Novel DNA bis-intercalators of isoquinolino[4,5-bc]acridines: Design, synthesis and evaluation of cytotoxic activity

Article abstract of DOI:10.1016/j.tet.2005.09.065

Mono- and dinuclear isoquinolino[4,5-bc]acridine derivatives were designed and facilely synthesized, their DNA-binding affinities and cytotoxic activities were evaluated. A4 induced unwinding of supercoiled plasmid pBR 322 DNA by (36±2)°while A6 induced that by (41±1)°, both of which were higher than the mono-analogue A1 ((19±2)°). A6 exhibited the highest in vitro antitumor activity against human lung cancer cell (A549) and A4 was the most active one against murine leukemia cell (P388). DNA binding constant and molecular model indicated that both the length of linker chain and the distance of interchromophore were key impact factors for DNA binding affinity and biological activity.

Full text of DOI:10.1016/j.tet.2005.09.065

Tetrahedron 61 (2005) 11895–11901
Novel DNA bis-intercalators of isoquinolino[4,5-bc]acridines:
design, synthesis and evaluation of cytotoxic activity
Peng Yang,a Qing Yangb and Xuhong Qiana,c,
*
aState Key Laboratory of Fine Chemicals, Dalian University of Technology, PO Box 158, Dalian 116012, China
bDepartment of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116012, China
cShanghai Key Laboratory of Chemical Biology, East China University of Science and Technology, Shanghai 200237, China
Received 27 June 2005; revised 12 September 2005; accepted 16 September 2005
Available online 19 October 2005
Abstract—Mono- and dinuclear isoquinolino[4,5-bc]acridine derivatives were designed and facilely synthesized, their DNA-binding
affinities and cytotoxic activities were evaluated. A4 induced unwinding of supercoiled plasmid pBR 322 DNA by (36G2)8 while A6
induced that by (41G1)8, both of which were higher than the mono-analogue A1 ((19G2)8). A6 exhibited the highest in vitro antitumor
activity against human lung cancer cell (A549) and A4 was the most active one against murine leukemia cell (P388). DNA binding constant
and molecular model indicated that both the length of linker chain and the distance of interchromophore were key impact factors for DNA
binding affinity and biological activity.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
heterocycle modified bis-naphthalimide and bis-acridine
derivatives have been extensively studied for their
cytotoxicity, antitumor, and antiparasitic activities.5,6
However, the reports that could take full advantage of the
structural characteristics of both naphthalimide and acridine
chromophores seldom appeared. In our continuous attempt7
to develop high DNA binding affinity and highly active
antitumor agents, we designed and synthesized novel mono-
and bis-isoquinolino[4,5-bc]acridine derivatives A1–A6
(Figure 2). In this study, naphathalimide active group was
DNA intercalators have received much attention due to their
therapeutic potential in anticancer treatment.1 The recent
molecular design of novel antitumor agents is focused on
bisintercalating compounds, which present higher DNA
binding affinities and slower dissociation rates than the
corresponding monomers.2 Over the past two decades, a
great number of dimeric forms of DNA intercalators, such
as bis-acridinecarboxamides, bis-naphthalimides, and bis-
imidazoacridones (see Fig. 1), have been developed as
potential anticancer drugs.3
DNA intercalators usually exhibit a planar structure with at
least two annelated aromatic rings, also termed as
chromophores, and have one or two flexible basic side
chains such as polyamides. Many of these compounds were
reported to function as DNA-targeted topoisomerase I
and/or II inhibitors.4 Both naphthalimide and acridine
derivatives have proved valuable chromophores in anti-
tumor activity and various modifications have been
attempted to promote their bioactivities.4
The heterocyclic-fused larger ring system, in particular in
dimeric forms, was found to account for the high DNA
affinity and antitumor activity. As we know well, bis-
Keywords: Acridine; DNA-intercalation; Cytotoxicity.
*
Corresponding author. Tel.: C86 411 83673466; fax: C86 411
83673488; e-mail addresses: xhqian@dlut.edu.cn; xhqian@ecust.edu.cn
Figure 1. Structure of bis-naphthalimides (a), bis-acridinecarboxamides
(b), and bis-imidazoacridones (c).
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.09.065

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P. Yang et al. / Tetrahedron 61 (2005) 11895–11901
and directly carried out the ring closure reaction in
phosphorus oxychloride.10 The ring closure was proceeded
through the formation of a mixed anhydride followed by
intramolecular acylation and elimination of hydrochloric
acid.10 And then, it was poured into the ice-cold aqueous
solution of NH4OH, filtered, dried, and separated on silica
gel chromatography (CHCl3/MeOHZ9:1, v/v), to give
yellow product 3. Mention should be made here that this
ring closure reaction could reach satisfactory yields (88%)
of the desired product. Since the compound 3 was of the
characteristics of the strong electron-deficiency, the sub-
stituent chloride would be a good leaving group, which
made the compounds A2–A6 easily obtained by the reaction
with different polyamines and the yields were acceptable
(from 90 to 33%). However, polyamines were highly
hygroscopic and could adopt a different salt, which made
elementary analysis an inadequate method of measuring
the purity of these compounds.11 Thus, structures of the
obtained novel mono- and bisintercalators were confirmed
by IR, 1H NMR and H RMS after purification by silica gel
chromatography (CHCl3, MeOH, and NH4OH). And the
UV–vis and fluorescent spectra for these compounds were
measured and the data were shown in Table 1.
