Synthesis, in vitro evaluation and DNA interaction studies of N-allyl naphthalimide analogues as anticancer agents

Article abstract of DOI:10.1039/c5ra00925a

A novel series of 2-allyl-6-substituted-benzo[de]isoquinoline-1,3-diones has been synthesized and evaluated against 60 human tumor cell lines for their in vitro antitumor activities. Compound 6b proved to be the most active member at a single dose concentration of 10 μM and broad spectrum of antitumor activity with GI₅₀, TGI and LC₅₀ values of 84.2 nM, 27.6 μM and 89.3 μM respectively at five dose concentration levels. The DNA binding properties of this compound has been investigated by a UV-Vis and fluorescence spectrophotometer as well as thermal denaturation experiments. Molecular docking studies of compound 6b have also supported the corresponding biological data.

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ARTICLE TYPE
Synthesis, in vitro evaluation and DNA interaction studies of N-allyl naphthalimide
analogues as anticancer agents
Meenakshi Verma, Vijay Luxami and Kamaldeep Paul*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
14
Abstract:
A
novel
series
of
2ꢀallylꢀ6ꢀsubstitutedꢀ
lines. Another example of such modification is UNBS5162,
which proved to be a good anticancer agent that presently at
benzo[de]isoquinolineꢀ1,3ꢀdiones has been synthesized and
evaluated against 60 human tumor cell lines for their in vitro 55 clinical trials. It showed lesser toxic side effects which decreased
antitumor activities. Compound 6b proved to be the most active
member at single dose concentration of 10 µM and broad
spectrum of antitumor activity with GI , TGI and LC values of
CXCL chemokine expression in advanced solid tumors or
1
5
1
0
lymphoma. The probable mechanism of these anticancer
activities of naphthalimide derivatives is supposed to be capable
of either intercalation with base pairs or alkylation, or groove
5
0
50
8
4.2 nM, 27.6 µM and 89.3 µM respectively at five dose
concentration levels. DNA binding properties of this compound 60 binding of DNA as the substituted naphthalimides are
1
6
has been investigated by UVꢀVis and fluorescence
spectrophotometer as well as thermal denaturation experiments.
Molecular docking studies of compound 6b has also been
supported the corresponding biological data.
characterized by presence of planar chromophore portion. Thus,
naphthalimides are not only interact with DNA, but members of
this class also offer various sites for numerous modifications
which provide a hope in the field of improvement of their
1
5
17
65
antitumor activity. Synthesis of such analogues is the high
priority for medicinal research because DNA is one of the
important targets for cancer treatment. Inspired by its promising
antitumor activities and keeping in mind for their possibility of
DNA intercalation, we have decided to synthesize Nꢀallyl
1
. Introduction
18
In the area of anticancer research, the development of small
molecules capable of interacting with deoxyribonucleic acid
2
0
5
0
5
0
(
DNA) and exhibiting anticancer activities has received 70 naphthalimide analogues substituted with primary and secondary
1
enormous attention in recent years. Amongst these it has been
found that 1,8ꢀnaphthalimides (benz[de]isoquinolinꢀ1,3ꢀdiones)
and its derivatives possess high anticancer activity towards
amines in order to improve their anticancer activities and
selectivity profile. Calf thymus (ct)ꢀDNA studies have also been
performed with the most active compound of this series in order
to observe its interaction with DNA.
2
ꢀ7
2
3
3
4
various human and murine cells.
The development of
functional 1,8ꢀnaphthalimide derivatives as anticancer agents and
DNA targeting is a fast growing area and has resulted in several
such derivatives like amonafide, mitonafide, elinafide and
75
N
N
N
O
N
O
O
N
O
O
N
O
8
bisnafide (Figure 1) that entered into clinical trials. Additionally
O
the naphthalimide ring can also be substituted, for example, at the
NH
Amonafide
2
NO
2
N
NH
2
3
ꢀ or 4ꢀposition with amino, bromo or nitro groups. This not only
H
Mitonafide
UNBS5162
allows the introduction of other active functional groups, which
can be used for targeting biomolecules, but can have a major
effect on the electronic properties with a consequent influence on
the chemical, photochemical and spectroscopic properties. In
literature, many examples are known where anticancer activities
of naphthalimides have been significantly affected via fusing
O
O
O
O
N
N
N
N
HN
NO
2
N
N
O
2
N
N
H
O
H
H
O
O
O
Elinafide
Bisnafide
Figure 1 Chemical structure of some naphthalimide analogues
9ꢀ10
aromatic rings
or varying the position and size of side
11
chains. Qian and coꢀworkers have synthesized a series of
naphthalimides with the substitution of 1,2,3ꢀtriazole ring at 3 or
2
2
. Results and Discussion
.1. Chemistry
4
position. These newly synthesized compounds showed better
cytotoxic activity against breast cancer cell line MCFꢀ7 than
12ꢀ13
amonafide.
Wang and coꢀworkers have synthesized
80
Target naphthalimide analogues 6a-n has been prepared via
multistep reactions using commercially available starting material
acenaphthene (1) (Scheme 1). Oxidation of acenaphthene was
naphthalimide analogues substituted with amino acids, and
dichloroacetamide functionalizations at 3ꢀposition and evaluated
their cytotoxic activities against Hela, A549 and K562 cancer cell
4
5
5
0
o
done with acetic acid and sodium dichromate at 75 C for 8 h to
School of Chemistry and Biochemistry, Thapar University, Patiala-147 001, India
obtain 1,8ꢀnaphthalic anhydride (2) with 75% yield. Compound 2
*
Corresponding author. Tel.: +91 9465670595; fax +91 175 236 4498 E-mail
address: kpaul@thapar.edu (K. Paul)
Electronic Supplementary Information (ESI) available: [NMR data]. See
85
was then treated with bromine in presence of KOH solution at 60
o
C for 6 h to get white solid of 6-bromoꢀbenzo[de]isochromeneꢀ
,3ꢀdione (3) with 77% yield (m.p. = 117ꢀ119 C). Refluxing of
†
o
1
DOI: 10.1039/b000000x/
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compound 3 with allylamine in the presence of ethanol for 8 h
resulted in the formation of white solid of 2ꢀallylꢀ6ꢀbromoꢀ
lines at single dose concentration of 10 µM which includes nine
tumor subpanels and their output was reported as a mean graph of
benzo[de]isoquinolineꢀ1,3ꢀdione (4) in 80% yield (m.p. = 128ꢀ 35 the percent growth of the treated cells and presented as
o
1
30 C).
percentage growth inhibition (GI %). Compound 6b exhibited
significant growth inhibition and evaluated as 60 cell panels at
five dose concentration levels.
a
5
Scheme 1 Synthesis of 2ꢀallylꢀ6ꢀsubstituted ꢀbenzo[de]isoquinolineꢀ1,3ꢀ
dione
Preliminary in vitro antitumor screening revealed that only
40 compounds belonging to secondary amines especially morpholine
showed significant inhibition for almost all the cancer cell lines.
The percentages of inhibition for these cancer cells were more
than 60% of tested derivative while primary amines substituted
H
O
N
O
(
iv)
O
O
O
O
O
O
5
Br
(
i)
(ii)
(v)
6
f-i showed weak activities with percentages of inhibition less
1
2
3
Br
45 than 40% except few cell lines (Table 2). These variations could
be correlated to the difference in C6 substituents on the core
naphthalimide moiety, in which the presence of morpholine
moiety is an important factor affecting for antitumor activity. On
the contrary; compound 6c with pyrrolidine, showed potency
50 towards nonꢀsmall lung cancer cell line EKVX and renal cancer
cell line A498 with GI values of 40.31% and 33.55%
respectively. In series of primary amines 6f-i, significant growth
inhibitions were observed for renal cancer cell (UOꢀ31; 26.10–
32.85%), lung cancer cell (HOPꢀ92; 21.32–50.37%) and prostate
(iii)
O
N
O
O
N
O
(vi)
4
Br
1 2
NR R
a-n
6
a
o
Reagents and conditions (i) acetic acid, Sodium dichromate, 75 C, (ii)
potassium hydroxide, bromine, 60 C, (iii) allyl amine, ethanol, reflux,
(iv) NH
o
10
4
OH, ethanol, reflux, (v) allyl bromide, EtOH, reflux, (vi)
CO , TBAHSO , acetonitrile, reflux.
