Oxoisoaporphine alkaloid derivatives: Synthesis, DNA binding affinity and cytotoxicity

Article abstract of DOI:10.1016/j.ejmech.2007.07.004

A series of novel oxoisoaporphine alkaloid derivatives, 9-aminoalkanamido-1-azabenzanthrone (general formula Ar-NHCO(CH₂)_nNR₂, Ar = 1-azabenzanthrone, n = 1, 2 or 3), had been synthesized. Compared with 1-azabenzanthrone, the derivatives had significantly higher DNA binding affinity with calf thymus DNA, and higher potent cytotoxicity against different tumor cell lines. The cytotoxicity and the structure-activity relationship of the prepared compounds were studied. The derivatives with two methylene groups (n = 2), and piperidine or ethanolamine functional group in the side chain exhibited highest DNA binding affinity and cytotoxicity.

Full text of DOI:10.1016/j.ejmech.2007.07.004

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 973e980
http://www.elsevier.com/locate/ejmech
Original article
Oxoisoaporphine alkaloid derivatives: Synthesis, DNA binding
affinity and cytotoxicity
a,b
Huang Tang , Xiao-Dong Wang , Yong-Biao Wei , Shi-Liang Huang , Zhi-Shu Huang
b
b
b
b,
*
,
Jia-Heng Tan , Lin-Kun An , Jian-Yong Wu , Albert Sun-Chi Chan , Lian-Quan Gu a,b,**
b
b
c
c
a
School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China
b
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510080, China
c
State Key Laboratory of Chinese Medicine and Molecular Pharmacology (Shenzhen) and Department of Applied Biology and Chemical Technology,
The Hong Kong Polytechnic University, Hong Kong, China
Received 26 October 2006; received in revised form 30 June 2007; accepted 2 July 2007
Available online 15 July 2007
Abstract
2 n 2
A series of novel oxoisoaporphine alkaloid derivatives, 9-aminoalkanamido-1-azabenzanthrone (general formula AreNHCO(CH ) NR ,
Ar ¼ 1-azabenzanthrone, n ¼ 1, 2 or 3), had been synthesized. Compared with 1-azabenzanthrone, the derivatives had significantly higher
DNA binding affinity with calf thymus DNA, and higher potent cytotoxicity against different tumor cell lines. The cytotoxicity and the
structureeactivity relationship of the prepared compounds were studied. The derivatives with two methylene groups (n ¼ 2), and piperidine
or ethanolamine functional group in the side chain exhibited highest DNA binding affinity and cytotoxicity.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Oxoisoaporphine alkaloid derivatives; Synthesis; DNA binding; Cytotoxicity
1
. Introduction
Fig. 1), also possessed a 1-azabenzanthone moiety and were
reported to display cytotoxic activities against a small panel
of cancer cell lines in preliminary screening [8]. The recent
studies showed that oxoaporphine alkaloids such as Liriode-
nine (D in Fig. 1) [9], which possessed a positional isomeric
moiety of oxoisoaporphine, could intercalate with DNA and
inhibit topoisomerase II [10]. From medicinal chemist’s view-
point, the 1-azabenzanthone moiety of oxoisoaporphine alka-
loids could also interact with DNA as an intercalator and
responsible for the cytotoxic behavior of oxoisoaporphine
alkaloids.
Some analogs of oxoisoaporphines [11e15] had been
synthesized to investigate their potential antitumor activities.
We found that a planar polycyclic chromophore, and at least
one basic side chain in these compounds could increase their
binding affinity with DNA, and in some cases increase
solubility under physiological conditions [16].
The interaction of small molecules with DNA plays an
important role in many biological processes. These associative
interactions with the DNA molecules can cause dramatic
changes in the physiological functions of DNA, that might be
responsible for the cytotoxic behavior of the small molecules.
Oxoisoaporphine alkaloids were isolated from the rhizome
of Menispermum dauricum DC. (Menispermaceae) which
occured widely in the People’s Republic of China. The
rhizomes of the plant were used in traditional Chinese
medicine as an analgesic and antipyretic [1e8]. Oxoisoapor-
phine alkaloids possessed a 1-azabenzanthrone moiety in their
structures (A in Fig. 1). Daurioxoisorphines (B and C in
*
* Corresponding author. School of Pharmaceutical Sciences, Sun Yat-sen
Corresponding author. Tel.: þ86 20 39332679, fax: þ86 20 39332678.
*
University, Guangzhou 510080, China.
In view of the above reasons, a series of oxoisoaporphine
derivatives (9e18 in Fig. 2) with different basic side chain
E-mail addresses: ceshzs@mail.sysu.edu.cn (Z.-S. Huang), cesglq@mail.
sysu.edu.cn (L.-Q. Gu).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.07.004

974
H. Tang et al. / European Journal of Medicinal Chemistry 43 (2008) 973e980
ꢀ
HO
O
anhydrous aluminium chloride (1:5) at 220 C for 2 h to yield
o-isoquinolin-2-ylbenzoic acid 2. Cyclization of 2 in concen-
4
3
9
2
5
6
N
ꢀ
N
H
trated sulfuric acid at 230 C for 2 h afforded 3 in 25% yield.
9-Nitro-1-azabenzanthrone (4) was obtained by nitration of 3
with fuming nitric acid in the presence of concentrated sulfuric
N 1
7
O
1
1
O
acid. Reaction of 4 with Na S led to the 9-amino-1-azabenzan-
2
8
10
O
throne 5 in 95% yield. The u-haloalkanamides 6e8 were pre-
pared in essentially quantitative yield by acylation of 5 with
the appropriate acid halide (n ¼ 1e3). Subsequent aminolysis
of 6e8 by reflux treatment with the appropriate secondary
amines or primary amines gave the target compounds 9e18.
