Distinct mechanism of action for antitumoral neutral cyclometalated Pt(II)-complexes bearing antifungal imidazolyl-based drugs

Jou nal o Ino ganic Biochemis y 226 (2022) 111663 A ailable online 11 No embe 2021 This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). Dis inc mechanism o ac ion o an i umo al neu al cyclome ala ed P (II)-complexes bea ing an i ungal imidazolyl-based d ugs Na alia Fe n´ andez-Pampín a , 1 , M´ onica Vaque o a , 1 , Tania Gil a , Gus a o Espino a , Da ío Fe n´ andez b , c , Bego˜ na Ga cía a , Na alia Bus o a , * a Depa amen o de Química, Facul ad de Ciencias, Uni e sidad de Bu gos, Plaza Misael Ba˜ nuelos s/n, 09001 Bu gos, Spain b Depa amen o de Ciencias de la Salud, Facul ad de Ciencias de la Salud, Uni e sidad de Bu gos, Paseo de los Comendado es, s/n, 09001 Bu gos, Spain c Consejo Nacional de In es igaciones Cien í icas y T´ ecnicas, CONICET, Godoy C uz 2290, C1425FQB, Buenos Ai es, A gen ina ARTICLE INFO Keywo ds: Cyclome ala ed pla inum(II) complexes Clo imazole Bi onazole Reac i e oxygen species (ROS) An i umo al ABSTRACT Th ee neu al P (II) complexes con aining 1-Me hylimidazole and he an i ungal imidazolyl d ugs Clo imazole and Bi onazole ha e been p epa ed. The gene al o mula o he new de i a i es is [P (κ 2 -(C^N)Cl(L)], whe e C^N s ands o ppy =2-phenylpy idina e, and L =1-Me hylimidazole (MeIm) o [P -MeIm]; L =Clo imazole (CTZ) o [P -CTZ] and L =Bi onazole (BFZ) o [P -BFZ]). The complexes ha e been comple ely cha ac e ized in solu ion and he c ys al s uc u es o [P -BFZ] and [P -CTZ] ha e been esol ed. Complexes [P -MeIm] and [P -BFZ] p esen highe cy o oxici y han cispla in in SW480 (colon adenoca cinoma), A549 (lung adenoca - cinoma) and A2780 (o a ian cance ) cell lines. [P -MeIm] shows he highes accumula ion in A549 cells, in ag eemen wi h i s inabili y o in e ac wi h se um albumin. By con as , [P -CTZ] and [P -BFZ] in e ac wi h se um p o eins, a ac ha educes hei bioa ailabili y. The s onges in e ac ion wi h bo ine se um albumin (BSA) is ound o [P -BFZ], which is he leas in e nalized inside he cells. All he complexes a e able o co alen ly in e ac wi h DNA. The mos cy o oxic complexes, [P -MeIm] and [P -BFZ] induce cellula accu- mula ion in G0/G1 and apop osis by a simila pa hway, p obably in ol ing a eac i e oxygen species (ROS) gene a ion mechanism. [P -BFZ] u ns ou o be he mos e icien complex ega ding ROS gene a ion and causes mi ochond ial memb ane depola iza ion, whe eas [P -MeIm] induces he opposi e e ec , hype pola - iza ion o he mi ochond ial memb ane. On he con a y, he leas cy o oxic complex, [P -CTZ] canno block he cell cycle o gene a e ROS and he mechanism by which i induces apop osis could be a di e en one. 1. In oduc ion Clo imazole (CTZ) and Bi onazole (BFZ) a e well known an i ungal agen s used in myco ic in ec ions like a hle e's oo , ul o aginal and o opha yngeal candidiasis, among o he s [1,2,3]. Due o he high e i- ciency and he un ema kable side e ec s o CTZ [4], his d ug was es ed agains o he in ec ious diseases (Mala ia (Plasmodium spp.) and Chagas (T ypanosoma c uzi)) and cance In 1995, in i o inhibi ion o umo g ow h was desc ibed o mel- anoma and glioblas oma cells ea ed wi h CTZ and BFZ. This inhibi o y e ec is a consequence o hei ac i i y as calmodulin inhibi o s [5]. Subsequen ly, hese d ugs ha e also been epo ed as inhibi o s o he glycoly ic enzymes [6]. Indeed, hey p e en binding o kinases o he ex e nal memb ane o mi ochond ia a oiding he phospho yla ion o hexoses and accele a ing he cellula apop osis. This ac i i y has been obse ed in di e en umo cell lines (b eas , ce ical, p os a e and melanoma cance cell lines among o he s) [5,6]. Coo dina ion o d ugs o ansi ion me als is a s a egy ha in ends enhancing hei biological ac i i y and allows educing he side e ec s o modi ying hei mechanism o ac ion. Se e al Ru(II) and Ru(III) complexes bea ing CTZ wi h biological ac i i y as an ipa asi ic [7,8,9,10,11,12,13,14,15], an i ungal [7,13,16], an i umo al [17,18] and an ibac e ial [19] agen and o he me al complexes con aining Au (I) [20], Ag(I) [21], Pd(II) [22], Mn(I) [23], Mn(II) [24], Cu(II) [20,23,25,26], Co(II) [23,24,25], Zn(II) [23,24,25,27], and Ni(II) [23,24,25] ha e been desc ibed in he li e a u e, al hough he e a e * Co esponding au ho . E-mail add ess: [email p o ec ed] (N. Bus o). 1 N. F – P and M. V. con ibu ed equally. The manusc ip was w i en h ough con ibu ions o all au ho s. All au ho s ha e gi en app o al o he inal e sion o he manusc ip . Con en s lis s a ailable a ScienceDi ec Jou nal o Ino ganic Biochemis y jou nal homepage: www.else ie .com/loca e/jino gbio h ps://doi.o g/10.1016/j.jino gbio.2021.111663 Recei ed 21 Sep embe 2021; Recei ed in e ised o m 4 No embe 2021; Accep ed 5 No embe 2021 Jou nal o Ino ganic Biochemis y 226 (2022) 111663 2 sca ce examples o P (II)-CTZ complexes and no examples o P (II)-BFZ de i a i es. The i s clo imazole-P (II) complex K 2 [P Cl 4 (CTZ) 2 ] was desc ibed by S´ anchez-Delgado and U bina [8] and es ed as an ipa asi ic agen agains T ypanosoma c uzi wi h simila ac i i y han [Ru(bpy)(CTZ) 3 ] (PF 6 ) 2 (50 % o inhibi ion). Following wi h his s a egy, Na a o e al., desc ibed he CTZ-pla inum(II) complexes, cis and ans-[P X 2 (CTZ) 2 ] (X=I and Cl), ha p esen an i umo al ac i i y [8,28]. The au ho s es ed hei ac i i y agains six umo cell lines (p os a e, panc eas, b eas and colon among o he s) obse ing ha bo h complexes showed lowe cy o oxici y han cispla in bu highe han anspla in. DNA is iden i ied as he biological a ge o hese complexes, in e ac ing by mino -g oo e [28]. O ganome allic P (II) complexes con aining monoden a e N-dono ligands o cyclome ala ed ligands ha can modi y hei an icance mechanism o ac ion ha e been ecen ly epo ed [29,30,31] being DNA and se um p o eins he mos common a ge s. Se e al neu al cyclo- me ala ed complexes desc ibed in he li e a u e displayed a di e en an ip oli e a i e mechanism o DNA binding, being eac i e oxygen species (ROS) gene a ion a sui able s a egy o induce p og ammed cell dea h in an icance he apy [32,33]. Among he neu al cyclome ala ed complexes, we ha e syn hesized a amily o chi al neu al cyclome ala ed pla inum(II) complexes wi h gene al o mula [P (κ 2 -(C^N))Cl(κ 