Synthesis of novel C4-benzazole naphthalimide derivatives with potent anti-tumor properties against murine melanoma

Article abstract of DOI:10.1016/j.bmc.2016.11.057

Novel C4-benzazole naphthalimide derivatives were synthesized and tested in vitro and in vivo as anti-cancer drugs. Among these synthetic molecules, compounds 9 and 10 exhibited cytotoxicity against murine B16F10 melanoma cells. In addition, the above-mentioned compounds significantly suppressed lung tumor metastasis with no visible sign of toxicity.

Full text of DOI:10.1016/j.bmc.2016.11.057

Accepted Manuscript
Synthesis of novel C4-benzazole naphthalimide derivatives with potent anti-
tumor properties against murine melanoma
Yen-Ta Lu, Tien-Ling Chen, Kuo-Song Chang, Chia-Ming Chang, Tsai-Yin
Wei, Jen-Wei Liu, Chih-Ang Hsiao, Tzenge-Lien Shih
PII:
S0968-0896(16)31141-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.057
BMC 13424
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
7 November 2016
29 November 2016
Please cite this article as: Lu, Y-T., Chen, T-L., Chang, K-S., Chang, C-M., Wei, T-Y., Liu, J-W., Hsiao, C-A., Shih,
T-L., Synthesis of novel C4-benzazole naphthalimide derivatives with potent anti-tumor properties against murine
melanoma, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.11.057
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Synthesis of novel C4-benzazole naphthalimide derivatives with potent anti-tumor properties against
murine melanoma
Yen-Ta Lua§, Tien-Ling Chenb§, Kuo-Song Changd,e, Chia-Ming Changc, Tsai-Yin Weic,
Jen-Wei Liuf, and Chih-Ang Hsiaof ,Tzenge-Lien Shih.f,*
a Chest Division, Medical Department, MacKay Memorial Hospital, Zhongshan Dist., 10449 Taipei, Taiwan.
b
Division of Allergy, Immunology and Rheumatology, Department of Internal Medicine, MacKay Memorial
Hospital, Zhongshan Dist., 10449 Taipei, Taiwan.
c
Department of Medical Research, MacKay Memorial Hospital, Tamsui Dist., 25173 New Taipei City,
Taiwan.
d Department of Emergency Medicine, MacKay Memorial Hospital, Zhongshan Dist., 10449 Taipei, Taiwan.
e Department of Medicine, MacKay Medical College, New Taipei City, 25245, Taiwan
f Department of Chemistry, Tamkang University, Tamsui Dist., 25137 New Taipei City, Taiwan.
§ These authors contributed equally to this work.
Abstract
Novel C4-benzazole naphthalimide derivatives were synthesized and tested in vitro and in vivo as
anti-cancer drugs. Among these synthetic molecules, compounds 9 and 10 exhibited cytotoxicity against
murine B16F10 melanoma cells. In addition, the above-mentioned compounds significantly suppressed lung
tumor metastasis with no visible sign of toxicity.
Keywords: benzazole, B16F10 cells; melanoma; MTS assay; naphthalimide; topoisomerase II.
1. Introduction
Malignant melanoma, an aggressive type of skin cancer with high metastatic potential, is a cause of growing
public concern worldwide. Malignant melanoma can be cured by a number of treatments, such as surgery,
radiation, and chemotherapy.1-3 However, once it metastasizes to other parts of the body, effective therapies
1

