Novel naphthalimide derivatives as potential apoptosis-inducing agents: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2009.07.011

A series of novel naphthalimide derivatives with flexible alkyl/aryl moieties were designed and synthesized. Their antitumor activities were evaluated against HeLa, A549, P388, HL-60, MCF-7, HCT-8 and A375 cancer cell lines in vitro. The preliminary resul

Full text of DOI:10.1016/j.ejmech.2009.07.011

European Journal of Medicinal Chemistry 44 (2009) 4674–4680
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel naphthalimide derivatives as potential apoptosis-inducing agents:
Design, synthesis and biological evaluation
*
Aibin Wu, Yufang Xu, Xuhong Qian , Jin Wang, Jianwen Liu
Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel naphthalimide derivatives with flexible alkyl/aryl moieties were designed and
synthesized. Their antitumor activities were evaluated against HeLa, A549, P388, HL-60, MCF-7, HCT-8
and A375 cancer cell lines in vitro. The preliminary results showed that most of the derivatives had
comparable antitumor activities over Amonafide with the IC50 values of 10ꢀ6 to 10ꢀ5 M. More impor-
tantly, flow cytometric analysis indicated that the derivatives could effectively induce G2/M arrest and
progress to apoptosis in HL-60 cell line after double staining with annexin V–FITC and propidium iodide.
The present work provided a novel class of naphthalimide-based derivatives with potent apoptosis-
inducing and antitumor activities for further optimization.
Received 17 April 2009
Received in revised form
30 May 2009
Accepted 3 July 2009
Available online 16 July 2009
Keywords:
Naphthalimide
Antitumor
Ó 2009 Elsevier Masson SAS. All rights reserved.
Cytotoxicity
G2/M arrest
Apoptosis
1. Introduction
apoptosis was also an evolutionarily conserved and highly regu-
lated process [10], which used to eliminate defective and unnec-
The design and synthesis of highly efficient antitumor agents
had attracted a lot of attention in organic and medicinal chemistry.
Naphthalimide-based anticancer drugs constituted an indispens-
able part in the development of antitumor agents. Although this
kind of chemotherapeutic agent was the mainstay for cancer
therapy for decades [1–4], the medical need was largely unmet due
to its unexpected toxicity such as Amonafide (1, Fig. 1), which
limited the use and development of this kind of drug [5]. Another
crucial limitation was that malignant stem cells could escape
treatment of this medicine [6]. These defects conferred cancer cells
resistance to therapeutic agents and made current anticancer
therapies less effective, leading ultimately to their failure [7]. So it
was necessary for us to attempt alternative structural modification
of naphthalimide to achieve favorable or different biological
activities.
essary cells [11]. It was recognized that dysfunction of the apoptosis
machinery was a hallmark of cancer [12–14], and induction of
apoptosis was arguably the most potent defense against cancer [5].
Therefore, much attention was paid to the design and discovery of
apoptotic inducers during recent years [15–34], and promising
results have been obtained in this way. Compounds 2 [5] and 3 [34]
were the representative reported apoptotic inducers (Fig. 1), and
had the characteristics of a conformational flexible aryl moiety.
These inspired us to assume that naphthalimide derivatives with
alkyl/aryl moiety linked by various chains might have some
improved or different biological activities.
As shown in Scheme 1, the naphthalimide scaffold was utilized
as key prototype structural unit, and the aliphatic amine and aryl
functional groups were conjugated to the naphthalene ring. Intro-
duction of basic hydrophilic amine chain to the position-2 of
naphthalimide ring might contribute to increase its cytotoxicity
against tumor cell lines [35]. The amino substituents introduced to
the position-6 of naphthalimide ring were difficult to be acetylated
[36] and might involve arrest of cell cycle [37]. Various linkers were
designed between naphthalimide scaffold and flexible aryl moiety
in order to investigate their effects on the biological activities.
Therefore, the target compounds 5a–c, 7a–e and 9a–b were
prepared, and their in vitro cytotoxicities and apoptosis-inducing
activities were also evaluated.
In parallel with the growth of knowledge concerning the side
effects of this medicine, the activation of apoptosis pathways has
become an alternative and promising method of cancer treatment
[8,9]. Apoptosis was a cellular process critical to normal develop-
ment and homeostasis of multicellular organisms [7]. Meanwhile,
* Corresponding author. Tel.: þ86 21 64253589; fax: þ86 21 64252603.
E-mail address: xhqian@ecust.edu.cn (X. Qian).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.07.011

A. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 4674–4680
4675
NO2
[34]. The structures of all the newly synthesized compounds were
well identified by 1H NMR, 13C NMR, HRMS and IR spectra.
O
N
H
N
NC
N
H
N
O
O
N
O
S
S
O2
2.2. Cytotoxic effects
The in vitro antitumor activities of the target compounds were
evaluated by examining their cytotoxic effects using sulforhod-
amine B (SRB) assay [39] against A549 and MTT tetrazolium dye
assay [40] against HeLa, P388, HL-60, MCF-7, HCT-8 and A375,
HN
NH2
N
N
Amonafide,1
S
3
respectively. The IC50 represented the drug concentration (mM)
required to inhibit cell growth by 50% and the results were
summarized in Table 1.
2
Ph
Ph
From Table 1, it could be seen that the target compounds
showed comparable cytotoxicities over Amonafide against tested
cancer cell lines except for HCT-8 with the IC50 values of 10ꢀ6 to
10ꢀ5 M. Compounds 5b and 7a exhibited the highest cytotoxicities
against HeLa, A549 and P388, MCF-7 cell lines among each group
Fig. 1. The structures of some reported compounds.
