since the discovery of the cytotoxic activity of cis - platin 
 ( cis - diamminedichloroplatinum(ii ) ) 
 , the efforts have been directed towards elucidation of the molecular mechanism of its action [ 24 ] and synthesis of new platinum compounds with improved antitumor activity   and side effects . till now , a large library of platinum complexes has been synthesized and their antitumour activity has been examined but only cis - platin , carboplatin , and oxaliplatin have received a worldwide approval and achieved routine clinical use .   at present ,   the cytotoxicity of cis - platin and other platinum(ii ) complexes is thought to originate from their interaction with dna as well as with non - dna targets [ 2 , 68 ] , and subsequent induction of cell death through apoptosis and/or necrosis [ 9 , 10 ] . 

 despite its wide application as an antitumor drug for treatment of different kinds of cancer , cis - platin has several inherent shortcomings as limited solubility and side toxic effects including nausea , nephrotoxicity , vomiting , and so forth . in addition , many tumors display 
 natural resistance to cis - platin and others develop resistance after the 
 initial treatment . thus the 
 application of the drug is restricted to a relatively narrow class of tumors 
 and the efforts were focused on the design of new platinum compounds with 
 broader spectrum of antitumor activity and improved pharmacological properties 
 with respect to toxicity and resistance . several complexes structurally related 
 to cis - platin , such as oxaliplatin and carboplatin , nedaplatin ( japan ) , 
 lobaplatin ( china ) , heptaplatin ( south korea ) [ 5 , 13 , 14 ] , are currently in 
 clinical trials and use , but till now they have not demonstrated significant 
 advantages over cis - platin . it seems possible that the complex 
 structural analogs of cis - platin designed on the basis of structure - activity relationships   could scarcely offer serious advantages over the existing drugs . analyzing the current status of the platinum - based antitumor drugs , 
 it can be concluded that the search for improved platinum antitumor agents 
 continues [ 1618 ] mainly in direction to modulate the dna - binding mode and 
 dna damage by changing the model structure of the platinum drug used . one of the strategies for a design of new platinum antitumor compounds is to combine platinum(ii ) moiety with proper carrier groups , and thus to target 
 selectively the tumor cells . the porphyrin ligands provide preferable accumulation in neoplastic tissue [ 1921 ] , whereas platinum complexes such as cis - platin and carboplatin penetrate unselectively [ 3 , 17 ] . besides , the porphyrins and , in particular , hematoporphyrin derivatives ( hpd ) are known with their widespread application in the photodynamic therapy and diagnosis 
 [ 22 , 23 ] . during the last decades , a considerable interest has been paid to the synthesis and characterization of pt(ii ) complexes with hematoporphyrin ix and hematoporphyrin - type ligands [ 21 , 2428 ] . the porphyrin ligand could be coordinated to pt(ii ) as a bidentate or tetradentate one by the pyrrole nitrogens [ 24 , 25 ] . recently , the preparation and structural characterization of a large number of 
 platinum - porphyrin conjugates have been discussed [ 21 , 2629 ] . in these compounds , platinum(ii ) fragment is attached to the propionic acid side chains out of the porphyrin macrocycle . the cytostatic activity of these platinum(ii ) porphyrin conjugates towards different kinds of mammary and bladder cancer cell lines has been tested in dark and after irradiation . a high activity was reported , especially for the water - soluble conjugates [ 27 , 28 ] . other factor of great importance for the enhancement of the antitumor activity and circumvention of the drug resistance is the oxidation state of platinum in the antitumor drug . the higher oxidation platinum complexes possess increased opportunity to be delivered to the cellular target as an active agent . it is well known that in physiological media , platinum(iv ) anticancer agents are easily reduced to active platinum(ii ) products [ 5 , 18 ] . an advantage of the pt(iv ) complexes is their lower reactivity which decreases the loss of the active drug and the incidence of the side reactions . in addition , the axial ligands in the octahedral platinum ( iv ) complexes could alter the lipophilicity and the redox potential of the complex , and thus improve the cellular uptake and control of the reduction rate . in the present paper , we propose a new approach for design of cytotoxic agents with improved properties , namely to combine platinum in the unusual oxidation state + 3 with a proper ligand system which could stabilize this oxidation state and serve as a specific carrier group . this approach is based on the idea for 
 the intermediate behavior of pt(iii ) complexes between the complexes of pt(ii ) 
 and pt(iv ) with respect to kinetic inertness and thermodynamic stability . hematoporphyrin ix was used as a ligand system . as a further development of our 
 studies on the synthesis of stable pt(iii ) complexes 
 [ 2931 ] with proper ligand systems 
 [ 3234 ] , we have investigated the pt(ii)-hematoporphyrin 
 ix interaction in aqueous - alkaline medium and aerobic conditions on light . three stable octahedral pt(iii)-hematoporphyin 
 complexes have been synthesized and 
 structurally characterized by spectroscopic and magnetic methods . the cytotoxic 
 activity study showed that despite the violation of the empirical structure  activity relationship rules , the complexes show a promising cytotoxicity comparable 
 with that of cis - platin . although the high antitumor activity of mixed oxidation pt(ii)pt(iii ) polymeric complexes of  platinum blues  type was recognized long ago , this is the first report on the cytotoxic effect of stable monomer pt(iii ) species . all reagents and solvents were of analytical grade and were obtained by commercial sources and used without further purification . a sample of 0.030  g ( 0.050  mmol ) hp was dissolved by stirring in 8  ml 
 5102  m koh ( 0.40  mmol ) and a 
 ph value of 11.011.5 was finally obtained . 
