Synthesis, characterization and in vitro biological evaluation of [Ru(η6-arene)(N,N)Cl]PF6 compounds using the natural products arenes methylisoeugenol and anethole

Article abstract of DOI:10.1016/j.jorganchem.2014.09.005

Abstract Five new organometallic Ru(II) compounds (VI-X) with the general formula [Ru(η⁶-arene)(N,N)Cl]PF₆, where arene-N,N correspond to methylisoeugenol-bipyridine (VI); anethole-bipyridine (VII); methylisoeugenol-ethylenediamine (VIII); anethole-ethylenediamine (IX) and methylisoeugenol-1,2-diaminobenzene (X), have been synthesized, fully characterized and biologically evaluated in vitro. The reaction conditions based on the reduction of [Ru(1,5-COD)Cl₂]_n in situ with methyleugenol and estragole, which are natural ligands, induced an alkene isomerization on the allylic substituent of coordinated arenes. The Ru(II)-arene bond formation and isomerization of the CC bond on the allyl substituent was confirmed using ¹H NMR spectroscopy; this result was validated for compound VIII by X-ray diffraction. An XRD analysis revealed the presence of both enantiomers of the complex in the single-crystal. Compounds IX and X exhibited a better cytotoxic activity in vitro than carboplatin, which is a commercial drug, against three human tumor cell lines (MCF-7, PC-3 and HT-29).

Full text of DOI:10.1016/j.jorganchem.2014.09.005

Journal of Organometallic Chemistry 782 (2015) 131e137
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis, characterization and in vitro biological evaluation of
6
[Ru(h
-arene)(N,N)Cl]PF compounds using the natural products
6
arenes methylisoeugenol and anethole
a
b
c
a
Ricardo A. Delgado , Antonio Gald aꢀ mez , Joan Villena , Patricio G. Reveco ,
a, *
Franz A. Thomet
a
ꢀ
Laboratory of Organometallics, Department of Chemistry, Universidad T ꢀe cnica Federico Santa María, Avenida Espa n~ a N 1680, Valparaíso, Chile
Department of Chemistry, Faculty of Science, Universidad de Chile, Las Palmeras N 3425, Nu n~ oa, Santiago, Chile
School of Medicine, Centro de Investigaciones Biom ꢀe dicas (CIB), Universidad de Valparaíso, Avenida Hontaneda N 2664, Valparaíso, Chile
b
c
ꢀ
~
ꢀ
a r t i c l e i n f o
a b s t r a c t
Five new organometallic Ru(II) compounds (VIꢁX) with the general formula [Ru(h6-arene)(N,N)Cl]PF6,
where arene-N,N correspond to methylisoeugenol-bipyridine (VI); anethole-bipyridine (VII);
methylisoeugenol-ethylenediamine (VIII); anethole-ethylenediamine (IX) and methylisoeugenol-1,2-
diaminobenzene (X), have been synthesized, fully characterized and biologically evaluated in vitro. The
Article history:
Received 6 February 2014
Received in revised form
3
September 2014
Accepted 4 September 2014
Available online 16 September 2014
2 n
reaction conditions based on the reduction of [Ru(1,5-COD)Cl ] in situ with methyleugenol and estra-
gole, which are natural ligands, induced an alkene isomerization on the allylic substituent of coordinated
arenes. The Ru(II)-arene bond formation and isomerization of the C]C bond on the allyl substituent was
confirmed using 1H NMR spectroscopy; this result was validated for compound VIII by X-ray diffraction.
An XRD analysis revealed the presence of both enantiomers of the complex in the single-crystal. Com-
pounds IX and X exhibited a better cytotoxic activity in vitro than carboplatin, which is a commercial
drug, against three human tumor cell lines (MCF-7, PC-3 and HT-29).
Keywords:
Bioorganometallic
Ru(II) complexes
Natural products
Cytotoxicity
©
2014 Elsevier B.V. All rights reserved.
Introduction
is related to the reductive capabilities of the Ru(III) to Ru(II) in the
complex [4]. After a chemical reduction, NAMI-A is more active
against metastatic growth than the non-reduced complex [5]. For
some KP1019 derivatives and a structurally related series of
indazole-based Ru(III) compounds, the antiproliferative activity
against colon carcinoma (SW-480) was correlated to the increased
reductive potential of the corresponding complexes [6]. In addition,
the low oxygen concentration levels (hypoxia) of tumor cells pro-
mote a more reductive environment than in normal tissue, favoring
the active reduced form [7].
Since 1978, cisplatin has been broadly employed to treat
different types of cancer. The use of cisplatin, however, has become
limited due to its deleterious secondary effects (nephro-, neuro-
and ototoxicity) [1]. New compounds have been developed using
different design strategies that aimed either to diminish the sec-
ondary toxicity and/or to have activity against cell lines resistant to
cisplatin. Ruthenium-based compounds have been studied for this
purpose in recent years [2]. Ruthenium can mimic iron when
binding certain biological molecules, particularly transferrin, and
Ru(II) and Ru(III) complexes can display ligand exchange kinetics
similar to Pt(II). Because these attributes are combined with a lower
toxicity, ruthenium compounds are suitable for medical applica-
tions [3]. Currently, NAMI-A and KP1019, which are ruthenium
compounds, are in phase II clinical trials. The mechanism of action
for these Ru(III) compounds remains unknown; however, new ev-
idence has indicated that the cytotoxic activity of these complexes
“Piano-stool” organometallic Ru(II) type compounds, such as
6
[Ru(
h
-arene)(PTA)Cl
2
]
(PTA:
1,3,5-triaza-7-phosphatricyclo-
6
[3.3.1.1]decane) and [Ru(
h
-arene)(en)Cl]PF (en: ethylenedi-
6
amine), have also been evaluated [8,9]. These complexes undergo
an intracellular hydrolysis of the metal-chloride bond(s), similar to
cisplatin, to generate the aquo-metabolite that binds to DNA or
proteins, forming mono- or bifunctional adducts [10]. However, the
hydrolysis of these metal-based complexes is not essential during
6
reactions with biomolecules. In [Ru(
h
-p-cymene)(PTA)(oxalate)]
6
and [Ru(
h
-p-cymene)(PTA)(1,1-cyclobutanedicarboxylate)],
a
considerable decrease in the rate of hydrolysis was detected, and
the cytotoxicity of these compounds against several tumor cells
*
Corresponding author. Tel.: þ56 322654236; fax: þ56 322654782.
