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Water-Soluble Mixed-Ligand Ruthenium(II) and Osmium(II) Arene Complexes with High Antiproliferative Activity
Organometallics 2008, 27, 6587–6595
6587
Water-Soluble Mixed-Ligand Ruthenium(II) and Osmium(II) Arene
Complexes with High Antiproliferative Activity
Raffael Schuecker, Roland O. John, Michael A. Jakupec, Vladimir B. Arion,* and
Bernhard K. Keppler*
Institute of Inorganic Chemistry, UniVersity of Vienna, Währinger Str. 42, A-1090 Vienna, Austria
ReceiVed August 12, 2008
The synthesis of ruthenium(II) and osmium(II) arene complexes of the general formula [(η6-p-cymene)M(oxine)(Hazole)]X, where M ) Ru, Os; oxine ) deprotonated 8-hydroxyquinoline, and Hazole ) azole
heterocycle, i.e., pyrazole (Hpz), indazole (Hind), imidazole (Him), benzimidazole (Hbzim), or 5,6dimethylbenzimidazole (Hdmbzim); X ) CF3SO3-, PF6-, or Cl-, combining ligands involved in metalbased complexes that are currently in clinical development, is reported. The compounds have been
comprehensively characterized by elemental analysis, ESI mass spectrometry, spectroscopy (IR, UV-vis,
NMR), and X-ray crystallography. The synthesis of these complexes was performed in order to achieve
a balance between aqueous solubility and lipophilicity. The ruthenium(II) and osmium(II) compounds
exhibit excellent cytotoxic effects in the tumor cell lines CH1 and SW480, with IC50 values ranging
from 3.3 to 9.4 µM. As expected, the compounds are water soluble and show no evidence of hydrolysis
or ligand exchange in aqueous media, which makes them noteworthy candidates for further development.
The complexes [(η6-p-cymene)M(oxine)(Hazole)]+ do not react with DNA purine bases, even if the latter
are present in excess. However, the complex [(η6-p-cymene)Os(oxine)Cl] reacts with 9-methyladenine
(meade) to form [(η6-p-cymene)Os(oxine)meade]+, which was isolated and characterized by X-ray
diffraction as a hexafluorophosphate salt.
Introduction
Coordination chemistry offers great opportunities for the
development of metal-containing anticancer agents.1 Besides the
platinum compounds used routinely in the clinic,2,3 two
ruthenium-based complexes, namely, (H2ind)[trans-RuCl4(Hind)2] (KP1019)4 and (H2im)[trans-RuCl4(Him)(DMSO)]
(NAMI-A),5 are currently in clinical studies. In addition, the
gallium complex Ga(oxine)3 (KP46), where oxine ) deprotonated 8-hydroxyquinoline, showed promising results in a phase
I clinical trial.6 Organometallic compounds of “three-leg pianostool geometry” of the general formula [(η6-arene)M(XY)(Z)]
(where arene is biphenyl, benzene, or p-cymene; M ) Ru, Os;
XY is a bidentate chelating ligand; and Z a monodentate ligand)
and [(η6-arene)Ru(PTA)Cl2] complexes, where PTA ) 1,3,5triaza-7-phosphaadamantane,7 have also gained much attention
* To whom correspondence should be addressed. E-mail: vladimir.arion@
univie.ac.at (V.B.A.), bernhard.keppler@univie.ac.at (B.K.K.). Fax: +43
1 4277 52630 (V.B.A.), +43 1 4277 52680 (B.K.K.).
(1) Guo, Z.; Sadler, P. J. Angew. Chem., Int. Ed. 1999, 38, 1512–1531.
(2) Lippert, B., Ed. Cisplatin. Chemistry and Biochemistry of a Leading
Anticancer Drug; VHCA & Wiley-VCH: Zurich, 1999.
(3) Galanski, M.; Arion, V. B.; Jakupec, M. A.; Keppler, B. K. Curr.
Pharm. Des. 2003, 9, 2078–2089.
(4) Hartinger, C. G.; Zorbas-Seifried, S.; Jakupec, M. A.; Kynast, B.;
Zorbas, H.; Keppler, B. K. J. Inorg. Biochem. 2006, 100, 891–904.
(5) Alessio, E.; Mestroni, G.; Bergamo, B.; Sava, G. Curr. Top. Med.
Chem. 2004, 4, 1525–1535.
(6) Collery, P.; Jakupec, M. A.; Kynast, B.; Keppler, B. K. Met. Ions
Biol. Med. 2006, 9, 521–524.
(7) (a) Ang, W. H.; Dyson, P. J. Eur. J. Inorg. Chem. 2006, 20, 4003–
4018. (b) Ang, W. H.; Daldini, E.; Scopelliti, R.; Juillerat-Jeannerat, L.;
Dyson, P. J. Inorg. Chem. 2006, 45, 9006–9013. (c) Gossens, C.; Dorcier,
A.; Dyson, P. J.; Rothlisberger, U. Organometallics 2007, 26, 3965–3975.
(d) Dyson, P. J.; Sava, G. Dalton Trans. 2006, 1929–1933.
as promising antitumor agents.8 Recent studies have shown that
the aqueous behavior of [(η6-arene)M(XY)(Z)] compounds is
highly dependent on the identity of the ligands and especially
of the chelating one.9,10
Our idea for the design of novel metal-based compounds with
anticancer activity explored in the present study is based on a
combination of typical features of the above-mentioned compounds in order to achieve both efficacy and a balance of
aqueous solubility and lipophilicity. We found the half-sandwich
piano-stool geometry with Ru(II) and Os(II) as metal ions and
p-cymene as the arene ligand well suited for further development, because this coordination mode stabilizes the metal ion
in its low oxidation state. As suggested for various Ru(III)
complexes including KP1019, the reduced Ru(II) complex11
might be the biologically relevant species. Thus, KP1019 acts
as a prodrug, being reduced in tumor tissue to its active species.
The use of a half-sandwhich piano-stool complex enables the
synthesis of air-stable Ru(II) and Os(II) complexes. The choice
of the remaining components seems to be crucial. If only
(8) (a) Aird, R. E.; Cummings, J.; Ritchie, A. A.; Muir, M.; Morris,
R. E.; Chen, H.; Sadler, P. J. Br. J. Cancer 2002, 86, 1652–1657. (b)
Melchart, M.; Sadler, P. J. Bioorganometallics 2006, 39–64. (c) Morris,
R. E.; Aird, R. E.; Murdoch, P. D. S.; Chen, H.; Cummings, J.; Hughes,
N. D.; Parsons, S.; Parkin, A.; Boyd, G.; Jodrell, D. I.; Sadler, P. J. J. Med.
Chem. 2001, 44, 3616–3621. (d) Scolaro, C.; Bergamo, A.; Brescacin, L.;
Delfino, R.; Coccietto, M.; Laurenczy, G.; Geldbach, T. J.; Sava, G.; Dyson,
P. J. J. Med. Chem. 2005, 48, 4161–4171. (e) Dorcier, A.; Ang, W. H.;
Bolano, S.; Gonsalvi, L.; Juillerat-Jeannerat, L.; Laurenczy, G.; Peruzzini,
M.; Phillips, A. D.; Zanobini, F.; Dyson, P. J. Organometallics 2006, 25,
4090–4096.
(9) Wang, F.; Chen, H.; Parsons, S.; Oswald, I. D. H.; Davidson, J. E.;
Sadler, P. J. Chem.-Eur. J. 2003, 9, 5810–5820.
(10) Chen, H.; Parkinson, J. A.; Novakova, O.; Bella, J.; Wang, F.;
Dawson, A.; Gould, R.; Parsons, S.; Brabec, V.; Sadler, P. J. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 14623–14628.
(11) Clarke, M. J. Coord. Chem. ReV. 2003, 236, 209–233.
10.1021/om800774t CCC: $40.75 2008 American Chemical Society
Publication on Web 11/14/2008
6588 Organometallics, Vol. 27, No. 24, 2008
Chart 1. Compounds Reported in This Worka
Schuecker et al.
diffraction characterization, hydrolytic stability, reactivity toward
purine bases, and high antiproliferative activity in two human
cancer cell lines in Vitro.
Experimental Section
a Underlined complexes have been characterized by X-ray crystallography; atom labeling was introduced for assignment of resonances
in NMR spectra.
monodentate ligands are employed, this can generate hydrolysis
products that make administration difficult.12,13 Taking into
account the composition of KP46, we chose 8-hydroxyquinoline
as an N,O-chelating ligand in order to prevent unwanted
hydrolysis reactions. The sixth coordination site is occupied by
an azole heterocycle, such as those acting as a ligand in KP1019
or NAMI-A. We expected to generate complexes capable of
penetrating the cell membrane readily, at the same time
possessing sufficient water solubility. To this end, azole
heterocycles differing in their aqueous solubility and lipophilicity
(Chart 1) were chosen. Since Os(II) as the heavier congener of
Ru(II) is expected to be kinetically more inert,14,17-21 it was
obvious to compare the hydrolytic behavior and the cytotoxicity
of Ru(II) and Os(II) complexes with identical sets of ligands.
