Tunable Anticancer Activity of Furoylthiourea-Based Ru^II -Arene Complexes and Their Mechanism of Action.

Chemistry—A European Journal Full Paper doi.org/10.1002/chem.202004954 & Antitumor Agents Tunable Anticancer Activity of Furoylthiourea-Based RuII–Arene Complexes and Their Mechanism of Action Srividya Swaminathan,[a] Jebiti Haribabu,[a] Naveen Kumar Kalagatur,[b] Maroli Nikhil,[c] Nithya Balakrishnan,[a] Nattamai S. P. Bhuvanesh,[d] Krishna Kadirvelu,[b] Ponmalai Kolandaivel,[e] and Ramasamy Karvembu*[a] Abstract: Fourteen new RuII–arene (p-cymene/benzene) complexes (C1–C14) have been synthesized by varying the N-terminal substituent in the furoylthiourea ligand and satisfactorily characterized by using analytical and spectroscopic techniques. Electrostatic potential maps predicted that the electronic effect of the substituents was mostly localized, with some influence seen on the labile chloride ligands. The structure–activity relationships of the Ru–p-cymene and Ru– benzene complexes showed opposite trends. All the complexes were found to be highly toxic towards IMR-32 cancer cells, with C5 (Ru–p-cymene complex containing C6H2(CH3)3 Introduction The growth of research in the field of medicinal chemistry has been highly beneficial for the well-being of humans. In a study that proved to be a boon to mankind, Barnett Rosenberg accidentally discovered the cytotoxic nature of cisplatin,[1] a platinum-based compound that gained approval from the Food and Drug Administration (FDA) in 1978 and went on to become a widely used anticancer drug. After its discovery, the research on platinum-based anticancer drugs flourished, which led to the discovery of various new therapeutics, a couple of which were approved for the global market (Figure 1).[1, 2] [a] S. Swaminathan, Dr. J. Haribabu, N. Balakrishnan, Prof. R. Karvembu Department of Chemistry, National Institute of Technology Tiruchirappalli 620015, Tamil Nadu (India) E-mail: kar@nitt.edu [b] Dr. N. K. Kalagatur, Dr. K. Kadirvelu DRDO-BU Centre for Life Sciences, Bharathiar University Campus Coimbatore 641046, Tamil Nadu (India) [c] Dr. M. Nikhil Centre for Condensed Matter Theory, Department of Physics Indian Institute of Science, Bangalore 560012, Karnataka (India) as N-terminal substituent) and C13 (Ru–benzene complex containing C6H4(CF3) as N-terminal substituent) showing the highest activity among each set of complexes, and hence they were chosen for further study. These complexes showed different behavior in aqueous solutions, and were also found to catalytically oxidize glutathione. They also promoted cell death by apoptosis and cell cycle arrest. Furthermore, the complexes showed good binding ability with the receptors Pim-1 kinase and vascular endothelial growth factor receptor 2, commonly overexpressed in cancer cells. Although platinum-based drugs gained importance in cancer treatment, they came with serious drawbacks.[3] In a search for alternatives, scientists began to explore options with other metal ions. Ruthenium complexes came foremost with desirable properties, which made them exciting candidates for medicinal applications. They were found to be selectively active toward cancer cells over normal cells, and to mimic iron in binding to biological molecules.[4] The antineoplastic ruthenium frameworks that have entered clinical trials mostly involve RuIII species, imidazolium (imidazole)(dimethyl sulfoxide)tetrachlororuthenate(III) (NAMI-A), indazolium trans-tetrachlorobis(1H-indazole)ruthenate(III) (KP1019) and sodium trans-tetrachlorobis(1H-indazole)ruthenate(III) (KP1339; Figure 2).[5] Numerous pharmaceutical chemists are exploring RuII scaffolds in preclinical studies that are at several stages of development. Namely, RuII–arene compounds containing ethylenediamine (en) or 1,3,5-triaza-7-phosphaadamantane (PTA) ligand have shown efficient antineoplastic activity. Another promising RuII therapeutic, TLD1433, has entered phase 1 and phase 2a clinical trials for non-muscle invasive bladder cancer treatment by photodynamic therapy (PDT).[6] [d] Dr. N. S. P. Bhuvanesh Department of Chemistry, Texas A & M University College station, Texas 77842 (USA) [e] Prof. P. Kolandaivel Periyar University, Salem 636011, Tamil Nadu (India) Supporting information and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.org/10.1002/chem.202004954. Chem. Eur. J. 2021, 27, 7418 – 7433 Figure 1. Globally approved platinum-based anticancer drugs. 7418 T 2021 Wiley-VCH GmbH Chemistry—A European Journal Full Paper doi.org/10.1002/chem.202004954 Figure 3. Aroylthiourea-based RuII–arene complexes reported by our group. Results and Discussion Figure 2. Ruthenium compounds in clinical trials. Synthesis Metals combined with organic motifs are becoming increasingly dominant with their vast number of applications.[7–10] In the pharmaceutical industry, although many drugs in the market are seemingly organic in nature, organometallic compounds offer various benefits over their organic counterparts. They demonstrate a wide range of oxidation states, lipophilic character, redox properties, structural diversity, kinetic stability, an ability to bind to biomolecules and the opportunity to tune ligands to control the kinetic properties. In this regard, ligand selection needs to be carefully planned for the development of organometallic drugs.[11–14] Aroylthioureas are known to exhibit antibacterial, antifungal, and anticancer properties. Our group has been actively investigating the biological properties of NiII, PdII and CuII complexes of aroylthiourea ligands.[15, 16] More recently, we have diverted our efforts towards aroylthiourea-based RuII–arene complexes, which have resulted in potential anticancer agents (Figure 3).[17–19] Although the literature contains a significant number of reports on RuII–arene acylthiourea complexes, the structure–activity relationships of these compounds have seldom been investigated. The structures of these compounds are similar to ruthenium–arene PTA (RAPTA) species, which make them interesting candidates for anticancer applications, because the latter are rapidly evolving as lead compounds for the treatment of cancer. Hence, in this study, we have explored the RuII–arene complexes of furoyl-based thiourea ligands for their anticancer properties, with the perspective that furan derivatives are important pharmaceutical compounds. Various substituents (electron-donating/electron-withdrawing/steric/different aliphatic chain length) on the terminal nitrogen were selected to study their effects.[20, 21] As a result, 14 RuII–p-cymene or RuII–benzene complexes with furoylthiourea ligands were synthesized. They were characterized and evaluated for their solution behavior, GSH binding and anticancer activity. Chem. Eur. J. 2021, 27, 7418 – 7433 www.chemeurj.org The ligands L1–L7 were synthesized from 2-furoyl chloride, potassium thiocyanate and the respective amines according to a previously reported procedure (Scheme 1).[16] Among these ligands, L6 and L7 are new, and they were characterized by elemental analysis and UV/Vis, FTIR and NMR spectroscopy. The RuII–arene complexes were synthesized by adding the [Ru(arene)Cl2]2 (arene = p-cymene or benzene) precursor to a solution of the corresponding ligand in toluene (Scheme 2). A few drops of methanol were added to promote the solubility of the metal precursor.[19] The reaction was allowed to proceed for 4 h, after which the ligand spot in TLC had completely disappeared. The resulting reaction mixture was concentrated Scheme 1. Synthesis of the furoylthiourea ligands. Scheme 2. Synthesis of the RuII–arene complexes. 