Synthesis, characterization, and anticancer activities of lipophilic pyridinecarboxaldimine platinum(II) complexes

Article abstract of DOI:10.1016/j.poly.2015.07.039

We have prepared eleven pyridinecarboxaldimines (N-N'R) from the condensation of 2-pyridinecarboxaldehyde and the corresponding lipophilic primary amines. Addition of these ligands to [PtCl₂(η²-coe)]₂ (coe = cis-cycloocten

Full text of DOI:10.1016/j.poly.2015.07.039

Accepted Manuscript
Synthesis, Characterization, and Anticancer Activities of Lipophilic Pyridine-
carboxaldimine Platinum(II) Complexes
Brittany A. Miles, Alyssa E. Patterson, Christopher M. Vogels, Andreas Decken,
Jeffrey C. Waller, Pier Jr. Morin, Stephen A. Westcott
PII:
S0277-5387(15)00394-0
DOI:
http://dx.doi.org/10.1016/j.poly.2015.07.039
POLY 11425
Reference:
Polyhedron
To appear in:
Received Date:
Accepted Date:
28 May 2015
15 July 2015
Please cite this article as: B.A. Miles, A.E. Patterson, C.M. Vogels, A. Decken, J.C. Waller, P.J. Morin, S.A.
Westcott, Synthesis, Characterization, and Anticancer Activities of Lipophilic Pyridinecarboxaldimine Platinum(II)
Complexes, Polyhedron (2015), doi: http://dx.doi.org/10.1016/j.poly.2015.07.039
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Synthesis, Characterization, and Anticancer Activities of Lipophilic
Pyridinecarboxaldimine Platinum(II) Complexes
Brittany A. Miles,a Alyssa E. Patterson,a Christopher M. Vogels,a Andreas
Decken,b Jeffrey C. Waller,a,* Pier Jr. Morinc,*, and Stephen A. Westcotta,*
a Department of Chemistry and Biochemistry, Mount Allison University, Sackville,
NB E4L 1G8, Canada
b Department of Chemistry, University of New Brunswick, Fredericton, NB E3B
5A3, Canada
c Département de chimie et biochimie, Université de Moncton, Campus de Moncton,
Moncton, NB E1A 3E9, Canada
* Corresponding authors. Tel.: +1 506 364 2310 (J. C. Waller); fax.: +1 506 364
2351 (J. C. Waller and S. A. Westcott). Tel.: +1 506 858 4355; fax.: +1 506 858
4541 (P. Morin). Tel.: +1 506 364 2351 (S. A. Westcott).
E-mail addresses: jwaller@mta.ca (J. C. Waller), pier.morin@umoncton.ca (P.
Morin), swestcott@mta.ca (S. A. Westcott).