Herein, we report on the synthesis of Ru(II) and Os(II) arene
complexes of the general formula [(η6-p-cymene)M(oxine)(Hazole)]X (where M ) Ru, Os; oxine ) deprotonated
8-hydroxyquinoline; Hazole ) azole heterocycle, i.e., indazole,
pyrazole, imidazole, benzimidazole, or 5,6-dimethylbenzimidazole; X ) [PF6]- of OTf-), their spectroscopic and X-ray
(12) Yan, Y. K.; Melchart, M.; Habtemariam, A.; Sadler, P. J. Chem.
Commun. 2005, 38, 4764–4776.
(13) Peacock, A. F. A.; Melchart, M.; Deeth, R. J.; Habtemariam, A.;
Parsons, S.; Sadler, P. J. Chem.-Eur. J. 2007, 13, 2601–2613.
(14) (a) Shriver, D. F.; Atkins, P. W. Inorganic Chemistry, 3rd ed.;
Oxford University Press: Oxford, 1999; p 245. (b) Griffith, W. P. In
ComprehensiVe Coordination Chemistry, Vol. 4; Pergamon: Oxford, 1987;
pp 519-633. (c) Ashby, M. T.; Alguindigue, S. S.; Khan, M. A.
Organometallics 2000, 19, 547–552. (d) George, R.; Andersen, J.-A. M.;
Moss, J. R. J. Organomet. Chem. 1995, 505, 131–133. (e) Halpern, J.; Cai,
L.; Desrosiers, P. J.; Lin, Z. J. Chem. Soc., Dalton Trans. 1991, 717–723.
(15) Armarego, W. L. Purification of Laboratory Chemicals; Elsevier
Science: Zurich, 2003.
(16) Bennett, M. A.; Smith, A. K. J. Chem. Soc., Dalton Trans. 1974,
233–241.
(17) Kiel, W. A.; Ball, R. G.; Graham, W. A. G. J. Organomet. Chem.
1990, 383, 481–496.
(18) Sue, P.; Wetroff, G. Bull. Soc. Chim. 1935, 2, 1002–1007.
(19) Gemel, C.; John, R.; Slugovic, C.; Mereiter, K.; Schmid, R.;
Kirchner, K. J. Chem. Soc., Dalton Trans. 2000, 2607–2612.
(20) Peacock, A. F. A.; Parsons, S.; Sadler, P. J. J. Am. Chem. Soc.
2007, 129, 3348–3357.
(21) (a) Otwinowski, Z.; Minor, W. In Methods in Enzymology, Vol.
276 (“Macromolecular Crystallography”, part A); Carter, C. W., Jr., Sweet,
R. M., Eds.; Academic Press: New York, 1997; pp 307-326. (b) SAINTPlus, version 7.06a and APEX2; Bruker-Nonius AXS Inc.: Madison, WI,
2004.
Starting Materials. All chemicals were standard reagent grade
and used without further purification. The solvents were purified
according to standard procedures.15 RuCl3 and OsO4 were purchased
from Johnson Matthey. AgCF3SO3, 8-hydroxyquinoline, and the
azole compounds (imidazole, benzimidazole, 5,6-dimethylbenzimidazole, pyrazole, and indazole) were purchased from Fluka. The
deuteriated solvents were from Aldrich and dried over 4 Å
molecular sieves. The precursors [(η6-p-cymene)RuCl2]2,16 [(η6-pcymene)OsCl2]2,17 K(oxine),18 [(η6-p-cymene)(oxine)RuCl],19 and
[(η6-p-cymene)(oxine)OsCl]20 were prepared according to literature
protocols.
Preparation of Aqueous Solutions of [(η6-p-cymene)Ru(oxine)(H2O)]NO3 (2a) and [(η6-p-cymene)Os(oxine)(H2O)]NO3 (2b) for a Cytotoxicity Assay. In an Eppendorf tube to [(η6p-cymene)(oxine)RuCl] (1a) (20 µM) or [(η6-p-cymene)(oxine)OsCl] (1b) (20 µM) were added AgNO3 (20 µM) and H2O (1 mL).
The tubes were treated in an ultrasound bath for 5 min and then
centrifuged at 14000 rpm for 5 min. The clear solutions were used
for cytotoxicity tests.
Synthesis of Metal Complexes. All manipulations were performed under argon by using standard Schlenk techniques.
Complexes with Azole Ligands, General Procedure A. A
suspension of 1a (100 mg, 0.24 mmol) and silver triflate (65 mg,
0.25 mmol) in THF (5 mL) was stirred at room temperature for
2 h, and then the azole ligand (0.24 mmol) was added. After stirring
the reaction mixture for 2 h, the solution was transferred to a
centrifuge tube and centrifuged at 4000 rpm for 5 min. The
supernatant was transferred into a Schlenk tube, and the solvent
was removed under reduced pressure. The residue was dissolved
in dichloromethane (2 mL). Addition of diethyl ether (15 mL)
resulted in precipitation of a yellow product, which was filtered
off, washed with diethyl ether (2 × 5 mL), and dried in Vacuo.
Complexes with Azole Ligands, General Procedure B. To a
suspension of 1b (250 mg, 0.5 mmol) and silver triflate (128 mg,
0.5 mmol) in methanol (5 mL), stirred at ambient temperature for
30 min, was added the azole ligand (0.5 mmol), and stirring
continued for 30 min. The solvent was removed under reduced
pressure and the remaining solid suspended in dichloromethane (5
mL). Filtration of the suspension over Celite and concentration of
the filtrate to 0.5 mL, followed by addition of diethyl ether (5 mL),
gave rise to a yellow precipitate. The mixture was stirred for 18 h,
and the product was filtered off, washed with diethyl ether (2 × 2
mL), and dried in Vacuo.
[(η6-p-cymene)Ru(oxine)(Hpz)]CF3SO3 (3a). General Procedure
A. Yield: 96 mg, 67% of a bright yellow powder. Anal. Calcd for
C23H24F3N3O4RuS (Mr 596.16): C, 46.31; H, 4.06; N, 7.04; S, 5.37.
Found: C, 46.14; H, 4.04; N, 6.94; S, 5.21. ESI-MS: positive, m/z
447 [(η6-p-cymene)Ru(oxine)(Hpz)]+, 380 [(η6-p-cymene)Ru(oxine)]+; negative, m/z 149 [CF3SO3]-. Solubility in water: 3.4 mM.
IR spectrum (selected bands, KBr, νmax, cm-1): 523, 631, 749, 799,
1033, 1054, 1109, 1167, 1251, 1324, 1376, 1468, 1496, 1566, 2965,
3056, 3138. UV-vis spectrum [methanol, λmax, nm (ε, M-1 cm-1)]:
263 (19 330), 240 (17 130) 420 (4100), 321 (3090), 335 (3050).
1
H NMR [400.13 MHz, CDCl3, δH, ppm]: 13.00 [bs (broad singlet),
1H, pyrazoleNH], 9.60 (d, 3JHH ) 4.5 Hz, 1H, hc2), 8.07 (d, 3JHH )
8.5 Hz, 1H, hc4), 7.56 (d, 3JHH ) 2.0 Hz, 1H, pyrazole3/5), 7.48 (q,
3
JHH ) 4.5 Hz, 1H, hc3), 7.39 (d, 3JHH ) 2.0 Hz, 1H, pyrazole3/5),
7.32 (t, 3JHH ) 8.0 Hz, 1H, hc6), 7.03 (d, 3JHH ) 8.0 Hz, 1H, hc7),
6.85 (d, 3JHH ) 8.0 Hz, 1H, hc5), 6.12 (m, 1H, pyrazole4), 6.04 [d,
3
JHH ) 6.0 Hz, 1H, cy (cymene)], 5.83 (t, 3JHH ) 7.0 Hz, 2H, cy),
5.79 (d, 3JHH ) 6.0 Hz, 1H, cy), 2.39 (m, 1H, CHMe2), 1.87 (s,
3H, CH3), 1.11 (d, 3JHH ) 6.5 Hz, 3H, CHMe2), 0.89 (d, 3JHH )
Ru(II) and Os(II) Complexes with High AntiproliferatiVe ActiVity
Organometallics, Vol. 27, No. 24, 2008 6589
7.0 Hz, 3H, CHMe2). 13C{1H} NMR [100.63 MHz, CDCl3, δC,
ppm]: 167.5 (C8), 152.2 (C2), 143.4 (C8a), 137.8 (C4), 136.7
(pyrazole-C3/5), 134.0 (pyrazole-C3/5), 131.0 (C6), 130.1 (C4a), 123.0
(C3), 114.8 (C7), 113.2 (C5), 105.2 (pyrazole-C4), 92.4 (cy-C1), 91.7
(cy-C4), 75.6, 72.4, 72.1, 71.2 (cy-C2,3,5,6), 30.6 (CHMe2), 23.0, 22.1
(CHMe2), 17.8 (Me).
[(η6-p-cymene)Os(oxine)(Hpz)]CF3SO3 (3b). General Procedure
B. Yield: 207 mg, 61% of a yellow powder. Anal. Calcd for
C23H24F3N3O4OsS (Mr 685.74): C, 40.28; H, 3.53; N, 6.13; S, 4.68.
