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Synthesis and DNA-binding properties of apoptosis-inducing cytotoxic half-sandwich rhodium(III) complexes with methyl-substituted polypyridyl ligands
JournalofOrganometallicChemistry696(2011)1023e1031
ContentslistsavailableatScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis and DNA-binding properties of apoptosis-inducing cytotoxic
half-sandwich rhodium(III) complexes with methyl-substituted
polypyridyl ligands
Yvonne Geldmachera, Riccardo Rubbianib, Pascal Wefelmeierc, Aram Prokopc, Ingo Ottb,
William S. Sheldricka,*
aFakultätfürChemieundBiochemie,Ruhr-UniversitätBochum,Universitätsstrasse150,D-44780Bochum,Germany
bInstituteofPharmaceuticalChemistry,TechnischeUniversitätBraunschweig,Beethovenstr.55,D-38106Braunschweig,Germany
cDepartmentofPediatricOncology,CologneChildren’sHospital,AmsterdamerStr.59,D-50735Cologne,Germany
a r t i c l e i n f o a b s t r a c t
Articlehistory: Half-sandwichorganorhodium(III)complexesofthetype[(h5-C5Me5)RhCl(pp)](CF3SO3)containingpoly-
Received14July2010 pyridylligands(pp)representapromisingclassofcytostaticagents.Replacementofthepolypyridylligands
Receivedinrevisedform ofcomplexes1(pp¼phen)and6(pp¼dppz)bymethyl-substitutedderivativesin2e5(pp¼4-Mephen,
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5-Mephen,4,7-Me2phen,5,6-Me2phen)and7(pp¼Me2dppz)leadstoasignificantimprovementintheir
antiproliferativeactivitytowardshumanMCF-7andHT-29cancercells.Forinstance,theIC50valuetowards
HT-29cellsdecreasesfrom4.3(cid:2)0.2mMfor6to0.98(cid:2)0.49mM forcomplex7.Incontrast,noactivity
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ds: (IC50 >100mM)wasobservedfortheHOOCandn-BuNHCOsubstituteddppzcomplexes8and9.UV/vis,CD
andNMRspectraformixturesofcomplexes7e9withCTDNAwereinaccordancewithintercalationofthe
Polypyridylligands
Cytotoxicity substituteddppzligandsbetweenthebasepairsofthedoublehelixanddirectevidenceforthisbinding
Apoptosis
modewasalsoprovidedbya2DNOESYstudyforcomplex7withthehexanucleotided(50-CGTCGG-30).Each
DNAbinding ofthemethyl-substitutedphencomplexes2e5issignificantlymoreactivetowardsimmortalizedHEK-293
cells(IC50values0.40(cid:2)0.02to0.94(cid:2)0.02mM)thantowardsthecancercells.Flowcytometricmeasure-
mentsofDNAfragmentationinBJABcellsfollowinganincubationperiodof72hwith1,5and6indicatethat
thecomplexesinducespecificapoptoticcelldeathinthenon-adherentlymphomacells.
(cid:1)2010ElsevierB.V.Allrightsreserved.
1. Introduction reported for complexes of the type [(h5-C5Me5)MCl(pp)](CF3SO3)
(M¼Ir,pp¼dppz,dppn[3,6];M¼Rh,pp¼phen,dpq,dppz,dppn
Investigationsofthecytotoxicpropertiesofareneruthenium(II) [7])containingthe polypyridylligands1,10-phenanthroline(phen)
complexes with amino acidato [1], diamine [2], diimine [3] and dipyrido[3,2-f:20,30-h]quinoxaline (dpq), dipyrido[3,2-a:20,30-c]
phosphaneligands[4]haveestablishedpromisinganticanceractivity phenazine (dppz) and benzo[i]dipyrido[3,2-a:20,30-c]phenazine
for this class of half-sandwich compounds. In striking contrast, it (dppn)(Fig.S6).Acleardependenceonthesizeofthepolypyridyl
is only very recently that similar pentamethylcyclopentadienyl ligand is apparent for the cytotoxicity of these rhodium(III)
complexesoftheneighbouringGroup9transitionmetals,rhodium compoundstowardsthehumanMCF-7(breastcarcinoma)cellline.
(III)andiridium(III),haveattractedanyinterestinthisrespect.Afirst TheirIC50valuesdecreaseintheligandorderphen,dpq>dppz>
articlebyDysonetal.in2006[5]reportedaninvitroevaluationofthe dppnfrom4.7/5.1over1.5to0.8mM[7].Markedinvitrocytotoxicity
RAPTA [4] analogues [(h5-C5Me5)RhCl2(pta)] and [(h5-C5Me5) has also recently been reported for analogous (h5-C5Me5)M(III)
RhCl(pta)2] þ (pta¼1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane) (M¼Rh,Ir)complexescontainingthechelatingligands2-(pyridine-
towardsthehumancelllinesHT-29(coloncarcinoma),A549(lung 2-yl)thiazole (towards the A2780 and A2780cisR cell lines) [8]
carcinoma)andT47D(breastcarcinoma),butestablishedonlyvery and 1,2-naphthoquinone-1-oximate (towards the HeLa and HL60
limited cytotoxicity, as indicated by the determined IC50 values of celllines)[9].
greater than 380mM. A much higher activity was subsequently CelldeathinducingpropertiesforthehumancancercelllinesMCF-
7andHT-29(coloncarcinoma)havealsobeenestablishedforrhodium
(III)complexesofthetype[(coligand)RhCl(pp)](CF3SO3)containingthe
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343214420. facial coligands 1,4,7-trithiacyclononane ([9]aneS3) [10] and tris
0022-328X/$eseefrontmatter(cid:1)2010ElsevierB.V.Allrightsreserved.
doi:10.1016/j.jorganchem.2010.10.034
1024 Y.Geldmacheretal./JournalofOrganometallicChemistry696(2011)1023e1031
Scheme 1. Cations and the employed numbering scheme of the compounds
[(h5-C5Me5)RhCl(pp)](CF3SO3)1e5withphenandsubstitutedphenanthrolineligands.
(pyrazolyl)methane(tpm)[11].Severalinterestingstructureeactivity
relationships are apparent on comparison of the IC50 values for
rhodium(III) complexes containing different tridentate coligands. In
particular,theorderoftheIC50values(hereforMCF-7cells)changesin
Fig.1. Molecularstructureofthecationofthe5-methylphenanthrolinecomplex3.
dependenceonthecoligandfromtpm>[9]aneS3 >[h5-C5Me5] (cid:3) for
pp¼phen (51.7>36.3>4.7mM) over [9]aneS3 >[h5-C5Me5] (cid:3) , tpm the cell death induced by these half-sandwich complexes. In
forpp¼dpq(19.1>5.1,4.0mM)to[9]aneS3 >[h5-C5Me5] (cid:3)>tpmfor
contrast to ([9]aneS3)Rh(III) half-sandwich complexes and tri-
pp¼dppz (4.7>1.5>0.43mM). Based on these results, it can be chlorido-rhodium(III) complexes of the type mer-[RhCl3(DMSO)
concludedthattheextentoftheinfluenceofpolypyridylligandsizeon (pp)], which induce specific apoptotic cell death via the intrinsic
compound cytotoxicity increases in the order of the coligands [h5- mitochondrialpathway[10,16],thepronouncedcytotoxicityof(h5-
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compounds(n¼1,2)containingderivativesofthesmallerpolypyridyl
nowreportstudiesofpossiblenecrosisand/orapoptosisinduction
ligand 1,10-phenanthroline should concentrate on employing pen- in lymphoma BJAB cells following their incubation with the
tamethylcyclopentadienylasthefacialcoligand. complexes1(pp¼phen),5(pp¼5,6-Me2phen),6(pp¼dppz)and
Onepromisingstrategyforenhancingthebiologicalactivityof 8(pp¼HOOC-dppz).
phenanthroline complexes is to replace phen by its methyl-
substituted derivatives, as first demonstrated by McFadyen et al.
