Homodinuclear organometallics of ditopic N,N-chelates: Synthesis, reactivity and in vitro anticancer activity

Article abstract of DOI:10.1016/j.ica.2020.120220

In some instances, multimetallic complexes have shown higher anticancer activity than mononuclear analogues, possibly by interacting with target molecules through a different binding mode. Therefore, a series of novel bis-pyridylimine-based homodinuclear

Full text of DOI:10.1016/j.ica.2020.120220

Inorganica Chimica Acta 518 (2021) 120220
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Research paper
Homodinuclear organometallics of ditopic N,N-chelates: Synthesis,
reactivity and in vitro anticancer activity
a
a
a
b
a
Tasha R. Steel , Kelvin K.H. Tong , Tilo S o¨ hnel , Stephen M.F. Jamieson , L. James Wright ,
c
a,
*
,
1
a,*,1
James D. Crowley , Muhammad Hanif
, Christian G. Hartinger
a
School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
b
c
Auckland Cancer Society Research Centre, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
Department of Chemistry, University of Otago, PO Box 56, Dunedin, New Zealand
A R T I C L E I N F O
A B S T R A C T
In some instances, multimetallic complexes have shown higher anticancer activity than mononuclear analogues,
possibly by interacting with target molecules through a different binding mode. Therefore, a series of novel
Dedicated to Prof. Maurizio Peruzzini on the
th
occasion of his 65 birthday.
II/III
6
5
bis-pyridylimine-based homodinuclear M
(cym/Cp*)Cl (cym =
η
-p-cymene: M = Ru, Os; Cp* =
η
-pentam-
ethylcyclopentadienyl: M = Rh, Ir) complexes were synthesized and studied. The dinuclear complexes were
Keywords:
characterized by 1H, C{ H}, and P{ H} NMR spectroscopy, ESI-MS, and elemental analysis. Additionally,
13
1
31
1
Bioorganometallics
Cancer chemotherapy
Ditopic ligands
the molecular structures of several complexes were investigated by single crystal X-ray diffraction analysis.
2
6
[
{N,N’-(1,4-Phenylene)(bis(1-(pyridin-2-yl)(methanimine)-ᴋ N,N’))}bis{chlorido(
η
-p-cymene)ruthenium(II)}]
Polynuclear complexes
hexafluorophosphate, 1a, was used in stability and binding studies to 9-ethylguanine (EtG) as a DNA nucleobase
model and L-histidine (His), L-cysteine (Cys) and L-methionine (Met) as protein models. However, compared to
structurally related Ru(arene) complexes, the investigations were inconclusive in terms of the nature of
hydrolysis product(s) and EtG adducts formed, while reactions to His and Cys but not Met were observed for 1a.
The in vitro cytotoxicity of the ligands and dinuclear complexes was determined against a small panel of human
cancer cell lines. Some of the complexes showed moderate antiproliferative activity but were less potent than the
bis-pyridylimine-based bridging ligands.
1
. Introduction
The field of medicinal inorganic chemistry gained significant mo-
resistant cancer models, probably because of alternative DNA interac-
tion modes [15]. However, BBR3464 was found to be toxic to patients
with gastric or gastro-esophageal adenocarcinomas in a phase II clinical
trial [16]. The concept of multinuclearity has been developed further to
include Ru [12,14,17–22] Au [19,23,24] Fe [25] and Ti [26] centers in
homo- and heteromultimetallic complexes, affording diverse mecha-
nisms of action.
mentum after the serendipitous discovery of the cytotoxic properties of
the platinum-based complex cisplatin [1–3]. Although several successful
new generations of cisplatin derivatives have since been developed,
traditional platinum-based chemotherapy is commonly associated with
severe dose-limiting side effects and drug resistance, either acquired or
intrinsic [4–9]. As a result, research efforts have sought alternative
treatment options and focus has turned to complexes formed from a
variety of other transition metals [3,10].
II
We have previously reported a series of dinuclear Ru (arene) anti-
cancer agents with in vitro anticancer activity dependent on the length of
the aliphatic linker, where 1,12-bis{chlorido[3-(oxo-κO)-2-methyl-4-
6
pyridinonato-κO](
η
2
-p-cymene)ruthenium(II)}dodecane (Ru ) was the
II
Incorporating multiple transition metal centers in inorganic anti-
cancer agents has in several cases shown an improvement in anticancer
activity when compared to their monometallic analogues [11–14]. One
such compound is the tri-nuclear Pt complex BBR3464 which showed
superior in vitro and in vivo activity compared to cisplatin in cisplatin-
most potent derivative [17,18,21,27]. These dinuclear Ru (arene)
compounds underwent fast chlorido/aqua ligand exchange in aqueous
solution, and were able to bind to both proteins and DNA [18]. A notable
feature of their mode of action was that they cross-linked DNA duplexes
as well as DNA and proteins [27]. The same ditopic pyridone-based
*
Corresponding authors.
E-mail addresses: m.hanif@auckland.ac.nz (M. Hanif), c.hartinger@auckland.ac.nz (C.G. Hartinger).
1
http://hartinger.auckland.ac.nz/.
https://doi.org/10.1016/j.ica.2020.120220
Received 15 September 2020; Received in revised form 13 December 2020; Accepted 17 December 2020
Available online 12 January 2021
0
020-1693/© 2021 Elsevier B.V. All rights reserved.

T.R. Steel et al.
Inorganica Chimica Acta 518 (2021) 120220
′
ligand system was used to prepare Rh(Cp*)Cl and Ir(Cp*)Cl analogues
pyridine carboxaldehyde (2 equiv.) with 1,4-diaminobenzene or 4,4 -
(
Rh
compared to the Ru analogue. Rh
and Ir counterparts with IC50 values of ca. 50 nM in non-small cell lung
carcinoma NCI-H460 cells. Rh and Ir showed DNA damaging ability
similar to Ru as well as cisplatin [28,29], while both were capable of
2
and Ir
2
), which resulted in similar or superior cytotoxic activity
methylene dianiline (1 equiv.), respectively [38,39]. These ditopic li-
gands with a bidentate N,N-chelating group at each coordination pocket
were employed to prepare a series of homodimetallic complexes. Com-
2
was slightly more active than the Ru
2
2
2
2
plexes 1a–1d and 2a–2d were obtained by adding 1 or 2 (1 equiv.) to
II/III
2
stirred solutions of the dimeric metal precursors [M
2 2
(cym/Cp*)Cl ]
inducing the formation of reactive oxygen species in cancer cells.
