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. Liang, C.-C. Kuo, P. Shieh, D.-H. Kuo, C.-R. Jan,
J. Recep . Signal T ansduc . 34 (2014) 449–493.
[6] J. Penso, R. Bei ne , Eu . J. Pha macol. 342 (1998) 113–117.
[7] T. Gagini, L. Colina-Vegas, W. Villa eal, L.P. Bo ba-San os, C. De Souza Pe ei a, A.
A. Ba is a, M. Kneip Fleu y, W. De Souza, S. Rozen al, L.A.S. Cos a, e al., New J.
Chem. 42 (2018) 13641–13650.
[8] R. S´
anchez-Delgado, M. Na a o, K. Laza di, R. A encio, M. Cappa elli, F. Va gas,
J. U bina, A. Bouillez, A.F. Noels, D. Masi, Ac a 275–276 (1998) 528–540.
[9] M. Na a o, T. Lehmann, E.J. Cisne os-Faja do, A. Fuen es, R.A. S´
anchez-Delgado,
P. Sil a, J.A. U bina, Polyhed on 19 (2000) 2319–2325.
[10] A. Ma ínez, T. Ca eon, E. Iniguez, A. Anzello i, A. S´
anchez, M. Tyan, A. Sa le ,
L. He e a, R.A. Maldonado, R.A. S´
anchez-Delgado, J. Med. Chem. 55 (2012)
3867–3877.
[11] R.A. S´
anchez-Delgado, K. Laza di, L. Rinc´
on, J.A. U bina, A.J. Hube , A.N. Noels,
J. Med. Chem. (1993) 2041–2043.
[12] E. Iniguez, A. Va ela-Rami ez, A. Ma ínez, C.L. To es, R.A. S´
anchez-Delgado, R.
A. Maldonado, Ac a T op. 164 (2016) 402–410.
[13] J. Kljun, A.J. Sco , T. Laniˇ
snik Riˇ
zne , J. Keise , I. Tu el, O ganome allics 33
(2014) 1594–1601.
[14] L. Colina-Vegas, J. Lima P ado Godinho, T. Cou inho, R.S. Co ea, W. De Souza, J.
Cola Fe nandes Rod igues, A.A. Ba is a, M. Na a o, New J. Chem. 43 (2019)
1431–1439.
[15] E. Rod íguez A ce, C. Sa nigue , T.S. Mo aes, M. Viei es, A.I. Tomaz, A. Medei os,
M.A. Comini, J. Va ela, H. Ce ece o, M. Gonz´
alez, F. Ma ques, M.H. Ga cía,
L. O e o, D. Gambino, J. Coo d. Chem. 68 (2015) 2923–2937.
[16] M. Na a o, I. Colmena es, H. Co eia, A. He n´
andez, Y. Ching, Y. Mill´
an, L. Ojeda,
M. Vel´
asquez, G. F aile, A zneimi el o schung 54 (2012) 746–751.
[17] E. Robles-Escajeda, A. Ma ínez, A. Va ela-Rami ez, R.A. S´
anchez-Delgado, R.
J. Aguile a, Cell Biol. Toxicol. 29 (2013) 431–433.
[18] M. S asbe g Riebe , A. Anzello i, R.A. S´
anchez-Delgado, M. Riebe , In . J. Cance
112 (2004) 376–384.
[19] L. Colina-Vegas, J.L. Du a, W. Villa eal, J.H. De A. Ne o, M.R. Comine i,
F. Pa an, M. Na a o, A.A. Ba is a, J. Ino g. Biochem. 162 (2016) 135–145.
[20] M. Na a o, E.J. Cisne os-Faja do, T. Lehmann, R.A. S´
anchez-Delgado, R. A encio,
P. Sil a, R. Li a, J.A. U bina, Ino g. Chem. 40 (2001) 6879–6884.
[21] H.A. Mohamed, S. Shephe d, N. William, H.A. Blundell, M. Das, C.M. Pask, B.R.
