coumarin ( 1,2-benzopyrone ) is structurally the least complex 
 member of a large class of compounds known as benzopyrones 
 . the biological activities of coumarin and related 
 compounds are multiple and include antithrombotic activity 
  and antimicrobial properties . in addition , coumarins have been shown to inhibit n - methyl - n - nitrosourea , aflatoxin b1 and 
 7,12-dimethylbenz(a)anthracene - induced mammary carcinogenesis in 
 rats [ 4 , 5 ] . more recently , coumarin derivatives have been 
 evaluated in the treatment of human immunodeficiency virus , due to 
 their ability to inhibit human immunodeficiency virus integrase 
 [ 6 , 7 ] . since the late 1980s , a number of in vitro and in vivo studies have investigated the possible use of coumarins in the treatment of cancer . the in vitro effects of coumarins on the growth of renal cell carcinoma that derived cell 
 lines showed that coumarin and 7-hydroxycoumarin were potent cytotoxic and cytostatic agents . several authors have 
 reported on the use of coumarin ( 1,2-benzopyrone ) , or its 
 metabolite 7-hydroxycoumarin , for the treatment of some human 
 carcinomas [ 1013 ] . there are several reports indicating that some coumarin compounds , including coumarin and 7-hydroxycoumarin , inhibit cell growth of cell lines of various 
 types of cancer [ 1418 ] . the coumarin derivatives have been the focus of our recent 
 research concerning the design of new cytotoxic agents . it is well 
 known that many investigations have proved that binding of a drug 
 to a metalloelement enhances its activity and , in some cases , the 
 complex possesses even more healing properties than the parent 
 drug . this has prompted us to investigate the metal binding 
 properties of several coumarin derivatives . we have recently 
 reported the synthesis of lanthanide(iii ) complexes with some 
 coumarins and the study of their anticancer activity 
 [ 1929 ] . in previous works [ 1929 ] , we investigated the coordination behavior of some 4-hydroxycoumarins with cerium(iii ) , lanthanum(iii ) , and neodymium(iii ) . in the course of these studies , considering that lanthanides(iii ) have an interesting but 
 not well - known biological role in living organisms as trace 
 elements , we have investigated the coordination properties of a 
 series of other lanthanides(iii ) with coumarin derivatives . thus , the aim of this work is to synthesize and characterize 
 complexes of samarium(iii ) , gadolinium(iii ) , and dysprosium(iii ) 
 with coumarin-3-carboxylic acid ( see figure 1 ) and to 
 determine the antiproliferative effects of these complexes against 
 the chronic myeloid leukemia - derived k-562 . the cell line is 
 characterized with a strong expression of bcr - abl fusion protein ( a constitutive nonreceptor tyrosine kinase ) which 
 determines the low responsiveness of these cells to proapoptotic 
 stimuli . grade : sm(no3)3  6h2o , gd(no3)3  6h2o , and dy(no3)3  5h2o . coumarin-3-carboxylic acid ( figure 1 ) was used for the preparation of metal complexes as a ligand . the complexes were synthesized by reaction of samarium(iii ) , 
 gadolinium(iii ) , and dysprosium(iii ) salts and the ligand , in 
 amounts equal to metal : ligand molar ratio of 1 : 2 . the synthesis 
 of the complexes was made in different ratios ( 1 : 1 , 1 : 2 , 1 : 3 ) 
 but in all the cases the product was with the composition 1 : 2 . the complexes were prepared by adding ethanol solutions of 
 ln(iii ) salts to ethanol solutions of the ligand . the 
 reaction mixture was stirred with an electromagnetic stirrer at 
 25c for one hour . at the moment of mixing of the 
 solutions , the precipitates were 
 filtered , washed several times with water and ethanol , and dried 
 in a desiccator to constant weight . the complexes were insoluble in water , methanol , and ethanol and 
 well soluble in dmso . the carbon , hydrogen , and nitrogen contents of the compounds were 
 determined by elemental analysis . the water content was determined 
 by metrohn herizall e55 karl fisher titrator and was confirmed by 
 tga . ir spectra ( nujol ) were recorded on ir - spectrometer ftir-8101 m 
 shimadzu . the raman spectra of the ligand and their new 
 ln(iii ) complexes were recorded with a dilor 
 microspectrometer ( horiba - jobin - yvon , model labram ) equipped with 
 a 1800 grooves / mm holographic grating . the 514.5 nm line 
 of an argon ion laser ( spectra physics , model 2016 ) was used for 
 the probes excitation . the spectra were collected in a 
 backscattering geometry with a confocal raman microscope equipped 
 with an olympus lmplanfl 50x objective and with a resolution of 
 2 cm . the detection of raman signal was carried out 
 with a peltier - cooled ccd camera . the antiproliferative effects of the tested lanthanide complexes 
