New molecular markers for prostate tumor imaging: A study on 2-methylene substituted fatty acids as new AMACR inhibitors

Article abstract of DOI:10.1002/chem.201003176

The development of prostate carcinoma is associated with alterations in fatty acid metabolism. α-Methylacyl-CoA racemase (AMACR) is a peroxisomal and mitochondrial enzyme that catalyses interconversion between the (S)/(R)-isomers of a range of α-methylacyl-CoA thioesters. AMACR is involved in the β-oxidation of the dietary branched-chain fatty acids and bile acid intermediates. It is highly expressed in prostate (more than 95 %), colon (92 %), and breast cancers (44 %) but not in the respective normal or hyperplastic tissues. Thus, targeting of AMACR could be a new strategy for molecular imaging and therapy of prostate and some other cancers. Unlabeled 2-methylenacyl-CoA thioesters (12 a-c) were designed as AMACR binding ligands. The thioesters were tested for their ability to inhibit the AMACR-mediated epimerization of (25R)-THC-CoA and were found to be strong AMACR inhibitors. Radioiodinated (E)-¹³¹I-13-iodo-2-methylentridec-12-enoic acid ( ¹³¹I-7 c) demonstrated preferential retention in AMACR-positive prostate tumor cells (LNCaP, LNCaP C4-2wt and DU145) compared with both AMACR-knockout LNCaP C4-2 AMACR-siRNA and benign BPH1 prostate cell lines. A significant protein-bound radioactive fraction with main bands at 47 (sum of molecular weights of AMACR plus 12 c), 70, and 75 kDa was detected in LNCaP C4-2 wt cells. Copyright

Full text of DOI:10.1002/chem.201003176

DOI: 10.1002/chem.201003176
New Molecular Markers for Prostate Tumor Imaging: A Study on
2-Methylene Substituted Fatty Acids as New AMACR Inhibitors**
Agnieszka Morgenroth,[a, b] Elizaveta A. Urusova,[b] Cornelia Dinger,[b]
Ehab Al-Momani,[b] Thomas Kull,[b] Gerhard Glatting,[b] Holm Frauendorf,[c]
Olaf Jahn,[d] Felix M. Mottaghy,[a] Sven N. Reske,[b] and Boris D. Zlatopolskiy*[a, b]
Abstract: The development of prostate
carcinoma is associated with alterations
in fatty acid metabolism. a-Methylacyl-
CoA racemase (AMACR) is a peroxi-
somal and mitochondrial enzyme that
catalyses interconversion between the
(S)/(R)-isomers of a range of a-methyl-
acyl-CoA thioesters. AMACR is in-
volved in the b-oxidation of the dietary
branched-chain fatty acids and bile
acid intermediates. It is highly ex-
pressed in prostate (more than 95%),
colon (92%), and breast cancers
(44%) but not in the respective normal
or hyperplastic tissues. Thus, targeting
of AMACR could be a new strategy
for molecular imaging and therapy of
prostate and some other cancers. Unla-
beled 2-methylenacyl-CoA thioesters
(12a–c) were designed as AMACR
binding ligands. The thioesters were
tested for their ability to inhibit the
AMACR-mediated epimerization of
(25R)-THC-CoA and were found to be
strong AMACR inhibitors. Radioiodi-
ed preferential retention in AMACR-
positive prostate tumor cells (LNCaP,
LNCaP C4-2wt and DU145) compared
with both AMACR-knockout LNCaP
C4-2 AMACR-siRNA and benign
BPH1 prostate cell lines. A significant
protein-bound radioactive fraction with
main bands at 47 (sum of molecular
weights of AMACR plus 12c), 70, and
75 kDa was detected in LNCaP C4-2
wt cells. In contrast, only negligible
amounts of protein-bound radioactivity
were found in LNCaP C4-2 AMACR-
siRNA cells.
