cancer is a disease 
 that is difficult to treat , and novel drugs 
 are still highly demanded . synthesis of metal complexes with 
 biologically active ligands is a promising approach in developing 
 anticancer drugs , as metal ions can significantly alter the physical 
 and biological properties of these ligands . indolo[3,2-d]benzazepines , 
 also referred to as paullones , are one class of potential cyclin - dependent 
 kinase ( cdk ) inhibitors , identified in a comparative database search 
 at the national cancer institute ( nci ; nci60 screen ) . thereby , the 
 lead compound kenpaullone exhibited an activity profile similar to 
 that of flavopiridol , the first clinically studied cdk 
 inhibitor . within a series of paullones , however , the antiproliferative 
 activity did not parallel the cdk inhibitory potencies . as a result , other intracellular targets for 
 this class of compounds , e.g. , glycogen synthase kinase 3 ( gsk3 ) 
 or mitochondrial malate dehydrogenase ( mmdh ) , have been suggested . despite marked efforts to develop these compounds 
 as anticancer drugs , paullones remain at an early preclinical stage 
 mainly because of their low aqueous solubility and bioavailability . however , the original paullones did not contain suitable binding sites 
 for metal ions , and these had to be introduced by chemical modification . a library of paullone - based ligands with a broad structural diversity 
 and the respective complexes with copper(ii ) , gallium(iii ) , ruthenium(ii ) , 
 and osmium(ii ) have been reported . in an effort to elucidate novel structure activity 
 relationships 
 ( sars ) , the folded seven - membered azepine ring of paullones has been 
 replaced by a pyridine ring , leading to another class of biologically 
 active compounds , namely , indolo[3,2-c]quinolines , 
 with an essentially planar structure . indolo[3,2-c]quinolines and the structurally related indolo[3,2-b]quinolines are known phytochemicals found in the roots of the west 
 african climbing shrub cryptolepis sanguinolenta that 
 is used in traditional african medicine . both exhibit a broad spectrum 
 of biological properties , including antibacterial , antitumor , as well 
 as anti - inflammatory activity . in contrast 
 to indolo[3,2-b]quinolines , few studies have addressed 
 indolo[3,2-c]quinolines . like paullones , they can , however , be 
 introduced with synthetic tools essentially different from those applied 
 for paullones . the first ruthenium(ii ) , osmium(ii ) , and copper(ii ) 
 complexes with modified indolo[3,2-c]quinoline ligands 
 were derived from structurally related paullone complexes using distinct 
 chemical transformations . in particular , it has been found that complexes of 
 indolo[3,2-c]quinolines exhibit higher cytotoxicity 
 than their paullone counterparts , thus clearly establishing the effect 
 of replacing the azepine ring in paullones by a pyridine ring in indoloquinolines . in addition , it was shown that sars of complexes with modified paullones 
 do not necessarily apply to indolo[3,2-c]quinoline - based 
 compounds . current efforts by us are focused 
 on investigation of the underlying mechanisms of their antiproliferative 
 activity by exploiting the intrinsic fluorescence of indolo[3,2-c]quinolines . recently , 
 we reported on the syntheses of highly antiproliferative 
 copper(ii ) complexes with modified indolo[3,2-c]quinolines . herein , we report on the synthesis of a more 
 elaborate bioconjugate hl with two distinct binding sites and 
 the dinuclear copper(ii ) and zinc(ii ) complexes 1 and 2 , respectively . the new ligand is sufficiently soluble in 
 biological media and intrinsically fluorescent when light irradiated 
 at ex = 395 nm . these properties permitted us to 
 track the intracellular distribution of hl and 2 . moreover , the ligand design led to assembly of homometallic dinuclear 
 complexes with distinct compartments ( scheme 1 ) , a feature not explored by us so far in the development of anticancer 
 metal complexes . hcl ; ( ii ) diethyl-2,2-iminodiacetate , 
 triethylamine , dry thf , room temperature , 3 h ( 95% ) ; ( iii ) methanol , 
 room temperature , 2 h ( 95% ) ; ( iv ) copper(ii ) acetate monohydrate or 
 zinc(ii ) acetate dihydrate , methanol , room temperature , 30 min [ 1 
 ( 37% ) , 2 ( 33% ) ] . hydrochloric acid , 
 2-hydroxy-5-methylbenzaldehyde ( a ) , diethyl-2,2-iminodiacetate , 
 4-((2-hydroxyethyl)-piperazin-1-yl)ethanesulfonic acid ( hepes ) , and 
 guanosine 5-triphosphate were received from sigma - alrdich . l - histidine , formaldehyde solution ( 35% ) , copper(ii ) acetate 
 monohydrate , and zinc(ii ) acetate dihydrate were received from merck , 
 while tetrahydrofuran ( thf ) and methanol ( both analytical reagent 
 grade ) were received from fisher scientific . dimethyl sulfoxide ( dmso ) was received 
 from acros , ammonium bicarbonate , formic acid , and l - glutamic 
 acid were received from fluka , and l - aspartic acid was received 
