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Anticancer Organometallic Osmium(II)-p-cymene Complexes.
DOI: 10.1002/cmdc.201500221 Full Papers
Anticancer Organometallic Osmium(II)-p-cymene
Complexes
Emilia Pa˘unescu,[a] Patrycja Nowak-Sliwinska,[a,b] Catherine M. Clavel,[a] Rosario Scopelliti,[a]
Arjan W. Griffioen,[b] and Paul J. Dyson*[a]
Osmium compounds are attracting increasing attention as po- non-cancerous human embryonic kidney (HEK-293) cells and
tentialanticancerdrugs.Inthiscontext,aseriesofbifunctional human endothelial (ECRF24) cells. Two of these three cancer-
organometallic osmium(II)-p-cymene complexes functionalized cell-selective compounds induce cell death largely via apopto-
with alkyl or perfluoroalkyl groups were prepared and sis and were also found to disrupt vascularization in the chick-
screened for their antiproliferative activity. Three compounds en embryo chorioallantoic membrane (CAM) model. Based on
from the series display selectivity toward cancer cells, with these promising properties, these compounds have potential
moderate cytotoxicity observed against human ovarian carci- clinicalapplications.
noma (A2780) cells, whereas no cytotoxicity was observed on
Introduction
Organometallic complexes based on ruthenium and, to Numerous organometallic osmium-based compounds, par-
alesserextent,osmium,rhodium,andiridium,showpromising ticularly half-sandwich complexes, have been prepared and
antitumor properties that differ from those of classical plati- studied for potential anticancer activity,[15–23] and some of
num-based derivatives.[1] Despite the current focus on rutheni- these compounds have been evaluated in vivo.[24–26] Despite
umcompounds,osmium-baseddrugsalsoappeartobeprom- having structures similar to ruthenium compounds, differences
ising, due, in part, to slower metal–ligand exchange kinetics inthechemicalandphysicalpropertiesofosmiumcompounds
that are highly compatible with physiological environments.[2] were observed as well as notable differences in biological ac-
The prospective clinical applications of the ruthenium com- tivity, including cellular uptake and accumulation, which influ-
pounds (Him)[trans-RuCl(Him)(DMSO)] (NAMI-A)[3,4] and ence the cell cycle and consequently anticancer activity.[24,27]
2 4
(Hind)[trans-RuCl(Hind)] (KP1019),[5–7] as anti-metastatic and Some of these differences have been tentatively ascribed to
2 4 2
anticancer agents, respectively, as well as advances in the pre- a slower ligand exchange (aquation) rate, which is very much
clinical evaluation of [Ru(h6-p-cymene)(PTA)Cl] (PTA=1,3,5- influenced bythenature oftheligands.[2]Forcompoundswith
2
triaza-7-phosphatricyclo[3.3.1.1]decane, RAPTA-C)[8,9] have in- chloride co-ligands, aquation is usually considered to take
spired research of osmium-based structural analogues place following uptake into a cell, activating the complex, al-
(Figure1).[10–14] lowing the compound to undergo reaction with biomolecular
targets.[20,22,28–30]
The potencies of some organometallic osmium(II)–arene
complexes are similar to carboplatin and cisplatin,[19,20] and
structure–cytotoxicity relationships including mechanistic stud-
ies have been reported.[18–20,30,31] Notably, a number of osmium
complexes have been evaluated in vivo including osmium(IV)–
indazole complexes,[32] a nitridoosmium(VI) complex,[33] osmiu-
m(II)–arene complexes,[24–26] and an osmium–carbonyl cluster
(Figure2).[34]
Figure1.OsmiumanaloguesofNAMI-A(left),KP1019(middle)andtheor- The anticancer activity of [HInd][OsIVCl(1H-indazole)] and
2 5
ganometalliccompoundRAPTA-C(right). [HInd][OsIVCl(2H-indazole)] (Figure2) was evaluated on
2 5
aHep3B SCID mouse xenotransplantation model.Overall, both
complexes were well tolerated, and the mice did not display
[a] Dr.E.Pa˘unescu,Dr.P.Nowak-Sliwinska,Dr.C.M.Clavel,Dr.R.Scopelliti,
Prof.P.J.Dyson any symptoms of toxicity. The 1H-indazole complex was found
InstitutdesSciencesetIngƒnierieChimiques to inhibit tumor growth, whereas the 2H-indazole analogue
EcolePolytechniqueFƒdƒraledeLausanne,1015Lausanne(Switzerland) did not inhibit tumor growth, but reduced tumor necrosis.[32]
E-mail:paul.dyson@epfl.ch
The organometallic compound [Os(h6-p-cymene)(N,N-dimethyl-
[b] Dr.P.Nowak-Sliwinska,Prof.A.W.Griffioen
4-(pyridine-2-yldiazenyl)aniline))I][PF] (Figure2) was found to
AngiogenesisLaboratory,DepartmentofMedicalOncology 6
VUUniversityMedicalCenter,1081HVAmsterdam(TheNetherlands) retard tumor growth in a HCT116 human colorectal cancer
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using a route adapted from the
literature.[39] The osmium dimer
[Os(h6-p-cymene)Cl] was initial-
22
lyreactedwithsilveroxalateand
subsequentlywithL6(Scheme1)
toafford8inhighyield.
Compounds 1–8 were fully
characterized by spectroscopic
methods (see Experimental Sec-
tion below), and 6 additionally
by single-crystal X-ray diffraction
analysis (see below). Coordina-
tion of the modified pyridine li-
gands to the osmium(II) center
in 1–7 is accompanied by a shift
Figure2.Osmium-basedanticancercompoundsevaluatedinvivo. of ~Dd =0.4ppm to higher fre-
H
xenograft model.[25] In vivo data on murine mammary carcino-
ma (MCa) are available for a pair of complexes, one based on
ruthenium and the other osmium, namely [M(h6-biphe-
nyl)(N,N’-ethylenediamine)Cl][PF] (Figure2).[24] The ruthenium
6
complexledtoslightdecreasesinbothprimary tumorvolume
and in lung metastasis formation, whereas the osmium conge-
nerwaslessactiveonboththeprimaryandsecondarytumors.