2. Results and discussion
2.1. Chemistry
with IC50 values of 6.06, 1.27 and 4.53, 8.65 mM, respectively. For
A375 and HL-60 cell lines, the highest cytotoxic compounds were
7d and 5a, respectively. Among them, the IC50 values of compounds
5a and 5b were 2.1 and 10.2-fold lower than the values found for
Amonafide against HL-60 and A549 cell lines, respectively. In most
cases, the cytotoxicities increased in sequence of 9, 7 and 5, which
indicated that the magnitude and conformation of alkyl/aryl
substituents had intense influence on the cytotoxic activities of
these compounds. For example, compound 7a was more cytotoxic
than compound 9b but less cytotoxic than compound 5b. Moreover,
the linkers in position-6 influenced its cytotoxicity. We found that
compounds 5c and 9a bearing flexible alkyl linker showed
a striking contrast compared to compound 7e bearing semi-rigid
piperazin linker. Additionally, the cytotoxic activities of compounds
7a–d were also influenced by the substituents on aryl moiety.
The synthetic routes of the designed compounds 2-(2-(dime-
thylamino)ethyl)-6-(substituted amino)-1H-benzo[de]isoquino-
line-1,3(2H)-dione derivatives 5a–c, 7a–e and 9a–b were shown in
Scheme 1. 6-Bromo-2-(2-(dimethylamino)ethyl)-1H-benzo[de]iso-
quinoline-1,3(2H)-dione 4 [38] was treated with various aliphatic
amines in CH3OCH2CH2OH, affording the target compounds 5a–c
with moderate yields of 40–60% and intermediates 6 and 8,
respectively. The subsequent nucleophilic addition/reduction
reaction of 6 and 8 with substituted cinnamaldehyde led to the
target compounds 7a–d and 9b with satisfied yields of 85–90%.
Compounds 7e and 9a were prepared by the condensation reaction
of 6 and 8 with 4-(4-(3,3-diphenylallyl)piperazin-1-yl)benzoic acid
N
N
N
N
N2
O
O
N
O
1
O
O
N
O
O
N
O
3
6
4
b,c
a
a
5
Br
N
N
N
R1 R2
4
N
H
N
5a-5c
R3
6
a
7a-7e
R5
N
O
O
H2N
NH
NH
R4
b,c
N
O
N
O
N
N
8
9a-9b
3
: R1 = -H; R2 = -CH2-2-thiophene
5b : R1 , R2 = -CH2CH2SCH2CH2-
7a
5a
: R =-CH2CH=C(C6H5)2
7b : R3=-CH2CH=CH(C6H4-p-CN)
3
: R1 = -H; R2 = -CH2CH2SC6H5
7c
5c
: R =-CH2CH=CH(C6H4-p-N(CH3)2)
9a : R4 = -H; R5 = -COC6H4-p-N(CH2CH2)2NCH2CH=C(C6H5)2
7d : R3=-CH2CH=CHC6H5
: R4 = R5 = -CH2CH=C(C6H5)2
7e : R3=-COC6H4-p-N(CH2CH2)2NCH2CH=C(C6H5)2
9b
Scheme 1. Reagents and conditions: (a) corresponding amine, HOCH2CH2OCH3, reflux 3 h, 40–88% yield; (b) corresponding cinnamaldehyde, NaBH(OAc)3, ClCH2CH2Cl, r.t. 3 h,
85–90% yield; (c) 4-(4-(3,3-diphenylallyl) piperazin-1-yl)benzoic acid, CHCl3, EDCI, DMAP, r.t. 48 h, 60% yield.

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A. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 4674–4680
Table 1
Table 2
Cytotoxicities of naphthalimide derivatives against HeLa, A549, P388, HL-60, MCF-7,
HCT-8 and A375 cell lines.
Cell cycle distribution of HL-60 cell line in the presence of IC50 concentration of the
target compounds.
Compound Cytotoxicity (IC50
HeLa A549
1.40 13.00
,
m
M)
P388
4.56
Compound
Concentration (
m
M)
Cell cycle distribution (%)
HL-60
MCF-7
11.88
13.02 >50
16.04
16.37 >50
8.65 >50
16.77 >50
HCT-8
9.26
A375
7.51
46.10
11.45
6.66
11.93
7.41
>50
6.62
>50
G1
S
G2/M
sub-G1
Amonafide
17.96
8.55
16.99
37.63
22.09
10.86
Control
Amonafide
0
18
9
52.86
52.26
54.34
30.64
55.52
52.36
37.07
34.93
40.33
52.04
51.15
66.73
33.86
36.68
27.76
42.54
27.70
32.68
29.84
51.34
41.06
25.34
32.41
0.3
13.29
11.06
17.90
26.81
16.78
14.96
33.08
13.73
18.61
22.62
16.44
32.97
0.72
2.69
5a
5b
5c
7a
7b
7c
7d
7e
9a
9b
12.59
6.06
6.89
7.64 >50
6.89 11.73
>50 4.88
11.4 >50
>50
7.19
36.01
6.24
1.27 >50
3.37 7.23
26.71
43.79
5a
5b
5c
7a
7b
7c
7d
7e
9a
9b
11.70
13.20
10.31
20.02
12.41
18.77
14.52
13.20
17.98
17.31
17
38
22
11
50
12
50
9
4.53
6.61
11.29 >50
9.12
13.81 >50
>50
>50
12.21 >50
20.68 >50
21.74 >50
12.21
25.58
36.25
22.01
7.67
24.45
8.65
31.66 >50
6.81
36.90
>50
32
All data are expressed as means from three separate determinations. IC50 values
were given only if they were less than 50
tration tested.
mM, which was the maximum concen-
3. Conclusion
When the substituents were phenyl and nitrile groups, relatively
favorable cytotoxic activities were obtained. Therefore, manipula-
tion of naphthalimide derivatives to achieve potent antitumor
activities, seemed to be closely dependent on structures and
conformation of the aryl groups and the linkers, which played
important roles in the chemical and biological functions [41].
