A potent ruthenium(II) antitumor complex bearing a lipophilic levonorgestrel group

Article abstract of DOI:10.1021/ic201388n

The novel steroidal conjugate 17-α-[2-phenylpyridyl-4-ethynyl]-19- nortestosterone (LEV-ppy) (1) and the steroid-C,N-chelate ruthenium(II) conjugate [Ru(η⁶-p-cymene)(LEV-ppy)Cl] (2) have been prepared. At 48 h incubation time, complex 2 is more

Full text of DOI:10.1021/ic201388n

ARTICLE
pubs.acs.org/IC
A Potent Ruthenium(II) Antitumor Complex Bearing a Lipophilic
Levonorgestrel Group
Josꢀe Ruiz,*,† Venancio Rodríguez,† Natalia Cutillas,† Arturo Espinosa,‡ and Michael J. Hannon§
†Departamento de Química Inorgꢀanica and ‡Departamento de Química Orgꢀanica, Universidad de Murcia, 30071, Murcia, Spain
§School of Chemistry, University of Birmingham, Edgbaston, B15 2TT, U.K.
S Supporting Information
b
ABSTRACT: The novel steroidal conjugate 17-R-[2-phenylpyridyl-
4-ethynyl]-19-nortestosterone (LEV-ppy) (1) and the steroidꢀC,N-
chelate ruthenium(II) conjugate [Ru(η6-p-cymene)(LEV-ppy)Cl]
(2) have been prepared. At 48 h incubation time, complex 2 is more
active than cisplatin (about 8-fold) in T47D (breast cancer) and also
shows an improved efficiency when compared to its nonsteroidal
analogue [Ru(η6-p-cymene)(ppy)Cl] (ppy = phenylpyridine) (3) in
the same cell line. The act of conjugating a levonorgestrel group to a
ruthenium(II) complex resulted in synergistic effects between the
metallic center and the steroidal ligand, creating highly potent
ruthenium(II) complexes from the inactive components. The inter-
action of 2 with DNA was followed by electrophoretic mobility.
Theoretical density functional theory calculations on complex 2
show the metal center far away from the lipophilic steroidal moiety and a labile RuꢀCl bond that allows easy replacement of Cl
by N-nucleophiles such as 9-EtG, thus forming a stronger RuꢀN bond. We also found a minimum energy location for the chloride
counteranion (4+ Clꢀ) inside the pseudocavity formed by the R side of the steroid moiety, the phenylpyridine chelating subsystem,
3
and the guanine ligand, i.e., a hostꢀguest species with a rich variety of nonbonding interactions that include nonclassical
CꢀH anion bonds, as supported by electrospray ionization mass spectra.
3 3 3
’ INTRODUCTION
On the other hand, targeting the drugs to specific organs or
tumor types is also desirable as is maximizing the delivery of the
cytotoxic agent into the cell and onto the (nuclear) DNA.19
Thus, natural and synthetic estrogens and androgens have been
attached to a range of different organometallic and coordination
units with the aim of targeting the steroidal receptors.20ꢀ22
Jaouen has demonstrated that organometallics could be attached
to the 17R-position of estradiol, preserving some estrogen
receptor binding ability.23,24 An ethynyl group was a successful
linker, as it provided adequate separation between the steroid
and the molecule. Yet estrogen is not necessarily the most
interesting of the hormone steroids. Thus, Quiroga et al. have
recently shown that the conjugation of the testosterone confers
relatively high activity to otherwise nonactive platinum(II)
complexes.25,26 Some anticancer organoplatinum(II) bioconju-
gates of the type [Pt(ET-dmba)Cl(L)] (L = dimethyl sulfoxide
(DMSO) and PTA; dmba = dimethylbenzylamine) with an
ethisterone tag (ET) have been reported by us.27
The discovery of cisplatin has stimulated huge efforts to
develop new anticancer platinum complexes.1ꢀ3 Cisplatin
(Platinol), carboplatin (Paraplatin), and recently oxaliplatin have
received worldwide approval for clinical uses,1ꢀ6 with annual
sales over $2 billion (USD).7,8 These agents are believed to act by
binding to DNA, with the three structurally related drugs having
similar molecular-level actions. The clinical use of cisplatin,
however, has a number of drawbacks, including neurotoxicity,
nephrotoxicity, acquired or inherent resistance in some cancer
cells, and a limited spectrum of activity against different types of
cancer. For these reasons, the potential of other transition metal-
based agents as anticancer agents is being actively explored.9,10
Thus, two ruthenium(III) based drugs, KP101911 and NAMI-A12
(Scheme 1a and 1b, respectively), have currently completed
phase I clinical trials and are either in or set to enter phase II
trials. Organoruthenium complexes of the type [Ru(η6-arene)-
(en)Cl]+ (arene = e.g., biphenyl- or tetrahydroanthracene;
en = ethylenediamine) show promising in vitro and in vivo activity,
including toward cisplatin-resistant cell lines (Scheme 1c).13ꢀ15
It should be noted that the anticancer properties of ruthenium
complexes of the type [Ru(η6-arene)Cl2(PTA)] (PTA = 1,3,
5-triaza-7-phosphatricyclo-[3.3.1.1]decane) are also currently
attracting considerable attention.16ꢀ18
We report herein the synthesis of the novel steroidal conjugate
17-R-[2-phenylpyridyl-4-ethynyl]-19-nortestosterone (LEV-ppy)
(1) and the steroidꢀC,N-chelate ruthenium(II) conjugate
Received: June 29, 2011
Published: August 10, 2011
r
2011 American Chemical Society
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|

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ARTICLE
Scheme 1. Structures of Some Ruthenium Drugs
Scheme 2
[Ru(η6-p-cymene)(LEV-ppy)Cl] (2). Values of IC50 were stu-
died for the new ruthenium complexes against a panel of human
tumor cell lines representative of ovarian (A2780 and
A2780cisR) and breast cancers (T47D, cisplatin resistant and
AR+; AR = androgen receptor). At 48 h incubation time, complex 2
is more active than cisplatin (about 8-fold) in T47D (breast cancer)
and shows an improved efficiency when compared to its non-
steroidal analogue [Ru(η6-p-cymene)(ppy)Cl] (ppy = C6H4-2-
C5H4N-k-C,N) (3) in the same cell lines. The DNA adduct
formation of 2 has been studied by electrophoretic mobility, and
the reaction of 2 with the model nucleobase 9-EtG has been studied
by means of both ESI-MS and density functional theory (DFT).