7419 T 2021 Wiley-VCH GmbH Chemistry—A European Journal Full Paper doi.org/10.1002/chem.202004954 under reduced pressure, and then hexane was added until a solid formed. The precipitate was filtered, washed thoroughly with hexane and dried under vacuum. The complexes were obtained in good yields (70–79 %). Crystals of C1–C4, C6, C8 and C14 were grown in DMF/chloroform solution, and their structures were solved by single-crystal XRD analysis. Spectroscopic characterization The compounds were initially characterized by elemental analysis and UV/Vis and FTIR spectroscopy (see Figures S1–S16 and Tables S1 and S2 in the Supporting Information).[17–19] The 1H and 13C NMR spectra of the ligands L6 and L7, aside from their aromatic protons and carbon atoms being observed in their respective regions, showed amide and thioamide N@H protons as singlets in the ranges 12.53–12.50 and 11.62–11.45 ppm, respectively. The thioamidic and amidic carbon signals were seen at around 179.5–178.4 and 157.9–156.9 ppm, respectively. Each of the trifluoromethyl carbon atoms showed four signals in the 13 C NMR spectra in the range 126.3–119.9 ppm as a result of spin–spin coupling between the carbon and fluorine atoms (see Figures S17–S22).[17, 18] In the 1H NMR spectra of the complexes, the three aromatic protons of the furan ring were observed as two doublets and a doublet of doublets in the ranges 8.11–7.60 and 6.79– 6.49 ppm, respectively. The terminal N substituents of the ligands, apart from the aromatic protons, which were observed in their respective regions, displayed signals as follows: the methylene protons were seen as a doublet at 4.86–4.79 ppm (C2 and C9), the CH2CH2 protons as a doublet of doublets (3.94–3.92 ppm) and triplet (3.03 ppm; C3 and C10), and the CH2CH3 protons as a multiplet and triplet at 2.62 and 1.22 ppm (C4 and C11), respectively. The signals due to the ortho- and para-methyl protons of C5 and C12 appeared at 2.25 and 2.14 ppm as singlets, respectively. The aliphatic segments of the p-cymene ligand showed characteristic signals, that is, a doublet corresponding to the six protons of the isopropyl methyl groups at 1.35–1.19 ppm, a multiplet at 3.02–1.63 ppm due to the C@H proton and a singlet at 2.29–2.09 ppm caused by the methyl protons. Similarly, the aromatic protons of the p-cymene ring appeared as doublets in the ranges 5.83–5.32 and 5.79–5.21 ppm (C1–C7). The benzene complexes (C8–C14) each showed a sharp singlet corresponding to the six protons in the range 5.98–5.62 ppm. The 13C NMR spectra of the complexes showed a signal due to the thioamidic carbon (C=S) in the most shielded region (185.4–178.8 ppm), whereas the amidic carbon (C=O) signal was observed in the range 162.6–157.7 ppm. All the aromatic carbon atoms were observed in the expected region. The aliphatic carbon atoms of the p-cymene, that is, the methyl carbon attached to the aromatic ring, the isopropyl carbon and the two methyl carbons attached to the isopropyl carbon, displayed signals in the ranges 30.5–30.3, 22.3–21.9 and 18.4– 18.2 ppm, respectively (C1–C7). In the spectra of the complexes with benzene as the arene moiety (C8–C14), an intense signal corresponding to the six carbon atoms was observed at around 92.8–85.3 ppm (see Figures S23–S54 in the Supporting Information). The 19F NMR spectra of the compounds containing CF3 were recorded and a single sharp peak was observed for all in the range 60.5–61.5 ppm (see Figures S19, S22, S35, S38, S51 and S54). The mass spectra of the complexes showed a base peak corresponding to the [M@2 H + @2 Cl@+H + ] + fragment, in agreement with the data reported for similar types of complexes in the literature (see Figures S55–S68 in the Supporting Information).[17–19] Molecular structures The structures of C1–C4 and C6 (Ru–p-cymene complexes), and C8 and C14 (Ru–benzene complexes) were determined by single-crystal XRD analysis. The complexes showed a pseudooctahedral geometry around the ruthenium ion with a piano stool form (Figure 4 and Tables S3 and S4 in the Supporting Information).[22–24] On going from C1 (2.405 a) to C3 (2.4206 a), there was a noticeable increase in the Ru@S bond length, owing to the distancing of the phenyl group from the thiocarbonyl (C=S) moiety. This increase was also observed in the case of C4 (2.4065 a), in which electron-donating ethyl groups are attached at the ortho positions of the terminal phenyl moiety, from which it can be concluded that the Ru@S bond becomes weaker in such cases. In contrast, the Ru@S bond was Figure 4. Crystal structures of the complexes C1–C4, C6, C8 and C14. Chem. Eur. J. 2021, 27, 7418 – 7433 www.chemeurj.org 7420 T 2021 Wiley-VCH GmbH Full Paper doi.org/10.1002/chem.202004954 Chemistry—A European Journal shorter in C6 (2.4025 a), and still shorter in C14 (2.3875 a) compared with their parent complexes C1 and C8 (2.406 a), respectively, which signifies the strengthening of the bond in the presence of electron-withdrawing group(s). The Ru@C bond lengths in the arene rings showed similar trends. It is interesting to note that the Ru@S bond in benzene complex C8 was longer than that in its p-cymene analogue C1. This may be because the electron-donating groups on pcymene reduce its p-accepting ability, which result in longer Ru@arene and shorter Ru@S bonds (Table 1). In addition, an increase in the Cl-Ru-Cl angle was observed upon moving from C1 (87.548) to C3 (87.968), which might be attributed to the fact that the bulky phenyl group is further away from the metal center, thereby allowing the chloride ligands to assemble freely in space. Further proof for this was observed in a contrasting manner when the same angle was lower in C4 (86.958) due to the steric effect of the ethyl groups present in both of the ortho positions of the phenyl substituent. Comparing the Cl-Ru-Cl angles of C1 (87.548) and C6 (91.968), and C8 (86.858) and C14 (87.798), the angle showed a noticeable increase with the electron-withdrawing group(s) attached to the terminal N of C6 and C14, which may be due to the shortening of the Ru@S bond in these complexes placing the sulfur atom and chloride ligands in a crowded environment. The N@H bond distances (0.88 a) in all the complexes were almost the same. All the bond lengths and angles of these complexes are in good agreement with those of similar compounds reported in the literature.[17–19] Table 1. Selected bond lengths in C1–C4, C6, C8 and C14. Ru1@Cl1 Ru1@Cl2 Ru1@S1 Ru1@C13 Ru1@C14 Ru1@C15 Ru1@C16 Ru1@C17 Ru1@C18 N1@H1 N2@H2 S1@C1 O1@C8 Bond lengths [a] C4 C6 C1 C2 C3 2.416 2.431 2.405 2.206 2.203 2.195 2.200 2.185 2.175 0.880 0.880 1.704 1.220 2.418 2.434 2.416 2.189 2.169 2.207 2.169 2.189 2.197 0.880 0.880 1.709 1.225 2.410 2.426 2.420 2.189 2.172 2.187 2.223 2.199 2.162 0.880 0.880 1.709 1.223 2.412 2.439 2.406 2.205 2.177 2.183 2.219 2.188 2.166 0.880 0.880 1.700 1.232 2.403 2.426 2.402 2.189 2.165 2.172 2.212 2.179 2.193 0.880 0.880 1.689 1.218 C8 C14 2.420 2.425 2.406 2.230 2.170 2.173 2.197 2.160 2.183 0.880 0.880 1.713 1.226 2.414 2.408 2.387 2.189 2.183 2.164 2.176 2.173 2.206 0.880 0.880 1.683 1.228 Theoretical studies using DFT The results of density functional theory (DFT) calculations verified the structures obtained from single-crystal XRD analysis as well as the diamagnetic character of RuII in the studied complexes (see Figures S69–S71 and Tables S5 and S6 in the Supporting Information).[25–30] The electrostatic potential (ESP) surface diagrams (see Figure S70) revealed that the electron density is highest on the two chloride ligands, the carbonyl oxygen and the oxygen of furan.[27, 28] The effect of the electron-donating/withdrawing substituents on the furoylthiourea Chem. Eur. J. 2021, 27, 7418 – 7433 www.chemeurj.org ligand is mostly observed as a localized effect.