Steric and Electronic Effects Responsible for N, O- or N, N-Chelating Coordination of Pyrazolones Containing a Pyridine Ring in Ruthenium Arene Systems

Steric and Electronic Effects Responsible for N,O- or N,N‑Chelating Coordination of Pyrazolones Containing a Pyridine Ring in Ruthenium Arene Systems Lorenzo Pietracci, Riccardo Pettinari,*Alessia Tombesi, Fabio Marchetti, Claudio Pettinari, Agustín Galindo, Farzaneh Fadaei-Tirani, Mouna Hadiji, and Paul J. Dyson* Cite This: Organometallics 2023, 42, 1495−1504 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Structural and electronic factors are crucial to rationalize the different N,Oor N,Nchelating coordination of pyrazolones containing a pyridine ring. The reactivity of proligand 3-phenyl-1-(pyridin-2-yl)-5-pyrazolone (HLpy,ph) with the (arene)- Ru(II) fragment was explored. Neutral and ionic (arene)Ru(II) complexes were obtained and fully characterized, also by X-ray diffraction, revealing the ligand to coordinate in an unusual N,O- chelating fashion. Other ruthenium complexes were also synthesized with 3-methyl-1-(pyridin-2-yl)-5-pyrazolone (HLpy,me) and 3- methyl-1-(pyridin-2-yl)-4-trifluoroacetyl-5-pyrazolone (HQpy,CF3). In these complexes the ligands adopt the preferred N,N-chelating mode. Ligands and complexes were theoretically analyzed by density functional theory (DFT). The most stable tautomer of HLpy,ph matched well with the experimental behavior of this proligand and the structures of Ru-complexes were well described by calculations. The thermodynamic stability of the N,O- and N,N-coordination modes was analyzed and a proposal for the achievement of the N,O-coordination mode in complexes 1−4was proposed. Cytotoxicity tests were performed against human ovarian carcinoma (A2780 and Cisplatin-resistant A2780cis) and nontumorigenic human embryonic kidney (HEK293T) cell lines, showing the free ligands to be more cytotoxic that the ensuing (arene)Ru(II) complexes. ■INTRODUCTION Pyrazole is a unique five-membered heterocycle, containing two adjacent nitrogen atoms, and is considered a very important ligand in coordination chemistry because it can form a variety of coordination complexes with several metal ions, providing varying coordination geometries and nuclearities. Pyrazole and its synthetic derivatives have a broad spectrum of applications, 1,2 and in particular two classesof compounds have been intensively studied, pyrazolones and, by acylating the C-4 position, acylpyrazolones (Chart 1). Our group has contributed to this field, through the design and synthesis of numerous metal complexes using chelating acylpyrazolones and pyrazolonesbased ligands. 3−5 Recently, we expanded our studies on arene- Ru(II) chemistry of two proligands, namely 3-methyl-1- (pyridin-2-yl)-5-pyrazolone (HLpy,me) and 3-methyl-1-(pyridin-2-yl)-4-trifluoroacetyl-5-pyrazolone (HQpy,CF3,Chart 2). 6 The two ligands were found able to coordinate (in the deprotonated form) to Ru(II) in a bidentate N,N-fashion, 6 which is structurally analogous to some very cytotoxic Ru(II)- and Os(II)-arene phenylazopyridine complexes. 7−9 Received: March 6, 2023 Published: May 12, 2023 Chart 1. Pyrazolone and Acylpyrazolone Ligands Chart 2. Proligands Used in This Work Article pubs.acs.org/Organometallics © 2023 The Authors. Published by American Chemical Society 1495 https://doi.org/10.1021/acs.organomet.3c00121 Organometallics 2023, 42, 1495−1504 Downloaded via UNIV DE SEVILLA on May 10, 2024 at 13:53:24 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. Both proligands and their neutral arene-Ru(II) complexes (arene = cymene and hexamethylbenzene) were tested against A2780 and A2780cis tumor cell lines and compared to HEK293T nontumor cell lines, giving some promising results. 6 On this basis, here we expand our investigation on another pyrazolone proligand, namely 5-phenyl-2-(pyridin-2-yl)-2,4- dihydro-3H-pyrazol-3-one, with a phenyl replacing the methyl group in position 3 of the pyrazolone ring (HLpy,ph in Chart 2), and to the corresponding neutral arene-ruthenium complexes. The proligand HLpy,ph with binding potential has been previously reported by others, 10−12 and its crystal structure solved by Watkins et al., 13 but its coordinating ability toward metal ions is still unexplored. Moreover, we have introduced 1,3,5-triaza-7-phosphaadamantane (PTA) phosphine in the ruthenium environment by replacing the chloride ligand, affording cationic complexes with the aim to improve solubility in water. ■RESULTS AND DISCUSSION The proligand HLpy,ph is similar to HLpy,me (Chart 2) but with a phenyl replacing the methyl in 3-position of pyrazole. In principle, four tautomeric forms, two of which have additional rotamers, are possible for HLpy,ph (Chart 3). In the solid state the structure previously determined by X-ray crystallography 13 corresponds to rotamer I-r, which is stabilized by the intramolecular hydrogen bond O−H···N. Rotamer I-r is the only observed in chlorinated solvents, as indicated by the broad resonance at 12.86 ppm found at room temperature in the 1H NMR spectrum. N−H coupling was not observed in the HSQC 1H−15N NMR spectrum at room temperature, providing additional evidence for the presence of enol group in CDCl3. Arene-Ru(II) complexes 1and 2were synthesized from reaction of HLpy,ph with [Ru(arene)Cl2]2[where arene = p-cymene (cym) or hexamethylbenzene (hmb)] in methanol in the presence of KOH (Scheme 1). Complexes 3and 4were obtained by reaction of 1and 2in methanol with aqueous AgBF4 to remove the chloride from the Ru(II) coordination sphere, followed by addition of PTA (Scheme 1). Note that in these complexes the Lpy,ph ligand acts as N,O- chelating donor toward the organometallic fragment (see also below in the X-ray diffraction study), whereas in previous studies the analogous Lpy,me ligand preferred to coordinate to ruthenium in a N,N-chelating fashion. The different behavior of the two ligands may be ascribed to the electron-withdrawing phenyl ring in Lpy,ph which depletes of electron density the N2 atom of pyrazole, causing it to coordinate through the oxygen. The ionic nature of 3and 4is confirmed by conductivity measurements in DMSO with Λmvalues in the range 23−26 cm2mol−1, typical of 1:1 electrolytes. 14 The solubility in polar solvents increases from neutral 1and 2to ionic 3and 4, the latter being slightly soluble in water. ESI-MS of 1and 2performed in acetonitrile/methanol show main peaks due to [Ru(arene)(Lpy,ph)]+generated by the loss of the chloride ligand, whereas for 3−4both [Ru(arene)- (Lpy,ph)]+and [Ru(arene)(Lpy,ph)(PTA)]+species are observed. The most relevant feature in the IR spectra is the progressive decrease of the ν(C�O) vibration mode from 1654 cm−1in HLpy,ph to 1632−1635 cm−1in the neutral complexes 1and 2, in accordance with coordination in N,O-chelating mode by deprotonation of tautomer I of proligand, and to 1626 and 1587 cm−1in the spectra of 3and 4respectively, due to the positive charge of the complexes which further strengths the Ru−O bonding thus reducing the C�O bonding order. 