Library of second-generation cycloruthenated compounds and evaluation of their biological properties as potential anticancer drugs: Passing the nanomolar barrier

Article abstract of DOI:10.1039/c1dt10322a

A library of 32 organoruthenium compounds has been synthesised. Known and novel C-N cyclometalated compounds as well as N-C-N and N-N-C pincer derivatives of this metal have been used in this purpose. Most of the compounds have been tested for their in vi

Full text of DOI:10.1039/c1dt10322a

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Cleavage of unreactive bonds with pincer metal complexes
Martin Albrecht and Monika M. Lindner
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Pincer Ru and Os complexes as efficient catalysts for racemization and deuteration of alcohols
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Mechanistic analysis of trans C–N reductive elimination from a square-planar macrocyclic aryl-
copper(III) complex
Lauren M. Huffman and Shannon S. Stahl
Dalton Trans., 2011, DOI: 10.1039/C1DT10463B
CSC-pincer versus pseudo-pincer complexes of palladium(II): a comparative study on
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PAPER
Library of second-generation cycloruthenated compounds and evaluation of
their biological properties as potential anticancer drugs: Passing the
nanomolar barrier†
Ludivine Fetzer,a Bastien Boff,a Moussa Ali,a Meng Xiangjun,b Jean-Paul Collin,a Claude Sirlin,a
Christian Gaiddonb and Michel Pfeffer*a
Received 24th February 2011, Accepted 7th June 2011
DOI: 10.1039/c1dt10322a
A library of 32 organoruthenium compounds has been synthesised. Known and novel C–N
cyclometalated compounds as well as N–C–N and N–N–C pincer derivatives of this metal have been
used in this purpose. Most of the compounds have been tested for their in vitro antitumoral behaviours,
good to excellent activities have thus been found. Several of the newly synthesized compounds pass the
symbolic barrier of the nanomolar range for their IC50 indicating a critical improvement. The level of
activity is tentatively correlated to physicochemical properties of the compounds such as their RuIII/II
redox potential and their lipophilicity (log P).
possible cycloruthenated derivatives. This has led to the selection
Introduction
of several candidates as anticancer drugs that were selected from
a library of 20 cycloruthenated compounds.5 The in vivo results
It is now well accepted that molecular ruthenium derivatives can
we have accumulated so far on one of them, RDC11,4 have indeed
convinced us that further studies on this class of compounds is
worthwhile.
display antiproliferative effects against tumor cells with a fairly
good selectivity,1 the lethality of the tumour cells treated with
some ruthenium derived molecules being significantly higher than
that of the normal cells. Several ruthenium(III) or ruthenium(II)
containing molecules have thus been studied, some of them
displaying activities against tumour cells analogous to that of
cisplatin. The benefit of the ruthenium-containing molecules is to
be found in their intrinsic toxicities that are often lower than that
of cisplatin. We have been involved in this field since we found that
some cyclometalated ruthenium compounds are good candidates
for becoming anticancer drugs.2 We have indeed demonstrated
that cycloruthenated phenylpyridine derivatives have several qual-
ities required for such a purpose since these compounds are
thermodynamically and kinetically stable (against substitution
reactions), they have an interesting RuIII/II redox potential3 that
allow them to efficiently interfere with oxido-reductase enzymes,
they have good lipophilicities that will allow their entries into
the cells, and finally they seem to have mechanisms of action
against tumor cells that are very likely to be different from that
of the more classical platinum derivatives.4 So far, we have indeed
investigated the biological properties of only a few of the many
In this paper we wish to publish further studies that enlarge our
library of organoruthenium molecules and show that the premises
found earlier are indeed confirmed with most of the new or known
molecules of the present study.
Results
Syntheses
Since many cycloruthenated derivatives can be formed with 2-
phenylpyridine (2-PhPy) ligands and since the other ligands
on the Ru atom are also important to promote the biological
activity we are interested in, we decided to investigate further
structures potentially available in which the 2-(Ph-H)py unit is
found (except 6 and 11, see below). In this purpose we have defined
three classes of compounds. The first class (1–3 and 7) consists
in cycloruthenated 2-(Ar-H)Py derivatives, which have several
coordination sites available on the Ru atom, by substitution of a
aInstitut de Chimie, UMR 7177, CNRS, Universite´ de Strasbourg, 4, rue
Blaise Pascal, 67000, Strasbourg, France
bSignalisations Mole´culaires et Neurode´ge´ne´rescence, UMRS 692, IN-
SERM, Universite´ de Strasbourg, 11, rue Humann, 67000, Strasbourg,
France
† Electronic supplementary information (ESI) available: Syntheses of 1b,c,
3a–c, 4a–d,g, 7b and 8c. The CIF of 3c and tables of bond lengths and
angles. CCDC reference number 814874. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c1dt10322a
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©

6
priori weakly bound ligands on Ru such as one h -arene or one or
terpy, for instance 8–12, or the neutral bidentate phen ligand and
one acetonitrile, 7. Herein we briefly present the synthesis, most
of the time straightforward, of the novel compounds and examine
their in vitro activities by analysing the viability of cancer cell lines
in contact to these molecules.
The library of the compounds used in this study is shown in
Scheme 1.
All but two compounds, namely 6 and 11, have a Ru–C bond
which is part of a ruthenated heterocyclic unit in their structure.
two acetonitrile ligands. The second class are those compounds in
which three bidentate chelates (one monoanionic N–C chelate and
two neutral N–N chelates) are found on the ruthenium atom (4 and
5), the coordination on Ru by external ligands being in principle
impossible or less likely than with the compounds of the previous
class. The third class of compounds derive from pincer ligands
in which N–C–N or N–N–C terdentate monoanionic chelates are
present in conjunction to either neutral terdentate chelates such as
Scheme 1
8870 | Dalton Trans., 2011, 40, 8869–8878
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We have chosen to incorporate these two compounds, 6 and 11,
in our library in order to, hopefully, emphasise the role of the
C–Ru bond in the various compounds used. Around half of the
compounds (1,3–7 and 8c) are new, whereas several compounds (2,
4b, 8a,b,d–f, 9–12) described more or less recently have been chosen
because their structure was related to that of the biologically active
compounds.
