Novel naphthalimide-amino acid conjugates with flexible leucine moiety as side chain: Design, synthesis and potential antitumor activity

Article abstract of DOI:10.1016/j.bmc.2008.11.080

A series of novel naphthalimide derivatives with flexible leucine side chains 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.bmc.2008.11.080

Bioorganic & Medicinal Chemistry 17 (2009) 592–599
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel naphthalimide–amino acid conjugates with flexible leucine moiety
as side chain: Design, synthesis and potential antitumor activity
*
Aibin Wu, Yufang Xu, Xuhong Qian
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 leucine side chains were designed and synthe-
sized. 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 moderate anti-
tumor activities with the IC50 values of 10ꢀ6–10ꢀ5 M. More importantly, compounds 8a–c exhibited
exclusive antitumor activities against MCF-7 cell line. The interaction between compound 8b and BSA
was also investigated. DNA binding experiments showed that these derivatives behaved as DNA interca-
lating agents. This work provided a novel class of naphthalimide-based lead compounds with exclusive
antitumor activities against MCF-7 cell line for further optimization.
Received 17 October 2008
Revised 24 November 2008
Accepted 26 November 2008
Available online 7 December 2008
Keywords:
Naphthalimide
Leucine
Antitumor
MCF-7
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
uptake of antitumor agents.25 Therefore, a possible strategy for
increasing the transport of lipophilic compounds of biological inter-
The design of highly efficient antitumor agents is of significant
importance in organic and medicinal chemistry. Naphthalimide-
based anticancer drugs constitute an indispensable part in the
development of anticancer drugs. Since the first lead compound,
Amonafide (Fig. 1), was discovered by Braña et al.,1,2 plenty of
naphthalimide derivatives were synthesized and their antitumor
activities were evaluated upon a variety of murine and human tu-
mor cell lines.3–7 However, most of them were abandoned because
of poor therapeutic index and unpredictable toxicity.5,8–10 From
then on, much attention was paid to the modification of naphthal-
imide skeleton. The modified derivatives were characterized by
fused multi-aromatic ring system and one or two flexible basic side
chains.6,7,11–15 Although the significant results have been obtained
in this way, there were some problems on the poor solubility and
complicated synthesis of the derivatives. It was necessary for us to
attempt alternative structural modification of naphthalimide.
In recent years, various drug–amino acid conjugates were
reported, such as anthraquinone (Fig. 1),16,17 doxorubicin,18,19
metal complex,20–25 nitrosourea/nitrogen mustard,26 vinblastin,27
hydroxymethylacylfulvene,28 benzothiazole,29 imidazotetrazines,30
methionine-enkephalin,31 camptothecins,32 bile acid,33 and
9-hydroxyellipticinium (Fig. 1)34. The promising results were
obtainedinthisway. Itwas wellknownthataminoacidswere essen-
tial starting materials for cell growth and building blocks for protein
synthesis. Furthermore, amino acids could also improve the cell
est across cell membrane, was the conjugation with amino acids.34
This inspired us to assume that naphthalimide conjugated with
flexible leucine moiety as side chain might have some improved or
different biological activity.
As shown in Figure 2, the naphthalimide scaffold was utilized as
key prototype structural unit, and the leucine, arylamine and
aliphatic amine functional groups were conjugated to the naphtha-
lene ring. The amino substituents introduced to the position-6 of
naphthalimide ring, contrary to Amonafide, would be difficult to
be acetylated35 and the side effects might be reduced. The intro-
duction of arylamine to leucine moiety might result in favorable
solubility and pharmacologic profiles.36,37 Furthermore, the intro-
duction of thio group, known to facilitate the interaction of naph-
thyl heterocycles with DNA,38 might lead to a concomitant increase
in the cytotoxicity against tumor cell lines. Therefore, the 6-amino
substituted benzo[de]isochromene-1,3-dione-leucine conjugates 7
and 8 were prepared and their antitumor activities were evaluated
against a variety of cancer cell lines. The preliminary results
showed that most of the compounds had moderate antitumor
activities with the IC50 values of 10ꢀ6–10ꢀ5 M. More importantly,
compounds 8a–c exhibited the exclusive antitumor activities
against MCF-7 cell line. As all knew that serum albumin, the most
abundant protein in plasma, functioned in the binding and
transportation of various ligands such as fatty acids, hormones,
and drugs.39 The distribution, free concentration, and metabolism
of these ligands strongly depended on their binding properties
with serum albumin.40,41 Therefore, the interaction between
compound 8b and bovine serum albumin (BSA) has been
* Corresponding author. Tel./fax: +86 021 64252603.
E-mail address: xhqian@ecust.edu.cn (X. Qian).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.080

A. Wu et al. / Bioorg. Med. Chem. 17 (2009) 592–599
593
investigated in order to understand its unique antitumor property.
Their spectra and calf thymus DNA (ct-DNA) binding properties
were also studied.
and MTT tetrazolium dye assay46 against HeLa (human cervical
carcinoma cell line), P388 (murine leukemia cell line), HL-60 (Hu-
man promyelocytic leukemia cell line), MCF-7 (Human caucasian
breast adenocarcinoma cell line), HCT-8 (human ileocecal adeno-
carcinoma cell line) and A375 (human melanoma cell line), respec-
2. Results and discussion
tively. The IC50 represented the drug concentration (lM) required
to inhibit cell growth by 50% and the results were summarized in
2.1. Synthesis and spectra data
Table 2.
