Synthesis, in vitro evaluation and molecular modelling of naphthalimide analogue as anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2013.07.027

A series of amine substituted naphthalimide analogue were synthesized and evaluated for in vitro anti-tumour activities against 60 tumour cell lines at a single dose concentration of 10 μM. These new analogue showed potential anticancer activities against various cancer cell lines. Compound 5d exhibited significant growth inhibition and was evaluated as 60 cell panel at five dose concentration levels. Compound 5d proved to be fivefold more active than standard antitumour drug 5-fluorouracil (5-FU) with MG-MID GI₅₀ and TGI values of 5.05 and 38.71 respectively. ct-DNA binding studies of most active compound 5d revealed strong interacting properties. Molecular docking studies in the active binding site provided complementary theoretical support for the experimental biological data acquired.

Full text of DOI:10.1016/j.ejmech.2013.07.027

European Journal of Medicinal Chemistry 68 (2013) 352e360
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, in vitro evaluation and molecular modelling of
naphthalimide analogue as anticancer agents
*
Meenakshi Verma, Vijay Luxami, Kamaldeep Paul
School of Chemistry and Biochemistry, Thapar University, Patiala 147004, Punjab, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of amine substituted naphthalimide analogue were synthesized and evaluated for in vitro anti-
Received 18 January 2013
Received in revised form
7 July 2013
Accepted 15 July 2013
Available online 11 August 2013
tumour activities against 60 tumour cell lines at a single dose concentration of 10
mM. These new
analogue showed potential anticancer activities against various cancer cell lines. Compound 5d exhibited
significant growth inhibition and was evaluated as 60 cell panel at five dose concentration levels.
Compound 5d proved to be fivefold more active than standard antitumour drug 5-fluorouracil (5-FU)
with MG-MID GI50 and TGI values of 5.05 and 38.71 respectively. ct-DNA binding studies of most active
compound 5d revealed strong interacting properties. Molecular docking studies in the active binding site
provided complementary theoretical support for the experimental biological data acquired.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Naphthalimide analogue
Anticancer activity
DNA intercalation
Molecular modelling
Physicochemical parameters
1. Introduction
side effects and the risk of developing secondary cancers, thereby
supporting the discovery of nongenotoxic DNA-binding small
The design of highly efficient antitumour agents is of significant
importance in organic and medicinal chemistry. Naphthalimide
based anticancer drugs constitute an indispensable part in the
development of anticancer drugs. Due to the presence of a coplanar
molecules as anticancer therapies [16].
Naphthalimide derivatives modified at different positions pro-
vided an effective way to improve the anticancer activity and
decrease the toxic side effects [17e19]. Moreover, existing research
results confirmed that the substituent at 4-position of the naph-
thalene ring is key to antitumour activity [20,21]. As the amino
group at 4-position of Amonafide showed extensive metabolism
and the nitro-group of Mitonafide was made responsible for toxic
side effects, the modified derivatives were characterized by fused
multi-aromatic ring system and one or two flexible basic side
chains [22e27]. Thus the fusion of one or more aromatic hetero-
cycles with naphthalene core would be an efficient approach to
enhance the antitumour activity. Although the significant results
have been obtained in this way, there were some problems on the
poor solubility and complicated synthesis of naphthalimide de-
rivatives. In the present manuscript, we have focused on fusion of
benzimidazole to the naphthalimide moiety and substitution of
primary and secondary amines at 4-position of naphthalimide.
These would reduce therapeutically problematic functions and lead
to the discovery of several naphthalimides containing enlarged
fused aromatic ring systems whose planar tri-cyclic ring system
may also intercalate into DNA to show medical or biological po-
tential. Among the evaluated compounds, it has been significantly
noted that especially sulphur-containing derivatives showed
promising antiproliferative efficiency [6a,15,21,27]. One of the most
fluorophore, p-deficient aromatic system and one or two basic side
chains, naphthalimide constitutes an important class of drugs in
antitumour therapy and thus characterized by high cytotoxicity
activity against a variety of tumour cells [1e8]. Naphthalimide
compounds are an important class of DNA intercalators and have
been demonstrated to inhibit topo II [9,10]. Amonafide and Mito-
nafide (Fig. 1) were two of the most active naphthalimide-based
topo II inhibitors and had been tested in clinical trials. But, due to
central neurotoxicity and limited efficacy in solid tumours, the
clinical developments of these drugs were terminated [11]. To
improve the efficacy and toxicological profiles, considerable efforts
have been attempted on the design of more active naphthalimides
[12e14], such as Elinafide and Bisnafide and most of the efforts
were focused on the analogue with higher DNA-binding affinity
[15]. Biological studies of these molecules showed that intercalat-
ing reagents are capable of targeting DNA without inducing DNA
damage and mutations in cellular genes. This phenomenon reduced
* Corresponding author. Tel.: þ91 94656 70595; fax: þ91 175 236 4498.
E-mail addresses: kpaul@thapar.edu, kamal.paul2001@gmail.com (K. Paul).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.07.027

M. Verma et al. / European Journal of Medicinal Chemistry 68 (2013) 352e360
353
O
O
O
O
O
O
N
CH3COOH,
Na2Cr2O7
75 0C, 8 hrs
KOH, Br2
60 0
6 hrs
C
O
N
O
O
O
1
3
2
Br
N
N
N
H
N
H
NH2
NH2
NH2
O
O
(1)
THF, Reflux
6 hrs
(3)
N
NO2
O
O
O
N
O
N
N
O2N
N
N
H
H
N
O
N
N
O
O
HNR1R2
O
N
(4)
NO2
K2CO3, CH3CN
TBAHSO4
Reflux
(2)
Fig. 1. Chemical structures of Amonafide (1), Mitonafide (2), Elinafide (3) and Bisna-
fide (4).
NR1R2
Br
4
5
active agents found within our study is thio-heterocyclic naph-
thalimide 5d, which exhibited superior selectivity towards several
tumour cell lines and intercalating properties with ct-DNA.
SH
NH2
H2N
HO
f
O
NR1R2=
NH
N
H
N
2. Results and discussion
H
b
NH2
d
F
a
c
e
2.1. Chemistry
Br
F
NH2
The target naphthalimide analogue was synthesized via multi-
step reactions from commercially available starting material ace-
naphthene 1 (Scheme 1). Oxidation of acenaphthene with acetic
acid and sodium dichromate at 75 ꢀC for 8 h gave 1,8-naphthalic
anhydride (2) with 80% yield followed by bromination with Br2
and KOH at 60 ꢀC for 6 h gave yellow powder of 4-bromo-1,8-
naphthalic anhydride (3) with 82% yield (m.p. ¼ 117e119 ꢀC).