Treating 1a,b with 2 equivalents of phenanthroline (phen) in
methanol at reflux led to the formation of 4b,c, whereas 3a
and 3b were obtained pure by the substitution of two MeCN
by one phenanthroline ligand from the corresponding tetrakis-
acetonitrile species in refluxing dichloromethane. It is important
to note that there exists an apparent contradiction between
the ease of substitution of the four acetonitrile ligands by two
phenanthrolines in methanol to afford compounds 4, whereas
the same reaction in CH2Cl2 led to mono-phenanthroline adduct
compounds 3. As for RDC11, see above, we experienced an
unexpected behaviour for this class of compounds as the two
remaining acetonitrile ligands were difficult to substitute, to
such an extent that once these compounds having two MeCN
ligands were isolated, we could not add the second phenanthroline
(or another bidentate ligand such as 4,4¢-bipyridine) to obtain
4b,d, whatever the conditions. This led us to conclude that the
compounds studied in this work display a common behaviour
with respect to their ease of substitution whether they contain
acetonitrile ligands or not, hence compounds such as 2, 3 or 7 are
not basically different from the others which have no coordination
sites available. Note also that 3a was obtained in very low yields
(6%) because its tetrakis-acetonitrile precursor (see Scheme 1) was
rather unstable: it decomposed readily during the synthesis and
work-up of the reaction product.
The synthesis of the compounds containing at least one MeCN
ligand was achieved with the dimer starting materials having an
6
arene h -bound to the Ru centre, according to Scheme 2.
Compounds 1a–c were obtained through the reaction between
6
the appropriate 2-arylpyridine and [(h -p-cymene)RuCl(m-Cl)]2,
in the presence of KPF6 and NaOH in acetonitrile at 45 ◦C
for 24 h, following a published procedure.6 A similar synthesis
of a compound closely related to 1a, in which the acetonitrile
ligand was substituted by a chloride ion, was reported recently.7
In marked contrast to what was found when the same reac-
tion was conducted using the other Ru(II) derivative, namely
6
[(h -benzene)RuCl(m-Cl)]2, the para-cymene unit was here not
substituted by the acetonitrile ligands. In fact, we found that
6
the h -coordinated para-cymene ligand was strongly bound to
the Ru atom, as neither prolonged reflux in MeCN (up to
24 h), nor the use of UV light and prolonged heating led to
the substitution of the arene unit by MeCN. This behaviour is in
contrast to that observed by Berlinguette et al.8 who found that the
above reaction, using 2-(arene)pyridine ligands substituted at the
arene ring by electron-withdrawing substituents, led to a mixture
of two compounds one of them being the tetrakis-acetonitrile
cycloruthenated species, the second one being closely related to
1a but in which two NCMe ligands instead of one were bound to
the Ru atom. The formation of this latter compound is however
doubtful as it would lead to a 20-electron ruthenium atom, unless
3c was obtained directly from RDC11 by oxidative substitution
of the H para to the C–Ru bond using a well-known procedure,
which involved the addition of silver nitrate on RDC11 in the
presence of PhCOCl (eqn (1)).9
6
the arene ligand is not h -coordinated to the Ru atom, which is also
unlikely.
We therefore ascertained the structure of this class of com-
pounds by performing a three-dimensional structure determina-
tion by means of an X-ray diffraction study on a single crystal of
1c. The results of this study are gathered in the ESI†and confirm
that the structure of 1c is as shown in Scheme 1.
(1)
This reaction emphasises the chemical stability of the C–Ru
bond even in an oxidising environment. The bis-4,4¢-bipyridine
(bipy) analogue of 4a (i.e. in which two bipy have substituted
Scheme 2 (i) MeCN, NaOH (2 eq.), KPF6 (4 eq.), 45 ◦C; (ii) CH2Cl2, phen (1 eq.), reflux; (iii) MeOH, phen (2 eq.), reflux.
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the two phen ligands) has been known for 25 years since its
synthesis was reported by Constable in the 1980s.10 However,
the protocol used necessitated the use of a large excess (up to
5 eq. per Ru atom) of 2-phenylpyridine with the Ru(4,4¢-bipy)2Cl2
starting material, this leading to the desired product in only 47%
yield. As these compounds display high antitumor activities (see
below), we had in mind to synthesise a large selection of related
compounds with different 2-arylpyridine ligands, some of which
are of high value, see below. We thus sought a method that would
use a 1 : 1 stoichiometry of 2-ArPy to Ru. We first obtained high
yields of Ru(phen)2Cl2 by performing its synthesis in a microwave
oven, instead of using the classical synthesis.11 This method, that
was adapted from a recent synthesis of Ru(bipy)2Cl2,12 led to
the synthesis of pure Ru(phen)2Cl2 in up to 80% yields. We then
reacted this intermediate in the presence of 2 eq. of silver triflate,
1 eq. of 2-Phpy and 1 eq. of NMe4OH in CH2Cl2 at reflux for
24 h (eqn (2)). This led to the synthesis of 4a in up to 87%
yields.
operative to get 6, starting with Ru(phen)2Cl2 that was reacted with
8-quinolinol. As for the synthesis of 3a–c, 7a was obtained via the
cyclometalation of 2,6-(p-toldiyl)bis(2-pyridine) (N–CH–N)) with
6
[(h -benzene)RuCl(m-Cl)]2 that led to the cyclometalated (N–C–
N)Ru(MeCN)3 cation that was treated with one equivalent of phen
affording 7a. 7b was obtained from the related tris-acetonitrile
intermediate which was synthesised previously by Wadman et al.
according to the same procedure as that used for 7a.16
The remaining compounds of the library were kindly provided
to us by the laboratories who published their synthesis first,
except 8c that was made according to a procedure similar to that
used to obtain of 8b by changing the nature of the terpyridine
ligand.
Properties of the compounds
As the compounds described above were synthesised for evaluating
their in vitro cytotoxicities towards cancer cell lines, we checked
whether their solutions (in neat DMSO diluted with the required
amount of cell culture media) were stable at least for two days. We
found that only the complexes 1a–c showed obvious instability in
solution as their 1H NMR spectra in DMSO-d6 were significantly
modified after 3 h.17 These latter three compounds were thus
discarded from the in vitro tests as the data that we would have
had obtained would not have been representative of the initial
composition of the compounds. Also 3a which was obtained in
very low yields was not used for these in vitro tests. All other
compounds showed good stability towards ligand substitution
reactions in the absence of light, which was checked by either
1H NMR or UV-visible spectroscopy, and all other compounds,
2–12, were thus considered to be good candidates for the cell
proliferation assays.
(2)
Hence this method is much superior to the previous one as much
time and ligands have been saved by using this new protocol. 4e
was synthesised from 4a, using the same oxidative substitution
reaction as used for 3c (see above) with 95% yields. 4f was obtained
R
by reducing the nitro function in 4e with hydrazine and RANEYꢀ
Nickel, again with an excellent yield of 95%; note that the C–Ru
bond of the cyclometalated unit of this compounds is also stable
in a strongly reducing medium.
