New [(η5-C5H5)Ru(N-N)(PPh3)][PF6] compounds: Colon anticancer activity and GLUT-mediated cellular uptake of carbohydrate-appended complexes

Article abstract of DOI:10.1039/c6dt01571a

Eight ruthenium(ii) compounds of the general formula [(η⁵-C₅H₅)Ru(N-N)(PPh₃)][PF₆] were rationally designed, exhibiting high cytotoxicity against HCT116 human colon cancer cells, with IC₅₀

Full text of DOI:10.1039/c6dt01571a

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New [(η5-C5H5)Ru(N–N)(PPh3)][PF6] compounds:
colon anticancer activity and GLUT-mediated cellular
uptake of carbohydrate-appended complexes†
Cite this: DOI: 10.1039/c6dt01571a
Received 22nd April 2016,
Accepted 12th May 2016
Pedro R. Florindo,*a Diane M. Pereira,b Pedro M. Borralho,b Paulo J. Costa,c
DOI: 10.1039/c6dt01571a
M. F. M. Piedade,a,d Cecília M. P. Rodriguesb and Ana C. Fernandes*a
www.rsc.org/dalton
Eight ruthenium(II) compounds of the general formula loped an immense library of organometallic compounds with
[(η5-C5H5)Ru(N–N)(PPh3)][PF6] were rationally designed, exhibiting improved affinity and selectivity for proto-oncogenic protein
high cytotoxicity against HCT116 human colon cancer cells, with kinases such as GSK3, Pim-1 and PI3K.4
IC50 between 14.56 and 1.56 µM; importantly, compounds 5Ru and
Within the same [(η5-C5H5)Ru(N–N)(L)] scaffold, but har-
6Ru are the first reported ruthenium glycoconjugates exploiting bouring L = PPh3 and 2,2′-bipyridyl derivatives as nitrogen
glucose transporters, widely overexpressed in cancer, for cellular bidentate donors, Garcia and coworkers developed a set
uptake.
of powerful anticancer agents.5 The parent and most
studied compound [(η5-C5H5)Ru(2,2′-bipyridyl)(PPh3)][CF3SO3]
(TM34),5a,b consistently revealed IC50 values in the low micro-
molar range against a large spectrum of cancer cell lines,
including cisplatin-resistant lines, being mechanistically shown
to strongly inhibit PARP-1, an enzyme involved in DNA repair
mechanisms and apoptosis pathways. Derivatives of TM34 have
been reported, bearing either methyl groups (TM102)5c or poly-
lactide polymeric chains (RuPMC)5d at the 4,4′-positions of the
2,2′-bipyridyl scaffold; while the former has revealed slight
improvements in cytotoxicity, particularly in MCF7 breast
adenocarcinoma cells, the latter has shown a completely
different cellular distribution profile in the same cell line.
Carbohydrates such as glucose are the most important
energy source for cell growth, and fast-growing cancer cells
have higher carbohydrate demand than normal ones, a hall-
mark of cancer known as “Warburg effect”.7 Glucose Transpor-
ters (GLUTs) are transmembrane proteins involved in the
cellular uptake of monosaccharides such as glucose, mediat-
ing the passage of these highly hydrophilic biomolecules
through hydrophobic membranes.8 GLUT1, the most common
glucose transporter, is widely overexpressed in many human
cancers, including colorectal cancer, and its expression levels
are correlated with the invasiveness and metastatic potential
of cancers.9 The differential rate of glucose uptake has been
used in positron emission tomography (PET), where 2-[18F]-2-
deoxy-D-glucose (FDG, a glucose analogue) accumulates in
cancer cells, allowing tumor imaging.10 This strategy was
also successfully employed in the development of inorganic
glycoconjugates, both for cancer diagnostics11 and platinum
chemotherapeutics.12
Among metals studied as non-platinum chemotherapeutic
alternatives, ruthenium presents unique features, such as the
capacity to mimic iron when interacting with biomolecules,
selectivity for cancer tissues due to transferrin receptor over-
expression in cancer cells, low general toxicity, and activity
against cisplatin-resistant cell lines,1 making ruthenium com-
pounds attractive anticancer drug candidates.
