Ru(II) trithiacyclononane 5-(2-hydroxyphenyl)-3-[(4-methoxystyryl)pyrazole], a complex with facile synthesis and high cytotoxicity against PC-3 and MDA-MB-231 cells

The ruthenium(II) complex [Ru([9]aneS 3)(phpz)Cl 2] (1) ([9]aneS 3=trithiacyclononane, phpz=5-(2- hydroxyphenyl)-3-[(4-methoxystyryl)pyrazole]) was readily isolated by reacting [Ru([9]aneS 3)(DMSO)Cl 2] with one equivalent of the ligand phpz. A combination of MS, FT–IR and solution NMR studies (1-D and 2-D) was employed to determine the structural formula of the complex 1, in which phpz coordinates in a monodentate mode to Ru(II) by a simple replacement of the leaving group DMSO of the precursor. The cytotoxic properties of 1 in vitro were investigated by determination of the half-maximal growth inhibition on the human prostate (PC-3) and breast cancer cells (MDA-MB-231).


Introduction
The preparation and properties of pyrazole and its analogues are a subject of consistent interest due to their wide range of applications in various areas, namely in pharmacy [1]. Known examples of pyrazole-derived active pharmaceutical ingredients include the non-steroidal antiinflammatory celecoxib [2,3] and sildenafil citrate for the treatment of erectile dysfunction [4]. Several pyrazole compounds also feature cytotoxic and anti-retroviral properties [5]. Crizotinib, a pyridine-pyrazole derivative, is already in use against non-small cell lung cancer [6,7], and several new pyrazole compounds developed between 2002 and 2011 showed promising antitumor activity [8]. * Corresponding author. Email: sbraga@ua.pt Within these, 3(5)-(2-hydroxyphenyl)pyrazoles are of particular relevance due to their potent inhibition of the heat shock protein 90 (Hsp90) ATPase [9], a biomolecule essential for cell growth and viability which causes resistance to chemotherapy in cancer cells [10]. Copper complexes of (2-hydroxyphenyl)pyrazoles were reported to inhibit the WM-115 melanoma cancer cell line at only half of the active concentration of cisplatin [11,12].

Materials and methods
The syntheses of phpz and [Ru( [9]aneS 3 )(DMSO)Cl 2 ] were previously reported [13,14]. All other chemicals were purchased from Sigma-Aldrich (Poole, UK) and used as received. Reactions were executed using standard Schlenk techniques under nitrogen atmosphere. Microanalyses for CHNS were performed by M. Marques on a TruSpec 630-200-200 CNHS Analyzer.
Mass spectra were performed using methanol as solvent and electrospray ionization (ESI-MS). Results were recorded in a Micromass R Q-ToF 2 mass spectrometer.
Nuclear magnetic resonance (NMR) studies in dimethylsulfoxide (DMSO) solution comprised 1-D ( 1 H and 13 C) and 2-D experiments, namely 1 H-13 C heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond coherence (HMBC) NMR spectra were recorded at ambient temperature in a Bruker Avance 300 (frequencies: 300.13 MHz for 1 H, 75.47 MHz for 13 C) or in a Bruker DRX 500 spectrometer (500.13 MHz for 1 H and 125.77 MHz for 13 C), using the residual protic solvent signal as an internal reference (DMSO-d 6 : 1 H 2.50 ppm and 13 C 39.5 ppm). Chemical shifts are quoted in parts per million (ppm).

