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Synthesis, structural analysis, solution equilibria and biological activity of rhodium(iii) complexes with a quinquedentate polyaminopolycarboxylate
RSC Advances
PAPER
Synthesis, structural analysis, solution equilibria
and biological activity of rhodium( ) complexes
III
Citethis:RSCAdv.,2017,7,5282 with a quinquedentate polyaminopolycarboxylate †
Marija S. Jeremic´,a Hubert Wadepohl,b Vesna V. Kojic´,c Dimitar S. Jakimov,c
Ratomir Jelic´,d Suzana Popovic´,d Zoran D. Matovic´ *a and Peter Comba*b
Two rhodium(III) complexes [Rh(ed3a)(OH
2
)]$H
2
O (1) and Na[Rh(ed3a)Cl]$H
2
O (2) with ethylenediamine-
N,N,N0-triacetate (ed3a) have been synthesized and characterized by elemental, spectroscopic and
structural analyses. The crystal structure of (1) and (2) and the spectroscopic analysis of the two
rhodium(III)–ed3a complexes are discussed in detail. The protonation constants of H
3
ed3a and the
conditional stability constants of its RhIII complexes have been determined in aqueous solution by pH
potentiometry and UV-Vis spectrophotometry. Molecular mechanics (MM) and density functional theory
(DFT)havebeenusedtomodelallpossiblegeometricisomers,determinetheglobalenergyminimumand
comparethecomputedwiththeexperimentallyobservedstructures.ThecytotoxicactivityofthenewRhIII
complexes was evaluated by an MTT assay against four human cancer lines (MCF-7, A549, HT-29 and
HeLa)andanormalhumancellline(MRC-5).A549,HT-29andHeLacellsweresensitivetoallcompounds
tested, while the breast carcinoma cell line MCF-7 was only sensitive to the reference compounds
(doxorubicinandcisplatin).Westernblot(WB)analysisoftheeffectsofthetestedcompoundsindicatesthat
Received2ndNovember2016
bothcomplexesincreasetheexpressionofcaspase3andconsequentlytheinvolvementofthisenzymein
Accepted23rdDecember2016
apoptotic processes of the treated cells. WB also demonstrates proteolytic cleavage ofpoly-(ADP-ribose)
DOI:10.1039/c6ra26199j polymerase (PARP) in HeLa cells after treatment with both tested substances. Flow cytometry confirmed
www.rsc.org/advances apoptoticcelldeathandshowedtheinductionofcellcycleterminationasapossiblepromoterofapoptosis.
Introduction (Fig. 1). Most of the reported [M(ed3a-type)X] complexes (M ¼
CoIII,CrIII,CuII,NiIIandX¼monodentateligand)havethecis-
For a multitude of reasons, transition metal complexes with
equatorialconguration.3–5
edta-type aminopolycarboxylate ligands have attracted consid- The success of cisplatin as antitumor agent has stimulated
erable attention and have been extensively investigated and enormous efforts to designing and preparing other clinically
reviewed.1,2 The best studied quinquedentate ligand system is useful metal complexes.6–10 RhIII coordination compounds are
ethylenediamine-N,N,N0-triacetate(ed3a)anditsderivatives.For isoelectronic with RuII and PtIV complexes, which provide
octahedral metal complexes with pentadentate coordinated a range of active antitumor agents.11,12 Generally, they are
symmetrical edta- (ethylenediamine-N,N,N0,N0-tetraacetate) octahedralandinertbutmanyRhIIIcomplexesshowconsider-
or ed3a-type ligands three possible geometric isomers,
ableantitumorandantimicrobialactivities.11,12Therstreport
cis-equatorial, trans-equatorial and cis-polar are possible of an antitumor active RhIII complex, RhCl 3 $3H 2 O,13 appeared
before Rosenberg's discovery of cisplatin.6,7 Simple complexes
aUniversityofKragujevac,FacultyofScience,DepartmentofChemistry,R.Domanovi´ca
12,34000Kragujevac,Serbia.E-mail:zmatovic@kg.ac.rs
bUniversita¨tHeidelberg,Anorganisch-ChemischesInstitutandInterdisciplinaryCenter
for Scientic Computing (IWR), Im Neuenheimer Feld 270, D-69120, Heidelberg,
Germany.E-mail:Peter.Comba@aci.uni-heidelberg.de
cOncology Institute of Vojvodina, Faculty of Medicine, University of Novi Sad, Dr
Goldmana4,21204SremskaKamenica,Serbia
dUniversity of Kragujevac, Faculty of Medical Sciences, S. Markovi´ca 69, 34000
Kragujevac,Serbia
†Electronicsupplementaryinformation(ESI)available:Furthersolutionstudy
details, IR,1H NMR, 13CNMR data. CCDC 1412103 and 1412104 for thenew
compounds are included. For ESI and crystallographic data in CIF or other Fig. 1 Geometrical isomerism of six-coordinate [M(ed3a-type)Xn]
electronicformatseeDOI:10.1039/c6ra26199j complexes:n¼1.
5282 | RSCAdv.,2017,7,5282–5296 Thisjournalis©TheRoyalSocietyofChemistry2017
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Paper RSCAdvances
suchasmer-[RhCl (NH ) ]ormer,cis-[RhCl (DMSO) (NH )]are
3 33 3 2 3
alsoknowntobeanticanceractive.14,15Earlyworkupto2002on
rhodiumanticancercomplexesintheoxidationstates+1to+3
was summarized in two review articles.16,17 A large number of
complexes of rhodium has been explored for antitumor
activity.18 Numerous half-sandwich complexes of Rh such as
[(h5-C Me )Rh(LL)Cl], where (LL) is a bidentate polypyridyl
5 5
ligand were prepared and studied in terms of their anti-
proliferative properties in human cancer cell lines.19–22 Rho-
dium(III) complexes appeared to be a good inhibitors of the
kinase,inhibitors ofotherenzymes andinhibitors ofprotein–
protein interactions.22 Data on the antimalignant activity of
rhodium(III)chelatedbyEDTA-typeligandsisveryscarce.
Here,wereportonthesynthesisandcoordinationchemistry
of RhIII complexes of H 3 ed3a and their medicinal/biological Scheme1 Synthesisofed3a3(cid:2)andthecorrespondingRhIIIcomplexes
properties.Thecharacterizationismainlybasedontheoctahe- (1)and(2).
dral complexes [Rh(ed3a)(OH )]$H O (1) and Na[Rh(ed3a)Cl]$ 2 2
H O (2). These contain fully deprotonated quinquedentate
2 ligands,whichmayformfourve-memberedchelaterings.The Descriptionofthecrystalstructures
IR,NMRandelectronicspectraofthesecomplexesarediscussed Astructuraldiagramofthecis-equatorial-[Rh(ed3a)(OH 2 )]$H 2 O
in relation to their geometry, and the formation constants are (1)andcis-equatorial-Na[Rh(ed3a)Cl]$H 2 O(2)withtheadopted
used to estimate the in vivo stabilities of the rhodium(III) atom-numbering scheme is shown in Fig. 2, along with the
complexes. Also reported are the results of the two complexes' packinginthecrystalsof(1)and(2).Selectedbondlengthsand
cytotoxicity in vitro towards diverse tumour cell lines and rst valenceanglesarelistedinTable1.
resultsonthemechanismsofantiproliferativeactivityofthenew
RhIII complexes against human cervix adenocarcinoma cells.
Theeffectsonexpressionofproteinsincludedinapoptoticsig-
nalling pathways (Bcl-2, Bax, caspase-3, and poly(ADP-ribose)
polymerase, PARP) as well as cell cycle phase distribution of
HeLacellsweremonitored.AWBanalysiswasusedtoevaluate
theexpressionlevelsofapoptosis-associatedproteins.
Results and discussion
Coordinationchemistry
Chelates of ed3a-type ligands can be prepared: (a) by the
condensation method, starting from a neutralized a- or b-
monohalogencarboxylic acid and the corresponding diamine,
(b) by condensation ofacrylic acid and a diamine forchelates
with propionate arms or (c) by condensation of dihalogen
derivatives of the diamine with various amino acids. The
quinquedentate
ed3a3(cid:2)
was prepared by the condensation
method,from1,2-diaminoethaneandneutralizedchloroacetic
acid.5 The ligand was isolated as the calcium salt Ca (ed3a) -
3 2
$12H O and transformed in the two complexes (1) and (2) as
2
outlined in Scheme 1. The reaction of RhCl $H O and the
3 2
quinquedentate
ed3a3(cid:2)
produced a mixtureoftwocomplexes,
and chromatography was used to separate the two complexes.
