Ru-Based NSAIDs as Potential Anticancer Therapeutics

TL;DR

This paper explores new ruthenium-based compounds with anti-inflammatory drugs that show potential as cancer treatments.

Contribution

The study introduces new Ru-based NSAID complexes with promising anticancer properties.

Findings

Two Ru-NSAID complexes were structurally characterized using X-ray diffraction.

A Ru-salicylic acid complex showed antiproliferative activity against HeLa cells.

The complexes were synthesized with molecular hydrogen release.

Abstract

The use of metal-based species bearing existing pharmaceuticals as ligands—often resulting in enhanced bioactivity—represents an attractive strategy for the development of novel therapeutic formulations. In this context, five well-known non-steroidal anti-inflammatory drugs (NSAIDs) were employed to substitute both PPh3 and hydride ligands in [Ru(H)2(CO)(PPh3)3] (1), thereby selectively affording neutral κ2-(O,O)–chelate complexes in satisfactory yields via molecular hydrogen release. Among the obtained species, two complexes coordinating diclofenac (4) and aspirin (5) were further investigated by single-crystal X-ray diffraction (SCXRD). Preliminary biological studies were conducted on the ruthenium–salicylic acid species 2 and ibuprofen 6. The former showed promising antiproliferative activity against HeLa cancer cells, consistent with the well-established role of NSAID–ruthenium(II) complexes as a platform for the development of novel anticancer metallotherapeutics.

Figures

Ruthenium–NSAID complexes encompassing phosphine ligands. (a) Graminha (2022) [25], (b) Correa (2025) [27], (c) Tabrizi (2019) [30].

Full text

1. Introduction

The increasing societal impact of cancer has prompted the scientific community to seek novel chemotherapeutic agents with reduced side effects. The search for novel and effective anticancer agents has fuelled a growing interest in the development of transition metal complexes encompassing biologically relevant molecules. Metal-based species containing versatile ruthenium coordinated to multi-functionalized ligands undoubtedly occupy a prominent position in this field [1,2,3,4,5,6]. Non-steroidal anti-inflammatory drugs (NSAIDs) constitute a well-known, broad class of medications used to treat pain and inflammation and exhibit similar pharmacological properties, mechanisms of action, and side effects [7]. These compounds possess a variety of biological activities due to their ability to bind plasma proteins, a feature primarily ascribed to amphiphilic properties. Their structure includes hydrophilic groups, such as carboxyl or enol functions, together with lipophilic groups, such as aromatic rings or halide units [8,9].

Cyclooxygenase enzymes are known to play a crucial role in inflammation and carcinogenesis by catalyzing the conversion of arachidonic acid into prostaglandins [10]. NSAIDs have been demonstrated to reduce the incidence of several cancers, including breast, lung, and colorectal [11], through non-selective inhibition of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and lipoxygenase (LOX) metabolism, which plays a significant role in angiogenesis by promoting migration of endothelial cells [12].

In recent decades, multiple studies have demonstrated the existence of a strong correlation between inflammation and cancer development. A combinatory action, ascribable to the synergistic therapeutic effects arising from the combination of anti-inflammatory properties with Ru-based anticancer activities, has been recently demonstrated to be more than mere speculation [13,14]. NSAID–ruthenium (II) complexes represent a promising area of research in the development of novel anticancer treatments that leverage the combined benefits of anti-inflammatory and anticancer effects. In fact, this dual functionality (Ru site and NSAID) offers an alternative approach, targeting both inflammation and neoplastic growth and, in certain cases, improving efficacy and selectivity compared with fragments alone. Recently, Martling’s group reported a remarkable result, relating low-dose aspirin to the control of localized colorectal cancer [15]. It is noteworthy that aspirin, as an anti-inflammatory molecule, can also exhibit anticancer activity alone. In the case of the Ru-coordinating species 5, this aptitude is expected to be enhanced, according to the trend of analogous Ru compounds. These types of organo–inorganic systems are reported to exhibit advantages by enhancing therapeutic value by the combined ability of anti-inflammatory features with Ru-anticancer potential [16]. Sáez’s group reported that the presence of PPh_3_ moieties induces intercalation reactions with DNA [17].