are lacking.4 At present, alkylating agents such as dacarbazine, cisplatin, and temozolomide are the most
common chemotherapy drugs for the treatment of stage IV melanoma.5 However, the long-term response
rate with systematic chemotherapeutic agents is poor, while incidence rate and number of deaths from
melanoma have been increasing continuously. Therefore, there remains an unmet need for novel medicines
for effective treatment of melanoma.
Figure 1. Naphthalimide-based antitumor agents.
Naphthalimide is a main scaffold in various luminophores owing to its flat and stable conjugated aromatic
system.6 Therefore, its derivatives are widely applied in dyes,7 liquid crystals,8 chemosensors,9 fluorescent
probes,10 etc. In particular, the flat structure of naphthalimide derivatives allows then intercalate into DNA.11
Thus these derivatives, such as amonafide12 and mitonafide,12b,13 (Figure 1) have shown promise against
various cancer cell lines in vitro and have been tested in clinical trials. However, amonafide and mitonafide
have failed in trials due to toxicity.14 Recently, some sulphonamide and sulphonyl-hydrazone naphthalimide
derivatives have shown potential cytotoxic properties against murine B16F10 melanoma cells with IC50
around 50 μM and 300 μM.15
Some naphthalimide analogs have been demonstrated to inhibit melanoma tumor growth in a subcutaneous
2

xenograft model with fewer side effects.16 In particular, it has been reported that the naphthalimide
derivatives produced by modifying the N,N-dimethyl portion of mitonafide,16,17 have been utilized in
treatment of melanoma. We are aware that core structures with incorporating benzoxazole, benzothialole or
benzoimidazole moieties are pervasively found in potential drugs,18 and that these benzazole-based
molecules are used as clinical drugs with therapeutic potency.19 We are interested in combining of
benzazoles at the C-4 position of naphthalimide derivatives in order to provide a wider spectrum of
applications. In order to quickly assemble the two portions, we coupled benzazoles and
4-bromonaphthalimide derivatives via palladium-copper catalysis.20 Our main goal is pursuing novel
potential chemotherapeutic agents for treatment of melanoma.
2. Results and discussion
2.1 Chemistry-Synthesis of C4-benzazole naphthalimide derivatives compounds 5-10
The syntheses of compounds 5-10 are depicted in Scheme 1. We utilized commercially available compound
1 and modified its anhydride portion by reaction with n-butylamine, 2-hydroxyethylamine, and
N,N-dimethylethylenediamine under reflux in EtOH to afford 2, 3, and 4, respectively, in high yields. It is
worthwhile to point out that the antitumor agents amonafide and mitonafide were also possess an
N,N-dimethylethylenediamine portion. Compounds 2, 3 and 4 were directly coupled with either
benzoxazoles or benzothialoles by palladium-copper catalysis20 to provide compounds 510.
3

Scheme 1. Syntheses of C4-benzazole naphthalimide derivative compounds 510
2.2 Biological evaluation
2.2.1 In vitro cytotoxic effect of compounds 5-10
To evaluate the effect of synthetic compounds on cell viability, B16F10 cells were treated with various
concentrations of compounds 510 for 2 days. The cytotoxicity of compounds 510 against murine B16F10
melanoma cells was examined by MTS assay. As shown in Figure 2, the cytotoxicity of these compounds
was highly dependent on the structure of the side chain. Compounds 5-8 at concentrations ranging from 0.01
to 40 µM did not exhibit cytotoxic activity. In contrast, compounds 9 and 10 dramatically decreased cell
viability in a dose-dependent manner (IC50 of compound 9: 7.8  0.05 µM; IC50 of compound 10: 4.5  0.06
µM) showing almost the same degree of cytotoxicity as amonafide (IC50 of amonafide: 6.7  1.11 µM).
Therefore, compounds 9 and 10 were selected for further studies.
4

