Design, synthesis and biological evaluation of 3,5-diaryl-isoxazoline/ isoxazole-pyrrolobenzodiazepine conjugates as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2010.05.047

A series of 3,5-diaryl-isoxazoline/isoxazole linked pyrrolo[2,1-c][1,4] benzodiazepine (PBD) conjugates were prepared. These conjugates showed potent anticancer activity with GI₅₀ values in the range of <0.1-3.6 μM. Some of these PBD conjugates (6a-c) with promising anticancer activity were further investigated on the cell cycle distribution. Moreover, these PBD conjugates exhibited G0/G1 arrest, enhancement in the levels of p53 protein as well as mitochondrial-mediated intrinsic pathway, leading to release of cytochrome c, activation of caspase-3, cleavage of PARP and subsequent apoptotic cell death. Hence these PBD conjugates with 6a being the most potent one could be be taken up for preclinical studies either alone or in combination with existing therapies. New class of 3,5-diaryl-isoxazoline/isoxazole- pyrrolobenzodiazepine conjugates were prepared and evaluated for their anticancer activity. Further, some of the biological assays related to mechanism aspects were also carried out.

Full text of DOI:10.1016/j.ejmech.2010.05.047

European Journal of Medicinal Chemistry 45 (2010) 3924e3937
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of
3,5-diaryl-isoxazoline/isoxazole-pyrrolobenzodiazepine conjugates
as potential anticancer agents
,
a
a
a
a
Ahmed Kamal a , J. Surendranadha Reddy , M. Janaki Ramaiah , D. Dastagiri , E. Vijaya Bharathi ,
*
M. Ameruddin Azhar a, Farheen Sultana a, S.N.C.V.L. Pushpavalli a, Manika Pal-Bhadra a , Aarti Juvekar ,
,
b
*
Subrata Sen b, Surekha Zingde b
a Division of Organic Chemistry, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 607, India
b Advanced Centre for Treatment, Research & Education in Cancer, Navi Mumbai, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3,5-diaryl-isoxazoline/isoxazole linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD) conjugates
Received 16 April 2010
Received in revised form
19 May 2010
Accepted 21 May 2010
Available online 31 May 2010
were prepared. These conjugates showed potent anticancer activity with GI50 values in the range of
<0.1e3.6
mM. Some of these PBD conjugates (6aec) with promising anticancer activity were further
investigated on the cell cycle distribution. Moreover, these PBD conjugates exhibited G0/G1 arrest,
enhancement in the levels of p53 protein as well as mitochondrial-mediated intrinsic pathway, leading
to release of cytochrome c, activation of caspase-3, cleavage of PARP and subsequent apoptotic cell death.
Hence these PBD conjugates with 6a being the most potent one could be be taken up for preclinical
studies either alone or in combination with existing therapies.
Keywords:
Pyrrolobenzodiazepine
3,5-Diaryl-isoxazoline/isoxazole
p53
Ó 2010 Elsevier Masson SAS. All rights reserved.
Cytochrome c
Procaspase-3
1. Introduction
cancer cell lines [6]. From the previous comparative studies of the
combretastatins itappears thatthe cis orientationof thetwo aromatic
Chemotherapy is often the treatment of choice for many types of
cancer as a result the search for newer chemotherapeutic agents
still plays a major role in the fight against cancer. In recent years
there has been increasing interest in the design of conjugate
molecules that could act in a specific manner on more than one
target. The development of such conjugates lowers the risk of
drugedrug interaction in comparison to cocktails but could also
enhance the efficacy as well as improve the safety aspects in rela-
tion to the drugs that interact on a single target, [1e3] and one such
recent example is bleomycin [4].
rings plays an important role in cytotoxicity. Accordingly, a number of
cis-restricted analogues of CA-4 have been prepared using five-
membered heterocycles [7e9] to avoid the stability problem. Many of
them showed potent cytotoxicity against various cancer cells
compared to CA-4. In a recent study, cis-restricted analogues of CA-4
(diaryl substituted isoxazoline/isoxazole derivatives) are reported to
possess potent apoptosis-inducing activity [10,11].
Similarly, pyrrolo[2,1-c][1,4]benzodiazepines (PBD’s) like
anthramycin, tomaymycin and DC-81 belong to a family of potent
tricyclic anticancer antibiotics, that are isolated from various Strep-
tomyces species and they bind in the minor groove of double-
stranded DNA forming a covalent bond to the exocyclic amino group
ofa central guaninewithin a three base pairrecognition site[12e14].
The cytotoxicity and antitumour effects of these compounds are
believed to arise from this modification of DNA, which leads to
inhibition of nucleic acid synthesis and production of excision
dependent single and double strand breaks in cellular DNA (Fig. 1).
In the past few years, several hybrid compounds, in which
a known antitumour compound or some simple active moiety
Combretastatins, naturally occurring stilbenes, are isolated from
Combretum caffrum by Pettit’s group [5]. Among them, com-
bretastatin A-4 (CA-4) showed most potent cytotoxicity against
a variety of human cancer cell lines including multiple drug-resistant
* Corresponding authors. 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 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.047

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3924e3937
3925
O N
O N
OH
MeO
MeO
MeO
MeO
OH
OMe
OH
MeO
MeO
OMe
3
1
OMe
2
OMe
OMe
OMe
N O
N
O
O
n
MeO
MeO
MeO
N
HO
MeO
H
( )
H
N
OMe
N
5a: n = 3
OMe
OMe
O
O
4
5b: n = 4
5c: n = 5
N O
O N
O N
O N
OMe
MeO
MeO
N
O
O
H
N
( )
n
OMe
OMe
OMe
OMe
MeO
5d: n = 4
5e: n = 5
O
OMe
MeO
MeO
N
O
O
H
( )
n
6a: n = 3
6b: n = 4
6c: n = 5
N
N
MeO
O
N
O
O
MeO
MeO
( )
H
n
MeO
OMe
O
7a: n = 3
7b: n = 4
7c: n = 5
OMe
MeO
MeO
N
O
O
H
( )
n
OMe
N
MeO
7d: n = 3
O
7e: n = 5
Fig. 1. Structures of combretastatin (CA-4) (1), 3,5-diarylisoxazoline (2), 3,5-diarylisoxazole (3), DC-81(4), and 3,5-diaryl-isoxazoline/isoxazole-PBD conjugates (5aee, 6aec and
7aee).
tethered to PBD, have been designed, synthesized and evaluated for
their biological activity [15e18]. Recently, Wang and co-workers
have synthesized indole-PBD conjugates as potential antitumour
agents and a correlation between antitumour activity and apoptosis
has been well explained [19]. For the last few years, we have been
involved in the development of new synthetic strategies for the
preparation of PBD ring system [20,21] and also in the design as
well as synthesis of structurally modified PBDs and their conjugates
[22e26]. More recently, we have also reported some of the PBD
conjugates that demonstrated potent apoptotic activity through
mitochondrial-mediated pathway [27,28].
In continuation of these efforts, we herein report the synthesis
and biological evaluation of some new conjugates where in 3,5-
diaryl-isoxazoline/isoxazole moieties have been linked to the pyr-
rolo[2,1-c][1,4]benzodiazepine ring system. Initially the anticancer
activity has been carried out for these new PBD conjugates. In view
of promising activity it has been considered of interest to investi-
gate their role in cell proliferation and apoptosis by using the
human melanoma cell line A375. The main aim of this study is to
explore whether these conjugates possess better anticancer activity
than DC-81 (4) and to substantiate these results with detailed
biological studies.
2. Chemistry
The synthesis of 3,5-diaryl-isoxazoline/isoxazole derivatives
(13a,b, 18 and 22a,b) [10,11] was carried out from aldehydes 8, 10a,
b and 14 as the starting materials. Reaction of these aldehydes with
hydroxylamine in a MeOH/H2O (3:1) solution produced the corre-
sponding oximes 9, 20a,b and 15. Olefins 11a,b and 16 were
obtained by the reaction of aldehydes 10a,b and 8 with methyl-
triphenylphosphoniumbromide, in the presence of sodium hydride.
Then these olefins 11a,b and 16 were coupled to oximes 9 and 15 to
provide the corresponding TBDMS protected isoxazolines 12a,
b and 17. Similarly, TBDMS protected isoxazoles 21a,b were
prepared by employing alkyne 19 and oximes 20a,b. Then, these
compounds 12a,b, 17 and 21a,b upon deprotection with tetrabu-
tylammonium fluoride gave the desired precursors (13a,b, 18 and
22a,b) as shown in Schemes 1 and 2.
The synthesis of C8-linked 3,5-diaryl-isoxazoline/isoxazole-PBD
conjugates (5aee, 6aec and 7aee) was carried out from the (2S)-N-
{4-[3-bromoalkoxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-car-
boxaldehyde diethyl thioacetal (23aec), and these were prepared by
the methods reported in our earlier studies [20e28]. These upon
etherificationwiththe3,5-diaryl-isoxazoline/isoxazoleintermediates

3926
A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3924e3937
R1
MeO
MeO
CHO
OHC
R2
R3
10a b
OMe
a
8
b
R1
R3
R1
N O
R2
MeO
MeO
MeO
MeO
NOH
c
R2
+
12a b
R3
11a b
OMe
9
OMe
12a: R1 = OTBDMS, R2 = OMe, R3 = H
12b: R1 = OMe, R2 = OTBDMS, R3 = OMe
d
13a: R1 = OH , R2 = OMe, R3 = H
13b: R1 =OMe, R2 = OH, R3 = OMe
O N
17: R = OTBDMS
18: R = OH
OMe
OR
MeO
MeO
d
OMe
17
c
OMe
OTBDMS
MeO
MeO
+
HON
15
OMe
16
b
a
OMe
OTBDMS
8
OHC
14
Scheme 1. Synthesis of 3,5-diaryl-isoxazoline analogues (13a,b, 18). Reagents and conditions: a) NH2OH.HCl, NaHCO3, CH3OH:H2O (3:1), 0 ꢂC, then rt, 6 h; b) CH3PPh3þBrꢁ, NaH, THF,
ꢂC, then rt, 5 h; c) 13% aq NaOCl, Et3N, CH2Cl2, 0 ꢂC, then rt, 24 h; d) TBAF, THF, rt, 2 h.
0
using K2CO3 in acetone provided the corresponding nitro thioacetals
(24aee, 25aec and 26aee). Further, reduction of nitro compounds by
using SnCl2.2H2O in MeOH followed by deprotective cyclization
employing HgCl2/CaCO3 afforded the desired PBD conjugates (5aee,
6aec and 7aee) as shown in Scheme 3.
colon, oral, ovarian and prostate by using Sulforhodamine B (SRB)
method. The compounds that exhibiting GI50 ꢀ 10ꢁ5
mM were
considered to be active on the respective cell lines. Table 2 reveals
that compounds 5a, 5cee, 6a, 6c, and 7aee exhibit significant
anticancer activity against some of the cell lines with GI50 values
ranging from <0.1 to 2.15
adriamycin demonstrated highly significant activity with the GI50
in the range from <0.1 to 14.0 M and for DC-81 the GI50 ranged
from 0.11 to 2.37 M. Further, it was observed that among all the
conjugates prepared, compounds 6a and 6c showed better anti-
cancer activity against number of cancer cell lines with
GI50 < 0.1 M. Similarly compounds 5a, 5cee, and 7aee also
exhibited significant activity against respective cell lines with GI50
values ranging from <0.1 to 2.37 M.
MTT assay was also carried out to identify the cytotoxic effect
of the most active compounds 6aec on A375 cells at 4
mM. The positive control compound
3. Results and discussion
m
m
3.1. Evaluation of biological activity
a
3.1.1. Anticancer activity
m
The anticancer activity of compounds 5b and 6b was evaluated
against a panel of sixty human cancer cell lines derived from nine
cancer cell types as shown in Table 1. The mean GI50 value for
m
compound 6b is 0.25 mM, which exhibits an interesting profile of
m
M
activity for various cell lines, indicating that this compound 6b has
potent broad-spectrum anticancer activity. The promising activity
shown by compounds 5b and 6b prompted us to evaluate the
anticancer activity of the other analogues (5a, 5cee, 6a, 6c, and
7aee) in selected human cancer cell lines of breast, cervix, lung,
concentration. Moreover, the anticancer activity of compound 1
(CA-4), 2 (3,5-diaryl-isoxazoline) and 4 (DC-81) was examined to
substantiate the potentiality of these conjugates (6aec) as anti-
cancer agents. Conjugates 6a and 6b with the exception of 6c