NOESY, distortionless enhancement by polarization transfer
(DEPT) and HSQC. The previously reported28 nonsteroid
phenylpyridine ruthenium complex [Ru(η6-p-cymene)(ppy)Cl]
(3) has also been prepared (Scheme 2) in order to compare their
biological properties in the same cell lines.
’ RESULTS AND DISCUSSION
Molecular Design. Our aim was to design a levonorgestrel-
linked complex and explore the effect of this conjugation on the
activity of nonconventional ruthenium(II) drugs. Our design has
three features: a nortestosterone derivative moiety, a linker, and a
ruthenium(II) center. Linking the steroid and metal complex via
an alkyne linker is attractive due to its synthetic feasibility and
because, as a spacer unit, it introduces distance without steric
bulk. As shown in Scheme 2, the use of the commercially available
levonorgestrel (17R-ethynyl-19-nortestosterone) and 4-bromo-
2-phenylpyridine as starting materials allowed us to prepare in
very good yield the functionalized 1 in a palladium-catalyzed
Sonogashira coupling. 1 is an air- and moisture-stable white
compound. 1H and 13C NMR, electrospray ionization (ESI) mass
(positive mode) analysis spectrometry (M + H+ = 466.4), and
elemental analysis were used to characterize 1. Correlation spectro-
scopy (COSY), nuclear Overhauser effect spectroscopy (NOESY),
and heteronuclear single quantum coherence (HSQC) were used
for the assignment. In the 1H NMR, the resonances that are easily
observed correspond to the aromatic region of the ppy fragment
and correspond to the Ha, which appears at δ 5.8 ppm. They are
in the appropriate integral ratios. Furthermore, the ethynyl
proton (δ 2.6 ppm) from the starting steroid is absent.
Theoretical Calculations on Complex 2. Further insight
regarding the structure and binding in the above-mentioned
complex 2 has been extracted from DFT calculations (see
Computational Details). The initial geometry was made up by
assembling the RuII(η6-p-cymene)(ppy)Cl core, obtained after
optimization at the current level of the reported X-ray
structure,29 and the steroid unit, which was directly derived from
the ethisterone (17-R-ethynyl-testosterone) fragment whose
substructure was described in steroidꢀC,N-chelate platinum(II)
conjugates recently reported by us.27 The conformational space
was checked by rotation around the CꢀC triple bond axis to
ensure the conformational minimum. The diastereomer with
epimeric configuration at the Ru(II) chiral center was found to be
0.52 kcal/mol less stable at its conformational minimum in the
potential energy surface at the working level of theory (Figure S1,
Supporting Information). The resulting most stable structure
(Figure 1) features the metal center far away from the lipophilic
steroidal moiety (distance from the Ru atom to its projection into
the 17-carbons tetracyclic mean plane, 8.446 Å). The Ru atom is
surrounded by four different donor atoms (Do). Assessment of
RuꢀDo bond strengths has been achieved by means of fre-
quently-used bond strength descriptors, such as the Mayer’s
bond order (MBO)30 as well as within the framework of Bader’s
atoms-in-molecules (AIM) theory,31 by computing the electron
density at the respective bond critical points (BCPs). According
to these criteria, the strongest bond to ruthenium is the
Design and Synthesis of the New Ruthenium Compound.
The new ruthenium(II) monomer [Ru(η6-p-cymene)(LEV-
ppy)Cl] (2) was synthesized through an adaptation of the Davies
route28 by stirring, at room temperature, [Ru(p-cymene)Cl2]2
with 2 equiv of 1 for 24 h in dichloromethane in the presence of
NaOAc (Scheme 3). The structure was assigned on the basis of
1
microanalytical, IR, and H and 13C NMR data and ESI mass
η6-interaction to the p-cymene ligand (dRu arene = 1.707 Å;32
spectra. Most of the NMR resonances of complex 2 were
duplicated in CDCl3 solution (see Experimental Section) as a
consequence of the existence of two diastereomers (Figure S1,
Supporting Information) in comparable amounts (see below).
The assignments were unambiguously confirmed by COSY,
3 3 3
ΣMBO = 2.529; ΣF(rc) = 35.35 ꢁ 10ꢀ2 e/a0 ) featuring a high
3
ellipticity (εaver = 1.437 for the four BCPs found), characteristic
of bonds involving π-electron density. Two other coordination
positions around Ru(II) are occupied by the chelate ppy ligand,
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Scheme 3
Table 1. IC50 (μM) and Resistance Factors for Cisplatin and
Compounds 1ꢀ3
compound
T47D 48 h
A2780 48 h
A2780cisR 48 h (RF)a
1
>100
>100
>100
2
7.4 ( 0.1
>100
3.7 ( 0.04
66 ( 1
3.1 ( 0.03 (0.8)
79 ( 1 (1.2)
22 ( 2 (15.7)
3
cisplatin
60 ( 8
1.4 ( 0.05
a The numbers in parentheses are the resistance factors RF (IC50 resistant/
IC50 sensitive).
cisplatin, the free ligand 1, and the nonsteroidal ruthenium
complex 3 was also evaluated under the same experimental
conditions. Because of low aqueous solubility, the test com-
pounds were dissolved in DMSO first and then serially diluted in
complete culture medium, such that the effective DMSO content
did not exceed 1%. Complex 2 was more active than cisplatin in
T47D (about 8-fold), with the antiproliferative activity of the
nonsteroidal compound 3 being much poorer (IC50(μM) > 100)
than that of 2. On the other hand, A2780cisR encompasses all of
the known major mechanisms of resistance to cisplatin: reduced
drug transport,33 enhanced DNA repair/tolerance,34 and ele-
vated glutathione levels.35 The ability of complex 2 to circumvent
acquired resistance of cisplatin was determined from the resis-
tance factor (RF), defined as the ratio of IC50 (resistant line) to
IC50 (parent line), a very low RF value being observed at 48 h
(RF = 0.8, Table 1). An RF of <2 was considered to denote non-
cross-resistance.36 The IC50 value for the free ligand 1 was higher
than 100 in all the cancer cell lines studied.
