in the past six years several research groups reported on the synthesis and screening for antiproliferative activity in vitro of osmium coordination compounds and osmium(ii)-arene species , as well as on studies of their reactivity with dna model compounds.(4 ) these investigations showed that osmium is another metal that deserves attention for the development of effective inorganic antitumor drugs .
osmium analogues that are equipotent to their ruthenium congeners have the advantage of being more inert under conditions relevant for drug formulation.(2c ) of particular academic interest are also the observed or established differences between ruthenium and osmium compounds in stabilization of higher oxidation states , magnitude of spin  orbit coupling , metal  ligand exchange reactions , and so forth . in pursuing the goal of establishing structure - cytotoxicity relationships for osmium - azole - chlorido series and creating active metal - based antitumor drugs , we already reported the synthesis of trans-[oscl2(hazole)4]cl , cis-[oscl2(him)4]cl , and mer-[oscl3(hazole)3 ] .
compounds of the last type were insoluble in water , rendering tests for antiproliferative activity in vitro impossible , while trans-[oscl2(hazole)4]cl showed noteworthy antiproliferative effects in the cell lines a549 ( nonsmall cell lung cancer ) , ch1 ( ovarian carcinoma ) , and sw480 ( colon carcinoma ) , with ic50 values in the 10 to 10 m concentration range.(3b)trans-[oscl2(hpz)4]cl was found to be more active than the ruthenium counterpart , whereas the osmium(iii ) imidazole complex was less cytotoxic than the ruthenium analogue in all three cell lines .
quite recently , anderson type rearrangement reactions(11 ) of ( h2azole)2[oscl6 ] in alcohols in the presence of tetrabutylammonium chloride enabled the synthesis of the next family of compounds , namely , ( n - bu4n)[oscl5(hazole ) ] , where hazole = 1h - pyrazole , 1h - indazole , 1h - imidazole , 1h - benzimidazole , or 1h,2,4-triazole.(5 ) the ( n - bu4n ) was also substituted by biologically relevant or nontoxic cations such as na and ( h2azole ) .
comparison of these osmium complexes with related ruthenium analogues , such as ( h2azole)2[rucl5(hazole ) ] ( 1215 ) and ( h2azole)[ru cl4(hazole)2](14 ) showed a clear preference for higher oxidation states of the heavier congener .
the efficacy of the observed transformation is dependent on the electron - donating potency of the azole heterocycle and the solvent used . a deeper substitution at osmium(iv )
was first observed in the case of imidazole and benzimidazole ligands with highest basicity and further investigated for other azole heterocycles . in this paper
, we report on the synthesis , x - ray diffraction studies , and spectroscopic characterization of osmium(iv ) complexes , namely , trans-[oscl4(hazole)2 ] , where hazole = 1h - pyrazole , 2h - indazole , 1h - imidazole , and 1h - benzimidazole . of these compounds
the osmium indazole complex is the first example in which the ortho - quinoid tautomer is stabilized upon coordination to the transition metal ion .
the cytotoxic activity of all complexes has been assessed in three human cancer cell lines originating from different solid malignancies , namely , ch1 ( ovarian carcinoma ) , a549 ( nonsmall cell lung carcinoma ) , and sw480 ( colon carcinoma ) .
in addition , attempts to encapsulate the complexes prepared into the macrocyclic host molecule cucurbituril are also described ( chart 1 ) .
aall complexes have been characterized by x - ray crystallography ; atom labeling was introduced for assignments of resonances in nmr spectra .
all complexes have been characterized by x - ray crystallography ; atom labeling was introduced for assignments of resonances in nmr spectra .
the starting compounds [ ( dmso)2h]2[oscl6 ] and ( h2azole)2[oscl6 ] ( hazole = hpz , him , hind , hbzim ) were synthesized as previously reported in the literature .
oso4 ( 99.8% ) and n2h42hcl were purchased from johnson matthey and fluka , respectively .
