Novel synthetic isoquinolino[5,4-ab]phenazines: Inhibition toward topoisomerase I, antitumor and DNA photo-cleaving activities

Article abstract of DOI:10.1016/j.bmc.2005.07.029

The novel DNA interactive isoquinolino[5,4-ab]phenazine derivatives were designed and synthesized. Their inhibitory abilities toward topoisomerase I, antitumor activities and DNA photo-cleaving abilities were examined. The substituents at peri sites of tw

Full text of DOI:10.1016/j.bmc.2005.07.029

Bioorganic & Medicinal Chemistry 13 (2005) 5909–5914
Novel synthetic isoquinolino[5,4-ab]phenazines: Inhibition
toward topoisomerase I, antitumor and DNA
photo-cleaving activities
Peng Yang,a Qing Yang,b Xuhong Qiana,c, and Jingnan Cui
a
*
aState Key Laboratory of Fine Chemicals, Dalian University of Technology, 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 15 June 2005; revised 10 July 2005; accepted 12 July 2005
Available online 22 August 2005
Abstract—The novel DNA interactive isoquinolino[5,4-ab]phenazine derivatives were designed and synthesized. Their inhibitory
abilities toward topoisomerase I, antitumor activities and DNA photo-cleaving abilities were examined. The substituents at peri sites
of two phenazine N atoms played very important roles for all these biological activities. At a concentration of 100 lM, all these
phenazine derivatives (but A2 and A6) exhibited an inhibitory activity toward topoisomerase I. A6 had efficient antitumor activities
against both human lung cancer cell (A549) and murine leukemia cell (P388). A1, A5, and A6 exhibited antitumor activities
selectively only against P388. A2 was the most efficient DNA photocleaver, which had converted supercoiled DNA from form
I to form II at <1 lM. Under anaerobic conditions, the electron transfer mechanism mainly contributed to DNA photo-induced
cleavage, while under aerobic conditions, superoxide anion was also involved in this process.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
on incorporating substituents or fusing five- or six-mem-
bered (phenyl or heterocyclic) rings to the naphthalene
skeletons.3 BranaÕs group made a lot of effort to improve
naphthalimide derivativesÕ topoisomerase inhibitory and
DNA intercalators have received much attention due to
their therapeutic potential in anticancer treatment.1
Some of these compounds, known as photonucleases,
cleave DNA under light irradiation of a certain wave-
length,2 and also exhibit inhibitory activity against
DNA regulatory enzymes, such as topoisomerase I.3
Either DNA damage or topoisomerase inhibition is
believed to disrupt DNA replication in vivo and thus,
in turn, to show the cytotoxicity against tumor cells.
Therefore, any development in the design of DNA inter-
calators with high topoisomerase inhibition, antitumor
and DNA photo-cleaving activities is of the importance.
4
5
´
antitumor abilities. Fernandez et al. reported a photo-
sensitive DNA cleaver containing the phenazine bisin-
tercalator. Gamage et al.6 found that the heterocyclic
phenazinecarboxamides could be used as topoisomer-
ase-targeted anticancer agents. Studying the DNA-bind-
ing property of dipyrido [3,2-a: 20,30-c]phenazine (dppz)
unit, Phillips et al.7 found that intramolecular charge
transfer accounted for unstructured luminescence. How-
ever, examples that took advantage of the structural
characteristics of both naphthalimide and phenazine
chromophores seldom appeared (see Figure 1).
Various attempts have been made to modify the
naphthalimide and phenazine units to promote their
topoisomerase I inhibitory, antitumor and DNA pho-
to-damaging abilities. Most of these works are focused
N
N
N
N
N
N
Keywords: Phenazine naphthothiazole carboxamides; Photocleavage;
Cytotoxicity.
