Synthesis, DNA-binding ability and anticancer activity of benzothiazole/benzoxazole-pyrrolo[2,1-c][1,4]benzodiazepine conjugates

Article abstract of DOI:10.1016/j.bmc.2010.05.007

A series of benzothiazole and benzoxazole linked pyrrolobenzodiazepine conjugates attached through different alkane or alkylamide spacers was prepared. Their anticancer activity, DNA thermal denaturation studies, restriction endonuclease digestion assay and flow cytometric analysis in human melanoma cell line (A375) were investigated. One of the compounds of the series 17d showed significant anticancer activity with promising DNA-binding ability and apoptosis caused G0/G1 phase arrest at sub-micromolar concentrations. To ascertain the binding mode and understand the structural requirement of DNA binding interaction, molecular docking studies using gold program and more rigorous 2 ns molecular dynamic simulations using Molecular Mechanics-Poisson-Boltzman Surface Area (MM-PBSA) approach including the explicit solvent were carried out. Further, the compound 17d was evaluated for in vivo efficacy studies in human colon cancer HT29 xenograft mice.

Full text of DOI:10.1016/j.bmc.2010.05.007

Bioorganic & Medicinal Chemistry 18 (2010) 4747–4761
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, DNA-binding ability and anticancer activity of
benzothiazole/benzoxazole–pyrrolo[2,1-c][1,4]benzodiazepine conjugates
a
a
a
Ahmed Kamal a, , K. Srinivasa Reddy , M. Naseer A. Khan , Rajesh V. C. R. N. C. Shetti ,
M. Janaki Ramaiah a, S. N. C. V. L. Pushpavalli a, Chatla Srinivas a, Manika Pal-Bhadra a, Mukesh Chourasia b,
G. Narahari Sastry b, Aarti Juvekar c, Surekha Zingde c, Madan Barkume c
*
a Chemical Biology Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 607, India
b Molecular Modeling Group, Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 607, India
c Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Navi Mumbai 410 208, 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 benzothiazole and benzoxazole linked pyrrolobenzodiazepine conjugates attached through
different alkane or alkylamide spacers was prepared. Their anticancer activity, DNA thermal denaturation
studies, restriction endonuclease digestion assay and flow cytometric analysis in human melanoma cell
line (A375) were investigated. One of the compounds of the series 17d showed significant anticancer
activity with promising DNA-binding ability and apoptosis caused G0/G1 phase arrest at sub-micromolar
concentrations. To ascertain the binding mode and understand the structural requirement of DNA bind-
ing interaction, molecular docking studies using GOLD program and more rigorous 2 ns molecular dynamic
simulations using Molecular Mechanics-Poisson–Boltzman Surface Area (MM-PBSA) approach including
the explicit solvent were carried out. Further, the compound 17d was evaluated for in vivo efficacy stud-
ies in human colon cancer HT29 xenograft mice.
Received 23 March 2010
Revised 1 May 2010
Accepted 4 May 2010
Available online 7 May 2010
Keywords:
Pyrrolobenzodiazepine
Benzothiazole
Benzoxazole, DNA-binding affinity
Anticancer activity
Molecular modelling
In vivo efficacy
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
ing ability with promising anticancer activity. One of the
analogues, SJG-136 is under clinical evaluation.5
DNA has been considered for a long time as a favoured target for
cancer chemotherapeutic agents. On the other hand, achievement
of the desired sequence selectivity with DNA-binding agents is
considered to be one of the most difficult tasks. Meanwhile, for
the past few years certain molecules are being developed and
one such class are naturally occurring pyrrolo[2,1-c][1,4]benzodi-
azepines. These tricyclic molecules have been isolated from various
Streptomyces species and typical examples of which include DC-81,
anthramycin, tomaymycin, sibiromycin and neothramycins.1 From
a mechanistic point of view, these molecules exert their biological
activity by covalently binding to NH2 of guanine in the minor
groove of DNA through the imine or imine equivalent functionality
at N10–C11 position of the PBD ring system.2 Moreover, the PBDs
bind to DNA sequence selectively and have potential not only as
antitumour agents but also as gene regulators and probes of DNA
structure.3 The excellent antitumour potential of these molecules
has stimulated the synthesis of a large number of hybrids and di-
mers.4 Some of these analogues have shown significant DNA-bind-
N
S
N
S
NHCOCH3
NH2
B
D
A
C
OCH3
N
HO
H
N
S
OCH3
N
H3CO
F
O
N
N
Y
O
n
H
X
S
R
N
H3CO
X = NH, O
Y = CH2, CO
R = H, F; n = 3, 4
O
17a-d, 18a
* Corresponding author. Tel.: +91 40 27193157; fax: +91 40 27193189.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
Figure 1. Chemical structures of anticancer active molecules.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.007

4748
A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 4747–4761
Benzothiazoles demonstrated interesting pharmacological
(Scheme 3). The synthesis of the desired benzothiazole/benzoxaz-
ole–pyrrolo[2,1-c][1,4] benzodiazepine hybrids (17a–f, 18a,b) was
carried out by the synthetic sequence illustrated in Schemes 4
and 5. The synthesis of the starting material 2-nitrobenzoic acid
(10) was prepared by employing the commercially available vanil-
lin. Oxidation of vanillin, nitration, followed by benzylation as re-
ported in the literature15 provided 2-nitrobenzoic acid (10). This
activities and in the past two decades they have been extensively
studied for their anticancer activity.6 2-(4-Aminophenyl)benzo-
thiazoles7 and their corresponding N-acetylated derivatives
(Fig. 1A and B)8 have showed surprisingly remarkable anticancer
activity against certain cancer cell lines particularly against breast,
colon and ovarian cell lines in in vitro anticancer screening pro-
gram of the National Cancer Institute (NCI). The anticancer activity
of these molecules is assumed to be due to the formation of reac-
tive intermediates that can bind covalently to DNA.9 Some other
related class of benzothiazoles have also shown good antitumour
activity.10 Surprisingly, among an extended library of very close
structural analogues, only a compound possessing a 2-(3,4-dime-
thoxyphenyl) group and a fluoro substituent in the benzothiazole
ring, especially at the 5-position exhibited potent anticancer activ-
ity (Fig. 1C). However, compounds with no substituent in the ben-
zothiazole moiety retained sub-micromolar activity against certain
cancer cell lines. The definitive molecular target responsible for the
antitumour activity of this series has not been identified and mech-
anistically, this new series of agents contrasts with the 2-(4-ami-
nophenyl)benzothiazoles. Similarly, isosteric benzoxazoles have
also demonstrated good anticancer activity.11
was further coupled with L-proline methyl ester hydrochloride to
afford the nitro ester derivative (11). The ester group of this com-
pound was then reduced with DIBAL-H to produce the correspond-
ing aldehyde derivative 12, which upon protection with EtSH/
TMSCl yielded 13. Compound 13 upon debenzylation afforded
(2S)-N-(4-hydroxy-5-methoxy-2-nitrobenzoyl) pyrrolidine-2-car-
boxaldehyde diethylthioacetal (14), nitrothioacetal intermediates
upon reduction with SnCl2ꢀ2H2O in refluxing methanol gave the
aminothioacetal precursors (15). This upon thiol deprotection by
HgCl2/CaCO3 in CH3CN–H2O at room temperature afforded the
required DC-81 (16). This intermediate was treated with 2-[4-(n-
alkyloxy)-phenyl) benzothiazoles (5a–d) or 2-[4-(n-alkyloxy)-phe-
nyl) benzoxazoles (5e,f) or bromoalkoxyamido benzothiazoles
(5e,f) by employing KI/K2CO3 in acetone at room temperature to af-
ford the required PBD conjugates 17a–f and 18a,b (Schemes 4 and
5). However, when bromobutyrylamide of benzothiazole (9c) was
coupled with the PBD precursor (16) the expected product 18c
was not obtained and instead benzothaizole derivative was cyclized
to the corresponding pyrrolidinone derivative 19 (Scheme 6).16
In continuation of our previous studies12 on the structural mod-
ifications of pyrrolo[2,1-c][1,4]benzodiazepines and encouraging
antitumour activity of benzothiazole as well as isosteric benzoxaz-
oles we herein wish to report the synthesis, DNA-binding ability,
molecular modelling, anticancer activity and in vivo efficacy study
of C8-coupled benzothiazole/benzoxazole–PBD conjugates. In order
to identify agents with better potency, systematic structural modi-
fications were made with different alkoxyamide and alkane spacers.
3. Results and discussion
3.1. Thermal denaturation studies
2. Chemistry
The DNA binding activity for these new C8-linked benzoxazole
or benzothiazole–PBD conjugates (17a–f, 18a,b) was examined by
thermal denaturation studies using calf thymus (CT) DNA.17 Melt-
ing studies showed that these compounds stabilize the thermal he-
The synthesis of benzoxazole intermediates 3a,b was carried out
by the condensation of 2-aminophenol with 4-benzyloxybenzalde-
hyde precursors to provide 1a,b. Compounds 1a,b were converted
to 2a,b by oxidative cyclization, which were then subjected to deb-
enzylation to afford 3a,b (Scheme 1). The other key intermediates
4a,b were obtained by the condensation of 2-aminothiophenol with
substituted benzaldehydes as previously reported.10,13 The benzo-
thiazole/benzoxazole bromoalkyl spacers were prepared by treat-
ing hydroxyl benzoxazoles/benzothiazole (3a,b and 4a,b) with
dibromo alkanes by potassium carbonate in acetone reflux affords
the desired bromoalkoxy benzothiazole/benzoxazoles (5a–f,
Scheme 2). The bromoamide derivatives of benzothiazoles (9a–c)
were prepared according to Scheme 3. The synthesis of precursors
9a–c was carried out by Jacobson’s cyclization approach. The treat-
ment of 4-fluoroaniline/aniline with 4-nitrobenzoyl chloride in pyr-
idine gave 6a,b. These compounds were further converted to its
corresponding thio-derivative 7a,b using Lawesson’s reagent by
refluxing in toluene. Finally, the compounds 7a,b were cyclized
by potassium ferricyanide according to Jacobson’s method14,7b
and were reduced to their amino precursors (8a,b). These were cou-
pled to the bromopentanoyl chloride resulting to produce 9a–c
lix?coil or melting stabilization (DTm) for the CT-DNA duplex at pH
7.0, incubated at 37 °C, where PBD/DNA molar ratio is 1:5. The data
for the compounds 17a–f, 18a,b is included in Table 1 along with
naturally occurring DC-81. It is observed that some of the prepared
compounds have shown good DNA binding property compared to
naturally occurring PBD-like DC-81. It is also observed that benzo-
thiazole–PBD conjugates with five-carbon spacer (17c,d) exhibited
better DNA-binding affinity than their corresponding four-carbon
spacer benzothiazole–PBD conjugates (17a,b). The amide conju-
gates (18a,b) show less binding affinity than their counter part
ether derivatives (17c,d). The substitution of fluorine atom on ben-
zothiazole moiety of 18a elevates the DNA melting temperature
considerably. Another observation is that the replacement of sulfur
atom with oxygen does not alter the binding ability.
3.2. Restriction endonuclease digestion assay
The restriction endonuclease inhibition studies (RED assay)
were carried out to further confirm the binding ability of these
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7 8
DC-81
17b
17d
Figure 2. Restriction endonuclease digestion assay (RED100-assay) for A-C8-linked PBD with CT-DNA inhibitory activity of DC-81, 17b and 17d on the cleavage of plasmid
pBR322 by restriction endonuclease BamH1 (20 units in 2 L) for 1 h at 37 °C. The products were separated by agarose gel electrophoresis and visualized by ethidium bromide
staining under UV illumination. Lane 1: control pBR322; lane 2: complete digest of pBR322 by BamH1; lanes 3–8: increasing concentration of DC-81, 17b and 17d ranging
from 5, 10, 15, 20, 25 and 30 M.
l
l

