Copper(II/I) complexes of 5-pyridin-2-yl-[1,3]dioxolo[4,5-g]isoquinoline: Synthesis, crystal structure, antitumor activity and DNA interaction

Article abstract of DOI:10.1016/j.ejmech.2013.10.031

Three new copper(II) complexes of 5-pyridin-2-yl-[1,3]dioxolo[4,5-g] isoquinoline (PYP), i.e. [Cu₂(PYP)₂Cl₄] (1), [Cu₄(PYP)₄(ClO₄)₂(H ₂O)₂](ClO₄)

Full text of DOI:10.1016/j.ejmech.2013.10.031

European Journal of Medicinal Chemistry 70 (2013) 640e648
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Copper(II/I) complexes of 5-pyridin-2-yl-[1,3]dioxolo[4,5-g]
isoquinoline: Synthesis, crystal structure, antitumor activity and DNA
interaction
,
,
a
a
a
Ke-Bin Huang a b, Zhen-Feng Chen a , Yan-Cheng Liu , Meng Wang , Jian-Hua Wei ,
**
*
Xiao-Li Xie a, Jian-Lian Zhang a, Kun Hu a, Hong Liang a
,b,
a Guangxi Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmacy of Guangxi Normal
University, Guilin 541004, PR China
b School of Chemistry of Nankai University, Tianjin 300071, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three new copper(II) complexes of 5-pyridin-2-yl-[1,3]dioxolo[4,5-g]isoquinoline (PYP), i.e. [Cu2(PYP)2Cl4]
(1), [Cu4(PYP)4(ClO4)2(H2O)2](ClO4)2$2H2O (2), and [Cu2(PYP)2Cl4]n (3), were synthesized and fully char-
acterized. In comparison to free PYP, complexes 1e3 exhibited enhanced cytotoxicity against tested hu-
man tumor cell lines BEL-7404, SK-OV-3, A549, A375, MGC-803 and NCI-H460, with IC50 values ranging
Received 11 July 2013
Received in revised form
11 October 2013
Accepted 12 October 2013
Available online 22 October 2013
from 0.31 to 30.76 mM. Complexes 1e3 exhibited lower cytotoxicity to HL-7702 than them to cancer
cells. Complex 1 induced apoptotic death of BEL-7404, which involved mitochondria in the process.
Caspase-3 activation assay indicated that 1 could be an efficient activator of caspase-3. DNA binding
studies by UVevis, DNA-melting, competitive binding, CD, viscosity measurement and agarose gel elec-
trophoresis, revealed that intercalation might be the most likely binding mode of 1 with DNA.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Isoquinoline
Copper(II) complexes
Synthesis
Crystal structure
Cytotoxicity
1. Introduction
complexes have been reported as potential anticancer agents both
active in vitro and in vivo [14e18].
Transition metal complexes with anticancer activity have been
extensively studied following the discovery of cisplatin and its
analogs as transcription inhibitors [1e3]. However, treatment fail-
ure is often caused by the development of side effects, drug resis-
tance, etc [4]. As a result, it is necessary to design new metallodrugs
that are less toxic and highly active in small dosages, especially
against cell lines that have acquired high resistance to cisplatin. In
this context, much attention has been drawn toward antitumor
complexes based on non-platinum metals [5e9]. Ru(II) and Cu(II)
complexes are regarded as the best alternatives to platinum-based
complexes as anticancer agents [10,11]. Copper(II), which is known
to play a significant role in biological systems, has also been used as
a pharmacological agent [12,13]. A number of synthetic copper(II)
Isoquinolines, a type of alkaloids widely existing in traditional
Chinese medicine, exhibit variety of biological activities
a
especially inhibition of cellular proliferation and cancer develop-
ment [19e21]. For example, berberrubine, a protoberberine alka-
loid, shows antitumor activity in animal models [22]. Most of
aporphine and oxoaporphine alkaloids which contain isoquinoline
skeleton exhibit antitumor activity [23]. Various bioactivities have
been found in [1,3]dioxolo[4,5-g]isoquinoline (papraline), a typical
isoquinoline alkaloid isolated from the aerial parts of Fumaria indica
which is regarded as a laxative, diuretic, alterative and mild anal-
gesic, beneficial for dyspepsia and scrofulous skin infections
[24,25]. Moreover, 2-substituted isoquinolines with aryl have
shown enhanced anticancer activity [21]. Because of the extensive
biological effects of isoquinolines, a series of platinum(II) com-
plexes of isoquinoline and derivatives with potent anticancer ac-
tivity have been reported by Osella and Farrell et al. in the past two
decades [26e28]. However, very few examples of isoquinoline as
chelating, nonleaving ligands in cis-platinum(II) antitumor com-
plexes have been documented [29]. Previously, we reported
promising anticancer activity in transition metal complexes of
* Corresponding author. Tel./fax: þ86 773 2120958.
** Corresponding author. Guangxi Key Laboratory for the Chemistry and Molec-
ular Engineering of Medicinal Resources, School of Chemistry and Pharmacy of
Guangxi Normal University, Guilin 541004, PR China.