NHR , K
1
R
2
2
3
4
Table 1 Physical data of synthesized compounds
5
5
cancer cell (PCꢀ3; 23.41–39.40%). Compound 6i with allylamine
at C6 position also showed selectivity towards lung cancer cell
line HOP 92 and breast cancer cell line MCF 7 with GI values of
50.37% and 66.05% respectively. On the other hand,
naphthalimide analogue 6b proved to be the most active
compound of these series. It showed broad spectrum of activity
against all nine subpanels of cancer cell lines at primary single
dose concentration level (Table 3).
o
Entry
NR
I
R
2
Product Time (h) Yield (%) m.p. ( C)
1
2
3
4
5
6
7
piperidinꢀ1ꢀyl
6a
6b
6c
6d
6e
6f
9
8
78
87
76
60
59
63
53
115ꢀ117
173ꢀ174
155ꢀ158
185ꢀ188
205ꢀ208
210ꢀ212
205ꢀ207
morpholinꢀ4ꢀyl
pyrrolidinꢀ1ꢀyl
9
60
4ꢀaminoꢀbenzenethiol
2ꢀaminoꢀbenzenethiol
2ꢀaminoꢀpyridinꢀ3ꢀol
11
12
10.5
10
Table 2 Percentage growth inhibition (GI%) of the selected compounds
over the most sensitive tumor cell lines at single concentration of 10 µM
5ꢀbromoꢀpyridinꢀ2ꢀ
ylamine
6g
65
8
9
2ꢀaminoꢀethanol
6h
6i
8.5
9
57
50
65
62
59
51
49
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Cell lines
MOLTꢀ4
RPMIꢀ8226
SR
6c
ꢀ
ꢀ
22.00
40.31
28.67
24.02
6f
ꢀ
ꢀ
6g
6h
ꢀ
ꢀ
ꢀ
6i
ꢀ
ꢀ
25.25
ꢀ
50.37
ꢀ
allylamine
27.75
21.90
20.73
ꢀ
43.52
ꢀ
22.83
24.71
ꢀ
1
1
1
1
1
0
1
2
3
4
nꢀpropylamine
nꢀbutylamine
nꢀpentylamine
nꢀhexylamine
nꢀoctylamine
6j
8.5
8
25.51
31.68
36.81
ꢀ
25.75
30.78
28.97
22.66
ꢀ
31.16
26.10
39.40
ꢀ
6k
6l
EKVX
ꢀ
8.5
9
HOPꢀ92
NCIꢀH522
HCTꢀ116
SNBꢀ75
UACCꢀ62
OVCARꢀ4
A498
CAKIꢀ1
UOꢀ31
PCꢀ3
21.32
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
20.17
27.36
25.20
ꢀ
6m
6n
ꢀ
ꢀ
ꢀ
ꢀ
8
21.85
ꢀ
33.55
ꢀ
24.18
ꢀ
ꢀ
27.05
28.50
30.66
66.05
ꢀ
Alternatively, compound 4 was also synthesized by the refluxing
compound 3 with ammonium hydroxide in ethanol to get 6ꢀ
bromoꢀbenzo[de]isoquinolineꢀ1,3ꢀdione (5)
treatment with allyl bromide in the presence of ethanol at reflux
condition for 8 h. The crude product was purified through column
chromatography
benzo[de]isoquinolineꢀ1,3ꢀdione (4) in 65% overall yield.
Compound 4 was further refluxed with primary and secondary
amines in the presence of K CO and CH CN using TBAHSO as
catalyst for 8ꢀ12 h to get the crude product. The crude was
purified by column chromatography to afford pure target product
2ꢀallylꢀ6ꢀsubstitutedꢀbenzo[de]isoquinolineꢀ1,3ꢀdione (6a-n) in
moderate to good yields (Table 1). Structures of all these final
ꢀ
ꢀ
15
20
25
followed by
34.13
32.85
28.38
ꢀ
ꢀ
23.41
ꢀ
ꢀ
to
get
pure
2ꢀallylꢀ6ꢀbromoꢀ
MCF7
MDAꢀMBꢀ
34.58
23.72
ꢀ
2
31/ATCC
2
3
3
4
Tꢀ47D
MDAꢀMBꢀ468
25.27
ꢀ
29.71
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
39.71
From the above data, it is clear that compound 6b is the most
active member of the series. Consequently, this active compound
was carried over and tested against a panel of different tumor cell
lines at five dose concentration level (Figure S31ꢀS34) where it
exhibited remarkable anticancer activity against nonꢀsmall cell
lung cancer cell line HOPꢀ92, CNS cancer cell line SNBꢀ75 and
breast cancer cell line HS 578T with GI50 values in the nano
molar range. Compound 6b also showed specificity towards
central nervous system (CNS), melanoma, renal and breast cancer
1
13
compounds were confirmed by H and C NMR as well as mass
spectrometry (Supporting Information).
70
2
.2. In vitro screening for antitumor activities
30
All compounds were submitted to National Cancer Institute
NCI) for evaluation of their in-vitro antitumor activities.
Compounds 6b-c and 6f-i were evaluated against 60 human cell
1
9ꢀ21
(
2
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Table 3 The percentages of growth inhibition of compound 6b over the full panel of tumor cell lines at a single concentration of 10 µM
Subpanel Cell lines
Inhibition (%) Subpanel Cell lines
Inhibition (%)
Leukemia
CCRFꢀCEM
HLꢀ60(TB)
Kꢀ562
MOLTꢀ4
RPMIꢀ8226
SR
Ovarian Cancer
IGROV1
ꢀ7.18
ꢀ5.15
90.22
ꢀ21.21
ꢀ25.79
ꢀ9.38
ꢀ14.55
ꢀ66.42
85.64
78.58
79.30
93.94
OVCARꢀ3
OVCARꢀ4
OVCARꢀ5
OVCARꢀ8
NCI/ADRꢀRES
Non-Small Cell Lung
Cancer
SKꢀOVꢀ3
86.12
A549/ATCC
EKVX
HOPꢀ62
HOPꢀ92
96.95
ꢀ7.17
73.56
78.89
ꢀ2.43
91.99
ꢀ56.58
91.48
ꢀ1.11
Renal Cancer
786ꢀ0
A498
ACHN
CAKIꢀ1
RFX 393
SN12C
TKꢀ10
UOꢀ31
Melanoma
LOX IMVI
MALMEꢀ3M
M14
MDAꢀMBꢀ435
SKꢀMELꢀ2
SKꢀMELꢀ28
SKꢀMELꢀ5
UACCꢀ257
UACCꢀ62
Breast Cancer
MCF7
73.84
ꢀ57.04
81.42
ꢀ39.46
ꢀ29.91
83.04
ꢀ12.91
ꢀ3.07
NCIꢀH226
NCIꢀH23
NCIꢀH322M
NCIꢀH460
NCIꢀH522
Colon Cancer
COLO 205
HCCꢀ2998
HCTꢀ116
HCTꢀ15
HT29
KM12
SWꢀ620
CNS Cancer
SFꢀ268
ꢀ21.78
ꢀ33.96
ꢀ25.42
ꢀ9.11
93.24
ꢀ56.35
92.76
98.87
ꢀ3.28
99.61
99.64
ꢀ28.12
94.33
ꢀ62.71
ꢀ3.63
ꢀ23.87
97.13
99.50
75.05
87.23
94.63
ꢀ24.66
SFꢀ295
SFꢀ539
SNBꢀ19
SNBꢀ75
ꢀ0.41
MDAꢀMBꢀ231/ATCC
HS 578T
BTꢀ549
76.09
95.42
ꢀ47.77
ꢀ40.05
ꢀ2.10
U251
Prostate Cancer
PCꢀ3
DUꢀ145
MDAꢀMBꢀ468
Tꢀ47D
ꢀ21.28
89.72
5
Table 4 Compounds 6b, naphthalimide analogue and oxaliplatin of median growth inhibitory (GI50, µM), total growth inhibitory (TGI, µM) and median
lethal concentrations (LC50, µM) of in vitro subpanel tumor cell line
a
Compds.
Activity
GI50
TGI
LC50
GI50
I
II
III
0.112
b17.04
IV
V
VI
VII
VIII
0.095
b7.174
IX
0.018
b3.962
MGꢀMID
0.084
27.69
89.38
5.05
6
b
b0.098
0.115
21.3
97.35
3.94
0.043
10.53
92.41
4.09
0.073
15.61
87.55
6.93
0.132
40.18
80.14
6.97
0.072
33.42
89.48
3.30
b
Naphthalimide
analogue
2.90
b44.51
6.58
3.23
5.20
TGI
LC50
GI50
TGI
LC50
32.51
93.94
20.86
31.11
79.40
60.81
98.84
2.58
44.17
73.96
45.41
93.98
8.91
55.87
63.10
35.07
83.93
3.50
38.18
72.07
37.44
97.94
11.76
b42.12
15.41
88.94
6.61
40.24
79.40
17.9
b45.98
b38.71
80.15
13.13
b59.62
Oxaliplatin
0.844
b15.84
42.96
b6.30
12.35
37.05
73.58
I, leukemia; II, nonꢀsmall cell lung cancer; III, colon cancer; IV, central nervous system (CNS) cancer; V, melanoma; VI, ovarian cancer; VII, renal
cancer; VIII, prostate cancer; IX, breast cancer.