The mixture was passed through a column of QAE A-25
Sephadex in the Cl (cid:2) form. The yellow bands with different
charges, i.e. neutral cis-equatorial-[Rh(ed3a)H O]$H O (1) and
2 2
anioniccis-equatorial-Na[Rh(ed3a)Cl]$H O(2), wereseparated.
2
Aer desalting by passage through a Sephadex G-10 column,
Fig.2 Ortep diagramof the [Rh(ed3a)(OH )] molecular complex (a)
we were able to isolate crystals of (1) and (2) suitable for and[Rh(ed3a)Cl](cid:2)complexanion(b)andcr 2 ystalpackingviewsalong
X-ray analysis, and the complexes were also characterized by aaxis(for[Rh(ed3a)(OH )])andbaxis(for[Rh(ed3a)Cl](cid:2))(50%proba-
2
elementalanalysis,IR,UV-VisandNMRspectroscopy. bilityellipsoids).
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Table1 Selectedbonddistancesandanglesfor[Rh(ed3a)(OH 2 )]$H 2 O conformation are q2 > 0 A ˚ , 42 ¼ 0(cid:3), and for a twisted confor-
(1)andNa[Rh(ed3a)Cl]$H 2 O(2) mationq2>0A ˚ ,42¼90(cid:3)[p/2]),24whichrelatetodeviationsof
theringatomsfromthemeanplane,areq2¼0.443(1)A ˚ and42
(1) (2) ¼281.0(2)(cid:3)for(1)andq2¼0.455(2)A ˚ and42¼259.1(3)(cid:3)for(2).
M–Lbondlengths(A ˚ ) The three ve-membered acetate rings have a twisted confor-
Rh–O(7) 2.072(2) Rh–Cl 2.353(1) mationin(1).Theirpuckeringparametersareq2¼0.433(5)A ˚ ,
Rh–O(2) 2.018(2) Rh–O(2) 2.008(3) 42 ¼ 153.1(7)(cid:3) (RhO C C N ); q2 ¼ 0.301(0) A ˚ , 42 ¼ 159.1(6)(cid:3)
Rh–O(4) 2.050(2) Rh–O(4) 2.074(3) (RhO C C N )andq2 4 ¼ 6 0 5 .08 2 7(4)A ˚ ,42¼228.8(0)(cid:3) (RhO C C -
Rh–O(6) 2.010(2) Rh–O(6) 2.013(3) 2 1 2 1 6 8 7
Rh–N(1) 2.027(2) Rh–N(1) 2.011(4) N 2 ).Incomplex(2),oneoftheaxialve-memberedacetaterings
Rh–N(2) 1.996(2) Rh–N(2) 2.031(4) isnearlyplanarandthesecondadoptsanenvelopeconforma-
tion(Rrings).Theequatorialacetatering(G)isinanenvelope
Valenceangles((cid:3)) conformation.Thepuckeringparametersare:q2¼0.416(8)A ˚ ,
cisangles 42 ¼ 147.6(2)(cid:3) (RhO C C N ); q2 ¼ 0.290(8) A ˚ , 42 ¼ 171.3(4)(cid:3)
O(2)–Rh–O(4) 92.01(7) O(2)–Rh–Cl 91.11(10) 4 4 3 1
O(2)–Rh–O(7) 89.03(7) O(2)–Rh–O(4) 90.05(12) (RhO 6 C 8 C 7 N 2 ).
O(2)–Rh–N(1) 83.06(7) O(2)–Rh–N(1) 85.55(14)
O(4)–Rh–O(7) 98.62(7) O(2)–Rh–N(2) 95.39(13)
O(6)–Rh–O(4) 86.64(7) O(4)–Rh–Cl 97.46(10) Spectroscopicanalysis
O(6)–Rh–O(7) 91.45(7) O(6)–Rh–Cl 89.99(10)
IR spectroscopy. The asymmetric stretching frequencies of
O(6)–Rh–N(1) 98.21(8) O(6)–Rh–O(4) 91.05(12)
N(1)–Rh–O(7) 91.14(8) O(6)–Rh–N(2) 83.30(14) the carboxylate have been used as criterion for distinguishing
N(2)–Rh–O(2) 93.66(7) N(1)–Rh–O(4) 82.97(14) between protonated (1700–1750 cm (cid:2)1) and coordinated
N(2)–Rh–O(4) 83.09(7) N(1)–Rh–O(6) 93.34(14) carboxylate(1560–1680cm (cid:2)1).5Thevibrationofve-membered
N(2)–Rh–O(6) 85.90(7) N(1)–Rh–N(2) 86.90(16) rings is at higher energy (1600–1680 cm (cid:2)1) than the corre-
N(2)–Rh–N(1) 87.42(8) N(2)–Rh–Cl 93.02(12) sponding vibration of six-membered chelate rings (1560–1600
t
O
ra
(6
n
)
s
–R
an
h
g
–
l
O
es
(2) 178.62(7) O(2)–Rh–O(6) 178.33(13) cm
(cid:2)1).5Complex(1)showsonestrongpeakat1631cm (cid:2)1,and
N(2)–Rh–O(7) 176.77(8) N(1)–Rh–Cl 176.63(10) this is assigned to an asymmetric vibration of the ve-
N(1)–Rh–O(4) 169.01(8) N(2)–Rh–O(4) 168.09(14) membered acetate rings (Fig. S1, ESI†). Complex (2) shows
oneverystrongpeakat1630cm
(cid:2)1,assignedtothecarboxylate
group of the ve-membered rings, and the weak peak at 1680
A coordination number of six is attained by RhIII in both cm (cid:2)1 indicates a cis-equatorial isomer (Fig. S2, ESI†). The
complexes, via three deprotonated oxygen atoms of the absence of other vibrations in the 1700–1750 cm (cid:2)1 area
carboxylategroups,twonitrogenatomsofthediamine,andthe suggeststhatallcarboxylategroupsarecoordinated.
sixthcoordinationplaceoccupiesawater(1)orchlorideligand Electronic spectroscopy. Data of the UV-Vis spectra of (1)
(2).Inthecrystalsof(1)ahydrogenbondingnetworkisformed, and (2) are presented in Table 2 and Fig. 3. For comparison,
whichinvolvesthecarboxylateandaminogroupsoftheed3a3(cid:2) the transitions of [Rh(Hedta)(OH 2 )] (3), with a 5-coordinate
ligandaswellasthecoordinatedandsolventwatermolecules. Hedta3(cid:2) , are also tabulated.23 The shapes of the electronic
In (2) apolymeric structure is formed,with twoofthe carbox- spectra of (1), (2) and (3) are different to previously examined
ylate groups bridging RhIII and Na+. The water molecules are RhIII complexes25 and show two almost symmetrical bands,
primarilyinvolvedinNa–O–Nabridgingviatheiroxygenatoms arisingfromspin-allowedtransitions.Althoughthecomplexes
but further hydrogen bridges are formed to the coordinated have C 1 symmetry, their electronic spectra have only one
chloride and carboxylate ligands. Na+ is seven coordinate; component in the region of the lower energy 1T 1g (O h ) transi-
intermolecular hydrogen bridges are also formed between tion, and there is no splitting of the higher energy 1T 2g (O h )
amino(Hdonor)andcarboxylategroups(Hacceptor). absorption band. In holohedrized (O h ) symmetry only one
Thepositionsofthecarboxylategroupsdenethecis-equa- component is predicted in this region for complexes of edta-
torialgeometry.TwoacetateRrings(out-of-planeglycinatering)
occupy axial positions and the equatorial plane includes one
ve-memberedacetateGring(in-planeglycinatering),theve- Table2 UV-VisdataofRh–ed3a-typeofcomplexes
memberedethylenediamineEringandawatermolecule(1)or
chlorideion(2).Complex(1)includesonelongerequatorialRh– Wavelength/energy
˚
O(7) bond (2.072(2) A, to the water ligand), while complex (2) nm 103n(cm(cid:2)1) 3(Lmol(cid:2)1cm(cid:2)1) Assignmenta
hasonelongerRh–Clbond(2.353(1)A ˚ ).Theothermetal–donor
distancesarewithintheexpectedrangeof1.996(2)A ˚ to2.050(2) (1) 353b 28.33 405.59 O h
A ˚ (1)and2.008(3)A ˚ to2.074(3)A ˚ (2).3–5,23Thecisanglesareinthe 294 34.01 326.51 I1A 1g /1T 1g
(2) 373b 26.81 512.13 II1A /1T
rangeof83.0(7)(cid:3)to98.6(7)(cid:3)for(1)and83.0(1)(cid:3)to97.4(1)(cid:3)for(2), 1g 2g
307 32.57 447.77 D
andthetransanglesvarybetween169.0(8)(cid:3)and178.6(7)(cid:3)for(1) (3) 371c 26.95 275.00 IO 4h /1A ,1E a
h 2g g
and 168.1(1)(cid:3) and 178.3(1)(cid:3) for (2). The equatorial ethylenedi- 301 33.22 265.00 IIO /1B ,1E b
h 2g g
amine(E)ringisinanenvelopeconformation.Thepuckering aForallcomplexes.bThiswork.cRef.23.