 from serva . milli - q water ( 18.2 m , millipore advantage a10 , 
 185 uv ultrapure water system , molsheim , france ) and methanol ( fisher , 
 hplc grade ) were used for esi - ms experiments . 6-hydrazinyl-11h - indolo[3,2-c]quinolone ( d , scheme 1 ) was synthesized according to the 
 published protocol . details of the synthesis 
 and h nmr characterization of d are given 
 in the supporting information . 3-(chloromethyl)-2-hydroxy-5-methylbenzaldehyde 
 ( b ) was obtained from 5-methyl-2-hydroxybenzaldehyde 
 ( a ) via a previously described chloromethylation reaction . to a stirred solution of 3-(chloromethyl)-2-hydroxy-5-methylbenzaldehyde 
 ( 0.10 g , 0.54 mmol ) in dry thf ( 30 ml ) was added diethyl-2,2-iminodiacetate 
 ( 85 l , 0.48 mmol ) under argon atmosphere . upon addition of 
 triethylamine ( 281 l , 2.0 mmol ) the colorless solution turned 
 bright yellow and a white precipitate formed . after stirring for 3 
 h at room temperature , the yellow 
 filtrate was concentrated under reduced pressure to give an orange 
 oil , which was dried in vacuo overnight . h nmr 500.13 mhz ( dmso - d6 , h , ppm ) : 10.60 ( s , 1h , oh ) , 10.23 ( s , 1h , 
 c1 ) , 7.43 ( d , 1h , j(hc5 ) = 
 2.0 hz , c7 ) , 7.32 ( d , 1h , j(hc7 ) = 2.1 hz , c5 ) , 4.12 ( q , 4h , j(hc16 ,  c18 ) = 7.1 hz , c15 , c17 ) , 3.90 ( s , 2h , c9 ) , 3.54 
 ( s , 4h , c11 , c13 ) , 2.26 ( s , 3h , c8 ) , 1.20 ( t , 6h , j(hc15 ,  c17 ) = 7.1 hz , c16 , c18 ) . c{h } nmr 125.76 mhz ( dmso - d6 , c , ppm ) : 192.9 ( c1 ) , 171.2 ( c12 , c14 ) , 158.3 
 ( c3 ) , 137.9 ( c5 ) , 129.1 ( c7 ) , 128.6 ( c6 ) , 125.2 ( c4 ) , 122.3 ( c2 ) , 
 60.8 ( c15 , c17 ) , 54.4 ( c11 , c13 ) , 53.6 ( c9 ) , 20.3 ( c8 ) , 14.5 ( c16 , 
 c18 ) . to a solution of c ( 0.78 g , 2.3 mmol ) in methanol ( 40 ml ) the reaction mixture turned into a bright yellow 
 suspension , which was stirred for 2 h under argon atmosphere and allowed 
 to stand at 4 c overnight . the yellow product was filtered off , 
 washed with cold methanol ( 2  4 ml ) , and dried in vacuo overnight . calcd for c32h33n5o50.75h2o ( mr = 581.15 ) : c , 66.14 ; h , 5.98 ; n , 12.05 . h nmr 500.13 mhz ( dmso - d6 , h , ppm ) : 12.46 ( s , 
 1h , n11 ) , 10.72 ( s , 1h , n5 ) , 10.63 ( s , 1h , oh ) , 8.77 ( s , 1h , c14 ) , 
 8.43 ( d , 1h , j(hc8 ) = 7.8 
 hz , c7 ) , 8.11 ( d , 1h , j(hc2 ) = 7.8 hz , c1 ) , 7.80 ( d , 1h , j(hc3 ) = 8.3 hz , c4 ) , 7.61 ( d , 1h , j(hc9 ) = 8.3 hz , c10 ) , 7.59 ( s , 1h , c20 ) , 7.507.44 
 ( m , 1h , c3 ) , 7.407.35 ( m , 1h , c9 ) , 7.307.22 ( m , 2h , 
 c8 , c2 ) , 7.10 ( s , 1h , c18 ) , 4.13 ( q , 4h , j(hc29 ,  c31 ) = 7.1 hz , c28 , c30 ) , 3.94 ( s , 2h , c22 ) , 
 3.57 ( s , 4h , c24 , c26 ) , 2.30 ( s , 3h , c21 ) , 1.21 ( t , 6h , j(hc28 ,  c30 ) = 7.1 hz , c29 , c31 ) . c{h } nmr 125.76 mhz ( dmso - d6 , c , ppm ) : 171.2 ( c25 , 
 c27 ) , 154.0 ( c16 ) , 152.1 ( c14 ) , 150.1 ( c6 ) , 138.6 ( c10a ) , 138.5 ( c11a ) , 
 138.3 ( c4a ) , 132.1 ( c18 ) , 129.5 ( c3 ) , 129.2 ( c20 ) , 127.8 ( c19 ) , 124.3 
 ( c9 ) , 124.2 ( c17 ) , 124.1 ( c6b ) , 122.9 ( c7 ) , 122.2 ( c1 ) , 121.9 ( c2 ) , 
 121.2 ( c8 ) , 120.8 ( c15 ) , 117.1 ( c4 ) , 113.6 ( c11b ) , 112.0 ( c10 ) , 105.2 
 ( c6a ) , 60.6 ( c28 , c30 ) , 54.5 ( c24 , c26 ) , 53.1 ( c22 ) , 20.7 ( c21 ) , 14.6 
 ( c29 , c31 ) . esi - ms ( methanol ) , positive m / z 379 [ hl n(ch2cooet)2 ] , 568 [ hl + h ] , 590 [ hl + na ] ; negative m / z 566 [ hl  
 h ] , 603 [ hl + cl ] . vis ( methanol ) , 
 max (  , m cm ) : 226 ( 43  300 ) , 260 ( 32  350 ) , 273 sh ( 25  300 ) , 
 306 ( 22  400 ) , 348 ( 15  250 ) , 365 sh ( 15  900 ) , 
 382 ( 17  300 ) . atr - ir , selected bands , cm : 3640 , 3375 , 2976 , 1730 , 1608 , 1461 , 1192 , 1002 . to 
 a suspension of hl ( 0.20 g , 0.35 mmol ) in methanol ( 15 ml ) was added 
 copper(ii ) acetate monohydrate ( 0.16 g , 0.78 mmol ) . after stirring 
 for 30 min the dark - green solution was allowed to stand at 25 c 
 to evaporate slowly . after 3 days , green crystals formed were filtered 
 off , dried in vacuo overnight , and stored under argon atmosphere . calcd for c33h31cu2n5o91.5h2o ( mr = 795.72 ) : c , 49.81 ; h , 4.31 ; n , 8.80 . found : 
 c , 49.57 ; h , 4.30 ; n , 8.64 . esi - ms ( methanol ) : positive m / z 604 unidentified , 648 [ 1  
 ( hoac )  ( oac ) ] , 680 [ 1  ( oac)2 + ( ch3o ) ] . uv  vis ( methanol ) , 
 max (  , m cm ) : 235 ( 60  800 ) , 272 ( 41  000 ) , 296 ( 24  540 ) , 
 354 ( 18  900 ) , 420 sh ( 20  800 ) , 441 ( 22  700 ) . atr - ir , selected bands , cm : 1737 , 1583 , 1540 , 
 1385 , 1217 , 1028 . x - ray diffraction - quality single crystals were picked 
 from the reaction vessel prior to filtration . to a 
 suspension of hl ( 0.14 g , 0.25 mmol ) in methanol ( 15 ml ) was added 
 zinc(ii ) acetate dihydrate ( 0.12 g , 0.57 mmol ) . after stirring for 
 30 min after 4 days cold pentane was added and the mixture allowed 
 to stand at 4 c for 3 h. the yellow precipitate formed was filtered 
 off , dried in vacuo overnight , and stored under argon atmosphere . calcd for c33h31n5o9zn2ch3ohh2o ( mr = 822.46 ) : c , 49.65 ; h , 