1h - pyrazole , 1h - indazole , 1h - imidazole , 1h - benzimidazole , and 1-hexanol were from aldrich and fluka .
trans-[oscl4(hazole)2 ] ( hazole = hpz , him , hbzim ) complexes were prepared under argon atmosphere using standard schlenk techniques . the synthesis of trans-[oscl4(2h - ind)2 ] was performed in the solid state in an evacuated glass oven .
cucurbituril was generously offered by marcel wieland , institute of organic chemistry , university of vienna . a suspension of ( h2pz)2[oscl6 ] ( 100 mg , 0.18 mmol ) in 1-hexanol ( 2 ml ) was stirred at 160 c for 2 h. the red precipitate formed was filtered off , washed with ethanol and dried in vacuo .
calcd for c6h8cl4n4os0.1c2h5oh ( mr = 472.80 g / mol ) : c , 15.75 ; h , 1.83 ; n , 11.85 .
esi - ms in meoh ( negative ) : m / z 332 [ oscl4 ] , 467 [ oscl4(hpz)2h ] , esi - ms in meoh ( positive ) : m / z 491 [ oscl4(hpz)2+na ] , 513 [ oscl4(hpz)2 + 2na  h ] .
ei - ms ( positive ) : m / z 192 [ os ] , 329 [ oscl2(hpz ) ] , 364 [ oscl3(hpz ) ] , 432 [ oscl3(hpz)2 ] , 467 [ oscl4(hpz)2 ] .
mir , cm : 570 , 593 , 529 , 662 , 775 , 904 , 1052 , 1072 , 1113 , 1165 , 1263 , 1345 , 1398 , 1482 , 1507 , 2014 , 2166 , 2363 , and 3331 .
fir , cm : 159 , 178 , 265 , 287 , 325 , 345 , 571 .
vis ( meoh ) , max , nm (  , m cm ) : 215 ( 11884 ) , 245 ( 5 898 ) , 277 sh ( 2578 ) , 350 sh ( 4796 ) , 382 ( 7981 ) .
vis ( dmso ) , max , nm (  , m cm ) : 350 sh ( 5576 ) , 385 ( 8404 ) .
h nmr ( dmso - d6 , 500.32 mhz ) :  5.59 ( d , 1h , j = 2.40 hz , h3 ) , 2.97 ( s , 1h , h5 ) , 6.48 ( brs , 1h , h4 ) , 19.21 ( s , 1h , h1 ) ppm . c{h } nmr ( dmso - d6 , 125.81 mhz ) :  73.52 ( c4 ) , 181.14 ( c5 ) , 218.41 ( c3 ) ppm .
n nmr ( dmso - d6 , 50.69 mhz ) :  115.5 ( n1 ) ppm .
suitable crystals for x - ray diffraction study were grown from a solution of 1 in acetone .
( h2ind)2[oscl6 ] ( 166 mg , 0.25 mmol ) was heated in the solid state at 150 c in a glass oven for 45 h. the black product was extracted with methanol in a soxhlet extractor for 24 h. the dark blue residue collected as a solid from the extraction thimble was recrystallized from dimethylformamide ( dmf ) .
calcd for c14h12cl4n4os ( mr = 568.31 g / mol ) : c , 29.59 ; h , 2.13 ; n , 9.86 .
esi - ms in meoh ( negative ) : m / z 567 [ oscl4(hind)2h ] .
mir , cm : 591 , 626 , 734 , 752 , 838 , 863 , 895 , 980 , 1084 , 1131 , 1174 , 1234 , 1314 , 1362 , 1387 , 1440 , 1485 , 1511 , 1620 , 1665 , 3108 , and 3346 .
fir , cm : 157 , 181 , 194 , 217 , 249 , 262 , 310 , 324 , 334 , 429 , 459 , 550 , 595 , and 627 .
vis ( dmf ) , max , nm (  , m cm ) : 283 ( 9539 ) , 362 ( 9194 ) , 459 sh ( 2342 ) , 491 ( 2767 ) , 628 ( 12055 ) .
vis ( dmso ) , max , nm (  , m cm ) : 265 ( 9658 ) , 289 ( 9025 ) , 369 ( 7785 ) , 619 ( 12978 ) .
h nmr ( dmso - d6 , 500.32 mhz ) :  4.44 ( s , 1h ) , 3.28 ( s , 1h ) , 0.58 ( d , 1h , j = 8.51 hz ) , 5.18 ( s , 1h ) , 13.68 ( s , 1h ) ppm .
suitable crystals for x - ray diffraction study were grown from a solution of 2 in dmf . a suspension of ( h2im)2[oscl6 ] ( 100 mg , 0.18 mmol ) in 1-hexanol ( 2 ml ) was stirred at 160 c for 6 h. the brown precipitate formed was filtered off , washed with methanol and diethyl ether , and dried in vacuo .