parameters q2 and 42 (the ideal values for an envelope
5284 | RSCAdv.,2017,7,5282–5296 Thisjournalis©TheRoyalSocietyofChemistry2017
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(R and R above and below the Rh–ed3a nitrogen plane) and
1 2
oneGringintheRh–ed3anitrogenplane,shouldresultinthree
differentacetate-typeABsignals.
If the chemical shis of two acetate protons on one of the
rings are very similar, as might be expected for R , the AB
1
patterncollapsesintoastrongsignalwithveryweaksidepeaks.
TwoacetateprotonsonR (Fig.4)aresymmetricwithrespectto
2
the C–N bond in this ring and experience nearly identical
shieldingbytheC–NbondsoftheGandErings.Therefore,the
splittingofthesetwoprotonsshouldbeminimal.Thecollapsed
AB pattern appearing as a single absorption at 3.99 ppm is
attributedtothiseffect,andtheweaksidepeakscaninfactbe
observed. The 1H NMR spectrum of (1) does not differ signi-
cantly from that in Fig. 4, except that the collapsed singlet at
Fig. 3 Electronic absorption spectra of RhIII complexes in aqueous 4.05 ppm along with the weak side peaks at 4.02 ppm shows
solution: _____ [Rh(ed3a)(OH 2 )]$H 2 O (1), _____ [Rh(ed3a)Cl](cid:2)$H 2 O reversed shis (Fig. S3, ESI†). Further, Fig. S4 (ESI†) and S5
(2),_____[Rh(Hedta)(OH 2 )](3). (ESI†)showthecorresponding13CNMRspectra.
type ligands.1 The absorption maxima of (1) are shied to Solutionstudies
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(2)
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,
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m
ad
t
in
h
g
e Protonationequilibriaofed3a3(cid:2)
.Theprotonationconstants
(logKH)ofed3aweredeterminedbypHpotentiometryandare toalowerin-planeligandeldforthechloridocomplex. i
reportedinTable 3togetherwiththoseofedtafor comparison
NMR spectroscopy. The NMR spectra are discussed in
(standarddeviationsinparentheses).Theprotonationconstants
accordancewithpublisheddata.1Atypical1HNMRspectrumof
aredenedbyeqn(1):
Na[Rh(ed3a)Cl]$H O(2)isgiveninFig.4.Itconsistsofasinglet
2 foracetateat3.99ppmandtwoABsignals(4.08–3.38ppm)for
KH¼
½HiL(cid:4)
; i¼1; 2; 3. (1)
acetate as well as several resonances at lower eld for the i ½LHi(cid:2)1 (cid:4)½Hþ(cid:4)
ethylenegroup.
Someofthelatterresonances(acomplexABCDpattern)are Thersttwoprotonationeventsoccuratthenitrogenatoms
superimposed on the AB-type resonances of acetate. The andthelogKHandlogKHvalueswerefoundtobe9.72and5.81.
tentative assignment of the low-eld part of the AB pattern is The logKH v 1 alue (2.89 2 ) corresponds to the protonation of
3
complicated by the superposition with the singlet, obscuring a carboxylate. The logKH and logKH values of two additional
4 5
oneoftheresonances.Thesymmetryofthemolecule,assuming
carboxylate groups are below 2 and could therefore not be
cis-equatorialconguration(seeFig.1),withtwoacetateRrings
determined potentiometrically. A comparison of the
Fig.4 1HNMRspectrumof[Rh(ed3a)Cl](cid:2).
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Table3 Protonationconstantsofed3a Table 4 Conditional stability constants of RhIII–ed3a3(cid:2) complexes
formedin0.1MNaClionicmediuma
ed3a edtaa
logb (cid:5)s
p,q,r
Ionicstrength 0.1MNaCl 0.1MKCl
logKH 9.72(2) 10.19 Potentiometric Spectrophotometric
1
logKH 5.81(3) 6.16
logK 2 H 2.89(3) 2.69 25(cid:3)C 25(cid:3)C Aerheating
3
aRef.26. [Rh(Hed3a)]+ 12.16(4) 12.39(8) 12.54(9)
[Rh(ed3a)] 5.18(3) 5.26(6) 7.26(3)
protonationconstantsofed3a3(cid:2) andedta4(cid:2) obtainedinsimilar [ [ R R h h ( O e H d3 (e a d ) 2 3 ]3 a (cid:2) )](cid:2) 8 (cid:2) . 2 8 . 7 9 ( 3 8 ( ) 5) 8 (cid:2) . 3 1 . 8 0 ( 4 1 ( 2 7 ) ) — —
media reveals that the logKH 3 values are similar but the Statistics c2¼11.28,s¼1.50 s¼0.032 s¼0.024
logKH and logKH are signicantly different. The rst and
secon
1
dprotonatio
3
n
constantsofed3a3(cid:2)
arelowerby 0.47and
a
ac
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i
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at
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(cid:2)m
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ur
i
,
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1
t
8
e
d
d
,30
.
0.35logKunitsthanthoseofedta4(cid:2) ,whichcanbeexplainedby
oneadditionalacetategroupinedta4(cid:2) .Theacetategroupwith
itspositiveinductiveeffectreducestheacidityoftheprotonat
anaminenitrogenatom.
Thedistributiondiagramofed3a3(cid:2) isgiveninFig.S6(ESI†).
Thefullydeprotonatedspecies,ed3a3(cid:2)
existsinsolutionatpH
higher than 7. Protonated species
Hed3a2(cid:2)
, H ed3a
(cid:2)
and
2
H ed3aarepresentinsolutionfrompH2to12. 3
ComplexformationequilibriaofRhIIIwithed3a3(cid:2)
Potentiometric titrations. The conditional stability constants
of the RhIII complex with H ed3a were determined potentio-
3
metrically at 25 (cid:3)C in 0.1 M aqueous NaCl. The experimental
dataobtainedareshowninFig.S7(ESI†).
Tondthemodelthatgivesthebestttotheexperimental
data, various complexes and combinations thereof were
includedinHyperquad2006calculations.27Themodelselected
was that which gave the best statistical t and which was
chemically consistent with the titration data.28 The sample
standarddeviation,s,andthec2-statisticswereusedascriteria
for the selection of the complex models. The results obtained
arelistedinTable4.
Spectrophotometric titrations. Spectrophotometric data were
obtainedfromRhIII–ed3asolutions,cooledto25(cid:3)C,whereboth
RhIIIanded3aconcentrationswerekeptconstant,whilethepH
wasvariedbyadditionofstandardHClorNaOHsolutions,as
appropriate.AllcorrespondingUV-Visspectraarepresentedin
Fig. S8–S12, ESI.† The spectroscopic data were evaluated with
the HypSpec2014 program,29 where the complexes found by
potentiometrywereincludedinHypSpeccalculations,andthe
corresponding conditional stability constants were optimized.
TheresultingparametersaregiveninTable4.
