Unprecedented synthesis, in vitro and in vivo anti-cancer evaluation of novel triazolonaphthalimide derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.11.025

An efficient synthesis method for fusing triazole ring onto the naphthalimide core was described. The anti-cancer activities of the generated triazolonaphthalimide derivatives were evaluated with five cancer cell lines. The compounds generally displayed higher potency than amonafide. 4d,4e carrying two amino side chains showed the strongest cytotoxicities. N-oxide 5, a prodrug of 4a, was designed and synthesized. The agent was expected to be activated under the hypoxic condition in tumor tissue. Compared with 4a, 5 manifested much lower cytotoxicity both in cancer cell lines and human normal cells in the in vitro assays. However, N-oxide 5 performed potent anti-cancer activity in vivo using S-180 sarcoma bearing mice. All the results suggested that 5 was a promising anti-cancer agent.

Full text of DOI:10.1016/j.ejmech.2011.11.025

European Journal of Medicinal Chemistry 47 (2012) 546e552
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Unprecedented synthesis, in vitro and in vivo anti-cancer evaluation of novel
triazolonaphthalimide derivatives
*
*
Shasha Li, Wenhe Zhong, Zhongjun Li , Xiangbao Meng
State Key Laboratory of Natural and Biomimetic Drugs; Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient synthesis method for fusing triazole ring onto the naphthalimide core was described. The
anti-cancer activities of the generated triazolonaphthalimide derivatives were evaluated with five cancer
cell lines. The compounds generally displayed higher potency than amonafide. 4d,4e carrying two amino
side chains showed the strongest cytotoxicities. N-oxide 5, a prodrug of 4a, was designed and synthe-
sized. The agent was expected to be activated under the hypoxic condition in tumor tissue. Compared
with 4a, 5 manifested much lower cytotoxicity both in cancer cell lines and human normal cells in the
in vitro assays. However, N-oxide 5 performed potent anti-cancer activity in vivo using S-180 sarcoma
bearing mice. All the results suggested that 5 was a promising anti-cancer agent.
Received 27 August 2011
Received in revised form
18 October 2011
Accepted 11 November 2011
Available online 20 November 2011
Keywords:
Naphthalimide
Triazole
Ó 2011 Elsevier Masson SAS. All rights reserved.
N-oxide
DNA intercalator
Anti-cancer
1. Introduction
modification of employing different aromatic heterocycles to the
naphthalimide core; such investigation generally obtained
Since 1973, naphthalimides have received most attention and
been investigated broadly as DNA intercalators, generally showed
high anti-cancer activities against a broad spectrum of cell lines,
and also displayed manifold mechanisms of action besides acting as
improved anti-cancer activities and/or lower side effect [5e11].
Some representative modifications were showed in Fig. 2.
Triazole is an important class of heterocycles and occupies
a momentous position in the field of organic and pharmaceutical
chemistry, owning to its broad spectrum of biological activities
[12e14]. Derivatives of benzotriazole were broadly employed in
antifungal [15], antidepressant [16], antilipolysis [17], antibacterial
[18], anti-cancer agents [19,20], etc. The introduction of triazole
moiety to naphthalene core can lead to a larger intercalating plane
and higher capacity of hydrogen bonding because of the nitrogen
atoms in the triazole ring, which increased the affinity for DNA to
obtain higher cytotoxic activity. The synthesis of benzotriazole is
rather difficult, and mainly through azide-alkyne cycloaddition
[21e24] or metal catalyzed triazene intramolecular cyclization
[25,26]. Herein we designed and synthesized a series of tri-
azolonaphthalimides (4aee, 40aee, Scheme 1) via a novel method,
which was treating amonafide and its analog using nitrosonium
tetrafluoroborate (NOBF4) and amines under mild conditions.
Furthermore, we focused on enhancing the selectivity between
cancer cells and normal cells, which existed as a fundamental cause
for low therapeutic efficacy in the chemotherapeutic field. One
tactic was to develop tumor-activated prodrug, which can be trig-
ged in tumor tissue but stayed non-toxic in normal cells [27]. A
unique property of solid tumor cells was its hypoxia because of
topoisomerase
Ⅱ poisons. Amonafide, elinafide and bisnafide
(Fig. 1) were three comparatively successful naphthalimide deriv-
atives which acceded to different phases in clinical trial as potential
anti-cancer agents. Superior to most anti-tumor agents, naph-
thalimides haven’t been affected by multi-drug resistance accord-
ing to all clinical trials. However, heretofore no naphthalimide
derivative has ever reached the antineoplastic market because of
dose-limiting toxicity [1,2]. It is thus meaningful and valuable for
more investigation with naphthalimide.