Cytotoxicity studies
The complex 1, phpz and cisplatin (used as positive control) were tested on the human PC-3 prostate (androgen-independent) and the MDA-MB-231 (estrogenindependent) breast cancer cell lines, using the mitochondrial dehydrogenase activity assay (MTT) for cell viability evaluation. The two cell lines were grown in monolayers in 75 cm 2 tissue culture flasks at 37 • C in a humidified atmosphere with 5% CO 2 , sub-cultured every 2-3 days and harvested upon addition of trypsin/EDTA (0.05% trypsin/EDTA solution). The culture medium was RPMI 1640, supplemented with sodium bicarbonate (1.5 g L −1 ), 10% heat-inactivated fetal bovine serum (FBS) and penicillin-streptomycin (100 units mL −1 penicillin and 100 µg mL −1 streptomycin) for PC-3 cells

Results and discussion
The complex [Ru( [9]aneS 3 )(phpz)Cl 2 ] (1) precipitated as a yellow microcrystalline solid (powder diffractogram available as supplementary information) from the reaction of [Ru( [9]aneS 3 )(DMSO)Cl 2 ] with one molar equivalent of phpz. It is partially soluble in polar solvents such as dimethylsulfoxide, methanol or water. As will be described below, solution-phase NMR studies of the complex 1 revealed that the coordination of the ligand phpz occurred in a monodentate fashion at the N2 of the pyrazole moiety ( Figure 1). The FT-IR spectrum of 1, though not showing the Ru-N vibration which lies below 400 cm −1 , is in accordance with this structure since a redshift is observed for the pyrazole's N-H and C=N bands, associated with metal coordination [15]. The N-H stretch suffers a redshift and the O-H stretch, absent from the spectrum of phpz, appears as a broad band at 3447 cm −1 .
The positive ESI-MS spectrum of 1 (in methanol) shows the most intense peak at m/z = 581, corresponding to [Ru([9]aneS 3 -CH 2 CH 2 )(phpz)Cl] + , a fragment generated by the loss of one ethylene unit of trithiacyclononane Solution-phase NMR studies determined the structure and geometry of the coordinated phpz in complex 1. Free phpz, like most pyrazoles, has two tautomers ( Figure 2), along with a trans-vinylic system (Cα=Cβ). Its 1 H NMR spectrum, represented in Figure 3 (bottom), presents four deshielded broad singlets attributed to the NH and C2 -OH resonances of the two prototropic isomer forms. The hydroxyl proton usually appears at a higher frequency than the NH because it is involved in an intramolecular hydrogen bond with the N2 (OH· · ·N tautomer, phpz-I).
In a DMSO-d 6 solution, this OH· · ·N2 bridge is partially replaced by another with DMSO and the resonances of the NH· · ·O tautomer (phpz-II) can be observed, although with less intensity. Indeed, the two most intense signals (C2 -OH at 13.32 and NH at 10.91 ppm) of the spectrum of the phpz ligand are ascribed to phpz-I and the two less intense (2 -OH -12.77 and NH -10.25 ppm) to phpz-II.
In the 1 H NMR spectrum of the complex 1 (Figure 3, top), the Hβ signal of coordinated phpz is slightly shifted downfield (0.17 ppm) and the Hα affords a doublet ( 3 J α,β 16.0 Hz), indicating a trans configuration for Cα=Cβ in the Ru-coordinated phpz. The NH proton appears deshielded to 12.82 ppm (shift of 2.57 ppm), which is associated with N2-coordination of phpz to the ruthenium cation (details in the next paragraph) and with the presence of an NH · · · O bridge with the hydroxyl (i.e. ruthenium-coordinated phpz-II). The resonances of the methoxybenzene moiety are unshifted, and the [9]aneS 3 macrocycle appears as a multiplet at 3.10-2.58 ppm. A summary of the 1 H NMR spectral data for the complex 1 and the pure ligand is presented in Table 1.

Conclusion
The new complex herein presented, [Ru( [9]aneS 3 )(phpz) Cl 2 ] (1), combines a facile one-step synthesis with a notable cytotoxic action towards the MDA-MB-231 and PC-3 human cancer cell lines. Given its potential value, both phpz and the complex 1 warrant further studies on healthy human cells, regarding toxicological safety. Should these studies demonstrate that coordination to ruthenium plays a key role in the selectivity of action of phpz towards cancer cells, the coordination of phpz to other ruthenium scaffolds will be contemplated as a follow-up study.