[12] for [Pt(en)(3,4,7,8-Me4phen)]2þ (IC50 ¼0.7mM) against the 2. Resultsanddiscussion
murine leukemia L1210 cell line in comparison to [Pt(en)(phen)] 2.1. Synthesisof1e9
(IC50 ¼2mM). This approach was subsequentlyextended to mon-
omethyl- (4-Mephen, 5-Mephen) and dimethyl-substituted phe- The half-sandwich complexes [(h5-C5Me5)RhCl(pp)](CF3SO3)
n [1 a 3 n ,1 th 4] r , o w lin h e o s d ( i 4 s , c 7 o - v M er e e 2 d ph th en at ,5 th ,6 e - 5 M ,6 e - 2 M ph e e 2 n p ) he b n y c A o ld m r p ic l h ex -W ex r h ig i h bi t ts et th a e l. 1 R e hC 9 l( w ac e e re to p n r e e ) p 2] a ( r C e F d 3S b O y 3) re w flu it x h in t g he th a e p s p o r l o v p e r n i t at c e om po p l l y e p x y [ r ( i h dy 5- l C l 5 ig M a e n 5 d )
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l (pp) in CH3OH/CH2Cl2 (1:1) for 5h. [(h5-C5Me5)RhCl(acetone)2] þ
d of iffe m re e n th c y e l s i s n ub t s h t e itu ge e o n m ts et a r t y v o a f r t y h in ei g r D p N os A iti b o i n n s din c g au t s h e a s tm sig a n y ifi af c f a e n ct t R w a h a C s s o l} l o 2 u b ( t m i t o - a C n in l) e 2 o d ] f [ i 1 n t 7 h ] e si i t n d u in a b c u y e c t l o e a a n d r e dit a s i t n o a n d rti s o n u f g b 2 se c e o q q m u u e p i n v o t u o c n f e d n A t g r [{ i ( f ( C u h F g 5 3 a - S C t O i 5 o 3 M n ) e t o 5 o ) f
theircytotoxicity[13].
precipitated AgCl after stirring in the dark for 2h. Reaction of
We have now investigated the biological activity and DNA- dipyrido[3,2-a:20,30-c]phenazine-7-carbonic acid (HOOC-dppz)
bindingpropertiesofthecomplexes2e5(Scheme1)withmethyl-
[18]withn-butylaminefor2hat80(cid:4)Cinapyridinesolutioninthe
substituted phenanthroline ligands in comparison to[(h5-C5Me5) presence of the coupling agent 1,10-carbonyldiimidazole led to
RhCl(phen)](CF3SO3)1[7],andthecomplexes7e9(Scheme2)with
formation of the novel ligand (7-n-butylamido)dipyrido[3,2-
substituteddppzligandsincomparisonto[(h5-C5Me5)RhCl(dppz)]
a:20,30-c]phenazine(n-BuNHCO-dppz),whichwascharacterizedby
(CF3SO3)6[15].Asecondpointofinterestwasthemechanismof
elemental analysis, LSIMS (liquid secondary ion mass spectrom-
etry)and1HNMRspectroscopy,aswerecomplexes1e9.
The structures of compounds 3 and 5 were determined by
X-raystructuralanalysisandtheirorganorhodium(III)cations are
depicted in Figs. 1 and 2. Crystal and refinement data are
summarizedinTable1.Bondlengthsandanglesinthecationsof
3 and 5 are similar to those previously reported for [(h5-C5Me5)
RhCl(pp)]X(pp¼bpy,phen;X (cid:3)¼ClO4 (cid:3) ,CF3SO3 (cid:3) )[7,19,20]and[(h5-
C5Me5)RhCl(pp)](CF3SO3)(pp¼dpq,dppz,dppn)[7].Forinstance,
the 5,6-Me2phen complex 5 exhibits RheN distances of 2.102(2)
and 2.121(2) (cid:2) A, RheC distances in the range 2.151(2)e2.180(2) (cid:2) A
andanRheClbondlengthof2.398(1) (cid:2) A.Theanalogousdistances
in [(h5-C5Me5)RhCl(phen)](CF3SO3) [7] are 2.100(2) and 2.121(2),
2.141(3)e2.171(3) and 2.406(1) (cid:2) A. Pronounced envelope confor-
mationsareadoptedbythefive-memberedNRhNCCchelaterings
in both 3 and 5, as can be gauged by the respective interplanar
angles of 8.3(2)(cid:4) and 10.3(2)(cid:4) between their N1eRh1eN10 and
N1eCeCeN10 planes. Contrasting angles of 14.7(4) and 1.7(5)(cid:4)
Scheme 2. Cations and the employed numbering scheme of the compounds
[(h5-C5Me5)RhCl(pp)](CF3SO3)6e9withdppzandsubstituteddppzligands. were observed for the analogous compounds [(h5-C5Me5)RhCl
Y.Geldmacheretal./JournalofOrganometallicChemistry696(2011)1023e1031 1025
Fig.2. Molecularstructureofthecationofthe5,6-dimethylphenanthrolinecomplex5. Fig.3. CDspectraofCTDNAandmixturesofcomplexes3and5withCTDNA(r¼0.2)
ina10mMphosphatebuffer(pH7.2)aftera2hincubationperiod.Molarellipticities
(pp)](CF3SO3) with the larger polypyridyl ligands dpq and dppz [q]aregivenintheunitsdegcm2dmol(cid:3)1(cid:5)10(cid:3)3.
[7], which exhibit interplanar angles of 76.6(2) and 58.5(3)(cid:4)
1e5at1:5complex/DNAmolarratios([DNA]¼M(basepairs))leads
between their cyclopentadienyl and polypyridyl ring systems.
Somewhat lower interplanar angles of 50.1(2) and 49.4(2)(cid:4) are torelativelyminorchangesinthel maxvaluesandmolarellipticities
[q]oftheseCDbands.Fig.3depictstheCDspectraformixturesofthe
foundinthecationsof3and5,respectively.
