Impact of the central atom and halido ligand on the structure, antiproliferative activity and selectivity of half-sandwich Ru(ii) and Ir(iii) complexes with a 1,3,4-thiadiazole-based ligand

Dal on T ansac ions PAPER Ci e his: Dal on T ans., 2023, 52, 12717 Recei ed 2nd June 2023, Accep ed 10 h Augus 2023 DOI: 10.1039/d3d 01696j sc.li/dal on Impac o he cen al a om and halido ligand on he s uc u e, an ip oli e a i e ac i i y and selec i i y o hal -sandwich Ru(II) and I (III) complexes wi h a 1,3,4- hiadiazole-based ligand† Radka Křika o á, * a Michaela Romano o á, b Zuzana Jendželo ská, b Ma in Maje ník, b LukášMasa yk, a Pa el Zou alý, a Da id Milde, c Jan Moncol, d Rado an He chel, a Ras isla Jendželo ský b and I an Nemec a,e Hal -sandwich complexes [Ru(η 6 -pcym)(L1)X]PF 6 (1,3) and [I (η 5 -Cp*)(L1)X]PF 6 (2,4) ea u ing a hiadia- zole-based ligand L1 (2-( u an-2-yl)-5-(py idin-2-yl)-1,3,4- hiadiazole) we e syn hesized and cha ac e - ized by a ied analy ical me hods, including single-c ys al X- ay diff ac ion (X = Cl o I, pcym = p-cymene, Cp* = pen ame hylcyclopen adienyl). The s uc u es o he molecules we e analysed and in e p e ed using compu a ional me hods such as Densi y Func ional Theo y (DFT) and Quan um Theo y o A oms in Molecules (QT-AIM). A 1 H NMR spec oscopy s udy showed ha complexes 1–3exhibi ed hyd oly ic s abili y while 4unde wen pa ial iodido/chlo ido ligand exchange in phospha e-buffe ed saline. Mo eo e , 1–4demons a ed he abili y o oxidize NADH ( educed nico inamide adenine dinucleo ide) o NAD + wi h I (III) complexes 2and 4displaying highe ca aly ic ac i i y compa ed o hei Ru(II) analogues. None o he complexes in e ac ed wi h educed glu a hione (GSH). Addi ionally, 1–4exhibi ed g ea e lipophilici y han cispla in. In i o biological analyses we e pe o med in heal hy cell lines (CCD-18Co colon and CCD-1072Sk o eskin fib oblas s) as well as in cispla in-sensi i e (A2780) and - esis an (A2780cis) o a ian cance cell lines. The esul s indica ed ha I (III) complexes 2and 4had no effec on human fib oblas s, demons a ing hei selec i i y. In con as , complexes 1and 4exhibi ed mode a e inhibi o y effec s on he me abolic and p oli e a ion ac i i ies o he cance cells es ed (selec i i y index SI > 3.4 o 4and 2.6 o cispla in; SI = IC 50 (A2780)/IC 50 (CCD-18Co)), including he cispla in- esis an cance cell line. Based on hese findings, i is possible o emphasize ha mainly complex 4could ep- esen a u he s ep in he de elopmen o selec i e and highly effec i e an icance agen s, pa icula ly agains esis an umou ypes. In oduc ion Con en ional pla inum-based an icance d ugs ep esen one o he mos widely used g oups o chemo he apeu ics which ha e been used in clinical p ac ice o mo e han 40 yea s. 1,2 Howe e , due o hei la ge numbe o side effec s and low efficacy agains some ypes o umou s, one o he main goals o medicinal chemis s is o de elop new agen s wi h highe an ip oli e a i e ac i i y, lowe gene al oxici y and he abili y o kill cance cells esis an owa ds he biological ac ion o con en ional d ugs. 3–5 Cu en ly in es iga ed p omising g oups o po en ial non- pla inum d ugs include u henium complexes (BOLD-100 6,7 and TLD1433 8,9 ), which ha e al eady en e ed clinical ials. Indeed, BOLD-100 is p esen ly he mos clinically ad anced u henium-based agen , which has al eady ecei ed O phan D ug Designa ions (ODDs) om he FDA in bo h gas ic and †Elec onic supplemen a y in o ma ion (ESI) a ailable: NMR, ESI+ mass spec a, 1 H NMR s abili y and in e ac ion s udies, c ys allog aphic da a, c ys al s uc u es and non-co alen in e ac ions, and cellula expe imen al (me abolic ac i i y, ia- bili y, MMP) esul s. CCDC 2266585–2266588. Fo ESI and c ys allog aphic da a in CIF o o he elec onic o ma see DOI: h ps://doi.o g/10.1039/d3d 01696j a Depa men o Ino ganic Chemis y, Facul y o Science, Palacký Uni e si y Olomouc, 17. lis opadu 12, CZ-771 46 Olomouc, Czech Republic. E-mail: adka.k ika o[email p o ec ed] b Depa men o Cellula Biology, Ins i u e o Biology and Ecology, Facul y o Science, Pa ol Joze Ša á ik Uni e si y in Košice, Š obá o a 2, 041 54 Košice, Slo akia c Depa men o Analy ical Chemis y, Facul y o Science, Palacký Uni e si y Olomouc, 17. lis opadu 12, CZ-771 46 Olomouc, Czech Republic d Depa men o Ino ganic Chemis y, Facul y o Chemical and Food Technology, Slo ak Uni e si y o Technology in B a isla a, B a isla a SK-81237, Slo akia e Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, Pu kyňo a 123, 61200 B no, Czech Republic This jou nal is © The Royal Socie y o Chemis y 2023 Dal on T ans.,2023,52, 12717–12732 | 12717 Open Access A icle. Published on 11 Augus 2023. Downloaded on 2/19/2024 1:17:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online View Jou nal | View Issue panc ea ic cance s. 10 Based on he success o such agen s, i seems a ional o u he in es iga e po en ially effec i e an i- cance complexes o no only u henium bu also o he noble me als, such as i idium which ends o o m complexes wi h simila s uc u al ea u es. 11–13 One o he mos in e es ing s uc u al ypes o bioac i e Ru and I complexes is undoub - edly ep esen ed by hal -sandwich complexes o he gene al o mula [M(η 6 /η 5 -a ene/a enyl)(L)(X)] 0/n+ , which ha e been widely s udied o hei high cy o oxici y, accep able selec i i y and diffe en mechanisms o ac ion compa ed o pla inum- based an icance d ugs. 14 The coo dina ion sphe e o biologi- cally ele an hal -sandwich coo dina ion compounds is ypi- cally composed o h ee s uc u al elemen s: (i) a η 5/6 -a ene/yl ligand which s abilizes he oxida ion s a e o he me al ca ion and can acili a e anspo h ough a cell memb ane; (ii) a monoden a e ligand, o en (bu no necessa ily) a lea ing g oup (X, ypically a halido ligand bu also o he s) 15 which eadily dissocia es o allow coo dina ion o he me al a om by he a ge biomolecules (iii) and an auxilia y ligand L which can egula e he eac i i y o he complex molecule o a ious biomolecules (DNA, enzymes) and e en play a key ole in he in e ac ions wi h hem h ough hyd ogen bonds o in e cala- ion. Fu he mo e, he o e all cha ge and coun e ion iden i y a e o he ac o s which could affec solubili y, cell up ake, in acellula me abolism and gene ally, he a e o he com- plexes in he biological en i onmen . 