cis -Tetrachlorido-bis(indazole)osmium( IV ) and its osmium( III ) analogues: paving the way towards the cis -isomer of the ruthenium anticancer drugs KP1019 and/or NKP1339 †‡

The relationship between cis – trans isomerism and anticancer activity has been mainly addressed for square-planar metal complexes, in particular, for platinum( II ), e.g. , cis - and trans -[PtCl 2 (NH 3 ) 2 ], and a number of related compounds, of which, however, only cis -counterparts are in clinical use today. For octahedral metal complexes, this e ﬀ ect of geometrical isomerism on anticancer activity has not been investigated systematically, mainly because the relevant isomers are still unavailable. An example of such an octahedral complex is trans -[RuCl 4 (Hind) 2 ] − , which is in clinical trials now as its indazolium (KP1019) or sodium salt (NKP1339), but the corresponding cis -isomers remain inaccessible. We report the synthesis of Na[ cis -Os III Cl 4 ( κ N2-1 H -ind) 2 ]·(Na[ 1 ]) suggesting a route to the cis -isomer of NKP1339. The procedure involves heating (H 2 ind)[Os IV Cl 5 ( κ N1-2 H -ind)] in a high boiling point organic solvent resulting in an Anderson rearrangement with the formation of cis -[Os IV Cl 4 ( κ N2-1 H -ind) 2 ] ([ 1 ]) in high yield. The transformation is accompanied by an indazole coordination mode switch from κ N1 to κ N2 and stabilization of the 1 H -indazole tautomer. Fully reversible spectroelectrochemical reduction of [ 1 ] in acetonitrile at 0.46 V vs. NHE is accompanied by a change in electronic absorption bands indicating the formation of cis -[Os III Cl 4 ( κ N2-1 H -ind) 2 ] − ([ 1 ] − ). Chemical reduction of [ 1 ] in methanol with NaBH 4 followed by addition of n Bu 4 NCl a ﬀ orded the osmium( III ) complex n Bu 4 N[ cis -Os III Cl 4 ( κ N2-1 H -ind) 2 ] ( n Bu 4 N [ 1 ]). A metathesis reaction of n Bu 4 N[ 1 ] with an ion exchange resin led to the isolation of the water-soluble salt Na[ 1 ]. The X-ray di ﬀ raction crystal structure of [ 1 ]·Me 2 CO was determined and compared with that of trans -[Os IV Cl 4 ( κ N2-1 H -ind) 2 ]·2Me 2 SO ( 2 ·2Me 2 SO), also prepared in this work. EPR spectroscopy was performed on the Os III complexes and the results were analyzed by ligand-ﬁ eld and quantum chemical theories. We furthermore assayed e ﬀ ects of [ 1 ] and Na[ 1 ] on cell viability and proliferation in comparison with trans -[Os IV Cl 4 ( κ N1-2 H -ind) 2 ] [ 3 ] and cisplatin and found a strong reduction of cell viability at concentrations between 30 and 300 µM in di ﬀ erent cancer cell lines (HT29, H446, 4T1 and HEK293). HT-29 cells are less sensitive to cisplatin than 4T1 cells, but more sensitive to [ 1 ] and Na[ 1 ], as shown by decreased proliferation and viability as well as an increased late apoptotic/necrotic cell population.

After the discovery of the antiproliferative activity of cisplatin in 1965, 17 the cis-geometry was long believed to be a prerequisite for the anticancer activity of metal compounds. 18,19ven though this situation changed over the years as several trans-isomers were synthesized and discovered to exhibit higher antiproliferative potency than their cis-congeners, 20 with some of them having the advantage of not showing cross resistance to cisplatin, 21 there are no trans-isomers in clinical use today.However, all these examples are dealing with square-planar metal complexes.The importance of the cistrans isomerism in octahedral coordination complexes has been known since Alfred Werner's time. 22In the case of octahedral ruthenium and osmium compounds, some cases of isomeric pairs are well-documented for azole heterocycles 23,24 and bis-picolinamide ruthenium(III) dihalide complexes. 25The structurally related osmium(III) pyridine derivatives, cis-and trans-[OsCl 4 (Py) 2 ] − are also known, and both were crystallographically characterized. 26Overall, the number of cytotoxic Werner's type osmium complexes is increasing. 27However, elucidation of structure-activity relationship remains an important task for the understanding of the mode of action of such complexes and the development of more effective anticancer agents than those exploited today.
The present study of [1] adds to the growing family of osmium(IV) and osmium(III) azole compounds. 28The complexes presented in this work are stable in aqueous and/or Me 2 SO solution for at least 24 h (osmium(III) compounds in the absence of air oxygen).Upon one-electron reduction of the metal center the tautomer coordinated to metal remains intact as also reported for trans-[Os III Cl 4 (κN1-2H-ind) 2 ] − . 29Quite recently, a pH dependent indazole tautomerization has been reported, wherein the stabilization of one of the tautomers was achieved by coordination of an indazole-pendant azamacrocyclic scorpiand to Cu II or Zn II . 30erein, we describe the synthesis of cis-[Os IV Cl 4 (κN2-1Hind) 2 ] ( [1]) and its one electron reduced species (nBu 4 N) [1] and Na [1] (Chart 2).These complexes are spectroscopically characterized, in particular by electron paramagnetic resonance (EPR), a technique which has been shown to be very useful in unravelling the solution structure of Ru III anti-cancer drug candidates, 31 as well as other Ru III complexes. 32EPR has been less widely applied to Os III complexes of any type because of the paucity of such compounds and frequently their inherently more challenging experimental EPR properties. 33,34In addition, the antiproliferative activity of the two octahedral cis-isomers [1]  and Na [1], along with the X-ray diffraction structures of cis-and trans-[Os IV Cl 4 (κN2-1H-ind) 2 ] ([1] and [2], respectively) are reported.This discovery offers the pathway to cis-isomers of investigational anticancer drugs KP1019 and NKP1339 and a subsequent opportunity to investigate structure-activity relationships for octahedral cis/trans-[RuCl 4 (κN2-1H-ind) 2 ] − isomers.