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[GRAPHICAL ABSTRACT]
Eleven platinum dichloride pyridinecarboxaldimine complexes containing
aliphatic groups have been prepared and characterized fully. All platinum
complexes were examined for their in vitro cytotoxic properties against two
human glioblastoma multiforme cell lines LN18 and LN405.
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ABSTRACT
We have prepared eleven pyridinecarboxaldimines (N-N’R) from the
condensation of 2-pyridinecarboxaldehyde and the corresponding lipophilic
primary amines. Addition of these ligands to [PtCl2(η2-coe)]2 (coe = cis-
cyclooctene) gave complexes of the type cis-PtCl2(N-N’R) (2a-k) in moderate to
high yields. The molecular structure of the N-butylphenyl derivative (2h) has
been confirmed by an X-ray diffraction study. Crystals of 2h were monoclinic
with a = 8.5414(9) Å, b = 11.6774(12) Å, c = 16.1235(16) Å, β = 92.7610(10)° in
the space group P2(1)/c. All platinum complexes were examined for their in
vitro cytotoxic properties against two human glioblastoma multiforme cell lines
LN18 and LN405. The DNA binding properties of one of the most cytotoxic
compounds was compared with that of cisplatin, but no effect was observed
under the conditions tested.
Key
words:
anticancer,
cytotoxicities,
glioma,
liophilicity,
pyridinecarboxaldimines, platinum
HIGHLIGHTS
ꢀ
ꢀ
ꢀ
ꢀ
Eleven novel platinum(II) pyridinecarboxaldimine complexes synthesized.
Lipophilic side chains derived from cyclic and acyclic hydrocarbons.
In vitro cytotoxicities against two human glioblastoma cell lines.
Two families of complexes displayed significant cytotoxicities.
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1. Introduction
The use of metals in medicine is replete with many wondrous examples dating
back several thousands of years. Indeed, the ancient Chinese and Egyptians
had the wherewithal to employ gold and copper, respectively, for medicinal
applications and in the sterilization of water [1].
The first modern
chemotherapeutic inorganic agent, however, came in the unlikely form of the
organometallic arsenic compound Salvarsan, or arsphenamine, discovered by
Erlich and Hata in the early twentieth century for the treatment of syphilis [2].
While the following century gave birth to a whole new pantheon of medicinally-
important inorganic compounds, the biggest impact in this area came from the
serendipitous observation that a small platinum complex, cisplatin (cis-
[PtCl2(NH3)2]), displayed potent antitumor properties in various cancer models
[3-7]. Since this remarkable discovery, cisplatin and related platinum-based
complexes are currently used world-wide for the treatment of testicular and
ovarian malignancies. The primary mechanism of action in these platinum
complexes is believed to arise from the metal’s interaction with DNA, whereby
the platinum atom binds preferentially with the N-7 positions of the purine
bases. Unfortunately, several, yet severe, side effects such as neural and
kidney toxicity, as well as intrinsic and acquired resistance of tumor cells to the
drugs, limit the efficacy of current platinum-based therapy.
These
complications arise from the ability of cisplatin to interfere with healthy cells
and, as such, have provided incentive for a considerable body of research into
the development of platinum-based complexes with enhanced specificity
towards cancer cells.
The cisplatin derivative cis-aminedichlorido(2-
methylpyridine)platinum(II) (picoplatin or ZD0473, Figure 1a) was designed
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specifically to overcome glutathione-mediated drug resistance and has found
remarkable promise in the treatment of lung cancer [8-18].
Fig. 1. Structure of picoplatin (ZD0473) (a) and dichlorido-N-(pyridin-2-
ylmethylene)anilineplatinum(II) (b).
As part of our research designed at generating bioactive metal complexes we
have been making picoplatin analogues using iminopyridines [19]. Previous
studies have shown that the platinum complex derived from 2-
pyridinecarboxaldehyde and aniline (Fig. 1b) has shown promising activity
against the hormone independent human mammary carcinoma cell line MDA-
MB 231 [20]. Variation of the amine group allows for the design of a large
family of compounds with a wide range of physical and chemical properties. As
well, the use of bidentate ligands prevents trans labilization and undesired
displacement of the ligands by sulfur and nitrogen donors in biomolecules,
commonly believed to be responsible for some of the adverse side effects
associated with cisplatin. We report herein our findings on the synthesis and
initial biological testing of cis-dichlorido(pyridin-2-ylcarboxaldimine)platinum(II)
compounds containing a variety of aliphatic and aromatic groups, which we
propose will improve lipophiliticities and aid in drug delivery. We further
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demonstrate promising cytotoxic effects for such compounds in two brain
tumor cell lines.
2. Experimental
2.1. Materials and methods
Reagents and solvents used were obtained from Aldrich Chemicals. [PtCl2(η2-
coe)]2 (coe = cis-cyclooctene) [21] and 2a [22] were prepared as previously
reported. NMR spectra were recorded on a JEOL JNM-GSX400 FT NMR
1
spectrometer. H NMR chemical shifts are reported in ppm and are referenced
to residual protons in deuterated solvent at 400 MHz. 13C NMR chemical shifts
are reported in ppm and are referenced to solvent carbon resonances as
internal standards at 100 MHz. Multiplicities are reported as singlet (s),
doublet (d), triplet (t), quintet (quint), multiplet (m), and overlapping (ov) with
coupling constants (J) reported in hertz. FTIR spectra were obtained with a
Thermo Fisher Scientific Nicolet iS5 FT-IR spectrometer in ATR mode and are
reported in cm-1. Melting points were measured uncorrected with a Stuart
SMP30 apparatus. Elemental analyses for carbon, hydrogen, and nitrogen were
carried out at Laboratoire d’Analyse Élémentaire de l’Université de Montréal
(Montréal, QC).
2.2. Synthesis of iminopyridine ligands
A CH2Cl2 (10 mL) solution of the appropriate amine (10 mmol) was added to a
CH2Cl2 (15 mL) solution of 2-pyridinecarboxaldehyde (1.07 g, 10.0 mmol) and
activated 4Å molecular sieves (10 g). The reaction mixture was allowed to stand
at RT for 2 days at which point the molecular sieves were separated by filtration
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and the solvent was removed under vacuum to afford the desired
iminopyridines 1a-k as orange-red oils.
2.3. Synthesis of platinum iminopyridine complexes
To a stirred CH2Cl2 (10 mL) suspension of [PtCl2(η2-coe)]2 (225 mg, 0.30 mmol)
was added a CH2Cl2 (5 mL) solution of the appropriate iminopyridine (0.60
mmol). The reactions were allowed to proceed at RT for 18 h. Removal of
solvent under vacuum afforded oily, orange solids which were triturated with
hexane (3 x 5 mL) to give the desired platinum iminopyridine complexes as
orange solids.
2.3.1. Synthesis of dichlorido(N-cyclopropanyl-pyridyl-2-methanimine)platinum(II)
(2b)
1
Yield: 207 mg (84%); mp: 252-254 oC. H NMR (DMSO-d6): δ 9.32 (d, JHH = 6.0
Hz, 1H, Ar), 8.93 (s, JHPt = 47.9 Hz, 1H, CH=N), 8.32 (ov dd, JHH = 7.8, 7.8 Hz,
1H, Ar), 7.99 (d, JHH = 7.8 Hz, 1H, Ar), 7.84 (ov dd, JHH = 7.8, 6.0 Hz, 1H, Ar),
4.12 (m, 1H, NCH), 1.25-1.13 (ov m, 4H, CH2). 13C{1H} NMR (DMSO-d6): δ
166.2, 158.0, 149.2, 141.1, 129.2, 128.8, 41.1, 12.0. FTIR: 3038 (m), 1618 (m,
νCN), 1478 (m), 1435 (w), 1298 (m), 1245 (m), 1131 (m), 1036 (m), 890 (m), 777
(s). Anal. Calc. for C9H10N2Cl2Pt (412.17 g/mol) (%): C 26.23, H 2.45, N 6.80.
Found: C 26.18, H 2.32, N 6.69.
2.3.2. Synthesis of dichlorido(N-cyclopentanyl-pyridyl-2-methanimine)platinum(II)
(2c)
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1
Yield: 230 mg (87%); mp 283-285 °C. H NMR (DMSO-d6): δ 9.33 (d, JHH = 6.0
Hz, 1H, Ar), 9.07 (s, JHPt = 49.0 Hz, 1H, CH=N), 8.35 (ov ddd, JHH = 7.8, 7.8, 1.4
Hz, 1H, Ar), 8.12 (dd, JHH = 7.8, 0.9 Hz, 1H, Ar), 7.84 (ov ddd, JHH = 7.8, 6.0, 1.4
Hz, 1H, Ar), 4.93 (quint, JHH = 6.4 Hz, 1H, NCH), 2.08 (m, 2H, CH2), 1.87 (m,
2H, CH2), 1.72 (m, 2H, CH2), 1.62 (m, 2H, CH2). 13C{1H} NMR (DMSO-d6): δ
167.9, 158.4, 149.2, 141.1, 129.3, 129.1, 68.9, 33.2, 23.8. FTIR: 3045 (m),
2948 (m), 2876 (m), 1620 (m, νCN), 1475 (m), 1435 (w), 1293 (m), 1240 (s), 1170
(m), 933 (m). Anal. Calc. for C11H14N2Cl2Pt (440.23 g/mol) (%): C 30.01, H 3.21,
N 6.36. Found: C 30.06, H 3.21, N 6.27.
2.3.3. Synthesis of dichlorido(N-cyclohexanyl-pyridyl-2-methanimine)platinum(II)
(2d)
1
Yield: 251 mg (92%); mp 262-263 °C. H NMR (DMSO-d6): δ 9.33 (d, JHH = 6.0
Hz, 1H, Ar), 9.11 (s, JHPt = 50.2 Hz, 1H, CH=N), 8.34 (ov ddd, JHH = 7.8, 7.8, 1.4
Hz, 1H, Ar), 8.08 (dd, JHH = 7.8, 0.9 Hz, 1H, Ar), 7.84 (m, 1H, Ar), 4.36 (m, 1H,
NCH), 2.16 (m, 2H, CH2), 1.79 (m, 2H, CH2), 1.64 (m, 1H, CH), 1.48-1.25 (ov m,
4H, CH2), 1.11 (m, 1H, CH). 13C{1H} NMR (DMSO-d6): δ 168.7, 158.4, 149.2,
141.2, 129.4, 129.1, 65.5, 33.3, 25.6 (2C). FTIR: 3058 (m), 2926 (m), 2845 (m),
1620 (m, νCN), 1480 (m), 1444 (m), 1317 (m), 1241 (m), 1160 (m), 1063 (m), 893
(m), 761 (s). Anal. Calc. for C12H16N2Cl2Pt (454.25 g/mol) (%): C 31.73, H 3.55,
N 6.17. Found: C 31.35, H 3.24, N 6.01.
2.3.4. Synthesis of dichlorido(N-cyclooctanyl-pyridyl-2-methanimine)platinum(II)
(2e)
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1
Yield: 218 mg (75%); mp: 263-264 oC. H NMR (DMSO-d6): δ 9.34 (d, JHH = 6.0
Hz, 1H, Ar), 9.16 (s, JHPt = 47.2 Hz, 1H, CH=N), 8.34 (ov dd, JHH = 7.8, 7.8 Hz,
1H, Ar), 8.06 (d, JHH = 7.8 Hz, 1H, Ar), 7.85 (ov dd, JHH = 7.8, 6.0 Hz, 1H, Ar),
4.76 (m, 1H, NCH), 1.99-1.87 (ov m, 4H, CH2), 1.68-1.52 (ov m, 10H, CH2).
13C{1H} NMR (DMSO-d6): δ 169.0, 158.3, 149.0, 141.0, 129.1, 128.8, 65.3, 32.7,
26.4, 25.7, 24.2. FTIR: 3045 (w), 2918 (m), 2847 (m), 1622 (m, νCN), 1474 (m),
1446 (m), 1241 (m), 1154 (w), 1241 (m), 936 (m), 772 (s). Anal. Calc. for
C14H20N2Cl2Pt (482.31 g/mol) (%): C 34.86, H 4.18, N 5.81. Found: C 34.56, H
3.96, N 5.71.
2.3.5. Synthesis
of
dichlorido(N-(2-phenylethanyl-pyridyl-2-
methanimine))platinum(II) (2f)
1
Yield: 237 mg (83%); mp: 228-231 oC. H NMR (DMSO-d6): δ 9.34 (d, JHH = 6.0
Hz, 1H, Ar), 8.95 (s, JHPt = 46.8 Hz, 1H, CH=N), 8.32 (ov dd, JHH = 7.8, 7.8 Hz,
1H, Ar), 8.03 (d, JHH = 7.8 Hz, 1H, Ar), 7.87 (ov dd, JHH = 7.8, 6.0 Hz, 1H, Ar),
7.27-7.19 (ov m, 5H, Ph), 4.18 (t, JHH = 7.4 Hz, 2H, NCH2), 3.12 (t, JHH = 7.4 Hz,
2H, CH2Ph). 13C{1H} NMR (DMSO-d6): δ 171.6, 157.2, 149.6, 141.3, 138.2,
129.7, 129.5, 129.1, 128.8, 127.2, 61.1, 37.0. FTIR: 3066 (w), 2880 (w), 1614
(m, νCN), 1495 (w), 1476 (m), 1300 (m), 1382 (m), 1300 (m), 1241 (m), 1155 (m),
910 (w), 771 (s), 702 (s). Anal. Calc. for C14H14N2Cl2Pt (476.26 g/mol) (%): C
35.31, H 2.96, N 5.88. Found: C 35.04, H 2.83, N 5.75.
2.3.6. Synthesis
of
dichlorido(N-(3-phenylpropanyl-pyridyl-2-
methanimine))platinum(II) (2g)
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1
Yield: 256 mg (87%); mp 238-241 °C. H NMR (DMSO-d6): δ 9.31 (d, JHH = 6.0
Hz, 1H, Ar), 9.12 (s, JHPt = 46.5 Hz, 1H, CH=N), 8.33 (ov dd, JHH = 7.8, 7.8 Hz,
1H, Ar), 8.06 (d, JHH = 7.8 Hz, 1H, Ar), 7.85 (ov dd, JHH = 7.8, 6.0 Hz, 1H, Ar),
7.26-7.18 (ov m, 4H, Ar), 7.13 (m, 1H, Ar), 4.00 (t, JHH = 7.1 Hz, 2H, NCH2), 2.62
(t, JHH = 7.1 Hz, 2H, CH2Ph), 2.12 (quint, JHH = 7.1 Hz, 2H, NCH2CH2). 13C{1H}
NMR (DMSO-d6): δ 171.5, 157.5, 149.4, 141.5, 141.2, 129.5, 128.9, 128.8 (2C),
126.5, 59.7, 32.5, 32.3. FTIR: 3023 (w), 2981 (m), 2914 (w), 1600 (m, νCN),
1479 (m), 1448 9m), 1300 (m), 1239 (m), 1155 (m), 1239 (m), 1030 (w), 751 (s),
698 (s). Anal. Calc. for C15H16N2Cl2Pt (490.28 g/mol) (%): C 36.75, H 3.29, N
5.71. Found: C 36.23, H 3.09, N 5.56.
2.3.7. Synthesis
of
dichlorido(N-(4-phenylbutanyl-pyridyl-2-
methanimine))platinum(II) (2h)
1
Yield: 273 mg (90%); mp: 149-152 oC. H NMR (DMSO-d6): δ 9.32 (d, JHH = 6.0
Hz, 1H, Ar), 9.14 (s, JHPt = 44.4 Hz, 1H, CH=N), 8.32 (ov dd, JHH = 7.8, 7.8 Hz,
1H, Ar), 8.06 (d, JHH = 7.8 Hz, 1H, Ar), 7.87 (ov dd, JHH = 7.8, 6.0 Hz, 1H, Ar),
7.24-7.09 (ov m, 5H, Ph), 3.99 (t, JHH = 7.4 Hz, 2H, NCH2), 2.56 (t, JHH = 7.4 Hz,
2H, CH2Ph), 1.82 (quint, JHH = 7.4 Hz, 2H, NCH2CH2), 1.57 (quint, JHH = 7.4 Hz,
2H, CH2CH2Ph). 13C{1H} NMR (DMSO-d6): δ 171.4, 157.5, 149.5, 142.3, 141.2,
129.5, 128.9, 128.8 (2C), 126.3, 59.5, 35.3, 30.6, 28.3. FTIR: 3037 (w), 2934
(w), 2853 (w), 1619 (m, νCN), 1480 (m), 1449 (m), 1304 (m), 1285 (m), 936 (w),
772 (s), 707 (s). Anal. Calc. for C16H18N2Cl2Pt (504.31 g/mol) (%): C 38.11, H
3.60, N 5.55. Found: C 38.04, H 3.61, N 5.50.
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2.3.8. Synthesis
of
dichlorido(N-(4-hexylphenyl-pyridyl-2-
methanimine))platinum(II) (2i)
1
Yield: 262 mg (82%); mp: 244-247 oC. H NMR (DMSO-d6): δ 9.43 (d, JHH = 6.0
Hz, 1H, Ar), 9.26 (s, JHPt = 40.3 Hz, 1H, CH=N), 8.40 (ov dd, JHH = 7.8, 7.8 Hz,
1H, Ar), 8.17 (d, JHH = 7.8 Hz, 1H, Ar), 7.87 (ov dd, JHH = 7.8, 6.0 Hz, 1H, Ar),
7.35 (d, JHH = 8.7 Hz, 2H, Ar), 7.26 (d, JHH = 8.7 Hz, 2H, Ar), 2.60 (t, JHH = 7.4
Hz, 2H, PhCH2), 1.55 (quint, JHH = 7.4 Hz, 2H, PhCH2CH2), 1.31-1.20 (ov m, 6H,
CH2), 0.83 (t, JHH = 6.4 Hz, 3H, CH3). 13C{1H} NMR (DMSO-d6): δ 172.6, 157.7,
149.5, 145.4, 144.0, 141.2, 130.1, 130.0, 128.5, 124.7, 35.3, 31.6, 31.3, 28.9,
22.6, 14.5. FTIR: 3023 (w), 2925 (m), 2855 (w), 1615 (m, νCN), 1503 (w), 1473
(m), 1353 (m), 1244 (m), 1205 (m), 951 (m), 854 (m), 772 (s). Anal. Calc. for
C18H22N2Cl2Pt (532.36 g/mol) (%): C 40.61, H 4.17, N 5.26. Found: C 40.24, H
4.20, N 5.14.
2.3.9. Synthesis
of
dichlorido(N-(4-octylphenyl-pyridyl-2-
methanimine))platinum(II) (2j)
1
Yield: 296 mg (88%); mp 252-254 °C. H NMR (DMSO-d6): δ 9.43 (d, JHH = 6.0
Hz, 1H, Ar), 9.26 (s, JHPt = 40.3 Hz, 1H, CH=N), 8.40 (ov ddd, JHH = 7.8, 7.8, 1.4
Hz, 1H, Ar), 8.17 (dd, JHH = 7.8, 0.9 Hz, 1H, Ar), 7.94 (ov ddd, JHH = 7.8, 6.0, 0.9
Hz, 1H, Ar), 7.35 (d, JHH = 8.2 Hz, 2H, Ar), 7.26 (d, JHH = 8.2 Hz, 2H, Ar), 2.60 (t,
JHH = 7.4 Hz, 2H, PhCH2), 1.56 (quint, JHH = 7.4 Hz, 2H, PhCH2CH2), 1.26-1.22
(ov m, 10H, CH2), 0.82 (t, JHH = 7.0 Hz, 3H, CH3). 13C{1H} NMR (DMSO-d6): δ
172.6, 157.7, 149.5, 145.4, 144.0, 141.2, 130.1, 130.0, 128.5, 124.7, 35.2,
31.8, 31.4, 29.4, 29.2, 29.1, 22.6, 14.5. FTIR: 3025 (w), 2921 (m), 2852 (w),
1614 (m, νCN), 1504 (m), 1353 (w), 1242 (m), 1158 (w), 1017 (m), 937 (m), 772
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(s), 720 (w). Anal. Calc. for C20H26N2Cl2Pt (560.42 g/mol) (%): C 42.86, H 4.68,
N 5.00. Found: C 42.52, H 4.69, N 4.78.
2.3.10.
Synthesis
of
dichlorido(N-(4-decylphenyl-pyridyl-2-
methanimine))platinum(II) (2k)
o
Yield: 303 mg (86%); mp: 255-257 C (decomposition). 1H NMR (DMSO-d6): δ
9.44 (d, JHH = 6.0 Hz, 1H, Ar), 9.27 (s, JHPt = 41.2 Hz, 1H, CH=N), 8.40 (ov dd,
JHH = 7.8, 7.8 Hz, 1H, Ar), 8.18 (d, JHH = 7.8 Hz, 1H, Ar), 7.94 (ov dd, JHH = 7.8,
6.0 Hz, 1H, Ar), 7.35 (d, JHH = 8.7 Hz, 2H, Ar), 7.26 (d, JHH = 8.7 Hz, 2H, Ar),
2.60 (t, JHH = 7.4 Hz, 2H, PhCH2), 1.55 (quint, JHH = 7.4 Hz, 2H, PhCH2CH2),
1.34-1.20 (ov m, 14H, CH2), 0.82 (t, JHH = 6.4 Hz, 3H, CH3). 13C{1H} NMR
(DMSO-d6): δ 172.6, 157.7, 149.5, 145.4, 144.0, 141.2, 130.1, 130.0, 128.4,
124.7, 35.3, 31.8, 31.4, 29.6 (2C), 29.4, 29.3 (2C), 22.6, 14.5. FTIR: 2956 (w),
2920 (m), 2851 (w), 1615 (m, νCN), 1470 (m), 1353 (w), 1242 (m), 1016 (w), 819
(m), 773 (s), 719 (m). Anal. Calc. for C22H30N2Cl2Pt (588.47 g/mol) (%): C 44.90,
H 5.14, N 4.76. Found: C 44.61, H 5.21, N 4.61.
2.4. X-ray crystallography
o
Crystals of 2h were grown from a saturated CH2Cl2 solution stored at 5 C.
Single crystals were coated with Paratone-N oil, mounted using a polyimide
MicroMount and frozen in the cold nitrogen stream of the goniometer.
A
hemisphere of data was collected on a Bruker AXS P4/SMART 1000
diffractometer using ω and φ scans with a scan width of 0.3o and 10 s exposure
times. The detector distance was 5 cm. The data were reduced (SAINT) [23]
and corrected for absorption (SADABS) [24]. The structure was solved by direct
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methods and refined by full-matrix least squares on F2(SHELXTL) [25]. All non-
hydrogen atoms were refined using anisotropic displacement parameters.
Hydrogen atoms were included in calculated positions and refined using a
riding model.
2.5. Cell cultures
Human glioma cells LN18 and LN405 were maintained in an incubator at 37 °C
and 5% CO2 in DMEM supplemented with 10% FBS (foetal bovine serum) and
antibiotics (Invitrogen) and have been described elsewhere [26]. All cell lines
were kind gifts of Adrian Merlo (Laboratory of Molecular Neuro-oncology,
University of Basel, Basel, Switzerland).
2.6. MTT assays
10,000 cells were seeded in triplicate wells in 96-well plates in 200 µL of cell
medium and incubated for 24 hrs at 37 °C and 5% CO2. Medium was replaced
with medium containing 1 µM, 10 µM or 100 µM of the selected complexes
dissolved in DMSO (or DMF for the case of cisplatin) and further incubated for
48 hrs. Complexes were substituted for DMSO in control wells. Following
incubation, 20 µL of a 5 mg/mL solution of MTT in PBS was added to each well.
Cells and MTT were incubated for 3 hrs and removed afterwards. Stained cells
were re-suspended in 100 µL of a 1:24 1M HCl/95% EtOH and read at 560 nm
on a Varioskan (Scanlab). All data presented are mean ± standard error of the
mean (SEM).
2.7. IC50 cytotoxicity assay
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MTT assays as described above were used to determine the experimental IC50
values for selected novel platinum complexes and cisplatin in glioblastoma
multiforme cell lines. Cells were treated with the control compound cisplatin
and the selected novel platinum complexes at final concentrations of 0.1 µM,
0.5 µM, 1 µM, 10 µM, 100 µM and 250 µM. Data were converted to the
percentage of cell viability compared to solvent control (DMSO only) wells from
the same experiment.
Graphs of log (%viable cells) vs. final treatment
concentration (µM) were analyzed with linear regression to determine the final
treatment concentration that would give 50% viable cells (IC50).
2.8. DNA isolation and purification
pBluescript SK(+) (Stratagene, Genbank accession number X52328) plasmid
DNA was purified with a Plasmid Midi Kit (Geneaid). Eluted plasmid DNA was
washed by first adding chilled isopropanol, adjusting to a final concentration of
1M NaCl, and centrifuging at 7200 X g for 30 min at 4 °C. After incubating
overnight at -20 °C, DNA was then re-centrifuged at 7200 X g for 30 min at 4
°C. The supernatant was discarded, and the DNA pellet washed with pre-chilled
75% (v/v) EtOH and centrifuged at ~1800 X g for 10 min at 4 °C. The
supernatant was discarded and the DNA pellet was dried in a centrifugal
o
evaporator (Savant Speed Vac Plus SC110A) at 43 C before dissolution in
sterile ultrapure water. DNA was quantified and purity assessed by measuring
the absorbance at 260 nm and 280 nm, respectively. A 260 nm to 280 nm
absorbance ratio of ≥1.8 was taken to be pure. An absorbance at 260 nm of 1.0
14

