6.0 Hz, OCH2), 3.95
(s, 3H, OCH3), 3.55e3.60 (t, 2H, J ¼ 6.2 Hz, eCH2), 3.17e3.32 (m, 2H,
eCH2e), 2.60e2.90 (m, 4H, eCH2e), 1.74e2.48 (m, 8H, eCH2e),
1.19e1.43 ppm (m, 6H, eCH3). ESI-MS: m/z 557 [M þ Na]
2
1
0
4.1.9. (2S)-N-[4-(5-Bromopentoxy-5-methoxy-2-nitrobenzoyl)]
pyrrolidine-2-carboxaldehyde diethylthioacetal (15c)
The compound 15c was prepared following the method
described for the compound 15a, employing 1,5-dibromopentane
(282 mg,1.2 mmol) and anhydrous K2CO3 (565 mg, 4 mmol) and the
crude product was purified bycolumn chromatography to afford the
compound 15c as yellow liquid (Yield 512 mg, 91%): Rf ¼ 0.59
G1
G0
S
G2/M
Fig. 5. Effect of nocodozole, podophyllotoxin and the compounds 5b and 6b on
distribution of cells at various stages of cell cycle.
(EtOAcehexane 2:8); 1H NMR (300 MHz, CDCl3):
d 7.73 (s, 1H, ArH),

3878
A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3870e3884
Fig. 6. Analysis of nuclear morphology: MCF-7 cells were grown on cover slips in 6 well plates were treated with compounds 5b (1e3
podophyllotoxin (1 M) for 48 h. Following the termination of incubation, cells were incubated with Hoechst (5 M) for 30 min. Later, cells were washed thrice with PBS and
photographs were taken using Olympus fluorescence microscope equipped with DAPI filter settings. Data is the representative of five different fields of view.
mM), 6b (1e3 mM), nocodozole (1 mM) and
m
m
6.97 (s, 1H, ArH), 4.77e4.82 (d, 1H, J ¼ 4.3 Hz, eCHe), 4.59e4.69 (m,
1H, eCHe), 3.96e4.09 (t, 2H, J ¼ 6.0 Hz, OCH2), 3.94 (s, 3H, OCH3),
3.52e3.60 (t, 2H, J ¼ 6.2 Hz, eCH2), 3.18e3.33 (m, 2H, eCH2e),
2.62e2.98 (m, 4H, eCH2e), 1.73e2.49 (m, 8H, eCH2e),
1.19e1.45 ppm (m, 8H, eCH2e and eCH3). ESI-MS: m/z 571 [M þ Na]
After completion of the reaction, K2CO3 was removed by suction
filtration and the solvent was evaporated under reduced pressure to
get the crude product. This was further purified by column chroma-
tography (98:2 CHCl3eMeOH) to afford the pure compound 16a
(Yield 672 mg, 75%): Rf ¼ 0.61 (MeOHeCHCl3 2:98); Light yellow
solid. mp 130e132 ꢁC; 1H NMR (300 MHz, CDCl3)
d: 7.66 (d, 1H,
4.1.10. (2S)-N-[4-(6-Bromohexyloxy-5-methoxy-2-nitrobenzoyl)]
pyrrolidine-2-carboxalde-hyde diethylthioacetal (15d)
J ¼ 14.95Hz, olefinic-H);7.50 (s, 2H, ArH); 7.02 (s,1H, ArH);6.87e6.97
(m, 2H, ArH); 6.81 (s, 2H, ArH); 6.40 (d, 1H, J ¼ 15.42 Hz, olefinic-H);
4.86 (d,1H, J ¼ 3.67 Hz, eCHe); 4.61e4.76 (m,1H, eCHe); 4.24e4.38
(m, 4H, OCH2); 3.92 (s, 6H, OCH3); 3.88 (m, 8H, NCH2); 3.84 (s, 9H,
OCH3); 3.16e3.32 (m, 2H, eCH2e); 2.63e2.88 (m, 4H, eSCH2);
1.76e2.45(m, 4H, eCH2e);1.56e1.67 (m, 2H,eCH2e);1.22e1.40 (m,
The compound 15d was prepared following the method described
for the compound 15a, employing 1,6-dibromohexane (285 mg,
1.2 mmol) and anhydrous K2CO3 (507 mg, 4 mmol) and the crude
product was purified by column chromatography to afford the
compound 15d as yellow liquid (Yield 512 mg, 93%): Rf ¼ 0.60
6H, eCH3).13C NMR (75 MHz, CDCl3):
d 170.3,154.3,148.7,143.7,137.1,
(EtOAcehexane 2:8); 1H NMR (300 MHz, CDCl3):
d
7.69 (s, 1H, ArH),
130.1, 129.3, 128.1, 127.3, 126.7, 121.8, 119.7, 112.7, 115.4, 114.0, 112.2,
110.0, 109.4, 108.6, 66.1, 65.1, 60.9, 55.2, 56.3, 52.5, 50.5, 30.9, 29.5,
26.7, 24.7, 22.3, 14.8. ESI-MS: m/z 897 [Mþ]
6.94 (s, 1H, ArH), 4.71e4.82 (d, 1H, J ¼ 4.3 Hz, eCHe), 4.51e4.67 (m,
1H, eCHe), 3.98e4.11 (t, 2H, J ¼ 6.0 Hz, OCH2), 3.93 (s, 3H, OCH3),
3.54e3.62 (t, 2H, J ¼ 6.2 Hz, eCH2), 3.14e3.31 (m, 2H, eCH2e),
2.60e2.97 (m, 4H, eCH2e), 1.75e2.50 (m, 8H, eCH2e),
1.19e1.50 ppm (m, 10H, eCH2e andeCH3). ESI-MS:m/z585[Mþ Na]
4.1.12. (2S)-N-{4-[4-[2-Methoxy-4-(E)-3-oxo-3-[4-(3,4,5-
trimethoxybenzoyl)piperazino]-1-propenylphenoxy]butyloxy}-5-
methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde
4.1.11. (2S)-N-{4-[3-[2-Methoxy-4-(E)-3-oxo-3-[4-(3,4,5-
trimethoxybenzoyl)piperazino]-1-propenylphenoxy]propoxy}-5-
methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde
diethylthioacetal (16b)
The compound 16b was prepared following the method
described for the compound 16a, employing compound 10
(456 mg, 1 mmol) and the compound (2S)-N-[4-(4-bromobutyloxy-
5-methoxy-2-nitrobenzoyl)]pyrrolidine-2-carboxaldehyde diethyl-
thioacetal 15b (642 mg, 1.2 mmol) and the crude product was
purified by column chromatography to afford the compound 16b.
Yield 728 mg (80%): Rf ¼ 0.61 (MeOHeCHCl3 2:98); Light yellow
diethylthioacetal (16a)
Toastirredsolutionof(E)-3-(4-hydroxy-3-methoxyphenyl)-1-[4-
(3,4,5-trimethoxy benzoyl)piperazino]-2-propen-1-one 10 (456 mg,
1 mmol) in dry acetonitrile (20 mL) was added anhydrous K2CO3
(544 mg, 5.0 mmol) and compound (2S)-N-[4-(3-bromopropoxy-
5-methoxy-2-nitrobenzoyl)]pyrrolidine-2-carboxaldehyde diethyl-
thioacetal15a (625mg,1.2mmol). Thereactionmixturewasstirredat
reflux temperature for 12 h and the reaction was monitored by TLC.