5-Mephen and 5,6-Me2phen complexes 3 and 5 with CT DNA at
r¼0.2(r¼[complex]/[DNA]).Itisapparentthattheinteractionwith
2.2. DNA-bindingstudies thecomplexesleadstoabathochromicshiftforl maxofthepositive
band to about 280nm in both cases. Whereas complex 3 causes
Stable intercalative binding into DNA has previously been a small increase in the molar ellipticity [q] of this band, a 10%
confirmed by induced CD bands and large viscosity increases for decrease is observed in the presence of 5. Similar changes were
half-sandwich rhodium(III) polypyridyl complexes with pp¼dpq recordedforcomplex/DNAmixturescontainingthe4-Mephenand
anddppz(6)[7].Asboththesecomplexesarehighlycytotoxicitis 4,7-Me2phencomplexes2and4(Fig.S1).Bathochromicshiftsand
plausible that their activity may be related to DNA distortion or smalldecreasesinmolarellipticityhaveoftenbeenregisteredforCT
damage caused by this binding interaction. It was, therefore, of DNAspectrafollowingtheformationofcovalentadductsthrough
interest to study the effects of both electron-donating methyl metalenucleobasebinding[3].Incontrast,thesignificantincreases
groups(complexes2e5,7)andelectron-withdrawingCOOHandn-
inmolarellipticityandpronouncedblueshiftsforthepositiveCD
BuNHCOgroups(complexes8and9)ontheDNAbindingofsuch bands of complex/DNA mixtures (r¼0.2) containing 7e9 (Fig. 4)
organometallicrhodium(III)complexes. maybeattributedtochangesinnucleobasestackingduetopoly-
pyridylligandintercalation[3,6,7].Additionalstrongevidenceforan
2.2.1. Electronicspectra intercalativebindingmodeisprovidedbythepresenceofinduced
AnegativeCDbandat246nmduetothehelicalBconformation negativeCDbandsataboutl¼296nm.Thelowermolarellipticity
and a positive band at 275nm caused by nucleobase stacking of(cid:3)2.7(cid:5)10 (cid:3)3(l¼297nm)forthemixtureofcomplex8withCT
representthecharacteristicfeaturesinthecirculardichroism(CD) DNA suggests, in comparison to the [q] values of (cid:3)4.6(cid:5)10 (cid:3)3
spectrumofcalfthymusDNA(CTDNA)[21].Additionofcomplexes (l¼301nm)and(cid:3)3.9(cid:5)10 (cid:3)3degcm2dmol (cid:3)1(l¼296nm)forthe
other complexes 7 and 9 (Table 2), that the interaction may be
Table1
Crystalandrefinementdatafor3and5. weakerinthecaseoftheHOOC-dppzcompound8.A[q]valueof
(cid:3)4.5(cid:5)10 (cid:3)3degcm2dmol (cid:3)1 (l¼296nm), very similar to that
Compound 3 5
observedfortheMe2dppzcomplex7,waspreviouslyreportedfor
Formula C24H25ClF3N2O3RhS C25H27ClF3N2O3RhS theunsubstituteddppzcomplex6[7].
M[gmol(cid:3)1] 616.9 630.9
ThermaldenaturationmeasurementsforCTDNAinthepresence
T[K] 115 115
Radiation MoKa MoKa oftransitionmetalcomplexesprovideasimplemeansofgauging
Crystalsystem Triclinic Triclinic
Spacegroup P1 P1
(cid:2)
a[A] 8.0012(4) 7.9010(3)
(cid:2)
b[A] 11.8641(6) 12.1663(8)
(cid:2)
c[A] 13.1047(6) 13.2542(9)
a[(cid:4)] 87.742(4) 90.180(6)
b[(cid:4)] 79.559(4) 100.962(4)
g[(cid:4)] 81.405(4) 98.884(4)
V( (cid:2) A3) 1209.56(10 1235.13(13)
Z 2 2
D[gcm(cid:3)3] 1.694 1.696
F(000) 624 640
m[mm(cid:3)1] 0.956 0.938
2q max[(cid:4)] 50.0 57.5
Collectedrefl. 6974 11,906
Independentrefl. 4263 5517
Refinedparameters 328 332
R1[I>2s(I)] 0.031 0.027
wR2(alldata)a 0.057 0.059
S(goodness-of-fit) 0.800 0.920
Max./min.Dr[e (cid:2) A(cid:3)3] 0.54/(cid:3)0.44 0.56/(cid:3)0.51 Fig.4. CDspectraofCTDNAandmixturesofcomplexes7e9withCTDNA(r¼0.2)in
a10mMphosphatebuffer(pH7.2)aftera2hincubationperiod.Molarellipticities[q]
a wR2 ¼[Sw(Fo 2(cid:3)Fc 2)2/Sw(Fo 2)2]1/2. aregivenintheunitsdegcm2dmol(cid:3)1(cid:5)10(cid:3)3.
1026 Y.Geldmacheretal./JournalofOrganometallicChemistry696(2011)1023e1031
Table2 DA/Avaluesof(cid:3)47,(cid:3)44and(cid:3)44%fortheabsorptionbandatabout
Characteristicparametersfor theinteractionofcomplexes6e9withCTDNA in 370nmbelongingtocomplexes6,8and9arethereforetypicalfor
a10mMphosphatebufferatpH7.2.
thisbindingmodeandareaccompaniedbyredshiftsof5,4 and
Complex DTm a((cid:4)C) [q]b DA/Ac(%) 4nm, respectively. In contrast, a much lower decrease in absor-
6 þ12 (cid:3)4.5(cid:5)10(cid:3)3 (cid:3)47 banceof(cid:3)23%withanassociatedredshiftof5nmisobservedfor
7 8 þ þ 7 4 (cid:3) (cid:3) 4 2 . . 6 7 (cid:5) (cid:5) 1 1 0 0 (cid:3) (cid:3) 3 3 (cid:3) (cid:3) 2 4 3 4 t in he th a e na o l u o t g e o r u b s e b n a z n e d ne of ri t n h g e l M ea e d 2 s d t p o p i z n c c o re m a p se le d x e 7 le .M ctr e o th n y d l e s n u s b i s t t y it i u n t t io h n e
9 þ8 (cid:3)3.9(cid:5)10(cid:3)3 (cid:3)44
dppzringsystemandalsocausesasignificantdisruptionofsolvent
a Meltingtemperatureshift. hydrogen bonding to the quinoxaline nitrogen atoms [23]. It is,
b Molarellipticity(degcm2dmol(cid:3)1)ofthenegativeICDataboutl¼296nm.
therefore,possiblethatsolventeffectsorchangesinthepolypyridyl
c Absorptionshiftofthecomplexabsorptionmaximumataboutl¼370nm.
ligandorientationrelativetothebasestackmayberesponsiblefor
anystabilizationofthedoublehelixduetoabindinginteraction. the much lower decrease in absorption at the 370 and 390nm
Whereas the intercalation of polypyridyl ligands between the maximaoftheMe2dppzcomplex7followingitsintercalationinto
duplexnucleobasepairsgenerallyleadstolargeincreasesintheDNA the double helix. An analogous trend has been reported for the
meltingtemperature,covalentmetalenucleobasebindingtypically complexcations [Ru(phen)2 (Mendpq)]2þ (n¼0,2). Whereas DNA
causesonlyminorchangesinTmandcaneveninvokeadestabiliza- interaction causes a hypochromic shift DA/A of (cid:3)21% for n¼0,
tionoftheBconformation[7].TheDTmvalueslistedforcomplexes amuchsmallerDA/Ashiftofonly(cid:3)7%wasobservedforn¼2[24].