Miles et al.
submitted to Polyhedron.
was taken to be a concentration of 50 ng/µL and the molar mass of pBluescript
SK(+) was calculated to be 1,936,296 grams per moles.
2.9. Chloride abstraction reactions
To facilitate increased water solubility for examining the interaction of cisplatin
and 2e with DNA, chloride abstraction reactions were performed using a
modified protocol obtained from Sengupta et al [27]. Cisplatin (49.0 mg, 0.163
mmol) was partially dissolved in water, to which silver nitrate (55.0 mg, 0.326
mmol) was added.
The abstraction reaction proceeded for 1h at room
temperature to give AgCl and the corresponding diaquated platinum dication.
The solution was then centrifuged at 20 000 X g for 1h at 4 °C to separate the
resulting precipitated silver chloride. Complex 2e (30.0 mg, 0.0619 mmol) was
abstracted as above with silver nitrate (21.0 mg, 0.124 mmol).
2.10. DNA binding assays
The interaction between cisplatin and 2e and pBluescript SK(+) plasmid DNA
was examined by performing DNA binding assays using a modified protocol
obtained from Urquiola et al. [28]. Stock solutions of abstracted cisplatin and
abstracted complex 2e were made using water to concentrations of 0.0815 M
and 0.0310 M, respectively. The solutions were adjusted to 10 mM TRIS-Cl and
0.1 mM EDTA (pH 7.5) to give varying molar ratios (ri, moles of complex:moles
of DNA) when incubated with DNA (at a final volume of 20 µL). The reactions
were incubation for 24 h at 37 °C. Two control reactions in absence the metal
complexes were incubated as above and the other at 4 °C to verify that any
mobility shift was due to metal complexes and not thermal damage. DNA
15

