Synthesis, characterization, DNA binding ability and cytotoxicity of the novel platinum(II), copper(II), cobalt(II) and nickel(II) complexes with 3-(1H-benzo[d]imidazol-2-yl)-β-carboline

Article abstract of DOI:10.1016/j.ica.2014.05.036

3-(1H-benzo[d]imidazol-2-yl)-β-carbolin (Y-5) and its Pt(II), Cu(II), Co(II) and Ni(II) complexes 1-4 were synthesized and characterized by ¹H NMR, MS, IR, elemental analyses and TG. Crystal structure of Y-5 and its Pt(II) complex were reported. The DNA binding and DNA cleavage ability of complexes with Cu(II) ion and Pt(II) ion as the metal center showed enhanced activities than that of the other two, which were evaluated by absorption spectra, EB displacement experiments and agarose gel electrophoresis. Assay on the cytotoxicity of the above complexes against A549 and Hela tumor cells and Helf normal cells revealed that the complexes 1 and 2 are toxic against tumor cells but relatively safe to normal cell.

Full text of DOI:10.1016/j.ica.2014.05.036

Accepted Manuscript
Synthesis, characterization, DNA binding ability and cytotoxicity of the novel
Platinum(II), Copper(II), Cobalt(II) and Nickel(II) complexes with 3-(1H-ben-
zo[d]imidazol-2-yl)-β-carboline
Qiao-Mei Jin, Yi Lu, Jian-Lin Jin, Hao Guo, Guo-Wu Lin, Yue Wang, Tao Lu
PII:
S0020-1693(14)00328-4
http://dx.doi.org/10.1016/j.ica.2014.05.036
ICA 16018
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
21 February 2014
16 May 2014
17 May 2014
Please cite this article as: Q-M. Jin, Y. Lu, J-L. Jin, H. Guo, G-W. Lin, Y. Wang, T. Lu, Synthesis, characterization,
DNA binding ability and cytotoxicity of the novel Platinum(II), Copper(II), Cobalt(II) and Nickel(II) complexes
with 3-(1H-benzo[d]imidazol-2-yl)-β-carboline, Inorganica Chimica Acta (2014), doi: http://dx.doi.org/10.1016/
j.ica.2014.05.036
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Synthesis, characterization, DNA binding ability and cytotoxicity of the
novel Platinum(II), Copper(II), Cobalt(II) and Nickel(II) complexes with
3
-(1H-benzo[d]imidazol-2-yl)-β-carboline
a
a
a
a
a
a,*
b,*
Qiao-Mei Jin , Yi Lu , Jian-Lin Jin , Hao Guo , Guo-Wu Lin , Yue Wang , Tao Lu
a
School of Sciences, China Pharmaceutical University, Nanjing 210009, P.R. China
b
Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Ministry of
Education, Nanjing 210009, P.R. China
*
Corresponding author. Email: zwy_1115@126.com; lut163@163.com
Abstract
3
-(1H-benzo[d]imidazol-2-yl)-β-carbolin (Y-5) and its Pt(II), Cu(II), Co(II) and Ni(II) complexes
1
1
-4 were synthesized and characterized by H NMR, MS, IR, elemental analyses and TG. Crystal
structure of Y-5 and its Pt(II) complex were reported. The DNA binding and DNA cleavage ability
of complexes with Cu(II) ion and Pt(II) ion as the metal center showed enhanced activities than that
of the other two, which were evaluated by absorption spectra, EB displacement experiments and
agarose gel electrophoresis. Assay on the cytotoxicity of the above complexes against A549 and
Hela tumor cells and Helf normal cells revealed that the complexes 1 and 2 are toxic against tumor
cells but relatively safe to normal cell.
Key words: β-Carboline, Metal complexes, Crystal structure, DNA interaction, Cytotoxicity
1. Introduction
Because of the coordination ability with different ligands, transition metals have been regarded
as important scaffolds for construction of compounds with desired functions [1, 2]. Since the
discovery of the anticancer activity of cisplatin and its derivatives, there has been a great deal of
1

interest in developing new metallodrugs [3, 4]. Numerous biological studies have proved that
organometallic complexes exert different modes of anticancer activities depending on their
intracellular targets, which includes nucleic acids and kinases. DNA is the primary intracellular
target of anticancer drugs (including cisplatin and its derivatives) due to the interaction between
small molecules and DNA which causes DNA damage in cancer cells, thus blocking the division of
cancer cells and resulting in cell death [5].
During the construction of transition metal complexes, ligands containing multidentate aromatic
plane have gained much attention. The β-carboline derivatives, containing a planar tricyclic system,
are common to many natural alkaloids, which are originally isolated from seeds of Pegannum
harmala L. [6, 7]. Recent researches demonstrated that β-carboline derivatives show potential
cytotoxic activities against human tumor cell lines [8-11]. Their anticancer activities may be
attributed to their various pharmacological properties, such as intercalating into DNA [12],
inhibiting cyclin-dependent kinases (CDKs) [13, 14], topoisomerase [15] or monoamine oxidase
[
16]. Recently, our research group focused on modifying β-carboline scaffold at 1-, 3-, 6-positions
on the basis of the structure-activity relationship and the combination principle of drug molecular
design. It should be noted that the metal complexes of these compounds with potential biological
activity were seldomly reported.
Based on the above facts, 3-(1H-benzo[d]imidazol-2-yl)-β-carboline (Y-5) and its four
complexes, [PtL] (1), [CuL] (2), [Co(L)
2
] (3), and [Ni(L) ] (4) (where L is Y-5), were designed and
2
synthesized in this paper what is shown in Scheme 1. Single crystal X-ray structure of complex 1
was determined in order to discern the mode of coordination of the β-carboline ligand to the
transition metal ions. We have investigated the interactions of these complexes with calf thymus
DNA using absorption spectroscopy and ethidium bromide (EB) displacement experiments, also the
DNA cleavage study was done by agarose gel electrophoresis. The in vitro cytotoxic activities of
complexes 1-4 were tested against two tumor cell lines (A549 and Hela) and one normal cell line
(
human embryonic lung fibroblasts, Helf) in order to evaluate the selectivity between the tumor
cells and healthy cells.
2

