Fine Tuning of Cholinesterase and Glutathione-S-Transferase Activities by Organoruthenium(II) Complexes

Cholinesterases (ChEs) show increased activities in patients with Alzheimer’s disease, and remain one of the main therapeutic targets for treatment of this neurodegenerative disorder. A library of organoruthenium(II) complexes was prepared to investigate the influence of their structural elements on inhibition of ChEs, and on another pharmacologically important group of enzymes, glutathione S-transferases (GSTs). Two groups of organoruthenium(II) compounds were considered: (i) organoruthenium(II) complexes with p-cymene as an arene ligand, and (ii) organoruthenium(II) carbonyl complexes as CO-releasing molecules. Eight organoruthenium complexes were screened for inhibitory activities against ChEs and GSTs of human and animal origins. Some compounds inhibited all of these enzymes at low micromolar concentrations, while others selectively inhibited either ChEs or GSTs. This study demonstrates the importance of the different structural elements of organoruthenium complexes for their inhibitory activities against ChEs and GSTs, and also proposes some interesting compounds for further preclinical testing as ChE or GST inhibitory drugs.


Introduction
For at least 3500 years, precious metals have been used for different medicinal purposes, and it is now known that the medicinal properties of metals are linked to their specific biological effects. As many metal ions (e.g., zinc, copper, iron) are involved in several physiological processes, there is great scope for designing metal-based therapeutic agents [1]. Factors that have critical influences on the biological activities of metal complexes include the nature and oxidation state of the metal ion, the number and types of bonded ligands and the coordination geometry [2][3][4].
[RuCym(L1)Br] (2). For the preparation of complex 2, complex 1 was initially prepared according to the procedure reported previously [33]. Then, a mixture of complex 1 (0.202 mmol, 1 equiv.) and AgNO 3 (0.524 mmol, 2.6 equiv.) was stirred in MeOH at room temperature in the dark for 1 h. The precipitated AgCl was filtered off through fine Celite powder. KBr (3.361 mmol, 16.7 equiv.) was added to the filtrate and the mixture was stirred further at room temperature in the dark for 45 min. The solvent was then evaporated and dichloromethane was added, resulting in the precipitation of KNO 3 , which was filtered off through fine Celite powder. The filtrate was concentrated on a rotary evaporator to around 2 mL. The complex was precipitated by addition of n-heptane, with the dark red-brown solid filtered off and dried at 45 • C. Yield: 44 mg, 49%. 1   For the preparation of the complex 3, [Ru(p-cymene)I 2 ] 2 was initially prepared. A mixture of ruthenium precursor 9 (0.262 mmol, 1 equiv.) and AgNO 3 (2.070 mmol, 7.9 equiv.) was stirred in MeOH at room temperature in the dark for 1 h. Then, KI (3.352 mmol, 12.8 equiv.) was added and the mixture was left to stir for an additional 15 min. The solvent was then evaporated, and the crude product dissolved in dichloromethane. The by-product salts that precipitated were filtered off through fine Celite powder. The filtrate was concentrated to around 2 mL, and after addition of hexane, the complex precipitated, and was filtered off and dried at 45 • C. 1  [RuCym(L1)I] (3). A mixture of [Ru(p-cymene)I 2 ] 2 (0.083 mmol, 1 equiv.), ligand 1-hydroxypyridine-2(1H)-thione (L1; 0.246 mmol, 3 equiv.) and the base NaOMe (0.261, 3.1 equiv.) was stirred in acetone at room temperature overnight. The next day, the solvent was evaporated, and the crude product was purified by column chromatography using silica gel as stationary phase (mobile phase, 5% acetone in dichloromethane). After combining the appropriate fractions, the mobile phase was removed on a rotary evaporator, and the dark red-brown solid was precipitated from a dichloromethane/n-heptane solvent combination, and filtered off and dried at 45 • C. Yield: 20 mg, 25%. 1  [RuCym(L2)Cl] (4). The syntheses of ruthenium(II) chlorido complexes 4 and 5 followed a previously published, but slightly modified, procedure [45]. Ruthenium precursor 9 (0.163 mmol, 1 equiv.), β-diketonate ligand 1-(2-bromophenyl)-4,4,4-trifluorobutane-1,3dione (L2; 0.346 mmol, 2.1 equiv.) and NaOMe base (0.359 mmol, 2.2 equiv.) were stirred in 10% MeOH in dichloromethane at room temperature overnight. The solvent was removed under reduced pressure, and the residue was dissolved in dichloromethane. The NaCl and other insoluble impurities precipitated in this solvent and were removed by filtration through fine Celite powder. The filtrate was concentrated on a rotary evaporator, and the product precipitated by addition of hexane. The orange solid was filtered off and dried at 45 • C, with no further purification required. Yield: 145 mg, 79%. 