Synthesis, anticancer activity and mitochondrial mediated apoptosis inducing ability of 2,5-diaryloxadiazole-pyrrolobenzodiazepine conjugates

Article abstract of DOI:10.1016/j.bmc.2010.07.067

A series of 2,5-diaryloxadiazole linked pyrrolo[2,1-c][1,4]benzodiazepine conjugates have been prepared and evaluated for their anticancer activity. These conjugates have shown promising activity with GI₅₀ values ranging from <0.1 to 0.29 μΜ. It is observed that some of these conjugates particularly 4a, 4d, 4i and 4l exhibit significant anticancer activity. Some detailed biological assays relating to the cell cycle aspects associated to Bax and caspases have been examined with a view to understand the mechanism of action of these conjugates.

Full text of DOI:10.1016/j.bmc.2010.07.067

Bioorganic & Medicinal Chemistry 18 (2010) 6666–6677
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, anticancer activity and mitochondrial mediated apoptosis
inducing ability of 2,5-diaryloxadiazole–pyrrolobenzodiazepine conjugates
a
a
a
a
Ahmed Kamal a, , D. Dastagiri , M. Janaki Ramaiah , E. Vijaya Bharathi , J. Surendranadha Reddy ,
*
G. Balakishan a, Pranjal Sarma a, S. N. C. V. L. Pushpavalli a, Manika Pal-Bhadra a, , Aarti Juvekar ,
b
*
Subrata Sen b, Surekha Zingde b
a Chemical Biology Laboratory, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 607, India
b Advanced Centre for Treatment, Research & Education in Cancer, Navi Mumbai 400 611, 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 2,5-diaryloxadiazole linked pyrrolo[2,1-c][1,4]benzodiazepine conjugates have been prepared
Received 20 May 2010
Revised 28 July 2010
Accepted 29 July 2010
Available online 1 August 2010
and evaluated for their anticancer activity. These conjugates have shown promising activity with GI50 val-
ues ranging from <0.1 to 0.29 lV. It is observed that some of these conjugates particularly 4a, 4d, 4i and
4l exhibit significant anticancer activity. Some detailed biological assays relating to the cell cycle aspects
associated to Bax and caspases have been examined with a view to understand the mechanism of action
of these conjugates.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Pyrrolobenzodiazepine
2,5-Diaryloxadiazoles
Anticancer activity
Apoptosis
p53
Caspases
Cyclin dependent kinase-2 (CDK-2)
1. Introduction
heterocyclics are very good bioisosters of amide and ester func-
tionalities with substantial improvement in biological activity
Currently cancer is one of the causes of death and it is likely to
become the most common disease in the near future. It is well
established that DNA is the target1,2 for many anticancer drugs that
are presently being used in the clinic. However, there are very few
DNA-interactive agents that bind to DNA with high sequence selec-
tivity. DC-81 (1), an antitumor antibiotic produced from Streptomy-
ces species,3 belongs to the pyrrolo[2,1-c][1,4]benzodiazepine
(PBD) class of compounds that are potent inhibitors of nucleic acid
synthesis. These compounds exhibit their antitumor activity by
binding in the minor groove of double stranded DNA and forming
a covalent bond to the exocyclic amino group of a central guanine
within a three base pair recognition site.4–6
1,3,4-Oxadiazoles are an important class of heterocyclic com-
pounds7 with a broad range of biological activities such as anti-
viral,8 antimicrobial,9 fungicidal,10 antineoplastic,11 anticancer,12
and inhibition of tyrosinase.13 Moreover, it is considered that
the presence of toxophoric –N@C–O– linkage7 is responsible for
their potent pharmacological activity. Further, 1,3,4-oxadiazole
by participating in hydrogen bonding interactions with different
receptors.14,15 In recent years, some PBD conjugates have been
reported as potential anticancer agents not only from this labora-
tory, but as well from other research groups.16–19 Recently, Wang
and co-workers have synthesized DC-81–indole and DC-81–ened-
iyne conjugates to explore their anticancer potential. In this
study, a correlation between antitumor activity and apoptosis
has been well explained in such conjugates.20,21 For the last
few years, we have been involved in the development of new
synthetic strategies22,23 for the PBD ring system and also in the
design of structurally modified PBDs including their conju-
gates.24–28 Some representative PBD’s, oxadiazoles, and their con-
jugates are illustrated in Figure 1.
In continuation of our efforts in search of new effective antican-
cer agents, we have synthesized a series of PBD conjugates by link-
ing a diaryloxadiazole moiety to the PBD (DC-81) scaffold through
the stable alkane spacers and evaluated their anticancer activity.
The promising activity obtained, prompted us to investigate their
role in the cell proliferation and apoptosis of human melanoma cell
line (A375). Further, it is considered of interest to investigate the
effect of these compounds on regulatory proteins of cell cycle
progression.
* Corresponding authors. Tel.: +91 40 27193157; fax: +91 40 27193189.
E-mail addresses: ahmedkamal@iict.res.in (A. Kamal), manika@iict.res.in (M. Pal-
Bhadra).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.067

A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 6666–6677
6667
N
N
HO
HO
N
N
H
H
N
O
N
H3CO
H3CO
N
O
O
N
N
O
2
O
1
3
N
N
R1
R2
( )
O
H
O
n
N
OCH3 H3CO
R3
O
4a−l n = 1−3
4a−c R1 = H; R2 = F; R3 = H
4d−f R1 = H; R2 = CF3; R3 = H
4g−i R1 = OCH3; R2 = OCH3; R3 = OCH3
4j−l R1 = H; R2 = OCH3; R3 = OCH3
Figure 1. Structures of DC-81 (1), tomaymycin (2), 2,5-diaryloxadiazole (3), 2,5-diaryloxadiazole–PBD conjugates (4a–l).
oral, colon, prostate, ovarian and cervix by using sulforhodamine
B (SRB) method. The compounds that exhibit GI50 6 10ꢀ5 V are
considered to be active on the respective cell lines and the results
are illustrated in Table 1. All the compounds (4a–l) exhibited sig-
nificant anticancer activity with GI50 values ranging from <0.1 to
2. Results and discussions
2.1. Chemistry
The synthesis of 2,5-diaryloxadiazoles (9a–d) was carried out
from isovanilic ester by using the reported procedures29,30 as
shown in Scheme 1. Reaction of benzylated isovanilic ester (5) with
hydrazine hydrate in ethanol gave isovanilic acid hydrazide (6)
which upon treatment with substituted benzoyl chloride in dry
pyridine provided the intermediates 7a–d. Then these upon reac-
tion with phosphorus oxychloride yielded the corresponding com-
pounds 8a–d which on further debenzylation provided the
precursors 9a–d in good yields.
The synthesis of C8-linked 2,5-diaryloxadiazole–PBD conjugates
(4a–l) was carried out from the (2S)-N-{4-[n-bromoalkoxy-5-meth-
oxy-2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioac-
etal (10a–c), which were prepared by the methods reported in
our earlier studies.22–28 These upon etherification with the 2,5-diar-
yloxadiazole intermediates (9a–d) using K2CO3 in acetone provided
the corresponding nitrothioacetal intermediates (11a–l) in 80ꢀ92%
yield. Further, reduction of nitro compounds by using SnCl2.2H2O in
methanol followed by deprotection using HgCl2/CaCO3 afforded the
desired PBD conjugates 4a–l as shown in Scheme 2.
0.29
demonstrated the GI50 in the range of 0.1ꢀ7.25
M, respectively, in the cell lines employed. Interestingly,
lV, while the positive controls, adriamycin and DC-81 (1)
l
M
and
0.1ꢀ2.37
l
all the compounds showed promising anticancer activity, particu-
larly against A2780, Gurav, MCF-7, Colo205, DWD cell lines. How-
ever, 4a, 4d, 4i and 4l exhibited significant activity.
Additionally, cell viability assay was carried out to investigate
the cytotoxic effect mediated by the four most promising PBD con-
jugates (4a, 4d, 4i and 4l) in A375 cells. These PBD conjugates
exhibited cytotoxicity at 4 lM concentration, moreover these were
more effective than DC-81 (1) and 9d as shown in Figure 2.
Cells develop multiple DNA repair pathways such as base exci-
sion repair system (BER), nucleotide repair system (NER) and mis-
match repair system (MMR) to protect themselves from different
types of DNA damage.31 TK6 are the human lymphoblastoid cells
that have proficient mismatch repair system. Hence, in order to
understand the potent anticancer activity for these conjugates
against tumor cell lines with proficient DNA repair system, an
MTT cell viability assay was carried out with these PBD conjugates
(4a, 4d, 4i and 4l) in TK6 cells. The results suggest that these com-
pounds are functioning in those cells in which there is a DNA repair
system (Fig. 3a). Further Western blot analysis showed upregula-
tion of Chk2 protein32 level as illustrated in Figure 3b. These stud-
2.2. Biological results
2.2.1. Anticancer activity
The synthesized compounds 4a–l were evaluated for their anti-
cancer activity in selected human cancer cell lines of lung, breast,
R1
R2
O
O
O
H
BnO
BnO
NH2
BnO
N
R3
(i)
OMe
(ii)
N
H
N
H
O
H3CO
H3CO
H3CO
6
7a−d
(iii)
5
N
N
N N
R1
R2
R1
R2
(iv)
OH
OCH3
OBn
OCH3
O
O
R3
R3
8a−d
9a−d
1 = H; R2 = F; R3 = H
9a:
9b:
R
R1 = H; R2 = CF3; R3 = H
9c: R1 = OCH3; R2 = OCH3; R3 = OCH3
9d: R1 = H; R2 = OCH3; R3 = OCH3
Scheme 1. Reagents and conditions: (i) NH2ꢁNH2ꢁH2O, EtOH, reflux; (ii) substituted benzoyl chlorides, pyridine, reflux, 30 min, 85ꢀ95%; (iii) POCl3, reflux, 1 h, 80ꢀ87%; (iv)
EtSH–BF3OEt2, CH2Cl2, 12 h, rt, 80ꢀ89%.

