TCM active ingredient oxoglaucine metal complexes: Crystal structure, cytotoxicity, and interaction with DNA

Article abstract of DOI:10.1021/ic200443p

The alkaloid oxoglaucine (OG), which is a bioactive component from traditional Chinese medicine (TCM), was synthesized by a two-step reaction and used as the ligand to react with transition metal salts to give four complexes: [OGH][AuCl₄]·DMSO (1), [Zn(OG)₂(H ₂O)₂]- (NO₃)₂ (2), [Co(OG) ₂(H₂O)₂](ClO₄)₂ (3), and [Mn- (OG)₂(H₂O)₂](ClO₄)₂ (4). The crystal structures of the metal complexes were confirmed by single crystal X-ray diffraction. Complex 1 is an ionic compound consisting of a charged ligand [OGH]⁺ and a gold complex [AuCl₄] ^-. Complexes 2-4 all have similar structures (inner-spheres), that is, octahedral geometry with two OG coordinating to one metal center and two aqua ligands occupying the two apical positions of the octahedron, and two NO₃^- or ClO₄ ^- as counteranions in the outer-sphere. The complexation of OG to metal ion was confirmed by ESI-MS, capillary electrophoresis and fluorescence polarization. The in vitro cytotoxicity of these complexes toward a various tumor cell lines was assayed by the MTT method. The results showed that most of these metal-oxoglaucine complexes exhibited enhanced cytotoxicity compared with oxoglaucine and the corresponding metal salts, with IC₅ values ranging from 1.4 to 32.7 μM for sensitive cancer cells, which clearly implied a positive synergistic effect. Moreover, these complexes appeared to be selectively active against certain cell lines. The interactions of oxoglaucine and its metal complexes with DNA and topoisomerase I were investigated by UV-vis, fluorescence, CD spectroscopy, viscosity, and agarose gel electrophoresis, and the results indicated that these OG-metal complexes interact with DNA mainly via intercalation. Complexes 2-4 are metallointercalators, but complex 1 is not. These metal complexes could effectively inhibit topoisomerase I even at low concentration. Cell cycle analysis revealed that 1-3 caused S-phase cell arrest.

Full text of DOI:10.1021/ic200443p

Article
pubs.acs.org/IC
TCM Active Ingredient Oxoglaucine Metal Complexes: Crystal
Structure, Cytotoxicity, and Interaction with DNA
Zhen-Feng Chen,* Yan-Fang Shi, Yan-Cheng Liu, Xue Hong, Bo Geng, Yan Peng, and Hong Liang*
State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry &
Chemical Engineering, Guangxi Normal University, Guilin, 541004, China
S
* Supporting Information
ABSTRACT: The alkaloid oxoglaucine (OG), which is a
bioactive component from traditional Chinese medicine
(TCM), was synthesized by a two-step reaction and used as
the ligand to react with transition metal salts to give four
complexes: [OGH][AuCl4]·DMSO (1), [Zn(OG)2(H2O)2]-
(NO3)2 (2), [Co(OG)2(H2O)2](ClO4)2 (3), and [Mn-
(OG)2(H2O)2](ClO4)2 (4). The crystal structures of the
metal complexes were confirmed by single crystal X-ray
diffraction. Complex 1 is an ionic compound consisting of a
charged ligand [OGH]+ and a gold complex [AuCl4]−.
Complexes 2−4 all have similar structures (inner-spheres),
that is, octahedral geometry with two OG coordinating to one
metal center and two aqua ligands occupying the two apical
−
−
positions of the octahedron, and two NO3 or ClO4 as
counteranions in the outer-sphere. The complexation of OG to metal ion was confirmed by ESI-MS, capillary electrophoresis and
fluorescence polarization. The in vitro cytotoxicity of these complexes toward a various tumor cell lines was assayed by the MTT
method. The results showed that most of these metal−oxoglaucine complexes exhibited enhanced cytotoxicity compared with
oxoglaucine and the corresponding metal salts, with IC50 values ranging from 1.4 to 32.7 μM for sensitive cancer cells, which
clearly implied a positive synergistic effect. Moreover, these complexes appeared to be selectively active against certain cell lines.
The interactions of oxoglaucine and its metal complexes with DNA and topoisomerase I were investigated by UV−vis,
fluorescence, CD spectroscopy, viscosity, and agarose gel electrophoresis, and the results indicated that these OG−metal
complexes interact with DNA mainly via intercalation. Complexes 2−4 are metallointercalators, but complex 1 is not. These
metal complexes could effectively inhibit topoisomerase I even at low concentration. Cell cycle analysis revealed that 1−3 caused
S-phase cell arrest.
ceae,19 and Papveraceae.20,21 It is also commonly found in
INTRODUCTION
■
many traditional Chinese medicines, such as aquilegia ecalcarata
Maxim (Ranunculaceae), which is mainly distributed in the
Sichuan and Yunnan provinces of China and used to treat
necrotic boils, pustulosis, and other infections.22 The primary
screening results reveal that oxoglaucine possesses strong
anticancer activity, for example, against HCT-8 (ED50 = 2.85
μM) and KB (ED50 = 5.69 μM).23 In addition, oxoglaucine
exhibits other important pharmacological properties including
antiplatelet aggregation,13,24 immunomodulatory activity,25
amelioration of adjuvant arthritis,26 anti-inflammatory activity,27
antifungal activity.28
Intercalators are small organic molecules or metal complexes
that unwind DNA to π-stack between two neighboring base
pairs. Metallointercalators are metal complexes that bear at least
one intercalating ligand. Since the preparation of the first
platinum metallointercalator by S. J. Lippard and co-workers,29
Since the successful use of cisplatin and related platinum
complexes as anticancer agents, developing other transition
metal complexes as active anticancer agents with better
efficiency and new mechanisms of action has become a central
research theme in bioinorganic chemistry.1−5 The discovery of
nonplatinum metal-based anticancer complexes with potent
anticancer activity, such as gold(III),6 zinc(II),7 cobalt(II)/
(III),8 and manganese(II)9 complexes, has been extensively
investigated during the past two decades. Recently some new
coordination compounds based on traditional Chinese
medicines (TCMs) were found to afford novel potential
(pro)drugs.10,11 Our group previously reported a series of metal
complexes bearing liriodenine/plumbagin/matrine ligands as
anticancer compounds,12 and in our continuing research, we
now focus on the oxoaporphine−metal complex as anticancer
agents. Oxoglaucine (OG, Scheme 1) is an oxoaporphine
alkaloid that has been isolated from the overground parts of
plants from different families, such as Annonaceae,13,14
Lauraceae,15 Magnoliaceae,16 Fumariaceae,17,18 Menisperma-
Received: March 4, 2011
Published: February 6, 2012
© 2012 American Chemical Society
1998
dx.doi.org/10.1021/ic200443p | Inorg. Chem. 2012, 51, 1998−2009

Inorganic Chemistry
Article
spectra for the stability studies on the complexes 1−4 were recorded
on a Bruker HCT Electrospray Ionization Mass Spectrometer.
Capillary electrophoresis was recorded on Agilent HP3D High-
performance capillary electrophoresis. Fluorescence polarization was
performed on Horiba Jobin Jvon FL3-P-TCSPC Time-Resolved
Fluorescence spectrometer. UV−vis absorption titration was per-
formed on a Varian Cary100 UV−visible spectrophotometer.
Fluorescence emission titration was performed on a Shimadzu RF-
5301/PC spectrofluorophotometer. The Circular dichroic spectra of
DNA in the region between 200 and 400 nm were obtained by using a
JASCO J-810 automatic recording spectropolarimeter operating at 25
°C. Viscosity of DNA solution was measured on a Brookfield LVDV-II
+ Pro digital viscometer equipped with a ULV adapter, as well as a
Brookfield TC-502D digital controlled circulating bath for constant
temperature.
