Novel naphthalimide-indomethacin hybrids as potential antitumor agents: Effects of linkers on hypoxic/oxic cytotoxicity and apoptosis-inducing activity

Article abstract of DOI:10.1007/s00706-010-0337-x

A series of novel naphthalimide-indomethacin hybrids with different linkers were designed and synthesized. Their antitumor activity was evaluated against HeLa, A549, P388, HL-60, MCF-7, HCT-8, and A375 cancer cell lines in vitro. Preliminary results showe

Full text of DOI:10.1007/s00706-010-0337-x

Monatsh Chem (2010) 141:893–899
DOI 10.1007/s00706-010-0337-x
ORIGINAL PAPER
Novel naphthalimide–indomethacin hybrids as potential antitumor
agents: effects of linkers on hypoxic/oxic cytotoxicity
and apoptosis-inducing activity
•
Aibin Wu Yufang Xu Xuhong Qian
•
Received: 13 January 2010 / Accepted: 29 May 2010 / Published online: 24 July 2010
Ó Springer-Verlag 2010
Abstract A series of novel naphthalimide–indomethacin
hybrids with different linkers were designed and synthe-
sized. Their antitumor activity was evaluated against HeLa,
A549, P388, HL-60, MCF-7, HCT-8, and A375 cancer cell
lines in vitro. Preliminary results showed that the hybrids
had moderate cytotoxic activity with 50% inhibition con-
centration (IC50) values of *10-5 M, and could effectively
induce apoptosis in HeLa cells. More importantly, the amide
derivatives had better cytotoxic and proapoptotic activity
than their ester counterparts, whereas the ester derivatives
had hypoxic preferred cytotoxicity and might be used as
promising candidates of prodrug in hypoxic tumor cells.
This work provides a novel class of naphthalimide–indo-
methacin hybrids with unique antitumor activity for further
optimization.
are specifically activated in tumor tissue to improve their
therapeutic index [3, 4]. However, the majority of clini-
cally used anticancer drugs are not truly selective for
cancer cells, and usually they have side-effects to some
extent, which made their therapy less effective, leading
ultimately to their failure [5]. It was, therefore, imperative
that innovative approaches be employed to circumvent
these defects. To fulfill this need, one strategy was the
development of hybrids (Fig. 1) composed of two antitu-
mor moieties with different mechanisms of action [6, 7].
These derivatives might carry a significant advantage in
cancer therapy because tumor growth is caused by multiple
mutations [8]. Therefore, activation of more than one sig-
naling pathway could frequently define cancerous cell
phenotype [9] and possibly augment the potency of both
compounds or reduce side-effects and drug resistance
development [10].
Keywords Apoptosis Á Cytotoxicity Á Hybrid Á
Indomethacin Á Naphthalimide
Naphthalimides, first discovered by Bran˜a and
co-workers [11, 12], are DNA-targeted chemotherapeutic
agents acting primarily by attacking DNA at some level
(synthesis, replication, or processing) [13]. Heretofore,
plenty of naphthalimide-based anticancer drugs have been
synthesized [9, 14–25], and promising results have been
obtained. Meanwhile, indomethacin is of particular inter-
est [26], being a member of the nonsteroidal anti-
inflammatory drugs, which are widely applied in treatment
of arthritis [27] and cardiovascular diseases [28], cancer
prevention [29, 30], etc. [31–35]. It has been proven that
indomethacin can induce G1 arrest and apoptosis of
human colorectal cancer cells by influencing the Wnt
signaling pathway [36], downregulate transcriptional
activity of the peroxisome proliferation-activated receptor
[37], and inhibit angiogenesis [29]. Based on the charac-
teristic structure of multitargeted drugs [9] and our
previously reported results [38], we speculated that
Introduction
The search for novel antitumor agents has been focused
on compounds that can induce apoptosis selectively in
malignant cells [1, 2], or that could be used as prodrugs that
A. Wu Á Y. Xu Á X. Qian (&)
Shanghai Key Laboratory of Chemical Biology,
School of Pharmacy, East China University of Science
and Technology, Shanghai 200237, People’s Republic of China
e-mail: xhqian@ecust.edu.cn
A. Wu
School of Chemistry and Environmental Engineering,
Yangtze University, Jingzhou 434023,
People’s Republic of China
123

894
A. Wu et al.
Cl
Fig. 1 Structures of some
reported hybrids
OH
HN
N
N
N
N
Cl
H
N
N
O
Cl
N
H
HO
O
indomethacin moiety
Results and discussion
Cl
O
N
The synthetic routes of the designed compounds 4a–4d are
shown in Scheme 1. 6-Bromobenzo[de]isochromene-1,3-
dione (1) was treated with dimethylamine in N,N-dimethyl-
formamide (DMF) catalyzed by CuSO4Á5H2O to afford
intermediate 2 [49] with satisfactory yield of 85%. Sub-
sequent nucleophilic substitution of 2 with corresponding
amines in EtOH led to key intermediates 3a–3d, which were
subjected to condensation with indomethacin, affording the
target compounds 4a–4d with moderate yields of 70–80%.
The structures of all newly synthesized compounds were
confirmed by using 1H nuclear magnetic resonance (NMR),
13C NMR, high-resolution mass spectroscopy (HRMS),
infrared (IR), and elemental analysis.
