Encapsulation of pyrene-functionalized poly(benzyl ether) dendrons into a water-soluble organometallic cage

Article abstract of DOI:10.1002/asia.201100136

Two generations of lipophilic pyrenyl functionalized poly(benzyl ether) dendrimers (P₁ and P₂) have been synthesized. The thermal properties of the two functionalized dendrimers have been investigated, and the pyrenyl group of the dendritic molecules encapsulated in the arene-ruthenium metalla-cage, [Ru₆(p-cymene)₆(tpt)₂(donq) ₃]⁶⁺ ([1]⁶⁺) (tpt=2,4,6-tri(pyridin-4-yl)-1,3, 5-triazine; donq=5,8-dioxydo-1,4-naphthoquinonato). The host-guest properties of [P₁1]⁶⁺ and [P₂1]⁶⁺ were studied in solution by NMR and UV/Vis spectroscopic methods, thus allowing the determination of the affinity constants. Moreover, the cytotoxicity of these water-soluble host-guest systems was evaluated on human ovarian cancer cells. Copyright

Full text of DOI:10.1002/asia.201100136

DOI: 10.1002/asia.201100136
Encapsulation of Pyrene-Functionalized Poly(benzyl ether) Dendrons into a
Water-Soluble Organometallic Cage
Anaꢀs Pitto-Barry,[a] Nicolas P. E. Barry,[a] Olivier Zava,[b] Robert Deschenaux,[a] and
Bruno Therrien*[a]
Abstract: Two generations of lipophilic
pyrenyl functionalized poly(benzyl
ether) dendrimers (P1 and P2) have
been synthesized. The thermal proper-
ties of the two functionalized dendrim-
ers have been investigated, and the
pyrenyl group of the dendritic mole-
cules encapsulated in the arene–ruthe-
N
G
[P2ꢀ1]6+ were studied in solution by
NMR and UV/Vis spectroscopic meth-
ods, thus allowing the determination of
the affinity constants. Moreover, the
cytotoxicity of these water-soluble
host–guest systems was evaluated on
human ovarian cancer cells.
nium metalla-cage, [Ru6ACTHUNTRGNEU(GN p-cymene)6
Introduction
based on polyamidoamine (PAMAM),[4] poly-(l-lysine),[12]
polyamide,[3] polypropylenimine,[13] and poly-(2,2-bis(hy-
droxymethyl) propionic acid (bis-MPA)[14] dendrimers. The
properties of some dendrimers have been harnessed as drug
carriers,[15] and their combination with metal ions has led to
the discovery of new putative anticancer agents with novel
modes of action.[16]
As far as we are aware, and despite considerable interest
in biological applications of dendrimers, the well-known and
well-documented poly(benzyl ether) dendrimers, based on
benzyloxy-core with dodecanyloxy-end-groups,[17] have not
been reported in this respect. The reason that these den-
drimers has not been tested as biological agents probably
stems from their insolubility in water, which prevents their
formulation in biological media.[18]
Dendrimers are polymeric compounds that possess different
shapes, sizes, and structures (spherical, ellipsoidal, or cylin-
drical), depending on the dendrimerꢀs generation, core, and
peripheral groups,[1] leading to a plethora of different physi-
cal and chemical properties.[2] Since their conception, many
efforts have been made to facilitate the synthesis of these
macromolecules. In particular the groups of Newkome,[3]
Tomalia,[4] Frꢁchet,[5] and others[6] have developed numerous
strategies for the preparation of dendritic systems. The ver-
satility and the accessibility of dendrimers have led to appli-
cations in liquid-crystal chemistry,[7] as materials,[8] and also
in biochemistry.[9,1b] Indeed, many examples of dendrimers
being used as biological agents are known. Selected exam-
ples include antibacterial drugs based on polypropylenimine
(PPI) dendrimers,[10] antiviral drugs comprising poly-(phos-
phorhydrazone) dendrimers with terminal phosphonic acid
and alkyl chain groups,[11] as well as drug-delivery systems
Recently, we have reported water-soluble arene–rutheni-
um metalla-assemblies that are able to encapsulate planar
guest molecules with[19] or without easy release of the
guest.[20] Three generations of hydrophobic pyrenyl-function-
alized cyanobiphenyl dendrimers were encapsulated into the
hexacationic water-soluble arene–ruthenium metalla-prism,
[a] A. Pitto-Barry, N. P. E. Barry, Prof. R. Deschenaux, Dr. B. Therrien
Institut de Chimie
(donq)3]6+ ([1]6+) (tpt=2,4,6-tri(pyri-
[Ru6ACHTUNGRTNE(NNUG p-cymene)6AHCTUNRTGEG(NNUN tpt)2ACHTNUGTRENNUGN
din-4-yl)-1,3,5-triazine; donq=5,8-dioxydo-1,4-naphthoqui-
nonato). The cytotoxicity of the host–guest systems was
evaluated on human ovarian cancer cell lines, which indi-
cates that the metalla-cage [1]6+ was able to deliver the hy-
drophobic guest molecules into cancer cells.[21] Consequent-
ly, to allow poly(benzyl ether) dendrimers with dodecany-
loxy-end-groups to be biologically evaluated, we have pre-
Universitꢁ de Neuchꢂtel
Ave de Bellevaux 51, CH-2000, Neuchꢂtel (Switzerland)
Fax : (+41)32-7182511
E-mail: bruno.therrien@unine.ch
[b] Dr. O. Zava
Institut des Sciences et Ingꢁnierie Chimique
Ecole Polytechnique Fꢁdꢁrale de Lausanne (EPFL)
CH-1015 Lausanne (Switzerland)
Chem. Asian J. 2011, 6, 1595 – 1603
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FULL PAPERS
pared pyrenyl-containing den-
drimers (Pn), based on 3,5-di-
substituted benzyl ether re-
peated units functionalized at
their periphery. These mole-
cules have been fully charac-
terized and their thermal prop-
erties studied. Moreover, the
pyrenyl end-group of P1 and P2
has been encapsulated into the
hydrophobic cavity of prism
[1]6+, giving rise to water-solu-
ble host–guest systems. The
synthesis, host–guest proper-
ties, stability, and cytotoxicity
of [P1ꢀ1]6+ and [P2ꢀ1]6+
.