nated
(E)-131I-13-iodo-2-methylentri-
dec-12-enoic acid (131I-7c) demonstrat-
Keywords: antitumor agents · fatty
acids · imaging agents · inhibitors ·
metabolism · radiopharmaceuticals
Introduction
In the United States of America, about 27360 deaths have
been estimated to result from PCa in 2009.[1] 18F-2-Deoxy-2-
fluoroglucose (FDG), which is an indicator of glycolytic ac-
tivity in cancer cells, is generally ineffective in the diagnosis
of localized PCa due to the low metabolic glucose activity of
PCa compared with other cancer types.[2] Numerous studies
revealed that PCa is associated with changes to fatty acid
metabolism. Therefore, 11C-choline, 18F-fluoromethyl-, or
18F-fluoroethylcholine, which target upregulated lipid syn-
thesis, have been employed in molecular imaging of PCa.[3]
However, normal and hyperplastic prostatic tissues may also
accumulate choline-derived tracers, that consequently leads
to false positive diagnoses.[4,5]
Prostate cancer (PCa) is one of the most commonly diag-
nosed cancers and the second leading cause of cancer-relat-
ed death among men in the western industrialized nations.
[a] Dr. A. Morgenroth, Prof. F. M. Mottaghy, Dr. B. D. Zlatopolskiy
Klinik fꢀr Nuklearmedizin, Aachen University
RWTH, Pauwelsstr. 30, 52074 Aachen (Germany)
Fax : (+49)241-80-82520
E-mail: bzlatopolskiy@ukaachen.de
a-Methylacyl-CoA racemase (AMACR) is a peroxisomal
and mitochondrial enzyme involved in b-oxidation of a-
branched fatty acids that catalyses the interconversion of
(S)/(R)-isomers of a range of a-methylacyl-CoA thioesters
(Scheme 1).[6,7] AMACR is overexpressed in various cancers
such as colon, pulmonary, renal, and liver cancers.[8–10] In
PCa, AMACR expression level is high in more than 95% of
cases, whereas more than 99% of normal and benign pros-
tate tissues demonstrate either no or only low AMACR ex-
pression.[11–13] Furthermore, inhibition of AMACR expres-
sion was shown to induce the reversion of androgen depend-
ency in advanced prostate cancer, which may present a new
treatment option for hormone-refractory prostate cancer.[14]
However, in spite of the potential of AMACR as an anti-
[b] Dr. A. Morgenroth, E. A. Urusova, C. Dinger, E. Al-Momani,
Dr. T. Kull, Prof. G. Glatting, Prof. S. N. Reske,
Dr. B. D. Zlatopolskiy
Klinik fꢀr Nuklearmedizin, Ulm University
Albert-Einstein-Allee 23, 89081 Ulm (Germany)
[c] Dr. H. Frauendorf
Institut fꢀr Organische und Biomolekulare Chemie
Gçttingen University, Tammann-Str. 2
37077 Gçttingen (Germany)
[d] Dr. O. Jahn
Max-Planck-Institute of Experimental Medicine
Hermann-Rein-Str. 3, 37075 Gçttingen (Germany)
[**] AMACR = a-Methyl-CoA-racemase.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201003176.
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Chem. Eur. J. 2011, 17, 10144 – 10150

FULL PAPER
or another nucleophilic group
to give covalent adducts with
the enzyme (Scheme 2B).
To investigate this approach,
we prepared three 2-methyl-
enacyl-CoA compounds, includ-
ing those with fluorine or
iodine substituents. The synthe-
sis commenced with acylation
of Meldrumꢂs acid with the cor-
responding carboxylic acid 4a–
c, followed by reduction with
NaBH4 to give monosubstituted
Meldrumꢂs
acids
5a–c
Scheme 1. Structures of some substrates of AMACR: pristanoyl-CoA (1), 3a,7a,12a-trihydroxycholestanoyl-
CoA (THC-CoA; 2), ibuprofenyl-CoA (3), and coenzyme A. AMACR = a-Methyl-CoA-racemase.
(Scheme 3) by using a previous-
ly reported method.[18] The
latter compounds were treated
cancer target, the development of high-affinity AMACR-
specific substances has so far attracted little attention.[15]
Not surprisingly, no AMACR-selective markers for molecu-
lar imaging have been published so far. Here, we present
the synthesis and in vitro evaluation of new AMACR-inhibi-
tors as potential molecular tracers for PCa and other
AMACR-positive tumors.
with Eschenmoserꢂs iodide salt according to the published
protocol[19] to give a-substituted methyl acrylates 6a–c in
Results and Discussion
As demonstrated[16,17] for M. tuberculosis and human homo-
logues, AMACR catalyses the chiral inversion of (2R)- and
(2S)-2-methylacyl-CoA through a 1,1-proton transfer mech-
anism. 2-Methylacyl-CoA undergoes deprotonation and
forms a planar enolate with His122 (number refer to human
AMACR IA) acting as a base to deprotonate the substrate,
followed by addition of H+ with Asp152 as a proton donor
(Scheme 2A). Glu237 is also involved in epimerization.
