Synthesis, structure, properties and biological behaviour of the complex [RuIV (H2L) CI2] 2H2O (H4L- 1,2-cyclohexanediamminetetraacetic acid)

Syn hesis, S uc u e, P ope ies And Biological Beha iou O The Complex [Ru i (HL) CI] 2HO (H4L- 1,2-Cyclohexanediammine e aace ic Acid) Rosa io A. Vilaplana," A. Cas i lei as b and F ancisco Gonzb.lez-Vilchez aDepa amen o de Quimica Ino gdnica, Secci6n de Quimica Bioino gdnica, Facul ad de Quimica, Ap do. Co eos 553, E-41071 Se illa, Spain. hDepa amen o de Quhnica Ino gdnica, Facul ad de Fa macia, Uni e sidad de San iago de Compos ela, E- 15 782 San iago de Compos ela, Spain GRAPHICAL ABSTRACT The oc ahed al complex [Ru(Hzcd a)CI2]’2H20 con ains Ru(IV) su ounded by wo ni ogen a oms, N(I) and N(2), and wo chlo ide ions c/s o each o he , all in he equa o ial plane. Two oxygen a oms, O(11) and O(21) a e loca ed in ans axial oc ahed al posi ions. The complex shows ema kable in i o and in i o ac i i y agains di e en ca cinomas. Cll OI 1 o whom co espondence should be add essed; phone: +34 95 4557159; ax: +34 95 4557081; e-mail: [email p o ec ed] 275 Vol. 2, Nos. 3-4, 2004 Syn hesis, S uc u e, P ope ies and Biological Beha iou O he Complex ABSTRACT The highly wa e -soluble u henium complex [Ru(H2L)CI2]’2H20, in which H4L is he seques e ing ligand ans-l,2-cyclohexanediammine e aace ic acid (cd a) has been syn hesized, s uc u ally cha ac e ized and i s p ope ies s udied. The X- ay c ys allog aphic s udy shows ha he chela ing coo dina ed ligand is e aden a e while he u henium en i onmen is oc ahed al and sligh ly dis o ed, wi h wo chlo ide anions coo dina ed in cis posi ions. Po en iome ic, conduc ime ic and in a ed s udies con i m he p esence o wo ee ca boxylic g oups, while elec onic and ol amme ic s udies show ha he cen al ion is Ru(IV). The es ing o he cy o oxic ac i i y o his complex agains h ee di e en human cance cell lines indica es ha [Ru(H2L)CIz].2HzO shows a ema kable and selec i e an ip oli e a i e e ec agains he human u e ine neck ca cinoma HeLa and he malign adenoca cinoma ADLD, showing only a disc e e u nou cell inhibi ion ac i i y agains colon adenoca cinoma HT-29. The impo an an ip o i e a i e beha iou o complex agains he human adenoca cinoma ADLD, indica es ha [Ru(HL)CI].2HzO migh be conside ed as po en ial an ineoplas ic compound. INTRODUCTION Me al-based an i umou d ugs cons i u e in ou days a b oad esea ch a ea o inc easing in e es /1-4/. Conc e ely, cispla in, cis- [P (N H3)2C12], ca bopla in [cis-diammine- I, -cyclobu anedica boxyla o- pla inum(II)] and oxalipla in [ ans-(R,R)-l,2-diamminecyclohexaneoxala opla inum (II)], a e cu en ly being used clinically /5-10/. Howe e , he p oblem o bo h acqui ed and inhe en esis ance o umou cells p esen s a main limi a ion o he mo e widesp ead clinical use o pla inum complexes/3,7,9/. In he hope o o e coming hese limi a ions, o he pla inum-based an i umou d ugs ha e been syn hesized and es ed o an i umou ac i i y/11-15/. Fu he mo e, new an icance d ugs con aining ansi ion me al ions o he han pla inum /16/ha e been also assayed. Possible ad an ages may in ol e di e en coo dina ion geome ies, se e al me al-ion oxida ion s a es and biological a ge s o he han DNA. In he design o hese new d ugs, u henium complexes ha e aised g ea in e es /17-24/. As an