15−18 In the IR spectra of 3and 4strong and sharp absorptions at ca. 1054 and 1028−1034 cm−1confirm the presence of the BF4 − anion. 19 In the far-IR region strong bands due to ν(Ru−N), ν(Ru−O), and ν(Ru−Cl) stretching modes in 1and 2were tentatively assigned below 500 cm−1. 20 The proton and carbon assignments of the free ligand and complexes 1−4have been made based on {1H−1H}-COSY, {1H−13C}-HSQC, and {1H−13C}-HMBC spectroscopy (Figures S6−S37). 21 The 1H and 13C NMR spectra of 1−4recorded in CDCl3or CD3CN show the expected shift in frequency for the resonances of the pyrazolone and pyridyl ring protons and carbon atoms in comparison to the free ligand. Moreover, in the 31P NMR spectra of 3and 4, the phosphorus of PTA affords a singlet at −37.7 and −40.7 ppm, respectively. On the basis of the different coordination observed with HLpy,ph ligand with respect to HLpy,me and HQpy,CF3, we decided to verify if these differences persisted also in their corresponding cationic complexes with PTA. The new complexes 5−8were prepared using a procedure similar to that employed for 3and 4, starting from [Ru(arene)- Chart 3. Possible Tautomers (I−IV) and Rotamers (I-r and IV-r) of HLpy,ph a a Rotamer I-r is observed in the solid state and chlorinated solvents. Scheme 1. Synthesis of Complexes 1−4 Organometallics pubs.acs.org/Organometallics Article https://doi.org/10.1021/acs.organomet.3c00121 Organometallics 2023, 42, 1495−1504 1496 (Lpy,me)Cl] (Scheme 2) and [Ru(arene)(Qpy,CF3)Cl] (Scheme 3). 6 In 5−8the ligands act in a N,N-chelating fashion, and in general their solubility in common solvents is lower than 1−4. Complexes 5−8are all 1:1 electrolytes in DMSO, in accordance with their ionic formulation. The IR spectra of 5−8display a shift of the ν(C�O) to higher frequencies upon coordination, in accordance with noninvolvement of the O atom(s) of the pyrazolone moiety. The presence of BF4 −in 5and 6is confirmed by the medium-to-strong bands at ca. 1050 and 1030 cm−1, and a very strong absorption at 834 cm−1due to PF6 −is observed in the IR spectra of 7and 8. 22,23 ESI-MS of 5and 6display peaks due to [Ru(arene)(Lpy,me)]+and [Ru(arene)(Lpy,me)PTA]+ fragments, whereas in the spectra of 7and 8a unique peak is observed corresponding to [Ru(arene)(Qpy,CF3)PTA]+.In1H and 13C NMR spectra of 5−8the expected change of resonances was observed for the proton and carbon atoms of the pyrazolone ligands upon coordination (Figures S38−S67). The 31P NMR spectra of 7and 8contain a singlet at −32 and −40 ppm, respectively, with the PF6 −anion giving a multiplet (hept) at ca. −143 ppm, due to coupling with fluorine atoms. By comparison of the {1H−15N}-HMBC spectra of the free ligands with their metal complexes it is possible to assign indirect 15N NMR chemical shifts (Table 1). A general feature involving a shift of the resonances of N1 and N2 atoms of pyrazolone and that of Npy of the pyridine ring upon coordination to ruthenium, with the shift of Npy being much larger when PTA is present as a coligand. X-ray Diffraction Study. As stated before, although the structure of the HLpy,ph ligand was previously reported, 13 we reobtained its crystal structure to confirm the rotamer I-r configuration, which is the most stable according to DFT calculations (see below). X-ray data obtained are completely similar to the previous study but with a better R factor (see Table S4). For this reason, no further discussion of the structure of HLpy,ph is needed. Complex 3is crystallized in a monoclinic system with a space group P21/n, including 8 molecules in the unit cell. In the X-ray structure analysis of 3, it is noteworthy to mention that there are two independent salts with similar structural parameters and that the crystal shows both enantiomers. One of two crystallographically independent molecules of complex 3in the asymmetric unit is shown in Figure 1. Generally, the coordination environment around Ru(II) center possesses octahedral geometry in which the p-cymene molecule is bonded in η6coordination mode to the metal ion center, while other coordination sites are occupied by deprotonated bidentate L ligand with N7 pyridine atom and deprotonated O2 atom, and P atom of the triaza ligand. Pyrazole ligand bind to the metal center at N and O forming a sixmembered chelate ring with bite angles N(7)−Ru(2)−O(2) 83.38(7)°and N(1)−Ru(1)−O(1) 83.10(7)°, bond lengths O(1)−Ru(1) 2.0892(15) and O(2)−Ru(2) 2.0883(15); N(1)−Ru(1) 2.1258(18) and N(7)−Ru(1) 2.1210(18) Å for the complexes. Also, the Ru(II) atom is π-bonded to the arene Scheme 2. Synthesis of Complexes 5 and 6 Scheme 3. Synthesis of Complexes 7 and 8 Table 1. 15N Chemical Shifts (ppm) in the Free Ligands and Complexes Obtained from {1H−15N} HMBC NMR Spectroscopy a N1 N2 Npy NPTA HLpy,ph 263.1 194.5 251.9 1205.3 157.0 202.9 2206.1 n.o. n.o. 3206.8 131.5 176.5 42.5 4205.6 n.o. 186.5 39.6 HLpy,me 191.4 322.2 252.4 5205.6 135.0 177.1 43.4 6205.4 139.9 180.4 40.0 HQpy,CF3 n.o. 267.9 229.6 7n.o. 167.3 179.7 42.8 8n.o. 172.6 182.8 40.0 a n.o. = not observed. Figure 1. X-ray molecular structure of complex 3with thermal ellipsoids at the 50% probability level. Organometallics pubs.acs.org/Organometallics Article https://doi.org/10.1021/acs.organomet.3c00121 Organometallics 2023, 42, 1495−1504 1497 ring with an average Ru(1)−C and Ru(2)−C distance of 1.746 and 1.716 Å respectively. These results show that the complexes have a distorted octahedron structure. Selected bond angles and bond lengths are presented in Table 2. DFT Study. The possible tautomers and rotamers of the HLpy,ph proligand (Chart 2) were examined using density functional theory (DFT) at the B3LYP/6-311+G** level of theory. The resulting optimized structures and their relative energies are collected in Figure S1 (Supporting Information). The most stable form is rotamer I-r, and the theoretical data obtained match well with the experimental behavior of the HLpy,ph proligand, both in solution and in the solid state. The calculated NMR spectrum of rotamer I-r correlates well with the experimental spectrum (coefficient of determination, R2, of 0.9963; see Figure S3) and, additionally, a good fit was found in the comparison of the structural parameters of the calculated HLpy,ph molecule with those determined by X-ray crystallography (Figure S2). 