This general procedure was successful for compounds with
n ¼ 1 or 2, but attempted aminolysis of compounds where
n ¼ 3 (e.g. 17 and 18) gave low yields of the required products.
A
B
O
O
O
N
N
O
H N
2
O
2.2. DNA binding properties
O
Spectroscopic methods had been used to ascertain the DNA
C
D
binding features of compounds 9e18. The DNA binding
affinity of oxoisoaporphine chromophores with various types
of amino groups and the length of the side chain were
evaluated.
Fig. 1. Chemical structures of 1-azabenzanthrone (A), two daurioxoisophines
B and C) and Liriodenine (D).
(
Fig. 3 showed the representative absorption spectra of
compound 15 (20 mM) in the presence of increasing concen-
tration of CT DNA (0e400 mM). Other compounds exhibited
a similar absorption spectra pertaining to the chromophore but
with the hypochromicity and red shifts depending on the
types of amino side chains. It was observed that the addition
of CT DNA to the solutions of 9e18 at the DNA/compound
molar ratios varying from 0 to 20 induced large bathochromic
shifts (7e14 nm) and hypochromicities (17e26%) (Table 1).
Large hypochromism and the red shift in the absorption
spectra indicated a strong electronic interaction between the
1-azabenzanthrone moiety of oxoisoaporphines and the DNA
bases, that suggested a close proximity of the oxoisoaporphine
2chromophore to the DNA bases through a strong overlap of
the pep* states [18]. On the other hand, an isosbestic point
was also observed in the spectra. These various spectral
changes were consistent with the intercalation of these
derivatives into the DNA base stack [18].
at 9-position of 1-azabenzanthrone (general formula Are
NHCO(CH ) NR , Ar ¼ 1-azabenzanthrone, n ¼ 1, 2 or 3)
2
n
2
were designed and synthesized. Their DNA binding capacity
was evaluated based on interaction with calf thymus (CT)
DNA. The cytotoxicity of the synthetic componds were
evaluated against different tumor cell lines, such as human
breast cancer MCF-7, human hepatocellular carcinoma
HepG2, and NCI-H460 cells. Their structure and bioactivity
relationships were discussed.
2
. Results and discussion
2
.1. Synthesis of oxoisoaporphine derivatives
The synthetic steps of the target compounds are shown in
Scheme 1. Preparation of 1-azabenzanthrone 3 was carried out
by a reported method [17]. Acylation of b-phenylethylamine
with phthalic anhydride gave phenylethylphthalimide 1 in
The selection of ionic strength (150 mM NaCl) in the
absorption titration experiments was mainly based on the
8
0% yield. Compound 1 was heated in sodium chloride and
N
NHCO(CH ) NR
2
2
n
O
Compound
NR2
9: n=1
10: n=1
16: n=2
18: n=3
11: n=2
12: n=2
13: n=2
14: n=2
15: n=2
17: n=3
N
N(CH3)2
N
NEt2
NH(CH ) N(CH )
3 2
NH(CH ) OH
2 2
2
2
Fig. 2. Structures of oxoisoaporphine derivatives 9e18.

H. Tang et al. / European Journal of Medicinal Chemistry 43 (2008) 973e980
975
O
O
N
NH2
a
b
+
O
N
O
O
HOOC
1
2
N
N
N
c
d
e
NO2
NH2
O
O
O
3
4
5
N
O
N
f
g
NHCO(CH )nCl
NHCO(CH )nNR2
2
2
O
6: n=1
7: n=2
8
: n=3
9-18
ꢀ
ꢀ
Scheme 1. Synthesis of oxoisoaporphine derivatives. Reagents: (a) EtOH (reflux); (b) anhydrous AlCl
nitric acid/concentrated H SO ; (e) Na S$9H O/N OH/EtOH (reflux); (f) COCl(CH Cl (reflux); (g) R
3
(220 C); (c) concentrated H
NH/EtOH/KI (reflux).
2
SO
4
(230 C); (d) fuming
2
4
2
2
a
2
)
n
2
avoidance of DNA deposition (or precipitation) in all drug
solutions (20 mM) [19].
fluorescence intensity of the compound decreased rapidly
with increasing concentrations of calf thymus DNA. The titra-
tion spectra and peaks for other compounds (9e12, 14, and
16) exhibited a similar trend of changes with the CT DNA
concentration titration. Interesting phenomenon was found
where emission spectra showed reverse trend when titrated
for compounds 13, 17 and 18. Fig. 4(b) showed the fluores-
cence emission spectra of compound 17. The compounds 13
In the fluorescence spectrometric titration, a much lower
drug concentration (3 mM) could be used at a lower NaCl
concentration (50 mM) to measure the appropriate binding
constants. Despite the similarity in terminal amine, the fluores-
cence emission spectra of the compounds with n ¼ 1 and n ¼ 2
(
except compound 13) showed different phenomenon to the
n ¼ 3 compounds (17 and 18) and 13. Fig. 4(a) showed the
fluorescence emission spectra of compound 15 in the presence
of various concentrations of calf thymus DNA. The
Table 1
Binding constants (K
tives 9e18 with CT DNA
i
) and photometric properties of oxoisoaporphine deriva-
Compound
K
i
Red shift
b,d
(nm)
Hypochromicity
b,d
(%)
Isosbestic
point (nm)
0
0
0
0
0
0
0
0
0
.16
.14
.12
.10
.08
.06
.04
.02
.00
ꢂ5
ꢂ1 a
(
ꢁ10
M
)
CT DNA
c
3
9
1.04
1.79
1.68
2.55
2.39
0.49
3.17
2.54
2.70
1.45
1.23
1
5
9%
20
21
17
23
23
16
19
22
17
15
Unclear
434
437
c
c
c
c
e
c
c
c
e
e
10
11
12
13
14
15
16
17
18
a
5
8
438
439
12
7
Unclear
440
440
8
9
10
7
440
440
8
440
Drug of 3 mM in 10 mM NaH
0 mM NaCl at room temperature.