A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 4747–4761
4749
CHO
R
N
OH
OH
i
OCH2Ph
+
R
NH2
1a,b
OCH2Ph
ii
R
R
N
N
O
iii
OH
OCH2Ph
O
3a,b
2a,b
a
: R = H
b: R = OCH3
Scheme 1. Reagents and conditions: (i) ethanol, reflux, 1 h; (ii) Pb(OAc)4, CHCl3, reflux, 1 h, (iii) Pd/C, H2, ethyl acetate, rt, 30 min.
R
R
N
Z
N
Z
( )
i
Br
OH
O
n
5a-f
5
3a: Z = O, R = H
3b: Z = O, R = OCH3
4a: Z = S, R = H
a: n = 4, Z = S, R = H
b: n = 4, Z = S, R = OCH3
c: n = 5, Z = S, R = H
4b: Z = S, R = OCH3
d: n = 5, Z = S, R = OCH3
e: n = 5, Z = O, R = H
f: n = 5, Z = O, R = OCH3
Scheme 2. Reagents and conditions: (i) dibromoalkanes, K2CO3, acetone, 24 h, reflux.
NO2
NO2
NH2
COCl
H
H
N
N
ii
i
+
O
S
7a,b
X
X
6a,b
iii, iv
X
NO2
N
S
N
v
9a; X = F, n = 4
9b; X = H, n = 4
9c; X = H, n = 3
NH
NH2
( )
n
S
X
X
Br
O
9a-c
8a,b
Scheme 3. Reagents and conditions: (i) pyridine, reflux, 1 h; (ii) Lawesson’s reagent, HMPA, 100 °C, 5 h; (iii) K3Fe(CN)6, aq NaOH, 90 °C, (iv) SnCl2ꢀ2H2O, methanol, reflux, 5 h;
(v) n-alkylbromoacid chloride, Et3N, THF, 0 °C to rt, 30 min.
PBD conjugates to DNA particularly at G-rich sites. The experimen-
tal protocol described in the previous studies has been employed.18
The dose dependent inhibition (0–30 lM) of the compounds with
DNA cleavage activity of BamH1 was studied using pBR 322 vector
DNA. The results of this experiment clearly showed that 17d is the
most effective DNA binding compound among all the compounds
tested in this series. The order of binding to DNA is 17d > 17b >
DC-81 and also dose dependent inhibition of BamH1 was observed
with these PBD conjugates.
About eight different DNA sequences were examined to investigate
the most suitable DNA sequence for carrying out the docking stud-
ies. Among these, sequence A (50-CGCAGAAATTTCTGCG-30) pro-
vides the best correlation with the experimental results which
may be traced to the possession of optimal AT rich region in
this model strand. Molecular Mechanics-Poisson–Boltzman Sur-
face Area (MM-PBSA) approach of AMBER molecular dynamics
(MD) package was used to investigate the free binding energies
(D
Gbind,solv) of the DNA–ligand complex.20
A quick analysis of the docking results reveals that the binding
3.2.1. Molecular modelling studies
affinities of DNA–ligand complexes have dependence on the linker
length (Table 2 and Fig. 3). Compound 17d has showed the highest
docking score of 73.04. The linker of heterodimers localized in the
span of six AT sites (50-CGCAGAAATTTCTGCG-30) are observed in
each DNA–ligand complex. The final docked conformation of
The variations in the DNA-binding affinities with respect to lin-
ker length and substitution of sulfur, oxygen and methoxy in this
class of molecules were investigated by molecular modelling ap-
proaches employing docking19 and molecular dynamic studies.21

4750
A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 4747–4761
BnO
NO2
H3COOC
BnO
NO2
CHO
COOCH3
BnO
NO2
OH
i
ii
+
N
ClH.HN
N
H3CO
H3CO
H3CO
O
O
O
10
12
11
iii
HO
NO2
HO
NH2
N
CH(SEt)2
BnO
NO2
CH(SEt)2
CH(SEt)2
v
iv
N
N
H3CO
H3CO
H3CO
O
O
O
14
13
15
vi
R
N
O
O
n
H3CO
N
HO
H
H
N
vii
N
N
H3CO
Z
O
O
17a-f
16
17
a: n = 4, R = H, Z = S
b: n = 4, R = OCH3, Z = S
c: n = 5, R = H, Z = S
d: n = 5, R = OCH3, Z = S
e: n = 5, R = H, Z = O
f: n = 5, R = OCH3, Z = O
Scheme 4. Reagents and conditions: (i) (1) SOCl2, benzene, rt; (2) THF, Et3N; (ii) DIBAL-H, DCM, ꢁ70 °C; (iii) TMSCl, EtSH, DCM; (iv) EtSH, BF3–OEt2, CH2Cl2, 12 h, rt; (v)
SnCl2ꢀ2H2O, MeOH, 2 h, reflux; (vi) HgCl2–CaCO3, CH3CN–H2O (4:1); (vii) compounds 5a–f, KI, K2CO3, acetone, 24 h.
DNA (50-CGCAGAAATTTCTGCG-30)–ligand complex from the GOLD
docking has been opted for the input preparation of molecular dy-
namic simulations.
N
S
N
HO
H
NH
O
+
( )
4Br
X
N
H3CO
9b, c
3.2.2. Molecular dynamic simulations
O
Further, more rigorous molecular dynamic simulations were
employed to quantitatively understand the dynamical nature of
the complex, and the interaction energy as well as assessing the ef-
fect of temperature and solvent. The effect of water as solvent was
taken into account through the GBSA implicit solvent model.21,22
The molecular dynamic results show that the minor groove of
the DNA structure is preferred over the major groove and linker re-
gion of the heterodimers preferred to bind in the sequence com-
posed mainly of adenine (A) and thymine (T) bases. Ligand
binding to the DNA shows that the linker stretched over the AT re-
gion instead of making bend conformation.
16
i
X
S
N
O
H
O
4
N
( )
N
N
H
H3CO
18a: X = H; 18b: X = F
O
Scheme 5. Reagents and condition: (i) KI, K2CO3, acetone, 24 h.
Scheme 6. Reagents and condition: (i) DC-81, KI, K2CO3, acetone, 24 h.

A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 4747–4761
4751
Table 1
3.2.3. MM-PBSA calculations
Thermal denaturation data for benzothiazole, benzoxazole–PBD hybrids (17a–f,
18a,b) with calf thymus (CT) DNA
The MM-PBSA approach has emerged as a useful approach to
calculate binding affinities of biomolecular complexes for distin-
guishing between good and weak binders, but rarely to reproduce
smaller free energy differences.23 Binding of the ligands in the min-
or groove does not have a large impact on the conformation of the
DNA, as revealed by the qualitative (trajectory) and quantitative
agreement with RMSD of the complex. In the last 500 ps (confor-
mations in the trajectory 500–550) of 2 ns production run (confor-
mations in the Fig. 3, 350–550) the RMSD fluctuates from the
equilibrium and steady at certain level. The fluctuation in RMSD
has been observed when DNA strands relaxed from the ends of
the ligand binding.
a
PBD hybrids
[PBD]/[DNA] molar ratiob
D
Tm (°C) after incubation at
37 °C for
0 h
18 h
17a
17b
17c
17d
17e
17f
18a
18b
DC-81
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
1:5
0.5
0.5
4.1
6.2
4.1
4.1
0.5
2.1
0.3
0.5
0.5
4.3
6.3
4.3
4.2
0.5
2.3
0.7
In the bound structure, the molecules form interactions with
the AT rich sequence of the DNA duplex, locally displace the water
molecules of the spine of hydration (Fig. 4B). The different energy
terms of DNA, 17d and complex is given in Table 3. The total bind-
a
For CT-DNA alone at pH 7.00 0.01, Tm = 69.1 °C 0.01 (mean value from 10
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
D
b
lM
l
ing free energy (DGbind,solv) shows that the DNA–17d complex
sodium phosphate + 1 mM EDTA, pH 7.00 0.01).
(ꢁ39.92 kcal/mol) is slightly more stable than other compounds
(Table 4). The flanking pentameric ring of these compounds is
found to be parallel with the ribose sugar of nucleotide which ap-
Table 2
GOLD docking fitness scores of the DNA (A)–ligand interaction
pears to make a
p-stacking type of arrangement. The comparison
Mol Exp.
rank
Fitness Fitness vdw_ext vdw_ext vdw_int vdw_int
rank
Rank
Rank
between GOLD score, the binding free energy in gas and solvent
phase are consistent with the experimental observations (Fig. 4).
The double bonded nitrogen of the seven-membered ring of
compound 17d interacts (ꢂ3 Å) with the N3 nitrogen of A7 and
A8 nucleotide. The nitrogen of sulfur containing pentameric ring
is found to be close with the A23 and A24 in the trajectory analysis.
The methoxy groups of dimer always protrude out from the
DNA axis. In this series of compounds, the benzothiazole shows
17a
17b
17c
17d
17e
17f
5
3
4
1
68.10
69.47
68.52
73.04
67.68
68.44
71.05
70.97
7
4
5
1
8
6
2
3
54.62
58.22
57.28
61.54
56.82
58.41
58.24
57.98
8
4
6
1
7
2
3
5
ꢁ7
8
3
5
2
4
1
6
7
ꢁ10.58
ꢁ10.24
ꢁ11.58
ꢁ10.46
ꢁ11.87
ꢁ9.02
ꢁ8.76
18a
18b
2
Figure 3. DNA–17d complex: the density, temperature, total energy and RMSD plots have all clearly converged by the end of equilibration and production period.

4752
A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 4747–4761
significantly higher binding strengths than the benzoxazole, with
the DNA. It is interesting to note that the Gbind,solv decreases as
(SRB) protein assay was used to estimate cell viability or growth
(Table 5).24 Specifically, compound 17d has exhibited excellent
cytotoxicity against all tested leukaemia cell lines and also against
Hop-62, Hop-92, NCI-H23, NCI-H460, NCI-H522 (non-small cell
lung cancer), COLO 205, HT-116, HT29, SW-620 (colon cancer),
SF-295, SF-539, U251 (CNS cancer), LOX IMVI, M14, SK-MEL-5,
UACC-62 (melanoma cancer), prostate, some of the renal, breast
and ovarian cell lines in the range of 15–25 nM. While the other
compounds (17a–c) have also shown significant anticancer activity
in the sub-micromolar range against all the cell lines tested. Inter-
estingly, the amide derivative 18a has shown comparatively less
activity than the corresponding ether derivatives. It is noticed that
the acetylated benzothiazoles7a which are more active towards
breast cancer cell lines have not retained their activity in benzothi-
azole–pyrrolobenzodiazepine conjugates (18a).
D
the linker length increases, indicating that the increase in the al-
kane spacer length from three to five enhances the stability of
the DNA–ligand complex by about 9 kcal/mol (Table 4). As the
covalent binding with the guanine nucleotide of AGA sequence is
important for the stability of DNA–ligand complex, the longer
chain length in the PBD ligands is desirable as this facilitates the
ligand to come closer to guanine. Both strands of DNA come closer
together when the compound is introduced in the minor groove
but rest of the DNA seems to be more relaxed as depicted in Fig-
ure 4B. This partitioning of anionic charge by heterodimer into
the minor groove of AT base pairs intended in the attempt toward
ligand design.
3.3. In vitro anticancer activity
3.4. Cell cycle effects in A375 cells
The C8-linked benzothiazole–PBD conjugates (17a–d, 18a) have
been evaluated against 60 human cancer cell lines derived from
nine cancer cell types (leukaemia, non-small cell lung cancer, colon
cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, pros-
tate cancer and breast cancer). For each compound, dose–response
curves for individual cell lines have been measured at a minimum
of five concentrations at 10-fold dilutions. A protocol of 48 h con-
tinuous drug exposure has been used, and a sulforhodamine B
The anticancer activity data has revealed that these compounds
17b, 17d and 18a caused pronounced and efficient cytotoxicity
against 60 cancer cell lines tested, among the above, compound
17d was found to be highly effective in causing cytotoxicity. These
results prompted us to investigate the effect of these compounds
on cell cycle progression and apoptosis in A375 human melanoma
cancer cell line by using DC-81 as positive control. The optimized
Figure 4. GOLD docking and MD trajectory pose of DNA(A)–ligand (17d) complex: (A) binding of ligand in the minor groove of DNA as observed in GOLD docking. (B) Distortions
of the double helix of DNA on the binding of ligand observed on molecular dynamics simulation of the docked pose revealing the local displacement of water molecules of the
spine of hydration and the effect of binding of inhibitor on DNA conformation showing the uncoiling of the DNA helix from the sites flanking the bound inhibitor, in contrast
to the shrinking tendency exhibited by the region of DNA strands bound by the inhibitor may be due to non-covalent interaction.