Importantly, they were found to be less toxic than cisplatin in hemolysis
and in vivo zebrafish assays whilst retaining cytotoxicity [29]. Other
examples include dinuclear Ru compounds based on N,N’-bis(2-amino-
ethyl)-hexane-1,6-diamine linkers with biphenyl arene moieties which
have shown in vitro anticancer activity similar to or lower than the
mononuclear analogues towards A2780 ovarian cancer cells [30].
In line with our interest in designing multinuclear metal complexes
for biological applications, we report the synthesis and characterization
of a series of homodinuclear metal complexes of two different ditopic
pyridylimine ligands. We also report the investigation of aqueous and
DMSO stability, reactivity towards biomolecules and in vitro cytotoxic
properties.
a–d (1 equiv.) in dichloromethane (DCM)/methanol (MeOH) (1:1) at
room temperature (r.t.) for ca. 20 h. Following anion exchange using
excess NH
4
PF6, the homodimetallic complexes 1a–1d and 2a–2d were
obtained as pure solids in good to excellent yields (73–95%) (Scheme 1).
Complex 1a , an analogue of 1a which retains the chloride counter-
Cl
BF4
anions [37], and the tetrafluoroborate derivative (1a ) [40] have been
reported previously.
Characterization of the homodinuclear complexes was achieved
1
31
1
13
1
through analysis of H, P{ H} and C{ H} NMR spectroscopic data
(Supporting Information) as well as electrospray ionization mass spec-
1
trometry (ESI-MS) and elemental analysis. The H NMR spectra revealed
the expected peaks originating from the cym/Cp* moieties and the bis-
pyridylimine ligands. The signals assigned to the protons of the central
phenylene ring(s) appeared as distinct singlets at ca. 8.1 ppm for com-
plexes 1a–1d (H-6) and as two doublets at ca. 7.6 ppm for complexes
2
. Results and discussion
2
a–2d (H-6/7), due to the presence of the methylene group in the latter.
Ligands featuring the pyridylimine coordination motif have been
Protons H-1 and H-5 adjacent to the endocyclic nitrogen atoms of the
bis-pyridylimine ligands saw the most significant downfield shifts upon
coordination of the ligands to the corresponding metal centers,
appearing for all complexes between ca. 8.9 and 9.6 ppm. For complexes
containing the Cp* moiety, the methyl groups appeared as a singlet at
around 1.6 ppm. Exchange of the chloride counterions for hexa-
widely studied due to their extreme versatility, accessible syntheses, and
formation of stable coordination complexes with many transition metals
[
31]. Additionally, several pyridylimine transition metal complexes
have shown promising biological and anticancer activity [32–37]. The
syntheses of bis-pyridylimine ligands 1 and 2 (Scheme 1) followed
modified literature preparations using condensation reactions of 2-
fluorophosphate (PF-
) was indicated by P{ H} NMR spectroscopy,
31
1
6
II/III
Scheme 1. Preparation of dinuclear [{M
(cym/
Cp*)Cl} (pyridylimine ligand 1/2)]PF complexes
2
6
13
1
1
1
a–1d and 2a–2d, as well as the H and C{ H} NMR
numbering scheme used. Conditions: (i) 1,4-diamino-
′
benzene, EtOH, 20 h, reflux; (ii) 4,4 -methylene dia-
II/III
niline, MeOH, 20 h, r.t.; (iii) [M
(cym/Cp*)Cl
2 2
] ,
DCM : MeOH, 20 h, r.t.; (iv) NH
4
6
PF , MeOH, 1 h, r.t.
The syntheses of ligands 1 and 2 have been previously
reported [38,39] as well as those of the analogous
Cl
BF4
complexes 1a and 1a
[37,40].
2

T.R. Steel et al.
Inorganica Chimica Acta 518 (2021) 120220
where a septet at ca. –140 ppm was observed for all complexes.
ESI-mass spectra recorded in positive ionization mode generated
2
+
base peaks that corresponded to [M – 2PF
6
]
cations for complexes 1a,
1
b, and 2a–2d. In contrast, the base peaks for complexes 1c and 1d were
found as the singly charged mononuclear Rh /Ir species following
hydrolysis at one of the Schiff bases and subsequent loss of a metal
III III
center, pyridyl unit and chlorido ligand during MS analysis to give
+
cations assignable to [M – C
6
H
5
N – Rh/Ir(Cp*)Cl – 2PF
6
] .
The molecular structures of 1b–1d, 2a and 2c were determined by
single crystal X-ray diffraction analysis (Table S1). Single crystals were
grown by the slow diffusion of toluene into saturated solutions of the
respective complex in acetonitrile. All complexes display the charac-
teristic pseudooctahedral piano-stool configuration around the metal
centers, with the pyridylimine and Cl ligands forming the legs of the
6
5
stool and the
π
-bound cym or Cp* rings acting as the seat via
η
- or
η
-
coordination, respectively. Complexes 1b–1d, and 2c crystallized as
mixtures of enantiomers with both metal centers in each molecule
having the same configuration. In contrast, the two metal centers in each
molecule of 2a had opposite configurations. Complexes 1b–1d adopted
a triclinic crystal system and crystallized in the P-1 space group (Fig. 1,
Fig. S1 for 1d). Complexes 2a and 2c adopted a monoclinic crystal
system in the I2/a space group (Fig. 2, Table S1). The presence of a five-
membered ring, that forms upon metal coordination, confirms the N,N-
bidentate coordination mode of the bis-pyridylimine chelate ligands in
1
b–1d (as well as for 2a and 2c), which was also observed for the tet-
Fig. 2. ORTEP representations of one of the molecules of the complex cations
2a (top) and 2c (bottom) drawn at 50% probability levels. Co-crystallized
solvent molecules, and hexafluorophosphate counteranions have been
omitted for clarity.
BF4
rafluoroborate derivative of 1a, i.e., 1a
[40]. Epimerization can occur
in complexes closely related to these through chlorido ligand dissocia-
tion and re-coordination on the opposite side of the metallacyclic ring to
form a mixture of isomers in solution [41]. The presence of a single set of
1
peaks in the H NMR spectrum suggests that the epimerization process is
although the NMR data suggest that in solution rotation occurs rapidly
about the N2-C(X) and N4-C(Y) bonds. The pyridylimine units are not
co-planar to the central phenylene ring and have torsion angles of
1
fast relative to the H NMR timescale.
