Synthesis and antitumour evaluation of mono- and multinuclear [2+1] tricarbonylrhenium(I) complexes

Article abstract of DOI:10.1016/j.ejmech.2018.08.011

A series of mono- and multinuclear [2 + 1] Re(I) tricarbonyl complexes were synthesised and evaluated as antiproliferative agents against the epithelial carcinoma (A431), colon carcinoma (DLD-1) and ovarian cancer (A2780) tumour cell lines and a healthy fibroblast (BJ) cell line. The compounds have moderate to good activity against the tumour cell lines, with the trinuclear and tetranuclear complexes showing selective cytotoxicity towards the tumour cell lines. The Re(I) complexes were found to influence programmed cell death mechanisms in vitro. They were able to inhibit the soluble form of the Fas receptor in malignant cells, allowing the Fas domain to receive the apoptotic signal through an extrinsic pathway. The complexes augmented the pro-apoptotic Bax-α concentrations, with the tetranuclear complex more superior at modulating the Bax-α molecular target in all tested cell lines, which influenced its cell growth inhibitory activity. The tetranuclear complex has the best activity against all tumour cell lines.

Full text of DOI:10.1016/j.ejmech.2018.08.011

Accepted Manuscript
Synthesis and antitumour evaluation of mono- and multinuclear [2+1]
tricarbonylrhenium(I) complexes
Dylan Giffard, Eva Fischer-Fodor, Catalin Vlad, Patriciu Achimas-Cadariu, Gregory S.
Smith
PII:
S0223-5234(18)30669-X
10.1016/j.ejmech.2018.08.011
EJMECH 10622
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 June 2018
Revised Date: 3 August 2018
Accepted Date: 4 August 2018
Please cite this article as: D. Giffard, E. Fischer-Fodor, C. Vlad, P. Achimas-Cadariu, G.S. Smith,
Synthesis and antitumour evaluation of mono- and multinuclear [2+1] tricarbonylrhenium(I) complexes,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.011.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and antitumour evaluation of mono- and multinuclear [2+1]
tricarbonylrhenium(I) complexes
Dylan Giffarda, Eva Fischer-Fodorb, Catalin Vladd,e, Patriciu Achimas-Cadariud,e
Gregory S. Smith*a
a
Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
b
Tumour Biology Department, The Oncology Institute „Prof. Dr. Ion Chiricuţă”, Cluj-Napoca 400015,
Romania
c
Medfuture- The Research Center for Advanced Medicine, “Iuliu Hatieganu” University of Medicine and
Pharmacy, 400337 Cluj Napoca, Romania
d Department of Surgery, The Oncology Institute „Prof. Dr. Ion Chiricuţă”, 400015 Cluj Napoca, Romania
e
Department of Surgery and Gynecological Oncology, “Iuliu Hatieganu” University of Medicine and
Pharmacy, 400337 Cluj Napoca, Romania
* Corresponding author. Tel.: +27-21-6505279.
E-mail address: gregory.smith@uct.ac.za
Keywords: Tricarbonylrhenium(I) complexes; Metallodendrimers; Cytotoxicity; Antitumour
activity

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Abstract
A series of mono- and multinuclear [2 + 1] Re(I) tricarbonyl complexes were synthesised and
evaluated as antiproliferative agents against the epithelial carcinoma (A431), colon
carcinoma (DLD-1) and ovarian cancer (A2780) tumour cell lines and a healthy fibroblast
(BJ) cell line. The compounds have moderate to good activity against the tumour cell lines,
with the trinuclear and tetranuclear complexes showing selective cytotoxicity towards the
tumour cell lines. The Re(I) complexes were found to influence programmed cell death
mechanisms in vitro. They were able to inhibit the soluble form of the Fas receptor in
malignant cells, allowing the Fas domain to receive the apoptotic signal through an extrinsic
pathway. The complexes augmented the pro-apoptotic Bax-α concentrations, with the
tetranuclear complex more superior at modulating the Bax-α molecular target in all tested cell
lines, which influenced its cell growth inhibitory activity. The tetranuclear complex has the
best activity against all tumour cell lines.

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1. Introduction
Since the discovery of the anticancer activity of the metallodrug, cisplatin and related
platinum-based drugs, metal-based complexes have been at the forefront of rational cytotoxic
drug design [1]. Metal-based complexes possess notable physicochemical properties when
compared to their purely organic counterparts, many of which are somewhat unique and of
interest in the context of medicinal chemistry. These interesting properties are due to the
inclusion of a metal which allows structural diversity, redox properties, and ligand exchange,
all of which can be advantageous in a biological setting [1]. However, cisplatin and related Pt
complexes display physiological side-effects, such as toxicity towards healthy, non-
tumourigenic cells, and inherent or developed chemoresistance [2-4]. New metal-based
complexes are therefore needed to overcome the failings observed in current anti-cancer drug
regimens and to meet the targeted drug discovery challenges [5].
Rhenium carbonyl complexes have been shown to display a wide range of biological
properties, including anticancer [6-13], antimalarial [14] and antitrypanosomal [15]
properties. Most noteworthy are Re(I) tricarbonyl complexes that show potent
antiproliferative activity against a variety of cancerous cell lines. In particular, reports of a
series of Re(I) tricarbonyl complexes have low micromolar IC50 values (1 – 5 µM) against
HeLa cells [7, 16], while [2 + 1] Re(I) tricarbonyls show potent activity (IC50 = 2 – 10 µM)
against the breast cancer MDA-MB-468 [10] and HeLa [8, 12, 17] cell lines. The Re(I)
tricarbonyl complexes are also capable of triggering apoptosis in tumour cells [18] as a
possible mechanism of action. It is therefore important to elucidate the in vitro cell death
mechanisms by studying the interaction with intrinsic and extrinsic apoptotic targets. Two
members of the Bcl-2 protein family of value to this study are the pro-apoptotic Bax-α (B cell
leukemia/lymphoma-2 associated X), and the anti-apoptotic Bcl-xL (B-cell lymphoma-extra
large) proteins. These molecules were first studied in lymphoma and later identified in almost
all cell types, including solid tumours and normal cells. The proteins exert antagonistic (pro-
apoptotic versus prosurvival) effects on intrinsic mitochondrial programmed cell death
pathways, but also on invasion and metastasis [19] in tumour cell populations. Another
participant in cell death mechanism is the Fas (first apoptosis signal) receptor, a member of
the large group of tumour necrosis factor receptors on the cell membrane. Together with its
ligand, FasL, is involved in the extrinsic regulation of cell death in tumours via the Fas/FasL
pathway. This pathway is targeted by metal-based chemotherapy agents which act against the
colon [20], ovarian [21] or epidermal [22] carcinoma.