 a cis - diammine(diaqua)platinum(ii ) hydroxide solution was prepared by stirring cis - platin ( 0.2  mmol , 0.060  g for the complex  
 1 ; and 0.1  mmol , 0.030  g for the 
 complex 2 ) with 1.95 molar equivalents of 
 agno3 
 in water ( 5  ml ) . the mixtures were kept in dark for 24 hours and the 
 agcl formed was then removed by a centrifuge . aqueous solution of k2ptcl4 
 ( 1.10  m ) was used for the synthesis . the ph values together with the uv - vis and epr spectra were monitored during the reaction course at regular intervals . the uv - vis spectra were recorded after dilution of samples from the reaction mixtures with distilled water to 6.25105  m ligand concentration . the epr spectra 
 of frozen samples ( 100270 k ) of the reaction systems were recorded without 
 dilution . aqueous solution of diammine(diaqua)platinum(ii ) hydroxide ( 1.10  m , 0.2  mmol ) was added in excess to a ( 0.05  mmol ) solution of hp ( molar ratio pt : hp = 4 ) at permanent stirring . a solution of 5102  m koh was added immediately until ph 10.811.0 is obtained . during this period , the acidity of the reaction solution decreases further spontaneously , and at ph  8 , the complex [ pt(nh3)2(hp3h 
 ) ( h2o)2]h2o 1 precipitates as a dark - violet powder . the powder was filtered , washed with 
 water and alcohol , and dried over p4o10 . the mass losses of 2.02% , 4.16% , and 3.67% in the temperature ranges 95130c , 
 130260c , 
 and 260295c , 
 respectively , correspond to 1h2o molecule , 
 2h2o molecules , and 2nh3 molecules per mol of the complex ( theoretical contents 2.05% , 4.10% , and 3.88% , resp . ) . c 46.46 , h 5.39 , n   9.56 , pt 22.19 , found c 46.51 , h 5.48 , n 9.62 , pt 
 22.38% . ir ( cm ) : 3303 
                          (=nh ) ; 3235 , 3218 , 3172 , 3093 
                             (nh3 ) ; 2964 , 2924 , 2855 (ch3 , ch2 ) ; 1552 , 
 1407 (coo ) . uv - vis ( 8.10  m in concentrate ch3cooh ) max(log   ) : 395 ( 4.87 ) , 510 ( 3.86 ) , 545 ( 3.92 ) , 565 ( 3.90 ) , 630sh ( 3.53 ) . a procedure similar to that described for 1 was used for 
 the preparation of the complex 2 , but the aqueous solution of 
 diammine(diaqua)platinum(ii ) hydroxide ( 1.10  m , 0.1  mmol ) was added in twofold excess ( molar ratio pt : hp = 2 ) and a ph value of 10.310.5 was obtained . the complex with composition [ pt(hp3h 
                         ) ( h2o)2]h2o 2 was isolated as a dark - violet precipitate by adding 3  ml 0.2 m hno3 . the precipitate was filtered , washed with water and alcohol , and dried over 
 p4o10 . the mass losses 
 of 2.00% in the temperature range 100130c and 4.25% in the temperature range 130250c correspond to 1h2o molecule and 2h2o molecules ( theoretical contents of 2.13% and 4.26% , resp . ) . c 48.33 , h 4.89 , n 6.63 , pt 23.09 , found c 48.26 , h 4.79 , n 6.70 , pt 23.15% . ir ( cm ) : 2968 , 2915 , 2875 , 2820sh 
 (ch3 , ch2 ) ; 1704 (c = o ) ; 1563 , 1352 , 
                          (coo ) . uv - vis ( 8.10  m in concentrate ch3cooh ) max ( log   ) : 400 ( 4.89 ) , 520 ( 3.92 ) , 560 ( 3.97 ) , 635 sh ( 3.56 ) ; ( 8.10  m in dmf ) 
 max(log   ) : 405 ( 4.88 ) , 515 ( 3.94 ) , 540 ( 3.94 ) , 630 sh ( 3.49 ) . aqueous solution of k2ptcl4 ( 0.0208  g , 0.05  mmol ) and a solution of hp ( 0.05  mmol ) were mixed in equimolar ratio ( pt : hp = 1 ) and the basicity of the reaction mixture was adjusted to ph = 11.5 by addition of 
  5102  m koh . the system was kept at ambient temperature for 2 weeks . a dark violet complex with composition 
  [ pt((o , o)hp2h 
                         ) cl(h2o)3 ] the mass losses in the temperature ranges 100161c ( 4.09% ) and 225250c ( 2.04% ) correspond to 2h2o and 1h2o molecules ( theoretical contents 4.09% and 2.04% , resp . ) . a mass 
 loss of 4.14% observed in the temperature range 160225c corresponds to removal of cl as hcl ( theoretical 4.14% ) . c 46.34 , h 4.80 , n 6.36 , pt 22.14 , cl 4.02% , found c 46.66 , h 4.72 , n 6.69 , pt 22.26 , cl , 4.33% . ir 
 ( cm ) 3319 (=nh ) ; 2984 , 2913 , 2862 (ch3 , ch2 ) ; 
 1620 , 1350 , (coo ) . uv - vis ( 8.10 m in 
 dmf ) max ( log   ) : 375 ( 4.04 ) 410 ( 4.89 ) , 520 ( 3.83 ) , 560 ( 3.74 ) , 590 ( 3.70 ) , 635 ( 3.57 ) . c , h , n , and cl analyses were performed in the elemental analyses laboratory in the university of sofia . the pt content was determined gravimetrically after treatment of the sample with concentrate 