 4.53 ; n , 8.52 . h nmr 
 500.13 mhz ( dmso - d6 , 
 h , ppm ) : 12.3311.60 ( bs , 2h , n11 , n12 ) , 
 8.626.71 ( bm , 11h , c14 , 710 , 14 , 18 , 20 ) , 
 4.003.53 ( bm , 9h , c22 , 24 , 26 , 28 ) , 2.22 ( s , 3h , c21 ) , 1.88 
 ( bs , 6h , ch3coo ) . esi - ms ( methanol ) , positive : m / z 668 [ 2  ( oac)2  ( ch3 ) + ( ch3oh ) ] , 682 [ 2  ( oac)2 + ( ch3o ) ] , 710 [ 2  ( oac ) ] , 
 724 unidentified , 784 unidentified . uv vis ( methanol ) , max (  , m cm ) : 
 230 ( 44  400 ) , 258 ( 45  700 ) , 290 ( 27  900 ) , 309 
 ( 31  000 ) , 330 ( 17  900 ) , 346 ( 18  200 ) , 394 ( 18  900 ) . atr - ir , selected bands , cm : 1744 , 1706 , 1583 , 
 1407 , 1216 , 1012 . x - ray diffraction - quality single crystals were picked 
 from the reaction vessel prior to addition of pentane . c hsqc , and h  c hmbc nmr spectra were recorded on a bruker 
 avance iii spectrometer 
 ( ultrashield magnet ) in dmso - d6 at 25 c using standard pulse programs at 500.13 ( h ) and 125.76 ( c ) mhz . h and c nmr chemical shifts are quoted relative to the residual 
 solvent signals . elemental analyses were carried out at the microanalytical 
 service of the faculty of chemistry , university of vienna . electrospray 
 ionization mass spectrometry was performed on a bruker esquire 3000 
 instrument ( bruker daltonic , bremen , germany ) on samples dissolved 
 in methanol . vis spectra were recorded with an agilent 8453 
 spectrophotometer in the 1901000 nm window using samples dissolved 
 in methanol at 10 m concentrations . ir spectra were measured 
 with a bruker vertex 70 fourier transform ir spectrometer by means 
 of the attenuated total reflection ( atr ) technique . fluorescence excitation 
 and emission spectra were recorded with a horiba floromax-4 spectrofluorimeter 
 and processed using the fluoressence v3.5 software package . samples 
 of hl and 2 were prepared from a 1 mm solution of each 
 in dmso and dilution with hepes buffer ( 20 mm , ph = 7.4 ) to give samples 
 at 10 m concentrations with a maximum content of 1% dmso ( v / v ) . x - ray diffraction 
 measurements were performed on a bruker x8 apexii ccd diffractometer . single crystals were positioned at 40 mm from the detector , and 1312 
 and 722 frames were measured , each for 60 and 90 s over 1 scan 
 width for 13ch3oh and 22ch3oh , correspondingly . crystal data , data collection 
 parameters , and structure refinement details are given in table 1 . structures were solved by direct methods and refined 
 by full - matrix least - squares techniques . non - hydrogen atoms were refined 
 with anisotropic displacement parameters , while h atoms were inserted 
 in calculated positions and refined with a riding model . the following 
 software programs were used : structure solution , shelxs-97 ; refinement , 
 shelxl-97 ; molecular diagrams , ortep ; computer , intel coreduo . wr2 = 
 { [w(fo  fc)]/[w(fo)]}. gof = { [w(fo  fc)]/(n  p ) } , where n is the number 
 of reflections and p is the total number of parameters 
 refined . magnetic measurements were carried 
 out on a microcrystalline sample of 1 with a quantum 
 design squid magnetometer ( mpms - xl ) . variable - temperature ( 2300 
 k ) direct current ( dc ) magnetic susceptibility was measured under 
 an applied magnetic field of 0.1 t. all data were corrected for the 
 contribution of the sample holder and diamagnetism of the samples 
 estimated from pascal s constants . analysis of the magnetic data was carried out by fitting the mt(t ) and m(t ) thermal variations including temperature - independent 
 paramagnetism ( tip ) , impurity contribution (  ) , 
 and intermolecular interaction ( zj ) according to the expression ( eq 1)1 complex formation was 
 studied by uv  vis titration of 10 and 250 m solutions 
 of hl in methanol with 10 l aliquots of 0.5 and 6.25 mm stock 
 solutions of copper(ii ) acetate monohydrate , respectively . one aliquot 
 was added at 2 min intervals followed by homogenization of the solutions 
 as within this period the equilibrium could be reached . an agilent 
 8453 spectrophotometer was used to record uv  vis 
 spectra in the 1901000 nm window . stability constants and molar absorbance spectra of the individual 
 copper(ii ) complexes were calculated by the computer program psequad . electrospray 
 ionization mass 
 spectra were recorded on an amazon sl ion trap mass spectrometer ( bruker 
 daltonics gmbh , bremen , germany ) . experimental data and provided simulations 
 were acquired using compass 1.3 software and processed using data 
 analysis 4.0 ( bruker daltonics gmbh , bremen , germany ) . the experimentally 
 obtained mass signals include a maximum standard deviation of m / z  0.06 for each species . general 
 instrument parameters were set as follows : positive - ion mode ( hv 4.5 
 kv , rf level 89% , trap drive 74.4 , dry temperature 250 c , nebulizer 
 8 psi , dry gas 6 l / min and average accumulation time 144 s ) , 
 negative - ion mode ( hv 4.5 kv , rf level 89% , trap drive 63.8 , dry temperature 
