cisplatin and the follow - on 
 drugs carboplatin ( paraplatin ) and 
 oxaliplatin ( eloxatin ) have been used to treat different cancers for 
 the past 40 years .
however , their effectiveness 
 is still hindered by clinical problems , including acquired or intrinsic 
 resistance , a limited spectrum of activity , and high toxicity , leading 
 to side effects . in the search for more effective 
 and selective potential anticancer metallodrugs ,
evidence showing that organometallic 
 compounds of platinum perform better than their non - organometallic 
 derivatives was reported . in general , 

organometallic compounds are kinetically more inert and lipophilic 
 than coordination metal complexes , which may offer opportunities in 
 the design of anticancer metallodrugs with improved properties .
several 
 reviews on the anticancer activity of organometallic compounds from 
 a number of different transition metals have appeared in the past 
 5 years .
more specifically , gold(iii ) and platinum(ii ) organometallic 
 compounds have been studied as potential anticancer agents .
a number 
 of complexes containing the [ pt(cod ) ] fragment and different ligands , 
 such as alkyls , alkynyls , and nucleosides , have been described .
platinum cod alkynyl compounds showed high toxicity against ht-29 
 colon carcinoma and mcf-7 breast adenocarcinoma cell lines , while [ ptme(r - cod)l ] compounds with 
 different ligands ( halides , alkyl , aryl , alkynyl ) revealed higher 
 toxicity to hela cells in comparison to that of cisplatin . in the 
 case of gold(iii ) , it is well known that pincer ligands containing 
 carbon and nitrogen stabilize the metal center against reduction to 
 gold(i ) and gold(0 ) species in physiological media .
the anticancer activities of cyclometalated gold(iii ) and 
 platinum(ii ) compounds with bidentate c , n- or terdentate c , n , n - pincer 
 ligands have been recently reviewed .
some cyclometalated 
 gold(iii ) complexes based on c , n , n- and c , n , c - pincer complexes have displayed impressive 
 anticancer activity in vitro and in vivo by a mode of action different from that shown by cisplatin .
it has 
 been proposed that , for these complexes , the presence of the (m 
c ) 
 bond increases the stability of the compounds allowing the organometallic 
 fragment to reach the cell unaltered .
in addition , it has been postulated 
 that in platinum compounds the presence of aromatic groups in the 
 cyclometalated ligand might favor intercalative binding to dna (  
 stacking ) , while the labile positions in the coordination sphere may 
 favor covalent coordination for dna as in cisplatin .
very recently , 
 a luminescent dna intercalator cyclometalated platinum(ii)complex , 
 [ pt(c^n^n)(c - ntbu)]clo4 ( hc^n^n = 6-phenyl-2,2-bipyridyl ) 
 with a potent inhibitory effect in human cancer cells in vitro and in a xenograft model in mice has been described .
the stabilization of the topoisomerase i  dna 
 complex with resulting dna damage by the cyclometalated compound is 
 suggested to contribute to its anticancer activity .
multinuclear ( sscs ) 
 fluorescent rhomboidal pt(ii ) metallacycles have also been reported 
 recently showing a potent tumor growth 
 inhibitory effect on mda - mb-231 xenograft models in mice as well as 
 high stability in media and in cancer cells in vitro .
we have reported that nontoxic iminophosphorane or iminophosphane 
 ( i m ) compounds ( r3p = n - r , i m ) are useful 
 precursors for the preparation of coordination ( n , n- ) or cyclometalated 
 ( c , n- ) complexes of d ( au(iii ) , pd(ii ) , and pt(ii ) ) and 
 d ( ru(ii ) ) metals ( selected compounds a 
these i m metal complexes display high cytotoxicity in 
 vitro ( low micromolar to nanomolar ) against a variety of 
 human cancer cell lines with different degrees of selectivity .
organogold(iii ) complexes containing iminophosphorane ligands ( e.g. , a ) exert cell death with pathways involving mitochondrial 
 production of reactive oxygen species .
we have studied 
 the interaction of the i m metal compounds with ( pbr322 ) dna , calf 
 thymus ( ct ) dna , and human serum albumin ( hsa ) .
we have confirmed that some compounds ( such as f ) inhibit 
 parp-1 proteins .
more recently , we have described a water - soluble ruthenium(ii ) 
 i m compound ( g in chart 1 ) which has displayed high activity against a number 
 of cancer cell lines in vitro .
this compound was 
 also highly active on mda - mb-231 xenografts in mice , with an impressive 
 tumor reduction ( shrinkage ) of 56% after 28 days of treatment ( 14 
 doses of 5 mg / kg every other day ) , with low systemic toxicity , quick 
 absorption in plasma , and preferential accumulation in breast tumor 
 tissues . in most cases ( including some 
 pd(ii ) and pt(ii ) derivatives ) , we have demonstrated that dna is not 
 the target for these compounds and that most complexes are highly 
 active against cisplatin - resistant cancer cell lines , pointing to 
 a mode of action different from that of cisplatin .
we also evaluated the stability of the compounds in solution and 
 proved that , for gold(iii ) and palladium(ii ) metal centers , cyclometalated 
 c , n - im compounds were more stable than those in which the i m ligand 
 was n , n - coordinated . in this context , we aimed to 
 prepare cyclometalated i m compounds 
 of gold(iii ) and platinum(ii ) in which the aryl group of the imino 
 fragment is coordinated to the metal center ( exo derivatives 
 such as palladium compounds d and e in chart 1 ) as opposed to an 
 aryl group of the phosphine fragment ( endo derivatives 
 like a and b ) in order to expand the range 
 of phosphines incorporated into the final molecule to tune electronic / steric 
 properties of the resulting complexes .
the synthesis of the exo cyclometalated palladium starting material containing 
 a water - soluble phosphine ( d ) was achieved by oxidative 
 addition of pd(0 ) to the c  br bond in the i m bromide - containing 
 ligand , a method that can not be used 
 to generate gold(iii ) and platinum(ii ) analogues .
we report 
 here on the synthesis of novel exo cyclometalated 
 c , n - im compounds of gold(iii ) and platinum(ii ) containing the water - soluble 
 phosphine 1,3,5-triaza-7-phosphaadamantane ( pta ) and the synthesis 
 of endo - c , n - im compounds of platinum(ii ) derivatives 
 never described before .
all these complexes , along with the previously 
 described exo derivative [ au(2-c6h4c(o)n = pph3)cl2 ] ( 1 ) and cisplatin , have been evaluated 
 against a number of human cancer cell lines in vitro , and initial cell death mechanistic insights are discussed .
we have 
 studied the interaction of these compounds with relevant biomolecules 
 such as plasmid ( pbr322 ) dna as a model for nucleic acids and hsa 
 ( the most abundant carrier protein in plasma ) .
the platinum(ii ) compounds 
 for which effects on dna may be expected ( 35 ) have been further evaluated for their interaction with 
 ct dna by circular dichroism ( cd ) .
all these studies point out that 
 cationic cycloplatinated compounds 4 and 5 ( differing only in the anion ) are the most active and have a mode 
 of action different from that of cisplatin . additionally , we report 
 on the permeability of 4 and 5 evaluated 
 by two different assays , in vitro caco-2 monolayers 
 and rat perfusion assay , in order to make comparisons with cisplatin 
 and drugs or compounds that can be orally administered .
the 
 synthesis of the exo cyclometalated gold(iii ) 
 and platinum(ii ) compounds was based on the preparation of [ hg(ph3p = n - co-2-c6h4)cl ] by nicholson 
 et al .
the c  h activation at the 
 n - co - ph fragments takes place at a manganese center ; thus , by transmetalation 
 of the resulting cyclometalated iminophosphorane manganese compounds 
 to hgcl2 , the organomercury derivatives with pph3 , [ hg(ph3p = n - co-2-c6h4)cl ] , or water - soluble phosphine pta , [ hg(pta = n - co-2-c6h4)cl ] , described 
 by us , are obtained in high yields ( scheme 1 ) .
transmetalation reactions of [ hg(pr3=n - co-2-c6h4)cl ] ( pr3 = pph3 ; pta ) with nme4[aucl4 ] or [ ptcl2(cod ) ] afforded previously 
 described compound [ au(2-c6h4c(o)n = pph3)cl2 ] ( 1 ) and new cyclometalated exo - iminophosphorane complexes 
 of gold(iii ) and platinum(ii ) of the type [ au(2-c6h4c(o)n = pta)cl2 ] ( 2 ) and [ pt(2-c6h4c(o)n = pta)(cod)]2[hg4cl10 ] ( 3 ) ( scheme 2 ) in moderate to high yields . compound [
the reaction of [ hg(pph3=n - co-2-c6h4)cl ] with [ ptcl2(cod ) ] did not afford 
 a pure 
 cycloplatinated compound .
different synthetic conditions were tried , 
 and in most cases abundant pt(0 ) decomposition took place , while unreacted 
 [ hg(pph3=n - co-2-c6h4)cl ] and 
 pph3=o were the observed products along with free 
 cod .
longer refluxing times in polar solvents afforded small amounts 
 ( 410% ) of a possible cyclometalated product along with [ hg(pph3=n - co-2-c6h4)cl ] and pph3=o .
new compounds 2 and 3 are obtained as 
 air - stable yellow and white solids , respectively .
compound 2 is neutral , whereas the pt(ii ) derivative 3 is cationic 
 ( 2:1 ions ) , as confirmed by conductivity measurements ( see experimental section ) .
we found that the cod 
 ligand in 3 is immediately exchanged by dmso molecules 
 in dmso - d6 solution at rt and that the 
 new im - cycloplatinated species did not change in dmso - d6 over time ( see supporting information 
 ( si ) ) .
this was surprising since a cod ligand is not easily 
 replaceable ( usually requires thermal activation ) .
the structures 
 of these compounds have been proposed on the basis of elemental analysis , 
 nmr and ir spectroscopy , and mass spectrometry ( ms ) .
both compounds 
 are soluble in mixtures of 1:99 dmso : h2o at micromolar 
 concentrations ( relevant for biological studies ) . the structure 
 of 2 has been determined by an x - ray 
 analysis , and it is very similar to that previously reported for compound [ au(2-c6h4c(o)n = pph3)cl2 ] ( 1 ) , with very similar distances and angles . the 
 molecular structure of 2 is depicted in figure 1 , while selected structural 
 parameters are collected in table 1 .
the analysis 
 confirms the square - planar arrangement around the 
 gold(iii ) center with a bite angle of 81.68(8). like in other 
 c , n - im cycloaurated complexes , the au 
cl(1 ) bond trans to the carbon is 
 longer ( 2.3834(5 )  ) than the au 
cl(2 ) bond trans to the nitrogen ( 2.2798(5 )  ) due to the higher trans influence of the c donor atom .
as observed in compound 1 , upon coordination to the gold there is an increase in both the 
 p 
n and n  c bond lengths when compared to the uncoordinated 
 ligand ( p  n : 1.626(3 )  in 
 ligand , 1.6658 ( 18 )  in 2 ; n  c : 1.353(5 ) 
  in ligand , 1.401(3 )  in 2 ) .
this effect 
 is also observed in the ir spectra of compound 2 , for 
 which the band corresponding to the p 
n bond appears at a lower 
 frequency than that for the free ligand ( 1289 cm versus 1374 cm ) . as described in the structure 
 of compound 1 , a decrease of the c = o bond length 