Scheme 1. Synthesis Route of Oxoglaucine
Caution! Perchlorate complexes are potentially explosive. The experi-
ments were carried out in an isolated room and the operator must be
protected with blast shield and other necessary equipments. The perchlorate
complexes should be prepared only in small amount and handled with
extreme care.
Synthesis of (+)-Glaucine. To a solution of (+)-boldine (1.80 g,
5.498 mmol) and trimethylphenylammonium chloride (3.303 g, 19.24
mmol) in dry DMF (100 mL) was added anhydrous K2CO3 (3.78 g,
27.292 mmol), and the suspension was heated to 90 °C under N2 for
16 h. After removal of the organic solvent by vacuum distillation, the
residue was dissolved in CHCl3 and washed with 5% sodium
hydroxide solution (100 mL × 2) followed by H2O (100 mL × 3).
The organic layer was dried over anhydrous sodium sulfate and
concentrated under reduced pressure. The residue was purified by
silica gel chromatography using gradient elution with CHCl3−CH3OH
(200:1 to 30:1) to afford (+)-glaucine (1.1 g, 60%). mp: 112−114 °C.
1H NMR (CDCl3, 500 MHz): δ 8.07 (1H, s, H-11), 6.74 (1H, s, H-8),
6.55 (1H, s, H-3), 3.90 (3H, s, 9-OMe), 3.89 (3H, s, 10-OMe), 3.85
(3H, s, 2-OMe), 3.64 (3H, s, 1-OMe), 2.53 (s, N-Me). APCI-MS: [M
+ H] 356.
over the past three decades, there has been rising interest on
octahedral transition metal complexes as metallointercalator30
and new dual-function metal complexes that can interact with
DNA by both coordination and intercalation,31 which may have
potential diagnostic and therapeutic applications. Because OG
is an aromatic planar ligand with N and carbonyl O donors, it
can coordinate to metal to form metal intercalator.
We previously noted the potent anticancer activity of the
gold(III),6 zinc(II),7 cobalt(II)/(III),8 and manganese(II)9
complexes with different N/O donor ligands. To explore
alkaloid−metal complexes as anticancer agents, we synthesized
oxoglaucine and reacted it with the gold(III), zinc(II),
cobalt(II) and manganese(II) salts to afford four metal
complexes. Herein, we report the synthesis and characterization
of oxoglaucine and its metal complexes: [OGH]-
[AuCl4]·DMSO (1), [Zn(OG)2(H2O)2](NO3)2 (2), [Co-
(OG)2(H2O)2](ClO4)2 (3), and [Mn(OG)2(H2O)2](ClO4)2
(4). Their cytotoxicity and interactions with DNA and
topoisomerase I were also investigated.
Synthesis of Oxoglaucine. To a solution of glaucine (1.18 g, 3.30
mmol) in acetic acid (50 mL) was added manganese(III) acetate
hydrate (26.20 mmol), and the mixture was stirred at 70 °C for 2 h.
After the completion of the reaction (monitored by TLC), acetic acid
was distilled off under high vacuum, and the residue was dissolved in
chloroform (100 mL) and washed with saturated NaHCO3 solution
(50 mL), followed by water (3 × 50 mL). The organic layer was dried
over anhydrous sodium sulfate and then concentrated under reduced
pressure. The residue was purified by silica gel chromatography using
gradient elution of CHCl3/CH3OH (200:0 to 200:1) to afford
oxoglaucine as yellow fine needles (0.285 g, 25%). The crystals of
oxoglaucine suitable for single-crystal X-ray diffraction analysis were
obtained by recrystallization from CHCl3/CH3OH at room temper-
ature. mp: 196−198 °C. IR(KBr, cm−1): 2938 (m, Ar−H), 1647 (s,
CO), 1594(s), 1571(s), 1510 (m), 1463(s), 1277(m, C−O),
EXPERIMENTAL SECTION
■
Materials. All chemicals, unless otherwise noted, were purchased
from Sigma and Alfa Aesar. All materials were used as received without
further purification unless noted specifically. Tris-HCl-NaCl (TBS)
buffer solution (5 mM Tris, 50 mM NaCl, pH adjusted to 7.35 by
titration with hydrochloric acid using a Sartorius PB-10 pH meter, Tris
= tri(hydroxymethyl)aminomethane) was prepared using double
distilled water. The TBE buffer (1×) and DNA loading buffer (6×)
were commercially available. Calf thymus DNA (ct-DNA) was
purchased from Sino-American Biotech Co., Ltd. (Beijing). Ct-DNA
gave a UV absorbance ratio at 260−280 nm of ∼1.85:1, indicating that
the DNA was effectively free of protein.32 The DNA concentration per
nucleotide was determined spectrophotometrically by employing a
molar absorption coefficient (6600 M−1cm−1) at 260 nm.33 TOPO I
was purchased from Sigma. The stock solution of pUC19 plasmid
DNA (250 μg/mL) was purchased from Takara Biotech Co., Ltd.
Instrumentation. Infrared spectra were obtained on a Perkin-
Elmer FT-IR Spectrometer. 1H NMR and 13C NMR spectra were
recorded on a Bruker AV-500 NMR spectrometer with (CD3)2SO as
solvent. Elemental analyses (C, H, and N) were carried out on a
PerkinElmer Series II CHNS/O 2400 elemental analyzer. ESI-MS
spectra for the characterization of complexes 1−4 were performed on
Thermofisher Scientific Exactive LC-MS Spectrometer. ESI-MS
1
1243(s), 1068(m, C−N). H NMR (CDCl3, δH,500 MHz): δ4.02(s,
3H, OCH3), 4.06(s, 6H, 2 × OCH3), 4.0(s, 3H, OCH3), 7.15(s, 1H,
C3−H), 7.72d, 1H, J = 5.2 Hz, C4−H), 7.99(s, 1H, C8−H), 8.76(s,
1H, C11−H), 8.86(d, 1H, J = 5.2 Hz, C5−H). 13C NMR(CDCl3,
δC,125 MHz): 181.2, 157.2, 152.7, 151.3, 148.5, 145.6, 145.0, 136.2,
128.8, 126.4, 123.7, 120.5, 118.9, 110.2, 109.5, 105.3, 60.5, 56.7, 56.7,
56.7.
General Procedure for the Synthesis of Complexes 1−4. To
a mixture of the corresponding metal salt, oxoglaucine, methanol (1
mL), and CHCl3 (0.25 mL) was placed in a thick Pyrex tube (∼20 cm
long). The mixture was frozen by liquid N2 and evacuated under
vacuum, then sealed with a torch, warmed to room temperature, and
heated at 80 °C for a few days to give the corresponding block crystals
of the complexes 1−4 suitable for X-ray diffraction analysis.
