JIB-09029; No of Pages 7

Journal of Inorganic Biochemistry 111 (2012) xxx-xxx

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Journal of Inorganic Biochemistry

j o u r n a l h o m e p a ge : w w w . e l s e v i e r . c o m / l o c a t e / j i n o r gb i o

Synthesis, characterization, and anticancer activity of ruthenium-pyrazole complexes

Solene David a, Richard S. Perkins a, Frank R. Fronczek b, Sahba Kasiri c, Subhrangsu S. Mandal c, Radhey S. Srivastava a,⁎ a Department of Chemistry, University of Louisiana at Lafayette, LA 70504

b Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803 c Department of Chemistry and Biochemistry, University of Texas at Arlington, TX 76019

a r t i c l e i n f o

Article history:Received 26 November 2011Received in revised form 23 February 2012 Accepted 24 February 2012Available online 4 March 2012

Keywords:Ruthenium(III)pyrazole complexesCrystal structure cyclic voltammetryin vitro cytotoxicity

1. Introduction

a b s t r a c t

A series of new water soluble Ru(III) pyrazole complexes mer-[RuCl3(DMSO-S)(pyz)2] 1, mer-[RuCl3(DMSO-S) (DMSO-O)(pyz)] 2, mer-[RuCl3(bpy)(dmpyz)] 3, and mer-[RuCl3(DMSO-S)(dmpyz)2] 4 (pyz =pyrazole; dmpyz = 3,5-dimethylpyrazole, bpy = 2,2’-bipyridine) have been synthesized and characterized by use of a combination of spectroscopy (IR and UV-visible), X-ray diffraction, and cyclic voltammetry. The molecular X-ray structure of all reported compounds (1-4) revealed distorted octahedral coordination aroundruthenium. The cytotoxicity assay on human breast cancer cells (MCF7) demonstrated that compounds 1 and 4 affect cell viability, whereas compounds 2 and 3 do not show appreciable activity. The IC50 values for 1 and 4 lie within the range of 71-32 μM in MCF7 cells.

© 2012 Elsevier Inc. All rights reserved.

effective means for ruthenium complexes to accumulate in cancercells [22]. Two ruthenium complexes, KP1019 {[IndH][trans-RuCl4(-

The success of platinum-derived anticancer drugs has stimulated a

renaissance of medicinal inorganic chemistry. Cisplatin is one of the most widely used drugs in clinical use. However, its effectiveness is limited due to its severe toxicity, resistance to the drug, and unwanted side effects [1-5]. This has led to the search for complexes of other transition metals with interesting biological properties [6-17], wider ranges of activity, and lower systematic toxicities. Ruthenium com-plexes are increasingly gaining interest as potential alternatives to platinum-based chemotherapeutic agents. Some ruthenium com-plexes have been shown to be effective against cancers not readily treated by cisplatin [18]. Among all ruthenium-based anticanceragents, ruthenium-DMSO complexes are believed to have great poten-tial because of their selectivity for solid tumor metastases and low host toxicity [19]. Furthermore, several ruthenium complexes have been found to display a significantly higher degree of selectivity towards cancerous cells than the leading commercially available platinum derived drugs, which results in reduced damage to healthy tissues [19]. The mechanism of selective delivery of Ru(III) complexes to specific cancer cells is widely argued. One widely accepted possibility is the ability of ruthenium(III) complexes to mimic iron in

reversible binding to plasma proteins such as transferrin, which is correlated with the overexpressed concentration of receptors for this protein on the surface of cancer cells [20-22]. This mechanism provides an

⁎ Corresponding author. Tel.: + 1 337 482 5677; fax: + 1 337 482 5676.

E-mail address: rss1805@louisiana.edu (R.S. Srivastava).

0162-0134/$ - see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2012.02.022

Ind)2] (Ind = indazole)} [23] and NAMI-A {[ImH][trans-RuCl4(-DMSO)(Im)] (Im = imidazole)} [24], are known to bind to iron(III) binding sites of transferrin [25].

NAMI-A and KP1019 are under clinical trial against metastatic and colon cancers, respectively [26,27]. They are scheduled for phase 2 trials [28-30]. The low general toxicity of these complexes is ascribed to the ability of ruthenium to collect specifically in cancer tissues, possibly via the transferrin mechanism [20-22,31,32].