From Table 2, it could be seen that the conjugates showed mod-
erate cytotoxicities against tested cancer cell lines except for HCT-
8. Compounds 7a, 7b and 7c demonstrated the highest cytotoxici-
ties against MCF-7, A375 and A375 cell lines with IC50 values of
The synthetic routes of the designed compounds N-(substituted
aryl)-2-(6-(2-(dimethylamino) ethylamino)-1,3-dioxo-1H-benzo
[de]isoquinolin-2(3H)-yl)-4-methylpentanamide 7a–c and N-
(substituted aryl)-2-(1,3-dioxo-6-(2-(phenylthio)ethylamino)-1H-
benzo[de]isoquinolin-2(3H)-yl)-4-methylpentanamide 8a–c were
shown in Scheme 1. 6-Nitrobenzo[de]isochromene-1,3-dione 142
8.64, 6.51 and 6.60
pounds 8a–c showed the exclusive cytotoxicities against MCF-7
with IC50 values of 24.55, 9.45 and 12.56 M, respectively. Com-
lM, respectively. More importantly, com-
l
was treated with L-leucine methyl ester hydrochloride in EtOH to
pounds 8a–c with phenylthioethylamino substituent instead of
dimethylaminoethylamino substituent at the position-6 of naph-
thalimide ring, showed a striking contrast compared to 7a–c, indi-
cating that the position-6 of naphthalimide was a crucial active
site. The introduction of appropriate substituents to the position-
6 of naphthalimide could obviously improve the antitumor selec-
tivity. Moreover, the cytotoxicities of compounds 8a–c were weak-
er than those of 7a–c against cancer cell lines. The reason was
possibly that the nitrogen atoms at amino side chain of 7a–c could
be protonated to different extent under physiological pH, and form
hydrogen bond with DNA bases, which might contribute to their
cytotoxicities.47–49 Additionally, we found that the antitumor
activities of the target compounds were influenced by the arylami-
no substituents on the leucine moiety. When the substituents were
o-dianisidine and sulfanilamide groups, relatively favorable antitu-
mor activities were obtained. It was well known that antitumor
activity of one compound was determined by many factors includ-
ing cell membrane crossing ability, protein binding and transporta-
tion, DNA binding ability, drug metabolism etc., once these factors
were effectively balanced, higher antitumor potency was possi-
ble.13 Therefore, manipulation of naphthalimide–leucine conju-
gates to achieve a appreciable properties for potent antitumor
drugs, seemed to be closely dependent on structures and confor-
mation of the side chain substituents.
afford intermediate 2. The following nucleophilic substitution reac-
tionbetween 4-nitronaphthalimideandvarious aliphaticaminesled
to the substrates 3 and 4, which were subjected to hydrolysis to
obtain the key intermediates 5 and 6, respectively. The subsequent
condensation reaction of 5 and 6 with various arylamines afforded
the target compounds 7a–c and 8a–c with moderate yields of 55–
65%, respectively. The structures of all the newly synthesized com-
pounds were well identified by 1H NMR, 13C NMR, HRMS, EI and IR
spectra.
The UV–vis and fluorescent data of the target compounds were
measured and shown in Table 1. It was found that the compounds
with different substituents had slight difference on absorption but
significant effects on emission. The absorption and emission max-
ima of these compounds were around 436 nm and 525 nm, respec-
tively, while the fluorescence quantum yields of these compounds
were dramatically changed with different substituents. This phe-
nomenon could be well explained by photo-induced electron
transfer (PET) effects.43
2.2. Cytotoxic evaluation in vitro
The in vitro antitumor activities of the target compounds were
evaluated by examining their cytotoxic effects using sulforhoda-
mine B (SRB) assay45 against A549 (human lung cancer cell line)
N
H
N
HO
X
X
O
O
HN
HN
R
R
O
N
O
O
HO
N
+
N
N
H
N
H
NH2
anthraquinone-amino acid conjugate
amonafide
9-hydroxyellipticinium-Leu conjugate
Figure 1. The structures of some representative antitumor compounds.
hydrophobic chain
Leucine moiety
O
O
O
R
N
N
HN
O
O
conjugate with leucine scaffold
R2
different aromatic ring changes
HN
R1
naphthalimide
6-position amino group substitution
Figure 2. The design strategy of naphthalimide–leucine conjugates.

594
A. Wu et al. / Bioorg. Med. Chem. 17 (2009) 592–599
COOH
O
COR
O
COOCH3
O
N
O
O
N
O
N
c
d
b
HN
HN
HN
COOCH3
N O
N
N
N
O
O
O
O
3
5
7a-c
a
COOCH3
N O
COOH
O
COR
NO2
NO2
1
2
O
N
O
N
O
O
b
c
d
O2
S
HN
HN
HN
7a, 8a : R = HN
HN
NH2
NH2
S
S
S
7b, 8b : R =
H3CO
7c, 8c : R = HN
OCH3
SO2NH2
6
8a-c
4
Scheme 1. Reagents and conditions: (a)
L-leucine methyl ester hydrochloride, EtOH, Et3N, reflux 1 h, 90% yield; (b) corresponding amine, DMF, rt, 24 h, 60% yield; (c)
LiOHꢂH2O, THF–MeOH–H2O, 50 °C overnight, 96% yield; (d) corresponding arylamine, CHCl3, EDCI, DMAP, rt, 48 h, 55–65% yield.
Table 1
ment of representative compound 7c were illustrated in Figure 3A,
B and Figure 4, respectively.