1  [RuCym(L3)Cl] (5). Ruthenium precursor 9 (0.163 mmol, 1 equiv.), β-diketonate ligand 1-phenylicosane-1,3-dione (L3; 0.357 mmol, 2.2 equiv.) and NaOMe (0.359 mmol, 2.2 equiv.) were stirred in 10% MeOH in dichloromethane at room temperature overnight. The solvent was removed under reduced pressure, and the residue was dissolved in dichloromethane. The NaCl and other insoluble impurities precipitated in this solvent and were removed by filtration through fine Celite powder. The filtrate was evaporated to dryness on a rotary evaporator. The crude orange product was purified by column chromatography using silica gel as stationary phase (mobile phases, dichloromethane, 10% MeOH in dichloromethane). The appropriate fractions were combined, the solvent was removed, and the orange product precipitated after drying at 45 • C. Yield: 68 mg, 32%. 1  [RuCym(L1)pta]PF 6 (6). Complex 6 was synthesised as reported previously [33].
[RuCym(L2)pta]PF 6 (7). Ruthenium(II) pta complex 7 was synthesised according to a procedure published previously [30]. A mixture of ruthenium(II) chlorido complex 4 (0.177 mmol, 1 equiv.), pta (0.267 mmol, 1.5 equiv.) and silver salt AgPF 6 (0.267 mmol, 1.5 equiv.) was stirred in acetone at room temperature for approximately 48 h in the dark. The solvent was removed under reduced pressure, and the residue was dissolved in dichloromethane. The insoluble AgCl salt that formed as a by-product of the reaction was removed by filtration through fine Celite powder. The filtrate was concentrated on a rotary evaporator, and the product was precipitated by addition of hexane. The crude product was filtered off and purified by column chromatography using silica gel as stationary phase (mobile phases, 5% MeOH in dichloromethane; 10% MeOH in dichloromethane). The appropriate fractions were combined and concentrated, and the product was precipitated with hexane, with the orange solid filtered off and dried at 45 • C. Yield: 53 mg, 36%. 1  [Ru(L1) 2 (CO) 2 ] (8). Ruthenium precursor 10 (0.073 mmol, 1 equiv.), ligand L1 (0.292 mmol, 4 equiv.) and the base NaOMe (0.292 mmol, 4 equiv.) were stirred in a mixture of MeOH and chloroform (3 mL, 5 mL, respectively) at room temperature for 30 min. Then, the solvents were evaporated, and dichloromethane was added. The NaCl that precipitated was filtered off through fine Celite powder. The filtrate was evaporated, and the crude product was purified by column chromatography using aluminium oxide (mobile phase, 2% acetone in dichloromethane). After combining the appropriate fractions, the mobile phase was removed on a rotary evaporator, and the pale-yellow solid that precipitated from the dichloromethane/n-heptane added was filtered off and dried at 45 • C. Yield: 37 mg, 62%. 1   The activities of the ChEs were determined using a modification of the Ellman method [46] adapted for microtiter plates, as described in [47]. Stock solutions of complexes 4, 5, 6, 7, 8 and 10, as well as of ligands L2 and L3 (1 mg/mL) were prepared in 100% MeOH, whereas stock solutions of complexes 2 and 3 (1 mg/mL) were prepared in 5% DMSO in deionised water. Positive control (1 mg/mL neostigmine bromide; Sigma-Aldrich, St. Louis, MO, USA) was also prepared in 100% MeOH. The stock solutions of the potential inhibitors and the positive and negative controls were added to the wells, and progressively diluted in 100 mM potassium phosphate buffer (pH 7.4) to the final volume of 50 µL. Then, 100 µL acetylthiocholine chloride (1 mM) and 5,5 -dithiobis-2nitrobenzoic acid (0.5 mM) in 100 mM potassium phosphate buffer (pH 7.4) were added into the microtiter plate wells. Three ChEs were used as the enzyme sources: electric eel AChE (eeAChE); human recombinant AChE (hrAChE), and horse serum BChE (hsBChE) (all Sigma-Aldrich, St. Louis, MO, USA). These were dissolved in the same buffer to the final concentration of 0.0075 U/mL. Finally, 50 µL of each ChE solution was added into the microtiter plate wells to start the reaction, which was followed spectrophotometrically at 405 nm at 25 • C over 5 min using a kinetic microplate reader (Dynex Technologies Inc., Chantilly, VA, USA). The blank reactions without the inhibitors were run with the appropriate dilutions of the solvents in which the tested compounds were initially diluted (100% MeOH or 5% aqueous DMSO), and the readings were corrected according to the appropriate blanks. Each measurement was repeated at least three times. To determine the inhibitory constants (K i ), the kinetics were monitored using three different final substrate concentrations (0.125, 0.25, 0.5 mM). The data were analysed using the OriginPro software (OriginPro 2020, OriginLab Corporation, Northampton, MA, USA).