Figure 2. The structures of mono- and dinuclear isoquinolino[4,5-
bc]acridine derivatives A1–A6.
remained and the acridine unit was effectively fused with
electron-deficiency group. Also, several polyamine chains
The linkers of A2–A6 were designed to enable the bis-
acridine chromophores to extend over a distance of 7.3–
˚
ranging from 7.3–12.3 A were used as linkers to bridge two
˚
heterocyclic-fused acridine chromophores at the 8-site of
these compounds.8 This resulted structure therefore
distinguished our work from the common bisintercalating
mode of naphthalimide derivatives and was expected to
show higher DNA intercalative property and better
antitumor activity due to the presence of two intercalator
groups, naphthalimide and acridine.
12.3 A and thus could form the bisintercalated DNA
complex.8
2.2. DNA unwinding property
To illustrate the intercalating mode by the bis-acridine
derivatives, we investigated their unwinding ability of
supercoiled closed circular DNA pBR322 by electrophor-
etic mobility measurements on 2% agarose gels. Figure 3
showed the difference of electrophoretic mobility between
the intact supercoiling pBR322 and the treated pBR322
DNA by A1, A4, and A6 in the range of 0.005–0.090 molar
ratios of intercalator per nucleotide (r). The DNA
unwinding angle was calculated from the Eq. (1):12
2. Results and discussion
2.1. Synthesis and spectra
The facile syntheses of these compounds were outlined in
Scheme 1. To avoid the imidation of naphthalic anhydride
by 2-aminobenzoic acid, 4-bromo-1, 8-naphthalic anhy-
dride, the starting material, was reacted with N,N-
dimethylethylenediamine in ethanol for 2 h as the initial
reaction step. Then, the naphthalimide derivative 1 was
coupled with 2-aminobenzoic acid in DMF, catalyzed by
CuI and Cu.9 The obtained intermediate 2 was not purified
f Z 18 s=rðcÞ (1)
where s is the superhelical density, r(c) is the molar ratio of
intercalator per nucleotide at the coalescence point.
In Figure 3, the DNA unwinding angles for compounds A1,
A4, and A6 were (19G2)8, (36G2)8, (41G1)8, respectively.
Scheme 1. Synthesis of mono- and bis-isoquinolino[4,5-bc]acridines derivatives (a) N,N-dimethylethylenediamine, ethanol, reflux, 2 h, 85% yield; (b) 2-
aminobenzoic acid, Cu/CuI, DMF, 100 8C, 24 h, 86% yield; (c) phosphorus oxychloride, 110 8C,12 h, 88% yield; (d) corresponding polyamine, acetonitrile,
reflux, 10 h, 90–33% yield.

P. Yang et al. / Tetrahedron 61 (2005) 11895–11901
11897
Table 1. Spectra dataa,b, properties of the linkers and cytotoxicity (A-549,c P388d) of compounds
Compounds
UV lmax (nm)
(log 3)
FL lmax (nm) (F)
Interchromophore
Cytotoxicity [IC50 (mM)]
˚
Distance (A)
Linker
A549
P388
A1
A2
A3
A4
A5
A6
462(4.04)
472(3.94)
473(4.19)
473(4.20)
474(4.27)
470(3.88)
544(0.056)
558(0.024)
550(0.015)
567(0.010)
557(0.024)
562(0.012)
NAe
(CH2)2NH(CH2)2
(CH2)3NH(CH2)3
(CH2)3NCH3(CH2)3
(CH2)2NH(CH2)2NH(CH2)2
(CH2)2NH(CH2)3NH(CH2)2
NA
7.3
9.8
9.8
11.0
12.3
3.71
56.2
0.150
0.333
0.547
0.025
0.275
122
0.472
0.246
1.03
0.281
a In absolute ethanol.
b With rhodamine B in ethanol as quantum yield standard (fZ0.97).
c Cytotoxicity (CTX) against human lung cancer cell (A549) was measured by sulforhodamine B dye-staining method.
d CTX against murine leukemia cells (P388) was measured by microculture tetrazolium–formazan method.
e NA, not applicable.
Figure 3. Effects of A1, A4, and A6 on the supercoiled pBR 322 DNA.
Panel (a) Drug to DNA ratios (r) in lanes 1–12 for A1 are 0.090, 0.085,
0.079, 0.075, 0.072, 0.065, 0.055, 0.04, 0.025, 0.01, 0.005, 0; Panel
(b) Drug to DNA ratios (r) in lanes 1–9 for A4; 0.060, 0.050, 0.043, 0.040,
0.037, 003, 0.02, 0.01, 0; Panel (c) Drug to DNA ratios (r) in lanes 1–9 for
A6: 0.050, 0.045, 0.039, 0.035, 0.033, 0.025, 0.02, 0.01, 0.