A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 4747–4761
4753
Table 3
Calculated interaction energy and solvation free energy for complex (DNA + 17d) and average the results to obtain an estimate of the final binding free energy (
D
Gbind,solv)
contributions of all the snapshots from the complex trajectory using MM-PBSA
Component
Complex
Receptor
Ligand
DGbind,solv
Mean
2444.83
ꢁ346.37
1598.82
3697.28
44.57
ꢁ9606.59
ꢁ9562.02
ꢁ7161.76
ꢁ5864.75
44.57
ꢁ9833.25
ꢁ9788.68
ꢁ7388.42
ꢁ6091.4
STD
Mean
2471.02
ꢁ291.07
1506.53
3686.48
45.57
ꢁ9633.17
ꢁ9587.61
ꢁ7162.15
ꢁ5901.13
45.57
ꢁ9868.87
ꢁ9823.3
ꢁ7397.84
ꢁ6136.82
STD
Mean
STD
Mean
STD
ELE
VDW
INT
GAS
62.87
12.35
23.98
61.91
0.28
57.64
57.73
14.79
23.08
0.28
62.64
11.28
23.32
61.3
ꢁ2.63
18.98
92.29
108.64
6.91
ꢁ28.68
ꢁ21.76
ꢁ31.31
86.87
1.1
ꢁ23.56
ꢁ74.28
0.00
ꢁ97.84
ꢁ7.91
55.26
47.35
31.7
ꢁ50.49
ꢁ7.91
65.82
57.92
42.26
ꢁ39.92
4.39
2.94
0.00
5.65
0.19
4.05
3.98
1.8
2.95
0.19
4.79
4.7
2.19
6.34
6.04
0.05
0.72
0.7
0.86
5.92
0.05
0.79
0.76
0.72
5.91
GBSUR
GBTOT
GBSOL
GBELE
GBTOT
PBSUR
PBCAL
PBSOL
PBELE
PBTOT
0.23
57.56
57.63
14.53
21.95
0.23
6.91
58.02
58.11
14.85
23.24
58.1
ꢁ30.21
ꢁ23.29
ꢁ32.84
85.34
58.19
14.05
21.63
3.87
4.23
All values are in kcal/mol.
ELE: electrostatic energy; VDW: van der Waals energy; INT: internal energy (this term always amounts to zero in the single trajectory approach); GAS: total gas-phase energy
(sum of ELE, VDW and INT); PBSUR/GBSUR: non-polar contribution to the solvation free energy calculated by an empirical model; PBCAL/GB: electrostatic contribution to the
solvation free energy calculated by PB or GB, respectively; PBSOL/GBSOL: sum of non-polar and polar contributions to salvation; PBELE/GBELE: sum of the electrostatic
solvation free energy and MM electrostatic energy; PBTOT/GBTOT: final estimated binding free energy.
Table 4
scid mice. The dose determination studies demonstrated the max-
imum tolerated dose of compound 17d as <50 mg/Kg. Hence, com-
pound 17d was administered at 5 mg/kg and 10 mg/kg,
Calculated free binding energy (kcal/mol) of DNA (A)–ligand interaction using MM-
PBSA method (AMBER)a
Component
Binding free energy
D
Gbind,solv
intraperitoneal (ip), on days 1, 5 and 9 (q4d) (total drug adminis-
tered was 15 and 30 mg/kg, respectively). With these doses no tox-
icity was observed in terms of weight loss or mortality of the
experimental mice (Fig. 6i and ii). The tumour volume, body
weight and signs of overt toxicity following test compound dosing
were monitored and recorded for the duration of the experiment
(35 days). Tumour growth was expressed in terms of relative tu-
mour volume (RTV) (Fig. 6iii) which is the ratio of tumour volume
on a particular day to the tumour volume on day 1 of the study. Tu-
mour growth inhibition index T/C (Fig. 6iv) was calculated as
follows:
17a
17b
17c
17d
18a
ELE
VDW
INT
GAS
GBSUR
GB
GBSOL
GBELE
GBTOT
PBSUR
PBCAL
PBSOL
PBELE
PBTOT
ꢁ12.93
ꢁ68.69
0.00
ꢁ81.62
ꢁ7.20
44.89
37.70
31.97
ꢁ43.92
ꢁ7.20
58.16
50.96
45.24
ꢁ30.65
ꢁ20.25
ꢁ67.45
0.00
ꢁ20.57
ꢁ73.54
0.00
ꢁ23.56
ꢁ74.28
0.00
ꢁ97.84
ꢁ7.91
55.26
47.35
31.70
ꢁ50.49
ꢁ7.91
65.82
57.92
42.26
ꢁ39.92
ꢁ28.81
ꢁ67.92
0.00
ꢁ96.73
ꢁ7.26
58.35
51.09
29.53
ꢁ45.64
ꢁ7.26
65.71
58.45
36.90
ꢁ87.7
ꢁ7.24
49.91
42.66
29.66
ꢁ45.04
ꢁ7.24
57.69
50.45
37.44
ꢁ37.26
ꢁ94.11
ꢁ7.76
51.98
44.22
31.41
ꢁ49.89
ꢁ7.76
65.54
57.78
44.97
T/C = Average RTV of test group mice/Average RTV of control
group mice.
ꢁ36.33
ꢁ38.28
The T/C data demonstrated that administration of 17d under
the above conditions did not cause significant inhibition of tumour
weight relative to the vehicle control during the period of experi-
ment (35 days). However, a trend of tumour inhibition was ob-
served with a minimum T/C value of 0.58 was attained on day 21
in the 30 mg/kg treatment group. Probably metabolic instability
or poor bioavailability is responsible for limiting the in vivo effi-
cacy. Current studies are ongoing to understand the metabolism
and distribution of this series and to design newer generation of
compounds with more stability and bioavailability aspects.
a
For the definition of these terms refer to footnote in Table 3.
concentration, at which good apoptotic events occur, was found to
be at 2 M (Table 6.1). Subsequently, these compounds were
tested at 2 M concentration, and the percentage of G0/G1 phase
l
l
cells were found to be 54.55, 77.93, 90.55, 93.79 and 88.44 for con-
trol, DC-81, 17b, 17d and 18a, respectively (Figs. 5.1 and 5.2).
An increase in the percentage of cells in the G0 phase was ob-
served which indicated the cells have undergone programmed cell
death (apoptosis) for all the tested compounds (Fig. 5.3). Com-
pound 17d was found to be the most effective one among the
tested conjugates. Increase of apoptotic cells (G0 cells) with in-
4. Conclusion
In conclusion, the synthesis and DNA-binding affinity of new
C8-linked benzothiazole/benzoxazole–PBD conjugates attached
through varying alkane and alkylamide spacers have been investi-
gated. Among the four PBD conjugates evaluated, conjugate 17d
has shown pronounced in vitro anticancer activity against 60 hu-
man tumour cell lines. Molecular modelling studies suggest that
the sulfur atom in benzothiazole ring system and an increase of
the linker length to a five-carbon chain significantly enhances the
DNA-binding ability of the minor groove binders with the DNA se-
quence A. The MD simulations using MM-PBSA methodologies pro-
vide valuable insights in the ligand DNA binding. Further, the effect
of these compounds on cell cycle progression was studied with
PBD conjugates such as 17b, 17d, 18a and DC-81 against A375 cell
crease of concentration (0–2 lM) of compounds (DC-81 and 17d)
was observed and this increase was found to be synergistic
(Fig. 5.4). The different phases of cell cycle were analyzed and
the results are summarized in Table 6.2.
3.5. In vivo tumour xenograft studies of 17d
The preliminary in vitro anticancer activities, thermal denatur-
ation and FACS studies revealed that compound 17d has shown
significant anticancer activity among the series. These encouraging
results provided an impetus to carry out in vivo efficacy studies
using xenograft model of human colon cancer cells (HT29) in male