The two Cp* rings in each of the complexes 1c and 1d sit on opposite
◦
◦
sides of the bridging phenylene group in the solid state structures,
ꢀ 60.38 and ꢀ 72.95 for 1c, and ꢀ 106.72 and +60.85 for 1d. These
angles are presumably determined by a combination of steric repulsions,
π
-bond formation with the phenylene unit, intermolecular interactions
and crystal packing forces. A similar molecular geometry was reported
BF4
for structurally related dinuclear Ru complex 1a
[40]. In contrast, the
two cym rings in the solid state structure of the di-osmium derivative 1b
were oriented on the same side of the bridging phenylene group, in a
staggered geometry. The torsion angles between the pyridylimine units
◦
and the central phenylene ring are +48.21 and +48.40 for 1b. In
general, the other aspects of the structures do not differ significantly
upon altering the metal center. The differences in metal–ligand bond
lengths of 1b–1d are no greater than 0.01, 0.05 and 0.02 Å for the
M1–Cl1, M1–Npyridyl and M1–Nimine bonds, respectively (Table 1), and
the interatomic distance between the two metal centers (M1⋅⋅⋅M2) was
calculated to be ca. 8.4 Å. The Mꢀ Cl bond lengths were very close to the
average values for these types of complexes [42]. -Stacking between
π
the cym ring of one molecule of Os complex 1b and the phenylene bridge
of another was found at 3.35 Å.
Table 1
◦
Key bond lengths (Å) and angles ( ) in the molecular structures of complexes
BF4
1
b–1d, 2a, and 2c, in comparison to 1a
BF4 a
.
Bond/
angle
1a
1b
1c
1d
2a
2c
M1–Cl1
M1–N1
2.3863
2.3991
(15)
2.4082
(7)
2.4096
(8)
2.3972
(14)
2.4050
(5)
(
14)
2.099
4)
2.055
4)
76.50
14)
2.079
(5)
2.125
(2)
2.106
(3)
2.099
(4)
2.104
(2)
(
M1–N2
2.087
(5)
2.110
(2)
2.090
(3)
2.077
(4)
2.113
(2)
(
N1–M1–N2
76.0(2)
76.51
(9)
76.18
(11)
76.84
(17)
77.09
(7)
Fig. 1. ORTEP representations of one of the molecules of the complex cations
of 1b (top) and 1c (bottom) drawn at 50% probability levels. Co-crystallized
solvent molecules, and hexafluorophosphate counteranions have been
omitted for clarity.
(
a
Taken from Ref. [40].
3

T.R. Steel et al.
Inorganica Chimica Acta 518 (2021) 120220
Complex 2c with the central methylene bridge is structurally flexible
secondary peak pertaining to the H-8 of unbound EtG. Instead, a pre-
1
and its Cp* units sit on the same internal face of ligand 2. The distances
between the two metal centers were found to be ca. 13.3 and 9.2 Å for
complexes 2a and 2c, respectively, and were therewith significantly
greater than for complexes of 1. There were only slight differences in the
torsion angles involving the diphenylmethylene moieties and in the
cipitate formed that gave a H NMR spectrum identical to that of 1a.
Titration of EtG with DCl resulted in the H-8 signal of EtG to shift to
lower field, with the shift dependent on the amount of DCl added. This
may point to the possible formation of hydroxido ligands that prevent
binding of EtG to 1a.
metal–ligand bond lengths. In the structure of 2a, intermolecular
π
-in-
Furthermore, we investigated the reaction of 1a with the amino acids
L-methionine (Met), L-histidine (His) and L-cysteine (Cys) in 10%
teractions between the terminal pyridyl rings of adjacent molecules
were found at 3.38 Å.
6 2
/D
O by 1H NMR spectroscopy (data not shown). Similar
DMSO‑d
behavior as in the stability studies with DMSO‑d
6
2
/D O was observed in
2
.1. Stability studies in dimethylsulfoxide and aqueous solution
the reaction between 1a and Met. The spectra recorded in the first 2 h of
the reaction were virtually unchanged compared to those of 1a, but after
72 h minor peaks at around 9.0 and 9.8 ppm were observed, while only
minor changes in the region indicative of the cym protons were detected.
Many transition metal complexes undergo aquation under physio-
logical conditions, i.e., halido/aqua ligand exchange, which is important
for facilitating metal binding to biomolecular targets [43,44]. However,
many organometallic complexes have low aqueous solubility and
therefore, in these cases, biological assays usually require prior disso-
lution of the complex in a water-miscible solvent such as dimethylsulf-
oxide (DMSO). A complication of using DMSO as a solvent is that it has
the ability to coordinate to metal centers, for example, through halido
ligand exchange, and this may alter the structure and nature of the
pharmacophore and sometimes even lead to decomposition [45].
Consequently, preliminary 1H NMR spectroscopic studies were con-
1
In case of Cys and His, more significant changes in the H NMR spectra
were observed. Similar to Met, in case of reaction with Cys, the cym
protons did not change significantly, which is unusual as cym complexes
often decompose in the presence of Cys [52,53]. Moreover, additional
signals in the aromatic and aliphatic regions of the spectra indicated
coordination of Cys. Similarly, the reaction with His resulted in signif-
icant changes in the peak pattern in the aromatic region.
2
.3. In vitro cytotoxic activity
6 2
ducted for 1a in 10% DMSO‑d /D O as a representative example over a
period of 72 h (Fig. S2). In particular, we were interested in whether any
evidence could be obtained that indicated the Mꢀ Npyridyl and Mꢀ Nimine
bonds were retained during this time. Unfortunately, 1a had low solu-
bility in this solvent mixture and furthermore, a small amount of pre-
cipitate formed in the NMR tube over the timeframe of analysis. These
factors resulted in a low signal-to-noise ratio in the 1H NMR spectra,
especially those taken after 24 h. Comparison of these spectra with those
Ligands 1 and 2, and complexes 1a–1d and 2a–2d were subjected to
a sulforhodamine B cytotoxicity assay in the human cancer cell lines
HCT116 (colorectal carcinoma), NCI-H460 (non-small cell lung carci-
noma), SiHa (cervical carcinoma), and SW480 (colon adenocarcinoma)
(
Table 2). Compounds that gave mean half maximal inhibitory con-
centrations (IC50) of >100 µM were deemed to be inactive in that
particular cell line. In most cell lines, ligands 1 and 2 showed better
antiproliferative properties than their corresponding metal complexes.