 250 c , nebulizer 8 psi , dry gas 6 l / min and average accumulation 
 time 2 ms ) . samples were diluted with water : methanol ( 50:50 ) or water : methanol : formic 
 acid ( 50:50:0.2 ) to a final metal concentration of 510 m 
 and measured by direct infusion into the mass spectrometer at a flow 
 rate of 4 l / min . stock solutions of 1 and 2 in dmso ( 10 mm ) were prepared and stored at 20 c 
 in the dark . each compound was diluted in ammonium carbonate buffer 
 ( 20 mm , ph = 7.95 ) to give a solution of 100 m of each compound 
 ( with 1% dmso content ) . furthermore , a solution containing l - histidine ( his ) , l - aspartic acid ( asp ) , l - glutamic 
 acid ( glu ) , and guanosine 5-triphosphate ( gtp ) in equimolar 
 amounts ( 100 m each ) and a solution containing his , asp , glu , 
 and gtp ( each 100 m ) and ascorbic acid ( asc , 400 m ) 
 were prepared in the same buffer . metal - containing solutions were 
 diluted with buffer or mixed with the solutions containing the amino 
 acids and asc at equimolar ratios to give a final metal concentration 
 in each incubation mixture of 50 m . reaction mixtures were 
 incubated at 37 c , and aliquots were measured directly after 
 mixing and after 1 , 3 , 5 , and 24 h after 10-fold dilution of each 
 with water : methanol ( 1:1 ) . the slightly acidic tetramethylammonium 
 acetate buffer ( 20 mm , ph = 6 ) was avoided because partial release 
 of the metal was observed . finally , dilution with water only resulted 
 in a low ionization in the positive- and negative - ion modes . for cytotoxicity 
 determination , three different human cancer cell lines were used : 
 a549 ( nonsmall cell lung cancer ) and sw480 ( colon carcinoma ) from 
 the american type culture collection ( atcc , manassas , va ) , both kindly 
 provided by brigitte marian , institute of cancer research , department 
 of medicine i , medical university vienna , austria , as well as ch1 
 ( ovarian carcinoma ) , established and kindly provided by the laboratory 
 of lloyd r. kelland , crc centre for cancer therapeutics , institute 
 of cancer research , sutton , u.k . cells were grown as adherent monolayer 
 cultures in 75 cm culture flasks ( starlab , cytoone ) in 
 minimal essential medium supplemented with 10% heat - inactivated fetal 
 bovine serum ( invitrogen ) , 1 mm sodium pyruvate , 1% ( v / v ) nonessential 
 amino acids ( from 100 ready - to - use stock ) , and 4 mm l - glutamine but without antibiotics at 37 c under a moist atmosphere 
 containing 5% co2 and 95% air . all cell culture media and 
 reagents were purchased from sigma - aldrich austria unless indicated 
 otherwise . cytotoxicity 
 was determined by the 
 colorimetric mtt assay ( mtt = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h - tetrazolium bromide ) as described previously . briefly , cells were harvested by trypsinization 
 and seeded in medium ( vide supra ) into 96-well plates in volumes of 
 100 l / well . depending on the cell line , different cell densities 
 were used to ensure exponential growth of the untreated controls during 
 the experiment : 1.0  10 ( ch1 ) , 2.0  10 ( sw480 ) , and 3.0  10 ( a549 ) cells per well . 
 in the first 24 h , the cells were allowed to settle and resume exponential 
 growth . then the test compounds were dissolved in dmso , serially diluted 
 in medium , and added to the plates in volumes of 100 l / well 
 so that the dmso content did not exceed 1% . due to limited solubility 
 of hl and 1 , the highest concentration was applied 
 in volumes of 200 l / well after replacing the original medium . after continuous exposure for 96 h ( in the incubator at 37 c 
 and under 5% co2 ) , the medium was replaced with 100 l / well 
 rpmi 1640 medium ( supplemented with 10% heat - inactivated fetal bovine 
 serum and 4 mm l - glutamine ) and mtt solution ( mtt reagent 
 in phosphate - buffered saline , 5 mg / ml ) in a ratio of 6:1 and plates 
 were incubated for further 4 h. then the medium / mtt mixture was removed 
 and the formed formazan was dissolved in dmso ( 150 l / well ) . optical densities at 550 nm were measured ( reference wavelength 690 
 nm ) with a microplate reader ( elx880 , biotek ) . the quantity of viable 
 cells was expressed as a percentage of untreated controls , and 50% 
 inhibitory concentrations ( ic50 ) were calculated from the 
 concentration  effect curves by interpolation . every test was 
 repeated in at least three independent experiments , each consisting 
 of three replicates per concentration level . sw480 cells were seeded in 
 medium on coverslips in 6-well plates and allowed to settle and resume 
 exponential growth for 24 h. then cells were incubated for 12 
 h with 5 m of 2 or 10 m of hl in medium . co - staining 
 with er - tracker red and lyso - tracker red ( invitrogen ) was performed 
 according to the manufacturer s instructions . after staining , 
 each slide was washed three times in pbs . a fluorescence microscope 
 bx40 ( olympus ) with f - view ccd camera ( olympus ) , cellf fluorescence 
