Synthesis of β-carboline-benzimidazole conjugates using lanthanum nitrate as a catalyst and their biological evaluation

Article abstract of DOI:10.1039/c3ob42236d

A series of β-carboline-benzimidazole conjugates bearing a substituted benzimidazole and an aryl ring at C3 and C1 respectively were designed and synthesized. The key step of their preparation was determined to involve condensation of substituted o-phenylenediamines with 1-(substituted phenyl)-9H-pyrido[3,4-b]indole-3-carbaldehyde using La(NO₃) ₃·6H₂O as a catalyst and their cytotoxic potential was evaluated. Conjugates 5a, 5d, 5h and 5r showed enhanced cytotoxic activity (GI₅₀ values range from 0.3 to 7.1 μM in most of the human cancer cell lines) in comparison to some of the previously reported β-carboline derivatives. To substantiate the cytotoxic activity and to understand the nature of interaction of these conjugates with DNA, spectroscopy, DNA photocleavage and DNA topoisomerase I inhibition (topo-I) studies were performed. These conjugates (5a, 5d and 5r) effectively cleave pBR322 plasmid DNA in the presence of UV light. In addition, the effect of these conjugates on DNA Topo I inhibition was studied. The mode of binding of these new conjugates with DNA was also examined by using both biophysical as well as molecular docking studies, which supported their multiple modes of interaction with DNA. Moreover, an in silico study of these β-carboline-benzimidazole conjugates reveals that they possess drug-like properties. the Partner Organisations 2014.

Full text of DOI:10.1039/c3ob42236d

Volume 12 Number 15 21 April 2014 Pages 2325–2490
Organic &
Biomolecular
Chemistry
www.rsc.org/obc
ISSN 1477-0520
PAPER
Ahmed Kamal, Narayana Nagesh et al.
Synthesis of β-carboline–benzimidazole conjugates using lanthanum
nitrate as a catalyst and their biological evaluation

Organic &
Biomolecular Chemistry
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PAPER
Synthesis of β-carboline–benzimidazole
conjugates using lanthanum nitrate as a
catalyst and their biological evaluation†
Cite this: Org. Biomol. Chem., 2014,
12, 2370
Ahmed Kamal,*a,c M. P. Narasimha Rao,a P. Swapna,a Vunnam Srinivasulu,a
Chandrakant Bagul,c Anver Basha Shaik,a Kishore Mullagiri,a Jeshma Kovvuri,a
Vangala Santhosh Reddy,a K. Vidyasagarb and Narayana Nagesh*b
A series of β-carboline–benzimidazole conjugates bearing a substituted benzimidazole and an aryl ring at
C3 and C1 respectively were designed and synthesized. The key step of their preparation was determined
to involve condensation of substituted o-phenylenediamines with 1-(substituted phenyl)-9H-pyrido-
[3,4-b]indole-3-carbaldehyde using La(NO3)3·6H2O as a catalyst and their cytotoxic potential was
evaluated. Conjugates 5a, 5d, 5h and 5r showed enhanced cytotoxic activity (GI50 values range from
0.3 to 7.1 μM in most of the human cancer cell lines) in comparison to some of the previously reported
β-carboline derivatives. To substantiate the cytotoxic activity and to understand the nature of interaction
of these conjugates with DNA, spectroscopy, DNA photocleavage and DNA topoisomerase I inhibition (topo-
I) studies were performed. These conjugates (5a, 5d and 5r) effectively cleave pBR322 plasmid DNA in the
presence of UV light. In addition, the effect of these conjugates on DNA Topo I inhibition was studied. The
mode of binding of these new conjugates with DNA was also examined by using both biophysical as well as
molecular docking studies, which supported their multiple modes of interaction with DNA. Moreover, an
in silico study of these β-carboline–benzimidazole conjugates reveals that they possess drug-like properties.
Received 11th November 2013,
Accepted 11th February 2014
DOI: 10.1039/c3ob42236d
www.rsc.org/obc
and synthetic β-carboline derivatives have been reported as
Introduction
potential anticancer agents.6–13 These compounds exhibit their
The β-carboline alkaloids are a large group of natural and syn- anticancer activity through multiple mechanisms, such as inter-
thetic indole alkaloids that possess a common tricyclic pyrido- calation into DNA,7,14 inhibiting topoisomerase I and II,9,10
[3,4-b]indole ring structure. The β-carboline alkaloids were origi- CDK,15 MK-2,16,17 and kinesin Eg5.18 Among these, intercalation
nally isolated from the seeds of Peganum harmala (Zygophilla- is of particular importance in the clinical oncology as some of
ceae, Syrian Rue) that has been traditionally used for hundreds them are valuable drugs currently used for the treatment of
of years to treat the alimentary tract cancers and malaria in various cancers.19,20 These have been characterized as DNA
Northwest China.1 Some of these alkaloids are widely found in intercalators due to the presence of polycyclic aromatic planar
nature, including various plants, foodstuffs, marine creatures, pharmacophore, which is capable of stacking between DNA
insects, mammalian as well as human tissues and body base pairs.10,21 For example, harmane and norharman have
fluids.2–5 The well-known members of this β-carboline family been reported to intercalate into DNA leading to altered DNA
are harmane, harmine and norharman. Recently there has been replication fidelity or leading to an influence on enzymatic
increased interest in β-carboline derivatives due to their poten- activities in DNA-repair processes apart from inhibiting DNA
tial biological activities. In particular a large number of natural topoisomerase I22–24 (Fig. 1).
On the other hand, the benzimidazole moiety is structurally
related to purine bases and is found in a variety of natural pro-
ducts, such as vitamin B12. In addition, the benzimidazole
derivatives exhibited potential antitumor/anticancer
aMedicinal Chemistry and Pharmacology, CSIR – Indian Institute of Chemical
Technology, Hyderabad 500 007, India. E-mail: ahmedkamal@iict.res.in;
Fax: (+) 91-40-27193189; Tel: (+) 91-40-27193157
bCSIR – Centre for Cellular and Molecular Biology, Hyderabad-500 007, India.
E-mail: nagesh@ccmb.res.in; Fax: (+) 91-40-27193189; Tel: (+) 91-40-27192568
cDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education
activity,25–28 antibacterial,29 anti-fungal,30 antiviral including
anti-HIV31 and antioxidant32 activities. A series of 2-substi-
tuted benzimidazole-4-carboxamide derivatives have been syn-
thesized and evaluated for in vitro and in vivo anticancer
activities and DNA binding affinity.33 The well-known bis-
benzimidazole derivative Hoechst 33258 is widely used as a
and Research (NIPER), Hyderabad 500 037, India
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ob42236d
2370 | Org. Biomol. Chem., 2014, 12, 2370–2387
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comparison to some of the previously reported β-carboline
derivatives39–42 with GI50 values ranging from 0.3 to 7.1 μM in
most of the cancer-cell lines of the NCI panel.
All these β-carboline–benzimidazole conjugates were evalu-
ated for their cytotoxic activity, DNA intercalation, DNA topo-
isomerase I inhibition and photocleavage studies. The in silico
study of the ADME properties of these conjugates was carried
out by investigating Lipinski’s parameters, topological polar
surface area (TPSA) and percentage of absorption (% ABS).
Results and discussion
Chemistry
Fig. 1 Chemical structures of harmine (A), harmane (B), norharman (C),
Hoechst 33258 (D), carbohydrazide linked β-carboline derivatives (E),
N9-arylated alkyl substituted β-carbolines (F) and designed C3 substi-
tuted β-carboline–benzimidazole conjugates (5a–z & 6a–c).
These β-carboline–benzimidazole conjugates (5a–z, 6a–c) were
prepared as shown in Scheme 1. The Pictet–Spengler conden-
sation reaction of L-tryptophan methyl ester was obtained by
esterification of L-tryptophan using SOCl2 and MeOH, with
various benzaldehydes in the presence of a catalytic amount of
PTSA to yield the corresponding methyl tetrahydro-β-carboline-
3-carboxylates (2a–e). Dehydrogenation of 2a–e with sulfur in
refluxing xylene affords the fully unsaturated methyl-β-carbo-
line-3-carboxylates 3a–e. Then reduction of 3a–e with LiAlH4 in
dry THF under a nitrogen atmosphere followed by oxidation
with Dess Martin periodinane in CH2Cl2 provides the corres-
ponding 1-aryl substituted-9H-pyrido[3,4-b]indole-3-carbalde-
hydes 4a–e. Finally, condensations of subsequent β-carboline
aldehydes 4a–e with various o-phenylenediamines afford the
required substituted benzimidazole linked β-carboline conju-
gates (5a–z, 6a–c).
fluorescent dye to stain DNA; it has undergone Phase I clinical
evaluation and shows its activity by inhibiting DNA topoiso-
merase I and helicase.34,35 There are currently a number
of synthetic methodologies available for the synthesis of
benzimidazoles. Generally, the condensation of o-phenylene-
diamines and carboxylic acids (or their derivatives such as
nitriles, imidates, and ortho esters) has been widely used for
the synthesis of the benzimidazole scaffold under harsh de-
hydrating conditions (170–180 °C).36 Alternative approaches such
as palladium or rhodium catalyzed reactions and solid-phase
supported synthesis,37 etc., have also been developed to
prepare functionalized benzimidazoles. However, directly
employing the condensation and aromatisation reaction of
o-phenylenediamines with aldehydes under oxidative conditions
is considered to be a facile and effective method to prepare
2-substituted benzimidazole.38 Herein, we describe a new
and efficient synthetic methodology for the preparation of
C3 substituted β-carboline–benzimidazole conjugates using
La(NO3)3·6H2O as a catalyst.
A number of synthetic methodologies45 are available in the
literature for the synthesis of benzimidazoles; most of these
Previous reports on β-carboline derivatives revealed that the
presence of various substituents at 1, 3 and 9 positions was
related to their cytotoxic activity against numerous trans-
planted animal tumours.39–42 Chen and coworkers reported
that 3-chlorobenzyl and 3-phenylpropyl substituents at posi-
tion 9 of β-carbolines showed significant antitumor activity.41
Ikeda and coworkers recently reported 3-benzylamino-β-carbo-
line derivatives as potential antitumor agents.39 Their SAR ana-
lysis revealed that (i) the common β-carboline moiety is very
important for the antitumor activity; (ii) the introduction of
appropriate substituents at positions 1, 3 and 9 of the β-carbo-
line nucleus enhanced the antitumor activity. Our earlier
efforts toward the discovery of new synthetic molecules led to
the development of a number of hybrid/conjugate based
different heterocyclic scaffolds as potent antitumor agents.43,44 Scheme 1 Synthesis of β-carboline–benzimidazole conjugates.
Reagents and conditions: (a-i) SOCl2, MeOH, rt, 6–8 h; (a-ii) Ar-CHO,
toluene, cat. PTSA, reflux, 10–12 h; (b) sulfur, xylene, reflux, 10–12 h;
(c-i) LAH, dry THF, 0 °C to rt, 4 h; (c-ii) DMP, CH2Cl2, rt, 2 h; (d) respective
o-phenylenediamine, EtOH, catalyst (La(NO3)3·6H2O), 60 °C,
In continuation of these efforts, we have designed and syn-
thesized a series of β-carboline C3 linked benzimidazole
conjugates with aryl substitution at C1 position as potential
cytotoxic agents. Gratifyingly, among these, 5a, 5d, 5h
30–40 min; (e) pyridine-2,3-diamine, EtOH, catalyst (La(NO3)3·6H2O),
and 5r showed significantly enhanced antitumor activity in 60 °C, 30 min.
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require longer reaction times and higher temperatures. In products were obtained in the ratio of 65 : 35. In order to
addition, these methods produce toxic as well as inseparable improve the selectivity of the reaction, we have studied the
by-products that often require laborious workup and purifi- reaction conditions by screening various catalysts as well as
cation processes,46 resulting in poor isolated yields of the solvents. Among the conditions screened, the one in which
desired products. Therefore, there is still a need for the intro- La(NO3)3·6H2O (10 mol%) was used in EtOH as a solvent
duction of milder and efficient methods to overcome the draw- produced the best results as shown in Table 1. It was observed
backs of existing procedures. In this investigation, we report that a further increase in the amount of catalyst had no effect
an efficient method for the synthesis of benzimidazoles using on the yield (Table 1, entry 7), whereas a reduction in the
La(NO3)3·6H2O as a catalyst under aerobic conditions. This amount of catalyst resulted in a significant decrease in the iso-
reaction proceeds via condensation followed by aerobic oxi- lated yield of the product (Table 1, entry 6). However, there
dation. Initially, a reaction of o-phenylenediamine (1 mmol) was no considerable increase in the yield even after prolonged
with 4a (1.2 mmol) was performed using Na2S2O5 (4–5 mmol) reaction time (1 h). It was observed that there was no for-
as an oxidant in an EtOH–H2O (8 : 2) mixture heated at 70 °C mation of the required product in the absence of catalyst
for about 4–6 h. Both the required as well as N-benzylated (Table 1, entry 9). Earlier reports have also demonstrated the
Table 1 Optimization of reaction conditions for the chemoselective formation of 2-aryl benzimidazole derivatives
Entry
1
R1
H
R2
Catalyst/oxidant
Yields (%) III
65
Yields (%) IV
35
Solvent
Time
8 h
Na2S2O5 (5 equiv.)
EtOH–H2O (8 : 2)
2
3
H
Na2S2O5 (5 equiv.)
Na2S2O5 (5 equiv.)
