Synthesis, characterization and cytotoxicity of cyclopentadienyl ruthenium(II) complexes containing carbohydrate-derived ligands Dedicated to Prof. Maria José Calhorda on the occasion of her 65th birthday.

Article abstract of DOI:10.1016/j.jorganchem.2013.09.004

We here report the synthesis of new cyclopentadienyl ruthenium(II) complexes of general formula [(η⁵-C₅H ₅)Ru(PP)(L)]⁺ (PP = two triphenylphosphine, 1,2-diphenylphosphinoethane), isolated as PF6- salts, with L b

Full text of DOI:10.1016/j.jorganchem.2013.09.004

Accepted Manuscript
Synthesis, Characterization and Cytotoxicity of Cyclopentadienyl Ruthenium(II)
Complexes Containing Carbohydrate-Derived Ligands
Pedro Florindo, Inês J. Marques, Carla D. Nunes, Ana C. Fernandes
PII:
S0022-328X(13)00672-4
10.1016/j.jorganchem.2013.09.004
JOM 18250
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 24 July 2013
Revised Date: 2 September 2013
Accepted Date: 3 September 2013
Please cite this article as: P. Florindo, I.J. Marques, C.D. Nunes, A.C. Fernandes, Synthesis,
Characterization and Cytotoxicity of Cyclopentadienyl Ruthenium(II) Complexes Containing
Carbohydrate-Derived Ligands, Journal of Organometallic Chemistry (2013), doi: 10.1016/
j.jorganchem.2013.09.004.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
The
synthesis,
characterization
and
cytotoxic
evaluation
of
new
cyclopentadienylruthenium(II) complexes of general formula [(η5-C5H5)Ru(PP)L][PF6],
(PP = two triphenylphosphine, 1,2-diphenylphosphinoethane), L being galactose and
fructose carbohydrate derivative ligands, N-coordinated to the metal centre by nitrile,
tetrazole and 1,3,4-oxadiazole moieties, is described.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, Characterization and Cytotoxicity of Cyclopentadienyl Ruthenium(II)
Complexes Containing Carbohydrate-Derived Ligands
Pedro Florindo,a Inês J. Marques,b Carla D.Nunes,b Ana C. Fernandesa,*
aCentro de Química Estrutural, Complexo I, Instituto Superior Técnico, Universidade de
Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal, Tel: 351 218419264; Fax: 351
218464457, E-mail:anacristinafernandes@ist.utl.pt
bCentro de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa,
Campo Grande 1749-016 Lisboa, Portugal
Abstract
We here report the synthesis of new cyclopentadienyl ruthenium (II) complexes of
general formula [(η5-C5H5)Ru(PP)(L)]+(PP
=
two triphenylphosphine, 1,2-
-
diphenylphosphinoethane), isolated as PF6 salts, with L being galactose and fructose
carbohydrate derivative ligands, N-coordinated to the metal centre by nitrile, tetrazole
and 1,3,4-oxadiazole moieties. The ten new organometallic compounds were fully
1
characterized by FT-IR, H, 13C, and 31P NMR spectroscopies, and by elemental
analysis. The cytotoxicity of the ruthenium(II) compounds was tested on HeLa cancer
cells (cervical carcinoma), unveiling IC50 values in the low micromolar range.
Keywords: Ruthenium(II), Cyclopentadienyl, Carbohydrates, Organometallic,
Cytotoxicity
1

ACCEPTED MANUSCRIPT
1. Introduction
Organometallic complexes containing monosaccharide ligands represent a small but
challenging field in modern chemistry. Carbohydrates are the largest class of natural
compounds and thereby readily available and renewable. They provide a large number
of functional groups and several stereogenic centres per molecule, and each of the
hydroxyl groups offers the opportunity of selective modification and coordination [1, 2].
They can act as monodentate as well as polydentate chelating ligands with pronounced
three-dimensional characteristics [3] and their coordination capability is not limited to
oxophilic metal centers: the change of donor atoms from oxygen to others, e.g.,
nitrogen, enables the coordination to almost every metal atom [4]. They allow also some
control over the lipophilicity/aqueous solubility of the complexes, by selective
modification of the carbohydrate moiety.
Since the accidental discovery of the anticancer drug cisplatin by Rosenberg and co-
workers in 1965 [5], metal complexes have attracted much interest as
metallopharmaceuticals. Although cisplatin is still nowadays successfully used in the
treatment of many cancer types, problems such as toxicity, side effects and drug
resistance lead to investigation of alternative anticancer drugs.
Among the metal atoms used in anticancer metal complexes, ruthenium is most unique.
Despite being a rare noble metal, unknown to living systems, ruthenium compounds
show remarkable features, such as low general toxicity, the ability to mimic iron
binding to biomolecules (transferrin, albumin) and stronger affinity for cancer tissues
over normal tissues [6,7]. In particular, the families of half-sandwich organometallic
complexes [(η6-C6H6)Ru(L)3] [8-17] and [(η5-C5H5)Ru(L)3] [18-23] in which three
coordination sites are occupied by the aromatic rings, have been studied for their
anticancer properties, evidencing cytotoxic properties in cisplatin resistant cancer cell
2

ACCEPTED MANUSCRIPT
lines, with IC50 values in nanomolar range. Apart from applications as anticancer drugs,
other medical applications of ruthenium compounds have been explored. Uses include
immunosupressants [24], nitric oxide scavengers [25], antimicrobial agents [26, 27],
malaria [28] and Chaga’s disease treatment [29].
The synthesis of ruthenium compounds bearing carbohydrate derived ligands is a
relatively unexplored area: our bibliographic search revealed some examples of
ruthenium carbonyl clusters containing carbohydrate moieties [30-34], ruthenium-arene
complexes containing a carbohydrate phosphite derivative with anticancer properties
[35-37], and a report of ruthenium cyclopentadienyl complexes with coordinated
thiomonosaccharides concerning their promising anti-inflammatory effects [38].
As part of our endeavour to produce a library of carbohydrate-containing
organometallic compounds, we here report the synthesis and cytotoxic evaluation
against human HeLa cells (cervical carcinoma) of ten new η5-cyclopentadienyl
-
ruthenium(II) complexes of general formula [(η5-C5H5)Ru(PP)(L)]+, isolated as PF6
salts, in which L are galactose and fructose carbohydrate derivative ligands,
functionalized with nitrile, tetrazole and 1,3,4-oxadiazole N-coordinating moieties. The
electronic density and the stereochemichal environment of the metal centre are played
by using two different phosphanes were used as co-ligands, PPh3 and Dppe. All new
compounds were characterized by IR, 1H, 13C, 31P-NMR spectroscopies and by
elemental analysis.
2. Results and Discussion
2.1. Synthesis of the carbohydrate derivative ligands
The aldehyde precursors P2 and P4 (Scheme 1) were obtained by oxidation of the
commercially available 1,2:3,4-Di-O-isopropylidene-α-D-galactopyranose (P1) and
3

ACCEPTED MANUSCRIPT
2,3:4,5-Di-O-isopropylidene-β-D-fructopyranose (P3), prepared as described in the
literature [39], respectively. The corresponding nitrile derivatives L1 and L4 were
obtained in good yields by reaction with hydroxylamine hydrochloride and subsequent
dehydration of the oximes with dicyclohexylcarbodiimide (DCC).
The tetrazole derivatives L2 and L5 were obtained quantitatively by 1,3-dipolar cyclo-
addition of the corresponding nitriles with sodium azide, in DMF. Finally, acylation in
boiling acetic anhydride of L2 and L5 afforded the 1,3,4-oxadiazole derivatives L3 and
L6, respectively, in excellent yields.
1
Compounds P2, L1, L2and L3 were fully characterized H-, 13C-NMR and FTIR
spectroscopies, and by elemental analysis. Compounds P4, L4, L5 and L6 were obtained
and its 1H-, 13C-NMR spectra compared with the data described in the literature [40].
4