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A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 6666–6677
N
N
R1
R2
( )
OH
Br
(i)
O
NO2
CH(SEt)2
O
n
+
N
OCH3
H3CO
R3
O
9a−d
10a−c n=1−3
N
N
R1
R2
( )
NO2
O
O
CH(SEt)2
O
n
N
OCH3 H3CO
11a−l n=1−3
R3
O
(ii),(iii)
Figure 2. Effect of PBD conjugates (4a, 4d, 4i and 4l) on cell viability (in vitro
cytotoxicity). A375 cells were treated with 4 V concentration of PBD conjugates as
l
indicated for 24 h in 96-well plates seeded with 10,000 cells per well. OD readings
were taken at 420 nm wavelength to measure the percentage of cell viability after
treatment with the respective compound. DC-81 (1) was used as the positive
control. Control: control cells (untreated cells).
N
N
R1
R2
( )n
N
O
O
H
O
N
OCH3 H3CO
4a−l n=1−3
R3
O
MTT Assay
4a−c: R1 = H; R2 = F; R3 = H
4d−f: R1 = H; R2 = CF3; R3 = H
1.2
4g−i: R1 = OCH3; R2 = OCH3; R3 = OCH3
4j−l: R1 = H; R2 = OCH3; R3 = OCH3
1
0.8
0.6
0.4
0.2
0
Scheme 2. Reagents and conditions: (i) K2CO3, acetone, reflux, 24 h, 80ꢀ92%; (ii)
SnCl2ꢁ2H2O, MeOH, reflux, 1ꢀ2 h, 70%; (iii) HgCl2–CaCO3, CH3CN/H2O (4:1), rt, 8–
12 h, 55ꢀ60%.
ies indicate that these conjugates actively function in the DNA
damage and repair pathway.
Control
1
4a
4d
4i
4l
9d
Prediction of lipophilicity (log P) and aqueous solubility (log S)
of compounds 4a–l was calculated using A LOGPS 2.1 software.33,34
Compounds 4a–l showed lipophilicity with log P values in the
range of 3.76–5.84 (Table 2) and aqueous solubility with log S val-
ues in the range of 5.65–6.46 mg/L. These studies indicate that the
aqueous solubility of these compounds is better than the DC-81
(1).
Compounds
Figure 3a. Effect of PBD conjugates (4a, 4d, 4i and 4l) on cell viability (in vitro
cytotoxicity). TK6 cells were treated with 4 V concentration of PBD conjugates as
l
indicated for 24 h in 96-well plates seeded with 10,000 cells per well. OD readings
were taken at 420 nm wavelength to measure the percentage of cell viability after
treatment with the respective compound. DC-81 (1) was used as the positive
control. Control: control cells (untreated cells).
2.2.2. Cell cycle effects
To investigate the effect of these PBD conjugates on the cell cy-
cle progression of human cancerous cell line A375, the DNA
content of the cell nuclei was measured by flow cytometric (FACS)
analysis. Drug induced hypodiploid DNA (i.e., sub-G1 phase) is the
Table 1
GI50 valuesa (in
lM) for compounds 4a–l in selected human cancer cell lines
Compound
A549b
Guravb
HOP62b
MCF7c
Zr-75-1c
A2780d
DWDe
KBe
Colo205f
PC-3g
SiHah
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
0.15
0.29
0.14
<0.1
0.11
<0.1
0.11
<0.1
0.14
<0.1
<0.1
<0.1
7.25
0.16
0.16
0.1
0.16
0.15
0.12
<0.1
0.16
0.13
0.17
0.13
<0.1
0.13
0.13
<0.1
0.14
0.15
<0.1
0.16
0.12
0.11
0.14
<0.1
<0.1
0.15
<0.1
<0.1
<0.1
<0.1
0.17
0.17
<0.1
0.17
0.15
0.11
0.12
0.11
0.12
0.16
0.12
<0.1
0.12
<0.1
1.79
2.37
0.14
<0.1
<0.1
<0.1
0.14
<0.1
0.14
<0.1
<0.1
0.11
<0.1
<0.1
0.16
0.14
<0.1
0.13
<0.1
0.11
0.15
<0.1
<0.1
0.12
<0.1
<0.1
<0.1
<0.1
0.10
1.49
0.15
<0.1
<0.1
<0.1
0.15
0.11
0.14
<0.1
<0.1
0.11
0.11
<0.1
0.17
0.17
<0.1
0.15
<0.1
<0.1
0.16
0.11
<0.1
<0.1
0.12
<0.1
<0.1
<0.1
0.14
0.11
<0.1
0.17
0.16
0.14
0.16
0.13
0.14
0.16
0.15
<0.1
0.13
<0.1
1.81
0.20
0.17
<0.1
0.12
<0.1
0.17
0.12
0.18
0.13
<0.1
0.15
0.13
<0.1
0.17
0.17
<0.1
<0.1
0.14
<0.1
0.14
<0.1
<0.1
0.11
<0.1
<0.1
0.17
0.16
ADRi
DC-81 (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

A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 6666–6677
6669
Table 3
Cell cycle distribution of A375 cell line at 4
4i, 4l and DC-81 (1)
l
M concentration of compounds 4a, 4d,
Compound
Sub G1
G1
S
G2/M
Control
DC-81 (1)
4a
4d
4i
1.53 0.05
15.33 1.24
6.79 0.46
17.93 0.33
13.76 0.29
36.66 0.73
53.9 1.35
73.66 2.05
74 0.92
72.28 0.4
73.5 0.28
59.66 0.47
20.12 0.66
8.02 0.81
14.14 0.20
7.51 0.614
10.4 0.43
2.68 0.32
24.44 0.84
2.98 0.02
5.06 0.821
2.26 0.13
2.34 0.28
0.99 0.13
Figure 3b. Effect of PBD conjugates on the expression of chk2 protein levels. TK6
cells were treated with PBD conjugates (4a, 4d, 4i, 4l) and DC-81 (1) at 4
lM
4l
concentration for 24 h. The cell lysates were collected and expression levels chk2
was determined by Western blot analysis, b-actin was used as loading control.
characteristic feature of apoptosis.35,36 The majority of the control
cells exposed to DMSO were in the G1 phase. Cells (1.53%) were in
sub-G1, 53.9% in G1, 20.12% in S and 24.44% in G2/M phase. Treat-
ment of A375 cells with the most promising compounds 4a, 4d, 4i
and 4l at 4 lM concentration for 24 h induced apoptosis effects up
to 6.79%, 17.93%, 13.76% and 36.66%, respectively, whereas, DC-81
(1) showed 15.33% of sub-G1 DNA peak. Simultaneously there was
a concomitant decrease in the G2/M phase in A375 cells when trea-
ted with 4a (5.06%), 4d (2.26%), 4i (2.34%) and 4d (0.99%) com-
pounds. Increased cells of sub-G1 phase, decrease of G2/M phase
cells clearly showed that all the compounds are effective in causing
apoptosis in case of A375 cells as shown in Table 3.
Figure 4. Effect of PBD conjugates on the expression of p53, p21 and pRb protein
levels. A375 cells were treated with PBD conjugates (4a, 4d, 4i, 4l) and DC-81 (1) at
4 lM concentration for 24 h. The cell lysates were collected and expression levels
p53, p21 and pRb were determined by Western blot analysis, b-actin was used as
loading control.
2.2.3. Effect on tumor suppressor proteins
release of cytochrome c.43–45 cytosolic cytochrome c is known to
cause activation of caspase-3.46 To investigate the effect of Bax
and release of cytochrome c, A375 cells were treated with PBD con-
To understand the mechanism underlying the G1 cell cycle ar-
rest in these conjugates, we examined the G1/S check point associ-
ated tumor suppressor proteins such as p53, p21 and pRb. The
tumor suppressor protein p53 regulates the transcription of p21
which in turn increases the retinoblastoma protein (pRb) leading
into cell cycle arrest at G1 phase.37,38 In order to understand the ef-
fect of PBD conjugates on p53, p21 and pRb dependent apoptotic
jugates (4a, 4d, 4i and 4l) at 4 lM concentration for 24 h and Wes-
tern blot analysis was carried out. It was observed that
upregulation of Bax which inturn causes an increase in cytochrome
c protein levels as shown in Figure 5a.
One of the important characteristic features of apoptosis is acti-
vation of caspase-3 leads to the cleavage of DNA repair enzyme
PARP(poly(ADP-ribose) polymerase)47 and eventually leads to
apoptosis.48 Release of cytosolic cytochrome c is known to cause
activation of caspase-3.46 To assess the effect of PBD conjugates
on the activation of caspase-3 and cleavage of PARP, A375 cells
were treated with PBD conjugates (4a, 4d, 4i and 4l) at a concen-
pathway, A375 cells were treated with these conjugates at 4 lM
concentration for 24 h and Western blot analysis was carried out.
It was observed that, the p53, p21 and pRb levels were upregulated
in the case of compounds 4d and 4l compared to the untreated
controls. Predominantly, increase of these protein levels was more
prominent in compound 4l, as shown in Figure 4.
tration of 4 lM for 24 h and the expression levels of caspase-3
2.2.4. Effect on apoptotic proteins
was analyzed by Western blot analysis. An increase in the levels
of caspase-3 was observed. An increase in the expression of cleaved
PARP product specific to apoptosis was observed and the levels
were more pronounced in the case of compound 4l. This data
clearly indicated that these PBD conjugates induce apoptosis
through intrinsic pathway as shown in Figure 5a and b.
According to previous studies apoptosis can be induced in two
major pathways, including extrinsic and intrinsic apoptosis signal-
ing.39 Extrinsic pathway is probably mediated through the regula-
tion of death receptors such as Fas and TNF-
a located on the cell
surface.40 In order to know whether these conjugates are inducing
apoptosis in an extrinsic pathway, Western blot analysis was car-
ried out in relation to TNF-
change in the level of TNF-
observation, it was clear that the apoptosis caused by these conju-
gates is independent of extrinsic pathway mediated by TNF-
An important consequence of intrinsic apoptotic pathway is the
mitochondrial dysfunction and cytochrome c release.41 The Bcl-2
family of proteins plays a crucial role in regulating the protein re-
lease from mitochondria.42 This family is divided into two sub-
groups; pro-apoptotic and anti-apoptotic members. The pro-
apoptotic proteins like Bax, induces cell apoptosis through mito-
chondrial membrane permeabilization (MMP), which leads to the
a
and it was found that there was no
2.2.5. Effect of PBD conjugates on the expression of Cdk2
Further, to understand the molecular events involved in G1
phase arrest, the effects on the expression level of CDKs particu-
larly Cdk249 (G1 related protein) was also investigated in A375
cells that were treated with these conjugates. Accordingly, Wes-
tern blot analysis was carried out and it was observed that there
was a reduction of Cdk2 levels as seen in Figure 6. The reduction
of Cdk2 protein level is significantly more in case of 4l compared
to the other conjugates. Thus confirm the blockade of G1/S transi-
tion there by resulting in cell cycle arrest.
a
(data not shown). Therefore, from this
a
.
Table 2
Lipophilicity (log P) and aqueous solubility (log S) of compounds 4a–l
Compd
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
DC-81(1)
Log P
Log S (gm/L)
4.39
5.96
4.78
6.11
5.17
6.24
5.07
6.19
5.44
6.34
5.84
6.46
3.76
5.72
4.13
5.86
4.53
5.98
3.92
5.65
4.28
5.80
4.68
5.91
0.88
2.01