1
0
a
Full panel meanꢀgraph midpoint (ꢁM)
b
Compounds showed values >100 ꢁM
2
7.6 µM and 89.3 µM respectively. Compound 6b was also
2
2
compared with approved chemotherapeutic drug, oxaliplatin
cell lines with GI50 values in the range of 18 nMꢀ73 nM (Table 4,
Figure 2). Compound 6b also showed almost sixty fold more
20
which also showed interaction with DNA (Table 4). It was
observed that compound 6b showed almost one forty seven fold
1
5
activity
than
naphthalimide
derivative
3ꢀ(4ꢀaminoꢀ
more activity than oxaliplatin (MG MID GI , TGI and LC
phenylsulfanyl)ꢀbenzo[de]benzo[4,5]imidazo[2,1ꢀa]isoquinolinꢀ
50
50
10
values of 12.35 µM, 37.05 µM and 73.58 µM respectively). It is
7
ꢀone, with MG MID GI , TGI and LC values of 84.2 nM,
50 50
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1
1
20
00
8
6
4
2
0
0
0
0
0
ꢀ
ꢀ
ꢀ
ꢀ
20
40
60
80
Leukemia
NonꢀSmall Cell LungCancer
Colon Cancer
CNS Cancer
Melanoma
Cell line type
Ovarian Cancer
Renal Cancer
Prostate Breast Cancer
Cancer
5
F
igure 2 The percentage growth inhibition (GI%) of compound 6b over the full panel cancer cell lines at concentration of 10 µM
clear that replacement with morpholine group at C6 position than
effects showed selectivity towards cancer cells while
other secondary or primary amines, and allyl group at N2 position 35 exhibiting minimal/no toxicity towards normal cells.
leads to excellent increase in antitumor activity.
2
.3. Cytotoxicity (MTT and LDH)
10
Cytotoxicity of compound 6b in human normal cell line
(
(
Hek293) was performed by means of a colorimetric assay
MTT assay). The results showed that there was only 12%,
8
.5%, 8%, 6.5% and 6% cytotoxicity of compound 6b to
ꢀ4 ꢀ5 ꢀ6 ꢀ7 ꢀ8
Hek293 cells at 10 , 10 , 10 , 10 and 10 M concentrations,
respectively (Figure 3). Compound 6b showed only 12%
toxicity to Hek293 cells even at 100 µM. These indicated that
compound 6b showed potent anticancer activity and low
toxicity to normal cells.
15
Figure 4 LDH release of compound 6b at one and two doses after 12 h
blue bar) and 24 h (red bar).
(
2
.4. DNA binding studies
4
4
5
5
0
5
0
5
DNA binding studies of the most active compound 6b has
been evaluated using both UVꢀVisible and fluorescence
spectrophotometer in order to determine the interaction of
compound with ctꢀDNA. The addition of ctꢀDNA (4ꢀ100
µM) to the phosphate buffered solution of 6b (20 µM) caused
decrease in absorption intensity at 405 nm (Figure 5).
24
20
25
30
Figure 3 Effect of compound 6b on Hek 293 cells
Fluorescence titrations were also performed with ctꢀDNA
in phosphate buffered solution of compound 6b. The
incremental addition of ctꢀDNA caused emission
enhancement with concomitant blue shift of emission band
from 555 nm to 520 nm. The apparent binding constant of 6b
with ctꢀDNA was determined to be log β = 5.03 calculated
from titration data using BenesiꢀHildebrand method which is
comparable with amonafide having log β = 5.15. The
intercalative nature of compound 6b with ctꢀDNA 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
Lactate dehydrogenase (LDH) leakage is an indicator of
2
3
cell membrane integrity. When the cell membrane is
disrupted, the membraneꢀbound LDH leaks into the medium
and hence, LDH leakage is considered a hallmark of
cytotoxicity. Cell death leads to a collapse in membrane
integrity, thereby releasing LDH into the medium. In the
present study, a significant level of LDH leakage was
observed in the cell culture medium of the A549 cell line at
one (IC ) and two doses (2xIC ), when they were treated
50
50
with the respective IC50 concentrations of the compound for a
period of 12 h and 24 h (Figure 4). It has been suggested that
compound 6b exhibited significant membraneꢀdamaging
25
increases thermal melting temperature (Tm). The derivative
4
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o
melting curve shows an increase of 18.2 C in thermal melting
emission intensities of EtBr bound to ctꢀDNA at 610 nm
o
o
temperature of ctꢀDNA (60 C) upon addition of 6b (78.2 C). 25 enhanced indicating that they cannot effectively displace EtBr
Thus, both UVꢀVisible and fluorescence titrations as well as
thermal denaturation experiments indicated intercalative
nature of compound 6b with ctꢀDNA.
from the EtBr:DNA complex (Figure S36). This observation
is often considered to be due to that they can bind weakly to
the DNA, probably by electrostatic interactions. It is generally
agreed that strong fluorescence enhancement accompanies
30 intercalation of the dye into the double helix conformation of
the nucleic acid, but there is also evidence for additional nonꢀ
intercalative, less fluorescenceꢀenhanced sites, which are
5
2
.5. Ethidium bromide (EtBr) displacement study
In order to prove the reversibility of the compound 6b:DNA
complex, fluorescence quenching experiments of ethidium
bromide:DNA were carried out by adding 0–20 ꢁM of the
compound to samples containing 10 ꢁM EtBr, 10 ꢁM DNA
and a phosphate (pH = 7). Before the measurements, the
26
presumed to involve electrostatic binding.
1
1
2
0
5
0
2.6. ADME Prediction
system was shaken and incubated at room temperature for ~5 35 In order to determine the properties and drugꢀlike characteristics
of compounds 6b-c and 6f-i, we carried out the calculation with
Molinspiration software of the lipophilicity (expressed as the
octanol/water partition coefficient and herein called logP), and
the theoretical prediction of other ADME properties (molecular
weight, TPSA, number of hydrogen donors and acceptors, and
volume) for Lipinski’s rule of five. Drug likeness seems to be a
promising standard for the properties of a molecule which
min. The emission was recorded at 100–750 nm. On addition
of 100 µM solution of ethidium bromide to compound
6b:DNA complex, the emission band at 520 nm disappeared
with reappearance of the fluorescence maxima at 610 nm
typically due to EtBr:DNA complex. It confirmed the
reversible binding of ligand to DNA (Figure S35).
Competitive ethidium bromide binding studies were
carried out in order to examine the binding mode of each
complex with ctꢀDNA. The emission spectra of EtBr bound to
ctꢀDNA in the absence and presence of each complex have
been recorded at [EtBr] = 10 ꢁM, [DNA] = 10 ꢁM. The
40
27
influences its pharmacodynamics and pharmacokinetics. This
rule is based upon the prediction that if a molecule have
molecular weight ≤ 500, log P ≤ 5, ≤ 5 hydrogen bond donor sites
and ≤ 10 hydrogen bond acceptor sites (N or O atoms), only then
45
28
it can be accepted as a member of biological active drug family.
0
.4
.35
.3
.25
.2
.15
.1
b
a
0
0
0
0
4
05 nm
0
0
0
.05
0
2
00
300
400
500
600
Wavelength (nm)
Wavelength (nm)
50
Figure 5 Effect of addition of ctꢀDNA (4ꢀ100 µM) on (a) absorption (b) emission spectra of compound 6b (20 µM, pH 7.0) in phosphate buffer
1
0
Not considering this criteria may lead the poor absorption or
permeation of the drug like molecule. Compounds selected for in
vitro anticancer studies showed good physicochemical properties
having no violation with any of the parameters. The results
obtained are concluded in table 5. A comparison among the
values leave us with the result that higher activity of compound
analogue
which is an estimate of compound’s overall lipophilicity
(supported by low lipophilicity of amonafide). This parameter
might be influence the factors like solubility and permeability
through biological membrane.
5
5
65
6
b may be described on the basis of lower value of log P factor,
6
0
Table Structural properties of 2ꢀallylꢀ6ꢀsubstitutedꢀ
5
2.7. Molecular docking studies
benzo[de]isoquinolineꢀ1,3ꢀdione 6b-c and 6f-i, and the reference
compounds.
Molecular docking is an interesting tool to predict the possible
drug–DNA interactions because the mode of action of various
drugs like anticancer, antiviral, antibacterial, is directly associated
7
0
Compds
m.wt. logP TPSA nON nOHNH nviolations volume
29
with their binding to DNA. To explore the most feasible
binding site, interaction mode and binding affinity docking
studies have been performed on compound 6b with DNA (PDB
6
b
322.36 2.60 51.54
306.36 3.15 42.31
345.35 3.47 84.22
408.25 4.72 63.99
296.32 1.87 71.33
292.33 3.15 51.10
5
4
6
5
5
4
5
4
0
0
2
1
2
1
2
2
0
0
0
0
0
0
0
0
294.65
285.67
300.15
310.01
266.50
275.04
258.74
333.61
6
c
6
f
30
6
6
g
h
75 ID: 1BNA). Most stable binding conformation of 6b fits into
the major groove comfortably without disrupting the double
helical structure of DNA and stabilized by electrostatic
interaction. The planarity of naphthalimide core is comfortable
for strong ᴫꢀᴫ stacking interactions and fits inside the DNA
6
i
Amonafide 283.33 1.11 68.33
Naphthali 393.47 4.90 60.40
mide
80
strands by van der Waals interaction and hydrophobic contacts
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5

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DOI: 10.1039/C5RA00925A
while morpholine moiety moved outside of the DNA strand
Figure 6). These results are consistent with UVꢀVis and
(
fluorescence studies. Therefore, docking of compound 6b with
DNA indicated the probable mode of action for anticancer
activities.