Scheme 1. Structures of four metal complexes.
2. Experimental
2.1. Materials and spectral measurements
All solvents and other reagents were all of chemical grade and used as received. Water for
solution preparation was distilled water. Calf thymus DNA (CT DNA) and pBR322 plasmid DNA
were purchased from Sigma.
Reaction progress was monitored using analytical thin layer chromatography (TLC) on
percolated Merck silica gel Kiesegel 60 F254 plates, and the spots were detected under UV light
(
254 nm). Melting points were determined with a digital melting point apparatus and were reported
uncorrected. NMR spectra were recorded on a Bruker ARX 600 spectrometers. IR spectra were
measured on a Jasco FT/IR-430 spectrophotometer. Mass spectra were recorded on an a Quattro
microMS Micromass UK mass spectrometer, and was recorded on an electrospray ionization mass
spectrometer as the value m/z. Thermal analysis was carried out on a NETZSCH TG 209 analyser
2
under dry N atmosphere. The X-ray measurements were made on a Rigaku RAXIS RAPID
diffractometer with a graphite monochromatised Mo-Kα radiation (λ = 0.71069 Å) using ω scan
mode. All the UV absorption spectra were recorded on a Shimadzu UV-1800 spectrophotometer
after through mixing for 5 min at 25 °C. Fluorescence spectra were performed with a Shimadzu
FR-5301 spectrofluorometer at 25 °C, respectively. The gel was photographed on a capturing
system gel printer plus TDI. Bands were quantified using the Scion Image for Windows software
program based on NHI Image.
2.2. Synthesis
3

2.2.1. Preparation of 3-(1H-benzo[d]imidazol-2-yl)-β-carboline (Y-5)
The ligand 2, 3-(1H-benzo[d]imidazol-2-yl)-β-carboline (Y-5) was prepared with
(
±)-tryptophan as starting material via total five steps. The synthetic methods were outlined in
Scheme 2.
Scheme 2. Synthesis routine of Y-5.
Reagents and conditions: (a) HCHO, NaOH; (b) EtOH, SOCl ; (c) KMnO
0% EtOH, then 4 M HCl; (e) PPA, o-phenylenediamine, 180 °C, 6 hr.
2
4
, DMF, r.t.; (d) NaOH,
5
2.2.1.1. Preparation of 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (Y-1). The preparation of
1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid was achieved by the method of Synder et al [17].A
2
mixture of (±)-tryptophan (102 g, 0.5 mol), NaOH (20 g, 0.5 mol), and H O (200 mL) was stirred
until clear, and then formalin (30%; 50 g, 0.5 mol) was added. The mixture was stirred at room
temperature for 3 hours, refluxed for 3 hours until there were no starting materials left (TLC
control), and then neutralized (pH 5) with glacial acetic acid. The precipitate was collected by
filtration, washed with water (50 mL × 2), and dried in vacuum to give the compound as a pale
white solid Y-1, m.p. 308-310 °C.
2.2.1.2. Preparation of ethyl 1,2,3,4-tetrahydro-β-carboline-3-carboxylate (Y-2). Y-1 (4 g, 4.6
4

mmol) and ethanol (100 mL) were mixed and cooled to 0 °C with ice bath, and thionyl chloride (5
mL) was added gradually for 30 min. Then the resulting mixture was refluxed for 6 hours until there
were no starting materials left (TLC control). After removal of the solvent, the residue was treated
with water (100 mL), adjusted pH to 9 with saturated NaOH solution and extracted with ethyl
acetate (3 × 100 mL). The combined organic layers were washed with brine (1 × 100 mL), dried
with MgSO
4
and removed the solvent under vacuum to provide the crude product, which was
crystallized from ethyl acetate to provide white solid Y-2, m.p. 319-321 °C.
4
2.2.1.3. Preparation of ethyl β-carboline-3-carboxylate (Y-3). KMnO (44.5 g, 100 mmol) was
added gradually for 30 min to the cooled solution of Y-2 (11.5 g, 50 mmol) in DMF (200 mL), and
then the resulting mixture was stirred for 24 hours at room temperature. The suspension was filtered
and the precipitate was washed with DMF (100 mL × 2). The solvent was removed under reduced
pressure to provide a brown residue, which was crystallized from ethyl acetate to get white solid
Y-3, m.p. 230-231 °C.
2
.2.1.4. Preparation of β-carboline-3-carboxylic acid (Y-4). In a 250 ml single-necked,
round-bottomed flask equipped with a magnetic stirrer, crude Y-3 (2 g, 8.3 mmol), sodium
hydroxide solid (1.3 g, 32.5 mmol), EtOH (75 mL) and H O (75 mL) were placed. The stirred
2
mixture was refluxed for 2 hours until there were no starting materials left (TLC control). Ethanol
was removed by evaporation under reduced pressure, and then the remaining solution was
neutralized to pH 5 with acetic acid to afford a solid, which was filtered to provide Y-4 as a pale
yellow solid, m.p. 307-309 °C.
2.2.1.5. Preparation of 3-(1H-benzo[d]imidazol-2-yl)-β-carboline (Y-5). β-Carboline-3-carboxylic
acid (2.0 g) was dissolved in polyphosphoricacid (80 mL) at 180 °C. o-Phenylenediamine (10mL)
was added and the resulting solution stirred at 180 °C for 6 hours until there were no starting
materials left (TLC control). After cooling, the reaction mixture was poured into ice-cold aqueous
ammonia (100 mL). The precipitate was collected by filtration and washed with water (50 mL × 2).
The product was dried and crystallized from mixture solution (acetoacetate : MeOH = 1 : 1) to give
5