Following the success of ruthenium(III) anticancer drugs,
NAMI-A ([ImH][trans-Ru(III)Cl4Im(Me2SO)]; Im = imidazole)2
and KP1019 ([Hind][trans-Ru(III)Cl4(Ind)2], Ind = indazole),3
ruthenium(II) organometallic compounds have appeared as
alternative anticancer drug candidates.1 Cyclopentadienyl-
ruthenium(II) compounds have been a subject of intensive
research as anticancer agents, showing significant toxicity
against a large variety of cancer cell lines, including cisplatin
resistant histo-types.4–6 Unlike most of the platinum chemo-
therapeutics, these compounds target biomolecules other
than DNA. By replacing the sugar moiety in staurosporine with
a CpRu organometallic center, Meggers and coworkers deve-
aCentro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa,
Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
E-mail: anacristinafernandes@tecnico.ulisboa.pt, pedro.florindo@tecnico.ulisboa.pt
bResearch Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy,
Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal
cCentro de Química e Bioquímica, DQB, Faculdade de Ciências da Universidade de
Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
dDepartamento de Química e Bioquímica, Faculdade de Ciências da Universidade de
Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
†Electronic supplementary information (ESI) available. CCDC 1444128–1444130.
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c6dt01571a
Similarly to GLUTs, Nucleoside Transporters (NTs) are inte-
gral membrane glycoproteins that regulate cellular proliferation,
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neurotransmission, and cardiovascular activity, mediating the
passage of natural and non-natural nucleosides in and out of
mammalian cells.13 NT down-regulation is one of the resist-
ance mechanisms developed by cancer cells during treatment
with nucleoside analog (NA) anticancer drugs, such as
5-fluoro-2′-deoxyuridine and gemcitabine, limiting their clini-
cal efficacy.14 Interestingly, fluorescent iridium(III) NA com-
plexes were found capable of NT cellular uptake.15
In our approach to cancer problematics, we have success-
fully developed libraries of anticancer ruthenium(II) and
iron(II) organometallic compounds with carbohydrate-derivative
ligands,6 lately focusing on colorectal cancer, a high profile
cancer type displaying both high incidence and mortality.16
Our leading compound was shown to be highly cytotoxic
against HCT116 colon cancer cells (IC50 = 0.45 µM), triggering
significantly higher levels of caspase-3/-7 activity and apoptosis
compared to oxaliplatin.6b
Targeting the same cancer phenotype, we herein report
eight new ruthenium compounds of the general formula
[(η5-C5H5)Ru(N–N)(PPh3)][PF6], in which the N–N ligand is varied.
Two distinct sets of bidentate ligands were used (Scheme 1);
dioctyl-2,2′-bipyridine-4,4′-dicarboxylate (L1), 2,2′-biquinoline
(L2), [1,10]phenanthroline (L3) and bathophenanthroline (L4)
were chosen to test the effect of well-defined extensions of the
N–N bidentate ligand, and the corresponding increase in the
lipophilicity of the complexes, in the biological activity of
derivative organometallic compounds. L1 was prepared by a
reaction of 1-octanol with 2,2′-bipyridine-4,4′-dicarbonyl
dichloride in the presence of trimethylamine, and L2–L4 were
commercially obtained. On the other hand, we were interested
in the development of [(η5-C5H5)Ru(N–N)(PPh3)]+ complexes
bearing carbohydrate- and nucleoside-derivative N–N ligands.
Pyridyl-2-triazole was chosen as an N–N structural motif:
it contains the structural features needed for N–N chelation of
the ruthenium centre, being easily accessed by “clicking” the
appropriate azide to 2-ethynyl pyridine, and allows the appen-
dage of “unprotected” bio-molecules. The Cu(I)-catalysed
[3 + 2] cycloaddition was first attempted with AZT (Zidovudine,
anti-HIV drug), affording L8 in high yield. The strategy was
Scheme 1 Synthesis of the organometallic compounds 1Ru–8Ru
(all with PF6 counterion) and ligands L1–L8; reagents and conditions:
(a) TlPF6, DCM, r.t., 1 h; then L1–L8, overnight.