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3924e3937
3927
R1
MeO
R2
+
MeO
HON
R3
OMe
19
b
a
20a b
c
10a b
8
20a: R1 = OTBDMS , R2 = OMe, R3 = H
20b: R1 =OMe, R2 = OTBDMS, R3 = OMe
R1
O N
R2
MeO
MeO
R3
OMe
21a b
21a: R1 = OTBDMS, R2 = OMe, R3 = H
21b: R1 = OMe, R2 = OTBDMS, R3 = OMe
d
22a: R1 = OH , R2 = OMe, R3 = H
22b: R1 = OMe R2 = OH, R3 = OMe
Scheme 2. Synthesis of 3,5-diaryl-isoxazole analogues (22a,b). Reagents and conditions: a) (i) CBr4, PPh3, CH2Cl2, 0 ꢂC, 10 min; (ii) n-BuLi, THF, ꢁ78 ꢂC to rt; b) NH2OH.HCl, NaHCO3,
CH3OH:H2O (3:1), 0 ꢂC, then rt, 6 h; c) 13% aq NaOCl, Et3N, CH2Cl2,
0
ꢂC, then rt, 24 h; d) TBAF, THF, rt, 2 h.
showed a higher cytotoxicity than 1, 2 and 4 on A375 cells as
known to cause activation of caspase-3 [31] and to investigate the
shown in Fig. 2.
effect on cytochrome c, A375 cells were treated with PBD conjugates
at 4
mM concentration for 24 h. It was observed that there is an
3.1.2. Cell cycle effects
increase in cytochrome c protein level and this enhancement in
cytochrome c level is more pronounced in case of compound 6a as
shown in Fig. 4. This clearly shows the involvement of mitochondria
in the apoptotic pathway.
Another important consequence of apoptosis is the procaspase-
3 degradation to active caspase-3 and also cleavage of DNA repair
enzyme PARP (poly ADP ribose polymerase) by caspase-3 [32].
Treated and untreated cell lysates have been analyzed for the cas-
pase-3 expression levels by fluorometric based caspase-3 assay. The
order of performance of the production of active caspase-3 is
6a > 6b > 6c as shown in Fig. 5.
Asweobserved anincreaseinthelevelsofcaspase-3, itisexpected
that there will be an enhancement in the levels of cleaved PARP [33].
An increase in the expression of cleaved PARP (85 kDa) specific to
apoptosis was observed in all the conjugates (6aec), where in
compound 6a showed the maximum increase as seen in Fig. 4.
To investigate the effect of these PBD conjugates on the cell cycle
progression of human cancerous cell line A375, the DNA content of
the cell nuclei was measured by flow cytometric analysis (FACS
analysis). Treatment of A375 cells with these compounds (6aec) at
4 mM concentration for 24 h induced apoptosis up to 98.5%, 95.25%
and 92.45% respectively of G0/G1 and there is a concomitant
decrease in the G2/M phase. Whereas, DC-81 (4) showed 88.05% in
G0/G1 phase, conjugate partner 3,5-diaryl-isoxazoline (2), and CA-
4 (1) have shown 82.59% and 49.08% respectively in G0/G1 phase.
Therefore, increase in cells of G0/G1 phase and decrease of G2/M
phase cells clearly shows that all these conjugates cause G1 arrest
and are effective in causing apoptosis in case of cancerous A375
cells. Moreover, compound 6a could be considered as the most
effective conjugate to produce cell cycle arrest (Fig. 3a and b).
3.1.3. Effect of 3,5-diaryl-isoxazoline-pyrrolobenzodiazepine
conjugates on the expression of apoptotic proteins
3.1.4. Effect on cell cycle proteins
It is well known that p53, a tumor suppressor gene, stops the
formation of tumors. It exerts antiproliferation effects through its
ability to function as a sequence-specific DNA-binding transcription
factor. Activation of certain genes such as p53 is found to be
important in the regulation of apoptotic pathway induced byvarious
stimuli [29]. In order to understand the effect of PBD conjugates on
p53 dependent apoptotic pathway, the cells were treated with these
conjugate compounds and the cell lysate was used for Western blot
analysisusingp53 specific antibody. Itis observedthat thep53 levels
are upregulated in case of compounds 6aec when compared to the
untreated controls and interestingly, the levels of p53 are markedly
high in compound 6a as seen in Fig. 4.
Cell cycle progression is regulated by cyclin dependent kinases
(CDKs) that are activated by the binding of cyclins such as cyclin A,
B, C and D and inhibited by CDK inhibitors, moreover CDK2 protein
plays a pivotal role in G1eS interphase transition of cell cycle [34].
It was considered of interest to know the levels of CDK2 upon
treatment with PBD conjugates (6aec) and it was observed that
there was a reduction of CDK2 levels as seen in Fig. 4. The reduction
of Cdk2 protein level is slightly more in case of 6a and 6b than 6c.
This indicates that there is a possibility of involvement of cell cycle
regulatory proteins in this event, however the intricacies involved
further needs to be evaluated.
Further, it was considered of interest to understand the nature of
apoptotic pathway and the effect involved in regulating this activity.
In this context the intrinsic apoptotic pathway was examined which
is an important consequence of the mitochondrial dysfunction and
cytochrome c release [30]. Moreover, cytosolic cytochrome c is
4. Conclusion
In the present study, a series of 3,5-diaryl-isoxazoline/iso-
xazoleepyrrolobenzodiazepine conjugates were synthesized.
Compounds 5b and 6b that were evaluated at the National Cancer

3928
A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3924e3937
N O
13a
NO2
O
O
MeO
MeO
CH(SEt)2
( )
n
N
MeO
OMe
OMe
24a c
O
b, c
5a: n = 3
5b: n = 4
5c: n = 5
N O
OMe
MeO
MeO
13b
NO2
O
O
CH(SEt)2
( )
n
OMe
OMe
N
MeO
24d e
O
b, c
5d: n = 4
5e: n = 5
O N
Br
O
NO2
N
CH(SEt)2
( )
OMe
O
MeO
MeO
n
a
18
MeO
NO2
O
CH(SEt)2
( )
O
n
OMe
23a: n = 3
23b: n = 4
23c: n = 5
N
MeO
25a c
O
b, c
6a: n = 3
6b: n = 4
6c: n = 5
O N
NO2
O
O
MeO
( )
CH(SEt)2
22a
n
N
OMe MeO
MeO
OMe
O
26a c
b, c
7a: n = 3
7b: n = 4
7c: n = 5
O N
OMe
MeO
MeO
22b
NO2
O
O
CH(SEt)2
( )
n
OMe
OMe
N
O
MeO
26d e
b, c
7d: n = 3
7e: n = 5
Scheme 3. 3,5-diaryl-isoxazoline/isoxazole-pyrrolobenzodiaze conjugates (5aee, 6aec and 7aee). Reagents and conditions: a) K2CO3, dry CH3COCH3, 48 h, 80e89%; b) SnCl2.2H2O,
CH3OH, reflux, 2 h; c) HgCl2, CaCO3, CH3CNeH2O, (4:1), 8 h, 52e59%.
Institute (NCI), Bethesda, USA exhibited potent anticancer activity
against various cancer cell lines indicating that these compounds
have the potential for their development as broad-spectrum anti-
cancer agents. Further the other PBD conjugates also showed
promising anticancer activity against the number of human cancer
cell lines tested. Moreover, from the MTT proliferation assay it was
observed that the conjugates 6a and 6b are more effective as
antiproliferative agents than the naturally occurring PBD 4 (DC-81)
conjugates (6aec), thus indicating the involvement of mitochon-
drial pathway. Concomitant with this an increase in the levels of
active caspase-3 and cleavage of PARP was also observed with all
these conjugates, particularly it was more pronounced in case of 6a.
Further, all the conjugates (5a, 5cee, 6a, 6c, and 7aee) in which 3,5-
diaryl-isoxazoline/isoxazole moiety linked to PBD ring system
showed significant anticancer activity with GI50 values in the range
of <0.1e2.15
conjugates, like C-8 linked triazolobenzothiadiazine-PBD conju-
gates [24] (GI50 values in the range of 0.22e30.30 M), anthraqui-
none-PBD conjugates [23] (GI50 values in the range of <0.1e10 M),
1,2,3-triazole-PBD conjugates [25] (GI50 values in the range of
0.13e30.50 M), and phosphonate linked PBD conjugates [26] (GI50
values in the range of 0.17e30.50 M) against 9e11 human cancer
mM in comparison to the previous prepared PBD
in A375 cells 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. It was also
observed from the results of detailed biological assays that the p53
levels were enhanced when treated with compounds 6aec.
Moreover, release of cytochrome c was as well observed for the
m
m
m
m

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3924e3937
3929
Table 1
Anticancer activity of compounds 5b and 6b and in selected cancer cell linesa
Cancer panel/cell line GI50
(
mM)
Cancer panel/cell line GI50
(
m
M)
5b
6b
5b
6b
Leukemia
Ovarian
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
1.41
1.90
1.26
1.85
1.43
1.12
0.16
0.18
0.12
0.20
0.24
0.14
IGROV1
1.57 0.24
1.94 0.27
2.07 0.40
3.13 0.42
2.55 0.37
3.37 0.47
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Non-smallcell lung
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Renal
786e0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
2.33
3.68
1.68
2.47
1.67
1.83
3.46
1.85
0.61
0.26
0.46
0.27
0.31
0.17
0.27
0.34
0.21
0.12
1.57 0.28
1.72 0.18
2.41 0.28
3.09 0.25
1.39 0.21
1.58 0.30
3.51 0.28
2.09 0.29
Fig. 2. Effect of PBD conjugates (6aec) on cell viability. A375 cells were treated with
4 mM concentration of PBD conjugates as indicated for 24 h in 96-well plates seeded
with 10,000 cells per well. O.D readings were taken at 420 nm wavelength which is
a measure of cell viability after treatment with the respective compounds. DC-81 was
used as the positive control, left side conjugate partner (2) and reference compound
CA-4 (1) are also used. C: control cells (untreated cells).
Colon
Breast
MCF7
MDA-MB-
HS 578T
MDA-MB-435
BT-549
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
1.96
1.58
1.70
2.27
2.75
1.74
1.55
0.21
0.19
0.18
0.18
0.36
0.22
0.23
1.75 0.20
1.15 0.19
1.10 0.16
3.54 0.17
1.23 0.09
1.31 0.28
1.34 0.22
cell lines in SRB assay. Hence from this data we can conclude that
linking of 3,5-diaryl-isoxazoline/isoxazole moiety to the PBD ring
system improved the anticancer activity. Finally, it may be
concluded that one of the PBD conjugate 6a prepared in the present
investigation has the potential to be taken up for preclinical studies
either alone or in combination with existing therapies.
KM12
SW-620
T-47D
MDA-MB-468
CNS
Prostate
PC-3
DU-145
SF-268
SF-539
SNB-19
SNB-75
U251
1.89
1.80
1.96
1.71
1.73
0.34
0.22
0.34
0.32
0.30
0.32 0.58
0.32 2.48
5. Experimental protocols
Melanoma
LOX IMVI
MALME-3M
M14
Melanoma
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
1.47
2.88
1.73
2.03
0.22
0.21
0.18
0.23
2.07 0.20
1.47 0.16
2.33 0.27
1.52 0.16
All chemicals and reagents were purchased from Aldrich (Sig-
maeAldrich, 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 per-
formed with Merck (60e120) mesh silica gel. 1H NMR spectra were
SK-MEL-2
Meanb
1.90(5b) 0.25(6b)
a
Data obtained from NCI’s in vitro anticancer activity cells screen.
Mean values over 60 cell lines tested.
b
Table 2
GI50 values (in m
M) for compounds (5a, 5cee, 6a, 6c and 7aee) in selected human cancer cell linesa
Compound
A549b
HOP62b
Zr-75-1c
MCF7c
A2780d
KBe
0.16
0.16
0.18
0.18
<0.1
<0.1
0.17
0.15
0.13
0.15
0.16
0.17
0.16
Colo205f
PC3g
0.17
0.16
0.17
0.18
<0.1
<0.1
0.16
0.14
0.13
0.14
0.17
0.20
SiHah
5a
5c
5d
5e
6a
6c
7a
7b
2.15
0.15
0.17
0.16
<0.1
<0.1
0.17
0.12
0.11
0.12
0.11
0.16
13.0
0.17
0.18
0.17
0.18
<0.1
<0.1
0.19
0.17
0.14
0.17
0.17
0.15
<0.1
0.13
0.12
0.15
0.18
<0.1
<0.1
0.13
0.10
<0.1
0.18
0.10
2.37
<0.1
1.94
1.85
0.15
0.16
<0.1
<0.1
0.14
0.12
<0.1
<0.1
0.10
0.17
<0.1
0.15
0.14
0.15
0.15
<0.1
<0.1
0.15
0.14
0.12
0.13
0.14
0.14
<0.1
1.45
1.32
0.13
0.13
<0.1
<0.1
0.15
0.13
0.12
0.13
0.14
0.11
14.0
0.18
0.16
0.17
0.18
<0.1
<0.1
0.15
0.15
0.13
0.15
0.16
0.17
1.9
7c
7d
7e
DC-81(4)
ADRi
<0.1
a
50% growth inhibition and the values are mean of three determinations.
b
c
d
e
f
Lung cancer.
Breast cancer.
Ovarian cancer.
Oral cancer.
Colon cancer.
Prostate cancer.
Cervix cancer.
Adriamycin.
g
h
i

3930
A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3924e3937
Fig. 3. (a). DNA histograms obtained by flow cytometry and the percentages of cells in G0/G1, S, and G2/M cell cycle phase, after the treatment of A375 cells with compounds 6aec,
left side conjugate partner (2), reference compound CA-4 (1) and positive control DC-81 (4) at 4 M for 24 h. (b). FACS analysis of cell cycle distribution of A375 cells after treatment
M. DC-81 was used as the positive control, left side conjugate partner (2) and reference compound CA-4 (1) are also used. C: control cells
m
with PBD conjugates (6aec) for 24 h at 4
m
treated with DMSO.
recorded on Bruker UXNMR/XWIN-NMR (300 MHz) instruments.
Chemical shifts ( ) are reported in ppm downfield from internal
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.
d
TMS standard. ESI spectra were recorded on Micromass, Quattro LC
using ESIþ software with capillary voltage 3.98 kV and ESI mode