70
71
30
29
EtOH–H2O (8 : 2)
EtOH–H2O (8 : 2)
8 h
8 h
OCH3
4
5
6
7
8
H
H
H
H
H
K4[Fe(CN)6] (20 mol%)
71
29
Neat
20 min
La(NO3)3·6H2O (10 mol%)
La(NO3)3·6H2O (5 mol%)
La(NO3)3·6H2O (20 mol%)
La(NO3)3·6H2O (10 mol%)
69, 95
75, 77
95
Trace
Trace
Trace
Trace
EtOH
EtOH
EtOH
DMF
15 min, 30 min
30 min, 1 h
30 min
85
1 h
9
H
No catalyst
0
0
EtOH
30 min
2372 | Org. Biomol. Chem., 2014, 12, 2370–2387
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chemoselective properties of La(NO3)3·6H2O, which have
allowed the selective deprotection of acetonides,47 primary
alcohols48 and preparation of 1,5-benzodiazepines from
ketones.49
Table 3 Cytotoxicity of β-carboline–benzimidazole conjugates (5a, 5b,
5c, 5d, 5h, 5r and 5w) in 60 human cancer cell lines
Growth inhibition : GI50a [μM]
Cancer panel/cell line 5ab
5bc
5cd
5de
5hf
5rg
5wh
Cytotoxicity
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
0.88
4.08
1.52
1.98
0.36
4.29
2.94 2.51 2.29 2.36 3.37 3.96
4.11 2.59 2.83 2.78 4.32 4.37
3.09 3.36 2.79 3.35 3.32 4.33
2.31 2.37 1.56 2.03 2.48 3.93
1.85 1.77 1.16 1.55 1.84 2.15
2.68 2.33 3.34 1.97 3.71 9.50
Most of these conjugates were initially tested at a single dose
at higher concentration (10 μM) in the sixty-cell line panel of
NCI (One-Dose Screen). This panel is organized into subpanels
representing leukemia, melanoma, cancers of lung, colon,
kidney, ovary, breast, prostate, and central nervous system. The
compounds that satisfy predetermined threshold inhibition
criteria in a minimum number of cell lines are taken up for
the five-dose assay. The threshold inhibition criteria for pro-
gression to the five-concentration screen were selected to
efficiently capture compounds with antiproliferative activity
based on the analysis of historical DTP screening data. The
Non-small cell lung cancer
A549/ATCC
HOP-62
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
1.92
3.43 3.94 2.21 4.05 2.89 3.20
5.78 7.63 3.22 10.9 3.87 5.87
2.17 3.32 3.12 8.92 3.34 0.52
4.81 11.0 3.01 6.56 3.66 7.19
5.36 8.11 2.77 4.93 3.54 4.40
2.99 3.49 1.98 2.97 2.97 44.1
4.39 3.00 2.00 10.0 2.74 3.96
4.24
4.37
5.80
3.85
2.05
2.70
Colon cancer
results are expressed as the percent growth of treated cells rela- COLO 205
6.76
6.57
2.48
2.88
3.46
2.57
4.83
2.83 1.79 2.10 1.88 1.89 4.58
7.61 3.88 3.71 8.96 5.56 7.32
3.55 3.22 2.69 2.05 3.29 3.95
3.20 2.68 2.82 3.41 3.63 4.14
5.29 3.76 2.96 3.74 4.31 6.87
3.26 3.50 2.78 2.93 3.46 3.66
6.23 7.54 3.73 4.82 4.01 7.14
HCC-2998
tive to the control following 48 h of incubation. Amongst these
HCT-116
conjugates, 5a–d, 5h, 5r and 5w were active in the preliminary
test and progressed to the five-concentration (0.01, 0.1, 1.0, 10
and 100 μM) assay. Table 3 summarizes the results obtained as
HCT-15
HT29
KM12
SW-620
CNS cancer
SF-268
4.90
9.29
3.37
2.89
6.80 12.7 3.24 5.80 3.66 4.81
3.92 1.72 2.80 15.5 3.55 23.1
7.28 68.9 2.94 8.57 4.28 9.45
3.42 3.04 2.42 2.77 3.31 4.76
SF-539
Table 2 Synthesis of β-carboline–benzimidazole conjugates using
La(NO3)3·6H2O as a catalyst
SNB-19
U251
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
3.14
27.6
3.34
4.31
3.31
100
2.18
4.34
2.75
3.34 3.08 2.22 2.86 2.41 4.72
5.10 4.60 2.32 16.3 4.54 4.31
3.09 4.16 2.54 4.02 3.05 4.18
3.94 3.97 2.58 3.69 3.59 3.65
3.11 3.28 2.11 12.1 2.76 3.58
5.00 2.89 3.40 12.7 4.25 6.89
3.12 2.90 1.80 4.91 3.52 2.99
3.94 3.47 2.01 5.68 2.41 3.57
3.34 2.25 1.86 2.76 2.28 3.38
Time Yield
(min) (%)
Compound
R
R1
R2
X
Ovarian cancer
IGROV1
2.83
3.51
3.58 9.99 2.49 4.62 2.89 2.94
3.59 2.25 2.23 1.94 2.74 6.20
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
OCH3
CH3
CH3
F
H
H
4-OCH3C6H4
4-OCH3C6H4
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
N
40
40
30
40
30
40
40
40
40
30
30
30
30
30
40
30
30
30
30
30
30
40
30
30
30
30
40
40
40
80
82
85
85
87
85
80
90
90
95
90
96
88
95
83
85
82
86
89
88
85
92
93
94
91
89
90
89
85
OVCAR-3
OVCAR-4
>100 4.09 6.98 5.10 6.30 4.81 47.4
>100 6.83 4.01 4.46 17.8 6.13 >100
CH3 4-OCH3C6H4
OVCAR-5
H
H
H
H
H
H
4-OCH3C6H4
4-OCH3C6H4
4-OCH3C6H4
4-OCH3C6H4
3,4-F2C6H3
OVCAR-8
1.04
2.10
3.02 3.46 1.78 3.08 2.88 2.79
3.72 6.37 2.77 4.22 3.42 3.59
H
NCI/ADR-RES
SK-OV-3
COC6H4
CF3
OCH3
COC6H4
CH3
Br
CH3
Cl
H
F
H
Cl
Cl
H
CH3
Cl
COC6H4
H
CH3
Cl
OCH3
H
H
>100 6.10 13.0 4.00 17.1 7.43 >100
Renal cancer
786-0
A498
ACHN
4.62
3.34
2.33
6.07
2.77
3.49
3.14
1.66
1.93 2.60 3.33 10.2 5.71 50.7
2.36 14.4 1.92 9.85 2.01 2.43
3.05 2.60 2.87 3.20 3.06 4.86
3.28 2.44 3.87 2.41 3.22 7.03
3.82 1.72 1.72 3.46 1.62 3.25
3.96 5.54 2.97 3.97 3.04 6.27
5.45 7.85 2.20 4.29 3.35 4.70
2.99 2.53 2.54 1.46 2.40 3.62
3,4-F2C6H3
CH3 3,4-F2C6H3
H
H
H
H
H
H
H
H
H
H
Cl
H
H
3,4-F2C6H3
3,4-F2C6H3
3,4-F2C6H3
3,4-F2C6H3
4-CF3C6H4
4-CF3C6H4
4-CF3C6H4
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
3,4,5-(OCH3)3C6H2
3,4,5-(OCH3)3C6H2
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate cancer
PC-3
DU-145
1.52
2.06
2.74 2.77 1.69 3.25 2.09 2.64
5.02 8.53 2.99 4.13 3.41 6.27
5s
5t
Breast cancer
MCF-7
MDA-MB 231/ATCC
HS 578 T
BT-549
T-47D
MDA-MB-468
2.55
8.51
2.86
7.10
2.27
3.16
3.06 3.22 2.53 3.23 3.27 4.11
6.88 2.54 2.30 10.4 2.88 8.38
4.94 4.60 2.82 8.68 3.38 4.44
6.88 2.96 3.18 6.30 4.62 5.48
3.67 3.09 2.56 3.76 2.68 2.92
2.69 1.99 2.08 1.70 2.43 3.32
5u
5v
5w
5x
5y
5z
6a
6b
6c
CH3 3,4,5-(OCH3)3C6H2
H
H
H
H
H
3,4,5-(OCH3)3C6H2
3,4,5-(OCH3)3C6H2
4-OCH3C6H4
3,4-F2C6H3
4-CF3C6H4
a Compound concentration required to decrease cell growth to half that
of untreated cells. b 5a (NSC765800). c 5b (NSC764568). d 5c (NSC764567).
e 5d (NSC765814). f 5h (NSC764570). g 5r (NSC765810). h 5w (NSC765815).
N
N
H
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percentage of growth inhibition (GI50) determined relative to in Table 4 corroborated the NCI-60 cell line screen; it has been
that of untreated control cells. observed that the conjugates which contain a 4-methoxy
The tested compounds showed GI50 values in the range of phenyl ring at C1, electron donating groups like methoxy (5a),
0.3 to 63 μM. The conjugates which contain a 4-methoxyphenyl methyl (5b), 5,6-dimethyl (5c), and unsubstituted (5e) and
ring at C1 and 6-methoxy (5a), 6-fluoro (5d) substituted benz- weak ring deactivating groups like fluoro (5d) on the benzimi-
imidazole at C3; a 3,4-difluorophenyl ring at C1, 6-methoxy (5h) dazole moiety at C3 of a β-carboline ring possess significant to
benzimidazole at C3; and a 4-fluorophenyl ring at C1, 6-chloro- moderate cytotoxicity, whereas the presence of electron with-
benzimidazole (5r) at C3 possess significant cytotoxicity. In drawing groups like trifluoromethyl (5g) on benzimidazole
contrast, a 4-methoxyphenyl ring at C1 and 6-methyl benzimi- decreases the cytotoxicity. In addition a 3,4-difluorophenyl
dazole (5b), 5,6-dimethyl benzimidazole (5c) at C3; and a 3,4,5- ring at C1, 6-methoxy benzimidazole (5h) at C3; and a 4-fluoro-
trimethoxyphenyl group at C1 and unsubstituted benzimida- phenyl ring at C1, 6-chlorobenzimidazole (5r) at C3 also exhibi-
zole (5w) at C3 displayed moderate activity. The other conju- ted promising cytotoxicity.
gates 5g, 5k, 5l, 5m, 5q, 5u, 5v, 5y, 5z, 6a and 6c displayed
Based on the results that depict the cytotoxic potential, it
weak activity. The conjugate 5a showed promising cytotoxic was considered of interest to gain insights into the interaction
activity with GI50 values of 0.3 and 0.8 μM against RPMI-8226 of these conjugates.
and CCRF-CEM cancer cell lines (leukemia). Conjugates 5d, 5r
DNA binding studies
and 5h also showed significant cytotoxic activity against most
of the tested human cancer cell lines with mean GI50 values of
2.4, 3.1 and 5.3 μM.
Further, to understand the cytotoxicity potential of the con-
jugates that were not evaluated in the sixty cell line panel of
NCI screening, an MTT assay was performed for all conjugates
5a–z, 6a–c against three human cancer cell lines HeLa, DU145
and A549. Taken together, results from our cytotoxicity assays
CD studies. Circular dichroism studies provide information
on changes in DNA conformation upon small molecule inter-
action which in turn gives further insight into the mode of
DNA–ligand binding (Fig. 2).
Among several biological macromolecules, DNA is known
to play a crucial role in cell proliferation and other biological
activities. The CD spectrum of CT DNA exhibits a positive
a
Table 4 IC50 (μM) for the 48 h of action of the investigated compounds and (std) on the HeLa, DU145, A549, BHK-21 and L929 cells determined
by the MTT assay
Compound
HeLab
DU145c
A549d
BHK-21e
L929f
5a
5b
5c
5d
5e
1.8 2.3
3.1 1.5
4.3 1.9
1.9 1.1
4.3 1.9
22.9 2.5
12.6 1.9
1.9 1.1
8.5 4.2
18.5 3.2
14.3 2.5
12.6 2.2
15.9 5.1
7.8 1.2
8.5 4.1
6.4 2.7
8.3 1.9
2.6 0.4
9.4 1.8
4.0 3.8
13.5 3.2
24.6 1.8
3.6 2.1
11.7 3.3
16.4 4.7
15.5 3.4
13.9 4.1
16.9 2.6
21.0 3.6
16.0 1.1
2.4 1.8
5.5. 2.6
7.8 1.1
3.0 3.7
6.3 1.8
28.3 2.2
13.0 2.4
3.6 1.7
10.7 1.1
19.8 1.3
15.5 2.2
11.5 4.8
13.7 7.4
7.2 1.1
11.1 6.0
6.7 2.4
20.6 4.3
3.2 1.1
22.4 1.2
3.4 1.2
16.1 1.5
37.6 1.4
6.5 2.4
3.5 6.4
5.6 1.5
21.9 5.3
14.1 2.4
27.4 3.9
21.5 1.7
12.5 1.7
2.0 0.4
3.7 2.2
3.1 2.6
2.4 2.9
2.0 1.0
19.4 1.0
23.7 3.8
4.3 1.9
6.4 1.4
8.3 1.7
15.4 2.8
13.7 3.7
15.8 3.3
10.7 3.1
4.4 2.0
6.0 3.6
18.2 6.6
2.4 1.2
18.4 0.4
2.5 3.3
9.8 1.7
16.4 2.5
3.6 1.5
17.0 1.9
13.4 0.9
19.5 2.2
12.0 2.0
23.3 4.2
15.5 3.2
6.5 2.0
>100 1.2
>100 1.2
>100 2.0
85 1.4
>100 2.6
>100 1.5
63 1.1
>100 1.8
>100 0.7
>100 2.0
>100 1.1
>100 1.5
>100 1.7
>100 1.2
>100 3.7
>100 2.1
>100 2.0
>100 2.7
>100 2.0
>100 2.5
>100 2.2
>100 1.8
>100 0.7
49 1.2
>100 1.5
>100 1.3
>100 1.3
>100 1.4
>100 1.2
>100 2.0
>100 1.5
>100 1.7
>100 2.7
>100 1.1
>100 1.2
>100 2.1
>100 1.8
>100 1.3
>100 1.6
>100 2.8
>100 1.6
>100 1.1
>100 1.1
>100 1.3
>100 1.8
>100 2.0
>100 1.8
>100 1.6
>100 3.3
>100 2.9
>100 2.0
>100 1.5
>100 1.7
>100 1.5
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
5y
5z
6a
6b
6c
>100 1.5
>100 1.9
>100 1.2
>100 1.8
>100 0.8
>100 1.2
Harmine
a Each datum represents mean + S.D. from three different experiments performed in triplicate. b HeLa: human cervix cancer cell line. c DU145:
human prostate cancer cell line. d A549: human lung adenocarcinoma epithelial cell line. e BHK-21: Hamster kidney cells. f L929: mouse
connective tissue fibroblast cells.
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Fig. 2 CD spectra of CT DNA (15 μM) at two concentrations of ligands
(5a, 5d, 5h and 5r).
band at 275 nm and a negative band at 245 nm due to π–π
base stacking and right-hand helicity which is the character-
istic profile of B form DNA. In the present study, the positive
band exhibited hyperchromicity on addition of 5a to CT DNA
in a 1 : 0.5 DNA : ligand (5a) ratio, manifesting stabilization of
the DNA structure on ligand interaction. The negative band
intensities gradually reduced, indicating a decrease in the
DNA helicity on complex interaction with DNA. On further
increasing the concentration of the ligand, the positive band
at 275 nm exhibited hypochromicity, indicating unfolding of
the DNA structure. A similar type of interaction was also
noticed in the case of ligands 5h, 5r and 5d. The results indi-
cate that these ligands stabilize the DNA at lower concen-
tration and, upon increasing the ligand concentration, they
start unfolding of the CT DNA structure.50
Fig. 3 The UV-visible absorption changes with the titration of ligands
(5a, 5d, 5h and 5r) by CT DNA.
UV-visible spectral studies. UV-visible spectroscopic titra-
tion studies were carried out to obtain preliminary information
about the mode of DNA–ligand interactions. The UV-visible
spectra of ligand 5a demonstrate a prominent absorption
band at 285 nm and 342 nm. On addition of equal increments
of 2 µM CT DNA to 25 µM ligand solution, the ligand absorp-
tion band intensities were reduced continuously. The hypo-
chromicity of the ligand Soret band with the addition of DNA
is a characteristic feature of ligand intercalation with DNA.
The reduction in the intensities of the ligand Soret band is
usually attributed to the interaction between the electronic
states of the compound and those of the DNA bases.51 More-
over the extent of hypochromism of the Soret band generally
indicates the intercalative binding strength.52 The interaction
of ligands 5h, 5r and 5d was also similar to 5a, manifesting
their intercalative mode of binding with DNA.
Fig. 4 Agarose gel picture showing the intercalation of 5a, 5h, 5r and
5d (2 µM) with DNA (5 µM) in Tris buffer (pH 7.0). Lane 1, (5a + DNA);
lane 2, (5 h + DNA); lane 3, (5r + DNA); and lane 4 (5d + DNA).
maybe due to the intercalation. The intercalation nature of
these ligands was further confirmed by performing the DNA
intercalation assay. The pBR322 plasmid DNA was incubated
with these ligands for 1 h at 37 °C and then loaded on a 0.8%
agarose gel (Fig. 4).
After running the DNA–ligand complex for 1 h, DNA bands
were seen in the agarose gel indicating that 5a, 5h, 5r and 5d
ligands exhibit intercalative mode of DNA binding.
Photocleavage studies
DNA photocleavage reaction was carried out in the presence of
5a, 5d and 5r to find the efficiency of these conjugates in gen-
erating free radicals in the system. Generally, photocleavage
reactions proceed through the generation of molecular oxygen
or hydroxyl radical species. These reactions involving the
triplet oxygen state (3O2) are known to proceed by two major
mechanistic pathways. In the first one, the singlet excited elec-
tronic state of the hybrid through intersystem crossing gener-
ates an excited triplet state of the hybrid which in turn
In the case of ligand 5a, an isosbestic point was observed at
325 nm indicating the existence of at least one 5a-DNA
complex with identical molar absorptivity. In the case of other
ligands, a sharp isosbestic point was observed at around
335 nm and this shows that their interaction with DNA occurs
in a single step (Fig. 3).