One approach to enhance the cytotoxicity of naphthalimides
was to fuse one or more aromatic rings to naphthalene core. This
modification was first explored by Sami et al in 1993 [3] who re-
ported
a series of compounds synthesized from anthracene
nucleus, which showed significantly improved potency than amo-
nafide, such as the representative compounds azonafide and
ethonafide [4]. Since then, many groups were devoted into the
* Corresponding authors. Tel.: þ86 10 82801714; fax: þ86 10 82805496.
E-mail addresses: zjli@bjmu.edu.cn (Z. Li), xbmeng@bjmu.edu.cn (X. Meng).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.11.025

S. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 546e552
547
[29]. Thus, to fulfill this requirement, we chose 4a to prepare the
corresponding N-oxide derivative 5 to evaluate its activity in cancer
cells, normal cells in vitro and S-180 sarcoma bearing ICR mice
in vivo.
2. Chemistry
To obtain the target compounds (4aee, 40aee), we first
synthesized the precursor 3 and 30 following three steps according
to the reported methods [31,32] (Scheme 1). First, commercially
available 1,8-naphthalic acid anhydride was treated with 65% HNO3
in concentrate H2SO4 to give compound 1. The reduction of 1 can be
achieved by adding in portion to a stirring SnCl2eHCl (conc.)
solution and refluxing at 80 ꢀC for several hours. Then the mixture
was filtrated and purified by washing with 1M HCl and water
subsequently to obtain amine 2. Amonafide 3 and its analog 30 were
prepared after treatment of 2 with N,N-dimethylethylenediamine
or N,N-dimethyl-1,3-propanediamine in refluxing ethanol,
respectively.
Fig. 1. Representative naphthalimide derivatives in clinical trial.
poorly organized tumor vasculature [28], this feature was often
employed in developing prodrugs which depending on hypoxia to
be activated. Oxidation of tertiary amine was a common strategy to
afford anti-cancer prodrug, because N-oxide can be bioreduced to
the corresponding amine under the hypoxic condition in tumor
cells and displayed cytotoxicity, while stable in normal cells
[11,29,30]. Qian et al reported a novel series of N-oxide of naph-
thalimides as prodrug in 2007 [11], and another article about novel
aliphatic N-oxide of naphthalimides as fluorescent makers recently
The formation of compound 4 from 3 was in fact a happy acci-
dent. At the beginning, we were trying to synthesize triazene using
Fig. 2. Aromatic heterocycle fused naphthalimide derivatives.

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S. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 546e552
Scheme 1. Reagents and conditions: (i) HNO3 (65%), conc. H2SO4, 0ꢀC-rt; (ii) SnCl2, conc. HCl (37%), 80ꢀC; (iii) corresponding amine, EtOH, reflux; (iv) a: NOBF4, CH3CN, N2, -5ꢀC;
b: corresponding amine, Et2O/H2O, -5ꢀC- 0ꢀC.
NOBF4 to produce nitrene intermediate, which would be trapped by
amine to give triazene product [33e36], but instead we only got the
ring-fused structure 4a. The mechanism of this reaction was still
not clear, we can only speculate that triazene was first formed as
intermediate, then through electrophilic intramolecular cycliza-
tion, triazole was produced. Because of steric hindrance, the
angularly fused isomer was predominant over the linear one, which
was verified by the NMR data. Considering the various bioactivities
of triazole, we decided to synthesize a series of triazolonaph-
thalimide derivatives taking advantage of this unique trans-
formation by employing various amines (4aee, 40aee). The
reactions underwent smoothly with aliphatic amines, but were
frustrated when anilines were used. N-aryl substituted product
6a,b, instead of the triazole derivatives, were obtained (Scheme 2).
The mechanism and scope of this reaction are still under
investigation.
Scheme 3. Reagents and conditions: CH2Cl2, m-CPBA, 0ꢀC, 0.5 h; then rt, 0.5 h.
Table 1
IC50 values of triazolonaphthalimide compounds over five cancer cell lines.a
Compound
IC50 (mM)
N-oxide 5 can be readily synthesized from 4a by oxidation with
m-CPBA in CH2Cl2. It should be noted that 1 eq K2CO3 and MeOH
should be added to quench the reaction after the reaction was
finished, otherwise, the yield would be very low (Scheme 3).
