Design, synthesis and anticancer activity of oxoaporphine alkaloid derivatives

Article abstract of DOI:10.3109/14756366.2013.845818

A series of new oxoaporphine derivatives were synthesized and their inhibitory activity of topoisomerase I, cytotoxicity and DNA-binding properties were studied. Oxoaporphine can strongly inhibit topoisomerase I at concentrations of 5-50'M and the cytotoxicity of the derivatives are more potent than their lead compound. Hypochromism, broadening and red shift in the absorption spectra were observed when these compounds bind to calf thymus DNA (CT DNA). These spectral characteristics were consistent with the intercalative binding of these compounds.

Full text of DOI:10.3109/14756366.2013.845818

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2014; 29(5): 722–727
2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.845818
!
ORIGINAL ARTICLE
Design, synthesis and anticancer activity of oxoaporphine alkaloid
derivatives
Yong-Biao Wei, Ying-Xin Li, Hui Song, and Xian-Jin Feng
Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Guangxi Medical University, Nanning, Guangxi, PR China
Abstract
Keywords
A series of new oxoaporphine derivatives were synthesized and their inhibitory activity of
topoisomerase I, cytotoxicity and DNA-binding properties were studied. Oxoaporphine can
strongly inhibit topoisomerase I at concentrations of 5–50 mM and the cytotoxicity of the
derivatives are more potent than their lead compound. Hypochromism, broadening and red
shift in the absorption spectra were observed when these compounds bind to calf thymus DNA
(CT DNA). These spectral characteristics were consistent with the intercalative binding of these
compounds.
Cytotoxicity, DNA binding, oxoaporphine
alkaloids, synthesis, topoisomerase I
inhibitor
History
Received 3 June 2013
Revised 26 August 2013
Accepted 4 September 2013
Published online 18 June 2014
Introduction
Chemical shifts are reported in parts per million (ꢀ) relative to
tetramethylsilane (ꢀ 0.00). UV/visible absorbance spectra were
measured on a Shimadzu UV-2450 spectrophotometer (Tokyo,
Japan). ESI-MS spectra were obtained using a LCMS-2010A
Mass Spectrometer (Tokyo, Japan). Elemental analysis was
carried out on an Elementar Vario EL CHNS Elemental
Analyzer (Hanau, Germany). All reagents were purchased from
either the Aldrich Chemical Co. or the Lancaster Synthesis Co.
and used without further purification unless otherwise stated.
Oxoaporphine alkaloids have been isolated from plant species of
many genera1. These alkaloids exhibited remarkable biological
activities such as potent anticancer2,3, antiviral4, antimicrobial5,6
and lowering blood pressure7 properties. Recent studies showed
that Liriodenine8 and Dicentrinone9 (Figure 1) could inhibit
topoisomerase II and topoisomerase I, respectively. Although
oxoaporphine alkaloids have broad biological activity, the activity
is weak to moderate even for the most active compound10.
Therefore, these alkaloids are interesting substrates for chemical
modification (Figure 1).
Dimethyl 7H-dibenzo[de,g]quinolin-7-one -4,5-dicarboxy-
late (2)
A great deal of studies indicated that intercalation of planar
aromatic molecules into the DNA double helix results in dramatic
changes in DNA conformation and can inhibit DNA replication,
transcription11 and/or topoisomerase activities. These associative
interactions with the DNA molecules can cause dramatic changes
in the physiological functions of DNA, which might respond for
the cytotoxic behavior of the small molecules. Additionally,
previous studies demonstrated that introducing at least one basic
side chain into chromophore could increase DNA-binding affin-
ity12. Enlightened by the studies above, a series of oxoaporphine
derivatives with different basic side chain were synthesized. Their
DNA-binding properties, inhibition of topoisomerase I and
antitumor activity were studied likewise.
A solution of 1 (23.7 g, 0.1 mol) and dimethyl acetylenedicarbox-
ylate (DMAD) (31.2 g, 0.22 mol) in toluene (400 ml) was refluxed
for eight days and then set aside overnight, during which yellow
crystals separated. This crystalline solid was filtered off and was
further purified by recrystallization from chloroform to give
dimethyl 7H-dibenzo[de,g]quinolin-7-one-4, 5-dicarboxylate 2
1
(25%), m.p. 271–273 ꢀC. H NMR (CDCl3): 4.09 (s, 3H), 4.14
(s, 3H), 7.60 (t, 1H, J ¼ 7.3 Hz), 7.78 (t, 1H, J ¼ 7.3 Hz), 7.93–
8.04 (m, 2H), 8.25 (d, 1H, J ¼ 8.1 Hz), 8.47–8.54 (m, 2H). ESI-
MS m/z: 348 [M þ H]þ. Anal. Calcd for C20H13NO5: C, 69.16; H,
3.77; N, 4.03. Found: C, 69.32; H, 3.57; N, 4.11.