13 This confirms the appropriateness of the selected combination of method and basis sets. As previously noted for the related proligand HLpy,me, 6 the existence in rotamer I-r of an intramolecular hydrogen bond (O−H···N, 1.781 Å; O···N, 2.649 Å; and O−H···N angle of 144.2°) is the key stabilizing factor. Complexes 1−4were also studied by DFT calculations. The selected combination of the method and basis sets provides a good structural description of these complexes based on the comparison of the calculated and experimental structural parameters of complex 3(Table S1). The resulting optimized structures (Figure 2) show the typical three-legged piano-stool structure, where Lpy,ph acts as a N,Obidentate ligand. The six-membered [Ru(Lpy,ph)] metallacycle in these complexes displays a half-chair conformation. The angles between the pyrazolone plane and the plane defined by Ru and the N and O donor atoms are around 45°for 1and 3(ca. 48°in the X-ray structure) and 41°for 2and 4. To rationalize this difference, the structure of the anion [Lpy,ph]−was also optimized. Two rotamers with similar energies were located, corresponding to the potential N,Oor N,Nbidentate ligands (Figure S4). The deprotonation of rotamer I-r affords a C−O bond of 1.23 Å for the N,Orotamer. Upon coordination, this distance increases to approximately 1.29 Å (complexes 1and 2) and to around 1.31 Å (3and 4), suggesting a bond order greater than one, 24 and delocalization along the [Ru(Lpy,ph)] metallacycle. These values agree with the observed decrease in the νCO vibration mode from neutral complexes 1and 2to cationic complexes 3and 4discussed above. The MOs of [Lpy,ph]− involved in the in-plane N,Ocoordination to the ruthenium center are HOMO−1, HOMO−4 and HOMO−6 (Figure 3), these being MOs in which the lone pairs of the donor N and O atoms participate in the in-phase and out-of-phase contributions of σtype that provide the M−O and M−N bonds in 1−4. However, considering the conformation of the [Ru(Lpy,ph)] metallacycle, some supplementary contribution to the Ru−O bond comes from the πpart of the HOMO of the ligand (Figure 3). Since the [Lpy,ph]−ligand also exists as a N,Nrotamer, the possible ruthenium isomers of 1−4with the N,Nbidentate ligand were also optimized (Figure S5). Surprisingly, despite the steric hindrance of the Ph substituent, the N,Nisomers are not clearly destabilized, except for complex 4(Table S2). This can be explained on the basis of a reinforcement of the Ru-X bonds in the N,Nisomers that compensates for the steric pressure, which is observed in the calculated Mayer indexes for the Ru−X bonds. An increase in these indexes was observed for the N,O complexes compared to the N,Nisomers (Table S3). On the basis of the similar relative energies shown in Table S2 for complexes 1and 2and their isomers, the N,Ocoordination is not clearly favored from a thermodynamic point of view. Therefore, the formation of 1and 2is assumed to occur via a mechanism involving nucleophilic attack of the oxygen atom (Mulliken charge of −0.41) on the deprotonated [Lpy,ph]− species toward the Ru center. Thus, the only possible bidentate coordination of the ligand involves the formation of the Ru−N bond through the Npy atom. For comparison, complexes 5−8 were also analyzed by DFT calculations and similar three-legged piano-stool structures were observed in optimized molecules Table 2. Selected Bond Lengths (Å) and Bond Angles (°) in Complex 3 bond lengths bond angles Ru1−P1 2.3051(6) O1−Ru1−P1 80.62(4) Ru1−O1 2.0892(15) O1−Ru1−N1 83.10(7) Ru1−N1 2.1258(18) N1−Ru1−P1 89.95(5) Ru2−P2 2.3072(6) N7−Ru2−P2 89.15(5) Ru2−O2 2.0883(15) O2−Ru2−P2 78.61(4) Ru2−N7 2.1210(18) O2−Ru2−N7 83.38(7) Figure 2. Optimized structures of complexes 1−4. Figure 3. MOs of anionic ligand [Lpy,ph]−involved in the N,O- coordination to Ru center. Organometallics pubs.acs.org/Organometallics Article https://doi.org/10.1021/acs.organomet.3c00121 Organometallics 2023, 42, 1495−1504 1498 (Figure 4). The C−O bond lengths (1.225 Å for 5and 6and 1.220 Å for 7and 8) are shorter than those observed for complexes 1−4and, consequently, higher νCO frequencies were observed in agreement with the experimental IR spectra. These complexes are characterized by a HOMO constituted by a dπ ruthenium orbital that displays an antibonding combination with the πpart of the Lpy or Qpy,CF3 ligands. This situation is similar to that previously reported for the neutral counterparts, [Ru(arene)(Lpy,me)Cl] and [Ru(arene)(Qpy,CF3)Cl]. 6 Cytotoxicity Studies. To investigate the stability of 1−8a series of 1H (for the neutral complexes 1−2) and 31P NMR spectra (for the cationic 3−8) were recorded in DMSO-d6 solution over time (Figures S68−S75). Complexes 1−2 undergo partial dissociation of the pyrazolone ligand immediately after dissolution while 3−8are stable in DMSO-d6and their 31P NMR spectra remain unchanged within 72 h. Based on these data, we decided to investigate only the cytotoxicity of compounds 3−8against the human ovarian carcinoma cell line (A2780) and its Cisplatin resistant form (A780cis) as well as nontumorigenic human embryonic kidney (HEK293T) cells over an incubation period of 72h using the MTT assay. The resulting IC50 values of the compounds are presented in Table 3 together with the values for Cisplatin and Rapta-C used as positive and negative controls, respectively. The proligands are more potent than their corresponding complexes on the ovarian cell line A2780, most notably with HLpy,ph with an IC50 = 1.7 ± 0.5 μM. It is possible that the active part of the free ligand is the part that coordinates to the metal center and hence it is deactivated unless it is released slowly from the complex after it reaches the cancer cell. In particular, cationic complexes 3and 4, resulting from the substitution of the chloride ligand in complexes 1and 2by PTA, are essentially inactive (IC50 > 100 μM) on the three cell lines. A similar loss of cytotoxicity is observed with complexes 7and 8compared to their precursors [Ru(cym)(Qpy,CF3)Cl] and [Ru(hmb)(Qpy,CF3)Cl], respectively. As observed for related Ru(II) half sandwich compounds, the introduction of hydrophilic PTA clearly reduces the lipophilicity of such complexes, which can be disadvantageous to cross cell membranes thus reducing the uptake of complexes in tumor cells. 25 In contrast, complexes 5and 6have a higher potency than [Ru(cym)(Lpy,me)Cl] and [Ru(hmb)(Lpy,me)Cl] with IC50 values of 36 ±19 and 35 ±7μM, respectively. Interestingly, 5is completely inactive on the nontumorigenic HEK293T cell line (IC50 > 100 μM), while being cytotoxic to the ovarian cancer A2780 cell line. Additionally, complex 6shows similar activity to both A2780 cells and Cisplatin-resistant A2780cis cells (IC50 = 35 ±7 and 28 ±3μM, respectively), similar to that observed for related compounds, 26,27 indicating that the mechanism of action is different than Cisplatin. 