Synthesis and: In vitro evaluation of naphthalimide-benzimidazole conjugates as potential antitumor agents

Article abstract of DOI:10.1039/c8ob02973c

A series of novel naphthalimide-benzimidazoles was designed and synthesized for the first time and studied for their effect on antiproliferative activity. Some of these compounds possessed good antitumor activity towards the tested cancer cell lines. Noticeably, (diethylamino)ethyl 15 and (dimethylamino)ethyl 23 derivatives displayed superior antiproliferative activity towards human cancer cell lines with MG-MID GI₅₀ values of 1.43 and 1.83 μM, respectively. Preliminary investigation revealed that compounds 15 and 23 might bind with ct-DNA through the intercalation mode which is responsible for potent bioactivity. Moreover, transportation behaviour indicated that these molecules could efficiently bind to and be carried by bovine albumin, and the hydrogen bonding and hydrophobic interactions played important roles in interaction with serum albumin.

Full text of DOI:10.1039/c8ob02973c

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Synthesis and in vitro evaluation of naphthalimide-benzimidazole conjugates
as potential antitumor agents
Iqubal Singh, Vijay luxami and Kamaldeep Paul*
School of Chemistry and Biochemistry, Thapar University, Patiala- 147 004, India
E-mail: kpaul@thapar.edu
Abstract
A series of novel naphthalimide-benzimidazole has been designed and synthesized for first time
and studied for its effect on antiproliferative activity. Some of these compounds possessed good
antitumor activity towards the tested cancer cell lines. Noticeably, (diethylamino)ethyl 15 and
(dimethylamino)ethyl 23 derivatives displayed superior antiproliferative activity towards human
cancer cell lines with MG_MID GI50 values of 1.43 and 1.83 μM, respectively. Preliminarily
investigation reveals that compounds 15 and 23 might bind with ct−DNA through intercalation
mode which is responsible for potent bioactivity. Moreover, transportation behaviour indicates
that these molecules could efficiently bind and carried by bovine albumin, and the hydrogen
bonding and hydrophobic interactions play important roles in interaction with serum albumin.
INTRODUCTION
DNA-damaging agent constitutes a cornerstone of cancer therapy and contributes to the
subsistence of cancer patients in binding with drugs having different modes of interaction.
However, the severe toxicity and drug resistance in clinic is the Achilles’ heel of DNA-damaging
agents.1,2 Thus, by targeting DNA-associated processes, enhanced selectivity for cancer cells could
be acquired. Naphthalimides are highly versatile functionalized moiety, and continuously
receiving attention to bind with DNA,3 thus establishing pronounced therapeutic importance in
pharmaceutical and medicinal chemistry.4,5 Some of the naphthalimides such as mitonafide,
amonafide, and aristolochic acid showed effective antitumor activity towards the growth of various
murine and human cancer cell lines. These naphthalimides exerted their anticancer function by
interacting with DNA and inhibiting directly the function of replication and transcription, and/or
topoisomerase (TOPO-II) to stabilize the supramolecular TOPO-II and DNA complex.6 On
account of limited efficacy and central neurotoxicity in solid tumors, the clinical developments of
these compounds were regrettably terminated.7,8 To increase the efficiency and toxicological
1

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profile, significant efforts have been attempted for the development of more potent
naphthalimides,9-12 but most of these efforts were concentrated only on the binding affinity with
DNA.13 On the other hand, benzimidazole is an important structural motif for bioactivity and
widely present in broad range of therapeutic drugs14 having an electron-accepting group (C=N)
and an electron-donating group (NH), that could rapidly interact with DNA and other useful
biomacromolecules.15 A large number of derivatives based on this moiety has been designed and
evaluated for antitumor activity. Amongst these compounds, nocodazole, a 2-thienyl carbonyl
benzimidazole, carbendazim, a benzimidazole carbamate and Veliparib, a pyrrolidine-2-yl benzimidazole
are used in the clinic while mebendazole is currently undergoing clinical trials.16 The biological potential
of benzimidazoles against cancer cells has been reported with different mechanism of action. The success
of these drugs and many other clinical trial derivatives17 has provoked wide range of studies to
construct more benzimidazole based bioactive molecules in the field of cancer. In view of these
investigations, we have introduced benzimidazole fragment into C-4 position of naphthalimide
ring to develop novel naphthalimide−benzimidazole conjugates. It is well known that functional
groups present at the naphthalimide moiety especially at N-position are significantly affected the
bioactivities of naphthalimide,18 which might be beneficial for DNA and other targeting
biomolecules. Aliphatic and aromatic amines viz., (diethylamino)ethyl, (dimethylamino)ethyl,
allylamine etc. are important structural appendages in improving and regulating the biological
activities19 and commonly present in number of clinical antitumor agents.20 Rationally, in the
present study, these moieties have been introduced at N-position of naphthalimide skeleton to
investigate their effect on activity profile (Figure 1). The antitumor screenings with 60 human
cancer cell lines were executed to naphthalimide-benzimidazoles 11-26. Additionally, to study the
effect of naphthalimide, the mode of action for anticancer activity was also evaluated through
interaction with ct-DNA using different spectroscopic techniques. Cell cycle arrest was also
checked to investigate the effect of benzimidazole using flow cytometer. The transportation
behaviour of bovine serum albumin (BSA) to most active derivatives has also been done using
UV-visible and fluorescence spectroscopy.
2

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Modification on N-position of naphthalimide is
to explore the influence of substituents on
antitumor activity
R
N
O
O
Crucial segment as DNA-targeting agents
Important fragment in clinical antitumor
drugs, like nocodazole, carbendazim,
bendamustin etc
N
N
Alicyclic substituent is helpful to regulate
the physicochemical properties and
improve pharmacokinetic profile
Figure-1 Design of novel naphthalimide-benzimidazoles
RESULTS AND DISCUSSION
Chemistry: Naphthalimide-benzimidazoles 11-26 have been synthesized via multistep reactions
starting with commercial available 1,4- and 1,3-dibromobenzene according to Scheme 1. Nitration
of 1,4- and 1,3-dibromobenzene 1a-b with nitric acid and sulphuric acid in the ratio of 4:1, yielded
1,4-dibromo-2-nitro-benzene 2a and 2,4-dibromo-1-nitro-benzene 2b in 90% and 95% yields, both
of which were subjected to regioselective nucleophilic substitution with cyclohexylamine to afford
3a-b. Boronation of 3a-b with bis(pinacolato)diboran in the presence of Pd(PPh3)2Cl2 and KOAc,
generated 4a-b. Suzuki-Miyaura cross coupling of 4a-b was carried out with 6-bromo-1H,3H-
benzo[de]isochromene-1,3-dione 5 (obtained from bromination of acenaphthene followed by
oxidation with potassium dichromate in glacial acetic acid) to obtain 6a-b. Compound 6a-b was
further reduced with sodium dithionate in the presence of ammonia to afford mixture of 7a-b and
8a-b which were subsequently cyclized with triethylorthoformate in acetic acid at room
temperature to afford the requisite 9a-b and 10a-b, respectively. The yields of both the
intermediates were depended upon the amount of ammonia used in reduction. On refluxing of 9a-b
with alkyl and aryl amines in ethanol, compounds 11-26 were achieved in the yields of 71-86%
(Table 1). These novel synthesized compounds were well characterized by NMR and mass
spectrometry (Figures S1-S71).
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Scheme 1. Synthesis of 6-(1-cyclohexyl-1H-benzo[d]imidazol-5/6-yl)-2-substituted-1H-
benzo[de]isoquinoline-1,3(2H)-dione
O
O
O
O
O
O
NO2
Br
NO2
NH
NO2
NH
a
O
O
Br
c
b
5
Br
Br
Br
B
Br
d
1a-b
2a-b
NO2
3a-b
NH
4a-b
Br
1a =
1b =
Br
Br
Br
6a-b
e
R
N
H
N
H
O
O
O
O
O
O
O
O
O
O
N
O
O
O
O
O
g
f
+
+
NH2
NH2
N
N
NH
N
N
N
NH
N
N
N
7a-b
8a-b
9a-b
11-26
9a-b
10a-b
Reagents and conditions: (a) HNO3, H2SO4, DCM, 0 °C, 30 min., 90-95%; (b) Cyclohexyl amine, K2CO3,
DMF, 100 °C, 18 h, 70-75%; (c) Bis(pinacolato)diboron, Pd(PPh3)2Cl2, KOAc, dioxane, reflux, 10 h, 78-
82%; (d) Pd(PPh3)4, K2CO3, CH3CN : water (9:1), N2, reflux, 10-12 h, 70-73%; (e) Na2S2O4, aq. NH3, THF
: water, 1 h, rt; (f) Triethylorthoformate, AcOH, 30 min., rt; (g) RNH2, ethanol, reflux, 12-15 h, 71-86%.
Table 1. Photophysical properties of naphthalimide-benzimidazole conjugates
Compound
Starting
R
% yield
M.Pt. (oC)
Molecular
Formulae
Material
10a
10b
11
8a
8b
9a
9a
H
H
20
25
76
82
208-211
213-216
261-263
258-261
C25H21N3O2
C25H21N3O2
C28H25N3O2
C28H23N3O2
12
13
14
9a
9a
86
80
254-257
260-263
C29H29 N3O2
C29H30N4O2
N
4

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15
9a
81
265-268
C31H34N4O2
N
OH
16
17
9a
9a
76
275-278
270-273
C27H25N3O3
C27H26N4O2
71
81
NH2
18
19
9a
9a
259-262
261-263
C31H32N4O3
C32H27N3O2
N
O
83
20
21
9a
9a
78
75
262-264
269-272
C31H24FN3O2
C32H24N4O2S
F
S
N
N
22
23
9a
9b
80
81
281-283
260-263
C31H24N4O
C29H30N4O2
N
N
24
25
9b
9b
OH
76
83
275-278
259-262
C27H25N3O3
C31H32N4O3
N
O
26
9b
77
266-269
C32H27N3O2
Biological Activities. Antiproliferative activity. We are now interested to find out the effect of
alkyl/aryl groups on naphthalimides towards the antiproliferative activity. National Cancer
Institute (NCI)21 has selected eighteen naphthalimide-benzimidazoles (9a-b, 10b, 11-23, 25 and
26) for one-dose screening in the panel of 60 human cancer cell lines (Tables S1-S2). The results
indicated that many of these naphthalimides exhibited equal or better antiproliferative activity than
amonafide or mitonafide (drugs used in clinical trials). A comparison of the antiproliferative
activities of two isomers of benzimidazole with naphthalimide revealed that derivatives substituted
with aromatic rings were definitely less active than the aliphatic groups. In fact, amongst these
eighteen naphthalimide-benzimidazoles, five showed antitumor effects in a broad range of cell
lines at 10-5 M level and two of them showed antiproliferative activity with cytostatic and cytotoxic
effects against all the 58 tested cell lines (Table S3). Compounds 15 and 23 fulfilled the selection
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criteria set by NCI for antiproliferative activity in this assay and thus, selected for further
evaluation at the full panel of cancer cell lines at five dose concentrations (10-4 – 10-8 M).