2
PO
4
eNa
2
HPO
4
buffer (pH 7.0) containing
5
1
l
b
Drug of 20 mM in 10 mM NaH
50 mM NaCl at room temperature.
2
PO
4
eNa
2
HPO
4
buffer (pH 7.0) containing
360
380
400
420
440
Wavelength (nm)
460
480
500
c
Obtained from spectrofluorimetric titration at
em ¼ 550 nm.
l
ex ¼ 418 nm and
ex ¼ 418 nm and
d
Fig. 3. Absorbance titration of 15 with CT DNA. Compound 15 (20 mM) in
0 mM sodium phosphate buffer (pH 7.0) with 150 mM NaCl at increasing
CT DNA concentration (arrow: 0e400 mM).
Obtained at lmax.
e
1
Obtained from spectrofluorimetric titration at
em ¼ 540 nm.
l
l

976
H. Tang et al. / European Journal of Medicinal Chemistry 43 (2008) 973e980
1
.7
.6
1.0
0.9
0.8
0.7
0.6
0.5
0.4
(
a) 650
(b)
1
1
80
6
5
5
4
4
3
3
2
2
1
1
00
50
00
50
00
50
00
50
00
50
00
1.5
1.4
160
CT DNA
1.3
1.2
1.1
1.0
1
40
20
CT DNA
1
100
0.9
0
20 40 60 80 100 120
0
20 40 60 80 100 120
8
6
0
0
DNA Concentration ( M)
DNA Concentration ( M)
40
2
0
50
0
0
-
50
-20
4
60 480 500 520 540 560 580 600 620 640 660 680 700
460 480 500 520 540 560 580 600 620 640 660 680 700
Wavelength (nm)
Wavelength (nm)
Fig. 4. (a) Fluorescence spectra of compound 15 (3 mM) in 10 mM sodium phosphate buffer (pH 7.0) with 50 mM NaCl at increasing CT DNA concentration
arrow: 0e138 mM); Inset: ratio of the fluorescence intensities in the presence of DNA I and in absence of DNA I versus CT DNA concentration with excitation
at 418 nm and monitoring at 550 nm. (b) Fluorescence spectra of compound 17 (3 mM) in 10 mM sodium phosphate buffer (pH 7.0) with 50 mM NaCl at increas-
(
0
0
ing CT DNA concentration (arrow: 0e138 mM). Inset: ratio of the fluorescence intensities in the presence of DNA I and in absence of DNA I versus CT DNA
concentration with excitation at 418 nm and monitoring at 540 nm.
and 18 had the similar emission spectra that fluorescence in-
tensity of compound increased with increasing concentrations
of calf thymus DNA. Either an increase or a decrease of the
emission intensity was suggestive of a strong association of
the compound molecules with the DNA molecules. Increase
in the fluorescence intensity might be due to significant sup-
pression of the conformational flexibility of longer side chain
when the compound interacts with DNA to form complex [20].
Table 1 summarizes the DNA binding constants and related
properties of compounds 3 and 9e18 after interaction with CT
DNA. The relative binding affinities as indicated by the
be responsible for the lower binding affinity. Compounds 13
and 14 which possessed the longest side chain showed marked
distinctness in DNA binding affinity. The lowest and the
highest DNA binding constant were found for compounds
13 and 14, respectively. The high binding affinity of
compound 14 was attributed to hydroxyl group in the end of
the side chain [21]. The alkyl chain of compound 14 could
fold onto itself to position the hydroxyl group close to the
hydrogen bonding acceptor sites of the adjacent base pairs.
Hydrogen bonding interactions between the hydroxyl group
of 14 and oxygen-2 of thymine were most likely responsible
for the high DNA binding affinity.
binding constants K were in the order of 14 > 11 > 16 > 15 >
i
1
2 > 9 > 10 > 17 > 18 > 3 > 13. It was clear that most of
derivatives had a higher DNA binding constant than their
lead compound 3. This result indicated that introduction of
side chains with amino group could increase the DNA binding
capacity of original chromophore.
Among the compounds, large differences in binding
affinities were found. The length and flexibility of the linker
connecting the fused ring and amino functional groups were
expected to be important in capacity for the ligand binding
to DNA. Compounds 11, 12, 14, 15 and 16 with two
methylene groups in the side chains (n ¼ 2) had larger DNA
binding constant. Compounds 9 and 10 with shorter side chain
2.3. Cytotoxicity against tumor cells
The in vitro cytotoxicity of oxoisoaporphine derivatives
was evaluated using MTT method. The concentration of
oxoisoaporphine derivatives for 50% inhibition (IC ) on the
human hepatocellular carcinoma HepG2 cell line, human
breast cancer MCF-7 cell line and NCI-H460 cell line were
determined and the results are summarized in Table 2. Accord-
ing to the data of Table 2, it was clear that the oxoisoaporphine
derivatives exhibited significant higher cytotoxicity than that
of the lead compound 3.
5
0
(
n ¼ 1) showed weaker binding properties, resulting in
The derivatives 11, 14 and 16, with the side chain n ¼ 2,
decrease of the electrostatic interactions between terminal
amino group and DNA phosphates. Compounds 17 and 18
with longer and more flexible side chain (n ¼ 3), obstructed
the chromophore accessing the DNA base pairs, which might
had lower IC values for all tumor cells. Compounds 17
5
0
and 18, with the side chain n ¼ 3, displayed higher IC
5
0
values. Other compounds, with the side chain n ¼ 1, showed
moderate cytotoxicity.