In vitro cell growth inhibition
To evaluate the antitumor potential of the various ruthenium-
derived compounds we analyzed their effect on cell proliferation.
HCT-116 cell lines derived from a human colon cancer were
thus treated with different doses of complexes from our library
and the cells’ viability was determined by measuring a specific
cellular enzymatic activity of the remaining living cells after 48 h
(MTT test). Reduction of cell number observed by the MTT test
was validated by observation of the cells under a microscope.
The results obtained are summarized in Table 1. As for the
With the aim of improving the solubility in water of the
cycloruthenated compounds we wished to functionalise the cy-
clometalated unit of 4f with either a sugar or an amino acid unit.
With this objective in mind we treated 4f with either gluconic
acid,13 whose alcohol functions have been previously protected
with benzyl (Bn) groups, or with aspartic acid selectively protected
at the amino acid function.14 This led to the formation of 4g and
4h in 67 and 89% yields respectively (eqn (3)).
Unfortunately, all our attempts to deprotect the alcohol or
the amino acid functions in these compounds failed. We thus
followed a different strategy to obtain the spermine derivative
5b. We first synthesised the protected spermine substituted 2-
phenylpyridine ligand by classical peptidic coupling between
6-phenylnicotinic acid and (N1,N4,N9-tri-tert-butoxycarbonyl)-
1,12-diamino-4,9-diazadodecane.15 This new ligand was then
successfully cycloruthenated by Ru(phen)2Cl2 using the method
described above for 4a, affording 5a in 78% yield (eqn (4)).
The deprotection of the amido functions in this latter product
to give 5b, was achieved in good yields by treating 5a with
trimethylsilyl trifluoromethane sulfonate (TMSOTf). The new
product 5b was nicely soluble in water as we found that at least
25 mM aqueous solutions could be obtained. The same procedure
as that used for the cyclometalation of 2-phenylpyridine was
5
cycloruthenated compounds of the previous library we found
that almost all compounds of the present library display good to
very good cytotoxicities against the HCT-116 cell line. One can
classify the obtained results in three categories: 12 compounds
have a IC50 in the nanomolar range, i. e. lower than 1 mM, for 12 of
them 1 < IC50 < 5 mM, whereas for 4 of them, IC50 > 5 mM
(5b seems to be inactive). It is apparent at first sight that all
compounds having a genuine cycloruthenated unit have a good
cytotoxicity.
We first note that the substitution of the phenylpyridine had
little effect upon the cytotoxicity of the molecule; for instance 8d
and 8f which differ from each other by the presence of a CO2Me
group on the metallated ring displayed the same effect.
Thus, as we did not find an obvious relationship between
the activity of the compounds and their structure, we
8872 | Dalton Trans., 2011, 40, 8869–8878
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©

(3)
(4)
Table 1 Cytotoxicity, RuIII/II redox potentials and lipophilicity of the compounds of the library
Compound
IC50/mM (HCT-116)
E◦(RuIII/II)/mV ( 5 mV vs. SCE)
log P ( 0.1)
2
0.5 0.1
590
620
795
410
410
430
540
690
200
nr
n.r.
2.05
0.8
2.35
2.35
2.2
n.r.
2.05
0.9
nr
nr
5.5
<0
0.85
2.4
1.75
2.2
3.45
2.8
2.25
2.05
2.00
n.r.
2.9
3b
3c
4a
4b
4c
4d
4e
4f
1
10
0.5
2
0.1 0.05
0.1 0.05
0.2 0.05
0.2 0.03
0.5 0.2
5
4
2
1
0.5
1
1
4g
4h
5a
5b
6
nr
0.5
540
520
530
380
595
520
490
310
600
720
650
540
710
660
620
770
570
> 50
15
5
7a
7b
8a
8b
8c
8d
8e
8f
1.4 0.3
2.9 0.4
0.7 0.1
0.5 0.1
2.5 0.5
1.3 0.3
2.8 0.3
1.2 0.1
9
1
0.5
10a
10b
10c
11
12
1.2 0.2
2.8 0.3
1.3 0.2
4.0 0.5
0.3 0.1
2.3
2.5
n.r.
n.r.
checked whether the redox potential and the lipophilicities
of our compounds could be correlated with their in vitro
activity.
namely 3c, 4f and 5b show indeed significantly larger IC50
values than the other compounds, regardless of their redox
data.
These data show clearly that all molecules having a redox
potential in the range 0.4–0.6 V (vs. SCE) and log P above 2.0
display the lowest IC50 values, i.e. in the nanomolar region. The
compounds showing some hydrophilic behaviour with log P < 1
A three-dimensional graphical representation of activity is
displayed in Fig. 1. After having verified that the optimization
problem admits a unique solution, the polynomial defining the
optimized surface is:
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Dalton Trans., 2011, 40, 8869–8878 | 8873
©

Fig. 1 Three-dimensional representation of the correlation between IC50, the redox potential (E◦(RuIII/II)) and the lipophilicity (log(Po/w)) of the
cyclometallated molecules. Regression shows a non-linear determination coefficient R2 = 0.90.
IC50 = 10,7157 –32.9217E◦(RuIII/II) - 0.5258log(Po/w) +
Discussion
45.8467E◦(RuIII/II)2 + 0.5095(log(Po/w))2 -
5.9293E◦(RuIII/II)log(Po/w
)
Although the importance of the redox potential for ruthenium
containing compounds displaying anticancer properties has been
recognized as early as in the late 1970s,19 we did not found many
correlations analogous to ours, between the E◦ value and the IC50
of a series of compounds. In 1996, Clarke et al. found that the most
active ruthenium complexes as metalloimmunosuppressants have
reduction potentials between 100 and 400 mV.20 These authors
hypothesized that electron transfer could explain their activity and
that a likely site of metabolic interference could be the respiratory
redox pathway producing the ATP essential to cell growth.
It is now well accepted that most of the Ru(III) derivatives such
as in NamiA or Kp1019 that display antitumor activities are in
The behaviour of 5b, which is hydrophilic and which apparently
showed no cytoxicity at concentration below 50 mM, is reminiscent
to that of a related water-soluble derivative built up with the 1,3,5-
triaza-7-phospha-adamantane ligand (PTA). Indeed, [Ru(C6H4-
2-C5H4N)(PTA)(NCMe)3]PF6,5 also did not show any activity
against the same cell line for the same range of concentrations.
