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Ruthenium-arene complexes of curcumin: X-ray and density functional theory structure, synthesis, and spectroscopic characterization, in vitro antitumor activity, and DNA docking studies of (p-cymene)Ru(curcuminato)chloro.
Article
pubs.acs.org/jmc
Ruthenium−Arene Complexes of Curcumin: X-Ray and Density
Functional Theory Structure, Synthesis, and Spectroscopic
Characterization, in Vitro Antitumor Activity, and DNA Docking
Studies of (p-Cymene)Ru(curcuminato)chloro
Francesco Caruso,*,† Miriam Rossi,*,‡ Aidan Benson,‡ Cristian Opazo,§ Daniel Freedman,∥ Elena Monti,⊥
Marzia Bruna Gariboldi,⊥ Jodi Shaulky,# Fabio Marchetti,▽ Riccardo Pettinari,○ and Claudio Pettinari○
†
Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, c/o University of Rome “La Sapienza”, Istituto Chimico,
Piazzale Aldo Moro 5, 00185, Rome, Italy
‡
Vassar College, Department of Chemistry, Poughkeepsie, New York 12604, United States
§
Vassar College, Academic Computing Service, Poughkeepsie, New York 12604, United States
∥
State University of New York, Department of Chemistry, New Paltz, New York 12561, United States
⊥
University of Insubria, Department of Structural and Functional Biology, Via A. da Giussano 10, 21052 Busto Arsizio, Varese, Italy
#
Accelrys, Inc., 10188 Telesis Court, Suite 100, San Diego, California 92121, United States
▽
School of Science and Technology, Università degli Studi di Camerino, via S. Agostino 1, 62032 Camerino MC, Italy
○
School of Pharmacy, Università degli Studi di Camerino, via S. Agostino 1, 62032 Camerino MC, Italy
S Supporting Information
*
ABSTRACT: The in vitro antiproliferative activity of the title compound
on five tumor cell lines shows preference for the colon−rectal tumor
HCT116, IC50 = 13.98 μM, followed by breast MCF7 (19.58 μM) and
ovarian A2780 (23.38 μM) cell lines; human glioblastoma U-87 and lung
carcinoma A549 are less sensitive. A commercial curcumin reagent, also
containing demethoxy and bis-demethoxy curcumin, was used to
synthesize the title compound, and so (p-cymene)Ru(demethoxycurcuminato)chloro was also isolated and chemically characterized. The
crystal structure of the title compound shows (1) the chlorine atom
linking two neighboring complexes through H-bonds with two O(hydroxyl), forming an infinite two-step network; (2) significant twist in
the curcuminato, 20° between the planes of the two phenyl rings. This was
also seen in the docking of the Ru-complex onto a rich guanine B-DNA
decamer, where a Ru−N7(guanine) interaction is detected. This Ru−N7(guanine) interaction is also seen with ESI-MS on a Rucomplex-guanosine derivative.
■
drug resistance.3 The search for other metal drug anticancer
agents drives much current research interests,4 with particular
attention to ruthenium.5
Two Ru[III] species, NAMI-A(antimetastatic)6and KP1019,7
are in clinical trials. Ru(II) arene complexes appear to have an
altered profile of biological activity in comparison with metalbased anticancer complexes currently in clinical use.8 The
ligand exchange kinetics of Pt(II) and Ru(II) complexes in
aqueous solution, crucial for anticancer activity, are very
similar.9 Moreover, ruthenium compounds are not very toxic
and some are quite selective for cancer cells, likely due to the
ability of ruthenium to mimic iron in binding to biomolecules.10
Sadler and Dyson have explored the activity of neutral or
INTRODUCTION
Cancer is the second leading cause of death in economically
developed countries and the third in emergent nations.1
Although survival rates have increased due to efficient
anticancer drugs and prevention, many types of cancer still
have no effective cure. Most new therapeutic candidates are
organic compounds, yet metal complexes are of interest
because metal ions are essential in many natural biological
processes. Notably, metal complexes offer mechanisms of
action that are unavailable to organic compounds. The nature
of the metal ion, its oxidation state (including multiple redox
modifications), and the type and number of bound ligands can
exert a critical influence on the biological activity of a metal
complex.2 A turning point for the use of metal antitumor agents
was the development of the platinum-based drugs that provide
well established treatments, although limited by toxicity and
© 2011 American Chemical Society
Received: July 11, 2011
Published: December 29, 2011
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cationic “half-sandwich” arene Ru(II) complexes;11,12 these
often possess good aqueous solubility (useful for clinical use)
and the arene ligand is relatively inert toward displacement
under physiological conditions.13,14 Cationic arene ruthenium
ethylenediamine complexes reported by Sadler show very high
activity both in vitro and in vivo.15−17 Their interaction with
DNA model compounds and other biologically relevant
molecules has been established through simultaneous covalent
coordination, intercalation of extended aromatic groups and
stereospecific hydrogen bonding.18−21 Arene ruthenium
complexes containing phosphine ligands, such as pta (1,3,5triaza-7-phosphatricyclo[3.3.1.1]decane), as well as N,O- or
O,O-chelating ligands, such as carboxylates, resist hydrolysis, yet
this phenomenon does not diminish cytotoxicity.22−25 However, limited efforts have been made in conjugating the metal
center with ligands that themselves show biological activity. It
was for this reason that we are investigating the chemistry and
potential antitumor activity of novel Ru(II)−(arene) compounds with a variant of β-diketonato ligands, curcumin, a wellknown natural compound whose coordination chemistry has
been only partially explored.26−29 Systematic investigations of
curcumin reveals a wide spectrum of beneficial properties
including antioxidant, anti-inflammatory, antimicrobial, and
anticancer activities.30,31 In addition, it protects neurons against
β-amyloid peptide toxicity and binds to β-amyloid plaques of
transgenic mouse models of Alzheimer’s disease.32,33 In
continuation of our previous exploration of Ru(II)−(arene)
chemistry with β-diketones,34 and considering the interesting
biological properties of curcumin, we report the chemical
characterization of several Ru−(arene)−curcuminato complexes and the molecular structure of [(p-cymene)Ru(curcuminato)chloro] along with its antitumor activity on five
tumor cell lines in vitro. We also describe docking experiments
of this complex onto a guanosine rich DNA substrate.
Ru-Cur is rather insoluble in water. Upon prolonged
standing in deuterated-water suspension, a small portion
(5%) decomposed toward neutral curcumin and Ru(pcymene)(Solv)xCl species, the remaining 95% being unaltered.
