Ru(II)-Arene Complexes of Curcumin and Bisdesmethoxycurcumin Metabolites

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ "This document is the Accepted Manuscript version of a Published Work that appeared in final form in Inorganic Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.inorgchem.4c00970 ." 1 Ru(II)-Arene Complexes of Curcumin and Bisdesmethoxycurcumin Metabolites Noemi Pagliaricci,a Riccardo Pettinari,a* Fabio Marchetti,b Alessia Tombesi,a Sara Pagliaricci,a Massimiliano Cuccioloni,c Agustín Galindo,d Farzaneh Fadaei-Tirani,e Mouna Hadiji,e Paul J. Dyson.e* a School of Pharmacy, bSchool of Science and Technology, and cSchool of Biosciences and Veterinary Medicine, University of Camerino, via Madonna delle Carceri, 62032 Camerino, MC, Italy; dDepartamento de Química Inorgánica, Facultad de Química, Universidad de Sevilla, 41012 Sevilla, Spain; e Institut des Sciences et Ingénierie Chimiques, École Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland. KEYWORDS. Curcumin, Bisdesmethoxycurcumin, Ruthenium complexes, Tetrahydrocurcumin, Bisdesmethoxytetrahydrocurcumin, Bioorganometallic chemistry. 2 ABSTRACT In this decades, curcuminoids are attracting much scientific attention as well as their complexes but only little attention was given to their metabolic derivatives. Here the first examples of (arene)Ru(II) complexes with curcuminoid metabolites, tetrahydrocurcumin (THcurcH) and bisdesmethoxytetrahydrocurcumin (THbdcurcH), have been prepared and characterized. The neutral complexes [Ru(arene)(THcurc)Cl] and [Ru(arene)(THbdcurc)Cl] (arene = cymene, benzene or hexamethylbenzene) were characterized by NMR spectroscopy and ESI mass spectrometry, and the crystal structures of three complexes were determined by X-ray diffraction analysis. Compared to curcuminoids, these metabolites lose their conjugated double bond system responsible for their planarity, showing unique closed conformation structures. Both closed and open conformations have been analyzed and rationalized using density functional theory (DFT). The cytotoxicity of the complexes was evaluated in vitro against human ovarian carcinoma cells (A2780 and A2780cisR), human breast adenocarcinoma cells (MCF-7 and MCF-7CR), as well as against nontumorigenic human embryonic kidney cells (HEK293) and human breast (MCF-10A) cells and compared to the free ligands, cisplatin, and RAPTA-C. There is a correlation between cellular uptake and cytotoxicity of the compounds, suggesting that cellular uptake and binding to nuclear DNA may be the major pathway for cytotoxicity. However, the levels of the complexes binding to DNA do not strictly correlate with the cytotoxic potency, indicating that other mechanisms are also involved. In addition, treatment of MCF-7 cells with [Ru(cym)(THcurc)Cl] showed a significant decrease in p62 protein levels, which is generally assumed as a non-cisplatin- like mechanism of action involving autophagy. Hence, a cisplatin- and a non-cisplatin-like concerted mechanism of action, involving both apoptosis and autophagy, is possible. 3 INTRODUCTION The turmeric plant (Curcuma longa), a perennial herb belonging to the ginger family, is cultivated extensively in south and southeast tropical Asia.1,2 The main polyphenol constituents of turmeric’s rhizome are curcuminoids that have three main chemical components, including curcumin (curcH), desmethoxycurcumin, and bisdemethoxycurcumin (bdcurcH).3 Recently, interesting results have emerged from clinical studies of curcumin in various pathologies. In particular, positive effects have been observed on inflammation, skin, eye, central nervous