In summary, a series of novel naphthalimide derivatives with
flexible alkyl/aryl moiety were designed and synthesized, and their
antitumor activities were evaluated against a variety of cancer cell
lines in vitro. The preliminary results showed that most of the
derivatives had comparable antitumor activities over Amonafide
with the IC50 values of 10ꢀ6 to 10ꢀ5 M. More importantly, flow
cytometric analysis indicated that these derivatives could effectively
induce G2/M arrest and progress to apoptosis in HL-60 cell line after
double staining with annexin V and propidium iodide. This sug-
gested that the target compounds might inhibit the growth of HL-60
cell line by induction of apoptosis. The present work demonstrated
that incorporating the biological active unit of naphthalimide and
flexible alkyl/aryl moiety might be able to result in a novel class of
lead compounds with potential apoptosis-inducing and antitumor
activities. Further structural optimization and detailed biological
studies on the molecular mechanism of action about the designed
naphthalimide derivatives were under the way.
2.3. Cell cycle profile and apoptosis in HL-60 cells
In the search of a possible mechanism of action responsible for
antitumor activity of the target compounds, we have investigated their
effects on the cell cycle by fluorescence activated cell sorting (FACS)
[17–19,30,31]. HL-60 cell line was used in the assay. Cells were treated
with these compounds, and after 24 h were fixed and labeled with
propidium iodide. The different phases of cell cycle were analyzed by
flow cytometry and the results were summarized in Table 2.
As shown in Table 2, the target compounds were found to
effectively induce G2/M arrest and progress to apoptosis in HL-60
cell line [42]. After incubation with IC50 concentration of these
compounds, the sub-G1 portions were increased from 9.59% to
19.30% or from 7.62% to 17.33% versus the untreated control or the
control treated with Amonafide, respectively. Meanwhile, the G2/M
population increased from 13.29% in the control to 13.73–33.08% in
cells treated with the target compounds. Moreover, the compounds
treatment HL-60 cell line could notably induce morphological
changes into rounding form (data not listed), which was a primary
indication of apoptosis and cell death [21]. The results in Tables 1
and 2 indicated that there was no obvious relationship between
cytotoxicity and apoptosis-inducing activity. Cell cycle distribution
of HL-60 cell line in the presence of representative compounds 5b,
7b and 7e were displayed in Fig. 2.
4. Experimental protocols
All reagents were of the commercial quality and were used
without purification. 1H and 13C NMR were obtained with a Bruker
AV-400 spectrometer with chemical shifts reported as ppm (in
CDCl3/DMSO-d6/CD3COCD3, TMS as internal standard). IR was
obtained using a Perkin–Elmer 2000 FTIR instrument. High-reso-
lution mass spectra (HRMS) were obtained on a HPLC-Q-Tof MS
(Micro) spectrometer. Melting points were determined by an X-6
micro-melting point apparatus and uncorrected. Column chroma-
tography was performed using silica gel 200–300 mesh.
To further verify their pro-apoptotic function, FACS analysis was
carried out after double staining cells with propidium iodide and
annexin V–FITC [7,21,29]. As shown in Fig. 3, representative
compound 5b was very effective in induction of apoptosis in a dose–
4.1. Synthesis of naphthalimide derivatives
4.1.1. General procedure for the synthesis of 6, 8 and 5a–5c
Compound 4 (50 mg, 0.144 mmol), prepared by using the
previously reported method [38], was dissolved in ethylene glycol
monomethyl ether (2 mL), then piperazin or corresponding amine
(1.44 mmol) was added. The solution was stirred and refluxed
under nitrogen for 3 h, cooled, and concentrated under vacuum.
The residue was subjected to column chromatography on silica gel.
Compounds 6, 8 and 5a–5c were separated with CH2Cl2/CH3OH
50:1 (v/v) as orange solids, respectively.
dependent manner. TreatmentofHL-60cell lineby 4.3, 8.5 and17 mM
of5b for36 h resulted in 48.92%, 85.05%and90.88% of apoptoticcells,
respectively, as compared to 2.84% of apoptotic cells in an untreated
control. Also, compounds 7b and 7e had significant effects on
induction of apoptosis, and the data were summarized in Table 3.
(Early apoptosis corresponded to annexin V single-positive cells and
late apoptosis/necrosis corresponded to double-positive cells.) These
results collectively suggested that the target compounds might
inhibit the growth of HL-60 cell line by induction of apoptosis.
Detailed interaction mechanism, structure–antitumor activity rela-
tionships and further biological experiments to distinguish late
apoptosis from necrosis, were well under way in our laboratory.
4.1.1.1. 2-(2-(Dimethylamino)ethyl)-6-(piperazin-1-yl)-1H-benzo[de]
isoquinoline-1,3(2H)-dione (6). Orange solid. Yield: 88%; m.p.