Complex 1c was the only derivative of 1 with IC50 values less than 100
µM in all cell lines under the conditions used. Complexes 2a and 2b
showed minor activity only in SiHa and NCI-H460 cells, respectively,
whereas 2c and 2d showed activity in all cell lines except NCI-H460. 2c
and 2d were most potent in SiHa cells with IC50 values of 44 ± 1 and 45
taken of 1a in the presence of 100 mM NaCl in 10% DMSO‑d
suppress chlorido/aqua ligand exchange), or those taken after the
addition of 2 equiv. AgNO in 10% DMSO‑d /D O, (to remove the
6 2
/D O (to
3
6
2
chlorido ligand as AgCl) showed almost no differences between them at
all (Fig. S2). It is reasonable to assume the same product was formed in
each case, and this is most likely to be a derivative of 1a in which both
metal-bound chlorido ligands have been replaced either by DMSO, aqua
or hydroxido ligands. Formation of a derivative with aqua ligands is less
likely as this would result in a quadruply-charged complex for which
spontaneous deprotonation of the aqua ligands would be expected to
readily occur to give less labile hydroxido ligands [46]. In summary, on
the basis of the limited changes observed in the NMR spectra described
above, we assume that any substitution product of complex 1a formed
immediately upon dissolution and showed reasonable stability for 72 h
under the conditions investigated. The results indicate that, in biologi-
cally relevant solutions, the complexes retain their structural integrity
with the Mꢀ Npyridyl, Mꢀ Nimine and Mꢀ arene/Cp* bonds remaining
intact. Therefore, this was sufficient to allow for biological assays to be
carried out (Figs. S2 and S3).
±
4 µM, respectively. Notably, both Rh-based complexes 1c and 2c
showed moderate activity in almost all cell lines, while only the Ir
complex of 2, i.e., 2d, was moderately active.
3
. Conclusions
A series of homodinuclear MII/III(cym/Cp*)Cl complexes derived
from ditopic pyridylimine ligands have been synthesized. The syntheses
Table 2
IC50 values (µM) for ligands 1 and 2 and complexes 1a–1d and 2a–2d against
HCT116 (human colorectal carcinoma), NCI-H460 (human non-small cell lung
carcinoma), SiHa (human cervical carcinoma), and SW480 (human colon
adenocarcinoma) cancer cells as compared to cisplatin, expressed as mean ±
standard error (n = 3).
2
.2. Biomolecule interaction
Compound
HCT116
NCI-H460
SiHa
SW480
Many metal complexes are known to covalently bind to DNA, where
IC50
/μM
the nucleophilic N7 atom of guanine is often a preferential binding site
1
2
55 ± 20
21 ± 6
57 ± 6
31 ± 4
88 ± 4
42 ± 3
>100
[
47–50]. The potential for 1a to bind to DNA over 72 h was assessed by
H NMR spectroscopy using 9-ethylguanine (EtG) as a model compound
46 ± 5
1
1
1
1
a
b
c
>100
>100
>100
>100
>100
>100
>100
>100
in a 1a:EtG ratio of 1:2 in 10% DMSO‑d
chlorido/aqua/DMSO exchange that occurred could lead to activation of
a for biomolecule coordination and may allow for formation of 1a–EtG
6 2
/D O (Figs. S4 and S5). Any
70 ± 29
>100
61 ± 13
>100
70 ± 1
>100
73 ± 4
>100
1
1d
2a
adducts. In contrast, a hydroxido complex may be more inert towards
further substitution with EtG. The most prominent indication of EtG
binding to a metal center is usually the downfield shift of the signal
assigned to H-8 of EtG [51]. In this case, upon addition of EtG to 1a, a
downfield shift of only ca. 0.09 ppm was immediately observed. How-
ever, addition of a further 2 equiv. of EtG to this sample did not yield a
>100
>100
83 ± 23
>100
>100
2
2
2
b
c
>100
81 ± 6
>100
>100
78 ± 21
59 ± 31
44 ± 1
45 ± 4
52 ± 9
73 ± 5
d
>100
a
cisplatin
2.5 ± 0.3
0.8 ± 0.03
3.0 ± 0.6
8.1 ± 2.9
a
Taken from Ref. [54].
4

T.R. Steel et al.
Inorganica Chimica Acta 518 (2021) 120220
were facile and generated pure products in high yields. All complexes
were characterized by 1D and 2D NMR spectroscopy, ESI-MS, and
elemental analysis. Single crystals of complexes 1b–1d, 2a, and 2c were
analyzed by X-ray diffraction crystallography for structural validation.
The complexes crystallized as a mixture of isomers with the molecules
showing a variety of conformations, depending on both the ligand
structures and the metal center. In 1c and 1d, the Cp* rings sat on
opposite sides of 1, while in the structure of 1b both cym rings were
arranged on the same side of 1. Stability studies conducted as a prelude
for biological experiments indicated that complex 1a, or a rapidly
(40 mL), filtered and the solvent removed from the filtrate under
reduced pressure. However, in the case of complex 1b, the crude product
was dissolved in DCM : acetone (1 : 1, 40 mL), filtered, and the solvent
was removed under reduced pressure. The resulting residue was dried in
vacuo to obtain pure complexes in high yields.
2
4.3. [N,N’-(1,4-phenylene)(bis(1-(pyridin-2-yl)(methanimine)-ᴋ N,N’)
6
bis{chlorido(
η
-p-cymene)ruthenium(II)}] hexafluorophosphate 1a
The synthesis was performed according to the general procedure
formed hydrolysis product, was adequately stable in 10% DMSO‑d
6
/D
2
O
using 1 (104 mg, 0.36 mmol), [RuCl (cym)] (244 mg, 0.36 mmol), and
2
2
and DMSO‑d over a period of 72 h. Surprisingly, studies on the reac-
6
4 6
NH PF (1.174 g, 7.2 mmol) to afford the product 1a as an orange solid
◦
1
tivity with the DNA model EtG were inconclusive, while 1a formed
adducts with Cys and His but not with Met. The in vitro cytotoxicity
assays demonstrated limited cytotoxicity of the homodinuclear com-
plexes with the ligands being more potent than the derived complexes.