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3924e3937
3931
aldehyde 10b (1.96 g, 10 mmol). After the suspension was stirred for
5 h at room temperature, Et2O (30 mL) was added, and the mixture
was poured into ice water and extracted with Et2O. The combined
organic extracts were dried and evaporated, and this was further
purified by column chromatography using ethyl acetate-hexane (5%)
as eluent to obtain the pure product 11b as oil. Yield 2.2 g, 75%; 1H
NMR(CDCl3, 300MHz):
d
6.60(dd,J¼ 17.6,10.5Hz,1H, olefineH), 6.55
(s, 2H, ArH), 5.53 (d, J ¼ 17.6 Hz,1H, olefineH), 5.16 (d, J ¼ 10.5 Hz, 1H,
olefineH), 3.82 (s, 6H, 2ꢃ eOCH3), 1.00 (s, 9H, eC(CH3)3), 0.16 (s, 6H,
eSi(CH3)3); MS (ESI): m/z 295 [M þ 1]þ.
Fig. 4. Effect of 3,5-diaryl-isoxazoline/isoxazole-pyrrolobenzodiaze conjugates on the
expression of apoptotic proteins. A375 cells were treated with compounds 6aec, DC-
5.1.3. Synthesis of 5-(4-(tert-butyldimethysilyloxy)-3,5-
dimethoxyphenyl)-3-(3,4,5-trimethoxy-phenyl)-4,5-
dihydroisoxazole (12b)
81 (4) and 2 at 4
expression levels of p53, cytochrome c, cleaved PARP and Cdk2 were determined by
Western blot analysis. -actin was used as loading control.
mM concentrations for 24 h. The cell lysates were collected and
b
To dipolarophile 11b (10 mmol) and Et3N (1 mmol) in CH2Cl2
were added, under argon atmosphere, a 13% aqueous solution of
NaOCl (16 mmol) and dropwise (over a period of 1 h) at 0 ꢂC, the
appropriate oxime 9 (2.81 g, 10 mmol) in CH2Cl2. After being stirred
at room temperature for 24 h, water was added to the reaction
mixture and the aqueous layer was extracted with CH2Cl2. The
combined organic layers were washed with water, brine, dried
(Na2SO4), filtered and concentrated under reduced pressure. This
was further purified by column chromatography using ethyl
acetate-hexane (15%) as eluent to obtain the pure product 12b as
5.1. Synthesis of 3,5-diaryl-isoxazoline/isoxazole-PBD conjugates
5.1.1. Synthesis of 4-(tert-butyl-dimethyl-silyloxy)-3,5-
dimethoxybenzaldehyde oxime (20b)
Toasolutionofhydroxylaminehydrochloride(12mmol)dissolved
in H2O, NaHCO3 (18 mmol) was added portion wise at 0 ꢂC, and the
mixture was stirred for 30 min at room temperature. The compound
10b (2.96 g, 10 mmol), dissolved in MeOH, was then added to the
solution, and stirring was continued for an additional 6 h. MeOH was
evaporated in vacuum, and the residue extracted with diethyl ether.
The organic extracts were washed with brine, dried, and evaporated
under reduced pressure. This was further purified by column chro-
matography using ethyl acetate-hexane (8%) as eluent to afford
yellow oil. Yield 3.0 g, 60%; 1H NMR (CDCl3, 300 MHz):
d 6.87 (s, 2H,
ArH), 6.56 (s, 2H, ArH), 5.60 (dd, J ¼ 10.5, 8.3 Hz, 1H, iso-
xazolineeH), 3.90 (s, 6H, 2ꢃ eOCH3), 3.88 (s, 6H, 2ꢃ eOCH3), 3.84
(s, 3H, eOCH3), 3.67 (dd, J ¼ 16.4, 10.5 Hz, 1H, isoxazolineeH), 3.24
(dd, J ¼ 16.4, 8.3 Hz, 1H, isoxazolineeH), 1.02 (s, 9H, eC(CH3)3), 0.15
(s, 6H, eSi(CH3)3); MS (ESI): m/z 504 [M þ 1]þ.
compound 20b. Yield 1.8 g, 60%; 1H NMR (CDCl3, 300 MHz):
d 7.98 (s,
1H, ArH), 6.74 (s, 2H, ArH), 3.83 (s, 6H, 2ꢃ eOCH3), 0.99 (s, 9H, eC
5.1.4. 3-(4-(tert-Butyldimethylsilyloxy)-3-methoxyphenyl)-5-
(3,4,5-trimethoxyphenyl)-4,5-dihydroisoxazole (17)
(CH3)3), 0.12 (s, 6H, eSi(CH3)3); MS (ESI): m/z 312 [M þ 1]þ.
This compound was prepared according to the method
described for compound 12b, employing alkene 16 (2.94 g,
10 mmol) and oxime 15 (2.11 g,10 mmol) to obtain the pure product
5.1.2. Synthesis of tert-butyl-(2,6-dimethoxy-4-vinylphenoxy)-
dimethyl silane (11b)
NaH (55%) (20 mmol), previously washed with dry hexane, was
added to a stirred suspension of methyltriphenylphosphoniumbr
omide (10 mmol) in dry THF (15 mL) containing the appropriate
17 as yellow oil. Yield 2.5 g, 55%; 1H NMR (CDCl3, 300 MHz):
d 6.89
(d, 1H, J ¼ 2.1 Hz, ArH), 6.82 (d, 1H, J ¼ 8.3, 2.1 Hz, ArH), 6.77 (d, 1H,
J ¼ 8.3 Hz, ArH), 6.44 (s, 2H, ArH), 5.68 (dd, 1H, J ¼ 10.6, 8.4 Hz,
isoxazolineeH), 3.93 (s, 3H, eOCH3), 3.84 (s, 6H, 2ꢃ eOCH3), 3.65
(s, 3H, eOCH3), 3.75 (dd, 1H, J ¼ 16.6, 10.6 Hz, isoxazolineeH), 3.31
(dd, 1H, J ¼ 16.6, 8.3 Hz, isoxazolineeH), 1.03 (s, 9H, eC(CH3)3), 0.17
(s, 6H, eSi(CH3)3); MS (ESI): m/z 474 [M þ 1]þ.
5.1.5. 3-(4-(tert-Butyldimethylsilyloxy)-3,5-dimethoxyphenyl)-5-
(3,4,5-trimethoxy-phenyl) isoxazole (21b)
This compound was prepared according to the method
described for compound 12b, employing alkyne 19 (1.92 g,
10 mmol) and oxime 20b (2.81 g, 10 mmol). This was further
purified by column chromatography using ethyl acetate-hexane
(15%) as eluent to obtain the pure product 21b as yellow oil. Yield
2.25 g, 45%; 1H NMR (CDCl3, 300 MHz):
d 7.01 (s, 4H, ArH), 6.63 (s,
1H, isoxazoleeH), 3.95 (s, 6H, 2ꢃ eOCH3), 3.89 (s, 6H, 2ꢃ eOCH3),
3.88 (s, 3H, eOCH3), 1.01 (s, 9H, eC(CH3)3), 0.15 (s, 6H, eSi(CH3)3);
MS (ESI): m/z 502 [M þ 1]þ.
5.1.6. Synthesis of 2,6-dimethoxy-4-(5-(3,4,5-trimethoxyphenyl)-
4,5-dihydroisoxa-zol-3-yl) phenol (13b)
A solution of the silyl ether 12b (1.50 g, 3 mmol) in dry THF was
treated with a 1 M solution of tetrabutylammonium fluoride in THF
(3 mmol). The mixture was stirred at room temperature during 2 h,
then diluted with water and extracted with ethyl acetate. The
organic layers were washed with water, dried (Na2SO4), filtered and
concentrated under reduced pressure. This was further purified by
column chromatography using ethyl acetate-hexane (50%) as
Fig. 5. Effect of PBD compounds on caspase-3 activity in A375 cells. The graph
represents the increased enzymatic activity of caspase-3, during apoptotic event that
occurs after the treatment of PBD conjugates (6aec) at 4 mM concentration in A375
cells and was determined by fluorometery . Here DC-81 (4) is positive control and 2 is
the conjugate partner. The cleavage of peptide by caspase-3 releases the fluorophore,
AFC that was quantified at excitation wavelength of 400 nm and emission wavelength
of 505 nm. ‘I’ represent the inhibitor used. C þ I: Control treated with caspase-3
inhibitor. DEVD-CHO is the inhibitor here.