was observed ( from 1.245(5 )  in the ligand to 1.213(3 )  
 in the cycloaurated complex ) .
we had described the biological 
 activity of the endo - iminophosphorane compound [ au{-c , n - c6h4(pph2=n(c6h5))-2}cl2 ] and some of its 
 cationic derivatives , like [ au{-c , n - c6h4(pph2=n(c6h5)-2}(s2cn(ch3)2)]pf6 ( a in chart 1 ) , but we had never synthesized pt(ii ) endo compounds with the i m ph - n = pph3 ligand . 

we carried out the reaction of [ hg{c6h4(pph2=n(c6h5))-2}cl ] with [ ptcl2(cod ) ] and obtained ( as in the case of the exo compound 3 ) a cationic species ( 4 ) with a mercury chloride - containing anion ( in this case [ hg2cl6 ] ) . in order to avoid the 
 use of organomercury compounds and the presence of mercury in the 
 resulting compound , a 
synthetic approach based on transmetalation with an organogold(i)phosphine 
 compound [ au{c6h4(pph2=n(c6h5))-2}(pph3 ) ] , described previously , was employed ( scheme 3 ) .
we had used this mercury - free approach to obtain the endo gold(iii ) cyclometalated compound [ au{-c , n - c6h4(pph2=n(c6h5))-2}cl2 ] .
the 
 reaction 
 proceeds much faster and in much milder conditions than that for the 
 synthesis of 4 ( 25 min at rt in ch2cl2 instead of 5 days in refluxing acetone ) , and compound 5 is obtained in moderate yield ( 58% ) . in order to avoid the 
 formation of a neutral platinum(ii ) dimer with chloride bridges , [ pt{c6h4(pph2=n(c6h5))-2}cl]2 , observed while performing this reaction , 
 nh4pf6 was added . in this way , we obtained compound 5 , an analogue of cationic compound 4 with a 
 mercury - free anion ( pf6 ) .
the structures 
 of these compounds have been confirmed by elemental analysis , nmr 
 ( including pt nmr ) and ir spectroscopy , and ms studies . 
 in this case the compounds do not exchange the cod ligand by dmso 
 molecules at rt in dmso - d6 solution as 
 it happened for compound 3 , which may have some connotations 
 for the biological activity of the compounds .
compounds 4 and 5 are soluble in mixtures of 1:99 dmso : h2o solutions at micromolar concentrations ( relevant for biological 
 studies ) . a mercury - free analogue of compound 3 could 
 not be obtained , since the preparation of the appropriate au(i ) transmetalation 
 agent from the organomanganese compound ( scheme 1 ) was not successful .
the number of 
 cycloplatinated iminophosphorane compounds described 
 previously is limited to two examples of endo neutral 
 derivatives , [ pt(c6h4 - 2-pph2=n - c(o)-2-nc5h4--c , n , n)cl ] and [ pt{-c , n , n - c6h4(pph2=n-8-c9h6n}cl ] , in which the iminophosphorane fragment acts as a c , n , n - pincer 
 ligand . in compounds 35 ,
the i m 
 ligand is cyclometalated in either an exo ( 3 ) or endo ( 4 , 5 ) position , acting as a c , n - pincer ligand .
the other two coordination 
 positions for the pt(ii ) center are occupied by the cod ligand .
the 
 molecular structure for compound 4 was determined by 
 x - ray crystallography , confirming the proposed structure .
the molecular 
 structure of the cation in 4 is depicted in figure 2 , while selected 
 structural parameters are collected in table 2 .
a complete drawing of the crystal structure 
 of 4 , including the [ hg2cl6 ] anion and crystallization molecules , along with a 
 more complete table of distances and angles are provided in the si .
the coordination geometry around the platinum 
 atom is slightly distorted from square - planarity , with the c(1)pt(1)n(1 ) 
 angle of 85.31(9) suggesting a rigid  bite  angle . 

n(1 ) 2.039(2 )  is shorter than those 
 for other c , n - cyclometalated pt(ii ) derivatives , such as dimethylbenzylamine 
 ( dmba ) compounds ( ca .
2.12302.1340  ) ( see , e.g. , refs ( 19 , 46 , and 47 ) ) .
the 
 distances pt  n(1 ) and pt  c(1 ) , both 2.039(2 )  , 
 are almost identical to those found for au 
x(2 ) 
 to the centroids of the cod ligand are 2.169(3 ) and 2.039(3 )  , 
 respectively , which reflects the higher trans influence 
 of c versus n ( longer pt 
the stability of 1 and of the 
 new compounds 25 was evaluated in 
 dmso - d6 solution by p{h } and h nmr spectroscopy .
all the complexes are 
 stable for months in dmso - d6 solution 
 ( see spectra and stability table 
 in the si ) .
as mentioned before , compound 3 exchanges the cod ligand by dmso immediately when dissolved 
 in dmso - d6 ( free cod is clearly visible 
 along with coordinated dmso ) . in the case of compounds 4 and 5
, this exchange is extremely slow , and after 1 
 week the percentage of free un - coordinated cod observed is around 
 6% ( see si ) .
compounds 4 and 5 are stable in mixtures of 1:99 dmso : pbs for 24 h , as established 
 by vis 
the stability of mercury - free compound 5 in acidic media 
 over time was studied by h and p{h } nmr spectroscopy . due to the lack of solubility of 5 in mixtures of 1:99 dmso : pbs in concentrations high enough to obtain 
 a meaningful p{h } nmr spectrum
, these experiments 
 were performed in a 2:1 dmso - d6/pbs-1x(d2o ) solution at ph 6 ( see experimental section for details ) . in
these conditions compound 5 is stable 
 for at least 5 days , as can be observed by comparison with its h and p{h } nmr spectra in the same 
 deuterated mixture at ph 7.4 ( figures s9s12 in the si ) .
the 
 anti - proliferative properties of the gold(iii ) and platinum(ii ) 
 complexes 15 and ligand cod were 
 assessed by monitoring their ability to inhibit cell growth using 
 the mtt assay ( see experimental section ) . 