5
0
1
15
Figure 6 Molecular docked model of compound 6b with DNA (pdb ID 1BNA)
3
. Conclusion
were monitored by thin layer chromatography (TLC) with silica
plate coated with silica gel HFꢀ254 and column chromatography
was performed with silica gel mesh size 60ꢀ120. Hexane:
ethylacetate were adopted solvent systems.
The present work has led to the development of novel Nꢀallyl
naphthalimide analogues with different substitution of primary
and secondary amines at C6 position. These compounds were
2
2
3
0
5
0
evaluated towards 60 human tumor cell lines for their in vitro 55 4.2. General procedure for synthesis of 2-allyl-6-bromo-
activities and some of which shown promising antitumor
activities. Compound 6b showed more active in most of the
cancer cell lines at 10 µM concentration range and showed broad
spectrum of antitumor activity with MG MID GI50 value of 84
nM at five dose concentration levels. LDH leakage into the
medium also confirmed the cytotoxic effect of this compound.
Compound 6b showed strong intercalating properties with ctꢀ
DNA determined by UVꢀVisible and fluorescence spectroscopy.
Molecular docking studies indicating considerable interactions of
these compounds with DNA that also favor the intercalation of
synthesized compounds. These preliminary encouraging results
of biological screening could offer an excellent framework in this
field and thus further studies of these molecules are in progress.
benzo[de]isoquinoline-1,3-dione (4)
Sodium dichromate (14.50 g, 55.34 mmol) was added to a
solution of acenaphthene (3 g, 19.7 mmol) and acetic acid (60 ml)
with continue stirring at room temperature. This suspension was
o
6
6
7
7
8
8
0
5
0
5
0
5
then heated to 75 C for 8 h. Reaction was monitored with TLC.
On complete reaction, cold water was added to the reaction
mixture, resulted in precipitation. After filtration and washed off
with water, yellow coloured solid of 1,8ꢀnaphthalic anhydride
o
(
2.9 g, 75%, m.p. 266ꢀ268 C) 2 was collected and vacuum dried.
A solution of 1,8ꢀnaphthalic anhydride (2.57 g,12.9 mmol) and
hot KOH (3.5 g in 15 ml water) was prepared and cooled to room
temperature. To this solution, bromine was added dropꢀwise with
vigorous stirring. After complete addition, reaction mixture was
o
4
. Experimental
heated to 60 C for 6 h. The resulted solution was acidified with
HCl. Brown coloured solid separated out and filtered. This solid
residue was further heated with 60 ml of 5% NaOH solution,
filtered and treated with HCl solution till complete neutralization.
Off white precipitates separated out, filtered and washed with
cold water, dried to get off white solid of 6ꢀbromoꢀ
benzo[de]isochromeneꢀ1,3ꢀdione (3) (2.75 g, 77%, m.p. 117ꢀ119
35
4.1. Chemistry
All chemicals and solvents of commercial grade were used
without further purification and were supplied by
spectrochemicals and SigmaꢀAldrich. Melting points were
determined in open capillaries and were uncorrected. H and
NMR spectra were recorded on Jeol ECSꢀ400 MHz spectrometer
o
1
13
C). 6ꢀBromoꢀbenzo[de]isochromeneꢀ1,3ꢀdione (3.00 g, 10.8
C
mmol) was then reacted with allylamine (0.62 g, 10.8 mmol) in
ethanol at reflux temperature for 8 h. After the completion of
reaction (TLC), cooled the reaction mixture to get crude solid,
filtered and washed with ethanol to afford pure white compound
of 2ꢀallylꢀ6ꢀbromoꢀbenzo[de]isoquinolineꢀ1,3ꢀdione (2.4 g, 80%,
40
45
50
at 400 MHz and 100 MHz respectively, using CDCl as solvent.
3
The chemical shifts were expressed in parts per million with TMS
as internal reference and J values are given in Hz. Mass Spectra
of the synthesized compounds were recorded at Waters
Micromass QꢀTof Micro. Elemental Analysis has been done with
Thermo Scientific (Flash 2000) analyzer. UVꢀVisible spectra
were recorded using Chamnion UV/Vis spectrometer.
Fluorescence measurements were performed on a Varian Cary
Eclipse fluorescence spectrometer. Thermal denaturation
experiments were performed on Shimadzu UVꢀ2450. Reactions
o
m.p. 128ꢀ130 C) (4).
An alternative method for synthesizing 2ꢀallylꢀ6ꢀbromoꢀ
benzo[de]isoquinolineꢀ1,3ꢀdione has also been followed where 6ꢀ
bromoꢀbenzo[de]isochromeneꢀ1,3ꢀdione (3) (2.00 g, 7.20 mmol)
was refluxed with ammonium hydroxide (21.6 mmol) and
ethanol. The reaction mixture was then cooled and solid product
6
| Journal Name, [year], [vol], 00–00
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DOI: 10.1039/C5RA00925A
was filtered to get 6ꢀbromoꢀbenzo[de]isoquinolineꢀ1,3ꢀdione 5
2H, NꢀCH ), 4.03 (t, J = 4.12 Hz, 4H, morꢀCH ), 3.27 (t, J = 4.60
2 2
o
13
(m.p. 134ꢀ136 C). 5 (7.25 mmol) was further refluxed with
Hz, 4H, morꢀCH2); C NMR (100 MHz, CDCl ): δ 161.3 (C=O),
3
allylbromide (7.50 mmol) in presence of EtOH to yield crude
160.8 (C=O), 152.9, 129.8, 129.5, 128.5, 127.3, 127.1, 123.3,
product, washed with ethanol to get white solid of 2ꢀallylꢀ6ꢀ 60 123.0, 120.4, 114.5, 114.2, 112.1 (ArC), 64.1 (morꢀOCH ), 50.6
2
+
5
bromoꢀbenzo[de]isoquinolineꢀ1,3ꢀdione (4) (1.5 g, 65% overall,
m.p. 128ꢀ130 C).
(morꢀNCH ), 39.5 (NꢀCH ); MS (EI) : m/z 323.1 (M +1). Anal.
Calc. for C H N O : C, 70.79; H, 5.63; N, 8.69. Found: C,
19 18 2 3
2 2
o
7
0.55; H, 5.89; N, 8.83.
2
-Allyl-6-bromo-benzo[de]isoquinoline-1,3-dione (4)
2
-Allyl-6-pyrrolidin-1-yl-benzo[de]isoquinoline-1,3-dione (6c)
o
1
White solid (80 %); m.p. 128ꢀ130 C; H NMR (400 MHz,
2
3
o
1
CDCl ): δ 8.66 (dd, J = 7.32 Hz, J = 0.92 Hz, 1H, ArH), 8.56 65 Yellow solid (76 %); m.p. 155ꢀ158 C; H NMR (400 MHz,
3
2
3
1
0
(dd, J = 8.68 Hz, J = 0.92 Hz, 1H, ArH), 8.41 (d, J = 7.80 Hz,
H, ArH), 8.04 (d, J = 7.76 Hz, 1H, ArH), 7.86 (dd, J = 8.24 Hz,
J = 0.92 Hz, 1H, ArH), 6.04ꢀ5.94 (m, 1H, CH), 5.35ꢀ5.30 (dq, J
CDCl ): δ 8.59ꢀ8.55 (m, 2H, ArH), 8.42 (d, J = 8.68 Hz, 1H,
ArH), 7.54 (dd, J = 8.68 Hz, J = 1.32 Hz, 1H, ArH), 6.80 (d, J =
8.68 Hz, 1H, ArH), 6.05ꢀ5.96 (m, 1H, CH), 5.31ꢀ5.26 (dq, J =
3
2
2
3
1
3
2
2
3
2
3
2
=
17.4 Hz, J = 1.36 Hz, 1H, CH ), 5.24ꢀ5.20 (dq, J = 10.3 Hz,
16.96 Hz, J = 1.36 Hz, 1H, CH ), 5.19ꢀ5.16 (dq, J = 10.54 Hz,
2
2
3
2
3
3
2
3
J = 1.36 Hz, 1H, CH ), 4.80ꢀ4.78 (dt, J = 5.96 Hz, J = 1.36 Hz, 70 J = 1.36 Hz, 1H, CH ), 4.81ꢀ4.79 (dt, J = 5.48 Hz, J = 1.36 Hz,
2
2
13
1
5
2H, NꢀCH2); C NMR (100 MHz, CDCl ): δ 163.3 (C=O),
33.4, 132.2, 131.9, 131.4, 131.1, 130.6, 130.5, 129.0, 128.1,
23.0, 122.1, 117.9 (ArC), 42.6 (NꢀCH ); MS (EI) : m/z 316.0
M +1). Anal. Calc. for C H BrNO : C, 56.99; H, 3.19; N, 4.43.
2H, NꢀCH ), 3.78 (t, J = 6.44 Hz, 4H, pyrrꢀCH ), 2.11ꢀ2.08 (m,
4H, pyrrꢀCH2); C NMR (100 MHz, CDCl ): δ 164.4 (C=O),
163.6 (C=O), 152.5, 133.4, 132.6, 131.9, 131.0, 122.8, 122.3,
122.2, 116.8, 110.1, 108.3 (ArC), 53.0 (pyrrꢀCH ), 42.0 (NꢀCH ),
75 25.9 (pyrrꢀCH ); MS (EI) : m/z 307.1 (M +1). Anal. Calc. for
3
2
2
13
1
1
(
3
2
+
1
5
10
2
2
2
+
Found: C, 56.71; H, 3.33; N, 4.59.
