Fast Hydrolysis and Strongly Basic Water Adducts Lead to Potent Os(II) Half-Sandwich Anticancer Complexes

{"full_text": " pubs.acs.org/IC Article\n\n\n\n Fast Hydrolysis and Strongly Basic Water Adducts Lead to Potent\n Os(II) Half-Sandwich Anticancer Complexes\n Sonia Infante-Tadeo, Vanessa Rodr\u00edguez-Fanjul, Cintia C. Vequi-Suplicy, and Ana M. Pizarro*\n Cite This: Inorg. Chem. 2022, 61, 18970\u221218978 Read Online\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\n\n\n ABSTRACT: Complexes of the formula [Os(\u03b7 6 -arene)(C,N-\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n phenylpyridine)Z] (where Z is chlorido or a tethered oxygen) undergo\n very fast Os\u2212Z hydrolysis (<5 min), and the high basicity of the coordinated\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 12:10:19 (UTC).\n\n\n\n\n water molecule of the aqua adducts (Os\u2212OH2; pKa > 8) very much contrasts\n with previously reported Os-aqua adducts bearing NN- and NO-chelating\n ligands (pKa < 6). The Os\u2212Cl bond is unreactive in pure DMSO, yet the\n complexes readily form DMSO adducts upon aquation when dimethyl\n sulfoxide is present. Such a peculiar aqueous behavior is directly related to the\n negatively charged CN ligand. Potent Os-CN compounds (but not their Os-\n NN analogues) are particularly reactive; they bind to cysteine in vitro and\n decrease the activity of thioredoxin reductase (TrxR) in living cancer cells. By revealing some interesting structure\u2212activity\n relationship on Os-CN vs Os-NN complexes, we start uncovering the molecular rationale for the successful biological applications of\n osmium(II) half-sandwich compounds.\n\n\n \u25a0 INTRODUCTION\n Cisplatin, or cis-diamminedichloridoplatinum(II), is one of the\n metal center (formation of tethered alkoxy complexes), thus\n ensuring kinetic reactivation of osmium in water.5\n most widely used drugs in cancer chemotherapy. Its mode of In the past years, potent hydrolyzable Os-chlorido\n action to stop cell replication involves coordinative binding to complexes [Os(\u03b76-arene)(CN)Cl]+, where CN is an anionic\n DNA upon aquation of the Pt\u2212Cl bonds inside the cell, that is, \u03ba2-C,N-binding ligand such as a phenylazole (including\n a water molecule replaces each chlorido ligand, and a further phenylbenzimidazole) derivative9\u221212 or phenylpyridine12\u221215\n substitution reaction results in Pt-guanine coordination.1 and \u03b76-arene is benzene or p-cymene, have been reported by\n Initial interest in anticancer osmium half-sandwich com- Ruiz et al. and Keppler et al. Such complexes present IC50\n plexes of the formula [Os(\u03b76-arene)(\u03ba2-XY)Cl]+ that fit into values in several cancer cell lines up to one order of magnitude\n the activation-through-hydrolysis paradigm was sparked by lower than cisplatin. Interestingly, the Os(II)-arene-CN half-\n Peacock et al., who successfully activated Os reactivity in water sandwich structure is reminiscent of those of Ru(II)-arene-\n by carefully selecting the XY chelating ligand.2\u22124 A relationship CN,16\u221219 Ir(III)-Cp*-CN,20\u221222 and Rh(III)-Cp*-CN,23 and it\n was established between the coordinative nature of XY, Os\u2212Cl might seem that when looking for cytotoxic potency of\n aquation, and the acidity of the subsequently Os-\u03ba1-bound metallodrug candidates, it is the anionic CN ligand that\n water molecule. Complexes with neutral NN ligands hardly ensures such a goal. Nevertheless, the molecular rationale\n hydrolyzed (and were thus unreactive toward substitution behind why half-sandwich Os(II) compounds bearing anionic\n reactions and inactive toward cancer cells),5\u22127 while anionic CN-chelates remain some of the most potent organo-osmium\n OO ligands had the opposite effect, producing fast hydrolyzing compounds to date is elusive, despite being fundamental to\n complexes.3,8 Rapid aquation combined with high acidity of understanding how potent organo-osmiums exert their intra-\n Os-bound water triggered the formation of noncytotoxic inert cellular effect.\n hydroxido-bridged dimers, [Os(\u03b76-arene)(\u03bc2-OH)3Os(\u03b76- Dimethyl sulfoxide is a common solvent used in cell studies\n arene)]+, concomitant to the XY ligand loss.2 Os-picolinate to aid dissolution for the testing of lipophilic drugs. In\n complexes (NO-chelating ligands), with faster hydrolysis and addition, DMSO can be considered a strongly coordinating\n pKa(Os\u2212OH2) closer to physiological pH, showed moderate\n in vitro cytotoxicity, approaching that of carboplatin.2,8\n In a quest to find hydrolyzable, and intracellularly reactive, Received: September 12, 2022\n organo-osmium compounds, we recently reported Os-arenes Published: November 15, 2022\n bearing a pendant alcohol functionality to bypass the inert\n hydroxido-bridge dimer formation, as the Os-hydroxido (Os\u2212\n OH) adduct triggers intramolecular rearrangement that\n culminates in the binding of the pendant alcohol to the\n\n \u00a9 2022 American Chemical Society https://doi.org/10.1021/acs.inorgchem.2c03246\n 18970 Inorg. Chem. 2022, 61, 18970\u221218978\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nmolecule, and a few interesting studies have demonstrated that PhpyH), 5.85 (d, J = 4.9 Hz, 1H, ArH), 5.79 (d, J = 5.3 Hz, 1H, ArH),\nit may be a noninnocent vehicle during biological studies as it 5.49 (d, J = 5.5 Hz, 1H, ArH), 5.06 (d, J = 4.8 Hz, 1H, ArH), 4.97 (t, J\ncan mediate metal-based unexpected reactivity.24,25 Sulfur (and = 5.0 Hz, 1H, ArH), 3.57 (d, J = 17.1 Hz, 1H, ArCH2), 3.41 (d, J =\nselenium) coordination to transition metals has not been 17.1 Hz, 1H, ArCH2). 13C{1H} NMR (101 MHz, CDCl3, \u03b4): 192.80\n (s, CO), 167.33 (s, PhpyC), 164.27 (s, PhpyC), 154.40 (s, PhpyCH),\noverlooked in the interaction of metallodrugs with the 144.80 (s, PhpyC), 139.05 (s, PhpyCH), 137.85 (s, PhpyCH), 130.56\nthioredoxin reductase (TrxR) system. TrxR inhibition by (s, PhpyCH), 124.22 (s, PhpyCH), 123.95 (s, PhpyCH), 122.52 (s,\nmetal-based compounds is well-documented and expectedly PhpyCH), 119.32 (s, PhpyCH), 90.11 (s, ArC), 88.13 (s, ArCH),\ninvolves ligand exchange reactions at a labile position in the 84.56 (s, ArCH), 79.68 (s, ArCH), 64.43 (s, ArCH), 56.73 (s, ArCH),\nfirst coordination sphere of the metal with SeCys and Cys 42.23 (s, ArCH2). MS (ESI): m/z calcd for C19H15NO2Os [M +\nresidues at the enzyme active site.26\u221230 Recent studies have H+]+, 482.1; found, 482.2. Elemental analysis (EA): calcd for\nindicated that the TrxR activity is weakly affected by cytotoxic C19H15NO2Os\u00b7H2O (497.55): C, 45.87; H, 3.44; N, 2.82%. Found:\ncompounds based on osmium,31 while it is strongly inhibited C, 45.53; H, 3.45; N, 2.78%. Melting point (MP): 130\u2212167 \u00b0C.\nby Au-,32,33 Ru-,34,35 and Ir-based22 anticancer candidates. [Os(\u03b76:\u03ba1-C6H5CH2CH2COO)(Phpy)] (2). Synthesis as for 1, using\n [Os(\u03b76-C6H5CH2CH2COOH)(\u03bc-Cl)Cl]2 (28 mg, 0.034 mmol),\n We present here a series of experiments in water on very\n NaOAc (45 mg, 0.17 mmol), and Phpy (10.2 \u03bcL, 0.07 mmol).\npotent Os(II)-CN complexes that appear to unveil, at the Yield: 28 mg, 0.057 mmol, 83%. 1H NMR (400 MHz, DMSO-d6, \u03b4):\nmolecular level, what are the principles for maintaining Os- 9.30 (d, J = 5.9 Hz, 1H, PhpyH), 8.14\u22128.08 (m, 2H, PhpyH), 7.92 (s,\nbased chemical reactivity in aqueous media and thus Os- 1H, PhpyH), 7.84\u22127.80 (m, 1H, PhpyH), 7.23 (s, 1H, PhpyH), 7.03\nmediated anticancer potency. Our results on the aqueous (dd, J = 8.6, 3.3 Hz, 2H, PhpyH), 6.08 (t, J = 5.4 Hz, 1H, ArH), 5.89\nbehavior of complexes of the type [Os(\u03b76-arene)(C,N- (d, J = 5.9 Hz, 1H, ArH), 5.76 (t, J = 4.9 Hz, 1H, ArH), 5.62 (d, J =\nphenylpyridine)Z]+ successfully relate their strong in vitro 5.0 Hz, 1H, ArH), 4.91 (t, J = 5.0 Hz, 1H, ArH), 3.57 (s, 2H, ArCH),\ncytotoxicity to the reactivity of the Os\u2212Z bond and the basicity 3.04\u22122.92 (m, 1H, CH2O), 2.41\u22122.37 (m, 1H, CH2O). 13C{1H}\nof the Os-coordinated water upon aquation. The persistence of NMR (101 MHz, CDCl3, \u03b4): 177.31 (s, CO), 167.18 (s, PhpyC),\nthe Os\u2212OH2 bond in water appears to drive Os-based 164.86 (s, PhpyC), 154.47 (s, PhpyCH), 145.28 (s, PhpyC), 138.36\n (s, PhpyCH), 137.85 (s, PhpyCH), 130.62 (s, PhpyCH), 124.39 (s,\nreactivity, in particular with S-containing molecules, outside PhpyCH), 123.72 (s, PhpyCH), 122.71 (s, PhpyCH), 119.36 (s,\nand inside the cancer cell. PhpyCH), 95.79 (s, ArC), 86.99 (s, ArCH), 82.79 (s, ArCH), 81.11\n\n\u25a0 EXPERIMENTAL SECTION\n\u03b76-Bound arenes containing electron-withdrawing groups such as\n (s, ArCH), 64.20 (s, ArCH), 58.18 (s, ArCH), 36.75 (s, CH2CO),\n 28.84 (s, ArCH2). MS (ESI): m/z calcd for C20H18NO2Os [M +\n H+]+, 496.1; found, 496.3. EA: calcd for C20H17NO2Os\u00b7H2O\nesters are thermally labile when coordinated to Ru(II), which has (511.58): C, 46.96; H, 3.74; N, 2.74%. Found: C, 47.00; H, 3.75;\nbeen extensively exploited in the preparation of Ru(II) dimers N, 2.6%. MP: 90\u2212120 \u00b0C.\ncontaining mono- and disubstituted arenes for a number of [Os(\u03b76-C6H5CH2COOEt)(Phpy)Cl] (3). Synthesis as for 1, using\napplications.36\u221241 However, arene exchange reactions proved [Os(\u03b76-C6H5CH2COOEt)(\u03bc-Cl)Cl]2 (30 mg, 0.035 mmol), NaOAc\nineffective for osmium using [Os(\u03b76-Et/Me-benzoate)(\u03bc-Cl)Cl]2 as (7.8 mg, 0.097 mmol), and Phpy (9.6 \u03bcL, 0.07 mmol). The yellow-\nthe starting material. The preparation of the complexes described in green powder precipitated after the addition of hexane into the\nthis work thus required to reduce the arene hemilabile ligands 2- solution, and this powder was washed with hexane and dried. Yellow-\nphenylacetic acid and 3-phenylpropanoic acid to the corresponding orange crystals suitable for X-ray diffraction were obtained of the\ncyclohexadienes, prior to the reaction with OsCl3\u00b73H2O to obtain complex [Os(\u03b76-C6H5CH2COOEt)(Phpy)Cl]\u00b70.5H2O (3\u00b70.5H2O)\ndimers [Os(\u03b76-C6H5(CH2)nCOOH)(\u03bc-Cl)Cl]2, precursors of 1 (n = by slow evaporation of a triple-layer diffusion of DCM/toluene/\n1) and 2 (n = 2). For dimers [Os(\u03b76-C6H5(CH2)nCOOEt)(\u03bc- hexane (2:3:1) at 253 K. Yield: 25 mg, 0.046 mmol, 66%. 