HCT-116
PC-3
1.58
4.77
4.29
0.73
0.71
U87 MG
Hep G2
SK-OV-3
4a
4b
4c
4d
0.65
1.82
1.42
0.19
0.15
1.35
6.34
7.73
0.39
0.35
1.24
0.62
>10.0
4.55
0.45
1.46
1.22
0.13
0.14
1.14
6.32
4.31
0.37
0.30
2.52
0.62
>10.0
2.80
0.68
1.01
1.07
0.17
0.18
0.97
5.77
5.21
0.78
0.29
0.90
0.41
>10.0
1.41
1.17
2.55
2.53
0.35
0.40
2.00
8.93
9.64
0.64
0.46
2.34
1.32
>10.0
8.83
4e
3. Results and discussion
40a
3.21
40b
15.49
24.98
3.28
1.20
1.62
0.41
>10.0
8.05
40c
3.1. Cytotoxic activity
40d
40e
We determined the cytotoxic potency of compounds 4, 40, 5 and
6 against five cancer cell lines, which were HCT-116 (colorectal
carcinoma), PC-3 (prostate carcinoma), U87 MG (brain tumor), Hep
G2 (liver cancer), SK-OV-3 (ovarian cancer),with the in vitro cell
growth assay. The results were listed in Table 1. All the compounds
6a
6b
5
amonafide
a
MTT assays were used for evaluation, and values were expressed as mean IC50 of
the triplicate experiment.
except 40b,c and 5 exhibited much stronger anti-cancer activities
than the parental amonafide by several to dozens folds. The results
demonstrated that fusing one more ring onto the naphthalene core
increased the DNA binding potency and enhanced anti-cancer
ability of the amonafide derivatives.
Table 2
IC50 values of compounds 4a and 5 against human normal cells L-02 and HK-2
proliferation.a
Compound
IC50 (mM)
L-02
HK-2
0.99
107.7
4a
5
1.77
141.9
a
Scheme 2. Synthesis condition of N-aryl substituted amonafide: a. NOBF4, CH3CN, N2,
MTT assays were used for evaluation, and values were expressed as mean IC50 of
the triplicate experiment.
-5ꢀC; b. corresponding amine, Et2O/H2O, -5ꢀC- 0ꢀC.

S. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 546e552
549
Fig. 3. Inhibitory effects of 4a and 5 against sarcoma S-180 bearing ICR mice. Each compound was injected intraperitoneally every day over seven consecutive days: model (0.9% phys-
iological saline), ST (amonafide, 30 mg/kg), 4a (7.5 mg/kg, 15 mg/kg, 30 mg/kg), 5 (30 mg/kg, 45 mg/kg, 60 mg/kg). The weights of mice were recorded every two days (left line graphs).
Sarcoma S-180 were excised and weighed 24h after the last time of injection (right column graphs). Each value was the meanꢁS.E.of ten data. TTest: **, p<0.01;***, p<0.001 vs Model.
Comparing to compounds 40aee, 4aee generally showed better
anti-cancer activities, respectively. The result indicated that the
length of the side chain is important for the cytotoxicity of the
compounds. It also proved the best length of the side chain is two
carbons between the two nitrogen atoms [1]. Compared to 4a
which has only a methyl group attached to the triazole moiety, 4b
and 4c have longer alkyl chains (n-butyl and hydroxyethyl) and
showed weaker cytotoxicities. 4d,e, which possessed a terminal
amino group (N,N-dimethylethanamine and N,N-dimethylpropan-
1-amine) to the triazole moiety, exhibited better potency than
4aec. These results indicated that two basic amino side chains can
significantly improve the cytotoxicity of the naphthalimides.
Compounds 6a and 6b which are N-phenyl amonafide also showed
delightful activities [37,38]; especially 6b which has a comparable
activity with 4a. Further investigation is undertaking by synthe-
sizing more analogs and evaluating their cytotoxicities.
possibly transformed to the active form. A study using sarcoma S-
180 bearing mice was performed; mice body weights and S-180
tumor weights were used as evaluating indicators which were
illustrated in Fig. 3. To examine the efficiency of prodrug 5, both 4a
and 5 were evaluated in three dose levels (7.5, 15 and 30 mg/kg for
4a, 30, 45 and 60 mg/kg for 5, intraperitoneal). Compared with
amonafide (ST, 30 mg/kg), 4a showed similar tumor growth
suppression activity when administered at low dose (7.5 mg/kg,
26.25% suppression). However, when the dose was increased to 15
and 30 mg/kg, mice were died before termination of the tests.
Obviously, 4a was more toxic to the animals than the parental
amonafide. 5 displayed dose-dependent tumor suppression activity
when administered at 30, 45 or 60 mg/kg once a day, with 4.83%,
46.18% or 62.74% tumor growth suppression compared with the
control group, respectively.