The presence of the metal is crucial and implies several accessible and stable oxidation states under physiological conditions. The Ru site acts as a carrier species due to its analogue Fe redox potentials and its affinity for Fe–transferrin, thereby slowing ligand exchange in vivo and allowing greater chemotherapeutic durability and improved drug internalization. The iron-mimicking ability to bind specific proteins, such as albumin, results in higher accumulation of ruthenium inside tumor cells compared to healthy cells. [18,19,20,21,22]. The biological responses are indeed attributable to the synergistic effects of the metal ion core interacting with NSAID pharmacophores [10]. Several studies have reported the success of [Ru(η^6^-p-cymene)(NSAID)Cl], which coordinates ibuprofen, aspirin, naproxen, and diclofenac as k^2^-(O,O)-chelate ligands (Figure 1) [21,23]. The obtained complexes demonstrate significant antiproliferative activity against different cancer cell lines.

The above class of Ru complexes demonstrates promising antiproliferative activity against various cancer cell lines, such as cervical (HeLa), breast (MCF-7), and lung (A549), as well as tumors, with growth-inhibition values comparable to those of the most efficient current antineoplastic drugs [23]. The results reported from other authors for similar phosphine compounds indicate higher activity, corresponding to a lower dose (IC_50_) for fighting cancer cells. Specifically, Graminha (2022) indicated 3.46 µM for [Ru(dppe)2(A)] (Figure 2a), compared to the 8.91 µM for cis-platin on the MCF-7 breast cancer cell line, while Correa (2025) reported a value of 0.66 µM for [Ru(bipy)(dppp)(A)] (Figure 2b) compared with 11.80 µM for cis-Pt on the A2780 ovarian cell line [24,25,26,27].

Further, the cyclometalated Ru complex [Ru(CCC-Nap)(Ibu)(PTA)] (Figure 2c), concomitantly incorporating ibuprofen and naproxen-derived ligand, displays significant cancer cell cytotoxicity [28,29,30]. The structural similarity to the trans-phosphines complexes described by Correa—[Ru(PPh_3_)2(Th)(bipy)]PF_6_, in which Th indicates thiourea derivatives [31]—and by Baratta’s group—[Ru(OAc)(acac)(PPh_3_)2] [32]—has prompted the assumption of analogous amphiphilic properties for our class of metal systems.

Ruthenium–NSAID complexes encompassing phosphine ligands. (a) Graminha (2022) [25], (b) Correa (2025) [27], (c) Tabrizi (2019) [30].

In the present report, we described the chemistry of five different NSAIDs (salicylic acid 2, naproxen 3, diclofenac 4, acetylsalicylic acid 5, and ibuprofen 6) with the ruthenium hydride precursor [Ru(H)2(CO)(PPh_3_)3] 1, achieving the correspondent adducts [RuH(CO)(PPh_3_)2(NSAID)] by thermal solicitation in satisfactory yields. The selected NSAIDs span a broad range of polarities, as reflected by their log p values (salicylic acid/acetylsalicylic acid ≈ 1.1 < naproxen ≈ 3.2 < ibuprofen ≈ 4.0 < diclofenac ≈ 4.5), as shown in Figure 3. Variations in ligand polarity may influence the physical–chemical properties of the corresponding Ru–NSAID complexes, potentially affecting cellular uptake and intracellular distribution.

2. Results and Discussion

Complexes of the general formula [RuH(CO)(PPh_3_)2(NSAID)] were obtained in a 49–79% range by the reaction of the parent compound (1) [Ru(H)2(CO)(PPh_3_)3] with a slight excess of the NSAID to obtain the expected Ru(II)–NSAID complexes as unique species via selective H_2_ release and elimination of a PPh_3_ ligand. All the synthesized complexes were treated with Et_2_O extractions and recrystallized. Their proposed structures were supported by analytical and spectroscopic data, including ESI-MS, FTIR, ^1^H, ^31^P{^1^H}, ^13^C{^1^H} NMR, and UV–Vis spectra (Scheme 1).