the cytotoxicity activity of the compounds was determined in several 
 human cancer cell lines , i.e. , leukemia jurkat - t , lung a549 , prostate 
 du-145 , pancreas miapaca2 , and triple - negative breast mda - mb-231 , 
 in comparison to cisplatin .
the cod ligand is 
 poorly cytotoxic in all tested cell lines ( ic50 > 125 
 m ) . 

the i m ligands are known to be poorly cytotoxic ( ic50 
 > 100500 m in different cell lines ) .
all compounds were 
 dissolved in 
 1% dmso and diluted with water before addition to cell culture medium 
 for a 24 h incubation period .
cyclometalated neutral gold(iii ) 
 showed cytotoxicity similar to 
 that of cisplatin , while compound 2 was less cytotoxic 
 for all the studied cell lines , with the exception of the leukemia 
 jurkat cell line .
we have found previously that replacement of pph3 by pta in im - cyclometalated complexes decreases the cytotoxicity .
the ic50 value for jurkat for compound 1 is very similar to that obtained for the neutral iminophosphorane endo derivative [ au{-c , n - c6h4(pph2=n(c6h5))-2}cl2 ] .
cationic gold(iii ) 
 complexes containing i m ligands are more cytotoxic than neutral derivatives .
the cationic cyclometalated platinum compounds described here , 3 and especially 4 and 5 , were considerably 
 more cytotoxic than cisplatin in all the cell lines studied . 4 and 5 ( same cation ) display almost identical 
 ic50 values , with the exception of a549 and mda - mb-231 , 
 for
the data indicate that cytotoxicity for these compounds comes mainly 
 from the cationic platinum fragment . in order to assess the 
 compounds selectivity for cancerous 
 cells with respect to normal cell lines
, they were also screened for 
 their anti - proliferative effects on the non - tumorigenic human embryonic 
 kidney cells hek293 t . in most cases
all compounds are more toxic 
 to leukemia than to hek293 t cell lines ( 212 times ) , and compound 4 is more toxic to all the cell lines than to the hek293 t 
 cell lines .
the toxicity of mercury - free compound 5 to 
 hek293 t is comparable to that in the human cancer cell lines . as hek293 t 
 cell lines ( immortalized cells )
can display a higher sensitivity to 
 chemicals , we measured the effect of compound 5 on human 
 renal proximal tubular cells ( rptc ) .
rptcs in primary culture have 
 been described as an in vitro model to study nephrotoxicity .
the ic50 value ( xtt assay 24 h ; see si for details ) for 5 in this cell 
 line was 2.77  0.83 m , making 5 more sensitive 
 to cancerous cell lines than to rptcs .
in addition , we have described 
 recently an i m ruthenium compound , [ ( -p - cymene)ru{(ph3p = n - co-2-n - c5h4)--n , o}cl]cl , which displayed similar ic50 values in vitro for all the human cancer 
 cell lines described above and hek293 t but which was very effective in vivo on mda - mb-231 xenografts in nod.cb17-prkdc scid / j 
 mice while having low toxicity .
the mechanism 
 of cell death induced by mercury - free cytotoxic cycloplatinated compound 5 was analyzed in two cell lines of different origin : a549 
 lung carcinoma and jurkat t - cell leukemia .
phosphatidyl serine exposure , 
 plasma membrane damage , and nuclear morphology were assessed in both 
 cell lines after treatment with 5 .
caspase implication 
 in the toxicity of 5 was studied using the general caspase 
 inhibitor z - vad - fmk . in a549 cells we found that z - vad - fmk protected 
 cells from 5 at doses up to 0.5 m ( figure 3 ) , inhibiting both phosphatidylserine 
 exposure ( annexin v binding ) and plasma membrane permeabilization 
 ( 7-add uptake ) .
as expected , phosphatidylserine exposure was more 
 dependent on caspase activity . at higher concentrations 7-aad staining , 
 but
cells were cultured for 24 h in the presence of 5 at the indicated concentrations , alone ( solid lines ) or 
 combined with the general caspase inhibitor z - vad - fmk ( dashed lines ) . 
 subsequently , phosphatidylserine exposure ( triangles ) and cell membrane 
 permeabilization ( squares ) were analyzed by flow cytometry after staining 
 with annexin v - dy634 and 7-aad , respectively .
the apparent decrease in the percentage of annexin 
 v - positive cells 
 could reflect cell disintegration caused by necrosis .
consistently , 
 z - vad - fmk did not inhibit cell death at 1 m ( figure 3 ) .
when an early event of apoptosis , 
 loss of mitochondrial transmembrane potential , was analyzed , we also 
 observed that caspase inhibition by z - vad - fmk only partially reduced 
 m loss caused by 5 ( figure 4 ) , further suggesting that compound 5 can induce caspase - dependent and caspase - independent cell 
 death in a549 cells .
cells were cultured for 24 h in the presence 
 of compound 5 at the indicated concentrations , alone 
 ( solid line ) or combined with the general caspase inhibitor z - vad - fmk 
 ( dashed line ) .
then , transmembrane mitochondrial potential was analyzed 
 by flow cytometry after cells were stained with the probe dioc6(3 ) . 

jurkat cells were more sensitive 
 to 5 than a549 cells , 
 with an ic50 of 0.6 m , even though this cell line 
 does not express functional p53 , discarding an essential role of this 
 protein in the activity of compound 5 . in these cells , 

the percentages of 7-aad ( figure 5 ) and annexin v - positive ( data not shown ) cells were 
 the same in every assay .
high sensitivity of jurkat cells was confirmed 
 in short - term experiments , as we observed that 5 at 0.5 
 m induced cell death in almost 100% of the cells even at 6 
 h. caspase inhibition by z - vad - fmk completely avoided cell death at 
 6 h. however , longer treatment with 5 induced both caspase - dependent 
 and caspase - independent cell death ( figure 5 ) .
cells were treated with 
 compound 5 for 6 or 24 h in the presence or in the absence 
 of the general 
 caspase inhibitor z - vad - fmk .
membrane integrity was analyzed by flow 
 cytometry after the cells were stained with 7-aad , as indicated in 
 the experimental section .
mitochondrial damage was also analyzed in jurkat cells ( figure 6 ) . at 24 h ,
treatment 
 with 5 caused a decrease in m in 80% of 
 cells , compared to 32% in cells treated with 5 in the 
 presence of the general caspase inhibitor z - vad - fmk . in order to determine 
 whether mitochondrial damage caused by 5 was irreversible 
 and committed cells to death ,
cells were washed and resuspended in 
 fresh medium . after a further 24 h incubation in fresh medium , m 
 collapse
these results indicate that caspase inhibition 
 only delays cell death in jurkat cells , and 5 induces 
 cell damage , leading to cell death independently of caspase activation . 

thus , these experiments confirm that alternative caspase - independent 
 cell death mechanisms are activated by this compound , as observed 
 in a549 cells .
jurkat cells were treated with 5 or 5+z - vad for 24 h and then harvested , washed , and seeded in 
 fresh medium . 
 after further 24 h in fresh medium ,
mitochondrial transmembrane potential 
 ( m ) was analyzed as indicated in the experimental section .
results are mean  sd 
 of three independent experiments . on the other hand ,
analysis of nuclear morphology indicated 
 that 5 induced typical apoptotic features ( chromatin 
 condensation 
 and fragmentation ) that were prevented by z - vad - fmk in both cell lines 
 ( figure 7 ) .
however , 
 some nuclei of cells treated with 5+zvad displayed an 
 altered morphology when compared to controls .
this morphology could 
 be caused by necroptosis or aif - mediated 
 cell death .
compound 5 induces apoptosis in jurkat ( upper panels ) 
 and a549 cells ( bottom panels ) .
cells were cultured for 24 h in the 
 presence of compound 5 ( 0.5 m ) , alone or combined 
 with the general caspase inhibitor z - vad - fmk or left untreated ( control ) . 