M. Lake, R.M. Phillips, A. Nelson, C.E. Willans, O ganome allics 39 (2020)
1318–1331.
[22] M. Na a o, N.P. Pe˜
na, I. Colmena es, T. Gonz´
alez, M. A senak, P. Taylo , J. Ino g.
Biochem. 100 (2006) 152–157.
[23] P.V. Simpson, C. Nagel, H. B uhn, U. Scha zschneide , O ganome allics 34 (2015)
3809–3815.
[24] H.F. Abd El-Halim, F.A. Nou El-Dien, G.G. Mohamed, N.A. Mohmed, Syn h. Reac .
Ino ganic, Me . Nano-Me al Chem. 41 (2011) 544–554.
[25] S. Be anzos-La a, N.P. Chmel, M.T. Zimme man, L.R. Ba ´
on-Sosa, C. Ga ino,
L. Salassa, A. Rodge , J.L. B umaghim, I. G acia-Mo a, N. Ba ba-Beh ens, Dal on
T ans. 44 (2015) 3673–3685.
[26] S. Be anzos-La a, C. G´
omez-Ruiz, L.R. Ba ´
on-Sosa, I. G acia-Mo a, M. Flo es-
´
Alamo, N. Ba ba-Beh ens, J. Ino g. Biochem. 114 (2012) 82–93.
[27] V. Midlej, F. Rubim, W. Villa eal, ´
E.S. Ma ins-Dua e, M. Na a o, W. de Souza,
M. Benchimol, Pa asi ology 146 (2019) 1206–1216.
[28] M. Na a o, A.R. Higue a-Padilla, M. A senak, P. Taylo , T ansi . Me . Chem. 34
(2009) 869–875.
[29] A. Annunzia a, M.E. Cuccioli o, R. Esposi o, G. Fe a o, D.M. Mon i, A. Me lino,
F. Ru o, Eu . J. Ino g. Chem. (2020) 918–929.
[30] G. Facche i, I. Rimoldi, Bioo g. Med. Chem. Le . 29 (2019) 1257–1263.
[31] M.V. Babak, M. P a enede -Kmen, S.M. Meie -Menches, M.S. Legina, S. Theine ,
C. Licona, C. O ain, M. Hejl, M. Hani , M.A. Jakupec, B.K. Kepple , C. Gaiddon, C.
G. Ha inge , Ino g. Chem. 57 (2018) 2851–2864.
[32] A.T. Dha ma aja, J. Med. Chem. 60 (2017) 3221–3240.
[33] B. Pe illo, M. Di Dona o, A. Pezone, E. Di Zazzo, P. Gio annelli, G. Galasso,
G. Cas o ia, A. Migliaccio, Exp. Mol. Med. 52 (2020) 192–203.
[34] M. Vaque o, N. Bus o, N. Fe n´
andez-Pampín, G. Espino, B. Ga cía, Ino g. Chem. 59
(2020) 4961–4971.
[35] M. Ro hemund, S.I. B¨
a , T. Rehm, H. Kos huno a, V. B abec, R. Schobe , Dal on
T ans. 49 (2020) 8901–8910.
[36] M. Clemen e, I. Halil Pola , J. Albe , R. Bosque, M. C espo, J. G anell, C. L´
opez,
M. Ma ínez, J. Qui an e, R. Messegue , C. Cal is, J. Badía, L. Baldoma, M. Fon -
Ba dia, M. Cascan e, O ganome allics 37 (2018) 3502–3514.
[37] SAINT A ea-De ec o In eg . P og am, SAINT+ 7.12a, B uke AXS Inc, Madison,
WI, 2004.
[38] G.M. Sheld ick, SADABS A P og . Empi . Abso p . Co ec . Ve sion 2004/ 1, Uni .
Go ingen, Go ingen, Ge many, 2004.
[39] L. Fa ugia, J. IUC . WinGX and ORTEP o windows: an upda e, J. Appl.
C ys allog . 45 (2012) 849–854.
[40] O.V. Dolomano , L.J. Bou his, R.J. Gildea, J.A.K. Howa d, H. Puschmann, J. Appl.