Table 2
IC50 cytotoxicity values (mM) of oxoisoaporphine derivatives against tumor cells (data derived from the mean of three independent assays)
Compound
3
5
9
10
9.22
11
12
4.99
13
14
15
16
17
9.08
18
NCI-H460
HepG2
MCF
n.d.
>100
>100
35.53
19.68
20.4
2.09
8.14
4.23
6.36
5.07
30.26
21.02
28.08
2.36
3.88
4.3
16.72
8.18
7.96
6.71
7.74
9.11
19.29
29.83
11.48
9.69
17.46
12.1
12.67
12.94
27.39
11.81

H. Tang et al. / European Journal of Medicinal Chemistry 43 (2008) 973e980
977
3
3
2
2
2
2
2
1
1
1
1
1
2
0
8
6
4
2
0
8
6
4
2
0
8
6
4
2
30
28
26
(a)
(b)
HepG2 cells
MCF-7 cells
2
2
2
1
1
1
1
1
4
2
0
8
6
4
2
0
8
6
4
2
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0.0
0.5
1.0
1.5 2.0
-5 -1
Ki (×10 M )
2.5
3.0
3.5
-
5
-1
Ki (×10 M )
Fig. 5. Scatter plots representing DNA binding constant versus biological effect for a series of oxoisoaporphine derivatives (9e18). (A) Correlation between in vitro
cytotoxicity (for human hepatocellular carcinoma HepG2 cells) and binding affinities with double-stranded CT DNA. (B) Correlation between in vitro cytotoxicity
(for human breast cancer MCF-7 cells) and binding affinities with double-stranded CT DNA.
There was a clear correlation between the DNA binding
300 NMR spectrometer with tetramethylsilane (TMS) as an
internal standard. Elemental analysis was carried out on an
Elementar Vario EL CHNS elemental analyzer.
affinities and the IC50 values. Fig. 5 shows a scatter plot of
in vitro cytotoxicity (IC50 values determined for HepG2 cells
and MCF-7 cells, respectively) versus binding constant with
CT DNA for a series of oxoisoaporphine derivatives (9e18).
Using a linear fitting procedure, a statistically significant fit
was obtained, and it was clearly shown that the cytotoxic
effects and DNA binding affinities were positively correlated
3.1.1. Phenylethylphthalimide (1)
A mixture of phthalic anhydride (30 g, 0.2 mol) and
b-phenylethylamine (26 g, 0.2 mol) in anhydrous ethanol
ꢀ
was refluxed for 6 h. After cooling to 0e5 C, crystals of
phenylethylphthalimide (1, 43 g, 78%) separated. These were
[
22]. Compound 14 in the side chain with terminal hydroxyl
group had the highest DNA binding constant that responded
to the lowest IC50 for both HepG2 and MCF-7. Compound
filtered off, washed with ethanol. The product is pure enough
ꢀ
ꢀ
for the next reaction. Mp 130e131 C (lit. [17] 124e126 C);
1
1
3 possessing the lowest DNA binding affility showed the
H NMR (CDCl , 300 MHz): d 2.98 (t, 2H, J ¼ 7.6 Hz), 3.92
3
H
highest IC50 against NCI-H460 and MCF-7. The results
pointed out that the cytotoxicity of derivatives could be
attributed to the interaction of compounds with DNA.
(t, 2H, J ¼ 7.6 Hz), 7.17e7.29 (m, 4H), 7.67e7.71 (m, 2H),
þ
7.78e7.82 (m, 2H); ESI-MS m/z: 252 [M þ H] .
In conclusion, we have synthesized a new series of
oxoisoaporphine alkaloid derivatives (9e18), and all of the
prepared products with different basic side chain at 9-position
of 1-azabenzanthrone showed higher in vitro cytotoxicity
against three human carcinoma cells than the parent
compounds 1-azabenzanthrone (3). The results are consistent
with the viewpoint that the introduction of basic side chain
into a planar polycyclic chromophore might strengthen the
cytotoxic action. Furthermore, the fact that derivatives
showing higher cytotoxicity than the parent compounds also
provide a good lead for finding more potent anticancer agents.
3.1.2. 1-Azabenzanthrone (3)
To a mixture of anhydrous aluminium chloride (75 g,
0.56 mol) and sodium chloride (15 g, 0.26 mol) was slowly
ꢀ
added phenylethylphthalimide (1, 53 g, 0.2 mol) at 180 C
for 30 min. The reaction was allowed to continue at
ꢀ
220e230 C for 2 h. The product was cooled, finely ground
and poured slowly into concentrated sulfuric acid (550 mL)
ꢀ
ꢀ
at 90 C. The mixture was stirred and heated at 230e240 C
for 2 h. After being cooled, the solution was poured into ice.
Sodium hydroxide was added until pH ¼ 3 was obtained and
the resultant precipitate was filtered off and washed in turn
with dilute aqueous sodium hydroxide and water to give the
crude product, 18 g, which was extracted with acetic acid.
The extract was condensed under reduced pressure and the
3
. Experimental
3
.1. Synthesis
resultant precipitate was washed off, dried, and sublimed at
ꢀ
130e140 C at a pressure of 1 mm mercury to give the prod-
uct 3 (15.3 g, 32%) as light yellow solid, mp 182e183 C. (lit.
ꢀ
UV/visible absorbance spectra were measured on a Shima-
ꢀ
1
[17] 181e183 C); H NMR (CDCl , 300 MHz): dH 7.64
dzu UV-2450 spectrophotometer. Fluorometric measurements
were performed on a Shimadzu RF-5301PC spectrofluoropho-
tometer. Melting points (mp) were determined using a WRR
melting point instrument without correction. ESI-MS spectra
were obtained using an LCMS-2010A mass spectrometer.
