Isolation, characterization and antitumour properties of the 1,2-propylenediaminetetraacetate-trans-diaqua-copper(II)

Me al Based D ugs Vol. 7, No. 4, 2000 ISOIATION, CHARATION AND ANIII’UMOUR PROPIi IES OFTHE 1,2-PROPYLENEDIAMINETETRAACETATE ans-DIAQUA-COPPER (II) S. Kamah , R. Vilaplana , J. Mo enC, K. Akdi , G. Ga cia-He dugo’ and F. Gonzlez-Vilchez * Uni e sidad de Se illa, 41012 Se i la, Spain Dp o. de Quimica Ino ginica, F. de Quimica Dp o. de Biologia Celula , F. de Biologia Abs ac A ans-diaquacomplex o med by coppe (ll) sulpha e and he seques e ing polyammino- polyca boxylic ligand 1,2-p opylenediamine e aace ic acid (PDTA) has been isola ed and cha ac e ized by chemical analysis, i ime y, FT-IR and elec onic spec oscopy. Po en iome ic and elec onic measu emen s iden i ied he ligand as e aden a e, wo ni ogen and wo oxygen a oms being bonded o he Cu(II) in plana posi ions. This oc ahed al monome ic soluble compound, is an unusual example o a coppe (I|) subs ance showing signi ican in i o an i umou ac i i y agains he human o a ian umou cells T G (IDs0 2.29 k M a 48 h) and impo an in i o an i umou ac i i y agains solid Sa coma 180 wi h comple e eg ession o he umou a a dose o 12.5 mg/Kg body weigh . In oduc ion Se e al.compounds o ansi ion pla inum g oup me als wi h 1,2-p opylenediamine-N,N,N’,N’- e aace ic acid (PDTA), a me hyl de i a i e o EDTA, ha e been isola ed and es ed o biological ac i i y agains human umou s cells du ing ecen yea s by ou esea ch g oup[l’3]. Consequen ly, ou in e es has also been add essed o he complexes o med by me al ions o he han pla inum as po en ial me allopha maceu icals showing an i umou ac ion ha di e ed signi ican ly om ha o cis-pla in. In his con ex , ou wo k on he chemis y and biological ac ion o new PDTA-Ru(III) complexes and he in e es ing esul s ecen ly ob ained [4-8] conce ning hei in e ac ion agains DNA and human cells, encou aged us o unde ake new s udies ocused on he biological beha iou shown by PDTA complexes o med wi h coppe (II), an essen ial ion p esen ing no el in i o an ibac e ial, an i i al and an i ungal ac i i ies on a b oad ange o mic oo ganisms[ 9-11], besides i o ms a pa o he Cu-Zn SOD enzymes. Ou in e es on hese ype o complexes is also suppo ed by he impo an ole played by Cu(ll) complexes wi h chela ing ligands as models o he associa i e complexes in ol ed in he subs i u ion eac ions a he coppe si e o hese enzymes: indeed s ongly seques e ing ligands should a ou he o ma ion o six-coo dina e coppe complexes p esen ing he same coo dina ion numbe as he associa i e complex[ 12]. In e es ingly, complexes o he hexaden a e PDTA ha e been ound wo o de s o magni ude mo e s able han hose o med by analogous pa en ligand (EDTA). Mo eo e , Cu(II) complexes wi h poly unc ional ligands such as PDTA has also been impo an because PDTA can eadily o m di e en shapes wi h di e se coo dina ion numbe s and hus adap o he subs a e[13]. This communica ion deals wi h he syn hesis o he solid oc ahed al compound [Cu(PDTA- Hz)(H20)2]H20 and i s cha ac e iza ion using chemical analyses, po en iome y, ib a ional spec oscopic and he mal s udies. Finally, he ac i i ies o his complex agains he o a ian umou cell line TG (in i o) and he solid umou Sa coma 180 (in i o ha e also been es ed. Ma