7-Oxo-7H-dibenzo[de,g]quinoline-4,5-dicarboxylic
acid (3)
Experimental
Proton (1H) nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker 300 spectrometer (Rheinstetten, Germany).
To a stirred suspension of 2 (3.5 g, 0.01 mol) in methanol (40 ml)
was added a solution of KOH (3 g, 0.05 mol) in water (100 ml).
The mixture was heated at reflux for 24 h and left to stand
overnight. The methanol was removed under vacuum and the
aqueous solution was added HCl until pH ¼ 1–2. The yellow solid
was filtered off and was purified by crystallization from
nitrobenzene to afford 3 as an amorphous powder (92%).
Address for correspondence: Yong-Biao Wei, Department of Pharma-
ceutical Chemistry, School of Pharmaceutical Sciences, Guangxi Medical
University, No. 22, Shuangyong Road, Nanning 530021, Guangxi, PR
China. E-mail: ybwei2008@163.com

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DOI: 10.3109/14756366.2013.845818
Oxoaporphine alkaloid 723
N4,N5-bis(2-(dimethylamino)ethyl)-7-oxo-7H-dibenzo[-
de,g]quinoline-4,5-dicarboxamide (2b)
O
O
O
O
N
O
N
O
N
O
Compound 5 was treated with N1,N1-dimethylethane-1,2-diamine
according to general acylation procedure to give 2b (26%) as
H3CO
1
yellow solid. H-NMR (CDCl3, 300 MHz): 2.4 (s, 12H), 2.7 (t,
OCH3
2H, J ¼ 5.7), 2.8 (t, 2H, J ¼ 6.0), 3.7 (dd, 2H, J1 ¼ 5.1, J2 ¼ 6.1),
4.0 (dd, 2H, J1 ¼ 5.1, J2 ¼ 6.7), 7.6 (t, 1H, J ¼ 7.6), 7.8 (m, 2H),
8.3 (d, 1H, J ¼ 7.7), 8.5 (d, 1H, J ¼ 8.9), 8.6 (m, 2H), 9.2 (s, 1H),
10.9 (s, 1H) ESI-MS m/z: 460 [M þ H]þ. Anal. Calcd for
C26H29N5O3: C, 67.95; H, 6.36; N, 15.24. Found: C, 68.31; H,
6.73; N, 15.92.
Oxoaporphine
Liriodenine
Dicentrinone
Figure 1. Structure of oxoaporphine, liriodenine and dicentrinone.
1H-NMR (DMSO-d6, 300 MHz): 7.69 (t, 1H, J ¼ 7.5 Hz), 7.91 (t,
1H, J ¼ 7.5 Hz), 8.09–8.18 (m, 2H), 8.31 (d, 1H, J ¼ 7.7 Hz), 8.61
(d, 1H, J ¼ 8.0 Hz), 8.90 (d, 1H, J ¼ 7.0 Hz); ESI-MS m/z: 318
[M ꢁ H]ꢁ. Anal. Calcd for C18H9NO5: C, 67.72; H, 2.84; N, 4.39.
Found: C, 67.61; H, 2.52; N, 4.56.
N4,N5-bis(2-(diethylamino)ethyl)-7-oxo-7H-dibenzo[de,g]-
quinoline-4,5-dicarboxamide (2c)
Compound 5 was treated with N1,N1-diethylethane-1,2-diamine
according to general acylation procedure to give 2c (38%) as
yellow solid. 1H-NMR (CDCl3, 300 MHz): 1.1 (t, 6H, J ¼ 7.1), 1.2
(t, 6H, J ¼ 6.9), 2.6 (q, 4H, J1 ¼ 6.9, J2 ¼ 14.0) 2.7 (q, 4H,
J1 ¼5.9, J2 ¼ 12.6), 2.8 (t, 4H, J ¼ 6.2), 2.6 (dd, 2H, J1 ¼ 5.7,
J2 ¼ 12.0), 3.9 (dd, 2H, J1 ¼ 5.5, J2 ¼ 12.1), 7.6 (t, 1H, J ¼ 7.5),
7.7 (m, 2H), 8.3 (d, 1H, J ¼ 8.1), 8.6 (m, 3H), 9.1 (s, 1H), 10.7
(s, 1H); ESI-MS m/z: 516 [M þ H]þ. Anal. Calcd for
C30H37N5O3: C, 69.88; H, 7.23; N, 13.58. Found: C, 70.11; H,
7.77; N, 13.09.