E-mail addresses: chenzfubc@yahoo.com, chenzfgxnu@yahoo.com (Z.-F. Chen),
hliang@gxnu.edu.cn (H. Liang).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.031

K.-B. Huang et al. / European Journal of Medicinal Chemistry 70 (2013) 640e648
641
isoquinoline alkaloids having the active ingredients liriodenine and
oxoglaucine found in traditional Chinese medicine [30e32]. To
further our search for new metal-based anticancer agents, we
designed and synthesized a new isoquinoline derivative, 5-pyridin-
2-yl-[1,3]dioxolo[4,5-g]isoquinoline (PYP), with papraline as the
leading compound. PYP contains a DNA intercalation moiety and a
pyridine ring. It can act as a chelating, nonleaving ligand (Scheme
1). Herein, we report the synthesis, crystal structure and in vitro
antitumor activity of three copper(II/I) complexes with the
chelating ligand PYP. The mechanism of their cytotoxicity and their
interactions with DNA were also investigated.
m2-Cl, and two N atoms from PYP. It is worth pointing out that the
dimeric structures of 1 and 2 as well as the polymeric structure of 3
can disassociate into mononuclear species in solution, as confirmed
by their ESI-MS spectra.
2.3. In vitro cytotoxicity
The in vitro cytotoxicity of PYP and complexes 1e3 against BEL-
7404, SK-OV-3, A549, A375, MGC-803, NCI-H460 and HL-7702 cell
lines was investigated with cisplatin as the positive control. As
shown in Table 1, the IC50 values of complexes 1e3 are lower than
that of cisplatin. Significantly enhanced activities can be seen for
these complexes compared to free PYP and the corresponding
copper(II) salts, suggesting the synergistic effect between PYP and
copper ions. It should be noted that these copper(II) complexes to-
wards the normal human liver HL-7702 cells displayed lower
cytotoxicity than that of them to the tested cancer cells. Thereby,
complexes 1e3 exhibited a certain extent selectivity to cancer cells.
Among these complexes, complex 1 has the highest cytotoxicity
against BEL-7404, A549, A375, and NCI-H460, with the IC50 values of
2. Results and discussion
2.1. Synthesis
Dihydroisoquinoline (MPDQ) was synthesized by Bischlere
Napieralski cyclization of the acylation product (ALP) of phomopi-
peronylamine and picolinic acid. Then 5-pyridin-2-yl-[1,3]dioxolo
[4,5-g]isoquinoline (PYP) was obtained by treating MPDQ with
activated MnO2 in toluene. Both were fully characterized by ESI-MS,
1H NMR and 13C NMR spectroscopy.
The corresponding copper complexes [Cu2(PYP)2Cl4] (1) and
[Cu4(PYP)4(ClO4)2(H2O)2](ClO4)2$2H2O (2) were obtained by
reacting PYP with CuCl2$2H2O and Cu(ClO4)2$6H2O in CH3OH/
CHCl3 (2:1) respectively under solvothermal conditions.
[Cu2(PYP)2Cl4]n (3) was obtained in the same manner in CH3OH/
H2O (4:1) (Scheme 2). These complexes were characterized by
elemental analysis, ESI-MS and single crystal X-ray diffraction
analysis.
ranging from 0.31 to 4.29
toxicity against BEL-7404, A549, MGC-803 and NCI-H460, with the
IC50 values in the range of 1.32e8.36 M. Based on their structures
mM. Complex 3 also exhibits strong cyto-
m
and the molecular species in solution (ESI-MS data), it can be
concluded that their cytotoxicity originates from the copper ion and
the approximately planar benzo[1,3]dioxole moiety of PYP that can
intercalate between adjacent base pairs of DNA [12]. Since complex
1 displays the highest cytotoxicity in most cases, it is used as a
representative compound in the following studies on the mecha-
nism of cell cytotoxicity against the BEL-7404 cancer cell lines.
2.4. Apoptosis study by flow cytometry
2.2. Crystal structure
To determine whether the observed cell death induced by the
complexes was due to apoptosis or necrosis, the interactions of BEL-
7404 cells with the complexes were further investigated using an
Annexin V-FITC/propidium iodide assay. As phosphatidylserine
(PS) exposure usually precedes loss of plasma membrane integrity
in apoptosis, the presence of annexin Vþ/PIꢀ cells can be consid-
ered as an indicator of apoptosis. In the case of complex 1 (Fig. 4),
the population of annexin Vþ/PIꢀ cells (Q4) is 36.8%, which sug-
gests that apoptotic death was induced in BEL-7404 cells.
The molecular structures of the three complexes [Cu2(PYP)2Cl4]
(1), [Cu4(PYP)4(ClO4)2(H2O)2](ClO4)2$2H2O (2) and [Cu2(PYP)2Cl4]n
(3) are depicted in Fig. 1e3. Complexes 1 and 3 have dinuclear
structures, whereas complex 2 has a tetranuclear structure. As
shown in Fig.1, each copper(II) in 1 is chelated by one bidentate PYP
with the two Cu(II) centers bridged by two m2-Cl anions. Each Cu(II)
center adopts a distorted square pyramid geometry and is sur-
rounded by three Cl and two N atoms from PYP. Complex 2 in Fig. 2
resembles [(LCu2)2(CO3)(H2O)2(ClO4)](ClO4)3 (L ¼ bis(tridentate)
pyrazolate-based ligand) [33]. Cu(1), Cu(2), Cu(1A) and Cu(1A),
Cu(2), Cu(2A) are each spanned by one m3
-k
O,
k
O0,
k
O00-bridging
2.5. Changes in the mitochondrial membrane potential
perchlorate anion, respectively. The Cu(1) and Cu(1A) centers adopt
a distorted tetrahedron geometry and are surrounded by one O
atom from the perchlorate anions, one water molecule, and two N
atoms from PYP. The distorted tetrahedron geometry of the Cu(2)
and Cu(2A) centers are formed by two O atoms from two different
m3-perchloride anions and two N atoms from PYP. As depicted in
Fig. 3, complex 3 has a one-dimensional infinite zigzag chain
structure, in which the adjacent two PYPCuCl2 building blocks are
bridged by one m2-Cl anion and each Cu(II) center adopts a distorted
square pyramid geometry completed by one terminal Clꢀ, two
Mitochondria act as a point of integration for apoptotic signals
originating from both extrinsic and intrinsic apoptotic pathways.