Figure 4. The dose–response curve of the compound A6 for both A549 and
P388.
(micromolar) required to inhibit cell growth by 50%. The
IC50 values were calculated based on the parameters in
Table 2. Figure 4 presented the dose–response curve of the
representative compound A6 for both A549 and P388. The
structure parameters of these compounds and their
cytotoxicities (as IC50 values) were listed in Table 1. It
was showed that these polyamine systems except A2 were
active in the described panels, although their activities were
in every case different. The dinuclear NH substituted
compound A6 was found to be the most effective antitumor
agent against A549 (IC50, 0.025 mM) and A4 was more
cytotoxic against P388 (IC50, 0.246 mM). However, A1, the
mononuclear analogue exhibited higher activity towards
P388 (IC50, 0.275 mM) than against A549 (IC50, 3.71 mM).
For A4 and A6, the unwinding angles were 1.9–2.2-fold
greater than that of the monomer, 8-(dimethylamino)ethyl-
amino-5-(2-(dimethylamino)ethyl)-5H-isoquinolino[4,5-
bc]acridine-4,6-dione, indicating that A4 and A6 may have
bis-intercalated with pBR 322 DNA. In addition, the
calculated helix-unwinding angles also revealed that linkers
played a key role on unwinding supercoiling of DNA.
2.3. Structure–activity relationships for cellular growth
inhibition
SRB (Sulforhodamine B) assay against A549 (human lung
cancer cell) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay against P388 (murine
leukemia cell) were used to evaluate the antitumor activities
of these compounds. IC50 represents the drug concentration
For A549, the dimeric forms of isoquinolino[4,5-bc]acri-
dine exhibited 10–20 fold more active cytotoxicity than its
Table 2. Cytotoxic activities of A1–A6 against A549 and P388 at different concentrations (mol/L)
Compounds (M)
Inhibition of tumor growth (%)
A549
Inhibition of tumor growth (%)
P388
10K4
10K5
10K6
10K7
10K8
10K4
10K5
10K6
10K7
10K8
A1
A2
A3
A4
A5
A6
98.4
50.7
84.3
99.6
94.3
89.5
97.2
13.8
97.8
99.0
82.2
98.9
38.4
4.2
80.2
43.9
73.8
82.7
0
3.4
39.9
11.2
15.2
59.2
0
0
13.8
8.5
12.2
33.0
100.0
30.8
100.0
100.0
100.0
100.0
100.0
8.9
100.0
100.0
80.9
39.7
5.1
39.5
91.6
6.1
10.7
2.1
9.0
10.5
5.4
10.3
4.2
0
0.4
1.2
5.2
1.8
100.0
70.4

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P. Yang et al. / Tetrahedron 61 (2005) 11895–11901
mono-derivative. Compound A4, the N-methylation of the
potent cytotoxic agent A3, resulted in approximately a
2-fold less activity. A5, with shorter CH2 spacer between
two N atoms, led to nearly 22-fold loss of cytotoxic potency
than that of A6. Evaluation of the polyamine bridge
indicated that A2–A5 did not exhibit good cytotoxic activity
˚
as A6 (12.3 A) did, which may be caused by their relatively
˚
shorter linker (from 7.3 to 11.0 A) not allowing both inter-
calator moieties intercalating DNA at the same time. The
aminoalkyl linker chain [(CH2)2–NH–(CH2)3–NH–(CH2)2]
of A6, exhibited decent antitumor activity against both
A549 and P388.
Figure 6. Images of A5 and A6 intercalated into d (CGCGC). Carbon atoms
are colored in green, nitrogens in blue, oxygens in red and hydrogens in
white, phosphorus in yellow and sodium counterions is in pink. All the
atoms of A5 and A6 are colored in green.
There was one interesting trend listed in Table 1 that
deserved further discussion. We could see that for both
A549 and P388, the alkyl chain between the aminicimidic
nitrogens was propylene (A6) rather than ethylene (A5)
exerted greater activity (22-fold and 5-fold, respectively). In
order to analyze the difference between A5 and A6, their
Scatchard binding constants to calf thymus DNA (in 20 mM
Tris–HCl buffer, pH 7.0) were determined based on the
method of fluorescence quenching technique.13 Figure 5
presented the Scatchard plots of spectrophotometric
titrations of CT-DNA to the representative compound A6.
The calculated Scatchard binding constants for A5 and A6
were 3.56!104 MK1 and 2.37!105 MK1, respectively,
indicating that A6 could form a more stable DNA complex.
The order of Scatchard constants and the binding site size
were similar to those in the reference.14a–c
could be caused by the sufficient long interchromophore
˚
distance (12.3 A).
16
3. Conclusion
The design, facile synthesis, DNA-binding affinity and the
cytotoxic activity of novel mono- and dinuclear isoquino-
lino[4,5-bc]acridine derivatives A1–A6 were demonstrated.