99.1–101.1 ꢁC; 1H NMR (CDCl3, 400 MHz) dH: 8.26–8.22 (m, 2H,
Ar–H), 8.18 (d, J ¼ 8.0 Hz,1H, Ar–H), 7.62 (t, J ¼ 8.0 Hz,1H, Ar–H), 7.10

A. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 4674–4680
4677
Fig. 2. Cell cycle distribution of HL-60 cell line in the presence of representative compounds 5b, 7b and 7e, respectively. HL-60 cell line was treated with IC50 concentration of the
compounds or with 0.1% DMSO. Cells were harvested, fixed and stained with propidium iodide. 20,000 stained cells were then subjected to FACScalibur analysis to determine the
distribution of cells.
(d, J ¼ 8.0 Hz, 1H, Ar–H), 4.02 (t, J ¼ 6.8 Hz, 2H, NCH2), 3.04 (s, 4H,
CH2NCH2), 2.97 (s, 4H, CH2NHCH2), 2.41 (t, J ¼ 6.8 Hz, 2H,
CH2N(CH3)2), 2.16 (s, 6H, N(CH3)2); MS (EI) m/z: 352.2 (M)þ.
148.76, 140.28, 134.17, 131.13, 129.52, 127.21, 126.14, 126.08, 125.37,
124.80, 122.88, 120.28, 110.87, 104.84, 57.22, 45.69, 42.94, 37.60;
FTIR (KBr, cmꢀ1): 3288, 2940, 2770, 1673, 1643, 1580, 1547, 1387,
1365, 1320, 1287, 1238, 1120, 1090, 774, 693; HRMS (ESþ) calcd for
C21H22N3O2S ([M þ H])þ 380.1433, found 380.1404.
4.1.1.2. 6-(6-Aminohexylamino)-2-(2-(dimethylamino)ethyl)-1H-ben-
zo[de]isoquinoline-1,3(2H)-dione (8). Orange solid. Yield: 60%; m.p.
141.9–143.9 ꢁC; 1H NMR (CDCl3, 400 MHz) dH: 8.56 (d, J ¼ 7.2 Hz,
1H, Ar–H), 8.44 (d, J ¼ 8.4 Hz, 1H, Ar–H), 8.09 (d, J ¼ 8.0 Hz, 1H, Ar–
H), 7.60 (t, J ¼ 8.0 Hz, 1H, Ar–H), 6.70 (d, J ¼ 8.8 Hz, 1H, Ar–H), 5.35
(s, br, 1H, ArNH), 4.32 (t, J ¼ 7.2 Hz, 2H, NCH2), 3.41 (q, J1 ¼6.8 Hz,
J2 ¼12.4 Hz, 2H, ArNHCH2), 2.75 (t, J ¼ 6.4 Hz, 2H, CH2N), 2.66 (t,
J ¼ 6.4 Hz, 2H, CH2N(CH3)2), 2.38 (s, 6H, N(CH3)2), 1.84–1.83 (m, 2H,
CH2CH2N), 1.56–1.43 (m, 6H, CH2CH2CH2); MS (EI) m/z: 382.2 (M)þ.
4.1.1.4. 2-(2-(Dimethylamino)ethyl)-6-thiomorpholino-1H-benzo[de]i-
soquinoline-1,3(2H)-dione (5b) [43]. Orange yellow solid. Yield: 40%;
m.p. 122.5–124.5 ꢁC; 1H NMR (CDCl3, 400 MHz) dH: 8.58 (d,
J ¼ 6.8 Hz, 1H, Ar–H), 8.51 (d, J ¼ 8.0 Hz, 1H, Ar–H), 8.36 (d,
J ¼ 8.4 Hz,1H, Ar–H), 7.70 (t, J ¼ 7.6 Hz, 1H, Ar–H), 7.23 (d, J ¼ 8.0 Hz,
1H, Ar–H), 4.33 (t, J ¼ 6.8 Hz, 2H, CH2N), 3.50 (s, 4H, CH2NCH2), 2.97
(s, 4H, CH2SCH2), 2.69 (t, J ¼ 2.4 Hz, 2H, CH2N(CH3)2), 2.39 (s, 6H,
N(CH3)2); 13C NMR (CDCl3, 100 MHz) dC: 164.43, 163.95, 156.74,
132.48, 131.23, 129.99, 129.89, 126.55, 125.93, 123.24, 117.18, 115.95,
56.88, 55.55, 45.58, 37.79, 28.14; FTIR (KBr, cmꢀ1): 2970, 2911, 2814,
2762, 1691, 1654, 1584, 1509, 1446, 1391, 1339, 1283, 1231, 1127,
1038, 949, 852, 778, 756; HRMS (ESþ) calcd for C20H24N3O2S
([M þ H])þ 370.1589, found 370.1588.