[21] The ESP of the ligand exposed enhanced/diminished electronic charge density on the terminal N to which the substituent was attached. There was no significant effect observed for complexes C1–C3 and C8–C10. The electron-donating groups in complexes C4, C5, C11 and C12 generated a more negative ESP within the terminal N-substituted aromatic ring, whereas the complexes with electron-withdrawing substituents (C6, C7, C13 and C14) provided a more positive ESP within the ring. The calculations also showed that the chloride ligands of C6, C7, C13 and C14 (containing electron-withdrawing substituents) displayed a less negative ESP than those of C4, C5, C11 and C12 (containing an electron-donating substituent). Anticancer potential The in vitro anticancer activities of the complexes were evaluated by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in five different cancer cell lines (A431epidermoid carcinoma, HeLa-cervix, HepG2-liver, IMR-32-neuroblastoma and SW40-colorectal cancer; Figure 5) and one normal cell line (Vero; see Figure S72 in the Supporting Information).[31, 32] The IC50 values of the complexes are compiled in Table 2. The complexes showed highest activity in the human neuroblastoma cell line (IMR-32) with the lowest IC50 values determined for complexes C5 (11.34 : 0.42 mm) and C13 (11.84 : 0.66 mm). Although the anticancer potential of the complexes was lower than that of the positive control cisplatin, it was comparable to or even greater than those of other reported complexes after an incubation period of just 24 h.[33–36] We could see that the IC50 values of the complexes mostly decreased as the N-substituted phenyl moved away from the furoylthiourea group. Interestingly, for the p-cymene complexes (C1–C7), electron-donating substituent(s) in the phenyl ring attached to the terminal N lowered the IC50 values of the complexes. In contrast, the electron-withdrawing groups exerted the same influence in the benzene analogues (C8–C14). The IC50 values of the complexes in the normal cell line (Vero) were almost three-fold greater than those observed in the cancer cell lines, which reveals the selectivity of the complexes. Owing to their highest activities and contrasting behavior, C5 and C13 were chosen from each set and subjected to further mechanistic studies. Since the discovery of metal-based anticancer drugs, it has been of the utmost importance to find the mechanism of operation of these compounds inside the human body.[37–39] Thus, the mechanistic pathway by which the most active complexes C5 and C13 cause cell death has been investigated, as detailed in the following. Solution behavior of C5 and C13 The solution behavior of C5 and C13 was studied by using UV/ Vis spectrophotometry under a variety of conditions. The UV/ Vis spectra of C5 and C13 at 298 K immediately after dissolution in water (water/DMSO, 9.9:0.1, v/v; see Figure S74 in the 7421 T 2021 Wiley-VCH GmbH Chemistry—A European Journal Full Paper doi.org/10.1002/chem.202004954 Figure 5. Variation in the cell viability [%] of the complexes with change in the N-terminal substituent. Table 2. IC50 values of the complexes C1–C14 in various cancer and normal cell lines. Complex C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 cisplatin IC50 [mm] A431 HeLa HepG2 28.5 : 1.2 42.2 : 0.6 43.5 : 0.4 25.1 : 0.5 21.3 : 1.0 50.5 : 0.4 53.8 : 1.6 36.1 : 0.3 39.1 : 1.1 31.3 : 0.7 37.7 : 1.7 33.2 : 1.3 22.6 : 0.7 45.5 : 1.0 9.8 : 0.8 23.3 : 0.8 28.2 : 1.2 26.6 : 1.3 19.2 : 0.6 17.0 : 1.0 31.3 : 0.4 39.0 : 0.9 28.8 : 1.2 29.9 : 0.7 32.2 : 0.1 29.5 : 0.6 27.8 : 0.6 21.8 : 0.7 27.4 : 0.2 6.1 : 0.5 25.5 : 0.1 33.1 : 0.6 31.9 : 0.3 23.3 : 0.5 17.2 : 0.6 34.7 : 1.2 40.1 : 0.2 38.4 : 0.9 28.3 : 1.3 34.6 : 1.5 37.3 : 0.4 35.3 : 0.5 21.7 : 0.6 38.9 : 1.1 7.9 : 0.2 Supporting Information) exhibited a strong absorption band at around 280 nm that underwent a small hypsochromic shift while decreasing in intensity over time, concomitant with the appearance of a new shoulder peak at around 330 nm, the intensity of which increased with time, indicating rapid hydrolysis of the complexes, although the exact nature of the hydrolysis products was not known until later.[40–42] The hydrolysis process seemed to attain equilibrium after 25 min for both complexes. Chem. Eur. J. 2021, 27, 7418 – 7433 www.chemeurj.org IMR-32 SW40 Vero 15.0 : 1.0 19.6 : 0.6 15.1 : 0.9 13.9 : 0.9 11.3 : 0.4 18.1 : 0.8 22.3 : 0.1 23.0 : 0.5 20.9 : 0.6 22.2 : 0.9 23.0 : 0.5 17.0 : 0.6 11.8 : 0.7 24.9 : 1.1 5.7 : 0.6 31.4 : 0.1 47.1 : 0.4 54.0 : 1.5 28.2 : 0.7 26.2 : 0.1 52.8 : 1.7 54.9 : 1.3 39.5 : 0.6 56.2 : 1.8 46.4 : 1.0 33.6 : 0.7 38.0 : 1.3 28.8 : 0.4 39.2 : 1.3 7.12 : 0.3 107.2 : 2.3 121.1 : 2.0 119.6 : 3.8 95.9 : 4.0 91.8 : 4.6 121.9 : 3.9 126.3 : 1.7 130.0 : 3.7 112.0 : 2.0 133.3 : 0.4 117.1 : 2.1 134.5 : 1.3 93.8 : 4.1 116.7 : 2.6 16.7 : 1.3 To investigate the suppression of hydrolysis by chloride ions, the UV/Vis spectra of C5 and C13 (0.5 mm) were recorded at 298 K in 4 mm NaCl solution. The spectra were essentially the same as those observed in water, which indicates that a low chloride concentration did not suppress hydrolysis. However, the spectra of the complexes dissolved in a higher concentration of NaCl (100 mm) revealed that the hydrolysis was suppressed (Figure 6). These results indicate that these complexes, unless metabolized in a different way, for example, by protein 7422 T 2021 Wiley-VCH GmbH Chemistry—A European Journal Full Paper doi.org/10.1002/chem.202004954 Figure 6. Time-dependent UV/Vis absorption spectra of C5 and C13 in 4 and 100 mm NaCl at 298 K, recorded at 1 min intervals over a period of 30 min. binding, may be able to survive the conditions found in the bloodstream, and that hydrolysis may take place inside the cells in which the chloride concentration decreases to around 4 mm.[41] The time-dependent absorption spectra (difference spectra) of C5 and C13 in 100 mm aqueous NaClO4 at 298 K are shown in Figures S75 and S76 in the Supporting Information. In each case, the presence of two isosbestic points (C5: 274 and 326 nm; C13: 281 and 324 nm) suggested the hydrolysis of the chloro complexes. The maximum change in absorption occurred at 285 and 295 nm for C5 and C13, respectively, and hence these wavelengths were selected for subsequent kinetic studies.[42] The hydrolysis of C5 and C13 at 298 K in aqueous NaClO4 (0.015–0.5 m) was monitored at the selected wavelengths (see Figure S77). In each case, the time-dependent absorbance followed first-order kinetics; the corresponding rate constants (kobs) are listed in Table 3. For both complexes, the rate of hydrolysis was almost independent of ionic strength. The reverse reaction was too rapid to be studied by UV/Vis spectrophotometry. The aqueous solution chemistry of C5 and C13 was studied in detail at 298 K over 24 h by 1H NMR spectroscopy and ESIMS spectrometry. Due to the low solubility of the complexes in water, the NMR studies were conducted in 3:7 (v/v) [D6]DMSO/ Chem. Eur. J. 2021, 27, 7418 – 7433 www.chemeurj.org Table 3. Rate constants and half-lives for the hydrolysis of complexes C5 and C13 in various strengths of NaClO4. [NaClO4] [m] 0.015 0.1 0.25 0.5 kobs [10@3 s@1] 1.38 : 0.001 1.41 : 0.005 1.35 : 0.002 1.39 : 0.002 t1/2,obs [min] 8.4 8.2 8.5 8.3 C13 kobs [10@3 s@1] 1.67 : 0.005 1.79 : 0.005 1.63 : 0.006 1.64 : 0.004 t1/2,obs [min] 6.9 6.5 7.1 7.0 C5 D2O. The exchangeable protons present in the complexes disappeared immediately after the addition of D2O. Further analysis of the 1H NMR spectra revealed rapid hydrolysis of the complexes, leading to a mixture of compounds with arene loss (that is, products in which the three coordination sites originally occupied by the arene and chloride ligands were occupied by solvent molecules). Separate sets of peaks were observed for the chloride, aqua and arene-loss species (Figure 7). The loss of the arene was inferred by the presence of resonances for free p-cymene (d & 7.23 ppm (dd)) and benzene (d & 7.32 ppm (s)) in the aromatic region for C5 and C13, respectively. Almost 85 % of the complexes were hydrolyzed within 30 min, and the major hydrolyzed product constituted 75 % of the products. The equilibria appeared to be reached quickly, 7423 T 2021 Wiley-VCH GmbH Chemistry—A European Journal Full Paper doi.org/10.1002/chem.202004954 Figure 7. 