calcd for c6h8cl4n4os ( mr = 468.20 g / mol ) : c , 15.39 ; h , 1.72 ; n , 11.97 . found : c , 15.70 ; h , 1.56 ; n , 11.65 .
esi - ms in meoh ( negative ) : m / z 468 [ oscl4(him)2h ] , 934 [ { oscl4(him)}2h ] , esi - ms in meoh ( positive ) : m / z 490 [ oscl4(him)2+na ] , 956 [ { oscl4(him)2}2+na ] . mir , cm : 584 , 604 , 641 , 682 , 738 , 830 , 1065 , 1119 , 1503 , 2041 , 2351 , and 3359 .
fir , cm : 147 , 178 , 271 , 281 , 316 , 343 , 605 , 643 .
vis ( meoh ) , max , nm (  , m cm ) : 251 sh ( 2696 ) , 280 ( 1967 ) , 343 ( 5838 ) , 374 ( 6458 ) , 452 ( 1011 ) .
vis ( dmso ) , max , nm (  , m cm ) : 344 ( 7209 ) , 374 ( 7045 ) , 450 sh ( 1422 ) .
h nmr ( dmso - d6 , 500.32 mhz ) :  0.51 ( s , 1h ) , 7.29 ( s , 1h ) , 7.86 ( s , 1h ) , 12.29 ( s , 1h1 ) ppm .
c{h } nmr ( dmso - d6 , 125.81 mhz ) :  119.52 { 7.29 } , 114.45 { 7.86 } , 188.36 { 0.51 } ppm .
n nmr ( dmso - d6 , 50.69 mhz ) :  157.08 ( n1 ) ppm .
suitable crystals of 32dmso for x - ray diffraction study were grown from a solution of 3 in dimethylsulfoxide ( dmso ) .
a suspension of ( h2bzim)2[oscl6 ] ( 200 mg , 0.31 mmol ) in 1-hexanol ( 3 ml ) was stirred at 160 c for 8 h. the blue precipitate formed was filtered off , washed with methanol and diethyl ether , and dried in vacuo .
calcd for c14h12cl4n4os ( mr = 568.31 g / mol ) : c , 29.59 ; h , 2.13 ; n , 9.86 .
esi - ms in meoh ( negative ) : m / z 331 [ oscl4 ] , 448 [ oscl4(hbzim)h ] , 567 [ oscl4(hbzim)2h ] .
mir , cm : 591 , 658 , 689 , 733 , 775 , 988 , 1117 , 1246 , 1422 , 1490 , 1945 , 2018 , 2150 , 2189 , 2235 , 2364 , and 3338 .
vis ( dmso ) , max , nm (  , m cm ) : 354 sh ( 6318 ) , 373 ( 6176 ) , 454 ( 3362 ) , 513 ( 4075 ) .
h nmr ( dmso - d6 , 500.32 mhz ) :  5.58 ( s , 1h , h2 ) , 4.58 ( d , 1h , j = 8.17 hz , h4 or 7 ) , 5.55 ( t , 1h , j = 7.27 hz , h5 or 6 ) , 6.71 ( d , 1h , j = 8.18 hz , h4 or 7 ) , 6.74 ( t , 1h , j = 7.26 hz , h5 or 6 ) , 9.00 ( s , 1h , h1 ) ppm . c{h } nmr ( dmso - d6 , 125.81 mhz ) :  114.64 ( c8 or 9 ) , 122.64 { 6.71 } ( c4 or 7 ) , 127.45 { 6.74 } ( c5 or 6 ) , 133.73 { 4.58 } ( c4 or 7 ) , 135.48 { 5.55 } ( c5 or 6 ) , 159.43 ( c8 or 9 ) , 200.46 ( c2 ) ppm
. suitable crystals for x - ray diffraction study were grown from a solution of 4 in dmso .
a suspension of 1 ( 4 mg , 0.009 mmol ) and cucurbituril ( 10 mg , 0.009 mmol ) in water ( 3 ml ) was heated at 100 c for 2.5 h in a closed schlenk vial . the hot solution was filtered .