2.1. Infrared Spectroscopy

IR spectral analyses are in agreement with the proposed structures. The IR spectra of 3–6 demonstrate asymmetric carboxy absorptions in the 1634–1521 cm^−1^ interval, close to the lower frequency set of bands (1526–1455 cm^−1^) attributable to the symmetric stretching. These signals are diagnostic for a dihapto coordination mode that allows the formation of four-membered metallacycles. An intermolecular H-bond network can be invoked, promoted by aryl-substituent lone pairs, such as those from hydroxyl or methoxy groups in the salicylate class of ligands, as well as the chloride in coordinated diclofenac. In the case of complex 2 (Scheme 1), the coordination of the salicylate ligand is supported by the occurrence of an OH stretching band. Useful comparisons are provided in Table 1.

2.2. NMR Spectra of the Prepared Complexes

^1^H, ^31^P{^1^H}, ^13^C{^1^H}, and bidimensional COSY, HSQC, and HMBC NMR spectra support the predicted molecular structure for complexes 2–6. Multiplets in the range of 7.64–7.14 ppm demonstrate the presence of two triphenylphosphine ligands. In the precursor spectrum, two ^1^H NMR signals are observed at −6.50 and −8.30 ppm, respectively. These are attributed to the highly shifted hydride ligands, whereas single hydride triplets are observed in the range −16.33/−16.91 ppm in the spectra of complexes 2–6, due to the coupling with the equivalent trans-PPh_3_, as confirmed by the ^31^P{^1^H} NMR resonances in the 43.09–44.60 ppm interval. The ^13^C NMR spectra of 2–6 display a downfield triplet at about 205 ppm, assigned to Ru-CO, while the singlets of the carboxylic carbon atoms fall in the range 186.05–178.53 ppm (Table 2).

2.3. ESI-MS and UV Spectra

Mass spectrometry provides fundamental information regarding the structure of complexes in solution. In the ESI-MS spectra of complexes 2 to 6, acquired in MeCN, the most relevant peak is the one related to the [M–H]^+^ composition. Other common fragments detected are [M–H + MeCN]^+^, [M–L + 2 MeCN]^+^, [M–L + MeCN]^+^, and [M–L]^+^ (where L is the coordinated NSAID ligand). The latter appears as a high-intensity peak, and it is related to the loss of O-donor ligands, confirming major metal affinity for softer carbonyl and triphenylphosphine ligands. The carboxylate coordinative mode of the NSAID’s dihapto k^2^-species indicates displacement by the acetonitrile solvent used in the ESI-MS technique, since MeCN shows softer and more nucleophilic aptitude compared to the delocalized k^2^-C(O)O moiety, typical of all NSAID ligands. In the absence of mechanistic studies, it would be difficult to confirm the integrity of the metal organometallic structure along the targeted path. Isotopic peak patterns are all in good agreement with the simulated spectra.

The UV–Vis spectra of the complexes have been acquired in DMSO solution. A band in the range 259–275 nm has been observed in the analyzed species (Table 2) and attributed to intra-ligand charge transfer transition (ILCT). All recorded spectra are shown in Supplementary Information (Figures S7, S16, S24, S32 and S41).