Physical measurements
UV-vis spectra were recorded on a Jasco V-630 UV-vis spectrophotometer using samples dissolved in Me 2 CO, MeCN, DMF, Me 2 SO, EtOH, and THF.MIR spectra in the region 4000-600 cm −1 were recorded with a Thermo Nicolet NEXUS 470 FT-IR spectrometer (Thermo Fisher Scientific, USA), in KBr pellets.Electrospray ionization mass spectrometry was carried out with a Thermo Scientific LCQ Fleet ion trap instrument (Thermo Scientific, Waltham, MA, USA) by using methanol as a solvent.High-resolution mass spectrometry (HRMS) was performed on a LTQ Orbitrap Velos instrument (Thermo Scientific, Waltham, MA, USA).Expected and measured isotope distributions were compared.The cyclic voltammetric studies were performed in a home-made miniature electrochemical cell using a platinum wire as working and counter electrodes, and a silver wire as a pseudoreference electrode.Ferrocene served as the internal potential standard.The redox potentials in Me 2 SO solutions were recalculated vs.Standard Hydrogen Electrode (NHE) using the known redox potential of ferrocene (0.64 V) vs. NHE.A Heka PG310USB (Lambrecht, Germany) potentiostat with a PotMaster 2.73 software package was used in cyclic voltammetric and spectroelectrochemical studies.In situ spectroelectrochemical measurements were performed on a spectrometer Avantes, Model AvaSpec-2048x14-USB2. Halogen and deuterium lamps were used as light sources (Avantes, Model AvaLight-DH-S-BAL) under an argon atmosphere in a spectroelectrochemical cell kit (AKSTCKIT3) with the Pt-microstructured honeycomb working electrode, purchased from Pine Research Instrumentation.The cell was positioned in the CUV-UV Cuvette Holder (Ocean Optics) connected to the diode-array UVvis-NIR spectrophotometer by optical fibers.UV-vis-NIR spectra were processed using the AvaSoft 7.7 software package.The 1 H and 13 C NMR spectra were recorded at 500 and 125 MHz on a Bruker Avance III 500 MHz (Ultrashield Magnet) instrument by using MeOH-d 4 and Me 2 SO-d 6 as solvents.The solvent residual peak for 1 H and 13 C was used as an internal reference.EPR spectra were recorded using an EMXplus X-band EPR spectrometer (Bruker, Germany) equipped with a high-sensitivity resonator ER 4119 HS and an ER 4141 VT variable temperature unit as well as with a modified Bruker EMX spectrometer equipped with an Oxford Instruments cryostat for liquid helium temperature (10-20 K) X-band spectra and a modified Varian spectrometer for superfluid helium temperature (2 K) Q-band (∼35 GHz) spectra.The EPR spectra were analyzed and simulated with the Easyspin toolbox. 36Ligand-field parameters for LS d 5 systems were calculated using a fitting program originally provided by B. R. McGarvey 33b and modified by J. Telser. 32ystallographic structure determination X-ray diffraction measurements of [1]•Me 2 CO and [2]•2Me 2 SO were performed on a Bruker X8 APEX-II CCD diffractometer at 199 and 100 K, respectively.The data were processed using SAINT software. 37The structures were solved by direct methods and refined by full-matrix least-squares techniques.Non-hydrogen atoms were refined with anisotropic displacement para-meters.H atoms were placed at calculated positions and refined as riding atoms in the subsequent least squares model refinements.The isotropic thermal parameters were estimated to be 1.2 times the values of the equivalent isotropic thermal parameters of the non-hydrogen atoms to which hydrogen atoms are bonded.The following computer programs and equipment were used: solution SHELXS-2014 and refinement, SHELXL-2014; 38 molecular diagrams, ORTEP; 39 computer, Pentium IV.Crystal data, data collection parameters, and structure refinement details are given in Table 1.CCDC 1544885 and 1544886 contain the CIFs for this manuscript.

Computational details
The Gaussian09 package 40 has been used for the geometry optimizations of the anion 2 [1] − (total charge minus one and doublet spin state).The unrestricted B3LYP 41 /def2-TZVP 42 level of theory has been employed and the core electrons of osmium were treated via the SDD pseudo potential. 43ibrational analysis showed that the optimized geometry of 2 [1] − is free of imaginary (negative) frequencies.
The g-tensor values 44 have been theoretically determined using two different approaches.The first attempt employed the ORCA software package 45 at the DFT (with either BLYP or B3LYP functional) and complete active space self-consistent field (CASSCF) levels of theory.Here the scalar ZORA Hamiltonian 46 and uncontracted double zeta (UDZ) basis sets 47 have been used together with inclusion of solvent effects via the conductor-like screening model (COSMO) 48 of Me 2 SO.The spin-orbit coupling (SOC) effects have been included as a perturbation (with ORCA input settings: grid 7; soctype 3; socflags 1,3,3,1).The CASSCF calculation used five electrons in five orbitals and has been run as state specific (accounting formally for 10 roots).SOC treatment in CASSCF has been performed under default settings of the relativity module (dosoc true; g-tensor true).In the second approach, the ReSpect program package 49 has been employed utilizing the four-component relativistic Dirac-Kohn-Sham (DKS) methodology. 50,513][54] The non-collinear Kramers unrestricted formalism was used as the best method currently available for description of spin polarization at the relativistic DFT level of theory. 51,54,55The recent implementation of solvent models in the ReSpect program 51 allowed us to take into account the effects of the environment (Me 2 SO) in the framework of the COSMO model.

