Synthesis, X-ray diffraction study, and cytotoxicity of a cationic p-cymene ruthenium chloro complex containing a chelating semicarbazone ligand

Article abstract of DOI:10.1002/zaac.201300142

An organometallic salt composed of a new cationic p-cymene ruthenium chloro complex containing a chelating benzaldehyde semicarbazone ligand and of the known anionic p-cymene ruthenium trichloro complex, [(η⁶-p- cymene)Ru(bzsc)Cl]⁺[(η⁶-p-cymene)RuCl ₃]^- (1) (bzsc = benzaldehyde semicarbazone) was synthesized and further characterized by IR, ¹H NMR, and UV/Vis spectroscopy HR-ESI mass spectrometry, and elemental analysis. The single-crystal structure of 1 was also determined. The in vitro anticancer activities of the complex was evaluated against three human cancer cell lines (SGC-7901, BEL-7404 and CNE-1), and the IC₅₀ values were 20.7, 71.1 and 42.6 μM, respectively. Copyright

Full text of DOI:10.1002/zaac.201300142

ARTICLE
DOI: 10.1002/zaac.201300142
Synthesis, X-ray Diffraction Study, and Cytotoxicity of a Cationic p-Cymene
Ruthenium Chloro Complex Containing a Chelating Semicarbazone Ligand
Quan Zhou,[a,b] Peiyuan Li,*[a] Rumei Lu,[a] Quanquan Qian,[c] Xiaolin Lei,[c]
Qi Xiao,[b,c] Shan Huang,[b,c] Lifeng Liu,[c] Chusheng Huang,[b] and Wei Su*[b,c]
Keywords: Semicarbazones; Ruthenium; Cytotoxicity; Antitumor agents
Abstract. An organometallic salt composed of a new cationic troscopy HR-ESI mass spectrometry, and elemental analysis. The sin-
p-cymene ruthenium chloro complex containing
a
chelating
gle-crystal structure of 1 was also determined. The in vitro anticancer
benzaldehyde semicarbazone ligand and of the known anionic p-cy- activities of the complex was evaluated against three human cancer
mene ruthenium trichloro complex, [(η6-p-cymene)Ru(bzsc)Cl]+[(η6-
cell lines (SGC-7901, BEL-7404 and CNE-1), and the IC50 values
were 20.7, 71.1 and 42.6 μM, respectively.
p-cymene)RuCl3]– (1) (bzsc = benzaldehyde semicarbazone) was syn-
1
thesized and further characterized by IR, H NMR, and UV/Vis spec-
arene complexes which also contain semicarbazone ligand.has
rarely been reported.
Introduction
Half-sandwich ruthenium(II) arene complexes have been
widely developed in the past few years, one of the driving
forces being their promising biological properties.[1–3] Note-
worthy, one of the most interesting representatives, the com-
plex [(η6-C6H5C6H5)RuCl(en)]PF6, developed by Sadler et al.,
shows high anti-cancer activity including activity against
cisplatin-resistant cell lines.[4] At the same time, complex
(η6-p-cymene)RuCl2(1,3,5-triaza-7-phosphaadamantane), the
so-called RAPTA compound from the Dyson group, has low
in vitro cytotoxicity but shows in vivo antimetastic behavior.[5]
Semicarbazones and its derivatives are of considerable phar-
macological interest for their wide range of biological activi-
ties, i.e. antitumor, antibacterial, and antiviral properties.[6] In
addition, as a class of N,O chelating ligands for coordination
to metals, the complexes with semicarbazone ligands have at-
tracted attention for their chemotherapeutic activity.[7] Re-
cently, the biological evaluation of copper and nickel semi-
carbazone complexes has been investigated widely.[8–10] How-
ever, the investigation of the biological activities of the Ru-
Herein, we report on the synthesis, X-ray structure, and in
vitro antitumor activity of an unusual organometallic salt com-
posed of a new cationic p-cymene ruthenium chloro complex
containing a chelating benzaldehyde semicarbazone ligand and
of the known anionic p-cymene ruthenium trichloro complex,
[(η6-p-cymene)Ru(bzsc)Cl]+[(η6-p-cymene)RuCl3]– (1) (bzsc
= benzaldehyde semicarbazone).