Refluxing of compound 3 with o-phenylenediamine in THF for 6 h
to obtain brown powder of bromo-benzo[de]benzo[4,5]imidazo
[2,1-a]isoquinoline-7-one (4) in 63% yield (m.p. ¼ 190e192 ꢀC). The
proposed mechanism for the synthesis of compound 4 has been
shown in Scheme 2. Compound 4 was refluxed with primary and
secondary amines in the presence of phase transfer catalyst (K2CO3,
CH3CN and TBAHSO4) for 8e12 h to give compounds 5aek. After
separation with careful column chromatography, each pure target
product of substituted-benzo[de]benzo[4,5]imidazo[2,1-a]iso-
quinolin-7-one (5aek) was obtained in moderate to good yields.
NH2
H2N
N
N
NH2
F
NH2
g
j
h
i
k
Scheme 1. Synthesis of substituted-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-
one.
cells/well depending on the doubling time of individual cell lines.
The microtiter plates are then incubated at 37 ꢀC, 5% CO2, 95% air
and 100% relative humidity for 24 h.
After 24 h, two plates of each cell line are fixed in situ with TCA,
to represent a measurement of the cell population for each cell line.
Experimental drugs are solubilized in DMSO at 400 fold the desired
H2N
Structures of all these final products were confirmed by 1H and 13
C
H2N
H2N
H2N
N
NMR (Supplementary material) as well as mass spectroscopy.
H
N
O
O
O
O
O
O
O
OH
2.2. In vitro cancer screen at NCI-USA
-H2O
Compounds 5aek were submitted to National Cancer Institute
(NCI) for evaluation of their in vitro antitumour activities [28e30]
(Table 1). Ten compounds (5a, 5cek) were evaluated against 60
Br
Br
Br
cell lines at a single dose of 10 mM which included nine tumour
subpanels and their output was reported as a mean graph of the
percent growth of treated cells, and presented as percentage
growth inhibition (GI %). Compound 5d exhibited significant
growth inhibition and was evaluated for further 60 cell panel at five
dose concentration levels.
H
OH N
H
N+
N
O
HO
-O
N
O
N
N
-H2O
2.2.1. Pharmacology
The human tumour cell lines of the cancer screening panel are
grown in RPMI 1640 medium containing 5% foetal bovine serum
Br
Br
Br
and 2 mM
L-glutamine. Cells are inoculated into 96 well microtiter
plates in 100 ml at plating densities ranging from 5000 to 40,000
Scheme 2. Proposed mechanism for the synthesis of compound 4.

Table 1
Percentage growth inhibition (GI%) of in vitro subpanel tumour cell lines at 10 mM concentration of compounds 5a, 5cek.
Panel/cell line
5a
5c
5d
5e
5f
5g
5h
5i
5j
5k
5-FU
Leukaemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
e
e
e
e
e
e
46.30
27.96
24.01
97.43
24.26
79.81
55.12
81.83
39.70
L
50.00
83.87
e
e
e
24.98
36.60
21.21
30.59
e
27.47
e
48.24
37.74
53.46
38.16
50.97
29.77
34.56
37.35
33.09
50.16
24.36
98.89
57.1
47.9
42.3
43.1
41.4
24.8
e
e
e
e
e
e
e
e
e
e
25.87
24.64
78.49
42.60
NT
28.57
NT
43.83
47.13
84.42
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
e
67.38
24.06
33.85
59.33
44.68
31.82
e
79.38
NT
e
e
e
23.35
NT
e
29.13
NT
33.46
NT
34.2
58.4
47.8
50.6
69.5
39.0
59.5
13.0
58.0
61.72
e
NT
e
NT
e
NT
e
NT
e
48.83
71.29
59.41
62.45
38.20
90.53
39.95
e
32.19
45.10
30.35
34.45
25.46
47.30
43.37
e
40.79
e
46.77
e
39.99
e
37.60
e
38.79
26.30
40.02
e
35.79
24.92
e
51.55
28.02
54.00
e
23.05
e
e
22.23
e
e
32.23
22.14
e
22.08
e
e
e
83.94
28.85
42.76
e
51.62
28.13
27.34
e
65.28
38.51
e
e
Colon cancer
COLO-205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
e
36.39
29.65
45.30
21.41
22.19
e
49.22
e
e
e
e
e
e
e
e
e
e
e
e
29.00
31.46
35.00
40.79
35.42
25.24
22.93
24.42
48.30
e
40.2
L
26.84
e
23.91
e
25.19
e
26.03
e
27.28
e
53.20
59.54
33.26
26.41
53.75
17.8
26.5
27.1
40.7
50.1
e
28.87
e
28.01
e
23.84
e
24.48
e
25.18
23.15
e
e
21.87
e
25.60
e
22.83
e
22.34
e
21.07
e
40.47
e
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
e
e
40.60
88.03
51.45
53.74
56.29
60.10
e
e
e
e
e
e
e
e
e
e
e
e
59.0
69.1
L
65.9
65.9
50.3
e
40.22
e
e
e
NT
e
NT
e
NT
e
NT
e
e
e
e
e
24.00
26.97
35.29
22.44
29.25
e
e
e
e
e
e
20.9
e
56.00
e
26.56
NT
20.05
NT
21.44
NT
e
NT
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
e
48.31
29.56
e
70.85
65.97
54.61
e
e
e
e
24.11
e
e
30.38
e
26.63
e
30.4
58.2
NT
36.6
95.5
NT
33.7
19.5
39.7
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
NT
e
NT
e
NT
e
NT
e
e
e
26.26
60.60
28.99
68.09
e
e
27.07
23.78
e
26.55
e
35.45
e
29.60
e
20.19
e
24.48
NT
e
e
48.16
NT
35.70
33.91
NT
e
NT
e
37.86
37.45
32.24
23.56
e
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
41.68
e
42.85
e
65.49
22.24
26.02
e
NT
e
63.48
e
51.13
e
58.18
e
46.61
e
43.37
30.01
30.92
e
58.81
e
51.2
47.4
59.4
44.3
NT
30.08
e
21.16
e
24.01
e
e
46.26
e
e
e
e
e
e
e
e
e
33.96
e
50.00
43.42
46.12
e
e
e
e
e
24.58
25.40
e
e
e
e
e
e
e
e
e
47.6
77.5
e
20.92
e
e
e
e
e
e
Renal cancer
786-0
A498
e
e
62.66
41.22
77.92
94.10
27.44
80.78
21.42
90.73
e
e
e
e
e
e
e
48.7
L
e
61.96
47.65
48.66
e
33.84
e
32.85
e
e
e
e
e
e
ACHN
e
e
e
e
23.94
48.16
e
27.11
52.78
e
39.3
39.4
34.3
54.0
66.9
41.3
CAKI-1
RXF 393
SN12C
TK-10
30.37
e
20.13
e
e
e
56.82
e
38.00
e
e
e
e
35.19
e
e
e
e
e
e
28.28
34.20
53.45
e
28.19
34.11
24.68
50.78
e
21.91
47.89
29.84
41.38
NT
49.37
22.63
58.62
UO-31
43.81
38.32
Prostate cancer
PC-3
DU-145
e
e
32.07
21.86
49.58
60.67
22.79
e
e
e
23.33
e
e
e
e
e
35.99
e
20.05
e
58.2
35.5
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
76.03
e
50.00
22.86
32.80
28.77
27.85
e
76.42
45.60
40.37
40.94
60.37
56.48
89.91
29.13
26.39
e
57.46
e
95.85
33.82
e
89.72
20.17
e
79.64
e
71.34
29.40
e
86.51
e
11.5
78.1
L
37.8
56.7
NT
59.82
e
e
e
e
e
e
e
e
e
e
61.88
L
76.56
L
31.68
81.08
81.64
L
64.33
L
47.34
L
53.38
L
67.46
L
MDA-MB-468
Prominent GI values are bolded.