However, the Rh and Ir complexes 2c and 2d showed the best anti-
proliferative effect toward human cervical carcinoma cells, despite at
moderate IC50 values compared to the anticancer drug cisplatin.
(387 mg, 95%). M.p. 220 C (decomp.). H NMR (399.89 MHz, ace-
3
tone‑d , 298 K): δ 9.56 (d, J = 3 Hz, 2H, H-1), 8.93 (s, 2H, H-5),
6
3
4
8.34–8.24 (m, 4H, H-3,4), 8.06 (s, 4H, H-6), 7.81 (td, J = 6 Hz, J = 1
3
Hz, 2H, H-2), 6.05 (d, J = 4 Hz, 2H, H-8), 5.76–5.69 (m, 4H, H-8,9),
3
3
3
5.62 (d, J = 3 Hz, 1H, H-9), 5.57 (d, J = 3 Hz, 1H, H-9), 2.64 (sept, J
= 7 Hz, 2H, H-10), 2.19 (s, 3H, H-7), 2.18 (s, 3H, H-7), 1.06–0.99 ppm
(m, 12H, H-11). 13C{ H}DEPT-Q (100.55 MHz, acetone‑d , 298 K): δ
1
6
1
69.1 (C-5), 157.1 (C-1), 154.0 (C-4‘), 153.9 (C-6‘), 141.1 (C-3), 131.5
4
. Experimental section
(C-4), 130.3 (C-2), 125.0 (C-6), 107.0 (C-9‘), 104.9 (C-8‘), 87.9–86.6 (C-
3
1
1
8
,9), 32.0 (C-10), 22.3 (C-11), 19.0 ppm (C-7). P{ H} NMR (161.85
3
-
4
.1. Materials and methods
MHz, acetone‑d
6
, 298 K): δ –144.2 ppm (sept, J = 703 Hz, PF
6
). HRMS
+
2+
(
ESI ): m/z 414.0434 [M – 2PF
6
]
(mcalc = 414.0437). EA calculated
All air and moisture-sensitive reactions were carried out under a
for C38
H42Cl
2
F
12
N
4
P
2
2
Ru ⋅0.75CH
2
Cl
2
: C 39.39%, H 3.71%, N 4.74%.
2
nitrogen (N ) atmosphere, and light sensitive reactions were protected
Found: C 39.10%, H 3.93%, N 5.09%.
from photolytic degradation by covering the apparatus in aluminum foil.
Chemicals and solvents purchased from commercial suppliers were used
without further purification. Solvents were dried prior to use when
necessary. Solvents were evaporated under reduced pressure using a
2
4.4. [N,N’-(1,4-phenylene)(bis(1-(pyridin-2-yl)(methanimine)-ᴋ N,N’)
6
bis{chlorido( -p-cymene)osmium(II)}] hexafluorophosphate 1b
η
rotary evaporator. The precursor complexes [RuCl
OsCl (cym)] (b) [56], [RhCl (Cp*)] (c) [57,58], and [IrCl
d) [57,59], as well as the pyridylimine ligands 1 and 2 were prepared
according to literature procedures [38,39].
2
(cym)]
2
(a) [55],
The synthesis was performed according to the general procedure
[
2
2
2
2
2
(Cp*)]
2
using 1 (35 mg, 0.12 mmol), [OsCl (cym)] (98 mg, 0.12 mmol), and
2
2
(
4 6
NH PF (400 mg, 2.5 mmol) to afford the product 1b as a dark red solid
(131 mg, 82%). Single crystals suitable for Xray diffraction analysis were
grown by slow diffusion of toluene into a saturated solution of the
1
13
1
31
1
1
1
Both 1D ( H, and C{ H}DEPT-Q, P{ H}) and 2D ( H– H COSY,
1
1
1
13
1
13
◦
1
H– H NOESY, H C HSQC, H– C HMBC) NMR spectra were recor-
ded on Bruker DRX 400 MHz NMR spectrometers at ambient tempera-
complex in acetonitrile. M.p. 207–210 C. H NMR (399.89 MHz, ace-
3
3
tone‑d , 298 K): δ 9.64 (d, J = 3 Hz, 2H, H-1), 9.44 (d, J = 1 Hz, 2H, H-
6
1
13
1
31
1
3
ture. The measurement frequencies for H, C{ H}, and P{ H} NMR
were 399.89, 100.55 and 161.85 MHz, respectively. Deuterated chlo-
5), 8.58–8.54 (m, 2H, H-4), 8.38 (t, J = 8 Hz, 2H, H-3), 8.10 (s, 4H, H-
3
6), 7.94–7.88 (m, 2H, H-2), 6.45 (d, J = 3 Hz, 2H, H-8), 6.10 (m, 4H, H-
3
3
roform, acetone‑d
6
, D
2
O, and DMSO‑d
6
were used as NMR solvents. High
8,9), 5.89 (d, J = 3 Hz, 1H, H-9), 5.86 (d, J = 3 Hz, 1H, H-9), 2.64
3
resolution mass spectrometry (HRMS) data were recorded on a Bruker
microTOF-Q II mass spectrometer in positive ion electrospray ionization
(sept, J = 7 Hz, 2H, H-10), 2.42–2.33 (m, 6H, H-7), 1.15–1.02 ppm (m,
12H, H-11). 13C{ H}DEPT-Q (100.55 MHz, acetone‑d , 298 K): δ 170.2
1
6
(
ESI) mode. Melting and decomposition points were recorded on a Stuart
(C-5), 157.0 (C-4‘), 156.6 (C-1), 154.1 (C-6‘), 141.1 (C-3), 131.2 (C-4),
SMP50 automatic melting point apparatus. X-ray diffraction measure-
ments of single crystals were conducted on a Rigaku Oxford Diffraction
XtaLAB Synergy-S single-crystal diffractometer with a PILATUS 200 K
131.0 (C-2), 125.2 (C-6), 99.5 (C-9‘), 98.9 (C-8‘), 79.9 (C-8), 79.2 (C-8),
3
1
1
77.2 (C-9), 76.8 (C-9), 32.1 (C-10), 22.5 (C-11), 18.8 ppm (C-7). P{ H}
3
NMR (161.85 MHz, acetone‑d , 298 K): δ –144.2 ppm (sept, J = 705 Hz,
6
-
+
2+
6
hybrid pixel array detector using Cu K
α
radiation (λ = 1.54184 Å). The
PF ). HRMS (ESI ): m/z 503.1021 [M – 2PF ] (m
= 503.0980). EA
6
calc
structure solution and refinements were performed with the SHELXS-97,
SHELXL-2016 [60] and Olex2 program packages [61,62]. Molecular
structures were visualized using Mercury 4.0.0. Elemental analyses (EA)
were carried out on the vario EL cube CHNOS Elemental analyzer at the
University of Auckland for Ru, Rh, and Ir complexes, and at the Camp-
bell Microanalytical Laboratory, the University of Otago for Os
complexes.