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A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 6666–6677
Figure 6. Effect of PBD conjugates on Cdk2 levels. A375 cells were treated with
compounds 4a, 4d, 4i, 4l and DC-81 (1) at 4 V concentrations for 24 h. The cell
l
lysates were collected and expression levels of Cdk2 were determined by Western
blot analysis. b-actin was used as a loading control.
Figure 5a. Effect of PBD conjugates on cytochrome c, cleaved PARP and Bax levels.
A375 cells were treated with compounds 4a, 4d, 4i, and 4l and DC-81 (1) at 4 lM
Table 4
concentration for 24 h. The cell lysates were collected and expression levels of
cytochrome c, cleaved PARP and Bax were determined by Western blot analysis.
b-Actin was used as loading control.
Thermal denaturation data for 2,5-diaryloxadiazolineꢀPBD conjugates (4a–l) with
calfthyms (CT)-DNA
Compound
[PBD]: [DNA]
Molar ratiob
D
Tm (°C)a after incubation at 37 °C for
0 h
18 h
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1.9
2.3
1.5
2.3
1.7
1.6
1.4
1.9
1.1
1.4
2.1
3.0
0.3
2.0
2.5
1.8
4.0
2.1
1.7
1.9
2.1
1.6
1.5
2.3
5.8
0.7
DC-81 (1)
a
For CT-DNA alone at pH 7.00 0.01,
separate determinations), all Tm values are 0.1–0.2 °C.
For a 1:5 molar ratio of [PBD]/[DNA], where CT-DNA concentration = 100
and ligand concentration = 20 M in aqueous sodium phosphate buffer [10 mM
sodium phosphate + 1 mM EDTA, pH 7.00 0.01].
DTm = 68.5 0C 0.01 (mean value from 10
D
b
l
M
l
Figures 5b. Effect of PBD conjugates on caspase-3 activity in A375 cells. The
increased enzymatic activity of caspase-3, in apoptosis after the treatment of PBD
conjugates (4a, 4d, 4i and 4l) at 4 lV concentration was determined by fluorimetry.
% Survival Data: PC-3 Xenograft
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. DEVD-CHO is the inhibitor of caspase-3. DC-81 (1)
was used as the positive control.
105
100
95
90
2.2.6. Thermal denaturation studies
Many previous reports by our group24,17 showed strong binding
of the PBD hybrids with DNA molecules that is proved both by RED
assay and by computational studies. To show the DNA binding
activity for these new C8-linked 2,5-diaryloxadiazole–PBD conju-
gates (4a–l) thermal denaturation studies were carried out using
calf thymus (CT) DNA.50 Melting studies showed that these com-
pounds stabilize the thermal helixcoil or melting stabilization
Control
ADR, 5mg/kg
4l
85
80
75
1
4
8
11
15
18
21
25
28
31
Days
(DTm) for the CT-DNA duplex at pH 7.0, incubated at 37 °C, where
Figure 7a. NOD-SCID male mice treated with compound 4l, Survival data of
compound 4l on PC-3 xenograft mice.
PBD/DNA molar ratio is 1:5. The data for compounds 4a–l is in-
cluded in Table 4 and it is observed that some of these compounds
have shown good DNA binding affinity compared to naturally
occurring PBD-like DC-81. It is also observed that 2,5-diarylox-
adiazole–PBD conjugates (4d and 4l) exhibited better DNA binding
affinity than other PBD conjugates and DC-81 (1).
Animal Weight Data: PC-3 Xenograft
30.0
25.0
20.0
15.0
2.2.7. In vivo tumor xenograft studies of compound 4l
The preliminary in vitro anticancer activities revealed that com-
pound 4l has shown significant anticancer activity among the ser-
ies. These encouraging results provided an impetus to carry out
in vivo efficacy studies using xenograft model of human prostate
cancer cells (PC-3) in male NOD-SCID mice. The dose determina-
tion studies demonstrated the maximally tolerated dose (MTD)
of compound 4l as <50 mg/Kg. Hence, compound 4l was adminis-
tered at 20 mg/kg intraperitoneal (ip), on days 1, 5 and 9 (q4d) (to-
tal drug administered was 20 mg/kg). With these doses no toxicity
was observed in terms of weight loss or mortality of the experi-
mental mice (Fig. 7a and b).
Control
10.0
ADR, 5mg/kg
5.0
4l
0.0
1
4
8
11
15
18
21
25
28
31
Days
Figure 7b. Tumors weight curves in NOD-SCID male mice treated with compound
4l.

A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 6666–6677
6671
4.2. General procedures
3. Conclusion
4.2.1. Synthesis of 3-(benzyloxy)-N0-(4-fluorobenzoyl)-4-meth-
oxybenzohydrazide (7a)
In conclusion, a new class of 2,5-diaryloxadiazoleꢀPBD conju-
gates have been synthesized and these exhibit promising anti-
cancer activity against a number of human cancer cell lines.
The cell viability assay of the two most promising compounds
A mixture of methyl 3-(benzyloxy)-4-methoxy benzoate 5
(272 mg, 1 mmol), ethanol (100 mL), and hydrazine hydrate
(1.3 mL, 0.34 mol) was heated to reflux and left overnight. The so-
lid formed was filtered and washed with cold water and ether, to
obtain compound 3-(benzyloxy)-4-methoxy benzohydrazide (6)
and as such taken for the next step.
4d and 4l showed cytotoxicity in A375 cells at 4 lM concentra-
tion. It is interesting to observe that these compounds exhibit a
strong effect against cancer cells with a proficient DNA repair
system. The FACS analysis also showed more population in
sub-G1 phase indicating that these two PBD conjugates have
apoptosis inducing ability. It is also observed from the detailed
biological assays that the p53, p21 and pRb levels have been en-
hanced when treated with compounds 4a, 4d, 4i and 4l, interest-
ingly it is more pronounced in case of compound 4l. Further,
release of cytochrome c has been observed when a Bcl-2 family
protein (Bax) regulates the mitochondrial mediated pathway for
these conjugates (4a, 4d, 4i and 4l). Therefore, these conjugates
have been examined for their effects on other mitochondrial
mediated apoptotic proteins such as Bax, cytochrome c, cas-
pase-3 and PARP. Upregulation of Bax, release of cytochrome c,
increase in the levels of active caspase-3 and cleavage of PARP
is noted, and is more pronounced in case of compound 4l. Simi-
larly, in case of 4l a drastic decrease in the levels of Cdk2 has
been observed. Moreover, thermal denaturation studies are also
carried out, these conjugates showed better DNA binding ability.
The in vivo efficacy study of compound 4l revealed that the abil-
ity to delay the tumor growth and retention of body weight in
the PC-3 human prostate cancer xenograft model suggested that
2,5-diaryloxadiazole–PBD conjugates have promising anticancer
activity in the treatment of human cancer.
A
mixture of 3-(benzyloxy)-4-methoxy benzohydrazide 6
(272 mg, 1 mmol) and 4-fluorobenzoyl chloride (158 mg, 1 mmol),
in dry pyridine (10 mL), was heated under reflux for 30 min. On
cooling, the mixture was poured onto cold water (50 mL) and stir-
red for 10 min. The separated solid was filtered, washed thor-
oughly with cold water and dried to yield compound 7a as white
solid. The products were pure enough to be used in the next step
without further purification (355 mg, 90%). Mp: 186–189 °C; 1H
NMR (300 MHz, DMSO-d6): d 3.92 (s, 3H, –OCH3), 5.18 (s, 2H, ben-
zyl –CH2–), 6.93 (d, 1H, J = 8.9 Hz, ArH), 7.15 (d, 1H, J = 8.9 Hz, ArH),
7.24ꢀ7.61 (m, 8H, ArH), 8.03 (dd, 2H, J = 12.8 Hz, J = 2.5 Hz, ArH),
10.24 (d, 2H, J = 8.9 Hz, ꢀCONHNHCOꢀ); MS (ESI): m/z 395 [M+1]+.
4.2.2. 3-(Benzyloxy)-4-methoxy-N0-(4-(trifluoromethyl)
benzoyl)benzohydrazide (7b)
Compound 7b was prepared following the method described for
the preparation of compound 7a, employing 6 (272 mg, 1 mmol)
and 4-(trifluoromethyl)benzoyl chloride (208 mg, 1 mmol) to af-
ford compound 7b as a white solid (413 mg, 93%). Mp: 162–
165 °C; 1H NMR (300 MHz, DMSO-d6): d 3.88 (s, 3H, –OCH3), 5.22
(s, 2H, benzyl –CH2–), 7.22 (d, 1H, J = 8.7 Hz, ArH), 7.39ꢀ7.61 (m,
6H, ArH), 7.97 (d, 3H, J = 8.0 Hz, ArH), 8.17 (d, 2H, J = 8.0 Hz,
ArH), 10.64 (d, 2H, J = 8.0 Hz, ꢀCONHNHCOꢀ); MS (ESI): m/z 445
[M+1]+.
All these new conjugates showed significant anticancer activity
with GI50 values in the range of <0.1ꢀ0.29
lM. Whereas, some of
the PBD conjugates previously synthesized showed GI50 values in
the range of 0.13–30.50
l
M (1,2,3-triazoleꢀPBD conjugates),28
4.2.3. N0-(3-(Benzyloxy)-4-methoxybenzoyl)-3,4,5-trimethoxy-
benzohydrazide (7c)
0.22–30.30
0.17–30.50
l
l
M
(triazolobenzothiadiazineꢀPBD conjugates),51
M (phosphonate linked PBD conjugates)27 and 1.48–
26.2
l
M (some of the quinazolinone linked PBD conjugates).17 In
Compound 7c was prepared following the method described for
the preparation of compound 7a, employing 6 (272 mg, 1 mmol)
and 3,4,5-trimethoxybenzoyl chloride (230 mg, 1 mmol) to afford
compound 7c as a white solid (396 mg, 85%). Mp: 172–174 °C;
1H NMR (300 MHz, DMSO-d6): d 3.73 (s, 3H, –OCH3), 3.84 (s, 9H,
3 ꢂ –OCH3), 5.17 (s, 2H, benzyl –CH2–), 7.17 (d, 1H, J = 8.3 Hz,
ArH), 7.26 (s, 2H, ArH), 7.34ꢀ7.50 (m, 5H, ArH), 7.55 (d, 2H,
J = 6.0 Hz, ArH), 10.40 (d, 2H, J = 9.0 Hz, ꢀCONHNHCOꢀ); MS
(ESI): m/z 467 [M+1]+.
this article we highlight that by linking the 2,5-diaryloxadiazole
moiety to the PBD ring system has dramatically improved the anti-
cancer activity. These studies are likely to provide an important
mechanistic insight into the action of such conjugates.
4. Experimental section
4.1. Chemistry
4.2.4. 3-(Benzyloxy)-N0-(3,4-dimethoxybenzoyl)-4-
methoxybenzohydrazide (7d)
All chemicals and reagents were obtained from Aldrich (Sig-
ma–Aldrich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson
Matthey Company, Ward Hill, MA, USA) or Spectrochem Pvt.
Ltd (Mumbai, India) and were used without further purification.
Reactions were monitored by TLC, performed on silica gel glass
plates containing 60 GF-254, and visualization on TLC was
achieved by UV light or iodine indicator. Column chromatogra-
phy was performed with Merck 60–120 mesh silica gel. 1H spec-
tra were recorded on Gemini Varian-VXR-unity (200 MHz) or
Bruker UXNMR/XWIN-NMR (300 MHz) instruments. Chemical
shifts (d) are reported in ppm downfield from internal TMS stan-
dard. ESI spectra were recorded on Micro mass, Quattro LC using
ESI+ software with capillary voltage 3.98 kV and ESI mode posi-
tive ion trap detector. High-resolution mass spectra (HRMS) were
recorded on QSTAR XL Hybrid MS/MS mass spectrometer. Melt-
ing points were determined with an Electro thermal melting
point apparatus, and are uncorrected.
Compound 7d was prepared following the method described for
the preparation of compound 7a, employing 6 (272 mg, 1 mmol)
and 3,4-dimethoxybenzoyl chloride (200 mg, 1 mmol) to afford
compound 7d as a white solid (414 mg, 95%). Mp: 130–133 °C;
1H NMR (300 MHz, DMSO-d6): d 3.94 (s, 9H, 3 ꢂ –OCH3), 5.29 (s,
2H, benzyl –CH2–), 7.19 (d, 1H, J = 8.0 Hz, ArH), 7.45ꢀ7.72 (m,
10H, ArH), 10.43 (d, 2H, J = 9.0 Hz, ꢀCONHNHCOꢀ); MS (ESI):
m/z 436 [M+1]+.
4.2.5. Synthesis of 2-(3-(benzyloxy)-4-methoxyphenyl)-5-(4-
fluorophenyl)-1,3,4-oxadiazole (8a)
Compound 7a (395 mg, 1 mmol) was added to phosphorus oxy-
chloride (6 mL) and the mixture was heated under reflux for 1 h.
On cooling, crushed ice was added cautiously and the mixture
was stirred for 30 min. The separated crude product was filtered,
washed with water then with saturated sodium hydrogen carbon-