suitable crystals of 511.25h2o for x - ray diffraction study were grown from the filtrate at room temperature over 24 h. elemental analyses were performed by the microanalytical laboratory of the faculty of chemistry of the university of vienna .
mir spectra were obtained by using an atr unit with a perkin - elmer 370 ftir 2000 instrument ( 4000400 cm ) .
vis spectra were recorded on a perkin - elmer lambda 20 uv  vis spectrophotometer using samples dissolved in dmso , dmf , methanol , or water containing 1% dmso .
electrospray ionization mass spectrometry was carried out with a bruker esquire 3000 instrument ( bruker daltonics , bremen , germany ) by using methanol as solvent .
cyclic voltammogramms were measured in a three - electrode cell using a 2 mm diameter glassy carbon disk working electrode , a platinum auxiliary electrode , and an ag|ag reference electrode containing 0.1 m agno3 .
measurements were performed at room temperature using a eg&g parc potentiostat / galvanostat model 273a .
deareation of solutions was accomplished by passing a stream of argon through the solution for 5 min prior to the measurement and then maintaining a blanket atmosphere of argon over the solution during the measurement .
the potentials were measured in 0.2 m ( n - bu4n)[bf4]/dmso using [ fe(-c5h5)2 ] ( e1/2 = + 0.68 v vs nhe)(18 ) as internal standard and are quoted relative to nhe .
the h , c and n nmr spectra were recorded at 500.32 , 125.81 , and 50.69 mhz on a bruker dpx500 ( ultrashield magnet ) in dmso - d6 .
2d c , h hsqc , n , h hsqc , c , h hmbc and h , h cosy experiments were performed for 1 , 3 , and 4 , while a homonuclear decoupling experiment only for 1 .
single crystals were positioned at 35 , 35 , 40 , 40 , and 50 mm from the detector , and 1150 , 1200 , 3557 , 1758 , and 1569 frames were measured , each for 30 , 80 , 20 , 30 , and 60 s over 1 scan width for 1 , 2 , 32dmso , 4 , and 511.25h2o , correspondingly .
the data were processed using the saint software.(19 ) the structures were solved by direct methods and refined by full - matrix least - squares techniques .
the following software programs and computer were used : structure solution , shelxs-97;(20 ) refinement , shelxl-97;(21 ) molecular diagrams , ortep-3;(22 ) computer , intel coreduo .
crystal data , data collection parameters , and structure refinement details for 1 , 2 , 32dmso , 4 , and 511.25h2o are given in table 1 .
ch1 ( ovarian carcinoma , human ) cells were a gift from lloyd r. kelland ( crc centre for cancer therapeutics , institute of cancer research , sutton , u.k . ) .
a549 ( nonsmall cell lung cancer , human ) and sw480 ( colon carcinoma , human ) cells were kindly provided by brigitte marian ( institute of cancer research , department of medicine i , medical university of vienna , austria ) .
cells were grown in 75 cm culture flasks ( iwaki / asahi technoglass ) as adherent monolayer cultures in minimal essential medium ( mem ) supplemented with 10% heat - inactivated fetal bovine serum , 1 mm sodium pyruvate , and 2 mm l - glutamine ( all purchased from sigma - aldrich ) without antibiotics .
cultures were maintained at 37 c in a humidified atmosphere containing 5% co2 and 95% air .
fc/fo. wr2 = { w(fo  fc)/w(fo)}. gof = { [w(fo  fc)]/(n  p ) } , where n is the number of reflections and p is the total number of parameters refined .
cytotoxicity in the cell lines mentioned above was determined by the colorimetric mtt assay ( mtt = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h - tetrazolium bromide , purchased from fluka ) . for this purpose
, cells were harvested from culture flasks by trypsinization and seeded in 100 l / well aliquots in mem supplemented with 10% heat - inactivated fetal bovine serum , 1 mm sodium pyruvate , 4 mm l - glutamine , and 1% nonessential amino acids ( 100  stock solution ) into 96-well microculture plates ( iwaki / asahi technoglass ) in the following densities to ensure exponential growth of untreated controls throughout the experiment : 1.5  10 ( ch1 ) , 4.0  10 ( a549 ) , and 2.5  10 ( sw480 ) viable cells per well .
cells were allowed to settle and resume proliferation for 24 h and were then exposed to the test compounds by addition of 100 l / well aliquots of appropriate dilutions in the same medium . for this purpose
, compounds were dissolved in dmso first and diluted in medium such that the effective dmso content did not exceed 0.5% whenever necessary .