2.4. Description of the X-Ray Crystal Structures of 4 and 5

X-ray-quality crystals of 4 and 5 were grown by a double-layer crystallization technique (DCM-hexane = 1:10), and their structures were determined using Single Crystal X Ray Diffraction (SCXRD). In both structures, the ruthenium atom adopts a distorted octahedral geometry in which the PPh_3_ ligands are in a mutual trans-position (Figure 4a,b) and one hydride, one carbonyl, and one carboxylate ligand occupy equatorial coordination sites. The molecular structure of 4 shows an asymmetric chelate coordination of the carboxyl group of the diclofenac ligand [Ru-O1 2.183(2) and Ru-O2 2.310(2) Å, respectively]. This is presumably due to the presence of the very bulky substituent (2,6-dichlorophenylamine) in the ortho-position of the benzyl ring, which is on the same side as O2. In addition, the conformation of the coordinated diclofenac ligand shows some differences compared to that found in the crystal structure of the diclofenac acid [37], with the dihedral angle between the least-squares planes of the two aromatic rings being wider in the latter. Another significant effect attributable to steric crowding is the deviation from linearity of the P-Ru-P angle [171.94(3)°], which relieves the steric strain between the phenyl groups of the axial phosphines and the bulky diclofenac ligand. The two Ru-P distances are almost identical [Ru-P 2.3455(8) and 2.3458(9) Å]. Intramolecular hydrogen bonds in the diclofenac ligand also play an important role: in fact, the carboxylic oxygen O2 is involved in both classical [NH…O2] and nonclassical hydrogen bonds [C23H23…O2 and C45H45…O2] with one phenyl ring from the two PPh_3_ ligands, the most significant being the NH…O2 interaction [N1…O2 2.904(4) Å and N1-H1N…O2 150°]. In the crystal packing, intermolecular H bonds are established through one of the two chlorine atoms (Cl2) and one H atom of the methylene moiety of the diclofenac belonging to adjacent molecules, thus forming dimeric units (Figure S41). Furthermore, an important role is played by π–π interactions between two 2,6-dichlorophenyl rings [centroid–centroid distance 3.555 Å] of neighboring molecules, generating dimers (Figure S42) different from the ones generated by H bonds. To the best of our knowledge, very few ruthenium complexes bearing the diclofenac ligand coordinated in a chelate fashion have been crystallographically investigated [38].

In the crystal structure of 5, the Ru atom, the hydride, the CO, and most of the chelating acetylsalicylate ligand lie on a crystallographically imposed symmetry plane. This symmetry is broken by the carbonyl [C8 and O4] of the acetylsalicylate AcO group that is disordered over two positions below and above the aromatic ring plane with half occupancy. Also, in complex 5, the two Ru-O distances are significantly different [Ru-O1 2.318(5) and Ru-O2 2.165(5) Å], while the Ru-P distance [2.353(1) Å] is almost identical to those reported for 4. The asymmetry in the carboxylate coordination is likely caused by steric hindrance, as the weaker Ru-O interaction involves O1, which is located in the ortho-position and on the same side as the bulky AcO- substituent. In the crystal packing of 5, in addition to intramolecular H bonds, nonclassical intermolecular H bonds [C24-H24…O1, C6-H6…O2] are at work (Figure S43).

2.5. Stability of the Tested Complexes

None of the complexes is moisture sensitive. The salicylic acid–Ru 2 complex is slightly air sensitive.

2.5.1. Stability in Aqueous Solution of 2

A preliminary evaluation of the antiproliferative properties shown by complex 2 was performed using the MTT assay. Prior to that, the stability in solution was evaluated by recording UV–Vis spectra in phosphate-buffered solution (PBS-5% DMSO, pH 7.4) over a period of 48 h. The spectra showed no wavelength shifts, indicating preserved structural integrity throughout the experiment duration (Figure S8, Supplementary Material).

2.5.2. Stability of 6

The stability of complex 6 was evaluated by the ^1^H NMR spectroscopy in CDCl_3_ solution observed over 48 h, confirming both the chemical shift position and invariant shape of the equivalent Me group signals of the isobutyl unit (0.84 ppm) and the distinct α−carboxylic Me unit (0.53 ppm).

2.6. Antiproliferative Activity of 2 and 6

The anticancer ability of the ruthenium complexes 2 and 6 was evaluated using an MTT proliferation assay for HeLa cells. Dose–response graphs were constructed to determine the IC_50_ concentrations of various treatments, and the results are shown in Figure 5A,B. Complex 6 shows no toxic activity at any of the concentrations tested, thus confirming that cytotoxicity is heavily dependent on the coordinated NSAID. In contrast, complex 2 shows an IC_50_ of 75.0 ± 0.8 µM, although it is less efficient than the cis-platin results in analogous conditions (12.3 µM) [39]. Interestingly, compound 2 shows an antiproliferative effect already at a concentration of 20 µM, reducing cell growth by 18.8 ± 5.8%. The in vitro antiproliferative properties of 2 are in line with those of other reported NSAID–ruthenium complexes [20,38].