Several platinum and ruthenium complexes with N-heterocyclic ligands have been reported of which some showed significant cytotoxicity. For example one of the ligands in NAMI-A is imidazole,

a heterocyclic amine. Pyrazole, a five membered N-heterocycle, also

belongs to the same class of compound as imidazole. Pyrazole is a poor π-electron acceptor and a better π-donor and hence acts as a hard donor site [33]. Pyrazoles have long been applied in agrochemical and pharmaceutical industries as herbicides and active pharmaceuti-cals. Pyrazole moieties are known for their antimicrobial,

anticancer, ACE (angiotensin-converting-enzyme) inhibitory, antiviral and anti-inflammatory activities. However, pyrazole complexes are less ex-plored. Pyrazole type heterocycles also represent the important class of non-leaving ligands because of their rich electronic property which can be altered by appropriate choice of substituents on the pyrazole ring. This in turn enables optimization of the electronic properties on the metal. Also, pyrazole ligands are less basic than imidazole. This reduces loss of DMSO from the complex and increases the antitumor activity [34]. Additionally, all the complexes reported herein are water soluble, which makes them attractive for being developed as drugs. It is therefore pertinent to explore the chemistry of ruthenium

Please cite this article as: S. David, et al., Synthesis, characterization, and anticancer activity of ruthenium-pyrazole complexes, J. Inorg. Biochem. (2012), doi:10.1016/j.jinorgbio.2012.02.022

2 S. David et al. / Journal of Inorganic Biochemistry 111 (2012) xxx-xxx

(III) pyrazole complexes with regard to anti-cancer properties. Here-in, we report synthesis, structure, and cytotoxic effects of these com-plexes (1-4) on human breast cancer cells MCF7.

2. Experimental section

2.1. Materials

All reagents were of analytical grade. Hydrated ruthenium(III) chloride and silver triflate were purchased from Strem chemicals. DMSO, pyrazole, 3,5-dimethylpyrazole, and solvents, includinganhydrous solvents, were purchased from Sigma-Aldrich. Reagent grade solvents were dried and stored over 4 Å molecular sieves.

2.2. Cyclic voltammetry

Cyclic voltammetry was carried out with a three electrode system described by us previously [35]. Redox potentials were calculated using Gaussian 03. DFT (Density functional theory) calculationswere done using the BP3LP method and a LANL2DZ basis set. Optimized gas phase structures were used to calculate energies in solution using the IEFCPM solvation model.

2.3. Physical measurements

The IR spectra were measured on a JASCO 480 Plus spectropho-tometer with samples in compressed KBr discs. UV-visible (UV-vis) spectra were obtained on a JASCO V-550 spectrophotometer.Elemental analysis was performed by Atlantic Microlab, Norcross, Georgia. The diffraction data were collected at low temperature on a Nonius KappaCCD equipped with Mo Kα (λ = 0.71073 Å) and a Bruker Kappa Apex II equipped with Cu (λ = 1.54178 Å) radiation source diffractometer, a graphite monochromator, and an Oxford Cryostream low-temperature device. Absorption collections were made by the multi-scan method. Crystal data and details of data collection and refinement are given in Tables 1-3.

Table 1Crystal data and structure refinement for compounds 1 and 2.

1 2

Formula C8H14Cl3N4ORuS C7H16Cl3N2O2RuS2.CH2Cl2Formula weight 421.71 516.68Crystal system Monoclinic MonoclinicSpace group Cc P21/nT (K) 90 90a (Å) 8.921 (2) 13.2179 (10)b (Å) 13.054 (2) 8.3150 (5)c (Å) 12.856 (3) 16.5031 (14)β (°) 98.090 90.084 (3)V (A3) 1482.2 (5) 1813.8 (2)Z 4 4Dx (Mg m- 3) 1.890 1.892μ (mm- 1) 1.73 1.83F(000) 836 1028Crystal size (mm) 0.15 x 0.10 x 0.03 0.35 x 0.22 x 0.12ϕ range (°) 4.8-57.1 2.5- 33.1h, k, l limits -12 → 12, -18 → 17, -18 → 18 -20→20, -12→12, -25→25Reflections measured 10419 67756Unique data with 3765 6176

I > 2σ(I)Refinement method full-matrix least squares on F2

R [F² > 2σ(F²)] 0.031 0.022Rint 0.011 0.019wR(F²) 0.058 0.052

Table 2Crystal data and structure refinement for compounds 3 and 4.