For compound 5 to achieve a comparable tumor suppression
activity to amonafide, a much higher dosage (60 mg/kg) is needed.
This phenomenon is possible due to the insufficient in vivo bio-
reduction of the prodrug [11]. Low bioreductive efficiency resulted
low transformation of compound 5 to 4a, which is actually the
effective agent to inhibit hypoxic tumor growth. However, taking
account of the complexity of in vivo metabolism process, the exact
cause for low tumor suppression activity of prodrug 5 still need
further investigation. Nevertheless, all the data suggest that N-
oxide 5 is an efficient and low toxic anti-cancer prodrug and has the
value for more investigation to improve the therapeutic efficacy.
3.2. Prodrug designing and testing
Compound 4a displayed stronger cytotoxicity than amonafide,
which encouraged us to prepare its prodrug analog N-oxide 5 and
measure the potency of 5. N-oxide 5 showed poor cytotoxicity
against all five cell lines as we predicted (Table 1), revealed that it
was unable to be bioreduced to amine form in the cancer cells. We
subsequently conducted a proliferative inhibition assay with human
normal liver cells (L-02) and renal proximal tubular epithelial cells
(HK-2). As shown in Table 2, compound 4a with a tertiary amine side
chain manifested obvious toxic effect on L-02 and HK-2 with IC50s of
4. Conclusion
1.77 and 0.99 mM, respectively. The N-oxide 5 showed a remarkable
decrease in cytotoxicity by about 100 folds. It indicated that N-oxide
5 would be a promising prodrug if only N-oxide can be bioreduced
to compound 4a in the hypoxic condition of tumor tissues.
In summary, a series of triazolonaphthalimide derivatives were
synthesized via a novel method and displayed much potent cyto-
toxicities than the lead compound amonafide. The preliminary SAR
revealed that 1) by fusing a triazole ring onto the naphthalene core
can increase the intercalating plane and significantly improve the
cytotoxic activity and 2) activity would be enhanced if the triazole
ring possessed another basic amino side chain. The in vitro and
in vivo invistigations suggest that N-oxide 5, a prodrug synthesized
from 4a, could be bioreduced to its precursor amine form 4a and
3.3. In vivo anti-cancer activity evaluation
Since N-oxide 5 showed low cytotoxic activity in the in vitro
assay, the next step was to employ an in vivo assay to investigate if
compound 5 has the in vivo anti-cancer activity, since it could be

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S. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 546e552
displayed dose-dependent tumor growth suppression activity. All
the data demonstrated that this novel series of triazolonaph-
thalimides merited further investigation and derivatization, and
the N-oxide 5 could be used as promising prodrug which possessed
selectivity for hypoxic cancer cells, so an improved therapeutic
efficacy.
J ¼ 7.8 Hz), 7.97 (1H, t, J ¼ 7.9 Hz), 5.09 (2 H, t, J ¼ 7.3 Hz), 4.34
(2 H, t, J ¼ 7.0 Hz), 2.69 (2 H, t, J ¼ 7.0 Hz), 2.37 (6 H, s), 2.16e2.06
(2 H, m), 1.52 (2 H, m), 1.04 (3 H, t, J ¼ 7.4 Hz). 13C NMR (100 MHz,
DMSO): 163.3, 162.9, 142.9, 132.0, 130.1, 129.3, 127.9, 126.9, 124.2,
122.9, 119.6, 118.3, 56.2, 52.8, 45.1 (2C), 37.6, 30.7, 19.1, 13.4. HR-
ESI-MS: Calcd for C20H23N5O2 [M
366.1925.
þ
H]þ: 366.1927; Found:
5. Experimental section
5.1.5. 5-[3-(Dimethylamino)propyl]-10-butylbenzo[de] [1e3]
triazolo[4,5-g]isoquinoline-4,6(5H,10H)-dione (40b)
5.1. Synthesis
Brown solid, yield: 67%; m.p. 197e199 ꢀC. 1H NMR (400 MHz,
CDCl3) dH(ppm): 9.17 (1H, s), 8.73 (1H, d, J ¼ 7.4 Hz), 8.58 (1H, d,
J ¼ 8.1Hz), 7.97 (1H, t, J ¼ 7.9 Hz), 5.09 (2 H, t, J ¼ 7.3 Hz), 4.25
(2 H, t, J ¼ 7.3 Hz), 2.48 (2 H, t, J ¼ 7.2 Hz), 2.35e2.18 (6 H, m),
2.19e2.03 (2 H, m), 2.03e1.86 (2 H, m), 1.59e1.46 (2 H, m), 1.04
(3 H, t, J ¼ 7.4 Hz). 13C NMR (100 MHz, CDCl3): 163.8, 163.4,
144.0, 131.2, 130.5, 127.9, 127.8, 127.3, 125.8, 124.0, 120.2, 118.4,
57.2, 50.7, 45.2 (2C), 39.1, 31.2, 25.9, 19.8, 13.5. HR-ESI-MS: Calcd
for C21H25N5O2 [M þ H]þ: 380.2079; Found: 380.2081.