Figure 2. The in vitro cytotoxicity of a range of doses of compounds 5-10 to B16F10 melanoma cells. Cell
viability was examined after 2 days incubation by MTS assay. Optical density of DMSO control groups was
taken as 100% of cell viability. Values are presented as the mean  SEM from three independent
experiments. Data are the results of three independent experiments with each assay performed in duplicate
within each experiment.
Figure 3. Inhibitory effects of compounds 9 and 10 on topoisomerase II mediated DNA relaxation.
Supercoiled pHOT-1 plasmid DNA was incubated with topoisomerase II enzyme in the presence of various
concentrations of indicated compounds. Intercalation was evaluated by conversion of nicked oc DNA and
relaxed DNA into supercoiled DNA. Lane 1: supercoiled pHOT-1 DNA, lane 2: no drug, lane 3: 1% DMSO,
lane 4: 100M of VP16, lane 5: 10M of compound 9, lane 6: 100 M of compound 9, lane 7: 10 M of
compound 10, lane 8: 100 M of compound 10. oc, open circular; sc, supercoiled.
Because some amonafide derivatives exert their anti-cancer activity by poisoning topoisomerase II,21 we
investigated the mechanism of B16F10 melanoma cells death induced by compounds 9 and 10. We used in
vitro DNA relaxation assay to investigate topoisomerase II inhibitory activity. As shown in Figure 3,
intercalation by compounds 9 and 10 into DNA was detectable at 10 M. In addition, complete intercalation
into samples could be observed by treating with compound 10 (100 M), but not with compound 9. This
indicated that compound 10 at high concentration might have a better inhibitory effect on topoisomerase II
activity in vitro.
5

2.2.2 Effect on mouse melanoma lung metastatic models
To examine the therapeutic effect of compounds 9 and 10 against pre-established B16F10 lung metastases,
mice were intravenously (iv) injected with 2 x 105 B16F10 tumor cells and treated with 2 mg/kg of
compound 9 or 10, respectively, or 5% DMSO vehicle control. The number of lung nodules in each
individual mouse in each group is shown in Figure 4. Mice treated with compound 9 showed a reduction in
the number of lung metastases (mean 41; range 2-90) compared with mice receiving the DMSO vehicle
control (mean 60; range 37-94). In contrast, 2 mg/kg of compound 10 had a marked effect in reducing
metastasis to the lung, with a mean of 30 lung nodules (range 3-59).
To investigate the toxicity of compounds 9 and 10, mice were intraperitoneally (ip) injected daily with 2
mg/kg of compound 9 or 10 or 5% DMSO vehicle control. As shown in Figure 5, no significant decrease in
body weight was observed in mice treated with high doses of compound 9 or 10. We conclude that
compounds 9 and 10 are very safe.
Figure 4. Lung metastases after i.v. injection of B16F10 tumor cells. 2x105 B16F10 cells were injected into
each mouse via tail vein on day 0. Compounds 9 and 10 2 mg/kg vs 5% DMSO vehicle control was given ip
6

daily on days 114 (n=11/group). The amount of tumor seeding was counted as total numbers of black
nodules presented in the lungs under microscopy. Both compounds 9 and 10 significantly reduced tumor
seeding and growth on day 14. Ctrl 5% DMSO vehicle control; #9 compound 9; #10 compound 10.
Figure 5. Average body weights of C57BL/6 mice during 9-day toxicity study. Mice (n=5/group) were ip
injected daily with 2 mg/kg of compound 9 or 10 or 5% DMSO vehicle control, and their body weights were
measured on the indicated days. There was no difference in the weights of 5% DMSO vehicle control, or
treated compounds 9 and compound 10 mice. Ctrl, 5% DMSO vehicle control; #9 compound 9; #10
compound 10.
3. Conclusion
Compounds 9 and 10 possess the same naphthalimide moiety as the anti-cancer agent amonafide and
exhibited comparable biological activity against B16F10 melanoma cells in vitro. Their differences in
structure are at the C-4 benzazoles of 9 and 10 and the nitro as well as the amine groups in amonafide and
mitonafide at C-3, respectively. We believe these functional groups to contribute a certain degree of their
activity at both sides. Our work is based on a syngeneic lung metastatic model which models cancers that
are more difficult to treat. Compound 9 and 10 significantly reduced B16F10 lung metastasis in C57BL/6
mice without obvious weight loss. Recently, some clinically approved chemotherapeutic drugs not only
directly killed tumors but also elicited an immune response against tumors.25 Our unpublished results
demonstrate that compounds 9 and 10 also enhance innate cell function in vitro. Whether these cells play
7