calculated for C38
H
Cl F N P Os ⋅0.5NH PF : C 33.13%, H 3.22%, N
42
2
12
4
2
2
4
6
4.58%. Found: C 33.31%, H 3.27%, N 4.73%.
2
4.5. [N,N’-(1,4-phenylene)(bis(1-(pyridin-2-yl)(methanimine)-ᴋ N,N’)
5
bis{chlorido( -pentamethylcyclopentadienyl)rhodium(III)}]
η
hexafluorophosphate 1c
The synthesis was performed according to the general procedure
4
.2. General procedure for the synthesis of homodinuclear complexes
2 2
using 1 (98 mg, 0.34 mmol), [RhCl (Cp*)] (211 mg, 0.34 mmol), and
1
a–1d and 2a–2d
NH PF (1.117 g, 6.9 mmol) to afford the product 1c as a bright yellow
4
6
solid (319 mg, 82%). Single crystals suitable for X-ray diffraction anal-
ysis were grown by slow diffusion of toluene into a saturated solution of
Pyridylimine ligands 1 or 2 (1.0 equiv.) were added to a solution of
◦
1
dimeric metal precursor complex (a–d, 1.0 equiv.) in DCM : MeOH (60
mL, 1 : 1) and stirred at r.t. overnight. The solvent was removed under
reduced pressure. The crude product was dissolved in MeOH (20 mL)
the complex in acetonitrile. M.p. 221 C (decomp.). H NMR (399.89
3
3
MHz, acetone‑d
6
, 298 K): δ 9.23 (d, J = 2 Hz, 2H, H-1), 9.13 (dd, J = 4
4
Hz, J = 1 Hz, 2H, H-5), 8.50–8.42 (m, 4H, H-3,4), 8.12–8.03 (m, 6H, H-
2,6), 1.66 ppm (s, 30H, H-7). 13C{ H}DEPT-Q (100.55 MHz, acetone‑d
1
and a solution of NH
4
PF
6
(20 equiv.) in MeOH (20 mL) was added. The
6
,
resulting mixture was stirred for 1 h, was concentrated under reduced
pressure and chilled overnight. The precipitate was filtered, washed
with cold MeOH, and dried. The crude product was dissolved in DCM
298 K): δ 170.0 (C-5), 155.0 (C-4‘), 154.2 (C-1), 150.5 (C-6‘), 141.7 (C-
3
1
3), 131.6 (C-4), 131.3 (C-2), 125.1 (C-6), 98.7 (C-7‘), 9.4 ppm (C-7).
P
1
3
{ H} NMR (161.85 MHz, acetone‑d
6
, 298 K): δ –144.3 ppm (sept, J =
5

T.R. Steel et al.
Inorganica Chimica Acta 518 (2021) 120220
-
+
7
15 Hz, PF
6
). HRMS (ESI ): m/z 470.0887 [M – C
6
H
5
N – Rh(Cp*)Cl –
K): δ 168.6 (C-5), 157.3 (C-4‘), 156.5 (C-1), 151.6 (C-6‘), 144.1 (C-7‘),
141.0 (C-3), 130.9 (C-4), 130.7 (C-2), 124.2 (C-6,7), 99.0 (C-11‘), 98.7
(C-12‘), 79.8, (C-11) 79.2 (C-11), 77.1 (C-12), 76.9 (C-12), 41.3 (C-8),
+
2
PF
6
]
(mcalc
Rh
=
470.0865).
EA
calculated for
C
38
H
44Cl
2
F
12
4
N P
2
2
⋅0.25CH
2
Cl : C 40.14%, H 3.92%, N 4.89%.
2
3
1
1
Found: C 40.12%, H 4.24%, N 4.88%.
32.1 (C-10), 22.6 (C-9), 18.8 ppm (C-13). P{ H} NMR (161.85 MHz,
3
-
+
acetone‑d
6
, 298 K): δ –144.2 ppm (sept, J = 701 Hz, PF
6
). HRMS (ESI ):
2
2+
4
.6. [N,N’-(1,4-phenylene)(bis(1-(pyridin-2-yl)(methanimine)-ᴋ N,N’)
bis{chlorido(
m/z 548.1233 [M – 2PF
6
]
(mcalc = 548.1214). EA calculated for
5
η
-pentamethylcyclopentadienyl)iridium(III)}]
C
45
H48Cl
2 12 4 2 2
F N P Os : C 38.99%, H 3.49%, N 4.04%. Found: C 38.86%,
hexafluorophosphate 1d
H 3.48%, N 4.07%.