Found: C, 40.04; H, 3.42; N, 6.17; S, 4.72. ESI-MS: positive, m/z
538 [(η6-p-cymene)Os(oxine)(Hpz)]+, 470 [(η6-p-cymene)Os(oxine)]+; negative, m/z 149 [CF3SO3]-. Solubility in water: 1.2 mM.
IR spectrum (selected bands, KBr, νmax, cm-1): 520, 636, 760, 822,
1031, 1051, 1112, 1164, 1247, 1289, 1323, 1374, 1466, 1500, 1575,
2966, 3058, 3141. UV-vis spectrum [water, λmax, nm (ε, M-1
cm-1)]: 230 (20 430), 261 (19 370), 418 (4220), 324 (3190), 336
(3070).
NMR [100.63 MHz, CDCl3, δC, ppm]: 170.4 (C8), 151.7 (indazoleC3a), 149.5 (C2), 144.8 (C8a), 138.0 (C4), 130.5 (C6), 130.4 (C4a),
128.2 (indazole-C3), 122.6 (indazole-C7a), 122.3 (C3), 122.2 (indazole-C6), 119.6 (indazole-C7), 117.9 (indazole-C5), 115.8 (indazole-C4), 114.8 (C7), 112.2 (C5), 94.0 (cy-C1), 93.6 (cy-C4), 77.4,
74.2, 72.9, 71.2 (cy-C2,3,5,6), 31.6 (CHMe2), 23.5, 22.9 (CHMe2),
18.1 (Me). Single crystals of 5b · 3CH3OH suitable for X-ray
diffraction measurements were grown in methanol.
[(η6-p-cymene)Ru(oxine)(Him)]CF3SO3 (6a). General Procedure A. Yield: 98 mg, 68% of a pale yellow powder. Anal. Calcd
for C23H24F3N3O4RuS (Mr 596.16): C, 46.31; H, 4.06; N, 7.04; S,
5.37. Found: C, 45.90; H, 3.97; N, 6.95; S, 5.19. ESI-MS: positive,
m/z 447 [(η6-p-cymene)Ru(oxine)(Him)]+, 380 [(η6-p-cymene)Ru(oxine)]+; negative, m/z 149 [CF3SO3]-. Solubility in water: 2.8
mM. IR spectrum (selected bands, KBr, νmax, cm-1): 414, 520, 637,
748, 787, 824, 906, 1029, 1166, 1270, 1318, 1382, 1467, 1501,
1577, 1926, 2694, 2969, 3109. UV-vis spectrum [methanol, λmax,
nm (ε, M-1 cm-1)]: 231 (20 430), 266 (19 640). 1H NMR [400.13
MHz, CDCl3, δH, ppm]: 11.41 (bs, 1H, imidazoleNH), 9.37 (d, 3JHH
) 4.5 Hz, 1H, hc2), 7.48 (m, 2H, hc4, imidazole2), 7.41 (dd, 3JHH
) 5.2 Hz, 4JHH ) 3.4 Hz, 1H, hc3), 7.29 (t, 3JHH ) 8.0 Hz, 1H,
hc6), 6.98 (d, 3JHH ) 7.5 Hz, 1H, hc7), 6.90 (s, 1H, imidazole4),
6,83 (m, 2H, hc5, imidazole5), 5.90 (d, 3JHH ) 6.0 Hz, 1H, cy),
5.75 (d, 3JHH ) 5.5 Hz, 1H, cy), 5.65 (d, 3JHH ) 6.0 Hz, 1H, cy),
5.61 (d, 3JHH ) 5.5 Hz, 1H, cy), 2.45 (m, 3JHH ) 7.0 Hz, 1H,
CHMe2), 1.85 (s, 3H, CH3), 1,07 (d, 3JHH ) 6.5 Hz, 3H, CHMe2)
0.96 (d, 3JHH ) 7.0 Hz, 3H, CHMe2). 13C{1H} NMR [100.63 MHz,
CDCl3, δC, ppm]: 168.0 (C8), 151.0 (C2), 143.6 (C8a), 138.5 (C4),
138.6 (imidazole-C2), 130.5 (C6), 130.2 (C4a), 127.7 (imidazoleC4), 123.3 (C3), 118.2 (imidazole-C5), 115.0 (C7), 112.1 (C5), 103.5
(cy-C1), 101.5 (cy-C4), 85.9, 84.7, 82.0, 81.5 (cy-C2,3,5,6), 31.4
(CHMe2), 22.9, 22.3 (CHMe2) and 18.3 (Me). X-ray diffraction
quality single crystals were obtained by slow diffusion of diethyl
ether into a CH2Cl2 solution of 6a.
[(η6-p-cymene)Os(oxine)(Him)]CF3SO3 (6b). General Procedure B. Yield: 211 mg, 62% of a yellow powder. Anal. Calcd for
C23H24F3N3O4OsS (Mr 685.74): C, 40.28; H, 3.53; N, 6.13; S, 4.68.
Found: C, 40.03; H, 3.47; N, 6.04; S, 4.55. ESI-MS: positive, m/z
470 [(η6-p-cymene)Os(oxine)]+; negative, m/z 149 [CF3SO3]-.
Solubility in water: 0.9 mM. IR spectrum (selected bands, KBr,
νmax, cm-1): 520, 637, 749, 824, 1028, 1070, 1107, 1164, 1272,
1318, 1383, 1467, 1502, 1577, 2635, 2781, 2928, 2964, 3107.
UV-vis spectrum [methanol, λmax, nm (ε, M-1 cm-1)]: 233
(17 220), 261 (16 560), 421 (4260), 324 (3160), 338 (3030).
[(η6-p-cymene)Os(oxine)(Him)]Cl (6c). A mixture of 1b (200
mg, 0.4 mmol) and imidazole (27 mg, 0.4 mmol) in methanol (3
mL) was stirred for 18 h at ambient temperature. The resulting
clear solution was filtered over Celite and concentrated under
reduced pressure to 0.5 mL. Addition of diethyl ether (5 mL)
resulted in a brown-yellow oil, which solidified upon vigorous
stirring for 5 h. The bright yellow solid was collected by filtration,
washed with diethyl ether (2 × 2 mL), and dried in Vacuo. Yield:
189 mg, 83% of yellow hygroscopic powder. Anal. Calcd for
C22H24ClN3OOs (Mr 572.13): C, 45.47; H, 4.34; N, 7.23. Found:
C, 45.30; H, 4.08; N, 7.11. ESI-MS: positive, m/z 470 [(η6-pcymene)Os(oxine)]+. Solubility in water: 52.3 mM. IR spectrum
(selected bands, KBr, νmax, cm-1): 526, 753, 824, 1076, 1111, 1283,
1320, 1377, 1465, 1499, 1573, 2625, 2800, 2909. UV-vis spectrum
[water, λmax, nm (ε, M-1 cm-1)]: 232 (18 110), 261 (17 480), 422
(4410), 442 (3940), 325 (3320), 337 (3190).
[(η6-p-cymene)Ru(oxine)(Hbzim)]CF3SO3 (7a). General Procedure A. Yield: 116 mg, 75%. Anal. Calcd for C27H26F3N3O4RuS
(Mr 646.64): C, 50.15; H, 4.05; N, 6.50; S, 4.96. Found: C, 49.65;
H, 3.95; N, 6.47; S, 4.79. ESI-MS: positive, m/z 647 [(η6-pcymene)Ru(oxine)(Hbzim)]+, 380 [(η6-p-cymene)Ru(oxine)]+; negative, m/z 149 [CF3SO3]-. Solubility in water: 0.9 mM. IR spectrum
(selected bands, KBr, νmax, cm-1): 521, 563, 640, 775, 837, 1112,
[(η6-p-cymene)Ru(oxine)(Hind)]CF3SO3 · CH2Cl2 (4a · CH2Cl2). General Procedure A. Purification by crystallization from
dichloromethane/diethyl ether gave bright orange crystals of X-ray
diffraction quality. Yield: 114 mg, 65%. Anal. Calcd for
C27H26F3N3O4RuS · CH2Cl2 (Mr 731.58): C, 45.97; H, 3.86; N, 5.74;
S, 4.38. Found: C, 46.04; H, 3.87; N, 5.79; S, 4.39. ESI-MS:
positive, m/z 646 [(η6-p-cymene)Ru(oxine)(Hind)]+, 380 [(η6-pcymene)Ru(oxine)]+; negative, m/z 149 [CF3SO3]-. IR spectrum
(selected bands, KBr, νmax, cm-1): 519, 639, 749, 823, 1031, 1111,
1170, 1285, 1321, 1375, 1467, 1500, 1574, 2972, 3073, 3225.