C ys allog . 42 (2009) 339–341.
[41] M. Ma ínez-Alonso, N. Bus o, L.D. Agui e, L. Be langa, M.C. Ca i´
on, J.V. Cue as,
A.M. Rod íguez, A. Ca bayo, B.R. Manzano, E. O í, F.A. Jal´
on, B. Ga cía,
G. Espino, Chem. Eu .J. 24 (2018) 17523–17537.
[42] H.M.E. Azzazy, R.H. Ch is enson, Clin. Chem. 43 (1997) 2014–2016.
[43] G. Felsen eld, S.Z.A. Hi schman, S. Z. A., J. Mol. Biol. 13 (1965) 407–427.
[44] F. Niede mai , K. Waich, S. Kappaun, T. May , G. T immel, K. Me ei e , C. Slugo c,
Ino g. Chim. Ac a 360 (2007) 2767–2777.
[45] A. Esmaeilbeig, H. Samouei, S. Abedanzadeh, Z. Ami gho an, J. O ganome .
Chem. 696 (2011) 3135–3142.
[46] T. Okada, I.M. El-Mehasseb, M. Kodaka, T. Tomohi o, K. Okamo o, H. Okuno,
J. Med. Chem. 44 (2001) 4661–4667.
[47] R.R. Pa ke , J.P. Sa ju, A.C. Whi wood, J.A.G. Williams, J.M. Lynam, D.W. B uce,
Chem. – A Eu . J. 24 (2018) 19010–19023.
[48] N. Godbe , T. Pugliese, I. Aiello, A. Bellusci, A. C ispini, M. Ghedini, Eu . J. Ino g.
Chem. (2007) 5105–5111.
[49] J.-Y. Cho, K.Y. Suponi sky, J. Li, T.V. Timo ee a, S. Ba low, S.R. Ma de ,
J. O ganome . Chem. 690 (2005) 4090–4093.
[50] P.-I. K am, M.V. Puzyk, K.P. Balashe , Songs ad, Ac a Chem. Scand. 49 (1995)
335–343.
[51] D.M. Jenkins, J.F. Senn, S. Be nha d, Dal on T ans. 41 (2012) 8077–8085.
[52] S. Chen, R. Fn, X. Wang, Y. Yang, Ino g. Chem. Commun. 44 (2014) 101–106.
[53] S. Cu y, D ug Me ab. Pha macokine . 24 (2009) 342–357.
[54] G. Fanali, A. di Masi, V. T ezza, M. Ma ino, M. Fasano, P. Ascenzi, Mol. Asp. Med.
33 (2012) 209–290.
[55] J.J. Gullo, C.L. Li e s , P.J. Magui e, B.I. Sikic, D.F. Ho h, P.V. Woolley, Cance
Chemo he . Pha macol. 5 (1980) 21–26.
[56] D. T achoo ham, J. Alexand e, P. Huang, Na . Re . D ug Disco . 8 (2009) 579–591.
[57] J. Li, X. He, Y. Zou, D. Chen, L. Yang, J. Rao, H. Chen, M.C.W. Chan, L. Li, Z. Guo,
L.W. Zhang, C. Chen, Me allomics 9 (2017) 726–733.
[58] K.-B. Huang, F.-Y. Wang, H.-W. Feng, H. Luo, Y. Long, T. Zou, A.S.C. Chan, R. Liu,
H. Zou, Z.-F. Chen, Y.-C. Liu, Y.-N. Liu, H. Liang, Chem. Commun. 55 (2019)
13066–13069.
[59] M.J.R. Tham, M.V. Babak, W. Ang, Angew. Chem. In . Ed. 59 (2020) 19070–19078.
[60] B. Pe illo, M. Di Dona o, A. Pezone, E. Di Zazzo, P. Gio annelli, G. Galasso,
G. Cas o ia, A. Migliaccio, Exp. Mol. Med. 52 (2020) 192–203.
N. Fe n´
andez-Pampín e al.