R
N
O
N
*
Corresponding author. Tel.: +86 411 83673466; fax: +86 411
83673488; e-mail addresses: xhqian@dlut.edu.cn; xhqian@ecust.
edu.cn
H
Figure 1. Structures of the reported phenazine derivatives.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.07.029

5910
P. Yang et al. / Bioorg. Med. Chem. 13 (2005) 5909–5914
Table 1. UV–vis and fluorescent data of A1–A6a,b
Compounds
UV kmax, nm (lge)
FL kmax, nm (U)
N
O
A1, R=H
A1
A2
A3
A4
A5
A6
374(3.66)
384(3.04)
423(3.33)
410(3.38)
420(3.28)
378(3.31)
430(0.00065)
454(0.00082)
483(0.02265)
467(0.00100)
480(0.00717)
455(0.00145)
1
4
A2, R=1-CH3
A3, R=1-OCH3
A4, R=2-CH3
A5, R=2-OCH3
A6, R=4-CH3
N
N
2
3
N
O
R
a In absolute ethanol.
b With rhodamine B in ethanol as quantum yield standard (U = 0.97).
Figure 2. Structures of the novel isoquinolino[5,4-ab]phenazine.
Table 1 shows the UV–vis and fluorescent data of A1–
A6. It can be seen that the fluorescence quantum yields
of these compounds were relatively low, which may have
been caused by the rapid IST from a singlet to the
triplet. The compound A3, due to the existence of a
methoxyl group at the 1-site, had a relatively higher
fluorescence quantum yield compared to other
compounds.
In our continuous attempt8 to develop compounds with
high DNA-binding affinity and highly active antitumor
activities, we designed and synthesized novel isoquinoli-
no[4,5-bc]phenazine derivatives A1–A6 (Fig. 2). In this
study, naphthalimide active group remained in the struc-
ture and the quinoxaline unit was effectively fused with
the electron-deficient naphthalimide.
The Scatchard-binding constant between the compound
A2, used as an example, and calf thymus DNA was deter-
mined using the fluorescence technique method (Fig. 3).10
,
which indicated that A2 could effectively intercalate into
the calf thymus DNA.
These compounds were designed to include the obvious
electron-deficient characteristics for the sake of strong
electron-withdrawing effects of two carbonyl groups of
naphthalimide. In addition, their asymmetrical elec-
tron-withdrawing effects exert different actions on the
electrons of phenazine N heteroatoms. Hence, highly
DNA intercalative abilities of these novel phenazine
derivatives, their antitumor activities, and DNA pho-
to-damaging properties were anticipated.
Its Scatchard-binding constant was 3.63 · 105 Mꢀ1
2.2. Topoisomerase I inhibitory and antitumor activities
The inhibitory activities of these compounds toward
topoisomerase I were evaluated by 2% agarose-gel
2. Results and discussion
2.1. Synthesis and spectra
F4
F4-CT
F3
F3-CT
F2
F2-CT
F1
F1-CT
6×105
4×105
2×105
0
The synthesis of compounds A1–A6 is shown in Scheme
1. Take compound A1 as an example: the starting mate-
rial, 4-bromo-3-nitro-1, 8-naphthalic anhydride, reacted
with aniline in DMF at room temperature for 5 h. And
then, the ring closure reaction was carried out based on
the reported procedure.9 The obtained naphthalic anhy-
dride was condensed with N,N-dimethylethylenediamine
in ethanol for 2 h to give the designed compounds. After
separation with careful column chromatography, each
pure targeted product was obtained. All their structures
420
460
500
540
Wavelenth (nm)
Figure 3. Fluorescent spectra before and after interaction of com-
pound A2 with calf thymus DNA. Curves F and F-CT correspond to
compound A2 before and after being mixed with DNA.
1
were confirmed by H NMR, HRMS, and IR.
O
O
O
O
O
O
O
O
O
b
c
a
A1~A6
NO2
N
NO2
HN
N
Br
R
R
Scheme 1. Synthesis of A1–A6. Reagents and conditions: (a) aniline derivatives, DMF, room temperature, 5 h; (b) NaBH4, NaOH, H2O, reflux for
24 h; (c) N,N-dimethylethylenediamine, ethanol, reflux for 2 h.