e, GI < 20%; L, compounds proved lethal to the cancer cell line; NT, not tested.

M. Verma et al. / European Journal of Medicinal Chemistry 68 (2013) 352e360
355
final maximum test concentration and stored frozen prior to use. At
the time of drug addition, an aliquot of frozen concentrate is
thawed and diluted to twice the desired final maximum test con-
leukaemia cancer cell line MOLT-4 while breast cancer cell line
MDA-MB-468 is lethal for compounds 5a, 5e, and 5gek. Compound
5d exhibited more than 50% inhibition for most of the tumour cell
lines and much better than 5-fluorouracil (positive control) in most
of the tested derivatives.
centration with complete medium containing 50
Additional four,10-fold or ½ log serial dilutions are made to provide
a total of five drug concentrations plus control. Aliquots of 100 L of
mg/ml gentamicin.
m
From above results, it is clear that compound 5d is the most
active member of the series. This compound was tested against a
panel of 60 different tumour cell lines at a 5-log dose range. Three
response parameters, GI50, TGI, and LC50 were monitored for each
cell line, using the known drug 5-fluorouracil (5-FU) as a positive
control (Table 2). A comparison of values of MG-MID of GI50, TGI,
and LC50 for compounds 5d and 5-fluorouracil conclude that
compound 5d is fivefold more active than 5-fluorouracil, with GI50
and TGI values of 5.05 and 38.71 respectively as compared to GI50
and TGI values (22.6 and >100) for 5-fluorouracil (Table 2, Fig. 2).
The obtained results revealed that compounds 5a, 5i and 5j
were devoid of any antitumour potency. Compounds bearing pyr-
rolidine, 5-bromo-2-aminopyridine, butylamine and cyclohexyl-
amine showed antitumour activity as exemplified by 5c, 5g, 5h and
5k respectively. Substitution with 4-aminothiophenol in 5d
showed broad spectrum antitumour activity. Thus, thio group in
naphthalimide derivative imparts a key role for the anticancer
activity.
these different drug dilutions are added to the appropriate micro-
titer wells, resulting in the required final drug concentrations.
Following drug addition, the plates are incubated for an addi-
tional 48 h at 37 ꢀC, 5% CO2, 95% air, and 100% relative humidity. For
adherent cells, the assay is terminated by the addition of cold TCA.
Cells are fixed in situ by the gentle addition of 50 mL of cold 50% (w/
v) TCA and incubated for 60 min at 4 ꢀC. The supernatant is dis-
carded, and the plates are washed five times with tap water and air
dried. Sulforhodamine B (SRB) solution (100 mL) at 0.4% (w/v) in 1%
acetic acid is added to each well, and plates are incubated for
10 min at room temperature. After staining, unbound dye is
removed by washing five times with 1% acetic acid and the plates
are air dried and then subsequently solubilized with 10 mM trizma
base, and the absorbance is read on an automated plate reader at a
wavelength of 515 nm. Using the seven absorbance measurements
[time zero (Tz), control growth (C), and test growth in the presence
of drug at the five concentration levels (Ti)], the percentage growth
is calculated at each of the drug concentrations levels. Percentage
growth inhibition is calculated as:
2.3. DNA binding studies
[(Ti ꢁ Tz)/(C ꢁ Tz)] ꢂ 100 for concentrations for which Ti ꢃ Tz;
[(Ti ꢁ Tz)/Tz] ꢂ 100 for concentrations for which Ti < Tz.
DNA binding properties of most active compound 5d have been
evaluated using both UVevisible spectrophotometer and fluores-
cence spectrophotometry in order to determine the interaction of
Three dose response parameters are calculated for each exper-
imental agent. Growth inhibition of 50% (GI50) is calculated from
[(Ti ꢁ Tz)/(C ꢁ Tz)] ꢂ 100 ¼ 50. The drug concentration resulting in
total growth inhibition (TGI) is calculated from Ti ¼ Tz. The LC50 is
calculated from [(Ti ꢁ Tz)/Tz] ꢂ 100 ¼ ꢁ50 [28,30,31].
compounds with DNA. The addition of ct-DNA (4e100
mM) to the
phosphate buffered solution of 5d (50 M) induced bathochromic
m
shift (w20 nm) of absorption band from 410 nm to 430 nm with
hypochromic effect (Fig. 3a). The observed hypochromic shifts in
absorption bands of 5d upon addition of DNA result from the
intercalation of 5d with ct-DNA as suggested in the literature [32].
The intercalative nature of interaction of compound 5d with ct-
DNA has been additionally supported by thermal denaturation
experiment. Intercalation of molecules into the double helix is
known to stabilize the DNA against thermal strand separation and
thus increases thermal melting temperature (Tm) [33]. The deriv-
ative melting curve shows an increase of 8.2 ꢀC in thermal melting
temperature of ct-DNA (60 ꢀC) upon addition of 5d (68.2 ꢀC). Thus,
both UVevisible titrations and thermal denaturation experiments
indicate strong intercalative nature of interactions between com-
pound 5d and ct-DNA.
2.2.2. Regarding the activity towards individual cell lines
In the present investigation, most of the tested compounds
showed broad spectrum antitumour activity. Compounds 5a, 5eeg
and 5i displayed modest potency against the tested tumour cell
lines and considered to be the least effective members. Leukaemia
cancer cell line HL-60 (TB) proved to be sensitive towards com-
pound 5d with GI value of 81.83%, while leukaemia cell line MOLT-4
proved to be sensitive towards compound 5c with GI value of
97.43% and leukaemia cell line SR showed sensitivity to compounds
5d, 5h and 5k with GI values of 83.87%, 84.42% and 98.89%
respectively. Compounds 5c and 5d exhibited sensitivity towards
non-small cell lung cancer cell line NCI-H460 with GI values of
83.94% and 90.53% respectively. CNS cancer line SF-295, renal
cancer cell lines SN-12C and UO-31 of compound 5d showed
sensitivity with GI values of 88.03%, 80.78% and 90.73% respectively.