4754
A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 4747–4761
Table 5
Table 6.1
Anticancer activity of benzothiazole–pyrrolo[2,1-c][1,4]benzodiazepine conjugates
(17a–d, 18a) in human cancer cell lines
Cell cycle distribution of A375 cell line using 17b, 17d, 18a and DC-81 at 2 lM
concentrations
Panel/cell line
Growth inhibition Log10GI50 (M)
Compound
Concn (lM)
Cell cycle distribution (%)
17a
17b
17c
17d
18a
G0
0.99
20.86
26.75
15.10
10.70
G1
S
G2/M
Leukaemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
Control
17b
17d
18a
DC-81
0
2
2
2
2
53.56
69.69
67.04
73.34
67.23
15.93
5.00
29.52
4.45
3.10
5.41
8.53
ꢁ6.55
ꢁ6.42
ꢁ6.45
ꢁ6.63
ꢁ6.67
ꢁ7.66
ꢁ6.47
ꢁ6.29
ꢁ6.47
ꢁ6.17
ꢁ6.51
ꢁ6.53
ꢁ6.57
ꢁ6.25
ꢁ6.44
ꢁ6.55
ꢁ6.40
ꢁ7.72
ꢁ7.66
ꢁ7.48
ꢁ7.69
ꢁ7.36
ꢁ7.13
ꢁ7.76
ꢁ5.92
ꢁ5.75
ꢁ5.76
ꢁ5.79
ꢁ5.84
ꢁ6.26
3.11
6.15
13.54
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
ꢁ5.58
ꢁ5.90
ꢁ6.39
ꢁ6.75
ꢁ5.84
ꢁ6.13
ꢁ5.66
ꢁ6.39
ꢁ6.49
ꢁ5.57
ꢁ5.76
ꢁ5.85
ꢁ6.58
ꢁ5.62
ꢁ5.83
ꢁ5.59
ꢁ6.19
ꢁ6.26
ꢁ5.59
ꢁ5.81
ꢁ6.39
ꢁ6.57
ꢁ5.82
ꢁ6.05
ꢁ5.71
ꢁ6.46
ꢁ6.26
ꢁ6.62
ꢁ6.69
ꢁ7.34
ꢁ7.35
ꢁ6.67
ꢁ7.21
ꢁ6.52
ꢁ7.40
ꢁ7.38
ꢁ5.47
ꢁ5.61
ꢁ5.61
ꢁ5.76
ꢁ5.54
—
ꢁ5.55
ꢁ5.78
ꢁ6.01
Percentage of cells in each phase
100
90
80
70
60
50
40
30
20
10
0
Control
DC-81
17b
17d
18a
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
ꢁ6.47
ꢁ6.44
ꢁ6.53
ꢁ6.19
ꢁ6.42
ꢁ6.26
ꢁ6.37
ꢁ6.16
ꢁ6.19
ꢁ6.28
ꢁ5.88
ꢁ6.27
ꢁ5.99
ꢁ6.10
ꢁ6.41
ꢁ6.20
ꢁ6.46
ꢁ6.03
ꢁ6.34
ꢁ5.81
ꢁ5.99
ꢁ7.22
ꢁ6.90
ꢁ7.41
ꢁ6.65
ꢁ7.39
ꢁ6.78
ꢁ7.25
ꢁ5.77
ꢁ5.80
ꢁ5.73
ꢁ5.55
ꢁ5.67
ꢁ5.53
ꢁ5.79
G0
G1
S
G2/M
G0/G1
Figure 5.1. Flow cytometric analysis for the cell cycle distribution of A375 cells
after exposure to hybrids 17b, 17d, 18a and DC-81 (2 lM) for 24 h (G0 = apoptotic
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
sub-G1 area; G1 area; S area; G2/M area).
ꢁ6.18
ꢁ6.17
ꢁ6.46
ꢁ5.82
ꢁ5.97
ꢁ6.64
ꢁ5.91
ꢁ5.72
ꢁ6.27
ꢁ5.79
ꢁ5.91
ꢁ6.38
ꢁ5.49
ꢁ6.11
ꢁ6.32
ꢁ5.53
ꢁ5.92
ꢁ6.34
ꢁ6.46
ꢁ7.17
ꢁ7.40
ꢁ6.66
ꢁ6.84
ꢁ7.47
ꢁ5.69
ꢁ6.26
ꢁ5.80
ꢁ5.57
ꢁ5.74
ꢁ5.72
line and 17d was found to be most effective at a low concentration
of 0.5 M which is indicated by the increase of sub-G1 apoptotic
l
cells. The order of effect of these compounds in causing apoptosis
is 17d > 17b > 18a > DC-81. The in vivo efficacy study of compound
17d revealed that the ability to delay the tumour growth and
retention of body weight apart from no other adverse side-effects
in the HT29 human colon cancer xenograft model suggested that
PBD-bezothiazole/benzoxazole conjugates have promising anti-
cancer activity in the treatment of human cancer.
Melanoma
LOX IMVI
MALME 3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
ꢁ6.39
ꢁ5.90
ꢁ6.27
ꢁ6.41
ꢁ5.67
ꢁ6.46
ꢁ5.66
ꢁ6.10
ꢁ6.14
ꢁ5.81
ꢁ5.91
ꢁ5.91
ꢁ5.67
ꢁ6.39
ꢁ5.66
ꢁ5.95
ꢁ6.62
ꢁ5.91
ꢁ6.49
ꢁ5.81
ꢁ5.41
ꢁ6.47
ꢁ5.65
ꢁ5.93
ꢁ7.74
ꢁ6.84
ꢁ7.40
ꢁ6.75
ꢁ6.48
ꢁ7.61
ꢁ6.73
ꢁ7.09
ꢁ5.81
ꢁ5.72
ꢁ5.81
ꢁ5.71
ꢁ5.65
ꢁ5.75
ꢁ5.76
ꢁ5.76
5. Experimental
5.1. General
Ovarian cancer
IGROV1
ꢁ5.62
ꢁ6.42
ꢁ5.94
ꢁ6.48
ꢁ6.12
ꢁ5.91
ꢁ5.51
ꢁ6.14
ꢁ5.79
ꢁ6.30
ꢁ5.75
ꢁ5.62
ꢁ5.57
ꢁ6.13
ꢁ5.67
ꢁ6.18
ꢁ6.09
ꢁ5.59
ꢁ6.46
ꢁ7.01
ꢁ6.54
ꢁ7.15
ꢁ6.85
ꢁ6.75
ꢁ6.16
ꢁ5.73
ꢁ5.55
ꢁ5.45
ꢁ5.81
ꢁ5.35
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
1H NMR spectra were recorded on a Bruker UXNMR/XWIN-NMR
(300 MHz) or Varian VXR-Unity (200 MHz). Chemical shifts have
been expressed in (ppm) down field from TMS. Coupling constants
are reported in hertz (Hz). EI mass spectra were recorded on a VG-
7070H Micromass mass spectrometer at 200 °C, 70 eV, with a trap
Renal cancer
786-0
A498
ꢁ6.67
ꢁ5.69
ꢁ6.44
ꢁ6.45
ꢁ6.72
ꢁ6.20
ꢁ6.32
ꢁ6.18
ꢁ6.05
ꢁ5.79
ꢁ6.11
ꢁ6.24
ꢁ6.62
ꢁ6.02
ꢁ5.82
ꢁ5.91
ꢁ6.58
ꢁ5.65
ꢁ5.96
ꢁ6.29
ꢁ6.70
ꢁ6.09
ꢁ5.46
ꢁ6.14
ꢁ7.56
ꢁ6.76
ꢁ7.27
ꢁ6.93
ꢁ7.29
ꢁ6.70
ꢁ6.48
ꢁ6.70
ꢁ5.81
ꢁ5.73
ꢁ5.52
ꢁ5.66
ꢁ5.79
ꢁ5.55
ꢁ5.49
ꢁ5.78
current of 200
lA and 4 kV of acceleration voltage. FAB mass
ACHN
spectra were recorded on
a
LSIMS-VG-AUTOSPEC Micromass
CAKI-1
RXF 393
SN12C
TK-10
spectrometer. LC mass spectra and ESI mass spectra were recorded
on LC-MSD-Trap-SL spectrometer and Q-STAR-XL Hybrid spec-
trometer, respectively. Elemental analysis was within 0.4% of
the theoretical values. All reactions were monitored by thin-layer
chromatography (TLC) carried out on 0.25 mm E. Merck silica gel
plates (60F-254) with UV light, iodine as probing agents. Column
chromatography was performed using Acme silica gel (100–200
mesh). Yields were not optimized. All solvents and reagents were
used without further purification unless otherwise specified.
UO-31
Prostate cancer
PC-3
DU-145
ꢁ6.53
ꢁ6.57
ꢁ6.39
ꢁ6.52
ꢁ6.63
ꢁ6.45
ꢁ7.38
ꢁ7.47
ꢁ5.69
—
Breast cancer
MCF-7
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
BT-549
T-47D
ꢁ6.49
ꢁ5.94
ꢁ6.57
ꢁ6.30
ꢁ6.35
ꢁ6.74
ꢁ6.66
ꢁ6.30
ꢁ6.39
ꢁ5.72
ꢁ6.28
ꢁ5.99
ꢁ5.87
ꢁ6.55
ꢁ6.59
ꢁ6.07
ꢁ6.48
ꢁ5.95
ꢁ6.58
ꢁ5.88
ꢁ5.86
ꢁ6.64
ꢁ6.63
ꢁ6.15
ꢁ7.72
ꢁ6.57
ꢁ7.05
ꢁ6.85
ꢁ7.07
ꢁ7.66
ꢁ7.85
ꢁ7.09
ꢁ6.14
ꢁ4.96
ꢁ5.68
ꢁ5.56
ꢁ5.75
ꢁ5.95
ꢁ5.71
ꢁ5.72
5.2. General procedures
5.2.1. 2-[4-(Benzyloxy)phenyl]-1,3-benzoxazole (2a)
A mixture of 2-aminophenol (545 mg, 5 mmol) and 4-benzyl-
oxy benzaldehyde (1.06 g, 5 mmol) was refluxed in ethanol
MG_MID

A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 4747–4761
4755
Figure 5.2. FACS analysis of cell cycle distribution of A375 cells after treatment with benzothiazole/benzoxazole–pyrrolo[2,1-c][1,4]benzodiazepine conjugates at 2 lM
concentration for 24 h. DC-81 was used as the positive control. Control: cells treated with DMSO.
(35 mL). After few minutes, a yellow precipitate was observed. The
obtained precipitate 1a was filtered and used without further puri-
fication (1.2 g, 80%). The product 1a (500 mg, 1.65 mmol) was dis-
solved in chloroform (20 mL). To this solution, 1.1 g of lead tetra
acetate (2.5 mmol) was added in portionwise. The resulting mix-
ture was refluxed for about 1 h. After cooling to room temperature,
the reaction mixture was filtered and concentrated under reduced
pressure. The residue thus obtained was purified by column