Results and Discussion
Synthesis and Characterization of Complexes
As shown in Scheme 1, compound 1 was synthesized by
reaction of [Ru(η6-p-cymene)Cl2]2 and benzaldehyde semi-
carbazone in ethanol.
* Dr. P. Li
Fax: +86-771-227-9416
E-Mail: lipearpear@yahoo.cn
* Prof. Dr. W. Su
Scheme 1. Reaction scheme for the preparation of compound 1.
Fax: +86-771-390-8308
E-Mail: aaasuwei@yahoo.com.cn
[a] College of Pharmacy
Guangxi University of Chinese Medicine
Nanning 530001, P. R. China
[b] Key Laboratory of Beibu Gulf Environment Change and Resources
Utilization
The 1H NMR spectrum of 1 contains all the expected signals
for (p-cymene)RuII fragment and semicarbazone ligand, in ac-
cordance with the two ionic forms in solution. The electrospray
ionization (ESI) mass analysis spectrum of 1 in methanol dis-
plays the peaks corresponding to [(p-cymene)Ru(bzsc)]+ and
[(p-cymene)RuCl3]–. The compound was further characterized
by IR, UV/Vis spectrometry, and elemental analysis.
Ministry of Education
Guangxi Teachers Education University
Nanning 530001, P. R. China
[c] College of Chemistry and Life Science
Guangxi Teachers Education University
Nanning 530001, P. R. China
Z. Anorg. Allg. Chem. 2013, 639, (6), 943–946
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
943

P. Li, W. Su et al.
Table 2. Selected bond lengths /Å and angles /° for 1.
ARTICLE
Structural Data
Ru1–centroid
Ru2–centroid
Ru1–O1
Ru1–N1
Ru1–Cl1
Ru2–Cl2
Ru2–Cl3
Ru2–Cl4
O1–Ru1–Cl1
O1–Ru1–N1
1.665
1.668
2.108(4)
2.113(4)
The single-crystal X-ray diffraction experiment yielded the
molecular structure of 1 show that the cation, [(η6-p-cymene)-
Ru(bzsc)Cl]+, and the anion, [(η6-p-cymene)RuCl3]–, approach
each other pairwise as depicted in Figure 1. Crystallographic
data and details of refinement are reported in Table 1, selected
bond lengths and angles are presented in Table 2. The com-
pound crystallized in the monoclinic crystal system and the
two organometallic counterions adopt the archetypical piano-
2.4011(15)
2.4169(15)
2.4429(15)
2.4334(15)
85.74(12)
76.76(15)
89.15(12)
87.534(55)
86.267(53)
85.930(59)
stool shape of ruthenium half-sandwich arene complexes. Cl1–Ru1–N1
Compared with the analogue [(η6-cymene)Ru(thiosemicarba-
Cl2–Ru2–Cl3
Cl2–Ru2–Cl4
zone)Cl]+,[11] the length of Ru1–O1 is shorter (2.108 Å for
Cl4–Ru2–Cl3
Ru1–O1, compared to 2.351 Å for Ru1–S1) and the chelate
angle is smaller (76.76° for O1–Ru1–N1, compared to 82.5°
for S1–Ru1–N1), which may be ascribed to the low steric hin-
drance of the oxygen atom.
C4 and Cl1 (2.694 Å). T-stacking (edge-to-face) π interaction
between C15–H protons of p-cymene and the benzene ring
[R = C(3)/C(4)/C(5)/C(6)/C(7)/C(8)] (distance 3.125 Å) was
identified (Figure 1) with regard to the non-bonded interac-
tions between the aromatic rings.[12,13] In the [(η6-p-cymene)-
RuCl3]– counterion, the Ru–Cl distances are 2.417, 2.443, and
2.433 Å, and the Cl–Ru–Cl angles are 87.53, 86.27, and
85.93°, respectively, harmonious with those of analogous rho-
dium and iridium complexes.[14] A prominent structural feature
is the presence of an intramolecular hydrogen bond between
the methyl proton of η6-p-cymene and Cl3 with an intramolec-
ular Cl3···H19a distance of 2.735 Å.
Figure 2 shows the packing of compound 1. In the asymmet-
ric unit of 1, a crystallization water molecule is located in the
vicinity of the anion thus establishing a weak hydrogen bond,
C19–H···Ow (2.968 Å).