Three parameters for each cell line viz. GI50, TGI and LC50 have been reported for derivatives
15 and 23 towards antitumor activity and average of mean graph midpoint (MG_MID) was
determined for these parameters (Figure S72-S73). In vitro evaluation of these compounds
indicated that derivatives 15 and 23 exhibited antitumor activity towards most of the human cell
lines, showing MG_MID GI50 values of 1.43 and 1.83 µM, respectively (Table 2). Both the
derivatives showed particular efficacy against colon and leukemia subpanels having GI50 values
in the range of 0.42-1.03 µM. The most sensitive leukemia and colon cell lines are MOLT-4 (GI50
= 0.44-0.47 µM) and HCT-116 (GI50 = 0.40-0.42 µM) for derivative 15 and SR (GI50 = 0.41-0.49
µM) and HT-29 (GI50 = 0.54-0.93 µM) in case of compound 23, respectively. Moreover, derivative
15 exhibited good selectivity towards HOP-92 (GI50 = 0.37 µM) of non-small cell lung cancer,
UO-31 (GI50 = 0.58 µM) of renal cancer and BT-549 (GI50 = 0.92 µM) of breast cancer while
compound 23 showed selectivity towards LOX IMVI (GI50 = 0.82 µM) of melanoma cancer and
ACHN (GI50 = 0.53 µM) of renal cancer. Thus, in the series of 5-benzimidazole series,
(diethylamino)ethyl (15) indicated the better antitumor activity while in 6-benzimidazole series,
(dimethylamino)ethyl (23) exhibited good activity. Overall, the naphthalimide substituted with 5-
benzimidazole (15) led to more potency than the 6-benzimidazole analogue (23). These results
were also compared with clinical trial drug, amonafide (NSC: 308847), a naphthalimide derivative
and marketed drug nocadozaole (NSC: 238159), a benzimidazole analogue. It has been observed
that compounds 15 and 23 are more active than amonafide but less potent than nocadozaole
towards cancer cell lines.
Table 2: GI50 (µM), TGI (µM) and LC50 (µM) of derivatives 15 and 23 in vitro tumor cell lines
Comp.
15
Activity
(µM)
I
II
III
IV
V
VI
VII
VIII
IX
MG_
MIDa
GI50
1.09 1.50
4.94 3.39
1.18
1.36
1.67
1.61
1.32
1.61
1.56
1.43
TGI
LC50
GI50
TGI
3.07
6.74
1.29
8.89
2.89
5.72
2.02
11.6
3.15
5.85
1.98
5.47
4.14
13.8
1.92
12.1
3.39
11.2
1.39
7.05
3.93
53.2
2.38
9.62
3.97
5.84
2.68
14.2
3.65
14.1
1.83
9.24
b
10.8
23
1.21 1.63
7.87 6.19
b
LC50
44.6
58.5
55.3
35.6
67.2
29.5
53.1
66.4
51.3
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Amonafide GI50
Nocodazole GI50
2.09 3.35
0.18 0.39
2.93
0.09
4.15
0.25
3.43
0.22
4.24
0.34
2.76
0.86
2.57
0.28
3.96
0.23
3.32
0.60
Leukemia (I), non-small cell lung cancer (II), colon cancer (III), CNS cancer (IV), melanoma (V), ovarian cancer (VI), renal cancer
(VII), prostate cancer (VIII), breast cancer (IX). a(MG_MID): The average sensitivity of all cell lines towards the test agent (µM),
bCompounds showed values > 100 µM. GI50 = concentration for 50% of maximal inhibition of cell proliferation, TGI =
concentration of the compound resulting in total growth inhibition, LC50 = lethal concentration required to kill 50% of the
population.
DNA interaction studies. The binding properties of DNA with compounds 15 and 23 as a
model of naphthalimide-benzimidazole conjugates have been studied with calf thymus (ct)-DNA
using UV-visible and fluorescence spectroscopy as well as circular dichroism experiment.
UV-visible spectroscopic studies. Absorption spectroscopy is an effective technique to
investigate the interaction mode of DNA with small molecule. Thus, in order to provide evidence
for the possibility of binding of naphthalimide-benzimidazoles to calf-thymus DNA, UV-visible
titrations of a solution of 15 and 23 with DNA have been performed. The absorption spectra of
compounds 15 and 23 were exhibited absorption band at 370 nm in phosphate buffer (pH 7.4) at
room temperature. The changes observed in the absorption spectra of compounds 15 and 23 (20.0
µM) with incremental addition of ct-DNA (0-110 µM for 15; 0-85 µM for 23), showed
hypochromism at 370 nm (29.92 % for 15 and 20% for 23), indicating the interaction of derivatives
with double-helical ct-DNA (Figure 2). The absorption intensity at 370 nm has been decreased
due to the fact that purine and pyrimidine bases of DNA are exposed because of binding of planar
naphthalimide moiety with DNA. To access the stability of an adduct formed between DNA and
substrate, Benesi-Hildebrand equation (Equation-1)22 has been used to calculate the intrinsic
binding constant (Kb) for compounds 15 and 23 (Figure S74) and were found to be 3.62 × 105 M-1,
R = 0.9847 and 2.23 × 105 M-1, R = 0.9963, respectively. The results indicated that compound 15
with (diethylamino)ethyl showed strong interaction with ct-DNA than compound 23.
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Figure 2. Absorption spectra of the compounds (a) 15 and (b) 23 (20.0 µM) in the presence of ct-
DNA in phosphate buffer (pH 7.4) at 298 K
Thermal denaturation studies. Thermal behaviour of DNA in the presence of 15 and 23 might
be given an insight into DNA conformation changes on rising the temperature, and thus offered
information about the interaction strength of compounds with DNA. Generally, the intercalation
of natural or synthetic organic intercalators results in considerable increase in melting temperature
which is strongly correlated with the stability of double-helical ct-DNA.23 The thermal
denaturation experiment was carried out for DNA in the absence of compounds that revealed a Tm
value of 75.6 ± 0.2 ˚C under our experimental conditions, whereas the melting temperature of
DNA in the presence of compounds 15 and 23 were successively increased to 89.8 ± 0.2 ˚C and
81.5 ± 0.3 ˚C, respectively (Figure S75). The observed changes in UV-visible and Tm were
consistent with the antitumor activity, with potency improving from compound 23 to 15.
Fluorescence studies. Fluorescence emission of compounds 15 and 23 (5.0 µM) in aqueous
solution showed a band at around 530 nm, at the excitation of 370 nm. Quenching of fluorescent
intensity of compounds 15 (56.16 % at 298 K, 54.38 % at 308 K and 39.11 % at 318 K) and 23
(55.10 % at 298 K, 36.30 % at 308 K and 29.37 % at 318 K) at three different temperatures has
been observed while maximum emission wavelength was slightly blue shifted to 20 nm with
increase in the concentration of ct-DNA (Figures 3 and S76-S77). Quantitative estimation of
quenching with DNA in terms of fluorescence quenching data was done by Stern-Volmer equation
(Equation 2)24 where Ksv and Kq have been calculated at different temperatures (Table 3 and
Figures S78). It has been observed that Ksv and Kq were decreased on increasing the temperature,
indicated that the mechanism of quenching might be a static process. Moreover, the Kq is much
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larger than diffusion controlled (1.0 × 1010 M-1 s-1)25, also suggested that interaction involved the
stating quenching. To calculate the binding constant (Kb) and the average number of binding sites
per DNA molecule (n) of compound-DNA interaction, fluorescence titrations were performed at
different temperatures using modified Stern-Volmer equation (Equation 3)26 (Figure S79). The
values of Kb were observed to decrease with increasing temperature for compounds 15 and 23,
indicating a reduction in the stability of the DNA-compound adduct at higher temperatures. The
values of n have been obtained for both the compounds from the slope which were found to be
close to one.
The non-covalent interactions between small molecule and ct-DNA are hydrogen bonding,
electrostatic forces, hydrophobic interactions and van der Waals forces.27 To determine these
binding forces, thermodynamic parameters such as entropy change (ΔS), enthalpy change (ΔH)
and free energy change (ΔG) have been calculated using van't Hoff equation (equations 4 and 5)
(Table 4, Figure S80). The observed negative values of ΔG for compounds revealed that the
binding process is spontaneous and favourable. The type of interaction between compound and
DNA has been identified by evaluating the values of ΔH and ΔS and in the present study found to
be -38.59 Kcal M-1 and -102.75 cal M-1 K-1 for compound 15, and -27.36 Kcal M-1 and -67.40 cal
M-1 K-1 for compound 23, respectively. The negative values of ΔH and ΔS for 15 and 23 indicated
that the interaction with DNA is mainly enthalpy driven by which hydrogen bonding and van der
Waals contacts contributed towards the stability of complexes.
Figure 3. Emission spectra of compounds (a) 15 and (b) 23 in the presence of ct-DNA in phosphate
buffer (pH 7.4) at 298 K
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Table 3. Quenching and binding parameters for the interaction of compounds 15 and 23 with ct-
DNA at three different temperatures
Compd T (K) KSV (×104) (M-1) Kq (×1012) (M-1s-1)
R2
Kb (×105) (M-1)
n
R2
15
298
308
318
298
308
318
4.72
2.92
1.72
5.98
2.26
1.88
4.72
2.92
1.72
5.98
2.26
1.88
0.9563
0.9597
0.9805
0.9479
0.9497
0.9894
9.26
0.49
0.15
2.83
0.29
0.15
1.37 0.9856
1.06 0.9609
0.97 0.9686
1.18 0.9352
1.03 0.9770
0.98 0.9931
23
Table 4. Thermodynamic parameters for the interaction of compounds 15 and 23 with ct-DNA at
three different temperatures
Compd
15
T (K)
298
308
318
298
308
318
ΔH (Kcal M-1) ΔS (cal M-1 K-1) ΔG (Kcal M-1)
R2
-38.59
-102.75
-7.9705
-6.943
0.9502
-5.9155
-7.2748
-6.6008
-5.9268
23
-27.36
-67.40
0.9178
Competitive displacement assay. To investigate the mode of binding of 15 and 23 to ct-DNA,
a competitive binding experiment with ethidium bromide (EB) was performed. Ethidium bromide
acts as one of the sensitive fluorescent probes having a planar structure that interacts with DNA
by an intercalative mode.28 Therefore, the relative affinity of compounds 15 and 23 to DNA was
compared by examining their ability to displace EB from the DNA helix. Ethidium bromide shows
an emission maximum at 600 nm on excitation at 520 nm. Fluorescence spectra of fixed
concentration of EB-DNA complex (3 µM : 30 µM) in the presence of increasing concentration of
compounds (15: 0-150 µM; 23: 0-120 µM) in phosphate buffer (pH 7.4) were recorded (Figure
S81). On subsequent addition of compounds, a decrease in fluorescence emission (15: 62.03% and
23: 55.72%) at 600 nm of EB-DNA complex was observed indicated that these compounds were
effectively competing with EB for the occupation at the same binding sites on DNA through
intercalation.
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Circular Dichroism (CD) studies. In order to get insight into the variations of properties
induced by small molecule binding, CD spectroscopy is helpful in analysing the changes in DNA
structure during DNA-drug interactions. The CD spectrum of DNA consists of a negative band at
247 nm because of helicity and a positive band at 277 nm due to base stacking, which are
characteristics of DNA in the right handed B form.29 As seen in Figure 4 and S82, on the addition
of compounds 15 and 23, the intensities of positive and negative bands of DNA increased
significantly; this observation is a strong indicator of classical intercalation. Moreover, these
compounds did not yield positive induced CD (ICD) band at the region of (λ) 350 and 400 nm,
instead of weak negative ICD band was observed, thus excluding binding into the minor groove
of ct-DNA.30-32 This data shows good agreement with increasing Tm and decreasing EB
fluorescence intensity.