roles in the anti-cancer effects mediated by compounds 9 and 10 is a topic that is currently under
investigation.
4. Experimental
1
All chemicals were purchased from commercial providers and used without purification. H (600 MHz)
13
and C (150 MHz) NMR were recorded on a Bruker 600 MHz spectrometer. Chemical shifts were reported
1
in parts per million (ppm) and referred to the residual of deuterium solvent: H NMR (CDCl3, 7.26 ppm;
CD3OD, 3.3ppm; d6-DMSO, 2.5 ppm); 13C NMR (CDCl3, 77.0 ppm; CD3OD, 49.15 ppm; d6-DMSO, 39.51
ppm). Melting points were determined on a Fargo MP-2D apparatus and not corrected. HRMS were
recorded on Finnigan MAT-95S. Purification conducted by flash column chromatography on either SiO2
(Chromatorex GS 60-40 (230-400 mesh) Fuji Silysia Chemical LTD) or NH-SiO2 (Chromatorex, MB
100-40) except as otherwise stated. All solid products were filtrated by suction manner and washed with
appropriate solvents.
4.1. Synthetic methods
6-Bromo-2-butyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (2). Compound 1 (0.415 g, 1.5 mmol) and
o
n-butylamine (0.163 mL, 1.65 mmol) in EtOH (8 mL) was heated at 70 C under nitrogen atmosphere for 3
h. This mixture was cooled to room temperature and filtered. The solid was collected and washed with 95%
o
EtOH to afford a pale-yellow, needle-like solid (0.422 g, 1.27 mmol) in 86% yield. Mp 196198 C. (lit.22
o
1
109110 C from chlorobenzene) H NMR (600 MHz, CD3OD): δ 8.65 (d, J=7.2 Hz, 1H), 8.56 (d, J=8.4
Hz, 1H), 8.41 (d, J=7.8 Hz, 1H), 8.03 (d, J=7.8 Hz, 1H), 7.84 (t, J=8.4 Hz, 1H), 4.17 (t, J=7.8 Hz, 2H),
1.71 (quint, J=7.2 Hz, 2H), 1.45 (sixtet, J=7.2 Hz, 2H), 0.98 (t, J=7.2 Hz, 3H). 13C NMR (150 MHz,
8

CD3OD): 163.6 (x2), 133.2, 132.0, 131.2, 131.1, 130.6, 130.2, 129.0, 128.1, 123.2, 122.3, 40.4, 30.2, 20.4,
13.8. HRMS (EI) calculated for C16H14BrNO2 ([M]+): 331.0208. Found: 331.0204.
6-Bromo-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (3). Compound 1 (1.00 g, 3.61
mmol) and 2-aminoethanol (0.244 g, 4.322 mmol) in EtOH (20 mL) was refluxed under nitrogen
atmosphere for 6 h. At the end of reaction time, the mixture was filtrated and the solid was washed with 95%
o
o
EtOH to afford a pale-yellow solid (0.979 g, 3.07 mmol) in 85% yield. Mp 209211 C. (lit.23 203204 C)
1H NMR (600 MHz, CDCl3): δ 8.68 (dd, J=7.2, 1.0 Hz, 1H), 8.59 (dd, J=8.5, 1.0 Hz, 1H), 8.43 (t, J=7.8 Hz,
1H), 8.06 (d, J=7.8 Hz, 1H), 7.86 (dd, J=8.5, 8.5 Hz, 1H), 4.45 (t, J=10.8 Hz, 2H), 3.99 (t, J=10.8 Hz, 2H),
13
2.08 (s, 1H). C NMR (150 MHz, CDCl3): δ 164.5, 164.4, 133.6, 132.4, 131.5, 131.2, 130.7, 129.1, 128.2,
122.8, 121.2, 61.7, 42.9. HRMS (EI) calculated for C14H10BrNO3 ([M]+): 318.9844, Found: 318.9840.
6-Bromo-2-(2-(dimethylamino)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (4). Compound 1 (1.00 g,
3.61 mmol) and N,N-dimethylethylenediamine (0.382 g, 4.332 mmol) was refluxed under nitrogen
atmosphere for 4 h. The mixture was filtrated and the solid was washed with 95% EtOH to provide a
1
pale-yellow solid (1.025 g, 2.952 mmol) in 82% yield. Mp 152154 oC. (lit.24 142.5–143.5 °C) H NMR
(600 MHz, CD3OD): δ 8.61 (d, J=7.2 Hz, 1H), 8.60 (d, J = 8.4 Hz, 1H), 8.37 (d, J=7.8 Hz, 1H), 8.13 (d,
13
J=7.8 Hz, 1H), 7.91 (t, J=8.1 Hz, 1H), 4.30 (t, J=7.2 Hz, 2H), 2.69 (t, J=7.2 Hz, 2H), 2.35 (s, 6H). C
NMR (150 MHz, CD3OD): 165.3, 165.2, 134.4, 133.1, 132.6, 132.4, 132.0, 131.2, 130.4, 129.6, 124.6,
123.8, 57.9, 45.9 (x2), 38.9. HRMS (EI) calculated for C16H15BrN2O2 ([M]+): 346.0317, Found: 346.0309.
9