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loading buffer containing 30% (v/v) glycerol, 25 mM EDTA (pH 8), and 0.015%
(w/v) Orange G dye was added to the plasmid DNA incubation reactions. The
samples were then resolved on a 0.7% (w/v) agarose gel in a 1X TBE (TRIS -
borate - EDTA) running buffer at 70V for 1 h with a 1Kb DNA ladder RTU
(GeneDireX). After electrophoresis, agarose gels were stained with 0.5 µg/mL
ethidium bromide and destained in water with agitation. DNA was visualized
using a Versa Doc Imaging System and the Quantity One image software (Bio-
Rad).
3. Results and discussion
3.1. Synthesis and structure
Addition of lipophilic primary amines to commercially available 2-
pyridinecarboxaldehyde gave the corresponding pyridin-2-ylcarboxaldimine
ligands 1a-k in high yields [29-31]. Variation of the starting amine allowed for
the facile design of a number of lipophilic ligands. The corresponding platinum
complexes 2a-k (Scheme 1) were readily prepared by addition of the
pyridinecarboxaldimine ligands to CH2Cl2 solutions of dimeric [PtCl2(η2-coe)]2
(coe = cis-cyclooctene).
Scheme 1. Synthesis of platinum complexes 2a-k.
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The use of lipophilic ligands to deliver platinum complexes in anticancer
research is a rapidly growing area of interest [32-35]. In this study, complexes
2a-k have been characterized by a number of physical methods, including
multinuclear NMR and FTIR spectroscopy. A significant downfield shift in the
1H NMR spectra is observed for the N=CH sp2 proton upon coordination of the
ligand to the platinum center. For example, the singlet at 8.48 ppm for the free
ligand derived from 2-pyridinecarboxaldehyde and cyclopropylamine shifts to
8.93 ppm in complex 2b. Likewise, platinum satellites are also observed for
these resonances (JH-Pt = 47.9 Hz for 2b) upon complexation of the ligand to the
metal. Similar trends are observed for the pyridine hydrogen alpha to the
nitrogen atom which is observed at 8.57 ppm in the free ligand and shifts
downfield to 9.32 ppm upon complexation to platinum.
Complex 2h has also been characterized by an X-ray diffraction study (Fig. 2)
with crystallographic data given in Table 1. The nitrogen-platinum bonds of
2.002(2) Å (imine) and 2.018(2) Å (pyridine) are similar to those reported in
other iminopyridine platinum systems [36-48]. The imine C(7)-N(8) distance is
1.291(4) Å and in the range of accepted carbon-nitrogen double bonds. The
N(8)-Pt(1)-N(1) angle is 80.12(10)o and is considerably less than 90o, as is often
observed in related complexes owing to the bidentate nature of the
iminopyridine ligand. The platinum chloride distances are Pt(1)-Cl(2) 2.2915(7)
Å and Pt(1)-Cl(1) 2.2921(8) Å, with a typical angle between these three atoms of
Cl(2)-Pt(1)-Cl(1) 89.27(3)o.
17