Thus, the 1H NMR (D2O) spectrum of Ru-Cur shows only
signals due to p-cymene moiety, in accordance with the
observations reported in the literature.35
The IR spectrum of 1 shows the typical ν(O−H) and ν(C
O, CC) bands of curcumin shifted to lower wavenumbers as
a consequence of its metal coordination through both carbonyl
arms. The 1H and 13C{1H} NMR spectra of 1 contain all the
expected resonances of the (p-cymene)Ru(II) fragment and of
the curcuminato ligand (the numbering of C atoms in curcumin
is indicated in Scheme 2), in a 1:1 ratio in accordance with the
Scheme 2
stoichiometry of proposed derivatives. The positive ESI-MS
spectra of Ru-Cur, carried out in methanol or acetonitrile,
display the peak corresponding to [(p-cymene)Ru(curcuminato)]+, while those carried out on the crude product
prior the TLC separation contain also the peaks due to
derivatives containing demethoxycurcumin and bis(demethoxy)curcumin.
Using the same previously described TLC separation
procedure, we were able to isolate derivative 2 (p-cymene)Ru(demethoxycurcuminato)chloro which originated from the
small fraction of the demethoxycurcumin (d-curcH) in the
commercial ligand. It displays similar spectral features with
some differences in the proton spectrum due to the absence of
a methoxy group in the O2-chelating ligand.
To ascertain the ability of Ru-Cur to interact with nucleic
acid bases, we have performed an ESI-MS study on a methanol
solution containing a 1:1 mixture of Ru-Cur and 9-ethylguanine. The positive ESI spectrum shows a m/z peak at 782
corresponding to [(p-cymene)Ru(curcuminato)(9-ethylguanine)]+, and an additional peak at 450 due to [(p-cymene)RuCl(9-ethylguanine)]+, thus confirming the ability of the
ruthenium(II) fragment to coordinate the nucleobase. Similarly,
the ESI-MS spectrum on a methanol solution, containing a 1:1
mixture of derivative 2 and 9-ethylguanine, showed a peak at
752 corresponding to [(p-cymene)Ru(demethoxycurcuminato)
(9-ethylguanine)]+, which further confirmed the coordination
of the nucleobase to ruthenium.
Structural Data. The single crystal X-ray diffraction
experiment yielded the molecular structure of the title
compound and is depicted in Figure 1. Crystals gave weak
diffraction and provided limited number of useful reflections; as
a result, only some atoms could be refined anisotropically. The
metal is bound to 2 O donors from the chelating β-diketone
curcuminato, one Cl anion, and the p-cymene aromatic ring,
thus forming a piano-stool arrangement, where both O(curcuminato) and Cl are the legs. The curcumin ligand is
twisted with a torsion angle about the coordination sphere
O(6)−Ru(1)−O(5)−C(9) of 11.2°(1), while the angle
between the planes of the two phenyl rings in curcumin is
■
RESULTS AND DISCUSSION
Synthesis and Characterization of Complexes. Derivative 1, Ru-Cur, is obtained by interaction of commercial
curcumin and [(p-cymene)RuCl2]2 in methanol in the presence
of NaOMe; consequent TLC separation using a mixture of
chloroform/methanol (9:1) as eluent gave its composition as
[(p-cymene)Ru(curcuminato)Cl] (Scheme 1).
Scheme 1
The compound is an air and moisture stable orange solid,
soluble in chlorohydrocarbon, alcohol, acetone, acetonitrile,
and DMSO solvents, where they are partially dissociated, as
indicated by eq 1:
[(p‐cymene)Ru(curcuminato)chloro] + S(solvent)
⇆ [(p‐cymene)Ru(curcuminato)(S)]+ + Cl−
(1)
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Figure 1. (a) X-ray molecular structure of the title compound, Ru-Cur. Disorder on the i-propyl goup of p-cymene is evident. (b) Profile view with
H atoms omitted showing piano-stool geometry as well as the twist of the curcumin ligand; torsion angle about the coordination sphere O(6)−
Ru(1)−O(5)−C(9) is 11.2°(1), while the angle between the planes of the two phenyl rings in curcumin is 20°. (c) In the packing of the molecule,
the intermolecular interactions are such that each chlorine (green) atom forms two strong O···Cl-hydrogen bonds with neighboring complexes
through their hydroxyl oxygen (red) atoms O(1) and O(3), thereby forming a parallel two-step ladder infinite network. One of the steps is shown in
bold.
20° (see Figure 1b). Disorder of the i-propyl moiety in the pcymene ring is seen by the large spherical isotropic displacement parameters on the lower left side of Figure 1a.
Using all non-H atom coordinates experimentally obtained
from the X-ray study and the generated H atoms, we performed
a DFT geometry-optimization on Ru-Cur. The converged
structure, shown in Figure 2, is at a minimum of energy as all its
calculated frequencies are positive. Specific comparison
between the coordination sphere of the experimental and
calculated structures indicates an excellent agreement of bond
angles and slightly overestimated bond distances in the latter.
Thus, the Ru−Cl bond length, 2.473 Å (DFT) is longer than
2.436(2) Å (X-ray), as are Ru−O data (DFT), around 2.10 Å
and 2.07 Å, respectively; the Ru arene centroid is also slightly
elongated: Ru−centroid of 1.747 Å and 1.65(2) Å, respectively.
This DFT structure was later used for docking onto a DNA
substrate.