In all cases, the NSAID ligands coordinate via oxygen atoms of the carboxylate groups, except for salicylate in complex 2, which anchors the carbonyl unit together with the phenolate group. Despite the difference shown in the coordination mode, remarkable bioactivity may suggest that pharmacokinetic pathways could be similar to those evidenced by the analogous buffered aspirin derivative [40].

3. Materials and Methods

3.1. General

All the Ru–hydride adducts with NSAIDs were obtained using the analogous procedure by optimizing the synthesis through the selection of the solvent so that the obtained adducts are insoluble. This triggers an almost complete selective precipitation, avoiding undesirable side or subsequent reaction pathways. The yield of complexes 3–6 is ca. 50%. Under the reaction conditions, conversion goes to completion only upon thermal activation; therefore, the formed coproducts are difficult to analyze because of adsorption on the solid stationary phase during chromatography in the purification procedure.

NMR measurements were taken at 298 K on a Mercury Plus 400 instrument (Oxford Instruments, Abingdon-on-Thames, UK). Frequencies are reported in Hz, and the chemical shifts are referenced to the solvent. The chemical shifts are expressed in parts per million (ppm). The multiplet lists are reported with the aid of two-dimensional COSY, HSQC, and HMBC NMR spectra. All the chemicals were of reagent grade and were used as received from commercial suppliers. Commercially available [RuCl_3_·xH_2_O] was purchased from Strem (Bischheim, France).

Elemental analyses were performed using a Thermo Fischer Scientific Flash 2000 CHNS instrument (Walter, MA, USA). In the elemental analyses (E, %), the small discrepancies are likely due to traces of adsorbed solvents in the microcrystalline powders.

Infrared spectra (4000–400 cm^−1^) were recorded at 298 K on a PerkinElmer Spectrum 2000 Ftir (Fourier transform infrared) spectrophotometer (Waltham, MA, USA), and ESI-MS (electrospray ionization mass spectrometry) spectra were recorded on a Waters Micromass ZQ 4000 (Milford, MA, USA), with samples dissolved in MeCN. The spectra acquired for the powders were compared to those obtained for the crystals, and no significant differences were observed.

3.2. Synthesis of Ru–Salicylic Acid Complex 2

A small excess of salicylic acid (37 mg, 0.268 mmol) and [Ru(H)2(CO)(PPh_3_)3] 1 (220 mg, 0.240 mmol) were dissolved in methanol (15 mL) and refluxed overnight. Upon cooling to room temperature, a grey powder precipitate formed. The solid was then filtered and washed with methanol (3 mL aliquots for 3 times), then dried under vacuum.

Yield: 79%. Elemental analysis (%) Calc. for RuC_44_H_36_P_2_O_4_ Found (calc): C: 65.61 (65.75); H: 4.61 (4.58). ATR-FTIR (ν, cm^−1^): 3053 (aromatic CH), 2001 (RuH), 1927 (C≡O), 1625 (COOH), 1598 (C=C-C), 1464 (C=C-C), 1432 (CH, PPh_3_), 1254 (C-O), 1095 (PPh). ^1^H NMR (400 MHz, CDCl_3_) (δ, ppm) 10.59 (1H, COOH, s), 7.53–7.30 (30H, PPh_3_, m), 7.05 (1H, C-H, m), 7.03 (1H, C-H, m), 6.53 (1H, C-H, dd), 6.45 (1H, C-H, dt), −16.91 (1H, Ru-H, t, ^2^J_H–P_ = 20.04 Hz), ^13^C NMR (101 MHz, CDCl_3_) (δ, ppm): 205.24 (Ru-C≡O, t), 178.53 (COOH), 159.81 (C-H), 133.08 (PPh_3_), 133.05 (C-H), 129.97 (PPh_3_), 129.79 (C-H), 128.25 (PPh_3_), 117.52 (C-H), 116.03 (C-H), 115.91 (C-H). ^31^P NMR (162 MHz, CDCl_3_) (δ, ppm): 43.09 (2P, PPh_3_). ESI-MS^+^ (MeCN) (m/z): 791 [M-H]^+^.