3 4

Formula C15H16Cl3N4Ru C12H22Cl3N4ORuS.H2OFormula weight 459.74 495.83Crystal system Monoclinic MonoclinicSpace group P21/n P21/nT (K) 90 90a (Å) 7.8024 (16) 8.9841 (10)b (Å) 9.965 (2) 13.7983 (15)c (Å) 21.753 (5) 30.823 (3)β (°) 92.801 (19) 95.453 (5)V (A3) 1689.3 (6) 3803.7 (7)Z 4 8Dx (Mg m- 3) 1.808 1.732μ (mm- 1) 11.90 11.69F(000) 916 2008Crystal size (mm) 0.03 x 0.02 x 0.02 0.28 x 0.10 x 0.04ϕ range (°) 2.5-30.5 4.1-68.2h, k, l limits -8→8, -11→10, -23→13 -10→9, -16→16, -36→36Reflections measured 12076 32623Unique data with I > 2σ(I) 13496195Refinement method full-matrix least squares on F2

R [F² > 2σ(F²)] 0.058 0.035

Rint 0.172 0.044wR(F²) 0.163 0.073

2.4. Cell viability assay and IC50 measurements

Human breast cancer cells (MCF7 purchased from ATCC) were grown in Dulbeco's modified Eagles media (DMEM) that wassupplemented with 10% heat inactivated fetal bovine serum (FBS, Sigma-Aldrich), 2 mM L-glutamine and penicillin/streptomycin (100 units and 0.1 mg/mL) in humidified CO2 incubators with 5% CO2

as described by us previously [36,37]. The cytotoxicity of ruthenium metal complexes was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay described previously [38,39]. In brief, approximately 10,000 cells were seeded into each well of a 96-well micro titer plate and incubated for 24 h in 150 μL DMEM. An additional 50 μL DMEM containing the required amount

of each metal complex to obtain 0.05 to 60 μM final concentration (in 200 μL final volume) were added in each well. Each experiment was carried out in 5 replicate wells. Control wells were treated with equivalent amounts of DMSO (the solvent used for dissolving the metal-complex stock solution). After 96 h of incubation, 20 μL of MTT (5 mg/mL) was added into each well and cell viability was assayed by measuring the formazan absorption at 560 nm by using a 96 well plate reader, Flowstar-Omega (BMG labtech). The absor-bance (at 560 nm) values were plotted against concentration of metal-complexes to determine the IC50. The concentration of the metal-complexes at which the conversion of MTT to formazan by viable cells is reduced by 50% compared to control cells is defined as the IC50. The experiments were repeated at least twice with 5replicates each time.

2.5. Synthesis of starting complexes

Complexes [H(DMSO)2]trans-[RuCl4(DMSO)2] and mer-[RuCl3(DMSO-S)2(DMSO-O)] were prepared by the literature method [40].

2.6. Synthesis of ruthenium-pyrazole complexes

2.6.1. Synthesis of mer-[RuCl3(DMSO-S)2(DMSO-O)] (1): A solution of mer-[RuCl3(DMSOS)2(DMSO-O)] (0.1

g,0.23 mmol) and pyrazole (0.118 g, 1.74 mmol) in dichloromethane (20 mL) was heated to reflux for 24 h. The volume of solution was reduced to 10 mL and then a small amount of diethyl ether was added. The mixture was kept refrigerated to yield a dark red crystal-line solid (yield 0.172 mmol, 76%). Anal. Calcd. for C8H14Cl3N4ORuS

Please cite this article as: S. David, et al., Synthesis, characterization, and anticancer activity of ruthenium-pyrazole complexes, J. Inorg. Biochem. (2012), doi:10.1016/j.jinorgbio.2012.02.022

S. David et al. / Journal of Inorganic Biochemistry 111 (2012) xxx-xxx 3

Table 3 dimethylpyrazole. However, the X-ray structure revealed that thebond length and bond angle of compounds 1, 2, 3, and 4.

Compound Bond length (Å) Bond angle (°)

1 Ru1-N3 2.064 (3) N3-Ru1-N1 86.54 (13)Ru1-N1 2.114 (4) N3-Ru1-S1 94.69 (9)Ru1-S1 2.2783 (12) N1-Ru1-S1 178.57 (11)Ru1-Cl1 2.3378 (9) N3-Ru1-Cl1 87.35 (9)Ru1-Cl2 2.3439 (10) N1-Ru1-Cl1 90.15 (8)Ru1-Cl3 2.3508 (9) S1-Ru1-Cl1 89.19 (3)

N3-Ru1-Cl2 176.78 (9)N1-Ru1-Cl2 90.27 (11)S1-Ru1-Cl2 88.49 (4)Cl1-Ru1-Cl2 92.20 (3)N3-Ru1-Cl3 87.97 (9)N1-Ru1-Cl3 88.71 (8)S1-Ru1-Cl3 92.05 (3)Cl1-Ru1-Cl3 175.25 (4)Cl2-Ru1-Cl3 92.42 (3)