6672
A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 6666–6677
ate (NaHCO3) solution and finally with water, dried and recrystal-
lized from ethanol to afford compound 8a as a white solid (327 mg,
87%). Mp: 142–144 °C; 1H NMR (200 MHz, CDCl3): d 4.00 (s, 3H,
ꢀJCH3), 5.21 (s, 2H, benzyl ꢀCH2ꢀ), 6.95 (d, 1H, J = 8.8 Hz, ArH),
7.21 (t, 2H, J = 8.8 Hz, ArH), 7.30ꢀ7.47 (m, 4H, ArH), 7.55 (d,
J = 1.4 Hz, 1H, ArH), 7.59 (d, J = 2.2 Hz, 1H, ArH), 7.65 (d, 1H,
J = 2.2 Hz, ArH), 8.12 (dd, 2H, J = 13.9 Hz, J = 3.6 Hz, ArH); MS
(ESI): m/z 377 [M+1]+.
(300 mg, 89%). Mp: 183–186 °C; 1H NMR (200 MHz, CDCl3): d
4.02 (s, 3H, ꢀJCH3), 7.07 (d, 1H, J = 8.3 Hz, ArH), 7.65 (d, 1H,
J = 2.2 Hz, ArH), 7.67 (s, 1H, ArH), 7.80 (d, 2H, J = 8.3 Hz, ArH),
8.26 (d, 2H, J = 8.3 Hz, ArH); MS (ESI): m/z 337 [M+1]+.
4.2.11. 2-Methoxy-5-(5-(3,4,5-trimethoxyphenyl)-1,3,4-
oxadiazol-2-yl) phenol (9c)
Compound 9c was prepared following the method described for
the preparation of compound 9a, employing 8c (449 mg, 1 mmol)
and the crude product was purified by column chromatography
(70% EtOAc–hexane) to afford compound 9c as a white solid
(235 mg, 82%). Mp: 138–142 °C; 1H NMR (200 MHz, CDCl3): d
3.92 (s, 3H, ꢀJCH3), 3.97 (s, 6H, 2 ꢂ ꢀJCH3), 4.01 (s, 3H, ꢀOCH3),
7.05 (d, 1H, J = 8.0 Hz, ArH), 7.34 (s, 2H, ArH), 7.64ꢀ7.70 (m, 2H,
ArH); MS (ESI): m/z 359 [M+1]+.
4.2.6. 2-(3-(Benzyloxy)-4-methoxyphenyl)-5-(4-(trifluoro
methyl)phenyl)-1,3,4-oxadiazole (8b)
Compound 8b was prepared following the method described for
the preparation of compound 8a, employing 7b (445 mg, 1 mmol)
to afford compound 8b as a white solid (354 mg, 83%). Mp: 137–
139 °C; 1H NMR (200 MHz, CDCl3): d 4.00 (s, 3H, –OCH3), 5.20 (s,
2H, benzyl –CH2–), 6.95 (d, 1H, J = 8.0 Hz, ArH), 7.29ꢀ7.47 (m,
5H, ArH), 7.56 (d, 1H, J = 2.2 Hz, ArH), 7.66 (d, 1H, J = 2.2 Hz,
ArH), 7.79 (d, 2H, J = 8.0 Hz, ArH), 8.24 (d, 2H, J = 8.0 Hz, ArH);
MS (ESI): m/z 427 [M+1]+.
4.2.12. 5-(5-(3,4-Dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)-2-
methoxy phenol (9d)
Compound 9d was prepared following the method described for
the preparation of compound 9a, employing 8d (419 mg, 1 mmol)
and the crude product was purified by column chromatography
(60% EtOAc–hexane) to afford compound 9d as a white solid
(279 mg, 85%). Mp: 181–183 °C; 1H NMR (200 MHz, CDCl3): d
3.96 (s, 3H, ꢀJCH3), 3.99 (s, 3H, ꢀJCH3), 4.01 (s, 3H, ꢀJCH3),
6.97 (d, 1H, J = 8.4 Hz, ArH), 7.04 (d, 1H, J = 8.4 Hz, ArH),
7.58ꢀ7.70 (m, 4H, ArH); MS (ESI): m/z 329 [M+1]+.
4.2.7. 2-(3-(Benzyloxy)-4-methoxyphenyl)-5-(3,4,5-trimethoxy
phenyl)-1,3,4-oxadiazole (8c)
Compound 8c was prepared following the method described for
the preparation of compound 8a, employing 7c (467 mg, 1 mmol)
to afford compound 8c as a white solid (358 mg, 80%). Mp: 133–
135 °C; 1H NMR (400 MHz, CDCl3): d 3.92 (s, 3H, ꢀJCH3), 3.97 (s,
6H, 2 ꢂ ꢀOCH3), 4.01 (s, 3H, ꢀJCH3), 5.24 (s, 2H, benzyl ꢀCH2ꢀ),
6.98 (d, 1H, J = 8.6 Hz, ArH), 7.32 (s, 2H, ArH), 7.36ꢀ7.41 (m, 3H,
ArH), 7.45 (d, 2H, J = 7.0 Hz, ArH), 7.60 (d, 1H, J = 7.0 Hz, ArH),
7.68 (s, 1H, ArH); MS (ESI); m/z 449 [M+1]+.
4.2.13. Synthesis of (2S)-N-{4-[3-[5-(5-(4-fluorophenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]propyl)oxy-5-methoxy-
2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal
(11a)
4.2.8. 2-(3-(Benzyloxy)-4-methoxyphenyl)-5-(3,4-dimethoxy
phenyl)-1,3,4-oxadiazole (8d)
To a solution of (2S)-N-[4-(3-bromopropoxy)-5-methoxy-2-
nitrobenzoyl] pyrrolidine-2-carboxaldehyde diethylthioacetal 10a
(521 mg, 1 mmol) in acetone (10 mL) was added anhydrous
K2CO3 (552 mg, 4 mmol) and 5-(5-(4-fluorophenyl)-1,3,4-oxa-
diazol-2-yl)-2-methoxyphenol 9a (286 mg, 1 mmol). The reaction
mixture was heated to reflux for 24 h. After completion of the reac-
tion as indicated by TLC, potassium carbonate was removed by suc-
tion filtration and the solvent was removed under vacuum. The
crude product thus obtained was purified by column chromatogra-
phy using 50% EtOAc–hexane as eluant to afford pure compound of
11a as a yellow solid (581 mg, 80%). Mp: 118–120 °C; 1H NMR
(300 MHz, CDCl3): d 1.29ꢀ1.40 (m, 6H, ꢀ(CH3)2), 1.68ꢀ2.15 (m,
4H, ꢀ(CH2)2ꢀ), 2.41ꢀ2.49 (m, 2H, ꢀCH2ꢀ), 2.66ꢀ2.84 (m, 4H,
2 ꢂ ꢀSCH2ꢀ), 3.17ꢀ3.29 (m, 2H, ꢀNCH2ꢀ), 3.93 (s, 3H, ꢀJCH3),
3.97 (s, 3H, ꢀJCH3), 4.29ꢀ4.38 (m, 4H, 2 ꢂ ꢀJCH2ꢀ), 4.65ꢀ4.72
(m, 1H, ꢀNCHꢀ), 4.85 (d, 1H, J = 3.7 Hz, ꢀCHS2ꢀ), 6.80 (s, 1H,
ArH), 7.01 (d, 1H, J = 8.3 Hz, ArH), 7.21 (d, 1H, J = 8.3 Hz, ArH),
7.26 (s, 1H, ArH), 7.62 (s, 1H, ArH), 7.65 (d, 1H, J = 2.2 Hz, ArH),
7.72 (s, 1H, ArH), 8.14 (dd, 2H, J = 14.3 Hz, J = 3.7 Hz, ArH); MS
(ESI): m/z 727 [M+1]+.
Compound 8d was prepared following the method described for
the preparation of compound 8a, employing 7d (436 mg, 1 mmol)
to afford compound 8d as a white solid (355 mg, 85%). Mp: 163–
165 °C; 1H NMR (400 MHz, CDCl3): d 3.96 (s, 3H, ꢀJCH3), 3.99 (s,
3H, ꢀOCH3), 4.01 (s, 3H, ꢀJCH3), 5.21 (s, 2H, benzyl ꢀCH2ꢀ), 6.93
(d, 1H, J = 2.8 Hz, ArH), 6.96 (d, 1H, J = 2.2 Hz, ArH), 7.27ꢀ7.46 (m,
5H, ArH), 7.54ꢀ7.66 (m, 4H, ArH); MS (ESI); m/z 419 [M+1]+.
4.2.9. Synthesis of 5-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)-
2-methoxyphenol (9a)
To a stirred solution of EtSH (1.18 g, 19.0 mmol) and BF3ꢁOEt2
(1.41 g, 10 mmol) in dichloromethane was added dropwise to the
solution of compound 8a (377 mg, 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 and then extracted with ethyl acetate. The
combined organic phases were washed with saturated brine, dried
over anhydrous Na2SO4 and the solvent removed under vacuum to
afford the crude product. This was further purified by column chro-
matography using (50% EtOAc–hexane) as eluant to afford com-
pound 9a as a white solid (286 mg, 80%). Mp: 199–202 °C; 1H
NMR (200 MHz, CDCl3): d 3.99 (s, 3H, –OCH3), 7.02 (d, 1H,
J = 7.8 Hz, ArH), 7.23 (t, 2H, J = 8.5 Hz, ArH), 7.37 (s, 1H, ArH),
7.59 (d, 1H, J = 7.8 Hz, ArH), 8.13 (dd, 2H, J = 14.0 Hz, J = 3.9 Hz,
ArH); MS (ESI): m/z 287 [M+1]+.
4.2.14. (2S)-N-{4-[4-[5-(5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-
yl)-2-methoxyphenyl) oxy]butyl)oxy-5-methoxy-2-
nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal
(11b)
This compound was prepared according to the method de-
scribed for compound 11a employing compound 10b (535 mg,
1 mmol) and compound 9a (286 mg, 1 mmol). The crude product
was purified by column chromatography (50% EtOAc–hexane) to
afford compound 11b as a yellow solid (607 mg, 82%). Mp: 97–
99 °C; 1H NMR (300 MHz, CDCl3): d 1.21ꢀ1.42 (m, 6H, ꢀ(CH3)2),
1.60ꢀ1.72 (m, 4H, ꢀ(CH2)2ꢀ), 2.03ꢀ2.19 (m, 4H, 2 ꢂ ꢀCH2ꢀ),
2.67ꢀ2.87 (m, 4H, 2 ꢂ ꢀSCH2ꢀ), 3.18ꢀ3.32 (m, 2H, ꢀNCH2ꢀ),
3.92 (s, 3H, ꢀOCH3), 3.96 (s, 3H, ꢀOCH3), 4.16ꢀ4.30 (m, 4H,
2 ꢂ ꢀOCH2ꢀ), 4.64ꢀ4.75 (m, 1H, ꢀNCHꢀ), 4.86 (d, 1H, J = 4.1 Hz,
4.2.10. 2-Methoxy-5-(5-(4-(trifluoromethyl)phenyl)-1,3,4-
oxadiazol-2-yl) phenol (9b)
Compound 9b was prepared following the method described for
the preparation of compound 9a, employing 8b (427 mg, 1 mmol)
and the crude product was purified by column chromatography
(40% EtOAc–hexane) to afford compound 9b as a white solid