A6 exhibited the highest in vitro antitumoral activity against
human lung cancer cell (A549) and A4 was the most active
against murine leukemia cell (P388). DNA binding study
and molecular modeling of the A5/A6 DNA complexes
indicated that A6 with the long enough linker could exhibit
the higher DNA affinity than A5, which contributed to its
higher antitumor activity.
4. Experimental
4.1. Materials
All the solvents were of analytic grade. 1H NMR was
measured on a Bruker AV-400 spectrometer with chemical
shifts reported as parts per million (in DMSO-d6/CDCl3,
TMS as an internal standard). Mass spectra were measured
on a HP 1100 LC-MS spectrometer. Melting points were
determined by an X-6 micro-melting point apparatus and
uncorrected. Absorption spectra were determined on
PGENERAL TU-1901 UV–vis spectrophotometer.
Figure 5. Scatchard plots of spectrophotometric titration of CT-DNA to A6
in Tris–HCl buffer. A plot of rb/c versus rb gives the association constant
(slope, 2.37!105 MK1) and the apparent number of binding sites per
nucleotide (x-intercept, 0.04) for the agent.
To further illustrate the difference between A5 and A6 DNA
complexes, hyperchem 7.0 package was used to build a
simple molecular model. The properly modified AMBER
method was selected in this study. The intercalation
4.2. Synthesis
4.2.1. 8-(Dimethylamino)ethylamino-5-(2-(dimethyl-
amino)ethyl)-5H-isoquinolino[4,5-bc]acridine-4,6-dione
(A1):. 4-Bromo-1, 8-naphthalic anhydride (2.77 g, 10 mmol)
and N,N-dimethylethylenediamine (1.0 g, 11.4 mmol) were
added to 10 mL ethanol, the reaction mixture was stirred at
reflux temperature for 2 h, then cooled, filtered, and dried,
the crude product was obtained as yellow solid 1 (2.95 g,
8.5 mmol). APCI-MS (positive) m/z: 348.2 ([MCH]C).
(b) The obtained 4-bromo-1,8-naphthalimide derivative
(2 g, 5.76 mmol) and 2-aminobenzoic acid (0.8 g, 6 mmol),
copperbronze(0.038 g, 0.6 mmol), CuI (0.101 g,0.53 mmol),
were added to 10 mL DMF. The reaction mixture was
stirred at 100 8C for 24 h, filtered while it was hot, and the
filtrate was cooled and poured into the ice water, filtered,
energies (DE) were calculated using DEZEcomplex
K
[EcompdCEDNA], where Ecomplex, Ecompd, and EDNA were
the computed potential energies for the minimized average
A5/A6-DNA complex, free A5/A6 and free DNA.15
The images of A5 and A6 DNA complexes obtained by
molecular modeling were presented in Figure 6. The
calculated intercalation energies (DE) for A5 and A6 were
K2.91 and K23.78 kcal/mol, respectively. According to
the stable geometries of the bisintercalation complexes and
the calculated intercalation energies (DE), we concluded
that A6 could exhibit higher DNA affinity than A5, which

P. Yang et al. / Tetrahedron 61 (2005) 11895–11901
11899
and dried to get the red product 2. (2.0 g, 4.95 mmol, 86%
yield) APCI-MS (positive) m/z: 404.3 ([MCH]C). This
product was not purified and used directly in the next step.
(c) 1 g of 2 was stirred in phosphorus oxychloride at 110 8C
for 12 h, then cooled and poured into the ice water, N(Et)3
was added when stirred vigorously, filtered, and dried to
give yellow solid 3 (0.88 g, 88% yield).
3HC/3): 289.4766, Found: 289.4767. IR (KBr): 3399, 2924,
2853, 1689, 1650, 1376 cmK1
.
4.3.3. Compound A4. Purified by silica gel chromato-
graphy (CHCl3/MeOH/NH4OH Z1:2:0.03, v/v/v), 85%
yield, Mp: 213.6–213.8 8C. H NMR (d6-DMSO) d (ppm):
1
2.15 (s, 4H, 2CH2), 2.30 (s, 4H, 2CH2), 2.32 (s, 3H, NCH3),
2.42 (s, 12H, 2NCH3), 2.74 (s, 4H, 2CH2), 3.66 (s, 4H,
2CH2), 4.01 (s, 4H, 2CH2), 7.33–7.37 (t, J1Z7.6 Hz, J2Z
7.6 Hz, 2H), 7.33–7.37 (t, J1Z7.6 Hz, J2Z7.6 Hz, 2H),
7.56–7.69 (m, 6H), 8.12–8.14 (d, JZ7.2 Hz, 2H), 8.28–8.30
(d, JZ8.0 Hz, 2H), 8.71 (s, 2H), 8.85–8.87 (d, JZ7.2 Hz,
2H), ESI-HRMS: calcd for C17.7H18.7N3O1.3 (MC3HC/3):
294.1485, Found: 294.1483. IR (KBr): 3431, 2922, 2854,
Purified by silica gel chromatography (CHCl3/MeOHZ9:1,
v/v) to get pure product 3, Mp: 204.8–205.2 8C. H NMR
1
(CDCl3) d (ppm): 2.99 (s, 6H, NCH3), 3.51 (s, 2H, NCH2),
4.69–4.72 (t, J1Z6.0 Hz, J2Z6.0 Hz, 2H, CONCH2), 7.77–
7.81 (t, J1Z7.6 Hz, J2Z7.6 Hz, 1H), 7.97–8.04 (m, 2H),
8.39–8.42 (d, J1Z8.8 Hz, 1H), 8.52–8.54 (d, JZ8.4 Hz,
1H), 8.72–8.74 (d, JZ7.6 Hz, 1H), 9.52 (s, 1H), 9.77–9.79
(d, JZ8.0 Hz, 1H), ESI-HRMS: calcd for C23H19ClN3O2
(MCHC): 404.1166, Found: 404.1166, Found: 404.1158.