Figure 4. CD spectra of free ct-DNA (40 µM) (blue line), 15-DNA complex (red line) and 23-
DNA complex (green line) at ratio r[compound/ct-DNA] = 0.025 in Tris HCl buffer (pH 7.4)
Cell cycle analysis. Numerous anticancer compounds exhibit their effect by blockade of cell cycle
process at a particular phase.33 We hypothesized that naphthalimide-benzimidazole conjugates
have the mechanism of action that they also arrest the process of mitosis. To test this hypothesis,
we performed the cell cycle distribution using flow cytometer on the compounds 15 and 23 on
MDA-MB-468 breast cancer cells. In our study, MDA-MB-468 cells were treated with compounds
15 and 23 at 1 µM concentration for 24 h along with control (without compound). The results
indicated that compound 15 had no significant effect at cell cycle whereas compound 23 arrested
the cell cycle at G2/M phase (Figure 5, Table 5).
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In the vehicle treated group, about 13.47% of MDA-MB-468 cells were distributed in G2/M
phase. Only compound 23 increased the proportion of cells in G2/M phase. About 25.23% of cells
were found in G2/M phase when treated with 1 µM of compound 23 for 24 h.
Table 5. Cell cycle distribution of MDA-MB-468 cell line treated with compounds 15 and 23.
Compound
Control
15
Sub G1%
1.88
G0/G1%
51.98
S%
G2/M%
13.47
32.67
22.01
28.84
1.91
62.55
13.53
23
1.88
44.05
25.23
Figure 5. Flow cytometry analysis on cell cycle progression on MDA-MB-468 (breast cancer) cell
line: (a) control, (b) compound 15 and (c) compound 23.
BSA binding studies. Binding of a prospective drug to plasma protein is recognized as a
crucial step in accessing its bioavailability,34 and the reports on plasma protein binding are required
in screening potential therapeutic agents.35 Albumin binding, in particular, plays a decisive role
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for in vivo bioavailability of any drug. Within this rationale, we examined the interaction of
naphthalimide-benzimidazoles with bovine serum albumin (BSA), structural homology with
human serum albumin (HSA), using absorption and emission titration experiments.
UV-visible spectroscopic studies. Absorption spectroscopy is used to explore the possibility
of ground state interaction between drug and protein. Therefore, the interactions of derivatives
with protein have been inferred from the changes in absorption spectra of serum albumin on
gradual addition of compounds. UV-visible spectrum of BSA showed an intense absorption band
at about 279 nm in phosphate buffer at pH 7.4, mainly due to the presence of tyrosine (Tyr),
tryptophan (Trp) and phenylalanine (Phe) residues. On subsequent addition of compounds 15 and
23 (0-15 µM) to BSA solution (7.0 µM), gradual increase in intensity of band at 279 nm along
with enhancement of new band at 370 nm was observed (Figure 6). These variations originated
from changes in the conformation and the polarity of the microenvironment around aromatic
residues of BSA. Moreover, the absorption maxima of BSA remains unchanged, suggested that
the interactions between compounds and BSA are non-covalent in nature and likely to occur
through π-π stacking between aromatics of naphthalimide-benzimidazole and phenyl rings of Trp,
Tyr and Phe residues located in the binding cavity of BSA. Binding constants (Kb) for the
interaction of compounds with BSA have been calculated using Benesi-Hildebrand equation
(Equation 1)22 and were found to be 6.96 × 104 M-1 for 15 and 5.64 × 104 M-1 for 23 (Figure S83),
signifying better binding affinity of compound 15 with serum albumin than 23.
Figure 6. Absorption spectra of BSA in the presence of compounds (a) 15 and (b) 23 in phosphate
buffer (pH 7.4) at 298 K.
Fluorescence studies. The observations obtained from absorption studies are not sufficient to
determine the interaction of compounds in detail. Therefore, emission spectroscopy has been
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preferred for reviewing binding mode of interactions. The emission of BSA is observed from two
Trp residues; one of them is located on the surface and the other residue present in the hydrophobic
pocket of protein molecule.36 Figure 7 depicts that on progressive addition of different
concentration of compounds (0-45 µM) to BSA (7.0 µM), the emission spectrum of Trp residues
of serum protein at around 350 nm was gradually quenched (82.49% for 15 and 79.11% for 23)
with the appearance and enhancement of new band at 485 nm. The λmax of the emission of Trp
residues in BSA was also blue shifted from 350 nm (in free BSA) to 330 nm. This suggests that
the emitting Trp residue(s) in the complex of compound-BSA are in less polar or more
hydrophobic environment as compared with free BSA.
Figure 7. Emission spectra of BSA in the presence of compounds (a) 15 and (b) 23 in
phosphate buffer (pH 7.4) at 298 K
The steady-state fluorescence quenching of Trp emission in serum albumin with addition of
compound has been characterized by Stern-Volmer equation (equation 2)24 that showed the
relation between the quenching extent for each compound and the strength of their interactions
with BSA. The values of Stern-Volmer constant (KSV) and bimolecular quenching constant (Kq)
were calculated from the slope of linear portion of regression curve using equation 2 (Table 6 and
Figure S84a). A linear Stern-Volmer plot was obtained with compounds 15 and 23, suggesting
that solely one type of quenching or binding process occur, either stating or dynamic quenching.
The values obtained for bimolecular quenching constant (Kq) of compounds 15 (1.41 ×1013 M-1s-
1) and 23 (1.52 ×1013 M-1s-1) were much greater than the diffusion control limited value of 1 ×1010
M-1 s-1, which is the largest possible value reported in aqueous medium.25 Thus, the binding of
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compounds to BSA probably involves the stating quenching with formation of complex at ground
state.
Modified Stern-Volmer equation (Equation 3)26 has been used to determine the binding
constant (Kb) and the average number of binding sites (n) for compound-BSA complexes (Figure
S84b). These complexes exhibited excellent binding parameters, suggesting their binding to the
albumins and their possible transfer as well as their release ability upon arrival at their target sites.
As these values are significantly lower than 1015 M-1, which is the value of the association constant
of the protein albumin with diverse compounds; their interactions are considered significant among
the known non-covalent ones.37
Table 6. Quenching and binding parameters for interaction of BSA with compounds 15 and 23
Compd Ksv (×105)(M-1) Kq (×1013) (M-1s-1)
R2
Kb (×105)(M-1)
9.34
n
R2
15
23
1.41
1.52
1.41
1.52
0.9808
0.9903
1.23 0.9971
1.02 0.9956
1.41
CONCLUSION
A series of isomeric benzimidazole with substituted naphthalimides has been synthesized in
moderate to good yields and studied their effects on antiproliferative activity. Both the isomeric
groups of 18 compounds were assessed over 60 human tumor cell line panel at 10-5
M
concentration, where most of the examined compounds showed superior activity. Compounds 15
and 23 were identified higher activity against leukemia and colon cancer subpanels at five dose
concentration levels. The interaction between these compounds with DNA was studied by UV-
visible, fluorescence and CD spectroscopy as well as DNA melting experiment. The results
suggested that the fluorescence of compounds 15 and 23 has been quenched significantly by DNA
and the probable mechanism was a stating quenching process. The association constant (Kb) value
between compound and DNA was in the order of 105 mol-1, which is better than other reported
DNA intercalators. The thermodynamic parameters, ∆Ho and ∆So, were calculated in the range of
-38.59 to -27.36 Kcal M-1 and -102.75 to -67.40 cal M-1 K-1, respectively, indicated that the binding
of compounds to DNA were driven mainly by hydrogen bonds and van der Waals interactions.
Mechanism of action studies with cell cycle arrest confirmed that compound 23 maintained their
ability to arrest cells in G2/M phase and induce cell apoptosis. Compounds 15 and 23 have also
been effectively bound to BSA protein with good binding constant values of 9.34 x 105 M-1 and
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1.41 x 105 M-1, respectively, determined through emission spectroscopy. Thus, naphthalimide-
benzimidazole molecules with a superior bioactivity profile and mode of interaction with DNA
and BSA have been described, and objective is completely satisfied.
EXPERIMENTAL SECTION
Chemistry. General Methods. All commercially available compounds (Spectrochem, Aldrich,
Merck etc.) were used without purification. Melting points were determined in an open capillary
1
13
and were uncorrected. H and C NMR spectra have been performed on Jeol ECS 400 NMR
spectrophotometer, which were operated at 400 MHz for 1H nuclei and 100 MHz for 13C nuclei,
using CDCl3 as solvent. Chemical shifts are reported in parts per million (ppm) with TMS as an
internal reference. Water Micromass-Q-T of Micro has been used to determine the mass Spectra.
CHN analysis was done using Thermo Scientific (Flash 2000) analyser. Reactions have been
observed by TLC with plate coated with silica gel HF-254 and column chromatography was done
with silica gel 60-120/100-200 mesh. UV-visible absorption and fluorescence emission spectra
were measured with 1 cm quartz cell. Shimadzu-2400 PC spectrometer instrument was used to
determine the UV-visible spectra. Emission spectra were recorded with Varian Cary Eclipse
fluorescence spectrometer. Circular dichroism spectra were recorded with an Applied
Photophysics CD spectrophotometer.
Synthesis of dibromonitrobenzene (2a-b):38 Dibromobenzene (1a-b) (5 g, 21.27 mmol) in
dichloromethane (15 ml) and sulphuric acid (10 ml) was treated with cooled solution of nitric acid
and sulphuric acid (4:1) at 0˚C for 30 min. The reaction mixture was allowed to warm at room
temperature with stirring. On completion of reaction (monitored by TLC), quenched the reaction
mixture with ice. The precipitate formed was collected and dried in an oven to afford desired
product (2a-b) in 90-95% yields.
Synthesis of 4/5-bromo-N-cyclohexyl-2-nitroaniline (3a-b): Dibromonitrobenzene (3 g, 10.71
mmol), cyclohexyl amine (1.27 g, 12.85 mmol) and K2CO3 (1.47 g, 10.71 mmol) were taken in
DMF (20 ml) in 100 ml dried round bottom flask and stirred the reaction mixture at 100˚C for 18
h. On completion of reaction, 50 mL water was added and extracted with ethyl acetate. The extract
was then dried over anhydrous sodium sulphate, filtered and concentrated to get the crude product.
The residue was column purified on silica gel (Hexane/EtOAc = 20:1) to obtain the desired
product.
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o
1
4-Bromo-N-cyclohexyl-2-nitroaniline (3a): Yellow solid; 75% yield; mp 93-96 C; H NMR
(CDCl3, 400 MHz):  (ppm) 8.27 (d, J = 2.32 Hz, 1H, ArH), 8.12 (d, J = 7.36 Hz, 1H, NH), 7.44
2
3
(dd, J = 9.16 Hz, J = 2.28 Hz, 1H, ArH), 6.79 (d, J = 9.16 Hz, 1H, ArH), 3.52-3.43 (m, 1H,
cyclohex-CH), 2.04-2.02 (m, 2H, cyclohex-CH2), 1.82-1.78 (m, 2H, cyclohex-CH2), 1.69-1.62 (m,
13
1H, cyclohex-CH2), 1.47-1.25 (m, 5H, cyclohex-CH2); C NMR (CDCl3, 100 MHz): δ (ppm)
143.5, 138.6, 131.6, 128.8, 115.8, 105.6 (ArC), 51.0 (CH), 32.4 (CH2), 25.3 (CH2), 24.3 (CH2).
o
1
5-Bromo-N-cyclohexyl-2-nitroaniline (3b): Yellow solid; 70% yield; mp 90-93 C; H NMR
(CDCl3, 400 MHz):  (ppm) 8.13 (d, J = 6.88 Hz, 1H, NH), 8.00 (d, J = 9.16 Hz, 1H, ArH), 7.01
2
3
(d, J = 1.84 Hz, 1H, ArH), 6.69 (dd, J = 9.16 Hz, J = 2.28 Hz, 1H, ArH), 3.49-3.42 (m, 1H,
cyclohex-CH), 2.05-2.02 (m, 2H, cyclohex-CH2), 1.82-1.74 (m, 2H, cyclohex-CH2), 1.68-1.64 (m,
13
1H, cyclohex-CH2), 1.49-1.28 (m, 5H, cyclohex-CH2); C NMR (CDCl3, 100 MHz): δ (ppm)
144.8, 131.4, 130.3, 128.1, 117.9, 116.4 (ArC), 50.9 (CH), 32.4 (CH2), 25.3 (CH2), 24.3 (CH2).