A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 6666–6677
6673
ꢀCHS2ꢀ), 6.80 (s, 1H, ArH), 6.98 (d, 1H, J = 8.3 Hz, ArH), 7.25 (d, 2H,
J = 8.3 Hz, ArH), 7.61ꢀ7.71 (m, 3H, ArH), 8.14 (dd, 2H, J = 14.1 Hz,
J = 4.1 Hz, ArH); MS (ESI): m/z 741 [M+1]+.
1 mmol) and compound 9b (336 mg, 1 mmol). The crude product
was purified by column chromatography (50% EtOAc–hexane) to
afford compound 11f as a yellow solid (658 mg, 82%). Mp: 91–
93 °C; 1H NMR (300 MHz, CDCl3): d 1.23ꢀ1.41 (m, 6H, ꢀ(CH3)2),
1.68ꢀ1.84 (m, 2H, ꢀCH2ꢀ), 1.89ꢀ2.08 (m, 6H, 3 ꢂ ꢀCH2ꢀ),
2.21ꢀ2.36 (m, 2H, ꢀCH2ꢀ), 2.66ꢀ2.86 (m, 4H, 2 ꢂ ꢀSCH2ꢀ),
3.19ꢀ3.27 (m, 2H, ꢀNCH2ꢀ), 3.94 (s, 3H, ꢀOCH3), 3.98 (s, 3H,
ꢀOCH3), 4.05ꢀ4.19 (m, 4H, 2 ꢂ ꢀOCH2ꢀ), 4.62ꢀ4.74 (m, 1H,
ꢀNCHꢀ), 4.84 (d, 1H, J = 3.9 Hz, ꢀCHS2ꢀ), 6.79 (s, 1H, ArH), 6.96
(d, 1H, J = 8.5 Hz, ArH), 7.62ꢀ7.69 (m, 3H, ArH), 7.80 (d, 2H,
J = 8.5 Hz, ArH), 8.26 (d, 2H, J = 7.81 Hz, ArH); MS (ESI): m/z 805
[M+1]+.
4.2.15. (2S)-N-{4-[5-[5-(5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-
yl)-2-methoxyphenyl) oxy]pentyl)oxy-5-methoxy-2-nitro
benzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal (11c)
This compound was prepared according to the method de-
scribed for compound 11a employing compound 10c (549 mg,
1 mmol) and compound 9a (286 mg, 1 mmol). The crude product
was purified by column chromatography (50% EtOAc–hexane) to af-
ford compound 11c as a yellow solid (679 mg, 90%). Mp: 99–101 °C;
1H NMR (300 MHz, CDCl3): d 1.22ꢀ1.40 (m, 8H, ꢀ(CH3)2, ꢀCH2ꢀ),
1.61ꢀ1.80 (m, 4H, 2 ꢂ ꢀCH2ꢀ), 1.89ꢀ2.07 (m, 4H, 2 ꢂ ꢀCH2ꢀ),
2.66ꢀ2.84 (m, 4H, 2 ꢂ ꢀSCH2ꢀ), 3.19ꢀ3.31 (m, 2H, ꢀNCH2ꢀ),
3.94 (s, 3H, ꢀOCH3), 3.97 (s, 3H, ꢀOCH3), 4.07ꢀ4.17 (m, 4H,
2 ꢂ ꢀOCH2ꢀ), 4.66ꢀ4.74 (m, 1H, ꢀNCHꢀ), 4.86 (d, 1H, J = 3.7 Hz,
ꢀCHS2ꢀ), 6.81 (s, 1H, ArH), 6.97 (d, 1H, J = 8.3 Hz, ArH), 7.24 (d,
2H, J = 8.3 Hz, ArH), 7.27 (s, 1H, ArH), 7.62ꢀ7.67 (m, 2H, ArH),
8.14 (dd, 2H, J = 13.5 Hz, J = 3.0 Hz, ArH); MS (ESI): m/z 755 [M+1]+.
4.2.19. (2S)-N-{4-[3-[5-(5-(3,4,5-Trimethoxyphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]propyl)oxy-5-methoxy-
2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal
(11g)
This compound was prepared according to the method de-
scribed for compound 11a employing (2S)-N-[4-(3-bromoprop-
oxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxycarbalde-
hyde diethylthioacetal 10a (521 mg, 1 mmol) and 2-methoxy-5-(5-
(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-2-yl)phenol 9c (358 mg,
1 mmol).The crude product was purified by column chromatogra-
phy (50% EtOAc–hexane) to afford compound 11g as a yellow solid
4.2.16. (2S)-N-{4-[3-[5-(5-(4-Trifluoromethylphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]propyl)oxy-5-methoxy-
2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal
(11d)
(717 mg, 90%). Mp: 93–95 °C; 1H NMR (300 MHz, CDCl3):
d
This compound was prepared according to the method de-
scribed for compound 11a employing (2S)-N-[4-(3-bromoprop-
oxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxycarbalde-
hyde diethylthioacetal 10a (521 mg, 1 mmol) and 2-methoxy-5-(5-
(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl) phenol 9b (336
mg, 1 mmol). The crude product was purified by column chroma-
tography (50% EtOAc–hexane) to afford compound 11d as a yellow
solid (654 mg, 85%). Mp: 101–103 °C; 1H NMR (300 MHz, CDCl3): d
1.22ꢀ1.35 (m, 6H, ꢀ(CH3)2), 1.90ꢀ2.13 (m, 2H, ꢀCH2ꢀ), 2.20ꢀ2.35
(m, 2H, ꢀCH2ꢀ), 2.40ꢀ2.49 (m, 2H, ꢀCH2ꢀ), 2.67ꢀ2.84 (m, 4H,
2 ꢂ ꢀSCH2ꢀ), 3.18ꢀ3.29 (m, 2H, ꢀNCH2ꢀ), 3.93 (s, 3H, ꢀOCH3),
3.98 (s, 3H, ꢀOCH3), 4.29ꢀ4.38 (m, 4H, 2 ꢂ ꢀOCH2ꢀ), 4.65ꢀ4.73
(m, 1H, ꢀNCHꢀ), 4.85 (d, 1H, J = 3.7 Hz, ꢀCHS2ꢀ), 6.80 (s, 1H,
ArH), 7.02 (d, 1H, J = 8.3 Hz, ArH), 7.63ꢀ7.69 (m, 2H, ArH), 7.72
(s, 1H, ArH), 7.80 (d, 2H, J = 8.3 Hz, ArH), 8.26 (d, 2H, J = 8.3 Hz,
ArH); MS (ESI): m/z 777 [M+1]+.
1.24ꢀ1.38 (m, 6H, ꢀ(CH3)2), 1.48ꢀ1.84 (m, 4H, ꢀCH2)2ꢀ),
2.38ꢀ2.50 (m, 2H, ꢀCH2ꢀ), 2.65ꢀ2.85 (m, 4H, 2 ꢂ ꢀSCH2ꢀ),
3.14ꢀ3.31 (m, 2H, ꢀNCH2ꢀ), 3.92 (s, 6H, ꢀOCH3), 3.93 (s, 3H,
ꢀOCH3), 3.98 (s, 6H, ꢀOCH3), 4.28ꢀ4.40 (m, 4H, 2 ꢂ ꢀOCH2ꢀ),
4.64ꢀ4.73 (m, 1H, ꢀNCH), 4.84 (d, 1H, J = 3.7 Hz, ꢀCHS2ꢀ), 6.80
(s, 1H, ArH), 7.01 (d, 1H, J = 8.3 Hz, ArH), 7.32 (s, 2H, ArH), 7.64
(s, 2H, ArH), 7.72 (s, 1H, ArH); MS (ESI): m/z 799 [M+1]+.
4.2.20. (2S)-N-{4-[4-[5-(5-(3,4,5-Trimethoxyphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]butyl)oxy-5-methoxy-2-
nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal
(11h)
This compound was prepared according to the method de-
scribed for compound 11a employing compound 10b (535 mg,
1 mmol) and compound 9c (358 mg, 1 mmol). The crude product
was purified by column chromatography (50% EtOAc–hexane) to
afford compound 11h as a yellow solid (689 mg, 85%). Mp: 88–
90 °C; 1H NMR (300 MHz, CDCl3): d 1.22ꢀ1.39 (m, 6H, ꢀ(CH3)2),
1.73ꢀ1.86 (m, 4H, ꢀ(CH2)2ꢀ), 2.07ꢀ2.16 (m, 4H, 2 ꢂ ꢀCH2ꢀ),
2.64ꢀ2.82 (m, 4H, 2 ꢂ ꢀSCH2ꢀ), 3.18ꢀ3.28 (m, 2H, ꢀNCH2ꢀ),
3.90 (s, 3H, ꢀOCH3), 3.91 (s, 3H, ꢀOCH3), 3.95 (s, 3H, ꢀOCH3),
3.97 (s, 6H, 2 ꢂ ꢀOCH3), 4.16ꢀ4.27 (m, 4H, 2 ꢂ ꢀOCH2ꢀ),
4.63ꢀ4.70 (m, 1H, ꢀNCHꢀ), 4.81 (d, 1H, J = 3.7 Hz, ꢀCHS2ꢀ), 6.74
(s, 1H, ArH), 6.93 (d, 1H, J = 9.0 Hz, ArH), 7.30 (s, 2H, ArH), 7.61
(d, 2H, J = 6.7 Hz, ArH), 7.65 (s, 1H, ArH); MS (ESI): m/z 813 [M+1]+.
4.2.17. (2S)-N-{4-[4-[5-(5-(4-Trifluoromethylphenyl)-1,3,4-oxa
diazol-2-yl)-2-methoxyphenyl)oxy]butyl)oxy-5-methoxy-2-
nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal
(11e)
This compound was prepared according to the method de-
scribed for compound 11a employing compound 10b (535 mg,
1 mmol) and compound 9b (336 mg, 1 mmol). The crude product
was purified by column chromatography (50% EtOAc–hexane) to
afford compound 11e as a yellow solid (726 mg, 92%). Mp: 95–
97 °C; 1H NMR (300 MHz, CDCl3): d 1.23ꢀ1.35 (m, 6H, ꢀ(CH3)2),
1.89ꢀ2.17 (m, 4H, 2 ꢂ ꢀCH2ꢀ), 2.21ꢀ2.34 (m, 2H, ꢀCH2ꢀ),
2.64ꢀ2.82 (m, 4H, 2 ꢂ ꢀSCH2ꢀ), 3.17ꢀ3.30 (m, 2H, ꢀNCH2ꢀ),
3.91 (s, 3H, ꢀOCH3), 3.95 (s, 3H, ꢀOCH3), 4.16ꢀ4.30 (m, 4H,
2 ꢂ ꢀOCH2ꢀ), 4.62ꢀ4.69 (m, 1H, ꢀNCHꢀ), 4.80 (d, 1H, J = 3.7 Hz,
ꢀCHS2ꢀ), 6.73 (s, 1H, ArH), 6.94 (d, 1H, J = 8.3 Hz, ArH), 7.61 (d,
2H, J = 4.5 Hz, ArH), 7.64 (s, 1H, ArH), 7.80 (d, 2H, J = 8.3 Hz, ArH),
8.26 (d, 2H, J = 8.30 Hz, ArH); MS (ESI): m/z 791 [M+1]+.
4.2.21. (2S)-N-{4-[5-[5-(5-(3,4,5-Trimethoxyphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]pentyl)oxy-5-methoxy-
2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal
(11i)
This compound was prepared according to the method de-
scribed for compound 11a employing compound 10c (549 mg,
1 mmol) and compound 9c (358 mg, 1 mmol). The crude product
was purified by column chromatography (50% EtOAc–hexane) to
afford compound 11i as a yellow solid (677 mg, 82%). Mp: 78–
90 °C; 1H NMR (300 MHz, CDCl3): d 1.22ꢀ1.40 (m, 8H, ꢀ(CH3)2,
ꢀCH2ꢀ), 1.68ꢀ1.84 (m, 4H, 2 ꢂ ꢀCH2ꢀ), 1.90ꢀ2.08 (m, 4H,
2 ꢂ ꢀCH2ꢀ), 2.64ꢀ2.88 (m, 4H, 2 ꢂ ꢀSCH2ꢀ), 3.18ꢀ3.29 (m, 2H,
ꢀNCH2ꢀ), 3.91 (s, 6H, 2 ꢂ ꢀOCH3), 3.95 (s, 3H, ꢀOCH3), 3.99 (s,
6H, 2 ꢂ ꢀOCH3), 4.07ꢀ4.18 (m, 4H, 2 ꢂ ꢀOCH2ꢀ), 4.60ꢀ4.73 (m,
1H, ꢀNCHꢀ), 4.82 (d, 1H, J = 3.9 Hz, ꢀCHS2ꢀ), 6.77 (s, 1H, ArH),
4.2.18. (2S)-N-{4-[5-[5-(5-(4-Trifluoromethylphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]pentyl)oxy-5-methoxy-
2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal
(11f)
This compound was prepared according to the method de-
scribed for compound 11a employing compound 10c (549 mg,