AMACR exhibits broad substrate selectivity (Scheme 1).
However, all substrates and inhibitors of AMACR known to
date are 2-methyl- or 2-difluoromethyl- and 2-trifluorome-
thylacyl-CoA thioesters. We envisaged that the substrate se-
lectivity of AMACR could be broader than assumed, and
that 2-methylenacyl-CoA substrates could also fit the cata-
lytic pocket. In this case, the substrates may undergo Mi-
chael addition with the imidazole ring of the active site base
Scheme 3. Synthesis of 7a–c. Eschenmoserꢂs salt=N,N-dimethylmethyl-
enammonium iodide; MsCl=methanesulfonyl chloride.
Scheme 2. A) Proposed[16, 17] intermediate for the AMACR-catalyzed epimerization. B) Hypothetical mechanisms of possible AMACR inhibition by
2-methylenacyl-CoA thioesters.
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10145

B. D. Zlatopolskiy et al.
52–87% over three steps. It is worth noting that concomi-
tant quantitative deprotection of the O-TBS group was ob-
served in the case of 5b.
cording to the published procedure for its preparation.[15] In-
stead, compounds 16–18 were obtained. Therefore, the
CoA-thioester of 17 or 18 and not 13 was prepared and
shown to be a strong AMACR inhibitor. Consequently, we
decided to synthesize these compounds and test their
AMACR inhibitory activity.
Treatment of 17 with Im2CO, followed by reaction of the
intermediate acylimidazolide with CoA-SH as described for
preparation of 13,[15] gave the desired product 19 in 45%
isolated yield (Scheme 6). However, attempts to prepare do-
2-Methylenmyristic acid (7a)[20] was obtained by base-
mediated hydrolysis of the respective ester 6a in 85% yield
(Scheme 3). 14-Fluoro-2-methylentetradecanoic acid (7b)
was prepared from 6b in 55% yield over three steps by me-
sylation, followed by treatment with tetrabutylammonium
fluoride (TBAF) and, finally, base-mediated hydrolysis of
the intermediate ester 9. Compound 6c was hydrostannylat-
ed according to Zhang et al.,[21] followed by iodination with
I2 to give, after base-mediated hydrolysis, iodo-2-methylen-
tridecen-12-oic acid (IVCrot, 7c) as a mixture of regioisom-
ers (13E/12=60:40) in 40% yield over three steps.
Reaction of coenzyme A with the mixed anhydrides of
acrylic acids 7a–c and ethyl chloroformate gave CoA-thio-
esters 12a–c in 46–51% yield and more than 90% purity
(HPLC) (Scheme 4).
Scheme 6. Preparation of 13 and 19 as well as myristoyl-CoA.
Scheme 4. Synthesis of 12a–c. CoA-SH=coenzyme A.
decylmalonyl-CoA (20)[24] by the same route gave myristoyl-
CoA in 55% yield (HPLC) as the only detectable product
of thioesterification, presumably due to extensive decarbox-
ylation of the intermediate monoimidazolide. Compound 13
could be obtained, albeit in moderate yield, from 15 via the
mixed anhydride but, again, not from the acylimidazolide.
Inhibition of the AMACR-catalyzed epimerization of
(25R)-THC-CoA [(25R)-2][15,25] by 2-substituted acryloyl-
CoAs 12a–c was studied to evaluate the potential of the
newly prepared compounds as AMACR inhibitors. Gratify-
ingly, thioesters 12a–c were capable of inhibiting the epime-
rization of (25R)-2 up to 80–
2-Trifluoromethylmyristoyl-CoA (13), which is the most
effective AMACR inhibitor described to date, inhibits epi-
merization of 2 in low micromolar range.[15] We decided to
synthesize 13 to compare its AMACR inhibition activity
with those of 12a–c (Scheme 4). Unexpectedly, base-mediat-
ed hydrolysis of methyl 2-trifluoromyristinate (14) under the
described conditions[15] gave, instead of acid 15, a mixture of
dimethyl- and monomethyl dodecyl malonates as well as do-
decylmalonic acid (16–18) (Scheme 5).