1 -(L))] whe e C^N =2-phenyl- py idina e and L s ands o 2-(2-Py idyl)benzimidazole de i ed ligands unc ionalized wi h CH 2 -A (A =phenyl, naph yl and py enyl) moie ies on he imidazole agmen [34]. These neu al complexes we e es ed as an ip oli e a i e d ugs in SW480 cance cell lines, being he complex wi h he un unc ionalized 2-(2-Py idyl)benzimidazole ligand he mos cy o oxic complex in spi e o ha DNA was no i s biological a ge . The an i umo neu al pla inum complex, [P (II)(cis-[bis-(NHC)Cl (Cl)], con aining a N-he e ocyclic ca bene (NHC, being NHC =1,3- dibenzylimidazol-2-ylidene) showed a s ongly in e ac ion wi h DNA in i o [35]. mo eo e , his complex educed he mi ochond ial mem- b ane po en ial and also inc eased he cellula ROS le els. As a conse- quence, his complex showed cy o oxici y in 518A2 melanoma cells. On he o he hand, highly cy o oxic neu al ca ionic pla inacycles based on biden a e phosphines we e ac i e agains se e al b eas and colon cance cells. Thei biological ac i i y is ela ed o ROS gene a ion ins ead o DNA co alen binding [36]. Taking in o accoun he abo e desc ibed p eceden s, he an i umo al ac i i y o he imidazolyl an i ungical agen s CTZ and BFZ, he sca ce examples o P (II)-CTZ complexes and he lack o P (II)-BFZ complexes, we ackle he syn hesis o new neu al cyclome ala ed P (II) complexes bea ing imidazolyl an i ungal agen s CTZ and BFZ, and 1-Me hylimida- zole (MeIm) as a model imidazolyl ligand. I wo h men ioning ha [P - MeIm] was p e iously es ed in leukaemia umo wi h p omising esul s [34]. The coo dina ion o he monoden a e imidazolyl ligands o pla inum could inc ease he cy o oxici y o he complexes espec o he ee imidazolyl ligands, and he cyclome ala ed ligand ppy − could con e in e es ing p ope ies ha could modi y he mechanism o ac ion o he classical P (II)-complexes a oiding umo esis ance. 2. Expe imen al Sec ion. 2.1. Gene al syn he ic p ocedu e o he P (II) complexes Pla inum p ecu so [P (κ 2 -(ppy)Cl(κ 1 -(Hppy)] whe e ppy =2-phe- nylpy idina e and Hppy =2-phenylpy idine named as [P -0] (0.185 mmol) and he co esponding imidazolyl ligand (MeIm, 0.222 mmol; CTZ, 0.203 mmol and BFZ 0.185 mmol) we e dissol ed in 20 mL o DMF and he mix u e was s i ed o 24 h a 65 ◦C. The sol en was e apo- a ed unde educed p essu e, washed unde s i ing wi h E 2 O (2 ×7 mL) il e ed and d ied unde acuum. De ailed syn he ic p ocedu es and cha ac e iza ion o he complexes a e compiled in he Suppo ing In o ma ion. 2.2. X- ay c ys allog aphy Da a collec ion and e inemen pa ame e s o [P -CTZ] and [P - BFZ] a e summa ized in Tables SI1 in he Suppo ing In o ma ion. A single c ys al o he complexes was coa ed wi h high- acuum g ease, moun ed on a glass ibe , and ans e ed o a B uke SMART APEX CCD- based di ac ome e equipped wi h a g aphi e-monoch oma ed Mo Ka adia ion sou ce (λ =0.71073). The highly edundan da a se s we e in eg a ed wi h SAINT [37] and co ec ed o Lo en zian and pola iza- ion e ec s. The abso p ion co ec ion was based on he unc ion i ing o he empi ical ansmission su ace as sampled by mul iple equi alen measu emen s wi h he p og am SADABS [38]. The so wa e package WingX [39] was used o space-g oup de e mina ion and s uc u e so- lu ion, and OLEX 2 1.2.10 [40] was used o e inemen by ull-ma ix leas -squa es me hods based on F2. A success ul solu ion by di ec me hods p o ided mos non‑hyd ogen a oms om he E map. The emaining non‑hyd ogen a oms we e loca ed in an al e na ing se ies o leas -squa es cycles and di e ence Fou ie maps. All non‑hyd ogen a oms we e e ined wi h aniso opic displacemen coe icien s. Hyd ogen a oms we e placed by using a iding model and included in he e inemen a calcula ed posi ions. CCDC 2091796 ([P -CTZ]) and 2,091,795 ([P -BFZ]) con ain he supplemen a y c ys allog aphic da a o his pape . These da a a e p o ided ee o cha ge by The Camb idge C ys allog aphic Da a Cen e. 2.3. Gene al p ocedu e o s abili y by NMR spec oscopy S abili y in DMSO ‑ d 6 : Complexes we e dissol ed in DMSO‑d 6 (300 μ L, 1.42 ×10 −3 M), and 1 H NMR spec a we e eco ded o e 24 h in a 400 MHz spec ome e . S abili y in H 2 O (3% DMSO): Complexes we e dissol ed i s in DMSO‑d 6 (150 μ L) and wa e was added (4.850 mL, 1.5 ×10 −2 M), 1 H NMR spec a we e eco ded o e 24 h in a 300 MHz spec ome e . Addi ion o AgNO 3 : O e a solu ion coming om he s a- bili y in H 2 O (3% DMSO), AgNO 3 was added and he 1 H NMR spec a we e eco ded a e 24 h and 48 h in a 300 MHz spec ome e . 2.4. Gene al p ocedu e o s abili y by HR-MS ESI spec oscopy Samples o he complexes P (II) complexes we e p epa ed in NaCaC (2.5 mM, pH =7, 3% o DMSO) in concen a ions o 100 ppm. The sample was injec ed a e 24 h in solu ion in a 6545 Q-TOF (Agilen ; V inj =0.1 μ L). Mobile phase: H 2 O- o mic acid/MeOH (30:70) (0.1% o o - mic acid). Flow: 0.1 mL/min. 2.5. Cell cul u e SW480 (colon adenoca cinoma) and A549 (lung ca cinoma) cells om he ECACC (Eu opean Collec ion o Au hen ica ed Cell Cul u es, Salisbu y, UK) we e cul u ed in Dulbecco's Modi ied Eagle's Medium (DMEM) and A2780 (o a ian ca cinoma) cells, also om he ECACC, we e cul u ed in Roswell Pa k Memo ial Ins i u e (RPMI-1640). DMEM and RPMI we e supplemen ed wi h 10% e al bo ine se um (FBS) and 1% ampho e icin-penicillin-s ep omycin solu ion (all om Sigma Ald ich). Cell we e main ained a 37 ◦C in a humidi ied a mosphe e con aining 5% CO 2 . 2.6. MTT an ip oli e a i e assay Cell p oli e a ion was de e mined by he 3-(4, 5-dime hyl hiazol- 2- yl)-2, 5-diphenyl e azolium b omide (MTT) assay (Sigma Ald ich) using he ollowing p o ocol: SW480 we e seeded a a densi y o 1 ×10 4 , A549 cells a 5 ×10 3 and A2780 a 2 ×10 4 cells pe well in 96 well pla es. A e 24 h o incuba ion, cells we e ea ed wi h di e en con- cen a ions o he complexes unde s udy o o he 24 h. Then, ea men N. Fe n´ andez-Pampín e al. Jou nal o Ino ganic Biochemis y 226 (2022) 111663 3 was emo ed, and cells we e incuba ed o 3 h wi h 100 μ L o MTT (5 mg/mL) in cul u e medium a 37 ◦C and 5% CO 2 . Then, 100 μ L o sol- ubilizing solu ion (10% SDS (sodium dodecyl sul a e) and 0.01 M HCl) was added o each well o dissol ing he o mazan c ys als. Finally, pla es we e incuba ed o e nigh a 37 ◦C wi h so agi a ion, and abso bance was measu ed a 590 nm using a mic opla e eade (Cy a ion 5 Cell Imaging Mul i-Mode Reade -Bio ek Ins umen s, USA). Fou eplica es pe dose we e included in each expe imen and a leas wo independen expe imen s we e pe o med. Then, hal -maximal inhibi- o y concen a ion (IC 50 ) alues we e calcula ed using he G aph- PadP ism So wa e Inc. ( e sion 6.01) (USA). Pho o oxici y expe imen s we e ca ied ou as p e iously desc ibed [41]. 