28−30 ■CONCLUSIONS In this study we have shown that in pyrazolone ligands containing a pyridine ring, the presence of a phenyl in position 3 of the pyrazole ring in HLpy,ph in place of a methyl as in HLpy,me induces electronic and structural changes that determine the preference for a bidentate N,O-coordination, instead of the N,N- coordination observed in ruthenium complexes with HLpy,me. The presence of an acyl moiety in the ligand HQpy,CF3 also leads to a preference for an N,N-chelated coordination. For complexes 1and 2the N,Ocoordination is not clearly thermodynamically favored, according to DFT studies, and their formation maybe occurs via a mechanism involving nucleophilic attack of the oxygen atom in the deprotonated [Lppy,ph]−ligand toward the Ru center. DFT results rationalize the isolation of HLpy,ph as the most stable enol tautomer I in the form of rotamer I-r and describes well the structures of Ru-complexes. These theoretical studies support the spectroscopic assignments (IR and NMR) and, based on the MO analyses of these ligands, provide explanation about their coordination to the Ru center. Anticancer studies performed against A2780, A2780cis, and nontumor HEK293T unexpectedly showed the proligands being more efficient than their corresponding ruthenium complexes. Introduction of a PTA ligand brings the formation of cationic complexes, which are however less active than neutral parents with chloride or completely inactive. In any case, the ligand HLpy,ph is quite interesting, being more potent and selective than Cisplatin, and this result may open further developments by exploring the coordination chemistry of such ligand toward other metal acceptors. ■EXPERIMENTAL SECTION Materials and Methods. Solvents were used as supplied or distilled using standard methods. All chemicals were purchased from Aldrich (Milwaukee) and used as received. The dimers [Ru(arene)- Figure 4. Optimized structures of the cations of complexes 5−8. Table 3. IC50 Values (μM) of the Compounds Tested on Human Ovarian Carcinoma (A2780), Its Cisplatin Resistant Form (A2780cis), and Human Embryonic Kidney Cells (HEK293T) a A2780 A2780cis HEK293T HLpy,ph 1.7 ±0.5 1.3 ±0.4 8 ±4.4 3>100 >100 >100 4>100 >100 >100 HLpy,me 9.1 ±1.1 17.4 ±1.7 14.9 ±0.1 536 ±19 >100 >100 635 ±7 28 ±3 33 ±6 HQpy,CF3 8.9 ±0.5 10.4 ±2.6 12.9 ±0.3 7>100 >100 >100 8>100 >100 >100 Cisplatin 0.9 ±0.2 6.7 ±4 2.2 ±0.8 Rapta-C >200 >200 >200 a Values are given as the mean obtained from 3 independent experiments ±standard deviation. Organometallics pubs.acs.org/Organometallics Article https://doi.org/10.1021/acs.organomet.3c00121 Organometallics 2023, 42, 1495−1504 1499 Cl2]2(arene = p-cymene (cym) or hexamethylbenzene (hmb)) were purchased from Aldrich. The ligands HLpy,me and HQpy,CF3 were synthesized by a procedure similar to that previously reported. 6 IR spectra were recorded on a PerkinElmer Frontier FT-IR instrument. 1H, 13C, 31P NMR spectra were recorded on a 500 Bruker Ascend instrument operating at room temperature relative to TMS. Positive ion electrospray mass spectra were obtained on a Series 1100 MSI detector HP spectrometer, using methanol and acetonitrile as solvent for all complexes 1−8. Solutions (3 mg/mL) for electrospray ionization mass spectrometry (ESI-MS) were prepared using reagent-grade methanol. Masses and intensities were compared to those calculated using IsoPro Isotopic Abundance Simulator, version 2.1.28. Melting points are uncorrected and were recorded on a STMP3 Stuart scientific instrument and on a capillary apparatus. Samples for microanalysis were dried in vacuo to constant weight (20 °C, ca. 0.1 Torr) and analyzed on a Fisons Instruments 1108 CHNS-O elemental analyzer. X-ray Crystallography. The diffraction data of HLpy,ph and 3were collected, at 140 K, using Cu Kαradiation. Suitable crystals of HLpy,ph and 3were selected and mounted on an XtaLAB Synergy R, DW system, HyPix-Arc 150 diffractometer. The data sets were reduced and corrected for absorption, with the help of a set of faces enclosing the crystals as snugly as possible, with the latest available version of CrysAlisPro. 30 The solution and refinement of the structures were performed by the latest available version of ShelXT 31 and ShelXL 32 using Olex2−1.5 33,34 as the graphical interface. All non-hydrogen atoms were refined anisotropically using full-matrix least-squares based on |F|2. The hydrogen atoms were placed at calculated positions employing the “riding” model, where each H atom was assigned a fixed isotropic displacement parameter with a value equal to 1.2 Ueq of its parent C atom. In HLpy,ph, the hydrogen atom bound to O1 was found in a difference map and refined freely. Crystallographic and refinement data for HLpy,ph and 3are summarized in Table S4. The CCDC numbers 2211175 and 2204821 contain the crystallographic data for compounds HLpy,ph and 3, respectively. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif. Computational Details. The electronic structures and geometries of the HLpy,ph proligand and the [Lpy,ph]−anion, their tautomers, rotamers and ruthenium complexes 1−8and some of their isomers were investigated by using density functional theory at the B3LYP level. 35,36 For the proligand, tautomers, rotamers and its anion the 6- 311+G** basis set was used for the optimization, while for Ru compounds the optimization was carried out using LANL2DZ, 37 for the Ru atom and the 6-31G*basis set for the remaining atoms. Molecular geometries were optimized without symmetry restrictions. Frequency calculations were carried out at the same level of theory to identify all the stationary points as minima (zero imaginary frequencies) and to provide the thermal correction to free energies at 298.15 K and 1 atm. The GIAO method was used for the NMR calculations (1H, 13C, and 15N NMR isotropic shielding tensors), which were carried out at the 6-311+G(2d,p) level of theory. The computed IR spectra were scaled by a factor of 0.96. 38,39 The DFT calculations were executed using the Gaussian 09 program package. 