Results and Discussion
Two generations of dendritic
ꢁ
Scheme 1. Synthesis of P1 and P2 from 1-pyrenebutyric acid and the Gn OH precursors.
precursors
developed
by
Percec et al.,[17b] 3,4,5-tris[p-(n-
dodecan-1-yloxy)benzyloxy]-
ꢁ
benzyl alcohol (G1 OH) and
3,5-bis{3’,4’,5’-tris[p-(n-dodec-
an-1-yloxy)benzyloxy]benzyl-
ꢁ
oxy}benzyl alcohol (G2 OH),
were used for the synthesis of
the
pyrenyl-functionalized
dendrimers P1 and P2. These
two pyrenyl derivatives (Pn)
were obtained in good yield by
an esterification reaction be-
tween 1-pyrenebutyric acid
and the dendritic precursors
ꢁ
Scheme 2. Encapsulation of P1 and P2 in the metalla-prism [1]6+
.
(Gn OH) (Scheme 1).
The H NMR signals for the
1
aliphatic protons of the pyren-
yl unit were used to monitor the progress of the esterifica-
tion reactions. After coupling, these signals were shifted
slightly upfield by up to 0.03 ppm relative to 1-pyrenebutyric
acid. Moreover, the chemical environments of protons H22
in P1 and H27 in P2 (for numbering, see Scheme 1), were
strongly modified and led to a significant downfield shift of
the associated signals (0.46 and 0.48 ppm, respectively). In
their 13C{1H} NMR spectra, an upfield shift was also ob-
served for the signals of carbons C21 in P1 (4.87 ppm) and
C26 in P2 (5.18 ppm). All chemical shifts were consistent
with the formation of P1 and P2 and full assignments are
given in the Experimental Section.
pounds. The resulting hexacationic host–guest systems were
isolated in approximately 80% yield as their triflate salts
[Pnꢀ1]
ACHTNUGRTNE[NUGN CF3SO3]6.
The dinuclear intermediate, mentioned above, can be iso-
lated prior to the formation of the metalla-prism. If the re-
action is performed in water, the aqua complex [Ru
2ACHTUNGTRENNUNG(p-
cymene)2A(donq)(OH2)2][CF3SO3]2 is isolated, which has
C
R
ACHTUNGTRENNUNG
been characterized by IR and NMR spectroscopy, mass
spectrometry, and by single-crystal X-ray diffraction analy-
sis. The molecular structure of the dication [Ru2ACHTUNGTRNEN(UNG p-cymene)2
A
ACHTUNGTRENNUNG
geometrical parameters. The dication adopts a syn geometry
in the crystal, the required pre-orientation for construction
of the metalla-prism,[22] although the existence of the anti
isomer in solution cannot be ruled out. Indeed, an anti ge-
ometry was observed in the crystalline structure of the oxa-
The host–guest systems [Pnꢀ1]6+ were prepared using a
two-step strategy (Scheme 2). The dinuclear complex [Ru2
ACHTUNGTRENNUNG(p-cymene)2ACHTUNGTRENNUNG(donq)Cl2] was first reacted with AgCF3SO3 to
afford a dinuclear intermediate, and then 0.66 equivalent of
the tpt panels and 0.33 equivalent of the guest molecule (Pn)
were added to obtain the corresponding inclusion com-
lato
A
derivatives
[Ru2(hexamethylbenzene)
2ACHTUNGTRENN(UNG oxalato)-
[23]
(p-cymene)2A(oxalato) ,
C
R
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Chem. Asian J. 2011, 6, 1595 – 1603

Encapsulation of Pyrene-Functionalised Dendrons into an Organometallic Cage
(OH2)2]2+ at the
Figure 1. ORTEP drawing of [Ru2ACHTUNGTREN(NNGU p-cymene)2AHCTUNRTGENN(GUN donq)CAHTNUGTRNENGUN
50% probability level. Hydrogen atoms and CF3SO3 anions are omitted
for clarity. Selected bond lengths (ꢄ) and angles (8): Ru1–O1 2.067(10),
Ru1–O2 2.017(10), Ru1–O5 2.157(6), Ru2–O3 1.993(11), Ru2–O4
2.071(9), Ru2–O6 2.150(8); O1-Ru1-O2 86.1(4), O1-Ru1-O5 81.6(3), O2-
Ru1-O5 77.8(3), O3-Ru2-O4 87.2(4), O3-Ru2-O6 81.4(4), O4-Ru2-O6
81.2(3).
while a syn geometry was observed in [Ru
(ON\NO)Cl2] (ON\NO=2,5-di-[2-(trifluoromethyl)-anili-
no]-1,4-benzoquinone).[24]
In the dinuclear complex, each ruthenium center is bound
to a para-cymene ligand and two of the four oxygen atoms
of the dianionic OO\OO donq ligand. A water molecule
completes the coordination sphere of the octahedral ruthe-
2ACHTUNGTRENN(UNG p-cymene)2
ACHTUNGTRENNUNG
Figure 2. HyperChem simulations of [P1ꢀ1]6+ and [P2ꢀ1]6+, hydrogen
atoms and triflate anions being omitted for clarity and carbon atoms of
[1]6+ being dark grey.
nium atoms. In the structure, the ruthenium ruthenium in-
matic ethers at 1244 cmꢁ1 and C=O stretching vibrations of
the ester at 1734 cmꢁ1.
ꢁ
tramolecular separation is 8.363(1) ꢄ, which is slightly
longer than those found in analogous ON\NO dinuclear
complexes.[24] The presence of coordinated water molecules
generates an extensive hydrogen-bonding network between
the oxygen atoms of the triflate anions and the OH2 ligands:
the shortest OgO distance being 2.62(1) ꢄ.
The electronic absorption spectra of metalla-prism [1]6+
and the inclusion complexes [Pnꢀ1]6+ are characterized by
an intense high-energy band centered at 250 nm, which may
be assigned to a localized-ligand or intra
G
N
tion. Two broad high-energy bands associated to metal-to-
ligand charge transfer (MLCT) transitions are observed at
350 and 440 nm and two low-energy MLCT bands occurred
at 640 and 700 nm. In the [Pnꢀ1]6+ spectra, additional
bands, owing to the presence of the guest molecules are ob-
served at 317, 329, and 346 nm. The characteristic pattern of
the electronic absorption spectra of [Pnꢀ1]6+ was used to
study the stability of the inclusion complexes in biological
media. Absorption spectra of [Pnꢀ1]6+ (10ꢁ5 m) are moni-
tored in a solution of 10% of dimethyl sulfoxide and 90%
of RPMI (Roswell Park Memorial Institute medium) 1640
with GlutaMAXꢅ containing 5% fetal calf serum (FCS)
and antibiotics (penicillin and ciproxin). The systems did not
show any signs of degradation or loss of the guest molecule,
even after 24 hours at 408C (Figure 3). For comparison, ab-
sorption spectra of P1 and P2 in dichloromethane at 10ꢁ5 m,
and absorption spectrum of [1]6+ at 10ꢁ5 m in a solution of
10% of dimethyl sulfoxide and 90% of RPMI are also pro-
vided in Figure 3.