2.7. Cellula up ake A549 cells we e seeded in 12 well pla es a a densi y o 1.5 ×10 5 cells in 2 mL o cul u e medium pe well. Cells we e ea ed wi h 2 μ M o he es ed d ugs and incuba ed o 24 h. Then, cells we e washed wice wi h DPBS (Dulbecco's Phospha e Bu e ed Saline), ha es ed and cen i uged. The pelle s we e esuspended in 1 mL o DPBS and 10 μ L pe sample we e used o coun he cells in an au oma ed cell coun e (TC20 – Bio ad). Then, samples we e diges ed wi h 65% HNO 3 a oom em- pe a u e du ing 24 h o ICP-MS. Finally, solu ions we e analyzed in an 8900 T iple Quad upole ICP-MS (Agilen Technologies). 2.8. Binding o se um albumin and DNA Bo ine se um albumin (BSA) as c ys allized and lyophilized powde was dissol ed in double deionized wa e om a Pu ani y TU sys em (VWR) and i s concen a ion was spec opho ome ically de e mined ( ε 278 nm =45,000 M −1 cm −1 ) [42]. Cal hymus DNA (c DNA) as lyophilized sodium sal was dissol ed in double deionized wa e and sonica ed, p oducing sho polynucleo ide agmen s (ca. 1000 base pai s). The mola DNA concen a ion was spec opho ome ically de e mined ( ε 260 nm =13,200 M −1 cm −1 ) and exp essed in base pai s [43]. Plasmid pUC18 (2686 bp) was ex ac ed om Esche ichia coli DH5 α and pu i ied by a HP Plasmid Midi ki (Omega Bio ek, VWR). Sodium cacodyla e, (CH 3 ) 2 AsO 2 Na, om now named NaCaC, was used as a bu e solu ion o keep pH =7.0. All hese eagen s we e pu chased om Sigma Ald ich. Measu emen s o pH we e made by a Me ohm 713 pH-me e equipped wi h a combined glass elec ode. 2.9. Na i e polyac ylamide gel elec opho esis (PAGE) o bo ine se um albumin (BSA) PAGE expe imen s we e done a e o e nigh incuba ion a 37 ◦C o 1 μ M BSA in he absence o in he p esence o di e en concen a ions o he P complexes. Then, 5 μ L o sample bu e 2×0.01% b omophenol blue and 20% glyce ol in T is HCl bu e (0.5 M, pH 6.8) we e added o 5 μ L o he sample solu ions and loaded on o 10% polyac ylamide gels. Gels we e un in na i e PAGE bu e (250 mM T is Base, 1.92 M glycine, pH =8.3) a 6.6 V/cm o 6 h a 4 ◦C o a oid he mal dena u a ion o he p o ein. Finally, gels we e s ained wi h Coomassie b illian blue R- 250 and isualized wi h a Gel Doc XR+Imaging Sys em (Bio Rad). 2.10. Aga ose gel elec opho esis o plasmid DNA (pUC18) Aga ose gel elec opho esis o plasmid was pe o med a e o e - nigh incuba ion a 37 ◦C o he plasmid (3.7 μ M, base pai s) in he p esence o di e en concen a ions o he P (II) complexes in 2.5 mM NaCaC, pH =7.0. Samples we e loaded on o 1% aga ose gel and elec- opho esis was un a 6.5 V/cm du ing 150 min. A e he un, he gel was s ained wi h a solu ion o e hidium b omide 1 μ g/mL in T is-bo a e- EDTA (TBE) 1× o 30 min. Finally, he gel was isualized in a Gel Doc XR+Imaging Sys em (Bio-Rad). Da a Analysis. All da a we e exp essed as mean ±s anda d de ia ion (SD). S a is ical signi icance was e alua ed using by s a is ical analysis pe o med wi h G aphPadP ism So wa e Inc. ( e sion 6.01, USA). 2.11. Ci cula dich oism (CD) CD spec a we e eco ded on a MOS-450 Biologic spec ome e (Claix, F ance) o samples a a P (II) complex/BSA concen a ions a io o 5 and a P (II) complex/c DNA (cal hymus DNA) concen a ions a io o 1 incuba ed o e nigh , in 2.5 mM NaCaC bu e a pH =7.0 and T =25 ◦C. 2.12. In acellula eac i e oxygen species (ROS) gene a ion A549 cells we e seeded in a clea bo om black side 96 well pla e a a densi y o 3 ×10 4 cells pe well and incuba ed o 24 h. Then, media was emo ed and o ROS quan i ica ion by he p obe 2′,7′-dichlo odihy- d o luo escein diace a e (H 2 DCFDA), 100 μ L o 25 μ M o H 2 DCFDA in DMEM wi hou phenol- ed was added o each well. Cells we e incuba ed o o he 30 min and hen, cells we e ea ed wi h 100 μ L o he ehicle and wi h he co esponding P (II) complex wi h he equi ed concen- a ion o ob ain a inal concen a ion equal o he IC 50 alue. Cells ea ed wi h 20 μ M o e abu ylammonium hyd oxide (TBH) we e included as posi i e con ol. A e 2 h o ea men , cells we e washed wice wi h DPBS and emission was measu ed a λ em =530 nm wi h λ exc =490 nm du ing 2 h in a mic opla e eade (Cy a ion 5 Cell Imaging Mul i-Mode Reade -Bio ek Ins umen s, USA). Measu emen s a 4 h a e ea men we e selec ed a he inal poin o da a analysis. Cell images we e also aken, and he collec ed esul s we e co ec ed by he numbe o cells. Two independen expe imen s wi h 4 eplica es pe ea men we e pe o med. Fo he dihyd oe hidium (DHE) assay, ins ead o H 2 DCFDA, cells we e incuba ed wi h 0.5 μ M o DHE (dihy- d oe hidium) o 20 min. Then, cells we e washed wice wi h phospha e bu e saline (PBS) and luo escence in ensi y was measu ed a e 2 h a λ em =535 nm wi h λ exc =635 nm in a mic opla e eade (Cy a ion 5 Cell Imaging Mul i-Mode Reade -Bio ek Ins umen s, USA). The emission o each well was co ec ed by he numbe o cells. Resul s a e exp essed as he mean and s anda d de ia ion o wo independen expe imen s wi h 4 eplica es pe dose. The le els o cy osolic supe oxide anion in un ea ed A549 cells and a e 2 h o ea men wi h he ehicle (0.5% DMSO) o he imidazolyl-P (II) complexes we e also e alua ed by low cy ome y. Fo his aim, A549 cells we e seeded a a densi y o 30,000 cells/well and incuba ed o 24 h. Then, cells we e incuba ed wi h 1 μ M DHE du ing 20 min in DMEM wi hou phenol ed. Then, cells we e washed wice wi h PBS and ea ed a he IC 50 alues o he P (II) complexes unde s udy. A e 2 h o incuba ion, cells we e ha es ed, washed wi h PBS, and collec ed in 100 μ L o PBS. Finally, cells we e analyzed by low cy ome y (No oCy e Flow cy ome e , ACEA Biosciences, Inc., USA). Two eplica es pe expe imen al condi ion and wo independen expe imen s we e pe - o med and analyzed by No oExp ess 1.4.0 So wa e. 2.13. Mi ochond ial memb ane po en ial (MMP) assay by TMRM ( e ame hyl hodamine me hyl es e ) The e ec o he imidazolyl P (II)-complexes on he MMP o A549 cells was e alua ed by means o e ame hyl hodamine me hyl es