Whilst planning the synthesis of 5b, we thought that locating
the hydrophilic property of the ligand away from the ruthenium
centre as with the spermine unit in 5b, would have allowed a
better activity, the observed results proved, however, that this is
unfortunately not the case. The results obtained for 6 whose IC50
is two orders of magnitude larger than that of the related 4a or
4b, and in which the C–Ru bond is absent, illustrates nicely the
importance that this latter bond might play for the activity of
these monocationic molecules. It is also obvious from the results
reported in Table 1 that the compounds having a N–C type ligand
are not significantly superior to those having a N–C–N or a N–N–
C ligand, as the IC50 of compounds 7–12 are analogous (within one
order of magnitude) to those of the other molecules studied here.
This observation lets us conclude that the coordination sphere
of the compounds which is of the type Ru(NNNN,NC), is likely
to be responsible for their activity rather than the substitution
of the cyclometallated ligand. The result obtained for 11, the
second compound not displaying a C–Ru bond, is somewhat more
puzzling. Its IC50 is only one order of magnitude larger than that
of the closely related 12 but it is definitely similar to that of 10a–c.
To emphasise the importance of the presence of a C–Ru
bond and that of the RuIII/II redox potential in the cytotox-
icity of the compounds, it is interesting to compare the re-
sults of the library above with those of structurally related
compounds such as [Ru(bipy)3](PF6)2,5,18 [Ru(phen)3](PF6)2 or
[Ru(phen)2(MeCN)2](PF6)2. These latter all have IC50 > 50 mM
and E◦(RuIII/II) > 1.3 V. However one has to take into account
that they are dicationic species and thus might be less lipophilic
than the monocationic organoruthenium derivatives studied here.
fact prodrugs that are activated by reduction.21
22
Recently, Keppler et al.
found that the most active
compounds in a series of ruthenium indazole (ind) adducts
([RuIIICl(6-n)(ind)n](3-n)-) were those whose redox potential was
between 0.1 and 0.58 V vs. NHE. These compounds (with
n = 3 and 4) presented IC50 just above (2.6 mM, for n = 3) or
below (0.69 mM, for n = 4) the nanomolar range for the inhibition
of colon cancer cells (SW480) and ovarian cancer cells (CH1),
whereas the compounds with n = 0–2, whose E◦ varied from -1.36
to -0.39 V (estimated value),22 had cytoxicities up to 2 orders of
magnitude higher.
A relation between the value of the redox potential and the
cytotoxicity of our cycloruthenated molecules is established in
this work but its rationalisation is not clear yet. A reasonable
hypothesis however could be that the ruthenium compounds alter
somehow the behaviour of certain enzymes in the cells based on
the following grounds.
Some of us have shown earlier3,23 that cycloruthenated com-
pounds closely related to those described here displayed remark-
able redox catalytic activity towards oxido-reductase enzymes. For
instance in situ electrochemically-generated Ru(III) in 4b was able
to very efficiently oxidise the reduced form of glucose oxidase
enzyme in the absence of dioxygen. Also high rates of the horse
radish peroxidase-catalysed-oxidation of Ru(II) into Ru(III) by
H2O2 have been measured for the same compound, showing that
cycloruthenated species are strikingly reactive electron donors for
8874 | Dalton Trans., 2011, 40, 8869–8878
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The Royal Society of Chemistry 2011
©

this enzyme. Electron transfer akin to that postulated by Clarke
et al. might therefore be invoked to tentatively explain the im-
species. The study of the biological activity of these compounds
in vivo as well as their toxicity studies on normal cells is therefore
worthwhile and will be actively pursued in the future.
19
portant in vitro cytoxicity of our compounds. DFT calculations on
compounds such as 4b 24 have shown that its HOMO and LUMO
molecular orbitals are mainly located close to the ruthenium–
aryl unit. The electron transfer could thus well take place at this
part of the molecule, this emphasising somehow the role of the C–
Ru bond in our molecules. However, we are far from being able to
give a rational explanation about the exact role the C–Ru bond is
playing in our molecules and much further work will be necessary
to propose a satisfactory mode of action of these cyclometallated
species. Some data we obtained recently might however shed light
on possible mechanisms of action of our molecules. We have
indeed had recently evidences that the activity of these ruthenium-
derived compounds in tumor cells induced some oxidative stress
with various degrees. For example, using dihydroethidium, we
observed that 4b and RDC11 show a production of radical
oxygen species as early as 1 h after treatment (data not shown).25
Dihydroethidium is the chemically reduced form of the DNA
intercalating dye ethidium bromide (B-ring reduction) able to
penetrate live cells. This reduced dye is therefore very useful for
detection of oxidative activities in viable cells, including superoxide
generation in mitochondria. Dihydroethidium itself shows a blue
fluorescence (absorption/emission: 355/420 nm) in cell cytoplasm
until oxidization (dehydrogenation) to form ethidium, which
becomes red fluorescent (absorption/emission: 518/605 nm) upon
DNA intercalation. Therefore elevated red fluorescence in cells
treated with RDC is an indication of reactive oxygen species (ROS)
production.
Experimental
Experiments were carried out under an argon atmosphere using
a vacuum line. Diethyl ether and pentane were distilled over
sodium/benzophenone, dichloromethane and acetonitrile over
calcium hydride and methanol and ethanol over magnesium under
argon immediately before use. Chromatography columns were
carried out on Merck aluminium oxide 90 standardized.
The other starting materials were purchased from Sigma–
Aldrich or Alfa Aesar and used as received without further
purification.
The compounds listed hereafter were synthesized following
reported procedures: [(h -benzene)RuCl2]2,28 [(h -p-cymene)-
RuCl2]2,29 (N,N-dimethyl-4-(pyridin-2-yl)benzenamine),30 4-
amino-2-phenylpyridine,31 2-(3-bromophenyl)pyridine,32 2,3,4,5,
6-penta-O-benzyl-D-glucose,33 2,3,4,5,6-penta-O-benzyl-D-glu-
conic acid,34 (N1,N4,N9-tri-tert-butoxycarbonyl)-1,12-diamino-
6
6
4,9-diazadodecane,35
pyridyl)toluene.37
Compounds 2,38 8a,b,d–f, 10a,b,39 9,40 11 and 12
6-phenylnicotinic
acid,36
3,5-di(2-
41
were
synthesised according to published procedures.