ACCEPTED MANUSCRIPT
Scheme 1- Synthesis of the carbohydrate-derived ligands.I) PCC, CH2Cl2; ii) 1- H2NOH·HCl,
Pyridine; 2- CuSO4·5H2O, Et3N, DCC, CH2Cl2; iii) NaN3, NH4Cl, DMF, 100 ºC; iv) Ac2O, ꢀ.
L2 and L3 are derivatives of topiramate, an anticonvulsant used in epilepsy treatment,
and were in this case proposed as less toxic, more efficient alternative anticonvulsant
drugs.
2.2. Synthesis of the Ru(II) complexes
The novel cationic complexes of general formula [(η5-C5H5)Ru(PP)(L)]+((PP) = 2PPh3
-
or Dppe), isolated as PF6 salts, were prepared by halide abstraction with TlPF6 from the
parent neutral complexes [(η5-C5H5)Ru(PP)Cl] in the presence of a slight excess of the
corresponding carbohydrate-derived ligand, in dichloromethane at room temperature
(Scheme 2). The compounds were recrystallized by slow diffusion of n-pentane or n-
hexane in dichloromethane or acetone solutions.
The
synthesis
of
compounds
[(η5-C5H5)Ru(PPh3)2(L3)][PF6]
and
[(η5-C5H5)Ru(PPh3)2(L6)][PF6] was unsuccessfully attempted, resulting in product
mixtures. Stereochemical hindrance, due to the methyl group in α position relatively to
the coordinated nitrogen and the larger cone angle of PPh3 over Dppe, may be the
reason for the unsuccessful attempts. The same reactions were attempted in refluxing
toluene, with similar results.
The ten new organometallic compounds were fully characterized by FT-IR, 1H, 13C, and
31P NMR spectroscopies, and by elemental analysis, corroborating the proposed
formulations and structures. The solid state FT-IR spectra of the complexes present the
characteristic band of the cyclopentadienyl ring (3055-3059cm-1), the
hexafluorophosphate anion (~840 and 560 cm-1) and the coordinated carbohydrate
moieties.
5

ACCEPTED MANUSCRIPT
Scheme 2- Synthesis of the Ru(II) organometallic complexes.
2.2.1. NMR Spectrocopies
Scheme 3 presents the numbering models and Tables 1 and 2 present selected 1H NMR
data for the galactose and fructose series compounds.
Scheme 3- Numbering models for NMR purposes.
6

ACCEPTED MANUSCRIPT
Table 1- Selected 1H NMR data for the galactose series compounds, in CDCl3.
Compound
H1
H2
H3
4.64-4.67
m
H4
H5
4.64-4.67
m
Cp
L1
5.53, d
4.36, dd
4.33, dd
J = 7.6, 2.4
4.63, dd
_
J = 5.4 J = 4.8, 2.8
5.67, d 4.53,dd
L2*
4.83, dd
5.38, d
_
J = 4.8 J = 4.8, 2.4 J = 7.8, 2.6 J = 8.0, 2.0 J = 2.0
5.65, d 4.36, dd 4.80, dd 4.58, dd 5.14, d
J = 5.2 J = 4.8, 2.8 J = 7.0, 2.4 J = 7.6, 2.0 J = 2.4
L3*
_
[1][PF6]
[2][PF6]
[3][PF6]
5.16, d
J = 4.8 J = 4.8, 2.7 J = 7.8, 2.7
5.51 d 4.24, dd 4.48, dd
J = 4.8 J = 4.8, 2.7 J = 7.8, 2.4
4.11, dd
4.32, dd
3.27, d
J = 7.5
3.60, d
J = 7.5
3.36, d
J = 8.0
4.37, br
4.34, s
4.26, s
4.72, s
4.71, s
4.66, s
4.48, s
4.38, s
5.44, d 4.25 - 4.28
4.36, dd
J = 7.6, 2.0
4.62, dd
J = 4.8
[7][PF6] 5.44, d,
m
4.28, dd
4.45 – 4.46 5.18, br
m
J = 4.8 J = 4.8, 2.7 J = 7.8, 2.4
5.65, d 4.37 ,dd 4.64, dd,
J = 4.8 J = 5.1, 2.1 J = 7.8, 2.7 J = 7.8, 2.1 J = 1.8
[8][PF6]
4.15, dd
4.81, d
*In acetone D6
Table 2- Selected 1H NMR data for the fructose series compounds, in CDCl3.
Compound
H2
H3
H4
H5a
H5b
3.78, dd
Cp
L4
4.61, d
4.64, dd
4.26, dd,
J = 7.9, 1.3
4.34, dd
3.82, dd
_
J = 2.0 J = 7.4, 2.3
4.98, d 4.67dd,
J = 13.0, 1.2 J= 13.0, 1.8
4.05, dd 3.94, d
L5
_
7

ACCEPTED MANUSCRIPT
J= 2.2
J = 7.9, 2.2
4.69, dd
J = 7.9, 1.0
J = 13.0, 1.0
4.02, dd
J = 13.0
L6
5.03, d
4.31, ddd
3.91, dd
_
J = 2.4 J = 8.0, 2.4 J = 8.0, 1.6, 0.9 J = 13.0, 1.6 J = 13.0, 0.9
[4][PF6]
[5][PF6]
[6][PF6]*
[9][PF6]
[10][PF6]
3.32, d
J = 2.1 J = 8.1, 2.7
3.66, d 4.40, dd
J = 2.4 J = 7.8, 2.4
3.42, d 4.48, dd
J = 2.4 J = 8.0, 2.4
4.36, d 4.66, dd
J = 1.8 J = 8.1, 2.7
4.19, d 4.60, dd
4.33, dd
4.05, d
J = 7.8
3.43, d
3.50, d
4.71, s
4.72, s
4.89, s
4.44, s
4.37, s
J = 12.9
J = 12.9
4.11, d
3.72, s
J = 7.8
4.20, dd
J = 8.0, 1.6
4.25, d
3.81, dd
3.67, d
J = 13.2
J = 13.2, 1.6
3.78, d
3.76, dd
J = 7.8
J = 12.6
J = 13.2, 1.5
4.26, d
3.92,s
J = 2.4 J = 7.8, 2.4
*In acetone D6
J = 7.8
The resonances of the cyclopentadienyl ring are within the characteristic range of
monocationic ruthenium (II) complexes [18,41,42]. The carbohydrate-derived ligands
display a general up-field shift of its protons upon coordination, with special relevance
for the ones contiguous to the coordinating moiety (N≡C-, tetrazole or 1,3,4-
oxadiazole), and in compounds with Dppe as co-ligand. Up-field shifts up to 1.2 ppm
for H4 in compound [2][PF6] and up to 1.4 for H2 in [6][PF6] upon coordination (see
Scheme 3 for numbering), are probably due to the anisotropic effect of the neighbour
phosphine aromatic rings, since the its aliphatic nature of the ligands excludes the
possibility of π-backdonation throughout the carbohydrate backbone, this considered as
the major contribute to this phenomenon in other [(η5-C5H5)Ru(PP)(L)]+ derivative
8

ACCEPTED MANUSCRIPT
complexes [18,41,42]. Also, the effect of the σ-donation upon coordination should lead
to the opposite effect.
Selected 13C NMR data for the organometallic complexes is presented in Table 3. The
cyclopentadienyl ring chemical shifts are in the range usually observed for Ru(II)
cationic derivatives. Chemical shifts of the carbohydrate-derived ligands carbon atoms
remained almost unchanged upon coordination, exception made for the ones of the
coordinated nitriles, with low-field shifts from 9.1 to 13.6 ppm, this further confirming
the stereochemical nature of the shielding effect verified for the corresponding protons.
Table 3- Selected 13C NMR data, in CDCl3.
Compound C1
C2 C3 C4 C5
C6 (η5-C5H5)
__
L1
L2*
96.3 70.0 70.5 71.0 60.3 115.3
97.2 71.4 71.3 73.0 64.7 154.4
97.4 71.2 71.6 73.1 65.1 163.8
93.8 74.4 69.3 69.4 61.4 116.6
100.4 74.5 68.6 70.0 61.1 156.8
97.8 73.0 69.8 70.0 61.6 164.5
95.7 69.5 69.9 70.5 61.4 125.0
96.0 70.3 70.3 71.5 62.7 154.0
96.2 70.2 70.3 70.5 62.5 161,0
94.4 73.3 68.7 69.2 61.6 125.7
96.7 74.2 69.0 69.4 61.3 157.5
97.8 74.0 70.2 70.6 62.7 164.5
96.3 70.3 70.0 70.9 62.5 128.9
96.3 70.5 70.5 71.4 63.3 155.2
__
__
__
__
__
L3*
L4
L5
L6
[1][PF6]
[2][PF6]
[3][PF6]
[4][PF6]*
[5][PF6]*
[6][PF6]*
[7][PF6]
[8][PF6]
82.4
82.5
80.6
82.5
82.8
81.8
84.3
83.2
9