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submitted to Polyhedron.
Fig. 2. A ball and stick diagram of 2h with hydrogen atoms omitted for clarity.
Selected bond distances (Å): Pt(1)-N(8) 2.002(2), Pt(1)-N(1) 2.018(2), Pt(1)-Cl(2)
2.2915(7), Pt(1)-Cl(1) 2.2921(8), C(2)-C(7) 1.450(4), C(7)-N(8) 1.291(4); Selected
bond angles (o): N(8)-Pt(1)-N(1) 80.12(10), N(8)-Pt(1)-Cl(2) 95.49(7), N(1)-Pt(1)-
Cl(2) 175.47(7), N(8)-Pt(1)-Cl(1) 175.23(7), N(1)-Pt(1)-Cl(1) 95.13(7), Cl(2)-Pt(1)-
Cl(1) 89.27(3), C(6)-N(1)-C(2) 119.2(2), C(6)-N(1)-Pt(1) 127.0(2), C(2)-N(1)-Pt(1)
113.83(19), N(1)-C(2)-C(3) 122.1(3), N(1)-C(2)-C(7) 113.8(3).
3.2. Cytotoxicities
Recent reports have shown that platinum Schiff base complexes can also be
used to inhibit the growth of human breast, colon and cervical cancer cells
[49,50]. These results led us to examine representative examples of new
complexes 2a-k, using cisplatin and DMSO (control) as standards, for their
cytotoxic properties against three glioma cell lines using the MTT method. The
therapeutic use of cisplatin in high-grade gliomas has been recently
documented [51,52]. The cell lines used in this study, LN18 and LN405, are
two glioblastoma multiforme cell lines with distinct PTEN, p14/ARF and p16
tumor suppressors expression status [26]. Significant DNA fragmentation
following cisplatin treatment has been reported in LN18 cells [53] while
sensitivity to this platinum complex is unknown in LN405. Cytotoxic activities
18