3
2
1
9
-(1H-benzo[d]imidazol-2-yl)-β-carboline (Y-5) as a pale yellow solid (6.4 g, yield 45%), m.p.
-1
83-285 °C. IR (KBr, cm ) 3412, 3221, 1622, 1494, 1442, 1406, 1238, 990. MS m/z 285.42 (M +
+
1
) . H NMR (300MHz, DMSO-d
6
) δ (ppm): 12.90 (1H, s, benzimidazole), 11.85 (1H, s, indole),
.12 (1H, s, ArH), 9.00 (1H, s, ArH), 8.43 (2H, d, J = 6.5 Hz, ArH), 7.57-7.67 (4H, m, ArH), 7.33
1
3
(
1H, m, ArH), 7.16-7.20 (1H, m, ArH). C NMR (300MHz, DMSO-d
42.0, 141.8, 135.7, 134.8, 124.6, 124.1, 123.0, 122.9, 122.1, 121.7, 119.8, 119.6, 116.5, 115.4,
(%): C 76.04, H 4.25, N 19.71. Found (%): C 76.00, H 4.20,
6
) δ (ppm): 155.3, 154.3,
1
1
12.6, 111.0. Anal. Calcd for C18
H
12
N
4
N 19.80.
2
.2.2. Preparation of Pt (II) complex of 3-(1H-benzo[d]imidazol-2-yl)-β-carboline (1)
The DMF solution (5 mL) containing cis-[Pt(DMSO) Cl ] (0.2 mmol) was added dropwise to
stirred methanol solution (5 mL) of Y-5 (0.2 mmol) at room temperature. Cis-[Pt(DMSO) Cl ] was
prepared according to the published method [18]. After stirring for 30 min, the resulting mixture
was standing at 25 °C to evaporate the solvent. Yellow platelet crystals of cis-[Pt(L)Cl ·DMF](L =
Y-5) (1) were obtained by a slow evaporation for several weeks. Yield: 67%. H NMR (300MHz,
DMSO-d ) δ (ppm): 14.71 (1H, s, benzimidazole), 12.60 (1H, s, indole), 9.90 (1H, s, ArH), 9.08
1H, s, ArH), 8.78 (1H, d, J = 7.8 Hz, ArH), 8.33 (1H, d, J = 7.8 Hz, ArH), 7.74-7.78 (3H, m, ArH),
2
2
2
2
2
1
6
(
7.44-7.50 (2H, m, ArH), 7.37 (1H, t, ArH).
2.2.3. Preparation of Cu (II) complex of 3-(1H-benzo[d]imidazol-2-yl)-β-carboline (2)
2 2 2
Y-5 and CuCl ·2H O (molar ratio 1 : 1) were mixed in the solution (DMF : H O = 1 : 1) at
room temperature for 4 hours. The suspension was filtered and the precipitate was washed with H
2
O
(
2 2
10mL × 3) and MeOH (10 mL × 3). Blue precipitation of [Cu(L)Cl ·2H O](L = Y-5) (2) was
1
obtained. Yield: 58%. H NMR (300MHz, DMSO-d
6
) δ (ppm): 14.49 (1H, s, benzimidazole), 12.23
(
1H, s, indole), 9.27 (1H, s, ArH), 8.85 (1H, s, ArH), 7.96 (1H, d, J = 7.9 Hz, ArH), 7.12-7.35 (4H,
m, ArH), 6.32-6.78 (3H, m, ArH).
2.2.4. Preparation of Co (II) complex of 3-(1H-benzo[d]imidazol-2-yl)-β-carboline (3)
Y-5 and CoCl ·6H O (molar ratio 2 : 1) were mixed in the solution (THF : H O = 4 : 1) at room
2
2
2
6