40 The coordinates of all optimized compounds are collected in a separate associated XYZ file attached to the Supporting Information. Cytotoxicity Tests on A2780, A2780cis, and HEK293T Cell Lines. The human ovarian carcinoma cell line and its Cisplatin resistant form, A2780 and A2780cis, were purchased from the European Collection of Cell Cultures (ECACC, United Kingdom). The human embryonic kidney 293T cell line (HEK293T) was kindly provided by the biological screening facility (EPFL, Switzerland). Fetal bovine serum (FBS) was obtained from Sigma, Switzerland. RPMI 1640 GlutaMAX and DMEM GlutaMAX media were purchased from Life Technologies. The cells were cultured in RPMI 1640 GlutaMAX supplemented for the ovarian cancer cell lines A2780 and A2780cis and in DMEM GlutaMAX supplemented for HEK293T with 10% heatinactivated FBS at 37 °C and CO2(5%). To uphold Cisplatin resistance, the A2780cis cell line was routinely treated with Cisplatin at a final concentration of 2 μM in the media. MTT (3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay was used to evaluate the cytotoxicity of the compounds. Stock solutions were prepared in DMSO and sequentially diluted in cell culture grade water to obtain a concentration range of 0−1 mM. Ten μL aliquots of these prepared compound solutions were added in triplicates to a 96-well plate to which 90 μL of the cell suspension (approximately 1.4 ×104 cells/well) were added (final volume 100 μL/concentrations range 0− 100 μM). Cisplatin and RAPTA-C were used as positive (0−100 μM) and negative (0−100 μM) controls, respectively, and the plates were incubated for 72 h. Ten microliters of an MTT solution prepared at a concentration of 5 mg/mL in Dulbecco’s phosphate buffered saline (DPBS) was added to the cells, and the plates were incubated for additional 4 h. The culture media was carefully aspirated to preserve the purple formazan crystals that were dissolved in DMSO (100 μL/well). The absorbance of the resulting solutions, which is directly proportional to the number of surviving cells, was measured at 590 nm using SpectroMax M5e microplate reader and the data was analyzed with GraphPad Prism software (version 9.3.1). The reported IC50 values are based on the means of three independent experiments, each comprising three tests per concentration level. Synthesis of Proligand HLpy,ph and Complexes 1−8. Proligand HLpy,ph.The proligand HLpy,ph was synthesized with a different procedure from those reported in the literature, 10−13 which increases its yield. HLpy,ph was synthesized by reacting equimolar amounts of 2- hydrazineylpyridine (2.29 g, 21 mmol), and ethyl 3-oxo-3-phenyl- propanoate (4.04 g, 21 mmol) at room temperature. A solutionofKOH (87.9%) (100 mg, 1.57 mmol) in methanol (about 20 mL) was added to the mixture. The initial violet mixture was stirred 1 h at room temperature, changing to dark blue. The solution was dried on a rotavapor until an oil was obtained, which was dissolved in hot acetonitrile. The final product crystallized by slow cooling and evaporation (4.52 g, 0.019 mol, yield 90.7%). It is a dark blue solid opaque crystal, highly soluble in alcohols, DMSO, DMF, acetone, acetonitrile, and chlorinate solvents. Anal. Calcd for C14H11N3O (MW: 237 g/mol): C, 70.87; H, 4.67; N, 17.71%. Found: C, 70.51; H, 4.58; N, 17.83%. mp 121−122 °C. IR (cm−1): 3056w ν(C−H aromatics), 1656m ν(C�O), 1624w ν(C�N), 1596m and 1579m ν(C�C), 1537w, 1489m, 1469s, 1456s, 1441s, 1385m, 1330m, 1296m, 1280m, 999m, 943m, 840m, 814m, 783vs, 749vs, 691s, 655s, 677m, 652m, 590w, 523m, 441s, 407m, 326m, 266w, 194w, 151vs 1H NMR (500 MHz, CDCl3, 298 K): δ5.98s (1H, C4−H of HLpy,ph), 7.17t (1H, 3J(H−H) = 7.5 Hz, C9−H of HLpy,ph), 7.38t (1H, 3J(H−H) = 7.2 Hz, C14− H of HLpy,ph), 7.45t (2H, 3J(H−H) = 7.5 Hz, C13,13′-H of HLpy,ph), 7.90m (3H, C12,12′-H and C8−H of HLpy,ph), 8.08d (1H, 3J(H−H) = 8.4 Hz, C7−H of HLpy,ph), 8.30d (1H,, 3J(H−H) = 4.6 Hz, C10−H of HLpy,ph), 12.84s (1H, OH of HLpy,ph). 13C NMR (CDCl3, 298 K): δ8.8 (C4 of HLpy,ph), 112.3 (C7 of HLpy,ph), 119.9 (C9 of HLpy,ph), 125.9 (C13−13′of HLpy,ph), 128.6 (C14 of HLpy,ph), 128.6 (C12−12′of HLpy,ph), 133.0 (C11 of HLpy,ph), 140.0 (C8 of HLpy,ph), 145.1 (C10 of HLpy,ph), 152.7 (C3 of HLpy,ph), 154.5 (C6 of HLpy,ph), 157.3 (C5 of HLpy,ph). {1H−15N}-g-HMBC NMR (CDCl3, 51 MHz, 3J(N−H) = 3 Hz, 298 K): δN194.5 (N2 of Lpy,ph), 251.9 (Npy of Lpy,ph), 263.1 (N1 of Lpy,ph). ESI-MS(−) (CH3OH) (m/z, relative intensity%): 236 [100] [Lpy,ph]−. [Ru(cym)(Lpy,ph)Cl] (1). HLpy,ph (222.7 mg, 0.94 mmol) was dissolved in 20 mL of methanol, KOH (87.9%) was added (65.1 mg, 0.94 mmol) and the mixture was stirred 1 h at room temperature. Then a methanol solution (30 mL) of [Ru(cym)Cl2]2(287.8 mg, 0.47 mmol) was slowly added affording a dark green mixture, which was left under stirring at room temperature overnight. The solvent was removed by rotary evaporator, and the crude solid was dissolved in 30 mL of dichloromethane. The mixture was filtered to remove the byproduct KCl, and the volume of filtrate reduced to ca. 4 mL. Then, 30 mL of nhexane were added affording a dark green precipitate, which was filtered off and dried to constant weight (261.4 mg, 0.51 mmol, yield 54.8%). It is soluble in alcohols, DMSO, DMF, acetone, acetonitrile, and chlorinated solvents. Anal. Calcd for C24H24ClN3ORu (MW: 508 g/ mol): C, 56.86; H, 4.77; N, 8.29%. Found: C, 56.59; H, 4.64; N, 8.19%. Λm(DMSO, 295 K, 9.8 ×10−4mol/L): 2.2 S cm2mol−1. It decomposes gradually with temperature, starting from about 234 °C. IR (cm−1): Organometallics pubs.acs.org/Organometallics Article https://doi.org/10.1021/acs.organomet.3c00121 Organometallics 2023, 42, 1495−1504 1500 3419vb (O−H···O, hydrogen bond), 3058w ν(C−H aromatics), 2965w ν(C−H aliphatic), 1635vs ν(C�O), 1483vs and 1461vs δ(C− H), 1370s ν(C−N), 1189w, 1145w, 1091w, 1029w, 940m, 872m, 769vs, 740s, 662s, 520m ν(Ru−N), 450m, 440m ν(Ru−O), 403m, 287vs ν(Ru−Cl), 246vs, 228s, 202m. 1H NMR (500 MHz, CD3CN, 298 K): δ0.94d, 0.97d (6H, 3J(H−H) = 6.9 Hz, CH3−C6H4−CH− (CH3)2of cym), 2.17s (3H, CH3−C6H4−CH−(CH3)2of cym), 2.32hept (1H, 3J(H−H) = 6.9 Hz, CH3−C6H4−CH−(CH3)2of cym), 4.76d, 4.88d (2H, 3J(H−H) = 6.0 Hz, CH3−C6H4−CH−(CH3)2of cym), 5.14s (1H, C4−H of Lpy,ph), 5.17d, 5.43d (2H, 3J(H−H) = 6.0 Hz, CH3− C6H4−CH−(CH3)2of cym), 5.14s (1H, C4−H of Lpy,ph), 7.16ddd (1H, 3J(H−H) = 7.2, 5.8 Hz, 4J(H−H) = 1.3 Hz, C9−H of Lpy,ph), 7.58m (3H, C13,13′,14-H of Lpy,ph), 7.92ddd (1H, 3J(H−H) = 8.7, 7.3 Hz, 4J(H−H) = 1.5 Hz, C8−H of Lpy,ph), 7.99dd (2H, 3J(H−H) = 7.9 Hz, 4J(H−H) = 1.7 Hz, C12,12′-H of Lpy,ph), 8.61dd (1H, 3J(H−H) = 8.5 Hz, 4J(H−H) = 1.3 Hz, C7−H of Lpy,ph), 8.86dd (1H, 3J(H−H) = 5.8 Hz, 4J(H−H) = 1.6 Hz, C10−H of Lpy,ph). 