All the solvents are of analytical grade. CH3CN used in the
reaction was distilled with P2O5 according to the reported method.
1H NMR and 13C NMR spectra were recorded with Bruker AM-
400 MHz spectrometer. The chemical shifts were reported in ppm
using TMS as internal standard. High resolution mass spectrometry
data were measured on Bruker Apex IV FTMS. Melting points were
measured on an X-5 micro-melting point apparatus and were
uncorrected. Thin-layer chromatography (TLC) was developed on
commercial silica gel HSGF254 plates. Column chromatography
was conducted on silica gel 60 (E. Merck, 0.063e0.200 mm).
5.1.6. 5-[2-(Dimethylamino)ethyl]-10-(2-hydroxyethyl)benzo[de]
[1e3]triazolo[4,5-g]isoquinoline-4,6(5H,10H)-dione (4c)
5.1.1. General procedure for synthesis and purification of 4aee and
40aee
Pale yellow solid, yield: 63%; m.p. 202e204 ꢀC. 1H NMR
(400 MHz, CDCl3) dH(ppm): 8.86 (1H, s), 8.82 (1H, d, J ¼ 8.0 Hz), 8.62
(1H, d, J ¼ 7.0 Hz), 7.89 (1H, t, J ¼ 7.9 Hz), 5.15 (2 H, t, J ¼ 5.1Hz), 4.38
(1H, t, J ¼ 5.2 Hz), 4.15 (1H, t, J ¼ 6.9 Hz), 2.59 (2 H, t, J ¼ 6.8 Hz), 2.31
(7 H, br). 13C NMR (100 MHz, DMSO): 163.6, 163.2, 142.8, 132.0,
130.2, 129.4, 128.0, 127.0, 124.2, 122.9, 119.6, 118.3, 59.9, 56.2, 52.8,
45.1 (2C), 37.6. HR-ESI-MS: Calcd for C18H19N5O3 [M þ H]þ:
354.1561; Found: 354.1561.
To obtain compounds 4aee and 40aee, we first synthesized the
precursor compound 3 and 30 from commercially available 1,8-
naphthalic acid anhydride following three steps according to the
reference methods [31,32], illustrated above in Chemistry section.
Then compound 3 or 30 (0.5 mmol) was suspended in 5 mL dry
CH3CN at ꢂ5 ꢀC and cooled for 10 min under nitrogen atmosphere.
Then nitrosonium tetrafluoroborate (NOBF4, 1 mmol) was added
and stirred at ꢂ5 ꢀC for 1 h to obtain a clear solution. The resulting
mixture was added dropwise into a solution of corresponding
amine (4 mmol) in 3 mL Et2O/H2O (v/v, 10:1), and then raised the
temperature to 0 ꢀC. After the reaction was finished monitored by
TLC, 1 mmol Na2CO3 was added and the solution was stirred for
additional 30 min at room temperature. After removal of the
solvent in vacuum, the crude product was purified by column
chromatography (CH2Cl2/MeOH, 1e5& Et3N) to obtain compound
4 or 40, respectively (yield 58e95%).
5.1.7. 5-[3-(Dimethylamino)propyl]-10-(2-hydroxyethyl)benzo[de]
[1e3]triazolo[4,5-g]isoquinoline-4,6(5H,10H)-dione (40c)
Pale yellow solid, yield: 61%; m.p. 200e201 ꢀC. 1H NMR
(400 MHz, DMSO) dH(ppm): 8.98 (1H, d, J ¼ 8.3 Hz), 8.85 (1H, s),
8.52 (1H, d, J ¼ 7.3 Hz), 7.98 (1H, t, J ¼ 7.9 Hz), 5.19 (2 H, t, J ¼ 4.9 Hz),
4.09e4.00 (4 H, m), 2.89 (2 H, br), 2.54 (6 H, s), 2.07e1.89 (2 H, m).
13C NMR (100 MHz, DMSO): 164.0, 163.5, 143.3, 132.4, 130.6, 129.8,
128.4, 127.5, 124.6, 123.4, 120.2, 118.7, 59.9, 55.1, 52.8, 43.0 (2C), 37.7,
23.7. HR-ESI-MS: Calcd for C19H21N5O3 [M þ H]þ: 368.1716; Found:
368.1717.