4.1.1.3. 2-(2-(Dimethylamino)ethyl)-6-(thiophene-2-ylmethylamino)-
1H-benzo[de]isoquinoline-1,3(2H)-dione (5a). Orange solid. Yield:
60%; m.p. 154.9–156.9 ꢁC; 1H NMR (CDCl3, 400 MHz) dH: 8.46 (d,
J ¼ 7.6 Hz, 1H, Ar–H), 8.35 (d, J ¼ 8.4 Hz, 1H, Ar–H), 8.06 (d,
J ¼ 8.4 Hz,1H, Ar–H), 7.53 (t, J ¼ 8.0 Hz,1H, Ar–H), 7.27 (d, J ¼ 4.4 Hz,
1H, thiophene-H), 7.11 (d, J ¼ 3.2 Hz, 1H, thiophene-H), 7.02–7.00
(m, 1H, thiophene-H), 6.70 (d, J ¼ 8.4 Hz, 1H, Ar–H), 5.98 (t,
J ¼ 5.2 Hz, 1H, Ar–NH), 4.76 (d, J ¼ 5.2 Hz, 2H, NHCH2-thiophene),
4.34 (t, J ¼ 6.8 Hz, 2H, CH2N), 2.74 (t, J ¼ 6.8 Hz, 2H, CH2N(CH3)2),
2.42 (s, 6H, N(CH3)2); 13C NMR (CDCl3, 100 MHz) dC: 164.63, 164.07,
4.1.1.5. 2-(2-(Dimethylamino)ethyl)-6-(2-(phenylthio)ethylamino)-
1H-benzo[de]isoquinoline-1,3(2H)-dione (5c). Orange red solid.
Yield: 55%; m.p. 142.3–144.3 ꢁC; 1H NMR (CDCl3, 400 MHz) dH: 8.54
(d, J ¼ 7.2 Hz, 1H, Ar–H), 8.39 (d, J ¼ 8.4 Hz, 1H, Ar–H), 7.94

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A. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 4674–4680
Fig. 3. Compound 5b induced apoptosis of HL-60 cell line. HL-60 cell line was treated with 4.3, 8.5 and 17.0
mM of 5b or with vehicle solvent (0.1% DMSO) for 36 h and stained with
annexin V–FITC and propidium iodide. Stained cells then were subjected to FACScalibur analysis to determine the distribution of cells.
(d, J ¼ 8.4 Hz, 1H, Ar–H), 7.53 (t, J ¼ 8.0 Hz, 1H, Ar–H), 7.47 (d,
J ¼ 7.2 Hz, 2H, Ar–H), 7.35–7.29 (m, 3H, Ar–H), 6.57 (d, J ¼ 8.4 Hz,
1H, Ar–H), 5.91 (s, br, 1H, Ar–NH), 4.44 (t, J ¼ 6.8 Hz, 2H, CH2N), 3.60
(q, J1 ¼6 Hz, J2 ¼12 Hz, 2H, NHCH2), 3.33 (t, J ¼ 6 Hz, 2H,
CH2N(CH3)2), 3.02 (s, br, 2H, CH2S), 2.64 (s, 6H, N(CH3)2); 13C NMR
(CDCl3, 100 MHz) dC: 164.91, 164.10, 134.57, 134.34, 131.33, 130.76,
129.91, 129.32, 127.15, 124.71, 120.40, 104.15, 56.14, 44.27, 42.43,
33.05, 29.69; FTIR (KBr, cmꢀ1): 3296, 3051, 2918, 2844, 1680, 1632,
1576, 1547, 1394, 1361, 1290, 1246, 1179, 1112, 771, 752, 698; HRMS
(ESþ) calcd for C24H26N3O2S ([M þ H])þ 420.1746, found 420.1730.
phase was washed twice with saturated NaCl solution and dried
over sodium sulfate and evaporated to dryness. The residue was
purified on silica gel column chromatography by using CH2Cl2/
CH3OH 50:1 (v/v) as the eluent, affording 7a–7d and 9b as bright
yellow–khaki solids.
4.1.2.1. 2-(2-(Dimethylamino)ethyl)-6-(4-(3,3-diphenylallyl)piperazin-
1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (7a). Bright yellow
solid. Yield: 90%; m.p. 160.6–161.9 ꢁC; 1H NMR (CDCl3, 400 MHz)
dH: 8.57 (d, J ¼ 7.2 Hz, 1H, Ar–H), 8.52 (d, J ¼ 8.0 Hz, 1H, Ar–H), 8.37
(d, J ¼ 8.4 Hz, 1H, Ar–H), 7.67 (t, J ¼ 8.0 Hz, 1H, Ar–H), 7.45–7.37
(m, 3H, Ar–H), 7.29–7.28 (m, 5H, Ar–H), 7.23–7.22 (m, 3H, Ar–H),
6.29 (t, J ¼ 6.8 Hz, 1H, C]CH), 4.33 (t, J ¼ 7.2 Hz, 2H, CH2N), 3.31
(s, 4H, CH2NCH2), 3.25 (d, J ¼ 6.8 Hz, 2H, CHCH2N), 2.79 (s, 4H,
CH2NCH2), 2.66 (t, J ¼ 7.2 Hz, 2H, CH2N(CH3)2), 2.37 (s, 6H,
N(CH3)2); 13C NMR (CDCl3, 100 MHz) dC: 164.52, 164.03, 155.98,
144.64, 141.91, 139.57, 132.61, 131.11, 130.29, 129.94, 129.80, 128.27,
128.20, 127.43, 127.32, 127.27, 126.18, 125.57, 123.23, 116.68, 114.93,
4.1.2. General procedure for the synthesis of 7a–7d and 9b
Compound 6/8 (0.142 mmol) was dissolved in 1,2-dichloro-
ethane (2 mL) in the presence of NaBH(OAc)3 (36 mg, 0.171 mmol),
then the corresponding cinnamaldehyde (0.171 mmol) was added.
The solution was stirred at room temperature under nitrogen for
3 h, then diluted with saturated NaHCO3 solution (15 mL) and
extracted twice with ethyl acetate (30 mL). The combined organic
Table 3
Apoptosis data of representative compounds 7b and 7e treatment HL-60 cells after double staining with annexin V–FITC and propidium iodide.