[OGH][AuCl4]·DMSO (1). HAuCl4·4H2O (0.041 g, 0.1 mmol),
oxoglaucine (0.0351 g, 0.1 mmol). Yield: 0.050 g, 65%. Anal. Found
(%): C, 34.32; H, 3.12; N, 1.86. Calcd for C22H24AuCl4NO6S: 34.35;
H, 3.14; N, 1.82. Main IR (KBr, cm−1) peaks: 3422 m (−NH), 3080 m
(Ar−H), 1657s (CO), 1622s, 1588s (CC), 1477s, 1287 m (C−
O), 1254s, 1065s (C−N). 1H NMR(DMSO-d6, δH, 500 MHz):
1999
dx.doi.org/10.1021/ic200443p | Inorg. Chem. 2012, 51, 1998−2009

Inorganic Chemistry
Article
Table 1. Crystal Data and Structure Refinement Details for OG and Complexes 1−4
formula
C20H17NO5
C22H24AuCl4NO6S
C40H38N4O18Zn
C40H38Cl2CoN2O20
C40H38Cl2MnN2O20
fw
351.35
296(2)
monoclinic
P2(1)/n
7.1197(15)
15.374(3)
15.031(3)
90
769.25
928.11
293(2)
triclinic
996.55
296(2)
triclinic
992.56
223(2)
triclinic
T, K
296(2)
monoclinic
P2(1)/n
8.5115(6)
12.3968(9)
25.1060(18)
90
cryst syst
space group
P1
̅
P1
̅
P1
̅
a, Å
8.323(6)
11.010(7)
11.838(8)
101.369(12)
104.335(16)
104.810(8)
976.1(11)
1
8.6032(16)
11.174(2)
12.212(2)
65.314(2)
74.206(2)
78.120(2)
1020.7(3)
1
8.6702(6)
11.1589(6)
12.2521(6)
63.838(8)
74.356(9)
77.682(10)
1018.40
1
b, Å
c, Å
α, deg
β, deg
92.461(3)
90
96.1080(10)
90
γ, deg
V, Å3
1643.7(6)
4
2634.0(3)
4
Z
Dc, g cm−3
μ, mm−1
1.420
1.937
1.579
1.621
1.618
0.103
6.109
0.718
0.641
0.545
GOF on F2
reflns (collected/unique)
Rint
1.009
1.024
1.070
1.040
1.056
8193/2929
0.0657
0.0649
12869/4624
0.0255
9225/3549
0.0762
5126/3563
0.0235
8788/3757
0.0354
a
R1 (I > 2σ(I))
0.0295
0.0912
0.0640
0.0514
b
R2 (I > 2σ(I))
0.2230
0.0692
0.2429
0.2055
0.1314
a
b
2
2
R1 = Σ ∥Fo| − |Fc∥/Σ|Fo|. R2 = [Σw(Fo − Fc2)2/Σw(Fo )2]1/2
.
δ3.794(s, 3H, OCH3), 3.848(s, 3H, OCH3), 4.041(s, 6H, 2 × OCH3),
7.269 (s, 2H, C3−H), 7.401(s, 2H, C11−H), 8.187(d, 2H, J =5.2 Hz,
C4−H), 8.384(d, 2H, J =5.2 Hz, C8−H), 8.462(s, 2H, C5−H). 13C
NMR(DMSO-d6, δC, 125 MHz): δ 206.8, 173.8, 158.7, 158.1, 154.8,
148.3, 139.4, 133.2, 132.1, 131.0, 124.5, 122.8, 120.7, 109.8, 107.6,
107.5, 105.3, 57.4, 55.9, 55.7, 40.0. ESI-MS: m/z 338.84 [AuCl4]−,
352.12 [OGH]+.
refinement was performed by full-matrix least-squares methods with
anisotropic thermal parameters for non-hydrogen atoms on F2. The
hydrogen atoms were added theoretically and riding on the concerned
atoms. The crystallographic data and refinement details of the
structures are summarized in Tables 1. The selected bond distances
and angles for OG, 1−4 are given in the corresponding figure captions.
Stability of Complexes 1−4 in Aqueous Solution. Complexes
1−4 are soluble at 2 × 10−5 M concentration level in the TBS at 25 °C
containing 1% DMSO. The kinetic stability of all four complexes was
evaluated by UV−vis absorption under this condition. The kinetic
[Zn(OG)2(H2O)2](NO3)2 (2). Zn(NO3)2·6H2O (0.030 g, 0.1 mmol),
oxoglaucine (0.070 g, 0.2 mmol). Yield: 0.060 g, 65%. Anal. Found
(%): C, 51.72; H, 4.18; N, 6.12. Calcd for C40H38N4O18Zn: 51.76; H,
4.13; N, 6.04. Main IR (KBr, cm−1): 3443s (−OH), 2946 m (Ar−H),
−
1620w (CO), 1574 m (CC), 1508 m 1469 m, 1384s (NO3 ),
1
1283 m (C−O), 1260 m, 1023(m) (C−N). H NMR(DMSO-d6, δH,
500 MHz): δ3.807(s, 6H, OCH3), 3.970(s, 12H, 2 × OCH3), 4.01(s,
6H, OCH3), 8.307 (s, 2H, C3−H), 8.409(s, 2H, C11−H), 8.881(d, 2H,
J = 5.2 Hz, C4−H), 8.927(d, 2H, J = 5.2 Hz, C8−H), 9.184 (s, 2H,
C5−H). 13C NMR(DMSO-d6, δC, 125 MHz): δ 206.9, 188.7, 157.9,
156.6, 154.9, 150.2, 148.8, 146.4, 134.9, 130.3, 126.7, 123.4, 121.6,
117.5, 110.5, 108.9, 61.6, 57.3, 56.6, 56.2, 45.9, 44.9. ESI-MS: m/z
828.14 [Zn(OG)2 + NO3]+, 558.63 [Zn(OG)3]2+, 383.07 [Zn-
(OG)2]2+.
[Co(OG)2(H2O)2](ClO4)2 (3). Co(ClO4)2·6H2O (0.037 g, 0.1 mmol),
oxoglaucine (0.070 g, 0.2 mmol). Yield: 0.068 g, 68%. Anal. Found
(%): C, 48.28; H, 3.81; N, 2.89. Calcd for C40H38Cl2CoN2O20: 48.21;
H, 3.84; N, 2.81. Main IR (KBr, cm−1): 3410s (−OH), 2846 m (Ar−
H), 1641 m (CO), 1591 m (CC), 1571 m, 1508s, 1283 m (C−
−
O), 1260 m, 1109s (ClO4 ), 1008 m (C−N). ESI-MS: m/z 860.10
[Co(OG)2 + ClO4]+, 556.13 [Co(OG)3]2+, 380.58 [Co(OG)2]2+.
[Mn(OG)2(H2O)2](ClO4)2 (4). Mn(ClO4)2·6H2O (0.036 g, 0.1
mmol), oxoglaucine (0.070 g, 0.2 mmol). Yield: 0.056 g, 56%. Anal.
Found (%): C, 48.46; H, 3.81; N, 2.85. Calcd for C40H38Cl2MnN2O20:
48.40; H, 3.86; N, 2.82. Main IR (KBr, cm−1): 3423s (−OH), 2942 m
(Ar−H), 1643 m (CO), 1592 m (CC), 1567 m, 1510s, 1282 m
Figure 1. Time-dependent stability studies on complex 2 in TBS
monitored by UV−vis absorption spectra.
UV−vis spectra of complex 2 are shown in Figure 1 (for the spectra of
OG and complexes 1, 3 and 4, see Supporting Information, Figure S1).
Complexes 2, 3, and 4 showed similar changes in their absorption
spectra, most probably because of their similar chemical structures
(inner-spheres). Over the time course, the characteristic absorption of
each complex all showed hypochromicity but no bathochromic shift.
The hypochromicity can be attributed to the gradual formation of
aggregates of the complexes in solution, which will decrease their
effective concentration for UV−vis absorption.36 While the UV−vis
absorption of OG remained unchanged in 2 h but decreased obviously
after 8 and 16 h. No further changes were observed in the UV−vis
spectra of complex 1 after 2 h, which suggests a rapid hydrolysis
−
(C−O), 1259 m, 1109s (ClO4 ), 1008 m (C−N). ESI-MS: m/z
856.11 [Mn(OG)2 + ClO4]+, 554.13 [Mn(OG)3]2+, 378.58 [Mn-
(OG)2]2+.