−
successfully extended to the synthesis of ligands L5–L7, from and 4Ru were further confirmed by X-ray diffraction studies.†
methyl α-D-mannopyranoside, methyl α-D-glucopyranoside and Due to the pro-chiral nature of an organo-ruthenium metal
thymidine, respectively, by tosylation of the parent primary center, compounds 5Ru–8Ru are obtained as diastereoiso-
alcohols, –OTs displacement with NaN3, and a final reaction meric mixtures, reflected by multiple peaks in the 1H NMR
with 2-ethynyl pyridine. Methyl α-D-pyranosides were chosen spectra of these compounds; the structures of organometallic
to avoid further isomer mixtures in the derivative complexes cations were thus further confirmed by ESI-MS.†
(see below).17
The cytotoxic activity of organometallic compounds 1Ru–
Organometallic compounds 1Ru–8Ru were synthesized by 8Ru and TM34[PF6] was evaluated in HCT116 human colon
halide abstraction from the parent neutral complex cancer cells (Table 1). Extensions of the N–N ligand in com-
[(η5-C5H5)Ru(PPh3)2Cl] and subsequent chelation of the pounds 1Ru–4Ru all resulted in increased cytotoxicity toward
respective N–N ligands L1–L8, and obtained in good yields HCT116 colon cancer cells, compared to the 2,2′-bipyridyl
(77–92%, Scheme 1). The compound [(η5-C5H5)Ru(2,2′-bipy) parent structure of TM34[PF6], similarly to the extension of the
(PPh3)][PF6] (TM34[PF6]) was synthesized and studied along η6-arene “seat” in RAPTA compounds.1 The introduction of
with our newly designed compounds. Structures of the organo- octyl carboxylate chains into the 2,2′-bipyridyl scaffold (1Ru)
metallic compounds were confirmed by 1H-, 13C- and resulted in an approximately 6-fold decrease in IC50. 2,2′-Biqui-
31P-NMR, and formulations and purity were confirmed by noline “side wards” extension in 2Ru led to a nonsignificant
elemental analysis. The structures of compounds 2Ru, 3Ru decrease in IC50. Phenanthroline (3Ru) and bathophenanthroline
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Table 1 IC50 values of compounds 1Ru–8Ru, TM34[PF6] and oxalipla-
tina in HCT116 colon cancer cells (72 h)
Compound
IC50 (µM)
95% CIb
1Ru
2Ru
3Ru
4Ru
5Ru
6Ru
7Ru
8Ru
1.69
7.25
2.43
1.53
14.16
7.41
13.37
9.85
9.62
0.45
1.15–2.48
4.31–12.20
1.68–3.52
1.32–1.77
8.42–23.82
6.50–8.46
12.47–14.32
9.16–10.59
5.35–17.30
0.41–0.48
TM34
Oxaliplatina
a Positive control. b 95% confidence interval.
(4Ru) led to approximately 4- and 6-fold increase, respectively,
in the cytotoxic activity of the corresponding compounds, the
latter proving as the most active herein reported. The cyto-
toxicity order within these simple compounds is thus: IC50
(4Ru) < IC50 (1Ru) < IC50 (3Ru) < IC50 (6Ru) < IC50 (TM34[PF6])
(Table 1).
Challenging the trend verified for 1Ru–4Ru, compounds
5Ru–8Ru, harboring hydrophobic carbohydrate or nucleoside
moieties, revealed IC50 values ranging from 7.41 to 14.16 µM.
The least active compound 5Ru, presents an IC50 only 1.5-fold
higher than TM34[PF6] in HCT116 cells, and its glucose
counterpart 6Ru presents an IC50 1.3-fold lower, 2-fold lower
than its isomer 5Ru. Compound 8Ru presents an IC50 1.4-fold
lower than its thymidine isomer 7Ru.
Fig. 1 Evaluation of the influence of glucose in the cytotoxicity of com-
pounds 5Ru, 6Ru and 4Ru in HCT116 colon cancer cells. Cells were
exposed for 72 h to IC50 or 2-fold IC50 of each compound in low
glucose medium (5 mM D-glucose) supplemented with either (A) 50 mM
D-glucose; or (B) 50 mM D-glucose or 50 mM L-glucose. DMSO was
used as a vehicle control. Cell viability was evaluated according to MTS
metabolism. Results are expressed as mean
SEM fold-change to
vehicle-treated cells, from at least three independent experiments. *p <
0.05 and **p < 0.01 from control vehicle-treated cells.
To inspect the role of GLUTs in cellular internalization of
carbohydrate derivative compounds 5Ru and 6Ru, we per-
formed an uptake competition experiment using D-glucose
(GLUT substrate) and L-glucose (non-substrate). For this
purpose, we evaluated the changes in the cytotoxicity of these
compounds exposing cells to IC50 and 2-fold IC50 concen-
trations in low glucose medium (5 mM D-glucose) sup-
plemented with either 50 mM D-glucose or 50 mM L-glucose.