The distribution diagram of the
RhIII–ed3a3(cid:2)
system
(concentrationratio[ed3a]/[Rh]¼3:1)isshowninFig.5.The Fig. 5 Concentration distribution diagrams of RhIII–ed3a3(cid:2)
dominatingcomplexatlowpHvaluesis[Rh(Hed3a)]+withthe complexesatconcentrationsof2.0mMforRhIII,6.0mMfored3a3(cid:2)
maximumconcentrationof45%atpH¼6.3. and100.0mM NaCl,obtainedby: (top)potentiometryandspectro-
photometry at 25 (cid:3)C; (bottom) spectrophotometry after heating in
RhIIIþH ed3a(cid:2)$½RhðHed3aÞ(cid:4)þþHþ; b aclosedvesselto145(cid:3)C(seeExperimental).
2 (2)
logb¼logb (cid:2)logKH¼12:16(cid:2)9:72¼2:44
1;1;1 1
%4and1%2%3.Therstpathwayemergesfromtheresults
Theconditionalstabilityconstant(logb¼2.44)impliesthat of the potentiometric and spectrophotometric titration, while
the second describes the results of the spectrophotometric
onenitrogenandcarboxylateoxygendonorsdonotcoordinate
titrationaerheatinginaclosedvesselto145(cid:3)C.
torhodiumin[Rh(Hed3a)]+(Scheme2).Here,twopathwaysare
proposedfortheformationofcomplexesinsolution,i.e.1%2 With increasing pH [Rh(Hed3a)]+ releases a proton (the
equilibrium 1 % 2 % 4), and forms [Rh(ed3a)] in which the
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ed3a3(cid:2)
is fully deprotonated, with a maximum of 52% the two complexes of rhodium(III) and possible isomers.
concentrationat pH ¼7.6(Fig. 5andScheme 2).The equilib- Therefore, we have optimized the geometries of the three
riumconstant,K ,forthisreactionmaybecalculatedfromthe geometric isomers (cis-equatorial, cis-polar and trans-equato-
1
overall conditional stability constants of [Rh(Hed3a)]+ and rial,Fig.1)ofthetwocomplexes,usingDFT(densityfunctional
[Rh(ed3a)],logK ¼logb (cid:2)logb ¼12.16(cid:2)5.18¼6.98. theory asimplemented inGaussian 09).32The results forboth
1 1,1,1 1,0,1
This value is similar to the protonation constant logKH. This complexes determine the cis-equatorial isomer as the most
2
meansthattheaminenitrogenlosesaprotonandcoordinates energeticallystableone,by13.4kJmol
(cid:2)1forthechlorocomplex
to rhodium(III). Since the stability of the [Rh(ed3a)] is small (1)andby14.6kJmol
(cid:2)1fortheaquacomplex(2)(seeTable5).
(logb ¼ 5.18), the remaining free carboxylate group of Table 6 contains selected geometric parameters for the 1,0,1
ed3a3(cid:2)
isonlyweaklycoordinatedtorhodium(III).Theequilib- optimized structures of (1) and (2), comparing experimental,
riumconstant,logK,forthemodel1%2%3is5.28whichis DFTandforceelddataofRh–Nin-plane,Rh–Oin-plane,Rh–O
similar to logKH (see Table 3). Aer heating the solution of axialbonds,theaveragedcoordinationcis-andtrans-anglesand
2
complex2(Scheme2),themonodentatedonorXisreplacedby theaveragedRh–O–Cangles.Itemergesthattheobservedbond
(cid:2) theCOO group,andtheformationofaquinquedentateligand lengthsandanglesareinexcellentagreementwiththeexperi-
complex with rhodium(III) and ed3a occurs. The equilibrium mentalstructures.
constant(equilibrium2%3)logK ¼2.08canbecompared Molecular mechanics (force eld) calculations. Molecular
aq
with the formation constant of a hexacoordinated complex mechanics (MM) is based on a classical parameterization of
betweenrhodium(III)andEDTA.31 non-classicaleffectsforthecalculationofmolecularstructure.
Upon increasing the pH (the model 1 % 2 % 4), The relative energies of the three isomers each of the two
[Rh(ed3a)(H O)X]releasesaproton andforms [RhOH(ed3a)X], complexes, obtained by MM and using the MOMEC soware
2
which begins to form at pH ¼ 6.2, and its concentration and force eld (see Table 5; see Experimental section for
increases with further increase of pH. The complex [RhO- modications of the published force eld),33,34 and the corre-
H(ed3a)X], upon increasing of pH, binds another
Hed3a2(cid:2)
sponding structural data (see Table 6) are in very good
ligandandforms[Rh(ed3a) ]3(cid:2) (seeFig.5andS6,ESI†):
2
[RhOH(ed3a)X]+Hed3a2(cid:2)¼[Rh(ed3a) 2 ]3(cid:2)+H 2 O+X (3) Table 5 Comparison of DFT and MM calculated energies for
complexes(1)and(2)
[Rh(ed3a)
]3(cid:2)
starts to form at pH 6.5 and has a maximum
2
concentration at pH ¼ 9. Our current research deals with DFT MM
complexeswithpd3a3(cid:2)
ligand(pd3astandsforpropanediamine-
Geometricalisomer (1) (2) (1) (2)
N,N,N0-triacetate). The ligand pd3a3(cid:2) belongs to ed3a-type
chelates.The [Rh(pd3a) ]3(cid:2) complexwas isolated and its struc- cis-Equatorial 0a 0a 0a 0a
2
turewasconrmedbyX-rayanalysis.However,theseresultshave trans-Equatorial 13.4 14.6 1.4 1.9
cis-Polar 33.5 21.8 4.8 4.7
not been published yet (Jeremi´c et al., unpublished results).
Bearingthatinmind,webelievethatexistenceof[Rh(ed3a) ]3(cid:2) is aThecis-equatorialisomerattheglobalminimumisassignedanenergy
2 of0kJmol(cid:2)1.
highlyprobableaswell.
Computationalchemistry
Table6 Comparisonofexperimental(X-ray),DFT(B3LYP/def2-TZVP)
DFT calculations. Computational methods have been used andMM(MOMEC)structuraldatafor[Rh(ed3a)(OH 2 )]$H 2 O(1)andNa
[Rh(ed3a)Cl]$H O(2)
tointerprettheexperimentallyobservedstructuralpropertiesof 2
X-ray:DFT:MM
(1) (2)
Rh–N(A ˚ )in-plane 1.996:2.015:2.055 2.011:2.055:2.054
2.027:2.084:2.061 2.033:2.075:2.062
Rh–O(A ˚ )in-plane 2.050:2.043:2.004 2.074:2.063:2.004
2.072:2.143:2.018a —
Rh–O(A ˚ )axial 2.018:2.039:2.001 2.008:2.046:1.999
2.010:2.020:1.996 2.013:2.035:1.994
Rh–Cl(A ˚ ) — 2.353:2.399:2.363
Rh–O–C((cid:3))b 112.7:114.3:113.0 112.8:114.5:112.9
cis-ang.((cid:3))b 90.0:90.0:90.1 90.0:90.0:90.1
trans-ang.((cid:3)) 178.6:178.0:176.3 178.3:176.2:176.1
176.8:177.4:174.6 176.6:176.9:173.2
169.0:168.1:167.0 168.2:166.8:166.8
RMSD(A ˚ )c 0.0700 0.0910
aWatermolecule.bAveragevalue.cRMSDvalueshavebeencalculated
Scheme2 Dissociationof[Rh(Hed3a)X ]+.X¼H OorCl(cid:2). onheavyatomsofoverlaidX-rayandMOMECoptimizedgeometries.
3 2
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agreement with the experimental data and the DFT results. thereforeincreasethenumberofpossibleisomersbyafactorof
CorrespondingoverlayplotsareshowninFig.6. 2. Also, the diaminoethane ve-membered chelate ring may
FromMMaswellasfromDFTitemergesthat,asexpected adopt l or d conformation, and this may also increase the
from a large body of data of aminocarboxylate ligands coordi- number of isomers. However, changing the absolute congu-
nated to transition metal complexes (see Introduction), the rationrequirestheinversionofNatomsandthereforealigand
observedcis-equatorialisomeristhemoststable.MMsuggests exchange,whichisnotlikelytooccuraroundaninertrhodiu-
thattheminimaaremoreshallowthanthosepredictedbyDFT m(III) center. Conformational exibility is not unlikely but is
andthatitmightbepossibletoisolateotherisomers.However, afastprocesswithenergybarriersinthe20kJmol
(cid:2)1range.It
isomerconversionisslowforaninertmetalcentersuchasRhIII, therefore appears that these isomeric possibilities are not of
andforthebiologicaltestsreportedbelow,theisomerobserved importanceforthebiologicaltestsreportedbelow,i.e.theonly
experimentallyinthesolidstateistheonlyrelevantspecies. relevantstructuresarethoseobservedbyexperiment.