e (TMRM). A549 cells we e seeded in a clea bo om black side 96 well pla e a a densi y o 3 ×10 4 cells pe well and incuba ed o 24 h. Then, media was emo ed and eplaced by 100 μ L o assay bu e (25 mM D- glucose, 80 mM NaCl, 75 mM KCl, 25 mM Hepes (4-(2-hyd oxye hyl)-1- pipe azinee hanesul onic acid), pH =7.4) and cells we e ea ed wi h he ehicle (0.5% DMSO) and he P (II) complexes a hei IC 50 alues du ing 1 h. Then, 10 μ L o TMRM (2 μ M) we e added and a e 15 min o incuba ion a oom empe a u e and ligh p o ec ed, cells we e washed wice wi h PBS. A e wa ds, 100 μ L o PBS we e added and luo escence o TMRM was measu ed a e 15 min a λ em =535 nm wi h λ exc =590 N. Fe n´ andez-Pampín e al. Jou nal o Ino ganic Biochemis y 226 (2022) 111663 4 nm in a mic opla e eade (Cy a ion 5 Cell Imaging Mul i-Mode Reade -Bio ek Ins umen s, USA). The luo escence in ensi y o each well was co ec ed by he co esponding numbe o cells. Two independen ex- pe imen s wi h 4 eplica es pe ea men we e pe o med. 2.14. Cell cycle a es To s udy he pe u ba ions in he cell cycle o A549 cells induced by pla inum complexes, cells we e s ained wi h p opidium iodide (PI) and analyzed by low cy ome y. B ie ly, 1.5 ×10 5 cells pe well we e seeded in 6 well pla es. Cells we e ea ed a he IC 50 alue o each complex. Cells wi hou any ea men and ehicle- ea ed cells (0.5% DMSO) we e used as con ol. A e 24 o ea men , cells we e ha es ed and cen i- uged, esuspended in cold PBS and inally ixed in 70% E OH (CH 3 CH 2 OH) o e nigh a 4 ◦C. A e ixa ion, samples we e ea ed wi h a solu ion con aining 0.1 mg/mL PI, 0.1 mM EDTA (e hyl- enediamine e aace ic acid) and 0.1% T i on- ×100 in PBS o pe - meabilize and s ain cells, and wi h RNAse (2 mg/mL) o 30 min in ice and p o ec ed om ligh . Finally, cells we e analyzed using he No o- Cy e Flow cy ome e (ACEA Biosciences, Inc., USA). Cell cycle dis i- bu ion was e alua ed by No oExp ess 1.4.0 So wa e. Two eplica es pe expe imen al condi ion and h ee independen expe imen s we e pe o med. 2.15. Apop osis de ec ion by low cy ome y Apop osis was e alua ed by an Annexin V:FITC Assay Ki (Bio ad) acco ding o he manu ac u e 's ins uc ions. B ie ly, A549 cells we e seeded a a densi y o 2 ×10 5 cells pe well in 12 wells pla es and incuba ed o 24 h. Then, cells we e ea ed a he IC 50 alue o each complex. A e o he 24 h, cells we e washed wi h cold PBS, ha es ed, and esuspended in binding bu e . A e wa ds cells we e s ained wi h Annexin V:FITC conjuga e, ha is, annexin V conjuga ed wi h luo es- cein iso hiocyana e (FITC) du ing 10 min (ligh p o ec ed) a oom empe a u e. Then, cells we e washed wi h binding bu e , cen i uged, and esuspended in 190 μ L o binding bu e , 10 μ L o p opidium iodide (PI) (20 μ g/mL) we e added jus be o e da a collec ion in a No oCy e Flow cy ome e (ACEA Biosciences, Inc., USA). 10,000 e en s we e coun ed and analyzed by No oExp ess 1.4.0 So wa e. Two eplica es and wo independen expe imen s we e pe o med. 3. Resul s and Discussion 3.1. Syn hesis o he neu al cyclome ala ed Pla inum(II) complexes 1-Me hylimidazole (MeIm), Clo imazole (CTZ) and Bi onazole (BFZ) a e comme cially a ailable. All he complexes, isola ed as pale- yellow solids, a e ai - and mois u e- s able. Neu al Pla inum(II) com- plexes [P -MeIm], [P -CTZ] and [P -BFZ] o gene al o mula [P (ppy) Cl(L)], being L =MeIm, CTZ and BFZ, espec i ely, and ppy =2-phe- nylpy idina e, we e syn hesized by di ec eac ion o he pla inum p e- cu so [P (κ 2 -(ppy)Cl(κ 1 -(Hppy)] [34,44], om now [P -0] (Hppy =2- phenylpy idine), in DMF a 65 ◦C (Scheme 1 and syn he ic p ocedu es in SI). Complex [P -MeIm] has been p e iously syn hesized [45], bu he syn he ic p ocedu e desc ibed he ein a oids he syn hesis o he in e - media e [P (ppy)Cl(DMSO)], a o ding he desi ed [P -MeIm] complex in simila yields. 3.2. Cha ac e iza ion in solu ion and in solid s a e 3.2.1. Cha ac e iza ion in solu ion P (II)-complexes we e comple ely cha ac e ized in solu ion by NMR spec oscopy in CDCl 3 , IR, elemen al analysis as well as high esolu ion mass spec ome y, and, in he solid s a e, by X- ay di ac ion o single c ys al. Spec oscopic da a ob ained o [P -MeIm] a e in conco dance wi h hose desc ibed by Esmaeilbeig e al. [45] (see cha ac e iza ion in S⋅I). 1 H NMR spec a o he P (II) complexes [P -CTZ] and [P -BFZ] showed cha ac e is ic sa elli es due o he expec ed 195 P – 1 H couplings o he p o ons H1 (J P -H =35–39 Hz) and H5 (J P -H =45–47 Hz) o he 2-phe- nylpy idina e ligand and o he p o on Ha (J P -H =18–22 Hz) o he imidazolyl agmen (see spec a o [P -BFZ], Figu e SI1, as ep esen- a i e P (II) complex). The las ea u e co obo a es he coo dina ion o he imidazolyl ligands o he Pla inum cen e . The HR-MS ESI(+) spec a eco ded o he neu al cyclome ala ed P (II) complexes exhibi ed in each case a peak ully compa ible wi h he espec i e ca ionic agmen [P (C^N)(L)] + (m/z a io and iso opic pa e n), which co espond o he loss o he chlo ide ligand (see cha ac e iza ion in Suppo ing in o ma ion). 3.2.2. Solid s a e cha ac e iza ion Sui able single c ys als o X- ay s uc u al de e mina ion we e ob- ained o he complexes [P -CTZ] and [P -BFZ] by slow e apo a ion o a solu ion o he complexes in ace one. Bo h ORTEP diag ams a e depic ed in Fig. 1, selec ed bond leng hs and angles wi h es ima ed s anda d de ia ions a e ga he ed in, and c ys allog aphic e inemen pa ame e s a e gi en in he Suppo ing In o ma ion (Table SI1). S uc- u e o he [P -MeIm] has been p e iously desc ibed by Esmaeilbeig e al. [45] (da a a e compiled in Table 1 o compa a i e pu pose). Complex [P -CTZ] c ys allizes in he monoclinic space g oup P2 1 /c and shows ou molecules in he uni cell, while [P -BFZ] c ys allizes in a iclinic space g oup P-1 wi h wo molecules in he uni cell which di e om each o he in he o ien a ion o he imidazole agmen , along wi h an ace one molecule. In bo h s uc u es, he pla inum cen e displays a sligh ly dis o ed squa e plane coo dina ion geome y wi h a ans-N,N disposi ion o he N a om o he 2-phenylpy idina e ligand and he N a om o he imidazole agmen o he co esponding imida- zolyl ligands. The P – N dis ances ha de ine he dis o ed squa e plana Scheme 1. Syn he ic p ocedu e o he neu al cyclome ala ed P (II) complexes. Fig. 1. ORTEP diag am o [P -CTZ] (le ) and [P -BFZ] ( igh ) o ming pa o he asymme ic uni s. Hyd ogen a oms ha e been omi ed o cla i y. The - mal ellipsoids a e shown a he 50% p obabili y le el. N. Fe n´ andez-Pampín e al. Jou nal o Ino ganic Biochemis y 226 (2022) 111663 5 geome y p esen alues o app ox. 2 Å, as was desc ibed o [P -MeIm] (see Table 1) [45]. The P -Cl(1) dis ance (app ox. 2.4 Å) obse ed o he complexes [P -CTZ] and [P -BFZ] a e s anda d o a P – Cl bond in which he Cl − a e loca ed in ans posi ion o he sp 2 ca bon o he 2-phe- nylpy idina e ligand [34,45,46,47,48,49]. The bi e angles C(9A)-P (1)- N(1) o he P (II) complexes a e a ound 81◦, while he opposi e an- gles N(2)-P (1)-Cl(1) p esen alues o 87–88◦(see Table 1). The 3D-s uc u es o [P -CTZ] and [P -BFZ] a e s abilized by di e en spa ial in e ac ions. In he s uc u e o [P -CTZ] a chlo ide a om o one molecule is in ol ed in hyd ogen bonding in e ac ions wi h wo hyd ogen a oms (H3 and H4) o wo ppy − ligands ha belong o wo di e en molecules (see Figu e SI2 and Table SI2). Besides, each mole- cule o [P -CTZ] pa icipa es in wo chlo ide bond in e ac ions C-Cl—C be ween he C20 – Cl and C16 o a di e en molecule (See Figu e SI3 and Table SI4). Addi ionally, wo CH- π in e ac ions a e obse ed o he same molecule o [P -CTZ], (see Figu e SI4 and Table SI3). In he case o he complex [P -BFZ] he chlo ide a om is in ol ed in hyd ogen bonding in e molecula in e ac ions wi h he H6 a om o a ppy − o a neighbo molecule, he Ha and CH o ano he di e en neighbo mole- cule, and a H o a me hyl g oup o he ace one molecule (see Figu e SI5 and Table SI2). In addi ion, be ween he same molecules o [P -BFZ] di e en CH- π in e ac ions a e obse ed in he c ys al s uc u e. These in e ac ions a e esumed in he suppo ing in o ma ion (Figu e SI6, and Table SI3) along wi h he in e ac ions in which one molecule o ace one is implica ed (Figu e SI7). 3.3. S abili y in solu ion Fi s o all, he s abili y in solu ion o he P (II)-complexes in DMSO‑d 6 (1.42 10 −3 M, 300 μ L) was s udied o e 24 h by 1 H NMR spec oscopy ( he s ock solu ions o he complexes we e s o ed in DMSO). The imidazolyl P (II) complexes showed a low deg ee o ligand dissocia ion a e 24 h, 3% o dissocia ion o he MeIm o [P -MeIm], 17% and 28% o he co esponding ligands o [P -BFZ] and [P -CTZ], espec i ely (see Figu es SI8-SI10). In any case, he subs i u ion o he Cl − anion by DMSO‑d 6 was obse ed. This beha io was p e iously desc ibed o [P (ppy)Cl(Hpybzi)] de i a i es [34]. The s abili y o he P (II) complexes in DMSO solu ion was also s udied by UV measu e- men s and he eco ded spec a showed no changes du ing 24 h (Figu e SI11). Addi ional s abili y s udies o he P (II) complexes we e pe o med in D 2 O/DMSO‑d 6 (97:3) by moni o ing he e olu ion o he 1 H NMR spec a du ing 24 h. DMSO‑d 6 was used o ensu e he comple e disso- lu ion o he complexes (see Figu es SI12-SI14). Indeed, his sol en mix u e is commonly employed in biological s udies o wa e -insoluble me al complexes. Then, AgNO 3 was added o o ce he elease o he chlo ide anion and he o ma ion o he co esponding aqua-complex (aqua ion p ocess). In pa icula , he 1 H NMR spec a o [P -MeIm] in a D 2 O (3% DMSO‑d 6 ) emained essen ially unchanged o e 24 h, con i ming he s abili y o his complex in he medium (compa e spec a a and b in Figu e SI12). Howe e , he addi ion o AgNO 3 ga e place o a new se o signals, ha we en a i ely assigned o he aqua-complex [P (ppy)(D 2 O)(MeIm)] + (compa e spec a b, c and d in Figu e SI12). This ac con i med us ha he chlo ide is s ill bound o he P cen e a e 24 h in aqueous solu ion. A simila beha io is obse ed o [P -CTZ] and [PT-BFZ] complexes unde analogous condi ions. Hence, hese wo complexes a e also s able in wa e solu ion (spec a a and b in Figu e SI13 o [P -CTZ] and spec a a and b in Figu e SI14 [PT-BFZ]). Fu he mo e, we could conclude ha he aqua ion p ocess did no ake place in aqueous media. The s abili y o he imidazolyl-P (II) complexes was also checked by HR-MS ESI spec ome y in bu e ed media (Sodium Cacodyla e (NaCaC), 2.5 mM, pH =7, 3% DMSO) (see expe imen al sec ion). All he complexes a e s able a e 24 h o incuba ion in a solu ion o sodium cacodyla e NaCaC (DMSO 3%; see NaCaC ch oma og am in Figu e SI15 and ch oma og ams o he Pla inum complexes in Figu es SI16 −SI18). F ee ligands MeIm, CTZ and BFZ we e no obse ed in any case. 3.4. Pho ophysical p ope ies UV–Vis abso p ion spec a o he neu al cyclome ala ed P (II) complexes we e eco ded in H 2 O (3% DMSO) (10 −5 M) and in ace o- ni ile solu ions o compa ison wi h o he P (II) complexes desc ibed in he li e a u e. The abso p ion spec a o he P (II)-imidazolyl complexes in H 2 O p esen a b oad band wi h he maximum loca ed be ween 235 and 255 nm (Fig. 2, da a in Table 2). These abso p ion bands could be a ibu able o single spin-allowed ligand cen e ed ansi ions ( π → π *, 1 LC) ha akes place in bo h he imidazolyl (MeIm, CTZ and BFZ) and he ppy − ligands. In he a ea be ween 373 and 433 nm, ypical o he 1 MLCT ansi ions, he complexes p esen low in ense bands, simila o hose obse ed o ela ed cyclome ala ed ppy-P complexes [45,50,51]. The abso p ion p o ile o he P (II) complexes did no p esen ema kable sol a och omism e ec s when compa ing he spec a measu ed in aqueous solu ion wi h hose eco ded in ace oni ile (see Figu e SI19 and Table SI5). The pho oluminescence p ope ies o he complexes ha e been eco ded in 10 −5 M solu ions in bo h deoxygena ed H 2 O (3% DMSO) (see Fig. 2, da a in Table 2) and ace oni ile (see Figu e SI20, da a in Table SI6). In bo h sol en sys ems, he new cyclome ala ed P (II) complexes showed pho oluminescence in he cyan-g een hue unde i adia ion wi h λ exc =375 nm, abso p ion wa eleng h ha co esponds o 1 MLCT ansi ions. The spec a p o iles o he pla inum complexes [P -MeIm] and [P -CTZ] a e qui e simila in bo h sol en sys ems wi h he maximum o he emission bands cen e ed a 480 nm and 514 nm ha a e assigned o spin-allowed 1 MLCT and spin- o bidden and, o [P - MeIm] 3 MLCT (single o iple d π (P ) → π *(N^N)), 3 LLCT (single o iple , 3 π (N^N) → π *(C^N)) and 3 LC (single o iple 3 π → π *) ansi- ions. 51 The complex [P -BFZ] showed a di e en emission spec um wi h b oad emission bands (491 nm and 529 nm) and a pla eau ha Table 1 Selec ed