Mitochondrial dysfunction and the release of apoptogenic factors
are critical events in triggering various apoptotic pathways. Loss of
mitochondrial membrane potential is an important indicator of cell
mitochondrial dysfunction. To evaluate the function of mitochon-
dria in complex 1 induced apoptosis, the changes in mitochondrial
membrane potential were measured by the JC-1 probe, which
would dissociate from the aggregated form (red fluorescence) to
Scheme 1. Synthesis of PYP ligand. (a) SOCl2, 2-Picolinic acid, K2CO3, CH2Cl2. (b) POCl3, Toluene, 5%NaOH. (c) Activated MnO2, Toluene.

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K.-B. Huang et al. / European Journal of Medicinal Chemistry 70 (2013) 640e648
Scheme 2. Synthesis of the Cu(II/I) complexes of PYP.
the monomeric form (green fluorescence) when mitochondrial
membrane potential was reduced. The protocol is shown in the
materials and methods section and the pictures are shown in Fig. 5.
Cells treated with complex 1 have a significant decrease in mito-
chondrial membrane potential as indicated by a notable shift in the
ratio of green/red fluorescence versus the control. The results
suggest that mitochondrial is also involved in the process of
apoptosis.
neighboring base pairs of DNA and covalent binding of the metal
cations, e.g. platinum(II) or zinc(II), to the DNA. In this study, DNA
interactions of our newly synthesized isoquinoline metal com-
plexes were characterized by a number of methods including UVe
vis absorption, fluorescence, CD, viscosity measurements, and
agarose gel electrophoresis assay.
2.7.1. UVevis absorption spectral analysis
The UVevis absorption spectra of complex 1 in the presence of
different concentrations of DNA are shown in Fig. 7 and the spectra
for PYP shown in Fig. S1, ESI. Complex 1 exhibits three absorption
bands at around 260, 330 and 370 nm, respectively, with the
maximum absorbance at 330 nm, which is significantly enhanced
as compared to that of PYP. As the amount of DNA increases, the
absorption intensities of complex 1 at 330 and 370 nm gradually
decrease. The hypochromic effect in the UV spectra upon binding of
a small molecule to DNA is characteristic of an intercalating mode
[35]. Therefore, it can be concluded that the binding mode between
complex 1 and DNA might be intercalation. Compared with com-
plex 1, the free ligand PYP shows a maximum absorbance at around
372 nm and the addition of ct-DNA could not induce significant
decrease in this peak (see Fig. S1). This suggests that the inter-
calative binding of PYP is weaker than that of complex 1.
2.6. Caspase-3 activation assay
The caspase family is well characterized and plays a crucial role
in modulating programmed cell death, which is a genetically
regulated and evolutionarily conserved process with numerous
links to many human diseases, most notably cancer. Caspase-3, the
executioner caspase, is able to directly degrade multiple substrates
including structural and regulatory proteins. Thus, therapeutic
strategies designed to stimulate apoptosis by activating caspase-3
may help in combating cancer caused by apoptosis deficiency.
Some small molecules have been developed to be selective acti-
vators of caspase-3. We have therefore investigated whether the
effector caspase-3 is activated when BEL-7404 cells are exposed to
complex 1. In Fig. 6, cells treated with complex 1 show a significant
increase in the activity of caspase-3 as indicated by a notable shift
in the ratio of green/dark fluorescence versus the control. These
results suggest that the tested complex 1 is an efficient activator of
caspase-3.
2.7.2. DNA-melting analysis
The melting temperature (Tm) of DNA, defined as the temper-
ature at which half of the dsDNA is dissociated into single strands, is
strictly related to the stability of the DNA macromolecule. The Tm of
DNA is usually altered by the interaction with chemicals. In general,
the classic intercalation of small molecules into the double helix
can increase the stability of the DNA helical structure resulting in an
increase of Tm by 5e8 ꢁC, while the non-intercalation binding
causes no visible enhancement in Tm [36]. As shown in Fig. 8, the
Tm value of DNA in the absence of the added compounds is
71.97 ꢁC, while those in the presence of PYP and complex 1 are
2.7. 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 [34]. Our previous work indicated
that metal complexes of isoquinolines alkaloids (oxoaporphine
alkaloid) might act as bifunctional DNA-binding compounds. The
binding involved intercalation of isoquinoline ligand between the
Fig. 2. ORTEP drawing of complex 2, and theꢁhydrogen atoms have been omitted for
ꢀ
clarity. Selected bond lengths (A) and angles ( ), for 2: Cu(1)eO(8) 1.9198(62), Cu(1)e
N(4) 1.9779(77), Cu(1)eN(3) 2.0054(71), Cu(1)eO(3W) 2.3123(35), Cu(2)eO(6A)
1.9308(63), Cu(2)eO(5) 1.9438(60), Cu(2)eN(2) 1.9819(82), Cu(2)eN(1) 2.0133(79);
N(4)eCu(1)eO(8) 167.67(31), N(3)eCu(1)eO(8) 89.68(29), O(8)eCu(1)eO(3W)
89.68(29), N(3)eCu(1)eN(4) 80.42(31), N(4)eCu(1)eO(3W) 98.01(24), O(3W)eCu(1)e
N(3) 161.19(24); O(5)eCu(2)eO(6A) 92.88(26), N(2)eCu(2)eO(6A) 90.43(29), N(1)e
Cu(2)eO(6A) 165.66(30), N(2)eCu(2)eO(5) 174.57(31), N(1)eCu(2)eO(5) 95.88(30),
N(2)eCu(2)eN(1) 80.05(32).