2
temperature for 2 hours. The suspension was filtered and the precipitate was washed with H O
(
2 2 2
10mL × 3) and MeOH (10 mL × 3). Yellow precipitation of [Co(L) Cl ·2H O](L = Y-5) (3) was
1
obtained. Yield: 64%. H NMR (300MHz, DMSO-d
6
) δ (ppm): 14.83 (1H, s, benzimidazole), 12.64
(
1H, s, indole), 9.24 (1H, s, ArH), 8.93 (1H, s, ArH), 8.51 (1H, d, J = 7.8 Hz, ArH), 7.65-7.80 (3H,
m, ArH), 7.33-7.48 (3H, m, ArH), 7.19 (1H, t, ArH).
2
.2.5. Preparation of Ni (II) complex of 3-(1H-benzo[d]imidazol-2-yl)-β-carboline (4)
Y-5 and NiCl ·6H O (molar ratio 2 : 1) were mixed in the solution (THF : H O = 4 : 1) at room
temperature for 4 hours. The suspension was filtered and the precipitate was washed with H
2
2
2
2
O
(
2 2 2
10mL × 3) and MeOH (10 mL × 3). Gray precipitation of [Ni(L) Cl ·2H O](L = Y-5) (4) was
1
obtained. Yield: 66%. H NMR (300MHz, DMSO-d
6
) δ (ppm): 14.94 (1H, s, benzimidazole), 12.78
(
1H, s, indole), 10.13 (1H, s, ArH), 9.89 (1H, s, ArH), 9.60-9.72 (2H, m, ArH), 7.08-7.37 (4H, m,
ArH), 6.60-6.84 (3H, m, ArH).
2.3. Biological studies
2.3.1. Absorption spectroscopic studies
The absorption spectra of the interaction of four metal complexes with CT DNA have been
recorded at a constant complex concentration with increasing amount of CT DNA. Solutions of CT
DNA in Tris-HCl buffer (5 mM Tris-HCl/50 mM NaCl buffer, pH 7.4) gave a ratio of UV
absorbance at 260 nm and 280 nm of 1.8-1.9: 1, indicating that the DNA was sufficiently free of
protein [19]. The concentration of CT DNA was determined spectrophotometrically by employing
-
1
-1
an extinction coefficient of 6600 M cm at 260 nm [20].
2.3.2. Ethidium bromide (EB) displacement experiments
Ethidium bromide (EB) emits intense fluorescence light in the presence of DNA, due to strong
intercalation between the adjacent DNA base pairs. The competitive binding of the complexes to
DNA reduces the emission intensity of EB with DNA [21, 22]. In the binding study, the
fluorescence spectra (λex = 520 nm, λem = 590 nm) were recorded at room temperature and
-6
complexes solution with original concentration of 6.9×10 M were added into a volume of 2 ml of
7

-5
the EB-DNA solution (C DNA = 6.9 × 10 M) to get the concentration ratio of C complex/C EB-DNA
varying from 0 to 1/1 respectively. Five minutes later after adding the complexes, the fluorescence
intensity of the mixture was recorded.
2.3.3. Research on the cleavage of pBR 322 by complex 1
Reactions were carried out in a total volume of 18 µL by mixing 1 µL of supercoiled (SC) pBR
22 plasmid DNA (0.25 µg/µL), a buffer solution (containing 50 mM Tris-HCl and 50 mM NaCl,
3
pH 7.4) and complex solution with ascorbate at a 10-fold molar excess relative to the complex to
yield a total volume of 18 µl. The complex with different concentration was dissolved in DMSO.
After mixing, the sample was incubated at 37 °C for 90 min. The reaction was quenched by the
addition of sodium ethylene diamine tetraacetic acid (EDTA-2Na) and 5 µL of loading buffer
(
0.25% bromphenol blue, 50% glycerol). Then the solution was subjected to electrophoresis.
Electrophoresis was achieved at 80 V for 70 min in apt TAE buffer (40 mM Tris acetate/1 mM
EDTA). The gel was photographed on a capturing system gel printer plus TDI. Bands were
quantified using the Scion Image for Windows software based on NHI Image.
2.3.4. Cytotoxicity assay
To determine potential in vitro antitumor activities of the metal complexes, we have
investigated their inhibition effects on human lung adenocarcinoma cell A549 and human cervical
cancer cell Hela by a MTT [3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide]
assay. We also tested cytotoxicity of the complexes on human embryonic lung fibroblasts cell Helf
to confirm the selectivity towards tumor cells.
Cytotoxicity assays in vitro were carried out using 96 microtiter plate cultures according to the
procedures described by Al-Allaf with a slight modification [23]. Three cell lines were grown in
RPMI-1640 (A549) medium and DMEM (Hela and Helf) containing 10% (v/v) fetal calf serum,
100 µg/ml penicillin, and 100 µg/mL streptomycin. Cultures were propagated for 48 hours at 37 °C
in a humified atmosphere containing 5% CO . Cell lines were obtained from Nanjin keyGen
2
Biotech Company. Drug stock solutions were prepared in DMSO. The final concentration of
DMSO in the growth medium was 2% (v/v) or lower, concentration without effect on cell
8