3
(t, 1H, J ¼ 7.7 Hz), 7.74 (d, 1H, J ¼ 5.8 Hz), 7.80 (t, 1H,
J ¼ 7.3 Hz), 7.90 (t, 1H, J ¼ 7.3 Hz), 8.15 (d, 1H, J ¼ 8.2 Hz),
8.41 (d, 1H, J ¼ 7.8 Hz), 8.66 (d, 1H, J ¼ 7.2 Hz), 8.77 (d, 1H,
J ¼ 5.6 Hz), 8.90 (d, 1H, J ¼ 7.9 Hz); ESI-MS m/z: 232
1
þ
H NMR spectra were performed on a Varian Mercury-Plus
[M þ H] .

978
H. Tang et al. / European Journal of Medicinal Chemistry 43 (2008) 973e980
3
.1.3. 9-Nitro-1-azabenzanthrone (4)
mL of 62% nitric acid was added to a mixture of concen-
trated sulfuric acid (12 mL) and 3 (2.3 g 10 mmol) in a round-
3.1.7. 9-(4-Chlorobutyramido)-1-azabenzanthrone (8)
1
9-Amino-1-azabenzanthrone (5) (1.0 g, 4 mmol) was
treated with 4-chlorobutanoyl chloride according to the
general acylation procedure, except that reflux was for only
2.5 h, to give butyramide 8 (1.0 g, 74%) as orange/yellow
solid. (Found: C, 68.8; H, 4.7; N, 8.3%. C H N O Cl
ꢀ
bottom flask, and the mixture was stirred at 50 C for 7 h.
After being cooled, the solution was poured into ice. Ammonia
was added until pH ¼ 8e9 was obtained and the precipitate
was filtered off and washed with water to give the crude
product. Recrystallization from benzene afforded 4 (1.4 g,
2
0 15 2 2
ꢀ
1
requires C, 68.5; H, 4.3; N, 8.0%.) Mp 230e232 C;
H
NMR (DMSO, 300 MHz): dH 2.09 (m, 2H), 2.58 (t, 2H,
J ¼ 7.3 Hz), 3.74 (t, 2H, J ¼ 6.5 Hz), 7.98 (d, 1H,
J ¼ 5.6 Hz), 8.01e8.10 (m, 2H), 8.41 (d, 1H, J ¼ 8.1 Hz),
8.54e8.56 (m, 2H), 8.72e8.78 (m, 2H), 10.46 (s, 1H,
ꢀ
ꢀ
1
5
1%) as yellow solid. Mp > 280 C (lit. [23] 284e286 C); H
NMR (CDCl , 300 MHz): dH 7.89 (d, 1H, J ¼ 5.5 Hz), 7.99
3
(
t, 1H, J ¼ 8.1 Hz), 8.26 (d, 1H, J ¼ 8.2 Hz), 8.58 (dd, 1H,
1 2
þ
J ¼ 8.7 Hz and J ¼ 2.4 Hz), 8.75 (d, 1H, J ¼ 7.3 Hz), 8.88
eCONH); ESI-MS m/z: 352 [M þ H] .
(
d, 1H, J ¼ 5.5 Hz), 9.15 (d, 1H, J ¼ 8.8 Hz), 9.23 (d, 1H,
þ
J ¼ 2.5 Hz); ESI-MS m/z: 277 [M þ H] .
3.1.8. 9-(Pyrrolidinoacetamido)-1-azabenzanthrone (9)
General aminolysis procedure: To a stirred refluxing
3
.1.4. 9-Amino-1-azabenzanthrone (5)
To a stirred suspension of 4 (2.8 g, 10 mmol) in ethanol
suspension of 9-(2-chloroacetamido)-1-azabenzanthrone (6)
0.5 g, 1.5 mmol) and NaI (0.15 g) in EtOH (40 mL) was
added dropwise pyrrolidine (1.0 mL) in EtOH (10 mL). The
(
(
(
40 mL) was added a solution of sodium sulfide nonahydrate
5.5 g, 22.5 mmol) and sodium hydroxide (2.0 g, 50 mmol)
ꢀ
mixture was stirred at reflux for 3 h, cooled to 0 C, filtered,
in water (100 mL). The mixture was heated at reflux for 5 h
and left to stand overnight. The ethanol was removed in vacuo
and the residue cooled to 0e5 C. The resulting precipitate
was collected by filtration, washed with water, and dried.
and washed with ether and water. The crude solid was purified
by column chromatography with chloroform/methanol (50:1)
elution to afford a yellow solid compound 9 (0.48 g, 87%).
ꢀ
(Found: C, 70.5; H, 5.6; N, 11.5%. C H N O $H O requires
C, 70.4; H, 5.6; N, 11.2%.) Mp 186e188 C; H NMR
22 19 3 2 2
ꢀ
Recrystallization from ethanol afforded the product 5 as
ꢀ
1
a red solid (2.2 g, 88%). Mp w265 C (darkened). (lit. [23],
(
3
CDCl , 300 MHz): d 1.93 (m, 4H), 2.75 (t, 4H, J ¼ 6.7 Hz),
3
H
ꢀ
1
mp 278e281 C); H NMR (DMSO, 300 MHz): dH 7.03
d, 1H, J ¼ 8.6 Hz), 7.42 (d, 1H, J ¼ 2.4 Hz), 7.82 (d, 1H,
.35 (s, 2H), 7.69 (d, 1H, J ¼ 5.6 Hz), 7.87 (t, 1H,
(
J ¼ 7.3 Hz), 8.13 (d, 1H, J ¼ 8.2 Hz), 8.17 (d, 1H,
J ¼ 2.3 Hz), 8.45 (dd, 1H, J ¼ 8.7 Hz and J ¼ 2.3 Hz), 8.62
J ¼ 5.6 Hz), 7.99 (t, 1H, J ¼ 7.7 Hz), 8.34 (d, 1H, J ¼ 8.2 Hz),
1
2
8
.48e8.51 (m, 2H), 8.66 (d, 1H, J ¼ 5.6 Hz), 6.05 (s, 2H
(
d, 1H, J ¼ 7.2 Hz), 8.73 (d, 1H, J ¼ 5.6 Hz), 8.84 (d, 1H,
þ
eNH ); ESI-MS m/z: 247 [M þ H] .