3.3. Synthesis of Ru–Naproxen Complex 3

Naproxen (63 mg, 0.272 mmol) and [Ru(H)2(CO)(PPh_3_)3] 1 (250 mg, 0.272 mmol) were dissolved in toluene (15 mL) and refluxed until the IR Ru-CO absorption of 1 at 1940 cm^−1^ disappeared. After 1 h, the mixture was cooled down to room temperature, and the solvent evaporated under vacuum. The powder was then dissolved in 1 mL of DCM, and hexane (15 mL) was added to precipitate a red powdered microcrystalline solid, which was then filtered, washed with hexane (3 times with 10 mL aliquots), and finally dried under vacuum.

Yield: 49%. Elemental analysis (%) Calc. for RuC_51_H_44_P_2_O_4_ Found (calc): C: 69.60 (69.30); H: 5.16 (5.02). ATR-FTIR (ν, cm^−1^): 3055 (aromatic CH), 2929 (aliphatic CH), 1923 (C≡O), 1634 (asym. COO), 1606 (C=C-C), 1526 (asym. COO), 1455 (C=C-C), 1434 (CH, PPh_3_), 1267 (C-O), 1095 (PPh). ^1^H NMR (400 MHz, CDCl_3_) (δ, ppm), 7.55–7.29 (30 H, PPh_3_, m) 7.06–6.96 (5H, m), 6.60 (1H, CH, d), 3.90 (3H, OCH_3_, s) 2.56 (1H, CH, q), 0.64 (3H, CH_3_, d), −16.45 (1H, Ru-H, t, ^2^J_H–P_ = 21.45 Hz). ^13^C NMR (101 MHz, CDCl_3_) (δ, ppm): 205.54 (Ru-C≡O), 185.84 (COO), 157.19 (CH), 134.41 (PPh_3_), 133.89 (CH), 132.20 (PPh_3_), 129.79 (PPh_3_), 128.72 (CH), 128.59 (CH), 128.15 (PPh_3_), 127.53 (CH), 127.22 (CH), 126.20 (CH), 118.15 (CH), 105.61 (CH), 55.41 (CH), 47.85 (CH_3_), 17.89 (OCH_3_). ^31^P NMR (162 MHz, CDCl_3_) (δ, ppm): 43.29 (2P, PPh_3_). ESI-MS^+^ (MeCN) (m/z): 883 [M-H]^+^.

3.4. Synthesis of Ru–Diclofenac Complex 4

A small excess of diclofenac (107 mg, 0.361 mmol) and [Ru(H)2(CO)(PPh_3_)3] 1 (276 mg, 0.301 mmol) were dissolved in 2-propanol (40 mL) and refluxed for 45 h. A grey powder precipitated by cooling down to room temperature. The solid was then filtered, washed with 2-propanol (3 mL aliquots × 3 times), and dried under vacuum.

Yield: 49%. Elemental analysis (%) Calc. for RuC_51_H_40_P_2_NO_3_Cl_2_ Found (calc): C: 64.72 (64.56); H: 4.30 (4.25). ATR-FTIR (ν, cm^−1^): 3263 (NH), 3053 (aromatic CH), 2079 (RuH), 1915(C≡O), 1578 (C=C-C), 1557 (asym. COO), 1455 (sym. COO), 1431 (CH, PPh_3_), 1093 (PPh). ^1^H NMR (400 MHz, CDCl_3_) (δ, ppm), 7.64 (1H, NH, s), 7.44–7.14 (30 H, PPh3, m), 7.35 (1H, C-H, d), 6.96 (1H, CH, t), 6.87 (1H, CH, t), 6.52 (1H, CH, d), 6.32 (1H, CH, d), 6.24 (1H, CH, d), 2.51 (2H, CH_2_, s), −16.69 (1H, Ru-H, t, ^2^J_H–P_ = 20.3 Hz) ^13^C NMR (101 MHz, CDCl_3_) 205.41 (Ru-C≡O, t), 183.99 (COO), 142.83 (CH), 138.72 (CH), 134.34 (PPh_3_), 133.33 (PPh_3_), 130.46 (CH), 129.88 (PPh_3_), 129.73 (CH), 128.90 (CH), 128.23 (PPh_3_), 126.68 (CH), 125.09 (CH), 123.29 (CH), 120.97 (CH), 117.28 (CH), 41.57 (CH_2_). ^31^P NMR (162 MHz, CDCl_3_) (δ, ppm): 44.60 (2P, PPh_3_). ESI-MS^+^ (MeCN) (m/z): 948 [M-H]^+^.