UV-vis spectrum [methanol, λmax, nm (ε, M-1 cm-1)]: 265
(21 440), 230 (20 040), 408 (4760), 320 (3370), 334 (3150). 1H
NMR [400.13 MHz, CDCl3, δH, ppm]: 12.99 (bs, 1H, indazoleNH),
9.58 (d, 3JHH ) 4.0 Hz, 1H, hc2), 8.05 (d, 3JHH ) 8.0 Hz, 1H, hc4),
7.90 (s,1H, indazole3), 7.64 (d, 3JHH ) 8.5, 1H, indazole4), 7.48
(m, 2H, hc3, indazole7), 7.34 (m, 2H, hc6, indazole6), 7.07 (t, 2H,
hc7, indazole5), 6.83 (d, 3JHH ) 8.0 Hz, 1H, hc5), 6.12 (d, 3JHH )
6.0 Hz, 1H, cy), 5.90 (m, 3H, cy), 2.39 (m, 3JHH ) 6.8 Hz, 1H,
CHMe2), 1.87 (s, 3H, CH3), 1.12 (d, 3JHH ) 7.0 Hz, 3H, CHMe2),
0.88 (d, 3JHH ) 6.5 Hz, 3H, CHMe2). 13C{1H} NMR [100.63 MHz,
CDCl3, δC, ppm]: 167.8 (C8), 152.2 (C2), 142.6 (C8a), 138.8 (C4),
135.5 (indazole-C3), 130.4 (C6), 130.3 (C4a), 129.0 (indazole-C6),
123.5 (C3), 122.5 (indazole-C5), 122.1 (indazole-C3a), 120.3 (indazole-C7), 118.1 (indazole-C7a), 115.1 (C7), 112.6 (C5), 111.5
(indazole-C4), 104.4 (cy-C1), 101.6 (cy-C4), 85.5, 85.4, 83.1, 82.9
(cy-C2,3,5,6), 31.5 (CHMe2), 23.1, 21.9 (CHMe2) 18.6 (Me).
[(η6-p-cymene)Os(oxine)(ind)] (5b). A suspension of 1b (100
mg, 0.2 mmol) and Ag2CO3 (28 mg, 0.1 mmol) in methanol (10
mL) was stirred for 2 h at room temperature. Then indazole (24
mg, 0.2 mmol) was added, and the mixture was stirred again for
2 h. The resulting white solid was filtered off, and half of the solvent
was removed by rotary evaporation under reduced pressure. The
solution was left to stand at 4 °C overnight. The crystals obtained
were washed with diethyl ether (10 mL), recrystallized from
acetonitrile, and dried in Vacuo. Yield: 50 mg, 43% of a yellow
powder. Anal. Calcd for C26H25N3OOs (Mr 585.73): C, 53.31; H,
4.30; N, 7.17. Found: C, 53.21; H, 4.34; N, 7.17. ESI-MS: positive,
m/z 587 [(η6-p-cymene)Os(oxine)(ind)]+, 470 [(η6-p-cymene)Os(oxine)]+. IR spectrum (selected bands, KBr, νmax, cm-1): 524, 750,
778, 818, 1064, 1111, 1192, 1322, 1374, 1466, 1499, 1570, 2957,
3014, 3044. UV-vis spectrum [methanol, λmax, nm (ε, M-1 cm-1)]:
265 (23 400), 296 (9570), 304 (8420), 432 (3840), 342 (3480). 1H
NMR [400.13 MHz, CDCl3, δH, ppm]: 9.05 (d, 3JHH ) 4.8 Hz,
1H, hc2), 7.79 (d, 3JHH ) 8.3 Hz, 1H, hc4), 7.66 (s, 1H, indazole3),
7.61 (d, 3JHH ) 8.6 Hz, 1H, indazole4), 7.43 (d, 3JHH ) 8.1 Hz,
1H, indazole7), 7.31 (m, 1H, hc6), 7.08 (d, 3JHH ) 7.9 Hz, 1H, hc7),
6.95 (m, 2H, hc3, indazole6), 6.73 (m, 2H, hc5, indazole5), 5.99 (d,
3
JHH ) 5.6 Hz, 1H, cy), 5.95 (d, 3JHH ) 5.4 Hz, 1H, cy), 5.78 (m,
2H, cy), 2.38 (m, 1H, CHMe2), 1.91 (s, 3H, Me), 1.07 (d, 3JHH )
6.9 Hz, 3H, CHMe2), 0.93 (d, 3JHH ) 6.9 Hz, 3H, CHMe2). 13C{1H}
6590 Organometallics, Vol. 27, No. 24, 2008
1264, 1285, 1322, 1379, 1431, 1504, 1576, 2965, 3061, 3109.
UV-vis spectrum [water, λmax, nm (ε, M-1 cm-1)]: 232 (18 540),
266 (17 736) 428 (4520), 328 (3380), 346 (3210). 1H NMR [400.13
MHz, CDCl3, δH, ppm]: 12.00 (bs, 1H, benzimidazoleNH), 9.62 (d,
3
JHH ) 4.5 Hz, 1H, hc2), 8.29 (s, 1H, benzimidazole2), 8.04 (d,
3
JHH ) 8.0 Hz, 1H, benzimidazole4/7), 7.96 (d, 3JHH ) 8.5 Hz, 1H,
hc4), 7.44 (m, 2H, hc3, benzimidazole4/7), 7.29 (t, 3JHH ) 7.8, 1H,
hc6), 7.15 (m, 2H, hc7, benzimidazole5/6), 6.89 (d, 3JHH ) 7.5 Hz,
1H, benzimidazole5/6), 6.71 (d, 3JHH ) 7.6 Hz, 1H, hc5), 6.08 (d,
3
JHH ) 6.0 Hz, 1H, cy), 5.88 (d, 3JHH ) 6.0, 1H, cy), 5.71 (d, 3JHH
) 6.0 Hz, 1H, cy), 5.68 (d, 3JHH ) 6.0, 1H, cy), 2.48 (m, 3JHH )
7.0 Hz, 1H, CHMe2), 1.65 (s, 3H, CH3), 1.03 (d, 3JHH ) 7.0 Hz,
3H, CHMe2) 0.94 (d, 3JHH ) 7.0 Hz, 3H, CHMe2). 13C{1H} NMR
[100.63 MHz, CDCl3, δC, ppm]: 167.8 (C8), 151.9 (C2), 143.9 (C8a),
143.3 (benzimidazole-C2), 140.8 (benzimidazole-C4a/7a), 138.7 (C4),
132.9 (benzimidazole-C4a/7a), 130.5 (benzimidazole-C5/6), 130.3
(C4a), 124.9 (C7), 124.4 (C6), 123.2 (C3), 118.3 (benzimidazoleC4/7), 115.0 (benzimidazole-C5/6), 113.8 (benzimidazole-C4/7), 112.0
(C5),102.7 (cy-C1), 102.5 (cy-C4), 85.9, 84.7, 82.0, 81.5 (cy-C2,3,5,6),
31.4 (CHMe2), 22.9, 22.3 (CHMe2) and 18.3 (Me). Single crystals
of 7a · 0.5(C2H5)2O were grown by slow diffusion of diethyl ether
into a CH3OH solution of 7a.
[(η6-p-cymene)Os(oxine)(Hbzim)]CF3SO3 (7b). General Procedure B. Yield: 251 mg, 69% of a yellow powder. Anal. Calcd
for C27H26F3N3O4OsS (Mr 735.80): C, 44.07; H, 3.56; N, 5.71; S,
4.36. Found: C, 44.28; H, 3.48; N, 5.75; S, 4.27. ESI-MS: positive,
m/z 586 [(η6-p-cymene)Os(oxine)(Hbzim)]+, 470 [(η6-p-cymene)Os(oxine)]+; negative, m/z 149 [CF3SO3]-. Solubility in water:
0.3 mM. IR spectrum (selected bands, KBr, νmax, cm-1): 521, 639,
755, 827, 1031, 1112, 1162, 1264, 1322, 1379, 1466, 1501, 1574,
2924, 2965, 3109. UV-vis spectrum [water, λmax, nm (ε, M-1
cm-1)]: 232 (21 740), 263 (20 490), 426 (4420), 325 (3170), 338
(3030).
[(η6-p-cymene)Os(oxine)(Hbzim)]PF6 (7c). To a suspension of
1b (200 mg, 0.4 mmol) in methanol (10 mL) was added dropwise
a solution of silver hexafluorophosphate (100 mg, 0.4 mmol) in
methanol (2 mL), and the mixture was stirred for 2 h at room
temperature. Benzimidazole (47 mg, 0.4 mmol) was added, and
the mixture was stirred for an additional 2 h. The resulting white
solid was filtered off, and half of the solvent was removed by rotary
evaporation under reduced pressure. The concentrated solution was
left to stand at 4 °C overnight. The crystals obtained were filtered
off, washed with diethyl ether (5 mL), recrystallized from acetonitrile, and dried in Vacuo. Yield: 209 mg, 72% of brown-yellow
crystals. Anal. Calcd for C26H26F6N3OOsP (Mr 731.70): C, 42.68;
H, 3.58; N, 5.74. Found: C, 42.74; H, 3.38; N, 5.83. ESI-MS:
positive, m/z 470 [(η6-p-cymene)Os(oxine)]+; negative, m/z 145
[PF6]-. IR spectrum (selected bands, KBr, νmax, cm-1): 523, 555,
636, 737, 776, 840, 1113, 1247, 1281, 1320, 1377, 1425, 1465,
1501, 1574, 2965, 3058, 3157, 3335. UV-vis spectrum [methanol,
λmax, nm (ε, M-1 cm-1)]: 233 (21 670), 266 (21 090), 435 (4350),
329 (3340), 341 (3180). X-ray diffraction quality single crystals
of 7c · 2CH3OH were grown from a saturated solution of 7c in
methanol.