bond leng hs (Å) o he molecula s uc u e o complexes [P -CTZ] and [P -BFZ]. Dis ances (Å) [P -MeIm] [45] [P -CTZ] [P -BFZ] Angles (◦) [P -MeIm] [45] [P -CTZ] [P -BFZ] P (01)-Cl(1) 2.4105(13) 2.3807(18) 2.3876(10) N(2)-P (01)-Cl(1) 87.45(13) 87.72(15) 88.76(10) P (01)-N(2) 2.026(5) 2.028(5) 2.028(4) C(9)-P (01)-N(1) 81.3(2) 82.1(3) 81.51(15) P (01)-C(9) 1.978(5) 1.967(6) 1.979(4) N(1)-P (01)-Cl(1) 96.60(12) 96.31(16) 95.69(10) P (01)-N(1) 2.017(4) 2.023(5) 2.013(4) C(9)-P (01)-N(2) 94.9(2) 93.8(2) 94.67(15) Fig. 2. Abso p ion (le ) and emission ( igh ) spec a in deoxygena ed H 2 O (3% DMSO) o he P (II) complexes (10 −5 M). N. Fe n´ andez-Pampín e al. Jou nal o Ino ganic Biochemis y 226 (2022) 111663 6 en e in o he ed spec um egion cen e ed a 602 nm. The spec um o he complex in ace oni ile showed a g ea blue-shi ed o he bands up o 54 nm. As o he exci ed s a e hal li e ime, τ 1/2 , he complexes exhibi ed highe hal -li e imes in H 2 O (3% DMSO) (3 μ s o [P -MeIm], 166 ns and 267 ns o [P -CTZ] and [P -BFZ], espec i ely, Table 2) han in ace oni ile (28.97 ns o [P -MeIm], 28.90 ns o [P -CTZ] and 43.29 ns [P -BFZ], see Table SI6). We should highligh he e ha [P - MeIm] is a phospho escen complex in deoxygena ed H 2 O. The di e - ences on he hal -li e imes in bo h sol en sys ems a e ela ed o he s abiliza ion o he exci ed s a e in p o ic pola sol en s compa ed o hose in ace oni ile solu ion [52]. Rega ding o he pho oluminescence quan um yields (ϕ PL ), he alues dec ease acco ding he ollowing sequence [P -MeIm] > [P -CTZ] > [P -BFZ]. Hence, [P -MeIm] exhibi he maximum alue in bo h sol en sys ems (6.43% in H 2 O (3% DMSO) and 15.81% in ace oni ile). Despi e hei emission p ope ies, hey ha e no been isualized inside he cells. 3.5. Biological beha io 3.5.1. Cy o oxici y o he P (II) complexes The iabili y o he human cell lines A549 (lung adenoca cinoma), SW480 (colon adenoca cinoma) and A2780 (o a ian cance ) was s ud- ied a e 24 h o ea men o he cells wi h he pla inum complexes by means o he MTT assay (Table 3). Ou p esen s udies ha e demons a ed ha [P -CTZ] is he less cy o oxic complex whe eas [P -MeIm] and [P -BFZ] a e mo e ac i e han cispla in agains all he s udied umo cells. While [P -MeIm] is he mos ac i e de i a i e in he SW480 cell line, [P -BFZ] is as cy o- oxic as [P -MeIm] in he A549 and A2780 cells. Pho oac i a ion o hese P (II) complexes by UV (λ =365 nm, 8 mWcm −2 ) and blue (λ = 460 nm, 5.5 mWcm −2 ) ligh i adia ion ha e also been e alua ed wi h nega i e esul s since no a ia ion o he hal maximal inhibi o y con- cen a ion was obse ed. 3.5.2. Cellula up ake A e 24 h o exposu e o lung adenoca cinoma A549 cells o 2 μ M o he P -complexes, he alues ob ained o he cellula up ake by ICP mass spec ome y we e compa ed wi h hose obse ed o cispla in (CDDP) (Fig. 3). [P -MeIm] was he mos in e nalized compound in he A549 cells, in ag eemen wi h i s high cy o oxic ac i i y, in high ex en han CDDP. By con as , [P -BFZ] exhibi s a low deg ee o accumula ion in his cell line, lowe han cispla in, in spi e o i s high cy o oxici y. The opposi e end is obse ed o [P -CTZ], which displays a high cellula up ake inside A549 cells al hough is almos inac i e. 3.5.3. Binding s udies o he P (II) complexes o BSA and DNA Cellula up ake depends, among o he ac o s, on d ug seques a ion by plasma p o eins be o e eaching he umo cells. Since human se um albumin (HSA) is he main p o ein o blood and ce eb ospinal luid and plays a key ole in bio-dis ibu ion o essen ial me al ions and a la ge a ie y o endogenous and exogenous molecules [53], HSA binding s udies a e essen ial o unde s and ADME (Abso p ion, Dis ibu ion, Me abolism, and Exc e ion) p ope ies o he d ugs [54]. Bo ine se um albumin (BSA) is o en used as a model o HSA binding s udies due o i s simila i y and i s lowe cos . I is known ha upon cispla in adminis- a ion, mo e han 90% o he P is co alen ly bound o he plasma p o eins as albumin, ans e in, and γ-globulin [55]. The e o e, na i e polyac ylamide gel elec opho esis (PAGE) expe imen s o e BSA incuba ed wi h di e en a ios o he imidazolyl-P complexes we e pe o med (Fig. 4). [P -BFZ] s ongly a ec s he BSA na i e con o - ma ion a low concen a ion o his complex ([P -BFZ]/[BSA] =25). On he con a y, [P -CTZ] only a ec s BSA con o ma ion a high concen- a ion a ios ([P -CTZ]/[BSA] a ios o 25 and 50) and in a less ex en han [P -BFZ]. In o de o con i m his in e ac ion, ci cula dich oism expe imen s o BSA incuba ed o e nigh in p esence o he cyclome ala ed P (II)- complexes we e pe o med (Figu e SI21). No signi ican di e ences in he BSA in insic band (cen e ed a 220 nm) we e obse ed o [P - MeIm]. Howe e , [P -BFZ] and [P -CTZ] induce con o ma ional Table 2 Elec onic and pho ophysical da a o he P (II) complexes in deoxygena ed H 2 O (3% DMSO, 10 −5 M) a 25 ◦C. a Complex λ abs (nm)/ ε 10 −3 (M −1 ⋅cm −1 ) λ em [nm] Δλ [nm] [d] ϕ PL [%] [c] τ 1/2 [ns] [d] π → π * MLCT [P -MeIm] 232(19.87), 247(21.90) ◆ , 322(5.71) s 364(1.88)* 478, ◆ 512 ◆ 103, 137 6.43 3014 [P -CTZ] 235(34.13) ◆ , 235(21.81) s , 322(7.13) s 390(1.13)* 476, 510 101, 135 4.21 166 [P -BFZ] 232(37.45), 256(41.96) ◆ , 326 s (10.78), 355 (7.98) 392(6.75)* 491, 529, 602* 116, 154, 227 2.58 267 a λ exc =375 nm, (s) Shoulde , *B oad band, ◆ =maximum. Table 3 IC 50 ( μ M) o he P (II) complexes in di e en cell lines a e 24 h o exposu e ime. SW480 A549 A2780 [P -MeIm] 11.6 ±3.5 22.6 ±1.8 7.0 ±0.6 [P -CTZ] 61.5 ±4.0 50.6 ±1.6 68.7 ±4.0 [P -BFZ] 38.3 ±3.3 19.0 ±1.4 6.1 ±0.4 cispla in 45.1 ±9.1 38.8 ±1.2 10.5 ±0.5 [P -M e Im ] [P -C T Z ] [P -B F Z ] [C D D P ] 0 .0 0 .5 1 .0 1 .5 2 .0 n m o le s P /1 0 6ce lls Fig. 3. Me al accumula ion de e mined by ICP-MS in A549 cells a e 24 h o ea men wi h 2 μ M o he P complexes. Fig. 4. Na i e PAGE o BSA incuba ed o e nigh wi h P -complexes. C BSA =1.5 μ M, [P -complex]/[BSA] a ios: 10, 25 and 50, C DMSO =0.2%. N. Fe n´ andez-Pampín e al. Jou nal o Ino ganic Biochemis y 226 (2022) 111663 7 changes in he BSA s uc u e acco ding o he na i e PAGE esul s. These esul s allowed us o explain quali a i ely he la ge di e ences in me al accumula ion obse ed in he up ake s udies (see Fig. 3). The highe he in e ac ion o he complexes wi h BSA, he lowe hei accumula ion inside cells. Al hough he an