Despite obtaining crystals of the dinuclear intermediate,
we were unable to grow crystals of the inclusion systems
[Pnꢀ1]
ACHTUNGTRENNUNG[CF3SO3]6 suitable for X-ray analysis. Consequently,
molecular dynamic simulation using HyperChem[25] provided
a 3D representation of these hexacationic host–guest sys-
tems. The simulations showed, as expected, the dendritic
arm extending from the cavity with the pyrenyl moiety
being encapsulated inside the prism (Figure 2). The [P1ꢀ1]6+
and [P2ꢀ1]6+ systems are estimated to be approximately
4.2 nm long if they are considered to be cylindrical.
The [P1ꢀ1]
N
ACHTNUGTRNEN[UGN CF3SO3]6 host–guest
located between 1550–1600 cmꢁ1, and absorptions of the ar-
ꢁ
omatic C H groups at 3050 cm . Moreover, the bands asso-
1
ciated with the donq bridge, including the strong C=O
The formation of [Pnꢀ1]6+ is also monitored by H NMR
stretching vibration (ca. 1630 cmꢁ1), are near to those ob-
spectroscopy. The signals of the different protons of the pyr-
idyl groups of the tpt panels are shifted upfield compared to
the empty metalla-prism upon formation of the inclusion
systems, whereas the signals of the CH protons of the donq
bridging ligands are shifted downfield. Moreover, broaden-
ing of the signals of the tpt panels is observed, which is char-
served in the dinuclear complex [Ru2ACTHUNTRGNEU(GN p-cymene)2
ACHTUNGTRENNUNG
(donq)Cl2].[19a] In addition to these peaks, strong absorptions
attributed to the triflate anions (1264, 1030, 639 cmꢁ1) are
observed as well as peaks that may be assigned to the guest
ꢁ
molecule, that is, the C O stretching vibration of the aro-
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1597

B. Therrien et al.
FULL PAPERS
influenced by encapsulation (Figure 4); similar behavior is
observed in the NMR spectrum of [P2ꢀ1]6+
.
Under electro-spray mass spectrometry conditions the cat-
ionic host–guest complexes showed remarkable stability.
The peaks corresponding to [P1+1+
(CF3SO3)3]3+ and
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
at m/z 1432.7 and 1794.6, respectively. These peaks are un-
ambiguously assigned on the basis of their characteristic Ru6
isotope patterns (Figure 5).
Thermal Studies
The thermal properties of the pyrenyl derivatives P1 and P2
have been investigated by polarized optical microscopy and
differential scanning calorimetry. During the first heating
run, viscous and birefringent fluids are observed between 53
and 608C for P1 and between 59 and 748C for P2 (Figure 6).
Figure 3. UV/Vis spectra of [1]6+ and [Pnꢀ1]6+ (10ꢁ5 m concentration) in
biological medium (P1 and P2 in CH2Cl2 at 10ꢁ5 m concentration).
acteristic of encapsulation.[20]
As expected, the signals of the
protons of the para-cymene li-
gands located on the periphery
of the prism are not signifi-
cantly affected by the presence
of a guest molecule in the
cavity of [1]6+. The signals of
the pyrenyl moiety broadened
to such an extent that they are
not observed in CD2Cl2, in ac-
cordance with previously re-
ported
encapsulations
in
arene–ruthenium metalla-as-
semblies.[21] The signals of the
CH2 protons of the butyric
chain, connecting the pyrenyl
and dendritic parts, are strong-
ly influenced by encapsulation,
with an upfield shift of
Figure 5. ESI-MS of the peak envelopes corresponding to [P1+1+ACTHNUGRTNENUG ACHUTNGTRENNUGN
(CF3SO3)3]3+ and [P2+1+(CF3SO3)3]3+ and
simulations of their isotopic pattern.
0.6 ppm for the protons of the CH2 group directly attached
to the pyrenyl moiety and 0.2 ppm for the b-CH2 group. The
signals of the protons located on the dendritic arms are not
However, no typical textures are obtained which prevent-
ed the identification of the mesophases. From consideration
of the nature and structure of P1 and P2, the mesophases
could be of columnar type,[17b] although this can only be con-
firmed by X-ray studies. The texture observed for P2 is pre-
sented in Figure 7 as an illustrative example. On the other
hand,
upon
heating,
both
host–guest
systems
Figure 4. 1H NMR spectra of P1 (a), [P1ꢀ1]
[CF3SO3]6 (b) and
Figure 6. Differential scanning calorimetry thermograms of P1 (a) and
P2 (c) recorded during the first heating run.
[1]ACHTUNGTRENNUNG[CF3SO3]6 (c) in CD2Cl2 at 218C.
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Chem. Asian J. 2011, 6, 1595 – 1603

Encapsulation of Pyrene-Functionalised Dendrons into an Organometallic Cage
Figure 8. 1H NMR chemical-shift changes for the Hb proton of the tpt li-
gands vs. the molar ratio of P1
(
) and P2 (&) to [1]6+ (4.0 mm) in
^
CD2Cl2 at 218C.
Figure 7. A thermal-polarized optical micrograph of the texture displayed
by P2 in the unidentified phase upon heating the sample from the solid
state to 668C.
mined from the corresponding association constants ob-
tained at 218C in CD2Cl2 and, in both cases, the values of
DG8 are inferior to ꢁ6.19 kcalmolꢁ1. To confirm the values
of Ka and DG8 for [P1ꢀ1]6+ and [P2ꢀ1]6+, the thermody-
namic properties of the host–guest systems have been also
studied by UV/Vis spectroscopy. Aliquots of a CH2Cl2 solu-
tion of guest molecule Pn are added to a CH2Cl2 solution of
A
E
ACHTNUGTRNEN[NUG CF3SO3]6 show decomposition
(>2008C) under polarized optical microscopy.