Figure 1. Calculated (RI-BP86/def2-TZVP) most stable structure for
complex 2.
which forms a strong bond with the C atom (dRuꢀC = 2.029 Å;
3
MBO = 0.941; F(rc) = 13.86 ꢁ 10ꢀ2 e/a0 ) and a moderate
bond with the N atom (dRuꢀN = 2.061 Å; MBO = 0.702; F(rc) =
10.53 ꢁ 10ꢀ2 e/a0 ). The Cl ligand lies almost orthogonal to the
3
chelate ring (NꢀRuꢀCl 87.0°, CꢀRuꢀCl 88.7°) and forms a
weaker bond to ruthenium (dRuꢀCl = 2.391 Å; MBO = 0.719;
F(rc) = 7.64 ꢁ 10ꢀ2 e/a0 ).
3
Biological Activity: Cytotoxicity Studies. To analyze the
potential of the bioconjugate 2 as an antitumor agent, its
cytotoxicity was evaluated (Table 1) toward the T47D human
breast cancer cell line (cisplatin resistant) and epithelial ovarian
carcinoma cells A2780 and A2780cisR (acquired resistance to
cisplatin), and for comparison purposes, the cytotoxicity of
Biological Assays: Gel Electrophoresis of Compoundꢀ
pBR322 Complexes. The influence of the compounds on the
tertiary structure of DNA was determined by their ability to
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Figure 2. Electrophoretic mobility pattern of pBR322 plasmid DNA
incubated with the following compounds: lane 1, pBR322; lane 2,
compound 1; lane 3, compound 2; lane 4, compound 3; lane 5, cisplatin.
modify the electrophoretic mobility of the covalently closed
circular (ccc) and open (oc) forms of pBR322 plasmid DNA.
The compounds 1ꢀ3 were incubated at the molar ratio ri = 0.50
with pBR322 plasmid DNA at 37 °C for 24 h. A representative gel
obtained for the new compounds 1 and 2 is shown in Figure 2.
The behavior of the gel electrophoretic mobility of both forms,
ccc and oc, of pBR322 plasmid and DNA/cisplatin adducts is
consistent with previous reports.37 When the pBR322 was
incubated with the ruthenium compound 2 (lane 3), a single
footprinting for both forms, ccc and oc, coalescent form, was
observed, indicating that some conformational changes occurred.
This means that the degree of superhelicity of the DNA
molecules has been altered. Noteworthy, both the free ligand 1
and the nonsteroidal ruthenium compound 3 do not seem to
modify the tertiary structure of DNA.
Reactions of the Ruthenium Complex 2 with 9-Ethylgua-
nine. To gain further insight into the mechanism of the inter-
action of the new ruthenium complex with DNA, a DFT study
was undertaken. Reaction of the model nucleobase 9-ethylgua-
nine (9-EtG) with 2 displaces the weakly linked chloride ligand
and affords the cationic complex [Ru(η6-p-cymene)(LEV-ppy)-
(9-EtG)]+ (4+) (see the Supporting Information) as the most
stable stereoisomer. The conformer obtained by rotation of the
9-EtG group around the RuꢀN7 bond was found to be 9.21 kcal/
mol less stable at the working level of theory.
Figure 3. Calculated (RIJCOSX-X3LYP/def2-TZVP) most stable
structure for complex 4+ Clꢀ.
3
(dH ring-mean-plane = 2.473 Å; MBO < 0.1; F(rc) = 1.23 ꢁ 10ꢀ2
3 3 3
e/a0 ; ε = 1.294). The Clꢀ anion displays anion-π type
3
interactions39 with both the six-membered pyrimidine
(dCl
= 3.164 Å; MBO = 0.111; F(rc) = 0.89 ꢁ
ring-mean-plane
3 3 3
10ꢀ2 e/a0 ; ε = 2.454) and pyridine rings (dCl
=
3
ring-mean-plane
3 3 3
3
3.222 Å; MBO = 0.117; F(rc) = 1.02 ꢁ 10ꢀ2 e/a0 ; ε = 0.401),
presumably cooperatively giving rise to hydrogen bonds40 with
two H atoms at the steroid inner (R) face (dCl H = 2.628 and
3 3 3
2.708 Å; ΣMBO = 0.260; ΣF(rc) = 2.32 ꢁ 10ꢀ2 e/a0 ) and a
3
terminal H atom on the 9-EtG ethyl group (dCl H = 2.894 Å;
3 3 3
MBO < 0.1; F(rc) = 0.74 ꢁ 10ꢀ2 e/a0 ). All noncovalent
3
interactions were evidenced and further analyzed as real-space
isosurfaces by means of the recently implemented NCIplot
software41 (see the Supporting Information and Figure S2).
Experimental evidence of the possibility for the formation of a
9-EtG monoadduct came from the ESI-MS analysis of the
mixture obtained after incubating 9-ethylguanine with 2 (5:1
ratio) in H2O/DMSO at 37 °C (Figure 4). The cationic [Ru(η6-
p-cymene)(LEV-ppy)(9-EtG)]+ (4+), its neutral complex with
the chloride counteranion (4+ Clꢀ), and the cationic species
3
[Ru(η6-p-cymene)(LEV-ppy)(DMSO)]+ (5+) were observed
(see inset of Figure 4).