Allthreepossiblestructuresofthe(1)and(2)arechiral(see
Fig. 1) and therefore may adopt L or D conguration and
Biologicaltests
Antiproliferative activity may results from cytostatic (effect on
cell cycle) or cytotoxic effects and both can contribute to
apoptosis.TheBcl-2familyofproteinsarecrucialintheregu-
lationofapoptoticprocesses.Apoptosisdependsonthebalance
between pro- and anti-apoptotic Bcl-2 proteins. The anti-
apoptotic protein Bcl-2 plays a key role in apoptosis. Its
suppressiveactivityinapoptoticprocessmaycontributetodrug
resistanceoftumorcells.35–37
Invitroantitumoractivity. Thecytotoxicactivityofthenew
RhIIIcomplexes,doxorubicinandcisplatinwasevaluatedaer
48hbytheMTTassayagainstfourhumancancer(MCF-7,A549,
HT-29andHeLa)andonehumannormalcellline(MRC-5).The
commercial antitumor agents doxorubicin and cisplatin were
Fig. 6 An overlay of X-ray and MOMEC optimized structures for
[Rh(ed3a)(OH )](a)and[Rh(ed3a)Cl](cid:2)(b)complexes:blue¼MOMEC usedasreference.Theantiproliferativeeffectswerelinearand 2
optimizedstructures,green¼X-raystructures. dose-dependent. The cytotoxicity of (1) and (2) against HeLa
Fig.7 Linearanddose-dependentcytotoxiceffectofcomplexes(1)and(2)againstHeLacellsthroughtherangeofappliedconcentrationsand
graphicalpresentationofIC values(50%growthinhibitoryconcentration,mM)ofselectedRhIIIcomplexes,doxorubicinandcisplatinforasetof
50
malignantcellsandMRC-5cellline;eachpointisthemeanoftwoindependentexperiments,eachdoneinquadruplicate.
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cellsthroughtherangeofappliedconcentrationsandtheIC V-FITC/7-AADstainedcellsisconsideredasgoldenstandardfor
50
valuesarepresentedinFig.7.ThenewRhIIIcomplexes(1)and detecting apoptosis. Our results show that (1) and (2) induce
(2),incontrasttothereferencecompounds,showedselectivity apoptosisinHeLacells(Fig.8).Themajorityofcellswereearly
betweentumorcelllinesandnon-tumorMRC-5cells(Table7). apoptotic(39.38%and28.71%,respectively),asmallpercentage
Cell lines HT-29 and A549 were moderately sensitive to of cells were in late apoptosis (2.1% and 1.88%, respectively),
compound (2). The aqua complex (1) was 8-fold more active whileminorapercentageofcellswerenecrotic(0.17;0.19).
against the HT-29 than A549 cell lines. The human colon Although exposure of PS on the outer leaet of the cell
adenocarcinoma cells HT-29 were found very sensitive to (1). membrane is considered as hallmark of apoptosis, trans-
OnlyHeLacellsweresensitivetoallcomplexes,whilethebreast locationofPScanoccurinothertypesofcelldeath.39Therefore,
carcinoma cell line MCF-7 was only sensitive to doxorubicin. more than one method has to be utilized to verify apoptosis.
Noneofthetestedcompoundsinhibitedcellgrowthofnormal Cellmorphologyassessmentmaybethemostreliablemethod
fetalbroblasts(MRC-5)tomorethan50%aer48hoftreat- fordiscriminationofapoptosis.40Whentheapoptoticprogram
ment within the applied range of concentrations (Fig. 7). The is started, cells shrink and become rounded, chromatin is
ligandexchangekineticsforrhodiumisratherslowbutweare
not sure that what we observe in the proliferation inhibition
experiments isnota kineticeffectoriginatedfromtheconver-
sion of (2) into (1); this means that the activity of both
compounds may arise due to aqua complex (1) only. Doxoru-
bicinandcisplatinwereconsistentlyandnon-specicallycyto-
toxictoalltreatedcelllines.
Exposure of phosphatidylserine at the outer surface of cell
membraneisoneofthersteventswhenacelliscommittedto
apoptosis.DuetothefactthatAnnexinV(acalcium-dependent
phospholipid-bindingprotein)showsaffinitytobindtothecell
membrane, this molecule was used for detecting apoptotic
cells.387-AAD,auorescentdyewithhighaffinityforDNA,was
employedfordistinguishingbetweenviableandlateapoptotic
ornecroticcells,sincethislargemoleculecannotpassthrough
anintactcellmembrane.Therefore,owcytometryofAnnexin
Table7 Cytostaticactivityofcomplexes(1)and(2)anded3a3(cid:2)ligand
againsttumorstrains
IC ,mM
50
Compound MRC-5 MCF-7 A549 HT-29 HeLa Fig.9 ChangesinmorphologyofHeLacellsaftertreatmentvisualized
by AO/EB staining. Both (1) and (2) induced alterations typical for
(1) >100 >100 16.83 1.96 1.10 apoptosis. Nuclei of viable cells are green with organized structure,
(2) >100 >100 24.28 37.64 20.54 whereas nuclei of early apoptotic cells are bright green and of late
Na Hed3a >100 >100 18.01 >100 13.86 apoptotic cells bright orange to red with condensed chromatin.
2
Doxorubicin 0.12 0.75 7.86 0.32 1.17 Necroticcellshavenormalmorphologyandorangetorednucleiwith
Cisplatin 0.45 1.5 36.12 22.05 2.02 organizedstructure.
Fig.8 FlowcytometricanalysisofAnnexinV-FITC/7-AADstaining.Dotplotspresentpercentagesofviable(AnnexinV(cid:2)7-AAD(cid:2)),earlyapoptotic
(AnnexinV+7-AAD(cid:2)),lateapoptotic(AnnexinV+7-AAD+)andnecroticcells(AnnexinV(cid:2)7-AAD+)inuntreatedHeLacells(A)andcellstreatedwith
IC concentrationof(1)(B)and(2)(C).
50
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condensing and fragmenting. Using AO/EB staining, we
conrmedthatcellstreatedwiththetestedsubstancesshowall
morphological changes typical for apoptosis (Fig. 9). AO/EB
staining enabled discrimination between viable, early
apoptotic,lateapoptoticandnecroticcellsaer24htreatment
withthetestedsubstances.
WBanalysisoftheeffectsofthetestedcompounds(Fig.9and
10)indicatesthat,incomparisonwiththecontrol,theydecrease
the amount of the Bcl-2 protein, similar to doxorubicin; Bax
protein expression is only increased with [Rh(ed3a)(OH )]$H O
2 2
(1). Both RhIII complexes increase the expression of caspase 3
(Fig.10and11),whichindicatestheinvolvementofcaspase3in
apoptotic processes of the investigated cell line. WB also
demonstrateproteolyticcleavageofpoly-(ADP-ribose)polymerase
(PARP)inHeLacells,aertreatmentwithboth(1)and(2)(Fig.9
and10).Actinwasusedasaninternalcontrolandshowsuniform
expressioninallsamples.Thevariationswerewithinarangeof
(cid:5)5%,comparedtothecontrol.
The expression pattern of the investigated proteins of the
apoptoticsignalingpathwayofthemostaffectedtumorcellline
HeLa reveals a conducted apoptosis. This is conrmed by the
Fig.10 TheexpressionofapoptoticproteinsinvestigatedbyWestern
blotanalysis.(1)Controlsample;(2)doxorubicin;(3)Na[Rh(ed3a)Cl]$ detectionofPARPproteincleavageinsamplestreatedwithboth
H O(2);(4)[Rh(ed3a)(OH )]$H O(1). RhIII complexes (1) and (2). The reduction of Bcl-2 protein 2 2 2
Fig. 11 Graphical presentation of protein expression densitometry data obtained by WB analysis and processed with ImageJ [http://
imagej.nih.gov].Expressionofproteinsbelongingtoanapoptoticsignalingpathwayinthesamplesarecomparedtountreatedsamplesand
presentedaspercentageofcontrol.Thedensitometryofactinexpression,whichservesasinternalcontrol,ispresentedasmeasured.(1)Control;
(2)doxorubicin;(3)Na[Rh(ed3a)Cl]$H O(2);(4)[Rh(ed3a)(OH )]$H O(1).