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Several research groups have revealed that using the [2 + 1] mixed-ligand approach,
consisting of a metal ion with one bidentate ligand and one monodentate ligand, is a useful
tool in the art of rational drug design. Mundwiler et al. described the [2+1] approach as
comprising a bidentate ligand (displacing two of the aqua ligands of a fac-[Re(CO)3(OH2)3]+
entity) which can influence the overall lipophilicity of the tricarbonyl complex, whilst the
third [+1] aqua site is occupied by a monodentate ligand (acting as a linker to a biomolecule)
[20, 21]. The advantage of a [2+1] mixed-ligand complexation is that the apical [+1] site can
be changed to modulate the physical characteristics of a compound, such as solubility,
lipophilicity, and permeability. Cationic bipyridyl Re(I) tricarbonyl complexes display great
potential in this regard, particularly, in the field of anticancer research [10, 12, 23]. With this
approach, either of the two ligands can be modified to achieve the desired properties, such as
introducing water-solubilising groups on the 2,2’-bipyridyl moiety. It is important to note that
Re(I) tricarbonyl diimine complexes have been studied extensively as potential
electrochemiluminescent probes, based on their lowest excited state properties and high
quantum emission efficiencies from metal-to-ligand charge-transfer excited state.[17, 24, 25]
In this work, we designed, synthesised and characterised three different pyridine-based
ligands with [Re(bpy)(CO)3] to yield mono-, tri- and tetranuclear [2 + 1] complexes. The
dendritic nature allows for the exploitation of the multivalent effect exhibited by dendrimers,
which may result in improved site-specific drug delivery and reduced systemic exposure [26,
27]. The complexes were evaluated in vitro as potential antitumour agents against A431
(epithelial carcinoma), DLD-1 (colon carcinoma), and A2780 (ovarian carcinoma) cancer cell
lines, in addition to normal fibroblast cells (BJ). The mechanism of cell death was assessed
quantifying their capacity to trigger apoptosis, by measuring the intracellular level of Bax-α
and Bcl-xL, and by evaluating the influence of the complexes on extracellular Fas/FasL
signalling.
2. Results and discussion
2.1 Synthesis and characterisation
Metal precursor 1 and ligand precursors 3-7 were prepared following published literature
methods [28-31]. After a successful Schiff-base condensation reaction, ligands 6, 7 and 8
were prepared (from 1-3 respectively) via reduction of the corresponding ligand precursors
using sodium borohydride in methanol (Scheme 1). The corresponding complexes 9, 10 and
11 were prepared in a one-pot reaction by first abstracting the halide using silver triflate,

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followed by addition of the corresponding ligand in the appropriate stoichiometric ratio, to
afford the corresponding [2 + 1] complex. The rhenium(I) complexes were isolated as the
hexafluorophosphate salts through an exchange metathesis reaction using NH4PF6. The
complexes 9 – 11 were isolated in very low yields, likely due to the need to use the metal
precursor in excess to ensure functionalisation of all dendritic arms, and in part, due to the
similar solubilities of the metal precursor and the products. The complexes are air-stable, and
soluble in DCM, methanol, ethanol, acetone, and DMSO.
Scheme 1. (i) 2,2’-bipyridyl, toluene, 110 °C, 2 h. (ii) AgOTf, DCM, room temperature, 3
h. (iii) n-propylamine, tris-(2-aminoethyl)amine or DAB-AM-4, DCM, room temperature,
24 h. (iv) NaBH4, MeOH, room temperature, 24 h. (v) DCM, 24 h, followed by addition of
NH4PF6 and another hour of stirring.
The new cationic complexes 9 – 11 were fully characterised using standard analytical and
1
spectroscopic techniques, such as H, 13C{1H} and 31P{1H} NMR, infrared spectroscopy,
1
high-resolution electrospray ionisation mass spectrometry, and elemental analysis. The H
NMR spectra of complexes 9 – 11 show similar signals for the aromatic peaks, notably with
the main differences being in the aliphatic signals of the respective cores. Integration of the
1H NMR spectra of complexes 9 – 11 show that the bipyridyl moiety has been added to the