The opposite trend was observed for the in vivo evaluation of
a ruthenium(II)-p-cymene complex and its osmium(II)-p-
cymene analogue containing modified indolo[3,2-c]quinolones
([Ru(h6-p-cymene)[(11H-indolo[3,2-c]quinolin-2-yl)(1-kN-pyridin-
2-ylmethylidene)-kN-amine]Cl]Cl and [Os(h6-p-cymene)[(11H-
indolo[3,2-c]quinolin-2-yl)(1-kN-pyridin-2-ylmethylidene)-kN-
amine]Cl]Cl (Figure2).[26] Compounds were applied both intra-
peritoneally (i.p.) and orally against the murine colon carcino- Scheme1.Synthesisof1–8.Reagentsandconditions:a)L1–L7,CHCl,RT,
2 2
mamodelCT-26,andtheosmium(II)complexdisplayedsignifi- dark,2d;b)Ag 2 C 2 O 4 ,H 2 O,RT,dark,24h;c)L6,CH 2 Cl 2 ,RT,dark,48h.
cant growth-inhibitory activity in contrast to its ruthenium
counterpart, which, at equimolar concentrations, was inactive
in this model. The mitochondria-targeting organometallic tri- quency for the two protons in the aposition of the pyridine
nuclear osmium cluster [Os(CO) (m-H)(m-S)CHN] (Figure2), ring nitrogen atom, and by ~Dd =5ppm for the respective
3 10 9 6 C
whenadministeredbyi.p.injection,significantlyinhibitstumor carbon atoms, relative to the equivalent peaks of the free li-
formation in a murine HCT116 carcinogenesis model, and pre- gands(spectrarecordedinCDCl).Incomplex8thesechanges
3
treatment with the tris-osmium cluster significantly enhances are less pronounced. The 13CNMR spectrum of 8 contains
tumorcellsensitivitytocisplatinanddoxorubicin.[34] a characteristic peak at 165.1ppm that may be attributed to
Herein we describe the synthesis, characterization, and in theoxalateligand,andthepropionateC=Ogroupgivesriseto
vitro and in vivo properties of a series of osmium(II)-p-cymene a resonance at 171.5ppm, this latter value being similar to
complexes derivatized with lipophilic alkyl and perfluoroalkyl that observed in 1–7 (171.6–172.3ppm), also corroborated by
chains, which are structurally related to a series of rutheniu- IR spectroscopy. Moreover, the 19F and 13CNMR and IR spectra
m(II) half-sandwich complexes.[35,36] Comparisons between the of1–8containdistinctivesignalsforthefluorouschain(seeEx-
osmiumandrutheniumcomplexesaremade. perimental Section). Complex 8 is stable in[D]DMSO,whereas
6
1–7 react over time, presumably involving substitution of the
chlorideligandsby[D]DMSO.Theelectrosprayionizationmass
Results and Discussion 6
spectra of 1–7 are dominated by species assigned to [M¢Cl]+
Reaction of the osmium dimer [Os(h6-p-cymene)Cl] with the ions, and the ESI mass spectrum of 8 contains a parent ion
22
appropriate ligand, L1–L7,[35] affords complexes 1–7 peakthatmaybeassignedtothe[M+H]+ ion.
(Scheme1). Compound 8, in which the two chloride ligands in Crystals of 6 suitable for X-ray diffraction were obtained
6 are substituted by an oxalate ligand, an approach used to from chloroform by slow evaporation. The structure of 6 com-
modulate the biological activity of platinum anticancer prisestheexpectedhalf-sandwichthree-leggedpiano-stoolge-
drugs[30,37,38] and ruthenium(II)–arene compounds[39,40] by de- ometry (Figure3), with the h6-p-cymene ring forming the seat
creasing the rate of chloride ligand hydrolysis, was prepared and the pyridine and two chloride ligands the legs of the
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cellselectivity.Incontrast,6isslightlymorecytotoxictoA2780
cells than the ruthenium analogue (IC values of 38mm vs.
50
44mm), but it is less selective. Compound 8 is less cytotoxicto
A2780 cancer cells than the ruthenium analogue (IC =22mm
50
against A2780 cells, 80mm against ECRF24 cells, and >100mm
againstHEK-293cells;Table2).
Compounds 1–8 are less cytotoxic than other osmium(II)–
arene complexes that contain hydrophobic chains, such as
those with 1,2,3-triazolylidene-N-heterocyclic carbine ligands
modifiedwithn-hexylandn-dodecylaliphaticchainswhichex-
hibited low-micromolar IC values against a range of human
50
Figure3.Solid-statestructureof6;thermalellipsoidsare30%equiprobabili- cancer cell lines.[17] However, weakly cytotoxic and even non-
tyenvelopes,andhydrogenatomsarerepresentedbyspheresofarbitrary
cytotoxic ruthenium compounds have, to date, been those
diameter.
that have advanced to clinical trials,[3–7,41] and of the
ruthenium(II)-p-cymene complexes, RAPTA-C is not
cytotoxicandyetinvivohasnumerous antitumor ef-
Table1. Comparison of key bond lengths and angles of complex 6 with those of
[Os(h6-p-cymene)(pta)Cl][14]andRu(h6-benzene)Cl(1H,1H,2H,2H-perfluorodecyl-3-(pyri- fects.[8,9,42,43] Therefore, we decided to investigate the
2 2
din-3-yl)propanoate)(A).[36] mechanism of cell death elicited by 4, 7, and 8, as
these compounds are moderately cytotoxic to A2780
l[(cid:230)]or][8] 6(M=Os) [Os(h6-p-cymene)(pta)Cl](M=Os) ComplexA[a]
2
cells and inactive on HEK-293 and ECRF24 cells.
M¢h6[(cid:230)] 1.651(6) 1.698(3) 1.662,1.663
A2780 cells treated with 4, 7, or 8 were analyzed for
M¢P[(cid:230)] – 2.3324(16) –
M¢N[(cid:230)] 2.133(11) – 2.121(4),2.125(4) DNA content by flow cytometry to investigate cell-
M¢Cl[(cid:230)] 2.425(3),2.427(3) 2.4344(15),2.4194(15) 2.415[b] cycle characteristics. This was done by permeabiliza-
tion of the cells and staining with propidium iodide
Cl-M-Cl[8] 86.32(11) 86.27(5) 88.25(4),88.64(4)
(PI-FACS).[44]Thisrevealedthatasignificantsub-popu-
P-M-Cl[8] – 86.75(5),81.61(6) –
N-M-Cl[8] 83.8(3),86.8(3) – 86.72,86.47[b] lation of the cells undergoes apoptosis (Table3). In-
duction of apoptosis was most pronounced for 4
(58.3%) and 7 (52.4%), administrated at a dose of
50mm. These values are similar to apoptosis induc-
tionbysunitinib(at10mm),ananticancerandantian-
giogenic compound in clinical use[45] which induced
[a]Ru(h6-benzene)Cl(1H,1H,2H,2H-perfluorodecyl-3-(pyridin-3-yl)propanoate) (shown),
containingtwoinde 2 pendentmoleculesintheasymmetricunit.[36][b]Averagevalues. 62.6% of A2780 cells to undergo apoptosis. In con-
trast,8administeredata50mmdoseinducesconsid-
erably less apoptosis (14.1%), indicating that cell
stool.Thebondparametersaroundtheosmium(II)ionaresim-
ilar to those of [Os(h6-p-cymene)(pta)Cl] (pta=1,3,5-triaza-7-
2
phosphaadamantane),[14]andaruthenium(II)–benzenecomplex
Table2. IC valuesfor1–8againstA2780,HEK-293,andECRF24cellsin
containing the same ligand, that is, Ru(h6-benze- 50
cell proliferation inhibition assays (72h). Values for reported ruthenium
ne)Cl(1H,1H,2H,2H-perfluorodecyl-3-(pyridin-3-yl)propanoate), analoguesaregiveninparentheses.[35,36]
2
denotedcompoundAinTable1.