Figure 1. X-ray structure of 1. Thermal ellipsoids are at the 50% prob-
ability level.
Table 1. Crystal data and structure refinement for 1·H2O.
Formula
C30H43Cl4N3ORu2·H2O
Mr
823.65
Crystal system
Space group
a /Å
b /Å
c /Å
monoclinic
P21/c
7.3596(3)
37.5252(14)
12.0264(5)
98.959(4)
3280.8(2)
4
1.607
0.30ϫ0.15ϫ0.10
1600
β /°
V /Å3
Z
Dcalcd /Mg·m–3
Crystal size /mm3
F(000)
Figure 2. Packing arrangement and hydrogen bonding for 1.
μ /mm–1
1.276
Rint
0.0595
2θ range /°
Reflns collected
Reflns independent
GOF (S)
R1 / wR2 [I Ն 2σ (I)]
R1 / wR2 [all data]
5.54 to 52.74°
14492
6676
1.014
0.0572 / 0.0765
0.1007 / 0.0910
Biological Activity
To analyze the potential of compound 1 as an antitumor
agent, its cytotoxicity was evaluated (Table 3) toward the
SGC-7901 human gastric carcinoma, BEL-7404 liver carci-
noma, and CNE-1 nasopharyngeal carcinoma cell lines, and
The hydrogen atoms in semicarbazone ligand of the for comparison purposes, the cytotoxicity of cisplatin was also
cation are hydrogen bonded to the Cl3 component of the anion: evaluated under the same experimental conditions. The IC50
N2–H2a···Cl2 (2.637 Å); C2–H2b···Cl3 (2.619 Å), and values for the compound are in the micromolar range with
N3–H3a···Cl4 (2.682 Å). In the cation, a feature of the struc- 20.7, 71.1, and 42.6 μM against the three cell lines, respec-
ture is an intramolecular hydrogen bond between the proton of tively. Moreover, under the experimental conditions 1 shows
944
www.zaac.wiley-vch.de
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2013, 943–946

p-Cymene Ru Chloro Complex with Chelating Semicarbazone Ligand
comparable activity to cisplatin (42.6 μM to 30.1 μM) against benzaldehyde semicarbazone ligand and of the known anionic
BEL-7404, indicating the good cytotoxicity of 1. Compared to p-cymene ruthenium trichloro complex, [(η6-p-cymene)-
the Ru-arene analogue, [(η6-cymene)Ru(thiosemicarb- Ru(bzsc)Cl]+[(η6-p-cymene)RuCl3]– (1) was prepared. The
azone)Cl]+,[11,15] the cytotoxicity of 1 is not high, indicating molecular structure of 1 was characterized by X-ray crystal-
that the anion moiety of 1, [(η6-p-cymene)RuCl3]–, is probably lography. In addition, the compound was screened for in vitro
not contributed to the cytotoxic behavior. In general, however, anticancer activity against the SGC-7901 gastric carcinoma,
the complexes do not reach the activity of the standard against BEL-7404 liver carcinoma, and CNE-1 nasopharyngeal carci-
the cancer cell lines. This is in itself not an unusual issue. noma cells, with the IC50 values of 20.7, 71.1, and 42.6 μM,
Some Ru-arene complexes have lower in vitro toxicity but ex- respectively.
hibit higher in vivo anticancer characteristics, i.e. complexes
of the type [(η6-cymene)Ru(PTA)Cl]+ exhibited lower activity
against cancer cells but showed higher anti-metastatic activity
Experimental Section
in vivo.[15,16]
Materials: The benzaldehyde semicarbazone ligand and [(η6-p-cy-
mene)RuCl2]2 were prepared according to literature methods.[4,21] All
chemicals were purchased from commercial sources and used as re-
Table 3. IC50 (μM) values of the compound 1 and cisplatin in three
tumor cell lines.
1
ceived. H NMR spectra were recorded with a Bruker AV-300 spec-
SGC-7901
BEL-7404
CNE-1
trometer at working frequencies 300 MHz. Mass spectra for the com-
plexes were recorded with a Waters UPLC XEVO G2 TOF mass spec-
trometer using electrospray ionization probe. Elemental analyses were
carried out with an Elementar Vario EL Cube. The UV/Vis absorption
spectra were measured with an Agilent 8453 spectrophotometer.