O
N
O
N
Linker
O
X
O
amide/ester moiety
naphthalimide moiety
Fig. 2 Design strategy of naphthalimide–indomethacin hybrids
naphthalimide–indomethacin hybrids with different linkers
might have improved or different biological activity.
As shown in Fig. 2, the naphthalimide scaffold was
utilized as the key prototype structural unit, and dimeth-
ylamine and indomethacin functional groups were
conjugated to the naphthalene ring. The dimethylamino
substituent introduced at position 6 of naphthalimide is
difficult to acetylate [39] and might be involved in DNA
synthesis arrest [40]. Various (hydrophobic or hydrophilic)
linkers were designed between naphthalimide and indo-
methacin in order to investigate their effects on biological
activity, which might lead to concomitant increase in
cytotoxicity against tumor cell lines [41–44]. Introduction
of an amide/ester moiety might result in different phar-
macologic profiles and optimal therapeutic window for
hybrids [45–48]. Therefore, four naphthalimide–indo-
methacin hybrids 4a–4d were prepared and their antitumor
activity evaluated against a variety of cancer cell lines.
Their hypoxic cytotoxicity and apoptosis-inducing activity
were also studied in this work.
The in vitro antitumor activity of the target compounds
was evaluated by examining their cytotoxic effects using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) tetrazolium dye assay [50]. IC50 represents
the drug concentration (lM) required to inhibit cell growth
by 50%, as summarized in Table 1.
As shown in Table 1, the hybrids had cytotoxicity better
than indomethacin and 3a against the cancer cell lines
tested. Under oxic condition, IC50 values were *10-5
M
against HeLa, HL-60, HCT-8, and A375 cell lines. For
A549, P388, and MCF-7 cell lines, there was no activity. It
was interesting that amide derivatives had better cytotoxic
activity than their ester counterparts, which indicated that
different kinds of compounds had important effects on
cytotoxicity. Furthermore, the length of the linker also
influenced its bioactivity. It was obvious that compounds
XH
Cl
O
O
O
O
O
O
O
O
N
O
O
N
a
b
c
N
N
O
X
O
Br
N
N
O
1
2
3a-3d
4a-4d
4b:
Reagents and conditions: (a) dimethylamine, CuSO4·5H2O, DMF, reflux 1.5 h, 85% yield;
(b) corresponding amine, EtOH, reflux 1.5 h, 85-90% yield; (c) indomethacin, EDCI, DMAP,
CHCl3, r.t. 48 h, 70-80% yield
4a:
X=-O-;
X=-O(CH2)2O-;
4c: X=-NH-; 4d: X=-NH(CH2)4-;
Scheme 1
123

Novel naphthalimide–indomethacin hybrids as potential antitumor agents
895
Table 1 Cytotoxicity of naphthalimide–indomethacin hybrids against HeLa, A549, P388, HL-60, MCF-7, HCT-8, and A375 cell lines
Compound
Cytotoxicity [IC50 (lM)]a
Oxic
Hypoxic
HeLa
HCRb (O2/N2)
HeLa
A549
P388
HL-60
MCF-7
HCT-8
A375
Indomethacin
74.5 10.1
87.3 10.5
57.3 6.9
76.3 11.5
13.9 2.5
24.4 2.9
NAc
NAc
NAc
NAc
NAc
NAc
NAc
NAc
NAc
NAc
NAc
NAc
63.2 9.5
50.5 7.7
47.4 6.6
57.4 8.6
20.3 3.1
24.7 3.0
NAc
NAc
NAc
NAc
NAc
NAc
58.7 7.9
39.2 4.9
27.4 3.3
37.2 5.6
47.4 7.1
29.6 3.6
92.6 13.9
82.1 12.6
64.3 7.7
68.7 10.3
44.2 6.6
26.9 3.2
NAc
NAc
–
3a
4a
4b
4c
4d
–
44.3 5.3
19.7 3.0
NAc
1.3
3.9
–
NAc
–
Cancer cell lines: A549 human lung cancer cell line, HeLa human cervical carcinoma cell line, P388 murine leukemia cell line, HL-60 human
promyelocytic leukemia cell line, MCF-7 human Caucasian breast adenocarcinoma cell line, HCT-8 human ileocecal adenocarcinoma cell line,
A375 human melanoma cell line
a
Cytotoxicity values are means of three experiments
b
HCR, hypoxic/oxic cytotoxicity ratio
c
NA, not active, IC50 [ 100 lM
Table 2 Cell-cycle distribution of HeLa cells in the presence of IC50 concentration of the hybrids
Compound
IC50 conc. (lM)
Cell cycle distribution (%)a
G1
S
G2/M
Sub-G1
Control
0
50
57
76
14
24
52.86 7.93
55.20 6.63
52.62 6.31
52.67 6.32
47.71 7.16
43.12 5.17
33.86 5.08
30.17 3.62
35.78 4.29
29.66 3.56
31.93 4.79
43.03 5.16
13.29 1.99
14.63 2.87
11.59 1.39
17.67 2.12
20.36 3.05
13.85 1.66
0.72 0.11
8.92 1.78
11.88 1.43
16.11 1.93
38.43 5.76
49.84 5.98
Indomethacin
4a
4b
4c
4d
a
Values are means of three experiments
4a and 4c with CH2CH2 linker had relatively lower activity
than compounds 4b and 4d with CH2CH2OCH2CH2 and
hexyl linker. More importantly, ester derivatives demon-
strated unique hypoxic preferred cytotoxicity compared
with their amide counterparts against HeLa cells. For
compounds 4a and 4b, the hypoxic/oxic cytotoxicity ratio
(HCR) was 1.3 and 3.0, respectively, whereas compounds
4c and 4d exhibited no hypoxic cytotoxicity. The results
indicated that the ester bond easily underwent a bioreduc-
tion process and that the molecule presented better hypoxic
cytotoxicity than with an amide bond [48]. It was also
demonstrated that different kinds of linkers could signifi-
cantly impact the bioactivity of the hybrids [42, 43].