6e9 in Table 2 were recorded under equilibrium conditions in
a 10mM phosphate buffer at pH 7.2 for 1:5 complex/CT DNA 2.2.2. Viscositymeasurements
mixturesandareindicativeofDNAintercalationforthecationsof ThemostconvincingevidenceforDNAintercalationisprovided
the substituted dppz complexes 7 and 9. It is interestingto note, by viscosity titrations [25,26]. Insertion of extended aromatic
however,thattheDTmvalueofþ12(cid:4)Cfortheunsubstituteddppz
ligands such as dppz between adjacent nucleobase pairs causes
complex6issignificantlyhigherthanthevaluesdeterminedforthe lengtheningandstiffeningofthedoublehelixandsuchchangesare
substituteddppzcomplexes.ThissuggestsweakerDNAbindingfor reflected in an increase in DNA viscosity. The reduced viscosity
7e9duepossiblytostericcontactstothesubstitutedgroupscausing function(h/h 0)1/3isequalto(L/L0),wherehandLarethereduced
alessfavourablepolypyridylligandorientationrelativetothebase viscosityandthelengthoftheDNAatagivencomplex/DNAratio
pairs. The lower DTmvalue of þ4(cid:4)C for the HOOC-dppz complex r,andh 0andL0arethecorrespondingvaluesforDNAalone[25].At
8 correlates well with the lower [q] value of (cid:3)2.6(cid:5)10 (cid:3)3 low complex/DNA ratios (0(cid:6)r(cid:6)0.15), ideal monointercalation
degcm2dmol (cid:3)1 for its negative ICD at l¼297nm. Although shouldleadtoavaluecloseto1.0forthefunction(L(cid:3)L0)/(L0 $r),on
theincreasesintheDNAmeltingtemperatureforthepresumably thebasisoftheclassicalmodelofintercalation,wherethehelixis
non-intercalatingcompounds2e5aremuchlowerthanfor6,7and lengthenedbyabout3.4 (cid:2) Aperintercalatedmoiety.Infact,amuch
9,theirrespectivevaluesofþ4,þ3,þ5andþ4(cid:4)Careatthesame widerrangeofhelixextensionsfrom2.0 toabout3.7 (cid:2) A hasbeen
timesignificantlylargerthanthatof(cid:3)1(cid:4)Cpreviouslyrecordedfor established for aromatic molecules by electric dichroism
the phen complex 1 [7]. This suggests that hydrophobic contacts measurements [27]. On plotting experimental (h/h 0)1/3 values
involvingthemethylsubstituentsmaypossiblyhaveastabilizing against the complex/DNA ratio r for r(cid:6)0.15, slope values for
influenceontheCTDNAdoublehelix. (L(cid:3)L0)/(L0 $r) of 0.71,1.02 and 0.93 were obtained for complexes
As depicted in Fig. 5 for complex 9, the (h5-C5Me5)Rh(III) 7, 8 and 9, respectively. All three values are indicative of strong
complexesofsubstituteddppzligandsexhibitcharacteristicbands intercalativebinding.
atabout370and390nm,whichareduetopep*transitioncentred
inthephenazinepartof theligand[22].Followinganincubation 2.3. NOESYstudies
periodof2min,UV/visspectraofbufferedsolutionsofcomplexes
6e9(10mMphosphatebuffer,pH7.2)withCTDNAat25(cid:4)Cand ThebindingmodeoftheMe2dppzcomplex7toaB-typedouble
r¼0.2 exhibit no further significant changes over the next 24h, helixwasalsoinvestigatedby1HNMRspectroscopy.Aminimumof
thereby indicating that achievement of equilibrium conditions six base pairs is necessary to enable the adoption of a normal
mustberelativelyrapid.Pronounceddecreasesinabsorbancefor B-typedoublehelixwithamajorandminorgroove[28].Thishas
themaximaatabout370and390nmandbathochromicshiftsof led to the palindromic hexanucleotide d(50-GTCGAC-30)2 being
these maxima are generally indicative of intercalation of dppz often employed to study the interaction of metal-containing
ligandsbetweenthenucleobasepairsofthedoublehelix[6,7].The monointercalators with a model system for DNA [15,29,30]. Both
the possibility of competition between a number of preferred
intercalative binding sites and extensive peak overlapping can
prevent a detailed 2D NMR spectroscopic analysis of interactions
withlongeroligonucleotides [30].Theemploymentofnon-palin-
dromicoligonucleotidesavoidstheassignmentambiguitiescaused
bysymmetry-relatedbindingssites[31]andwe,therefore,chosed
(50-CGTCGG-30)forourNOESYstudies.Thishexanucleotideoffers
pyrimidineepyrimidine, pyrimidineepurine and purineepurine
sequences for intercalative binding and was selected to contain
a GTC sequence on the basis of the previous observation of
respectively T2/C3 and G1/T2 intercalation for D-[Ru(phen)2
(dpq)]2þ [30]and[(h5-C5Me5)Ir(dppz)(H2-Met-OMe)]3þ
[15]with
palindromicd(50-GTCGAC-30)2.
1H NMR analysis of changes in the exchange kinetics of the
hydrogen-bonded nucleobase imino protons on metal complex
bindingtoDNAoftenprovidesusefulinsightsintothenatureofthe
Fig.5. UV/visspectrumofthen-BuNHCO-dppzcomplex9anditsinteractionmixture
withCTDNAatr¼0.2ina10mMphosphatebuffer(pH7.2)after2min. interaction with the double helix [32,33]. Upfield shifts and
Y.Geldmacheretal./JournalofOrganometallicChemistry696(2011)1023e1031 1027
Fig.6. Changesintheiminoprotonregion(12.2e14.0ppm)ofthe1HNMRspectrum
at288Kofd(50-CGTCGG-30)uponadditionofthecomplex[(h5-C5Me5)RhCl(Me2dppz)]
Cl(7)atr¼0.2,0.4,0.6and1.0. Fig.7. Two-dimensionalNOESYspectrumofthearomaticMe2dppzproton/nucleobase
H6,H8andsugarH20,H200þT3-CH3regionofthe1.1mixtureof7andd(50-CGTCGG-
30)atpH7.2and288K.ThenucleotidesarenumberedfromC1toG6inthefirststrand
pronouncedpeakbroadeningarecharacteristicforcompoundsthat
andC7toG12inthesecondstrand.
intercalateintotheduplex[34]andbothphenomenaareobserved
fortheG2,T3,G5andG9iminoprotonsformixturesofcomplex Inthesecases,NOEcrosspeakswereinaccordancewithdppzinsertion
7andd(50-CGTCGG-30)atr¼0.2,0.4and0.6(Fig.6).Anassignment
betweenA5/T8andC6/G7.
of theGiminoprotonswasnotpossibleowingtotheabsenceof
suitable NOE cross peaks. The signals for the T3 and two of the 2.4. Cytotoxicitymeasurements
G imino protons fully disappeared at r¼1.0 leaving only a very
broad resonance for the remaining G imino proton. A tentative Invitrocytotoxicitystudiesofcompounds1e9wereperformed
assignmentofthisresonancetoG2ispossibleonthebasisofthe withtheadherentcancercelllinesMCF-7andHT-29andwiththe
lowerdegreeofbroadeningobservedforG2eH6incomparisonto
adherent immortalized cell line HEK-293 (human embryonal
G5eH6andG9eH6.Thewideningoftheiminoprotonsignalsisin
kidney).TheresultingIC50valuesarelistedinTable3.Stocksolu-
accordance with rapid solvent exchange due to weakening or tionsofthecompoundsinDMFwerefreshlypreparedanddiluted
interruption of the WatsoneCrick hydrogen bonds. Both the with cell culture medium prior to incubation for 72h (HT-29) or
broader signals and the upfield shifts are clearly indicative of 96h (MCF-7 and HEK-293) with the cells. Reproducible results
intercalativebinding. could notbe obtained for HT-29 cells in the presence of the 5,6-
Both dppz/nucleotide and Cp*eCH3 /nucleotide NOEs could Me2phen complex, presumably owing to the relatively low solu-
be detected in the two-dimensional NOESY spectrum of the bilityofthishalf-sandwichcompoundinthecellculturemedium.