1H NMR\nCl)Cl]2, which are the dimer precursors of 3 (n = 1) and 4 (n = 2), (400 MHz, CDCl3, \u03b4): 9.13 (d, J = 5.6 Hz, 1H, PhpyH), 8.02 (d, J =\nOsCl3\u00b73H2O was also reacted with the same reduced versions of 2- 7.4 Hz, 1H, PhpyH), 7.80 (d, J = 8.1 Hz, 1H, PhpyH), 7.72\u22127.62 (m,\nphenylacetic acid and 3-phenylpropanoic acid, yet in these reactions, 2H, PhpyH), 7.16 (t, J = 6.9 Hz, 1H, PhpyH), 7.10\u22126.94 (m, 2H,\nesterification of the carboxylic groups was achieved by using ethanol PhpyH), 5.71 (dd, J = 10.4, 5.3 Hz, 2H, ArH), 5.53 (t, J = 5.2 Hz, 1H,\nas the solvent, as previously described for similar ester-derivatized ArH), 5.44 (d, J = 5.4 Hz, 1H, ArH), 5.25 (t, J = 5.1 Hz, 1H, ArH),\nruthenium(II) dimers,42 in a one-pot microwave-assisted synthesis.43 4.16 (q, J = 7.1 Hz, 2H, OCH2), 3.32 (d, J = 6.0 Hz, 2H, ArCH2),\nThe dimer precursor of complex 5, [Os(\u03b76-C6H5(CH2)3OH)(\u03bc- 1.26 (t, J = 7.1 Hz, 3H, CH3). 13C{1H} NMR (101 MHz, CDCl3, \u03b4):\nCl)Cl]2, was obtained by reacting OsCl3\u00b73H2O with the reduced 171.14 (s, CO), 166.85 (s, PhpyC), 165.32 (s, PhpyC), 155.15 (s,\nversion of 3-phenylpropanol, as previously described by us.5 The PhpyCH), 144.23 (s, PhpyC), 139.11 (s, PhpyCH), 137.22 (s,\ndetailed syntheses of the organic ligands and the individual dimers are PhpyCH), 130.76 (s, PhpyCH), 124.28 (s, PhpyCH), 123.10 (s,\nincluded in the Supporting Information. PhpyCH), 122.51 (s, PhpyCH), 119.15 (s, PhpyCH), 87.60 (s, ArC),\n Synthesis of Os(II) Complexes 1\u22125. [Os(\u03b76:\u03ba1-C6H5CH2COO)- 83.43 (s, ArCH), 79.23 (s, ArCH), 77.16 (s, ArCH), 74.64 (s, ArCH),\n(Phpy)] (1). [Os(\u03b76-C6H5CH2COOH)(\u03bc-Cl)Cl]2 (22 mg, 0.028 72.06 (s, ArCH), 61.33 (s, OCH2), 39.31 (s, ArCH2), 14.30 (s, CH3).\nmmol) and sodium acetate (NaOAc; 37 mg, 0.14 mmol) were stirred MS (ESI): m/z calcd for C21H20NO2Os [M\u2212Cl\u2212]+, 510.11; found,\nfor 1 h in 3 mL of DCM at 333 K. After that, 2-phenylpyridine (Phpy; 509.9. EA: calcd for C21H20ClNO2Os\u00b7H2O (562.07): C, 44.88; H,\n8 \u03bcL, 0.056 mmol) was added to the solution, and the mixture was 3.95; N, 2.49%. Found: C, 45.07; H, 3.87; N, 2.45%. MP: 125\u2212149\nstirred at 333 K for 3 days. The black suspension was filtered over a \u00b0C.\npad of Celite, the yellow-green solvent was reduced in a rotary [Os(\u03b76-C6H5CH2CH2COOEt)(Phpy)Cl] (4). Synthesis as for 3, using\nevaporator until it was one-fourth of the initial volume, and then, a [Os(\u03b76-C6H5CH2CH2COOEt)(\u03bc-Cl)Cl]2 (30 mg, 0.034 mmol),\nyellow-green powder precipitated after the addition of Et2O. The NaOAc (7.5 mg, 0.094 mmol), and Phpy (9.6 \u03bcL, 0.07 mmol).\npowder was washed with diethyl ether and dried under reduced Yield: 32 mg, 0.057 mmol, 84%. Yellow crystals suitable for X-ray\npressure. Yield: 21 mg, 0.044 mmol, 78%. Yellow-green crystals diffraction were obtained in a solution of Et2O at RT. 1H NMR (400\nsuitable for X-ray diffraction were obtained of the complex [Os(\u03b76:\u03ba1- MHz, CDCl3, \u03b4): 9.13 (d, J = 5.6 Hz, 1H, PhpyH), 8.05 (d, J = 7.4\nC6H5CH2COO)(Phpy)]\u00b71.5CH2ClCH2Cl (1\u00b71.5DCE) by slow evap- Hz, 1H, PhpyH), 7.81 (d, J = 8.1 Hz, 1H, PhpyH), 7.67 (dd, J = 15.1,\noration of a layer diffusion of DCE/pentane at 253 K. 1H NMR (400 7.5 Hz, 2H, PhpyH), 7.15 (t, J = 7.2 Hz, 1H, PhpyH), 7.03 (dd, J =\nMHz, CDCl3, \u03b4): 9.07 (d, J = 5.7 Hz, 1H, PhpyH), 8.09 (d, J = 7.5 11.8, 6.5 Hz, 2H, PhpyH), 5.67 (t, J = 5.2 Hz, 1H, ArH), 5.58 (d, J =\nHz, 1H, PhpyH), 7.82 (d, J = 8.1 Hz, 1H, PhpyH), 7.74\u22127.66 (m, 2H, 5.3 Hz, 1H, ArH), 5.49 (t, J = 5.2 Hz, 1H, ArH), 5.38 (d, J = 5.3 Hz,\nPhpyH), 7.14 (td, J = 7.3, 1.3 Hz, 1H, PhpyH), 7.11\u22127.01 (m, 2H, 1H, ArH), 5.24 (t, J = 5.0 Hz, 1H, ArH), 4.12 (q, J = 7.1 Hz, 2H,\n\n 18971 https://doi.org/10.1021/acs.inorgchem.2c03246\n Inorg. Chem. 2022, 61, 18970\u221218978\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nOCH2), 2.73\u22122.56 (m, 4H, ArCH2 overlapped with CH2CO), 1.24 functionality, cannot form a tether ring, while complex 5\n(t, J = 7.1 Hz, 3H, CH3). 