Synthesis, characterization, and antitumor activity of water-soluble (arene)ruthenium(II) derivatives of 1,3-dimethyl-4-acylpyrazolon-5-ato ligands. First example of Ru(arene)(ligand) antitumor species involving simultaneous Ru-N7(guanine) bonding and ligand intercalation to DNA

Article abstract of DOI:10.1021/ic403170y

We report on the synthesis of novel water-soluble [(arene)Ru ^II(Q)Cl] and [(arene)Ru^II(Q)(X)]BF₄ compounds (arene = p-cymene, benzene, hexamethylbenzene; HQ = 1,3-dimethyl-4-R-(C=O)-5- pyrazolone, HQ^Me, R = methyl, HQ^Ph, R = phenyl, HQ ^Naph, R = naphthyl; X = H₂O, 9-ethylguanine), and their in vitro antitumor activity toward the cell lines MCF7 (HTB-22, human breast adenocarcinoma), HCT116 (CCL-247, human colorectal carcinoma), A2780 (human ovarian carcinoma), A549 (CCL-185, human lung carcinoma), and U87 MG (HTB-1, human glioblastoma). The X-ray crystal structures of two complexes were determined. One of them, {chlorido-(p-cymene)-[(1,3-dimethyl-4-(1-naphthoyl)- pyrazolon-5-ato]ruthenium(II)}, was also studied with density functional theory methods and was selected for docking on a DNA octamer showing intercalation between DNA bases by the naphthyl moiety and for Ru-N7(guanine) bonding.

Full text of DOI:10.1021/ic403170y

Article
pubs.acs.org/IC
Synthesis, Characterization, and Antitumor Activity of Water-Soluble
(Arene)ruthenium(II) Derivatives of 1,3-Dimethyl-4-acylpyrazolon-5-
ato Ligands. First Example of Ru(arene)(ligand) Antitumor Species
Involving Simultaneous Ru−N7(guanine) Bonding and Ligand
Intercalation to DNA
Francesco Caruso,*,† Elena Monti,‡ Julian Matthews,† Miriam Rossi,† Marzia Bruna Gariboldi,‡
Claudio Pettinari,§ Riccardo Pettinari,⊥ and Fabio Marchetti*,⊥
†Department of Chemistry, Vassar College, Poughkeepsie, New York 12604-0484, United States
‡Department of Theoretical and Applied Sciences, Division of Biomedical Sciences, University of Insubria, Via A. da Giussano 10,
21052 Busto Arsizio, Varese, Italy
§School of Science and Technology, Chemistry Section, University of Camerino, Via S. Agostino 1, 62032 Camerino Italy
⊥School of Pharmacy, Chemistry Section, University of Camerino, Via S. Agostino 1, 62032 Camerino Italy
S
* Supporting Information
ABSTRACT: We report on the synthesis of novel water-soluble [(arene)RuII(Q)Cl]
and [(arene)RuII(Q)(X)]BF4 compounds (arene = p-cymene, benzene, hexamethyl-
benzene; HQ = 1,3-dimethyl-4-R-(CO)-5-pyrazolone, HQMe, R = methyl, HQPh, R
= phenyl, HQNaph, R = naphthyl; X = H2O, 9-ethylguanine), and their in vitro
antitumor activity toward the cell lines MCF7 (HTB-22, human breast
adenocarcinoma), HCT116 (CCL-247, human colorectal carcinoma), A2780
(human ovarian carcinoma), A549 (CCL-185, human lung carcinoma), and U87
MG (HTB-1, human glioblastoma). The X-ray crystal structures of two complexes
were determined. One of them, {chlorido-(p-cymene)-[(1,3-dimethyl-4-(1-naphthoyl)-pyrazolon-5-ato]ruthenium(II)}, was also
studied with density functional theory methods and was selected for docking on a DNA octamer showing intercalation between
DNA bases by the naphthyl moiety and for Ru−N7(guanine) bonding.
dependent on the nature of X, YZ, and the arene.9,10 [(η6-
1. INTRODUCTION
arene)Ru(ethylenediamine)Cl]+ complexes exhibit anticancer
The design of antitumor ruthenium complexes has been of
increasing interest after two RuIII species, imidazolium-
activity both in vitro and in vivo, including cisplatin-resistant
cancer cells.11 The hydrolysis of the metal-chloride bond
(imidazole)(dimethylsulfoxide)tetrachlororuthenate(III)
appears to be important for activation, giving aqua adducts9
(NAMI-A) and indazolium trans-[tetrachlorobis(1H-indazole)-
[(η6-arene)Ru(ethylenediamine)(H2O)]2+ that can bind to
DNA (or proteins), forming monofunctional adducts. Studies
in aqueous solution have shown that [(η6-biphenyl)Ru-
(ethylenediamine)]2+ binds specifically to guanine when in
competition with adenine, cytosine, and thymine nucleotides,11
and similarly to plasmid DNA.12 Moreover, guanine binds
strongly through N7 with additional formation of a strong H-
bond between C6O(guanine) and NH(ethylenediamine).13
The possibility of arene intercalation between the DNA bases
has also been recognized.13,14 Replacement of neutral ethyl-
enediamine by anionic β-diketonates such as acetylacetonato
(acac) showed that the chelating ligand increases the rate and
extent of hydrolysis as well as raising the pKa of the aqua
complex.15
ruthenate(III)] (KP1019) entered clinical trials.1,2 Some
features of these compounds suggested potential metal
reduction to RuII, and compounds of the type RuII(η6-arene)
explored by Sadler also were found to be active.3 Compounds
incorporating the 1,3,5-triaza-7-phosphaadamantane (PTA)
ligand, for example, [Ru(η6-p-cymene)(PTA)Cl2] (RAPTA-
C), show activity against metastases, and a pH-dependent
interaction with DNA was suggested as a key component.4
Recent studies on arene−RuII−chloroquine complexes have
demonstrated their interaction with DNA and their ability to
induce apoptosis on human lymphoid cell lines.5 Studies
involving natural ligands included in the Ru coordination
sphere have been undertaken as well.6,7
Compounds of the type [(η6-arene)Ru(X)(YZ)] (where YZ
is a bidentate chelating ligand and X a good leaving group, e.g.
Cl−) exhibit both in vitro and in vivo anticancer activity, in some
cases even comparable to that of cisplatin.8 The aqueous
reactivity of [(η6-arene)Ru(X)(YZ)] complexes is highly
Received: December 30, 2013
Published: March 11, 2014
© 2014 American Chemical Society
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Inorganic Chemistry
Article
We recently reported the synthesis of (arene)RuII curcumin
derivatives displaying antitumor activity,16 in which not only
the metal fragment but also the chelating ligand is known to
possess biological activity. The same approach guided us to
synthesize similar (arene)RuII systems17 containing a variant of
classic β-diketonates that are well-known molecules with
biological properties,18 namely, the acylpyrazolonate ligands
(Scheme 1),19 where a pyrazole ring is fused to the O,O′-
mmol) were dissolved in methanol (20 mL) and stirred for 30 min at
room temperature; then, [Ru(η6-p-cymene)Cl2]2 (0.306 g, 0.5 mmol)
was added to the red solution, which immediately changed to orange.