 and of the corresponding nitrate salts were assessed on the 
 chronic myeloid leukemia - derived k-562 . the cells were maintained 
 as suspension - type cultures in a controlled environment : rpmi 1640 
 medium ( sigma ) , with 10% heat inactivated fetal bovine serum 
 ( sigma ) and 2 mm l - glutamine ( sigma ) , in a incubator with humidified atmosphere and 5% carbon dioxide , at 37c . in order to keep the cells in log phase , cell 
 suspension was discarded 2 or 3 times per week and the cell 
 culture was refed with fresh rpmi-1640 aliquots . briefly , exponentially growing cells were seeded in 96-well 
 microplates ( 100 l / well ) at a density of 1  10 
 cells per ml and after 24-hour incubation at 37c they were 
 exposed to various concentrations of the lanthanide complexes for 
 48 hours . for each concentration after the incubation with the test compounds mtt solution 
 ( 10 mg / ml in pbs ) was added ( 10 l / well ) . the plates 
 were further incubated for 4 hours at 37c and the formazan 
 crystals formed were dissolved through addition of 
 100 l / well 5% solution of formic acid in 2-propanol 
 ( merck ) . the absorption of the samples was then measured using an 
 elisa reader ( uniscan titertec ) at wavelength of 580 nm . the 
 blank solution consisted of 100 l rpmi 1640 medium 
 ( sigma ) , 10 l mtt stock , and 100 l 5% formic 
 acid in 2-propanol . the survival fractions were calculated as 
 percentage of the untreated control using the formula 
 ( 1)sf  % = atestacontrol100 , 
 where atest is the average value for the 
 absorption at a given concentration and acontrol is the average absorption of the untreated control , respectively . the stock solutions of the tested lanthanide complexes ( at 
 20 mm ) were freshly prepared in dmso , and thereafter 
 consequently diluted in rpmi-1640 medium , in order to achieve the 
 desired final concentrations . at the final dilutions obtained , the 
 concentration of dmso never exceeded 1% . the stock solutions 
 ( 20 mm , in water ) of the nitrate salts of the lanthanides were 
 freshly prepared and following antibacterial filtration they were 
 accordingly diluted in rpmi-1640 medium . data processing , generation of dose - response curves , and ic50 
 calculations were performed using microsoft excel and microcal 
 origin software for pc . the 
 metal ions were determined after mineralization . the water content 
 in the complexes was determined by karl fisher analysis . the data of the elemental analysis of the compounds obtained 
 serving as a basis for the determination of their empirical 
 formulas and the results of the karl fisher analysis are presented 
 in table 1 . the mode of bonding of the ligand to samarium(iii ) , 
 gadolinium(iii ) , and dysprosium(iii ) ions was elucidated by 
 recording the ir and raman spectra of the complexes as compared 
 with those of the free ligand . the vibrational spectra of the 
 complexes showed new bands in comparison with these of the free 
 ligand which have been assigned to the rocking , waggling , and 
 metal - oxygen stretching vibrations . depending on the orientation of the two donor groups , 
 c = o and coo , different binding of the anion of coumarin-3-carboxylic acid ( cca ) is possible . however , in most of the known lanathanide complexes , the metal - ligand interaction is mainly electrostatic by nature . to help further the 
 binding mode elucidation in the new sm(iii ) , 
 gd(iii ) , and dy(iii ) complexes of hcca , 
 detailed vibrational analysis was performed on the basis of 
 comparison of experimental vibrational spectra of hcca and its 
 sm(iii ) , gd(iii ) , and dy(iii ) complexes with 
 those theoretically predicted by us earlier   as 
 well as with literature data about related compounds . the data of 
 the experimental ft - ir and ft - raman spectra of hcca and its 
 sm(iii ) , gd(iii ) , and dy(iii ) complexes are 
 given in table 2 . the ft - raman spectra of the ligand and its ln(iii ) complexes are presented in 
 figure 2 . the broadband at 3176 cm in the ir spectrum of the ligand was assigned to the (oh ) vibrational mode . this band was not detected in the spectra of the complexes , indicating that the deprotonated ligand form participates in the complexes . the bands in the 30602920 cm region were assigned to (ch ) modes of hcca . in the ir spectra of sm(iii ) , gd(iii ) , and dy(iii ) complexes they remain almost unchanged . the strong ir bands at 1746 cm and 1685 cm and the medium raman bands at 1729 , 1676 , and 
 1663 cm were assigned to (c = o ) modes of 
 the carboxylic and carbonylic groups , respectively . the high ir 