complex7/d(50-CGTCGG-30)mixtureat r¼1.0(Fig.7).The nucle- Anincreaseintheantiproliferativepropertiesofthe(h5-C5Me5)Rh
obase H6/H8 resonances were assigned on the basis of intra- (III)complexesonreplacementofthephenordppzligandsof1and
molecular nH20/and nH200/(nþ1) H6/H8 cross peaks. An 6bymethyl-substitutedderivativesisapparentforcomplexes2e5
upfieldshiftto8.95/8.99ppmwasobservedfortheMe2dppz-H2/9 and7.IncontrasttothefindingsofAldrich-Wrightetal.forPt(II)
protonsatr¼1.0inPBSincomparisontothedoubletat9.29ppm
complexesofthetype[Pt(en)(pp)]Cl2[13],thesechangesarenow
forthecomplexonitsowninCD2Cl2.Nosignalscouldbeidentified
morepronouncedforthe4-Mephenand4,7-Me2phencompounds.
for the Me2dppz-H4/H7 protons at r¼1.0, presumably due to Forinstance,the4-Mephenand4,7-Me2phencomplexes2 and4
pronouncedbroadeningcausedbyintercalativebinding.Addition aresome2.6and3.5timesmoreactivetowardsHT-29cellsthan
of the Me2dppz complex to the hexanucleotide also induced thephencomplex1,whereasonlyatwofoldincreasewasobserved
significantwideningofthearomaticH6/H8resonancesoftheC4,
forthe5-Mephencomplex3.Inthe(en)Pt(II)series,theIC50values
G5, G6, C7 and C8 nucleobases, which suggests that the metal towards murine leukemia L1210 cells improve from 9.7(cid:2)0.4mM
complexmaypreferentiallyintercalatebetweenbasepairsinthis over 2.8(cid:2)0.8 to 1.5(cid:2)0.3mM for pp¼phen, 5-Mephen, 5,6-
partoftheoligonucleotide.
TheintermolecularNOEcrosspeakslinkingG6eH8(7.60ppm)and
Table3
C7eH6(7.53ppm)withtheCp*eCH3protonsofcomplex7(1.65ppm) IC50values(mM)aforthecomplexes[(h5-C5Me5)RhCl(pp)](CF3SO3)1e9towardsthe
andtheNOEbetweenthesugarC7eH20proton(1.98ppm)andtheH2/ celllinesMCF-7,HT-29andHEK-293.
9protons(8.95,8.99ppm)ofitsdppzligand(Fig.7)areinaccordance Compound pp MCF-7 HT-29 HEK-293
withthisconclusion.Thesecontactsprovideevidencefordppzinter-
1 Phen 4.7(0.01) 8.0(1.6) n.d.
calationbetweenthebasepairsG5/C8andG6/C7.TheweakerNOE 2 4-Mephen 1.95(1.30) 3.1(1.1) 0.94(0.02)
betweentheH2/H9protonsandC11eH20(1.74ppm)andthecontact 3 5-Mephen 3.79(0.25) 3.96(0.15) 3.18(0.06)
betweenC1eH6(7.43ppm)andtheCp*eCH3protonsindicatethat 4 4,7-Me2phen 2.50(0.28) 2.27(1.80) 0.40(0.02)
aminorintercalationmodebetweenC1/G12andG2/C11mayalsobe
5 5,6-Me2phen 3.97(0.04) n.d. 0.85(0.14)
6 dppz 1.5(0.4) 4.3(0.2) 2.80(0.10)
involved.Suchasequence-selectiveintercalationbetweenthefirst/last
7 Me2dppz 0.93(0.09) 0.98(0.49) 2.48(0.69)
two base pairs of the octanucleotide would be analogous to that 8 HOOC-dppz >100 >100 >100
previously reported for [(h5-C5Me5)Ir(dppz)(H2-Met-OMe)]3þ and 9 n-BuNHCO-dppz >100 >100 >100
[(h6-C6Me6)Ru(Ac-Met-OH)(dppz)]2þ withd(50-GTCGAC-30)2[15,35]. a Thevaluesinbracketsrepresentthestandarderrors;n.d.¼notdetermined.
1028 Y.Geldmacheretal./JournalofOrganometallicChemistry696(2011)1023e1031
Fig.8. InhibitionofcellproliferationinBJABcellsaftertreatmentwithcomplexes5(a)
and6(b)for24hasmeasuredbyaCASYcellcounter.N¼numberofcellsinunitsof Fig. 9. DNA fragmentation of BJAB cells after incubation for 72h with different
105cells/ml(cid:2)ESD(n¼3);I,thepercentageinhibitionofcellproliferationisdefinedas concentrationsofcomplexes5(a)and6(b).Dataaregivenin%hypoploidy(subG1)
I¼[{(Nc)t (cid:3)(Ns)t}/{(Nc)t (cid:3)(Nc)0}](cid:5)100%, where (Nc)0 and (Nc)t are the numbers of whichreflectsthenumberofapoptoticcells.
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r complex6towardsHEK-293cellsissimilartothevaluesof4.3(cid:2)0.2
24h. and1.5(cid:2)0.4recordedforHT-29andMCF-7cells.
M ina e c 2 t p i h ve en ( , IC b 5 u 0 t > bo 5 t 0 h m t M he ) 4 [ - 1 M 3] e . p I h t e s n ho a u n l d d, 4 h ,7 o - w M e e v 2 e p r h , e b n e c n o o m te p d lex th e a s t ar in e 2.5. Apoptosisinduction
contrastto2e5theplatinum(II)complexesareDNAintercalators
andthatCDmeasurementswereconsistentwithdifferingconfor-
Compounds 1, 5 (pp¼phen, 5,6-Me2phen) and 6 (pp¼dppz)
were selected as biologically active compounds to ascertain
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whetherthecelldeathinvokedby(h5-C5Me5)Rh(III)complexesis
duetoapoptosisand/ornecrosis.Thecomplex8,whichisinactive
(s)andthenumberofmethylsubstituentshavelessinfluenceon
towards MCF-7 and HT-29 cells, was studied for comparison
theactivityoftheorganometallicrhodium(III)complexes.Eachof
purposes.Necroticcelldeathischaracterizedbyanearlyreleaseof
the methyl-substituted phenanthroline complexes 2e5 is signifi-
lactatedehydrogenase(LDH),whereasapoptoticcellsretaintheir
cantly more active towards HEK-293 cells in comparison to the
membrane integrity and do not exhibit an early release of large
cancercelllinesMCF-7andHT-29.
intracellularproteinsincludingLDH[37].NosignificantLDHrelease
Respectively 4.4- and 1.6-fold improvements in its cytostatic
wasobservedfornon-adherentBJABlymphomacells(Burkitt-like
activity towards HT-29 and MCF-7 cells are observed for the
lymphoma) following 3h incubation periods with complexes
Me2dppz complex 7 when compared to the dppz complex 6.