X-ray Crystallography. Crystal data were collected on a Bruker
Smart Apex II CCD or Rigaku Mercury CCD diffractometer equipped
with graphite monochromated Mo Kα radiation (λ = 0.71073 Å) at
room temperature. Absorption correction were applied by using the
multiscan program SADABS.34 The structures were solved with direct
methods and refined using SHELX-97 programs.35 The non-hydrogen
atoms were located in successive difference Fourier synthesis. The final
2000
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Inorganic Chemistry
Article
process and deficient aggregation of 1 within 2 h. Since both OG and
its complexes (except for complex 1) have the UV absorption peaks at
same wavelength of 245 and 272 nm, the absence of bathochromic
shift cannot unambiguously prove the dissociation of OG ligands from
the complexes. As shown in Figure S2 (see Supporting Information),
the kinetic ESI-MS spectra (performed on a Bruker HCT Electrospray
Ionization Mass Spectrometer) of 1 in TBS indicate that 1 underwent
some change while the cation remained unchanged (m/z 352 [OGH]+
and reduction of Au(III) to Au(I), showing the [AuCl2]− anion, m/z
267). However, the kinetic ESI-MS spectra of 2−4 (see Supporting
Information, Figure S2) in TBS are almost unchanged, and mainly
contain species [M(OG)3]2+ (m/z 558.6, 556, 554), [OG2 + H]+ (m/z
703), [M(OG)2 + Cl]+ (m/z 801, 796, 792), where M = Zn(II),
Co(II), Mn(II). These results indicate that the inner-spheres of
complexes 2−4 are almost stable in TBS, although there exist a small
amount of dissociated species and polymeric species ([M(OG)3]2+) in
the mass spectrometer, similar to copper(II) complexes with modified
indolo[3,2-c]quinoline ligands.37 The ESI-MS results generally agree
well with that of UV−vis spectra analysis. In fact, these OG−metal
complexes contain various species in TBS that all contribute to the
cytotoxicity.
Cytotoxicity Assay. The cell lines BEL7404, SGC7901, HeLa,
MCF-7, A549 were obtained from the Shanghai Cell Bank in the
Chinese Academy of Sciences. Tumor cell lines were grown in the
RPMI-1640 medium supplemented with 10% (v/v) fetal bovine
serum, 2 mM glutamine, 100 U/mL penicillin, and 100 U/mL
streptomycinin at 37 °C, in a highly humidified atmosphere of 95%
air/5% CO2. The cytotoxicity of MT and the title compounds against
BEL7404, SGC7901, HeLa, MCF-7, A549 cell lines were examined by
the microculture tetrozolium (MTT) assay.38 The experiments were
carried out using reported procedure.12 The growth inhibitory rate of
treated cells was calculated using the data from three replicate tests as
(ODcontrol − ODtest)/ODcontrol × 100%. The compounds were
incubated with various cell lines for 48 h at five different
concentrations of complex dissolved in fresh media; the range of
concentrations used is dependent on the complex. The final IC50
values were calculated by the Bliss method (n = 5). All tests were
independently repeated at least three times.
Cell Cycle Analysis. BEL7404 cell lines were maintained in
Dulbecco’s modified Eagle’s medium with 10% fetal calf serum in
5% CO2 at 37 °C. Cells were harvested by trypsinization and rinsed
with PBS. After centrifugation, the pellet (105−106 cells) was
suspended in 1 mL of PBS and kept on ice for 5 min. The cell
suspension was then fixed by the dropwise addition of 9 mL precooled
(4 °C) 100% ethanol with violent shaking. Fixed samples were kept at
4 °C until use. For staining, cells were centrifuged, resuspended in
PBS, digested with 150 mL of RNase A (250 μg/mL), and treated with
150 mL of propidium iodide (PI) (0.15 mM), then incubated for 30
min at 4 °C. PI-positive cells were counted with a FACScan
fluorescence-activated cell sorter (FACS). The population of cells in
each cell cycle phase was determined using Cell Modi FIT software
(Becton Dickinson).
Spectroscopic Studies on DNA Interactions. The synthesized
complexes and ligand were dissolved in DMSO to make 2.0 mM stock
solutions for DNA binding studies. The 2 × 10−3 M ct-DNA stock
solution was stored at 4 °C for no more than 4 days before use. The
final working solutions of the complexes for DNA binding studies are
diluted in the TBS and the containing DMSO is limited in 1%. In
UV−vis absorption spectrometry, the working solutions of OG and
the complexes are 20 μM. The ct-DNA stock solution was added 15
μL per scan at a [DNA]/[compound] ratio of 0.5:1 and gradually
increased up to a sufficient concentration to achieve saturation for
analysis. After each addition, the solution was allowed to incubate for
10 min before the absorption spectra were recorded. The intrinsic
binding constant Kb was determined by the following equation:
[DNA]/(εa − εf) = [DNA]/(εa − εf) + 1/[Kb(εa − εf)],32 where
[DNA] is the concentration of the DNA, εa is the apparent absorption
coefficient (εa = Aobsd/[compound]), εf is the extinction coefficient for
the free compound, and εb is the absorption coefficient of the
compound fully bound to DNA. The intrinsic binding constant, Kb, of
the compounds are calculated as the ratio of the slope to the Y
intercept by linear fitting of [DNA]/(εa − εf) with [DNA] from the
above equation. The absolute values of Kb are presented in the inset of
each linear fitting plot.
In the CD absorption spectrometry, the working solution of each
sample was prepared by using 1 × 10−4 M DNA and titrating the
complexes into the DNA solution stepwise with the [DNA]/
[compound] ratio ranging from 10:0.5 to 10:5. The working solution
was incubated for 10 min after each addition and then its CD spectrum
was recorded at 100 nm/min scan rate. The CD signals of the TBS
were subtracted as the background. A solution containing 10−4
M
DNA and 10−5 M EB ([DNA]/[EB] = 10:1) was prepared for EB-
DNA competitive binding studies. The quenching constants of the test
compounds were calculated according to the classic Stern−Volmer
equation: I0/I = 1 + Kq × [Q],39 where I0 and I are the peak emission
intensity of the EB-DNA system in the absence and presence of the
quencher, [Q] is the concentration of each quencher compound, and
Kq is the Stern−Volmer quenching constant, which is obtained by the
linear fit of plotting I0/I versus [Q] and determines the efficiency of
the quencher. All the spectroscopic experiments were kept at 25 °C. In
DNA viscosity measurements, ct-DNA is dissolved in BPE buffer (6
mM Na2HPO4, 2 mM NaH2PO4, 1 mM Na2EDTA, pH = 7.2) to
prepare 1 × 10−3 M working solution. Compounds were added with
increasing concentrations from 2 × 10−5 M to 2 × 10−4 M to reach the
[compound]/[DNA] ratios range from 0.02 to 0.20. Circulating bath
temperature was maintained at 35.0
0.1 °C. Viscosity values, η,
(unit: cP) were directly obtained by running 0# spindle in working
samples at 30 rpm. Data were presented as η/η0 versus [compound]/
[DNA] ratio, in which η0 and η refers to viscosity of each DNA
working sample in the absence and presence of added compound.
Influence of DMSO as solvent on viscosity was eliminated based on its
viscosity value of 2.03 cP measured at 35.0 °C.
Agarose Gel Electrophoresis Assay.40 In plasmid DNA
unwinding experiments, all compounds were prepared as 2 × 10−3
M stock solutions of DMSO and diluted to 10 and 100 μM by 1× TBE
buffer. Compounds of various concentrations were mixed with 0.5 μg
DNA and made up to a total 25 μL by TBE buffer so that the same
experiment can be repeated twice. All samples were incubated at 25 °C
in dark for 4 h. Then 12 μL of each sample mixed with 2 μL DNA
loading buffer was electrophoresed at 5 V/cm through 0.8% agarose
gel immersed in 1× TBE buffer solution for 60 min. Finally, the gel
was stained with EB (1.27 μM) in dark for 30 min, followed by
visualized on a BIO-RAD imaging system with a UV−vis trans-
illuminator.