4756
A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 4747–4761
ture for about 1 h. After this time, the reaction mixture was filtered
Apoptosis percentage (G0)
30
25
20
15
10
5
and concentrated under reduced pressure. The residue thus ob-
tained was purified by column chromatography using ethyl acetate
and hexane (1:9) as eluant affords compound 3a as light brown
colour solid (165 mg, 95%). 1H NMR (CDCl3, 300 MHz): d 8.08 (td,
2H, J = 8.5, 2.3 Hz), 7.62–7.68 (m, 1H), 7.53 (m, 1H), 7.25–7.31
(m, 2H), 6.92 (td, 2H, J = 8.5, 2.3 Hz); ESIMS: m/z 212 (M++1).
5.2.4. 4-(1,3-Benzoxazol-2-yl)-2-methoxyphenol (3b)
The compound 3b was prepared according to the method de-
scribed for 3a by employing 2b (250 mg, 0.75 mmol) and Pd/C
(0.075 mmol) (yield 172 mg, 95%). 1H NMR (CDCl3, 300 MHz): d
7.66–7.83 (m, 3H), 7.50–7.55 (m, 1H), 7.25–7.32 (m, 2H), 7.03 (d,
1H, J = 7.8 Hz), 5.95 (br s, 1H, exchangable with DMSO-d6), 4.05
(s, 3H); ESIMS: m/z 242 (M++1).
0
Control
DC-81
17b
17d
18a
% of cells in G0 phase
Figure 5.3. Apoptosis percentage of A375 cells treated with compounds 17b, 17d,
18a and DC-81 at 2 lM concentration.
Percentage of apotosis
5.2.5. 2-[4-(4-Bromobutyloxy)-phenyl]-1,3-benzothiazole (5a)
To a solution of compound 4a (227 mg, 1 mmol) in dry acetone
(15 mL) was added, anhydrous K2CO3 (553 mg, 4 mmol), 1,4-dibro-
mobutane (324 mg, 1.5 mmol) and the reaction mixture was stir-
red at reflux temperature for 48 h. the reaction mixture was
monitored by TLC. After completion of the reaction as indicated
by TLC, K2CO3 was removed by filtration and the solvent was evap-
orated under reduced pressure, diluted with water and extracted
with ethyl acetate. The combined organic phases were dried over
Na2SO4 and evaporated under vacuum. The residue, thus obtained
was purified by column chromatography using ethyl acetate and
hexane (2:8) to afford compound 5a as white solid (340 mg,
95%). 1H NMR (CDCl3, 300 MHz); d 7.90–7.98 (m, 3H, J = 9.0 Hz),
7.83 (d, 1H, J = 7.5 Hz), 7.42 (d, 1H, J = 8.3 Hz), 7.30 (d, 1H,
J = 7.5 Hz), 6.94 (d, 2H, J = 9.0 Hz), 4.2 (t, 2H, J = 6.0 Hz), 3.58 (t,
2H), 1.5–2.1 (m, 4H); EIMS: m/z 363 (M++1).
30
25
20
15
10
5
DC-81
17 d
0
0 uM
0.5 uM
1 uM
2 uM
Concentrations
Figure 5.4. Comparative study of apoptosis in A375 cells treated with compound
DC-81 and 17d. The percentages of accumulation of sub-G0 phase cells of the
positive control (DC-81) and compound 17d (0.5, 1 and 2 lM) were 2.13%, 2.23%;
2.7%, 14.41%; and 10.7%, 26.75%, respectively.
5.2.6. 2-[4-(4-Bromobutyloxy)-2-methoxyphenyl]-1,3-
benzothiazole (5b)
Table 6.2
Cell cycle distribution of A375 cell line with variable concentration of the 17d and DC-
The compound 5b was prepared according to the method de-
scribed for 5a by employing hydroxyl benzothiazole (4b) (257
mg, 1 mmol) and 1,4-dibromobutane (324 mg, 1.5 mmol) as a
white coloured solid (yield 372 mg, 96%).
81 (0, 0.5, 1 and 2
lM) compounds
Compound
Concn (
lM)
Cell cycle distribution (%)
G0
0.99
G1
S
G2/M
G0/G1
1H NMR (CDCl3, 300 MHz); d 7.98 (d, 1H, J = 8.3 Hz), 7.83 (d, 1H,
J = 7.5 Hz), 7.67 (s, 1H), 7.52 (dd, 1H, J = 7.5, 2.5 Hz), 7.44 (t, 1H,
J = 7.5 Hz), 7.32 (t, 1H, J = 7.5 Hz), 6.91 (d, 1H, J = 8.3 Hz), 4.2 (t,
2H, J = 6.0 Hz), 3.92 (s, 3H), 3.58 (t, 2H), 2.1 (m, 4H); EIMS: m/z
393 (M++1).
Control
DC-81
17d
DC-81
17d
0
53.56
64.95
79.42
61.56
74.22
67.23
67.04
15.93
16.96
10.40
15.42
5.03
29.52
15.96
6.45
20.32
6.34
54.55
67.08
83.15
64.26
88.63
77.93
93.79
0.5
0.5
1
2.13
3.73
2.70
1
2
2
14.41
10.70
26.75
DC-81
17d
13.54
3.11
8.53
3.10
5.2.7. 2-[4-(5-Bromopentyloxy)-phenyl]-1,3-benzothiazole (5c)
The compound 5c was prepared according to the method
described for 5a by employing hydroxyl benzothiazole (4a) (227
mg, 1 mmol) and 1,5-dibromopentane (345 mg, 1.5 mmol) as a
white colour solid (yield 360 mg, 96%).
chromatography using ethyl acetate and hexane (5:95) as eluant
affords compound 2a as a brown solid (350 mg, 70%). 1H NMR
(CDCl3, 300 MHz): d 8.22 (d, 2H, J = 9.0 Hz), 7.68–7.72 (m, 1H),
7.44–7.52 (m, 1H), 7.27–7.51 (m, 7H), 7.12 (d, 2H, J = 9.0 Hz),
5.20 (s, 2H); ESIMS: m/z 302 (M++1).
1H NMR (CDCl3, 300 MHz); d 7.90–7.98 (m, 3H, J = 9.0 Hz), 7.83
(d, 1H, J = 7.5 Hz), 7.42 (d, 1H, J = 8.3 Hz), 7.30 (d, 1H, J = 7.5 Hz),
6.94 (d, 2H, J = 9.0 Hz), 4.2 (t, 2H, J = 6.0 Hz), 3.43 (t, 2H,
J = 6.0 Hz), 1.97 (q, 2H, J = 7.5 Hz), 1.87 (q, 2H, J = 7.3 Hz), 1.72–
1.63 (m, 2H); EIMS: m/z 377 (M++1).
5.2.2. 2-[4-(Benzyloxy)-3-methoxyphenyl]-1,3-benzoxazole (2b)
The compound 2b was prepared according to the method de-
scribed for 2a by employing 2-aminophenol (545 mg, 5 mmol)
and 4-benzyloxy-3-methoxybenzaldehyde (1.2 g, 5 mmol) fol-
lowed by oxidative cyclization with lead tetra acetate affords com-
pound 2b as light brown coloured solid (yield 70%). 1H NMR
(CDCl3, 300 MHz): d 7.62–7.78 (m, 3H), 7.24–7.55 (m, 8H), 6.95
(d, 1H, J = 8.5 Hz), 5.20 (s, 2H), 4.02 (s, 3H); ESIMS: m/z 332 (M++1).
5.2.8. 2-[4-(5-Bromopentyloxy)-2-methoxyphenyl]-1,3-
benzothiazole (5d)
The compound 5d was prepared according to the method de-
scribed for 5a by employing hydroxyl benzothiazole (4b) (257
mg, 1 mmol) and 1,5-dibromopentane (345 mg, 1.5 mmol) as a
white coloured solid (yield 380 mg, 96%).
1H NMR (CDCl3, 300 MHz); d 7.98 (d, 1H, J = 8.3 Hz), 7.83 (d, 1H,
J = 7.5 Hz), 7.67 (s, 1H), 7.52 (dd, 1H, J = 7.5, 2.5 Hz), 7.44 (t, 1H,
J = 7.5 Hz), 7.32 (t, 1H, J = 7.5 Hz), 6.91 (d, 1H, J = 8.3 Hz), 4.2 (t,
2H, J = 6.0 Hz), 3.94 (s, 3H), 3.43 (t, 2H, J = 6.0 Hz), 1.97 (q, 2H,
5.2.3. 4-(1,3-Benzoxazol-2-yl)phenol (3a)
To a solution of 2a (250 mg, 0.82 mmol) in ethyl acetate (20 mL)
under hydrogen atmosphere, was added 10% Pd/C (87 mg,
0.082 mmol). The resulting mixture was stirred at room tempera-

A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 4747–4761
4757
(i)
(ii)
graft
Animal Weight Data: HT29 xenograft
% Survival Data: HT29 xeno
200
150
100
50
120
100
80
60
40
20
0
0
1
5
9
14 17 21 25 28 32 35
1
5
9
14 17 21 25 28 32 35
Days
Days
V. Control
17d, 15 mg/kg
ADR 15 mg/kg
Control
17d, 15 mg/Kg
17d, 30 mg/Kg
ADR, 15mg/kg
17d, 30 mg/kg
(iii)
(iv)
Relative Tumor Volume (RTV) Data: HT29
xenograft
Days
*T/C
17d, 30
mg/Kg
40
30
20
10
0
17d, 15
mg/Kg
ADR,
15mg/kg
1
5
9
14
17
21
25
28
32
35
1
0.65
0.49
0.58
0.65
0.65
0.72
0.73
0.76
0.82
1
0.85
0.78
0.66
0.62
0.49
0.30
0.28
0.20
0.20
0.18
0.16
0.14
0.81
0.58**
0.67
1
5
9
14 17 21 25 28 32 35
Days
Control
17d, 15 mg/Kg
ADR, 15mg/kg
0.75
0.76
0.68
17d, 30 mg/Kg
Figure 6. (i) Scid mice treated with 17d, Survival data of compound 17d on HT29 xenograft mice. (ii) tumour weight curves in scid mice treated with 17d. (iii) Relative tumour
volume (RTV), T/C values derived from RTV data, *T/C 6 0.2 indicates activity (values in red colour in the above table), **minimum T/C attained by compound 17d,
ADR = adriamycin (doxorubicin).
J = 7.5 Hz), 1.87 (q, 2H, J = 7.3 Hz), 1.72–1.63 (m, 2H); EIMS: m/z
1H NMR (CDCl3, 300 MHz); d 7.77 (d, 1H, J = 8.30 Hz), 7.67–7.73
(m, 3H, J = 9.0 Hz), 7.52 (d, 1H, J = 9.0 Hz), 6.96 (d, 2H, J = 8.3 Hz),
4.2 (t, 2H, J = 6.0 Hz), 3.94 (s, 3H), 3.43 (t, 2H, J = 6.0 Hz), 1.97 (q,
2H, J = 7.5 Hz), 1.87 (q, 2H, J = 7.3 Hz), 1.72–1.63 (m, 2H); EIMS:
m/z 390 (M++1).
407 (M++1).
5.2.9. 2-[4-(5-Bromopentyloxy)-phenyl]-1,3-benzoxazole (5e)
The compound 5e was prepared according to the method de-
scribed for 5a by employing hydroxyl benzoxazole (3a) (211 mg,
1 mmol) and 1,5-dibromopentane (345 mg, 1.5 mmol) as a white
coloured solid (yield 340 mg, 96%).
5.2.11. N1-(4-Fluorophenyl)-4-nitrobenzamide (6a)
A mixture of 4-fluoroaniline (556 mg, 5 mmol) and 4-nitro-
benzoyl chloride 927.5 mg, 5 mmol) in pyridine (20 mL) was stir-
red under reflux. After completion of reaction, the reaction
mixture was poured into water (100 mL). The precipitate was col-
lected and washed with water (2 ꢃ 50 mL), ice-cold methanol
(2 ꢃ 20 mL) to afford compound 6a (1.24 g, 95%). 1H NMR (CDCl3,
300 MHz): d 8.33 (d, 2H, J = 8.6 Hz), 8.09 (d, 2H, J = 8.6 Hz), 7.66
(m, 2H, J = 4.7 Hz), 7.07 (t, 2H, J = 8.6 Hz); EIMS: m/z 260 (M+).
1H NMR (CDCl3, 300 MHz); d 8.02 (d, 2H, J = 9.0 Hz), 7.73 (d, 1H,
J = 9.0 Hz), 7.50 (d, 1H, J = 9.0 Hz), 7.29 (m, 2H, J = 9.0 Hz), 6.98 (d,
2H, J = 8.3 Hz), 4.2 (t, 2H, J = 6.0 Hz), 3.43 (t, 2H, J = 6.0 Hz), 1.97 (q,
2H, J = 7.5 Hz), 1.87 (q, 2H, J = 7.3 Hz), 1.72–1.63 (m, 2H); EIMS: m/z
361 (M++1).
5.2.10. 2-[4-(5-Bromopentyloxy)-2-methoxyphenyl]-1,3-
benzoxazole (5f)
The compound 5d was prepared according to the method de-
scribed for 5f by employing hydroxyl benzoxazole (3b) (241 mg,
1 mmol) and 1,5-dibromopentane (345 mg, 1.5 mmol) as a white
colour solid (yield 370 mg, 96%).
5.2.12. N1-(4-Fluorophenyl)-4-nitro-1-benzenecarbothioamide
(7a)
A
mixture of nitrobenzanilide (6a, 1 g, 3.84 mmol) and
Lawesson’s reagent (775.7 mg, 1.92 mmol) in HMPA (10 mL)