Synthesis of N-cyclohexyl-2-nitro-4/5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline
(4a-b): 4/5-Bromo-N-cyclohexyl-2-nitroaniline (3a-b) (2.0 g, 6.68 mmol), bis(pinacolato)diboron
(2.03 g, 8.01 mmol), KOAc (0.98 g, 10.01 mmol), palladium(II)bis(triphenylphosphine) dichloride
(5.0 mol%) in 1,4-dioxane (30 mL) were charged in 100 ml oven-dried round bottom flask. The
reaction mixture was refluxed for 10 h until the halide was completely consumed as determined
by TLC. The mixture was then cooled to room temperature. Solvent of reaction was concentrated
under reduced pressure and the crude was extracted with chloroform and water (3 × 50 mL). The
extract was dried over anhydrous Na2SO4, filtered and concentrated. The crude material obtained
was column purified on silica gel using ethyl acetate and hexane to obtain the desired reddish
yellow product.
N-Cyclohexyl-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline
(4a):
Reddish yellow solid; 82% yield; mp 125-127 oC; 1H NMR (CDCl3, 400 MHz):  (ppm) 8.63 (d,
J = 1.36 Hz, 1H, ArH), 8.29 (d, J = 6.88 Hz, 1H, NH), 7.75 (dd, 2J = 8.68 Hz, 3J = 0.92 Hz, 1H,
ArH), 6.83 (d, J = 8.72 Hz, 1H, ArH), 3.58-3.51 (m, 1H, cyclohex-CH), 2.06-2.03 (m, 2H,
cyclohex-CH2), 1.82-1.76 (m, 2H, cyclohex-CH2), 1.67-1.63 (m, 1H, cyclohex-CH2), 1.48-1.23
13
(m, 17H, cyclohex-CH2 & boronate-CH3); C NMR (CDCl3, 100 MHz): δ (ppm) 146.1, 141.3,
134.5, 131.4, 113.2 (ArC), 83.7 (C), 50.8 (CH), 32.5 (CH2), 25.4 (CH2), 24.7 (CH3), 24.4 (CH2).
N-Cyclohexyl-2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (4b): Reddish
yellow solid; 78% yield; mp 120-122 oC; 1H NMR (CDCl3, 400 MHz):  (ppm) 8.13 (d, J = 8.68
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Hz, 2H, NH & ArH), 7.28 (s, 1H, ArH), 6.96 (d, J = 8.72 Hz, 1H, ArH), 3.71-3.63 (m, 1H,
cyclohex-CH), 2.07-2.02 (m, 2H, cyclohex-CH2), 1.83-1.76 (m, 2H, cyclohex-CH2), 1.67-1.63 (m,
1H, cyclohex-CH2), 1.53-1.28 (m, 17H, cyclohex-CH2 & boronate-CH3); 13C NMR (CDCl3, 100
MHz): δ (ppm) 143.8, 135.5, 132.6, 125.7, 120.8, 119.9 (ArC), 84.3 (C), 50.3 (CH), 32.7 (CH2),
25.5 (CH2), 24.7 (CH3), 24.3 (CH2).
Synthesis of 6-(4/3-(cyclohexylamino)-3/4-nitrophenyl)-1H,3H-benzo[de]isochromene-1,3-
Dione (6a-b): To a solution of 6-bromo-1H,3H-benzo[de]isochromene-1,3-dione (5) (2 g, 7.22
mmol) in a mixture of acetonitrile and water (9:1) in 100 ml round bottom flask, N-cyclohexyl-2-
nitro-4/5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (4a-b) (2.51 g, 7.22 mmol) and
K2CO3 (1.0 g, 7.22 mmol) were added under inert atmosphere. Then, Pd(PPh3)4 (5 mol%) was
added with continued N2 purging and refluxed the reaction mixture for 10-12 h. On completion of
reaction (determined by TLC), solvents were evaporated under reduced pressure followed by water
(50 ml) was added and then extracted with chloroform. Chloroform layer was dried over sodium
sulphate. The crude product has further been purified by column chromatography with hexane and
ethylacetate to get the yellowish solid product.
6-(4-(Cyclohexylamino)-3-nitrophenyl)-1H,3H-benzo[de]isochromene-1,3-dione
(6a):
Reddish yellow solid; 70% yield; mp 83-86 oC; 1H NMR (CDCl3, 400 MHz):  (ppm) 8.67-8.63
(m, 2H, ArH), 8.42 (dd, 2J = 8.72 Hz, 3J = 0.92 Hz, 1H, ArH), 8.39 (d J = 2.28 Hz, 1H, ArH), 8.35
(d, J = 7.36 Hz, 1H, ArH), 7.82-7.78 (m, 1H, ArH), 7.76 (d, J = 7.76 Hz, 1H, ArH), 7.61 (dd, 2J =
8.72 Hz, 3J = 1.84 Hz, 1H, ArH), 7.11 (d, J = 9.16 Hz, 1H, NH), 3.68-3.59 (m, 1H, Cyclohex-CH),
2.16-2.12 (m, 2H, Cyclohex-CH2), 1.89-1.82 (m, 2H, Cyclohex-CH2), 1.73-1.69 (m, 1H,
Cyclohex-CH2), 1.53-1.43 (m, 4H, Cyclohex-CH2), 1.41-1.33 (m, 1H, Cyclohex-CH2); 13C NMR
(CDCl3, 100 MHz): δ (ppm) 160.6, 160.4, 146.3, 144.6, 137.0, 133.5, 133.4, 133.1, 131.4, 131.0,
130.0, 128.2, 128.0, 127.4, 124.2, 118.9, 117.4, 114.9 (ArC), 51.3 (CH), 32.6 (CH2), 25.4 (CH2),
24.5 (CH2).
6-(3-(Cyclohexylamino)-4-nitrophenyl)-1H,3H-benzo[de]isochromene-1,3-dione
(6b):
Reddish yellow solid; 73% yield; mp 81-84 oC; 1H NMR (CDCl3, 400 MHz):  (ppm) 8.65 (t, J =
6.44 Hz, 2H, ArH), 8.35 (t, J = 7.32 Hz, 2H, ArH), 8.31 (d, J = 7.80 Hz, 1H, ArH), 7.83-7.78 (m,
2
3
2H, ArH), 6.99 (s, 1H, NH), 6.72 (dd, J = 8.72 Hz, J = 1.36 Hz, 1H, ArH), 3.60-3.52 (m, 1H,
Cyclohex-CH), 2.10-2.06 (m, 2H, Cyclohex-CH2), 1.82-1.77 (m, 2H, Cyclohex-CH2), 1.68-1.62
(m, 1H, Cyclohex-CH2), 1.50-1.32 (m, 5H, Cyclohex-CH2); 13C NMR (CDCl3, 100 MHz): δ (ppm)
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160.3, 160.1, 146.7, 145.7, 144.5, 133.5, 132.8, 131.3, 130.5, 129.8, 127.8, 127.7, 127.5, 118.8,
118.4, 116.1, 115.2 (ArC), 51.0 (CH), 32.6 (CH2), 25.4 (CH2), 24.3(CH2).
Synthesis of 6-(3/4-amino-4/3-(cyclohexylamino)phenyl)-1H,3H-benzo[de]isochromene-1,3-
dione (7a-b) and 6-(3/4-amino-4/3-(cyclohexylamino)phenyl)-1H-benzo[de]isoquinoline-
1,3(2H)-dione (8a-b): A 100 ml round bottom flask was charged with 6-(4/3-(cyclohexylamino)-
3/4-nitrophenyl)-1H,3H-benzo[de]isochromene-1,3-dione (6a-b) (2 g, 4.80 mmol) and sodium
dithionite (4.18 g, 24.03 mmol) in THF:water (3:2). Ammonia solution (5 ml) has been added to
the reaction mixture and stirred for 1 h at room temperature. Completion of reaction was monitored
by TLC and the reaction mixture was extracted with ethyl acetate. Ethyl acetate layer was dried
over Na2SO4, filtered and concentrated to get the crude product. Mixture of crude brown product
(7 and 8) was directly used further without purification.
Synthesis of 6-(1-cyclohexyl-1H-benzo[d]imidazol-5/6-yl)-1H,3H-benzo[de]isochromene-
1,3-dione (9a-b) and 6-(1-cyclohexyl-1H-benzo[d]imidazol-5/6-yl)-1H-benzo[de]isoquinoline-
1,3(2H)-dione (10a-b): Mixture of 6-(3/4-amino-4/3-(cyclohexylamino)phenyl)-1H,3H-
benzo[de]isochromene-1,3-dione (7a-b) and 6-(3/4-amino-4/3-(cyclohexylamino)phenyl)-1H-
benzo[de]isoquinoline-1,3(2H)-dione (8a-b) (1 mmol) was stirred with triethylorthoformate (1
mmol) in acetic acid (30 ml) for 30 min at room temperature. The reaction mixture was then poured
into water and then treated with NaHCO3 followed by extracted with chloroform. Sodium sulphate
was used to dry the chloroform layer. Crude was purified by column chromatography using ethyl
acetate and hexane as elutents to obtain light brown solid.
6-(1-Cyclohexyl-1H-benzo[d]imidazol-5-yl)-1H,3H-benzo[de]isochromene-1,3-dione (9a):
Light brown solid; 72% yield; mp 191-193 oC; 1H NMR (CDCl3, 400 MHz):  (ppm) 8.65 (d, J =
7.32 Hz, 1H, ArH), 8.62 (d, J = 7.32 Hz, 1H, ArH), 8.46 (d, J = 8.68 Hz, 1H, ArH), 8.16 (s, 1H,
ArH), 7.92 (s, 1H, ArH), 7.81 (d, J = 7.32 Hz, 1H, ArH), 7.74 (t, J = 8.44 Hz, 1H, ArH), 7.66 (d,
J = 8.24 Hz, 1H, ArH), 7.45 (dd, 2J = 8.28 Hz, 3J = 1.40 Hz, 1H, ArH), 4.36-4.28 (m, 1H, cyclohex-
CH), 2.33 (d, J = 11.00 Hz, 2H, cyclohex-CH2), 2.08-2.03 (m, 2H, cyclohex-CH2), 1.95-1.85 (m,
3H, cyclohex-CH2), 1.64-1.52 (m, 2H, cyclohex-CH2), 1.44-1.32 (m, 1H, cyclohex-CH2); 13C
NMR (CDCl3, 100 MHz): δ (ppm) 160.8, 160.5, 149.1, 143.8, 141.7, 134.5, 133.6, 133.2, 132.9,
131.9, 130.8, 130.4, 128.6, 127.1, 124.3, 121.6, 118.7, 117.1, 110.5 (ArC), 55.7 (cyclohex-CH),
33.2 (cyclohex-CH2), 25.5 (cyclohex-CH2), 25.2 (cyclohex-CH2); MS (ESI): m/z 397.0 (M++1);
Anal Calcd for C25H20N2O3: C, 75.74; H, 5.09; N, 7.07; found C, 75.78; H, 5.05; N, 7.09.