DNA intercalation assay
It is known that the unfolding of DNA takes place due to small activates the molecular oxygen in its stable triplet oxygen state
molecule intercalation between the bases.53 From the spectro- (3O2) to a more reactive singlet oxygen state (1O2).54 In the
scopic studies it is evident that these ligands unfold the DNA second pathway, the excited state molecule could reduce the
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molecular oxygen to generate the highly reactive hydroxyl the irradiation time. After 2 h, complete disappearance
radical. of supercoiled DNA was observed. This indicates that 5a
When pBR322 plasmid DNA in the presence of ligands was has a higher photocleavage capacity, which increases with
subjected to electrophoresis, relatively fast migration was irradiation time (Fig. 5B).
observed for the supercoiled form (Form I). If scission occurs
on one strand, the supercoiled form will relax to generate a
relaxed circular form (Form II). In the present study, the
pBR322 plasmid DNA was irradiated with UV light (365 nm) in
the presence and absence of ligands and subjected to electro-
phoresis. The gel electrophoresis separation of pBR322 DNA
after UV light irradiation in the presence of two different con-
centrations (100 µM and 200 µM) of the 5a, 5r and 5d ligands
is shown in Fig. 5A. It is evident that the control samples as
well as DNA + ligands (100 µM) incubated in the absence of
light did not show any considerable cleavage (lanes 8–10,
Fig. 5A). However, the pBR322 DNA samples irradiated along
with ligands exhibited remarkable photocleavage as indicated
by the decrease in the band intensity of supercoiled DNA. The
photocleavage studies show that all the three ligands are
efficient in cleaving pBR322 plasmid DNA; however, the effect
of photocleavage is prominent in the cases of 5a followed by
5r and 5d.
Moreover, the photocleavage of pBR322 DNA was enhanced
on increasing the concentration (200 μM) of the ligand. On the
other hand, 5a is considerably active even at lower concen-
tration (100 µM) demonstrating its higher ability to generate
free radicals for the effective cleavage of the DNA. The photo-
cleavage activity of these hybrids may be due to the presence
of –NH and polyaromatic rings, which could be involved in
n–π* and π–π* transitions upon their intercalation with DNA.
The photocleavage efficiency of these ligands depends on their
relative orientation upon interaction with DNA and the proxi-
mity of the –NH group to DNA. From the DNA binding studies
it was also found that the 5a, 5r and 5d ligands intercalate
with DNA. The above results suggest that these ligands inter-
calate with DNA and generate the free radicals on exposure to
UV light.
DNA topoisomerase I inhibition
It is well known that DNA topoisomerase I (Topo I) binds to
double stranded supercoiled DNA and cuts the single stranded
portion, which releases the superhelical tension in DNA and
transforms it to a relaxed form. Topo I inhibitors have gained
importance in cancer chemotherapy treatment as they inhibit
Topo I and leave single stranded breaks in DNA, thereby dama-
ging the genome integrity. Topo I has been identified as a
potential target for several anticancer drugs that are clinically
in use even today. Since Topo I is involved in the replication
and proliferation process, overproduction of Topo I was
observed in cancer cells compared to normal cells. The Topo I
inhibition occurs in two ways. The inhibitors may bind topo-
isomerase directly or they may bind to DNA and alter its struc-
ture, so that it cannot be recognised by topoisomerases.
Cao and coworkers reported that camptothecin inhibits
Topo I at 100 µM concentration whereas harmine derivatives
inhibit Topo I at 150 µM55.
Interestingly, the present β-carboline–benzimidazole
scaffolds have also shown significant Topo I inhibition at 100
as well as 200 µM concentrations (Fig. 6). The observed low
IC50 values for the conjugates 5a, 5d, 5h and 5r may be the
result of effective Topo I inhibition as well as better DNA
intercalation.
Molecular docking studies
Docking studies were performed to obtain an insight into the
mode of binding of the C1 and C3 substituted β-carboline con-
jugates to the protein and the DNA ternary complex. Geome-
tries of all the conjugates (5a, 5h, 5r and 5d) were optimized by
the PM3 method using the Gaussian 09 package.56 The coordi-
nates of the protein were obtained from the Protein Data Bank
(PDB ID: 1SC7)57 and necessary corrections to the protein
structure were carried out using the Protein Preparation
Wizard in Schrodinger package. The docking studies were per-
formed using GOLD docking software; docking poses showed
that these conjugates intercalate with DNA at the binding site
of the ternary complex (Fig. 7). The docking pose for 5a shows
that there is a hydrogen-bonding interaction between the N2
With a view to understanding the effect of DNA photoclea-
vage as a function of irradiation time, 100 µM of 5a was
irradiated with UV light at different time intervals (15 min,
30 min, 60 min, 90 min and 120 min). It was observed that the
intensity of the band corresponding to Form I diminishes
gradually, whereas that of Form II increases upon increasing
Fig. 5 Lane 1 (A & B), DNA solution irradiated in the absence of com-
pounds; (A) lanes 2–4, conjugates 5a, 5r and 5d (100 μM) + pBR 322
plasmid DNA; lanes 5–7, conjugates 5a, 5r and 5d (200 μM) + pBR 322
plasmid DNA; lanes 8–10, DNA + conjugates in the absence of light. (B)
Effect of 5a as a function of the irradiation time (0.15, 0.30, 1, 1.5, 2 h;
lanes 2–6). Form II, relaxed circular DNA; Form I, supercoiled DNA.
Fig. 6 Effects of 5a, 5r, 5d and 5h on the activity of DNA topoisomerase
I in a cell free system. Lane 1, DNA alone; lane 2, DNA + Topo I; lane 3,
DNA + Topo I + camptothecin (100 μM); lanes 4–7, DNA + Topo I +
compounds 5a, 5h, 5r and 5d (100 μM) (A) and the same in (B) (200 μM);
Form II, relaxed circular DNA; Form I, supercoiled DNA.
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Fig. 7 Docking pose for 5a in DNA Topo I; hydrogen bonds are shown
in red dotted lines, and hydrogen bonding residues are shown in green
colour; nucleic acid residues which are showing π–π stacking are shown
in brown colour; amino acids having hydrophobic interactions are
shown in gray colour.
Fig. 8 Docking pose for 5a in DNA showing the intercalation-binding
mode.
In silico computational studies
of a β-carboline ring and the guanidine group of Arg364
similar to the cocrystal ligand; besides this, it also possesses
an additional hydrogen bonding interaction between the
methoxy group of the benzimidazole ring and the amino
group of the side chain of Lys374. The benzimidazole ring
shows π–π* interactions with the C112 and A113 DNA base
pairs; on the other hand, the β-carboline ring shows π–π* inter-
actions with the C10 and G11 DNA base pairs, whereas C1 sub-
stitution extends outwards to the minor groove. In addition to
this, 5a possesses hydrophobic interactions with amino acids
close to the binding site, in which the C1 substitution shows
interaction with the Asp533 and Ile535, the β-carboline ring
with Asn722 and Arg364 and the benzimidazole ring with
Glu365 and Lys425. As compared to the cocrystal ligand, these
conjugates exhibit a high gold score. These studies indicate
that the C1 and C3 substituted β-carbolines are capable of
fitting properly in the binding site of the DNA topoisomerase I.
The biophysical studies have already shown that these con-
jugates possess DNA intercalation properties. Therefore,
docking studies were performed to obtain a better insight into
the binding mode of these conjugates to the DNA. The coordi-
nates of the DNA were obtained from the Protein Data Bank
(PDB ID 1NAB).58 Necessary corrections to the crystal structure
were carried out using the protein preparation wizard in Schro-
An in silico computational study of the representative C-3 sub-
stituted β-carboline–benzimidazole conjugates (5a–d, 5h, 5r,
5w) was performed for determining the Lipinski’s parameters,
topological polar surface area (TPSA) and percentage of
absorption (% ABS).60–62 Calculations were performed using
the Molinspiration online Property Calculation Toolkit (http://
www.molinspiration.com)61 and OSIRIS Property Explorer
(http://www.organic-chemistry.org/prog/peo).62 The percentage
of absorption was estimated using the equation: % ABS = 109
− 0.345 × TPSA60 and the data generated are shown in Table 5.
In vivo absorption of the new synthesized derivatives was
tentatively assessed by means of theoretical calculations fol-
lowing Lipinski’s rule of five, which establishes that the
absorption or permeation of an orally administered compound
is more likely to be good if the molecule satisfies the following
criteria: (a) hydrogen bond donors ≤5 (OH and NH groups);
(b) hydrogen bond acceptors ≤10 (N and O atoms); (c) mole-
cular weight < 500; (d) calculated log P < 5.60–62 Compounds
that violate more than one of these parameters could have pro-
blems related to bioavailability.
Conclusion
dinger. Docking studies showed that all conjugates (5a, 5h, 5r In summary, we have synthesized a series of β-carboline–benz-
and 5d) bind to the DNA through intercalation. The docking imidazole conjugates bearing the substituted benzimidazole
pose of 5a shows that the β-carboline ring lies in the central moiety at C3 and the substituted aryl group at C1. The final
part of the DNA intercalation cavity and is stacked between the key step of the benzimidazole ring formation was carried out
C5, G6 of chain A and C7, G8 of chain B, while C1 and C3 sub- using La(NO3)3·6H2O as a catalyst that resulted in higher yields
stitution extended outwards. The phenyl substitution extended with chemoselectivity. In general, this method may be highly
towards the minor groove and the benzimidazole substitution useful for the chemoselective formation of various 2-sustituted
towards the major groove (Fig. 8).
benzimidazole derivatives. The SAR analysis reveals that
Docking studies on DNA topoisomerase-I and CT DNA amongst the conjugates synthesized, the ones having
showed that these conjugates have the potential to bind to the 4-methoxy phenyl substitution at C1 (5a–e) exhibit significant
DNA Topo-I and also intercalate with DNA. Images for docking activity. Surprisingly, some conjugates with 3,4-difluoro phenyl
poses were generated using Pymol visualization software.59
and 4-fluoro phenyl rings at C1 (5h and 5r) are also equally
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Table 5 Lipinski’s parameters and % ABS, TPSA, log S for compounds 5a–d, 5h, 5r and 5w
Lipinsk’s parameters
nHBA
(ON)
nHBD
(OHNH)
n
Compound
% ABS
TPSAa (A2)
Log Pb
MW
violations
Log Sb
5a
5b
5c
5d
5h
5r
82.84
86.02
86.02
86.02
86.02
89.21
79.65
75.8
66.6
66.6
66.6
66.6
57.3
85.0
6
5
5
5
5
4
7
2
2
2
2
2
2
2
5.27
5.69
6.01
5.44
5.51
6.16
5.17
420
404
418
408
426
412
450
1
1
1
1
1
1
1
−6.71
−7.06
−7.38
−7.01
−7.32
−7.72
−6.73
5w
a http://www.molinspiration.com; b http://www.organic-chemistry.org/prog/peo; % ABS = 109 − 0.345 × TPSA; number of hydrogen bond acceptors
(NO) = nHBA ≤ 10; number of hydrogen bond donors (OHNH) = nHBD ≤ 5; MW ≤ 500; octanol–water partition coefficient = log P < 5; solubility =
log S > −4.
active irrespective of the SAR analysis. The representative con- melting point apparatus, and are uncorrected. 1H NMR and
jugates (5a, 5d, 5h and 5r) showed potential cytotoxic activity 13C NMR spectra of the final compounds 5a–z, 6a–c are pro-
with GI50 values ranging from 0.36 to 7.1 μM in most of the vided in the ESI.†
human cancer cell lines panel of the NCI. These conjugates
also showed promising DNA topoisomerase-I inhibition
General procedure for the preparation of compounds (2a–e)
activity. Further, biophysical studies speculated that these con-
jugates could intercalate into the DNA, which is supported by
molecular docking studies. The DNA photocleavage activity of
these conjugates was measured using pBR322 plasmid DNA in
the presence of UV light. This study suggests that 5a has the
potential to generate singlet oxygen species which cleaves DNA
by converting the supercoiled form to a relaxed/circular form
and can be used in photodynamic therapy. Based on the above
results it is evident that these β-carboline–benzimidazole con-
jugates (particularly 5a) have the potential to be developed as
a new class of cancer therapeutics. Detailed cellular and mole-
cular biology as well as in vivo studies are actively in progress
in our laboratory.
To a stirred solution of L-tryptophan (0.1 mol) in methanol
(50 mL), 8.02 mL (0.11 mol) of thionyl chloride was added
dropwise at 0 °C and stirring was continued for 6.0 h at room
temperature. Then, an excess amount of solvent was removed
under vacuum and the crude product was co-distilled with
toluene (2 × 10 mL) to obtain a solid. Then, the resulting solid
was dissolved in CH2Cl2, washed with saturated NaHCO3 solu-
tion, and extracted with an excess amount of CH2Cl2. Then,
the combined organic layers were dried over anhydrous
sodium sulphate and concentrated under vacuum to obtain a
white solid product. To
a mixture of tryptophan ester
(0.023 mol) and substituted benzaldehyde (0.023 mol) in
toluene (50 mL), a catalytic amount of PTSA was added and
the mixture was refluxed for 24 h. After completion of the reac-
tion, the solvent was removed under vacuum. The crude
mixture was extracted with ethyl acetate and the combined
organic layers were dried over anhydrous sodium sulphate and
concentrated under vacuum. Then, the resulting diastereo-
meric mixture (2a–e) obtained was used for the next step
without further purification.
Materials and methods
Synthesis data
Chemistry. All chemicals and reagents were obtained from
Aldrich (Sigma-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
General procedure for the preparation of compounds (3a–e)
further purification. TLC was performed on silica gel glass The suspension of the above compounds (2a–e, 5 gr) and
plates containing 60 GF-254 and visualization was achieved by sulphur (0.075 mol) in xylene (100 mL) was refluxed for 12 h.
UV light or iodine indicator monitored reactions. Column Then, the mixture was cooled to room temperature and kept at
chromatography was performed with Merck 60–120 mesh 4 °C for 3 h to obtain a precipitate. Then, the precipitate was
silica gel. 1H and 13C spectra were recorded on Bruker filtered, washed with petroleum ether and dried. The obtained
UXNMR/XWIN-NMR (300 MHz) or Inova Varian-VXR-unity solid was recrystallized using ethyl acetate to afford products
(400, 500 MHz) instruments. Chemical shifts (δ) are reported (3a–e) with high purity.
in ppm downfield from a TMS internal standard. ESI spectra
Methyl
1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-car-
were recorded on a Micromass Quattro LC using ESI+ software boxylate (3a). White solid; 80% yield; mp: 228–230 °C; 1H
with a capillary voltage of 3.98 kV and an ESI mode positive NMR (300 MHz, CDCl3) δ (ppm): 9.18 (bs, 1H), 8.80 (s, 1H),
ion trap detector. High-resolution mass spectra (HRMS) were 8.17 (d, J = 7.7 Hz, 1H), 7.78 (d, J = 8.8 Hz, 2H), 7.57–7.55 (m,
recorded on a QSTAR XL Hybrid MS-MS mass spectrometer. 2H), 7.36–7.33 (m, 1H), 6.88 (d, J = 8.6 Hz, 2H), 4.03 (s, 3H),
Melting points were determined with an electrothermal 3.77 (s, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm): 165.8, 159.6,
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141.8, 141.2, 136.2, 134.2, 129.8, 129.7, 129.1, 128.7, 128.0, EtOAc–n-hexane (1 : 1) to afford products (4a–e) with high
121.2, 120.9, 119.9, 115.7, 113.7, 113.1, 112.4, 54.9, 51.6; MS purity.
(ESI): m/z 333 [M + H]+.