2
C H N O : C, 74.49; H, 5.92; N, 9.14. Found: C, 74.78; H,
19
18
2
2
5
.77; N, 9.36.
2
0
4.3. General procedure for synthesis of 2-allyl-6-substituted-
benzo[de]isoquinoline-1,3-dione (6a-n)
2
-Allyl-6-(4-amino-phenylsulfanyl)-benzo[de]isoquinoline-1,3-
A mixture of 2ꢀallylꢀ6ꢀbromoꢀbenzo[de]isoquinolineꢀ1,3ꢀdione
dione (6d)
(
0.20 g, 0.65 mmol) and corresponding amine (0.80 mmol) was
o
1
refluxed for 8ꢀ12 h in the presence of potassium carbonate (0.059 80 Brown solid (60 %); m.p. 185ꢀ188 C; H NMR (400 MHz,
2
5
g, 1.05 mmol), TBAHSO (catalytic amount) in acetonitrile. After
CDCl ): δ 8.65ꢀ8.61 (m, 2H, ArH), 8.31 (d, J = 8.24 Hz, 1H,
3
4
2
3
completion of reaction, reaction mixture was extracted with
chloroform, separated the organic layer, dried over Na SO ,
ArH), 7.79 (dd, J = 8.24 Hz, J = 7.32 Hz, 1H, ArH), 7.40 (d, J =
8.72 Hz, 2H, ArH), 7.06 (d, J = 7.76 Hz, 1H, ArH), 6.81 (d, J =
2
4
2
filtered and concentrated to get solid crude product of 2ꢀallylꢀ6ꢀ
substitutedꢀbenzo[de]isoquinolineꢀ1,3ꢀdione (6a-n) which were 85 17.42 Hz, J = 1.36 Hz, 1H, CH ), 5.21ꢀ5.17 (dq, J = 10.08 Hz,
purified using column chromatography on silica gel using
hexane:ethylacetate as eluents.
8.68 Hz, 2H, ArH), 6.01ꢀ5.95 (m, 1H, CH), 5.32ꢀ5.26 (dq, J =
3
2
2
3
2
3
3
0
J = 1.36 Hz, 1H, CH ), 4.79ꢀ4.77 (dt, J = 5.48 Hz, J = 1.36 Hz,
2
1
3
2H, NꢀCH ), 4.01 (bs, 2H, NH ); C NMR (100 MHz, CDCl ): δ
2
2
3
1
1
63.8 (C=O), 163.7 (C=O), 148.8, 148.3, 137.4, 132.1, 131.6,
31
31.0, 129.8, 128.3, 126.5, 122.9, 122.8, 118.5, 117.3, 116.3,
2
-Allyl-6-piperidin-1-yl-benzo[de]isoquinoline-1,3-dione (6a)
+
90
115.5 (ArC), 42.3 (NꢀCH ); MS (EI) : m/z 361.1 (M +1). Anal.
2
o
1
Calc. for C H N O S: C, 69.98; H, 4.47; N, 7.77; S, 8.90.
Found: C, 69.83; H, 4.69; N, 7.52; S, 8.73.
21
16
2
2
Yellow solid (78 %); m.p. 115ꢀ117 C; H NMR (400 MHz,
2
3
CDCl ): δ 8.59 (dd, J = 7.36 Hz, J = 1.40 Hz, 1H, ArH), 8.51
(d, J = 8.24 Hz, 1H, ArH), 8.40 (dd, J = 8.24 Hz, J = 1.36 Hz,
H, ArH), 7.70 (dd, J = 8.72 Hz, J = 1.32 Hz, 1H, ArH), 7.19
d, J = 8.24 Hz, 1H, ArH), 6.03ꢀ5.95 (m, 1H, CH), 5.32ꢀ5.27 (dq,
J = 17.2 Hz, J = 1.40 Hz, 1H, CH ), 5.20ꢀ5.17 (dq, J = 10.32
Hz, J = 1.36 Hz, 1H, CH ), 4.81ꢀ4.78 (dt, J = 5.52 Hz, J = 1.36 95 Yellow solid (59 %); m.p. 205ꢀ208 C; H NMR (400 MHz,
3
2
3
3
4
4
5
0
5
2
3
1
2-Allyl-6-(2-amino-phenylsulfanyl)-benzo[de]isoquinoline-1,3-
dione (6e)
(
2
3
2
2
3
2
3
o
1
2
Hz, 2H, NꢀCH ), 3.24 (t, J = 5.04 Hz, 4H, pipꢀCH ), 1.91ꢀ1.86
CDCl ): δ 8.49 (q, J = 8.28 Hz, 2H, ArH), 8.11 (d, J = 7.80 Hz,
1H, ArH), 7.72ꢀ7.62 (m, 3H, ArH), 7.57 (d, J = 8.24 Hz, 1H,
ArH), 7.47 (t, J = 7.76 Hz, 1H, ArH), 6.82 (d, J = 8.24 Hz, 1H,
2
2
3
1
3
(
m, 4H, pipꢀCH ), 1.75ꢀ1.71 (m, 2H, pipꢀCH ); C NMR (100
2
2
MHz, CDCl ): δ 164.3 (C=O), 163.8 (C=O), 157.3, 132.8, 132.4,
3
1
31.1, 130.7, 129.9, 126.2, 125.3, 122.9, 117.1, 115.7, 114.6
ArH), 5.90ꢀ5.82 (m, 1H, CH), 5.24 (d, J = 16.96, 1H, CH ), 5.17
2
(
ArC), 54.4 (pipꢀNCH ), 42.1 (NꢀCH ), 26.1 (pipꢀCH ), 24.3 100 (d, J = 10.08 Hz, 1H, CH ), 4.64 (d, J = 5.96 Hz, 2H, NꢀCH );
2
2
2
2
2
+
13
(pipꢀCH ); MS (EI) : m/z 321.1 (M +1). Anal. Calc. for
C NMR (100 MHz, CDCl ): δ 161.4 (C=O), 161.3 (C=O),
3
2
C H N O : C, 74.98; H, 6.29; N, 8.74. Found: C, 74.79; H,
148.9, 143.1, 135.7, 130.7, 130.5, 129.7, 129.0, 128.2, 126.7,
2
0
20
2
2
6
.43; N, 8.95.
126.4, 125.3, 120.8, 120.5, 116.8, 116.0, 115.0, 113.9, 106.6
+
(
ArC), 42.3 (NꢀCH ); MS (EI): m/z 361.1 (M +1). Anal. Calc. for
2
1
05 C H N O S: C, 69.98; H, 4.47; N, 7.77; S, 8.90. Found: C,
2
-Allyl-6-morpholin-4-yl-benzo[de]isoquinoline-1,3-dione
21 16 2 2
3
2
6
9.79; H, 4.64; N, 7.99; S, 8.65.
(6b)
o
1
5
0
Yellow solid (87 %); m.p. 173ꢀ174 C; H NMR (400 MHz,
2-Allyl-6-(3-hydroxy-pyridin-2-ylamino)-benzo[de]
isoquinoline-1,3-dione (6f)
2
3
CDCl ): δ 8.61 (dd, J = 7.32 Hz, J = 1.40 Hz, 1H, ArH), 8.55
d, J = 8.24 Hz, 1H, ArH), 8.44 (dd, J = 8.68 Hz, J = 1.36 Hz,
H, ArH), 7.73 (dd, J = 8.24 Hz, J = 7.32 Hz, 1H, ArH), 7.27 (t,
J = 9.36 Hz, 1H, ArH), 6.04ꢀ5.94 (m, 1H, CH), 5.33ꢀ5.28 (dq, J 110 CDCl ): δ 8.70 (d, J = 8.72 Hz, 1H, ArH), 8.65 (d, J = 7.36 Hz,
3
2
3
(
1
2
3
o
1
Yellow solid (63 %); m.p. 210ꢀ212 C; H NMR (400 MHz,
2
3
3
2
2
3
5
5
= 17.42 Hz, J = 1.36 Hz, 1H, CH ), 5.21ꢀ5.18 (dq, J = 10.08 Hz,
1H, ArH), 8.48 (dd, J = 8.24 Hz, J = 0.92 Hz, 1H, ArH), 8.06 (d,
J = 5.04 Hz, 1H, ArH), 7.82 (t, J = 7.56 Hz, 1H, ArH), 7.29ꢀ7.27
2
3
2
3
J = 1.36 Hz, 1H, CH ), 4.81ꢀ4.78 (dt, J = 5.96 Hz, J = 1.36 Hz,
2
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 7

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2
3
2
3
(
6
m, 1H, ArH), 6.94 (dd, J = 8.24 Hz, J = 0.92 Hz, 1H, ArH),
ArH), 8.11 (d, J = 7.80 Hz, 1H, ArH), 7.61 (dd, J = 8.28 Hz, J =
0.92 Hz, 1H, ArH), 6.72 (d, J = 8.72 Hz, 1H, ArH), 6.03ꢀ5.97 (m,
2
.79ꢀ6.75 (m, 1H, ArH), 6.02ꢀ5.94 (m, 1H, CH), 5.33ꢀ5.29 (dt, J
16.96 Hz, J = 1.36 Hz, 1H, CH ), 5.22ꢀ5.20 (dt, J = 10.08 Hz,
J = 1.40 Hz, 1H, CH ), 4.80ꢀ4.77 (m, 2H, NꢀCH ); C NMR 60 1.40 Hz, 1H, CH ), 5.19ꢀ5.16 (dq, J = 10.52 Hz, J = 1.40 Hz,
(100 MHz, CDCl ): δ 163.8 (C=O), 163.2 (C=O), 158.1, 151.8,
3
2
2
3
=
1H, CH), 5.36 (bs, 1H, NH), 5.31ꢀ5.26 (dq, J = 17.16 Hz, J =
2
3
13
2
3
2
2
2
2
3
5
1H, CH ), 4.80 (dt, J = 5.48 Hz, J = 1.36 Hz, 2H, NꢀCH ), 3.40ꢀ
3
2
2
1
1
45.0, 136.4, 132.9, 132.1, 129.6, 128.4, 128.1, 126.8, 123.5,
3.35 (m, 2H, propꢀNCH ), 1.87ꢀ1.81 (m, 2H, propꢀCH ), 1.11 (t,
2 2
13
22.5, 117.5, 117.1, 114.5, 109.8 (ArC), 42.3 (NꢀCH ); MS (EI) :
J = 7.36 Hz, 3H, propꢀCH3); C NMR (100 MHz, CDCl ): δ
2
3
+
m/z 346.1 (M +1). Anal. Calc. for C H N O : C, 69.56; H,
4
164.4 (C=O), 163.8 (C=O), 149.5, 134.6, 132.6, 131.2, 129.8,
65 125.8, 124.6, 122.9, 120.0, 116.9, 109.9, 104.3 (ArC), 45.4 (Nꢀ
CH ), 42.1 (NꢀCH ), 22.1 (CH ), 11.6 (CH ); MS (EI) : m/z 295.1
2
0
15
3
3
.38; N, 12.17. Found: C, 69.33; H, 4.54; N, 12.43.