Compd IC [mm][a]
50
A2780 HEK-293 ECRF24
Invitrostudies 1 >100 >100 >100
(>500) (155(cid:156)17)
Complexes 1–8 were evaluated in vitro for their antiprolifera- 2 >100 65(cid:156)3 >100
tive activity using the MTTassay against human ovarian carci- (>500) (>500)
noma (A2780), immortalized human embryonic kidney (HEK- 3 >100 >100 >100
(113(cid:156)2) (86(cid:156)10)
293), and human endothelial (ECRF24) cells (Table2). Com-
4 59(cid:156)1 >100 >100
plexes 1–3 and 5 are not cytotoxic to the A2780 cancer cells (>500) (>500)
(IC >100mm),whereas4and6–8displaymodestcytotoxicity, 5 >100 >100 >100
50
withIC valuesintherangeof38–59mm.Notably,withtheex- (>500) (270(cid:156)18)
50 6 38(cid:156)5 56(cid:156)1 >100
ception of 2 and 6, all the other derivatives are not cytotoxic
(44(cid:156)1) (>500) (56(cid:156)4)
toward HEK-293 cells, which are used as a non-cancerous cell 7 56(cid:156)6 >100 >100
model,andnoneofthecomplexesarecytotoxictothehuman (>500) (>500)
endothelial (ECRF24) cells. Note that the fluorous ligands are 8 56(cid:156)2 >100 >100
(22.4(cid:156)0.1) (>100) (80(cid:156)4)
not cytotoxic to A2780 and HEK-293 cells.[35] Compounds 4
and 7 are more cytotoxic to A2780 cells than the correspond- [a]Valuesaregivenasthemean(cid:156)SDofn=2independentexperiments
carriedoutintriplicate.
ing ruthenium analogues,[35] while retaining a degree of tumor
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CAMs were treated with 4 or 8 (administration of
Table3. Apoptosisinductionandcell-cyclephasesinA2780cellsevaluatedbyFACS
50mm), or with the vehicle control 0.1% DMSO
analysisafterDNAstainingwithpropidiumiodide.[a]
(80mLday¢1 for four consecutive days, days 11–14).
Compd Dose[mm] Apoptosis G/G S G/M On EDD 15, that is, 24h after administration of the
0 1 2
control 0 4.6(cid:156)1.5 33.2(cid:156)0.2 14.0(cid:156)2.5 16.6(cid:156)0.5 last dose, fluorescence angiographies were made
4 25 32.0(cid:156)10.8 26.2(cid:156)5.2 10.2(cid:156)2.1 10.1(cid:156)3.8 after intravenous FITC–dextran injection. Under the
4 50 58.3(cid:156)2.3 17.9(cid:156)0.5 5.5(cid:156)0.1 6.6(cid:156)0.1 applied conditions, avascular zones (Figure4A, white
7 25 13.6(cid:156)4.2 32.0(cid:156)3.0 11.1(cid:156)2.2 12.7(cid:156)2.3
arrows) were observed after treatment with 4 and 8.
7 50 52.4(cid:156)4.0 20.7(cid:156)5.1 10.2(cid:156)3.0 6.9(cid:156)1.3
8 25 2.6(cid:156)0.3 32.5(cid:156)1.8 12.5(cid:156)0.5 6.6(cid:156)0.1 Using image analysis[48,49] the number of branching
8 50 14.1(cid:156)9.0 28.8(cid:156)4.9 11.8(cid:156)2.1 14.6(cid:156)2.9 points per mm2 was compared, confirming that 4
sunitinib 10 62.6(cid:156)8.1 13.8(cid:156)3.3 5.0(cid:156)1.4 4.2(cid:156)1.0 and8significantly decrease thenumber ofcapillaries
[a]Measurementswereperformedafter24hincubationwithtwodosesof4,7,or8, within the treated areas (Figure4B). Complexes 8
aswellassunitinibat10mmasapositivecontrol;valuesarethemean(cid:156)SEMofn=6 and 4 exhibit a great vascular-disrupting activity on
independentexperimentscarriedoutinduplicate.
the CAM with perfused vasculature reductions of 53
and 49%, respectively. Intravenous administration of
the ruthenium analogue of 8 at a dose of 50mm in-
death induced by this compound takes place via mechanisms duced an antivascular effect represented by a decrease in the
not involving apoptosis. There is no evidence that these com- number of branching points per mm2 on the CAM capillary
pounds cause a synchronization of cells in a certain phase of bed by 42%,[36] less than that observed for 4. A similar de-
the cell cycle (Table3); instead the effect of 4, 7, and 8 is crease in the number of branching points per mm2 on the
throughalossofcellsinG andMcell-cyclephases. CAMcapillarybed(by39%)wasobtainedaftertreatmentwith
2
sunitinib at 30mm, that is, at a dose sixfold higher (topical ad-
ministrationbetweenEDD7and9.[50]Variousotherorganome-
talliccompounds,somewithbioactiveligandsknowntoinhib-
InvivoactivityinthechickenCAMmodel
it vascularization, have been shown to have antiangiogenic
The most active alkyl complex 4, that leads to the largest per- properties,includingcompoundsbasedontitanium,[51–54]vana-
centage of apoptotic cell death, and the oxalate derivative 8, dium,[55,56] cobalt,[57] ruthenium,[42,44,58–61] iridium,[62,63] plati-
which induces only limited apoptosis, were evaluated in vivo num,[64] and gold.[65] However, direct comparisons between
for their antivascular activity in the chicken embryo chorioal- thesecompoundstendstobeproblematicduetothedifferent
lantoicmembrane(CAM)model.[46]Thisanalysiswasperformed modelsandprotocols usedtodetermineantiangiogenicactivi-
during embryo development days (EDDs) 11 and 14, the ty. A notable difference in the morphology of the remaining
period during which vascular remodeling takes place.[46,47] vasculature was observed with 4, leading to the complete dis-
appearance of capillaries, with only small perfused vessels ob-
served. The behavior of 4 and 8 on the CAM, together with
the absence of activity in ECRF24 cells in vitro, suggests that
the compounds disrupt vascular activity; that is, they induce
vaso-occlusion of already existing vasculature. Note that no
significantlossofembryobodyweightwasobserved,suggest-
ingalackoftoxicityofboth4and8totheembryoatthecon-
centrationstested.