replication. In all of these experiments, three replicate wells were used to determine each point.
3. Results and discussion
3.1. ESI-MS and NMR spectra
The ligand (Y-5) and complexes 1-4 were characterized by electrospray mass spectra (ESI).
+
ESI-MS spectra of Y-5 showed peaks at m/z 285.1 corresponding to the fragments [Y-5 + H] .
2
Complex 1 displayed strong peaks centered at m/z 573 corresponding to the fragments [PtLCl +
+
Na] . Two major peaks were observed for complex 2 at m/z 459 and 384 corresponding to the
+
+
fragments [CuLCl
prominent peaks at m/z 627 which could be assigned to [CoL
27 in the spectrum of the complex 4 were assigned to [NiL
2 2
+ K] and [CuLCl − Cl] , respectively. Complex 3 also displayed two
2
+
2
2
Cl − 2Cl] . The peaks at m/z 661 and
+
+
6
2
Cl
2
− Cl] and [NiL
2
Cl − 2Cl] ,
2
respectively.
1
H NMR spectra of free ligand (Y-5) and complexes 1-4 were recorded in DMSO-d
6
and show
1
the signals of the coordinated ligands. NMR data are reported in the experimental section. In the H
NMR spectrum of Y-5, the multiplets at 7.12-9.12 ppm correspond to the aromatic protons of the
carboline and benzimidazole rings. The NH signal appears at 12.90 ppm corresponds to the
benzimidazole ring of Y-5 and moves downfield in its complexes (14.71 ppm for complex 1, 14.49
ppm for complex 2, 14.83 ppm for complex 3 and 14.94 ppm for complex 4) [24, 25]. This shift is
related to the charge density change in the benzimidazole ring, which supports the formation of
N-M coordination bonds between nitrogen atom and metal atoms.
3.2. Elemental analysis
The molecular composition of four complexes were supported with elemental analysis.
Elemental analysis and physical data are listed in Table 1.
9

Table 1. Elemental analysis and physical data for complexes 1-4.
Complex
No.
Found (calcd.) (%)
Empirical formula
Color
C
H
N
1
2
3
4
Pt(C18
Cu(C18
Co(C18
H
H
H
H
12
N
4
)Cl
2
·DMF
)Cl ·2H
Cl ·2H
Cl ·2H
Yellow
Blue
O Yellow
Gray
40.32 (40.46) 3.15 (3.07) 11.21 (11.23)
47.91 (47.54) 3.85 (3.55) 12.07 (12.32)
58.98 (58.87) 4.15 (3.84) 15.14 (15.26)
59.08 (58.89) 4.16 (3.84) 15.27 (15.26)
12
N
N
4
2
2
O
12
4
)
2
2
2
Ni(C18
12
N
4
)
2
2
2
O
3.3. IR
The IR spectra of the complexes 1-4 were compared with that of the free ligand (Y-5) in the
−1
region 4000-200 cm . The spectrum of the free ligand displayed the characteristic absorption bands
−1
at 3412, 3221, 1623 cm due to ν (N-H, benzimidazole), ν (N-H, carboline), ν (C=N), respectively.
−1
−1
In the metal complexes, sharp bands of ligand at 1623 cm shift to lower frequencies (1619 cm in
−1
−1
−1
complex 1, 1620 cm in complex 2, 1618 cm in complex 3, 1620 cm in complex 4), which can
be attributed to the coordination of benzimidazole nitrogen and carboline nitrogen to Pt(II), Cu(II),
−1
Co(II) and Ni(II) [26, 27]. Besides, two new bands in the region 600-400 cm for each complex
−1
−1
−1
(
597 and 454 cm in complex 1, 586 and 479 cm in complex 2, 560 and 463 cm in complex 3,
−1
and 575 and 456 cm in complex 4) indicated the bond formation between metal and nitrogen
bonds [28, 29].
3.4. Thermogravimetric Analysis (TG)
The thermal properties of the prepared complexes 1-4 were examined by TG. For complex 1,
the TG curve (Fig. 1(a)) shows one step at 150-210 °C corresponding to the loss of one DMF
molecule (calcd 11.73 %, found 11.67 %). The TG curves of complexes 2-4 were shown in Fig. 1(b).
It is found that complexes 2 and 3 exhibit two separate weight loss steps in the range 25-300 °C
corresponding to the loss of one H
.45 % for 3, found 2.51 % for 3) and then to the loss of another H
found 4.03 % for 2 and calcd 2.45 % for 3, found 2.47 % for 3). Complex 4 shows one weight loss
step in the range 25-300 °C corresponding to the loss of two H O molecules (calcd 4.90 %, found
.98 %). The TG results were in agreement with the formula suggested from the analytical data.
2
O molecule (calcd 4.00 % for 2, found 3.98 % for 2 and calcd
2
2
O molecule (calcd 4.00 % for 2,
2
4
1
0

Figure 1. (a) TG curve of complex 1.
Figure 1. (b) TG curves of complexes 2-4.
3.5. X-ray Crystal Analysis
The X-ray measurements were made on a Rigaku RAXIS RAPID diffractometer with a
graphite monochromatised Mo-K radiation (λ = 0.71069 Å) using ω scan mode. A summary of the
α
crystallographic data and structure refinements are given in Table 2. The data were corrected for
Lorentz and polarization effects. The structures were solved by direct methods using the Crystal
Structure software package [30]. The refinement was performed using SHELXL-97 [31]. All H
atoms were located from difference Fourier maps and treated as riding, with C-H distance of 0.93,
phenol N-H distance of 0.86 and Uiso(H) values equal to 1.2 Ueq(C) and Uiso(water H) values equal
11