Absorption and emission dataa,b of naphthalimide–leucine conjugates
It was obvious that both absorption and emission of compound
7c exhibited dramatic changes with increasing ct-DNA concentra-
tion (Fig. 3). From the analyses of the relationship between the
fluorescence intensities and ct-DNA concentrations by nonlinear
curve fitting methods,50–53 these significant fluorescence changes
made the association steady constants of 7c with ct-DNA available
with 4.92 ꢁ 104 Mꢀ1; Also, absorption spectra demonstrated slight
bathochromic shifts (1–3 nm) and strong hypochromicities (12.8–
57.6%). Furthermore, the increase in viscosity of ct-DNA solution
was observed versus the increase in concentration of 7c (Fig. 4),
indicating that the lengthening, unwinding and stiffening of the
helix which increased the viscosity of the solution, resulted from
the intercalation of compound 7c into ct-DNA.11,49 These collec-
tively demonstrated that the derivatives behaved as DNA interca-
lating agents. Derivatives that strongly binding ct-DNA, were
high cytotoxic agents.49 (data not listed). The roles of leucine moi-
ety in the DNA binding were well under way.
Compound
UV kmax (log
e
)
FL kmax (U)
7a
7b
7c
8a
8b
8c
436 (4.31)
437 (4.31)
436 (4.28)
436 (4.35)
440 (4.33)
434 (3.87)
527 (0.0151)
525 (0.0073)
524 (0.0126)
530 (0.2820)
529 (0.0230)
533 (0.2600)
a
In absolute ethanol.
b
With quinine sulfate in 0.1 mol/L sulfuric acid as quantum yield standard
(
U
= 0.58).44
Table 2
Cytotoxicity of naphthalimide–leucine conjugates against HeLa, A549, P388, HL-60,
MCF-7, HCT-8 and A375 cell lines
Cytotoxicity (IC50 lM)
HeLaa
A549b
P388a
HL-60a
MCF-7a
HCT-8a
A375a
7a
7b
7c
8a
8b
8c
>50
6.74
8.09
>50
>50
>50
17.20
25.08
>50
>50
>50
>50
7.67
7.23
>50
>50
>50
>50
8.64
15.02
8.65
24.55
9.45
12.56
>50
>50
>50
>50
>50
>50
>50
6.51
6.60
>50
>50
>50
22.79
17.83
>50
>50
>50
2.4. BSA binding properties
The interaction between compounds 8a–c and BSA have been
investigated in order to understand their unique antitumor proper-
ties. The UV–vis and fluorescence spectra of representative com-
pound 8b were illustrated in Figure 5A and B.
As shown in Figure 5, slight increase in absorption and signifi-
cant increase in emission occurred with increasing BSA concentra-
tion. The variations of absorbance and emission intensity were
14.9% and 676%, respectively. The dramatic increase of fluores-
cence intensity showed that the albumin proteins introduced
motional restriction on the drug molecule in the proteinous envi-
ronments54 and such a ‘rigidification’ decreased the vibrational–
rotational processes that coupled the excited and ground states,
thereby decreasing the radiationless decay rate and increasing
>50
a
Cytotoxicity (CTX) against cancer cell line was measured by microculture tet-
razolium–formazan method after 48 h.46
b
CTX against cancer cell line was measured by sulforhodamine B dye-staining
method after 72 h.45
2.3. DNA binding properties
The affinities of the target compounds for ct-DNA were
determined by spectroscopic technique50–53 and viscosity measure-
ment.11,49 The fluorescence, UV–vis spectra and viscosity measure-

A. Wu et al. / Bioorg. Med. Chem. 17 (2009) 592–599
595
250
200
150
100
50
220
200
180
160
140
120
100
80
0.15
0.10
0
60
0
0
20 40 60 80 100 120 140 160 180 200
[ct-DNA] /uM
DNA
DNA
180
180
0.05
7c
7c
0.00
0
500
550
600
650
700
750
300
400
500
600
Wavelength (nm)
Wavelength (nm)
Figure 3. Titration curves of compound 7c (10
l
M) in 20 mM Tris–HCl buffer (pH 7.5, 25 °C) containing different concentrations of ct-DNA from 0 to 180
l
M. (A)
Fluorescence emission spectra. Inset: fluorescence intensity as a function of ct-DNA concentration. (B) UV–vis absorption spectra.
interaction.57 It was obvious that hydrophobic substituents of
compounds 8a–c proved to be a key structural unit for their bind-
ing abilities to BSA, which might provide some suggestions on the
understanding of their exclusive antitumor selectivity against
MCF-7 cell line. Detailed interaction mode, mechanism and inter-
action–antitumor activity relationships were still under way.
1.025
1.020
1.015
1.010
1.005
1.000
3. Conclusion
In summary, a series of novel naphthalimide derivatives with
flexible leucine side chains were designed and synthesized, and
their antitumor activities were evaluated against a variety of can-
cer cell lines in vitro. The preliminary results showed that most of
the derivatives had moderate antitumor activities. More impor-
tantly, compounds 8a–c exhibited the exclusive antitumor activi-
ties against MCF-7 cell line. The interaction between compound
8b and BSA might provide some suggestions on the understanding
of their exclusive antitumor selectivity against MCF-7 cell line.