After a thorough search in the gas-phase potential energy
surface of 4+, we found a minimum energy location for the
chloride counteranion (4+ Clꢀ) inside the pseudocavity formed
’ CONCLUSIONS
3
by the R side of the steroid moiety, the phenylpyridine chelating
A novel steroidal conjugate 17-R-[2-phenylpyridyl-4-ethynyl]-
19-nortestosterone (LEV-ppy) (1) and the steroidꢀC,N-chelate
ruthenium(II) complex [Ru(η6-p-cymene)(LEV-ppy)Cl] (2) have
been prepared. The ruthenium bioconjugate 2 was more active
than cisplatin in T47D human breast cancer cell line (about 8-fold),
the antiproliferative activities of both 1 and the nonsteroidal
related compound [Ru(η6-p-cymene)(ppy)Cl] (3) being much
poorer (IC50(μM) > 100). Especially noteworthy is the very low
resistance factor (RF) of 2 at 48 h (RF = 0.8) against an A2780 cell
line that has acquired resistance to cisplatin, indicating efficient
circumvention of cisplatin resistance. Reactions of the new ruthe-
nium complex 2 with 9-ethylguanine, as followed by ESI-MS,
gave the correponding monoadduct to [Ru(η6-p-cymene)(LEV-
ppy)(9-EtG)]+ (4+). The cationic derivative [Ru(η6-p-cymene)-
(LEV-ppy)(DMSO)]+ (5+) and the chloride counteranion
subsystem, and the guanine ligand, i.e., a hostꢀguest complex
with a nonclassical CꢀH anion interaction (see Computa-
3 3 3
tional Details). The resulting energy minimum (Figure 3) shows
a similar bonding environment38 to that in 2, in addition to the
newly formed strong RuꢀN7 bond with the nucleobase model
(dRuꢀN = 2.128 Å; MBO = 0.611; F(rc) = 8.96 ꢁ 10ꢀ2 e/a0 ), the
3
latter being oriented so as to approach the steroid carbonyl group
and giving rise to two moderately strong hydrogen bonds with
the amino (dNH O = 1.880 Å; MBO = 0.129; F(rc) = 3.22 ꢁ
3 3 3
10ꢀ2 e/a0 ) and the ring NH group (dNH O = 2.284 Å; MBO <
3
3 3 3
0.1; F(rc) = 1.18 ꢁ 10ꢀ2 e/a0 ). Such an approximation entails a
3
little distortion at the acetylenic carbon atoms (C—CtC angles
170.1 and 168.4°). In turn, the guanine carbonyl group forms two
additional hydrogen bonds with the p-cymene moiety at either
3
ring (dO HAr = 2.395 Å; MBO < 0.1; F(rc) = 1.23 ꢁ 10ꢀ2 e/a0 )
(4+ Clꢀ) were also observed. Theoretical DFT calculations on
3 3 3
3
and benzylic protons (dO HCAr = 2.433 Å; MBO < 0.1; F(rc) =
complex 2 show the metal center far away from the lipophilic
steroidal moiety and a labile RuꢀCl bond that allows easy replace-
ment of Cl by N-nucleophiles such as 9-EtG, which forms a
3 3 3
1.15 ꢁ 10ꢀ2 e/a0 ), whereas the H at C8 is involved in a T-stacking
3
(edge-to-face) interaction with the phenyl ring at the C,N-chelate
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Figure 4. Experimental ESI mass spectrum of the reaction of complex 2 with 9-ethylguanine in H2O/DMSO after 10 min at 37 °C. Inset: Expanded
spectrum highlighting the chloride inclusion complex 4+ Clꢀ.
3
stronger RuꢀN bond. We also found a minimum energy location
for the chloride counteranion inside the pseudocavity formed by
the R side of the steroid moiety, the phenylpyridine chelating
subsystem, and the guanine ligand, thus yielding a hostꢀguest
levonorgestrel (1.50 g, 4.8 mmol) were mixed with PdCl2(PPh3)2
(182.0 mg, 0.256 mmol), CuI (48.76 mg, 0.256 mmol), and K2CO3
(0.995 g, 7.2 mmol) in a 250 mL Schlenk tube. Dry and degassed tetra-
hydrofuran (80 mL) was added. The resulting suspension was stirred at
65 °C under nitrogen for 72 h, treated with charcoal, and then filtered
through a short pad of Celite. The filtrate was evaporated under reduced
pressure to dryness. The residue was treated with Et2O to give a white solid,
which was collected by filtration, washed with Et2O (2 ꢁ 5 mL), and air-
dried (yield 1.3 g, 87%). Found (%): C, 82.37; H, 7.71; N, 2.96.
C32H35NO2 requires C, 82.54; H, 7.58; N, 3.01. Mp: 305 °C (dec).
1H NMR (400 MHz, CDCl3): δ 8.63 (d, 1H, H6 of pyridyl ring of ppy,
JH5H6 = 5.0 Hz), 7.97 (m, 2H, H2 and H6 of phenyl ring of ppy), 7.70
(s, 1H, H3 of pyridyl ring of ppy), 7.46 (m, 3H, H3, H4, and H5 of phenyl
ring of ppy), 7.21 (dd, 1H, H5 of pyridyl ring of ppy, JH5H6 = 5.0 Hz,
assembly (4+ Clꢀ) that displays a complex pattern of noncova-
3
lent interactions including nonclassical CꢀH anion bonding.
3 3 3
’ EXPERIMENTAL SECTION
Instrumental Measurements. The C, H, N, and S analyses were
performed with a Carlo Erba model EA 1108 microanalyzer. Decom-
position temperatures were determined with a SDT 2960 simultaneous
DSC-TGA of TA Instruments at a heating rate of 5 °C minꢀ1 and the
solid samples under nitrogen flow (100 mL minꢀ1). The 1H, 13C, and 31
P
J
H4H6 = 1.2 Hz), 5.83 (s, 1H, Ha of levonorgestrel), 2.52ꢀ0.85 (m, 22H,
NMR spectra were recorded on a Bruker AV 400 or 600 spectro-
meter, using SiMe4 as standard. ESI mass spectra (positive mode) analyses
were performed on a LC-MS Agilent VL system or HPLC/MS TOF 6220.
Materials. Solvents were dried by the usual methods. [Ru(p-
cymene)Cl2]2, ethylenediaminotetracetic acid (EDTA), and tris-
(hydroxymethyl)-aminomethane-hydrochloride (Tris-HCl) used were
obtained from SigmaꢀAldrich (Madrid, Spain); 4-bromo-2phenylpyr-
idine from Small Molecules, Inc.; Levonorgestrel from OChem, Inc.;
and pBR322 plasmid DNA was obtained from BoehringerꢀMannheim
(Mannheim, Germany).