7H-dibenzo[de,g]quinolin-7-one (4)
Finely ground diacid 3 (3.0 g, 0.02 mol) in diphenyl ether (10 ml)
was heated to approximately 250 ꢀC, and heating was discon-
tinued after a few minutes when the obvious reaction had ceased.
The residue was purified by column chromatography (dichlor-
omethane as eluent) to afford compound 4 (60% yield) as yellow
needle solid. 1HNMR (DMSO-d6, 300 MHz) ꢀ 8.9 (d, l H,
J ¼ 5.3 Hz), 8.7 (d, l H, J ¼ 7.0 Hz), 8.5 (d, l H, J ¼ 8.0 Hz), 8.3
(m, 2 H), 8.2 (d, l H, J ¼ 8.1 Hz), 7.9 (d, 1 H, J ¼ 7.5 Hz), 7.8 (t, l
H, J ¼ 6.9 Hz), 7.6 (t, l H, J ¼ 7.1 Hz); ESI. MS m/z: 232[M þ H]þ
Anal. Calcd for C16H9NO: C, 83.10; H, 3.92; N, 6.06. Found: C,
83.21; H, 3.87; N, 6.23.
7-oxo-N4,N5-bis(2-(piperidin-1-yl)ethyl)-7H-dibenzo[de,g]-
quinoline-4,5-dicarboxamide (2d)
Compound 5 was treated with 2-(piperidin-1-yl)ethanamine
according to general acylation procedure to give 2d (18%) as
7-oxo-7H-dibenzo[de,g]quinoline-4,5-dicarbonyl
dichloride (5)
1
yellowish red solid. HNMR (CDCl3) ꢀ (ppm): 1.6 (m, 4 H), 1.9
(m, 4 H), 2.4 (dd, 12 H, Ji1 ¼ 15.9 Hz, J2 ¼ 8.4 Hz), 3.5 (dd, 4 H,
J1 ¼ 12.9 Hz, J2 ¼ 6.4 Hz), 3.7 (s, 2 H), 7.5 (m, 2 H), 7.6 (t, 1 H),
8.1 (d, 1 H, J ¼ 5.2 Hz), 8.2 (dd, 2 H, J ¼ 6.7 Hz), 8.4 (d, 1 H), 9.4
(t, 1 H, J ¼ 5.8 Hz), 10.8 (t, 1 H, J ¼ 4.6 Hz), ESI-MS m/z: 540
[M þ H]þ. Anal. Calcd for C32H37N5O3: C, 71.22; H, 6.91; N,
12.98. Found: C, 72.14; H, 6.53; N, 13.32.
A suspension of 3 (2.07 g, 1 mmol) in benzene (70 ml) with
thionyl chloride (3.6 ml, 5 mmol) was heated at 95 ꢀC for 5 hours.
After cooling, the solvent was removed under reduced pressure,
benzene was added in three 25 ml aliquots and removed, and
70 ml of hexane was added. The precipitate was collected by
filtration, washed with 20 ml of hexane and dried afford 5 (96%
yield). ESI-MS m/z: 356 [M þ H]þ.
7-Oxo-N4,N5-bis(2-(pyrrolidin-1-yl)ethyl)-7H-dibenzo[-
de,g]quinoline-4,5-dicarboxamide (2e)
General procedure for the preparation of the amines
A suspension of the 5 (0.354 g, 0.1 mmol) in 1,4-dioxane (25 ml)
was stirred at room temperature, then 0.5 ml triethylamine and
0.3 mmol appropriate amines added thereto. After stirring for 30
minutes at room temperature, the temperature was then raised to
80 ꢀC and held at this temperature for about 1 hour. After cooling,
the solvent was removed under reduced pressure. The residue was
taken up with dichloromethane and the solution was washed with
a solution of sodium carbonate, dried over sodium sulfate and
concentrated under vacuum. The crude product was purified by
column chromatography with chloroform:methanol to afford the
title compound.