2
2
2
3
+
(
M +1). Anal. Calc. for C H N O : C, 73.45; H, 6.16; N, 9.52.
10
2-Allyl-6-(5-bromo-pyridin-2-ylamino)-benzo[de]isoquinoline
1,3-dione (6g)
18 18 2 2
Found: C, 73.72; H, 6.30; N, 9.31.
-
o
1
Brown solid (53 %); m.p. 205ꢀ207 C; H NMR (400 MHz,
2-Allyl-6-butylamino-benzo[de]isoquinoline-1,3-dione (6k)
2
3
CDCl ): δ 8.75 (dd, J = 8.68 Hz, J = 1.36 Hz, 1H, ArH), 8.67
dd, J = 7.32 Hz, J = 1.36 Hz, 1H, ArH), 8.45 (d, J = 8.24 Hz, 70 Yellow liquid (62 %); H NMR (400 MHz, CDCl ): δ 8.58 (dd, J
1H, ArH), 7.79 (dd, J = 8.68 Hz, J = 1.36 Hz, 1H, ArH), 7.01
d, J = 1.96 Hz, 2H, ArH), 6.86 (d, J = 8.24 Hz, 1H, ArH), 6.79
d, J = 8.68 Hz, 1H, ArH), 6.04ꢀ5.95 (m, 1H, CH), 5.33ꢀ5.28 (dq,
J = 17.18 Hz, J = 1.36 Hz, 1H, CH ), 5.22ꢀ5.18 (dq, J = 10.08
Hz, J = 1.36 Hz, 1H, CH ), 4.81ꢀ4.79 (dt, J = 5.96 Hz, J = 1.36 75 1.36 Hz, 1H, CH ), 5.19ꢀ5.16 (dq, J = 10.08 Hz, J = 1.40 Hz,
Hz, 2H, NꢀCH2); C NMR (100 MHz, CDCl ): δ 164.2 (C=O),
3
2
3
1
2
(
3
2
3
3
15
20
25
= 7.32 Hz, J = 0.92 Hz, 1H, ArH), 8.47 (d, J = 8.24 Hz, 1H,
2
3
(
ArH), 8.10 (dd, J = 8.24 Hz, J = 0.92 Hz, 1H, ArH), 7.62 (dd,
2
3
(
J = 8.72 Hz, J = 1.32 Hz, 1H, ArH), 6.72 (d, J = 8.68 Hz, 1H,
ArH), 6.03ꢀ5.97 (m, 1H, CH), 5.31ꢀ5.26 (dq, J = 17.18 Hz, J =
2
3
2
2
3
2
3
2
3
2
3
2
2
1
3
2
3
1H, CH ), 4.80ꢀ4.78 (dt, J = 5.96 Hz, J = 1.36 Hz, 2H, NꢀCH ),
3
2
2
1
1
63.5 (C=O), 161.1, 146.1, 144.2, 133.1, 132.3, 131.9, 129.6,
3.43ꢀ3.38 (m, 2H, butꢀNCH ), 1.84ꢀ1.75 (m, 2H, butꢀCH ), 1.56ꢀ
2 2
1
3
28.8, 126.2, 123.6, 122.3, 122.0, 117.2, 116.3, 115.6, 109.3
1.50 (m, 2H, butꢀCH ), 1.04 (t, J = 7.32 Hz, 3H, butꢀCH );
C
2
3
+
(ArC), 42.2 (NꢀCH ); MS (EI) : m/z 345.1 (M +1). Anal. Calc.
NMR (100 MHz, CDCl ): δ 164.4 (C=O), 163.8 (C=O), 149.5,
2
3
for C H BrN O : C, 58.84; H, 3.46; N, 10.29. Found: C, 58.49; 80 134.6, 132.6, 131.2, 129.8, 125.8, 124.6, 122.9, 120.0, 116.9,
H, 3.65; N, 10.07.
20
14
3
2
109.9, 104.2 (ArC), 43.3 (butꢀNCH ), 42.1 (NꢀCH ), 30.9 (butꢀ
2 2
CH ), 20.3 (butꢀCH ), 13.8 (butꢀCH ); MS (EI) : m/z 309.2
2
2
3
+
(
M +1). Anal. Calc. for C H N O : C, 74.00; H, 6.54; N, 9.08.
2
-Allyl-6-(2-hydroxy-ethylamino)-benzo[de]isoquinoline-1,3-
19 20 2 2
Found: C, 74.33; H, 6.29; N, 9.37.
dione (6h)
1
Brown liquid (57 %); H NMR (400 MHz, CDCl ): δ 8.56 (d, J = 85 2-Allyl-6-pentylamino-benzo[de]isoquinoline-1,3-dione (6l)
3
7
.32 Hz, 1H, ArH), 8.44 (d, J = 8.68 Hz, 1H, ArH), 8.35 (d, J =
1
2
30
35
40
8.28 Hz, 1H, ArH), 7.61 (t, J = 8.24 Hz, 1H, ArH), 6.70 (d, J =
Yellow liquid (59 %); H NMR (400 MHz, CDCl ): δ 8.59 (dd, J
3
3
8
1
5
.72 Hz, 1H, ArH), 6.34 (t, J = 4.36 Hz, 1H, NH), 6.01ꢀ5.96 (m,
= 7.32 Hz, J = 0.92 Hz, 1H, ArH), 8.47 (d, J = 8.24 Hz, 1H,
ArH), 8.10 (dd, J = 8.24 Hz, J = 0.92 Hz, 1H, ArH), 7.63 (dd,
J = 8.68 Hz, J = 7.32 Hz, 1H, ArH), 6.72 (d, J = 8.24 Hz, 1H,
2
3
2
3
H, CH), 5.31ꢀ5.25 (dq, J = 17.4 Hz, J = 1.40 Hz, 1H, CH ),
2
2
3
2
3
.19ꢀ5.16 (dq, J = 10.3 Hz, J = 1.40 Hz, 1H, CH ), 4.79ꢀ4.77
2
2
3
2
3
(dt, J = 5.48 Hz, J = 1.84 Hz, 2H, NꢀCH ), 4.21 (bs, 1H, OH), 90 ArH), 6.03ꢀ5.97 (m, 1H, CH), 5.31ꢀ5.26 (dq, J = 17.18 Hz, J =
2
2
3
4.00 (t, J = 4.60 Hz, 2H, CH ), 3.53 (q, J = 5.04 Hz, 2H, CH );
1.36 Hz, 1H, CH ), 5.19ꢀ5.16 (dq, J = 10.08 Hz, J = 1.40 Hz,
2
2
2
1
3
2
3
C NMR (100 MHz, CDCl ): δ 164.3 (C=O), 163.8 (C=O),
1H, CH ), 4.80ꢀ4.78 (dt, J = 5.48 Hz, J = 1.36 Hz, 2H, NꢀCH ),
3
2
2
1
1
1
63.9, 163.2, 158.0, 151.7, 149.4, 144.9, 136.4, 134.4, 132.9,
32.5, 132.1, 131.3, 129.7, 128.4, 128.1, 126.8, 126.1, 124.8,
23.5, 122.8, 122.5, 117.5, 117.2, 117.0, 114.5, 110.4, 109.9, 95 C NMR (100 MHz, CDCl ): δ 164.4 (C=O), 163.8 (C=O),
3.42ꢀ3.37 (m, 2H, pentꢀNCH ), 1.85ꢀ1.78 (m, 2H, pentꢀCH ),
2 2
1.50ꢀ1.38 (m, 4H, pentꢀCH ), 0.97 (t, J = 7.36 Hz, 3H, pentꢀCH );
2
3
13
3
104.4 (ArC), 60.4 (OꢀCH ), 45.2 (NꢀCH ), 42.3 (NꢀCH ); MS
149.5, 134.6, 132.6, 131.2, 129.8, 125.8, 124.6, 122.9, 120.1,
116.9, 109.9, 104.3 (ArC), 43.6 (pentꢀNCH ), 42.1 (NꢀCH ), 29.2
2
2
2
+
(EI) : m/z 297.0 (M +1). Anal. Calc. for C H N O : C, 68.91;
17 16 2 3
2
2
H, 5.44; N, 9.45. Found: C, 68.76; H, 5.79; N, 9.22.