The NMR spectra were obtained at room temperature on
Bruker spectrometers. 1H NMR spectra were recorded at
300.13 MHz (AC-300), 400.13 MHz (AM-400) or 500.13 (AM-
500), and referenced to SiMe4. 13C{ H} NMR spectra were
1
recorded at 75.48 MHz (AC-300), 100.62 MHz (AC-400) or
125.54 MHz (AC-500) and referenced to SiMe4. The NMR
assignments were supported by COSY spectra for 1H NMR. The
chemical shifts are referenced to the residual solvent peak.
Chemical shifts (d) and coupling constants (J) are expressed in
ppm and Hz respectively. Multiplicity: s = singlet, d = doublet, t =
triplet, q = quadruplet, hept = heptet, m = multiplet.
Using this tool, we are performing comparative studies on
relative ROS production between ruthenium-derived compounds.
How exactly the intrinsic redox potential of these ruthenium
derivatives are responsible of the cellular oxidative stress is still
under investigation.
In addition, we remarked that various copper complexes
displaying CuII/I redox couples in the same range of potential also
show efficient cytostatic activity.26 This observation is certainly
strongly connected to the basic role of the oxidative stress in the
processes of the cells.27
Numeration used for the assignments of the NMR spectra:
Conclusion
This study of the biological activity of a library of cycloruthenated
2-phenylpyridine derivatives has confirmed that organometallic
ruthenium compounds having a genuine C–Ru bond stabilised
by one or two intramolecular N–Ru bonds display important
cytoxicity a◦gainst cancer cell lines. The activity is somewhat related
to a low E (RuIII/II) of these compounds provided that they are
also reasonably lipophilic. The coordination sphere around the
ruthenium atom is stable as a substitution reaction of a N-ligand
at the ruthenium centre cannot easily take place (at least in
the absence of light for acetonitrile containing compounds), this
being especially true for the complexes of the library having no
monodentate ligands. Hence, if exchange of electrons between the
organoruthenium cations and biological macromolecules (such as
oxido-reductase enzymes for example) are taking place within the
cells, it should occur through an outer-sphere mechanism. More
insight into the role of the red-ox potential will probably become
clearer as we progress into the mechanism of action of these
ES-MS spectra and elemental analyses were carried out by
the corresponding facilities at the Institut de Chimie, Universite´
de Strasbourg and at the Service Central d’Analyse du CNRS,
Vernaison, France.
Synthesis of the compounds
We only report below typical syntheses of some of the products
described in this paper. The syntheses of all other new compounds
are given in ESI†.
6
1a. [RuCl2(h -C10H14)]2 (0.239 g, 0.388 mmol), phenyl-2-
pyridine (0.111 ml, 0.777 mmol), NaOH (0.031 g, 0.777 mmol)
and KPF6 (0.143 g, 0.777 mmol) were mixed in CH3CN (5 mL).
The yellow suspension was heated at 45 ◦C for 24 h. The desired
This journal is
The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 8869–8878 | 8875
©

3
product was purified by column chromatography over Al2O3 using
CH2Cl2–CH3CN 95/5 as eluent. The yellow fraction was collected
and the solvent removed in vacuo. 1a was obtained as a yellow solid
(0.199 g, 89% yield).
1H NMR (400 MHz, CD3CN): d = 8.56 (dd, 1H, JHH = 5.3,
4JHH = 1.4, Ho), 8.43 (dd, 1H, JHH = 8.24, JHH = 1.4, Hp), 8.34
3
4
3
4
(dd, 1H, JHH = 8.08, JHH = 1.4, Hp), 8.24 (m, 3H), 8.10 (d, 2H,
4JHH = 1.7, 2H, Hphen), 8.02 (s, 2H, 2H, Hphen), 7.90 (d, 1H, 3JHH
=
Anal. Calc. for C23H25N2F6PRu: C, 48.00; H, 4.38; N, 4.87.
Found: C,47.60; H, 4.35; N, 5.51%.
8.2, H12), 7.84 (dd, 1H, 3JHH = 5.3, 4JHH = 1.5, Ho), 7.60 (m, 3H),
7.54 (d, 1H, 3JHH = 5.8, H9), 7.53 (dd, 1H, 3JHH = 8.2, 4JHH = 5.5,
3
3
4
4
1H NMR (400 MHz, CD3CN): d = 9.23 (dd, 1H, JHH = 5.7,
Hm), 7.37 (dd, JHH = 8.1, JHH = 5.3, Hm), 7.29 (d, 1H, JHH =
4JHH = 2.3, H12), 8.17 (dd, 1H, 3JHH = 7.5, 4JHH = 1.5, H5), 7.96 (m,
2.3, H8), 6.76 (ddd, 1H, 3JHH = 7.3, 4JHH = 1.4, H10), 6.23 (dd, 1H,
3JHH = 7.9, 4JHH = 2.3, H6), 5.98 (d, 1H,3JHH = 7.9, H5), 3.67 (s, 2H,
NH2).
3
4
2H, H10, H9), 7.80 (dd, 1H, JHH = 7.5, JHH = 1.5, H8), 7.30 (m,
3
4
1H, H11), 7.25 (dd, 1H, JHH = 7.5, JHH = 1.5, H6), 7.18 (td, 1H,
4
3
4
1
3JHH = 7.5, JHH = 1.5, H7), 5.98 (dd, 1H, JHH = 6.2, JHH = 1.2,
13C{ H} NMR (100 MHz, CD3CN): d = 176.8, 168.7, 156.1,
3
4
Harom), 5.96 (dd, 1H, JHH = 6.1, JHH = 1.1, Harom), 5.69 (dd, 1H,
3JHH = 6.2, 4JHH = 1.3, Harom), 5.45 (dd, 1H, 3JHH = 6.1, 4JHH = 1.2,
Harom), 2.4 (hept, 1H, CHMe2¢), 2.1 (s, 3H, CH3CN), 2.02 (s, 3H,
CH3), 0.96 (d, 3H, 3JHH = 6.9, CH3), 0.93 (d, 3H, 3JHH = 6.9, CH3).
151.8, 151.7, 151.3, 150.8, 150.8, 149.1, 148.9, 147.4, 146.5, 143.5,
136.6, 136.4, 136.1, 134.7, 133.3, 132.9, 131.7, 131.5, 131.3, 131.2,
128.7, 128.6, 128.5, 128.4, 126.7, 126.1, 126.0, 125.9, 122.8, 119.6,
118.7, 112.4.
13C{ H} NMR (100 MHz, CD3CN): d = 174.9, 165.5, 155.7,
1
144.4, 140.1, 138.7, 129.7, 124.3, 123.7, 122.6, 119.4, 117.4, 103.9,
102.3, 92.4, 92.3, 88.2, 84.7, 30.9, 21.4, 21.2, 17.9, 2.9.