3932
A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3924e3937
eluent to obtain the pure product 13b as a light yellow solid. Yield
0.99 g, 85%; mp 137e139 ꢂC; 1H NMR (CDCl3, 300 MHz):
6.89 (s,
1 mmol) and compound 13a (1 mmol) to obtain the pure product
24b as a yellow solid. Yield 666 mg, 82%; mp 64e66 ꢂC; 1H NMR
(CDCl3, 300 MHz): 7.62 (s, 1H, ArH), 6.86 (s, 2H, ArH), 6.84 (dd, 1H,
J ¼ 8.4, 2.0 Hz, ArH), 6.82 (d, 1H, J ¼ 2.0 Hz, ArH) 6.79 (d, 1H,
J ¼ 8.1 Hz, ArH), 6.73 (s, 1H, ArH), 5.60 (dd, 1H, J ¼ 10.1, 9.4 Hz,
isoxazolineeH), 4.82 (d, 1H, J ¼ 3.7 Hz, eCHS2e), 4.70e4.61 (m, 1H,
eNCHe), 4.10 (t, 4H, J ¼ 6.4 Hz, 2ꢃ eOCH2e), 3.90 (s, 3H, eOCH3),
3.87 (s, 6H, 2ꢃ eOCH3), 3.83 (s, 3H, eOCH3), 3.82 (s, 3H, eOCH3),
3.66 (dd, 1H, J ¼ 16.6, 10.1 Hz, isoxazolineeH), 3.25 (dd, 1H, J ¼ 16.2,
9.4 Hz, isoxazolineeH), 3.23e3.17 (m, 2H, eNCH2e), 2.85e2.65 (m,
4H, 2ꢃ eSCH2e), 2.15e1.99 (m, 2H, eCH2e), 1.96e1.91 (m, 2H,
eCH2e), 1.85e1.73 (m, 4H, 2ꢃ eCH2e), 1.40e1.29 (m, 6H, e(CH3)2);
MS (ESI): m/z 814 [M þ 1]þ.
d
2H, ArH), 6.54 (s, 2H, ArH), 5.80 (br, 1H, eOH), 5.60 (dd, 1H, J ¼ 10.5,
8.3 Hz, isoxazolineeH), 3.91 (s, 6H, 2ꢃ eOCH3), 3.88 (s, 6H, 2ꢃ
eOCH3), 3.85 (s, 3H, eOCH3), 3.68 (dd, 1H, J ¼ 16.6, 10.5 Hz, iso-
xazolineeH), 3.26 (dd, 1H, J ¼ 16.6, 8.3 Hz, isoxazolineeH); MS
(ESI): m/z 390 [M þ 1]þ.
5.1.7. 2-Methoxy-4-(5-(3,4,5-trimethoxyphenyl)-4,5-
dihydroisoxazol-3-yl)phenol (18)
This compound was prepared according to the method
described for compound 13b, employing silyl ether 17 (1.42 g,
3 mmol). This was further purified by column chromatography
using ethyl acetate-hexane (60%) as eluent to obtain the pure
product 18 as light yellow solid. Yield 0.93 g, 86%; mp 130e132 ꢂC;
5.1.11. (2S)-N-{4-[5-[(2-Methoxy-5-(3-[2,3,4-triMethoxyphenyl]-
4,5-dihydro-5-isoxazolylphenyl)oxy]pentyl)oxy-5-methoxy-2-
nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal (24c)
This compound was prepared according to the method described
for compound 24a, employing compound 23c (549 mg,1 mmol) and
compound 13a (1 mmol) to obtain the pure product 24c as a yellow
solid. Yield 736 mg, 89%; mp 68e70 ꢂC; 1H NMR (CDCl3, 300 MHz):
1H NMR (CDCl3, 300 MHz):
d
6.91 (d,1H, J ¼ 2.1 Hz, ArH), 6.85 (d,1H,
J ¼ 8.3, 2.1 Hz, ArH), 6.73 (d, 1H, J ¼ 8.3 Hz, ArH), 6.42 (s, 2H, ArH),
5.85 (br, 1H, eOH), 5.67 (dd, 1H, J ¼ 10.6, 8.4 Hz, isoxazolineeH),
3.94 (s, 3H, eOCH3), 3.83 (s, 6H, 2ꢃ eOCH3), 3.64 (s, 3H, eOCH3),
3.74 (dd, 1H, J ¼ 16.6, 10.6 Hz, isoxazolineeH), 3.35 (dd, 1H, J ¼ 16.6,
8.3 Hz, isoxazolineeH); MS (ESI): m/z 360 [M þ 1]þ.
d
7.66 (s,1H, ArH), 6.93 (d,1H, J ¼ 3.0 Hz, ArH), 6.92 (s, 2H, ArH), 6.91
5.1.8. 2,6-Dimethoxy-4-(5-(3,4,5-trimethoxyphenyl)isoxazol-3-yl)
phenol (22b)
(d, 1H, J ¼ 8.3, 3.0 Hz, ArH), 6.86 (d, 1H, J ¼ 8.3 Hz, ArH), 6.81 (s, 1H,
ArH), 5.68 (dd, 1H, J ¼ 10.5, 9.0 Hz, isoxazolineeH), 4.88 (d, 1H,
J ¼ 3.7 Hz, eCHS2e), 4.74e4.67 (m, 1H, eNCHe), 4.44e4.04 (m, 4H,
2ꢃ eOCH2e), 3.93 (s, 3H, eOCH3), 3.88 (s, 9H, 3ꢃ eOCH3), 3.86 (s,
3H, eOCH3), 3.73 (dd,1H, J ¼ 16.4,10.5 Hz, isoxazolineeH), 3.32 (dd,
1H, J ¼ 16.4, 9.0 Hz, isoxazolineeH), 3.26e3.18 (m, 2H, eNCH2e),
2.86e2.67 (m, 4H, 2ꢃ eSCH2e), 2.00e1.76 (m, 4H, 2ꢃ eCH2e),
1.73e1.62 (m, 4H, 2ꢃ eCH2e), 1.39e1.30 (m, 8H, e(CH3)2, eCH2e);
MS (ESI): m/z 828 [M þ 1]þ.
This compound was prepared according to the method described
for compound 13b, employing silyl ether 21b (1.50 g, 3 mmol). This
was further purified bycolumn chromatography usingethylacetate-
hexane (60%) as eluent toobtain the pure product 22b as light yellow
solid. Yield 1.02 g, 88%; mp 195e197 ꢂC; 1H NMR (CDCl3, 300 MHz):
d
7.05 (s, 2H, ArH), 7.00 (s, 2H, ArH), 6.64 (s, 1H, isoxazoleeH), 5.62
(br,1H, eOH), 3.98 (s, 6H, 2ꢃ eOCH3), 3.95 (s, 6H, 2ꢃ eOCH3), 3.87 (s,
3H, eOCH3); MS (ESI): m/z 388 [M þ 1]þ.
5.1.12. (2S)-N-{4-[4-[(2,6-Dimethoxy-4-(3-[2,3,4-
5.1.9. Synthesis of (2S)-N-{4-[3-[(2-methoxy)-5-(3-[2,3,4-
trimethoxyphenyl]-4,5-dihydro-5-isoxazolylphenyl)oxy]butyl)oxy-
5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal (24d)
This compound was prepared according to the method
described for compound 24a, employing compound 23b (535 mg,
1 mmol) and compound 13b (1.0 mmol) to obtain the pure product
24d as a yellow solid. Yield 676 mg, 80%; mp 68e70 ꢂC; 1H NMR
trimethoxyphenyl]-4,5-dihydro-5-isoxazolylphenyl)oxy]propyl)oxy-
5-methoxy-2-nitrobenzoyl} pyrrolidine-2-carboxaldehyde diethyl
thioacetal (24a)
To a solution of 2S-N-[4-(3-bromopropoxy)-5-methoxy-2-nitro-
benzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal (23a)
(521 mg, 1.0 mmol) in dry CH3COCH3 (20 mL) was added anhydrous
K2CO3 (5.0 mmol) and 4-(4,5-dihydro-5-(3,4,5-trimethoxyphenyl)
isoxazol-3-yl)-2-methoxyphenol of formula 13a (1.0 mmol). The
reaction mixture was refluxed in an oil bath for 24 h and the reaction
was monitored by TLC using ethyl acetate-hexane (60%) as a solvent
system. The K2CO3was then removed by suction filtration and the
solvent was evaporated under vacuum to afford the crude product.
This was further purified by column chromatography using ethyl
acetate-hexane (60%) as a solvent system to obtain the pure product
24a as a yellow solid. Yield 647 mg, 81%; mp 65e67 ꢂC; 1H NMR
(CDCl3, 300 MHz):
d 7.70 (s, 1H, ArH), 6.94 (s, 2H, ArH), 6.82 (s, 1H,
ArH), 6.60 (s, 2H, ArH), 5.68 (dd,1H, J ¼ 10.7, 8.6 Hz, isoxazolineeH),
4.85 (d, 1H, J ¼ 3.7 Hz, eCHS2e), 4.76e4.64 (m, 1H, eNCHe), 4.01 (t,
4H, J ¼ 6.2 Hz, 2ꢃ eOCH2e), 3.94 (s, 3H, eOCH3), 3.89 (s, 6H, 2ꢃ
eOCH3), 3.88 (s, 6H, 2ꢃ eOCH3), 3.84 (s, 3H, eOCH3), 3.76 (dd, 1H,
J ¼ 16.6, 10.9 Hz, isoxazolineeH), 3.34 (dd, 1H, J ¼ 16.6, 8.8 Hz,
isoxazolineeH), 3.28e3.18 (m, 2H, eNCH2e), 2.85e2.68 (m, 4H, 2ꢃ
eSCH2e), 2.14e2.07 (m, 4H, 2ꢃ eCH2e), 1.99e1.90 (m, 4H, 2ꢃ
eCH2e), 1.38e1.31 (m, 6H, e(CH3)2); MS (ESI): m/z 844 [M þ 1]þ.
(CDCl3, 300 MHz):
d
7.67 (s,1H, ArH), 6.90 (d,1H, J ¼ 2.1 Hz, ArH), 6.86
(s, 2H, ArH), 6.83 (d, 1H, J ¼ 2.1 Hz, ArH), 6.78 (d, 1H, J ¼ 8.4 Hz, ArH),
6.73 (s, 1H), 5.58 (dd, 1H, J ¼ 10.5, 8.3 Hz, isoxazolineeH), 4.80 (d, 1H,
J¼ 3.7 Hz, eCHS2e), 4.68e4.62(m,1H, eNCHe), 4.32(t, 4H, J ¼ 5.2Hz,
2ꢃ eOCH2e), 3.90 (s, 3H, eOCH3), 3.87 (s, 6H, 2ꢃ eOCH3), 3.83 (s, 3H,
eOCH3), 3.82 (s, 3H, eOCH3), 3.65 (dd, 1H, J ¼ 16.6, 10.1 Hz, iso-
xazolineeH), 3.26 (dd,1H, J ¼ 16.2, 9.0 Hz, isoxazolineeH), 3.23e3.17
(m, 2H, eNCH2e), 2.85e2.63 (m, 4H, 2ꢃ eSCH2e), 2.39e2.32 (m, 2H,
eCH2e), 2.15e2.04 (m, 2H, eCH2e), 1.98e1.89 (m, 2H, eCH2e),
1.38e1.30 (m, 6H, e(CH3)2); MS (ESI): m/z 800 [M þ 1]þ.
5.1.13. (2S)-N-{4-[5-[(2,6-Dimethoxy-4-(3-[2,3,4-
trimethoxyphenyl]-4,5-dihydro-5-isoxazolylphenyl)oxy]pentyl)oxy-
5-methoxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl
thioacetal (24e)
This compound was prepared according to the method described
for compound 24a, employing compound 23c (549 mg,1 mmol) and
compound 13b (1.0 mmol) to obtain the pure product 24e. Yield
686 mg, 80%; mp 67e69 ꢂC; 1H NMR (CDCl3, 300 MHz):
d 7.62 (s,1H,
ArH), 6.87 (s, 2H, ArH), 6.76 (s, 1H, ArH), 6.53 (s, 2H, ArH), 5.61 (dd,
1H, J ¼ 10.9, 8.3 Hz, isoxazolineeH), 4.82 (d,1H, J ¼ 3.7 Hz, eCHS2e),
4.71e4.61 (m,1H, eNCHe), 4.10 (t, 4H, J ¼ 6.9 Hz, 2ꢃ eOCH2e), 3.92
(s, 3H, eOCH3), 3.88 (s, 9H, 3ꢃ eOCH3), 3.84 (s, 3H, eOCH3), 3.83 (s,
3H, eOCH3), 3.70 (dd,1H, J ¼ 16.4,10.9 Hz, isoxazolineeH), 3.27 (dd,
1H, J ¼ 16.4, 8.3 Hz, isoxazolineeH), 3.24e3.20 (m, 2H, eNCH2e),
2.83e2.65 (m, 4H, 2ꢃ eSCH2e), 2.15e2.04 (m, 2H, eCH2e),
5.1.10. (2S)-N-{4-[4-[(2-Methoxy-5-(3-[2,3,4-trimethoxyphenyl]-
4,5-dihydro-5-isoxazolylphenyl)oxy]butyl)oxy-5-methoxy-2-
nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal (24b)
This compound was prepared according to the method
described for compound 24a, employing compound 23b (535 mg,