1-(4-Methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carbaldehyde
Methyl 1-(3,4-difluorophenyl)-9H-pyrido[3,4-b]indole-3-car- (4a). Pale yellow solid; 80% yield; mp: 218–220 °C; 1H NMR
boxylate (3b). White solid; 71% yield; mp: 236–239 °C; (300 MHz, CDCl3) δ (ppm): 10.62 (s, 1H), 9.18 (bs, 1H), 8.80
1H NMR (300 MHz, DMSO[d6]) δ (ppm): 12.05 (bs, 1H), 8.97 (s. 1H), 8.17 (d, J = 7.7 Hz, 1H), 7.78 (d, J = 8.8 Hz, 2H),
(s, 1H), 8.46 (d, J = 7.9 Hz, 1H), 7.60–7.78 (m, 4H), 7.45 (t, J = 7.57–7.55(m, 2H), 7.36–7.33 (m, 1H), 6.88 (d, J = 8.6 Hz, 2H),
9.4 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 3.94 (s, 3H); 13C NMR 4.03 (s, 3H); 13C NMR (75 MHz, CDCl3 + DMSO[d6]) δ (ppm):
(75 MHz, DMSO[d6]) δ (ppm): 163.6, 143.0, 141.5, 135.2, 134.3, 192.5, 159.7, 142.9, 142.2, 141.3, 135.0, 131.6, 130.7, 129.6,
131.4, 130.9, 130.2, 129.1, 126.1, 122.1, 121.1, 120.7, 114.4, 128.8, 128.2, 128.1, 121.3, 120.1, 113.7, 112.6, 54.9; MS (ESI):
112.8, 111.9, 111.5, 52.1; MS (ESI): m/z 339 [M + H]+.
m/z 303 [M + H]+.
Methyl1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indole-
1-(3,4-Difluorophenyl)-9H-pyrido[3,4-b]indole-3-carbalde-
3-carboxylate (3c). Pale yellow solid; 72% yield; mp: hyde (4b). Pale yellow solid: 84% yield; mp: 225–228 °C; 1H
252–254 °C; 1H NMR (300 MHz, DMSO[d6]) δ (ppm): 11.99 (bs, NMR (300 MHz, DMSO[d6]) δ (ppm): 10.16 (s, 1H), 8.88 (s, 1H),
1H), 8.93 (s, 1H), 8.36 (d, J = 7.7 Hz, 1H), 8.23 (d, J = 7.7 Hz, 8.48 (d, J = 7.9 Hz, 1H), 7.92–8.03 (m, 2H), 7.62–7.80 (m, 2H),
2H), 7.92 (d, J = 8.1 Hz, 2H), 7.68 (d, J = 8.1 Hz, 1H), 7.59 (t, J = 7.44–7.49 (m, 1H), 7.37 (t, J = 7.9 Hz, 1H); 13C NMR (75 MHz,
7.9 Hz, 1H), 7.32 (t, J = 7.5 Hz, 1H), 3.94 (s, 3H); 13C NMR DMSO[d6]) δ (ppm): 192.6, 143.0, 141.5, 135.2, 134.3, 132.2,
(75 MHz, CDCl3 + DMSO[d6]) δ (ppm): 163.9, 139.7, 138.4, 130.9, 130.2, 129.1, 126.1, 122.1, 120.7, 114.4, 112.8, 111.9,
134.9, 132.8, 127.7, 127.5, 127.1, 126.8, 124.1, 123.6, 119.9, 111.5; MS (ESI): m/z: 309 [M + H]+.
118.5, 115.2, 110.8, 50.1; MS (ESI): m/z 371 [M + H]+.
1-(4-(Trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indole-3-carb-
Methyl 1-(4-fluorophenyl)-9H-pyrido[3,4-b]indole-3-carboxy- aldehyde (4c). Pale yellow solid: 84% yield; mp 280–283 °C;
late (3d). White solid: 76% yield; mp: 197–198 °C; 1H NMR 1H NMR (300 MHz, DMSO[d6]) δ (ppm): 12.15 (s, 1H), 10.16 (s,
(300 MHz, CDCl3) δ (ppm): 8.68–8.76 (bs, 1H), 8.84–8.90 (s, 1H), 8.86 (s, 1H), 8.44 (d, J = 7.9 Hz, 1H), 8.28 (t, J = 8.1 Hz,
1H), 7.88 (d, J = 5.4 Hz, 2H), 7.60–7.68(t, J = 5.4 Hz, 1H), 2H), 8.01 (d, J = 8.1 Hz, 2H), 7.61–7.71 (m, 2H), 7.36 (t, J = 7.9
7.52–7.58 (d, J = 7.7 Hz, 1H), 7.36–7.44 (t, J = 7.9 Hz, 1H), 7.26 Hz, 1H); 13C NMR (75 MHz, CDCl3 + DMSO[d6]) δ (ppm):
(d, J = 8.6 Hz, 2H), 4.04 (s, 3H); 13C NMR (75 MHz, CDCl3
+
192.4, 143.1, 141.5, 140.9, 135.4, 129.8, 129.5, 129.1, 128.7,
DMSO[d6]) δ (ppm): 165.7, 164.0, 160.7, 141.3, 136.4, 134.3, 125.7, 125.3, 122.1, 121.8, 121.1, 120.4, 113.9, 112.6, 125.7; MS
133.8, 130.6, 130.5, 128.2, 121.5, 120.9, 120.1, 116.3, 115.4, (ESI): m/z: 341 [M + H]+.
115.1, 112.4, 51.7; MS (ESI): m/z 291 [M + H]+.
1-(4-Fluorophenyl)-9H-pyrido[3,4-b]indole-3-carbaldehyde
Methyl1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3- (4d). White solid of 90% yield; mp: 189–190 °C; 1H NMR
1
carboxylate (3e). Yellow solid: 76% yield; mp: 229–230 °C; H (300 MHz, DMSO[d6]) δ (ppm): 11.64 (bs, 1H), 10.24 (s, 1H),
NMR (300 MHz, CDCl3) δ (ppm): 9.38 (bs, 1H), 8.75 (s, 1H), 8.68 (s, 1H), 8.23 (d, J = 7.7 Hz, 1H), 8.04–8.16 (m, 2H), 7.68 (d,
8.21(d, J = 7.5 Hz, 1H), 7.60 (d, J = 7.5 Hz, 1H), 7.56–7.61 (m, J = 7.7 Hz, 1H), 7.56–7.62 (m, 1H), 7.37 (d, J = 7.7 Hz, 2H), MS
1H), 7.32–7.38 (m, 1H), 6.94 (s, 2H), 4.03 (s, 3H), 3.84 (s, 3H), (ESI): m/z 291 [M + H]+.
3.77 (s, 6H); 13C NMR (75 MHz, DMSO[d6]) δ (ppm): 166.1,
1-(3,4,5-Trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carbal-
1
159.8, 141.9, 141.3, 136.5, 134.3, 129.9, 128.9, 128.4, 121.8, dehyde (4e). Pale yellow solid: 80% yield; mp: 213–214 °C; H
121.1, 120.2, 116.1, 114.0, 112.7, 55.2, 51.9; MS (ESI): m/z 363 NMR (300 MHz, CDCl3 + DMSO[d6]) δ (ppm): 10.29 (s, 1H),
[M + H]+.
9.18 (bs, 1H), 8.72 (s, 1H), 8.23 (d, J = 7.9 Hz, 1H), 7.66–7.61
(m, 2H), 7.42–7.39 (m, 1H), 7.15 (s, 2H), 3.95 (s, 6H), 3.93 (s,
3H); 13C NMR (75 MHz, CDCl3 + DMSO[d6]) δ (ppm): 193.5,
General procedure for the preparation of compounds (4a–e)
The compounds (3a–e, 0.019 mol) taken in dry THF (100 mL) 153.7, 144.1, 143.4, 141.5, 140.9, 138.6, 136.0, 133.1, 131.5,
were cooled to −5 °C to 0 °C in an ice–salt (NaCl) bath. To the 129.7, 129.1, 122.2, 122.0, 121.3, 113.7, 112.2, 105.4, 60.8, 56.2;
reaction mixture, LAH (0.076 mol) was added slowly portion- MS (ESI): m/z 363 [M + H]+.
wise and stirring was continued at room temperature for about
4 h. After completion of the reaction, further the reaction
mixture was cooled to 0 °C and the excess of LAH was
General procedure for the preparation of compounds
(5a–z and 6a–c)
quenched with Na2SO4 paste, filtered on a Buckner funnel To a solution of 4a–e (1 equiv.) and the respective o-phenylene-
washed with MeOH (2 × 20 mL). The filtrate was dried over diamine (1 equiv.) in ethanol (20 mL), 10 mol% of catalyst
anhydrous Na2SO4 and concentrated under vacuum. The crude (La(NO3)3·6H2O) was added. The reaction mixture was heated
mixture obtained (0.014 mol) was taken in dry CH2Cl2 at 60 °C for a stipulated time mentioned in Table 2. After the
(100 mL). To that, DMP (0.021 mol) was added, and stirred at completion of the reaction, ethanol was removed under
room temperature for about 2 h. After the completion of the vacuum. The crude reaction was dissolved in ethyl acetate
reaction the reaction mixture was washed with water (2 × (50 mL) and washed with water (2 × 10 mL). The organic layer
100 mL), and the organic layer was dried over anhydrous was dried over anhydrous Na2SO4 and concentrated under
Na2SO4, and concentrated under vacuum. Then, the resulting vacuum. Then the crude product was purified by silica gel
crude obtained was purified by column chromatography using column chromatography using EtOAc–n-hexane (4 : 6) as the
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eluent to afford the final products (5a–z and 6a–c) with high CDCl3 + DMSO[d6]) δ (ppm): 10.59 (bs, 1H), 9.00 (s, 1H), 8.68
purity. (s, 1H), 8.15 (d, J = 7.5 Hz, 1H), 7.97 (d, J = 8.3 Hz, 2H),
3-(6-Methoxy-1H-benzo[d]imidazol-2-yl)-1-(4-methoxyphe- 7.61–7.51 (m, 3H), 7.34 (t, J = 6.7 Hz, 1H), 7.11 (d, J = 9.0 Hz,
nyl)-9H-pyrido[3,4-b]indole (5a). This compound was prepared 2H), 7.02 (dt, J = 6.7, 2.2 Hz, 1H), 3.91 (s, 3H); 13C NMR
according to the general procedure, employing 4a (200 mg, (175 MHz, CDCl3 + DMSO) δ (ppm): 159.6, 158.4 (d, J = 235.2
0.66 mmol) and 4-methoxybenzene-1,2-diamine (91 mg, Hz, ArC-F), 153.6, 141.5, 141.4, 137.2, 133.2, 130.1, 129.9,
0.66 mmol) to obtain pure product 5a as a pale yellow solid. 129.7, 121.0, 121.1, 119.6, 113.6, 112.3, 111.1, 109.5, 54.9; MS
Yield: 222 mg (80%); mp: 166–168 °C; IR (KBr): λmax/cm−1
=
(ESI, m/z): 409 [M + 1]+; HRMS (ESI, m/z) calculated for
3420, 2929, 2832, 1663, 1624, 1452, 1401, 1244, 1026, 834, 744, C25H18ON4F: 409.14592, found: 409.14500 [M + 1]+.
1
578, 507, 435; H NMR (300 MHz, CDCl3) δ (ppm): 10.49 (bs,
3-(1H-Benzo[d]imidazol-2-yl)-1-(4-methoxyphenyl)-9H-pyrido-
1H), 8.98 (s, 1H), 8.74 (s, 1H), 8.14 (d, J = 8.3 Hz, 1H), [3,4-b]indole (5e). This compound was prepared according to
7.98–7.94 (m, 2H), 7.58–7.49 (m, 2H), 7.35–7.29 (m, 2H), the general procedure, employing 4a (200 mg, 0.66 mmol) and
7.12–7.09 (m, 2H), 6.91 (dd, J = 2.26, 8.30 Hz, 1H), 3.89 (s, 3H), benzene-1,2-diamine (71 mg, 0.66 mmol) to obtain pure
3.87 (s, 3H); 13C NMR (125 MHz, CDCl3) δ (ppm): 160.2, 141.9, product 5e as a pale yellow solid. Yield: 224 mg (87%); mp:
140.7, 138.3, 133.7, 130.5, 130.3, 129.4, 128.6, 122.1, 122.0, 201–202 °C; IR (KBr): λmax/cm−1 = 3435, 3061, 2929, 1624,
120.6, 114.4, 111.6, 55.7, 55.3; MS (ESI, m/z): 421 [M + 1]+; 1608, 1563, 1513, 1497, 1468, 1453, 1427, 1407, 1321, 1298,
1
HRMS (ESI, m/z) calculated for C26H21O2N4: 421.16590, found: 1244, 1176, 1111, 1030, 838, 742, 613, 585; H NMR (300 MHz,
421.16486 [M + 1]+.
CDCl3) δ (ppm): 10.57 (bs, 1H), 9.05 (s, 1H), 8.74 (s, 1H), 8.15
1-(4-Methoxyphenyl)-3-(6-methyl-1H-benzo[d]imidazol-2-yl)- (d, J = 7.5 Hz, 1H), 7.96 (d, J = 9.0 Hz, 2H), 7.60–7.52 (m, 3H),
9H-pyrido[3,4-b]indole (5b). This compound was prepared 7.35–7.26 (m, 3H), 7.11 (d, J = 8.3 Hz, 2H), 3.91 (s, 3H); 13C
according to the general procedure, employing 4a (200 mg, NMR (75 MHz, CDCl3) δ (ppm): 159.1, 151.9, 141.1, 140.1,
0.66 mmol) and 4-methylbenzene-1,2-diamine (80 mg, 137.0, 132.9, 129.8, 129.3, 127.3, 121.1, 120.7, 120.5, 119.1,
0.66 mmol) to obtain pure product 5b as a pale yellow solid. 113.1, 111.7, 110.6, 54.4; MS (ESI, m/z): 391 [M + 1]+; HRMS
Yield: 219 mg (82%); mp: 188–190 °C; IR (KBr): λmax/cm−1
=
(ESI, m/z) calculated for C25H19ON4: 391.15534, found:
3420, 3054, 2920, 1719, 1607, 1564, 1510, 1454, 1401, 1316, 391.15518 [M + 1]+.
1
1243, 1174, 1109, 875, 802, 743, 576, 434; H NMR (300 MHz,
(2-(1-(4-Methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)-1H-
CDCl3 + DMSO [d6]) δ (ppm): 11.09 (bs, 1H), 9.00 (s, 1H), 8.13 benzo[d]imidazol-6-yl)(phenyl)methanone (5f). This com-
(d, J = 8.6 Hz, 3H), 7.66–7.46 (m, 4H), 7.27 (t, J = 7.3 Hz, 1H), pound was prepared according to the general procedure,
7.10 (d, J = 8.6 Hz, 2H), 7.05 (d, J = 8.1 Hz, 1H), 3.90 (s, 3H), employing 4a (200 mg, 0.66 mmol) and (3,4-diaminophenyl)-
2.49 (s, 3H); 13C NMR (75 MHz, CDCl3 + DMSO[d6]) δ (ppm): (phenyl)methanone (140 mg, 0.66 mmol) to obtain pure
159.6, 152.1, 141.5, 141.2, 137.5, 133.4, 131.5, 130.4, 130.0, product 5f as a pale yellow solid. Yield: 278 mg (85%); mp:
129.7, 127.9, 123.3, 121.3, 121.1, 119.6, 113.6, 111.9, 111.1, 186–188 °C; IR (KBr): λmax/cm−1 = 3366, 2924, 2356, 1644,
54.9, 21.2; MS (ESI, m/z): 405 [M + 1]+; HRMS (ESI, m/z) calcu- 1610, 1573, 1513, 1494, 1464, 1447, 1426, 1403, 1320, 1247,
1
lated for C26H21ON4: 405.17099, found: 405.17072 [M + 1]+.