5.1.2. 5-[2-(Dimethylamino)ethyl]-10-methylbenzo[de] [1e3]
triazolo[4,5-g]isoquinoline-4,6(5H,10H)-dione (4a)
5.1.8. 5,10-Bis[2-(dimethylamino)ethyl]benzo[de] [1e3]triazolo
[4,5-g]isoquinoline-4,6(5H,10H)-dione (4d)
Light yellow solid, yield: 95%; m.p. 214e216 ꢀC. 1H NMR
(400 MHz, DMSO) dH(ppm): 8.66 (1H, d, J ¼ 8.2 Hz), 8.55 (1H, s),
8.35 (1H, d, J ¼ 7.3 Hz), 7.86 (1H, t, J ¼ 7.8 Hz), 4.60 (3 H, s), 4.07 (2 H,
t, J ¼ 6.5 Hz), 2.58 (2 H, t, J ¼ 6.4 Hz), 2.30 (6 H, s). 13C NMR
(100 MHz, DMSO): 163.3, 162.9, 143.0, 131.7, 130.2, 129.0, 128.3,
126.6, 124.1, 122.8, 119.5, 118.3, 56.6, 45.6 (2C), 38.2, 38.1. HR-ESI-
MS: Calcd for C17H17N5O2 [M þ H]þ: 324.1455; Found: 324.1453.
Yellow solid, yield: 73%; m.p. 182 ꢀC. 1H NMR (400 MHz, CDCl3)
dH(ppm): 9.14 (1H, s), 8.70 (1H, dd, J ¼ 7.5, 0.8 Hz), 8.63 (1H, dd,
J ¼ 8.0, 0.8 Hz), 7.94 (1H, t, J ¼ 7.9 Hz), 5.16 (2 H, t, J ¼ 7.2 Hz), 4.32
(2 H, t, J ¼ 6.9 Hz), 2.99 (2 H, t, J ¼ 7.2 Hz), 2.67 (2 H, t, J ¼ 6.9 Hz),
2.36e2.35 (6 H, 2s). 13C NMR (100 MHz, CDCl3): 163.8, 163.4, 143.9,
131.5, 130.7, 127.9, 127.3, 125.9, 124.0, 120.3, 118.4, 57.8, 56.9, 49.5,
45.7, 38.5. HR-ESI-MS: Calcd for C20H24N6O2 [M þ H]þ: 381.2035;
Found: 381.2034.
5.1.3. 5-[3-(Dimethylamino)propyl]-10-methylbenzo[de] [1e3]
triazolo[4,5-g]isoquinoline-4,6(5H,10H)-dione (40a)
Yellow solid, yield: 68%; m.p. 207e208 ꢀC. 1H NMR (400 MHz,
CDCl3) dH(ppm): 9.11 (1H, s), 8.68 (2 H, dd, J ¼ 7.8, 0.8 Hz), 7.94 (1H,
t, J ¼ 7.7 Hz), 4.74 (3 H, s), 4.23 (2 H, t, J ¼ 7.6 Hz), 2.46 (2 H, t, J ¼
6.8 Hz), 2.27 (6 H, s), 2.00e1.81 (2 H, m). 13C NMR (100 MHz, CDCl3):
163.7, 163.3, 143.9, 131.9, 130.6, 127.9, 127.6, 127.2, 125.6, 123.9,
120.2, 118.6, 57.2, 45.2 (2C), 39.1, 38.0, 25.9. HR-ESI-MS: Calcd for
C18H19N5O2 [M þ H]þ: 338.1613; Found: 338.1612.
5.1.9. 10-[2-(Dimethylamino)ethyl]-5-[3-(dimethylamino)propyl]
benzo[de] [1e3]triazolo[4,5-g]isoquinoline-4,6(5H,10H)-dione
(40d)
Yellow solid, yield: 64%; m.p. 175e177 ꢀC. 1H NMR (400 MHz,
CDCl3) dH(ppm): 9.14 (1H, s), 8.72 (1H, d, J ¼ 7.8 Hz), 8.67 (1H, d,
J ¼ 7.8 Hz), 7.98 (1H, t, J ¼ 7.9 Hz), 5.18 (2 H, t, J ¼ 7.1Hz), 4.27
(2 H, t, J ¼ 7.3 Hz), 3.02 (2 H, t, J ¼ 7.1Hz), 2.85e2.71 (2 H, m), 2.52
(6 H, s), 2.38 (6 H, s), 2.16e2.01 (2 H, m). 13C NMR (100 MHz,
DMSO/CDCl3): 163.4, 163.0, 142.9, 131.1, 130.1, 128.8, 128.2, 127.0,
124.2, 123.1, 119.8, 118.0, 57.0, 54.7, 48.3, 45.4, 45.0, 42.4, 23.2. HR-
ESI-MS: Calcd for C21H26N6O2 [M þ H]þ: 395.2189; Found:
395.2190.