Compound 4b had the best hypoxic cytotoxicity and might
be used as a promising candidate prodrug against hypoxic
tumor cells for further optimization.
As shown in Table 2, the hybrids were found to induce
apoptosis effectively in the HeLa cell line, better than
indomethacin. After incubation with the IC50 concentration
of these hybrids, the sub-G1 portions were increased from
0.72% in the control to 11.88–49.84% in cells treated with
the target compounds. Compound 4d was the most potent
apoptosis-inducing agent among the hybrids and could
induce 49.84% cell apoptosis. It was obvious that amide
derivatives 4c and 4d had better proapoptotic activity than
ester derivatives 4a and 4b, which might be attributed to
their better partitioning into the lipid phase [47] and dif-
ferent absorption, bioconversion, biodistribution, and
pharmacodynamic profiles [35]. Moreover, length of the
linker also influenced proapoptotic activity; for example,
compound 4d with a hexyl linker had better activity than
compound 4c with an ethyl linker. In addition, the results
in Tables 1 and 2 indicate that there was no obvious
relationship between cytotoxicity and apoptosis-inducing
activity.
To investigate the possible mechanism of action
responsible for antitumor activity of the hybrids, we tested
their effects on cell cycle by fluorescence-activated cell
sorting (FACS) [51]. The HeLa cell line was used in this
assay, and the results are summarized in Table 2.
To further confirm their proapoptotic function, FACS
analysis was carried out after double-staining cells with
123

896
A. Wu et al.
Fig. 3 Compound 4c induced
apoptosis of HeLa cell line.
HeLa cells were treated with
5.0, 10.0, and 20.0 lM 4c or
with vehicle solvent [0.1%
dimethyl sulfoxide (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
propidium iodide and annexin V-fluorescein isothiocya-
nate (FITC) [51]. Early apoptosis corresponded to
annexin V single-positive cells and late apoptosis/
necrosis corresponded to annexin V double-positive
cells. As shown in Fig. 3, the representative compound
4c was very effective in inducing apoptosis in a dose-
dependent manner. Treatment of HeLa cells by 5, 10,
and 20 lM 4c for 36 h resulted in 72.12%, 52.04%, and
13.35% cell apoptosis, as compared with 0.81% in an
untreated control. Obviously, necrosis increased as the
concentration of 4c increasing. Also, the treatment
could notably induce morphological changes of HeLa
cells into round form (data not listed), which was a
primary indication of apoptosis and cell death. These
results collectively suggested that the target compounds
might inhibit growth of the HeLa cell line by induction
of apoptosis.
analogues, whereas the ester derivatives exhibited hypoxic
preferred cytotoxicity and might be used as promising
candidate prodrug against hypoxic tumor cells. The
results indicated that linkers played important roles in
biological activity. This work provides a novel class of
hybrid lead compounds with improved bioactivity for
further optimization. Detailed biological studies on the
molecular mechanism of action of the hybrids are in
progress.
Experimental
All reagents were of commercial quality and used without
purification. 1H and 13C NMR were obtained with a Bruker
AV-400 spectrometer; chemical shifts are reported as ppm
(in CDCl3/DMSO-d6, TMS as internal standard). IR spectra
were obtained using a Perkin-Elmer 2000 Fourier-transform
IR (FTIR) instrument. High-resolution mass spectra
(HRMS) were obtained on a HPLC-Q-Tof MS (Micro)
spectrometer. Melting points were determined with an X-6
micro-melting point apparatus and were corrected using
standard compounds. Elemental analysis was obtained on a
PE-2400 analyzer. Column chromatography was performed
on 200–300 mesh silica gel. 6-(Dimethylamino)benzo[de]-
In conclusion, a series of novel naphthalimide–indo-
methacin hybrids with different linkers were designed and
synthesized, and their antitumor activity was evaluated
against a variety of cancer cell lines in vitro. Preliminary
results showed that the hybrids had moderate cytotoxic
activity and could effectively induce apoptosis in HeLa
cells. More importantly, the amide derivatives had better
cytotoxic and proapoptotic activity than their ester
123

Novel naphthalimide–indomethacin hybrids as potential antitumor agents
897
6-(N,N-Dimethylamino)-2-[2-(2-hydroxyethoxy)ethyl]-1H-
benzo[de]isoquinoline-1,3(2H)-dione (3b, C18H20N2O4)
3b was prepared from 2 and 2-(2-aminoethoxy)ethanol by
using the procedure described for preparation of 3a; yield
85%, orange solid. M.p.: 100.1–102.1 °C; 1H NMR
(500 MHz, DMSO-d6): d = 8.49 (d, J = 8.5 Hz, 9-Ar–
H), 8.44 (d, J = 7.2 Hz, 4-Ar–H), 8.32 (d, J = 8.2 Hz, 7-
Ar–H), 7.74 (t, J = 7.6 Hz, 8-Ar–H), 7.19 (d, J = 8.2 Hz,
5-Ar–H), 4.20 (t, J = 6.6 Hz, NCH2CH2O), 3.61 (t,
J = 6.6 Hz, NCH2CH2O), 3.63–3.42 (m, OCH2CH2OH),
3.07 (s, N(CH3)2) ppm; 13C NMR (100 MHz, DMSO-d6):
d = 163.8, 157.4, 156.2, 139.6, 136.9, 134.5, 129.1, 125.3,
124.9, 123.0, 115.9, 68.9, 64.3, 62.5, 44.8, 38.7 ppm;
HRMS (ES?): m/z = 329.1503 (M ? H)?, required
329.1501.