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3924e3937
3933
2.00e1.87 (m, 4H, 2ꢃ eCH2e), 1.87e1.66 (m, 4H, 2ꢃ eCH2e),
1.39e1.36 (m, 6H, e(CH3)2); MS (ESI): m/z 858 [M þ 1]þ.
2.0 Hz, ArH), 7.00 (s, 2H, ArH), 6.86 (d,1H, J ¼ 8.3 Hz, ArH), 6.74 (s,1H,
ArH), 6.63 (s, 1H, isoxazoleeH), 4.81 (d, 1H, J ¼ 3.7 Hz, eCHS2e),
4.68e4.61 (m, 1H, eNCHe), 4.38e4.30 (m, 4H, 2ꢃ eOCH2e), 3.95 (s,
6H, 2ꢃ eOCH3), 3.92 (s, 3H, eOCH3), 3.91 (s, 3H, eOCH3), 3.86 (s, 3H,
eOCH3),3.26e3.12(m,2H, eNCH2e),2.78e2.65(m,4H, 2ꢃ eSCH2e),
2.45e2.27 (m, 4H, 2ꢃ eCH2e), 1.89e1.67 (m, 2H, eCH2e), 1.42e1.22
(m, 6H, e(CH3)2); MS (ESI) : m/z 798 [M þ 1]þ.
5.1.14. (2S)-N-{4-[3-[(2-Methoxy-4-(5-[3,4,5-trimethoxyphenyl]-
4,5-dihydro-3-isoxazolylphenyl)oxy]propyl)oxy-5-methoxy-2-
nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal (25a)
This compound was prepared according to the method described
for compound 24a, employing compound 23a (521 mg, 1.0 mmol)
and compound 18 (1 mmol) to obtain the pure product 25a as
a yellow solid. Yield 639 mg, 80%; mp 65e67 ꢂC; 1H NMR (CDCl3,
5.1.18. (2S)-N-{4-[4-[(2-Methoxy-5-(5-[3,4,5-trimethoxyphenyl]-3-
isoxazolyl-phenyl)oxy]butyl)oxy-5-methoxy-2-nitrobenzoyl}
pyrrolidine-2-carboxaldehyde diethyl thioacetal (26b)
This compound was prepared according to the method
described for compound 24a, employing compound 23b (535 mg,
1.0 mmol) and compound 22a (1.0 mmol) to obtain the pure
product 26b as a yellow solid. Yield 648 mg, 80%; mp 65e67 ꢂC; 1H
300 MHz):
d
7.63 (s, 1H, ArH), 7.38 (d, 1H, J ¼ 2.0 Hz, ArH), 7.06 (dd,
1H, J ¼ 8.2, 2.0 Hz, ArH), 6.87 (d,1H, J ¼ 8.2 Hz, ArH), 6.83 (s,1H, ArH),
6.63 (s, 2H, ArH), 5.58 (dd, 1H, J ¼ 10.5, 8.3 Hz, isoxazolineeH), 4.81
(d, 1H, J ¼ 3.7 Hz, eCHS2e), 4.68e4.61 (m, 1H, eNCHe), 4.32 (t, 4H,
J ¼ 5.2 Hz, 2ꢃ eOCH2e), 3.89 (s, 3H, eOCH3), 3.87 (s, 6H, 2ꢃ eOCH3),
3.83 (s, 6H, 2ꢃ eOCH3), 3.70 (dd,1H, J ¼ 16.6,10.5 Hz, isoxazolineeH)
3.27 (dd, 1H, J ¼ 16.6, 8.3 Hz, isoxazolineeH) 3.24e3.14 (m, 2H,
eNCH2e), 2.84e2.63 (m, 4H, 2ꢃ eSCH2e), 2.39e2.29 (m, 2H,
eCH2e), 2.16e2.05 (m, 2H, eCH2e), 1.83e1.71 (m, 2H, eCH2e),
1.38e1.29 (m, 6H, e(CH3)2); MS (ESI): m/z 800 [M þ 1]þ.
NMR (CDCl3, 300 MHz):
d
7.69 (s, 1H, ArH), 7.45 (d, 1H, J ¼ 1.8 Hz,
ArH), 7.29 (dd,1H, J ¼ 8.3, 2.0 Hz, ArH), 7.00 (s, 2H, ArH), 6.88 (d, 1H,
J ¼ 8.4 Hz, ArH), 6.73 (s, 1H, ArH), 6.65 (s, 1H, isoxazoleeH), 4.80 (d,
1H, J ¼ 3.7 Hz, eCHS2e), 4.69e4.61 (m, 1H, eNCHe), 4.49e4.29 (m,
4H, 2ꢃ eOCH2e), 3.95 (s, 6H, 2ꢃ eOCH3), 3.91 (s, 3H, eOCH3), 3.90
(s, 3H, eOCH3), 3.87 (s, 3H, eOCH3), 3.24e3.15 (m, 2H, eNCH2e),
2.79e2.68 (m, 4H, 2ꢃ eSCH2e), 2.33e2.24 (m, 2H, eCH2e),
1.92e1.83 (m, 2H, eCH2e), 1.80e1.69 (m, 4H, 2ꢃ eCH2e), 1.39e1.28
(m, 6H, e(CH3)2); MS (ESI) : m/z 812 [M þ 1]þ.
5.1.15. (2S)-N-{4-[4-[(2-Metloxy-4-(5-[3,4,5-trimethoxyphenyl]-
4,5-dihydro-3-isoxazolylphenyl)oxy]butyl)oxy-5-methoxy-2-
nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal (25b)
This compound was prepared according to the method described
for compound 24a, employing compound 23b (535 mg,1 mmol) and
compound 18 (1 mmol) to obtain the pure product 25b as a yellow
solid. Yield 683 mg, 84%; mp 68e70 ꢂC; 1H NMR (CDCl3, 300 MHz):
5.1.19. (2S)-N-{4-[5-[(2-Methoxy-5-(5-[3,4,5-trimethoxyphenyl]-3-
isoxazolyl- phenyl)oxy]pentyl)oxy-5-methoxy-2-nitrobenzoyl}
pyrrolidine-2-carboxaldehyde diethyl thioacetal (26c)
d
7.69 (s, 1H, ArH), 7.41 (d, 1H, J ¼ 2.2 Hz, ArH), 7.04 (dd, 1H, J ¼ 8.3,
This compound was prepared according to the method
described for compound 24a, employing compound 23c (549 mg,
1.0 mmol) and compound 22a (1.0 mmol) to obtain the pure
product 26c as yellow solid. Yield 701 mg, 85%; mp 68e70 ꢂC; 1H
2.2 Hz, ArH), 6.87 (d,1H, J ¼ 8.3 Hz, ArH), 6.81 (d,1H, J ¼ 2.2 Hz, ArH),
6.61 (s, 2H, ArH), 5.67 (dd, 1H, J ¼ 10.5, 9.0 Hz, isoxazolineeH), 4.88
(d, 1H, J ¼ 3.7 Hz, eCHS2e), 4.74e4.69 (m, 1H, eNCHe), 4.14 (t, 4H,
J ¼ 3.7 Hz, 2ꢃ eOCH2e), 3.92 (s, 3H, eOCH3), 3.89 (s, 3H, eOCH3),
3.87 (s, 6H, 2ꢃ eOCH3), 3.84 (s, 3H, eOCH3), 3.75 (dd, 1H, J ¼ 16.6,
10.5 Hz, I isoxazolineeH) 3.32 (dd, 1H, J ¼ 16.6, 9.0 Hz, iso-
xazolineeH) 3.28e3.19 (m, 2H, eNCH2e), 2.85e2.69 (m, 4H, 2ꢃ
eSCH2e), 2.14e2.06 (m, 4H, 2ꢃ eCH2e), 1.86e1.73 (m, 4H, 2ꢃ
eCH2e), 1.39e1.30 (m, 6H, e(CH3)2); MS (ESI): m/z 814 [M þ 1]þ.
NMR (CDCl3, 300 MHz):
d
7.65 (s, 1H, ArH), 7.40 (d, 1H, J ¼ 1.8 Hz,
ArH), 7.31 (dd, 1H, J ¼ 8.3, 2.0 Hz, ArH), 7.00 (s, 2H, ArH), 6.90 (d, 1H,
J ¼ 8.4 Hz, ArH), 6.85 (s, 1H, ArH), 6.70 (s, 1H, isoxazoleeH), 4.88 (d,
1H, J ¼ 3.7 Hz, eCHS2e), 4.74e4.66 (m, 1H, eNCHe), 4.16e4.02 (m,
4H, 2ꢃ eOCH2e), 3.95 (s, 3H, eOCH3), 3.89 (s, 6H, 2ꢃ eOCH3), 3.88
(s, 3H, eOCH3), 3.86 (s, 3H, eOCH3), 3.29e3.20 (m, 2H, eNCH2e),
2.86e2.67 (m, 4H, 2ꢃ eSCH2e), 2.00e1.89 (m, 4H, 2ꢃ eCH2e),
1.75e1.60 (m, 4H, 2ꢃ eCH2e), 1.40e1.22 (m, 8H, e(CH3)2, eCH2e);
MS (ESI): m/z 826 [M þ 1]þ.
5.1.16. (2S)-N-{4-[5-[(2-Methoxy-4-(5-[3,4,5-trimethoxyphenyl]-
4,5-dihydro-3-isoxazolylphenyl)oxy]pentyl)oxy-5-methoxy-2-
nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal (25c)
This compound was prepared according to the method described
for compound 24a, employing compound 23c (549 mg, 1 mmol) and
compound 18 (1 mmol) to obtain the pure product 25c as a yellow
solid. Yield 719 mg, 87%; mp 69e71 ꢂC; 1H NMR (CDCl3, 300 MHz):
5.1.20. (2S)-N-{4-[3-[(2,6-Dimethoxy-4-(5-[3,4,5-
trimethoxyphenyl]-5-isoxa-zolylphenyl)oxy]propyl)oxy-5-methoxy-
2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal
(26d)
d
7.70 (s, 1H, ArH), 7.38 (d, 1H, J ¼ 2.3 Hz, ArH), 7.14 (dd, 1H, J ¼ 8.4,
This compound was prepared according to the method
described for compound 24a, employing compound 23a (521 mg,
1.0 mmol) and compound 22b (1.0 mmol) to obtain the pure
product 26d as a yellow solid. Yield 694 mg, 84%; mp 78e80 ꢂC; 1H
2.3 Hz, ArH), 6.87 (d,1H, J ¼ 8.4 Hz, ArH), 6.83 (d,1H, J ¼ 2.2 Hz, ArH),
6.61 (s, 2H, ArH), 5.61 (dd,1H, J ¼ 10.6, 8.6 Hz, isoxazolineeH), 4.81 (d,
1H, J ¼ 3.7 Hz, eCHS2e), 4.69e4.62 (m, 1H, eNCHe), 4.10 (t, 4H,
J ¼ 3.7 Hz, 2ꢃ eOCH2e), 3.93(s, 3H, eOCH3), 3.89(s, 3H, eOCH3), 3.85
(s, 6H, 2ꢃ eOCH3), 3.82 (s, 3H, eOCH3), 3.71 (dd,1H, J ¼ 16.4, 10.6 Hz,
isoxazolineeH) 3.29 (dd, 1H, J ¼ 16.6, 8.6 Hz, I isoxazolineeH)
3.24e3.17 (m, 2H, eNCH2e), 2.84e2.66 (m, 4H, 2ꢃ eSCH2e),
2.36e2.19 (m, 4H, 2ꢃ eCH2e), 1.94e1.60 (m, 4H, 2ꢃ eCH2e),
1.39e1.28 (m, 8H, e(CH3)2, eCH2e); MS (ESI): m/z 828 [M þ 1]þ.
NMR (CDCl3, 300 MHz): d 7.68 (s, 1H, ArH), 7.09 (s, 2H, ArH), 7.06 (s,
2H, ArH), 6.82 (s, 1H, ArH), 6.72 (s, 1H, isoxazoleeH), 4.88 (d, 1H,
J ¼ 4.5 Hz, eCHS2e), 4.75e4.67 (m, 1H, eNCHe), 4.07 (t, 4H,
J ¼ 6.0 Hz, 2ꢃ eOCH2e), 3.96 (s, 6H, 2ꢃ eOCH3), 3.94 (s, 9H, 3ꢃ
eOCH3), 3.91 (s, 3H, eOCH3), 3.33e3.20 (m, 2H, eNCH2e),
2.85e2.67 (m, 4H, 2ꢃ eSCH2e), 2.15e2.06 (m, 2H, eCH2e),
2.01e1.83 (m, 2H, eCH2e), 1.81e1.55 (m, 2H, eCH2e), 1.39e1.28 (m,
6H, e(CH3)2); MS (ESI): m/z 828 [M þ 1]þ.
5.1.17. (2S)-N-{4-[3-[(2-Methoxy-5-(5-[3,4,5-trimethoxyphenyl]-3-
isoxazolyl-phenyl)oxy]propyl)oxy-5-methoxy-2-nitrobenzoyl}
pyrrolidine-2-carboxaldehyde diethyl thioacetal (26a)
5.1.21. (2S)-N-{4-[5-[(2,6-Dimethoxy-4-(5-[3,4,5-
This compound was prepared according to the method described
forcompound 24a, employing compound 23a (521 mg,1.0mmol)and
compound 22a (1.0 mmol) to obtain the pure product 26a as a yellow
solid. Yield 693 mg, 87%; mp 66e68 ꢂC; 1H NMR (CDCl3, 300 MHz):
trimethoxyphenyl]-5-isoxa-zolylphenyl)oxy]pentyl)oxy-5-methoxy-
2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethyl thioacetal (26e)
This compound was prepared according to the method described
for compound 24a, employing compound 23c (549 mg,1.0 mmol) and
compound 22b (1.0 mmol) to obtain the pure product 26e as a yellow
d
7.70 (s, 1H, ArH), 7.43 (d, 1H, J ¼ 2.0 Hz, ArH), 7.30 (dd, 1H, J ¼ 8.3,