1(Fig.S2a),5(Fig.S3),6(Fig.S2b)and8intheconcentrationrange
d Su p r p p z r ) is a in re gl i y n , a b c o ti t v h e 8 (IC ( 5 p 0 p > ¼ 1 H 00 OO mM C- ) dp to p w z) ar a d n s d th 9 e ( s p a p m ¼ e n c - a B n u c N e H r C c O el - l 5 ca e u 5 s 0 e m n M eg . li T g h ib is le i c n e d l i l c d a e te a s th th b a y t n t e h c e ro o si r s g . anorhodium(III) complexes
lines,astheyalsoaretowardsHEK-293cells.Thissuggeststhatthe
The in vitro inhibition of BJAB cell proliferation bycomplexes
presence of electron-withdrawing substituents mayhave a nega-
1,5,6and8wasalsoevaluated.Afteranincubationperiodof24h,
tiveeffectonthecytotoxicityofthesecomplexes.Wehadconsid-
theviabilityandcellcountweremeasuredwithaCASYCellCounter
ered introducing a biotin-reporter moiety [36] at the end of
andAnalyzerSystem,withthesettingsspecificallydefinedforthe
a polyalkane spacer attached to the dppz ligand but have now
requirementsoftheBJABcells.Thedose-dependentdecreaseincell
abandonedthisprojectowingtothelackofactivityofcomplex9.It
is interesting to note that the IC50 value of 2.80(cid:2)0.10mM for
1
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complexes 1, 5 and 6 are all effective at intermediate dose levels
Table4
(IC50 ¼9e18mM).Althoughinactivetowardstheadherentcelllines
MCF-7andHT-29,HOOC-dppzcomplex8doesinhibitproliferation
Biologicaldataforcomplexes1,5,6and8inBJABcells.
ofthenon-adherentBJABcells,albeitatasignificantlyhigherdose
Compound pp IC50 a(mM) AC50 b(mM) level(IC50 ¼31mM).
1 Phen 11 21 Apoptosis,incontrasttounspecificnecrosis,requiresacontrolled
5 5,6-Me2phen 9 17
andregulatedmechanismleadingtocelldeath.DNAfragmentation
6 dppz 18 19
8 HOOC-dppz 31 >50 (hypoploidy) is considered to be a typical effect of apoptotic cell
death,andwethereforequantifiedtheinductionofapoptosisfor1,5,
a Inhibitionofproliferationafter24h.
b Apoptosis induction after 72h; the AC50 values give concentrations of the
6and8byflowcytometricmeasurementsofDNAfragmentsafter
complexesforwhich50%ofthecellsareundergoingapoptosis. incubatingBJABcellswiththecomplexesfor72h.Thenumbersof
Y.Geldmacheretal./JournalofOrganometallicChemistry696(2011)1023e1031 1029
apoptotic cells for different concentrations of the complexes are RhCl}2(m-Cl)2] [17], [(h5-C5Me5)RhCl(phen)](CF3SO3) (1) [7], [(h5-
illustratedinFig.9for5and6andinFig.S5for1. C5Me5)RhCl(dppz)](CF3SO3)(6)[15]andthedipyrido[3,2-a:20,30-c]
AC50valuesfor50%apoptosisinductionafter72harelistedin phenazine 7,8-dimethyl (Me2dppz) and 2-carboxylic acid (HOOC-
Table4.NosignificantDNAfragmentationwasobservedforBJAB dppz) derivatives [38,18] were synthesized according to literature
cells after a 72h incubation period with concentrations of the procedures.
HOOC-dppzcomplex8intherange0.1e25mM,andonly4.1%after
treatment with 8 at a 50mM concentration. As can be seen from 4.1. Preparationof2e5and7e9
Fig.9andFig.S5,complexes1and5inducesignificantDNAfrag-
mentation levels of, respectively, 14.3 and 28.3% at a 5mM 4.1.1. [(h5-C5Me5)RhCl(4-Mephen)](CF3SO3)(2)
concentration. 38.8% of the BJAB cells are apoptotic following TwoequivalentsofAg(CF3SO3)(77.1mg,0.3mmol)wereadded
treatmentwiththedppz6complexata10mMconcentration. to [{(h5-C5Me5)RhCl}2(m-Cl)2] (92.7mg, 0.15mmol) in 10ml
acetone and stirred in the dark for 2h. Centrifugation of the
3. Conclusions resulting AgCl precipitate and subsequent solvent removal under
vacuum afforded [(h5-C5Me5)RhCl(acetone)2](CF3SO3), which was
Methyl substitution of phenanthroline and dppz ligands in stirredwith4-Mephen(58.3mg,0.3mmol)in10mlCH3OH/CH2Cl2
organorhodium(III) complexes of the type [(h5-C5Me5)RhCl(pp)] (1:1)for5hat75(cid:4)C.Theproductwassubsequentlyprecipitatedby
(CF3SO3)leadstoasignificantincreaseincytostaticactivitytowards addition of diethyl ether, washed and dried in vacuo. Yield: 69%
thehumancancercelllinesMCF-7andHT-29.Forphenanthroline (128mg).C24H25ClF3N2O3RhS:M¼616.9g/mol.Anal.Found(%):C,
derivatives, increases in the antiproliferative properties are more 46.3;H,4.4;N,4.9;Calc.(%):C,46.7;H,4.1;N,4.5.LSIMS:m/z467
pronouncedforthe4-Mephenand4,7-Me2phencomplexes2and4 (100%) [M(cid:3)CF3SO3] þ , 431 (70%) [M(cid:3)CF3SO3 (cid:3)HCl] þ . 1H NMR
than for the 5-Mephen and 5,6-Me2phen complexes 3 and 5. (CD2Cl2,200MHz,30(cid:4)C):d¼1.70(s,15H,Cp*eCH3),2.90(s,3H,4-
Respectively 1.6- and 4.4-fold improvements in activity towards CH3),7.87(d,1H,H3),8.03(dd,1H,H8),8.09(d,1H,H5),8.17(d,1H,
MCF-7andHT-29cellswereobservedfortheMe2dppzcomplex7 H6),8.62(d,1H,H7),8.95(d,1H,H2),9.12(d,1H,H9).
incomparisontothedppzcomplex6.Incontrast,theHOOC-dppz
and n-BuNHCO-dppz complexes 8 and 9 are inactive (IC50 > 4.1.2. [(h5-C5Me5)RhCl(5-Mephen)](CF3SO3)(3)
100mM)towardsboththeadherentcancercelllinesandadherent Preparation as for 2 with the ligand 5-Mephen (58.3mg,
immortalized HEK-293 cells. Whereas electron-donating methyl 0.3mmol). Yield: 74% (136mg). C24H25ClF3N2O3RhS: M¼616.9g/
substituents cause an increase in cytotoxicity for 2e5 and 7, the mol.Anal.Found(%):C,47.1;H,4.1;N,4.9;Calc.(%):C,46.7;H,4.1;N,
presence of electron-withdrawing groups in complexes 8 and 9 4.5. LSIMS: m/z 467 (100%) [M(cid:3)CF3SO3] þ , 431 (60%)
leadstodrasticlossinactivity.InducednegativeCDbandsatabout [M(cid:3)CF3SO3 (cid:3)HCl] þ .1HNMR(CD2Cl2,200MHz,30(cid:4)C):d¼1.84(s,
296nmandlargeviscosityincreasesareinaccordancewithstable 15H,Cp*eCH3),2.93(s,3H,5-CH3),7.97(d,1H,H6),8.19(2dd,2H,H3/
DNA intercalation bycomplexes 7e9 with their substituted dppz 8),8.64(d,1H,H7),8.84(d,1H,H4),9.17(d,1H,H9),9.26(d,1H,H2).