example, he an i umou ac i i y o he highly wa e soluble complex H[Ru(H2L)CI]-4H20 (H4L: 1,2- p opylenediammine-N,N,N’,N’- e aace ic acid, pd a) has been e alua ed in i o and in i o/23,25/. This complex apidly binds o se um p o eins p oducing s able adduc s in which he co e Ru(lII)(pd a) is p obably bound o his idines on he p o ein su ace/26-28/. On he o he hand, he complex damages nuclea DNA, inhibi s DNA ecogni ion and s imula es NADPH oxidase and a espi a o y bu s in phagocy ic neu ophils and elici s phospho yla ion o y osine esidues/25,29/. Ano he in e es ing ligand is he po en ial hexaden a e seques e ing agen ans-l,2- cyclohexanediammine e aace ic acid (cd a), in which he e hylenediammine g oup o ed a has been eplaced by he he e ocyclic moie y ans-l,2-cyclohexanediammine. The ligand cd a is widely used nowadays in a ied applica ions, i.e., as de oxi ie o hea y me als ha con amina e pa ien s/30/, o use in endodon ics /31/, o ex ac ion o pec in polyme s /32/ and o sepa a ion and quan i a ion by se e al echniques o 276 Rosa io A. Vilaplana e al. Bio&o ganic Chem& y and Applica ions ino ganic ions and anionic me al complexes/33/. Al hough he me al complexes o med by cd a a e known om ime ago/34/, ecen li e a u e has shown new syn hesis p ocedu es and impo an p ope ies o new isola ed complexes. Conc e ely, hose o med by cd a wi h Cu(ll) and Ni(ll) ha e been s udied by X- ay pho oelec on spec oscopy and X- ay c ys al di ac ion s udies /35,36/ and in e es ing esul s on he s uc u e, elec ochemis y, kine ics, pKa alues and in luence o chela e e ec s on he wa e -exchange mechanisms o complexes cd a/Fe(lll)/Fe(ll)/ ha e been epo ed /37,38/. Resea ch on he o ma ion cons an s and dissocia ion kine ic o cd a/lan hanide(lll)/ complexes /39,40/ as well as solu ion s udies and de e mina ion o he c ys al s uc u es o cd a complexes o med wi h he i alen ions o AI, Ga, In and Sc /41/ha e been also published. The esea ch de eloped so a on pla inum-g oup me al complexes o med wi h cd a conce ns i s he he X- ay c ys allog aphic s udy o he dip o ona ed ligand and i s complexes wi h [PdCI4] 2- and [P CI4] 2 sal s /42a/. Addi ionally, he eac ion kine ic p ocesses in ol ed in he o ma ion o Pd(ll) and P (ll) complexes o di e en composi ion wi h amminepolyca boxyla e ligands (i.e., {(H6L)[Pd/P CI4]} o [Pd(H2L)]) we e also s udied/42b/. Dini ogen complexes o med be ween Ru(ll) and cd a we e isola ed and cha ac e ized/42c/. On he o he hand, p omising esul s we e ob ained in he i s s udy de eloped on he an i umou ac i i y o new Pd(ll)(cd a) complexes/42d/. Di e en Ru(lll) complexes such as [Ru(H4L)CI] 3-n and [Ru(HL)Ci]3"-I), we e syn hesized and cha ac e ized by analy ical and elec ochemical echniques/43/. Finally, educ ion o molecula ni ogen o ammonia in aqueous solu ion unde ambien condi ions occu s in he p esence o an illumina ed RuO2/P /CdS sys em; his eac ion is ca alysed by se e al Ru(ll) complexes as, o example, [Ru(cd a)N] 2-/44/. As a esul o he a ac i e esea ch in p og ess on biological p ope ies o complexes o med by Ru(lll) wi h amminepolyca boxylic ligands, as well as he absence o s uc u al and an i umou ac i i y o Ru(cd a) complexes, we p esen in his pape he syn hesis, cha ac e iza ion by chemical and spec oscopic echniques, x- ay c ys al s uc u e and biological