98%, compared with approxi-
mately 60% inhibition by 2-
methylmyristoyl-CoA,[7,26]
which was used as a control
(Table 1). Compound 13 also
exhibited strong AMACR in-
hibition. In contrast, neither 19
nor myristoyl-CoA significantly
Scheme 5. Products of the base-mediated hydrolysis of 14 and synthesis of 15.
inhibited
racemase.
a-methylacyl-CoA-
The dependence of the base-mediated hydrolysis of 14 on
the concentration of NaOH (0.5–10 m), reaction time, and
temperature (0–408C) was studied. Under none of the
tested conditions even traces of 14 or 15 were found in the
reaction mixture after addition of more than two equivalents
of NaOH.[22] Consequently, we prepared an authentic
sample of 15 by transesterification of 14 with benzyl alcohol
using Bu2SnO as catalyst,[23] followed by hydrogenolysis of
the intermediate benzyl ester in 70% yield over two steps.
In our hands, it was not possible to synthesize acid 15 ac-
Because the presence of a metabolically stable iodovinyl
group allows switching between SPECT, PET, and radiother-
apy agents simply by using different iodine radioisotopes
(
123I, 124I and 131I, or 125I, respectively) with conveniently long
half-life times that permit prolonged biological studies,
IVCrot (7c) and its CoA-thioester 12c were chosen for fur-
ther study.
A kinetic study of AMACR inhibition by IVCrot-CoA
(12c) was carried out using different concentrations of in-
hibitor 12c (0–300 mm) and the substrate (25R)-2 (15–75 mm)
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Prostate Tumor Imaging
FULL PAPER
cell. Instead, 18F-FDG-6-phosphate accumulates (“traps”) in
cells with high sugar metabolism and high GLUTI and
HexII levels, although affinity of FDG to HEXII is not very
good and, consequently, the respective phosphorylation con-
stants are very high (normally more than 100 mm, often
more than 300 mm depending on the cell type).[30] Neverthe-
less, FDG is the most popular PET-tracer, and this com-
pound works very well in the majority of cases. We pro-
posed that, similar to FDG, 7c would also be metabolically
trapped within the cell. Compound 7c would be taken up
into the cell by fatty acid transporters, where it is thioesteri-
fied by long-chain acyl-CoA thioester synthetases (LACS)[31]
or xenobiotic-CoA ester ligases,[32] both of which have very
broad substrate specificity, to give thioester 12c,[33] which is
a reactive Michael-acceptor and, at the same time, can fit
the catalytic pocket of AMACR (Scheme 7). Consequently,
Table 1. Study of AMACR inhibition.
Inhibitor
Epim. [%][a–d]
–
100ꢀ1.8 (3)
2-methylmyristoyl-CoA
12a
12b
12c
13
19
40.8ꢀ9.9 (4)
<2 (3)
<5 (3)
20.1ꢀ1.8 (3)
<2 (3)
91.7ꢀ4.3 (3)
91.7ꢀ2.7 (3)
myristoyl-CoA
[a] Assay conditions: 60 mm (25R)-2, 200 mm inhibitor, 14.5 mgmLꢁ1 rat
liver homogenate was used as the AMACR source, 50 mm Na/K phos-
phate buffer pH 7.22, 358C, 15 min.; [b] (25R)/ACTHNUTRGNEUG(N 25S)-2 Ratio was deter-
mined by HPLC analysis. [c] Epimerization in the absence of inhibitor
was taken as 100%. [d] The number of runs is given in parentheses.
Scheme 7. Proposed concept of substituted acrylic acids as “pro-tracers”.
radioactive agents can be trapped in the AMACR-positive
cells through covalent binding with AMACR. In AMACR-
negative cells, thioester 12c (except of a relatively small pro-
portion that would be incorporated into the cellular lipid
fraction) would be partially hydrolyzed into the free acid,
which will leave the cell through passive diffusion and/or
efflux. Alternatively, 12c could be metabolized to give low
molecular weight metabolites (i.e., adduct with glutathione),
which will leave the cell. Therefore, radioiodinated 7c was
prepared and tested in vitro.
Figure 1. Kinetic plot for inhibitor 12c.