e ials and Me hods Syn hesis o he compound [CuL(H20)2]H20 (1) (L PDTA-H2) An aqueous solu ion o CuSO4.SH20 (3.80 g in 100 ml o dis illed wa e ) was passed epea edly h ough a il e ing lask su ace con aining solid PDTA (6.12 g) un il he ini ial o ma ion o a blue solu ion. This solu ion was slowly e apo a ed a oom empe a u e un il p ecipi a ion o a blue compound which was il e ed and washed wi h ace one ollowed by die hyl e he . The ob ained mic oc ys alline powde was Anal. Calcd. o C iH Ol IN-Cu: C, dissol ed in wa e , ec ys allized and s o ed in dessica o on CaC "1 31.2; H, 5.2; N, 6.6; Cu, 15.6; M + 421.8. Found: C, 31.4; H, N, 6.6; Cu 15.5; M /---- 422" Chemicals Coppe (ll)sulpha e pen ahyd a e was used as p o ided by Sigma. All chemicals ob ained om comme cial supplie s we e used wi hou u he pu i ica ion. The ligand PDTA was syn hesized in ou labo a o y ollowing a published me hod[14]. Elemen al analyses Elemen al mic oanalyses we e pe o med a he Mic oanaly ical Labo a o y o he Ba celona Uni e si y. The wa e con en was de e mined by Ka l-Fische i ime y whe eas ne al con en was de e mined by a omic abso p ion spec oscopy on a Pe kin Elme 2380, a l0 mA and ) o 349.9 nm. 219 F. Gonzalez-Vilchez e al Isola ion, cha ac e iza ion and An i umou P ope ies o 1,2-P opylenediamine e aace a e- ans-diaqua-coppe (II) Po en iome ic and conduc ime ic s udies Po en iome ic and conduc ime ic s udies we e pe o med using a C ison Mic oTT 2022 i ime e , p o ided wi h au obu e e Mic obu 2030. An aqueous solu ion o he complex (50 mg/100 ml) was i a ed agains NaOH 21.5 mM solu ion. Elec ical conduc ime y o he same solu ion was pe o med on a C ison 525 conduc ime e . Spec oscopic, magne ic and he mal s udies In a ed spec a we e eco ded on a Jasco FT-IR spec ome e in he 4000-400 cm -1 ange as KB discs while a -in a ed spec a we e eco ded as Nujol mulls suppo ed be ween polye hylene shee s. Elec onic u - isible spec a we e measu ed wi h a Jasco V-550 spec ome e while he magne ic measu emen was ca ied ou on a Gouy ype equipmen a 298K. Calib a ing balance wi h he s anda d compound Hg[Co(SCN)a and Pascal cons an s we e used o diamagne ic co ec ions. The he mal beha iou o he compl6x was s udied wi h a Me le DSC ins umen (un il 300C) and a S an on he mobalance (300-500C); measu emen s we e ca ied ou wi h 25 mg o sample and a hea ing a e o 10C/min. Cy o oxic and an i umo essays The cy o oxic e ec o he coppe complex has been e alua ed agains human cance cells o he o a ian umou cell line TG. The cells we e ou inely main ained as monolaye s in cell cul u e lasks (T-25 cm 2, Nunc, Sweden)con aining Dulbeco’s minimal essen ial media supplemen ed wi h 10% e al bo ine se um (FBS) 2 mM glu amin, 100 U/ml penicillin s ep omycin and 1% py u a e. The sample es ed was dissol ed in wa e a pH 7.0 and hen added o he app op ia e cell lasks o ob ain inal concen a ions o l, l0 and 100 k g/ml. Cells we e incuba ed o 120 h a 37C unde g ow h condi ions (5% CO2; 95% 02; He aeus incuba o ) and washed wice wi h p ewa med phospha e bu e ed saline (PBS, 37C). The co esponding cul u es we e exposed o ipsin/EDTA solu ion (0.1% ipsin, 0.4% EDTA in PBS) a 37C o 2 ain. Cul u e medium ( wice olume o ipsin solu ion) was added o he cells be o e cen i uga ion (1500 pm, 5 min) and