Cell lines and culture conditions
H446 small cell lung carcinoma (American Type Culture Collection, Manassas, VA, USA) and HEK293 embryonic kidney cells (a kind gift from the laboratory of Dr Ross Levine, MSKCC, New York, NY, USA) were maintained in minimal essential medium.4T1 breast carcinoma (American Type Culture Collection, Manassas, VA, USA) cells were grown in RPMIM.HT-29 colorectal adenocarcinoma cells (a kind gift from the laboratory of Dr Vladimir Ponomarev, MSKCC, New York, NY, USA) were grown in McCoy's medium.All media were supplemented with 10% fetal calf serum, penicillin (100 IU) and streptomycin (100 μg mL −1 ).All the cell lines were grown in 150 cm 2 culture flasks (Corning Incorporated, New York, NY, USA) under standard sterile cell culture conditions at 37 °C under a humidified atmosphere with 5% CO 2 and 95% air.

Cytotoxicity in cancer cell lines
Cell viability was tested by the Alamar Blue assay (Invitrogen, Carlsbad, CA, USA).Stock solutions of the osmium compounds in Me 2 SO were prepared at a concentration of 30 mM.Cisplatin was dissolved in 0.9% aqueous solution of NaCl.Na [cis-Os III Cl 4 (Hind) 2 ] (Na [1]) stock solution was prepared under an argon atmosphere to prevent direct contact with oxygen.Serial dilutions of the osmium compounds and cisplatin were prepared in the corresponding nutrient medium, ranging from 0.003 μM to 300 μM.All dilutions contained a final Me 2 SO concentration of 1%.For the Alamar Blue assay, cells were plated into 96-well plates (black, clear-bottom, Corning Incorporated) at a density of 4000 cells per well and allowed to adhere for 24 h.Then, the cells were incubated with osmium compounds or vehicle control (medium with Me 2 SO) for 48 h.The Alamar Blue reagent was used as described by the manufacturer.The Alamar Blue reagent was diluted 1 : 10 in full culture medium to achieve the working solution.The medium was removed from 96-well plates and 100 μl Alamar Blue reagent was added.
The plates were incubated at 37 °C for 3 h under protection from direct light.Fluorescence was read (bottom-read mode) using an excitation wavelength of 570 nm and an emission wavelength of 585 nm on a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA).The average reading values were analyzed by subtracting the background readings (wells with Alamar Blue but without cells) and compared against the untreated controls of each plate (defined as 100% viability) to determine the percentage of living cells.The experiment was carried out in six parallels of each compound.

Measurement of antiproliferative activity and apoptosis induction
We evaluated the potential of complexes [1] and Na [1] on cell viability, proliferation and apoptosis induction in comparison with trans-[Os IV Cl 4 (κN1-2H-ind) 2 ] ([3]), 15 cisplatin and untreated cells using an Annexin V Apoptosis Detection Kit (#640914; BioLegend).Thus, we plated 200 000 HT-29 or 4T1 cells per well in 6-well plates.After 24 h medium was removed and the cells were incubated with fresh medium or a 200 µM solution of [1], complex Na [1], [3] or cisplatin in medium for 48 h.After trypsinization, the supernatant and cells were collected in Falcon tubes. 1 ml of the suspension was analyzed in an automated cell counter (Beckman Coulter Counter) for cell count and viability.The remaining cells were stained with a FITC Annexin V (AV) and Propidium Iodide (PI) Kit as described by the manufacturer.The cells were washed twice with cell staining buffer (#420201; BioLegend), resuspended in Annexin binding buffer and transferred to a flow tube with a cell strainer cap (0.2-1 × 10 6 cells in 100 µl).Then, 5 µl Annexin V were added per sample, followed by 10 µl PI.The tubes were incubated for 15 min in the dark and then analyzed on a LSR Fortessa III Flow Cytometer (BD Biosciences).
Compensation was carried out using single stained controls.The cells that stained positive for Annexin V were considered early apoptotic, Annexin V and PI positive cells were considered late apoptotic or necrotic and the cells positive only for PI were considered necrotic.