ACCEPTED MANUSCRIPT
[9][PF6]1
95.2 73.6 68.9 69.3 62.5 129.2
84.5
84.3
[10][PF6]1 97.8 70.6 70.2 75.4 62.5 159.5
*In acetone D6
The intrinsic asymmetry of the chiral carbohydrate-derived ligands leads to the non-
equivalency of the coordinated phosphorus atoms (see below). This effect extends to the
phosphine aromatic rings, leading to an interesting multiplicity of signs in the 13C-NMR
spectra. Compounds with 2PPh3 show the non-equivalency of the coordinated
phosphines, with two signs for each type of carbon (Cipso, Cortho, Cmeta, Cpara). In the case
of Dppe compounds, the non-equivalency is not only between the phenyl rings bonded
to different phosphorus atoms, but also between the ones bonded to the same one, this
being explained by the fact that the rotation around the Ru-P axles is not possible.
31P NMR spectra of the complexes showed two doublets, at ~40 ppm for compounds
with the PPh3 co-ligand and ~80 ppm for compounds with Dppe, attributed to the
phosphine co-ligands, revealing the non-equivalence of the coordinated phosphorus
atoms, as a result of the asymmetry induced by the chiral carbohydrate-derived ligands
2
on the metal centre. JPP coupling constants of compound with two PPh3 are ~36 Hz,
while for compounds with Dppe as co-ligand it’s within the range 22.5-25.5 Hz. This
difference may be explained by the different P-Ru-P angles: PPh3 has a larger cone
angle, thus leading to a larger P-Ru-P angle and subsequently to a larger 2JPP[43].
Table 4- Selected 31P NMR data, in CDCl3.
Compound
[1][PF6]
[2][PF6]
[3][PF6]
δP (ppm),2JPP (Hz)
78.4, 79.0 (2d, 2JPP = 25.5)
83.3, 84.9 (2d, 2JPP = 25.5)
84.3, 85.9 (2d, 2JPP = 22.7)
10

ACCEPTED MANUSCRIPT
[4][PF6]
77.3, 77.8 (2d, 2JPP = 25.4)
[5][PF6]*
[6][PF6]*
[7][PF6]
83.3, 84.1 (2d, 2JPP = 25.3)
82.1, 84.3 (2d, 2JPP = 22.5)
40.2, 40.9 (2d, 2JPP = 35.5)
40.2, 41.2 (2d, 2JPP = 36.3)
38.9, 40.6 (2d, 2JPP = 35.4)
39.4, 40.8 (2d, 2JPP = 36.3)
[8][PF6]
[9][PF6]
[10][PF6]
*In acetone D6
2
Furthermore, the JPP coupling constants for compounds [3][PF6] and [6][PF6] are
approximately 3 Hz lower than for other complexes with Dppe. This difference might
be explained by a larger stereochemical constraint of the coordinated 1,3,4-oxadiazole
ring, due to the methyl group in α position to the coordinated nitrogen atom, which
leads to lower P-Ru-P angle and 2JPPvalue. This further supports the hypothesis pointed
out to the unsuccessful synthesis of compounds [(η5-C5H5)Ru(PPh3)(L3)][PF6] and [(η5-
C5H5)Ru(PPh3)(L6)][PF6].
All the compounds spectra showed also the characteristic septuplet signal of the
hexafluorophosphate anion, at approximately -144.1 ppm.
2.3. Cytotoxic studies
The effect of the ruthenium complexes on human cervical carcinoma cells (HeLa) was
assayed within the concentration range 5 to 500 ꢁM, using the MTT assay, a
colorimetric determination of cell viability during in vitro treatment with a drug,
developed as an initial stage drug screening. The effects of compounds [1][PF6]–
[10][PF6] on the growth of these cell lines were evaluated after 48 h of continuous
exposure to the compounds. The IC50 values (final concentration ≤0.5% DMSO) were
11

ACCEPTED MANUSCRIPT
calculated from dose–response curves obtained by nonlinear regression analysis and are
shown in Table 5
Table 5- IC50 values (mean ± SD of three replicates each) for ruthenium compounds
against HeLa cells.
Compound
[1][PF6]
[2][PF6]
[3][PF6]
[4][PF6]
[5][PF6]
[6][PF6]
[7][PF6]
[8][PF6]
[9][PF6]
[10][PF6]
Cisplatina,b
IC50 (µM)
3.58 ± 0.39
3.92 ± 0.51
6.81 ± 0.11
6.07 ± 0.30
10.61 ± 0.06
4.64 ± 0.22
2.63 ± 0.14
6.39 ± 0.04
9.26 ± 0.05
4.49 ± 0.29
12.4±0.85
a Ref. [44]; b72 h incubation
All ruthenium complexes showed higher cytotoxic activity than cisplatin against in vitro
growth of HeLa cancer cells, after 48 h incubations, with IC50 values ranging from 2.63
for [7][PF6] to 10.61 ꢁM for [3][PF6]), lower than cisplatin (12.4ꢁM), even though this
value concerns data obtained from longer cell incubations with the compound (72 h vs
48 h) [44].
12

ACCEPTED MANUSCRIPT
The best IC50 results were obtained for compounds [1][PF6] (3.58ꢁM) and [7][PF6]
(2.63ꢁM), both with L1 coordinated, the best result being obtained for the PPh3
complex. Other than this, no structure–activity relationship can be disclosed.
Direct comparison of the IC50 values with other [(η5-C5H5)Ru(PP)(L)][X] is not possible
since there are no reports concerning HeLa cells. AnIC50 of 1.4 ±0.07 is reported for
[(η6-p-cymene)RuCl(κ2-dppp)]Cl, slightly better than our best result, but obtained from
72 h incubation data.
3. Conclusions
A new family of Ru(II) three-legged piano stool complexes with N-coordinated
galactose and fructose derivative ligands, was synthesized in good yields and
1
characterized by FTIR, H, 13C and 31P-NMR spectroscopies. Cytotoxic studies on
HeLa cancer cell lines revealed very good activities, with IC50 values in the low
micromolar range, better than cisplatin.
4. Experimental Section
4.1. General Procedures
All the experiments were carried out under inert atmosphere (N2) using standard
Schlenk techniques. Commercial reagents were used without further purification. All
solvents were dried using standard methods [45]. Starting materials were prepared
following the methods described in the literature: [(η5-C5H5)Ru(Dppe)Cl] and
[(η5-C5H5)Ru(PPh3)2Cl] [46], 2,3:4,5-Di-O-isopropylidene-β-D-arabino-hexos-2-ulo-
2,6-pyranose [47], 2,3:4,5-Di-O-isopropylidene-β-D-arabino-hex-2-ulosonitrile, 1,2:3,4-
Di-O-isopropylidene-1-(tetrazol-5′-yl)-β-D-arabinopyranose
and
1,2:3,4-Di-O-
isopropylidene-1-(2′-methyl-1′,3′,4′-oxadiazol)-5′-yl-β-D-arabinopyranose [40]. Solid
13