after exposure for 96 h , the medium was replaced by 100 l / well rpmi 1640 medium ( supplemented with 10% heat - inactivated fetal bovine serum and 4 mm l - glutamine ) plus 20 l / well solution of mtt in phosphate - buffered saline ( 5 mg / ml ) ( all purchased from sigma - aldrich ) .
after incubation for 4 h , medium / mtt mixtures were removed , and the formazan precipitate formed by viable cells was dissolved in dmso ( 150 l / well ) .
optical densities at 550 nm ( corrected for unspecific absorbance at 690 nm ) were measured with a microplate reader ( tecan spectra classic ) to yield relative quantities of viable cells as percentage of untreated controls , and 50% inhibitory concentrations ( ic50 ) were calculated by interpolation .
evaluation is based on at least two ( in case of inactivity ) or three independent experiments , each comprising triplicate samples .
by exploring further the anderson type rearrangement reactions , which already enabled the synthesis of ( azole)pentachloridoosmate(iv ) complexes,(5 ) we obtained trans - bis(azole)tetrachloridoosmium(iv ) complexes following the reaction scheme : the reactions were performed in suspensions of the starting compounds ( h2azole)2[oscl6 ] , where hazole = 1h - pyrazole , 1h - imidazole , and 1h - benzimidazole , in 1-hexanol at 160 c to yield 1 , 3 , and 4 .
the indazole product 2 was obtained by heating the indazolium hexachloridoosmate(iv ) in the solid state at 150 c .
the liberation of two molecules of hydrochloric acid was followed by thermogravimetric analysis and the formation of bis - azole complexes by nmr spectroscopy .
the trans-[oscl4(hazole)2 ] complexes can also be prepared starting from ( h2azole)[oscl5(hazole ) ] providing further evidence for anderson type rearrangement reaction in the solid state ( supporting information , figure s1 ) .
it is worth noting that in the case of indazole the only product isolated was trans-[oscl4(2h - ind)2 ] .
therefore , attempts to encapsulate them in a cucurbituril host molecule to improve the aqueous solubility and use the latter as a vehicle for drug delivery ( 23 ) to the cell , as reported for platinum ( 24,25 ) and metallocene(26 ) anticancer agents , have been undertaken .
however , instead of inclusion complexes adduct 5 resulted from reaction of 1 with cucurbituril , as described for other metal chlorido complexes .
( 2730 ) the results of x - ray diffraction studies of complexes 1 , 2 , 32dmso , 4 , and 511.25h2o are shown in figures 1 and 2 , respectively .
selected bond lengths and angles are quoted in table 2 . to our knowledge 14 are the first bis(azole)tetrachloridoosmium(iv ) complexes characterized by x - ray crystallography as individual compounds .
ortep view of [ oscl4(hpz)2 ] ( 1 ) , [ oscl4(hind)2 ] ( 2 ) , [ oscl4(him)2 ] in 32dmso , and [ oscl4(hbzim)2 ] ( 4 ) .
complex 1 crystallized in the triclinic centrosymmetric space group p1 , while compounds 2 , 32dmso , and 4 crystallized in the monoclinic space group p21/c .
there is one molecule of 1 and two molecules of 24 in the corresponding unit cells .
the osmium ion in all four complexes lies on a center of symmetry , and the centrosymmetric molecules are of point group symmetry ci .
the osmium atom displays distorted octahedral coordination geometry with four chlorido ligands in the equatorial positions and two azole ligands bound axially .
comparison of the bond lengths in the coordination environment of osmium in 14 shows ( table 2 ) that the longer the axial bonds , the shorter are the equatorial ones .
both trans - azole ligands in 14 lie in one plane in contrast to what was found in ( ph4p)[trans - rucl4(1h - ind)2 ] , where the 1h - indazole ligands are significantly twisted.(31 ) the plane through both azole ligands crosses the equatorial plane between chloride ligands , the torsion angles cl1os  n1n2 , cl2os 
n1c2 , and cl1os  n1c1 being 30.4(4 ) , 33.2(6 ) , 31.6(2 ) , and 47.8(2) , respectively . of particular interest is the structure of indazole derivative 2 ( figure 1 ) , in which the indazole is stabilized in a quinoid tautomeric form coordinating to osmium via nitrogen atom n1 .
