Cyclopentadienyl-ruthenium(II) and iron(II) organometallic compounds with carbohydrate derivative ligands as good colorectal anticancer agents

Article abstract of DOI:10.1021/acs.jmedchem.5b00403

New ruthenium(II) and iron(II) organometallic compounds of general formula [(η⁵-C₅H₅)M(PP)Lc][PF₆], bearing carbohydrate derivative ligands (Lc), were prepared and fully characterized and the crystal structures of five of those compounds were determined by X-ray diffraction studies. Cell viability of colon cancer HCT116 cell line was determined for a total of 23 organometallic compounds and SAR's data analysis within this library showed an interesting dependency of the cytotoxic activity on the carbohydrate moiety, linker, phosphane coligands, and metal center. More importantly, two compounds, 14Ru and 18Ru, matched oxaliplatin IC₅₀ (0.45 μM), the standard metallodrug used in CC chemotherapeutics, and our leading compound 14Ru was shown to be significantly more cytotoxic than oxaliplatin to HCT116 cells, triggering higher levels of caspase-3 and -7 activity and apoptosis in a dose-dependent manner.

Full text of DOI:10.1021/acs.jmedchem.5b00403

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Article
Cyclopentadienyl-Ruthenium(II) and Iron(II) Organometallic Compounds with
Carbohydrate Derivative Ligands as Good Colorectal Anticancer Agents.
Ana Cristina Fernandes, Pedro Florindo, Diane M Pereira, Pedro M.
Borralho, Cecília M.P. Rodrigues, and Maria Fátima Minas da Piedade
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00403 • Publication Date (Web): 29 Apr 2015
Downloaded from http://pubs.acs.org on May 1, 2015
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CyclopentadienylꢀRuthenium(II) and Iron(II)
Organometallic Compounds with Carbohydrate
Derivative Ligands as Good Colorectal
Anticancer Agents.
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Pedro R. Florindo,a* Diane M. Pereira,b Pedro M. Borralho,b Cecília M.P. Rodrigues,b
Maria F.M. Piedadea,c and Ana C. Fernandes a*.
a Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av.
Rovisco Pais, 1049ꢀ001 Lisboa, Portugal.
b Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade
de Lisboa, Av. Prof. Gama Pinto, 1649ꢀ003 Lisboa, Portugal.
cDepartamento de Química e Bioquímica, Faculdade de Ciências da Universidade de
Lisboa, Campo Grande, 1749ꢀ016 Lisboa, Portugal.
Ruthenium(II); Iron(II); Carbohydrates; Colon Anticancer Agents; MetalloꢀDrugs;
Cytotoxicity.
ABSTRACT: New ruthenium(II) and iron(II) organometallic compounds of general
formula [(η5ꢀC5H5)M(PP)Lc][PF6], bearing carbohydrate derivative ligands (Lc) were
prepared and fully characterized, and the crystal structures of five of those compounds
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were determined by Xꢀray diffraction studies. Cell viability of colon cancer HCT116
cell line was determined for a total of twentyꢀthree organometallic compounds and
SAR’s data analysis within this library showed an interesting dependency of the
cytotoxic activity on the carbohydrate moiety, linker, phosphane coꢀligands and metal
center. More importantly, two compounds, 14Ru and 18Ru, matched oxaliplatin IC50
(0.45 ꢁM), the standard metalloꢀdrug used in CC chemotherapeutics, and our leading
compound 14Ru was shown to be significantly more cytotoxic than oxaliplatin to
HCT116 cells, triggering higher levels of caspaseꢀ3 and ꢀ7 activity, and apoptosis in a
doseꢀdependent manner.
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1. Introduction
Since the discovery of cisplatin in 1965,1 the quest for metalꢀbased drugs has been
driven by the hope that it might be replaced by a lessꢀexpensive metal complex, with
fewer side effects and improved therapeutic value. Metalꢀbased drugs are very attractive
due to its great versatility in terms of coordination number, oxidation state and
geometric orientation around the metal center, and to the metal itself. Among other
metals studied for their anticancer properties, ruthenium compounds present special
properties, such as capacity to mimic iron by binding to biological molecules, reduced
general toxicity and strong affinity to cancer tissues over normal tissues, making them
appealing candidates as anticancer drugs.2 Two ruthenium(III) compounds, NAMIꢀA
([ImH][transꢀRu(III)Cl4Im(Me2SO)]; Im = imidazol)3 and KP1019 ([Hind][transꢀ
Ru(III)Cl4(Ind)2], Ind = indazol)4 were featured in clinical trials as antiꢀmetastatic and
anticancer agents, respectively, but exposed problems concerning their delivery and
associated side effects. Clinical studies have shown that NAMIꢀA application may
cause skin blisters lasting up to several months and resulting in intense pain.5 In turn,
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KP1019 presents low solubility, which makes it challenging to obtain proper dosage in
clinical trials.6
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Organoꢀruthenium(II) complexes seem suitable drug candidates. Ruthenium(II)ꢀarene
complexes, of general formula [(η6ꢀC6H6)Ru(L1)(L2)(L3)], constitute the most studied
class of ruthenium compounds, exhibiting promising anticancer activity both in vitro
and in vivo. The complexes are stable and their framework provides a considerable
scope for design optimization, in terms of biological activity and of minimizing sideꢀ
effects, through variations in the arene and in the other coordinated ligands.2 Two
compounds of this class, RM175 and RAPTAꢀT (Figure 1), entered preꢀclinical
development stages, due to its activity against primary and metastasis tumors,
respectively. 2d
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Isoelectronic
cyclopentadienylꢀruthenium(II)
compounds
[(η5ꢀ
C5H5)Ru(L1)(L2)(L3)][X] have been also subject to posterior research. Compounds of
general formulas [Ru(η5ꢀC5H5)(PP)(L)][X] (PP= monoꢀ or bidentate phosphanes; L= Nꢀ
donor ligand; X= counterꢀion) and [Ru(η5ꢀC5H5)(P)(NꢀN)][X] (P= phosphane ligand; Nꢀ
N= bidentate ligand; X= counterꢀion) showed significant toxicity against a variety of
cancer cell lines, namely LoVo (human colon adenocarcinoma), MiaPaCa (pancreatic
cancer), HLꢀ60 (human leukemia), A2780 and A2780CisR (human ovarian, cisplatinꢀ
sensitive and cisplatinꢀresistant, respectively), MCF7 and MDAMB231 (human breast,
estrogen dependent and independent, respectively), PC3 (human prostate) and HeLa
(cervical carcinoma) cancer cell lines, with IC50 values consistently lower than
cisplatin’s, up to 200ꢀfold in some cases.7 Stausporine mimetic cyclopentadienylꢀ
ruthenium compounds, exhibited affinity and selectivity for protoꢀoncogenic enzymes
such as GSK3, Pimꢀ1, PI3K.7aꢀd In particular, compound DW1/2 (Fig. 1) the most
studied within this subꢀclass, inhibits either GSK3beta and PI3K in human melanoma
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cells, leading to apoptotic cell death mediated by p53 and the mitochondrial pathway.