Compound
Concentration (
mM)
Viable cells (%)
Early apoptosis (%)
Late apoptosis and necrosis (%)
7b
0
95.33
28.11
0.28
0.51
64.60
70.49
18.43
3.92
5.98
27.68
79.03
2.7
5.5
11.0
1.16
7e
0
95.33
49.16
0.34
0.51
43.69
79.60
40.18
3.92
5.71
18.64
58.07
12.5
25.0
50.0
0.16
Extent of apoptosis was measured through annexin V–FITC apoptosis detection kit (Invitrogen, USA) as described by the manufacture’s instruction. Early apoptosis corre-
sponded to annexin V single-positive cells and late apoptosis/necrosis corresponded to double-positive cells.

A. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 4674–4680
4679
57.28, 56.99, 53.21, 53.10, 45.70, 37.95; FTIR (KBr, cmꢀ1): 3422,
2938, 2814, 2762, 1688, 1647, 1584, 1450, 1383, 1235, 1135, 1045,
786, 756, 696; HRMS (ESþ) calcd for C35H37N4O2 ([M þ H])þ
545.2917, found 545.2917.
J ¼ 5.2 Hz, 1H, Ar–NH), 6.78 (d, J ¼ 8.8 Hz, 1H, Ar–H), 6.17 (d,
J ¼ 6.8 Hz, 2H, 2C]CH), 4.23 (t, J ¼ 6.8 Hz, 2H, CH2N), 3.45 (q,
J ¼ 6.4 Hz,, 2H, Ar–NHCH2), 3.18 (d, J ¼ 6.8 Hz, 4H, 2NCH2CH), 2.56
(t, J ¼ 6.8 Hz, 2H, CH2N(CH3)2), 2.45 (d, J ¼ 6.8 Hz, 2H,
CH2N(CH2CH])2), 2.27 (s, 6H, N(CH3)2), 1.83–1.76 (m, 2H, CH2),
1.62–1.59 (m, 2H, CH2), 1.49–1.43 (m, 4H, CH2CH2); 13C NMR
(CD3COCD3, 100 MHz) dC: 164.01, 163.24, 150.40, 143.36, 142.36,
139.74, 134.08, 130.48, 129.90, 129.74, 128.13, 128.09, 127.37, 127.17,
127.10, 124.20, 122.82, 120.52, 109.06, 103.78, 57.02, 53.79, 52.67,
45.16, 43.35, 37.44, 31.77, 27.00, 26.88, 24.76, 22.47, 13.52; FTIR
(KBr, cmꢀ1): 2925, 2844, 1680, 1643, 1580, 1535, 1439, 1361, 1342,
1242, 1123, 767, 696; HRMS (ESþ) calcd for C52H55N4O2 ([M þ H])þ
767.4325, found 767.4337.
4.1.2.2. (E)-4-(3-(4-(2-(2-(Dimethylamino)ethyl)-1,3-dioxo-2,3-dihy-
dro-1H-benzo[de]isoquinolin-6-yl)piperazin-1-yl)prop-1-enyl)benzo-
nitrile (7b). Orange solid. Yield: 90%; m.p. 128.2–130.2 ꢁC; 1H NMR
(CDCl3, 400 MHz) dH: 8.58 (d, J ¼ 7.2 Hz, 1H, Ar–H), 8.52 (d,
J ¼ 8.0 Hz, 1H, Ar–H), 8.41 (d, J ¼ 8.4 Hz, 1H, Ar–H), 7.69 (t,
J ¼ 8.0 Hz, 1H, Ar–H), 7.62 (d, J ¼ 8.4 Hz, 2H, Ar–H), 7.49 (d,
J ¼ 8.4 Hz, 2H, Ar–H), 7.23 (d, J ¼ 8.0 Hz, 1H, Ar–H), 6.64 (d,
J ¼ 16.0 Hz, 1H, C]CH), 6.51–6.44 (m, 1H, Ar–H), 4.33 (t, J ¼ 7.2 Hz,
2H, CH2N), 3.36–3.33 (m, 6H, CHCH2N and CH2NCH2), 2.85 (s, 4H,
CH2NCH2), 2.67 (t, J ¼ 7.2 Hz, 2H, CH2N(CH3)2), 2.37 (s, 6H,
N(CH3)2); 13C NMR (CDCl3, 100 MHz) dC: 164.50, 164.01, 155.84,
141.22, 132.59, 132.47, 131.62, 131.15, 130.59, 130.21, 129.95, 126.82,
126.19, 125.64, 123.28, 118.85, 116.83, 114.97, 110.93, 60.73, 56.96,
53.32, 53.02, 45.66, 37.93; FTIR (KBr, cmꢀ1): 2933, 2814, 2762,
2214, 1695, 1651, 1584, 1569, 1450, 1368, 1231, 1131, 1038, 975, 778;
HRMS (ESþ) calcd for C30H32N5O2 ([M þ H])þ 494.2556, found
494.2542.
4.1.3. General procedure for the synthesis of 7e and 9a
To a solution of compound 6/8 (0.142 mmol) in CHCl3 (2 mL), 4-(4-
(3,3-diphenylallyl)piperazin-1-yl)benzoic acid (68 mg, 0.170 mmol),
prepared by using the previously reported method [27,34,44], EDCI
(65 mg, 0.340 mmol) and DMAP (41 mg, 0.340 mmol) were added.
The solutionwas stirred at room temperature under nitrogen for 48 h
and then concentrated. The residue was purified on silica gel column
chromatography by using CH2Cl2/CH3OH 50:1 (v/v) as the eluent,
affording 7e/9a as bright yellow–orange red solids.