Conclusions
Significantadvancesinthefieldofruthenium-basedanticancer
agents have initiated interest in related osmium complexes. In
recentyearsmanyosmiumanaloguesofrutheniumcomplexes
havebeenpreparedandevaluatedinvitroandinvivo.Ingen-
eral, the osmium analogues tend to be slightly more cytotoxic
than their ruthenium counterparts in cell culture assays, but in
vivo there are contrasting results which leads to very low pre-
Figure4.Antivascularactivityof4and8intheCAMmodel.A)Angiographic
dictabilityofbehavior:inonepairofcongenerstheruthenium
imagesoftheCAMatembryodevelopmentday15(EDD15)treatedwith
derivative is active in vivo, whereas the osmium derivative is
0.1%DMSO(control),4,or8(50mm,80mLday¢1forfourconsecutivedays,
EDD11–14).ThevasculatureisvisualizedbyFITC–dextranfluorescencean- inactive, and vice versa.[24–26] For the compounds studied
giography(25mgkg¢1,20kDa,l =470(cid:156)20nm,l >520nm).Scalebar: herein,improvedinvitroandinvivoeffectswereobservedrel-
ex em
200mm;whitearrowsindicatetheavascularzones.B)Quantificationbydigi-
ative to related ruthenium compounds. While more in-depth
talanalysisofthefluorescenceangiographyimages:numberofbranching
structure–activity relationships would not be valid at this
pointspermm2;*p<0.05:statisticallysignificant.Errorbarsrepresentstan-
darderrorofthemean. stage, strikingly, the osmium compounds reported herein out-
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perform sunitinib in the invivo CAM model,exerting agreater mentalisotopicpatternfitsthecalculatedonewell;Anal.calcd
antivascular effectat a lower dose. Therefore, these complexes for C H ClNOOs: C 47.41, H 5.97, N 2.13, found: C 47.20, H
26 39 2 2
warrantfurtherevaluationinotherpreclinicalmodels. 5.98,N2.08.
[Os(h6-p-cymene)Cl (decyl-3-(pyridin-3-yl)propanoate)] (2):
Experimental Section 2
According to the general procedure, [Os(h6-p-cymene)Cl]Cl
2 2
(0.120g, 0.152mmol), decyl-3-(pyridin-3-yl)propanoate, L2
Materials and methods: Reagents were purchased from com-
(0.097g,0.334mmol),CHCl (35mL),2d.Theproductwasiso-
mercial sources (Aldrich, Alfa Aesar, and Acros Chemicals), and 2 2
lated as a pale-yellow solid (0.182g, 87%): mp: 81–828C;
OsCl hydrate was obtained from ABCR chemicals. All reagents
3 1HNMR (400MHz, CDCl) d=0.88 (t, J=6.6Hz, 3H), 1.22–1.35
were used without further purification. Reactions were per- 3
(brm, 14H), 1.30 (d, J=6.8Hz, 6H), 1.60 (quint, J=6.8Hz, 2H),
formed in solvents dried using a drying columnapparatus and
2.06 (s, 3H), 2.64 (t, J=7.3Hz, 2H), 2.82 (sept, J=6.8Hz, 1H),
collected and manipulated under N using Schlenk techniques.
2 2.93 (t, J=7.3Hz, 2H), 4.06 (t, J=6.8Hz, 2H), 5.56 (d, J=
The dimer [Os(h6-p-cymene)Cl] was prepared according to
22 5.6Hz, 2H), 5.81 (d, J=5.6Hz, 2H), 7.19 (dd, J=7.7, 5.8Hz,
apublishedprocedure.[66]LigandsL1–L7werepreparedaspre-
1H), 7.53 (ddd overlapped, J=7.7, 1.7Hz, 1H), 8.80 (d, J=
viously described.[35] Silver oxalate was prepared following
5.8Hz, 1H), 8.83ppm (d, J=1.7Hz, 1H); 13CNMR (100MHz,
a published procedure.[39] 1H (400.13MHz), 19F (376.46MHz),
CDCl): d=14.2, 18.2, 22.8, 22.8, 26.0, 27.9, 28.7, 29.4, 29.4,
and 13C (100.62MHz) NMR spectra were recorded on a Bruker 3
29.7, 31.0, 32.0, 34.8, 65.2, 73.2, 75.1, 88.8, 93.9, 124.3, 137.4,
AvanceII400spectrometerat298K.Chemicalshiftsarereport-
137.9, 152.4, 154.6, 172.2ppm; IR: n˜=3052, 2957–2854, 1723,
ed in parts per million and referenced to deuterated solvent
1577, 1467–1427, 1185–1151cm¢1; ESI-MS(+): m/z calcd for
residual peaks (CDCl : 1H d=7.26, 13C{1H} d=77.16ppm;
3 C H ClNOOs+: 652.26, found: 652.42 [M¢Cl]+, the experi-
CDCl : 1H d=5.32, 13C{1H} d=53.84ppm),[67] and coupling 28 43 2
2 2 mentalisotopicpatternfitsthecalculatedonewell;Anal.calcd
constants (J) are reported in Hertz. IR spectra were recorded
for C H ClNOOs: C 48.97, H 6.31, N 2.04, found: C 49.01, H
on a PerkinElmer Spectrum One FT-IR spectrometer. Electro- 28 43 2 2
6.52,N2.01.
spray ionization mass spectra (ESI-MS) were obtained on
a Thermo-Finnigan LCQ Deca XP Plus quadrupole ion-trap in-
[Os(h6-p-cymene)Cl (dodecyl-3-(pyridin-3-yl)propanoate)] (3):
2
strumentoperatedinpositive-ionmodeasdescribedprevious-
According to the general procedure, [Os(h6-p-cymene)Cl]Cl
2 2
ly.[68] Elemental analysis was carried out by the microanalytical
(0.080g, 0.101mmol), dodecyl-3-(pyridin-3-yl)propanoate, L3
laboratory at the Institute of Chemical Sciences and Engineer-
(0.071g,0.222mmol),CHCl (35mL),2d.Theproductwasiso-
2 2
ing (EPFL). Melting points were determined using a SMP3
lated as a pale-yellow solid (0.094g, 65%): mp: 83–848C;
StuartMeltingPointApparatusandareuncorrected.