Fig. 1. ORTEP drawing of complex 1. The hydrogen atoms have been omitted for clarity.
ꢁ
ꢀ
Selected bond lengths (A) and angles ( ) for complex 1: Cu(1)eN(1) 2.2138(39), Cu(1)e
N(2) 2.2688(40), Cu(1)eCl(1) 2.3561(15), Cu(1)eCl(2) 2.5405(14); N(2)eCu(1)eCl(2)
163.72(11), N(2)eCu(1)eCl(1) 90.92(12), N(1)eCu(1)eCl(1) 125.26(11), N(2)eCu(1)e
Cl(2) 91.63(11), N(2)eCu(1)eN(1) 72.72(14), Cl(1)eCu(1)eCl(2) 102.12(5).

K.-B. Huang et al. / European Journal of Medicinal Chemistry 70 (2013) 640e648
643
Fig. 4. AnnexinV/propidium iodide assay of BEL-7404 cells treated by complex 1 (at
IC50 concentrations) measured by flow cytometry.
corresponding quenching data of EB fluorescence intensity and the
calculated competitive binding ability. The EB fluorescence can be
quenched by complex 1 to a certain extent, which suggests that
complex 1 may compete with EB to bind DNA through intercalation
at similar binding sites [39]. Table 2 also shows the quenching
constants Kq calculated by restricted linear fitting of [I0/I] to [Q]
from the SterneVolmer equation. The calculated Kq values for
complex 1 and PYP are 4.29 ꢂ 105 and 6.34 ꢂ 102, respectively. Both
complex 1 and PYP exhibit moderate quenching ability, but com-
plex 1 is a stronger quencher than PYP.
Fig. 3. ORTEP drawing of complex 3, and theꢁhydrogen atoms have been omitted for
ꢀ
clarity. Selected bond lengths (A) and angles ( ), for 3: Cu(1)eN(1) 2.0530(42), Cu(1)e
2.7.4. CD analysis
At a low concentration of complex 1 (5 ꢂ 10ꢀ5 M), there is a
significant increase in the positive absorbance in the CD spectrum
of DNA, which indicates the intercalative binding of complex 1 with
DNA (Fig. 10) [40,41]. At the same concentration of PYP
(5 ꢂ 10ꢀ5 M), however, there is less variation in the CD absorbance
of DNA and the characteristic DNA absorption peaks (Fig. S3, ESI).
These results confirm stronger interactions of complex 1 with DNA
than PYP.
N(2) 1.9924(42), Cu(1)eCl(1) 2.2393(15), Cu(1)eCl(2) 2.2776(14), Cu(1)eCl(4A)
2.6057(14); N(1)eCu(1)eN(2) 79.41(17), Cl(1)eCu(1)eN(2) 174.51(13), Cl(1)eCu(1)e
N(1) 95.46(12), N(1)eCu(1)eCl(2) 152.70(12), Cl(1)eCu(1)eCl(2) 92.23(6), Cl(4A)e
Cu(1)eN(2) 90.06(12), Cl(4A)eCu(1)eN(1) 99.94(12), Cl(1)eCu(1)eCl(4A) 92.78(5),
Cl(4A)eCu(1)eCl(2) 105.81(5), Cl(3)eCu(1)eCl(4) 91.45(6), Cl(3)eCu(1)eCl(2)
101.38(5), Cl(4)eCu(1)eCl(2) 86.10(5).
75.82 ꢁC and 80.93 ꢁC, respectively. The Tm increases indicate
enhanced DNA stability in the presence of the two compounds. The
results strongly support the previous conclusion of intercalative
binding of complex 1 with DNA. The Tm increase of 8.96 ꢁC for
complex 1 is smaller than 13 ꢁC, the literature value of Tm increase
for ethidium bromide (EB), suggesting weaker intercalative binding
capacity of both as compared to that of EB [37].
2.7.5. Viscosity measurement
Viscosity change of the ct-DNA solution was measured upon the
binding of complex 1 and PYP with EtBr as the reference (Fig. 11).
The viscosity of the DNA solution shows a small increase of about
9.5% as the [PYP]/[DNA] ratio increases from 0.02 to 0.20. In
contrast, the viscosity shows a significant increase with the
increasing [complex 1]/[DNA] ratio from 0.02 to 0.20, which con-
firms the intercalative binding mode between complex 1 and ct-
DNA [42]. The intercalation is very likely due to the approxi-
mately planar benzo[1,3]dioxole moiety of PYP after its coordina-
tion with copper(II). Based on the observation of a greater viscosity
increase induced by complex 1 than PYP ligand, it may be
concluded that the central copper(II) cation may also play an
important role in facilitating the complex-DNA interaction. In
particular, there may exist electrostatic attraction with the poly-
anionic backbone of DNA [43,44]. Nevertheless, the intercalative
2.7.3. Competitive binding studies using fluorescence
In a competitive binding experiment, complex 1 competes with
EB as an intercalative probe to bind ct-DNA [38]. As expected, the
EB-ct-DNA system with [EB]/[DNA] ¼ 1:10 ([EB] ¼ 1 ꢂ 10ꢀ5 M,
[DNA] ¼ 1 ꢂ10ꢀ4 M) shows the characteristic strong emission at ca.