Breast cancer cell line MCF7 exhibited sensitivity towards com-
pounds 5e, 5g, 5h and 5k with GI values of 89.91%, 95.85%, 89.72%
and 86.51% respectively. Compound 5d proved to be lethal to
Fluorescence titrations of 5d were also performed with ct-DNA
in phosphate buffered solution, showed decrease in the intensity of
the emission band of 5d at 500 nm (Fig. 3b) upon addition of
increasing concentration of ct-DNA. Binding constant of 5d with ct-
DNA was log
b
¼ 5.32 calculated from titration data using Benesie
Hildebrand method. The apparent binding constant with ct-DNA
Table 2
Compounds 5d and 5-FU median growth inhibitory (GI50, mM), total growth inhibitory (TGI, mM) and median lethal concentrations (LC50, mM) of in vitro subpanel tumour cell
lines.
Compounds
Activity
I
II
III
IV
V
VI
VII
VIII
IX
MG-MIDa
5.05
5d
GI50
TGI
LC50
2.9
3.94
32.51
93.94
6.58
60.81
98.84
4.09
45.41
93.98
6.93
35.07
83.93
6.97
37.44
97.94
3.30
15.41
88.94
3.23
17.9
80.15
5.20
44.51
45.98
38.71
b
b
b
b
b
b
5-FU
GI50
TGI
LC50
1.51
8.4
72.1
70.6
61.4
45.6
22.7
76.4
22.6
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
I, Leukaemia; II, non-small cell lung cancer; III, colon cancer; IV, CNS cancer; V, melanoma; VI, ovarian cancer; VII, renal cancer; VIII, prostate cancer; IX, breast cancer.
a
Full panel mean-graph midpoint (
Compounds showed values >100
m
m
M).
M.
b

356
M. Verma et al. / European Journal of Medicinal Chemistry 68 (2013) 352e360
Fig. 2. Compounds 5d and 5-FU median growth inhibitory (GI50
lines (A) and comparison of full panel mean-graph midpoint (
,
m
M), total growth inhibitory (TGI,
mM) and median lethal concentration (LC50, mM) of in vitro subpanel tumour cell
mM) of compounds 5d and 5-FU (B).
was determined to be log
indicate that compound 5d intercalates more strongly than
Amonafide.
b
¼ 5.15 for Amonafide. The results clearly
compound 5d showed more electronic affinity and less steric hin-
dered for DNA intercalation. Comparison of these parameters with
5-fluorouracil also proved that compound 5d is superior in all
aspects.
2.4. ADME-T prediction
2.5. Structureeactivity relationship
As a part of our study, the compliance of compounds 5a, 5cek
was also evaluated with different physico-chemical parameters;
drug bioavailability parameters (Log P, Total Polar Surface Area) and
other parameters such as molar refractivity, dipole moment, elec-
tron affinity and steric energy, determined by SciGress software
[34]. The results disclosed in Table 3 showed that all the com-
pounds exhibited good passive oral absorption and thus difference
in their bioactivity cannot be attributed to this property. The vari-
ations of alkyl or aryl fragments on the naphthalimide and the
substitution on these fragments have allowed us to evaluate the
influence of different parameters at the pharmacophoric part of the
molecules. Within the series of compounds, we have observed that
antitumour activity of compound 5d (5-dose studies) increased
with increase in molar refractivity (117.0), electron affinity (1.7 eV)
and Total Polar Surface Area (66.4) and decreased in steric energy
(15.3). Although lipophilicity did not exert any effect on activity in
all compounds but hydrophilicity produced an important param-
eter for the activity (5d showed higher TPSA value). Steric param-
eters have a clear influence of molar refractivity and steric energy
on antitumour activity compared with lipophilicity and TPSA. Thus,
The structureeactivity relationship emerged from the activity
results revealed that combination of naphthalimide and benz-
imidazole was well tolerated for the in vitro anticancer activity. We
then turned our attention to exploring the SAR profile of the C4-
position of naphthalimide ring (5aek) in hope of finding a
permissive region. (i) The primary and secondary amines at C4-
position of naphthalimide play a key role in varying the efficiency of
antitumour activities. (ii) The primary amines positively
influence in the antitumour effectiveness, inducing good activity
against most of the cancer cell lines. (iii) The introduction of 4-
amino-phenylsulfanyl group at the C4-position of naphthalimide
ring (5d) led to a broad spectrum of antitumour activity with
respect to other primary amines. The role of sulphur atom in
improving antitumour activities had been reported in the literature
[15,21,27]. (iv) Presence of 5-bromopyridine (5g) and 2,6-
difluoroaniline (5i) has slightly improved the activity. (v) Com-
pounds with acyclic aliphatic amines (5f and 5h) showed the least
activity at 10
mM concentrations. (vi) Amongst the secondary
amines, five membered ring (5c) has improved the antitumour
Fig. 3. Effect of addition of ct-DNA on (a) absorption (b) emission spectra of compound 5d (50 mM, pH 7.0) in phosphate buffer.

M. Verma et al. / European Journal of Medicinal Chemistry 68 (2013) 352e360
357
Table 3
Calculated physicochemical parameters of compounds 5a, 5cek and 5-FU.
Parameters
5a
5c
5d
4.9
46.2
117.0
3.7
377.8
91.8
1.7
5e
5f
5g
5h
4.7
43.0
103.7
5.1
352.8
38.4
1.4
5i
5j
5k
5.1
46.4
106.3
4.9
372.7
33.0
1.4
5-FU
Log P
Polarizability
3.9
4.5
4.9
5.1
5.4
5.5
4.6
ꢁ1.3
43.0
103.2
2.7
346.1
20.1
1.5
42.5
101.6
4.4
339.1
50.0
1.4
45.8
108.4
2.6
366.9
41.0
1.6
46.4
110.9
4.9
372.7
33.0
1.4
48.9
113.9
5.2
381.9
100.1
1.5
45.8
108.6
4.8
368.5
1.4
45.0
106.3
4.9
357.2
91.7
1.5
10.7
26.1
Molar refractivity
Dipole moment (Debye)
Solvent accessibility surface area (A m )
Conformation minimum energy (kcal/mole)
Electron affinity (eV)
3.4
2
ꢀ
138.9
ꢁ107.4
0.8
1.5
Steric energy (kcal/mole)
Total polar surface area (TPSA)
68.1
46.8
40.6
37.6
15.3
66.4
56.5
46.4
39.5
60.6
50.9
59.2
39.5
46.4
67.2
46.4
47.5
59.2
39.5
46.4
ꢁ13.4
65.7
activity. (vii) The hydrophilicity and steric parameters of the com-
pounds may also play a crucial role on the effectiveness of the
compounds tested.
have proven useful for refining the structure of the molecules and
improving their efficiencies (Table 4).