 imaging software ( olympus ) , and 60 magnification oil immersions 
 objective lens were used . syntheses of the ligand hl and the copper(ii ) 
 and zinc(ii ) complexes 1 and 2 , respectively , 
 were carried out as shown in scheme 1 . we prepared 
 a potentially hexadentate nonsymmetric 
 ligand consisting of two chelating arms , one flexible able to provide 
 facial coordination to an octahedral metal ion , while the second is 
 rigid and provides a meridional binding . ester functionalities are 
 frequently introduced into the structure of organic molecules to improve 
 their aqueous solubility and bioavailability . recently , our group reported on the conjugation of l- and d - proline to 3-(chloromethyl)-2-hydroxy-5-methylbenzaldehyde 
 ( b ) after chloromethylation of 2-hydroxy-5-methylbenzaldehyde 
 ( a ) ( scheme 1 ) . similarly , we reacted 3-(chloromethyl)-2-hydroxy-5-methylbenzaldehyde 
 ( b ) with diethyl-2,2-iminodiacetate and triethylamine 
 in dry thf at room temperature , obtaining diethyl-2,2-((3-formyl-2-hydroxy-5-methylbenzyl)azanediyl)-diacetate 
 ( c ) as an orange oil in excellent yield ( 95% ) . the ligand hl was obtained by reacting c with 6-hydrazinyl-11h - indolo[3,2-c]quinoline ( d ) in methanol at room temperature , again 
 in excellent yield ( 95% ) . complexes 1 and 2 were synthesized in 37% and 33% yields starting from the ligand hl and copper(ii ) acetate monohydrate and zinc(ii ) acetate dihydrate , 
 respectively , in methanol at room temperature . the complexation reaction 
 is in both cases accompanied by hydrolysis of one ethyl ester group 
 and transesterification of another ethyl ester function with formation 
 of a new ligand hl . both generated donor arms are involved in coordination 
 to copper(ii ) and zinc(ii ) in 1 and 2 , respectively , 
 via the deprotonated carboxylate group and the carbonyl oxygen of 
 the methyl ester group ( see scheme 1 , figures 1 and 2 ) . the ligand hl and its zinc(ii ) complex 2 have been 
 characterized by one- and two - dimensional nmr spectroscopy , esi mass 
 spectrometry , elemental analysis , uv  vis , and atr - ir spectroscopy , 
 while copper(ii ) complex 1 was studied by magnetic susceptibility 
 measurements , esi mass spectrometry , and optical spectroscopy . h and c nmr spectral data of intermediate c , ligand hl , and zinc(ii ) complex 2 along 
 with their assignments are given in the experimental 
 section . the presence of a proton at n in the h nmr spectra and the chemical shift of neighboring c in the c nmr spectra indicate that the ligand 
 adopts a configuration with an exocyclic c = n double bond . moreover , esi mass spectra of 1 and 2 in methanol showed peaks that confirmed formation 
 of dimetal complexes . the most abundant peaks at m / z 680 and 682 for 1 and 2 , correspondingly , were assigned to [ 1/2  ( oac)2 + ( ch3o ) ] . uv vis spectra of hl , 1 , and 2 in methanol are depicted in figure s1 ( supporting 
 information ) . metal coordination led to pronounced changes 
 in the visible range of the ligand spectrum , namely , to evolution 
 of an absorption band at ca . 400 nm for 2 and formation 
 of a broad charge - transfer band at 440 nm for 1 . results of x - ray diffraction 
 studies of 13ch3oh and 22ch3oh shown in figures 1 and 2 , respectively , confirm formation of 
 dinuclear complexes with the two copper(ii ) ions and zinc(ii ) ions 
 bridged by the phenolate oxygen and two exogenous 2-: acetato ligands . both copper(ii ) ions in 1 are distorted 
 square pyramidal with  = 0.27 for 
 cu1 with the bridging phenolate oxygen o1 in an apical position and 
 tertiary amine n23 , atoms o6 and o8 of the two bridging acetates , 
 and one aminoacetate o2 in the basal plane . we do not describe the 
 coordination environment around cu1 as octahedral , since the interaction 
 between cu1 and o5 of the dangling methyl ester group is extremely 
 weak ( cu1o5 2.946(2 )  ) . for cu2 a distorted 
 square - pyramidal coordination geometry (  = 0.22 ) was realized 
 with a bridging phenolate oxygen o1 , quinoline nitrogen n5 , hydrazinic 
 nitrogen n13 , one oxygen atom o9 of bridging acetate in a basal plane , 
 and another bridging acetate oxygen atom o7 in apical position . unlike 1 , the coordination environments of zinc(ii ) 
 ions in 2 differ from each other . zn1 has an octahedral 
 environment comprised of the bridging phenolate oxygen o1 , tertiary 
 amine donor n23 , methyl ester oxygen o5 , and atom o6 of the bridging 
 acetate in equatorial positions and two oxygen atoms , one aminoacetate 
 o2 , and a second o8 of a bridging acetate in apical positions . zn2 
 in contrast to cu2 shows a more pronounced tendency toward a trigonal - bipyramidal 
 coordination geometry (  = 0.47 ) of the same donor atoms . cu2 
 lies in the mean plane through cu2n5c6n12n13 in 1 , while 
 cu1 comes out from this plane by 1.307  . in 2 the 
 deviation of zn1 from the mean plane through zn2n5c6n12n13 is markedly 