Tin precursor 10 was radioiodinated using chloramine T
(CAT) as an oxidant to give, after base-mediated hydrolysis
in EtOH at reflux and purification by HPLC, no-carrier-
added (n.c.a.) 131I-7c as a single (13E)-regioisomer in 56%
radiochemical yield over two steps and in more than 98%
radiochemical purity (Scheme 8).[34]
(Figure 1). Fitting the data to competitive, uncompetitive
and non-competitive inhibition models using GraphPad
Prism[27] and selecting the model most consistent with the
data using the corrected Akaike information criterion,[28]
showed that the kinetics were best described by competitive
enzyme inhibition; this process yielded the following param-
eters: Michaelis–Menten constant Km =29.0ꢀ2.8 mm, maxi-
mum enzyme velocity vmax =0.919ꢀ0.028 mmminꢁ1, and in-
hibition constant Ki =19.0ꢀ1.2 mm (Figure 1).
High affinity of the tracer to the target is a prerequisite
for successful radiological imaging. However, for metabolic
pathway tracers that work on the principle of “metabolic
trapping”[29] (FDG, FLT and other nucleosides, methionine,
FDOPA, and other amino acids), this parameter is not as
important as for receptor targeting tracers (DOTA-TOC,
bombesine conjugates, and other peptide tracers). For exam-
ple, 18F-FDG enters the cell with the assistance of glucose
transporter proteins (mainly GLUT I and Glut III) and is
phosphorylated by hexokinase II to give 18F-FDG-6-phos-
phate, which cannot be further metabolized due to the fluo-
rine substituent in the 2-position. The high polarity of 18F-
FDG-6-phosphate prevents its passive diffusion out of the
Scheme 8. Radiosynthesis of 131I-7c.
The cellular uptake of 131I-7c was studied in AMACR-
positive LNCaP, LNCaP C4-2, and DU-145 prostate tumor
cell lines. As a negative control, the benign hyperplasia
prostate cell line BPH-1 and siRNA-AMACR knockout
LNCaP C4-2 cell line [LNCaP C4-2(ꢁ)], which was specially
generated for this study, were used (Figure 2A).[35]
Incubation for 2 h resulted in only moderate selectivity
for radioactivity uptake and accumulation in the LNCaP
C4-2 wt cell line compared with the LNCaP C4-2 AMACR-
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B. D. Zlatopolskiy et al.
siRNA cell line [percent injected dose (ID%) per 5·105
cells: 27.0ꢀ1.5 vs. 13.6ꢀ0.4]. Moreover, no significant dif-
ferences between AMACR-positive malignant and
AMACR-negative benign cells were observed (ID%/5·105
cells: 12.7–27.1 vs. 17.5ꢀ0.9). However, after 24 h incuba-
tion, the radioactivity retention in LNCaP C4-2 wt cells was
more than four times higher as those in AMACR knockout
cells (23.3ꢀ0.9% vs. 5.7ꢀ0.1%). Importantly, at this time
point, the accumulation of radioactivity was significantly
lower in benign BPH-1 than in AMACR-positive tumor
cells (ID%/5·105 cells: 3.7ꢀ0.4 vs. 13.1–23.2) (Figure 2A)
and correlated with the expression level of AMACR as de-
termined by western blot analysis (Figure 2B). In contrast,
the cellular uptake and accumulation of 1-14C-palmitic acid
was virtually the same in both, wt and AMACR-siRNA,
LNCaP cell lines, indicating that the knockout of AMACR
does not affect fatty acid metabolism in general (Figure 2C).
The protein-bound radioactivity in lysates obtained from
LNCaP C4-2 wt and LNCaP C4-2 AMACR-siRNA cells
after incubation with the inhibitor was studied using SDS-
gel electrophoresis, followed by phosphorimager analysis
(Figure 2D). In contrast to AMACR knockout LNCaP C4-2
cells, a significant protein-bound radioactivity fraction with
a distinct band at about 47 kDa (corresponding to the mo-
lecular weight of full length AMACR-IVCrot-CoA conju-
gate) was detected in LNCaP C4-2 wt cells. Interestingly,
two additional radioactive fractions with increased molecu-
lar weights of approximately 70 and 75 kDa were visualized.