indazole acts mainly as a monodentate neutral ligand in metal complexes binding to metal ions via n2 . in a few cases , it was found to be deprotonated , acting as a bridging ligand in polynuclear metal complexes(32 ) or even more rarely as a monodentate indazolate ligand coordinated via n1 or n2 .
the molecules of 1 in the crystal structure form a chain running almost parallel to axis a ( supporting information , figure s2 ) .
hydrogen bonding interactions between the pyrazole nitrogen atom n2 as a proton donor and chloride ligand cl2 as a proton acceptor are evident [ n2cl2 3.270 , n2h 0.88 , hcl2 2.511  , n2hcl2 144.86 ; symmetry code i : x  1 , y , z ] .
the unit cell packing diagram for 2 is shown in supporting information , figure s3 .
a bifurcated hydrogen bond between n2 as proton donor and chloride ligands cl1 and cl2 as proton acceptors is present in the crystal structure [ n2cl1 3.444 , n2cl2 3.143  ] .
in addition , stacking interaction between indazole ligands of neighboring molecules of 2 is also evident .
intermolecular hydrogen bonding interaction between the imidazole nitrogen n4 and the oxygen atom o1 ( 2.698  ) of cocrystallized dmso is shown in supporting information , figure s5 . a bifurcated hydrogen bonding interaction between atom n2 of benzimidazole ligand in one molecule with chloride ligands cl1 and cl2 of two adjacent molecules of 4 [ n2cl1 3.541 , n2cl2 3.504  ] , along with  * stacking interactions between heteroaromatic rings separated at about 3.4 
in contrast to 14 , trans-[oscl4(hpz)2]cucurbituril ( 511.25h2o ) crystallizes in a noncentrosymmetric orthorhombic space group pca21 .
the result of x - ray diffraction study of this complex is shown in figure 2 .
the bond lengths os  n1 and os  n6 of 2.076(7 ) and 2.077(7 )  , respectively , along with os 
[ 2.386(2 ) , 2.357(2 ) , 2.382(2 ) and 2.386(2 ) , correspondingly ] are significantly longer than the os 
the atom n2 is involved in a hydrogen bond n2hcl3 with the following geometrical parameters : n2cl3 3.242  , n2hcl3 133.6 , while atom n7 forms a hydrogen bond with a cocrystallized water molecule [ n7o11 2.899  , n7ho11 133.6 ] .
passage of trans-[oscl4(hpz)2 ] through the portal of cucurbituril could experience strong repulsive forces at distances less than the sums of van der waals radii ( portal diameter 5.4 ,(23 ) cavity width 7.3 ,(23 ) and cl2cl4 vector in 5 of 4.74  ) .
although the cucurbituril does form an inclusion complex with a metal halide , namely , cis-[sncl4(h2o)2 ] , a more sophisticated mechanism of portal passage and formation of the complex was suggested.(34 ) first a tetrahedral sncl4 molecule enters the host molecule , which then undergoes hydration .
partial inclusion of oxaliplatin into cucurbituril has been also reported.(35 ) the h , c and n nmr spectra show signals due to coordinated azole heterocycles ( 14 ) .
the assignment of the protons and carbon atoms of the azole heterocycle is in some cases hindered by the temperature - independent paramagnetism of low - spin d osmium(iv ) which causes some broadening of the signals and large shifts relative to uncoordinated azoles at room temperature .
however , it should be noted that signals are remarkably sharp for the majority of the compounds studied .
we succeeded to obtain two - dimensional nmr spectra for the compounds 1 , 3 , and 4 ( c , h hsqc , n , h hsqc ( 1 , 3 ) , c , h hmbc , h , h cosy ) .
the h1 proton in 1 at 19.21 ppm was identified from the n , h hsqc plot with a chemical shift for n1 at 115.50 ppm .
because of the cs local symmetry of the coordinated pyrazole , the h3 and h5 signals do not overlap as is the case for the metal - free pyrazole .
the resonance signal h5 is observed as a singlet at 2.97 ppm , while h3 is observed as a doublet at 5.59 ppm .
the assignment of the h3 and h5 signals was enabled by a homonuclear decoupling experiment .
nmr spectra of 1 the carbon resonances of c4 , c5 , and c3 appear at 73.52 , 181.14 , and 218.41 ppm , respectively .