4h. a-tert-Butyl-(S)-N-[(tert-butoxy)carbonyl]-L-aspartate
(23.7 mg, 0.082 mmol) was dissolved in dry DMF–CH2Cl2 (1 : 2,
3 ml) and then EDAC (18.8 mg, 0.098 mmol) and HOBT (13.2 mg,
0.098 mmol) were added at 0 ◦C under argon. After 30 min,
diisopropylethylamine (34.3 mL, 0.197 mmol) and 4f (80 mg,
0.102 mmol) were added and the reaction mixture was stirred
for 18 h. The solution was concentrated in vacuo. The solid was
purified by column chromatography under aluminium oxide using
CH2Cl2–MeOH (95 : 5) as eluent to give 77 mg of purified product
(89%, yield).
4e. To a deep purple solution of 4a (175 mg, 0.23 mmol) in
CH2Cl2 (10 mL), was added AgNO3 (39.11 mg, 0.23 mmol) and
PhCOCl (39 ml, 0.23 mmol). The solution was stirred at room
temperature for 2 h. TLC revealed only a red spot which was
collected by column chromatography over Al2O3 using CH3CN
as eluent and the solvents were removed in vacuo. The resulting
powder was dissolved in minimum of CH3CN–Et2O and after the
addition of n-hexane, a dark red solid was precipitated (177 mg,
95% yield).
Anal. Calc. for C36H23F3N6O5RuS: C, 53.40; H, 2.86; N, 10.38.
Found: C, 52.47; H, 2.98; N, 10.13%.
MS (ES, m/z): Calc. for C35H23N6O2101Ru: 661.09; found:
661.13.
Anal. Calc. for C49H46F3N7O8RuS: C, 55.99; H, 4.41; N, 9.33.
Found: C, 54.55; H, 4.53; N, 9.11%.
HRMS (ES, m/z): calc. for C48H46N7O5101Ru: 902.2604; found:
902.2622.
1H NMR (400 MHz, CD3CN): d 8.49–8.47 (m, 2H), 8.39 (dd,
1H, 3JHH = 8.3, 4JHH = 1.3, Hp), 8.34 (dd, 1H, 3JHH = 8.3, 4JHH = 1.3,
1H NMR (500 MHz, CD3CN): d = 8.64 (d, 1H, 4JHH = 2.3, H8),
8.52 (dd, 1H, 3JHH = 8.2, 4JHH = 1.4, Hp), 8.44 (dd, 2H, 3JHH = 8.1,
4JHH = 1.1, Hp), 8.38 (dd, 1H, 3JHH = 8.2, 4JHH = 1.2, Hp), 8.33 (dd,
1H, 3JHH = 5.2, 4JHH = 1.2, Ho), 8.24 (d, 1H, 3JHH = 8.4, H12), 8.18
(m, 1H), 8.16 (d, 2H, 3JHH = 4.0, 2H, Hphen), 8.12 (d, 2H, 4JHH = 1.1,
2H, Hphen), 8.06 (dd, 1H, 3JHH = 5.0, 4JHH = 1.4, Ho), 7.86 (dd, 1H,
3JHH = 5.2, 4JHH = 1.1, Ho), 7.75 (t, 1H, 3JHH = 7.8, H10), 7.66–7.62
Hp), 8.30 (dd, 1H, 3JHH = 8.3, 4JHH = 1.2, Hp), 8.22 (dd, 1H, 3JHH
=
4
3
5.3, JHH = 1.0, Ho), 8.21–8.17 (m, 2H), 8.14 (d, 2H, JHH = 0.8,
3
4
H, Hphen), 8.11 (dd, 1H, JHH = 5.0, JHH = 1.0, Ho), 8.08 (d, 2H,
3JHH = 0.8, H, Hphen), 7.95 (d, 1H, 3JHH = 8.0), 7.86 (dd, 1H, 3JHH
=
5.3, 4JHH = 1.0, Ho), 7.69–7.56 (m, 5H), 7.41 (dd, 1H, 3JHH = 8.2,
3JHH = 5.3, Hm), 6.82 (m, 1H), 6.75 (m, 1H), 6.23 (d, 1H, JHH
=
3
8.0), 5.81 (d, 1H, 3JHH = 8.0, NH), 4.34 (m, 1H, CH), 2.72 (m, 2H,
3
4
(m, 3H, H9), 7.60 (dd, 1H, JHH = 8.2, JHH = 5.3, Hm), 7.48 (dd,
CH2), 1.39 (s, 18H, tBu + tBoc).
3
4
3
4
1H, JHH = 8.4, JHH = 2.4, H6), 7.45 (dd, 1H, JHH = 8.2, JHH
=
13C{ H} NMR (100 MHz, CD3CN): d 187.88, 171.41, 168.93,
1
3
3
4
5.5, Hm), 6.94 (ddd, 1H, JHH = 7.2, JHH = 5.6, JHH = 1.2, H11),
6.64 (d, 1H, 3JHH = 8.4, H5).
167.93, 155.71, 151.64, 151.56, 151.20, 150.54, 149.64, 148.73,
148.52, 146.88, 146.29, 136.52, 136.11, 135.98, 134.75, 133.47,
133.34, 131.45, 131.24, 131.03, 128.45, 128.37, 128.24, 126.35,
125.89, 125.82, 125.75, 122.99, 121.62, 119.57, 116.77, 81.92,
79.66, 52.07, 39.00, 28.25, 27.86.
13C{ H} NMR (125 MHz, CD3CN): d = 167.1, 156.3, 152.7,
1
152.7, 152.4, 151.3, 150.0, 149.3, 149.1, 148.4, 147.1, 144.3, 137.7,
137.6, 137.4, 136.5, 135.7, 135.3, 132.3, 132.1, 132.0, 131.9, 129.3,
129.2, 129.1, 127.2, 126.9, 126.8, 124.9, 122.1, 121.2, 118.3.
4f. To a deep purple solution of 4e (170 mg, 0.211 mmol) in
5a. Ru(phen)2Cl2 (200 mg, 0.37 mmol), Boc3SperPhNic (see
ESI†) (257 mg, 0.37 mmol), tetramethylamonium hydroxide
(67.0 mg, 0.37 mmol) and AgOTf (190.1 mg, 0.74 mmol) were
refluxed in CH2Cl2 (10 mL) for 2 days at 40 ◦C. The reaction
mixture was cooled at r.t. and the solvents were then removed in
vacuo. The complex was purified by column chromatography over
Al2O3 using CH2Cl2–MeOH (95/5) as eluent and concentrated in
vacuo. 5a was obtained as a deep purple solid (375 mg, 78% yield).