[1]ACHTNUTGRNEUNG
[CF3SO3]6 ([Pn]/[1]6+ =0–2 equiv), at 218C. Based on
Host—Guest Studies
changes to the absorbance and applying the Rose and
Drago equation,[28] the association constants of [P1ꢀ1]6+ and
[P2ꢀ1]6+ are estimated (Table 1). This method is widely
used to study binding phenomena, in particular for 1:1 host–
guest systems.[29] The Ka values for [P1ꢀ1]6+ and [P2ꢀ1]6+
obtained using UV/Vis spectroscopy are consistent with the
With the stability of the inclusion systems confirmed, the
host–guest properties of the systems have been further stud-
ied in solution by 1H NMR and UV/Vis spectroscopy.
1H NMR titrations of P1 and P2 in the presence of [1]6+ are
performed in CD2Cl2 at room temperature. Upon gradual
addition of the guest molecule P1 or P2 (0.1–3.0 equivalents)
1
values estimated by H NMR titrations.
to a CD2Cl2 solution of [1]ACHTNUGRTNEUNG[CF3SO3]6 (4.0 mm), chemical
changes to some of the protons of both the host and the
guest are observed in the H NMR spectrum. These changes
Cytotoxicity Studies
1
indicate that rapid inclusion of the guest molecule into the
Metal-based drugs are widely used in clinical applications;
notably, platinum compounds are used for the treatment of
cancer,[30] although ruthenium compounds are now progress-
ing through clinical trials.[31] In addition to the classical
ruthenium compounds in clinical trials, organometallic
arene–ruthenium(II) compounds are attracting considerable
interest as antitumor agents.[32] One of the main limitations
of metal drugs is their lack of selectivity, and consequently
methods to target such drugs for tumor tissue could improve
their therapeutic index by reducing damage to healthy tissue
and lowering drug side-effects. Dendrimers are interesting
drug candidates[9a] because their macroscopic properties
cavity of [1]6+ takes place, as previously observed with
[pyreneꢀ1]
ACHTUNGTRENNUNG
[CF3SO3]6.[19a] Plots of the chemical shifts (Dd)
for the Hb proton of the tpt ligands versus the molar ratio of
P1 or P2 to the prism [1]6+ indicate a 1:1 stoichiometry of
the host–guest systems because no changes to the chemical
shifts are noticed after reaching
a 1:1 stoichiometry
(Figure 8).[26]
From these plots, stability constants of association (Ka)
have been estimated by using the non-linear least-square fit-
ting program winEQNMR2[27] (see Table 1). The binding
free energies (DG8) for [P1ꢀ1]6+ and [P2ꢀ1]6+ are deter-
Table 1. Association constants (Ka) and free energies (DG8) for the encapsulation of P1 and P2 in [1]6+, determined by 1H NMR titration (CD2Cl2 at
218C; 4.0 mm concentration of [1]6+) and UV/Vis spectroscopy (CH2Cl2 at 218C).
Ka [104 Mꢁ1
]
DG8 [kcalmolꢁ1
]
NMR data
[P1ꢀ1]6+
[P2ꢀ1]6+
UV/Vis data
[P1ꢀ1]6+
[P2ꢀ1]6+
G
4.2
3.5
ꢁ6.30
ꢁ6.19
ACHTUNGTRENNUNG
E
4.9
3.9
ꢁ6.39
ꢁ6.25
ACHTUNGTRENNUNG
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B. Therrien et al.
FULL PAPERS
Table 2. IC50 values of Pn, [Pnꢀ1]
ACHTUGNTRNEGUN[CF3SO3]6, [1]ACHUTNTGREN[NUGN CF3SO3]6 and cisplatin on human ovarian A2780 and A2780cisR cell lines.
A2780 [mm]
A2780cisR [mm]
P1
P2
ND
ND
ND
ND
A
G
2.4 (ꢂ0.5)
2.5 (ꢂ0.8)
3.1 (ꢂ1.0)
1.6 (ꢂ0.6)
2.9 (ꢂ0.7)
3.2 (ꢂ0.9)
4.6 (ꢂ0.5)
8.6 (ꢂ0.6)
E
ACHTUNGTRENNUNG
take advantage of passive targeting of tumors via enhanced
permeability and retention effect.[33] Essentially, the normal
endothelial layer surrounding the blood vessels feeding
healthy cells restricts the size of molecules that can diffuse
from the blood, whereas the endothelial layer of blood ves-
sels in diseased tissues is more porous, providing access for
macromolecules to the surrounding cancer cells. In addition,
because diseased tissue does not usually have a lymphatic
drainage system, once macromolecules have entered, they
are retained. Indeed, metallo-dendrimers have been shown
to have promising anticancer properties.[16a,34]
dritic host–guest systems [P1ꢀ1]6+ and [P2ꢀ1]6+ were shown
to exhibit similar cytotoxicities to those of the metalla-prism
alone, which suggests that the dendrimers do not have a det-
rimental effect on the in vitro activity, at least in the ovarian
cancer cell lines studied herein, and should help to target
tumor tissue in vivo by the enhanced permeability and re-
tention effect.
Experimental Section
2ACHTNUTRGNENUG(p-cymene)2AHCUTNGTRNENGUN
2,4,6-Tris(4-pyridyl)-1,3,5-triazine (tpt),[39] [Ru (donq)Cl2],[19a]
Although poly(benzyl ether)-type dendrons have been
used to functionalize metal nanoparticles,[35] and have been
connected with platinum,[36] to the best of our knowledge
the use of such dendrimers as anticancer agents has not
been reported. The cytotoxicity of the water-soluble host–
4-(dimethylamino)pyrimidium para-toluenesulfonate,[40] G1 OH and G2
OH[17b] were prepared according to published methods. 1-Pyrenebutyric
acid and all other reagents were commercially available (Sigma–Aldrich)
and used as received. 1H and 13C{1H} NMR spectra were recorded on a
Bruker AMX 400 spectrometer using the residual protonated solvent as
an internal standard (CD2Cl2: dH =5.32 ppm). Infrared spectra were re-
corded as KBr pellets on a Perkin–Elmer FTIR 1720 X spectrometer.
UV/Vis absorption spectra were recorded on a Uvikon 930 spectropho-
tometer using precision cells made of quartz (1 cm). Microanalyses were
performed at the Mikroelementarisches Laboratorium, ETH Zꢇrich
(Switzerland). Electrospray ionisation mass spectra were obtained in pos-
itive-ion mode on a Bruker FTMS 4.7T BioAPEX II mass spectrometer.