P. Yang et al. / Bioorg. Med. Chem. 13 (2005) 5909–5914
5911
antitumor activities of A1, A5, and A6 was also ob-
served. A1 was 11-fold more cytotoxic against P388 than
against A549, A5 was 12-fold, and A6 was 5-fold,
reflecting their different activities toward human or lung
cell type.
a
1
2
3
4
5
6
7
8
Form II
Form I
1
2
3
4
5
b
Based on these results, we did not find any obvious rela-
tionship between the cytotoxic potency of A1–A6 and
their topoisomerase I inhibitory activities. However,
there was a trend that was worth noting here. The com-
pound A6 with a methyl group at the 4-site exhibited de-
cent activity for both A549 and P388. Rewcastle9 has
once showed that introduction of a methyl group peri
to the phenazine N atoms enhanced antitumor activity
in their assay toward P388 leukemia and Lewis lung car-
cinoma cell lines. For phenazine N atoms in this study,
there were two peri sites, 1-site (A2 and A3) and 4-site
(A6). Against the two tested cancer cells, A6 exhibited
a higher cytotoxicity than what A2 and A3 did, suggest-
ing that these two peri-substituted compounds exhibited
quite different effects on their antitumor activities.
Form II
Form I
Figure 4. (a) Inhibition of the relaxation activity of topoisomerase I by
A1–A6 (100 lM). Supercoiled pBR322 DNA was relaxed by incubat-
ing with topoisomerase I. Lane 1, DNA alone; lanes 2–7, compounds
A1, A2, A3, A4, A5, A6 + DNA + Topo I, respectively; lane 8,
DNA + Topo I; (b) inhibition of the relaxation activity of topoiso-
merase I by compound A3. Lane 1, DNA + Topo I + A3 (50 lM); lane
2 DNA + Topo I + A3 (20 lM); lane 3, DNA + Topo I + A3 (10 lM);
lane 4, DNA alone; lane 5, DNA + Topo I.
electrophoresis (Fig. 4). As shown in Figure 4a, A1–A6
(100 lM) displayed a different inhibitory activity in the
order: A1 ꢁ A3 > A4 ꢁ A5 > A6 ꢁ A2. A2 was almost
inactive, even at a concentration of 100 lM (Fig. 4a,
lane 3). However, A3 could inhibit topoisomerase I at
the minimized concentration of 20 lM (Fig. 4b). Com-
pared with A2, A3 displayed a stronger inhibitory activ-
ity toward topoisomerase I. This result indicated that
the oxygen atom at the 1-site might favor the binding
between A3 and its corresponding receptor. However,
for 2-site-substituted compounds (A4 and A5), there
was no obvious difference between their weaker inhibito-
ry activities. These results suggested that substituted
sites affected greatly the inhibitory activity toward topo-
isomerase I.
2.3. DNA photo-damaging property
The photo-induced DNA cleaving activities of A1–A6
were assayed using supercoiled pBR322 DNA. As
shown in Figure 5a, A1–A6 caused DNA cleavage under
the irradiation of 365 nm-UV light, confirming that the
UV light functioned as a trigger to initiate DNA strand
scission. A2 with a methyl group at the 1-site, a peri site
of phenazine N atoms, had the highest DNA cleavage
activity and it cleaved DNA at the minimized concentra-
a
b
1
2
3
4
5
6
7
1
1
2
2
3
4
4
5
5
6
6
7 8
The antitumor activities in vitro of all these isoquinoli-
no[5,4-ab]phenazine derivatives were evaluated against
cell lines of human lung cancer cell (A549) and murine
leukemia cell (P388), respectively. The results are given
in Table 2. The IC50 value represents the drug concen-
tration (lM) required to inhibit the cell growth by
50%. A6 showed the most efficient activities against
P388 (IC50, 0.33 lM) cell, while A4 exhibited the highest
activities against A549 (IC50, 1.51 lM). For A549, the
order of cytotoxicity exhibited by these compounds
was: A4 > A6 > A2 > A1 > A3 > A5, while the order of
their cytotoxic potency against P388 was as follows:
A6 > A1 > A4 ꢁ A5 > A3 > A2. The selectivity of the
Form II
Form I
Form II
Form I
c
1
2
3
4
5 6
d
3
3
7 8
Form II
Form I
Form II