588 nm when excited by 332 nm light, indicating that the inter-
calated EB molecules are sufficiently protected by the neighboring
base pairs in the DNA from the polar solvent molecule quenchers. In
Fig. 9, the characteristic emissions of EB show decreases to different
extents upon the addition of the complex from [compound]/
[EB] ¼ 1:1 to 8:1 (for PYP see Fig. S2, ESI). Listed in Table 2 are the
Table 1
IC50 (mM) values for complexes 1e3 and PYP against six human tumor cell lines.
Complexes
BEL-7404
SK-OV-3
A549
A375
MGC-803
NCI-H460
HL-7702
PYP
1
2
3
27.57 ꢃ 1.13
1.48 ꢃ 0.73
17.45 ꢃ 1.26
3.22 ꢃ 0.15
74.38 ꢃ 5.68
63.26 ꢃ 4.14
64.22 ꢃ 1.21
56.32 ꢃ 1.23
17.67 ꢃ 0.19
30.76 ꢃ 0.36
16.38 ꢃ 0.51
e
54.61 ꢃ 0.53
0.31 ꢃ 0.25
10.24 ꢃ 0.28
1.32 ꢃ 0.12
81.31 ꢃ 2.17
87.54 ꢃ 6.22
17.21 ꢃ 0.58
28.15 ꢃ 0.70
4.29 ꢃ 0.14
15.29 ꢃ 1.24
18.57 ꢃ 0.91
60.26 ꢃ 3.04
69.13 ꢃ 1.12
78.54 ꢃ 2.25
38.26 ꢃ 1.37
3.376 ꢃ 0.14
9.68 ꢃ 0.25
2.25 ꢃ 0.46
72.15 ꢃ 3.17
52.24 ꢃ 2.29
23.58 ꢃ 0.43
88.45 ꢃ 3.34
3.65 ꢃ 0.17
28.43 ꢃ 0.24
8.36 ꢃ 0.45
41.07 ꢃ 1.54
53.19 ꢃ 4.10
48.52 ꢃ 0.32
>100
21.31 ꢃ 0.14
35.22 ꢃ 0.04
54.77 ꢃ 0.07
>100
>100
74.25 ꢃ 0.11
CuCl2$2H2O
Cu(ClO4)2$6H2O
Cisplatina
e
84.21 ꢃ 0.24
a
Cisplatin was used as positive control. “e” represents no activity.

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K.-B. Huang et al. / European Journal of Medicinal Chemistry 70 (2013) 640e648
Fig. 5. Loss of DJm induced by complex 1. Cells were treated with complex 1 for 2 h
and examined by JC-1 under a fluorescence microscope.
binding capacity of the copper(II)/PYP complex is still somewhat
weaker than that of EtBr. A viscosity increase of 24.81% corresponds
to the [EtBr]/[DNA] ratio of only 0.14:1.
Fig. 7. UVevis absorption spectra of complex 1 in the absence (—) and presence (d) of
ct-DNA with increasing [DNA]/[complex 1] ratios in the range from 2:1 to 10:1.
2.7.6. Agarose gel electrophoresis assay
The binding modes of complex 1 to pUC19 plasmid DNA were
further examined by agarose gel electrophoresis assay. As shown in
Fig.12 (for PYP, see Fig. S4, ESI), the migration rate of DNA decreases
significantly in both the major supercoiled form and the minor
relaxed form with increasing concentrations of complex 1 from 10
by pH adjustment to 7.35 by titration with hydrochloric acid using a
Sartorius PB-10 pH meter. The TBE buffer (1ꢂ) and DNA loading
buffer (6ꢂ) were obtained commercially. ct-DNA was purchased
from Sino-American Biotech Co., Ltd. (Beijing). The UV absorbance
ratio 260 nm/280 nm of w1.85:1 indicated that the DNA was
effectively free of protein. The DNA concentration per nucleotide
was determined spectrophotometrically by employing a molar
absorption coefficient of 6600 Mꢀ1 cmꢀ1 at 260 nm. The stock so-
to 50
mM in the absence of any external reagent or light. This
phenomenon is similar to that of EB [43]. The migration rate of DNA
only shows a slight decrease in the case of PYP (Fig. S4, ESI). This
once again confirms a stronger intercalative effect of complex 1 to
DNA than PYP.
lution of pUC19 plasmid DNA (250
Takara Biotech Co., Ltd.
mg/mL) was purchased from
3. Conclusions
Three new copper(II/I) complexes of PYP were synthesized and
fully characterized. These isoquinoline-copper(II/I) complexes
exhibited enhanced cytotoxicity against tested human tumor cell
lines compared to free PYP and its corresponding copper(II) salts,
which suggested synergistic effect between PYP and copper(II/I)
ions. Complex 1 induced the apoptotic death in BEL-7404, which
involved mitochondria in the process. Caspase-3 activation assay
indicated that complex 1 could be an efficient activator of caspase-
3. Based on DNA binding studies using UVevis absorption, DNA-
melting analysis, fluorescence, CD, viscosity measurements and
agarose gel electrophoresis assay, it may be concluded that inter-
calation is the most likely binding mode of complex 1 with DNA.
4.2. Instrumentation
1H NMR and 13C NMR spectra were recorded on a Bruker AV-600
NMR spectrometer with DMSO-d6 as the 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 complexes were collected
on a Bruker HCT Electrospray Ionization Mass Spectrometer.