2 Ru1-N1 2.0958 (11) O2-Ru1-S1 94.59 (3)Ru1-S1 2.2777 (3) N1-Ru1-Cl2 88.95 (3)Ru1-O2 2.0656 (9) N1-Ru1-S1 179.75 (3)Ru1-Cl1 2.3477 (3) O2-Ru1-N1 85.59 (4)Ru1-Cl2 2.3400 (3) N1-Ru1-Cl3 89.48 (3)Ru1-Cl3 2.3456 (3) S1-Ru1-Cl1 90.083 (13)S2-O2 1.5592 (10) O2-Ru1-Cl2 174.28 (3)

O2-Ru1-Cl1 86.42 (3)S1-Ru1-Cl2 90.856 (12)

3 Ru1-N1 2.024 (11) N1-Ru1-N2 79.4 (4)Ru1-N2 2.063 (9) N1-Ru1-N3 98.2 (4)Ru1-N3 2.095 (9) N2-Ru1-N3 177.5 (4)Ru1-Cl1 2.352 (4) N1-Ru1-Cl3 84.6 (3)Ru1-Cl2 2.366 (3) N2-Ru1-Cl3 91.7 (3)Ru1-Cl3 2.327 (3) N3-Ru1-Cl3 88.6 (3)

N1-Ru1-Cl1 92.4 (3)N2-Ru1-Cl1 88.1 (3)N3-Ru1-Cl1 91.5 (3)Cl3-Ru1-Cl1 177.03 (12)N1-Ru1-Cl2 171.5 (3)N2-Ru1-Cl2 94.1 (3)N3-Ru1-Cl2 88.4 (3)Cl3-Ru1-Cl2 90.24 (12)Cl1-Ru1-Cl2 92.73 (12)

4 Ru1-N1 2.095 (3) N1-Ru1-N3 88.81 (12)Ru1-N3 2.106 (3) N1-Ru1-S1 92.28 (9)Ru1-S1 2.3050 (9) N3-Ru1-S1 176.62 (9)Ru1-Cl1 2.3356 (9) N1-Ru1-Cl1 91.14 (9)Ru1-Cl2 2.3466 (9) N3-Ru1-Cl1 91.36 (9)Ru1-Cl3 2.3411 (9) N3-Ru1-Cl3 90.11 (9)

Cl3-Ru1-Cl1 178.38 (3)N1-Ru1-Cl2 176.83 (9)Cl3-Ru1-Cl2 89.24 (3)Cl1-Ru1-Cl2 90.13 (3)

(424.71): C, 22.78; H, 3.35; N, 13.29. Found: C, 22.93; H, 3.24; N,13.25. Selected IR absorption bands in KBr (cm- 1): νSO1054(S-DMSO), νC=C (pyz) 1629, νC=N (pyz) 1400. UV-vis (H2O):364 nm (ε = 32.60 M- 1 cm- 1); 426 nm (ε = 10.60 M- 1 cm- 1).

2.6.2. Synthesis of mer-[RuCl3(DMSO-S)(DMSO-O)(pyz)] (2): mer-[RuCl3(DMSO-S)2(DMSO-O)] (0.1 g, 0.23 mmol)

andpyrazole (0.017 g, 0.25 mmol) were dissolved in dichloromethane (10 mL) and stirred at room temperature for 20 h. The solution was evaporated to 3 mL and 10 drops of hexane were added. The flask was kept in a refrigerator. The resulting solution yields red crystals (yield 0.184 mmol, 80%). Anal. Calcd. for C7H16Cl3N2O2RuS2·CH2Cl2

(516.68) C,18.6; H, 3.5; N, 5.4. Found: C, 18.87; H, 3.82; N, 5.11. Selected IR absorption bands in KBr (cm- 1): νSO1103, (S-DMSO), 909 (O-DMSO), νC=C (pyz) 1627, νC=N (pyz) 1409. UV-vis

(H2O):386 nm (ε = 27.11 M- 1 cm- 1); 525 nm (ε = 8.34 M- 1 cm- 1).

2.6.3. Synthesis of mer-[RuCl3(bpy)(dmpyz](3). Our aim was to synthesize mer-[RuCl2(bpy)

(DMSO-S) (dmpyz)]CF3SO3 by refluxing mer-[RuCl3(DMSO-S)(bpy)] with equi-molar amounts of silver triflate and an excess amount of 3,5-

isolated product is mer-[RuCl3(bpy)(dmpyz)] 3, not what weexpected. Compound 3 was also synthesized by the direct reaction of mer-[RuCl3(DMSO-S)(bpy)] with an excess amount of 3,5-dimethylpyr-azole. mer-[RuCl3(DMSO-S)(bpy)] (0.1 g, 0.23 mmol), 3,5-dimethylpyr-azole (0.167 g, 1.74 mmol) and acetone (35 mL) were placed in a flask, which was heated to reflux for 24 h. The volume of solution was reduced to 3 mL and 10 drops of hexane were added. On cooling at