6674
A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 6666–6677
6.92 (d, 1H, J = 7.8 Hz, ArH), 7.30 (s, 2H, ArH), 7.58 (d, 1H, J = 1.5 Hz,
ArH), 7.63 (s, 2H, ArH); MS (ESI): m/z 827 [M+1]+.
was then carefully adjusted to pH 8 with 10% NaHCO3 solution
and then extracted with ethyl acetate. The combined organic phase
was dried over anhydrous Na2SO4 and evaporated under vacuum to
afford the amino diethylthioacetal, which due to potential stability
problems preceded for the next step. A solution of amino diet-
hylthioacetal (1 mmol), HgCl2 (2.26 mmol) and CaCO3 (2.46 mmol)
in CH3CN–water (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 supernatant 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 chromatogra-
phy (MeOH/CHCl3, 3%) to give the final product 4a as a white solid
4.2.22. (2S)-N-{4-[3-[5-(5-(3,4-Dimethoxyphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]propyl)oxy-5-methoxy-
2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal
(11j)
This compound was prepared according to the method de-
scribed for compound 11a employing (2S)-N-[4-(3-bromoprop-
oxy)-5-methoxy-2-nitrobenzoyl]pyrrolidine-2-carboxycarbalde-
hyde diethylthioacetal 10a (521 mg, 1 mmol) and 5-(5-(3,4-
dimethoxyphenyl)-1,3,4-oxadiazol-2-yl)-2-methoxyphenol
9d
(328 mg, 1 mmol). The crude product was purified by column chro-
matography (50% EtOAc–hexane) to afford compound 11j as a yel-
low solid (660 mg, 86%). Mp: 106–108 °C; 1H NMR (300 MHz,
(332 mg, 58% yield). Mp 102–104 °C; ½a D27
¼ þ118:5 (c = 1 in
ꢃ
CDCl3):
d
1.22ꢀ1.39 (m, 6H, ꢀ(CH3)2), 1.74ꢀ1.98 (m, 4H,
CHCl3). 1H NMR (400 MHz, CDCl3): d 1.60–1.82 (m, 2H, –CH2–),
2.01–2.14 (m, 2H, –CH2–), 2.37–2.51 (m, 2H, –CH2–), 3.48–3.88
(m, 3H, –NCH2–, –NCH–), 3.94 (s, 3H, –OCH3), 3.98 (s, 3H, –OCH3),
4.26–4.38 (m, 4H, 2 ꢂ –OCH2–), 6.88 (s, 1H, ArH), 7.03 (d, 2H,
J = 9.1 Hz, ArH), 7.25 (s, 2H, ArH), 7.52 (s, 1H, ArH), 7.64 (t, 1H,
J = 1.6 Hz, ArH), 7.69 (d, 1H, J = 4.1 Hz, imine–H), 8.15 (dd, 2H,
J = 14.1 Hz, J = 3.3 Hz, ArH); 13C NMR (75 MHz, CDCl3): d 166.2,
164.5, 163.8, 162.9, 162.3, 159.1, 151.4, 150.6, 149.5, 147.6, 140.5,
129.0, 128.9, 120.1, 116.4, 116.1, 112.2, 111.4, 110.3, 109.6, 68.4,
56.0, 53.6, 46.5, 29.5, 25.7, 24.1, 22.5 ppm; IR (KBr) (Umax/cmꢀ1):
ꢀ(CH2)2ꢀ), 2.38ꢀ2.52 (m, 2H, ꢀCH2ꢀ), 2.70ꢀ2.87 (m, 4H,
2 ꢂ ꢀSCH2ꢀ), 3.19ꢀ3.30 (m, 2H, ꢀNCH2ꢀ), 3.94 (s, 3H, ꢀOCH3),
3.98 (s, 6H, 2 ꢂ ꢀOCH3), 4.00 (s, 3H, ꢀOCH3), 4.28ꢀ4.41 (m, 4H,
2 ꢂ ꢀOCH2ꢀ), 4.65ꢀ4.77 (m, 1H, ꢀNCHꢀ), 4.88 (d, 1H, J = 3.9 Hz,
ꢀCHS2ꢀ), 6.82 (s, 1H, ArH), 6.99 (d, 1H, J = 8.5 Hz, ArH), 7.04 (s,
1H, ArH), 7.63ꢀ7.72 (m, 4H, ArH), 7.75 (s, 1H, ArH); MS (ESI): m/
z 769 [M+1]+.
4.2.23. (2S)-N-{4-[4-[5-(5-(3,4-Dimethoxyphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]butyl)oxy-5-methoxy-2-
nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal
(11k)
m
3359 (br), 2930, 1606, 1499, 1463, 1432, 1271, 1226, 1025, 846,
743, 656, 611; MS (ESI): m/z 573 [M+1]+; HRMS (ESI m/z) for
31H30N4O6F calcd 573.2149, found 573.2152 [M+1]+.
C
This compound was prepared according to the method described
for compound 11a employing compound 10b (535 mg, 1 mmol) and
compound 9d (328 mg, 1 mmol). The crude product was purified by
column chromatography (50% EtOAc–hexane) to afford compound
11k as a yellow solid (640 mg, 82%). Mp: 102–104 °C; 1H NMR
(300 MHz, CDCl3): d 1.24ꢀ1.39 (m, 6H, ꢀ(CH3)2), 1.56ꢀ1.87 (m,
4H, ꢀ(CH2)2ꢀ), 2.08ꢀ2.17 (m, 4H, 2 ꢂ ꢀCH2ꢀ), 2.68ꢀ2.86 (m, 4H,
2 ꢂ ꢀSCH2ꢀ), 3.18ꢀ3.30 (m, 2H, ꢀNCH2ꢀ), 3.92 (s, 3H, ꢀOCH3),
3.96 (s, 6H, 2 ꢂ ꢀOCH3), 4.00 (s, 3H, ꢀOCH3), 4.18ꢀ4.28 (m, 4H,
2 ꢂ ꢀOCH2ꢀ), 4.66ꢀ4.74 (m, 1H, ꢀNCHꢀ), 4.86 (d, 1H, J = 3.7 Hz,
ꢀCHS2ꢀ), 6.80 (s, 1H, ArH), 6.97 (d, 2H, J = 8.3 Hz, ArH), 7.64 (d,
2H, J = 6.7 Hz, ArH) 7.68 (s, 2H, ArH); MS (ESI): m/z 783 [M+1]+.
4.2.26. 7-Methoxy-8-{4-[5-(5-(4-fluorophenyl)-1,3,4-oxadiazol-
2-yl)-2-methoxyphenyl)oxy]butyloxy}-(11aS)-1,2,3,11a-
tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one (4b)
Compound 4b was prepared according to the method described
for compound 4a, employing compound 11b (740 mg, 1.0 mmol).
The crude product was purified by column chromatography
(MeOH/CHCl3, 3%) to afford compound 4b as a white solid
(329 mg, 56% yield). Mp: 114ꢀ116 °C; ½a D27
¼ þ159:5 (c = 1 in
ꢃ
CHCl3); 1H NMR (400 MHz, CDCl3): d 1.56–1.68 (m, 4H, 2 ꢂ
–CH2–), 2.03–2.15 (m, 4H, 2 ꢂ –CH2–), 3.71–3.86 (m, 3H, –NCH2–,
–NCH–), 3.93 (s, 3H, –OCH3), 3.96 (s, 3H, –OCH3), 4.18–4.27 (m,
4H, 2 ꢂ –OCH2–), 6.82 (s, 1H, ArH), 7.01 (d, 1H, J = 8.0 Hz, ArH),
7.24 (d, 2H, J = 8.0 Hz, ArH), 7.26 (s, 1H, ArH), 7.51 (s, 1H, ArH),
7.61 (s, 1H, ArH), 7.68 (d, 1H, J = 4.3 Hz, imine–H), 8.14 (dd, 2H,
4.2.24. (2S)-N-{4-[5-[5-(5-(3,4-Dimethoxyphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]pentyl)oxy-5-methoxy-
2-nitrobenzoyl}pyrrolidine-2-carboxaldehyde diethylthioacetal
(11l)
J = 13.9 Hz, J = 3.6 Hz, ArH); IR (KBr) (Umax/cmꢀ1):
m 3342 (br),
2936, 1606, 1498, 1464, 1430, 1272, 1225, 1025, 846, 743, 655,
610, 520 cmꢀ1; MS (ESI): m/z 587 [M+1]+; HRMS (ESI m/z) for
This compound was prepared according to the method de-
scribed for compound 11a employing compound 10c (549 mg,
1 mmol) and compound 9d (328 mg, 1 mmol). The crude product
was purified by column chromatography (50% EtOAc–hexane) to af-
ford compound 11l as a yellow solid (700 mg, 88%). Mp: 101–
103 °C; 1H NMR (300 MHz, CDCl3): d 1.24ꢀ1.39 (m, 6H, ꢀ(CH3)2),
1.68ꢀ1.86 (m, 2H, ꢀCH2ꢀ), 1.92ꢀ2.11 (m, 6H, ꢀCH2)3), 2.22ꢀ2.35
(m, 2H, ꢀCH2ꢀ), 2.68ꢀ2.85 (m, 4H, 2 ꢂ ꢀSCH2ꢀ), 3.19ꢀ3.33 (m,
2H, ꢀNCH2ꢀ), 3.94 (s, 3H, ꢀOCH3), 3.97 (s, 3H, ꢀOCH3), 3.98 (s,
3H, ꢀOCH3), 4.00 (s, 3H, ꢀOCH3), 4.10ꢀ4.18 (m, 4H, 2 ꢂ ꢀOCH2ꢀ),
4.68ꢀ4.76 (m, 1H, ꢀNCHꢀ), 4.88 (d, 1H, J = 3.7 Hz, ꢀCHS2ꢀ), 6.83 (s,
1H, ArH), 6.99 (d, 2H, J = 8.3 Hz, ArH), 7.64ꢀ7.69 (m, 5H, ArH); MS
(ESI): m/z 796 [M+1]+.
C
32H32N4O6F calcd 587.2305, found 587.2276 [M+1]+.
4.2.27. 7-Methoxy-8-{5-[5-(5-(4-fluorophenyl)-1,3,4-oxadiazol-
2-yl)-2-methoxyphenyl)oxy]pentyloxy}-(11aS)-1,2,3,11a-
tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one (4c)
Compound 4c was prepared according to the method described
for compound 4a, employing compound 11c (754 mg, 1.0 mmol).
The crude product was purified by column chromatography
(MeOH/CHCl3, 3%) to afford compound 4c as
a
white solid
(402 mg, 55% yield). Mp: 95ꢀ97 °C; ½a D27
ꢃ
¼ þ147:5 (c = 1 in CHCl3);
1H NMR (400 MHz, CDCl3): d 1.58–1.77 (m, 4H, 2 ꢂ –CH2–), 1.91–
2.06 (m, 6H, 3 ꢂ –CH2–), 3.69–3.86 (m, 3H, –NCH2–, –NCH–),
3.94 (s, 3H, –OCH3), 3.97 (s, 3H, –OCH3), 4.10–4.16 (m, 4H, 2 ꢂ
–OCH2–), 6.80 (s, 1H, ArH), 6.98 (d, 1H, J = 8.0 Hz, ArH), 7.24 (d,
2H, J = 8.0 Hz, ArH), 7.26 (s, 1H, ArH), 7.51 (s, 1H, ArH), 7.59 (s,
1H, ArH), 7.67 (d, 1H, J = 3.6 Hz, imine–H), 8.14 (dd, 2H,
4.2.25. Synthesis of 7-methoxy-8-{3-[5-(5-(4-fluorophenyl)-
1,3,4-oxadiazol-2-yl)-2-methoxyphenyl)oxy]propyloxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepine-5-
one (4a)
To a solution of compound 11a (726 mg, 1.0 mmol) in methanol
(20 mL), SnCl2.2H2O (5 mmol) was added and refluxed for 1–2 h.
The methanol was evaporated in vacuum and the aqueous layer
J = 13.5 Hz, J = 3.0 Hz, ArH); IR (KBr) (Umax/cmꢀ1):
2933, 1607, 1498, 1464, 1273, 1225, 1024, 847, 741, 654, 610,
520 cmꢀ1 MS (ESI): m/z 601 [M+1]+; HRMS (ESI m/z) for
33H34N4O6F calcd 601.2462, found 601.2476 [M+1]+.
m 3380 (br),
;
C