to 1.5 Ueq(C) (Ueq is the equivalent isotropic displacement parameter for the pivot atom).
2
2 2
2
2
The function of ∑w(F
o
− F
c
) was minimized by using the weight scheme of w = 1/[σ (F
o
) +
2
2
2
(
aP) + bP], where P = (F
o
+ 2F
c
)/3. Final R [=∑(|F
o
| − |F
c
o
|)/∑|F |], Rw [=(∑w(|F
o
| −
2
2 1/2
2
1/2
c o o c
|F |) /∑w|F | ) ] and S (goodness of fit) [=(∑w(|F | − |F |) /(M − N) ), where M = no. of
reflections and N = no. of variables used for the refinement] and anisotropic displacement
coefficients were refined for all non-hydrogen atoms are listed in Table 2.
Table 2. Crystal data and structure refinement for Y-5 and complex 1.
Chemical formula
2C18
H
12
N
4
·C
2
H
5
OH·H
2
O
C
C
18
H
H
12
N
N
4
5
Pt·C
OPt
3 7
H NO
(
C
38
H
32
N
8
O
2
)
21
19
Formula weight
632.72
623.40
Crystal system
Space group
Triclinic
P-1
Monoclinic
P21/n
Unit cell dimensions (Å,°)
a
7.7570(16)
13.583(3)
15.325(3)
81.87(3)
81.89(3)
77.23(3)
8.6960(17)
15.336(3)
15.983(3)
90.00
100.61 (3)
90.00
b
c
α
β
γ
Volume (Å), Z
Crystal size (mm )
1548.8(5), 2
0.3×0.2×0.1
Prism
Yellow
1.357
2095.1(7), 4
0.2×0.1×0.1
Prism
Yellow
1.976
3
Shape
Color
-3
D
Calcd (g cm )
Reflections collected
Unique reflections
Absorption coefficient (mm )
5650
3860
0.088
3805
2790
6.976
-1
F(000)
664
1200
0
.0597
0.0426
2
2
R(F ＞ 2σ (F ))
2
wR(F )
0.1490
1.000
436
0.0974
1.002
271
Goodness-of-fit
No. of variables
3
.5.1. X-ray structure of 3-(1H-benzo[d]imidazol-2-yl)-β-carboline (Y-5)
The molecular structure of Y-5 together with the atom numbering scheme is shown in Fig. 2(a).
Angle between β-carboline ring and benzimidazole ring is 4.6°, which indicates β-carboline and
1
2

benzimidazole rings are approximately coplanar.
Figure 2. (a) ORTEP drawing of crystal structure of Y-5.
3.5.2. X-ray structures of cis-[Pt(L)Cl
2
] (1)
The crystal structure of the complex 1 has also been determined and depicted in Fig. 2(b). A
summary of the crystallographic data and structure refinements are shown in Table 2. Selected bond
distances and angles of complex 1 are listed in Table 3. As shown in X-ray structure, complex 1 is a
four-coordinated Pt(II) mononuclear complex with the central atom chelated by two nitrogen atoms
of a neutral ligand Y-5 and two chloride ions. The sum of the four valence angles around the central
atom (Table 3) is 360.07° and the distance between Pt atom and N(1)-Cl(2)-Cl(1)-N(4) plane is
0.002 Å, indicating that PtN
2
Cl core is nearly planar. In complex 1, the dihedral angle between
2
β-carboline and benzimidazole rings is 3.9°, which is smaller than the angle (4.6°) in free ligand Y-5
due to the chelate formation of metal complex, thus stretching those two planes. After the
coordination with Pt(II) ion, the whole molecule of complex 1 is approximately coplanar.
1
3

Figure 2. (b) ORTEP drawing of crystal structure of complex 1.
Table 3. Selected bond distances (Å) and angles (°) for complex 1.
Bond distances (Å)
Bond angles (°)
N4-Pt-N1
Pt-N4
Pt-N1
Pt-Cl2
Pt-Cl1
2.036(7)
2.019(7)
2.300(2)
2.291(3)
80.6(3)
Cl1-Pt-Cl2
Cl2-Pt-N4
89.33(9)
94.74(18)
95.4(2)
N1-Pt-Cl2
3.6. Study with electronic absorption spectroscopy
The binding modes of complexes to DNA are characterized classically through electronic
absorption titrations. With complexes intercalating to DNA, the π* orbital of the intercalated ligands
on the complexes could match with π orbitals of the base pair and decrease the π-π* transition
energies [32]. Therefore, these interactions can result in obvious hypochromism and red shift as
reported previously [33]. The UV-Vis spectra of the complexes 1-4 consist of one or two bands in
the range of 260-450 nm. Maximum absorption of complexes 1, 2, 3 and 4 occurs in 309 nm, 297
nm, 296 nm and 295 nm respectively. All metal complexes showed obvious red shift compared with
free ligand Y-5 (λmax = 289 nm). With the addition of DNA, the above bands corresponding to four
metal complexes all showed significant hypochromism accompanied with a red shift, indicating the
interaction between complexes and DNA. The UV-Vis titration curve were showed in Fig. 3(a), Fig.
3
(b), Fig. 3(c) and Fig. 3(d).
In order to compare quantitatively the binding strength of four complexes, the intrinsic binding
constants K
of complexes 1, 2, 3 and 4 respectively, with increasing concentration of DNA: [DNA]/[ε
DNA]/[ε - ε ] + 1/K [ε - ε ]; where [DNA] is the concentration of DNA in base pairs, the apparent
absorption coefficients ε , ε , and ε corresponding to Aobsd/[Complex], the extinction coefficient for
the complex in free solution, and the extinction coefficient for the complex in the fully bound form,
respectively [34]. [DNA]/[ε - ε ] vs [DNA] gave a slope and the intercept which are equal to 1/[ε
] and (1/K ) [ε - ε ], respectively; K is the ratio of the slope to the intercept. Therefore, we
calculated K with the value of 5.90 × 10 M , 4.60 × 10 M , 2.06 × 10 M and 1.73 × 10 M
b
of them with CT-DNA were obtained by monitoring the changes in lower energy bands
a
- ε ] =
f
[
b
f
b
b
f
a
f
b
a
f
b
-
ε
f
b
b
f
b
5
-1
5
-1
5
-1
5
-1
b
1
4