16–18 The main aim o his s udy was o p epa e new Ru(II) and I (III) hal -sandwich complexes wi h he gene al o mulas [Ru(η 6 -pcym)(L1)X]PF 6 and [I (η 5 -Cp*)(L1)X]PF 6 , whe e pcym is 1-me hyl-4-(p opan-2-yl)benzene (pa a-cymene), Cp* is pen a- me hylcyclopen adienyl, X = Cl − o I − and L1 is a biden a e N-dono ligand de i ed om hiadiazole, 2-( u an-2-yl)-5- (py idin-2-yl)-1,3,4- hiadiazole (Fig. 1). Compounds in ol ing hiadiazole ings as scaffolds ha e been o g ea in e es as co e s uc u es o an i umo agen s due o hei high eac i i y and he p esence o a oxopho ic N–C–S moie y. 19 Di e se modi ica ions o he hiadiazole ings in a ious posi ions ha e led o a a ie y o no el compounds wi h a wide spec um o pha macological ac i i ies, such as an i ungal, 20 an ibac e ial, 21 an i i al, 22 an i-in lamma o y, 23 analgesic, 24 an ihelmin ic 25 and o pa icula in e es is he imp essi e an icance /an i umo ac i i y. 26–29 Se e al pa en s ha e been egis e ed since 2008 conce ning new hiadiazole ing-con ain- ing de i a i es use ul o he de elopmen o new an icance d ug molecules. 30 To he bes o ou knowledge, only wo wo ks ha e epo ed on he biological in es iga ion o Ru(II) hal -sandwich complexes in ol ing hiadiazole-based ligands. The s udies ocused on Ru(II) complexes wi h a ca bonic anhy- d ase inhibi o ace azolamide, which we e ound o be inac i e in i o on all es ed cell lines, 31 howe e showed o be e y po en inhibi o s o umou -associa ed ca bonic anhyd ase iso- o ms. 32 The hiadiazole de i a i e in his wo k con ains wo he e ocyclic subs i uen s, i.e. he py idine and u an ings. Py idine is p esen o enable a biden a e N,N-coo dina ion mode. This mo i has been equen ly used in hal -sandwich u henium/i idium complexes as an N-dono pa o biden a e ligands, such as bipy idine, azopy idine, 2-phenylpy idine, picolina e, o as a e minal monoden a e ligand as a pa o s uc u e–ac i i y s udies, pa icula ly, in he pionee ing wo ks o Sadle e al. 14,33–35 The u anyl moie y b ings ano he he e ocyclic unc ionali y and as he non-coo dina ing one, i could enable a a ied a ay o non-co alen con ac s due o i s a oma ic ing and he e oa om. Addi ionally, he u an ing is also an in e es ing moie y om he medicinal poin o iew, as mul iple clinically app o ed pha maceu icals, e.g. wi h an i- mic obial, an i i al, an i-in lamma o y, an i-ageing, and an i- cance p ope ies, con ain his he e ocycle in hei s uc u es. 36 Fu he mo e, ano he objec i e o his s udy was o in es i- ga e changes in he biological and chemical p ope ies (e.g. solu ion s abili y, an ip oli e a i e ac i i y and cy o oxici y) o he p epa ed complexes upon eplacemen o he chlo ido by he iodido ligand. In p e ious wo ks on a ious hal -sandwich complexes, 33,37,38 i was shown ha a ele an diffe ence in biological p ope ies can be achie ed by such s uc u al a i- a ion. This wo k hus epo s on hal -sandwich complexes [M(η 6 /η 5 -a ene/yl)(L1)X]PF 6 in which he in luence o wo a i- ables, i.e. M = Ru/I , X = Cl/I, on s uc u al p ope ies, solu ion s abili y and an ip oli e a i e ac i i y was in es iga ed. Resul s Syn hesis and basic cha ac e iza ion The hiadiazole-based compound L1 (2-( u an-2-yl)-5-(py idin- 2-yl)-1,3,4- hiadiazole) used in his wo k was p epa ed by a wo-s ep syn he ic p ocedu e (Scheme 1), which was inspi ed by p e iously published p o ocols. 39,40 Fi s , picolinic acid and 2- u oic hyd azide we e con e ed ia a 1,1′-ca bonyldiimidazole coupling eagen in o N-2- u anyl-N‘-picolinoylhyd azine(I) in dichlo ome hane a oom empe a u e. Second, Lawesson’s eagen was employed in he cycliza ion eac ion o I unde e lux and a ni ogen a mo- sphe e in chlo o o m o yield L1 (Scheme 1). Compounds 1–4we e p epa ed by he eac ion be ween he co esponding dime ic complex p ecu so s [M(μ-Cl)(η 6 /η 5 - a ene/yl)Cl] 2 and L1, ollowed by he addi ion o he s abilizing PF 6− coun e anions (NH 4 PF 6 ). Du ing he p epa a ion o 3 Fig. 1 S uc u al o mulas o [Ru(η 6 -pcym)(L1)X]PF 6 (le ) and [I (η 5 -Cp*) (L1)X]PF 6 ( igh ) complexes, whe e X = Cl − (1,2)o I − (3,4). Pape Dal on T ansac ions 12718 |Dal on T ans.,2023,52, 12717–12732 This jou nal is © The Royal Socie y o Chemis y 2023 Open Access A icle. Published on 11 Augus 2023. Downloaded on 2/19/2024 1:17:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online and 4, he chlo ide anions we e p ecipi a ed by he addi ion o sil e i la e, and hen we e subs i u ed by iodide anions (KI) and again, mic oc ys alline p oduc s we e isola ed a e he addi ion o PF 6− coun e anions. Compounds 1–4we e sligh ly soluble in wa e bu we e well soluble in o ganic media, such as N,N-dime hyl o mamide (DMF), dime hyl sul oxide (DMSO), me hanol, e hanol o ace one. The pu i y and s uc- u e o L1 and esul ing complexes 1–4we e s udied and con- i med by elemen al analysis, mass spec ome y, FTIR, and NMR spec oscopy and c ys al s uc u es we e de e mined by single c ys al X- ay diff ac ion. The ESI+ mass spec a con ained peaks wi h m/z alues and iso opic dis ibu ions a ibu able o he ions (e.g. {[Ru(pcym) (L1)]-H} + , {[I (Cp*)(L1)]-H} + , [Ru(pcym)(L1)X] + , [I (Cp*)(L1)X] + ) ag eeing well wi h he p oposed o mulas o he complex ca ions in 1–4(Fig. S1–S4†). The iden i y and pu i y o he ligand and complexes we e in es iga ed using high esolu ion 1 H and 13 C NMR spec- oscopy (see he ESI, Fig. S5–S12†). The 1 H signals in L1 we e all signi ican ly shi ed down ield upon coo dina ion wi h he me al a oms (see ESI, Fig. S10†). The mos signi ican change was obse ed o he signal assigned o he C13H hyd ogen, adjacen o he coo dina ion si e, i.e. py idine ni ogen, whe e he coo dina ion shi , Δδ=δ complex −δ ligand , equalled 0.91 (1), 0.36 (2) 0.86 (3) and 0.38 ppm (4). La