A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 6666–6677
6675
4.2.28. 7-Methoxy-8-{3-[5-(5-(4-trifluoromethylphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]propyloxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepine-5-
one (4d)
The crude product was purified by column chromatography
(MeOH/CHCl3, 3%) to afford compound 4g as a white solid
(368 mg, 57% yield). Mp: 86–88 °C; ½a D27
¼ þ112:5 (c = 1 in CHCl3);
ꢃ
1H NMR (400 MHz, CDCl3): d 1.51–1.78 (m, 4H, 2 ꢂ –CH2–), 1.92–
2.14 (m, 2H, –CH2–), 3.73–3.88 (m, 3H, –NCH2–, –NCH–), 3.96 (s,
9H, 3 ꢂ –OCH3), 4.00 (s, 6H, 2 ꢂ –OCH3), 4.20–4.39 (m, 4H, 2 ꢂ
–OCH2–), 6.88 (s, 1H, ArH), 7.03 (d, 1H, J = 8.7 Hz, ArH), 7.24 (d,
1H, J = 8.0 Hz, ArH), 7.34 (s, 1H, ArH), 7.63 (s, 1H, ArH), 7.66 (d,
1H, J = 4.2 Hz, imine–H), 8.14 (dd, 2H, J = 5.1 Hz, ArH; IR (KBr)
Compound 4d was prepared according to the method described
for compound 4a, employing compound 11d (776 mg, 1.0 mmol).
The crude product was purified by column chromatography
(MeOH/CHCl3, 3%) to afford compound 4d as a white solid
(361 mg, 58% yield). Mp: 98ꢀ100 °C; ½a D27
¼ þ158:5 (c = 1 in
ꢃ
CHCl3); 1H NMR (400 MHz, CDCl3): d 1.53–2.19 (m, 4H, 2 ꢂ
–CH2–), 2.29–2.52 (m, 2H, –CH2–), 3.79–3.95 (m, 3H, –NCH2–,
–NCH–), 3.97 (s, 3H, –OCH3), 3.98 (s, 3H, –OCH3), 4.22–4.42 (m,
4H, 2 ꢂ –OCH2–), 6.87 (s, 1H, ArH), 7.04 (d, 2H, J = 8.5 Hz, ArH),
7.51 (s, 1H, ArH), 7.66 (d, 1H, J = 3.8 Hz, imine–H), 7.69 (d, 1H,
J = 6.2 Hz, ArH), 7.81 (d, 2H, J = 7.7 Hz, ArH), 8.26 (d, 2H,
(Umax/cmꢀ1):
m 3404 (br), 2928, 1603, 1497, 1460, 1464, 1421,
1337, 1245, 1179,1127, 1024, 868, 813, 738, 652 cmꢀ1; MS (ESI):
m/z 645 [M+1]+; HRMS (ESI m/z) for C34H37N4O9 calcd 645.2560,
found 645.2553 [M+1]+.
4.2.32. 7-Methoxy-8-{4-[5-(5-(3,4,5-trimethoxyphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]butyloxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepine-5-
one (4h)
J = 7.7 Hz, ArH); IR (KBr) (Umax/cmꢀ1):
m 3415 (br), 2932,
1609,1502,1465,1427, 1325, 1275, 1172, 1129, 1065, 1020, 853,
752, 597 cmꢀ1; MS (ESI): m/z 623 [M+1]+; HRMS (ESI m/z) for
C32H30N4O6F3 calcd 623.2117, found 623.2098 [M+1]+.
Compound 4h was prepared according to the method described
for compound 4a, employing compound 11h (812 mg, 1.0 mmol).
The crude product was purified by column chromatography
(MeOH/CHCl3, 3%) to afford compound 4h as a white solid
4.2.29. 7-Methoxy-8-{4-[5-(5-(4-trifluoromethylphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]butyloxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepine-5-
one (4e)
(362 mg, 55% yield). Mp: 76ꢀ78 °C; ½a D27
¼ þ123:5 (c = 1 in CHCl3);
ꢃ
1H NMR (400 MHz, CDCl3): d 1.56–1.71 (m, 4H, 2 ꢂ ꢀCH2ꢀ), 2.02–
2.16 (m, 4H, 2 ꢂ ꢀCH2ꢀ), 3.70–3.89 (m, 3H, ꢀNCH2, ꢀNCHꢀ), 3.93
(s, 6H, 2 ꢂ ꢀOCH3), 3.97 (s, 9H, 3 ꢂ ꢀOCH3), 4.13–4.26 (m, 4H,
2 ꢂ ꢀOCH2ꢀ), 6.82 (s, 1H, ArH), 7.00 (d, 2H, J = 8.7 Hz, ArH), 7.34
(s, 2H, ArH), 7.52 (s, 1H, ArH), 7.65 (d, 1H, J = 1.4 Hz, ArH), 7.68
Compound 4e was prepared according to the method described
for compound 4a, employing compound 11e (790 mg, 1.0 mmol).
The crude product was purified by column chromatography
(MeOH/CHCl3, 3%) to afford compound 4e as a white solid
(d, 1H, J = 4.3 Hz, imineꢀH); IR (KBr) (Umax/cmꢀ1):
m 3364 (br),
(357 mg, 56% yield). Mp: 103ꢀ105 °C; ½a D27
¼ þ171:5 (c = 1 in
ꢃ
CHCl3); 1H NMR (400 MHz, CDCl3): d 1.72–1.98 (m, 4H, 2 ꢂ ꢀCH2ꢀ),
2.05–2.18 (m, 4H, 2 ꢂ ꢀCH2ꢀ), 3.53–3.88 (m, 3H, ꢀNCH2ꢀ,
ꢀNCHꢀ), 3.94 (s, 3H, ꢀOCH3), 3.98 (s, 3H, ꢀOCH3), 4.09–4.29 (m,
4H, 2 ꢂ ꢀCH2ꢀ), 6.83 (s, 1H, ArH), 7.01 (d, 2H, J = 8.7 Hz, ArH),
7.51 (s, 1H, ArH), 7.67 (d, 1H, J = 4.1 Hz, imine-H), 7.69 (d, 1H,
J = 5.8 Hz, ArH), 7.81 (d, 2H, J = 8.0 Hz, ArH), 8.27 (d, 2H, J = 8.0 Hz,
2933, 1600, 1497, 1463, 1422, 1338, 1313, 1245, 1179, 1127,
1009, 865, 737, 653 cmꢀ1; MS (ESI): m/z 659 [M+1]+; HRMS (ESI
m/z) for C34H39N4O9 calcd 659.2717, found 659.2695 [M+1]+.
4.2.33. 7-Methoxy-8-{5-[5-(5-(3,4,5-trimethoxyphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]pentyloxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepine-5-
one (4i)
Compound 4i was prepared according to the method described
for compound 4a, employing compound 11i (826 mg, 1.0 mmol).
The crude product was purified by column chromatography
ArH); IR (KBr) (Umax/cmꢀ1):
m 3417 (br), 2930, 1608, 1502, 1466,
1429, 1324, 1275, 1225, 1172, 1129, 1068, 1017, 853, 786, 751,
727, 650, 598, 471 cmꢀ1; MS (ESI): m/z 637 [M+1]+; HRMS (ESI
m/z) for C33H32N4O6F3 calcd 637.2273, found 637.2283 [M+1]+.
4.2.30. 7-Methoxy-8-{5-[5-(5-(4-trifluoromethylphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]pentyloxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepine-5-
one (4f)
Compound 4f was prepared according to the method described
for compound 4a, employing compound 11f (804 mg, 1.0 mmol).
The crude product was purified by column chromatography
(MeOH/CHCl3, 3%) to afford compound 4i as
a
white solid
(390 mg, 58% yield). Mp: 79ꢀ80 °C; ½a D27
ꢃ
¼ þ152:5 (c = 1 in CHCl3);
1H NMR (400 MHz, CDCl3): d 1.55–1.78 (m, 4H, 2 ꢂ ꢀCH2ꢀ), 1.87–
2.12 (m, 6H, 3 ꢂ ꢀCH2ꢀ), 3.55–3.86 (m, 3H, ꢀNCH2ꢀ, ꢀNCHꢀ),
3.93 (s, 6H, 2 ꢂ ꢀOCH3), 3.98 (s, 9H, 3 ꢂ ꢀOCH3), 4.07–4.18 (m,
4H, 2 ꢂ ꢀOCH2ꢀ), 6.80 (s, 1H, ArH), 6.98 (d, 2H, J = 9.1 Hz, ArH),
7.35 (s, 2H, ArH), 7.51 (s, 1H, ArH), 7.65 (d, 1H, J = 2.4 Hz, ArH),
7.67 (d, 1H, J = 4.1 Hz, imineꢀH); 13C NMR (75 MHz, CDCl3): d
164.1, 163.7, 161.9, 153.2, 151.1, 150.3, 149.2, 145.1, 141.8,
126.9, 119.9, 118.6, 115.8, 111.8, 111.1, 109.9, 109.5, 103.7, 68.2,
60.5, 56.0, 55.7, 53.2, 46.2, 29.1, 28.1, 23.7, 22.0 ppm; IR (KBr)
(MeOH/CHCl3, 3%) to afford compound 4f as
a
white solid
(384 mg, 59% yield). Mp: 100ꢀ102 °C; ½a D27
ꢃ
¼ þ162:5 (c = 1 in
CHCl3); 1H NMR (400 MHz, CDCl3): d 1.62–1.70 (m, 4H, 2 ꢂ
–CH2–), 2.01–2.12 (m, 6H, 3 ꢂ –CH2–), 3.79–3.95 (m, 3H, –NCH2–
, –NCH–), 3.93 (s, 3H, ꢀOCH3), 3.96 (s, 3H, ꢀOCH3), 4.15–4.22 (m,
4H, 2 ꢂ –OCH2–), 6.87 (s, 1H, ArH), 7.04 (d, 1H, J = 8.5 Hz, ArH),
7.14 (d, 2H, J = 8.0 Hz, ArH), 7.24 (s, 1H, ArH), 7.26 (s, 1H, ArH),
7.69 (s, 1H, ArH), 7.41 (d, 1H, J = 3.9 Hz, imineꢀH), 8.16 (d, 2H,
(Umax/cmꢀ1):
m 3415 (br), 2937, 2677, 1602, 1497, 1463,
1421,1337, 1245, 1178, 1127, 1006, 868, 767, 737, 616 cmꢀ1; MS
(ESI): m/z 673 [M+1]+; HRMS (ESI m/z) for C34H41N4O9 calcd
673.2873, found 673.2861 [M+1]+.
J = 7.7 Hz, ArH); IR (KBr) (Umax/cmꢀ1):
m 3414 (br), 2928, 2875,
1691, 1603, 1507, 1460, 1431, 1380, 1244, 1176, 1090, 1055,
1023, 870, 833, 756 cmꢀ1; MS (ESI): m/z 651[M+1]+; HRMS (ESI
m/z) for C34H34N4O6F3 calcd 651.2430, found 651.2411 [M+1]+.
4.2.34. 7-Methoxy-8-{3-[5-(5-(3,4-dimethoxyphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]propyloxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzo- diazepine-5-
one (4j)
4.2.31. 7-Methoxy-8-{3-[5-(5-(3,4,5-trimethoxyphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]propyloxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepine-5-
one (4g)
Compound 4g was prepared according to the method described
for compound 4a, employing compound 11g (798 mg, 1.0 mmol).
Compound 4j was prepared according to the method described
for compound 4a, employing compound 11j (768 mg, 1.0 mmol).
The crude product was purified by column chromatography
(MeOH/CHCl3, 3%) to afford compound 4j as
a white solid
(351 mg, 57% yield). Mp: 74ꢀ76 °C; ½a D27
¼ þ135:5 (c = 1 in CHCl3);
ꢃ
1H NMR (400 MHz, CDCl3): d 1.57–1.81 (m, 4H, 2 ꢂ –CH2–), 1.99–