1176, 1112, 1028, 893, 741, 718, 587, 456; H NMR (500 MHz,
3-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)-1-(4-methoxyphe- CDCl3 + DMSO[d6]) δ (ppm): 10.80 (bs, 1H), 9.08 (d, J = 18.3
nyl)-9H-pyrido[3,4-b]indole (5c). This compound was prepared Hz, 1H), 8.76 (s, 1H), 8.18 (s, 1H), 7.98 (d, J = 8.3 Hz, 2H),
according to the general procedure, employing 4a (200 mg, 7.89–7.79 (m, 3H), 7.60–7.53 (m, 4H), 7.48 (t, J = 7.7 Hz, 2H),
0.66 mmol) and 4,5-dimethylbenzene-1,2-diamine (90 mg, 7.35 (t, J = 7.3 Hz, 1H), 7.12 (d, J = 8.0 Hz, 2H), 3.91 (s, 3H); 13
C
0.66 mmol) to obtain pure product 5c as a pale yellow solid. NMR (125 MHz, CDCl3 + DMSO[d6]) δ (ppm): 194.8, 158.7,
Yield: 235 mg (85%); mp: 246–248 °C; IR (KBr): λmax/cm−1
140.8, 140.5, 137.2, 135.9, 132.6, 130.4, 129.8, 129.2, 129.0,
=
3461, 3058, 2920, 2852, 1723, 1607, 1510, 1455, 1399, 1315, 128.8, 128.2, 127.0, 126.7, 120.1, 120.0, 118.8, 112.6, 111.4,
1
1241, 1174, 1109, 1024, 836, 746, 578, 435; H NMR (300 MHz, 110.7, 54.0; MS (ESI, m/z): 495 [M + 1]+; HRMS (ESI, m/z) calcu-
CDCl3) δ (ppm): 10.49 (bs, 1H), 9.02 (s, 1H), 8.65 (s, 1H), 8.16 lated for C32H23O2N4: 495.18155, found: 495.18005 [M + 1]+.
(d, J = 7.5 Hz, 1H), 7.97 (d, J = 9.0 Hz, 2H), 7.59–7.50 (m, 3H),
1-(4-Methoxyphenyl)-3-(6-(trifluoromethyl)-1H-benzo[d]imi-
7.33 (t, J = 6.7 Hz, 2H), 7.16 (d, J = 8.3 Hz, 2H), 3.92 (s, 3H), dazol-2-yl)-9H-pyrido[3,4-b]indole (5g). This compound was
2.40 (s, 6H); 13C NMR (125 MHz, CDCl3) δ (ppm): 160.3, 151.6, prepared according to the general procedure, employing 4a
141.9, 140.7, 138.6, 133.7, 132.4, 130.6, 130.5, 129.4, 128.7, (200 mg, 0.66 mmol) and 4-(trifluoromethyl)benzene-1,2-
122.2, 122.1, 120.7, 114.6, 111.6, 55.4, 29.6, 20.4; MS (ESI, m/z): diamine (116 mg, 0.66 mmol) to obtain pure product 5g as a
419 [M + 1]+; HRMS (ESI, m/z) calculated for C27H23ON4: pale brown colour solid. Yield: 242 mg (80%); mp: 296–298 °C;
419.18446, found: 419.18588 [M + 1]+.
IR (KBr): λmax/cm−1 = 3440, 3086, 2930, 2835, 1626, 1608, 1566,
3-(6-Fluoro-1H-benzo[d]imidazol-2-yl)-1-(4-methoxyphenyl)- 1549, 1513, 1501, 1471, 1455, 1406, 1371, 1328, 1244, 1217,
1
9H-pyrido[3,4-b]indole (5d). This compound was prepared 1161, 1114, 1051, 1029, 975, 932, 819, 666, 611, 589; H NMR
according to the general procedure, employing 4a (200 mg, (300 MHz, CDCl3 + DMSO[d6]) δ (ppm): 11.33 (bs, 1H), 9.03 (d,
0.66 mmol) and 4-fluorobenzene-1,2-diamine (83 mg, J = 6.6 Hz, 1H), 8.23–8.15 (m, 3H), 7.96 (s, 1H), 7.73–7.21 (m,
0.66 mmol) to obtain pure product 5d as a pale yellow solid. 5H), 7.18–7.05 (m, 2H), 3.92 (s, 3H); 13C NMR (175 MHz,
Yield: 229 mg (85%); mp: 232–234 °C; 1H NMR (300 MHz, CDCl3 + DMSO) δ (ppm): 159.6, 154.8, 146.2, 143.3, 141.6,
2380 | Org. Biomol. Chem., 2014, 12, 2370–2387
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136.8, 134.1, 133.4, 130.0, 129.9, 129.6, 128.0, 124.7 (q, J = 19.87; MS (ESI, m/z): 425 [M + 1]+; HRMS (ESI, m/z) calculated
271.4 Hz, ArC-CF3), 122.7, 121.1, 120.9, 119.7, 118.1(m), 115.4, for C26H19N4F2: 425.15723, found: 425.15678 [M + 1]+.
113.6, 112.3, 111.8, 108.9, 54.9; MS (ESI, m/z): 459 [M + 1]+;
3-(6-Bromo-1H-benzo[d]imidazol-2-yl)-1-(3,4-difluorophe-
HRMS (ESI, m/z) calculated for C26H18ON4F3: 459.14032, nyl)-9H-pyrido[3,4-b]indole (5k). This compound was prepared
found: 459.14099 [M + 1]+.
according to the general procedure, employing 4b (200 mg,
1-(3,4-Difluorophenyl)-3-(6-methoxy-1H-benzo[d]imidazol-2- 0.64 mmol) and 4-bromobenzene-1,2-diamine (121 mg,
yl)-9H-pyrido[3,4-b]indole (5h). This compound was prepared 0.64 mmol) to obtain pure product 5k as a pale brown colour
according to the general procedure, employing 4b (200 mg, solid. Yield: 277 mg (90%); mp: 156–158 °C; IR (KBr): λmax
/
0.64 mmol) and 4-methoxybenzene-1,2-diamine (89 mg, cm−1 = 3420, 3059, 2921, 1724, 1607, 1563, 1510, 1457, 1430,
0.64 mmol) to obtain pure product 5h as a pale yellow solid. 1407, 1399, 1315, 1242, 1176, 1109, 1026, 838, 742, 613, 580;
Yield: 248 mg (90%); mp: 173–175 °C; IR (KBr): λmax/cm−1
=
1H NMR (300 MHz, CDCl3 + DMSO[d6]) δ (ppm): 10.06 (bs,
3452, 3059, 2930, 1719, 1625, 1515, 1492, 1451, 1403, 1341, 1H), 7.78 (s, 1H), 7.02–6.82 (m, 2H), 6.78 (s, 1H), 6.57 (s, 1H),
1
1272, 1200, 1153, 1025, 745, 603; H NMR (500 MHz, CDCl3 + 6.43–6.22 (m, 4H), 6.09–6.04 (m, 2H); 13C NMR (75 MHz,
DMSO[d6]) δ (ppm): 10.42 (bs, 1H), 9.00 (s, 1H), 8.85 (s, 1H), CDCl3 + DMSO[d6]) δ (ppm): 152.3, 140.8, 138.2, 136.3, 134.1,
8.10 (d, J = 7.7 Hz, 1H), 7.88–7.84 (m, 1H), 7.77–7.74 (m, 1H), 132.6, 129.7, 129.3, 127.4, 127.3, 124.1, 123.8, 120.2, 119.1,
7.59–7.53 (m, 2H), 7.39–7.30 (m, 3H), 6.94 (dd, J = 8.6, 2.4 Hz, 117.1, 116.8, 116.1, 115.9, 114.4, 114.1, 113.5, 111.4, 111.1; MS
1H), 3.89 (s, 3H); 13C NMR (75 MHz, CDCl3 + DMSO[d6]) δ (ESI, m/z): 475 [M + 1]+; HRMS (ESI, m/z) calculated for
(ppm): 155.3, 151.3, 141.1, 138.3, 137.2, 134.5, 132.7, 130.1, C24H14N4F2Br: 475.03644, found: 475.03595 [M + 1]+.
127.6, 124.3, 120.5, 119.3, 118.6, 117.3, 117.1, 116.4, 116.1,
1-(3,4-Difluorophenyl)-3-(6-methyl-1H-benzo[d]imidazol-2-
111.6, 111.2, 110.8, 54.7; MS (ESI, m/z): 427 [M + 1]+; HRMS yl)-9H-pyrido[3,4-b]indole (5l). This compound was prepared
(ESI, m/z) calculated for C25H17ON4F2: 427.13649, found: according to the general procedure, employing 4b (200 mg,
427.13549 [M + 1]+.
0.64 mmol) and 4-methylbenzene-1,2-diamine (79 mg,
(2-(1-(3,4-Difluorophenyl)-9H-pyrido[3,4-b]indol-3-yl)-1H- 0.64 mmol) to obtain pure product 5l as a yellow colour solid.
benzo[d]imidazol-6-yl)(phenyl)methanone (5i). This com- Yield: 255 mg (96%); mp: 286–288 °C; IR (KBr): λmax/cm−1
=
pound was prepared according to the general procedure, 3451, 3061, 2921, 1625, 1606, 1565, 1517, 1497, 1468, 1453,
employing 4b (200 mg, 0.64 mmol) and (3,4-diaminophenyl) 1436, 1404, 1319, 1274, 1240, 1201, 1155, 1144, 936, 880, 804,
(phenyl)methanone (137 mg, 0.64 mmol) to obtain pure 746, 604, 570, 516, 435; 1H NMR (300 MHz, CDCl3 + DMSO
product 5i as a pale brown colour solid. Yield: 292 mg (90%); [d6]) δ (ppm): 10.39 (bs, 1H), 9.03 (s, 1H), 8.91 (s, 1H), 8.09 (d,
mp: 182–184 °C; IR (KBr): λmax/cm−1 = 3269, 3059, 1721, 1643, J = 8.3 Hz, 1H), 7.89–7.82 (m, 1H), 7.76–7.74 (s, 1H), 7.62–7.50
1614, 1574, 1542, 1517, 1495, 1467, 1446, 1404, 1340, 1242, (m, 2H), 7.39–7.29 (m, 3H), 7.11 (d, J = 8.3 Hz, 1H), 2.51 (s,
1114, 1043, 936, 891, 737, 717, 642, 606, 509, 936; 1H NMR 3H); 13C NMR (75 MHz, CDCl3 + DMSO[d6]) δ (ppm): 151.4,
(300 MHz, CDCl3 + DMSO[d6]) δ (ppm): 12.88 (bs, 1H), 11.59 141.1, 138.5, 137.4, 132.8, 130.9, 130.2, 127.7, 124.4, 122.9,
(s, 1H), 9.06 (s, 1H), 8.33–8.25 (m, 1H), 8.15 (d, J = 7.9 Hz, 1H), 120.6, 119.4, 117.4, 117.2, 116.4, 116.2, 114.7, 111.7, 111.4,
8.11–8.08 (bs, 1H), 7.83–7.77 (m, 4H), 7.67 (d, J = 8.3 Hz, 1H), 111.0, 20.9; MS (ESI, m/z): 411 [M + 1]+; HRMS (ESI, m/z) calcu-
7.64–7.38 (m, 5H), 7.35–7.27 (m, 1H); 13C NMR (75 MHz, lated for C25H17N4F2: 411.14158, found: 411.14059 [M + 1]+.
CDCl3 + DMSO[d6]) δ (ppm): 195.2, 140.9, 140.8, 140.0, 138.4,
3-(6-Chloro-1H-benzo[d]imidazol-2-yl)-1-(3,4-difluorophe-
137.4, 136.3, 134.1, 133.2, 133.0, 132.8, 130.7, 130.3, 129.6, nyl)-9H-pyrido[3,4-b]indole (5m). This compound was pre-
129.9, 129.4, 128.6, 127.6, 127.4, 127.0, 124.2, 123.4, 120.3, pared according to the general procedure, employing 4b
119.3, 119.1, 117.2, 116.9, 116.2, 116.0, 114.5, 114.2, 111.9, (200 mg, 0.64 mmol) and 4-chlorobenzene-1,2-diamine
111.7; MS (ESI, m/z): 501[M + 1]+; HRMS (ESI, m/z) calculated (92 mg, 0.64 mmol) to obtain pure product 5m as a pale
for C31H19ON4F2: 501.15214, found: 501.15126 [M + 1]+.
orange colour solid. Yield: 246 mg (88%); mp: 180–182 °C; IR
1-(3,4-Difluorophenyl)-3-(5,6-dimethyl-1H-benzo[d]imidazol- (KBr): λmax/cm−1 = 3436, 3062, 1721, 1624, 1606, 1563, 1517,
2-yl)-9H-pyrido[3,4-b]indole (5j). This compound was prepared 1495, 1467, 1452, 1401, 1318, 1272, 1239, 1201, 1108, 1056,
according to the general procedure, employing 4b (200 mg, 923, 885, 845, 804, 721, 746, 606, 569; 1H NMR (300 MHz,
0.64 mmol) and 4,5-dimethylbenzene-1,2-diamine (88 mg, CDCl3 + DMSO[d6]) δ (ppm): 11.44 (bs, 1H), 9.04 (s, 1H),
0.64 mmol) to obtain pure product 5j as a pale yellow solid. 8.33–8.14 (m, 2H), 8.08 (s, 1H), 7.74–7.51 (m, 4H), 7.48–7.40
Yield: 261 mg (95%); mp: 186–184 °C; IR (KBr): λmax/cm−1
=
(m, 1H), 7.35–7.25 (m, 1H), 7.20 (s, 1H); 13C NMR (75 MHz,
3420, 3059, 2921, 1724, 1607, 1563, 1510, 1457, 1430, 1407, CDCl3 + DMSO[d6]) δ (ppm): 141.2, 141.2, 138.7, 136.9, 133.2,
1399, 1315, 1242, 1176, 1109, 1026, 838, 742, 613, 580; 1H 133.1, 130.2, 130.1, 127.9, 127.7, 124.4, 121.7, 120.7, 119.5,
NMR (500 MHz, CDCl3 + DMSO[d6]) δ (ppm): 10.38 (bs, 1H), 119.4, 117.5, 117.3, 116.5, 116.3, 114.8, 114.5, 111.9, 111.8; MS
9.08 (s, 1H), 8.85 (s, 1H), 8.18 (d, J = 7.9 Hz, 1H), 7.63 (bs, 1H), (ESI, m/z): 431[M + 1]+; HRMS (ESI, m/z) calculated for
7.60–7.54 (m, 2H), 7.34 (dt, J = 6.8, 1.0 Hz, 1H), 7.27 (s, 1H), C24H14N4F2Cl: 431.08696, found: 431.08591 [M + 1]+.