13C{1H} NMR (101 MHz, CDCl3, \u03b4): readily affords the tethered structure [Os(\u03b76:\u03ba1-C6H5(CH2)3O-\n172.65 (s, CO), 166.88 (s, PhpyC), 165.69 (s, PhpyC), 155.11 (s, (H))(Phpy)]0/+, whereby the alcohol pendant from the arene\nPhpyCH), 144.25 (s, PhpyC), 139.15 (s, PhpyCH), 137.12 (s,\nPhpyCH), 130.67 (s, PhpyCH), 124.25 (s, PhpyCH), 122.97 (s,\n is \u03ba1-O-binding to Os (forming a tether ring in the same\nPhpyCH), 122.46 (s, PhpyCH), 119.10 (s, PhpyCH), 93.77 (s, ArC), fashion as complexes 1 and 2) in basic aqueous solution (vide\n80.95 (s, ArCH), 79.89 (s, ArCH), 77.03 (s, ArCH), 74.36 (s, ArCH), infra).\n71.98 (s, ArCH), 60.78 (s, OCH2), 35.01 (s, CH2CO), 28.77 (s, Compounds 1\u22125 were synthesized in good yield (66\u221284%)\nArCH2), 14.36 (s, CH3). MS (ESI): m/z calcd for C22H22NO2Os by the reaction of the corresponding dimer with deprotonated\n[M\u2212Cl\u2212]+, 524.13; found, 523.9. EA: calcd for C22H22ClNO2Os\u00b7 phenylpyridine, using procedures similar to those reported\n0.5H2O (567.09): C, 46.60; H, 4.09; N, 2.47%. Found: C, 46.57; H, previously (Chart S1).10,12,13,19 Experimental details as well as\n4.09; N, 2.54%. MP: 158\u2212187 \u00b0C. individual characterization are detailed in the Experimental\n [Os(\u03b76-C6H5(CH2)3OH)(Phpy)Cl] (5). Synthesis as for 1, using\n[Os(\u03b76-C6H5(CH2)3OH)(\u03bc-Cl)Cl]2 (33 mg, 0.04 mmol), NaOAc Section and Supporting Information of this manuscript\n(9 mg, 0.11 mmol), and Phpy (11 \u03bcL, 0.08 mmol). Yield: 31 mg, 0.06 (Figures S1\u2212S14 and Table S1).\nmmol, 75%. 1H NMR (400 MHz, CDCl3-d, \u03b4): 9.13 (d, J = 5.6 Hz, Stodt et al. successfully synthesized Ru-arenes bearing a\n1H, PhpyH), 8.05 (d, J = 7.4 Hz, 1H, PhpyH), 7.81 (d, J = 8.1 Hz, pendant carboxylate, demonstrating that the pendant-arm\n1H, PhpyH), 7.67 (dd, J = 17.5, 7.9 Hz, 2H, PhpyH), 7.15 (t, J = 7.3 fragment {Ru(\u03b76-C6H5(CH2)3COOH)} was suitable for both\nHz, 1H, PhpyH), 7.03 (dd, J = 13.7, 6.8 Hz, 2H, PhpyH), 5.69 (t, J = N-terminal labeling of amino acids and peptides and\n5.2 Hz, 1H, ArH), 5.56 (d, J = 5.3 Hz, 1H, ArH), 5.49 (t, J = 5.2 Hz, esterification when the dimer formation was carried out in\n1H, ArH), 5.34 (d, J = 5.3 Hz, 1H, ArH), 5.19 (t, J = 5.0 Hz, 1H,\nArH), 3.68 (t, J = 6.1 Hz, 2H, CH2OH), 2.62\u22122.42 (m, 2H, ArCH2),\n ethanol.42 In fact, a number of Ru(II) half-sandwich complexes\n1.93\u22121.75 (m, 2H, CH2). 13C{1H}c NMR (101 MHz, CDCl3-d, \u03b4): are reported that bear derivatized arenes for further tagging of\n166.91 (s, PhpyC), 165.79 (s, PhpyC), 155.12 (s, PhpyCH), 144.32 proteins,44 specific protein inhibitors,45 drugs,46 or fluoro-\n(s, PhpyC), 139.19 (s, PhpyCH), 137.08 (s, PhpyCH), 130.62 (s, phores. 47 Derivatization of Os(II) arenes is far less\nPhpyCH), 124.22 (s, PhpyCH), 122.92 (s, PhpyCH), 122.44 (s, exploited,48,49 perhaps due to the lack of access to arene\nPhpyCH), 119.06 (s, PhpyCH), 96.82 (s, ArC), 80.39 (d, J = 16.8 Hz, exchange reactions in Os(II) arene dimers bearing electron-\nArCH), 73.26 (s, ArCH), 70.49 (s, ArCH), 62.14 (s, CH2OH), 32.87 withdrawing groups such as esters.36 Murray recently reported\n(s, CH2), 29.57 (s, ArCH2). MS (ESI): m/z calcd for C20H20NOOs the formation of [Os(\u03b76-3-(p-tolyl)propanoic acid)Cl2]2,\n[M\u2212Cl]+, 482.1; found, 481.7. EA: calcd for C20H20ClNOOs\u00b7H2O\n(534.06): C, 44.98; H, 4.15; N, 2.62%. Found: C, 44.80; H, 3.98; N, following acidic hydrolysis of undesired [Os(\u03b76-ethyl-3-(p-\n2.74%. tolyl)propanoate)Cl2]2.49 Here, we avoided the esterification\n Synthesis of metal complexes bearing bipyridine for comparison of the dimer by reacting the corresponding cyclohexadiene\npurposes and details of chemicals and instrumentation, chemical with OsCl3\u00b7H2O in acetone/water (to obtain the precursors of\ncharacterization, pH* measurements, X-ray crystallographic analysis, complexes 1 and 2) and purposely triggered esterification by\ncell culturing techniques, and cell-based assays are included in detail carrying out the reaction in ethanol (to obtain the precursors\nin the Supporting Information. of 3 and 4). However, to the best of our knowledge, 1 and 2\n\n\u25a0 RESULTS AND DISCUSSION\nWe present for the first time complexes 1\u22125 (Chart 1) of\n are the first Os-tethered complexes with a chelating hemilabile\n arene-carboxylate. Likewise, X-ray crystal structures of Os-\n tethered complexes are scarce.5,50 Here, we determined the\ngeneral formulae [Os(\u03b7 6 :\u03ba 1 -C 6 H 5 (CH 2 ) n COO)(Phpy)] first X-ray examples of Os(II) half-sandwich complexes with a\n tethered carboxylate (1\u00b71.5DCE; Figure 1A) or an ester\nChart 1. Structures of Open and Closed Tethered functionality pendant from the arene (3\u00b70.5H2O and 4, Figure\nOsmium(II) Arene Compounds 1\u22125 Included in This Work 1B,C). Full X-ray analysis and detailed crystallographic data\n (Tables S2 and S3) are included in the Supporting\n Information.\n The in vitro cytotoxic activity of 1\u22125 was investigated in\n triple negative MDA-MB-231 breast cancer cells. All complexes\n consistently impaired cell survival at a lower concentration\n than cisplatin, ranging within 1.9\u22126.6 \u03bcM, while\n IC50(cisplatin) was 13.7 \u00b1 0.6 \u03bcM (Figure 2 and Table S4).