Compound 5 was treated with 2-(pyrrolidin-1-yl)ethanamine
according to general acylation procedure to give 2e (20%) as
yellowish red solid. 1HNMR (CDCl3) ꢀ (ppm): 1.8 (q, 4 H,
J ¼ 6.2 Hz), 1.9 (q, 4 H, J ¼ 6.2 Hz), 2.6 (t, 4 H, J ¼ 5.0 Hz), 2.8
(s, 4 H), 2.9 (dd, 4 H, J1 ¼ 6.5 Hz, J2 ¼ 6.6 Hz), 3.7 (dd, 2 H,
J1 ¼ 12.7 Hz, J2 ¼ 6.4 Hz), 4.0 (dd,
2
H, Ji1 ¼ 12.1 Hz,
J2 ¼ 6.6 Hz), 7.6 (t, 1 H, J ¼ 7.5 Hz), 7.7 (td, 2 H, J1 ¼ 16.5 Hz,
J2 ¼ 7.7 Hz), 8.3 (d, 1 H, J ¼ 8.1 Hz), 8.4 (d, 1 H, J ¼ 8.6 Hz), 8.5
(t, 2 H, J ¼ 7.9 Hz), 9.2 (t, 1 H),10.7 (t, 1 H). ESI-MS m/z: 512
[M þ H]þ. Anal. Calcd for C30H33N5O3: C, 70.43; H, 6.50; N,
13.69. Found: C, 69.85; H, 6.02; N, 13.17.
N4,N5-bis(2-morpholinoethyl)-7-oxo-7H-dibenzo[de,g]qui-
noline-4,5-dicarboxamide (2a)
N4,N5-bis(3-morpholinopropyl)-7-oxo-7H-dibenzo[de,g]-
quinoline-4,5-dicarboxamide (2f)
Compound 5 was treated with 2-(morpholine)ethylamine accord- Compound
5 was treated with 3-(morpholino)propylamine
ing to general acylation procedure to give 2a (35%) as yellow solid. according to general acylation procedure to give 2f (32%) as
1
1H-NMR (CDCl3, 300 MHz): 2.6 (m, 8H), 2.7 (t, 2H, J ¼ 6.1), 2.8 yellow solid. HNMR(CDCl3): 1.93 (m, 2H), 2.04 (m, 2H), 2.46
(t, 2H, J ¼ 6.4), 3.6 (q, 2H, J1 ¼ 5.8, J2 ¼ 11.7), 3.7 (t, 4H, J ¼ 3.2), (t, 4H), 2.55 (m, 8H), 3.56 (q, 2H, J ¼ 6.7), 3.67 (t, 4H, J ¼ 4.58),
3.8 (s, 4H), 3.9 (q, 2H, J1 ¼ 4.7, J2 ¼ 10.3), 7.6 (t, 1H, J ¼ 7.4), 7.7 3.78 (t, 4H, J ¼ 4.58), 3.90 (q, 2 H, J ¼ 5.36), 7.58 (dd, 1H,
(m, 2H), 8.3 (d, 1H, J ¼ 8.6), 8.4 (d, 2H, J ¼ 8.6), 8.5 (t, 1H, J ¼ 8.0, J ¼ 1.0), 7.7 (m, 2H,), 8.3 (d, 1H, J ¼ 8.0), 8.4 (d, 1H,
J ¼ 8.6), 9.3 (t, 1H, J ¼ 4.6), 10.8 (t, 1H, J ¼ 4.8) ESI-MS m/z: 544 J ¼ 8.6), 8.5 (m, 2H), 9.1 (t, 1H, J ¼ 6.0), 10.7 (t, 1H, J ¼ 1.0);
[M þ H]þ. Anal. Calcd for C30H33N5O5: C, 66.28; H, 6.12; N, ESI-MS m/z: 572 [M þ H]þ. Anal. Calcd for C32H37N5O5: C,
12.88. Found: C, 65.35; H, 6.89; N, 12.07.
67.23; H, 6.52; N, 12.25. Found: C, 68.31; H, 6.92; N, 12.74.

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724 Y.-B. Wei et al.
J Enzyme Inhib Med Chem, 2014; 29(5): 722–727
Topoisomerase I inhibition
where r is the molar ratio of bound ligand to DNA, Cf the free
ligand concentration, Ki the binding constant and n the binding
A modified relaxation assay was carried out as described13.