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6-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)-1H,3H-benzo[de]isochromene-1,3-dione (9b):
Light brown solid; 70% yield; mp 193-196 oC; 1H NMR (CDCl3, 400 MHz):  (ppm) 8.69 (d, J =
7.80 Hz, 1H, ArH), 8.67 (dd, 2J = 7.32 Hz, 3J = 0.92 Hz, 1H, ArH), 8.42 (d, J = 7.76 Hz, 1H, ArH),
8.15 (s, 1H, ArH), 7.97 (d, J = 8.24 Hz, 1H, ArH), 7.85 (d, J = 7.76 Hz, 1H, ArH), 7.79-7.75 (m,
1H, ArH), 7.56 (s, 1H, ArH), 7.41 (dd, 2J = 8.24 Hz, 3J = 1.36 Hz, 1H, ArH), 4.30-4.22 (m, 1H,
cyclohex-CH), 2.29 (d, J = 11.00 Hz, 2H, cyclohex-CH2), 2.01-1.96 (m, 2H, cyclohex-CH2), 1.90-
1.80 (m, 3H, cyclohex-CH2), 1.56-1.45 (m, 2H, cyclohex-CH2), 1.42-1.30 (m, 1H, cyclohex-CH2);
13C NMR (CDCl3, 100 MHz): δ (ppm) 160.8, 160.6, 149.1, 141.7, 134.4, 133.4, 132.9, 132.1,
131.9, 130.8, 130.6, 128.7, 128.5, 128.4, 127.3, 124.1, 120.4, 118.8, 117.4, 111.4 (ArC), 55.6
(cyclohex-CH), 33.3 (cyclohex-CH2), 25.6 (cyclohex-CH2), 25.2 (cyclohex-CH2); MS (ESI): m/z
397.0 (M++1); Anal Calcd for C25H20N2O3: C, 75.74; H, 5.09; N, 7.07; found C, 75.72; H, 5.11;
N, 7.05.
6-(1-Cyclohexyl-1H-benzo[d]imidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (10a):
Light yellow solid; 20% yield; mp 208-211 oC; 1H NMR (CDCl3, 400 MHz):  (ppm) 8.86 (s, 1H,
NH), 8.66 (d, J = 7.32 Hz, 1H, ArH), 8.63 (d, J = 7.32 Hz, 1H, ArH), 8.39 (d, J = 8.72 Hz, 1H,
ArH), 8.15 (s, 1H, ArH), 7.96 (s, 1H, ArH), 7.79 (d, J = 7.32 Hz, 1H, ArH), 7.71 (t, J = 7.80 Hz,
1H, ArH), 7.62 (d, J = 8.24 Hz, 1H, ArH), 7.44 (d, J = 8.24 Hz, 1H, ArH), 4.34-4.26 (m, 1H,
cyclohex-CH), 2.32 (d, J = 12.80 Hz, 2H, cyclohex-CH2), 2.05 (d, J = 13.20 Hz, 2H, cyclohex-
CH2), 1.94-1.85 (m, 3H, cyclohex-CH2), 1.62-1.52 (m, 2H, cyclohex-CH2), 1.42-1.32 (m, 1H,
cyclohex-CH2); 13C NMR (CDCl3, 100 MHz): δ (ppm) 164.2, 164.0, 148.2, 143.8, 141.6, 133.7,
133.4, 132.7, 130.9, 130.7, 130.6, 130.0, 128.2, 126.6, 124.6, 122.5, 121.8, 121.1, 110.3 (ArC),
55.7 (cyclohex-CH), 33.3 (cyclohex-CH2), 25.6 (cyclohex-CH2), 25.3 (cyclohex-CH2); MS (ESI):
m/z 396.1 (M++1); Anal Calcd for C25H21N3O2: C, 75.93; H, 5.35; N, 10.63; found C, 75.91; H,
5.37; N, 10.68.
6-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (10b):
Light yellow solid; 25% yield; mp 213-216 oC; 1H NMR (CDCl3, 400 MHz):  (ppm) 8.78 (s, 1H,
NH), 8.67-8.63 (m, 2H, ArH), 8.35 (d, J = 8.24 Hz, 1H, ArH), 8.14 (s, 1H, ArH), 7.97 (d, J = 8.24
Hz, 1H, ArH), 7.81 (d, J = 7.80 Hz, 1H, ArH), 7.74 (t, J = 7.80 Hz, 1H, ArH), 7.55 (s, 1H, ArH),
7.41 (d, J = 7.80 Hz, 1H, ArH), 4.29-4.21 (m, 1H, cyclohex-CH), 2.29 (d, J = 11.00 Hz, 2H,
cyclohex-CH2), 2.01-1.96 (m, 2H, cyclohex-CH2), 1.89-1.79 (m, 3H, cyclohex-CH2), 1.55-1.42
(m, 2H, cyclohex-CH2), 1.37-1.30 (m, 1H, cyclohex-CH2); 13C NMR (CDCl3, 100 MHz): δ (ppm)
20

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164.1, 163.8, 148.3, 133.6, 133.2, 131.9, 131.0, 130.8, 130.5, 129.9, 128.5, 128.4, 128.2, 126.8,
124.3, 122.6, 121.3, 120.3, 111.4 (ArC), 55.6 (cyclohex-CH), 33.3 (cyclohex-CH2), 25.6
(cyclohex-CH2), 25.2 (cyclohex-CH2); MS (ESI): m/z 396.1 (M++1); Anal Calcd for C25H21N3O2:
C, 75.93; H, 5.35; N, 10.63; found C, 75.89; H, 5.39; N, 10.61.
Synthesis
of
2-aryl/alkyl-6-(1-cyclohexyl-1H-benzo[d]imidazol-yl)-1H-
benzo[de]isoquinoline-1,3(2H)-dione (11-26): 6-(3/4-Amino-4/3-(cyclohexylamino)phenyl)-
1H,3H-benzo[de]isochromene-1,3-dione (7a-b) (1 mmol) and corresponding amines (1.2 mmol)
in 5 ml ethanol were taken in oven dried round bottom flask and refluxed the reaction mixture for
12-15 h. After the completion of the reaction, solvent was evaporated under pressure followed by
water (50 ml) was added and extracted with chloroform. Chloroform layer was dried over sodium
sulphate. Crude product was further column purified using ethyl acetate and hexane as eluents to
get the desired solid products.
2-Allyl-6-(1-cyclohexyl-1H-benzo[d]imidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(11): Reddish brown solid; 76% yield; mp 261-263 oC; 1H NMR (CDCl3, 400 MHz):  (ppm) 8.68
(d, J = 7.80 Hz, 1H, ArH), 8.65 (d, J = 7.32 Hz, 1H, ArH), 8.36 (d, J = 8.68 Hz, 1H, ArH), 8.12
(s, 1H, ArH), 7.95 (s, 1H, ArH), 7.77 (d, J = 7.76 Hz, 1H, ArH), 7.70-7.67 (m, 1H, ArH), 7.61 (d,
2
3
J = 8.24 Hz, 1H, ArH), 7.44 (dd, J = 8.24 Hz, J = 1.40 Hz,1H, ArH), 6.08-5.98 (m, 1H, allyl-
2
3
2
3
CH), 5.36 (dd, J = 17.44 Hz, J = 1.40 Hz,1H, allyl-CH2), 5.24 (dd, J = 10.08 Hz, J = 0.92
Hz,1H, allyl-CH2), 4.85 (d, J = 5.52 Hz, 2H, allyl-CH2), 4.33-4.26 (m, 1H, cyclohex-CH), 2.32 (d,
J = 11.00 Hz, 2H, cyclohex-CH2), 2.05 (d, J = 13.32 Hz, 2H, cyclohex-CH2), 1.93-1.83 (m, 3H,
13
cyclohex-CH2), 1.62-1.51 (m, 2H, cyclohex-CH2), 1.43-1.32 (m, 1H, cyclohex-CH2); C NMR
(CDCl3, 100 MHz): δ (ppm) 164.1, 163.9, 147.5, 143.8, 141.5, 133.4, 132.7, 132.0, 131.2, 130.9,
130.3, 128.7, 128.4, 128.2, 126.7, 124.6, 122.6, 121.7, 121.2, 117.3, 110.3 (ArC), 55.7 (cyclohex-
CH), 42.3 (allyl-CH2), 33.3 (cyclohex-CH2), 25.6 (cyclohex-CH2), 25.3 (cyclohex-CH2); MS
(ESI): m/z 436.1 (M++1); Anal Calcd for C28H25N3O2: C, 77.22; H, 5.79; N, 9.65; found C, 77.20;
H, 5.80; N, 9.63.
6-(1-Cyclohexyl-1H-benzo[d]imidazol-5-yl)-2-(prop-2-yn-1-yl)-1H-benzo[de]isoquinoline-
o
1
1,3(2H)-dione (12): Yellow solid; 82% yield; mp 258-261 C; H NMR (CDCl3, 400 MHz): 
(ppm) 8.71 (d, J = 7.80 Hz, 1H, ArH), 8.68 (d, J = 7.36 Hz, 1H, ArH), 8.37 (d, J = 8.28 Hz, 1H,
ArH), 8.13 (s, 1H, ArH), 7.94 (s, 1H, ArH), 7.79 (d, J = 7.32 Hz, 1H, ArH), 7.71 (t, J = 8.24 Hz,
1H, ArH), 7.62 (d, J = 8.28 Hz, 1H, ArH), 7.44 (dd, 2J = 8.24 Hz, 3J = 0.92 Hz,1H, ArH), 5.00 (d,
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J = 2.72 Hz, 2H, propagyl-CH2), 4.33-4.26 (m, 1H, cyclohex-CH), 2.32 (d, J = 11.00 Hz, 2H,
cyclohex-CH2), 2.21 (t, J = 2.32 Hz, 1H, propagyl-CH), 2.05-2.02 (m, 2H, cyclohex-CH2), 1.94-
1.83 (m, 3H, cyclohex-CH2), 1.62-1.51 (m, 2H, cyclohex-CH2), 1.43-1.35 (m, 1H, cyclohex-CH2);
13C NMR (CDCl3, 100 MHz): δ (ppm) 163.6, 163.3, 147.9, 143.9, 141.6, 133.5, 132.6, 131.5,
131.2, 130.4, 128.7, 128.2, 126.7, 124.6, 122.4, 121.8, 121.0, 110.3 (ArC), 78.6 (propargyl-C),
70.4 (propargyl-CH), 55.7 (cyclohex-CH), 33.3 (cyclohex-CH2), 29.4 (propargyl-CH2), 25.6
(cyclohex-CH2), 25.3 (cyclohex-CH2); MS (ESI): m/z 434.1 (M++1); Anal Calcd for C28H23N3O2:
C, 77.58; H, 5.35; N, 9.69; found C, 77.61; H, 5.32; N, 9.68.