\n The high potency of 5 (2.1 \u00b1 0.4 \u03bcM) strikingly contrasts with\n the moderate potency of analogues [Os(\u03b76-C6H5(CH2)3OH-\n (XY)Cl], bearing a variety of NN- and NO-chelating ligands.5\n For the latter, the lowest IC50 values in MDA-MB-231 cells\n were 90.6 \u00b1 4.0 and 93.0 \u00b1 4.3 \u03bcM, when XY was 4-Me-\n picolinate or quinolinate, respectively, that is, the most active\n(where Phpy = C,N-phenylpyridine; n = 1, 1; n = 2, 2), complexes were over 40-fold less active than complex 5 in this\n[Os(\u03b76-C6H5(CH2)nCOOEt)(Phpy)Cl] (n = 1, 3; n = 2, 4), work. The cytotoxicity of 5 was also evaluated in MCF7 and\nand [Os(\u03b76-C6H5(CH2)3OH(Phpy)Cl] (5). Complexes 1 and HCT116 cancer cells with comparable results (IC50 = 2.8 \u00b1\n2 are tethered complexes, that is, there is no chlorido in the 0.1 and IC50 = 5.5 \u00b1 1.5 \u03bcM, respectively; Table S4).\nstructure and the monodentate position (Z in the general Intracellular accumulation of 1\u22125 ([Os] = 10 \u03bcM, 8 h drug\nstructure [Os(\u03b76-arene)(Phpy)Z]) is occupied by the pendant exposure) was determined in MDA-MB-231 cells by means of\n\u03ba1-O-bound carboxylate (COO\u2212). The Os\u2212O bond in tether ICP-MS. Results were in the range of 61\u221283 ng Os/106 cells\ncomplexes is prone to cleavage in water in a process akin to for all but 2, which showed a lower accumulation than the\nOs\u2212Cl aquation. Complexes 3 and 4, containing an ester others (29 ng Os/106 cells), correlating well with its slightly\n 18972 https://doi.org/10.1021/acs.inorgchem.2c03246\n Inorg. Chem. 2022, 61, 18970\u221218978\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 1. ORTEP diagrams and atom numbering schemes for compounds: (A) 1\u00b71.5DCE, (B) 3\u00b70.5H2O, and (C) 4 (50% probability ellipsoids).\nThe H atoms and the solvent molecules are omitted for clarity.\n\n chlorido complex 5, based on 1H NMR peak integration, was\n plotted against time, fitted to pseudo-first-order kinetics, and\n their half-life was calculated (Figure 3A,B, insets). The 1H\n NMR spectra of 1 and 2 showed three species coexisting in\n solution over time: the initial tethers (1 and 2), the aqua\n adducts (1A and 2A), and the DMSO adducts (1DMSO and\n 2DMSO; Figure S15). For open chlorido complexes 3\u22125, two\n species are observed in solution, the aqua and DMSO adducts,\n as interconversion occurred and a new sharp singlet appears\n over time at ca. 2.2 ppm in the 1H NMR spectra, attributable\nFigure 2. Antiproliferative activity and intracellular accumulation of to the coordinated DMSO ligand (Figure S16).52\u221254\nOs(II) complexes in MDA-MB-231 cells. Both the Os\u2212O(tether) bond (in 1 and 2) and the Os\u2212Cl\n bond (in 3\u22125) undergo similar fate in DMSO:D2O when\n NaCl (ca. 300 mM) is present, as complexes 1\u22125 rapidly\nlower potency (Figure 2). Accumulation of 5 in MCF7 and evolve to DMSO adducts 1DMSO\u22125DMSO (Figure 3A,B,\nHCT116 cells afforded similar results (Table S5). orange lines) even with excess chloride ions in solution.\n We aimed at unraveling the molecular basis that relates the\n Likewise, incorporation of DMSO into the first coordination\nstrong in vitro cytotoxicity of Os-arenes 1\u22125 with the presence\n sphere of Os prevailed even in the presence of cell culture\nof a phenylpyridine in the structure, for which we explored Os-\n media components, such as inorganic salts, amino acids, and\nmediated chemical reactivity. The cleavage of the Os\u2212\n vitamins (Table S6), even containing fetal bovine serum\nO(tether) bond in 1 and 2 and the Os\u2212Cl bond in 3\u22125\ncomplexes was monitored by 1H NMR ([Os] = 5 mM) at (FBS), for over 24 h by 1H NMR. An interaction with media\nvarious time intervals in pure DMSO-d6, DMSO-d6/D2O components was not observed as the DMSO adduct was still\n(5:95), and 300 mM NaCl DMSO-d6/D2O (5:95). We favored in 5% DMSO-d6/D2O solutions (Figure S17).\nexpected that, albeit slowly, complexes 1\u22125 interacted with Importantly, when analogous experiments were run in 5%\npure DMSO readily forming metal\u2212DMSO adducts.12,51 DMF-d7/D2O, the aqua adduct was the major species (Figure\nHowever, no significant changes were observed in the first S18). This encouraged the use of DMF, instead of DMSO, as\n24 h for 2\u22125 in pure DMSO, while complex 1 was not totally the vehicle to help dissolve our complexes in aqueous media\nunreactive toward DMSO, as it slowly evolved to form during the cell studies.\n1DMSO (22% conversion after 17 h) as determined by 1H Our results indicate that the aqua adduct acts as an\nNMR (Figure 3A, black dots and diamonds for 1 and 2, intermediate in the DMSO adduct formation (Figure 3C).