ge shi s we e also calcu- la ed o he hyd ogen C16H wi h Δδ anging be ween 0.39 and 0.49 ppm. In he ca bon NMR spec a, which also showed signi ican shi ing o mos o he signals wi h espec o he spec um o L1, he highes Δδs we e obse ed o he signal o C16, i.e. 6.3–7.0 ppm down ield. Then he chemical shi o C13 also changed ma kedly, ye mo e so o Ru(II) complexes 1 and 3(∼6 ppm) han o I (III) complexes 2and 4(∼3 ppm). Simila ly, signi ican up ield shi s o ca. 4.5 ppm, and 2.5 ppm in he spec a o Ru(II) and I (III) complexes, espec - i ely, we e obse ed o he signals co esponding o he qua- e na y ca bon C5, which lies in he icini y o ni ogen N4, i.e. he coo dina ion si e on he 1,3,4- hiadiazole ing. C ys al s uc u es Single c ys als we e ob ained o all he he ein epo ed coo di- na ion compounds 1–4and hei c ys al s uc u es we e de e - mined using single-c ys al X- ay diff ac ion analysis (Table S1†). The coo dina ion compounds consis o he complex ca ions and PF 6− anions. All he complex ca ions con ain he biden a e L1 ligand, η 6 -pcym (1and 3)o η 5 -Cp* (2 and 4) ligands and monoden a e halido ligands X (X = Cl − in 1 and 2,I − in 3and 4). The o e all coo dina ion geome y can be desc ibed bes as h ee-legged piano s ool pseudooc ahed al. The longes me al–ligand (M–L) bond leng hs we e obse ed o bonds wi h he halides (in Å, 2.394(2) in 1, 2.3857(11) in 2, 2.6986(4) in 3and 2.6445(9) in 4), while he M–N bonds we e signi ican ly sho e (2.05–2.12 Å, Fig. 2). The dis ances be ween he cen oids o he a ene/yl ligands and me al a oms a e sho e in he Ru complex ca ions (1.69 in 1and 3 s. 1.78 Å in 2and 4). The non-co alen in e ac ions in he c ys al s uc u es o 1–4a e mos ly o weak na u e, mainly he C–H⋯π,C–H⋯S, C–H⋯F, C–H⋯Cl (1and 2), C–H⋯I(3and 4) hyd ogen bonds. Signi ican non-co alen in e ac ions a e summa ized in ESI (Fig. S13–S16†) and some o he selec ed in e ac ions a e discussed in g ea e de ail in he Discussion pa ag aph ( ide in a). Lipophilici y s udies Cy o oxici y and he abili y o d ugs o en e cells o en co e- la e wi h hei lipophilici y (hyd ophobici y). Thus, one o he Scheme 1 P epa a ion o 2-( u an-2-yl)-5-(py idin-2-yl)-1,3,4- hiadi- azole (L1) ia a wo-s ep eac ion p ocedu e: (a) 2- u oic hyd azide, di- chlo ome hane, oom empe a u e and (b) Lawesson’s eagen , chlo o- o m, eflux, o e nigh , ni ogen a mosphe e, gi en wi h he a om num- be ing scheme. PA = py idine-2-ca boxylic acid and I = N‘-( u an-2-ca - bonyl)py idine-2-ca bohyd azide. Fig. 2 A pe spec i e iew illus a ing he molecula s uc u es o he complex ca ions [Ru(η 6 -pcym)(L1)Cl] + (A, complex 1), [I (η 5 -Cp*)(L1)Cl] + (B, 2), [Ru(η 6 -pcym)(L1)I] + (C, 3), and [I (η 5 -Cp*)(L1)I] + (D, 4). Hyd ogen a oms ha e been omi ed o cla i y. The colou code used is as ollows: ligh g ey (ca bon), g een (chlo ine), iole (iodine), da k blue (i idium), u quoise ( u henium), ligh blue (ni ogen), ed (oxygen), yellow (sulphu ). Selec ed bond leng hs (in Å): 1(A), d(Ru1–N1) = 2.052(10), d(Ru1–N3) = 2.116(10), d(Ru1–Cl1) = 2.394(2); 2(B), d(I 1–N1) = 2.063 (4), d(I 1–N3) = 2.114(4), d(I 1–Cl1) = 2.3857(11); 3(C), d(Ru–N1) = 2.063 (3), d(Ru1–N3) = 2.120(3), d(Ru1–I1) = 2.6986(4); and 4(D), d(I 1–N1) = 2.054(8), d(I 1–N3) = 2.093(8), d(I 1–I1) = 2.6445(9). Dal on T ansac ions Pape This jou nal is © The Royal Socie y o Chemis y 2023 Dal on T ans.,2023,52, 12717–12732 | 12719 Open Access A icle. Published on 11 Augus 2023. Downloaded on 2/19/2024 1:17:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online possible explana ions o diffe en cy o oxici y o 1–4(see below) may be based on hei diffe en lipophilici y, which is ela ed o hei abili y o en e cance cells. Lipophilici y can be de e mined by he oc anol/wa e pa i ion coefficien (log P), which was calcula ed o all s udied compounds: log P =−0.96 ± 0.03 ( o 1), −0.25 ± 0.01 ( o 2), −0.03 ± 0.02 ( o 3) and −0.07 ± 0.01 ( o 4). The ob ained esul s showed ha he iodido compounds (3and 4) we e mo e lipophilic han he chlo ido ones (1and 2). Compounds 1–4we e mo e lipophilic han cispla in (CDDP, −2.21 ± 0.1). 41 Solu ion s abili y Solu ion s abili y o 1–4was in es iga ed by 1 H NMR spec- oscopy in wa e con aining sol en mix u es o 150 µL o MeOD-d 4 and 350 µL o D 2 O (SM1) and in 150 µL o MeOD-d 4 and 350 µL o D 2 O wi h he addi ion o PBS (SM2, pH = 7.4, PBS s ands o phospha e-buffe ed saline, concen a ion o he chlo ide anions in SM2 was 98 mM) (Fig. S17–S24†). In he sol en mix u es SM1 and SM2, 1–3showed o e all hyd oly ic s abili y because hei spec a did no change o e ime. No new signals eme ged up o 48 h. To exclude immedi- a e hyd olysis, he ob ained spec a we e compa ed o hose o dehalogena ed complexes 1 h and 2 h , which unambiguously p o ed no occu ence o hyd olysis. The ob ained signals co e- sponded nei he o he signals o 1 h and 2 h , no o hose o ligand L1 measu ed unde he same condi ions. Analogically, in SM1 compound 4was hyd oly ically s able. In con as , hough, new signals appea ed in he spec um o 4dissol ed in SM2. The chemical shi s o he new se o signals ag eed pe ec ly wi h he signals o he chlo ido analogue, i.e. complex 2(Fig. 3). Many signals we e o e lapping, esul ing in b oad un esol ed mul iple s, howe e , disc e e new signals appea ed o C15H a 8.33 ppm (a 8.27 ppm o he iodido complex and 8.34 ppm o 2) and o C14H a 7.91 ppm (a 7.82 ppm o 4and 7.92 ppm o 2) as well as o Cp* hyd o- gens a 1.77 ppm (a 1.87 ppm o 4and 1.77 ppm o 2). The e o e, in he p esence o chlo ide anions in solu ion, complex 4unde goes g adual iodido/chlo ido ligand exchange. The con e sion a e o he ligand exchange o 4in SM2 was 40% a e s anding a oom empe a u e o 48 h. In e ac ion wi h GSH Reduced glu a hione (GSH) is a ipep ide, which o en coo di- na es o me al cen es o complex molecules and is in ol ed in he de oxi ica ion o many an icance me allod ugs. 42,43 Impo an ly, i plays a i al ole in he edox balance in he cell, he e o e any in e e ence wi h he equilib ium be ween GSH and i s oxidized o m GSSG (GSSG = glu a hione disul- phide) can esul in pa hological changes in cellula me abolism. 