E-mail addresses: fthometi@yahoo.com, franz.thomet@usm.cl (F.A. Thomet).
http://dx.doi.org/10.1016/j.jorganchem.2014.09.005
022-328X/© 2014 Elsevier B.V. All rights reserved.
0

132
R.A. Delgado et al. / Journal of Organometallic Chemistry 782 (2015) 131e137
remained unchanged [8]. The structureeactivity relationships of
calculated as follows: Uiso(H) ¼ 1.2Ueq(N) and Uiso(H) ¼ k Ueq(C),
where k ¼ 1.5 and 1.2. The crystallographic data and refinement
parameters for VIII are summarized in Table 1. The data were
collected with a Bruker SMART (BRUKER 1996); the cell refinement
was performed with Bruker SAINTPLUS V6.02 (BRUKER 1997); the
data were reduced with Bruker SHELXTL V6.10 (BRUKER 2000); the
SHELXS97 program (Sheldrick, 1990) was used to solve and refine
the structure (Sheldrick, 1997) [23,24]. The molecular graphics
were produced using DIAMOND (Brandenburg, 1999); the software
used to prepare material for publication was PLATON (Spek, 2003)
[25,26].
6
[
Ru(
h
-arene)(XY)Cl]Z complexes where XY is an N,N-, N,O- or O,O-
revealed that polar sub-
chelating ligand and Z is usually PF
6
stituents in the coordinated aromatic ring tended to decrease the
cytotoxic activity of the complex. Incorporating N,N-chelating li-
gands, such as ethylenediamine and 1,2-diaminobenzene, increase
considerably the cytotoxicity of the complexes [11].
Increasing evidence suggests that combination therapy
involving natural products and synthetic drugs can enhance the
effectiveness of the treatment through a synergic process [12e15].
This work aimed to synthesize new drugs with ruthenium-bound
natural products, improving their pharmacological effects. To-
ward this purpose, phenylpropanoids, such as methyleugenol (I),
Synthesis
estragole (II) and saphrole (III) were used to obtain compounds
6
with the general formula of [Ru(
h
-arene)(N,N)Cl]PF
6
(N,N: ethyl-
6-arene)
2 2 2
Cl ]
General procedure for synthesizing the [Ru(
h
enediamine, bipyridine and 1,2-diaminobenzene). These types of
natural products possess interesting antioxidant, anti-
compounds (IV and V) [11]
To a dry, 100 ml round-bottom flask connected to a dry N
was added 1.04 g of [Ru(1,5-COD)Cl (3.69 mmol of Ru),
4.4 mmol of methyleugenol (I) or estragole (II) and 9.43 g of zinc
144 mmol). Freshly distilled anhydrous THF (25 ml) was added and
2
inlet
inflammatory and antimicrobial activities [16,17].
A series
2 n
]
composed of five organometallic compounds (VIeX) was synthe-
sized, fully characterized and evaluated for cytotoxicity in vitro.
Moreover, the single-crystal X-ray diffraction data for compound
VIII is reported. Previously, an in vitro cell viability assay involving
boldiplatin, which is a platinum(II) compound synthesized by our
research group with a naturally occurred ligand and recognized
antioxidant activity, showed that this compound was more active
against tumor cell lines than non-tumor cell lines [18]. This dif-
ference in activity, which is not observed for the commercial drug
oxaliplatin, motivated the synthesis of the above mentioned
complexes.
1
(
the reaction mixture was refluxed for 24 h. The solvent was
removed under vacuum, and the oily residue was washed with dry
hexane (3 ꢂ 30 ml). The organic extracts were passed through a
glass filter and combined. The organic solvent was removed under
vacuum, and the oily product was redissolved in 5 ml of dry
acetonitrile. Fifteen ml of a 1 M solution of HCl in ether was added,
and the mixture was stirred at room temperature overnight. The
solvents were removed under vacuum and the deep red solid was
washed with 30 ml of diethyl ether for 1 h. The solid was filtrated
6
and dried under vacuum. Finally, 229 mg of [Ru(
isoeugenol) Cl
le) Cl (V, yield: 22%) were obtained.
h
-methyl-
Experimental section
(IV, yield: 18%) and 263 mg of [Ru(h6-anetho-
2 2
]
2
2
2 2
]
General considerations
1
6
h (VI)
6-methylisoeugenol)(bipy)Cl]PF
The H NMR experiments were performed using an Avance 400
Synthesis of [Ru(
In a 100 ml round-bottom flask, 100 mg of [Ru(h6-methyl-
isoeugenol) Cl (0.14 mmol) was dissolved in 15 ml of MeOH. A
solution of 50 mg of bipyridine (0.32 mmol) in 5 ml of MeOH was
added dropwise, and the mixture was stirred at room temperature
1
Digital Bruker NMR spectrometer operating at 400.13 MHz for H.
The chemical shifts (
J) in Hz. The chemical shifts are reported relative to the proton
signal of incompletely deuterated DMSO-d 2.49). The mass
spectra were recorded on a Bruker MALDI-TOF Microflex spec-
trometer using the flexControl 3.0 software. -Cyano-4-
hydroxycinnamic acid was employed as a matrix and the spectra
were obtained in positive mode. The elemental analyses were
performed on
cyclooctadiene (1,5-COD), bipyridine (bipy), ethylenediamine (en),
,2-diaminobenzene (dab) and 4-allylanisole (estragole) were
purchased from Aldrich. The eugenol was extracted through
hydrodistillation from cloves (Eugenia caryophyllata) and methyl-
ated according to an established method [19]. The [Ru(1,5-COD)
2 n
Cl ] was prepared according to a published procedure [20]. The
methylisoeugenol and anethole were obtained after isomerizing
methyleugenol and estragole, respectively [21], with a catalyst
d) are given in ppm, and the coupling constant
2
2 2
]
(
6
(d
a
Table 1
Crystallographic data and structural refinement parameters for compound VIII.