4°C the resulting solution yields orange crystalline solid (yield0.156 mmol, 68%). Calcd. for C15H16Cl3N4Ru (459.74) C, 39.2; H, 3.5; N, 12.2. Found: C, 38.8; H, 3.41; N, 11.81. Selected IR absorption bands

in KBr (cm- 1): νC=C, 1573 (m), νC=N, 1448 (s) (pyz), νC-N (bpy) 1607 (m), UV-vis (H2O): 435 nm (ε = 47.89 M- 1 cm- 1); 375 nm(ε = 8.29 M- 1 cm- 1). Unfortunately, our attempt to synthesize the sim-ilar complex with pyrazole ligand was unsuccessful.

2.6.4. Synthesis of mer-[RuCl3(DMSO-S)(dmpyz)2](4): A solution of mer-[RuCl3(DMSO-S)2(DMSO-O)] (0.2 g,

0.45 mmol) and 3,5-dimethylpyrazole (0.334 g, 3.48 mmol) indichloromethane (15 mL) was heated to reflux for 9 h. The brown solution was evaporated to 3 mL and then 1 mL of hexane was

added. On cooling at 4°C a reddish-orange solid was separated

(yield 0.262 mmol, 58%). Product was purified by column chromatog-raphy with ethyl acetate and hexane (40/60) as eluent. Anal. Calcd. C12H20Cl3N4ORuS∙H2O (495.83): C, 29.06; H, 4.95, N, 11.35. Found: C, 29.18.; H, 5.24; N, 10.95. Selected IR absorption bands in KBr(cm- 1): νSO1086 (S-DMSO), νC=C (pyz) 1632, νC=N (pyz) 1398. UV-vis (H2O): 362 nm (ε = 12.20 M- 1 cm- 1).

3. Results and discussion

3.1. Syntheses and general properties

All the complexes were formed by the displacement of either one or two DMSO ligands from the precursor, mer-[RuCl3(DMSO-S)2 (DMSO-O)] with pyrazole or 3,5-dimethylpyrazole ligands. Thespectroscopic features of the sulfoxides in these complexes are comparable to those found for the corresponding complexes with N-monodentate ligands [41]. The pyrazole (Fig. 1) as a ligand, coordi-nates to metals and metalloids through the N2 [42]. The presence of a strong IR band in the range assignable to N-H vibrations in the spectra of all the complexes (1-4) confirms the coordination of pyrazole via N2 (Fig. 1) in the neutral monodentate form. The characteristic IR bands of C =C and C =N stretching modes are observed at ca. 1640, and 1558 cm- 1 in uncomplexed pyrazole and are shifted to consider-ably lower frequencies. This is fairly consistent with longer C =C and C =N bonds in 1-4 compared with the same distances in the uncom-plexed ligand [43]. The composition of all the complexes reported herein has been determined by various physical methods, especially single crystal analysis.

4 3

5 N 2

N 1H

Fig. 1. Structure of pyrazole.

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4 S. David et al. / Journal of Inorganic Biochemistry 111 (2012) xxx-xxx

Fig. 2. Ortep diagram of mer-[RuCl3(DMSO)(pyz)2] 1.

3.2. Crystallographic analysis

The single crystals suitable for X-ray diffraction of the complexes 1,2,3, and 4 were obtained by slow diffusion of hexane into CH2Cl2

and diethyl ether into acetone solutions. Figs. 2-5 depict themolecular structures of these complexes. Data collection parameters and selected bond angles and lengths are listed in Tables 1-3 for all the complexes.

Compound 1 crystallizes in the monoclinic space group Cc that adopts a distorted octahedral geometry with meridional arrangement (Fig. 2). Both pyrazole ligands are coordinated to ruthenium through the tertiary ring nitrogen atom N2 (Fig. 1), The average Ru-Cl bond length, 2.344(9) Å is comparable to other related Ru(III) complexes [41,44,45]. The Ru-S bond distance, 2.2783(12) is appreciably shorter than the average value of 2.34 (1) Å found for the Ru(III)-Scompounds trans to DMSO-S in the precursor [46,47]. Likewise the average bond length of Ru-N, 2.089 Å is comparable to Ru(III)-N (imidazole), 2.079 Å [41]. The bond angles of N1Ru1S1, 178.57(11)°;

Fig. 3. Ortep diagram of mer-[RuCl3(DMSO)2(pyz)] 2.

Fig. 4. Ortep diagram of mer-[RuCl3(bpy)(dmpyz)2] 3.

and Cl2Ru1N3, 176.78(9)° are slightly smaller than the known values of octahedral complexes of ruthenium (III).