system, respiratory, cardiovascular, gastrointestinal, urogenital and metabolic disorders.4 Curcumin has also shown therapeutic potential in chemoprevention and for the treatment of cancer.5–7 Although curcuminoids have been successfully evaluated for a wide range of biological activities, they show two main limitations related to the poor bioavailability and rapid metabolism.8 The metabolism of curcumin in humans produces several products using three main pathways, degradation, O- conjugation and reduction.9,10 The reduction products are mainly tetrahydrocurcumin (THcurcH) (Scheme 1), hexahydrocurcumin and octahydrocurcumin.11–13 THcurcH has been reported to show a wide range of therapeutic properties similar to curcumin such as anti-oxidant, radicalscavenging, anti-metastatic and anti-carcinogenic activities, but unlike curcumin, THcurcH has greater bioavailability.14,15 Having additional hydrogens, THcurcH is more hydrophilic than curcumin16 and pharmacokinetic assessments reveal that it is more stable than curcumin in phosphate buffers at neutral pH and plasma.17 Following our recent studies on the anticancer activity of Ru(II) half-sandwich complexes of curcuminoids,18 we decided to extend our research to the synthesis and characterization of new organometallic Ru(II) complexes containing the more important curcuminoid metabolites, i.e. tetrahydrocurcumin (THcurcH) and bisdesmethoxytetrahydrocurcumin (THbdcurcH). 4 Scheme 1. Tetrahydrocurcumin obtained from curcumin after oral administration following metabolization by enzymatic reduction. RESULTS AND DISCUSSION The curcumin metabolite ligands, tetrahydrocurcumin (THcurcH) and tetrahydrobisdemethoxycurcumin (THbdcurcH), were synthesized as reported in Scheme 2, starting from the commercially available curcumin and bisdemethoxycurcumin. Curcumin and bisdemethoxycurcumin were dissolved in methanol and then, the catalyst Pd/BaSO4 was added a to the chilled solution. The reaction mixture was stirred at room temperature for 10 h under H2 (2 atm). Work up of the reaction mixture and purification by chromatography column gave THcurH and THbdcurc as white powders (77% and 60 % yield, respectively). The IR spectra of the two ligands possess aliphatic C-H absorption in the region of 3066-2841 cm-1 and no longer show the typical ν (C=C) vibration around 1590-1530 cm-1, characteristic of the α-β unsaturated chain. Scheme 2. Synthesis of THcurcH and THbdcurcH. 5 They were fully characterized by mass spectrometry and 1H, 13C NMR and bidimensional spectroscopy (see Supporting Information). Complexes 1–6 were prepared from the reaction of the appropriate dimer, [Ru(arene)Cl2]2, (arene = cymene, benzene or hexamethylbenzene) with THcurcH and THbdcurcH and potassium hydroxide in methanol (Scheme 3). Scheme 3. Synthesis of ruthenium(II)-arene complexes 1-6. Complexes 1–6 are air-stable and soluble in acetone, acetonitrile, DMSO, chlorinated and alcoholic solvents and are slightly soluble in water. The IR spectra of 1–6 show the typical ν(C=O, C=C) bands of THcurc and THbdcurc at lower wavenumbers than the corresponding bands in the free ligands, as a consequence of coordination through both the carbonyl arms to the metal. In the far-IR region, absorptions in the range 230-295 cm−1 may be assigned to ν(Ru–Cl) stretches.19 Electrospray ionization (ESI) mass spectra of 1–6 in positive ion mode, recorded in CH3CN, show the expected isotopic patterns and display peaks that correspond to [Ru(arene)(THcurc/THbdcurc)]+ arising from the dissociation of the chloride ligand. 