4.1.2.3. (E)-2-(2-(Dimethylamino)ethyl)-6-(4-(3-(4-(dimethylamino)-
phenyl)allyl)piperazin-1-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(7c). Orange red solid. Yield: 85%; m.p. 125.7–127.7 ꢁC; 1H NMR
(CDCl3, 400 MHz) dH: 8.58 (dd, J1 ¼7.2 Hz, J2 ¼ 0.8 Hz, 1H, Ar–H),
8.52 (d, J ¼ 8.0 Hz, 1H, Ar–H), 8.42 (dd, J1 ¼8.4 Hz, J2 ¼ 0.8 Hz, 1H,
Ar–H), 7.68 (q, J1 ¼7.6 Hz, J2 ¼ 0.8 Hz, 1H, Ar–H), 7.32 (d, J ¼ 8.8 Hz,
2H, Ar–H), 7.22 (d, J ¼ 8.0 Hz, 1H, Ar–H), 6.70 (d, J ¼ 8.8 Hz, 2H, Ar–
H), 6.52 (d, J ¼ 15.6 Hz, 1H, C]CH), 6.16–6.09 (m, 1H, Ar–H), 4.33 (t,
J ¼ 7.2 Hz, 2H, CH2N), 3.33–3.29 (m, 6H, CHCH2N and CH2NCH2),
2.97 (s, 6H, Ar–N(CH3)2), 2.84 (s, br, 4H, CH2NCH2), 2.66 (t,
J ¼ 7.2 Hz, 2H, CH2N(CH3)2), 2.37 (s, 6H, N(CH3)2); 13C NMR (CDCl3,
100 MHz) dC: 164.54, 164.04, 150.19, 133.63, 132.64, 131.11, 130.37,
129.96, 127.31, 126.18, 125.56, 125.25, 123.22, 121.35, 116.61, 114.92,
112.45, 61.34, 56.97, 53.12, 53.09, 45.68, 40.49, 37.94; FTIR (KBr,
cmꢀ1): 2933, 2807, 2755, 1688, 1654, 1584, 1521, 1446, 1346, 1231,
1116, 1042, 964, 782, 756; HRMS (ESþ) calcd for C31H38N5O2
([M þ H])þ 512.3026, found 512.3026.
4.1.3.1. 2-(2-(Dimethylamino)ethyl)-6-(4-(4-(4-(3,3-diphenylallyl)
piperazin-1-yl)benzoyl)piperazin-1-yl)-1H-benzo[de]isoquinoline-
1,3(2H)-dione (7e). Bright yellow solid. Yield: 60%; m.p. 99.5–
101.5 ꢁC; 1H NMR (CDCl3, 400 MHz) dH: 8.61 (d, J ¼ 7.2 Hz, 1H,
Ar–H), 8.54 (d, J ¼ 8.0 Hz, 1H, Ar–H), 8.43 (d, J ¼ 8.4 Hz, 1H, Ar–H),
7.73 (t, J ¼ 8.0 Hz, 1H, Ar–H), 7.43–7.34 (m, 5H, Ar–H), 7.32–7.28 (m,
5H, Ar–H), 7.25–7.22 (m, 1H, Ar–H), 7.18 (d, J ¼ 6.8 Hz, 2H, Ar–H),
6.91 (d, J ¼ 8.8 Hz, 2H, Ar–H), 6.26 (t, J ¼ 7.2 Hz, 1H, C]CH), 4.33 (t,
J ¼ 7.2 Hz, 2H, CH2N), 3.98 (s, 4H, CH2NCH2), 3.30–3.27 (m, 8H,
N(CH2CH2)2N), 3.15 (d, J ¼ 6.8 Hz, 2H, CHCH2N), 2.67 (t, J ¼ 7.2 Hz,
2H, CH2N(CH3)2), 2.61 (s, 4H, CH2NCH2), 2.37 (s, 6H, N(CH3)2); 13C
NMR (CDCl3, 100 MHz) dC: 170.94, 164.40, 163.94, 155.28, 153.44,
144.55, 141.89, 139.53, 132.44, 131.28, 129.96, 129.84, 129.76, 129.18,
128.25, 128.19, 127.41, 127.28, 127.25, 126.28, 126.03, 125.84, 124.96,
123.87,117.52,115.38, 114.57, 57.28, 56.92, 53.23, 52.92, 48.19, 45.72,
38.00; FTIR (KBr, cmꢀ1): 2933, 2814, 1695, 1654, 1584, 1509, 1387,
1368, 1279, 1235, 1135, 1019, 786, 756, 696; HRMS (ESþ) calcd for
C46H49N6O3 ([M þ H])þ 733.3866, found 733.3851.