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submitted to Polyhedron.
of seven novel platinum complexes were assessed in LN18 and LN405 using
cisplatin as a control. The results (Fig. 3A and 3B) showed that all seven
complexes displayed greater cytotoxicity than cisplatin in both cell lines at 100
µM. In addition, complexes 2e and 2h appeared to be the most cytotoxic of the
seven complexes tested. It is unclear which factors are responsible for the
enhanced activities in these two disparate complexes and further structure-
activities studies will be required based on these two structural motifs.
19

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submitted to Polyhedron.
A)
B)
Fig. 3. Cytotoxic effects of selected platinum complexes in LN18 (A) and LN405
(B) glioblastoma multiforme cell lines. Results display % Cell Viability ±
standard error.
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submitted to Polyhedron.
We then determined the IC50 values for each platinum complex, along with the
control, cisplatin. All compounds were tested at final concentrations of 0.1, 0.5,
1, 10, 100, and 250 µM and assays were done in triplicate, the results of which
are shown in Table 2. Once again, all seven platinum complexes displayed
lower IC50 values against both LN405 and LN18 than cisplatin, indicating these
new complexes are better at reducing cell viability. Complex 2i exhibited the
lowest IC50 value against cell line LN405 (17 times lower than that of cisplatin)
and 2e exhibited the lowest IC50 value against cell line LN18 (16 times lower
than that of cisplatin).
Table 2. IC50 Values (µM) of Novel Platinum Complexes and Cisplatin in
Glioblastoma Cell Lines LN405 and LN18
Complex
LN405
50 ± 1
20 ± 2
36 ± 1
4 ± 7
LN18
66 ± 9
22 ± 4
20 ± 7
11 ± 1
15 ± 1
20 ± 8
18 ± 2
31 ± 1
Cisplatin
2a
2b
2e
2f
16 ± 2
11 ± 1
3 ± 4
2h
2i
2k
16 ± 1
3.3. DNA binding assays
To examine the basis of cytotoxicity of the novel complexes, interaction
experiments were performed with cisplatin complexes and a model DNA
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molecule. The interaction between 2e, cisplatin, and pure pBluescript SK(+)
plasmid DNA was examined by performing DNA binding assays consisting of
incubating a fixed amount of plasmid DNA with varying concentrations of
chloride-abstracted cisplatin (used as a standard) and chloride-abstracted 2e.
Complex 2e was chosen as a representative complex due to its potent cytotoxic
effects (Figure 3).
As observed in Figure 4A, an effect on the plasmid DNA is evident upon
exposure to cisplatin at certain ri values. The three ‘bands’ in each lane are
likely attributable to (from low to high mobility, or top to bottom):
concatemerized, relaxed, and supercoiled pBluescript SK(+) plasmid DNA. A
decrease in the relative mobility of supercoiled plasmid DNA becomes evident
when ri=1000, likely a result of DNA binding by cisplatin to the supercoiled
plasmid. This damage would cause the plasmid DNA to relax from its
negatively supercoiled state and thus decrease its mobility. When ri=2500,
nearly all supercoiled plasmid DNA was converted to its relaxed form. When
ri=6000 (and higher), the presence of supercoiled DNA is again apparent. A
previous study suggested this to be indicative of positive plasmid DNA
supercoiling induced by high concentrations of cisplatin causing unwinding of
the negatively supercoiled plasmid and eventually, positive supercoiling [28].
The reversibility of plasmid DNA supercoiling indicates that the plasmid DNA
remained intact and was not nicked by cisplatin. Nicking would lead to
irreversible unwinding of the plasmid DNA, as one nicked (and as a result, free)
strand would lead to that strand spinning freely around the other intact strand.
This would render plasmid DNA unable to become supercoiled at the highest
22