 intensity of these bands retained in the spectra of sm(iii ) , gd(iii ) , and dy(iii ) complexes , the (c = o)carboxylic band was shifted to lower frequency ( 1703 cm , 1703 cm , 1705 cm for sm(iii ) , gd(iii ) , and dy(iii ) complexes , resp . ) , and the (c = o)carbonylic band showed also a position change ( 1672 cm , 1672 cm , 1674 cm for sm(iii ) , gd(iii ) , and dy(iii ) complexes , resp . ) . the same shift effects were observed in the 
 raman spectra of the complexes . in agreement with the literature data , the bands 
 observed in the 16501330 cm frequency range are due 
 to the (cc ) stretching vibrations of hcca coumarin ring . the bands that are typical for the coumarin vibrations were 
 not shifted significantly in the spectra of sm(iii ) , gd(iii ) , and dy(iii ) complexes , which indicated that the ln(iii ) cations did not produce substantial polarization on the coumarin ring . the strong ir ( at 1613 and 1569 cm ) and raman ( at 1608 and 1559 cm ) bands are attributed to the (c = c ) stretching vibrations of hcca coumarin fragment . their positions and intensities are almost retained and the second band is split in 
 the complexes . the bands at 1489 , 1453 , and 1374 cm 
  ( ir ) and at 1483 , 1442 , and 1363 cm ( raman ) , which also are assigned to the (cc ) modes of hcca , show shifts in the ir and raman spectra of sm(iii ) , gd(iii ) , and dy(iii ) complexes and at the same time the intensity of these bands increases . the induced polarization by ln(iii)cca interaction produces electron 
 density distribution in the conjugated coumarin ring and as a result the (cc ) frequencies change their positions and intensity . the strong bands at 1228 cm ( ir spectrum of hcca ) and at 1216 cm ( raman spectrum of hcca ) and the medium one at 989 cm , in the ir spectrum of hcca , were assigned to the lactone (co ) modes , respectively . in the complexes , these modes were shifted to lower frequency . in agreement with ln(iii)ocarbonyl interaction , the induced polarization on cca leads to changes of the co lactone bond lengths as well as of their frequencies in a direction mentioned above . the following bands , observed in the ir spectra of the 
 complexes , are assigned to the vibrational modes of the no3 group : 1263 cm ( sm complex ) , 1262 cm ( gd complex ) , 1262 cm ( dy complex ) for (no)bonded ; 1053 cm ( sm complex ) , 1049 cm ( gd complex ) , 1049 cm ( dy complex ) for (ono ) ; 786 cm ( sm complex ) , 791 cm ( gd complex ) , 781 cm ( dy complex ) for (ono ) ; and 725 cm ( sm complex ) , 725 cm ( gd complex ) , 713 cm ( dy complex ) for (ono ) . some of them also appear in the raman spectra of the complexes : 1040 cm ( sm complex ) , 1040 cm ( gd complex ) , 1043 cm ( dy complex ) for (ono ) ; 777 cm ( sm complex ) , 772 cm ( gd complex ) , 777 cm ( dy complex ) for (ono ) . because of the predominant electrostatic character of the lno bonding , the bands corresponding to the (lno ) modes have low intensities , they are coupled with other modes and hence , their assignment is unreliable . the doublet bands observed in the ir spectra of the complexes at 767 , 748 cm ( sm complex ) , 768 , 749 cm ( gd complex ) , 766 , 750 cm ( dy complex ) , the bands at 459 cm ( sm complex ) , 457 cm ( gd complex ) , 457 cm ( dy complex ) , and the bands at 449 cm for sm(iii ) , gd(iii ) , and dy(iii ) complexes were assigned to (lno)carboxylic and (lno)carbonylic modes , respectively . on the basis of the above - detailed vibrational study we can 
 conclude that the metal - ligand bonding in ln(iii ) complexes 
 of coumarin-3-carboxylic acid appeared to be strongly ionic with 
 very small donor - acceptor character . the vibrational study gave 
 evidence for bidentate coordination of cca to sm(iii ) , gd(iii ) , and dy(iii ) ions through the carbonylic oxygen and the carboxylic oxygen . a survey of the h nmr spectral data reveals downfield chemical shifts of the protons in the ln(iii ) complexes spectra relative to the free ligand . the resonances due to protons 
 of the ligand are considerably broadened and shifted indicating 
 complexation . this peak is absent in the spectra of 
 the complexes due to the deprotonation of the carboxylic group . in 
 the c nmr spectra of the complexes , the largest upfield chemical shifts are observed for the carbon atoms which 
 are neighbors of the carboxylic and carbonylic oxygens and this 
 finding confirms their participation in ln(iii)cca interaction . the solvent dmso was used for the nmr measurements because the solubility of the complexes in noncoordinating 
 solvents was too low . dmso molecule could indeed bind to the metal and 
 give rise to equilibrium , fast on the nmr time scale , 
 involving the dmso molecule as an additional ligand , 
 because of the high coordination number of ln(iii ) . taking 
 into consideration the obtained data , we can say the following . 