 smaller ( 0.820  ) , while distortion from planarity of the indoloquinoline 
 moiety is more evident than in 1 . the bridging of copper(ii ) 
 ions via phenolate oxygen results in distinct cu1o1 and cu2o1 
 bond distances . the difference between them ( 0.39  ) is larger 
 than in other nonsymmetrically -phenoxido bridged dicopper(ii ) 
 complexes , in which the two copper(ii ) ions in addition are bridged 
 by at least one exogenous 2-: acetato group . the cu1o6 bond 
 distance is markedly shorter than cu2o7 , and cu1o8 
 is also shorter than cu2o9 ( see caption to figure 1 ) . the cu1cu2 distance in the 
 complex is 3.2897(6 )  , which is comparable with cucu 
 distances of 3.297(3 ) and 3.263(2 )  in dicopper(ii ) complexes with symmetric dinucleating 
 ligands , containing a di--acetato--phenolatodicopper(ii ) 
 core . ortep view of [ cu2(l)(ch3coo)2 ] with thermal ellipsoids drawn at the 50% 
 probability level . selected bond distances ( angstroms ) and bond angles 
 ( degrees ) : cu1o1 2.323(2 ) , cu1o2 1.937(2 ) , cu1o6 
 1.946(2 ) , cu1o8 1.936(2 ) , cu1n23 2.093(3 ) , cu1o5 
 2.946(2 ) , cu2o1 1.913(2 ) , cu2o7 2.194(2 ) , cu2o9 
 2.002(2 ) , cu2n5 2.038(3 ) , cu2n13 1.956(3 ) , cu1o1cu2 
 101.47(10 ) . the zn1o1 bond 
 distance is only slightly longer than zn2o1 , 
 as also observed in other complexes with nonsymmetrical dinucleating 
 ligands with a di--acetato--phenolatodizinc(ii ) core . the zn1o6 bond distance is only slightly 
 longer than zn2o7 , while the difference between zn1o8 
 and the shorter bond zn2o9 is more pronounced ( see caption 
 to figure 2 ) . the interaction between zn1 and 
 o5 of the dangling methyl ester group is markedly stronger than comparable 
 interaction in 1 . the zn1zn2 distance 
 in the complex is at 3.2154(7 )  , which is similar to the znzn 
 distance of 3.29(1 )  in a dizinc(ii ) 
 complex with a nonsymmetrical hybrid ligand . ortep view of [ zn2(l)(ch3coo)2 ] with thermal ellipsoids drawn at the 50% 
 probability level . selected bond distances ( angstroms ) and bond angles 
 ( degrees ) : zn1o1 2.057(3 ) , zn1o2 2.105(3 ) , zn1o6 
 2.004(3 ) , zn1o8 2.079(3 ) , zn1n23 2.149(4 ) , zn1o5 
 2.322(3 ) , zn2o1 2.027(3 ) , zn2o7 1.991(3 ) , zn2o9 
 1.993(3 ) , zn2n5 2.087(4 ) , zn2n13 2.097(4 ) , zn1o1zn2 
 103.86(14 ) . the magnetic behavior of a polycrystalline 
 sample of 13ch3oh in the temperature 
 range 2300 k in a field of 0.1 t is shown in figure 3 . the value of mt is 0.952 cm k mol at 300 k. this 
 value is slightly higher than the expected mt value ( 0.750 cm k mol ) 
 for two noninteracting copper(ii ) ions ( d , g = 2.0 , s = 1/2 ) . the value of mt continuously increases with decreasing temperature 
 and reaches a value of 1.174 cm k mol at 3 k. this behavior suggests the presence of ferromagnetic interactions 
 in 13ch3oh . according to x - ray diffraction 
 data , complex 13ch3oh has a dinuclear 
 structure , in which the two copper(ii ) ions are connected by a phenolate 
 oxygen atom and two bidentate bridging acetato ligands ( figure 1 ) . therefore , the magnetic behavior can be analyzed 
 by using the classical spin hamiltonian ( eq 2):2where j is the exchange coupling 
 constant and s1 = s2 = 1/2 . plots of mt vs t and magnetization vs h ( inset ) at 2 and 
 3 k for 13ch3oh . solid lines correspond 
 to the best 
 fit with parameters quoted in the text . in this case , the van vleck equation leads to the following 
 analytical 
 expression ( eq 3)3the fitting procedure results in 
 an excellent 
 agreement between the experimental data and the calculated curve ( r = 1.4  10 ; figure 3 ) . the parameters extracted from the fit are j = 3.49(3 ) cm , g = 2.24(1 ) , 
 and zj = 0.08(1 ) cm and correspond to ferromagnetic interaction between copper(ii ) ions . the temperature - independent paramagnetism ( tip ) and 
 impurity contribution (  ) have values close to zero , and both 
 were fixed at zero in the final fit . the presence of ferromagnetic 
 interaction was confirmed by magnetization measurements at low temperature 
 ( see inset picture in figure 3 ) . the fitting 
 of magnetization vs field using the brillouin function indicates the 
 presence of spin ground state s = 1 ( g = 2.203(2 ) ) in 13ch3oh , which is consistent 
 with the results obtained from analysis of the temperature dependence 
 of magnetic susceptibility . the nature of magnetic interaction 
 in dinuclear copper(ii ) complexes 
 has been extensively studied from both theoretical and experimental 
 points of view . the magnetic interaction in 13ch3oh 
 occurs via three bridges : two 2-: acetato ligands and one bridging phenolate . . according to the literature , the acetate 
 bridges mediate the antiferromagnetic interactions , while the phenolate 
 bridge in dinuclear copper(ii ) complexes can promote both antiferromagnetic 
 as well as ferromagnetic interactions . the character of magnetic interaction 
 depends on geometrical features , especially on the cu o  cu 
 angle  , out - of - plane deviation angle  ( see figure 4 ) , and torsion angle cu  o  cu  o . 