i umo al ac i i y o [P -MeIm] has been p e iously desc ibed in leukaemia umo cells [45], he e is no e idence o i s in e ac ion wi h DNA. To explo e he po en ial in e ac ions o he P (II)- imidazolyl complexes wi h DNA hey we e incuba ed wi h he plasmid DNA (pUC18) a 37 ◦C o e nigh and, hen, hei elec opho e ic mobili y h ough an aga ose gel was e alua ed (Fig. 5). The p esence o he imidazolyl-P (II) complexes a ec s he DNA mig a ion in a concen- a ion dependen manne , sugges ing con o ma ional modi ica ions in he s anda d double s anded DNA. All o hem induced condensa ion o he Open Ci cula (OC) band and e a da ion in he mig a ion o he Supe -coiled (SC) band. These changes a e compa ible wi h DNA co a- len binding. In o de o con i m hese esul s, Ci cula Dich oism (CD) expe i- men s we e pe o med. The P (II)-complexes modi ied he mola ellip- ici y o he DNA double helix as a unc ion o he incuba ion ime (Figu e SI22). This ea u e oge he wi h he obse ed changes in he mig a ion pa e ns o he elec opho esis expe imen s indica e a co a- len P -DNA binding. Mo e speci ically [P -MeIm] induces he mos p onounced s uc u al changes in DNA con o ma ion, while [P -CTZ] causes he lowes impac in DNA con o ma ion. The e o e, he co alen P -DNA binding could be one o he mechanisms o cy o oxici y ope - a ing in he case o hese P -complexes. Ne e heless, i is insu icien o jus i y he pa adoxical high cy o oxici y o [P -BFZ] conside ing i s educed cellula up ake and mode a e in e ac ion wi h DNA. 3.5.4. ROS p oduc ion The gene a ion o eac i e oxygen species (ROS) is o en in ol ed in he mechanism o ac ion o many cy o oxic agen s [56], some P (II) de i a i es among hem [35,57,58,59]. Al hough ROS play impo an oles in li ing cells, an abno mal inc ease o hese species can gene a e damage on DNA, lipids and p o eins. These oxygen species can p oduce oxida i e s ess al e ing he edox s a us o he umo cells and ig- ge ing hei p og ammed dead [60]. Thus, he le els o in acellula ROS in A549 umo cells was measu ed by means o wo di e en p obes: H 2 DCFDA (2′,7′-dichlo - odihyd o luo escein diace a e), ha allows o measu e hyd ogen pe oxide (H 2 O 2 ) and o he adical oxida i e species, and DHE (dihy- d oe hidium), o supe oxide anion (O 2•- ) de ec ion [61]. Cy osolic supe oxide le els we e e alua ed in A549 cells ea ed wi h he imidazolyl-P (II) complexes a hei co esponding IC 50 alues du - ing 2 h by luo escence measu emen s. As a esul , only [P -BFZ] was able o signi ican ly inc eased O 2•- le els (Fig. 6A). These esul s we e also con i med by low cy ome y expe imen s (Fig. 6B) in which a dose dependen inc ease in he O 2•- p oduc ion was obse ed o [P -BFZ], whe eas [P -MeIm] and [P -CTZ] induce no e ec e en a wo- old he IC 50 alue. As a o e-men ioned, o e alua e he gene a ion o o he ROS, A549 cells we e ea ed wi h H 2 DCFDA and wi h he P (II) complexes a hei co esponding IC 50 alues o wi h TBH ( e -bu yl hyd ope oxide) as posi i e con ol du ing 4 h. [P -CTZ] do no induce signi ican di e - ences in ROS le els compa ed o un ea ed cells (Fig. 7). By con as , he gene a ion ROS was clea ly con i med o [P -MeIm] and [P -BFZ]. Indeed, [P -BFZ] is he mos po en H 2 O 2 and o he eac i e oxygen species gene a o explaining is high cy o oxici y. Thus, he capaci y o [P -BFZ] o induce oxida i e s ess seems o be he dominan ac o in i s an icance ac i i y. The e o e, a dual mechanism, ha is o say co- alen binding o DNA and ROS gene a ion, could be esponsible o he high cy o oxici y o [P -MeIm] and [P -BFZ]. 3.5.5. E alua ion o mi ochond ial memb ane po en ial (MMP) The main sou ce o in acellula ROS a e he mi ochond ia, and hei accumula ion on hem can al e mi ochond ia dynamics p o oking mi ochond ia memb ane depola iza ion, along wi h mi ochond ial dys unc ion and apop osis igge ed by he mi ochond ial pa hway. As a ma e o ac , apop osis induced by oxida i e s ess on mi ochond ia is a sui able app oach o cance ea men [62]. One o he e ec s o ROS gene a ion may be he al e a ion o he mi ochond ial memb ane po- en ial (MMP). So, we ha e e alua ed changes o he MMP (ΔѰ) by using he e ame hyl hodamine me hyl es e (TMRM) p obe, which is accumula ed inside he mi ochond ia due o i s ca ionic na u e [63]. T ea men o A459 cells wi h [P -CTZ] seems o ha e no e ec in MMP, Fig. 5. Aga ose elec opho esis o he plasmid DNA (pUC18) incuba ed o e - nigh in he absence o P (II)-complexes (lane 1) and in he p esence o di e en concen a ions o [P -MeIm] (lanes 2–5), [P -CTZ] (lanes 6–9), [P -BFZ] (lanes 10–13) and CDDP (lanes 14 and 15). C D /C P s ands o he P (II) complex/ pUC18 concen a ions a io. Cispla in (CDDP) was included as posi i e con ol. Fig. 6. De ec ion o supe oxide anion by DHE a e 2 h exposu e o A549 cells o he imidazolyl-P (II) complexes. A) Rela i e luo escence o ea ed cells a he IC 50 alues o he P (II) complexes ela i e o con ol cells. ** S a is ical signi icance p- alue <0.05 ( - es ). B) Flow cy ome y plo s a he IC 50 and 2- old he IC 50 alues. Fig. 7. De ec ion o ROS by H 2 DCFDA a e 4 h exposu e o A549 cells o he imidazolyl-P (II) complexes. ** S a is ical signi icance p- alue <0.05 (ANOVA es wi h Dunne 's co ec ion). N. Fe n´ andez-Pampín e al. Jou nal o Ino ganic Biochemis y 226 (2022) 111663 8 whe eas [P -BFZ] led o a dec eased in ΔѰ ela i e o ehicle- ea ed cells in a dose dependen manne (Fig. 8). Consequen ly, his ΔѰ is hough o a ec bo h mi ochond ial in eg i y and unc ionali y. By con as , he opposi e end is obse ed in cells ea ed wi h [P -MeIm]. The hype pola iza ion o he mi ochond ial memb ane is la ge as he concen a ion is inc eased, being ex emely high a 2- old he IC 50 alue. An enhanced ΔѰ indica es ha he mi ochond ial unc ion is inc eased and a collapse in he mi ochond ia bioene ge ics may occu . 