(pentꢀCH ), 28.6 (pentꢀCH ), 22.4 (pentꢀCH ), 13.9 (pentꢀCH );
2 2 2 3
+
MS (EI) : m/z 323.2 (M +1). Anal. Calc. for C H N O : C,
20
22
2
2
100 74.51; H, 6.88; N, 8.69. Found: C, 74.68; H, 6.59; N, 8.57.
2
-Allyl-6-allylamino-benzo[de]isoquinoline-1,3-dione (6i)
1
Brown liquid (50 %); H NMR (400 MHz, CDCl ): δ 8.59 (d, J =
2-Allyl-6-hexylamino-benzo[de]isoquinoline-1,3-dione (6m)
3
45
7.32 Hz, 1H, ArH), 8.47 (d, J = 8.72 Hz, 1H, ArH), 8.14 (d, J =
1
2
8
8
5
.72 Hz, 1H, ArH), 7.64 (t, J = 8.28 Hz, 1H, ArH), 6.73 (d, J =
Yellow liquid (51 %); H NMR (400 MHz, CDCl ): δ 8.61 (dd, J
3
3
.72 Hz, 1H, ArH), 6.04ꢀ5.96 (m, 2H, CH), 5.49 (bs, 1H, NH),
= 7.76 Hz, J = 1.36 Hz, 1H, ArH), 8.49 (d, J = 8.24 Hz, 1H,
ArH), 8.09 (d, J = 7.80 Hz, 1H, ArH), 7.65 (dd, J = 8.24 Hz, J
NꢀCH ), 4.09 (t, J = 5.28 Hz, 2H, NꢀCH ); C NMR (100 MHz, 105 = 1.32 Hz, 1H, ArH), 6.74 (d, J = 8.28 Hz, 1H, ArH), 6.04ꢀ5.95
2
3
2
3
.41ꢀ5.16 (m, 4H, CH ), 4.80 (dd, J = 5.96 Hz, J = 1.36 Hz, 2H,
2
1
3
2
2
2
3
50
CDCl ): δ 164.4 (C=O), 163.8 (C=O), 149.0, 134.5, 132.9, 132.5,
1
MS (EI) : m/z 293.1 (M +1). Anal. Calc. for C H N O : C,
(m, 1H, CH), 5.32ꢀ5.26 (dq, J = 17.2 Hz, J = 1.36 Hz, 1H, CH ),
5.20ꢀ5.16 (dq, J = 10.08 Hz, J = 1.36 Hz, 1H, CH ), 4.80ꢀ4.78
2
(dt, J = 5.52 Hz, J = 1.36 Hz, 2H, NꢀCH ), 3.43ꢀ3.38 (m, 2H,
3
2
2
3
31.2, 118.0, 116.9, 104.9 (ArC), 46.0 (NꢀCH ), 42.1 (NꢀCH );
2
2
+
2
3
1
8
16
2
2
2
7
3.95; H, 5.52; N, 9.58. Found: C, 73.63; H, 5.70; N, 9.34.
hexꢀNCH ), 1.85ꢀ1.78 (m, 2H, hexꢀCH ), 1.52ꢀ1.46 (m, 2H, hexꢀ
2
2
1
10 CH ), 1.38ꢀ1.36 (m, 4H, hexꢀCH ), 0.94ꢀ0.91 (m, 3H, hexꢀCH );
2
2
3
13
C NMR (100 MHz, CDCl ): δ 164.4 (C=O), 163.8 (C=O),
2
-Allyl-6-propylamino-benzo[de]isoquinoline-1,3-dione (6j)
3
1
49.4, 134.6, 132.6, 131.2, 129.8, 125.8, 124.6, 123.0, 120.1,
1
2
116.9, 110.0, 104.3 (ArC), 43.7 (hexꢀNCH ), 42.1 (NꢀCH ), 31.5
(hexꢀCH ), 28.9 (hexꢀCH ), 26.8 (hexꢀCH ), 22.5 (hexꢀCH ), 14.0
2 2 2 2
2 2
55
Yellow liquid (80 %); H NMR (400 MHz, CDCl ): δ 8.58 (dd, J
=
3
3
7.32 Hz, J = 0.92 Hz, 1H, ArH), 8.46 (d, J = 8.24 Hz, 1H,
8
| Journal Name, [year], [vol], 00–00
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Page 9 of 11
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View Article Online
DOI: 10.1039/C5RA00925A
+
(
hexꢀCH ); MS (EI) : m/z 337.2 (M +1). Anal. Calc. for
acid and the plates are air dried. Bound stain is subsequently
solubilized with 10 mM trizma base, and the absorbance is read
on an automated plate reader at a wavelength of 515 nm. For
suspension cells, the methodology is the same except that the
assay is terminated by fixing settled cells at the bottom of the
wells by gently adding 50 ꢁL of 80% TCA (final concentration,
16% TCA). 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:
3
C H N O : C, 74.97; H, 7.19; N, 8.33. Found: C, 74.67; H,
2
1
24
2
2
7
.32; N, 8.47.
6
5
2
-Allyl-6-octylamino-benzo[de]isoquinoline-1,3-dione (6n)
1
5
0
5
0
Yellow liquid (49 %); H NMR (400 MHz, CDCl ): δ 8.54 (d, J =
3
7
8
8
1
.36 Hz, 1H, ArH), 8.43 (d, J = 8.24 Hz, 1H, ArH), 8.13 (d, J =
.24 Hz, 1H, ArH), 7.57 (t, J = 8.04 Hz, 1H, ArH), 6.68 (d, J =
.72 Hz, 1H, ArH), 6.03ꢀ5.96 (m, 1H, CH), 5.53 (t, J = 4.8 Hz,
H, NH), 5.31ꢀ5.26 (dq, J = 17.42 Hz, J = 1.36 Hz, 1H, CH ),
5.19ꢀ5.15 (dq, J = 10.08 Hz, J = 1.36 Hz, 1H, CH ), 4.79 (d, J =
70
2
3
2
2
3
1
1
2
2
[
(Ti ꢀ Tz)/(CꢀTz)] × 100 for concentrations for which Ti>/= Tz
5
.52 Hz, 2H, NꢀCH ), 3.40ꢀ3.35 (m, 2H, octylꢀNCH ), 1.83ꢀ1.76
2 2
(
8
m, 2H, octylꢀCH ), 1.51ꢀ1.44 (m, 2H, octylꢀCH ), 1.38ꢀ1.25 (m,
2
3
13
H, octylꢀCH ), 0.89 (t, J = 6.88 Hz, 3H, octylꢀCH ); C NMR
[(Ti – Tz)/Tz] × 100 for concentrations for which Ti < Tz:
2
3
(100 MHz, CDCl ): δ 164.3 (C=O), 163.7 (C=O), 149.6, 134.5,
3
132.5, 131.1, 129.7, 126.0, 124.4, 122.7, 120.0, 116.9, 109.6,
1
2
Three dose response parameters are calculated for each
75 experimental agent. Growth inhibition of 50% (GI ) is calculated
50
from [(TiꢀTz)/(CꢀTz)] ×100 = 50, which is the drug concentration
resulting in a 50% reduction in the net protein increase (as
measured by SRB staining) in control cells during the drug
incubation. The drug concentration resulting in total growth
04.1 (ArC), 43.6 (octylꢀNCH ), 42.0 (NꢀCH ), 31.7 (octylꢀCH ),
2 2 2
9.2 (octylꢀCH ), 29.1 (octylꢀCH ), 28.8 (octylꢀCH ), 27.1 (octylꢀ
2
2
2
CH ), 22.5 (octylꢀCH ), 14.0 (octylꢀCH ); MS (EI) : m/z 365.2
2
2
3
+
(M +1). Anal. Calc. for C H N O : C, 75.79; H, 7.74; N, 7.69.
23 28 2 2
Found: C, 75.95; H, 7.89; N, 7.53.