1HNMR (400MHz, CDCl) d=0.87 (t, J=7.0Hz, 3H), 1.22–1.34
3
General procedure for the synthesis of complexes 1–7: To (brm, 18H), 1.29 (d, J=6.9Hz, 6H), 1.60 (quint, J=6.8Hz, 2H),
a solution of [Os(h6-p-cymene)Cl] (1equiv) in CHCl (10mL), 2.05 (s, 3H), 2.64 (t, J=7.3Hz, 2H), 2.82 (sept, J=6.9Hz, 1H),
22 2 2
a solution of the appropriate ligand (L1–L7, 2.1equiv) was 2.93 (t, J=7.3Hz, 2H), 4.05 (t, J=6.8Hz, 2H), 5.56 (d, J=
added in CHCl (25mL), and the resulting mixture was stirred 5.7Hz, 2H), 5.81 (d, J=5.7Hz, 2H), 7.19 (dd, J=7.6, 5.8Hz,
2 2
at room temperature for two days. The reaction mixture was 1H), 7.53 (ddd overlapped, J=7.6, 1.7Hz, 1H), 8.80 (d, J=
then concentrated under reduced pressure to near dryness, 5.8Hz, 1H), 8.83ppm (s, J=1.7Hz, 1H); 13CNMR (100MHz,
and the complex was precipitated with Et 2 O (5mL), washed CDCl 3 )d=14.3,18.2,22.8,22.8,26.0,27.9,28.7,29.4,29.5,29.7,
successively with EtO (5(cid:213)25mL) and hexane (2(cid:213)25mL), re- 29.7, 29.8, 31.0, 32.0, 65.2, 34.8, 73.1, 75.1, 88.8, 93.9, 124.3,
2
coveredbyfiltration,anddriedunderhighvacuum. 137.4, 137.9, 152.4, 154.6, 172.2ppm; IR: n˜=3066, 2956–2849,
1724, 1579, 1466–1426, 1188–1161cm¢1; ESI-MS(+): m/z calcd
[Os(h6-p-cymene)Cl (octyl-3-(pyridin-3-yl)propanoate)] (1):
2 forC H ClNOOs+:680.29,found:680.42[M¢Cl]+,theexperi-
According to the general procedure, [Os(h6-p-cymene)Cl]Cl 30 47 2
2 2 mentalisotopicpatternfitsthecalculatedonewell;Anal.calcd
(0.120g, 0.152mmol), octyl-3-(pyridin-3-yl)propanoate), L1
for C H ClNOOs: C 50.41, H 6.63, N 1.96, found: C 50.53, H
(0.088g,0.334mmol),CHCl (35mL),2d.Theproductwasiso- 30 47 2 2
2 2 6.70,N1.92.
lated as a pale-yellow solid (0.146g, 73%): mp: 113–1148C;
1HNMR (400MHz, CDCl): d=0.87 (t, J=6.5Hz, 3H), 1.21–1.36 [Os(h6-p-cymene)Cl (octadecyl-3-(pyridin-3-yl)propanoate)]
3 2
(brm, 10H), 1.29 (d, J=6.9Hz, 6H), 1.60 (quint, J=6.8Hz, 2H), (4): According to the general procedure, [Os(h6-p-cyme-
2.05 (s, 3H), 2.63 (t, J=7.3Hz, 2H), 2.81 (sept, J=6.9Hz 1H), ne)Cl]Cl (0.150g, 0.190mmol), octadecyl-3-(pyridin-3-yl)pro-
2 2
2.92 (t, J=7.3Hz, 2H), 4.05 (t, J=6.8Hz, 2H), 5.56 (d, J= panoate, L4 (0.176g, 0.436mmol), CHCl (35mL), 2d. The
2 2
5.6Hz, 2H), 5.80 (d, J=5.6Hz, 2H), 7.19 (dd, J=7.6, 5.7Hz, productwasisolatedasapale-yellowsolid(0.215g,71%):mp:
1H), 7.53 (dd overlapped, J=7.6, 1.3Hz, 1H), 8.79 (dd, J=5.7, 77.5–78.58C; 1HNMR (400MHz, CDCl) d=0.87 (t, J=6.7Hz,
2 2
1.3Hz, 1H), 8.82ppm (d, J=1.3Hz, 1H); 13CNMR (100MHz, 3H), 1.20–1.55 (brm, 30H), 1.27 (d, J=7.0Hz, 6H), 1.59 (quint,
CDCl): d=14.2, 18.2, 22.8, 22.8, 26.0, 27.9, 28.7, 29.3, 29.3, J=6.8Hz, 2H), 1.96 (s, 3H), 2.63 (t, J=7.4Hz, 2H), 2.76 (sept,
3
31.0, 31.9, 34.8, 65.1, 73.1, 75.1, 88.8, 93.8, 124.3, 137.4, 137.9, J=7.0Hz, 1H), 2.92 (t, J=7.4Hz, 2H), 4.03 (t, J=6.8Hz, 2H),
152.4, 154.5, 172.2ppm; IR: n˜=3062, 2958–2854, 1724, 1577, 5.53(d,J=5.7Hz,2H),5.79(d,J=5.7Hz,2H),7.21(dd,J=7.6,
1481–1421, 1196–1165cm¢1; ESI-MS(+): m/z calcd for 5.7Hz, 1H), 7.55 (ddd overlapped, J=7.6, 1.7Hz, 1H), 8.74 (d,
C H ClNOOs+: 624.23, found: 624.33 [M¢Cl]+, the experi- J=5.7Hz,1H),8.78ppm (d,J=1.9Hz,1H);13CNMR(100MHz,
26 39 2
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CDCl) d=14.3, 18.2, 22.7, 23.1, 26.3, 28.1, 29.0, 29.7, 29.8, [Os(h6-p-cymene)Cl]Cl (0.150g,0.190mmol),1H,1H,2H,2H-per-
2 2 2 2
29.9, 30.0, 30.1, 30.1, 32.3, 35.1, 65.3, 73.2, 75.7, 89.0, 93.7, fluorododecyl-3-(pyridin-3-yl)propanoate, L7 (0.304g,
124.5,137.9,138.3,152.5,154.7,172.3ppm;IR:n˜=3057,2954– 0.436mmol), CHCl (35mL), 2d. The product was isolated as
2 2
2850, 1729, 1578, 1469–1428, 1179–1153cm¢1; ESI-MS(+): m/z a pale-yellow solid (0.266g, 64%): mp: 116–1178C; 1HNMR
calcd for C H ClNOOs+: 764.38, found: 764.52 [M¢Cl]+, the (400MHz, CDCl): d=1.28 (d, J=7.0Hz, 6H), 1.98 (s, 3H),