3. Conclusions
2.6. Molecular modelling studies
The present work has led to the development of novel naph-
thalimide molecules and some of which shown promising anti-
tumour activities and even more active than 5-fluorouracil at 10 mM
Molecular modelling studies were also carried out for com-
pound 5d, which has been proved to be most active compound.
Although the cellular targets were not defined in the experimental
anticancer investigation of these molecules, to look into the
possible interactions at the enzymatic level, we have carried out the
docking studies [35] of compound 5d in the active site of topo-
isomerase _ (Topo I, pdb ID 1A36), topoisomerase II (Topo II, pdb ID
1BJT, enzymes responsible for DNA replication) and ribonucleotide
reductase (RNR, pdb ID 4R1R).
concentration. Compound 5d proved to be fivefold more active
than 5-FU in five dose concentration levels, with MG-MID GI50 and
TGI values of 5.05 and 38.71 respectively and also have strong
intercalation properties with ct-DNA. The experimental as well as
theoretical studies clearly predicts that substitution at 4-position of
naphthalimide and sulphur linkage is essential for the antitumour
activity. The new naphthalimide compounds prepared in this work
have good physical properties that qualify them to have good
pharmacokinetic and drug bioavailability. These preliminary
encouraging results of biological screening and docking of tested
compounds could offer an excellent framework in this field and
may lead to discovery of potent antitumour agents.
Docking of compound 5d in the active site of Topo I (1A36)
showed hydrogen bonding interaction between terminal NH group
ꢀ
of naphthalimide with carbonyl of Gln 692 (d ¼ 2.79 A) amino acid
residue and nitrogen atom of imidazolone ring of 5d showed
ꢀ
hydrogen bonding with amine group of Arg 622 (d ¼ 2.94 A) amino
acid residue. Aminothiophenol moiety was surrounded by amino
acid residues Glu 695, Glu 696 and Leu 694 (Fig. 4A).
4. Experimental section
Docking of compound 5d with active site of Topo II (1BJT)
showed hydrogen bonding interaction between terminal amine
4.1. Chemistry
ꢀ
group of naphthalimide with Asn 431 (NH2.O]C, d ¼ 2.66 A) and
ꢀ
All chemicals and solvents of commercial grade were used
without further purification and were supplied by loba, spec-
trochemicals and Aldrich. Melting points were determined in open
capillaries and are uncorrected. 1H and 13C NMR spectra were
recorded on Bruker 400 MHz and 100 MHz NMR spectrometer
respectively, using CDCl3 and DMSO-d6 as solvents. The chemical
shifts were expressed in parts per million with TMS as internal
reference and J values are given in Hz. Reactions were monitored by
thin layer chromatography (TLC) with silica plate coated with silica
gel HF-254 and column chromatography was performed with silica
gel 60e120/100e200 mesh. Hexane/ethyl acetate and chloroform/
methanol were adopted solvent systems.
Tyr 518 (NH2.O]C, d ¼ 1.89 A) amino acid residues. Carbonyl
group of imidazolone (5d) showed hydrogen bonding interaction
ꢀ
ꢀ
with amino group of Leu 447 (d ¼ 2.49 A) and Thr 448 (d ¼ 1.90 A)
amino acid residues. Nitrogen atom of amide group of imidazolone
ꢀ
also showed H-bonding with amino group of Leu 447 (d ¼ 1.75 A)
amino acid residue. Imidazolone nitrogen atom of this compound
showed hydrogen bonding interaction with amino group of Met
ꢀ
ꢀ
522 (d ¼ 2.49 A) and Ile 531 (d ¼ 2.62 A) amino acid residues. The
aromatic rings of 5d were surrounded by rings of Tyr 518, Tyr 472
and Tyr 469 amino acid residues. These residues might help in the
formation of pep interaction with DNA for binding (Fig. 4B).
Molecular docking of terminal amine group of compound 5d
showed H-bonding interaction with Gln 415 (NH2.O]C,
ꢀ
d ¼ 2.13 A) amino acid residue of RNR (4R1R, another enzyme
4.2. General procedure for synthesis of 3-bromo-benzo[de]benzo
[4,5]imidazo[2,1-a]isoquinolin-7-one 4
involved in the propagation of cancer, synthesis of raw material for
replication of DNA). Imidazolone nitrogen atom of this compound
ꢀ
showed hydrogen bonding with amino group of Ile 414 (d ¼ 2.64 A)
To a solution of acenaphthene (2.31 g,15.0 mmol) and acetic acid
(60 ml), sodium dichromate (11.17 g, 40.0 mmol) was added with
stirring at room temperature. The suspensionwas heated to75 ꢀC for
8 h. After the completion of reaction, cold water was added to the
reaction mixture to get solid, filtered and washed with water. Off
white product of 1,8-naphthalic anhydride (2) (2.6 g, 80%, m.p. 266e
268 ꢀC) was obtained. To a cooled solution of 1,8-naphthalic anhy-
dride (1.98 g,10.0 mmol) in KOH (2.8 g in 12 ml water), bromine was
charged with dropping funnel. The reaction mixture was heated to
60 ꢀC for 6 h and then acidified with HCl solution. Solid was sepa-
rated out, filtered to get crude product. The solid residue was heated
ꢀ
and Tyr 413 (d ¼ 1.19 A) amino acid residue. Imidazolone amide
nitrogen atom also showed hydrogen bonding interaction with
ꢀ
amino group of Tyr 413 (d ¼ 1.58 A) amino acid residue (Fig. 4C).
Compound 5d has been surrounded by aromatic rings of residues
Phe 305, Tyr 306 and Tyr 413. Thus, these amino acid residues
showed pep interactions with 5d which helps in the intercalation
with DNA and effect the anticancer activity. Therefore, docking of
compound 5d in the active site of these enzymes indicated the
probable mode of action for anticancer activities. Remarkably,
correlations between the experimental data and docking studies

358
M. Verma et al. / European Journal of Medicinal Chemistry 68 (2013) 352e360
Table 4
Hydrogen bonding of compound 5d with topoisomerase I (1A36), topoisomerase II
(1BJT) and ribonucleotide reductase (4RIR).