1
20.7Ϯ2.3
6.7Ϯ0.4
71.1Ϯ6.2
23.2Ϯ2.6
42.6Ϯ3.5
30.1Ϯ2.9
Cisplatin
DNA Unwinding
As plasmid DNA is generally a potential target for Ru-arene
Synthesis of [(η6-p-cymene)Ru(bzsc)Cl]+[(η6-p-cymene)RuCl3]–
drugs,[17,18] the interaction between compound 1 and plasmid (1): [Ru(η6-p-cymene)Cl2]2 (0.05 mmol, 31.5 mg) and benzaldehyde
semicarbazone (0.10 mmol, 16.4 mg) were dissolved in ethanol
(4 mL). The reaction mixture was stirred for 6 h at room temperature.
Afterwards, the solution was reduced to 2 mL and 6 mL n-hexane was
slowly layed on, which afforded clusters of red products. The red prod-
uct was filter out, washed with hexane (2ϫ40 mL) and dried under
DNA was investigated by agarose gel electrophoresis. The un-
winding of the supercoiled plasmid pBR322 DNA induced on
binding compound 1 was determined by incubating the plas-
mid pBR322 DNA with compound 1 in the concentration
range of 25–150 μM for 30 min at 37 °C. As shown in Fig-
ure 3, SC DNA (form I) and NC DNA (form II) were cleaved
with all DNA migrating in the form III band when the concen-
tration of compound 1 was higher. The cleavage process of the
pBR322 plasmid DNA seems to start when the concentration
1
vacuum. Yield 25 mg, 43%. H NMR (300 MHz CDCl3): δ = 12.09
(br., 1 H, NH), 8.77 (s, 1 H, CH=N), 8.03 (t, J = 7.8 Hz, 2 H, –phenyl),
3
7.53 (m, J = 6.9 Hz, 3 H, –phenyl), 5.52, 5.30{2ϫd, JAB = 5.7 Hz,
4 H, [(p-cymene)RuCl3]– AA’BB’ spin system}, 5.53, 4.96, 4.69, 4.59
3
{4ϫd, JAB = 5.7 Hz, 4 H, [(p-cymene)Ru(bzsc)Cl]+ AA’BB’ spin
of the compound was higher than 50 μM (line 3). The SC system}, 2.80, 2.69 (2ϫm, J = 6.9 Hz, 2 H, 2ϫ–CH(CH3)2), 2.31,
2.21 (2ϫs, 6 H, 2ϫ–CH3), 1.37 (d, J = 6.9 Hz, 6 H, anion-
CH(CH3)2), 1.22 (dd, J = 6.9 Hz and 17.2 Hz, 6 H, cation-CH(CH3)2)
ppm. HR-ESI-MS (MeOH, 6 eV, 120 °C) (+) m/z (%) = 398.0812
(100) {[(η6-p-cymene)Ru(bzsc)Cl] – HCl}+. HR-ESI-MS (MeOH,
6 eV, 120 °C) (–) m/z (%) = 342.9189 (12) [(η6-p-cymene)RuCl3]–. IR
(KBr): ν˜ = (NH2, NH) 3424, 3211, ν(–CH) 2958, 2868, v(Phenyl,
=CH) 3051, ν(–C=N–) 1646 cm–1. UV/Vis (methanol): λmax (lg ε) =
328 nm (4.477), 280 nm (4.602). C28H37Cl4N3ORu2: calcd. C 43.36;
H 4.81; N 5.42%; found: C 43.46; H 4.87; N 5.53%. Single crystals
suitable for X-ray diffraction were obtained by recrystallization in
ethanol/hexane (30/70) solution.