nuclei were stained with hoechst 33342 ( 10 g / ml ) , and cells 
 were photographed under uv light .
finally , we analyzed the implication of mitochondria 
 in the toxicity 
 of compound 5 .
since jurkat cells do not 
 express bax , the jurkat - shbak cell line constitutes a model of human 
 leukemia cells deficient in the intrinsic ( mitochondrial ) pathway 
 of apoptosis .
a cell line transfected with a nonspecific shrna was 
 used as a control ( jurkat plvthm ) .
as shown in figure 8 , jurkat - shbak cells were less sensitive 
 to 5 than control cells .
however , high concentrations 
 of 5 induced bax / bak - independent cell death in jurkat - shbak 
 cells , suggesting that this compound could be useful in the treatment 
 of tumors with alterations in the intrinsic pathway of apoptosis .
jurkat - plvthm 
 ( control ) and jurkat - shbak cells were treated with 
 compound 5 for 24 h. mitochondrial transmembrane potential 
 was analyzed as indicated in the experimental section .
we also analyzed the type of cell 
 death for the cycloaurated exo compounds 1 and 2 , the cycloplatinated exo compound 3 , and the cycloplatinated endo compound 4 ( analogue of 5 containing the mercury anion 
 hg2cl6 ) in jurkat cells 
 in the presence or absence of z - vad - fmk ( caspase 
 inhibitor ) .
this analysis showed that the gold compounds 1 and 2 were less toxic than platinum complexes 3 and 4 , as already indicated by the ic50 values ( table 3 ) . 

moreover , these results show that gold compounds 1 and 2 induced mainly caspase - independent cell death .
organogold(iii ) endo compounds also activated caspase - independent pathways that lead 
 to cell death , as the addition of z - vad - fmk did not significantly 
 reduce the percentage of annexin v - pe or pi in jurkat cells treated with these derivatives .
organogold(iii ) 
 ( both endo and exo ) compounds is 
 basically the same and also similar to that of other cyclometalated 
 gold(iii ) anticancer agents .
the 
 toxicity for the cycloplatinated exo compound 3 ( figure 9 ) and for the endo compound 4 , differing 
 from 5 only in the anion ( data not shown ) , was only partially 
 dependent on caspase activity . at 24 h , 3 caused cell 
 death in around 50% of total jurkat cells , and caspase inhibition 
 reduced this percentage to 25% .
these data indicate that cycloplatinated 
 compounds 3 , 4 , and 5 can activate 
 alternative caspase - independent mechanisms of death
. however , at short 
 incubation times , cell death seems to be mainly caspase dependent 
 ( figure 5 ) , suggesting 
 that the main mechanism of cell death for these compounds is apoptosis .
jurkat 
 cells were treated with dmso ( control ) or compound 1 ( 10 
 m ) , 2 ( 20 m ) , or 3 ( 10 m ) 
 for 24 h , in the absence or in the presence of 50 
 m z - vad - fmk .
, from these 
 initial mechanistic studies it seems clear 
 that the cell death type for the most active mercury - free cycloplatinated 
 compound 5 is mainly through caspase - dependent apoptosis 
 but that 5 triggers caspase - independent cell death when 
 apoptosis is blocked , pointing to a mode of action different from 
 that of cisplatin .
the lipophilicity 
 of the most active cycloplatinated compounds 4 and 5 was determined by calculating the partition coefficients 
 ( see table 4 and experimental section ) between n - octanol and phosphate buffer ( ph 7.00 ) .
partition coefficients have 
 been used to predict the permeability of drugs since there is a good 
 correlation between intestinal permeability and physicochemical parameters 
 such as lipophilicity .
we wanted to study the influence of the two 
 different anions ( hg2cl6 , 4 , and pf6 in 5 ) on the lipophilicity and permeability of these cationic 
 cycloplatinated compounds .
metoprolol 
 was chosen as the reference compound for permeability 
 since it is known that 95% of the drug is absorbed from the gastrointestinal 
 tract .
thus , drugs that exhibit partition coefficients and human intestinal 
 permeability values greater than or equal to the corresponding values 
 for metoprolol are considered high - permeability drugs .
drugs with 
 estimated partition coefficients and human intestinal permeability 
 values less than the corresponding values for metoprolol are classified 
 as low - permeability drugs .
this type of correlation is a suitable 
 source of information on the passive and also possible carrier - mediated 
 absorption mechanism . from these data we can state
subsequently , the permeabilities 
 of cisplatin as commercialized 
 parent compound , cycloplatinated 4 and 5 as test compounds , and metoprolol , cimetidine , and atenolol / lucifer 
 yellow as reference compounds of high , intermediate , and low permeability , 
 respectively , were determined using an in vitro cell 
 model based on the measurement of the permeabilities of the compounds 
 through caco-2 monolayers and an in situ method by performing a rat perfusion assay .
results from the in vitro cell assay are shown 
 in figure 10 and data 
 collected in table 5 , while the results in the rat model are depicted in figure 11 and data collected in table 6 .
permeability values 
 obtained from apical to basal ( pab ) and from 
 basal to apical ( pba ) of cisplatin ( at different concentrations ) , 
 cycloplatinated 4 and 5 , and permeability 
 reference compounds metoprolol , cimetidine , and lucifer yellow at 
 20 m in caco-2 cells .
metoprolol , 
 cimetidine , and lucifer 
 yellow were used as model compounds of high , medium , and low oral 
 permeability , respectively .
pab is the value corresponding to the permeability 
 from the apical 
 to the basolateral chamber that simulates the permeability in the 
 physiological sense from intestine to plasma .
this pba 
 value would be the hypothetical value for the permeability  from 
 plasma to intestine  .
although the pba value has no physiological 
 sense , this parameter and the ratio pab / pba can help to elucidate 
 the mechanism of drug transport across the intestinal barrier .
cell transport assays reveal that cisplatin 
 is a compound with 
 very low permeability .
this permeability value ( 5.44  10cm / s ) indicates that cisplatin is not a suitable drug 
 for oral administration if the objective is to obtain therapeutic 
 plasma values . however , organoplatinum compounds 4 and 5 show higher permeability values than cisplatin .
in fact , 
 the permeability value of compound 4 is 10-fold higher 
 than that of cisplatin at the same concentration , and the permeability 
 of compound 5 is 50-fold higher .
the permeability of 
 compound 4 is higher than those of lucifer yellow and 
 cimetidine but lower than that of metoprolol .
4 can be 
 considered a compound of medium oral permeability . however , the permeability 
 of compound 5 is higher than that of compound 4 and even higher than that of metoprolol ( in accordance with the 
 lipophilicity data ) , indicating that it can be considered a highly 
 permeable compound .
metoprolol , cimetidine , and atenolol 
 were used as model compounds of high , medium , and low oral permeability , 
 respectively .
results from the in 
 situ rat model assays confirm 
 those obtained by the in vitro cell experiments . 

the permeability of compound 4 is higher than that of 
 atenolol , slightly higher that that of cimetidine , but lower than 
 that of metoprolol .
compound 5 exhibits permeability 
 higher than that of compound 4 and slightly higher than 
 that of metoprolol , indicating that 5 is a highly permeable 
 compound . both compounds 4 and 5 display 
 a much better absorption profile than cisplatin .
in addition , 
 we have validated the relationship between caco-2 
 cells permeability and oral fraction absorbed in our experimental 
 system ( represented in figure 12 ) and previously used for fraction absorbed predictions .
the permeabilities of cisplatin and derivatives 4 and 5 have been included in this correlation . 

the predicted oral fraction absorbed is more than 60% for compound 4 and almost 100% for compound 5 , demonstrating 
 its improved absorbability properties with respect to cisplatin . in 
 the absence of solubility or dissolution limitations
the absorption 
 of these compounds would be almost complete ; thus , with the adequate 
 formulation strategy , they represent promising candidates for oral 
 administration .
correlation between oral fractions absorbed vs permeability 
 values 
 obtained from caco-2 cell monolayers transport assay in apical to 
 basal direction ( pab ) .
triangles 
 correspond to permeability reference compounds ( metoprolol / caffeine 
 for high permeability , cimetidine for intermediate permeability , and 
 lucifer yellow for low permeability ) .
since dna replication is the 
 key event for cell division , it is among the critically important 
 targets in cancer chemotherapy .
most cytotoxic platinum drugs form 
 strong covalent bonds with dna bases . however , a variety of platinum compounds act as dna intercalators 
 upon coordination to the appropriate ancillary ligands .
it has been reported that most gold(iii ) compounds 
 display reduced affinity for dna , although 
 there are a number of au(iii ) porphyrin complexes and cyclometalated species with c , n , c - pincer 
 ligands that act as 
 dna intercalators and , in some cases , as dna topoisomerase inhibitors . 

we investigated the interactions of the gold(iii ) and platinum(ii ) 
 complexes with plasmid pb322 dna and with ct dna and directly compared 
 them to the same interactions of cisplatin . to 
 gain insight into the nature 
 of the compound 
dna interactions , gel electrophoresis studies 
 were performed with gold(iii ) ( 1 and 2 ) 
 and platinum(ii ) ( 35 ) complexes 
 on plasmid ( pbr322 ) dna ( figure 13 ) .
plasmid pbr322 presents two main forms , oc ( open 
 circular or relaxed ) and ccc ( covalently closed or supercoiled ) , which 
 display different electrophoretic mobility .
changes in the electrophoretic 
 mobility of any of the forms upon incubation of the plasmid with a 
 compound are usually interpreted as evidence of interaction .
generally , 
 a drug that induces unwinding of the ccc form will produce a retardation 
 of the electrophoretic mobility , while coiling of the oc form will 
 result in increased mobility .
figure 13 shows the effect of cisplatin and compounds 15 on plasmid ( pbr322 ) dna after incubation 
 at 37 c for 20 h in tris - hcl buffer at different drug / dna ratios . 