the h1 proton in 3 at 12.29 ppm was identified from the n , h hsqc plot with the chemical shift for n1 at 157.08 ppm .
the h4 and h5 resonances do not overlap as is the case for the metal - free imidazole .
the singlets at 0.51 , 7.29 , and 7.86 ppm were attributed to h2 , h4 , or h5 ; a more precise assignment failed .
the corresponding carbon resonances of c2 , c4 , and c5 were found at 119.52 { 7.29 } , 114.45 { 7.86 } , and 188.36 { 0.51 } ppm .
the c , h hmbc plot does not enable the assignment of c2 , c4 , c5 and h2 , h4 , h5 because of the same number of cross peaks .
compound 4 shows two singlets , two doublets , and two triplets in the h nmr spectrum .
the h1 proton is found as a singlet at 9.00 ppm , while a strongly shifted singlet at 5.58 ppm was assigned to h2 , which is in close proximity to the coordination site .
the two doublets were attributed to h4 and h7 , whereas the two triplets to h5 and h6 . in the c{h } nmr
spectra the carbon resonances of c4 and c7 are seen at 122.64 { 6.71 } and 133.73 { 4.58 } ppm , while those for c5 , c6 at 127.45 { 6.74 } , 135.48 { 5.55 } and c2 at 200.46 ppm , respectively .
there are two signals at 114.64 and 159.43 ppm for the quarternary carbons c8 and c9 . neither the c , h hmbc nor the h , h cosy allowed to differentiate between h4/h7 ( c4/c7 ) and h5/h6 ( c5/c6 ) . the cyclic voltammograms ( cvs ) of the complexes 14 in dmso ( 0.2 m
( n - bu4n)[bf4]/dmso ) at a carbon disk working electrode , recorded with a scan rate of 0.2 v / s , display a reversible one - electron reduction wave attributed to the os  os process with potential values ranging from 0.31 to 0.50 v and an irreversible single electron reduction wave ( i ) attributed to the os  os process with ep potential values between 1.17 and 1.55 v versus nhe ( figure 3 , table 3 ) .
the redox waves os / os are characterized by a peak - to - peak separation ( ep ) of 6871 mv and an anodic peak current ( ipa ) that is almost equal to the cathodic peak current ( ipc ) , as expected for reversible electron transfer processes .
the one - electron nature of these processes was verified by comparing the peak current height ( ip ) with that of standard ferrocene / ferrocenium couples under identical experimental conditions .
the reduction potentials os / os are in the following order : e1/2(1 ) > e1/2(2 ) > e1/2(4 ) > e1/2(3 ) , which agrees quite well with the relative electron - donating character of the azole ligands [ el(hpz ) > el(2h - ind ) > el(hbzim ) > el(him ) ] .
the reduction potential for os  os was calculated using lever s equation(38 ) ( eq 1 ) [ el(cl ) = 0.24,(37)el(2h - ind ) = 0.18,(39)el(hpz ) = 0.20,(37)el(hbzim ) = 0.1,(37)el(him ) = 0.09,(40)sm(os / os ) = 1.01,(37)im(os / os ) = 0.40(37 ) ] .
the values of ecalc for os / os obtained by using this formula are quoted for comparison with the ep values measured for os / os in table 3
( e1/2 = ( epa + epc)/2 , where epa and epc are the anodic and cathodic peak potentials ) are given in v and measured at a scan rate of 0.2 v / s in dmso , using ferrocene as internal standard , and are quoted relative to nhe .
cyclic voltammogram of 0.2 m solution of 3 in dmso at a carbon disk working electrode at a scan rate of 0.2 v / s , starting the scan in cathodic direction .
the solubility of the complexes 14 in water containing 1% dmso at 298 k varies from 1.9 ( 1 and 3 ) to 2.1 mm ( 4 ) .
the complexes remain intact in this medium for at least 24 h as is shown for complex 4 in figure 4 by uv  vis spectroscopy .
the optical spectra of 14 in dmso are shown in supporting information , figure s7 .
vis spectra of an aqueous solution ( containing 1% dmso ) of 4 , measured immediately after dissolution and 24 h thereafter .
the cytotoxicity of compounds 14 was assessed by means of the mtt assay in three human cell lines originating from different malignant tumors .