7.16 (s, 2H), 2.40 (s, 6H); 13C NMR (75 MHz, CDCl3 + DMSO
3-(1H-Benzo[d]imidazol-2-yl)-1-(3,4-difluorophenyl)-9H-
[d6]) δ (ppm): 151.0, 147.9, 145.9, 141.1, 140.7, 138.8, 138.0, pyrido[3,4-b]indole (5n). This compound was prepared accord-
134.9, 132.5, 131.7, 131.4, 130.7, 130.4, 127.9, 127.5, 120.9, ing to the general procedure, employing 4b (200 mg,
120.7, 120.3, 119.5, 118.7, 117.1, 116.8, 111.7, 111.5, 30.1, 0.64 mmol) and benzene-1,2-diamine (70 mg, 0.64 mmol) to
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obtain pure product 5n as a pale orange colour solid. Yield: 11.36 (bs, 1H), 9.08 (s, 1H), 8.36 (d, J = 7.5 Hz, 2H), 8.18 (d, J =
244 mg (95%); mp: 178–179 °C; IR (KBr): λmax/cm−1 = 3436, 7.5 Hz, 1H), 7.87 (d, J = 7.5 Hz, 2H), 7.67–7.52 (m, 4H), 7.32 (t,
3062, 1721, 1624, 1606, 1563, 1517, 1495, 1467, 1452, 1401, J = 7.5 Hz, 1H), 7.19 (dd, J = 8.3, 2.2 Hz, 1H); 13C NMR
1318, 1272, 1239, 1201, 1108, 1056, 923, 885, 845, 804, 721, (75 MHz, CDCl3 + DMSO[d6]) δ (ppm): 152.6, 140.9, 140.7,
746, 606, 569; 1H NMR (300 MHz, CDCl3 + DMSO[d6]) δ (ppm): 139.1, 136.7, 133.1, 129.8, 129.0, 128.5, 128.1, 127.6,
10.77 (bs, 1H), 9.10 (s, 1H), 8.21–8.05 (m, 3H), 7.95 (s, 1H), 124.2, 121.3, 120.4, 120.2, 119.2, 111.8, 111.5; MS (ESI, m/z):
7.66–7.48 (m, 4H), 7.32–7.22 (m, 3H); 13C NMR (75 MHz, 463 [M + 1]+; HRMS (ESI, m/z) calculated for C25H14N4F3Cl:
CDCl3 + DMSO[d6]) δ (ppm): 151.7, 143.4, 141.1, 137.2, 132.8, 463.13216, found: 463.13076 [M + 1]+.
130.1, 129.7, 127.7, 127.5, 124.3, 121.6, 120.9, 120.6, 119.3,
3-(6-Chloro-1H-benzo[d]imidazol-2-yl)-1-(4-fluorophenyl)-9H-
118.0, 117.4, 117.2, 116.4, 116.1, 111.6, 111.5, 110.7; MS (ESI, pyrido[3,4-b]indole (5r). This compound was prepared accord-
m/z): 397 [M + 1]+; HRMS (ESI, m/z) calculated for C24H15N4F2: ing to the general procedure, employing 4d (200 mg,
397.12593, found: 397.12534 [M + 1]+.
0.68 mmol) and 4-chlorobenzene-1,2-diamine (97 mg,
3-(6-Fluoro-1H-benzo[d]imidazol-2-yl)-1-(4-(trifluoromethyl)- 0.68 mmol) to obtain pure product 5r as a pale yellow solid.
phenyl)-9H-pyrido[3,4-b]indole (5o). This compound was pre- Yield: 244 mg (86%); mp: 157–159 °C; IR (KBr): λmax/cm−1
=
pared according to the general procedure, employing 4c 3429, 3061, 2924, 1717, 1622, 1603, 1506, 1450, 1397, 1318,
(200 mg, 0.58 mmol) and 4-fluorobenzene-1,2-diamine (74 mg, 1224, 1154, 1094, 1052, 973, 922, 840, 801, 744, 700, 590, 565,
0.58 mmol) to obtain pure product 5o as a pale yellow solid. 504, 434; 1H NMR (300 MHz, CDCl3 + DMSO[d6]) δ (ppm):
Yield: 217 mg (83%); mp: 302–304 °C; IR (KBr): λmax/cm−1
=
10.51 (bs, 1H), 9.07 (s, 1H), 8.62 (s, 1H), 8.20 (d, J = 7.9 Hz,
3456, 3067, 1623, 1598, 1566, 1549, 1999, 1455, 1396, 1321, 1H), 8.03–8.00 (m, 2H), 7.75 (d, J = 8.3 Hz, 1H), 7.62–7.59 (m,
1251, 1202, 1164, 1109, 1064, 844, 750, 607, 573; 1H NMR 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.39–7.32
(300 MHz, CDCl3 + DMSO[d6]) δ (ppm): 10.47 (bs, 1H), 9.13 (s, (m, 3H); 13C NMR (75 MHz, CDCl3 + DMSO[d6]) δ (ppm):
1H), 8.61 (s, 1H), 8.23 (d, J = 8.1 Hz, 1H), 8.16 (d, J = 8.1 Hz, 162.7, 161.5, 159.4, 152.1, 140.2, 139.2, 135.9, 132.5, 132.1,
2H), 7.94 (d, J = 8.1 Hz, 2H), 7.66–7.52 (m, 3H), 7.40 (t, J = 7.9 129.4, 129.3, 128.7, 126.8, 125.1, 120.5, 119.8, 119.6, 118.5,
Hz, 1H), 7.06 (dt, J = 9.4, 2.2 Hz, 1H); 13C NMR (175 MHz, 113.9, 113.6, 111.0, 110.6; MS (ESI, m/z): 413[M + 1]+; HRMS
CDCl3 + DMSO) δ (ppm): 158.5 (dd, J = 244.9 Hz, 54.6 Hz, (ESI, m/z) calculated for C24H15N4FCl: 413.09638, found:
ArC-CF), 153.6, 152.6, 144.2, 141.6, 141.3, 140.4, 139.6, 137.6, 413.09602 [M + 1]+.
133.5, 130.5, 129.3 (q, J = 32.4 Hz, CF3Ar-C), 129.2, 128.3,
3-(1H-Benzo[d]imidazol-2-yl)-1-(4-fluorophenyl)-9H-pyrido-
125.0, 123.8 (q, J = 272.1 Hz, ArC-CF3), 121.3, 120.8, 119.8, [3,4-b]indole (5s). This compound was prepared according to
119.0, 112.2 (m), 109.6 (m), 103.6, 97.8; MS (ESI, m/z): the general procedure, employing 4d (200 mg, 0.68 mmol) and
447 [M + 1]+; HRMS (ESI, m/z) calculated for C25H15N4F4: benzene-1,2-diamine (74 mg, 0.68 mmol) to obtain pure
447.12274, found: 447.12133 [M + 1]+.
product 5s as a pale yellow solid. Yield: 232 mg (89%); mp:
3-(1H-Benzo[d]imidazol-2-yl)-1-(4-(trifluoromethyl)phenyl)- 178–180 °C; IR (KBr): λmax/cm−1 = 3456, 3291, 3058, 2918,
9H-pyrido[3,4-b]indole (5p). This compound was prepared 1625, 1554, 1510, 1498, 1469, 1454, 1425, 1408, 1275, 1241,
1
according to the general procedure, employing 4c (200 mg, 1217, 1158, 1112, 1092, 837, 797, 730, 665, 577, 501; H NMR
0.58 mmol) and benzene-1,2-diamine (63 mg, 0.58 mmol) to (300 MHz, CDCl3) δ (ppm): 10.51 (bs, 1H), 9.10 (s, 1H), 8.65 (s,
obtain pure product 5p as a pale yellow solid. Yield: 214 mg 1H), 8.21 (d, J = 7.5 Hz, 1H), 8.06–7.99 (m, 2H), 7.87 (s, 1H),
(85%); mp: 299–301 °C; IR (KBr): λmax/cm−1 = 3458, 3067, 7.62–7.50 (m, 3H), 7.40–7.29 (m, 5H); 13C NMR (175 MHz,
2925, 2358, 1622, 1566, 1548, 1498, 1471, 1455, 1414, 1321, CDCl3 + DMSO) δ (ppm): 162.9 (d, J = 247.5 Hz, ArC-F), 152.8,
1
1168, 1149, 1106, 1064, 1017, 849, 750, 737, 622, 583, 561; H 142.1, 141.0, 138.2, 134.5, 133.9, 131.3, 131.2, 130.7, 128.7,
NMR (300 MHz, CDCl3 + DMSO[d6]) δ (ppm): 10.52 (bs, 1H), 122.6, 121.7, 121.5, 120.3, 115.7, 115.6, 112.9, 112.2; MS (ESI,
9.14 (s, 1H), 8.77 (s, 1H), 8.21 (s, 1H), 8.15 (d, J = 8.1 Hz, 2H), m/z): 379 [M + 1]+; HRMS (ESI, m/z) calculated for C24H16N4F:
7.90 (d, J = 7.9 Hz, 3H), 7.65–7.51 (m, 3H), 7.40–7.30 (m, 2H); 379.13535, found: 379.13529 [M + 1]+.
13C NMR (175 MHz, CDCl3 + DMSO) δ (ppm): 152.0, 141.6,
1-(4-Fluorophenyl)-3-(6-methyl-1H-benzo[d]imidazol-2-yl)-
141.4, 139.6, 137.9, 133.5, 130.4, 129.2, 129.1 (q, J = 31.7 Hz, 9H-pyrido[3,4-b]indole (5t). This compound was prepared
CF3Ar-C), 128.5, 128.3, 124.9, 123.8 (q, J = 272.1 Hz, ArC-CF3), according to the general procedure, employing 4d (200 mg,
112.3, 112.2; MS (ESI, m/z): 429 [M + 1]+; HRMS (ESI, m/z) cal- 0.68 mmol) and 4-methylbenzene-1,2-diamine (84 mg,
culated for C25H16N4F3: 429.13216, found: 429.13076 [M + 1]+.
0.68 mmol) to obtain pure product 5t as a pale yellow solid.
3-(6-Chloro-1H-benzo[d]imidazol-2-yl)-1-(4-(trifluoromethyl)- Yield: 237 mg (88%); mp: 236–238 °C; IR (KBr): λmax/cm−1
=
phenyl)-9H-pyrido[3,4-b]indole (5q). This compound was pre- 3454, 3252, 3053, 2920, 1626, 1604, 1509, 1455, 1470, 1425,
1
pared according to the general procedure, employing 4c 1404, 1320, 1278, 1218, 1157, 844, 806, 745, 608, 572, 504; H
(200 mg, 0.58 mmol) and 4-chlorobenzene-1,2-diamine NMR (300 MHz, CDCl3 + DMSO[d6]) δ (ppm): 10.37 (bs, 1H),
(83 mg, 0.58 mmol) to obtain pure product 5q as a pale yellow 9.08 (s, 1H), 8.63 (s, 1H), 8.19 (d, J = 7.5 Hz, 1H), 8.05–8.00 (m,
solid. Yield: 223 mg (82%); mp: 326–328 °C; IR (KBr): λmax
/
2H), 7.62–7.50 (m, 2H), 7.41–7.28 (m, 4H), 7.11 (d, J = 7.5 Hz,
cm−1 = 3453, 3065, 2923, 1722, 1625, 1565, 1547, 1466, 1454, 1H), 2.52 (s, 3H); 13C NMR (175 MHz, CDCl3 + DMSO) δ (ppm):
1396, 1321, 1248, 1165, 1108, 1064, 924, 882, 850, 747, 732, 162.3 (d, J = 247.9 Hz, ArC-F), 151.9, 141.4, 140.3, 137.7, 133.9,
596, 576; 1H NMR (300 MHz, CDCl3 + DMSO[d6]) δ (ppm): 133.2, 131.0, 130.6 (d, J = 8.2 Hz), 130.0, 128.0, 123.1, 120.9
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(d, J = 8.2 Hz), 119.6, 115.0, 114.9, 112.2, 111.3, 21.2; MS (ESI, 4e (200 mg, 0.55 mmol) and 4,5-dimethylbenzene-1,2-diamine
m/z): 393 [M + 1]+; HRMS (ESI, m/z) calculated for C25H18N4F: (75 mg, 0.55 mmol) to obtain pure product 5x as a pale brown
393.15100, found: 393.15062 [M + 1]+.
colour solid. Yield: 248 mg (94%); mp: 284–286 °C; IR (KBr):
3-(5,6-Dichloro-1H-benzo[d]imidazol-2-yl)-1-(4-fluorophe- λmax/cm−1 = 3444, 3201, 2925, 2852, 1625, 1585, 1505, 1454,
1
nyl)-9H-pyrido[3,4-b]indole (5u). This compound was prepared 1467, 1402, 1309, 1234, 1127, 1003, 845, 743, 692, 630, 558; H
according to the general procedure, employing 4d (200 mg, NMR (500 MHz, CDCl3 + DMSO[d6]) δ (ppm): 10.41 (bs, 1H),
0.68 mmol) and 4,5-dichlorobenzene-1,2-diamine (121 mg, 9.07 (s, 1H), 8.95 (s, 1H), 8.18 (d, J = 7.6 Hz, 1H), 7.65–7.54 (m,
0.68 mmol) to obtain pure product 5u as a pale yellow solid. 3H), 7.35–7.31 (m, 1H), 7.15 (s, 2H), 3.92 (s, 9H), 2.40 (s, 6H);
Yield: 262 mg (85%); mp: 158–160 °C; IR (KBr): λmax/cm−1
=
13C NMR (75 MHz, CDCl3 + DMSO[d6]) δ (ppm): 152.3, 151.0,
3417, 3324, 2921, 2356, 1625, 1607, 1562, 1540, 1510, 1499, 141.1, 141.0, 137.3, 137.2, 132.9, 129.9, 129.5, 127.4, 120.7,
1468, 1448, 1392, 1319, 1228, 1183, 1156, 1097, 874, 835, 735, 120.5, 119.0, 111.7, 110.7, 105.4, 59.7, 55.3, 19.4; MS (ESI, m/z):
1
659, 573, 548, 507; H NMR (300 MHz, CDCl3 + DMSO[d6]) δ 479 [M + 1]+; HRMS (ESI, m/z) calculated for C29H26O3N4:
(ppm): 10.33 (bs, 1H), 8.15 (s, 1H), 7.34 (s, 3H), 6.92 (s, 1H), 479.20647, found: 479.20536 [M + 1]+.
6.85–6.55 (m, 4H), 6.53–6.39 (m, 3H); 13C NMR (75 MHz,
3-(6-Chloro-1H-benzo[d]imidazol-2-yl)-1-(3,4,5-trimethoxy-
CDCl3 + DMSO[d6]) δ (ppm): 154.1, 141.1, 140.4, 136.5, 133.6, phenyl)-9H-pyrido[3,4-b]indole (5y). This compound was pre-
133.3, 130.2, 130.1, 129.7, 127.8, 124.6, 120.7, 119.5, 114.9, pared according to the general procedure, employing 4e
114.6, 111.8; MS (ESI, m/z): 447 [M + 1]+; HRMS (ESI, m/z) (200 mg, 0.55 mmol) and 4-chlorobenzene-1,2-diamine
calculated for C24H14N4Cl2F: 447.05741, found: 447.05731 (78 mg, 0.55 mmol) to obtain pure product 5y as a dark brown
[M + 1]+.
colour solid. Yield: 243 mg (91%); mp: 232–234 °C; IR (KBr):
Phenyl(2-(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol- λmax/cm−1 = 3389, 3149, 2931, 1624, 1587, 1562, 1504, 1468,
3-yl)-1H-benzo[d]imidazol-6-yl)methanone (5v). This com- 1452, 1404, 1272, 1236, 1181, 1130, 1058, 1005, 924, 849, 804,
1
pound was prepared according to the general procedure, 743, 690; H NMR (500 MHz, DMSO [d6]) δ (ppm): 10.48 (bs,
employing 4e (200 mg, 0.55 mmol) and (3,4-diaminophenyl) 1H), 8.19 (s, 1H), 7.29 (d, J = 7.3 Hz, 1H), 6.84–6.61 (m, 5H),
(phenyl)methanone (117 mg, 0.55 mmol) to obtain pure 6.48 (s, 2H), 6.40 (t, J = 7.5 Hz, 1H), 6.31 (d, J = 8.4 Hz, 1H),
product 5v as a pale yellow solid. Yield: 281 mg (92%); mp: 3.14 (s, 6H), 3.04 (s, 3H); 13C NMR (75 MHz, CDCl3 + DMSO
208–210 °C; IR (KBr): λmax/cm−1 = 3323, 3060, 2926, 1644, [d6]) δ (ppm): 152.7, 152.1, 141.2, 140.7, 137.2, 135.9, 132.9,
1615, 1585, 1542, 1497, 1468, 1446, 1405, 1347, 1320, 1296, 132.4, 129.1, 127.3, 126.1, 121.4, 120.3, 119.0, 116.6, 115.6,
1
1235, 1178, 1126, 1002, 892, 829, 791, 719, 642, 434; H NMR 111.3, 105.3, 59.8, 59.5, 55.1; MS (ESI, m/z): 485 [M + 1]+; HRS
(300 MHz, CDCl3 + DMSO[d6]) δ (ppm): 10.88 (bs, 1H), 9.12 (d, (ESI, m/z) calculated for C27H22O3N4Cl: 485.13749, found:
J = 10.5 Hz, 1H), 8.85 (d, J = 8.3 Hz, 1H), 8.22 (t, J = 6.7 Hz, 485.13742 [M + 1]+.