3.5. Synthesis of Ru–Acetylsalicylic Acid Complex 5

Acetylsalicylic acid (39 mg, 0.218 mmol) and [Ru(H)2(CO)(PPh_3_)3] 1 (220 mg, 0.218 mmol) were dissolved in ethanol (15 mL) and refluxed overnight. A grey powder precipitated after cooling down the mixture solution to room temperature. The obtained solid was then filtered, washed with ethanol (3 times with 3 mL aliquots), and dried under vacuum.

Yield: 54%. Elemental analysis (%) Calc. for RuC_46_H_38_P_2_O_5_ Found (calc): C: 66.42 (66.26); H: 4.62 (4.59). ATR-FTIR (ν, cm^−1^): 3056 (aromatic CH), 2011 (RuH), 1909 (C≡O), 1761 (COOCH_3_), 1587 (C=C-C), 1571 (asym. COO), 1465 (sym. COO), 1433 (CH, PPh_3_), 1200 (C-O). ^1^H NMR (400 MHz, CDCl_3_) (δ, ppm) 7.54–7.30 (30 H, PPh_3_), 7.16 (1 H, CH, dt), 7.05 (1 H, CH, dd), 6.84 (1H, CH, dt), 6.69 (1 H, CH, dd), 2.10 (3 H, CH_3_, s), −16.32 (1 H, RuH, t, ^2^J_H–P_ = 20.34 Hz), ^13^C NMR (101 MHz, CDCl_3_) (δ, ppm): 205.64 (C≡O), 185.08 (COO), 175.02 (C(O)OCH_3_), 169.55 (C-O), 149.82 (C), 134.79 (PPh_3_), 133.50 (PPh_3_), 131.62 (CH), 131.50 (CH), 129.78 (PPh_3_), 129.15 (PPh_3_), 124.57 (CH), 122.75 (CH), 21.29 (CH_3_). ^31^P NMR (162 MHz, CDCl_3_) (δ, ppm): 44.58 (2P, PPh_3_). ESI-MS^+^ (MeCN) (m/z): 833 [M-H]^+^.

3.6. Synthesis of Ru–Ibuprofen Complex 6

A small excess of ibuprofen (31 mg, 0.150 mmol) was dissolved in EtOH (40 mL) solution of Ru(H)2(CO)(PPh_3_)3] 1 (113 mg, 0.123 mmol) and refluxed overnight. The cooling down to room temperature caused white powder precipitation. The solid was filtered, washed with hexane, and subsequently with H_2_O (10 mL aliquots × 3 times). The obtained powder was dried under vacuum.

Yield: 53%. Elemental analysis (%) Calc. for RuC_50_H_48_P_2_O_3_ Found (calc): C: 70.01 (69.84); H: 5.70 (5.63). ATR-FTIR (ν, cm^−1^): 3056 (aromatic CH stretch), 2955 (aliphatic CH stretch), 2867 (aliphatic CH stretch), 1995 (Ru-H), 1924 (C≡O), 1521 (asym. COO), 1480 (C=C-C), 1458 (sym. COO), 1432 (CH, PPh_3_), (PPh), 1095 (PPh). ^1^H NMR (400 MHz, CDCl_3_) (δ, ppm): 7.45–7.30 (30 H, PPh_3_, m), 6.69 (2 H, CH, d), 6.40 (2 H, CH, d), 2.41 (1 H, CH, q), 2.32 (2 H, CH_2_, d), 1.75 (1 H, CH, dt), 0.84 (6 H, CH_3_, dd), 0.53 (3 H, CH_3_, d), −16.44 (1 H, Ru-H, t, ^2^J_H–P_ = 21.00 Hz). ^13^C NMR (101 MHz, CDCl_3_) (δ, ppm): 205.59 (C≡O), 186.05 (COO), 139.00 (C), 138.92 (C), 134.44 (PPh_3_), 133.91 (PPh_3_), 129.79 (PPh_3_), 128.64 (CH), 128.18 (PPh_3_), 127.56 (CH), 47.61 (CH), 45.23 (CH_2_), 30.31 (CH), 22.61 (CH_3_), 17.90 (CH_3_). ^31^P NMR (162 MHz, CDCl_3_) (δ, ppm): 43.30 (2P, PPh_3_). ESI-MS^+^ (MeCN) (m/z): 859 [M-H]^+^.