36 59 2 2 2
experimental isotopic pattern fits the calculated one well; 2.43–2.56 (m, 2H), 2.69 (t, J=7.3Hz, 2H), 2.77 (sept, J=7.0Hz,
Anal. calcd for C H ClNOOs: C 54.12, H 7.44, N 1.75, found: 1H), 2.95 (t, J=7.3Hz, 2H), 4.38 (t, J=6.4Hz, 2H), 5.54 (d, J=
36 59 2 2
C54.24,H7.66,N1.72. 5.6Hz, 2H), 5.79 (d, J=5.6Hz, 2H), 7.23 (dd overlapped, J=
7.7, 5.8Hz, 1H), 7.56 (ddd overlapped, J=7.7Hz, 1H), 8.76 (dd
[Os(h6-p-cymene)Cl (1H,1H,2H,2H-perfluorooctyl-3-(pyridin-
2 overlapped, J=5.8Hz, 1H), 8.79ppm (d, J=1.5Hz, 1H);
3-yl)propanoate)] (5): According to the general procedure,
13CNMR (100MHz, CDCl): d=18.2, 22.7, 27.6, 30.6 (t, J=
[Os(h6-p-cymene)Cl]Cl (0.150g,0.190mmol),1H,1H,2H,2H-per- 3
2 2 21Hz), 31.0, 34.5, 56.8 (t,J=4Hz),73.2, 75.0, 88.8, 93.9, 107.3–
fluorooctyl-3-(pyridin-3-yl)propanoate, L5 (0.217g,
120.4 (m series), 124.3, 137.0, 137.9, 152.6, 154.5, 171.6ppm;
0.436mmol), CHCl (35mL), 2d. The product was isolated as
2 2 19FNMR (376MHz, CDCl): d=¢126.10 (m, 2F), ¢123.51 (m,
a pale-yellow solid (0.234g, 69%): mp: 94–95.58C; 1HNMR 3
2F),¢122.68(m,2F),¢121.51-(¢121.99)(m,10F),¢113.61(m,
(400MHz,CDCl):d=1.29(d,J=7.0Hz,6H),2.06(s,3H),2.40–
3 2F), ¢80.74ppm (t, J=9.7Hz, 3F); IR: n˜=3048, 2974–2876,
2.53 (m, 2H), 2.67 (t, J=7.2Hz, 2H), 2.82 (sept, J=7.0Hz, 1H),
1739,1472–1423,1373,1344,1243–1111,1192cm¢1;ESI-MS(+):
2.93 (t, J=7.2Hz, 2H), 4.38 (t, J=6.4Hz, 2H), 5.56 (d, J=
m/z calcd for C H ClF NOOs+: 1058.09, found: 1058.10
5.3Hz, 2H), 5.80 (d, J=5.3Hz, 2H), 7.19 (dd overlapped, J= 30 26 21 2
[M¢Cl]+, the experimental isotopic pattern fits the calculated
7.8, 5.6Hz, 1H), 7.52 (d, J=7.8Hz, 1H), 8.80 (d, J=5.6Hz, 1H),
one well; Anal. calcd for C H ClF NOOs: C 32.98 H 2.40, N
8.83ppm (s, 1H); 13CNMR (100MHz, CDCl): d=18.2, 22.7, 30 26 2 21 2
3 1.28,found:C32.88,H2.64,N1.25
27.6, 30.6 (t, J=22Hz), 31.0, 34.5, 56.8 (t, J=4Hz), 73.2, 75.0,
88.8, 93.9, 102.6–120.8 (m series), 124.3, 137.0, 137.9, 152.6,
[Os(h6-p-cymene)oxalate(1H,1H,2H,2H-perfluorodecyl-3-(pyri-
154.5, 171.6ppm; 19FNMR (376MHz, CDCl) d=¢126.10 (m,
3 din-3-yl)propanoate)] (8): To a solution of [Os(h6-p-cyme-
2F), ¢123.55 (m, 2F), ¢122.84 (m, 2F), ¢121.85 (m, 2F),
ne)Cl] (0.236g, 0.299mmol) in degassed water (50mL), silver
¢113.62 (m, 2F), ¢80.75ppm (t, J=9.9Hz, 3F); IR: n˜=3085– 22
oxalate(0.236g,0.779mmol)wasaddedandtheresultingsus-
2878, 1740, 1472–1423, 1234–1123, 1191cm¢1; ESI-MS(+): m/z
pensionwasstirredatroomtemperaturefor24h.Thereaction
calcd for C H ClF NOOs+: 858.11, found: 858.26 [M¢Cl]+,
26 26 13 2 mixture was filtered on a Celite pad, washed with water (2(cid:213)
the experimental isotopic pattern fits the calculated one well;
30mL), and the resulting filtrate was concentrated under re-
Anal. calcd for C H ClF NOOs: C 34.99, H, 2.94, N, 1.57,
26 26 2 13 2 duced pressure. The resulting intermediate was dissolved in
found:C34.83,H2.75,N1.55.
CHCl (10mL) and added dropwise to a solution of
2 2
1H,1H,2H,2H-perfluorododecyl-3-(pyridin-3-yl)propanoate, L6
[Os(h6-p-cymene)Cl (1H,1H,2H,2H-perfluorodecyl-3-(pyridin-
2 (0.500g, 0.837mmol) in CHCl (25mL). The reaction mixture
3-yl)propanoate)] (6): According to the general procedure, 2 2
was stirred at room temperature for 48h, the reaction mixture
[Os(h6-p-cymene)Cl]Cl (0.189g,0.239mmol),1H,1H,2H,2H-per-
2 2 was filtered, and the filtrate was then concentrated under re-
fluorodecyl-3-(pyridin-3-yl)propanoate, L6 (0.300g,
duced pressure to near dryness, and the complex was precipi-
0.502mmol), CHCl (35mL), 2d. The product was isolated as
2 2 tated with EtO (5mL), washed successively with EtO (5(cid:213)
a pale-yellow solid (0.314g, 66%): mp=107.5–1098C; 1HNMR 2 2
25mL) and hexane (2(cid:213)25mL), thendried under highvacuum.