DNA binding studies showed that these derivatives behaved as
DNA intercalating agents. The present work demonstrated that
assembling the biological active unit of naphthalimide and leucine
might be able to result in a novel class of lead compounds with po-
tential antitumor activities. This was the first report about the
naphthalimide–leucine conjugates and their exclusive antitumor
activities against MCF-7 cell line as we have known. Further struc-
tural optimization and structure–antitumor activity relationships
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
[7c] / [ct-DNA]
Figure 4. Effects of increasing amount of compound 7c on the relative viscosities of
ct-DNA at 25 ( 0.1) °C. [ct-DNA] = 100 M in 20 mM Tris–HCl buffer (pH 7.5). and
0 represented the viscosity of ct-DNA in the presence and absence of compound 7c,
respectively.
l
g
g
the fluorescence quantum yield of the protein–bound mole-
cule.55,56 Also, the slight increase of absorbance proved the moder-
ate interaction between naphthalimide ring and BSA. Therefore,
these spectral changes were caused by compound–BSA complex
formation, in which compound 8b was strongly bound at the
hydrophobic positions of the BSA supported by the hydrophobic
120
100
0.4
80
100
60
40
80
0.3
y = 101.8 -82.49exp(-x/435.5)
R2 = 0.9695
20
1200
60
0
0
200 400 600 800 1000 1200
[BSA] / (ug/mL)
0.2
0.1
0.0
BSA
1200
BSA
0
40
20
0
0
8b
8b
300
400
500
600
500
550
600
650
700
750
Wavelength (nm)
Wavelength (nm)
Figure 5. Titration curves of compound 8b (10
lM) in 20 mM HEPES buffer (pH 7.2, 25 °C) containing different concentrations of BSA from 0 to 1200 lg/mL. (A) UV–vis
absorption spectra, (B) fluorescence emission spectra. Inset: fluorescence intensity as a function of BSA concentration.

596
A. Wu et al. / Bioorg. Med. Chem. 17 (2009) 592–599
about the designed naphthalimide–leucine conjugates were under
the way.
tively, in 20 mM HEPES buffer (pH 7.2) and DMSO (1% by volume).
The solution in a final volume was 10 mL, which was used for
spectrophotometric titration experiments. An equilibrium period
of 15 min for constant oscillation at 25 °C in the dark of the mixed
solution was allowed before recording each spectrum.
4. Experimental
4.1. Materials and methods
4.4. Synthesis
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, TMS as internal standard). IR spectra were ob-
tained using a Perkin–Elmer 2000 FTIR instrument. High-resolu-
tion 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. Absorption spec-
tra were determined on PGENERAL TU-1901UV–VIS spectropho-
tometer. Fluorescence spectroscopic studies were performed with
a Hitachi F-4500. Column chromatography was performed using
silica gel 200–300 mesh.
4.4.1. Methyl 4-methyl-2-(6-nitro-1,3-dioxo-1H-benzo[de]
isoquinolin-2(3H)-yl)pentanoate (2)
Compound 1 (300 mg, 1.234 mmol), prepared by using the pre-
viously reported method,42 was dissolved in EtOH (5 mL), then
L-
leucine methyl ester hydrochloride (269 mg, 1.480 mmol) and
Et3N (150 mg, 1.480 mmol) were added. The solution was stirred
and refluxed under nitrogen for 60 min, and then cooled and con-
centrated under vacuum. The residue was subjected to column
chromatography on silica gel by using CH2Cl2/CH3OH 60:1 (v/v)
as eluent, affording 2 (411 mg, 1.111 mmol, 90%) as orange-yellow
solid. mp 105.2–107.3 °C; 1H NMR (400 MHz, CDCl3): d 8.863 (d,
J = 8.8 Hz, 1H), 8.753 (d, J = 7.2 Hz, 1H), 8.707 (d, J = 8.0 Hz, 1H),
8.420 (d, J = 8.0 Hz, 1H), 8.012 (t, J = 8.0 Hz, 1H), 5.774 (q,
J1 = 4.0 Hz, J2 = 8.8 Hz, 1H), 3.732 (s, 3H), 2.282–2.214 (m, 1H),
2.150–2.082 (m, 1H), 1.584 (s, br, 1H), 1.014 (d, J = 6.0 Hz, 3H),
0.933 (d, J = 6.0 Hz, 3H); MS (EI+) calcd for C19H18N2O6 [M+]
370.4, found 370.1.
4.2. DNA binding experiments
4.2.1. Preparation of ct-DNA solution
Ct-DNA (highly polymerized) was purchased from Sigma Al-
drich. Solutions of ct-DNA in 10 mM Tris–HCl, 1 mM EDTA buffer
(pH 7.5) gave a ratio of UV absorbance at 260 nm and 280 nm of
1.8–1.9:1, indicating that the ct-DNA was sufficiently free of pro-
tein. The concentration of ct-DNA was determined spectrophoto-
metrically assuming the molar absorption was 6600 Mꢀ1 cmꢀ1
(260 nm).58 The solution was stored at 4 °C and used after no more
than four days. Doubly distilled water was used to prepare the buf-
fer solution.
4.4.2. General procedure for the preparation of methyl 2-(6-(2-
substituted amino)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-
yl)-4-methylpentanoate
Compound 2 (200 mg, 0.540 mmol) was dissolved in DMF
(2 mL), the corresponding amine (5.400 mmol) was added. The
solution was stirred at room temperature under nitrogen for
24 h, and then concentrated under vacuum. The residue was sub-
jected to column chromatography on silica gel by using CH2Cl2/
CH3OH 40:1 (v/v) as eluent, affording the corresponding products
as orange solid.
4.2.2. DNA titration experiments
The concentrations of compounds and ct-DNA were 10
lM and
0–180 M, respectively, in 20 mM Tris–HCl, 50 mM NaCl buffer
l
(pH 7.5) and DMSO (1% by volume). The solution in a final volume
was 10 mL, which was used for spectrophotometric titration
experiments. An equilibrium period of 6 h for constant oscillation
at 25 °C in the dark of the mixed solution was allowed before
recording each spectrum. The association constants (Ka) were
4.4.2.1. Methyl 2-(6-(2-(dimethylamino)ethylamino)-1,3-dioxo-
1H-benzo[de]isoquinolin-2(3H)-yl)-4-methylpentanoate (3).