Compound [(η5ꢀC5H5)Ru(bipy)(PPh3)]+, TM34, and derivatives, such as its water
soluble analogue TM85 (Figure 1) were later studied for its anticancer properties,
revealing highly cytotoxic against a large spectra of cancer cell lines. Similarly to
DW1/2, TM34 revealed as a strong PARPꢀ1 inhibitor, an enzyme involved in DNA
repair mechanisms and apoptosis pathways,7h and TM85 was found to induce cell death
involving the Golgi apparatus.7k
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Figure 1. Most studied anticancer “halfꢀsandwich” ruthenium(II) compounds.
What about iron, the bioꢀmetal mimicked by ruthenium? Curiously enough, this metal
has attracted much less interest regarding anticancer research. Ferrociffens, “sandwich”
ferrocene derivatives of Tamoxifen, a drug used to treat breast cancer, revealed
anticancer activity against estrogenꢀdependent (MCF7) and independent (MDAꢀ
MB231) breast cancer cell lines, while the parent drug is inactive in the hormoneꢀ
independent cell line.8 Other classes of ferrocene derivatives have been studied, also
providing promising results.9 Ferrocene derivatives suffer, however, from
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bioavailability problems, restricting its clinical use.10 More recently, “halfꢀsandwich”
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cyclopentadienylꢀiron(II)
compounds
of
general
formula
[Ru(η5ꢀ
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C5H5)(Dppe)(L)][CF3SO3] revealed good cytotoxic activities against A2780 (ovarian),
MCF7 (breast), HeLa (cervical carcinoma),11a and HLꢀ60 (human leukemia)11b cancer
cell lines, with IC50 values up to 30 x lower than cisplatin. Similarly to ruthenium
analogues, these early results envision this class of compounds as very promising to the
development of anticancer metalloꢀdrug candidates.
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We have been focusing in developing anticancer organometallic compounds bearing
bioꢀderivative ligands. Carbohydrates are the largest class of natural compounds, readily
available and renewable, providing a large number of functional groups and several
stereogenic centers per molecule, and each hydroxyl group offers the opportunity of
selective modification and coordination, also granting some control over the
lipophilicity/aqueous solubility of derivative complexes. Recently we reported the
synthesis and characterization of cyclopentadienylꢀruthenium(II) complexes bearing
carbohydrate (galactose and fructose) derivative ligands, which revealed outstanding
cytotoxic activities against HeLa (cervical carcinoma) cancer cells, much better than
cisplatin.7m
We now turn our attention to colon cancer (CC), a high profile cancer type, displaying
both high incidence and associated mortality.12 So far, synthetic chemical compounds
such as 5ꢀfluorouracil (5ꢀFU) and oxaliplatin, a secondꢀgeneration platinum
chemotherapeutic drug used specifically for CC, are still the first choice of treatment for
CC patients in advanced stages (Figure 2). Nonetheless, problems such as
innate/acquired resistance, lack of selectivity for cancer tissues and severe associated
sideꢀeffects, constitute major obstacles for this cornerstone chemotherapeutic agents.13
Thus, the discovery of new agents with fewer side effects and improved therapeutic
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value together with the clarification of the pathways by which they operate, is of great
interest to the development of anticancer drug.
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Figure 2. Standard CC chemotherapeutic agents.
Here, we disclose the extension of our library of Group 8 anticancer organometallic
compounds, with the development of new cyclopentadienylꢀRu(II) and Fe(II)
compounds bearing Dꢀribose, Dꢀxylose, Dꢀgalactose and Dꢀglucose derivative ligands,
obtained in oneꢀpot synthesis from commercial raw sugars. The cytotoxicity of twenty
organometallic compounds studied was evaluated in HCT116 CC cells, allowing the
determination of StructureꢀActivity Relationships (SAR’s), concerning the variation of
metal centers, carbohydrate derivative ligands and phosphane coꢀligands. In vitro
studies of our leading compound, 14Ru, indicate improved therapeutic properties when
compared to oxaliplatin, the standard CC chemotherapeutic metalloꢀdrug.
2. Results and Discussion
2.1. Synthesis and Characterization
The carbohydrate derivative ligands L1ꢀL4 were prepared from commercially available
Dꢀribose, Dꢀxilose, Dꢀgalactose and Dꢀglucose, respectively, in a straightforward oneꢀ
pot procedure, by reaction of the raw sugars with hydroxylamine hydrochloride in
pyridine and in situ dehydration of the oximes with acetic anhydride, resulting in the
corresponding Oꢀacetylꢀprotected nitrile ligands (Scheme 1). The synthesis of ligands
L5ꢀL8 had already been previously reported by some of us7m (Scheme 1).
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Scheme 1. Synthesis of the carbohydrate derivatives Lc and organometallic compounds
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1ꢀ23 (all with PF6 counterion).