Compound 2 was crystallized in the monoclinic space group P21/n with distorted octahedral geometry having meridional arrangement (Fig. 3). The pyrazole ligands coordinated to ruthenium through N2 (Fig. 1). The average Ru-Cl bond length, 2.344(9) Å is similar tocompound 1. The Ru-S bond distance, 2.2777(3) is very close to compound 1. The bond length of Ru-N, 2.0958 Å is slightly smaller than Ru(III)-N (imidazole), 2.079 Å [41] but significantly shorter than 1 (2.114 Å). The Ru-O bond length 2.065 Å is slightly shorter than the precursor, mer-[RuCl3(DMSO-S)2(DMSO-O)] (2.077 Å). The S-O bond distance (1.559 Å) of the O-bonded DMSO is noticeably longer than those of the S- bonded DMSO ligands (1.479 Å), which

shows a considerable decrease of the double-bond character of the sulfur-oxygen bond upon coordination to the metal atom via oxygen.

Fig. 5. Ortep diagram of mer-[RuCl3(DMSO)(dmpyz)2] 4.

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S. David et al. / Journal of Inorganic Biochemistry 111 (2012) xxx-xxx 5

Compound 3 was crystallized in the monoclinic space group P21/c.

The bite angle in compound 3 (Fig. 4) involving coordinatedbipyridine is N1RuN2, 79.4(4)°, typically found in 2, 2 ’-bipyridine coordinated to second and third row transition metal ions [48]. The bond angle of the nonchelating ligand, N3Ru1Cl2 is 88.4 (3)°and is much larger than the chelating ligand bond angle, N1RuN2, 79.4 (4)°. The average Ru-N (bipyridine) bond length, 2.0435 Å, isshorter than the Ru-N(pyrazole) bond length, 2.095 Å, and distinctive bond length of 2.099 Å reported for Ru(II, III) complexes [47-49]. This is probably due to chelation through bipyridine nitrogens. The average Ru-Cl bond length of 2.348 Å is comparable to the related Ru(III) complexes. The bond angles of N2RuN3, 177.5(4)°; N1RuCl2, 171.5 (3)° are smaller than the known values of octahedralcomplexes. Likewise, the bond angles N2RuCl3, 91.7(3)°; N1RuCl1, 92.4 (3)°; Cl1RuN3, 91.5(3)°; N2RuCl2, 94.1(3)° vary from 91° to94°. These distortions from ideal octahedral geometry are probably caused by the acute (~ 79.4°) bite angle of the chelating bipyridine ligand.

Compound 4 was crystallized in the monoclinic space group P21/n. The X-ray structure of 4 shows a distorted octahedral geometry (Fig. 5) with three chlorine atoms in a mer configuration. The average Ru-Cl bond length, 2.3411(9) Å is slightly shorter than that of compound 1. The Ru-S bond distance, 2.3050(9) Å is shorter than the average value of 2.34(1) Å found for the Ru(III)-S compounds trans to DMSO-S in the precursor [40,46]. The average bond length of Ru-N, 2.1005(3) Å, is longer than Ru(III)-N (imidazole), 2.079 Å [44,45], and the average bond length (Ru-N) of 1 (2.089 Å). The bond angles of N1Ru1S1, 176.62(9)° and N1RuCl2, 176.83(3)° are close to the known values of octahedral complexes of ruthenium (III).

The average values of the Ru-Cl bond lengths are comparable in all four complexes (2.344(9) Å for 1; 2.344(9) Å for 2; 2.348 Å for 3, and 2.3411 (9) Å for 4) and are close to the average value of 2.357 Å foundin related ruthenium (III) complexes [44]. These distances are shorter than those found in the trans-Cl-Ru(II)-Cl (av. 2.41 Å) [40,44,50].

3.3. Electrochemical study

Figures of cyclic voltammetry (Figures S1-S7) are given in

supporting information. Cyclic voltammetry results were

obtained for 1 in both aqueous and DMSO solutions at sweep rates ranging from 100 mV/s to 3011 mV/s. Sweeps were similar at all rates. A dom-inant reduction and corresponding oxidation are evident. In addition there is a weak and broad oxidation peak. The values of the separation of the dominant oxidation and reduction peaks (ΔEp) ranged from 79 mV at 100 mV/s to 137 mV at 3011 mV/s and are assumed to be due to a quasireversible oxidation-reduction couple. The formal potential (E°’) calculated as the average of the oxidation andreduction peak values ranged from 0.059 to 0.061 V with an average value of 0.060 V vs. SCE (saturated calomel electrode). Semiderivative analysis [51-53] was also done with these sweeps. The average formal potential obtained this way was 0.061 V.