1H NMR spectra of C5 (bottom) and C13 (top) in 3:7 [D6]DMSO/D2O at 298 K recorded over a period of 24 h. Peak assignments: a, exchangeable protons of intact chloride complex; b, intact chloride complex; c, predominant hydrolyzed species; d, free arene; *, other aquated/hydrolyzed species. because the spectra recorded after 12 and 24 h showed insignificant changes in comparison with those recorded after 1 h. After 24 h, the original complex species accounted for less than 10 % of species, and arene loss of around 7 and 27 % was observed for C5 and C13, respectively (based on peak integrals).[43–46] Chem. Eur. J. 2021, 27, 7418 – 7433 www.chemeurj.org ESI-MS analysis of the complexes in 3:7 (v/v) DMSO/H2O after 24 h revealed the mononuclear [Ru(h6-p-cymene)(L5)(Cl)(H2O)] + to be the major hydrolysis species deriving from C5. However, the hydrolysis product of C13 was observed to be a dimer, [{Ru(h6-benzene)}2(L6)(OH)2(H2O)]2 + , formed along with other minor products (see Figures S78 and S79 in the Support- 7424 T 2021 Wiley-VCH GmbH Chemistry—A European Journal Full Paper doi.org/10.1002/chem.202004954 ing Information). This might be because benzene as an arene ligand is known to exhibit a strong trans labilizing effect on aqua ligands.[44] The rate of hydrolysis of C13 was higher than that of C5, although the more electron-accepting arene ligand in the former should lower the hydrolysis rate. However, the electron-withdrawing CF3 substituent in C13 weakens the donor ability of the ligand, thereby leading to an increase in the hydrolysis rate. In summary, the hydrolysis rates are tunable within this family of ruthenium–arene complexes, which is potentially useful in the design of anticancer drugs.[37] Binding with GSH The tripeptide glutathione (g-l-Glu-l-Cys-Gly; GSH) is an abundant (mm) intracellular thiol that is known to cause the detoxification of heavier transition-metal ions, including several platinum and ruthenium anticancer compounds, which have an affinity towards sulfur.[47] However, Wang et al. reported a possibly conflicting role of GSH in the mechanism of action of RuII– arene complexes, which may contribute to the lack of cross-resistance for platinum-based therapeutics.[48, 49] The interaction of complexes C5 and C13 with GSH was studied by 1H NMR spectroscopy; the spectra were recorded over a period of 24 h, after the addition of a known amount of the respective complex in [D6]DMSO to a 10-fold excess concentration of GSH in D2O (see Figure S80 in the Supporting Information). The experimental conditions were the same as used in the solution stability studies. The complexes immediately bound to GSH, as indicated by the observed spectral changes in comparison with the spectrum of free GSH. This could have an important impact on the biological activity of the RuII–arene complexes.[50] After 12 h, new peaks attributable to the oxidized product GSSG arose in the spectra of the complexes.[51] However, the intensity of these signals was lower in the spectrum of C5 than in the spectrum of C13. This is perhaps not surprising because the electron-donating groups attached to furoylthiourea decreases its ability to oxidize GSH.[52] On the basis of these studies, it is plausible that these complexes act as catalysts for the oxidation of GSH to GSSG. This also could explain why the Ru–p-cymene and Ru–benzene complexes show reverse activity on varying the substituents. As benzene is already a good p-acceptor ligand, the addition of electron-withdrawing group(s) to the ligand makes the metal ion less electron-dense, which makes it more susceptible to bind to GSH, and may reduce the level of GSH/GSSG in the cell, and hence promote apoptosis. However, the methyl and isopropyl groups in p-cymene tend to decrease its p-acidic properties, so the electron-withdrawing substituent in the ligand does not have much influence. Nevertheless, the presence of electron-donating groups in the ligand may increase the p-electron cloud of the planar rings, and subsequently the intercalation and distortion of DNA, leading to apoptosis. Cell death pathway Complexes C5 and C13 were subjected to bright-field microscopy, intracellular reactive oxygen species (ROS), mitochondrial Chem. Eur. J. 2021, 27, 7418 – 7433 www.chemeurj.org membrane potential (MMP), DNA damage and caspase-3 analyses to further elucidate their mechanism of action on IMR-32 cancer cells. Bright-field microscopy was preliminarily employed to explore the micromorphology of the cells in the presence and absence of the complexes. Cisplatin was employed as a positive control. The control cells were seen to be healthy, with cell bodies and dendrites characteristic of neuronal cells. On treating the IMR-32 cells with IC50 and IC90 concentrations of the complexes, changes such as the leakage of cellular debris, damage to the cellular membrane and the formation of apoptotic bodies were visualized (Figure 8).[19] Cancer cells, which are metabolically altered, have higher ROS levels than normal cells. Therefore, they tend to reach the ROS threshold faster and are at a greater risk of undergoing apoptosis. Hence, a complex that can further promote ROS production in a cancer cell is probably an excellent drug candidate to induce cancer cell death. Keeping this in mind, the ROS levels in IMR-32 cells in the presence and absence of the complexes were measured by using the dichlorodihydrofluorescein diacetate (DCFH-DA) staining assay (Figure 9).[53–55] Cisplatin was used as a positive control. An increase in fluorescence intensity was observed when the cancer cells were treated with IC50 and IC90 concentrations of the complexes as compared with the control cells. The fluorescence intensity was higher with the IC90 concentration for both complexes. Notably, the complexes showed dose-dependent generation of ROS (see Figure S81 in the Supporting Information). Figure 8. Bright-field microscopic images. (A) IMR-32 control cells. Cells treated with IC50 and IC90 concentrations of (B,C) cisplatin, (D,E) C5 and (F,G) C13, respectively. The arrows indicate the leakage of cellular debris, damage to the cellular membrane and the formation of apoptotic bodies. 7425 T 2021 Wiley-VCH GmbH Chemistry—A European Journal Full Paper doi.org/10.1002/chem.202004954 Figure 9. DCFH-DA-stained images. (A) IMR-32 control cells. Cells treated with IC50 and IC90 concentrations of (B,C) cisplatin, (D,E) C5 and (F,G) C13, respectively. *GFP = green fluorescent product. Having proved that the complexes generate ROS species in vitro, we next carried out the MMP analysis. A crucial step in intrinsic apoptosis is the increase in the permeability of the mitochondria, which results in a dramatic loss of its electrical potential, following which cytochrome c may be released.[19, 56] Rhodamine 123 (3,6-diamino-9-[2-(methoxycarbonyl)phenyl]xanthylium chloride) is a cationic dye used to measure MMP. The green fluorescence of rhodamine 123 should decrease after the addition of a complex, which means that the electrical potential of the mitochondria has reduced. This was observed when the cancer cells were treated with both the complexes and the stain (Figure 10). Again, a dose-dependent de- crease in the fluorescence intensity was observed. Also, the fluorescence was lower with the IC90 concentration of the complexes (see Figure S81 in the Supporting Information). After the loss of MMP, which may have led to the release of cytochrome c, the next step in the intrinsic apoptosis pathway would be a cascade of caspases. Cytochrome c is known to activate caspase-9 by promoting nucleotide binding to apoptotic protein activating factor-1 (APAF-1), which in turn leads to the activation of caspase-3. Hence, caspase-3 analysis was carried out to prove the overexpression of caspases, which would confirm the apoptotic process. We observed a dose-dependent increase in caspase-3 for C5 and C13, which indicates that the apoptotic process might have occurred in the IMR-32 cancer cell line.