 h2so4 and 30% h2o2 . the absorption electronic , reflectance , and ir spectra ( kbr - disks , 4000400  cm , csi - disk , 
 40050  cm ) were recorded on a uv - vis  carl - zeiss , jena , lambda 17 uv - vis , and ftir - bruker ifs 113  v and  perkin elmer 983  spectrometers , respectively . bruker 
 b - er 420  spectrometer in the temperature range 100298 k. magnetic susceptibility was measured between 2  k and 300  k in magnetic 
 field of 1 and/or 5 keys using commercial squid magnetometer ( quantum design 
 mpms - xl ) with sensitivity of 10  emu . the data were corrected for the diamagnetic response of the sample holder and for the diamagnetic 
 contribution of the sample ( pascal 's constants ) . x - ray photoemission 
 spectra were recorded on an escalab - mkii ( vg scientific ) electron spectrometer escalab - mkii ( vg scientific ) with a base pressure of 
 1.10  pa . all xps spectra were calibrated using the c1s - core level at 285.0  ev as a reference . in this study , the following human cell lines were used : 
 skw-3 ( dsmz no . : acc 53)t - cell leukemia  a derivative of ke-37 established from a patient with acute lymphoblastic leukemia ; bv-173 
 ( dsmz no . : acc 20)chronic myeloid leukemia established from a cml 
 patient in a lymphoblastic crisis ; lama-84 ( dsmz no . the cell lines were obtained from the german collection of microorganisms and cell cultures ( dsmz 
 gmbh , germany ) . rpmi-1640 medium , supplemented with 10% fbs 
 and 2  mm l - glutamin , in cell culture flasks at 
 37c with humidified atmosphere and 5% co2 . cells were refereed with fresh medium two or three times a week in order to maintain logarithmic growth . the cell viability was assessed using the standard mtt ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) 
 reduction assay as described by mosmann   with minor modifications . stock solutions of cis - platin and the new platinum complexes were freshly prepared in dmso and then diluted with corresponding growth medium . at the final dilutions , horizontal gel - electrophoresis of cytosolic dna , isolated from skw-3 cells treated with [ pt(hp3h 
                         ) ( h2o)2 ] , 2 was performed in order to test the ability of the compounds under investigation to trigger programmed cell death 
 ( apoptosis ) . the dna fragmentation was monitored by a  cell death detection  elisa ( roche diagnostics gmbh , germany ) 
 as well . cytosolic fractions of 1104 cells per group ( treated or untreated ) were used as antigen source in a 
 sandwich elisa , applying primary antihistone antibody - coated microplate and a 
 secondary peroxidase - conjugated anti - dna - antibody . the photometric immunoassay 
 for histone - associated dna fragments was executed according to the 
 manufacturers instructions at 405  nm , using elisa reader ( unican titertec ) . the results are presented as the oligonucleosomal enrichment factor ef(% ) : ef(%)=(atr / aco)100 , where atr is 405  nm absorption of treated samples ; a 
                             co 
                          is 405  nm absorption of control samples . the pt(ii)-hematoporphyrin ix interaction was studied in aqueous - alkaline solution 
 and aerobic conditions on light . water solutions of 
 cis - diammine(diaqua)platinum(ii)hydroxide 
 ( obtained from cis - platin   after precipitation of cl ) and k2ptcl4 were used as initial pt(ii ) 
 complexes . the ph value of the reaction systems , obtained at different metal - to - ligand ratios was adjusted in the range 10.511.5 by addition of 5102  m koh 
 solution . in all cases studied , the pt(ii)hematoporphyrin interaction started with an increase of the acidity ( ph = 24 ) . the changes in the electronic absorption spectra ( soret and q - bands ) during the interactions are depicted on figures 1(a ) and 1(b ) . the mixing of hematoporphyrin with diammine(diaqua)platinum(ii)hydroxideat metal excess ( pt : hp  2 ) is connected with a slight hypsochromic 