\nrespectively; Figure 3B, black triangles for 5). The different Assignment of the rapidly forming aqua adduct was supported\nbehavior of 1 at longer times is attributed to the higher tether- by titration experiments in DMF/water solutions (vide infra).\nring tension, as the shorter tether arm generates a 5-membered This rapid aquation (<5 min) only parallels with that of\ntether ring (see the large C7 offset toward osmium in the X-ray complexes [Os(\u03b76-p-cymene)(quinol)Cl], reported by Sadler,2\nstructure of 1\u00b71.5DCE, Figure 1 and Table S2), ultimately and [Os(\u03b76-C6H5(CH2)3OH)(quinol)Cl], by us.5 Regarding\naffording an Os\u2212O bond more prone to dissociation. the water-mediated interaction with DMSO, a similar\n The lack of solvent coordination in pure DMSO was observation has been reported before by Keppler et al., who\nsurprising when contrasted to the readiness to form Os\u2212 stated that complexes of the formula [Ru(\u03b76-p-cym)(Phtz)Cl]\nDMSO adducts when the complexes were dissolved in aqueous (Phtz = phenyltriazole derivatives) rapidly evolve to their\nsolution containing 5% DMSO (that is, an excess of about 140 DMSO counterparts only in the presence of water.11\nmol equivalents of DMSO per mol of Os(II) complex). When In an attempt to better understand the biological relevance\nwater was present, 1\u22125 readily converted into 1DMSO\u2212 of the Os(II)-DMSO adduct formation, cell viability data were\n5DMSO. The percentage of disappearance of closed tethered 1 also obtained using DMSO to aid dissolution of the Os(II)\nand 2, and the percentage of newly formed DMSO adducts in complexes. The experiments were run in two different\n 18973 https://doi.org/10.1021/acs.inorgchem.2c03246\n Inorg. Chem. 2022, 61, 18970\u221218978\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n [Os(\u03b76-C6H5(R))(Phpy)(OH2/OH)] was determined by\n means of 1H NMR. Dissolution of neutral and highly\n hydrophobic 1\u22125 in water required a cosolvent. As for\n biological studies, DMSO was disfavored to avoid DMSO\n adduct formation, so noncoordinative DMF was used instead\n (5% DMF-d7/D2O). The Os\u2212Z bond in 1\u22125 hydrolyzed\n affording aqua adducts 1A\u22125A. The pKa values assignable to\n coordinated water in 1A\u22125A (pKa1) ranged from 8.73 to 9.38\n (Figure 4, blue lines, Table 1, and Figure S19). To the best of\n\n\n\n\nFigure 3. Percentage of (A) tethered Os complexes 1 (dots) and 2\n(diamonds) and (B) Os\u2212DMSO adduct formation (5DMSO;\ntriangles) over time, as determined by 1H NMR. For (A) and (B),\nthe solutions are as follows: black, pure DMSO; blue, 5% DMSO-d6\nand 95% D2O; orange, 5% DMSO-d6 and 95% D2O, containing 300\nmM NaCl. Kinetic constants (k) and half-life (t1/2) for the\ndisappearance of tethered complexes 1 and 2 (A) and formation of\n5DMSO (B) are shown in the same color coding. (C) Scheme of\nspeciation into the aqua adduct to readily afford the DMSO adduct.\nIn order to assign formal charges to the organo-osmium compounds\nthat bear ligands prone to acid/base equilibria (containing COO(H)\nand OH(H) groups), we assume a pH of ca. 7. Figure 4. Dependence on pH* (pH in D2O solutions) of the 1H\n NMR chemical shifts of osmium complexes. Plots of the chemical\nconditions: (i) using freshly prepared DMSO/media solutions shift against pH are fitted to the Henderson\u2212Hasselbalch equation,\nand (ii) allowing the complexes to stand in the mixture for 24 which afforded the pKa* values in DMF-d7/D2O or DMSO-d6/D2O\nh at 37 \u00b0C prior to addition of the Os(II)-containing solutions solutions for (A) 1DMSO, 1A, and 3A, (B) 2DMSO, 2A, and 4A, and\n (C) 5A and 5C.\nto the cells. While the former produced similar data to those\nusing DMF to aid compound dissolution, the latter indicated\nthat DMSO-aged Os(II) solutions failed to impair cell death our knowledge, these are the first pKa(Os\u2212OH2) reported for\n(reduction in cell viability was not observed at osmium Os(II) (and even Ru(II)) half-sandwich complexes containing\nconcentrations up to 200 \u03bcM; Table S7), further corroborating CN-chelates. Interestingly, they lie in the range of (highly\nthat the aqua adduct Os-(arene)(C,N)(OH2) is the active potent) Ir(III)-Cp* complexes also bearing phenylpyridine\nspecies that ultimately results in cell damage. derivatives (pKas ranging within 8.3\u22128.9)20 and are the most\n Intrigued by the high in vitro cytotoxic potency of rapidly basic pKas reported to date for Os(II) arenes, with the\nhydrolyzing complexes 1\u22125 and failing to observe inert exception of an Os(\u03b76-p-cym) complex bearing an anionic NN-\nhydroxido dimeric species in solution, we were prompted to chelate (deprotonated pyridinylindol).51 The high basicity of\nbetter understand the behavior of the Os\u2212OH2 aqua adduct. the coordinated water can be attributed to the strong \u03c3-donor\nThe acid\u2212base equilibrium constant (pKa) of species 1A\u22125A CN