as previously reported , cisplatin is able to both increase and decrease 
 the mobility of the oc and the ccc forms , respectively .
treatments with increasing amounts of compounds 1 , 2 , 4 , and 5 do not 
 cause any shift for either form , consistent with no unwinding or other 
 changes in topology under the chosen conditions .
treatment with increasing 
 amounts of 3 retards the mobility of the faster - running 
 supercoiled form ( form i ) , especially at higher molar ratios . in order 
 to understand the interaction of 3 with dna , platinum 
 compounds 35 were incubated with 
 ct dna and analyzed by cd .
electrophoresis mobility shift assays for cisplatin 
 and compounds 15 ( see experimental 
 section for details ) .
a , b , c , and d correspond to metal / dnabp ratios of 0.25 , 0.5 , 1.0 , 
 and 2.0 , respectively .
more detailed dna 
 conformational changes can be detected by means of cd spectroscopy . 

cd spectral technique is very sensitive to diagnose alteration on 
 the secondary structure of dna that results from dna  drug interactions . 

a typical cd spectrum of ct dna shows two conservative bands , a positive 
 band with a maximum at 273 nm due to base stacking and a negative 
 band with a minimum at 242 nm due to helipticity , characteristic of 
 the b conformation of dna .
therefore , 
 changes in the cd signals can be assigned to corresponding changes 
 in dna secondary structures .
in addition , it is known that simple 
 grove binding or electrostatic interaction of small molecules causes 
 little or no alteration to any of the cd bands when compared to major 
 perturbation induced by covalent binding or intercalation .
the most 
 dramatic changes in ct dna can be observed with compound 3 ( figure 14a ) . upon 
 addition of increasing amounts of the complex ,
the intensity of the 
 positive band diminishes , and a new negative band at 287 nm and a 
 positive band at 251 nm appear .
this type of modification in the cd 
 spectrum of ct dna is characteristic of conformational changes in 
 dna from b , the usual right - handed form of dna , to z , the left - handed 
 form of dna .
the formation of left - handed 
 helix of z - form dna structure is similar to the transition seen in 
 purely electrostatic environments such as those provided by hgcl2 and hg(clo4)2 .
thus , the presence of [ hg4cl10 ] anion in compound 3 seems to lead to the conformational change from b form to 
 z form .
cd spectra of ct dna ( 195 m ) and ct dna incubated with 0.1 , 
 0.25 , 0.5 , and 1.0 equiv of compounds 3 ( a ) , 4 ( b ) , 5 ( c ) , and cisplatin ( d ) for 20 h at 37 c .
organoplatinum endo compound 4 leads 
 to minor changes of the b - type cd spectrum ( figure 14b ) with slight decrease of the intensities 
 of the positive bands and with no modification in the negative region . 

this points out that the dna binding of complex 4 induces 
 conformational changes including conversion from a more b - like to 
 a more c - like structure within the dna molecule .
this conformational change is indicative of a non - intercalative 
 mode of binding of the complex and offers support that the complex 
 is either grove binding or electrostatic in nature , and the change might be due to the lower concentration of hg released by compound 4 , [ hg2cl6 ] ( compared to that released by compound 3 containing a [ hg4cl10 ] anion ) , although the influence of the more lipophilic pt(ii ) cation 
 in compound 4 , could not be completely ruled out .
finally , as shown in figure 14c , compound 5 does not lead to any modification 
 of the dna bands with respect to untreated ct dna , suggesting that 
 the interaction of compound 5 with ct dna is almost nonexistent . 

this is in good agreement with our findings described above about 
 the influence of the mercury anion in compounds 3 and 4 in their interaction with ct dna since the anion in compound 5 is pf6 . in conclusion
, 
 the experiments of dna  drug interactions 
 have shown that compound 3 induces the formation of left - handed 
 helix of z - form dna through strong electrostatic interactions and 
 compound 4 appears to be either grove binding or electrostatic 
 in nature .
this is supported by two main facts : ( 1 ) retardation of 
 the plasmid ( pbr322 ) dna electrophoretic mobility observed only for 
 compound 3 and ( 2 ) results obtained by cd spectroscopy . 
 importantly , the mercury - free cationic organoplatinum compound 5 does not seem to interact with dna , indicating that , as 
 for other transition - metal i m complexes , its antitumor properties are due to non - dna - related mechanisms / factors .
hsa is the most 
 abundant carrier protein in plasma and is able to bind a variety of 
 substrates , including metal cations , hormones , and most therapeutic 
 drugs .
it has been demonstrated that the distribution , the free concentration , 
 and the metabolism of various drugs can be significantly altered as 
 a result of their binding to this protein .
hsa possesses three fluorophores , namely tryptophan ( trp ) , tyrosine 
 ( tyr ) , and phenylalanine ( phe ) residues , with trp214 being the major 
 contributor to the intrinsic fluorescence of hsa .
this trp fluorescence 
 is sensitive to the environment and binding of substrates , as well 
 as changes in conformation that can result in quenching ( either dynamic 
 or static ) .
thus , the fluorescence spectra of hsa in the presence 
 of increasing amounts of the compounds 15 and cisplatin were recorded in the 300450 nm range 
 upon excitation of the tryptophan residue at 295 nm .
the compounds 
 caused a concentration - dependent quenching of fluorescence without 
 changing the emission maximum or the shape of the peak , as seen in figure 15a for compound 3 .
 volmer plot is indicative of a single 
 quenching mechanism , either dynamic or static , the positive deviation 
 observed in the plots of f0/f versus [ q ] of compounds 24 ( figure 15c ) suggests the 
 presence of different binding sites in the protein with different 
 binding affinities . of note
, a similar 
 behavior was observed in the case of coordination iminophosphorane 
 complexes of d metals , for which we also reported a concentration - dependent 
 fluorescence quenching . on the other hand , the stern 
volmer plot for complexes 1 and 5 shows a linear relationship ( figure 15d ) , suggesting 
 the existence of a single quenching mechanism , most likely dynamic , 
 and a single binding affinity .
volmer constants for 
 complexes 1 and 5 are 4.58  10 and 3.67  10 m , respectively . in general , higher quenching by the iminophosphorane complexes 
 was observed compared to that of cisplatin under the chosen conditions , 
 most likely due to the faster reactivity of our compounds with hsa , 
 as compared to cisplatin .
volmer plot for hsa fluorescence quenching observed 
 with compounds 15 and cisplatin 
 ( b ) , 24 and cisplatin ( c ) , and 1 , 5 , and cisplatin ( d ) .
we have reported on the synthesis and anticancer 
 properties of 
 cyclometalated neutral gold(iii ) and cationic platinum(ii ) derivatives 
 containing iminophosphorane ligands .
most compounds are more cytotoxic 
 to a number of human cancer cell lines than cisplatin .
the gold compounds 
 induced mainly caspase - independent cell death , as previously described 
 for related cycloaurated iminophosphorane compounds . cycloplatinated 
 compounds 3 , 4 , and 5 can also 
 activate alternative caspase - independent mechanisms of death .
however , 
 at short incubation times cell death seems to be mainly caspase dependent , 
 suggesting that the main mechanism of cell death for these compounds 
 is apoptosis .
the most promising candidate is the mercury - free lipophilic 
 cationic cycloplatinated compound 5 .
this derivative 
 is much more active ( 25300-fold ) than cisplatin against a 
 number of cancer cell lines while being less toxic on human renal 
 proximal tubular cell lines .
these facts , along with the lack of interaction 
 observed for 5 with plasmid ( pbr322 ) and ct dna , point 
 to a mode of action different from that of cisplatin .
permeability 
 studies of 5 by two different assays , in vitro caco-2 monolayers and a rat perfusion model , have revealed its high 
 permeability profile ( comparable to that of metoprolol or caffeine ) 
 and an estimated oral fraction absorbed of 100% , which potentially 
 makes it a good candidate for oral administration .
iminophosphorane 
 compound highly active in vivo against breast cancer warrant further advanced preclinical studies 
 with selected organometallic iminophosphorane compounds .
the work 
 described in this paper supports the idea that nontoxic iminophosphorane 
 molecules are excellent ligands for the synthesis of organometallic 
 compounds of d and d metals ( especially cationic 
 species ) with relevant anticancer properties , high permeability , and , 
 in some cases , water - solubility .
all manipulations involving air - free syntheses were 
 performed using 
 standard schlenk - line techniques under a nitrogen atmosphere or in 
 a glovebox ( mbraun mod system ) .
solvents were purified by use of a 
 puresolv purification unit from innovative technology , inc .
the phosphine 
 substrates tpa and pph3 were purchased from sigma - aldrich , 
 [ mn2(co)10 ] and [ ptcl2(cod ) ] were 
 purchased from strem chemicals , and na / hg were purchased from fisher 
 scientific and used without further purification .
compounds [ phch2mn(co)5 ] , [ hg(2-c6h4c(o)n = pph3)cl ] , and [ hg(2-c6h4c(o)n = pta)cl ] and i m ligands ph3p = n - co-2-n - c5h4 were prepared by 
 reported methods .
nmr spectra were recorded on a bruker 
 av400 instrument ( h nmr at 400 mhz , c nmr 
 at 100.6 mhz , p nmr at 161.9 mhz , pt nmr 
 at 85.7 hz ) . chemical shifts (  ) are given in ppm using cdcl3 or dmso - d6 as solvent , unless 
 otherwise stated .
elemental analyses were performed on a perkinelmer 
 2400 chns / o analyzer , series ii .
high - resolution electrospray ionization 
 ( hr - esi ) and matrix - assisted laser desorption / ionization ( maldi ) mass 
 spectra were obtained on an agilent analyzer or a bruker analyzer . 