beha iou o he wa e -soluble complex [Ru(HzL)CI].2H:O (I), in which he ligand cd a (H4L) is ac ing as e aden a e molecule/45/. Ou esul s demons a e ha he complex con ains Ru(IV) and shows ac i i y agains se e al in i o and in i o umou s. This esea ch open a new way o he medical and pha maceu ical de elopmen o po en ial an ineoplas ic complexes o med by he chela ing agen cd a wi h pla inum me als. EXPERIMENTAL X- ay c ys allog aphy A p isma ic yellow-ambe c ys al o compound was moun ed (glass ibe ) on an En a Nonius CAD4 au oma ic di ac ome e /46/ and 2388 unique e lec ions we e measu ed. Cell cons an s and o ien a ion ma ix o da a collec ion we e ob ained by leas -squa es e inemen o he 20 alues o 25 e lec ions. In ensi ies o 3123 e lec ions wi hin he ange 2 <20 <50 we e measu ed and collec ed a 293 K using monoch oma ic MoK, adia ion (2 0.71073 A) and he a/20 scan echnique. In ensi ies we e co ec ed o Lo en z and pola iza ion e ec s/47/and 2388 {(1>2o->(I)} we e conside ed as obse ed. A semiempi ical 277 Vol. 2, Nos. 3-4, 2004 Syn hesis, S uc u e, P ope ies and Biological Beha iou O he Complex abso p ion co ec ion (q-scans) was made/48/. The s uc u e was sol ed by he Pa e son me hod/49/ and subsequen di e ence Fou ie maps, and e ined on F by a ull ma ix leas -squa es p ocedu e using aniso opic displacemen pa ame e s/50/. All hyd ogen a oms we e loca ed in di e ence map and included as ixed con ibu ions iding on a ached a oms wi h iso opic he mal pa ame e s 1.2 imes hose o hei ca ie .a oms. The H a oms o wo wa e molecules, O(1) and O(2), we e no loca ed. The e o e, he con ibu ion o he densi y o a diso de ed wa e molecule was sub ac ed om he measu ed s uc u e ac o s wi h use o he SQUEEZE op ion /51/. Subsequen e inemen hen con e ged wi h R ac o s and pa ame e e o s signi ican ly be e han o all a emp s o model he sol en diso de . The Flack x pa ame e (absolu e s uc u e pa ame e ) was calcula ed o be 0.05(6) o he p esen s uc u e and 0.95(6) o he in e ed s uc u e, hus p o iding s ong e idence ha he absolu e s uc u e has been assigned co ec ly/52/. C i e ia o a sa is ac o y comple e analysis we e he a ios o ms shi o s anda d de ia ion less han 0.001 and no signi ican ea u es in inal di e ence maps. A omic sca e ing ac o s, om "In e na ional Tables o C ys allog aphy"/53/. Molecula g aphics, om PLATON /51/and SCHAKAL/54/. Chemicals Hyd a ed u henium (III) chlo ide (Sigma) was dissol ed in e hanol and e luxed o 30 min. A e concen a ion o d yness, he compound was s o ed unde CaCI_ (RuCI3). The ligand cd a was used as pu chased (Sigma). All o he chemicals and sol en s we e analy ical g ade eagen p oduc s. Analy ical, po en iome ic and conduc ime ic s udies 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. Me al con en was de e mined by a omic abso p ion spec oscopy using a Pe kin Elme 2380 model, a 10 mA and 349.9 im. Hyd a ion wa e molecules we e de e mined by he mal analysis. Po en iome ic and conduc ime ic s udies we e ca ied ou wi h a C ison Mic oTT 2022 i ime e , p o ided wi h au obu e e Mic obu 2030. Aqueous solu ions o he complex (50-100 mg/100 ml) we e i a ed agains a 30 mM NaOH solu ion. Elec ical conduc ime y o he same solu ion was pe o med on a C ison 525 conduc ime e . Elec onic and in a ed spec oscopy The elec onic spec a o solu