þ
2
J ¼ 8.8 Hz), 9.50(s, 1H, eCONH); ESI-MS m/z: 358 [M þ H] .
3
.1.5. 9-(2-Chloroacetamido)-1-azabenzanthrone (6)
3
.1.9. 9-[(Dimethylamino)acetamido]-1-azabenzanthrone (10)
Acetamide 6 was treated with excess dimethylamine in
General acylation procedure: A suspension of 9-amino-1-
azabenzanthrone (5) (1.0 g, 4 mmol) in chloroacetyl chloride
EtOH according to the general aminolysis procedure used
for 9 above, to give 10 (80%), after column chromatography
with chloroform/methanol (50:1) elution, as yellow/orange
solid. (Found: C, 72.3; H, 5.3; N, 12.9%. C H N O requires
C, 72.5; H, 5.2; N, 12.7%.) Mp 167e169 C; H NMR
(CDCl , 300 MHz): d 2.43 (s, 6H), 3.14 (s, 2H), 7.70 (d,
3 H
(
0
15 mL) was heated at reflux for 4 h. After cooling to
ꢀ
e5 C, the mixture was filtered and the crude solid was
washed with ether. Recrystallization from EtOHeDMF (4:1
v/v) afforded chloroamide 6 (1.1 g, 85%) as yellow/brown
solid. (Found: C, 66.8; H, 3.7; N, 8.4%. C H N O Cl
2
0
17 3 2
ꢀ
1
1
8 11 2 2
ꢀ
1
requires C, 67.0; H, 3.4; N, 8.7%.) Mp 262e264 C;
H
1
H, J ¼ 5.6 Hz), 7.88 (t, 1H, J ¼ 7.3 Hz), 8.12 (d, 1H,
NMR (DMSO, 300 MHz): dH 4.35 (s, 2H), 7.98 (d, 1H,
J ¼ 5.6 Hz), 8.01e8.09 (m, 2H), 8.41 (d, 1H, J ¼ 8.2 Hz),
J ¼ 8.2 Hz), 8.21 (d, 1H, J ¼ 2.3 Hz), 8.44 (dd, 1H,
1
J ¼ 2.3 Hz and J ¼ 8.7 Hz), 8.65 (d, 1H, J ¼ 7.2 Hz), 8.74
1
2
8
8
[
.51 (d, 1H, J ¼ 2.2 Hz), 8.54 (d, 1H, J ¼ 7.2 Hz), 8.73e
(
d, 1H, J ¼ 5.6 Hz), 8.87 (d, 1H, J ¼ 7.3 Hz), 9.50(s, 1H,
,78 (m, 2H), 10.76 (s, 1H, eCONH); ESI-MS m/z: 324
þ
þ
eCONH); ESI-MS m/z: 332 [M þ H] .
M þ H] .
3
.1.6. 9-(3-Chloropropionamido)-1-azabenzanthrone (7)
-Amino-1-azabenzanthrone (5) (1.0 g, 4 mmol) was
3.1.10. 9-(3-Piperidinopropionamido)-1-azabenzanthrone (11)
Chloroamide 7 was treated with excess piperidine
according to the general aminolysis procedure to give 11
(75%), after column chromatography with chloroform/metha-
nol (100:3) elution, as yellow solid. (Found: C, 71.3; H, 6.3; N,
10.5%. C H N O $H O requires C, 71.4; H, 6.3; N, 10.4%.)
9
treated with 3-chloropropanoyl chloride according to the gen-
eral acylation procedure to give chloroamide 7 (1.0 g, 74%) as
orange/yellow solid. (Found: C, 68.1; H, 4.2; N, 8.2%.
C H N O Cl requires C, 67.8; H, 3.9; N, 8.3%.) Mp
1
9
13
ꢀ
2
2
24 23
3
2
2
1
ꢀ
Mp 156e159 C; H NMR (CDCl , 300 MHz): d 1.75 (m,
3 H
1
w242 C (darkened); H NMR (DMSO, 300 MHz): d 2.92
H
(
t, 2H, J ¼ 6.2 Hz), 3.93 (t, 2H, J ¼ 6.2 Hz), 7.98 (d, 1H,
2H), 1.80 (m, 4H), 2.56e2.60 (m, 6H), 2.73 (t, 2H,
J ¼ 6.2 Hz), 7.68 (d, 1H, J ¼ 5.6 Hz), 7.87 (t, 1H, J ¼ 7.3 Hz),
8.12 (d, 1H, J ¼ 8.2 Hz), 8.18 (d, 1H, J ¼ 2.3 Hz), 8.35 (dd,
1H, J ¼ 8.7 Hz and J ¼ 2.3 Hz), 8.62 (d, 1H, J ¼ 7.2 Hz),
J ¼ 5.6 Hz), 8.01e8.11 (m, 2H), 8.42 (d, 1H, J ¼ 8.2 Hz),
8
.54e8.56 (m, 2H), 8.73e8.78 (m, 2H), 10.55(s, 1H,
þ
eCONH); ESI-MS m/z: 338 [M þ H] .
1
2

H. Tang et al. / European Journal of Medicinal Chemistry 43 (2008) 973e980
979
8
.72 (d, 1H, J ¼ 5.6 Hz), 8.83 (d, 1H, J ¼ 8.7 Hz), 11.82 (s, 1H,
J ¼ 8.0 Hz), 8.72 (d, 1H, J ¼ 5.6 Hz), 8.80 (d, 1H, J ¼ 8.7),
þ
þ
eCONH); ESI-MS m/z: 386 [M þ H] .