ACCEPTED MANUSCRIPT
state IR spectra were recorded on a Jasco FTIR-4100 spectrophotometer with KBr
1
pellets; only significant bands are cited in the text. H, 13C and 31P NMR spectra were
recorded on a Bruker Avance II 400 spectrometer operating at 400, 100, 162 MHz,
respectively; or on a Bruker Avance II 300 spectrometer operating at 300, 75, 121 MHz,
1
respectively, at probe temperature. The H and 13C chemical shifts are reported in parts
per million (ppm) downfield from the residual solvent peak; the 31P NMR spectra are
reported in ppm downfield from external standard H3PO4 85%. Coupling constants are
reported in Hz. Spectral assignments of the carbohydrate derivative ligands follow the
1
numbering scheme shown in Scheme 3.Assignments of the H and 13C NMR spectra
1
were confirmed with the aid of two dimensional techniques H, 13C (COSY, HSQC).
Microanalyses were performed using a Fisons Instruments EA1108 system; data
acquisition, integration and handling were performed using the software package Eager-
200 (Carlo Erba Instruments).
4.2. Synthesis of the carbohydrate derivatives
4.2.1. Precursor P2
A solution of 1,2:3,4-Di-O-isopropylidene-α-D-galactopyranose (1.30 g, 5.0mmol) in
CH2Cl2 (10 mL) was added to a suspension of PCC (2.50 g, 11.5 mmol) and powder
molecular sieves 4Å (5.00 g) in CH2Cl2 (20 mL). After 16 h stirring, AcOEt (40 mL)
was added. The mixture was filtered through celite and the solvent removed under
reduced pressure. The crude was purified by column chromatography (eluent: hexane to
AcOEt:hexane 1:4), affording pure product P2 (0.72 g, 56%) as a colourless oil. FTIR
1
(KBr, cm-1): 1742 (νC =O). H NMR (CDCl3, 400 MHz): 1.30, 1.33, 1.42, 1.49 (four s,
12H, -C(CH3)2), 4.17 (d, 1H, J =2.0, H5), 4.36 (dd, 1H, J =5.0, 2.6, H2), 4.58 (dd, 1H, J
=7.6, 2.0, H4), 4.63 (dd, 1H, J =7.6, 2.4, H3), 5.65 (d, 1H, J =5.2, H1), 9.62 (s, 1H,
14

ACCEPTED MANUSCRIPT
13
CHO). C NMR (CDCl3, 100 MHz): 24.3, 24.9, 25.9, 26.1 (-C(CH3)2), 70.5 (C3), 70.6
(C2), 71.8 (C4), 73.3 (C5), 96.4 (C1), 109.1, 110.1 (-C(CH3)2), 200.4 (CHO). Anal.
Calcd. for C12H18O6: C, 55.81; H, 7.02. Found: C, 54.25; H, 7.13.
4.2.2. Ligand L1
To a solution of P2 (517 mg, 2.00mmol) in pyridine (2 mL) was added a solution of
hydroxylamine hydrochloride (167 mg, 2.40mmol) in water (1 mL). After stirring for 1
h, copper sulphate pentahydrate (1.00 g, 4.00mmol), a solution of DCC (495 mg,
2.40mmol) and Et3N (0.56 mL, 4.0mmol) in CH2Cl2 (10 mL) were added to the
mixture. After stirring for 2 h more, formic acid (0.40 mL) was added, the mixture was
filtered, the phases separated and the aqueous phase further extracted with CH2Cl2 (3 x
20 mL). The organic phase was washed with HCl 10% (20 mL), dried with MgSO4,
filtered and pumped to dryness. The crude obtained was purified by column
chromatography (eluent: AcOEt:hexane 1:9), affording the pure ligand L1 (388 mg,
76%) as a white crystalline solid. FTIR (KBr, cm-1): 2262 (νC≡N). 1H NMR (CDCl3, 400
MHz): 1.33, 1.38, 1.53, 1.54 (four s, 12H, -C(CH3)2), 4.33 (dd, 1H, J =7.6, 2.4, H4),
4.36 (dd, 1H, J =4.8, 2.8, H2), 4.64 - 4.67 (m, 2H, H3+H5), 5.53 (d, 1H, J =5.4, H1).
13C NMR (CDCl3, 100 MHz): 24.7, 24.8, 26.0, 26.2 (-C(CH3)2), 60.3 (C5), 70.0 (C2),
70.5 (C3), 71.0 (C4), 96.3 (C1), 109.7, 111.2 (-C(CH3)2), 115.3 (C≡N). Anal. Calcd. for
C12H17O5N: C, 56.46; H, 6.71; N, 5.49. Found: C, 56.40; H, 6.68; N, 5.48.
4.2.3.Ligand L2
To a solution of ligand L1 (383 mg, 1.50mmol) in DMF (5 mL) were added NaN3 (117
mg, 1.8 mmol) and NH4Cl (120 mg, 2.20mmol), and the mixture was heated to 100 ºC.
After stirring for 3 h, the solvent was removed and the crude obtained was extracted
with AcOEt (3 x 20 mL), filtered and pumped to dryness. The crude was purified by
column chromatography (eluent: AcOEt:hexane 1:1), affording the pure ligand L2(425
15

ACCEPTED MANUSCRIPT
mg, 95%) as a white crystalline solid. FTIR (KBr, cm-1): 3434 (νN-H); 1559 (νN =N); 1388
(νN =C). 1H NMR ((CD3)2CO, 400 MHz): 1.31, 1.35, 1.38, 1.55 (four s, 12H, -C(CH3)2),
4.53 (dd, 1H, J =4.8, 2.4, H2), 4.63 (dd, 1H, J =8.0, 2.0, H4), 4.83 (dd, 1H, J =7.8, 2.6,
H3), 5.38 (d, 1H, J =2.0, H-5). 5.67 (d, 1H, J =4.8, H1), 15.11 (br, 1H, N-H). 13C NMR
((CD3)2CO, 100 MHz): 24.2, 25.0, 26.0, 26.3 (-C(CH3)2), 64.7 (C5), 71.3 (C3), 71.4
(C2), 73.0 (C4), 97.2 (C1), 109.8, 110.3 (-C(CH3)2), 154.4 (C6). Anal. Calcd. for
C12H18O5N4: C, 48.32; H, 6.08; N, 18.78. Found: C, 48.52; H, 6.22; N, 18.52.
4.2.4. Ligand L3
A solution of ligand L2 (298 mg, 1.00mmol) was dissolved in Ac2O and heated to
reflux. After 3 h, the reaction was stopped by addition of EtOH and the solvent removed
under reduced pressure. The remains of AcOH were removed by consecutive additions
of toluene and evaporation, affording the ligand L3 (300 mg, 96%) as a white crystalline
solid. FTIR (KBr, cm-1): 1386(νN=C);.1H NMR ((CD3)2CO, 400 MHz): 1.31, 1.37, 1.39,
1.53 (four s, 12H, -C(CH3)2), 2.50 (s, 3H, CH3) 4.52 (dd, 1H, J =5.0, 2.6, H2), 4.58 (dd,
1H, J =7.6, 2.0, H4), 4.80 (dd, 1H, J =7.0, 2.4, H3), 5.14 (d, 1H, J =2.4, H-5). 5.65 (d,
1H, J =5.2, H1). 13C NMR ((CD3)2CO, 100 MHz): 10.7 (CH3), 24.7, 25.0, 26.2, 26.3 (-
C(CH3)2), 65.1 (C5), 71.2 (C2), 71.6 (C3), 73.1 (C4), 97.4 (C1), 109.7, 110.5 (-
C(CH3)2), 163.8, 164.8 (C6, C8). Anal. Calcd. for C14H20O6N2: C, 53.84; H, 6.46; N,
8.97. Found: C, 53.52; H, 6.48; N, 8.67.
4.3. Synthesis of the complexes [(η5-C5H5)Ru(P–P)(L)][PF6]
Complexes of general formula [(η5-C5H5)Ru(P–P)(L)][PF6] were prepared by halide
abstraction from the parent neutral complexes [(η5-C5H5)Ru( P–P)Cl] (0.20 mmol) with
TlPF6 (0.20 mmol) in dichloromethane, in the presence of a slight excess of the ligands
L (0.22 mmol), at room temperature, under inert atmosphere for 48 h. The solutions
16