Yield: 60%. mp 57.5–59.5 °C; 1H NMR (400 MHz, CDCl3): d 8.576
(d, J = 7.2 Hz, 1H), 8.461 (d, J = 8.4 Hz, 1H), 8.179 (d, J = 8.4 Hz, 1H),
7.633 (t, J = 8.4 Hz, 1H), 6.678 (d, J = 8.4 Hz, 1H), 6.372 (s, br, 1H),
5.798 (q, J1 = 4.8 Hz, J2 = 9.2 Hz, 1H), 3.703 (s, 3H), 3.396 (q,
J1 = 4.8 Hz, J2 = 11.2 Hz, 2H), 2.750 (t, J = 6.0 Hz, 2H), 2.350 (s, 6H),
2.258–2.187 (m, 1H), 2.142–2.071 (m, 1H), 1.633–1.532 (m, 1H),
1.011 (d, J = 6.4 Hz, 3H), 0.916 (d, J = 6.8 Hz, 3H); MS (EI+) calcd
for C23H29N3O4 [M+] 411.5, found 412.2.
derived according to the equation I = I0 + {(I
ꢀ I0)/2[Q]0}.
1
{([DNA]0 + [Q]0 + 1/Ka) ꢀ {([DNA]0 + [Q]0 + 1/Ka)2 ꢀ 4[DNA]0[Q]0}1/2},
wherein I0, I, and I represented the fluorescence intensities of
1
compounds alone, the sample, and DNA totally bound, respec-
tively. [DNA]0 and [Q]0 were the initial analytical concentrations
of ct-DNA and the agents, respectively.50
4.4.2.2. Methyl 2-(1,3-dioxo-6-(2-(phenylthio)ethylamino)-1H-
benzo[de]isoquinolin-2(3H)-yl)-4-methylpentanoate (4).
Yield: 60%. mp 67.6–69.6 °C; 1H NMR (400 MHz, CDCl3): d 8.689
(s, 1H), 8.566 (s, 1H), 8.131 (s, 1H), 7.741 (s, 1H), 7.652 (s, 2H),
7.519–7.471 (m, 4H), 6.775 (s, 1H), 5.999 (s, 1H), 3.920 (s, 3H),
3.807 (s, 2H), 3.532 (s, 2H), 2.394–2.313 (m, 2H), 1.781 (s, 1H),
1.199 (s, 3H), 1.105 (s, 3H); MS (EI+) calcd for C27H28N2O4S [M+]
476.6, found 476.2.
4.2.3. Viscosity measurements
Viscosity measurements were carried out using an Ubbelodhe
viscometer at 25 ( 0.1) °C in thermostated bath. Flow times were
measured with a digital stopwatch and each sample was measured
three times and an average flow time was calculated. Data were pre-
sented as (
to that of ct-DNA, where
g/g0)
1/3 versus the ratio of the concentration of compound
g
and g0 represented the viscosityof ct-DNA
solution in the presence and absence of compound 7c, respectively.
4.4.3. General procedure for the preparation of methyl 2-(6-(2-
substituted amino)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-
yl)-4-methylpentanoic acid
4.3. BSA binding experiments
BSA (lyophilized powder) was purchased from Sigma Aldrich.
The stock solution was prepared at concentration of 40 mg/mL
in 20 mM HEPES (N-[2-hydroxyethyl]-piperazine-N0-[2-ethanesul-
phonic acid]) buffer (pH 7.2, 25 °C) and stored at 4 °C and used
after no more than four days for equilibrium. The concentrations
Compound 3/4 (0.540 mmol) was dissolved in THF/MeOH/H2O
4:1:1 (v/v/v), then LiOHꢂH2O (45 mg, 1.080 mmol) was added.
The solution was heated at 50 °C overnight and concentrated to
half of the volume, neutralized with 1 M HCl. The precipitate was
collected by filtration and dried in vacuum to give the desired
product (0.518 mmol, 96%) as orange solid.
of compounds and BSA were 10 lM and 0–1200 lg/mL, respec-

A. Wu et al. / Bioorg. Med. Chem. 17 (2009) 592–599
597
4.4.3.1. 2-(6-(2-(Dimethylamino)ethylamino)-1,3-dioxo-1H-benzo-
[de]isoquinolin-2(3H)-yl)-4-methylpentanoic acid (5). Yield: 96%.
J = 8.4 Hz, 1H), 6.491 (s, 1H), 5.952 (dd, J1 = 6.0 Hz, J2 = 8.8 Hz,
1H), 3.911 (s, 3H), 3.791 (s, 3H), 3.394 (dd, J1 = 4.8 Hz,
J2 = 10.4 Hz, 2H), 2.764 (t, J = 6.0 Hz, 2H), 2.361 (s, 6H), 2.322–
2.215 (m, 2H), 1.687–1.620 (m, 1H), 1.053 (d, J = 6.8 Hz, 3H),
0.989 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 168.33,
164.72, 164.04, 149.99, 148.25, 147.48, 137.23, 135.54, 135.09,
131.79, 131.63, 130.00, 127.03, 126.33, 124.67, 122.57, 120.33,
120.06, 119.57, 119.19, 115.03, 109.49, 109.30, 108.45, 104.40,
67.96, 56.84, 55.93, 55.60, 54.06, 44.95, 40.17, 37.84, 25.79,
23.23, 22.33; FTIR (KBr, cmꢀ1): 3355, 2955, 2859, 1680, 1647,
1576, 1535, 1509, 1450, 1357, 1238, 1112, 1031, 774; HRMS
(ES+) calcd for C36H41N5O5Na ([M+Na])+ 646.3005, found 646.3005.