13C{1H} NMR (500 MHz, CD3CN, 298 K): δ18.0 ((CH3- C6H4−CH−(CH3)2of cym), 20.8, 21.4 ((CH3−C6H4−CH− (CH3)2), 81.8, 83.0, 84.3, 84.5 (CH3−C6H4−CH−(CH3)2of cym), 88.0 (C4 of Lpy,ph), 101.9, 103.1 (CH3−C6H4−CH−(CH3)2of cym), 111.1 (C7 of Lpy,ph), 119.4 (C9 of Lpy,ph), 128.4 (C13,13′oh Lpy,ph), 129.2 (C14,12,12′of Lpy,ph), 135.0 (C11 oh Lpy,ph), 140.0 (C8 of Lpy,ph), 150.7 (C6 of Lpy,ph), 152.6 (C10 of Lpy,ph), 161.5 (C3 of Lpy,ph), 165.3 (C5 of Lpy,ph). {1H−15N}-g-HMBC NMR (CD3CN, 51 MHz, 3J(N−H) = 3 Hz, at 298 K): δN157.0 (N2 of Lpy), 202.9 (Npy of Lpy), 205.3 (N1 of Lpy). ESI-MS (+) (CH3CN) (m/z, relative intensity%): 472 [100] [Ru(hmb)Lpy,ph]+. [Ru(hmb)(Lpy,ph)Cl] (2). Complex 2was prepared using a method similar to that of 1, using HLpy,ph (184.9 mg, 0.78 mmol), KOH (87.9%) (49 mg, 0.78 mmol), and [Ru(hmb)Cl2]2(260.7 mg, 0.39 mmol). It was isolated as an orange powder (310 mg, 0.58 mmol, yield 74.3%). It is slightly soluble in alcohols and acetonitrile and soluble in DMSO, DMF, and chlorinated solvents. Anal. Calcd for C26H28ClN3ORu (MW: 535 g/mol): C, 58.37; H, 5.28; N, 7.85%. Found: C, 58.26; H, 5.27; N, 7.72%. Λm(DMSO, 296 K, 8.3 ×10−4mol/L): 2.8 S cm2 mol−1. It decomposes gradually with temperature starting from about 295 °C. IR (cm−1): 3105w νs(C−H aromatics), 3054w νa(C−H aromatics), 2921w ν(C−H aliphatic), 1633vs ν(C�O), 1593s ν(C� C), 1578m ν(C�N), 1558m, 1485vs and 1405m δ(C−H), 1356s ν(C−N), 1289m, 1235w, 1183w, 1147w, 1087w, 1075w, 1025m, 992m, 762vs, 749s, 676w, 666s, 627m, 529m, 507m ν(Ru−N), 473m, 455m, 439m ν(Ru−O), 401m, 355w, 304s, 291s ν(Ru−Cl), 264m, 227m, 202s. 1H NMR (500 MHz, CDCl3, 298 K) δ, 2.00s (18H, C6(CH3) of hmb), 5.86s (1H, C4−H of Lpy,ph), 7.13ddd (1H, 3J(H−H) = 7.3, 5.8 Hz; 4J(H−H) = 1.5 Hz, C9−H of Lpy,ph), 7.37m (1H, C14−H of Lpy,ph), 7.44dd (2H, 3J(H−H) = 8.3, 6.9 Hz, C13,13′-H of Lpy,ph), 7.80ddd (1H, 3J(H−H) = 8.7, 7.1 Hz, 4J(H−H) = 1.8 Hz, C8−H of Lpy,ph), 7.90m (3H, 3J(H−H)= 8.3, 1.6 Hz, C12,12′-H of Lpy,ph and C7−H of Lpy,ph), 8.47dd (1H, 3J(H−H)= 5.9 Hz, 4J(H−H) = 1.8 Hz, C10−H of Lpy,ph). 13C NMR (500 MHz, CDCl3, 298 K): δ15.2 (CH3of hmb), 87.2 (C4 of Lpy,ph), 91.4 (Carom of hmb), 115.3 (C7 of Lpy,ph), 119.8 (C9 of Lpy,ph), 125.8 (C12−12′of Lpy,ph), 128.3 (C14 of Lpy,ph) 128.4 (C13−13′of Lpy,ph), 133.9 (C11 of Lpy,ph), 139.1 (C8 of Lpy,ph), 150.7 (C6 of Lpy,ph), 152.0 (C10 of Lpy,ph), 155.3 (C3 of Lpy,ph), 163.7 (C5 of Lpy,ph). {1H−15N}-g-HMBC NMR (CDCl3, 51 MHz, 3J(N−H) = 3 Hz, 298 K): δN206.1 (N1 of Lpy,ph), Npy and N2 of Lpy,ph not observed. ESI-MS (+) (CH3CN) (m/z, relative intensity%): 500 [100] [Ru(hmb)Lpy,ph]+. [Ru(cym)(Lpy,ph)PTA]BF4(3). Complex 1(107.5 mg, 0.21 mmol) was dissolved in 30 mL of methanol and 3 mL of an aqueous solution of AgBF4(41.7 mg, 0.21 mmol) were added. PTA (0.034 mg, 0.21 mmol) was then added, and the mixture stirred 2 h at room temperature. The initial brown-green solution turned to pale green within time. After removal of the byproduct AgCl by filtration, the volume of filtrate was reduced to ca. 4 mL and Et2O (about 30 mL) was added, with formation of a dark green precipitate, which was identified as complex 3 (101 mg, 0.14 mmol, yield 67%). It is very soluble in alcohols, acetonitrile, DMSO, DMF and acetone, and slightly soluble in water and chlorinated solvents. Anal. Calcd for C30H36BF4N6OPRu (MW: 715 g/mol): C, 50.36; H, 5.07; N, 11.75%. Found: C, 50.13; H, 5.17; N, 11.65%. Λm(DMSO, 294 K, 9.9.10−4mol/L): 23.4 S cm2mol−1. It decomposes gradually with temperature starting from about 235 °C. IR (cm−1): 3074w ν(C−H aromatics), 2924w ν(C−H aliphatic), 1626s ν(C�O), 1483s, 1447s, 1373s. 973s, 947s, 803m, 742s, 698s, 662m, 581vs, 519s, 476s, 451s, 389s, 336m, 305m, 229m, 203m. 1H NMR (500 MHz, CD3CN, 298 K): δ0.82d, 0.94d (6H, 3J(H−H) = 6.9 Hz, CH3−C6H4−CH−(CH3)2of cym), 2.22s (3H, CH3−C6H4−CH− (CH3)2), 2.33hept (1H, 3J(H−H) = 6.9 Hz, CH3−C6H4−CH−(CH3)2of cym), 4.01m (6H, (P−CH2−N)3of PTA phosphine), 4.45s (6H, (N− CH2−N)3of PTA phosphine), 4.90d, 5.16d (2H, 3J(H−H) = 6.2 Hz, CH3−C6H4−CH−(CH3)2of cym), 5.22s (1H, C4−H of Lpy,ph), 5.69d, 5.72d (2H, 3J(H−H) = 6.2 Hz, CH3−C6H4−CH−(CH3)2of cym), 7.25ddd (1H, 3J(H−H) = 7.3, 5.9 Hz, 4J(H−H) = 1.4 Hz, C9−H of Lpy,ph), 7.57m (2H, C13,13′-H of Lpy,ph), 7.64m (1H, C14−H of Lpy,ph), 7.68m (2H, C12,12′-H of Lpy,ph), 8.10ddd (1H, 3J(H−H) = 8.8, 7.2 Hz, 4J(H−H) = 1.6 Hz, C8−H of Lpy,ph), 8.40dd (1H, 3J(H−H) = 6.0, 4J(H−H) = 1.5 Hz, C10−H of Lpy,ph), 8.78dd (1H, 3J(H−H) = 8.7, 4J(H−H) = 1.4 Hz, C7−H of Lpy,ph). 13C NMR (500 Hz, 298 K, CD3CN): δ18.7 (CH3−C6H4− CH−(CH3)2of cym), 20.4, 21.5 (CH3−C6H4−CH−(CH3)2of cym), 30.9 (CH3−C6H4−CH−(CH3)2of cym), 50.7d (1J(P−C) = 13.6 Hz, (P−CH2−N)3of PTA phosphine), 72.1 (3J(P−C) = 7.5 Hz, (N−CH2− N)3of PTA phosphine), 86.6, 87.6, 89.5 (CH3−C6H4−CH−(CH3)2of cym), 89.8 (C4 of Lpy,ph), 90.6, 103.4 (CH3−C6H4−CH−(CH3)2of cym), 112.4 (C7 of Lpy,ph), 120.4 (CH3−C6H4−CH−(CH3)2of cym), 121.0 (C9 of Lpy,ph), 128.3 (C13,13′of Lpy,ph), 129.0 (C12,12′of Lpy,ph), 129.8 (C14 of Lpy,ph), 133.9 (C11 of cym), 141.5 (C8 of cym), 151.3 (C3 of Lpy,ph), 153.3d (1J(N−C)= 5.6 Hz C10 of Lpy,ph), 164.1 (C6 of Lpy,ph), 165.5 (C5 of Lpy,ph). 31P NMR (500 Hz, 298 K, CD3CN): δ −37.7. {1H−15N}-g-HMBC NMR in (CD3CN, 51 MHz, 3J(N−H) = 3 Hz, at 298 K): δN42.5 (NPTA), 131.5 (N2 of Lpy,ph), 176.5 (Npy of Lpy,ph), 206.8 (N1 of Lpy,ph). ESI-MS (+) (CH3CN) (m/z, relative intensity%): 472 [100] [Ru(cym)Lpy,ph]+, 629 [91] [Ru(cym)(Lpy,ph)- PTA]+. [Ru(hmb)(Lpy,ph)PTA]BF4(4). Complex 4has been synthesized similarly to 3, using complex 2(75.4 mg, 0.14 mmol), AgBF4(98% of purity) (28.8 mg, 0.14 mmol), and PTA (97%) (23.5 mg, 0.14 mmol). It is an orange solid (78.0 mg, 0.10 mmol, yield 72.3%) which is very soluble in alcohols, DMSO, DMF, and acetone and chlorinated solvents and slightly soluble in water and acetonitrile. Anal. Calcd for C32H40BF4N6OPRu (MW: 743 g/mol): C, 51.69; H, 5.42; N, 11.30%. Found: C, 51.53; H, 5.37; N, 11.21%. Λm(DMSO, 297 K, 1 ×10−3mol/L): 25.9 S cm2mol−1. It decomposes gradually with temperature after 249 °C. IR (cm−1): 2920w ν(C−H), 1602w ν(C− N), 1587s ν(C�O), 1576s, 1554s, 1469s, 1447m, 1411m, 1365m, 974vs, 950vs, 944m, 785s, 773s, 684w, 671w, 643w, 609w, 582m, 572m, 557s, 524w, 475m, 453m, 392w, 329m, 203m. 1H NMR (500 Hz, 298 K, CD3CN): δ2.16s (18H, CH3of hmb), 4.04m (6H, (P−CH2−N)3of PTA phosphine), 4.45m (6H, (N−CH2−N)3of PTA phosphine), 5.91s (1H, C4−H of Lpy,ph), 7.30ddd (1H, 3J(H−H) = 7.3, 5.9 Hz, 4J(H−H) = 1.7 Hz, C9−H of Lpy,ph), 7.42m (1H, C14−H of Lpy,ph), 7.48m (2H, C13,13′-H of Lpy,ph), 7.90m (2H, C12,12′-H of Lpy,ph), 8.05dd (1H, 3J(H−H) = 7.5 Hz, 4J(H−H) = 1.6 Hz, C7−H of Lpy,ph), 8.10ddd (1H, 3J(H−H) = 8.7, 7.1, 4J(H−H) = 1.6 Hz, C8−H of Lpy,ph), 8.13dd (1H, 3J(H−H) = 6.0 Hz, 4J(H−H) = 1.6 Hz, C10−H of Lpy,ph). 