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The organometallic compounds of general formula [(η5ꢀC5H5)M(PP)Lc][PF6] were
prepared by halide abstraction from the parent neutral complexes [(η5ꢀC5H5)M(PP)X] in
the presence of a slight excess of the corresponding carbohydrate derivative ligand Lc,
in dichloromethane at room temperature (Scheme 1). Compounds were recrystallized by
slow diffusion of nꢀhexane in dichloromethane solutions.
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All new compounds were fully characterized by FTꢀIR, 1H, 13C, and 31P NMR
spectroscopies, and by elemental analysis, corroborating the proposed formulations and
structures. The solid state FTꢀIR spectra of the complexes presents characteristic bands
of Cp rings (3055ꢀ3075 cmꢀ1), the hexafluorophosphate anion (~ 840 and 560 cmꢀ1) and
the coordinated carbohydrate moieties (~ 1750 (νC=O), ~ 1210 (νCꢀO)). The νC≡N band is
not visible in the FTꢀIR spectra of the free ligands, but appears as a medium intensity
band at ~ 2250 cmꢀ1 for Ru(II) compounds and at ~ 2230 cmꢀ1 for Fe(II) analogues.
1H and 13C resonances of the cyclopentadienyl ring are within the characteristic range
for monocationic Ru(II) and Fe(II) complexes, at 4.50 – 4.60 ppm for Fe and Ru(P(pꢀ
YPh)3)2 (Y = H, F, Cl) compounds and at ~ 4.80 ppm for [(η5ꢀC5H5)Ru(Dppe)]+
analogues.7a,14 In compounds with Dppe as coꢀligand, the protons of Lc ligands display
a general upꢀfield upon coordination; for P(pꢀYPh)3 analogues, chemical shifts remain
almost unchanged. Upꢀfield shifts up to 1.2 ppm for H2 of 8Ru and up to 0.9 ppm for
H4 of 5Ru are attributed to the anisotropic effect of the neighbor phosphine aromatic
rings. As discussed for compounds 13Ruꢀ20Ru,7m the aliphatic nature of the ligands
excludes the possibility of πꢀbackdonation throughout the carbohydrate backbone.7a,14
This shielding effect is apparently larger for [(η5ꢀC5H5)Ru(Dppe)Lc]+ compounds than
to their Fe(II) analogues, but a direct comparison cannot be made, since the spectra were
determined in different solvents.
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13C NMR spectra show the chemical shifts of Lc ligands carbon atoms almost unꢀ
shifted upon coordination, which further supports the stereochemical nature of the
shielding effect verified for the respective protons. Nitrile carbon atoms are the only
exception, with lowꢀfield shifts upon coordination ranging from ~ 9 ppm for [(η5ꢀ
C5H5)Ru(Dppe)Lc]+ compounds, to ~ 16 ppm for the Fe(II) counterparts. Concerning
Ru(II) compounds, the nitrile carbon of [(η5ꢀC5H5)Ru(P(pꢀYPh)3)2Lc]+derivatives is
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more deꢀshielded upon coordination (~ 13 ppm) than in the Dppe counterparts,
consistent with the lower electronic density of the metal center in the [(η5ꢀC5H5)Ru(pꢀ
YPPh3)2Lc]+ moiety.12 The higher lowꢀfield shifts verified for Fe(II) compounds can be
explained by the harder metal center and a consequently stronger σꢀdonation from the
nitrile. 31P NMR spectra of the complexes present two doublets, attributed to the
phosphine coꢀligands, due to a nonꢀequivalence of the coordinated phosphorus atoms, as
a result of the asymmetry induced in the metal center by the chiral carbohydrateꢀderived
ligands; one exception is observed for 4Ru and 22Ru, which presents an
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“uncharacteristic” singlet. 2JPP coupling constants of [(η5ꢀC5H5)Ru(P(pꢀ
YPh)3)2Lc]+complexes lie within the range 34.0ꢀ36.9 Hz, while for [(η5ꢀ
C5H5)Ru(Dppe)Lc]+derivatives it lies within the range 22.5ꢀ25.5 Hz. This difference is
explained attending the different PꢀRuꢀP angles: P(pꢀYPh)3 has larger cone angles, thus
leading to a larger PꢀRuꢀP angle and subsequently to a larger 2JPP.15 For the Fe(II)
compounds, the 2JPP coupling constants lie within the range 31.4ꢀ33.5 Hz, larger than
their Ru(II)ꢀDppe analogues. FeꢀP bonds are shorter than RuꢀP bonds and PꢀFeꢀP angles
are consequently larger than PꢀRuꢀP angles, explaining the larger 2JPP coupling
constants verified for Fe analogues. 31P NMR spectra of the complexes also display a
septupletat ~ ꢀ144 ppm, a characteristic of hexafluorophosphate counterꢀion.
Stability of organometallic compounds in stock solution conditions, used in cytotoxic
studies, was accessed by 31P NMR spectroscopy. The spectra were determined in
DMSOꢀd6, following sample preparation and after two weeks of air and moisture
exposure, revealing no decomposition within this period. Stability studies in aqueous
media were attempted using 31P NMR and UVꢀVis spectroscopies, but failed due to
extremely low solubility of the compounds in this media.
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Suitable crystals for Xꢀray diffraction studies were obtained by slow diffusion of nꢀ
hexane in dichloromethane solutions of 3Fe (Figure 3A), 6Fe (B), 10Ru (C), 11Ru (D)
and 12Fe (E). Compound 3Fe crystallizes in orthorhombic crystalline system, space
group P212121, and all others in monoclinic system, space group P21.
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A
B
C
E
D
Figure 3. Crystal structure of organometallic cations 3Fe (A), 6Fe (B), 10Ru (C), 11Ru
(D) and 12Fe (C), with atom labeling. Displacement ellipsoids are drawn at the 50%
probability level. Hydrogen atoms are omitted for picture clarity.