42,44 Possible in e ac ions wi h GSH o 1–4we e s udied in a sol en mix u e (SM3) o 150 µL o MeOD-d 4 and 350 µL o D 2 O wi h PBS (pH = 7.4) wi h 5 mola equi alen s o GSH. The s uc u al ea u es o complexes 1–3 emained unal- e ed because he posi ions o signals in hei spec a did no change o e ime. In con as , new signals appea ed in he spec um o 4, as i exhibi ed he same changes as desc ibed abo e in SM2 (i.e. he iodido/chlo ido ligand exchange). The p esence o GSH did no affec he con e sion a e o he ligand exchange signi ican ly, which was ca. 35% a e 48 h. In addi ion, as a as any co alen in e ac ions and/o GSH- ela ed ligand exchange eac ions a e conce ned, he esul s showed ha none o hese occu ed in he in e ac ion sys em, as e idenced by he unal e ed alipha ic pa o he 1 H NMR spec a o e 48 h. Simila ly, complexes 1–4we e ca aly ically inac i e in he GSH- o-GSSG oxida ion eac ion, since negli- gible (1) o no GSH ans o ma ion was e idenced in he NMR s udy (Fig. S25–S32†). In e es ingly, wi h espec o he spec- um o GSH alone in he same medium, he posi ion o wo signals o GSH be ween 3.5 and 4.0 ppm in he spec a o all he in e ac ion mix u es was shi ed al eady a 0 h. These wo shi ed signals belong o he p o ons a ached o ca bons di ec ly neighbou ing wi h he wo e minal COOH g oups o GSH. Since no co alen in e ac ions and/o GSH- ela ed ligand exchange eac ions we e de ec ed in he spec a, i could be sugges ed ha he shi o he signals is ela ed o he immedi- a e o ma ion o a diffe en a ay o non-co alen in e ac ions o he COOH g oups o GSH wi h he complexes p esen in he mix u es. Analogical shi s o he same GSH p o ons we e obse ed by Y. Q. Hao e al. 45 In e ac ion wi h NADH The NADH/NAD + sys em is indispensable o cellula me ab- olism, as i plays mul iple c ucial oles especially in many enzyma ic e en s in cells. The abili y o u henium and i idium complexes o oxidize NADH o o m NAD + has been epo ed in se e al s udies. 46–48 Diso de ing o he NADH o NAD + a io can lead o he dis up ion o a ious me abolic e en s e en ually esul ing in cell dea h. 49 In a mix u e (SM4) Fig. 3 1 H NMR s abili y s udy o complex 4in SM2 (30% MeOD-d 4 /70% D 2 O wi h PBS), as obse ed a diffe en ime poin s (0 h o 48 h). The g ey a ea shows he 1 H NMR spec um o 4a e 48 h confi ming he occu ence o I − →Cl − ligand exchange wi h ligh blue colou deno ing he signals o he o iginal iodido complex and wi h yellow o he chlo - ido analogue. O he signals a e no colou ed due o o e lap. Fo com- pa a i e pu poses, he 1 H spec um o chlo ido complex 2in he same sol en mix u e is shown ( op). Pape Dal on T ansac ions 12720 |Dal on T ans.,2023,52, 12717–12732 This jou nal is © The Royal Socie y o Chemis y 2023 Open Access A icle. Published on 11 Augus 2023. Downloaded on 2/19/2024 1:17:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online o 150 µL o MeOD-d 4 and 350 µL o D 2 O wi h PBS (pH = 7.4) and 5 mola equi alen s o NADH, complexes 1–3 emained in ac and he posi ions o hei signals in he spec a did no change o e ime up o 48 h (Fig. S33–S35†). On he o he hand, simila o he abo e p esen ed esul s, due o he p es- ence o chlo ide anions in he solu ion, he s abili y o 4was lowe and again, new signals co esponding o chlo ido complex 2 esul ing om he ligand exchange we e obse ed in he ob ained spec a. No ably, he p esence o NADH in he in e ac ion mix u e somewha affec ed he iodido/chlo ido exchange, whose con e sion a e was 47% a e 48 h (Fig. 4). In he spec a o all compounds new signals con i ming he oxida ion o NADH o NAD + we e obse ed. In e es ingly, u henium complexes 1and 3exhibi ed signi ican ly lowe abili y o oxidize NADH (i.e. 14 and 10% NADH oxida ion a e 48 h, espec i ely) han hei i idium congene s 2and 4(28% and 32%). In o he wo ds, he ca aly ic efficiency o 1–4 owa ds he NADH oxida ion can be exp essed as being ca. 0.7, 1.4, 0.5 and 1.6 mola equi . pe mol o a complex a e 48 h, espec i ely. We did no de ec he cha ac e is ic hyd ido signal in he high- ield egion o he 1 H NMR spec a. P e iously epo ed hal -sandwich Ru(II) and I (III) complexes (wi h py idine de i ed ligands) we e also obse ed as po en oxidan s o NADH. 47,50,51 De e mina ion o IC 50 alues and effec s o es ed complexes on me abolic ac i i y in heal hy CCD-18Co and CCD-1072Sk human ib oblas s To de e mine he IC 50 (hal maximal inhibi o y concen a ion) alues o es ed complexes in he CCD-18Co colon and CCD-1072Sk o eskin ib oblas s we used he well-es ablished MTT assay. The MTT assay was pe o med 24 and 48 h a e he exposu e o he cells o es ed complexes. As he e e ence d ug, we used CDDP, a well-known chemo he apeu ic agen , which is gene ally used in he ea men o o a ian cance . The es ima ed IC 50 alues de i ed om mean me abolic ac i i y a e shown in Table 1. Based on he ob ained esul s, i idium complexes 2and 4which had he weakes ac i i y agains ib oblas s we e chosen o subsequen expe imen s ealized on o a ian cance cells. Complexes 1and 3showed an inhibi o y effec agains heal hy cells; ne e heless, complex 1 was also in ol ed in u he s udies on he cance cell lines o compa a i e pu poses. De e mina ion o IC 50 alues and effec o es ed complexes on he p oli e a ion o A2780 and A2780cis o a ian ca cinoma cells Complexes 1,2and 4we e chosen o a s udy o a po en ial effec on p oli e a ion and induc ion o cell dea h in CDDP- sensi i e A2780 and CDDP- esis an A2780cis o a ian ca ci- noma cells. A i s , we de e mined he IC 50 alues o selec ed complexes by he MTT assay. The MTT assay was pe o med 24 and 48 h a e exposu e o he cells o es ed complexes. We used CDDP as he e e ence d ug and he es ima ed IC 50 alues de i ed om mean me abolic ac i i y a e shown in Table 2. The lowes IC 50 alues we e ob ained o 1and 4. The IC 50 alues o 2we e no de ined; he e o e, his compound was excluded om subsequen analyses. De e mina ion o IC 50 alues in heal hy and cance