3934
A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3924e3937
1
solid. Yield 710 mg, 83%; mp 79e81 ꢂC; H NMR (CDCl3, 300 MHz):
5.1.24. 7-Methoxy-8-{5-[(2-methoxy-5-(3-[3,4,5-
d
7.69 (s, 1H, ArH), 7.09 (s, 2H, ArH), 7.05 (s, 2H, ArH), 6.83 (s, 1H, ArH),
trimethoxyphenyl]-4,5-dihydro-5-isoxazolylphenyl)oxy]pentyloxy}-
(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-
5-one (5c)
The compound 5c was prepared according to the method
described for the compound 5a, employing the compound 24c
6.72 (s, 1H, isoxazoleeH), 4.88 (d, 1H, J ¼ 4.5 Hz, eCHS2e), 4.73e4.67
(m, 1H, eNCHe), 4.16e4.04 (m, 4H, 2ꢃ eOCH2e), 3.95 (s, 6H, 2ꢃ
eOCH3), 3.94 (s, 9H, 3ꢃ eOCH3), 3.90 (s, 3H, eOCH3), 3.32e3.15 (m,
2H, eNCH2e), 2.85e2.65 (m, 4H, 2ꢃ eSCH2e), 2.35e2.21 (m, 2H,
eCH2e), 2.01e1.92 (m, 2H, eCH2e), 1.88e1.61 (m, 4H, 2ꢃ eCH2e),
1.39e1.30 (m, 8H, e(CH3)2, eCH2e); MS (ESI): m/z 856 [M þ 1]þ.
(827 mg, 1 mmol) to obtain the pure product 5c as a white solid.
25
Yield 397 mg, 59%; mp 65e67 ꢂC; [
a
]
þ 140.0ꢂ (c0.1, CHCl3); 1H
D
NMR (CDCl3, 300 MHz):
d
7.65 (d, 1H, J ¼ 4.3 Hz, imineeH), 7.50
5.1.22. Synthesis of 7-methoxy-8-{3-[(2-methoxy-5-(3-[3,4,5-
trimethoxyphenyl]-4,5-dihydro-5-isoxazolylphenyl)oxy]propyloxy}-
(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-
5-one (5a)
(s, 1H, ArH), 6.93 (s, 3H, ArH), 6.90 (d, 1H, J ¼ 2.3 Hz, ArH), 6.85
(d, 1H, J ¼ 8.7 Hz, ArH), 6.79 (s, 1H, ArH), 5.67 (dd, 1H, J ¼ 10.2,
8.7 Hz, isoxazolineeH), 4.04 (t, 4H, J ¼ 6.5 Hz, 2ꢃ eOCH2e), 3.92
(s, 3H, eOCH3), 3.87 (s, 9H, 3ꢃ eOCH3), 3.85 (s, 3H, eOCH3), 3.70
(dd, 1H, J ¼ 16.2, 10.2 Hz, isoxazolineeH), 3.61e3.54 (m, 3H,
eNCH2e, eNCHe), 3.32 (dd, 1H, J ¼ 16.4, 8.7 Hz, isoxazolineeH),
2.33e2.28 (m, 2H, eCH2e), 2.08e2.02 (m, 2H, eCH2e), 1.96e1.87
(m, 2H, eCH2e), 1.69e1.62 (m, 4H, 2ꢃ eCH2e); 13C NMR (CDCl3,
To a solution of compound 24a (799 mg, 1 mmol) in MeOH
(20 mL), SnCl2.2H2O (5 mmol) was added and refluxed for 1e2 h.
The MeOH 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 diethyl thioacetal, which due to
potential stability problems proceeded for the next step. A solution
of amino diethyl thioacetal (1 mmol), HgCl2 (2.26 mmol) and CaCO3
(2.46 mmol) in CH3CN/H2O (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) and brine
(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, 2%) to obtain the pure
product 5a as a white solid. Yield 335 mg, 52%; mp 59e61 ꢂC;
75 MHz):
d 164.4, 162.2, 155.9, 153.1, 150.6, 149.3, 148.6, 147.6,
140.4, 139.7, 132.9, 124.7, 119.9, 118.4, 111.5, 111.4, 110.6, 110.2,
103.9, 82.6, 68.6, 60.7, 56.1, 55.9, 55.9, 53.5, 46.4, 43.0, 29.4, 28.7,
23.9, 22.3; IR (KBr) (Umax/cmꢁ1): 3381, 2933, 1601, 1511, 1458,
1424, 1371, 1258, 1172, 1127, 1020, 898, 817, 764, 636; MS (ESI): m/
z 674 [M þ 1]þ; HRMS (ESI m/z) for C37H44N3O9, calcd 674.3077,
found 674.3063 [M þ 1]þ.
5.1.25. 7-Methoxy-8-{4-[(2,6-dimethoxy-4-(3-[2,3,4-
trimethoxyphenyl]-4,5-dihydro-5-isoxazolylphenyl)oxybutyloxy}-
(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepine-
5-one (5d)
The compound 5d was prepared according to the method
described for the compound 5a, employing the compound 24d
[
a
]
25 þ 143.5ꢂ (c0.1, CHCl3); 1H NMR (CDCl3, 300 MHz):
d
7.65 (d,1H,
(844 mg, 1.0 mmol) to obtain the pure product 5d as a white solid.
D
25
J ¼ 4.3 Hz, imineeH), 7.50 (s, 1H, ArH), 6.95 (d, 1H, J ¼ 8.4 Hz, ArH),
6.93 (s, 3H, ArH), 6.86 (d, 1H, J ¼ 2.6 Hz, ArH), 6.85 (s, 1H, ArH), 5.65
(dd, 1H, J ¼ 10.4, 8.3 Hz isoxazolineeH), 4.32e4.19 (m, 4H, 2ꢃ
eOCH2e), 3.89 (s, 6H, 2ꢃ eOCH3), 3.88 (s, 6H, 2ꢃ eOCH3), 3.84 (s,
3H, eOCH3), 3.69 (dd, 1H, J ¼ 16.3, 10.4 Hz, isoxazolineeH),
3.61e3.52 (m, 3H, eNCH2e, eNCHe), 3.31 (dd, 1H, J ¼ 8.6, 7.3 Hz,
isoxazolineeH), 2.42e2.27 (m, 2H, eCH2e), 2.11e2.01 (m, 2H,
eCH2e), 1.81e1.68 (m, 2H, eCH2e); IR (KBr) (Umax/cmꢁ1): 3380,
2925, 1599, 1511, 1461, 1425, 1371, 1260, 1176, 1126, 1023, 896, 816,
761, 631; MS (ESI): m/z 646 [M þ 1]þ; HRMS (ESI m/z) for
C35H40N3O9, calcd 646.2764, found 646.2745 [M þ 1]þ.
Yield 392 mg, 57%; mp 65e67 ꢂC; [
a
]
þ 152.4ꢂ (c0.1, CHCl3); 1H
D
NMR (CDCl3, 300 MHz):
d
7.65 (d, 1H, J ¼ 4.3 Hz, imineeH), 7.51 (s,
1H, ArH), 6.93 (s, 2H, ArH) 6.82 (s, 1H, ArH), 6.60 (s, 2H, ArH), 5.67
(dd, 1H, J ¼ 10.9, 8.7 Hz, isoxazolineeH), 4.01 (t, 4H, J ¼ 6.5 Hz, 2ꢃ
eOCH2e), 3.92 (s, 3H, eOCH3), 3.88 (s, 9H, 3ꢃ eOCH3), 3.83 (s, 6H,
2ꢃ eOCH3), 3.73 (dd, 1H, J ¼ 16.8, 10.9 Hz, isoxazolineeH),
3.61e3.54 (m, 3H, eNCH2e, eNCHe), 3.33 (dd, 1H, J ¼ 16.9, 8.7 Hz,
isoxazolineeH), 2.36e2.28 (m, 2H, eCH2e), 2.13e1.90 (m, 2H,
eCH2e), 1.69e1.59 (m, 4H, 2ꢃ eCH2e); 13C NMR (CDCl3, 75 MHz):
d
164.5, 162.2, 155.9, 153.6, 153.2, 150.8, 147.6, 140.5, 139.7, 136.7,
136.2, 124.6, 119.9, 111.3, 110.3, 103.9, 102.5, 82.74, 68.5, 60.8, 56.1,
56.0, 53.6, 46.5, 43.3, 30.7, 29.5, 24.0; IR (KBr) (Umax/cmꢁ1): 3380,
2930, 1597, 1507, 1460, 1419, 1371, 1237, 1125, 1002, 895, 831, 763,
5.1.23. 7-Methoxy-8-{4-[(2-methoxy-5-(3-[3,4,5-
trimethoxyphenyl]-4,5-dihydro-5-isoxazolylphenyl)oxy]butyloxy}-
(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-
5-one (5b)
635; MS (ESI): m/z 690 [M þ 1]þ; HRMS (ESI m/z) for C37H44N3O10
,
calcd 690.3026, found 690.3016 [M þ 1]þ.
The compound 5b was prepared according to the method
described for the compound 5a, employing the compound 24b
5.1.26. 7-Methoxy-8-{5-[(2,6-dimethoxy-4-(3-[2,3,4-
trimethoxyphenyl]-4,5-dihydro-5-isoxazolylphenyl)oxypentyloxy}-
(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepine-
5-one (5e)
The compound 5e was prepared according to the method
described for the compound 5a, employing the compound 24e
(813 mg, 1 mmol) to obtain the pure product 5b as a white solid.
25
Yield 382 mg, 58%; mp 72e74 ꢂC; [
a
]
þ 148.3ꢂ (c0.1, CHCl3); 1H
D
NMR (CDCl3, 300 MHz):
d
7.67 (d, 1H, J ¼ 4.3 Hz, imineeH), 7.50
(s, 1H, ArH), 7.02 (d, 1H, J ¼ 8.6 Hz, ArH), 6.87 (d, 1H, J ¼ 2.2 Hz,
ArH), 6.84 (d, 1H, J ¼ 8.6 Hz, ArH), 6.82 (s, 1H, ArH), 6.61 (s, 2H,
ArH), 5.71 (dd, 1H, J ¼ 11.2, 8.6 Hz, isoxazolineeH), 4.18e4.08 (m,
4H, 2ꢃ eOCH2e), 3.92 (s, 3H, eOCH3), 3.89 (s, 3H, eOCH3), 3.86
(s, 6H, 2ꢃ eOCH3), 3.84 (s, 3H, eOCH3), 3.71 (dd, 1H, J ¼ 16.5,
11.2 Hz, isoxazolineeH), 3.61e3.51 (m, 3H, eNCH2e, eNCHe),
3.38e3.25 (dd, 1H, J ¼ 16.5, 8.6 Hz, isoxazolineeH), 2.38e2.25 (m,
2H, eCH2e), 2.11e2.01 (m, 2H, eCH2e), 1.71e1.61 (m, 4H, 2ꢃ
eCH2e); IR (KBr) (Umax/cmꢁ1): 3418, 2924, 2853, 1602, 1511, 1459,
1372, 1258, 1173, 1127, 1021, 903, 818, 766, 637; MS (ESI): m/z 660
[M þ 1]þ; HRMS (ESI m/z) for C36H42N3O9, calcd 660.2921, found
660.2898 [M þ 1]þ.
(858 mg, 1.0 mmol) to afford the compound 5e as a white solid.
25
Yield 407 mg, 58%; mp 62e64 ꢂC; [
a]
þ 154.6ꢂ (c0.1, CHCl3); 1H
D
NMR (CDCl3, 300 MHz):
d
7.67 (d, 1H, J ¼ 4.3 Hz, imineeH), 7.50 (s,
1H, ArH), 6.93 (s, 2H, ArH) 6.80 (s,1H, ArH), 6.61 (s, 2H, ArH), 5.68 (t,
1H, J ¼ 10.1, 8.7 Hz, isoxazolineeH), 4.13e3.98 (m, 4H, 2ꢃ eOCH2e),
3.94 (s, 3H, eOCH3), 3.89 (s, 9H, 3ꢃ eOCH3), 3.85 (s, 6H, 2ꢃ eOCH3),
3.74 (dd, 1H, J ¼ 16.8, 10.1 isoxazolineeH), 3.61e3.49 (m, 3H,
eNCH2e, eNCHe), 3.33 (dd, 1H, J ¼ 16.8, 8.7 Hz, isoxazolineeH),
2.39e2.25 (m, 2H, eCH2e), 2.13e1.76 (m, 4H, 2ꢃ eCH2e), 1.74e1.58
(m, 4H, 2ꢃ eCH2e); IR (KBr) (Umax/cmꢁ1): 3417, 2925, 2855, 1599,
1507, 1460, 1420, 1371, 1238, 1124, 1019, 894, 829, 764, 632; MS