RESULTS AND DISCUSSION
■
Synthesis and Characterization of Oxoglaucine. On
the basis of the relate references,41,42 oxoglaucine was
synthesized from (+)-boldine in two steps (Scheme 1). A
facile large-scale semisynthesis of (+)-glaucine (1,2,9,10-
tetramethoxyaporphine) from the commercially available
(+)-boldine (2,9-dihydroxy-1,10-dimethoxyaporphine) by treat-
ment of (+)-boldine with the conventional O-methylation
reagents for the phenolic group such as PhN + (CH3)3Cl−
under basic conditions afforded quaternary ammonium salt
predominantly for (+)-boldine being a tertiary amine. But the
raw product is a mixture and need to be purified on a silica gel
column four times, the yield is lower than that of reference.43
The (+)-glaucine product was confirmed by elemental analysis,
1
APCI-MS, H NMR, and 13C NMR spectroscopy, which agree
well with the reference values.44,45
On the basis of the reported method by Lee et al.,
(+)-glaucine was oxidized with manganese(III) acetate
(MTA) under mild conditions to give oxoglaucine, but our
yield was lower than that reported. The identity of the
oxoglaucine was confirmed by elemental analysis, APCI-MS, 1H
NMR, and 13C NMR. In particular, we for the first time report
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the crystal structure of oxoglaucine. The crystal data and
refinement details of oxoglaucine are summarized in Table 1,
and the important bond lengths and angles are listed in the
caption of Figure 2. As shown in Figure 2, similar to
phoresis and fluorescence polarization. The capillary electro-
phoresis results of 2 and OG in DSMO−methanol−H2O are
shown in Figure S4 (see Supporting Information), which
indicate that 2 differs strongly from that of OG; moreover 2
displays a strong single peak which confirms the formation
zinc(II) complex.48 In addition, since the fluorescence
polarization P value is relevant to the bulk of fluorescent
molecule. The P enhances with the bulk of fluorescent
molecule increasing. Therefore the polarization value is
expected to enhance after OG ligated to metal ion to form
complex. Fluorescence polarization P values were determined
in DMSO−methanol (v/v = 1:1) at 298 K exemplarity for
complex 2 and OG using fluorescence polarization assay49
(λmax(ex) = 379 nm, λmax(em) = 570 nm) under the same molar
concentrations and experimental conditions. Comparison of
POG (0.148) and Pcomplex2 (0.171) reveals that the P value of
complex 2 significantly enhanced, which further confirms the
formation of zinc(II) complex and is agreement with their
results of ESI-MS and capillary electrophoresis.
Crystal Structures of Complexes 1−4. The molecular
structures of gold(III) complex 1 is shown in Figure 3. Selected
Figure 2. ORTEP view of oxoglaucine showing atom labeling, thermal
ellipsoids are drawn at the 30% probability. Selected bond distances
(Å) and angles (deg): C(1)−N(1) 1.338(5), C(15)−N(1) 1.328(4),
C(14)−O(5) 1.367(4), C(5)−O(1) 1.367(4), C(6)−O(2) 1.373(4),
C(10)−O(3) 1.365(4), C(11)−O(4) 1.364(4); N(1)−C(15)−C(14)
115.3(3), C(15)−C(14)−O(5) 121.1(4), C(5)−O(1)−C(17)
116.5(3), C(6)−O(2)−C(18) 115.8(3), C(10)−O(3)−C(19)
118.0(3), C(11)−O(4)−C(20) 117.4(3).
liriodenine,12 oxoglaucine is a planar molecule with N(1) and
carbonyl O(5) as potential donors. The structure of
oxoglaucine is comparable with O-acetylisomoschatoline46
and oxocrebanine.47 In oxoglaucine, the four fused rings are
essentially coplanar, while the four −OMe groups not coplanar
with the ring system.
Characterization of Metal Complexes. Analytical data
1
are in agreement with their formulations. The H and 13C
Figure 3. ORTEP view of [OGH][AuCl4]·DMSO (1) showing atom
labeling. Thermal ellipsoids are drawn at the 30% probability, and
DMSO solvent molecules have been omitted for clarity. Selected bond
distances (Å) and angles (°): C(1)−N(1) 1.344(6), C(15)−N(1)
1.338(5), C(14)−O(1) 1.225(5), Au(1)−Cl(1) 2.2695(13), Au(1)−
Cl(2) 2.2735(14), Au(1)−Cl(3) 2.2753(14), Au(1)−Cl(4)
2.2760(13); N(1)−C(15)−C(14) 116.2(4), C(15)−C(14)−O(1)
121.1(4), C(5)−O(1)−C(17) 119.1(4), Cl(1)−Au(1)−Cl(2)
90.30(5), Cl(1)−Au(1)−Cl(3) 179.53(5), Cl(2)−Au(1)−Cl(4)
178.02(6).
NMR spectral data of complexes 1 and 2 along with their
assignments are given in the Experimental Section. From the
elemental analysis results, the desired metal complexes of OG
were of sufficient purity.
Formation of metal complexes was confirmed by ESI mass
spectrometry (see Supporting Information, Figure S3), capillary
electrophoresis as well as fluorescence polarization. The spectra
of complex 1 in DMSO−methanol showed peaks with m/z
338.84 (negative) and 352.12 (positive), which were attributed
to [AuCl4]− and [OGH]+. The spectra of complexes 2−4 in
DMSO−methanol showed peaks with m/z 828.14 (2), 860.10
(3), 856.11 (4) were assigned to [Zn(OG)2+NO3]+, [Co-
(OG)2+ClO4]+, [Mn(OG)2+ClO4]+, respectively. In addition
peaks with m/z 383.07 (2), 380.58 (3), 378.58 (4), which were
attributed to [Zn(OG)2]2+, [Co(OG)2]2+, and [Mn(OG)2]2+,
respectively, indicating that two aqua ligands easily disasso-
ciated. In some cases, peaks with medium m/z values of 558.63
(1), 556.13 (2), and 554.13 (3) were attributed to the
formation of [Zn(OG)3]2+, [Co(OG)3]2+, and [Mn(OG)3]2+ in
the mass spectrometer, similar to copper(II) complexes with
modified indolo[3,2-c]quinoline ligands.37 The observed
isotopic patterns fit well with the theoretical isotopic
distributions.
bond distances (Å) and bond angles (deg) are given in the
legend to the figure. Complex 1 crystallized in the monoclinic
space group P2(1)/n. 1 is an ionic compound and comprises
one oxoglaucine cation, one [AuCl4]− anion, and one DMSO
solvent. In 1, oxoglaucine (OG) is protonated at N(1) to form
a cation and does not contact Au(III) directly, and gold(III)
tetrachloride serves as the counterion. The [AuCl4]− anion has
the square planar structure commonly observed for gold(III).50
The Au−Cl bond lengths in the anion (2.2719−2.2760 Å) are
very similar to those previously reported for other chlorogold-
(III) complexes.51 The geometric parameters of oxoglaucine
cation are comparable to those of oxoglaucine, and oxoglaucine
still retains its planar structure, which can intercalate between
the neighboring base pairs of DNA. But 1 is not a metallo-
intercalator since Au is not involved in the intercalation.
The results of X-ray diffraction studies of complexes 2−4 are
shown in Figure 4 (for complexes 3 and 4 see Supporting
Due to complexes 2−4 having similar structures (inner-
spheres), to confirm the formation of complexes, we selected
complex 2 as representative to measure its capillary electro-
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enhanced cytotoxicity. The inhibitory rates of 1 toward
BEL7404, SGC 7901, MCF-7, and A549 were 82.91 1.36,
45.15 3.55, 31.05 2.50, and 73.50 0.70%, respectively.
The inhibitory rates of 2 and 3 against HeLa were 53.85 3.61
and 60.08
7901 was 54.57
3.14%, respectively; and that of 4 against SGC
1.54%. From Table S2 (see Supporting
Information), it was found that without the OG ligand, none of
the metal salts could exhibit an inhibition rate of >56% toward
the five tested tumor cell lines even at 100 μM concentration of
the metal salts. These results clearly suggested that the metal
salts alone did not play a key role in the antitumor function,
and the combination of OG with the metal produced
appreciable synergetic effect. The IC50 values in Table 2
further showed various cytotoxicity of these OG-metal
complexes. The IC50 value (6.1 μM) of 1 toward BEL7404
was lower than that of the divalent late transition metal
complexes of liriodenine (IC50= 8.4−112.1 μM)12b and
[Cu(PLN)(bipy)(H2O)]2(NO3)2·4H2O (IC50 = 12.9 μM,
PLN = plumbagin, bipy =2,2′-bipyridine),12c as well as cisplatin.