Crystal structures
The results of X-ray diffraction studies of complexes [1]•Me 2 CO and [2]•2Me 2 SO are shown in Fig. 1.Crystallographic data are summarized in Table 1, while selected bond lengths and angles are quoted in Table 2. To the best of our knowledge these are the first tetrachlorido-bis(1H-indazole)osmium(IV) complexes to be crystallographically characterized.[1]•Me 2 CO and [2]•2Me 2 SO crystallized in the monoclinic centrosymmetric space groups P2 1 /c and P2 1 /n, respectively.The first contains one molecule of cis-[Os IV Cl 4 (1H-ind) 2 ] and one molecule of Me 2 CO, while the second contains one molecule of trans-[Os IV Cl 4 (1H-ind) 2 ] and two molecules of Me 2 SO in the asymmetric unit.The osmium atom in [1] displays a distorted octahedral coordination geometry with two chlorido ligands and two 1H-indazole ligands in equatorial positions, and two chlorido ligands bound axially.In [2] osmium(IV) adopts a compressed octahedral coordination geometry with four chlorido ligands occupying the equatorial plane and two 1H-indazole ligands in the axial positions.In a few cases reported in the literature, 1H-indazole was found to be deprotonated, acting as a bridging ligand in polynuclear metal complexes, 61 or even more rarely it operates as a monodentate indazolato ligand, such as when coordinated via N2 to platinum and deprotonated at N1 in the square planar complex [PtCl(N-indazolato)(PPh 3 ) 2 ]. 62 A comparison of the bond lengths in the coordination sphere of osmium in [1], [2], and [3] is shown in Table 2. Inspection of geometric parameters in complexes [1] and [2]  with the same coordination mode of indazole shows that the Os-N2 and Os-N11 bonds are significantly longer (0.02 Å; 5.5 × σ) in the cis-than in the trans-isomer (see Fig. 1 and Table 2).The same trend is clear when the Os-N bonds in ciscomplex [1] are compared to those in trans-isomer [3] with a dissimilar coordination mode of indazole (see Chart 2).This is presumably due to a stronger trans-influence of the chlorido ligand compared to nitrogen atom N2 or N1 (using the numbering for indazole in Chart 1) of indazole.Interestingly, an insignificant (<3 × σ) shortening of axial Os-N bonds in transcomplex [3] compared to that in trans-complex [2] (coordination modes are different) results in a significant (14 × σ) lengthening of equatorial Os-Cl bonds by ca.0.03 Å (see Table 2) in [3] vs. those in [2].
A salient structural feature is the stabilization of indazole in its usual 1H-indazole form in [1] and [2] (Fig. 1) and coordination to osmium via nitrogen atom N2.This mode of coordination is typical of other metal ions, e.g.ruthenium, but rare for osmium(IV).The only previous example for osmium, namely H 2 ind[Os IV Cl 5 (1H-ind)], was reported by some of us recently. 57ntermolecular hydrogen bonding interaction between complexes [1] and [2] and co-crystallized solvent molecules are shown in Fig. S2 and S3 in the ESI.‡

NMR spectra
All compounds were characterized by 1 H NMR spectra showing signals due to the coordinated indazole.In the spectrum of nBu 4 N [1], signals of the Bu 4 N + cation could also be assigned.The paramagnetic osmium(IV) in [1] caused some line broadening and spread of chemical shifts from −9.42 to +16.49 ppm (Fig. S4 ‡).It should, however, be noted that despite the paramagnetism of osmium(IV) in [1], the NMR lines remain sharp enough such that coupling constants could be easily determined.Qualitatively, the fact that the nuclear paramagnetic resonance spectrum of [1] is readily interpretable is unsurpris-Fig. 1 ORTEP views of [1] and [2] thermal ellipsoids are drawn at the 50% probability level.
Table 2 Selected bond lengths (Å) and angles (°) in the coordination polyhedron of osmium(IV) in [1], [2] and [3]  ing for Os IV (low-spin (LS) 5d 4 , S = 1) because this ion is expected to have very fast electron spin relaxation 64 due to the substantial orbital angular momentum of its ground state and the extremely large spin-orbit coupling (SOC) constant of Os IV (free-ion Os 4+ (g) (Os(V) in spectroscopic notation) has ζ d = 4133.7 cm −1 ); 65 ions with slow electronic relaxation, such as high-spin (HS) 3d 5 S = 5/2 (e.g., Fe III ), in contrast, typically give unusable NMR spectra. 64The number of carbon resonances in 13 C NMR spectra of [1] is in agreement with C 2 molecular symmetry of the complex.The broadening of resonances due to paramagnetism of osmium(III) (low-spin 5d 5 S = 1/2) in nBu 4 N [1] and Na [1] is more obvious compared to that for [1] and signal splitting for indazole protons is no more discernible.All signals are significantly upfield shifted being in the δ range from 3.55 to −22.16 ppm.

Electrochemical behavior
The cyclic voltammogram of [1] in a solution of 0.2 M nBu 4 NPF 6 in acetonitrile starting from the anodic part using a platinum wire as the working electrode and a scan rate of 0.2 V s −1 is shown in Fig. 3.
The first reduction step is reversible (E 1/2 = 0.69 V vs. NHE or +0.01 V vs. Fc + /Fc) at all scan rates used (from 10 mV s −1 to 500 mV s −1 ), and is followed by the less reversible one at a more negative potential (E 1/2 = −0.78V vs. NHE).Upon in situ reduction at a scan rate of 10 mV s −1 in the region of the first reversible cathodic peak in acetonitrile, the UV-vis absorption bands at 408, 370 and 272 nm decreased, and, simultaneously, a new dominating band at 318 nm appeared (Fig. 4a).Fully reversible spectroelectrochemical behavior confirmed the high stability of cathodically generated cis-[Os III Cl 4 (κN2-1H-ind) 2 ] − ([1] − ) species.A very similar reversible cyclic voltammetric and spectroelectrochemical response was observed for [1] also in Me 2 SO at the first reduction peak even at a very low scan rate of 5 mV s −1 as illustrated in Fig. S5.‡ Here the UV-vis absorption bands of [1] in Me 2 SO at 415, 370 and 280 nm decreased, and simultaneously, a new dominating band at 325 nm was observed.The spectroelectrochemical data indicated that the isolation of the osmium(III) species in the solid state after reduction of the parent osmium(IV) complex is feasible.The chemically prepared and isolated osmium(III) complex nBu 4 N [1] can be easily oxidized in Me 2 SO back to [1] 0 at anodic potential E = +1.0V vs. NHE as shown in Fig. S6.‡ Fig. 4c shows the absorption spectra of the initial cisisomer [1] (cyan trace) and one-electron reduced species, namely ([1] − ) (dark yellow trace).The UV-vis spectrum of the osmium(III) species [1] − generated electrochemically exhibits an absorption maximum at a slightly higher wavelength (318 nm in acetonitrile and 325 nm in Me 2 SO) compared to its analogue trans-[Os III Cl 4 (κN1-2H-ind) 2 ] − ([3] in Me 2 SO) reported previously 29 with λ max at 301 nm.Of note is a more negative reduction potential with a shift of −200 mV reported recently for [3] 29 with E 1/2 = +0.49V vs. NHE or E 1/2 = −0.19V vs. Fc + /Fc for the first reduction step.A significant difference in the E 1/2 values for [1] and [3] indicates a stronger effect of cis/trans isomerism on reduction potentials than that of the coordination mode of indazole in the compared species.
As mentioned previously, the Os III/II couple is much less reversible than the Os IV/III couple and takes place at much more negative potentials.Upon repetitive redox cycling of sample 1 going up to the voltage of the second reduction peak, Fig. 2 UV-vis spectra of solutions of [1] in Me 2 CO (black), acetonitrile (red), DMF (blue), Me 2 SO (green), ethanol ( purple) and THF (orange).irreversible changes were observed in cyclic voltammetric experiments in the thin layer cell at a scan rate of 10 mV s −1 both for Me 2 SO and acetonitrile solutions as illustrated in Fig. S7.‡ New redox active species are formed during the reduction of [1] − in the region of the second electron transfer indicating a lower stability of the generated osmium(II) complex.The changes in UV-vis spectra observed during cyclic voltammetry of [1] − going to the region of the second reduction in the Me 2 SO solution (scan rate 5 mV s −1 ) are shown in Fig. 5.A new optical band arises at ca. 440 nm in the region of the second electron transfer (see Fig. 5c and Fig. S8 and S9 ‡) which again disappears upon reoxidation.The irreversible changes in the initial optical spectra of [1] were observed confirming the irreversible chemical changes of the corresponding osmium(II) species [1] 2− formed upon cathodic reduction of [1] at the second reduction step.Interestingly, a newly formed product can be reversibly reduced at E 1/2 = +0.27V vs. NHE (see blue trace in Fig. S7 ‡).Cathodically induced metal dechlorination was previously mentioned for the trans-[Ru III Cl 4 (Hind) 2 ] − complex 71 upon reduction of ruthenium(III) to ruthenium(II) and for the trans-Os III analogues. 29Similarly, our findings indicate that a chlorido ligand is replaced by a solvent molecule upon reduction of osmium(III) species and a new redox active osmium(II) complex containing a coordinated solvent molecule is formed at the second reduction potential of [1] 0 (respectively at the first reduction peak for [1] − ).