Cationic complexes present the usual distorted threeꢀlegged piano stool geometry for η5ꢀ
monocyclopentadienyl complexes, with (η5ꢀC5H5)CentꢀMꢀX angles (X = N, P) ranging
from 119.38(17)º to 131.86(14)º, within the range found for analogous compounds.14
Compound 10Ru presents a PꢀRuꢀP angle of 98.26(4) whereas for complexes 3Fe, 6Fe,
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11Ru and 12Fe, the geometry is restricted by the bite angle of Dppe, with PꢀMꢀP angles
of 86.31(4), 86.44(6), 83.83(5) and 86.41(5), respectively; the smaller PꢀRuꢀP angle for
11Ru is rationalized attending to longer RuꢀP distances (~ 2.29 Å), in comparison to Feꢀ
P distances (~ 2.21 Å), in perfect agreement with 31P NMR results interpretation (see
above).
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Although an exhaustive Xꢀray data discussion falls outside the scope of this article, it is
important to notice that, due to the structural similarity of the compounds studied, the
differences in cytotoxic activity must be closely linked to the 3D arrangement of the
cationic complexes. A structural comparison of the three iron compounds confirms the
stereochemistry and chain lengths of the different carbohydrate derivative ligands. The
pentose derivative ligands in 3Fe and 6Fe adopt the same relative orientation, with the
C2ꢀH2 bonds oriented “upwards”, roughly towards the (η5ꢀC5H5) ring, confirming the
different configurations at C3 stereocenter (Figure 3A and B, respectively). The
conformation adopted by the glucose derivative ligand L4 in compounds 10Ru, 11Ru
and 12Fe seems to depend mainly on the stereochemical environment created by the
phosphane coꢀligands. In compound 10Ru, the C(2)ꢀC(6) chain extends sideward in
relation to Ruꢀ(η5ꢀC5H5) direction, “accommodating” between two Ph rings of the same
PPh3 (Figure 3C). On the other hand, in compounds 11Ru and 12Fe (Figure 3D and E,
respectively) L4 extends upwards, minimizing the steric interaction with Ph rings of
different P atoms and hindering the metal centers.
2.2. Cytotoxic studies
The cytotoxic activity of the organometallic compounds 1ꢀ23 was evaluated in HCT116
CC cells. The IC50 values determined are presented in Table 1.
When analyzing compounds 1ꢀ12, bearing the linear Oꢀacetylated carbohydrate
derivative ligands L1ꢀL4, the variation of the cytotoxic activity with the organometallic
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moiety, within the same ligand, is IC50 ([(η5ꢀC5H5)Ru(PPh3)2]+) < IC50 ([(η5ꢀ
C5H5)Ru(Dppe)]+) < IC50 ([(η5ꢀC5H5)Fe(Dppe)]+), except for the galactose derivative
L3, in which case the order of Ru(II) moieties is reversed. The best IC50 value within
this series of compounds was obtained for 10Ru, bearing the carbohydrate derivative
L4 (IC50= 1.30 ꢁM).
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Table 1. IC50 values of the organometallic compounds 1ꢀ23, oxaliplatin (positive
control) and 5ꢀFU17 in HCT116 CC cells, after 72h of compound exposure.
IC50 (µM) 95% CI
IC50 (µM) 95% CI
Compound
1Ru
Compound
13Ru
14Ru
15Ru
16Ru
17Ru
18Ru
19Ru
20Ru
21Ru
22Ru
23Ru
1.52
3.37
1.68ꢀ1.81
3.08ꢀ4.03
1.32
0.45
1.60
6.88
1.16
0.44
1.31
3.95
4.10
3.71
9.30
0.45
3.80
1.22ꢀ1.43
0.44ꢀ0.46
1.52ꢀ1.68
6.54ꢀ7.24
1.10ꢀ1.23
0.43ꢀ0.46
1.24ꢀ1.39
3.79ꢀ4.12
2.70ꢀ6.68
1.67ꢀ9.65
4.38ꢀ18.90
0.41ꢀ0.48
2Ru
36.53 30.82ꢀ43.30
3Fe
1.96
5.42
1.88ꢀ2.05
5.12ꢀ5.84
4Ru
5Ru
26.77 21.03ꢀ34.07
6Fe
2.46
1.50
2.23ꢀ3.25
1.42ꢀ1.59
7Ru
8Ru
25.81 20.94ꢀ31.81
9Fe
1.30
2.14
4.08
1.22ꢀ1.38
1.88ꢀ2.44
10Ru
11Ru
12Fe
3.64ꢀ4.58 Oxaliplatin
5ꢀFU
Although [(η5ꢀC5H5)Fe(Dppe)]+ derivatives have recently been studied for their
anticancer properties,11 this is the first time where a direct comparison between isoꢀ
structural Ru(II) and Fe(II) compounds can be established. All Fe(II) compounds
revealed less cytotoxic against HCT116 cancer cells than their Ru(II)Dppe counterparts.
Compounds 3Fe, 6Fe and 9Fe, with ligands L1ꢀL3 revealed much higher IC50 than its
Ru(II) analogues, while compound 12Fe, bearing the glucose derivative L4, revealed an
IC50 of 4.08 ꢁM, only ~ 2x higher than the isoꢀstructural 11Ru. This value lies within
the IC50 range spanned by Ru(II) compounds and roughly matches 5ꢀFU IC50 (3.80
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ꢁM),16 a current standard in CC chemotherapeutics. As discussed above for Xꢀray
diffraction and NMR spectroscopic data, 3D structural features imposed by the different
carbohydrate and organometallic moieties may be at the origin of these differences in
cytotoxicity. Moreover, L3 and L4, obtained from galactose and glucose, respectively,
differ only in the stereocenter at C4, giving rise to a huge difference in the cytotoxic
activity of the corresponding iron complexes 9Fe (IC50 25.81 ꢁM) and 12Fe (IC50 4.08
ꢁM), further supporting the importance of stereochemical features for the high cytotoxic
activity of 12Fe.