isochromene-1,3-dione (2) was prepared by using a previ-
ously reported method [49].
The target compounds were submitted to the Chinese
National Center for Drug Screening and School of Phar-
macy in East China University of Science and Technology
for in vitro antitumor activity assays. Growth inhibitory
effects on the cancer cell lines were measured by MTT
assay [50]. Hypoxic cytotoxicity was tested according to
our reported procedure [52].
Cell cycle analysis was analyzed on FACScalibur
(Becton–Dickinson, San Jose, CA). HeLa cells were
incubated with different concentration of the compounds.
After centrifugation at 1,000 rpm for 5 min at room tem-
perature, the supernatant was removed. Then the cells were
washed twice with phosphate-buffered saline (PBS) solu-
tion and fixed with 0.3 cm3 PBS and 0.7 cm3 ice-cold 75%
EtOH overnight. Fixed cells were harvested by centrifu-
gation at 1,000 rpm for 10 min and washed twice with
PBS. Collected cells were resuspended in 1 cm3 PBS and
treated with 5 9 10-4 cm3 RNase A at 37 °C for 30 min.
Propidium iodide was then added to final concentration of
50 lg cm-3 for DNA staining, and 20,000 fixed cells were
analyzed using the Modifit program.
2-(2-Aminoethyl)-6-(N,N-dimethylamino)-1H-benzo[de]-
isoquinoline-1,3(2H)-dione (3c, C16H17N3O2)
3c was prepared from 2 and ethane-1,2-diamine by using
the procedure described for preparation of 3a; yield 85%,
orange-red solid. M.p.: 153.3–155.3 °C; 1H NMR
(500 MHz, CDCl3): d = 8.57 (d, J = 7.3 Hz, 9-Ar–H),
8.47 (d, J = 8.2 Hz, 4-Ar–H), 8.44 (d, J = 8.5 Hz, 7-Ar–
H), 7.66 (t, J = 7.9 Hz, 8-Ar–H), 7.11 (d, J = 8.2 Hz, 5-
Ar–H), 4.28 (t, J = 6.5 Hz, NCH2CH2NH2), 3.12 (s,
N(CH3)2), 3.08 (t, J = 6.5 Hz, NCH2CH2NH2) ppm; 13C
NMR (100 MHz, CDCl3): d = 164.6, 158.8, 136.3, 133.0,
129.5, 124.8, 122.9, 115.8, 55.4, 44.7, 38.5 ppm; HRMS
(ES?): m/z = 284.1395 (M ? H)?, required 284.1399.
Extent of apoptosis was measured through annexin
V-FITC apoptosis detection kit as described by the man-
ufacturer’s instructions. Briefly, HeLa cells were collected
36 h after target compound treatment, and washed twice
with PBS, then resuspended in 0.4 cm3 19 binding buffer.
Cells were transferred to a 5-cm3 culture tube containing
5 9 10-3 cm3 annexin V-FITC and 0.01 cm3 propidium
iodide, and then incubated for 15 min at room temperature
in the dark. After 19 binding buffer was added to each
tube, stained cells were analyzed by flow cytometry.
2-(6-Aminohexyl)-6-(N,N-dimethylamino)-1H-benzo[de]-
isoquinoline-1,3(2H)-dione (3d, C20H25N3O2)
3d was prepared from 2 and hexane-1,2-diamine by using
the procedure described for preparation of 3a; yield 90%,
1
orange solid. M.p.: 100.6–102.7 °C; H NMR (500 MHz,
6-(N,N-Dimethylamino)-2-(2-hydroxyethyl)-1H-benzo[de]-
isoquinoline-1,3(2H)-dione (3a, C16H16N2O3)
CDCl3): d = 8.56 (d, J = 6.9 Hz, 9-Ar–H), 8.47 (d,
J = 8.2 Hz, 4-Ar–H), 8.43 (d, J = 8.4 Hz, 7-Ar–H), 7.65
(t, J = 7.8 Hz, 8-Ar–H), 7.11 (d, J = 8.2 Hz, 5-Ar–H),
4.16 (t, J = 7.6 Hz, NCH2), 3.10 (s, N(CH3)2), 2.68 (t,
J = 6.7 Hz, CH2NH2), 1.77–1.68 (m, NCH2CH2), 1.53–
1.40 (m, NCH2CH2CH2CH2CH2CH2NH2) ppm; 13C NMR
(100 MHz, CDCl3): d = 164.6, 157.9, 157.3, 139.4, 136.3,
133.7, 129.1, 125.5, 124.7, 123.0, 115.0, 44.8, 41.6, 38.3,
31.9, 29.1, 27.3, 26.5 ppm; HRMS (ES?): m/z = 340.2029
(M ? H)?, required 340.2025.