DNA and NC DNA were all converted into form III com-
pletely when 150 μM compound 1 was present, which indi-
cated that some conformational changes of pBR322 plasmid
DNA occurred and the degree of superhelicity of the DNA
molecules has also been altered.[19,20]
X-ray Crystallography: Crystal data and details of data collection
and structure refinement are given in Table 1. The crystallographical
data of 1 were determined by X-ray diffraction analysis at 100 K with
an Oxford Diffraction Gemini E system with Cu-Kα radiation (λ =
1.5418 Å). The structures were solved by the direct method (SHELXS-
97) and refined by full-matrix least-squares (SHELXL-97) on F2.[22]
Anisotropic thermal parameters were used for the non-hydrogen atoms
and isotropic parameters for the hydrogen atoms. Hydrogen atoms
were added geometrically and refined using a riding model.
Figure 3. Electrophoretic mobility pattern of pBR322 plasmid DNA
incubated with the compound 1: lane 1, DNA control; lane 2, DNA +
1 (25 μM); lane 3, DNA + 1 (50 μM); lane 4, DNA + 1 (75 μM); lane
5, DNA + 1 (100 μM); lane 6, DNA + 1 (125 μM); lane 7, DNA + 1
(150 μM).
Crystallographic data (excluding structure factors) for the structure in
this paper have been deposited with the Cambridge Crystallographic
Data Centre, CCDC, 12 Union Road, Cambridge CB21EZ, UK.
Conclusions
An organometallic salt composed of a new cationic
p-cymene ruthenium chloro complex containing a chelating Copies of the data can be obtained free of charge on quoting the de-
Z. Anorg. Allg. Chem. 2013, 943–946
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de 945

P. Li, W. Su et al.
ARTICLE
pository number CCDC-860118 (Fax: +44-1223-336-033; E-Mail:
deposit@ccdc.cam.ac.uk, http://www.ccdc.cam.ac.uk).
References
[1] Y. K. Yan, M. Melchart, A. Habtemariam, P. J. Sadler, Chem.
Commun. 2005, 4764–4776.
[2] B. Therrien, Coord. Chem. Rev. 2009, 253, 493–519.
[3] G. Süss-Fink, Dalton Trans. 2010, 39, 1673–1688.
[4] R. E. Morris, R. E. Aird, P. del S. Murdoch, H. Chen, J. Cum-
mings, N. D. Hughes, S. Parsons, A. Parkin, G. Boyd, D. I. Jod-
rell, P. J. Sadler, J. Med. Chem. 2001, 44, 3616–3621.
[5] C. Scolaro, A. Bergamo, L. Brescacin, R. Delfino, M. Cocchietto,
G. Laurenczy, T. J. Geldbach, G. Sava, P. J. Dyson, J. Med. Chem.
2005, 48, 4161–4171.
[6] G. Aguirre, E. Cabrera, H. Cerecetto, R. Di Maio, M. González,
G. Seoane, A. Duffaut, A. Denicola, M. J. Gil, V. Martínez-
Merino, Eur. J. Med. Chem. 2004, 39, 421–431.
[7] M. Pagano, B. Demoro, J. Toloza, L. Boiani, M. González, H.
Cerecetto, C. Olea-Azar, E. Norambuena, D. Gambino, L. Otero,
Eur. J. Med. Chem. 2009, 44, 4937–4943.
Cell Culture and Assay for Cell Viability: SGC-7901 (gastric carci-
noma), BEL-7404 (liver carcinoma), and CNE-1 (nasopharyngeal car-
cinoma) cells were obtained from commercial. Cells were grown in
RPMI-1640 supplemented with 10% cosmic calf serum (Hyclone) and
antibiotics in a humidified atmosphere of 5% CO2 at 37 °C. The via-
bility of these cells was determined using the colorimetric Cell Titer 96
aqueous Cell Proliferation Assay (MTT) according to the instructions
provided by the manufacturer (Promega, Madison, WI). Briefly, cells
(ca. 1–3ϫ104 cells per well) were seeded in 96 wells plates. One day
after seeding, the cells were treated with or without different concen-
tration of each compound and reincubated for 72 h. After the cells
were washed with sterile phosphate buffer saline (PBS), 190 μL of
RPMI-1640 and 10 μL of the tetrazolium dye (MTT) (5 mg·mL–1)
solution were added to each well, and the cells were incubated for an
additional 4 h. The medium was discarded; 200 μL of DMSO was
added to dissolve the purple formazan crystals formed. The absorbance
(A) at 492 nm was measured using a Biocell ELISA analysis spectrom-
eter.