Glutathione S-Transferase Inhibition Assay
The activities of the GSTs were determined according to the method described by Habig et al. (1974) [48] using a cell imaging multi-mode reader (Cytation 3; BioTek, Winooski, VT, USA). The stock solutions of inhibitors were prepared as described for the ChE inhibition assays. Then the stock solutions of the potential inhibitors and negative controls were added to the wells, and progressively diluted in 100 mM sodium phosphate buffer (pH 6.5) to the final volume of 50 µL. 1-Chloro-2,4-dinitrobenzene (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in ethanol to 50 mM, and then diluted with 100 mM sodium phosphate buffer (pH 6.5) to a final concentration of 4 mM. This solution (50 µL) and 2 mM reduced glutathione (100 µL) in the same buffer were added into the microtiter plate wells. Two GSTs were used as the enzyme sources: horse liver GST (hlGST) and human placenta GST (hGST) (Sigma-Aldrich, St. Louis, MO, USA). These were dissolved in 100 mM sodium phosphate buffer (pH 6.5), and 50 µL of these enzyme solutions were added into the wells to start the reaction. The final enzyme concentration was 0.044 U/mL. The blank reactions without the inhibitors were run with the appropriate dilutions of the solvents in which the tested compounds were initially diluted (100% MeOH or 5% aqueous DMSO), and the readings were corrected according to the appropriate blanks. The reactions were followed spectrophotometrically at 340 nm at 25 • C over 4 min. Each measurement was repeated at least three times. For determination of the inhibitory constants (K i ), the kinetics were monitored using three different final substrate concentrations (200, 400, 800 µM). The data were analysed using the OriginPro software (OriginPro 2020, OriginLab Corporation, Northampton, MA, USA).
The organoruthenium(II) chlorido complex 1 with O,S-ligand pyrithione has been tested previously for its AChE, BChE and GST inhibition [12]. To further evaluate the influence of the monodentate halide ligands on biological activity of the ChEs and GSTs, the bromido 2 and iodido 3 analogues were prepared. Furthermore, to examine the influence of another type of bidentate ligand on the investigated system, the organoruthenium(II) chlorido complexes 4 and 5 with O,O-ligands were prepared following a modified procedure reported previously [45], using the chlorido ruthenium precursor 9 and the appropriate β-diketonate ligand L2 or L3. Additionally, halide ligand Z was substituted by monodentate bulky phosphine pta ligand. Two organoruthenium(II) pta complexes with pyrithione ligand L1 and β-diketonate ligand L2 were prepared following a modified procedure published previously [30], to yield cationic complexes 6 and 7, respectively. In order to also evaluate the activity of ruthenium complexes, derived from other ruthenium precursors than ruthenium precursor 9, CORM complex 8 was synthesised.

Crystal Structures
Over the course of the study, new crystal structures of complexes 4 and 8 were obtained. Single crystals of complex 4 were prepared by liquid-liquid diffusion from a mixture of dichloromethane and hexane, and single crystals of complex 8 were prepared from a mixture of acetone and diethyl ether at room temperature. The crystallographic data and geometric parameters are given in Tables S1-S3.