.4. In vitro evaluation assay
In vitro anticancer screening at NCI is a twoꢀstage process,
8
0
inhibition (TGI) is calculated from Ti = Tz. The LC50
4
(concentration of drug resulting in a 50% reduction in the
measured protein at the end of the drug treatment as compared to
that at the beginning) indicating a net loss of cells following
treatment is calculated from [(TiꢀTz) / Tz] ×100 = ꢀ50. Values are
calculated for each of these three parameters if the level of
activity is reached; however, if the effect is not reached or is
exceeded, the value for that parameter is expressed as greater or
less than the maximum or minimum concentration tested.
beginning with the evaluation of all compounds against the 60
cell lines at a single dose of 10 ꢁM. The output from the single
dose screen is reported as a mean graph and is available for
analysis by the COMPARE program. Compounds which exhibit
significant growth inhibition are evaluated against the 60 cell
panel at five concentration levels. The human tumor cell lines of
the cancer screening panel are grown in RPMI 1640 medium
containing 5% fetal bovine serum and 2 mM Lꢀglutamine. For a
typical screening experiment, cells are inoculated into 96 well
microtiter plates in 100 mL at plating densities ranging from 5000
to 40,000 cells/well depending on the doubling time of individual
85
2
3
3
4
4
5
5
6
5
0
5
0
5
0
5
0
4.5. MTT assay
90
Hek293 (Human embryonic kidney) cells, DMEM with
50mM glutamine, 10% FBS, 100 U/ml pencillin and 100
mg/ml streptomycin. The test was performed against Hek293
cell lines. After cell inoculation, the microtiter plates are
incubated at 37 C, 5% CO , 95% air and 100% relative humidity
for 24 h prior to addition of experimental drugs.
(
Human embryonic kidney) cells. Cells were seeded in 96
o
ꢀ5
2
well plates at the density of 1x10 cells/well in DMEM media
95
supplemented with 10% FBS cells. Cells were incubated at 37
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 at the time of drug addition (Tz). Experimental drugs are
solubilized in dimethyl sulfoxide 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 ꢁg/ml gentamicin.
Additional four, 10ꢀfold or ½ log serial dilutions are made to
provide a total of five drug concentrations plus control. Aliquots
of 100 ꢁL of these different drug dilutions are added to the
appropriate microtiter wells already containing 100 ꢁL of
medium, resulting in the required final drug concentrations.
Following drug addition, the plates are incubated for an
additional 48 h at 37 C, 5% CO , 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 ꢁL of cold 50% (w/v) TCA (final concentration,
10% TCA) and incubated for 60 min at 4 C. The supernatant is
discarded, and the plates are washed five times with tap water and 110 with DMEM (Biological Industries) media contains 10% FBS
o
C in 5% CO incubator. Cells were treated with compound
b at five concentrations (10 10 , 10 , 10 , 10 M) for 24 h
,
o
2
ꢀ4
ꢀ5
ꢀ6
ꢀ7
ꢀ8
6
at 37 C. 10 ꢁl of MTT (prepared in 1* PBS buffer) from 5
o
mg/ml stock was added in each well and incubated at 37 C
00 for 4 h in dark. The formazan crystals were dissolved using
1
1
00 ꢁl of DMSO. Further, the amount of formazan crystal
formation was measured as difference in absorbance by Bioꢀ
Red ELISA plate reader at 570 nm and 690 nm reference
wavelength. The relative cell toxicity (%) related to control
05 wells containing culture medium without test material was
calculated by using formula:
1
OD (Compound treated wells)
X 100
% Cell Toxicity = 100 ꢀ
o
OD (Untreated Wells)
2
4.6. LDH activity
o
6
A549 cells (5x10 cells/well) were seeded in 96 well plates
air dried. Sulforhodamine B (SRB) solution (100 ꢁl) at 0.4%
w/v) in 1% acetic acid is added to each well, and plates are
(SigmaꢀAldrich) and 1% PenStrep (SigmaꢀAldrich), incubated
o
(
at 37 C in 5% CO incubator. Cells were allowed to adhere.
2
incubated for 10 min at room temperature. After staining,
unbound dye is removed by washing five times with 1% acetic
After 24 h, containing media was replaced by fresh media and
treated with mentioned concentration of compound again
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 9

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Page 10 of 11
View Article Online
DOI: 10.1039/C5RA00925A
following incubation for 12 and 24 h respectively in 5%CO2
incubator at 37 C. Removal of aliquot of the culture medium
3. E. Van Quaquebeke, T. Mahieu, P. Dumont, J. Dewelle, F.
Ribaucour, G. Simon, S. Sauvage, J. F. Gaussin, J. Tuti, M. El
Yazidi, F. Van Vynckt, T. Mijatovic, F. Lefranc, F. Darro and R.
Kiss, J. Med. Chem., 2007, 50, 4122.
̊
form seeded cell followed by the addition of Lactate
Dehydrogenase assay mixture (LDH assay substrate solution,
LDH assay dye solution, and 1× LDH assay cofactor) into
each sample in a volume equal to twice. Plates were covered
with aluminum foil to protect from light and incubated at
room temperature for 20–30 minutes. After incubation
reaction was terminated by the addition of 1/10 volume of 1 N
HCl to each well. Absorbance measured at wavelength of 490
nm by ELISA reader. Measure absorbance of the blank was
subtracted from this value.
6
6
7
0
5
0
4
.
(a) V. K. Malviya, P. Y. Liu, D. S. Alberts, E. A. Surwit, J. B.
Craig and E. V. Hannigan, Am. J. Clin. Oncol., 1992, 15, 41. (b)
I. Ott, X. Qian, Y. Xu, D. H. W. Vlecken, I. J. Marques, D.
Kubutat, J. Will, W. S. Sheldrick, P. Jesse, A. Prokop and C. P.
Bagowski, J. Med. Chem., 2009, 52, 763.
5
5
.
R. Rosell, J. Carles, A. Abad, N. Ribelles, A. Barnadas, A.
Benavides and M. Martin, Invest. New Drugs, 1992, 10, 171.
10
6. M. F. Brana, M. Cacho, M. A. Garcia, B. de PascualꢀTeresa, A.
Ramos, N. Acero, F. Llinares, D. MunozꢀMingarro, C. Abradelo,
M. F. ReyꢀStolle and M. Yuste, J. Med. Chem., 2002, 45, 5813.
7
.
U. Hossain Sk, A. S. P. Gowda, M. A. Crampsie, J. K. Yun, T. E.
Spratt, S. Amin and A. K. Sharma, Eur. J. Med. Chem., 2011, 46,
4
.7. DNA binding studies
3
331.
8
9
1
.
.
M. J. Ratain, R. Mick, F. Berezin, L. Janisch, R. L. Schilsky, N.
J. Vogelzang and L.B. Lane, Cancer Res. 1993, 53, 2304.
Q. Yang, P. Yang, X. Qian and L. Tong, Bioorg. Med. Chem.
Lett., 2008, 18, 6210
Stock solution of compound 6b (1 mM) was prepared by
dissolving 6b in AR grade DMSO. The DNA binding
experiments were carried out by making dilution of stock solution
with phosphate buffer. Stock solution of ctꢀDNA was prepared by
dissolving DNA in phosphate buffer (10 mM, pH 7.0). The DNA
concentration was estimated from its absorbance intensity at 260
nm with a known molar absorption coefficient value of 6600
75
1
5
0. M. Verma, V. Luxami and K. Paul, Eur. J. Med. Chem., 2013,
8, 352.
6
8
8
0
5
11. (a) K. Wang, Y. Wang, X. Yan, H. Chen, G. Ma, P. Zhang, J. Li,
X. Li and J. Zhang, Bioorg. Med. Chem. Lett., 2012, 22, 937. (b)
L. Xie, J. Cui, X. Qian, Y. Xu, J. Liu and R. Xu, Bioorg. Med.
Chem., 2011, 19, 961.
12. X. Li, Y. Lin, Q. Wang, Y. Yuan, H. Zhang and X. Qian, Eur. J.
Med. Chem., 2011, 46, 1274.
20
3
ꢀ1
ꢀ1
dm M cm . The purity of DNA was established from ratio of
absorbance intensity at 260 nm and at 280 nm.
1
3. X. Li, Y. Lin, Y. Yuan, K. Liu and X. Qian, Tetrahedron, 2011,
7, 2299.
4. K.ꢀR. Wang, F. Qian, X.ꢀM. Wang, G.ꢀH. Tan, R.ꢀX. Rong, Z.ꢀ
R. Cao, H. Chen, P.ꢀZ. Zhang and X.ꢀL. Li, Chin. Chem. Lett.,
2014, 25, 1087.
UV-Vis and Fluorescence titrations: The titration experiments
were performed by varying the concentration of ctꢀDNA and
keeping the compound concentration constant (20 µM). All the
UV spectra were recorded after equilibration of solution for 5
min. Fluorescence titration were carried out on Cary Eclipse
Fluorescence Spectrophotometer at ambient temperature. A slit
width of 10 nm was used with λex = 400 nm. The titration
experiment was accomplished by varying the concentration of
DNA in cuvette (0.5ꢀ150 µM).
6
1
25
90
15. T. Mijatovic, T. Mahieu, C. Bruyère, N. De Nève, J. Dewelle, G.
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