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With regard to compounds 13ꢀ20, the best cytotoxic results were obtained for the [(η5ꢀ
C5H5)Ru(Dppe)]+ derivatives 14Ru (IC50= 0.45 ꢁM) and 18Ru (IC50= 0.44 ꢁM),
bearing the galactose and fructose nitrile derivative ligands L5 and L7, respectively,
matching the activity of the cornerstone CC metalloꢀdrug oxaliplatin; the corresponding
[(η5ꢀC5H5)Ru(PPh3)2]+ derivatives 13Ru and 17Ru, respectively, revealed it selves less
cytotoxic than the Dppe counterparts.
In all cases, organometallic compounds with tetrazole ligands L6 and L8 showed less
inhibitory effects than the compounds bearing the respective nitrile analogues, but while
[(η5ꢀC5H5)Ru(PPh3)2]+ derivatives show a minimum difference , for[(η5ꢀ
C5H5)Ru(Dppe)]+ derivatives, IC50’s varies from 0.45 ꢁM (14Ru) to 6.88 ꢁM (16Ru)
for compounds with L6, and slightly less for compounds with L8.
So far, Dppe and PPh3 were the only phosphane coꢀligands used in anticancer
compounds of general formula [(η5ꢀC5H5)Ru(PP)(L)][X]. Compounds 21ꢀ23Ru were
synthesized to evaluate the effect of halogenated 4ꢀphenylphosphanes in the cytotoxicity
of derivative complexes, when compared to PPh3. Fluorine is well known to impart
special physicochemical and pharmacological properties on drug candidates, being
present in 15–20% of currently approved drugs,17 including anticancer agents (e.g. 5ꢀ
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Fluorouracil, Fludarabine), and chlorine is also present in chemotherapeutic drugs (e.g.
Cladribine). The results were somehow disappointing, since all three compounds
revealed it selves less cytotoxic than the PPh3 analogues. Nonetheless, compounds
21Ru and 22Ru, bearing P(pꢀFPh)3 phosphane coꢀligands, revealed IC50 values within
the range spanned by Ru derivatives (4.10 and 3.71 ꢁM, respectively) while compound
23Ru, the chlorinated analogue of 13Ru and 22Ru, was revealed the least cytotoxic of
Ru derivatives (IC50 = 9.30 ꢁM).
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Compounds 14Ru and 18Ru revealed the same cytotoxicity against HCT116 CC cells
(IC50 = 0.45 and 0.44 ꢁM, respectively); for HeLa cancer cells (cervical carcinoma), the
galactose derivative 14Ru(IC50 = 3.58 ꢁM) proved more cytotoxic than its fructose
analogue 18Ru (IC50 = 6.07 ꢁM)7m and less cytotoxic than 13Ru, bearing PPh3 coꢀ
ligands (IC50 = 2.63 ꢁM).
Next, the cytotoxicity mechanisms triggered by compound 8Ru (IC50 = 1.50 ꢁM) and
14Ru (IC50 = 0.45 ꢁM), both [(η5ꢀC5H5)Ru(Dppe)]+ derivatives bearing galactose
derivative ligands in openꢀchain (L3) and cyclic (L5) forms, respectively, were further
explored.
First, cell viability and general cell death were evaluated following HCT116 cell
exposure to IC50, 1 and 2 µM of 8Ru, 14Ru and oxaliplatin, for 72 h, respectively by
ApoToxGlo Triplex Assay, and by lactate dehydrogenase (LDH) release assay. Our
results show that for IC50 concentrations, and similarly to oxaliplatin, exposure to both
8Ru and 14Ru led to significantly reduced cell viability (p < 0.01) (Figure 4A) and
increased general cell death (p < 0.01) (Figure 4B), whereas compared to vehicle
control (DMSO). Moreover, in HCT116 cells, exposure to 1 and 2 µM of 14Ru induced
a doseꢀdependent effect, further decreasing cell viability to 40 and 16 % (p < 0.01)
(Figure 4A) and enhancing cell death by 1.5ꢀ to 2.7ꢀfold (p < 0.01) (Figure 4B),
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respectively, whereas compared to vehicle control. In turn, no significant increase in the
cytotoxic effect of 8Ru was seen when exposing cells to 1 µM of this compound, while
2 µM exposure induced a modest 25% decrease in cell viability (Figure 4A), and a 1.18ꢀ
fold increase in in cell death (p < 0.01) (Figure 4B).
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Figure 4. Evaluation of the cytotoxic effects of compounds 8Ru, 14Ru and oxaliplatin,
on HCT116 human CC cells. Cells were exposed to IC50, 1 and 2 µM 8Ru, 14Ru or
oxaliplatin for 72 h. DMSO was used as a vehicle control. Cell viability (A), general
cell death (B) and percentage of dead cells (C), were evaluated using ApoToxGlo
Triplex Assay, LDH release assay and trypan blue exclusion assay, respectively. Results
are expressed as (A, B) mean ± SEM foldꢀchange to vehicle treated cells, or as (C)
percentage of death cells per sample ± SEM, from at least three independent
experiments. Dotted lines represent vehicle control results. § p < 0.05 and * p < 0.01
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from vehicle control treated cells; † p < 0.05 and ‡ p < 0.01 from oxaliplatin treated
cells.
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More importantly, exposure to 14Ru was significantly more cytotoxic than oxaliplatin
at 2 µM (p < 0.01) (Figure 4A and B), highlighting the antitumor potential of this
compound when compared to a current standard of care in CC chemotherapy, which
presented a similar IC50 value to 14Ru in HCT116 cells (Table 1). In parallel, the
cytotoxic effects of organometallic compounds were validated using the trypan blue
exclusion assay after 72 h of exposure to IC50, 1 and 2 µM concentrations of the
compounds. Our results confirmed the cytotoxicity of all test compounds, and also the
significantly increased cytotoxicity of 14Ru in HCT116 at 2 µM (68 % dead cells)
when compared to oxaliplatin at 2 µM (43 % dead cells) (p < 0.05) (Figure 4C).