4.2 General procedure of palladium-copper-catalyzed direct coupling of compounds 2-4 with
benzazoles.
6-(Benzo[d]oxazol-2-yl)-2-butyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (5). To a mixture of compound
2 (0.050 g, 0.150 mmol), benzoxazole (0.0536 g, 0.450 mmol), Pd(PPh3)4 (0.0017 g, 0.0015 mmol),
Cu(OAc)2・H2O (0.0060 g, 0.030 mmol) and Na2CO3 (0.0477 g, 0.4503 mmol) was added toluene (2 mL).
The solution was refluxed for 6 h under nitrogen atmosphere. The solid was removed by filtration through
celite. The filtrant was diluted with EtOAc and 20% HCl. The organic layer was separated, dried over
MgSO4 and purified by flash column chromatography (EtOAc/Hexane=1/81/4; Rf=0.55,
o
EtOAc/Hexane=1/6) to afford a yellow-green solid (0.0342 g, 0.0923 mmol). Yield: 60%. Mp 208211 C.
1H NMR (600 MHz, CDCl3) : 9.97 (d, J=8.6 Hz, 1H), 8.728.70 (m, 2H), 8.67 (d, J=7.8 Hz, 1H), 7.95 (t,
J=7.6 Hz, 1H), 7.92 (d, J=7.8 Hz, 1H), 7.69 (d, J=7.8 Hz, 1H), 7.48 (t, J=7.4 Hz, 1H), 7.46 (t, J=7.8 Hz,
1H), 4.21 (t, J=7.8 Hz, 2H), 1.76 (quint, J=7.3 Hz, 2H) , 1.47 (sextet, J=7.3 Hz, 2H), 1.00 (t, J=7.3 Hz, 3H).
13C NMR (150 MHz, CDCl3):164.0, 163.6, 160.9, 150.3, 142.1, 133.1, 131.6, 130.0, 129.1, 128.8, 128.7,
128.5, 126.4, 125.1, 125.0, 123.0, 120.8, 110.8, 40.4, 30.2, 20.4, 13.8. HRMS (EI) calculated for
C23H18N2O3 ([M]+) : 370.1317. Found: 370.1320.
6-(Benzo[d]thiazol-2-yl)-2-butyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (6). Same procedure as 5. To a
mixture of compound 2 (0.100 g, 0.3021 mmol), benzothiazole (0.034 g, 0.252 mmol), Pd(PPh3)4 (0.0030 g,
0.0025 mmol), Cu(OAc)2・H2O (0.010 g, 0.0503 mmol) and Na2CO3 (0.0533 g, 0.503 mmol) was added
10