Form I
e
1
2
4
5
6
Form II
Form I
Figure 5. Photocleavage of closed supercoiled pBR322 DNA in Tris–
HCl buffer (20 mM, pH 7.5) containing 20% acetonitrile. (a) DNA
cleavage by A1–A6 (100 lM) for 1 h. Lanes 1–6, A6, A5, A4, A3, A2,
A1 and DNA, respectively, lane 7, DNA alone (hv), and lane 8, DNA
alone (no hv); (b) DNA cleavage by A2 at various concentrations for
2 h. Lanes 1–5 A2 at concentrations of 50, 20, 10, 5, and 1 lM,
respectively, lane 6, DNA alone (hv), and lane 7, DNA alone (no hv);
(c) DNA cleavage by A2 (30 lM) in Tris–HCl buffer (20 mM, pH 7.5)
for 2 h. Lanes 1–5, DNA and A2 in the presence of DMSO, SOD
(100 mg/mL), DTT (30 mM), ethanol (1.7 M), and histidine (6 mM),
respectively, lane 6, DNA and A2, lane 7, DNA alone (hv), and lane 8,
DNA alone (no hv); (d) mechanistic experiment for A2 carried out in
phosphate buffers (20 mM, pH 7.5) under aerobic conditions. DNA
photocleavage by A2 (30 lM) for 2.5 h. Lanes 1–3, DNA and A2 in the
presence of DMSO, DTT (30 mM), ethanol (1.7 M), respectively, lane
4, DNA and A2, lane 5, DNA alone (hv), and lane 6, DNA alone (no
hv); (e) mechanistic experiment for A2 carried out in phosphate buffers
(20 mM, pH 7.5) under anaerobic conditions. DNA photocleavage by
A2 (30 lM) for 2.5 h. Lanes 1–3, DNA and A2 in the presence of
DMSO, DTT (30 mM), and ethanol (1.7 M), respectively, lane 4, DNA
and A2, and lane 5, DNA alone (hv), lane 6, DNA alone (no hv).
Table 2. Cytotoxicities of A1–A6
Compounds
Cytotoxicity (IC50, lM)
A549a
P388b
A1
A2
A3
A4
A5
A6
5.16
3.79
7.03
1.51
0.46
7.25
2.54
1.04
1.02
0.33
12.0
1.92
a Cytotoxicity (CTX) against human lung cancer cell (A549) was
measured by sulforhodamine B dye-staining method.11
b CTX against murine leukemia cells (P388) was measured by micro-
culture tetrazolium-formazan method.12

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P. Yang et al. / Bioorg. Med. Chem. 13 (2005) 5909–5914
tion of 1 lM (Fig. 5b). Compared to A2, A3 with a
methoxyl group at the 1-site and A6 with a methyl group
at the 4- site exhibited lower activities. These results
showed that the two peri sites exerted different influences
on DNA photo-cleaving activities. The order of their
the superoxide anion, which could be deduced from
the DNA form II/form I ratio under aerobic/anaerobic
conditions.
DNA
cleaving
abilities
was:
A2 > A5 > A1 >
3. Conclusion
A4 > A6 ꢁ A3.
In summary, this study has demonstrated the design of
novel DNA intercalative isoquinolino[5,4-ab]phenazine
derivatives A1–A6 and the evaluation of their biologi-
cal activities. The two peri sites of phenazine N atoms
played different roles on these bioactivities. At
100 lM, all these compounds (but A2 and A6) inhib-
ited the activity of topoisomerase I. A6 showed effi-
cient activities against both A549 and P388. A1, A5,
and A6 exhibited selective cytotoxicity against P388.
There was no obvious connection between the cytotox-
ic potency of A1–A6 and their topoisomerase I inhib-
itory activities. Under the irradiation of 365 nm-UV
light, the circular supercoiled pBR322 could be cleaved
by A2 at the lowest concentration of 1 lM. Under
anaerobic conditions, the electron transfer mechanism
contributed mainly to the DNA cleavage while the
superoxide anion also involved in this process under
aerobic conditions.
To verify the reactive species responsible for plasmid
DNA cleavage, A2 was chosen for the mechanistic
experiment. As shown in Figure 5c, histidine (singlet
oxygen quencher) and SOD (superoxide dismutase,
superoxide anion scavenger) did not inhibit the DNA
cleavage, DTT (dithiothreitol, superoxide anion scaven-
ger), ethanol (radical killer) and DMSO (radical scaven-
ger) inhibited the DNA damage to different extents, and
DTT acted as the strongest inhibitor.