[(η6-p-cymene)Os(oxine)(Hdmbzim)]PF6 (8b). To a suspension
of 1b (200 mg, 0.4 mmol) in methanol (10 mL) was added dropwise
a solution of silver hexafluorophosphate (100 mg, 0.4 mmol) in
methanol (2 mL), and the mixture was stirred for 2 h at room
temperature. Then 5,6-dimethylbenzimidazole (58 mg, 0.4 mmol)
in methanol (2 mL) was added, and the mixture was stirred for an
additional 2 h. The resulting white solid was filtered off, and half
of the solvent was removed by rotary evaporation under reduced
pressure. The concentrated solution was allowed to stand at 4 °C
overnight. The product formed was filtered off, washed with cooled
diethyl ether (2 mL), and dried in Vacuo. Yield: 251 mg, 83% of
yellow-brown crystals. Anal. Calcd for C28H30F6N3OOsP (Mr
759.75): C, 44.26; H, 3.98; N, 5.53. Found: C, 44.20; H, 3.69; N,
Schuecker et al.
5.49. ESI-MS: positive, m/z 616 [(η6-p-cymene)Os(oxine)(Hdmbzim)]+, 470 [(η6-p-cymene)Os(oxine)]+; negative, m/z 145 ([PF6]-.
IR spectrum (selected bands, KBr, νmax, cm-1): 527, 557, 749, 778,
845, 1113, 1282, 1319, 1377, 1466, 1501, 1574, 2962, 3371.
UV-vis spectrum [methanol, λmax, nm (ε, M-1 cm-1)]: 265
(21 930), 287 (11 190). 1H NMR [400.13 MHz, CDCl3, δH, ppm]:
10.19 (bs, 1H), 9.41 (d, 3JHH ) 5.0 Hz, 1H), 8.09 (m, 2H), 7.76 (s,
1H), 7.55 (dd, 3JHH ) 8.4 Hz, 3JHH ) 5.0 Hz, 1H), 7.25 (t, 3JHH )
8.0 Hz, 1H), 7.16 (s, 1H), 6.91 (d, 3JHH ) 8.0 Hz, 1H), 6.82 (d,
3
JHH ) 8.0 Hz, 1H), 6.23 (d, 3JHH ) 5.6 Hz, 1H), 6.20 (d, 3JHH )
5.6 Hz, 1H), 5.94 (d, 3JHH ) 5.6 Hz, 1H), 5.89 (d, 3JHH ) 5.6 Hz,
1H), 2.42 (m, 4H), 2.28 (s, 3H), 1.79 (s, 3H), 1.05 (d, 3JHH ) 6.9
Hz, 3H), 0.98 (d, 3JHH ) 6.9 Hz, 3H).
[(η6-p-cymene)Os(oxine)(meade)]PF6 (9b). A mixture of 1b
(100 mg, 0.2 mmol) and 9-methyladenine (30 mg, 0.2 mmol) in
methanol (5 mL) was stirred for 1 h in the dark at ambient
temperature. The resulting clear orange solution was filtered directly
into a solution of NH4PF6 (652 mg, 4.0 mmol) in water (10 mL).
The yellow solid formed was collected by filtration, washed with
water, and dried to receive the crude product, which was purified
by diffusion of diethyl ether into a saturated methanolic solution
of the compound. Yield: 70 mg, 47% of yellow-brown crystals
suitable for X-ray diffraction measurements. Anal. Calcd for
C25H27F6N6OOsP (Mr 752.13): C, 39.37; H, 3.57; N, 11.02. Found:
C, 39.31; H, 3.56; N, 10.92. ESI-MS: positive, m/z 617 [(η6-pcymene)Os(oxine)(meade)]+, 470 [(η6-p-cymene)Os(oxine)]+. IR
spectrum (selected bands, KBr, νmax, cm-1): 3307, 3139, 1652, 1576,
1504, 1467, 1378, 1320, 1279, 1240, 1112, 836, 785, 750, 557.
UV-vis spectrum [water, λmax, nm (ε, M-1 cm-1)]: 208 (50 580),
264 (29 380), 235 (24 260), 435 (4350), 324 (4200), 340 (3680).
1
H NMR [400.13 MHz, CDCl3, δH, ppm]: 9.60 (dd, 3JHH ) 5.1
Hz, 4JHH ) 1.1 Hz, 1H, hc2), 9.03 (bs, 1H, meade6-NH2), 8.33 (s,
1H, meade2), 8.31 (s, 1H, meade8), 8.23 (dd, 3JHH ) 8.5 Hz, 4JHH
) 1.1 Hz, 1H, hc4), 7.72 (dd, 3JHH ) 8.5 Hz, 3JHH ) 5.1 Hz, 1H,
hc3), 7.39 (t, 3JHH ) 7.9 Hz, 1H, hc6), 6.99 (m, 2H, hc5, hc7), 6.27
(d, 3JHH ) 5.6 Hz, 1H, cy), 6.19 (d, 3JHH ) 5.6 Hz, 1H, cy), 6.01
(d, 3JHH ) 5.6 Hz, 1H, cy), 5.94 (d, 3JHH ) 5.6 Hz, 1H, cy), 5.60
(bs, 1H, meade6-NH2), 3.80 (s, 3H, meade-Me), 2.42 (m, 1H,
CHMe2), 1.74 (s, 3H, Me), 1.06 (d, 3JHH ) 6.9 Hz, 3H, CHMe2),
0.91 (d, 3JHH ) 6.9 Hz, 3H, CHMe2). 13C{1H} NMR [100.63 MHz,
CDCl3, δC, ppm]: 167.1 (C8), 154.7 (meade-C6), 154.2 (meadeC2), 151.7 (C2), 150.2 (meade-C4), 144.1 (meade-C8), 143.8 (C8a),
139.2 (C4), 130.3 (C6), 130.0 (C4a), 124.3 (C3), 117.2 (meade-C5),
114.9 (C7), 114.2 (C5), 95.0 (cy-C4), 91.9 (cy-C1), 78.2, 76.2, 71.3,
71.1 (cy-C2,3,5,6), 31.4 (CHMe2), 30.5 (meade-Me), 23.3, 21.6
(CHMe2), 17.8 (Me). NMR shifts and coupling constants for
compounds 3b, 6b, 6c, 7b, and 7c are quoted as Supporting
Information.
Crystallographic Structure Determination. X-ray diffraction
measurements were performed on a Nonius Kappa CCD or Bruker
X8 APEXII CCD diffractometer. Single crystals were positioned
at 30, 40, 30, 30, 40, and 40 mm from the detector, and 502, 3001,
344, 344, 1145, and 5036 frames were measured, each for 40, 5,
40, 35, 2, and 10 s over 1.5, 1, 2, 2, 1, and 0.5° scan width for 4a,
5b, 6a, 7a, 7c, and 9b, correspondingly. The data were processed
using Denzo-SMN or SAINT software.21 Crystal data, data collection parameters, and structure refinement details are given in
Tables 1 and 2. The structures were solved by direct methods and
refined by full-matrix least-squares techniques. Non-H atoms were
refined with anisotropic displacement parameters. H atoms were
inserted in calculated positions and refined with a riding model.
The following computer programs were used: structure solution,
SHELXS-97;22 refinement, SHELXL-97;23 molecular diagrams,
ORTEP;24 computer, Pentium IV; scattering factors.25
Ru(II) and Os(II) Complexes with High AntiproliferatiVe ActiVity
Organometallics, Vol. 27, No. 24, 2008 6591
Table 1. Crystal Data and Details of Data Collection for 4a · CH2Cl2,
5b · 3CH3OH, and 6a
4a · CH2Cl2
5b · 3CH3OH
6a
chemical formula C28H28Cl2F3N3O4RuS C29H37N3O4Os C23H24F3N3O4RuS
fw
731.56
681.82
596.58
space group
P21/n
P1j
P21/c
a, Å
13.531(3)
10.4410(5)
12.819(3)
b, Å
11.219(2)
11.3821(5)
12.076(2)
c, Å
20.357(4)
13.4172(7)
15.823(3)
R, deg
90.499(3)
β, deg
106.48(3)
108.143(2)
105.74(3)
γ, deg
112.641(2)
V, Å3
2963.3(10)
1383.35(12)
2357.6(8)
Z
4
2
4
λ, Å
0.71073
0.71073
0.71073
-3
Fcalcd, g cm
1.640
1.637
1.681
cryst size, mm3 0.40 × 0.29 × 0.04 0.34 × 0.32 × 0.23 × 0.20 ×
0.32
0.14
T, K
120
100
120
µ, cm-1
8.38
46.47
8.14
R1a
0.0273
0.0123
0.0227
b
wR2
0.0688
0.0304
0.0606
GOFc
1.044
1.065
1.099
a
R1 ) ∑|Fo| - |Fc|/∑|Fo|. b wR2 ) {∑[w(Fo2 - Fc2)2]/∑[w(Fo2)2
]}1/2. c GOF ) {∑[w(Fo2 - Fc2)2] /(n - p)}1/2, where n is the number of
reflections and p is the total number of parameters refined.