5.1.4. 5-[2-(Dimethylamino)ethyl]-10-butylbenzo[de] [1e3]triazolo
[4,5-g]isoquinoline-4,6(5H,10H)-dione (4b)
Yellow solid, yield: 73%; m.p. 204e207 ꢀC. 1H NMR (400 MHz,
CDCl3) dH(ppm): 9.16 (1H, s), 8.72 (1H, d, J ¼ 7.5 Hz), 8.57 (1H, d,

S. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 546e552
551
5.1.10. 5-[2-(Dimethylamino)ethyl]-10-[3-(dimethylamino)propyl]
5.2. Materials and methods for bioactivity assays
5.2.1. Animals
Male ICR mice weighing from 16 to 18 g were purchased from
Sino-British Sippr/Bk Lab.Animal Ltd.,Co. They were raised at
controlled environment with a 12 h lightedark cycle, drinking and
eating were provided ad libitum.
benzo[de] [1e3]triazolo[4,5-g]isoquinoline-4,6(5H,10H)-dione (4e)
Yellowish white solid, yield: 70%; m.p. 214e216 ꢀC. 1H NMR
(400 MHz, CDCl3) dH(ppm): 9.21 (1H, s), 8.78 (1H, d, J ¼ 7.9 Hz), 8.73
(1H, d, J ¼ 7.6 Hz), 7.94 (1H, t, J ¼ 7.9 Hz), 5.17 (2 H, t, J ¼ 8.0 Hz), 4.35
(2 H, t, J ¼ 7.6 Hz), 2.67 (2 H, t, J ¼ 7.2 Hz), 2.42 (2 H, t, J ¼ 6.5 Hz),
2.35 (6 H, s), 2.31e2.21 (8 H, br). 13C NMR (100 MHz, DMSO): 164.3,
163.9, 143.6, 131.9, 130.9, 129.8, 129.0, 127.7, 124.9, 123.7, 120.6,
118.7, 55.5, 54.6, 48.3, 43.5 (2C), 43.1 (2C), 36.2, 24.5. HR-ESI-MS:
Calcd for C21H26N6O2 [M þ H]þ: 395.2190; Found: 395.2190.
5.2.2. Preparation of test solutions
Each test solution for in vitro assay was prepared by diluting
with DMSO. Solutions for in vivo assay were dissolved by 0.9%
physiological saline to obtain a series of concentrations: ST (3 mg/
mL), 4a (0.75 mg/mL, 1.5 mg/mL, 3 mg/mL), 5 (3 mg/mL, 4.5 mg/mL,
6 mg/mL).
5.1.11. 5,10-Bis[3-(dimethylamino)propyl]benzo[de] [1e3]triazolo
[4,5-g]isoquinoline-4,6(5H,10H)-dione (40e)
Yellow solid, yield: 58%; m.p. 193e195 ꢀC. 1H NMR (400 MHz,
CDCl3) dH(ppm): 9.10 (1H, s), 8.76 (1H, d, J ¼ 8.0 Hz), 8.69 (1H, d,
J ¼ 7.6 Hz), 7.95 (1H, t, J ¼ 8.0 Hz), 5.16 (2 H, t, J ¼ 7.2 Hz), 4.30 (2 H, t,
J ¼ 8.4 Hz), 2.50 (2 H, t, J ¼ 7.2 Hz), 2.44 (2 H, t, J ¼ 6.4 Hz), 2.30e2.23
(14 H, br), 1.99e1.94 (2 H, m). 13C NMR (100 MHz, CDCl3): 163.6,
163.2,143.6,131.2,130.4,127.8,127.8,127.5,125.4,123.6,112.0,118.3,
57.0, 55.8, 48.9, 45.9, 45.5 (2C), 45.1 (2C), 27.6, 25.7. HR-ESI-MS:
Calcd for C22H28N6O2 [M þ H]þ: 409.2344; Found: 409.2346.