Column chromatography used silica gel 60 (0.063–0.200 mm, Brunsch-
wig). Transition temperatures and enthalpies were determined on a Met-
tler–Toledo DSC1 STARe System differential-scanning calorimeter at a
rate of 108C minꢁ1 under N2. Optical studies were made using a Zeiss-
Axioskop polarising microscope equipped with a Linkam THMS-600 var-
iable-temperature stage.
ꢁ
ꢁ
guest systems [Pnꢀ1]
ACHTUNGTRENNUNG
drimers P1 and P2 and the empty metalla-cage [1]
AHCTUNGTRENNUNG
have been evaluated against A2780 (cisplatin sensitive) and
A2780cisR (cisplatin resistant) human ovarian cancer cells.
Their cytotoxicities, in comparison to cisplatin, are present-
ed in Table 2.
The two Pn dendrimers showed no cytotoxic effects on the
cell lines, presumably owing to their poor solubility in water.
The metalla-cage [1]6+ is water-soluble as well as the host–
guest systems [Pnꢀ1]6+ and in the A2780 cell line they all
exhibit a comparable cytotoxic effect. The cytotoxicities of
[1]6+ and [Pnꢀ1]6+ in the cisplatin-resistant cell line are es-
sentially unchanged from the sensitive cell line. This similar-
ity is not unexpected, because the targets and resistance
mechanisms for arene–ruthenium compounds are believed
to be distinct from those of cisplatin.[37] Despite the high sta-
bility of the host–guest systems in biological media, it is not
clear if the guest is released or not after cellular internaliza-
tion of the host–guest systems. Nevertheless, it is worth
noting that ꢆper metalꢀ [1]6+ and [Pnꢀ1]6+ are less cytotoxic
than cisplatin, but caution should be applied when making
this type of comparison as ruthenium compounds tend to be
better tolerated by the body.[38]
Synthesis of Pyrenyl-Containing Dendrimers P1 and P2
N,N’-dicyclohexylcarbodiimide (100 mg, 0.48 mmol) and 4-(dimethylami-
no)pyrimidium para-toluenesulfonate (45 mg, 0.16 mmol) were added to
ꢁ
ꢁ
a mixture of dendrimer (G1 OH, 361 mg; G2 OH, 665 mg; 0.32 mmol)
and 1-pyrenebutyric acid (93 mg, 0.32 mmol) in dry CH2Cl2 (30 mL) at
08C. The mixture was allowed to warm to RT and stirred for 24 h. The
solvent was removed and the residue purified by column chromatography
on silica gel (CH2Cl2). The solvent was then evaporated under reduced
pressure and the isolated product dried under vacuum, dissolved in
CH2Cl2 (ca. 3 mL), and precipitated with cold MeOH (ca. 80 mL). After
16 h at 58C, the solid was filtered and dried under vacuum.
P1: Yield=272 mg, 86%. UV/Vis (1.0ꢈ10ꢁ5 m, CH2Cl2): lmax 237 nm (e=
0.71ꢈ105 mꢁ1 cmꢁ1), lmax 244 nm (e=0.82ꢈ105 mꢁ1 cmꢁ1), lmax 267 nm (e=
0.34ꢈ105 mꢁ1 cmꢁ1), lmax 277 nm (e=0.56ꢈ105 mꢁ1 cmꢁ1), lmax 314 nm (e=
0.14ꢈ105 mꢁ1 cmꢁ1), lmax 328 nm (e=0.30ꢈ105 mꢁ1 cmꢁ1), lmax 345 nm (e=
0.43ꢈ105 mꢁ1 cmꢁ1). IR (KBr): n˜ =3039 (w, CHaryl), 2920 (s, CH2), 2851 (s,
1
CH3), 1728 (s, C=O), 1105 cmꢁ1 (m, C O). H NMR (400 MHz, CD2Cl2):
ꢁ
Conclusions
d=8.30 (d, 3JHꢁH =9.3 Hz, 1H, H28), 8.17 (d, 3JHꢁH =7.5 Hz, 1H, H34),
8.16 (d, 3JHꢁH =6.7 Hz, 1H, H36), 8.12 (d, 3JHꢁH =8.0 Hz, 1H, H38), 8.10
(d, 1H, H29), 8.04 (s, 2H, H31 and H32), 8.00 (t, 1H, H35), 7.87 (d, 1H,
H39), 7.28 (d, 3JHꢁH =8.6 Hz, 4H, H15), 7.23 (d, 3JHꢁH =8.6 Hz, 2H, H15’),
6.84 (d, 4H, H14), 6.73 (d, 2H, H14’), 6.67 (s, 2H, H20), 5.03 (s, 2H, H22),
4.97 (s, 4H, H17), 4.88 (s, 2H, H17’), 3.91 (t, 3JHꢁH =6.6 Hz, 4H, H12), 3.90
(t, 3JHꢁH =6.5 Hz, 2H, H12’), 3.40 (m, 2H, H26), 2.51 (t, 3JHꢁH =7.2 Hz,
2H, H24), 2.20 (m, 2H, H25), 1.75 (m, 6H, H11 and H11’), 1.42 (m, 6H, H10
Poly(benzyl ether)-type dendrimers with dodecanyloxy-end-
groups functionalized with a pyrenyl moiety were used to
generate host–guest systems in which the pyrenyl ring was
encapsulated within a hydrophilic hexanuclear metalla-
prism whilst the dendritic arm was standing out. The den-
1600
www.chemasianj.org
ꢃ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2011, 6, 1595 – 1603

Encapsulation of Pyrene-Functionalised Dendrons into an Organometallic Cage
and H10’), 1.30 (m, 48H, Haliphatic), 0.88 ppm (t, JHꢁH =6.8 Hz, 9H, H1 and
H1’). 13C{1H} NMR (100 MHz, CD2Cl2): d=173.4 (C23), 159.5 (C13), 159.4
(C13’), 153.4 (C19), 138.5 (C18), 136.4 (C27), 132.1 (C21), 131.8 (C33), 131.3
(C40), 130.5 (C15’), 130.4 (C30), 130.3 (C16’), 129.7 (C15), 129.3 (C16), 129.2
(C37), 127.9 (C31), 127.8 (C29), 127.7 (C39), 127.1 (C32), 126.3 (C35), 125.4
(C41 and C42), 125.3 (C36), 125.2 (C34), 125.1 (C38), 123.8 (C28), 114.8 (C14),
114.4 (C14’), 107.9 (C20), 75.1 (C17’), 71.3 (C17), 68.5 (C12), 68.4 (C12’), 66.6
(C22), 34.3 (C24), 33.1 (C26), 32.4 (C3 and C3’), 30.3 (Caliph), 30.2 (Caliph),
30.1 (Caliph), 30.0 (Caliph), 29.9 (Caliph), 29.8 (Caliph), 29.7 (Caliph), 29.6
(Caliph), 29.5 (Caliph), 27.3 (C25), 26.5 (C10’), 26.4 (C10), 23.1 (C2 and C2’),
14.3 ppm (C1 and C1’). ESI-MS: m/z 1271.8 [P1+Na]+. Elemental analysis
calcd (%) for C84H112O8: C 80.73, H 9.03; found: C 80.75, H 9.05.
data for this paper. These data can be obtained free of charge from the
3
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif.