Using an extended range in the anodic direction gives an additional anodic/cathodic pair of peaks. The characteristics of this voltammagram are consistent with an electron-chemical-electron (ECE) mechanism as shown generally though not exactly by simula-tion [54] as given in Fig. S3. The chemical reaction involved in the ECE mechanism may be due to ligand replacement by DMSO similar to that discussed for compound 2 below.

Compound 1 gives similar sweeps in aqueous solution. Voltamma-grams at rates from 100 to 3011 mV/s were similar. For the largest pair of peaks, an average value of ΔEp = 83 mV and an average E°’ = 0.333 V were found from the voltammagrams. Semiderivative analysis gives E°’ = 0.337 mV. The pair of peaks is assumed to be due to the quasireversible Ru(III)/Ru(II) redox couple.

The cyclic voltammagram for 2 shows a large reduction peak ataround − 0.20 V but only a small corresponding oxidation peak that is barely discernible at low sweep rates. Further oxidation to about 0.6 V on the anodic sweep produces a well defined oxidation peak and corresponding reduction on the following anodic sweep. When

cyclic voltammetry is initiated in the anodic direction the peak at 0.6 V does not appear on the first sweep. Only after reduction at − 0.2 V does the more positive redox couple appear (Fig. S5). This behavior is interpreted as being due to reduction of 2 as Ru(III) goes to Ru(II). Oxidation of 2 on the following anodic sweep competes

with chemical reactions between 2 and DMSO. The predominant

compound that forms is then the basis for the redox couple seen at

more positive potentials. There may be other compounds formed as well. These would account for redox couples that give small (but quite distinct when viewed using semiderivative analysis) oxidation and reduction peaks.

Lacking a large anodic signal, it is possible to give only an estimate of the characteristics of the redox couple of 2. The intermediate between Eox and Ered was calculated and extrapolated to zero sweep rate for both the CVs and the semiderivative graphs. These extrapola-tions give an average value of − 0.15 V. For the other large redox peaks, extrapolation of CV data gives a formal potential of 0.545 V and extrapolation of semiderivative data gives 0.546 V.

The identity of the main compound formed from 2 is assumed to

be the product of ligand displacement by DMSO. In 2, one to four

ligands could be displaced. In addition, DMSO can bond through S or O atoms. This gives rise to many possibilities. One possibility can

be checked with available CV data. Alessio et al. [40] obtained the CV of mer-[RuCl3(DMSO-S)2(DMSO-O)] and observed a redox couple at 0.070 V This then would not be responsible for our large peaks around 0.55 V but it may be responsible for one of the small peaks around zero volts in our data. The redox potentials of this compound

as well as 2 were calculated using Gaussian 03. The value obtained for

mer-[RuCl3(DMSO-S)2(DMSO-O)] was found to be only 0.3 V more positive than that for 2. Calculations were not done for every possible compound but calculation for mer-[RuCl3(DMSO-S)2(DMSO-O)] gives

a redox potential about 1.1 V more positive than calculated for 2.

Cyclic voltammetry was done for 3 in DMSO (Fig. S6) (No peaks were found in aqueous solution). Five subsequent segments were recorded and are nearly the same as the second and third segments in the figure. Voltammagrams in DMSO were done at rates of 100 to 3011 mV/s and were all similar in appearance. Two redox couples are seen. The formal potential averaged over all sweep rates for one couple was found to be − 0.175 V from the cyclic voltammagrams and − 0.180 V as determined from semiderivative analysis. For the other complex the formal potential was found to be 0.129 V from cyclic voltammetry and 0.125 V from semiderivative analysis. The couples are assumed to be due to sequential one-electron processes involving the + 1, + 2, and + 3 oxidation states of Ru.

Cyclic voltammetry was done for 4 in aqueous and DMSO solu-tions. In aqueous solution a single redox couple is observed. Anodic and cathodic peaks of the couple are broad and shallow. At some sweep rates the signal is only a shoulder to the water reduction area of the graph. Though only approximate, values obtained indicate a separation between anodic and cathodic peaks to be about 0.15 V and an average of the anodic and cathodic potentials is around − 0.1 V. Cyclic voltammetry in DMSO shows four cathodic peaks and five anodic peaks. Near-coincidence of potential values for cathodic and anodic peaks when using semiderivative analysis indicates redox couples at these potentials: -0.41 V; -0.15 V; + 0.44 V (Fig. S7). Many in the array of peaks for 4 are likely due to other com-pounds formed through exchange of dmpyz with dmso and through bonding rearrangement through the O and S atoms of DMSO ligands.