The synthesis was performed according to the general procedure
4.9. [N,N’-(methylenebis(4,1-phenylene))(bis(1-(pyridin-2-yl)
2
5
using 1 (85 mg, 0.30 mmol), [IrCl
NH PF (969 mg, 5.9 mmol) to afford the product 1d as a dark red solid
318 mg, 82%). Single crystals suitable for X-ray diffraction analysis
were grown by slow diffusion of toluene into a saturated solution of the
2
(Cp*)]
2
(237 mg, 0.30 mmol), and
(methanimine)-ᴋ N,N’)bis{chlorido(
rhodium(III)}] hexafluorophosphate 2c
η
-pentamethylcyclopentadienyl)
4
6
(
The synthesis was performed according to the general procedure
using 2 (110 mg, 0.07 mmol), [RhCl (Cp*)] (180 mg, 0.07 mmol), and
NH PF (954 mg, 1.5 mmol) to afford the product 2c as a bright yellow
◦
1
complex in acetonitrile. M.p. 242 C (decomp.). H NMR (399.89 MHz,
2
2
3
acetone‑d
6
, 298 K): δ 9.52–9.47 (m, 2H, H-5), 9.24 (d, J = 3 Hz, 2H, H-
4
6
3
3
4
1
8
3
), 8.55 (d, J = 5 Hz, 2H, H-3), 8.43 (td, J = 7 Hz, J = 1 Hz, 2H, H-4),
solid (334 mg, 94%). Single crystals suitable for X-ray diffraction anal-
ysis were grown by slow diffusion of toluene into a saturated solution of
.10–8.08 (m, 4H, H-6), 8.08–8.03 (m, 2H, H-2), 1.68–1.56 ppm (m,
0H, H-7). 13C{ H}DEPT-Q (100.55 MHz, acetone‑d
1
, 298 K): δ 170.7
the complex in acetonitrile. M.p. 198 C (decomp.). H NMR (399.89
◦
1
6
3
(
C-5), 156.5 (C-4‘), 153.3 (C-1), 151.0 (C-6‘), 141.6 (C-3), 131.7 (C-2),
MHz, acetone‑d
6
, 298 K): δ 9.11 (d, J = 3 Hz, 2H, H-1), 8.89 (s, 2H, H-
3
1
1
3
1
31.3 (C-4), 125.1 (C-6), 91.2 (C-7‘), 8.6 ppm (C-7). P{ H} NMR
5), 8.43–8.36 (m, 2H, H-3), 8.32 (d, J = 5 Hz, 2H, H-4), 8.06–7.97 (m,
3
-
3
3
(
161.85 MHz, acetone‑d
6
, 298 K): δ –144.3 ppm (sept, J = 704 Hz, PF
6
).
2H, H-2), 7.77 (d, J = 5 Hz, 4H, H-6), 7.60 (d, J = 4 Hz, 4H, H-7), 4.29
+
+
13 1
HRMS (ESI ): m/z 560.1461 [M – C
60.1431). EA calculated for C38
6
H
5
N – Ir(Cp*)Cl – 2PF
6
]
(mcalc
=
(s, 2H H-8), 1.55 ppm (s, 30H, H-9). C{ H}DEPT-Q (100.55 MHz,
acetone‑d , 298 K): δ 168.6 (C-5), 155.7 (C-4‘), 154.3 (C-1), 148.6 (C-6‘),
144.6 (C-7‘), 142.0 (C-3), 131.5 (C-4,7), 131.4 (C-2), 124.3 (C-6), 99.1
5
H
44Cl
2
F
12
4
N P
Ir
2 2
: C 35.05%, H 3.41%,
6
N 4.30%. Found: C 34.99%, H 3.56%, N 4.19%.
3
1
1
(
C-9‘), 41.9 (C-8), 9.3 ppm (C-9). P{ H} NMR (161.85 MHz, ace-
3
-
6
+
4
.7. [N,N’-(methylenebis(4,1-phenylene))(bis(1-(pyridin-2-yl)
tone‑d
6
, 298 K): δ –144.3 ppm (sept, J = 717 Hz, PF
). HRMS (ESI ): m/
2
6
2+
(
methanimine)-ᴋ N,N’)bis{chlorido(
η
-p-cymene)ruthenium(II)}]
z 461.0757 [M – 2PF
C
6
]
(mcalc = 461.0762). EA calculated for
hexafluorophosphate 2a
45
H
48Cl
2
F
12
N
P
4 2
Rh
2
⋅0.5CH
2 2
Cl : C 43.51%, H 4.09%, N 4.46%. Found:
C 43.42%, H 4.05%, N 4.49%.
The synthesis was performed according to the general procedure
using 2 (41 mg, 0.11 mmol), [RuCl (cym)] (67 mg, 0.11 mmol), and
NH PF (352 mg, 2.2 mmol) to afford the product 2a as a bright orange
2
2
4.10. [N,N’-(methylenebis(4,1-phenylene))(bis(1-(pyridin-2-yl)
2
5
4
6
(methanimine)-ᴋ N,N’)bis{chlorido(
iridium(III)}] hexafluorophosphate 2d
η
-pentamethylcyclopentadienyl)
solid (100 mg, 76%). Single crystals suitable for X-ray diffraction anal-
ysis were grown by slow diffusion of toluene into a saturated solution of
◦
1
the complex in acetonitrile. M.p. 206–213 C. H NMR (399.89 MHz,
The synthesis was performed according to the general procedure
using 2 (66 mg, 0.06 mmol), [IrCl (Cp*)] (139 mg, 0.06 mmol), and
NH PF (567 mg, 1.2 mmol) to afford the product 2d as a red solid (210
mg, 87%). M.p. 222–232 C. H NMR (399.89 MHz, acetone‑d
3
3
acetone‑d
6
, 298 K): δ 9.64 (d, J = 3 Hz, 2H, H-1), 8.92 (d, J = 3 Hz, 2H,
2
2
H-5), 8.39–8.32 (m, 4H, H-3,4), 7.95–7.87 (m, 6H, H-2,6), 7.65–7.56
4
6
3
3
◦
1
(
m, 4H, H-7), 6.11 (d, J = 3 Hz, 2H, H-12), 5.79 (d, J = 3 Hz, 2H, H-
6
, 298 K):
δ 9.34 (s, 2H, H-5), 9.18 (d, J = 3 Hz, 2H, H-1), 8.50 (d, J = 5 Hz, 2H,
3
3
3
3
1
2
1), 5.75 (d, J = 3 Hz, 2H, H-12), 5.63 (d, J = 3 Hz, 2H, H-11), 4.32 (s,
3
3
4
H, H-8), 2.72 (sept, J = 7 Hz, 2H, H-10), 2.30 (s, 6H, H-13), 1.11 ppm
H-4), 8.38 (td, J = 8 Hz, J = 1 Hz, 2H, H-3), 8.03–7.99 (m, 2H, H-2),
3
13
1
3 4
(
d, J = 4 Hz, 12H, H-9). C{ H}DEPT-Q (100.55 MHz, acetone‑d
6
, 298
7.79–7.75 (m, 4H, H-6), 7.60 (dd, J = 4 Hz, J = 1 Hz, 4H, H-7), 4.30 (s,
2H, H-8), 1.55 ppm (s, 30H, H-9). 13C{ H}DEPT-Q (100.55 MHz, ace-
1
K): δ 167.7 (C-5), 156.9 (C-1), 155.9 (C-4‘), 151.6 (C-6‘), 144.0 (C-7‘),
1
1
4
40.9 (C-4), 131.0 (C-7), 130.9 (C-3), 129.9 (C-2), 123.8 (C-6), 107.4 (C-
tone‑d
6
, 298 K): δ 169.0 (C-5), 156.9 (C-4‘), 153.3 (C-1), 148.6 (C-6‘),
1‘), 104.8 (C-12‘), 87.4 (C-11), 87.1 (C-12), 86.7 (C-11), 86.4 (C-12),
144.1 (C-7‘), 141.5 (C-3), 131.4 (C-2), 130.9 (C-4,7), 123.9 (C-6), 91.3
3
1
1
31
1
1.5 (C-8), 31.9 (C-10), 22.3 (C-9), 18.9 ppm (C-13). P{ H} NMR
(C-9‘), 41.4 (C-8), 8.6 ppm (C-9). P{ H} NMR (161.85 MHz, ace-
3
-
3
-
+
(
161.85 MHz, acetone‑d
6
, 298 K): δ –144.2 ppm (sept, J = 705 Hz, PF
6
).