3.5.6. Cell cycle analysis Cell-cycle dys egula ion is one o he p ime ea u es o cance cells. Since cell cycle a es ende s umo cells suscep ible o apop osis, a - ge ing cell cycle phases and checkpoin s is one p omising app oach in an icance he apy [64]. Indeed, he cy o oxici y o many an icance d ugs is ela ed o DNA damage and cell cycle pe u ba ion [65]. To be e unde s and he biological e ec o he imidazolyl-P (II) com- plexes, he impac o hese compounds a IC 50 alues on A549 cell cycle dis ibu ion was e alua ed a e 24 h o incuba ion ime. The esul s indica ed ha complexes [P -MeIm] and [P -BFZ] lead o accumula- ion o ea ed cells a he G 0 /G 1 phase (Fig. 9). Compa ed wi h he ehicle- ea ed con ol (DMSO), he pe cen age o cells in G 0 /G 1 phase inc eased by 15.4% and 9.4% o [P -MeIm] and [P -BFZ], espec- i ely, while he numbe o cells in he S phase dec eased acco dingly. G 0 /G 1 cell cycle a es was p e iously desc ibed o se e al o ganome- allic P (II) complexes [66] as well as o imidazole an i ungal d ugs, such as Clo imazole, in some cance cell lines [67,68,69]. By con as , ea men wi h [P -CTZ] does no he block cell cycle p og ession. 3.5.7. Apop osis induc ion The abili y o inducing apop osis (p og ammed cell dea h) was e alua ed o he P (II) complexes by low cy ome y using Annexin V s aining, being his p obe able o bind he anionic phospholipid phos- pha idylse ine in he cell su ace o apop o ic cells [70]. The ga he ed esul s in Fig. 10 show ha ea men o A549 cells wi h hal he maximal inhibi o y concen a ion o he P (II) complexes caused he highes accumula ion in ea ly apop osis o [P -BFZ] and [P -MeIm]. In he case o [P -CTZ] ea men , simila amoun s o cells in la e apop osis and nec osis we e obse ed, while he pe cen age o cells in ea ly apop osis is smalle . Thus, he apop osis induc ion pa hway o [P -CTZ] may be di e en om ha o [P -BFZ] and [P -MeIm]. The obse ed esul s sugges ha [P -BFZ] and [P -MeIm] p obably induce oxida i e s ess since ROS le els a e inc eased and he MMP is a ec ed. Hence, we specula e ha apop osis may be igge ed by he mi ochon- d ial pa hway upon ea men wi h hese wo complexes. 4. Conclusion In his wo k h ee neu al P (II) complexes con aining imidazolyl d ugs ha e been syn hesized and cha ac e ized. The c ys al s uc u es o [P -CTZ] and [P -BFZ] ha e been esol ed by X-Ray di ac ion. These complexes exhibi high s abili y in wa e and in bu e ed solu ions. The elec onic and pho ophysical s udies ha e shown ha [P -MeIm] is phospho escen in deoxygena ed H 2 O ( τ 1/2 =3 μ s), whe eas he o he wo cyclome ala ed P (II) complexes con aining an i ungal imidazolyl ligands, [P -CTZ] and [P -BFZ], a e also pho oluminescen bu exhibi lowe exci ed s a e li e imes. [P -MeIm] and [P -BFZ] p esen highe cy o oxici y han [P -CTZ] and cispla in agains colon adenoca cinoma (SW480), o a ian cance (A2780) and lung adenoca cinoma (A549) cell lines. Despi e hei simila IC 50 alues, [P -MeIm] and [P -BFZ] ea u e dissimila cellula up ake beha io s, [P -MeIm] is highly accumula ed bu [P -BFZ] is poo ly in e nalized inside he cells a e 24 h o incuba ion. Indeed, we pos ula e ha he cellula up ake o he la e is s ongly hinde ed by i s in e ac ion wi h plasma p o eins. DNA may no be he main a ge o hese complexes since all o hem a e able o in e ac wi h DNA by co alen binding inducing simila con o ma- ional changes on he DNA double helix, and he cy o oxici y o [P - Fig. 8. Rela i e MMP (ΔѰ) o A549 cells ea ed wi h he P (II) complexes a hei IC 50 and 2- old IC 50 alues ela i e o un ea ed cells. Vehicle ea ed (0.5% DMSO). Da a we e ob ained om duplica es o wo di e en expe imen s and plo ed as mean alues wi h s anda d de ia ion. **** (p- alue <0.001) and ** (p- alue <0.05) (ANOVA es wi h Dunne 's co ec ion). Fig. 9. A549 cells dis ibu ion in G 0 /G 1 , S and G 2 /M phases upon ea men wi h he ehicle (0.5% DMSO) and he pla inum complexes unde s udy a he IC 50 alues. Da a we e ob ained om duplica es o h ee di e en expe imen s and plo ed as mean alues wi h s anda d de ia ion. **** (p- alue <0.0001) and *** (0.0001 ≥p- alue <0.001) ex emely signi ican and ** (0.001 ≥p- alue <0.01) e y signi ican di e ences (ANOVA es wi h Dun- ne 's co ec ion). % o C e lls A li e Ea ly a p o p o s is La e A p o p o s is N e c o s is 0 2 0 4 0 6 0 8 0 100 V e h ic le [P -M e -Im ] [P -C T Z ] [P -B F Z ] Fig. 10. Flow cy ome y esul s exp essed as pe cen age o A549 cells in ali e, ea ly apop o ic, la e apop o ic and nec o ic phases. Da a we e ob ained om duplica es o wo di e en expe imen s and plo ed as mean alues wi h s an- da d de ia ion. N. Fe n´ andez-Pampín e al. Jou nal o Ino ganic Biochemis y 226 (2022) 111663 9 CTZ] is low. Howe e , only [P -MeIm] and [P -BFZ] cause cell cycle pe u ba ion wi h a signi ican accumula ion o cells in G 0 /G 1 . Bo h induce changes in he mi ochond ial memb ane po en ial, and hey seem o induce apop osis by he mi ochond ial pa hway whe eas he pa hway o [P -CTZ] in apop o ic induc ion may be a di e en one. On he o he hand, only [P -MeIm] and [P -BFZ] a e able o cause ROS inc ease. The less accumula ed, bu high cy o oxic complex, [P -BFZ] shows a ma ked inc ease in ROS le els in compa ison o [P - MeIm]. In ac , [P -BFZ] is he only de i a i e ha inc eases O 2•- le els. Thus, ROS gene a ion seem o be he key o hei biological ac i i y. Au ho Con ibu ions ‡N. F – P and M. V. con ibu ed equally. The manusc ip was w i en h ough con ibu ions o all au ho s. All au ho s ha e gi en app o al o he inal e sion o he manusc ip . Decla a ion o Compe ing In e es The au ho s decla e ha hey ha e no known compe ing inancial in e es s o pe sonal ela ionships ha could ha e appea ed o in luence he wo k epo ed in his pape .. Acknowledgmen s The au ho s g a e ully acknowledge he inancial suppo by La Caixa Founda ion (LCF/PR/PR12/11070003), Conseje ía de Educaci´ on- Jun a de Cas illa y Le´ on-FEDER (BU042U16-BU305P18), Minis e io de Ciencia, Inno aci´ on y Uni e sidades (RTI2018-102040-B-100). M.V. is g a e ul o he inancial suppo ecei ed om he Conseje ía de Edu- caci´ on-Jun a de Cas illa y Le´ on-FEDER (BU042U16-BU305P18). We a e indeb ed o P. Cas o iejo and M. Mansilla (PCT o he Uni e sidad de Bu gos) o he echnical suppo . Appendix A. Supplemen a y da a Supplemen a y da a o his a icle can be ound online a h ps://doi. o g/10.1016/j.jino gbio.2021.111663. Re e ences [1] P.M. Buchel, W. D abe , E. Regel, A zneimi el o schung 22 (1972) 1260–1272. [2] J.M. To es-Rod íguez, A ch. Med. Res. 24 (1993) 371–375. [3] P.D. C owley, H.C. Gallaghe , J. Appl. Mic obiol. 117 (2014) 611–617. [4] S. Kada akollu, C. S ailey, C.S. Kunapa eddy, S. Whi e, Med. Chem. 4 (2014) 722–724. [5] J.S. Cheng, C.-T. Chou, W.-Z. 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