Comparison between X-ray structures of the title compound
and the related Ru complex of dimethoxy-curcumin, bis((1,7bis(3,4-dimethoxyphenyl)-hept-1,6-diene-3,5-dione)-(η 6 -pcymene)-chloro-ruthenium(II),36 in Table 1 indicates that
replacement of both peripheral hydroxyls in the title compound
by methoxy groups does not affect the coordination sphere, as
expected; this confirms that our X-ray structure is reliable
Figure 2. DFT molecular structure of Ru-Cur. Both H(hydroxyl)
atoms were pointed toward their methoxy neighbors. Ru, both
O(keto−enol) and Cl are ball and stick style, all other atoms are stick
style. The p-cymene ring center is also indicated (yellow). The initial
coordinates for all non-H atoms in this DFT minimized species were
obtained from the X-ray study.
notwithstanding its high Rf crystallographic factor. There are 15
hits in the CSD database for (arene)Ru(chloro) β-diketonates
and, compared to them, the title compound shows normal
structural features. For instance, the Ru−arene centroid mean
value is 1.648 Å, which compares well with our 1.65(2) Å,
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Biological Activity. The title compound was initially tested
on five tumor cell lines. Figure 3 shows these results for the
Table 1. Structural Data in the Coordination Sphere of the 2
Molecules in the Asymmetric Unit of Bis((1,7-bis(3,4dimethoxyphenyl)-hept-1,6-diene-3,5-dione)-(η6-p-cymene)chloro-ruthenium(II),36 Named Ru-Dim-Cur, and the Title
Compound (p-cymene)Ru(curcuminato)chloro, Named RuCur, and the DFT Minimized Structure of the Latter
name/
technique
Ru-Dim-Cur
X-ray (1)
Ru-Dim-Cur
X-ray (2)
Ru-Cur
X-ray
Ru-Cur
DFT
Ru−Cl
2.435(1)
2.422(1)
2.436(4)
2.473
Ru−O
2.067(4)
2.071(3)
2.066(4)
2.073(3)
2.06(1)
2.07(1)
2.098
2.100
Ru−centroid
1.646(4)
1.649(5)
1.65(2)
1.747
Ru−C(max)
2.198(5)
2.189(5)
2.20(2)
2.297
Ru−C(min)
2.142(4)
2.138(5)
2.07(2)
2.234
O−C
1.284(6)
1.285(6)
1.274(7)
1.277(6)
1.41(2)
1.33(2)
1.294
1.294
C−C
1.381(7)
1.397(7)
1.392(8)
1.407(6)
1.38(2)
1.40(2)
1.413
1.415
Cl−Ru−O
85.1(1)
84.6(1)
84.3(1)
84.7(1)
83.4(3)
84.5(3)
84.7
85.6
O−Ru−O
88.3(1)
88.0(1)
87.3(4)
88.7
Cl−Ru−
centroid
128.1(1)
128.1(1)
130.6(5)
128.5
O−Ru−
centroid
128.1(1)
127.9(2)
126.2(6)
126.1
127.8(1)
128.6(2)
129.2(6)
128.8
Figure 3. Effect of 72 h exposure of MCF7 (breast), A2780 (ovarian),
and HCT116 (colon) cancer cells to Ru-Cur (empty bars) and
cisplatin (CDDP, hatched bars). Means ± SE of 4−6 independent
experiments. *** p < 0.001 vs CDDP; ** p < 0.01 vs CDDP; # p <
0.001 vs HCT116.
most sensitive three lines while the dose-dependent behavior is
shown in Supporting Information Figure S8, specific IC50
values are presented in Table 2. The activity of the title
Table 2. IC50 (μM) Values of the Title Compound Ru-Cur
and Cisplatin in Several Tumor Cell Lines (n = Number of
Experiments Performed)
cancer line
MCF7
HCT116
A2780
CP8
A549
Table 1. The symmetric nature of the curcumin ligand is
reflected in the coordination sphere, as both Ru−O(curcuminato) bond lengths are equal [2.06(1), 2.07(1) Å],
in contrast with the asymmetric β-diketonato ligand in (pcymene)-chloro-(4-(trifluoroacetyl)-3-methyl-1-phenyl-pyrazol-5-onato)-ruthenium(II) that shows significantly different
Ru−O bond lengths [2.095(1), 2.104(1) Å].34
As seen in Table 1, our structure shows a Ru−Cl bond length
(2.436(4)Å) similar to that in the dimethoxycurcumin
derivative. The packing of our molecule shows the chlorine
atom having a key role in the intermolecular interactions
because it links two neighboring complexes through two strong
O−H···Cl-hydrogen bonds with their hydroxyl oxygen atoms
O(1)···Cl 3.100(5) Å and O(3)···Cl 3.128(5) Å and thereby
forming an infinite two-step network, as seen in Figure 1c. The
step distance is the Ru−Cl bond distance while the separation
between the two-step network is graphite-like, with the shortest
(C(4)···C(15′)) stacking interaction distance of 3.374(5) Å.
This stacking occurs down the crystallographic b axis between
two curcumin-phenyl rings. The solvent water molecule, which
is on a 2-fold axis of symmetry, lies at the apex of a distorted
square pyramid with two hydrogen bonded (2.902(5) Å)
methoxy O(2) atoms at opposite corners of the square base
and two weaker interactions with the two hydroxyl oxygen
O(1) atoms at the remaining two corners at 3.358(5) Å.
U87
breast
adenocarcinoma
colon
adenocarcinoma
ovarian carcinoma
ovarian platinumresistant
lung
adenocarcinoma
glioblastoma
Ru-Cur
mean ± SD (n)
cisplatin
mean ± SD (n)
19.58 ± 2.367 (5)
1.835 ± 0.237 (5)
13.98 ± 1.503 (6)
5.217 ± 0.348 (5)
23.38 ± 3.334 (4)
27.00 ± 2.332 (5)
1.325 ± 0.196 (5)
9.918 ± 1.155 (5)
62.33 ± 8.934 (4)
29.36 ± 1.842 (3)
compound on the ovarian tumor cell line A2780 yields an IC50
value similar to that reported for the cationic complex
[(hexamethylbenzene)Ru(ethylendiamine)(NCS)]+ (23.38 ±
3.334 μM vs 24 μM, respectively).37 A more specific
comparison with related β-diketonato Ru complexes, that
contain the same p-cymene arene, shows an IC50 of 19 μM for
[(p-cymene)Ru(R1C(O)CHC(O)R2)Cl] (R1 = CH3, R2 =
H), 14 μM (R1 = t-Bu3, R2 = H), 11 μM (R1 = Ph, R2 = H),23
24 μM (R1 = CH3, R2 = Cl).38 Also the lung carcinoma A549
cell line shows values similar to those reported in the literature
for [(p-cymene)Ru(R1C(O)CHC(O)R2))(Cl)], R1 = CH3,
R2 = Cl.38 The highest activity for Ru-Cur was observed in the
human colorectal carcinoma cell line HCT116 (CCL-247),
13.98 ± 1.503 μM, which can be compared to 8 μM obtained
for the ethylenediamine−Ru chelate cationic species
[(C6H5C6H5)RuCl (H2NCH2CH2NH2-N,N′)]+ PF6− in the
same cell line.39 Interestingly, ethylenediamine Ru chelates are
considered better antitumor agents than Ru-β-diketonates.13
SW480 is another colon−rectal cell line in which Ru
compounds of the type [(p-cymene)Ru(oxine)(Hazole),
oxine = deprotonated 8-hydroxyquinoleine, Hazole = azole
heterocycle, were investigated. For these highly active
antitumor compounds, the range of activity is 3.3−15.3 μM,
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Comparison of Ru-Cur activity with curcumin (Figure 5)
shows that, while curcumin is significantly more potent on
with the most active species having Hazole = imidazole; similar
activity was found for the ovarian carcinoma CH.40 Moreover,
related compounds have submicromolar range of activity for
the A549 lung carcinoma cell line.41 In contrast, the title
compound shows the least activity toward A549, 62.33 ± 8.934
μM, whereas glioblastoma U87 has intermediate activity, 29.36
± 1.842 μM.