 the results of nmr spectra , discussed above , and the 
 results of the pharmacological activity , presented below , all made 
 in dmso , give us reason to suggest that in these conditions 
 ( in the solution of dmso ) the complexes are present . the 
 stability of the complexes is of great interest with respect to 
 their further pharmacological properties ( not cytotoxicity test ) 
 and will be the subject of coming investigations which are in 
 progress . our previous molecular electrostatic potential ( mep ) study on the 
 preferred reactive sites of cca in the gas phase and in solution revealed two regions suitable for electrophilic attack 
 and binding : between the deprotonated carboxylic and the 
 carbonylic oxygens and between the carboxylic oxygens 
 [ 31 , 32 ] . to suggest the binding mode of hcca , a detail theoretical and vibrational investigation based on raman , ftir , and dft / b3lyp / svp studies of hcca , its deprotonated form 
 ( cca ) , kcca , and ln(cca)2(no3)(h2o ) species , two bidentate binding modes of cca to ln(iii ) were modeled : ( 1 ) through the deprotonated carboxylic and the carbonylic oxygens and ( 2 ) through both carboxylic oxygens . the vibrational analysis and the electronic energy calculations pointed to the first binding as more probable . on the basis of 
 detailed dft study of the vibrational behavior of hcca , cca , kcca , and ln(cca)2(no3)(h2o ) species and comparison of the theoretical and experimental vibrational spectra , it was established that cca is bidentate bound to ln(iii ) through the carboxylic and the carbonylic atoms . as seen from the vibrational spectra , the no3 group is bidentate coordinated , the calculated and experimental no3 modes for the complexes were found at very similar wavenumbers , and the assignment of the 
 no3 vibrational modes is in good agreement with literature data [ 31 , 32 ] . moreover , the metal - ligand binding mode of coumarin-3-carboxylic 
 acid ( hcca ) was recently explained by us through modeling 
 of the ln(iii)-coumarin-3-carboxylic acid structures , where 
 ln = la , ce , nd [ 31 , 32 ] . it was suggested that 
 coumarin-3-carboxylic acid binds to the ln(iii ) ions 
 through both oxygen atoms of the carboxylic and carbonylic groups 
 from the ligands and through the oxygen atoms of 
 no3 , and thus , the central ion ln(iii ) is six - coordinated . the nbo analysis of the complexes suggests predominantly ionic character of the lncca bond with slight ligand - metal charge transfer [ 31 , 32 ] . nevertheless , we have to take into consideration that the 
 coordination number 6 for these central metal ions indeed is too 
 low , but not impossible for lanthanide(iii ) ions 
 [ 1929 , 31 , 32 ] . one plausible mode of coordination might involve also the water molecules , which is 
 typical for coordination compounds of lanthanides . on the bases of our experimental spectral data and our theoretical 
 density function calculations [ 31 , 32 ] , we were able to suggest the most probable structure of these ln(iii ) complexes . the preliminary pharmacological screening performed revealed that 
 all of the lanthanide complexes exerted antiproliferative effects 
 against the chronic myeloid leukemia - derived k-562 line in a 
 concentration - dependent matter , which enabled the construction of 
 concentration response curves as depicted on figures 3 , 4 , 5 , 6 , 7 , 8 and table 3 . in contrast to the sm(iii ) and gd(iii ) complexes with coumarin-3-carboxylic acid , dy(cca)2(no3 )  h2o failed to induce 50% 
 inhibition of the cellular proliferation within the concentration range under investigation . interestingly , the gd(iii ) and 
 dy(iii ) nitrates were 
 found to exert considerable antiprolferative effects ( figures 
 6 , 7 , 8) , superior to 
 those of the complexes thereof . in contrast , despite the 
 considerable activity of the samarium complex , the corresponding 
 nitrate salt sm(no3)3  6h2o ( figure 6 ) caused only marginal inhibitory effects against k-562 ( figure 3 ) . the results from this study demonstrate the antiprolferative 
 potential of three novel lanthanide coordination compounds of 
 coumarin-3-carboxylic acid derivatives , in line with our preceding 
 papers concerning the activity of lanthanide 
 ( ce(iii ) , la(iii ) , and nd(iii ) ) coordination compounds with diverse coumarin ligands [ 1929 ] . in our hands , the samarium(iii ) complex of coumarin-3-carboxylic acid proved to be the most active antiproliferative agent among the novel complexes and thus it necessitates further more detailed pharmacological evaluation . the complex formation proved to be 
 detrimental for the efficacy of gd(iii ) and dy(iii ) compounds as in both cases the nitrates exerted superior efficacy 
 versus the corresponding coordination compounds .