Non-supplemented low glucose medium was used as a control.
Aglycone 4Ru was used as a negative control. As shown in
Fig. 1, for both carbohydrate derivative compounds, cyto-
toxicity was significantly reduced at a high D-glucose concen-
tration, while for 4Ru there were no significant differences in
cytotoxicity between D-glucose poor and rich media. Specifi-
cally, the cytotoxicity of 5Ru and 6Ru in HCT116 colon cancer
cells was reduced by 1.4- and 1.3-fold at IC50, and by 3.5 and
3.0 at 2-fold IC50, respectively. In turn, no significant changes
in cytotoxicity were detected for any of the compounds in
L-glucose rich media (Fig. 1B). Together, these results demon-
strate that the cellular uptake of 5Ru and 6Ru depends on
[D-glucose] but not on [L-glucose], suggesting that these glyco-
conjugates enter the cell through a GLUT-mediated uptake
system.17
GLUT1 homolog. Both complexes are able to fit the binding
cavity. In spite of the potential steric clashes (vide ref. 12b),
significant docking poses were obtained for both 6Ru′ and 6Ru″
in which the glucose moiety was positioned similarly to the
sugar unit in the X-ray structure (Fig. 2), but with different rela-
tive orientations. Interestingly, a similar glucose orientation
was depicted for a recently reported C6-platinum glycoconjugate,
Fig. 2 Complex 6Ru’ docked into XylE (PDB 4GBZ) at the glucose
binding site (6Ru’’ is shown in the ESI†). The protein is represented as a
cartoon with flexible side chains depicted as dark grey sticks. The sugar
moiety is shown as orange sticks whereas the glucose in the X-ray struc-
ture is drawn as yellow lines.
GLUT-mediated transport was further validated by docking
studies with AutoDock Vina,19 using the structures of both
6Ru diastereoisomers (6Ru′ and 6Ru″, Fig. S2†) on the cavity
of the bacterial xylose transporter XylE (PDB 4GBZ),20
a
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studied using rigid docking.12b These docking studies further
support the GLUT-internalization hypothesis herein
proposed.21
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Next, we evaluated the potential role of NTs in the cellular
uptake of nucleoside derivative compounds 7Ru and 8Ru,
bearing appended 5′- and 3′-thymidine moieties, respectively.
In this framework, we performed uptake competition experi-
ments using 200 µM 2′-deoxyadenosine (2′-DAd) or 10 µM
dipyridamole to block the internalization of the nucleoside
derivative compounds. Once again, 4Ru was used as a negative
control. Surprisingly, no significant changes in cell viability
were observed for any of the compounds in neither inhibition
setting tested (data not shown). These results suggest that thy-
midine derivative compounds 7Ru and 8Ru are not interna-
lized via NT, an advantage over organic NA chemotherapeutics
(vide supra).
In summary, new cyclopentadienyl-ruthenium(II) com-
pounds were designed and synthesized, and their cytotoxicity
in HCT116 colon cancer cells was evaluated with good results.
The cellular uptake mechanism of bio-appended compounds
was evaluated, revealing that 5Ru and 6Ru were most readily
internalized through GLUT transmembrane proteins and that
this facilitated diffusion has a major contribution to their tox-
icity. Although several carbohydrate-containing ruthenium
anticancer compounds have been reported in the last few
years,5d,6,18 the question whether they were actually taken up
by the GLUTs is herein addressed for the first time. Thus,
these are, to the best of our knowledge, the first examples of
ruthenium anticancer glycoconjugates with GLUT-mediated
cellular uptake.
This research was supported by the Portuguese Foundation
for Science and Technology (FCT) through projects PTDC/
QUI-QUI/102114/2008 and PTDC/QUI-QUI/110532/2009 and
annual funds UID/QUI/00100/2013 and UID/MULTI/00612/ 10 (a) H. Parekh and H. Simpkins, Gen. Pharmacol., 1997, 29,
2013. PRF and DMP (SFRH/BD/96517/2013) thank FCT for
grants. ACF (IF/00849/2012) and PJC (IF/00069/2014) acknowl-
edge FCT for the “Investigador FCT” Program. Authors thank
the Portuguese NMR and X-Ray Networks (IST-UTL Centers)
for access to facilities.
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