solid. mp: 127e129 ꢁC; 1H NMR (300 MHz, CDCl3)
d: 7.67 (d, 1H,
J ¼ 14.57 Hz olefinic-H); 7.38 (s, 1H, ArH); 7.01e7.14 (m, 2H, ArH);
6.80e6.93 (m, 3H, ArH and olefinic-H); 6.64e6.76 (m, 2H, ArH);

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3870e3884
3879
4.88 (d, 1H, J ¼ 3.39 Hz, eCHe); 4.68e4.75 (m, 1H, eCHe);
4.13e4.26 (m, 4H, OCH2); 3.92 (s, 6H, OCH3); 3.88 (m, 9H, OCH3);
3.69e3.83 (m, 8H, NCH2); 3.19e3.33 (m, 2H, eCH2e); 2.67e2.88
(m, 4H, eCH2e); 2.23e2.34 (m, 2H, eCH2e); 2.04e2.15 (m, 2H,
eCH2e); 1.91e2.02 (m, 2H, eCH2e); 1.75e1.86 (m, 2H, eCH2e);
acetonitrile (20 mL) was added anhydrous K2CO3 (1.08 g, 5.0 mmol)
and compound (2S)-N-[4-(3-bromopropoxy-5-methoxy-2-nitro-
benzoyl)]pyrrolidine-2-carboxaldehyde diethyl thioacetal 15a
(1.25 g, 1.2 mmol). The reaction mixture was stirred at reflux
temperature for 12 h and the reaction was monitored by TLC. After
completion of the reaction, K2CO3 was removed by suction filtra-
tion and the solvent was evaporated under vacuum to get the crude
product. This was further purified by column chromatography
(99:1 CHCl3eMeOH) to afford the pure compound 17a.Yield: 1.09 g,
(78%); Rf ¼ 0.63 (MeOHeCHCl3 1:99); Light yellow solid. mp:
1.23e1.38 (m, 6H, eCH3). 13C NMR (75 MHz, CDCl3):
d 169.5, 166.1,
165.2,154.0, 152.7,149.3,148.8,147.7,142.7,138.2,136.2,130.1,128.7,
127.8, 121.8, 115.2, 113.2, 111.9, 109.6, 108.7, 107.9, 103.7, 68.2, 67.8,
60.3, 55.6, 52.1, 59.2, 35.5, 29.0, 26.6, 25.5, 24.0, 14.6. ESI-MS: m/z
911 [Mþ]
137e139 ꢁC; 1H NMR (300 MHz, CDCl3)
d: 7.71 (s, 1H, ArH); 7.60 (d,
4.1.13. (2S)-N-{4-[5-[2-Methoxy-4-(E)-3-oxo-3-[4-(3,4,5-
trimethoxybenzoyl)piperazino]-1-propenylphenoxy]pentoxy}-5-
methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde
1H, J ¼ 15.86 Hz, olefinic-H); 7.06 (d, 1H, J ¼ 8.30 Hz, ArH); 7.0
(s, 1H, ArH); 6.87 (d, 1H, J ¼ 8.30 Hz, ArH); 6.77 (s, 1H, ArH); 6.66 (d,
1H, J ¼ 15.10 Hz, olefinic-H); 4.84 (d, 1H, J ¼ 3.77 Hz, eCHe);
4.65e4.72 (m, 1H, eCHe); 4.35 (t, 2H, J ¼ 6.04 Hz, OCH2e); 4.26 (t,
2H, J ¼ 6.04 Hz, OCH2e) 3.94 (s, 3H, OCH3); 3.89 (s, 3H, OCH3);
3.61e3.77 (m, 8H, NCH2); 3.16e3.33 (m, 2H, eCH2e); 2.64e2.86
(m, 4H, eCH2e); 2.24e2.45 (m, 2H, eCH2e); 1.75e2.15 (m, 2H,
diethylthioacetal (16c)
The compound 16c was prepared following the method
described for the compound 16a, employing compound 10
(456 mg, 1 mmol) and the compound (2S)-N-[4-(5-bromopentoxy-
5-methoxy-2-nitrobenzoyl)]pyrrolidine-2-carboxaldehyde diethyl-
thioacetal 15c (658 mg, 1.2 mmol) and the crude product was
purified by column chromatography to afford the compound 16c.
Yield 721 mg (78%); Rf ¼ 0.61 (MeOHeCHCl3 2:98); Light yellow
eCH2e); 1.43e1.58 (m, 2H, eCH2e); 1.23e1.41 (m, 6H, eCH3). 13
NMR (75 MHz, CDCl3): 166.7, 165.7, 154.4, 149.9, 149.2, 148.1,
C
d
143.0, 137.2, 128.3, 121.8, 114.7, 112.9, 110.3, 109.2, 108.1, 66.8, 65.8,
64.9, 61.1, 56.4, 55.9, 52.9, 50.9, 28.9, 26.8, 24.6, 15.0. ESI-MS: m/z
704 [Mþ]
solid. mp: 124e126 ꢁC; 1H NMR (300 MHz, CDCl3)
d: 7.67 (d, 1H,
J ¼ 14.92 Hz, olefinic-H); 7.51 (s, 2H, ArH); 7.04 (s, 1H, ArH);
6.88e6.96 (m, 2H, ArH); 6.83 (s, 2H, ArH); 6.42 (d, 1H, J ¼ 15.39 Hz,
olefinic-H); 4.85 (d, 1H, J ¼ 3.64 Hz, eCHe); 4.62e4.77 (m, 1H,
eCHe); 4.21e4.36 (m, 4H, eCH2e); 3.93 (s, 6H, OCH3); 3.89 (m, 8H,
NCH2); 3.85 (s, 9H, OCH3); 3.15e3.33 (m, 2H, eCH2e); 2.64e2.87
(m, 4H, eCH2e); 1.77e2.48 (m, 4H, eCH2e); 1.50e1.72(m, 4H,
eCH2e); 1.21e1.42 (m, 6H, eCH3). 13C NMR (75 MHz, CDCl3):
4.1.16. (2S)-N-{4-[4-[2-Methoxy-4-[(E)-3-morpholino-3-oxo-1-
propenyl]phenoxy]butyloxy}-5-methoxy-2-nitrobenzoyl}
pyrrolidine-2-carboxaldehyde diethylthioacetal (17b)
The compound 17b was prepared following the method
described for the compound 17a, employing compound 11
(526 mg, 1 mmol) and the compound (2S)-N-[4-(4-bromobuty-
loxy-5-methoxy-2-nitrobenzoyl)]pyrrolidine-2-carboxaldehyde
diethylthioacetal 15b (1.28 g, 1.2 mmol) and the crude product was
purified by column chromatography to afford the compound 17b.
Yield 1.06 g (74%); Rf ¼ 0.63 (MeOHeCHCl3 1:99); Light yellow
d
169.8, 159.6, 153.6, 147.4, 144.5, 136.9, 131.3, 129.8, 128.5, 127.9,
126.2, 122.3, 120.3, 115.7, 114.6, 112.5, 112.4, 110.2, 109.8, 108.2, 66.7,
65.4, 60.5, 56.8, 53.3, 50.7, 31.7, 29.8, 26.8, 24.9, 22.3, 21.0, 15.4, 14.8.
ESI-MS: m/z 925 [Mþ]
solid. mp: 134e136 ꢁC; 1H NMR (300 MHz, CDCl3)
d: 7.69 (s, 1H,
4.1.14. (2S)-N-{4-[6-[2-Methoxy-4-(E)-3-oxo-3-[4-(3,4,5-
trimethoxybenzoyl)piperazino]-1-propenylphenoxy]hexyloxy}-5-
methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde
ArH); 7.64 (d, 1H, J ¼ 15.26 Hz, olefinic-H); 7.09 (d, 1H, J ¼ 6.78 Hz,
ArH); 7.04 (s, 1H, ArH); 6.87 (d, 1H, J ¼ 8.46 Hz, ArH); 6.81 (s,
1H, ArH); 6.71 (d, 1H, J ¼ 15.26 Hz, olefinic-H); 4.87 (d, 1H,
J ¼ 3.39 Hz, eCHe); 4.68e4.74 (m, 1H, eCHe); 4.13e4.26 (m, 4H,
OCH2e); 3.92 (s, 3H, OCH3); 3.89 (s, 3H, OCH3); 3.64e3.81 (m, 8H,
NCH2); 3.18e3.34 (m, 2H, eCH2e); 2.67e2.88 (m, 4H, eCH2e);
1.92e2.33 (m, 6H, eCH2e); 1.73e1.87 (m, 2H, eCH2e); 1.29e1.42
diethylthioacetal (16d)
The compound 16d was prepared following the method
described for the compound 16a, employing compound 10
(456 mg,1 mmol) and the compound (2S)-N-[4-(6-bromohexyloxy-
5-methoxy-2-nitrobenzoyl)]pyrrolidine-2-carboxaldehyde diethyl-
thioacetal 15d (675 mg, 1.2 mmol) and the crude product was
purified by column chromatography to afford the compound 16d.