ions we e eco ded on a Jasco V550 spec ome e in e aced wi h a PC. IR spec a we e eco ded on an FT-IR Jasco 300E ins umen in he 200-4000 cm - ange, using ei he Nujol mulls suppo ed be ween polye hylene pla es o KB pelle s. Vol ammme ic s udies Cyclic oi amme y measu emen s we e ca ied ou using a P ince on Applied Resea ch analyse . A 278 Rosa io A. l,71al)lana e aL Bioinol2,anic Chem& y and Applica ions glassy ca bon elec ode was used as wo king elec ode on aqueous solu ions (NaCIO4 0.15 M) o complex a concen a ions anging be ween 1.0 and 6.0 mM. Po en ials we e measu ed agains a sa u a ed (NaC1) calomel elec ode (SCE) as e e ence elec ode. Vol ammog ams (CV) we e ob ained a po en ial alues be ween +1.5 and -1.5 V. Scan a e was equal o 50 m V/s. Biological in i o and in i o assays Complex was dissol ed in a mixed 1:1 DMSO:H20 solu ion and used as concen a ed s ock solu ion om which dilu ed solu ions (I/10 dil / ion ac o ) we e p epa ed and added o g owing cul u e medium o each one o he cellula ypes included in he s udy. The in i o assays we e done agains h ee di e en human cance cell cul u es: malignan melanoma (ADLD), u e ine neck ca cinoma (HeLa) and colon adenoca cinoma (HT-29). Fo de e mina ion o pla ing e iciency and colo ime ic eading, we p epa ed pla es o 24 and 96 wells, espec i ely, ha we e hen inocula ed wi h 5,000 o 30,000 cells/well. A e 24 h om he inocula ion, di e en concen a ions o he es ing p oduc we e added o he cance cells. A e 48 h o he named addi ion, he cells we e washed and hen ixed. Finally, he colo ing and eading o he pla es we e ca ied ou . In all cases we de e mined he numbe o cells a he beginning (To ), jus be o e he addi ion o he es ing p oduc s (To) and 48 h la e (T con ols and T es s), acco ding o Scheme 1. The an i umou ac i i y o was es ed in i o agains Eh lich asci ic umou (EAT), he in ape i oneally-implan ed P388 lymphocy ic leukemia and he sub enal capsule ( ansplan ed human mamma y ca cinoma) MX-I xenog a . The expe imen s we e pe o med acco ding o he NIH p o ocols a ONI Cen e, Mad id, Spain. CD2F emale mice wi h weigh s wi hin a 3 g alue ange and a minimum weigh o 18 g we e used o all expe imen s excep o MX-1 ca cinoma, whe e a hymic swiss mice we e used (4 g alue ange and weigh o 17 g). The es g oups was o med by 6 animals and con ol g oup by 12. Syn hesis o he complex [RuV(HL)CI].2HO [1] Solid cd a (228 mg, 1.0 mmol) was added o a clea solu ion o RuCI3 (0.230 mg, 1.1 e ool) dissol ed in a 50 ml o HCI 0.1 M, while s i ing. The deep ed mix u e was in oduced in o a sealed p essu e eac o and hea ed o 16 h a 120C in elec ic o en. A e cooling, he pink- ed ob ained solu ion was slowly concen a ed o abou 5 ml by e apo a ion a oom empe a u e. Yellow-ambe p isma ic c ys als sui able o X- ay di ac ion s udies we e eco e ed and he solid analysed. Two hyd a ion wa e molecules pe mol o compound we e ound by he mal analysis. Anal. (%), Calc. o C4H2008N2CI2Ru’2H20: C, 30.4; H, 4.5; N, 5.1; CI, 12.8; Ru, 18.3. Found: C, 30.6; H, 4.1; N, 5.3; CI, 12.2; Ru, 17.9. RESULTS AND DISCUSSION X- ay s uc u e Table p esen s a summa y o he ele an c ys al da a and e inemen esul s o complex I. The 279 Vol. 2, Nos. 3-4, 2004 Syn hesis, S uc u e, P ope ies and Biological Beha iou O he Complex Table 1 C ys al da a and s uc u e e inemen