conductivity was measured in an oakton ph / conductivity meter in acetone 
 solution ( 10 m ) .
x - ray collection was performed 
 at room temperature ( rt ) using graphite - monochromated and 0.5 mm monocap - collimated 
 mo k radiation (  = 0.71073  ) with the  
 scan method .
cd spectra were recorded using a chirascan cd spectrometer 
 equipped with a thermostated cuvette holder .
electrophoresis experiments 
 were carried out in a bio - rad mini subcell gt horizontal electrophoresis 
 system connected to a bio - rad power pac 300 power supply .
photographs 
 of the gels were taken with an alpha innotech fluorchem 8900 camera . 

fluorescence intensity measurements were carried out on a pti qm-4/206 
 se spectrofluorometer ( pti , birmingham , nj ) with right angle detection 
 of fluorescence using a 1 cm path length quartz cuvette .
[ hg(2-c6h4c(o)n = pta)cl ] 
 ( 0.15 g , 0.3 mmol ) , [ nme4][aucl4 ] ( 0.12 g , 0.2 
 mmol ) , and [ nme4]cl ( 0.035 g , 0.32 mmol ) were stirred at 
 rt in ch2cl2 ( 15 ml ) for 1 day in a foil - covered 
 flask .
the fraction 
 containing compound 2 was then extracted from the solid 
 residue with chcl3 ( 3  10 ml ) , and the resulting 
 yellow solution was filtered through celite . the volume was reduced 
 ( <3 ml ) , and upon addition of et2o ( 20 ml ) a pale yellow 
 solid was precipitated .
calcd for c13h16n4opcl2au ( 543.14 ) : c , 28.75 ; 
 h , 2.97 ; n , 10.32 . found : c , 28.32 ; h , 3.07 ; n , 9.93 .
esi - ms : m / z 507.04 ( 100% , [ m  cl ] , calcd 507.04 ) .
p{h } nmr ( cdcl3 ) :  7.66 ( s ) ; ( dmso - d6 ) :  2.68 ( s ) .
h nmr ( cdcl3 ) : 
  4.54 ( 6h , ab system , nch2n ) , 5.10 ( 6h , d , jph = 9.1 hz , pch2n ) , 
 7.34 ( 1h , d , jhh = 7.0 hz , 
 6-c6h4 ) , 7.38 ( dd , jhh = 7.8 , jhh = 
 7.8 hz , 4-c6h4 ) , 7.42 ( dd , jhh = 7.2 , jhh = 7.1 hz , 5-c6h4 ) , 8.04 ( d , jhh = 8.1 hz , 3-c6h4 ) .
c{h } nmr :  55.78 ( d , jpc = 36.8 hz , pch2n ) , 72.37 
 ( d , jpc = 10.8 hz , nch2n ) , 128.64 ( s , 2-c6h4 ) , 129.73 ( s , 3-c6h4 ) , 130.74 ( s , 5-c6h4 ) , 
 134.42 ( s , 4-c6h4 ) , 143.16 ( s , au 
signals corresponding to nc = o and c1 not observable . ir 
 ( cm ) :  352 ( au  cl ) , 1299 ( n = p ) 
 1654 ( c = o ) .
[ hg(2-c6h4c(o)n = pta)cl ] ( 0.225 g , 0.44 mmol ) and 
 [ ptcl2(cod ) ] ( 0.165 g , 0.44 mmol ) were refluxed in ch3cn ( 20 ml ) for 2 h , affording a white solid that was filtrated 
 off and washed with et2o ( 3  10 ml ) , benzene ( 2  
 5 ml ) , and hexane ( 2  10 ml ) . after drying in vacuo , complex 3
calcd for c42h56cl10hg4n8o2p2pt2 ( 2313.94 ) : c , 21.80 ; h , 2.44 ; n , 4.84 . found : c , 21.72 ; h , 2.58 ; 
 n , 4.72 .
hg4cl10 ] , calcd 470.4 ) , 578.2 ( 100% , [ m  hg4cl10 ] , calcd 577.9 ) , 1275.0 ( 100% , 
 [ 2m  hg4cl6 ] + ccl3 , calcd 1275.4 ) .
p{h } 
 nmr ( cdcl3 ) :  7.66 ( s ) ; ( dmso - d6 ) : 10.25 ( s ) .
pt{h } 
 nmr ( dmso - d6 ) :  3652.87 
 ( s ) .
h nmr ( dmso - d6 ) :  
 2.30 ( 8h , s , cod ) , 4.39 ( 6h , s , nch2n ) , 4.91 ( 6h , d , jph = 10.3 hz , pch2n ) , 5.51 ( 4h , s , cod ) , 7.07 ( 1h , m , 4-c6h4 ) , 
 7.16 ( 1h , m , 5-c6h4 ) , 7.29 ( 1h , d , jhh = 7.3 hz , 6-c6h4 ) , 8.15 ( 1h , d , jhh = 8.0 
 hz , 3-c6h4 ) .
c{h } nmr 
 ( dmso - d6 ) :  27.98 ( s , cod ) , 54.27 
 ( d , jpc = 39.2 hz , pch2n ) , 71.30 ( d , jpc = 
 10.2 hz , nch2n ) , 124.6 ( s , 4-c6h4 ) , 128.1 ( s , 6-c6h4 ) , 128.9 ( s , cod ) , 131.9 
 ( s , 5-c6h4 ) , 132.7 ( s , 3-c6h4 ) , 135.0 ( s , 1-c6h4 ) , 138.0 ( d , jcptc = 10.2 hz , 2-ptc ) , 182.1 
 ( d , jpc = 5.18 hz , c = o ) 
 ppm .
ir ( cm ) :  1300 ( n = p ) , 1643 
 ( c = o ) .
[ hg{c6h4(pph2=n(c6h5)}cl ] 
 ( 0.18 g , 0.3 mmol ) and [ ptcl2(cod ) ] ( 0.11 g , 0.3 
 mmol ) were refluxed in acetone ( 30 ml ) for 5 d. the solvent was removed 
 under reduced pressure .
the final product was extracted with ch2cl2 and the resulting solution filtered through 
 celite , giving a light yellow solution . the solution was concentrated 
 ( <3 ml ) , and upon addition of et2o ( 20 ml ) the final 
 product was precipitated as a white solid , isolated by filtration , 
 and dried in vacuo .
calcd 
 for c64h62n2p2cl6pt2hg2ch2cl2 ( 1961.24 ) : 
 c , 38.84 ; h , 3.21 ; n , 1.39 .
esi - ms : m / z 655.18 ( 100% , [ m ] , calcd 
 655.18 ) . p{h } nmr ( cdcl3 ) :  
 64.51 ( s ) ; ( dmso - d6 ) :  63.19 ( s ) . pt{h } nmr ( cdcl3 ) :  3622.47 
 ( d , jppt = 404 hz ) .
h nmr ( cdcl3 ) :  2.66 ( 8h , m , cod ) , 5.125.30 
 ( 4h , m , cod ) , 6.88 ( 2h , m , 2-,6-nar ) , 7.12 ( 1h , m , 4-nar ) , 7.21 ( 2h , 
 m , 3-,5-nar ) , 7.35 ( 1h , m , 4-c6h4 ) , 7.567.67 
 ( 10h , m , o-,m-,p - c6h5 ) , 7.75 
 ( 2h , m , 5-c6h4 ) . c{h } nmr ( cdcl3 ) :  28.24 ( s , cod ) , 31.23 ( s. cod ) , 
 89.30 ( s , cod ) , 116.12 ( s , cod ) , 124.3 ( d , jpc = 91.6 hz , ci ) , 127.1 ( d , jpc = 2.7 hz , 4-nar ) , 127.8 ( d , jpc = 13.9 hz , 4-c6h4 ) , 
 129.5 ( d , jpc = 2.9 , hz , m - c6h5 ) , 129.7 ( s , 3-,5-nar ) , 130.1 
 ( d , j = 4.8 hz , 2-,6-nar ) , 132.7 ( d , jpc = 14.3 hz , 3-c6h4 ) , 133.3 ( m , o-,p - c6h5 ) , 134.3 ( d , jpc = 2.3 hz , 5-c6h4 ) , 147.8 ( s , pt 
c ) . 
 ir ( cm ) :  529 ( pt  n ) , 1310 ( n = p ) . 