ACCEPTED MANUSCRIPT
were double filtered to remove the TlCl formed and pumped to dryness. The
compounds were washed with n-pentane and recrystallized by slow diffusion of n-
pentane or n-hexane in acetone or dichloromethane solutions, affording crystalline
products.
4.3.1.[1][PF6]
Light yellow; recrystallized from CH2Cl2/pentane; η = 81 %. FTIR (KBr, cm-1): 3056
-
1
(νC-H,η5-C5H5), 2264 (νC≡N), 840 (νP-F, PF6 ). H NMR (CDCl3, 300 MHz): 1.00, 1.04,
1.23, 1.48 (four s, 12H, -C(CH3)2), 2.54-2.71 (m, 4H, -CH2CH2-, Dppe), 3.27 (d, 1H, J=
7.5, H4), 4.11 (dd, 1H, J= 4.8, 2.7, H2), 4.32 (dd, 1H, J= 7.8, 2.7, H3), 4.37 (br, 1H,
H5), 4.72 (s, 5H, η5-C5H5), 5.16 (d, 1H, J= 4.8, H1), 7.13-7.84 (m, 20H, C6H5, Dppe).
13C NMR (CDCl3, 100 MHz): 24.1, 24.7, 25.5, 25.8 (-C(CH3)2), 27.8 (m, -CH2CH2-),
61.4 (C5), 69.5 (C2), 69.9 (C3), 70.5 (C4), 82.4 (η5-C5H5), 95.7 (C1), 109.8, 110.0 (-
C(CH3)2), 125.0 (C≡N) 128.9-129.1 (m, Cmeta), 129.3 (d, Cmeta, 3JCP = 9.8), 130.3, 130.5
2
(two s, Cpara), 130.6 (d, Cortho, JCP = 10.5), 130.8, 131.0 (two s, Cpara), 131.1 (d, Cortho
,
2
2
2JCP = 10.3), 133.1 (d, Cortho, JCP = 10.8), 133.7 (d, Cortho, JCP = 11.0), 137.7, 138.1
-
(two d, Cipso, 1JCP = 45.6). 31P NMR (CDCl3, 121 MHz): -144.2 (quint., JPF = 714, PF6 ),
2
2
78.4 (d, JPP = 25.4, Dppe), 79.0 (d, JPP = 25.6, Dppe). Anal. Calcd. for
C43H46O5NRuP3F6: C, 53.53; H, 4.81; N, 1.45. Found: C, 53.45; H, 4.41; N, 1.45.
4.3.2.[2][PF6]
Light yellow; recrystallized from CH2Cl2/hexane; η = 86 %. FTIR (KBr, cm-1): 3056
-
1
(νC-H,η5-C5H5), 839 (νP-F, PF6 ). H NMR (CDCl3, 300 MHz): 1.18, 1.30, 1.31, 1.46
(four s, 12H, -C(CH3)2), 2.51-3.21 (m, 4H, -CH2CH2-, Dppe), 3.60 (d, 1H, J= 7.5, H4),
4.24 (dd, 1H, J= 4.8, 2.7, H2), 4.34 (s, 1H, H5), 4.48 (dd, 1H, J= 7.8, 2.4, H3), 4.71 (s,
5H, η5-C5H5), 5.51 (d, 1H, J= 4.8, H1), 7.16-7.46 (m, 20H, C6H5, Dppe). 13C NMR
1
(CDCl3, 100 MHz): 24.5, 24.8, 25.6, 26.3 (-C(CH3)2), 28.5 (t, -CH2CH2-, JCP = 29.9),
17

ACCEPTED MANUSCRIPT
62.7 (C5), 70.3 (C2, C3), 71.5 (C4), 82.5 (η5-C5H5), 96.0 (C1), 108.9, 110.0 (-
3
3
C(CH3)2), 128.5, 128.8 (two d, Cmeta, JCP = 9.5), 128.9 (d, Cmeta, JCP = 9.9), 129.9,
2
130.0, 130.2, 130.3 (four s, Cpara), 130.5, 130.7 (two d, Cortho, JCP = 10.2), 132.2 (d,
2
2
1
Cortho, JCP = 10.7), 132.7 (d, Cortho, JCP = 10.9), 139.5 (d, Cipso, JCP = 45.1), 140.2 (d,
1
Cipso, JCP = 44.8), 154.0 (C6). 31P NMR (CDCl3, 121 MHz): -144.2 (quint., JPF = 712,
-
2
2
PF6 ), 83.3 (d, JPP = 25.4, Dppe), 84.9 (d, JPP = 25.6, Dppe). Anal. Calcd. for
C43H47O5N4RuP3F6·0.5C6H14: C, 52.57; H, 5.12; N, 5.33. Found: C, 52.54; H, 4.88; N,
5.66.
4.3.3.[3][PF6]
Yellow; recrystallized from CH2Cl2/hexane; η = 74 %. FTIR (KBr, cm-1): 3055 (νC-H,η5-
-
C5H5), 841 (νP-F, PF6 ). 1H NMR (CDCl3, 300 MHz): 1.28, 1.30, 1.38, 1.47 (four s, 12H,
-C(CH3)2), 2.12 (s, 3H, -CH3, OxD), 2.51-2.71 (m, 2H, -CH2CH2-, Dppe), 2.85-2.96 (m,
1H, -CH2CH2-, Dppe), 3.05-3.12 (m, 1H, -CH2CH2-, Dppe), 3.36 (d, 1H, J= 8.0, H4),
4.25 – 4.28(m, 2H, H2+H5), 4.46 (dd, 1H, J= 7.6, 2.0, H3), 4.66 (s, 5H, η5-C5H5), 5.44
(d, 1H, J= 4.8, H1), 7.11-7.71 (m, 20H, C6H5, Dppe). 13C NMR (CDCl3, 100 MHz):
12.5 (-CH3, OxD), 24.3, 24.8, 25.9, 26.2 (-C(CH3)2), 28.4-28.5 (m, -CH2CH2-), 29.6-
29.7 (m, -CH2CH2-), 62.5 (C5), 70.3, 70.5, 70.6 (C2, C3, C4), 80.6 (η5-C5H5), 96.2
(C1), 109.5, 109.9 (-C(CH3)2), 128.6-129.1 (m, Cmeta), 129.9, 130.3, 130.4 (three s,
Cpara), 130.7 (d, Cortho, 2JCP = 10.0), 130.4 (s, Cpara), 131.2 (d, Cortho, 2JCP = 10.0), 131.6
2
2
1
(d, Cortho, JCP = 10.7), 132.7 (d, Cortho, JCP = 10.5), 138.7 (d, Cipso, JCP = 45.4), 140.3
1
(d, Cipso, JCP = 45.9), 161.0, 169.5 (C6, C8). 31P NMR (CDCl3, 121 MHz): -144.3
-
2
2
(sept., JPF = 713, PF6 ), 84.3 (d, JPP= 22.2, Dppe), 85.9 (d, JPP= 23.2, Dppe). Anal.
Calcd. for C45H49O6N2RuP3F6: C, 52.89; H, 4.83; N, 2.74. Found: C, 52.54; H, 4.68; N,
2.59.
4.3.4. [4][PF6]
18