Compound 2 (500 mg, 2.07 mmol) was dissolved in
10 cm3 EtOH, then 190 mg ethanolamine (3.11 mmol)
was added. The solution was stirred and refluxed under
nitrogen for 1.5 h, and then cooled and filtered. The residue
was subjected to column chromatography on silica gel by
using CH2Cl2/CH3OH 30:1 (v/v) as eluent, affording 3a
(529 mg, 1.86 mmol, 90%) as orange solid. M.p.: 130.6–
132.6 °C; 1H NMR (500 MHz, CDCl3): d = 8.57 (dd,
J1 = 0.7 Hz, J2 = 3.8 Hz, 9-Ar–H), 8.48 (d, J = 8.3 Hz,
4-Ar–H), 8.55 (dd, J1 = 0.7 Hz, J2 = 3.8 Hz, 7-Ar–H),
7.66 (t, J = 7.6 Hz, 8-Ar–H), 7.11 (d, J = 8.3 Hz, 5-Ar–
H), 4.45 (t, J = 5.1 Hz, NCH2CH2OH), 3.97 (t, J =
5.2 Hz, NCH2CH2OH), 3.13 (s, N(CH3)2) ppm; 13C NMR
(100 MHz, CDCl3): d = 164.4, 159.0, 158.0, 139.6, 139.0,
135.6, 129.0, 125.3, 123.5, 115.0, 57.2, 44.9, 39.9 ppm;
HRMS (ES?): m/z = 285.1233 (M ? H)?, required
285.1239.
2-[6-(Dimethylamino)-1,3-dioxo-1H-benzo[de]isoquinolin-
2(3H)-yl]ethyl 2-[1-(4-chlorobenzoyl)-5-methoxy-2-
methyl-1H-indol-3-yl]acetate (4a, C35H30ClN3O6)
To a solution of 100 mg 3a (0.352 mmol) in 5 cm3 CHCl3,
151 mg indomethacin (0.422 mmol), 135 mg 1-(3-dimeth-
ylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)
(0.704 mmol), and 86 mg 4-dimethylaminopyridine
(DMAP) (0.704 mmol) were added. The solution was
123

898
A. Wu et al.
1
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 60:1
(v/v) as eluent, affording 4a as orange-yellow solid. Yield
solid. M.p.: 91.8–93.8 °C; H NMR (500 MHz, CDCl3):
d = 8.41 (d, J = 8.4 Hz, 9-naphthalene-H), 8.32 (d, J =
7.2 Hz, 7-naphthalene-H), 8.20 (d, J = 8.2 Hz, 4-naphtha-
lene-H), 7.84 (d, J = 8.3 Hz, 2,6-Ph-H), 7.59 (t,
J = 7.8 Hz, 8-naphthalene-H), 7.49 (d, J = 8.3 Hz, 3,5-
Ph-H), 7.02 (d, J = 8.2 Hz, 5-naphthalene-H), 6.82 (d,
J = 9.0 Hz, 7-indole-H), 6.70 (d, J = 1.6 Hz, 4-indole-H),
6.54 (br s, CONH), 6.46 (dd, J1 = 1.8 Hz, J2 = 8.8 Hz,
6-indole-H), 4.28 (t, J = 4.8 Hz, NCH2CH2), 3.63 (d,
J = 4.1 Hz, NCH2CH2), 3.57 (s, CH3O), 3.54 (s,
CH2CONH), 3.11 (s, N(CH3)2), 2.31 (s, 2-indole-CH3)
ppm; 13C NMR (100 MHz, CDCl3): d = 170.6, 156.0,
134.1, 133.0, 131.5, 131.4, 131.3, 131.0, 130.4, 129.1, 124.8,
122.3, 115.0, 113.2, 112.7, 112.3, 100.2, 55.4, 44.7, 38.5,
1
80%; m.p.: 189.5–191.5 °C; H NMR (500 MHz, CDCl3):
d = 8.49 (d, J = 7.1 Hz, 9-naphthalene-H), 8.43 (d,
J = 8.3 Hz, 7-naphthalene-H), 8.40 (d, J = 8.2 Hz, 4-
naphthalene-H), 7.65–7.62 (m, 8-naphthalene-H and 2,6-
Ph-H), 7.44 (d, J = 8.5 Hz, 3,5-Ph-H), 7.09 (d,
J = 8.2 Hz, 5-naphthalene-H), 6.94 (d, J = 2.5 Hz, 4-
indole-H), 6.81 (d, J = 9.0 Hz, 7-indole-H), 6.53 (dd,
J1 = 2.5 Hz, J2 = 9.0 Hz, 6-indole-H), 4.49 (s,
COOCH2CH2N), 3.74 (s, CH3O), 3.63 (s, CH2COO),
3.11 (s, N(CH3)2), 2.28 (s, 2-indole-CH3) ppm; 13C NMR
(100 MHz, CDCl3): d = 170.8, 168.2, 164.6, 164.0, 157.0,
156.0, 139.0, 135.8, 134.0, 132.7, 131.3, 131.2, 131.1,
130.7, 130.3, 129.0, 125.3, 124.9, 122.7, 114.8, 114.6,
113.3, 112.6, 111.7, 101.0, 62.4, 55.6, 44.8, 38.8, 30.2,
ꢀ
32.1, 13.3 ppm; FTIR (KBr): m = 3,325, 2,925, 1,680,
1,647, 1,513, 1,472, 1,450, 1,350, 1,316, 1,224, 1,083, 1,057,
778, 752 cm-1; HRMS (ES?): m/z = 645.1864 (M ?