The ruthenium(II) chlorido complex 4 has a pseudo-octahedral "piano-stool" geometry, which is typical for organoruthenium(II) arene complexes with O,O-chelating ligands [49]. Crystal structure of complex 4 is shown in Figure 3. The ruthenium(II) ion was bound to the neutral p-cymene, chlorido ligand and a bidentate chelating β-diketonate ligand L2. The η 6 -arene ligand represents the "seat" of the piano stool, while the three remaining coordination sites have the roles of the "legs". Complex 4 has bond lengths between the ruthenium(II) ion and the oxygen donor atoms of the β-diketonate ligand of 2.0810 (19) Å and 2.0862(19) Å. A survey of the Cambridge Structural Database was performed for comparisons with known crystal structures. This included a number of ruthenium(II) compounds with β-diketonate ligands, and therefore only those compounds in which the p-cymene was coordinated to the ruthenium together with the β-diketonate ligand containing a -CF 3 group were considered (structure codes: CUZZEE, CUZZII, KIMGAQ, KIMGEU, KIMGIY, KIMJEY, KIMJIC, KIMJOI, KIMLID, KIMLOJ, MIDNIX, NAYPEL, NAYPIP, NAYPOV, NAYPUB, WUNGIX, WUNGOD, WUNGUJ, WUNHAQ, WUNHEU, WUNHIY). The ruthenium-to-oxygen bond lengths ranged from 2.066 Å to 2.111 Å [50]. The bond lengths defined in the present study fit very well into the middle of this range.
The ruthenium pyrithione complex 8 has an octahedral geometry. Crystal structure of complex 8 is shown in Figure 3. The six-numbered coordination sphere of ruthenium(II) consists of two bidentate chelating pyrithione (L1) ligands, bound via the sulphur and oxygen donor atoms, and two neutral monodentate carbonyl ligands, bound via carbon atoms. The sulphur atoms from deprotonated pyrithione are positioned trans to each other. Only a few compounds were reported where pyrithione or its analogues were bound to ruthenium, and most of these were synthesised by the Turel research group. Pyrithione is usually bound in a deprotonated form in a bidentate manner via both oxygen and sulphur donor atoms. Therefore, only these structures were used for comparisons (structure codes: TOXVEK, TOXVIO, TOXVOU, TOXVUA, TOXWAH, TOXWEL, TOXWIP, TOXWOV, UQUZUD, URABAS). The bonds between ruthenium(II) and sulphur in complex 8 had lengths of 2.3711(6) Å and 2.3598(6) Å, which agrees well with the range from 2.334 Å to 2.370 Å in the structures from the Cambridge Structural Database [50]. In complex 8, the distances between ruthenium and oxygen were 2.0859(16) Å and 2.1023(16) Å, which are also comparable to the lengths in the pyrithione-type organoruthenium(II) complexes in the Cambridge Structural Database [50].

Inhibition of Cholinesterases and GSTs by the Ruthenium-Based Complexes
In the present study, six organoruthenium(II) arene complexes with β-diketonate-type (4, 5, 7) or pyrithione-type ligands (2-3 and 6) were newly tested, along with the ruthenium precursor 10 and its new complex with pyrithione 8 towards eeAChE, hrAChE, hsBChE, hlGST and hGST. The compounds were first screened for the IC 50 determination, and for those with IC 50 < 33 µM, the inhibitory constants (K i ) were determined. This threshold was chosen since currently approved and used anticholinesterase drugs exert their activity mostly in the low micromolar and in submicromolar range [51]. This paper also includes our previously published results of complex 1 and precursor 9 towards these enzymes [12]. The main objective of this study was to investigate the significance of the various structural elements of the newly prepared library of various organoruthenium complexes on the activities of selected ChEs as potential therapeutic drug targets that are involved in the pathogenesis of Alzheimer's disease, and thus to further expand our previous data on ruthenium compounds with interesting activities [12,34]. Therefore, complexes were prepared from the ruthenium precursors 9 and 10 with various bidentate ligands (i.e., pyrithione L1, β-diketonates L2-L3) with different steric/electronic properties (i.e., different substituents on β-diketonates) together with various monodentate Z ligands (i.e., Cl -, Br -, I -, pta). Through this fine-tuning, insight could be gained into which structural elements are essential for the inhibition of the ChEs to determine the structure-activity relationship, and consequently to plan further synthesis optimisation. In addition, we investigated the possible inhibitory effects of these complexes on GST activities, as GSTs have essential roles in the development of anticancer drug resistance [25,26].
The discovery of new compounds that can simultaneously inhibit ChEs and GSTs would be interesting for treatment of patients who suffer from both Alzheimer's disease and certain cancers, although some studies have suggested mutual exclusion of these two diseases in the same patient [52]. Tested ruthenium complexes have shown various activities on ChEs and GSTs described below, whereas all three ligands, i.e., L1 [12], L2 and L3 showed no activities against these ChEs and GSTs.