4.3. Synthesis
4.3.1. Preparation of the ligands
4.3.1.1. The acylation product (ALP). Picolinic acid (0.1 mol) was
treated with thionyl chloride in dry CH2Cl2. The resulting solution
4. Experimental
4.1. Materials and reagents
All chemicals, unless otherwise noted, were purchased from
Sigma and Alfa Aesar. All materials were used as received without
further purification unless noted specifically. To make TriseHCle
NaCl (TBS) buffer, 5 mM tri(hydroxymethyl)aminomethane (Tris)
and 50 mM NaCl, were dissolved in double distilled water followed
Fig. 6. Activation of caspase-3 induced by complex 1, examined by FITC-DEVD-FMK
under a fluorescence microscope.
Fig. 8. Melting curves of DNA in the absence and presence of complex 1 and PYP.

K.-B. Huang et al. / European Journal of Medicinal Chemistry 70 (2013) 640e648
645
Fig. 9. Fluorescence emission spectra of EB bound with ct-DNA in the absence (dashed
Fig. 10. Circular dichroism spectra of ct-DNA bound by complex 1 with [DNA]/[com-
pound] ratios 2:1 (DNA alone of 1 ꢂ 10ꢀ4 M, dashed line; DNA bound by complex 1,
colored solid line).
line ——) and presence (solid lines d) of complex
increasing [complex 1]/[EB] ratios of 1:1 to 8:1.
1 as competitive agent with
was stirred at 70 ꢁC for 12 h. Excess thionyl chloride was removed
under reduced pressure. The resulting acid chloride was dissolved
in dry benzene, and added dropwise to a stirred solution of pho-
mopiperonylamine (0.1 mol) and pyridine (10 mL) in dry benzene.
The mixture was continuously stirred overnight at room temper-
ature and then poured into water (200 mL). The solution was
washed and the organic layer was collected. The crude product
was recrystallized from CH3OH/n-hexane. 1H NMR (600 MHz,
149.23 (s), 147.13 (s), 138.28 (s), 133.01 (s), 130.02 (s), 127.35 (s),
118.83 (s), 111.95 (s), 109.37 (s), 103.39 (s), 41.91 (s), 25.60 (s). ESI-
MS: m/z 253.81 [MPDQ þ H]þ.
4.3.1.3. 5-Pyridin-2-yl-[1,3]dioxolo[4,5-g]isoquinoline (PYP). To
a
refluxing solution of dihydroisoquinoline (MPDQ) (10.0 mmol) in
dry toluene was added a portion of activated MnO2 (0.5 g) every
30e50 min. After the completion of the reaction (2 h) as indicated
by TLC, the hot reaction mixture was filtered, and the filter cake was
washed with CHCl3 (5 ꢂ100 mL). The combined filtrates were dried
(Na2SO4) and the remaining particles of MnO2 removed by filtration
over Celite. Evaporation of the solvent yielded PYP that was
recrystallized upon the addition of CH3OH. 1H NMR (600 MHz,
DMSO-d6)
d
8.80 (t, J ¼ 5.6 Hz, 1H), 8.62 (d, J ¼ 4.4 Hz, 1H), 8.04 (d,
J ¼ 7.7 Hz, 1H), 8.01e7.95 (m, 1H), 7.61e7.55 (m, 1H), 6.82 (s, 1H),
6.81 (d, J ¼ 7.9 Hz, 1H), 6.68 (t, J ¼ 10.1 Hz, 1H), 5.96 (s, 2H), 3.52
(dd, J ¼ 13.9, 6.8 Hz, 2H), 2.81e2.74 (m, 2H). 13C NMR (151 MHz,
DMSO-d6) d164.13 (s), 150.45 (s), 148.82 (s), 147.69 (s), 145.99 (s),
138.21 (s), 133.62 (s), 126.88 (s), 122.26 (s), 121.94 (s), 109.42 (s),
108.58 (s), 101.12 (s), 40.96 (s), 35.25 (s). ESI-MS: m/z 271.45
[ALP þ H]þ.
DMSO-d6)
d
8.75 (d, J ¼ 4.6 Hz, 1H), 8.44 (d, J ¼ 5.4 Hz, 1H), 8.08 (s,
1H), 8.02 (dd, J ¼ 12.1, 5.0 Hz, 2H), 7.75 (d, J ¼ 5.4 Hz, 1H), 7.52 (ddd,
J ¼ 6.7, 4.9, 1.7 Hz, 1H), 7.44 (s, 1H), 6.28e6.04 (m, 2H). 13C NMR
(151 MHz, DMSO-d6)
d 158.35 (s), 154.82 (s), 150.66 (s), 149.01 (s),
4.3.1.2. 4,5-Methylenedioxy-1-pyridinedihydroisoquinoline(MPDQ).