2-Butyl-6-(1-cyclohexyl-1H-benzo[d]imidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione (13): White solid; 86% yield; mp 254-257 oC; 1H NMR (CDCl3, 400 MHz):  (ppm) 8.66
(d, J = 7.80 Hz, 1H, ArH), 8.63 (dd, 2J = 7.32 Hz, 3J = 0.92 Hz, 1H, ArH), 8.34 (dd, 2J = 8.24 Hz,
3J = 0.92 Hz, 1H, ArH), 8.12 (s, 1H, ArH), 7.95 (s, 1H, ArH), 7.77 (d, J = 7.32 Hz, 1H, ArH), 7.69
(t, J = 7.32 Hz, 1H, ArH), 7.61 (d, J = 8.72 Hz, 1H, ArH), 7.44 (dd, 2J = 8.68 Hz, 3J = 1.36 Hz,
1H, ArH), 4.33-4.25 (m, 1H, cyclohex-CH), 4.24 (t, J = 7.32 Hz, 2H, butyl-CH2), 2.32 (d, J =
11.44 Hz, 2H, cyclohex-CH2), 2.06-2.00 (m, 2H, cyclohex-CH2), 1.93-1.83 (m, 3H, cyclohex-
CH2), 1.77-1.71 (m, 2H, cyclohex-CH2), 1.61-1.35 (m, 5H, cyclohex-CH2 & butyl-CH2), 1.01 (t,
3H, J = 7.32 Hz, butyl-CH3); 13C NMR (CDCl3, 100 MHz): δ (ppm) 164.3, 164.1, 147.3, 143.9,
141.5, 133.4, 132.9, 132.8, 131.0, 130.7, 130.3, 128.6, 128.1, 126.6, 124.6, 122.8, 121.8, 121.4,
110.2 (ArC), 55.7 (cyclohex-CH), 40.2 (butyl-CH2), 33.3 (cyclohex-CH2), 30.1 (butyl-CH2), 25.6
(cyclohex-CH2), 25.3 (cyclohex-CH2), 20.3 (butyl-CH2), 13.8 (butyl-CH3); MS (ESI): m/z 452.1
(M++1); Anal Calcd for C29H29N3O2: C, 77.14; H, 6.47; N, 9.31; found C, 77.18; H, 6.51; N, 9.38.
6-(1-Cyclohexyl-1H-benzo[d]imidazol-5-yl)-2-(2-(dimethylamino)ethyl)-1H-
o
1
benzo[de]isoquinoline-1,3(2H)-dione (14): Light yellow solid; 80% yield; mp 260-263 C; H
NMR (CDCl3, 400 MHz):  (ppm) 8.59 (d, J = 7.76 Hz, 1H, ArH), 8.56 (d, J = 6.88 Hz, 1H, ArH),
8.33 (d, J = 8.28 Hz, 1H, ArH), 8.13 (s, 1H, ArH), 7.90 (s, 1H, ArH), 7.73 (d, J = 7.32 Hz, 1H,
ArH), 7.65 (t, J = 8.24 Hz, 1H, ArH), 7.60 (d, J = 8.24 Hz, 1H, ArH), 7.39 (d, J = 8.24 Hz, 1H,
ArH), 4.73 (t, J = 6.64 Hz, 2H, ethyl-CH2), 4.33-4.25 (m, 1H, cyclohex-CH), 3.86 (t, J = 6.40 Hz,
2H, ethyl-CH2), 3.51 (s, 6H, N-CH3), 2.31 (d, J = 11.00 Hz, 2H, cyclohex-CH2), 2.04 (d, J = 13.32
Hz, 2H, cyclohex-CH2), 1.93-1.78 (m, 3H, cyclohex-CH2), 1.61-1.51 (m, 2H, cyclohex-CH2),
13
1.42-1.33 (m, 1H, cyclohex-CH2); C NMR (CDCl3, 100 MHz): δ (ppm) 164.2, 164.0, 148.1,
143.8, 141.6, 133.6, 133.5, 132.4, 131.5, 131.2, 130.3, 128.6, 128.3, 126.8, 124.6, 122.0, 121.7,
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120.5, 110.4 (ArC), 66.5 (ethyl-CH2), 57.7 (N-CH3), 55.7 (cyclohex-CH), 34.8 (ethyl-CH2), 33.3
(cyclohex-CH2), 25.6 (cyclohex-CH2), 25.3 (cyclohex-CH2); MS (ESI): m/z 467.1 (M++1); Anal
Calcd for C29H30N4O2: C, 74.65; H, 6.48; N, 12.01; found C, 74.62; H, 6.51; N, 12.05.
6-(1-Cyclohexyl-1H-benzo[d]imidazol-5-yl)-2-(2-(diethylamino)ethyl)-1H-
o
1
benzo[de]isoquinoline-1,3(2H)-dione (15): Light yellow solid; 81% yield; mp 265-268 C; H
NMR (CDCl3, 400 MHz):  (ppm) 8.66 (d, J = 7.36 Hz, 1H, ArH), 8.63 (d, J = 7.32 Hz, 1H, ArH),
8.35 (d, J = 8.24 Hz, 1H, ArH), 8.12 (s, 1H, ArH), 7.94 (s, 1H, ArH), 7.77 (d, J = 7.80 Hz, 1H,
ArH), 7.69 (t, J = 7.80 Hz, 1H, ArH), 7.61 (d, J = 8.28 Hz, 1H, ArH), 7.44 (d, J = 8.24 Hz, 1H,
ArH), 4.37 (t, J = 7.36 Hz, 2H, ethyl-CH2), 4.32-4.25 (m, 1H, cyclohex-CH), 2.90 (t, J = 7.76 Hz,
2H, ethyl-CH2), 2.78 (q, J = 7.32 Hz, 4H, ethyl-CH2), 2.32 (d, J = 11.48 Hz, 2H, cyclohex-CH2),
2.05 (d, J = 13.32 Hz, 2H, cyclohex-CH2), 1.93-1.83 (m, 3H, cyclohex-CH2), 1.62-1.50 (m, 2H,
cyclohex-CH2), 1.43-1.34 (m, 1H, cyclohex-CH2), 1.17 (t, J = 6.88 Hz, 6H, ethyl-CH3); 13C NMR
(CDCl3, 100 MHz): δ (ppm) 164.3, 164.1, 147.5, 143.9, 141.5, 133.4, 133.1, 132.7, 131.1, 130.8,
130.3, 128.6, 128.2, 126.6, 124.6, 122.6, 121.7, 121.2, 110.2 (ArC), 55.7 (cyclohex-CH2), 49.4
(ethyl-CH2), 47.5 (ethyl-CH2), 37.4 (ethyl-CH2), 33.3 (cyclohex-CH2), 25.6 (cyclohex-CH2), 25.3
(cyclohex-CH2), 11.86 (ethyl-CH3); MS (ESI): m/z 495.1 (M++1); Anal Calcd for C31H34N4O2: C,
75.28; H, 6.93; N, 11.33; found C, 75.25; H, 6.90; N, 11.36.
6-(1-Cyclohexyl-1H-benzo[d]imidazol-5-yl)-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-
1,3(2H)-dione (16): Light brown solid; 76% yield; mp 275-278 oC; 1H NMR (CDCl3, 400 MHz):
 (ppm) 8.67 (d, J = 7.32 Hz, 1H, ArH), 8.64 (dd, 2J = 7.36 Hz, 3J = 0.92 Hz, 1H, ArH), 8.36 (d, J
= 8.72 Hz, 1H, ArH), 8.12 (s, 1H, ArH), 7.94 (s, 1H, ArH), 7.77 (d, J = 7.76 Hz, 1H, ArH), 7.70
(t, J = 8.24 Hz, 1H, ArH), 7.61 (d, J = 8.24 Hz, 1H, ArH), 7.44 (dd, 2J = 8.72 Hz, 3J = 1.40 Hz,
1H, ArH), 4.51 (t, J = 4.05 Hz, 2H, ethyl-CH2), 4.32-4.26 (m, 1H, cyclohex-CH), 4.02 (t, J = 5.04
Hz, 2H, ethyl-CH2), 2.31 (d, J = 11.00 Hz, 2H, cyclohex-CH2), 2.05-2.02 (m, 2H, cyclohex-CH2),
1.93-1.83 (m, 3H, cyclohex-CH2), 1.61-1.52 (m, 2H, cyclohex-CH2), 1.42-1.35 (m, 1H, cyclohex-
13
CH2); C NMR (CDCl3, 100 MHz): δ (ppm) 165.3, 165.1, 147.8, 143.7, 141.6, 133.4, 132.5,
131.4, 131.1, 130.2, 128.7, 128.2, 126.7, 124.6, 122.4, 121.7, 120.9, 110.3 (ArC), 61.7 (ethyl-
CH2), 55.2 (cyclohex-CH), 42.8 (ethyl-CH2), 33.3 (cyclohex-CH2), 25.6 (cyclohex-CH2), 25.3
(cyclohex-CH2); MS (ESI): m/z 440.0 (M++1); Anal Calcd for C27H25N3O3: C, 73.79; H, 5.73; N,
9.56; found C, 73.84; H, 5.79; N, 9.59.
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2-(2-Aminoethyl)-6-(1-cyclohexyl-1H-benzo[d]imidazol-5-yl)-1H-benzo[de]isoquinoline-
o
1
1,3(2H)-dione (17): Brown solid; 71% yield; mp 270-273 C; H NMR (CDCl3, 400 MHz): 
(ppm) 8.53-8.47 (m, 2H, ArH), 8.23 (t, J = 8.72 Hz, 1H, ArH), 8.11 (s, 1H, ArH), 7.93 (s, 1H,
ArH), 7.68 (d, J = 6.40 Hz, 1H, ArH), 7.60-7.56 (m, 2H, ArH), 7.43 (d, J = 8.72 Hz, 1H, ArH),
4.31-4.19 (m, 5H, cyclohex-CH, ethyl-CH2), 2.31 (d, J = 9.60 Hz, 2H, cyclohex-CH2), 2.05 (d, J
= 13.76 Hz, 2H, cyclohex-CH2), 1.92-1.82 (m, 3H, cyclohex-CH2), 1.61-1.51 (m, 2H, cyclohex-
13
CH2), 1.41-1.32 (m, 1H, cyclohex-CH2); C NMR (CDCl3, 100 MHz): δ (ppm) 164.4, 164.1,
147.6, 143.8, 141.5, 133.3, 133.0, 131.1, 130.7, 130.4, 128.6, 128.2, 126.8, 124.3, 122.7, 121.4,
120.3, 111.3 (ArC), 56.1 (ethyl-CH2), 55.5 (cyclohex-CH), 37.1 (ethyl-CH2), 33.3 (cyclohex-
CH2), 25.6 (cyclohex-CH2), 25.2 (cyclohex-CH2); MS (ESI): m/z 439.0 (M++1); Anal Calcd for
C27H26N4O2: C, 73.95; H, 5.98; N, 12.78; found C, 73.91; H, 5.92; N, 12.74.