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ligand in the desired ratio. The characteristic peak attributed to the picolyl methylene protons
(Hd) appear at ca. δ 3.80 ppm and display no significant shift upon coordination, suggesting
that coordination occurs via the pyridyl nitrogen and not the secondary amine nitrogen. A
downfield shift of the aromatic pyridyl signals (Hb) from ca. δ 7.30 ppm to ca. δ 7.43 ppm
upon complexation further confirm the bonding modes of the complexes. The 13C{1H} NMR
spectra of the complexes 9 – 11 all display signals for the carbonyls in the range of 190 – 196
ppm, aromatic carbon signals in the range of 120 – 160 ppm, and aliphatic signals in the
range of 10 – 55 ppm. The infrared spectra show a distinct shift of the aromatic C=N
stretching
band
from
1600
cm-1
to
1619 cm-1 upon coordination. This shift to higher wavenumbers is due to backdonation from
the Re(I) centre to the pyridyl ring, increasing electron density in the C=N bond. This
confirms coordination of the Re(I) centre to the ligand via the pyridyl nitrogen. In addition,
high-resolution ESI-mass spectra for the complexes 9 – 11 confirm the presence of the
desired structures. Peaks for complexes 9 and 10 correspond to the molecular ion in the
-
absence of the PF6 counter ions ([M-PF6]+ and [M-3PF6]3+, respectively), m/z = 577.12 and
566.07, respectively. The tetranuclear complex displays a peak for the fragment [M-CO-
4PF6]4+ with an m/z value of 590.08. Elemental analysis was used to confirm the structural
integrity and purity of the complexes. All results were within reasonable limits, confirming
high purity of the samples. Several solvent molecules were present in the sample of complex
11, as confirmed by 1H NMR, as dendrimers are known to incorporate solvent molecules and
are notoriously difficult to dry [32].
2.2 Antiproliferative studies
The Re(I) tricarbonyl complexes 9, 10 and 11 were tested for their in vitro cytotoxicity
against epithelial carcinoma (A431), colon carcinoma (DLD-1) and ovarian cancer (A2780)
cancerous cell lines, in addition to normal fibroblast cells (BJ). The MTT assay was used to
determine the cytotoxicity of the compounds. The extent of inhibition is displayed as an IC50
value, which is defined as the concentration required to inhibit cell growth to half. In parallel,
three standard platinum-based cytotoxic drugs were tested, in the same concentration range as
the Re(I) complexes; the results are presented in Table 1.

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Table 1
IC50 values of Re(I) complexes 9, 10 and 11 and reference platinum-based drugs against
cancerous (A431, DLD-1, A2780) and normal (BJ) cell lines
IC50 (µM)b
Compound
na
A431
DLD-1
A2780
BJ
1
3
4
1
1
1
57.84 ± 3.67
46.64 ± 21.89
14.09 ± 2.23
17.17± 2.97
17.85 ± 1.79
15.04 ± 2.20
10.18 ± 0.47
58.85± 6.55
26.61 ± 3.66
36.45 ± 3.54
6.38 ± 1.18
37.30 ± 5.69
81.57 ± 5.18
18.97 ± 1.76
58.82 ± 7.65
17.69 ± 1.50
27.05 ± 4.14
86.50 ± 14.53
9
10
11
Cisplatinc
Oxaliplatinc
Carboplatinc
106.61 ± 10.16 93.58 ± 14.79
138.73 ± 15.09 175.70 ± 13.01 101.13 ± 19.05 120.07 ± 21.99
a number of metal centres per molecule
b IC50 value ± standard error
c Standard clinically approved Pt(II) drugs
The tested complexes show moderate to good cytotoxicity against the A2780, A431 and
DLD-1 tumour cell lines, and are toxic to some extent against the normal BJ cells. Complex
11 show the best inhibitory effects against all the tested cell lines. No selectivity is observed
in the case of complex 9, as the IC50 values are similar against the tumour and normal cells.
Complexes 10 and 11 showed selectivity, as significantly higher IC50 values were observed
against the healthy BJ cell line, which indicates a lower toxicity towards normal cells. In
the DLD-1 cells, complexes 10 and 11 revealed a good therapeutic window. The selectivity
was moderated in A431 cells, and complex 11 was particularly selective against the A2780
cells as well. In general, the complexes are the most effective inhibitors against the colon
carcinoma cell line (DLD-1) and have the least potent cytotoxicity against the epithelial
carcinoma cell line (A431). In the treated DLD-1 tumour cell population, there is a
discernible relationship between the IC50 values and the number of Re metal centres in the
complex, i.e. complex 11 is the most cytotoxic, followed by complex 10, while complex 9 is
the least cytotoxic. In the A431 cell line, complex 10 showed a higher standard deviation from
the best-fit IC50 value calculated by the software, indicating an inferior dose-relationship. In
the A2780 cells and normal fibroblasts, complex 10 was not aligned with the correlation
tendency between the IC50 values and the number of metal centres present per molecule,
while complexes 9 and 11 seem to follow a trend; complex 11 with four metal centres having

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a higher toxicity in A2780 as well. Both complexes 10 and 11 are more selective against
normal BJ cells, in comparison with the standard drugs oxaliplatin and carboplatin.