 for  angles < 99 and  angles > 30 , 
 a 
 strong ferromagnetic interaction can be expected . in the case of 13ch3oh with 
  = 101.47 and = 30.04 , the presence of 
 a weak ferromagnetic interaction ( j = 3.49 cm ) is justified . we can conclude that due to the out - of - plane 
 deviation of the phenol group relative to cu o  cu plane 
 the resulting magnetic interaction between the triple - bridged cu(ii ) 
 ions is weakly ferromagnetic . comparable weakly ferromagnetic interactions 
 were reported for other dinuclear copper(ii ) complexes with two or three different bridges . to elucidate whether the 
 two binding sites in hl show different affinities to copper(ii ) , vis 
 titrations of the ligand hl at two different concentrations with 
 copper(ii ) acetate monohydrate in methanol at room temperature ( figures 5 and s2 , supporting information ) . uv  vis absorbance spectrum of the ligand hl ( dashed trace ) 
 and its changes by addition of copper(ii ) acetate monohydrate ( solid 
 traces ) in methanol ( cl = 10 m ; ccu = 022.5 m ; t = 298 k ; l = 1 cm ) . 440 nm 
 was observed 
 upon addition of up to 1.5 mol equiv of copper(ii ) . a wide band with max at 664 nm overlapped partly with the charge - transfer band 
 was seen upon addition of copper(ii ) . this absorption band is slightly 
 red shifted upon addition of more than 1 equiv of copper(ii ) . on the 
 basis of the spectral changes in the wavelength range 230520 
 nm ( figure 5 ) , overall stability constants 
 have been calculated for the mono- [ cul ] ( log  = 7.17  
 0.08 ) and dinuclear [ cu2l ] species ( log  = 13.13 
  0.24 ; log k = 5.96 ) . the molar absorbance 
 spectra of the ligand , [ cul ] , and [ cu2l ] complexes were 
 also calculated ( figure 5 ) . the goodness - of - fit between measured and calculated absorbance values is 
 shown in figure 6 . d transition bands were in good 
 agreement with those obtained by monitoring the charge - transfer band 
 within 0.2 log unit . stepwise formation constant of the [ cu2l ] species is merely 1 log unit lower than that of the [ cul ] 
 showing the overlapping binding of the metal ions . therefore , it can 
 be concluded that both binding sites in ligand hl coordinate with 
 a similar affinity and no preference for either of them can be perceived . measured 
 and calculated ( dashed lines ) absorbance values at 382 
 (  ) and 440 nm (  ) at various hl -to - copper(ii ) ratios 
 ( cl = 10 m ; ccu = 022.5 m ; t = 298 k ; l = 1 cm , methanol ) . the stability of complexes 1 and 2 in aqueous solution and their reactivity toward 
 small biomolecules was studied by esi mass spectrometry ( esi - ms ) since 
 it proved to be effective for characterizing also complex metallodrug 
 interactions with biomolecules . both complexes display a very 
 similar aqueous solution behavior , which is characterized by ester 
 hydrolysis of the ligand and partial metal release over time . products 
 of ester hydrolysis are detected directly after dissolving the compounds 
 in buffer , and the ester is quantitatively hydrolyzed within 24 h. 
 the major thermodynamic products after this period correspond to ions 
 [ m2(l  me)(oh )  h ] and [ m(l  me ) h ] , 
 where m = cu or zn and l = l , detected 
 in the negative - ion mode ( figure 7 ) . the latter 
 mass signal suggests that release of specifically one metal can occur 
 from both 1 and 2 . interestingly , these 
 signals are detected at 95% and 38% intensities relative to [ m2(l  me)(oh )  h ] for 1 and 2 , respectively , i.e. , the cu 
 complex 1 releases the metal to a greater extent . therefore , 
 complex 2 appears to be slightly more stable in aqueous 
 solution , which also seems to be of relevance for the cytotoxicity . additionally , 2 does not ionize in the positive - ion mode , 
 suggesting stable bonds between zn ions and the acetato ligands ( figure 
 s3 , supporting information ) . note that 
 acetato complexes were not detected in the mass spectra of 1 or 2 . furthermore , the isotopic distributions of the 
 major mass signals of 1 and 2 are in good 
 agreement with simulated patterns ( figure s4 , supporting information ) . both complexes were exposed to mixtures 
 containing equimolar amounts of l - histidine ( his ) , l - aspartic acid ( asp ) , l - glutamic acid ( glu ) , and guanosine 
 5-triphosphate ( gtp ) . the complexes did not react with any 
 of the biological nucleophiles , and similar mass spectra were observed 
 compared to solutions containing only the respective metal . addition 
 of 4 equiv of ascorbic acid ( asc ) to the amino acids resulted in transient 
 formation of glu and asc adducts with 1 in a small amount ; 
 however , they were only detected immediately after mixing ( figure 7c ) and absent for 2 . free ascorbate 
 was consumed within 1 h but had no impact on the overall reactivity 
 of the complexes . esi mass spectra in negative - ion mode are shown for 1 ( a ) and 2 ( b ) in methanol over a period of 
 24 h. ( c ) 
 glu- and asc - adducts of 1 , which were only detected directly 
 after mixing . an interesting feature of both 
 compounds is their ability to release 
 a metal ion also in a ph - dependent manner ( figure s5 , supporting information ) . the samples incubated 
 at ph = 7.95 for 24 h displayed only partial metal release . lowering 
 the ph of this incubation solution by dilution with 0.1% formic acid 
 resulted in immediate and quantitative release of one metal from both 
 dimetallic complexes . it is suggested that the carboxylates are prone 
 to protonation under these conditions , leading to release of the coordinated 
 metal . fluorescence spectra of hl and 2 were recorded in hepes - buffered solutions ( 20 
 mm ; ph = 7.4 ) with a 1% ( v / v ) content of dmso ( figure s6 , supporting information ) . fluorescence excitation 
 spectra ( em = 470 nm ) were measured in the range 
 between 260 and 460 nm and emission spectra ( ex = 
 395 nm ) in the range from 410 to 710 nm . coordination to zinc(ii ) led to a blue 
 shift of the emission band by 54 nm , with the maximum at 466 nm in 
 the spectra of 2 . hl was found fluorogenic , as 
 excitation and emission spectra strongly increased in intensity upon 
 binding to zinc(ii ) . the 
 cytotoxicity of hl , 1 , and 2 was determined 
 by the mtt assay 
 in three human cancer cell lines , namely , a549 ( nonsmall cell lung 
 carcinoma ) , ch1 ( ovarian carcinoma ) , and sw480 ( colon adenocarcinoma ) , 
 all yielding ic50 values in the micromolar concentration 
 range ( table 2 ) . values for a simple copper(ii ) 
 salt , cucl2 , are given for comparison . fifty percent 
 inhibitory concentrations 
 ( means  standard deviations from at least three independent 
 experiments ) , as obtained by the mtt assay using exposure times of 
 96 h. ch1 is the most sensitive cell line to all tested 
 compounds , whereas 
 a549 , a more chemoresistant cell line equipped with multidrug - resistance - mediating 
 proteins , is the least sensitive one , 
 with ic50 values up to 13 times higher than in ch1 cells . 