6-(1-Cyclohexyl-1H-benzo[d]imidazol-5-yl)-2-(2-morpholinoethyl)-1H-
o
1
benzo[de]isoquinoline-1,3(2H)-dione (18): Brown solid; 81% yield; mp 259-262 C; H NMR
(CDCl3, 400 MHz):  (ppm) 8.66 (d, J = 7.32 Hz, 1H, ArH), 8.63 (dd, 2J = 7.36 Hz, 3J = 0.92 Hz,
1H, ArH), 8.36 (dd, 2J = 8.24 Hz, 3J = 0.92 Hz, 1H, ArH), 8.12 (s, 1H, ArH), 7.95 (s, 1H, ArH),
7.78 (d, J = 7.32 Hz, 1H, ArH), 7.70 (t, J = 8.72 Hz, 1H, ArH), 7.62 (d, J = 8.28 Hz, 1H, ArH),
7.44 (dd, 2J = 8.24 Hz, 3J = 0.92 Hz, 1H, ArH), 4.40 (t, J = 6.88 Hz, 2H, ethyl-CH2), 4.33-4.26 (m,
1H, cyclohex-CH), 3.71 (t, J = 4.60 Hz, 4H, morph-CH2), 2.76 (t, J = 7.32 Hz, 2H, ethyl-CH2),
2.63 (bs, 4H, morph-CH2), 2.32 (d, J = 11.44 Hz, 2H, cyclohex-CH2), 2.05-2.02 (m, 2H, cyclohex-
CH2), 1.94-1.85 (m, 3H, cyclohex-CH2), 1.61-1.52 (m, 2H, cyclohex-CH2), 1.42-1.35 (m, 1H,
cyclohex-CH2); 13C NMR (CDCl3, 100 MHz): δ (ppm) 164.4, 164.1, 147.5, 143.9, 141.6, 133.4,
133.1, 132.7, 131.1, 130.8, 130.3, 128.7, 128.2, 126.7, 124.6, 122.7, 121.8, 121.3, 110.3 (ArC),
66.9 (morph-CH2), 56.1 (ethyl-CH2), 55.7 (cyclohex-CH), 53.7 (morph-CH2), 37.1 (ethyl-CH2),
33.3 (cyclohex-CH2), 25.6 (cyclohex-CH2), 25.3 (cyclohex-CH2); MS (ESI): m/z 509.1 (M++1);
Anal Calcd for C31H32N4O3: C, 73.21; H, 6.34; N, 11.02; found C, 73.18; H, 6.31; N, 11.05.
2-Benzyl-6-(1-cyclohexyl-1H-benzo[d]imidazol-5-yl)-1H-benzo[de]isoquinoline-1,3(2H)-
o
1
dione (19): Off white solid; 83% yield; mp 261-263 C; H NMR (CDCl3, 400 MHz):  (ppm)
8.67 (d, J = 7.36 Hz, 1H, ArH), 8.64 (dd, 2J = 7.32 Hz, 3J = 0.92 Hz, 1H, ArH), 8.34 (d, J = 8.24
Hz, 1H, ArH), 8.12 (s, 1H, ArH), 7.93 (s, 1H, ArH), 7.76 (d, J = 7.32 Hz, 1H, ArH), 7.68 (t, J =
7.32 Hz, 2H, ArH), 7.60-7.55 (m, 3H, ArH), 7.43 (dd, 2J = 8.24 Hz, 3J = 0.92 Hz, 1H, ArH), 7.32
(t, J = 7.36 Hz, 2H, ArH), 5.41 (s, 2H, benzyl-CH2), 4.32-4.24 (m, 1H, cyclohex-CH), 2.31 (d, J
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= 12.84 Hz, 2H, cyclohex-CH2), 2.04-2.01 (m, 2H, cyclohex-CH2), 1.92-1.82 (m, 3H, cyclohex-
CH2), 1.61-1.50 (m, 2H, cyclohex-CH2), 1.42-1.37 (m, 1H, cyclohex-CH2); 13C NMR (CDCl3, 100
MHz): δ (ppm) 164.3, 164.1, 147.5, 143.8, 141.5, 137.2, 133.1, 132.7, 132.0, 131.9, 131.3, 131.0,
130.3, 128.7, 128.3, 128.1, 127.3, 126.6, 124.6, 122.6, 121.7, 121.2, 110.3 (ArC), 55.7 (cyclohex-
CH), 43.5 (benzyl-CH2), 33.3 (cyclohex-CH2), 25.6 (cyclohex-CH2), 25.2 (cyclohex-CH2); MS
(ESI): m/z 486.1 (M++1); Anal Calcd for C32H27N3O2: C, 79.15; H, 5.60; N, 8.65; found C, 79.11;
H, 5.65; N, 8.61.
6-(1-Cyclohexyl-1H-benzo[d]imidazol-5-yl)-2-(4-fluorophenyl)-1H-benzo[de]isoquinoline-
o
1
1,3(2H)-dione (20): Brown solid; 78% yield; mp 262-264 C; H NMR (CDCl3, 400 MHz): 
(ppm) 8.70 (d, J = 7.32 Hz, 1H, ArH), 8.68 (dd, 2J = 7.32 Hz, 3J = 0.88 Hz, 1H, ArH), 8.42 (dd, 2J
= 8.24 Hz, 3J = 0.92 Hz, 1H, ArH), 8.13 (s, 1H, ArH), 7.97 (d, J = 0.88 Hz, 1H, ArH), 7.81 (d, J
= 7.36 Hz, 1H, ArH), 7.73 (t, J = 8.72 Hz, 1H, ArH), 7.63 (d, J = 8.24 Hz, 1H, ArH), 7.46 (dd, 2J
= 8.68 Hz, 3J = 1.84 Hz, 1H, ArH), 7.35-7.30 (m, 2H, ArH), 7.27-7.23 (m, 2H, ArH), 4.34-4.26
(m, 1H, cyclohex-CH), 2.33 (d, J = 11.88 Hz, 2H, cyclohex-CH2), 2.06 (d, J = 13.76 Hz, 2H,
cyclohex-CH2), 1.94-1.84 (m, 3H, cyclohex-CH2), 1.63-1.51 (m, 2H, cyclohex-CH2), 1.43-1.35
(m, 1H, cyclohex-CH2); 13C NMR (CDCl3, 100 MHz): δ (ppm) 164.5, 164.3, 148.0, 143.9, 141.6,
133.5, 133.4, 132.6, 131.6, 131.3, 131.1, 130.5, 130.4, 130.3, 129.0, 128.3, 126.8, 124.6, 122.7,
121.8, 121.2, 116.5, 116.2, 110.3 (ArC), 55.7 (cyclohex-CH), 33.3 (cyclohex-CH2), 25.6
(cyclohex-CH2), 25.3 (cyclohex-CH2); MS (ESI): m/z 490.1 (M++1); Anal Calcd for
C31H24FN3O2: C, 76.06; H, 4.94; N, 8.58; found C, 76.11; H, 4.97; N, 8.61.
2-(Benzo[d]thiazol-2-yl)-6-(1-cyclohexyl-1H-benzo[d]imidazol-5-yl)-1H-
o
1
benzo[de]isoquinoline-1,3(2H)-dione (21): Light yellow solid; 75% yield; mp 269-272 C; H
2
3
NMR (CDCl3, 400 MHz):  (ppm) 8.74 (d, J = 7.32 Hz, 1H, ArH), 8.71 (dd, J = 7.32 Hz, J =
0.92 Hz, 1H, ArH), 8.45 (dd, 2J = 8.72 Hz, 3J = 1.40 Hz, 1H, ArH), 8.17 (d, J = 7.80 Hz, 1H, ArH),
8.14 (s, 1H, ArH), 7.97 (d, J = 7.32 Hz, 2H, ArH), 7.82 (d, J = 7.80 Hz, 1H, ArH), 7.75 (t, J = 7.32
Hz, 1H, ArH), 7.63 (d, J = 8.04 Hz, 1H, ArH), 7.58-750 (m, 2H, ArH), 7.48-7.45 (m, 1H, ArH),
4.34-4.26 (m, 1H, cyclohex-CH), 2.32 (d, J = 11.00 Hz, 2H, cyclohex-CH2), 2.05 (d, J = 14.24 Hz,
2H, cyclohex-CH2), 1.94-1.84 (m, 3H, cyclohex-CH2), 1.62-1.52 (m, 2H, cyclohex-CH2), 1.42-
1.33 (m, 1H, cyclohex-CH2); 13C NMR (CDCl3, 100 MHz): δ (ppm) 163.8, 163.6, 155.8, 150.4,
148.6, 143.9, 141.7, 136.7, 134.2, 133.5, 132.5, 132.0, 131.6, 130.6, 129.2, 128.4, 126.8, 126.3,
126.0, 124.6, 124.2, 122.1, 121.8, 120.6, 110.4 (ArC), 55.7 (cyclohex-CH), 33.3(cyclohex-CH2),
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25.6 (cyclohex-CH2), 25.3 (cyclohex-CH2); MS (ESI): m/z 529.1 (M++1); Anal Calcd for
C32H24N4O2S: C, 72.71; H, 4.58; N, 10.60; S, 6.06; found C, 72.66; H, 4.65; N, 10.71; S, 6.09.
3-(1-Cyclohexyl-1H-benzo[d]imidazol-5-yl)-7H-benzo[de]benzo[4,5]imidazo[2,1-
a]isoquinolin-7-one (22): Yellow solid; 80% yield; mp 281-283 oC; 1H NMR (CDCl3, 400 MHz):
 (ppm) 8.87 (t, J = 8.24 Hz, 1H, ArH), 8.82 (t, J = 7.80 Hz, 1H, ArH), 8.58-8.55 (m, 1H, ArH),
8.43-8.24 (m, 1H, ArH), 8.13 (d, J = 2.28 Hz, 1H, ArH), 7.99 (d, J = 3.68 Hz, 1H, ArH), 7.90-7.87
(m, 1H, ArH), 7.81 (q, J = 4.12 Hz, 1H, ArH), 7.73-7.67 (m, 1H, ArH), 7.63 (dd, 2J = 8.24 Hz, 3J
= 3.20 Hz, 1H, ArH), 7.50-7.45 (m, 3H, ArH), 4.34-4.26 (m, 1H, cyclohex-CH), 2.33 (d, J = 11.44
Hz, 2H, cyclohex-CH2), 2.05 (d, J = 13.72 Hz, 2H, cyclohex-CH2), 1.93-1.85 (m, 3H, cyclohex-
CH2), 1.63-1.51 (m, 2H, cyclohex-CH2), 1.43-1.35 (m, 1H, cyclohex-CH2); 13C NMR (CDCl3, 100
MHz): δ (ppm) 160.8, 160.7, 149.5, 148.6, 145.3, 143.9, 141.6, 134.3, 133.3, 132.9, 131.7, 131.6,
131.2, 131.0, 128.6, 128.3, 127.0, 126.7, 125.7, 125.1, 124.9, 121.9, 119.8, 115.8, 110.2 (ArC),
55.7 (cyclohex-CH), 33.3 (cyclohex-CH2), 25.6 (cyclohex-CH2), 25.3 (cyclohex-CH2); MS (ESI):
m/z 469.0 (M++1); Anal Calcd for C31H24N4O: C, 79.46; H, 5.16; N, 11.96; found C, 79.55; H,
5.13; N, 11.99.