[8] W. Y. Lee, P. P. F. Lee, Y. K. Yan, M. Lau, Metallomics 2010, 2,
694–705.
[9] D. S. Raja, N. S. P. Bhuvanesh, K. Natarajan, Inorg. Chem. 2011,
50, 12852–12866.
[10] E. Ramachandran, D. S. Raja, J. L. Mike, T. R. Wagner, M. Zeller,
K. Natarajan, RSC Adv. 2012, 2, 8515–8525.
[11] W. Su, Q. Zhou, Y. Huang, Q. Huang, L. Huo, Q. Xiao, S. Huang,
C. Huang, R. Chen, Q. Qian, L. Liu, P. Li, Appl. Organomet.
Chem. 2013, 27, 307–312.
[12] M. O. Sinnokrot, C. D. Sherrill, J. Am. Chem. Soc. 2004, 126,
7690–7697.
DNA Cleavage Studies Using Agarose Gel Electrophoresis: In the
gel electrophoresis experiments, supercoiled plasmid pBR322 DNA
was treated with compound, and the mixture was incubated for 30 min
at 37 °C. The samples were analyzed by 1.5% agarose gel electropho-
resis in Tris/acetic acid/ethylenediamine tetraacetic acid (TAE, 1.0ϫ,
pH 8.3) buffer. The gel was stained with 0.5 μg·mL–1 ethidium brom-
ide before migration. After electrophoresis at 50 V for 3 h, the gel was
illuminated and the digital images were analysized by gel documenta-
tion system (Junyi-Dongfang Co., JY02G).
[13] S. Catak, M. D’hooghe, N. De Kimpe, M. Waroquier, V.
Van Speybroeck, J. Org. Chem. 2010, 75, 885–896.
[14] C. Pettinari, R. Pettinari, F. Marchetti, A. Macchioni, D. Zuccac-
cia, B. W. Skelton, A. H. White, Inorg. Chem. 2007, 46, 896–906.
[15] F. Beckford, D. Dourth, M. Shaloski Jr., J. Didion, J. Thessing, J.
Woods, V. Crowell, N. Gerasimchuk, A. Gonzalez-Sarrías, N. P.
Seeram, J. Inorg. Biochem. 2011, 105, 1019–1029.
[16] M. A. Jakupec, M. Galanski, V. B. Arion, C. G. Hartinger, B. K.
Keppler, Dalton Trans. 2008, 183–194.
[17] O. Novakova, J. Kasparkova, V. Bursova, C. Hofr, M. Vojtiskova,
H. Chen, P. J. Sadler, V. Brabec, Chem. Biol. 2005, 12, 121–129.
[18] P. Govender, A. K. Renfrew, C. M. Clavel, P. J. Dyson, B. Ther-
rien, G. S. Smith, Dalton Trans. 2011, 40, 1158–1167.
[19] J. Ruiz, V. Rodríguez, N. Cutillas, K. G. Samper, M. Capdevila,
Ò. Palacios, A. Espinosa, Dalton Trans. 2012, 41, 12847–12856.
[20] L. Liu, P. Li, Q. Qian, X. Lei, Y. Huang, Q. Zhou, S. Huang, Q.
Xiao, W. Su, Chin. J. Org. Chem. 2013, 33, 854–859.
[21] F. Basuli, S.-M. Peng, S. Bhattacharya, Inorg. Chem. 2001, 40,
1126–1133.
Acknowledgements
This research was supported by the National Natural Science Founda-
tion of China (20961001, 21261005, 21203035, 51263002), Key Pro-
ject of Chinese Ministry of Education (2010168), Guangxi Natural Sci-
ence Foundation (2010GXNSFB013014, 2012GXNSFAA053194),
Scientific Research Fund of Guangxi Provincial Education Department
(201106LX273), Scientific Research Fund of Guangxi University of
Traditional Medicine (2012024) and Key Laboratory of Beibu Gulf
Environment Change and Resources Utilization (Guangxi Teachers
Education University), Chinese Ministry of Education.
[22] G. M. Sheldrick, SHELX97, Program System for Crystal Structure
Determination; University of Göttingen, Göttingen, Germany,
1997.
Received: March 7, 2013
Published Online: May 6, 2013
946
www.zaac.wiley-vch.de
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2013, 943–946