Na)?, required 645.1881.
ꢀ
13.3 ppm; FTIR (KBr): m = 2,948, 1,736, 1,688, 1,651,
2-[1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]-N-[6-[6-(dimethylamino)-1,3-dioxo-1H-benzo[de]-
isoquinolin-2(3H)-yl]hexyl]acetamide
1,584, 1,472, 1,376, 1,354, 1,324, 1,231, 1,172, 1,071,
912, 834, 778 cm-1; HRMS (ES?): m/z = 624.1898 (M ?
H)?, required 624.1901.
(4d, C39H39ClN4O5)
2-[2-[6-(Dimethylamino)-1,3-dioxo-1H-benzo[de]isoquino-
lin-2(3H)-yl]ethoxy]ethyl 2-[1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl]acetate
4d was prepared from 3d and indomethacin by using the
procedure described for preparation of 4a; yield 70%,
orange-yellow solid. M.p.: 164.6–166.6 °C; 1H NMR
(500 MHz, CDCl3): d = 8.49 (d, J = 7.2 Hz, 9-naphtha-
lene-H), 8.43 (d, J = 8.4 Hz, 7-naphthalene-H), 8.39 (d,
J = 8.2 Hz, 4-naphthalene-H), 7.65 (d, J = 8.2 Hz,
8-naphthalene-H), 7.61 (d, J = 8.5 Hz, 2,6-Ph-H), 7.44
(d, J = 8.5 Hz, 3,5-Ph-H), 7.10 (d, J = 8.2 Hz, 5-naphtha-
lene-H), 6.91 (d, J = 2.3 Hz, 4-indole-H), 6.83 (d,
J = 9.0 Hz, 7-indole-H), 6.66 (dd, J1 = 2.4 Hz, J2 =
9.0 Hz, 6-indole-H), 5.89 (br s, CONH), 4.06 (t,
J = 7.3 Hz, NCH2), 3.81 (s, CH3O), 3.65 (s, CH2CONH),
3.19 (dd, J1 = 6.7 Hz, J2 = 13.0 Hz, CONHCH2), 3.10
(s, N(CH3)2), 2.39 (s, 2-indole-CH3), 1.68–1.62 (m,
NCH2CH2), 1.46–1.41 (m, NHCH2CH2), 1.33–1.29 (m,
CH2CH2CH2CH2CH2CH2) ppm; 13C NMR (100 MHz,
CDCl3): d = 169.8, 168.2, 164.6, 164.1, 156.9, 156.3,
139.4, 136.3, 133.7, 132.6, 131.1, 131.0, 130.9, 130.4,
130.2, 129.1, 125.3, 124.9, 123.0, 115.1, 115.0, 113.3, 113.1,
112.3, 100.8, 55.7, 44.8, 39.6, 39.3, 32.3, 29.1, 27.7, 26.1,
(4b, C37H34ClN3O7)
4b was prepared from 3b and indomethacin by using the
procedure described for preparation of 4a; yield 70%,
orange-yellow solid. M.p.: 155.3–157.3 °C; 1H NMR
(500 MHz, CDCl3): d = 8.57 (d, J = 7.2 Hz, 9-naphtha-
lene-H), 8.48–8.45 (m, 4,7-naphthalene-H), 7.68–7.65 (m,
8-naphthalene-H and 2,6-Ph-H), 7.46 (d, J = 8.4 Hz, 3,5-
Ph-H), 7.12 (d, J = 8.2 Hz, 5-naphthalene-H), 6.95 (d,
J = 2.3 Hz, 4-indole-H), 6.85 (d, J = 9.0 Hz, 7-indole-H),
6.63 (dd, J1 = 2.4 Hz, J2 = 9.0 Hz, 6-indole-H), 4.39 (t,
J = 6.2 Hz, NCH2CH2O), 4.24 (t, J = 4.5 Hz, COOCH2),
3.82 (s, CH3O), 3.79 (t, J = 6.2 Hz, NCH2CH2O), 3.76 (t,
J = 4.7 Hz, CH2CH2OCH2CH2N), 3.62 (s, CH2COO),
3.11 (s, N(CH3)2), 2.33 (s, 2-indole-CH3) ppm; 13C NMR
(100 MHz, CDCl3): d = 170.8, 168.3, 164.6, 164.0, 157.0,
156.0, 139.2, 135.9, 134.0, 132.7, 131.3, 131.2, 131.1,
130.8, 130.7, 130.3, 129.1, 125.3, 124.9, 123.0, 114.9,
113.3, 112.6, 111.7, 101.2, 68.5, 68.0, 64.3, 55.7, 44.8,
ꢀ
25.9, 13.3 ppm; FTIR (KBr): m = 3,281, 3,074, 2,925,
ꢀ
38.7, 30.1, 29.7, 13.3 ppm; FTIR (KBr): m = 2,948, 2,851,
2,844, 2,770, 1,688, 1,651, 1,584, 1,472, 1,450, 1,354, 1,316,
1,220, 1,142, 1,083, 1,060, 845, 778, 752 cm-1; HRMS
(ES?): m/z = 701.2497 (M ? Na)?, required 701.2507.