Synthesis of [Pnꢀ1]CAHTUGNTERN[NUNG CF3SO3]6
A mixture of Ag(CF3SO3) (144 mg, 0.56 mmol) and [Ru CHTUTGNREN(NUNG p-cymene)2
A
2A
AHCTUNGTREG(NNNU donq)Cl2] (198 mg, 0.27 mmol), in MeOH (80 mL) was stirred at RT for
6 h. Then the guest molecule (P1, 114 mg; P2, 213 mg; 0.09 mmol) and tpt
(56 mg, 0.18 mmol) were added and the solution was stirred at 608C for
24 h. The solvent was removed under vacuum and the residue dissolved
in CH2Cl2 (40 mL), and filtered to eliminate AgCl. After evaporation of
CH2Cl2, the solid was washed with diethyl ether and pentane before
being dried under vacuum.
P2: Yield=527 mg, 70%. UV/Vis (1.0ꢈ10ꢁ5 m, CH2Cl2): lmax 236 nm (e=
1.14ꢈ105 mꢁ1 cmꢁ1), lmax 244 nm (e=0.96ꢈ105 mꢁ1 cmꢁ1), lmax 267 nm (e=
0.37ꢈ105 mꢁ1 cmꢁ1), lmax 277 nm (e=0.61ꢈ105 mꢁ1 cmꢁ1), lmax 314 nm (e=
0.13ꢈ105 mꢁ1 cmꢁ1), lmax 328 nm (e=0.29ꢈ105 mꢁ1 cmꢁ1), lmax 345 nm (e=
A
[CF3SO3]6: Yield=389 mg, 82%. UV/Vis (1.0ꢈ10ꢁ5 m, CH2Cl2):
ACHTUNGTRENNUNG
lmax 249 nm (e=2.44ꢈ105 mꢁ1 cmꢁ1), lmax 278 nm (e=1.17ꢈ105 mꢁ1 cmꢁ1),
lmax 321 nm (e=0.71ꢈ105 mꢁ1 cmꢁ1), lmax 331 nm (e=0.83ꢈ105 mꢁ1 cmꢁ1),
lmax 345 nm (e=0.89ꢈ105 mꢁ1 cmꢁ1), lmax 440 nm (e=0.39ꢈ105 mꢁ1 cmꢁ1),
lmax 643 nm (e=0.09ꢈ105 mꢁ1 cmꢁ1), lmax 701 nm (e=0.10ꢈ105 mꢁ1 cmꢁ1).
IR (KBr): n˜ =3050 (w, CHaryl), 1734 (s, C=Oester), 1630 (s, C=Odonq), 1595
(s, C=Ctpt), 1558 (s, C=Ntpt), 1264 (s, CF3), 1242 (s, C-Oaromatic ether), 1030 (s,
CF3), 640 cmꢁ1 (s, CF3). 1H NMR (400 MHz, CD2Cl2): d=8.46 (m, 12H,
Ha), 8.01 (m, 12H, Hb), 7.42 (s, 12H, Hdonq), 7.38 (d, 3JHꢁH =8.4 Hz, 4H,
H15), 7.29 (d, 3JHꢁH =8.4 Hz, 2H, H15’), 6.99 (s, 2H, H20), 6.89 (d, 4H,
0.42ꢈ105 mꢁ1 cmꢁ1). IR (KBr): n˜ =3040 (w, CHaryl), 2922 (s, CH2), 2851 (s,
1
CH3), 1735 (s, C=O), 1107 cmꢁ1 (m, C O). H NMR (400 MHz, CD2Cl2):
ꢁ
d=8.28 (d, 3JHꢁH =9.2 Hz, 1H, H33), 8.14 (d, 3JHꢁH =7.5 Hz, 1H, H39),
8.13 (d, 3JHꢁH =5.4 Hz, 1H, H41), 8.09 (d, 3JHꢁH =7.9 Hz, 1H, H43), 8.06
(d, 1H, H34), 8.00 (s, 2H, H36 and H37), 7.97 (t, 1H, H40), 7.85 (d, 1H,
H44), 7.30 (d, 3JHꢁH =8.6 Hz, 8H, H15), 7.23 (d, 3JHꢁH =8.6 Hz, 4H, H15’),
6.86 (d, 8H, H14), 6.74 (d, 4H, H14’), 6.72 (s, 4H, H20), 6.64 (d, JHꢁH
=
4
2.1 Hz, 2H, H25), 6.58 (t, 1H, H23), 5.10 (s, 2H, H27), 4.96 (s, 8H, H17),
3
4.94 (s, 4H, H22), 4.88 (s, 4H, H17’), 3.93 (t, JHꢁH =7.0 Hz, 12H, H12), 3.91
H14), 6.77 (d, 2H, H14’), 5.64 (d, JHꢁH =6.3 Hz, 12H, Hcym), 5.44 (d, 12H,
3
(t, 3JHꢁH =7.0 Hz, 6H, H12’), 3.38 (m, 2H, H31), 2.53 (t, 3JHꢁH =7.1 Hz,
2H, H29), 2.20 (m, 2H, H30), 1.76 (m, 12H, H11 and H11’), 1.45 (m, 12H,
H10 and H10’), 1.32 (m, 96H, Haliphatic), 0.88 ppm (t, 3JHꢁH =6.8 Hz, 18H,
H1 and H1’). 13C{1H} NMR (100 MHz, CD2Cl2): d=173.4 (C28), 160.5
(C24), 159.6 (C13), 159.4 (C13’), 153.5 (C19), 139.2 (C26), 138.3 (C18), 136.4
(C32), 132.7 (C21), 131.8 (C38), 131.3 (C45), 130.5 (C15’), 130.4 (C16ꢀ and
C35), 129.7 (C15), 129.3 (C16), 129.2 (C42), 127.9 (C36), 127.8 (C34), 127.7
(C44), 127.1 (C37), 126.3 (C40), 125.4 (C46 and C47), 125.3 (C41 and C39),
125.2 (C43), 123.8 (C33), 114.8 (C14), 114.4 (C14’), 107.5 (C25), 107.3 (C20),