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3924e3937
3935
(ESI): m/z 704 [M þ 1]þ; HRMS (ESI m/z) for C38H46N3O10, calcd
704.3183, found 704.3185 [M þ 1]þ.
isoxazolineeH), 3.62e3.51 (m, 3H, eNCH2e, eNCHe), 3.33 (dd, 1H,
J ¼ 8.7, 8.0 Hz, isoxazolineeH), 2.43e2.23 (m, 2H, eCH2e),
2.10e2.01 (m, 4H, 2ꢃ eCH2e), 1.78e1.64 (m, 4H, 2ꢃ eCH2e); IR
(KBr) (Umax/cmꢁ1): 3380, 2928, 1599, 1511, 1461, 1426, 1370, 1241,
1176, 1125, 1022, 896, 816, 759, 624; MS (ESI): m/z 674 [M þ 1]þ;
HRMS (ESI m/z) for C37H44N3O9, calcd 674.3077, found 674.3066
[M þ 1]þ.
5.1.27. 7-Methoxy-8-{3-[(2-methoxy-4-(5-[3,4,5-
trimethoxyphenyl]-4,5-dihydro-3-isoxazolylphenyl)oxy]propyloxy}-
(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-
5-one (6a)
The compound 6a was prepared according to the method
described for the compound 5a, employing the compound 25a
5.1.30. 7-Methyloxy-8-{3-[(2-methoxy-5-(5-[3,4,5-
trimethoxyphenyl]-3-isoxazolylphenyl)oxy]propyloxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one
(7a)
The compound 7a was prepared according to the method
described for the compound 5a, employing the compound 26a
(799 mg, 1.0 mmol) to afford the compound 6a as a white solid.
25
Yield 374 mg, 58%; mp 63e65 ꢂC; [
a
]
D
þ 144.5ꢂ (c0.1, CHCl3); 1H
NMR (CDCl3, 300 MHz):
d
7.66 (d, 1H, J ¼ 4.3 Hz, imineeH), 7.51 (s,
1H, ArH), 7.00 (d, 1H, J ¼ 8.0 Hz, ArH), 6.93 (d, 1H, J ¼ 2.1 Hz, ArH),
6.88 (d, 1H, J ¼ 8.0 Hz, ArH), 6.85 (s, 1H, ArH), 6.61 (s, 2H, ArH), 5.65
(dd, 1H, J ¼ 10.3, 9.3 Hz, isoxazolineeH), 4.26 (t, 4H, J ¼ 5.8 Hz, 2ꢃ
eOCH2e), 3.92 (s, 3H, eOCH3), 3.89 (s, 3H, eOCH3), 3.86 (s, 6H, 2ꢃ
eOCH3), 3.83 (s, 3H, eOCH3), 3.72 (dd, 1H, J ¼ 16.4, 10.3 Hz, iso-
xazolineeH), 3.61e3.53 (m, 3H, eNCH2e, eNCHe), 3.31 (dd, 1H,
J ¼ 16.4, 9.3 Hz, isoxazolineeH), 2.42e2.26 (m, 2H, eCH2e),
2.08e2.01 (m, 2H, eCH2e), 1.84e1.73 (m, 2H, eCH2e); 13C NMR
(797 mg, 1.0 mmol) to obtain the pure product 7a as a white solid.
25
Yield 360 mg, 56%; mp 59e61 ꢂC; [
a]
þ 147.6ꢂ (c0.1, CHCl3); 1H
D
NMR (CDCl3, 300 MHz):
d
7.66 (d, 1H, J ¼ 4.4 Hz, imineeH), 7.50 (s,
1H, ArH), 7.47 (d, 1H, J ¼ 2.2 Hz), 7.39 (dd, 1H, J ¼ 8.0, 2.2 Hz, ArH),
7.05 (s, 2H, ArH), 6.94 (d, 1H, J ¼ 8.0 Hz, ArH), 6.87 (s, 1H, ArH), 6.72
(s, 1H, isoxazoleeH), 4.32 (t, 4H, J ¼ 5.8 Hz, 2ꢃ eOCH2e), 3.95 (s,
6H, 2ꢃ eOCH3), 3.92 (s, 6H, 2ꢃ eOCH3), 3.91 (s, 3H, 2ꢃ eOCH3),
3.83e3.50 (m, 3H, eNCH2e, eNCHe), 2.50e2.25 (m, 2H, eCH2e),
2.12e1.98 (m, 2H, eCH2e), 1.69e1.59 (m, 2H, eCH2e); IR (KBr)
(Umax/cmꢁ1): 3385, 2923, 2852, 1600, 1575, 1504, 1459, 1428, 1379,
1334, 1260, 1177, 1127, 1022, 870, 765, 612; MS (ESI): m/z 644
[M þ 1]þ; HRMS (ESI m/z) for C35H38N3O9, calcd 644.2608, found
644.2638 [M þ 1]þ.
(CDCl3, 75 MHz):
d 164.5, 162.3, 155.9, 153.4, 150.5, 150.1, 149.5,
147.7, 140.3, 137.6, 136.5, 122.2, 120.2, 114.5, 112.3, 111.5, 110.6, 109.1,
102.6, 82.4, 65.3, 60.7, 56.1, 56.0, 55.9, 53.6, 46.5, 43.4, 29.5, 28.9,
24.0; IR (KBr) (Umax/cmꢁ1): 3378, 2928, 1597, 1511, 1461, 1425, 1367,
1335, 1241, 1123, 1019, 893, 814, 628; MS (ESI): m/z 646 [M þ 1]þ;
HRMS (ESI m/z) for C35H40N3O9, calcd 646.2764, found 646.2748
[M þ 1]þ.
5.1.28. 7-Methoxy-8-{4-[(2-methoxy-4-(5-[3,4,5-
5.1.31. 7-Methyloxy-8-{4-[(2-methoxy-5-(5-[3,4,5-
trimethoxyphenyl]-3-isoxazolylphenyl)oxy]butyloxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one
(7b)
trimethoxyphenyl]-4,5-dihydro-3-isoxazolylphenyl)oxy]butyloxy}-
(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-
5-one (6b)
The compound 6b was prepared according to the method
described for the compound 5a, employing the compound 25b
The compound 7b was prepared according to the method
described for the compound 5a, employing the compound 26b
(813 mg, 1 mmol) to obtain the pure product 6b as a white solid.
(811 mg, 1.0 mmol) to obtain the pure product 7b as a white solid.
25
25
Yield 362 mg, 55%; mp 72e74 ꢂC; [
a
]
þ 145.6ꢂ (c0.1, CHCl3); 1H
Yield 381 mg, 58%; mp 65e67 ꢂC; [
a
]
þ 149.4ꢂ (c0.1, CHCl3); 1H
D
D
NMR (CDCl3, 300 MHz):
d
7.66 (d, 1H, J ¼ 4.3 Hz, imineeH), 7.50 (s,
NMR (CDCl3, 300 MHz):
d
7.66 (d, 1H, J ¼ 4.3 Hz, imineeH), 7.50 (s,
1H, ArH), 6.90e6.97 (m, 4H, ArH) 6.87 (s, 1H, ArH), 6.81 (s, 1H, ArH),
5.67 (dd, 1H, J ¼ 10.1, 9.3 Hz, isoxazolineeH), 4.10 (t, 4H, J ¼ 5.4 Hz,
2ꢃ eOCH2e), 3.92 (s, 3H, eOCH3), 3.88 (s, 6H, eOCH3), 3.85 (s, 6H,
2ꢃ eOCH3), 3.72 (dd, 1H, J ¼ 16.4, 10.1 Hz, isoxazolineeH),
3.61e3.51 (m, 3H, eNCH2e, eNCHe), 3.32 (dd, 1H, J ¼ 16.4, 9.3 Hz,
isoxazolineeH), 2.36e2.24 (m, 2H, eCH2e), 2.11e1.99 (m, 2H,
eCH2e), 1.71e1.56 (m, 4H, 2ꢃ eCH2e); 13C NMR (CDCl3, 75 MHz):
1H, ArH), 7.45 (d, 1H, J ¼ 2.2 Hz, ArH), 7.37 (d, 1H, J ¼ 8.0, 2.2 Hz,
ArH), 7.04 (s, 2H, ArH), 6.93 (d,1H, J ¼ 8.0 Hz, ArH), 6.82 (s, 1H, ArH),
6.72 (s, 1H, isoxazoleeH), 4.24e4.18 (m, 4H, 2ꢃ eOCH2e), 3.94 (s,
9H, 3ꢃ eOCH3), 3.90 (s, 6H, 2ꢃ eOCH3), 3.85e3.68 (m, 1H,
eNCHe), 3.62e3.53 (m, 2H, eNCH2e), 2.34e2.28 (m, 2H, eCH2e),
2.13e2.02 (m, 2H, eCH2e), 1.75e1.67 (m, 4H, 2ꢃ eCH2e); 13C NMR
(CDCl3, 75 MHz):
d 169.8, 164.5, 162.6, 162.2, 153.4, 150.8, 150.6,
d
164.3, 162.1, 155.8, 153.2, 150.4, 150.1, 149.2, 147.4, 140.3, 136.4,
148.5, 147.6, 140.4, 139.6, 122.7, 121.4, 119.9, 119.8, 111.3, 111.2, 110.7,
110.2, 102.9, 96.9, 68.4, 60.8, 56.1, 53.4, 46.5, 29.4, 25.6, 24.0; IR
(KBr) (Umax/cmꢁ1): 3383, 2933, 1606, 1505, 1458, 1378, 1333, 1258,
1176, 1127, 1018, 865, 793, 626; MS (ESI): m/z 658 [M þ 1]þ; HRMS
(ESI m/z) for C36H40N3O9, calcd 658.2764, found 658.2740 [M þ 1]þ.
137.4, 121.8, 120.1, 119.9, 111.7, 111.3, 110.2, 108.8, 102.5, 82.4, 68.4,
60.7, 56.1, 56.0, 55.9, 53.6, 46.5, 43.4, 29.5, 25.8, 24.1; IR (KBr) (Umax
/
cmꢁ1): 3388, 2924, 2854, 1602, 1511, 1459, 1371, 1257, 1172, 1125,
1018, 900, 816,763, 634; MS (ESI): m/z 660 [M þ 1]þ; HRMS (ESI m/
z) for C36H42N3O9, calcd 660.2921, found 660.2914 [M þ 1]þ.
5.1.32. 7-Methyloxy-8-{5-[(2-methoxy-5-(5-[3,4,5-
trimethoxyphenyl]-3-isoxazolylphenyl)oxy]pentyloxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one
(7c)
The compound 7c was prepared according to the method
described for the compound 5a, employing the compound 26c
5.1.29. 7-Methoxy-8-{5-[(2-methoxy-4-(5-[3,4,5-
trimethoxyphenyl]-4,5-dihydro-3-isoxazolylphenyl)oxy]pentyloxy}-
(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-
5-one (6c)
The compound 6c was prepared according to the method
described for the compound 5a, employing the compound 25c
(825 mg, 1.0 mmol) to obtain the pure product 7c as a white solid.
þ 150.5ꢂ (c0.1, CHCl3); 1H
25
(828 mg, 1 mmol) to obtain the pure product 6c as a white solid.
Yield 382 mg, 57%; mp 70e72 ꢂC; [
a]
D
25
Yield 376 mg, 56%; mp 66e68 ꢂC; [
a]
þ 138.2ꢂ (c0.1, CHCl3); 1H
NMR (CDCl3, 300 MHz):
d
7.64 (d, 1H, J ¼ 4.3 Hz, imineeH), 7.49 (s,
D
NMR (CDCl3, 300 MHz):
d
7.65 (d, 1H, J ¼ 4.3 Hz, imineeH), 7.51 (s,
1H, ArH), 7.43 (d, 1H, J ¼ 2.1 Hz, ArH), 7.34 (dd, 1H, J ¼ 8.7, 2.1 Hz,
ArH), 7.03 (s, 2H, ArH), 6.93 (d,1H, J ¼ 8.0 Hz, ArH), 6.78 (s, 1H, ArH),
6.69 (s, 1H, isoxazoleeH), 4.12 (t, 4H, J ¼ 5.1 Hz, 2ꢃ eOCH2e), 3.93
(s, 6H, 2ꢃ eOCH3), 3.91 (s, 6H, 2ꢃ eOCH3), 3.89 (s, 3H, eOCH3),
3.83e3.67 (m, 1H, eNCHe), 3.60e3.51 (m, 2H, eNCH2e), 2.32e2.25
(m, 2H, eCH2e), 2.03e1.89 (m, 2H, eCH2e), 1.72e1.62 (m, 6H, 3ꢃ
1H, ArH),7.05 (d, 1H, J ¼ 8.0 Hz, ArH), 6.90 (d, 1H, J ¼ 2.4 Hz, ArH),
6.86 (d, 1H, J ¼ 8.1 Hz, ArH) 6.84 (s, 1H, ArH), 6.62 (s, 2H, ArH), 5.63
(dd, 1H, J ¼ 10.5, 9.5 Hz, isoxazolineeH), 4.13 (t, 4H, J ¼ 5.8 Hz, 2ꢃ
eOCH2e), 3.93 (s, 3H, 2ꢃ eOCH3), 3.87 (s, 3H, 2ꢃ eOCH3), 3.84 (s,
6H, 2ꢃ eOCH3), 3.82 (s, 3H, 2ꢃ eOCH3), 3.74 (dd, 1H, J ¼ 7.3, 5.8 Hz,