Among the prepared library of compounds, the organoruthenium(II) pyrithione complexes 2 and 3 with bromide and iodide ligands, respectively, showed inhibitory activities in the low micromolar range against all of the ChEs and GSTs tested (i.e., eeAChE, hrAChE, hsBChE, hlGST, hGST). Instead, the organoruthenium(II) chlorido complexes with the β-diketonate ligands, i.e., complexes 4 and 5, selectively inhibited only hsBChE and hlGSTs in the pharmacologically relevant micromolar range. Interestingly, the organoruthenium(II) pta complex 6 with pyrithione and the organoruthenium(II) pta complex 7 with β-diketonate ligand selectively inhibited only hsBChE, and the CORM complex 8 with pyrithione selectively inhibited only GSTs. The inhibition parameters for these compounds against ChEs and GSTs (i.e., IC 50 , K i ) are shown in Table 1, Table 2, Table 3.
5.98 ± 1.0 / / / a , Inhibition of eeAChE and hrAChE by 1 and 9 was previously reported in [12]; b , The synthesis of the complex 6 was previously published in [33]; IC 50 , Concentration required to induce 50% inhibition of enzyme activity; K i , Inhibition constants determined for compounds with IC 50 < 33 µM. Data are means ± SEM of three independent measurements; /, No activity.  1 and 9 was previously reported in [12]; b , The synthesis of the complex 6 was previously published in [33]; IC 50 , Concentration required to induce 50% inhibition of enzyme activity; K i , Inhibition constants determined for compounds with IC 50 < 33 µM. Data are means ± SEM of three independent measurements; /, No activity. Table 3. Inhibition of horse liver (hlGST) and human placenta glutathione S-transferase (hGST) by the ruthenium compounds 1-10 and the free ligands L1-L3.

Enzyme Inhibition (µM) hlGST hGST
Compared with the organoruthenium(II) pyrithione complex with the chlorido ligand, as complex 1 [12], complexes 2 and 3 showed slightly lower inhibitory potential against eeAChE, but comparable inhibitory activity against hlGST, and even better inhibitory activities against hrAChE, hsBChE and hGST. However, it is not disputed that changes in Z ligands (e.g., Br -, Iinstead of Cl -) play major roles in increasing inhibitory potential of organoruthenium(II) pyrithione complexes 2 and 3 with bromido or iodido ligands, respectively, against hsBChE and hGST. This effect is particularly striking against hGST, where the bromido ligand of complex 2 promoted an IC 50 lower by approximately a factor of 10 compared to the chlorido ligand of complex 1. The differences in inhibition of complexes 1-3 might be partly a consequence of various hydrolysis rates of the monofunctional halido leaving groups, but might also be related to changes in hydrophobicity, as well as solubility. Importantly, the hydrolysis rates of halido ions are reported to be connected to the activation of the complexes, as the substitution of the negatively charged halido ligands with neutral water ligand results in positively charged metal species that can further interact with biological targets via electrostatic interactions [9,[58][59][60].
In addition, this study also investigated the inhibitory activity of organoruthenium(II) pyrithione complex 6 with the pta ligand, which effectively inhibited only hsBChE with IC 50 value of 0.5 µM. The IC 50 value of complex 6 is lower by approximately a factor of 15 compared to complexes 1 and 2, and a factor 7 for complex 3. This compound compared to previously reported pyrithione compounds (Cl -, Br -, I -) shows us that the changes in the Z ligands might result in alterations of the inhibitory activities or in alterations to the specificities towards the enzymes used in the present study. As a selective BChE inhibitor, this compound could be of interest for further preclinical studies; however, its activity should be tested also on BChE of a human origin that was not commercially available during the course of this study.
The combined results here thus demonstrate the importance of a suitable Z ligand choice in such organoruthenium(II) pyrithione complexes for the fine tuning of their inhibitory potentials against enzymes of human and other animal origins.
Other compounds studied here were the organoruthenium(II) complexes in which the bidentate ligands were β-diketonates with various substituents and the monodentate Z ligands Cl -(complexes 4 and 5) or pta (complex 7). The data given in Table 2 show that all of these compounds effectively inhibited hsBChE in low micromolar range, with IC 50 values of 30.98 µM for complex 4, 31.99 µM for complex 5, and 19.2 µM for complex 7. The IC 50 values of complexes 4, 5, and 7 are about 2.5 to 9 times higher compared to the IC 50 values of complexes 1 and 2 or 3, respectively. Moreover, complex 7 expressed selective inhibitory activity towards animal BuChE. On the other hand, the chlorido compounds 4 and 5 also inhibited hlGST with IC 50 values of 16.11 µM and 18.28 µM, respectively. However, complex 4 did not inhibit hGST in the concentration range of interest, and another compound, complex 5, did not inhibit hGST at all. In all of these cases, the inhibition was reversible and competitive, with K i values in the low micromolar range ( Figure S3).