The activity of 2 and 3 (IC50 = 16.4 and 11.4 μM) against HeLa
was lower than that of cisplatin, but is higher than that of the
divalent late transition metal complexes of liriodenine (IC50=
6.4−9.8 μM).12b In addition, 3 and 4 (IC50 = 5.1 and 7.8 μM)
were more active toward MCF-7 than OG does (IC50 = 11.1
μM) and similar to cisplatin, and 1 (IC50 = 1.4 μM) exhibits
higher activity toward A549 than free ligand OG and cisplatin.
To sum up, these OG-metal complexes have different activity
profile against the tested cell lines even though complexes 2−4
possess similar structure (inner-sphere). Such phenomenon is
difficult to explain. In fact, except for cisplatin, there is relatively
little mechanistic information on how metal anticancer drugs
function, but it is clear that different metal ions can work
through different routes that lead to different cellular
responses.52
DNA Binding Studies. Despite the presence of other
biological targets in tumor cells, including RNA, enzyme, and
protein, it is generally accepted that DNA is the primary target
for many metal-based anticancer drugs such as cisplatin.53 Here
we only consider DNA as the main target in the cell and leave
the possible interaction of the OG−metal complexes with other
targets, such as RNA, enzyme, and protein for future studies.
The interactions between small molecules and DNA are known
to be the primary action mechanisms of antitumor activity. In
general, the interactions between small molecules and DNA
include covalent binding and noncovalent binding, and the
noncovalent bindings include intercalation, insertion, groove
binding, electrostatic interaction, etc.30b,54 The typical
oxoaporphine alkaloids have been proven to realize antitumor
activity by intercalating the neighboring base pairs of DNA to
block DNA replication.55 Our previous work indicated that the
Figure 4. ORTEP view of [Zn(OG)2(H2O)2](NO3)2 (2) showing
atom labeling. Thermal ellipsoids are drawn at the 30% probability,
and hydrogen atoms, as well as two NO3− or ClO4− anions, have been
omitted for clarity. Selected bond distances (Å) and angles (deg) in 2:
Zn(1)−N(1) 2.079(5), Zn(1)−O(1) 2.103(6), Zn(1)−O(6)
1.367(4), N(1)−Zn(1)−O(1) 78.68(18), N(1)−Zn(1)−O(6)
90.7(2), O(1)−Zn(1)−O(6) 95.1(2), O(6)−Zn(1)−O(6A)
180.000(1), N(1)−Zn(1)−O(1A) 101.32(18), N(1)−Zn(1)−N(1A)
180.000(1) (A: −x + 2, −y + 1, −z + 1).
Information, Figures S5, S6), and selected bond lengths and
bond angles are given in the caption. Three complexes
crystallized in the triclinic space group P1. Since complexes
̅
2−4 possess general formula [M(OG)2(H2O)2](NO3/ClO4)2
(M = Zn, Co, Mn) and similar structure (inner-sphere), except
for their different metal center and outer-sphere anion,
therefore, herein only the structure of 2 is discussed. As
shown in Figure 4, in one symmetry unit, 2 consists of one
complex cation [Zn(OG)2(H2O)2]2+ and two NO3 anions.
−
The zinc ion exhibits a distorted octahedral environment
surrounded by the two N and two O atoms from the two
bidentate oxoglaucine (OG) ligands and the two O atoms of
the aqua ligands in trans position. Atoms O(1), N(1), O(1A),
and N(1A) constitute the equatorial plane of the octahedron.
Such structural characteristics was also observed in the
liriodenine−cobalt(II) complex,12 which retains an approximate
planarity and can behave like a metallointercalator to intercalate
between the neighboring base pairs of DNA to give high
cytotoxicity.
In Vitro Cytotoxicity Assay. The in vitro cytotoxicity of
oxoglaucine and its gold(III), zinc(II), cobalt(II), and
manganese(II) complexes 1−4 were evaluated by the MTT
method against five typical human tumor cell lines (using
cisplatin as the positive control) involving liver, gastric
adenocarcinoma, cervix, breast adenocarcinoma, and lung
cancers. As shown in Table S1 (see Supporting Information),
after the tumor cells were incubated with 20 μM test
compounds for 48 h, each compound exhibited different
antitumor activity. The inhibition rates of 20 μM OG ranged in
8%−15% toward all selected cells except HeLa, for which OG
showed no inhibitory effect. In contrast to the free OG, the
OG−metal complexes 1−4 generally exhibited significantly
a
Table 2. IC50 Values (μM) for Compounds 1−4 and Cisplatin in Panel of Five Human Cancer Cell Lines
BEL7404
>150
SGC7901
HeLa
>150
MCF-7
A549
>150
OG
>150
11.1 0.6
>150
1
6.1 0.5
>150
>150
>150
1.4 0.7
>150
2
>150
16.4 4.5
11.4 5.0
>150
>150
3
13.1 8.1
>150
32.7 0.8
No data
>150
5.1 0.8
7.8 1.0
8.3 1.0
>150
4
>150
cisplatin
132.8 1.2
>150
25.3 3.1
a
IC50 values are presented as the mean SD (standard error of the mean) from five separated experiments. Cisplatin was used as a reference
metallodrug and DMSO was used as a solvent control.
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metal complexes of liriodenine, which is also an oxoaporphine
alkaloid, might act as bifunctional DNA-binding compounds in
which the liriodenine ligand intercalates between the
neighboring base pairs of DNA and the metal cations such as
platinum(II) and zinc(II) covalently bind to DNA. To
investigate the DNA binding properties of oxoglaucine and
its metal complexes, a series of spectroscopic studies were
carried out, including UV−vis, fluorescence, CD spectra,
viscosity measurements, and agarose gel electrophoresis assay.
Because 3 and 4 are iso-structures, complex 4 was evaluated
only by CD spectra.
UV−vis Spectral Analysis. It is well documented that
intercalative π−π stacking of the aromatic rings in the metal
complexes with the DNA bases affects the transition dipoles of
the molecules and usually leads to a reduction in their
absorbance.56 The UV−vis absorption was primarily used to
discuss the binding mode of 2 to ct-DNA.
that complexes 1, 2, and 3 have stronger intercalative binding
ability to DNA than OG.55 According to their crystal structures,
1 is not a metallointercalator, whereas 2 and 3 are
metallointercalators that have reinforced binding ability to
DNA. However, because of the different nature of the metals,
the binding abilities of 2 and 3 to DNA are different.
Competitive Binding Studies by Fluorescence Spec-
tra. The binding abilities of OG and complexes 1−3 to ct-DNA
were further investigated by competing with ethidium bromide
(EB) as an intercalative probe.57 In the competitive binding
experiments, the EB-DNA system with [EB]/[DNA] = 1:10
([EB] = 1 × 10−5 M, [DNA] = 1 × 10−4 M) showed the
characteristic strong emission at ∼588 nm when excited by 332
nm light, indicating that the intercalated EB molecules had
been sufficiently protected by the neighboring base pairs in the
DNA from being quenched by polar solvent molecules. When
increasing the concentration of 2 from [2]/[EB] = 1:1 to 4:1,
the characteristic emission of EB was decreased significantly, as
shown in Figure 7 (for OG, 1 and 3 see Supporting
The UV−vis absorption spectra of 2 in the absence and
presence of calf thymus DNA (ct-DNA) are shown in Figure 6
Figure 7. Fluorescence emission spectra of EB bound with ct-DNA in
the absence (dashed line ----) and presence (solid lines ) of 2 as
competitive agent with increasing [2]/[EB] ratios of 1:1 to 4:1.
Figure 6. UV−vis absorption spectra of 2 in the absence (···) and
presence () of ct-DNA with increasing [DNA]/[1] ratios range
from 0.5:1 to 6:1.