102.1 (C23), 75.1 (C17’), 71.3 (C17), 70.7 (C22), 68.5 (C12), 68.4 (C12’), 66.4
(C27), 34.3 (C29), 33.1 (C31), 32.4 (C3 and C3’), 30.2 (Caliph), 30.1 (Caliph),
30.0 (Caliph), 29.9 (Caliph), 29.8 (Caliph), 29.7 (Caliph), 29.6 (Caliph), 27.3 (C30),
26.5 (C10 and C10’), 23.1 (C2 and C2’), 14.3 ppm (C1 and C1’). ESI-MS: m/z
Hcym), 5.27 (s, 2H, H22), 5.12 (s, 4H, H17), 4.96 (s, 2H, H17’), 3.95 (t,
3JHꢁH =6.5 Hz, 4H, H12), 3.92 (t, 3JHꢁH =5.5 Hz, 2H, H12’), 2.75 (sept,
ACTHNUTRGNEUNG
3JHꢁH =7.0 Hz, 6H, CH(CH3)2), 2.54 (m, 2H, H26), 1.96 (s, 18H, CH3),
1.81 (m, 4H, H24 and H25), 1.76 (m, 6H, H11 and H11’), 1.45 (m, 6H, H10
and H10’), 1.28 (m, 84H, Haliphatic and CH(CH3)2), 0.88 ppm (m, 9H, H1
AHCTUNGTRENNUNG
and H1’). 13C{1H} NMR (100 MHz, CD2Cl2): d=173.7 (C23), 171.3 (CO),
169.0 (Ctpt), 159.6 (C13), 159.5 (C13’), 153.5 (Ca), 153.0 (C19), 143.8 (Ctpt),
138.4 (C18), 138.1 (CHdonq), 132.0 (Cpyr), 130.7 (C15’), 130.2 (C16’), 129.8
(C15), 129.3 (C16), 127.9 (Cpyr), 127.6 (CHpyr), 127.4 (CHpyr), 126.7 (Cpyr),
126.3 (Cb), 114.8 (C14), 114.5 (C14’), 112.0 (Cdonq), 108.2 (C20), 104.2 (Ccym),
100.7 (Ccym), 85.2 (CHcym), 83.0 (CHcym), 75.3 (C17’), 71.5 (C17), 68.5 (C12),
68.4 (C12’), 66.9 (C22), 32.3 (C26), 31.1 (C3 and C3’), 30.1 (CH
(Caliph), 26.4 (C10 and C10’), 23.1 (C2 and C2’), 22.4 (CH
(CH3)2), 17.4
(CH3), 14.3 ppm (C1 and C1’). ESI-MS: m/z 1432.7 [P1+1+(CF3SO3)3]3+
.
ACHTUGNTRNEN(UNG CH3)2), 29.8
AHCTUNGTRENNUNG
2355.6 [(P2+1)+Na]+. Elemental analysis calcd (%) for C155H214O16
:
G
C 79.78, H 9.24; found: C 80.02, H 9.27.
Elemental analysis calcd (%) for C216H232F18N12O38Ru6S6: C 54.68, H 4.93,
N 3.54; found: C 54.93, H 5.19, N 3.54.
Synthesis of [Ru
G
2ACHTGNUTERN(NUGN donq)ACHNUTTRGEG(NNUN OH2)2]CAHTNUGRTEN[NNGU CF3SO3]2
A
[CF3SO3]6: Yield=484 mg, 83%. UV/Vis (1.0ꢈ10ꢁ5 m, CH2Cl2):
ACHTUNGTRENNUNG
lmax 245 nm (e=2.78ꢈ105 mꢁ1 cmꢁ1), lmax 278 nm (e=1.18ꢈ105 mꢁ1 cmꢁ1),
lmax 319 nm (e=0.81ꢈ105 mꢁ1 cmꢁ1), lmax 329 nm (e=0.88ꢈ105 mꢁ1 cmꢁ1),
lmax 345 nm (e=0.85ꢈ105 mꢁ1 cmꢁ1), lmax 440 nm (e=0.47ꢈ105 mꢁ1 cmꢁ1),
lmax 643 nm (e=0.15ꢈ105 mꢁ1 cmꢁ1), lmax 701 nm (e=0.17ꢈ105 mꢁ1 cmꢁ1).
IR (KBr): n˜ =3050 (w, CHaryl), 1733 (s, C=Oester), 1630 (s, C=Odonq), 1582
(s, C=Ctpt), 1564 (s, C=Ntpt), 1264 (s, CF3), 1242 (s, C-Oaromatic ether), 1030 (s,
CF3), 639 cmꢁ1 (s, CF3). 1H NMR (400 MHz, CD2Cl2): d=8.53 (m, 12H,
Ha), 8.29 (m, 12H, Hb), 7.34 (s, 12H, Hdonq), 7.32 (m, 8H, H15), 7.23 (d,
3JHꢁH =8.5 Hz, 2H, H15’), 6.87 (d, 3JHꢁH =8.8 Hz, 8H, H14), 6.85 (s, 4H,
H20), 6.74 (d, 4H, H14’), 6.73 (m, 2H, H25), 6.70 (t, 1H, H23), 5.64 (d,
3JHꢁH =6.3 Hz, 12H, Hcym), 5.45 (d, 12H, Hcym), 5.26 (s, 2H, H27), 5.06 (s,
4H, H22), 4.99 (s, 8H, H17), 4.88 (s, 4H, H17’), 3.94 (m, 18H, H12 and H12’),
A mixture of Ag
ACHTUNGTRENNUNG(donq)Cl2] (204 mg, 0.28 mmol), in MeOH (80 mL) was stirred at RT for
6 h. After filtration of AgCl, the filtrate was evaporated to dryness to
give a green solid. Recrystallisation from CH2Cl2/H2O afforded nicely
shaped green crystals of [Ru2ACTHUGNETRN(NUGN p-cymene)2CAHTUNGTERN(NGU donq)AHCTNUGRETG(NNUN OH2)2]ACHTNUGRTEN[NNGU CF3SO3]2.