tone‑d
6
, 298 K): δ –144.4 ppm (sept, J = 715 Hz, PF
6
). HRMS (ESI ): m/
+
2+
2+
HRMS (ESI ): m/z 459.0668 [M – 2PF
calculated for C45
6
]
(mcalc = 459.0672). EA
z 551.1333 [M – 2PF
6
]
(mcalc = 551.1319). EA calculated for
H
48Cl
2
F
12
N
4
P
Ru
2 2
⋅1H
2
O: C 44.09%, H 4.11%, N
C
45
H48Cl
2
F
12
N
P
4 2
Ir
2
⋅0.5CH
2 2
Cl : C 38.09%, H 3.58%, N 3.91%. Found: C
4
.57%. Found: C 43.72%, H 4.173%, N 4.75%.
37.89%, H 3.48%, N 3.96%.
4
.8. [N,N’-(methylenebis(4,1-phenylene))(bis(1-(pyridin-2-yl)
4.11. DMSO and aqueous stability studies
2
6
(
methanimine)-ᴋ N,N’)bis{chlorido(
η
-p-cymene)osmium(II)}]
hexafluorophosphate 2b
DMSO Stability studies were conducted by dissolving ca. 1 mg of the
1
complex in DMSO‑d
6
(0.5 mL). H NMR spectra were recorded at t = 0,
The synthesis was performed according to the general procedure
using 2 (78 mg, 0.05 mmol), [OsCl (cym)] (164 mg, 0.05 mmol), and
NH PF (675 mg, 0.92 mmol) to afford the product 2b as a dark red solid
2, 6, 24, 48, and 72 h to determine compound stability in 100% DMSO.
Hydrolysis stability studies were conducted similarly by dissolving
2
2
4
6
ca. 1 mg of the complex in DMSO‑d
(0.45 mL) to form a 10% DMSO‑d /D
recorded at t = 0, 2, 6, 24, 48, and 72 h to determine the stability of the
complex in aqueous solutions, as modelled by D O. After 72 h, AgNO
3
6 2
(0.05 mL) and diluting it with D O
◦
1
1
(
209 mg, 73%). M.p. 204–211 C. H NMR (399.89 MHz, acetone‑d
6
,
6
2
O solution. H NMR spectra were
3
3
2
98 K): δ 9.46 (d, J = 3 Hz, 2H, H-1), 9.19 (d, J = 3 Hz, 2H, H-5), 8.36
3
3
4
(
d, J = 3 Hz, 2H, H-4), 8.20 (td, J = 6 Hz, J = 1 Hz, 2H, H-3),
2
3
4
7
7
2
4
0
.77–7.72 (m, 2H, H-2), 7.70 (dd, J = 4 Hz, J = 1 Hz, 4H, H-6),
(2 equiv.) was added to force hydrolysis. After vigorous shaking, the
3
3
1
.48–7.44 (m, 4H, H-7), 6.27 (d, J = 3 Hz, 2H, H-11), 5.86 (d, J = 3 Hz,
yellow suspension was filtered and a H NMR spectrum of the filtrate
3
3
H, H-12), 5.82 (d, J = 3 Hz, 2H, H-11), 5.65 (d, J = 3 Hz, 2H, H-12),
was recorded for the hydrolyzed complex as a comparison to the pre-
vious samples. In addition, compounds that demonstrated reasonable
levels of hydrolysis over the first 6 h were tested by the addition of 100
3
.20 (s, 2H, H-8), 2.46 (sept, J = 7 Hz, 2H, H-10), 2.23 (s, 6H, H-13),
1
3
1
6
.93 ppm (m, 12H, H-9). C{ H}DEPT-Q (100.55 MHz, acetone‑d , 298
6

T.R. Steel et al.
Inorganica Chimica Acta 518 (2021) 120220
mM NaCl in D
2
O (0.45 mL) to a solution of ca. 1 mg of the complex in
Appendix A. Supplementary data
ꢀ
DMSO‑d
6
(0.05 mL) to approximate the standard [Cl ] in blood.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ica.2020.120220.
4
.12. Reactivity studies with biomolecules
References
The biomolecule interactions of complexes with 9-ethylguanine
EtG, AK Scientific, 98%), L-methionine (Met), L-histidine (His) and L-
(
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cysteine (Cys) were studied by H NMR spectroscopy. Stock solutions of
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lution of ca. 1 mg of 1a in DMSO‑d (0.05 mL) and D O (0.2 mL) to form
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2
[
[
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a 10% DMSO‑d
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/D
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[
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α
-MEM supplemented with 5% FCS at
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◦
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3
7 C in a humidified incubator with 5% CO
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(
HCT116, NCI-H460), 4000 (SiHa) and 5000 (SW480) cells per well in
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2
688–2702.
9
[13] E´ .A. Enyedy, O. D o¨ m o¨ t o¨ r, C.M. Hackl, A. Roller, M.S. Novak, M.A. Jakupec, B.
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plates in a series of 3-fold dilutions in 0.5% DMSO at the highest con-
centration for 72 h before the assay was terminated by addition of 10%
[
14] B.T. Elie, J. Fern ´a ndez-Gallardo, N. Curado, M.A. Cornejo, J.W. Ramos, M. Contel,
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