After 4 h of stirring at room temperature, the clear orange solution was
evaporated to dryness, and the residue was dissolved in dichloro-
methane (10 mL), from which a precipitate slowly formed, which was
filtered off and dried under reduced pressure. The powder was
identified as derivative 1. Recrystallization in methanol at +4 °C slowly
yielded orange-red crystals. The compound is soluble in water,
alcohols, acetone, acetonitrile, DMSO, and halogenated organic
solvents. Yield 66%. Mp 161−163 °C. Anal. Calcd. for
C17H23ClN2O2Ru: C, 48.17; H, 5.47; N, 6.61%. Found: C, 47.88; H,
5.62, N, 6.36%. Λm (acetonitrile, 298K, 10−3 mol/L) 11.7 Ω−1 cm2
mol−1. Λm (water, 298K, 10−3 mol/L) 114.9 Ω−1 cm2 mol−1. IR (nujol,
cm−1): 1590vs ν(CO), 1522m ν(CN + CC), 440s, 369m
ν(Ru−O), 279s ν(Ru−Cl). 1H NMR (CDCl3, 298 K, δ): 1.27d, 1.34d
(6H, J = 6.6 Hz, CH3−C6H4−CH(CH3)2), 2.22s (3H, C(O)CH3,
Scheme 1. 4-Acyl-5-pyrazolone Ligand, Keto-Enol Form,
Used in This Work (QMe, QPh, and QNaph
)
Q
Q
Me), 2.27s (3H, CH3−C6H4−CH(CH3)2), 2.37s (3H, C3−CH3,
Me), 2.95sp ((septet), 1H, CH3−C6H4−CH(CH3)2), 3.45s (3H,
N1−CH3, QMe), 5.25d, 5.52d (4H, AA′BB′ system, CH3−C6H4−
CH(CH3)2). 1H NMR (D2O, 298 K, δ): 0.99d, 1.08d (6H, J = 6.6 Hz,
CH3−C6H4−CH(CH3)2), 1.93s (3H, C(O)CH3, QMe), 2.05s (3H,
CH3−C6H4−CH(CH3)2), 2.16s (3H, C3−CH3, QMe), 2.68sp
((septet), 1H, CH3−C6H4−CH(CH3)2), 3.53s (3H, N1−CH3, QMe),
5.58d, 5.74d (4H, AA′BB′ system, CH3−C6H4−CH(CH3)2). 13C{1H}
(CDCl3, δ): 17.4 (C3−CH3, QMe), 20.3 (CH3−C6H4−CH(CH3)2),
21.8 (CH3−C6H4−CH(CH3)2), 26.5 (C(O)CH3, QMe), 30.5
(CH3−C6H4−CH(CH3)2), 32.6 (N1−CH3, QMe), 78.8, 80.7, 81.4,
82.3, 96.8, 99.4 (C6H6, η6-p-cymene), 105.1 (C4, QMe), 147.2 (C3,
chelating moiety. Here we extend our studies to the design,
synthesis, and characterization of novel (arene)RuII acylpyr-
azolonates, which are more soluble in water than the curcumin
compounds, and test their antitumor activity on five tumor cell
lines in vitro. We also describe successful docking features of
one derivative onto a guanosine-containing DNA fragment.
Q
Me), 161.9 (C5, QMe), 189.6 (CO, QMe). 13C{1H} (D2O, δ): 17.4
(C3−CH3, QMe), 20.3 (CH3−C6H4−CH(CH3)2), 21.8 (CH3−C6H4−
CH(CH3)2), 26.5 (C(O)CH3, QMe), 30.5 (CH3−C6H4−CH-
(CH3)2), 32.6 (N1−CH3, QMe), 80.4, 81.0, 81.3, 82.5, 98.6, 103.6
(C6H6), 104.4 (C4, QMe), 155.8 (C3, QMe), 161.2 (C5, QMe), 193.0
(CO, QMe). ESI-MS (+) CH3OH (m/z, relative intensity %): 388
EXPERIMENTAL SECTION
■
Chemicals and Reagents. All reactants were purchased from Alfa
(Karlsruhe) and Aldrich (Milwaukee) and used as received. The
samples for microanalyses were dried in vacuo to constant weight (20
°C, ca. 0.1 Torr). Elemental analyses (C, H, N, S) were performed in
house with Fisons Instruments 1108 CHNS-O Elemental Analyzer. IR
spectra were recorded from 4000 to 600 cm−1 with a Perkin-Elmer
Spectrum 100 FT-IR instrument and from 600 to 200 cm−1 with a
Perkin-Elmer System 2000 FT-IR instrument. 1H and 13C NMR
spectra were recorded on a 400 Mercury Plus Varian spectrometer
+
[100] [Ru(η6-p-cymene)(QMe)] .
(2), [Chlorido-(p-cymene)-(4-benzoyl-1,3-dimethyl-pyrazolon-5-
ato)ruthenium(II)], [Ru(η6-p-cymene)(QPh)Cl]. Compound 2 was
prepared following a procedure similar to that reported for 1, starting
from [Ru(η6-p-cymene)Cl2]2 (0.306 g, 0.5 mmol), HQPh (0.216 g, 1
mmol), and NaOMe (0.054 g, 1 mmol). The compound is soluble in
water, alcohols, acetone, acetonitrile, DMSO, and halogenated organic
solvents. Yield 68%. Mp 187−191 °C. Anal. Calcd. for C22H25Cl-
N2O2Ru: C, 54.37; H, 5.19; N, 5.76%. Found: C, 54.02; H, 5.35, N,
5.45%. Λm (acetonitrile, 298K, 10−3 mol/L) 8.4 Ω−1 cm2 mol−1. Λm
(water, 298K, 10−3 mol/L) 121.6 Ω−1 cm2 mol−1. IR (nujol, cm−1):
1594vsν (CO), 1572, 1522, 1501 ν(CC, CN), 443s, 427m,
367m ν(Ru−O), 281s ν(Ru−Cl). 1H NMR (CDCl3, 298 K, δ): 1.29d,
1.35d (6H, CH3−C6H4−CH(CH3)2), 1.57s (3H, C3−CH3, QPh),
2.27s (3H, CH3−C6H4−CH(CH3)2), 2.95sp ((septet), 1H, CH3−
C6H4−CH(CH3)2), 3.43s (3H, N1−CH3, QPh), 5.25d, 5.55d (4H,
AA′BB′ system, CH3−C6H4−CH(CH3)2), 7.42m (5H, C(
O)C6H5). 1H NMR (D2O, 298 K, δ): 1.09d, 1.12d, 1.21d, 1.24d
(6H, CH3−C6H4−CH(CH3)2), 1.86s, 2.06s (3H, CH3−C6H4−
CH(CH3)2), 1.95s, 2.14s (3H, C3−CH3), 2.74m (1H, CH3−C6H4−
CH(CH3)2), 3.42s (3H, N1−CH3), 5.50t, 5.59d, 5.74t, 5.84d (4H,
AA′BB′ system, CH3−C6H4−CH(CH3)2), 7.41m, 7.52m (5H, C(
O)C6H5). 13C{1H} (CDCl3, δ): 16.2 (C3−CH3), 18.1 (CH3−C6H4−
CH(CH3)2), 22.1, 22.3, 22.5 (CH3−C6H4−CH(CH3)2), 30.8, 31.0
(CH3−C6H4−CH(CH3)2), 32.1 (N1−CH3), 78.7, 79.0, 80.7, 81.5,
82.4, 82.8, 96.9, 99.5 (CH3−C6H4−CH(CH3)2), 127.9, 128.1, 130.4,
138.9 (C(O)C6H5), 106.1 (C4), 147.7 (C3), 164.0 (C5), 187.8
(CO). ESI-MS (+) CH3OH (m/z, relative intensity %): 450 [100]
[Ru(η6-p-cymene)(QPh)]+.