Yield 694 mg (74%); Rf ¼ 0.65 (MeOHeCHCl3 2:98); Light yellow
(m, 6H, eCH3). 13C NMR (75 MHz, CDCl3):
d 166.8, 166.0, 154.2,
149.9, 149.4, 148.4, 142.9, 137.0, 128.3, 121.8, 114.3, 112.2, 110.0,
109.0, 107.7, 69.3, 68.3, 66.7, 60.9, 56.5, 55.7, 52.9, 49.9, 45.8, 27.1,
26.0, 24.5, 14.8. ESI-MS: m/z 718 [Mþ]
solid mp: 121e123 ꢁC; 1H NMR (300 MHz, CDCl3):
d 7.66 (d, 1H,
J ¼ 14.87 Hz, olefinic-H); 7.41 (s, 1H, ArH); 7.03e7.14 (m, 2H, ArH);
6.83e6.94 (m, 3H, ArH); 6.62e6.75 (m, 2H, ArH and olefinic-H);
4.87 (d, 1H, J ¼ 3.37 Hz, eCHe); 4.64e4.73 (m, 1H, eCHe);
4.18e4.29 (m, 4H, eCH2e); 3.91 (s, 6H, OCH3); 3.89 (m, 9H, OCH3);
3.68e3.84 (m, 8H, NCH2); 3.20e3.34 (m, 2H, eCH2e); 2.68e2.89
(m, 4H, eCH2e); 2.25e2.37 (m, 2H, eCH2e); 2.06e2.17 (m, 4H,
eCH2e); 1.90e2.05 (m, 4H, eCH2e); 1.75e1.86 (m, 2H, eCH2e);
4.1.17. (2S)-N-{4-[5-[2-methoxy-4-[(E)-3-morpholino-3-oxo-1-
propenyl]phenoxy]pentyloxy}-5-methoxy-2-nitrobenzoyl}
pyrrolidine-2-carboxaldehyde diethylthioacetal (17c)
The compound 17c was prepared following the method
described for the compound 17a, employing compound 11 (526 mg,
1
mmol) and the compound (2S)-N-[4-(5-bromopentyloxy-5-
methoxy-2-nitrobenzoyl)]pyrrolidine-2-carboxaldehyde dieth-
1.23e1.38 (m, 6H, eCH3). 13C NMR (75 MHz, CDCl3):
d
169.2, 167.2,
ylthioacetal 15c (1.31 g, 1.2 mmol) and the crude product was
purified by column chromatography to afford the compound 17c.
Yield 1.05 g (72%); Rf ¼ 0.61 (MeOHeCHCl3 1:99); Light yellow
164.8, 153.9, 152.4, 150.2, 149.8, 148.7, 143.1, 137.8, 136.6, 131.2,
128.3, 127.4, 122.1, 115.6, 113.7, 112.7, 109.3, 108.4, 107.5, 103.6, 68.6,
67.5, 60.1, 55.4, 52.5, 51.2, 36.5, 29.8, 26.3, 25.7, 24.8, 24.2, 23.0, 14.6.
ESI-MS: m/z 939 [Mþ]
solid. mp: 131e133 ꢁC; 1H NMR (200 MHz, CDCl3)
d: 7.71 (s, 1H,
ArH); 7.62 (d, 1H, J ¼ 15.41 Hz, olefinic-H); 7.04 (d, 1H, J ¼ 8.10 Hz,
ArH); 7.03 (s, 1H, ArH); 6.86 (d, 1H, J ¼ 8.10 Hz, ArH); 6.75 (s, 1H,
ArH); 6.64 (d, 1H, J ¼ 15.41 Hz, olefinic-H); 4.84 (d, 1H, J ¼ 3.75 Hz,
eCHe); 4.63e4.71 (m, 1H, eCHe); 4.34 (t, 2H, J ¼ 6.32 Hz, OCH2e);
4.27 (t, 2H, J ¼ 6.32 Hz, OCH2e) 3.93 (s, 3H, OCH3); 3.90 (s, 3H,
OCH3); 3.59e3.75 (m, 8H, NCH2e); 3.15e3.35 (m, 2H, eCH2e);
2.61e2.84 (m, 4H, eCH2e); 2.25e2.47 (m, 2H, eCH2e); 1.78e2.17
4.1.15. (2S)-N-{4-[3-[2-Methoxy-4-[(E)-3-morpholino-3-oxo-1-
propenyl]phenoxy]propoxy}-5-methoxy-2-nitrobenzoyl}
pyrrolidine-2-carboxaldehyde diethylthioacetal (17a)
To a stirred solution of (E)-3-(4-hydroxy-3-methoxyphenyl)-1-
morpholino-2-propen-1-one 11 (526 mg,
1 mmol) in dry

3880
A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3870e3884
(m, 4H, eCH2e); 1.40e1.56 (m, 4H, eCH2e); 1.22e1.43 (m, 6H,
eCH3). 13C NMR (75 MHz, CDCl3):
167.2, 165.3, 154.7, 150.7, 149.7,
Yield 984 mg (73%); Rf ¼ 0.64 (MeOHeCHCl3 4:96); Light yellow
solid. mp: 138e140 ꢁC; 1H NMR (200 MHz, CDCl3):
7.71 (s, 2H,
d
d
148.4, 143.6, 137.5, 128.6, 121.4, 114.6, 112.7, 110.4, 109.5, 108.4, 66.7,
65.5, 64.8, 60.9, 56.6, 55.6, 52.7, 51.2, 28.7, 26.9, 24.8, 24.3, 23.2, 15.0.
ESI-MS: m/z 732 [Mþ]
ArH), 7.64 (d, 2H, J ¼ 15.30 Hz, olefinic-H), 6.97e7.14 (m, 4H, ArH),
6.79e6.96 (m, 4H, ArH), 6.73 (d, 2H, J ¼ 15.46 Hz, olefinic-H), 4.84
(d, 2H, J ¼ 3.77 Hz, eCHe), 4.61e4.74 (m, 2H, eCHe), 4.17e4.47
(m, 8H, OCH2e), 3.90 (s, 12H, OCH3), 3.56e3.84 (m, 8H, NCH2e),
3.11e3.32 (m, 4H, eCH2e), 2.62e2.87 (m, 8H, eCH2e), 1.72e2.44
(m, 6H, eCH2e), 116e1.47 (m, 12H, eCH3). 13C NMR (75 MHz,
4.1.18. (2S)-N-{4-[6-[2-methoxy-4-[(E)-3-morpholino-3-oxo-1-
propenyl]phenoxy]hexyloxy}-5-methoxy-2-nitrobenzoyl}
pyrrolidine-2-carboxaldehyde diethylthioacetal (17d)
CDCl3):
d 154.3, 150.1, 149.6, 147.7, 143.6, 136.8, 129.5, 128.4, 121.4,
The compound 17d was prepared following the method
described for the compound 17a, employing compound 11 (526 mg,
114.6, 112.8, 110.3, 109.7, 108.5, 66.7, 64.6, 60.8, 56.5, 52.7, 50.3,
31.8, 28.4, 26.9, 24.8, 23.4, 22.8, 15.2. ESI-MS: m/z 1347 [Mþ].
1
mmol) and the compound (2S)-N-[4-(6-bromohexyloxy-
5-methoxy-2-nitrobenzoyl)]pyrrolidine-2-carboxaldehyde dieth-
ylthioacetal 15d (1.35 g, 1.2 mmol) and the crude product was
purified by column chromatography to afford the compound 17d.
Yield 1.11 g (75%); Rf ¼ 0.64 (MeOHeCHCl3 1:99); Light yellow solid.
4.1.21. (2E,20E)-1,10-(Piperazine-1,4-diyl)bis(4-pentyloxy-3-
methoxy-phenyl-)prop-2-en-1-one dioxy]-bis [(2-nitro-5-methoxy-
1,4-phenylene)carbonyl]-bis[pyrrolidine-2-carboxaldehyde diethyl
thioacetal] (20c)
mp: 129e131 ꢁC; 1H NMR (200 MHz, CDCl3)
d: 7.70 (s,1H, ArH); 7.60
The compound 20c was prepared following the method
described for the compound 20a, employing compound 12
(439 mg, 1 mmol) and the compound (2S)-N-[4-(5-bromopenty-
loxy-5-methoxy-2-nitrobenzoyl)]pyrrolidine-2-carboxaldehyde
diethylthioacetal 15c (1.13 g, 2 mmol) and the crude product was
purified by column chromatography to afford the compound 20c.
Yield 1.01 g (74%); Rf ¼ 0.62 (MeOHeCHCl3 4:96); Light yellow solid.