(400MHz,CDCl):d=1.29(d,J=6.9Hz,6H),2.06(s,3H),2.41–
3 Theproduct was isolatedas a pale-yellow solid (0.529g, 88%):
2.53 (m, 2H), 2.67 (t, J=7.3Hz, 2H), 2.82 (sept, J=6.9Hz, 1H),
mp: 184–1858C; 1HNMR (400MHz, CDCl): d=1.28 (d, J=
2.94 (t, J=7.3Hz, 2H), 4.39 (t, J=6.4Hz, 2H), 5.56 (d, J= 3
6.9Hz, 6H), 2.15 (s, 3H), 2.40–2.55 (m, 2H), 2.66 (t, J=7.0Hz,
5.5Hz, 2H), 5.81 (d, J=5.5Hz, 2H), 7.20 (dd overlapped, J=
2H), 2.71 (sept, J=6.9Hz, 1H), 2.90 (t, J=7.0Hz, 2H), 4.38 (t,
7.8, 5.8Hz, 1H), 7.52 (d, J=7.8Hz, 1H), 8.81 (d, J=5.8Hz, 1H),
J=6.3Hz, 2H), 5.59 (d, J=5.5Hz, 2H), 5.83 (d, J=5.5Hz, 2H),
8.84ppm (s, 1H); 13CNMR (100MHz, CDCl): d=18.2, 22.7,
3 7.27 (dd overlapped, J=7.8, 5.4Hz, 1H), 7.61 (ddd overlapped,
27.7, 30.6 (t, J=22Hz), 31.0, 34.5, 56.8 (t, J=5Hz), 73.2, 75.0,
J=7.8Hz, 1H), 8.30 (d, J=5.4Hz, 1H), 8.38ppm (s, 1H);
88.8, 93.9, 104.9 ¢121.2 (m series), 124.3, 137.0, 137.9, 152.6,
13CNMR (100MHz, CDCl): d=18.1, 22.9, 27.5, 30.5 (t, J=
154.5, 171.7ppm; 19FNMR (376MHz, CDCl): d=¢126.06 (m, 3
3 22Hz), 31.5, 34.3, 56.8 (t, J=4Hz), 71.0, 73.3, 87.7, 92.1, 104.4
2F), ¢123.49 (m, 2F), ¢122.67 (m, 2F), ¢121.87 (m, 4F),
¢120.5 (m series), 125.6, 138.7, 139.0, 150.6, 152.8, 165.1,
¢121.61 (m, 2F), ¢113.59 (m, 2F), ¢81.71ppm (t, J=9.9Hz,
171.5ppm; 19FNMR (376MHz, CDCl): d=¢126.10ppm (m,
3F); IR: n˜=3044, 2975–2879, 1739, 1480, 1423, 1372, 1333, 3
2F), ¢123.52 (m, 2F), ¢122.71 (m, 2F), ¢121.82 (m, 4 F),
1113–1239, 1192cm¢1; ESI-MS(+): m/z calcd for
¢121.51 (m, 2F), ¢113.64 (m, 2F), ¢80.75ppm (t, J=9.8Hz,
C H ClF NOOs+: 958.10, found: 958.11 [M¢Cl]+, the experi-
28 26 17 2 3F); IR: n˜=3047, 2962–2870, 1736, 1699–1665,1479, 1437,
mentalisotopicpatternfitsthecalculatedonewell;Anal.calcd
1363, 1240–1115, 1198cm¢1; ESI-MS(+): m/z calcd for
for C H ClF NOOs: C 33.88, H 2.64, N 1.41, found: C 33.96,
28 26 2 17 2 C H F NOOs: 1011.11, found: 1012.38 [M+H]+, 1034.37
H2.78,N1.39. 30 26 17 6
[M+Na]+,the experimental isotopic pattern fits the calculated
[Os(h6-p-cymene)Cl (1H,1H,2H,2H-perfluorododecyl-3-(pyri- one well; Anal. calcd for C H F NOOs: C 35.69, H 2.60, N
2 30 26 17 6
din-3-yl)propanoate)](7):Accordingtothegeneralprocedure, 1.39;found:C35.54,H2.36,N1.45.
ChemMedChem2015,10,1539–1547 www.chemmedchem.org 1544 (cid:211)2015Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim
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X-ray structure determination: Data collection and crystallo- mentary crystallographic data for this paper. These data are
graphic data for 6 are listed in Table4. Diffraction data of 6 provided free of charge by The Cambridge Crystallographic
were measured at low temperature [100(2)K] using Mo radi- DataCentre.
Ka
ation on a Bruker APEXII CCD diffractometer equipped with
a kappa geometry goniometer. The datasets were reduced by Cell culture: Human A2780 ovarian carcinoma cells were ob-
EvalCCD[69] and then corrected for absorption.[70] The solution tained from the European Centre of Cell Cultures (ECACC, UK).
and refinement were performed by SHELX.[71] Thecrystal struc- A2780 cells were routinely grown in RPMI 1640 medium sup-
ture was refined using full-matrix least-squares based on F2 plemented with GlutaMAX (Gibco), containing heat-inactivated
withallnon-hydrogenatomsanisotropicallydefined.Hydrogen fetalcalfserum(FCS,10%;Sigma,USA)andantibiotics(penicil-
atoms were placed in calculated positions by means of the lin/streptomycin)at378CandCO (5%).Non-tumorigenicHEK-
2
“riding” model. Because of the extensive disorder displayed, 293 cells were provided by the Institute of Pathology, CHUV,
particularly by the fluorous chain, some geometrical restraints Lausanne (Switzerland), and immortalized human endothelial
were applied to C···C distances (SADI cards used for 1,2- cells (ECRF24) were a gift from VUMC, Amsterdam (Nether-
bonded and 1,3-nonbonded carbon atoms), to C¢F bond lands). HEK-293 cells were routinely grown in DMEM (Gibco),
lengths and to F···F distances (DFIX cards employed in this containing heat-inactivated FCS (10%) and antibiotics (1%
case). To improve the behavior of the anisotropic refinement, penicillin/streptomycin) at 378C and CO (5%), whereas
2
all carbon and fluorine atoms of the chain were submitted to ECRF24 cells were grown in gelatin-coated (0.2%) flasks con-
restraints(SIMU0.02 card). CCDC1410467 contains thesupple- taining RPMI 1640 cell culture medium supplemented with
GlutaMAX and DMEM (50:50) supplemented with heat-inacti-
vated FCS (10%), and antibiotics (1% penicillin/streptomycin)
at378CandCO (5%).
2
Table4. Datacollectionandcrystallographicdatafor6.
Antiproliferative activity in vitro: Cytotoxicity was determined
Parameter Value
by MTT assay (MTT=3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
Chemicalformula C H ClF NOOs
28 26 2 17 2 2H-tetrazolium bromide). Cells were seeded in 96-well plates
FW[gmol¢1] 992.6
T[K] 100(2) as monolayers with 100mL cell solution per well and pre-incu-
l[(cid:230)] 0.71073 bated for 24h in the cell medium. Compounds were prepared
Crystalsystem Monoclinic as DMSO solutions that were rapidly dissolved in the culture
Spacegroup P2/c
1 medium and serially diluted to the appropriate concentration
Unitcelldimensions: to give afinal DMSO concentration of 0.5%. The drugsolution
a[(cid:230)] 24.990(4) (100mL) was added to each well, and the plates were incubat-
b[(cid:230)] 9.2741(13) ed foranother 72h.MTT (5mgmL¢1solution) was then added
c[(cid:230)] 14.993(4)
to the cells, and the plates were incubated for a further 4h.
a[8] 90
b[8] 106.210(13) The culture medium was aspirated, and the purple formazan
g[8] 90 crystals formed by mitochondrial dehydrogenase activity of
V[(cid:230)3] 3336.5(11) vitalcellsweresolubilizedinDMSO.Theopticaldensity,direct-
lyproportionaltothenumberofsurvivingcells,wasquantified
Z 4
D [gcm3] 1.976 at l=540nm using a multi-well plate reader, and the fraction
calcd
m[mm¢1] 4.107 of surviving cells was calculated from the absorbance of un-
F(000) 1920 treated control cells. Evaluation is based on the mean of two
Crystalsize 0.34(cid:213)0.22(cid:213)0.16
independent experiments, each comprising three microcul-
Vrangefordatacollection[8] 3.17–27.50
turesperconcentrationlevel.