3936
A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3924e3937
eCH2e); IR (KBr) (Umax/cmꢁ1): 3378, 2937, 1601, 1505, 1458, 1426,
1380, 1334, 1260, 1128, 1019, 867, 772, 621; MS (ESI): m/z 672
[M þ 1]þ; HRMS (ESI m/z) for C37H42N3O9, calcd 672.2921, found
672.2904 [M þ 1]þ.
were again incubated for 60 min at 4 ꢂC. The plates were washed
five times with tap water and air-dried. Sulforhodamine B (SRB)
solution (50 mL) at 0.4% (w/v) in 1% acetic acid was added to each of
the wells, and plates were incubated for 20 min at room temper-
ature. The residual dye was removed by washing five times with 1%
acetic acid. The plates were air-dried. 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 refer-
ence wavelengths. Percent growth was calculated on a plate-by-
plate basis for test wells relative to control wells. The above
determinations were repeated three times.
5.1.33. 7-Methoxy-8-{3-[(2,6-dimethoxy-4-(5-[3,4,5-
trimethoxyphenyl]-3-isoxazolylphenyl)oxy]propyloxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one
(7d)
The compound 7d was prepared according to the method
described for the compound 5a, employing the compound 26d
(827 mg, 1.0 mmol) to obtain the pure product 7d as a white solid.
25
Yield 370 mg, 55%; mp 76e78 ꢂC; [
a
]
þ 154.5ꢂ (c0.1, CHCl3); 1H
5.3. Cell culture
D
NMR (CDCl3, 300 MHz):
d
7.67 (d,1H, J ¼ 4.3 Hz, imineeH), 7.51 (s,1H,
ArH), 7.06 (s, 2H, ArH), 7.04 (s, 2H, ArH), 6.90 (s,1H, ArH), 6.71 (s,1H,
isoxazoleeH), 4.26 (t, 4H, J ¼ 6.7 Hz, 2ꢃ eOCH2e), 3.95 (s, 9H, 3ꢃ
eOCH3), 3.91 (s, 6H, 2ꢃ eOCH3), 3.85 (s, 3H, eOCH3), 3.76e3.69 (m,
1H, eNCHe), 3.63e3.52 (m, 2H, eNCH2e), 2.34e2.27 (m, 2H,
eCH2e), 2.10e2.01 (m, 2H, eCH2e), 1.73e1.64 (m, 2H, eCH2e); IR
(KBr) (Umax/cmꢁ1): 3383, 2927, 1600, 1503, 1463, 1426, 1382, 1332,
1240,1126,1002, 847, 783; MS (ESI): m/z 674 [M þ 1]þ; HRMS (ESI m/
z) for C36H40N3O10, calcd 674.2713, found 674.2740 [M þ 1]þ.
The human melanoma cell line A375 purchased from American
Type culture collection was maintained in Dulbecco’s modified
Eagle’s medium (DMEM) (Invitrogen), supplemented with 10% fetal
calf serum and 100 U/mL Pencillin and 100 mg/mL streptomycin
sulfate (Sigma). These cell lines were maintained at 37 ꢂC in
a humidified atmosphere containing 5% CO2 in the incubator.
5.4. MTT cell proliferation assay
5.1.34. 7-Methoxy-8-{5-[(2,6-dimethoxy-4-(5-[3,4,5-
trimethoxyphenyl]-3-isoxazolylphenyl)oxy]pentyloxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one
(7e)
The compound 7e was prepared according to the method
described for the compound 5a, employing the compound 26e
Cell viability was assessed by the MTT based assay using WST-1
(premix WST-1 cell proliferation Assay system, Takara), is more
sensitive than MTT. Briefly, A375 cells were seeded in a 96-well
plate (TPP) at a cell density of 10,000 cells/well. After overnight
incubation, the cells were treated with compounds 6aec, DC-81
(4), 2 and CA-4 (1) incubated for 24 h. The medium was then dis-
(856 mg, 1.0 mmol) to obtain the pure product 7e as a white solid.
carded and replaced with fresh 100
mL media followed by addition
25
Yield 406 mg, 58%; mp 66e68 ꢂC; [
a
]
þ 157.5ꢂ (c0.1, CHCl3); 1H
of 10
m
L of WST-1 dye. Plates were incubated at 37 ꢂC for 30 min.
D
NMR (CDCl3, 300 MHz):
d
7.66 (d,1H, J ¼ 4.3 Hz, imineeH), 7.51 (s,1H,
Optical density (O.D.) was read at 420 nm using Multimode Vari-
oskan Flash (Thermo scientifics).
ArH), 7.08 (s, 2H, ArH), 7.04 (s, 2H, ArH), 6.81 (s,1H, ArH), 6.72 (s,1H,
isoxazoleeH), 4.06 (t, 4H, J ¼ 4.9 Hz, 2ꢃ eOCH2e), 3.94 (s, 6H, 2ꢃ
eOCH3), 3.92 (s, 9H, 3ꢃ eOCH3), 3.90 (s, 3H, eOCH3), 3.83e3.69 (m,
1H, eNCHe), 3.65e3.52 (m, 2H, eNCH2e), 2.26e2.37 (m, 2H,
eCH2e), 2.10e1.79 (m, 2H, eCH2e), 1.66e1.59 (m, 6H, 3ꢃ eCH2e);
5.5. Cell cycle analysis
5 ꢃ105 A375 Cells were seeded in 60 mm dish and were allowed
to grow for 24 h. Compounds 6aec, DC-81 (4), 2 and CA-4 (1) at
13C NMR (CDCl3, 75 MHz):
d 170.2, 164.5, 162.8, 162.2, 153.7, 153.4,
150.7,147.6, 140.4, 139.4, 139.0, 124.0,122.4, 119.9,111.3, 110.2, 103.9,
103.0, 97.0, 68.8, 60.8, 56.1, 56.0, 53.6, 46.5, 29.5, 28.5, 24.0, 22.2; IR
(KBr) (Umax/cmꢁ1): 3418, 2934, 1601, 1503, 1464, 1429, 1384, 1333,
1242,1127, 850, 792, 615; MS (ESI): m/z 702 [M þ 1]þ; HRMS (ESI m/
z) for C38H44N3O10, calcd 702.3026, found 702.3035 [M þ 1]þ.
4 mM concentration were added to the culture media and the cells
were incubated for an additional 24 h. Harvesting of cells was done
with Trypsin-EDTA, fixed with ice-cold 70% ethanol at 4 ꢂC for
30 min, washed with PBS and incubated with 1 mg/mL RNaseA
solution (Sigma) at 37 ꢂC for 30 min. Cells were collected by
centrifugation at 2000 rpm for 5 min and further stained with
5.2. Anticancer activity screening
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 dis-
solved 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.
The synthesized compounds (5a, 5cee, 6a, 6c and 7aee) were
evaluated for their in vitro cytotoxicity in selected human cancer
cell lines of breast (Zr-75-1, MCF7), lung (A-549, HOP62), colon
(Colo205), oral (KB), prostate (PC-3), ovarian (A2780) and cervix
(SiHa) origin. A protocol of 48 h continuous drug exposure was used
and a sulforhodamine B (SRB) protein assay was used to estimate
cell viability or growth. The cell lines were grown in RPMI 1640
medium containing 10% fetal bovine serum and 2 mML-glutamine,
5.6. Protein extraction and Western blot analysis
and were inoculated into 96-well microtiter plates in 90
m
L at
Total cell lysates were isolated from cultured A375 cells after
compound treatments as mentioned earlier were obtained by
lysing the cells in ice-cold RIPA buffer (1XPBS, 1% NP-40, 0.5%
sodium deoxycholate and 0.1% SDS containing protease inhibitors).
After centrifugation at 12,000 rpm for 10 min, the protein in
supernatant was quantified by Bradford method (BIO-RAD) using
Multimode varioskan Flash instrument (Thermo Scientifics). Thirty
micrograms of protein per lane was applied in 12% SDS poly-
acrylamide gel. After electrophoresis, the protein was transferred to
polyvinylidinedifluoride (PVDF) membrane (Amersham Biosci-
ences). The membrane was blocked at room temperature for 2 h in
plating densities depending on the doubling time of individual cell
lines. The microtiter plates were incubated at 37 ꢂC, 5% CO2, 95% air
and 100% relative humidity for 24 h prior to addition of experi-
mental drugs. Aliquots of 10
the appropriate microtiter wells already containing 90
resulting in the required final drug concentrations. Each compound
was evaluated for four concentrations (0.1, 1, 10 and 100 M) and
each was done in triplicate wells. Plates were incubated further for
48 h, and assay was terminated by the addition of 50 L of cold
trichloro acetic acid (TCA) (final concentration, 10% TCA) and plates
mL of the drug dilutions were added to
m
L of cells,
m
m

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 3924e3937
3937
[11] J. Kaffy, R. Pontiks, D. Carrez, A. Croisy, C. Monnereta, J.C. Floreta, Bioorg. Med.
Chem. 14 (2006) 4067e4077.
[12] D.E. Thurston, Molecular Aspects of Anticancer Drug-DNA Interactions. The
Macmillan Press Ltd., London, UK, 1993, pp. 54e88.
[13] R.L. Petrusek, E.L. Uhlenhopp, N. Duteau, L.H. Hurley, J. Biol. Chem. 257 (1982)
6207e6216.
[14] L.H. Hurley, T. Reck, D.E. Thurston, D.R. Langley, K.G. Holden, R.P. Hertzberg, J.
R.E. Hoover, G.J. Gallagher, L.F. Faucette, S.M. Mong, R.K. Johnson, Chem. Res.
Toxicol. 1 (1988) 258e268.
[15] S.C. Wilson, P.W. Howard, S.M. Forrow, J.A. Hartley, L.J. Adams, T.C. Jenkins, L.
R. Kelland, D.E. Thurston, J. Med. Chem. 42 (1999) 4028e4041.
[16] M. Tercel, S.M. Stribbling, H. Sheppard, B.G. Siim, K. Wu, S.M. Pullen, K.
J. Botting, W.R. Wilson, W.A. Denny, J. Med. Chem. 46 (2003) 2132e2151.
[17] P.G. Baraldi, G. Balboni, B. Cacciari, A. Guiotto, S. Manfredini, R. Romagnoli,
G. Spalluto, D.E. Thurston, P.W. Howard, N. Bianchi, C. Rutigiiano, C. Mischiati,
R. Gambari, J. Med. Chem. 42 (1999) 5131e5141.
TBS þ 0.1% Tween 20 (TBST) containing 5% blocking powder (Santa
Cruz). The membrane was washed with TBST for 5 min, and
primary antibody was added and incubated at 4 ꢂC overnight (O/N).
The primary antibodies p53, Cdk2 were purchased from Santa Cruz
and cytochrome c and PARP antibodies from Imgenex. After three
TBST washes, the membrane was incubated with corresponding
horseradish peroxidase-labeled secondary antibody (1:2000)
(Santa Cruz) at room temperature for 1 h. Membranes were washed
with TBST three times for 15 min and the protein blots were
visualized with chemiluminescence reagent (Thermo Fischer
Scientifics Ltd.). The X-ray films were developed with developer
and fixed with fixer solution.
[18] Y. Damayanthi, B.S.P. Reddy, J.W. Lown, J. Org. Chem. 64 (1999) 290e292.
[19] J.J. Wang, Y.K. Shen, W.P. Hu, M.C. Hsieh, F.L. Lin, M.K. Hsu, M.H. Hsu, J. Med.
Chem. 49 (2006) 1442e1449.
Acknowledgments
[20] A. Kamal, E. Laxman, P.S.M.M. Reddy, Tetrahedron Lett. 41 (2000) 7743e7746.
[21] A. Kamal, N. Shankaraiah, V. Devaiah, K.L. Reddy, Tetrahedron Lett. 47 (2006)
6553e6556.
[22] A. Kamal, G. Ramesh, N. Laxman, P. Ramulu, O. Srinivas, K. Neelima, A.
K. Kondapi, V.B. Srinu, H.M. Nagarajaram, J. Med. Chem. 45 (2002)
4679e4688.
[23] A. Kamal, R. Ramu, T. Venkatesh, G.B. Ramesh Khanna, M.S. Barkume,
A. Juvekar, S. Zingde, Bioorg. Med. Chem. 15 (2007) 6868e6875.
[24] A. Kamal, M.N. Khan, Y.V.V. Srikanth, K.S. Reddy, A. Juvekar, S. Sen, N. Kurian,
S. Zingde, Bioorg. Med. Chem. 16 (2008) 7804e7810.
We thank the National Cancer Institute, Bethesda, for in vitro
anticancer assay in human cell lines. J.S.N.R., D.D.G., E.V.B., M.A.A.,
and F. S. are thankful to CSIR, New Delhi, for the award of research
fellowships. We are also thankful to the Department of Biotech-
nology (BT/PR/7037/Med/14/933/2006), New Delhi; for financial
assistance.
[25] A. Kamal, N. Shankaraiah, V. Devaiah, K.L. Reddy, A. Juvekar, S. Sen, N. Kurian,
S. Zingde, Bioorg. Med. Chem. Lett. 18 (2008) 1468e1473.
[26] A. Kamal, P.P. Kumar, B.N. Seshadri, O. Srinivas, M.S. Kumar, S. Sen, N. Kurian,
A. Juvekar, S. Zingde, Bioorg. Med. Chem. 16 (2008) 3895e3906.
[27] A. Kamal, E.V. Bharathi, M.J. Ramaiah, D. Dastagiri, J.S. Reddy, A. Viswanath,
F. Sultana, S.N.C.V.L. Pushpavalli, M.P. Bhadra, H.K. Srivastava, G.N. Sastry,
A. Juvekar, S. Sen, S. Zingde, Bioorg. Med. Chem. 18 (2010) 526e542.
[28] A. Kamal, K. Sreekanth, P.P. Kumar, N. Shankaraiah, G. Balakishan, M.
J. Ramaiah, S.N.C.V.L. Pushpavalli, P. Ray, M.P. Bhadra, Eur. J. Med. Chem. 45
(2010) 2173e2181.
[29] T. Taguchi, Y. Kato, Y. Baba, G. Nishimura, Y. Tanigaki, C. Horiuchi,
I. Mochmatsu, M. Tsukuda, Oncol. Rep. 11 (2004) 421e426.
[30] X. Wang, X. Jiang, Ann. Rev. Biochem. 3 (2004) 87e106.
[31] R.M. Kluck, E.B. Wetzel, D.R. Green, D.D. Newmeyer, Science 275 (1997)
1132e1136.
References
[1] R. Morphy, Z. Rankovic, J. Med. Chem. 48 (2005) 6523e6543.
[2] R. Morphy, C. Kay, Z. Rankovic, Drug Discov. Today 9 (2004) 641e651.
[3] A.B. Alexis, J.E. Peter, W.H. Nicole, S.L. Margaret, E.R. Joseph Leha’ r, S. George,
R.Z. Grant, A.F. Michael, R.S. Brent, T.K. Curtis, PNAS 100 (2000) 7977e7982.
[4] B. Meunier, Acc. Chem. Res. 41 (2008) 69e77.
[5] G.R. Pettit, G.M. Cragg, S.B. Singh, J. Nat. Prod. 50 (1987) 386e391.
[6] C.M. Lin, H.H. Ho, G.R. Pettit, Biochemistry 28 (1989) 6984e6991.
[7] L. Wang, K.W. Woods, Q. Li, K.J. Barr, R.W. McCroskey, S.M. Hannick, L. Gherke,
R.B. Credo, Y.A. Hui, K. Marsh, R. Warner, J.Y. Lee, N. ZielinskiMozng, D. Frost, S.
H. Rosenberg, H.L. Sham, J. Med. Chem. 45 (2002) 1697e1711.
[8] K. Ohsumi, T. Hatanaka, K. Fujita, R. Nakagawa, Y. Fukuda, Y. Nihei, Y. Suga,
Y. Morinaga, Y. Akiyama, T. Tsuji, Bioorg. Med. Chem. Lett. 8 (1998) 3153e3158.
[9] N.H. Nam, Y. Kim, Y.J. You, D.H. Hong, H.M. Kim, B.Z. Ahn, Bioorg. Med. Chem.
Lett. 11 (2001) 3073e3076.
[32] W.P. Hu, H.S. Yu, P.J. Sung, F.Y. Tsai, Y.K. Shen, L.S. Chang, J.J. Wang, Chem. Res.
Toxicol. 20 (2007) 905e912.
[33] L. Tyas, V.A. Brophy, A. Pope, J.A. Rivett, J.M. Tavare, EMBO Rep. 3 (2000)
266e270.
[34] B.A. Carlson, M.M. Dubay, E.A. Sausville, L. Brizuela, P.J. Worland, Cancer Res.
56 (1996) 2973e2978.
[10] D. Simoni, G. Grisolia, G. Giannini, M. Roberti, R. Rondanin, L. Piccagli,
R. Baruchello, M. Rossi, R. Romagnoli, F.P. Invidiata, S. Grimaudo, M.K. Jung,
E. Hamel, N. Gebbia, L. Crosta, V. Abbadessa, A. DiCristina, L. Dusonchet,
M. Meli, M. Tolomeo, J. Med. Chem. 48 (2005) 723e736.