POCl3 (128 mL) was added dropwise to a stirred solution of the
acylation product 1 (200 mmol) in dry CH3CN (800 mL). The
stirring continued for 1 h at room temperature. The resulting
mixture was refluxed for approximate 5 h until all starting ma-
terial was consumed as monitored by TLC. After removal of the
solvent in vacuo, CHCl3 (400 mL) and H2O (400 mL) were added,
and the aqueous phase was adjusted with aqueous 10 N NaOH to
pH 10. The organic layer was washed with saturated aqueous
NaHCO3 (4 ꢂ 100 mL), dried over anhydrous Na2SO4, and
concentrated under reduced pressure. 4,5-methylenedioxy-1-
pyridine-dihydroisoquinoline (MPDQ) precipitated upon the
148.58 (s), 141.48 (s), 137.68 (s), 135.92 (s), 125.37 (s), 123.88 (s),
123.60 (s), 121.31 (s), 103.35 (s), 103.06 (s), 102.47 (s). ESI-MS: m/z
251.48 [PYP þ H]þ.
addition of Et2O. 1H NMR (600 MHz, DMSO-d6)
d 8.85 (d,
J ¼ 4.4 Hz, 1H), 8.14 (td, J ¼ 7.7, 0.9 Hz, 1H), 8.02 (d, J ¼ 7.8 Hz, 1H),
7.77 (dd, J ¼ 7.3, 4.9 Hz, 1H), 7.23 (s, 1H), 7.05 (s, 1H), 6.22 (d,
J ¼ 9.7 Hz, 2H), 3.92 (t, J ¼ 7.8 Hz, 2H), 3.09 (t, J ¼ 7.8 Hz, 2H). 13
C
NMR (151 MHz, DMSO-d6)
d 168.65 (s), 154.31 (s), 150.43 (s),
Table 2
Calculated quenching ratios of EB by titration of complex 1 to EB-ctDNA system.
Compound
aOne-step bFI
Total FI
Kq
Complex 1
PYP
40.05%
2.36%
97.36% at [complex 1]/[EB] ¼ 8:1
19.62%[PYP]/[EB] ¼ 8:1
4.29 ꢂ 105
6.34 ꢂ 102
Fig. 11. Relative viscosity increments of ct-DNA solution bound with complex 1 and
PYP with increasing [compound]/[DNA] ratios in the range from 0.02 to 0.20. EtBr as
intercalative indicator was set for comparison.
a
One-step refers to [compound]/[EB] ¼ 1:1.
FI, fluorescence intensity.
b

646
K.-B. Huang et al. / European Journal of Medicinal Chemistry 70 (2013) 640e648
Table 3
Crystal data and structure refinement details for complexes 1e3.
Formula 30H20Cl4Cu2N4O4 C60H48Cl4Cu4N8O28 C30H20Cl4Cu2N4O4
C
fw
T/K
Crystal system Triclinic
769.38
293(2)
1725.06
293(2)
Triclinic
P-1
11.2659(10)
12.8269(6)
13.4367(14)
69.278(7)
76.648(8)
71.236(6)
1704.5(2)
1
769.40
293(2)
Orthorhombic
Pna21
14.2612(4)
15.3518(4)
12.8356(3)
90.00
90.00
90.00
2810.15(13)
4
1.771
Space group
P-1
ꢀ
a, A
7.4731(4)
9.0689(5)
12.0496(7)
78.178(5)
83.738(5)
66.129(5)
730.62(7)
1
ꢀ
b, A
ꢀ
Fig. 12. Electrophoresis in agarose gel of pUC19 plasmid DNA (0.02 mg/mL, 30
m
M base
c, A
ꢁ
ꢁ
ꢁ
pair) incubated for 2 h at 37 ꢁC with complex 1. “C” represents control; the concen-
tration of copper(II) complex is indicated in the figure.
a,
b
,
g,
3
ꢀ
V, A
Z
Dc, g cmꢀ3
1.749
1.867
1.602
1.479
1.058
14,533/7041
4.3.2. Preparation of the copper(II/I) complexes
, mmꢀ1
1.939
1.037
22,050/5726
Typical procedures for the synthesis of complexes 1e3 are as
follows. For complexes 1 and 2: to a mixture of the corresponding
metal salt, PYP and methanol (1 mL)/CHCl3 (0.5 mL) were placed in
a thick Pyrex tube (w20 cm long) (for complex 3, methanol (1 mL)/
H2O (0.25 mL) was used instead of methanol (1 mL)/CHCl3
(0.5 mL)). The mixture was frozen by liquid N2 and evacuated under
vacuum, then sealed with a torch, warmed to room temperature,
and heated at 110 ꢁC for a few days to give the corresponding block
crystals of complexes 1e3 suitable for X-ray diffraction analysis.
m
GOF on F2
Reflns
1.100
7391/2994
(collected/
unique)
Rint
0.0229
0.0575
0.0369
0.1193
0.4352
0.0644
0.0467
0.0842
R1a (I > 2
s(I))
wR2b (all data) 0.2049
a
R1 ¼ SjjFoj ꢀ jFcjj/SjFoj.
b
½
wR2 ¼ [Sw(Fo2 ꢀ Fc2)2/Sw(Fo2)2]
.
4.3.2.1. Complex 1. CuCl2$2H2O (0.1 mmol), PYP (0.1 mmol),
methanol (1 mL)/CHCl3 (0.5 mL). Anal. Found(%): C, 46.83; H, 2.62;
N, 7.28. Calcd for: C, 46.32; H, 2.56; N, 7.23. ESI-MS: m/z 347.9
[Cu(PYP)Cl]þ.
were carried out following the reported procedure [32]. The
growth inhibitory rate of treated cells was calculated using
the data from three replicate tests as (ODcontrol
ꢀ
ODtest)/
ODcontrol ꢂ 100%. The complexes were incubated with various cell
lines for 48 h at five different concentrations of the complexes
dissolved in fresh media. The range of concentrations used is
dependent on the complexes. The final IC50 values were calculated
by the Bliss method (n ¼ 5). All tests were independently repeated
at least three times.