1H- and 13C- 6 NMR spectra were assigned based on 1H−1H and 1H−13C couplings, arising from the bidimensional {1H−1H}-COSY, {1H−13C}-HSQC and {1H−13C}-HMBC experiments. The 1H and 13C NMR spectra of 1–6 in CDCl3 and CD3CN display the expected signals due to the coordinated arene ligands. Furthermore, in tetrahydrocurcumin and bisdesmethoxytetrahydrocurcumin there are diagnostic protons (H1 and H3-3') and carbons (C1, C2-2' and C3-3') which, being closer to the coordination site, are shifted to higher fields compared to those of free ligands (Supporting Information). The stability profile of complexes 1-6 and ligands THcurcH and THbdcurcH has been evaluated under physiologically relevant conditions in phosphate-buffered solution (PBS, pH = 7.4). The solutions, with a concentration of about 10-6 M, were monitored over time using UV- Visible spectroscopy. The compounds were initially solubilized in DMSO and then diluted to 10% DMSO with PBS. The absorbance spectra were collected after 24, 48 and 72 h. The hydrogenated ligands THcurcH and THbdcurcH were shown to be more stable than their parent unmodified curcuminoids, as expected from the literature.20,21 The absorbance profile of curcumin (curcH) and bisdemethoxycurcumin (bdcurcH) (Figure S51 a and c) gradually decrease with time indicating their instability under physiological conditions. In contrast, the reduced ligands THcurcH and THbdcurcH maintain their structure for a longer time with the absorbance profile of THcurcH remaining unchanged over 72 hours and THbdcurcH being stable for at least 48 hours (Figure S51 b and d). The complexes show good stability with 1-3 that contain the THcurc moiety generally being more stable than their analogue with THbdcurc, 4-6 (Figure S52). Compound 1 is the most stable for which changes in the absorption spectra were not observed. The least stable complexes are those with a benzene ligand. With the exception of 1, all complexes show a hypochromism in the maximum absorption intensity at about 270 nm, accompanied by a 3-5 nm red shift after 72 h, presumably due to aquation phenomena derived from the replacement of chloride ligand with a 7 H2O molecule. This behavior represents a crucial step in the activation of ruthenium-arene anticancer compounds.22 The stability of the complexes was also investigated using 1H-NMR spectroscopy (conducted for 1 and 4). The δ values of the characteristic peaks in all the spectra remained unchanged over 5 days, indicating that the complexes are stable (Figure S48 and S49). However, additional peaks appear in the 1H-NMR spectra of compound 4 which could be due to secondary species formed by the hydrolysis of Ru-Cl bond. Moreover, 1H-NMR spectra of compound 4 recorded in CD3CN do not show any additional peaks over time (Figure S50). X-ray crystallography Complexes 2-4 were structurally characterized by X-ray crystallography (see Experimental). Their structures show the expected piano stool geometry around the Ru(II) center (Figures 1-3). Selected structural parameters are collected in Table S1. The Ru-Cl bond lengths are within the typical 2.40- 2.43 Å range, and the Ru-arene centroid distances are close to 1.65 Å as usual in neutral complexes. The THcurc and THbdcurc ligands coordinate the ruthenium center in a similar fashion, with Ru- O distances between 2.06 and 2.09 Å. These values are similar those found for related Ru complexes with curcumin (curc) and bisdemethoxycurcumin (bdcurc) ligands in which this distance is close to 2.06 Å.23 The curcuminoid metallacycle is delocalized and the C-O distances in 2-4 are around 1.27-1.28 Å, which are longer than those calculated for the localized C=O bonds in the THcurc and THbdcurc precursors (1.24 Å, see Figure S1 and discussion below). 