4 CH and 9 CH2 of levonorgestrel), 1.05 (t, 3H, Me of levonorgestrel,
J
HH =7.6 Hz). 13C{1H} NMR (100.8 MHz, CDCl3): δ 149.4 (CH6 of
pyridyl ring of ppy), 129.4, 128.8 (CH3, CH4, and CH5 of phenyl ring of
ppy), 126.9 (CH2 and CH6 of phenyl ring of ppy), 124.6 (CHa of
levonorgestrel), 123.9 (CH4 of pyridyl ring of ppy), 122.6 (CH3 of
pyridyl ring of ppy), 51.2, 48.9, 42.4, and 40.9 (CH of levonorgestrel),
39.7, 36.5, 35.4, 30.6, 28.9, 26.5, 26.2, 22.6, and 19.0 (CH2 of
levonorgestrel), 9.6 (CH3 of levonorgestrel). ESI+ mass spectra
(CH3CN): m/z = +466.4 [[(LEV-ppyH) + H]+, 32%].
Synthesis of 17-r-[2-Phenylpyridyl-4-ethynyl]-19-nortes-
tosterone (1). 4-Bromo-2-phenylpyridine (0.749 g, 3.2 mmol) and
Synthesis of [Ru(η6-p-cymene)(LEV-ppy)Cl] (2). 1 (155.5 mg,
0.334 mmol), NaOAc (33.50 mg, 0.408 mmol), and [Ru(p-cymene)Cl2]2
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(100 mg, 0.163 mmol) were mixed in a 100 mL Schlenk tube. Freshly
distilled CH2Cl2 (15 mL) was added. The resulting mixture was stirred
at room temperature for 24 h under nitrogen. The solution was filtered
through a short pad of Celite. The filtrate was evaporated to dryness,
and the residue was treated with Et2O to give a brown solid, which
was collected by filtration and air-dried (yield 125 mg, 52%). Found (%):
C, 68.43; H, 6.72; N, 1.79. C42H48ClNO2Ru requires C, 68.60; H, 6.58;
generated with the def2-TZVPP basis set and the Gaussian09 software
package.55 Figures 1 and 3 were generated with VMD.56
Cell Line and Culture. The T-47D human mammary adenocarci-
noma cell line used in this study was grown in RPMI-1640 medium
supplemented with 10% (v/v) fetal bovine serum (FBS) and 0.2 unit/
mL bovine insulin in an atmosphere of 5% CO2 at 37 °C. The human
ovarian carcinoma cell lines (A2780 and A2780cisR) used in this study
were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal
bovine serum (FBS) and 2 mM L-glutamine in an atmosphere of 5%
CO2 at 37 °C.
Cytotoxicity Assay. Cell proliferation was evaluated by assays of
crystal violet. T-47D cells were plated in 96-well sterile plates at a density
of 5 ꢁ 103 cells/well with 100 μL of medium and were then incubated
for 48 h. After attachment to the culture surface, the cells were incubated
with various concentrations of the tested compounds freshly dissolved in
DMSO and diluted in the culture medium (DMSO final concentration
1%) for 48 h at 37 °C. The cells were fixed by adding 10 μL of 11%
glutaraldehyde. The plates were stirred for 15 min at room temperature
and then washed 3ꢀ4 times with distilled water. The cells were stained
with 100 μL of 1% crystal violet. The plates were stirred for 15 min and
then washed 3ꢀ4 times with distilled water and dried. Acetic acid (100
μL, 10%) was added, and the mixture was stirred for 15 min at room
temperature. Absorbance was measured at 595 nm in a Tecan Ultra
Evolution spectrophotometer.
1
N, 1.90. Mp: 194 °C (dec). H NMR (600 MHz, at 25 °C, CDCl3):
δ (SiMe4) 9.13 (d, 2H, H6 of pyridyl ring of ppy, JH5H6 = 2.8 Hz),
8.14 (d, 2H, H3 of phenyl ring of ppy, JH3H4 = 7.2 Hz), 7.66
(s, 1H, H3 of pyridyl ring of ppy), 7.64 (s, 1H, H3 of pyridyl ring of
ppy), 7.59 (d, 1H, H6 of phenyl ring of ppy, JH6H5 = 7.6 Hz), 7.56 (d, 1H,
H6 of phenyl ring of ppy, JH6H5 = 7.6 Hz), 7.17 (m, 2H, H4 of phenyl ring
of ppy), 7.00 (m, 4H, 2H5 of phenyl ring and 2H5 of pyridyl ring of ppy),
5.84 (s, 2H, Ha of levonorgestrel), 5.58 (m, 2H, H5 of p-cymene), 5.55
(m, 2H, H2 of p-cymene), 5.17 (m, 2H, H3 of p-cymene), 5.00 (m, 2H,
H6 of p-cymene), 2.50ꢀ0.85 (m, 44H, 8 CH and 18 CH2 of
levonorgestrel), 2.4 (m, 2H, H8 of p-cymene), 2.03 (s, 6H, H7 of p-
cymene), 1.05 (t, 6H, CH3 of levonorgestrel, JHH = 7.3 Hz), 0.96 (m, 6H,
H10 of p-cymene), 0.87 (d, 6H CH3 of p-cymene, JHH = 6.6 Hz).