ACCEPTED MANUSCRIPT
Light yellow; recrystallized from CH2Cl2/hexane; η = 76 %. FTIR (KBr, cm-1): 3056
-
1
(νC-H,η5-C5H5), 2248 (νC≡N), 840 (νP-F, PF6 ). H NMR (CDCl3, 300 MHz): 0.72, 1.28,
1.30, 1.33 (four s, 12H, -C(CH3)2), 2.48-2.72 (m, 4H, -CH2CH2-, Dppe), 3.32 (d, 1H, J
= 2.1, H2), 3.43 (d, 1H, J = 12.9, H5b), 3.50 (d, 1H, J = 12.9, H5a), 4.05 (d, 1H, J = 7.8,
H4), 4.33 (dd, 1H, J = 8.1, 2.7, H3), 4.71 (s, 5H, η5-C5H5), 7.15-7.77 (m, 20H, C6H5,
Dppe). 13C NMR ((CD3)2CO, 100 MHz): 23.0, 23.7, 24.4, 25.7 (-C(CH3)2), 27.4 (t, -
CH2CH2-, 1JCP = 23.8), 61.6 (C5), 68.7 (C3), 69.2 (C4), 73.3 (C2), 82.5 (η5-C5H5), 94.4
(C1), 109.1, 111.5 (-C(CH3)2), 125.7 (C≡N), 128.9 (d, Cmeta, 3JCP = 9.8), 129.0 (d, Cmeta
3JCP = 10.1), 129.2 (d, Cmeta, 3JCP = 9.8), 129.5 (d, Cmeta, 3JCP = 9.9), 130.2, 130.5 (two d,
,
4
2
4
Cpara, JCP = 2.7), 130.7 (d, Cortho, JCP = 10.3), 130.8 (d, Cpara, JCP = 2.2), 130.2 (d,
4
4
2
Cpara, JCP = 2.2), 131.0 (d, Cpara, JCP = 2.3), 131.3 (d, Cortho, JCP = 10.9), 133.0 (d,
2
2
1
Cortho, JCP = 10.8), 133.5 (d, Cortho, JCP = 10.9), 136.8 (d, Cipso, JCP = 45.0), 137.9 (d,
1
-
Cipso, JCP = 45.5). 31P NMR (CDCl3, 121 MHz): -144.2 (quint., JPF = 708, PF6 ), 77.3
2
(d, JPP = 25.4, Dppe), 77.8 (d, 2JPP = 25.4, Dppe). Anal. Calcd. for C43H46O5NRuP3F6:
C, 53.53; H, 4.81; N, 1.45. Found: C, 53.35; H, 4.53; N, 1.46.
4.3.5. [5][PF6]
Yellow; recrystallized from CH2Cl2/hexane; η = 82 %. FTIR (KBr, cm-1): 3056 (νC-H,η5-
-
C5H5), 842 (νP-F, PF6 ). 1H NMR (CDCl3, 300 MHz): 1.16, 1.18, 1.26, 1.45 (four s, 12H,
-C(CH3)2), 2.54-2.73 (m, 2H, -CH2CH2-, Dppe), 2.94-3.05 (m, 2H, -CH2CH2-, Dppe),
3.66 (d, 1H, J = 2.4, H2), 3.72 (s, 2H, H5), 4.11 (d, 1H, J = 7.8, H4), 4.40 (dd, 1H, J =
7.8, 2.4, H3), 4.72 (s, 5H, η5-C5H5), 7.16-7.51 (m, 20H, C6H5, Dppe). 13C NMR
((CD3)2CO, 100 MHz): 23.1, 24.1, 25.0, 25.1 (-C(CH3)2), 27.6-28.0 (m, -CH2CH2-),
61.3 (C5), 69.0 (C3), 69.4 (C4), 74.2 (C2), 82.8 (η5-C5H5), 96.7 (C1), 108.4, 110.6 (-
C(CH3)2), 128.4 (d, Cmeta, 3JCP = 9.5), 128.6 (d, Cmeta, 3JCP = 9.4), 128.6, 128.7 (d, Cmeta
,
3JCP = 9.6), 129.6 (d, Cpara, 4JCP = 2.6), 129.7, 130.1 (two d, Cpara, 4JCP = 2.2), 130.2 (d,
19

ACCEPTED MANUSCRIPT
Cpara, 4JCP = 2.5), 130.4 (d, Cortho, 2JCP = 9.8), 130.6 (d, Cortho, 2JCP = 10.2), 132.4, 132.5
2
1
1
(two d, Cortho, JCP = 10.8), 139.3 (d, Cipso, JCP = 44.8), 139.7 (d, Cipso, JCP = 45.2),
157.5 (C6). 31P NMR ((CD3)2CO, 162 MHz): -144.2 (quint., JPF = 706, PF6 ), 83.3
-
2
(d,2JPP
=
25.1, Dppe), 84.1 (d, JPP = 25.4, Dppe). Anal. Calcd. for
C43H47O5N4RuP3F6·0.2CH2Cl2: C, 50.62; H, 4.66; N, 5.46. Found: C, 50.59; H, 4.68; N,
5.04.
4.3.6. [6][PF6]
Yellow; recrystallized from CH2Cl2/hexane; η = 80 %. FTIR (KBr, cm-1): 3057 (νC-H,η5-
-
1
C5H5), 842 (νP-F, PF6 ). H NMR ((CD3)2CO, 400 MHz): 0.94, 1.35, 1.38, 1.42 (four s,
12H, -C(CH3)2), 2.30 (s, 3H, CH3), 2.88-3.15 (m, 2H, -CH2CH2-, Dppe), 3.42 (d, 1H, J
= 2.4, H2), 3.67 (d, 1H, J = 13.2, H5b), 3.81 (d, 1H, J = 13.2, 1.6, H5a), 4.20 (dd, 1H, J
= 8.0, 1.6, H4), 4.48 (dd, 1H, J = 8.0, 2.4, H3), 4.89 (s, 5H, η5-C5H5), 7.25-7.60 (m,
13
20H, C6H5, Dppe). C NMR ((CD3)2CO, 100 MHz): 13.1 (CH3), 24.4, 25.5, 26.0, 26.4
(-C(CH3)2), 28.6 (t, -CH2CH2-, 1JCP = 23.8), 62.7 (C5), 70.2 (C3), 70.6 (C4), 74.0 (C2),
3
81.8 (η5-C5H5), 97.9 (C1), 109.7, 111.4 (-C(CH3)2), 129.6 (d, Cmeta, JCP = 9.5), 129.7
(d, Cmeta, 3JCP = 9.9), 129.8-129.9 (m, Cmeta), 130.9 (d, Cpara, 4JCP = 2.2), 131.0 (d, Cpara
,
2
4JCP = 2.1), 131.2, 131.5 (two s, Cpara), 132.2 (d, Cortho, JCP = 10.1), 132.4, 132.9 (two
2
2
1
d, Cortho, JCP = 10.2), 133.5 (d, Cortho, JCP = 10.7), 140.2 (d, Cipso, JCP = 47.0), 140.5
1
(d, Cipso, JCP = 46.0), 164.5, 170.9 (C6, C8). 31P NMR ((CD3)2CO, 162 MHz): -144.2
-
2
2
(sept., JPF = 708, PF6 ), 82.1 (d, JPP = 23.3, Dppe), 84.3 (d, JPP = 21.7, Dppe). Anal.
Calcd. for C45H49O6N2RuP3F6: C, 52.89; H, 4.83; N, 2.74. Found: C, 52.59; H, 4.89; N,
2.22.
4.3.7. [7][PF6]
Yellow; recrystallized from acetone/hexane; η = 77 %. FTIR (KBr, cm-1): 3058 (νC-H,η5-
-
C5H5), 2260 (νC≡N), 841 (νP-F, PF6 ). 1H NMR (CDCl3, 300 MHz): 0.99, 1.13, 1.31, 1.63
20