It is well established that chemotherapeutic agents used against tumor cells act by
triggering mechanisms of apoptotic cell death. In this context, activation of caspases has
been shown to be a hallmark of drugꢀinduced cellular apoptosis.18 In addition, apoptotic
cell death is characterized by distinctive morphological changes, including cell
shrinkage, loss of intercellular membrane contact, progressive condensation of
chromatin and cytoplasm, and subsequent nuclear fragmentation. These events
culminate in a characteristic formation of apoptotic bodies, consisting of nuclear
fragments and intact cell organelles surrounded by a plasma membrane.19 Therefore, we
assessed caspaseꢀ3/7 activation using CaspaseꢀGlo 3/7 Assay (Promega), and apoptosis
induction by evaluation of nuclear morphology under fluorescent microscopy following
staining with the DNAꢀbinding stain Hoechst, upon HCT116 cell exposure to IC50, 1
and 2 µM of 8Ru, 14Ru and oxaliplatin for 24 h and 72 h, respectively. Our data
showed that similarly to oxaliplatin, both 8Ru and 14Ru triggered apoptotic cell death,
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leading to a significant increase in caspaseꢀ3 and ꢀ7 activity (p < 0.05) (Figure 5A) and
apoptotic cells (p < 0.01) (Figure 5B and C), whereas compared to vehicle control cells.
Moreover, our results established that 14Ru induced a 1.4ꢀfold increase in caspaseꢀ3
and ꢀ7 activity at IC50, which was significantly higher than oxaliplatin’s (p < 0.05),
which increased caspaseꢀ3 and ꢀ7 activity only by 1.27ꢀfold (p < 0.01) (Figure 5A).
Hereupon, 14Ru exposure induced a doseꢀdependent increase in HCT116 apoptosis,
leading to up to 16 , 22 and 43 % of apoptotic cells at IC50, 1 and 2 µM, respectively,
whereas compared to vehicle control exposure (p < 0.01) (Figure 5B). In contrast,
exposure to 2 µM 8Ru led to less than 25 % of apoptotic events (p < 0.01) (Figure 5B).
It should be emphasized that 14Ru also induced significantly higher levels of apoptosis
than oxaliplatin at 2 µM (p < 0.01), which was shown to trigger apoptosis in 30 % of
HCT116 cells (p < 0.01) (Figure 5B).
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Figure 5. Evaluation of the apoptotic effects of compounds 8Ru, 14Ru and oxaliplatin
on HCT116 human CC cells. Cells were exposed to IC50, 1 and 2 µM of 8Ru, 14Ru or
oxaliplatin. DMSO was used as a vehicle control. (A) Caspaseꢀ3/7 activity was
determined at 24 h of compound exposure using the CaspaseꢀGlo 3/7 Assay. (B)
Nuclear morphology after Hoechst staining was evaluated by fluorescence microscopy
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at 72 h of compound exposure. Dotted lines represent vehicle control results. (C)
Representative images of Hoechst staining at 400x magnification. Arrows indicate
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nuclear fragmentation and chromatin condensation Results are expressed as (A) mean ±
.
9
SEM foldꢀchange to vehicle treated cells, or as (B) percentage of apoptotic cells per
field ± SEM, from at least three independent experiments. § p < 0.05 and * p < 0.01
from vehicle control treated cells; † p < 0.05 and ‡ p < 0.01 from oxaliplatin treated
cells.
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Collectively, our data indicate that 8Ru and 14Ru are capable of markedly decreasing
cell viability and inducing apoptotic cell death in HCT116 human CC cells. More
importantly, 14Ru was shown to trigger significantly higher levels of apoptosis when
compared to oxaliplatin, the cornerstone colon cancer chemotherapeutic agent.
3. Experimental Section
All experiments were carried out under inert atmosphere (N2) using standard Schlenk
techniques. Commercial reagents were bought from SigmaꢀAldrich and used without
further purification. All solvents were dried using standard methods.20 Starting materials
were prepared following the methods described in the literature for the synthesis of [(η5ꢀ
C5H5)Ru(Dppe)Cl], [(η5ꢀC5H5)Ru(PPh3)2Cl]21 and [(η5ꢀC5H5)Fe(Dppe)I].22 Compounds
[(η5ꢀC5H5)Ru(P(pꢀFPh)3)2Cl] and [(η5ꢀC5H5)Ru(P(pꢀClPh)3)2Cl] are here first described,
and were synthesized adopting the procedure used for the synthesis of [(η5ꢀC5H5)
Ru(PPh3)2Cl], as described below. Solid state IR spectra were recorded in a Jasco FTIRꢀ
4100 spectrophotometer with KBr pellets; only significant bands were cited in the text.
1H, 13C and 31P NMR spectra were recorded on Bruker Avance II 400 or Bruker Avance
II 300 spectrometers, at probe temperature. The 1H and 13C NMR chemical shifts are
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reported in parts per million (ppm) downfield from the residual solvent peak; 31P NMR
spectra are reported in ppm downfield from external standard H3PO4 85%. Coupling
constants are reported in Hz. Assignments of 1H and 13C NMR spectra were confirmed
with the aid of two dimensional techniques 1H, 13C (COSY, HSQC). Microanalyses
were performed at Laboratório de Análises do Instituto Superior Técnico, using a Fisons
Instruments EA1108 system and data acquisition, integration and handling were
performed using the software package Eagerꢀ200 (Carlo Erba Instruments), confirming
≥ 95% purity for all the tested compounds.
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General procedure for the synthesis of ligands Lc: To a solution of carbohydrate (10
mmol) in pyridine (5 mL) was added H2NOH·HCl (12 mmol) and the mixture was
stirred for 2h. Ac2O was then added (5 mL) and the mixture stirred at r.t. for further 2h.
The solvent was then removed and the crude was extracted with AcOEt (3 x 20 mL),
filtered and pumped to dryness. The crude obtained was purified by column
chromatography (AcOEt:nꢀhexane), affording the pure Oꢀacetylated nitrile ligands as a
white crystalline solids.