EPR of osmium(III) complexes
As mentioned previously, the NMR resonances of the osmium(III) complexes nBu 4 N [1] and Na [1] were considerably broadened due to the rapid nuclear relaxation induced by the paramagnetism of these compounds, which could be expected from the LS d 5 electron configuration of the central metal.33b,c EPR is a valuable technique to unravel the underlying mechanisms of action of paramagnetic anticancer drugs and, in particular, those of ruthenium(III). 31The X-band EPR spectra of frozen solutions of nBu 4 N [1] (in Me 2 SO at 94 K) and Na [1] (in H 2 O/ethylene glycol 5 : 1 v/v at 12 K) are shown in Fig. 6 and  confirm the paramagnetic nature of the complexes, while a Q-Band EPR spectrum of Na [1] and a powder EPR spectrum of nBu 4 N [1] are shown in Fig. S10 and S11, ‡ respectively.
Both spectra show rather broad lines with ΔB pp > 150 mT and not a very well resolved spectral pattern, characterized by a rhombic g-tensor. 72The g values obtained by the simulation of the spectra (Table 3, g max ≈ 2.50, g mid ≈ 1.45 and g min ≈ 1.35) are very similar and can be considered virtually identical for both complexes, if we take into account the uncertainty of g-value determination induced by the large linewidth.Thus the cis-[Os III Cl 4 (κN2-1H-ind) 2 ] − complex anion geometry in frozen solution as well as the electronic configuration are not influenced by the different counterions that govern the solubility of nBu 4 N [1] and Na [1] in an organic or aqueous environment, respectively.An unambiguous conclusion about the spin state of a complex cannot be made based solely on a single frequency cw-EPR experiment. 73However, the interactions between the unpaired electrons in the high spin states usually result in a facilitated electron spin relaxation, and the consequent line broadening renders their EPR spectra often undetectable above liquid helium temperatures.The fact that we were able to record the spectrum of nBu 4 N [1] at 94 K and a comparable linewidth was observed in the spectrum of Na [1] at 12 K, indicate the absence of such electron-electron interactions.Moreover, the 35 GHz EPR spectrum of Na [1]  (Fig. S10 ‡) confirms the g max value from X-band.The osmium(III) ion is thus in the usual low spin d 5 (S = 1 2 ) configuration, in both nBu 4 N [1] and Na [1], as reported for the vast majority of Os III complexes in the literature.33a,b Ligand field theory discussion of 2 [1] − g-tensor The g values of LS nd 5 complexes have been extensively analyzed over the years because of the importance of this electronic configuration in heme proteins and other bioinorganic systems. 74The model that we shall use for a simple ligandfield theory (LFT) analysis of the g tensor of 2 [1] − is that developed by McGarvey.33b,c The details need not be discussed here, but the model treats this t 5 2 e 0 g configuration (strong-field notation) by ignoring any effects of excited states involving the e g orbitals and using the "hole" in the t 2g orbitals as a basis set as follows: 75 The (axial) splitting between the d xy and d xz,yz orbitals is given by Δ and the (rhombic) splitting between the d xz and d yz orbitals is given by V.This analysis has been used previously for Ru III complexes of general type trans-[Ru III (NH 3 ) 4 (L) (L′)] n+ , 76 which could be considered as cationic analogues to the equatorially tetrachlorido coordinated anionic Ru/Os III complexes.To the best of our knowledge, no EPR spectra of complexes of general formula cis-[Os III X 4 (L)(L′)] have been reported.Clarke and co-workers have reported EPR spectra for cis-[Ru III (NH 3 ) 4 (Him) 2 ]Br 3 . 77It is possible to use analytical equations given by McGarvey to extract an internally consistent set of LFT parameters from the experimental g values. 78This has been done here and the results are given in Table 4.In each case, there are two solutions, but usually, one can be discarded as it requires unreasonable parameters, e.g., the orbital reduction factor, k, is much too far from 1.0, or the splitting among the t 2g orbitals is much too large.Note that in this model it is not possible to provide the absolute energy splitting among the t 2g orbitals, only their energies normalized by the spin-orbit coupling (SOC) constant, ζ.This parameter has recently been accurately determined for free-ion Os 3+ (g) (Os IV in spectroscopic notation) in its 5d 5 electronic configuration: 79 The reduction of this parameter due to covalency in these complexes is unknown, but values of ζ = 2530 and 2220 cm −1 in trans-[OsCl 4 (CO)( py)] − ( py = pyridine) are found respectively for Cs + and tetraethylammonium countercations. 34The fact that the SOC constant is so sensitive even to counterions indicates the difficulty of quantifying this interaction, but an estimate of ζ ≈ 2500 cm −1 seems reasonable in Fig. 6 (Top) X-band (9.436 GHz) EPR spectrum of nBu 4 N [1] in Me 2 SO recorded at 94 K (solid trace) together with the simulation (dashed trace).(Bottom) X-band (9.362 GHz) EPR spectrum of Na [1] in H 2 O/ ethylene glycol 5 : 1 v/v recorded at 12 K (solid line) together with the simulation (dashed line).Spin Hamiltonian parameters are listed in Table 3. * denotes the signal from an impurity., where L = Me 2 SO 80 or various azole ligands, e.g., pyrazole, as well as pyridine. 81The key point, however, is not that whether a (Ru,Os) III species exhibits a rhombic or axial spectrum, but whether g ∥ (i.e., the unique feature) is greater (at lower resonant magnetic field) or lesser (at higher field) than g ⊥ (averaging a rhombic signal if necessary).In general, the (Ru,Os) III species listed in Table 4 shows the reversed g value ordering.
McGarvey has explained the electronic structure of trans-[MX 4 (Y 2 ,YZ)] complexes (where M = Ru III most commonly) in terms of an angular overlap model (AOM) wherein the z axis is along Y-M-Y(Z) and the relevant AOM parameter is π-bonding between the metal ion and each ligand type, ε π (X, Y, Z), so that McGarvey showed that this model works reasonably well for simple complexes such as trans-[RuCl 2 (NH 3 ) 4 ] + .More relevant here, the model also works qualitatively for trans-[OsBr 4 (CO) 2 ] − : the bromido ligands are expected to be π-donors (i.e.,ε π (X) > 0) and the carbonyl ligands are expected to be strong π-acceptors (i.e., ε π (Y) < 0).This would give Δ > 0, consistent with what is obtained from the g value analysis.No cis-[MX 4 (Y 2 ,YZ)] complexes were known to McGarvey, but he proposed that Δ = −ε π (X) + ε π (Y) for this geometry.33b The chlorido ligands (X) are expected to be π-donors and the indazole nitrogen atoms (Y) are expected to be weak π-acceptors ε π (Y) ≲ 0, which would give Δ < 0, again consistent with the g values.
A further discussion of the LFT parameters of these complexes is beyond the scope of this study, and would be in any case risky due to the overall relatively small number of Os III coordination complexes characterized by EPR, and in particular, the lack of data on other Os III cis/trans isomers.However, it is clear that the cis-Os III complex, [1] − , as manifest in its "reversed" EPR spectrum, exhibits an electronic structure distinctly different from almost all of the related species listed in Table 4, in particular the trans-Os III complexes.The wavefunction coefficients resulting from this analysis show that the "hole" is primarily in the d   80 The g values reported are for the solid material; in aqueous or methanol solution, multiple species are reported due to replacement of Me 2 SO and/or chlorido ligands by solvent.e Taken from Clarke et al. 77 The values for |g min | were not observed (hence they are given in parentheses), but were proposed by their LFT analysis.In these systems, the proper choice of sign of the g values is not apparent based on standard criteria, namely the magnitudes of k or Δ, so both choices are given.The results of additional calculations are given; for the trans complex, using a value for |g min | that would likely be the largest possible, but difficult to observe experimentally: |g min | = 0.9; for the cis complex using the a value for |g min | that would likely be the smallest possible: |g min | = 0.15 (a value of zero is possible, but does not provide enough information for the fitting process).We favor the first entry listed in each case, which is in agreement with the model originally favored by Clarke et al. electronic ground state. 32A quantum chemical theory (QCT) treatment presented in the next section provides further insight into [1] − .
Theoretical determination of 2 [1] − g-tensor QCT calculations of EPR parameters of compounds containing heavy elements are challenging due to a dominating importance of spin-orbit relativistic effects.2][53][54][55] Furthermore, the solvent polarization effects will be considered for further consistency via the COSMO approach.The calculated g-tensor values are shifted from the experimental ones in the case of scalar ZORA DFT calculations with the perturbative inclusion of SOC.The solvent effects are large within the ZORA DFT treatment but do not lead to improvement with respect to experiments (Table 5).Considerably better agreement is achieved in the case of ZORA CASSCF calculations which yield a semi quantitative agreement with experiment [both in vacuo and via the COSMO(Me 2 SO) solvent model].Nevertheless, the CASSCF setup has been kept under conservative settings for the rotations out of the active space to enforce the d-electronic configuration of the open shell and/or Fig. 7 Cell viability of human cancer cell lines after a 48 h treatment with cisplatin, [1], Na [1] and [3], in relation to the untreated control (1% Me 2 SO) using the Alamar Blue assay kit.Displayed are means ± standard deviation of six parallels.
in the active space.Although the rigorous DKS method does not yield the best agreement in vacuo, introducing a solvent model leads to a large improvement of the calculated results providing almost quantitative agreement with the experiment.This once again shows the need for a suitable treatment of both relativistic and spin polarization effects in the calculation of EPR parameters for 5d ions.As such, the calculated solvent effects on the electronic g-tensor parameters are so far the largest ones observed at the DKS level of theory and to the best of our knowledge, they are the largest ones calculated for systems containing heavy elements.Furthermore, the excellent agreement of DKS/COSMO theory with experimentally determined values is promising for a more systematic theoretical exploration of experimentally determined g-tensors for osmium(III) compounds, 34 and potentially other paramagnetic 5d 5 ion complexes, such as of rhenium(II) 84 or iridium(IV). 85fective concentrations to affect cell viability Having now the availability of well-characterized cis-as well as trans-osmium(III) complexes it is possible to make a direct comparison of their anti-cancer efficacy.Specifically, first to determine in which concentration range these complexes mediate cytotoxic effects, we conducted a cytotoxicity screen in four different cancer cell lines (HT29, 4T1, HEK293 and H446) and compared the effects to an untreated control and cispla-tin.The results for select drug concentrations are displayed in Fig. 7. Strong effects on cell viability were only observed for the highest concentration of 300 µM, where all cell lines showed a viability of below 30% compared to the control, with the exception of 4T1 cells to treatment with trans-[Os IV Cl 4 (kN1-2H-ind) 2 ] ( [3]).At 30 µM cisplatin reduced cell viability to 30-60%, while the osmium compounds did not reduce viability.HT29 cells were the only cells where the osmium compounds led to a stronger reduction of cell viability than cisplatin.The corresponding IC 50 values are quoted in Table S1.‡