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Fig. 1. Comparison between the cell survival rates of tumor cells subjected to different
concentrations of complexes 9, 10, 11 and the reference platinum-based clinical drugs.
For each cell line, the activity of the Re complexes was compared with the same
concentration range of the platinum drug; oxaliplatin is the Pt(II) chemotherapeutic drug
of choice in colorectal cancer, carboplatin in ovarian cancer, and cisplatin is the most used
metal-based drug in epidermoid carcinoma.
In A431 epidermoid carcinoma, the parallel of survival rates at different concentrations
proved once again that the toxicity of Complex 11 was comparable with cisplatin (Figure 1),
and this most visible at higher concentrations. Despite complexes 9 and 10 being less toxic
than cisplatin, in vitro studies reveal that they show higher toxicity than the platinum drugs
oxaliplatin and carboplatin (Table 1). In the DLD-1 colon carcinoma, all complexes trigger
cell death more than oxaliplatin, at each studied concentration, and this tendency was
observed for the comparison of 9-11 with the other two standard drugs. For the A2780
ovarian carcinoma, above the concentration of 10 µM, carboplatin toxicity exceeds the
inhibitory capacity of complex 11, and above 50 µM became more active than both
complexes 9 and 10 as well.
The in vitro activity of the three platinum drugs was not consistent; there are significant
differences between the IC50 values. This phenomenon was largely described in the
literature [33,34] and although the antitumor activity of the rhenium compounds operates
via an alternate mechanism compared to platinum-based drugs, the magnitude of their
antiproliferative activity was not always convergent in the cell populations that originated
from different tissues, namely, colon, ovary and epidermal. Nonetheless, in comparison with

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the standard metal-based drugs, the in vitro activity of the compounds was remarkable, even
in the resistant A431 cell line. The dose-effect relationship points towards the prodrug
potential of complex 9 and especially complex 11, while for complex 10 the dose/structure-
activity relationship was less adequate.
In tumor cells, the ABCB1 and ABCG2 multidrug transporter molecules are responsible for
the efflux of the drugs from the cell, which causes resistance to chemotherapeutic agents.
These are widely expressed on many tumour populations, including A2780 [32], DLD-1 [35])
and A431 [36]. Nonetheless, there are some significant differences between the cell lines
used in our experiment. For the A2780 cell line, there is a characteristic presence of ABCB4
and ABCA8 transporters [32], therefore these cells are more susceptible to a stronger drug
resistance. For the A431 epidermal carcinoma, the role of ABCC6 multidrug transporter is
also important [37], whilst in normal BJ cells, the above-mentioned transporter’s role was not
mentioned in previous studies. The differences between the multidrug resistance genetic
patterns might allude to why the number of the Re metal centres is not directly related to the
antiproliferative effect in the A2780 and BJ cell lines.

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Fig. 2. The influence of Re compounds against the metabolic activity of tumor cells.
The linear regression of dose-effect relationship data (provided by the GraphPad prism
software) shows a decrease in the cells metabolism and proliferative capacity, where
the hill slopes were negative in the 95% confidence interval; the dashed line indicates
the standard error of the linear regression curve.
The metabolic rate of the treated cells, quantified using the Alamar Blue method, was
proven to be dose-dependent and significantly decreased by complexes 9 and 11 in all cell
lines (Fig. 2). Complex 10 was capable to slow down the metabolism of DLD-1 and A2780
cells but does not significantly influence the A431 and BJ populations. In these two cell
lines the cytotoxicity of 10 was also less pronounced.

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2.3 Effect of Re(I) Complexes on Cell Death Mechanisms
Complexes 9, 10 and 11 influence the intracellular level of the two molecules implicated in
programmed cell death pathways: the pro-apoptotic Bax-α and the pro-survival Bcl-xL
(Fig. 3). The in vitro concentration of the pro-apoptotic Bax-α increases significantly in all
tumour types following a 24-hour treatment with complex 11 (p = 0,0185 in DLD-1 cells, p
= 0,0050 in A2780 cells and p = 0,0148 in A431 cells). In the normal BJ cells, no
significant variation was observed. The anti-apototic Bcl-xL levels are not significantly
influenced by complexes 9 and 10.
a.
b.
c.
Fig. 3. Complexes 9, 10 and 11 can modulate the apoptotic signal transduction through the
soluble Bax-α and the anti-apoptotic Bcl-xL molecules. (a) The soluble pro-apoptotic Bax-
α level inside the treated cells. (b) The intracellular anti-apoptotic Bcl-xL level. (c) The
proportion between the two molecules, expressed as a ratio of Bax-α/Bcl-xL.

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The anti-apoptotic Bcl-xL concentration was not modified significantly (one-way analysis
of variance, p > 0.05), except in the A431 cell line where treatment with complexes 9, 10
and 11 results in an increase of Bcl-xL (Fig. 3) at approximately the same intensity (one-
way analysis of variance, Bonferroni post-test, p = 0.0026). The Bax-α/Bcl-xL ratio, a
reliable apoptosis indicator, is higher following treatment with complex 11, as for the
untreated control cells, in all cell lines, except in the case of the normal BJ cells. An
elevated Bax-α/Bcl-xL ratio indicates a sensitivity to treatment and a good transduction of
the apoptotic streamline from extrinsic apoptotic signals such as the Fas/FasLigand towards
mitochondrial functions [35].
It has been shown that overexpression of the anti-apoptotic Bcl-2 family does not influence
the Re complexes activity [36], and these results confirm this assertion. Rhenium
accumulates preferentially in the mitochondria [37] and lysosomes, and therefore the
secretion of mitochondrial proteins which regulates the apoptotic processes will be strongly
influenced by rhenium complexes. As described previously in the literature [37], the Re(I)
tricarbonyl [2 + 1] multinuclear complexes are more cytotoxic than their mononuclear
counterparts. This is very likely to be reflected in the apoptosis regulation. Bcl-xL is an
inhibitor of the cell cycle progression and can arrest the cell cycle before the mitotic
phase [38]. In A431 cells treated with complexes 9, 10 and 11, the lowest cytotoxicity
values of the studied Re(I) series were recorded, and an increase of Bcl-xL secretion
observed. Therefore, the augmentation of Bcl-xL observed in the A431 cells points to the
assumption that, in this case, the cell death mechanism was triggered through a non-
apoptotic pathway.
2.4 Influence of Re(I) Complexes on Fas/FasL Signalling
The Fas (First apoptotic factor) membrane protein or tumour necrosis factor receptor 6, is a
receptor implicated in cell differentiation, proliferation and apoptosis. Fas receptors have
some soluble isoforms, which lack the transmembrane domain. They bind the Fas ligand
(FasL) before it reaches the Fas receptor on the cell membrane, and thereby exert an
inhibitory effect on the Fas/FasL signalling pathway and inhibit apoptosis [39]. We
measured the soluble form of both Fas and its ligand FasL (Fig. 4). The secreted Fas
receptor level showed a decreasing tendency in DLD-1, A2780 and A431 tumour cell lines,
following a 24-hours exposure to complexes 9,10 and 11. The decline was statistically
significant (one-way analysis of variance, in the 95% confidence interval, p value 0.0008