(for OG, 1 and 3 see Supporting Information, Figure S7). In
the absence of DNA, complexes 2, 3, and the free OG all
showed characteristic absorption peaks at ∼245 and 272 nm
(for complex 1, absorption peaks were shown at 253 and 307
nm), which is ascribed to the π → π* electronic transition of
the conjugated system of oxoglaucine. Considering the
absorption influence of DMSO at ∼250−260 nm, the
absorbance at 272 nm for OG, complexes 2, 3 and 307 nm
for complex 1 was analyzed upon addition of ct-DNA. When
the ratio of [DNA]/[OG] was increased from 0.5:1 to 6:1, a
moderate hypochromicity of 17.6% and a slight red-shift were
observed, suggesting the characteristic intercalation of OG to
DNA with a moderate degree of binding. The steric hindrance
of the four methoxyl groups in OG may have prevented more
effective intercalation. Under similar conditions, evident
hypochromicity for 1−3 (18.7% for 1, 34.0% for 2, 46.5% for
3) were also observed along with the corresponding red-shifts.
Especially, the 46.5% hypochromicity of 3 was obtained only at
a relatively lower [DNA]/[3] ratio of 4:1. For further
quantitative comparison, the intrinsic binding constant Kb was
also calculated. The Kb values for OG and complexes 1−3 are
2.71 × 103, 1.62 × 104, 8.24 × 104, and 5.82 × 104 M−1,
respectively (inset plot of Figure 6; for OG, 1 and 3 see
Supporting Information, inset plot of Figure S7), suggesting
Table 3. Calculated Quenching Ratios of EB by Titration of
OG/Complexes to EB-ctDNA System
one-step FI quenching
total FI quenching
ratio
a
compound
OG
ratio (%)
Kq
7.1 × 103
13.9
18.1
29.1
28.0
54.7% at [OG]/[EB]
= 6:1
1
2
3
69.0% at [1]/[EB] =
9:1
1.3 × 104
2.7 × 104
2.2 × 104
78.1% at [2]/[EB] =
4:1
71.6% at [3]/[EB] =
6:1
a
FI, fluorescence intensity; one-step refers to [compound]/[EB] =
1:1.
Information Figure S8). Table 3 lists the corresponding
quenching data of EB fluorescence intensity and the calculated
competitive binding ability. It was found that the metal
complexes and OG could all effectively quench the fluorescence
emission of EB, which strongly suggests that they may compete
with EB to bind to DNA through intercalation at the similar
binding site.58
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Furthermore, the quenching constants Kq were also
calculated by restricted linear fitting of [I0/I] to [Q] from the
Stern−Volmer equation and listed in Table 3. The calculated
Kq for OG is 7.1 × 103, while those for complexes 1, 2, 3 are 1.3
× 104, 2.7 × 104, and 2.2 × 104, respectively. All the three
complexes exhibited stronger quenching ability than OG, which
is consistent with the UV−vis spectral titration results. It seems
that combining OG with the metal ions will reinforce its
binding to ct-DNA and the metal ions play important roles in
the high cytotoxicity of the OG-metal complexes. Even though
the binding of these complexes can only induce small structural
changes in the DNA duplex, they lead to a different cellular
response.59
Circular Dichroism Spectral Analysis. Circular dichroism
(CD) is a useful technique to assess whether nucleic acids
undergo conformational changes as a result of complex
formation or changes in environment.60 The normal ct-DNA
has a right-handed chiral conformation and exists in B-form in
solution. The CD spectrum of DNA is very sensitive to its
conformational changes. It is generally accepted that covalent
binding and intercalative binding can influence the tertiary
structure of DNA and induce changes in the CD spectra of
DNA, whereas other noncovalent binding modes such as
electrostatic interaction or groove binding cannot significantly
perturb the CD spectra.61
The CD absorption of ct-DNA shows a positive absorbance
peak at ∼270 nm and a negative absorbance peak at ca. 245 nm
(shown as dashed line in CD spectra), due to the π−π base
stacking of DNA and the right-hand helicity of B-form DNA,
respectively.62 The addition of OG did not perturb the CD
spectrum of DNA until [OG] reached 5 × 10−5 M, when both
the positive and the negative absorbance of DNA evidently
decreased (for OG, 1, 3, and 4, see Supporting Information,
Figure S9). Intercalation is the most probable binding mode
between OG and DNA because of the planar aromatic structure
of OG, but the intercalative ability of OG to DNA appears
weak. However, the addition of complex 2 induced significant
changes in the characteristic absorption of DNA, which
indicates the formation of a steady complex between DNA
and 2, as shown in Figure 8.63 At lower concentrations of 2 (5
the negative absorption in the CD spectra, which represents the
intercalative binding of 2 to DNA. At higher concentrations of
2 (from 2 × 10−5 M to 5 × 10−5 M), there were dramatic
changes in the CD absorption of DNA and the characteristic
DNA absorption peaks disappeared, which indicates the
transformation from B-form to non-B-form DNA secondary
structure.64 Furthermore, induced positive and negative CD
signals respectively at ∼380 and 345 nm could be observed in
the CD spectrum, which resulted from the binding of complex
2 to DNA as a metallointercalator.65
The changes in the CD spectra of the DNA upon addition of
complex 4 are very similar to those of complex 2 (see
Supporting Information, Figure S9), which strongly suggests
that 4 and 2 have the same binding mode toward ct-DNA,
especially considering their similar structure and coordination
mode. Because complex 3 has poor solubility in water and
precipitates at higher concentration, only two CD spectra of
DNA binding with 3 were scanned at [DNA]/[3] molar ratios
of 10:0.5 and 10:1 respectively. Nevertheless, the significant
decreases in the positive absorbance and the slight decreases in
the negative absorbance, along with the unchanged CD
absorption curves, also suggests the intercalative binding of 3
at low concentration (≤1 × 10−5 M) toward ct-DNA.66 Under
the same conditions, complex 1 did not induce such dramatic
changes in the CD spectrum of DNA as complexes 2−4 did,
which should be due to the different structure of 1 to those of
2−4. Similar to OG, complex 1 also induced slight but regular
decreases in the positive absorption without changing the
negative absorption at lower concentration, and induced more
significant decreases in both the positive and the negative
absorption at higher concentration, which evidently indicates a
weak intercalative binding of 1 to DNA without DNA
cleavage.67
DNA Viscosity Experiments. It is generally accepted that
besides X-ray crystallography and high-resolution NMR, the
viscosity measurement of DNA solution can also serve as a
quite unambiguous method to distinguish the binding modes of
small molecules to DNA. When small planar aromatic
molecules intercalate between the neighboring base pairs of
DNA, the double helix loosens to accommodate the
intercalation, which increases the length of the DNA helix.
Since the viscosity of DNA solution is very sensitive to the
changes of DNA length, the increased viscosity of DNA
solution can be associated with the specific intercalation
binding mode.68
The viscosity of the ct-DNA solution increased notably after
the addition of OG, OG-metal complex or EtBr, as shown in
Figure 9. When increasing the [compound]/[DNA] ratios from
0.02 to 0.20, the viscosity of the DNA solution bound with OG
or its three complexes 1−3 all increased regularly. Complexes 2
and 3 exhibited similar behavior and both increased the
viscosity slightly more than OG did when the [compound]/
[DNA] ratio was in the range of 0.02−0.16. Despite the
apparent differences in the strength of interaction, OG, 2, and 3
all exhibited similar viscosity enhancement profile compared
with EtBr, which clearly suggests that intercalation is the main
binding mode for their interaction with ct-DNA. In contrast to
2 and 3, 1 increased the viscosity of DNA solution much less
effectively, which suggests that other binding modes, such as
electrostatic interaction may exist between 1 and DNA and play
important role.69
Figure 8. Circular dichroism spectra of ct-DNA bound by 2 with
[DNA]/[2] ratios range from 10:0.5 to 10:5 (DNA alone of 1 × 10−4
M, dashed line; DNA bound by 2 with increasing concentrations,
colored solid lines).
× 10−6 M and 1 × 10−5 M), there was significant decrease in
the positive absorbance (with 18.4%−53.6% hypochromicity
and 3−5 nm bathochromic shift, respectively) but no change in
In summary, the UV−vis spectral titration revealed that the
binding of 1−3 to ct-DNA was mainly through the intercalation
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the results from the spectral analyses and DNA viscosity
experiment.