[57] 4’,6-Diamidino-2-phenylindole (DAPI) is a wellknown bright-blue fluorescent, the intensity of which increases 20-fold on binding to double-stranded DNA preferentially at adenine–thymine (A-T) abundant zones. The fluorescence intensity exhibited by the cells treated with IC50 and IC90 concentrations of the complexes was higher than that exhibited by the control cells, which evidences conclusively that the complexes induced DNA damage and nuclear leakage in the IMR32 cancer cells (Figure 11 and Figure S81 in the Supporting Information).[58, 59] Figure 11. DAPI-stained images. (A) IMR-32 control cells. Cells treated with IC50 and IC90 concentrations of (B,C) cisplatin, (D,E) C5 and (F,G) C13, respectively. Cell cycle arrest Figure 10. Rhodamine 123-stained images. (A) IMR-32 control cells. Cells treated with IC50 and IC90 concentrations of (B,C) cisplatin, (D,E) C5 and (F,G) C13, respectively. Chem. Eur. J. 2021, 27, 7418 – 7433 www.chemeurj.org A cell that undergoes division renews itself in four phases, denoted G0-G1, S, G2 and M. The amount of DNA in a cell over varying periods can be determined by cell cycle analysis. The IMR-32 cell line was subjected to such analysis for 24 h following the addition of complexes C5 and C13 at their IC50 concentration, with cisplatin being used as a positive control.[59] The cell cycle diagrams depict the progression of the cell from G0-G1 to the G2-M phase (Figure 12). The percentages of 7426 T 2021 Wiley-VCH GmbH Chemistry—A European Journal Full Paper doi.org/10.1002/chem.202004954 Figure 13. Flow cytometric analysis of (A) control cells and cells treated with the IC50 concentration of (B) cisplatin, (C) C5 and (D) C13. early apoptosis and viable cells are limited to each quadrant. It can be seen that cisplatin achieved an apoptosis rate of 10.75 % (4.91 % of early apoptosis and 5.84 % of late apoptosis), whereas C5 and C13 achieved 10.08 % (3.20 % of early apoptosis and 6.88 % of late apoptosis) and 14.17 % of apoptosis (4.78 % of early apoptosis and 9.39 % of late apoptosis), respectively. Although both complexes gave comparable percentages of early and late apoptotic cells to those of cisplatin, C13 exhibited better activity than the other two. These results confirm that the complexes prompted cancer cell death through an apoptotic pathway.[59] Figure 12. Cell cycle analysis of (A) control cells and cells treated with the IC50 concentration of (B) cisplatin, (C) C5 and (D) C13. (E) Comparison of the percentages of cell cycle phases of cells treated with the complexes. the G0-G1, S and G2-M phases in the control were found to be 74.86, 18.63 and 6.52 %, respectively. When the cells were treated with the IC50 concentration of the complexes, the percentages decreased for G0-G1 and increased for the S and G2-M phases with respect to the control. It was observed that the complexes showed comparable activity to that of cisplatin. Notably, the changes in the percentages of cells in the S and G2M phases on going from the control to the complexes were almost two- and four-fold, respectively, which indicates that the complexes mainly arrest the cell cycle in the latter phases of the process. Apoptotic mode of cell death Cellular apoptosis of C5 and C13 was studied at their IC50 concentration, with cisplatin as a positive control. As depicted in Figure 13, clockwise from top left, dead cells, late apoptosis, Chem. Eur. J. 2021, 27, 7418 – 7433 www.chemeurj.org Interaction with protein receptors Meggers and co-workers described how the unique shapes of metal complexes can be exploited for targeting enzymes and proteins. The RuII–arene complexes are known to exert a “multi-targeted” approach, that is, not only do they target DNA, but they also contain vectors to enable them to target cancer cells selectively and/or moieties that target enzymes, peptides and intracellular proteins.[60, 61] The Pim-1 kinase receptor has direct involvement in the regulation of cell cycle progression and apoptosis, and is overexpressed in various cancers such as prostate cancer, Burkitt’s lymphoma and oral cancer, in addition to several hematopoietic lymphomas, whereas its deficiency leads to failure in cell survival and growth.[62] The principal responder to the vascular endothelial growth factor signal, which regulates endothelial migration and proliferation, is vascular endothelial growth factor receptor 2 (VEGFR2).[63] The VEGFR2 receptor has been reported to be 7427 T 2021 Wiley-VCH GmbH Chemistry—A European Journal Full Paper doi.org/10.1002/chem.202004954 expressed in carcinomas and lymphomas, in addition to its expression in endothelial cells and vascular tumors, which makes it a crucial cell type marker. Hence, these two receptors were chosen for this study to explore the possible interactions between complexes C5 and C13 and the receptors. The optimized geometries of complexes C5 and C13 were used to perform molecular docking experiments with the Pim-1 kinase and VEGFR2 receptors (Figure 14). Figure 14. Molecular docking images for C5 and C13. The complexes formed hydrogen-bonding interactions with the surrounding amino acid residues of Pim-1 kinase, as well as strong electrostatic attractions, namely p–alkyl, p–p and p– sulfur interactions, at the binding site. The presence of the metal ion and high-affinity atoms such as chlorine contributed to the higher interaction energies of C5. Hydrogen-bonding interactions were also observed with residues of VEGFR2, along with strong alkyl and p–alkyl interactions at the active binding sites of the receptor. All the interactions of the complexes with the individual proteins are listed in Table 4. The high binding energy and presence of electrostatic energy clouds in the binding sites will lead to structural changes in the receptors, which inhibit the functions of the proteins. Conclusions Fourteen new RuII–p-cymene/benzene complexes C1–C14 have been synthesized and characterized by UV/Vis, FTIR and NMR spectroscopy as well as by mass spectrometry. The pseudo-octahedral geometries of the RuII–arene complexes were confirmed by a single-crystal XRD study, which revealed that the Ru@S bond weakened as the phenyl ring moved away from the furoylthiourea core or when there was an electrondonating group on the terminal N. On the other hand, an electron-withdrawing group on the terminal N strengthened the coordination bond. Theoretical calculations revealed that changes in the N substituent of the furoylthiourea ligand have a localized effect on the complex system, with minor changes in the ESPs of the chloride ligands. All the complexes were screened for their anticancer potential by means of the MTT assay in five different cancer cell lines and were found to be highly toxic towards the IMR-32 cell line, while showing lower toxicity towards a normal cell line. To study their mechanism of action, complexes C5 and C13 were subjected to stability studies, and a preliminarily investigation showed that the complexes underwent rapid hydrolysis. The reaction followed pseudo-first order kinetics, and the rate for C13 was found to be higher than that for C5. The dominant species obtained from the hydrolysis of the former turned out to be a dinuclear complex, whereas the latter gave a mononuclear complex. Complex C13 also proved to be a better catalyst for the oxidation of GSH to GSSG. A series of staining assays revealed the presence of ROS species, a reduction in the MMP and nuclear damage. Furthermore, the complexes could arrest the cell cycle. Flow cytometry studies confirmed the mechanism of cell death to be apoptotic. Interestingly, the complexes were capable of binding to receptors such as Pim kinase-1 and VEGFR2, which may help to block their functions. Table 4. Interactions of C5 and C13 with the receptors Pim-1 kinase and VEGFR2. C5 Protein