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for DLD-1, 0.0109 for A431 and 0.0138 for A2780). In normal BJ cells the values are also
reduced, but not significantly (p > 0.5).
Fig. 4. The modulation of soluble FasReceptor and FasLigand concentration following the
treatment of DLD-1, A2780, A431 tumor cells and BJ normal cell line with complexes 9,
10 and 11.
The Fas receptor is ubiquitously expressed in normal or tumour human cells, whereas its
ligand, FasL, is expressed mainly in activated immune players such lymphocytes. The
soluble FasL levels were very low in the studied cells, close to 5 pg, the sensitivity limit of

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the reagent kit we used. The variations were not significant following the treatment with the
rhenium compounds in any of the studied cell lines.
The Fas-mediated extrinsic apoptotic pathway can crosstalk to the intrinsic pathway through
caspases, cleavage of BID (a BH3-ONLY member of the BCL2 family of proteins), which
results in Bcl family protein cleavage, and the activation of Bax proteins in mitochondria. In
the DLD-1 and A2780 cells, no correlation was found between the level of soluble Fas
receptor and the other molecules linked to apoptosis: Bax-α, Bcl-xL or their ratio. Instead, in
A431 cells, the Fas receptor modulation was inversely correlated to Bcl-xL (Pearson
correlation, r value -0.925; p value 0.0374). Therefore, the cell death signal exerted through
Fas receptor can be counterbalanced by the increase of Bcl-xL, indicating once again that in
this cell line, the cell death mechanisms were directed in a non-apoptotic manner. In the DLD-
1 and A2780 cell populations, the Bax/Bcl signalling and the soluble Fas receptor decline or
converge to apoptosis induction, with each Re(I) treatment.
3. Conclusions
Three new mono- and multinuclear Re(I) tricarbonyl [2 + 1] complexes (9 – 11) were
prepared and characterised using various spectroscopic and analytical techniques. Complexes
9 – 11 were evaluated for their antiproliferative activity against three tumour cell lines and
one normal cell line. All of the complexes displayed moderate biological activity against the
tested cancer cell lines (IC50 < 60 µM). The tetrameric complex 11 displayed the best activity
against all cancer cell lines, as well as significant selectivity towards inhibiting malignant
over normal cell lines, while complex 10 needs further structural improvements before further
testing. In vitro, the rhenium complexes 9, 10 and 11 can influence programmed cell death
mechanisms in three different types of tumors. The Re complexes inhibit the soluble form of
the Fas receptor, leaving the membrane Fas domain available to capture the apoptotic signal.
The cells are driven towards cell death through pro-apoptotic Bax-α augmentation, with the
influence on anti-apoptotic Bcl-xL being less important. In this study, complex 11 was the
more prominent Bax-α modulator in all cell lines, which is linked to its cell growth inhibitory
activity.

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4. Experimental
4.1 General information
All solvents, reagents and deuterated solvents were purchased from Sigma-Aldrich and used
without further purification. Metal precursor (1 [28]) and ligands (3 [29], 4 [30], 5 [31], 6
[29] and 7 [30]) were prepared using literature or modified literature procedures.
4.2 Instruments
All reagents were purchased from Sigma-Aldrich and were used as received unless otherwise
stated. Solvents were purchased from KIMIX and were used as received. Nuclear magnetic
resonance (NMR) spectra were recorded on a Bruker Topspin GmbH (1H at 400.22 MHz,
13C{1H} at 100.65 MHz, 31P{1H} at 162.01 MHz) or a Varian Mercury 300 (1H at 300.08
MHz) spectrometer, with a Bruker Biospin GmbH casing and sample injector at 30 °C.
Tetramethylsilane was used as the internal standard for the chemical shift reports. Infrared
(IR) absorption spectra were recorded using a Perkin-Elmer Spectrum One FT-IR
spectrometer using attenuated total reflectance (ATR). Mass spectrometry was carried out
using a JEOL GC Mate II single magnetic mass spectrometer in the positive-ion mode. High
resolution mass spectrometry was carried out using a Waters Synapt G2 ESI probe. Elemental
analyses were carried out using a Fission EA 110 CHNS analyser or an Elementar Vario EL
Cube Analyser. Melting points were determined using a Buchi B-540 apparatus.
4.3 Chemistry
4.3.1 Synthesis of tetrameric DAB-G1 ligand (8)
Ligand precursor 5 (0.063 g, 0.093 mmol) was stirred in methanol (30 mL) in an ice bath,
under argon for ten minutes. NaBH4 (0.057 g, 1.51 mmol) was added and the ice bath
removed. The mixture was stirred at room temperature for 22 hours. The reaction was
quenched with cold distilled water (40 mL), and the solvent volume reduced to 30 mL. The
product was extracted with DCM (40 mL), and the organic layer was washed with distilled
water (2 × 20 mL). The organic layer was then dried with MgSO4, which was removed by
gravity filtration before removal of the solvent to afford the product 8 as a brown oil. Yield:
1
3
4
0.0464 g, 0.0681 mmol, 72 %. H NMR (acetone-d6): δ (ppm) = 8.48 (dd, J = 4.4, J = 1.5
Hz, 8H, Ha), 7.33 (d, 3J = 5.9 Hz, 8H, Hb), 3.77 (s, 8H, Hd), 2.63 (t, 3J = 6.7 Hz, 8H, Hf), 2.47
3
3
3
(t, J = 6.9 Hz, 8H, Hh), 2.38 (t, J = 5.7 Hz, 4H, Hi), 1.63 (p, J = 6.8 Hz, 8H, Hg), 1.50 –
13
1.39 (m, 4H, Hj). C{1H} NMR (acetone-d6): δ (ppm) = 150.4 (Cc), 149.5 (Ca), 122.9 (Cb),
54.1 (Ci), 52.4 (Cd/h), 47.8 (Cf), 27.6 (Cg), 25.1 (Cj). FTIR v (cm-1) = 3270 (br, NH), 1602 (s,
C=Naromatic). Elemental analysis for C40H60N10·5H2O: Found C, 61.87 %, H, 8.90 %, N, 18.23