cell lines allowed he calcula ion o he selec i i y index, SI = (IC 50 (CCD-18Co)/IC 50 (A2780), which was mo e a ou able o complexes 1and 4wi h SI > 5.8 and SI > 3.4, espec i ely, as compa ed o 2.6 o CDDP. On he o he hand, wi h he second used heal hy cell line, i.e. SI 2 = (IC 50 (CCD-1072Sk)/ IC 50 (A2780), complex 1was ound non-selec i e wi h SI 2 = 0.4, in con as wi h >3.4 (4) and 6.2 (CDDP). Addi ionally, diffe - ences in IC 50 alues agains sensi i e and esis an cell lines enabled he calcula ion o he esis ance ac o RF, de ined as RF = IC 50 (A2780cis)/IC 50 (A2780), which equals 1.4 (1), 1.6 (4) and 2.8 (CDDP). The impac o complexes 1and 4on cell p oli e a ion was assessed by e alua ion o me abolic ac i i y (Fig. S36†), cell cycle dis ibu ion (Table 3) and o al cell numbe (Fig. 5). Tes ed complexes showed a ime- and dose-dependen inhibi- o y effec on he me abolic ac i i y o bo h cance cell lines. Howe e , a s onge effec o es ed complexes was obse ed in CDDP-sensi i e A2780 cance cells (Table 2, Fig. S36A and C†). Fig. 4 1 H NMR in es iga ion o ep esen a i e compound 1in SM4 (30% MeOD-d 4 /70% D 2 O wi h PBS+ 5 mola equi alen s o NADH), as obse ed a diffe en ime poin s (0 h o 48 h). As e isks deno e he signals co esponding o NAD + o igina ing om he oxida ion o NADH in he mix u e wi h 1(blue a eas). Fo compa ison pu poses, he 1 H spec a o NADH and NAD + a e shown ( op). Table 1 The IC 50 alues (µM) o es ed complexes in CCD-18Co and CCD-1072Sk fib oblas s CCD-18Co CCD-1072Sk 24 h 48 h 24 h 48 h 1 4.31 ± 1.83 >50 1.92 ± 1.02 3.29 ± 0.88 2 >50 >50 >50 >50 3 3.18 ± 1.33 >50 2.18 ± 1.27 2.63 ± 0.12 4 >50 >50 >50 >50 CDDP >50 11.03 ± 0.62 >50 26.39 ± 8.22 Dal on T ansac ions Pape This jou nal is © The Royal Socie y o Chemis y 2023 Dal on T ans.,2023,52, 12717–12732 | 12721 Open Access A icle. Published on 11 Augus 2023. Downloaded on 2/19/2024 1:17:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online Analyses o he o al cell numbe and cell cycle dis ibu ion we e pe o med 48 h a e incuba ion o he cells wi h 5 and 25 µM o complexes 1and 4, and CDDP. Analogously o he esul s om he me abolic ac i i y s udies, we obse ed a dose- dependen dec ease in he o al cell numbe in bo h cance cell lines. The effec o es ed complexes was weake in CDDP- esis an A2780cis cells in compa ison wi h CDDP-sensi i e A2780 cance cells (Fig. 5). Flow cy ome ic analysis o cell cycle dis ibu ion e ealed ha he obse ed an ip oli e a i e effec o es ed complexes agains CDDP- esis an cells was accompanied by inc eased accumula ion o cells in he G1 phase o he cell cycle. These changes we e a ended by he educ ion o cell popula ion in he S phase o he cell cycle. In he case o complex 1, dec eased pe cen age o cells in he G2/M phase o he cell cycle was also obse ed. A e he exposu e o CDDP-sensi i e A2780 cance cells o es ed com- plexes we did no obse e signi ican changes in he cell cycle dis ibu ion (Table 3). Effec o complexes 1 and 4 on he induc ion o cell dea h in A2780 and A2780cis o a ian ca cinoma cells To de e mine whe he he an ip oli e a i e effec o complexes 1and 4was associa ed wi h he onse o cell dea h, we ana- lysed cell iabili y and mi ochond ial memb ane depola iz- a ion. The analyses we e pe o med 48 h a e incuba ion o he cells wi h 5 and 25 µM o complexes 1and 4, and CDDP. Howe e , we did no obse e any effec on he iabili y o mi o- chond ial memb ane po en ial o used cance cells a e exposu e o es ed complexes. Signi ican changes in iabili y and mi ochond ial memb ane depola iza ion we e achie ed only a e he ea men o bo h cance cell lines wi h CDDP (Fig. S37†). Discussion Compu a ional e alua ion o s uc u es I is well es ablished ha he s uc u al and elec onic p o- pe ies o molecules de e mine hei biological and pha maco- logical p ope ies. 52 Fu he mo e, any s uc u al modi ica ion leads o he o ma ion o a diffe en a ay o non-co alen in e ac ions. This may be o c ucial impo ance in he biologi- cal en i onmen , as a ied non-co alen in e ac ions o a sui - able d ug wi h i s a ge molecule esul in unc ional modi i- ca ion o he biologically ele an molecules wi h diffe en con- sequences o cellula me abolism. 53 The e o e, we ook a close look a s uc u es and ene ge ically a ailable s uc u al modi ica ions o 1–4. The c ys al s uc u es we e de e mined by single c ys al X- ay analysis o all he epo ed compounds, which enabled mu ual compa ison be ween o ganic compound L1/complexes, Table 2 The IC 50 alues (µM) o selec ed complexes in A2780 and A2780cis o a ian ca cinoma cell lines A2780 A2780cis 24 h 48 h 24 h 48 h 1 14.90 ± 5.21 8.69 ± 1.75 16.11 ± 5.79 12.48 ± 4.83 2 >25 >25 >25 >25 4 >25 14.70 ± 6.72 >25 23.36 ± 1.03 CDDP 20.35 ± 1.39 4.27 ± 0.70 >25 11.96 ± 2.71 Fig. 5 The effec o 1,4and CDDP on he o al cell numbe o A2780 and A2780cis o a ian ca cinoma cell lines. The o al cell numbe was analysed 48 h a e ea men o cells wi h selec ed complexes. The expe imen al g oups we e compa ed wi h he un ea ed con ol (*p< 0.05, ** p< 0.01, *** p< 0.001). Table 3 The effec o 1,4and CDDP on cell cycle dis ibu ion o A2780 and A2780cis o a ian ca cinoma cell lines. Changes in he cell cycle dis i- bu ion [%] we e analysed 48 h a e ea men o cells wi h he selec ed complexes. The expe imen al g oups we e compa ed wi h he un ea ed con ol (* p< 0.05, ** p< 0.01, *** p< 0.001) A2780 A2780cis G0/G1 S G2/M G0/G1 S G2/M Con ol 64.60 ± 4.27 23.34 ± 2.33 12.06 ± 2.09 58.10 ± 1.07 28.69 ± 1.30 13.21 ± 0.24 DMSO (0.25%) 64.33 ± 5.36 24.14 ± 2.81 11.53 ± 2.99 59.01 ± 0.70 27.94 ± 0.49 13.04 ± 0.51 1 (5 µM) 65.88 ± 6.10 23.50 ± 3.39 10.63 ± 2.77 58.59 ± 1.65 28.25 ± 1.14 13.16 ± 0.51 1 (25 µM) 71.42 ± 5.56 18.40 ± 3.74 10.18 ± 2.06 63.66 ± 0.96** 25.50 ± 0.84* 10.84 ± 0.73* 4 (5 µM) 62.41 ± 6.01 26.53 ± 3.25 11.07 ± 2.96 58.02 ± 0.56 29.23 ± 0.25 12.76 ± 0.50 4 (25 µM) 72.61 ± 5.51 19.50 ± 2.94 7.90 ± 2.61 63.51 ± 1.22** 24.89 ± 0.47* 11.60 ± 0.83 CDDP (5 µM) 9.49 ± 1.02*** 85.21 ± 7.22*** 5.30 ± 7.20 35.77 ± 3.22* 30.63 ± 0.31 33.60 ± 3.07*** CDDP (25 µM) 66.23 ± 6.05 27.39 ± 1.99 6.38 ± 5.27 24.49 ± 15.82*** 58.28 ± 17.79** 17.23 ± 2.14 Pape Dal on T ansac ions 12722 |Dal on T ans.,2023,52, 12717–12732 This jou nal is © The Royal Socie y o Chemis y 2023 Open Access A icle. Published on 11 Augus 2023. Downloaded on 2/19/2024 1:17:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online Ru/I complexes and chlo ido/iodido complexes. A e analys- ing he c ys al s uc u es o compounds 1–4, he mos s iking diffe ence was ound in he con o ma ion o ligand L1 in Ru/I complexes, which is e idenced by he o ien a ion o he u anyl ings. In I (III) compounds 2and 4, he u anyl ings a e o ien ed owa ds he me al cen e and he hiadiazole ni ogen a oms ( u he abb e ia ed as he Zcon o ma ion), whe eas in Ru(II) compounds 1and 3, hey a e o ien ed away om he me al cen e (Econ o ma ion, see Fig. 2). The diffe - ence in he o ien a ion could po en ially be a ibu ed o he diffe en a yl ligands p esen in compounds 1–4(η 6 -pcym in 1 and 3,η 5 -Cp* in 2and 4). Howe e , i is appa en ha he a yl ligands do no induce any s e ic hind ance. In ac , some o he me hyl g oups on he a yl ligands pa icipa e in weak in a- molecula C–H⋯N hyd ogen bonding wi h he hiadiazole ni ogen a om, s abilizing he s uc u e o he molecule by one (1and 3) o by a bi u ca ed pai (2and 4) o hyd ogen bonds. The s eng hs o he non-co alen in e ac ions we e e alua ed employing he quan um heo y o a oms in molecules (QT-AIM) by means o in e ac ion ene gy (E in ), and wa e unc- ions we e calcula ed by DFT heo y using he ORCA 4.2.1 p og am. 54 The calcula ions we e pe o med as single-poin con e gence on he app op ia e agmen s o he c ys al s uc- u e a B3LYP and ZORA-de 2-TZVP le els o heo y (special basis se s: old-ZORA-TZVP o Ru and I, SARC-ZORA-TZVP o I ). 55 All QT-AIM calcula ions we e pe o med using he Mul iw n package. 56 I was e ealed ha E in o hese con ac s is compa able o all he in amolecula C–H⋯N hyd ogen bonds (1.5–2.3 kcal mol −1 ) wi h he weake second in e ac ions in he bi u ca ed pai s (kcal mol −1 , 0.86 in 2and 0.72 in 4). Impo an ly, he p esence o he PF 6− anion induces signi i- can non-co alen in e ac ions in 1–4. Apa om he weak C–H⋯F in e ac ions obse ed in all compounds, each PF 6− anion o ms ei he one (3) o wo (1,2, and 4)F⋯S in e ac ions wi h he hiadiazole sulphu a om om he L1 ligand (Fig. 6A and he ESI, Fig. S13†). Al hough he F⋯S dis ances in hese con ac s a e ela i ely long, hey a e mos ly sho e han he sum o he an de Waals adii (∑R dw (F,S) = 3.27 Å, see ESI Fig. S13†): d(F⋯S, in Å) = 1, 3.246(7), 3.422(6); 2, 3.024(3), 3.156(4); 3, 3.212(4); and 4, 3.025(7), 3.156(8). The in e ac ion ene gies o hese con ac s ange om 0.7 o 1.9 kcal mol −1 . The calcula ions pe o med using he non-co alen in e ac ion (NCI) me hod 57 con i m ha hese in e ac ions a e indeed weakly a ac i e (see ESI Fig. S13†). In e es ingly, in he mole- cules adop ing he Zcon o ma ion (2, and 4) he hyd ogen a om in he i h posi ion o he u anyl ing o ms a weak C–H⋯F in e ac ion wi h he PF 6− anion (in Å): 2,d(C19⋯F5) = 3.434(6), 4,d(C19⋯F3) = 3.459(8). Ano he signi ican dis inc- ion be ween he c ys al packing o he Eand Zcompounds is he p esence o he cen osymme ic R 22 (6) 58 syn hon, which is o med by hyd ogen bonding be ween neighbou ing u anyl moie ies h ough C–H⋯O in e ac ions (Fig. 6B and see ESI Fig. S14†). These in e ac ions a e ela i ely weak wi h long dono ⋯accep o dis ances (in Å, 3.406(7) in 2, 3.580(18) in 4) Fig. 6 (A) Pe spec i e iew illus a ing he non-co alen in e ac ions (black dashed lines) be ween he PF 6− anion and he complex ca ion in he c ys al s uc u e o 4. (B) Pe spec i e iew highligh ing he in e ac ions be ween he u anyl ings and iodido ligands. Hyd ogen a oms, excep o hose in ol ed in non-co alen in e ac ions, ha e been omi ed o cla i y. (C) G aphical compa ison displaying he ela i e elec onic ene gies o he g ound s a es o E/Z-isome s o L1 and 1–4, along wi h he ansi ion s a es (TS #1 and TS #2 ) de i ed om DFT calcula ions. The colou code used is as ollows: ligh g ey (ca bon), g een (chlo ine), iole (iodine), da k blue (i idium), u quoise ( u henium), ligh blue (ni ogen), ed (oxygen), yellow (sulphu ). Selec ed leng hs o non-co alen in e ac ions (in Å): (A), d(C7⋯N2) = 3.422(8), d(C14⋯F5) = 3.660(6), d(C19⋯F3) = 3.459(8), d(F3⋯S1) = 3.156(8), d(F5⋯S1) = 3.025(7) and (B) d(C21⋯O1) = 3.580(18), d(I1⋯O1) = 3.633(8). Dal on T ansac ions Pape This jou nal is © The Royal Socie y o Chemis y 2023 Dal on T ans.,2023,52, 12717–12732 | 12723 Open Access A icle. Published on 11 Augus 2023. Downloaded on 2/19/2024 1:17:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online esul ing in low E in (1.10 kcal mol −1 in 2and 0.50 kcal mol −1 in 4). No ably, he o ma ion o his syn hon is also suppo ed by in e ac ions wi h he halido ligands. In 2, a pai o Cl⋯O con ac s a e o med wi h d(Cl⋯O) = 3.670(4) Å, while I⋯O con- ac s in 4a e sligh ly sho e wi h a dis ance o d(I⋯O) = 3.633 (8) Å. Howe e , calcula ions using he elec on localiza ion unc ion (ELF) 59 indica e ha hese in e ac ions a e no elian on he o ma ion o a σ-hole on he halogen a om (see ESI Fig. S15†). 60 Consequen ly, i is unsu p ising ha hese in e - ac ions a e only weakly a ac i e, as e iden om he NCI plo s (see ESI Fig. S15†), and exhibi ela i ely low calcula ed E in (0.40 kcal mol −1 in 2and 0.86 kcal mol −1 in 4). As he o ien a ion o he u anyl ing is diffe en in he c ys al s uc u es o 1–4, he e a e E(dihed al angle S–C–C–O close o 0°) and Z(dihed al angle S–C–C–O close o 180°) isome s o L1 obse ed in hese complexes, we decided o in es iga e his phenomenon also by heo e ical me hods employing densi y unc ional heo y (DFT). He ein, ORCA 5.0 so wa e was u ilized, 61 and 2 SCAN unc ional 62 was applied o all calcula ions oge he wi h he a om-pai wise dispe sion co ec ion (D4). 