4758
A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 4747–4761
was stirred at 100 °C for 6 h and poured into water (100 mL).
The precipitate was collected, washed with water and then
ice-cold methanol to afford 7a (955 mg, 90%). 1H NMR (DMSO-
d6 + CDCl3, 200 MHz): d 11.78 (br s, 1H), 8.26 (d, 2H, J = 8.8 Hz),
8.02 (d, 2H, J = 8.8 Hz), 7.89 (m, 2H), 7.13 (t, 2H, J = 8.8 Hz); EIMS:
m/z 276 (M+).
5.2.17. N1-[4-(1,3-Benzothiazol-2-yl)phenyl]-5-bromopen-
tanamide (9b)
The compound 9b was prepared according to the method de-
scribed for 9a by employing 8b (250 mg, 1.1 mmol) and 5-bromo-
pentanoyl chloride (329 mg, 1.65 mmol) affords compound 9b as a
pale yellow coloured solid (371 mg, yield 95%). 1H NMR (CDCl3,
300 MHz): d 8.00–8.10 (m, 3H), 7.88 (d, 1H, J = 8.3 Hz), 7.65 (d,
2H, J = 8.3 Hz), 7.45 (dt, 1H, J = 1.5, 8.3 Hz), 7.35 (dt, 1H, J = 1.5,
8.3 Hz), 3.45 (t, 2H, J = 6.0 Hz), 2.45 (t, 2H, J = 6.8 Hz), 2.0 (m,
4H); LCMS: m/z 389 (M++1), 391 (M++3).
5.2.13. 4-(6-Fluoro-1,3-benzothiazol-2-yl)aniline (8a)
The fluoro substituted nitrothiobenzanilide 7a (900 mg,
3.25 mmol) was dissolved in a solution of NaOH (1.3 g, 32.5 mmol)
in water (30 mL) and ethanol (3 mL). The mixture was added drop-
wise to a solution of potassium ferricyanide (13 mmol) in water
(20 mL) at 90 °C, stirred for 30 min, and then allowed to cool. The
precipitate was collected, washed with water (100 mL ꢃ 2) and
purified by column chromatography using chloroform and hexane
(1:1) as eluant (534 mg, 60%). 1H NMR (DMSO-d6 + CDCl3,
200 MHz): d 8.34 (m, 4H), 8.09 (m, 1H), 7.76 (m, 1H), 7.32 (m,
1H); EIMS: m/z 274 (M+).
The above obtained compound (500 mg, 1.82 mmol) and
SnCl2ꢀ2H2O (1.65 g, 7.3 mmol) were taken in methanol (25 mL)
and refluxed for about 5 h. After completion of the reaction,
the reaction mixture was concentrated and the resulting com-
pound was suspended in 10% NaHCO3 solution (100 mL), ex-
tracted with ethyl acetate (2 ꢃ 50 mL). The organic layer was
concentrated under reduced pressure and purified by column
chromatography using ethyl acetate and hexane (1:4) to afford
compound 8a (356 mg, 90%). 1H NMR (DMSO-d6 + CDCl3,
200 MHz): d 7.85–7.72 (m, 3H), 7.48–7.57 (m, 1H), 7.08–7.16
(m, 1H), 6.67 (d, 2H, J = 8.8 Hz), 5.08 (br s, 2H); EIMS: m/z
244 (M+).
5.2.18. N1-[4-(1,3-Benzothiazol-2-yl)phenyl]-4-bromobutana-
mide (9c)
The compound 9c was prepared according to the method de-
scribed for 9a by employing 8b (250 mg, 1.1 mmol) and 4-bromob-
utanoyl chloride (309 mg, 1.65 mmol) affords compound 9c as a
pale yellow colour solid (358 mg, yield 95%).
1H NMR (CDCl3, 300 MHz): d 9.25 (br s, 1H), 7.92–8.00 (m, 3H),
7.82 (d, 1H, J = 8.6 Hz), 7.72 (d, 2H, J = 8.6 Hz), 7.40 (dt, 1H, J = 8.6,
1.6 Hz), 7.32 (dt, 1H, J = 8.6, 1.6 Hz), 3.48 (t, 2H, J = 6.3 Hz), 2.54 (t,
2H, J = 7.0 Hz), 2.18–2.24 (m, 2H); LCMS: m/z 375 (M++1), 377
(M++3).
5.2.19. 7-methoxy-8-{4-[4-(1,3-benzothiazol-2-yl)phenoxy]-
butyl}oxy-(11aS)1,2,3,11a-tetra-hydro-5H-pyrrolo[2,1-c][1,4]-
benzodiazepin-5-one (17a)
To a solution of 16 (246 mg, 1 mmol) in acetone (10 mL) was
added K2CO3 (200 mg, 1.5 mmol) at 0 °C and stirred for 30 min
2-[4-(4-bromobutyloxy)-phenyl]-1,3-benzothiazole (5a) (433 mg,
1.20 mmol, generated freshly in acetone (10 mL) and KI
(160 mg, 1 mmol), was added to the solution dropwise. The
resulting solution was stirred at room temperature for 24 h.
The reaction mixture was poured into ice-water (30 mL) and ex-
tracted with ethyl acetate. The combined organic phases were
washed with H2O and brine dried over Na2SO4 and concentrated
under vacuum. The residue was subjected to column chromatog-
raphy (CHCl3/MeOH = 24:1) to give product as a white coloured
5.2.14. N1-[4-(6-Fluoro-1,3-benzothiazol-2-yl)phenyl]-5-
bromopentanamide (9a)
To a mixture of 8a (244 mg, 1.1 mmol) and triethyl amine
(0.23 mL, 1.65 mmol) in THF (10 mL) at 0 °C, was added 5-bromo-
pentanoyl chloride (300 mg, 1.65 mmol) in THF (10 mL) and the
mixture was stirred at room temperature for 1 h. After completion
of the reaction, the reaction mixture was concentrated and sus-
pended in water (100 mL). The resulting precipitate was extracted
with ethyl acetate (25 ꢃ 2 mL) and washed with 2 N HCl (25 mL),
followed by brine. Finally, this compound was purified by column
chromatography using ethyl acetate and hexane (1:4) as eluant af-
fords compound 9a as a pale yellow solid (300 mg, 1.5 mmol)
(386 mg, yield 95%).
solid (yield 295 mg, 56%). Mp 90–91 °C. [
a]
+154.00 (c 0.5,
D
CHCl3). 1H NMR (CDCl3, 300 MHz): d 8.04 (d, 2H, J = 8.8 Hz),
7.90 (d, 1H, J = 8 Hz)), 7.65 (d, 1H, J = 4.0 Hz), 7.50 (s, 1H),
7.30–7.47 (m, 3H), 6.98 (d, 2H, J = 8.8 Hz), 6.82 (s, 1H), 4.05–
4.21 (m, 4H), 3.94 (s, 3H), 3.50–3.85 (m, 3H), 1.90–2.30 (m,
8H); 13C NMR (75 MHz, CDCl3): d 167.7, 164.6, 162.3, 161.2,
154.0, 150.8, 147.7, 140.3, 134.7, 129.0, 126.0, 124.6, 122.6,
121.3, 120.0, 114.6, 111.5, 110.1, 68.5, 67.3, 56.0, 53.8, 53.6,
46.6, 29.5, 25.9, 25.6, 24.0: FABMS: m/z 528 (M++1). Anal. Calcd
for C30H29N3O4S: C, 68.29; H, 5.54; N, 7.96. Found: C, 68.12; H,
5.42; N, 7.85.
1H NMR (CDCl3 + DMSO-d6, 200 MHz): d 9.39 (s, 1H), 7.74–7.85
(m, 3H), 7.58–7.64 (m, 2H), 7.39–7.44 (m, 1H), 7.01–7.11 (m, 1H),
3.26–3.32 (m, 2H), 2.25–2.32 (m, 2H), 1.68–1.85 (m, 4H); LCMS: m/
z 407 (M++1), 409 (M++3).
5.2.15. N1-4-Nitrobenzamide (6b)
5.2.20. 7-methoxy-8-{4-[4-(1,3-benzothiazol-2-yl)-2-metho-
xyphenoxy]butyl}oxy-(11aS)1,2,3,11a-tetra-hydro-5H-pyrrolo
[2,1-c][1,4]benzodiazepin-5-one (17b)
The compound 6b was prepared according to the method de-
scribed for 6a by employing aniline (250 mg, 1.1 mmol) and 4-
nitrobenylchloride (329 mg, 1.65 mmol) affords compound 6b
as a pale yellow coloured solid (229 mg, yield 95%). 1H NMR
The compound 17b was prepared according to the method
described for the compound 17a by employing compound 5b
(460 mg, 1.2 mmol) as a white coloured solid (yield 334 mg,
(CDCl3, 300 MHz):
d 8.33 (d, 2H, J = 8.6 Hz), 8.09 (d, 2H,
J = 8.6 Hz), 7.66 (m, 3H, J = 4.7 Hz), 7.07 (t, 2H, J = 8.6 Hz); EIMS:
56%). Mp 95–96 °C. [a]
+159.00 (c, 0.5, CHCl3). 1H NMR (CDCl3,
D
m/z 242 (M+).
300 MHz): d 8.05 (d, 1H, J = 7.8 Hz), 7.90 (d, 1H, J = 7.8 Hz),
7.64–7.72 (m, 2H), 7.30–7.62 (m, 4H), 6.96 (d, 1H, J = 7.8 Hz),
6.82 (s, 1H), 4.12–4.25 (m, 4H), 4.00 (s, 3H), 3.94 (s, 3H),
3.50–3.85 (m, 3H), 1.94–2.32 (m, 8H); 13C NMR (75 MHz,
CDCl3): d 167.8, 164.6, 162.3, 154.2, 150.8, 149.6, 147.7, 140.3,
134.8126.7, 126.1, 124.8, 122.7, 121.4, 121.0, 120.1, 115.5,
112.5, 110.6, 110.1, 68.5, 67.3, 56.0, 56.1, 53.8, 53.6, 46.7,
29.5, 25.9, 25.6, 24.0; FABMS: m/z 558 (M++1). Anal. Calcd for
C31H31N3O5S: C, 66.77; H, 5.60; N, 7.54. Found: C, 66.75; H,
5.58; N, 7.55.
5.2.16. 4-(1,3-Benzothiazol-2-yl)aniline (8b)
The compound 8b was prepared according to the method de-
scribed for 8a by employing 6b (250 mg, 1.1 mmol) affords com-
pound 8b as a pale yellow colour solid (214 mg, yield 95%).
1H NMR (CDCl3, 300 MHz): d 8.00 (d, 1H, J = 7.1), 7.92 (d, 2H,
J = 8.6 Hz,), 7.81 (d, 1H, J = 7.6 Hz), 7.44 (dt, 1H, J = 7.6, 1.3 Hz),
7.34 (dt, 1H, J = 7.5, 1.2 Hz), 6.71 (d, 2H, J = 8.6 Hz), 4.04 (br s, 2H,
NH2); EIMS: m/z 226 (M+).