1
operating at room temperature (400 MHz for H and 100 MHz for
13C). Melting points were taken on an IA 8100 Electrothermal
Instrument. The electrical conductance of the acetonitrile and water
solutions was measured with a Crison CDTM 522 conductimeter at
room temperature. 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.
Synthesis of Acylpirazolone Ligands: HQNaph. 1,3-Dimethyl-4-
(1-naphthoyl)-5-pyrazolone has been synthesized following a related
procedure previously reported.19 It is soluble in all common organic
solvents, such as alcohols, acetone, acetonitrile, dimethyl sulfoxide
(DMSO), and halogenated organic solvents. Yield 92%. Mp 177−180
°C. Anal. Calcd. for C16H14N2O2: C, 72.17; H, 5.30; N, 10.52%.
Found: C, 71.84; H, 5.40, N, 10.34%. IR (nujol, cm−1): 3370br ν(O−
H···O), 1685m δ(O−H···O), 1614vs ν(CO), 1588s ν(CN + C
1
C). H NMR (CDCl3, 298 K, δ): 2.05s (3H, C3−CH3), 3.68s (3H,
N1−CH3), 5.30br (1H, O−H), 7.60m, 7.68dd, 7.92m, 8.11dd, 8.70d
(7H, CHaromatic). 13C{1H} (CDCl3, δ): 15.8 (C3−CH3), 32.6 (N1−
CH3), 103.5 (C4), 124.5, 126.9, 127.1, 128.0, 128.1, 128.4, 128.6,
128.7, 129.0, 129.7 (Caromatic), 147.2 (C3), 160.8, 170.1 (C5), 193.0
(CO). The other HQ ligands, namely, HQMe (1,3-dimethyl-4-acetyl-5-
pyrazolone) and HQPh (1,3-dimethyl-4-benzoyl-5-pyrazolone), were
synthesized as previously reported.19
(3), {Chlorido-(p-cymene)-[(1,3-dimethyl-4-(1-naphthoyl)-pyra-
zolon-5-ato]ruthenium(II)}, [Ru(η6-p-cymene)(QNaph)Cl]. It was pre-
pared following a procedure similar to that reported for 1, starting
from [Ru(η6-p-cymene)Cl2]2 (0.306 g, 0.5 mmol), HQNaph (0.266 g, 1
mmol), and NaOMe (0.054 g, 1 mmol). The compound is soluble in
water, alcohols, acetone, acetonitrile, DMSO, and halogenated organic
Synthesis of Metal Complexes 1−9. (1), [Chlorido(p-cymene)-
(1,3-dimethyl-4-acetyl-5-pyrazolonato)ruthenium(II)], [Ru(η6-p-
cymene)(QMe)Cl]. HQMe (0.154 g, 1 mmol) and NaOMe (0.054 g, 1
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Inorganic Chemistry
Article
solvents. Yield 77%. Mp 104−106 °C. Anal. Calcd. for C26H27Cl-
N2O2Ru: C, 58.26; H, 5.08; N, 5.23%. Found: C, 57.97; H, 5.18, N,
5.14%. Λm (acetonitrile, 298K, 10−3 mol/L) 5.7 Ω−1 cm2 mol−1. Λm
(water, 298K, 10−3 mol/L) 108.4 Ω−1 cm2 mol−1. IR (nujol, cm−1):
acetonitrile/water (9:1) (10 mL); then, AgBF4 (0.0.97 g, 0.5 mmol)
was added to the solution, which was stirred for 2 h at room
temperature. The solution was filtered to remove silver chloride; the
solvent was removed in vacuo, and the residue was dried under
reduced pressure. The powder was identified as derivative 7, and it is
soluble in acetone, acetonitrile, DMSO, and slightly soluble in water,
alcohols, and halogenated organic solvents. Yield 85%. Mp 250 °C dec.
Anal. Calcd. for C17H25BF4N2O3Ru: C, 41.39; H, 5.11; N, 5.68%.
Found: C, 41.74; H, 5.31, N, 5.36%. Λm (acetonitrile, 298K, 10−3 mol/
L) 116.4 Ω−1 cm2 mol−1. IR (nujol, cm−1): 3520br ν(H2O), 1603vs
ν(CO), 1524m ν(CC, CN), 1047vs, 1030vs ν(BF4), 986s,
453s br, 376m ν(Ru−O). 1H NMR (CD3CN, 298 K, δ): 1.34d, 1.37d
(6H, J = 6.8 Hz, CH3−C6H4−CH(CH3)2), 2.19s (3H, C(O)CH3,
1
1589vs ν(CO), 479s, 456s, 363m ν(Ru−O), 280s ν(Ru−Cl). H
NMR (CDCl3, 298 K, δ): 1.37d, 1.40d (6H, CH3−C6H4−
CH(CH3)2), 1.59s (3H, C3−CH3, QNaph), 2.28s (3H, CH3−C6H4−
CH(CH3)2), 2.99sp ((septet), 1H, CH3−C6H4−CH(CH3)2), 3.52s
(3H, N1−CH3, QNaph), 5.31d, 5.58d (4H, AA′BB′ system, CH3−
C6H4−CH(CH3)2), 7.57m, 7.90m (6H, C−Hnaph, QNaph), 8.62d (1H,
C8−Hnaph, QNaph). 13C{1H} (CDCl3, δ): 16.4 (C3−CH3, QNaph), 18.1
(CH3−C6H4−CH(CH3)2), 22.5 (CH3−C6H4−CH(CH3)2), 31.10
(CH3−C6H4−CH(CH3)2), 32.2 (N1−CH3, QNaph), 78.8, 79.1, 82.5,
82.7, 96.9, 99.5 (C6H6, η6-p-cymene), 105.8 (C4, QNaph), 125.1, 126.9,
127.5, 128.0, 128.2, 128.8, 129.8, 131.9, 132.5, 134.3 (Cnaph, QNaph),
136.2 (C5, QNaph), 147.8 (C3, QNaph), 187.7 (CO, QNaph). ESI-MS
(+) CH3OH (m/z, relative intensity %): 501 [100] [Ru(η6-p-
cymene)(QNaph)]+.