Empirical formula
13 22 2 2 6
C H N O RuCl,PF
a
3 2
Flash EA™ 1112. The RuCl $xH O, 1,5-
Crystal system
Space group
Unit cell dimensions
a (Å)
Triclinic
P-1 (N 2)
ꢀ
1
9.2862(7)
9.5321(7)
b (Å)
c (Å)
11.6940(9)
110.247(1)
98.322(1)
91.148(1)
958.19(13)
2
ꢀ
a
b
g
( )
ꢀ
( )
ꢀ
( )
3
Volume (Å )
3
3
[
1
{RuCl(
m-Cl)(
h
:h
-C10
H16)}
2
] (C10
H16: 2,7-dimethylocta-2,6-diene-
Z
m
(mm 1
ꢁ
)
1.107
,8-diyl) prepared according to an established procedure [22]. The
anhydrous solvents were dried and freshly distilled as follows:
ethanol (Mg/I
nitrile (CaH ) and hexane (Na). All of the other reagents were ob-
tained from commercial suppliers and were used without further
purification.
Temperature
Wavelength ()
Crystal size (mm)
F(000)
Theta minemax range collected ( )
Measured reflections
293 K
0.71073
2
), THF and diethyl ether (Na/benzophenone), aceto-
0.10 ꢂ 0.14 ꢂ 0.20
2
520
ꢀ
1.9e29.0
9095 (Rint ¼ 0.0252)
Independent reflections
4387
Reflections with I > 2
s
(I)
(F )]
3776
X-ray crystallography
Final R indices [(F2 > 2
2
s
R1 ¼ 0.0321, wR2 ¼ 0.0772
R indices (all data)
Goodness-of-fit on F2
Parameters
R1 ¼ 0.0398, wR2 ¼ 0.0817
All of the H-atoms were positioned geometrically and treated as
riding atoms with NeH distances of 0.90 Å and CeH distances from
1.045
475
Drmax
,
Drmim (eꢁ3)
0.68/ꢁ0.40
0
.98 to 0.93 Å. The isotopic displacement parameters were

R.A. Delgado et al. / Journal of Organometallic Chemistry 782 (2015) 131e137
133
for 2 h. To the mixture was added 79 mg of NH
4
PF
6
(0.45 mmol),
(m, 2H, CH
NH
2
), 2.30 (m, 2H, CH
), 5.36 (d, J ¼ 6 Hz, 2H, AreH), 6.04 (d, J ¼ 6 Hz, 2H, AreH), 6.08
2
), 3.82 (s, 3H, OCH
3
), 4.20 (m, 2H,
and the mixture was stirred for 15 min. A yellow solid appeared,
2
ꢀ
and the reaction mixture was left at ꢁ18 C overnight. The pre-
(d, J ¼ 16 Hz, 1H, trans HC]C), 6.30 (dd, J ¼ 16, 7 Hz, 1H, C]
þ
cipitate was filtrated, washed with 2 ml of cold MeOH and small
CHeCH
3
); EM m/z 344.72 (C12
20
H N
2
ORuCl ); Anal. Calc. for
subsequently portions of diethyl ether and dried under vacuum.
Finally,119 mg of yellow product was obtained (yield: 69%). H NMR
C
12
H
20
N
2
ORuClPF
6
: C, 29.41%; H, 4.11%; N, 5.72%. Found: C, 29.37%;
1
H, 4.02%; N, 5.96%.
(
3
DMSO-d
H, OCH
HC]C), 6.16 (s, 1H, AreH), 6.19 (d, J ¼ 6 Hz, 1H, AreH), 6.69 (dd,
J ¼ 16, 7 Hz,1H, C]CHeCH ), 7.80 (m, 2H, H-5), 8.25 (dd, J ¼ 8, 8 Hz,
H, H-4), 8.61 (d, J ¼ 7 Hz, 2H, H-3), 9.20 (d, J ¼ 5 Hz, 1H, H-6), 9.26
6
)
d
1.77 (d, J ¼ 7 Hz, 3H, CH
3 3
), 3.74 (s, 3H, OCH ), 3.83 (s,
), 5.91 (d, J ¼ 6 Hz, 1H, AreH), 6.02 (d, J ¼ 16 Hz, 1H, trans
Synthesis of [Ru(
h
6-methylisoeugenol)(dab)Cl]PF
In a 100 ml round-bottom flask, 99 mg of [Ru(h6-methyl-
isoeugenol) Cl (0.14 mmol) was dissolved in 20 ml of MeOH and
6
(X)
3
3
2
2 2
]
2
4 ml of water. The mixture was refluxed for 1 h and cooled to room
temperature before 32 mg of 1,2-diaminobenzene (0.29 mmol) in
4 ml MeOH was added dropwise. The reaction mixture was refluxed
þ
22 2 2
H N O RuCl ); Anal. Calc.
(
d, J ¼ 5 Hz,1H, H-6); EM m/z 470.82 (C21
for C21 RuClPF : C, 40.95%; H, 3.60%; N, 4.55%. Found: C,
1.19%; H, 3.42%; N, 4.54%.
H
22
N
2
O
2
6
4
for 15 min and cooled to room temperature before 74 mg of NH
4 6
PF
(
0.44 mmol) was added. The mixture was stirred for 20 min, the
Synthesis of [Ru( 6
In a 100 ml round-bottom flask, 93 mg of [Ru(h6-anethole)
0.14 mmol) was dissolved in 20 ml of MeOH. A solution of 50 mg of
h
-anethole)(bipy)Cl]PF
(VII)
solution was concentrated under reduced pressure until approxi-
6
ꢀ
2
Cl
2
]
2
mately 7 ml remained, and the mixture was left al ꢁ18 C overnight.