Briefly, inhibitory activity of tested compounds on human DNA
Topo I was evaluated by monitoring the enzyme-catalyzed
relaxation of a supercoiled DNA substrate. Standard assays
were performed in 20 mL of topoisomerase I relaxation buffer
(10 mM Tris-HCl (pH 7.9), 150 mM NaCl, 0.1% bovine serum
albumin, 1 mM spermidine, 5% glycerol). The reaction mixture
contained 1 unit of the enzyme, 200 ng of supercoiled pBR322
and various concentrations of tested compound and incubated for
30 min at 37 ꢀC. The reactions were terminated by addition of
5 ml of gel loading buffer containing 10% SDS, 100 mM EDTA
and 0.125% bromophenol blue. The samples were subjected to
electrophoresis in 1% agarose in 45 mM Tris-borate (pH 8.0),
1 mM EDTA at 5 V/cm and then stained with 0.5 mg Lꢁ1 of
ethidium bromide.
size in base pairs16,17
.
Cytotoxicity assay
All tumor cell lines, human breast cancer MCF-7 cell, NCI-H460
cell and GLC-82 cell (from ATCC, Tockville, MD) were cultured
on RPMI-1640 medium supplemented with 10% fetal bovine
serum, 100 U mlꢁ1 penicillin and 100 g/ml streptomycin in 25 cm2
culture flasks at 37 ꢀC in humidified atmosphere with 5% CO2.
For the cytotoxicity tests, cells in exponential growth stage
were harvested from culture by trypsin digestion and centrifuging
at 180 g for 3 min, then resuspended in fresh medium at a cell
density of 5 ꢂ 104 cells per ml. The cell suspension was dispensed
into a 96-well microplate at 100 ml per well, and incubated in
humidified atmosphere with 5% CO2 at 37 ꢀC for 24 h, and then
treated with the compounds at various concentrations (0, 1, 10,
100 mmol). After 48 h of treatment, 50 ml of 1 mg mlꢁ1 MTT
solution was added to each well, and further incubated for 4 h. The
reaction product was then dissolved with 100 ml of DMSO and the
optical density (OD) at 570 nm was recorded. All drug doses were
tested in triplicate and the IC50 values were derived by data
regression of the OD values versus drug concentrations using a
curve-fitting program.
Spectrometric titration and DNA-binding assay
The calf thymus DNA (purchased from Sigma Chemical Co.) was
dissolved in double-distilled deionized water with 50 mM NaCl
and dialyzed for 48 h against a buffer solution of 5 mM of Tris-
HCl, at pH 7.2, containing 50 mM NaCl at 4 ꢀC. The purity of the
DNA was checked by monitoring the value of the ratio of the 260
and 280 nm absorbances. The ratio of 1.9 indicated that DNA
was sufficiently free of protein14. The DNA concentrations per
nucleotide were determined by absorption spectroscopy using the
molar extinction coefficient value of 6600 Mꢁ1 cmꢁ1 at 260 nm15.
The solution of DNA was stored at 4 ꢀC for a short time and
then used.
Absorption titration was performed at a fixed concentration of
drugs (30 mM) in a sodium phosphate buffer (20 mM sodium
phosphate, 150 mM NaCl, pH 6.3). Small aliquots of concentrated
CT DNA (3.9 mM) were added into the solution to final
concentrations ranged from 0 to 165 mM, and stirred for 5 min
before measurement. The parameters, ꢁmax, red shift, hypochro-
micity and isosbestic point were found from the absorption
spectra.
Results and discussion
Chemistry
Preparation of 2 was carried out by a reported method18.
Hydrolysis of 2 by potassium hydroxide gave 3 in 92% yield19.
Synthesis of 7 4 was by decarboxylation of 3. Attention to detail
was required in the decarboxylation process to obtain good yield
(Scheme 1). The solid was then recrystallized in toluene to give a
yellow needle solid with an yield of 60%.
Oxoaporphine alkaloids are coplanar isoquinoline alkaloids,
which possess a 7H-dibenzo[de,g]quinolin-7-one moiety in their
structures (Figure 1). The alkaloids can intercalate into the DNA
double helix, thus they can inhibit DNA replication, transcription
and/or topoisomerase activities11. To improve their DNA-binding
ability, the length of the intermediate chain, and consequently its
flexibility, was varied in some cases. A series of oxoaporphine
derivatives with different basic side chain at 4,5-position of
7H-dibenzo[de,g]quinolin-7-one (2a–f in Figure 2) (general
formula Ar-(CONH(CH2)nNR2)2, Ar ¼ 7H-dibenzo[de,g]quino-
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 concentrated
CT DNA (3.0 mM) were added into the solution to final
concentrations ranged from 0 to 150 mM, and stirred for 5 min.