Q
Q
Me), 2.25s (3H, CH3−C6H4−CH(CH3)2), 2.44s (3H, C3−CH3,
Me), 2.95sp ((septet), 1H, CH3−C6H4−CH(CH3)2), 3.30br (2H,
H2O), 3.44s (3H, N1−CH3, QMe), 5.52d, 5.83d (4H, AA′BB′ system,
CH3−C6H4−CH(CH3)2). ESI-MS (+) CH3OH (m/z, relative
intensity %): 388 [100] [Ru(η6-p-cymene)(QMe)]+.
(8), {Aqua-(η6-p-cymene)-[(1,3-dimethyl-4-(1-naphthoyl)-pyrazo-
lon-5-ato]ruthenium(II)} tetrafluoroborate, [Ru(η6-p-cymene)-
(QNaph)(H2O)]BF4. It was prepared following a procedure similar to
that reported for 7, starting from compound 3 (0.536 g, 1 mmol) and
AgBF4 (0.194 g, 1 mmol). The compound is soluble in water, alcohols,
acetone, acetonitrile, DMSO, and partially soluble in halogenated
organic solvents. Yield 70%. Mp 250 °C dec. Anal. Calcd. for
C26H29BF4N2O3Ru: C, 51.58; H, 4.83; N, 4.63%. Found: C, 51.30; H,
4.70, N, 4.48%. Λm (acetonitrile, 298K, 10−3 mol/L) 118.2 Ω−1 cm2
mol−1. IR (nujol, cm−1): 3514sbr ν(H2O), 1598vs ν(CO), 1516m
(4), [Chlorido-(η6-benzene)-(1,3-dimethyl-4-acetyl-5-
pyrazolonato)ruthenium(II)], [Ru(η6-benzene)(QMe)Cl]. It was pre-
pared following a procedure similar to that reported for 1, starting
from [Ru(benzene)Cl2]2 (0.250 g, 0.5 mmol), HQMe (0.154 g, 1
mmol), and NaOMe (0.054 g, 1 mmol). The compound is soluble in
water, alcohols, acetone, acetonitrile, DMSO, and slightly soluble in
halogenated organic solvents. Yield 74%. Mp 196−200 °C dec. Anal.
Calcd. for C13H15ClN2O2Ru: C, 42.45; H, 4.11; N, 7.62%. Found: C,
42.21; H, 4.25, N, 7.85%. Λm (acetonitrile, 298K, 10−3 mol/L) 7.3 Ω−1
cm2 mol−1. Λm (water, 298K, 10−3 mol/L) 109.7 Ω−1 cm2 mol−1. IR
(nujol, cm−1): 1587vs ν(CO), 1528m ν(CC, CN), 435vs,
369vs ν(Ru−O), 277vs ν(Ru−Cl). 1H NMR (CDCl3, 298 K, δ): 2.23s
(3H, C(O)CH3, QMe), 2.38s (3H, C3−CH3, QMe), 3.47s (3H, N1−
CH3, QMe), 5.69s (6H, C6H6, η6-benzene). 1H NMR (D2O, 298 K, δ):
2.11s, 2.20s (3H, C3−CH3), 2.33s, 2.44s (3H, C(O)−CH3), 3.32s,
3.57s (3H, N1−CH3), 5.78s, 5.91s (6H, C6H6, η6-benzene). ESI-MS
(+) CH3OH (m/z, relative intensity %): 332 [100] [Ru(η6-
benzene)(QMe)]+.
1
ν(CC, CN), 1003vsbr ν(BF4). H NMR (CD3OD, 298 K, δ):
1.41d, 1.43d (6H, CH3−C6H4−CH(CH3)2), 1.64s (3H, C3−CH3,
Q
Naph), 2.31s (3H, CH3−C6H4−CH(CH3)2), 2.97sp ((septet), 1H,
CH3−C6H4−CH(CH3)2), 3.59s (3H, N1−CH3, QNaph), 5.70d, 5.95d
(4H, AA′BB′ system, CH3−C6H4−CH(CH3)2), 7.61m, 8.02m (6H,
C−Hnaph, QNaph), 8.60d (1H, C−H8naph, QNaph). 13C{1H} (CDCl3, δ):
16.3 (C3−CH3, QNaph), 18.0 (CH3−C6H4−CH(CH3)2), 22.5 (CH3−
C6H4−CH(CH3)2), 32.4 (CH3−C6H4−CH(CH3)2), 32.5 (N1−CH3,
Q
Naph), 79.6, 79.9, 80.1, 81.9, 98.9, 101.8 (C6H6, η6-p-cymene), 105.8
(5), [Chlorido-hexamethylbenzene-(1,3-dimethyl-4-acetyl-5-
pyrazolonato)ruthenium(II)] [Ru(η6-hmb)(QMe)Cl]. It was prepared
following a procedure similar to that reported for 1, starting from
[Ru(η6-hexamethylbenzene)Cl2]2 (0.334 g, 0.5 mmol), HQMe(0.154 g,
1 mmol), and NaOMe (0.054 g, 1 mmol). The compound is soluble in
water, alcohols, acetone, acetonitrile, DMSO, and halogenated organic
solvents. Yield 76%. Mp 268−269 °C dec. Anal. Calcd. for
C19H27ClN2O2Ru: C, 50.49; H, 6.02; N, 6.20%. Found: C, 50.23; H,
5.93, N, 6.09%. Λm (acetonitrile, 298K, 10−3 mol/L) 14.0 Ω−1 cm2
mol−1. Λm (water, 298K, 10−3 mol/L) 129.1 Ω−1 cm2 mol−1. IR (nujol,
cm−1): 1592vs ν(CO), 1526m ν(CC, CN), 458s, 431vs, 365s
ν(Ru−O), 281vs ν(Ru−Cl). 1H NMR (CDCl3, 298 K, δ): 2.07s (3H,
C3−CH3, QMe), 2.21s (18H, CH3, η6-hexamethylbenzene), 2.38s (3H,
C(O)−CH3, QMe), 3.45s (3H, N1−CH3, QMe). ESI-MS (+)
CH3OH (m/z, relative intensity %): 416 [100] [Ru(η6-hmb)(QMe)]+.