(
The solid was collected by filtration, washed with small portions of
bipyridine (0.32 mmol) in 5 ml of MeOH was added dropwise, and
the mixture was stirred at room temperature for 3 h. The solvent
was removed under reduced pressure until approximately 10 ml of
diethyl ether and dried under vacuum. Finally, 82 mg of product
1
was obtained (yield: 52%). H NMR (DMSO-d
3H, CH ), 3.78 (s, 3H, OCH ), 3.85 (s, 3H, OCH
3 3 3
6
)
d
1.80 (d, J ¼ 6 Hz,
), 5.50 (d, J ¼ 6 Hz, 1H,
AreH), 5.73 (d, J ¼ 6 Hz, 1H, AreH), 5.87 (s, 1H, AreH), 6.11 (d,
J ¼ 16 Hz, 1H, trans HC]C), 6.44 (m, 1H, C]CHeCH ), 6.51 (d,
J ¼ 13 Hz, 2H, NH ), 7.14 (m, 2H, AreH), 7.23 (m, 2H, AreH), 7.65 (d,
J ¼ 13 Hz, 1H, NH); EM m/z 422.75 (C17
for C17 RuClPF : C, 35.95%; H, 3.90%; N, 4.93%. Found: C,
4 6
solution remained before 79 mg of NH PF (0.45 mmol) was added.
The mixture was stirred for 1 h. A yellow solid appeared, and the
3
ꢀ
reaction mixture was left at ꢁ18 C for 2 days. The precipitate was
2
þ
22 2 2
H N O RuCl ); Anal. Calc.
filtrated, washed with small portions of diethyl ether and dried
under vacuum. Finally, 99 mg of yellow product was obtained
22
H N
2
O
2
6
1
(
3
yield: 60%). H NMR (DMSO-d
6
)
d
1.65 (d, J ¼ 6 Hz, 3H, CH
3
), 3.77 (s,
36.15%; H, 4.10%; N, 4.84%.
H, OCH
3
), 5.88 (d, J ¼ 6 Hz, 2H, AreH), 5.97 (d, J ¼ 16 Hz, 1H, trans
HC]C), 6.36 (dd, J ¼ 16, 7 Hz, 1H, C]CHeCH
3
), 6.51 (d, J ¼ 6 Hz, 2H,
Determination of the octanol/water partition coefficient
AreH), 7.80 (m, 2H, H-5), 8.27 (dd, J ¼ 8, 8 Hz, 2H, H-4), 8.62 (d,
J ¼ 8 Hz, 2H, H-3), 9.43 (d, J ¼ 5 Hz, 2H, H-6); EM m/z 440.79
The log P values were determined using the shaken flask
method [27] with previously described modifications [28]. Octanol
was presaturated with water (containing 0.2 M HCl), and the
aqueous phase was saturated with octanol. The aqueous samples
(5 ml) were vortexed with octanol (5 ml) for 2 h. The mixtures were
centrifuged for 10 min at 3000 rpm to separate both phases. The
ruthenium compounds were quantified in both phases with a
Unicam UV/Vis Spectrometer (UV4).
þ
(
C
20
20
H N
2
ORuCl ); Anal. Calc. for C20
H
20
N
2
ORuClPF
6
: C, 40.99%; H,
3
.44%; N, 4.78%. Found: C, 41.33%; H, 3.28%; N, 4.62%.
Synthesis of [Ru( 6
h -methylisoeugenol)(en)Cl]PF (VIII)
6
In a 100 ml round-bottom flask, 100 mg of [Ru(h6-methyl-
isoeugenol) Cl (0.14 mmol) was dissolved in 10 ml of MeOH. To
the solution was added 30 l of ethylenediamine (0.45 mmol), and
2
2 2
]
m
the mixture was stirred at room temperature for 3 h. The reaction
mixture was filtrated and the filtrate was concentrated under
vacuum until 5 ml of solution remained. Afterward, 120 mg of
Cell lines
NH
4
PF
6
(0.74 mmol) was added, and the mixture was shaken for
The experimental cell cultures were obtained from the American
Type Culture Collection (Rockville, MD, USA). The HT-29 colon
cancer cell line, PC-3 prostate cancer cell line, MCF-7 breast
adenocarcinoma cell line and CCD-841 CoN human colon epithelial
cell line were grown in Dulbecco's modified Eagle's medium
ꢀ
1
h. The reaction mixture was left at ꢁ18 C for 2 days. The pre-
cipitate was filtrated, washed with small portions of diethyl ether
and dried under vacuum. Finally, 82 mg of yellow product was
obtained (yield: 56%). H NMR (DMSO-d
1
6
)
d
1.82 (d, J ¼ 6 Hz, 3H,
), 3.76 (s, 3H, OCH ), 3.79 (s,
), 5.40 (d, J ¼ 6 Hz, 1H, AreH), 5.64 (d,
CH
3
), 2.18 (m, 2H, CH
2
), 2.38 (m, 2H, CH
), 4.28 (m, 2H, NH
2
3
(DMEM) containing 10% FCS, 100 U/ml penicillin, 100
mg/mL strep-
3
H, OCH
3
2
tomycin and 1 mM glutamine. The cells were seeded into 96 well
3
J ¼ 6 Hz, 1H, AreH), 5.78 (s, 1H, AreH), 6.07 (d, J ¼ 15 Hz, 1H, trans
microtiter plates in 100
m
l volumes at a plating density of 5 ꢂ 10
ꢀ
HC]C), 6.45 (dd, J ¼ 15, 7 Hz, 1H, C]CHeCH
3
); EM m/z 374.77
cells/well. After 24 h of incubation at 37 C under a humidified 5%
þ
(
C
13
H
22
N
2
O
2
RuCl ); Anal. Calc. for C13
H
22
N
2
O
2
RuClPF : C, 30.03%;
6
2
CO atmosphere to allow cell attachment, the cells were treated
H, 4.26%; N, 5.39%. Found: C, 30.01%; H, 4.30%; N, 5.52%.
with different concentrations of the drugs (compounds VIeXII and
carboplatin) and incubated for 72 h under the same conditions.