 shift of the soret band ( 369  nm ) in comparison with the free ligand spectrum ( 374  nm ) ( figure 1(a)a , b ) . further spectral changes ( figure 1(a)c , d ) in the course of the reaction followed the drop of the ph value . the soret band ( 376  nm ) decreased and broadened and a shoulder at 400  nm arose . a new band with growing intensity appeared at 270  nm during the interaction ( figure 1(a)c , d ) . at the end of the reaction , all absorption bands 
 underwent a red shift and became less intensive and an additional decrease of 
 ph value to 7.5 was established . a sequence of iv > iii > ii > i for the satellite 
 q - band intensity is observed . these spectral changes suggest that the porphyrin 
 ring in the reaction product is distorted , and hence formation of 
 sitting - atop- ( sat- ) type complex with an asymmetrical coordination of the ligand through two adjacent pyrrole n atoms is most probably realized   [ 25 , 38 ] . a narrow signal with parameters typical for a free radical ( h 
                         pp 
                       3 
 g , with g = 2.0090.001 ) 
 was recorded in the epr spectrum of a frozen sample taken one hour after the 
 mixing of the reagents . the signal remains unchanged for a week in these 
 conditions and indicates that a redox process takes also place in the system . several hours after the start of the reaction together with the first signal , a 
 new one appeared with increasing intensity 
 ( figure 2(a)b , c ) . nine superhyperfine lines due to the interaction of the unpaired electron of pt(iii ) with 
 four n - nuclei ( i = 1 ) were readily observed in the perpendicular region ( figure 2 ) . the signal most probably corresponds to a rhombic symmetry as follows from the slight splitting observed in the perpendicular 
 region ( figure 2(a)d ) . a week later , an intensive signal with parameters closed to the former one could be only found in the spectrum ( figure 2(a)e ) . nine superhyperfine lines from four n - nuclei 
 ( i = 1 , a(n ) = 21.0  10  cm ) 
 were observed in the perpendicular region . the superhyperfine structure in the 
 parallel region due to n ( a(n ) = 24.9  10  cm ) 
 overlaps the hyperfine structure due to pt(i = 1/2 , a  50  10  cm ) . the hyperfine structure ( a(pt ) ) was better resolved in the parallel region than 
 in the perpendicular one ( a(pt )  60  10  cm ) . hence , a stable pt(iii ) complex is the product of the 
 cis - diammine(diaqua)platinum(ii)hydroxide interaction with 
 hematoporphyrin in aqueous alkaline medium , in which complex platinum(iii ) is 
 surrounded by four n donor atoms ( figure 2(a)e ) . the axial signal symmetry is in accordance with the symmetrical coordination of the ligand to pt(iii ) via the four pyrrole n - atoms in the porphyrin macrocycle . an intermediate pt(iii ) complex with a lower symmetry signal was recorded in the course of this reaction . in this complex , the lower symmetry is most probably due to a sat complex formation where two of the nitrogens come from adjacent porphyrin pyrroles and the other two from the ammine groups of the initial pt(ii ) complex . the interaction of ptcl42 with hp in aqueous alkaline solution was studied at equimolar ratio of the reagents . it was found that the acidity of the reaction mixture increases faster than in the case when diammine(diaqua)platinum(ii)hydroxide was used . during the reaction course , the intensity of the characteristic absorption bands decreased ( figure 1(b)a  c ) . a narrow epr 
 signal ( hpp  8  g ) with 
 g = 1.986 was recorded in 
 the epr spectra of the frozen reaction system ( 130  k ) again due to a stable free radical ( figure 2(b ) ) . in addition , a second broad 
 ( hpp 
                       60  g ) and low intensive signal appeared in a magnetic field about 3280  g ( g  2.06 ) 
 several hours later most probably due to a pt(iii)-complex formation . in the 
 spectrum , no superhyperfine splitting from n was observed , and 
 hence the ligand in this complex is coordinated through the carboxylic acid 
 groups outside the porphyrin   macrocycle . the neutral complex 
 cis-[pt(nh3)2(hp3h)(h2o)2]h2o 1 was precipitated from the reaction mixture of 
 cis - diammine(diaqua)platinum(ii ) hydroxide and hematoporphyrin 
 ( pt : hp = 4 ) after a spontaneous decrease of ph down to 8 . the complex [ pt(hp3h 
                     ) ( h2o)2]h2o 2 is the main product from the 
 reaction mixture at pt : hp = 2 molar ratio . 