(d, 1H, J ¼ 15.36 Hz, olefinic-H); 7.07 (d, 1H, J ¼ 6.23 Hz, ArH); 7.05
(s, 1H, ArH); 6.87 (d, 1H, J ¼ 8.26 Hz, ArH); 6.80 (s, 1H, ArH); 6.74 (d,
1H, J ¼ 15.36 Hz, olefinic-H); 4.85 (d, 1H, J ¼ 3.34 Hz, eCHe);
4.66e4.72 (m, 1H, eCHe); 4.15e4.27 (m, 4H, OCH2e); 3.92 (s, 3H,
OCH3); 3.88 (s, 3H, OCH3); 3.60e3.82 (m, 8H, NCH2); 3.19e3.36 (m,
2H, eCH2e); 2.67e2.89 (m, 4H, eCH2e); 1.93e2.31 (m, 6H,
eCH2e); 1.52e1.88 (m, 6H, eCH2e); 1.25e1.44 (m, 6H, eCH3). 13C
mp: 134e136 ꢁC; 1H NMR (200 MHz, CDCl3)
d: 7.69 (s, 2H, ArH), 7.63
NMR (75 MHz, CDCl3):
d
166.6, 165.7, 154.6, 150.7, 149.1, 148.7, 143.0,
(d, 2H, J ¼ 15.17 Hz, olefinic-H), 6.98e7.16 (m, 4H, ArH), 6.76e6.92
(m, 4H, ArH), 6.74 (d, 2H, J ¼ 15.47 Hz, olefinic-H), 4.85 (d, 2H,
J ¼ 3.77 Hz, eCHe), 4.62e4.75 (m, 2H, eCHe), 4.18e4.49 (m, 8H,
OCH2e), 3.92 (s, 12H, OCH3), 3.57e3.86 (m, 8H, NCH2), 3.14e3.35
(m, 4H, eCH2e), 2.64e2.88 (m, 8H, eCH2e), 1.67e2.48 (m, 6H,
eCH2e), 114e1.47 (m, 12H, eCH3). 13C NMR (75 MHz, CDCl3):
137.3, 128.7, 121.4, 114.7, 112.6, 110.3, 109.2, 107.5, 69.6, 68.2, 66.5,
60.7, 56.8, 55.4, 52.5, 49.6, 45.6, 28.3, 26.1, 24.5, 24.3, 23.6, 14.8. ESI-
MS: m/z 746 [Mþ]
4.1.19. (2E,20E)-1,10-(Piperazine-1,4-diyl)bis(4-propoxy-3-methoxy
phenyl)prop-2-en-1-one dioxy]-bis [(2-nitro-5-methoxy-1,4-
phenylene)carbonyl]-bis[pyrrolidine-2-carboxaldehyde
diethylthioacetal] (20a)
d
155.1, 149.8, 148.9, 147.6, 143.2, 137.5, 129.6, 128.2, 121.5, 114.3,
112.4, 110.3, 109.7, 108.6, 65.9, 64.7, 61.2, 56.7, 52.4, 50.3, 31.6, 28.8,
27.4, 26.3, 24.9, 24.1, 23.8, 15.1. ESI-MS: m/z 1375 [Mþ]
To a stirred solution of compound 12 (439 mg, 1 mmol) in dry
acetonitrile (20 mL) was added anhydrous K2CO3 (1.3 g, 5.0 mmol)
and compound (2S)-N-[4-(3-bromopropoxy-5-methoxy-2-nitro-
4.1.22. (2E,20E)-1,10-(Piperazine-1,4-diyl)bis(4-hexyloxy-3-
methoxy- phenyl-)prop-2-en-1-one dioxy]-bis[(2-nitro-5-methoxy-
1,4-phenylene)carbonyl]-bis[pyrrolidine-2-carboxaldehyde diethyl
thioacetal] (20d)
benzoyl)]pyrrolidine-2-carboxaldehyde
diethylthioacetal
15a
(1.08 g, 2 mmol). The reaction mixture was stirred at reflux
temperature for 12 h and the reaction was monitored by TLC. After
completion of the reaction, K2CO3 was removed by suction filtra-
tion and the solvent was evaporated under vacuum to get the crude
product. This was further purified by column chromatography
(96:4 CHCl3eMeOH) to afford the pure compound 20a. Yield: 1.0 g,
(76%); Rf ¼ 0.64 (MeOHeCHCl3 4:96); Light yellow solid. mp:
The compound 20d was prepared following the method
described for the compound 20a, employing compound 12
(439 mg,1 mmol) and the compound (2S)-N-[4-(6-bromohexyloxy-
5-methoxy-2-nitrobenzoyl)]pyrrolidine-2-carboxaldehyde dieth-
ylthioacetal 15d (1.16 g, 2 mmol) and the crude product was puri-
fied by column chromatography to afford the compound 20d. Yield
1.05 g (75%); Rf ¼ 0.60 (MeOHeCHCl3 4:96); Light yellow solid. mp:
140e142 ꢁC; 1H NMR (200 MHz, CDCl3)
d: 7.72 (s, 2H, ArH), 7.65 (d,
2H, J ¼ 15.10 Hz, olefinic-H), 6.99e7.15 (m, 4H, ArH), 6.78e6.94 (m,
4H, ArH), 6.72 (d, 2H, J ¼ 15.86 Hz, olefinic-H), 4.87 (d, 2H,
J ¼ 3.77 Hz, eCHe), 4.63e4.76 (m, 2H, eCHe), 4.16e4.48 (m, 8H,
OCH2e), 3.91 (s, 12H, OCH3), 3.58e3.84 (m, 8H, NCH2e), 3.12e3.33
(m, 4H, eCH2e), 2.63e2.89 (m, 8H, eCH2e), 1.70e2.45 (m, 2H,
eCH2e), 115e1.48 (m, 12H, eCH3). 13C NMR (75 MHz, CDCl3):
132e134 ꢁC; 1H NMR (200 MHz, CDCl3):
d 7.72 (s, 2H, ArH), 7.66 (d,
2H, J ¼ 15.48 Hz, olefinic-H), 6.97e7.13 (m, 4H, ArH), 6.78e6.95
(m, 4H, ArH), 6.72 (d, 2H, J ¼ 15.44 Hz, olefinic-H), 4.86 (d, 2H,
J ¼ 3.77 Hz, eCHe), 4.64e4.77 (m, 2H, eCHe), 4.20e4.49 (m, 8H,
OCH2e), 3.91 (s, 12H, eOCH3), 3.57e3.85 (m, 8H, NCH2e),
3.13e3.35 (m, 4H, eCH2e), 2.61e2.87 (m, 8H, eCH2e), 1.63e2.45
(m, 8H, eCH2e), 117e1.46 (m, 12H, eCH3). 13C NMR (75 MHz,
d
154.1, 149.6, 149.3, 148.0, 143.4, 137.0, 129.2, 128.0, 121.7, 114.0,
112.5, 110.0, 109.0, 108.2, 65.8, 64.9, 60.9, 56.3, 52.7, 50.1, 31.5, 28.7,
CDCl3): d 155.0, 149.4, 148.7, 148.1, 143.5, 137.3, 129.4, 128.6, 121.5,
26.5, 24.5, 22.6, 14.9. ESI-MS: m/z 1319 [Mþ]
114.4, 112.3, 110.5, 109.3, 108.7, 66.8, 64.7, 61.0, 56.4, 52.6, 50.5, 31.6,
28.9, 27.3, 26.5, 24.9, 23.8, 23.0, 21.4, 14.9. ESI-MS: m/z 1403 [Mþ]
4.1.20. (2E,20E)-1,10-(Piperazine-1,4-diyl)bis(4-butyloxy-3-methoxy
phenyl-)prop-2-en-1-one dioxy]-bis [(2-nitro-5-methoxy-1,4-
phenylene)carbonyl]-bis[pyrrolidine-2-carboxaldehyde diethyl
thioacetal] (20b)
The compound 20b was prepared following the method
described for the compound 20a, employing compound 12
(439 mg, 1 mmol) and the compound (2S)-N-[4-(4-bromobuty-
loxy-5-methoxy-2-nitrobenzoyl)]pyrrolidine-2-carboxaldehyde
diethylthioacetal 15b (1.11 g, 2 mmol) and the crude product was
purified by column chromatography to afford the compound 20b.