General procedure for the synthesis of complexes [(η5ꢀC5H5)Ru(P(pꢀYPh)3)2Cl] (Y = F,
Cl): To a boiling solution of RuCl3·xH2O (1.0 g) and freshly distilled cyclopentadiene (5
mL) in ethanol (80 mL), was added P(pꢀYPh)3 (12 mmol) and the mixture was stirred
for 2h. After cooling to r.t., the solvent was removed by filtration and the solid products
were washed with ethanol (3 x 20 mL) and nꢀhexane (3 x 20 mL). The products were
recrystallized from dichloromethane solutions, by addition of nꢀhexane, affording
crystalline compounds.
General procedure for the synthesis of complexes [(η5ꢀC5H5)M(P–P)(Lc)][PF6]: To a
schlenck charged with [(η5ꢀC5H5)M(P–P)X] (0.20 mmol), TlPF6 (0.20 mmol) and Lc
(0.22 mmol) was added dichloromethane (20 mL), and the mixture was stirred
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overnight at room temperature, under inert atmosphere. The solutions were double
filtered and pumped to dryness, and the crude compounds were then washed with nꢀ
hexane and recrystallized by slow diffusion of nꢀhexane in dichloromethane solutions,
affording crystalline products.
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Xꢀray Diffraction Studies. Suitable crystals for Xꢀray diffraction studies were obtained
by slow diffusion of hexane into dichloromethane solutions of 3Fe, 6Fe, 10Ru, 11Ru
and 12Fe. Data were collected at 150 K using a Bruker AXSꢀKAPPAAPEX II
diffractometer. Structure resolution was performed with SHELXS97 and refinement
with SHELXL97. H atoms were calculated and constrained as riding on their bound
atoms. CCDC 1050669 to 1050673 contains the supplementary crystallographic data for
this paper (3Fe, 6Fe, 10Ru, 11Ru and 12Fe, respectively). These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif or by emailing
data_request@ccdc.cam.ac.uk or by contacting The Cambridge Crystallographic Data
Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax +44 1223 336033.
Cell culture. HCT116 human colon carcinoma cells were grown in McCoy’s 5A
modified medium supplemented with 10% heatꢀinactivated fetal bovine serum (FBS)
and 1 % antibiotic/antimycotic solution (Gibco, Life Technologies, Paisley, UK), and
cultured at 37 °C under a humidified atmosphere of 5 % CO2. Cells were seeded in 96ꢀ
well plates at 1 x 104 cells/well for doseꢀresponse curves, cell viability and cell death
assays; and at 1.5 x 104 cells/well for caspase activity studies. Additionally, cells were
seeded in 24ꢀwell plates at 5x104 cells/well for trypan blue exclusion assay, and in
35 mm dishes at 3 x 105 cells/dish for morphological assessment of apoptosis.
Exposure to organometallic compounds. Stock solutions of the organometallic
complexes 1-23 were prepared in sterile DMSO. Prior to all treatments, the cells were
allowed to adhere for 24 h, and then exposed to test compounds for the mentioned time.
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To plot doseꢀresponse curves, cells were exposed to 0.1–100 µM test compounds for
72 h. For cell viability, cell death and apoptosis studies, metalꢀbased compounds were
tested at IC50, 1 and 2 µM, for 24 to 72 h. All experiments were performed in parallel
with DMSO vehicle control. Oxaliplatin, a cytotoxic agent used in colon cancer
treatment, was used as a positive control in all assays. The final DMSO concentration
was always of 0.1 %.
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Doseꢀresponse curves. To plot doseꢀresponse curves, cell viability was evaluated using
the CellTiter 96 AQueous NonꢀRadioactive Cell Proliferation Assay (Promega, Madison,
WI, USA), according to the manufacturer’s instructions. This colorimetric assay is
based on the bioꢀreduction of 3ꢀ(4,5ꢀdimethylthiazoꢀ2ꢀyl)ꢀ5ꢀ(3ꢀcarboxymethoxyphenyl)ꢀ
2ꢀ(4ꢀsulfophenyl)ꢀ2Hꢀtetrazolium inner salt (MTS) to formazan by dehydrogenase
enzymes found within metabolically active cells. The amount of water soluble formazan
product can be measured by the amount of 490 nm absorbance, correlating with the
number of living cells in culture. For this purpose, changes in absorbance were assessed
using a Model 680 microplate reader (BioꢀRad, Hercules, CA, USA). Bestꢀfit IC50
values from at least three independent experiments were calculated using GraphPad
Prism software (version 5.00; San Diego, CA, USA), using the log (inhibitor) vs
response (variable slope) function.
Evaluation of cell viability. Cell viability was evaluated using the ApoToxGlo Triplex
Assay (Promega), according to the manufacturer’s instructions. This assay uses a
fluorogenic, cellꢀpermeant, peptide substrate (glycylphenylalanylꢀaminofluorocoumarin;
GFꢀAFC) that may be cleaved by a protease found exclusively within intact cells to
release AFC, generating a fluorescent signal proportional to the number of living cells.
In brief, 20 µL of GFꢀAFC substrate solution was added to each well, and plates were
incubated at 37 °C for 1 h. Fluorescence emission was detected using a GloMaxꢀMulti+
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Detection System (Promega), with an at 405 nm excitation filter and at 495ꢀ505 nm
emission filter.
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Evaluation of cell death. General cell death was evaluated using the Cytotoxicity
Detection KitPLUS (Roche Diagnostics GmbH, Mannheim, Germany) to measure the
amount of cytoplasmic lactate dehydrogenase (LDH) released from plasma membraneꢀ
damaged cells into the extracellular medium. Thereby, the amount of enzyme activity on
supernatants can be proportionally determined by a coupled enzymatic reaction whereby
the pꢀiodonitrotetrazolium salt is reduced to a red formazan product that can be
spectrophotometrically quantified at 490 nm. For the LDH assay, 50 µL of culture
supernatant was collected from each well into a new 96ꢀwell plate to evaluate LDH
release. In parallel, the remaining cells attached to the original plate were lysed in 50 µL
of medium to release the intracellular LDH. Subsequently, supernatant samples and total
cell lysates were incubated with 50 µL of assay substrate for 10 to 30 min, at room
temperature, protected from light. Absorbance readings were measured
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spectrophotometrically at 490 nm, with a 620 nm reference wavelength, using a Model
680 microplate reader (BioꢀRad). The percentage of LDH release was determined as the
ratio between the released LDH (supernatant) and the total LDH (supernatant + cell
lysate).