The effects of Ru-Cur on the three most sensitive cell lines
tested, MCF7, A2780, and HCT116, were compared with those
elicited by cisplatin, the prototypical metal-based anticancer
drug, also shown in Table 2. It should be noted that HCT116
cells are deficient in mismatch repair function, due to a
hemizygous mutation in the hMLH1 gene, resulting in a
truncated, nonfunctional protein;42 this defect plays a major
role in determining the relative resistance of this cell line to
cisplatin.43 The results of this comparative evaluation indicate
that while cisplatin is significantly more potent than Ru-Cur on
all three cell lines, its effects on the colon carcinoma cell line are
somewhat inferior to those observed for the other cell lines
tested, as expected; in contrast, the effects of Ru-Cur do not
seem to depend on mismatch repair proficiency of the tumor
cells, as indicated by the absence of significant differences in the
IC50 values obtained for the three cell lines (Figure 3). To
compare the therapeutic windows of Ru-Cur and cisplatin, we
tested the two compounds on the nonmalignant human breast
cell line MCF-10A and compared the results with those
obtained for MCF7 cells. The following IC50 values were
obtained: 19.06 ± 2.37 μM in MCF7 vs 102.1 ± 6.5 μM in
MCF-10A for Ru-Cur; 1.83 ± 0.24 μM in MCF7 vs 7.27 ± 0.56
μM in MCF-10A for cisplatin. Thus, both compounds are
approximately 4- to 5-fold more potent on carcinoma cells as
compared to nonmalignant cells derived from the same tissue.
Interestingly, when Ru-Cur and cisplatin were tested on a
variant cell line selected from A2780 for its resistance to
cisplatin (CP8),44 the effects of Ru-Cur were similar to those
observed in the parental cell line, thus confirming the absence
of cross-resistance between the two compounds (Figure 4,
Figure 5. Effect of 72 h exposure of MCF7 (breast), HCT116 (colon),
and A2780 and CP8 (both from ovary) cancer cells to RuCUR (empty
bars), and curcumin (hatched bars). Means ± SE of 3−6 independent
experiments. * p < 0.05 vs curcumin ** p < 0.01 vs curcumin. # p <
0.01 vs all other cell lines exposed to curcumin.
MCF7 and A2780 cells, the IC50 values obtained for the two
compounds in HCT116 cells and in the cisplatin-resistant
ovarian line do not significantly differ from one another.
Interestingly, this latter cell line is significantly less responsive
to curcumin as well as to cisplatin, the resistance index (RI =
IC50[A2780]/IC50[CP8]) for curcumin being approximately 3;
in contrast, as already mentioned above, Ru-Cur is roughly as
potent on resistant as on sensitive cells (R.I. ≈1).
Docking on DNA. Bonding between Ru and N7(guanine)
is considered the predominant mode of action with DNA for
Ru antitumor compounds.46 Indeed, this interaction is shown
to be specific as only some guanine N7 sites are preferred.
Thus, in the self-complementary d(CGGCCG) nucleotide,
only G3 and G6 are ruthenated by a (p-cymene)Ru(ethylenediamine) species, while in the equivalent single strand
nucleotide all guanines were reactive.47 Therefore we decided
to study a rich-guanine polynucleotide as a receptor to explore
a docking of the title compound. Using Discovery Studio 3.0,
we built an ab initio double helix B DNA decamer containing
only alternant C-G bases, whose helices were named as “A” and
“S”. Counterions were imposed to hold a neutral system
receptor. The docking process suggests that indeed Ru−
N7(guanine) interaction is feasible, as depicted in Figure 6, see
also Experimental Section.
In addition, interaction between Ru and O(phosphate) for
the same Guanosine6 “(A)” nucleotide was also apparent,
shown in Figure 7, and this (p-cymene)Ru(curcuminato)decamer has a molecular energy very similar to that of the
corresponding Ru−N7(guanine) species shown in Figure 6.
However, the literature has a large amount of data supporting
Ru−N7(guanine) binding. For instance, the non-natural
species 9-ethylguanine was used to mimic the DNA N7(guanine)-Ru bonding, as shown crystallographically in the
cationic compound [(biphenyl)Ru(ethylethylenediamineN,N′)(9-ethylguanine)].46 This interaction was also confirmed
using the natural nucleoside guanosine in the crystalline
cationic species [(biphenyl)Ru(ethylenediamine)-(guanosine)],
whereas the use of guanosinemonophosphate, GMP, allowed
verification of the Ru−N7(guanine) bonding using NMR.46
Moreover, in a more recent study of Ru-β-diketonates, the
cationic compound [(p-cymene)Ru(Ph2acac)(9-ethyl-guanineN7)]+ also shows Ru−N7(guanine) bonding.48
Figure 4. Effect of 72 h exposure to Ru-Cur and cisplatin (CDDP) in
platinum-sensitive (A2780, empty bars) and -resistant (CP8, filled
bars) human ovarian cancer cells. Means ± SE of 4−5 independent
experiments. ** p < 0.01 vs A2780.
Table 2). This observation fits nicely with what has been
reported above about the effects of cisplatin and Ru-Cur on
HCT116 cells. Mismatch repair defects are among the major
mechanisms involved in acquired resistance to cisplatin in
A2780 cells;45 thus, the observation that Ru-Cur is about as
potent on cisplatin-sensitive as on cisplatin-resistant A2780
cells supports the notion that its activity is independent of
mismatch repair status.