63 The geome y op imiza ion was ca ied ou in wa e wi h he C-PCM implici sol a ion model 64,65 and again, Ahl ichs de 2-TZVP basis se was used o all a oms, wi h ECP o Ru, I and I. 66 The ela i e ene gies we e in es i- ga ed o bo h E- and Z-isome o L1 and complexes 1–4.I is e iden ha he lowes elec onic ene gy is ound o he E-isome s o all compounds, and he Z-isome s ha e ene gy highe by 2–3 kJ mol −1 (Fig. 6c and see ESI Fig. S16†). The selec ed bond dis ances o op imized molecula geome ies a e lis ed in Table S2†and show nice ag eemen wi h he X- ay da a, con i ming good pe o mance o he selec ed heo e ical me hod. The da a a e also supplemen ed by Maye bond o de s. Gene ally, he e is a e y small a ia ion in Ru–N and I –N bond dis ances and Maye bond o de s wi hin 1–4. Howe e , Maye bond o de s a e signi ican ly lowe o I –X(X = Cl and I) bonds in 2(0.774) and 4(0.668) compa ed o Ru–X bonds in 1(0.937) and 3(0.934), espec i ely. In pa icula , he I –I bond in 4is e iden ly much weake , which is in acco d- ance wi h he lowe solu ion s abili y o his complex o halogen-ligand subs i u ion (see he Solu ion s abili y sec ion). Mo eo e , in all cases, he ansi ion s a es (TS #1 and TS #2 ) we e iden i ied and con i med by he p esence o one imagin- a y equency. The ac i a ion ene gy (ene gy ba ie ) o he E–Zisome ic eac ion was ound close o 34–35 kJ mol −1 o 1–4and sligh ly lowe o he compound L1 i sel , close o 29–30 kJ mol −1 , which means ha he kine ics o such eac ion is slowed down by he coo dina ion o L1. In summa y, he s uc u al and compu a ional in es i- ga ions demons a e ha he o ien a ion o he u anyl ings in 1–4is dominan ly go e ned by he collec i e in luence o weak non-co alen in e ac ions wi hin he c ys al s uc u e, a he han in amolecula in e ac ions. Biological s udies As demons a ed p e iously, he s uc u e–ac i i y modi i- ca ions o his ype o Ru(II) and I (III) wi h he gene al o mula [M(η 6 /η 5 -a ene/yl)(L)X] 0/n+ can be achie ed by he choice o bo h a sui able biden a e ligand L and a monoden a e ligand X. 18,34,67 The o iginal idea behind such a design was ha an a ene/yl ligand should con ol lipophilici y and s abilize he oxida ion s a e o he me al, a chela ing ligand was p esen o ensu ing addi ional s abili y and a monoden a e ligand, X, was ini ially included as a si e o ac i a ion, expec edly by aqua ion. 33,68,69 Hyd olysis o hal -sandwich complexes was sugges ed o be an impo an ac i a ion s ep leading o he o ma ion o co alen bonds wi h biomolecula a ge s. 70 Howe e , i has been p o en ha , in iguingly, i is no only he iden i y o X, bu also he speci ic combina ion o monoden a e X and che- la ing ligands ha in luences he hyd olysis a e signi ican ly. A he same ime, e idence has shown ha o his ype o o ganome allic complexes, hyd olysis does no necessa ily ha e o be he ac i a ion s ep. 33,71 The e o e, we ho oughly in es iga ed he solu ion s abili y o 1–4 owa ds hyd olysis and in e ac ions wi h selec ed biomolecules. I was con i med ha 1–4we e s able in wa e (D 2 O wi h he addi ion o MeOD o solubili y) o a pe iod o 48 h. This is impo an in o - ma ion because apid hyd olysis o halido (X) hal -sandwich [M(η 6 /η 5 -a ene/yl)(L)(X)] + complex ca ions o hei aqua species [M(η 6 /η 5 -a ene/yl)(L)(H 2 O)] 2+ can esul in s ong binding o biomolecules, which may lead o deac i a ion and dec ease in hei cy o oxici y. 33,71 Analogically, a e he addi ion o PBS buffe (in a MeOD/ D 2 O solu ion mix u e) solu ion s abili y was also obse ed, bu in his case only o 1–3. In e es ingly, complex 4unde wen pa ial I − o Cl − ligand exchange, due o he p esence o an excess o he chlo ide anions o igina ing om PBS in solu ion. Such ligand exchange has been obse ed o simila hal sand- wich complexes p e iously. 71,72 I is impo an o no e ha he concen a ion o he chlo ide anions in he s udied solu ions (98 mM) was e y close o ha in he ex acellula en i onmen (110 mM), bu signi ican ly highe han ha in he in acellu- la (4 mM) en i onmen o mammalian cells. 73 I is also o impo ance ha he ex en o he ligand subs i u ion was 40% a e 48 h hus indica ing slow eac ion kine ics. Simila ly o p e ious epo s on analogous hal -sandwich complexes, 49,51 1–4ca alysed he con e sion o NADH o NAD + , sugges ing ha he mechanism o ac ion is possibly ela ed o changes in he edox homeos asis in he cell. These ypes o ca aly ically ac i e complexes change he equilib ium ela ed o he impo an NADH/NAD + edox couple in cells. Such a change may ha e signi ican consequences o cellula me abolism, such as a subs an ial inc ease in ROS le els o in e e ence wi h he lac a e dehyd ogenase-ca alysed lac a e– py u a e con e sions. 34,35 He ein, he I complexes (2and 4) we e signi ican ly mo e efficien oxidize s han Ru complexes (1and 3). NADH oxida ion occu ed wi h he efficiency o ca. 0.7, 1.4, 0.5 and 1.6 mola equi . pe mol o a complex a e 48 h o 1–4, espec i ely. Simila ly, as in he solu ion s abili y s udy pe o med in he absence o NADH, complexes 1–3 emained s able and did no unde go any s uc u al changes in he mix u e con aining NADH (in MeOD/D 2 O wi h PBS). On Pape Dal on T ansac ions 12724 |Dal on T ans.,2023,52, 12717–12732 This jou nal is © The Royal Socie y o Chemis y 2023 Open Access A icle. Published on 11 Augus 2023. Downloaded on 2/19/2024 1:17:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online he o he hand, he chlo ide anion p esence again induced he I − o Cl − ligand exchange (47% a e 48 h) o 4also in he p esence o NADH. Fu he mo e, we also in es iga ed he in e ac ions o 1–4 wi h GSH and i was ound ha 1–4did no eac wi h GSH, no oxida ion o GSH o o ma ion o GSH adduc s wi h complex molecules was obse ed wha soe e . Again, 1–3we e s able in his solu ion mix u e bu 4unde wen pa ial I − o Cl − ligand exchange o a e y simila ex en as in o he expe imen s (35%). The in es iga ion o he effec o 1–4on me abolic ac i i y o heal hy ib oblas s CCD-18Co and CCD-1072Sk (Table 1) e ealed ha a e 48 h signi ican inhibi o y ac i i y was shown by Ru complexes only owa ds he CCD-1072Sk cell line and bo h 1and 3we e signi ican ly mo e ac i