4.1.23. General procedure for the synthesis of compounds 18aed,
19aed and 21aed
The Nitro compound (16aed, 17aed and 20aed) (1 mmol)
dissolved in methanol (30 mL) and added SnCl2$2H2O (4 mmol)
was refluxed for 2 h or until the TLC indicated that reaction was
complete. The methanol was evaporated under vacuum and the
aqueous layer was then carefully adjusted to pH 8 with 10% NaHCO3
solution and then extracted with EtOAc (2 ꢂ 30 mL). The combined
organic phase was dried over Na2SO4 and evaporated under

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3870e3884
3881
vacuum to afford the corresponding amino compounds (18aed,
19aed and 21aed) as yellow liquid, which due to potential stability
problems, directly used in the next step.
Rf ¼ 0.56 (MeOHeCHCl3 5:95); White solid. mp: 112e114 ꢁC;
25
[a
]
¼ þ274 (c ¼ 0.5 in CHCl3); 1H NMR (300 MHz, CDCl3):
D
d
7.63e7.75 (m, 2H, imine-H and olefinic-H); 7.50 (s, 1H, ArH);
6.98e7.16 (m, 2H, ArH); 6.82e6.93 (m, 3H, ArH); 6.62e6.68 (m, 2H,
ArH and olefinic-H); 4.10e4.25 (m, 4H, OCH2e); 3.91 (s, 6H, OCH3);
3.87 (s, 9H, OCH3); 3.45e3.86 (m, 11H, NCH2, eCH2e and eCHe);
2.25e2.45 (m, 2H, eCH2e); 1.91e2.17 (m, 4H, eCH2e); 1.16e1.38
4.1.24. 7-Methoxy-8-{3-[2-methoxy-4-(E)-3-oxo-3-[4-(3,4,5-
trimethoxybenzoyl)piperazino]-1-propenylphenoxy]propoxy}-
(11 aS)1,2,3,11a-tetra-hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-
5-one (4a)
(m, 4H, eCH2e). 13C NMR (75 MHz, CDCl3):
d 170.5, 166.2, 164.3,
A solution of 18a (867 mg, 1 mmol), HgCl2 (693 mg, 2.26 mmol)
and CaCO3 (246 mg, 2.46 mmol) in CH3CN$H2O (4:1) was stirred
slowly at room temperature until TLC indicates complete loss of
starting material. The reaction mixture was diluted with EtOAc
(25 mL) and filtered through a celitebed. The clear yellow organic
supernatant was extracted with saturated 5% NaHCO3 (20 mL),
brine (20 mL) and the combined organic phase was dried on
Na2SO4. The organic layer was evaporated under vacuum and
purified by column chromatography (95% CHCl3$CH3OH) to give
compound 4a. This material was repeatedly evaporated from
CHCl3 under vacuum to generate the imine form. Yield 482 mg
162.7, 154.6, 151.2, 150.5, 149.4, 148.1, 143.4, 140.7, 139.4, 130.7, 127.6,
121.5, 120.4, 114.0, 112.8, 111.7, 110.5, 110.8, 66.7, 60.8, 56.7, 55.3,
53.7, 46.3, 31.8, 29.9, 28.7, 25.4, 24.5, 23.8.; ESI-MS: m/z 771 [Mþ]; IR
(KBr) (Umax/cmꢀ1): 3419, 2941, 1578, 1510, 1465, 1426, 1330, 1256,
1150, 1025, 824, 764.
4.1.27. 7-Methoxy-8-{6-[2-methoxy-4-(E)-3-oxo-3-[4-(3,4,5-
trimethoxybenzoyl)piperazino]-1-propenylphenoxy]hexyloxy}-
(11aS)1,2,3,11a-tetra-hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-
one (4d)
The compound 4d was prepared following the method
described for the compound 4a, employing HgCl2 (2.26 mmol) and
CaCO3 (2.46 mmol) and the crude product was purified by column
chromatography to afford the compound 4d. Yield 478 mg (61%);
(65%); Rf
118e120 ꢁC; [
CDCl3)
¼
0.56 (MeOH- CHCl3 5:95); White solid. mp:
25
a
]
¼ þ255 (c ¼ 0.5 in CHCl3); 1H NMR (300 MHz,
D
d
: 7.61e7.69 (m, 2H, imine-H and olefinic-H); 7.50 (s, 1H,
ArH); 6.98e7.13 (m, 2H, ArH); 6.82e6.93 (m, 3H, ArH); 6.63e6.76
(m, 2H, ArH and olefinic-H); 4.20e4.36 (m, 4H, OCH2e); 3.91 (s,
6H, OCH3); 3.88 (s, 9H, OCH3); 3.65e3.85 (m, 9H, NCH2, eCHe);
3.50e3.63 (m, 2H, eCH2e); 2.26e2.45 (m, 2H, eCH2e); 1.98e2.12
(m, 2H, eCH2e); 1.63e1.80 (m, 2H, eCH2e). 13C NMR (75 MHz,
Rf ¼ 0.53 (MeOHeCHCl3 5:95); White solid.mp: 109e111 ꢁC;
25
[a
]
¼ þ247 (c ¼ 0.5 in CHCl3); 1H NMR (300 MHz, CDCl3)
d:
D
7.61e7.72 (m, 2H, imine-H and olefinic-H); 7.52 (s, 1H, ArH);
6.99e7.18 (m, 2H, ArH); 6.81e6.95 (m, 3H, ArH); 6.63e6.69 (m, 2H,
ArH and olefinic-H); 4.12e4.25 (m, 4H, OCH2e); 3.92 (s, 6H, OCH3);
3.88 (s, 9H, OCH3); 3.46e3.87 (m, 11H, NCH2, eCH2e and eCHe);
2.24e2.47 (m, 2H, eCH2e); 1.92e2.18 (m, 4H, eCH2e); 1.14e1.46
CDCl3):
d 170.4, 165.8, 164.5, 162.3, 153.3, 150.6, 150.0, 149.5, 147.6,
143.6, 140.3, 139.5, 130.3, 127.8, 121.7, 120.2, 113.9, 112.6, 111.5,
110.6, 110.4, 65.2, 60.7, 56.3, 55.8, 53.6, 46.6, 31.4, 29.4, 28.7, 23.8.;
(m, 6H, eCH2e). 13C NMR (75 MHz, CDCl3):
d 170.4, 167.5, 166.5,
ESI-MS: m/z 743 [Mþ]; HRMS (ESI): [M
C40H47N4O10 m/z 743.3327, found m/z 743.3292; IR (KBr)
(Umax/cmꢀ1): 3412, 2932, 1592, 1510, 1460, 1425, 1329, 1257, 1127,
1023, 812, 763..
þ
Na]þ calcd for
164.3, 162.7, 153.2, 151.8, 149.4, 143.6, 140.4, 139.3, 131.5, 129.7,
127.6, 121.5, 119.2, 113.1, 112.8, 111.6, 110.7, 104.6, 68.7, 60.4, 56.5,
53.7, 46.5, 31.5, 29.7, 25.8, 24.7, 24.1, 23.0, 22.3, 14.0.; ESI-MS: m/z
785 [Mþ]; IR (KBr) (Umax/cmꢀ1): 3397, 2929, 1584, 1512, 1464, 1425,
1329, 1258, 1169, 1122, 1020, 828, 763.