Antiproliferative activity in cancer cell lines
We further determined the potential of complexes [1] and Na [1]  to inhibit proliferation and induce apoptosis in comparison with trans-[Os IV Cl 4 (κN1-2H-ind) 2 ] ( [3]) and cisplatin in HT29 and 4T1 cells at a concentration of 200 µM.Here, we used an apoptosis detection kit to quantify the percentage of a cell population that has entered an early apoptotic (AV+/PI−) or late apoptotic/necrotic state (AV+/PI+) using flow cytometry (Fig. 8a).We found that the 4T1 (breast cancer) cells were very sensitive to cisplatin (more than 60% increase in AV+ and AV+/PI+ cells compared to untreated cells), while HT29 (colorectal) cells showed no significant difference between untreated and cisplatin treated cells (Fig. 8b).Interestingly, the cisplatin resistant HT29 cells were more sensitive to treatment with the osmium complexes than 4T1 cells.In particular, the AV+/PI+ population increased significantly to 40.3% ([1]), 36.0%(Na [1]) and 57.2% ( [3]) upon treatment (vs.15.1% untreated, p < 0.05), indicating that the trans-isomer induced apoptosis most effectively in HT29 cells (Fig. 8b).
In addition to flow cytometry data, we also measured cell viability and cell number of each sample in an automated cell counter.We found more pronounced effects on cell count than on cell viability, indicating that proliferation was affected, but the cells remained viable (Fig. 8c).In HT29 cells, cell viability was significantly affected by cisplatin, Na [1] and [3].The cell number was strongly reduced by over 75% by all osmium compounds and to 41% by cisplatin (Fig. 8c).In 4T1 cells, the strongest effect on cell count was observed for the cis-compound Na [1] (20.9% of untreated), followed by cisplatin at 39.5% (Fig. 8c).