2 2 2
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of the
RhIII–ed3a3(cid:2)
system consists of four species:
[Rh(Hed3a)]+, [Rh(ed3a)], [Rh(ed3a) ]3(cid:2) and [Rh(OH)(ed3a)] (cid:2) .
2
At physiological pH the dominant species in the system are
[Rh(Hed3a)]+ and [Rh(ed3a)] complexes. Knowledge of the
compositionandstabilityofthesecomplexesinthesystemcan
contributetoabetterunderstandingoftheirphysiologicalroles
in different tissues and cell system. Biological tests demon-
Fig.12 Cellcycleanalysis.Histogramspresentcellcycledistributionin stratedthattheRhIIIcomplexes(1)and(2)showaninteresting
untreatedHeLacells(A)andcellstreatedfor48hwith(1)(B)and(2)(C). cytotoxicity behaviour in comparison to the doxorubicin and
cisplatinreferencesystems:incontrasttothese,theyareinac-
tive against the MCF-7 human breast carcinoma and healthy expression aer the treatment was observed in samples. Also,
MRC-5 cell lines but are very active against the human cervix
amuchlargerincreaseofBaxproteinexpressionrelativetothe
adenocarcinomaHeLacellline.AgainstHT-29andA549cells,
controlwasobservedaertreatmentwith[Rh(ed3a)(OH 2 )]$H 2 O theaquacomplex(1)issignicantlymoreactivethanthechloro
(1)(sample4).Bcl-2andBaxproteinsareimportantmembersof
complex(2).FlowcytometryandWesternblotanalysisrevealed
the Bcl protein family and are located at the beginning of the
mechanismofantitumoractivityoftestedcomplexes:cytostatic
apoptoticsignalingpathway.41,42
as a result of DNA synthesis blockade and cytotoxic through
Factorsthatcaninuencetheirbalancemayinstigatecellsto
inductionofapoptosis.
survival or death. Antiapoptotic Bcl-2 proteins block the
intrinsicapoptosispathway.Theirconcentrationisincreasedin
Experimental
human cancer cells and they are important targets for new
therapies.41,42 Therefore, the observed decrease (down regu-
Materialsandmethods
lated)Bcl-2expressionasaresultofthetwonewRhIIIcomplexes
Reagent grade commercially available chemicals were used
(1) and (2), as well as the upregulation of Bax expression by
withoutfurtherpurication.Thepreparationofthecalciumsalt
complex(1)areofpharmacologicalimportance.
of ed3a3(cid:2) , Ca (ed3a) $12H O was reported previously.5 Mono- Proteinexpressionanalysisalsogivesapictureofincreased 3 2 2
chloroacetic acid, ethylenediamine, calcium hydroxide, RhIII
caspase 3 activity, which suggests that the caspase-dependent
apoptosisunderliestheobservedcytotoxiceffect. chloride hydrate, hydrochloric acid, sodium hydroxide and
sodium chloride were purchased from Sigma-Aldrich. A rho- Both cell cycle progression and apoptosis are crucial for
maintainingtissuehomeostasis.Thesesetsofeventsarecoupled
dium(III) chloride stock solution was prepared by dissolving
dried RhCl $H O, p.a. (Sigma-Aldrich), in doubly-distilled
andsharecertainregulatorymolecules.Cellulardamageandstress 3 2
water. Elemental microanalyses for C, H, N were performed
signalsresultincellcycleterminationthatprovidethecellatime
on a CHN-O-vario EL by the Microanalysis Laboratory at the
to repair the damage. If the cell cannot recover, the apoptotic
chemical institutes at Heidelberg University. IR spectra were
programisactivated.AnalysisofthecellcycleinHeLacellstreated
measured with a Perkin-Elmer 16 PC FTIR instrument as KBr
with (1) and (2) (Fig. 12) showed that both substances induced
pellets. NMR spectra were recorded at 200 MHz (1H) and 50
G0/G1cycletermination(from74.97%inuntreatedcellsto81.70%
MHz (13C) on a Bruker Advance I 200 instrument with deuter-
in cells treated with complex (1) and 81.52% when treated with
ated solvents as reference. Electronic absorption spectra were
complex(2)).Concomitantly,thepercentageofcellsintheSphase
obtainedfromaTidasIIJ&Mspectrophotometeratconcentra-
decreased from 5.04% in control cells to 0.65% and 0.85%,
tionsoftheRhIIIcomplexes(aqueoussolutions)ofapprox.1.0(cid:6)
respectively.TheseresultsindicatethatblockadeofDNAsynthesis
10
(cid:2)3
M. The potentiometric measurements were carried out
inducedbythetestedsubstancesisapossibletriggerofapoptosis.
usingaMethrom827pHmeterwithaTitronicuniversalpiston
buretteandcombinedglasselectrode.Spectroscopicmeasure-
Conclusions
ments were made with a double beam UV-Vis spectrophotom-
eter model Cary 300 (Agilent Technologies, Santa Clara, USA)
Two new
RhIII–ed3a3(cid:2)
complexes, the neutral cis-equatorial-
with1.0cmquartzcells.Meltingpointsweredeterminedusing
[Rh(ed3a)(OH
2
)]$H
2
O (1) and the anionic cis-equatorial-Na aStuartdigitalmeltingpointapparatuswithaccuracy(cid:5)1(cid:3)C.
[Rh(ed3a)Cl]$H O (2), were isolated and characterized using
2
experimentalandcomputationaltechniques.Thecis-equatorial
geometrywasveriedbyX-raycrystallographyfor(1)and(2)and Syntheticprocedures
solution spectroscopy (UV-Vis, IR, NMR) indicates that, for (1) Preparation of cis-eq-[Rh(ed3a)(OH )]$H O (1). Ca (ed3a) -
2 2 3 2
thisisretainedinsolutionandthatitisalsothegeometryof(2), $12H O(1.99g;2.5mmol)wasdissolvedinwater(15mL)and
2
as expected from published strain energy analyses of similar solutionofNaOH(0.6g,15mmol)inwater(5mL)wasadded.
systems.5 MM and DFT results are in agreement with the ThedepositedCa(OH) wasseparatedbyltration;totheltrate
2
experimental X-ray and spectroscopic data and the observed wasaddedasolutionofRhCl $H O(1.14g,5mmol)inwater(5
3 2
isomer preference. The composition and conditional stability mL). The resulting mixture was stirred at 145 (cid:3)C for 7 h in
constants of RhIII complexes were determined by pH potenti- a closed Pyrex tube. Aer cooling to room temperature the
ometryandUV-Visspectrophotometry. Thespeciationscheme yellow solution was ltered off and the ltrate was passed
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throughacolumnofQAEA-25SephadexintheCl (cid:2) form.The (447.77) and 373 (512.13). d 1H NMR (200 MHz, D O, Me Si):
2 4
columnwaselutedwith100mMNaCl.Threeyellowbandswith 4.05(ABpattern,Gring),3.99(s,R ring),3.34(ABpattern,R
1 2
different charges appeared; the rst band was evaporated and ring)d13CNMR(50MHz,D O/CD OD,Me Si):184.67,183.71,
2 3 4
desalted by passage through a Sephadex G-10 column with 182.10(C]O),66.60,63.72,61.84,57.37,57.04(CH ).Thethird
2
distilledwateraseluent.Theeluatewasevaporatedtoca.1mL band, remaining on top of the column aer elution with
and neutral cis-equatorial-[Rh(ed3a)(OH )]$H O complex was 100 mM NaCl, was eluted with concentrated NaCl and was
2 2
crystallized aer adding ethanol and cooling the solution in foundtobeamixtureofdifferenthydroxospecieswithcharges
a refrigerator. The yellow crystals were collected, washed with higherthan(cid:2)2.