3.7. X-Ray Crystallography

The X-ray intensity data for 4 and 5 were collected on a Bruker APEX CCD diffractometer (Bruker, Karlsruhe, Germany) using Mo−Kα or Cu-Kα radiation (for 5). All data were processed using the Bruker suite of programs [41,42,43], and the structures were solved with SHELXT [44] and refined with the SHELXL programs [45]. All non-hydrogen atoms were assigned to anisotropic displacement parameters. Most of the hydrogen atoms were located in the Fourier map, placed in idealized positions, and included as riding with constrained isotropic displacement parameters for the aromatic and methyl protons and refined as riding with Uiso(H) = 1.2Ueq(C) or Uiso(H) = 1.3Ueq(C)methyl. Molecular graphics were generated using the program Mercury [46]. Table S1 reports crystal data and refinement parameters for 4 and 5.

3.8. MTT Assay

HeLa Cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were seeded at 1.5 × 10^4^ cells/well in a 96-well plastic culture plate (Sarstedt, Milan, Italy), and after 24 h of growth, were exposed to increasing concentrations of each distinct compound (from 0.25 μM to 10 μM) solubilized in RPMI 1640 medium. Cells were treated with DMSO (vehicle control). For the CDDP, the cells were treated at the same concentration range as the complexes. Treatments were left for 48 h to ensure efficient cellular uptake.

The MTT assay was performed according to the literature [47]. The absorbance at 570 nm was measured using a multiwell plate reader (Tecan, Männedorf, Switzerland), and data were analyzed by Prism GraphPad 8 software. Percent cell viability was determined with respect to the control. All concentrations were tested in triplicate, and the experiment was repeated three times.

4. Conclusions

Metal coordination aims to combine the anti-inflammatory effects of NSAIDs with the anticancer activities of the Ru species, potentially leading to novel synergistic therapeutic effects. The synthesized compounds contain both carboxylic groups from NSAIDs and phosphine ligands, therefore imparting amphiphilic features to the Ru core and enhancing its ability to be delivered to cancer targets. Herein, we report ruthenium complexes coordinating four non-steroidal anti-inflammatory drugs (NSAIDs) and the salicylate moiety as an aspirin precursor, which have been synthesized and spectroscopically and structurally investigated. The rationale for the synthesis of novel Ru–NSAID complexes consists of providing pharmacophore molecules for targeting cancer, while the ruthenium central core might provide further cytotoxic features, such as DNA binding to trigger apoptosis via ROS mediation [16,20,38].

The MTT assay on HeLa cancer cells performed with salicylic complex 2 suggests antiproliferative activity, albeit at relatively elevated doses. Future perspectives include extending investigations to additional biological targets, such as interaction measures with human serum albumin, calf-thymus DNA, and iron-carrier protein transferrin. In summary, we believe that Ru coordination is responsible for promoting synergistic effects by integrating complementary advantages, leading to improved tumor targeting and therapeutic outcomes by minimizing systemic toxicity, analogous to the findings in the case of small molecule–drug conjugates [48,49,50].

To address pharmacological resistance and toxic effects, novel therapeutics aim to enhance selectivity by penetrating exclusively into cancer cells while exhibiting steady and durable efficacy with minimal administered doses. However, many issues have yet to be overcome to allow Ru species to be tested as therapeutics in the next clinical phases. Aiming at this ambitious health target, it would be aspirational, for instance, to improve both the hydrolysis and metabolic absorption features of future synthesized Ru species [28].