a 96 h exposure yielded the concentration  effect curves depicted in figure 5 and ic50 values listed in table 4 .
ic50 values are mostly in the 10 m range in the rather chemosensitive ovarian cancer cell line ch1 , but mostly higher than 100 m or even higher than the maximally applicable concentrations ( because of limited solubility ) in the less chemosensitive colon cancer ( sw480 ) and nonsmall cell lung cancer ( a549 ) cells .
best of the four compounds , the indazole complex 2 retains cytotoxic properties in those two cell lines .
the cytotoxicity of this compound is in a range well comparable with that of the clinically studied ruthenium complex ( h2ind)[rucl4(hind)2 ] ( kp1019).(12 ) furthermore , this compound is similarly potent as or up to 2.4 times more potent than the corresponding monoindazole complex ( h2ind)[oscl5(2h - ind ) ] , depending on the cell line , while the cytotoxicity of the bispyrazole analogue 1 is comparable to that of the monopyrazole complex ( h2pz)[oscl5(hpz)],(5 ) all based on ic50 values . the fact that comparisons of 14 do not yield uniform structure  activity relationships in the three cell lines employed suggest that variation of the azole ligands in the compounds does not affect cytotoxic potency in a generally valid way but that the consequences for the biological effects are more complex .
50% inhibitory concentrations ( means  standard deviations from at least three independent experiments ) , as obtained by the mtt assay ( exposure time : 96 h ) . c. bartel , m. a. jakupec , unpublished results . concentration 
effect curves of compounds 14 in ( a ) a549 , ( b ) ch1 , and ( c ) sw480 cells , obtained by the mtt assay ( 96 h exposure ) .
anderson type transformation of ( h2azole)2[oscl6 ] in 1-hexanol and in the solid state resulted in complexes with the general formula trans-[oscl4(hazole)2 ] , where hazole = 1h - pyrazole , 1h - imidazole , 2h - indazole , or 1h - benzimidazole .
comparison of the described complexes with their ruthenium analogues ( h2azole)[rucl4(hazole)2 ] provides further evidence for the higher oxidation state preference of the heavier congener . despite the nonelectrolyte character of the prepared compounds , the solubility in water containing 1% dmso was borderline to allow the assessment of their antiproliferative activity in vitro against human cancer cell lines .
an attempt to improve the solubility of the complexes by encapsulation in cucurbituril was described .
the importance of this work consists in preparation of precursors , which can be suitable for the synthesis of osmium(iii ) analogues of kp1019 , a potent investigational anticancer drug in clinical trials . in addition
the complexes trans-[oscl4(hazole)2 ] are characterized for the first time as individual compounds by x - ray crystallography .
a remarkable finding is the stabilization of the quinoid - type 2h - indazole in 2 coordinated to metal via n1 .
further attempts to prepare osmium(iv ) complexes with 1h - indazole tautomer and inclusion complexes with cucurbituril and cucurbituril are underway in our laboratory .
 by controlled anderson type rearrangement reactions complexes of the general formula trans-[osivcl4(hazole)2 ] , where hazole = 1h - pyrazole , 2h - indazole , 1h - imidazole , and 1h - benzimidazole , have been synthesized . 
 note that 2h - indazole tautomer stabilization in trans-[osivcl4(2h - indazole)2 ] is unprecedented in coordination chemistry of indazole . 
 the metal ion in these compounds possesses the same coordination environment as ruthenium(iii ) in ( h2ind)[ruiiicl4(hind)2 ] , where hind = 1h - indazole , ( kp1019 ) , an investigational anticancer drug in phase i clinical trials . 
 these osmium(iv ) complexes are appropriate precursors for the synthesis of osmium(iii ) analogues of kp1019 . in addition 
 the formation of an adduct of trans-[osivcl4(hpz)2 ] with cucurbit[7]uril is described . 
 the compounds have been comprehensively characterized by elemental analysis , ei and esi mass spectrometry , spectroscopy ( ir , uv  vis , 1d and 2d nmr ) , cyclic voltammetry , and x - ray crystallography . 
 their antiproliferative acitivity in the human cancer cell lines ch1 ( ovarian carcinoma ) , a549 ( nonsmall cell lung carcinoma ) , and sw480 ( colon carcinoma ) is reported . 
Introduction
Experimental Section
Results and Discussion