The inhibition of DNA cleavage by both ethanol and
DMSO indicated that radicals might be involved in this
process. It became known that the excited chromophore
could oxidize the chloride anion (Tris–HCl buffer) to
form the chloride radicals.13 To exclude any Ôheavy atom
effect,Õ the same experiment was carried out with
phosphate buffers (Fig. 5d). The results showed that
both DMSO and ethanol did not inhibit DNA cleavage
anymore, which indicated that the radicals resulted from
the chloride anion in the Tris–HCl buffer.
4. Experimental
4.1. Materials
The inhibition of DNA cleavage by DTT has suggested,
that superoxide anions are likely to be involved in this
DNA photocleavage process. As we know, the irradiat-
ed naphthalimide chromophore could damage the DNA
via electron transfer3 and the exited phenazine unit14
could abstract H atoms from the environment. In addi-
tion, compounds with a C@N bond in aromatic rings
usually photocleaved DNA via electron transfer or a
H-abstraction mechanism3 due to the generation of
1
All the solvents were of analytical grade. H NMR was
measured on a Bruker AV-400 spectrometer, with chem-
ical 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 with an X-6 micro-melt-
ing point apparatus and are uncorrected. Absorption
spectra were determined on a PGENERAL TU-1901
UV–vis Spectrophotometer.
3
3
photo-excited (n–p*) and/or (p–p*) states. Moreover,
a superoxide anion could be produced during the
electron transfer process, in which the electrons first
get transferred from nucleobases to intercalators and
then to the oxygen. It should be noted that addition of
SOD did not obviously inhibit the DNA cleavage as
DTT did. This observation however did not rule out
the possibility that a superoxide anion was involved in
the DNA-cleaving process. It is on account of the
hydrogen peroxide produced by SOD from the superox-
ide anion under photo-irradiation, which then
contradicted the inhibition of superoxide anion by SOD.
5. Synthesis
5.1. Synthesis of A1
(a) 4-Bromo-3-nitro-1, 8-naphthalic anhydride (3.22 g,
10 mmol) and aniline (1.1 g, 11.8 mmol) were added to
7 mL DMF. The reaction mixture was stirred at room
temperature for 5 h, then cooled and poured into the
ice water, filtered, and dried. The crude product was ob-
tained as an orange solid (2.84 g, 8.5 mmol, 85% yield).
APCI-MS (positive) m/z: 335.1 ([M+H]+). (b) 4-aniline-
3-nitro-1, 8-naphthalic anhydride (2 g, 5.81 mmol) and
NaBH4 (1 g, 26.3 mmol) were dissolved in 100 mL of
2 M NaOH solution. The solution was refluxed for
24 h and then cooled, and HCl was added until the
pH became 6. Then, the mixture was filtered and dried
to give a red product. The red product was suspended
in boiling MeOH, and then sufficient Et3N was added
to just give a homogeneous solution. After being
acidified with AcOH, the precipitate was filtered and
dried (1.2 g, 3.9 mmol, 65% yield). This product was
To illustrate further the existence of electron transfer
process, a mechanistic experiment was performed under
anaerobic conditions. In this experiment, a phosphate
buffer was used to prevent the Ôheavy atom effect.Õ Figure
5e clearly shows that DTT did not inhibit the DNA
cleavage anymore under anaerobic conditions. Howev-
er, the supercoiled pBR322 DNA was still damaged,
although not as effectively as it was under aerobic con-
ditions. This result shows that superoxide anion was
likely to be a Ôside-productÕ of the electron transfer pro-
cess under aerobic conditions. In addition, the electron
transfer process favored the DNA damage more than

P. Yang et al. / Bioorg. Med. Chem. 13 (2005) 5909–5914
5913
1
not purified and was directly used in the next step. (c)
0.6 g of above-obtained solid was refluxed with N,N-
dimethylethylenediamine (0.3 mL) in ethanol (30 mL)
for 2 h, cooled, solvent removed, and separated on silica
gel chromatography to yield the pure product.