Stock solutions of compounds were prepared in ethanol and the
final concentration of this solvent was maintained at 1%. The control
cultures received only 1% ethanol.
Synthesis of [Ru( 6
h -anethole)(en)Cl]PF (IX)
6
In a 100 ml round-bottom flask, 93 mg of [Ru(h6-anethole)
0.14 mmol) was dissolved in 20 ml of MeOH. To the solution was
added 31 l of ethylenediamine (0.45 mmol), and the mixture was
2
2 2
Cl ]
(
m
stirred at room temperature for 3 h. The reaction mixture was fil-
trated, and the filtrate was concentrated under vacuum until 5 ml of
Cell viability: in vitro growth inhibition assay
solution remained. Afterward, 120 mg of NH
4
PF
6
(0.74 mmol) was
The sulforhodamine B assay was used according to the method
3
added, and the mixture was shaken for 20 min. An orange solid
of Skehan et al. [29,30]. Briefly, the cells were seeded at 3 ꢂ 10 cells
ꢀ
appeared, and the mixture was left at ꢁ18 C for 2 days. The pre-
per well in a 96-flat-bottomed, 200
were incubated at 37 C in a humidified 5% CO
m
l well microplate. The cells
ꢀ
cipitate was filtrated, washed with small portions of diethyl ether
2
/95% air mixture and
and dried under vacuum. Finally, 52 mg of product was obtained
treated with the compounds (VIeXII and carboplatin) at different
concentrations for 72 h. Afterward, the cells were fixed with 50%
1
(
yield: 38%). H NMR (DMSO-d
6
)
d
1.77 (d, J ¼ 6 Hz, 3H, CH
3
), 2.13

134
R.A. Delgado et al. / Journal of Organometallic Chemistry 782 (2015) 131e137
ꢀ
[32]. In addition, the 1H NMR spectra show that the pattern of
chemical shifts associated with the allyl moiety of the natural
products differ in the final Ru(II) complexes, indicating that an
isomerization process occurs upon coordination. The signals at
trichloroacetic acid at 4 C. After washing with water, the cells were
stained with 0.1% sulforhodamine B (SigmaeAldrich, St. Louis, MO,
USA), dissolved in 1% acetic acid (50
sequently washed with 1% acetic acid to remove any unbound stain.
The protein-bound stain was solubilized with 100 l of 10 mM
unbuffered Tris base, and the cell density was determined using a
spectrophotometric plate reader (wavelength 540 nm). The values
are reported as the means ± SD of three independent experiments.
The GraphPad software (GraphPad Software, San Diego, CA, USA)
was used to calculate the IC50 values.
ml/well) for 30 min, and sub-
m
d
3.75 (d, 2H, J ¼ 7 Hz, CH
CH ) and
6.40 (dddd, J ¼ 17, 10, 7, 7 Hz, 1H, CH
original methyleugenol (I) change to
1.77 (d, J ¼ 7 Hz, 3H, CH]
CHCH ), ) and 6.69 (dd, J ¼ 16,
6.02 (d, J ¼ 16 Hz, 1H, CH]CHCH
7 Hz, 1H, CH]CHCH ) in compound VI. The value of the coupling
2
CH]CH
2
),
d
5.40e5.55 (m, 2H, CH
2
CH]
2
d
2
CH]CH ) of the
2
d
3
d
3
d
3
constants of the alkene protons (J ¼ 16 Hz) indicated that only the
trans isomer of methylisoeugenol and anethole were present in the
final organometallic compounds.
Results and discussion
To confirm that the isomerization occurs on both precursors
after coordination, the methyleugenol (I) and estragole (II) were
isomerized using a catalyst [{RuCl(m-Cl)(h :h -C10H16)} ]. The
2
Synthesis and characterization of the compounds VIeX
3
3
The first procedure used to obtain this type of complex was based
methylisoeugenol (XI) and anethole (XII) were obtained in good
yields from I and II, respectively (Scheme 1); however longer re-
action times were required (approximately 90 min for I and
150 min for II) compared to the 10 min reported for compound II
6
on the arene ligand exchange reaction in complex [Ru(
h
-naph-
4
0
thalene)(h -1,5-cyclooctadiene)] [31]. Reacting this Ru complex
with methyleugenol (I) to promote naphthalene displacement with
1
the subsequent hydrochloric acid treatment did not produce the
[21]. The H NMR spectra of methyleugenol (I), methylisoeugenol
6
desired [Ru(
h
-methyleugenol)
Cl ]
2 2 2
dimeric complex in useful
in situ with zinc
(XI), compound VI, estragole (II), anethole (XII) and compound VII
clearly demonstrated that isomerization occurs in the final organ-
ometallic compounds (See Supplementary information).
2 n
yields. In contrast, reducing the [Ru(1,5-COD)Cl ]
dust and in the presence of the natural ligands I and II produced
dimericcomplexesIVand V, which had a characteristic deepredcolor
and were isolated in 18% and 22% yields, respectively (Scheme 1) [11].
For the structurally related natural product saphrole (III), no stable
0
The lack of experimental evidence (Ru intermediate were not
isolated) precludes the proposal of a mechanism for the isomeri-
zation of the ligands during coordination. However, reported Ru
6
6
[
Ru( -arene)
h
Cl ]
2 2 2
complex could be isolated. The [Ru(
h
-are-
2 3 3 3 3 3
complexes, including RuCl (PPh ) , RuCl (AsPh ) , ruthenium car-
ne) Cl ] complexes (IV and V) were treated with three different N,N-
2
2 2
bene catalyst (named Grubbs second-generation catalyst) and the
3
3
chelating ligands (bipyridine (bipy), ethylenediamine (en) and 1,2-
bis(allyl)-ruthenium(IV)
dimer
2
[{RuCl(m-Cl)(h :h -C10H16)} ],
diaminobenzene (dab)) to obtain five well-characterized complexes
employed in this work can perform this type of isomerization on
6
with the following general formula: [Ru(
h
-arene)(N,N-chelating)Cl]
eugenol and/or estragole [33e36]. The isomerizations promoted by
4
PF
6
. The complexes were crystalized either by slow evaporation in
the polymeric [Ru(
h
-1,5-COD)Cl
2
]
n
complex (our starting mate-
MeOH or a slow diffusion of ether into an MeOH solution. A crystal
suitable for X-ray diffraction was obtained for compound VIII.
rial) have not been reported in the literature.