Conclusion
In this work we reported on the synthesis and crystal structure of cis-[Os IV Cl 4 (κN2-1H-ind) 2 ] ( [1]), the first cis-configured isomer in the series of compounds of the general formula [Os IV Cl 4 (Hazole) 2 ], where Hazole = indazole, pyrazole, benzimidazole, and imidazole.In addition, we showed that trans-[Os IV Cl 4 (κN2-1H-ind) 2 ] ( [2]) is also available, although more effective synthetic routes still need to be developed.Both compounds are complementary to their elsewhere reported trans-isomer [Os IV Cl 4 (κN1-2H-ind) 2 ] ( [3]).Electrochemical and chemical one-electron reduction of [1] afforded cis-configured osmium(III) species isolated as tetrabutylammonium and sodium salts, nBu 4 N [1] and Na [1], respectively.X-band EPR spectra of nBu 4 N [1] in Me 2 SO at 94 K and Na [1] in H 2 O/ethylene glycol 5 : 1 v/v at 12 K are characterized by ill-resolved broad lines with a rhombic g-tensor indicating the low-spin 5d 5 (S = 1 2 ) electronic configuration of osmium(III) in these complexes.A simple ligand-field theory analysis of the g values indicates that in these cis-isomers, the electronic configuration differs from that of trans-analogs in that the electron "hole" is primarily in a d xz,yz orbital as opposed to a d xy orbital in the much more commonly found species with trans-geometry.The g-tensor of the Os III complexes of interest was further probed by quantum chemical theory.Specifically, the g-tensor values calculated by DKS/COSMO theory (with involvement of the solvent model) are in quite good agreement with those determined experimentally.Access to both cis-and trans-isomers of osmium(IV) and osmium(III) that are water soluble prompted us to investigate their in vitro anti-cancer activities.The complexes [1], Na [1] and [3] showed antiproliferative effects on cancer cells which were not coupled to their sensitivity to cis-platin.Cytostatic effects were stronger than actual cell killing at 48 h incubation time.HT29 cells showed a higher sensitivity to treatment with osmium complexes than cisplatin.4T1 cells showed lower rates of apoptosis induction by the osmium complexes than cisplatin, but a very strong cytostatic effect was observed for the cis-isomer Na [1].The unavailability of a larger scale procedure for the synthesis of the trans-isomer [2] makes the direct disclosure of the effect of geometrical isomerism on cytotoxicity in this case not possible.From the other side, a direct extrapolation of the results of the antiproliferative activity and cytostatic effects of the cis-isomer Na [1] on the ruthenium analogues is not possible as well, if one takes into account the body of accumulated evidence and cytotoxicity data for related ruthenium and osmium compounds 28,29,35,57,60,[86][87][88][89][90][91] and complexity of the problem. 92ven though the analogous cis-isomers of KP1019/NKP1339 remain unknown, their synthesis appears to be imminent, giving an opportunity that deserves to be exploited in the search for more effective metal-based anticancer drugs than those currently in clinical trials.75 An alternate basis set, which is analytically easier to handle, is that with a fictitious l = 1, where the d xy orbital corresponds to m l = 0 and the d xz,yz to linear combinations of m l = ± 1, so that the basis set is as follows: À d xy + : 76 K. J. LaChance-Galang, P. Doan, M. J. Clarke, U. Rao, in 1,1,2,2-tetrachloroethane at 128 °C starting from (H 2 ind)[Os IV Cl 5 (κN1-2H-ind)].This transformation is unprecedented in this family of compounds as it implies both a change of coordination mode from κN1 to κN2 of one indazole ligand and substitution of the chlorido ligand by an outer sphere indazole.The second indazole binds via N2 to osmium(IV) with the stabilization of the 1H-indazole tautomeric form (see Scheme 1).