13C NMR (500 Hz, 298 K, CD3CN): δ15.3 (CH3of hmb), 43.4d (1J(P−C) = 13.5 Hz, (P−CH2−N)3of PTA phosphine), 72.3d (3J(P−C) = 7 Hz, (N−CH2− N)3of PTA phosphine), 88.2 (C4 of Lpy,ph), 99.0 (Carom of hmb), 117.3 (C7 of Lpy,ph), 121.8 (C9 of Lpy,ph), 125.5 (C12,12′of Lpy,ph), 128.7 (C14,13,13′of Lpy,ph), 133.4 (C11 of Lpy,ph), 141.4 (C8 of Lpy,ph), 151.1 (C6 of Lpy,ph),153.4 (C10 of Lpy,ph), 154.7 (C3 of Lpy,ph), 162.0 (C5 of Lpy,ph). {1H−15N}-g-HMBC NMR in (CD3CN, 51 MHz, 3J(N−H) = 3 Hz, at 298 K): δN39.6 (NPTA), 186.5 (Npy of Lpy,ph), 205.6 (N1 of Lpy,ph), N2 of Lpy,ph not observed. ESI-MS (+) (CH3OH) (m/z, relative intensity%): 657 [100] [Ru(hmb)(Lpy,ph)PTA]+. [Ru(cym)(Lpy,me)PTA]BF4(5). Complex 5was prepared from [Ru(cym)(Lpy,me)Cl], previously reported. 6 82.4 mg (0.15 mmol) of the starting complex were dissolved in 30 mL of methanol and it was added to 1 mL of an aqueous solution of AgBF4(98% of purity) (29.8 mg, 0.15 mmol). PTA (97% of purity) was added (24.3 mg, 0.15 mmol) and the solution was stirred for 2h. An orange solution appeared. AgCl was formed as byproduct, and it was filtered. The remaining solution Organometallics pubs.acs.org/Organometallics Article https://doi.org/10.1021/acs.organomet.3c00121 Organometallics 2023, 42, 1495−1504 1501 was dried to about 4 mL and about 30 mL of Et2O was added, affording a brown precipitate which was shown to be complex 5(240 mg, 0.37 mmol, yield 75.1%). It is soluble in alcohols, acetonitrile, DMSO, DMF, and water. It is slightly soluble in acetone and chlorinated solvents. Anal. Calcd for C25H34BF4N6OPRu (MW: 653.44 g/mol): C, 45.95; H, 5.24; N, 12.86%. Found: C, 45.80; H, 5.28; N, 12.72%. Λm(DMSO, 297 K, 1 ×10−3M): 25.3 S cm2mol−1. It decomposes gradually with temperature starting from about 234 °C. IR (cm−1): 3596w, 3077w ν(C−H aromatics), 2930w ν(C−H aliphatic), 1625s ν(C�O), 1594m, 1474s, 1442m, 1418m, 1363m, 1013vs, 972vs, 947vs, 895m, 802m, 777m, 741s, 609s, 573s, 520m, 476m, 451m, 392m, 322w, 278m, 248w, 234w, 214w, 202s. 1H NMR (500 Hz, 298 K, DMSO): δ0.86d, 0.99 (6H, 3J(H−H) = 6.9 Hz, CH3−C6H4−CH−(CH3)2of cym), 2.17s (3H, C−CH3of Lpy,me), 2.45s (3H, CH3−C6H4−CH−(CH3)2of cym), 2.52m (1H, CH3−C6H4−CH−(CH3)2of cym), 3.75m (6H, (P− CH2−N)3of PTA phosphine), 4.36m (6H, (N−CH2−N)3of PTA phosphine), 4.85s (1H, C4−H of Lpy,me), 6.12d, 6.18d, 6.27d, 6.28d (4H, 3J(H−H) = 6.3 Hz, CH3−C6H4−CH−(CH3)2of cym), 7.22t (1H, 3J(H−H) = 6.6 Hz, H9 of Lpy,me), 8.08t (1H, 3J(H−H) = 7.9 Hz, C8−H of Lpy,me), 8.49d (1H 3J(H−H) = 8.6 Hz, C7−H of Lpy,me), 8.56d (1H, 3J(H−H) = 5.9 Hz, C10−H of Lpy,me). 13C{1H} NMR (500 Hz, 298 K, DMSO): δ17.4 (C3−CH3of Lpy,me), 19.5 (CH3−C6H4−CH−(CH3)2 of cym), 21.7, 22.8 (CH3−C6H4−CH−(CH3)2of cym), 31.5 (CH3− C6H4−CH−(CH3)2of cym), 51.0d (1J(P−C) = 14.0 Hz, (P−CH2−N)3 of PTA phosphine), 72.1d (3J(P−C) = 7.7 Hz, (N−CH2−N)3of PTA phosphine), 88.5 (C4 of Lpy,me), 88.6, 89.1, 89.1, 89.2, 89.6, 89.6 (CH3−C6H4−CH−(CH3)2of cym), 111.3 (C7 of Lpy,me), 120.9 (C9 of Lpy,me), 142.0 (C8 of Lpy,me), 151.1 (C3 of Lpy,me), 154.4 (C10 of Lpy,me), 160.6 (C6 of Lpy,me), 165.8 (C5 of Lpy,me). 31P{1H} NMR (500 Hz, 298 K, DMSO): δ−31.78. {1H−15N}-g-HMBC NMR (DMSO, 51 MHz, 3J(N−H) = 3 Hz, at 298 K): δN43.4 (NPTA), 135.0 (N2 of Lpy,me), 177.1 (Npy of Lpy,me), 205.6 (N1 of Lpy,me). ESI-MS (+) (CH3OH) (m/z, relative intensity%): 410 [100] [Ru(cym)(Lpy,me)]+, 567 [81] [Ru- (cym)(Lpy,me)PTA]+. [Ru(hmb)(Lpy,me)PTA]BF4(6). Complex 6was prepared using a method similar to that of 5from [Ru(hmb)(Lpy,me)Cl], previously reported. 6 213.7 mg (0.45 mmol) of the starting complex were dissolved in 30 mL of methanol. 89.7 mg (0.45 mmol) of AgBF4(98% of purity) and 73.2 mg (0.45 mmol) of PTA phosphine (97% of purity) were used, affording a brown precipitate which was shown to be complex 6(168.9 mg, 0.26 mmol, yield 57.5%). It is soluble in water, alcohols, DMSO, DMF, acetonitrile, and chlorinated solvents. It is slightly soluble in acetone. Anal. Calcd for C27H38BF4N6OPRu (MW: 681.49 g/mol): C, 47.59; H, 5.62; N, 12.33%. Found: C, 47.36; H, 5.59; N, 12.20%. Λm(DMSO, 297 K, 4.5 ×10−4M): 13.12 S cm2mol−1. It decomposes gradually with temperature starting from about 205 °C. IR (cm−1): 3118w ν(C−H aromatics), 2948w, 2919w, 2871w ν(C−H aliphatic), 1655s ν(C�O), 1603m ν(C−N), 1475s, 1441m, 1420m, 1358s, 1007vs, 972vs, 946vs, 893m, 799m, 779s, 734s, 708m, 670w, 609w, 572m, 519m, 475m, 452m, 392w, 355w, 322w, 277w, 248w, 202m. 1H NMR (500 Hz, 298 K, DMSO): δ2.11s (18H, CH3of hmb), 2.13s (C3−CH3of Lpy,me) 3.63dd, 3.79dd (6H, 2J(H−H) = 15.0, 3J(P−H) = 3.6 Hz, (P−CH2−N)3of PTA phosphine), 4.34s (6H, (N−CH2−N) of PTA phosphine), 4.82s (1H, C4−H of Lpy,me), 7.28ddd (1H, 3J(H−H) = 7.4, 6.0, 4J(H−H) = 1.5 Hz, C9−H of Lpy,me), 8.08ddd (1H, 3J(H−H) = 8.7, 7.2, 4J(H−H) = 1.5 Hz, C8−H of Lpy,me), 8.14dd (1H, 3J(H−H) = 6.0, 4J(H−H) = 1.6 Hz, C10−H of Lpy,me), 8.56dd (1H, 3J(H−H)= 8.6, 1.4 Hz, C7−H of Lpy,me). 13C{1H} NMR (500 Hz, 298 K, DMSO): δ16.7 (CH3 of hmb), 17.5 (C3−CH3of Lpy,me), 49.8 ((P−CH2−N)3of PTA phosphine), 72.0 ((N−CH2−N)3of PTA phosphine), 88.5 (C4 of Lpy,me), 102.3 (Carom of hmb), 110.7 (C10 of Lpy,me), 121.4 (C9 of Lpy,me), 141.7 (C7 of Lpy,me), 151.2 (C6 of Lpy,me), 152.0 (C8 of Lpy,me), 160.9 (C3 of Lpy,me), 166.7 (C5 of Lpy,me). 31P NMR (500 Hz, 298 K, DMSO): δ−38.71. {1H−15N}-g-HMBC NMR (DMSO, 51 MHz, 3J(N−H) = 3 Hz, at 298 K): δN40.0 (NPTA), 139.9 (N2 of Lpy,me), 180.4 (Npy of Lpy,me), 205.4 (N1 of Lpy,me). ESI-MS (+) (CH3OH) (m/z, relative intensity%): 438 [100] [Ru(cym)(Lpy,me)]+, 595 [55] [Ru- (cym)(Lpy,me)PTA]+. [Ru(cym)(Qpy,CF3)PTA]PF6(7). Complex 7was prepared using a method similar to that of 6from [Ru(cym)(Qpy,CF3)Cl], previously reported. 6 38.3 mg (0.07 mmol) of the starting complex were dissolved in 30 mL of methanol and an aqueous solution (1 mL) of AgPF6(18.5 mg, 0.07 mmol) was added. Then, 11.5 mg (0.07 mmol) of PTA (97% of purity) was added and the solution was stirred for 2 h. AgCl was formed as byproduct, and it was filtered. The remaining dark yellow solution was dried to about 4 mL and about 30 mL of Et2O was added, affording a yellow precipitate which was shown to be complex 7(42.5 mg, 0.05 mmol, yield 75.2%). It is soluble in DMSO, DMF. Anal. Calcd for C27H33F9N6O2P2Ru (MW: 807.61 g/mol): C, 40.16; H, 4.12; N, 10.41%. Found: C, 40.05; H, 4.24; N, 10.30%. It decomposes gradually with temperature, starting from about 281 °C. Λm(DMF, 298 K, 1.7 × 10−4M): 12.23 S cm2mol−1. IR (cm−1): 3081w ν(C−H aromatics), 2932w ν(C−H aliphatic), 1697m, 1682 ν(C�O), 1647vs ν(C−N), 1517m, 1464vs, 1340s, 1256s, 1190m, 1150s, 1054vs, 1011vs, 975m, 949m, 834vs ν(PF6), 773s, 728s, 685m, 611w, 582s,557vs, 525w, 479m, 453m, 384m, 281w. 1H NMR (500 Hz, DMSO, 298 K): δ0.77d (3H, 3J(H−H) = 6.8 Hz, CH3−C6H4−CH−(CH3)2of cym), 1.02d (3H, 3J(H−H) = 6.8 Hz, CH3−C6H4−CH−(CH3)2of cym), 2.46s (3H, CH3− C6H4−CH−(CH3)2of cym), 2.46m (1H, CH3−C6H4−CH−(CH3)2 of cym), 2.54s (3H, C3−CH3of Qpy,CF3) 3.89m (6H, (P−CH2−N)3of PTA phosphine), 4.40m (6H, (N−CH2−N)3of PTA phosphine), 6.12d, 6.31d, 6.33d 6.44d (4H, 3J(H−H) = 6.4 Hz, CH3−C6H4−CH− (CH3)2of cym), 7.37ddd (1H, 3J(H−H) = 7.4, 5.9 Hz, 4J(H−H) = 1.4 Hz, C9−H of Qpy,CF3), 8.20ddd (1H, 3J(H−H) = 8.6, 7.2 Hz, 4J(H−H) = 1.5 Hz, C8−H of Qpy,CF3), 8.47dd (1H, 3J(H−H) = 8.6 Hz, 4J(H−H) = 1.4 Hz, C7− H of Qpy,CF3), 8.63dd (1H, 3J(H−H) = 5.9 Hz, 4J(H−H) = 1.5 Hz, C10−H of Qpy,CF3). 