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%; calcd. C, 62.31 %, H, 9.15 %, N, 18.17 %. MS (EI, m/z): 121.07 (100 %, [M-C33H51N8]+),
149.05 (78.0 %, [M-C31H47N8]+).
4.3.2 Synthesis of monomeric Re(I) complex (9)
[Re(bpy)Cl(CO)3] 1 (0.105 g, 0.228 mmol) and silver triflate (0.075 g, 0.291 mmol) were
stirred in DCM (30 mL) for 3 hours. The resulting precipitate (AgCl) was removed by gravity
filtration. Ligand 6 was dissolved in DCM (5 mL) and added to the solution. The mixture was
stirred at room temperature for 20 hours. Excess NH4PF6 was added to the solution, after
which the solution was stirred for a further hour. Gravity filtration was employed to remove
insoluble particles, and the solution was washed with distilled water (2×20 mL). The organic
layer was dried with MgSO4, which was subsequently removed by gravity filtration, and the
solvent removed. The remaining residue was dissolved in acetone (5 mL) and distilled water
(10 mL) added to the solution. The solvent was reduced until it became cloudy and placed in
the fridge for 3 hours. The resulting precipitate was removed by gravity filtration and the
product extracted from the aqueous solution with DCM (2×20 mL). The organic layer was
dried with MgSO4, which was subsequently removed by gravity filtration. The solvent was
removed, and the product 9 dried in vacuo as an oily residue. Yield: 0.031 g, 0.0429 mmol,
19 %. Melting point: 104 – 107 °C. 1H NMR (acetone-d6): δ (ppm) = 9.49 (ddd, 3J = 5.5, 4J =
1.4, 5J = 0.7 Hz, 2H, Hm), 8.77 (d, 3J = 8.2 Hz, 2H, Hj), 8.52 – 8.45 (m, 2H, Hk), 8.44 (dd, 3J
4
3
= 5.2, J = 1.4 Hz, 2H, Ha), 8.08 – 7.97 (m, 2H, Hl), 7.46 (d, J = 6.6 Hz, 2H, Hb), 3.80 (s,
2H, Hd), 2.47 (t, 3J = 7.0 Hz, 2H, Hf), 1.51 – 1.36 (m, 2H, Hg), 0.86 (t, 3J = 7.4 Hz, 3H, Hh).
13C{1H} NMR (acetone-d6): δ (ppm) = 195.7 (CCO), 191.7 (CCO), 156.2 (Ci), 155.9 (Cc),
154.0 (Cm), 151.5 (Ca), 141.5 (Ck), 129.1 (Cl), 125.6 (Cb), 124.9 (Cj), 51.4 (Cd), 51.1 (Cf),
22.8 (Cg), 11.0 (Ch). 31P{1H} NMR (acetone-d6): δ (ppm) = -144.30 (hept, J = 708.2 Hz)
FTIR v (cm-1) = 2025 (s, CO), 1899 (br, CO), 1619 (s, C=Npyridyl) , 1604 (s, C=Nbpy).
Elemental analysis for C22H22N4O3Re·PF6: Found C, 36.98, H, 3.02, N, 7.47 %; calcd. C,
36.62, H, 3.07, N, 7.76 %. MS (HR-ESI, m/z): 577.12 (100 %, [M-PF6]+, calcd. 577.12),
427.01 (33 %, [M-L1-PF6]+, calcd. 427.01).
4.3.3 Synthesis of trimeric Re(I) complex (10)
[Re(bpy)Cl(CO)3] 1 (0.131 g, 0.284 mmol) and silver triflate (0.080 g, 0.311 mmol) were
stirred at room temperature in DCM for 3 hours. The resulting precipitate (AgCl) was filtered
and ligand 7 (0.036 g, 0.0863 mmol) was added to the flask. The reaction mixture was stirred
at room temperature for 19 hours, after which the solvent was removed and the resulting
residue taken up in minimum ethanol (4 mL) and ammonium hexafluorophosphate (excess in
H2O) was added to precipitate the crude product as a yellow powder. The powder was