8 Figure 1. Molecular structure of 2. Figure 2. Molecular structure of 3 (CH2Cl2 solvate is not shown). Figure 3. Molecular structure of 4. 15 THbdcurcH 56 ± 10 51 ± 10 >100 50 ± 9.2 >100 47 ± 16 1 47 ± 8 64 ± 18 72 ± 16 8.0 ± 3.9 19 ± 2 6.2 ± 1.7 2 26 ± 3 32 ± 3 32 ± 10 8.5 ± 1.6 19 ± 7 7.5 ± 1.6 3 74 ± 14 72 ± 13 >100 >100 >100 >100 4 77 ± 19 >100 >100 57 ± 2 29 ± 9 33.6 ± 7 5 72 ± 17 86 ± 17 70 ± 29 20 ± 7 37 ± 6 17 ± 7 6 64 ± 8 72 ± 8 88 ± 3 >100 >100 98 ± 9 cisplatin 1.1 ± 0.6 9.0 ± 3.5 3.0 ± 1.1 4.2 ± 2.3 49 ± 7 12 ± 4 RAPTA-C >100 >100 >100 >100 >100 >100 Cellular Uptake In order to investigate a possible relationship between the structure of the complexes and the observed cytotoxicity, the uptake of complexes 1-6 in MCF-7 cells was studied using fluorescence anisotropy measurements within 2 hours. In general, we observed three different internalization behavior over the time-frame. Specifically, the free ligand THbdcurcH and 2, 4 and 5 showed a comparable three-stage internalization mode (membrane entry, permanence in the membrane and intracellular release), but with significant differences in individual entry and release rates (kin and kout values found in Table 2). Complexes 1 and 6 were retained for a longer time in the membranes, with no evidence of release during the first 2 h. Conversely, the internalization of ligand THcurcH and complex 3 involve a peculiar mechanism which does not follow the mono-exponential models described in the Experimental section (Figure S54). The ruthenium complexes show faster cellular uptake compared to THcurcH and THbdcurcH (Table 2). Quantitative analysis of the internalization kinetics showed a direct correlation between the rate membrane entry with the cytotoxicity observed toward MCF-7 cells, with higher values 16 of kin (i.e., faster membrane entry) displayed by 1 and 4 (cymene) and 2 and 5 (hexamethylbenzene), which are associated with a higher cytotoxicity. Complexes 3 and 6 (benzene) showed slower or even not observable values of kin, consistent with their lower cytotoxicity. Table 2. Kinetic parameters for membrane entry and exit events. kin (M-1s-1) kout (s-1) IC50 MCF-7 THcurcH n.o. n.o. >100 [Ru(cym)(THcurc)Cl] (1) 0.105 ± 0.01 n.o. 8.0 ± 3.9 [Ru(hmb)(THcurc)Cl] (2) 0.158 ± 0.02 0.012 ± 0.003 8.5 ± 1.6 [Ru(benz)(THcurc)Cl] (3) n.o. n.o. >100 THbdcurcH 0.102 ± 0.01 0.023 ± 0.005 50 ± 9.2 [Ru(cym)(THbdcurc)Cl] (4) 0.156 ± 0.02 0.052 ± 0.002 57 ± 2 [Ru(hmb)(THbdcurc)Cl] (5) 0.133 ± 0.02 0.048 ± 0.002 20 ± 7 [Ru(benz)(THbdcurc)Cl] (6) 0.063 ± 0.01 n.o. >100 DNA Binding DNA is one of the most important biomolecules in living cells and it represents the primary target for metal-based anticancer agents, with the formation of drug-DNA adducts being an established apoptosis-triggering event.27 Here, the ds-DNA oligomer (3′- CCACCCACTACCCTGGTTGGATGCTAATGT-5′) binding ability of THcurcH, THbdcurcH, and complexes 1-6 was kinetically characterized using a standard biosensor-based approach, and the binding modes were further explored both spectrophoto-fluorometrically and by molecular docking studies. The compounds of interest showed low-to-moderate affinity for ds-DNA,28 with equilibrium dissociation constants in the submillimolar-to-micromolar range (Table S8), only THcurcH, 1, 3 and 4 showing similar affinity for ds-DNA compared with other previously tested 17 Ru compounds.29 All binding events followed mono-exponential binding kinetics (Figures S55a and b), compatible with the ability of the compounds to target ds-DNA on a single site, i.e. the minor groove (Table S9 and Figure S56). There is no clear correlation