1691, 1652, 1395 cmK1
.
4.3.4. Compound A5. Purified by silica gel chromato-
graphy (CHCl3/MeOH/NH4OH Z1:2:0.06, v/v/v), 52%
yield, Mp: 159.9–160.1 8C. H NMR (d6-DMSO) d (ppm):
IR (KBr): 2923, 2853, 1702, 1660, 1347 cmK1
.
1
The above obtained compound (1 g, 2.48 mmol) and N,N-
dimethylethylenediamine (0.26 g, 2.97 mmol) were added
to 10 mL acetonitrile. The solution was refluxed for 5 h,
cooled and filtered. Separated by silica gel chromatography
(CHCl3/MeOHZ5:1, v/v) to get pure A1 (1.01 g, 90%
2.35–2.39 (t, J1Z6.8 Hz, J2Z6.8 Hz, 4H, 2CH2), 2.40
(s, 6H, 2NCH3), 2.97 (s, 4H, 2CH2), 3.13 (s, 4H, 2CH2),
3.79 (s, 4H, 2CH2), 3.97 (s, 4H, 2CH2), 7.33 (s, 2H), 7.57–
7.64 (m, 6H), 8.12–8.14 (d, JZ6.8 Hz, 2H), 8.29–8.31
(d, JZ8.0 Hz, 2H), 8.85 (s, 2H), 8.92–8.94 (d, JZ8.4 Hz,
2H), ESI-HRMS: calcd for C26H27N5O2 (MC2HC/2):
441.2165, Found: 441.2179. IR (KBr): 3419, 2926, 2852,
1
yield), Mp: 148.9–150.4 8C. H NMR (CDCl3) d (ppm):
2.47 (s, 6H, N(CH3)2), 2.52 (s, 6H, N(CH3)2), 2.75–2.78
(t, J1Z6.0 Hz, J2Z5.2 Hz, 2H, CH2), 2.87 (s, 2H, CH2),
4.06–4.09 (t, J1Z5.6 Hz, J2Z6.0 Hz, 2H, CH2), 4.42–4.46
(t, J1Z6.8 Hz, J2Z7.2 Hz, 2H, CH2), 7.52–7.55 (t, J1Z
6.8 Hz, J2Z7.6 Hz, 1H), 7.79–7.88 (m, 2H), 8.13–8.15 (d,
JZ8.4 Hz, 1H), 8.20–8.22 (d, JZ8.4 Hz, 1H), 8.61–8.63
(d, JZ7.6 Hz, 1H), 9.35 (s, 1H), 9.63–9.65 (d, JZ8.8 Hz,
1H), ESI-HRMS: calcd for C13.5H15.5N2.5O (MC2HC/2):
228.6239, Found: 228.6231. IR (KBr): 3397, 2924, 2854,
1693, 1650, 1392 cmK1
.
4.3.5. Compound A6. Purified by silica gel chromato-
graphy (CHCl3/MeOH/NH4OH Z1:2:0.06, v/v/v), 33%
yield, Mp: 102.2–102.5 8C. H NMR (d6-DMSO) d (ppm):
1
2.11–2.14 (m, 2H, CH2), 2.37 (s, 4H, 2CH2), 2.54 (s, 12H,
2N(CH3)2), 2.89 (s, 4H, 2CH2), 3.03 (s, 4H, 2CH2), 3.82
(s, 4H, 2CH2), 3.90 (s, 4H, 2CH2), 7.40 (s, 2H), 7.60–7.69
(m, 6H), 8.17–8.19 (d, JZ7.6 Hz, 2H), 8.26–8.28 (d, JZ
8.0 Hz, 2H), 8.85 (s, 2H), 8.97–8.99 (d, JZ8.0 Hz, 2H),
ESI-HRMS: calcd for C26.5H28N5O2 (MC2HC/2):
448.2243, Found: 448.2260. IR (KBr): 3334, 2924, 2853,
1692, 1653, 1347 cmK1
.
4.3. Synthesis of dinuclear isoquinolino[4,5-bc]acridine
derivatives A2–A6
1695, 1654, 1391 cmK1
.
The preparation and purification procedure of A2–A6 was
similar to that of A1: several polyamine chains were
selected instead of N,N-dimethylethylenediamine.