1H), 7.91–7.77 (m, 4H), 7.61–7.51 (m, 4H), 7.51–7.45 (m, 2H),
3-(6-Methoxy-1H-benzo[d]imidazol-2-yl)-1-(3,4,5-trimethoxy-
7.40–7.33 (m, 1H), 7.16 (s, 2H), 3.95 (s, 9H); 13C NMR (75 MHz, phenyl)-9H-pyrido[3,4-b]indole (5z). This compound was pre-
CDCl3 + DMSO[d6]) δ (ppm): 159.0, 154.2, 141.2, 140.8, 136.2, pared according to the general procedure, employing 4e
133.0, 129.6, 129.3, 127.3, 120.5, 120.4, 119.1, 117.8, 113.0, (200 mg, 0.55 mmol) and 4-methoxybenzene-1,2-diamine
111.6, 111.1, 54.3; MS (ESI, m/z): 555 [M + 1]+; HRMS (ESI, m/z) (76 mg, 0.55 mmol) to obtain pure product 5z as a pale brown
calculated for C34H27N4O4: 555.20268, found: 555.20184 colour solid. Yield: 236 mg (89%); mp: 276–278 °C; IR (KBr):
[M + 1]+.
3-(1H-Benzo[d]imidazol-2-yl)-1-(3,4,5-trimethoxyphenyl)-9H-
λmax/cm−1 = 3425, 3062, 2933, 2830, 1626, 1546, 1504, 1468,
1453, 1408, 1326, 1289, 1235, 1154, 1127, 1012, 941, 847, 747,
1
pyrido[3,4-b]indole (5w). This compound was prepared 629; H NMR (300 MHz, CDCl3) δ (ppm): 10.60 (bs, 1H), 8.98
according to the general procedure, employing 4e (200 mg, (d, J = 21.1 Hz, 2H), 8.13 (d, J = 8.3 Hz, 1H), 7.56 (t, J = 6.7 Hz,
0.55 mmol) and benzene-1,2-diamine (59 mg, 0.55 mmol) to 2H), 7.35–7.30 (m, 2H), 7.14 (s, 2H), 6.91 (dd, J = 9.0, 2.2 Hz,
obtain pure product 5w as a pale yellow solid. Yield: 231 mg 1H), 3.91 (s, 3H), 3.89 (s, 9H); 13C NMR (75 MHz, CDCl3
+
(93%); mp: 224–226 °C; IR (KBr): λmax/cm−1 = 3407, 2937, DMSO[d6]) δ (ppm): 155.0, 152.0, 151.4, 140.9, 140.8, 136.9,
1623, 1587, 1505, 1452, 1410, 1384, 1325, 1292, 1178, 1127, 132.6, 129.2, 127.2, 120.4, 120.3, 118.8, 111.5, 110.6, 110.5,
1
740, 688; H NMR (500 MHz, CDCl3) δ (ppm): 10.55 (bs, 1H), 105.1, 59.5, 55.0, 54.4; MS (ESI, m/z): 481 [M + 1]+; HRMS (ESI,
9.13 (s, 1H), 8.22 (s, 1H), 7.88 (s, 1H), 7.62–7.47 (m, 3H), m/z) calculated for C28H25O4N4: 481.18703, found: 481.18573
7.38–7.30 (m, 1H), 7.30–7.26 (s, 2H), 7.17 (d, J = 8.3 Hz, 2H), [M + 1]+.
3.97 (s, 6H), 3.94 (s, 3H); 13C NMR (75 MHz, CDCl3 + DMSO
3-(3H-Imidazo[4,5-b]pyridin-2-yl)-1-(4-methoxyphenyl)-9H-
[d6]) δ (ppm): 152.9, 152.3, 141.8, 141.4, 137.9, 137.3, 133.6, pyrido[3,4-b]indole (6a). This compound was prepared accord-
133.3, 130.1, 128.0, 121.8, 121.3, 121.2, 119.7 112.0, 111.8, ing to the general procedure, employing 4a (200 mg,
105.7, 60.3, 55.8; MS (ESI, m/z): 451 [M + 1]+; HRMS (ESI, m/z) 0.66 mmol) and pyridine-2,3-diamine (72 mg, 0.66 mmol) to
calculated for C27H23 O3N4: 451.17647, found: 451.17549 obtain pure product 6a as a yellow solid. Yield: 233 mg (90%);
[M + 1]+.
mp: 298–300 °C; IR (KBr): λmax/cm−1 = 3074, 2928, 1624, 1608,
3-(5,6-Dimethyl-1H-benzo[d]imidazol-2-yl)-1-(3,4,5-tri- 1512, 1494, 1464, 1452, 1423, 1409, 1386, 1319, 1280, 1263,
methoxyphenyl)-9H-pyrido[3,4-b]indole (5x). This compound 1247, 1172, 1114, 1031, 832, 767, 755; 1H NMR (300 MHz,
was prepared according to the general procedure, employing CDCl3 + DMSO[d6]) δ (ppm): 11.47 (bs, 1H), 9.02 (s, 1H), 8.32
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(d, J = 4.1 Hz, 1H), 8.23 (t, J = 8.8 Hz, 3H), 7.93 (d, J = 6.9 Hz, complete medium, treated with compounds at desired concen-
1H), 7.83 (s, 1H), 7.64 (d, J = 8.3 Hz, 1H), 7.51 (t, J = 7.7 Hz, trations and harvested as required.63
1H), 7.27 (t, J = 7.5 Hz, 1H), 7.18–7.12 (m, 3H), 3.93 (s, 3H); 13
NMR (75 MHz, DMSO[d6]) δ (ppm): 159.9, 156.7, 154.0, 153.7,
C
Cytotoxicity
149.2, 143.7, 143.4, 141.5, 137.0, 133.4, 130.3, 130.1, 129.9, Cell proliferation and viability was determined by the 3-[4,5-
128.5, 127.2, 125.8, 121.9, 121.1, 120.0, 119.3, 117.7, 114.0, dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
112.6, 55.3; MS (ESI, m/z): 392 [M + 1]+; HRMS (ESI, m/z) calcu- assay. The pale yellow coloured tetrazolium salt (MTT) reduces
lated for C24H18ON5: 392.15059, found: 392.15003 [M + 1]+.
to a dark blue water-insoluble formazan by metabolically
1-(3,4-Difluorophenyl)-3-(3H-imidazo[4,5-b]pyridin-2-yl)-9H- active cells and this is measured quantitatively after soluble in
pyrido[3,4-b]indole (6b). This compound was prepared accord- DMSO. The absorbance of the soluble formazan is directly pro-
ing to the general procedure, employing 4b (200 mg, portional to the number of viable cells. Cells were seeded at a
0.64 mmol) and pyridine-2,3-diamine (70 mg, 0.64 mmol) to density of 1 × 104 cells in 200 µL of medium per well of a 96-
obtain pure product 6b as a pale brown colour solid. Yield: well plate. The 96-well microliter plates were incubated for
229 mg (89%); mp: 320–322 °C; IR (KBr): λmax/cm−1 = 3288, 24 h prior to addition of the experimental compounds. Cells
3061, 2919, 2850, 2360, 1677, 1624, 1518, 1496, 1464, 1452, were treated with vehicle alone (0.4% DMSO) or compounds
1438, 1422, 1409, 1388, 1317, 1274, 1204, 1138, 1107, 885, 828, (drugs were dissolved in DMSO previously) at different concen-
797, 772, 730, 640, 607, 524; 1H NMR (300 MHz, CDCl3
+
trations (1, 10 and 25 µM) of test compounds for 48 hours.
DMSO[d6]) δ (ppm): 11.42 (bs, 1H), 9.11 (s, 1H), 8.39 (s, 1H), The assay was completed with the addition of MTT (5%,
8.18 (d, J = 8.3 Hz, 1H), 8.11–7.97 (m, 2H), 7.67 (d, J = 7.5 Hz, 10 µL) and incubated for 60 min at 37 °C. The supernatant was
1H), 7.57 (m, 2H), 7.42 (q, J = 18.1 Hz, 1H), 7.32 (t, J = 7.5 Hz, aspirated and plates were air dried and the MTT–formazon
1H), 7.21 (m, 1H); 13C NMR (75 MHz, CDCl3 + DMSO[d6]) δ crystals dissolved in 100 µL of DMSO. The optical density was
(ppm): 153.0, 142.8, 140.9, 138.5, 136.4, 132.9, 129.9, 127.6, measured at 560 nm using a TECAN multimode reader. The
124.3, 120.4, 120.3, 119.3, 117.4, 117.2, 116.8, 116.2, 116.0, growth percentage of each treated well of a 96-well plate has
112.0, 111.6; MS (ESI, m/z): 398 [M + 1]+; HRMS (ESI, m/z) cal- been calculated based on test wells relative to control wells.
culated for C23H14N5F2: 398.10153, found: 398.10091 [M + 1]+.
The cell growth inhibition was calculated by generating dose–
3-(3H-Imidazo[4,5-b]pyridin-2-yl)-1-(4-(trifluoromethyl)- response curves as a plot of the percentage of surviving cells
phenyl)-9H-pyrido[3,4-b]indole (6c). This compound was pre- versus drug concentration. Antiproliferative activity of the
pared according to the general procedure, employing 4c cancer cells to the test compounds was expressed in terms of
(200 mg, 0.58 mmol) and pyridine-2,3-diamine (64 mg, IC50 value, which is defined as the concentration of the com-
0.58 mmol) to obtain pure product 6c as a pale yellow solid. pound that produced 50% absorbance reduction relative to the
Yield: 214 mg (85%); mp: 338–340 °C; IR (KBr): λmax/cm−1
=
control.64
3438, 3152, 1624, 1592, 1567, 1496, 1466, 1454, 1425, 1405,
1322, 1267, 1213, 1165, 1125, 1066, 1018, 848, 798, 773, 751,
CD studies
623; 1H NMR (300 MHz, CDCl3 + DMSO[d6]) δ (ppm): 10.91 Circular dichroism experiments were carried out using a
(bs, 1H), 9.96 (s, 1H), 7.87 (s, 1H), 7.17–7.05 (m, 2H), 6.96 JASCO 815 CD spectropolarimeter (Jasco, Tokyo, Japan). All the
(d, J = 8.1 Hz, 1H), 6.82 (d, J = 7.7 Hz, 1H), 6.62 (d, J = 7.7 Hz, CD titrations were performed in 100 mM KBPES buffer (pH
2H), 6.44–6.28 (m, 2H), 6.13–6.04 (m, 1H), 5.95 (s, 1H); MS 7.0) at 25 °C. CD spectra were recorded from 200 to 350 nm
(ESI, m/z): 430 [M + 1]+; HRMS (ESI, m/z) calculated for and for each experiment, 15 × 10−6 M of CT DNA was used
C24H15F3N5: 430.12741, found: 430.12640 [M + 1]+.
initially. Further, for the characterization of ligand–DNA inter-
action, CD spectra were recorded in 1 : 0.5 and 1 : 1 molar
ratios of CT DNA and ligand respectively. Each spectrum was
recorded three times and the average of three scans was taken.
In vitro cytotoxicity data
Cell culture and reagents. All the cell lines used in this
study were obtained from the American Type Culture Collec-
UV-visible titration studies
tion (ATCC). DU145 (human prostate carcinoma epithelial) UV-visible spectroscopic titrations were performed using an
and BHK-21 (Hamster kidney cells) cells were cultured in ABI Lambda 40 UV-Vis spectrophotometer (Foster City, USA) at
Eagle’s minimal essential medium (MEM) containing non- 25 °C using a 1 cm path length quartz cuvette. Stock solutions
essential amino acids, 1 mM sodium pyruvate, and 10% FBS. of 10 µM of ligand solution and 25 µM CT DNA were prepared
HeLa (human epithelial cervical cancer), A549 (human lung in 100 mM KBPES buffer (pH 7.0). Complex stock solution of
carcinoma epithelial) and L929 (mouse connective tissue fibro- the ligand was prepared in a 1 : 1 water–methanol mixture and
blast cells) were grown in Dulbecco’s modified Eagle’s diluted to the required concentration in suitable buffer solu-
medium (DMEM) containing non-essential amino acids and tions. The quartz cells were thoroughly cleaned with distilled
10% FBS. All the cells were maintained under a humidified water followed by nitric acid (∼0.1 N) after each experiment.
atmosphere of 5% CO2 at 37 °C. Cells were trypsinized when UV-visible absorption titrations were done by adding CT DNA
subconfluent from T75 flasks/90 mm dishes and seeded onto stock solution in 100 mM KBPES buffer (pH 7.0) to the quartz
96 well test plates at a concentration of 1 × 104 cells mL−1 in cuvette containing approximately 10 µM ligand solution
2384 | Org. Biomol. Chem., 2014, 12, 2370–2387
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prepared in the same buffer. Preparation of CT DNA and fellowships. The authors also acknowledge the Council of
ligands was done on the same day of performing the experi- Scientific & Industrial Research (CSIR), India, for financial
ment. Titrations were carried out until the ligand Soret band support under the 12th Five-Year Plan project “Affordable
remains at a fixed wavelength upon successive additions of CT Cancer Therapeutics (ACT)” (CSC0301). N. N. is thankful to the
DNA.
Indo Swiss Joint Research Programme (ISJRP) for partial finan-
cial support (grant number CH: 138844).
DNA intercalation
To verify the mode of ligand interaction with DNA, 2 µM of
each ligand was taken in Tris buffer (pH 7.0) and 5 µM of pBR
322 plasmid DNA was added. The mixture was incubated at
37 °C for 1 h. After incubation, the ligand–DNA mixture was
resolved using 0.8% agarose gel. The sample treated with 2 µM
ethidium bromide was considered as the control.
Notes and references
1 Q. Chen, R. Chao, H. Chen, X. Hou, H. Yan, S. Zhou,
W. Peng and A. Xu, Int. J. Cancer, 2004, 114, 675–682.
2 T. Herraiz and J. Galisteo, J. Agric. Food Chem., 2003, 51,
7156–7161.
DNA photocleavage
3 N. S. Buckholtz, Life Sci., 1980, 27, 893–903.
This experiment was carried out according to the protocol
reported by Toshima and coworkers.65 0.45 µg of pBR322
plasmid DNA was taken in Tris–HCl buffer (50 mM, pH 7.5)
and 100 and 200 µM of each ligand were added and the total
volume was maintained at 20 µL. The DNA samples with
complexes were taken in a TPP tissue culture test plate and
irradiated with UV light (8 W, 365 nm, 4 cm distance). After
irradiation, the samples were collected and mixed with 2 µL of
loading dye (50% sucrose and 0.25% bromophenol blue). For
the dark reaction, 100 µM ligand and 0.45 µg pBR322 plasmid
DNA were taken in a PCR tube and the samples were wrapped
with aluminium foil and placed in the dark. Then, samples
were analysed by gel electrophoresis on a 0.8% agarose hori-
zontal slab gel containing 0.5 µg mL−1 ethidium bromide in
Tris–EDTA buffer (40 mM Tris, 20 mM acetic acid and 1 mM
EDTA, pH 8.0) at 10 V cm−1. Gels were photographed under UV
light with a Bio-Rad digital camera and analysed with Gel-Pro
software.
4 M. M. Airaksinen and I. Kari, Med. Biol., 1981, 59, 21–34.
5 C. Melchior and M. A. Collins, CRC Crit. Rev. Toxicol., 1982,
10, 313–356.