 whereas complexation with copper(ii ) has either little effect on cytotoxicity 
 ( a549 , sw480 cells ) or yields 3-fold decreased potency ( ch1 cells ) , 
 complexation with zinc(ii ) results in about 2-fold enhancement of 
 cytotoxicity , compared to the metal - free ligand hl in all three cell 
 lines . in comparison to the dicopper(ii ) complex 1 , the 
 dizinc(ii ) complex 2 is up to three times more active 
 in sw480 and four times more active in ch1 cells ( see also figure 
 s7 , supporting information ) . this might 
 be directly related to the lower tendency of 2 to release 
 a metal in aqueous media compared to 1 as observed in 
 the esi - ms experiments . on the basis of these observations , it can 
 be concluded that complexation to zinc(ii ) results in higher cytotoxicity 
 but also better solubility in biocompatible media compared to the 
 metal - free ligand as well as copper(ii ) complex 1 . cytotoxic 
 potency of a simple copper(ii ) salt , cucl2 , is lower , with 
 ic50 values being at least five times higher than those 
 of complex 1 . on the basis 
 of the fluorescence 
 properties of hl and dizinc(ii ) complex 2 , their subcellular 
 localization was studied by fluorescence microscopy in human cancer 
 cells including their colocalization with organelle - specific dyes . 
 for visualization of the compounds in live sw480 cells , the u - mwu2 
 filter ( olympus japan , excitation filter bp330 - 385 , emission filter 
 ba420 ) was used , while the costaining dyes were recorded using the 
 u - mwg2 filter ( olympus japan , excitation filter bp510 - 550 , emission 
 filter ba590 ) . the compounds do not show interference in the u - mwg2 
 channel , and autofluorescence of the cells was not observed with the 
 used filters . microscopic images of cells treated with hl and 2 , as shown in figure 8 , revealed localization 
 of fluorescence in diffuse voluminous as well as distinct small cytoplasmic 
 structures but no discernible uptake into the nucleus . the highest 
 accumulation matches with both the er - tracker red and the lyso - tracker 
 red staining , suggesting that the endoplasmic reticulum as well as 
 lysosomes are potential target compartments of hl or that lysosomes 
 are involved in sequestration and/or detoxification of the compound . the same may apply to 2 , provided that the complex is 
 sufficiently stable throughout its passage through the cell , as it 
 can not be ruled out that the fluorescence distribution originates 
 from dissociated ligand molecules . cells were 
 costained with 10 m of hl ( a ) or 5 m of 2 ( b ) and er - tracker red ( 500 nm ) and lyso - tracker red ( 1 m ) , 
 respectively . condensation of 6-hydrazinyl-11h - indolo[3,2-c]quinoline with diethyl-2,2-((3-formyl-2-hydroxy-5-methylbenzyl)azanediyl)diacetate 
 afforded a new nonsymmetric dinucleating ligand hl with increased 
 aqueous solubility and fluorescence properties . complexes 1 and 2 were obtained upon treatment of the ligand with 
 2 equiv of cu(ch3coo)2h2o and 
 zn(ch3coo)22h2o in methanol , 
 respectively . complexation reaction in both cases is accompanied by 
 hydrolysis of one ethyl ester group and transesterification of another 
 ethyl ester function with formation of hl . dinuclear structure 
 in 13ch3oh and 23ch3oh is supported by three bridges : two acetato ligands and 
 one phenolato bridge from nonsymmetric hl ligand . the temperature 
 dependence and field dependence magnetic measurements for 13ch3oh indicate a weak ferromagnetic interaction 
 ( j = 3.49 cm ) between copper(ii ) 
 ions . all three compounds show respectable antiproliferative activity 
 in human cancer cell lines ( a549 , ch1 , sw480 ) with ic50 values in the low micromolar concentration range . it seems that 
 the increased resistance of 2 toward metal release in 
 aqueous solution compared to 1 may be responsible for 
 the higher cytotoxicity . localization of hl and 2 in 
 cytoplasmic structures has been found by fluorescence microscopy , 
 suggesting that the endoplasmic reticulum as well as the lysosomes 
 can be potential target compartments of these compounds .
dicopper(ii ) and 
 dizinc(ii ) complexes [ cu2(meooclcoo)(ch3coo)2 ] ( 1 ) 
 and [ zn2(meooclcoo)(ch3coo)2 ] ( 2 ) were synthesized by reaction of 
 cu(ch3coo)2h2o and zn(ch3coo)22h2o with a new nonsymmetric 
 dinucleating ligand etoochlcooet prepared by condensation of 6-hydrazinyl-11h - indolo[3,2-c]quinoline with diethyl-2,2-((3-formyl-2-hydroxy-5-methylbenzyl)azanediyl)diacetate . 
 the design and synthesis of this elaborate ligand was performed with 
 the aim of increasing the aqueous solubility of indolo[3,2-c]quinolines , known as biologically active compounds , and 
 investigating the antiproliferative activity in human cancer cell 
 lines and the cellular distribution by exploring the intrinsic fluorescence 
 of the indoloquinoline scaffold . 
 the compounds have been comprehensively 
 characterized by elemental analysis , spectroscopic methods ( ir , uv  vis , 1h and 13c nmr spectroscopy ) , esi mass spectrometry , 
 magnetic susceptibility measurements , and uv  vis complex formation 
 studies ( for 1 ) as well as by x - ray crystallography ( 1 and 2 ) . 
 the antiproliferative activity of etoochlcooet , 1 , and 2 was determined by the mtt assay 
 in three human cancer cell lines , namely , a549 ( nonsmall cell lung 
 carcinoma ) , ch1 ( ovarian carcinoma ) , and sw480 ( colon adenocarcinoma ) , 
 yielding ic50 values in the micromolar concentration range 
 and showing dependence on the cell line . 
 the effect of metal coordination 
 on cytotoxicity of etoochlcooet is also discussed . the subcellular distribution 
 of etoochlcooet and 2 was investigated by fluorescence microscopy , 
 revealing similar localization for both compounds in cytoplasmic structures .