4.4. CT-DNA binding studies
The solution of compounds A5 and A6 in DMSO (10K3
–
4.3.1. Compound A2. Purified by silica gel chromato-
graphy (CHCl3/MeOH/NH4OHZ1:2:0.03, v/v/v), 69%
yield, Mp: 191.3–191.5 8C. H NMR (d6-DMSO) d (ppm):
10K4 M) was diluted with 20 mM Tris–HCl (pH 7.0) to the
samples at the concentration of 1, 2.5, 5, 7.5, 10, 15, 20, 25 mM,
respectively. Then, it was separated to two parts: one
contained Calf-thymus DNA 30 mM, the other contained no
DNA but the same concentration of chemical as control. All
the above solutions were shaken for 3 days at 25 8C in the
dark. Fluorescence wavelength and intensity area of
samples were measured.
1
2.28 (s, 4H, 2CH2), 2.45 (s, 12H, 2N(CH3)2), 3.19 (s, 4H,
2CH2), 3.71 (s, 4H, 2CH2), 4.02 (s, 4H, 2CH2), 7.33 (s, 2H),
7.67–7.71 (m, 4H), 7.78–7.80 (d, JZ8.4 Hz, 2H), 8.23–8.25
(d, JZ8.0 Hz, 2H), 8.28–8.30 (d, JZ8.4 Hz, 2H), 8.96
(s, 2H), 9.08–9.10 (d, JZ8.4 Hz, 2H), ESI-HRMS: calcd for
C25H24.5N4.5O2 (MC2HC/2): 419.6954, Found: 419.6948.
IR (KBr): 3427, 2922, 2853, 1696, 1653, 1346 cmK1
.
4.5. DNA unwinding angle measurement
4.3.2. Compound A3. Purified by silica gel chromato-
graphy (CHCl3/MeOH/NH4OH Z1:2:0.03, v/v/v), 61%
yield, Mp: 195.9–196.1 8C. 1H NMR (CDCl3) d (ppm): 2.18
(s, 4H, 2CH2), 2.32 (s, 12H, 2N(CH3)2), 2.47 (s, 4H, 2CH2),
3.18 (s, 4H, 2CH2), 3.85 (s, 4H, 2CH2), 4.18 (s, 4H, 2CH2),
7.32–7.36 (t, J1Z7.2 Hz, J2Z8.0 Hz, 2H), 7.62–7.66 (m,
4H), 7.77–7.79 (d, JZ8.4 Hz, 2H), 8.02 (s, br, 2H), 8.32–
8.34 (d, JZ8.0 Hz, 2H), 8.86 (s, 2H), 8.97–8.99 (d, JZ
8.0 Hz, 2H), ESI-HRMS: calcd for C17.3H18N3O1.3 (MC
Covalently closed circular plasmid DNA, pBR 322, was
purchased from TaKaRa Co., Ltd as a 0.5 mg per mL. Prior
to application to the agarose gel, pBR322 DNA aliquots
were incubated with the compounds at 37 8C in TE buffer
(Tris–HCl 10 mm, pH 7.4, EDTA 0.1 mM) for 24 h at
several molar ratios of drug to nucleotide. The density of
supercoiling was d K0.08 under our experimental
conditions.17

11900
P. Yang et al. / Tetrahedron 61 (2005) 11895–11901
The fraction of unreacted drug was separated from the
mixture by precipitation of the DNA with 2.5 V of ethanol
and 0.3 M sodium acetate, pH 4.8. Two percentage of
agarose gel electrophoresis was carried out at 25 V in
40 mM TAE buffer (40 mM tris(hydroxymethyl)amino-
methane, 30 mM glacial acetic acid, 1 mM EDTA, pH 7.5).
After electrophoresis, the gel was stained with ethidium
bromide.
relative to that of the control sample. IC50 in Table 1 was
calculated based on these parameters.
Acknowledgements
Financial support by National Basic Research Program of
China(2003CB114400) and under the auspices of 863 plan
for High-Tech (2003AA2Z3520), is greatly appreciated.
This work is also supported by Dalian Excellent Young
Scientist Research Fund.
4.6. Molecular modeling methods
The d (CGCGC) was selected as the intercalation site
sequence, a preferential feature for cytostatic active prin-
ciples17b,18a,b and the similar intercalative mode by A5 and
A6 was supposed. According to the parameters of refer-
ence,18c–e AMBER method was properly modified. In all
cases, xZ4r was used to simulate the solvent effects NaC
References and notes
1. Kochevar, E. D. D. In Dunn in Bioorganic Photochemistry;
Morrison, H., Ed.; Wiley: New York, 1990; pp 273–283.
2. (a) Wakelin, L. P. G. Med. Res. Rev. 1986, 6, 275–340.
(b) Hsiang, Y. H.; Jiang, J. B.; Liu, L. F. Mol. Pharmacol.
1989, 36, 371–376. (c) Cholody, W. M.; Hernandez, L.;
Hassner, L.; Scudiero, D. A.; Djurickovic, D. B.; Michejda,
C. J. J. Med. Chem. 1995, 38, 3043–3052. (d) Spicer, J. A.;
Gamage, S. A.; Atwell, G. J.; Finlay, G. J.; Baguley, B. C.;
Denny, W. A. Anti-Cancer Drug Des. 1999, 14, 281–289.