13C{1H} NMR (150.90 MHz, at 25 °C, CDCl3): δ (SiMe4) 154.23 and
154.20 (C6 of pyridyl ring of ppy), 139.54 and 139.51(C3 of phenyl ring
of ppy), 129.73 and 129.70 (C4 of phenyl ring of ppy), 124.71 (Ca of
levonorgestrel), 124.04 (C6 of phenyl ring of ppy), 123.09 and 123.03
(C5 of pyridyl ring of ppy), 122.61 and 122.59 (C5 of phenyl ring of ppy),
120.79 (C3 of pyridyl ring of ppy), 90.87 and 90.85 (C2 of p-cymene),
89.84 and 89.80 (C5 of p-cymene), 84.50 and 84.36 (C3 of p-cymene),
82.54 (C6 of p-cymene), 51.34, 48.77, 42.47, and 40.95 (CH of
levonorgestrel), 39.70, 36.50, and 35.48 (CH2 of levonorgestrel),
30.80 (C8 of p-cymene), 30.68, 28.97, 26.52, 26.22, and 22.63 (CH2
of levonorgestrel), 22.52 and 22.50 (C9 of p-cymene), 21.74 and 21.73
(C10 of p-cymene), 19.07 (CH2 of levonorgestrel), 18.77 (C7 of p-
cymene), 9.60 (CH3 of levonorgestrel). ESI+ mass spectra (CH3CN):
m/z = +702.3 [[Ru(η6-p-cymene)(LEV-ppy)Cl] + 2H]+, 100%].
Reaction of the Ruthenium Complex 2 with 9-Ethylgua-
nine Followed by ESI-MS. The reaction was carried out in an NMR
tube with H2O and DMSO (5%) as solvents. 9-Ethylguanine was
incubated with the corresponding complex in a ratio of 5:1 in H2O at
37 °C. The concentration of complex 2 was 1.0 mM. After 15 min, a
mixture containing 2, [Ru(η6-p-cymene)(LEV-ppy)(9-EtG)]+ (4+),
[Ru(η6-p-cymene)(LEV-ppy)(DMSO)]+ (5+), and the chloride coun-
The effects of complexes were expressed as corrected percentage
inhibition values according to the following equation:
ð%Þinhibition ¼ ½1 ꢀ ðT=CÞꢂ ꢁ 100
where T is the mean absorbance of the treated cells and C the mean
absorbance in the controls.
The inhibitory potential of compounds was measured by calculating
concentrationꢀpercentage inhibition curves, and these curves were
adjusted to the following equation:
n
E ¼ Emax=½1 þ ðIC50Þ=CÞ ꢂ
where E is the percentage inhibition observed, Emax is the maximal
effects, IC50 is the concentration that inhibits 50% of maximal growth, C
is the concentration of compounds tested, and n is the slope of the
semilogarithmic doseꢀresponse sigmoid curves. This nonlinear fitting
was performed using GraphPad Prism 2.01, 1996 software (GraphPad
Software Inc.).
For comparison purposes, the cytotoxicity of cisplatin was evaluated
under the same experimental conditions. All compounds were tested in
two independent studies with triplicate points. The in vitro studies were
performed in the USEF platform of the University of Santiago de
Compostela (Spain).
Electrophoretic Mobility Study. pBR322 plasmid DNA of 0.25
μg/μL concentration was used for the experiments. Four microliters of
charge maker (LambdaꢀpUC Mix marker, 4) were added to aliquots of
20 μL of the drugꢀDNA complex. The platinum complexes were
incubated at the molar ratio ri = 0.50 with pBR322 plasmid DNA at
37 °C for 24 h. The mixtures underwent electrophoresis in agarose gel
1% in 1 ꢁ TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0) for 5 h
at 30 V. The gel was subsequently stained in the same buffer containing
ethidium bromide (1 μg/mL) for 20 min. The DNA bands were
visualized with an AlphaImager EC (Alpha Innotech).
teranion (4+ Clꢀ) was observed.
3
Computational Details. Quantum chemical calculations were
performed with the ORCA electronic structure program package.42
All geometry optimizations were run with tight convergence criteria,43
first at the BP8644 level using the RI approximation45 and the def2-
SVP46 basis set and thereafter refined with the def2-TZVP basis set.47
For Ru atoms, the [SD(28,MWB)] effective core potential48 (ECP) was
used. In all optimizations a semiempirical correction accounting for the
major part of the contribution of dispersion forces to the energy was
included.49 From these gas-phase optimized geometries, all reported
data were obtained by means of single-point (SP) calculations using the
B3LYP50 together with the new efficient RIJCOSX algorithm51 and the
more extense def2-TZVPP basis set.52 In order to better account for the
various weak interactions taking place, only the 4+ Clꢀ structure was
3
further refined with the X3LYP extended functional,53 developed for
accurate description of nonbond interactions and thermodynamic
properties of molecular systems and suggested for predicting ligand
binding in proteins and DNA. In this case all properties were computed
as SP calculations at the X3LYP/def2-TZVPP level with ECP for
ruthenium. Reported energies are uncorrected for the zero-point vibra-
tional term. The topological analysis of the electronic charge density was
conducted using the AIM2000 software,54 and the wave functions were
’ ASSOCIATED CONTENT
S
Supporting Information. NCIplot analysis. Structures,
b
energies, and Cartesian coordinates of all computed compounds.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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Inorganic Chemistry
ARTICLE
’ AUTHOR INFORMATION
(26) Sanchez-Cano, C.; Huxley, M.; Ducani, C.; Hamad, A. E.;
Browning, M. J.; Navarro-Ranninger, C.; Quiroga, A. G.; Rodger, A.;
Hannon, M. J. Dalton Trans. 2010, 39, 11365–11374.
(27) Ruiz, J.; Rodríguez, V.; Cutillas, N.; Espinosa, A.; Hannon, M. J.
J. Inorg. Biochem. 2011, 105, 525–531.
Corresponding Author
*E-mail: jruiz@um.es; fax: +34 868 884148; tel: +34 868 887455.
(28) Al-Duaij, Y. B.; Davies, D. L.; Griffith, G. A.; Sing, K. Organo-
metallics 2009, 28, 433–440.
’ ACKNOWLEDGMENT
(29) Djukic, J.-P.; et al. Organometallics 2004, 23, 5757–5767.
(30) (a) Mayer, I. Chem. Phys. Lett. 1983, 97, 270–274. (b) Mayer, I.
Int. J. Quantum Chem. 1984, 26, 151–154. (c) Mayer, I. Theor. Chim. Acta
1985, 67, 315–322.(d) Mayer, I. In Modelling of Structure and Properties
of Molecules; Maksic, Z. B., Ed.; John Wiley & Sons: New York, 1987.
(e) Bridgeman, A. J.; Cavigliasso, G.; Ireland, L. R.; Rothery, J. J. Chem.