This increase in mass from the original 47 kDa is likely due
to the dimerization of full length AMACR,[36] which is re-
ported to be the most abundant IA transcript, with its alter-
natively spliced variants IB (22 kDa) and IIA (28 kDa).[37]
Our assumption is supported by the presence of bands with
the same molecular weights in the Western blot analysis of
AMACR expression in LNCaP C4-2 cells using a polyclonal
AMACR-specific antibody.
The radiolabeled precursor of the AMACR substrate, 131I-
(E/Z)-13-iodo-2-methyltridec-12-enoic
acid
(
131I-IVC-
11MeMC; 131I-20),[26] which differs from 7c only by the pres-
Figure 2. Panel A: In vitro study with 131I-7c. Cellular uptake of n.c.a 131I-
7c (0.5 MBq + 3 mg FFA/well) in LNCaP (A), LNCaP C4-2 wt (B),
LNCaP C4-2 AMACR-siRNA (C), DU-145 (D), and BPH-1 (E) cell
lines. Panel B: Western blot analysis of AMACR expression using mono-
clonal antibody. GAPDH was used as a loading control. Panel C: Cellu-
lar uptake of n.c.a 1-14C-palmitic acid (20 kBq + 3 mg FFA/well) in
LNCaP C4-2 wt and LNCaP C4-2 AMACR-siRNA cell lines. Panel D:
SDS-gel electrophoresis of cell lysates (20 mg protein per lane) obtained
from LNCaP C4-2 wt and LNCaP C4-2 AMACR-siRNA cell lines incu-
bated with 131I-7c and visualized by phosphorimager (left) compared with
Western blot analysis of AMACR expression in LNCaP C4-2 cells using
polyclonal antibody (right). GAPDH was used as a loading control.
Panel E: Cellular uptake of n.c.a 131I-(E/Z)-13-iodo-2-methyltridec-12-
enoic acid (131I-IVC-11MeMC, 131I-20) (0.5 MBq + 3 mg FFA/well) in cell
lines 1–5. Error bars represent standard deviation (ꢀSD) for three ex-
periments. ID% (5e5)=% of injected dose per 5ꢃ105 cells; LNCaP C4-
2(ꢁ)=LNCaP C4-2 AMACR-siRNA; n.c.a=no carrier added; FFA=
fatty acid free albumin, GAPDH=glyceraldehyde-3-phosphate dehydro-
genase, SDS-gel electrophoresis=sodium dodecylsulfate polyacrylamide
gel electrophoresis.
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ence of a methyl instead of methylene group in the second
position, was prepared and tested in the same panel of cell
lines. Similarly, after 24 h incubation, a higher cellular
uptake was observed in LNCaP C4-2 wt cell line than in the
AMACR knockout cell line. The intracellular accumulation
of 131I-IVC-11MeMC was higher in AMACR-expressing
tumor cells than in benign AMACR-negative BPH-1 cells.
However, this effect was generally much less pronounced as
for inhibitor 7c (ID%/5·105 cells: 6.1ꢀ0.3 vs. 3.5ꢀ0.2 and
3.9–26.8 vs. 2.4ꢀ0.1, respectively). The relatively high cellu-
lar uptake and retention of 131I-20 in DU145 cells could be
explained by factors that are independent of AMACR ex-
pression level, for example, by the higher efficiency of the
CoA-thioesterification of 20 compared with 7c[38] and/or by
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in this cell line (acyl-CoA thioesters are polar and cannot be
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Conclusion
[18] M. Smrcina, P. Majer, E. Majerova, T. A. Guerassina, M. A. Eissen-
stat, Tetrahedron 1997, 53, 12867–12874.
In this work, we demonstrated that 2-methylene-substituted
acyl-CoA compounds are strong inhibitors of AMACR. The
in vitro study with radiolabeled IVCrot (131I-7c) showed sig-
nificant AMACR-specific accumulation of radioactivity in
prostate tumor cells. Moreover, after incubation with 131I-7c,
covalent binding of radioactivity to the intracellular
AMACR enzyme was observed in AMACR-positive cells.
Because AMACR overexpression starts early in the onco-
genesis and persists during progression to higher stages of
PCa,[40] IVCrot is a potential candidate for imaging of pros-
tate tumors. Furthermore, IVCrot and other 2-methylene-
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Chem. Eur. J. 2011, 17, 10144 – 10150
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10149

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Received: November 4, 2010
Revised: May 9, 2011
Published online: August 2, 2011
10150
www.chemeurj.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 10144 – 10150