4.1.25. 7-Methoxy-8-{4-[2-methoxy-4-(E)-3-oxo-3-[4-(3,4,5-
trimethoxybenzoyl)piperazino]-1-propenylphenoxy]butyloxy}-
(11aS)1,2,3,11a-tetra-hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-
one (4b)
4.1.28. 7-Methoxy-8-{3-[2-methoxy-4-[(E)-3-morpholino-3-oxo-1-
propenyl]phenoxy]propoxy}-(11aS)1,2,3,11a-tetra-hydro-5H-
pyrrolo[2,1-c][1,4]benzodiazepin-5-one (5a)
The compound 4b was prepared following the method
described for the compound 4a, employing HgCl2 (2.26 mmol) and
CaCO3 (2.46 mmol) and the crude product was purified by column
chromatography to afford the compound 4b. Yield 469 mg (62%);
A solution of 19a (741 mg, 1 mmol), HgCl2 (740 mg, 2.26 mmol)
and CaCO3 (270 mg, 2.46 mmol) in CH3CN$H2O (4:1) was stirred
slowly at room temperature until TLC indicates complete loss of
starting material. The reaction mixture was diluted with EtOAc
(25 mL) and filtered through a celitebed. The clear yellow organic
supernatant was extracted with saturated 5% NaHCO3 (20 mL),
brine (20 mL) and the combined organic phase was dried on
Na2SO4. The organic layer was evaporated in vacuum and purified
by column chromatography (95% CHCl3eCH3OH) to give
compound 5a. This material was repeatedly evaporated from CHCl3
in vacuum to generate the imine form. Yield: 368 mg, 61%;
Rf ¼ 0.56 (MeOHeCHCl3 5:95); White solid. mp: 113e115 ꢁC;
25
[
a]
¼ þ235 (c ¼ 0.5 in CHCl3); 1H NMR (300 MHz, CDCl3)
d:
D
7.62e7.71 (m, 2H, imine-H and olefinic-H); 7.49 (s, 1H, ArH);
6.99e7.15 (m, 2H, ArH); 6.80e6.92 (m, 3H, ArH); 6.61e6.67 (m, 2H,
ArH and olefinic-H); 4.07e4.24 (m, 4H, OCH2e); 3.91 (s, 6H, OCH3);
3.86 (s, 9H, OCH3); 3.46e3.82 (m, 11H, NCH2, eCH2e and eCHe);
2.24e2.39 (m, 2H, eCH2e); 1.92e2.14 (m, 4H, eCH2e); 1.17e1.34
(m, 2H, eCH2e). 13C NMR (75 MHz, CDCl3):
d
170.4, 168.8, 166.1,
Rf ¼ 0.58 (MeOHeCHCl3 4:95); White solid. mp: 110e112 ꢁC;
25
164.6, 162.3, 153.2, 150.3, 149.2, 143.2, 140.1, 139.2, 130.9, 129.4,
127.8, 121.8, 119.6, 113.8, 112.5, 111.3, 110.2, 104.2, 68.1, 60.6, 56.1,
53.2, 46.3, 31.6, 29.0, 25.4, 24.0, 22.3,14.0.; ESI-MS: m/z 757 [Mþ]; IR
(KBr) (Umax/cmꢀ1): 3383, 2927, 1587, 1510, 1462, 1425, 1329, 1258,
1169, 1126, 1019, 843, 763.
[a
]
¼ þ355 (c ¼ 0.5 in CHCl3);1H NMR (300 MHz, CDCl3)
d:
D
7.60e7.67 (m, 2H, imine-H and olefinic-H); 7.51 (s, 1H, ArH); 7.08
(d, 1H, J ¼ 7.77 Hz, ArH); 7.01 (s, 1H, ArH); 6.90 (d, 1H, J ¼ 8.63 Hz,
ArH); 6.85 (s, 1H, ArH); 6.69 (d, 1H, J ¼ 14.67 Hz, olefinic-H);
4.20e4.36 (m, 4H, OCH2e); 3.92 (s, 3H, OCH3); 3.88 (s, 3H, OCH3);
3.51e3.86 (m, 11H, morpholineeCH2, eCH2e and eCHe);
2.24e2.45 (m, 2H, eCH2e); 1.98e2.13 (m, 2H, eCH2e); 1.69e1.84
4.1.26. 7-Methoxy-8-{5-[2-methoxy-4-(E)-3-oxo-3-[4-(3,4,5-
trimethoxybenzoyl)piperazino]-1-propenylphenoxy]pentyloxy}-
(11aS)1,2,3,11a-tetra-hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-
one (4c)
The compound 4c was prepared following the method
described for the compound 4a, employing HgCl2 (2.26 mmol) and
CaCO3 (2.46 mmol) and the crude product was purified by column
chromatography to afford the compound 4c. Yield 462 mg (60%);
(m, 2H, eCH2e). 13C NMR (75 MHz, CDCl3):
d 165.7, 164.5, 162.3,
150.5, 149.9, 149.5, 147.7, 143.1, 140.5, 128.2, 121.6, 120.2, 114.2,
112.9, 111.6, 110.7, 66.8, 65.3, 56.0, 53.6, 46.5, 45.9, 42.3, 29.5, 28.9,
24.1.; ESI-MS: m/z 550 [Mþ]; HRMS (ESI): [M þ Na]þ calcd for
C30H36 N3O7 m/z 550.2566, found m/z 550.2553; IR (KBr)
(Umax/cmꢀ1): 3420, 2958, 1641, 1599, 1510, 1459, 1432, 1336, 1262,
1166, 1139, 1116, 980, 870, 762.

3882
A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3870e3884
4.1.29. 7-Methoxy-8-{4-[2-methoxy-4-[(E)-3-morpholino-3-oxo-1-
propenyl]phenoxy]butyloxy}-(11aS)1,2,3,11a-tetra-hydro-5H-
pyrrolo[2,1-c][1,4]benzodiazepin-5-one (5b)
4.1.32 (2E,20E)-1,10-(Piperazine-1,4-diyl)bis(4-propoxy-3-methoxy-
phenyl)prop-2-en-1-one dioxy]- bis[(11aS)-7-methoxy-1,2,3,11a-
tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one] (6a)
The compound 5b was prepared following the method
described for the compound 5a, employing HgCl2 (2.26 mmol) and
CaCO3 (2.46 mmol) and the crude product was purified by column
chromatography to afford the compound 5b. Yield: 396 mg, (64%);
A solution of 21a (644 mg,1 mmol), HgCl2 (1.8 g, 4.52 mmol) and
CaCO3 (540 mg, 4.98 mmol) in CH3CN$H2O (4:1) was stirred slowly
at room temperature until TLC indicates complete loss of starting
material. The reaction mixture was diluted with EtOAc (25 mL) and
filtered through a celitebed. The clear yellow organic supernatant
was extracted with saturated 5% NaHCO3 (20 mL), brine (20 mL)
and the combined organic phase was dried on NaSO4. The organic
layer was evaporated in vacuum and purified by column chroma-
tography (93% CHCl3eCH3OH) to give compound 6a. This material
was repeatedly evaporated from CHCl3 in vacuum to generate the
Rf ¼ 0.58 (MeOHeCHCl3 4:95); White solid. mp: 108e110 ꢁC;
25
[
a]
¼ þ372 (c ¼ 0.5 in CHCl3); 1H NMR (300 MHz, CDCl3)
d:
D
7.62e7.68 (m, 2H, imine-H and olefinic-H); 7.51 (s,1H, ArH); 7.09 (d,
1H, J ¼ 7.77 Hz, ArH); 7.02 (s,1H, ArH); 6.87 (d,1H, J ¼ 8.63 Hz, ArH);
6.82 (s, 1H, ArH); 6.70 (d, 1H, J ¼ 14.67 Hz, olefinic-H); 4.07e4.25
(m, 4H, OCH2e); 3.92 (s, 3H, OCH3); 3.88 (s, 3H, OCH3); 3.53e3.86
(m, 11H, morpholineeCH2, eCH2e and eCHe); 2.27e2.38 (m, 2H,
eCH2e); 1.98e2.17 (m, 4H, eCH2e); 1.67e1.82 (m, 2H, eCH2e).
imine form. Yield: 303 mg, (60%); Rf ¼ 0.56 (MeOHeCHCl3 7:93);
25
White solid. mp: 125e127 ꢁC; [
a
]
¼ þ125 (c ¼ 0.5 in CHCl3); 1H
D
13C NMR (75 MHz, CDCl3):
d
165.7, 164.5, 162.2, 150.6, 150.0, 149.3,
NMR (200 MHz, CDCl3) d: 7.59e7.81 (m, 4H, imine-H and olefinic-
147.6, 143.2, 140.4, 127.9, 121.7, 120.0, 114.0, 112.3, 111.3, 110.2, 68.4,
55.9, 53.6, 46.5, 42.5, 29.5, 25.7, 25.6, 24.0.; ESI-MS: m/z 564 [Mþ];
HRMS (ESI): [M þ Na]þ calcd for C31H38 N3O7 m/z 564.2719, found
m/z 564.2709; IR (KBr) (Umax/cmꢀ1): 3366, 2954, 1640, 1598, 1511,
1433, 1337, 1262, 1138, 1116, 1037, 979, 869, 806.