Indexranges:
h ¢32/32 Apoptosis assays: A2780 cells were seeded on six-well plates
k ¢12/11
(2(cid:213)105cellsperwell)andgrownfor24hincompletemedium
l ¢19/19
before treatment. Compounds 4, 7, 8, and sunitinib (10mm,
Measuredreflections 39910 Pfizer, used as a positive control) were freshly dissolved in
Independentreflections 7623[R(int)=0.0687] DMSO, diluted in complete medium, and added to the cells at
CompletenesstoV=27.508[%] 99.50
the final concentrations listed in Table3, with a final DMSO
Absorptioncorrection Semiempiricalfromequivalents
Max./min.transmission 0.7456/0.4660 concentration of 0.1%. After incubation for 24h, apoptosis
Refinementmethod Full-matrixleast-squaresonF2 wasmeasuredbyflowcytometricdeterminationofsub-diploid
Data/restraints/parameters 7623/207/464 cells after DNA extraction and subsequent staining with propi-
GooF2 1.18
dium iodide (PI) as described previously.[44] Briefly, cells were
R [I>2s(I)] 0.0862
w 1 R 0.2048 harvested and subsequently fixed in 70% ethanol at 208C.
2
R (alldata) 0.1039 After2h,thecellswerere-suspendedinDNAextractionbuffer
1
w La R r 2 gestdiff.peak/hole[(cid:230)¢3] 0 7 . . 2 1 1 4 5 9 0 /¢3.512e (45mm Na 2 HPO 4 , 2.5mm citric acid, 1% Triton X-100, pH7.4)
for 20min at 378C. PI was added to a final concentration of
ChemMedChem2015,10,1539–1547 www.chemmedchem.org 1545 (cid:211)2015Wiley-VCHVerlagGmbH&Co.KGaA,Weinheim
Full Papers
20mgmL¢1, and log scale red fluorescence was analyzed using [10] B. Cebri(cid:130)n-Losantos, A.A. Krokhin, I.N. Stepanenko, R. Eichinger, M.A.
aFACSCaliburinstrument(BDBiosciences,NJ,USA). Jakupec,V.B.Arion,B.K.Keppler,Inorg.Chem.2007,46,5023–5033.
[11] G.E.B(cid:236)chel,I.N.Stepanenko,M.Hejl,M.A.Jakupec,B.K.Keppler,V.B.
Developmental chicken chorioallantoic membrane (CAM) model: Arion,Inorg.Chem.2011,50,7690–7697.
The in vivo experiments were performed in accordance with [12] P.S. Kuhn, G.E. Buchel, K.K. Jovanovic, L. Filipovic, S. Radulovic, P.
Rapta,V.B.Arion,Inorg.Chem.2014,53,11130–11139.
ethical regulations in Swiss law under the guidance of the
[13] A. Dorcier, W.H. Ang, S. Bolano, L. Gonsalvi, L. Juillerat-Jeannerat, G.
local institution authority. The antiangiogenic efficacy of com- Laurenczy, M. Peruzzini, A.D. Phillips, F. Zanobini, P.J. Dyson, Organo-
plexes 4 and 8 was tested in the physiologically developing metallics2006,25,4090–4096.
chicken embryo chorioallantoic membrane (CAM) model[46] be- [14] A.Dorcier,P.J.Dyson,C.Gossens,U.Rothlisberger,R.Scopelliti,I.Taver-
nelli,Organometallics2005,24,2114–2123.
tween embryo development days (EDDs) 11 and 14. Com-
[15] W.F.Schmid,R.O.John,V.B.Arion,M.A.Jakupec, B.K.Keppler,Orga-
plexes 4 and 8 were administered by i.v. injection (50mm, nometallics2007,26,6643–6652.
80mLday¢1 forfour consecutive days),onEDDs 11,12,13,and [16] W.F.Schmid, R.O. John, G.Muhlgassner,P. Heffeter, M.A.Jakupec, M.
14. The control eggs were treated with 0.9% NaCl (80mL per Galanski, W. Berger, V.B. Arion, B.K. Keppler, J. Med. Chem. 2007, 50,
6343–6355.
day for four consecutive days). On EDD 15 the CAMs were vi-
[17] K.J. Kilpin, S. Crot, T. Riedel, J.A. Kitchen, P.J. Dyson, Dalton Trans.
sualized in ovo using FITC–dextran (20kDa, 20mL, 25mgmL¢1, 2014,43,1443–1448.
Sigma–Aldrich) epifluorescence angiography and subsequently [18] A.F.A. Peacock, S. Parsons, P.J. Sadler, J. Am. Chem. Soc. 2007, 129,
analyzed by the image-processing quantification method de- 3348–3357.
[19] Y. Fu, A. Habtemariam, A.M. Basri, D. Braddick, G.J. Clarkson, P.J.
scribed previously.[52] Briefly, based on FITC–dextran fluores-
Sadler,DaltonTrans.2011,40,10553–10562.
cence angiography, the skeleton of the vascular network is [20] Y. Fu, A. Habtemariam, A.M. Pizarro, S.H. vanRijt, D.J. Healey, P.A.
built, and defined descriptors, i.e., branching points per mm2, Cooper,S.D.Shnyder,G.J.Clarkson,P.J.Sadler,J.Med.Chem.2010,53,
give information on the vascular architecture. Five to six eggs 8192–8196.
[21] I.Romero-Canelßn,L.Salassa,P.J.Sadler,J.Med.Chem.2013,56,1291–
were tested per condition. Errors bars represent standard error
1300.
ofthemean. [22] S.H.vanRijt,A.J.Hebden,T.Amaresekera,R.J.Deeth,G.J.Clarkson,S.
Parsons,P.C.McGowan,P.J.Sadler,J.Med.Chem.2009,52,7753–7764.
Statistical analysis: Values are given as mean values (cid:156)SD (in
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