1,721, 1,688, 1,654, 1,584, 1,476, 1,368, 1,316, 1,231,
1,094, 782, 752 cm-1; HRMS (ES?): m/z = 668.2135
(M ? H)?, required 668.2164.
Acknowledgments This work is 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).
2-[1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl]-N-[2-[6-(dimethylamino)-1,3-dioxo-1H-benzo[de]iso-
quinolin-2(3H)-yl]ethyl]acetamide (4c, C35H31ClN4O5)
4c was prepared from 3c and indomethacin by using the
procedure described for preparation of 4a; yield 80%, orange
123

Novel naphthalimide–indomethacin hybrids as potential antitumor agents
899
26. Rosenbaum C, Baumhof P, Mazitschek R, Mu¨ller O, Giannis A,
Waldmann H (2004) Angew Chem Int Ed 43:224
References
27. Man CY, Cheung ITF, Cameron PA, Rainer TH (2007) Ann
Emerg Med 49:670
28. Rodriguez LA, Varas C, Patrono C (2000) Epidemiology 11:382
29. Jones MK, Wang HT, Peskar BM, Levin E, Itani RM, Sarfeh IJ,
Tarnawski AS (1999) Nat Med 5:1418
1. Thompson CB (1995) Science 267:1456
2. Sun S, Hail NJ, Lotan R (2004) J Natl Cancer Inst 96:662
3. Papadopoulos KP, Goel S, Beeram M, Wong A, Desai K,
Haigentz M, Milan ML, Mani S, Tolcher A, Lalani AS, Saran-
topoulos J (2008) Clin Cancer Res 14:7110
30. Liou J, Ghelani D, Yeh S, Wu KK (2007) Cancer Res 67:3185
31. Felts AS, Ji C, Stafford JB, Crews BC, Kingsley PJ, Rouzer CA,
Washington MK, Subbaramaiah K, Siegel BS, Young SM,
Dannenberg AJ, Marnett LJ (2007) ACS Chem Biol 2:479
32. Sato S, Kwon Y, Kamisuki S, Srivastava N, Mao Q, Kawazoe Y,
Uesugi M (2007) J Am Chem Soc 129:873
4. Hecker SJ, Erion MD (2008) J Med Chem 51:2328
5. Tang G, Nikolovska-Coleska Z, Qiu S, Yang C, Guo J, Wang S
(2008) J Med Chem 51:717
6. Corson TW, Aberle N, Crews CM (2008) ACS Chem Biol 3:677
7. Tietze LF, Bell HP, Chandrasekhar S (2003) Angew Chem Int Ed
42:3996
8. Sjoblom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD,
Mandelker D, Leary RJ, Ptak J, Silliman N, Szabo S, Buckhaults
P, Farrell C, Meeh P, Markowitz SD, Willis J, Dawson D,
Willson JK, Gazdar AF, Hartigan J, Wu L, Liu C, Parmigiani G,
Park BH, Bachman KE, Papadopoulos N, Vogelstein B, Kinzler
KW, Velculescu VE (2006) Science 314:268
9. Hariprakasha HK, Kosakowska-Cholody T, Meyer C, Cholody
WM, Stinson SF, Tarasova NI, Michejda CJ (2007) J Med Chem
50:5557
10. Nakagawa-Goto K, Nakamura S, Bastow KF, Nyarko A, Peng C,
Lee FY, Lee FC, Lee KH (2007) Bioorg Med Chem Lett 17:2894
11. Bran˜a MF, Ramos A (2001) Curr Med Chem Anticancer Agents
1:237
12. Bran˜a MF, Cacho M, Gradillas A, de Pascual-Teresa B, Ramos A
(2001) Curr Pharm Des 7:1745
13. Frei E, Teicher BA, Holden SA, Cathcart KNS, Wang Y (1988)
Cancer Res 48:6417
´
14. Sami SM, Dorr RT, Alberts DS, Solyom AM, Remers WA (2000)
¨
¨
33. Rosenbaum C, Rohrs S, Muller O, Waldmann H (2005) J Med
Chem 48:1179
34. Wey S, Augustyniak ME, Cochran ED, Ellis JL, Fang X, Garvey
DS, Janero DR, Letts LG, Martino AM, Melim TL, Murty MG,
Richardson SK, Schroeder JD, Selig WM, Trocha AM, Wexler
RS, Young DV, Zemtseva IS, Zifcak BM (2007) J Med Chem
50:6367
´
35. Velazquez CA, Chen Q, Citro ML, Keefer LK, Knaus EE (2008)