19F{1H} NMR (500 Hz, DMSO, 298 K): δ−69.4s, −70.9s (PF6 −), −74.5s (CF3). 31P{1H} NMR (500 Hz, DMSO, 298 K): δ −32.8 (PTA phosphine). 13C{1H} NMR (500 Hz, DMSO, 298 K): δ 19.1 (CH3−C6H4−CH−(CH3)2of cym), 20.0 (C3−CH3of Qpy,CF3), 20.9, 22.9 (CH3−C6H4−CH−(CH3)2of cym), 31.3 (CH3−C6H4− CH−(CH3)2of cym), 50.5d (1J(P−C) = 14.0 Hz, (P−CH2−N)3of PTA phosphine), 71.8 (4J(P−C) = 7.8 Hz (N−CH2−N)3of PTA phosphine), 88.5, 90.1, 90.4 (CH3−C6H4−CH−(CH3)2of cym), 99.7 (C4 of Qpy,CF3) 102.6 (CH3−C6H4−CH−(CH3)2of cym), 111.7 (C7 of Qpy,CF3), 117.2q (1J(C−F): 289.9 Hz, (C�O)CF3of Qpy,CF3), 122.2 (C9 of Qpy,CF3), 122.9 (CH3−C6H4−CH−(CH3)2of cym), 142.6 (C8 of Qpy,CF3), 150.0 (C6 of Qpy,CF3), 154.4 (C10 of Qpy,CF3), 161.5 (C5 of Qpy,CF3), 164.3 (C3 of Qpy,CF3), 170.7q (2J(C−F) = 35.9 Hz (C�O)CF3 of Qpy,CF3). {1H−15N}-g-HMBC NMR (DMSO, 51 MHz, 3J(N−H) = 3 Hz, at 298 K): δN42.8 (NPTA), 167.3 (N2 of Qpy,CF3), 179.7 (Npy of Qpy,CF3), N1 of Qpy,CF3 not observed. ESI-MS (+) (CH3CN) (m/z, relative intensity%): 663 [100] [Ru(cym)(Qpy,CF3)PTA]+, 506 [29] [Ru(cym)(Qpy,CF3)]+. [Ru(hmb)(Qpy,CF3)PTA]PF6(8). Complex 8was prepared using a method similar to that of 7from [Ru(hmb)(Qpy,CF3)Cl], previously reported. 6 96.1 mg (0.17 mmol) of the starting complex were dissolved in 30 mL of methanol and an aqueous solution (1 mL) of AgPF6(43.0 mg, 0.17 mmol) was added. Then, 27.5 mg (0.17 mmol) of PTA (97% of purity) was added and the solution was stirred for 2 h. AgCl was formed as byproduct, and it was filtered. The remaining dark yellow solution was dried to about 4 mL and about 30 mL of Et2O was added, affording a yellow precipitate which was shown to be complex 8. It is soluble in DMSO, DMF. Anal. Calcd for C29H37F9N6O2P2Ru (MW: 835.66 g/mol): C, 41.68; H, 4.46; N, 10.06%. Found: C, 41.55; H, 4.54; N, 9.97%. It decomposes gradually from 292 °C. Λm(DMF, 298 K, 2 × 10−4M): 11.88 S cm2mol−1. IR (cm−1): 3081w ν(C−H aromatics), 2935w ν(C−H aliphatic), 1674m, 1652s ν(C−N), 1516w, 1464s, 1341m, 1255m, 1192m, 1156s, 1048s, 1017m, 925s, 834vs ν(PF6), 784s, 725m, 684w, 608w, 582s, 557vs, 524w, 475m, 453w, 392w, 329m, 278w, 203w. 1H NMR (500 Hz, DMSO, 298 K): δ2.12s (18H, CH3of hmb), 2.49s (3H, C3−CH3of Qpy,CF3) 3.83m (6H, (P−CH2−N)3of PTA phosphine), 4.37m (6H, (N−CH2−N)3of PTA phosphine), 7.42ddd (1H, 3J(H−H) = 7.4, 5.9, 4J(H−H) = 1.5 Hz, C9−H of Qpy,CF3), 8.20ddd (1H, 3J(H−H) = 8.7, 7.3, 4J(H−H) = 1.5 Hz, C8−H of Qpy,CF3), 8.23dd (1H, 3J(H−H) = 6.0 Hz, 4J(H−H) = 1.4 Hz, C10−H of Qpy,CF3), 8.46dd (1H, 3J(H−H) = 8.7 Hz, 4J(H−H) = 1.3 Hz, C7−H of Qpy,CF3). 19F{1H} NMR (500 Hz, DMSO, 298 K): δ−69.4s, −70.9s (PF6 −), Organometallics pubs.acs.org/Organometallics Article https://doi.org/10.1021/acs.organomet.3c00121 Organometallics 2023, 42, 1495−1504 1502 −74.5s (CF3). 31P{1H} NMR (500 Hz, DMSO, 298 K): δ−40.3 (PTA phosphine). 13C{1H} NMR (500 Hz, DMSO, 298 K): δ16.7 (CH3of hmb), 20.7 (C3−CH3of Qpy,CF3), 49.6d (1J(P−C) = 13.4 Hz, (P−CH2− N)3of PTA phosphine), 71.9 (4J(P−C) = 7.5 Hz (N−CH2−N)3of PTA phosphine), 99.9 (C4 of Qpy,CF3), 103.1 (Caromatics of hmb) 111.5 (C7 of Qpy,CF3), 117.2q (1J(C−F) = 290.4 Hz, (C�O)CF3of Qpy,CF3), 122.8 (C9 of Qpy,CF3), 142.5 (C8 of Qpy,CF3), 150.1 (C6 of Qpy,CF3), 152.4 (C10 of Qpy,CF3), 162.3 (C5 of Qpy,CF3), 164.8 (C3 of Qpy,CF3), 170.5q (2J(C−F) = 35.8 Hz (C�O)CF3of Qpy,CF3). {1H−15N}-g-HMBC NMR (DMSO, 51 MHz, 3J(N−H) = 3 Hz, at 298 K): δN40.0 (NPTA), 172.6 (N2 of Qpy,CF3), 182.8 (Npy of Qpy,CF3), N1 of Qpy,CF3 not observed. ESI-MS (+) (CH3CN) (m/z, relative intensity%): 691 [100] [Ru(cym)- (Qpy,CF3)PTA]+. ■ASSOCIATED CONTENT * sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.organomet.3c00121. DFT calculations, figures and XYZ coordinates, crystal data and structure refinement, NMR spectra (PDF) Structure data (XYZ) Accession Codes CCDC 2204821 and 2211175 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. ■AUTHOR INFORMATION Corresponding Authors Riccardo Pettinari −School of Pharmacy, University of Camerino, 62032 Camerino, Macerata, Italy; orcid.org/ 0000-0002-6313-4431; Phone: +39 0737402338; Email: [email protected] Paul J. Dyson −Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland; orcid.org/0000-0003-3117-3249; Email: [email protected] Authors Lorenzo Pietracci −School of Science and Technology, University of Camerino, 62032 Camerino, Macerata, Italy Alessia Tombesi −School of Pharmacy, University of Camerino, 62032 Camerino, Macerata, Italy Fabio Marchetti −School of Science and Technology, University of Camerino, 62032 Camerino, Macerata, Italy; orcid.org/ 0000-0001-5981-930X Claudio Pettinari −School of Pharmacy, University of Camerino, 62032 Camerino, Macerata, Italy; orcid.org/ 0000-0002-2547-7206 Agustín Galindo −Departamento de Química Inorgánica, Facultad de Química, Universidad de Sevilla, 41071 Sevilla, Spain; orcid.org/0000-0002-2772-9171 Farzaneh Fadaei-Tirani −Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland; orcid.org/0000- 0002-7515-7593 Mouna Hadiji −Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland Complete contact information is available at: https://pubs.acs.org/10.1021/acs.organomet.3c00121 Notes The authors declare no competing financial interest. ■ACKNOWLEDGMENTS F.M. thanks the University of Camerino and the HP Composites. PhD scholarship of L. Pietracci is financed by Italian Ministry of University and Research; project “Composite Materials Hub of Valle del Tronto, Marche region”. A.G. thanks the Centro de Servicios de Informática y Redes de Comunicaciones (CSIRC), Universidad de Granada for providing the computing time. ■REFERENCES (1) Cruz, M. P. Edaravone (Radicava): A Novel Neuroprotective Agent for the Treatment of Amyotrophic Lateral Sclerosis. Pharm. Ther. 2018,43 (1), 25−28. (2) Faria, J. V.; Vegi, P. F.; Miguita, A. G. C.; dos Santos, M. S.; Boechat, N.; Bernardino, A. M. R. Recently Reported Biological Activities of Pyrazole Compounds. Bioorg. Med. Chem. 2017,25 (21), 5891−5903. (3) Pettinari, R.; Marchetti, F.; Di Nicola, C.; Pettinari, C. Half- Sandwich Metal Complexes with Diketone-Like Ligands and Their Anticancer Activity. Eur. J. Inorg. Chem. 2018,2018, 3521−3536. 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