4.1.2.4. (E)-6-(4-Cinnamylpiperazin-1-yl)-2-(2-(dimethylamino)ethyl)-
1H-benzo[de]isoquinoline-1,3(2H)-dione (7d). Khaki solid. Yield:
85%; m.p. 164.6–166.6 ꢁC; 1H NMR (CDCl3, 400 MHz) dH: 8.58 (dd,
J1 ¼7.6 Hz, J2 ¼ 0.8 Hz, 1H, Ar–H), 8.52 (d, J ¼ 8.0 Hz, 1H, Ar–H), 8.42
(dd, J1 ¼8.8 Hz, J2 ¼ 0.8 Hz, 1H, Ar–H), 7.69 (q, J1 ¼8.4 Hz,
J2 ¼ 0.8 Hz, 1H, Ar–H), 7.42 (d, J ¼ 7.2 Hz, 2H, Ar–H), 7.36–7.32 (m,
2H, Ar–H), 7.28–7.22 (m, 2H, Ar–H), 6.62 (d, J ¼ 16 Hz, 1H, C]CH),
6.38–6.31 (m,1H, Ar–H), 4.33 (t, J ¼ 7.2 Hz, 2H, CH2N), 3.34–3.32 (m,
6H, CHCH2N and CH2NCH2), 2.85 (s, br, 4H, CH2NCH2), 2.66 (t,
J ¼ 7.2 Hz, 2H, CH2N(CH3)2), 2.37 (s, 6H, N(CH3)2); 13C NMR (CDCl3,
100 MHz) dC: 164.52, 164.03, 155.98, 136.77, 133.52, 132.62, 131.12,
130.31, 129.95, 128.62, 127.66, 126.36, 126.18, 126.08, 125.59, 123.24,
116.69, 114.94, 61.04, 56.97, 53.21, 53.07, 45.68, 37.94; FTIR (KBr,
cmꢀ1): 2962, 2807, 2762, 1688, 1651, 1584, 1509, 1450, 1391, 1365,
1246, 1120, 1042, 971, 782, 760, 693; HRMS (ESþ) calcd for
C29H33N4O2 ([M þ H])þ 469.2604, found 469.2600.
4.1.3.2. N-(6-(2-(2-(Dimethylamino)ethyl)-1,3-dioxo-2,3-dihydro-1H-
benzo[de]isoquinolin-6-ylamino)hexyl)-4-(4-(3,3-diphenylallyl)piper-
azin-1-yl)benzamide (9a). Orange red solid. Yield: 60%; m.p. 85.6–
87.6 ꢁC; 1H NMR (DMSO-d6, 400 MHz) dH: 8.69 (d, J ¼ 8.4 Hz, 1H,
Ar–H), 8.40 (d, J ¼ 7.2 Hz, 1H, Ar–H), 8.23 (d, J ¼ 8.8 Hz, 1H, Ar–H),
8.14 (t, J ¼ 5.2 Hz, 1H, Ar–H), 7.74 (t, J ¼ 4.2 Hz, 1H, Ar–H), 7.70–7.62
(m, 3H, Ar–H), 7.41 (t, J ¼ 7.2 Hz, 2H, Ar–H), 7.35–7.22 (m, 4H, Ar–H),
7.21–7.19 (m, 2H, Ar–H), 7.14–7.12 (m, 2H, Ar–H), 6.88 (d, J ¼ 8.8 Hz,
2H, Ar–H), 6.72 (d, J ¼ 8.8 Hz, 1H, Ar–H), 6.18 (t, J ¼ 6.4 Hz, 1H,
C]CH), 4.10 (t, J ¼ 7.2 Hz, 2H, CH2N), 3.35 (d, J ¼ 6.4 Hz, 2H,
CONHCH2), 3.24–3.21 (m, 6H, CH2NCH2 and NHCH2), 3.00 (d,
J ¼ 6.8 Hz, 2H, NCH2CH), 2.46–2.45 (m, 6H, CH2NCH2 and
CH2N(CH3)2), 2.18 (s, 6H, N(CH3)2), 1.71–1.65 (m, 2H, CH2), 1.55–1.51
(m, 2H, CH2), 1.50–1.42 (m, 4H, CH2CH2); 13C NMR (DMSO-d6,
100 MHz) dC: 166.24, 164.21, 163.32, 153.03, 151.13, 143.62, 142.04,
139.62, 134.77, 131.11, 129.92, 129.85, 129.08, 128.80, 128.75, 127.77,
127.37, 126.76, 124.62, 124.42, 122.26, 120.55, 113.95, 107.87, 104.19,
57.12, 52.98, 47.69, 45.82, 43.25, 29.73, 28.26, 26.83, 26.73; FTIR
(KBr, cmꢀ1): 3280, 3044, 2925, 2844, 1680, 1639, 1576, 1543, 1502,
1450, 1391, 1361, 1290, 1238, 1120, 1005, 771, 695; HRMS (ESþ)
calcd for C48H55N6O3 ([M þ H])þ 763.4336, found 763.4332.
4.1.2.5. 6-(6-(Bis(3,3-diphenylallyl)amino)hexylamino)-2-(2-(dimethy-
lamino)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (9b). Viscous
orange solid. Yield: 85%; m.p. 76.8–78.8 ꢁC; 1H NMR (CD3COCD3,
400 MHz) dH: 8.56 (dd, J1 ¼0.8 Hz, J2 ¼ 8.4 Hz, 1H, Ar–H), 8.46 (dd,
J1 ¼1.2 Hz, J2 ¼ 7.6 Hz, 1H, Ar–H), 8.34 (d, J ¼ 8.8 Hz, 1H, Ar–H),
7.61 (dd, J1 ¼1.2 Hz, J2 ¼ 8.4 Hz, 1H, Ar–H), 7.39–7.31 (m, 6H, Ar–
H), 7.26–7.22 (m, 6H, Ar–H), 7.17–7.13 (m, 8H, Ar–H), 6.97 (t,

4680
A. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 4674–4680
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (20536010, 20746003) and Program of
Shanghai Subject Chief Scientist and Shanghai Leading Academic
Discipline Project (B507) and the National High Technology
Research and Development Program of China (863 Program
2006AA10A201).
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