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attractive ligand for metal coordination, so far almost
unexplored. Our Ru−curcuminato complex also shows good
antitumor activity in breast MCF7 (19.58 ± 2.367 μM) and
ovarian A2780 (23.38 ± 3.334 μM) cell lines, while human
glioblastoma U-87 and lung carcinoma A549 are less sensitive.
This indication of selectivity is considered a positive feature for
further development. A docking study of the title compound on
a guanine-rich DNA decamer shows metal interaction with
N7−guanine and O(phosphate); the former supported
experimentally by many studies, including this work. The
Ru−N7 binding is supported also by the fact that Ru−Cl bond
length of Ru curcuminates seems to be longer than the other
related structures in the database suggesting its weakeness. A
histogram of these Ru−Cl bond distances is depicted in
Supporting Information Figure S1, where the red hit belongs to
one of the two molecules in the asymmetric unit of the
dimethoxycurcumin derivative, shown in Table 1 [Ru−Cl
2.435(1)].36 The Pt chemotherapy mechanism of action has
established Pt−N7(guanine) as necessary for antitumor activity.
In addition, a strong deformation of curcumin planarity, similar
to that found for curcumin itself in the literature,49 is apparent
in the solid state structure of the title compound, and its
interaction with DNA. Using 9-ethylguanine as a model for
guanosine, the metal bonding with N7(guanine) in the
ruthenium complexes of curcumin and its demethoxylated
derivative was validated by ESI-MS studies. These results
further demonstrate that the presence or absence of peripheral
hydroxy or methoxy groups in curcumin do not greatly
influence the interaction of the metal with the β-diketone
moiety. This feature is promising for making suitable chemical
modifications of the ligand to achieve higher biological activity
and lower toxicity. The present study focuses on DNA
interaction because previous studies indicate that interaction
with DNA bases is highly probable and our results fit such
expectation. However, interaction with other targets such as
proteins has also been demonstrated50,51 and should not be
excluded for the title compound.
Figure 6. Minimization of conformer no. 155, from a standard
dynamic cascade where all nucleotides were not fixed (except those
terminal), showing a good approach of N7(guanine-“A”) to the metal
(2.824 Å). Only one pair of complementary nucleotides of the
receptor decamer C−G are shown, Guanosine6 (“A”) and its paired
Cytosine5 (“S”), labeled DG6 and DC5. The setting of this
minimization was the same for the standard dynamic cascade protocol,
shown in the Experimental Section; such process holds the complete
H-bond pairing, as shown. The Ru---O(phosphate) separation of 4.852
Å is also shown for later discussion.
■
Figure 7. Minimization of conformer no. 351, from a standard
dynamic cascade where only terminal nucleotides were fixed, showing
a potential interaction between Ru and O(phosphate) (2.390 Å). This
minimization process holds the H-bond pairing as depicted.
EXPERIMENTAL SECTION
Synthesis and Characterization. Samples for microanalysis were
dried in vacuo to constant weight (20 °C, about 0.1 Torr). Elemental
analyses (C, H) were performed in house with a Fisons Instruments
1108 CHNS-O elemental analyzer. Electrical conductivity measurements of solutions of the complexes were taken with a Crison CDTM
522 conductimeter at room temperature. IR spectra were recorded
from 4000 to 100 cm−1 with a Perkin-Elmer System 2000 FTIR
instrument. 1H NMR spectra were recorded with a VXR-300 Varian
spectrometer. Melting points were determined with an IA 8100
Electrothermal instrument. Positive and negative electrospray mass
spectra were obtained with a Series 1100 MSI detector HP
spectrometer. Solutions (3 mg/mL) were used for electrospray
ionization mass spectrometry (ESI-MS) and data, mass, and intensities
were compared to those calculated using IsoPro Isotopic Abundance
Simulator, version 2.1.16. Elemental combustion analyses of both
compounds confirmed >95% purity.
The commercial natural product curcumin, from Sigma-Aldrich,
which is a mixture of curcumin, demethoxycurcumin (d-curcH), and
bis(demethoxy)curcumin (bd-curcH), was dissolved (0.368 g, 1
mmol) in methanol (20 mL) and NaOMe (0.054 g, 1 mmol)
added. After 1 h stirring at room temperature, [(p-cymene)RuCl2]2
(0.306 g, 0.5 mmol) was added. The resulting orange solution was
stirred at reflux for 24 h. Then solvent was removed in vacuo, the
residue redissolved in dichloromethane (10 mL), and the mixture was
filtered to remove sodium chloride. The orange solution was
concentrated (2 mL) and an orange precipitate afforded, which was
We suggest that, as no experimental evidence exists, the Ru−
O(phosphate) interaction, although not absolutely excluded,
could be a consequence of the lack of appropriate Ru functions
in the molecular mechanics algorithm employed during the
docking technique. This is also suggested by the experimentally
Ru−N7 guanine derivative detected in this work using ESI-MS,
see above.
■
CONCLUSIONS
Cisplatin and other Pt antitumor drugs are well established in
the treatment of testes, ovarian, and lung cancer, but thus far,
other tumors resist treatment by metal complexes. Gastrointestinal tumors are increasingly of concern in humans, and
the title compound shows excellent activity on the colon−rectal
tumor cell line HCT116, IC50 = 13.98 ± 1.503 μM. This Ru[II]
complex contains the natural product curcumin as a βdiketonato ligand, which has been used extensively as a food
component in Asian cultures and as ethnic medicine; its
recognized benefits for health makes this natural product an
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separated, dried under a vacuum, and shown by ESI-MS to be a
mixture of (p-cymene)ruthenium derivatives, in detail [(p-cymene)Ru(curcuminato)Cl], [(p-cymene)Ru(d-curcH)Cl], and [(cymene)Ru(db-curc)Cl]. ESI-MS (+) CH3OH (m/z, relative intensity %): 603
[100] [(p-cymene)Ru(curcuminato)]+, 573 [20] [(cymene)Ru(dcurc)]+, 543 [5] [(p-cymene)Ru(db-curc)]+. ESI-MS (−) CH3OH
(m/z, relative intensity %): 367 [100] [curcuminato]−, 337 [20] [dcurc]−, 307 [5] [bd-curc]−. Hence, purification through preparative
TLC was carried out by using a mixture of chloroform/methanol in
9:1 ratio as eluent.
[(p-Cymene)Ru(curc)Cl] (1), Ru-Cur. The three fractions were
separated, and recrystallization in CHCl3 at 4 °C slowly yielded
orange−red crystals, which were identified as the pure compound 1;
mp 197−198 °C. Λm (CH3OH, 298 K, 10−3 mol/L): 22 S cm2 mol−1.