between the binding to DNA and the cytotoxicity of the compounds. Quantification of p62 expression levels In the absence of a correlation between the observed cytotoxicity and the DNA affinity, we evaluated the effects of the treatment on the expression levels of a protein biomarker, namely p62/sequestosome1, which is a multifunctional ubiquitinated binding protein and it is involved in signaling pathways of many cell life activities, including autophagy, and its abnormal expression and regulation are closely associated with development and progression of malignant tumors and mostly associated with breast cancer.30 In particular, p62 can be degraded in autophagolysosomes, and its expression levels correlate inversely with the autophagic activity.31 Accordingly, we evaluated the effects of complex 1 (the most cytotoxic compound of the series) and the related parent ligand, THcurcH, on p62 expression. In line with the activation of the autophagic flux, a significant decrease of the levels of p62 protein was observed in MCF-7 cells, this effect being more evident for complex 1, consistently with its higher cytotoxicity compared to THcurcH (Figure 5). 18 Figure 5. Autoradiographs and densitometric analyses of p62/SQSTM1 levels (A) in MCF7 cells after treatment with 5 μM of complex 1 for 24 h (*p < 0.01 compared with the control; #p < 0.01 compared with THcurcH). CONCLUSIONS We have reported the first examples of ruthenium(II)-arene complexes containing two curcuminoid metabolites, i.e. THcurc and THbdcurc. The complexes were fully characterized in solution and in the solid-state, with the X-ray structures obtained confirming that the THcurc and THbdcurc ligands coordinate the ruthenium center without significant differences between them and with respect to those found for related Ru complexes with curc and bdcurc. However, an unprecedent closed conformation of the THbdcurc ligand was found in 4, where both -OH groups are involved in intermolecular hydrogen bonding with the same chloro ligand. Both closed and open conformations were analyzed in gas phase by DFT studies which showed that the open conformation is the most stable. Hence, the closed conformation of THbdcurc found in the crystal of 4 may be attributed to the presence of intermolecular interactions in the solid state. Interestingly, the arene ligand plays an important role in cell internalization process of the complexes, which in turn contributes to their cytotoxicity. Complexes bearing cymene (1 and 4) or hexamethylbenzene 19 (2 and 5) displayed faster uptake into MCF-7 cells, and they also showed higher cytotoxicity than 3 and 6 bearing benzene. Moreover, the difference in cytotoxicity observed between MCF-7 and the cisplatin-resistant counterpart (MCF-7CR) was likely attributable to a concerted cisplatin-like and non-cisplatin-like mechanism of action that involves both apoptosis and autophagy, in line with the ability of the complexes to bind to ds-DNA and to lower the intracellular levels of p62/SQSTM1, respectively. EXPERIMENTAL SECTION Materials and Methods Curcumin and bisdemethoxycurcumin were purchased from TCI Europe and were used as received. All other reagents and solvents were purchased from Aldrich and used as received without further purification. All reactions for the syntheses of proligands and the corresponding ruthenium complexes were carried out in the air. The samples for microanalyses were dried in vacuo to constant weight (35 °C, ca. 0.1 Torr). Elemental analyses (C, H, N) were performed inhouse with a Fisons Instruments 1108 CHNS-O Elemental Analyser. IR spectra were recorded on a Perkin-Elmer Frontier FT-IR instrument. 