6-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)-2-(2-(dimethylamino)ethyl)-1H-
o
1
benzo[de]isoquinoline-1,3(2H)-dione (23): Light yellow solid; 81% yield; mp 260-263 C; H
NMR (CDCl3, 400 MHz):  (ppm) 8.66 (d, J = 7.32 Hz, 1H, ArH), 8.64 (d, J = 7.32 Hz, 1H, ArH),
8.30 (d, J = 7.80 Hz, 1H, ArH), 8.12 (s, 1H, ArH), 7.94 (d, J = 8.24 Hz, 1H, ArH), 7.78 (d, J =
7.80 Hz, 1H, ArH), 7.71 (t, J = 8.24 Hz, 1H, ArH), 7.54 (d, J = 0.68 Hz, 1H, ArH), 7.40 (dd, 2J =
8.28 Hz, 3J = 0.92 Hz, 1H, ArH), 4.44 (t, J = 6.68 Hz, 2H, ethyl-CH2), 4.28-4.21 (m, 1H, cyclohex-
CH), 2.88 (t, J = 7.12 Hz, 2H, ethyl-CH2), 2.49 (s, 6H, N-CH3), 2.28 (d, J = 11.00 Hz, 2H,
cyclohex-CH2), 2.00 (d, J = 13.72 Hz, 2H, cyclohex-CH2), 1.89-1.79 (m, 3H, cyclohex-CH2), 1.56-
1.44 (m, 2H, cyclohex-CH2), 1.40-1.31 (m, 1H, cyclohex-CH2); 13C NMR (CDCl3, 100 MHz): δ
(ppm) 164.4, 164.2, 147.6, 143.7, 141.5, 133.3, 133.0, 131.2, 130.8, 130.4, 128.7, 128.2, 126.8,
124.3, 122.6, 121.3, 120.2, 111.4 (ArC), 56.5 (ethyl-CH2), 55.5 (cyclohex-CH), 45.1 (N-CH3),
37.3 (ethyl-CH2), 33.3 (cyclohex-CH2), 25.5 (cyclohex-CH2), 25.2 (cyclohex-CH2); MS (ESI):
m/z 467.1 (M++1); Anal Calcd for C29H30N4O2: C, 74.65; H, 6.48; N, 12.01; found C, 74.71; H,
6.41; N, 12.12.
6-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-
o
1
1,3(2H)-dione (24): Brown solid; 76% yield; mp 275-278 C; H NMR (CDCl3, 400 MHz): 
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(ppm) 8.57-8.48 (m, 2H, ArH), 8.20-8.15 (m, 2H, ArH), 7.94 (d, J = 8.68 Hz, 1H, ArH), 7.70 (d,
J = 7.36 Hz, 1H, ArH), 7.64-7.59 (m, 1H, ArH), 7.54 (s, 1H, ArH), 7.41 (d, J = 9.16 Hz, 1H, ArH),
4.31-4.18 (m, 5H, cyclohex-CH & ethyl-CH2), 2.29 (d, J = 11.00 Hz, 2H, cyclohex-CH2), 1.99 (d,
J = 13.28 Hz,, 2H, cyclohex-CH2), 1.89-1.78 (m, 3H, cyclohex-CH2), 1.55-1.47 (m, 2H, cyclohex-
13
CH2), 1.45-1.42 (m, 1H, cyclohex-CH2); C NMR (CDCl3, 100 MHz): δ (ppm) 165.4, 165.2,
147.9, 141.4, 133.6, 132.7, 131.4, 131.1, 130.3, 128.5, 128.2, 126.8, 124.4, 122.3, 121.7, 120.7,
110.1 (ArC), 61.8 (ethyl-CH2), 55.7 (cyclohex-CH), 42.8 (ethyl-CH2), 33.3 (cyclohex-CH2), 25.6
(cyclohex-CH2), 25.2 (cyclohex-CH2); MS (ESI): m/z 440.0 (M++1); Anal Calcd for C27H25N3O3:
C, 73.79; H, 5.73; N, 9.56; found C, 73.72; H, 5.78; N, 9.64.
6-(1-Cyclohexyl-1H-benzo[d]imidazol-6-yl)-2-(2-morpholinoethyl)-1H-
o
1
benzo[de]isoquinoline-1,3(2H)-dione (25): Light brown solid; 83% yield; mp 259-262 C; H
NMR (CDCl3, 400 MHz):  (ppm) 8.67 (d, J = 7.32 Hz, 1H, ArH), 8.65 (d, J = 7.32 Hz, 1H, ArH),
8.32 (d, J = 8.24 Hz, 1H, ArH), 8.13 (s, 1H, ArH), 7.96 (d, J = 8.24 Hz, 1H, ArH), 7.79 (d, J =
7.36 Hz, 1H, ArH), 7.73 (t, J = 7.32 Hz, 1H, ArH), 7.54 (s, 1H, ArH), 7.41 (d, J = 8.24 Hz, 1H,
ArH), 4.41 (t, J = 6.64 Hz, 2H, ethyl-CH2), 4.29-4.21 (m, 1H, cyclohex-CH), 3.72 (t, J = 4.60 Hz,
4H, morph-CH2), 2.77 (t, J = 6.64 Hz, 2H, ethyl-CH2), 2.64 (bs, 4H, morph-CH2), 2.29 (d, J =
11.44 Hz, 2H, cyclohex-CH2), 2.00 (d, J = 13.76 Hz, 2H, cyclohex-CH2), 1.89-1.80 (m, 3H,
13
cyclohex-CH2), 1.56-1.44 (m, 2H, cyclohex-CH2), 1.37-1.33 (m, 1H, cyclohex-CH2); C NMR
(CDCl3, 100 MHz): δ (ppm) 164.3, 164.1, 147.6, 143.8, 141.5, 133.6, 133.3, 133.0, 131.1, 130.7,
130.4, 128.6, 128.2, 126.8, 124.3, 122.7, 121.4, 120.3, 111.3 (ArC), 66.9 (morph-CH2), 56.1
(ethyl-CH2), 55.5 (cyclohex-CH), 53.7 (morph-CH2), 37.1 (ethyl-CH2), 33.3 (cyclohex-CH2), 25.6
(cyclohex-CH2), 25.2 (cyclohex-CH2); MS (ESI): m/z 509.1 (M++1); Anal Calcd for C31H32N4O3:
C, 73.21; H, 6.34; N, 11.02; found C, 73.18; H, 6.42; N, 11.02.
2-Benzyl-6-(1-cyclohexyl-1H-benzo[d]imidazol-6-yl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione (26): Off whie solid; 77% yield; mp 266-269 oC; 1H NMR (CDCl3, 400 MHz):  (ppm) 8.68
(d, J = 7.32 Hz, 1H, ArH), 8.66 (d, J = 7.76 Hz, 1H, ArH), 8.30 (d, J = 8.24 Hz, 1H, ArH), 8.12
(s, 1H, ArH), 7.95 (d, J = 8.72 Hz, 1H, ArH), 7.78 (d, J = 7.80 Hz, 1H, ArH), 7.72 (t, J = 8.72 Hz,
1H, ArH), 7.57 (t, J = 7.32 Hz, 3H, ArH), 7.40 (d, J = 8.24 Hz, 1H, ArH), 7.34 (t, J = 7.36 Hz, 3H,
ArH), 5.42 (s, 2H, benzyl-CH2 ), 4.28-4.20 (m, 1H, cyclohex-CH), 2.28 (d, J = 11.44 Hz, 2H,
cyclohex-CH2), 1.99 (d, J = 13.72 Hz, 2H, cyclohex-CH2), 1.88-1.78 (m, 3H, cyclohex-CH2), 1.55-
1.44 (m, 2H, cyclohex-CH2), 1.37-1.33 (m, 1H, cyclohex-CH2); 13C NMR (CDCl3, 100 MHz): δ
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(ppm) 164.3, 164.1, 147.6, 141.5, 137.2, 133.3, 133.0, 131.4, 130.9, 130.3, 128.8, 128.4, 128.2,
127.4, 126.7, 124.3, 122.7, 121.3, 120.2, 111.2 (ArC), 55.5 (cyclohex-CH), 43.5 (benzyl-CH2),
33.3 (cyclohex-CH2), 25.6 (cyclohex-CH2), 25.2 (cyclohex-CH2); MS (ESI): m/z 486.1 (M++1);
Anal Calcd for C32H27N3O2: C, 79.15; H, 5.60; N, 8.65; found C, 79.13; H, 5.61; N, 8.78.
Procedure for in vitro anticancer screening
The human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium
containing 5% fetal bovine serum and 2 mM L-glutamine. Cells are inoculated into 96 well
microtiter plates in 100 ml at plating densities ranging from 5,000 to 40,000 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 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 concentration with complete medium containing 50 µg/ml gentamicin. Additional four, 10-
fold or ½ log serial dilutions are made to provide a total of five drug concentrations plus control.
Aliquots of 100 µL of these different drug dilutions are added to the appropriate microtiter wells,
resulting in the required final drug concentrations. Following drug addition, the plates are
°
incubated for an additional 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 µL of cold 50% (w/v) TCA and incubated for 60 min at 4 °C. The supernatant
is discarded, and the plates are washed five times with tap water and air dried. Sulforhodamine B
(SRB) solution (100 µL) 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 concentration levels. Percentage growth inhibition is calculated as:
[(Ti -Tz)/(C - Tz)] × 100 for concentrations for which Ti ≥ Tz; [(Ti -Tz)/Tz] × 100 for
concentrations for which Ti < Tz.
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Three dose response parameters are calculated for each experimental agent. Growth inhibition
of 50% (GI50) is calculated from [(Ti -Tz)/(C - Tz)] × 100 = 50. The drug concentration resulting
in total growth inhibition (TGI) is calculated from Ti = Tz. The LC50 is calculated from [(Ti-
Tz)/Tz]×100 = 50.
DNA and BSA binding experiments
Sample preparation for DNA and BSA. Calf thymus (ct)-DNA (Sigma Chemicals) was dissolved
in 10 mM Tris with 1 mM EDTA (pH 7.4). The purity of the DNA solution was checked by
determining the absorbance ratio at 260 nm to 280 nm. Concentration of stock solution of DNA
was measured by taking average extinction coefficient 6600 M-1 cm-1 of a single nucleotide at 260
nm. The stock solutions (10-3 M) of compounds (15 and 23) were prepared in DMSO. The stock
solution of BSA (Sigma-Aldrich Chemical Co., USA) has been formed by dissolving solid BSA
in 0.1 M phosphate buffer at pH 7.4 and was stored at 0-4 ˚C.
UV-Visible Spectroscopic. The DNA interactions were performed in the presence of fixed
concentration of compounds 15 and 23 (20 µM) and titrated with varying concentration of ct-DNA
(15; 0-110 µM, 23; 0–85 µM). Phosphate buffer (pH 7.4) has been used to make the final volume
to 1 ml.
The BSA interaction studies were performed in the presence of fixed concentration of BSA (7.0
µM) and titrated with different concentration of compounds 15 and 23 (0-15 µM). Phosphate
buffer (pH 7.4) has been used to make the final volume to 3 ml.
For both DNA and BSA studies, base line corrections were carried out with blank solution
containing phosphate buffer. All the UV-visible spectra have been recorded in the wavelength
range 200–800 nm. Benesi-Hildebrand equation (1) was used to calculate the binding constants
(Kb):
퐴₀
1
푓
푓


------------- (1)
=
+
푏

퐴 ― 퐴₀
푓퐾푏[퐴푛푎푙푦푡푒]
―

―
푏
푓


Where Ao is the initial absorbance of the free compound/BSA, A is the absorbance of the
compound/BSA in the presence of analyte (ct-DNA or compound), εf and εb are molar extinction
coefficients of the compound or BSA in its free and fully bound forms, respectively.
DNA melting experiments. DNA melting studies were performed by observing the absorption
spectra of ct-DNA (8.5 µM) at the wavelength of 260 nm in the presence of compounds (15 and
23) (10 µM) at different temperatures using UV-visible spectrophotometer coupled with a
thermostat. Phosphate buffer (pH 7.4) has been used to make the final volume to 1 ml. The
29