A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 4747–4761
4759
5.2.21. 7-methoxy-8-{5-[4-(1,3-benzothiazol-2-yl)phenoxy]
pentyl}oxy-(11aS 1,2,3,11a-tetra-hydro-5H-pyrrolo[2,1-c][1,4]
benzodiazepin-5-one (17c)
(465 mg, 1.2 mmol) as
277 mg, 50%). Mp 100–101 °C. [
a
pale yellow coloured solid (yield
a]
+168.00 (c 0.5, CHCl3). 1H
D
NMR (CDCl3, 300 MHz): d 8.80 (s, 1H), 8.05 (m, 3H), 7.90 (d, 1H,
J = 7.9 Hz), 7.68–7.76 (m, 3H), 7.60 (s, 1H), 7.30–7.56 (m, 2H),
6.90 (s, 1H), 4.08–4.28 (m, 2H), 3.96 (s, 3H), 3.50–3.88 (m, 3H),
2.64 (m, 2H), 1.62–2.38 (m, 8H); 13C NMR (75 MHz, CDCl3): d
186.0, 168.0, 164.6, 162.3, 161.1, 154.0, 151.0, 14.7.8, 140.3,
134.6, 129.8, 126.4, 126.1, 124.8, 122.7, 121.4, 121.0, 120.0,
112.3, 111.5, 110.3, 109.8, 68.7, 56.1, 53.7, 46.6, 29.5, 28.7, 24.2,
22.4, 20.7; LCMS: m/z 555 (M++1). Anal. Calcd for C31H30N4O4S:
C, 67.13; H, 5.45; N, 10.10. Found: C, 67.10; H, 5.08; N, 9.95.
The compound 17c was prepared according to the method de-
scribed for the compound 17a by employing compound 5c
(450 mg, 1.2 mmol) as a white coloured solid (yield 323 mg,
60%). Mp 95–96 °C. [a]
+149.00 (c 0.5, CHCl3). 1H NMR (CDCl3,
D
300 MHz): d 8.05 (d, 1H, J = 8.6 Hz), 7.85 (d, 1H, J = 8.6 Hz), 7.65
(d, 1H, J = 3.9 Hz), 7.30–7.55 (m, 5H), 7.0 (d, 2H, J = 8.6 Hz), 6.82
(s, 1H), 4.04–4.20 (m, 4H), 3.95 (s, 3H), 3.55–3.85 (m, 3H), 2.19–
2.38 (m, 2H), 1.80–2.10 (m, 5H), 1.62–1.80 (m, 3H); 13C NMR
(75 MHz, CDCl3): d 167.7, 164.6, 162.3, 161.2, 154.0, 150.8, 147.7,
140.3, 134.7, 129.0, 126.0, 124.6, 122.6, 121.3, 120.0, 114.6,
111.5, 110.1, 68.7, 67.6, 56.0, 53.5, 46.5, 29.5, 28.8, 28.4, 23.9,
22.3; FABMS: m/z 542 (M++1). Anal. Calcd for C31H31N3O4S: C,
68.74; H, 5.77; N, 7.76. Found: C, 68.70; H, 5.73; N, 7.74.
5.2.26. 7-methoxy-8-{5-[N1-(4-(6-fluoro-1,3-benzothiazol-2-
yl)phenyl)]pentane carboxa-mide}oxy-(11aS)1,2,3,11a-tetra-
hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (18b)
The compound 17b was prepared according to the method de-
scribed for the compound 17a by employing compound 9a
5.2.22. 7-methoxy-8-{5-[4-(1,3-benzothiazol-2-yl)-2-methoxy-
phenoxy]pentyl}oxy-(11aS)1,2,3,11a-tetra-hydro-5H-pyrrolo-
[2,1-c][1,4]benzodiazepin-5-one (17d)
(487 mg, 1.2 mmol) as
286 mg, 50%). Mp 102 °C. [
(CDCl3, 300 MHz): d 8.88 (br s, 1H), 7.90–8.05 (m, 4H), 7.64–7.76
(m, 3H), 7.58 (m, 1H), 7.20 (m, 1H), 6.84 (s, 1H), 4.12–4.36 (m,
2H), 3.94 (s, 3H), 3.50–3.88 (m, 3H), 2.50–2.70 (m, 2H), 1.65–2.40
(m, 8H); LCMS: m/z 573 (M++1). Anal. Calcd for C31H29FN4O4S: C,
65.02; H, 5.10; N, 9.78. Found: C, 65.00; H, 5.08; N, 9.73.
a
pale yellow coloured solid (yield
a]
D
+170.00 (c 0.5, CHCl3). 1H NMR
The compound 17d was prepared according to the method de-
scribed for the compound 17a by employing compound 5d
(486 mg, 1.2 mmol) as a white coloured solid (yield 314 mg,
55%). Mp 101–102 °C. [a]
+163.00 (c 0.5, CHCl3). 1H NMR (CDCl3,
D
300 MHz): d 8.05 (d, 1H, J = 8.2 Hz), 7.90 (d, 1H, J = 8.2 Hz), 7.64–
7.72 (m, 2H), 7.30–7.60 (m, 4H), 6.95 (d, 1H, J = 8.2 Hz), 6.80 (s,
1H), 4.06–4.18 (m, 4H), 4.00 (s, 3H), 3.95 (s, 3H), 3.50–3.80 (m,
3H), 1.62–2.34 (m, 10H); 13C NMR (75 MHz, CDCl3): d 167.8,
164.6, 162.3, 154.2, 150.8, 149.6, 147.7, 140.3, 134.8126.7, 126.1,
124.8, 122.7, 121.4, 121.0, 120.1, 115.5, 112.5, 110.6, 110.1, 68.6,
67.6, 56.0, 53.6, 46.6, 29.4, 28.8, 28.5, 24.0, 22.5; FABMS: m/z 572
(M++1). Anal. Calcd for C32H33N3O5S: C, 67.23; H, 5.82; N, 7.35.
Found: C, 67.20; H, 5.81, N, 7.30.
5.2.27. N-(4-(1,3-Benzothiazolyl)phenyl)-2-pyrrolidinone (19)
When two compounds 9c (375 mg, 1 mmol) and 16 (400 mg,
1 mmol) were reacted together according to the method described
for 17a, the expected 18c was not obtained, instead 9c was cyclized
to the compound 19 (yield 205 mg, 70%). 1H NMR (CDCl3,
300 MHz): d 8.10 (td, 2H, J = 2.26, 8.3 Hz), 8.05 (d, 1H, J = 8.3 Hz),
7.90 (d, 1H, J = 8.3 Hz), 7.80 (td, 2H, J = 2.26, 8.3 Hz), 7.49 (dt, 1H,
J = 6.8, 1.5 Hz), 7.39 (dt, 1H, J = 6.8, 1.5 Hz), 3.95 (t, 2H, J = 6.8 Hz),
2.67 (t, 2H, J = 8.3 Hz), 2.18–2.30 (m, 2H); ESIMS: m/z 295
(M++1).
5.2.23. 7-methoxy-8-{5-[4-(1,3-benzoxazol-2-yl)phenoxy]
pentyl}oxy-(11aS)1,2,3,11a-tetra-hydro-5H-pyrrolo[2,1-c][1,4]
benzodiazepin-5-one (17e)
5.3. Anticancer activity
The compound 17e was prepared according to the method de-
scribed for the compound 17a by employing compound 5e
(414 mg, 1.2 mmol) as a white coloured solid (yield 263 mg,
The compounds 17a–d and 18a were evaluated for in vitro
activity against selected human tumour cell lines, derived from
six cancer types (lung cancer, cervix cancer, breast cancer, prostate
cancer, colon cancer and ovarian cancer). For each compound,
dose–response curves against each cell line were measured. Sulfo-
rhodamine B (SRB) protein assay has been used to estimate cell
viability or growth.
50%). Mp 93–94 °C. [a]
+155.00 (c 0.5, CHCl3). 1H NMR (CDCl3,
D
300 MHz): d 8.20 (d, 2H, J = 8.6 Hz), 7.72 (m, 1H), 7.65 (d, 1H,
J = 3.9 Hz), 7.54–7.60 (m, 2H), 7.30–7.46 (m, 2H), 7.04 (d, 2H,
J = 8.6 Hz), 6.80 (s, 1H), 4.02–4.24 (m, 4H), 3.90 (s, 3H), 3.50–3.84
(m, 3H), 2.24–2.36 (m, 2H), 1.60–2.16 (m, 8H); LCMS: m/z 526
(M++1). Anal. Calcd for C31H31N3O5: C, 70.84; H, 5.94; N, 7.99.
Found: C, 70.80; H, 5.89; N, 7.94.
5.4. Thermal denaturation studies
5.2.24. 7-methoxy-8-{5-[4-(1,3-benzoxazol-2-yl)-2-methoxy-
phenoxy]pentyl}oxy-(11aS)1,2,3,11a-tetra-hydro-5H-pyrrolo-
[2,1-c][1,4]benzodiazepin-5-one (17f)
Compounds were subjected to thermal denaturation studies
with duplex-form CT-DNA using reported method. Working solu-
tions in aqueous buffer (10 mM NaH2PO4/Na2HPO4, 1 mM Na2ED-
The compound 20f was prepared according to the method de-
scribed for the compound 17a by employing compound 5f
(468 mg, 1.2 mmol) as a white coloured solid (yield 278 mg,
TA, pH 7.00 + 0.01) containing CT-DNA (100
lM in phosphate)
and the PBD (20 M) were prepared by addition of concentrated
l
PBD solutions in DMSO to obtain a fixed [PBD]/[DNA] molar ratio
of 1:5. The DNA–PBD solutions were incubated at 37 °C for 0, 18
and 36 h prior to analysis. Samples were monitored at 260 nm
using a Beckman–Coulter DU 800 spectrophotometer fitted with
high performance temperature controller, and heating was applied
at 1 °C minꢁ1 in the range of 40–90 °C. DNA helix?coil transition
temperatures I were obtained from the maxima in the d(A260)/dT
derivative plots. Results are given as means standard deviation
from three determinations and are corrected for the effects of
DMSO co-solvent using a linear correction term. Drug-induced
50%). Mp 98–99 °C. [a]
+149.00 (c 0.5, CHCl3). 1H NMR (CDCl3,
D
300 MHz): d 7.70–7.84 (m, 3H), 7.62 (d, 1H, J = 4.0 Hz), 7.43–7.58
(m, 2H), 7.30–7.38 (m, 2H), 6.90 (d, 1H, J = 8.6 Hz), 6.80 (s, 1H),
3.88–4.20 (m, 10H), 3.50–3.80 (m, 3H), 1.60–2.36 (m, 10H); LCMS:
m/z 556 (M++1). Anal. Calcd for C32H33N3O6: C, 69.17; H, 5.99; N,
7.56. Found: C, 69.15; H, 5.89, N, 7.54.
5.2.25. 7-methoxy-8-{5-[N1-(4-(1,3-benzothiazol-2-
yl)phenyl)]pentanecarboxamide}oxy-(11aS)1,2,3,11a-tetra-
hydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (18a)
The compound 18a was prepared according to the method de-
scribed for the compound 17a by employing compound 9b
alterations in DNA melting behaviour are given by
DTm = Tm
(DNA + PBD) ꢁ Tm(DNA alone), where the Tm value for the PBD-free
CT-DNA is 69.2 0.01 °C. The fixed [PBD]/[DNA] ratio used did not