Scheme 1
Scheme 1 Synthesis of 1 via Anderson rearrangement.

Fig. 5
Fig. 5 In situ UV-vis spectroelectrochemistry of [1] in 0.2 M nBuN 4 [PF 6 ] in Me 2 SO (scan rate 5 mV s −1 ): (a) UV-vis spectra observed upon cyclic voltammetry of [1] going to the second reduction peak.(b) The corresponding in situ cyclic voltammogram (selected colored UV-vis spectra in (a) correspond to the potentials marked with the colored circles in the voltammogram); (c) UV-vis spectra detected simultaneously upon the reduction of [1] in the region of the second cathodic peak (from −0.5 to −1.3 V vs. NHE).
our case (reduction by roughly one third of the free-ion value), so it can be used to convert the calculated Δ/ζ and V/ζ values into estimates of ligand-field splittings.It should also be noted that all of the trans-[OsX 4 (L)(L′)] − (X = Cl, Br and I; L = CO; L′ = CO and py) complexes studied by Kremer gave axial EPR spectra.This is a consequence of the linear (cylindrical π-bonding) binding of the CO ligand to osmium.Axial EPR spectra are also seen for complexes of type trans-[RuCl 4 (L) (Me 2 SO)] − xz and d yz orbitals in the present system, while it is primarily in the d xy orbital in most other cases.The exception is [Ru(NH 3 ) 4 (4-pic)(H 2 O)] 3+ (4-pic = 4-picoline (4-methylpyridine)) wherein competing π-bonding involving the trans ligands was proposed to lead to the change in the

f
Taken from McGarvey et al. 33c Two species are always observed in frozen aqueous solutions of [Os(NH 3 ) 5 (H 2 O)](CF 3 SO 3 ) 3 .g Taken from McGarvey et al. 33c The experimentally reported range for |g min | is 0.0 ≤ |g min | ≤ 0.6; we thus present calculations for both 0.6 and 0.0the minimum magnitude.h Taken from Souza et al.; 32 4-pic = 4-picoline (4-methylpyridine).The specific g values reported are those for frozen aqueous/ethylene glycol solution; those for a powder and in other solvents were similar.i The signs of the g values are not determined experimentally, but are determined here by the model of McGarvey, 33b which provides two choices of sign.Only the more physically plausible choice of signs is presented, except when both choices are acceptable.

Fig. 8
Fig.8Antiproliferative effects of treatment with 200 µM cisplatin,[1], Na[1] or[3], on HT29 and 4T1 cancer cell lines.(a) Example of discriminability of apoptotic and necrotic cell populations in HT29 cells after a 48 h treatment using a flow cytometry assay.(b) Quantification of early apoptotic and late apoptotic/necrotic cell populations in treated and untreated cells.(c) Relative cell viability and cell number after a 48 h treatment with the respective compound compared to untreated cells.* p < 0.05 (unpaired Student's t-test, always compared to untreated control).Displayed are the mean ± standard deviation values of at least six samples pooled from at least two biological replicates.

Table 3
The g-values of Os III complexes nBu 4 N[1], Na[1]obtained from simulation of their EPR spectra a While EPR is unable to provide the absolute sign of the g factor the ligand field theory analysis indicates that the g values are all negative for the investigated complexes (see Table4).The assignment of g-tensor component directions is not made here; only ordering by magnitude (see Table4for such assignments).bInMe2SO at 94 K.cIn H 2 O/ethylene glycol 5 : 1 v/v at 12 K.

Table 4
The g-values of Ru III and Os III complexes relevant to the present work with results from LFT analysis Counterions are not given except for the two Os III complexes reported here, but are noted for others below when relevant.b Taken from Kremer; 34 py = pyridine.The g values presented are all for complexes with tetraethylammonium countercation; for trans-Cs[OsCl 4 (CO)(py)]: g ⊥ = 2.46, g ∥ = 1.45.c Taken from Ni Dhubhghaill et al. 82 .
a d Taken from de Paula et al.

Table 5
Results of the theoretical determination by quantum chemical theory calculations of the g-tensor compared with experimental data