 the complex [ pt((o , o)hp2h)cl(h2o)3 ] 3 was obtained from the reaction system ptcl42-hp taken in equimolar ratio . the complexes 2 and 
 3 were isolated as powders after addition of hno3 and 
 hclo4 . the contents of water and nh3 for complex 1 and of cl for complex 3 
 were determined by thermogravimetric and calorimetric measurements 
 ( table 1 ) . the relatively high temperatures of 
 dehydration ( above 130c ) as well as the thermal effects indicated that some water molecules are bound in the inner coordination sphere . the processes of the removal of water and ammonia molecules are endothermic , while that for cl as hcl is an exothermic one . the bands in the uv - v is absorption and reflectance spectra of the complexes 
 obtained correspond to those from the electronic spectra recorded in the 
 solution during the interactions ( figure 1 ) . the three pt(iii)-hp complexes have shown the characteristic soret band about 400  nm with a molar absorptivity up to 1105  mol.l.cm . in the spectrum of the complex 1 , four 
 q - bands could readily be distinguished in the range of 500650 nm . the intensity of the bands iv < iii  ii > i differs from the etio - type free ligand spectrum and all bands 
 are red - shifted in comparison with the free ligand spectrum . the presence of 
 all four bands indicates a relatively low symmetry of the porphyrin ring , due 
 to an unsymmetrical coordination of the hematoporphyrin ligand to platinum as 
 it could be expected for sat - type complexes [ 25 , 38 ] . the isolation of the complex 2 
 by adding of acid resulted in a reduction of the number of the q - bands to two ( figure 1(a)e ) . a decrease of the 
 q - bands number was observed in the electronic and reflectance spectra of the 
 isolated complex . this fact could be explained with increasing of the symmetry 
 of the porphyrin ring through coordination of platinum to the four pyrrole 
 nitrogen atoms . the complex 3 showed 
 absorption spectrum of an etio type similar to that of the free ligand with a 
 sequence of the q - bands intensity of iv > iii > ii > i. in addition , a shoulder at 375  nm also appeared . the spectrum obtained indicates that platinum is most probably coordinated to the peripheral carboxylic groups of the hematoporphyrin ring . the parameters determined from the experimental spectra and those used in the simulation procedure are shown in table 2 . the complex 2 shows ( figure 3(a)a ) an anisotropic signal ( g=2.113 and g=2.038 ) with temperature - dependent intensity , which is due to pt(iii ) . nine superhyperfine lines due to interaction of uncoupled electron with four 
 n ( i = 1 ) were observed both in perpendicular and parallel regions . the hyperfine signal structure due to pt ( i = 1/2 ) overlaps the superhyperfine structure and is better resolved in the parallel region . simulated epr spectrum 
 ( figure 3(a)b ) was obtained using the parameters of the experimental epr spectrum ( figure 3(a)a ) . the model is based on the assumption that platinum is in oxidation state + 3   ( i = 1/2 ) , with 
 natural abundance of pt 33.8% . platinum(iii ) is assumed to be 
 coordinated via four nitrogens of the porphyrin macrocycle . n , i = 1 ) are present in two groups of magnetically equivalent nuclei , each group containing two opposite nitrogen nuclei from the porphyrin cycle . the simulation procedure was performed by variation of the 
 principal values of g - tensor , nuclear hyperfine tensors ( a(pt ) ) , and nuclear 
 superhyperfine tensors ( a(n ) ) . the epr signal of complex 1 is anisotropic but with a lower symmetry and an additional splitting in the perpendicular region . nine superhyperfine lines due to the interaction with 
 four n nuclei were observed . in the case of rhombic symmetry and 
 presence of superhyperfine coupling with four n nuclei , the number 
 of lines increases and the intensity of the superhyperfine lines from pt nuclei ( 33.8% ) decreases . hence , the principal values of nuclear hyperfine 
 tensor ( pt ) could not be determined from the experimental 
 spectrum . the analysis of the signal was based on the assumption for a rhombic 
 symmetry . the values of the rhombic g - tensor and rhombic nuclear superhyperfine 