(6) [Chlorido-hexamethylbenzene-(4-benzoyl-1,3-dimethyl-pyra-
zolon-5-ato)ruthenium(II)], [Ru(η6-hmb)(QPh)Cl]. It was prepared
following a procedure similar to that reported for 1, starting from
[Ru(η6-hexamethylbenzene)Cl2]2 (0.334 g, 0.5 mmol), HQPh (0.216 g,
1 mmol), and NaOMe (0.054 g, 1 mmol). The compound is soluble in
water, alcohols, acetone, acetonitrile, DMSO, and halogenated organic
solvents. Yield 81%. Mp 243−244 °C dec. Anal. Calcd. for
C24H29ClN2O2Ru: C, 56.08; H, 5.69; N, 5.45%. Found: C, 55.38; H,
5.48, N, 5.29%. Λm (acetonitrile, 298K, 10−3 mol/L) 6.2 Ω−1 cm2
mol−1. Λm (water, 298K, 10−3 mol/L) 102.6 Ω−1 cm2 mol−1. IR (nujol,
cm−1): 1588vs ν(CO), 1573m, 1520m ν(CC, CN), 460m,
(C4, QNaph), 125.6, 126.5, 127.9, 128.5, 128.9, 129.7, 130.5, 132.2,
133.9, 136.1 (Cnaph, QNaph), 149.3 (C5, QNaph), 163.9 (C3, QNaph),
191.0 (CO, QNaph). ESI-MS (+) CH3OH (m/z, relative intensity
%): 501 [100] [(p-cymene)Ru(QNaph)]+, 565 [60] [(p-cymene)Ru-
(QNaph)(CH3OH)2]+.
(9), {(η6-p-Cymene)-[(1,3-dimethyl-4-(1-naphthoyl)-pyrazolon-5-
ato]-(9-ethylguanine)ruthenium(II)} tetrafluoroborate, [Ru(η6-p-
cymene)(QNaph)(9-ethylguanine)]BF4. Compound 8 (0.294 g, 0.5
mmol) was dissolved in a mixture of methanol−water (16:4 v/v), and
9-ethylguanine (0.089 g, 0.5 mmol) was added to the red solution.
After 1 h of stirring at room temperature the solvent was removed in
vacuo, and the residue was dried under reduced pressure. The resulting
yellow powder was dried in air. The compound is soluble in acetone,
acetonitrile, DMSO, and slightly soluble in water, alcohols, and
halogenated organic solvents. Yield 85%. Mp 135 °C. Elem. Anal.
Calcd. for C33H36BF4N7O3Ru: C, 51.71; H, 4.73; N, 12.79%. Found:
C, 51.60; H, 4.41, N, 12.52%. IR (nujol, cm−1): 1579vs, 1569sh ν(C
O), 1542w, 1520w ν(CC, CN), 1051vs, 1000vs ν(BF4). 1H NMR
(CD3OD, 298 K, δ): 1.20t (3H, J = 7.2 Hz, CH3−CH2-9-
ethylguanine), 1.29d, 1.31d (6H, 3H, J = 7.6 Hz, CH3−C6H4−
CH(CH3)2), 1.43s (3H, CH3−C6H4−CH(CH3)2), 2.12s (3H, C3−
CH3, QNaph), 2.89sp (1H, J = 7.2 Hz, CH3−C6H4−CH(CH3)2), 3.51s
(3H, N1−CH3, QNaph), 4.06q (2H, J = 7.2 Hz, CH3−CH2-9-
ethylguanine), 5.67d, 5.92d (4H, AA′BB′ system, CH3−C6H4−
CH(CH3)2), 7.55m, 7.94m (6H, C−Hnaph), 7.80s (1H, 9-ethyl-
guanine), 8.60d (1H, C−H8naph, QNaph). 13C{1H} (CD3OD, δ): 15.8,
15.9 (C3−CH3 and CH3 of 9-ethylguanine), 18.2 (CH3−C6H4−
CH(CH3)2), 22.7, 22.8 (CH3−C6H4−CH(CH3)2 and CH2 of 9-
ethylguanine), 32.3 (CH3−C6H4−CH(CH3)2), 40.4 (N1−CH3,
1
449vs, 359m ν(Ru−O), 288vs ν(Ru−Cl). H NMR (CDCl3, 298 K,
δ): 2.10s (3H, C3−CH3, QPh), 2.06s (18H, CH3, η6-hexamethylben-
zene), 2.28s (3H, C(O)−CH3, QPh), 3.15s (3H, N1−CH3, QPh),
7.39m (5H, N1−C6H5, QPh). ESI-MS (+) CH3OH (m/z, relative
intensity %): 479 [100] [Ru(η6-hmb)(QPh)]+.
Q
Naph), 81.6, 82.2, 84.9, 85.2, 100.2, 103.5 (CH3−C6H4−CH(CH3)2),
(7). [Aqua-(η6 -p-cymene)-(1,3-dimethyl-4-acetyl-5-
pyrazolonato)ruthenium(II)] tetrafluoroborate, [Ru(η6-p-cymene)-
(QMe)(H2O)]BF4. Derivative 1 (0.212 g, 0.5 mmol) was dissolved in
105.8 (C4), 116.5 (9-ethylguanine), 125.8, 126.5, 127.9, 128.3, 128.9,
129.7, 132.1, 136.0, 136.8 (Cnaph, QNaph), 141.1 (9-ethylguanine), 148.9
(C3, QNaph), 153.5, 156.1, 156.8 (9-ethylguanine), 164.5 (C5, QNaph),
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Table 1. Crystal Data and Structure Refinement of Compounds 3 and 4
compound
empirical formula
3: [Ru(η6-p-cymene)(QNaph)Cl]
4: [Ru(η6-benzene)(QMe)Cl]
C26H27ClN2O2Ru·H2O
552.02
C13H15ClN2O2Ru
367.79
formula weight
crystal system
monoclinic
monoclinic
space group
P21/n
P21/n
unit cell dimensions, Å
a = 9.6986(4)
b = 20.2270(8)
c = 13.1742(5)
β = 102.788(1)
2520.32(17), red
4, 125
a = 7.8172(16)
b = 17.886(4)
c = 9.5912(19)
β = 97.37(3)
1330.0(5), red
4, 125
unit cell dimensions, deg
volume (Å3), crystal color
Z, T data collection (K)
density (calculated), Mg/m3
absorption coefficient, mm−1
crystal size, mm3
1.455
1.837
0.577
1.378
0.31 × 0.29 × 0.19
1.88 to 28.33
0.33 × 0.31 × 0.09
2.28 to 28.28
−10 ≤ h ≤ 10, −23 ≤ k ≤ 23,
−12 ≤ l ≤12
16701
theta range for data collection, deg
index ranges
−12 ≤ h ≤ 12, −26 ≤ k ≤ 27,
−17 ≤ l ≤ 17
67009
reflections collected
independent reflections
max. and min transmission
data/restraints/parameters
goodness-of-fit on F2
6263 [R(int) = 0.0278]
0.8695 and 0.7992
5541/0/386
5711 [R(int) = 0.0209]
0.8860 and 0.6592
3127/0/172
1.081
1.298
final R indices [I > 2σ(I)]
R indices (all data)
R1 = 0.0441, wR2 = 0.1055
R1 = 0.0522, wR2 = 0.1136
R1 = 0.0512, wR2 = 0.1492
R1 = 0.0531, wR2 = 0.1501
190.1 (CO, QNaph). ESI-MS (+) CH3OH (m/z, relative intensity
%): 501 [500] [(p-cymene)Ru(QNaph)]+, 680 [100] [(p-cymene)Ru-
(QNaph)(9-ethylguanine)]+.
nonessential amino acids (both from Sigma-Aldrich, Milan, Italy) were
also added to the culture medium. All experiments were performed
within 10 passages from thawing.