ethanol and air-dried. Yield: 0.4 g, 21.69%. Melting point:
>305 (cid:3)C (from EtOH). Anal. calc. for C H N O Rh (FW ¼
8 15 2 8
370.13gmol
(cid:2)1):C,25.96;H,4.08;N,7.57%.Found:C,25.37;H,
Crystalstructuredetermination
4.22;N,7.36%.IR(KBr,n cm (cid:2)1):1631n(COO (cid:2) ),3435n(N–H).
max
UV-Visl (H O)/nm(3/dm3mol (cid:2)1cm (cid:2)1):294(326.51)and353 Crystal data and details of the structure determinations are
max 2
(405.59).d1HNMR(200MHz,D O,Me Si):4.09(ABpattern,G listedinTable8.Fullshellsofintensitydatawerecollectedat
2 4
ring),4.05(s,R ring),3.30(ABpattern,R ring);d13CNMR(50 low temperature with an Agilent Technologies Supernova-E
MHz,D O/CD O 1 D,Me Si):184.26,182.84, 2 181.26(C]O),67.30, CCD diffractometer (Mo-Ka radiation for complex (1) and Cu-
2 3 4
Ka radiation for complex (2), microfocus tubes, multilayer
64.59,62.70,56.53,56.17(CH ). 2
Preparationofcis-eq-Na[Rh(ed3a)Cl]$H O(2). Fortheprep- mirror optics). Data were corrected for air and detector
aration of Na[Rh(ed3a)Cl]$H O, the same
2
procedure as for (1)
absorption,Lorentzandpolarizationeffects;43absorptionbythe
2
crystal was treated numerically (Gaussian grid).43,44 The struc-
was used. The second band was evaporated and desalted over
turesweresolvedbyintrinsicphasing45(complex(1))orbythe
a Sephadex G-10 column with distilled water as eluent. The
eluate was evaporated to ca. 3 mL and le to crystallize from heavy-atom method combined with structure expansion by
direct methods applied to difference structure factors46
ethanol overnight in a refrigerator. The yellow crystals of cis-
equatorial-Na[Rh(ed3a)Cl]$H O were collected, washed with
(complex(2))andrenedbyfull-matrixleastsquaresmethods
2 based on F2 against all unique reections.47 All non-hydrogen
ethanol and air-dried. Yield: 0.800 g, 39.10%. Melting point:
>305(cid:3)C(fromEtOH).Anal.calc.forC H ClN NaO Rh(FW¼ atoms were given anisotropic displacement parameters. 8 13 2 7
410.55gmol
(cid:2)1):C,23.40;H,3.19;N,6.82%.Found:C,23.55;H, Hydrogen atoms were generally input at calculated positions
3.39; N, 6.99%. IR (KBr, n cm (cid:2)1): 1630 and 1680 n(COO (cid:2) ), andrenedwitharidingmodel.
max 3426 n(N–H). UV-Vis l (H O)/nm (3/dm3 mol (cid:2)1 cm (cid:2)1): 307 The positions ofsome hydrogenatoms (those onN and O) max 2 were taken from difference Fourier syntheses and rened.
Table8 Detailsofthecrystalstructuredeterminationsof[Rh(ed3a)(OH )]$H O(1)andNa[Rh(ed3a)Cl]$H O(2)
2 2 2
(1) (2)
Formula C H N O Rh C H ClN NaO Rh
8 15 2 8 8 13 2 7
M 370.13 410.55
r
Crystalsystem Monoclinic Monoclinic
Spacegroup P2 /n P2 /c
1 1
˚
a/A 6.94313(8) 13.4003(6)
˚
b/A 15.71268(18) 6.9326(3)
˚
c/A 11.09315(11) 14.9772(8)
b/(cid:3) 96.9886(10) 115.328(6)
V/A
˚3
1201.22(2) 1257.62(12)
Z 4 4
F 744 816
000
d/Mgm(cid:2)3 2.047 2.168
c
X-radiation,l/A ˚ Mo-Ka,0.71073 Cu-Ka,1.5418
m/mm(cid:2)1 1.462 13.639
Max.,min.transmissionfactors 0.946,0.792 0.815,0.285
Datacollect.temp./K 110(1) 110(1)
qrange/(cid:3) 3.2to29.0 3.7to70.9
Indexranges(indep.set)h,k,l (cid:2)9.9,(cid:2)21.21,(cid:2)15.15 (cid:2)16.16,(cid:2)8.8,(cid:2)17.15
Reectionsmeasured 66435 24110
Unique[R ] 3126[0.0598] 2410[0.0938]
int
Observed[I$2s(I)] 2965 1861
Data/restraints/parameters 3126/0/87 2410/0/190
GooFonF2 1.276 1.030
Rindices[F>4s(F)]R(F),wR(F2) 0.0313,0.0518 0.0332,0.0739
Rindices(alldata)R(F),wR(F2) 0.0358,0.0527 0.0510,0.0807
Differencedensity:max,min/eA ˚(cid:2)3 0.520,(cid:2)0.671 1.334,(cid:2)0.647
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P
CCDC 1412103 (for (1)), CCDC 1412104 (for (2)) contain the U ¼ (E +E +E +E +E ) (4)
total b q F nb d
supplementarycrystallographicdataforthispaper.†
Inputcoordinateswereobtainedfromcrystalstructuresdata
or produced with HyperChem 7.01.50 Parameters not reported
Solutionstudies
beforearegiveninTable9.Allotherparametersaregiveninthe
All the equilibrium measurements were made at a constant
literature.34
ionicstrengthmaintainedby0.1MNaClat25(cid:3)C.Todetermine
the protonation constants of
ed3a3(cid:2)
and conditional stability
Biologicaltests
constants of the complexes formed with
ed3a3(cid:2)
were deter-
mined by potentiometric titrations. The metal-to-ligand ratios Celllines.Allhumansolidtumorandnormalcelllineswere
were1:1.5and1:3.TheconcentrationoftheRhIIIwas2mM.
purchasedfromAmericanTypeCultureCollection—ATCC.The
The potentiometric measurements were carried out using celllinesusedinthestudywereA549(humanlungcarcinoma,
a Methrom 827 pH meter with a Titronic universal Piston ATCCCCL185),MCF-7(humanbreastadenocarcinoma,ATCC
buretteand combinedglass electrode. Thetemperature ofthe HTB22),HT-29(humancolonadenocarcinoma,ATCCHTB38),
sample solutions (20 mL) was maintained at 25.0 (cid:3)C by circu- HeLa(humancervixadenocarcinoma,ATCCCCL2)andMRC-5
lating thermostatically controlled water through the jacket of
(normalhumanfetallungbroblasts,ATCCCCL171).Thecells
the titration vessel. The samples were stirred with a magnetic were grown in Dulbecco's modied Eagle's medium (DMEM,
stirrer,andtoavoidtheeffectofCO .Allofthemeasurements Sigma) with 4.5% of glucose, supplemented with 10% of fetal
2
wereperformedunderanitrogenatmosphere. bovine serum (FBS, Sigma) and antibiotics and antimicotics
Spectroscopic measurements were made on solutions in solution (Sigma). All cell lines were cultured at 37 (cid:3)C in the
whichtheconcentrationofRhIIIanded3a3(cid:2) wereconstant(C Rh 100% humidity atmosphere and 5% of CO 2 . Exponentially
¼2.0mM,C ¼6.2mMandC ¼3.1mM),whilethepH growingcellswereusedthroughouttheassays.