ACCEPTED MANUSCRIPT
(four s, 12H, -C(CH3)2), 4.28 (dd, 1H, J = 4.8, 2.7, H2), 4.46 (m, 1H, H4), 4.48 (s, 5H,
η5-C5H5), 4.62 (dd, 1H, J =7.8, 2.4, H3), 5.18 (br, 1H, H5), 5.44 (d, 1H, J = 4.8, H1),
7.04-7.30 (m, 30H, C6H5, PPh3). 13C NMR (CDCl3, 100 MHz): 22.8, 24.2, 24.9, 25.8 (-
C(CH3)2), 62.2 (C5), 70.0 (C3), 70.3 (C2), 70.9 (C4), 84.3 (η5-C5H5), 96.3 (C1), 109.9,
110.1 (-C(CH3)2), 128.3 (d, Cmeta, 3JCP = 9.5), 128.4 (d, Cmeta, 3JCP = 9.4), 128.9 (C≡N),
2
2
129.9 (m, Cpara), 133.2 (d, Cortho, JCP = 10.3), 133.5 (d, Cortho, JCP = 10.6), 135.9 (dd,
Cipso, 1JCP = 41.0, 3JCP = 2.8), 136.2 (dd, Cipso, 1JCP = 40.7, 3JCP = 2.4). 31P NMR (CDCl3,
-
2
2
121 MHz): -144.1 (quint., JPF = 711, PF6 ), 40.2 (d, JPP = 35.2, PPh3), 40.9 (d, JPP
=
35.7, PPh3). Anal. Calcd. for C53H52O5NRuP3F6: C, 58.35; H, 4.80; N, 1.28. Found: C,
58.24; H, 4.55; N, 1.31.
4.3.8. [8][PF6]
Yellow; recrystallized from CH2Cl2/hexane; η = 78 %. FTIR (KBr, cm-1): 3057 (νC-H,η5-
-
C5H5), 841 (νP-F, PF6 ). 1H NMR (CDCl3, 300 MHz): 1.25 (s, 3H, -C(CH3)2), 1.37(s, 6H,
-C(CH3)2), 1.50 (s, 3H, -C(CH3)2), 4.15 (dd, 1H, J = 7.8, 2.1, H4), 4.37 (dd, 1H, J = 5.1,
2.1, H2), 4.38 (s, 5H, η5-C5H5), 4.64 (dd, 1H, J = 7.8, 2.7, H3), 4.81 (d, 1H, J = 1.8,
H5), 5.65 (d, 1H, J = 4.8, H1), 6.96-7.33 (m, 30H, C6H5, PPh3). 13C NMR (CDCl3, 100
MHz): 24.5, 24.9, 25.8, 26.2 (-C(CH3)2), 63.3 (C5), 70.5 (C2, C3), 71.4 (C4), 83.2 (η5-
3
C5H5), 96.3 (C1), 109.3, 110.4 (-C(CH3)2), 128.0 (d, Cmeta, JCP = 9.4), 128.1 (d, Cmeta
,
2
3JCP = 9.3), 129.7, 129.8 (two s, Cpara), 133.5, 133.6 (two d, Cortho, JCP = 10.2), 136.3,
1
136.8 (two d, Cipso, JCP = 40.1), 155.2 (C6). 31P NMR (CDCl3, 162 MHz): -144.3
-
2
2
(quint., JPF = 712, PF6 ), 40.2 (d, JPP = 35.6, PPh3), 41.2 (d, JPP = 37.1, PPh3). Anal.
Calcd. for C53H53O5N4RuP3F6: C, 56.14; H, 4.71; N, 4.94. Found: C, 56.24; H, 5.06; N,
4.51.
4.3.9. [9][PF6]
21

ACCEPTED MANUSCRIPT
Yellow; recrystallized from CH2Cl2/hexane; η = 79 %. FTIR (KBr, cm-1): 3059 (νC-H,η5-
-
C5H5), 2241 (νC≡N), 840 (νP-F, PF6 ). 1H NMR (CDCl3, 300 MHz): 0.94, 1.29, 1.40, 1.50
(four s, 12H, -C(CH3)2), 3.76 (dd, 1H, J = 13.2, 1.5, H5b), 3.82 (d, 1H, J = 12.6, H5a),
4.25 (d, 1H, J = 7.8, H4), 4.36 (d, 1H, J = 1.8, H2), 4.44 (s, 5H, η5-C5H5), 4.66 (dd, 1H,
J = 8.1, 2.7, H3), 6.97-7.40 (m, 30H, C6H5, PPh3). 13C NMR ((CD3)2CO, 75 MHz):
23.2, 23.6, 24.6, 25.8 (-C(CH3)2), 62.1 (C5), 68.9 (C3), 69.3 (C4), 73.6 (C2), 84.5 (η5-
C5H5), 95.2 (C1), 109.3, 112.2 (-C(CH3)2), 128.5, 128.6 (two d, Cmeta, 3JCP = 9.7), 129.2
4
4
2
(C≡N), 130.2 (d, Cpara, JCP = 2.3), 130.3 (d, Cpara, JCP = 2.4), 133.0 (d, Cortho, JCP
=
2
3
10.4), 133.3 (d, Cortho, JCP = 10.6), 135.4 (dd, Cipso, 1JCP = 42.2, JCP = 1.7), 136.1 (dd,
1
3
Cipso, JCP = 42.2, JCP = 1.6). 31P NMR (CDCl3, 121 MHz): -144.3 (quint., JPF = 708,
-
2
2
PF6 ), 38.9 (d, JPP = 35.7, PPh3), 40.6 (d, JPP = 35.1, PPh3). Anal. Calcd. for
C53H52O5NRuP3F6: C, 58.35; H, 4.80; N, 1.28. Found: C, 58.35; H, 4.66; N, 1.34.
4.3.10. [10][PF6]
Yellow; recrystallized from CH2Cl2/hexane; η = 71 %. FTIR (KBr, cm-1): 3057 (νC-H,η5-
-
C5H5), 842 (νP-F, PF6 ). 1H NMR (CDCl3, 300 MHz): 1.01, 1.32, 1.35, 1.50 (four s, 12H,
-C(CH3)2), 3.92 (s, 2H, H5), 4.19 (d, 1H, J = 2.4, H2), 4.26 (d, 1H, J = 7.8, H4), 4.37 (s,
5H, η5-C5H5), 4.60 (dd, 1H, J = 7.8, 2.4, H3), 6.94-7.36 (m, 30H, C6H5, PPh3). 13C
NMR ((CD3)2CO, 75 MHz): 24.2, 25.3, 26.2 (-C(CH3)2), 62.5 (C5), 70.2 (C3), 70.6
(C2), 75.4 (C4), 84.3 (η5-C5H5), 97.8 (C1), 109.6, 111.8 (-C(CH3)2), 129.0 (d, Cmeta
,
4
2
3JCP = 9.4), 130.6, 130.7 (two d, Cpara, JCP = 2.3), 134.2 (d, Cortho, JCP = 10.3), 134.3
2
1
3
1
(d, Cortho, JCP = 10.6), 136.9 (dd, Cipso, JCP = 40.4, JCP = 2.1), 137.7 (dd, Cipso, JCP
=
=
40.0, JCP = 1.9), 159.5 (C6). 31P NMR (CDCl3, 162 MHz): -144.2 (quint., JPF
3
-
2
2
709,PF6 ), 39.4 (d, JPP = 35.6, PPh3), 40.8 (d, JPP = 36.9, PPh3). Anal. Calcd. for
C53H53O5N4RuP3F6: C, 56.14; H, 4.71; N, 4.94. Found: C, 56.54; H, 4.81; N, 4.43.
22

ACCEPTED MANUSCRIPT
4.4 .Cell viability assays in human HeLa tumor cells
HeLa cells were maintained in Roswell Park Memorial Institute medium (RPMI)
supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and
1% L-glutamine, at 37 °C in a humid incubator with 5% CO2. In order to detach cells
from the bottle, trypsin solution was used.
The MTT assay was used to determine the cell viability as an indicator for the
sensitivity of the cells to the complexes Ru (II). Exponentially growing cells were
seeded at a density of approximately 6×105 cells/mL, in a 96-well flat-bottomed
microplate, and 48 h later they were treated with the complexes. The complexes were
dissolved in DMSO and tested in concentrations ranging from 5 to 500 ꢁM.
Cytotoxicity of test compounds was evaluated by the MTT method [48]. The optical
density was measured at 570 nm using a 96-well multi-scanner auto-reader. The IC50
were calculated by nonlinear regression analysis using Origin.
Acknowledgements
This research was supported by FCT through projects PTDC/QUI-QUI/102114/2008
and PTDC/QUI-QUI/110532/2009 and pluriannual funds PEst-OE/QUI/UI0100/2013
and PEst-OE/QUI/UI0612/2013. PRF thanks FCT for grant. Authors thank the
Portuguese NMR Network (IST-UTL Center) for providing access to the NMR
facilities.
Bibliography
[1] H.-U. Blaser, Chem. Rev. 92 (1992) 935-952.
[2] P. Anastas, T. Williamson, Green Chemistry: Frontiers in Benign Chemical
Syntheses and Processes; Oxford University Press, New York, 1998.
23