The electrochemical study of metal complexes explains the change of structural arrangement of the ligand around metal ions. It is worth mentioning that the cyclic voltammetry of compounds 1-4 displayed

Please cite this article as: S. David, et al., Synthesis, characterization, and anticancer activity of ruthenium-pyrazole complexes, J. Inorg. Biochem. (2012), doi:10.1016/j.jinorgbio.2012.02.022

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120

100

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S. David et al. / Journal of Inorganic Biochemistry 111 (2012) xxx-xxx

increasing ease of reduction [63]. We observed that the reduction po-tential of compound 2 is − 2.0 V, which probably suggests that reduc-tion of Ru(III) to Ru(II) is slow compared to compounds 1 and 3.

4. Conclusions

We have reported, herein, the syntheses and characterization of 4new neutral water soluble ruthenium-DMSO-pyrazole complexes. Inall cases, the synthesis of these complexes involves substitution ofone or more DMSO molecules with pyrazoles. All members are fullycharacterized by various physical techniques as mononuclear dis-torted octahedral with meridonial arrangement. The result of invitro cytotoxicity testing showed that compounds 1 and 4 show sig-nificant cytotoxicity to the MCF7 breast cancer cells, whereas com-pounds 2 and 3 do not show appreciable anticancer activity to

40 60 80 100 tested cancer cell lines.

Concentration (M)

AcknowledgementsFig. 6. Effect of ruthenium-pyrazole complexes on cell viability- IC50 Values ofcompounds: 1. 71.0 μM (+ 2.1); 2. > 100 μM; 3. > 100 μM; 4. 32.2 μM (+ 1.3).

reversible Ru(III)/Ru(II) reduction waves. The observed redox poten-tials fall in the biologically relevant and accessible range. The known value of reduction potential is − 0.48 vs. SCE [55,56] in the proliferating cells, while inside the tumor cells it is up to 100 mV lower [57], suggest-ing that biological reducing agents (e.g., glutathione or ascorbic acid) are capable of reducing the compounds to the corresponding Ru(II) species [58]. Moreover, redox potentials, which are obtained by cyclic voltammetry were interconnected with protein binding and cytotoxic activity.

3.4. Cell viability assay and IC50 measurement

The metal ion coordination environment in biomolecules consists of nitrogen and oxygen-donor atoms that are provided by histidine, aspartates, or glutamates. Since pyrazole resembles imidazole, it has been incorporated in stable chelates to obtain ligand systems applicable in the design of new drugs.

In order to evaluate the antitumor potential of the metal com-plexes, we measured their cytotoxicity toward human breast cancer cells, MCF7. In brief, cells were incubated with varying concentrations of the metal complexes for 96 h at 37 °C and 5% CO2. Viable cells were then quantified using MTT assay as described previously [59,60]. The cell viability is plotted vs. concentration of ruthenium complexes (Fig. 6). These studies demonstrated that compounds 1 and 4 showed significant cytotoxicity toward MCF7 cells with IC50 values 71 (+ 2.1) μM and 32.2 (+ 1.3) μM respectively (Fig. 6). In contrast 2 and 3 were not effective (IC50 > 100 μM) in inducing cell death in MCF7 cells. We also determined the IC50 (~ 18 μM for MCF7) for cisplatin as a positive

control and it is in agreement with previously reported values.

It is interesting to note that compounds 1 and 4 showed significant cytotoxicity in MCF7 cells with IC50 values 71 (± 2) μM and 32.2 (± 1.3) μM whereas, the Ru-bipyridine complex 3 is not effective (IC50 > 100 μM) in inducing cell death in MCF7 cells. Some Ru- poly-pyridine complexes like mer-[Ru(terpy)Cl3] are known to form DNA-interstrand crosslinks, whereas the inactive cis-[Ru(bpy)2Cl2] appears to exhibit no such interactions [61,62]. We believe that com-pound 3, which contains polypyridine (bipyridine) ligand follows a similar trend. A possible explanation of discrimination between mer-[Ru-(terpy)Cl3] and cis-[Ru(bpy)2Cl2] and [Ru(terpy)(bpy)CI]CI or compound 3 is intracellular ruthenium uptake [61]. The biological diversity between compounds 1, 4 and 2, 3 may be due to their differ-ent redox properties [62]. There are multiple factors such as pKa values of the ligands, protein binding, and redox potential of com-pounds, etc. that are responsible for the cytotoxic effect of the com-pounds. However, cytotoxic ability is expected to increase with

We are grateful for financial support provided by the LouisianaBoard of Regents [LEQSF(2009-12)-RD-B-08]. Research in Dr. Mandal's lab is supported by the American Heart Association and the Texas Ad-vanced Research Program.

Appendix A. Supplementary data

Supplementary data to this article can be found online at doi:10. 1016/j.jinorgbio.2012.02.022.

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