 tensor a(n ) were determined from the experimental spectrum ( table 2 ) . the decrease of the symmetry is most probably due to the coordination of unequivalent n atoms in the pt equatorial plane of platinum . the lowfield signal corresponds to pt(iii ) complex with a rhombic symmetry and g - values given in table 2 . the epr linewidth is close to the hyperfine splitting constant , and for this reason the hyperfine structure 
 from pt is not resolved . the absence of a superhyperfine structure 
 from n nuclei could be related to the fact that pt(iii ) is 
 coordinated outside the porphyrin ring . the highfield signal corresponds to a 
 free radical with an axial symmetry and parameters g=2.000 and 
                     g = 1.980 . magnetic susceptibility (  ) decreases monotonically with increasing temperature 
 ( figure 4 ) , thus suggesting paramagnetic behavior in the range of 2300  k and octahedral structure for all complexes studied . the effective magnetic moments , eff , were estimated taking into account the diamagnetic corrections via the tabulated pascal constants 
                     ( 4.08104  emu / mol ; 
  3.72104  emu / mol ; 
 and 3.98104  emu / mol for complexes 1 , 2 , and 3 , resp . ) . the calculated values of eff=2.19  b ,  
 1.54  b and 1.73  b , for the complexes 1 , 2 , and 
 3 , respectively , are in an agreement with 
 the spin - only value so=1.73 . selected data from the x - ray photoemission spectra of the free ligand and the complexes studied are present in table 3 . the two peaks for the n 1s binding energy at 400.0 and 398  ev in the free ligand spectrum are usually assigned to the pyrrole ( hn < ) and the aza ( n= ) nitrogens . the higher energy peak belongs 
 to the pyrrole nitrogens because of their higher electronegativity 
 . the presence of only one n 1s peak in complex 2 indicates that all nitrogens in this compound are equivalent . the intermediate value of the n 1s binding energy ( 399.0  ev ) is due to coordination of the four n atoms to platinum and formation of metalloporphyrin - type complex by incorporation of platinum in the porphyrin ring . three different peaks could be distinguished in the n 1s spectrum of complex 
 1 . the two lower energy peaks at 399.2  ev and 398.4  ev were assigned to two pairs of equivalent hematoporphyrin 
 nitrogens . the n 1s binding energy peak at 399.2 is assigned to nitrogens coordinated to platinum . the lowest energy peak that is close to that of the free ligand 
 aza nitrogens is assigned to uncoordinated hematoporphyrin nitrogens . the 
 equivalence of these two nitrogens as well as the decrease of their 
 electronegativity in comparison with the free pyrrole nitrogens could be 
 explained with delocalization of electron density by h - bonding formation 
 ( scheme 1 ) . the highest binding energy peak at 400.5  ev shows the presence of nitrogens with a higher electronegativity with respect of the free ligand nitrogens and was assigned to coordinated ammine nitrogens . the coordination of the ligand by peripheral carboxylic groups in complex  
 3 was proved by the presence of low energy peak in the o 1s spectrum at 529.0  ev . the peaks in the interval of 
 532.2532.6  ev for the free ligand and the complexes o 1s spectra are due to uncoordinated ligand oxygens and the presence of water molecules . the peak at 400.3  ev in the n 1s spectrum of 
 complex 3 is assigned to the four equivalent uncoordinated hematoporphyrin nitrogens . the relatively high n 1s binding energy and the equivalence of the four nitrogens could be considered as a result of a significant metal to ligand electron - density transfer and delocalization by h - bonding formation ( scheme 2 ) . the pt 4f spectra of the complexes are resolved into spin - orbit pairs with 
 splitting of 3.3  ev for all complexes studied . the determined 
 pt 4f7/2 binding energies values for the three complexes in the range of 73.173.8  ev are in accordance with the formation 
 of the + 3 oxidation state . the ir data ( table 4 ) have shown identical 
 (oh ) and 
                     (ch3,ch2 ) 
 stretching vibrations for the hydroxyethyl , methyl , and propionic side chains 
 as well as in - plane and out - of - plane porphyrin skeletal vibrations . the 
 coordination of h2o in the complexes follows from the presence of 