3. Bousquet, P. F.; Brana, M. F.; Conlon, D.; Fitzgerald, K.;
Perron, M. D.; Cocchiaro, C.; Miller, R.; Moran, M.; George,
J.; Qian, X. D.; Keilhauer, G.; Romerdahl, C. A. Cancer Res.
1995, 55, 1176–1189.
˚
counterions were included, placed at 6 A distance from each
phosphate–oxygen bisector.
4.7. Cytotoxic activity in vitro
The prepared compounds have been submitted to Shanghai
Institute of Materia Medica for testing their cytotoxicities
in vitro.
A549 (human lung cancer cell) and P388 (murine leukemia
cell) were seeded into 96 well cell culture plates before
experimental manipulation, and then treated with a test
compound each at different concentrations for 72 h. At the
end of treatments, A549 and P388 cell numbers were
determined by SRB and MTT assay, respectively.19,20
4. (a) Laursen, J. B.; Nielsen, J. Chem. Rev. 2004, 104,
1663–1686. (b) Blanchard, S.; Rodriguez, I.; Tardy, C.;
Baldeyrou, B.; Bailly, C.; Colson, P.; Houssier, C.; Leonce,
S.; Kraus, B. L.; Pfeiffer, B.; Renard, P.; Pierre, A.;
Caubere, P.; Guillaumet, G. J. Med. Chem. 2004, 47,
978–987. (c) Hay, M. P.; Pruijn, F. B.; Gamage, S. A.;
Liyanage, H. D. S.; Kovacs, M. S.; Patterson, A. V.;
Wilson, W. R.; Brown, J. M.; Denny, W. A. J. Med. Chem.
2004, 47, 475–488. (d) Antonini, I.; Polucci, P.; Magnano,
A.; Gatto, B.; Palumbo, M.; Menta, E.; Pescalli, N.;
Martelli, S. J. Med. Chem. 2003, 46, 3109–3115. (e)
Rastogi, K.; Chang, J.-Y.; Pan, W.-Y.; Chen, C.-H.; Chou,
T.-C.; Chen, L.-T.; Su, T.-L. J. Med. Chem. 2002, 45,
4485–4493. (f) Gamage, S. A.; Spicer, J. A.; Rewcastle,
G. W.; Milton, J.; Sohal, S.; Dangerfield, W.; Mistry, P.;
Vicker, N.; Charlton, P. A.; Denny, W. A. J. Med. Chem.
2002, 45, 740–743. (g) Wang, S.; Miller, W.; Milton, J.;
Nigel, V.; Alistair, S.; Peter, C.; Prakash, M.; David, H.;
William, A. D. Bioorg. Med. Chem. Lett. 2002, 12,
415–418. (h) Brana, M. F.; Ramos, A. Curr. Med. Chem.:
Anti-Cancer Agents 2001, 1, 237–255. (i) Rogers, J. E.;
Weiss, S. J.; Kelly, L. A. J. Am. Chem. Soc. 2000, 122,
427–436. (j) Qian, X.; Huang, T. B.; Wei, D.-Z.; Zhu,
D. H.; Fan, M. C.; Yao, W. J. Chem. Soc., Perkin Trans. 2
2000, 715–718. (k) Rogers, J. E.; Kelly, L. A. J. Am.
Chem. Soc. 1999, 121, 3854–3861. (l) Brana, M. F.;
Castellano, J. M.; Perron, D.; Maher, C.; Conlon, D.;
Bousquet, P. F.; George, J.; Qian, X.-D.; Robinson, S. P.
J. Med. Chem. 1997, 40, 449–454.
For the MTT assay, briefly, a 20 mL of MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
solution (5 mg/mL) was added directly to all the appropriate
wells. The cultures were then incubated for 4 h. Then
100 mL of ‘triplex solution’ (10% SDS/ 5% isobutanol/
12 mM HCl) was added. After the plates were incubated at
37 8C overnight, they were measured by the absorbance at
570 nm using a multiwell spectrophotometer.
For the SRB assay, in brief, the cells were fixed with 10%
trichloroacetic acid for an hour at 4 8C. After they were
extensively washed, fixed cells were stained for 30 min with
0.4% sulforhodamine B (SRB) in 1% acetic acid. Unbound
SRB was washed away with 1% acetic acid. Then, the
cultures were air-dried. Bound dye was solubilized with
10 mM Tris (pH 10.5) prior to reading plates. The OD value
was read on a plate reader at a wavelength of 515 nm. The
ratio of the OD (optical density) value of a compound-
treated culture to the OD value of a mock-treated culture,
expressed in percentage, was used to quantify the
cytotoxicity of a compound. Results were expressed as
IC50 (the drug concentration that reduces by 50% the
absorbance in treated cells with respect to untreated cells).