1H and 13C NMR spectra were recorded on a 500 Bruker Ascend (500 MHz for 1H, 125 MHz for 13C) instrument operating at room temperature relative to TMS. Positive ion electrospray mass spectra were obtained on a Series 1100 MSI detector HP spectrometer, using acetonitrile as solvent for all complexes 1-5. 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 were recorded on a STMP3 Stuart scientific instrument and on a capillary apparatus. Samples for microanalysis were dried in vacuo to constant weight 20 (20°C, ca. 0.1 Torr) and analyzed on a Fisons Instruments 1108 CHNS-O elemental analyzer. Uvstability studies have been conducted with a Varian Caryl spectrometer. Synthesis of ligands and complexes THcurcH (1,7-bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-dione) Curcumin (curcH, 500 mg, 1.4 mmol) was dissolved in CH3OH (25 mL) and Pd/BaSO4 (50 mg) added. After 10 h agitation under a H2 atmosphere (2 atm) the reaction mixture was filtered and the remaining oil purified using column chromatography (cyclohexane: ethylacetate, 60:40) to obtain the ligand THcurcH as white powder, yield 78%. It is completely soluble in CH3OH, CH3CN, DMSO and CH3Cl and insoluble in H2O and hexane. Anal. Calcd. for C21H24O6: C, 67.73, H, 6.50. Found: C, 67.94; H, 6.78. m.p. 87-90 °C. IR (cm-1): 3407 mbr ν(-OH); 3064 w, 3023 w, 3004 w, 2961 w, 2933 m, 2844 w ν(aliphatic C-H); 1702 w, 1601 mbr ν(-C=O); 1450 m, 1429 m. 1H-NMR (CDCl3, 293 K): δ 2.55 (t, 4H, C(3-3’)H), 2.85 (t, 4H, C(4-4’)H), 3.86 (s, 6H, -OCH3), 5.43 (s, 1H, C(1)H), 5.51 (sbr, 2H, -OH ), 6.69 (m, 4H, C(6-6’) and C(10-10’)), 6.85 (d, 2H, C(9- 9’)H). 13C{1H}-NMR (CDCl3, 293 K): δ 31.31 [s, C(4-4’)], 40.37 [s, C(3-3’)], 55.88 [s, -OCH3], 99.79 (s, C1), 110.97 [s, C(6-6’)], 114.35 [s, C(9-9’)], 120.83 [s, C(10-10’)], 132.57 [s, C(5-5’)], 144.04 [s, C(7-7’)], 146.44 [s, C(8-8’)], 193.20 [s, C(2-2’)]. ESI-MS (-) CH3CN (m/z [relative intensity, %]): 371 [100] [(THcurc)]-. THbdcurcH (1,7-bis(4-hydroxyphenyl)heptane-3,5-dione) THbdcurcH was synthesized as reported for THcurH starting from bisdesmethoxycurcumin (bdcurcH, 500 mg, 1.6 mmol). The ligand was obtained as white powder, yield 60%. It is completely soluble in CH3OH, CH3CN, DMSO and CH3Cl and insoluble in H2O and hexane. Anal. Calcd. For C19H20O4: C, 73.06; H, 6.45. Found: C, 72.52; H, 6.53. m.p. 105-107 °C. IR (cm-1): 21 3270 mbr ν(-OH); 3024 w, 2963 sh, 2930 w, 2859 w ν(aliphatic C-H); 1072 w; 1629 sh, 1614 m, 1602 m ν(-C=O); 1514 vs, 1462 m.1H-NMR (CD3CN, 293 K): δ 2.56 (t, 4H, C(3-3’)H), 2.81 (t, 4H, C(4-4’)H), 5.60 (s, 1H, C(1)H), 6.73 (m, 4H, C(7-7’)H), 7.05 (m, 4H, C(6-6’)H).13C{1H}- NMR (CD3CN, 293 K): δ 30.28 [s, C(4-4’)], 39.74 [s, C(3-3’)], 99.51 [s, C(1)], 115.08 [s, C(7- 7’)], 129.33 [s, C(6-6’)], 155.17 [s, C8-8’)] 193.73 [s, C(5-5’)], 204.28 [s, C(2-2’)]. ESI-MS (-) CH3CN (m/z [relative intensity, %]): 311 [100] [(THbdcurc)]-. [Ru(cym)(THcurc)Cl] (1) THcurcH (74 mg, 0.2 mmol) and KOH (11 mg, 0.2 mmol) were dissolved in CH3OH (5 mL). After 1 h stirring at room temperature, [Ru(cymene)Cl2]2 (61 mg, 0.1 mmol) was added. The resulting solution was stirred at room temperature for 6 h, after which the solvent was removed under vacuum. The KCl salt was precipitated in a CH2Cl2 solution and 1 formed by adding n-hexane. The yellow precipitate (60 mg, 0.093 mmol, yield 93%) was filtered and characterized. It is completely soluble in CH3OH, CH3CN, DMSO and CH3Cl, it is partly soluble in H2O and insoluble in hexane. Anal. Calcd. for C31H37ClO6Ru: C, 57.98; H, 5.81. Found: C, 56.63; H, 5.67. mp: 135-138 °C. IR (cm-1): IR (cm-1): 3401 mbr ν(-OH); 3068 w, 2967 w, 2919 w, 2850 w ν(aliphatic C-H); 1557 m ν(-C=O); 1514 m; 287 ν(Ru-Cl). 