4760
A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 4747–4761
result in binding saturation of the host DNA duplex for any com-
pound examined.
solvation are then reintroduced by using a numerical Pois-
sonꢁBoltzmann calculation for the electrostatic portion and a sur-
face-area-dependent term for non-electrostatic contributions to
salvation.21
5.5. Restriction endonuclease inhibition
Stock solutions of each PBD (100 lM) were prepared by dissolv-
5.7. Cell cycle analysis
ing each compound in DMSO (Sigma). These were stored at ꢁ20 °C.
Plasmid (pBR 322) containing single BamHI site was used in this as-
say. Restriction endonuclease and the relevant buffer were ob-
tained from NEB. The DNA fragment (500 ng) was incubated with
each PBD (see Fig. 2 for PBD concentrations) in a final volume of
5 ꢃ 105 each of A375 cells were seeded in 60 mm dish and were
allowed to grow for 24 h. Compounds 17b, 17d, 18a and DC-81
were added at a final concentration 2 lM to the culture media,
and the cells were incubated for an additional 24 h. Harvesting of
cells was done with trypsin–EDTA, and fixed with ice-cold 70% eth-
anol at 4 °C for 30 min, washed with PBS and incubated with 1 mg/
mL Rnase A solution at 37 °C for 30 min. Cells were collected by
centrifugation at 2000 rpm for 5 min and were stained with propi-
dium iodide (PI) [10 mg of propidium iodide (PI), 0.1 mg of triso-
dium citrate and 0.03 mL of Triton X-100 were dissolved in
100 mL of sterile MilliQ water at room temperature for 30 min in
the dark]. The DNA contents of 20,000 events were measured by
flow cytometer (DAKO CYTOMATION, Beckman Coulter, Brea,
CA). Histograms were analyzed using Summit Software.
16
l
L for 16 h at 37 °C. Next 10ꢃ BamHI buffer (2
l
L) was added,
and the reaction mixture was made up to 20
lL with BamHI
(20 U) and then incubated for 1 h at 37 °C. Then loaded on to a
1% agarose gel electrophoresis in Tris–acetate EDTA buffer at
80 V for 2 h. The gels were stained with ethidium bromide and
photographed.
5.6. Molecular modelling
5.6.1. Docking
The GOLD 3.2 program protocol20 was used for the molecular
docking calculations of the cross-linked complex formed between
the synthesized molecules and the host DNA duplex. All molecules
were prepared using SYBYL6.9 (Tripos Inc., St. Louis, MO). Tripos
force field and Gasteiger–Hückel partial atomic charges were ap-
plied with distance dependent dielectric constant and Powell’s
conjugate gradient energy minimization method with a conver-
gence criterion of 0.001 kcal/mol was reached. The B-DNA duplex
was built and minimized using ‘nucgen’ and ‘sander’ modules of
AMBER, suit of programs, respectively. The parameter set for docking
was as follows: number of islands 5, population size of 100, num-
ber of operations was 100,000, a niche size of 2 and a selection
pressure of 1.1 and the van der Waals and hydrogen bonding were
set to 4.0 and 2.5, respectively.
Acknowledgements
The authors K.S.R, M.N.A.K. and R.V.C.R.N.C.S. thank CSIR and
the Department of Biotechnology, New Delhi (BT/PR/7037/Med/
14/933/2006); for financial assistance.
References and notes
1. (a) Thurston, D. E.; Bose, D. S. Chem. Rev. 1994, 94, 433; (b) Kamal, A.; Azeeza, S.;
Bharathi, E. V.; Malik, M. S.; Shetti, R. V. C. R. N. C. Mini.-Rev. Med. Chem., in
press.
2. Petrusek, R. L.; Uhlenhopp, E. L.; Duteau, N.; Hurley, L. H. J. Biol. Chem. 1982,
257, 6207.
3. (a) Dervan, P. B. Science 1986, 232, 464; (b) Hurley, L. H.; Boyd, F. L. TIPS 1988, 9,
402; (c) Hurley, L. H. J. Med. Chem. 1989, 32, 2027.
4. (a) Antonow, D.; Kaliszczak, M.; Kang, G.-D.; Coffils, M.; Tiberghien, A. C.;
Cooper, N.; Barata, T.; Heidelberger, S.; James, C. H.; Zloh, M.; Jenkins, T. C.;
Reszka, A. P.; Neidle, S.; Guichard, S. M.; Jodrell, D. I.; Hartley, J. A.; Howard, P.
W.; Thurston, D. E. J. Med. Chem. 2010, 53, 2927; (b) Rahman, K. M.; Thompson,
A. S.; James, C. H.; Narayanaswamy, M.; Thurston, D. E. J. Am. Chem. Soc. 2009,
131, 1375; (c) Rettig, M.; Weingarth, M.; Langel, W.; Kamal, A.; Kumar, P. P.;
Weisz, K. Biochemistry 2009, 48, 12223; (d) Kamal, A.; Rao, M. V.; Laxman, N.;
Ramesh, G.; Reddy, G. S. K. Curr. Med. Chem. Anticancer Agents 2002, 2, 215; (e)
Kamal, A.; Ramesh, G.; Laxman, N.; Ramulu, P.; Srinivas, O.; Neelima, K.;
Kondapi, A. K.; Srinu, V. B.; Nagarajaram, H. A. J. Med. Chem. 2002, 45, 4679; (f)
Wells, G.; Martin, C. R. H.; Howard, P. W.; Sands, Z. A.; Laughton, C. A.;
Tiberghien, A.; Woo, C. K.; Masterson, L. A.; Stephenson, M. J.; Hartley, J. A.;
Jenkins, T. C.; Shnyder, S. D.; Loadman, P. M.; Waring, M. J.; Thurston, D. E. J.
Med. Chem. 2006, 49, 5442; (g) Hu, W.-P.; Ling, J.-J.; Kao, C.-L.; Chen, Y.-C.; Chen,
C.-Y.; Tsai, F.-Y.; Wu, M.-J.; Chang, L.-S.; Wang, J.-J. Bioorg. Med. Chem. 2009, 17,
1172.
5. Alley, M. C.; Hollingshead, M. G.; Pacula-Cox, C. M.; Waud, W. R.; Hartley, J. A.;
Howard, P. W.; Gregson, S. J.; Thurston, D. E.; Sausville, E. A. Cancer Res. 2004,
64, 6693.
6. (a) Caleta, I.; Kralj, M.; Branimir Bertosa, B.; Sanja Tomic, S.; Pavlovic, G.;
Pavelic, K.; Karminski-Zamola, G. J. Med. Chem. 2009, 52, 1744; (b) Chung, Y.;
Shin, Y.-K.; Zhan, C.-G.; Lee, S.; Cho, H. Arch. Pharmacol. Res. 2004, 27, 893; (c)
Yoshida, M.; Hayakawa, I.; Hayashi, N.; Agatsuma, T.; Oda, Y.; Tanzawa, F.;
Iwasaki, S.; Koyama, K.; Furukawa, H.; Kurakata, S. Bioorg. Med. Chem. Lett.
2005, 15, 3328.
7. (a) Bradshaw, T. D.; Stevens, M. F. G.; Westwell, A. D. Curr. Med. Chem. 2001, 8,
203; (b) Hutchinson, I.; Chua, M.-S.; Browne, H. L.; Trapani, V.; Bradshaw, T. D.;
Westwell, A. D.; Stevens, M. F. G. J. Med. Chem. 2001, 44, 1446.
8. (a) Chua, M.-S.; Shi, D.-F.; Wrigley, S.; Bradshaw, T. D.; Hutchinson, I.; Shaw, P.
N.; Barrett, D. A.; Stanley, L. A.; Stevens, M. F. G. J. Med. Chem. 1999, 42, 381; (b)
Kashiyama, E.; Hutchinson, I.; Chua, M.-S.; Stinson, S. F.; Phillips, L. R.; Kaur, G.;
Sausville, E. A.; Bradshaw, T. D.; Westwell, A. D.; Stevens, M. F. G. J. Med. Chem.
1999, 42, 4172.
9. O’Brien, S. E.; Browne, H. L.; Bradshaw, T. D.; Westwell, A. D.; Stevens, M. F. G.;
Laughton, C. A. Org. Biomol. Chem. 2003, 1, 493.
10. Mortimer, C. G.; Wells, G.; Crochard, J.-P.; Stone, E. L.; Bradshaw, T. D.; Stevens,
M. F. G.; Westwell, A. D. J. Med. Chem. 2006, 49, 179.
5.6.2. Molecular dynamics simulations
Ligand parameters and charges were determined with the ante-
chamber module of AMBER 8 based on the general atom force field
(GAFF)25 and the AM1-BCC charge scheme.26,27 Since for most of
the ligands complete GAFF parameter set could not be generated,
we used an alternative, second parametrization in which parame-
ters for bonds, valence, dihedral and improper angles were ad-
justed using the prmchk command of AMBER
28
.
The complex is solvated with TIP3P water in the 10 Å periodic
box. Equilibration of the solvated complex has been done by carry-
ing out a minimization (500 steps of each steepest descent and
conjugate gradient method), 50 ps of heating and 50 ps of density
equilibration with weak restraints on the complex followed by
500 ps of constant pressure equilibration at 300 K. Twelve ang-
stroms cut off distance for the long range non-bonded interactions
is used. Shake method is applied on hydrogen atoms, a 2 fs time
step and Langevin dynamics for temperature control. The same
conditions as the final phase of equilibration are used for produc-
tion run. The final production run is performed for the 2 ns and
the coordinates are recorded in every 10 ps.
5.6.3. MM-PBSA calculations
Solute configurations DNA, ligand and complex (without water)
are sampled from the final MD trajectory. The energy terms are
averaged over 200 frames extracted from MD simulation trajecto-
ries, in the standard MM-PB(GB)SA protocol. For each solute con-
figuration, the gas-phase energy is estimated using the same
molecular mechanics potential that was used to perform the sim-
ulation, but all solvent molecules are ignored, and no cutoffs are
used in evaluating the non-bonded interactions. Free energies of
11. (a) Kumar, D.; Jacob, M. R.; Reynolds, M. B.; Kerwin, S. M. Bioorg. Med. Chem.
2002, 10, 3997; (b) Huagn, S. T.; Hsei, I.-J.; Chen, C. Bioorg. Med. Chem. 2006, 14,
6106.

A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 4747–4761
4761
12. (a) Kamal, A.; Khan, M. N. A.; Srikanth, Y. V. V.; Reddy, K. S.; Juvekar, A.; Sen, S.;
Kurian, N.; Zingde, S. Bioorg. Med. Chem. 2008, 16, 7804; (b) Kamal, A.; Khan, M.
N. A.; Reddy, K. S.; Ahmed, S. K.; Kumar, M. S.; Juvekar, A.; Sen, S.; Zingde, S.
Bioorg. Med. Chem. Lett. 2007, 17, 5345; (c) Kamal, A.; Ramulu, P.; Srinivas, O.;
Ramesh, G. Bioorg. Med. Chem. Lett. 2003, 13, 3517; (d) Kamal, A.; Srinivas, O.;
Ramulu, P.; Ramesh, G.; Kumar, P. P. Bioorg. Med. Chem. Lett. 2004, 14, 4107; (e)
Kamal, A.; Reddy, P. S. M. M.; Reddy, D. R.; Laxman, E. Bioorg. Med. Chem. 2006,
14, 385; (f) Kamal, A.; Bharathi, E. V.; Ramaiah, M. J.; Dastagiri, D.; Reddy, J. S.;
Vishwanath, V.; Sulthana, F.; Pushpavalli, S. N. C. V. L.; Bhadra, M.-P.;
Srivastava, H. K.; Sastry, G. N.; Juvekar, A.; Sen, S.; Zingde, S. Bioorg. Med.
Chem. 2010, 18, 526; (g) Kamal, A.; Sreekanth, K.; Kumar, P.; Shankaraiah, N.;
Balakishan, G.; Ramaiah, M. J.; Pushpavalli, S. N. C. V. L.; Ray, P.; Bhadra, M.-P.
Eur. J. Med. Chem. 2010, 45, 2173; (h) Kamal, A.; Rajender; Reddy, D. R.; Reddy,
M. K.; Balakishan, G.; Shaik, T. B.; Chourasia, M.; Sastry, G. N. Bioorg. Med. Chem.
2009, 17, 1557.
17. Jones, G. B.; Davey, C. L.; Jenkins, T. C.; Kamal, A.; Kneale, G. G.; Neidle, S.;
Webster, G. D.; Thurston, D. E. Anti-Cancer Drug Des. 1990, 5, 249.
18. Puvvada, M. S.; Hartley, J. A.; Jenkins, T. C.; Thurston, D. E. Nucleic Acids Res.
1993, 21, 3671.
19. (a) Srivani, P.; Sastry, G. N. J. Mol. Graphics Modell. 2009, 27, 676; (b) Srivani, P.;
Srinivas, E.; Raghu, R.; Sastry, G. N. J. Mol. Graphics Modell. 2007, 26, 378; (c)
Ravindra, G. K.; Achaiah, G.; Sastry, G. N. Eur. J. Med. Chem. 2008, 43, 830.
20. Ryckaert, J. P.; Ciccotti, G.; Berendsen, H. J. C. J. Comput. Phys. 1977, 23, 327.
21. Wilson, W. D.; Nguyen, B.; Tanious, F. A.; Mathis, A.; Hall, J. E.; Stephens, C. E.;
Boykin, D. W. Curr. Med. Chem. Anticancer Agents 2005, 5, 389.
22. Neidle, S. Nat. Prod. Rep. 2001, 18, 291.
23. Kollman, P. A.; Massova, I.; Reyes, C.; Kuhn, B.; Huo, S.; Chong, L.; Lee, M.; Lee,
T.; Duan, Y.; Wang, W.; Donini, O.; Cieplak, P.; Srinivasan, J.; Case, D. A.;
Cheatham, T. E., III Acc. Chem. Res. 2000, 33, 889.
24. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;
Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.
13. Centore, R.; Panunzi, B.; Roviello, A.; Villano, P. J. Polym. Sci., Part A: Polym.
Chem. 1996, 34, 3203.
14. Karlsson, J.; Bergqvist, M. H.; Lincoln, P.; Westman, G. Bioorg. Med. Chem. 2004,
12, 2369.
25. Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. J. Comput. Chem.
2004, 25, 1157.
15. Thurston, D. E.; Murty, V. S.; Langley, D. R.; Jones, G. B. Synthesis 1990, 81.
16. Sedlak, M.; Hejtmankova, L.; Kasparova, P.; Kavalek, J. J. Phys. Org. Chem. 2002,
15, 165.
26. Jakalian, A.; Bush, B. L.; Jack, D. B.; Bayly, C. I. J. Comput. Chem. 2000, 21, 132.
27. Jakalian, A.; Jack, D. B.; Bayly, C. I. J. Comput. Chem. 2002, 23, 1623.
28. Gerber, P. R.; Mueller, K. J. Comput. Aided Mol. Des. 1995, 9, 251.