ligand, which by having a formal negative charge increases\n 18974 https://doi.org/10.1021/acs.inorgchem.2c03246\n Inorg. Chem. 2022, 61, 18970\u221218978\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nTable 1. pKa Values of the Aqua Ligand (pKa1), the Pendant Carboxylic Group (pKa2), and the Tethered Alcohol/Alkoxy\nGroup (pKa3) Corrected from pKa* by Applying the Equation pH = 0.929 \u00d7 pH* + 0.42, Suggested by Kre\u0327ze\u0307 l and Bal58\n pKa1 pKa2 pKa3\n 6 +/0/\u2212\n [Os(\u03b7 -C6H5CH2COO(H))(Phpy)(OH(H))] (1A) 9.00 3.75\n [Os(\u03b76-C6H5CH2COO(H))(Phpy)(DMSO)]+/0 (1DMSO) 3.62\n [Os(\u03b76-C6H5(CH2)2COO(H))(Phpy)(OH(H))]+/0/\u2212 (2A) 8.95 4.45\n [Os(\u03b76-C6H5(CH2)2COO(H))(Phpy)(DMSO)]+/0 (2DMSO) 4.40\n [Os(\u03b76-C6H5CH2COOEt)(Phpy)(OH(H))]+/0 (3A) 8.57\n [Os(\u03b76-C6H5(CH2)2COOEt)(Phpy)(OH(H))]+/0 (4A) 8.53\n [Os(\u03b76-C6H5(CH2)3OH)(Phpy)(OH(H))]+/0 (5A) 9.13\n [Os(\u03b76:\u03ba1-C6H5(CH2)3O(H))(Phpy)]+/0 (5C) 8.21\n\n\n\n\nFigure 5. Percentage of the TrxR activity upon exposure of MDA-MB-231 cells to Os(II) complexes, where the blue bars represent Os-NN-\nbipyridine and red bars represent complexes 1\u22125 (bearing CN-phenylpyridine) described in this work.\n\nelectron density on the metal center. Conversely to the high agreement with our recent results on pH-dependent aqueous\npKa values of 1A\u22125A (>8) are those of recently reported aqua speciation of tethered Os(II) arenes bearing phenylpropanol.\nadducts [Os(\u03b76-C6H5(CH2)3OH)(NN/NO-chelate)(OH2)], In such a study, the pK a of compounds [Os(\u03b7 6 :\u03ba 1 -\nwhich are <6 for Os-NN and \u22647 for Os-NO complexes.5 C6H5(CH2)3OH/O)(NN/NO)] ranged within 4.35\u22125.65.5\nWe used DFT calculations on analogues [Os(\u03b76-C6H6)(Phpy)- The pKa value reported here for 5C (pKa3), 8.38, is\nOH2]+ and [Os(\u03b76-C6H6)(bipy)OH2]2+ to assess the elec- extraordinarily high, further supporting the strong effect by\ntronic distribution in both model complexes. For the Os-NN the CN-phenylpyridine ligand on the Os center and\nanalogue, osmium donates 0.23 electrons to the three consequently on the rest of the ligands in the first coordination\nsurrounding building blocks (all three ligands, including the sphere of the metal. Curiously, formation of 5C at a basic pH\nbipy). Interestingly, for the Os-CN analogue, the Os center occurred even in the presence of DMSO; in other words,\nreceives 0.08 electrons from its ligands, indicating that the osmium shielding by the tether arm occurs even in\nosmium center is significantly more basic (Tables S8 and S9). competition with strongly coordinating DMSO (Figure S21).\nThese results on distinct electronic fluxes of the first The rapid hydrolysis (activation) of these complexes, the\ncoordination sphere of NN versus CN coordination of Os(II) basicity of the water molecule (maintaining the organo-\nhalf-sandwich complexes agree with the observations of osmium in its activated form) together with the readiness of\nNeedham et al.55 the complexes to interact with DMSO on aquation, prompted\n We determined the pKa of the carboxylic acid groups (pKa2) us to probe the reactivity of the complexes against sulfur-\nin aqua adducts 1A and 2A and also in DMSO adducts containing biomolecules. For this experiment, we compared\n1DMSO and 2DMSO (Figure 4 and Table 1). Chart S2 Os-CN complexes 1\u22125 with their Os-NN counterparts\nsummarizes the speciation in aqueous solution of complexes (bearing bipyridine instead of phenylpyridine), as the Os\u2212Cl\n1\u22125 both in DMF-d7/D2O and in DMSO-d6/D2O. Titrations bond of the latter hardly hydrolyzes and presents a relatively\nin DMSO-d6/D2O helped us to unambiguously corroborate acidic coordinated water molecule (pK a1 < 6) upon\nOs\u2212DMSO adduct formation, as none of the titrations in 5% aquation.5,6 While the interaction with cysteine (Cys) was\nDMSO-d6/D2O yielded pKa data corresponding to the OH2\u21c6 easily followed over time for complex 5 by 1H NMR, the\nOH equilibria (pKa1), that is, the species in solution were analogue 5\u00b7NN [Os(\u03b76-C6H5(CH2)3OH)(bipy)Cl]+ did not\nindeed 1DMSO\u22125DMSO (Figure 4, Figure S20, and Table interact with the S-containing amino acid over 24 h (Figure\n1). Data of pKa2 demonstrated the acidity shift (ca. 0.4\u22120.8 S22). This observation corroborates the higher chemical\nunits) of the pendant \u2212COOH group upon metal-arene reactivity of Os-CN vs Os-NN complexes, and urged us to\ncoordination.56 Finally, the closed tethered complex [Os(\u03b76:\u03ba1- explore such Os-mediated reactivity inside the cell. To test our\nC6H5(CH2)3OH)(Phpy)], 5C, shows a protonation/deproto- hypothesis, we explored the intracellular activity of thioredoxin\nnation step for the alcohol/alkoxy group (Figure 4C orange) in reductase (TrxR) in MDA-MB-231 cells upon exposure to the\n 18975 https://doi.org/10.1021/acs.inorgchem.2c03246\n