Λm ((CH3)2SO, 298 K, 10−3 mol/L): 2 S cm2 mol−1. IR (nujol, cm−1):
3220 m br ν(OH), 1619 m, 1591s, 1500vs ν(CO, CC). 1H NMR
(CDCl3, 293K): δ, 1.39 (d, 6H, CH(CH3)2 of p-cymene), 2.34 (s, 3H,
CH3 of p-cymene), 2.98 (m, 1H, CH(CH3)2 of p-cymene), 3.93 (s,
6H, OCH3 of curc), 5.45 (s, 1H, C(1)H of curc), 5.55br, 5.84br (4H,
AA′BB′ system, CH3-C6H4-CH(CH3)2 of p-cymene), 6.42 (d, 2H, C(3,
3′)H of curc, 3Jtrans = 15 Hz), 6.91 (br, 2H, C(9, 9′)H of curc), 7.00
(br, 4H, C(10, 10′)H and C(6, 6′)H of curc), 7.52 (d, 2H, C(4, 4′)H of
curc, 3Jtrans = 16 Hz). 13C{1H} NMR (CDCl3, 293K): δ, 18.3 (s, CH3C6H4-CH(CH3)2), 22.7 (s, CH3-C6H4-CH(CH3)2), 31.1 (s, CH3C6H4-CH(CH3)2), 56.1 (s, O-CH3 of curc), 79.3, 83.2, 97.8, 99.8 (s,
CH3-C6H4-CH(CH3)2), 102.1 (s, C(1, 1′) of curc), 109.3 (s, C(6, 6′)
of curc), 114.9 (s, C(9, 9′) of curc), 122.7 (s, C(10, 10′) of curc), 125.6
(s, C(5, 5′) of curc), 128.7 (s, C(3, 3′) of curc), 138.9 (s, C(4, 4′) of
curc), 146.9 (s, C(7, 7′) of curc), 147.3 (s, C(8, 8′) of curc), 178.5 (s,
C(2, 2′)O of curc).
[(p-Cymene)Ru(d-curc)Cl] (2). Another fraction of the previous
separation procedure was identified as the pure compound 2. It is
soluble in alcohols, acetone, acetonitrile, DMSO, and chlorohydrocarbon solvents; mp 196−197 °C. Λm (CH3OH, 298 K, 10−3 mol/L):
19 S cm2 mol−1. Λm ((CH3)2SO, 298K, 10−3 mol/L): 3 S cm2 mol−1.
IR (nujol, cm−1): 3225 m br ν(OH), 1619 m, 1590s, 1503vs ν(CO,
CC). 1H NMR (CD3OD, 293K): δ, 1.40 (d, 6H, CH(CH3)2 of pcymene, 4J = 7 Hz), 2.32 (s, 3H, CH3 of p-cymene), 2.92 (m, 1H,
CH(CH3)2 of p-cymene), 3.90 (s, 3H, OCH3 of d-curc), 5.59d, 5.75d
(4H, AA′BB′ system, CH3-C6H4-CH(CH3)2 of p-cymene, 3J = 6 Hz),
5.65 (s, 1H, C(1)H of d-curc), 6.64 (d, 2H, C(3, 3′)H of d-curc, 3Jtrans
= 18 Hz), 6.89 (d, 2H, C(9, 9′)H of d-curc, 3J = 8 Hz), 7.15 (d, 1H,
C(6)H of d-curc, 3J = 8 Hz), 7.23 (s, 1H, C(6′)H of d-curc), 7.23 (s,
1H, C(6′)H of d-curc), 7.53 (d, 2H, C(4, 4′)H of d-curc, 3Jtrans = 8
Hz), 7.61 (d, 1H, C(7)H of d-curc, 3J = 8 Hz), 7.67 (d, 4H, C(10, 10′)
H of d-curc, 3J = 8 Hz). 13C{1H} NMR (CD3OD, 293K): δ, 18.6 (s,
CH3-C6H4-CH(CH3)2), 22.5 (s, CH3-C6H4-CH(CH3)2), 31.9 (s,
CH3-C6H4-CH(CH3)2), 56.5 (s, O-CH3 of d-curc), 79.7, 82.2, 99.0,
100.7 (s, CH3-C6H4-CH(CH3)2), 102.6 (s, C(1, 1′) of d-curc), 107.4
(s, C(6, 6′) of d-curc), 116.7 (s, C(9, 9′) of d-curc), 124.2 (s, C(10,
10′) of d-curc), 125.3 (s, C(5, 5′) of d-curc), 130.5 (s, C(3, 3′) of dcurc), 137.2 (s, C(4, 4′) of d-curc), 145.3 (s, C(7, 7′) of d-curc), 147.0
(s, C(8, 8′) of d-curc), 177.8 (s, C(2, 2′)O of d-curc).
Diffraction Study. Suitable crystals for X-ray diffraction data were
obtained by dissolving the samples in a mixture of 1:1 dichloromethane/ethanol solutions and on standing at room T for a week.
Data were collected at 125K using a Bruker SMART APEX II CCD Xray diffractometer. Structure resolution and refinement were
performed with SHELXTL,52 details are included in Table 3. H
atoms were calculated and constrained as riding on their bound atoms.
CCDC833577 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.