receptor Hydrogen-bonding interactions Pim-1 kinase Pro87, Met88, Val91, Leu192, Val90, Trp198, His157, Val91, Cys158, Cys161 Thr24, Ser23, Arg927, Ser1098, Gly1100, Pro1066, Pro1103, Leu1099, Arg930, Arg27 Ser261, Glu262, Cys263, Leu266, Trp269, Ala272, Glue283, Thr280, His287, Ile284 Cys1022, Leu910, Ser882, Met881, Arg1020, Leu1017, Phe1016, Leu828, Pro819, His877, Ile890 VEGFR2 C13 Pim-1 kinase VEGFR2 Chem. Eur. J. 2021, 27, 7418 – 7433 www.chemeurj.org Electrostatic interactions 7428 Binding Energy [kcal mol@1] Leu192, Met88, Leu92, Pro81, Lys94, Val90, Cys161 @10.2 Lys1060, Ala1101, Pro1066, Leu1027, Pro1105 @9.83 Leu152, Trp146, Phe148, Arg145, Met290, His25, Leu266, His265, Trp149 Cys1022, Arg1020, Leu1017, Phe1016, Leu828, Pro819, His877 @8.91 @9.66 T 2021 Wiley-VCH GmbH Chemistry—A European Journal Full Paper doi.org/10.1002/chem.202004954 The study concluded that the anticancer activity of a bifunctional RuII–arene furoylthiourea complex can be tuned by varying the substituent on the N-terminal moiety. Experimental Section Materials and measurements All the chemicals and solvents were of analytical reagent grade and used without further purification. Ligands L1–L5 were synthesized according to the previously reported procedure.[19] Elemental analyses was carried out on a PerkinElmer instrument. UV/Vis spectra were recorded on a Shimadzu UV2600 spectrophotometer. FTIR spectra were recorded as KBr pellets using a Thermo Scientific Nicolet iS5 spectrometer. 1H and 13C NMR spectra were recorded on a Bruker 500 MHz spectrometer. Mass spectra were recorded on a Thermo Exactive Plus UHPLC-MS spectrometer. A Bruker Quest Xray (fixed-Chi geometry) diffractometer was employed for crystal screening, unit cell determination and data collection. The goniometer was controlled using the APEX3 software suite. Olex2 was employed for the final data presentation and structure plots.[64–68] All the other methods related to the kinetic and biological studies are described in the Supporting Information. Deposition Numbers 1986311 (for C1), 1986316 (for C2), 1986315 (for C3), 1986313 (for C4), 1986317 (for C6), 1986310 (for C8), and 1986312 (for C14) contain the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service www.ccdc.cam.ac.uk/ structures. Synthesis of the ligands The ligands were synthesized according to the reported procedure with slight modifications.[15, 16, 19] Furoyl chloride (1.3052 g, 10 mmol) was added dropwise to a solution of potassium thiocyanate (0.971 g, 10 mmol) in acetone (15 mL), and the mixture was heated at reflux for 1 h. After cooling, a solution of the desired amine (0.931–2.29 g, 10 mmol; aniline (L1), benzylamine (L2), 2-phenylethanamine (L3), 2,6-diethylaniline (L4), 2,4,6-trimethylaniline (L5), 4-(trifluoromethyl)aniline (L6) or 3,5-bis(trifluoromethyl)aniline (L7)) in acetone (10 mL) was added dropwise to the mixture, which was stirred at room temperature for 3 h. The resulting solution was poured into 1 N HCl to neutralize any excess amine, and the solid product was filtered and washed with water to remove any traces of KCl. All the ligands were obtained in good yields of 80–90 %. N-{[4-(Trifluoromethyl)phenyl]carbamothioyl}furan-2-carboxamide (L6) Yield: 82 %; m.p. 135 8C; colorless; 1H NMR (500 MHz, [D6]DMSO): d = 12.50 (s, 1 H; OC-NH), 11.45 (s, 1 H; SC-NH), 8.10 (s, 1 H; aromatic), 7.95 (d, J = 8.3 Hz, 2 H; aromatic), 7.88 (d, J = 3.0 Hz, 1 H; aromatic), 7.79 (d, J = 8.4 Hz, 2 H; aromatic), 6.78 ppm (dd, J = 3.5, 1.7 Hz, 1 H; aromatic); 13C NMR (125 MHz, [D6]DMSO): d = 179.5 (C= S), 157.9 (C=O), 149.0, 145.1, 142.1, 126.8, 125.1, 123.5, 119.3, 113.2 (aromatic), 126.3, 126.3, 126.2, 126.2 ppm (CF3); 19F NMR (471 MHz, [D6]DMSO): d = 60.7 ppm; UV/Vis (CHCl3): lmax (e) = 285 (20 094), 310 nm (8333 dm3 mol@1 cm@1); FTIR (KBr): ñ = 3240 (m, n(thioamide N@H)), 3027 (m, n(amide N@H)), 1672 (s, n(C=O)), 1230 cm@1 (s, n(C=S)); elemental analysis calcd (%) for C13H9F3N2O2S (314.0336): C 49.68, H 2.89, N 8.91, S 10.20; found: C 49.81, H 2.74, N 8.99, S 10.32. Chem. Eur. J. 2021, 27, 7418 – 7433 www.chemeurj.org N-{[3,5-Bis(trifluoromethyl)phenyl]carbamothioyl}furan-2carboxamide (L7) Yield: 86 %; m.p. 161 8C; colorless; 1H NMR (500 MHz, [D6]DMSO): d = 12.53 (s, 1 H; OC-NH), 11.62 (s, 1 H; SC-NH), 8.43 (s, 2 H; aromatic), 8.10 (s, 1 H; aromatic), 7.98 (s, 1 H; aromatic), 7.90 (d, J = 3.5 Hz, 1 H; aromatic), 6.78 ppm (dd, J = 3.6, 1.8 Hz, 1 H; aromatic); 13 C NMR (125 MHz, CDCl3): d = 178.4 (C=S), 156.9 (C=O), 146.8, 144.5, 139.1, 132.4, 132.1, 123.8, 119.7, 113.6 (aromatic), 120.0, 120.0, 120.0, 119.9 ppm (CF3); 19F NMR (471 MHz, [D6]DMSO): d = 61.5 ppm; UV/Vis (CHCl3): lmax (e) = 286 (24 620), 317 nm (7963 dm3 mol@1 cm@1); FTIR (KBr): ñ = 3271 (m, n(thioamide N@H)), 3117 (m, n(amide N@H)), 1677 (s, n(C=O)), 1230 cm@1 (s, n(C=S)); elemental analysis calcd (%) for C14H8F6N2O2S (382.0210): C 43.99, H 2.11, N 7.33, S 8.39; found: C 43.75, H 2.27, N 7.21, S 8.46. Synthesis of the Ru–p-cymene and Ru–benzene complexes C1–C14 The metal precursors, namely Ru–p-cymene and Ru–benzene dimers, required for the preparation of the complexes, were prepared according to the reported procedures.[17–19] To prepare the complexes, the metal precursor (0.1 mmol, 500–612 mg) was added to toluene (10 mL) and the mixture stirred for a few minutes. A few drops of methanol were then added to promote the solubility of the precursor.[19] A solution of the ligand (0.2 mmol, 493–765 mg) in toluene (10 mL) was then added dropwise. The resulting mixture was stirred for 4 h to achieve completion of the reaction, after which it was reduced in volume, and the solid product was obtained by the addition of hexane. The solid was then filtered and washed with hexane. The complexes were obtained in good yields of 70–79 %. [Dichloro(p-cymene){N-(phenylcarbamothioyl)furan-2-carboxamide}ruthenium(II)] (C1) L1 (49 mg, 0.2 mmol) and [RuCl2(p-cymene)]2 (61 mg, 0.1 mmol) were used. Yield: 75 %; m.p. 180 8C; orange; 1H NMR (500 MHz, [D6]DMSO): d = 12.36 (s, 1 H; OC-NH), 11.28 (s, 1 H; SC-NH), 8.08 (d, J = 1.0 Hz, 1 H; aromatic), 7.86 (d, J = 3.5 Hz, 1 H; aromatic), 7.67 (d, J = 7.8 Hz, 2 H; aromatic), 7.42 (q, J = 7.9 Hz, 2 H; aromatic), 7.27 (t, J = 7.4 Hz, 1 H; aromatic), 6.77 (dd, J = 3.6, 1.7 Hz, 1 H; aromatic), 5.82 (d, J = 6.3 Hz, 2 H; aromatic-H of p-cymene), 5.78 (d, J = 6.3 Hz, 2 H; aromatic-H of p-cymene), 2.86–2.81 (m, 1 H; CH(CH3)2 of pcymene), 2.09 (s, 3 H; C-CH3 of p-cymene), 1.19 ppm (d, J = 6.9 Hz, 6 H; CH(CH3)2 of p-cymene); 13C NMR (125 MHz, [D6]DMSO): d = 179.1 (C=S), 158.0 (C=O), 148.9, 145.1, 138.4, 129.1, 126.8, 124.8, 119.1, 113.1 (aromatic), 106.8, 100.5, 86.8, 85.9 (aromatic-C of pcymene), 30.4, 21.9, 18.3 ppm (aliphatic-C of p-cymene); UV/Vis (CHCl3): lmax (e) = 288 (22 580), 366 (6082), 433 nm (1290 dm3 mol@1 cm@1); FTIR (KBr): ñ = 3232 (m, n(thioamide N@H)), 3171 (m, n(amide N@H)), 1678 (s, n(C=O)), 1178 cm@1 (s, n(C=S)); MS (ESI): m/z calcd for [C22H23N2O2RuS] + : 481.0524 [M @ 2 H + @ 2 Cl@ + H + ] + ; found: 481.0538; elemental analysis calcd (%) for C22H24Cl2N2O2RuS: C 47.83, H 4.38, N 5.07, S 5.80; found: C 47.98, H 4.51, N 5.21, S 5.61. [Dichloro(p-cymene){N-(benzylcarbamothioyl)furan-2-carboxamide}ruthenium(II)] (C2) L2 (52 mg, 0.2 mmol) and [RuCl2(p-cymene)]2 (61 mg, 0.1 mmol) were used. Yield: 72 %; m.p. 196 8C; orange; 1H NMR (500 MHz, CDCl3): d = 11.23 (s, 1 H; OC-NH), 10.86 (s, 1 H; SC-NH), 7.93 (d, J = 3.4 Hz, 1 H; aromatic), 7.60 (s, 1 H; aromatic), 7.41–7.32 (m, 5 H; aro- 7429 T 2021 Wiley-VCH GmbH Chemistry—A European Journal Full Paper doi.org/10.1002/chem.202004954 matic), 6.51–6.48 (m, 1 H; aromatic), 5.47 (d, J = 5.7 Hz, 2 H; aromatic-H of p-cymene), 5.30 (d, J = 5.7 Hz, 2 H; aromatic-H of p-cymene), 4.86 (d, J = 5.0 Hz, 2 H; CH2), 3.06–2.96 (m, 1 H; CH(CH3)2 of pcymene), 2.29 (s, 3 H; C-CH3 of p-cymene), 1.34 ppm (d, J = 6.9 Hz, 6 H; CH(CH3)2 of p-cymene); 13C NMR (125 