cell pelle s we e esuspended in he cul u e medium and coun ed in a Thomas chambe unde in e ed ligh mic oscope e e y 24 h. The da a we e s a is ically analysed using he s uden - es . Di e ences we e conside ed signi ican when p< 0.001. The an i umou ac i i y o 1 was es ed in i o agains Sa coma 180 (S180) by he NIO (Spain), acco ding o he p o ocol es ablished by he D ug Syn hesis and Chemical B anch o NCI, Be hesda, USA[ 15]. Animals we e BDF1 emale mice wi h weigh s wi hin a 3g alue ange and a minimum weigh o 17 g. The numbe o animals was 6 pe es g oup. The he apeu ic ac i i y o he complex was ob ained om he T/C pe cen age which is desc ibed as T/C % (100) x Mean li e span o ea ed mice/Mean li e span o un ea ed mice; umou ee su i o s we e excluded. The minimum alue o T/C o ac i i y is 115. I T/C > 125 he complex is conside ed as a candida e o u he es ing agains di e en selec ed u nou sys ems. Resul s and Discussion Po en iome ic S udy Figu e a co esponds o he po en iome ic i a ion o an aqueous solu ion o he coppe complex. A sha p inc ease o pH o he consump ion o 2 g-equi , o alkali wi h an in lec ion poin appea ing a pH 6.6 can be obse ed. Fu he mo e, a sudden change is also obse ed in he elec ical conduc i i y o he solu ion (Figu e lb). All hese ac s demons a ed he simul aneous neu aliza ion o wo ee -COOH g oups o he same s engh . These esul s suppo he e aden a e cha ac e o he PDTA, ha con ains wo coo dina ed ca boxyla e g oups and wo ee ca boxylic g oups. Molecula weigh deduced om he i a ion co esponds closely wi h he one (MW: 420) expec ed o he complex. In a ed spec oscopy The FT-in a ed spec um o compound I show in ense bands a 2920 cm "l and 2926 cm "1, assigned o symme ic s e ching ib a ions (Vs) o he C-H bond o me hylene g oups a ached o coo dina ed and ee ca boxylic g oups. The obse ed spli ing is demons a i e o he equal numbe o ee and coo dina ed ca boxylic g oups 16,17].. The la e assignemen is u he suppo ed in he complex by he s ong cha ac e is ic bands a 1730 cm "l and 1590 cm "l (Vas, C=O bond o ee and coo dina ed ca boxylic g oups) whe eas he s o C=O bond o coo dina ed ca boxyla e g oups appea ed a 1400 cm 1 The di e ence o 190 cm "l be ween Vas and s o coo dina ed ca boxyla e g oups indica ed ha he complex is o p edominan ly ionic cha ac e . The coo dina ed wa e in 1 p esen s di e en peaks a 990 cm -1 ( ocking), 760 cm l (wagging) and a ound 600 cm l (Vas) and 440 cm -1 (Vs) [18] whe eas none o hese ib a ions appea in he in i’a ed spec um o compound 1 when i was hea ed a 220C, he empe a u e a which bo h coo dina ed and la ice wa e a e los , as es ablished by he mal s udy o he complex. Simila ac s ha e been checked ecen ly in new u henium compounds o med wi h iminodiace ic acid I19]. 220 Me al Based D ugs Vol. 7, No. 4, 2000 equi , alk/mol o compound Figu e I. Po en iome ic (a) and conduc ime ic (b) s udy o [Cu(PDTA- H)(H20)21H,O. Mola conduc ance (298K) changes on A 190 S cm 2 (I g-equi . alkali) o A 142 S cm 2 (2 g equi , alkali). :-...., ,o " ’’"4 b 100 200 300 400 (C) Figu e 2. DSC s udy (cu e a), di e en ial he mal analysis (cu e b) and he mog a ime ic analysis (cu e c) o 1. Wa e no mally gi es b oad abso p ion a 3500 cm 1. 