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