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tested cisplatin concentrations. The concatemerized plasmid DNA followed the
same trend as the supercoiled plasmid DNA. This is to be expected, as the
concatemers would also be supercoiled in absence of high concentrations of
cisplatin. Relaxation of the concatemerized DNA is still observed at higher
concentrations of cisplatin, however, leading to a decrease in relative mobility.
Overall, the changes in the relative mobility of the DNA at varying ri values is
indicative of an interaction between plasmid DNA and cisplatin.
Fig. 4. Agarose gel electrophoresis patterns of plasmid DNA after incubation
with cisplatin compounds. pBluescript SK(+) DNA was incubated with varying
molar ratios (ri, moles of complex:moles of DNA) of cisplatin (A) or 2e (B).
Molecular mass marker sizes are indicated at left (in kb) and ri values (x100) at
top.
However, aquated 2e had no apparent effect on DNA supercoiling even with ri
values of up to 8000 (Figure 4B), indicating that 2e did not bind or nick DNA
under the observed conditions. No mobility shifts were observed in the control
DNA incubations lacking cisplatin compounds (data not shown). This may
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suggest that the cytotoxicity of 2e (Figure 3) is independent of DNA damage in
vivo and perhaps acts by another mechanism such as binding to key thiol
groups in proteins [16,54].
4. Conclusion
Pyridinecarboxaldimines
derived
from
the
condensation
of
2-
pyridinecaboxaldehyde and aliphatic amines have been prepared in high yields
and used as ligands for platinum. The resulting complexes have been examined
for their potential anticancer properties and have shown promise as lead
compounds for the potential treatment of brain tumours. Future work is
needed to design a metal drug candidate based on this structural motif and
additional studies in this area are currently in progress, the results of which
will be published in due course.
Appendix A. Supplementary data
Full crystallographic data in CIF format have been deposited with The Director,
CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44-1223-366033; e-
mail: deposit@ccdc.cam.ac.uk or www.ccdc.cam.ac.uk) and are available on
request, quoting deposition number 1046571 for compound 2h.
Acknowledgements
Thanks are gratefully extended to the Natural Sciences and Engineering
Research Council of Canada, Mount Allison University, and Université de
Moncton for financial support. We also thank Dan Keydrop Durant and Dr.
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Dylan Levac for their expert technical assistance and anonymous reviewers
thanked for their very helpful comments.
References
[1]
[2]
[3]
[4]
[5]
S. P. Fricker, Dalton Trans. (2007) 4903.
S. Gibaud, G. Jaouen, Top. Organomet. Chem. 31 (2010) 1.
Z. Guo, P. J. Sadler, Angew. Chem. Int. Ed. 38 (1999) 1513.
J. Reedijk, Naturwissenschaften 74 (1987) 71.
I. Warad, A. F. Eftaiha, M. A. Al-Nuri, A. I. Husein, M. Assal, A. Abu-
Obaid, N. Al-Zaqri, T. Ben Hadda, B. Hammouti, J. Mater. Environ. Sci. 4
(2013) 542.
[6]
[7]
[8]
S. H. van Rijt, P. J. Sadler, Drug Discov. Today 14 (2009) 1089.
A. Casini, J. Reedijk, Chem. Sci. 3 (2013) 3135.
C.-H. Tang, C. Parham, E. Shocron, G. McMahon, N. Patel, Cancer
Chemother. Pharmacol. 67 (2011) 1389.
[9]
M. F. Osborn, J. D. White, M. M. Haley, V. J. DeRose, ASC Chem. Biol. 9
(2014) 2404.
[10] J. D. White, M. F. Osborn, A. D. Moghaddam, L. E. Guzman, M. M.
Haley, V. J. DeRose, J. Am. Chem. Soc. 135 (2013) 11680.
[11] S. D. Brown, K. D. Trotter, O. B. Sutcliffe, J. A. Plumb, B. Waddell, N. E.
B. Briggs, N. J. Wheate, Dalton Trans. 41 (2012) 11330.
[12] B. Köberle, M. T. Tomicic, S. Usanova, B. Kaina, Biochim. Biophys. Acta
1806 (2010) 172.
25

Miles et al.
submitted to Polyhedron.
[13] M. Ravera, E. Gabano, I. Zanellato, I. Bonarrigo, E. Escribano, V.
Moreno, M. Font-Bardia, T. Calvet, D. Osella, Dalton Trans. 41 (2012)
3313.
[14] A. Chang, Lung Cancer 71 (2011) 3.
[15] A. Massaguer, A. González-Cantó, E. Escribano, S. Barrabés, G. Artigas,
V. Moreno, V. Marchán, Dalton Trans. 44 (2015) 202.
[16] N. J. Wheate, S. Walker, G. E. Craig, R. Oun, Dalton Trans. 39 (2010)
8113.
[17] S. Sharma, N. A. Shah, A. M. Joiner, K. H. Roberts, C. E. Canman, Mol.
Pharmacol. 81 (2012) 778.
[18] M. Malik, R. Wysokiński, W. Zierkiewicz, K. Helios, D. Michalska, J.
Phys. Chem. A 118 (2014) 6922.
[19] M. L. Conrad, J. E. Enman, S. J. Scales, H. Zhang, C. M. Vogels, M. T.
Saleh, A. Decken, S. A. Westcott, Inorg. Chim. Acta 358 (2005) 63.
[20] H. Brunner, M. Schmidt, H. Schönenberger, Inorg. Chim. Acta. 123
(1986) 201.
[21] M. P. Shaver, C. M. Vogels, A. I. Wallbank, T. L. Hennigar, K. Biradha, M.
J. Zaworotko, S. A. Westcott, Can. J. Chem. 78 (2000) 568.
[22] J. Cloete, S. F. Mapolie, J. Mol. Catal. A: Chem (2006) 221.
[23] SAINT 7.23A; Bruker AXS, Inc., Madison, WI (2006).
[24] G. M. Sheldrick, SADABS 2008; Bruker AXS, Inc., Madison, WI (2008).
[25] G. M. Sheldrick, Acta Crystallogr. A 64 (2008) 112.
[26] N. Ishii, D. Maier, A. Merlo, M. Tada, Y. Sawamura, A. C. Diserens, E. G.
Van Meir, Brain Pathol. 9 (1999) 469.
26