toluene (4 mL). Purification by flash column chromatography (EtOAc/Hexane=1/81/4; Rf=0.4,
o
EtOAc/Hexane=1/4) afforded a yellow-green solid (0.0342 g, 0.0923 mmol). Yield: 76%. Mp 154156 C.
1H NMR (600 MHz, CDCl3): 9.43 (d, J=8.6 Hz, 1H), 8.68 (t, J=7.6 Hz, 2H), 8.24 (d, J=8.2 Hz, 1H), 8.19
(d, J=7.6 Hz, 1H), 8.01 (d, J=8.0 Hz, 1H), 7.88 (t, J=7.9 Hz, 1H), 7.61 (t, J=7.6 Hz, 1H), 7.52 (t, J=7.6 Hz,
1H), 4.21 (t, J=7.7 Hz, 2H), 1.76 (quint, J=7.6 Hz, 2H), 1.47 (sextet, J =7.6 Hz, 2H), 1.00 (t, J=7.4 Hz, 3H).
13C NMR (150 MHz, CDCl3) :165.5, 164.0, 163.6, 154.2, 136.1, 135.4, 132.9, 131.7, 130.2, 129.6, 129.1,
128.9, 128.3, 126.8, 126.2, 124.2, 124.0, 122.9, 121.6, 40.4, 30.2, 20.4, 13.8. HRMS (ESI) calculated for
C23H19N2O2S ([M+H]+): 387.1167. Found: 387.1155.
6-(Benzo[d]oxazol-2-yl)-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (7). Compound 3
(0.100 g, 0.3124 mmol), benzoxazole (0.116 g, 0.937 mmol), Pd(PPh3)4 (0.0036 g, 0.0031 mmol), Cu(OAc)2
・H2O (0.0125 g, 0.0625 mmol) and Na2CO3 (0.0662 g, 0.635 mmol) in toluene (5 mL). Purification by flash
column chromatography (EtOAc/CH2Cl2=1/41/1; Rf=0.3, EtOAc/CH2Cl2=1/1) afforded a yellow-green
solid (0.072 g, 0.201 mmol). Yield: 64%. Mp 194198 oC. 1H NMR (600 MHz, CDCl3): 10.01 (d, J=8.4 Hz,
1H), 8.748.72 (m, 2H), 8.69 (d, J=7.7 Hz, 1H), 7.97 (t, J=8.0 Hz, 1H), 7.92 (d, J=7.6 Hz, 1H), 7.69 (d,
J=7.8 Hz, 1H), 7.49 (t, J=7.1 Hz, 1H), 7.46 (t, J=7.3 Hz, 1H), 4.49 (t, J=5.4 Hz, 2H), 4.02 (t, J=5.4 Hz, 2H),
1.59 (s, -OH). 13C NMR (150 MHz, CDCl3): 164.9, 164.5, 160.8, 150.3, 142.1, 133.6, 132.0, 130.4, 129.2,
129.1, 128.9, 128.5, 126.5, 126.5, 125.2, 124.6, 122.6, 120.9, 110.8, 61.7, 42.9. HRMS (EI) calculated for
C21H14N2O4 [M+H]+: 358.0954. Found: 358.0957.
11