ligands. The lack of cytotoxic activity for 8 and 9, despite their
ability to intercalate, suggests that DNA may well not be an 4.1.3. [(h5-C5Me5)RhCl(4,7-Me2phen)](CF3SO3)(4)
importantintracellulartargetforthesehalf-sandwichrhodium(III) Preparation as for 2 with the ligand 4,7-Me2phen (62.5mg,
complexes. This is most likely also the case for the Menphen 0.3mmol).Yield:75%(143mg).C25H27ClF3N2O3RhS:M¼630.9g/
complexes 2e5 (n¼1,2), for which noexperimental evidence for mol.Anal.Found(%):C,47.9;H,4.0;N,4.3;Calc.(%):C,47.6;H,4.3;
DNA intercalation was obtained. It is possible that improved N, 4.4. LSIMS: m/z 481 (95%) [M(cid:3)CF3SO3] þ , 445 (100%)
cellular uptake, increased electron density in the aromatic ring [M(cid:3)CF3SO3 (cid:3)HCl] þ .1HNMR(CD2Cl2,200MHz,30(cid:4)C):d¼1.84(s,
systems or specific hydrophobic interactions in target binding 15H,Cp*eCH3),2.93(s,6H,4-CH3/7-CH3),7.85(d,2H,H3/8),8.18(s,
pockets may lead to the observed increases in cytotoxicity on 2H,H5/6),8.94(d,2H,H2/9).
methylsubstitutionofboththephenanddppzligands.
Thenatureofcelldeathforlymphomacells(BJAB)wasstudied 4.1.4. [(h5-C5Me5)RhCl(5,6-Me2phen)](CF3SO3)(5)
forcomplexes1,5and6withpp¼phen,5,6-Me2phenanddppz.All Preparation as for 2 with the ligand 5,6-Me2phen (62.5mg,
three complexes induce significant apoptosis via the intrinsic 0.3mmol).Yield:65%(124mg).C25H27ClF3N2O3RhS:M¼630.9g/
mitochondrial pathway. The potential of these (h5-C5Me5)Rh(III) mol.Anal.Found(%):C,47.5;H,4.5;N,4.4;Calc.(%):C,47.6;H,4.3;
polypyridylcomplexesisfurtherunderlinedbythefactthatthey N, 4.4. LSIMS: m/z 481 (87%) [M(cid:3)CF3SO3] þ , 445 (100%)
causeonlynegligiblecelldeathbynecrosis. [M(cid:3)CF3SO3 (cid:3)HCl] þ .1HNMR(CD2Cl2,200MHz,30(cid:4)C):d¼1.69(s,
15H,Cp*eCH3),2.74(s,6H,5-CH3/6-CH3),8.05(dd,2H,H3/8),8.74
4. Experimental (d,2H,H4/7),9.05(d,2H,H2/9).
Solvents were dried and distilled before use. An Analytik Jena 4.1.5. 4.1.5[(h5-C5Me5)RhCl(Me2dppz)](CF3SO3)(7)
SPECORD200spectrometerwasemployedforUV/visiblemeasure- Preparation as for 2 with the ligand Me2dppz (93.1mg,
mentsandCDspectrawereregisteredonaJascoJ-715instrument. 0.3mmol). Yield: 35% (77mg). C31H29ClF3N4O3RhS: M¼733.0g/
LSIMS(LiquidSecondaryIonmassSpectrometry)datawereregis- mol.Anal.Found(%):C,51.3;H,4.0;N,7.5;Calc.(%):C,50.8;H,4.0;
teredwithaFisonsVGAutospecinstrumentemployingacesiumion N, 7.6. LSIMS: m/z 697 (10%) [M(cid:3)Cl] þ , 583 (96%) [M(cid:3)CF3SO3] þ ,
gun(voltage17kV)and3-nitrobenzylalcoholastheliquidmatrix. 548(100%)[M(cid:3)HCl(cid:3)CF3SO3] þ .1HNMR(CD2Cl2,200MHz,30(cid:4)C):
ElementalanalyseswereperformedonaVarioEl(ElementarAna- d¼1.88(s,15H,Cp*eCH3),2.72(s,6H,dppz-CH3),8.26(s,2H,H11/
lysensysteme). RhCl3 $3H2O and Ag(CF3SO3) were purchased from 14),8.32(2dd,2H,H3/8),9.29(d,2H,H2/9),9.92(d,2H,H4/7).
Chempur,1,10-phenanthroline(phen)andits4-methyl(4-Mephen),
5-methyl (5-Mephen), 4,7-dimethyl (4,7-Me2phen) and 5,6- 4.1.6. [(h5-C5Me5)RhCl(HOOC-dppz)](CF3SO3)(8)
dimethyl (5,6-Me2phen) derivatives together with 3,4-diamino Preparation as for 2 with the ligand HOOC-dppz (97.9mg,
benzoic acid, 4,5-dimethyl-1,2-phenyl-diamine, n-butylamine and 0.3mmol). Yield: 31% (70mg). C30H25ClF3N4O3RhS: M¼749.0g/
1,10-carbonyldiimidazole(CDI)fromAcros.Solventswerepurchased mol.Anal.Found(%):C,47.6;H,3.4;N,7.8;Calc.(%):C,48.1;H,3.4;
from TJ Baker and DEUTERO GmbH. The complexes [{(h5-C5Me5) N, 7.5. LSIMS: m/z 599 (100%) [M(cid:3)CF3SO3] þ , 563 (67%)
1030 Y.Geldmacheretal./JournalofOrganometallicChemistry696(2011)1023e1031
[M(cid:3)CF3SO3 (cid:3)HCl] þ .1HNMR(CD2Cl2,400MHz,30(cid:4)C):d¼1.86(s, removeparticulatematerialpriortouse.Reducedviscositieshwere
15H,Cp*eCH3),8.31(m,2H,H3/8),8.54(2d,2H,H13/14),9.11(s,1H, calculated by literature methods [25] and plotted as (h/h 0)1/3
H11),9.27(2d,2H,H2/9),9.92(2d,2H,H4/7). (h 0 ¼reduced viscosity of the DNA solution in the absence of
complex)againstrforrod-likeDNA(approximately600basepairs).
4.1.7. 2-(7-n-Butylamido)dipyrido[3,2-a:20,30-c]phenazine,
(n-BuNHCO-dppz) 4.4. NMRspectroscopy
HOOC-dppz(163.2mg,0.5mmol)and1,10-carbonyldiimidazole
(97.3mg, 0.6mmol) were stirred in 20ml pyridine for 1.5h at BrukerDPX400andDPX200spectrometerswereemployedfor
80(cid:4)C.Followingadditionofn-butylamine(49.6ml,0.5mmol),the the 1H NMR spectroscopic characterization of new compounds
solutionwasheatedforafurther 2hat80(cid:4)Candthenfor1hat withtheresidual1Hsignalofthedeuteratedsolventbeingusedfor
25(cid:4)C.75mldiethyletherwasaddedandthereactionmixturewas chemicalshiftcalculation.Thesplittingsofprotonresonancesare
left to stand for 18h at 4(cid:4)C. After solvent removal the resulting defined as s¼singlet, d¼doublet, t¼triplet and m¼multiplet.
precipitatewasdissolvedinCH2Cl2andseparatedfromunsoluble Thehexanucleotidespectraford(50-CGTCGG-30)withWATERGATE
materialbyfiltration.Theproductwasthenprecipitatedbyaddi- solventsuppressionwererecordedat600.13MHzprotonfrequency
tionofdiethyletherat4(cid:4)Candwashedanddriedinvacuo.Yield: and at 288K on a Bruker DRX 600 spectrometer equipped with
26%(50mg).C23H19N5O:M¼381.2g/mol.Anal.Found(%):C,72.6; apulsedfieldgradientandatripleresonanceprobehead[41e43].