6676
A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 6666–6677
2.18 (m, 2H, –CH2–), 3.74–3.85 (m, 3H, –NCH2–, –NCH–), 3.96 (s,
6H, 2 ꢂ –OCH3), 4.00 (s, 3H, 2 ꢂ –OCH3), 4.24–4.37 (m, 4H, 2 ꢂ
–OCH2–), 6.87 (s, 1H, ArH), 6.99 (d, 2H, J = 8.8 Hz, ArH), 7.02 (d,
2H, J = 8.0 Hz, ArH), 7.52 (s, 1H, ArH), 7.65 (d, 1H, J = 4.0 Hz,
4.4. Cell culture
A375 (Human melanoma cells) was obtained from ATCC, USA.
A375 cells were maintained in Dulbecco’s modified Eagle’s med-
ium (DMEM) (Invitrogen), supplemented with 10% fetal calf serum
and 100 U/mL penicillin and 100 mg/mL streptomycin sulfate (Sig-
ma). The cells were passaged and maintained at 37 °C in a humid-
ified atmosphere containing 5% CO2. TK6 cells were cultured in
imine–H), 7.68 (d, 2H, J = 8.8 Hz, ArH); IR (KBr) (Umax/cmꢀ1):
m
3424 (br), 2926, 1609, 1500, 1462, 1330, 1273, 1143, 1025, 868,
739, 603 cmꢀ1; MS (ESI): m/z 615 [M+1]+; HRMS (ESI m/z) for
C
33H35N4O8 calcd 615.2454, found 615.2432 [M+1]+.
RPMI 1640 medium supplemented with L-glutamine (2 mM) 10%
4.2.35. 7-Methoxy-8-{4-[5-(5-(3,4-dimethoxyphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]butyloxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-
one (4k)
fetal bovine serum (FBS). Cell lines were incubated at 37 °C in a
humidified atmosphere containing 5% CO2.
4.5. Cell cycle analysis
Compound 4k was prepared according to the method described
for compound 4a, employing compound 11k (782 mg, 1.0 mmol).
The crude product was purified by column chromatography
(MeOH/CHCl3, 3%) to afford compound 4k as a white solid
5 ꢂ 105 A375 cells were seeded in 60 mm dish and were al-
lowed to grow for 24 h. Compounds 4a, 4d, 4i, 4l and DC-81(1)
at 4 lM 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 °C for 30 min, washed with PBS and incubated with 1 mg/mL
RNaseA solution (Sigma) at 37 °C for 30 min. Cells were collected
by centrifugation at 2000 rpm for 5 min and further stained with
(352 mg, 56% yield). Mp: 76–78 °C; ½a D27
ꢃ
¼ þ139:5 (c = 1 in CHCl3);
1H NMR (400 MHz, CDCl3): d 1.59–1.77 (m, 4H, 2 ꢂ –CH2–), 2.03–
2.17 (m, 4H, 2 ꢂ –CH2–), 3.56–3.63 (m, 1H, –NCH–), 3.69–3.88
(m, 2H, –NCH2–), 3.93 (s, 3H, –OCH3), 3.97 (s, 6H, 2 ꢂ –OCH3),
4.00 (s, 3H, –OCH3), 4.15–4.26 (m, 4H, 2 ꢂ –OCH2–), 6.83 (s, 1H,
ArH), 6.99 (d, 3H, J = 8.3 Hz, ArH), 7.51 (s, 2H, ArH), 7.65 (d, 1H,
250 lL of DNA staining solution [10 mg of propidium iodide (PI),
J = 4.0 Hz, imine-H), 7.69 (d, 2H, J = 8.4 Hz, ArH); IR (KBr) (Umax
/
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 mea-
sured by flow cytometer (DAKO CYTOMATION, Beckman Coulter,
Brea, CA). Histograms were analyzed using Summit Software.
cmꢀ1):
m 3416 (br), 2935, 1605, 1500, 1464, 1336, 1273, 1178,
1140, 1104, 1023, 867, 814, 739, 656 cmꢀ1; MS (ESI): m/z 629
[M+1]+; HRMS (ESI m/z) for C34H37N4O8 calcd 629.2611, found
629.2568 [M+1]+.
4.6. Caspase-3 assay
4.2.36. 7-Methoxy-8-{5-[5-(5-(3,4-dimethoxyphenyl)-1,3,4-
oxadiazol-2-yl)-2-methoxyphenyl)oxy]pentyloxy}-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-
one (4l)
Compound 4l was prepared according to the method described
for compound 4a, employing compound 11l (795 mg, 1.0 mmol).
The crude product was purified by column chromatography
We have used Apo alert caspase-3 fluorescent assay kit (Clone-
tech, CA) according to manufacturer’s recommendations. A375
cells were treated with compounds 4a, 4d, 4i, 4l and DC-81(1) at
4 lM concentrations as obtained from FACS analysis. Here the sub-
strate and inhibitor (I) used are DEVD-AFC and DEVD-CHO, respec-
tively. The DEVD-AFC substrate, DEVD-AFC+ DEVD-CHO is added
to the cell lysate and incubation was carried out at 37 °C for 1 h.
Readings were taken at excitation wavelength 400 nm and emis-
sion wavelength 505 nm.
(MeOH/CHCl3, 3%) to afford compound 4l as
a
white solid
(386 mg, 60% yield). Mp: 78-80 °C; ½a D27
ꢃ
¼ þ142:5 (c = 1 in CHCl3);
1H NMR (400 MHz, CDCl3): d 1.65–1.76 (m, 4H, 2 ꢂ –CH2–), 1.94–
2.10 (m, 6H, 3 ꢂ –CH2–), 3.55–3.63 (m, 1H, –NCH–), 3.70–3.86 (m,
2H, – NCH2–), 3.94 (s, 3H, –OCH3), 3.97 (s, 3H, –OCH3), 3.98 (s, 3H,
–OCH3), 4.00 (s, 3H, –OCH3), 4.10–4.16 (m, 4H, 2 ꢂ –CH2–), 6.80 (s,
1H, ArH), 6.96–7.01 (m, 3H, ArH), 7.52 (s, 1H, ArH), 7.65 (d, 1H,
J = 3.6 Hz, imine–H), 7.67 (d, 2H, J = 4.6 Hz, ArH), 7.70 (d, 1H,
J = 1.8 Hz, ArH); 13C NMR (75 MHz, CDCl3): d 164.5, 164.1, 162.3,
153.7, 153.0, 151.8, 151.4, 149.2, 147.6, 146.2, 145.2, 140.4, 138.3,
120.2, 116.5, 116.3, 112.2, 111.4, 110.9, 110.3, 109.7, 109.3, 68.6,
4.7. Protein extraction and Western blot analysis
Total cell lysates were isolated from cultured A375 cells after
compound treatments as mentioned earlier were obtained by lys-
ing the cells in ice-cold RIPA buffer (1ꢂ PBS, 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
varioscan instrument (Thermo-Fischer Scientifics). Thirty micro-
grams of protein per lane was applied in 12% SDS–polyacrylamide
gel. After electrophoresis, the protein was transferred to polyvinyl-
idene difluoride (PVDF) membrane (Amersham Biosciences). The
membrane was blocked at room temperature for 2 h in
TBS + 0.1% Tween20 (TBST) containing 5% blocking powder (Santa
Cruz). The membrane was washed with TBST for 5 min, and pri-
mary antibody was added and incubated at 4 °C overnight (O/N).
Rabbit polyclonal b-actin, mouse monoclonal cytochrome c and
cleaved PARP were obtained from Imgenex. Rabbit polyclonal Bax
(p-19), TNFR1 (H271), Cdk2 (M2) and mouse monoclonal p53
(pab1801) were from Santa Cruz, p21 antibody was obtained from
upstate and Chk2 antibody was purchased from Imgenex. After
three TBST washes, the membrane was incubated with corre-
sponding 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
56.1, 55.9, 53.6, 46.5, 29.5, 28.6, 24.1, 22.4 ppm; IR (KBr) (Umax
/
cmꢀ1):
m 3418 (br), 2936, 1690, 1598, 1511, 1461, 1427, 1369,
1337, 1259, 1126, 1014, 895, 820, 757, 636 cmꢀ1; MS (ESI): m/z
643 [M+1]+; HRMS (ESI m/z) for C34H39N4O8 calcd 643.2767, found
643.2760 [M+1]+.
4.3. In vitro evaluation of cytotoxicity
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 and TK6 cells were seeded in a
96-well plate (TPP) at a cell density of 10,000 cells/well. After over-
night incubation, the cells were treated with compounds 4a, 4d, 4i,
4l and DC-81 (1) at 4
medium was then discarded and replaced with fresh 100
followed by addition of 10 L of WST-1 dye. Plates were incubated
l
M concentration and incubated for 24 h. The
l
L media
l
at 37 °C for 30 min. Optical density (OD) was read at 420 nm using
Multimode Varioskan FLASH (Thermo-Fischer Scientifics).

A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 6666–6677
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Compounds 4a–l subjected to DNA thermal melting (denatur-
ation) studies using duplex form calf thymus DNA (CT-DNA) using
modification reported procedure.48 Working solutions were pro-
duced by appropriate dilution in aqueous buffer (10 mM NaH2-
PO4/NaH2PO4, 1 mM Na2EDTA, pH 7.00+0.01) containing CT-DNA,
(100 lM in phosphate) and the PBD (20 lM) were prepared by
addition of concentrated PBD solutions in methanol to obtain a
fixed [PBD]/[DNA] molar ratio of 1:5 The DNA–PBD solutions were
incubated at 37 °C for 0 h prior to analysis sample were monitored
a 260 nm using a Beckman DU-7400 spectrophotometer fitted with
high performance temperature controller. Heating was applied at a
rate of 1 °C minꢀ1 in the 40–90 °C range. DNA helixcoil transition
temperatures (Tm) were determined from the maxima in the
d(A260)/dT derivative plots. Results for each compound are shown
as mean standard derivation from the least three determinations
and are corrected for the effects of methanol cosolvent using a lin-
ear correction term. Ligand-induced alteration in DNA melting
behavior are given by
D
Tm = Tm (DNA + PBD) ꢀ Tm (DNA alone),
where the Tm value for the PBD free CT-DNA is 68.5 0.001 the
fixed [PBD]/[DNA] ratio used did not result in binding saturation
of the host DNA duplex for any compound examined.
Acknowledgments
The authors D.D.G., E.V.B., J.S.N.R. are thankful to CSIR, New Del-
hi, for the award of research fellowships. We are also thankful to
the Department of Biotechnology (BT/PR/7037/Med/14/933/
2006), New Delhi; for financial assistance.
37. Sampath, D.; Plunkett, W. Curr. Opin. Oncol. 2001, 13, 484.
38. Kaelin, W. G. Mol. Cell. 2005, 18, 623.
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