11.71 (s, 1H, eCONH); ESI-MS m/z: 372 [M þ H] .
3
(
.1.11. 9-[3-(Diethylamino)propionamido]-1-azabenzanthrone
12)
Chloroamide 7 was treated with excess diethylamine ac-
cording to the general aminolysis procedure to give 12
78%), after column chromatography with chloroform/metha-
nol (100:3) elution, as yellow solid. (Found: C, 71.0; H, 6.6; N,
1.0%. C H N O $H O requires C, 70.6; H, 6.4; N, 10.7%.)
3.1.15. 9-[3-(Dimethylamino)propionamido]-1-azabenzanthrone
(16)
Chloroamide 7 was treated with excess dimethylamine in
EtOH according to the general aminolysis procedure to give
(
1
6 (about 70%), after column chromatography with chloro-
form/methanol (100:3) elution, as yellow/orange solid.
(Found: C, 69.9; H, 5.8; N, 11.7%. C21 $H O requires
1
2
3
23
3
2
1
2
H
19
N
3
O
2
ꢀ
2
ꢀ
1
Mp 166e168 C; H NMR (DMSO, 300 MHz): dH 1.21
C, 69.4; H, 5.8; N, 11.6%.) Mp 179e181 C; H NMR
(CDCl3, 300 MHz): dH 2.44 (s, 6H), 2.57 (t, 2H,
J ¼ 5.5 Hz), 2.70 (t, 2H, J ¼ 5.5 Hz), 7.66 (d, 1H,
J ¼ 5.6 Hz), 7.85 (t, 1H, J ¼ 7.3 Hz), 8.08e8.11 (m, 2H),
8.36 (dd, 1H, J ¼ 2.3 Hz and J ¼ 8.7 Hz), 8.59 (d, 1H,
(
(
(
t, 6H, J ¼ 7.0 Hz), 2.85 (t, 2H, J ¼ 6.4 Hz), 3.06e3.08
m, 4H), 3.31 (m, 2H), 7.98 (d, 1H, J ¼ 5.6 Hz), 8.01e8.08
m, 2H), 8.42 (d, 1H, J ¼ 8.2 Hz), 8.52e8.56 (m, 2H), 8.73e
8
.77 (m, 2H), 10.69 (s, 1H, eCONH); ESI-MS m/z: 374
1
2
þ
[
M þ H] .
J ¼ 7.2 Hz), 8.70 (d, 1H, J ¼ 5.6 Hz), 8.80 (d, 1H, J ¼ 8.7),
þ
11.43 (s, 1H, eCONH); ESI-MS m/z: 346 [M þ H] .
3
1
.1.12. 9-[3-[2-(Dimethylamino) ethylamino]propionamido]-
-azabenzanthrone (13)
Chloroamide 7 was treated with excess 2-(dimethylamino)
3.1.16. 9-(4-Pyrrolidinobutyramido)-1-azabenzanthrone (17)
Chloroamide 8 was treated with excess pyrrolidine accord-
ethylamine according to the general aminolysis procedure to
give 13 (about 70%), after column chromatography with chlo-
roform/methanol (25:1) elution, as yellow solid. (Found: C,
ing to the general aminolysis procedure, except that reflux was
for 4 days, to give 17 (about 20%), after column chromatogra-
phy with chloroform/methanol (100:3) elution, as yellow
solid. (Found: C, 67.9; H, 6.2; N, 9.6%. C H N O $2H O re-
6
2.4; H, 6.5; N, 12.3%. C H N O $3H O requires C, 62.4;
23 24 4 2 2
2
4
23
3
2
2
ꢀ
1
1
ꢀ
H, 6.8; N, 12.7%.) Mp 133e136 C; H NMR (DMSO,
00 MHz): dH 2.16 (s, 6H), 2.36 (t, 2H, J ¼ 6.2 Hz), 2.57
t, 2H, J ¼ 6.4 Hz), 2.67 (t, 2H, J ¼ 6.2 Hz), 2.90 (t, 2H,
J ¼ 6.4 Hz), 7.97 (d, 1H, J ¼ 5.6 Hz), 8.00e8.11 (m, 2H),
quires C, 68.4; H, 6.5; N, 10.0%.) Mp 138 C (darkened); H
NMR (DMSO, 300 MHz): d 1.82 (m, 2H), 1.95 (m, 4H), 2.52
3
(
H
(
t, 2H, J ¼ 7.3 Hz), 3.08 (t, 2H, J ¼ 7.2 Hz), 3.19 (t, 4H,
J ¼ 8.2 Hz), 7.95 (d, 1H, J ¼ 5.6 Hz), 7.99e8.04 (m, 2H),
8
.41 (d, 1H, J ¼ 8.2 Hz), 8.53e8.55 (m, 2H), 8.73 (d, 1H,
8
.39 (d, 1H, J ¼ 8.3 Hz), 8.50e8.53 (m, 2H), 8.69 (d, 1H,
J ¼ 8.8 Hz), 8.76 (d, 1H, J ¼ 5.6 Hz); ESI-MS m/z: 389
J ¼ 8.7 Hz), 8.71 (d, 1H, J ¼ 5.6 Hz), 10.46 (s, 1H,
þ
þ
[
M þ H] .
eCONH); ESI-MS m/z: 386 [M þ H] .
3
.1.17. 9-[4-(Dimethylamino) butyramido]-1-azabenzanthrone
(18)
Chloroamide 8 was treated with excess dimethylamine in
3
1
.1.13. 9-[3-(2-Hydroxyethylamino)propionamido]-
-azabenzanthrone (14)
Chloroamide 7 was treated with excess 2-hydroxy-ethyl-
amine according to the general aminolysis procedure to give
4 (90%) as yellow solid after recrystallization from ethanol.