Theoretical Study. The theoretical study involved calculations
using software programs from Accelrys.53 Density functional theory
code DMol3 was applied to calculate energy, geometry, and
frequencies implemented in Materials Studio 5.5 (PC platform).54
We employed the double numerical polarized (DNP) basis set that
includes all the occupied atomic orbitals plus a second set of valence
Table 3. Crystal Data and Structure Refinement for (pCymene)Ru(curcuminato)chloro·0.5H2O
empirical formula
formula weight
temperature
wavelength
crystal system
space group
unit cell dimensions
volume
Z
density (calcd)
absorption coefficient
F(000)
crystal size
θ range for data
collection
index ranges
reflns collected
independent reflections
completeness to θ =
19.61°
absorption correction
max and min
transmission
refinement method
data/restraints/
parameters
goodness-of-fit on F2
final R indices
[I > 2σ(I)]
R indices (all data)
largest diff. peak and
hole
C31H32ClO6Ru, 0.5H2O
1292.19
125(2)K
0.71073 Å
orthorhombic
Pbcn
a = 23.036(7) Å
b = 11.367(4) Å
c = 22.354(7) Å
5853(3) Å3
8
1.466 mg/m3
0.671 mm−1
2656
0.34 × 0.26 × 0.06 mm3
2.00−19.61°
−21 ≤ h ≤ 21, −10 ≤ k ≤ 10, −21 ≤ l ≤ 21
32685
2580 [R(int) = 0.1308]
99.8%
empirical
0.9609 and 0.8041
full-matrix least-squares on F2
2580/82/208
1.082
R1 = 0.0838, wR2 = 0.1921
R1 = 0.1268, wR2 = 0.2184
1.023 and −0.562 e·Å−3
atomic orbitals, and polarized d-valence orbitals,55 and correlation
generalized gradient approximation (GGA) was applied in the manner
suggested by Perdew−Burke−Ernzerhof (PBE),56 these are the
conditions for the highest-accuracy level in DMol3. The spin
unrestricted approach was exploited with all electrons considered
explicitly. The real space cutoff of 5 Å was imposed for numerical
integration of the Hamiltonian matrix elements. The self-consistentfield convergence criterion was set to the root-mean-square change in
the electronic density to be less than 10−6 electron/Å3. The
convergence criteria applied during geometry optimization were 2.72
× 10−4 eV for energy and 0.054 eV/ Å for force.
Cell Lines and in Vitro Culture Conditions. The cell lines
MCF7 (HTB-22, human breast adenocarcinoma), HCT116 (CCL247, human colorectal carcinoma), A549 (CCL-185, human lung
carcinoma), and U-87 MG (HTB-1, human glioblastoma) were
obtained from ATCC (American Type Culture Collection, Manassas,
VA, USA); A2780 human ovarian carcinoma were obtained from
ECACC (European Collection of Animal Cell Culture, Salisbury,
U.K.). They were maintained under standard culture conditions (37
°C; 5% CO2) in DMEM medium (Euroclone, Milan, Italy),
supplemented with 10% fetal calf serum (Euroclone, Milan, Italy),
1% glutamine, and 1% antibiotics mixture; for HCT116 and U-87 MG
cells, 1% sodium pyruvate and 1% nonessential amino acids (both
from Sigma-Aldrich, Milan, Italy) were also added to the culture
medium. CP8 cells (so-called because of their ability to grow in
medium containing 8 μM cisplatin), developed by chronic exposure of
the parental A2780 cisplatin-sensitive line to increasing concentrations
of cisplatin, were obtained from Dr. R. Ozols (Fox Chase Cancer
Center, Philadelphia, PA).57 MCF-10A (a cell line derived from
human mammary epithelium, spontaneously immortalized) was
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obtained from ATCC and maintained in a 1:1 mixture of Ham’s F12
and DMEM (Euroclone, Milan, Italy), supplemented with 5% horse
serum and 1% penicillin/streptomycin (Euroclone, Milan, Italy) and
with 0.5 mg/mL hydrocortisone, 10 μg/mL insulin,, and 20 ng/mL
recombinant human EGF (all three from Sigma-Aldrich, Milan, Italy).
All experiments were performed within 10 passages from thawing.
Drugs. The title compound was reconstituted in sterile DMSO at a
concentration of 1 M; stock solutions were then diluted to the desired
final concentrations with sterile complete medium immediately before
each experiment. The final DMSO concentration never exceeded
0.2%, which was not toxic to the cells under the drug exposure
conditions used in this study.
Growth Inhibition Assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was performed on all the
cell lines tested as described58 with minor modifications. Briefly,
according to the growth profiles previously defined for each cell line,
adequate numbers of cells were plated in each well of a 96-well plate in
0.1 mL of complete culture medium and allowed to attach for 24 h.
Cells were exposed at 37 °C for 72 h to the title compound at
concentrations ranging between 5 and 750 nm, bringing the final
volume in each well to 0.2 mL. Each experiment included 8
replications per concentration tested; control samples were run with
0.2% DMSO. At the end of the period of incubation with the title
compound, MTT (0.05 mL of a 2 mg/mL stock solution in PBS) was
added to each well for 3 h at 37 °C. Cell supernatants were then
carefully removed, the blue formazan crystals formed through MTT
reduction by metabolically active cells were dissolved in 0.1 mL of
DMSO, and the corresponding optical densities were measured at 570
nm, using a universal microplate reader EL800 (Bio-TekWinooski,
VT). IC50 values were estimated from the resulting concentration−
response curves by nonlinear regression analysis, using GraphPad
Prism software, version 4.03. (GraphPad, San Diego, CA, USA).
Differences between IC50 values were analyzed statistically by analysis
of variance with Bonferroni post-test for multiple comparisons.
Docking on DNA. Docking studies were performed with the
molecular mechanics CDOCKER package in Discovery Studio 3.0
from Accelrys.53 CDOCKER is a grid-based molecular docking
method that employs CHARMm.59 Curcumin was geometry
optimized using the density functional theory (DFT) code DMol3
included in Materials Studio version 5.5 from Accelrys, using the same
setting earlier described for the (p-cymene)Ru(curcuminato)chloro
complex. Initial docking of curcumin was performed on a DNA species
deposited in the Protein Data Bank www.pdb.org (code 1AU7).49
CDOCKER was used to dock curcumin into the double helix. The
simulation was performed using a binding site sphere of radius 12 Å
centered in the minor groove of the receptor. An excellent agreement
was obtained compared to published results as the best docked pose
showed important binding features mostly based on interactions due
to both peripheral moieties of curcumin including its twisted structural
features.49 Solvation of this receptor, including counterions, was
performed and 1610 molecules of water, 22 Na+ and 4 Cl− were
obtained. Therefore, the 18 negative DNA phosphate charges were
balanced with 18 positive charges, making the receptor neutral. The
center of a 12 Å radius sphere, defined as the midpoint between
O(phosphate- cytosine3-“S”) and O(phosphate- cytosine3-“A”), was
located in the major groove. The main purpose of the solvation
protocol was to include counterions, and so these added waters were
later removed. An initial docking of curcumin was performed
requesting 10 poses. In 9 out of 10 poses, the keto−enol curcumin
moiety was not positioned toward the DNA, rather the DNA−
curcumin interaction was based on peripheral O(curcumin) atoms and
curcumin loss of planarity, as indicated above for 1AU7.49 This feature
provides support for our next docking procedure for a Ru−curcumin
complex, e.g.. for the metal coordinated to the keto−enol moiety and
not, initially, involved in DNA binding while preserving peripheral
interactions of curcumin with DNA. Additional docking for 100 poses
was performed, confirming such statistics. Pose 21, suggested a
potential H(enol)−N7(guanine-“A”) approach and was selected. The
complex (p-cymene)Ru(curcuminato)chloro, previously geometry
optimized with DMol3, was curcuminato superimposed onto the
originally docked curcumin, while holding its Ru(p-arene)Cl environment, and the free curcumin was eliminated. Because the CHARMm
force field does not contain bonding parameters for Ru, the metal was
treated as an ion (Ru2+). To account for the Ru coordination sphere,
we set (1) a flat-bottomed distance restraint between Ru and the
center of p-cymene ring using a force constant of 200 kcal/(mol·Å2)
and an upper threshold of 1.90 Å, so that a closely related Ru-arene
distance could be kept while allowing p-cymene rotation during
docking; (2) a rigid-body harmonic restraint to Ru−curcuminato
chelate moiety using a force constant of 10 kcal/(mol·Å2), conserved
planarity of the 6-membered Ru−curcuminato ring, with one O atom
having a negative charge (anion) and the other set as a neutral
carbonyl; (3) the Cl as an anion. Throughout this process, we wanted
to be sure that the approach of the Ru complex to the DNA decamer
receptor was not chemically affected before entering the groove.