mp 205.5–207.5 °C; 1H NMR (400 MHz, DMSO-d6):
d 8.738 (d,
J = 8.4 Hz, 1H), 8.442 (d, J = 7.2 Hz, 1H), 8.278 (d, J = 8.4 Hz, 1H), 7.861
(s, br, 1H), 7.703 (t, J = 8.0 Hz, 1H), 6.859 (d, J = 8.8 Hz, 1H), 5.547 (d,
J = 6.8 Hz, 1H), 3.623 (d, J = 5.2 Hz, 2H), 2.957 (s, br, 2H), 2.491 (s, 6H),
2.010 (t, J = 6.8 Hz, 2H), 1.449–1.384 (m, 1H), 0.903 (d, J = 6.4 Hz, 3H),
0.819 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz, DMSO-d6): d 172.27,
164.05, 163.17, 150.99, 135.06, 131.61, 129.95, 129.53, 125.00,
122.02, 120.70, 108.12, 104.70, 56.25, 51.41, 44.45, 38.18, 25.46,
23.53, 22.43; MS (EI+) calcd for C22H27N3O4 [M+] 397.5, found 398.2.
4.4.3.2.
2-(1,3-Dioxo-6-(2-(phenylthio)ethylamino)-1H-benzo
[de]isoquinolin-2(3H)-4-methylpentanoic acid (6). Yield:
96%. mp 206.0–208.0 °C; 1H NMR (400 MHz, DMSO-d6): d 12.542
(s, br, 1H), 8.650 (d, J = 8.4 Hz, 1H), 8.443 (d, J = 7.2 Hz, 1H), 8.235
(d, J = 8.8 Hz, 1H), 7.989 (t, J = 5.2 Hz, 1H), 7.705 (t, J = 8.0 Hz, 1H),
7.408 (d, J = 8.0 Hz, 2H), 7.317 (t, J = 7.6 Hz, 2H), 7.193 (t,
J = 7.2 Hz, 1H), 6.759 (d, J = 8.8 Hz, 1H), 5.552 (t, J = 6.8 Hz, 1H),
3.636 (q, J = 6.4 Hz, 2H), 3.332 (m, 2H), 2.004 (t, J = 7.2 Hz, 2H),
1.451–1.384 (m, 1H), 0.903 (d, J = 6.8 Hz, 3H), 0.819 (d, J = 6.4 Hz,
3H); 13C NMR (100 MHz, DMSO-d6): d 172.13, 164.02, 163.13,
150.87, 135.93, 135.06, 131.62, 130.00, 129.59, 129.30, 129.12,
126.48, 125.01, 122.01, 120.60, 108.02, 104.57, 51.29, 42.71,
31.38, 25.42, 23.52, 22.42; MS (EI+) calcd for C26H26N2O4S [M+]
462.6, found 463.1.
4.4.4.3.
2-(6-(2-(Dimethylamino)ethylamino)-1,3-dioxo-1H-
benzo[de]isoquinolin-2(3H)-yl)-4-methyl-N-(4-sulfamoylphe-
nyl)pentanamide (7c). Yield: 60%. Orange red solid: mp 136.6–
138.6 °C.1H NMR (400 MHz, DMSO-d6): d 9.835 (s, 1H), 8.701 (d,
J = 8.4 Hz, 1H), 8.468 (d, J = 7.6 Hz, 1H), 8.287 (d, J = 8.4 Hz, 1H),
7.786 (s, br, 1H), 7.751–7.690 (m, 5H), 7.202 (s, 2H), 6.879 (d,
J = 8.8 Hz, 1H), 5.631 (dd, J1 = 4.8 Hz, J2 = 9.2 Hz, 1H), 3.659 (d,
J = 4.8 Hz, 2H), 3.062 (s, br, 2H), 2.596 (s, br, 4H), 2.596 (s, 6H),
2.245–2.176 (m, 1H), 1.940–1.871 (m, 1H), 1.488–1.404 (m, 1H),
0.926 (d, J = 6.4 Hz, 3H), 0.840 (d, J = 6.4 Hz, 3H); 13C NMR
(100 MHz, DMSO-d6): d 169.43, 164.51, 163.81, 150.69, 142.54,
138.73, 134.79, 131.44, 130.18, 129.18, 126.73, 124.98, 122.79,
120.79, 120.20, 109.07, 104.58, 52.89, 44.19, 37.45, 25.35, 23.62,
22.60; FTIR (KBr, cmꢀ1): 3281, 3096, 2948, 2859, 1680, 1648,
1580, 1535, 1450, 1361, 1242, 1153, 830, 774; HRMS (ES+) calcd
for C28H34N5O5S ([M+H])+ 552.2281, found 552.2244.
4.4.4. General procedure for the preparation of N-(substituted
aryl)-2-(6-(2-(dimethylamino)ethylamino)-1,3-dioxo-1H-
benzo[de]isoquinolin-2(3H)-yl)-4-methylpentanamide (7a–c)
To a solution of compound 5 (50 mg, 0.126 mmol) in CHCl3
(2 mL), the corresponding substituted arylamine (0.126 mmol),
EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride) (48 mg, 0.252 mmol) and DMAP (4-dimethylaminopyri-
dine) (31 mg, 0.252 mmol) were added. The solution was stirred
at room temperature under nitrogen for 48 h and concentrated.