X-ray Diffraction Study. Crystals of 3 and 4 suitable for X-ray
diffraction data collection were obtained by dissolving the samples in a
mixture of 1:1 dichloromethane/methanol and on standing at room
temperature for a week. Data were collected at 125 K using a Bruker
SMART APEX II CCD X-ray diffractometer. Structure resolution and
refinement were performed with SHELXTL;20 details are included in
Table 1. H atoms were calculated and constrained as riding on their
bound atoms.
Theoretical Study. The theoretical study involved calculations
using software programs from Accelrys.21 Density functional theory
(DFT) code DMol3 was applied to calculate energy, geometry, and
frequencies implemented in Materials Studio 5.5 (PC platform).22 We
employed the double numerical polarized (DNP) basis set that
includes all the occupied atomic orbitals plus a second set of valence
atomic orbitals and polarized d-valence orbitals,23 and correlation
generalized gradient approximation (GGA) was applied in the manner
suggested by Perdew-Burke-Ernzerhof (PBE);24 these are the
conditions for the highest-accuracy level available 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-consistent-
field 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 (CCL-
247, 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 Dulbecco's Modified Eagle's medium (DMEM)
(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%
Drugs. The four compounds under study were 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)-2,5-
diphenyltetrazolium bromide (MTT) assay was performed on all the
cell lines tested as described,25 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 four compounds at
concentrations ranging between 5 and 750 μM, bringing the final
volume to 0.2 mL/well. Each experiment included eight replications
per concentration tested; control samples were run with 0.2% DMSO.
At the end of the period of incubation, MTT (0.05 mL of a 2 mg/mL
stock solution in phosphate-buffered saline (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 0.120 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, v. 5.0 (GraphPad, San Diego, CA, USA). Differences
between IC50 values were analyzed statistically by analysis of variance
with Bonferroni post-test for multiple comparisons.
Docking Studies. These were performed with the molecular
mechanics CDOCKER package, a grid-based molecular docking
method that utilizes CHARMm26 in Discovery Studio 3.5 from
Accelrys.21 We took advantage of our previous work,16 where the
keto−enol fragment in curcumin had few interactions with the DNA
and was found to be energetically favored when pointing out of the
helix. Our general strategy was to replace the Ru-curcuminato complex
with [Ru(η6-p-cymene)(QNaph)Cl] (3) and minimize the system.
Compound 3 was geometrically optimized from original X-ray
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Figure 1. Development of potential energy (kcal/mol) vs conformation number for the dynamic cascade of the DNA model associated with
compound 3; conformation 952 has the minimum energy.
coordinates using the DFT program DMol3 (included in Materials
Studio version 6.1 from Accelrys21) using the same setting earlier
described for the (p-cymene)Ru(curcuminato)chloro complex.16
For the (p-cymene)Ru(curcuminato)chloro-DNA arrangement, the
binding site was shown to be between the ruthenium and the guanine
N7 helix. For the initial docking, complex 3 was graphically inserted
into the double helix of a DNA octamer species deposited in the
Protein Data Bank (www.Pdb.Org) (code 1N37).27 1N37 contains an
intercalating antibody (respinomycin D), which was excised and
replaced by 3 during the docking; one of the octamer’s bases facing
respinomycin D is guanine. We applied a solvation algorithm that
provided 1868 H2O molecules, 4 Cl−, and 18 Na+ ions. These, with
the 14 negatively charged phosphate groups, make up a neutral system.
We relocated the Cl− anion of 3 into the solvent area since the
expected reaction for the related Ru−Cl antitumor species is to cleave
the Ru−Cl bond after entering the cell.9
Since the CHARMm forcefield does not include ruthenium bonding
parameters, Ru was considered as an ion, similarly to our previous
work.16 Besides that, some constraints on bonds to optimize the
minimization and docking were imposed: the last two base pairs from
each end of the 1N37 were fixed, and various distances of the critical
interactions between the cationic Ru complex and 1N37 were
restrained: (1) Ru to N7 set at 2.1 Å; (2) distance restraint between
Ru and the p-cymene centroid with an upper threshold of 1.9 Å and
the lower of 1.8 Å; (3) all restraints included a force maximum and
force constant maximum at 200 kcal/mol and a rigid body harmonic
restraint at 10 kcal/mol.
In the Ru-curcuminato simulation16 the water molecules surround-
ing the double helix were removed, and the counterions were
maintained. For the present study we kept the solvent because the
water molecules better mimic a biological environment. The dielectric
constant was 4 as in the Ru-curcuminato simulation. Before our
successful dynamics cascade we found out that typing/placing a
forcefield on the molecules can modify the charge on specific atoms
and had to reassign the partial charges on the complex, making
O(carbonyl) {O(acyl) = Oa} as zero and O(anionic) {O(pyrazolonato
= Op} as −1, respectively. In addition, as mentioned above, we
assigned the Ru charge as +2, resulting in the remaining complex
having a +1 charge. The objective was to minimize the distance
between the ruthenium atom and N7(guanine) for ideal docking and
to verify whether the potentially intercalated naphthyl moiety would
stay in place. We first performed a 10 pose minimization as a
After removing the Cl− anion from 3, we placed the corresponding
cationic complex so that the Ru atom pointed to N7(guanine) along
the same direction where the Cl was originally placed, for example,
keeping the same bond angles for the coordination sphere. Then, we
rotated the Ru cationic complex along the Ru−N7(guanine) axis so
that the naphthyl moiety could be located in an area previously
occupied by respinomycin D, that is, potentially establishing π−π
interactions. During this process we rotated the arene moiety along the
Ru-arene centroid to avoid hindrance due to its isopropyl moiety.
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Scheme 2. Proligands HQR and Compounds 1−9
precautionary step for any obvious errors that may develop when
performing a minimization (as the dynamics cascade produces
hundreds of poses). The 10 poses were not very different from one
another; however, the distance between Ru and N7 lengthened.
Taking “bump” formation and chemically stable environments into
consideration, the cationic Ru complex was manually docked to fit our
aforementioned target criteria.
Subsequently, we performed the standard dynamics cascade. In the
initial simulation the Ru−N bond increased to about 2.7 Å, which is
consistent with the results obtained with Ru-curcuminato,16 and so we
increased the force maximum and force constant maximum to 1000
kcal/mol so as to afford a more realistic Ru−N bond distance. We also
changed production to be 200 000 with save results frequency at 1000,
10 000 steps for heating and equilibration with save results frequency
at 500. In summary, the standard dynamics cascade for 200 000 steps
at a save results frequency at 100, results in 1000 conformations.