Soc., Dalton Trans. 2001, 2095–2108.
(31) Bader, R. F. W. In Atoms in Molecules: A Quantum Theory;
Oxford University Press: Oxford, 1990.
(32) Distance between the metal ion and its projection onto the
arene mean plane.
(33) Loh, S. Y.; Mistry, P.; Kelland, L. R.; Abel, G.; Harrap, K. R. Br. J.
Cancer 1992, 66, 1109–1115.
This work was supported by the MICINN-Spain (Projects
CTQ2008-02178 and CTQ2008-01402) and Fundaciꢀon Seneca
(Projects 08666/PI/08 and 04509/GERM/06 (Programa de
Ayudas a Grupos de Excelencia de la Regiꢀon de Murcia, Plan
Regional de Ciencia y Tecnología 2007/2010)). We wish to
thank the Supercomputation Center at “Fundaciꢀon Parque
Científico de Murcia” (FPCMur) for their technical support
and the computational resources used in the supercomputer Ben-
Arabí. We also knowledge Dr. J. Contreras-García for technical
advice with the NCIplot analysis.
(34) Goddard, P. M; Orr, R. M.; Valenti, M. R.; Barnard, C. F.;
Murrer, B. A.; Kelland, L. R.; Harrap, K. R. Anticancer Res. 1996,
16, 33–38.
(35) Behrens, B. C.; Hamilton, T. C.; Masuda, H.; Grotzinger, K. R.;
Whang-Peng, J.; Louie, K. G.; Knutsen, T.; McKoy, W. M.; Young, R. C.;
Ozols, R. F. Cancer Res. 1987, 47, 414–418.
(36) Kelland, L. R.; Barnard, C. F. J.; Mellish, K. J.; Jones, M.;
Goddard, P. M.; Valenti, M.; Bryant, A.; Murrer, B. A.; Harrap, K. R.
Cancer Res. 1994, 54, 5618–5622.
’ REFERENCES
(1) Jakupec, M. A.; Galanski, M.; Arion, V. B.; Hartinger, C. G.;
Keppler, B. K. Dalton Trans. 2008, 183–194.
(2) Klein, A. V.; Hambley, T. W. Chem. Rev. 2009, 109, 4911–4920.
(3) Jung, Y. W.; Lippard, S. J. Chem. Rev. 2007, 107, 1387–1407.
(4) Kelland, L. Nat. Rev. Cancer 2007, 7, 573–584.
(5) O’Dwyer, P. J.; Stevenson, J. P.; Johnson, S. W. In Cisplatin.
Chemistry and Biochemistry of a Leading Anticancer Drug; Lippert, B., Ed.;
Wiley-VCH: Weinheim, Germany, 1999; p 3172.
(37) Ushay, H. M.; Tullius, T. D.; Lippard, S. J. Biochemistry 1981,
(6) Wang, D.; Lippard, S. J. Nat. Rev. Drug Discovery 2005, 4, 307–320.
(7) Hannon, M. J. Pure Appl. Chem. 2007, 79, 2243–2261.
(8) Sꢀanchez-Cano, C.; Hannon, M. J. Dalton Trans. 2009,
10702–10711.
(9) van Zutphen, S.; Reedijk, J. Coord. Chem. Rev. 2005, 249,
2845–2853.
(10) Gasser, G.; Ott, I.; Metzler-Nolte, N. J. Med. Chem. 2011, 54, 3–25.
(11) Lentz, F.; Drescher, A.; Lindauer, A.; Henke, M.; Hilger, R. A.;
Hartinger, C. G.; Scheulen, M. E.; Christian, D.; Keppler, B. K.; Jaehde,
U. Anti-Cancer Drugs 2009, 20, 97–103.
(12) Alessio, E.; Mestroni, G.; Bergamo, A.; Sava, G. Met. Ions Biol.
Syst. 2004, 42, 323.
(13) Bruijnincx, P. C. A.; Sadler, P. J. Curr. Opin. Chem. Biol. 2008,
12, 197–206.
(14) Bruijnincx, P. C. A.; Sadler, P. J. Adv. Inorg. Chem. 2009,
61, 1–62.
(15) Liu, H.-K.; Sadler, P. J. Acc. Chem. Res. 2011, 44, 349–359.
(16) Dyson, P. J.; Sava, G. Dalton Trans. 2006, 16, 1929–1933.
(17) Hartinger, C.; Dyson, P. J. Chem. Soc. Rev. 2009, 38, 391–401.
(18) Ang, W. H.; Parker, L. J.; De Luca, A.; Juillerat-Jeanneret, L.;
Morton, C. J.; Lo Bello, M.; Parker, M. W.; Dyson, P. J. Angew. Chem., Int.
Ed. 2009, 48, 3854–3857.
(19) Petrak, K. Drug Discovery Today 2005, 10, 1667.
(20) Jackson, A; Davis, J.; Pither, R. J.; Rodger, A.; Hannon, M. J.
Inorg. Chem. 2001, 40, 3964–3973.
(21) Hannon, M. J.; Green, P. S.; Fisher, D. M.; Derrick, P. J.; Beck,
J. L.; Watt, S. J.; Ralph, S. F.; Sheil, M. M.; Barker, P. R.; Alcock, N. W.;
Price, R. J.; Sanders, K. J.; Pither, R; Davis, J.; Rodger, A. Chem.—Eur. J.
2006, 12, 8000–8013.
(22) Sꢀanchez-Cano, C.; Hannon, M. J. Dalton Trans. 2009,
38, 10765–10773.
(23) Top, S.; Vessiꢁeres, A.; Cabestaing, C.; Laios, I.; Leclercq, G.;
Provot, C.; Jaouen, G. J. Organomet. Chem. 2001, 637, 500–506.
(24) Vessiꢁeres, A.; Top, S.; Pigeon, P.; Hillard, E. A.; Boubeker, L.;
Spera, D.; Jaouen, G. J. Med. Chem. 2005, 48, 3937–3940.