Fluorescence intensity was measured at Ex 410 nm and Ex/Em 5/
15 nm. Binding constants were derived from the modified
Scatchard equation, r/Cf ¼ Ki(1 ꢁ Nr)[(1 ꢁ Nr)/[1ꢁ(N ꢁ 1)r]]nꢁ1
,
lin-7-one,
n ¼ 2
or
3)
and
4-position
of
7H-
H CO
O
Scheme 1. Synthesis of 7H-dibenzo[de,g]qui-
nolin-7-one 4. Reagents and conditions:
(1) DMAD/toluene/reflux/8 days; (2) KOH/
CH3OH/H2O (85 ꢀC); (3) Diphenyl ether
(250 ꢀC).
3
O
O
O
reflux
PhMe
O
N
OCH
3
+
O
N
O
O
O
1
2
NaOH
92%
230∼240 °C
60
COOH
COOH
%
N
N
O
4
O
3

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DOI: 10.3109/14756366.2013.845818
Oxoaporphine alkaloid 725
CONH(CH )nNR
CONH(CH )nNR
Figure 2. Structures of oxoaporphine alkaloid
derivatives.
2
2
2
2
CONH(CH )nNR
2
2
N
O
N
O
NR
NR
2
Compound
Compound
2
N
O
N
O
2a: n=2; 2f: n=3
2b: n=2;
3a: n=2; 3e: n=3
N(CH )
N(CH )
3 2
3b: n=2; 3f: n=3
3c: n=2; 3g: n=3
3 2
NEt
NEt
2
2c: n=2;
2
2d:n=2
N
N
2e: n=2
Cl
O
O
CONH(CH )nNR
2
2
COOH
COOH
CONH(CH )nNR
2
2
Cl
N
O
N
O
SOCI
N
O
2
2a-f
5
Figure 3. Synthesis of disubstituted derivatives. Reagents and conditions: (1) SOCl2/C6H6; (2) NH2 (CH2)nNR2/Et3N.
dibenzo[de,g]quinolin-7-one (3a–g in Figure 2) (general formula
Ar-CONH(CH2)nNR2,
Ar ¼ 7H-dibenzo[de,g]quinolin-7-one,
n ¼ 2 or 3) were designed and synthesized.
7-Oxo-7H-dibenzo[de,g]quinoline-4,5-dicarbonyl dichloride 5
.
was obtained in 90% yield by 3 reacted with thionyl chloride20,21
The acid chlorides 5 was then submitted to amidification in 1,4-
dioxane to obtain amides 2a–2f (Figure 3)22.
Based on the favorable biological activity of disubstituted
derivatives, monosubstituted derivatives were chosen to avoid the
steric repulsion between the two side chains, because the steric
repulsion effect in these series of compounds plays an important
role. In the monosubstituted compounds, the side chains are
amenable to rotation and can easily become coplanar with the
oxoaporphine chromophore. Compounds 3a–3f were synthesized
using our previous method23.
Biology
Topoisomerase I inhibition study
Figure 4 Inhibition of Topo I-catalyzed DNA relaxation by 7-oxoapor-
phine and Camptothecin. Lane 1, pBR322 DNA only. Lane 2, pBR322
DNA and Topo I. Lanes 3–7, pBR322 DNA, Topo I, and various drug
concentrations.
The effect of 4 on human DNA Topo I was evaluated by the
Topo I relaxation assays24. Camptothecin, a well-known Topo I
inhibitor, was used as a positive control (Figure 4). The result
indicated that 4 could inhibit Topo I activity in dose-dependent
manner and completely eliminate Topo I at 50 mM. This is
consistent with another study by Woo et al.8,25 who showed that
oxoaporphine alkaloids, such as Liriodenine (in Figure 1), could
intercalate DNA and inhibit topoisomerase II26. Compared with
CPT that fully inhibited Topo I activity at 125 mM (Figure 4),
Topo I inhibitory activity of Compound 4 is somewhat two times
greater than that of CPT.
DNA-binding properties
The DNA-binding properties of compound 4 were evaluated by
using UV visible spectral absorbance analysis, which has been
used as a convenient tool to detect the interaction between drugs
and DNA27. It was observed that the addition of CT DNA to the