H), 7.51 (s, 2H, ArH), 6.67e7.22 (m, 8H, ArH), 6.52 (d, 2H,
J ¼ 16.05 Hz, olefinic-H), 4.18e4.40 (m, 8H, OCH2e), 3.91 (s, 12H,
OCH3), 3.46e3.87 (m, 11H, NCH2, eCH2e and eCHe), 1.94e2.48
(m, 8H, eCH2e), 1.57e1.83 (m, 4H, eCH2e). ESI-MS: m/z 1011 [Mþ].
4.1.33 (2E,20E)-1,10-(Piperazine-1,4-diyl)bis(4-butyloxy-3-methoxy-
phenyl-)prop-2-en-1-one dioxy]-bis[(11aS)-7-methoxy-1,2,3,11a-
tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one] (6b)
The compound 6b was prepared following the method
described for the compound 6a, employing HgCl2 (4.52 mmol) and
CaCO3 (4.98 mmol) and the crude product was purified by column
chromatography to afford the compound 6b. Yield: 316 mg, 61%;
4.1.30. 7-Methoxy-8-{5-[2-methoxy-4-[(E)-3-morpholino-3-oxo-1-
propenyl]phenoxy]pentyloxy}-(11aS)1,2,3,11a-tetra-hydro-5H-
pyrrolo[2,1-c][1,4]benzodiazepin-5-one (5c)
The compound 5c was prepared following the method
described for the compound 5a, employing HgCl2 (2.26 mmol) and
CaCO3 (2.46 mmol) and the crude product was purified by column
chromatography to afford the compound 5c. Yield: 380 mg, 60%;
Rf ¼ 0.58 (MeOHeCHCl3 7:93); White solid. mp: 121e123 ꢁC;
25
Rf ¼ 0.58 (MeOHeCHCl3 4:95); White solid. mp: 107e109 ꢁC;
[a
]
¼ þ122 (c ¼ 0.5 in CHCl3); 1H NMR (200 MHz, CDCl3)
d:
D
25
[
a]
¼ þ337 (c ¼ 0.5 in CHCl3); 1H NMR (200 MHz, CDCl3)
d:
7.56e7.82 (m, 4H, imine-H and olefinic-H), 7.50 (s, 2H, ArH),
6.65e7.23 (m, 8H, ArH), 6.53 (d, 2H, J ¼ 15.96 Hz, olefinic-H),
4.20e4.43 (m, 8H, OCH2e), 3.91 (s, 12H, OCH3), 3.43e3.88 (m, 11H,
NCH2, eCH2e and eCHe), 1.96e2.49 (m, 8H, eCH2e), 1.55e1.84
(m, 6H, eCH2e). ESI-MS: m/z 1039 [Mþ].
D
7.65e7.70 (m, 2H, imine-H and olefinic-H); 7.50 (s, 1H, ArH); 7.06
(d, 1H, J ¼ 7.75 Hz, ArH); 7.0 (s, 1H, ArH); 6.91 (d, 1H, J ¼ 8.66 Hz,
ArH); 6.84 (s, 1H, ArH); 6.70 (d, 1H, J ¼ 14.81 Hz, olefinic-H);
4.21e4.38 (m, 4H, OCH2e); 3.92 (s, 3H, OCH3); 3.89 (s, 3H, OCH3);
3.50e3.85 (m, 11H, morpholineeCH2, eCH2e and eCHe);
2.23e2.46 (m, 2H, eCH2e); 1.87e2.16 (m, 4H, eCH2e); 1.66e1.83
4.1.34 (2E,20E)-1,10-(Piperazine-1,4-diyl)bis(4-penyloxy-3-methoxy
phenyl-)prop-2-en-1-one dioxy]-bis[(11aS)-7-methoxy-1,2,3,11a-
tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one] (6c)
The compound 6c was prepared following the method described
for the compound 6a, employing HgCl2 (4.52 mmol) and CaCO3
(4.98 mmol) and the crude product was purified by column chro-
matography to afford the compound 6c. Yield: 330 mg, (62%);
(m, 4H, eCH2e). 13C NMR (75 MHz, CDCl3):
d 165.4, 164.6, 162.6,
150.4, 149.6, 149.1, 147.5, 143.3, 140.7, 128.6, 121.4, 120.5, 114.6,
112.7, 111.3, 110.5, 66.7, 65.4, 56.3, 53.7, 46.8, 46.1, 42.8, 29.8, 28.5,
26.4, 24.8, 24.1.; ESI-MS: m/z 578 [Mþ]; IR (KBr) (Umax/cmꢀ1): 3412,
2956, 1642, 1597, 1512, 1446, 1434, 1325, 1264, 1146, 1135, 1115, 978,
871, 764.
Rf ¼ 0.60 (MeOHeCHCl3 7:93); White solid. mp: 120e122 ꢁC;
25
4.1.31. 7-Methoxy-8-{6-[2-methoxy-4-[(E)-3-morpholino-3-oxo-1-
propenyl]phenoxy]hexyloxy}-(11aS)1,2,3,11a-tetra-hydro-5H-
pyrrolo[2,1-c][1,4]benzodiazepin-5-one (5d)
[a
]
¼ þ117 (c ¼ 0.5 in CHCl3); 1H NMR (200 MHz, CDCl3)
d:
D
7.57e7.80 (m, 4H, imine-H and olefinic-H), 7.52 (s, 2H, ArH),
6.67e7.24 (m, 8H, ArH), 6.54 (d, 2H, J ¼ 15.87 Hz, olefinic-H),
4.19e4.41 (m, 8H, OCH2e), 3.90 (s, 12H, OCH3), 3.45e3.86 (m, 11H,
NCH2, eCH2e and eCHe), 1.95e2.46 (m, 8H, eCH2e), 1.56e1.82
(m, 4H, eCH2e),1.35e1.49 (m, 4H, eCH2e). ESI-MS: m/z 1067 [Mþ].
The compound 5d was prepared following the method
described for the compound 5a, employing HgCl2 (2.26 mmol) and
CaCO3 (2.46 mmol) and the crude product was purified by column
chromatography to afford the compound 5d. Yield: 389 mg, (60%);
Rf ¼ 0.56 (MeOHeCHCl3 4:95); White solid. mp: 102e104 ꢁC;
4.1.35 (2E,20E)-1,10-(Piperazine-1,4-diyl)bis(4-hexyloxy-3-methoxy
phenyl-)prop-2-en-1-one dioxy]bis[(11aS)-7-methoxy-1,2,3,11a-
tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepine5-one] (6d)
The compound 6d was prepared following the method described
for the compound 6a, employing HgCl2 (4.52 mmol) and CaCO3
(4.98 mmol) and the crude product was purified by column chro-
matography to afford the compound 6d. Yield: 328 mg, 60%;
25
[
d
a
]
¼ þ355 (c ¼ 0.5 in CHCl3); 1H NMR (200 MHz, CDCl3):
D
7.64e7.69 (m, 2H, imine-H and olefinic-H); 7.49 (s, 1H, ArH); 7.05
(d, 1H, J ¼ 7.15 Hz, ArH); 7.01 (s, 1H, ArH); 6.86 (d, 1H, J ¼ 8.33 Hz,
ArH); 6.83 (s, 1H, ArH); 6.69 (d, 1H, J ¼ 14.81 Hz, olefinic-H);
4.15e4.26 (m, 4H, OCH2e); 3.91 (s, 3H, OCH3); 3.89 (s, 3H, OCH3);
3.52e3.87 (m, 11H, morpholineeCH2, eCH2e and eCHe);
2.26e2.37 (m, 2H, eCH2e); 1.93e2.13 (m, 6H, eCH2e); 1.64e1.81
Rf ¼ 0.60 (MeOHeCHCl3 7:93); White solid. mp: 118e121 ꢁC; [
D25 ¼ þ103 (c ¼ 0.5 in CHCl3); 1H NMR (200 MHz, CDCl3)
a
d
]
:
(m, 4H, eCH2e). 13C NMR (75 MHz, CDCl3):
d
166.1, 164.7, 162.4,
151.1, 150.3, 149.5, 147.7, 143.4, 140.6, 127.6, 121.8, 120.4, 114.2,
112.5, 111.7, 110.4, 68.7, 56.2, 53.8, 46.6, 42.3, 29.7, 26.5, 25.8, 24.7,
24.2, 23.4.; ESI-MS: m/z 592 [Mþ]; IR (KBr) (Umax/cmꢀ1): 3386,
2956, 1643, 1597, 1510, 1439, 1339, 1264, 1137, 1116, 1036, 978,
866, 804.