Table 2. Crystal Data and Details of Data Collection for
7a · 0.5(C2H5)2O, 7c · 2CH3OH, and 9b
7a · 0.5(C2H5)2O
7c · 2CH3OH
9b
chemical formula C29H31F3N3O4.5RuS C28H34F6N3O3OsP C25H27F6N6OOsP
fw
683.70
795.75
762.70
space group
C2/c
P1j
P21/c
a, Å
19.878(4)
9.2497(18)
17.261(4)
b, Å
22.441(4)
10.955(2)
10.044(2)
c, Å
14.179(3)
14.715(3)
16.238(3)
R, deg
77.78(3)
β, deg
115.89(3)
87.82(3)
103.90(3)
γ, deg
83.83(3)
V, Å3
5690.2(19)
1448.7(5)
2732.7(9)
Z
8
2
4
λ, Å
0.71073
0.71073
0.71073
Fcalcd, g cm-3
1.596
1.824
1.854
cryst size, mm3 0.28 × 0.17 × 0.11 0.50 × 0.40 ×
0.15 × 0.15 ×
0.18
0.03
T, K
120
100
296
µ, cm-1
6.87
45.31
47.97
R1a
0.0308
0.0215
0.0279
b
wR2
0.0779
0.0525
0.0705
GOFc
1.061
1.030
1.047
a
R1 ) ∑|Fo| - |Fc|/∑|Fo|. b wR2 ) {∑[w(Fo2 - Fc2)2]/∑[w(Fo2)2
]}1/2. c GOF ) {∑[w(Fo2 - Fc2)2] /(n - p)}1/2, where n is the number of
reflections and p is the total number of parameters refined.
Determination of Octanol/Water Partition Coefficients. The
log P values were determined using the shake flask method. Both
octanol and water were presaturated with water or octanol,
respectively. Due to the low stability of the compounds in water/
octanol mixture, the standard procedure was modified as follows:
The sample compound was shaken intensively in a mixture of water
and octanol for 2 h. Phase separation was achieved using a
centrifuge (Sorvall, RT6000B) at 20 °C for 5 min at 3000 rpm.
The compounds in the organic phase were quantified by using an
UV-vis spectrophotometer (Perkin-Elmer, Lambda 7).
Cell Lines and Culture Conditions. CH1 cells (ovarian
carcinoma) were kindly donated by Lloyd R. Kelland, CRC Centre
(22) Sheldrick, G. M. SHELXS-97, Program for Crystal Structure
Solution; Göttingen: Germany, 1997.
(23) Sheldrick, G. M. SHELXL-97, Program for Crystal Structure
Refinement; Göttingen, Germany, 1997.
(24) Johnson, G. K. Report ORNL-5138; Oak Ridge National Laboratory:
Oak Ridge, TN, 1976.
(25) International Tables for X-ray Crystallography; Kluwer Academic Press: Dordrecht, The Netherlands, 1992; Vol. C, Tables 4.2.6.8
and 6.1.1.
Figure 1. Structure of the complex cation in 4a (left) and of the
complex 5b (right).
for Cancer Therapeutics, Institute of Cancer Research, Sutton, UK.
SW480 cells (adenocarcinoma of the colon) were kindly provided
by Brigitte Marian, Institute of Cancer Research, Department of
Medicine I, Medical University of Vienna, Austria. Cells were
grown in 75 cm2 culture flasks (Iwaki) as adherent monolayer
cultures in complete culture medium, i.e., Minimal Essential
Medium (MEM) supplemented with 1 mM sodium pyruvate, 4 mM
L-glutamine, and 1% nonessential amino acids (100×) (all purchased from Sigma Aldrich) and 10% heat-inactivated fetal bovine
serum (purchased from Gibco/Invitrogen). Cultures were maintained
at 37 °C in a humidified atmosphere containing 5% CO2.
Antiproliferative Activity in Cancer Cell Lines. Antiproliferative activity was determined by means of a colorimetric
microculture assay (MTT assay, MTT ) 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, purchased from Fluka).
For this purpose, CH1 and SW480 cells were harvested from culture
flasks by trypsinization and seeded in 100 µL aliquots into 96-well
microculture plates (Iwaki). Cell densities of 1.5 × 103 cells/well
(CH1) and 2.5 × 103 cells/well (SW480) were chosen in order to
ensure exponential growth throughout drug exposure. Cells were
allowed to settle in drug-free complete culture medium for 24 h,
followed by the addition of dilutions of the test compounds in 100
µL/well complete culture medium and incubation for 96 h. At the
end of exposure, drug solutions were replaced by 100 µL/well
RPMI1640 culture medium (supplemented with 10% heatinactivated fetal bovine serum) plus 20 µL/well MTT solution in
phosphate-buffered saline (5 mg/ml PBS). After incubation for 4 h,
the medium/MTT mixtures were removed, and the formazan
product formed by vital cells was dissolved in 150 µL of DMSO
per well. Optical densities at 550 nm were measured with a
microplate reader (Tecan Spectra Classic), using a reference
wavelength of 690 nm to correct for unspecific absorption. The
quantity of vital cells was expressed in terms of T/C values by
comparison to untreated control microcultures, and IC50 values were
calculated from concentration-effect curves by interpolation.
Evaluation is based on means from at least three independent
experiments, each comprising six replicates per concentration level.
Results and Discussion
Synthesis. The reaction of [(η6-p-cymene)RuCl2]2 with 2
equiv of K(oxine) in ice-cold CH2Cl2 afforded the half-sandwich
complex 1a in 92% yield. The spectroscopic properties of 1a
were identical with those of [(η6-p-cymene)Ru(oxine)Cl] prepared by using THF as a solvent.19 Compound 1b was obtained
by reaction of 8-hydroxyquinoline with sodium methoxide and
[(η6-p-cymene)OsCl2]2 in approximately 2:2:1 molar ratio in
methanol in 84% yield.20 The monoaqua complexes 2a and 2b
were generated in situ by treatment of complexes 1a and 1b
with equivalent amounts of AgNO3 in water. The 1H NMR
spectra, which are similar to those of the starting compounds,
indicate that the complexes are rather stable in aqueous solution.
Compounds 3a, 4a, 6a, and 7a were synthesized in 67, 65,
68, and 75% yield, correspondingly, starting from equivalent
6592 Organometallics, Vol. 27, No. 24, 2008
Schuecker et al.
Figure 2. Structure of the ion pair in 6a.
Figure 4. Structure of the complex cation in 9b. Selected bond
distances (Å): Os-O1 2.076(3), Os-N7 2.124(3), Os-N8 2.089(3),
Os-C(1-6)av 2.187(9).
Figure 3. Structure of the complex cation in 7a · 0.5(C2H5)2O (left)
and in 7c (right).
Table 3. Selected Bond Distances (Å) and Angles (deg) for
Complexes 4a, 5a, 5c, 6a, and 7b
4a
5a
5c
6a
7b
M-O1
2.0853(14) 2.0771(15) 2.069(2) 2.0664(12) 2.0678(9)
M-N1
2.0911(16) 2.0926(14) 2.069(2) 2.0664(12) 2.0678(9)
M-N2
2.0950(17) 2.1027(14) 2.131(3) 2.1107(13) 2.0899(12)
M-C(1-6)av 2.198(7)
2.196(9)
2.190(7) 2.192(8)
2.188(9)
C-C(1-6)av 1.422(6)
1.419(3)
1.421(4) 1.417(3)
1.424(2)
O1-M-N1 78.55(6)
78.71(5)
78.83(10) 79.11(5)
78.34(4)
O1-M-N2 83.17(6)
86.22(5)
81.28(10) 83.01(5)
82.81(4)
N1-M-N2 85.08(6)
83.33(6)
83.89(10) 85.73(5)
82.46(4)
amounts of 1a and AgCF3SO3 in THF to which the corresponding azole ligand was added. The synthesis of osmium compounds 3b, 6b, and 7b was performed similarly in 61, 62, and
69% yield, respectively, by using methanol instead of THF as
a solvent. The noncharged species 5b with a deprotonated
indazole ligand was obtained in 43% yield by reacting 1b with
Ag2CO3 and indazole in 2:1:2 molar ratio in methanol. The
chloride salt 6c was synthesized from equivalent amounts of
1b and imidazole in methanol at room temperature in 83% yield,
while the hexafluorophosphate salt 7c was obtained by reaction
of 1b with AgPF6 in 1:1 molar ratio, followed by addition of
the equivalent quantity of benzimidazole at room temperature,
in 72% yield. When the mixture of 1b with 9-methyladenine
was allowed to react in methanol for 1 h and then added to an
aqueous solution of NH4PF6, the complex 9b was obtained.
Recrystallization of the latter from methanol saturated with
diethyl ether afforded large yellow-brown crystals of the pure
compound in 47% yield.