Miles et al.
submitted to Polyhedron.
[27] P. Sengupta, S. Basu, S. Soni, A. Pandey, B. Roy, M. Oh, K. Chin, A.
Paraskar, S. Sarangi, Y. Connor, V. Sabbisetti, J. Kopparam, A. Kulkarni,
K. Muto, C. Amarasiriwardena, I. Jayawardene, N. Lupoli, D. Dinulescu,
J. Bonventre, R. Mashelkar, S. Sengupta, Proc. Natl. Acad. Sci. U S A.
109 (2012) 11294.
[28] C. Urquiola, M. Vieites, M. H. Torre, M. Cabrera, M. Lavaggi, H.
Cerecetto, M. Gonzalez, A. Lopez de Cerain, A. Monge, P. Smircich, B.
Garat, D. Gambino, Bioorg. Med. Chem. 17 (2009) 1623.
[29] M. L. Ferrara, I. Orabona, F. Ruffo, M. Funicello, A. Panunzi,
Organometallics 17 (1998) 3832.
[30] A. Kundu, B. P. Buffin, Organometallics 20 (2001) 3635.
[31] S.-J. Kim, N. Yang, D.-H. Kim, S. O. Kang, J. Ko, Organometallics 19
(2000) 4036.
[32] Y. Qu, J. A. Fitzgerald, H. Rauter, N. P. Farrell, Inorg. Chem. 40 (2001)
6324.
[33] J. W. Williams, Y. Qu, G. H. Bulluss, E. Alvorado, N. P. Farrell, Inorg.
Chem. 46 (2007) 5820.
[34] S. Khiati, D. Luvino, K. Oumzil, B. Chauffert, M. Camplo, P. Barthélémy,
ACS Nano 5 (2011) 8649.
[35] V. Brabec, D. M. Griffith, A. Kisova, H. Kostrhunova, L. Zerzankova, C. J.
Marmion, J. Kasparkova, Mol. Pharmaceutics 9 (2012) 1990.
[36] X.-F. He, C. M. Vogels, A. Decken, S. A. Westcott, Polyhedron 23 (2004)
155.
[37] A. Benedetti, A. C. Fabretti, C. Preti, G. Tosi, P. Zannini, J. Crystallogr.
Spectrosc. Res. 17 (1987) 771.
27

Miles et al.
submitted to Polyhedron.
[38] M. Fettouhi, B. El Ali, M. Morsy, S. Golhen, L. Ouahab, B. Le Guennic,
J.-Y. Saillard, N. Daro, J.-P. Sutter, E. Amouyal, Inorg. Chem. 42 (2003)
1316.
[39] J. S. Casa, A. Castiñeiras, E. Garcia-Martinez, Y. Porajó, M. L. Pérez-
Parallé, A. Sánchez-González, J. Sordo, Z. Anorg. Allg. Chem. 631 (2005)
2258.
[40] S. Reinhardt, K. Heinze, Z. Anorg. Allg. Chem. 632 (2006) 1465.
[41] X. Zhao, P. P.-F. Lee, Y.-K. Yan, C.-K. Chu, J. Inorg. Biochem. 101 (2007)
321.
[42] D. W. Dodd, H. E. Toews, M. J. Trevail, M. C. Jennings, R. H. E. Hudson,
N. D. Jones, Can. J. Chem. 87 (2009) 321.
[43] S. Dehghanpour, A. Mahmoudi, S. Rostami, Polyhedron 29 (2010) 2190.
[44] M. E. Cucciolito, R. Del Litto, F. P. Fanizzi, D. Migoni, G. Roviello, F.
Ruffo, Inorg. Chim. Acta. 363 (2010) 741.
[45] N. Andrade-López, T. A. Hanna, J. G. Alvarado-Rodríguez, A. Luqueño-
Reyes, B. A. Martínez-Ortega, D. Mendoza-Espinosa, Polyhedron 29
(2010) 2304.
[46] D. Qiu, Y. Guo, H. Wang, X. Bao, Y. Feng, Q. Huang, J. Zeng, G. Qui,
Inorg. Chem. Commun. 14 (2011) 1520.
[47] Q.-M. Jin, Y. Lu, J.-L. Jin, H. Guo, G.-W. Lin, Y. Wang, T. Lu, Inorg.
Chim. Acta 421 (2014) 91.
[48] J. R. Pioqunito-Mendoza, D. Martínez-Ortero, N. Andrade-López, J. G.
Alvarado-Rodríguez, V. Salazar-Perada, G. Sánchez-Cabrera, F. J. Zuno-
Cruz, Polyhedron 50 (2013) 289.
[49] C. Shiju, D. Arish, N. Bhuvanesh, S. Kumaresan, Spectrochim. Acta A
28

Miles et al.
submitted to Polyhedron.
Mol. Biomol. Spectrosc. 145 (2015) 213.
[50] M. Proetto, W. Liu, A. Hagenbach, U. Abram, R. Gust, Eur. J. Med.
Chem. 53 (2012) 168.
[51] T. Boulikas, M. Vougiouka, Oncol. Rep. 11 (2004) 559.
[52] J. A. Carillo, C. A. Munoz, Neurosurg. Clin. N. Am. 23 (2012) 297.
[53] T. Shingu, K. Yamada, N. Hara, K. Moritake, H. Osago, M. Terashima, T.
Uemura, T. Yamasaki M. Tsuchiya, Cancer Res. 63 (2003) 4044.
[54] L. Kelland, Nat. Rev. Cancer. 7 (2007) 573.
29