o [Ru(H2cd a)Cl2].2H20 Iden i ica ion code Empi ical o mula Fo mula weigh Tempe a u e Waeleng h C ys al sys em, space g oup Uni cell dimensions Volume Z, Calcula ed densi y bso p ion coe icien F (000) C ys al size T..he a ange o da a collec ion Limi ing indices Re lec ions collec ed / unique Comple eness o he a 27.44 ?cbso p ion co ec ion Max.. and ain. ansmission Re inemen me hod Da a / es ain s / pa ame e s Goodness-o - i on Final R indices [I>2sigma(1)] R ndices (all da a) _bsolu e s uc u e pa ame e La ges di . peak and hole udc a Cl4 H24 C12 .|2 O10 Ru 552.32 293 (2) K 0.71073 A Hexagonal, P6(5) (No. 170) a 13. 567 (2) A b 13. 567 (2) A c 22. 286(6) A 3552.5(12) A^3 alpha 90 deg. be a 90 deg. gamma 120 deg. 6, i. 549 .Mg/m" 3 0. 935 mm"-I 1680 0.40 x 0.34 x 0.20 mm 1.73 o 27.44 deg. -14<=h<=0, 0<=k<=17, 0<=!<=28 3123 / 2779 [R(in ) 0.0103] i00.0 % Psi .-scans 0.8350 and 0.7061 Full-ma ix leas -squa es on 2779 / ! / 271 0.943 R1 0.0498, wR2 0.0518 R1 0.0498, wR2 0.0518 0.05(6) 0.515 and -0.380 e.A-3 molecula s uc u e ob ained by X- ay di ac ion analysis is shown in Fig. along wi h he numbe ing o a oms in he molecule. The e aden a e chela ing ligand (cd a) con ains wo ee ca boxylic g oups and is bonded o he cen al ion by wo ni ogen a oms, N(l) and N(2), and wo chlo ide anions cis o each o he , sha ing he equa o ial plane wi h he ni ogen a oms. The emaining coo dina ion posi ions a e occupied by wo oxygen a oms, O(11) and O(21), om coo dina ed ca boxyla e g oups, si ua ed in ans axial oc ahed al posi ions ha comple e in his way he oc ahed al coo dina ion sphe e a ound u henium a om. 280 Rosa io A. Vilaplana e al. .Bioino ganic Chemis y and Applica ions CI1 O12 O11 Rul Cl2 N2 O 22, N1 Fig. 1" O14 0,13 View o he molecula s uc u e o complex showing he oc ahed al en i onmen o he u henium cen al ion. Table 2 p esen s selec ed bonds leng hs and angles o complex 1. As expec ed, he chlo ide coo dina ed ions a e loca ed much u he om he u henium a om (2.37 A) han he wo ni ogen a oms (2.11 / and 2.13 A). A possible explana ion is ela ed o he no able nucleophilic cha ac e o he chela ing ligand ha induces a no able inc easing o he dis ance Ru-CI. Fu he mo e, he bulky chela ing ligand migh be he o igin o he sligh educ ion obse ed in he CI-Ru-CI angle alue (91.4), in compa ison wi h he same angle in cispla in (91.9). These wo e ec s ha e also been ound in simila complexes wi h o he aminopolyca boxylic ligands, i.e., Ru(ed a)CI2 [22] and Ru(pd a)Cb, [23]. Clea ly, he dis ance Ru-O(I 1) is sho e (2.03 /) han he dis ance Ru-O(21) (2.07 /). As a consequence, he oc ahed al con igu a ion a ound u henium a om is sligh ly dis o ed (Fig. I) wi h angles 281 Vol. 2, Nos. 3-4, 2004 Syn hesis, S uc u e, P ope ies and Biological Beha iou O he Complex Table 2 Selec ed bond leng hs [A] and angles [deg] o [Ru(H2cd a)Cl2].2H20 Ru (I) -O(II) 2. 028 (3) Ru (i) -O (21) 2.070 (3) Ru (i) -I., (I) 2.108 (2) Ru (i) -l.I (2) 2. 128 (2) Ru (!) -CI (2) 2.3658(9) Ru (I) -el (I) 2.3723(8) 0 (ll)-Ru (I)-O (21) o (11) -Ru (I) - , (i) o (21) - u ([) -u O (II) -Ru (l) -I.