6-(Benzo[d]thiazol-2-yl)-2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (8). Compound 3
(0.100 g, 0.3142 mmol), benzothiazole (0.1267 g, 0.937 mmol), Pd(PPh3)4 (0.0036 g, 0.0031 mmol),
Cu(OAc)2・H2O (0.010 g, 0.050 mmol) and Na2CO3 (0.0662 g, 0.625 mmol) in toluene (4 mL). Purification
by flash column chromatography (EtOAc/CH2Cl2=1/41/1; Rf=0.4, EtOAc/CH2Cl2=1/1) afforded a
o
1
yellow-green solid (0.082 g, 0.219 mmol). Yield: 70%. Mp 231234 C. H NMR (600 MHz, CD3OD): 
9.47 (d, J=8.3 Hz, 1H), 8.71 (t, J=7.4 Hz, 1H), 8.25 (d, J=8.0 Hz, 1H), 8.21 (d, J=7.4 Hz, 1H), 8.03 (d,
J=8.0 Hz, 1H), 7.90 (t, J=7.6 Hz, 1H), 7.62 (t, J=7.4 Hz, 1H), 7.53 (t, J=7.5 Hz, 1H), 4.50 (t, J=4.8 Hz, 2H),
13
4.02 (t, J=4.8 Hz, 2H), 1.84 (s, -OH). C NMR (150 MHz, CD3OD):164.9, 164.5, 154.1, 133.4, 132.1,
130.6, 129.7, 129.1, 129.0, 128.4, 126.9, 126.3, 124.1, 123.9, 122.5, 121.6, 61.8, 43.0. HRMS (EI) calculated
for C21H14N2O3S ([M]+): 374.0725. Found: 374.0717.
6-(Benzo[d]oxazol-2-yl)-2-(2-(dimethylamino)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(9).
Compound 4 (0.1000 g, 0.2889 mmol), benzoxazole (0.103 g, 0. 864 mmol), Pd(PPh3)4 (0.0033 g, 0.0029
mmol), Cu(OAc)2・H2O (0.0115 g, 0.0576 mmol) and Na2CO3 (0.061 g, 0.576 mmol) in toluene (3 mL).
Purification by flash column chromatography (NH-SiO2, EtOAc/Hexane=1/81/4; Rf=0.3,
1
EtOAc/Hexane=1/4) afforded a yellow-green solid (0.0742 g, 0.193 mmol). Yield: 67%. Mp 238242 oC. H
NMR (600 MHz, d6-DMSO): 9.86 (d, J=8.4 Hz, 1H), 8.70 (d, J=7.7 Hz, 1H), 8.65 (d, J=7.9 Hz, 1H), 8.62
(d, J=7.1 Hz, 1H), 8.09 (t, J=7.8 Hz, 1H), 7.99 (d, J=7.8 Hz, 7.91 (d, J=8.0 Hz, 1H), 7.56 (t, J=7.4 Hz, 1H),
13
7.51 (t, J=7.7 Hz, 1H), 4.18 (t, J=7.2 Hz, 2H), 2.57 (t, J=7.2 Hz, 2H), 2.23 (s, 6H). C NMR (150 MHz,
d6-DMSO):163.2, 162.8, 160.4, 149.7, 141.5, 132.4, 131.2, 130.0, 129.5, 128.9, 128.3, 128.1, 127.7, 126.8,
12