6 J. Ishida, H. K. Wang, K. F. Bastow, C. Q. Hu and K. H. Lee,
Bioorg. Med. Chem. Lett., 1999, 9, 3319–3324.
7 S. Xiao, W. Lin, C. Wang and M. Yang, Bioorg. Med. Chem.
Lett., 2001, 11, 437–441.
8 Y. C. Shen, C. Y. Chen, P. W. Hsieh, C. Y. Duh, Y. M. Lin
and C. L. Ko, Chem. Pharm. Bull., 2005, 53, 32–36.
9 A. M. Deveau, M. A. Labroli, C. M. Dieckhaus, M. T. Barthen,
K. S. Smith and T. L. Macdonald, Bioorg. Med. Chem. Lett.,
2001, 11, 1251–1255.
10 M. Zhao, L. Bi, W. Wang, C. Wang, M. Baudy-Floc’h, J. Ju
and S. Peng, Bioorg. Med. Chem., 2006, 14, 6998–7010.
11 A. S. N. Formagio, L. T. D. Dusman, M. A. Foglio,
C. Madjarof, J. E. Carvalho, W. F. Costa, F. P. Cardoso and
M. H. Sarragiott, Bioorg. Med. Chem., 2008, 16, 9660–9667.
12 J. Wu, M. Zhao, K. Qian, K.-H. Lee, S. Morris-Natschke and
S. Pen, Eur. J. Med. Chem., 2009, 44, 4153–4161.
13 Y. Boursereau and I. Coldham, Bioorg. Med. Chem. Lett.,
2004, 14, 5841–5844.
DNA topoisomerase I inhibition
The DNA Topo I inhibition study was carried out as described
in the previous paper.66 0.5 µg of pBR322 DNA was incubated
with 1 unit of Topo I (Invitrogen) in 1× NEB buffer (50 mM pot-
assium acetate, 20 mM Tris acetate buffer, 10 mM magnesium
acetate, 1 mM DTT). The ligands to be studied were added to
the Topo I-DNA complex and incubated at 37 °C for 30 min,
allowing the formation of ternary enzyme–DNA–ligand com-
plexes. Then, the enzyme was inactivated by increasing the
temperature to 65 °C. Next, the samples were resolved using
0.8% agarose gel electrophoresis which enables the visualisa-
tion of cleavage products. 100 µM camptothecin (CPT) treated
DNA was considered as the positive control.
14 K. Hayashi, M. Nagao and T. Sugimura, Nucleic Acids Res.,
1977, 4, 3679–3685.
15 (a) Y. Song, J. Wang, S. F. Teng, D. Kesuma, Y. Deng,
J. Duan, J. H. Wang, R. Z. Qi and M. M. Sim, Bioorg. Med.
Chem. Lett., 2002, 12, 1129–1132; (b) Y. Song, D. Kesuma,
J. Wang, Y. Deng, J. Duan, J. H. Wang and R. Z. Qi,
Biochem. Biophys. Res. Commun., 2004, 317, 128–132;
(c) Y. Li, F. Liang, W. Jiang, F. Yu, R. Cao, Q. Ma, X. Dai,
J. Jiang, Y. Wang and S. Si, Cancer Biol. Ther., 2007, 6,
1193–1199.
16 A. C. Castro, L. C. Dang, F. Soucy, L. Grenier,
H. Mazdiyasni, M. Hottelet, L. Parent, C. Pien, V. Palombella
and J. Adams, Bioorg. Med. Chem. Lett., 2003, 13, 2419–2422.
17 J. I. Trujillo, M. J. Meyers, D. R. Anderson, S. Hegde,
M. W. Mahoney, W. F. Vernier, I. P. Buchler, K. K. Wu,
S. Yang, S. J. Hartmann and D. B. Reitz, Bioorg. Med. Chem.
Lett., 2007, 17, 4657–4663.
Acknowledgements
M. P. N. S. R., P. S., V. S., A. B. S., K. M., V. S. R. and
J. K. acknowledge the Council of Scientific & Industrial
Research/University Grants Commission (CSIR-UGC), New
Delhi (India), for the award of senior research fellowships, and 18 P. A. Barsanti, W. Wang, Z. Ni, D. Duhl, N. Brammeier and
are thankful to DST (India) for the award of Inspire
E. Martin, Bioorg. Med. Chem. Lett., 2010, 20, 157–160.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 2370–2387 | 2385

View Article Online
Paper
Organic & Biomolecular Chemistry
19 M. F. Brana, M. Cacho, A. Gradillas, B. de Pascual-Teresa
and A. Ramos, Curr. Pharm. Des., 2001, 7, 1745–1780.
20 L. H. Hurley, Nat. Rev. Cancer, 2002, 2, 188–200.
21 R. Martinez and L. Chacon-Garcia, Curr. Med. Chem., 2005,
12, 127.
M. M. Khodaei and F. Naali, J. Org. Chem., 2008, 73, 6835;
(d) H. Sharghi, M. Aberi and M. M. Doroodmand, Adv.
Synth. Catal., 2008, 350, 2380; (e) Y. X. Chen, L. F. Qian,
W. Zhang and B. Han, Angew. Chem., Int. Ed., 2008, 47,
9330.
22 Y. Funayama, K. Nakasato, K. Wakabayashi, M. Nagao, 39 R. Ikeda, T. Iwaki, T. Iida, T. Okabayashi, E. Nishi,
K. Shimoi, T. Ohira, S. Hasegawa and N. Saijo, Effects of
beta-Caroline and gamma-Caroline derivatives on DNA
topoisomerase activities, Mutat. Res., 1996, 349, 183–191.
23 G. Duportail, Linear and circular dichroism of harmine
M. Kurosawa, N. Sakai and T. Konakahara, Eur. J. Med.
Chem., 2011, 46, 636–646.
40 R. Cao, Q. Chen, X. Hou, H. Chen, H. Guan, Y. Ma, W. Peng
and A. Xu, Bioorg. Med. Chem., 2004, 12, 4613–4623.
and harmaline interacting with DNA, Int. J. Biol. Macromol., 41 Z. Chen, R. Cao, L. Yu, B. Shi, J. Sun, L. Guo, Q. Ma, W. Yi,
1981, 3, 188–193.
X. Song and H. Song, Eur. J. Med. Chem., 2010, 45, 4740–
24 Z. Taira, S. Kanzawas, C. Dohara, S. Ishida, M. Matsumoto
4745.
and Y. Sakiya, Intercalation of six beta-carboline derivatives 42 X. Han, J. Zhang, L. Guo, R. Cao, Y. Li, N. Li, Q. Ma, J. Wu,
into DNA, Jpn. J. Toxicol. Environ. Health, 1997, 43, 83–91.
Y. Wang and S. Si, PLoS One, 2012, 7(10), e46546, http://
25 T. D. Penning, G. D. Zhu, V. B. Gandhi, J. Gong, X. Liu,
www.plosone.org.
Y. Shi, V. Klinghofer, E. F. Johnson, C. K. Donawho, 43 A. Kamal, D. Dastagiri, M. J. Ramaiah, J. S. Reddy,
D. J. Frost, V. B. Diaz, J. J. Bouska, D. J. Osterling,
A. M. Olson, K. C. Marsh, Y. Luo and V. L. Giranda, J. Med.
Chem., 2009, 52, 514–523.
E. V. Bharathi, C. Srinivas, D. Pal and M. Pal-Bhadra,
ChemMedChem, 2010, 5, 1937.
44 (a) A. Kamal, Y. V. V. Srikanth, T. B. Shaik, M. N. A. Khan,
M. Ashraf, M. K. Reddy, K. A. Kumar and S. V. Kalivendi,
Med. Chem. Commun., 2011, 2, 819; (b) A. Kamal,
F. Sultana, M. J. Ramaiah, Y. V. V. Srikanth, A. Viswanath,
C. Kishor, P. Sharma, S. N. C. V. L. Pushpavalli,
A. Addlagatta and M. Pal-Bhadra, ChemMedChem, 2012, 7,
292; (c) A. Kamal, M. Kashi Reddy, T. B. Shaik, Rajender,
Y. V. V. Srikanth, V. Santhosh Reddy, G. Bharath Kumar
and S. V. Kalivendi, Eur. J. Med. Chem., 2012, 50, 9–17.
26 D. Hao, J. D. Rizzo, S. Stringer, R. V. Moore, J. Marty,
D. L. Dexter, G. L. Mangold, J. B. Camden, D. D. Von-Hoff
and S. D. Weitman, Invest. New Drugs, 2002, 20, 261–270.
27 H. M. Refaat, Eur. J. Med. Chem., 2010, 45, 2949–2956.
28 R. Abonia, E. Cortes, B. Insuasty, J. Quiroga, M. Nogueras
and J. Cobo, Eur. J. Med. Chem., 2011, 46, 4062–4070.
29 B. V. S. Kumar, S. D. Vaidya, R. V. Kumar, S. B. Bhirud and
R. B. Mane, Eur. J. Med. Chem., 2006, 41, 599–604.
30 D. Sharma, B. Narasimhan, P. Kumar, V. Judge, R. Narang, 45 (a) A. Fazilinia, M. H. Mosslemin and H. Sadoughi,
E. D. Clercq and J. Balzarini, J. Enzyme Inhib. Med. Chem.,
2009, 24, 1161–1168.
31 S. Demirayak, A. Usama, A. C. Mohsen and K. Agri,
Eur. J. Med. Chem., 2002, 37, 255–260.
32 G. A. Kilcgil, C. Kus, T. Coban, B. C. Eke and M. Iscan,
J. Enzyme Inhib. Med. Chem., 2004, 19, 129–135.
33 W. A. Denny, G. W. Rewcastle and B. C. Baguly, J. Med.
Chem., 1990, 33, 814–819.
34 T. C. Jenkins, Curr. Med. Chem., 2000, 7, 99–115.
35 M. P. Singh, T. Joseph, S. Kumar, Y. Bathini and
J. W. Lown, Chem. Res. Toxicol., 1992, 5, 597–607.
36 (a) M. R. Grimmet, Comprehensive Heterocyclic Chemistry,
ed. A. R. Katritzky, C. W. Rees and K. T. Potts, Pergamon
J. Korean Chem. Soc., 2010, 54, 579–581; (b) C. G. Bletter,
W. A. Konig and G. Ruter, Synlett, 1999, 307–310;
(c) L. M. Dudd, E. Venardou, E. Garcia-Verdugo, P. Licence,
A. J. Blake, C. Wilson and M. Poliakoff, Green Chem., 2003,
5, 187–192.
46 (a) J. J. Vanden Eynde, F. Delfosse, P. Lor and Y. Van Haver-
beke, Tetrahedron, 1995, 51, 5813–5818; (b) I. Bhatnagar
and M. V. George, Tetrahedron, 1968, 24, 1293–1298.
47 S. M. Reddy, Y. V. Reddy and Y. Venkateswarlu, Tetrahedron
Lett., 2005, 46, 7439–7441.
48 T. S. Reddy, K. Ravinder, N. Suryakiran, M. Narasimhulu,
K. C. Mahesh and Y. Venkateswarlu, Tetrahedron Lett.,
2006, 47, 2341–2344.
Press, New York, 1984, vol. 5; (b) P. N. Preston, Chemistry of 49 N. Suryakiran, K. Rajesh, P. Prabhakar, J. J. P. Selvam and
Heterocyclic Compounds, ed. A. Weissberger and E. C. Taylor, Y. Venkateswarlu, CatCommun, 2007, 8, 1635–1640.
John Wiley and Sons, 1981, vol. 40; (c) L. M. Dudd, 50 G. Raju, R. Srinivas, V. Santhosh Reddy, M. M. Idris,
E. Venardou, E. Garcia-Verdugo, P. Licence, A. J. Blake,
A. Kamal and N. Nagesh, PLoS One, 2012, 7(4), e35920,
C. Wilson and M. Poliakoff, Green Chem., 2003, 5, 187.
DOI: 10.1371/journal.pone.0035920.
37 (a) R. J. Perry and B. D. Wilson, J. Org. Chem., 1993, 58, 51 B. D. Wang, Z. Y. Yang, P. Crewdson and D. Q. Wang,
7016; (b) C. T. Brain and S. A. Brunton, Tetrahedron Lett., J. Inorg. Biochem., 2007, 101, 1492.
2002, 43, 1893; (c) D. Anastasiou, E. M. Campi, H. Chaouk 52 J. K. Barton, J. J. Dennenberg and J. B. Chaires, Biochemis-
and W. R. Jackson, Tetrahedron, 1992, 48, 7467; (d) Z. Wu, try, 1993, 32, 2573–2584.
P. Rea and G. Wickam, Tetrahedron Lett., 2000, 41, 53 T. Berge, N. S. Jenkins, R. B. Hopkirk, M. J. Waring,
9871.
J. Michael Edwardson and R. M. Henderson, Nucleic Acids
Res., 2002, 30, 2980–2986.
38 (a) R. Trivedi, S. K. De and R. A. Gibbs, J. Mol. Catal. A:
Chem., 2006, 8, 245; (b) K. Bahrami, M. M. Khodaei and 54 J. M. Kelly, A. B. Tossi, D. J. McConnell and C. OhUigin,
I. Kavianinia, Synthesis, 2007, 547; (c) K. Baharami,
Nucleic Acids Res., 1985, 13, 6017–6037.
2386 | Org. Biomol. Chem., 2014, 12, 2370–2387
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
55 R. Cao, W. Peng, H. Chen, Y. Ma, X. Liu, X. Hou, H. Guan 58 C. Temperini, L. Messori, P. Orioli, C. Di Bugno, F. Animati
and A. Xu, Biochem. Biophys. Res. Commun., 2005, 338,
1557–1563.
and G. Ughetto, Nucleic Acids Res., 2003, 31, 1464–
1469.
56 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, 59 The PyMOL Molecular Graphics System, Version 1.5.0.4,
M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, Schrödinger, LLC, 2012.
B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, 60 C. A. Lipinski, F. Lombardo, B. W. Dominy and P. J. Feeney,
X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, Adv. Drug Delivery Rev., 1997, 23, 3–25.
J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, 61 P. Ertl, Calculation of Molecular Properties and Bioactivity
J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,
H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta,
Score, Available online: http://www.molinspiration.com
(accessed on 31 January 2012).
F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, 62 T. Sander, Molecular Property Explorer. Available online:
V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari,
A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi,
http://www.organic-chemistry.org/prog/peo (accessed on
31 January 2012).
N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, 63 M. A. Reddy, N. Jain, D. Yada, C. Kishore, J. R. Vangala,
V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts,
R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi,
P. R. Surendra, A. Addlagatta, S. V. Kalivendi and
B. Sreedhar, J. Med. Chem., 2011, 54, 6751.
C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, 64 A. S. Kumar, M. A. Reddy, N. Jain, C. Kishor, T. R. Murthy,
V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg,
S. Dapprich, A. D. Daniels, Ö. Farkas, J. B. Foresman,
D. Ramesh, B. Supriya, A. Addlagatta, S. V. Kalivendi and
B. Sreedhar, Eur. J. Med. Chem., 2013, 60, 305.
J. V. Ortiz, J. Cioslowski and D. J. Fox, Gaussian 09, Revision 65 K. Toshima, Y. Okuno, Y. Nakajima and S. Matsumura,
B.01, Gaussian, Inc., Wallingford CT, 2009. Bioorg. Med. Chem. Lett., 2002, 12, 671–673.
57 B. L. Staker, M. D. Feese, M. Cushman, Y. Pommier, 66 T. S. Dexheimer and Y. Pommier, DNA cleavage assay for
D. Zembower, L. Stewart and A. B. Burgin, J. Med. Chem.,
2005, 48, 2336–2345.
the identification of topoisomerase I inhibitors, Nat. Proto-
cols, 2008, 3, 1736–1750.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 2370–2387 | 2387