( 0.28 g , 0.3 mmol ) and ptcl2(cod ) ( 0.11 g , 0.3 mmol ) were 
 stirred in ch2cl2 ( 20 ml ) for 25 min at rt , 
 follow by addition of nh4pf6 ( 0.049 g , 0.3 mmol ) . 

the resulting solution was stirred for an additional 1 h. the solution 
 was concentrated ( <3 ml ) , dry et2o ( 20 ml ) was added , 
 affording a gray precipitate , and the solution was stirred for 10 
 min .
the precipitated gray solid was isolated by filtration and washed 
 with water ( 4  2 ml ) and a cold mixture ( 1:8 ) of ch2cl2/et2o ( 4  5 ml ) , yielding a white 
 solid that was dried in vacuo .
calcd for c32h31f6np2pt ( 800.63 ) : c , 48.01 ; h , 3.90 ; n , 1.75 . found : c , 47.74 ; h , 4.11 ; 
 n , 1.74 .
esi - ms : m / z 655.18 ( 100% , 
 [ m ] , calcd 655.18 ) .
p{h } nmr 
 ( cdcl3 ) :  64.65 ( s ) ; ( dmso - d6 ) :  63.19 ( s ) .
pt{h } nmr 
 ( cdcl3 ) :  3614.48 ( d , jppt = 406.1 hz ) .
h nmr ( cdcl3 ) :  2.64 ( 8h , m , cod ) , 5.20 ( 4h , m , cod ) , 6.88 ( 2h , 
 m , 2-,6-nar ) , 7.09 ( 1h , m , 4-nar ) , 7.20 ( 2h , m , 3-,5-nar ) , 7.23 ( 1h , 
 m , 3-c6h4 ) , 7.33 ( 1h , m , 4-c6h4 ) , 7.497.66 ( 10h , m , o-,m-,p - c6h5 ) , 7.72 ( 2h , m , 5-c6h4 ) . c{h } nmr ( cdcl3 ) :  28.05 ( s , 
 cod ) , 31.02 ( s , cod ) , 89.22 ( s , cod ) , 116.49 ( s , cod ) , 124.3 ( s , cipso ) , 125.2 ( s , cipso ) , 127.0 ( d , jpc = 2.9 hz , 4-nar ) , 127.6 ( d , jpc = 13.9 hz , 4-c6h4 ) , 
 129.5 ( d , jpc = 2.5 , hz , m - c6h5 ) , 129.6 ( s , 3-,5-nar ) , 130.1 
 ( d , jpc = 4.9 hz , 2-,6-nar ) , 
 132.6 ( d , jpc = 13.8 hz , 3-c6h4 ) , 133.3 ( m , o-,p - c6h5 ) , 134.2 ( d , jpc = 2.9 hz , 5-c6h4 ) , 147.8 ( s , pt 
c ) . 
 ir ( cm ) :  566 ( pt  n ) , 838 ( br , pf6 ) , 1298 ( n = p ) .
the stability of 5 in acidic media over time was studied by h and p{h } nmr in a 2:1 dmso - d6/pbs-1x(d2o ) solution at ph 6 .
the pbs-1x solution 
 was prepared using d2o as solvent and adjusting the ph 
 to 7.4 by addition of 0.1 n hcl .
the deuterated pbs-1x solution was 
 then used to prepare a 2:1 dmso - d6/pbs-1x(d2o ) solution for which the ph was adjusted to 6 by addition 
 of 0.1 n hcl .
complex 5 was then dissolved in the 2:1 
 dmso - d6/pbs-1x(d2o ) solution 
 ( ph 6 ) .
a 2:1 dmso - d6/pbs-1x(d2o ) solution at ph 7.4 was prepared to 
 run control nmr experiments . a gold block - like crystal with 
 the size of 0.10  0.18  0.18 mm was selected 
 for geometry and intensity data collection with a bruker smart apexii 
 ccd area detector on a d8 goniometer at 100 k. the temperature during 
 the data collection was controlled with an oxford cryosystems series 
 700 + instrument .
data were collected using 
 graphite - monochromated and 0.5 mm monocap - collimated mo k radiation 
 (  = 0.71073  ) with the  and  scan method . 

data were processed with the integrate program of the apex2 software 
 for reduction and cell refinement .
multiscan absorption corrections 
 were applied by using the scale program for the area detector .
the 
 structure was solved by the direct method and refined on f ( shelxtl ) .
non - hydrogen 
 atoms were refined with anisotropic displacement parameters , and hydrogen 
 atoms were placed in idealized positions ( c  h = 0.950.99 
  ) and included as riding with uiso(h ) = 1.2 or 1.5 ueq(non - h ) . for toxicity assays , cells ( 5  
 10 for jurkat cells and 10 for adherent cell 
 lines )
were seeded in flat - bottom 96-well plates ( 100 l / well ) 
 in complete medium .
adherent cells were allowed to attach for 24 h 
 prior to addition of cisplatin or tested compounds .
cells were incubated 
 with cisplatin or compounds for 24 h , and then cell proliferation 
 was determined by a modification of the mtt - reduction method . briefly , 

10 l / well of mtt ( 5 mg / ml in pbs ) was added , and plates were 
 incubated for 13 h at 37 c .
finally , formazan crystal 
 was dissolved by adding 100 l / well proh ( 0.05 m 
 hcl ) and gently shaking .
the optical density was measured at 570 nm 
 using a 96-well multiscanner autoreader ( elisa ) .
apoptosis / necrosis hallmarks of 
 cells treated with compound 5 were analyzed by measuring 
 mitochondrial membrane potential , plasma membrane integrity , and exposure 
 of phosphatidylserine .
cells were treated with different concentrations 
 and at different incubation times as indicated in figure legends . 
 in some experiments
the general caspase inhibitor z - vad - fmk was added 
 at 50 m , 1 h before compounds . for mitochondrial membrane potential 
 determination , cells ( 2.5  10 in 200 l ) after 
 treatment with 5 were incubated at 37 c for 15 
 min in medium containing 5 nm dioc6(3 ) ( molecular probes ) . 

phosphatidylserine exposure was quantified by labeling cells with 
 annexin v - dy634 ( invitrogen ) after treatment with 5 . 

annexin v was added at a concentration of 0.5 g / ml in annexin 
 binding buffer ( abb ) , and cells were incubated at room temperature 
 for 15 min .
plasma membrane integrity was evaluated by staining with 
 7-amino - actinomycin d ( 7-aad , inmunostep ) . at the end of the treatment 
 with 5
, cells were incubated for 15 min in 200 l 
 of pbs containing 50 ng/l 7-aad .
in all cases , cells were diluted 
 to 1 ml with abb or phosphate buffered saline ( pbs ) to be analyzed 
 by flow cytometry ( facscan , bd bioscience , spain ) .
caco-2 cells were 
 grown in dubelcco s modified eagle s medium containing l - glutamine , fetal bovine serum , and penicillin  streptomycin . 
 to obtain cells , monolayers of 250  000
cells / cm were seeded on each well with polycarbonate membrane with 4.2 cm area .
plates were incubated at standard conditions of 37 
 c temperature , 90% humidity , and 5% co2 until confluence . 