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redissolved in minimum acetone and added dropwise to isopropanol (80 mL, 75 °C). The
mixture was filtered through Celite and the product obtained by washing with DCM.
Removal of the solvent afforded product 10 as a brown powder. Yield: 0.022 g, 0.0103
mmol, 12 %. Melting point: 145 – 150 °C. 1H NMR (acetone-d6): δ (ppm) = 9.44 (d, 3J = 5.6
Hz, 6H, Hl), 8.78 – 8.62 (m, 6H, Hi), 8.51 – 8.33 (m, 12H, Hj/a), 8.07 – 7.91 (m, 6H, Hk), 7.41
3
(d, J = 6.5 Hz, 6H, Hb), 3.79 – 3.70 (m, 6H, Hd), 3.33 – 3.23 (m, 6H, Hf), 2.94 – 2.79 (m,
13
6H, Hg). C{1H} NMR (acetone-d6): δ (ppm) = 195.5 (CCO), 191.5 (CCO), 155.8 (Ch), 155.4
(Cc), 154.0 (Cl), 151.7 (Ca), 141.4 (Cj), 129.1 (Ck), 125.9 (Cb), 124.8 (Ci), 53.1 (Cf), 50.4
(Cd), 42.2 (Cg). 31P{1H} NMR (acetone-d6): δ (ppm) = -144.26 (hept, J = 708.7 Hz). FTIR v
(cm-1) = 2032 (s, CO), 1907 (s, CO), 1619 (s, C=Npyridyl) , 1605 (s, C=Nbpy). MS (HR-ESI,
m/z): 566.07 (15 %, [M-3PF6]3+, calcd. 566.10). Elemental analysis for C63H57N13O9·3PF6:
Found C, 35.37, H, 2.89, N, 8.18 %; calcd. C, 35.46, H, 2.69, N, 8.35 %.
4.3.4 Synthesis of tetrameric Re(I) complex (11)
[Re(bpy)Cl(CO)3] 1 (0.090 g, 0.194 mmol) and silver triflate (0.059 g, 0.229 mmol) were
stirred in DCM (30 mL) for 3 hours. A precipitate (AgCl) was formed and removed by
gravity filtration. Ligand 8 (0.033 mg, 0.0479 mmol) was dissolved in DCM (5 mL) and the
solution added to the reaction mixture. The solution was stirred for 24 hours at room
temperature, before the addition of NH4PF6 (excess) and subsequent stirring for one hour.
The solvent was removed by rotary evaporation and the residue dissolved in minimum DCM.
The solution was added to boiling isopropanol and stirred for five minutes. The solution was
allowed to cool to room temperature, precipitating a brown powder. The powder was filtered
using Celite and washed with isopropanol before being solubilised with DCM. The DCM was
removed to afford the product 11 as a brown powder. Yield: 0.016 g, 0.00527 mmol, 11 %.
Melting point: 134 – 137 °C. 1H NMR (acetone-d6): δ = 9.47 (ddd, 3J = 5.4, 4J = 1.4, 5J = 0.7
Hz, 8H, Ho), 8.74 (d, 3J = 8.1 Hz, 8H, Hl), 8.53 – 8.37 (m, 16H, Ha/m), 7.99 (ddd, 3J = 7.6, 3J
4
3
= 5.5, J = 1.2 Hz, 8H, Hn), 7.46 (d, J = 6.6 Hz, 8H, Hb), 3.83 (s, 8H, Hd), 3.38 – 3.06 (m,
16H, Hf/h), 2.77 – 2.64 (m, 4H, Hi), 1.98 – 1.77 (m, 12H, Hg/j). 13C{1H} NMR (acetone-d6): δ
= 195.6 (CCO), 191.6 (CCO), 155.8 (Ck), 154.0 (Co), 151.7 (Ca), 151.7 (Cc), 141.4 (Cm), 129.0
(Cn), 125.8 (Cb), 124.8 (Cl), 52.0 (Cf), 51.1 (Cd), 47.1 (Ch), 46.72 (Ci), 23.9 (Cg), 23.7 (Cj).
31P{1H} NMR (acetone-d6): δ = -144.26 (hept, J = 708.9 Hz). FTIR v (cm-1) = 2028 (s, CO),
1901 (s, CO), 1619 (s, C=Npyridyl), 1605 (s, C=Nbpy). MS (HR-ESI, m/z): 590.08 (26 %, [M-
CO-4PF6]4+),
547.05
(19
%,
[M-7CO-4PF6]4+).
Elemental
analysis
for
C92H92N18O12Re4·4PF6·5H2O·2DCM: Found C, 35.28, H, 3.68, N, 7.26 %; calcd. C, 34.80,
H, 3.36, N, 7.77 %.