H, 4.8; N,18.3; Calc. (%): C, 72.4; H, 5.0; N,18.4. LSIMS: m/z 404 Theone-dimensional1HNMRspectrawererecordedwithatime
(34%)[MþNa] þ ,382(100%)[MþH] þ ,327(43%)[M(cid:3)C4H7] þ .1H domainof32kdatapointsandaspectralwidthof12,019.23Hz.The
NMR(DMSO-d6,200MHz,30(cid:4)C):d¼0.89(t,3H,n-BueCH3),1.34, sweep width of the two-dimensional homonuclear spectra was
1.40(2dd,4H,n-Bu-Hc/Hd),2.20(m,2H,n-Bu-Hb),2.89(dd,1H,n- 12,019.23Hz in the direct and in the indirect 1H dimension. The
Bu-Ha),7.97(2dd,2H,H3/8),8.32(d,1H,H13)8.51(d,1H,H14),8.77 free-inductiondecaywasacquiredfor340.9msandthedwelltime
(s,1H,H11),9.24(2d,2H,H2/9),9.61(2d,2H,H4/7). wassetto41.6ms.HomonuclearandNOESYspectrawerezero-filled
prior to Fourier transformation and sine apodization functions
4.1.8. [(h5-C5Me5)RhCl(n-BuNHCO-dppz)](CF3SO3)(9) wereappliedinbothdimensions.Allspectrawereprocessedwith
Preparationasfor2withtheligandn-BuNHCO-dppz(114.4mg, NMR-PipeandanalyzedwithNRMView[44,45].Assignmentsand
0.3mmol). Yield: 20% (49mg). C34H34ClF3N5O4RhS: M¼804.1g/ datahandlingwereperformedusingNMRView[45].
mol.Anal.Found(%):C,50.5;H,3.9;N,8.7;Calc.(%):C,50.8;H,4.3;
N,8.7.LSIMS:m/z804(17%)[M(cid:3)H] þ ,713(76%)[M(cid:3)HCl(cid:3)C4H7] þ , 4.5. Cellcultures
563(100%)[M(cid:3)HCl(cid:3)H(CF3SO3)(cid:3)C4H7] þ .
MC7-7 breast cancer and HT-29 colon carcinoma cells were
4.2. X-raystructuralanalyses maintained in DMEM high glucose (PAA) supplemented with
gentamycin(50mgl
(cid:3)1)and10%(v/v)fetalcalfserum(FCS)at37(cid:4)C
Crystals of compounds 3 and 5 suitable for X-ray structural under 5% CO2 and passaged twice a week according to standard
analysis were obtained by slow evaporation of CH3OH/H2O solu- procedures.ForHEK-293cellsDulbecco’smodifiedeaglemedium
tions.TheircrystalandrefinementdataaresummarizedinTable1. (Invitrogen) supplemented with 10% fetal bovine serum and
Intensity datawere collected on an Oxford Diffraction Xcalibur 2 100units/mlpenicillinandstreptomycinwasemployedat37(cid:4)C/5%
diffractometerwithaSapphire2CCDdetectorusinguscansandMo CO2.Thecellsweresplitandaliquotswereseededin35mmculture
Karadiation(l¼0.71073 (cid:2) A).Semi-empiricalabsorptioncorrections dishesthreetimesaweek.BJAB(Burkitt-likelymphoma)cellswere
were applied to the intensities in all cases. The structures were maintained at 37(cid:4)C in RPMI 1640 (GIBCO, Invitrogen) supple-
solvedbydirectmethodswithSHELXSandrefinedagainstF2with mented with 10% heat-inactivated FCS, penicillin (100,000Ul (cid:3)1),
SHELXL[39].Anisotropictemperaturefactorswereintroducedfor streptomycin(0.1gl
(cid:3)1)andL-glutamine(0.56gl (cid:3)1).Thecellswere
non-hydrogen atoms and protons were refined at geometrically subcultured every 3e4 days by dilution of the cells to a concen-
calculatedpositionsasridingatoms. tration of 1(cid:5)105cells/ml. Twenty-four hours before the assay
setup,cellswereculturedataconcentrationof3(cid:5)105cells/mlto
4.3. DNA-bindingstudies ascertainstandardizedgrowthconditions.Fortheapoptosisassays,
the cells were then diluted to a concentration of 1(cid:5)105cells/ml
ThethermaldenaturationtemperaturesTmof1:5complex/DNA immediatelybeforeadditionofthecomplexes.
mixtures [DNA concentration¼M(base pairs)] were determined
for 1e9 in a 10mM phosphate buffer at pH¼7.2. Melting curves 4.6. Cytotoxicitymeasurements
wererecordedat1(cid:4) stepsforthewavelengthl¼260nmwithan
AnalytikJenaSPECORD200spectrometerequippedwithaPeltier Theantiproliferativeeffectsofthecompoundsweredetermined
temperaturecontroller.Tmvalueswerecalculatedbydetermining followingestablishedprocedures[46,47].Cellsweresuspendedin
the midpoints of melting curves from the first order derivatives. cell culture medium (HT-29: 2850cells/ml, MCF-7: 105cells/ml,
TheexperimentalDTmvaluesareestimatedtobeaccuratewithin HEK-293:2000cells/ml),and100mlaliquotsthereofwereplatedin
(cid:2)1(cid:4)C. Concentrations of CT DNA ([DNA]¼M(base pairs)) were 96well plates andincubated at 37(cid:4)C: 5%CO2 for 48h(HT-29or
determined spectrophotometrically using the molar extinction HEK-293) or 72h (MCF-7). Stock solutions of the compounds in
coefficient3 260 ¼13,200M (cid:3)1cm (cid:3)1[40]. DMFwerefreshlypreparedanddilutedwithcellculturemediumto
Viscositiesformixturesofcomplexes7e9withsonicatedDNA thedesiredconcentrations(finalDMFconcentration:0.1%v/v).The
weredeterminedusingaCannon-Ubbelhodesemi-microdilution medium in the plates was replaced with medium containing the
viscometer (Series No 75, Cannon Instruments Co) held at compoundsingradedconcentrations(sixreplicates).Afterfurther
a constant temperature of 25(cid:4)C in a water bath. The viscometer incubationfor72h(HT-29)or96h(MCF-7andHEK-293)thecell
initially contained 2ml of 0.4mM sonicated DNA solution in biomass was determined by crystal violet staining and the IC50
a 10mM phosphate buffer (pH¼7.2). 0.2mM complex solutions valueswereestablishedasthoseconcentrationscausing50%inhi-
alsocontaining0.4mMsonicatedDNAwereaddedinincrementsof bition of cell proliferation. Results were calculated from 2 to 3
100mlfromamicropipette.Solutionswerepassedthroughfiltersto independentexperiments.
Y.Geldmacheretal./JournalofOrganometallicChemistry696(2011)1023e1031 1031
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