after 1921 days , the integrity of the each cell monolayer 
 was evaluated by measuring the trans - epithelial electrical resistance 
 ( teer ) .
transport studies were performed using 
 an orbital environmental shaker at constant temperature ( 37 c ) 
 and agitation rate ( 50 rpm ) .
hank s balanced salt solution 
 ( hbss ) supplemented with hepes was used to fill the receiver chamber 
 and to prepare the drug solution placed in the donor chamber .
four 
 samples of 200 l each were taken from the receiver chamber 
 side at predefined times ( 15 , 30 , 60 , and 90 min ) and replaced with 
 the same volume of fresh buffer . moreover ,
two samples of the donor 
 side were taken at the beginning and the end of the experiment .
the 
 amount of compound in cell membranes and inside the cells was determined 
 at the end of the experiments in order to check the mass balance , 
 and the percentage of compound retained in the cell compartment was 
 always less than 5% .
transport studies were performed in both 
 directions , from apical - to - basal 
 ( a - to - b ) and from basal - to - apical ( b - to - a ) sides .
the volume of donor 
 compartment was 2 ml in a - to - b direction and 3 ml in b - to - a direction .
samples were analyzed by hplc 
 using a 5 m , 4  200 mm novapack c18 column .
samples of 
 cisplatin and compound 4 were analyzed with uv detection 
 (  = 240 nm ) .
the mobile phase was 95:5 acetonitrile : water , 
 with a flow rate of 1 ml / min , and the injected sample volume was 50 
 l .
samples of compound 5 were analyzed similarly 
 but using a uv detector at  = 215 nm and a mobile phase of 
 80:20 acetonitrile : water .
the apparent permeability 
 coefficient 
 was calculated following the equation1where creceiver , t is concentration of compound in the receiver chamber 
 at time t , qtotal is 
 the total amount of drug in both chambers , vreceiver and vdonor are the volumes 
 corresponding to receiver and donor compartment , respectively , in 
 each chamber , creceiver , t1 is the concentration of compound in the receiver chamber at the 
 previous time , f is the sample dilution factor due 
 to replaced volume , s is the surface area of the 
 monolayer , t is the time interval , and p is the permeability coefficient .
this equation takes into 
 account the continuous change of the donor and receiver concentrations , 
 i.e. , non - sink conditions .
however , when the transport rate is low , 
 there are not significant changes between the donor and the receiver 
 concentrations with time .
sink conditions are assumed , and a simpler 
 expression can be used to estimate the permeability coefficient:2where dq / dt is the apparent appearance 
 rate of drug in the receiver side calculated 
 using linear regression of amounts in the receiver chamber versus 
 time , s is the surface area of the monolayer , and c is the drug concentration in the donor chamber .
the permeability coefficient estimations in sink and non - sink conditions 
 were carried out in an excel worksheet .
studies were performed in 
 triplicate , and the data are presented as mean  sd .
student s t test was performed with spss 16.0 ( spss inc . ) in order 
 to determine statistically significant differences between a - to - b 
 and b - to - a permeabilities .
the study 
 was approved by the scientific committee of the faculty of pharmacy 
 and followed the guidelines described in the ec directive 86/609 , 
 the council of the europe convention ets 123 , and spanish national 
 laws governing the use of animals in research ( real decreto 223/1988 , 
 boe 67 , 18 - 3 - 98 : 8509 - 8511 ) .
previously to surgical procedure , 
 animals were anesthetized with diazepam ( valium , roche ) ( 1.67 mg / kg ) , 
 ketamine ( ketolar , parke - davis ) ( 50 mg / kg ) , and atropine ( atropine 
 sulfate , braun ) ( 1 mg / kg ) .
therefore , a midline abdominal 
 incision were performed , and a loop was isolated from the duodenal 
 and ileal region of each rat .
the proximal ligatures of the duodenal 
 and ileal regions were placed approximately 1 cm from the pylorus 
 and 2 cm above the ileocecal junction .
first , 50 ml of cleaning solution ( solution a 
 ( ph 7.4 ) : 9.2 g of nacl , 0.34 g of kcl , 0.19 g of cacl2h2o , and 0.76 g of nah2po42 h2o per liter ) was used to flushed out the content 
 of the loop , and then 20 ml of solution b ( nacl g , nah2po42h2o 1/15 m 3.9 ml , na2hpo4 1/15 m 6.1 ml , and water up to 1 l ) was perfused 
 to condition the intestinal mucosa prior to the experiments .
a catheter 
 was tight up at both intestinal ends and connected to a glass syringe 
 by the use of a stopcock type valve . under this setup ,
the intestinal 
 segment is an isolated compartment , and the drug solution can be perfused . 

the drug solutions were prepared freshly each day at 20 m using 
 solution b as solvent and perfused into the loop , and then the entire 
 intestine was restored into the abdominal cavity .
the apparent first - order 
 absorption rate coefficients ( kapp ) were 
 obtained by nonlinear fitting of a monoexponential equation to the 
 luminal concentrations versus time:3where c is the drug concentration 
 remaining in the lumen , kapp is the apparent 
 absorption rate constant , and c0 corresponds 
 to a calculated fraction of the initial perfusion concentration .
test 
 solutions suffer a slight dilution in the intestinal lumen due to 
 the remaining cleaning solution , the adsorption to the membrane , and 
 the loading process in the enterocyte .
the quasi - steady - state is achieved in the membrane when this process 
 is finished . under these conditions ,
the disappearance of the compound 
 from the lumen can be considered as a first - order process during the 
 sampling time interval . for these reasons , only the concentrations 
 obtained after 5 min were used for regression analysis . in order to 
 obtain good prediction data , water re - absorption correction
the intestinal permeability 
 values were calculated taking into account the relationship between ka and peff:4where r is the radius of 
 the intestinal segment , calculated as area / volume ratio .
the effective 
 intestinal permeabilities ( peff ) of the 
 tested compounds ( means of at least of three animals ) were used as 
 indexes of the absorption effectiveness .
first , 10 l aliquots of pbr322 plasmid 
 dna ( 20 g / ml ) in buffer ( 5 mm tris - hcl , 50 mm naclo4 , ph 7.39 ) were incubated with different concentrations of the compounds 15 ( in the range 0.252.0 metal 
 complex : dnabp ) at 37 c for 20 h in the dark .
the 
 samples were separated by electrophoresis for 1.5 h at 80 v in tris - acetate / edta 
 buffer ( tae ) .
afterward , the gel was stained for 30 min with a solution 
 of gelred nucleic acid stain .
stock solutions ( 5 mm ) of each complex 
 were freshly prepared in water 
 prior to use .
the right volume of those solutions was added to 3 ml 
 samples of an also freshly prepared solution of ct dna ( 195 m ) 
 in tris - hcl buffer ( 5 mm tris - hcl , 50 mm naclo4 , ph 7.39 ) 
 to achieve molar ratios of 0.1 , 0.25 , 0.5 , and 1.0 drug / dna .
the samples 
 were incubated at 37 c for a period of 20 h. all cd spectra 
 of dna and of the dna 
drug adducts were recorded at 25 c 
 over a range 220330 nm and finally corrected with a blank 
 and noise reduction .
a solution of each compound ( 8 mm ) in dmso was prepared , and 10 
 aliquots of 2.5 l were added successively to a solution of 
 hsa ( 10 m ) in phosphate buffer ( ph 7.4 ) to achieve final metal 
 complex concentrations in the range 10100 m .
the excitation 
 wavelength was set to 295 nm , and the emission spectra of hsa samples 
 were recorded at room temperature in the range of 300450 nm . 

the fluorescence intensities of all the metal compounds , the buffer , 
 and the dmso are negligible under these conditions .
 new 
 organometallic gold(iii ) and platinum(ii ) complexes containing 
 iminophosphorane ligands are described . 
 most of them are more cytotoxic 
 to a number of human cancer cell lines than cisplatin . 
 cationic pt(ii ) 
 derivatives 4 and 5 , which differ only in 
 the anion , hg2cl62 or pf6 respectively , display almost identical 
 ic50 values in the sub - micromolar range ( 25335-fold 
 more active than cisplatin on these cell lines ) . 
 the gold compounds 
 induced mainly caspase - independent cell death , as previously reported 
 for related cycloaurated compounds containing i m ligands . cycloplatinated 
 compounds 3 , 4 , and 5 can also 
 activate alternative caspase - independent mechanisms of death 
 . however , 
 at short incubation times cell death seems to be mainly caspase dependent , 
 suggesting that the main mechanism of cell death for these compounds 
 is apoptosis . 
 mercury - free compound 5 does not interact 
 with plasmid ( pbr322 ) dna or with calf thymus dna . 
 permeability studies 
 of 5 by two different assays , in vitro caco-2 monolayers and a rat perfusion model , have revealed a high 
 permeability profile for this compound ( comparable to that of metoprolol 
 or caffeine ) and an estimated oral fraction absorbed of 100% , which 
 potentially makes it a good candidate for oral administration . 
Introduction
Results and Discussion
Reactivity with Biomolecules
Conclusions
Experimental
Section