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4.4 Cytotoxicity and Antiproliferative Studies
The ovary carcinoma A2780, the epidermoid carcinoma A431 and the colon carcinoma DLD-
1 cell lines were acquired from the European Collection of Authenticated Cell Cultures
(ECACC) through Sigma Aldrich, St. Louis, USA. The normal human fibroblast cell line
(BJ) was acquired from the American Type Culture Collection (ATCC, Manasses, VA,
USA). The cells were cultured under conditions recommended by the cell bank using cell
culture media and supplements from Sigma Aldrich. The MTT reagent and the Hank’s media
to dissolve the salt (5 mg/mL final concentration) were purchased from Sigma Aldrich. The
cells were seeded on 96-well Nunclon Delta surface assay plates (from Thermo Scientific,
Waltham, USA) in wells containing 200 µL cell suspension. The compounds 9, 10 and 11
were diluted in DMSO, and serial dilutions were prepared using 0.01 M PBS solution, to
obtain concentrations in the range of 10 – 2500 µM. Cisplatin and oxaliplatin were
received from Actavis, through Sindan-Pharma Srl, Bucharest, Romania; carboplatin
was provided from Teva UK Limited, Castleford, United Kingdom. The platinum-
based drugs were diluted in physiological serum, and serial dilutions were made in
PBS, to obtain the same concentration range as for Re(I) compounds: 10 – 2500 µM.
For testing, each well containing cells was treated with the respective compound in a
proportion of 1:20 (compound:cell culture media), to obtain a final concentration range of 0.5
– 125 µM of the compound in the cell culture media. To assess the cytotoxicity of the
compounds, the cells were subjected to the treatment for 24 hours, than the MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was
performed, as described before [41]. Untreated cells were used as a reference, and cell
culture media without cells was used as a blank. Three independent experiments were
performed, each in duplicate. Measurements were made at 492 nm and 570 nm using a
Synergy 2.0 microplate reader (from BioTek Company, Winooski, USA), and the absorbance
data were implemented using GraphPad Prism 5 software (from GraphPad Software, La Jolla,
USA) to obtain the sigmoidal curves and the IC50 values. The antiproliferative effect was
evaluated using the cell-permeable fluorescent Alamar Blue dye (from Invitrogen, acquired
through Thermo Fisher Scientific, Waltham, MA, USA) which indicates the intracellular
reducing potential of the cells. The plates were loaded with tumor cells; they were incubated
and treated with the compounds in the same way as for MTT testing. After 24 hours
treatment, to each sample 20µL dye was added, and incubated for three hours. The
fluorescence of each well was measured at the 620nm emission (excitation wavelength
54nm), using the Synergy2 microplate reader. Three independent measurements were made,

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in duplicates. The data were processed with the GraphPad Prism 5 software to obtain a linear
regression between the doses and the degree of the metabolic activity reduction.
4.5 Cell Death Mechanistic Studies
The concentration of Bax-α and Bcl-xL were measured using the DuoSet IC Elisa kits from
R&D Systems (Minneapolis, MN, USA), for each molecule. The cells were cultivated were
seeded on 6-well plates at a density of 1.5x105 cells/mL, and they were treated for 24 hours
with the Rhenium compounds at the concentration of 10 µM in the cell culture media. Than
the cell culture media was removed, the plates were washed twice with cold PBS. The wells
were covered with 1 mL cell lysis solution (from MBL International Corporation, Woburn,
MA, USA) and kept 20 minutes on ice. The lysates were harvested from the cell culture
plates, centrifuged; the supernates aliquotes were placed in ultrafrezer. The total protein
level was measured in the lysates, and all samples were diluted with lysis buffer to
normalize the protein concentration. The Elisa plates were prepared by treating the each
well with Bax-α and respectively Bcl-xL capture antibody overnight at room temperature,
than the plates were washed 3 times with the automated plate washer (from DIAsource,
Louvain-la-Neuve, Belgium) using the buffer provided by the manufacturer. In each well
block buffer was added for two hours, and the washing steps were repeated. In each well
samples (duplicates), blank values or standard proteins were added: 8 different
concentrations and one blank, in the detection range of the kit. As reference we used
untreated cells for each cell line. After two hours the plates were washed, incubated with
125 ng/mL detection antibody for another two hours. The plates were washed and filled
with 100 µL Streptavidin-HRP for 20 minutes, than washed and incubated with substrate
solution for 20 minutes. Acid stop solution was added to each well after the blue coloration
development, and the samples color turned into yellow, the optical density of each well
was determined at 450 nm, with correction at 540nm, using a Sunrise microplate plate
reader (Tecan Group, Männedorf, Switzerland). The Magellan software provided
measurement data and the single concentrations for each sample.
4.6 Fas/FasL Signalling Studies
The soluble Fas receptor and FasLigand were evaluated using the Quantikine Elisa kits from
R&D Systems. The cells seeded for 24 hours on 6-well plates were treated with 10 µM 9, 10
or 11, then the cell culture media was harvested from the wells, centrifuged, and stored at -80

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°C. The 96-well plates precoated with anti-Fas or anti-FasL monoclonal antibodies were
filled with 100 µL of assay diluent provided by the kit, and 100 µL of samples were added to
each well, in duplicate. For quantitative measurements, the standard recombinant human Fas
or FasL proteins were used; 8 serial dilutions were prepared and added into the wells. The
samples and standards were incubated for 2 hours, washed 4 times with the wash buffer
provided by the kit, then horseradish peroxidase-conjugated polyclonal antibody was added
to each well and incubated at room temperature for 2 hours. After 3 wash steps 200 µL HRP
substrate solution was added into the wells, and the plates were incubated for 30 minutes.
Stop solution was added, and the optical density was measured immediately, in similar
condition and with the same instrument as described before. The soluble Fas and FasL
concentrations were calculated using the standard curves.
Acknowledgements
Financial support from the University of Cape Town (UCT), the National Research
Foundation of South Africa and NTeMBI is gratefully acknowledged. The National Research
Foundation of South Africa (UID: 104023) and the Romanian National Authority for
Scientific Research and Innovation, CCCDI – UEFISCDI Romanian-South African Bilateral
Cooperation grant PNIII-P3-116, 9BM/2016 is gratefully acknowledged for funding and
several short-term exchange visits between the two laboratories in South Africa and
Romania.
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Highlights
‹ Mono- and multinuclear [2+1] Re(I) tricarbonyl complexes have been
prepared.
‹ The complexes were characterized using several spectroscopic and analytical
techniques.
‹ The complexes were evaluated as antiproliferative agents against A431, DLD-
1 and A270 tumour cell lines.
‹ The Re(I) complexes all influence programmed cell death mechanisms.