A5: mp: 214.4–215.2 ꢁC. H NMR (CDCl3) d (ppm):
2.61(s, 6H, NCH3), 3.01 (s, 2H, NCH2), 4.09 (s,
3H, OCH3), 4.51 (s, 2H, CONCH2), 7.56–7.57
(d, J = 2.8 Hz, 1H), 7.65–7.65 (d, J = 2.4 Hz, 1H),
7.67–7.68 (d, J = 2.8 Hz, 1H), 8.01–8.05 (t,
J1 = 7.6 Hz,J2 = 8.0 Hz, 1H), 8.26–8.28 (d, J = 9.6 Hz,
2H), 8.73–8.75 (d, J = 7.6 Hz, 1H), 9.16 (s, 1H), 9.63–
9.65 (d, J = 8.0 Hz, 1H), ESI-HRMS: Calcd for
C23H20N4O3 (M+H+): 401.1614, Found: 401.1618. IR
Separated on silica gel chromatography (CHCl3/
MeOH = 9:1, v/v) to give pure A1 (0.629 g, 85% yield).
1
A1: mp: 198.3–198.5 ꢁC. H NMR (CDCl3) d (ppm):
2.60 (s, 6H, NCH3), 2.99 (s, 2H, NCH2), 4.51 (s, 2H,
CONCH2), 7.96–8.08 (m, 3H), 8.38–8.43 (m, 2H),
8.76–8.79 (d, J = 8.0 Hz, 1H), 9.22 (s, 1H), 9.69–9.71
(d, J = 8.0 Hz, 1H), ESI-HRMS: Calcd for
C22H18N4O2 (M+H+): 371.1508, Found: 371.1510. IR
(KBr): 2924, 2853, 1705, 1655, 1350 cmꢀ1
.
1
A6: mp: 185.2–185.5 ꢁC. H NMR (CDCl3) d (ppm):
2.98 (s, 6H, NCH3), 3.06 (s, 3H, CH3), 3.49 (s, 2H,
NCH2), 4.69 (s, 2H, CONCH2), 7.83–7.86 (m, 2H),
8.05–8.09 (t, J1 = 8.0 Hz, J2 = 8.0 Hz, 1H), 8.20–8.22
(d, J = 6.8 Hz, 1H), 8.75–8.77 (d, J = 7.2 Hz, 1H), 9.23
(s, 1H), 9.73–9.75 (d, J = 8.8 Hz, 1H). ESI-HRMS:
Calcd for C23H20N4O2 (M+H+): 385.1665, Found:
(KBr): 2924, 2854, 1708, 1662, and 1336, cmꢀ1
.
5.2. Synthesis of A2–A6
The preparation and purification procedure of A2–A6
were similar to those of A1; different aniline derivatives
were used here, instead of aniline.
385.1682. IR (KBr): 2924, 2854, 1704, 1667, 1345 cmꢀ1
.
5.3. Spectroscopic measurements and DNA-binding
studies
Separated on silica gel chromatography (CHCl3/
MeOH = 9:1, v/v) to give purified A4 (43% yield), A5
(55% yield), and A6 (35% yield). During the synthesis
of A2 and A3, two isomers at the 1-site and the 3-site
were produced and 1-site isomer was the main product.
The purified products could not be obtained after silica
gel chromatography. So, recrystallization from ethanol
after silica gel chromatography (CHCl3/MeOH = 9:1,
v/v) was necessary to give the purified A2 (32% yield)
and A3 (40% yield).
UV–vis absorption spectra were recorded on Shimadzu
UV and fluorescent spectra on a Perkin-Elmer LS 50
luminescence spectrophotometer.
A2 was dissolved in absolute ethanol to give 10ꢀ5 M
solutions and rhodamine B in ethanol was used as
quantum yield standard.
DNA-binding studies were performed in Tris buffer
(tris(hydroxymethyl)aminomethane)–HCl (20 mM, pH
7.0). 0.1 mL of A2 DMSO solution (10ꢀ3–10ꢀ4 M) was
diluted with buffer to 10 mL. Fluorescent wavelength
and intensity were measured.