-| (’2) 0 (21 -Ru ! -n (2) I- (i) -Ru (i) -l,I (2) o (I l -Ru (I) -cl. (2) O (2.) -Ru (I) -CI {2) I.i (I) -Ru (I) -el (2) I.I (2) -Ru (I) -CI (2) O (ll)-Ru (1)-Cl (i) O(21)-Ru (i) -CI (i) I..I ()-Ru ()-Cl () II(2)-Ru (1)-Cl (I) C1 (2) -Ru (1)-CI (1) 173.48 (8) 80.72(9) 94.89(8) 95.57 79.14(i0) 84.54(8) 93.05(7) 91.97(7) 92.69(7) 170.39(8) 91.83(6) 92.21(6) 171.69(8) 92.57(6) 9.1.39(3) signi ican ly educed om ideal oc ahed al alues, i.e., N(I)-Ru-N(2), 84.5; O(1 I)-Ru-N(I), 80.7 and 0(2 I)-Ru-N(2), 79.1 . I is impo an o ema k ha he nonbonding CI...C1 dis ance (bi e) in compound (3.35 A), is di ec ly co ela ed wi h he dis ance be ween adjacen o p oximal coo dina ion si es in biological a ge s such as DNA. This bi e dis ance is iden ical o ha o cispla in, and co esponds o he sepa a ion be ween wo app op ia e DNA-nucleobase dono a oms, his ac enabling c oss-linking o ma ion a e in e ac ion o complex wi h DNA inside he cell/29/. in he packing, he molecules o complex a e assembled in a 3D-ne wo k by in e molecula hyd ogen bonding ha in ol es wo pai s o oxygen a oms, O(13)...O(24), 2.65 /l and O(23)...O(14z), 2.67 A (symme y ans o ma ions; 1: x-y+ 1, x, z-l/6 and 2: y, -x+y+ 1, z+ 1/6), as ound in simila cd a complexes /42a/, i.e., he hyd oxyl g oups o he non-coo dina ed ca boxylic moie y ac as hyd ogen-a om dono s while he ca bonyl g oups a e he hyd ogen-a om accep o s in hese associa ions (Fig. 2). In addi ion, he wo wa e molecules loca ed be ween neighbou ing molecula uni s a e also H-bonded o oxygen ca boxyla o g oups {O(1)...O(213), 2.89/l and O(2)...O(113), 2.87 A (symme y ans o ma ions; 3: y, -x+y, z+l/6)}. This H- bonded pa e n can be desc ibed as a ne wo k o sup amolecula helices. In he c di ec ion he packing gene a es a six old sc ew axis and pa allel channels (Fig. 3), wi h ca i ies o adius 2.15 A. These channels a e hyd ophilic because he oxygen a oms o ca boxylic moie ies and hose o wa e molecules poin owa ds he ca i ies. 282 Rosa io A. Vilaplana e al. Bioino ganic Chemis ), and Applica ions Fig. 2: Packing d awing iew o showing he assembling o molecules in a 3D-ne wo k by in ennolecula hyd ogen bonding in ol ing wo pai s o oxygen a oms. 283 VoL 2. Nos. 3-4, 2004 Syn hesis, S uc u e, P ope ies and Biological Beha iou ()./’ he Complex The in i o an i umou ac i i y o complex agains EAT and P388 umou s was e alua ed a se e al ea men doses in he ange 20--240 mg/kg body weigh . 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; u nou ee su i o s we e excluded. The minimum alue o T/C o mode a e ac i i y is 120 (EAT and P388 umou s); i T/C > 125 he complex is conside ed a candida e o u he an i umou assays. T/C alues equi ed o ac i i y in MX-1 xenog a umou mus be lowe han 20 (T/C <20). Fo EAT, a T/C (%) o 350 was ob ained (25 mg/kg) and he comple e emission o he u nou obse ed a a dose o 50 mg/kg. The oxic .dose (LDs0) was equal o 100 mg/kg. In he case o he lymphocy ic leukemia P388, a T/C(%) alue o 140 was ob ained (60-120 mg/kg) while in MX-I xenog a ca cinoma, his pa ame e was equal o 16 a a dose o 240 mg/kg. These esul s e idence a no able and speci ic an ip oli e a i e e ec o complex [Ru(HzL)CI2]’2H20 agains he human umou cell lines ADLD and HeLa, wi h selec i e and impo an cy o oxic p ope ies in bo h cases. The in i o an i umou ac i i y is also ema kable in he s udied u nou s. A u he in es iga ion o he an ineoplas ic ac i i y o his new po en ial u henium d ug agains o he ypes o u nou is ad isable. 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