1
6-methylisoeugenol)(en)Cl]PF
The H NMR spectra of compounds VI to X exhibit the expected
X-ray diffraction of [Ru( (VIII)
h
6
upfield shifts of the aromatic protons from 7.1 to 7.3 ppm in the
natural precursors, to 5.5e6.3 ppm in the coordinated structure,
confirming the existence of the organometallic Ru(II)-arene bond
The molecular structure with atom-numbering scheme is
shown in Fig. 1. The X-ray diffraction analysis of VIII reveals the
Scheme 1. General synthetic procedures for compounds VI to XII.

R.A. Delgado et al. / Journal of Organometallic Chemistry 782 (2015) 131e137
135
Table 2
ꢀ
Selected bond lengths (Å) and angles ( ) in Compound
VIII.
Ruecentroid
RueN1
RueN2
RueCl
C4eC5
1.6817(2)
2.132(2)
2.137(2)
2.4148(9)
1.403(4)
1.355(4)
78.79(10)
85.64(8)
84.82(8)
125.6(3)
C3eO1
N1eRueN2
N1eRueCl
N2eRueCl
O2eC4eC5
2 6
cymene)RuCl ] and [(h ] [2.39e2.45 Å]
6-p-cymene)Ru(en)Cl][PF
are similar [38,39]. The Ruearene (Ru/Cg) bond length (see
Table 2) matches that in previous reports. Cg is the centroid of the
6
C1eC6 ring. The Ruearene bond length in [(
h
-p-cymene)Ru(OeN)
Cl] ranged from 1.655 to 1.689 Å [40]. The structure shows bond
lengths in C1eC2, C2eC3 and C3eC4, C4eC5 and C5eC6 in VIII
ranged from 1.403 Å to 1.437 Å.
The torsional C2eC1eC9eC10 angle of the corresponding alkyl
ꢀ
substituent group (at C1: C9eC10eC11) is ꢁ12.7(5) . The alkyl
group is essentially flat relative to the mean plane of the coordi-
nated arene with
a
C9]C10 bond distance of 1.309(5)
Å
Fig. 1. A view of the asymmetric unit showing the Ru(II) complex and PF-
displacement ellipsoids are drawn at the 50% probability level. H-atoms have been
omitted for clarity.
anion. The
[C9eC10eC11 ¼ 125.0(3) ]. The PeF bond lengths in PF
ꢀ
anion
6
6
ꢀ
ranged from 1.565(3) to 1.606 (2) Å [F1ePeF5 ¼ 90.68(16) and
ꢀ
F2ePeF5 ¼ 178.00(18) ].
-
In the crystal packing of VIII, the Ru(II) complex and PF
6
anions
are linked via NeH/F [2.24e2.55 Å] hydrogen bonds and CeH/F
2.46e2.47 Å] intermolecular contacts. The network is reinforced
presence of both enantiomers in the same single-crystal. The co-
crystallization of isomers is an important phenomenon for optical
resolution, as described in the literature. Brunner H. et al. suggested
that the stability of the inverted diastereomeric piano-stool com-
plex is attributed to molecular recognition [37]. The opposing ab-
solute configuration of both enantiomers in ruthenium complex
VIII compound (in this work) is shown in Fig. 2. The enantiomers
form an inverted piano-stool arrangement in a 1:1 ratio.
[
by the N2eH2A/O1 [2.300 Å] and N2eH2B/Cl [2.660 Å]
hydrogen bonds (see Fig. 5, Supplementary information). The H
atoms of the Nitrogen link each Cl atom forming a four-center
2
graph-set R ð4Þ motif [41]. The Ru/Ru distance of the two enan-
2
tiomers in the inverted piano-stool arrangement is 7.056 Å (see
Fig. 2). The supramolecular structure is additionally stabilized by
0
pe
p
interactions with Cg/Cg distance of 3.6489(18) Å.
All bond distances and angles in VIII are as expected (Table 2)
and are in acceptable agreement with the described analogs
Octanol/water partition coefficient
[
37e40]. The NeRueN bond angles in both enantiomers are com-
6
ꢀ
parable to those in [(
h
-p-cymene)Ru(en)Cl][PF
6
] [78.98(10) ]. The
The UVeVis spectra for compounds VI to X were recorded in
water and aqueous acidic media (0.2 M HCl) between 190 and
torsional N1eC13eC12eN2 angle of the corresponding bridge is
ꢀ
6
5
4.0(3) . The RueCl bond distance in VIII [2.4149(9) Å], [(h -p-
800 nm. All of the compounds were more stable in the aqueous
acidic media compared to water during the analysis (2 h). For each
compound, two acidic aqueous solutions with different concen-
trations were prepared (between 0.13 and 1.05 mM) and shaken
vigorously with octanol (presaturated with the acidic aqueous so-
lution). The equilibrium concentrations of the compounds were
measured in both phases at the following wavelengths for VI, VII,
VIII, IX and X, respectively: the organic phase was assessed at
292 nm, 296 nm, 220 nm, 260 nm, and 260 nm; the aqueous phase
was assessed at 412 nm, 344 nm, 312 nm, 300 nm, and 312 nm.
Table 3 summarizes the log P (log [ ]organic/[ ]aqueous) values for all of
the compounds.
In vitro cell viability assay
In vitro biological activity of organometallic compounds VI to X,
methylisoeugenol (XI), anethole (XII) and carboplatin against the
three human tumor cell lines MCF-7 (breast cancer), PC-3 (prostate
cancer) and HT-29 (colon cancer) and one human non-tumor cell
line CCD-841 (colon epithelial) are shown in Table 3. The results
indicated that the free ligands (XI and XII) exhibited no measurable
Fig. 2. A view of the two enantiomers. The H-atoms and PF-
6
anions have been omitted
for clarity.
activity (>100 mM) against any of the studied human cell lines.