The residue was purified on silica gel column chromatography by
using CH2Cl2/CH3OH 30:1 (v/v) as the eluent, affording 7a–c as or-
ange/khaki solid. Yield 55–65%.
4.4.5. General procedure for the preparation of N-(substituted
aryl)-2-(1,3-dioxo-6-(2-(phenylthio)ethylamino)-1H-benzo
[de]isoquinolin-2(3H)-yl)-4-methylpentanamide (8a–c)
To a solution of compound 6 (50 mg, 0.108 mmol) in CHCl3
(2 mL) the corresponding substituted arylamine (0.108 mmol),
EDCI (42 mg, 0.216 mmol) and DMAP (27 mg, 0.216 mmol) were
added. The solution was stirred at room temperature under nitro-
gen for 48 h and concentrated. The residue was purified on silica
gel column chromatography by using CH2Cl2/CH3OH 50:1 (v/v) as
the eluent, affording 8a–c as orange/khaki solid. Yield: 55–65%.
4.4.4.1. N-(4-(4-Aminophenylsulfonyl)phenyl)-2-(6-(2-(dimeth-
ylamino)ethylamino)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-
yl)-4-methylpentanamide (7a). Yield: 65%. Viscous orange
solid. 1H NMR (400 MHz, DMSO-d6): d 9.837 (s, 1H), 8.667 (d,
J = 8.4 Hz, 1H), 8.429 (d, J = 7.2 Hz, 1H), 8.249 (d, J = 8.8 Hz, 1H),
7.942 (s, 1H), 7.721–7.655 (m, 5H), 7.471 (d, J = 8.4 Hz, 2H), 6.803
(d, J = 8.8 Hz, 1H), 6.577 (d, J = 8.8 Hz, 2H), 6.093 (s, 2H), 5.595 (q,
J1 = 5.2 Hz, J2 = 8.8 Hz, 1H), 3.480 (dd, J1 = 6.0 Hz, J2 = 12 Hz, 2H),
2.575 (t, J = 6.8 Hz, 2H), 2.220 (s, 6H), 2.185–2.151 (m, 1H),
1.887–1.817 (m, 1H), 1.465–1.398 (m, 1H), 0.914 (d, J = 6.4 Hz,
3H), 0.824 (d, J = 6.4 Hz, 3H); 13C NMR (100 MHz, DMSO-d6): d
169.58, 164.48, 163.43, 153.82, 151.08, 143.37, 137.38, 134.97,
131.37, 130.27, 129.57, 128.99, 127.80, 126.54, 124.81, 122.74,
120.57, 120.45, 113.39, 108.34, 104.32, 57.36, 52.79, 45.79, 41.44,
38.02, 25.29, 23.60, 22.63; FTIR (KBr, cmꢀ1): 3355, 2948, 2859,
1680, 1647, 1580, 1535, 1450, 1357, 1290, 1242, 1142, 1101,
830, 771, 682, 548; HRMS (ES+) calcd for C34H37N5O5SNa
([M+Na])+ 650.2413, found 650.2392.
4.4.5.1. N-(4-(4-Aminophenylsulfonyl)phenyl)-2-(1,3-dioxo-6-
(2-(phenylthio)ethylamino)-1H-benzo[de]isoquinolin-2(3H)-yl)-
4-methylpentanamide (8a). Yield: 65%. Orange yellow solid.
mp 135.5–137.5 °C. 1H NMR (400 MHz, DMSO-d6): d 9.840 (s,
1H), 8.640 (d, J = 8.0 Hz, 1H), 8.431 (d, J = 7.2 Hz, 1H), 8.211 (d,
J = 8.4 Hz, 1H), 7.951 (t, J = 5.6 Hz, 1H), 7.713–7.674 (m, 5H),
7.470 (d, J = 8.8 Hz, 2H), 7.407 (d, J = 7.6 Hz, 2H), 7.321 (t,
J = 8.0 Hz, 2H), 7.198 (t, J = 7.2 Hz, 1H), 6.737 (d, J = 8.8 Hz, 1H),
6.577 (d, J = 8.8 Hz, 2H), 6.093 (s, 2H), 5.591 (q, J1 = 5.2 Hz,
J2 = 9.2 Hz, 1H), 3.631 (d, J = 6.0 Hz, 2H), 2.219–2.149 (m, 1H),
1.887–1.817 (m, 1H), 1.479–1.396 (m, 1H), 0.912 (d, J = 6.4 Hz,
3H), 0.816 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz, DMSO-d6): d
169.56, 164.45, 163.43, 153.82, 150.66, 143.33, 139.48, 138.93,
134.83, 130.41, 130.22, 129.81, 129.10, 128.98, 127.80, 126.50,
124.93, 122.73, 120.64, 120.45, 113.39, 108.78, 104.38, 52.60,
42.64, 37.99, 31.37, 25.28, 23.52, 22.63; FTIR (KBr, cmꢀ1): 3362,
2940, 2918, 2837, 1680, 1639, 1576, 1535, 1517, 1383, 1361,
1287, 1242, 1138, 1101, 830, 771, 685, 548; HRMS (ES+) calcd for
C38H36N4O5S2Na ([M+Na])+ 715.2025, found 715.2034.