Conformation 952 (−166 523 kcal/mol) showed the minimum
potential energy, see Figure 1, and was much lower than conformation
1(−155 010 kcal/mol). The greatest decrease of energy was found
after conformation 420, where the structure appears to find a region of
the conformational space with more favorable electrostatic inter-
actions.
Figure 3. X-ray crystal structure of [Ru(η6-benzene)(QMe)Cl] (4).
containing small amounts of water, affording the cationic
1
hydrated compounds 7 and 8 (Scheme 2). IR and H and 13C
NMR data confirm their proposed structure, in accordance with
similar compounds previously reported.17
RESULTS AND DISCUSSION
■
By interaction in methanol of proligands HQR and [(arene)-
RuCl2]2 in the presence of sodium methoxide, neutral
compounds 1−6 have been obtained (Scheme 2), which are
soluble in most organic solvents and also in water. Compounds
1 and 3 were then reacted with AgBF4 in acetonitrile,
Figures 2 and 3 show the molecular structures of compounds
3 and 4, determined using single-crystal X-ray diffraction,
displayed with ellipsoids at 50% probability. When comparing
the coordination sphere of both molecules (see Table 2), we
see that Ru−Cl distances are equivalent. In contrast, Ru−
O(pyrazolonato) {= Ru−Op} and Ru−O(acyl) {= Ru−Oa}
differ in [Ru(η6-p-cymene)(QNaph)Cl] (3) but are equal in
[Ru(η6-benzene)(QMe)Cl] (4). In a Ti-Q antitumor com-
pound,28 a Ti−Op bond distance shorter than Ti−Oa is
observed, which may be associated with a coordinative bond by
the acyl group. Both Ru−Cl bond distances are in agreement
with the related 40 structures stored in the crystallographic
database (CSD) having the framework “RuO2Cl(arene);” 34 of
them are p-cymene compounds. The mean Cl−Ru−O bond
angle (84.7°) in the CSD compares well with that observed in
both structures (85.2° (compound 3) and 84.3° (compound
4)). Also the CSD mean values of both the Ru−O (2.091 Å)
and the Ru−Cl (2.416 Å) bond distances are coincident with
values found for both structures.
The arene−centroid can be considered a fourth bond in a
potential tetrahedral arrangement, an alternative to the “piano-
stool” description by Sadler3 where Cl, Op, and Oa are the legs.
Figure 2. X-ray crystal structure of [Ru(η6-cymene)(QNapht)Cl] (3).
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Table 2. Geometrical Features in the Coordination Sphere of Compounds 3 and 4; Cen Stays for the Arene Ring Centroid; Op
= O(pyrazolonato), Oa = O(acyl)
Ru−Cl
Ru−Op
Ru−Oa
Ru−Cen
Cen−Ru−Cl
Cen−Ru−Op
Cen−Ru−Oa
3
4
2.4143(9)
2.418(2)
2.079(2)
2.095(5)
2.101(2)
2.091(4)
1.642
1.650
129.6
130.4
127.2
128.3
126.5
125.5
Figure 4. Antiproliferative effect of [Ru(η6-p-cymene)(QPh)Cl] (2), [Ru(η6-p-cymene)(QNaph)Cl] (3), [Ru(η6-hmb)(QMe)Cl] (5), and [Ru(η6-p-
cymene)(QMe)(H2O)]BF4 (7) on MCF-7, HCT116, A2780, A549, and U87 human cancer cells. IC50 values (μM) are the means SE of three or
four independent experiments and were extrapolated from dose response curves obtained at the end of 72 h of incubation in the presence of the
compounds. a, p < 0.001 vs 2, 5, and 7; b, p < 0.01 vs 2 and 5, p < 0.05 vs 7; c, p < 0.001 vs 2 and 5; d, p < 0.001 vs 2; e p < 0.001 vs 2, 5 and 7; f, p <
0.05 vs 2, p<0.01 vs 5 and 7.
arene group (see Table 2). In the 40 CSD stored structures, the
mean angles of (arene−centroid)−Ru−O and (arene−cent-
roid)−Ru−Cl are identical (129.5°); Table 2 shows equiv-
alence in structures 3 and 4 for (arene−centroid)−Ru−Cl and
smaller angles for (arene−centroid)−Ru−O. The (arene−
Table 3. Antiproliferative Effect of Four Ru-arene-Q
compounds on MCF-7, HCT116, A2780, A549, and U87
Human Cancer Cells. IC50 Values (μM) are the Means SE
of Three or Four Independent Experiments and Were
Extrapolated from Dose Response Curves Obtained at the
End of 72 h of Incubation in the Presence of the
Compounds
́
́
centroid)−Ru distance is 1.642 Å (compound 3) and 1.650 Å
(compound 4). This may suggest a stronger arene−Ru binding
in the former, probably due to the donor features of Me and
isopropyl substituents in p-cymene. However, the range of this
distance in CSD stored structures (1.63 Å−1.66 Å) also
includes the value shown by compound 4. No arene = benzene
compounds are included in the 40 stored structures to make a
comparison. Interestingly, even if p-cymene possesses larger
substituents than benzene, it does not affect the coordination
sphere geometry (except for the slightly stronger arene−Ru
bond). Since this ligand is a natural product it might be
preferable for interaction with DNA, especially since the
benzene ligand, used in 4, is carcinogenic.
2
5
7
3
a
MCF7
HCT116
A2780
A549
363 44
409 59
494 41
474 23
401 45
465 37
522 38
631 76
475 19
61
5
b
280 24
57 12
c
c
41
5
51
60
5
3
5
d
e
f
790
5
661 30
614 56
U87
437 73
64
a
b
c
p < 0.001 vs 2, 5, and 7. p < 0.01 vs 2 and 5, p < 0.05 vs 7. p < 0.001
d
e
f
vs 2 and 5. p < 0.001 vs 2. p < 0.001 vs 2, 5, and 7. p < 0.05 vs 2, p <
0.01 vs 5 and 7.
We were stimulated to explore the antitumor activity of Ru-
(4-acylpyrazolon-5-ato) compounds after seeing the pioneering
work by Sadler with classical diketones such as acetylacetona-
to15 and after studying the antitumor activity of an equivalent
This centroid subtends bond angles in the range of 126−129°
in compound 3 and 125−128° in compound 4, that is, making
the other 3 “tetrahedral” bond angles (Op−Ru−Cl, Op−Ru−
Oa, Oa−Ru−Cl) closer together, probably due to the bulky
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Figure 5. Conformation 952 for the whole DNA−Ru(p-cymene)QNaph system; water and counterions are omitted. In this model the cationic
coordination complex is shown as wide stick style, except for coordinated atoms, as ball style; N7(guanine) is purple ball style; bonds in the
coordination sphere are solid green. The p-cymene moiety is located on the right lower area of the complex, showing a green arene centroid bound
to Ru. Both DNA terminal pairs are blue marked, indicating that they were fixed during the dynamic cascade. One DNA helix is red, and the other is
aquamarine; π−π stacking interactions, involving DNA bases with the QNaph ligand, are depicted as solid green lines.
species containing a natural β-diketone, curcumin, coordinated
to a RuII(p-cymene) species.16 Moreover, recent studies have
shown that peripheral variation in the anionic ligand of
Ru(arene) compounds can establish interstrand DNA con-
nections,29 suggesting options for extended interaction far from
the metal coordination sphere. Indeed, our idea was to look for
some ligand moiety interaction with DNA through intercala-
tion, a widely explored structural feature associated with
significant biological activity.30 In Sadler’s work intercalation
due to special arenes was linked to increased antitumor
activity,12 but, to our knowledge, the role of the anionic ligands
as intercalating agents is much less explored. For instance,
antitumor studies in Ru structures having an anionic ligand with
a naphthalimide substituent were less efficient than those
having an arene-substituted naphthalimide.31 In addition,
although 9-aminoacridine was suggested as a potential ligand
for DNA intercalation and coordinated to a Ru(p-cymene)
framework, its interaction was not described in detail.32
Furthermore, a review describing the intercalation of Ru(arene)
compounds on DNA3 cited only data based on arenes
containing flexible substituents.