1H-NMR (CDCl3, 293 K): δ 1.31 (d, 6H, -CH(CH3)2 of cym, 3J = 7 Hz), 2.22 (s, 3H, -CH3 of cym), 2.51 (t, 4H, C3-3’H)), 2.84 (m, 5H, C(4-4’)H and CH(CH3)2 of cym), 3.90 (s, 6H, -OCH3), 5.15 (s, 1H, C(1)H),5.13 d, 5.38 d (4H, AA′BB′ system, CH3-C6H4-CH(CH3)2 of cym, 3J = 6 Hz), 5.51 (sbr, 2H, -OH), 6.69 (d, 2H, C(10- 10’)H, 3Jtrans = 8 Hz), 6.74 (s, 2H, (C-6-6’)H), 6.85 (d, 2H, C(9-9’)H, 3Jtrans = 8 Hz).13C{1H}-NMR (CDCl3, 293 K): δ 17.84 (s, -CH3 of cym), 22.29 (s, -CH(CH3)2 of cym), 30.67 [s, CH(CH3)2 of cym], 32.50 [s, C(4-4’)], 42.41 [s, C(3-3’)], 56.03 (s, -OCH3), 70.78 [s, C(b-b’)], 82.81 [s, C(a- a’)], 97.33 (s, Ci’), 98.01 (s, C1), 99.37 (s, Ci), 111.19 [s, C(6-6’)], 114.06 [s, C(9-9’)], 120.79 [s, 22 C(10-10’)], 133.50 [s, C(5-5’)], 143.84 [s, C(8-8’)], 146.38 [s, C(7-7’)], 188.54 [s, C(2-2’)=O]. ESI-MS (+) CH3CN (m/z [relative intensity, %]): 607 [100] [Ru(cym)(THcurc)]+. [Ru(hmb)(THcurc)Cl] (2) THcurcH (74 mg, 0.2 mmol) and KOH (11 mg, 0.2 mmol) were dissolved in CH3OH (5 mL). After 1 h stirring at room temperature, [Ru(hexamethylbenzene)Cl2]2 (67 mg, 0.1 mmol) was added. The resulting solution was stirred at room temperature for 6 h, after which the solvent was removed under vacuum. The KCl salt was precipitated in a CH2Cl2 solution and 3 crystallized by adding nhexane. The yellow precipitate (40 mg, 0.060 mmol, yield 60%) was filtered and characterized. It is completely soluble in in CH3OH, CH3CN, DMSO and CH3Cl, it is partly soluble in H2O and insoluble in hexane. Anal. Calcd. for C33H41ClO6Ru: C, 59.14; H, 6.17. Found: C, 59.04; H,.6.29. mp: 217-219 °C. IR (cm-1): 3278 mbr ν(-OH); 3004 w, 2960 w, 2931 w, 2856 w ν(aliphatic C-H); 1599 w; 1569 vs ν(-C=O); 1513 sbr; 295 ν(Ru-Cl). 1H-NMR (CDCl3, 293 K): δ 2.06 (s, 18H, - (CH3)6 of hmb), 2.50 (m, 4H, C(3-3’)H)), 2.79 m, 2.98 m, (4H, C(4-4’)H), 3.90 (s, 6H, -OCH3), 5.10 (s, 1H, C(1)H), 5.49 (sbr, 2H, -OH), 6.70 (d, 2H, C(10-10’)H, 3Jtrans = 8 Hz), 6.74 (s, 2H, (C- 6-6’)H), 6.84 (d, 2H, C(9-9’)H, 3Jtrans = 8 Hz).13C{1H}-NMR (CDCl3, 293 K): δ 15.03 (s, -(CH3)6 of hmb), 32.50 [s, C(4-4’)], 42.58 [s, C(3-3’)], 56.05 (s, -OCH3), 89.94 [s, C6 of hmb], 97.64 (s, C1), 111.14 [s, C(6-6’)], 114.10 [s, C(9-9’)], 120.76 [s, C(10-10’), 133.57 [s, C(5-5’)], 143.81 [s, C(8-8’)], 146.41 [s, C(7-7’)], 188.07 [s, C(2-2’)=O]. ESI-MS (+) CH3CN (m/z [relative intensity, %]): 635 [100] [Ru(hmb)(THcurc)]+. [Ru(benzene)(THcurc)Cl] (3) THcurcH (74 mg, 0.2 mmol) and KOH (11 mg, 0.2 mmol) were dissolved in CH3OH (5 mL). After 1 h stirring at room temperature, [Ru(benzene)Cl2]2 (50 mg, 0.1 mmol) was added. The resulting solution was stirred at room temperature for 6 h, after which the solvent was removed under 23 vacuum. The KCl salt was precipitated in a CH2Cl2 solution and 5 crystallized by adding n-hexane. The red precipitate (20 mg, 0.034 mmol, yield 34%) was filtered and characterized. It is completely soluble in in CH3OH, CH3CN, DMSO and CH3Cl, it is partly soluble in H2O and insoluble in hexane. Anal. Calcd. for C27H29ClO6Ru: C, 55.34; H, 4.99. Found: C, 55.34; H, 4.99. mp: 101-104 °C. IR (cm-1): 3414 mbr ν(-OH); 3071 w, 2963 w, 2934 w, 2845 ν(aliphatic C-H); 1614 sh, 1601 w; 1563 m, 1558 m ν(-C=O); 1514 sbr, 1506 s; 266 ν(Ru-Cl). 