corresponding to complexes 1, 2, 3 and 4 respectively (Fig. 3 inset). According to binding constants
, complex 1 and complex 2 showed stronger binding ability than complex 3 and complex 4.
K
b
Figure 3. (a) UV-Vis absorption spectra of complex 1 in the absence and presence of increasing
amounts of CT DNA. Arrows show the changes in absorbance with respect to an increase in the CT
5
-1
DNA concentration. (Inset: Plot of [DNA] vs [DNA]/ [ε
a
- ε
f
], K = 5.90 × 10 M ).
b
Figure 3. (b) UV-Vis absorption spectra of complex 2 in the absence and presence of increasing
amounts of CT DNA. Arrows show the changes in absorbance with respect to an increase in the CT
1
5

5
-1
DNA concentration. (Inset: Plot of [DNA] vs [DNA]/ [ε
a
- ε
f
], K = 4.60 × 10 M ).
b
Figure 3. (c) UV-Vis absorption spectra of complex 3 in the absence and presence of increasing
amounts of CT DNA. Arrows show the changes in absorbance with respect to an increase in the CT
5
-1
DNA concentration. (Inset: Plot of [DNA] vs [DNA]/[ε
a
- ε
f
], K = 2.06 × 10 M ).
b
Figure 3. (d) UV-Vis absorption spectra of complex 4 in the absence and presence of increasing
amounts of CT DNA. Arrows show the changes in absorbance with respect to an increase in the CT
1
6

5
-1
DNA concentration. (Inset: Plot of [DNA] vs [DNA]/[ε
a
- ε
f
], K = 1.73 × 10 M ).
b
3.7. Study with fluorescence spectroscopy
Fluorescent emission of EB bound to DNA in the absence and presence of complex 1 was
shown in Fig. 4. With the concentration of DNA increased, the fluorescence intensity was largely
decreased. Complexes 2-4 displayed similar hypochromism effect as complex 1. Such a
characteristic change is often observed in the typical intercalative DNA interaction.
To compare the binding ability of four complexes to CT DNA, it is apparent from the
quenching plot (shown in Fig. 5) that EB bound DNA was efficiently quenched by complex 1 and
complex 2. Deducing from the slope of the quenching plot with the complex concentration, the
DNA binding propensity can be reflected as the order: complex 2 > complex 1 > complex 4 >
complex 3. This observation suggests that complex 1 and complex 2 have better DNA interaction
compared with complex 3 and complex 4, which is coincident with the results of electronic
absorption titrations.
Figure 4. Fluorescence emission spectra (excited at 520 nm) of the CT DNA-EB system in the
absence and presence of increasing amounts of complex 1. Arrows show the changes in
fluorescence intensity with respect to an increase in the complex concentration.
1
7

Figure 5. The emission intensity of CT DNA- EB system with increasing amounts of four
complexes (complex 1 (●), complex 2 (★), complex 3 (▇), complex 4 (▲)).
3.8. Cleavage ability of pBR 322 by the complex 1 at different concentration
It is well known that the metal complexes cause random cuts to one of the DNA strands. As a
consequence, the supercoiled form opens to form an open circular after one nick and subsequently
to a linear form if two nicks on complementary strands are within a short distance. Finally, the DNA
degraded into small pieces of different size which cannot be detected in our assay. The cleavage
products were subjected to gel electrophoretic separation and the gels analyzed after ethidium
bromide staining [35-37].
Considering the favorable binding ability to CT DNA shown in UV and fluorescence study, we
chose complex 1 as example to carry out the cleavage research with pBR 322. The DNA cleavage
activity of the complex was studied by gel electrophoresis using SC pBR 322 DNA in Tris-HCl
buffer (pH 7.4). Control experiment under the same conditions does not show any apparent
cleavage activity. The conversion of the supercoiled pBR 322 DNA (SC DNA, Form I) to the
nicked DNA (NC DNA, Form II) and linear DNA (Form III) becomes more efficient when
increasing the concentration of the complex 1. With the increasing concentration of the complex 1,
the DNA cleavage propensity is apparently enhanced, as shown in Fig. 6 and Table 4. At the
1
8