Topoisomerase I Inhibition Studies. Topoisomerases are
ubiquitous molecules that relieve the torsional stress in the
DNA helix generated as a result of replication, transcription,
and other nuclear processes. They are also the specific targets
of a number of anticancer agents, including camptothecins,
indolocarbazoles, indenoisoquinolines, etoposide, and adriamy-
cin. These anticancer agents bind to a transient topoisomerase I
(TOPO I)-DNA covalent complex and inhibit the resealing of a
single-strand nick that the enzyme creates to relieve the
superhelical tension in duplex DNA.70
The TOPO I inhibition ability of OG and complexes 1−3 is
shown in Figure 11. Lanes 1 and 2 show the unwinding of
Figure 9. Relative viscosity increments of ct-DNA solution bound with
OG, complexes 1−3, and EtBr with increasing [compound]/[DNA]
ratios range from 0.02 to 0.20 every 0.02 interval.
of complexes 1−3. The OG metal complexes have stronger
binding ability than OG. Furthermore, the EB-competitive
binding studies indicated that complexes 1−3 had higher
competitive binding ability than OG against EB in the ct-DNA
system. The complexes 2−4 induced significant perturbation in
the CD spectra of ct-DNA but complex 1 did not, and
complexes 2 and 3 could increase the viscosity of DNA solution
much more effectively than 1. Thus it can be seen that the OG-
metal complexes bind to DNA more strongly than OG. These
findings agree well with the fact that the cytotoxicity of
complexes 1−4 toward most tested tumor cell lines are higher
than that of OG alone because the planar OG ligand in
complexes 1−4 can bind more powerfully to DNA than the free
OG and the metal ions also play key roles in their cytotoxicity.
Agarose Gel Electrophoresis Assay. The binding modes
of OG and complexes 1−3 to pUC19 plasmid DNA were
further examined by agarose gel electrophoresis assay. As
shown in Figure 10, upon addition of OG and increasing its
Figure 11. Inhibition of TOPO I-mediated DNA relaxation by OG
and complexes 1−3, respectively. Lane 0: DNA alone. Lane 1: DNA +
TOPO I. Lane 2: DNA + TOPO I + HCPT (50 μM). Lanes 3−4, 5−
6, 7−8 and 9−10 refer to DNA + TOPO I + OG/complexes 1−3 at
50, 100 μM, respectively.
supercoiled DNA by TOPO I and the inhibition ability of
camptothecin as a classic TOPO I inhibitor. The presence of
OG or 1−3 at 50 μM did not show obvious inhibition of
TOPO I, but at a higher concentration of 100 μM, they all
effectively inhibited TOPO I from unwinding supercoiled
DNA. The observed results suggest that the TOPO I inhibition
ability of these complexes should originate from the OG
moiety.
S-Phase Cell-Cycle Arrest. To determine whether cellular
DNA is a major target of the OG-metal complexes, we studied
the cell-cycle profiles of cancer cells treated with 1−3. Cell-
cycle analysis was performed by using flow cytometry to assess
the DNA content of cells, which was stained with propidium
iodine (PI). This enables the quantification of the total cellular
populations in different phases of the cell cycle (G0/G1, S, and
G2/M). The flow cytometric data for BEL7404 cells treated
with 1−3 are shown in Figure 12. Treatment of cells with 1−3
(10 μM) for 24 h enhanced cell-cycle arrest at the S phase,
resulting in concomitant population increase in the S phase and
population decrease in the G1 phase, the stage in which most
DNA replication occurs.71 The direct interaction of 1−3 with
DNA has been examined. We found that compounds 1−3
strongly interact with ct-DNA with binding constants of 1.62 ×
104, 8.24 × 104, and 5.82 × 104 M−1, respectively, based on the
results from spectrophotometric titration experiments. Com-
plexes 1−3 are able to intercalate between the neighboring base
pairs of DNA and have inhibitory activity on a DNA-binding
protein topoisomerase I. Taking these results altogether, DNA
may be a crucial cellular target for 1−3 to achieve cytotoxicity.
Figure 10. Agarose gel electrophoresis of pUC19 DNA treated with
OG and complexes 1−3 after incubation for 1 h at 37 °C. Lane 0:
DNA alone. Lanes 1−4, 5−8, 9−12 and 13−16 refer to DNA in the
presence of OG and complexes 1−3, respectively, at 10, 25, 50, and
100 μM; SC, supercoiled form, RLX, relaxed form.
concentration from 10 to 100 μM in the absence of any
external reagent or light, the DNA decreased significantly in
both the major supercoiled form and the minor relaxed form.
The addition of 2 and 3 did not significantly change the DNA
either in the supercoiled or the relaxed form, as shown by the
migration rate and proportions. Therefore, 2 and 3 have weak
unwinding ability for supercoiled DNA. But in the presence of
1 at 25 and 50 μM, more DNA in the relaxed form can be
clearly observed, which suggests that 1 has different binding
modes compared with other complexes and is consistent with
CONCLUSIONS
■
The TCM active component oxoglaucine was synthesized and
reacted with Au(III), Zn(II), Co(II), and Mn(II) salts to give
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Figure 12. Induction of cell-cycle arrest in BEL7404 cells after treatment with 1−3. Untreated cells and cells treated with 1−3 (10 μM) were
collected at 24 h, subjected to PI staining and flow cytometric analysis. G0/G1, G2/M, and S indicate the cell phase. Data shown is a representative
experiment repeated twice with similar results.
four oxoglaucine−metal complexes. The complexes were
characterized and their crystal structures were determined by
X-ray diffraction. The oxoglaucine−Au(III) compound 1 was
ionic and the Au metal was not directly bonded to oxoglaucine.
The other complexes 2−4 were coordination compounds with
chelated bonding between oxoglaucine and the metal, which
were further confirmed by ESI-MS, capillary electrophoresis
and fluorescence polarization; even though they might
dissociate and release positively charged species in solution.
The in vitro cytotoxicity of 1−4 against five selected human
tumor cell lines is different. In some cases, they exhibited
significant enhanced antitumor activity compared with those of
oxoglaucine and corresponding metal salts, such as 1 toward
BEL7404 and A549; 2 and 3 toward HeLa, 3 and 4 toward
MCF-7. It seems that these OG-metal complexes were
selectively active against certain cell lines. Although the
spectroscopic and agarose gel electrophoresis results showed
that the binding of these compounds to DNA could only
induce small structural changes in the duplex, it could lead to a
different cellular response.59 The cell cycle analyses showed
that 1−3 increased cell cycle arrest at the S phase. These OG−
metal compounds interacted with ct-DNA mainly through
intercalation and could effectively inhibit TOPO I, implying
that topoisomerase I may be yet another molecular target.
However, the exact molecular mechanism requires further
detailed investigation.
ASSOCIATED CONTENT
■
S
* Supporting Information
Further details are given in Figures S1−S9, Tables S1 and S2,
and X-ray crystallographic data is given in CIF format. These
materials are available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: chenzfubc@yahoo.com (Z.-F.C.); hliang@gxnu.edu.cn
(H.L.). Tel.: +86(0)773/2120998. Fax: +86(0)773/2120958.
ACKNOWLEDGMENTS
■
This work was supported by National Basic Research Program
of China (Nos. 2010CB534911, 2012CB723501), the National
Natural Science Foundation of China (No. 20861002), and
Natural Science Foundation of Guangxi Province
(Nos.0991012Z, 0991003, and 2010GXNSFF013001), as well
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Article
W.; Au-Yeung, S. C. F.; Wang, X.; Lin, G.; Han, X. J. Med. Chem. 2001,
44, 2065−2068.
as “BAGUI Scholar” program. We also thank Prof. Dr. Huai-
Ming Hu for the crystal structures determination, Prof. Dr. Z.
Guo from NJU and Prof. Dr. Chris Orvig from UBC for
valuable discussion.
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