We hypothesized that position 4 in 4-acyl-5-pyrazolone
ligands was potentially useful for DNA intercalation and
performed some preliminary ab initio and docking inves-
tigations when there was increased aromaticity in position 4. As
a result of our findings, we designed this series of Ru-Q-arene
complexes. Our experimentally found IC50 data (Figure 4 and
Table 3) confirm that compound 3, containing a 4-naphthyl
moiety in the ligand QNaph, has increased antitumor activity for
all cell lines, when compared to related 4-methyl and 4-Ph
substituted Ru complexes.
Metal affinity for N7(guanine) is widely accepted as a key
factor explaining the antitumor activity of cisplatin and Ru(II)
complexes.33,34 Our synthesized model complex [Ru(η6-p-
cymene)(QNaph)(9-ethylguanine)]BF4 (9) was easily obtained
at room temperature, as shown by ESI-MS data, after simple
mixing of [Ru(η6-p-cymene)(QNaph)(H2O)]BF4 (8) and 9-
ethylguanine (Scheme 2), and suggests the feasibility of Ru−
N(7) guanine binding. Indeed, the hydrolysis of the related Cl
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Figure 6. Details of Ru coordination sphere in conformation 952, the minimum energy obtained from the dynamic cascade, showing one helix in red
and the other helix in green. The terminal pairs, only one partially shown on the right (blue marked), were fixed during the process. The p-cymene
ring is on the right area. Light-green dashed lines, indicating H-bonds between DNA bases, are also shown. Some atoms of the red helix are omitted
for clarity. Oa = Oacyl and Op = Opyrazolonato correspond to O(carbonyl) and O(hydroxyl), respectively, in Scheme 1.
complex [Ru(η6-p-cymene)(QNaph)Cl] (3) to yield the cationic
species [Ru(η6-p-cymene)(QNaph)(H2O)]+ (8) is expected to
be formed inside the cell, where the chloride concentration is
lower. Therefore, a Cl derivative appears to be more protected,
as a neutral species, than a cationic complex as shown for
cisplatin; for example, it would allow the hydrolyzed “active
species” to be generated in situ. However, the only biologically
tested cationic species in this study (compound 7), containing a
pendant moiety variant (Et, Ph, and Naph) and so are related
to the Met, Ph, and Naph substituents in this study. These Ru
complexes were tested against A2780 and A2780R cell lines,
but their IC50 values did not show a preference for the Ru(p-
cymene)LNaph,PhCl, which suggests that the naphthyl moiety
does not intercalate with DNA. This appears in contrast with
the IC50 values for the HL ligands, which show higher activity
for HLNaph,Ph. It seems, therefore, that ligands containing
extended aromatic moieties, such as naphthyl, can intercalate in
DNA, but the intercalation mechanism through their Ru(arene)
complexes is more subtle.
Q
Me ligand, is also the most active (A2780 cell line), suggesting
that the complex [Ru(η6-p-cymene)(QNaph)(H2O)]BF4 (8) can
be even more active than its parent compound [Ru(η6-p-
cymene)(QNaph)Cl] (3), as it would combine the naphthyl
preference for intercalation, see infra, with the cationic feature
of 7. Compound 8 is currently being tested, and results will be
published in the near future.
CONCLUSIONS
■
Following our theoretical study of Ru(arene)(curcumin)Cl
complexes, we designed related potential antitumor compounds
Ru(arene)(β-diketonato) using 4-acyl-5-pyrazolone ligands
(HQ) to experimentally test ligand intercalation with DNA.
Compound Ru(p-cymene)QNaphCl, containing a peripheral
naphthyl moiety in position 4, shows in vitro antitumor activity
systematically higher than compounds having other substitu-
ents in all cell lines. Docking studies show that [Ru(p-
cymene)(QNaph)Cl] intercalation with DNA is feasible.
However, the curcumin ligand activity still is higher than the
most active species in the Q series. The present study may
stimulate the design of chelating ligands useful for DNA
intercalation in metal antitumor compounds
Consequently, compound 3 was selected for a docking study
on a DNA octamer, from which conformation 952 has the
lowest potential energy and is shown in Figure 5; additional
details of the Ru coordination sphere are depicted in Figure 6.
The guanine base is linked through N7 to Ru (2.565 Å) and
establishes a π−π interaction with the center of one ring of the
naphthyl moiety (3.394 Å); the Ru−Op bond distance is 2.404
Å, and the p-cymene ligand protrudes outside the DNA helix.
From our docking calculations, we felt that a larger arene such
as hexamethylbenzene would hinder the Ru−N7(guanine)
binding, although such a restraint would probably not be
present if interacting with a single helix.
The arene intercalation may induce strong deformation of
the double helix, as observed from NMR data of the 14-base
duplex (5′-ATACATGGTACATA-3′)/(3′-TATGTAC-
CAT17G18TAT-5′) ruthenated at N7(G18) by [(η6-biphenyl)-
Ru(ethylendiamine)]2+. One conformer has the biphenyl arene
intercalated between G18 and T17, while the other has no
biphenyl intercalation, but shows T17 flipped out of the helix.35
In contrast, our conformation 952 keeps all the DNA bases in
place, as seen in Figure 6 where H-bond interactions between
DNA bases are depicted. At present, there is not enough
information to determine whether better antitumor activity is
induced by more or less helix deformation after ruthenation.
ASSOCIATED CONTENT
■
S
* Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: caruso@vassar.edu (F.C.).
*E-mail: fabio.marchetti@unicam.it (F.M.).
A recent series of ketoamine ligands HL (HLet,Ph, HLPh,Ph
,
and HLNaph,Ph), closely related to 4-acyl-5-pyrazones, has been
Notes
used to synthesize Ru(p-cymene)LCl complexes.36 They have a
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Jodi Shaulky at Accelrys for helpful discussion. M.R.
thanks the HHMI Foundation through Grant 52006322 and
the U.S. National Science Foundation, through the Grant
0521237, for the X-ray diffractometer.
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