 (h2o ) in the range of 33503450  cmand of (h2o ) at 16001630  cm . the assignments of the bands are made in accordance with the ir data and nca published for metalloporphyrins . the (nh ) band at 3312  cm disappears in the spectrum of  
 2 , and thus indicates a metal insertion into the porphyrin 
 macrocycle and the formation of the metalloporphyrin - type complex . bands of 
 carboxylate ion ( (coo ) 1563 , 1352 ) and carbonyl stretch ( 1704  cm ) from the protonated cooh are present in the same spectrum . the coordination via 
 pyrrole n - atoms is supported also by the presence of a strong absorption band 
 at 441  cm assigned to ptn stretching vibrations . the bands in the far - infrared spectrum in the range 
 390230  cm were assigned to 
 stretching pto and deformation mode vibrations 
 of the coordinated h2o 
 molecules . conversely , in the ir spectrum of 3 , the presence of 
 (nh ) at 3319  cm shows coordination outside the porphyrin macrocycle . in addition , a shift of the c = o porphyrin - carboxylic acid bands at 1620 and 
 1350  cm indicates coordination through side chain deprotonated carboxylic groups . a value of 270  cm for  = ( (coo)(coo ) 
 proves unidentate coordination   of the carboxylic groups . the bands at 366 and 324  cm as well as those at 258 and 210  cm are assigned to stretching 
 vibrations of pto ' ( o ' belongs to coordinated carboxylic groups ) and ptcl . other far - infrared bands at 483 , 461 , 453 , 388 , and 231 could be assigned to stretching pto and deformation mode vibrations of the coordinated in - plane and in axial positions 
 h2o - molecules . the ir spectrum of 1 shows a characteristic 
 (nh ) absorption band of uncoordinated pyrrole at 
 3303  cm . the c = o absorption is 
 shifted to 1552 and 1407  cm for (coo ) stretching vibrations . the value of 
  = ( (coo)(coo ) = 145 cmcorresponds to the presence of deprotonated uncoordinated carboxylic groups . 
 besides , the bands due to coordination of nh3 molecules were 
 observed in the range 32353090  cm 
 and at 1605  cm . these spectral data correspond to a sat complex formation , where platinum is 
 coordinated partially to some of the porphyrin nitrogens , part of them being 
 still protonated . the pt(iii ) coordination sphere includes also 
 two nh3-molecules from the initial pt(ii)-complex in a cis - position as follows from the presence of two pairs of 
 antisymmetric and symmetric stretching vibrations of ptn2 and ptn'2(n ' belongs to nh3 ) at 444 , 425  cm and 394 , 
 364  cm . the other bands in the range 316160  cmcould be assigned to stretching pto and deformation mode vibrations of the coordinated h2o molecules . summarizing all experimental data obtained , it can be concluded that the interaction between pt(ii ) and hp in aqueous alkaline solution proceeds in a different way depending mainly on the type of the initial pt(ii ) species and the 
 metal - to - ligand ratio . the complexation process is accompanied by a parallel 
 redox process leading to formation of pt(iii ) species and organic radicals , together 
 with a considerable decrease of ph . the final products of the overall 
 pt(ii)-hp interaction are three hp - complexes of pt(iii ) . the first sat - type complex   1 precipitatesin alkaline medium 
 after spontaneous decrease of initial ph from 11 to 8 , 
 m : l molar ratio of 4 , platinum being introduced as 
 cis - diammine(diaqua)platinum(ii ) 
 hydroxide . in these conditions , the protons of the carboxylate groups 
 dissociate and the ligand reacts as twofold deprotonated species 
 . its coordination to pt(iii ) is realized through two adjacent porphyrin pyrrole nitrogens , substituting one pyrrole ' hydrogen . the ptn4-unit in the coordination sphere is formed with participation also of two nh3-molecules in 
 a cis - position ( scheme 1 ) . the metalloporphyrin - type complex 2the main product from the same reaction mixture but at pt : hp = 2 molar ratio  was isolated in a solid state using 
 hno3 ( scheme 3 ) . in acidic medium , the coordinated nh3 molecules leave 
 the inner coordination sphere as nh4 
                      and pt(iii ) coordinate to the other two pyrrole nitrogen atoms by substitution of a second proton . the complex   3 was obtained by the 
 reaction of ptcl42 and hematoporphyrin in equimolar 