ACCEPTED MANUSCRIPT
[3] Y. Pérez, S. Morante-Zarcero, I. delHierro, I. Sierra, I. López-Solera, M. Monari, M.
Fajardo, A. Otero, J. Organomet. Chem. 689 (2004) 3492-3500.
[4] S. Tschersich, M. Böge, D. Schwidom, J. Heck, Rev. Inorg. Chem. 31 (2011) 27–55.
[5] B. Rosenberg, L. Van Camp, T. Krigas, Nature 205 (1965) 698-699.
[6] R. Gagliardi, G. Sava, S. Pacor, G. Mestroni, E. Alessio, Clin. Exp. Metastasis 12
(1994) 93-100.
[7] C.S. Allardyce, P.J. Dyson, D.J. Ellis, S.L. Heath, Chem. Commun. (2001) 1936-
1937.
[8] Y.K. Yan, M. Melchart, A. Habtermariam, P.J. Sadler, Chem. Commun. 38 (2005)
4764–4776.
[9] S.J. Dougan, P.J. Sadler, Chimia 61 (2007) 704–715.
[10] C.S. Allardyce, P.J. Dyson, D.J. Ellis, S.L. Heath, Chem. Commun. (2001) 1396–
1397.
[11] C.S. Allardyce, P.J. Dyson, D.J. Ellis, P.A. Salter, R.J. Scopelliti, J. Organomet.
Chem. 668 (2003) 35–42.
[12] Y.N.V. Gopal, D. Jayaraju, A.K. Kondapi, Biochemistry 38 (1999) 4382–4388.
[13] Y.N.V. Gopal, N. Konuru, A.K. Kondapi, Arch. Biochem. Biophys. 401 (2002) 53-
62.
[14] R.E. Morris, R.E. Aird, P.S. Murdoch, H. Chen, J. Cummings, N.D. Hughes, J.
Med. Chem. 44 (2001) 3616–3621.
[15] C. Scolaro, A. Bergamo, L. Brescacin, R. Delfino, M. Cocchieto, G. Laurenczy,
T.J. Geldbach, G. Sava, P.J. Dyson, J. Med. Chem. 48 (2005) 4161–4171.
[16] G. Süss-Fink, Dalton Trans. 39 (2010) 1673–1688.
[17] M. Gras, B. Therrien, G. Süss-Fink, O. Zava, P.J. Dyson, Dalton Trans. 39 (2010)
10305–10313.
24

ACCEPTED MANUSCRIPT
[18] M.H. Garcia,; T.S. Morais, P. Florindo, M.F.M. Piedade, V. Moreno, C. Ciudad, V.
Noe, J. Inorg. Biochem. 103 (2009) 354–361.
[19] V. Moreno, J. Lorenzo, F.X. Aviles, M.H. Garcia, J.P. Ribeiro, T.S. Morais, P.
Florindo, M.P. Robalo, Bioinorg. Chem. Appl. (2010) 1–11.
[20] V. Moreno, M. Font-Bardia, T. Calvet, J. Lorenzo, F.X. Avilés, M.H. Garcia, T.S.
Morais, A. Valente, M.P. Robalo, J. Inorg. Biochem. 105 (2011) 241–249.
[21] T.S. Morais, T.J.L. Silva, F. Marques, M.P. Robalo, F. Avecilla, P.J.A. Madeira,
P.J.G. Mendes, I. Santos, M.H. Garcia, J. Inorg. Biochem. 114 (2012) 65–74.
[22] H. Bregman, E. Meggers, Org. Lett. 8 (2006) 5465–5468.
[23] R. Anand, J. Maksimoska, N. Pagano, E.Y. Wong, P.A. Gimotty, S.L. Diamond, E.
Meggers, R. Marmorstein, J. Med. Chem. 52 (2009) 1602–1611.
[24] D.S. Dwyer, K. Gordon, B. Jones, Int. J. Immunopharm. 17 (1995) 931-940.
[25] T.W. Hayton, P. Legzdins, W.B. Sharp, Chem. Rev. 102 (2002) 935-991.
[26] B. Cetinkaya, E. Cetinkaya, H. Kucukbay, R. Durmaz, Arzneimittel-
Forschung/Drug Res. 46 (1996) 821-823.
[27] M. Sulu, H. Kucukbay, R. Durmaz, S. Gunal, Arzneimittel-Forschung/Drug Res.
48 (1998) 291-293.
[28] R.A. Sanchez-Delgado, M. Navarro, H. Perez, J.A. Urbina, J. Med. Chem. 39
(1996) 1095-1099.
[29] N.S. Shailendra, N. Bharti, M.T.G. Garza, D.E. Cruz-Vega, J.C. Garza, K. Saleem,
F. Naqvi,; A. Azam, Bioorg. Med. Chem. Lett. 11 (2001) 2675-2678.
[30] T. Pill, W. Breu, H. Wagner, W. Beck, Chem. Ber. 124 (1991) 713-717.
[31] V.D. Reddy, D. Dayal, S.C. Cosenza, M.V.R. Reddy, W.C. Pearl Jr., R.D. Adams,
J. Organomet. Chem. 694 (2009) 959-967.
[32] P. DeShong, G. A. Slough, V. Elango, J. Am. Chem. Soc. 107 (1985) 7788-7790.
25

ACCEPTED MANUSCRIPT
[33] S. Bhaduri, N. Sapre, H. Khwaja, R. G. Jones, J. Organomet. Chem. 426 (1992)
C12-C15.
[34] V.D. Reddy, J. Organomet. Chem. 691 (2006) 27–34.
[35] I. Berger, M. Hanif, A.A. Nazarov, C.G. Hartinger, R.O. John, M.L. Kuznetsov, M.
Groessl, F. Schmitt, O. Zava, F. Biba, V.B. Arion, M. Galanski, M.A. Jakupec, L.
Juillerat-Jeanneret, P.J. Dyson, B.K. Keppler, Chem. Eur. J. 14 (2008) 9046-9057.
[36] M. Hanif, S.M. Meier, W. Kandioller, A. Bytzek, M. Hejl, C.G. Hartinger, A.A.
Nazarov, V.B. Arion, M.A. Jakupec, P.J. Dyson, B.K. Keppler, J. Inorg. Biochem. 105,
(2011) 224–231.
37] M. Hanif, A.A. Nazarov, C.G. Hartinger, Inorg. Chim. Acta 380 (2012) 211–215.
[38] Y. Zhou, W. Beck, J. Organomet. Chem. 479 (1994) 217-220.
[39] E. Beksan, P. Schieberle, F. Robert, I. Blank, L.B. Fay, H. Schlichtherle-Cerny, T.
Hofmann, J. Agric. Food Chem. 51 (2003) 5428
[40] M.A.M. Alho, N. D’Accorso, ARKIVOC vii (2011) 136-148.
[41] M.H. Garcia, P. Florindo, M.F.M. Piedade, S. Maiorana, E. Licandro, Polyhedron
28 (2009) 621.
[42] M.H. Garcia, P. Florindo, M.F.M. Piedade, M.T. Duarte, M.P. Robalo, E.
Goovaerts, W. Wenseleers, J. Organomet. Chem. 694 (2009) 433.
[43] O. Kühl, Phosphorous-31 NMR Spectroscopy: A Concise Introduction for the
Synthetic Organic and Organometallic Chemist, Springer-Verlag, Berlin, Germany,
2008.
[44] J.M. Walker, A. McEwan, R. Pycko, M.L. Tassotto, C. Gottardo, J.Th’ng, R.
Wang, G.J. Spivak, Eur. J. Inorg. Chem. 31 (2009) 4629-4633.
[45]D.D. Perrin, W.L.F. Amarego, D.R. Perrin, Purification of Laboratory Chemicals,
2nd ed., Pergamon, New York, 1980.
26

ACCEPTED MANUSCRIPT
[46] G.S. Ashby, M.I. Bruce, I.B. Tomkins, R.C. Walli, Aust. J. Chem. 32 (1979) 1003-
1016.
[47] I. Izquierdo Cubero, M. T. Plaza Lopez-Espinosa, Carbohydr. Res. 205 (1990) 293-
304.
[48] T. Mossman, J. Immunol. Meth. 65 (1983) 55-63.
27