ed3a ed3a
wasvariedbetween2.5and7.8.ThepHofthetestsolutionswas MTT assay. Growth inhibition was evaluated by tetrazolium
measuredwithaMethrom827pHmeter.Stablevalueswithin colorimetric MTT assay (SIGMA).51 Exponentially growing cells
0.02 pH and 0.01 absorbance units, were attained for the rst
seriesofsolutionsaer1hat25(cid:3)C(theseremainedstablefor
30min),andforthesecondseriesaerheatingfor3hat145(cid:3)C Table9 NewforcefieldparametersforRh–edtatypeofcomplexesa
(closedvessel,seeabove),andtheseremainedstablefor3hat
Bonddistanceparameters
the same temperature. Spectra of the test solutions were
recordedinthe250–600nmwavelengthrange.Tocalculatethe Forceconstant Strain-freebond
equilibriumconstantstheHYPERQUAD2006andHypSpec2014 Bondtype (mdynA
˚(cid:2)1)
distance(A
˚
)
programs were used.27,29 The concentration distribution
Rh–N 1.75 2.05 diagrams were obtained using the program HYSS2006 under Rh–Cl diamine 1 2.35
theexperimentalconditionsdescribed.48 Rh–O 1.75 1.99
carboxyl Rh–O 0.5 1.9
water
Computationaldetails
Valenceangleparameters
DFT calculations. Geometries for RhIII complexes were
Forceconstant Strain-freevalence
optimized usingGaussian09A01 program.32The Becke three- Valenceangletype (mdynA ˚ rad(cid:2)2) angle(rad)
parameter exchange functional was employed in this study in
conjunction with the Lee–Yang–Parr correlation hybrid func- O –Rh–O 0.026 1.571
carboxyl carboxyl
tional (B3LYP) and the Ahlrich's def2-TZVP basis set.49 The Cl–Rh–O carboxyl 0.026 1.571
systems were treated within the restricted formalism. All the O carboxyl –Rh–N diamine 0.026 1.571
O –Rh–O 0.026 1.571
calculationsweredoneunderthePolarizableContinuumModel carboxyl water
Cl–Rh–N(diamine) 0.026 1.571
(PCM) with the solute being water as implemented in G09 N –Rh–N 0.026 1.571
diamine diamine
package. All the calculated structures were veried to be local N –Rh–O 0.026 1.571
diamine water
minima (all positive eigenvalues by frequency analysis) for Rh–O water –H 0.100 1.915
ground state structures. Starting geometries were taken either Rh–O carboxyl –C carboxyl 0.026 1.970
fromexperimentalX-raystructuresorwerepre-optimizedusing
Torsionangleparameters
themolecularmechanics.
Molecular mechanics (force eld) calculation. Molecular Forceconstant Offsetangle
˚
Bondtorsionangletype (mdynA) (rad)
mechanicscalculationswereperformedusingthestrainenergy
minimizationprogramMOMEC.33Withintheframeworkofthe O –Rh 0.0000 0.0000
molecularmechanics,thestructureofamoleculewasmodied N carboxyl –Rh 0.0000 0.0000
diamine
tominimizeitstotalstrainenergy.Strainenergyincludes:bond O –Rh 0.0000 0.0000
water
lengths deformation (E b ), valence angle deformation (E q ), Rh–O carboxyl –C carboxyl –O carboxyl 0.0400 0.0570
torsion angle deformation (E
F
), nonbonded interactions (E
nb
) adyn¼10(cid:2)5N.
andout-of-planedeformation(E )(eqn(4)):
d
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wereharvestedandplatedinto96-wellmicrotiterplates(Costar) in PBS and treated with RNase A (500 mg mL (cid:2)1 PBS) for 30
at optimal seeding density of 10 (cid:6) 103 cells per well. Tested minutesat37(cid:3)C.5mLofpropidiumiodide(10mgmL (cid:2)1PBS)
substancesandreferencecompoundsdoxorubicinandcisplatin, wasaddedtoeachtubeandaer15minutesincubationindark
attenfoldtherequirednalconcentration,wereadded(10mLper samples were assayed by ow cytometer Cytomics FC500. The
well)toallwellsexcepttothecontrolonesandmicroplateswere datawereanalyzedusingFlowingSowareandtheresultswere
incubatedfor48h.Threehoursbeforetheendoftheincubation presentedbyhistograms.
period,10mLofMTTsolution(5mgmL (cid:2)1)wasaddedtoallwells. Western blot. The protein concentration in cell lysate was
Acid-isopropanol (100 mL of 0.04 N HCl in isopropanol) was determined by Bradford protein assay53 in a 96 well microtiter
addedtoallwellsandmixedthoroughlytodissolvethedarkblue plate (ThermoLab Systems, Multiscan Accent spectrophotom-
crystals.Aerafewminutesatroomtemperature,toensurethat eter) using bovine serum albumin as the standard. Molecular
allcrystalsweredissolved,theplateswerereadonaspectropho- mass markers for proteins were obtained from Amersham
tometer plate reader (Multiscan MCC340, Labsystems) at 540/ Biosciences.FortheWesternblot,50mgofproteinspersample
690 nm. Inhibition of growth was expressed as a percent of were separated by electrophoresis and electro-transferred to
a control and cytotoxicity was calculated according to the apolyvinylidenediuoride(PVDF)membraneHybond-P(Amer-
formula: (1 (cid:2) A /A ) (cid:6) 100. The substance potency was shamBiosciences,ArlingtonHeights,IL)andthenblottedwith
test control
expressedastheIC (50%inhibitoryconcentration).Twoinde- primary antibodies (Bcl-2, PARP, caspase-3, and actin). Mono-
50
pendent experiments were set out with quadruplicate wells for clonal antibodies against human Bcl-2 and Caspase 3 were ob-
each concentration of the compound. IC values were deter- tained from R&D Systems (Minneapolis, MN). Anti-poly(ADP-
50
minedbyMedianeffectanalysis.52 ribose)polymerase (PARP) was purchased from Santa Cruz
Celltreatmentforapoptosisstudy.Thecellswereseededin Biotechnology(SantaCruz,CA).Antibodyagainsta-,b-org-actin
6-well plates at a concentration of 5 (cid:6) 105 cells per well. was purchased from Sigma Chemical (St. Louis, MO). Proteins
Viabilitywasdeterminedusingtrypanbluedye-exclusionassay. weredetectedbyanenhancedchemiluminescence(ECLPlus)kit
Untreatedcellswereusedascontrol(sampleno.1).Cellswere (Amersham Biosciences), that includes peroxidase-labeled
treatedwithDoxorubicinasareferencecompound(sampleno. donkeyanti-rabbitandsheepanti-mousesecondaryantibodies.
2) and tested complexes (1) and (2) (sample no. 3 and 4, Blots were developed with an ECL Plus detection system and
respectively) for 48 h. Viable treated and control cell samples recorded on Hyperlm (Amersham Biosciences). Exposed lms
wereusedforapoptosisinvestigationbyWesternblotanalysis. wereprocessedwithKodakEX-OMATIIdeveloperreagentsand
Flow cytometric analysis. Thetypeofcelldeathinducedby photographed on a negatoscope with Canon 1100D camera on
testedsubstanceswasdeterminedusingAnnexinV-FITC/7-AAD mini-tripod. The protein expression images were analyzed by
kitaccordingtomanufacturer'sinstructions(BeckmanCoulter, densitometry in ImageJ computer program (NIH image, http://
USA). Briey, HeLa cells were treated with complex (1) and imagej.nih.gov)withonlyminor levelsadjustments.Expression
complex(2)inconcentrationscorrespondingtoIC valuesorin of apoptotic proteins in treated samples was compared to the 50
mediaalone(control).Aer48hincubation(37(cid:3)C,5%CO and control sample. Densitometry data processing was done in
2
absolute humidity) both attached and detached cells were MicrosoOfficeExcelprogram.
collected, washed in PBS and nally suspended in ice cold
binding buffer(1 (cid:6) 105 cells per100 mLbinding buffer). Cells
Acknowledgements
werestainedwith10mLofAnnexinV-FITCand20mL7-AADand
aer 15 minutes incubation in dark, 400 mL of binding buffer
The authors aregrateful totheSerbianMinistry ofEducation,
was added to each tube. Samples were assayed by ow
Science and Technological Development for the nancial
cytometer.
support (Project No. III41010). One of the authors (Marija
CytomicsFC500(BeckmanCoulter,USA)andthepercentof
Jeremi´c) is particularly grateful to the Erasmus Mundus Pro-
viable,apoptoticandnecroticcellswasevaluatedusingFlowing
grammefortheawardedscholarship.
Soware (http://www.owingsoware.com/). The results were
presentedbydotplots.
Cellmorphologyassessment.Untreated,controlHeLacells, References
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5294 | RSCAdv.,2017,7,5282–5296 Thisjournalis©TheRoyalSocietyofChemistry2017
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5296 | RSCAdv.,2017,7,5282–5296 Thisjournalis©TheRoyalSocietyofChemistry2017
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