1H-NMR (CDCl3, 293 K): 2.53 (t, 4H, C(3-3’)H)), 2.85 (m, 4H, C(4-4’)H), 3.90 (s, 6H, -OCH3), 5.17 (s, 1H, C(1)H), 5.32 (sbr, 2H, -OH), 5.50 (s, 6H, C6H6 of benzene), 6.69 (d, 2H, C(10-10’)H, 3Jtrans = 8 Hz), 6.75 (s, 2H, (C-6- 6’)H), 6.85 (d, 2H, C(9-9’)H, 3Jtrans = 8 Hz).13C{1H}-NMR (CDCl3, 293 K): δ 32.74 [s, C(4-4’)], 42.38 [s, C(3-3’)], 56.06 (s, -OCH3), 82.26 (s, C6H6 of benzene), 98.22 (s, C1), 111.31 [s, C(6- 6’)], 114.01 [s, C(9-9’)], 120.86 [s, C(10-10’), 133.37 [s, C(5-5’)], 143.88 [s, C(8-8’)], 146.34 [s, C(7-7’)], 188.82 [s, C(2-2’)=O]. ESI-MS (+) CH3CN (m/z [relative intensity, %]): 551 [100] [Ru(benzene)(THcurc)]+. [Ru(cym)(THbdcurc)Cl] (4) THbdcurc (62 mg, 0.2 mmol) and KOH (11 mg, 0.2 mmol) were dissolved in CH3OH (5 mL). After 1 h stirring at room temperature, [Ru(cymene)Cl2]2 (61 mg, 0.1 mmol) was added. The resulting solution was stirred at room temperature for 6 h, after which the solvent was removed under vacuum. The KCl salt was precipitated in a CH2Cl2 solution and 2 crystallized by adding nhexane. The yellow precipitate (40 mg, 0.069 mmol, yield 70%) was filtered and characterized. It is completely soluble in in CH3OH, CH3CN, DMSO and CH3Cl, it is partly soluble in H2O and insoluble in hexane. Anal. Calcd. for C29H33ClO4Ru: C,59.84; H, 5.71. Found: C, 60.06; H, 5.59. mp: 195-197 °C. IR (cm-1): 3406 mbr ν(-OH); 3069 w, 2963 m, 2923 m, 2875 w, 2856 w ν(aliphatic C-H); 1613 m ν(-C=O); 1592 m, 1564 vs; 294 ν(Ru-Cl). 1H-NMR (CD3CN, 293 K): δ 24 1.26 (d, 6H, CH(CH3)2 of cym, 3J = 7 Hz), 2.10 (s, 3H, -CH3 of cym), 2.41 (m, 4H, C(3-3’)), 2.70 (m, 1H, CH(CH3)2 of cym), 2.81 (m, 4H, C(4-4’)), 5.10 d, 5.34 d (4H, AA′BB′ system, CH3-C6H4- CH(CH3)2 of cym, 3J = 6 Hz), 5.17 (s, 1H, C(1)H), 6.77 (d, 4H, C(7, 7’)H, 3Jtrans = 8 Hz), 6.89 (sbr, 2H, -OH ), 7.07 (d, 4H, C(6-6’)H, 3Jtrans = 8 Hz). 13C{1H}-NMR (CD3CN, 293 K): δ 16.91 (s, - CH3 of cym), 21.42 (s, -CH(CH3)2 of cym), 30.37 (s, CH(CH3)2 of cym), 31.52 [s, C(4-4’)], 41.95 [s, C(3-3’)], 78.94 [s, C(b-b’)], 82.46 [s, C(a-a’)], 96.71 (s, Ci’), 97.62 (s, C1), 99.23 (s, Ci), 114.98 [s, C(7-7’)], 129.41 [s, C(6-6’)], 132.71 [s, C(5-5’)], 155.13 [s, C(8-8’)], 188.17 [s, C(2-2’)=O]. ESI-MS (+) CH3CN (m/z [relative intensity, %]): 547 [100] [Ru(cym)(THbdcurc)]+. [Ru(hmb)(THbdcurc)Cl] (5) THbdcurc (62 mg, 0.2 mmol) and KOH (11 mg, 0.2 mmol) were dissolved in CH3OH (5 mL). After 1 h stirring at room temperature, [Ru(hexamethylbenzene)Cl2]2 (67 mg, 0.1 mmol) was added. The resulting solution was stirred at room temperature for 6 h, after which the solvent was removed under vacuum. The KCl salt was precipitated in a CH2Cl2 solution and 4 crystallized by adding n-hexane. The orange precipitate (20 mg, 0.033 mmol, yield 33%) was filtered and characterized. It is completely soluble in in CH3OH, CH3CN, DMSO and CH3Cl, it is partly soluble in H2O and insoluble in hexane. Anal. Calcd. for C31H37ClO4Ru: C, 61.02; H, 6.11. Found: C, 60.74; H, 6.20. mp: 230-232 °C. IR (cm-1): 3296 mbr ν(-OH); 3012 w, 2955 w, 2923 w, 2857 w ν(aliphatic C-H); 1614 m ν(-C=O); 1574 sbr; 228 ν(Ru-Cl). 1H-NMR (CD3CN, 293 K): δ 1.98 (s, 18H, -(CH3)6 of hmb), 2.36 m, 2.44 m, 2.56 m (4H, C(3-3’)H)), 2.82 (4H, C(4-4’)H), 5.14 (s, 1H, C(1)H), 6.75 (d, 4H, C(7-7’)H, 3Jtrans = 8 Hz), 7.03 (m, 4H, (C-6-6’)H). 13C{1H}-NMR (CD3CN, 293 K): δ 14.28 (s, -(CH3)6 of hmb), 31.59 [s, C(4-4’)], 42.21 [s, C(3-3’)], 89.92 [s, C6 of hmb], 97.34 (s, C1), 115.03 [s, C(7-7’)], [s, C(6-6’)], 132.61[s, C(5-5’)], 155.16 [s, C(8-8’)], 31 Curcumin (curcH), bisdesmethoxycurcumin (bdcurcH), tetrahydrocurcumin (THcurcH), bisdesmethoxytetrahydrocurcumin (THbdcurcH), CCR2, CC chemokine receptor 2; CCL2, CC chemokine ligand 2; CCR5, CC chemokine receptor 5. REFERENCES (1) Goel, A.; Kunnumakkara, A. B.; Aggarwal, B. B. Curcumin as “Curecumin”: From Kitchen to Clinic. Biochem. Pharmacol. 2008, 75 (4), 787–809. (2) Priyadarsini, K. I. The Chemistry of Curcumin: From Extraction to Therapeutic Agent. 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