Groups of compound 1A36, amino 1BJT, amino acid
4RIR, amino acid
residue
5d
acid residue residue
ꢀ
ꢀ
Nitrogen atom of
terminal amine
group
Imidazolone
carbonyl oxygen
Imidazolone
nitrogen atom
Imidazolone amide
nitrogen atom
Q692
N431 (d ¼ 2.66 A), Q415 (d ¼ 2.13 A)
ꢀ
ꢀ
(d ¼ 2.79 A) Y518 (d ¼ 1.89 A)
ꢀ
L447 (d ¼ 2.49 A),
ꢀ
T448 (d ¼ 1.90 A)
ꢀ
ꢀ
R622
M522 (d ¼ 2.49 A), I414 (d ¼ 2.64 A),
ꢀ
ꢀ
ꢀ
(d ¼ 2.94 A) I531 (d ¼ 2.62 A)
T413 (d ¼ 1.19 A)
ꢀ
ꢀ
L447 (d ¼ 1.75 A)
T413 (d ¼ 1.58 A)
with 5% NaOH solution, filtered and neutralized with HCl solution.
Again filtered and washed with cold water, dried to get yellow
powder of 4-bromo-1,8-naphthalic anhydride (3) (2.05 g, 82%, m.p.
117e119 ꢀC). 4-Bromo-1,8-naphthalic anhydride (2.00 g, 5.0 mmol)
and o-phenylenediamine (0.618 g, 5.0 mmol) inTHF was refluxed for
6 h. After the completion of reaction (TLC), cooled the reaction
mixture to get crude solid, filtered and washed with THF to afford
pure 3-bromo-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-
one (4). Brown powder (2.51 g, 63%) m.p. 190e192 ꢀC. 1H NMR
(CDCl3 þ TFA);
d
8.90(dd, 2J¼ 8.48Hz, 3J ¼ 0.68 Hz,1H, ArH), 8.79 (dd,
2J ¼ 7.36 Hz, 3J ¼ 0.84 Hz,1H, ArH), 8.53 (d, J ¼ 7.92 Hz,1H, ArH), 8.24
(d, J ¼ 7.92 Hz,1H, ArH), 8.04 (dd, 2J ¼ 8.48 Hz, 3J ¼ 7.48 Hz,1H, ArH),
7.74 (m, 3H, ArH), 7.56 (m,1H, ArH).13C NMR (CDCl3 þ TFA);
d 165.76,
136.55, 134.50, 134.42, 133.44, 131.92, 131.75, 131.68, 131.15, 130.90,
129.30, 128.64, 128.20, 127.26, 124.97, 121.02, 119.98. EIMS, m/z: 350
(Mþ þ 1). Anal. Calc. for (C18H9BrN2O): C, 61.91; H, 2.60; N, 8.08.
Found: C, 61.76; H, 2.81; N, 8.45.
4.3. General procedure for synthesis of 3-substituted-benzo[de]
benzo[4,5]imidazo[2,1-a]isoquinolin-7-one 5aek
A mixture of 3-bromo-benzo[de]benzo[4,5]imidazo[2,1-a]iso-
quinolin-7-one (0.25 g, 0.7 mmol), potassium carbonate (0.059 g,
1.05 mmol), TBAHSO4 (catalytic) and corresponding amine
(0.84 mmol) in acetonitrile was heated to reflux for 8e12 h. After
the completion of reaction, reaction mixture was extracted with
chloroform, separated the organic layer, dried over Na2SO4, filtered
and concentrated to get solid crude product. The residue was col-
umn chromatographed on silica gel using hexane:ethylacetate as
eluents to afford pure compounds of 3-substituted-benzo[de]benzo
[4,5]imidazo[2,1-a]isoquinolin-7-one 5aek.
4.3.1. 3-Morpholin-4-yl-benzo[de]benzo[4,5]imidazo[2,1-a]
isoquinolin-7-one 5a
Orange powder (0.20 g, 66%) m.p. 250e252 ꢀC. 1H NMR (CDCl3);
d
8.85 (dd, 2J ¼ 7.28 Hz, 3J ¼ 0.96 Hz, 1H, ArH), 8.72 (d, J ¼ 8.08 Hz,
1H, ArH), 8.58 (m, 1H, ArH), 8.36 (dd, 2J ¼ 8.44 Hz, 3J ¼ 1.00 Hz, 1H,
ArH), 7.89 (m, 1H, ArH), 7.77 (dd, 2J ¼ 9.08 Hz, 3J ¼ 1.68 Hz,1H, ArH),
7.50 (m, 2H, ArH), 7.33 (dd, 2J ¼ 17.12 Hz, 3J ¼ 8.08 Hz, 1H, ArH), 4.05
(t, J ¼ 4.56 Hz, 4H, CH2), 3.33 (t, J ¼ 4.60 Hz, 4H, CH2); 13C NMR
(CDCl3 þ TFA);
d 159.71, 159.38, 147.53, 138.23, 136.28, 134.78,
134.70,132.31, 130.79, 129.96, 129.72,129.43, 129.29, 129.05, 128.74,
127.95, 127.53, 126.03, 117.78, 117.53, 66.94, 66.88, 53.60, 53.56.
EIMS, m/z; 356 (Mþ þ 1). Anal. Calc. for (C22H17N3O2): C, 74.35; H,
4.82; N, 11.82. Found: C, 74.03; H, 4.42; N, 11.45.
4.3.2. 3-Piperidin-1-yl-benzo[de]benzo[4,5]imidazo[2,1-a]
isoquinolin-7-one 5b
Fig. 4. Compound 5d docked in active site of Topo I (A), Topo II (B) and RNR (C). H-
bonds of compound 5d with different amino acids residues are visible. Carbon atoms
are given in different colour.
Yellow powder (0.17 g, 70%) m.p. 200e202 ꢀC. 1H NMR (CDCl3);
d
8.63 (dd, 2J ¼ 7.20 Hz, 3J ¼ 0.88 Hz, 1H, ArH), 8.55 (d, J ¼ 8.12 Hz,

M. Verma et al. / European Journal of Medicinal Chemistry 68 (2013) 352e360
359
1H, ArH), 8.45 (dd, 2J ¼ 8.44 Hz, 3J ¼ 0.96 Hz, 1H, ArH), 7.72 (dd,
2J ¼ 8.28 Hz, 3J ¼ 7.44 Hz, 1H, ArH), 7.29 (dd, 2J ¼ 7.76 Hz,
3J ¼ 1.48 Hz, 1H, ArH), 7.21 (d, J ¼ 8.16 Hz, 1H, ArH), 7.11 (dd,
2J ¼ 8.36 Hz, 3J ¼ 1.52 Hz, 1H, ArH), 6.94 (m, 2H, ArH), 3.27 (t,
J ¼ 4.96 Hz, 4H, CH2), 1.90 (m, 4H, CH2), 1.75 (t, J ¼ 6.12 Hz, 2H, CH2).