1
A2: mp: 178.1–178.5 ꢁC. H NMR (CDCl3) d (ppm):
2.47 (s, 6H, NCH3), 2.83 (s, 2H, NCH2), 2.99 (s, 3H,
CH3), 4.43–4.46 (t, J1 = 6.8 Hz, J2 = 7.2 Hz, 2H,
CONCH2), 7.77–7.79 (d, J = 6.4 Hz, 1H), 7.86–7.90 (t,
J1 = 8.0 Hz, J2 = 7.6 Hz, 1H), 8.01–8.05 (t, J1 = 7.6 Hz,
J2 = 7.6 Hz, 1H), 8.22–8.24 (d, J = 8.4 Hz, 1H), 8.75–
8.77 (d, J = 7.6 Hz, 1H), 9.24 (s, 1H), 9.66–9.68 (d,
J = 8.0 Hz, 1H), ESI-HRMS: Calcd for C23H20N4O2
(M+H+): 385.1665, Found: 385.1671. IR (KBr): 2924,
5.4. Topoisomerase I inhibitory
Topoisomerase I was purchased from TaKaRa Co.,
Ltd. The cleavage assays were performed as reported
in reference.15 The drug, DNA, and topoisomerase I
were incubated for 30 min at 37 ꢁC in Tris–HCl buffer
(20 mM, pH 7.5) before carrying out agarose-gel electro-
phoresis. 2% agarose-gel electrophoresis was carried out
at 25 V in 40 mM TAE buffer (40 mM tris(hydroxy-
methyl)aminomethane, 30 mM glacial acetic acid, and
1 mM EDTA, pH 7.5). After electrophoresis, the gel
was stained with ethidium bromide. The experiments
were repeated three times.
2853, 1702, 1660, 1340 cmꢀ1
.
1
A3: mp: 189.6–190.5 ꢁC. H NMR (CDCl3) d (ppm):
2.99 (s, 6H, NCH3), 3.52 (s, 2H, NCH2), 4.24 (s, 3H,
OCH3), 4.70 (s, 2H, CONCH2), 7.26–7.29 (t,
J1 = 7.6 Hz, J2 = 8.0 Hz, 1H), 7.91–8.05 (m, 3H), 8.77–
7.78 (d, J = 6.8 Hz, 1H), 9.39 (s, 1H), 9.69–9.71 (d,
J = 8.4 Hz, 1H), ESI-HRMS: Calcd for C23H20N4O3
(M+H+): 401.1614, Found: 401.1595. IR (KBr): 2924,
2853, 1702, 1660, 1340 cmꢀ1
.
5.5. Cytotoxicity in vitro evaluation
1
A4: mp: 209.6–210.1 ꢁC. H NMR (CDCl3) d (ppm):
2.49 (s, 6H, NCH3), 2.72 (s, 3H, CH3), 2.85 (s, 2H,
NCH2), 4.43–4.47 (t, 2H, J1 = 6.8 Hz,J2 = 7.2 Hz,
CONCH2), 7.82–7.84 (d, J = 10.0 Hz, 1H), 8.01–8.05
(t, J1 = 7.6 Hz, J2 = 8.0 Hz, 1H), 8.13 (s, 1H), 8.28–
8.30 (d, J = 8.8 Hz, 1H), 8.74–8.76 (d, J = 7.2 Hz,
1H), 9.18 (s, 1H), 9.67–9.67 (d, J = 8.0 Hz, 1H), ESI-
HRMS: Calcd for C23H20N4O2 (M+H+): 385.1665,
Found: 385.1664. IR (KBr): 2924, 2854, 1704, 1663,
The prepared compounds were submitted to Shanghai
Institute of Materia Medica with a view to get their
cytotoxicities tested.
5.6. Photocleavage of supercoiled pBR322 DNA
Irradiation was performed with a lamp (365 nm), placed
at 20 cm from the samples. The irradiated samples con-
tained pBR322 DNA (0.5 lg) dissolved in Tris–HCl
buffer (20 mM, pH 7.5) and the examined compounds.
1357 cmꢀ1
.

5914
P. Yang et al. / Bioorg. Med. Chem. 13 (2005) 5909–5914
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Acknowledgments
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.
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