Anal. Calc. for C62H72F3N9O10RuS + CH2Cl2: C, 54.90; H, 5.41;
N, 9.15. Found: C, 55.15; H, 5.38; N, 9.57%.
R
10 ml DMF was added RANEYꢀ Nickel (124 mg, 2.11 mmol)
and hydrazine (1.20 ml, 21.1 mmol). The solution was stirred
at room temperature for 2 h. TLC revealed only a purple band
which was collected using column chromatography over Al2O3
using CH3CN as eluent and the solvents were removed in vacuo.
The resulting powder was dissolved in a minimum of CH3CN and
after the addition of n-hexane a dark purple solid was precipitated
(177 mg, 95% yield).
Anal. Calc. for C35H25F3N6O3RuS: C, 55.45; H, 3.23; N, 10.78.
Found: C, 53.99; H, 3.48; N, 10.30%.
MS (ES, m/z): calc. for C35H25N6O101Ru: 631.11; found: 631.11.
HRMS (ES, m/z): calc. for C61H72N9O7101Ru: 1144.4610; found:
1144.4608.
8876 | Dalton Trans., 2011, 40, 8869–8878
This journal is
The Royal Society of Chemistry 2011
©

1H NMR (500 MHz, CD3CN): d 8.50 (dd, 1H, 3JHH = 8.1, 4JHH
1.2), 8.46 (dd, 1H, 3JHH = 5.3, 4JHH = 1.3), 8.40 (dd, 1H, 3JHH = 8.1,
=
HRMS (ES, m/z): calc. for C31H24101Ru: 568.1075; found:
568.1078.
4JHH = 1.3), 8.37 (dd, 1H, JHH = 8.2, JHH = 1.3), 8.32 (dd, 1H,
3JHH = 8.1, 4JHH = 1.2), 8.29 (dd, 1H, 3JHH = 5.3, 4JHH = 1.1), 8.17
(dd, 1H, 3JHH = 5.1, 4JHH = 1.3), 8.15 (d, 2H, 4JHH = 1.2), 8.10 (d,
2H, 4JHH = 2.5), 8.05 (m, 3H), 7.94 (dd, 1H, 3JHH = 7.8, 4JHH = 0.8),
7.85 (dd, 1H, 3JHH = 5.3, 4JHH = 1.2), 7.65–7.61 (m, 3H), 7.43 (dd,
1H, 3JHH = 8.1, 4JHH = 5.3), 6.92 (ddd, 1H, 3JHH = 7.3, 4JHH = 1.3),
1H NMR (400.13 MHz, CD3CN, 300 K): d 10.01 (dd, 1H, 3JHH
=
4 3 4
3
4
5.4, JHH = 1.5, Ho), 8.76 (dd, 1H, J = 8.1, JHH = 1.5, Hp), 8.31
3
3
3
(dd, 1H, JHH = 8.1, JHH = 5.4, Hm), (d, 1H, JHH = 8.8, Hphen),
7.99–8.04 (m, 3H, Ho + H1+ Hphen), 7.97 (s, 2H, H5), 7.65 (td, 2H,
3JHH = 7.7, 4JHH = 1.5, H2), (d, 2H, 3JHH = 7.7, H4), 7.37 (dd, 1H,
4
3
3
3JHH = 5.4, JHH = 1.5, Hp), 7.14 (dd, 2H, JHH = 6.2, JHH = 5.4,
3
4
3
3
4
6.77 (ddd, 1H, JHH = 7.4, JHH = 1.2), 6.39 (dd, 1H, JHH = 7.5,
4JHH = 0.9), 3.26–3.10 (m, 10H), 3.03 (q, 2H, 3JHH = 6.4), 169–1.59
(m, 4H), 1.48 (m, 4H), 1.44 (s, 9H), 1.42 (s, 9H), 1.38 (s, 9H).
Hm), 6.73 (td, 2H, JHH = 7.7, JHH = 1.5, H3), 2.66 (s, 3H, CH3),
2.09 (s, 3H, NCCH3).
1
13C{ H} NMR (100 MHz, CD3CN, 300 K): d 214.4, 169.7,
1
13C{ H} NMR (100 MHz, CD3CN): d 195.55, 171.25, 165.03,
155.7, 154.1, 154.1, 151.4, 146.9, 145.3, 137.1, 135.7, 134.3, 131.9,
131.3, 130.4, 129.0, 128.7, 127.3, 126.4, 125.9, 125.2, 122.9, 120.4,
22.4, 4.7.
157.14, 156.89, 156.42, 152.42, 151.68, 151.10, 150.41, 149.51,
149.33, 147.60, 146.48, 137.03, 136.69, 135.54, 134.80, 134.21,
134.09, 132.24, 131.89, 131.77, 130.12, 129.12, 128.95, 128.82,
126.88, 126.52, 126.41, 126.36, 126.30, 122.32, 119.22, 48.08
(C5/C8), 48.03 (C5/C8), 45.82 (C10), 45.53 (C3), 39.41 (C12),
37.99 (C1), 30.22 (C11), 29.29 (C2), 29.05 (3Boc), 27.12 (C6/C7),
27.09 (C6/C7).
X-Ray diffraction study of compound 1c
6
Single crystals of [Ru(4-NH2C6H3-2-C5H4N-kC,N)(h -1,4-
CH3C6H4CH(CH3)2)NCMe]PF6, 1c, were recrystallised from
CH2Cl2–pentane, mounted in inert oil and transferred to the cold
gas stream of the diffractometer.
5b. To a stirred solution of 5a (50 mg, 0.03 mmol) and 2,6-
lutidine (48.2 mL, 0.414 mmol) in dry CH2Cl2 (2 ml) at room
temperature was added trimethylsilyl trifluoromethanesulfonate
(TMSOTf; 56.6 mL, 0.313 mmol). The reaction mixture was stirred
for 22 h and concentrated in vacuo. The resulting solid was then
dissolved in MeOH (10 ml) and stirred at room temperature for 3 h.
The solution was concentrated in vacuo and purified by column
chromatography over C18-reversed phase using MeOH as eluent
to give (100 mg, 80% yield).
Crystal structure determination of complex 1c
Crystal data. C23H26F6N3PRu, M = 590.51, monoclinic, space
˚
group P21/c, a = 9.358(1), b = 10.311(1), c = 26.032(1) A, U =
3
˚
2418.4(2) A , T = 173 K, Z = 4, 24784 reflections measured, 8733
unique (Rint = 0.0153) which were used in all calculations. The final
wR(F2) was 0.0677 (all data).