MHz, CDCl3): d = 179.2 (C=S), 158.7 (C=O), 147.8, 144.6, 134.9, 129.0, 128.3, 127.8, 121.5, 112.6 (aromatic), 103.5, 100.0, 84.2, 82.6 (aromatic-C of p-cymene), 49.5 (CH2), 30.4, 22.2, 18.4 ppm (aliphatic-C of p-cymene); UV/Vis (CHCl3): lmax (e) = 282 (16 383), 323 (4874), 429 nm (1062 dm3 mol@1 cm@1); FTIR (KBr): ñ = 3244 (m, n(thioamide N@H)), 3179 (m, n(amide N@H)), 1679 (s, n(C=O)), 1202 cm@1 (s, n(C=S)); MS (ESI): m/z calcd for [C23H25N2O2RuS] + : 495.0680 [M@2 H + @2 Cl@+H + ] + ; found: 495.0656; elemental analysis calcd (%) for C23H26Cl2N2O2RuS: C 48.76, H 4.63, N 4.95, S 5.66; found: C 48.59, H 4.80, N 4.81, S 5.73. [Dichloro(p-cymene){N-(phenethylcarbamothioyl)furan-2-carboxamide}ruthenium(II)] (C3) L3 (54 mg, 0.2 mmol) and [RuCl2(p-cymene)]2 (61 mg, 0.1 mmol) were used. Yield: 71 %; m.p. 184 8C; orange; 1H NMR (500 MHz, CDCl3): d = 10.97 (s, 1 H; OC-NH), 10.74 (s, 1 H; SC-NH), 7.92 (d, J = 3.2 Hz, 1 H; aromatic), 7.60 (s, 1 H; aromatic), 7.36 (t, J = 7.3 Hz, 2 H; aromatic), 7.26 (d, J = 7.6 Hz, 3 H; aromatic), 6.49 (dd, J = 2.7, 1.1 Hz, 1 H; aromatic), 5.46 (d, J = 4.2 Hz, 2 H; aromatic-H of p-cymene), 5.29 (d, J = 3.6 Hz, 2 H; aromatic-H of p-cymene), 3.92 (dd, J = 12.6, 7.6 Hz, 2 H; HN-CH2CH2), 3.03 (t, J = 7.4 Hz, 2 H; HN-CH2CH2), 2.29 (s, 3 H; C-CH3 of p-cymene), 1.77–1.53 (m, 1 H; CH(CH3)2 of p-cymene), 1.35 ppm (d, J = 6.4 Hz, 6 H; CH(CH3)2 of p-cymene); 13C NMR (125 MHz, CDCl3): d = 179.0 (C=S), 158.6 (C=O), 147.7, 144.6, 137.4, 128.9, 128.7, 127.0, 121.4, 112.6 (aromatic), 103.4, 99.9, 84.2, 82.6 (aromatic-C of p-cymene), 46.9, 34.4 (CH2CH2), 30.4, 22.2, 18.3 ppm (aliphatic-C of p-cymene); UV/Vis (CHCl3): lmax (e) = 282 (21 313), 326 (6332), 428 nm (1351 dm3 mol@1 cm@1); FTIR (KBr): ñ = 3218 (m, n(thioamide N@H)), 3169 (m, n(amide N@H)), 1668 (s, n(C=O)), 1197 cm@1 (s, n(C=S)); MS (ESI): m/z calcd for [C24H27N2O2RuS] + : 509.0836 [M@2 H + @2 Cl@+H + ] + ; found: 509.0740; elemental analysis calcd (%) for C24H28Cl2N2O2RuS: C 49.52, H 4.95, N 4.77, S 5.46; found: C 49.66, H 4.83, N 4.92, S 5.60. [Dichloro(p-cymene)(N-{(2,6-diethylphenyl)carbamothioyl}furan-2-carboxamide)ruthenium(II)] (C4) L4 (60 mg, 0.2 mmol) and [RuCl2(p-cymene)]2 (61 mg, 0.1 mmol) were used. Yield: 72 %; m.p. 210 8C; orange; 1H NMR (500 MHz, CDCl3): d = 12.08 (s, 1 H; OC-NH), 11.17 (s, 1 H; SC-NH), 8.05 (d, J = 4.2 Hz, 1 H; aromatic), 7.65 (s, 1 H; aromatic), 7.36 (t, J = 8.1 Hz, 1 H; aromatic), 7.21 (d, J = 8.3 Hz, 2 H; aromatic), 6.54 (dd, J = 3.8, 1.5 Hz, 1 H; aromatic), 5.32 (d, J = 6.5 Hz, 2 H; aromatic-H of p-cymene), 5.21 (d, J = 6.5 Hz, 2 H; aromatic-H of p-cymene), 2.89–2.79 (m, 1 H; CH(CH3)2 of p-cymene), 2.67–2.58 (m, 4 H; CH2CH3), 2.21 (s, 3 H; CCH3 of p-cymene), 1.25–1.19 ppm (m, 12 H; CH(CH3)2 of p-cymene, CH2CH3); 13C NMR (125 MHz, CDCl3): d = 181.5 (C=S), 159.0 (C=O), 147.9, 144.7, 141.3, 133.32, 129.1, 126.6, 121.9, 112.7 (aromatic), 103.0, 99.8, 83.8, 82.8 (aromatic-C of p-cymene), 24.7, 14.3 (CH2CH3), 30.2, 22.0, 18.2 ppm (aliphatic-C of p-cymene); UV/Vis (CHCl3): lmax (e) = 283 (17 553), 342 (4645), 428 nm (1099 dm3 mol@1 cm@1); FTIR (KBr): ñ = 3215 (m, n(thioamide N@H)), 3129 (m, n(amide N@H)), 1664 (s, n(C=O)), 1168 cm@1 (s, n(C=S)); MS (ESI): m/z calcd for [C26H31N2O2RuS] + : 537.1149 [M@2 H + @2 Cl@+H + ] + ; found: 537.1052; elemental analysis calcd (%) for C26H32Cl2N2O2RuS: C 51.31, H 5.30, N 4.60, S 5.27; found: C 51.45, H 5.18, N 4.72, S 5.45. Chem. Eur. J. 2021, 27, 7418 – 7433 www.chemeurj.org [Dichloro(p-cymene){N-(mesitylcarbamothioyl)furan-2-carboxamide}ruthenium(II)] (C5) L5 (57 mg, 0.2 mmol) and [RuCl2(p-cymene)]2 (61 mg, 0.1 mmol) were used. Yield: 76 %; m.p. 197 8C; orange; 1H NMR (500 MHz, [D6]DMSO): d = 11.60 (s, 1 H; OC-NH), 11.30 (s, 1 H; SC-NH), 8.07 (d, J = 1.5 Hz, 1 H; aromatic), 7.86 (d, J = 3.6 Hz, 1 H; aromatic), 6.93 (s, 2 H; aromatic), 6.77 (dd, J = 3.6, 1.7 Hz, 1 H; aromatic), 5.82 (d, J = 6.3 Hz, 2 H; aromatic-H of p-cymene), 5.78 (d, J = 6.3 Hz, 2 H; aromatic-H of p-cymene), 2.90–2.76 (m, 1 H; CH(CH3)2 of p-cymene), 2.25 (s, 3 H; C-CH3 of p-cymene), 2.14 (s, 6 H; o-CH3), 2.09 ppm (s, 3 H; p-CH3), 1.20 ppm (d, J = 6.9 Hz, 6 H; CH(CH3)2 of p-cymene); 13 C NMR (125 MHz, [D6]DMSO): d = 180.4 (C=S), 157.9 (C=O), 148.8, 145.2, 137.0, 135.1, 134.1, 129.3, 118.9, 113.1 (aromatic), 106.8, 100.5, 86.8, 85.9 (aromatic-C of p-cymene), 30.4, 21.9, 18.2 (aliphatic-C of p-cymene), 21.0, 18.3 ppm (o,p-CH3); UV/Vis (CHCl3): lmax (e) = 285 (15 823), 346 (7685), 433 nm (1062 dm3 mol@1 cm@1); FTIR (KBr): ñ = 3226 (m, n(thioamide N@H)), 3115 (m, n(amide N@H)), 1671 (s, n(C=O)), 1184 cm@1 (s, n(C=S)); MS (ESI): m/z calcd for [C25H29N2O2RuS] + : 523.0993 [M@2 H + @2 Cl@+H + ] + ; found: 523.1011; elemental analysis calcd (%) for C25H30Cl2N2O2RuS: C 50.50, H 5.09, N 4.71, S 5.39; found: C 50.67, H 5.24, N 4.54, S 5.51. [Dichloro(p-cymene){N-({4-(trifluoromethyl)phenyl}carbamothioyl)furan-2-carboxamide}ruthenium(II)] (C6) L6 (62 mg, 0.2 mmol) and [RuCl2(p-cymene)]2 (61 mg, 0.1 mmol) were used. Yield: 79 %; m.p. 181 8C; orange; 1H NMR (500 MHz, [D6]DMSO): d = 12.50 (s, 1 H; OC-NH), 11.46 (s, 1 H; SC-NH), 8.10 (s, 1 H; aromatic), 7.95 (d, J = 8.3 Hz, 2 H; aromatic), 7.88 (d, J = 3.5 Hz, 1 H; aromatic), 7.79 (d, J = 8.3 Hz, 2 H; aromatic), 6.78 (dd, J = 3.9, 1.8 Hz, 1 H; aromatic), 5.82 (d, J = 6.2 Hz, 2 H; aromatic-H of pcymene), 5.78 (d, J = 6.1 Hz, 2 H; aromatic-H of p-cymene), 2.89– 2.78 (m, 1 H; CH(CH3)2 of p-cymene), 2.09 (s, 3 H; C-CH3 of pcymene), 1.20 ppm (d, J = 6.9 Hz, 6 H; CH(CH3)2 of p-cymene); 13 C NMR (125 MHz, [D6]DMSO): d = 179.5 (C=S), 157.9 (C=O), 149.0, 145.1, 142.1, 126.8, 125.1, 123.4, 119.3, 113.1 (aromatic), 126.3, 126.2, 126.2, 126.2 (CF3), 106.8, 100.5, 86.8, 85.9 (aromatic-C of pcymene), 30.4, 21.9, 18.3 ppm (aliphatic-C of p-cymene); 19F NMR (471 MHz, [D6]DMSO): d = 60.7 ppm; UV/Vis (CHCl3): lmax (e) = 291 (15 780), 359 (3561), 428 nm (849 dm3 mol@1 cm@1); FTIR (KBr): ñ = 3149 (m, n(thioamide N@H)), 3031 (m, n(amide N@H)), 1678 (s, n(C= O)), 1172 cm@1 (s, n(C=S)); MS (ESI): m/z calcd for [C23H22F3N2O2RuS] + : 549.0397 [M@2 H + @2 Cl@+H + ] + ; found: 549.0314; elemental analysis calcd (%) for C23H23Cl2F3N2O2RuS: C 44.52, H 3.74, N 4.51, S 5.17; found: C 44.69, H 3.90, N 4.37, S 5.34. [Dichloro(p-cymene){N-({3,5-bis(trifluoromethyl)phenyl}carbamothioyl)furan-2-carboxamide}ruthenium(II)] (C7) L7 (76 mg, 0.2 mmol) and [RuCl2(p-cymene)]2 (61 mg, 0.1 mmol) were used. Yield: 77 %; m.p. 185 8C; orange; 1H NMR (500 MHz, [D6]DMSO): d = 12.51 (s, 1 H; OC-NH), 11.64 (s, 1 H; SC-NH), 8.43 (s, 2 H; aromatic), 8.11 (d, J = 0.9 Hz, 1 H; aromatic), 8.01 (s, 1 H; aromatic), 7.90 (d, J = 3.5 Hz, 1 H; aromatic), 6.79 (dd, J = 3.6, 1.6 Hz, 1 H; aromatic), 5.83 (d, J = 6.2 Hz, 2 H; aromatic-H of p-cymene), 5.79 (d, J = 6.2 Hz, 2 H; aromatic-H of p-cymene), 2.89–2.79 (m, 1 H; CH(CH3)2 of p-cymene), 2.09 (s, 3 H; C-CH3 of p-cymene), 1.20 ppm (d, J = 6.9 Hz, 6 H; CH(CH3)2 of p-cymene); 13C NMR (125 MHz, [D6]DMSO): d = 180.4 (C=S), 157.7 (C=O), 149.2, 145.0, 140.6, 130.9, 130.6, 124.6, 119.4, 113.2 (aromatic), 120.0, 119.9 (CF3), 106.8, 100.5, 86.8, 85.9 (aromatic-C of p-cymene), 30.4, 21.9, 18.3 ppm (aliphaticC of p-cymene); 19F NMR (471 MHz, [D6]DMSO): d = 61.4 ppm; UV/ Vis (CHCl3): lmax (e) = 292 (19 260), 364 (4260), 429 nm 7430 T 2021 Wiley-VCH GmbH Chemistry—A European Journal Full Paper doi.org/10.1002/chem.202004954 (985 dm3 mol@1 cm@1); FTIR (KBr): ñ = 3228 (m, n(thioamide N@H)), 3118 (m, n(amide N@H)), 1683 (s, n(C=O)), 1180 cm@1 (s, n(C=S)); MS (ESI): m/z calcd for [C24H21F6N2O2RuS] + : 617.0271 [M@2 H + @2 Cl@+H + ] + ; found: 617.0297; elemental analysis calcd (%) for C24H22Cl2F6N2O2RuS: C 41.87, H 3.22, N 4.07, S 4.66; found: C 41.68, H 3.41, N 4.20, S 4.85. [Dichloro(benzene){N-(phenylcarbamothioyl)furan-2-carboxamide}ruthenium(II)] (C8) L1 (49 mg, 0.2 mmol) and [RuCl2(benzene)]2 (50 mg, 0.1 mmol) were used. Yield: 74 %; m.p. 250 8C; orange; 1H NMR (500 