136
R.A. Delgado et al. / Journal of Organometallic Chemistry 782 (2015) 131e137
Table 3
isomerization on the allylic chain of the coordinated arene was
verified by H NMR spectroscopy. The single-crystal X-ray diffrac-
Comparison of the biological activity of compounds VI to XII and carboplatin against
human breast cancer (MCF-3), human prostate cancer (PC-3), human colon cancer
1
tion data for complex VIII confirmed these features. The in vitro
(
HT-29) and human colon epithelial cells (CCD-841, a non-tumor cell line).
6
cytotoxicity measurements showed that [Ru(
h
-anethole) (en)Cl]
(X) were as
Compound
Log P
IC50
(
m
M)a
6
PF
6
(IX) and [Ru(
h
-methylisoeugenol)(dab)Cl]PF
6
MCF-7
PC-3
HT-29
CCD-841
cytotoxic as carboplatin, which is a commercial drug, against the
three human tumor cell lines studied. Compounds IX and X should
be tested against other human tumor cells, particularly cisplatin-
resistant cells, in a future study.
VI
VII
VIII
IX
X
XI
XII
ꢁ1.38
ꢁ0.83
ꢁ1.30
ꢁ0.99
ꢁ0.93
e
>100
>100
>100
40 ± 4
73 ± 9
>100
>100
78 ± 7
>100
>100
>100
58 ± 8
28 ± 4
>100
>100
87 ± 12
>100
>100
>100
18 ± 3
>100
>100
>100
>100
>100
>100
>100
79 ± 10
>100
>100
>100
>100
Acknowledgments
e
Carboplatin
e
The authors thank the Universidad T eꢀ cnica Federico Santa María
(DGIP 13.12.37) and the Universidad de Valparaíso (DIUV 50/2011)
for their financial support.
a
Inhibitory concentrations (50%) in the SRB assay (72 h exposure). Values are
means ± standard deviations of three independent experiments.
Furthermore, both complexes containing bipy [Ru(h6-arene)(bipy)
Appendix A. Supplementary material
Cl]PF
6
where the arene is methylisoeugenol for VI and anethole for
CCDC 955623 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
VII did not exhibit any measurable activity against the studied cell
lines. These results agree with previous reports on [(
Ru(bipy)Cl] complexes [11]. The lack of activity displayed by
6
h
-arene)
þ
6
6
[Ru(
h
-methylisoeugenol)(en)Cl]PF
6
(VIII) was unexpected. [Ru(
h
-
6
anethole)(en)Cl]PF
6
(IX) and [Ru(
h
-methylisoeugenol)(dab)Cl]PF
6
Appendix B. Supplementary material
(
X) are active compounds that are even better than carboplatin. The
6
lack of activity observed for [Ru(
h -methylisoeugenol)(en)Cl]PF
6
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jorganchem.2014.09.005.
(
VIII) can be correlated with the lower lipophilicity of VIII (see
Table 3) due to the additional methoxy substituent on the coordi-
nated aromatic ring compared to IX. The presence of an additional
polar group on the coordinated arene (methylisoeugenol vs anet-
hole) tends to decrease the lipophilicity of the organometallic
compounds (VI vs VII and VIII vs IX), disfavoring its biological ac-
tivity. The literature suggests that the presence of a polar substit-
uent on the coordinated arene tends to lower the cytotoxicity of the
organometallic complex [11]. When using ethylenediamine (en) or
References
[
[
1] L. Kelland, Nat. Rev. Cancer 7 (2007) 573e584.
2] A. Bergamo, C. Gaiddon, J.H.M. Schellens, J.H. Beijnen, G. Sava, J. Inorg. Bio-
chem. 106 (2012) 90e99.
[3] C.S. Allardyce, P.J. Dyson, Platin. Met. Rev. 45 (2001) 62e69.
[
4] E. Reisner, V.B. Arion, B.K. Keppler, A.J.L. Pombeiro, Inorg. Chim. Acta 361
2008) 1569e1583.
(
1,2-diaminobenzene (dab) as the N,N-chelating ligand, however,
[5] G. Sava, A. Bergamo, S. Zorzet, B. Gava, C. Casarsa, M. Cocchietto, A. Furlani,
V. Scarcia, B. Serli, E. Iengo, E. Alessio, G. Mestroni, Eur. J. Cancer 38 (2002)
organometallic compounds with similar biological activities were
6
427e435.
generated [11]. Therefore, the higher activity of [Ru(
h
-methyl-
-methylisoeugenol)(en)Cl]
(VIII) was attributed to the enhanced lipophilicity of the former
compound.
6
[6] M.A. Jakupec, E. Reisner, A. Eichinger, M. Pongratz, V.B. Arion, M. Galanski,
C.G. Hartinger, B.K. Keppler, J. Med. Chem. 48 (2005) 2831e2837.
[7] M.J. Clarke, Coord. Chem. Rev. 236 (2003) 209e233.
isoeugenol)(dab)Cl]PF
6
(X) versus [Ru(h
PF
6
[8] W.H. Ang, A. Casini, G. Sava, P.J. Dyson, J. Organomet. Chem. 696 (2011)
989e998.
Compound IX exhibited the highest biological activity against
the human tumor cells under study; its cytotoxicity was approxi-
mately 20 times greater than the value reported for anethole [42].
The coordination of the Ru(en)Cl moiety to this biologically active
natural product generated compound IX, which exhibited an
improved cytotoxic activity. Similarly, coordinating methyl-
[9] R.E. Aird, J. Cummings, A.A. Ritchie, M. Muir, R.E. Morris, H. Chen, P.J. Sadler,
D.I. Jodrell, Br. J. Cancer 86 (2002) 1652e1657.
[10] C. Scolaro, A. Bergamo, L. Brescacin, R. Delfino, M. Cocchietto, G. Laurenczy,
T.J. Geldbach, G. Sava, P.J. Dyson, J. Med. Chem. 48 (2005) 4161e4171.