3J ¼ 1.08 Hz, 1H, ArH), 8.63 (d, J ¼ 7.96 Hz, 1H, ArH), 8.57 (d,
J ¼ 7.92 Hz, 1H, ArH), 8.48 (m, 2H, ArH), 8.09 (dd, 2J ¼ 10.72 Hz,
3J ¼ 7.92 Hz, 2H, ArH), 7.87 (m, 4H, ArH). 13C NMR (CDCl3);
d 157.38,
155.92, 152.17, 145.86, 142.29, 141.42, 139.40, 134.32, 131.96, 131.77,
118.18, 117.27, 117.04, 116.83, 116.70, 114.33, 109.81, 106.63. EIMS, m/
z: 442 (Mþ þ 1). Anal. Calc. for (C23H13BrN4O): C, 62.60; H, 2.97; N,
12.70. Found: C, 62.55; H, 2.87; N, 12.45.
13C NMR (DMSO-d6);
d 163.55, 163.05, 156.61, 144.99, 131.78, 130.15,
130.08, 130.01, 129.27, 128.69, 125.79, 125.15, 123.62, 120.81, 116.32,
116.02, 115.60, 114.40, 54.01, 25.72, 23.84. EIMS, m/z; 354 (Mþ þ 1).
Anal. Calc. for (C23H19N3O): C, 78.16; H, 5.42; N, 11.89. Found: C,
78.32; H, 5.31; N, 11.45.
4.3.8. 3-Butylamino-benzo[de]benzo[4,5]imidazo[2,1-a]
isoquinolin-7-one 5h
Brown powder (0.1 g, 40%) m.p. 210e212 ꢀC. 1H NMR (CDCl3);
4.3.3. 3-Pyrrolidin-1-yl-benzo[de]benzo[4,5]imidazo[2,1-a]
isoquinolin-7-one 5c
d
8.56 (d, J ¼ 7.32 Hz, 1H, ArH), 8.45 (d, J ¼ 8.68 Hz,1H, ArH), 8.08 (d,
J ¼ 7.80 Hz, 1H, ArH), 7.57 (dd, 2J ¼ 8.28 Hz, 3J ¼ 7.36 Hz, 1H, ArH),
7.27 (m, 1H, ArH), 7.12 (dd, 2J ¼ 8.28 Hz, 3J ¼ 1.84 Hz, 1H, ArH), 6.93
(m, 2H, ArH), 6.70 (d, J ¼ 8.24 Hz, 1H, ArH), 5.36 (t, J ¼ 5.04 Hz, 2H,
NH), 3.38 (q, J ¼ 7.32 Hz, 2H, ArH), 1.83 (m, 2H, CH2), 1.58 (m, 2H,
Yellow powder (0.16 g, 67%) m.p. 190e192 ꢀC. 1H NMR (CDCl3);
d
8.65 (t, J ¼ 7.72 Hz, 2H, ArH), 8.48 (d, J ¼ 8.68 Hz, 1H, ArH), 7.58
(dd, 2J ¼ 8.52 Hz, 3J ¼ 7.36 Hz, 1H, ArH), 7.28 (m, 1H, ArH), 7.12 (dd,
2J ¼ 8.12 Hz, 3J ¼ 1.52 Hz, 1H, ArH), 6.93 (m, 2H, ArH), 6.85 (d,
J ¼ 8.72 Hz,1H, ArH), 3.83 (t, J ¼ 6.44 Hz, 4H, CH2), 2.13 (m, 4H, CH2).
CH2), 1.03 (t, J ¼ 7.36 Hz, 3H, CH3). 13C NMR (CDCl3);
d 164.34,
163.67, 149.72, 143.06, 135.00, 131.59, 130.30, 129.61, 129.52, 126.17,
124.62, 123.14, 122.41, 120.14, 119.41, 117.21, 109.89, 104.33, 43.41,
30.95, 20.32, 13.85. EIMS, m/z: 342 (Mþ þ 1). Anal. Calc. for
(C22H19N3O): C, 77.40; H, 5.61; N, 12.31. Found: C, 77.80; H, 5.31; N,
12.30.
13C NMR (DMSO-d6);
d 163.76, 162.88, 152.17, 145.19, 132.55, 131.97,
131.30, 130.22, 129.44, 128.47, 122.84, 122.13, 121.27, 115.80, 115.50,
110.45, 108.14, 52.81, 25.59. EIMS, m/z; 340 (Mþ þ 1). Anal. Calc. for
(C22H17N3O): C, 77.86; H, 5.05; N, 12.38. Found: C, 77.61; H, 4.84; N,
12.45.
4.3.9. 3-(2,6-Difluoro-phenylamino)-benzo[de]benzo[4,5]imidazo
[2,1-a]isoquinolin-7-one 5i
4.3.4. 3-(4-Amino-phenylsulfanyl)-benzo[de]benzo[4,5]imidazo
[2,1-a]isoquinolin-7-one 5d
Brown powder (0.17 g, 61%) m.p. 137e139 ꢀC. 1H NMR (CDCl3);
Pale brown powder (0.15 g, 56%) m.p. 137e139 ꢀC. 1H NMR
d
8.81 (m, 1H, ArH), 8.78 (d, J ¼ 6.88 Hz, 1H, ArH), 8.62 (d,
(DMSO-d6);
d
8.70 (d, J ¼ 7.92 Hz, 2H, ArH), 8.37 (d, J ¼ 8.00 Hz, 1H,
J ¼ 7.36 Hz, 2H, ArH), 8.59 (m, 2H, ArH), 8.25 (dd, 2J ¼ 11.0 Hz,
3J ¼ 8.24 Hz, 1H, ArH), 7.83 (m, 3H, ArH), 7.45 (m, 1H, ArH), 6.93 (t,
J ¼ 3.64 Hz, 1H, ArH). 13C NMR (CDCl3); 163.96, 160.81, 143.74,
143.07, 135.47, 134.43, 132.03, 131.80, 131.34, 131.26, 128.68, 127.50,
127.03, 125.92, 125.82, 125.55, 123.19, 122.78, 120.01, 119.69, 115.96.
EIMS, m/z: 398 (Mþ þ 1). Anal. Calc. for (C24H13F2N3O): C, 72.54; H,
3.30; N, 10.57. Found: C, 72.33; H, 3.32; N, 10.55.
ArH), 7.83 (t, J ¼ 7.88 Hz, 1H, ArH), 7.41 (d, J ¼ 6.60 Hz, 2H, ArH), 7.27
(d, J ¼ 1.48 Hz,1H, ArH), 7.11 (m, 2H, ArH), 6.94 (m, 2H, ArH), 6.80 (d,
J ¼ 6.60 Hz, 2H, ArH). 13C NMR (DMSO-d6);
d 163.21, 163.09, 150.19,
148.49, 144.66, 136.78, 130.70, 130.10, 129.11, 128.87, 128.40, 127.80,
126.23, 123.47, 122.35, 120.58, 119.00, 116.38, 115.77, 115.36, 111.72.