Obtained results show that the different substituents of β-diketonates in the organoruthenium(II) cholorido complexes 4 and 5 had no effects on the inhibitory activities or on the specificities against the ChEs and GSTs used in the present study. This was demonstrated by using different substituents on the β-diketonates with the same Z ligand as Cl -, where neither the inhibitory activity nor the avidity towards the different ChEs and GSTs changed. Both of these compounds inhibited hsBChE and hlGST with very similar IC 50 values.
Replacement of the Z ligand Clwith pta, as complex 7, resulted in a slight improvement of the inhibitory activity against hsBChE, but led to the loss of susceptibility against hlGST, and also to the other enzymes tested. This indicates that the nature of the Z ligand in the organoruthenium(II) β-diketonate complexes affects the inhibitory activities of the respective compounds on these ChEs and GSTs.
Nowadays, the development of safe and efficient CORMs as therapies for neurovascular diseases is very important [61]. In the central nervous system, a protective role of low-concentration dose CO has been reported, which has suggested beneficial effects in diseases such as Alzheimer's disease, traumatic brain injury and stroke [61,62]. Should a compound simultaneously inhibit ChEs and release CO, it might have a dual beneficial effect in the treatment of Alzheimer's disease. In the presented study, we investigated inhibition of tested enzymes by ruthenium precursor 10 and its complex with pyrithione 8 to evaluate the influence of the chosen metal precursor. Complex 8 was prepared from the ruthenium CORM precursor 10. The pyrithione ligand was chosen instead of the βdiketonate ligand because pyrithione complexes have generally shown better inhibition of ChEs and GSTs The ruthenium precursor 10 and its complex with pyrithione 8 were also included in the study to evaluate the influence of the chosen metal precursor. The pyrithione ligand was chosen instead of the β-diketonate ligand because pyrithione complexes have generally shown better inhibition of ChEs and GSTs. As shown by the data given in Table 3, complex 10 efficiently inhibited hlGST (IC 50 = 9.76 µM) and hGST only weakly, but did not have any effects on the ChEs which showed activity in the pharmaceutically interesting range only for hlGST inhibition (IC 50 = 9.76 µM). Meanwhile, unlike the precursor 9, when pyrithione was complexed with 10 to obtain complex 8, this showed effective inhibition of the GSTs of both animal and human origins, with IC 50 values of 3.66 µM for hlGST and 16.61 µM for hGST. These data show that complex 8 selectively inhibits GSTs, which makes it interesting for further preclinical studies. However, complex 8 showed no activity against ChEs. The inhibition of both of these GSTs was again reversible and competitive ( Figure S4). Future preclinical studies in cells and mammalian organisms would be necessary to confirm that these complexes selectively inhibit the enzymes of interest and can be considered as potential anticholinesterase and anti-GST drugs.

Conclusions
A small library of five novel organoruthenium(II) compounds with p-cymene as an arene ligand was synthesised, along with one organoruthenium(II) carbonyl complex CORM, and crystal structures of complexes 4 and 8 were determined. Further, ligands L1-L3, precursor 10 and complexes 2-8 were screened for inhibitory activities against AChEs, BChEs and GSTs of human and other animal origins. The arene-organoruthenium(II) pyrithione complexes with Br -(i.e., complex 2) and I -(i.e., complex 3) as monodentate ligands inhibited all of these ChEs and GSTs at low micromolar concentrations, with no selectivity observed. Furthermore, the organoruthenium(II) β-diketonate complexes 4 and 5 that contain Clinhibited hsBChE and hlGST, while pta complexes 6 and 7 selectively inhibited hsBChE in the low micromolar range. These data confirm that the organoruthenium(II) carbonyl complex with pyrithione (i.e., complex 8) is a selective GST inhibitor, without ChE inhibitory activity. These data also demonstrate the importance of the nature of the ligands in the structure of these organoruthenium(II) complexes for their inhibitory activities against ChEs and GSTs, and they provide some interesting compounds for further preclinical testing as ChE and GST inhibitory drugs.