(25) Huxley, M.; Sanchez-Cano, C.; Browning, M. J.; Navarro-
Ranninger, C.; Quiroga, A. G.; Rodger, A.; Hannon, M. J. Dalton Trans.
2010, 39, 11353–11364.
20, 3744–3748.
(38) η6-p-Cymene-Ru(II) bond (dRu
= 1.727 Å; ΣMBO =
arene
2.531; ΣF(rc) = 17.96 ꢁ 10ꢀ2 e/a0 ; εave3 r3 3= 0.649 for 2 BCPs). Two
3
bonds with ppy ligand at C atom (dRuꢀC = 2.051 Å; MBO = 0.983; F(rc)
= 13.18 ꢁ 10ꢀ2 e/a0 ) and N atom (dRuꢀN = 2.089 Å; MBO = 0.717;
3
3
F(rc) = 10.05 ꢁ 10ꢀ2 e/a0 ).
(39) (a) Qui~nonero, D.; Garau, C.; Rotger, C.; Frontera, A.;
Ballester, P.; Costa, A.; Deyꢁa, P. M. Angew. Chem., Int. Ed. 2002,
41, 3389–3392. (b) Frontera, A.; Gamez, P.; Mascal, M.; Mooibroek,
T. J.; Reedijk, J. Angew. Chem., Int. Ed. 2011in press.
(40) Escudero, D.; Frontera, A.; Qui~nonero, D.; Deyꢁa, P. M. J.
Comput. Chem. 2009, 30, 75–82.
(41) (a) Johnson, E. R.; Keinan, S.; Mori-Sꢀanchez, P.; Contreras
García, J.; Cohen, A. J.; Yang, W. J. Am. Chem. Soc. 2010,
132, 6498–6506. (b) Contreras García, J.; Johnson, E. R.; Keinan, S.;
Chaudret, R.; Piquemal, J.-P.; Beratan, D. N.; Yang, W. J. Chem. Theory
Comput. 2011, 7, 625–632.
(42) Neese, F. ORCA, Version 2.8.0; Universit€at Bonn: Bonn,
Germany, 2010. An ab initio, density functional, and semiempirical
program package. http://www.thch.uni-bonn.de/tc/orca/.
(43) Tight convergence criteria for optimizations in ORCA: energy
change 1.0 ꢁ 10ꢀ6 hartree; maximum gradient 1.0 ꢁ 10ꢀ4 hartree/a0;
rms gradient 3.0 ꢁ 10ꢀ5 hartree/a0; maximum displacement 1.0 ꢁ 10ꢀ3
a0; rms displacement 6.0 ꢁ 10ꢀ4 a0.
(44) (a) Becke, A. D. Phys. Rev. A: At., Mol., Opt. Phys. 1988,
38, 3098–3100. (b) Perdew, J. P. Phys. Rev. B: Condens. Matter Mater.
Phys. 1986, 33, 8822–8824.
€
(45) (a) Eichkorn, K.; Treutler, O.; Ohm, H.; H€aser, M.; Ahlrichs, R.
Chem. Phys. Lett. 1995, 240, 283–289. (b) Eichkorn, K.; Weigend, F.;
Treutler, O.; Ahlrichs, R. Theor. Chem. Acc. 1997, 97, 119–124.
(c) Weigend, F.; H€aser, M.; Patzelt, H.; Ahlrichs, R. Chem. Phys. Lett.
1998, 294, 143–152.
(46) Schaefer, A.; Horn, H.; Ahlrichs, R. J. Chem. Phys. 1992,
97, 2571–2577.
(47) Weigend, F.; Ahlrichs, R. Phys. Chem. Chem. Phys. 2005,
7, 3297–3305.
(48) Andrae, D.; H€aussermann, U.; Dolg, M.; Stoll, H.; Preuss, H.
Theor. Chim. Acta 1990, 77, 123–141.ECP parameters for Ru [SD(28,
9170
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Inorganic Chemistry
ARTICLE
MWB)] were obtained from the pseudopotential library of the
StuttgartꢀCologne group, at http://www.theochem.uni-stuttgart.de/
pseudopotentials/.
(49) (a) Grimme, S. J. Comput. Chem. 2004, 25, 1463–1476.
(b) Grimme, S. J. Comput. Chem. 2006, 27, 1787–1799.
(50) (a) Becke, A. D. J. Chem. Phys. 1993, 98, 5648–5652. (b) Lee,
C. T.; Yang, W. T.; Parr, R. G. Phys. Rev. B: Condens. Matter Mater. Phys.
1988, 37, 785–789.
(51) Neese, F.; Wennmohs, F.; Hansen, A.; Becker, U. Chem. Phys.
2009, 356, 98–109.
(52) (a) Sch€afer, A.; Huber, C.; Ahlrichs, R. J. Chem. Phys. 1994,
100, 5829–5835.(b) Basis sets may be obtained from the Basis Set
Exchange (BSE) software and the EMSL Basis Set Library: https://bse.
pnl.gov/bse/portal. (c) Feller, D. J. Comput. Chem. 1996, 17, 1571–
1586.
(53) (a) Xu, X.; Goddard, W. A., III Proc. Natl. Acad. Sci. U. S. A.
2004, 101, 2673–2677. (b) Xu, X.; Zhang, Q.; Muller, R. P.; Goddard,
W. A., III J. Chem. Phys. 2005, 122, 014105.
(54) (a) Biegler-K€onig, F. Scho€nbohm, J. AIM2000 v. 2.0; 2002;
http://www.aim2000.de/. (c) Biegler-K€onig, F.; Sch€onbohm, J.; Bayles,
D. J. Comput. Chem. 2001, 22, 545–559. (b) Biegler-K€onig, F.;
Sch€onbohm, J. J. Comput. Chem. 2002, 23, 1489–1494.
(55) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.;
Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.;
Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, Jr., J. A.;
Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin,
K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.;
Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.;
Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.;
Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.;
Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich,
€
S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.;
Fox, D. J. Gaussian 09, Revision A.02; Gaussian, Inc.: Wallingford, CT,
2009.
(56) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics 1996,
14, 33–38.VMD — Visual Molecular Dynamics. http://www.ks.uiuc.
edu/Research/vmd/.
9171
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