-5
concentration of 1.0 × 10 M in lane 4, most supercoiled DNA have degraded to nicked form
completely. At higher concentration, the linear form almost disappeared for it has been cut into
small pieces completely in lane 6. The result showed that complex 1 has a good cleavage activity to
SC DNA.
Figure 6. Agarose gel electro phoresis patterns for the cleavage of pBR 322 plasmid DNA (0.25 µg
-
1
µL ) by complex 1 for 90 min in the presence of 10-fold excess of ascorbate with buffer solution
(
containing 50 mM Tris-HCl and 50 mM NaCl, pH 7.4) at 37 °C. Key: lane 1, DNA control; lane2,
-
7
-6
-5
complex 1 (1 × 10 M); lane 3, complex 1 (1 × 10 M); lane 4, complex 1 (1 × 10 M); lane5,
-4
-3
complex 1 (1 × 10 M); lane 6, complex 1 (1 × 10 M).
Table 4. Cleavage data of SC pBR 322 by complex 1.
Concentration Supercoiled (Form I) Nicked (Form II) Linear
Lane
1
2
3
4
5
6
DNA control
100%
68.83%
65.30%
0.48%
0.02%
0%
0%
0%
0%
0%
0%
0%
0%
-7
-6
-5
-4
-3
1 × 10
1 × 10
1 × 10
1 × 10
1 × 10
31.17%
34.70%
99.52%
99.88%
0%
3.9. Cytotoxicity assay
Cytotoxic activities of complexes 1-4 were further investigated with two tumor cell lines (A549
and Hela) and one normal cell line (Helf). The results were analyzed by means of cell inhibition
curves and expressed as IC50 values. Cisplatin was used as positive drug. The IC50 values of
cisplatin for Hela, A549 and Helf were 3 µM, 7 µM and 9 µM, respectively. The inhibition effect of
1
9

complexes 1-4 on A549 and Hela at different concentrations (50 µM, 100 µM, 150 µM and 200 µM)
were shown in Fig. 7(a) and Fig. 7(b), respectively. The biological assays show that complex 1 and
complex 2 exhibit more significant activities than complex 3 and complex 4 against both tumor cell
lines. For A549, complex 2 (IC50 = 54 µM) exhibits the highest inhibitory ability compared with
complex 1 (IC50 = 152 µM), complex 3 (IC50 > 200 µM) and complex 4 (IC50 > 200 µM). For Hela,
complex 1 (IC50 = 77 µM) and complex 2 (IC50 = 92 µM) display similar antiproliferative activity.
Complex 1 and 2 have better antitumor activity which may be attributed to their higher DNA
binding ability compared with complex 3 and 4. Reviewing the structure of four metal complexes,
compound 1 and 2 are four-coordinate complexes with the central atom (Pt(II) and Cu(II)) chelated
by a neutral ligand Y-5 and two chloride ions. Hence, the whole molecule of complex 1 and 2 are
approximately coplanar. However, the Co(II) center of complex 3 and Ni(II) center of complex 4
are both located in a distorted octahedral coordination environment. As a result, complex 1 and 2
can intercalate into DNA more effectively than complex 3 and 4 which leads to the better
antiproliferative activity. Although the antitumor effect of four complexes are not superior to that of
cisplatin, we could focus on optimizing the structure of ligand due to its promising pharmacological
properties to improve cell inhibition activity. The investigation is under going by our group. Besides,
the inhibition effect of complexes 1 and 2 on Helf at 200 µM were shown in Fig. 8. For Helf,
complex 1 (IC50 > 200 µM) and complex 2 (IC50 > 200 µM) both display low antiproliferative
activity. The results of cytotoxicity assay imply that complex 1 and complex 2 can be used to arrest
the proliferation of tumor cells with relatively low toxicity to normal cells.
2
0

Figure 7. (a) Cytotoxic activity against A549 of complexes 1-4 under four concentration (50 µM,
00 µM, 150 µM, 200 µM).
1
Figure 7. (b) Cytotoxic activity against Hela of complexes 1-4 under four concentration (50 µM,
00 µM, 150 µM, 200 µM).
1
2
1

Figure 8. Cytotoxic activity of complex 1 and complex 2 against tumor cells (A549 and Hela) and
normal cells (Helf).
4. Conclusion
We described
a
convenient
and
efficient route for the synthesis of
3
-(1H-benzo[d]imidazol-2-yl)-β-carboline (Y-5) in moderate to good yields which was confirmed
1
13
by IR, H NMR, C NMR, mass spectrometry and elemental analysis together with the crystal
structure. Its platinum (II), copper (II), nickel (II) and cobalt (II) complexes were also prepared and
1
characterized with IR, ESI-MS, H NMR, TG and elemental analysis. Crystal structure of complex
1
was reported in this paper. Complex 1 and complex 2 showed stronger DNA binding and DNA
cleavage ability and better antitumor activity than complex 3 and complex 4. These properties,
together with relatively safe to the normal cells make this type of metal complexes exploitable in
search for potential chemotherapeutic agents, for example, for cancers.
Acknowledgements
We are grateful to the National Natural Science Foundation of China (No. 81172933) for
financial support. This study was also supported by the Project Program of State Key Laboratory of
Natural Medicines, China Pharmaceutical University (Program No. JKGP201105).
2
2

Supplementary material
CCDC 966491 and 966492 contains the supplementary crystallographic data of Y-5 and
complex 1. These data can be obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
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2
4

2
5

β-Carbolin and its Pt(II), Cu(II), Co(II) and Ni(II) complexes were synthesized, DNA binding
ability and cytotoxicity of four complexes has been investigated.
2
6

Research highlights
1. 3-(1H-benzo[d]imidazol-2-yl)-β-carbolin and its metal complexes were synthesized.
2. DNA binding and DNA cleavage ability of four metal complexes were tested.
3. Cytotoxicity of four complexes against A549, HeLa and Helf cells were tested.
4. Pt(II) complex and Cu(II) complex displayed antitumor activity with low toxicity.
2
7