MHz, [D6]DMSO): d = 12.34 (s, 1 H; OC-NH), 11.27 (s, 1 H; SC-NH), 8.07 (d, J = 1.0 Hz, 1 H; aromatic), 7.85 (d, J = 3.6 Hz, 1 H; aromatic), 7.65 (d, J = 8.9 Hz, 2 H; aromatic), 7.41 (t, J = 8.1 Hz, 2 H; aromatic), 7.26 (t, J = 7.6 Hz, 1 H; aromatic), 6.75 (dd, J = 3.8, 1.8 Hz, 1 H; aromatic), 5.96 ppm (s, 6 H; aromatic-H of benzene); 13C NMR (125 MHz, [D6]DMSO): d = 179.1 (C=S), 158.0 (C=O), 148.9, 145.1, 138.4, 129.1, 126.8, 124.8, 119.1, 113.1 (aromatic), 88.1 ppm (aromatic-C of benzene); UV/Vis (CHCl3): lmax (e) = 289 (25 888), 362 (6380), 424 nm (1500 dm3 mol@1 cm@1); FTIR (KBr): ñ = 3229 (m, n(thioamide N@H)), 3159 (m, n(amide N@H)), 1672 (s, n(C=O)), 1182 cm@1 (s, n(C=S)); MS (ESI): m/z calcd for [C18H15N2O2RuS] + : 424.9898 [M @ 2 H + @ 2 Cl@ + H + ] + ; found: 424.9902; elemental analysis calcd (%) for C18H16Cl2N2O2RuS: C 43.56, H 3.25, N 5.64, S 6.46; found: C 43.71, H 3.04, N 5.42, S 6.64. [Dichloro(benzene){N-(benzylcarbamothioyl)furan-2-carboxamide}ruthenium(II)] (C9) L2 (52 mg, 0.2 mmol) and [RuCl2(benzene)]2 (50 mg, 0.1 mmol) were used. Yield: 70 %; m.p. 242 8C; orange; 1H NMR (500 MHz, CDCl3): d = 11.06 (s, 1 H; OC-NH), 10.93 (s, 1 H; SC-NH), 7.97 (d, J = 1.6 Hz, 1 H; aromatic), 7.73 (d, J = 3.5 Hz, 1 H; aromatic), 7.30 (s, 2 H; aromatic), 7.29 (d, J = 3.3 Hz, 2 H; aromatic), 7.22 (dt, J = 8.2, 4.1 Hz, 1 H; aromatic-H), 6.66 (dd, J = 3.4, 1.5 Hz, 1 H; aromatic), 5.90 (s, 6 H; aromatic-H of benzene), 4.79 ppm (d, J = 5.7 Hz, 2 H; CH2); 13C NMR (125 MHz, CDCl3): d = 185.3 (C=S), 162.6 (C=O), 149.9, 142.1, 133.5, 132.6, 132.4, 123.4, 123.3, 117.6 (aromatic), 92.8 (aromatic-C of benzene), 53.5 ppm (CH2); UV/Vis (CHCl3): lmax (e) = 284 (18 403), 321 (6525), 425 nm (985 dm3 mol@1 cm@1); FTIR (KBr): ñ = 3222 (m, n(thioamide N@H)), 3131 (m, n(amide N@H)), 1675 (s, n(C=O)), 1201 cm@1 (s, n(C=S)); MS (ESI): m/z calcd for [C19H17N2O2RuS] + : 439.0054 [M@2 H + @2 Cl@+H + ] + ; found: 439.0264; elemental analysis calcd (%) for C19H18Cl2N2O2RuS: C 44.71, H 3.55, N 5.49, S 6.28; found: C 44.53, H 3.70, N 5.65, S 6.10. [Dichloro(benzene){N-(phenethylcarbamothioyl)furan-2-carboxamide}ruthenium(II)] (C10) L3 (54 mg, 0.2 mmol) and [RuCl2(benzene)]2 (50 mg, 0.1 mmol) were used. Yield: 71 %; m.p. 238 8C; orange; 1H NMR (500 MHz, CDCl3): d = 10.99 (s, 1 H; OC-NH), 10.67 (s, 1 H; SC-NH), 7.86 (d, J = 4.2 Hz, 1 H; aromatic), 7.62 (s, 1 H; aromatic), 7.36 (dd, J = 8.8, 5.7 Hz, 2 H; aromatic), 7.30–7.24 (m, 4 H; aromatic), 6.50 (dd, J = 3.9, 1.3 Hz, 1 H; aromatic), 5.73 (s, 6 H; aromatic-H of benzene), 3.94 (dd, J = 12.6, 6.9 Hz, 2 H; HN-CH2CH2), 3.03 ppm (t, J = 7.1 Hz, 2 H; HN-CH2CH2); 13C NMR (125 MHz, CDCl3): d = 178.8 (C=S), 158.5 (C= O), 147.8, 144.5, 137.3, 128.9, 128.7, 127.1, 121.4, 112.7 (aromatic), 85.5 (aromatic-C of benzene), 47.0, 34.4 ppm (CH2CH2); UV/Vis (CHCl3): lmax (e) = 284 (15 191), 322 (6510), 430 nm (936 dm3 mol@1 cm@1); FTIR (KBr): ñ = 3229 (m, n(thioamide N@H)), 3184 (m, n(amide N@H)), 1675 (s, n(C=O)), 1194 cm@1 (s, n(C=S)); MS (ESI): m/z calcd for [C20H19N2O2RuS] + : 453.0211 [M @ 2 H + @ 2 Cl@ + Chem. Eur. J. 2021, 27, 7418 – 7433 www.chemeurj.org H + ] + ; found: 453.0150; elemental analysis calcd (%) for C20H20Cl2N2O2RuS: C 45.81, H 3.84, N 5.34, S 6.11; found: C 45.69, H 3.97, N 5.23, S 6.26. [Dichloro(benzene)(N-{(2,6-diethylphenyl)carbamothioyl}furan-2-carboxamide)ruthenium(II)] (C11) L4 (60 mg, 0.2 mmol) and [RuCl2(benzene)]2 (50 mg, 0.1 mmol) were used. Yield: 73 %; m.p. 220 8C; orange; 1H NMR (500 MHz, CDCl3): d = 12.13 (s, 1 H; OC-NH), 11.12 (s, 1 H; SC-NH), 7.98 (d, J = 3.4 Hz, 1 H; aromatic), 7.66 (s, 1 H; aromatic), 7.37 (t, J = 7.5 Hz, 1 H; aromatic), 7.22 (d, J = 7.6 Hz, 2 H; aromatic), 6.54 (dd, J = 3.6, 1.3 Hz, 1 H; aromatic), 5.62 (s, 6 H; aromatic-H of benzene), 2.66–2.59 (m, 4 H; CH2CH3), 1.22 ppm (t, J = 7.5 Hz, 6 H; CH2CH3); 13C NMR (125 MHz, CDCl3): d = 181.1 (C=S), 159.0 (C=O), 148.1, 144.6, 141.2, 133.0, 129.3, 126.7, 122.0, 112.8 (aromatic), 85.3 (aromatic-C of benzene), 24.8, 14.4 ppm (CH2CH3); UV/Vis (CHCl3): lmax (e) = 284 (18 996), 322 (5501), 428 nm (1003 dm3 mol@1 cm@1); FTIR (KBr): ñ = 3204 (m, n(thioamide N@H)), 3136 (m, n(amide N@H)), 1671 (s, n(C= O)), 1166 cm@1 (s, n(C=S)); MS (ESI): m/z calcd for [C22H23N2O2RuS] + : 481.0524 [M@2 H + @2 Cl@+H + ] + ; found: 481.0395; elemental analysis calcd (%) for C22H24Cl2N2O2RuS: C 47.83, H 4.38, N 5.07, S 5.80; found: C 47.99, H 4.21, N 5.19, S 5.61. [Dichloro(benzene){N-(mesitylcarbamothioyl)furan-2-carboxamide}ruthenium(II)] (C12) L5 (57 mg, 0.2 mmol) and [RuCl2(benzene)]2 (50 mg, 0.1 mmol) were used. Yield: 76 %; m.p. 248 8C; orange; 1H NMR (500 MHz, [D6]DMSO): d = 11.60 (s, 1 H; OC-NH), 11.33 (s, 1 H; SC-NH), 8.08 (s, 1 H; aromatic), 7.87 (d, J = 3.8 Hz, 1 H; aromatic), 6.93 (s, 2 H; aromatic), 6.77 (dd, J = 3.6, 1.6 Hz, 1 H; aromatic), 5.97 (s, 6 H; aromatic-H of benzene), 2.25 (s, 3 H; p-CH3), 2.1 ppm (s, 6 H; o-CH3); 13 C NMR (125 MHz, [D6]DMSO): d = 180.4 (C=S), 157.9 (C=O), 148.8, 145.2, 137.0, 135.1, 134.1, 128.9, 118.9, 113.1 (aromatic), 88.1 (aromatic-C of benzene), 21.0, 18.2 ppm (o,p-CH3); UV/Vis (CHCl3): lmax (e) = 283 (21 285), 326 (6666), 424 nm (1095 dm3 mol@1 cm@1); FTIR (KBr): ñ = 3227 (m, n(thioamide N@H)), 3143 (m, n(amide N@H)), 1676 (s, n(C=O)), 1185 cm@1 (s, n(C=S)); MS (ESI): m/z calcd for [C21H21N2O2RuS] + : 467.0367 [M@2 H + @2 Cl@+H + ] + ; found: 467.0283; elemental analysis calcd (%) for C21H22Cl2N2O2RuS: C 46.84, H 4.12, N 5.20, S 5.95; found: C 46.67, H 4.29, N 5.35, S 5.72. [Dichloro(benzene){N-({4-(trifluoromethyl)phenyl}carbamothioyl)furan-2-carboxamide}ruthenium(II)] (C13) L6 (62 mg, 0.2 mmol) and [RuCl2(benzene)]2 (50 mg, 0.1 mmol) were used. Yield: 77 %; m.p. 241 8C; orange; 1H NMR (500 MHz, [D6]DMSO): d = 12.50 (s, 1 H; OC-NH), 11.46 (s, 1 H; SC-NH), 8.10 (s, 1 H; aromatic), 7.95 (d, J = 8.3 Hz, 2 H; aromatic), 7.88 (d, J = 3.5 Hz, 1 H; aromatic), 7.79 (d, J = 8.4 Hz, 2 H; aromatic), 6.78 (dd, J = 3.6, 1.7 Hz, 1 H; aromatic), 5.98 ppm (s, 6 H; aromatic-H of benzene); 13 C NMR (125 MHz, [D6]DMSO): d = 179.5 (C=S), 157.9 (C=O), 149.0, 145.1, 142.1, 128.7, 126.2, 126.2, 126.2, 126.2 (CF3), 125.1, 123.4, 119.3, 113.1 (aromatic), 88.1 ppm (aromatic-C of benzene); 19F NMR (471 MHz, [D6]DMSO): d = 60.5 ppm; UV/Vis (CHCl3): lmax (e) = 292 (15 326), 344 (4477), 426 nm (882 dm3 mol@1 cm@1); FTIR (KBr): ñ = 3149 (m, n(thioamide N@H)), 3031 (m, n(amide N@H)), 1678 (s, n(C= O)), 1172 cm@1 (s, n(C=S)); MS (ESI): m/z calcd for [C19H14F3N2O2RuS] + : 492.9771 [M@2 H + @2 Cl@+H + ] + ; found: 492.9701; elemental analysis calcd (%) for C19H15Cl2F3N2O2RuS: C 40.44, H 2.68, N 4.96, S 5.68; found: C 40.62, H 2.51, N 4.77, S 5.85. 7431 T 2021 Wiley-VCH GmbH Chemistry—A European Journal Full Paper doi.org/10.1002/chem.202004954 [Dichloro(benzene){N-({3,5-bis(trifluoromethyl)phenyl}carbamothioyl)furan-2-carboxamide}ruthenium(II)] (C14) L7 (76 mg, 0.2 mmol) and [RuCl2(benzene)]2 (50 mg, 0.1 mmol) were used. Yield: 78 %; m.p. 230 8C; orange; 1H NMR (500 MHz, [D6]DMSO): d = 12.36 (s, 1 H; OC-NH), 11.31 (s, 1 H; SC-NH), 8.09 (s, 1 H; aromatic), 7.87 (d, J = 4.2 Hz, 1 H; aromatic), 7.67 (d, J = 9.0 Hz, 2 H; aromatic), 7.28 (t, J = 7.8 Hz, 1 H; aromatic), 6.77 (dd, J = 3.9, 1.8 Hz, 1 H; aromatic), 5.98 ppm (s, 6 H; aromatic-H of benzene); 13 C NMR (125 MHz, [D6]DMSO): d = 179.1 (C=S), 158.0 (C=O), 148.9, 145.1, 138.4, 129.1, 126.8, 124.8, 119.1, 113.1 (aromatic), 88.1 ppm (aromatic-C of benzene); 19F NMR (471 MHz, [D6]DMSO): d = 61.4 ppm; UV/Vis (CHCl3): lmax (e) = 292 (19 476), 349 (4809), 423 nm (952 dm3 mol@1 cm@1); FTIR (KBr): ñ = 3221 (m, n(thioamide N@H)), 3114 (m, n(amide N@H)), 1675 (s, n(C=O)), 1181 cm@1 (s, n(C= S)); MS (ESI): m/z calcd for [C20H13F6N2O2RuS] + : 560.9645 [M@2 H + @2 Cl@+H + ] + ; found: 560.9841; elemental analysis calcd (%) for C20H14Cl2F6N2O2RuS: C 37.99, H 2.23, N 4.43, S 5.07; found: C 38.15, H 2.13, N 4.59, S 5.21. 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