Subsequently, we eliminated DNA counterions and performed a new
solvation-counterion generation, eliminated all water molecules and
performed a minimization of this counterion-decamer-Ru-complexpose-21 system, having the receptor and counterions fixed.
Conditions for minimizations were as follows: The Smart Minimizer
algorithm performed 1000 steps of Steepest Descent with a rms
gradient tolerance of 3, followed by 1000 steps of Conjugate Gradient
minimization with a rms gradient tolerance of 0.1. This simulation
used a Distance-Dependent Dielectric Constant of 4, and all other
parameters were default conditions.
This showed Ru−Cl of 2.72 Å, Ru−O(anionic) 2.158 Å, Ru−
O(carbonyl) 2.250 Å, and Ru−centroid 2.028 Å; potential energy was
−7.9 kcal/mol, electrostatic energy, −85.6 kcal/mol, and vdW energy
11.0 kcal/mol. The same constraints were applied to the standard
dynamic cascade protocol shown in Supporting Information Figure S2.
Conditions were set as follows: a first minimization used the Steepest
Descent algorithm with 500 steps and rms gradient tolerance of 0.1. A
second minimization used a Conjugate Gradient algorithm of 500
steps and rms gradient of 0.0001. The heating process used 100000
steps of time step 0.001, initial temperature was 50K, and target
temperature was 300K. The equilibration process used 10000 steps
with time step 0.001 and target temperature of 300K. The production
process was of 500000 steps, equivalent to 0.5 ns, with time step 0.001
and target temperature of 300K; the production stage was carried out
under an NVT setting, which is a constant temperature dynamics using
Berendsen weak coupling method. The simulation used a distancedependent dielectrics with a dielectric constant of 4; all other
parameters were default conditions. Supporting Information Figure S3
shows conformation no. 60, which has the lowest energy. A similar
standard dynamic cascade was performed for the receptor having more
flexibility: (1) DNA with all bases free, excepted both terminal pairs,
and all phosphate and ribose fixed; (2) Ru(II) cation; (3) Ru−centroid
distance restraint with force constant (100 kcal/(mol·Å2)) and
threshold (1.90 Å); (4) Ru−curcuminato chelate, harmonic restraint,
best-fit, force constant (10 kcal/(mol·Å2)), with one O atom
negatively charged (anion) and the other as a carbonyl; (5) Cl as an
anion taken out of the coordination sphere (Ru---Cl = 8.9 Å).
Supporting Information Figure S4 shows the pattern of potential
energy vs conformations. We selected conformer no. 155 as a good
candidate for further study, Supporting Information Figure S5. Its
minimized structure (Figure 6) shows a good approach of Ru to
N7(guanine)(2.824 Å). Comparing Supporting Information Figure S5
with Supporting Information Figure S3 one can see that one H-bond
between bases is lost, in the former, due to the greater flexibility
imposed.
Because some conformers show also some potential approach for
O(phosphate) of guanine6 (“A”) we also investigated this feature. A
longer simulation (1 ns) was performed, and the resulting 500
conformations are shown in Supporting Information Figure S6. We
focused on conformer no. 351 (Supporting Information Figure S7),
selected it, and performed a minimization (Figure 7). The energy of
both potential candidates of interaction for the Ru-complex with this
DNA decamer, Ru−N7 bond (Figure 6) and Ru−O(phosphate)
(Figure 7), were obtained using the same condition of minimizations,
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they are 263.8 and 263.4 kcal/mol, respectively. Such a small
difference cannot discriminate between these conformers.
■
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ASSOCIATED CONTENT
S Supporting Information
*
Histogram of the Ru−Cl bond length from the CSD data.
Docking plot of potential energy vs time for the production
stage for the Ru-complex on a B DNA C-G decamer.
Conformation no. 60 from Docking. Potential energy vs
conformations, showing conformation no. 155. Selected atoms
for conformation no. 155 from previous cascade. Standard
dynamic cascade search for potential Ru−O(phosphate)
interaction. Conformer no. 351, showing potential Ru−
O(phosphate) interaction. Dose-dependence of the title
compound on 5 tumor cell lines. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*(F.C.) Phone: 39 06 49913632. E-mail: caruso@vassar.edu.
(M.R.) Phone: 1 845 4377134. E-mail: rossi@vassar.edu.
■
ACKNOWLEDGMENTS
Financial support by Università degli Studi di Camerino,
Consiglio Nazionale delle Ricerche CNRRome, Research
Committee and URSI program at Vassar College. M.R. thanks
the U.S. National Science Foundation, through grant 0521237
for the X-ray diffractometer, and Howard Hughes Medical
Institute for grant 52006322.
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ABBREVIATIONS USED
DFT, density functional theory; ESI-MS, electron spray
resonance mass spectrometry; Ru-Cur, (1), (p-cymene)Ru(curcuminato)chloro; Ru-dim-Cur, (p-cymene)Ru(dimethoxycurcuminato)chloro; curcH, curcumine; d-curcH,
demethoxycurcumin; bd-curcH, bis-demethoxycurcumin
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