Crystal Structures. We determined the X-ray diffraction structures of [(η6-p-cymene)Ru(oxine)(Hind)]CF3SO3 ·
CH2Cl2 (4a · CH2Cl2), [(η6-p-cymene)Os(oxine)(ind)] · 3CH3OH
(5b · 3CH3OH, Figure 1), [(η6-p-cymene)Ru(oxine)(Him)]CF3SO3 (6a, Figure 2), [(η6-p-cymene)Ru(oxine)(Hbzim)]CF3SO3 · 0.5(C2H5)2O (7a · 0.5(C2H5)2O), and [(η6-p-cymene)Os(oxine)(Hbzim)]PF6 · 2CH3OH (7c · 2CH3OH, Figure 3). All
complexes have the characteristic “three-leg piano-stool” geometry of Ru(II) or Os(II) arene complexes, with an η6 π-bound
p-cymene ring forming the seat and three other donor atoms of
one bidentate oxine ligand and one azole ligand as the legs of
the stool. They are racemates due to the presence of the
stereogenic metal center. Selected bond distances and angles
are quoted in Table 3. The X-ray structure of complex
7c · 2CH3OH (Figure 3) is the first of a monoarene complex of
osmium, in which an oxine and an azole ligand are bound to
the same metal center, while that of compound 5b · 3CH3OH is
the first of a monoarene complex of osmium with a deprotonated
azole ligand.
A large number of transition metal complexes with pyrazoles
and/or pyrazolates have been documented in the literature.26-28
Coordinated pyrazole ligands are either protonated or deprotonated, depending on reaction conditions and the metal ion
identity.29 The proton in coordinated pyrazole is often involved
in hydrogen bonding.30 The deprotonated pyrazole normally acts
as a bridging ligand, enabling the formation of dinuclear31 or
cyclic polynuclear complexes.29,32 In contrast, indazole acts
mainly as a monodentate protonated ligand in metal complexes.
In a few cases, it was found to be deprotonated, acting as a
bridging ligand in polynuclear metal complexes33 or even more
rarely as a monodentate indazolate ligand.34
[(η6-p-cymene)Os(oxine)(meade)]PF6 (9b) crystallized in the
monoclinic space group P21/c. The structure of the complex
cation is shown in Figure 4. A part of the crystal structure of
9b, displaying the coordinated 9-methyladenine (meade) pairing
via intermolecular hydrogen bonding interactions, is shown in
Figure S1. Selected bond distances (Å) and angles (deg) are
quoted in the legends to Figures 4 and S1. The 9-methyladenine
ligand is coordinated to osmium through nitrogen atom N7 in
(26) Trofimenko, S. Prog. Inorg. Chem. 1986, 34, 115–210.
(27) Sadimenko, A. P. AdV. Heterocycl. Chem. 2001, 80, 157–240.
(28) La Monica, G.; Ardizzoia, G. A. Prog. Inorg. Chem. 1997, 46,
151–238.
(29) Umakoshi, K.; Yamaushi, Y.; Nakamiya, K.; Kojima, T.; Yamasaki,
M.; Kawano, H.; Onishi, M. Inorg. Chem. 2003, 42, 3907–3916.
(30) Stepanenko, I. N.; Cebrian-Losantos, B.; Arion, V. B.; Krokhin,
A.; Nazarov, A. A.; Keppler, B. K. Eur. J. Inorg. Chem. 2007, 400–411.
(31) (a) Beveridge, K. A.; Bushnell, G. W.; Dixon, K. R.; Eadie, D. T.;
Stobart, S. R.; Atwood, J. L.; Zaworotko, M. J. J. Am. Chem. Soc. 1982,
104, 920–922. (b) Coleman, A. W.; Eadie, D. T.; Stobart, S. R.; Zaworotko,
M. J.; Atwood, J. L. J. Am. Chem. Soc. 1982, 104, 922–923.
(32) Burger, W.; Straehle, J. Z. Anorg. Allg. Chem. 1985, 529, 111–
117.
(33) (a) Rendle, D. F.; Storr, A.; Trotter, J. Can. J. Chem. 1975, 53,
2930–2943. (b) Cortes-Llamas, S. A.; Hernández-Pérez, J. M.; Hô, M.;
Munoz-Hernández, M.-A. Organometallics 2006, 25, 588–595.
(34) Fackler, J. P., Jr.; Staples, R. J.; Raptis, R. G. Z. Kristallogr. 1997,
212, 157–158.
Ru(II) and Os(II) Complexes with High AntiproliferatiVe ActiVity
Organometallics, Vol. 27, No. 24, 2008 6593
Chart 2. Tautomeric Structures of 9-Methyladenine: the
Imine Form (left), the Amine Form (middle), and the
Zwitterionic Form (right)
Table 4. Maximum Absorption Wavelength and log Po/w Values for
Compounds 3a, 6b, and 7a
its amine form (Chart 2). The Os-N7 bond (see legend to Figure
4) is longer than that in [(η6-p-cymene)Os(pico)(9EtA)]PF6 ·
0.5Et2O at 150 K [2.094(7) Å], where pico ) picolinate.20
Coordination of 9-substituted adenine via the ring nitrogen atoms
is governed first of all by their basicity, which decreases in the
order N1 > N7 > N3. In some cases, metal ions can also bind
to the exocyclic amine group with concomitant loss of one
proton or its migration to the neighboring ring nitrogen atom
N1 and stabilization of the rare imine tautomer (Chart 2).35
Bridging and/or chelate interactions involving different combinations of exocyclic and endocyclic nitrogen atoms and even
the C8 of the imidazole ring have also been demonstrated by
X-ray diffraction.36 This is to our knowledge the second
crystallographic evidence for a monodentate coordination mode
of 9-alkyladenine to osmium documented so far.37 The amine
group of 9-methyladenine in 9b acts as a hydrogen donor and
N1 as a hydrogen acceptor, which are involved in complex
pairing through strong intermolecular hydrogen bonding as
shown in Figure S1. Similar purine base pairing was observed
for [(η6-p-cymene)Os(pico)(9EtA)]PF6 · 0.5Et2O20 and [RuIIICl3(Hind)2(meade)] · CH2Cl2 · CH3OH or [RuIIICl3(Hind)2(meade)] ·
1.1H2O · 0.9CH3OH.38 The same amine group of 9-methyladenine and the oxygen atom of the oxine ligand O1 are involved
in intramolecular hydrogen bonding (see Figure S1).
Aqueous Solubility, Lipophilicity, and Hydrolytic Stability. The aqueous solubility of the family of complexes prepared
varies from 0.3 mM (7b) to 52.3 mM (6c). This depends on
the metal ion, the identity of the azole heterocycle, and the
counteranion. Generally, the ruthenium complexes isolated are
more soluble in water than their osmium(II) congeners. The
effect of the azole heterocycle on the aqueous solubility of
the complexes is different, as anticipated from comparison of
their lipophilicity, expressed by the octanol-water partitioning
coefficients (log Po/w) for imidazole (-0.08),39 pyrazole (0.13),40
benzimidazole (1.3241 or 1.3842), indazole (1.77),39 and 5,6(35) (a) Kuo, L. Y.; Kanatzidis, M. G.; Sabat, M.; Tipton, A. L.; Marks,
T. J. J. Am. Chem. Soc. 1991, 113, 9027–9045. (b) Zamora, F.; Kunsman,
M.; Sabat, M.; Lippert, B. Inorg. Chem. 1997, 36, 1583–1587. (c) Clarke,
M. J. J. Am. Chem. Soc. 1978, 100, 5068–5075. (d) Arpalahti, J.; Klika,
K. D. Eur. J. Inorg. Chem. 1999, 8, 1199–1201. (e) Viljanen, J.; Klika,
K. D.; Sillampää, R.; Arpalahti, J. Inorg. Chem. 1999, 38, 4924–4925.
(36) (a) Trovó, G.; Bandoli, G.; Nicolini, M.; Longato, B. Inorg. Chim.
Acta 1993, 211, 95–99. (b) Olivier, M. J.; Beauchamp, A. L. Acta
Crystallogr. 1982, B38, 2159–2162. (c) Prizant, L.; Olivier, M. J.; Rivest,
R.; Beauchamp, A. L. J. Am. Chem. Soc. 1979, 101, 2765–2767.
(37) CSD version 5.29; November 2007.
(38) Egger, A.; Arion, V. B.; Reisner, E.; Cebrián-Losantos, B.; Shova,
S.; Trettenhahn, G.; Keppler, B. K. Inorg. Chem. 2005, 44, 122–132.
(39) Kibbey, C. E.; Poole, S. K.; Robinson, B.; Jackson, J. D.; Durham,
D. J. Pharm. Sci. 2001, 90, 1164–1175.
(40) (a) Chou, J. T.; Jurs, P. C. In Solubility and Partitioning in Drug
Design: Physical Chemical Properties of Drug; Yalkowsky, S. H., Sinkula,
A. A., Valvani, C. C., Eds.; Marcel Dekker: New York, 1980; pp 163199. (b) Bodor, N.; Hung, M.-J. J. Pharm. Sci. 1992, 81, 272–281.
(41) Mannhold, R.; Cruciani, G.; Dross, K.; Rekker, R. J. Comput.Aided Mol. Des. 1998, 12, 573–581.