1 shows a single peak a 3440 cm I, a ibu ed o he la ice wa e molecules mos likely bonded o anionic ligands h ough hyd ogen bonds [20]. This ac is he cause o he sa elli e peaks obse ed a a ound 2600 cm 1. Indeed, his is he case in compound 1, as checked in he in a ed spec a o se e al samples o he complex aken a e hea ing a 140C. In all cases, he spec a only show a weak abso p ion a abou 3540 cm -1. Table p esen s hese and o he peaks obse ed in he in a ed spec um o he 1. UV- isible spec al and magne ic s udies The elec onic spec a o an aqueous solu ion o he complex (8.0 mM) shows a sligh ly assymme ic single band a 13700 cm 1 (, 730 nm), wi h an ex inc ion coe icien alue o 67.75. These and o he pa ame e s o his absop ion a e simila o hose obse ed o he analogous Cu-EDTA complex[ 21] and his band may be consequen ly a ibu ed o he spin-allowed ansi ion 2E_ _.._> 2T2g, indica ing a e agonally dis o ed oc ahed al symme y o he complex. Two wa e molecules a e loca ed in ans posi ion on he oc ahed al Z axis a longe dis ances om he me al ion wi h he emaining ou coo dina ed dono a oms lying on he plane. Table 1. IR da a (cm l) o [CuL(H20)2]H20 COOH COO" CH2 H20 Cu-N Cu-OH2 (C=O) (C-H) h yd 1730 Vas 1590 Vas 2920 3440 s 400 600 Vas 840 1400 s 2926 1630 b 1120 440 s s and Vas" symme ic and asymme ic s e ching ib a ions; Vb: bending ib a ion. 221 F. Gonzalez-Vilchez e al Isola ion, cha ac e iza ion and An i umou P ope ies o 1,2-P opylenediamine e aace a e- ans-diaqua-coppe (II) The magne ic momen o he complex has been calcula ed a 298K and he ob ained alue (1.83 BM) di e s om ha heo e ical one expec ed o he spin-momen alue a his empe a u e (1.94 BM), as p edic ed o an E e m (second o de Zeeman e ec and pa ial cancela ion o he o bi al angula momen ). The expe imen al alue o he magne ic momen con i ms he e agonal dis o ion deduced om he elec onic spec a. 4 C on oi 100 0 54 48 72 96 120 Time (hou s) Figu e 3. De e mina ion o he e ec s exe ed by he coppe (ll) complex on he g ow h kine ics o he TG o a ian ca cinoma human cance cell line. TG cells we e exposed o di e en doses o he coppe (ll) complex (see expe imen al pa o de ails). The mal s udy The DSC s udy pe o med be ween 20C and 350C o he complex indica es a medium and ex ended endo he mic e ec a empe a u es be ween 85C and 140C (Fig. 2a). This e ec is absen in he PDTA DSC s udy and migh be a ibu ed o he dehyd a ion o he uncoo dina ed wa e molecules. Mo eo e , a mo e p onounced and spli ed endo he mic e ec appea ed a 200-220C, indica ing he elimina ion o wo coo dina ed wa e molecules. The deca boxila ion p ocess clea ly begins a 240C and con inues du ing all he empe a u e ange. Figu e 2b shows he DTA cu e ob ained be ween 20C and 500C while Fig. 2c p esen s he simul aneous TGA cu e a same empe a u es. In he i s case (cu e b), he elimina ion o he coo dina ed wa e obse ed in he DSC s udy is con i med by his echnique h ough an endo he mic spli ed e ec egis e ed be ween 200C and 230C. The deca boxyla ion p ocess appea s h ough an in ense exo he mic double e ec egis e ed be ween 230C and 330C. The i s acu e e ec a 240- 290C coincides wi h he weigh loss de ec ed in he TGA s udy (cu e c) o he deca boxila ion o he wo ee ca boxylic g oups and o e laps wi h a second sha p and less in ense e ec a 310C ha co esponds o he deca boxila ion o he coo dina ed ca boxyla