The cytotoxic activities of A1–A6 against the selected
tumor cell lines A549 and P388 at different concentrations
were listed in Table 2. The numbers 10K4–10K8 were the
concentrations of the test compounds while the numbers in
the matrix were the ratio of the OD of the treated sample
5. Brana, M. F.; Cacho, M.; Garcia, M. A.; Pascual, T. B.;
Ramos, A.; Dominguez, M. T.; Pozuelo, J. M.; Abradelo, C.;
Rey, S. M. F.; Yuste, M.; Banez, C. M.; Lacal, J. C. J. Med.
Chem. 2004, 47, 1391–1399.

P. Yang et al. / Tetrahedron 61 (2005) 11895–11901
11901
6. Girault, S.; Grellier, P.; Berecibar, A.; Maes, L.; Mouray, E.;
Lemiere, P.; Debreu, M. A.; Davioud, C. E.; Sergheraert, C.
J. Med. Chem. 2000, 43, 2646–2654.
15. Cairns, D.; Michalitsi, E.; Jenkins, T. C.; Mackay, S. P.
Bioorg. Med. Chem. 2002, 10, 803–807.
16. Bailly, C.; Carrasco, C.; Joubert, A.; Bal, C.; Wattez, N.;
Hildebrand, M. P.; Lansiaux, A.; Colson, P.; Houssier, C.;
Cacho, M.; Ramos, A.; Brana, M. F. Biochemistry 2003, 42,
4136–4150.
7. (a) Yang, Q.; Qian, X. H.; Xu, J. Q.; Sun, Y. S.; Li, Y. G.
Bioorg. Med. Chem. 2005, 13, 1615–1622. (b) Qian, X. H.; Li,
Y. G.; Xu, Y. F.; Liu, D.; Qu, B. Y. Bioorg. Med. Chem. 2004,
14, 2665–2668. (c) Xu, Y. F.; Qian, X. H.; Yao, W.; Mao, P.;
Cui, J. Bioorg. Med. Chem. 2003, 11, 5427–5433. (d) Qian,
X. H.; Yao, W.; Chen, G.; Huang, X. Y.; Mao, P. Tetrahedron
Lett. 2001, 42, 6175–6178.
17. (a) Wakelin, L. P. G.; Bu, X.; Eleftheriou, A.; Parmar, A.;
Hayek, C.; Stewart, B. W. J. Med. Chem. 2003, 46,
5790–5802. (b) Brana, M. F.; Casarrubios, L.; Dominguez,
G.; Fernandez, C.; Perez, J. M.; Quiroga, A. G.;
Navarro-Ranninger, C.; de Pascual-Teresa, B. Eur. J. Med.
Chem. 2002, 37, 301–313.
8. Wakelin, L. P.; Romanos, M.; Chen, T. K.; Glaubiger, D.;
Canellakis, E. S.; Waring, M. J. Biochemistry 1978, 17,
5057–5063.
18. (a) Ornstein, R. L.; Rein, R. Biopolymers 1979, 18,
2821–2847. (b) Chaires, J. B. Biophys. Chem. 1990, 35,
191–202. (c) Marinela, R. C.; Sanz, A. M.; Yunta, M. J. R.;
Fernando, G. C.; Navarro, P.; Fernandez, I.; Pardoa, M.;
Canoa, C. Bioorg. Med. Chem. 2003, 11, 2143–2148. (d) Fox,
T.; Scanlan, T. S.; Kollman, P. A. J. Am. Chem. Soc. 1997,
119, 11571–11577. (e) Grootenhuis, P. D.; Kollman, P. A.
J. Am. Chem. Soc. 1989, 111, 2152–2158.
9. Baudoin, O.; Teulade, F. P. M.; Vigneron, J. P.; Lehn, J. M.
J. Org. Chem. 1997, 62, 5458–5470.
10. Acheson, R. M.; Acridines, the chemistry of heterocyclic
compounds; Wiley: London, 1973; 9; pp 563–574.
11. Byk, G.; Dubertret, C.; Escriou, V.; Frederic, M.; Jaslin, G.;
Rangara, R.; Pitard, B.; Crouzet, J.; Wils, P.; Schwartz, B.;
Scherman, D. J. Med. Chem. 1998, 41, 224–235.
12. Keck, M. V.; Lippard, S. J. J. Am. Chem. Soc. 1992, 114,
3386–3390.
19. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.;
McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.;
Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107–1112.
13. Gupta, M.; Ali, R. J. Biochem. 1984, 95, 1253–1257.
14. (a) Wirth, M.; Buchardt, O.; Koch, T.; Nielsen, P. E.; Norden,
B. J. Am. Chem. Soc. 1988, 110, 932–939. (b) Boger, D. L.;
Ichikawa, S.; Tse, W. C.; Hedrick, M. P.; Jin, Q. J. Am. Chem.
Soc. 2001, 123, 561–568. (c) Antonini, I.; Polucci, P.;
Magnano, A.; Sparapani, S.; Martelli, S. J. Med. Chem.
2004, 47, 5244–5250.
20. Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.;
Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.;
Shoemaker, R. H.; Boyd, M. R. Cancer Res. 1988, 48,
589–601.