(42) Bodor, N.; Gabanyi, Z.; Wong, C.-K. J. Am. Chem. Soc. 1989,
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complex
λmax (nm)
log Po/w
3a
6b
7a
399
431
405
0.98 ( 0.02
1.18 ( 0.01
1.79 ( 0.08
dimethylbenzimidazole (2.35).41 Chlorides show the highest
aqueous solubility when compared to triflates and hexafluorophosphates. We also succeeded in determining the log Po/w
values for ruthenium and osmium complexes 3a, 6b, and 7a
by UV-vis spectroscopy, using the shake-flask method.43 The
partition of ions between octanol and aqueous phase is a more
complex process than that of neutral species. The observed
concentration ratio for the complex cation [(η6-p-cymene)Ru(oxine)(Hazole)]+ is related not only to the partition of the cation
but also to the partition of the ion pair.44 Nevertheless, we
believe that the data obtained (Table 4) can be used for relative
estimation of lipophilicity, since the concentration ratio [C]o/
[C]w was obtained at very low concentrations of solute, so that
the presence of only single ions is likely. Moreover, the log
Po/w value for compound 3a was found to be independent of
solute concentration. It should, however, be noted that the
determination of log Po/w values of the other compounds was
precluded by the observed slow decomposition of compounds,
evidenced by the appearance of a green color, upon shaking
the octanol/water mixture of compounds and separation of
octanol and aqueous phases, which requires prolonged times.
UV-vis spectra for complex 6a showing its degradation during
the measurement are given in the Supporting Information. The
differences in the spectra are similar to those of 3b, 6c, and 4a,
which undergo transformation as well. To reduce the timedependent effect of the octanol/water mixture on the decomposition of compounds 3a, 6b, and 7a, the separation of liquid
phases was accelerated by using centrifugation. As expected,
the lipophilicity of these complexes (Table 4) is mainly
determined by the lipophilicity of the ancillary azole ligands.
The hydrolytic stability of osmium complexes 3b, 6b, and
6c in D2O was studied by 1H NMR spectroscopy. The 1H NMR
spectra measured immediately and 96 h after dissolution at room
temperature were almost identical (Figures S1-S3), indicating
that they maintain their structural integrity in solution. The
stability of 6c in water and 7a in n-octanol was also confirmed
by electronic absorption spectra, which were recorded immediately and 24 h after dissolution at room temperature with
no change in the spectra. The ESI mass spectra of the solution
of 6c after 96 h showed a peak at m/z 447, attributed to the
intact complex cation [(η6-p-cymene)Os(oxine)(Him)]+.
Antiproliferative Activity. The ability of six ruthenium-based
(1a, 2a, 3a, 4a, 6a, 7a) complexes and their osmium-based (1b,
2b, 3b, 6b, 6c) analogues to inhibit cancer cell proliferation in
Vitro was investigated in the human cell lines CH1 (ovarian
carcinoma) and SW480 (colon carcinoma) by means of the MTT
assay with 96 h exposure. Concentration-effect curves are
depicted in Figure 5, and IC50 values are listed in Table 5.
In general, the investigated complexes show respectable
antiproliferative activity in low micromolar concentrations in
both cell lines. SW480 colon carcinoma cells are slightly (up
to 2.1 times, based on comparison of IC50 values) more sensitive
than CH1 ovarian carcinoma cells to the ruthenium compounds,
with the single exception of the pyrazole complex 3a. Differences between sensitivities to the osmium complexes are mostly
(43) Qiao, Y.; Xia, S.; Ma, P. J. Chem. Eng. Data 2008, 53, 280–282.
(44) Zhao, Y. H.; Abraham, M. H. J. Org. Chem. 2005, 70, 2633–2640.
6594 Organometallics, Vol. 27, No. 24, 2008
Schuecker et al.
Figure 5. Concentration-effect curves of the compounds in two human cancer cell lines (A1-3: CH1; B1-3: SW480).
Table 5. Antiproliferative Activity of Ruthenium Complexes (1a, 2a,
3a, 4a, 6a, and 7a) and Osmium Complexes (1b, 2b, 3b, 6b, and 6c)
in Two Human Cancer Cell Lines
IC50 (µM)a
compound
CH1
SW480
1a
1b
2a
2b
3a
3b
4a
6a
6b
6c
7a
24.2 ( 2.0
9.7 ( 1.8
17.0 ( 3.7
6.1 ( 1.3
5.5 ( 1.2
4.3 ( 0.9
9.4 ( 3.0
7.0 ( 2.7
4.5 ( 1.5
5.0 ( 0.6
7.4 ( 1.9
15.3 ( 2.1
7.7 ( 1.1
9.2 ( 0.9
6.0 ( 1.0
7.6 ( 1.9
7.5 ( 1.3
5.2 ( 0.8
3.3 ( 0.4
6.0 ( 0.6
6.5 ( 1.3
4.1 ( 0.2
a
50% inhibitory concentrations after exposure for 96 h in the MTT
assay. Values are means ( standard deviations, obtained from at least
three independent experiments.
less pronounced and within the ranges of variation, except for
the pyrazole complex 3b, which behaves similarly to 3a.
Since it is difficult to isolate monoaqua species in the solid
state, compounds 2a and 2b were generated in situ as described
in the Experimental Section. A comparison of the concentrationeffect curves of the chlorido complexes (1a, 1b) with their
activated counterparts (2a, 2b) (Figure 5, parts A1, B1) reveals
that the activated species show a higher antiproliferative activity
in both cases, in accordance with the expectation that forced
complete hydrolysis results in increased reactivity of the
complexes. Furthermore, the osmium species 1b and 2b are
2.5-2.8 times (CH1) and 1.5-2.0 times (SW480) more active
than the ruthenium congeners 1a and 2a, based on comparison
of IC50 values.
As can be seen in Figure 5, parts A2 and B2, all three
complexes with an imidazole ligand exert similar effects. The
change of the metal center from ruthenium (6a) to osmium (6b)
has a smaller impact than in the compounds mentioned above,
and the change of the counteranion of the complex [(η6-pcymene)Os(oxine)(Him)]+ from triflate (6b) to chloride (6c)
does not result in any meaningful differences. Likewise, the
ruthenium (3a) and osmium (3b) complexes with a pyrazole
ligand have similar activity (Figure 5, parts A3, B3). Variation
of the azole ligand, e.g., indazole (4a) or benzimidazole (7a),
results only in minor differences of antiproliferative activity,
Ru(II) and Os(II) Complexes with High AntiproliferatiVe ActiVity
Organometallics, Vol. 27, No. 24, 2008 6595
and no conclusive structure-activity relationships can be
deduced in this respect. Although a higher lipophilicity of the
azole ligand might favor cellular uptake and thereby biological
activity of the complex, no correlation can be found, neither
between IC50 values of the complexes and log P values of the
azole ligands (as reported in the literature cited in the previous
section) nor between IC50 values and log P values or aqueous
solubility of the complexes.
Within the ruthenium series, complexes containing a monodentate azole ligand show a 2.6-4.4 times (CH1) and 2.0-4.6
times (SW480) higher antiproliferative activity than the chlorido
analogue 1a, based on comparison of IC50 values. Their activity
even exceeds that of the aqua analogue 2a, although the less
easily exchangeable azole ligands should be less favorable for
coordination to target molecules. Within the osmium series,
differences are much less pronounced, and no consistent
structure-activity relationships are discernible.44
The osmium complexes are slightly less soluble in water than
the corresponding ruthenium compounds. The lipophilicity of
organometallic compounds, expressed by octanol/water partition
coefficients, can be to some extent controlled by the azole
heterocycle used as co-ligand. Both ruthenium and osmium
arene complexes with oxine and an azole heterocycle as ancillary
ligands are highly cytotoxic in the human tumor cell lines
SW480 (colon carcinoma) and CH1 (ovarian carcinoma), with
IC50 values ranging from 3.3 to 9.4 µM, and should therefore
be considered as potential novel anticancer drugs.
Conclusions
We reported the synthesis of ruthenium(II) and osmium(II)
arene complexes that combine ligands involved in metal-based
complexes that are currently in clinical development, namely,
oxine, which acts as a ligand in Ga(oxine)3 (KP46), as well as
azole ligands, which play a role in trans-[RuIIICl4(Hind)2](KP1019) and trans-[RuIIICl4(Him)(DMSO)]- (NAMI-A). The
aqueous solubility of the complexes reported in this study varies
from 0.3 mM (7b) to 53.3 mM (6c) and depends on the metal
ion, the identity of the azole heterocycle, and the counteranion.
Acknowledgment. We thank A. Roller for the collection
of X-ray data sets and Prof. M. Galanski for the measurement
of NMR spectra.
Supporting Information Available: Crystallographic data for
4a · CH2Cl2, 5b · 3CH3OH, 6a, 7a · 0.5(C2H5)2O, 7c · 2CH3OH, and
9b in CIF format, details of crystal structure of 9b showing the
coordinated 9-methyladenine pairing via intermolecular H-bonding
interactions (Figure S1), time-independent 1H NMR spectra of
compounds 3b, 6b, and 6c in D2O (Figures S2-S4), time-independent
UV-vis spectra of compounds 3a, 4a, 6a, and 7a in water (Figures
S5-S7, S9), time-independent UV-vis spectra of 7a in n-octanol
(Figure S10), and UV-vis spectra of 6a in n-octanol, measured
immediately after dissolution and after 2 h of shaking with water
followed by phase separation (Figure S8). This material is available
free of charge via the Internet at http://pubs.acs.org.
OM800774T