125.3, 124.6, 122.7, 120.5, 111.2, 59.4, 45.4, 37.8. HRMS (ESI) Calculated for C23H20N3O3 ([M+H]+)
386.1505. Found: 386.1492.
6-(Benzo[d]thiazol-2-yl)-2-(2-(dimethylamino)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(10).
Compound 4 (0.1595 g, 0.5 mmol), benzothiazole (0.212 g, 1.0 mmol), Pd(OAc)2 (0.001 g, 0.005 mmol),
PPh3 (0.0656, 1mmol), Cu(OAc)2・H2O (0.0199 g, 0.1 mmol) and Na2CO3 (0.111 g, 1.0 mmol) in toluene (8
mL). Purification by flash column chromatography (NH-SiO2, CH2Cl2/Hexane=1/61/41/2; Rf=0.35,
CH2Cl2/Hexane=1/3) afforded a yellow-green solid which was then washed with ether to produce 10 (0.151 g,
o
1
0.40 mmol). Yield: 81%. Mp 231234 C. H NMR (600 MHz, CD3OD): 9.41 (d, J=8.5 Hz, 1H), 8.64 ( t,
J=6.1 Hz, 2H), 8.25 (d, J=7.5 Hz, 1H), 8.15 (d, J=8.1 Hz, 1H), 8.08 (d, J=8.0 Hz, 1H), 7.91 (t, J=8.0 Hz,
1H), 7.61 (t, J=7.5 Hz, 1H), 7.53 (t, J=7.6 Hz, 1H), 4.55 (t, J=5.4 Hz, 2H), 3.57 (t, J=5.4 Hz, 2H), 3.05 (s,
13
6H). C NMR (150 MHz, CD3OD): 167.1, 166.0, 165.6, 155.5, 137.8, 136.9, 134.8, 133.1, 131.8, 131.2,
130.4, 130.3, 129.6, 128.3, 127.7, 125.1, 124.9, 123.8, 123.1, 57.7, 44.5, 36.9. HRMS (ESI) calculated for
C23H19N3O2S ([M+H]+) 402.1276. Found: 402.1264.
4.3. Anti-cancer assay
4.3.1. Cell lines and mice
Murine B16F10 cell lines maintained in Dulbecco’s modified Eagle’s medium (DMEM), 10%
heat-inactivated fetal calf serum, 2mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 g/ml)
o
were combined at 37 C in a 5% CO2 humidified atmosphere. Female C57BL/6 mice (6 to 8 weeks old)
13

were purchased from the National Laboratory Animal Center (Taipei, Taiwan). All animal experiments were
performed under specific pathogen-free conditions and in accordance with guidelines approved by the
Animal Care and Usage Committee of Mackay Memorial Hospital (Taipei, Taiwan). The body weight of
each mouse was measured at the beginning of treatment and every day during the treatment period.
4.3.2. Cell viability assay
5 x 103 B16F10 cells were seeded into 96-well plates and treated with the following: DMSO control or
combination of various concentrations of amonafide (purchased from Abcam) with compounds 5-10 for 2
days. For the MTS assay (Promega), 40 µl of MTS reagent was added into each well. Cells were incubated
at 37 oC for 4 h. Absorbance was detected at OD490 nm.
4.3.3. DNA intercalation assay
DNA intercalation was examined using topoisomerase II drug screening kit (TopoGen) according to the
manufacturer’s instructions. Intercalation reaction mixtures containing supercoiled pHOT-1 plasmid DNA
and topoisomerase II enzyme in the presence of various concentrations of indicated compounds were
incubated at 37 °C for 30 min. The sample contained supercoiled DNA and topoisomerase II in the presence
of 100 M etoposide VP-16 served as positive control. The reactions were terminated with 10% sodium
dodecyl sulfate, followed by proteinase K treatment for 15 min at 37 °C. Samples were separated by
electrophoresis on a 1% agarose gel and stained with EtBr. DNA bands were visualized by ultraviolet light.
14

4.3.4. Tumor treatment study
In order to generate lung metastases, 6- to 8-week-old female C57BL/6 mice were injected intravenously
with 2x105 B16F10 cells, a dose consistently yielding lung metastases in 100% of animals.
Compoundsor10 in an amount of 2mg/kg or 5% DMSO vehicle control was administered ip daily on
days 1-14 to test the effect of the compounds on tumor metastasis. The amount of tumor seeding was
determined based on the numbers of black nodules visible in the lungs under microscopy.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The authors gratefully acknowledged the Ministry of Science and Technology (MOST 104-2113-M-032-002)
and Tamkang University for financial support. The authors also thank the instrument centers of National
Taiwan University, National Tsing Hua University and National Chung Hsing University for their aid in
performing LRMS/HRMS experiments. Part of this project was funded with supports from MacKay
Memorial Hospital (Projects # MMH-E-103-08 and MMH-E-104-08)
Supplementary data
13
Supplementary data (1H and C NMR data) for all the compounds associated with this article can be found
in the online version.
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Table of Content
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