J Med Chem 51:1954
36. Hawcroft G, D’Amico M, Albanese C, Markham AF, Pestell RG,
Hull MA (2002) Carcinogenesis 23:107
37. He T, Chan TA, Vogelstein B, Kinzler KW (1999) Cell 99:335
38. Wu A, Xu Y, Qian X (2009) Bioorg Med Chem 17:592
39. Norton JT, Witschi MA, Luong L, Kawamura A, Ghosh S, Stack
MS, Sim E, Avram MJ, Appella DH, Huang S (2008) Anticancer
Drug 19:23
´
40. Noel G, Godon C, Fernet M, Giocanti N, Megnin-Chanet F,
¨
Favaudon V (2006) Mol Cancer Ther 5:564
41. Pignatello R, Spampinato G, Sorrenti V, Giacomo CD, Vicari L,
McGuire JJ, Russell CA, Puglisi G, Toth I (2000) Eur J Pharm
Sci 10:237
J Med Chem 43:3067
15. Bran˜a MF, Cacho M, Garcıa MA, de Pascual-Teresa B, Ramos A,
´
Domınguez MT, Pozuelo JM, Abradelo C, Rey-Stolle MF, Yuste
´
42. Hamann PR, Hinman LM, Beyer CF, Lindh D, Upeslacis J,
Flowers DA, Bernstein I (2002) Bioconjug Chem 13:40
43. Hamann PR, Hinman LM, Beyer CF, Greenberger LM, Lin C,
Lindh D, Menendez AT, Wallace R, Durr FE, Upeslacis J (2005)
Bioconjug Chem 16:346
44. Fang Y, Linardic CM, Richardson DA, Cai W, Behforouz M,
Abraham RT (2003) Mol Cancer Ther 2:517
45. Coleman RS, Burk CH, Navarro A, Brueggemeier RW, Diaz-
Cruz ES (2002) Org Lett 4:3545
46. Liu Z, Wang Y, Tang Y, Chen S, Chen X, Li H (2006) Bioorg
Med Chem Lett 16:1282
47. Tomic-Vatic A, EyTina J, Chapman J, Mahdavian E, Neuzil J,
Salvatore BA (2005) Int J Cancer 117:188
48. Weerapreeyakul N, Anorach R, Khuansawad T, Yenjai C, Isaka
M (2007) Chem Pharm Bull 55:930
49. Qian X, Zhu Z, Chen K (1992) J Prakt Chem 334:161
50. Kuroda M, Mimaki Y, Sashida Y, Hirano T, Oka K, Dobashi A
(1997) Tetrahedron 53:11549
´
M, Ban˜ez-Coronel M, Lacal JC (2004) J Med Chem 47:1391
16. Quaquebeke EV, Mahieu T, Dumont P, Dewelle J, Ribaucour F,
Simon G, Sauvage S, Gaussin J, Tuti J, Yazidi ME, Vynckt FV,
Mijatovic T, Lefranc F, Darro F, Kiss R (2007) J Med Chem
50:4122
17. Bailly C, Carrasco C, Joubert A, Bal C, Wattez N, Hildebrand M,
Lansiaux A, Colson P, Houssier C, Cacho M, Ramos A, Bran˜a
MF (2003) Biochemistry 42:4136
18. Carrasco C, Joubert A, Tardy C, Maestre N, Cacho M, Bran˜a MF,
Bailly C (2003) Biochemistry 42:11751
19. Li F, Cui J, Guo L, Qian X, Ren W, Wang K, Liu F (2007) Bioorg
Med Chem 15:5114
20. Qian X, Li Z, Yang Q (2007) Bioorg Med Chem 15:6846
21. Ott I, Xu Y, Liu J, Kokoschka M, Harlos M, Sheldrick WS, Qian
X (2008) Bioorg Med Chem 16:7107
22. Kamal A, Ramu R, Tekumalla V, Khanna GBR, Barkume MS,
Juvekar AS, Zingde SM (2008) Bioorg Med Chem 16:7218
23. Xie L, Xu Y, Wang F, Liu J, Qian X, Cui J (2009) Bioorg Med
Chem 17:804
24. Yang Q, Yang P, Qian X, Tong L (2008) Bioorg Med Chem Lett
18:6210
25. Zhu H, Huang M, Yang F, Chen Y, Miao Z, Qian X, Xu Y, Qin
Y, Luo H, Shen X, Geng M, Cai Y, Ding J (2007) Mol Cancer
Ther 6:484
51. Tang G, Ding K, Nikolovska-Coleska Z, Yang C, Qiu S, Shan-
gary S, Wang R, Guo J, Gao W, Meagher J, Stuckey J, Krajewski
K, Jiang S, Roller PP, Wang S (2007) J Med Chem 50:3163
52. Liu Y, Xu Y, Qian X, Liu J, Shen L, Li J, Zhang Y (2006) Bioorg
Med Chem 14:2935
123