HRMS (ES, m/z): Calc. for C46H48N9O101Ru: 844.3025; found:
844.3009.
Electrochemical measurements
1H NMR (300 MHz, CD3CN): d 8.51 (dd, 1H, 3JHH = 8.0, 4JHH
=
=
3
4
1.3), 8.43 (m, 1H), 8.40 (s, 1H), 8.38 (dd, 1H, JHH = 8.1, JHH
Electrochemical experiments were performed with a three-
electrode system consisting of a platinum working electrode, a
platinum wire counter electrode, and a silver wire as a pseudo-
reference electrode. The potentials were referenced to SCE us-
ing ferrocene or [Os(bipy)3](PF6)2 as an internal reference. The
measurements were carried out under argon, in degassed CH3CN
1.1), 8.33 (dd, 1H, 3JHH = 8.2, 4JHH = 1.1), 8.27 (dd, 1H, 3JHH = 5.3,
4JHH = 1.2), 8.18–8.08 (m, 5H), 8.00–7.97 (m, 2H), 7.94 (d, 1H,
3JHH = 7.7), 7.82 (m, 1H), 7.68–7.60 (m, 3H), 7.42 (dd, 2H, 3JHH
=
8.1, 3JHH = 5.3), 6.90 (ddd, 1H, 3JHH = 7.4, 4JHH = 1.1), 6.75 (ddd,
1H, 3JHH = 7.4, 4JHH = 1.1), 6.35 (d, 1H, 3JHH = 7.4), 3.27 (m, 2H),
3.04 (t, 2H, 3JHH = 6.6), 2.89 (t, 2H, 3JHH = 6.6), 2.83–2.71 (m, 6H),
1.84–1.64 (m, 8H)
n
at 298 K using 0.1 M Bu4NPF6 as the supporting electrolyte.
An EG&G Princeton Applied Research Model 273A potentiostat
connected to a computer was used for the cyclic voltammetry
experiments.
1
13C{ H} NMR (75 MHz, CD3CN): d 156.51, 154.16, 153.07,
152.59, 152.09, 151.81, 151.33, 150.28, 147.45, 146.26, 137.10,
136.77, 135.66, 135.05, 134.39, 134.30, 132.20, 131.83, 131.69,
130.36, 129.20, 129.04, 128.92, 128.68, 127.02, 126.83, 126.61,
126.54, 126.41, 122.35, 47.90, 46.20, 40.53, 37.15, 27.54, 25.84,
25.62, 25.18
Cell proliferation assays
The ruthenium samples for in vitro tests were obtained from 50 mM
solutions of the ruthenium complexes in neat DMSO. The mother
solutions were then sequentially diluted with the required amount
of cell culture media in order to obtain the studied solutions
whose concentration varies from 0.2 to 50 mM. HCT116 cells were
obtained from American Type Cell Culture Collection. Cells were
grown in 96-well plates and treated at 70% confluence. After 48 h
the medium was removed and MTT (0.5 mg mL-1) in DMEM was
added for 1 h. The medium was removed again and 0.04% HCl
in isopropanol was added to dissolve the crystals. Absorption
differences were quantified using an Elisa plate reader (Metertech
USA) at 490–650 nm. The experiments were repeated at least
2 times, the mean deviation was determined by considering the
extreme values found over all experiments.
7a. A solution of [Ru(MeC-NŸCŸN)(NCMe)3]PF6 (20 mg,
0.033 mmol) (see ESI†) with 1,10 phenanthroline (6.5 mg,
0.033 mmol) in methanol (2 mL) was refluxed for 12 h. The
solvent was evaporated under vacuum, and the dark brown residue
was dissolved in MeCN (5 mL) then filtered through Al2O3 using
NCMe as eluent. The purple fraction was collected and evaporated
to dryness under vacuum. Crystallization from CH2Cl2–pentane
or acetone–pentane (slow diffusion) gave dark purple crystals,
which were washed with diethyl ether and dried under vacuum
(20 mg, 85% yield).
Anal. Calc. for C31H24F6N5PRu: C, 52.25; H, 3.39; N, 9.83.
Found: C, 51.87; H, 3.21; N, 9.70%.
This journal is
The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 8869–8878 | 8877
©

Determination of log P
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MOPS (0.02 M) and n-decylamine amino modifier (0.15%, v/v) in
deionized 1-octanol saturated water and then adjusting the pH of
this solution to 7.4. The organic portion was prepared by diluting
1-octanol (0.20%, v/v) into analytical grade methanol. All mobile-
phase solvents were filtered through 0.45 mm filters before use and
degassed continuously during the experiment.
9 M. Gagliardo, D. J. M. Snelders, P. A. Chase, R. J. M. Klein Gebbink,
G. P. M. van Klink and G. van Koten, Angew. Chem., Int. Ed., 2007,
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12 S. Rau, B. Scha¨fer, A. Gru¨ssing, S. Schebesta, K. Lamm, J. Vieth, H.
Go¨rls, D. Walther, M. Rudolph, U. W. Grummt and E. Birkner, Inorg.
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Measurements were performed on a Varian Prostar 210 HPLC
equipped with a Prostar 335 photodiode array detector and a
Prostar 410 autosampler. The stationery phase was a 250 mm ¥
˚
4.6 mm column packed with 10 A Kromasil C-8. The different
compounds were dissolved into methanol (5 ¥ 10-5 M) and
then injected onto the column (5 mL). Column void volume was
estimated from the retention time of uracil, which was included
as a nonretained internal reference for each injection.44 The value
of log k¢w was determined by linear extrapolation of log k¢U vs.
Umethanol data acquired in the region 0.50 £ Umethanol £ 0.85.
Acknowledgements
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We are grateful to S. H. Wadham, and G. van Koten (Uni-
versity of Utrecht) for providing compounds 8d–f and 10a–c
and J. P. Sauvage for providing 8a,b, 9, 11 and 12. L. Brelot (UdS)
determined the X-ray diffraction study of 1c. L. F., A. M and
B. B. thank respectively ANR (ANR-05-EPB-019), INCa (8316F)
and the CNRS together with the Region Alsace for fellowships.
Partial financial support from ANR (PhotoBioMet, ANR-09-
BLAN-0191-01) and the RTRA (Strasbourg) through the project
‘synergie’ are also gratefully acknowledged.
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24 C. Daniel et al. unpublished data.
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