þ
[
11] A. Habtemariam, M. Melchart, R. Fern aꢀ ndez, S. Parsons, I.D.H. Oswald,
A. Parkin, F.P.A. Fabbiani, J.E. Davidson, A. Dawson, R.E. Aird, D.I. Jodrell,
P.J. Sadler, J. Med. Chem. 49 (2006) 6858e6868.
þ
[12] S. Hemalswarya, M. Doble, Phytoter. Res. 20 (2006) 239e249.
isoeugenol to the Ru(dab)Cl moiety produced compound X, which
[
[
13] F.H. Sarkar, Y. Li, Cancer Res. 66 (2006) 3347e3350.
14] M.U. Nessa, P. Beale, C. Chan, J.Q. Yu, F. Huq, Anticancer Res. 31 (2011)
3789e3798.
exhibited an increased biological activity compared to the natural
product alone. Whether the coordination of these ligands actually
improved the cytotoxic activity of the organometallic compound
should be established in future works. Previous studies have
established that combining anethole with platinum based drugs
such as cisplatin and oxaliplatin synergistically increases the
in vitro cytotoxicity against a human ovarian tumor cell line [15].
[
[
15] M.U. Nessa, P. Beale, C. Chan, J.Q. Yu, F. Huq, Anticancer Res. 32 (2012)
4843e4850.
16] G.P. Kamatou, I. Vermaak, A.M. Viljoen, Molecules 17 (2012) 6953e6981.
[17] I. Kubo, K. Fujita, K. Nihei, J. Sci. Food Agric. 88 (2008) 242e247.
[
[
[
[
18] F.A. Thomet, P. Pinyol, J. Villena, P.G. Reveco, Inorg. Chim. Acta 384 (2012)
55e259.
19] A. Basak, M.K. Nayak, A.K. Chakraborti, Tetrahedron Lett. 39 (1998)
883e4886.
20] M.O. Albers, T.V. Ashworth, H.E. Oosthuizen, E. Singleton, Inorg. Synth. 26
1987) 68e77.
2
4
Conclusions
(
21] B. Lastra-Barreira, J. Francos, P. Crochet, V. Cadierno, Green Chem. 13 (2011)
307e313.
The in situ reduction of the polymeric [Ru(1,5-COD)Cl
plex with methyleugenol and estragole generated two new dimeric
complexes: [Ru(
2 n
] com-
[
[
22] A. Salzer, A. Bauer, S. Geyser, F. Podewils, Inorg. Synth. 34 (2004) 59e65.
23] SMART, SAINTPLUS V6.02, SHELXTL V6.10 and SADABS, Bruker Analytical X-
ray Instruments Inc., Madison, Wisconsin, USA.
6
6
2 2 2
h -methylisoeugenol) Cl ] (IV) and [Ru(h -ane-
thole) Cl (V). Treating these complexes with N,N-chelating li-
2
2
]
2
[24] G.M. Sheldrick, SHELXL-97. Program for the Refinement of Crystal Structures,
University of G o€ ttingen, Germany, 1997.
gands (bipyridine, ethylendiamine and 1,2-diaminobenzene)
generates five new well-characterized “piano-stool” compounds:
VIeX. The Ru(II)-arene organometallic bond and the alkene
[25] K. Brandenburg, DIAMOND. Visual Crystal Structure Information System.
Version 2.1e, Crystal Impact GbR, Bonn, Germany, 1999.
[26] PLATON Program: A.L. Spek J. Appl. Cryst. 36 (2003) 7.

R.A. Delgado et al. / Journal of Organometallic Chemistry 782 (2015) 131e137
137
[
[
27] Y. Qiao, S. Xia, P. Ma, J. Chem. Eng. Data 53 (2008) 280e282.
28] R. Schuecker, R.O. John, M.A. Jakupec, V.B. Arion, B.K. Keppler, Organometallics
7 (2008) 6587e6595.
[35] C.R. Larsen, D.B. Grotjahn, J. Am. Chem. Soc. 134 (2012) 10357e10360.
ꢀ
[36] A.E. Díaz-Alvarez, P. Crochet, V. Cadierno, Tetrahedron 68 (2012) 2611e2620.
[37] H. Brunner, M. Weber, M. Zabel, Coord. Chem. Rev. 242 (2003) 3e13.
[38] A. Pastuszko, K. Niewinna, M. Czyz, A. J oꢀ zwiak, M. Malecka, E. Budzisz,
2
[29] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd, J. Natl. Cancer Inst. 82 (1990)
J. Organomet. Chem. 745e746 (2013) 64e70.
[39] R.E. Morris, R.E. Aird, P. Murdoch, H. Chen, J. Cummings, N.D. Hughes,
S. Parsons, A. Parkin, G. Boyd, D.I. Jodrell, P.J. Sadler, J. Med. Chem. 44 (2001)
3616e3621.
1107e1112.
[
[
30] V. Vichai, K. Kirtikara, Nat. Protoc. 1 (2006) 1112e1116.
31] G. Vitulli, P. Pertici, P. Salvadori, J. Chem. Soc. Dalton Trans. (1984)
2
255e2257.
[40] H. Brunner, M. Weber, M. Zabel, T. Zwack, Angew. Chem. Int. Ed. 42 (2003)
1859e1862.
[
[
[
32] P. Pertici, G. Vitulli, R. Lazzaroni, P. Salvadori, P.L. Barili, J. Chem. Soc. Dalton
Trans. (1982) 1019e1022.
33] S.K. Sharma, V.K. Srivastava, R.V. Jasra, J. Mol. Catal. A Chem. 245 (2006)
[41] J. Bernstein, R.E. Davis, L. Shimoni, N.-L. Chang, Angew. Chem. Int. Ed. Engl. 34
(1995) 1555e1573.
[42] Ch.H. Chen, L.A. deGraffenried, Phytomedicine 19 (2012) 763e767.
200e209.
34] S. Hanessian, S. Giroux, A. Larsson, Org. Lett. 8 (2006) 5481e5484.