4.3.2.2. Complex 2. Cu(ClO4)2$6H2O(0.1 mmol), PYP (0.1 mmol),
methanol (1 mL)/CHCl3 (0.5 mL). Anal. Found(%): C, 48.49; H, 2.7; N,
7.54. Calcd for: 48.31; H, 2.81; N, 7.68. ESI-MS: m/z 464.1[Cu(PYP)
ClO4þH2O þ CH3OH þ H]þ
4.3.2.3. Complex 3. CuCl2$2H2O(0.1 mmol), PYP (0.1 mmol),
methanol (1 mL)/H2O (0.25 mL). Anal. Found(%): C, 44.77; H, 2.50;
N, 6.96. Calcd for: 44.51; H, 2.44; N, 6.84. ESI-MS: m/z 347.9
[Cu(PYP)Cl]þ.
4.5. Apoptosis assay by flow cytometry
The ability of copper complex 1 to induce apoptosis was
evaluated in BEL-7404 cell lines using AnnexinV conjugated with
FITC and propidium iodide (PI) counterstaining by flow cytom-
etry. BEL-7404 cells of exponential growth were inoculated in 6-
well plates and cultured for 12 h before the tested compounds
were added to give the indicated final concentrations. After 48 h
incubation, cells were harvested, washed twice in phosphate-
4.3.3. X-ray crystallographic analysis and data collection
The suitable single crystals were mounted on glass fibers, and
crystal data were collected at room temperature on an Agilent
SuperNova CCD diffractometer equipped with graphite mono-
ꢀ
chromated Mo-K
a
radiation (
l
¼ 0.71073 A). Absorption correction
buffered saline (PBS), and resuspended in 100 mL binding buffer
was applied by using the multiscan program SADABS [45]. The
structures were solved with direct methods and refined using
SHELX-97 programs [46]. The non-hydrogen atoms were located in
successive difference Fourier synthesis. The final 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 Table 3.
(140 mmol/L NaCl, 2.5 mmol/L CaCl2 and 10 mmol/L Hepes/NaOH,
pH 7.4) at a concentration of 1 ꢂ 106 cells/mL. Then the cells were
incubated with 5
mL of Annexin V-FITC (in a buffer including
10 mmol/L NaCl, 1% bovine serum albumin, 0.02% NaN3 and
50 mmol/L Tris, pH 7.4) and 10 L PI (20 g/mL) for 15 min at
m
m
room temperature in the dark. Cells were kept shielded from light
before being analyzed by flow cytometry using a BectoneDick-
inson FACSCalibur.
4.6. Determination of caspase-3 activity
4.4. Cell lines, culture conditions and cytotoxicity assay
The activation of caspase-3 was examined using a caspase-3
(DEVD-FMK) conjugated to FITC as the in situ fluorescent marker
in living cells (catalog#QIA91, Merk). The cell-permeable and
nontoxic FITC-DEVD-FMK irreversibly binds to activated caspase-3
in apoptotic cells. After exposure of BEL-7404 cell lines to complex 1
for 12 h, the cultures were washed twice with HBSS containing 0.1%
Human tumor cell lines BEL-7404, SK-OV-3, A549, A375, MGC-
803 and NCI-H460 were obtained from the Shanghai Cell Bank in
the Chinese Academy of Sciences. The 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 humid atmosphere of 95% air/
5% CO2. The cytotoxicity of the title complexes against BEL-7404,
SK-OV-3, A549, A375, MGC-803 and NCI cell lines was examined
by the microculture tetrozolium (MTT) assay. The experiments
BSA and then incubated with FITC-DEVD-FMK (1 ml/ml in EBSS) for
1 h in a 37 ꢁC incubator with 5% CO2. The FITC label in apoptotic
cells was examined immediately under fluorescence microscope
with excitation and emission at 485 and 535 nm, respectively.

K.-B. Huang et al. / European Journal of Medicinal Chemistry 70 (2013) 640e648
647
4.7. Mitochondrial membrane potential measurement
Acknowledgments
Cells incubated with the IC50 concentration of complex 1 for 2 h
in poly-HEMA coated 6-wells were collected and re-suspended in
fresh medium. After the addition of 0.5 mL JC-1 working solution,
the cells were incubated in a 5% CO2 incubator for 40 min at 37 ꢁC.
The staining solution was removed by centrifugation and cells were
washed with JC-1 staining buffer twice. Cells having low membrane
potential were imaged under a fluorescence microscope (20ꢂ).
This work was supported by the National Natural Science
Foundation of China (No. 21271051), IRT1225, National Basic
Research Program of China (No. 2012CB723501), and Natural Sci-
ence Foundation of Guangxi Province (Nos. 2012GXNSFDA385001,
2012GXNSFDA053005) as well as “BAGUI Scholar” program of
Guangxi, China.
Appendix A. Supplementary data
4.8. DNA binding studies
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.10.031.
The 2 ꢂ 10ꢀ3 M ct-DNA stock solution was stored at 4 ꢁC for no
more than 5 days before use. The synthesized copper(II/I) com-
plexes were all prepared as 2 ꢂ 10ꢀ3 M DMSO stock solutions for
DNA binding studies. The final working solutions of the complexes
for DNA binding studies were diluted by TBS and the DMSO content
was less than 10%. A solution containing 2 ꢂ 10ꢀ4 M DNA and
2 ꢂ 10ꢀ5 M EB ([DNA]/[EB] ¼ 10:1) was prepared for EB-ct-DNA
competitive binding studies. For UVevis absorption experiments,
the working solutions of the complexes were kept constant at
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