 ratio . because of the faster base hydrolysis of ptcl42 and the pt(iii ) preference for o - donors ( in comparison with pt(ii ) ) , 
 the coordination here is realized via deprotonated carboxylic groups out of the 
 porphyrin macrocycle . the equatorial coordination plane of platinum includes 
 also cl and h2o molecules ( scheme 2 ) . a distorted octahedral structure with h2o molecules disposed in axial position the experimental data from the cytotoxicity investigation were fitted to sigmoidal 
 dose - response curves . the novel platinum(iii ) complexes under investigation 
 exerted cytotoxic effects against the panel of leukaemic cell lines in a 
 concentration - dependent manner . against both bv-173 and lama-84 cells , the 
 compounds   1 and   2 displayed significant cytotoxic efficacy with ic50 values comparable to those of the referent cytotoxic agent cis - ddp . furthermore , the maximal efficacy of 1 and   2 , estimated at 50  m , was superior 
 to that of cis - platin against both lama-84 and bv-173 . it is noteworthy 
 that despite the different cell types of lama-84 ( myeloid ) and bv-173 
 ( lymphoid ) , these lines share the same origin , being both isolated from chronic 
 myeloid leukemia ( cml ) patients in blast crisis . conversely , both cell lines 
 are characterized via the expression of the characteristic for cml bcr - abl protein , a nonreceptor tyrosine kinase whose constitutive activation renders the cells less responsive to proapoptotic stimuli , including chemotherapy agents [ 41 , 42 ] . the cytotoxicity 
 of cis - platin , being circa twofold less active on lama-84 than in bv-173 reflects the well - known discrepancies of the 
 degree of bcr - abl expression , being more pronounced in the former cell line 
 .   in a dissimilar fashion , both 1 and   2 share practically identical potency in both cell lines , which clearly indicates that the level of bcr - abl expression does not affect their cytotoxicity significantly . the complex 3 was found to be far less active on molar basis against lama-84 and bv-173 this complex causes 50% reduction of cell viability at 2 - 3 times higher concentrations as compared to 1 and 
 2 . the skw-3 cell line demonstrated higher sensitivity to the 
 complex 3 , with an ic50 value being twice that of 
 cis - ddp , whereas 1 and   2 induced 
 half - maximal effects at 2-fold higher concentrations . the detected dna - laddering showed that the observed cytotoxicity of the complex 2 is at least partly mediated via the 
 recruitment of the apoptotic cell signaling pathways in lymphoid skw-3 cells . in order to elucidate the proapoptotic activity of 2 in a more 
 quantitative manner , its ability to induce oligonucleosomal dna fragmentation 
 was analyzed by means of  cell death detection the obtained results have shown that a 24-hour treatment of skw-3 
 cells with   2 ( at 12.5 , 25 , or 50  m ) leads to a significant enrichment of the cytosole with histone - associated mono- and oligonucleosomal dna - fragments ( figure 6 ) . these findings together with the established dna - laddering 
 unambiguously indicate that the recruitment of the programmed cell death 
 signaling pathways plays a pivotal role for the cytotoxic mode of action of the 
 tested complex compound .
three novel stable pt(iii ) complexes with distorted octahedral structure and ( dz2)1 ground state have been obtained in the course of pt(ii)-hematoporphyrin ix ( ( 7,12-bis(1-hydroxyethyl)-3,8,13,17-tetramethyl-21h-23h - porphyn-2,18-dipropionic   acid ) , hp ) interaction in alkaline aqueous medium and aerobic conditions . 
 a redox interaction also takes place together with the complexation process leading to the formation of pt(iii ) species and organic radicals . 
 the processes in the reaction system and the structure of the complexes formed cis-[pt(iii)(nh3)2(hp3h)(h2o)2]h2o 
 1 , [ pt(iii)(hp3h)(h2o)2]h2o 
 2 , and [ pt((o , o)hp2h)cl(h2o)3 ] 3 , were studied by uv - vis , ir , epr and xps spectra , thermal ( tgs , dsc ) , potentiometric and magnetic methods . 
 the newly synthesized complexes show promising cytotoxic activity comparable with that of 
 cis - platin in in vitro tests against a panel of human leukemia cell lines . 
 the observed cytotoxicity of the complex 2 against skw-3 cells ( ke-37 derivative ) is due to induction of cell death through apoptosis .