Inorg. Chem. 2022, 61, 18970\u221218978\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nosmium compounds. TrxR is not only highly relevant in ROS\nhomeostasis (in particular in cancer metabolism) but also, and\n \u25a0 AUTHOR INFORMATION\n Corresponding Author\ncrucial to our purpose, is rich in cysteine (and SeCys) Ana M. Pizarro \u2212 IMDEA Nanociencia, Ciudad Universitaria\nresidues,57 which might be good nucleophiles in substitution de Cantoblanco, Madrid 28049, Spain; Unidad Asociada de\nreactions at the Os\u2212OH2 labile position. We thus compared Nanobiotecnolog\u00eda CNB-CSIC-IMDEA, 28049 Madrid,\nthe effect on the enzymatic activity of TrxR in MDA-MB-231 Spain; orcid.org/0000-0003-3037-9835;\ncells of 1\u22125 with four of their Os-NN analogues. While 1\u22125 Email: ana.pizarro@imdea.org\ndecreased the activity of TrxR by ca. 60% (50 \u03bcM, 6 h of drug\nexposure), osmium complexes bearing NN-chelates did not Authors\ndecrease the TrxR activity (Figure 5). This is, to the best of our Sonia Infante-Tadeo \u2212 IMDEA Nanociencia, Ciudad\nknowledge, the first study of Os-mediated TrxR inhibition in Universitaria de Cantoblanco, Madrid 28049, Spain;\nliving cancer cells. Present Address: Department of Cell and Tissue Biology,\n University of California San Francisco, San Francisco,\n\u25a0 CONCLUSIONS\nWe report for the first time complexes 1\u22125 of the general\n California 94122, United States; orcid.org/0000-0001-\n 9476-886X\n Vanessa Rodr\u00edguez-Fanjul \u2212 IMDEA Nanociencia, Ciudad\nformulae [Os(\u03b76:\u03ba1-C6H5(CH2)nCOO)(Phpy)] (where Phpy Universitaria de Cantoblanco, Madrid 28049, Spain;\n= C,N-phenylpyridine; n = 1, 1; n = 2, 2), [Os(\u03b76- orcid.org/0000-0002-3386-5874\nC6H5(CH2)nCOOEt)(Phpy)Cl] (n = 1, 3; n = 2, 4), and Cintia C. Vequi-Suplicy \u2212 IMDEA Nanociencia, Ciudad\n[Os(\u03b76-C6H5(CH2)3OH(Phpy)Cl] (5). Complexes 1 and 2 Universitaria de Cantoblanco, Madrid 28049, Spain;\nare tethered complexes, that is, the Z position in [Os(\u03b76- orcid.org/0000-0003-1980-5039\narene)(Phpy)Z] is occupied by the pendant \u03ba1-O-bound\ncarboxylate (COO\u2212). We have recounted the impact of the Complete contact information is available at:\nphenylpyridine CN coordination on osmium chemical https://pubs.acs.org/10.1021/acs.inorgchem.2c03246\nreactivity at the Os\u2212Z bond, from the lack of reactivity in\npure DMSO to the readiness to interact with the sulfur- Author Contributions\ncontaining molecule upon activation through hydrolysis. A.M.P. conceived and designed the study. S.I.-T. developed the\nAquation is extraordinarily fast (<5 min), and the coordinated synthetic methodology, synthesized the complexes, and carried\nwater molecule is extraordinarily basic (pKa > 8). The Os- out the aqueous studies. S.I.-T. and V.R.-F. carried out the\nmediated reactivity is proven inside cells as per inhibition of experiments in cells and analyzed the data. C.C.V.-S. carried\nTrxR, while osmium does not impact the in-cell TrxR activity out the theoretical calculations. S.I.-T., V.R.-F., C.C.V.-S., and\nwhen phenylpyridine is replaced by neutral bipyridine. We A.M.P. discussed the findings and contributed toward writing\nbelieve that these are the fundamental basis to design organo- the manuscript. All authors have given approval to the final\nosmium complexes whose intracellular chemical reactivity is version of the manuscript.\nunderstood, thus enabling the development of valid Os-based Notes\ntools in cancer research. The authors declare no competing financial interest.\n\n\n\u25a0 ASSOCIATED CONTENT \u25a0 ACKNOWLEDGMENTS\n We thank Dr. J. Perles and Dr. M. Ram\u00edrez (Universidad\n*\ns\u0131 Supporting Information\n Aut\u00f3noma de Madrid) for the collection of X-ray data and Dr.\nThe Supporting Information is available free of charge at Z. Pardo (IMDEA Nanociencia) for assistance with NMR\nhttps://pubs.acs.org/doi/10.1021/acs.inorgchem.2c03246. experiments. We acknowledge funding from the EC (FP7-\n PEOPLE-2013-CIG, no. 631396), from the Spanish MINECO\n Experimental details and synthetic procedures for the\n (RYC-2012-11231, SEV-2016-0686, CTQ2017-84932-P, and\n synthesis of ligands, intermediates, and additional metal\n PID2020-117766GB-I00), and the Comunidad Aut\u00f3noma de\n complexes for comparison purposes (1\u00b7NN\u22123\u00b7NN and\n Madrid (Scholarship PEJD-2016/IND-2608).\n 5\u00b7NN); crystallographic data for 1\u00b71.5DCE, 3\u00b70.5H2O, 4,\n 1\u00b7NN\u00b7PF6\u00b7MeOH, and 2\u00b7NN\u00b7PF6\u00b7MeOH; supporting\n figures, charts, and tables (PDF) \u25a0 REFERENCES\n (1) Wang, D.; Lippard, S. J. Cellular processing of platinum\n anticancer drugs. Nat. Rev. Drug Discovery 2005, 4, 307\u2212320.\nAccession Codes (2) Peacock, A. F. A.; Parsons, S.; Sadler, P. J. 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