e g oups. Final py olysis akes place a empe a u es o e 330C. An i umou ac i i y The inhibi ing e ec s o he 1 agains he human o a ian umou cell line TG a e shown in Fig. 3 which p esen s he e olu ion wi h ime o he o al numbe o cul u e cells in absence (con ol) o p esence o he complex. Th ough his s udy i is possible o de e mine he in luence o di e en doses o he coppe complex on he g ow h kine ics o he cells cul u e. In his way, he g ow h kine ic o hecul u e ea ed wi h doses o b g ml "1 and 10 b g ml "1 o complex is signi ican ly lowe (p< 0.001) han ha shown in con ol cul u es a e 48 h o he ea men . Simila esul s a e ob ained 24 h a e adminis a ion o he highe dose o compound (100 l. g ml’l). I can be obse ed ha he g ow h kine ics o he umou cells dec eases wi h inc easing doses o 1 in such a way ha he p oli e a ion p ocess is almos comple ely cancelled by his complex a e 96 h o ea men e en a he lowe doses handled. 222 Me al Based D ugs Vol. 7, No. 4, 2000 80 IG /,I /" !/" ,./ ;,, .nd 11E/ml 10 I/ml Dose Figu e 4. Va ia ion o he cellula cycle du a ion o he TG o a ian ca cinoma human cance cell line wi h a dose o 1. The e ec p oduced by he coppe complex on he i al cellula cycle was also s udied. Fig. 4 shows he cellula cycle du a ion co esponding o ea ed and con ol TG cell lines. A doses o l g ml "1, an impo an induced inc easing o he TG cellula cycle is obse ed (30 h) i_ compa ed o ha o he con ol cells (23 h). The leng h o he cycle o ea ed cells a a dose o 10 I. g ml-1 was also signi ican ly enhanced (62 h). Howe e , he dose o 100 I. g ml "1 weas s ongly cy o oxic and dea h o he ea ed cells was obse ed. The ob ained esul s demons a e ha a signi ican enhancemen o he i al cellula cycle is p oduced a all doses lowe han 100 g ml "1 On he basis o hese s udies one can conside ha 1 beha es as a po en ial an i umou compound showing p onounced in i o cy o oxici y. The an i umou ac i i y agains S 180 has been e alua ed a doses o 3.0, 6.0 and 12.5 mg/Kg body weigh . Table 2 shows he esul s ob ained. The oxic dose was ound equal o 35 mg/Kg body weigh . F om Table 2, a no ewo hy ac i i y agains S180 o he complex is deduced a all doses, wi h comple e emission o he umou a a dose o 12.5 mg/Kg body weigh wi h 100 % su i o s. The complex is well ole a ed and shows a lack o oxici y a doses lowe han 35 mg/Kg bodyweigh . Thus, 1 beha es as a ema kable an i umou subs ance agains S180 and should be es ed agains o he umou sys ems also because i s no able an ip oli e a i e e ec agains TG human umou cells line. The mechanism o he an i umou ac i i y cells is unde p og ess. Table 2. An i umou ac i i y o 1. agains S180 (In i o Dosage MLS(*) Numbe o mice T/C % (mg/Kg body weigh ) T/C su i o s a e 6 mon hs 3.0 28/40 70.0 6.0 48/40 120.0 12.5 All ali e Six (l 00%) (*) T/C =6/6; MLS: Mean li e span. Single injec ion doses we e adminis e ed i.p Acknowledgemen s We hanks inancial suppo om CICYT, Spain, p ojec PB97-0738. SK and KA a e indeb ed o AECI (Spain) o Ph.D. schola ships. Re e ences 1. Gonz ilez-Vilchez, F., Vilaplana, R., (1986), In: T ends in Cance Resea ch, Ba be a, E. (ed.) Vizcaya: UPV, pp. 194-198. 2. Vilaplana, R., Gonz ilez-Vilchez, F., Gu ie ez-Puebla, E., Ruiz-Vale o, C., lno g. Chim. 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