e han CDDP. In con as , I complexes 2and 4showed no ac i i y (IC 50 > 50 µM), which made hem in e es ing o he ollowing in es i- ga ions. In he case o s udies in cance cell lines A2780 and A2780cis (Table 2), only 1and 4exhibi ed an inhibi o y effec on me abolic ac i i y, which was u he compa able o ha o CDDP in he case o 1. Un o una ely, he esul s showed ha complex 1was mo e ac i e in heal hy cells han in cance cells. The e o e, aking in o conside a ion he p esen ed esul s, complex 4migh be unde s ood as he mos sui able candida e o u he s udies. Al hough he ac i i y o 4in o a ian ca ci- noma cells was weake han ha o 1and CDDP, i showed he bes ac i i y p o ile, as i was clea ly selec i e owa ds cance o e heal hy cells wi h he selec i i y index (SI = (IC 50 (CCD-18Co)/IC 50 (A2780)) > 3.4, as compa ed o 2.6 o CDDP. Diffe en ial selec i i y o an an ip oli e a i e agen owa d cance cells compa ed o heal hy cells is clea ly an impo an ac o as i inc eases he p obabili y o umou - speci ic cy o oxici y, which is ela ed o dec eased side-effec s du ing ea men . All in all, based on he esul s o complexes 1–4, i is e iden ha he change o he me al om Ru o I esul ed in lowe oxici y o no mal cells while halide swi ch om Cl o I led o highe ac i i y in cance cells. Highe cy o- oxici y o iodido wi h espec o chlo ido Ru, I , Rh o Os hal sandwich complexes has been p e iously epo ed in a ew s udies. 33,37,38 Howe e , his s udy epo s on he si ua ion, when he diffe ence in he de e mined IC 50 alues be ween 2 and 4is signi ican , and he IC 50 alue o 2could no be de e mined up o he highes es ed concen a ion, p o ing ha he halide swi ch indeed u ned on he desi ed ac i i y. Addi ionally, al hough he es ed complexes sha e pa ial c oss- esis ance wi h CDDP, complex 4exhibi ed somewha be e abili y o o e come esis ance in A2780cis cells han CDDP, as he esis ance ac o , de ined as RF = IC 50 (A2780cis)/ IC 50 (A2780), d opped om 2.8 o CDDP o 1.6 o 4. Las ly, in spi e o he no ably highe effec on me abolic ac i i y o 1, bo h 1and 4seem o in luence he cell cycle in a e y simila manne , (Table 3) leading o he accumula ion o cells in he G1 phase o he cell cycle and educ ion o hei numbe in he S and G2/M phases, which is in ag eemen wi h he p e ious epo s. 33 Because o he ac ha he analysed complexes affec ed he me abolic ac i i y s a us and cell cycle bu did no in luence he iabili y o he cells, i is possible o conclude ha he effec o bo h 1and 4is cy os a ic no cy o oxic. These indings ag ee wi h p e iously epo ed esul s. Sačko á e al. 74 showed ha despi e he dec eased me abolic ac i i y s a us analysed by he MTT assay, he cell iabili y could emain unchanged. Subsequen ly, cell dea h analyses ealized by Mikešo á e al. 75 and Babinčák e al. 76 also p o ed and en iched hese ind- ings. Thus, he dec ease in he me abolic ac i i y o he cells does no necessa ily esul om he cy o oxic ac ion o es ed compounds. In ela ion o ou analyses, cell dea h induc ion is no he subs an ial cause o lowe o al cell numbe obse ed 48 h a e applica ion o 1and 4. Mo e speci ically, his phenomenon seems o be p e e en ially associa ed wi h he an ip oli e a i e effec o he analysed complexes. The e o e, i can be highligh ed ha he esul s o ou s udy again con i med ha he MTT assay is a e y powe ul ool o assess p ima ily cell me abolic ac i i y. Un o una ely, his is o en o e looked in published s udies ha in e p e he esul s in ela ion o seconda y p ocesses o s a es o cells, such as iabili y and consequen ly d ug-induced cy o- oxici y. 77 Thus, in o de o d aw well- ounded conclusions, i should be emphasized ha ca e ul and a ional in e p e a ion o he da a in combina ion wi h diffe en ypes o cell-based assays is necessa y. In summa y, i was ound ha he p esen ed complexes did no equi e hyd olysis as an ac i a ion s ep. The esul s o lipo- philici y s udies (I complexes a e mo e lipophilic han Ru ana- logues) and NADH oxida ion efficiency in es iga ion (I com- plexes a e mo e efficien oxidan s) do no co ela e wi h he gene al indings o he an ip oli e a i e ac i i y s udy, in which Ru complex 1was labelled as he mos po en one. Howe e , looking a an ip oli e a i e ac i i y p o ile mo e closely, complex 1has o be excluded om u he s udies, since i was shown o nega i ely in luence me abolic ac i i y mo e in heal hy han in cance cells. The e o e, o e all bes esul s we e ound o I -iodido complex 4wi h possibly he mos p omising he apeu ic index. Complex 4was mo e lipophilic han i s Ru analogue and was he mos efficien ca alys o he NADH- o-NAD + oxida ion eac ion, which could indica e ha he mechanism o ac ion is likely ela ed o he dis up ion o cellula edox balance. These esul s highligh he impo ance o s udies pe o med no only in cance bu also in no mal cells o p ope ly e alua e he pha macological po en ial o an i- cance d ug candida es. Ne e heless, i should be poin ed ou ha he esul s o complex 4 ep esen a s epping s one o u he s udies, in which a ious s uc u al modi ica ions will be necessa y in o de o inc ease he s abili y o he iodido complex in he physiological en i onmen wi h high chlo ide concen a ions, as i was ound o complex 4 ha pa ial I − - o-Cl − ligand exchange occu ed in such an en i onmen . I migh be specula ed ha his eac ion could con ibu e o a gene al dec ease in an icance ac i i y o 4, which he eby pa ially ans o ms o complex 2(i.e. he chlo ido coun e pa o 4) which was e alua ed as inac i e up o he highes concen- a ion le el. Dal on T ansac ions Pape This jou nal is © The Royal Socie y o Chemis y 2023 Dal on T ans.,2023,52, 12717–12732 | 12725 Open Access A icle. Published on 11 Augus 2023. Downloaded on 2/19/2024 1:17:03 PM. 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Pape Dal on T ansac ions 12732 |Dal on T ans.,2023,52, 12717–12732 This jou nal is © The Royal Socie y o Chemis y 2023 Open Access A icle. Published on 11 Augus 2023. Downloaded on 2/19/2024 1:17:03 PM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online