Additionally, cell death was evaluated using the trypan blue dye exclusion test. This
method relies on the principle that live cells retain intact cytoplasmic membranes that
exclude trypan blue, remaining unstained, while dead cells incorporate this vital dye
into the cytoplasm due to loss of membrane selectivity. To evaluate trypan blue dye
intake upon loss of membrane integrity, cell culture supernatants and attached cells were
harvested to the same tube, and next collected by centrifugation at 500 g for 5 min.
Next, supernatants were discarded, and cells were reꢀsuspended and stained in 0.1 %
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trypan blue solution diluted in phosphateꢀbuffered saline (PBS). Subsequently, the
relative number of dead and live cells was obtained by optical microscopy by counting
the number of blueꢀstained (dead) and unstained (live) cells using a Neubauer chamber.
Evaluation of apoptotic cell death. Activity of effector caspaseꢀ3 and ꢀ7 was measured
using the CaspaseꢀGlo 3/7 Assay (Promega). This assay is based on the cleavage of a
proꢀluminescent substrate containing the specific DEVD sequence recognized by
caspaseꢀ3 and ꢀ7 to release aminoluciferin in cell lysates. The subsequent luciferase
cleavage of the unconjugated aminoluciferin generates a luminescent signal directly
proportional to the amount of caspase activity present in the sample. For this purpose,
75 µL of CaspaseꢀGlo 3/7 reagent was added to each well, and the mixture was
incubated at room temperature for 30 min, leading to complete cell lyses, stabilization
of substrate cleavage by caspases, and accumulation of luminescent signal. The
resulting luminescence was measured using the GloMaxꢀMulti+ Detection System
(Promega).
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Nuclear morphology was assessed using the DNAꢀbinding stain Hoechst to identify
apoptotic cells based on their typical morphological changes. In brief, attached cells
were fixed with 4 % paraformaldehyde in PBS for 20 min, stained with 5 mg/mL
Hoechst 33258 dye (Sigma Aldrich, St. Louis, MO, USA) in PBS for 15 min, washed
with PBS, and mounted with cover slips using PBS/glycerol (3:1). Nuclear morphology
was evaluated by fluorescence microscopy, under 400x magnification, and nuclei were
scored and categorized according to condensation and staining characteristics of
chromatin. Normal nuclei showed nonꢀcondensed chromatin dispersed over the entire
nucleus. Apoptotic nuclei were identified by condensed chromatin, contiguous to the
nuclear membrane, as well as nuclear fragmentation. A minimum of five random
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microscopic fields with approximately 100 nuclei were counted for each condition and
the results were expressed as the percentage of apoptotic nuclei per field.
Statistical Analysis. All data were expressed as mean ± standard error of mean (SEM)
from at least three independent experiments. Statistical analysis was performed using
Student’s tꢀtest. Values of p < 0.05 were considered significant.
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4. Conclusions
This study provides a first insight on cyclopentadienylꢀruthenium(II) and iron(II)
complexes with bioꢀderivative moieties as CC chemotherapeutic agents. The ligands Lc
are obtained from inexpensive raw carbohydrates using simple synthetic procedures,
and taking advantage of both cyclic and open chain forms of these biomolecules. The
cytotoxic activity of twenty three organometallic compounds of general formula [(η5ꢀ
C5H5)M(PP)Lc][PF6] was evaluated in HCT116 CC cells, with two ruthenium
compounds, 14Ru and 18Ru, matching the IC50 of oxaliplatin, and compound 12Fe
revealing high cytotoxicity, roughly matching the IC50 of 5ꢀFU, the nonꢀmetallic CC
chemotherapeutic cornerstone. SAR’s data analysis shows a dependency of the
cytotoxic activity on the carbohydrate moiety (sugar and openꢀchain/cyclic form), linker
(nitrile, tetrazole), metal (Ru, Fe) and coꢀligands (Dppe, PPh3, P(pꢀFPh)3, P(pꢀClPh)3).
Our leading compound, 14Ru, showed improved cytotoxic properties, when compared
to oxaliplatin, the cornerstone chemotherapeutic metalloꢀdrug used in CC treatment.
This compound induces doseꢀdependent HCT116 general cell death, triggering higher
levels of caspaseꢀ3 and ꢀ7 activities, and apoptosis in a dose dependent manner.
Taken together, these results highlight the potential of ruthenium and iron
organometallic compounds bearing carbohydrate moieties as rising opportunities to
develop alternative anticancer drugs, with very high potential for surpassing problems
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such as general toxicity, side effects and drug resistance, the current major obstacles in
colon cancer treatment.
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Supporting Information. Full spectroscopic characterization by FTꢀIR, 1Hꢀ, 13Cꢀ, 31P
NMR (spectra description and interpretation) and elemental analysis of compounds L1ꢀ
L4, [(η5ꢀC5H5)Ru(P(pꢀFPh)3)2Cl], [(η5ꢀC5H5)Ru(P(pꢀClPh)3)2Cl] and compounds 1ꢀ23,
and further Xꢀray experimental details. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* PRF email: pedro.florindo@tecnico.ulisboa.pt
* ACF email: anacristinafernandes@tecnico.ulisboa.pt
ACKNOWLEDGMENT
This research was supported by FCT through projects PTDC/QUIꢀQUI/102114/2008
and PTDC/QUIꢀQUI/110532/2009 and annual funds UID/QUI/00100/2013. PRF and
DMP (SFRH/BD/96517/2013) thank FCT for grants. Authors thank the Portuguese
NMR Network (ISTꢀUTL Center) for providing access to the NMR facilities..
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