EIMS, m/z; 394 (Mþ þ 1). Anal. Calc. for (C24H15N3OS): C, 73.26; H,
3.84; N, 10.68; S, 8.15. Found: C, 73.55; H, 3.48; N, 10.45; S, 8.08.
4.3.10. 3-(Pyridin-2-ylamino)-benzo[de]benzo[4,5]imidazo[2,1-a]
isoquinolin-7-one 5j
4.3.5. 3-(4-Fluoro-phenylamino)-benzo[de]benzo[4,5]imidazo[2,1-
a]isoquinolin-7-one 5e
Brownpowder (0.16 g, 63%) m.p.190e192 ꢀC.1H NMR (DMSO-d6);
Brown powder (0.21 g, 80%) m.p. 240e242 ꢀC. 1H NMR (CDCl3);
d
8.80 (d, J ¼ 24.24 Hz,1H, ArH), 8.60 (d, J ¼ 8.28 Hz,1H, ArH), 8.53 (dd,
d
8.80 (m, 2H, ArH), 8.57 (d, J ¼ 7.92 Hz, 2H, ArH), 8.50 (dd,
2J ¼ 7.28 Hz, 3J ¼ 0.96 Hz, 2H, ArH), 8.38 (dd, 2J ¼ 8.28 Hz, 3J ¼ 0.80 Hz,
2H, ArH), 7.84 (t, J ¼ 3.76 Hz, 2H, ArH), 7.45 (m, 1H, ArH), 7.24 (m, 1H,
ArH), 7.01(dd,2J¼ 7.76 Hz, 3J¼ 1.36 Hz,1H, ArH), 6.90(dd, 2J¼ 8.04Hz,
3J ¼ 1.00 Hz, 1H, ArH), 6.76 (t, J ¼ 6.56 Hz, 1H, ArH). 13C NMR (DMSO-
2J ¼ 7.28 Hz, 3J ¼ 4.00 Hz, 3H, ArH), 8.08 (dd, 2J ¼ 18.82 Hz,
3J ¼ 7.60 Hz, 1H, ArH), 7.87 (m, 4H, ArH). 13C NMR (DMSO-d6);
d
159.04, 158.82, 150.63, 150.44, 150.03, 140.61, 137.06, 134.28,
133.61, 129.73, 129.37, 127.74, 126.73, 126.80, 126.21, 123.66, 121.89,
121.21, 119.01, 116.20, 114.23, 113.72, 111.59, 106.96. EIMS, m/z; 380
(Mþ þ 1). Anal. Calc. for (C24H14FN3O): C, 75.98; H, 3.72; N, 11.08;
Found: C, 76.31; H, 4.03; N, 11.45.
d6); d 158.84, 152.84, 152.21, 139.34, 134.79, 132.31, 131.70, 126.68,
124.62, 124.31, 119.73, 117.17, 116.93, 116.37, 115.78, 113.43, 110.49,
109.38. EIMS, m/z: 363 (Mþ þ 1). Anal. Calc. for (C23H14N4O): C, 76.23;
H, 3.89; N, 15.46. Found: C, 76.21; H, 3.34; N, 15.45.
4.3.6. 3-(2-Hydroxy-ethylamino)-benzo[de]benzo[4,5]imidazo[2,1-
a]isoquinolin-7-one 5f
4.3.11. 3-Cyclohexylamino-benzo[de]benzo[4,5]imidazo[2,1-a]
isoquinolin-7-one 5k
Brown powder (0.12 g, 52%) m.p. 170e172 ꢀC. 1H NMR (DMSO-
Brown powder (0.15 g, 58%) m.p. 220e222 ꢀC. 1H NMR (DMSO-
d6);
d
8.78 (d, J ¼ 7.80 Hz, 1H, ArH), 8.67 (d, J ¼ 4.04 Hz, 1H, ArH),
d6);
d
8.22 (dd, 2J ¼ 23.32 Hz, 3J ¼ 8.04 Hz, 2H, ArH), 7.79 (m, 2H,
8.61 (m, 1H, ArH), 8.53 (d, J ¼ 8.80, 1H, ArH), 8.46 (d, J ¼ 6.92 Hz, 1H,
ArH), 8.30 (d, J ¼ 8.12 Hz, 1H, ArH), 7.80 (d, J ¼ 8.00 Hz, 1H, ArH),
7.70 (t, J ¼ 6.72, 1H, ArH), 7.63 (s, 1H, ArH), 3.86 (m, 2H, CH2), 3.56
ArH), 7.66 (dd, 2J ¼ 14.24 Hz, 3J ¼ 13.80 Hz, 2H, ArH), 7.44 (m, 1H,
ArH), 7.38 (m, 1H, ArH), 7.20 (d, J ¼ 8.08 Hz, 1H, ArH), 3.60 (m, 1H,
CH), 1.78 (t, J ¼ 5.08 Hz, 2H, CH2), 1.67 (m, 2H, CH2), 1.56 (m, 2H,
CH2), 1.31 (m, 4H, CH2). 13C NMR (DMSO-d6); 157.33, 156.97, 151.74,
151.64, 142.01, 133.83, 131.99, 131.34, 131.14, 124.42, 121.96, 119.96,
117.04, 116.68, 111.57, 110.23, 108.20, 46.31, 45.33, 25.05, 13.28.
EIMS, m/z: 368 (Mþ þ 1). Anal. Calc. for (C24H21N3O): C, 78.45; H,
5.76; N, 11.44. Found: C, 78.93; H, 5.88; N, 11.42.
(m, 2H, CH2); 13C NMR (DMSO-d6);
d 164.81, 160.27, 158.12, 157.38,
147.83, 140.67, 134.13, 133.03, 131.22, 122.91, 121.12, 119.80, 118.61,
108.69, 60.81, 60.33. EIMS, m/z: 330 (Mþ þ 1). Anal. Calc. for
(C20H15N3O2): C, 72.94; H, 4.59; N,12.76. Found: C, 72.78; H, 4.24; N,
12.45.
4.3.7. 3-(5-Bromo-pyridin-2-ylamino)-benzo[de]benzo[4,5]
imidazo[2,1-a]isoquinolin-7-one 5g
Acknowledgements
Brown powder (0.14 g, 45%) m.p. 220e222 ꢀC. 1H NMR (CDCl3);
We thank Department of Science and Technology, New Delhi
(SR/FT/CS-40/2010) for the research grant. We also thank Punjab
d
8.86 (dd, 2J ¼ 7.32 Hz, 3J ¼ 0.96 Hz, 1H, ArH), 8.82 (dd, 2J ¼ 7.32 Hz,

360
M. Verma et al. / European Journal of Medicinal Chemistry 68 (2013) 352e360
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