5.2.3. In vitro cytotoxicity evaluation
All cells used in the research were prepared at 3.5 ╳ 104 cells/mL
concentration and each 100 mL cells suspension was seeded in 96-
well cell imcroplate for 24 h (37 ꢀC, 5％CO2). Then each solution
was added and incubated for another 72 h. For the control group,
equivalent concentration of DMSO (final concentration 0.5%) was
added. MTT (3-[4,5-dimethylthiazol-2yl]-diphenyl tetrazolium
bromide) method was employed to measure the number of
surviving cells and recorded the OD value at 492nm/620 nm. The
IC50 values were calculated using Prism Graphpad software of the
triplicate experiment.
5.1.12. N,N-dimethyl-2-[10-methyl-4,6-dioxobenzo[de] [1e3]
triazolo[4,5-g]isoquinolin-5(4H,6H,10H)-yl]ethanamine oxide (5)
A solution of 4a (1 g, 3 mmol) in 50 mL CH2Cl2 was cooled for
5 min in an ice bath, added m-CPBA (0.57 g, 3.3 mmol) and stirred
for 0.5 h, then moved to room temperature for further 0.5 h. 1.1 eq
K2CO3 and 50 mL MeOH were added to quench the reaction and
stirred about 5 min, the solution became limpid. Then removed the
solvent and purificated with column chromatography (aluminum
oxide, neutral, 100e200 Å) can afford compound 5. Pale yellow
solid, yield: 95%; m.p. 236 ꢀC. 1H NMR (400 MHz, DMSO) dH(ppm):
9.29e8.89 (2 H, m), 8.64 (1H, d, J ¼ 7.3 Hz), 8.10 (1H, t, J ¼ 7.9 Hz),
4.77 (3 H, s), 4.58 (2 H, t, J ¼ 6.4 Hz), 3.99 (2 H, t, J ¼ 6.5 Hz), 3.56
(6 H, s). 13C NMR (100 MHz, DMSO): 163.1, 162.7, 142.6, 131.6, 130.1,
129.1, 128.1, 126.4, 124.1, 122.3, 119.1, 118.1, 64.7, 56.0, 42.8, 37.9,
33.9. HR-ESI-MS: Calcd for C22H28N6O2 [M þ H]þ: 340.1410; Found:
340.1411.
5.2.4. In vivo evaluation of anti-cancer activity
Sarcoma S-180 cells were inoculated hypodermically in male ICR
mice and grew for a week. The injection volume for the first batch
was 2.5 ╳ 106 cells/animal (used for compound 5), the second batch
3 ╳ 106 cells/animal (used for compound 4a). The rats were divided
into eight groups (ten rats/group) according to their weights:
model (0.9% physiological saline), ST (amonafide, dose 30 mg/kg,
positive control), 4a (three groups with 7.5 mg/kg,15 mg/kg, 30 mg/
kg, respectively), 5 (three groups with 30 mg/kg, 45 mg/kg, 60 mg/
kg, respectively). Each test compound was injected intraperitone-
ally once a day over seven consecutive days. The weights of rats
were recorded every two days. Sarcoma S-180 were excised and
weighed 24 h after the last time of injection. Both of the changes in
rats body weights and the sarcoma 180 tumor weights were
summarized for further analysis.
5.1.13. 2-[2-(dimethylamino)ethyl]-5-[(4-methoxylphenyl)amino]-
1H-benzo[de]isoquinoline-1,3(2H)-dione (6a)
Compound 6a and 6b was synthesized via the same procedure
for compounds 4 and 40. After the reaction completed, the product
was existed as precipitate in the resulted solution and could be
obtained by filtration then purified with column chromatography.
Yellow solid, yield: 41%; m.p. 168 ꢀC. 1H NMR (400 MHz, DMSO)
dH(ppm): 8.44 (1H, d, J ¼ 1.9 Hz), 8.36 (1H, d, J ¼ 8.2 Hz), 8.29 (1H, d,
J ¼ 7.2 Hz), 8.22 (1H, d, J ¼ 1.7 Hz), 7.78 (1H, t, J ¼ 7.8 Hz), 7.56 (2 H,
d, J ¼ 8.9 Hz), 7.04 (2 H, d, J ¼ 9.0 Hz), 4.15 (2 H, t, J ¼ 6.8 Hz), 3.81
(3 H, s), 2.52 (2 H, m), 2.22 (6 H, s). 13C NMR (100 MHz, DMSO):
163.8, 163.6, 158.9, 142.8, 142.1, 133.8, 133.1, 128.8, 128.2, 124.3,
123.7, 122.5, 121.8, 120.6, 116.1, 115.0, 56.9, 55.8, 45.8, 38.1. HR-ESI-
MS: Calcd for C23H23N3O3 [M þ H]þ: 390.1812; Found: 390.1812.
Acknowledgment
The authors thank a financial support from the PKUCare Phar-
maceutical R&D Center.
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