4.4.4.2. N-(40-Amino-3,30-dimethoxybiphenyl-4-yl)-2-(6-(2-(di-
methylamino)ethylamino)-1,3-dioxo-1H-benzo[de]isoquinolin-
2(3H)-yl)-4-methylpentanamide (7b).Yield: 55%. Viscous khaki
solid. 1H NMR (400 MHz, CDCl3): d 8.547 (d, J = 7.2 Hz, 1H),
8.434–8.402 (m, 2H), 8.314 (s, 1H), 8.231 (d, J = 8.4 Hz, 1H), 8.023
(s, 1H), 7.593 (t, J = 8.0 Hz, 1H), 7.113 (dd, J1 = 1.6 Hz, J2 = 8.4 Hz,
1H), 6.995–6.974 (m, 3H), 6.748 (d, J = 8.4 Hz, 1H), 6.624 (d,
4.4.5.2. N-(40-Amino-3,30-dimethoxybiphenyl-4-yl)-2-(1,3-
dioxo-6-(2-(phenylthio)ethylamino)-1H-benzo[de]isoquinolin-
2(3H)-yl)-4-methylpentanamide (8b). Yield: 55%. Viscous kha-
ki solid. 1H NMR (400 MHz, CDCl3): d 8.461 (d, J = 8.4 Hz, 1H), 8.415
(s, 1H), 8.264–8.216 (m, 2H), 8.030 (d, J = 8.0 Hz, 1H), 7.416 (d,

598
A. Wu et al. / Bioorg. Med. Chem. 17 (2009) 592–599
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J = 7.6 Hz, 2H), 7.325 (t, J = 8.0 Hz, 1H), 7.287–7.192 (m, 3H), 7.145
(d, J = 8.4 Hz, 1H), 7.042–7.007 (m, 3H), 6.781 (d, J = 8.0 Hz, 1H),
6.420–6.390 (m, 1H), 6.323 (s, 1H), 5.949 (q, J1 = 5.6 Hz,
J2 = 8.8 Hz, 1H), 3.918 (s, 3H), 3.904 (s, 3H), 3.530 (d, J = 5.6 Hz,
2H), 3.244 (t, J = 6.4 Hz, 2H), 2.407–2.338 (m, 1H), 2.199–2.130
(m, 1H), 1.670–1.604 (m, 1H), 1.063 (d, J = 6.8 Hz, 3H), 0.991 (d,
J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 168.77, 164.53,
163.89, 149.65, 148.37, 147.54, 137.59, 135.48, 134.75, 134.59,
131.83, 131.27, 130.44, 129.49, 129.17, 126.85, 126.10, 124.62,
122.01, 120.07, 119.98, 119.64, 119.20, 115.11, 109.46, 109.36,
108.56, 104.00, 56.00, 55.62, 53.90, 42.63, 38.06, 32.76, 25.83,
23.29, 22.33; FTIR (KBr, cmꢀ1): 3355, 2955, 2859, 1680, 1648,
1576, 1535, 1506, 1454, 1387, 1361, 1283, 1238, 1164, 1023,
771; HRMS (ES+) calcd for C40H40N4O5SNa ([M+Na])+ 711.2617,
found 711.2614.
4.4.5.3. 2-(1,3-Dioxo-6-(2-(phenylthio)ethylamino)-1H-benzo
[de]isoquinolin-2(3H)-yl)-4-methyl-N-(4-sulfamoylphenyl)pen-
tanamide (8c). Yield: 60%. Orange yellow solid. mp 120.9–
122.9 °C. 1H NMR (400 MHz, DMSO-d6): d 9.822 (s, 1H), 8.659 (d,
J = 8.4 Hz, 1H), 8.445 (d, J = 7.2 Hz, 1H), 8.225 (d, J = 8.4 Hz, 1H),
7.988 (t, J = 5.2 Hz, 1H), 7.939 (s, 1H), 7.723–7.685 (m, 5H), 7.409
(d, J = 7.6 Hz, 2H), 7.322 (t, J = 7.2 Hz, 2H), 7.216–7.179 (m, 3H),
6.748 (d, J = 8.8 Hz, 1H), 5.622 (dd, J1 = 4.8 Hz, J2 = 8.8 Hz, 1H),
3.636 (dd, J1 = 6.4 Hz, J2 = 12.8 Hz, 2H), 3.306 (s, 2H), 2.224–2.167
(m, 1H), 1.930–1.861 (m, 1H), 1.452–1.420 (m, 1H), 0.921 (d,
J = 6.4 Hz, 3H), 0.836 (d, J = 6.4 Hz, 3H); 13C NMR (100 MHz,
DMSO-d6): d 163.47, 158.69, 158.38, 158.08, 157.78, 150.68,
142.54, 138.72, 135.94, 134.82, 131.39, 130.04, 129.61, 129.09,
126.71, 126.49, 124.93, 122.78, 120.66, 120.23, 119.19, 116.80,
108.82, 104.37, 52.84, 42.65, 37.96, 31.36, 25.86, 23.68, 22.59; FTIR
(KBr, cmꢀ1): 3407, 3266, 2955, 2925, 1677, 1576, 1535, 1469, 1368,
1205, 1149, 849, 797, 722, 604, 518; HRMS (ES+) calcd for
C32H32N4O5S2Na ([M+Na])+ 639.1712, found 639.1704.
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4.5. Cytotoxic evaluation in vitro
The target compounds were submitted to the Chinese National
Center for Drug Screening and School of Pharmacy in East China
University of Science and Technology for in vitro antitumor activity
assays. Growth inhibitory effects on the cell lines (HeLa, P388,
HL-60, MCF-7, HCT-8 and A375) were measured by using MTT
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by sulforhodamine B (SRB) assay.45
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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).
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