7.54e7.79 (m, 4H, imine-H and olefinic-H), 7.51 (s, 2H, ArH),
6.66e7.21 (m, 8H, ArH), 6.51 (d, 2H, J ¼ 16.04 Hz, olefinic-H),
4.17e4.40 (m, 8H, OCH2-), 3.91 (s, 12H, OCH3), 3.47e3.86 (m, 11H,
NCH2, eCH2e and eCHe), 1.93e2.47 (m, 8H, eCH2e), 1.57e1.83
(m, 4H, eCH2e),1.28e1.50 (m, 6H, eCH2e). ESI-MS: m/z 1095 [Mþ].

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3870e3884
3883
4.2. Evaluation of anticancer activity
The reaction mixture in a total volume of 10
mL contained PEM
buffer, GTP (1 mM) and in the presence or absence of test
compounds 5b, 6b (2.5 M). The reaction was initiated by the
The synthesized compounds (4aed, 5aed and 6aed) have been
evaluated for their in vitro cytotoxicity in human cancer cell lines. A
protocol of 48 h continuous drug exposure has been used and
a sulforhodamine B (SRB) protein assay has been used to estimate
cell viability or growth. The cell lines were grown in RPMI 1640
m
addition of GTP to all the wells. Polymerization reaction was
monitored by the increase in absorbance at 340 nm using Dynex
multimode plate reader at 37 ꢁC. Absorbance was recorded at every
2 min intervals for up to 3 h. Nocodazole was used as positive
control in each assay. The IC50 value was defined as the drug
concentration required to inhibit 50% of tubulin assembly
compared to controls. The reaction mixture for these experiments
include: tubulin (3 mg/ml) in PEM buffer, GTP (1 mM), in the
medium containing 10% fetal bovine serum and 2 mM
L-glutamine
and were inoculated into 96 well microtiter plates in 90
mL at
plating densities depending on the doubling time of individual cell
lines. The microtiter plates were incubated at 37 ꢁC, 5% CO2, 95% air,
and 100% relative humidity for 24 h prior to addition of experi-
presence or absence of test compounds 5b, 6b at 2.5
mM, 5 mM,
mental drugs. Aliquots of 10
mL of the drug dilutions were added to
10 M, and 15 M concentrations, different concentrations of
m
m
the appropriate microtiter wells already containing 90
m
L of cells,
nocodozole was used as positive control in this study. Polymeri-
zation was monitored by increase in the absorbance at 340 nm
using Dynex multimode plate reader at37 ꢁC. Absorbance was
monitored at every 2 min for 3 h.
resulting in the required final drug concentrations. For each
compound four concentrations (0.1, 1, 10 and 100 uM) were eval-
uated and each was done in triplicate wells. Plates were incubated
further for 48 h and assay was terminated by the addition of 50 mL
of cold trichloro acetic acid (TCA) (final concentration, 10% TCA) and
incubated for 60 min at 4 ꢁC. The plates were washed five times
with tap water and airdried. Sulforhodamine B (SRB) solution
4.5. Immunohistochemistry and microscopy
MCF7 cells were seeded on glass cover slips, incubated for 48 h
in the presence or absence of test compounds 5b, 6b (2.5
(50 mL) at 0.4% (w/v) in 1% acetic acid was added to each of the
mM).
wells, and plates were incubated for 20 min at room temperature.
The residual dye was removed by washing five times with 1% acetic
acid. The plates were airdried. Bound stain was subsequently eluted
with 10 mM trizma base, and the absorbance was read on an ELISA
plate reader at a wavelength of 540 nm with 690 nm reference
wavelengths. Percent growth was calculated on a plateeby plate
basis for test wells relative to control wells. The above determina-
tions were repeated three times. Percentage growth was expressed
as the (ratio of average absorbance of the test well to the average
absorbance of the control wells) ꢂ 100. Growth inhibition of 50%
(GI50) was calculated from [(Ti ꢀ Tz)/(C ꢀ Tz)] ꢂ 100 ¼ 50, which is
the drug concentration resulting in a 50% reduction in the net
protein increase (as measured by SRB staining) in control cells
during the drug incubation. Where, Tz ¼ Optical density at time
zero, OD of control ¼ C, and OD of test growth in the presence of
drug ¼ Ti.
Following the termination of incubation, cells were fixed with 3%
paraformaldehyde, 0.02% glutaraldehyde in PBS and permiabilized
by dipping the cells in 100% methanol (ꢀ20 ꢁC). Later, cover slips
were blocked with 1% BSA in phosphate buffered saline for 1 h
followed by incubation with a primary anti tubulin (mouse
monoclonal) antibody and FITC conjugated secondary mouse anti
Ig G antibody. Photographs were taken using the confocal micro-
scope, equipped with FITC settings and the pictures were analyzed
for the integrity of microtubule network. In parallel experiments,
nocodozole (2.5 mM) and taxol (2.5 mM) were used as negative and
positive controls for analyzing microtubule integrity.
4.6. Analysis of cell cycle
Breast cancer cells (MCF7) in 6 well plates were incubated for
48 h in the presence or absence of test compounds 5b, 6b (2.5 mM),
nocodazole & podophyllotoxin of (1 mM). Cells were harvested with
4.3. Isolation of tubulin
TrypsineEDTA, fixed with ice-cold 70% ethanol at 4 ꢁC for 30 min,
ethanol was removed by centrifugation and cells were stained with
250 mL of DNA staining solution [10 mg of Propidium Iodide (PI),
Tubulin was purified according to the earlier protocol [54]. In
brief fresh bovine brain was obtained from slaughter house under
cold conditions and the cerebral hemispheres were dissected
immediately, washed in PBS (phosphate buffer saline) and
homogenized with appropriate amount of PEM buffer contain
80 mM PIPES, 0.5 mM MgCl2, and 1 mM EGTA. The homogenate was
centrifuged for 30 min at 4 ꢁC at 14,500 rpm. The supernatant was
collected and centrifuged in an ultra centrifuge at 4 ꢁC for 60 min at
1, 00,000g. The obtained pellet was discarded and the supernatant
was incubated with 1% glycerol and 3 mg GTP at 37 ꢁC for 30 min to
assist the polymerize the tubulin into microtubules. The superna-
tant was centrifuged in an ultra centrifuge at 25 ꢁC for 60 min at
1,00,000g. The pellet (polymerized tubulin) was suspended in ice-
cold PEM buffer and incubated on ice for 30 min to depolymerize
the tubulin. The suspension (depolymerized tubulin) is again
centrifuged in ultra centrifuge at 4 ꢁC for 60 min at 1,00,000g and
supernatant was collected. This polymerization and depolymer-
ization cycles are repeated for three times to get purified tubulin.
The purified tubulin was stored in MT buffer at ꢀ80 ꢁC.
0.1 mg of trisodium citrate, and 0.03 mL of Triton X-100 were dis-
solved in 100 mL of sterile water at room temperature for 30 min in
the dark. The DNA contents of 20,000 events were measured by
flowcytometer (DAKO CYTOMATION, Beckman Coulter, Brea, CA).
Histograms were analyzed using Summit Software.
4.7. Analysis of nuclear morphology
MCF7 cells were seeded on glass cover slip, incubated for 48 h in
the presence or absence of test compounds 5b, 6b (2.5
mM).
Following the termination of incubation, cells were fixed with 3%
paraformaldehyde, 0.02% glutaraldehyde in PBS; the cells were
incubated with the nuclear stain Hoechst, to analyze morphology of
nucleus. Photographs were taken in an olyumpus fluorescence
microscopy equipped with DAPI filter settings.
Acknowledgements
4.4. Tubulin polymerization assay
We thank the National Cancer Institute (NCI), Maryland, for
providing data on anticancer assay in human cancer cell lines and
the authors G. Balakishan, G.R.K, K.S and M.B.K. are grateful to CSIR,
New Delhi, for the award of research fellowships.
The assay was carried out in a 384 well plate in PEM buffer
[100 mM PIPES (pH-6.9), 1 mM MgCl2, 1 mM EGTA, 10% glycerol].

3884
A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3870e3884
[27] L.A. Masterson, S.J. Croker, T.C. Jenkins, P.W. Howard, D.E. Thurston, Bioorg.
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