2A
Yield=202 mg, 73%. IR (KBr): n˜ =3070 (w, CHaryl), 1536 (s, C=O),
1268 cmꢁ1 (s, CF3). 1H NMR (400 MHz, D2O): d=7.22 (s, 4H, Hdonq),
5.71 (d, 3JHꢁH =6.8 Hz, 4H, Hcym), 5.48 (d, 4H, Hcym), 2.68 (sept, JHꢁH
=
3
5.8 Hz, 2H, CHACHTUNGTRENNUNG(CH3)2), 2.12 (s, 6H, CH3), 1.19 ppm (d, 12H, CHACHTUNGTRENN(UNG CH3)2).
13C{1H} NMR (100 MHz, D2O): d=173.1 (CO), 139.2 (CHdonq), 114.7
(Cdonq), 102.1 (Ccym), 10.1 (Ccym), 84.3 (CHcym), 82.9 (CHcym), 30.6 (CH-
G
2.78 (sept, 3JHꢁH =7.0 Hz, 6H, CH
18H, CH3), 1.73 (m, 16H, H29, H30, H11 and H11’), 1.45 (m, 12H, H10 and
H10’), 1.28 (m, 132H, Haliphatic and CH(CH3)2), 0.88 ppm (m, 18H, H1 and
(CH3)2), 2.57 (m, 2H, H31), 2.01 (s,
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
H1’). 13C{1H} NMR (100 MHz, CD2Cl2): d=175.6 (C28), 171.3 (CO), 170.7
(Ctpt), 160.1 (C24), 159.6 (C13), 159.4 (C13’), 153.5 (C19’), 153.2 (Ca), 151.4
(Ctpt), 139.3 (C18), 139.2 (C26), 138.0 (CHdonq), 133.2 (C21), 130.5 (C15’),
130.1 (C16’), 129.8 (C15), 129.3 (C16), 124.8 (Cb), 123.1 (CHpyr), 122.6
(CHpyr), 119.9 (CHpyr), 119.7 (Cpyr), 119.5 (Cpyr), 114.8 (C14), 114.4 (C14’),
111.9 (Cdonq), 108.2 (C23), 108.0 (C25), 107.4 (C20), 104.4 (Ccym), 100.5
(Ccym), 85.0 (CHcym), 83.2 (CHcym), 75.4 (C17’), 71.4 (C17), 68.5 (C12), 68.4
X-ray Data for [Ru2ACHTUNGTRENNUNG(p-cymene)2ACHUTNGTERNNUG(donq)AHCNUTEGRTGNN(UN OH2)2]CAHTNUGTERN[NNGU CF3SO3]2
C32H36F6O12Ru2S2, M=992.89, Monoclinic, space group P n (No. 7), cell
parameters a=12.0325(15), b=12.8232(10), c=13.2027(16) ꢄ, b=
111.401(9)8, V=1896.7(4) ꢄ3, T=173(2) K, Z=2, 1cald =1.738 gcmꢁ3, l
(MoKa)=0.71073 ꢄ, 9031 reflections measured, 4269 unique (Rint
=
0.1283) which were used in all calculations. The structure was solved by
direct method (SHELXL-97)[41] and refined by full-matrix least-squares
methods on F2 with 463 parameters. R1 =0.0635 (I>2s(I)) and wR2 =
(C12’), 32.3 (C26), 31.1 (C3 and C3’), 30.1 (CH
(C10 and C10’), 23.1 (C2 and C2’), 22.4 (CH
(CH3)2), 17.5 (CH3), 14.3 ppm
(C1 and C1’). ESI-MS: m/z 1794.6 [P2+1+(CF3SO3)3]3+. Elemental analy-
CATHGNURTNE(UNNG CH3)2), 30.0 (Caliph), 26.5
AHCTUNGTRENNUNG
0.1621, GOF=0.829; max./min. residual density 1.036/ꢁ1.069 eꢄꢁ3
.
ACHTUNGTRENNUNG
Figure 1 was drawn with ORTEP-3.[42] CCDC 810124 [Ru
(p-cymene)2
sis calcd (%) for C287H334F18N12O46Ru6S6: C 59.14, H 5.78, N 2.88; found:
C 58.40, H 6.15, N 2.63.
ACHTUNGTRENNUNG(donq)ACHTUNGTRENNUNG(OH2)2]ACHTUNGTRENNUNG[CF3SO3]2 contains the supplementary crystallographic
Chem. Asian J. 2011, 6, 1595 – 1603
ꢃ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
1601

B. Therrien et al.
FULL PAPERS
Culture and Inhibition of Cell Growth
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Human A2780 and A2780cisR ovarian carcinoma cells were obtained
from the European Centre of Cell Cultures (ECACC, Salisbury, UK) and
maintained in culture as described by the provider. The cells were rou-
tinely grown in RPMI 1640 medium with GlutaMAXꢅ containing 5%
fetal calf serum (FCS) and antibiotics (penicillin and ciproxin) at 378C
and 5% CO2. To evaluate the growth inhibition, the cells were seeded in
96-well plates (25ꢈ103 cells per well) and grown for 24 h in complete
medium. Complexes were added to the required concentration and
added to the cell culture for 72 h incubation. Solutions of the compounds
were applied by diluting a freshly prepared stock solution of the corre-
sponding compound in aqueous RPMI medium with GlutaMAXꢅ
(20 mm). Following drug exposure, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) was added to the cells at a final concen-
tration of 0.25 mgmLꢁ1 and incubated for 2 h, then the culture medium
was aspirated and the violet formazan (artificial chromogenic precipitate
of the reduction of tetrazolium salts by dehydrogenases and reductases)
dissolved in dimethyl sulfoxide (DMSO). The optical density of each well
(96-well plates) was quantified three times in triplicates at 540 nm using
a multiwell plate reader (iEMS Reader MF, Labsystems, US), and the
percentage of surviving cells was calculated from the ratio of absorbance
of treated to untreated cells. The IC50 values for the inhibition of cell
growth were determined by fitting the plot of the logarithmic percentage
of surviving cells against the logarithm of the drug concentration using a
linear regression function. The median value and the median absolute de-
viation were obtained from the Microsoft Excel software and those
values are reported in Table 2.
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