Found: C, 69.6; H, 5.5; N, 11.6%. C H N O requires C,
EtOH according to the general aminolysis procedure, except
that reflux was for 4 days, to give 18 (about 15%), after col-
umn chromatography with chloroform/methanol (100:3) elu-
tion, as yellow/orange solid. (Found: C, 66.6; H, 6.7; N,
1
(
2
ꢀ
1 19 3 3
1
6
3
9.8; H, 5.3; N, 11.6%.) Mp 174e176 C; H NMR (DMSO,
00 MHz): dH 2.56 (t, 2H, J ¼ 6.4 Hz), 2.62 (t, 2H,
1
1
0.6%. C H N O $2H O requires C, 66.8; H, 6.4; N,
22 21 3 2 2
ꢀ
1
0.6%.) Mp 106e110 C; H NMR (CDCl , 300 MHz): d
H
J ¼ 5.6 Hz), 2.87 (t, 2H, J ¼ 6.4 Hz), 3.48 (t, 2H, J ¼ 5.6 Hz),
3
7
.94 (d, 1H, J ¼ 5.6 Hz), 7.98e8.07 (m, 2H), 8.38 (d, 1H,
1.96 (m, 2H), 2.46 (s, 6H), 2.59 (t, 2H, J ¼ 5.9), 2.64 (t, 2H,
J ¼ 6.2 Hz), 7.71 (d, 1H, J ¼ 5.6 Hz), 7.90 (d, 1H, J ¼ 7.5),
J ¼ 8.2 Hz), 8.50e8.53 (m, 2H), 8.68 (d, 1H, J ¼ 8.2 Hz),
þ
8
.73 (d, 1H, J ¼ 5.6 Hz); ESI-MS m/z: 362 [M þ H] .
8.14e8.18 (m, 2H), 8.40 (dd, 1H, J
1
¼ 2.3 Hz and J ¼
2
8
.7 Hz), 8.64 (d, 1H, J ¼ 7.2 Hz), 8.75 (d, 1H, J ¼ 5.6 Hz),
3
.1.14. 9-[3-Pyrrolidinopropionamido]-1-azabenzanthrone
8.85 (d, 1H, J ¼ 8.7 Hz), 10.81 (s, 1H, eCONH); ESI-MS
þ
(
15)
Chloroamide 7 was treated with excess pyrrolidine accord-
m/z: 360 [M þ H] .
ing to the general aminolysis procedure to give 15 (88%), after
column chromatography with chloroform/methanol (100:3)
elution, as yellow/orange solid. (Found: C, 70.4; H, 5.9; N,
3.2. Spectrometric titration and DNA binding assay
All types of spectrometric titration were conducted in
ꢀ
1
1
1
0.8%. C H N O $H O requires C, 70.9; H, 6.0; N,
2
a 1 cm quartz cuvette at room temperature (w25 C). The
3
21
3
2
2
ꢀ
1
0.8%.) Mp 175e177 C; H NMR (CDCl , 300 MHz): d
CT DNA (Sigma, St. Louis, MO) was dissolved in double
distilled deionized water with 50 mM NaCl, and dialyzed
against a buffer solution for 2 days. Its concentration was
determined by UV absorption spectrometry at 260 nm using
3
H
.98 (m, 4H), 2.61 (t, 2H, J ¼ 5.9 Hz), 2.75 (t, 4H,
J ¼ 5.9 Hz), 2.92 (t, 2H, J ¼ 5.9 Hz), 7.68 (d, 1H,
J ¼ 5.6 Hz), 7.87 (t, 1H, J ¼ 7.6 Hz), 8.09e8.14 (m, 2H),
ꢂ1 ꢂ1
a molar extinction coefficient of 6600 M cm . The ratio
8
.30 (dd, 1H, J ¼ 2.3 Hz and J ¼ 8.7 Hz), 8.62 (d, 1H,
1
2

980
H. Tang et al. / European Journal of Medicinal Chemistry 43 (2008) 973e980
A260/A280 > 1.80 was used as an indication of a protein-free
DNA. To the solutions of the drugs (20 mM) in a sodium
phosphate buffer (10 mM sodium phosphate, 150 mM NaCl,
pH 7.0) were added aliquots of CT DNA (2 mM) solution
containing drugs (20 mM) in a sodium phosphate buffer
(2006Z2-E402), State Education Ministry, the Shenzhen Key
Laboratory Fund, the University Grants Committee Areas of
Excellence Scheme in Hong Kong (Grant AoE P/10-01), and
the Hong Kong Polytechnic University Area of Strategic
Development Fund for financial support of this study.
(
10 mM sodium phosphate, 150 mM NaCl, pH 7.0). This
operation ensured that the concentration of CT DNA increased
gradually from 0 to 400 mM, while the concentrations of drugs
were kept constant. The mixing was stirred for 5 min before
measurement. The parameters, lmax, red shift, hypochromicity
and isosbestic point were found from the adsorption spectra.
Fluorescence titration was performed at a fixed concentration
of drugs (3 mM) in sodium phosphate buffer (10 mM sodium
phosphate, 50 mM NaCl, pH 7.0). Small aliquots of concen-
trated CT DNA (2 mM) were added into the solution at final
concentrations from 0 to 120 mM, and stirred for 5 min.
Fluorescence intensity was measured at Ex 418 nm and
Ex/Em 5/15 nm. Binding constants were derived from the
modified Scatchard equation, r/C ¼ K (1 ꢂ nr)[(1 ꢂ nr)/
Appendix. Supplementary material
1
Supplementary material ( H NMR spectra of compounds 1,
3e18) associated with this article can be found in the online
version at doi:10.1016/j.ejmech.2007.07.004.
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[
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[
(Grant PC20041131), and the Science Foundation of Guangzhou